Autism and Environmental Factors

Omar Bagasra Claflin University, Orangeburg, South Carolina, USA and Cherilyn Heggen Immunologist, Florida, USA

Illustrations by Muhammad I. Hossain Orangeburg, South Carolina, USA This edition first published 2018 © 2018 John Wiley & Sons, Inc. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions. The right of Omar Bagasra and Cherilyn Heggen to be identified as the author(s) of this work has been asserted in accordance with law. Registered Offices John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA Editorial Office 111 River Street, Hoboken, NJ 07030, USA For details of our global editorial offices, customer services, and more information about Wiley products visit us at www.wiley.com. Wiley also publishes its books in a variety of electronic formats and by print‐on‐demand. Some content that appears in standard print versions of this book may not be available in other formats. Limit of Liability/Disclaimer of Warranty The publisher and the authors make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties; including without limitation any implied warranties of fitness for a particular purpose. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for every situation. In view of on‐going research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of experimental reagents, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each chemical, piece of equipment, reagent, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. The fact that an organization or website is referred to in this work as a citation and/or potential source of further information does not mean that the author or the publisher endorses the information the organization or website may provide or recommendations it may make. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this works was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the publisher nor the author shall be liable for any damages arising here from. Library of Congress Cataloging‐in‐Publication Data Names: Bagasra, Omar, 1948– author. | Heggen, Cherilyn, 1973– author. Title: Autism and environmental factors / by Omar Bagasra, Cherilyn Heggen ; illustrations by Muhammad I. Hossain. Description: Hoboken, NJ : Wiley, 2018. | Includes bibliographical references and index. | Identifiers: LCCN 2017056771 (print) | LCCN 2017059203 (ebook) | ISBN 9781119042266 (pdf) | ISBN 9781119042273 (epub) | ISBN 9781119042259 (hardback) Subjects: | MESH: Autism Spectrum Disorder–etiology | Environmental Exposure–adverse effects | Gene-Environment Interaction Classification: LCC RC553.A88 (ebook) | LCC RC553.A88 (print) | NLM WS 350.8.P4 | DDC 616.85/882–dc23 LC record available at https://lccn.loc.gov/2017056771 Cover Design: Wiley Cover Image: Courtesy of Muhammad Hossain Set in 10/12pt Warnock by SPi Global, Pondicherry, India

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Contents

Acknowledgments xi Prologue xiii

1 Introduction to Autism Spectrum Disorders 1 Smell of Autism 5 The Roundup™ Conundrum 7 Testosterone and Male Gender Bias 9 Connecting the Dots 12 Why Is There a “Spectrum” in Autism? 20 Are Genetic Mutations the Cause of Autism? 23 ASD Parent and Affected Child Exome Sequencing Display De Novo Mutation 23 More Than 1,000 Genetic and Genomic Disorders and Still Counting 24 Why Do Certain Chemicals Induce Specific Depletions of Certain Brain Compartments? 30 Genesis of an ASD Brain 31 Pinpointing Critical Steps Where the Autistic Brain Emerges 32 Is Finding Mutations the Path to Discovering the Genesis of ASD? 34 Does Brain Size Matter? 36 How Autism Develops in a Fetal Brain 36 Why Is There a “Spectrum” in ASD? 39 ASD and Vaccines 41 Thimerosal Containing Mercury Stays in the Body and Is Very Toxic 42 Summary 43 References 43

2 What is Autism? 51 Legacy of Autism 51 A Short History of Autism 53 DSM‐5 and the ASD Spectrum 60 vi Contents

Changes in ASD diagnosis approved by the APA 61 The impact of the Recent DSM‐5 Changes on Diagnosis and Support Practices 61 A New ASD Diagnosis Category: Social Communication Disorder 62 ASD or a Giant Spectrum of Socioneuropsychological Disorders 62 Asperger Syndrome 63 Pervasive Developmental Disorder Not Otherwise Specified 63 Autistic Disorder 64 Rett Syndrome 64 Childhood Disintegrative Disorder 64 Is Autism a Genetic Disease? 65 Synthetic Chemicals Lack Coevolutionary Adaptation 66 Myth of the Genetic Origin of Autism 67 Phenotypic Heterogeneity in Autism 67 Why Fragile X Syndrome or Tuberous Sclerosis Should Not Be Included in ASD 70 Tuberous Sclerosis 71 Is Finding Mutation the Path to Discovering the Origin of ASD? 72 How Quickly Does Human DNA Mutate? 73 What is the Mutation Rate in the Whole Human Genome? 74 Does Brain Size Matter? 74 Genetics versus Environment 75 References 77

3 Olfaction and Autism 83 How Do We Smell? 87 Summary and Conclusions 92 References 92

4 Oxytocin, Arginine Vasopressin and Autism Spectrum Disorder 97 Oxytocin 97 Why Oxytocin Therapy May be Important for ASD? 99 Hormones, Neuropeptide Arginine Vasopressin and Oxytocin in ASD 102 Development of Oxytocin and AVP Neurons in Various Animals and in Man 103 Oxytocin and Social Experience in Development 104 Oxytocin and Developmental Neurological Disorders 106 Exogenous Oxytocin Treatments in Humans 108 Intranasal and Intravenous Oxytocin Studies in ASD 109 Oxytocin Trials in ASD: Beyond the Hype and Hope 110 Summary and Conclusions 112 References 113 Contents vii

5 Male Gender Bias and Levels of Male Hormones During Fetal Development 123 Association between 2D:4D Ratio and Brain Connection Development 129 Male and Female Estrogen and Testosterone Hormone Regulations 130 Are there Synthetic Chemicals that Humans Are Not Evolutionarily Exposed To? 130 Why Male Gender Bias? 131 Male and Female Brains in a Test Tube 132 Effects of Three Different Levels of Testosterone on Neuronal Morphology 133 Molecular Basis of Gender Bias in ASD 134 References 136

6 Maternal Twins and Male Gender Bias in Autism Spectrum Disorders 143 The Conundrum of ASD Discordance in Maternal Twins 146 Role of Environment in Maternal Twins revealed by Numerous Methods under Many Conditions 149 What Types of Discordance are Observed in Maternal Twins? 149 Differences in Frontal and Limbic Brain Activation in Monozygotic Twin Pairs Discordant for Severe Stressful Life Events 150 Structural Connectivity of the Brain of a Child with ASD and That of the Unaffected Identical Twin 150 Differences in Genomic and Epigenomic Expression in Monozygotic Twins Discordant for Rett Syndrome 150 Differences in CNV between Discordant Monozygotic Twins with Congenital Heart Defects 151 History of Autism Becoming a Genetic Disease 151 Many Diseases That Were Considered Genetic are Being Reassessed 152 De Novo Mutations 156 To find a Scientific Analysis of ASD Genesis 158 What are Neuroblastomas? 159 What Method Did We Use? 160 Possible Etiologies of Autism 160 Epigenetic Explained 160 Epigenetic Changes and the Environment: How Lifestyle Can Influence Epigenetic Change from One Generation to the Next 161 Factors Other Than Environment That May Be Contributing to ASD 161 Older Age of Mother and Increased Risk 161 Conclusion and Summary 163 References 163 viii Contents

7 Autism and Exposure to Environmental Chemicals 169 Contribution of Fragrances to ASD 178 Effects of Fragrances on Male Oxytocin‐Receptor Positive Neurons 184 Effects of Fragrances on Female Oxytocin‐Receptor Positive Neurons 186 How Synthetic Chemicals in Fragrances Affect Fetal Brain Development 186 Synthetic Musks 186 How Do Synthetic Musks Get Into the Food Chain? 189 How Are People Exposed to Synthetic Musks? 189 Musks in Food 191 Diethyl Phthalate 191 Octinoxate 193 Benzyl Benzoate and Benzyl Salicylate 194 d‐Limonene 194 α‐Pinene 195 Synthetic EDCs 195 Why Is It Important To Look at EDCs and Their Potential Effects on Our Next Generations? 196 Where in Fetal Life Are Androgen Receptors Expressed? 199 Why Testosterone is Essential for Engineering a Male Brain 200 Spatial Memory 207 Anxiety‐Related Behavior 207 Play Fighting and Aggression 208 Adverse Effects of EDCs and Their Mechanisms of Action 208 Effects of Testosterone or AR Mimicking EDCs 211 Early Puberty in Males 214 Change in Sex Ratio 215 Effects of EDCs on Neurodevelopmental and Neuroendocrine Systems 215 EDC Effects on Steroid Hormone Receptors in the Brain 217 Are the EDCs and other Synthetic Chemicals Depopulating the Human Race? 219 Summary 223 References 224

8 Maternal Antibodies to Fetal Brain Neurons and Autism 235 Link between Damage to the Fetal Brain and Maternal Antibodies: A Double Jeopardy 235 Are there Examples of Such an Immune Mechanism? 236 Rh Incompatibility 236 ABO Incompatibility 237 Why are Fetal Neuroantigens Immunogenic to the Maternal Immune System? 238 Is There Any Evidence of a Link Between Synthetic Chemicals Exposure, Neurotoxicity, and Autoimmunity? 241 Contents ix

The Relationship between Autoimmunity and ASD 241 The Detection of Fetal Brain Neuroantigens in the Maternal Blood 241 What are the Functions of the Neuroantigens that are Being Destroyed by the Maternal Antibodies? 243 Animal Models and Neuroantibodies to Autism 245 Rhesus Macaques Model 245 Studies Using Rodent Models 246 Why Do Some Autistic Children Have Bigger Brains? 246 Is There a Link between Autoimmunity and Other Forms of Neurodevelopmental or Neurodegenerative Disorders? 248 A Chicken and Egg Conundrum 250 Tourette Syndrome and Obsessive Compulsive Disorder 250 Other Contributing Factors in Neuropsychiatric Disorders 250 Infectious Flu Virus 250 Influenza Vaccine and Narcolepsy 250 Other Viral and Nonviral Infections 252 Blood–Brain Barrier 254 What is the BBB? 254 Summary and Conclusions 256 References 257

9 Vaccines and Autism 261 Childhood Vaccines and Regressive Autism 261 Politics Versus Science in the Vaccination Era 262 A Short Glimpse of the History of Vaccines: Justification for Using Vaccines 264 What is in the Vaccines? 265 Thimerosal 270 What is the Evidence That Organomercurial Vaccines Pose a Higher Risk of Regressive Autism? 272 Why Thimerosal‐Containing Vaccines are Harmful: A Scientific Narrative 273 How Much Mercury is Given to Children Before the Age of 3 years? 274 Why Do Only Small Numbers of Children Develop Regressive Autism After Vaccination? 274 Can Measurements of Abnormal Cytokines Identify Children Who Are at Increased Risk for Regressive ASD? 278 Summary of Contributing Immunological Factors to ASD 278 References 279

Epilogue 287 Index 291

Acknowledgments

We are very grateful to Dr Donald Gene Pace, former Dean of Humanities and Social Science at Claflin University, USA. He was the first co‐author of this book, before he retired. His contribution to this book cannot be under- stated. We are thankful for his early contributions to the first few chapters of this book. We are also very grateful to Karen Brady, who kindly edited, advised and made commentary to improve this manuscript. Omar Bagasra is thankful to his undergraduate and graduate students whose dedication to the Autism Project cannot be underestimated. They spent countless hours and occasional weekends to carry out some of the most critical experiments that contributed to this work. We are thankful to our friends and colleagues for their moral support and encouragement.

xiii

Prologue

We believe no one has presented the predicament of human life more accurately than Rachel Carson in her book when she stated:

As crude a weapon as the cave man’s club, the chemical barrage has been hurled against the fabric of life – a fabric on the one hand delicate and destructible, on the other miraculously tough and resilient, and capable of striking back in unexpected ways. These extraordinary capacities of life have been ignored by the practitioners of chemical control who have brought to their task no “high‐minded orientation,” no humility before the vast forces with which they tamper. Rachel Carson, 1962, Silent Spring

In 2014, according to the Centers for Disease Control and Prevention, USA, one in 68 children suffered from Autistic Spectrum Disorder (ASD); and, in 2015, one in 45 children were considered autistic. If a doomsday prediction is to become a reality, is one out of two children to be born autistic within a decade? For the literally millions affected by ASD, our silent offspring, and even more desperate parents who find themselves bewildered at the alarming rise of autism, we need to identify and curb the “chemical barrage” and further onslaught that harm mankind’s future. In this book, we have done our best to present scientific data supporting previous observations that were only anecdotal to attempt to understand the complex puzzle that is autism. We describe the rapid proliferation of synthetic chemicals in our modern world and the effects on the developing human brain, endocrine‐disturbing chemicals that alter DNA, epigenetics, and hormones. From chemical fragrances to herbicides, synthetic chemicals are abundant in everyday life and the chapters in this book examine the evidence surrounding these chemicals and their effects, including on the developing human brain, and how that might explain certain characteristics observed in autism. xiv Prologue

Yet, this only begins to scratch the surface and we do not claim to have all the answers to the autism puzzle. Our goal in this book is to try to bring together as many pieces of the puzzle as possible in one place to begin to clarify the picture and spark discussion to ensure a safe environment for all of us, especially our silent offspring. Orangeburg, SC, USA, August 2017 Omar Bagasra Cherilyn Heggen 1

Introduction to Autism Spectrum Disorders

The question is whether any civilization can wage relentless war on life without destroying itself, and without losing the right to be called civilized. Rachel Carson, 1962, Silent Spring

The belief that there is a single defining autism spectrum disorder brain dysfunction must be relinquished. The noise caused by the thorny brain‐ symptom inference problem must be reduced. Researchers must explore individual variation in brain measures within autism. Lynn Waterhouse and Christopher Gillberg, 2014, Why autism must be taken apart. J. Autism Dev. Disord., 44(7):1788–92

The transition from a paradigm in crisis to a new one from which a new tradition of normal science can emerge is far from a cumulative process, one achieved by an articulation or extension of the old paradigm. Rather it is a reconstruction of the field from new fundamentals, a reconstruction that changes some of the field’s most elementary theoretical generaliza- tions as well as many of its paradigm methods and applications. During the transition period there will be a large but never complete overlap between the problems that can be solved by the old and by the new paradigm. But there will also be a decisive difference in the modes of solution. When the transition is complete, the profession will have changed its view of the field, its methods, and its goals. Thomas Kuhn, 1969, The structure of scientific revolutions, in Foundations of the Unity of Science, Volume 2, pp.146–7. Originally published in 1939

In the process of scientific research, data occasionally emerge that cannot be easily correlated with present theories. Still, scientists, like others, are naturally reluctant to adopt new theories when established concepts still appear viable, although in need of major updates or revision. Typically, the effects of such scientific reluctance manifest themselves in enlivened discussions at profes sional conferences, in academic circles and journals, or in books written by journalists, scientists, and medical doctors. In the area of autism pathogenesis, however, the consequences of decades of inadequate progress in coming to grips with the basic underlying biology of autism spectrum disorder (ASD) may be calculated not in journal publications, but in the quality of human lives for literally millions of silent offspring, and even more desperate parents, who find themselves bewildered at the effects of the disorder on both their children and themselves. This alarming rise in autism is not a case of some sudden rise in avian flu from the 1950s, or the unexpected Ebola epidemic in West Africa a few years ago that could have rapidly killed millions but fortunately quickly burned itself out. Another case in point is the epidemic of Zika virus, which is harming unborn fetuses that are then born with small heads (microcephaly) but this is also bound to pass within a couple of years. These are the cases that we may hear about now but then not again for decades or even centuries until they rise again, like the proverbial phoenix. The traditional paradigm regarding the underlying causes of autism asserts that it is a genetic disease that emerges when children inherit two bad genes from their parents that show up in the toddler (in genetic terms, recessive genes). Although thousands of genes or genetic mutations have been suggested as links to autism by a growing number of scientists, none provides the causa tive link that is clearly authentic and replicable. As appealing as the bad genes explanation appears at a superficial level, this fails to withstand scientific scru tiny. A fundamental reason why autism cannot be labeled as simply a genetic disease is that genes simply do not change that fast. And so many genes (now numbering in the thousands) cannot be responsible for causing ASD – a spec trum of heterogeneous symptoms. Consider sickle cell anemia. It results from the replacement of a single nucle otide in the beta‐globin chain, which occurred among our ancestors, some where in Africa, in the far distant past. This genetic adaptation to the malaria‐infested environment produced a defective gene that has been inher ited. A child, who inherits just one gene (i.e., sickle trait) from just one parent, is protected against deadly malaria, but a minority of the children will inherit the defective gene from both parents and succumb to sickle cell disease. If a corrective major mutation could take place in decades, sickle cell anemia would have disappeared eons ago when people of African descent migrated to the Americas between two hundred and four hundred years ago. The emergence of sickle cell anemia gives additional evidence of natural selection and mutation that has affected countless individuals. The Introduction to Autism Spectrum Disorders 3 adaptation of sickled red blood cells is most common among people with origins in malaria‐infested regions, such as the Western parts of Africa and the Mediterranean. The people with sickle cell disease inherited a copy of the recessive gene from both parents. Like a genetic Trojan horse, they carry this seemingly harmless hemoglobin S (HbS) gene because one copy of it brings a great survival benefit by reducing susceptibility to the deadly form of malaria; two copies, however, threatens health and even life. Zero copies of the HbS gene commonly resulted in infant mortality from malaria, which increased the percentage of persons with at least one copy of the HbS gene. In this case more is not better: children of two par ents with the HbS gene are much more prone to die of sickle cell anemia, which, again, leaves a greater living population of those who have just one parent with the HbS gene [1]. Similar adaptations to new environments took place when our ancestors moved from Africa and faced varying temperatures, altitudes, and a lower degree of sun UV radiation than that of their progenitors in Africa. Our ances tors adapted to the new mutagenic plants and chemicals that they came across during their journey “out of Africa”. For example, skin pigmentation accompa nied geographic mobility as our ancestors migrated to Asia, Europe, Australia, and elsewhere. Adaptation of lactose tolerance is another example of genetic inheritance, where 65% of the human population is lactose intolerant. This appears to be a relatively new mutation. The gene that allows human children to digest milk shuts off after three years of age when they are weaned. Certain human popula tions, however, evolved to be lactose tolerant. This rise of lifelong lactose toler ance in particular is pronounced in populations whose ancestors experienced the time in history when the domestication of cows and other herd animals occurred, a time in which the populations needed supplemental proteins throughout their lives. Can autism be due to genetic mutations or deletions? Does this relate to survival mechanisms? In reality, human communication and patterns of behav ior are governed by a large number of genes and by a complex orchestra‐like communication between these genes that are formed and organized during the early stages of human fetal development. It appears unlikely that a large num ber of genes involved in human communications and survival mechanisms would just begin to mutate in a precise manner so suddenly. Counterintuitively, and “so suddenly,” from the epidemiological point of view, has this improbable mutation transpired and done so rapidly? Autism was relatively rare about four decades ago. In 1960, only one child in 10,000 was diagnosed with autism. By 2011, one in 100 children (1%) was diagnosed with ASD; in 2013, the ratio had worsened to one in 88; in 2014, according to the Centers for Disease Control and Prevention (CDC), USA, one in 68 children suffered from ASD; and, in 2015, one in 45 children were 4 Introduction to Autism Spectrum Disorders

considered autistic [2]. This sudden rise in ASD, should it continue its rapid increase, could potentially lead to even more devastating results [3,4]. Where will it end: one child in five; one in two? The genes in humans, or any other large living organism, do not change that rapidly! It is illogical to believe that literally thousands of genes suddenly arose in the human race to cause autism. In the cases of sickle cell anemia, lactose toler ance, and human skin pigmentation, these are each determined by a different single gene. But dozens or even hundreds of genes determine most of the traits that make us who we are. Human behavior and communication abilities – the majority of the traits that we perceive as causing differences among individuals and among populations – are complex traits. It is against the laws of nature that those genes involved in what “makes us a human” are developing mutations at the speed of light, in evolutionary terms, so we must look for another cause or causes of this disorder. The obvious culprit is the environment. As is often quoted in computer pro gramming circles: “garbage in, garbage out.” More elegantly, we are dealing with the law of the harvest. Consider three basic elements that have changed in the industrialized nations or so called developed world in the last 40–50 years. The first and foremost is the introduction of synthetic fragrances that initially were discovered by accident. For example, musk ketone was discovered by Albert Baur in 1888 when he was making TNT explosive by condensing tolu ene and isobutyl bromide in the presence of aluminum chloride [5]. Similarly, artificial vanilla was synthetized by two German chemists, Ferdinand Tiemann and Wilhelm Haarmann, in 1874 [6]. These were huge innovations for the flavor industry which would grow into the multibillion dollar industry it is today. Most of the synthetic chemicals are made from coal‐tar, petroleum by‐products and deadly phenolic ring compounds. The next infringement on our naturally adapted lives was the introduction of insecticides and herbicides that Rachel Carson wrote about in her groundbreaking book Silent Spring. Introduction of synthetic chemicals to kill insects and herbicides to kill unwanted weeds have permeated into our lives. Many of these chemicals produce endocrine disturb ing effects. They either mimic our own natural hormones or bind the receptors found on our cells imitating the natural biochemical and physiological signal ing processes. We will describe the effects of these chemicals in detail in Chapter 7 and in other appropriate sections. We are well aware that the use of these three elements has crept into our way of life, although this creeping has been so silent, so gradual, that we hardly noticed the invasion, and largely over looked the damage inflicted. Few pay even the slightest attention to these three major shifts that have quietly invaded our lives in the Western World, and are now quickly sweeping the rest of the developing and underdeveloped popula tions as they imitate the economic and behavioral patterns that have brought so much wealth, and so much anguish, to the developed world. The last one is the introduction of vaccines that contain organic mercury, a known Smell of Autism 5 neurotoxin, a real fetal brain damaging agent. Its use is rapidly decreasing due to public outcry but it is still in use. We will discuss this topic and the potential solution in Chapter 9.

Smell of Autism

The first of these three major elements is the global rise in the use of synthetic fragrances. These innocent, seemingly essential elements that we feel it neces sary to use every day, if not truly made from quality natural sources (and usually they are not), can cause genetic mutations in fetal brain cells at extremely miniscule concentrations. They selectively eliminate certain types of brain cells, a point we will discuss in Chapter 6. Could it be that the highly pleasant‐ smelling environment in which our society lives is one of the culprits in this unpleasant outcome? Could it be that the outcome for mothers and their babies would be more pleasant if mothers‐to‐be breathed air that was free from such pleasant smells? Should prenatal precaution involve not only abstinence from alcohol and other drugs that can be mutagenic to the fetus but also the sub stances of fragrant smell? Is more actually less in the aromatic arena of life? We have shown in our research that many synthetic chemicals found in fra grances can induce a huge number of genetic mutations in human fetal brain neurons (Figure 1.1). To find a major root cause of autism, we may need to look

1400 1200 1000 Number of mutated colonies 800 600 400 200 0 Number of mutated colonie s

Gap Dior Lust Curve Guess Bvlgari Jodore 4-NoPD Davidoff BCB girls Skin must Givenchy Unknown Rosewood Tommy girl Vera wana LDE toilette Burberry brit Juicy cauture

Champ elysees Victoria’s secret Sex and the city Victoria’s secret Body by victoria Sex and the city Halle pure orchid Spark for women Adagio very oudsy No mutagen added Bodycology brown... Bath and body works Bvlgari rose essentielle Private label by color...

Figure 1.1 Mutagenic/carcinogenic effects of various perfumes as assessed by the Ames test. Only perfumes with high mutagenic/carcinogenic activities are shown. Briefly, 1 μl of each perfume was added into 150 ml of liquid agar containing Salmonella typhimurium, and 1 μl of histidine, mixed well and plated on a Petri dish for 48 h. The number of bacterial colonies was counted visually. Each experimental variable was performed in triplicate, and plotted as shown. Source: Adapted from Ref. [9]. 6 Introduction to Autism Spectrum Disorders

no farther than our own noses. It should be noted that as a result of a giant loophole in the Federal Fair Packaging and Labeling Act of 1973 [7], fragrance and cosmetics producers were explicitly exempted from having to enumerate cosmetic ingredients on product labels. Fragrance concealment is not illegal and is often used by the industry to hide from the public the full list of ingredi ents, even substances that can cause grave health problems. It is common prac tice for businesses to list the chemicals as simply “fragrance,” which may mean that about half of the ingredients are never revealed to buyers. Even worse, people who use cologne, synthetic fragrances, body spray, and other scented cosmetics are blindly exposed to dangerous chemicals since the Food and Drug Administration lacks authority to control mandates to manufacturers that require testing of all synthetic chemicals in fragrances for safety before being released to the public We have shown that chemicals found in fragrances can be extremely harmful for developing fetal brain cells (neuroblastoma cells: Figure 1.2). When applied on the skin or sprayed, many chemicals from fragrances are either absorbed or inhaled, and many of the chemicals damage fetal brain cells [8–11]. Also, during pregnancy the use of fragrances and other cosmetics may expose the growing fetus to diethyl phthalate (DEP), a common fra grance solvent that can cause abnormal development of reproductive organs in infant males [11–28].The most commonly used fragrances contain thou sands of synthetic chemicals, many of them capable of changing the normal pattern of a fetus’s brain development. In Figure 1.3, some of the harmful chemicals are listed. One may question why these synthetic chemicals have not affected adult human brains and caused adults to get autism. As discussed below, in an adult brain, there is very little neural division going on. There are only two

100 90 Cytotoxicity assay: Neuroblastoma cell line 80 70 60 50 40

Cytotoxicity 30 20 10 0 Pior Lus t Ga p CB E BR E DA H D& G HP O BBW BBW Curve Gues s Jodore 4-NoP D Passion Davidof f Burberry Givench y Unknown Malachit e Perry ellis Skin must Rosewood Vera wang Bodycology Stress relie f Christia dior Neg. control Estee lauder Ralph lauren Ralph lauren Juicy coutur e Palmo picas o Heat-beyonc e Victoria secre t Victoria secre t The body shop White diamond Champ elysees Sex and the city Tommy baham a Lola marc jacob s

Perfume samples

Figure 1.2 Bar graph showing the cytotoxicity of various perfumes against a neuroblastoma cell line. Source: Adapted from Ref. [10]. The RoundupTM Conundrum 7

Popular fragrances contain 14 secret chemicals on average Chemicals found in lab tests but not listed on product labels 30

Chanel coco

Halle by halle berry Old spiceQuicksilver after hours (for men) Britney spears curious Hannah montona secret...Jennifer lopez J.Lo glow Dolce & gabbana light blue Calvin kleinBath eternity& body worksfor men japanese... American eagle seventy Giorgioseven armani di gio (for men) Victoria’sAbercrombie secret dream & Fitchangels...Axe bodyfierceClinique spray (for... for happy men-shock perfume spray Calvin klein eternity (for women)

Figure 1.3 Most popular fragrances and their number of secret ingredients. Source: Adapted from Ref. [64]. small parts of an adult human brain where one can detect neurogenesis (or replication of neurons). These two places participate in odor detection and building long‐term memory. Therefore, in an adult human only dentate gyrus (a small part of the hippocampus) and olfactory bulb neurons are constantly replicating. And, these two small brain parts are damaged in individuals with Alzheimer’s disease and other degenerative neurological diseases. We explain in Chapter 7 how these illnesses of the elderly may be connected to environ mental insults.

The RoundupTM Conundrum

The second most commonly used element that has crept into our lives, and that lingers for lengthy periods of time, is a chemical found in a widely used herbicide: glyphosate. This common ingredient in RoundupTM is also found in dozens of other related products. Recently, glyphosate has been shown to be an important factor in the development of ASD, as proposed by MIT scholar 8 Introduction to Autism Spectrum Disorders

Dr Seneff. The use of RoundupTM has been increasing steadily and stealthily since the 1990s [29–32]. Figure 1.4 depicts the correlation between the use of glyphosate and the rise in ASD [29–32]. Glyphosate is one of the most widely used herbicides, derived from glycine, an amino acid. It was introduced to agriculture in the 1970s. Glyphosate tar gets and blocks a plant metabolic pathway known as the Shikimate pathway that is not found in animals but occurs in certain plants and bacteria. The Shikimate pathway is required for the synthesis of aromatic amino acids in plants [29–33]. After four decades of commercial use, and multiple regulatory approvals including toxicology evaluations, literature reviews, and numerous human health risk assessments, the clear and consistent conclusions are that glyphosate is of low toxicological concern, and no concerns exist with respect to glyphosate use and the risk of cancer in humans. Mainly because of these reports and its inexpensiveness, its use has skyrocketed 6,504% from 1991 to 2010, according to data from the United States Department of Agriculture’s (USDA’s) National Agricultural Statistics Service [30]. Glyphosate is the most widely used herbicide in the world, mainly because it is believed to be nontoxic to humans and it is inexpensive. Most importantly, glyphosate is perceived to be harmless because its mechanism of action is to disrupt, in plants only, the biochemical pathway known as the Shikimate pathway [(32]. Interestingly,

Correlation of Autism prevalence with glyphosate applied to crops 35000

300 Glyphosate use 30000 Autism prevalence 250 25000

200 20000

150 15000

100 10000

50 Number of children diagnosed with autism 5000 Glyphosate applied to corn and soy crops (1,000 tons)

0 0 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 Year

Figure 1.4 Bar graph showing the number of children diagnosed with ASD versus the amount of glyphosate used on corn and soy crops in the USA from 1995 to 2010. Of note, this correlation does not prove causation and the correlation may be simply an unusual event. Source: Adapted from Ref. [9]. Testosterone and Male Gender Bias 9 the Shikimate pathway is absent from human cells. However, human gut bac teria do contain this pathway [34]. Inhibition of the Shikimate pathway by glyphosate can have an adverse effect on gut bacteria and the gut microbiome can change, and this has been associated with autism [35–37]. The Shikimate pathway is essential in the synthesis of aromatic amino acids in both plants and microbes. These amino acids are the precursors to all the monoamine neuro transmitters, including serotonin, dopamine, and melatonin. Seneff claims that this disruption of the Shikimate pathway in human gut microbes poses signifi cant threats to health. Seneff further argues that glyphosate may be a key con tributor to the autism epidemic in the USA [29–31]. She explains that glyphosate kills beneficial forms of bacteria in the gut and causes an overgrowth of pathogenic bacteria, including Bacteroides fragilis and Clostridium difficile [35]. This bacterium induces leaky gut syndrome and produces p‐cresol, which is a phenolic compound and a known biomarker for autism. A higher level of p‐cresol in urine is associated with autism [38]. Glyphosate exposure also disrupts sulfate synthesis, as well as sulfate trans port from the gut to the liver and pancreas. Serum sulfate deficiency is another known biomarker for autism. Defects in serotonin supply have been associ ated with various mood disorders. Seneff argues that glyphosate’s disruption in the synthesis of serotonin can lead to a defective serotonin transporter gene, which would decrease the bioavailability of serotonin for neuronal sign aling. This decreased supply of serotonin in the brain is a major feature of autism [38]. The absence of the Shikimate pathway in human cells and the presence of the pathway in beneficial forms of bacteria in the gut (Bacteroides fragilis) should not be taken lightly, since the total number of these bacteria in our gut is 10 times higher than the total number of cells in an adult human body. The vital questions are how does this mechanism cause autism and do various herbicides cause other adverse effects? The answers to these ques tions have been the center of numerous investigations and many respectable investigators are looking at these questions. In brief, we can state that glypho sate causes profound alteration of the gut microbial environment and studies have confirmed that this herbicide may be associated with ASD [36,37]. However, it should be noted that Seneff’s hypothesis does not explain the gender bias in ASD [29,39,40].

Testosterone and Male Gender Bias

The third element that could help explain male gender predominance in autism is still a hypothesis. There is a commonly recognized, but inexplica ble, bias toward males in classical autism, with a ratio of ~5:1 in ASD [1–3,41], and in Asperger syndrome, the ratio is ~10:1 [1–3]. Aside from the familiar dogma that genetic components form the basis of ASD, not a single gene has 10 Introduction to Autism Spectrum Disorders

been conclusively linked to ASD as the single leading causative factor. In fact, over 1,000 genes, or thousands of single nucleotide based mutations (single nucleotide polymorphisms, SNPs), have been suggested as possible causal factors of ASD. It should be noted that in the example we gave earlier of the sickle cell gene, it is an SNP, where a single nucleotide A is replaced with T. Therefore, looking for thousands of SNPs that might cause ASD is illogical. Moreover, several epigenetic influences have been identified as potential co‐ factors in ASD’s complex etiology. Numerous environmental factors can potentially induce genetic mutations [41–56]. We will cover this topic in detail in Chapter 5. It has been hypothesized that exposure to higher levels of male hormones during the fetal or perinatal periods of development could heighten the risk of autism [56–59]. The extreme male brain (EMB) theory of ASD argues that exposure to fetal testosterone could cause gender differences in various autistic traits. A causative link between the organizational impacts of fetal testosterone on cerebral development and on ASD development is often suggested using a 2D:4D digit [60] ratio (Figure 1.5), a presumed biomarker.

RIGHT HAND 2D ÷ 4D Ring nger Index nger (4D) (2D)

3 3 4 4 2 2

11 Low ration High ration

Figure 1.5 A loose association of abnormal 2D:4D ratio with autism. Source: Adapted from https://i1.wp.com/www.handresearch.com/news/pictures/finger‐digit‐ratio‐stock‐traders‐ income.jpg. Testosterone and Male Gender Bias 11

The leading popular hypothesis to explain why more males experience ASD than females is the EMB theory, which proposes that children with ASD show a heightened type of the male cognitive profile, and postulates that gestational exposure to testosterone produces biological effects that are a cause of autism. Some animal studies evidence indicates that testosterone could mediate differences in cognitive ability between the sexes through its organizational impact on the brain. Recent evidence that upholds the EMB theory reported that the levels of sex steroid found in amniocentesis sam ples were correlated with ASD diagnosis [61,62]. The ratio of index finger (second digit) to ring finger (fourth digit) (2D:4D) has been extensively uti lized in autism investigations as a proxy to determine levels of gestational testosterone exposure. One observation states that 2D:4D is sexually dimorphic, and explains that ASD males tend to have a lower 2D:4D ratio than females. In other words, normal males have an index finger (2D) that is shorter when com pared with the ring finger (4D) than do females. This finding is not univer sally accepted as valid [62]. EMB theory advocates usually argue that sexual dimorphism is evident beginning with the first trimester of pregnancy, that it seems later to be basically static following birth, and that it is not affected by the androgen that accompanies puberty. The 2D:4D based evidence of sexual dimorphism is also based on endocrine models with lower or higher levels of fetal testosterone exposure, associated with complete androgen insensitivity syndrome or congenital adrenal hyperplasia, respectively. There appears to be strong evidence to link elevated fetal levels of testoster one in amniotic fluid to autistic symptomatology, as well as an increase in rightward asymmetry of the corpus callosum [57–61]. We will discuss this in detail in Chapter 5. Figure 1.6 depicts a possible connection between fetal testosterone and 2D:4D ratio. Some of the most interesting studies, based on the research of various teams led by Baron‐Cohen, focus on elevated fetal hormones in males and the result ant development of autism. Taking advantage of the rich data available in the Danish Historic Birth Cohort and Danish Psychiatric Central Register, these researchers have analyzed the amniotic fluid samples of 128 males who were born during the 1993–1999 period and who were given diagnoses, consistent with the recommendations of the International Classification of Diseases, 10th Revision (ICD‐10), of autism, of Asperger syndrome, or of other related conditions lumped together under the acronym PDD‐NOS (pervasive devel opmental disorder not otherwise specified). These diagnosed individuals were subsequently compared with a control group of typically developing individuals. These investigators examined the concentration of four different sex steroid hormones (testosterone, androstenedione, progesterone, and 17α‐ hydroxyprogesterone) in amniotic fluids, and determined how the levels of these hormones correlated with later diagnosis of ASD. These findings have 12 Introduction to Autism Spectrum Disorders

4 2 0.98 1.00

High ration

Figure 1.6 Possible connection between 2D:4D ratio and autism and differences between male and female 2D:4D ratio, associated with fetal testosterone (see Chapter 5 for more detail). Source: Adapted from https://stynoski.wordpress.com/research/digit‐ratios/.

provided the first specifically demonstrated evidence that individuals with autism have higher fetal male hormonal activity than typically developing individuals. This research suggests a correlation that involves elevated levels of fetal testosterone [57]. However, it does not really explain the causation behind the surge of autism over the past few decades. More recent analyses of EMB hypothesis have not found persuasive digital ratio evidence. Therefore, Guyatt et al. [62] analyzed a large number (6,015) of children with ASD and a corresponding control group but were unable to find a 2D:4D association related to ASD for either males or females. Here, it must be emphasized that ASD is a spectrum and a clear‐cut answer to a spectrum that relates to fetal brain development may not be possible. We will further explain this conun drum in Chapter 5.

Connecting the Dots

As illustrated in Figure 1.7, over the past four decades, a startling rise in ASD prevalence has been reported, rising from ~1 per 10,000 in the 1960s, to about one in 45 children today. We maintain that the alarming 20‐fold increase in autism in recent years is due to exposure of the human popula tion to an increasingly diverse set of synthetic chemicals including fra grances, many of which involve endocrine disrupting chemicals (EDCs) (male and female hormone‐like chemicals) [63]. Published laboratory and Connecting the Dots 13

1990s Synthetic food/drink avors introduced

1980s 1970s Endrocrime Glyphosate Disturbing 1:110 introduce Chemicas as herbicide became part of our modern 1:150 lives 1975s 1960s Synthetic 1:166 Plastics Fragrance introduced introduced at 1:250 Mass-scale 1:500

1:2500 1:100000 1:100000

1920 1970 1975 1995 2001 2004 2007 2009 2012

Figure 1.7 The alarming rise in synthetic chemical use and autism. Source: Adapted from https://naturaltothecore.wordpress.com/2013/03/10/ autism‐head‐lag‐and‐the‐core‐of‐wellbeing/. epidemiological scholarship have indicated that undisclosed chemicals labeled as fragrance, including those that produce many different scents, and increase product shelf life, control the time‐release mechanisms of various fragrances and that improve stability, possess endocrine‐disrupting properties [64–80]. These disruptors have a troubling track record, with links to augmented cancer risks [8,64], negative effects on fetal development [9,10], and metabolic dis eases. For instance, chemicals with the capacity to increase the expression of human estrogen receptors include oxybenzone, octinoxate, benzyl salicylate, benzyl benzoate, benzophenone‐1, benzophenone‐2, butylphenyl methylpro pional, and various synthetic musks (e.g., musk ketone, tonalide, and galaxo lide). Of these, benzophenone‐1, tonalide oxybenzone, and galaxolide also have an effect on androgens. Moreover, octinoxate, benzophenone‐2, and butylated hydroxytoluene have links to hormonal disruption of the thyroid [8–10,64]. Even at very low concentration levels, fragrances with these chemi cals as ingredients can be carcinogenic and mutagenic [10,64]. One ingredient used under the elusive label fragrance is acetyl ethyl tetramethyl tetraline (AETT) [76]. It was used for over two decades in soaps and cosmetics, and it has been found to cause behavioral changes and to promote white matter degeneration in the brain, including substantial demyelination and troubling axonal degeneration within the central peripheral nervous systems. In 1978, its use was voluntarily discontinued. 14 Introduction to Autism Spectrum Disorders

Musk ambrette, a fragrance ingredient that was banned in the European Union although allowed in the USA, causes myelin sheath production in addi tion to distal axonal degeneration [18,70,72,73]. Even if one considers the high fetal testosterone levels present during the early stages of gestation, this still does not explain the rapid rise of ASD in recent decades; one must also consider the impact of synthetic chemicals that behave like hormones, particularly like fetal testosterone or that bind to andro gen receptors in the fetal brain and can enter the blood circulation of the devel oping fetus [63,64]. We maintain that numerous synthetic fragrances contain testosterone‐like hormones [63,64]. Chlordane was widely used in termite prevention in buildings in the USA starting in the 1960s. In 1979 its use inside homes was banned, and then in 1983 it was also banned as a soil treatment for areas underneath homes [78]. Then, in 1988, citing concerns about environ mental degradation and harm to human wellbeing, the United States Environmental Protection Agency (US EPA) proclaimed a complete ban on chlordane [78]. At this point it is pertinent to ask why the three factors mentioned in the preceding sections (synthetic fragrances, glyphosate, and fetal testoster one‐like chemicals or EDCs) may be contributing to ASD development in children while they do not seem to have any apparent adverse effects in adult brains. An adult body has a minuscule degree of cell differentiation taking place in the brain compared with the degree of differentiation in a rapidly developing fetal body, especially during weeks 4–24 of gestation. The majority of adult brain cells are already differentiated and only very few types of cells are still regenerating but no longer differentiating. These include olfactory neurons and a few cell types in the amygdala that are involved in solidifying our permanent memories (we will return to this later in Chapter 7 explaining why Alzheimer’s patients lose their olfaction ability and lose their memory); at birth our muscle cells are already dif ferentiated. The major cell types that are still regenerating are neutrophils, immune cells, glandular and other epithelial cells. Our immune cells are already built, and B and T lymphocytes are regenerating and differentiat ing from pre‐terminal to terminal phases constantly. These differentia tions are not the same as with the primordial and progenitor cells that differentiate in the early stages of human fetus development and especially in the fetal brain neurons. Therefore, any environmental agents that inter fere with fetal brain development can cause major adverse effects even after birth. The most vulnerable period is during weeks 4–24 of gestation. There are many external agents that can inflict damage to the DNA of dif ferentiating and rapidly replicating cells in an adult immune system but adult muscle cells have already completed their differentiation during the late fetal period. Thus, they are in no real danger, but glandular and epi thelial cells are vulnerable. There is little surprise that the incidence of Connecting the Dots 15 prostate and breast cancers, as well as leukemia and lymphoma, has increased dramatically since 1960, with no thanks to the endocrine disrupting synthetic chemicals [63]. A fetus, and especially a fetal brain, can be a major target for any synthetic chemical that can cause mutations and/or interrupt the well‐orchestrated pattern of fetal brain development. A human brain contains over 100 billion neurons, of which about one‐fifth are located in the cerebral cortex. Each cortical neuron has on average of 7,000 synaptic connections to other neu rons, resulting in a total of 0.15 quadrillion synapses, or 1,500,000,000,000,0 00,000,000,000 connections between various neurons. If you measure the distance that they cover, it translates to more than 150 km of nerve fibers. This whole sophisticated human brain system is caged into a skull in which the brain floats in a special fluid with a volume of 1,350 cm3 and a total sur face area of 1,820 cm2 [81]. The most conspicuous connective structure within the human brain is known as the corpus callosum, a flat and wide bundle composed of over one‐ fifth of a billion contralateral axons, which play the crucial role of connecting the brain’s right and left cerebral hemispheres (Figure 1.8). Corpus callosum abnormality, a congenital disorder that is both severe and rare, a condition in which the corpus callosum is either completely or partially missing, is

Cerebrum Corpus callosum

Hypothalamus

Thalamus

Pons

Amygdala Cerebellum

Medulla oblongata

Spinal cord

Figure 1.8 An illustration of a typical adult human brain structure. Source: Adapted from https://www.khemcorp.com/autism‐and‐social‐anxiety‐research‐summary‐and‐science/. (See insert for color representation of this figure.) 16 Introduction to Autism Spectrum Disorders

associated with profoundly disabled intellectual capacity [82]. In Figure 1.8, we have illustrated the corpus callosum in the normal brain. A recent review of normal versus ASD brain structural analyses by brain imaging has shown that total brain volume and amygdala overgrowth were increased in ASD brains, while corpus callosum and cerebellum volume were found to be decreased in ASD brains [83]. Let us take this a little further and realize that the human brain contains an unknown number of compartments to perceive and respond to environmental challenges. This starts when the fetus is the size of a little green pea. Then, hundreds or perhaps thousands of different compartments arise from a single progenitor cell that repeatedly divides and then differentiates into second lay ers of progenitor cells, each destined to become one or more compartments of our wonderful brain. If any chemical takes away a few of these secondary pro genitor cells, then there will be fewer or smaller compartments. When certain cell types disappear from a fetal brain at early stages of development, the vac uum created by the untimely death and degeneration of selected neurons invites replacement by another type of neuron. The growth and migration rate of each type of neuron is different and these new neurons may divide slightly faster than the ones that died due to environmental factors (we will describe the mechanism in Chapter 5 and provide recent evidence from our laboratory). Therefore, when certain neurons grow faster, the brain enlarges and the num ber of those filler neurons swell. Keep in mind that an autistic child’s brain is generally 30% bigger than that of a typical child. Thus, the brain matter may be more densely packed, meaning many more neurons are squeezed into an ASD‐affected child’s skull than into that of a typically developing child. We repeat that a typical human brain possesses 130 billion neurons and 1,500,000, 000,000,000,000,000,000 synapses. A 30% increase in size means that about 130 billion neurons, and perhaps 100 to 10,000 times more synapses, are pre sent, which can make the child highly sensitive to sound, touch, pressure, and light (or make the child totally unresponsive to those stimuli if connections are scrambled). This translates into a sensory overload with perhaps one excep tion, the paucity of olfaction, or sense of smell. In a significant number of ASD children, the sense of smell is reduced, suggesting the early death of olfactory neurons during the developmental pathways (this concept is further discussed in Chapter 3). One example of this sensory overload was provided in 2012 when ABC News published a video of a young woman who felt trapped by autism but who also, with extensive support from family and others, began to express herself in a remarkable way at age 11 through a computer keyboard. Pushed to communicate by keyboard in order to get privileges she wanted, Carly Fleishmann proved not only that she could communicate but also that she was an exceptionally gifted thinker and writer. “Autism has locked me inside a body I cannot control,” she explained (https://www.youtube.com/ Connecting the Dots 17 watch?v=xMBzJleeOno). For years, her parents had openly conversed in front of her as if she were not capable of understanding what they were saying. They learned that she had been listening and understanding. She longed for people to see her for who she really was: “I am autistic, but that is not who I am. Take time to know me, before you judge me.” Carly became an Internet blogger, began writing a novel, and came to see herself as some one who could accomplish things and make contributions. “You don’t know what it feels like to be me, when you can’t sit still,” Carly proclaimed by typing her feelings, “because your legs feel like they are on fire, or it feels like a hundred ants are crawling up your arms”. This sensory overload of the senses of sound, touch, and sight illustrate the huge number of synapses that an ASD child’s brain possesses [8]. As noted previously, fetal brain development involves a precisely, highly coordinated sequence of cellular activities. First, the neocortex forms; this is critical to human development. The central nervous system emerges from the neural tube, which begins in the ectoderm as a plate of cells. This neural plate experiences a process known as folding, which produces folds, ridges, and grooves. This proceeds from the center of the growing fetus; it results in two open ends. Neural plate bending or folding commences on the 22nd day in week 3. At a time when most women do not yet know for sure that they are pregnant, these momentous occurrences are taking place. The frontal portion of the tube closes on day 24, helping to enclose the place where the brain will continue development. On day 30, during week 5 of gestation, the closure of the neural tube’s other end takes place. An elementary ventricular system starts to form and inside the central canal amniotic fluid becomes trapped. The closure of the neural tube prompts an increase in intraven tricular fluid pressure; this signals the onset of swift brain enlargement (Figures 1.8–1.10). At this stage, the human brain develops with remarkable speed accompanied by an enormous degree of organization. How fast does the brain grow and for how long? This initial phase of brain development spans the first half of gesta tion and is accompanied by rapid neuron creation and differentiation, up to approximately one‐fourth of a million neurons per minute, and by the migra tion of these newly created neurons toward the surface of the outer brain, where they play a fundamental role in the formation of different compart ments, or pre‐compartments, within the brain. This establishment of vast numbers of brain faculties continues after birth, especially up to 6 years of age, and lasts until puberty. In size and organization, no other life form on planet earth comes close to the capacity of this magnificent instrument, which is all the more remarkable since the human brain weighs only about 3 lb (1,300– 1,400 g). The adult human brain comprises only about 2% of the body’s total weight, yet it contains a disproportionate percentage (10%) of the total number of human cells in the body. To better understand the brain is to marvel at its 18 Introduction to Autism Spectrum Disorders

Short-tailed Star-nosed Smoky Hamster Rat shrew Mouse mole Eastern mole shrew

1.802 g 0.176 g0.347 g 0.416g 1.020 g 0.802 g 0.999 g 36 M52M 71 M90M 131M 200M 204M

Guinea pig Marmoset Agouti Galago Owl monkey

1cm 1cm 1cm 3.759 g 7.78 g 10.15 g 18.365g 15.73 g 240 M 634 M 936M 857M 1468 M

Capybara Squirrel monkey Capuchin monkey

1cm

30.22 g 76.036 g 3246 M 53.21 g 1600 M 3690 M Human

Macaque monkey

1cm

87.35 g 6376 M 1508 g 86000 M

Figure 1.9 The size of the human brain compared with that of other mammals. Source: https://www.google.com/search?newwindow=1&hl=en&site=imghp&tbm=isch&source= hp&biw=1264&bih=576&q=relative+size+of+mammalian+brains&oq=relative+size+of+ mammalian+brains&gs_l=img.3...1668.11635.0.13335.34.34.0.0.0.0.66.1418.33.33.0....0... 1.1.64.img..1.19.861.0..0j35i39k1j0i30k1j0i24k1.isfvXm_tTjU#imgrc=BGRS6TbiE0ByLM. (See insert for color representation of this figure.) Connecting the Dots 19

(a)(b)

Figure 1.10 (a) A normal brain neural network versus (b) ASD brain neural network. (See insert for color representation of this figure.) compaction, its diverse functions, its rapid development, and its virtually inex haustible creative potential. The enormous quantity of neurons in such a tiny space, with neuronal connections that stretch about 150 km, is impressive; the coordinated functioning of this complex neuronal system is nothing short of miraculous. As shown in Figures 1.8–1.10), one can glance at the brain size and the unrealistic numbers of extra neurons in an autistic brain, and can readily per ceive how such an increase in size, and accompanying enormous increase in synaptic connections, can literally make an ASD person exceptional. If the communication centers are spared by the toxic environmental factors that kill selective types of neurons then these amazing increases can result in the brains of an Albert Einstein, Thomas Jefferson, Wolfgang Amadeus Mozart, Michael Jackson, Abraham Lincoln, Isaac Newton, Charles Darwin, Bill Gates, or another of the recognized geniuses. 20 Introduction to Autism Spectrum Disorders

One can surmise that a well‐organized, effectively connected larger brain could make an autistic child extraordinarily brilliant, with much higher computational, engineering, mathematical, artistic, and other abili ties. In fact, this is what one sometimes sees in people with Asperger syn drome. However, such a pattern is not universal; the abnormal, haphazardly connected brain results in an intellectual dysfunctional brain. In either case, the person with ASD would still be missing or have smaller compart ments of the brain that are involved in communication, empathy, eye con tact, and the so called abilities of mind‐reading and body language interpretation. Why? What may be the cause of essential missing pieces? We argue that this is the result of the early death or reduction of special ized progenitor neurons that did not live long enough to later give rise to these social communication faculties, resulting in socially awkward but at times brilliant people. What would cause the death or reduction of such highly specialized neurons at very early and critical stages of fetal development? What environmental factors can contribute to a premature demise of such fundamental neurons, and how does this relate to male brain development? Why are male fetal brains more susceptible to envi ronmental insults?

Why Is There a “Spectrum” in Autism?

It is well documented fact that teratogenic agents (chemicals or physical insults) interfere with normal fetal growth, homeostasis, development, dif ferentiation, and behavior. Teratogens are xenobiotics and other factors that cause malformations in the developing fetus. There are six basic principles of teratology that were proposed by James Wilson in 1959 and still hold to be true [84,85]:

1) Susceptibility to teratogenesis depends on the embryo’s genotype that inter acts with adverse environmental factors (G×E interaction). 2) The developmental stage of exposure to the conceptus (fetus) determines the outcome. 3) Teratogenic agents have specific mechanisms through which they exert their pathogenic effects. 4) The nature of the teratogenic compound or factor determines its access to the developing conceptus/tissue (fetus or specific fetal tissues, organs, or even specific progenitor cell types). 5) The four major categories of manifestations of altered development are: death; malformation; growth retardation; and functional deficits. 6) The manifestations of the altered development increase with increasing dose (i.e., ranging from no effect to death and demise of a fetus). Why Is There a “Spectrum” in Autism? 21

When describing a teratogen, one may think of three basic characteristics: 1) A given teratogen may be organ specific or cell receptor specific (i.e., like EDCs or synthetic chemicals) [63,64]. 2) It may be species specific meaning only certain species may express recep tors for those teratogens. 3) It can be dose specific meaning a very tiny dose may kill certain specific cells at a certain stage of gestation but may require much higher dose at a differ ent stage of gestation (many synthetic chemicals fall into this group). Figure 1.11 illustrates the concept. The last point is where we think the “spectrum” manifests in ASD. The syn thetic chemicals (i.e., fragrances, herbicides/pesticides and EDCs) hit specific brain progenitor cells in a developing fetus. For example, if a given chemicals enters a fetus brain and only binds to oxytocin receptors or olfactory neurons and kills those cells in the very early stages of gestation, it will result in total or almost complete deletion of those progenitor cells that are involved in social communication or olfaction (smell). In the early stage of gestation (i.e., 8 weeks of gestation), there may be only a few progenitor neurons that would ultimately represent the faculties of communication or olfaction. However, if the same chemical enters the fetal brain in the later stages of development (i.e., 24 weeks of gestation) at the same dose, then we would expect that there would be mil lions of partially differentiated second or third progenitor neurons for oxytocin receptors or olfaction receptors, so they may only be partially damaged – layers of creating a “spectrum” of deficit (see Chapters 3 and 4). One can think of this in terms of how an oak tree grows. If one kills the seed, it will be akin to a miscarriage; if one cuts off a large branch when the tree is just 30 cm tall (equivalent to 15 weeks of fetal gestation), then the tree will be a lopsided oak (equivalent to showing some degree of neurologic deficit). However, if one cuts off multiple branches when the tree is 1 year old (32 weeks of gestation), the tree may grow almost normally and those branches will regrow (equivalent toa relatively small adverse effect being observed). Presented in a simplistic fashion, Figure 1.12 represents the changes just described.

Extreme disability Autism Asperger syndrome

Figure 1.11 A metaphoric illusion of ASD to colors of light. 22 Introduction to Autism Spectrum Disorders

Typical child

Spinal cord Cerebral hemisphere

Hind brain Mid brain Mid brain 40 days Fore brain 35 days 50 days Cerebellum Pons

25 days Medulla 100 days

5 months

9 months

Autistic child

Spinal cord Cerebral hemisphere

Hind brain Mid brain Mid brain 40 days Fore brain 35 days 50 days Cerebellum Pons 25 days Medulla 100 days

5 months

9 months

Are Genetic Mutations the Cause of Autism?

Since 1977 the belief has persisted that autism is a genetic and heritable disease [8]. Both fathers of autism – Hans Asperger and Leo Kanner – thought that autism ran in families and was heritable (not necessarily genetic). They observed that the parents of autistic children were educated, elitists, and imparted some version of autism to their offspring in an inherited fashion. The most compelling evidence of the genetic origins of ASD is that maternal twins show a significant concordance, according to some studies over 90% concordance. According to this logic, both identical twins would show autism! A thorough inspection of outcomes and claims that support a strong genetic source of autism demonstrates that this interpretation is simply incorrect. It is the erroneous product of methodological biases, flawed approximations, and exaggerated media reports. Hallmayer et al. [42] in 2012 conducted one of the most extensive discordant twin‐pair studies to date. They undertook 192 studies of twin pairs, and found that a high degree of ASD risk (~50%) in maternal twins was the result of environmental factors, and that only a lesser risk was the result of heritability. Of importance, they did not incorporate into their analyses the single copy number variations (CNVs) and de novo muta tions, as noted below. De novo mutations occur within the womb during ges tation in the fetus only; thus, they are not inherited from a parent but caused by something else following fetal development. This suggests that some agents, such as chemicals, cause mutations during gestation in fetal brain cells. We will consider this mechanism further shortly. We have devoted a whole chapter to ASD and discordant twin‐pairs (see Chapter 6).

ASD Parent and Affected Child Exome Sequencing Display De Novo Mutation

A recent report in exome sequencing has revealed several fundamental aspects related to ASD. The exome is composed of exons of genes, which are basic coding units and stands for “expressed genes”. There are approximately 30 million base pairs in the exome, which is about 1% of the total human genome. Exome sequencing is carried out by choosing the exons using one of a number of new solution based or array methods. The cDNA selected through one of these processes is organized through massive parallel sequenc ing, and by comparison with the reference genome SNPs are identified [46,50–55] (Figure 1.13). Exome sequencing analysis based on data regarding trios (two parents plus an affected child, thus forming a trio) have found de novo mutations absent in the parental exome but present in their children, presumably due to in utero 24 Introduction to Autism Spectrum Disorders

Exome sequencing

Exons Transcription, elimination of intron transcript segments, and spilicing of exons

Exome

Figure 1.13 Illustration showing how the exomes are assembled and exome sequencing is carried out. Only ~1% of human genomes contain exons. In exome sequencing all the exons are joined together into one large assembled format and then sequenced. The exome represents all the genes that direct the production of thousands of proteins. Source: https:// d2gne97vdumgn3.cloudfront.net/api/file/AuHulxYbQ46mPuuqdJ6H. (See insert for color representation of this figure.)

exposure to environmental pollutants [8–13,43,49]. In contrast, studies involv ing genome‐wide and targeted microarrays have revealed substantial de novo CNVs that were meaningfully enriched among the proband (this term is used for the persons who are the original or index point for a genetic disease or a study subject) under consideration when they were compared with controls or siblings unaffected by ASD [85–88]. Comparative studies, using both subse quent and initial higher resolution methodology, have found a de novo CNV level of approximately 8% for sporadic ASD patients, while the level for unaf fected siblings is only about 2% [87]. Moreover, for children who experience general developmental delay and also ASD, the level of de novo CNVs may reach as high as 15% [8–12, 87]. Again, this points to a different cause or causes of ASD. We believe these are synthetic chemicals.

More Than 1,000 Genetic and Genomic Disorders and Still Counting

We have asserted that ASD is regarded as a disorder that is heritable; still, stud ies of candidate gene association, CNV, and genome‐wide association have not yet located a single causative gene, a single nucleotide, or a single CNV that can More Than 1,000 Genetic and Genomic Disorders and Still Counting 25 account for 85–90% or more of ASD cases [41,42]. Most significantly, trio exome sequencing investigation, where the complete exome (messenger RNA or the genes that carry out the daily functions of a living being) or exomes of both normal parents and their autistic child are fully sequenced. These sequencing studies have revealed that the child’s genes show de novo functional loss variants that do not exist in either parent [89–92]. ASD is complex, and it is estimated that there are hundreds of genetic loci or genes and a thousand or more SNPs that may have a causative impact on ASD development. It should be noted that SNPs are noted to be 10–20 times more common in ASD than in the normal control children. The higher the number of SNPs, the worse the degree of autism (i.e., higher prevalence of intellectual disability). If we look at the three characteristics of teratogenic effects, we can see that there appears to be a dose relationship as well as timing when a synthetic chemical imparts it effects on a developing fetus. In an effort to pull together all possible genes and recurring genomic imbal ances that have been posited to contribute to the etiology of ASD, a recent exhaustive summary of literature research, both of the basic research and clinical varieties, has identified 44 genomic loci and 103 disease‐implicated genes for subjects with autistic or ASD behaviors [93–103]. These loci and genes have each been in some way causally implicated in the intellectual disability associ ated with ASD or autism, which suggests that this related pair of neurodevel opmental disorders have genetic bases in common. For epilepsy and ASD, there is also genetic overlap in many instances. All things considered, these findings have demonstrated convincingly that autism cannot be categorized as a single unique clinical entity but rather as a complex behavioral manifestation of many, perhaps even hundreds, of genomic and genetic genomic disorders [104]. The exploration of these exhaustive analyses, with their supporting data, evidences the likelihood that such a large number of genetic mutations is not the sole cause of ASD. Rather, secondary effects seem to be at work, mutagenic agents that harm the developing fetus as gestation progresses [8–13]. Most of these thousands of suspect genes may, in fact, not relate to ASD. They may simply be part of abnormal developmental processes resulting from exposure to synthetic chemicals, and investigators may be pursuing false leads on unpro ductive trails. Similar false leads have occurred throughout recent human medical history; this is not a new phenomenon. A glaring example is the misinformation about the cause of AIDS. Initially, it was believed that AIDS was caused by homosexual activity and that the semen/ sperm of those involved caused AIDS [105]. Then, Professor Peter Duesberg put forward the hypothesis that “all AIDS diseases in America and Europe that exceed their long‐established, normal backgrounds are caused by the long‐ term consumption of illicit recreational drugs and by AZT and its analogs: Hemophilia‐AIDS, transfusion‐AIDS, and the extremely rare AIDS cases of the general population reflect the normal incidence of the AIDS‐defining 26 Introduction to Autism Spectrum Disorders

diseases, plus the AZT‐induced incidence of these diseases under a new name.” The key to this so called drug‐aids hypothesis was the assumption that chronic AIDS‐defining illnesses were only suffered by those who consumed illicit drugs, with an underlying sub‐thesis that HIV‐1 does not exist! Unfortunately, this hypothesis received support from the well‐known researcher Dr Kary B. Mullis, who received the Nobel Prize in 1993 for his discovery of the polymer ase chain reaction (PCR) method. Believing this false dogma, the South African government ignored the AIDS crisis and the evolving treatment that became available, thereby contributing to the spread of the AIDS pandemic. We recount these events to show that how sometimes the paradigm can be wrong, and that clinging to false paradigms can take a heavy toll on global health [105]. Removal of the shaded lenses of genetic bias and an unbiased examination of scientific facts invites us to turn the traditional genetic dogma with regard to ASD on its head, and focus on the environmental factors that have caused ASD cases to skyrocket in recent decades. Such pollutants can induce genetic muta tions in fetal brain cells at the early and most crucial stages of brain develop ment. Twin studies clearly refute the idea that genetic inheritance is primarily responsible for ASD; if it were, why do so many genetically identical (maternal) or monozygotic twins not share the same ASD experience? Figure 1.14 supports the idea that environmental causes of birth defects are well established, and these defects are not primarily genetic diseases. Literally hundreds of environ mental agents can cause genetically mediated malformations [63]. Therefore, ASD may well owe its complex origins to complex toxic interactions in a threatening chemical environment. ASD appears to be much more a matter of public pollutants than of genetic inheritance (Figure 1.14). Fetal malformations that result from a wide variety of drugs (i.e., valporic acid), numerous infectious agents, hormones (or synthetic chemicals that dis turb the hormonal balance), heavy metals, radiation, and alcohol are well known and documented. With few exceptions, these causal factors have been linked to specific malformations. Figure 1.15 summarizes various teratogenic agents (i.e., those that cause damage to a fetus). Due to astonishing progress in developmental biology and diagnostic proce dures, researchers can now precisely identify pathways that have been modi fied during the time of human fetal development (ontogenesis), and the precise timing of teratogenic interference that resulted in malformations. Figure 1.16 displays the times at which various parts of the developing fetus are highly susceptible to such interference (Figure 1.17). We maintain that ASD is the cumulative result of exposure to various toxic chemical agents that affect certain cell types in the brain during specific peri ods of fetal development. Considering this logically, we see that the agents mentioned below do, in fact, promote fetal brain malformation and do so in a patently obvious fashion. For autistic children, brain size is larger, and this has been traced to specific parts of the brain. The most significantly affected areas Figure 1.14 Various malformations resulting from teratogenic agents, including unique malformations that are organ specific. Among these are malformations caused by exposure to thalidomide. One does not see any of these types of mutations in ASD children. Source: Adapted from http://www.thalidomide.ca/recognition‐of‐thalidomide‐ defects/; http://www.merckmanuals.com/professional/pediatrics/congenital‐craniofacial‐ and‐musculoskeletal‐abnormalities/common‐congenital‐limb‐defects; http://www. dailymail.co.uk/news/article‐2198015/Paralympic‐swimmers‐incredible‐journey‐Iraqi‐ orphanage‐London‐2012‐Australia.html; and https://www.researchgate.net/ figure/26760442_fig6_FIGURE‐6‐Mirror‐hand‐attributed‐to‐ZPA‐cells‐in‐the‐anterior‐limb‐ margin‐and. http://keywordsuggest.org/gallery/573175.html. (See insert for color representation of this figure.)

Radiation Synthetic chemicals Alcohol

Drugs

Hormones

Placenta German measles Lead, Mercury

Figure 1.15 Common teratogenic agents that affect a developing fetus. Source: https:// www.google.com/search?newwindow=1&hl=en&site=imghp&tbm=isch&source=hp&biw= 1264&bih=576&q=teratogens+examples&oq=teratogens&gs_l=img.1.5.0l8j0i30k1j0i5i 30k1.1540.4164.0.10616.11.11.0.0.0.0.73.505.10.10.0....0...1.1.64.img..1.10.505.0..35i39k1. iV‐LM2I‐xgA#imgrc=UcRG‐EDY52CxhM. Reproduced with permission of S. Branch. 28 Introduction to Autism Spectrum Disorders

123 45 678916 20–36 38 Period of dividing zygote, Embryonic period, weeks Fetal period, weeks implantation and embryo, Brain Palate weeks Eye Ear Eye Ear Heart Eye Heart

External genitalia

Central nervous system

Heart

Upper limbs

Eyes

Lower limbs

Teeth

Palate

External genitalia

Not susceptible to teratogens Ear

Prenatal death Major congenital anomalies (red) Functional defects and minor congenital anomalies

Figure 1.16 Developmental progression and susceptibility to teratogens and fetal loss. The bars indicate periods when organs are most sensitive to damage from teratogenic agents. Note that in most literature development of the olfactory system is missing. The brain starts developing 18 days after fertilization. Often women do not know that they are pregnant until after a few weeks have passed. Therefore, pregnant women who are exposed to certain synthetic fragrances, either through inhalation or epidermal exposure, and food‐flavor chemicals, through ingestion (e.g., teas, chewing gum, and other food flavors), may put their embryo at risk without even knowing it. Source: Adapted from Ref. [8]. (See insert for color representation of this figure.)

of the ASD brain are the amygdala, frontal lobe, thalamus, hypothalamus, and hippocampus. Also, there is a typical loss of olfactory bulb capacity that results in a significant reduction in the ability to smell. What agents could potentially act as ASD causative factors? Our research indicates that every one of the 98 fragrances we tested using the Ames test, a gold standard to detect carcinogens and mutagens and required by FDA as one of the safety tests, induced fetal brain mutations at the cellular level, even with dilutions as small as 1:50,000 [10]. This suggests that synthetic chemical expo sure levels that may appear innocuous are not harmless to highly vulnerable fetal brain cells, and that significant fetal mutations may be linked to such exposure. However, we do not believe that these mutagenic agents are respon sible for ASD directly. We believe that these ASD unrelated mutations, thou sands of those SNPs and CNVs, are erroneously claimed to cause ASD. In reality, they are just associated mutations and not the cause of ASD. We emphasize the point that over 4,000 synthetic chemicals are used, collectively, More Than 1,000 Genetic and Genomic Disorders and Still Counting 29

Autism and the brain The areas of the brain affected by autism, which stems from abnormal brain development:

Hypothalamus Frontal lobe Thalamus

Amygdala Cerebellum Important to processing emotions, behavior

Hippocampus Involved in learning, memory

Spinal cord

Autism and the brain - Cells are smaller, more densely packed in certain areas - Have shorter, less developed branches

Figure 1.17 Area of an autistic child’s brain affected by neuromodifying agents during fetal development. Source: https://www.google.com/search?newwindow=1&hl=en&site= imghp&tbm=isch&source=hp&biw=1264&bih=576&q=Autism+brain&oq=Autism+brain &gs_l=img.3..0l10.2769.7284.0.9636.13.13.0.0.0.0.302.1133.11j3‐1.12.0....0...1.1.64. img..1.12.1133.0..35i39k1.‐vVn0071Vjw#imgrc=uP1nZdh1rl2osM. in the typical fragrances used in the western world: these chemicals are com monly produced from petroleum products, and many are benzene ring prod ucts, which is alarming given benzene’s well‐established reputation as a mutagenic agent (Figures 1.1 and 1.2). Then, what causes ASD, if these chemicals that induce mutations in the fetal brain cells do not directly cause ASD? The reasons for our opinion are two‐ fold: first, ASD is primarily not a genetic disease but a neurodevelopmental disease with a wide spectrum, hence the name ASD; and secondly, over 98% of our genome consists of noncoding DNA, and mutations in those parts of the DNA may not have any consequences. As part of our exploration of the molecular reasons why ASD has greater prevalence in male children than in their female counterparts [12], we dis covered that male‐derived fetal brain cell lines are much more sensitive to fragrances and consequently dramatically lose their arginine vasopressin‐ and oxytocin‐positive receptors that carry neurons. The loss of arginine 30 Introduction to Autism Spectrum Disorders

vasopressin‐ and oxytocin‐positive receptors positive neurons is not as pro nounced in the fetal neuronal cell lines of females as compared with males. These specific neuron types are known to play key roles in male brain develop ment. As we explain below, ASD results from degenerations of specific types of neurons at the early stages of human brain development when exposed to certain synthetic chemicals that are commonly found in fragrances and other chemicals. We will discuss this in detail in Chapter 4.

Why Do Certain Chemicals Induce Specific Depletions of Certain Brain Compartments?

The brain of ASD children is a complex mosaic. The typical newborn human brain possesses hundreds, perhaps even thousands, of different faculties, and is comparable with the Amazon Jungle in which millions of species thrive indi vidually. Yet this individualized existence takes place within a complex ecosys tem that involves complex patterns of interaction and communication. In the jungle that is the developing human brain, the speed of communication is exceptionally rapid. The human body develops from an extremely small genesis of a single ferti lized egg. Growth takes place quickly and through a series of cell divisions, body mass expands to include, at maturity, approximately 3.7 trillion cells. However, far from being uniformly homogeneous, these cells are beautifully apportioned throughout the various bodily organs, each of which contains various distinct tissue layers, which in turn contain one or more histologically recognizable cell types. The human brain is the leading organ in terms of het erogeneity and complexity, and includes the greatest cellular diversity in terms of length, shape, size, and function. In spite of modern molecular and immu nological characterization methods, the human brain is still to a significant degree a mystery to researchers – a true black box. During the period of early fetal development, stem cells proliferation and development facilitates distinc tive neural progenitor cells; these further differentiate into a variety of precur sor cells that then further differentiate and enable the creation of numerous specialized populations within the human brain. Eventually, the brain becomes home to over 100 billion neurons. Many cell divisions are requisite to the pro cess of manufacturing the large variety of neuronal types that make up the varied faculties present in the fully developed human brain. At any point in these complicated processes, mutations can occur. Fortuitously, most human DNA is composed of noncoding genes, and this renders many mutations of little or no significance. Thus, if the coding genes that represent only 1 or 2% of the entire human DNA genome are not damaged, then the countless other potential mutations can occur without producing negative phenotypic effects. Genesis of an ASD Brain 31

However, the human brain is uniquely vulnerable in the area of the regenera tion pattern of neurons, hence our notion that the genetic mutations associ ated with ASD are not the cause of ASD. However, the most important point in that during early fetal brain development, loss or untimely degeneration of certain progenitor cells can result in a permanent loss of associated faculties. The body has an enormous capacity to heal itself, but there are exceptions, and the loss of specific types of progenitor cells or mother neurons accompanying early fetal brain growth and differentiation can be permanent and life chang ing. Avoidance of exposure to chemicals that have the capacity to bind highly specialized progenitor cells is particularly crucial during early fetal develop ment. Why is it pertinent to explore such an argument? If one analyzes with just the simplest logic, the importance of the point becomes clear. Fragrances are designed to bind to specific odor neurons. As a matter of fact, all fragrances and food emit chemicals that bind adult human neurons, which facilitates the sense of smell. Fragrances (perfumes in particular) reach our blood stream due to their capacity to be absorbed via skin and inhalation, and this is by design. Once these chemicals enter the blood stream, even when highly diluted, they can still reach the brain of developing fetal brain neurons. If they bind only a few specific neurons, these neurons suffer spontaneous death. This concept has been proven to be a fact both in a recent report published in Science [106] and in findings of our research group (see below and Chapter 3 for details).

Genesis of an ASD Brain

Of course, the most dangerous stage of fetal brain development is the loss of specific progenitor cells that subsequently causes damage to complex human faculties and leads to aberrant behaviors linked to the ASD spectrum. If a large number of progenitor cells is lost in the very early stages of gestation, notably during the first 5–8 weeks of pregnancy, this may precipitate brain death, resulting in loss of the whole fetus through naturally occurring sponta neous abortion (i.e., miscarriage). In 97% of cases, the fetus develops fully but in 3% of cases the newborn has some type of mutation. It is estimated that perhaps 50% of pregnancies, many so early as to be unrecognized by the woman involved, are lost due to a variety of reasons that result in early miscar riages. This is nature’s way of keeping a healthy population. If only a few pro genitor cell types are lost at a later stage in the pregnancy, about 8–14 weeks of gestation, then the fetus may be born at full term with perhaps the loss of certain brain faculties but with also the enlargement of others. This is due to the fact that when a certain progenitor cell type is lost, it creates a mechanical and physical vacuum that needs to be filled, and in this case another pro genitor cell will grow into that newly created space and perhaps grow at a slightly faster replication rate, resulting in enlargement of certain brain 32 Introduction to Autism Spectrum Disorders

compartments. As we have shown earlier, in the majority of cases, an ASD child has a 30% larger frontal lobe, and an abnormally large amygdala, hip pocampus, thalamus, and hypothalamus. Similarly, the ASD child has lost olfactory capacity, the normal ability to smell, and also has damage to the part of the brain that controls communication in the frontal lobe. Note the obvious connection between the loss of olfactory capacity and the exposure to syn thetic fragrances [9]. This idea of partial missing pieces of human brain or other parts of the body due to selective damage is not a new concept. For example, Thalidomide was used in Europe from 1958 to 1961. This resulted in limb abnormalities, where the newborns’ limbs were partially developed (Figure 1.14). As seen in Figure 1.17, the part of the brain that controls emotions, empathy, and communication can show decreased capacity. In the case of Asperger’s, the vastly increased number of synapses can dramatically increase brain size and capacity in some ways, while presenting weaknesses in others.

Pinpointing Critical Steps Where the Autistic Brain Emerges

To ascertain at what stage of fetal development autism emerges, what types of approaches would reveal the potential extent of neuronal progenitor cell loss that results in ASD? One has to develop a precise map of each compartment of the human brain tree. The human brain represents an enormously intricate puzzle, assembled from neurons derived from a series of committed progeni tor cells, each cell representing a branch of a giant tree at the early stages of fetal brain development. Lineage‐tracing analyses have demonstrated that neuronal cohorts frequently derive from a common ancestor, and that com mon progenitor cell types give rise to specific subtypes of neurons. In specific regions of the brain, including the cortical columns, the assembling of func tional circuits occurs, and this assembling draws on the same cell types that grew out of specific progenitor cell types. Therefore, an all‐inclusive neuronal circuit could display the same developmental alterations that had their genesis in a common cell type progenitor. The skills and technology are becoming available to analyze such structures at molecular, cellular and subcellular lev els, and to characterize neurons with specific origins at various immunological levels. Ultimately, it would be possible to dissect out entire population of origi nal progenitor cells for the neurons from specific faculties of the brain; however, this exceeds current capacity [107–110]. Nevertheless, preliminary investigations are being pursued to ascertain the “leaves” and “branches” that stem from the so called human brain tree. One approach involves tracking the miniscule genetic alternations that accompany Pinpointing Critical Steps Where the Autistic Brain Emerges 33 every neuronal cell as transcription occurs. The tree, with its regularities and mutations, is a genealogical wonder that is not yet totally understood or ren dered predictable. The tree will be better comprehended as records of muta tions are assembled that document errors in DNA replication as new cell divisions occur. The rate of mutation may well be more rapid in cases of expo sure to agents not available in most centuries of world history, including syn thetic chemicals to which many of us are subjected to repeatedly. It is logically apparent that every cell could have a personal genomic fingerprint; when these cellular genealogical trees are better understood, insights regarding ASD and other health conditions will emerge. Because each mutation can potentially reveal a cell’s developmental ancestry record, a lineage tree could be con structed and developmental patterns noted. Scientists from MIT and Harvard have carried out whole‐genome sequencing of single cells to demonstrate how these processes unfold in the brains of human adults [87]. This methodological approach holds promise in terms of resolving perplexing biological and medi cal questions [104]. These investigators evaluated 36 single neurons taken from the prefrontal cortex of three normal human adults (15, 17 and 42 years of age), with no known brain disease, to assess whether these neurons had developed single nucleotide variants or SNPs (Figure 1.18). Since in the adult human brain, neurons do not divide or regenerate (except in a part of the amygdala and in the olfactory bulb, see Chapter 5), an examination of accumulated muta tions in the brain would be revealing. The MIT and Harvard researchers found such mutations, in fact over 1,000 mutations per cell [106]. They found that most of these mutations were unique to a particular cell. They observed promi nent mutation rates at specific areas of the DNA (i.e., DNase I hypersensitivity sites and at transcribed loci). This suggests that even nondividing cells are vulnerable to mutation. These are likely introduced to the adult brain

15 Yr 17 Yr 42 Yr

Figure 1.18 The sample sequencing alignment tracks were derived from one of the three brains on which the study was based (15, 17 and 42 year old normal male brains). A single nucleotide mutation appears uniquely in the 15 year old brain (shaded area, C7), and one single nucleotide mutation is displayed in the 17 year old brain, between neurons 2 and 77 (shaded area); however, this is nearly absent from the heart, and from other single cells as shown in the 42 year old brain (shaded area, c10). Source: Adapted from Ref. [106]. 34 Introduction to Autism Spectrum Disorders

bymutagenic agents such as electromagnetic radiation, oxygen free radicals, and endogenous transposons (segments of DNA capable of moving around spontenously and independently). By contrast, cells elsewhere in the body can experience mutation during the process of DNA replication [106]. Among the most critical and intriguing aspects of this creative and ground breaking research is the creation of nested lineage trees, which permits dating relative to landmarks of development and brings greater understanding of the cerebral cortex’s architecture that arrives from multiple progenitor cells. Somatic mutations found in the human brain constitute a solid and continuing documentation of the genealogy of neuronal life, from early development throughout the life of an adult person (i.e., mitosis and thereafter in the post‐ mitotic phase).

Is Finding Mutations the Path to Discovering the Genesis of ASD?

Since the beginning of contemporary science, scientists have utilized the mouse and numerous other small mammalian animal models to reproduce various theories of autism. However, it should be noted that the brain of Homo sapiens is unbelievably more complex and large. It has over 100 billion neu rons and 0.15 quadrillion synapses. If one is looking for genetic mutations and comparing research on humans with mouse animal models, then one has to realize that these comparisons are far from accurate. For example, the average adult human weighs 62 kg, is made up of 3.7 trillion cells (3.7 × 1013 cells), and lives to about 70 years of age. On the other hand, the weight, number of cells, and lifespan of an average mouse would be dramatically less: 20 g, or about 1/3,100 of an adult human’s weight; roughly 1.2×1010 cells, and a lifespan of 3 years. The human genome has about 3 billion base pairs; these reside in the 23 chromosome pairs that are in the nucleus of each cell. Every chromosome is home to between hundreds and thousands of genes; these contain the instructions needed to produce proteins. Comprised of 23 chromosome pairs, the mouse genome contains approximately 2.7 billion base pairs, about 15% less than that of the human genome. Using a conservative estimate of the mutation rate, per cell per generation, of 5×10−7, then the generation of 3.7×1013 cells would lead to more mutations, by several logarithms, in humans compared with mice. Similar reasoning may be applied to a comparison of the human and mouse brains. This is very simplistic, however, since the human brain is far more complex than that of a mouse, and the inference of findings from mouse studies to human studies is, at best, a study in gross approxima tions. This is not to imply that animal models lack usefulness in suggesting broader implications; it is to say that in the case of autism research, rodent or other small animal models are highly inadequate. It should also be noted that Is Finding Mutations the Path to Discovering the Genesis of ASD? 35 in a human body DNA replication continues for an average of 70 years whereas in a mouse such replication is only possible for about 3 years. Therefore, the degree of somatic cell mutations in humans may be hundreds of times higher than in mice [102]. Structurally speaking, the human brain, as well as those of nonhuman pri mates generally, are similar to those of other mammals. Also, the former are typically larger and proportional to their larger body mass. As far as making comparisons of brain size for various species, the generally accepted approach is to use EQ, the encephalization quotient. EQ adjusts for the nonlinear nature of the body to brain relationship. The EQ for humans averages 7–8; other pri mates usually have EQs that range from 2 to 3. As summarized in Table 1.1, the EQ for dolphins exceeds that of other primates, with the obvious exception of humans, whose EQ of 7.4–7.8 significantly surpasses that of dolphins and the other mammals listed. Primate brain enlargement is primarily the result of extensive grown in the cerebral cortex, and particularly in the prefrontal cortex and in those parts of the cortex that control vision. Visual processing in primates involves complex networks and interconnectedness that includes 30 or more identifiable areas of the brain. Estimates suggest that brain areas related to visual processing comprise over half of the entire surface area in the neocortex of primates. The activities of the prefrontal cortex are crucial in key functional capacities, including attention, working memory, execu tive control, motivation, and planning. This occupies a far larger space in the primate brain than in the brain of other species, and the percentage of space occupied by the prefrontal cortex in the human brain is particularly high (https://en.wikipedia.org/wiki/Brain).

Table 1.1 Encephalization quotients for a range of species.

Species Encephalization quotient (EQ)

Human 7.4 – 7.8 Chimpanzee 2.4 – 2.5 Rhesus monkey 2.1 Bottlenose dolphin 4.14 Elephant 1.13 – 2.36 Dog 1.2 Horse 0.9 Rat 0.4 36 Introduction to Autism Spectrum Disorders

Does Brain Size Matter?

Many continue to be blinded by a genetic dogma of autism that presents autism as an unusual genetic disorder. To understand how exposure to extremely small amounts of chemicals may interfere with the normal development of a human fetal brain, we have to avoid the lure and distraction of looking at mutations that occur as part of normal development. For example, from the moment of fertilization of a human egg by a sperm, as each cell divides, random mutations occur which are fixed and inher ited by daughter cells. Most of these variants have little, if any, physiological consequence but contribute to genetic diversity within tissues. A small propor tion will contribute to pathogenic processes such as ASD or other serious disor ders. Rather, our focus should be on the way the normal branching of the human brain tree is disturbed by the untimely degeneration of certain cell types that results in ASD. One approach is to utilize human fetal brain neurons that behave just like those in a typically developing fetal brain. These cells, known as neuro blastoma cells, arise from an anomaly in the development of a fetus as a result of the migration of fetal brain progenitor neurons that settle somewhere in the body as nonmalignant tumors. These progenitor groups of neurons behave much like the fetal brain neurons but are disorganized (for further details see Chapter 7). These neurons often behave like a developing fetal brain. They also differentiate when prompted by specific neuronal growth hormones; they respond to mutagenic and neuro‐modifying agents, and exhibit loss or gain of neuronal receptors similar to human fetal brain stem cells. We decided to use these neuroblastoma cell lines of male and female origin, and then evaluate if there are unique or differential responses to exposures of minuscule amounts of fragrances or testosterone or supernatants from human gut bacteria exposed to glyphosate. The amount or level of each of the proposed culprits was kept low to imitate what one would expect to find in the amniotic environment during the early stages of gestation. For example, in the case of fragrance, we exposed the neuroblastoma cells to 1:1,000,000 (1 to 1 million) or 1:10,000,000 (1 to 10 million) dilution. The rationale of using such an extremely small amount was to test the idea that if a pregnant woman was exposed to perfume and inhaled or applied the perfume on her skin, the absorbed chemicals would be diluted into six liters of her blood and minuscule amounts would reach the fetus’s brain. This would imitate the situation of a pregnant woman and the fetus inside her womb.

How Autism Develops in a Fetal Brain

Even though no proved neurophysiological biomarker has been associated with ASD, there have been reports of low plasma oxytocin and arginine vaso pressin levels. These “twin” nonapeptides are primarily produced within the How Autism Develops in a Fetal Brain 37 brain of mammals. The dysregulation of these two neuropeptides is associated with alterations in behavior, particularly social interactions. Therefore, we examined the neuromodifications of human fetal brain neurons of both males and females through immunohistochemistry methodology. We demonstrated that exposure to even extremely low (i.e., femtomolar level) concentrations of fragrances led to morphological changes, shown through light microscopy in the neuroblastoma cell lines. Of note, these fragrances significantly reduced the oxytocin‐ and arginine vasopressin‐receptor positive neurons in the male neuroblastoma cell lines. However, since they not do so in the female cell lines, this may help explain why there is a male bias in ASD cases. Our oxytocin– arginine vasopressin study was the first to demonstrate a plausible link between what appears to be three interrelated phenomena: fetal exposure to fragrances; depletion of oxytocin‐ and arginine vasopressin‐receptor positive neurons; and the widely established male bias in ASD cases [10–13]. We are also comparing testosterone exposure levels that are reported to have been found in the amniotic fluid of an experimental group of ASD children and a control group of normally developing children during gestational weeks 9–14. This investigation may show that ASD results from epigenetic factors, those that do not have direct genetic causation but rather emerge through environmental exposure. Significantly, we have identified in fragrances more than two dozen chemicals with testosterone and other properties that imitate natural sex hormones, including benzophenone‐1, oxybenzone, tonalide, and galaxolide (see Chapters 5 and 7). Other chemicals that have been linked to increased human estrogen receptor expression include benzophenone‐1, ben zophenone‐2, octinoxate, oxybenzone, benzyl salicylate, benzyl benzoate, butylphenyl methylpropional, and synthetic musks such as musk ketone, tonalide, and galaxolide. Moreover, thyroid hormone disruption has been asso ciated with butylated hydroxy toluene, octinoxate, and benzophenone‐2 [3,7,8]. Each of these chemicals is used in most fragrances commonly sold today. Recall that even at very small concentrations (at the femtomolar level), fragrances embedded with these chemicals can prove mutagenic and even carcinogenic to human fetal cell lines [8–13]. We discuss the roles of these two neurohormones in further detail in Chapter 5. Given the growing linkage of epigenetic factors to ASD causation, we pro pose that the genetic approach to ASD may continue to be less fruitful and productive in uncovering the genesis of autism than more careful examina tions of environmental factors, including fragrances and other toxins that lead to mutations, especially in fetal development, and that are carcinogens. ASD prevalence is an alarmingly increasing problem, not only because of greater public awareness but also because of actual diagnostic data. We argue that the “spectrum” is the result of interference in the normal fetal brain devel opment from the very early stages through to about two years after birth, as illustrated in Figure 1.19. 38 Introduction to Autism Spectrum Disorders

Low nasal bridge Epicanthal folds Small head Small eye Short palpebral openings ssures Short nose

Flat midface

Thin upper lip Smooth philtrum

Figure 1.19 The development of a fetus exposed to alcohol (fetal alcohol spectrum disorder or FASD). Source: Adapted from http://virtual‐lecture‐hall.com/ KRA2605cssCHAPTER3/images/Fetal‐Alcohol‐Syndrome‐Characteristics.jpg.

Figure 1.19 depicts how exposure to alcohol can manifest itself in the form of fetal alcohol spectrum disorder (FASD), which is characterized by intellectual deficiency as well as a combination of deformities of the face, brain, heart, genitals, bones and joints, and growth retardation. The head may be abnor mally small with inadequate development of the brain. In general, alcohol has a negative effect on every aspect of the early development of the nervous sys tem including the differentiation of new neurons, migration patterns of nerve cells, and the formation of synapses, and myelination of nerve pathways [78]. From FASD one can imagine how exposure to alcohol at different times of gestation and at different concentrations can result in a spectrum of pheno types with small head, low nasal bridge, epicanthal folds, small eye openings, flat midface, short nose, thin upper lip, smooth philtrum, and maldeveloped jaw. Most importantly, it ought to be emphasized that alcohol enters every cell in the fetal body, resulting in an overall retardation in brain size as opposed to fragrances and other synthetic chemicals that can selectively bind specific pro genitor neurons via receptors and can exert compartmentalized effects, killing some neurons and allowing the surviving cells to grow faster in the areas of the brain that are emptied due to the killing of certain neurons. Therefore, syn thetic chemical effects show a more subtle spectrum, difficult to visualize (unlike FASD), and even harder to diagnose at birth. Usually, ASD is not diag nosable until 3 years of age. One can image that depletion or significant reduction of some types of progenitor cells can prompt other types of progenitor cells to replace them, which can grow faster than the original cells and create a brain with a far higher number of neurons in the frontal cortex and an astronomical number of faulty neuronal synapses and connections that may function in an aberrant fashion. Why Is There a “Spectrum” in ASD? 39

Given that normotypic (typical or normal) brain function includes parts of the brain that control social functions and temporary memory, such faulty synapses and connections can be a very serious matter indeed, and may well be linked to ASD symptoms. Of course, one must keep in mind the “spectrum” associated with the disorder. When the depletion or reduction in the brain tree occurs is crucial. For example, if a fetal brain has been exposed to certain environmental factors at very early stage of the fetal brain development (i.e., between about 4 weeks and 8 weeks of gestation), then irreparable damage may have occurred to the undifferenti ated progenitor neurons (e.g., oxytocin‐ or arginine vasopressin‐receptor posi tive neurons). If environmentally introduced chemicals destroy these vital progenitor cells, then the newborn may lack key functional brain compart ments that control communication, and the infant would manifest classic autism symptoms. On the other hand, if the fetal brain is exposed to the same kinds of chemicals at later stages of the fetal brain development (about weeks 14–20) then the majority of these progenitor cells would survive and the fetal brain could still develop, albeit with reduced numbers of the neurons that participate in communication skills. This child may have weaker social skills but would manifest the symptoms of the spectrum and would be more like a “high functional autistic” child. Exposure to much later stages of development may result in an Asperger’s child. Some may possess the outstanding capabili ties of, say, an Albert Einstein, since the replaced neurons have made their brain much larger through a huge increase in the number of synapses. Such children may show the signs of Asperger syndrome, now considered part of the ASD syndrome, with a combination of troubling social awkwardness and dazzling intellectual ability.

Why Is There a “Spectrum” in ASD?

ASDs emerge very early in a child’s life, typically prior to the age of three, and are characterized by serious challenges in their communication skills, social interaction, and general behavior. Lack of reciprocal social interaction and lim ited verbal and nonverbal communication are common observations. These children will find it difficult to understand the feelings of others. This is central to a concept known as “theory of mind,” which focuses on the ASD individual’s diminished ability to recognize body language, relate to the emotions of others, and to make customary eye contact. These tell‐tale symptoms of children who suffer from ASD may contribute to the aggressive behavior of these children towards others. The term “spectrum” acknowledges the widely ranging skills, symptoms, communication disabilities, and impairment levels that children with ASD often exhibit. Some children suffer mildly from these symptoms; others live with severe disabilities. The latest edition of the widely accepted 40 Introduction to Autism Spectrum Disorders

Diagnostic and Statistical Manual of Mental Disorders (DSM‐5) [111] has dropped the category of Asperger syndrome; its characteristics are now included in the more encompassing category of ASD. Children with Asperger syndrome may show varying combinations of symptoms, with different degrees of severity, but these children are now included under the ASD definitional umbrella. While the precise causes of the ASD “spectrum” remain enigmatic, charac teristics in typical fetal brain development vary significantly from those seen in children with ASD, including Asperger’s. Here we elaborate on why there is a “spectrum” in autism and what potential causes have been suggested by health care providers and researchers. The pre‐DSM‐5 category of Asperger syndrome shared some basic charac teristics of autism, including restricted personal interests, inadequate recog nition of emotions in other people, and general social awkwardness. Children with Asperger’s, however, display no significant intellectual disability or meaningful delay in speech development, although they may at times speak in unusual ways and act socially clumsy. Children with Asperger’s may be extremely brilliant and turn out to be modern Einsteins, Jeffersons, and Mozarts, individuals capable of inspiring paradigm shifts in science, politics, and the arts. The use of male examples is particularly appropriate since Asperger’s is ten times more common in males than females. The development of the human fetal brain occurs via a remarkably complex interplay of precisely orchestrated fetal brain progenitor cell differentiation influenced by environmental factors. Every developmental stage is comparable with a tiny acorn that grows into an imposing oak tree. Each branch begins with a germinal center as its node, and this center is guided by specialized progenitor cells. Each mother cell, in turn, bears its own gifts and faces its own vulnerabilities. Most ASD children who also have psychiatric disorders experi ence the onset of ASD symptoms prior to 3 years old. These neurodevelop mental syndromes in classical autism and ASD have likely arisen due to the specific neuronal degenerations that occurred when they were exposed to harmful chemicals that depleted certain specialized types of progenitor cells. If this harmful chemical exposure occurred between gestation weeks 8–15, then depletion or significant reduction of the olfactory cells or oxytocin‐ or arginine vasopressin‐receptor bearing progenitor cells may, after three years, produce a tree of neuronal connections that is lopsided in shape and inade quate in function. This lopsided tree can be compared with a real tree that was pruned on a specific side or area and in the spring the pruned area grew faster and larger. The evidence of such lopsided growth is visible early on by using magnetic resonance imaging, as reported by Eric Courchesne and his cowork ers at the University of California, San Diego Autism Center of Excellence. These scientists compared ASD and normotypic infants from birth through age 5 and concluded that two phases of abnormal brain development precede ASD and Vaccines 41 the clinical onset of autism: a smaller than expected head size at birth; and then a sudden aberrational growth in head size beginning during the first two months of life and continuing through months 6–14. The Courchesne team suggested that abnormally accelerated head growth rates may signal that an infant is at risk of ASD development. Therefore, we believe that the ASD children’s brains are missing a large branch, the cerebral deficiency would be comparable with surgically removing a branch of a newly planted young plant. However, we speculate, after gesta tion week 24, if the fetal human brain is exposed to the same kinds of synthetic chemicals that cause degeneration of the specific kinds of progenitor cells just mentioned, the damage may be less, and although some branches may be smaller than normal, by age three, the neurological tree may appear fine, except for a larger brain and larger skull. In another case, if a fetus is exposed to the same kinds of chemical that removed the whole branch of the tree after 22 weeks or more of gestation, the result may be comparable with a much larger oak tree than usual, with lots of branches and extra fruit. The first scenario would be a case of classical autism, the middle case one of a child with ASD, and the third scenario a case of Asperger syndrome or an unusually brilliant child. A child with this syndrome may be missing some specific social skills, but overall will have a larger brain size and well‐formed synapses (Figure 1.10). Our goal is to understand how the process of normal fetal brain development is impacted by exposure to certain synthetic chemicals and how they interact with other factors to increase the risk of developing ASD, and to use this understanding to minimize potential damage (see Chapter 2).

ASD and Vaccines

Data show clearly that vaccines save lives. Health professionals recommend that all children routinely be given a variety of preventive vaccines in their early years as protection against infectious diseases that can be dangerous and even life threatening. Measles provides a case in point. Among earlier generations, measles was exceptionally common; given current vaccine practices, measles has become rare and could become nonexistent if universal compliance with vaccine recommendations was followed. In the USA, for instance, since pedia tricians began administering vaccines as a part of normal checkups, the results have been exceptionally positive, with nearly universal prevention of illness, death, and disability that plagued earlier generations (Chapter 11). In the USA, ~27 vaccines are administered to children by about age three. Because this is the age at which ASD symptoms commonly emerge and become apparent, some parents might have erroneously linked the ASD symptoms with the preventive vaccines their children have received. Thimerosal, used as an additive in vaccines to prevent dangerous growth of fungi and bacteria, 42 Introduction to Autism Spectrum Disorders

contains mercury. This has caused considerable concern, and has been linked to the rise of ASD. From several sources, including the CDC, the data show no apparent link between the type of mercury found in thimerosal and ASD. However, several recent highly comprehensive studies from unbiased, inde pendent and reliable investigators have clearly shown a higher risk of ASD in children who received organomercury containing vaccines. For example, Geier et al. [112} have explored the potential risk of thimerosal containing hepatitis B vaccine (or HepB). Three doses of this vaccine are given to children before the age of 18 months. They evaluated a large number of individuals (15,216) who received HepB vaccine and concluded that: “Cases diagnosed with atypi cal autism were statistically significantly more likely to have received greater overall and dose‐dependent exposures to thimerosal containing mercury from TM‐HepB vaccines administered within the first month of life, first two months of life, and first six months of life than the controls. Similar phenomena were observed when cases and controls were separated by gender.”

Thimerosal Containing Mercury Stays in the Body and Is Very Toxic

A documentary “Vaxxed” has been released that shows that the adverse effects of thimerosal were hidden and later the scientific data were destroyed by the CDC. This information came to light via the lead scientist who worked for the CDC on this specific project, suggesting that the MMR vaccine might have caused ASD‐like symptoms in children who were given the vaccine when under 12 months old. We have also investigated this issue and it appears that exposure to live MMR (which is the version given to children) causes severe damage to human fetal brain cell lines. We believe that since the adverse effects are generally reported in a very small percentage of young children (under 18 months of age), it is likely that these children were suffering from a leaky blood–brain barrier (BBB). The BBB becomes leaky if the young child is suffering from an asymptomatic underlying infection when administered a MMR vaccine. This can cause severe neurological damage to a young devel oping brain. We will provide details of this topic in Chapter 9). We also believe that pathology of ASD experienced after vaccination (so called regressive autism) is very different than the mechanism we have described above due to synthetic chemicals that interfere in neurodevelopment during the fetal development. Regressive autism may be the direct result of the toxic effects of actual vaccine ingredients and not a fetal developmental problem. There are numerous disclosed and undisclosed chemicals as well as live viruses that can penetrate the BBB if it is leaky and can directly damage the neurons. We will discuss this issue as well as a potential solution to prevent regressive autism in Chapter 9. References 43

Summary

In summary, ASDs are highly heterogeneous developmental conditions char acterized by deficits in social interaction, verbal and nonverbal communica tion, and obsessive/stereotyped patterns of behavior and repetitive movements. Social interaction impairments are the most characteristic deficits in ASD. There is also evidence of impoverished language and empathy, a profound inability to use standard nonverbal behaviors (eye contact, affective expres sion) to regulate social interactions with others, difficulties in showing empa thy, failure to share enjoyment, interests and achievements with others, and a lack of social and emotional reciprocity. In developed countries, it is now reported that 1–1.5% of children have ASD, and in the USA in 2015 one in 45 children suffered from ASD. Despite the intense research focus on ASD in the last decade, the underlying etiology remains unknown. Genetic research involving twins and family studies strongly supports a significant contribution of environmental factors in addition to genetic factors in ASD etiology. A com prehensive literature search has implicated several environmental factors asso ciated with the development of ASD. These include pesticides, phthalates, polychlorinated biphenyls, solvents, air pollutants, fragrances, glyphosate, and heavy metals, especially aluminum used in vaccines as adjuvant. Importantly, the majority of these toxicants are some of the most common ingredients in cosmetics and herbicides which almost all of us are regularly exposed to in the form of fragrances, face makeup, cologne, air fresheners, food flavors, deter gents, insecticides, and herbicides. We have also explained why ASD is a “spec trum” and propose mechanisms by which this “spectrum” becomes a reality.

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103 Muhle R, Trentacoste SV, Rapin I (2004). The genetics of autism. Pediatrics, 113(5):e472–86. 104 Waterhouse L, London E, Gillberg C (2016). ASD validity. Rev. J. Autism Dev. Disorders, 3:302–29. 105 Bagasra O, Pace DG (2017). A Guide to AIDS. CRC Press, Boca Raton, FL. 106 Lodato MA, Woodworth MB, Lee S (2015). Somatic mutation in single human neurons tracks developmental and transcriptional history. Science, 350(6256):94–8. 107 Wang M, Li H, Takumi T, et al. (2017). Distinct defects in spine formation or pruning in two gene duplication mouse models of autism. Neurosci. Bull., 33(2):143–52. 108 Ziats MN, Edmonson C, Rennert OM (2015). The autistic brain in the context of normal neurodevelopment. Front. Neuroanat., 9:115. 109 Bian WJ, Miao WY, He SJ, et al. (2015). Coordinated spine pruning and maturation mediated by inter‐spine competition for cadherin/catenin complexes. Cell, 162(4):808–22. 110 Chang YL, Chang SD, Chao AS, et al. (2009). Clinical outcome and placental territory ratio of monochorionic twin pregnancies and selective intrauterine growth restriction with different types of umbilical artery Doppler. Prenat. Diagn., 29(3):253–6. 111 Nuckols CC. The Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM‐5). http://dhss.delaware.gov/dsamh/files/si2013_dsm5 foraddictionsmhandcriminaljustice.pdf. Accessed October 17, 2017. 112 Geier D, Kern J, Geier M (2017). Neonatal factors among subjects diagnosed with pervasive developmental disorder in the US. J. Matern. Fetal Neonatal Med., 19:1–6. 51

What is Autism?

More than seventy years of research studying the arbitrary diagnosis of autism has not resulted in any targeted medical treatments. Now is the time to abandon the ASD diagnosis in research. Lynn Waterhouse, Eric London, and Christopher Gillberg, 2017, The ASD diagnosis has blocked the discovery of valid biological variation in neurodevelopmental social impairment. Autism Res., 10: 1182

We stand now where two roads diverge. But unlike the roads in Robert Frost’s familiar poem, they are not equally fair. The road we have long been traveling is deceptively easy, a smooth superhighway on which we progress with great speed, but at its end lies disaster. The other fork of the road – the one less traveled by – offers our last, our only chance to reach a destination that assures the preservation of the earth. Rachel Carson, 1962, Silent Spring

Legacy of Autism

In the last few decades billions of dollars have been spent, and are still being spent, in trying to prove that autism spectrum disorder (ASD) is genetic. Elite scientists around the globe have discovered hundreds of genes that may be associated with ASD; yet none can produce ASD‐like disorders in animal models. Many genetic diseases in which chromosomal aberrations and certain genetic mutations result in mental retardation have been added to the ASD group, skewing the obvious fact that besides select rare genetic mutational diseases (Table 2.1), the majority of ASDs do not show scientific proof that

Table 2.1 Most common chromosomal mutations associated with ASD.

Prader–Willi syndrome (deletion in paternal allele) 15q11–q13 Angelman syndrome(deletion) UBE3A Smith–Magenis syndrome 17p11.2 del Velocardiofacial/DiGeorge syndrome 22q11.2 del Ch 22q11 duplication syndrome 22q11.2 dup Potocki–upski syndrome 17p11.2 dup Down syndrome Trisomy of chromosome 21 Phelan–McDermid syndrome 22q13.3 del Williams syndrome 7q11.23

ASD is caused by specific genetic mutations. As we have mentioned in Chapter 1, human genes do not change that quickly. “Genetic drift,” the degree, to which genes change over time, is ~1% per 100 years. Therefore, with such a slow pace of change, there can be no such thing as a sudden “genetic pandemic.” While a rare type of genetic autism may exist (at the background rate of “1 in 10,000”), the autism epidemic (at about “1 in 45”) cannot be due to genetic drift. In addition, if autism were genetic, because it affects more males than females, geneticists teach us that it would have to be an X‐chromosome‐ linked disorder. (Girls, having two X chromosomes, would then have a redundant good chromosome, so the disorder would not manifest as often in females as in males who have only one X chromosome). For a girl to have autism, she would need to receive a defective X chromosome from each parent, meaning they both carried the defect. The problem lies in the fact that if the father has the defect to pass on to his daughter, and it is on his only X chromosome, then he should be affected by the disorder as well. Since autistic daughters rarely, if ever, have autistic fathers, the theory that “autism is genetic” fails because its claim does not match the empirical evidence [1]. There are two main recognized causes for any epidemic: an infectious agent (i.e., current Zika pandemic) or a widespread toxic exposure (i.e., PCB, DDT, DEP, and synthetic fragrances). Autism is due to the latter [2–15]. Corresponding to the sharply increasing level of mercury in the immunization schedule globally, which started in the late 1980s, there was an increasing rate of autism among children. This also explains why autism among 40‐, 50‐, 60‐, 70‐ and 80‐year‐olds (born before the 1980s) is not epidemic, but rather rare. Those over 30 did not routinely get levels of vaccine‐related mercury exposure high enough to cause regressive autism. A Short History of Autism 53

A Short History of Autism

Steve Silberman’s recent book, NeuroTribes [16], provides an excellent overview of people and themes that have been involved in the story of autism. According to Silberman, among the key people involved in the dramatic and moving story of autism are Henry Cavendish, Hans Asperger, Leo Kanner, Lorna Wing, Bruno Bettelheim, Bernard Rimland, Dustin Hoffman, and Temple Grandin. Autism involves traits that are out of balance with those present in a more typical human being. Although individuals on the autism spectrum are commonly lacking in certain areas, notably in the areas of social communication and interaction, they may excel in other areas. They are more prone to see things in new ways rather than being bound by preconceived notions, and they often have incredible memories. They also perceive the world visually with great sensitivity. One can easily recognize that these types of traits can be very important in scientific fields that depend on inventiveness and creative genius. An example given in NeuroTribes is of the great British scientist Henry Cavendish (1731–1810), who is particularly famous for discovering hydrogen and helium. It may be that Henry Cavendish’s ASD‐related weaknesses in some areas gave rise strengths in others. Well known to be exceedingly reclusive and exceptionally unassuming with regard to his discoveries, Cavendish may never have made the discoveries he did were it not for his autism. A scientist as well as a natural philosopher, Cavendish was afflicted with autism at a time when there was no official diagnosis available; however, a retrospective diagnosis of autism seems warranted. It was Cavendish who, from his own back yard, found a way to measure the density of the entire Earth. Numerous important scientists have also been unofficially diagnosed as being on the autism spectrum. Henry Cavendish typifies this pattern. Hans Asperger was an Austrian pediatrician and scientific researcher at the University of Vienna. It was he who first recognized that the disorder that came to bear his name was a condition that took various forms and appeared at different levels of severity. He published his seminal finding in 1944 that clearly described autistic symptoms. Asperger perceived a connection between high levels of intelligence and autism in such challenging intellectual areas as mathematics and music. Consequently, he referred to his patients as “little professors”. A devastating historical event under- mined Asperger’s progress in making his observations known: Hitler ordered the invasion of Austria at the same time as Asperger was making his groundbreaking contributions. Consequently, his prestigious University of Vienna, instead of becoming more renowned for quality research, came under the grip of the Hitler regime and its obsession with eugenics and 54 What is Autism?

racial purity. As the purposes of the institution were fundamentally compromised by useless and harmful studies by Nazi‐appointed researchers, the University of Vienna’s former international prestige was lost. Hans Asperger lived for decades after the end of World War II and the death of Hitler, his own death not coming until 1980. Meanwhile, his groundbreaking research and foundational ideas received little of the attention they merited, and so the needed progress in understanding and dealing with autism suffered. It is truly unfortunate that Asperger’s ideas did not flourish in a timely manner. Major positions taken by researchers and public health officials today parallel those that Asperger took so many decades ago. He realized autism had variations of severity and that a spectrum was appropriate in describing these levels. Asperger knew that autism was a rather uncommon condition, and found that it was often related to intelligence. Modern researchers have come to similar conclusions, but autism studies might well be more advanced today had precious time not been lost, a victim of the madness that was Nazism. Many people today know of Adolf Hitler and they have heard of Asperger syndrome, but they do not know of the impact of Hitler’s regime on Asperger’s life and research. At Asperger’s Clinic, researchers found that autism seemed to be connected to genius, involved a whole spectrum of conditions. Asperger was correct, but his ideas did not come to prominence for two historical reasons. First, as previously mentioned, Germany’s leading Nazi, Adolf Hitler, transformed the focus of the University of Vienna and did irreparable damage to the institution’s intellectual leadership. Secondly, in order to retain some measure of intellectual freedom, and to protect individual lives from suspicious Nazis, Asperger focused his scholarship on highly functioning individuals. This led to the incorrect view that Asperger was limited to high functioning individuals. His broad ranging interests were simply not understood because of the threat posed by Nazism and because of his understandable response to that threat. Hitler annexed Austria in 1938 and made it part of the infamous Third Reich. Reversing the previous pattern that featured careful selection of presidents and rectors at the University of Vienna, the Nazis simply appointed favorites who were political stooges, ideological cronies. Racial and political motives resulted in a massive removal of students and professors. In fact, some 45% of the scientific staff and over 50% of the university’s lecturers in the areas of law and medicine lost their jobs, uprooted by the National Socialists’ myopic obsession with eugenics. This destructive anti‐scientific barrage caused the previously illustrious reputation of the University of Vienna to quickly vanish. The impact was long lasting; it is estimated that biological excellence did not really return to the institution until the 1980s, decades after Hitler’s assault on the academic life of a great city and a great university. Asperger’s scholarship was outstanding and he wrote over 300 publications, mostly on autistic psychopathy [17]. A Short History of Autism 55

Asperger’s famous quotes include:

Not everything that steps out of line, and thus “abnormal” must necessarily be “inferior”. It seems that for success in science or art, a dash of autism is essential. The autistic personality is an extreme variant of male intelligence.

Some people may draw comparisons between Nazism and the current stance of the US government over climate crises or environmental insults to our offspring while still in their mothers’ wombs, with stooges appointed who have no scientific basis for ignoring the crises our next generation may face if carbon pollution is not controlled. Chaskel Leib (Leo) Kanner (generally pronounced as Conner), a Johns Hopkins University psychiatrist, has received credit as the discoverer of autism. Unlike Asperger, whose wisdom was buried by time, Kanner had a tendency to make diagnostic errors and to impose his ideas on others via a haughty personality. Kanner took a decidedly different view than that of Asperger, who emphasized a spectrum on which individuals could be placed. Kanner declared that autism was a disorder that began in infancy, was rare, and frequently struck with severity. His perspective proposed limited diagnostic inclusion, which varied from the position of Asperger who emphasized an inclusive spectral situation. Kanner viewed autism as a condition that began from the time of a child’s birth, and negatively impacted the individual’s intellectual and social capacity. At Johns Hopkins, he focused particularly on 11 patients; eight of these could hardly use oral language, and three showed no capacity for speech at all. Obviously, he dealt with patients on the left side (extreme disability) of the spectrum while Asperger observed patients on the right side (Asperger syndrome) of the spectrum, as illustrated in Figure 2.1. Kanner borrowed the word autism from research involving schizophrenia, where it was used to describe inwardness that had been perceived in adults with a schizophrenic condition. He realized that similar conditions were

Extreme disabilityAutism Asperger syndrome

Figure 2.1 Autism spectrum disorders. There is a growing body of opinion that we should view autism as largely independent sets of clinical features, each caused by different sets of environmental insults, with different dose and at different gestation periods. The alternative opinion is that autism is a coherent syndrome in which principal features of the disorder stand in intimate developmental relationship with each other. 56 What is Autism?

apparent in children, and correctly made the assertion that autism differs from schizophrenia because the latter has its onset later in life, whereas autism begins with infants. While his views differed from those of Asperger, there are key similarities in their views: both Asperger and Kanner observed a troubling paucity of emotional affection and an obsession with routine and predictability, characteristics of those on the ASD spectrum. They also both observed an unsettling preoccupation with objects that led to an exclusion of interest in social interactions with people. Still, the two scientists interpreted the same data from different perspectives. Kanner thought he had discovered a condition that could be precisely diagnosed and declared it to be rare. Asperger, on the other hand, saw the problem as spectral, that is existing along a spectrum, and he correctly perceived that autism was a rather uncommon problem in infants, that rarely occurred. Kanner wrote in 1943: “Autism is not a disease (or disorder), it is a Behavioral Constellation” [18]. Susie, the daughter of English psychiatrist Lorna Wing, proved to be the catalyst that helped to revive Asperger’s correct views of autism as part of a syndrome. Wing, who helped to reverse the more inflexible diagnostic patterns encouraged by Kanner, realized that a broadened diagnostic potential involved the opening of a veritable Pandora’s box. The question arose as to whether the number suffering from autism had actually increased or whether the increased numbers were simply due to changes in diagnostic decision making. With the expansion of the conditions allowed on the autism spectrum and with the broadening and sharpening of diagnoses of the attendant conditions, there has been a large increase in the number of children placed on the spectrum through diagnosis [16]. Misinformation has unfortunately accompanied this rise in interest and diagnosis, including the information that autism is the offshoot of tainted vaccines. A widely disseminated article from the prestigious journal The Lancet linked vaccinations to the onset of autism. Although this research was later renounced by the journal and many claimed that it was fraudulent, new findings support the view that thimerosal (an organic mercury) used in many vaccine appears to have links to ASD and increases the risk of ASD (see Chapter 9 for details). The confluence of public hesitancy about the growing use of chemical agents to treat human conditions, concerns about the size of the government, and suspicions about big medicine combined to provide fertile ground for the popularity of The Lancet article that was later retracted. A uniform and widespread vaccination program can virtually wipe out a disease, promoting so called herd immunity, and breaches in vaccination uni- formity can provide a foot in the door to diseases that otherwise would have no to hold. Unfounded paranoia about immunization not only puts unvaccinated children at risk but also puts others in danger, a situation that is particularly hazardous for those who due to serious health challenges already A Short History of Autism 57 have compromised immune systems. However, it should be noted that thimerosal poses serious risks to the young vaccinees, especially if they are younger than 18 months of age. We have addressed this issue in detail in Chapter 9. One of the more controversial characters in the autism story is Bruno Bettelheim [16]. The University of Chicago’s Sonia Shankman Orthogenic School was his creation, and he used it as a platform from which to argue his perspectives about how to treat children with disorders. He became popular and spread theories that were not only inaccurate but very hurtful to parents who were already hurting. It was he who blamed parents, particularly mothers, for toxic parenting, that is, for excessive coldness that drove children to autism. Bettelheim’s unfounded and guilt‐producing focus on parental culpability was abetted by the post World War II emphasis on the theories of Sigmund Freud. The climate for heaping guilt on mothers was also conditioned by controversy over whether mothers, especially now that the war was over, should primarily spend their time at home or continue in the workplace. During World War II, mothers at work outside the home were viewed as an emergency necessity and now that the war was over, there was more choice in the matter and substantial debate ensued. Bettelheim stated that institutions would need to provide the cures to com- pensate for these “refrigerator mothers.” He and others had found a convenient scapegoat and proclaimed a potential cure for autism that conveniently blamed parents, and especially mothers, for autistic conditions that afflicted their children. This unscientific approach was appealing because if the problem was as simple as bad parenting, then it could be reversed by teaching good parenting. One was a function of the other and improving one variable (parenting) would improve the other (autism). This emphasis on nurturing children to prevent a supposedly genetic problem was, of course, scientifically irresponsible and practically unrealistic. Still, humans have been found in many situations to tend to prize scapegoats over serious solutions when faced with seemingly intractable problems. Unscientific positions that parade as truth are unfortunate, dangerous, and harmful. Note that the genetic nature of autism is, itself, subject to considerable controversy currrently. A major thrust of our work is to persuade readers and researchers that while autism has a genetic base, it is not a hereditary base in the sense that it is passed down from generation to generation like sickle cell anemia or Huntingdon’s disease. Rather, we maintain, genetic alteration through rapid mutation due to environmental assaults on the fetus and developing child are what cause genetic alterations and autistic conditions [2–15]. The same unfortunate patterns of misperceptions regarding health conditions and relying on scapegoats has been seen in other areas, including HIV. Because initially HIV was so commonly found in gay men, and because there was considerable social prejudice against the behavior of these men, 58 What is Autism?

AIDS research did not receive as much attention as it might have received if, for instance, it had afflicted the wealthy and morally accepted segments of society [19]. Bernard Rimland served in the US Navy as a psychologist [16]. His book, Infantile Autism: The Syndrome and Its Implications for a Neural Therapy of Behavior, helped propel autism onto the public stage to a more pronounced degree [20]. Rimland was a guiding force behind the creation of the National Society for Autistic Children. He focused on genetics, and shifted the dialogue away from blaming parents to genetic forces that were not under human control. Unfortunately, he became so obsessed with the idea of curing individuals on the autism spectrum that he gave inadequate attention to schools and other services that could help autistic children in the here and now. This tension between searching for a cure/prevention and pressing for institutional assistance to ameliorate present challenges has continued. Certainly, one can see value in both. Finding the proper balance is the key. In dealing with serious health conditions, finding a cure has obvious appeal; so does providing quality services for those already afflicted. As obvious as is the need to provide support for both of these positions, finding the appropriately synchronized approach is difficult. Excessive emphasis on finding a cure can take much needed resources away from caring for those who presently suffer from an ASD condition. Excessive financial allocations to deal with present situations can, conversely, can lead to inadequate funding for the very research that could both prevent future health problems and lessen future budgetary demands that accompany the problems. In public policy decisions related to autism, this balance between funding for research and funding for services continues. We maintain that this is an important debate, and one that should continue, because neither area must be slighted. We also maintain that the two are not separate issues, and that both are served best when an interrelated approach is applied. Understanding the conditions that afflict those on the autism spectrum can inform research about causation. Likewise, discoveries about causation, for example in neurological fetal and infant development, can inform treatment or prevention possibilities. One specific example involves young adults, who are faced with educational needs and require employment support. Without appropriate social services (and many believe that such support is woefully lacking), this large group is falling significantly behind their peers in terms of successfully navigating the challenges of employment, family, and independence. Wise expenditures that enable individuals to be reasonably self‐sufficient can, in fact, prove cost‐effective. Moreover, there are many contributions that can be made, and have been made in the past, by those on the spectrum. However, if autistic individuals do not have reasonable support, they can easily be found among the unemployed, the homeless, and the ill rather than in the news headlines referring to discoveries, promotions, and creative achievements. A Short History of Autism 59

One major philosophical issue that has accompanied the debate over what to do with individuals on the autism spectrum revolves around adaptation: who should adapt? Is it the children who need to adapt to the general patterns of the world, or is it society that needs to appreciate the uniqueness and talents and gifts of those on the spectrum and adapt to them? As with most such questions the truth may lie somewhere in the middle of the two extremes. There are persuasive reasons to support both positions with a reasonable balance. As in the scenario of searching for a cure and pressing for institutional financing and support, balance is crucial. Professor Temple Grandin, a gifted Colorado State University professor of animal science, personifies the value of ASD individuals both adapting to society and society adapting to them [16]. She has found ways not only to cope but to contribute, and seems to have linked her sensitivity towards those with different neurological conditions to her theories about the care of animals. Her contributions appear to be due, at least in part, to her ASD, and not simply in spite of her ASD. We see a parallel in the deaf community, where a number of individuals with a hearing deficit argue that deafness is not something that needs to be cured; rather, it simply needs to be accepted and even appreciated. They speak their own language, something that most individuals with regular hearing capacity cannot do, and they have defined a world that most people do not or cannot fully enter. The struggles, sacrifices, and pain involving those with ASD take place primarily in homes. One innovative program has been launched by Professor Taryn Nicksic‐Springer, a certified behavioral analyst who works out of Salt Lake City, Utah. Arguing that a combination of professional guidance and family involvement is critical, she argues that parents are not simply peripheral therapists but the finest available in dealing with ASD since they spend large amounts of time with the children and can give context to what they observe. This is not to say that they simply work independently. Toys are offered to parents, and therapy is offered with limited guidance from experts, all in an effort to form a partnership through personal contact and internet‐based interaction. A technology based delivery system is used to help parents and foster care givers to train ASD children. Such an approach has merit from both resource allocation and human interaction standpoints. Technology has drastically altered the opportunities available to those on the autism spectrum. Individuals who are focused, even at times fanatically, on technologically based activities may make significant contributions without having to deal with real people in social settings. The same social weaknesses that make other types of work difficult are minimized in such circumstances. ASD can become a strength that opens doors and not simply a weakness known for closing doors of occupational opportunity for those affected. Grandin has been a leading advocate of rights for those on the autism spectrum, herself included. As an animal science professor, she has also advocated for the improved well‐being of animals. Her brilliance has been widely accepted. 60 What is Autism?

One can legitimately wonder if she could or would have made her significant contributions without her personal understanding of ASD and without the painful brilliance that accompanies her condition. Her life symbolizes a significant area of contemporary philosophical controversy about whether to encourage adaptation or whether to encourage acceptance. Another way of phrasing the debate is who should adapt, the individual on the autism spectrum or the surrounding institutions and society? People on the spectrum, as is widely known, do have intellectual gifts, and do share abilities that other humans share. Therefore, why not celebrate the talents and abilities of these humans just as the world celebrates the accomplishments and gifts of individuals who, though not on the autism spectrum, have other weaknesses and challenges? Here again, the need for balance is apparent. The media has a profound impact on public perceptions of autism. Through the Internet, information is quickly disseminated but the quality of information poses dilemmas regarding what is accurate and what is not. The Internet has opened up the possibility for chat rooms and other means of communication between caregivers that enables them to share experiences and share the loads of dealing with the innumerable problems associated with autism. The Internet also opens up similar opportunities for the individuals on the autism spectrum. By and large, it is individual homes, and individual families, that deal with autism and the burden it adds to the lives of those who care most about those who suffer with autism. Autism affects so many lives and homes that it has become a matter of significant debate in the healthcare and public policy arenas. A 1965 article that appeared in the popular magazine Life caused a setback in public comprehension of the challenges of ASD individuals. Its misinformation and lack of sympathy were unfortunate. The 1988 movie Rain Man, on the other hand, presented a more realistic and sympathetic view, and had a profound impact on public perceptions of autism. Dustin Hoffman starred in Rain Man, which helped to bring autism to the forefront of public consciousness. Other forms of media, whether accurate or not, have also had their influence. Even Disney’s recent movie, Frozen, involves the character Elsa who exhibits autistic‐like traits, and this character is portrayed as a hero.

DSM‐5 and the ASD Spectrum

The American Psychiatric Association (APA) periodically publishes updates to its widely referenced Diagnostic and Statistical Manual of Mental Disorders (DSM), a resource that provides recommend guidelines for the diagnosis of behavioral and mental disorders [21,22]. The DSM has become the standard for the definition, and at times redefinition, of diagnostic criteria for assessing psychiatric diseases, including those found among the ASDs. DSM‐5, the manual’s fifth edition, was made available in 2013. DSM‐5 and the ASD Spectrum 61

According to the new criteria introduced by DSM‐5, people with ASD demonstrate persistent deficits in two areas: (1) persistent social behaviors in the areas of general interaction and communication; and (2) behavioral patterns that are repetitive and restricted. Even more precisely, those with ASD need to display, either at present or in the past, deficits in: (1) reciprocal interaction in social–emotional settings; (2) the nonverbal behaviors they use to communicate in social interactions; and (3) the development, maintenance, and comprehension of interpersonal relationships. Moreover, individuals with ASD must exhibit at least two repetitive behavioral patterns, such as stereotyped motor motions, inflexible insistence on routines, excessive interest in sensory stimuli, or an exaggerated emphasis on sameness. DSM‐5 recommends that clinicians rate these deficits according to level of severity, and to base this decision on the amount of support patients require [22].

Changes in ASD diagnosis approved by the APA

In the new DSM‐5 issue, the APA approved five significant changes: First, earlier autism spectrum subcategories, such as Asperger syndrome, autistic disorder, and pervasive developmental disorder not otherwise specified (PDD‐NOS), have been brought together under the umbrella of the more general term ASD. Secondly, three autistic symptom areas were reduced to just two: impairment in social communication; and repetitive behaviors or restricted interests. Persons with ASD display a minimum of 6 deficits (out of 12) in their communication, social interaction, or repetitive behaviors. ASD diagnosis now requires that the individual shows three or more deficits in their social communication patterns, and two or more symptoms in a category focused on repetitive behaviors and a limited range of activities. Thirdly, the APA approved of diagnoses based on either past or present patient histories. Fourthly, besides the ASD diagnosis, healthcare professionals should provide a description regarding known genetic conditions (e.g., Rett or fragile X syndromes), disability level in the areas of intellect and language usage, and medical challenges such as depression, anxiety, seizures, or negative gastrointestinal (GI) conditions. Fifthly, social communication disorder (SCD) was added as a new category to facilitate diagnosis in cases where a social communication disability exists in the absence of a repetitive behavior disorder.

The impact of the Recent DSM‐5 Changes on Diagnosis and Support Practices

Anyone with a current ASD diagnosis, including PDD‐NOS or Asperger syndrome diagnoses, will retain their current ASD diagnosis. This diagnosis is valid for life, and entitles affected individuals to appropriate treatment for the remainder of their lives. 62 What is Autism?

The intent of the new guidelines is not to reduce qualifying cases but rather to capture more reliably everyone who merits ASD diagnosis. DSM‐5 is to be seen as a “living document,” one that can be altered as fresh information comes to light, for instance regarding two particular groups that require further study regarding diagnostic norms: infants and adults.

A New ASD Diagnosis Category: Social Communication Disorder

DSM‐5 introduced an additional diagnostic category called SCD. The intent of the new category is to include individuals who do not exhibit the repetitive behaviors or severely restricted interests so characteristic of many individuals with classic autism, and yet have serious deficits in the area of proper use of language in social settings. Many children can now receive this ASD diagnosis who previously were diagnosed with PDD‐NOS, a catch‐all category of individuals who were hard to diagnose under other categories. Those with a previous diagnosis will likely not be affected by this new category; basically, only newly diagnosed persons will receive the newly introduced diagnoses from the latest iteration of the DSM.

ASD or a Giant Spectrum of Socioneuropsychological Disorders

Before we get into the fallacy of ASD primarily being a genetic disease and not the result of environmental exposure to the fetus, it should be noted that until mid‐1970, the role of genetics and heritability was not mentioned much in the scientific literature with regards to autism. Let us first look at how we define ASD today. ASDs affect three different areas of a child’s life:

●● Social interaction ●● Communication – both verbal and nonverbal ●● Behaviors and interests

This so called phenotypic heterogeneity is the hallmark of ASD and why is that? If ASD was the result of genetic defects, then there would be a common denominator(s) by which to diagnose the disorders. We will discuss this very shortly below. Each child with an ASD diagnosis will have his or her own pattern of autism. This is a giant loophole in the definition. This will include many children who have serious chromosomal rearrangements, additions, and deletions as well as ones with outright well‐defined genetic diseases. This definition is like trying to put round pegs into square holes! This definition may include children who are normal but slightly delayed in their developmental chart. For example, sometimes, a child’s development is Pervasive Developmental Disorder Not Otherwise Specified 63 delayed from birth. Some children seem to develop normally before they suddenly lose social or language skills (called regressive autism). Others show normal development until they have enough language to demonstrate unusual thoughts and preoccupations. Granted, many of these children do have autism but all of them are not autistic! In some children, a loss of language is the major impairment. In others, unusual behaviors (like spending hours lining up toys) seem to be the dominant factors. Parents are usually the first to notice something is wrong. But a diagnosis of autism is often delayed. Parents or a physi- cian may downplay early signs of autism, suggesting the symptoms are “just a phase” or a sign of a minor delay in development. As we described above, until recently, the types of ASD were determined by guidelines in the diagnostic manual (DSM‐IV) of the APA. According to the Centers for Disease Control and Prevention (CDC), the three main types of ASD were: ●● Asperger syndrome ●● Pervasive developmental disorder not otherwise specified (PDD‐NOS) ●● Autistic disorder The DSM‐IV also included two rare but severe autistic‐like conditions – Rett syndrome and childhood disintegrative disorder. The new diagnostic manual (DSM‐5) has made some major changes in this list of disorders. It is unclear, though, how these changes will affect the way health professionals define exactly what is an ASD.

Asperger Syndrome

The mildest form of autism, Asperger syndrome, affects boys 10 times more often than girls. Children with Asperger syndrome become obsessively interested in a single object or topic. They often learn all about their preferred subject and discuss it nonstop. Their social skills, however, are markedly impaired, and they are often awkward and uncoordinated. Asperger syndrome is mild compared to other ASDs. Also, children with Asperger syndrome frequently have normal to above average intelligence. As a result, some doctors call it “high‐functioning autism.” As children with Asperger syndrome enter adulthood, though, they are at high risk of anxiety and depression.

Pervasive Developmental Disorder Not Otherwise Specified

This mouthful of a diagnosis applies to most children with ASD. Children whose autism is more severe than Asperger syndrome, but not as severe as autistic disorder, are diagnosed with PDD‐NOS. 64 What is Autism?

Autism symptoms in children with PDD‐NOS vary widely, making it hard to generalize. Overall, compared with children with other ASDs, children with PDD‐NOS have:

●● Impaired social interaction (like all children with ASD) ●● Better language skills than children with autistic disorder but not as good as those with Asperger syndrome ●● Fewer repetitive behaviors than children with Asperger syndrome or autistic disorder ●● A later age of onset

No two children with PDD‐NOS are exactly alike in their symptoms. In fact, there are no agreed‐upon criteria for diagnosing PDD‐NOS. In effect, if a child seems autistic to professional evaluators but does not meet all the criteria for autistic disorder, he or she has PDD‐NOS.

Autistic Disorder

Children who meet more rigid criteria for a diagnosis of autism have autistic disorder. They have more severe impairments involving social and language functioning, as well as repetitive behaviors. Often, they also have mental retardation and seizures.

Rett Syndrome

Almost exclusively affecting girls, Rett syndrome is rare. About one in 10,000– 15,000 girls develop this severe form of autism. Between 6 months and 18 months of age, the child stops responding socially, wrings her hands habitually, and loses language skills. Coordination problems appear and can become severe. Head growth slows down significantly and by the age of two is far below normal. Rett syndrome is usually caused by a genetic mutation in the X‐chromosome gene called MECP2 [23]. The mutation usually occurs randomly, rather than being inherited. Treatment focuses on physical therapy and speech therapy to improve function.

Childhood Disintegrative Disorder

The most severe ASD, childhood disintegrative disorder (CDD), is also very uncommon. Is Autism a Genetic Disease? 65

After a period of normal development, usually between 2 years and 4 years old, a child with CDD rapidly loses multiple areas of function. Social and language skills are lost, as well as intellectual abilities. Often, the child develops a seizure disorder. Children with CDD are severely impaired and do not recover their lost function. Fewer than two children per 100,000 with an ASD meet the criteria for CDD. Boys are affected by CDD more often than girls. Some of these cases may be “regressive autism” (see Chapter 9).

Is Autism a Genetic Disease?

One of the biggest ironies of ASD is that it is considered primarily a genetic or inherited disease. We believe that this is based on an incorrect assumption and it has become part of a dogma, the paradigm. It is very similar to the AIDS dogma that became established in at the early years of the tragic pandemic that it was a “gay disease” or a Haitian disease [19]. These are dogmas that need to be reassessed and revisited. Because of this incorrect dogma, scientists still desperately want to believe that some “gene” or “genes” will be linked to ASD or autism and will explain all the issues related to autism. We believe that classical autism results from environmental exposure to chemicals that humans are evolutionarily unfamiliar with [5– 15,24–31]. Let us explain this further. All the life forms on this earth have coevolved with hundreds of thousands or even millions of natural chemicals that can cause mutations in our genes. This is a more than 4 billion year long adaptation and evolutionary process. Two decades ago, Bruce Ames and his team of scientists tested thousands of natural and synthetic chemicals for their mutagenic and carcinogenic potential [32]. They catalogued cancer causing agents and showed that that cigarette smoke was mutagenic, as was tris‐BP, a flame retardant used in children’s pajamas. Using that database, the still‐active Carcinogenic Potency Database, Ames and Gold showed that nearly half of the chemicals tested, whether natural or synthetic, in the standard assay at the maximum tolerated dose, were carcinogens or mutagenic [33]. That made them suspicious, and they argued that the huge dose, not the chemical formulation itself, was responsible for inflammation, cell death, and cell proliferation. They concluded that animal cancer tests do not provide a good assessment of low‐dose cancer risk [34]. They also determined that many plant chemicals have higher carcinogenic capacity than synthetic chemicals [35,36]. However, we believe that Ames and Gold have missed one fundamental fact –coevolution of all life forms and their metabolic pathways that can neutralize the adverse carcinogenic effects of natural products – with one caveat. The dose of any chemical that may be considered tolerable can turn toxic if delivered in 66 What is Autism?

high doses. This is one of the fundamental principles (the six principles) that James Wilson established in 1956 [37,38]. A simple and obvious example is sugar. Sugar, or more precisely glucose, is an essential chemical for life. Almost all the life forms on earth need glucose for survival and glycolytic and Krebs cycle are integrated cycles of life where all the pathways that make up the engine of life run via the glycolytic pathway. But, if a human consumes too much sugar it will lead to diabetes, obesity, and cancer. High consumption of sugar is toxic to both men and women, it is harmful for liver, it leads to insulin resistance, disturbs hormonal balance, releases dopamine in the brain, it is the leading cause of obesity and cancer, and leads to high cholesterol and increased incidence of heart attacks. When a pregnant woman develops gestational diabetes during pregnancy (meaning her sugar level goes above the normal range), it can result in an overweight newborn that can develop serious illnesses after birth [39,40]. This is what happens when one consumes glucose – one of the most fundamental chemicals of life that perhaps evolved at the beginning of life and yet it can cause havoc when too much is consumed. Let us now discuss other chemicals that life on this earth consumes and has been consuming for over 4 billion years, yet has learned to balance. If we consume all the elemental food in balance it is fine, but if over or under consumed, it will result in adverse outcomes. Another, basic molecule that almost all life on earth consumes is alcohol. Here we are not referring to fermented ethanol but the molecules that result from glycolysis in our cells after glucose molecules are split into two. Our metabolic pathways efficiently consume alcohol molecules and convert them to energy. External consumption of alcohol is immediately utilized and converted into energy as long as the amount is sufficiently low. This molecule is so powerful that it has access to every cell in our body. However, exposure to this basic molecule after a certain threshold results in depression of brain functions and at high enough doses can result in death. We have discussed fetal alcohol syndrome already in Chapter 1. One more glaring example would be folic acid, which is found in green plants such as spinach. Folic acid deficiency can result in serious chromosomal breakage. This is due to massive incorporation of uracil into the DNA. Children born to folic acid deficient mothers have serious neural tube defect and are at increased risk of autism [41].

Synthetic Chemicals Lack Coevolutionary Adaptation

All the life forms on earth, all the flower and fauna on the planet, have a coevolutionary history except for the recently created synthetic chemicals. The majority of life forms lack the metabolic pathways to detoxify these chemicals [2–15,24–30]. Myth of the Genetic Origin of Autism 67

Myth of the Genetic Origin of Autism

The first description of autistic psychopathy was by Asperger in 1938 and in 1943 Kanner published a paper describing 11 autistic children; neither suggested that ASD was genetically inherited. Asperger identified in four boys a pattern of behavior and abilities that included “a lack of empathy, little ability to form friendships, one‐sided conversations, intense absorption in a special interest, and clumsy movements.” What he described were high functioning Autistic children (now called Asperger syndrome), two of whom went on to become university professors and one won a Nobel Prize. As late as the mid‐ 1970s there was little evidence of a genetic role in autism; now it is thought to be one of the most heritable of all psychiatric conditions [21]. Before 1970, it was considered that ASD was the result of bad mothering. Kanner’s description of autism led to decades of confused etiology and investigators believed that a lack of maternal passion was involved leading to misconceptions of autism as an infant’s response to “refrigerator mothers”. Starting in the late 1960s autism was established as a separate syndrome by demonstrating that it is lifelong, distinguishing it from intellectual disability and schizophrenia and from other developmental disorders [21,22].

Phenotypic Heterogeneity in Autism

ASD is characterized by broad heterogeneity in severity as well as in intellectual and functional communication ability not only between individuals, but also within the same individual over time [42]. In addition, a wide variety of medical and psychiatric conditions are associated with ASD, including intestinal disorders, epilepsy, and attention‐deficit/hyperactivity disorder (ADHD) [43,44]. It is surmised that complex interactions between genetics, increasing the risk for ASD, and environmental factors contribute to the broad heterogeneity observed in ASD [45]. This complexity makes determining the underlying causes of and developing treatments for ASD very challenging [46]. Recently, a large, multicenter, multidisciplinary observational study, the EU‐ AIMS Longitudinal European Autism Project (LEAP), was undertaken to examine biomarkers in ASD [47]. In the study, 437 children and adults with ASD and 300 controls between 6 years and 30 years of age and with IQs ranging from 50 to 148 were recruited from 6 research centers across 4 European countries. Broad heterogeneity in presentation of ASD symptoms was observed across the individuals in the study. Using a combination of clinical assessment and interviews, the investigators found lower social symptoms, severity of repetitive behavior, and inattentive and hyperactive/impulsive ADHD symptoms in adults. However, ASD symptom severity was higher in adults versus adolescents in self‐reported assessments. Differences were also observed between males and females. In diagnostic measures assessed through parent 68 What is Autism?

interviews, ASD symptom scores were higher in males than in females. ASD males also had increased levels of inattentive and hyperactive/impulsive ADHD symptoms. However, symptom severity was higher in females compared with males in self‐reports. There was some association between lower IQ and higher scores on ASD symptom measures. Overall, the individuals in the study reflected the phenotypic heterogeneity typically observed in ASD. Although associations were found between ASD symptoms severity and sex, age, and assessments in ASD symptoms differed between clinicians, parents, and individuals, highlighting the critical importance of selecting appropriate measurement approaches when examining ASD. Chaste and his collaborators have carried out one of the most comprehensive genome‐wide analyses of ASD [48]. This group carried out the full genetic analyses of 2,576 families to attempt to uncover phenotypic heterogeneity in autism that may be linked to certain genes. This approach is based on the hypothesis that phenotypic heterogeneity closely maps to genetic variation, which has not yet been shown. This study examined the impact of phenotyping and even subphenotyping of a well‐characterized ASD sample on genetic homogeneity and the ability to discover common genetic variants that may contribute to ASD. Their analyses of large numbers of families revealed no genome‐wide significant association signal. They concluded that analysis of subphenotypes is not a productive path forward for discovering genetic risk variants in ASD. Chaste et al. studied a large (N = 2,576 families) and behavio- rally well‐phenotyped ASD sample (the Simons Simplex Collection) to ask whether subgrouping to enhance phenotypic homogeneity increases the ability to make SNP findings in ASD. The results provide a clear “no.” The authors show that subphenotyping by many reasonable criteria, including IQ, extent of repetitive behaviors, insistence on sameness, and more severe ASD, failed to increase power substantially, and the subgroups showed very similar heritability estimates (~0.4). Most importantly, allele scores from the entire sample predicted case status equally well regardless of subgroup. In short, reducing phenotypic heterogeneity did nothing to increase genetic homogeneity. In a subsequent philosophic reflection, Chaste et al. [49] suggest that ASD is a complex polygenic disorder and rare de novo and inherited variations act within the context of a common‐variant genetic load, and this load accounts for the largest portion of ASD liability. Obviously, missing a real common sense point: these so called rare de novo mutations may be caused by environmental factors, while the fetal cells are going through enormous cellular differentiation and neurogenesis! It is useful to consider genetic variation as either common (e.g., an allele found in 0.5% of the population) or rare because these categories of variation are analyzed using different approaches and have different properties. Most importantly, for neurodevelopmental disorders, common genetic variation (e.g., SNP) is associated with very small effect sizes given evolutionary Myth of the Genetic Origin of Autism 69 constraints on deleterious variation (meaning if too much and too many changes are made in the genes the fetus would die in the womb, so called negative or purifying selection), whereas rare variation can be associated with a much wider range of effect sizes. Chaste and Leboyer [50] looked at common variation in ASD to answer questions about relationships between clinical and genetic heterogeneity. Enormous advances have been made in understanding the genomic architecture of ASD, including the role of common and rare variation. There is compelling evidence from multiple studies that common variation represents the major proportion of the genetic risk for ASD [49,50]. However, no SNP has been reliably associated with ASD to date because of small effect sizes. Until studies include many thousands of cases, it is exceedingly unlikely that many replicated common variation findings will be made in ASD. There has been much more success with gene discovery in ASD when focusing on rare variation. This increased success is due partly to the fact that there is a significant amount of de novo mutation in ASD: with de novo mutation being quite rare, even a few cases with de novo deleterious variation in a given gene are sufficient to provide statistically significant support for that gene in ASD [50]. Although the effect size for discoverable rare variation is higher than that of common variation, the total variance explained by rare variation is quite low. Gaugler et al. [51] showed that within a given family with ASD, a rare de novo copy number variant (CNV) or single nucleotide variant can often be the difference between an ASD diagnosis or no ASD diagnosis; however, there must be a “genetic background” in the family, defined by multiple inherited SNPs and other genetic variation, that is a critical part of the architecture in that family. In other words, there appears to be risk in the family in most cases in the form of a multiplicity of common variation, and higher risk rare variation pushes an individual in that family over a liability threshold to manifest with neurodevelopmental disorders. This model can explain why risk of family recurrence is high in ASD, while, at the same time, affected sibling or relative pairs within a family may have different rare risk variants, as first shown with CNV and, more recently, with single nucleotide variant [52,53]. Similar findings have been observed in studies of rare variation. Several rare ASD risk genes have been found in recent whole exome sequencing studies that examined >20,000 samples across approximately 4,000 cases of ASD [54–56]. Many recurrent CNVs have been linked to multiple phenotypes, such as with DiGeorge/velocardiofacial syndrome (22q11 deletion syndrome), which has been linked not only to autism, but also to epilepsy, schizophrenia, and congenital heart disease. In fact, as an example, chromatin remodeling genes appear to play a significant role in both ASD and congenital heart disease [57)]. Rare risk genes and CNV linked to intellectual disability have also been associated with ASD [54–56]. However, controversy persists in classification of ASD with and without intellectual disability, especially when social impairments result from other causes, such as a lack of social drive (Kanner autism) 70 What is Autism?

or other contributors to impairment of social interactions. Some maintain that ASD with and without intellectual disability represent distinct disorders. However, concrete genetic links have not been established to explain these distinct phenotypes. Perhaps if we understood more about common and rare risk loci, we could more accurately predict diagnosis. For example, the combination of the genetic background of a family and the presence of a high‐risk variant may contribute to the development of ASD. To understand this better, individuals with rare variants should be assessed for a broader range of disorders.

Why Fragile X Syndrome or Tuberous Sclerosis Should Not Be Included in ASD

Although persons with ASD may also have conditions knows as Fragile X syndrome or tuberous sclerosis, these conditions are not forms of ASD. Fragile X syndrome, which has symptoms that resemble those associated with ASD, is clearly a genetically caused disorder that passes on intellectual disability from one generation to the next (Figure 2.2).

An X chromosone affected by Fragile X syndrome 21 14 Translocation Normal carrier 14/21 CAUSE Gamete formation Tr inuoleotide repeat in the FMR-1 gene on APPEARANCE Gametes the X chromosone Portion of chromosone appears fragile and about to break

Normal TranslocationTr isomy 21 Monosomic 46 carrier (Down) (lethel) 45 46 45 Chromosone number

Figure 2.2 Fragile X syndrome and Down syndrome have genetic etiologies that are well‐defined and clear. Neither of them have environmental etiologies. Source: Adapted from: http://epilepsyu.com/blog/researchers‐propose‐new‐explanation‐for‐symptoms‐of‐ fragile‐x‐syndrome/; https://www.pinterest.se/pin/749990144165025587/. Myth of the Genetic Origin of Autism 71

The name Fragile X syndrome derives from a defective portion of the X chromosome that looks fragile and squeezed together under a microscope. It results from the mutation of one single gene and turns the gene off. Some individuals experience just a mutation and do not display symptoms; others have much more serious symptoms due to a larger mutation. About one‐third of children with Fragile X syndrome also meet ASD criteria, similar to individuals with Down syndrome who have extra pieces of chromosome 21. Conversely, about 1 in 25 ASD‐diagnosed children have experienced the same mutation that under- lies development of Fragile X syndrome. We maintain that Fragile X syndrome is inherited, a widely accepted position, but that ASD is largely due to environmental factors, arising from exposure to synthetic chemicals during gestation [58].

Tuberous Sclerosis

A rarely occurring genetic malady, tuberous sclerosis afflicts vital organs, including the brain, with noncancerous tumors (Figure 2.3). About 2 million

Brain: ∙ 90% Epilepsy Other: ∙ 80–90% SEN ∙ 50% oral fibromas ∙ 10–15% SEGA 50% retinal astrocytic 90% TAND ∙ ∙ hamartomas ∙ 50% intellectual disability ∙ 40% autism spectrum

Lung: Women Heart: ∙ 80% asymptomatic LAM Infants ∙ 5–10% symptomatic ∙ 90% cardiac rhabomyoma LAM, can lead to Adults respiratory failure Men and Women ∙ 20% cardiac rhabomyoma ∙ 10% MMPH

Kidney: Skin: 75% angiofibroma ∙ 70% angiomyolipoma ∙ ∙ 20–30% ungual fibroma ∙ 35% simple multiple cysts 25% fibrous cephalic ∙ 5% polycystic kindly ∙ disease plaques ∙ >50% shagreen patches 2–3% renal cell carcinoma ∙ ∙ 90% focal hypopigmen- tation

Figure 2.3 Key features of tuberous sclerosis. Source: Adapted from https://www.nature. com/articles/nrdp201635. Reproduced with permission of Springer Nature. 72 What is Autism?

individuals suffer from this genetic defect. TSc2 (also known as hamartin) and TSC2 (also known as tuberin) form the tuberous sclerosis protein complex that acts as an inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, which in turn plays a pivotal part in regulating cell growth, proliferation, autophagy and protein and lipid synthesis. Remarkable progress in basic and translational research has been made; some of its symptoms may be ameliorated through treatment, but tuberous sclerosis has no known cure. It is caused by a genetic disorder, and is linked to many mental and physical problems, including epilepsy and intellectual disability. As with Fragile X syndrome, we agree that tuberous sclerosis is an inherited genetic problem that should not be a part of the ASD spectrum [59–61]. We maintain that some of the leading genetic diseases that greatly increase the probability of developing autism are the result of chromosomal abnormalities (e.g., Down, Angelman, Fragile X, Rett, and Cohen syndromes), and as such should not be included in ASD because they are obviously genetic diseases, not diseases caused by epigenetic factors, environmental factors and other factors not primarily due to genetic causes. The inclusions of well‐established genetic diseases have greatly hindered the investigations that are attempting to find the real causes of ASD [62–64]. In the Table 2.1 we list other genetic diseases that are purely genetic disorders and are not really neurodevelopmental conditions arising from environmental factors. These impede finding the true causes of ASD and prevention efforts.

Is Finding Mutation the Path to Discovering the Origin of ASD?

Since the beginning of contemporary science, scientists have utilized mouse models, and numerous other small animal models, to produce various theories on autism. However, it should be noted that a Homo sapiens brain is both extremely complex and large. It has over 100 billion neurons and 0.15 quadrillion synapses. If one is looking for genetic mutations and comparing this with a mouse animal model, then one has to realize that these comparisons are not accurate. For example, an average adult human weighs 62 kg, contains 3.7 trillion cells (3.7×1013 cells), and lives to be about 70 years of age. On the other hand, an average mouse weighs about 20 g, or about 1/3,100 of an adult human, therefore on a proportional basis it would contain roughly 1.2×1010 cells, and live for 3 years. The human genome contains approximately 3 billion of these base pairs, which reside in the 23 pairs of chromosomes within the nucleus of all our cells. Each chromosome contains hundreds to thousands of genes, which carry the instructions for making proteins. The mouse genome is How Quickly Does Human DNA Mutate? 73 contained in 20 chromosome pairs and current results suggest that it is about 2.7 billion base pairs in size, or about 15% smaller than the human genome. If one takes a conservative estimation of the mutation rate of 5×10−7 mutations/ cell/generation, the generation of 3.7×1013 cells would cause several log higher mutations in a man than in a mouse. Similar logic can be applied to the human versus mouse brain. But, this is a very simplistic approach; a human brain is far more complex than a mouse brain and one cannot duplicate the findings in a mouse of a human by any means. This is not to say that the animal models are not useful in making broader implications but in the case of autism research a rodent or any small animal model is highly inadequate. If should also be noted that an adult animal body is constantly accumulating mutations while the cells are dividing and in a human body DNA replication happens across 70 years whereas in a mouse it lasts only for 3 years. Therefore, the degree of somatic cell mutations would be hundreds of times higher in a human than in in a mouse.

How Quickly Does Human DNA Mutate?

Every time human DNA is passed from one generation to the next it accumulates 100–200 new mutations, according to a DNA‐sequencing analysis of the Y chromosome. This number – the first direct measurement of the human mutation rate – is equivalent to one mutation in every 30 million base pairs, and matches previous estimates from species comparisons and rare disease screens [65]. The British–Chinese research team that developed the estimate sequenced 10 million base pairs on the Y chromosome from two men living in rural China who were distant relatives. These men had inherited the same ancestral male‐ only chromosome from a common relative who was born more than 200 years ago [66,67]. Over the subsequent 13 generations, this Y chromosome was passed faithfully from father to son, albeit with rare DNA copying mistakes. The researchers cultured cells isolated from the two men, and by utilizing next‐generation sequencing methods discovered 23 candidate mutations 12 of which were further validated by using traditional sequencing. Eight out of 12 of these mutations had arisen in their cell‐culturing process. Therefore, only 4 genuine, heritable mutations were left. Extrapolating that result to the whole genome gives a mutation rate of around one in 30 million base pairs. This direct measurement of the mutation rate can be used to infer events in our evolutionary past, such as when humans first migrated out of Africa, more accurately than previous methods. Most of the Y chromosome does not mix with any other chromosomes, which makes estimating its mutation rate easier. But the mutation rate might be somewhat different on other chromosomes. Also, there is a gender bias in mutations towards the male. 74 What is Autism?

What is the Mutation Rate in the Whole Human Genome?

For selfish reasons, we as humans are fundamentally interested in the mutation rate in our own species, and a number of questions are unresolved or still only partially answered. A central parameter is the average number of new genetic variants each of us has that our parents did not possess. How many of these mutations came from our father and our mother, how much does the mutation rate change with parental age, and is there significant variation in the mutation rate in the population as a consequence of other environmental or genetic factors? A more difficult question to answer concerns the frequency of mildly deleterious mutations and the distribution of their fitness effects. Along with the nature of selection on fitness in human populations, these parameters hold the key to understanding how a high genomic rate of deleterious mutation can be tolerated without causing an implausibly high rate of genetic death. Finally, if natural selection has been relaxed in current populations, what are the plausible consequences of mutation accumulation in our species? Mean estimates for mutation rates on a nucleotide site basis have been calculated at 1.8×10−8 and 1.3×10−8 per generation by Kondrashov [68], and Lynch and Conery [(69], respectively. These estimates align with other calculations based on the divergence of humans and chimpanzees as well as direct genome sequencing of parent–offspring trios. Deletions appear to occur approximately three times more often than insertions, whereas small insertion–deletions are relatively uncommon, occurring only in approximately 4% of mutations and only about 10% as often as single nucleotide events [68]. Thus, small insertion–deletions seem to have much lower importance in humans than in invertebrates (e.g., Caenorhabditis elegans, Drosophila) or flowering plants (e.g., Arabidopsis thaliana) [70].

Does Brain Size Matter?

The brains of humans and other nonhuman primates contain the same structures as the brains of other mammals, but are generally larger in proportion to body size. The most widely accepted method for comparing brain sizes across species is the encephalization quotient (EQ), which takes into account the non- linearity of the brain‐to‐body relationship. Humans have an average EQ in the 7–8 range, while most other primates have an EQ in the 2–3 range. Dolphins have values higher than those of primates other than humans, but nearly all other mammals have EQ values that are substantially lower (Table 2.2) [71]. Most of the enlargement of the primate brain comes from a massive expansion of the cerebral cortex, especially the prefrontal cortex and the parts of the Genetics versus Environment 75

Table 2.2 Encephalization quotient. This is a measure of relative brain size given as the ratio between actual brain mass and predicted brain mass for an animal of a given weight. EQ estimates the intelligence of an animal.

Species Encephalization quotient (EQ)

Human 7.44 Bottlenose dolphin 5.31 Orca 2.57 Chimpanzee 2.48 Rhesus monkey 2.09 Elephant 1.87 Whale 1.76 Dog 1.17 Cat 1.00 Horse 0.86 Sheep 0.81 Mouse 0.50 Rat 0.40 Rabbit 0.40

Source: Adapted from https://forum.bodybuilding.com/showthread.php?t=139128283. cortex involved in vision. The visual processing network of primates includes at least 30 distinguishable brain areas, with a complex web of interconnections. It has been estimated that visual processing areas occupy more than half of the total surface of the primate neocortex. The prefrontal cortex carries out functions that include planning, working memory, motivation, attention, and executive control. It takes up a much larger proportion of the brain in primates than in other species, and an especially large fraction of the human brain.

Genetics versus Environment

We are still stuck in the genetic dogma of autism and most researchers still believe that autism is a genetic disorder albeit an unusual one. It is not a single gene illness, but is caused by a cluster of genetic errors that occurs in an autistic child’s brain. ASD results in a distinctive constellation of behavior that manifests in the form of a varied number of disorders that are lumped together as a spectrum. To understand how exposure to extremely small amounts of chemicals may interfere in the normal development of a human fetus, we have to look at not 76 What is Autism?

the mutations that occur as part of the normal development as it is clear from the above discussion, but we have to look at how the normal branching of the human brain tree is disturbed by elimination or reduction in certain cell types that result in ASD. One approach is to utilize human fetal brain neurons that arise from an anomaly in the development of a fetus, resulting in migration of fetal brain‐progenitor neurons that settle somewhere in the body as nonmalignant tumors. These progenitor bundles of neurons behave much like fetal brain neurons but are disorganized. They also differentiate under specific neuronal growth hormones, respond to mutagenic and neuromodifying agents, and exhibit loss or gain of neuronal receptors similar to human fetal brain stem cells. We decided to use these neuroblastoma tumor cell lines from male and female origin and evaluate if there are unique or differential responses to exposures of minuscule amounts of fragrances or testosterone levels that one would expect to find in the amniotic environment during the early stages of gestation. Although no reliable neurophysiological network is associated with ASD, low levels of plasma oxytocin and arginine vasopressin have been reported. The “twin” nonapeptides oxytocin and arginine vasopressin are mainly produced in the brain of mammals, and dysregulation of these neuropeptides has been associated with changes in behavior, especially social interactions. Therefore, we analyzed the neuromodifications of three selected fragrances on male and female human fetal brain neurons, utilizing immunohistochemistry. We showed that exposure to even femtomolar concentrations of fragrances resulted in morphological changes by light microscopy in the neuroblastoma cell line (NBC). Importantly, these fragrances significantly reduced the oxytocin‐ and arginine vasopressin‐receptor positive neurons in male NBC but not in female NBC, possibly contributing to the development of male bias in ASD. This study is the first to show a potential link between fragrance exposure, depletion of oxytocin‐ and arginine vasopressin‐receptor positive neurons, and a male bias in autism. We are also evaluating the exposure of testosterone at the levels that are found in the amniotic fluids of normal as well as in ASD children during 9–14 weeks of gestation. The results from these lines of inquiry may show that ASD is the result of epigenetic factors, factors that are not genetic in origin but are due to exposure to environmental factors. Of note, we have identified over two dozen chemicals that show testosterone and other sex hormone‐like properties in fragrances. For example, numerous chemicals found to act like testosterone (i.e., oxybenzone, benzophenone‐1, galaxolide, and tonalide) are found in fragrances [2–15,24,25]. Other chemicals that have been shown to increase human estrogen receptor expression include octinoxate, oxybenzone, benzophenone‐1, benzophenone‐2, benzyl salicylate, benzyl benzoate, butylphenyl methylpropional, and synthetic musks (galaxolide, tonalide, and musk ketone). Butylated hydroxy toluene, benzophenone‐2 and octinoxate have been linked to thyroid hormone disruption [reviewed in References 77

Refs [15,72,73]. All of these chemicals are found in fragrances commonly used today. We have shown that even at femtomolar concentrations, fragrances with these chemicals can be mutagenic and carcinogenic to human fetal NBC [8– 10]. We will discuss the roles of these two neurohormones in detail in Chapter 4. We propose that a focus limited to proposed genetic causes to ASD may not be fruitful and or productive in solving the origin of the autism problem. This is an alarmingly growing problem – not only because of its public perception – but because of apparent increasing incidence based on diagnostic data. We argue that the “spectrum” is the result of interference in normal fetal brain development from the very early stages to 2 years after birth.

References

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Olfaction and Autism

It is also an era dominated by industry, in which the right to make a dollar at whatever cost is seldom challenged. Rachel Carson, 1962, Silent Spring

As with human beings, genes are classified by common characteristics, and grouped into units called families. In mammals, the largest of all gene families are the odorant receptors (ORs). Every olfactory sensory neuron selects only one such OR from over ~450 choices that are encoded in the genetic material available to it in the human genome. Many other mammals have up to 1,200 different ORs. However, these 450 ORs are akin to 450 different colors, combinations of which can be essentially unlimited [1–3]. Neurons are crucial to this process. Their shaft‐like and finger‐like extensions, axons and dendrites, facilitate the movement of electrical impulses and the attendant communication that helps the body function. Disruptions to the process can cause loss of a particular faculty, such as the ability to sense odors when ORs cannot carry out their customary functions, including the effective coordination of communica- tive nerve impulses. A complex circuit passes information from the nose to the brain [2,3]. Compared with other senses, such as sight or touch or hearing, olfaction (the capacity to smell) has commonly been regarded as less important. As more research links olfactory ability to various diseases, the importance of olfaction, including its relationship with the capacity to taste (gustation), may well deserve higher priority, if not equality, on research agendas. Individuals may survive better without the ability to smell than without the capacity to see, but the implications for having an impaired olfactory system, including the onset of disease in the elderly or prenatal or early childhood exposure to dangerous toxins, could be extremely serious in terms of quality of life for these individuals and others with whom they interact [4–19].

In recent years, research has focused increasingly on olfactory performance as an indicator of neurodegeneration. Viewed as a biomarker, such capacity, or lack thereof, has been linked to serious neurodegenerative conditions such as Parkinson’s disease, Alzheimer’s disease, Huntington’s chorea, and amyotrophic lateral sclerosis (ALS or Lou Gehrig’s disease). Olfactory dysfunction has, at times, been associated with severe psychiatric disorders, including schizophrenia, Alzheimer’s disease, Parkinson’s disease and many other diseases of the elderly, and in autism spectrum disorder (ASD) [4–19]. Moreover, loss of olfactory function may also be a meaningful predictor of 5‐year survival rates (5‐year mortality) for the elderly. Impaired olfactory ability has been observed in ASDs and other serious psychiatric disorders in adolescents and children, especially linked to tasks of identification [3–19]. Significantly, atypical capacity in the area of sensory processing is included as a diagnostic criterion in the latest version of the Diagnostic and Statistical Manual of Mental Disorders, DSM‐5. Atypical ability to process sensory information has been attributed to between 69% and 100% of ASD individuals in multiple studies. These sensory difficulties add to the observed symptoms in cognitive, social, and behavioral deficits identified with ASDs [3–19]. In anatomical terms, dysfunctions stemming from the brain’s orbitofrontal and medial temporal regions, which may be causative agents in olfactory deficits, are commonly associated with ASD symptoms. Although more difficult to examine through neuroimaging technology than areas of the brain that process auditory or visual responses, due to nearness of bony structures or cavities filled with air, progress has been made in the past decade in examining olfactory function. Particularly useful has been functional magnetic resonance imaging (fMRI), which has been utilized to assess the human brain’s olfactory function. The areas of the brain that process olfactory data have been more precisely identified, including the primary olfactory cortex, thalamus, hippocampus and parahippocampal cortex, entorhinal cortex, amygdala, insular cortex, hypothalamus, inferior lateral frontal region, and orbitofrontal cortex (Figure 3.1). Age is one of the primary influences on olfactory function with regard to ASD. Identification is more complex than mere detection. Odor detection capacity begins early in childhood (and according to some scientists in utero, in mid‐gestational period week 20). Odor detection ability occurs during adolescence but does so with less predictability for those with ASD than for normal (neurotypicals) genetic conditions not part of ASD but which commonly co‐present; this may shed light on olfactory realities but causation must be considered with caution [21,22]. More effective assessment of olfactory capacity has a number of possible clinical implications for ASD diagnosis and treatment. It could improve the accuracy and depth of ASD diagnosis. Standardized tools are still needed Olfaction and Autism 85

Tract Thalamus Amygdala

Olfactory bulb Hippocampus Odor/smell stimulation Raphe nucleus signaling Locus ceruleus Olfactory epithelium Pituitary gland Hypothalamus

Figure 3.1 Neural pathways of olfaction. Source: https://www.google.com/search? newwindow=1&hl=en&site=imghp&tbm=isch&source=hp&biw=1264&bih=576&q= OLFACTORY‐Sketch&oq=OLFACTORY‐Sketch&gs_l=img.3...1518.3940.0.5304.3.3.0.0.0.0.45.8 7.2.2.0....0...1.1.64.img..1.0.0.0.kQrt2CdsE_4#imgrc=uyYvJhZrR‐7EhM. Adapted from diy‐stress‐relief.com. (See insert for color representation of this figure.) for both olfactory and gustatory (sense of taste) diagnostic assessment. Specific olfactory environments that are favorable to ASD patients could potentially enhance the quality of their neurophysiological wellbeing. For example, olfaction has reinforcement potential. Those with ASD tend to be more effective with sorting tasks when a more pleasant smell, such as that of an orange, is present. One must be careful not to impose what is pleasant to those without ASD on those who are affected. For example, an experimental group of boys with either high functioning autism or Asperger syndrome, assessed via a test known as Sniffin’ Sticks, found the smell of cloves, pineap- ple, and cinnamon far less pleasant than did the control group of typical individuals [15]. Brain networks as they relate to olfactory ability involve carefully orchestrated coordination between central areas (important in odor identification tasks) and peripheral ones (important in sensitivity tasks). Since the same areas, especially the orbitofrontal cortex, are involved in ASD, olfactory research can inform ASD research, and vice versa. The orbitofrontal cortex is vital to higher level olfactory function and is crucial to flexible behavior and social control. Similarly, the improper functioning of the amygdala is strongly related to both ASD characteristics and loss of olfactory and gustatory capacity (Figure 3.1). 86 Olfaction and Autism

The human olfactory system is extensively represented in the cortical areas of the brain. This intricate system allows people to perceive odor and to remember it. When they inhale, or exhale, communication between cortical areas facilitates this perception and categorization of odors. The olfactory areas in the so called “gray matter” of the cerebral cortex are known to be sensitive to disease. Perception can be disrupted by many disorders, including autism, Parkinson’s disease, depression, and Alzheimer’s disease. Exposure to toxins early in life can also disrupt odor perception. Most of the cortices responsible for so called primary sensing are, in fact, multisensory. In olfaction in humans and other mammals, the sensory cortex is located near the brain’s periphery. The brain senses and remembers smell through coordinated assistance of olfactory sensory neurons (OSNs), the olfactory bulbs, and the olfactory cortex. Located between the eyes, the olfactory bulbs transmit signals that allow the brain to register the smell of odors that enter the nose (Figure 3.1). This intermediary role between nose and brain, as remarkable as it is, is just one reason why the olfactory bulb (the singular term bulb is often used to represent the plural reality of bulbs) is helpful to humans, and to researchers. It also plays a barometer‐like role in diagnosis of diseases. When an olfactory bulb does not function well, the chances are that the human involved has other serious problems besides an inadequate sense of smell. Given the obvious and visible importance of the senses of sight and hearing, it may come as no surprise that the sense of smell has been seriously understudied, but that has been changing in the recent past. As we will describe in detail, in a typical adult there are only two known areas in the brain that have the capacity to regenerate: olfactory cells; and dentate gyrus (see below). Our environment contains millions of odors, which are combinations of chemical odorants. Since there are so many faculties of the brain that are intimately involved in olfaction, there must be multiple mechanisms through which early sensory dysregulation in ASD could lead to social deficits throughout brain development, before and after birth. Future research is needed to clarify these mechanisms, and specific focus should be given to distinguish between deficits in primary sensory processing and altered top‐down attentional and cognitive processes. The olfactory system can detect and identify many thousands – and perhaps millions – of odorant molecules. No one knows exactly how many. Just imagine that each human possesses over 400 different odor recognition neurons. It is like there being over 400 different letters in an alphabet. How many words could be made from these 400+ letters? Odorants are small molecules that easily evaporate and become airborne. When one inhales the air, odorant molecules are drawn into the nose to enter the nasal cavity, entering a complex system of nasal passages (Figures 3.2). Lining a portion of the nasal cavity is the olfactory epithelium, a thin sheet of mucus‐coated sensory neurons that contains the olfactory receptor cells, along with supporting cells and basal (stem) cells. How Do We Smell? 87

+ Na 2+ Odorant Ca

Golf AC CI– Ca2+ GRK APR cAMP

Figure 3.2 Axons from the millions of olfactory receptor cells in the nose pass through a honeycomb‐like structure in the skull known as the cribriform plate as they travel to the olfactory bulb. Interaction of an odorant molecule with an olfactory receptor triggers a complex molecular cascade within the olfactory receptor cell. This process, known as transduction, translates chemical information from the odorant into electrical information that can be understood by the brain. Source:: Adapted from http://ars.els‐cdn.com/content/ image/3‐s2.0‐B9780123786302006769‐gr4.jpg.

Odorant molecules can reach the olfactory epithelium via the nose, the orthonasal olfaction process, and via the mouth, by the retronasal olfaction process. The odorants dissolve into and pass through a layer of mucous overly- ing the olfactory epithelium (Figure 3.2).

How Do We Smell?

Interaction of an odorant molecule with an olfactory receptor triggers a complex molecular cascade within the olfactory receptor cell. This process, known as transduction, decodes chemical information from the odorant into electrical information that can be understood by the brain. As an odorant molecule binds to cilia of the olfactory neurons (small protruding hair‐like structures) it activates a particular odor recognizing neuron and a signal is sent to various parts of the olfactory system. In this process, the primary neuron that recognizes the particular odor “dies”. However, shortly after it is regenerated by an identical neuron. Hence, regeneration of the olfactory system is essential for the sense of smell. This explains why when a person uses a perfume, after a few minutes the person cannot smell that particular fragrance. Also, we cannot smell our own bad odor! The receptors involved have died and someone else then has to tell us of our body odor (more later on this subject). Similarly, people working with garbage no longer smell the intense odors of the trash and the children, the so called “slumdogs,” who collect items from heaps of trash, do not smell the awful stench. Simply, this is because the neurons that sensed the odor have died and keep on dying and regenerating every day. In an adult 88 Olfaction and Autism

human, there are two types of neurons that constantly regenerate, olfactory odor neurons and the dentate gyrus neurons, which are involved in fortifying our memory [23]. The orbitofrontal cortex plays a particularly important role in the brain’s processing of olfactory input. This highly multisensory part of the brain is critical in the perception of flavor, an olfactory experience about which humans are intensely conscious. The perception of flavor is a particularly multisensory activity, with the taste (gustatory) component but one of several sensory contributors. If any of the odorant recognition systems becomes nonfunctional (sometimes due to an accident that results in a brain injury), food will no longer taste the same. Therefore, how an item of food feels in the mouth (somatosen- sory), smells (retronasal olfactory), and looks (visual) also contribute to an overall unitary perception of flavor. The orbitofrontal cortex, which receives input from the thalamus via the mediodorsal nucleus is crucial in information gathering and decision making; it is a highly developed multisensory and chemosensory area that helps humans perceive flavor and smell. Olfactory cortical areas, comprised of both six‐layer neocortical and three‐ layer archicortical parts, contribute to the processing of normal memory and odor perception. They help effect the seemingly fantastic transformation of a physicochemical substance into a mental perception. When the molecules of a substance are sufficiently volatile to travel into the nasal passages, the original substance typically loses an imperceptively small amount of mass. Our olfactory epithelium sensory neurons at the back of the nose, where millions of such neurons are located, start a bodily process that will initially detect that an odor is present, then discriminate, further recognize, and finally identify the odor. In addition, the brain will then remember what has been learned. It is not inconsequential that 5% of human coding DNA is tied to olfactory ability, and that humans have roughly 450 different kinds of receptors that, like locks that have met their odoriferous keys, assist in this complicated process. Dogs, noted for their olfactory capacity, have about double the number of receptors. They outperform their human owners at tracking because of these receptors, which bind the volatile odor molecules [1,2]. Odors are able to induce hedonic responses, which are based on a person’s perception of how pleasant or unpleasant an odor is. Two people can smell cinnamon and recognize it as cinnamon, yet their hedonic responses would vary if one finds the smell of cinnamon more pleasant than the other. During gestation, if a synthetic chemical found in fragrances and other substances enters the developing fetal brains they can be recognized by the primordial odorant neurons, just like they are recognized in the adult orthonasal olfaction process, or by the retronasal olfaction process. Once they bind any of these particular type(s) of odorant neurons they kill them (just as in the adult human where a particular neuron degenerates just after stimulation of an electrical signal). However, at early stages of gestation (in the developing fetal How Do We Smell? 89 brain), these primordial neurons may not be sufficient in number so that once they die, the brain may not have the capacity to regenerate that particular odor recognition neuron(s). These are the germinal centers of those particular odorant neurons. We have shown recently by utilizing male and female neuroblastoma cell lines (see details below) that these synthetic chemicals can also kill the neurons that are particularly important in the development of a male brain (the oxytocin‐ and arginine vasopressin‐receptor positive neurons) [24,25]. Oxytocin promotes social interactions and recognition in many species and particularly plays an important role in humans. Any disruption in the circuit mechanisms through which oxytocin modifies olfactory processing can be detrimental for normal social interaction [26]. Although anosmia (total loss of olfactory capacity) may not occur, since the synthetic fragrances do not kill all ~450 different kinds of odorant neurons found in man, the damage would be permanent. We maintain that the same toxins that can cause olfactory damage, in the areas behind the nose and those in the brain itself, can also lead to ASDs; hence the importance of taking olfactory capacity loss seriously, as something far more than loss of the normal ability to smell. Evidence of loss of selective odor capacity in ASD is overwhelming and explains our notion that synthetic chemicals as well as other environmental agents may be killing off certain odorant progenitor neurons during early fetal brain development. It should be noted that if our hypothesis is correct that only selected progenitor odorant neurons will be affected by the synthetic fragrant chemicals (as well as other chemicals that can be toxic to these kinds of neurons), then one can predict that there will be three groups of ASD children: one group would be supersensitive to certain odors (since the dead neurons have been replaced with surrounding neurons which have over grown to fill in the empty areas in the fetal brain that were destroyed by the selective killing of other odorant progenitor neurons); the second group would be deficient in certain odor sensing ability; and the third group would be nearly normal [27–41]. By utilizing the “sniff” test, all three groups have been identified and have been reported by numerous scientists. Just a reminder here that ASD is a “spectrum.” A keen observer would notice a true spectrum in the symptomology of odor research in ASD, with heightened odor sensitivity to the odor utilized in the ‘sniff’ test being reported, or abnormal response or no statistical differences, depending on the odor and methods employed and the ASD population chosen to test the assessments [4]. Heightened odor sense has been reported in ASD by some investigators [27,28,30,31,37], with low odor responses in ASD children and adults by other scientists [8,11–17,19,27,29–33], and no statistical differences by yet other investigators [28,39,42]. It should be noted that the degree of loss of olfaction would depend on how many kinds of synthetic odorant types and number of molecules actually reached a fetal brain and at what stages of gestation. Furthermore, the fetal 90 Olfaction and Autism

progenitor neurons must have receptors for those smell chemicals. Therefore, it is highly unlikely that all the odorant (~450 different kinds of ORs found in humans) will be present in early fetal brain development. This is the key to the autism spectrum. If only a few different types of odorants (fragrances) entered at 5 weeks of gestation, the elimination of the mother cells or progenitor cells for those particular odor neurons would be permanent. Moreover, other synthetic chemicals can enter and kill other types of nonodorant progenitor neurons. However, if the same degree of breach of the fetal brain occurred at later stages (i.e., weeks 11, 15, 22, 30, etc.), the degree of damage would be progressively less. Consequently, at any stage of breach, loss of odorant neurons as well as other types of neurons would be less obvious, but still causing ASD and in the smell area the symptoms would be highly heterogeneous. Of note, we want to reinforce the point that ASD is primarily not a genetic disease or disorder, but cellular damage by selective destruction of highly specialized neurons disrupting the highly organized evolutionary brain development cascade that is caused by manmade synthetic chemicals. These synthetic chemicals have no evolutionary history and our species (Homo sapiens) has never seen these chemicals (or rather, never previously smelled them during our evolution). There is not enough time to adapt to them. We have no efficient way to neutralize these chemicals. Back to the process of smelling: various output neurons in the olfactory bulb target different subregions of the cortex. Within just one or two synapses, information originating in the olfactory bulb spreads to diverse cortical regions that have diverse functional capacity. For example, signals sent from the olfactory bulb to the amygdala affect emotional processing; those to the olfactory tubercle affect motivated behavior; those to the entorhinal cortex‐hippocampus affect working memory and memory of episodic occurrences; and those to the orbitofrontal cortex affect the valuation of rewards and assistance with decision making, and autonomic regulation. The communication is not a one‐ way process. Extensive reciprocity is involved as seen, for instance, in the bidirectional flow of information between the entorhinal cortex and the piriform cortex, or between the orbitofrontal cortex and the piriform cortex. Moreover, the areas involved in olfactory processing are also often multisensory. The olfactory tubercle, for instance, helps process sound and odors, at least, and the piriform cortex deals with both taste and odor, at least. The entorhinal and orbitofrontal cortices deal with most of the senses. According to recent estimates, between 3% and 15% of the piriform cortical neurons are activated when a “puff” of odorant reaches them. While neuronal ensembles with only 50–100 neurons may be adequate for the encoding of particularly singular odors, most require more complex ensembles to process a particular odorant. Since the piriform cortex has a vast number of pyramidal neurons, perhaps as many as 50,000, shifting coalitions of overlapping ensembles are available to help identify the numerous odors extant in the modern world, which helps How Do We Smell? 91 explain the exceptional capacity of the human brain, and overall olfactory system, to identify and remember large numbers of smells. The multisensory processes in which the neurons in the piriform cortex participate not only help humans differentiate between one odor and another but also to discern the meaning associated with a particular odor. The piriform cortex may not only help one recall the pleasant smell of homemade bread but also evoke warm feelings about the place and circumstances where the olfactory memories originated. The same would be true of smells associated with highways, farms, restaurants, poisonous cyanide seed or mudroom, and gardens. An important passageway for information that enters and exits the hippocampal formation, the entorhinal cortex seems to be particularly sensitive to Alzheimer’s and other serious disorders. Its healthy functioning may therefore be useful as a predictor or validator of developing or advanced disorders. With regard to olfactory function, the entorhinal cortex (and especially the lateral entorhinal cortex) plays a vital role by accepting input from the piriform cortex and the principal olfactory bulb. In animals, entorhinal cortical response to odors is related to hunger and other internal states. In cases where an animal anticipates a reward, such as food that is consistently accompanied by a particular odor, the entorhinal cortex may actually be activated before a response from the olfactory bulb. This suggests that the odor identification process, which typically has been regarded as a bottom‐to‐top process, may at times be a top‐down process in which the piriform cortex and olfactory bulb are catalyzed by anticipation‐driven signals emanating from the entorhinal cortex. Olfactory impairment also results when children are exposed to alcohol during gestation. Heavy drinking during adolescence and in adulthood also leads to olfactory damage. To compound the tragedy of fetal alcohol spectrum disorders (FASDs), olfaction impairment may be accompanied by a lasting memory, and even preference for, the smell of alcohol. This, besides the insidious example of an alcohol‐abusing parent, can increase the probability of the continuity of excess alcohol consumption across generations. Impulsive drinking by a mother can, therefore, not only damage the olfactory system of the developing fetus but later make it harder for that same child to avoid impulsive drinking. Prenatal alcohol exposure has been widely studied and widely condemned due to the cognitive and behavioral damage it inflicts. Many regions of the brain affected by prenatal alcohol exposure have a simultaneous linkage to the processing and identification of odors. Damage to these regions of the brain are paralleled by documented neurological deficits associated with those same regions. Children exposed to alcohol prior to birth through maternal drinking were found to have significant olfactory deficits. Consequently, assessment of olfactory performance may well contribute to more accurate and thorough identification of persons at risk for deficits in cognitive and behavioral performance. Alcohol is toxic to mothers and fetuses; so are many ingredients in 92 Olfaction and Autism

fragrances. Therefore, while we applaud the extensive attention given to FASDs, we encourage more attention on other toxins. Such studies will be mutually reinforcing, deepen our understanding of olfactory and other sensory functioning, and promote greater public awareness of fragrance abuse, which, unlike alcohol abuse, may be done by individuals who do not knowingly imbibe or inhale substances that would damage them and their fetus. We further urge that public health officials, and particularly obstetricians and gynecologists, alert women who have conceived, or are attempting to do so, that alcohol, tobacco, and synthetic fragrances are dangerous to their future offspring, and that damage can take place even before they know they have conceived. When one reviews the types of behavioral problems commonly ascribed to gestational exposure to alcohol, it is easy to think of persons diagnosed with ASDs: poor adaptive abilities, inappropriate social interactions, weak communication skills, depression, hyperactivity, conduct problems, and attention deficits. Children on FASDs or ASDs have an increased likelihood of being unofficially labeled or officially classified as delinquent, impulsive, disruptive, and to perform poorly in structured academic settings.

Summary and Conclusions

Evidence has suggested that atypical sensory and, particularly, olfactory processing is present in many neurodevelopmental and neurodegenerative conditions, including ASDs, Alzheimer’s and Parkinson’s diseases, schizophrenia and depression. This chapter has outlined the mechanisms of olfaction and the organs and brain compartments involved in odor processing and suggested the possible involvement of olfactory impairment in ASDs, underlining the importance of olfactory evaluation in the clinical assessment of ASDs. Olfaction deficits are not universally reported in ASD patients since ASD is a spectrum and there is probably never going to be a biomarker or test that can clearly diagnose an ASD in a perfect fashion.

References

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Oxytocin, Arginine Vasopressin and Autism Spectrum Disorder

How could intelligent beings seek to control a few unwanted species by a method that contaminated the entire environment and brought the threat of disease and death even to their own kind? Rachel Carson, 1962, Silent Spring

Oxytocin

The neuropeptide oxytocin and its receptor have been predicted to be involved in the regulation of social functioning in autism spectrum disorders (ASDs). Produced in the hypothalamus, oxytocin is a neurotransmitter involved in social behaviors, including mammalian labor and nursing, maternal behavior, bonding, and social recognition and reward [1]. It binds to oxytocin receptors expressed by neurons across the brain, including in the amygdala, olfactory bulb, nucleus accumbens, brainstem, septum, and ventromedial hypothalamus [2]. Studies of mice in which the oxytocin receptor gene has been deleted revealed deficits in social interaction, including spending more time alone and self‐ grooming, compared with their wild‐type counterparts [3–5]. These behaviors are similar to those of individuals with ASD, suggesting that alterations in the oxytocin receptor during fetal development may help explain some of the social deficits characteristic of individuals with ASD [6–17]. In fact, decreased flow of oxytocin between mother and fetus has been associated with autistic‐ like characteristics in adulthood [6]. Autistic‐like behavior is observed when the receptor for oxytocin is down‐ regulated, thereby reducing binding of oxytocin in the central nervous system, resulting in social interaction deficiencies that are a hallmark of ASD [8–10]. Low levels of plasma oxytocin are associated with lower social and cognitive functioning [18–28].

Autism and Environmental Factors, First Edition. Omar Bagasra and Cherilyn Heggen. © 2018 John Wiley & Sons, Inc. Published 2018 by John Wiley & Sons, Inc. 98 Oxytocin, Arginine Vasopressin and Autism Spectrum Disorder

Although much remains to be learned about the oxytocin–oxytocin receptor system and its role in social behavior, low levels of oxytocin have also been associated with other neurodegenerative disorders. In Alzheimer patients, decreased oxytocin binding site density in the nucleus basalis of Meynert in the brain has been observed [8,10]. Similarly, individuals with Parkinson’s disease have fewer neurons with oxytocin receptors in the hypothalamus [8,10]. The exact causes of oxytocin receptor down regulation are not entirely clear; however, we have discovered that some fragrances have been linked to lower levels of the receptor [22,25]). Sex differences have also been observed, with females often exhibiting higher levels of oxytocin than males, which may help explain ASD male bias [22,29–44]. Environmental factors have been linked to oxytocin receptor expression changes during fetal development. Increased oxytocin receptor binding in the nucleus accumbens and CA3 region of the hippocampus has been observed with exposure to nicotine and ethanol [22,23,25,27,29,45–54]. Oxytocin receptors are also upregulated in mu‐opioid knockout mice, indicating a connection between oxytocin receptor expression and the opioid reward system [10]. Another factor that could suppress arginine vasopressin (AVP) and oxytocin receptor binding is underdevelopment of olfactory bulb neurons, which has been observed in children with ASD [52–54]. Fragrances are known to bind to olfactory neurons in the brain and could contribute to this underdevelopment [22,25,29]. Excessive exposure during critical periods of fetal development could overwhelm the natural system of regeneration, thereby inhibiting the normal development of the olfactory neurons [22,25,27,29,45–51]. Chemical fragrance exposure may also contribute to axon thinning/elongation and increased synapses [25,29]. Children with ASD are unable to eliminate excess neurons and synapses, resulting in excessive communication between different parts of the brain. The end result is inhibition of oxytocin and AVP binding, which can result in neurodevelopmental delays and social impairment, as is observed in ASD. Treatment with synthetic oxytocin hormone improves symptoms in children with autism. In a pilot study, treatment with 0.4 IU/kg/dose oxytocin for 12 weeks had positive impacts on social and behavioral parameters that persisted for 3 months following administration [55–57]. In a subsequent section we have provided a detail account of oxytocin treatment for ASD. Another study examining intranasal oxytocin at 12 IU for 5 weeks showed improvement in social interaction deficits with minimal side effects. These studies illustrate the important role of oxytocin in the regulation of social, emotional, and behavioral parameters and the potential amelioration of autistic symptoms by targeting this mechanism. In primates, oxytocin receptors are concentrated around the cholinergic regions that regulate visual and auditory processing, including response to visual cues [58–60]. Studies in nonhuman primates have shown that oxytocin delivered intranasally can increase oxytocin in cerebrospinal fluid and plasma and may, thereby, affect brain oxytocin signaling Why Oxytocin Therapy May be Important for ASD? 99

[61–64]. Therefore, if oxytocin administered intranasally can penetrate the brain, it may be able to improve social information processing. Alternatively, oxytocin release may be stimulated through melanocortin receptor activation, which has been shown to increase adult social bonding in prairie voles [62,63]. Overall, targeting the oxytocin system may be an effective mechanism for improving social cognition.

Why Oxytocin Therapy May be Important for ASD?

Oxytocin, the main neuropeptide of social communications, is expressed in neurons exclusively localized in the hypothalamus. Numerous neuroendocrine, metabolic, autonomic and behavioral effects of oxytocin have been reported over the last decade. In the rush to find treatments for syndromes such as autism, many clinical trials have been initiated evaluating oxytocin in adults, adolescents, and children. However, the impact of oxytocin on the developing brain, especially on the embryonic and early postnatal maturation of oxytocin neurons, has been poorly investigated. One of the most important points to understand is that oxytocin secretion in humans starts just after birth. Before this, during fetal development the oxytocin receptors develop in various parts of the brain, as shown in Figure 4.1. In the following section we will first summarize the available literature on the oxytocin and AVP neuropeptides and their receptors. Most importantly, we will address how and why oxytocin is important in human social development and in the human fetus where oxytocin and oxytocin receptor positive neurons emerge. We will also consider why it may be important to treat ASD infants with nasal oxytocin. The differentiation of oxytocin‐secreting neurons during fetal development and afterwards will be explored and we will provide evidence that early alterations, from birth, in the central oxytocin system can lead to severe neurodevelopmental diseases, including feeding deficit in infancy and severe defects in social behavior in adulthood, as described in ASD. Our review presents a hypothesis about developmental dynamics of central oxytocin pathways, which are essential for survival immediately after birth and for the acquisition of social skills later. A better understanding of the embryonic and neonatal maturation of the oxytocin system may lead to better oxytocin based treatments in autism and other social–behavioral conditions. An important consideration when comparing developmental trajectories in translational medicine approaches is that the neurodevelopmental stage of the human brain at birth corresponds to the rat and mouse brain at postnatal day 10 [10]. Consistently, the human infant pattern of oxytocin receptor expression is achieved before birth while, in the rat, oxytocin receptor expression is achieved around postnatal day 10, and, in mice, between postnatal day 7 100 Oxytocin, Arginine Vasopressin and Autism Spectrum Disorder

(a) Oxytocin receptor developmental expression

First appearance of Progressive appearance of OXR First transition to the Second transition to OXR in the brain in discrete brain regime adult pattern the adult pattern

Embryonic Embryonic Prenatal day 10 Prenatal Gestational Prenatal day 10 Age Gestational day 14 day 18 Prenatal day 13 day 19 week 5 week 8 Prenatal day 13

Infant pattern Adult pattern (b) Dorsal

Caudate nucleus PN-Postnatal

Hypothalamus ventro- Olfactory medial nucleus Periform Bed nucleus of the cortex Amygdala Nucleus accumens Lateral septum stria terminalis Anterior Transient OXR expression Permanent OXR expression

Figure 4.1 Developmental trajectories of oxytocin receptor in the rat brain. (a) Schematic time course of oxytocin receptor expression in the developing brain. (b) Oxytocin receptor expression in the infant brain around prenatal period P10–P13. Regions in which a transient oxytocin receptor expression is observed are colored in red. Regions in which oxytocin receptor expression is maintained to adult life are colored in blue. OXR, oxytocin receptor. Source: Ref. [10]. Copyright (c) 2015 Grinevich et al. (See insert for color representation of this figure.)

and postnatal day 14. To extrapolate the effects of pharmacologic and environmental manipulation of the oxytocin–oxytocin receptor system in the human newborn brain from experiments performed in mice and rats, treatments should thus be performed in rodents around postnatal day 10, when a comparable maturation of the brain and of the oxytocin receptor system has been reached in these species. Most importantly, we show that synthetic fragrances and many of the ingredients commonly found in many fragrances [i.e., diethyl phthalate (DEP), tonalide (musk ketone), octinoxate, d‐limonene, eugenol, benzyl benzoate, benzyl salicylate, (+)‐ α‐pinene] reduce the number of oxytocin receptor positive neurons in male neuroblastoma cell lines. We will provide detailed information below, but at this point it is suffice to mention that if an exposed offspring is born with significantly low numbers of oxytocin receptor expressing neurons, then delivery of a large amount of oxytocin may not be as effective since the “synthetic chemicals” have already damaged the corresponding receptors (see below for more detail). Several environmental factors have been reported to affect oxytocin receptor expressions in embryogenesis and the neonatal period, with social and Why Oxytocin Therapy May be Important for ASD? 101 sensorial experiences playing a predominant role, as more extensively discussed in the next paragraph. Furthermore, exposure to drugs and toxic agents can also modulate the oxytocin–oxytocin receptor system, as outlined by the interesting finding that nicotine and ethanol administration to pregnant rats increases oxytocin receptor binding in the hippocampus of male offspring, specifically in the nucleus accumbens and CA3 regions (Figure 4.2).

CA1

sch

Mol CA2

DG pp mf Glutamate CA3

Bregma-3.6

BNST CeA NAc BLA mPFC Glutamate

Figure 4.2 Neuronal projections in the hippocampus. Schematic representation of the coronal view of the hippocampus region indicating the subregions of the hippocampus and their location within the hippocampus. CA, cornu ammonis. Trisynaptic circuitry in the hippocampus is indicated with axons from the entorhinal cortex projecting unidirectionally to the apical dendrites of the hippocampal DG, CA1, and CA3 neurons (perforant path projection). DG neurons project to the apical dendrites of the CA3 pyramidal neurons (mossy fiber projection). CA3 neurons project to the apical dendrites of the CA1 neurons (Schaffer collateral projection). The CA1 neurons have bidirectional projections to and from the BLA. The BLA also sends projections to the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), bed nucleus of the stria terminalis (BNST), and central nucleus of the amygdala (CeA). mf, Mossy fiber projection; Mol, molecular layer; pp, performant path projection; and sch, Schaffer collateral projection. Source: https://www.researchgate.net/ publication/244482778_The_Interplay_between_the_Hippocampus_and_Amygdala_in_ Regulating_Aberrant_Hippocampal_Neurogenesis_during_Protracted_Abstinence_from_ Alcohol_Dependence/figures?lo=1. Reproduced with permission of Frontiers Media SA. 102 Oxytocin, Arginine Vasopressin and Autism Spectrum Disorder

Hormones, Neuropeptide Arginine Vasopressin and Oxytocin in ASD

The neuropeptides oxytocin and vasopressin increasingly have been identified as modulators of human social behaviors and have been associated with neuropsychiatric disorders characterized by social dysfunction, including autism [1–3,65– 68]. It has been shown that the measured volume of the olfactory bulb correlates with the functional capacity in normal individuals (e.g., larger olfactory bulbs exhibit finer olfactory discrimination). The ability to discriminate between distinct smells, which is dependent on intact functioning of the olfactory bulb, has been shown to be impaired in autism. It can be assumed, therefore, that a substantial reduction in size (or total absence) of the olfactory bulb, as observed in autism, may explain some of the symptoms. This abnormality may have impacts beyond olfactory discrimination due to utilization of the olfactory bulb in social and emotional processing. Of note, the neuropeptide AVP has been hypothesized to play a role in the etiology of autism based on demonstrated involvement in social bonding and in the regulation of a variety of socially relevant behaviors in animal models. AVP regulates male social behavior not just through higher expression in males but also through steroid‐sensitive brain sexual dimorphisms in AVP neurons. AVP may therefore influence sexually dimorphic social behaviors in a range of species. The role of sex hormones on AVP is of interest in the context of autism considering that the ratio of affected males with autism compared with females is markedly skewed (4:1). This may be due to the fact that a large number of fragrances have hormone‐like effects, perhaps partially explaining the underdevelopment of olfactory bulbs and male preference. Olfactory bulbs contain a high density of oxytocin and vasopressin receptors and the actions of these neurotransmitters are dependent on the proper functioning of this region of the brain. Any impairment of the olfactory bulbs during development would compromise a key mechanism of action mediating these social behaviors. As shown below, fragrances are designed to bind the smell receptors of olfactory neurons. Therefore, the binding of any substance that may hinder or modulate normal development of fetal brains may theoretically contribute to the development of autism. Although there is no reliable neurophysiologic marker associated with ASD, low levels of plasma oxytocin and AVP have been reported [8,20,45]. The twin nonapeptides oxytocin and AVP are mainly produced in the brain of mammals [18], and dysregulation of these neuropeptides has been associated with changes in behavior, especially social interactions. Both peptides are produced by nonoverlapping populations of neurons in the same hypothalamic nuclei, the supraoptic nucleus and the paraventricular nucleus. The supraoptic nucleus and paraventricular nucleus contain large magnocellular peptidergic neurons that send their axons to the posterior pituitary where they release AVP and oxytocin into the blood [1–3,64–66] (Figure 4.3). The AVP and oxytocin signals evoke their physiological effects via their respective receptors [8,10]. Development of Oxytocin and AVP Neurons in Various Animals and in Man 103

Paraventricular Supra-optic nucleus nucleus Hypothalamo- hypophysial tract

Optic chiasma

Inferior hypophysial artery

Vasopressin oxytocin

Figure 4.3 Illustration of the major neurons that produce oxytocin and AVP. Oxytocin is mainly synthesized in the paraventricular hypothalamic nucleus and supraoptic nucleus, stored in Herring bodies and released into systemic circulation from the posterior pituitary. In the pituitary gland, oxytocin is packaged in large, dense‐core vesicles, where it is bound to a large peptide fragment called neurophysin I.

Numerous studies have shown that children with autism have significantly lower levels of plasma oxytocin and AVP than their peers [8,20,45]. A recent study also illustrated that mothers of ASD children show low levels of oxytocin and AVP and high levels of testosterone. Furthermore, lower concentrations of oxytocin in plasma are associated with lower social and cognitive functioning in children.

Development of Oxytocin and AVP Neurons in Various Animals and in Man

In many species – from zebrafish to Homo sapiens – oxytocin producing neurons emerge from the proliferative (“convoluted”) neuroepithelium of the diamond shaped third ventricle during fetal development [64–66]. In the hypothalamus, oxytocin is produced in the specialized secretory cells called magnocellular neurons. Birth‐dating studies have discovered that the hypothalamic neurons that subsequently produce oxytocin are generated in the second half of the gestational period in rodents, within the first quarter of the gestational period (E30–43; length of pregnancy ~165 days) in macaques [10], and in the middle of pregnancy in humans [10]. In humans, the supraoptic and 104 Oxytocin, Arginine Vasopressin and Autism Spectrum Disorder

paraventricular nuclei are completely formed at 25 weeks of gestation and oxytocin‐immunoreactivity is first detected at 26 weeks of age. At that time, the number of stained oxytocin neurons is relatively similar in the fetal and adult hypothalamus [10], while the morphologic analysis of individual magnocellular neurons suggests that these cells are still immature, as indicated by the subsequent increase of their nuclear volume. A single oxytocin receptor and vasopressin receptor subtypes (V1α and V1β) are centrally expressed and distributed widely throughout the brain. Centrally, particularly high expressions of these receptors are found in regions underlying the control of many social behaviors, such as the nucleus accumbens, ventral tegmental area, amygdala, and hippocampus [10] (Figure 4.3). Although oxytocin and vasopressin exert a range of neuropeptide specific physiological functions, there is evidence that they may cross‐react at receptor levels, given similarities in structure, suggesting that increased activity of one peptide may also exert some influence over expressivity of the other [67–72]. Along with oxytocin, in males, AVP is also produced in the amygdala and the bed nucleus of the stria terminalis, transported to the pituitary and then released into the blood stream where they deliver hormone effects to tissues. Oxytocin and AVP also can also diffuse into the central nervous system [1–3,62–79]. The genes for these two neuropeptides are closely linked on chromosome 20p13, separated by only 12 kb, which allows interaction [5,27,46,48]. These neuropeptide hormones have receptors in various brain regions and throughout the body, including areas that are important for regulating social behavior and reactivity to stressors (Figure 4.4). As in humans, the transcriptomic analysis of oxytocin receptor revealed a progressive increase in oxytocin receptor mRNA during embryonic life in five out of six brain areas analyzed [73–75]. Remarkably, the receptor level appeared to reach a maximum prior to birth and remained quite stable thereafter, at least for the first 5 years of life, although with some individual variations. As we have shown in Figure 4.2, the human oxytocin receptor area in the brain is fully mature and ready to respond to oxytocin at birth.

Oxytocin and Social Experience in Development

Effects of oxytocin during embryogenesis and neonatal ontogenesis on social life have been extensively summarized in recent reviews [77,78], but the reverse effects (i.e., the effects of social stimuli on maturation of the oxytocin system) have been explored less. It has been reported that early social experience tre- mendously affects physiology of the oxytocin system [78]. Specifically, it has been demonstrated that early sensory experience (in the newborn) regulates development of sensory cortices via oxytocin signaling [63,64]. Oxytocin and Social Experience in Development 105

Exogenous OXT Reproductive experience: Exogenous OXT mating, pregnancy, and parturition OXT magnocellular neuronsin the Social memory hypothalamus: OXT production Social experience Social behavior

Autoregulation Dendritic OXT central release Dendritic OXT central release CSF

Posterior pituitary Anterior pituitary

Axonal OXT peripheral release

Figure 4.4 Effects of oxytocin on various human physiological responses including human reproduction, social behavior, social experiences, social memory, and overall social communications. Source: Adapted from http://journal.frontiersin.org/article/10.3389/ fnins.2012.00182/full. Reproduced with permission of Frontiers Media SA.

Early regulation of oxytocin may have long‐term consequences. It has been known for many years that exogenous oxytocin in neonates can reverse the long‐term behavioral effects of prenatal stress [80,81] and has consequences on other endocrine systems (i.e., the estrogen receptor) as well as on blood pressure [81] in adults. The effects of oxytocin, or of the environmental and familial circumstances resulting in increased production of mature neonatal oxytocin, on adult social behavior remain to be investigated. With respect to oxytocin receptor expression, increased levels have been observed in offspring after communal rearing [82], increased maternal licking/grooming, and social enrichment [83]. On the contrary, late weaning has been reported to reduce oxytocin receptor density in selected, socially relevant, brain regions [82]. Furthermore, maternal separation has been found to induce a complex, region‐ specific modulation of oxytocin receptor expression [10,84]. It is important to note that exploration of early life experience on the oxytocin–oxytocin receptor system in animals provides a scientific basis for new child care and new therapeutic approaches to ameliorating social alterations occurring in adult patients afflicted with ASDs or Prader–Willi syndrome (see below). 106 Oxytocin, Arginine Vasopressin and Autism Spectrum Disorder

Oxytocin and Developmental Neurological Disorders

Many reviews have exhaustively described the involvement of oxytocin in learning and memory, and in shaping and regulating the social brain. We believe one of the best reviews is by Grinevich et al. [10]. The strongest line of evidence supporting the oxytocin system in social behavior, feeding behavior, and maternal care has been generated via the study of knockout mice in which either the oxytocin or the oxytocin receptor genes are inactivated (Table 4.1). Mice constitutively lacking oxytocin (Oxt−/−) are unable to release milk and have impaired social memory [85]. Mice with constitu- tive ablation of the oxytocin receptor gene (Oxtr−/−) display a behavior very similar to that of a mouse where oxytocin producing neurons had been depleted. These animals display social deficits. In addition, although learning is normal in mice where the oxytocin receptor gene has been deleted (Oxtr−/− mice), reversal learning is strongly decreased, indicating impaired cognitive flexibility suggestive of ASD [86]. Importantly, even a 50% loss of

Table 4.1 Abnormal compartments in ASD that share the loci of oxytocin receptors.

Brain region Autistic abnormalities

Amygdala 13–16% enlargement; functional magnetic resonance imaging study showed that patients with autism did not activate the amygdala when making mentalistic inferences from the eyes, whilst people without autism did show amygdala activity Olfactory Individuals with ASD exhibited impaired, intact, or increased odor sensitivity. Abnormal responses to tastes, cold, heat, pain, tickle, and itch were also observed Nucleus This is involved in reward anticipation and appears to be defective in ASD accumbens BNST BNST processes information and readiness for response to a threat by maintaining information from a vast connectivity network. Anatomically, the BNST is sexually dimorphic; it may explain the gender disparity in the prevalence and treatment of stress‐related psychiatric diseases and as such should be investigated as a possible target for treatments. Indeed, drug targets already involve subpopulations of receptors abundant in the BNST such as serotonin Periform Cytoarchitecture changes in olfactory cortex may underlie olfactory cortex differences and sensory deficits seen in autism; increased glial cells Caudate Increased volume of caudate nucleus (enlargement) in medication naïve nucleus individuals

BNST, bed nucleus of the stria terminalis. Source: Adapted from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4341354/table/T4/. Oxytocin and Developmental Neurological Disorders 107 the oxytocin receptor positive neurons leads to an impairment of social behavior, suggesting that a fine tuning of the oxytocin system is necessary to develop and control social behavior. Other mouse models, in which the knockout of a specific gene induced a disruption in the oxytocin system that has been linked to a pathological phenotype, reinforce the role of oxytocin in neurodevelopmental disorders (Table 4.1). Recently, it has been shown that disruption of the neonatal surge of oxytocin coming from the mother during delivery results in autistic‐like features in the adults [87]. Together these data suggest that an early postnatal injury or dysfunction of the oxytocin system has consequences in infant behavior and subsequently in the adult behavior. As previously mentioned, in both the human and mouse genomes, oxytocin and AVP neuropeptide genes are located adjacently on the same chromosome. Often the blood levels of both hormones are highly correlated [88], suggesting a coordinated release. This close proximity facilitates coordinated regulation of social and adaptive behaviors, the hypothalamic–pituitary–adrenal axis (HPA), and autonomic nervous systems. At times, they may have opposing physiologic effects, such as in the autonomic nervous system where oxytocin may have parasympathetic effects while AVP has regulatory effects. However, at high levels the neuropeptides can be partial opposing effects for their homologous receptors, which may result in interactions between the AVP and oxytocin pathways. Of particular importance in neurodevelopmental disorders is the fact that oxytocin and AVP can modulate social and repetitive behavior and other manifestations of anxiety and state regulation [10]. Animal research has generally associated oxytocin release or exposure with positive sociality, reduced anxiety, and lower levels of reactivity to stressors. AVP influences anxiety, the regulation of the hypothalamic–pituitary–adrenal axis (Figure 4.4), and stress responses. In general, central AVP has stress and anxiety relaxing effects. However, evidence in rats has shown a dose related effect. For example, AVP may relieve anxiety if given in low doses. Oxytocin receptor and oxytocin regulator knockout mice exhibit decreased social memory, cognitive flexibility, and resistance to change a learned pattern of behavior in clinical studies [10]. Both social deficits and behavioral rigidity were ameliorated by oxytocin administration [86]. The finding that oxytocin continues to have effects in oxytocin receptor depleted (knockout) mice supports the notion that oxytocin can influence behavior through other receptors, especially the AVP receptors (e.g., AVPR1α and/or AVPR1β). Given the influence of these neuropeptides on brain regions affecting both social and repetitive behaviors, modulation of oxytocin and AVP pathways is being explored as a treatment target for disorders, including Fragile X syndrome and ASD. 108 Oxytocin, Arginine Vasopressin and Autism Spectrum Disorder

Exogenous Oxytocin Treatments in Humans

This and other research has set the stage for a series of recent studies on the effects of exogenous oxytocin treatments in humans (reviewed in Ref. [89]). For example, intranasal oxytocin administration in healthy human males has been shown to increase prosocial behaviors and trust, especially as measured by experimental economic games. Exogenous oxytocin may also increase gaze towards the eye region of the face, and has been associated with improved facial memory, enhanced salience of social cues, and improved performance in the “Reading the Mind in the Eyes” (or RMET) test. Multiple studies have demonstrated the positive effect of oxytocin on social interactions, fear reduction, and social cue interpretation and the negative effects that occur when oxytocin is downregulated, resulting in neuropsychiatric disorders. More recently, researchers have discovered defective oxytocin receptor genes in ASD patients [90] these are generally single nucleotide polymorphisms (SNPs; single nucleotide deletions or changes). Significant associations have been reported in ASD and oxytocin receptor SNPs. Of note, there are multiple different kinds of SNPs at different loci in the oxytocin receptor DNA, suggesting environmental insults have induced point mutations rather than a genetic basis of ASD [91–101]. There are many diseases that are caused by environmental insults [102–108]. A single oxytocin receptor and two subtypes of vasopressin receptor (i.e., V1α and V1β) are centrally expressed and distributed widely throughout the brain. Centrally, particularly high expressions of these receptors are found in regions underlying the control of many social behaviors, such as the nucleus accumbens, ventral tegmental area, amygdala, and hippocampus (Figure 4.2). Although oxytocin and vasopressin exert a range of neuropeptide specific physiological functions, there is a possibility that they also cross‐react at the receptor levels (given similarities in structure), which suggests that increased activity of one peptide may also can modulate the expressivity of the other. In summary, a large body of research suggests that lower concentrations of oxytocin in peripheral circulation may be causally associated with social impairments, particularly in ASD children but also in schizophrenia. Evidence in favor of this idea has often been used to support trials of oxytocin administration to increase oxytocin levels in individuals with an “oxytocin deficit”. However, inconsistent evidence in individuals with ASD does not favor such a treatment. Several studies have reported lower oxytocin levels in ASD [109–111], while others have not exhibited any differences [112–114] and one has reported increased oxytocin levels in ASD [115]. Here, again, we remind our reader that ASD is a spectrum, and it all depends when a fetus suffered the insult that disturbed the oxytocin and oxytocin receptor developmental pathways (Figure 4.2). We believe, the spectrum in ASD is due to exogenous insults (mainly the environmental chemicals). It is when, how much, and at what stage of brain development that a fetus encountered an insult that perturbs the balance of differentiation. Intranasal and Intravenous Oxytocin Studies in ASD 109

gistrations 5 re

3 Number of 2

10 11 12 13 14 15 2005 2006 2007 2008 2009 20 20 20 20 20 20 Year of registration

Figure 4.5 Some of the clinical studies registered on clinical trial registries by 2015. From 2005 to 2013 the number of studies involving intranasal delivery of oxytocin to ASD individuals had increased. For 2014 and 2015 the data are incomplete. Source: Adapted from Ref. [117].

By 2015 there were over 50 oxytocin administration studies, which included ASD, schizophrenia, postpartum depression, post‐traumatic stress disorder (PTSD), and irritable bowel syndrome. There have been a growing number of studies investigating the ability of intranasal oxytocin to treat a range of neurobehavioral disorders based on the associations between intranasal oxytocin and alterations in social decision‐making, processing of social stimuli, certain social behaviors such as eye contact, and social memory (Figure 4.5). This is briefly described below.

Intranasal and Intravenous Oxytocin Studies in ASD

Currently medications for ASD alleviate certain symptoms, but do not address the core features of ASD. Risperidone and aripiprazole may be used for irritability, whereas guanfacine and clonidine are used for aggression, and selective serotonin reuptake inhibitors are used to treat anxiety. Recently, oxytocin has been investigated to target ASD core symptoms, social deficits and restricted, 110 Oxytocin, Arginine Vasopressin and Autism Spectrum Disorder

repetitive patterns of behavior. Defined by the Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM‐5), restricted, repetitive patterns of behavior include stereotyped or repetitive motor movements, insistence on sameness, inflexible adherence to routines, or ritualized patterns of verbal or nonverbal behavior. Highly restricted, fixated interests that are abnormal in intensity or focus, and hyper‐ or hyporeactivity to sensory input or unusual interest in sensory aspects of the environment are also classified as restricted and repetitive behaviors (RRBs). Several studies, using intranasal and intravenous oxytocin, in patients with ASD have been conducted (reviewed in Ref. [89]). It has been shown that nonapeptides, like AVP, can be measured in cerebrospinal fluid after intranasal administration [116]. Ease of giving intranasal drugs makes it a preferred route for most ASD studies, although more research needs to be conducted on how intranasal oxytocin reaches the brain and how it regulates receptors and neural pathways with different or chronic dosing strategies. Several studies have measured oxytocin responses to single dose challenges in ASD, while few have examined longer term treatment effects. Studies have often focused on symptom subdomains or defined social tasks including: RRB, emotion recognition, affective speech comprehension, and facial recognition.

Oxytocin Trials in ASD: Beyond the Hype and Hope

In the preceding sections, we summarized some of the research that shows that potentially exogenous oxytocin may be helpful to ASD children. We have also cautioned that oxytocin may not be that helpful if oxytocin receptors are depleted during fetal development or due to SNPs that can render the receptors useless or with less function. However, this has not tempered the enthusi- asm of some physicians and investigators in trying to alleviate some of the ASD symptoms via exogenous oxytocin. In a recent comprehensive study, Alvares et al. [117] have reviewed the current status of oxytocin based therapy. After much hyped early findings, subsequent clinical trials of longer term administration of intranasal oxytocin have yielded mixed results. It is still unclear whether many of the disappointing results reflect no real beneficial effects of exogenous oxytocin or are diminished by methodological differences masking true effects. The evidence to date suggests a number of methodical or design differences that may be unified to improve research in this area. These include considering the choice of ASD outcome measures, the amount of oxytocin delivered by nasal spray devices, the types of delivery devices to be utilized, and the selection of ASD patients and normotypic controls. Despite these limitations in the field to date, there remains significant hope for delivery of exogenous oxytocin to improve social impairments observed in ASD patients. Alvares et al. [117] stated: “Given the considerable media hype around new Oxytocin Trials in ASD: Beyond the Hype and Hope 111 treatments for ASD, as well as the needs of eager families, there is an urgent need for researchers to prioritise considering such factors when conducting well‐designed and controlled studies to further advance this field.” Availability of intranasal oxytocin (most commonly Syntocinon) for the study of psychological and behavioral phenomena has attracted many physicians and other investigators to evaluate its effects on ASD patients. Kosfeld et al. (118) were the first to publish an influential paper reporting that acute intranasal oxytocin increased trusting behavior in socially deficit patients. This study led to the flood of subsequent studies and clinical trials utilizing a single‐dose administration suggesting that oxytocin was the panacea for improving individuals with deficits in social cognition, who were emotionally disturbed, and those with high stress and anxiety. Many studies were backed up by fMRI evidence that suggested oxytocin increases activity in brain regions associated with social cognition and modulates function connectivity between these regions, indicating a role in modulating not just social behaviors but the neural and network underlying social interaction. Subsequent studies to utilize oxytocin to alleviate ASD symptoms and identify effective therapeutics for ASD to reduce core social communication deficits were rapidly carried out. In the last decade or so, there have been numerous studies to investigate the use of either a single dose oxytocin or multi‐dose or long‐lasting oxytocin in ASD patients and the results have been mixed. The first, very promising study was by Guastella et al. [(119] who used intranasal oxytocin in adolescent ASD males, and found that oxytocin administration improved the accuracy of clas- sifying emotions, particularly with less challenging items; they were the first report to indicate feasibility and efficacy for intranasal oxytocin for improving social cognition in younger individuals with ASD. Subsequent studies demonstrated effects of intranasal oxytocin on a range of experimental outcomes. Andari et al. [110] first demonstrated oxytocin significantly increased eye gaze to social regions of faces in adults with ASD compared with placebo and normally developing control groups. Auyeung et al. [120] also reported that oxytocin modulated changes in eye gaze during a real‐time social interaction. Of note, in healthy individuals, a well‐replicated finding has been in the modulation of eye gaze to static facial image after oxytocin administration. Within the ASD group, this beneficial effect of oxytocin was particularly prominent for those individuals who spent less time looking at the eye region under placebo. These cumulative experimental findings from single‐dose studies suggest that the short term and immediate effects of oxytocin, mostly in males with ASD, with average‐to‐high cognitive functioning, imparted beneficial effects. Several studies that used fMRI reported significant changes in activation brain areas involved in social information processing, including the amygdala, medial prefrontal cortex, and anterior insula, as well as in broader areas involved in reward processing in a child study [117]. In all of these studies, the participants 112 Oxytocin, Arginine Vasopressin and Autism Spectrum Disorder

were relatively homogenous, restricted to high functioning males (i.e., a lack of comorbid intellectual disability), and there were consistent sample sizes (14–33 participants). In numerous investigations, where repeated administration of oxytocin was delivered over a 6‐ or 8‐week administration period, and ASDs were compared with a placebo group, the study individuals in the oxytocin group did not significantly improve in measures of social cognition, caregiver ratings of repetitive behaviors, and other clinical ratings of improvement. However, secondary outcome analyses suggested the oxytocin group showed improvements in the RMET test, caregiver reported quality of life, and lower order repetitive behaviors (stereotypy and self‐injury). Of particular note, there were two unexpected significant findings of the latter study. First, parents who believed their child had received oxytocin reported significant improvements in their child’s behavior over time irrespective of actual treatment assignment (oxytocin or placebo). This finding emphasizes the significant potential for expectancy biases to mask treatment efficacy within clinical trials of children with ASD. When investigators are asking parents or caregivers about the progress or conditions of their ASD children, one should be skeptical of the reporting and the biases that are embedded in the responses. Secondly, this study was the first that recruited a wider range of participants, not only high functioning ASD individuals, but including individuals with ASD and intellectual disability; the potential efficacy of oxytocin may have been masked by the greater amount of diagnostic heterogeneity within this sample. This brings us to the point of the spectrum again. As we have already mentioned, ASD is a spectrum and if oxytocin receptor neurons or other progenitor neurons are damaged early during fetal development (that results in extreme intellectual disability) then exogenous oxytocin may be of no real value, since the receptors are not there to carry out the signaling tasks. However, in high functioning ASD, oxytocin may bring good and positive effects.

Summary and Conclusions

Oxytocin is a neuropeptide (nonapeptide) made up of nine amino acids that is evolutionarily highly conserved. It is mainly synthesized in magnocellular neurons in the supraoptic and paraventricular nuclei of the mammalian hypothalamus. Oxytocin functions by binding to oxytocin receptors in specific areas of the brain and peripheral tissues. Oxytocin regulates coping with stresss, moods, and social behaviors. Most significantly, oxytocin is involved in attachment, pair bonding, sexual reproduction, maternal behavior, stress response, social memory, emotional cognition, and social interaction. Abnormalities of oxytocin due to either low levels of oxygen or genetic defects References 113 in oxytocin receptors show a significant association with impairment in social interaction and communication in ASD individuals. A large body of data suggest that oxytocin may play a role in the etiology of ASD, especially in social impairment. Several exogenous oxytocin treatments have shown normalization of impaired social functioning among ASD individuals. Adult ASD patients have displayed reduced repetitive behaviors and improvement in speech comprehension after infusion with oxytocin. However, the beneficial effects of oxytocin treatment have not been universal and there are numerous studies that report negative findings. We believe that there are several study limitations that might explain the discrepancies in results, including: ●● Exposure differences between the patients and control groups ●● Difference in oxytocin effects on lower order of intellectual disability ●● Noncomputerized measurement of eye gaze ●● Heterogeneity in ASD patients ●● Differences in age and diagnostic characteristics between studies Finally, results of a randomized, double‐blind, placebo controlled trial evaluating daily administration of oxytocin in patients with ASD demonstrated improved social cognition, quality of life, and emotional well‐being with no serious adverse reactions [117]. Additional studies are needed to further understand variations between ASD patients in response to intranasal oxytocin therapy and long‐term effects.

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123

Male Gender Bias and Levels of Male Hormones During Fetal Development

We are accustomed to look for the gross and immediate effects and to ignore all else. Unless this appears promptly and in such obvious form that it cannot be ignored, we deny the existence of hazard. Even research men suffer from the handicap of inadequate methods of detecting the beginnings of injury. The lack of sufficiently delicate methods to detect injury before symptoms appear is one of the great unsolved problems in medicine. Rachel Carson, 1962, Silent Spring

There is an inexplicable bias toward males in classical autism by a ratio of ~4:1, and ~10:1 in Asperger syndrome [1]. The diagnosis of autism spectrum disorder (ASD) is based on developmental history and the presence of abnormal behaviors. The clinical picture is heterogeneous and the etiology is unknown. We believe that the real genesis of the “spectrum” is based on three basic elements: (1) the timing of fetal testosterone (or other synthetic chemicals that a fetal brain would encounter during gestation), early higher than normal exposure may lead to a worse outcome; (2) the amount (dose) of testosterone (or other neuromodifying agents); and (3) the exposure to testosterone (or other male hormone‐like chemicals) has a selective neuromodifying effect. Of note, when we mention testosterone it does not imply that fetal testosterone is chemically unique from adult testosterone. It is the same chemical, secreted by the fetal cells or, according to some reports, from the placental part that is fetal in origin. We have discovered that testosterone (and numerous other synthetic chemicals) selectively kills oxytocin‐ and arginine vasopressin‐receptor positive neurons. The complete elimination of these neurons (if exposure occurred in the early stage of neurodevelopment) results in classical autism. This is due to the fact that in the early stage of gestation (i.e., weeks 8–24),

Autism and Environmental Factors, First Edition. Omar Bagasra and Cherilyn Heggen. © 2018 John Wiley & Sons, Inc. Published 2018 by John Wiley & Sons, Inc. 124 Male Gender Bias and Levels of Male Hormones During Fetal Development

there are only limited numbers of progenitor neurons that express oxytocin‐ and arginine vasopressin‐receptor positive neurons. Elimination or a significant variation in neurotoxicity most likely is due to timing of exposure to testosterone or similar acting chemicals found in fragrances and food flavors and our environment. We maintain that exposure of the human population to an increasingly diverse set of synthetic chemicals may provide the basis for the alarming 10‐fold increase in autism in recent years. It is possible that use of certain fragrances may be neuromodulatory to fetal brains, especially early in gestation. It is also probable that the neuromodulatory effects initiated during gestation may persist after birth. We are most intrigued by the observations that certain fragrances at fentomolar concentrations can be neuromodulatory considering the amount of fragrance that will reach a developing fetal brain during weeks 8–24 of gestation [the most vulnerable time of fetal brain development related to autism spectrum disorder (ASD)] [2,3]. One hypothesis that has been advanced, to account for the cognitive style in autism, suggests that it is best described as an extreme variant of male intelligence. Proponents of the “extreme male brain” theory or EMB theory, Baron‐ Cohen and colleagues [3,4] have also suggested that increased exposure to male hormones (i.e., androgen or testosterone) in utero may contribute to the development of ASD in both genders [3,4]. This is supported by an association between higher testosterone levels in amniotic fluid, poorer social skills at 3 years old, and higher scores on autism rating scales. Hormonal studies of children with ASD have shown elevated level of androgens, especially in girls. In fact, females with ASD often report androgen‐related conditions in adulthood. It has been suggested that fetal or perinatal exposure to elevated levels of male hormones may increase autism susceptibility [3–10]. In animal [11–16] and human [3–10,17–30] studies, variations in testosterone levels were associated with differences in behavior, cognition, and brain structure between males and females (reviewed in Ref. [3]). An association between prenatal testosterone levels and cognitive development has also been observed in recent studies looking at amniotic androgen [4,21,24,28]. The EMB theory of ASD suggests that fetal testosterone (testosterone) exposure may underlie sex differences in autistic traits [3–6]. Since it is difficult to directly measure prenatal testosterone due to cost and health risks, proxy measures such as maternal circulating testosterone levels during pregnancy and digit radio (second‐to‐fourth digit ratio, 2D:4D) have been utilized. In fact, a link has been drawn between the organizational effects of testosterone on the brain and ASD based on research using 2D:4D [3–6,31]. The EMB theory is the most popular hypothesis put forward to explain the sex difference in ASD [6]. This theory postulates that children with ASD exhibit an enhanced form of the male cognitive profile, and proposes that gestation exposure to testosterone causes biological effects associated with autism. Some evidence from animal studies suggests that testosterone may mediate cognitive differences between the sexes via organizational Male Gender Bias and Levels of Male Hormones During Fetal Development 125 effects on the brain. In the empathizing versus sympathizing theory differentiating genders in healthy brain development, male brains are built more to “systemize” by analyzing variables to determine a rule while female brains are built more to “empathize” by analyzing and responding to others’ emotions [3–6]. In EMB theory, Baron‐Cohen proposed that the male bias in ASD could be explained as an extreme manifestation towards the “systemize” characteristic of the male brain. Recent evidence supporting the EMB theory found that sex steroid levels in amniocentesis samples were correlated with diagnosis of ASDs [3–10]. The index to ring finger ratio (2D:4D) has been widely used as a proxy for testosterone exposure in autism research. Supporting a causal association between 2D:4D and fetal testosterone is the observation that 2D:4D appears to be sexually dimorphic, with males generally having lower 2D:4D (i.e., a relatively shorter index finger (2D) compared with their ring finger (4D). Although this is not a unanimously reported finding, lower 2D:4D ratios in children with ASD is supported through meta‐analyses (reviewed in Ref. [32]). This sexual dimorphism is apparent from the first trimester of pregnancy, and appears to be largely static after birth, with most, but not all, studies finding that it is unaffected by pubertal androgen. 2D:4D has also been shown to be sexually dimorphic in endocrine models of elevated (congenital adrenal hyperplasia) and reduced fetal testosterone exposure (complete androgen insensitivity syndrome). In studies in mice, the 2D:4D ratio has been shown to be affected by prenatal testosterone and estradiol [12,13]. Accordingly, there appears to be strong evidence linking elevated fetal levels of testosterone in amniotic fluid to autistic symptomatology, as well as an increase in rightward asymmetry of the corpus callosum [21]. One of the most interesting studies that examined elevated fetal male hormones and development of autism was that led by Baron‐Cohen [3–6]. Using the Danish Historic Birth Cohort and Danish Psychiatric Central Register, his team analyzed 128 amniotic fluid samples of males born between 1993 and 1999 who later received ICD‐10 (International Classification of Diseases, 10th revision) diagnoses of autism, Asperger syndrome or PDD‐NOS (pervasive developmental disorder not otherwise specified), then compared them with matched typically developing controls. Concentration levels of four sex steroids hormones (i.e. progesterone, 17α‐hydroxy‐progesterone, androstenedione, and testosterone) and cortisol were measured with liquid chromatography tandem mass spectrometry. The autism group showed elevations across all hormones on this latent generalized steroidogenic factor and this elevation was uniform across the ICD‐10 diagnostic label thereby providing the first direct evidence of elevated fetal steroidogenic activity in autism. Such elevations may be important as epigenetic fetal programming mechanisms and may interact with other important pathophysiological factors in autism. Previous studies that have examined the relationships between amniotic measurements of fetal testosterone and autistic traits employed Q‐CHAT (Quantitative Checklist for Autism in Toddlers). 126 Male Gender Bias and Levels of Male Hormones During Fetal Development

Sex differences were observed, with boys scoring higher on the Q‐CHAT than girls and a correlation was found between fetal testosterone and autistic traits [3,4]. These studies suggest that higher levels of fetal testosterone play a role. The connection between fetal testosterone and ASD has been supported by numerous independent studies, both in humans and rodents [3–41], but fail to explain the increased incidence of autism in the last three decades compared with several decades ago [42,43]. We will discuss the role of the environmental factors, especially the chemicals that act like testosterone or have similar effects as testosterone in a later section and in Chapter 7 [2,14,44–60]. More recently, several studies have analyzed the Baron‐Cohen hypothesis but found no evidence of a link with digital ratio. In an analysis of 6,015 ASD children and controls, Guyatt et al. (42) did not find an association between 2D:4D and ASD in males or females. Most significant is the paper by Kung and colleagues [43] which reported no relationship between prenatal androgen exposure and autistic traits. They took their evidence from studies of children with congenital adrenal hyperplasia and from studies of amniotic testosterone concentrations in typically developing children. They employed a parent‐ report questionnaire, the Childhood Autism Spectrum Test (CAST), to measure autistic traits in both studies. The first study examined autistic traits in young children with congenital adrenal hyperplasia, a condition causing unusually high concentrations of testosterone prenatally in girls. They assessed 81 children with congenital adrenal hyperplasia (43 girls) and 72 unaffected relatives (41 girls), aged 4–11 years. The second study examined autistic traits in relation to amniotic testosterone in 92 typically developing children (48 girls), aged 3–5 years. Findings from neither study supported the association between prenatal testosterone exposure and autistic traits. Specifically, young girls with and without congenital adrenal hyperplasia did not differ significantly in CAST scores and amniotic testosterone concentrations were not significantly associated with CAST scores in boys, girls, or the whole sample. These studies do not support a relationship between prenatal testosterone exposure and autistic traits. This study may appear to be a clear‐cut refutation of EMB theory. This is not the case. The most common form of congenital adrenal hyperplasia is due to 21‐hydroxylase deficiency that converts 17‐hydroxyprogesterone to 11‐deoxycortisol. Obviously, this is not testosterone. In the human fetus there are precise receptors for testosterone as well as for deoxycortisol and they have different physiological roles [43]. Furthermore, Kung et al.’s studies [43] had numerous methodological flaws: first, they measured testosterone concentration from umbilical cord blood. This is an inexpensive methodology that facilitates data collection on a large number of normal pregnancies. However, a limitation of this approach is that cord blood androgen levels may not reflect concentrations during the first and second trimesters of gestation, which is traditionally thought to be a critical period during which prenatal hormone exposure has its most pronounced Male Gender Bias and Levels of Male Hormones During Fetal Development 127 effect on neurodevelopment. Secondly, they used a very small sample size which they acknowledged as a limitation in their studies [43]. Thirdly, there was a lack of measurement of amniotic fluid from fetuses that were later diagnosed with ASD. Kung et al. [43] and Guyatt et al. [42] report that any link between prenatal androgen levels and the ASD phenotype is not clear cut. Any observer of ASD science over the past three decades should not be surprised by the mixed findings; all hypotheses seeking to describe the etiological pathways to ASD have endured similar inconsistent data. Here, we stress that ASD is a spectrum and exhibits a great degree of heterogeneity. The etiological variability observed in ASD exceeds any other known disorders [1]. The scientific response has been to reduce the emphasis on distinctly outlined diagnostic boundaries, and instead focus on understanding the simply definable causes which requires thinking outside the box. The heterogeneity of ASD is summarized in a recent article by Waterhouse et al. [61]:

“ASD research is at an important crossroads. The ASD diagnosis is important for assigning a child to early behavioral intervention and explaining a child’s condition. But ASD research has not provided a diagnosis‐specific medical treatment, or a consistent early predictor, or a unified life course. If the ASD diagnosis also lacks biological and construct validity, a shift away from studying ASD‐defined samples would be warranted…. The findings reviewed indicate that the ASD diagnosis lacks biological and construct validity.”

Hypothesis We believe that the severity of ASD depends on the time when the fetus was exposed to testosterone‐like chemicals during the gestation period. One of the reasons that autism manifest as a “spectrum” is due to the time of exposure of adverse neuromodifying synthetic chemicals during the fetal neurodevelopment. For example, if a fetus is exposed to a neuromodifying agent at an early stage of brain development (i.e., day 25, Figure 1.1) then the neuromodifying agent would have serious impact on the normal development of the exposed fetal primordial brain cells and may result in an autistic child. However, if the exposure to the same neurotoxic chemicals occurred at a later stage of brain development (i.e., days 35, 45, 55, etc.) the outcome will be different since at each of these stages of fetal development the damage would be different and significantly less adverse than it would be at day 25 (if the concentration of the neurotoxin was exactly the same at each stage). Elimination or a significant reduction in specific types of neurons can have widespread adverse effects on brain development. In humans, oxytocin‐ and arginine vasopressin (AVP)‐ receptor positive neurons play a central role in “social communication”. If a fetus is exposed to the same or similar oxytocin‐ and AVP‐receptor positive damaging neurotoxic agents, the result would be a “spectrum”. As shown in Figure 5.1, 128 Male Gender Bias and Levels of Male Hormones During Fetal Development

(a)

(b)

Figure 5.1 Effects of different levels of testosterone on (a) a female and (b) a male fetal brain cell line. The cells were exposed to 0.64, 0.82 and 1.2 nM/l concentrations of testosterone (representing normotypic, autistic and Asperger children, respectively) [3–6]. (A) Cell lines without testosterone exposure. (B) Exposure to 0.64 nM/l of testosterone. (C) Exposure to 0.82 nM/l of testosterone. (D) Exposure to 1.2 nM/l of testosterone.There were significant morphological changes in cells exposed to testosterone and significantly more changes in cells exposed to higher concentrations of testosterone (i.e., C and D) compared with normotypic testosterone concentration (i.e., B). There was no statistically significant difference between female and male neurons (Bagasra et al., unpublished data). Reproduced with permission of Bagasra et al. Association between 2D:4D Ratio and Brain Connection Development 129 testosterone has profound effects on fetal brain morphology, both in male and female fetal brain cells exposed to various concentrations of testosterone (unpublished data) [2, 62–66]. Maternal circulating testosterone has also been used in the past as a proxy measure for fetal testosterone. Since testosterone is lipid soluble, it can cross the placenta and bidirectional transfer between the mother and fetus is theoretically possible. Association between maternal circulating testosterone and fetal testosterone has been observed in numerous studies [3–30,63–66] and maternal circulating levels have been tied to gender role behavior in some young children (reviewed in Refs [3–6]). However, some studies suggest that maternal circulating testosterone may only affect female fetuses [42,43].

Association between 2D:4D Ratio and Brain Connection Development

Although associations have been observed between 2D:4D, maternal circulating testosterone [3–6,31], and ASD characteristics, variable results highlight the need for larger studies, particularly studies that include assessment of gender effects. In a recent analysis of 2D:4D ratio and social communication and emotional recognition issues characteristic of autism, the evidence did not strongly support a link between digit ratio and recognition of low intensity faces (reviewed in Ref. [67]). However, low right‐hand digit ratio was associated with those in the highest percentage for autistic traits and individuals who had difficulty recognizing facial expressions and correctly interpreting emotions. Lower 2D:4D ratios have also been linked to maternal circulating testosterone in certain populations and may be a valid proxy in certain populations, but evidence does not support an overall link between circulating maternal testosterone during pregnancy and subsequent social communication and recognition of emotion. However, it must be realized that these discrepancies are most likely due to timing of exposure to testosterone or similar acting chemicals found in fragrances and food flavors and our environment [33,34,44–60,63–66]. In Chapter 7 we describe some of the chemicals that either act like testosterone or act on androgen receptors and activate the receptors, essentially activating the signaling pathways that testosterone would do in a natural gestation environment, except the effect would be amplified. The authors proposed two possible explanations for the failure to replicate previous studies that showed a link between autistic traits and 2D:4D ratios. The first explanation revolved around potential bias due to gender influences that could have affected the evaluation of the systemizing and empathizing measures. The second explanation suggested the presence of a threshold effect in which in a nonlinear relationship between fetal testosterone and ASD traits, high levels of fetal testosterone may be required to predispose males to development of ASD traits [67]. 130 Male Gender Bias and Levels of Male Hormones During Fetal Development

Male and Female Estrogen and Testosterone Hormone Regulations

It is pertinent to mention here that females can convert testosterone into estrogen by the aromatase enzyme (CYP19A1 gene), however a male cannot convert estrogen into testosterone. Therefore, a high level of testosterone in females may not have any adverse effects. The expression levels of aromatase enzyme may be one of the reasons for the gender bias. The aim of hormones is to execute specific tasks precisely at specific times and with specific outcomes. Therefore, detrimental outcomes for offspring can result from exposure of a mother to any endocrine disruptive chemical(s) (EDCs) that can subsequently reach a fetus [33,34,44–60,63–66]. As shown in Figure 5.2, testosterone plays a central role in the development of the male brain, especially during weeks 8–24 of gestation. In female fetuses there appears to be no estrogen exposure during gestation. Estrogen exposure occurs later, after the postnatal period, when estrogen is required to lead a female towards puberty. As we have detailed in Chapter 4, fetal testosterone has a precise role in male brain development and any synthetic or natural chemical that can increase the levels of testosterone or bind to androgen receptors can modify the normal brain development (both male and female), as proposed in EMB theory [3–6]. Here we describe some of the endocrine disturbing chemicals that can have a potential detrimental effect on fetal development [33,34,44–60,63–66].

Are there Synthetic Chemicals that Humans Are Not Evolutionarily Exposed To?

Over the last four decades, a substantial increase in the prevalence of autism has been reported, from 4 to 5 children per 100,000 in the 1960s to around one in 45 children in 2015 [1,2]. We maintain that exposure of the human

Male (testosterone)

Female (estrogen) Hormone secretion

824 1 2 10 20 30 40 Age (years) Birth

Figure 5.2 Normal ranges of male and female sex hormones over the human lifespan. Why Male Gender Bias? 131 population to an increasingly diverse set of synthetic chemicals may provide the basis for this alarming 10‐fold increase in autism in recent years. Many of these synthetic chemicals, including fragrances, have steroidogenic (male and female hormone‐like chemicals) activity (reviewed in Ref. [68]). According to published laboratory and epidemiological studies, undisclosed chemicals in fragrances, such as those that provide different scents, increase shelf life, control release of fragrance and improve stability, and have endocrine‐disrupting properties [33,44,63–66]. Such endocrine disruptors and brain network modulators have been associated with increased risk for cancer [33,44,63–66,68], adverse effects on developing fetuses [33,44,63–66,68], and metabolic diseases. For example, chemicals that have been shown to increase human estrogen receptor expression include octinoxate, oxybenzone, benzophenone‐1, benzophenone‐2, benzyl salicylate, benzyl benzoate, butylphenyl methylpropional, and synthetic musks (galaxolide, tonalide, and musk ketone). Of these, oxybenzone, benzophenone‐1, galaxolide, and tonalide also affect androgens, while butylated hydroxytoluene, benzophenone‐2, and octinoxate have been linked to thyroid hormone disruption [33]. Even at very low concentrations, fragrances that contain these chemicals can be mutagenic and carcinogenic [33,44]. Of note, one of the fragrance ingredients, acetyl ethyl tetramethyl tetraline (AETT) was used for 22 years in fragrances, colognes, soaps, detergents and cosmetics [53]. However, it was voluntarily discontinued in 1978 after it was linked to behavioral changes and degeneration of the white matter of the brain including “widespread demyelination and scattered axonal degeneration in the central peripheral nervous systems.” Along with this, the fragrance ingredient musk ambrette (a fixative recently banned in the European Union but still allowed in the USA) also has been found to cause degeneration of the myelin sheath and distal axons. Even if one seriously considers high testosterone levels during the early stages of gestation, these data do not explain why the incidence of ASD is steadily going up, except that synthetic chemicals, behaving like hormones, can reach the fetal blood circulation [68]. We believe that many synthetic fragrances contain testosterone‐like hormones (see Chapter 7).

Why Male Gender Bias?

A question that remains is why are female neuronal cells relatively more protected than their male counterparts? Some investigators propose that the male bias for ASD may be partially due to under diagnosis of autism in females or the human female’s ability to mask some of the symptoms of ASD [1,2,66]. Even so, these investigators agree that male bias is observed in ASD and it is 3 times more common than the 4–5 times that is reported in many other studies. Several hypotheses have been advanced including: (1) epigenetic mechanisms 132 Male Gender Bias and Levels of Male Hormones During Fetal Development

in the EMB hypothesis of Baron‐Cohen [3–10] which postulates that elevated fetal testosterone is a risk factor for ASD (summarized in the preceding section); (2) genetic mechanisms which involve X or Y chromosome inactivation [1]; (3) and recently, Hu et al. [69] have shown that retinoic acid‐related orphan receptor alpha (RORA) is reduced in the brain and lymphoblastoid cell lines of multiple cohorts of individuals with ASD. This gene targets CYP19A1 (the gene that codes for aromatase enzyme), in a gender‐dependent manner that can also lead to elevated testosterone levels, a proposed risk factor for autism as mentioned in the above three hypotheses. These hypotheses have been comprehensively described in our recent review [2]. To date, none of these hypotheses have been either proven or disproven. Given the high clinical heterogeneity of ASD, it is possible that each of these mechanisms for gender bias may apply to specific cohorts of individuals with ASD, considering that ASD is a spectrum [1,2].

Male and Female Brains in a Test Tube

Since all of the above theories relate to early fetal brain development (weeks 4–24 of gestation), we hypothesize that the scientific community needs a robust in vitro model that can reveal the fundamental mechanism for gender bias in ASD. For the last decade we have been evaluating an in vitro model, utilizing human neuroblastoma cell lines (NBCs). A detail rationale and information is provided in Chapter 7. Briefly, these cell lines represent early fetal brain progenitor neurons, as has been shown previously [2,5]. In our preliminary studies we have evaluated two highly plausible hypotheses: (1) if testosterone levels that are found in the amniotic fluids of normotypic, classical autistic and Asperger syndrome children (as determined by Baron‐Cohen et al. [3]) affect the neurodevelopment of NBCs; and (2) examined expression levels of RORA and CYP19A1 (aromatase) genes in male and female NBCs exposed to three different levels of testosterone, equivalent to what Baron‐Cohen’s team found in the amniotic fluids of Danish children [3,4]. We have carried out preliminary studies to determine the feasibility of whether: (1) NBCs exposed to three different levels of testosterone (representing normal, autistic and Asperger syndrome children) differ at morphologic levels and exhibit signs of “connectome” dysregulation; (2) there is a differential expression of RORA and CYP19Aa?; and (3) there is a downregulation of oxytocin‐ and AVP‐receptor neurons in the exposed cell lines compared with the unexposed (control cell lines). For this purpose, we analyzed one pair of NBCs that represents and behaves similarly to developing fetal brain progenitor cells in vitro (i.e., CCL‐2266 (female) and CRL‐2267 (male)] to determine whether exposure to different levels of testosterone induces differential morphologic and immunologic changes (2). We also determined the differential Male and Female Brains in a Test Tube 133 expression levels of RORA and CYP19A1 (aromatase) gene expressions in cells exposed to 1.2 nM/l concentration of testosterone in vitro. The cell lines from one male and one female origin were exposed to 0.64 nM/l (representing normotypic), 0.82 nM/l (representing classical autism), and 1.2 nM/l (representing Asperger syndrome) as described by Baron‐Cohen et al. [3]. The studies were designed according to large retrospective studies where the elevated testosterone levels were seen by Baron‐Cohen’s group in amniotic fluids [3]. We exposed these NBCs for 5 days and evaluated for various parameters by histological and immunological means including central chromatolysis, axonal morphometric analyses, syncytia formation, and axonal changes as well as changes in size, shape and relative diameter measurements, as described by us recently [3]. We also analyzed these cell lines for degree and percentage of neurons positive for oxytocin receptor by immunological means [2]. First, we compared the effects of testosterone at normotypic versus autism levels and normotypic versus Asperger syndrome levels. In addition, we determined all three different levels of testosterone in both male and female NBCs and whether these testosterone levels had differential neuromodifications on male versus female NBCs, particularly with regards to oxytocin‐receptor positive neurons.

Effects of Three Different Levels of Testosterone on Neuronal Morphology

As shown in Figure 5.1, significant morphological neuromodifications were observed in neurons of both male and female NBCs. Exposure to 0.64 nM/l (representing normotypic) and 0.82 nM/l (representing autism) of testosterone led to significant morphologic changes as measured by central chromatolysis, enlargement of the neuronal cell body, shortening or abnormal increase of axonal length, change in cellular diameter, and syncytia formation. Similarly, exposure to 0.64 nM/l and 1.2 nM/l (representing Asperger syndrome) also induced significant changes in NBCs exposed to higher levels of testosterone. The most profound changes were increased neuronal length and thinning of axons in both 0.82 and 1.2 nM/l testosterone exposures compared with 0.64 nM/l, representing the normotypic level [3–6]. We also compared the morphological characteristics in testosterone unexposed control cell lines from both genders and determined that exposure to low levels of testosterone in cell lines from both genders induces significant morphological changes. One of the most agreed upon concepts in ASD is that it is associated with modifications in brain connectivity, even though the nature and extent of these neuromodifications remain controversial. Early analyses of connectivity derived from functional magnetic resonance imaging (fMRI) from various brain regions reported weaker functional connectivity between brain regions in individuals with ASD, leading to the long‐distance cortical “under‐ connectivity” theory [67,70–77]. Recent evidence utilizing fMRI suggest 134 Male Gender Bias and Levels of Male Hormones During Fetal Development

functional connectivity between brain regions may be stronger in ASD challenging the “under‐connectivity” theory [72]. These inconsistencies may be explained by taking into account the topographical nature of functional connectivity patterns, rather than their overall strength. We believe that significant elongations of axons in NBCs may provide some insight into connectivity discrepancies found in ASD patients. The significant elongation of axons in certain parts of brains with associated axonal thinning may result in increased connectivity but functionally weaker neurons in ASD. If this occurs in the area of brain that is involved in communication and social interaction, then the autism communication conundrum may be explained in morphological terms [67,75,76]. We hypothesize that one of the reasons for the contradictory functional connectivity findings in the literature may be reconciled by taking into account the fact that a developing fetal brain consists of hundreds of thousands of different progenitor neurons at 8 weeks of gestation, each destined to become a different compartment or faculty of an adult brain. However, one cannot expect each of these progenitor neurons to be equally susceptible to testosterone or other differentiation factors. Only the progenitor neurons that either have receptors for a particular chemical or neurons which are susceptible to the toxic effects of chemicals would be affected by the external (xenobiotic) or internal (endogenous) chemicals, including hormones [66]. Therefore, only certain progenitor neurons that express testosterone receptors would respond to higher levels of testosterone resulting in varied connectivity in different compartments of the ASD versus normotypic brain. For example, independent component analysis comparing within‐network connectivity in children with ASD and typically developing children reveals over‐connectivity of large‐scale brain networks in ASD, whereas no group differences are observed in similar analyses comparing adolescents [23] or adults [23,24]. Our in vitro data on elongation of axonal length may shed light on what may be taking place in vivo. However, we believe that despite the limitations of the in vitro model, we may be able to clarify the situation with regards to ASD pathogenesis [2,63–66].

Molecular Basis of Gender Bias in ASD

One of the hypotheses to explain potential underlying mechanisms is that in XX chromosome genes one of the silenced X chromosomes could play a role in sex ratios if the non‐silenced genes were protective. This means that either parental X chromosome or the maternal X chromosome could be protective. One of the hypotheses involves “genomic imprinting,” the process by which genetic effects are influenced by the father (Xf) or the mother (Xm). Skuse et al. [77] suggested that an imprinted X locus could explain sex differences in social and communication skills. Their theory was inspired by the finding that in Molecular Basis of Gender Bias in ASD 135 individuals with Turner syndrome), the rate of social difficulties varied according to whether their single X chromosome was Xf or Xm [77,78]. In Turner syndrome one of the X chromosomes is missing and a female could have acquired either the father’s or mother’s X chromosome. Typical females inherit an X chromosome from both parents (XfXm), but typical males have only a maternal X chromosome (XmY). Skuse et al. hypothesized that a gene expressed on the maternal Xm acts as a protective factor against the social problems seen in Turner syndrome and, by extrapolation, as a protective factor against ASD [1,2,77]. However, recent data from Creswell and Skuse [78], who reported five cases of ASD within an unselected sample of 150 subjects with Turner syndrome, showed that all cases were Xm, strongly suggesting that the maternal X chromosome is not protective. Also, given that 77% of Turner syndrome females are XmO, while only 23% are Xf [1,2,78], then, by probability one would expect to find ASD more often associated with Xm than with Xf, making the Xm hypothesis unlikely. It is well documented that ASD children have bigger overall brain size and larger amygdala and frontal lobes of the brains [1,63] than normally developing children. Also, many autistic children’s brains tend to grow faster, prenatally, than the typical child’s brain, and later brain growth is more normal, or even relatively slower, during childhood [1,63]. Therefore, it is possible that use of certain fragrances may be neuromodulatory to fetal brains, especially early in gestation. It is also probable that the neuromodulatory effects initiated during gestation may persist after birth. We are most intrigued by the observations that certain fragrances at fentomolar concentrations can be neuromodulatory considering the amount of fragrance that will reach a developing fetal brain during 8–24 weeks of gestation (the most vulnerable time of fetal brain development with regard to ASD) [1,2,63,64,66]. Expectant women may be exposed to fragrance not only from their own use (e.g., dermal application and inhalation), but also from the environment. The levels of fragrance components that would reach fetal brain neurons would be at fentomolar or even lower concentrations and unlikely to be at levels where cytotoxic effects would occur. However, these chemicals may interfere with the normal development of a fetal brain. Of note, the most vulnerable brain areas, where this excessively rapid growth seems to be most common, are the areas that are the foundations of superior cognitive specialization development [1,2,63,66]. We analyzed the male and female NBCs to determine if exposure to fentomolar concentrations of fragrances adversely affected these cell lines. Specifically, we examined OXY‐receptor (OXYR) and AVPR1‐receptor positive neurons to assess whether the fragrances had differential neurotoxic effects on female versus male neuroblastoma cells. In both cell types, 1:106 dilutions of three selected fragrances induced profound and significant neuro- modulations and caused central chromatolysis, enlargement of the neuronal cell body, shortening or abnormal increase and thinning of axonal length and 136 Male Gender Bias and Levels of Male Hormones During Fetal Development

neurostimulations or selective neurocytotoxicity (Figure 5.1). However, depletion of OXYR and AVPR1‐receptor positive neurons was only observed in male, but not female, NBCs following fragrance exposure (unpublished data). The question remains as to why would female neuronal cells be protective? Two potential mechanisms have been hypothesized. The first forwards the notion that X chromosome gene dosage could play a role in sex ratios if the non‐silenced genes were protective. It is increasingly recognized that learning difficulties are themselves a risk factor for ASD; therefore, any evaluation of how the X chromosome may be protective will need further evaluation [65]. It is still unknown which X‐linked genes may be involved; however, genes that regulate amygdala circuits (which are disrupted in ASD) are suspected. In a mouse model of Turner syndrome, the maternally expressed gene xlr3b was associated with cognitive flexibility, but a human orthologue has yet to be identified. Numerous hypotheses and thousands of articles have been published in the peer‐reviewed literature defining the illnesses and proposing potential causes of ASD. Through these studies it has been revealed that oxytocin‐ and AVP‐ positive neurons are underdeveloped in the brains of autistic children. Development of these neurons within the highly integrated metabolic, endocrine, and neuropeptide systems is influenced by the environment in which the brain develops, which may provide clues into the etiology of ASD. Our finding that testosterone concentrations can impact on the fetal neurodevelopment brings forth a new perspective to the pathogenesis of ASD and the potential role of testosterone or synthetic chemicals that act like testosterone (either directly or indirectly, and numerous other environmental agents) that may disturb the finely orchestrated fetal brain development. These chemicals are pervasive in modern society and may be important contributing factor(s) in ASD. Chemicals in fragrances may be harmful to developing fetal brains, as well as to adults [31–55]. Searching for effective biomarkers is one of the most challenging tasks in the research field of ASD. fMRI provides a noninvasive and powerful tool for investigating changes in the brain functions including structural changes, connectivity, differentiation, function, maturation, and metabolism of the brain of children with ASD. We believe that future studies will uncover some important biomarkers that may assist in the identification of ASD in children before they are 3 years old.

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61 Waterhouse L, London E, Gillberg C., et al. (2016). ASD validity. Rev. J. Autism Dev. Disord., 3:302–29. 62 Waterhouse L, London E, Gillberg C (2017). The ASD diagnosis has blocked the discovery of valid biological variation in neurodevelopmental social impairment. Autism Res., 10(7):1182. 63 Bagasra O, Pace DG (2013). Smell of autism: Synthetic fragrances and cause for allergies, asthma, cancer and autism. OA Autism, 1(2):15. 64 Golkar Z, Bagasra O, Jamil N (2013). Experimental phage therapy on multiple drug resistant Pseudomonas aeruginosa infection in mice. J. Antivir. Antiretrovir., S10‐005. DOI: 10.4172/jaa.S10‐005. 65 Sealey LA, BW Hughes, Steinemann A, et al. (2015). Role of environmental factors in autism development and male bias: Neuromodifying effects of fragrance. Environ. Res., 142:731–8. 66 Hughes BW, Sealey LA, Bagasra O (2016). Mechanism of male gender bias in neuroblastoma cell lines exposed to fragrances: A link to autism spectrum disorder. Expert Opin. Environ. Biol., 5:1–21. 67 Roine U, Roine T, Salmi J, et al. (2015). Abnormal wiring of the connectome in adults with high‐functioning autism spectrum disorder. Mol. Autism, 6:65. 68 Gore AC, Chappell VA, Fenton SE, et al. (2015). Executive Summary to EDC‐2: The Endocrine Society’s Second Scientific Statement on Endocrine‐ Disrupting Chemicals. Endocr. Rev., 36(6): 593–602. 69 Hu VW, Sarachana T, Sherrard RM, Kocher KM (2015). Investigation of sex differences in the expression of RORA and its transcriptional targets in the brain as a potential contributor to the sex bias in autism. Mol. Autism, 6:7. 70 Shu N, Wang X, Bi Q, et al. (2017). Disrupted topologic efficiency of white matter structural connectome in individuals with subjective cognitive decline. Radiology, 162696. 71 Wen H, Liu Y, Rekik I, Wang S, et al. (2017). Disrupted topological organization of structural networks revealed by probabilistic diffusion tractography in Tourette syndrome children. Hum. Brain Mapp., 38(8):3988–4008. 72 Meoded A, Huisman TAGM, Casamassima MGS, et al. (2017). The structural connectome in children: basic concepts, how to build it, and synopsis of challenges for the developing pediatric brain. Neuroradiology, 59(5):445–460. 73 Fields C, Glazebrook JF (2017). Disrupted development and imbalanced function in the global neuronal workspace: a positive‐feedback mechanism for the emergence of ASD in early infancy. Cogn. Neurodyn., 11(1):1–21. 74 Scheinost D, Sinha R, Cross SN, et al. (2017). Does prenatal stress alter the developing connectome? Pediatr. Res., 81(1–2):214–26. 75 Mevel K, Fransson P (2016). The functional brain connectome of the child and autism spectrum disorders. Acta Paediatr., 105(9):1024–35. 142 Male Gender Bias and Levels of Male Hormones During Fetal Development

76 Ameis SH, Catani M (2015). Altered white matter connectivity as a neural substrate for social impairment in autism spectrum disorder. Cortex, 62:158–81. 77 Skuse DH, James RS, Bishop DVM, et al. (1997). Evidence from Turner’s syndrome of the imprinted X‐linked locus affecting cognitive function. Nature, 287:705–08. 78 Creswell CS, Skuse DH (1999). Autism in association with Turner syndrome: Genetic implications for male vulnerability to pervasive developmental disorders. Neurocase, 5:511–18. 143

Maternal Twins and Male Gender Bias in Autism Spectrum Disorders

When the public protests, confronted with some obvious evidence of damaging results of pesticide applications, it is fed little tranquilizers pills of halftruth. Rachel Carson, 1962, Silent Spring

As we have discussed in previous chapters, many scientists believe that autism is a genetic disease. They believe genetics is the cause of the wide of spectrum of illness that we see in autism spectrum disorder (ASD) children and are impressed by numerous genetic defects found by the expert genetic analysts. Despite the high heritability estimates for ASD, which are claimed to be from 70 to 90% concordant in maternal twins (monozygotic twins) and a little less in fraternal twins (or dizygotic twins), there are notable differences in maternal twin pairs. So, what they are seeing? Where is this 70–90% similarity (concordance) coming from? These are highly trained experts who find genetic mutations in up to 25% of ASD children. Why then can none of the mutations in classic autism be directly linked to the spectrum? [1,2]. If we take out the well‐ characterized genetic diseases (which we believe should not be included as part of the spectrum) such as Rett syndrome (X‐linked dominant pattern of inheritance), Prader–Willi syndrome (deletion at 15q11‐q13), Angelman syndrome (deletion of maternal UBE3A), Smith–Magenis syndrome (deletion at 17p11.2), Down syndrome (trisomy of chromosome 21) and several other syndromes, then we can still observe over 1,000 mutations in ASD children. None of those mutations are linked to any specific disease, particularly not to ASD! They are occurring in a developing fetus, in utero, anew (de novo), in a relatively random fashion, not found in either of the parents. This suggests that those genetic mutations (now numbered in thousands) are due to unknown environmental factors and not inherited from either of the parents [3–6]. As we have shown previously, all of the fragrances that we have tested so far

Autism and Environmental Factors, First Edition. Omar Bagasra and Cherilyn Heggen. © 2018 John Wiley & Sons, Inc. Published 2018 by John Wiley & Sons, Inc. 144 Maternal Twins and Male Gender Bias in Autism Spectrum Disorders

cause mutations in fetal brain neurons [2]. We tested minuscule amounts of over 100 fragrances by the well‐recognized Ames test, approved by the US federal government and the scientific community to test for carcinogens and mutagens, and found significant mutations [2]. Are the synthetic chemicals found in fragrances, water, air, and food and drinks that we consume every day responsible for inducing mutations in a developing fetal brain? The answer is “definitely”. Furthermore, there are well documented and well researched scientific reports about the dissimilarity (discordance) within maternal twin pairs (monozygotic twin pair) who are genetically identical exhibiting significant variations in autism expression between the twin pairs [6–13]. In some cases, one twin was found to be normal (normotypic) and the other had severe autism. How can the same genetic framework explain these discrepancies? When we consider diseases that are assumed to be genetic or heritable in nature, we never think about the impact of the environment. In the last 40+ years, synthetic chemicals have silently penetrated our food, water, and air and are producing silent offspring. We are generally aware that autism is increasing in leaps and bounds, and schizophrenia and older age degenerative diseases such as Alzheimer’s, and other dementia also have increased beyond what we can blame on longer life span (a result of antibiotics and abundance of food supply) and better diagnosis, but we never mention the silent pandemic of synthetic chemicals. It is odd that we blame the alarming increase in Alzheimer‐like diseases on the fact that we are living longer or on our medical diagnostic system’s ability of earlier diagnosis. However, many people in other parts of the world have been living longer than those in the USA (Figure 6.1) but they have not experienced the disastrous prevalence of Alzheimer’s disease or dementia (Figure 6.2). One can see that there is no clear correlation between living longer and dementia. As a matter of fact, environmental factors and human lifestyle patterns play a significant role in both living longer and dementia [14]. Many in vitro and animal models have confirmed as well as identified the toxic effects of environmental factors at the cellular and molecular levels [6–13]. Scientists and the media also claim that the reason for the increased prevalence of ASD and schizophrenia is better diagnosis. This notion is false; even if we take into account the better diagnosis and broadening of the definition of these two diseases, there is no doubt that they are increasing very rapidly in industrialized nations. In most of the cases where the genetic causes of ASD are not known, one could argue that if maternal twins have the same genetic make‐up and have the same mother and so receive the same blood supply, how is it possible that one child has ASD and the other does not? Or, one has severe ASD and the other only a very mild case? Here, we forget to notice sometime startling but so obvious, namely, the unequal blood supplies from the same mother to each of the twins. This is where the environmental impact of the synthetic chemicals is involved. The maternal twins never receive the same Maternal Twins and Male Gender Bias in Autism Spectrum Disorders 145

(a) Countries with average age range

45+ 40–45 35–40 30–35 25–30 20–25 14–20

(b) Alzheimer’s disease incident rate

Set scale Reset 120 200 300 400 500 600 700 800 9 910

Figure 6.1 (a) Countries with average age range. There are many areas of the world where individuals have a long average age, including parts of Latin America, North Africa, and other small nations that cannot be seen (i.e., Bhutan and parts of Malaysia). Source: https:// en.wikipedia.org/wiki/List_of_countries_by_median_age (b) Global picture of Alzheimer’s disease incident rate. One can clearly see that the two maps do not completely coincide, suggesting there are other factors that determine the outcome other than just older age and early diagnosis. Source: http://www.alzforum.org/news/research-news/global-rise-total- ad-cases-dwarfs-falling-age-standardized-rate. 146 Maternal Twins and Male Gender Bias in Autism Spectrum Disorders

30,000

Ages 65–74 Ages 75–84 25,000 Ages 85 and over

20,000

15,000

10,000 Number of cases (in thousands) 5,000

0 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100 PhRMA projections calculated by applying current prevalence rates to population projections.

Figure 6.2 The projected increase in prevalence of Alzheimer’s disease. Source: http://www. silverbook.org/fact/projected‐alzheimers‐disease‐prevalence‐2000‐2100/.

amount of blood from their mother; the uneven supply of blood to each of the twins introduces uneven amounts of synthetic chemicals that the mother is inadvertently exposed to during pregnancy. The unequal supply of blood, mostly ignored in research, may explain the disconcordant twin children. We should just mention here that schizophrenia maternal twins also show discordance [15–17]. We are made to believe that schizophrenia is a “genetic” disease and inherited from one of the parents. The parents are made to feel guilty that they have transferred this illness to their children (just as mothers of ASD children were blamed for being bad mothers and “refrigerator mothers”). But, again, we forget to look at the influence of the environmental factors which may play an important role in manifestation of this horrific illness. We are not going to deal with this subject here, since it is beyond the scope of this book, but will refer to it again at the end of this chapter to further illustrate the mystery of discordant maternal twins. In Chapter 7, we will describe numerous endocrine disturbing chemicals (EDCs). If one was exposed to these in utero, the adverse effects may not manifest until much later in life.

The Conundrum of ASD Discordance in Maternal Twins

The fact is that almost all maternal twins receive different amounts of blood from their mother. How is that possible? As shown in Figures 6.3 and 6.4, there are five different kinds of maternal twins. One of the most unfortunate twin The Conundrum of ASD Discordance in Maternal Twins 147

Superficial anastomosis

Recipient

Donor

Deep anastomosis

Figure 6.3 Twin‐to‐twin transfusion syndrome. In this disease of the placenta, which affects maternal twins, the blood passes disproportionately from one baby to the other through connecting blood vessels within their shared placenta. One baby, the recipient twin, gets too much blood overloading his or her cardiovascular system, and may die from heart failure. The other baby, the donor twin or “stuck twin,” does not get enough blood and may die from severe anemia. If left untreated, mortality rates are nearly 100%. Source: http:// www.fetalhealthfoundation.org/fetal‐syndromes/twin‐to‐twin‐transfusion‐syndrome/. pairs is twin‐to‐twin transfusion syndrome (TTTS), also known as feto‐fetal transfusion syndrome (FFTS). This is a complication of highly disproportionate blood supply, resulting in high morbidity and mortality. It can affect monochorionic twins or triplets where two or more fetuses share a chorion and hence a single placenta. Chorion is the extraembryonic membrane that develops villi and becomes the fetal part of the placenta. TTTS is a very serious complication affecting approximately 15% of maternal twin pregnancies. It affects only monozygotic maternal twins sharing a single placenta and a single chorion. As the placenta develops, the blood vessels form in such a way that vessels from one umbilical cord connect up with vessels from the other. This causes a sort of “short circuit” between the babies. As a result of their differing blood supplies, each twin faces a set of challenges that require expert and experienced urgent care. The donor twin gets less blood than its sibling, causing it to become anemic (lacking in red blood cells) and produce less urine, the main ingredient of amniotic fluid. The fluid can continue to decrease (called oligo- hydramnios) to the point of disappearing completely, causing the amniotic sac to cling to the fetus. Because of this, the fetus may also appear to adhere to the 148 Maternal Twins and Male Gender Bias in Autism Spectrum Disorders

Monoamniotic monochorionic Diamniotic dichorionic (fused)

Diamniotic monochorionic Diamniotic dichorionic (separated)

Figure 6.4 Monochorionic monoamniotic twins have no separating membrane. They share a placenta and share an amniotic sac. Monochorionic diamniotic twins share the same placenta but have a separating membrane of amnion. Dichorionic diamniotic twins have a separating membrane consisting of both chorion and amnion. They may have separated or fused placentae. In each case, the blood flow would be unequal that may result in discordant monozygotic twins (see text). As shown, in each case the two monozygotic twins are receiving slightly different amounts of blood supply, making the ones on the left slightly more vulnerable to environmental factors than the ones on the right. Source: http://www. babymed.com/twins/twins‐monozygotic‐vs‐dizygotic‐and‐monochorionic‐vs‐dichorionic. Adapted from Dr Amos Grunebaum.

wall of the womb, so called “stuck twin.” The recipient twin, meanwhile, will struggle with opposite concerns. Too much blood will lead to excess urine production, resulting in a huge bladder and accumulation of fluid, a prenatal form of heart failure. Excess urine will increase the amniotic fluid around the recipient twin and cause the womb to over‐expand. If TTTS is untreated, premature labor and delivery often results, with the possible loss of one or both babies. If just one of the twins survives, that twin is at risk of brain and other organ Role of Environment in Maternal Twins revealed by Numerous Methods under Many Conditions 149 damage due to inadequate oxygen and blood flow. This can occur because the shared blood circulation increases the chance of the blood flowing in the opposite direction. Pumping blood back to the deceased twin deprives the surviving twin of oxygen, fluids, and nutrients. The surviving twin may also be at risk of restricted growth and other complications while in the womb. Most TTTS twins, whether they receive treatment or not, will be born prematurely. TTTS is an extreme form of unequal blood flow in maternal twins. There are four other conditions that are generally ignored but present subtle situations of unequal blood flows. There are four types of twins in Figure 6.4 showing that in all cases – maternal or fraternal – the blood supply is slightly uneven, making the exposure to nutritional and environmental factors unequal with differential outcomes. A comprehensive literature search has implicated several environmental factors associated with the development of ASD. These include pesticides, phthalates, polychlorinated biphenyls (PCBs), solvents, air pollutants, synthetic fragrances, glyphosate (herbicide) and heavy metals, especially aluminum and organomercury used in vaccines as adjuvant or preservative, respectively. Importantly, the majority of these toxicants are some of the most common ingredients in cosmetics and herbicides to which almost all of us are regularly exposed to in the form of synthetic fragrances, face makeup, cologne, air fresheners, food flavors, detergents, insecticides, herbicides, and vaccines, to name just a few. In this chapter we describe various scientific data to show the role of environmental factors in ASD.

Role of Environment in Maternal Twins revealed by Numerous Methods under Many Conditions

Monozygotic twin pairs provide a valuable opportunity to control for genetic and shared environmental influences while studying the effects of non‐shared or partially shared environmental influences, such as unequal blood supply to each of the maternal twins. This unequal sharing or exposure to an environmental chemical can result in unequal manifestation of numerous types in genetically identical twins. There are literarily hundreds of reports in peer‐ reviewed scientific journals that show discordant monozygotic twins with different phenotypes. In the following we describe only a few examples.

What Types of Discordance are Observed in Maternal Twins?

There are numerous dissimilarities that have been observed in identical twins. One of the most interesting studies in the literature is that of right‐ or left‐ handedness. This right‐ and left‐handedness plays a great role in language and learning. Häberling and colleagues [18] have examined the hemispherical asymmetry and callosal morphology in monozygotic twins. Some 20–25% of 150 Maternal Twins and Male Gender Bias in Autism Spectrum Disorders

monozygotic twins are of opposite handedness [19,20] with some even showing reversed cerebral asymmetries [21,22]. They evaluated arcuate fasciculus morphology in 35 pairs of monozygotic twins, of which 17 pairs were concordant for handedness and 18 pairs were discordant for handedness. Of note, the presence of arcuate fasciculus appears to be unique to humans and its connectivity has been shown to correspond to various functional areas within the temporal, parietal, and frontal lobes. Furthermore, this region of the brain plays an important role in language and speech development. Functional hemispheric language dominance was established for each twin member using functional magnetic resonance imaging (fMRI) that showed 26 twin pairs concordant and 9 twin pairs discordant for language dominance. Their data suggest that handedness and hemispheric dominance for speech production might be influenced by environmental factors.

Differences in Frontal and Limbic Brain Activation in Monozygotic Twin Pairs Discordant for Severe Stressful Life Events

Godinez et al. [23] looked at the discordance in maternal twins who suffered severe stressful life events during early development (i.e., before 18 years of age) and looked at their brain activation during performance of an emotional word‐face task (using functional MRI). Each of the twins was discordant in exposure to severe stress such that one twin who had suffered two or more severe events exhibited significant clusters of greater activation in the ventrolateral and medial prefrontal cortex, basal ganglia, and limbic regions as compared with their control co‐twin who had suffered no severe events.

Structural Connectivity of the Brain of a Child with ASD and That of the Unaffected Identical Twin

Conti et al. [24] studied the structural connectivity of the brain of a child with ASD, and of that of his unaffected identical twin, using high angular resolution diffusion imaging (HARDI) that can show neuronal tracks in brains. Around 11% of the discordant intra‐hemispheric tracts showed lower fractional anisotropy (FA) values in the ASD twin, while only 1% showed higher values. This difference was significant. Our findings in a disease‐discordant identical twin pair confirm previous literature consistently reporting lower FA values in children with ASD.

Differences in Genomic and Epigenomic Expression in Monozygotic Twins Discordant for Rett Syndrome

Tran and Miyake [25] reported the genomic and epigenomic sequences in skin fibroblasts of a discordant monozygotic twin pair with Rett syndrome. As we have mentioned previously, this X‐linked neurodevelopmental disorder is History of Autism Becoming a Genetic Disease 151 included in the ASD group of disorders. Children with Rett syndrome suffer from epileptic seizures, gait ataxia, and stereotypical hand movements. These twins shared the same de novo mutation in exon 4 of the MECP2 gene (G269AfsX288), which was paternal in origin and occurred during spermatogenesis. The X chromosome inactivation patterns in the twins did not differ in lymphocytes, skin fibroblasts, and hair cells (which originate from ectoderm as does neuronal tissue). No differences were detected between the twins in single nucleotide polymorphisms (SNPs), insertion‐deletion polymorphisms (indels), or copy number variations (CNVs). Differences in DNA methylation between the twins were detected in fibroblasts in the upstream regions of genes involved in brain function and skeletal tissues such as Mohawk Homeobox (MKX), Brain‐type Creatine Kinase (CKB), and FYN Tyrosine Kinase Protooncogene (FYN). The level of methylation in these upstream regions was inversely correlated with the level of gene expression. Thus, differences in DNA methylation patterns likely underlie the discordance in Rett phenotypes between the twins. In addition, they describe in detail the relationship between neurodevelopmental disorders and environmental toxicants, in particular plastic‐derived chemicals (bisphenol A and phthalates), persistent organic pollutants, heavy metals, and maternal smoking.

Differences in CNV between Discordant Monozygotic Twins with Congenital Heart Defects

Breckpot et al. [26,27] explored the occurrence of copy number differences in monozygotic twins discordant for the presence of a congenital heart defect (CHD). They carried out an array comparative genomic hybridization on peripheral blood‐derived DNA from six discordant monozygotic twin pairs and on sex‐matched reference samples. To identify somatic CNV/differences between both twin members as well as potential CNVs in both twins contributing to the CHD, they analyzed the DNA from each twin by hybridizing against its co‐twin, and against a normal control. They detected three copy number differences in one out of six monozygotic twin pairs, confirming the occurrence of somatic CNV events in monozygotic twins. This report emphasizes the postzygotic genetic, environmental and stochastic factors that can affect human heart development.

History of Autism Becoming a Genetic Disease

Until 1976, autism was not considered a genetic disease. In 1976, Hanson et al. published a comprehensive review [28]. They found no convincing evidence of a genetic role in the etiology of autism. “No strong evidence exists,” they 152 Maternal Twins and Male Gender Bias in Autism Spectrum Disorders

concluded. This was not a new idea but was the general view held at that time by scientists, the medical community, and the public [28]. Soon after the report, several developments changed the paradigm that is still held as scientific fact. In 1977, Folstein and Rutter [29] published a study that was based on information collected from same‐sex discordant autistic twin pairs. The collected data were not founded on a solid scientific basis but included data from the late Dr M. Carter, records of all children known to the UK National Society for Autistic Children and a request for cases published in the society’s newsletter. These efforts resulted in identification of 11 maternal twins and 10 fraternal twin pairs. Out of the 11 maternal twins, 4 pairs were found to be concordant. The collected data showed that there was 53% concordance in maternal twin pairs and 0% in fraternal twin pairs. One should be aware that in today’s rigorous scientific world these data may not have passed the scrutiny of the Institutional Review Board. In another study of twins published by Ritvo et al. [30], the authors obtained data by advertisements placed in the newsletter of the National Society for Autistic Children (USA). This resulted in 22 concordant out of 23 maternal twin pairs and 4 out of 17 fraternal pairs for fraternal twins giving a concordance of 98% and 38%, respectively. These data could be skewed since they were obtained from parents and not from medical doctors [30]. Smalley et al. [31] reviewed the combined results of Folstein and Rutter and Rivto et al. of 11 maternal twins and 9 fraternal twins. It concluded that maternal twins have concordance of 64% and fraternal twins 9%. An another extension of the above studies by Bailey et al. [32] found that in the combined sample 60% of maternal pairs were concordant for autism versus 0% of fraternal twins. From the above summary of information, readers will quickly realize that the belief currently held in the majority of the scientific community that autism is a genetic and heritable disease is based on a small number of twin studies. A thorough inspection of outcomes and claims that support a strong genetic source of autism reveals incorrect interpretations, methodological biases, and flawed approximations, not to mention the overstated media reports [1,2].

Many Diseases That Were Considered Genetic are Being Reassessed

On the basis that maternal twin‐pair twins can exhibit genetic differences, one could hypothesize that disease discordance in maternal twin‐pair twins can also derive from acquired genetic differences, or varied epigenetic influences. The discordance for the autosomal dominant disease neurofibromatosis 1 (NF1) in a maternal twin pair has been explained by the presence of an NF1 mutation; the affected twin carried the de novo NF1 mutation in all investigated cells, while the unaffected twin was mosaic [33]. Therefore, one way to Many Diseases That Were Considered Genetic are Being Reassessed 153 explain these observations is that the environmental factors induced genetic mutations, which would not be equally distributed due to slight variations in maternal blood delivery to the twins. The unequal blood flow in maternal twin pairs is well documented [34–37], see Figures 6.3 and 6.4. This may result in unequal genetic mutations in each twin [38–41]. Since certain environmental chemicals can interfere in neurodevelopment there will be discordant autism symptoms in some maternal twin‐pair twins [34–36]. This phenomenon may partially explain the paradox of discordant maternal twin‐pair twins, but also shed light on our hypothesis that even perhaps a few molecules of a harmful chemical reaching a fetal brain could deplete specific neurons and induce ASD. Why has it taken so many decades for us to realize that ASD is not only genetic but that environmental factors play a significant role? Part of the answer is that in the 1970s, genetic engineering was in vogue in scientific research. Everyone wanted to use the new tools for genetic sequencing and to determine the role of genetic codes in diseases. Scientists in the 1980s and 1990s began to analyze genetics that could be associated with a particular illness. In some cases, they offered great insight. But, after decades of searching, they were unable to identify any particular gene or genes that could be definitely liked to ASD. These efforts continue; scientists regularly publish articles in prestigious journals showing a “likely association” of a particular gene to autism! These efforts will most likely continue for decades since scientific culture is difficult to change, even though there are lonely voices that have challenged the single focus on genetics [42–48]. In a recent Letter to the Editor, Waterhouse et al. [48] wrote:

“Syndromic ASD brain impairments vary from syndrome to syndrome, and gene models of ASD brain impairment lack replication and coverage. The ASD diagnosis lacks boundary construct validity because 96% of those with ASD have significant non‐diagnostic symptoms, including many who have full comorbid disorders. In addition, the ASD spectrum of disorders lacks construct validity. The spectrum has no shared early brain or behavioral predictor, no shared consistent developmental course, no shared Broader Autism Phenotype, no replicated subgroups, and no shared recurrence risk rate. Crucially, the phenotypic brain and behavior links between ASD and other neurodevelopmental disorders remain unclear, and the real complexities of the interaction of developmental risk factors also remain unclear. Taken together, the preponder- ance of evidence argues that using the diagnosis of ASD in research is fruitless because the diagnosis is an arbitrary unscientific “convenient fiction” that has blocked the discovery of replicable neurobiological variation among individuals with serious neurodevelopmental social impairment. Equally important, maintaining the ASD diagnosis supports the impossible research goal of finding a unitary cause for ASD, and 154 Maternal Twins and Male Gender Bias in Autism Spectrum Disorders

supports the public’s belief that a single cure for ASD will be found. Maintaining the ASD diagnosis will also continue to support the reifying business of ASD research funding, journals, and societies. Unless the ASD diagnosis is clearly renounced as scientifically arbitrary, the wide ASD coverage of 1% or more of the population, along with the many errantly definitive diagnostic assessments will continue to reify and prop up the ASD diagnosis. More than seventy years of research studying the arbitrary diagnosis of autism has not resulted in any targeted medical treatments. Now is the time to abandon the ASD diagnosis in research.”

In addition to Waterhouse et al. [48] who have voiced their concerns, there are other scientists who are not fixated on the genes only concept and have begun to look outside of the box. Hallmayer et al. [6] published an important study in 2011. This was one of the largest twin‐pair studies up to that date. They completed 192 twin‐pair studies and reported that a large degree of risk for ASD in maternal twin pairs was due to environmental factors and a smaller risk was due to heritability or genetic factors. Of note, they did not consider the de novo mutations and single CNVs as we describe below. We would like to present scientific evidence that besides the few genetic diseases mentioned in Chapter 2, the rest of the ASDs are caused by environmental factors. The genetic mutations that are observed are caused by environmental factors that reach a developing fetal brain. The major culprits are synthetic chemicals that are introduced into our environment which are highly mutagenic. Their effects may not be obvious if they are introduced to an adult since an adult brain is fully developed but if introduced to a developing fetus they can cause significant mutations in a fetal brain [2]. In addition, if a fetus is exposed to these agents during the early stages of development they selectively deplete certain brain primordial cells, causing skewed brain development and can result in autism. The wide “spectrum” that is observed in ASD is most likely is due to the timing of the exposure to a synthetic chemical or chemicals that a fetus is exposed to during his or her fetal development. Figure 6.5 explains the potential mechanism of the “spectrum” seen in ASD children. In our studies we have shown that among hundreds of different cell types that are found in human fetal brain cell lines, olfactory‐, oxytocin‐ and arginine vasopressin (AVP)‐receptor positive cells are highly susceptible to certain components of synthetic fragrances. Human beings develop a highly elaborate network of oxytocin and A receptors during fetal development. Oxytocin and AVP are not produced until after birth. Therefore, until birth these receptors are not fully functional but become functional when oxytocin and AVP are being produced by the newborn brain cells. If oxytocin‐ and AVP‐receptor carrying neurons and compartments of the brain are reduced significantly, the newborn will have impaired social development and his or her social communication would be severely impaired. However, if a fetus is exposed to any Many Diseases That Were Considered Genetic are Being Reassessed 155

Figure 6.5 The hypothesis of why ASD is a “spectrum”. The brain of a fetus develops throughout pregnancy. Starting out with a few cells, the cells grow and divide until the brain contains billions of specialized cells (over 100 billion neurons, not including other supporting cells). Once in place, each neuron sends out long fibers that connect with other neurons. In this way, lines of communication are established between various compartments and faculties of the brain and between the brain and the rest of the body. As each neuron receives a signal it releases neurotransmitters, which pass the signal to the next neuron. By birth, the brain has evolved into a complex organ with several distinct compartments, faculties and foci (nuclei), each with a precise set of functions and responsibilities. If a fetal brain is exposed to a harmful synthetic chemical (such as synthetic fragrances, EDCs, insecticides or herbicides) at very early stages of pregnancy (i.e., day 25) then it may harm a specific kind of progenitor neuron or neurons which were destined to become a specific compartment, faculty or nuclei of an offspring, resulting in either complete elimination of that region or subregion of the brain or malformation of that particular faculty. Since at the early stages of brain development there are very few neurons, each one is a progenitor neuron (or mother neuron) for a specific brain compartment and elimination of that compartment would result in severe damage to the brain. In the case where a fetus is exposed to the same type of neuron‐damaging synthetic chemical (at the same dose) at later stages of development, the damage may be less severe and the result may be less obvious. We believe that this is the cause of the “spectrum” in ASD. This spectrum will never fit into a single “box”. neurotoxic agent that targets oxytocin or AVP receptors at later stages of fetal development, the adverse effects would depend on at what stage the destruction occurred. For example, if this occurred at day 14 of gestation, the consequences would be severe since there will be only a few neurons that would be positive for oxytocin and AVP receptors. If exposure took place at week 8 of gestation, there will be several hundred thousands of oxytocin‐ and AVP‐ receptor positive neurons and the damage may less significant (depending on the amount of the specific neurotoxin that reaches the brain). If exposure to a 156 Maternal Twins and Male Gender Bias in Autism Spectrum Disorders

synthetic chemical that targets oxytocin‐ or AVP‐ receptor positive neurons occurs at week 30 of gestation, then the neurotoxic agent will only harm a small population of that group of neurons, resulting in partial reduction in the social communication compartment of the brain. This hypothesis explains the concept of the “spectrum”. It all depends on at what stage of fetal brain development a neurotoxic agent was introduced, and what kinds of progenitor neurons it affected, and to what degree. In the early stages, the harm to the primary progenitor neurons may eliminate the whole faculty or faculties of the brain. In exposure at later stages, the harm may be partial. Furthermore, whenever a primary or secondary progenitor neuron is eliminated, the space left due to the death of a particular neuron is replaced with another progenitor neuron that may proliferate at a faster growth rate, resulting in a larger brain size, a hallmark of a typical ASD brain. The reduction in the oxytocin‐ and AVP‐receptor positive neurons results in damage to the areas of the brain that are generally observed in ASD children (Table 4.1). It should be noted that brain development does not stop at birth. The brain continues to change during the first few years of life, as new neurotransmitters become activated and additional lines of communication are established. Neural networks are forming and creating a foundation for processing language, emotions, and thoughts. We will discuss the potential damaging agents to this stage of brain development in the next chapter. More than 1000 genetic and genomic disorders have been linked to ASD and the number of genes implicated or are associated with ASD are still increasing, yet genome‐wide association studies, CNV, and candidate gene association have found no single genetic factor accounting for about 80% of ASD cases; the other 20% are actual genetic diseases and should not be included in ASD. Interestingly, trio analyses (where both the parent and the ASD child’s mRNAs were sequenced, called exome sequencing analyses) revealed genes that have suffered mutations only during fetal development and are not found in either of the parents. This clearly shows that ASD is not inherited from the parents but the new mutations are introduced during the fetal development. Hundreds of genetic loci and more than 1000 single nucleotide and short and long genes variants that are assumed to play a role in ASD may be the secondary outcome of unknown synthetic chemicals that a fetus was exposed to during the prenatal period [10,13,49]. In the attempt to collate all genes and recurrent genomic imbalances that have been implicated in the etiology of ASD, no definite answers have been found.

De Novo Mutations

The de novo (novel, new) mutations relate to mutations that are absent in either the biological father or the mother of an autistic child but arise during the fetal stages of development. There are two kinds of de novo mutation: the De Novo Mutations 157

CNVs, the very small gain or loss of genomic DNA (meaning there is an extra fragment of DNA found in the ASD child or small DNA fragments are lost). Usually, these DNA fragments are relatively small (i.e., <400 kb of DNA) that either disappear or emerge in an offspring. De novo CNVs are present in 4–7% of ASD children as compared with 1–2% in unaffected siblings or normal controls. Other than in ASD, larger fragments of DNA CNVs (>400 kb) affecting genes are present in 15% of patients with developmental delay or intellectual disability. The majority of these CNVs are personal to each individual, but some are repeatedly observed in independent patients, suggesting that these are rather random DNA mutations that appear in a child due to exposure to some harmful chemical during the fetal development. There have been 2,350 different CNVs found in ASD children, suggesting that these are chemically induced mutations that are present in the ASD children but mostly absent in the parents of these children [2]. Some studies have found no real differences in CNVs between ASD and controls [50]. The studies have also indicated that de novo CNVs identified in patients with ASD are most likely altering genes associated with synaptic functions. For example, Carreira and colleagues [51] analyzed 2,446 ASD‐affected families and confirmed an excess of genetic deletions and duplications in affected versus control groups that were statistically highly significant (1.41‐fold, p = 1.0×10−5). It is believed by many that CNVs increase the risk of having ASD in 5–10% of individuals. But, we believe that these CNVs are the result of mutations that are induced by the chemicals that a fetus is exposed to during prenatal development. Furthermore, we predict that there will be many thousands of mutations discovered as technological advancements able to identify slight variations in noncoding genes. CNVs do not necessarily mean “genetic defects” and are found in all of us as a natural variation phenomenon [51]. Few of the advanced technologies that are coming to light are whole exome or genome sequencing studies of the mRNAs or DNAs of ASD patients. One can identify even a single nucleotide variant or SNV. In particular, numerous de novo SNVs have been identified using whole exome sequencing of individuals with sporadic ASD. Among them, several disruptive mutations associated with sporadic ASD were reported, however, the genetic mutations in these genes account for only a small proportion of all cases. Several thousand SNVs have been found and the numbers will increase. Unfortunately, the functions of most candidate ASD‐associated genes, as well as the biological significance of the identified mutations in the central nervous system, remain unknown despite extensive bioinformatics resources. Accordingly, although the precise functional analysis of each disease‐associated gene and its mutation is important for understanding the etiology of ASDs, it is not easy to assign priorities to essentially thousands of mutations that are found in ASD children for further detailed biological analysis. We argue that these are mutations induced by synthetic chemicals and may or may not have any real direct function. 158 Maternal Twins and Male Gender Bias in Autism Spectrum Disorders

To find a Scientific Analysis of ASD Genesis

A scientist cannot intensely expose a pregnant woman to any synthetic chemical. It would be unethical and immoral. However, nature always provides means to uncover its secrets. We have developed a unique in vitro (test tube) model to determine if chemicals in fragrances can induce mutations. To figure out what is happening to a fetal brain, a neuroblastoma cell based rapid and convenient screening system for elucidating the effects of fragrances and their selected chemicals on a special kind of Salmonella bacteria was developed. This is called the Ames test. This was designed to elucidate the potential of any chemical to induce genetic mutations. By utilizing the Ames test, we have found that all the fragrances we tested induced significant mutations. This strongly suggests that when a pregnant woman is exposed to a fragrance (perfume) which is absorbed by the skin or inhaled by the lungs and enters the blood circulation, it can cause mutations in the rapidly developing fetus. We diluted the fragrances a million‐fold to mimic real‐life conditions. Even fragrance molecules diluted a million‐fold or so, with few molecules of the chemicals entering the fetal brain, can cause havoc in the fetus’s brain. Destroying only a few brain progenitor cells can, potentially, delete the whole faculty of the future fully grown brain (i.e., amygdala, communication compartments of the future brain) on fetal brain cells. Why are only selected cells of the brain destroyed? We will come to this shortly. If should be noted that whenever investigators report mutations in the ASD genomes or exomes, SNVs, CNVs, and so on, they are looking at all of the DNA of the ASD child, suggesting that these synthetic chemicals are inducing mutations in the fetus’s whole DNA, not just in the brain. However, there are unique mutations that are found specifically in ASD brains. For example, Uddin et al. [52] have analyzed mutation in “exons” that are specific to brain. Their analyses showed that specific critical exons were significantly enriched in individuals with ASD relative to their siblings without ASD. We think, at this stage, we should answer two crucial questions. We believe that besides inducing random mutations, fragrances and many other synthetic chemicals cause selective depletion or reduction in the developing fetal brain’s neurons. For example, Torres et al. [53] have shown in many neurodevelopmental disorders certain chromosomal regions (1q21.1, 3q29, 15q11.2, 15q13.3, 16p11.2, 16p13.1, and 22q11) harbor rare but recurrent CNVs that have been uncovered as being important risk factors for several of these disorders. These rearrangements may underlie a broad phenotypical spectrum, ranging from normal development, to learning problems, intellectual disability, epilepsy, ASDs, and schizophrenia. The highly increased risk of developing neurodevelopmental phenotypes associated with some of these CNVs makes them an unavoidable element in the clinical context in pediatrics, neurology, and psychiatry [53]. We hypothesize that over representation of certain chromosomes What are Neuroblastomas? 159 mentioned above may be due to the high frequency of open reading frames (ORFs) during early fetal brain development. As mentioned above [52], certain exons are more vulnerable to mutations in ASD and other neurodevelopmental disorders [53]. We believe this is due to the presence of a large numbers of ORFs during that particular stage of development in those chromosomes. In molecular biology, an ORF is the part of a reading frame that has the potential to be translated. An ORF is a continuous stretch of codons that contain a start codon (usually AUG) and a stop codon (usually UAA, UAG, or UGA). An ORF can be described as a handle of a knife; one has to hold that handle before starting to slice something.

What are Neuroblastomas?

In order to answer this particular question, we decided to use what nature has provided as a natural tool for ASD investigation. First, we would like to introduce a natural tool that can be used to investigate ASD. We have utilized neuroblastoma cells. A neuroblastoma is an embryonal tumor of the nervous system, arising during fetal or early post‐natal life from neuronal cells derived from the neural crest. Briefly, these cells behave like fetal brain neurons in the primordial brain, which at 5 weeks of gestation is the size of a grain of sand. There is no protective solid brain skull or even the meningeal protective thick membrane. And, most importantly, there is no blood–brain barrier. The brain stem cells at 5 weeks of gestation are vulnerable to any adverse synthetic chemicals that are evolutionarily new to the human detoxification system. The pregnant women who are carrying those babies, if exposed to a harmful chemical or chemicals with only even a few molecules reaching their fetuses’ brain, will turn them into “silent offspring”! Just a reminder here that a fragrance can contain up to 4,000 synthetic chemicals, many of them can selectively harm fetal brain neurons. Why selectively? Why do these chemicals not kill the whole brain? The fact is that about 10–25% of known pregnancies end in miscarriage, and more than 80% of these losses happen before 12 weeks. How many fetuses become victims to synthetic chemicals before week 12 is anyone’s guess. We believe that many factors play roles in the destruction of fetuses. In our studies we used neuroblastoma cell lines that originate from the neural crest. We have acquired numerous neuroblastoma cell lines that behave like fetal brain cells and most of the cell lines contain progenitor neurons that are destined to become various brain compartments in a newborn infant [54–57]. They can be differentiated under the influence of retinoic acid and contain numerous progenitor cells, representing many faculties of the human brain, including neurons that express oxytocin and AVP receptors. These receptors 160 Maternal Twins and Male Gender Bias in Autism Spectrum Disorders

and oxytocin and AVP neurohormones represent the center of the main human communication devices. Without these our social behaviors would be skewed and our communication abilities would be diminished [54–57].

What Method Did We Use?

We diluted 1 μl of a fragrance a million times and then exposed the diluted fragrance to fetal brain neurons to determine what it did to fetal brain cells.

Possible Etiologies of Autism

Accurate scientific data show that in industrialized areas of the world, where fragrance and other environmental synthetic chemical use are most common, there are serious mutagenic effects [15]. We maintain that these mutations increase the probability of ASD. Causation commonly involves multiple factors, including the time period of fetal development when pregnant mothers are exposed to potential environmental insults that may interfere with the developing fetal brain. Generally, weeks 4–18 of gestation are considered to be the most susceptible period. Previously, we have shown that all of the 98 fragrances we examined have significant mutagenic effects [2]. It should be noted that various environmental factors can induce not only changes in the DNA directly, but can induce epigenetic modifications which can change the DNA codes indirectly by interfering with gene regulation [56,57].

Epigenetic Explained

Being epigenetic is one of the new discoveries of modern science. As we have mentioned previously, only about 1% of human DNA codes for proteins and such regions are referred to as the coding (also exome) regions of the genome. The rest of the DNA was previously known as junk DNA or noncoding DNA. In reality, a large part of the so called noncoding DNA serves as a gene regulator. Epigenetic changes are changes to the noncoding DNA sequence — a change in phenotype without a change in genotype — which in turn affect how cells read the genes and respond to external and internal stimuli at a single cell level. Epigenetic change is a regular and natural occurrence but can also be influenced by several factors including age, the environment, lifestyle, and disease state. Epigenetic modifications can make one type of brain progenitor neuron change to another type. Epigenetic change can have more damaging effects and turn abnormal cells into cancer cells. At least three systems, including DNA methylation, histone modification, and Factors Other Than Environment That May Be Contributing to ASD 161 noncoding RNA (ncRNA), are currently considered to initiate and sustain epigenetic change. New and ongoing research is constantly uncovering the role of epigenetic changes in a variety of human disorders and fatal diseases. Most epigenetic studies are of microRNAs (miRNAs) that are known to control one‐third of human genes. These are small molecules that are known to regulate a large part of life‐related functions and are involved in many disease processes. Currently, two epigenetic areas are most broadly studied – DNA methylation and miRNAs. The latter is known to be involved in long term memory, chromatin remodeling, and histone modifications. The most interesting findings in the epigenetic studies are about the relationship between epigenetic changes and a host of disorders including various cancers, mental retardation associated disorders, immune disorders, and intellectual disorders.

Epigenetic Changes and the Environment: How Lifestyle Can Influence Epigenetic Change from One Generation to the Next

The growing understanding of interaction between the environment and genetics may influence epigenetic changes or vice versa. These changes may be reflected at various stages throughout a person’s life and even in later generations. For example, human epidemiological studies have provided evidence that gestation in utero and early postnatal environmental factors influence the adult risk of developing various chronic diseases and behavioral disorders. Studies have shown that children born during the period of the Dutch famine from 1944 to 1945 have increased rates of coronary heart disease and obesity as a result of maternal exposure to famine during early pregnancy, compared with those not exposed to famine. Similarly, adults who were prenatally exposed to famine conditions have also been reported to have significantly higher incidence of schizophrenia.

Factors Other Than Environment That May Be Contributing to ASD

Older Age of Mother and Increased Risk

During the investigation on the birth of children born in California during the 1990s, it was concluded that the risks for having autistic children were significantly higher for older women giving birth. It was concluded that it was 51% more likely for women older than age 40 to give birth to an autistic child, than mothers between the ages of 25 and 29 (Figure 6.6). Furthermore, it was also 162 Maternal Twins and Male Gender Bias in Autism Spectrum Disorders

20 0,000 per population ) 15

Rate (1 10

0 <25 25–29 30–34 35–39 40+ Maternal age

Figure 6.6 Maternal age and autism in California (1990–1999). Advanced maternal age is linked to a significantly elevated risk of having a child with autism, regardless of the father’s age, according to an exhaustive study of all births in California during the 1990s by UC Davis Health System researchers. The study found that advanced paternal age is associated with elevated autism risk only when the father is older and the mother is under 30. Source: Adapted from Refs [58, 59].

discovered that while a higher maternal age was consistently associated with higher risk for autism in children, the father’s age only had a negative impact when the mother was younger than 30 and the father was older. The reason that this is caused in older parents has yet to be solved, but genetic, environmental, and hormonal effects may be potential contributing factors [1,2,56–59]. We suggest that the repeated, cumulative exposure to synthetic chemicals that we are exposed to in the form of fragrances, herbicides and pollutants, in our food, water, and air, most of which have teratogenic and/or mutagenic effects over a long period of time, will increase the probability of parents to pass on accumulated germ cell mutations to the fetus. We speculate that these synthetic chemicals that cannot be broken down into innocuous chemicals remain bound to certain proteins and stay dormant in the human liver, where the majority of the toxic chemicals are detoxified. During pregnancy, a pregnant woman goes through enormous physiological and biochemical changes and large amounts of hormones are released and, unfortunately, also unleash the toxins that are present in the liver. This phenomenon may partially explain the paradox of discordant maternal twin pair twins, but also shed light on our hypothesis that even perhaps a few References 163 molecules of a harmful chemical reaching a fetal brain at the early stage of prenatal development could deplete specific progenitor neurons and induce ASD. We also confirmed the relative resistance of female neuroblastoma cell lines to certain fragrances with regards to oxytocin‐ and AVP‐receptor positive neuron depletion. Males, however, are highly vulnerable to fragrances when it comes to oxytocin‐ and AVP‐receptor positive neurons.

Conclusion and Summary

The discordant maternal twins provide an excellent opportunity to uncover the potential role of environmental factors such as synthetic fragrances, glyphosate and other EDCs derived from petrochemicals containing carcinogenic, mutagenic, hormone disturbing and neuromodifying capabilities at the molecular and cellular levels. The causes of ASD have not been evaluated. This is partly due to the 1973 Food and Drug Administration decision to exempt fragrances and cosmetics from appropriate testing, which is generally required for any consumer item that enters the human body and is metabolized by human metabolic pathways. Synthetic chemicals that are designed to enter a human body via respiratory and dermal routes and that appear to be harmless to adult humans and other mammals, with fully differentiated brain, may have severe consequences for the undifferentiated and still developing fetal brain. In addition, there are certain parts of the adult brain where brain cells regularly go through neurogenesis (i.e., piriform cortex, hippocampus, amygdala, and subventricular zone); these cells may be highly vulnerable to similar neurotoxic chemicals. Concerns about the potential neurotoxic effects of these chemicals are warranted.

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54 Lu KY, Tseng FW, Wu CJ, Liu PS (2004). Suppression by phthalates of the calcium signaling of human nicotinic acetylcholine receptors in human neuroblastoma SH‐SY5Y cells. Toxicology, 200(2–3):113–21. 55 Kovalevich J, Langford D (2013). Considerations for the use of SH‐SY5Y neuroblastoma cells in neurobiology. Methods Mol. Biol., 1078: 9–21. 56 Sealey LA, Hughes BW, Steinemann A, et al. (2015). J Role of environmental factors in autism development and male bias: Neuromodifying effects of fragrance. Environ. Res., 142:731–8. 57 Hughes BW, Sealey LA, Bagasra O (2016). Mechanism of male gender bias in neuroblastoma cell lines exposed to fragrances: A link to autism spectrum disorder. Expert Opin. Environ. Biol., 5:1–21. 58 Shelton JF, Tancredi DJ, Hertz‐Picciotto I (2010). Independent and dependent contributions of advanced maternal and paternal ages to autism risk. Autism Res., 3:30–9. 59 Sealey LA, Hughes BW, Sriskanda AN, et al. (2016). Environmental factors in the development of autism spectrum disorders. Environ. Int., 88:288–98.

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A Who’s Who of pesticides is therefore of concern to us all. If we are going to live so intimately with these chemicals eating and drinking them, taking them into the very marrow of our bones – we had better know something about their nature and their power. Rachel Carson, 1962, Silent Spring

Until recently, autism spectrum disorder (ASD) was considered essentially a heritable mental disorder, a notion based on surprisingly sparse data from small clinical studies. Population based studies of the heritability of other neu ropsychiatric disorders and comorbidities among them have also been limited. Now, rapidly accumulating evidence published in peer‐reviewed scientific journals has suggested that up to 40–59% of the variance observed in ASD might be due to environmental factors [1–7]. In this chapter, we will examine this dichotomy –environment versus heritability/genetics theories. The herit ability theory arose primarily from misinterpreted concordance in maternal twin pairs, which we have described in previous chapters. Since the genes in maternal twin pairs are identical, it was assumed that autism is inherited or genetic in origin; however, glaring dissimilarities in the maternal twins were overlooked [1–7]. Of note, when Leo Kanner first described the syndrome he assumed that it was a heritable illness. However, he noted that there was a common thread found in how each autistic child was raised, prompting him to suspect the potential influence of the environment. The concept of environ mental contributions in ASD is well regarded in some cases (i.e., exposure to valporic acid) and thoroughly debunked in other cases (Bettelheim’s “refrigera tor mother” theory, see Chapter 2). In contrast to single nucleotide mutational disorders such as Parkinson’s, Huntington disease, phenylketonuria, caustic fibrosis, neurofibromatosis, muscular dystrophy, and sickle cell anemia, or syndromes that are defined by a single microdeletion or several recurrent

Autism and Environmental Factors, First Edition. Omar Bagasra and Cherilyn Heggen. © 2018 John Wiley & Sons, Inc. Published 2018 by John Wiley & Sons, Inc. 170 Autism and Exposure to Environmental Chemicals

microdeletions and duplications (described in detail in Chapter 2), ASD has been associated with a wide range of phenotypic features and severity. As we have described in the previous chapter, wide variations observed in ASD chil dren are most likely due to the timing during the prenatal period when the fetus was exposed to a neurotoxic chemical and not the result of gene penetra tion or degree of expression of ASD‐associated genes [1–7]. Support for the possibility of an environmental contribution to the development of autism has come from two major sources: first, the current understanding of the extreme vulnerability of the developing human brain to toxic exposures in the environ ment; and secondly, proof‐of‐concept studies that specifically link autism to environmental exposure experienced prenatally [8]. There are numerous excel lent review articles that have covered these subjects [8–20], so we will just point out some of the types of agents involved: fragrances; lead; methylmer cury; polychlorinated biphenyls (PCBs); arsenic; manganese; organophosphate insecticides; DDT; and ethyl alcohol. Are synthetic chemicals that humans are not evolutionarily exposed to responsible for ASD? Over the last three decades, a substantial increase in the prevalence of autism has been reported, from 4 to 5 children per 100,000 in the 1960s to around one in 45 children in 2015 [4,5]. Investigators have examined many potential environmental factors including vaccination, maternal smok ing, thimerosal exposure, and most likely assisted reproductive technologies, but have decided that these factors are not related to risk for ASD. We maintain that the alarming 20‐fold increase in autism in recent years is due to exposure of the human population to an increasingly diverse set of synthetic chemicals including herbicides, insecticides, food and drink flavors, drinking water, air pollutants and fragrances, many of which have steroidogenic (male and female hormone‐like chemicals) activity [1–5]. One can easily oppose the claims that any of the chemicals in the food or liquid we consume are safe and that the US government makes sure that all the chemicals in these common sources are certified to be safe by independent Industrial Bio‐Test Laboratories (IBT). The irony is that, according to published reports by Bioscience Resource (https:// bioscienceresource.org/) many of the safety documents cannot be trusted. However, we do not want to get into the so called conspiracy realm and will only present information from mainstream laboratory and epidemiological studies, and show that undisclosed chemicals in fragrances, such as those that produce different scents, increase shelf life, control time release of fragrances and improve stability, have endocrine‐disrupting properties (reviewed in Ref. [7]). This disruption has been linked to increased risks for cancer (reviewed in Refs [4], [8], and [21]), adverse effects on developing fetuses [4,7], and meta bolic diseases. For example, chemicals that have been shown to increase human estrogen receptor (ER) expressions include octinoxate, oxybenzone, benzo phenone‐1, benzophenone‐2, benzyl salicylate, benzyl benzoate, butylphenyl methylpropional, and synthetic musks (galaxolide, tonalide, and musk ketone) Autism and Exposure to Environmental Chemicals 171

[22–28]. Of these, oxybenzone, benzophenone‐1, galaxolide, and tonalide also affect androgens, while butylated hydroxytoluene, benzophenone‐2, and octi noxate have been linked to thyroid hormone disruption [28–34]. Even at very low concentrations, fragrances that contain these chemicals can be mutagenic and carcinogenic [3,4,7,21]. Of note, one of the fragrance ingredients, acetyl ethyl tetramethyl tetraline, was used for 22 years in fragrances, colognes, soaps, detergents and cosmetics [33]. It resulted in behavioral changes and degenera tion of the white matter of the brain including “widespread demyelination and scattered axonal degeneration in the central peripheral nervous systems.” It was voluntarily discontinued in 1978. Along with this, the fragrance ingredient musk ambrette (a fixative recently banned in the European Union but still allowed in the USA) has been found to also cause degeneration of the myelin sheath and distal axon [33]. Even if one considers the high fetal testosterone levels during the early stages of gestation, these data do not explain the rising incidence of ASD, except that synthetic chemicals behaving like hormones can reach the fetal blood circulation [7,25,27,34,35]. We know that many fra grances, and especially perfumes, contain testosterone‐like hormones [21] (Table 7.1). Chlordane was a termite preventative chemical used in US homes in the 1960s–1980s. It was banned for use inside homes in 1979 and under neath homes as a soil treatment in 1983 [36–39]. Due to concerns about dam age to the environment and harm to human health, all use of chlordane was banned by the Environmental Protection Agency (EPA) in 1988 [37]. A question that remains is why are female neuronal cells relatively more protected than their male counterparts? It has been proposed by some investi gators that the male bias for ASD may be partially due to under diagnosis of autism in females or the human female’s ability to mask some of the symptoms of ASD [5]. Even so, these investigators agree that male bias is observed in ASD. While in the majority of cases, the exact etiology of ASD remains unknown, novel technologies and large population based studies have pro vided new insight into the risk architecture of ASD and the possible role of environmental factors in etiology. Recently, we have shown that one of the fac tors explaining why ASD is more common in boys than girls may be due to the preferential depletion of oxytocin‐ and arginine vasopressin (AVP)‐receptor positive neurons in developing fetal brains [40]. Utilizing neuroblastoma cell lines (NBCs) derived from both genders that were exposed to femtomolar con centrations of commonly used fragrances, we determined that both types of the neurons are more significantly depleted from the male neuroblastoma cells compared with female [2,5,40]. We already have discussed the potential role of fetal testosterone in ASD in Chapter 5. Here we present some of the alarming evidence regarding the fragrances we smell every day, the majority of which are synthetic chemicals that enter our brains via the olfactory system. However, the most important with respect to ASD are the ones that enter our blood stream via the lungs and are absorbed into our skin and then enter our blood via 172 Autism and Exposure to Environmental Chemicals

Table 7.1 Widespread pollutants with endocrine‐disrupting effects.

Compound(s) Hormone Mechanism

Persistent Organohalogens

Benzene hexachloride Thyroid Unknown 1,2‐Dibromoethane Reproductive Unknown Chloroform Reproductive Unknown Dioxins and furans Estrogen Work as anti‐estrogen through binding with Ah receptor, which then inhibits estrogen receptor binding to estrogen response elements, thereby inhibiting estrogen action Octachlorostyrene Thyroid Unknown PBBs Estrogen/Thyroid Unknown PCBs Estrogen/androgen/ Inhibits estrogen binding to Thyroid the receptor; works as Adverse outcomes in anti‐estrogen reproductive systems Anti‐androgenic via Ah receptor interaction PCB, hydroxylated Thyroid Binds to thyroid hormone binding protein, but not to the thyroid hormone receptor PBDEs Thyroid Interfere with thyroxine (T4) binding with transthryetin Pentachlorophenol Thyroid Reduces thyroid hormone possibly through a direct effect on the thyroid gland

Food Antioxidant

Butylated Estrogen Inhibits binding to the hydroxyanisole estrogen receptor.

Pesticide

Acetochlor Thyroid (decrease of Unknown thyroid hormone levels, increase in TSH) Alachlor Thyroid (decrease of Unknown thyroid hormone levels, increase in TSH) Aldrin Estrogen Binds to estrogen receptors; competes with estradiol Autism and Exposure to Environmental Chemicals 173

Table 7.1 (Continued)

Compound(s) Hormone Mechanism

Pesticide

Allethrin, d‐trans Estrogen Unknown Amitrol Thyroid Thyroid peroxidase inhibitors; inhibits thyroid hormone synthesis. Atrazine Neuroendocrine‐ Inhibits ligand binding to pituitary (depression of androgen and estrogen LH surge), testosterone receptors metabolism Carbaryl Estrogen and Unknown progesterone Chlofentezine Thyroid Enhances secretion of thyroid hormone Chlordane Testosterone and Unknown progesterone Cypermethrin Disruption of Unknown reproductive function DDT Estrogen DDT and related compounds act in a number of ways to disrupt endocrine function by binding with the estrogen receptor, including estrogen mimickry and antagonism, altering the pattern of synthesis or metabolism of hormones, and modifying hormone receptor levels DDT metabolite, Androgen Inhibits androgen binding to p,p′‐DDE the androgen receptor, androgen‐induced transcriptional activity, and androgen action in developing, pubertal and adult male rats Dicofol (Kelthane) Estrogen Unknown Dieldrin Estrogen Binds to estrogen receptor; competes with estradiol Endosulfan Estrogen Unknown Ethylene thiourea Thyroid Thyroid peroxidase inhibitor Fenarimol Estrogen Estrogen receptor agonist

(Continued) 174 Autism and Exposure to Environmental Chemicals

Table 7.1 (Continued)

Compound(s) Hormone Mechanism

Pesticide

Fenbuconazole Thyroid Enhances secretion of thyroid hormone Fenitrothion Antiandrogen Competitive androgen receptor antagonist Fenvalerate Estrogen Unknown Fipronil Thyroid Enhances secretion of thyroid hormone Heptachlor Thyroid unknown Heptachlor‐epoxide Thyroid/Reproductive Metabolite of heptachlor Iprodione Inhibition of Unknown testosterone synthesis Karate Thyroid A decrease of thyroid hormone in serum; direct effect on the thyroid gland? Kepone (Chlordecone) Estrogen Displays androgen and estrogen receptor‐binding affinities Ketoconazole Effects on reproductive Unknown systems Lindane Estrogen/Androgen Inhibits ligand binding to (Hexachlorocyclohexane) androgen and estrogen receptors Linuron Androgen Androgen receptor antagonist Malathion Thyroid Significant decrease of thyroid hormone in serum, with perhaps a direct effect on the thyroid gland Mancozeb Thyroid Thyroid peroxidase inhibitors Maneb Thyroid The metabolite ethylenthiourea inhibits thyroid hormone synthesis Methomyl Thyroid Unknown Methoxychlor Estrogen Through mechanisms other than receptor antagonism. Precise mechanism still unclear Metribuzin Thyroid Unknown Autism and Exposure to Environmental Chemicals 175

Table 7.1 (Continued)

Compound(s) Hormone Mechanism

Pesticide

Mirex Antiandrogenic Unknown activity; inhibits production of LH. Potentially thyroid Nitrofen Thyroid Structural similarities to the thyroid hormones; nitrofen or its metabolite may have thyroid hormone activities Nonachlor, trans‐ Estrogen Estrogen receptor agonist? Oxychlordane Reproductive Unknown Pendimethalin Thyroid Enhances secretion of thyroid hormone Pentachloronitrobenzene Thyroid Enhances secretion of thyroid hormone Permethrin Estrogenic Unknown Procymidone Androgen Androgen receptor antagonist Prodiamine Thyroid Enhances secretion of thyroid hormone Pyrimethanil Thyroid Enhances secretion of thyroid hormone Sumithrin Androgen Unknown Tarstar Thyroid A decrease of thyroid hormone in serum; direct effect on the thyroid gland? Thiazopyr Thyroid Enhances secretion of thyroid hormone Thiram Neuroendocrine‐ Unknown pituitary (depression of LH surge), thyroid (decrease of T4, increase of TSH Toxaphene Estrogen/Thyroid Unknown Triadimefon Estrogen Estrogen receptor agonist. Triadimenol Estrogen Estrogen receptor agonist Tributyltin Reproductive Unknown

(Continued) 176 Autism and Exposure to Environmental Chemicals

Table 7.1 (Continued)

Compound(s) Hormone Mechanism

Pesticide

Trifluralin Reproductive/ Unknown Metabolic Vinclozolin Androgen Anti‐androgenic. (Competes with androgens for the androgen receptor, inhibits androgen receptor–DNA binding, and alters androgen‐ dependent gene expression) Zineb Thyroid The metabolite ethylenthiourea inhibits thyroid hormone synthesis Ziram Thyroid Inhibits the iodide peroxidase. Structural similarities between ziram and thiram; ziram can be metabolized to thiram in the environment

Phthalate

Butyl benzyl phthalate Estrogen Inhibits binding to the estrogen receptor Di‐n‐butyl phthalate Estrogen Inhibits binding to the Androgen estrogen receptor Anti‐androgenic DEHP Estrogen Inhibits binding to the Androgen estrogen receptor Anti‐androgenic Diethyl phthalate Estrogen Aromatase inhibitor

Other Compounds

Benzophenone Estrogen Binds weakly to estrogen receptors; roles of its metabolite remain to be clarified. BPA Estrogen Estrogenic; binds to estrogen receptor Bisphenol F Estrogen Estrogenic; binds to estrogen receptor Benzo[a]pyrene Androgen Anti‐androgenic Autism and Exposure to Environmental Chemicals 177

Table 7.1 (Continued)

Compound(s) Hormone Mechanism

Other Compounds

Carbendazim Reproductive Unknown Ethane dimethane Reproductive Unknown sulfonate Perfluorooctane Thyroid, reproductive Suppression of T3,T4; sulfonate mechanism unknown Nonylphenol, Estrogen Estrogen receptor agonists; octylphenol reduces estradiol binding to the estrogen receptor Resorcinol Thyroid Unknown Styrene dimers and Estrogen Estrogen receptor agonists trimers Metals

Arsenic Glucocorticoid Selective inhibition of DNA transcription normally stimulated by the glucocorticoid‐GR complex Cadmium Estrogenic Activates estrogen receptor through an interaction with the hormone‐binding domain of the receptor Lead Reproductive Unknown Mercury Reproductive/ Thyroid Neurotoxic

BPA, bisphenol A; DDE, dichlorodiphenyldichloroethylene; DDT, dichlorodiphenyltrichloroethane; DEHP, di(2‐ethylhexyl) phthalate; LH, luteinizing hormone; PBBs, polybrominated biphenyls; PBDEs, polybrominated diphenyl ethers; PCBs, polychlorinated biphenyls; TSH, thyroid stimulating hormone. BPA is a chemical produced in large quantities for use primarily in the production of polycarbonate plastics and epoxy resins. The NTP Center for the Evaluation of Risks to Human Reproduction completed a review of BPA in September 2008. The NTP expressed “some concern for effects on the brain, behavior, and prostate gland in fetuses, infants, and children at current human exposures to bisphenol A.” DEHP is a high production volume chemical used in the manufacture of a wide variety of consumer food packaging, some children’s products, and some PVC medical devices. In 2006, the NTP found that DEHP may pose a risk to human development, especially critically ill male infants. Phytoestrogens are naturally occurring substances in plants that have hormone‐like activity. Examples of phytoestrogens are genistein and daidzein, which can be found in soy‐derived products. Source: Adapted from http://www.ourstolenfuture.org/Basics/chemuses.htm. 178 Autism and Exposure to Environmental Chemicals

tiny capillaries. These chemicals may not have any obvious adverse effects on an adult brain that is fully developed, but may have major detrimental effects on the fetal brain that is rapidly differentiating.

Contribution of Fragrances to ASD

Many modern companies do not disclose the industrial secrets in many of their fragrances that are, in reality, a complex concoction of synthetic chemicals and natural essences, which often have been found to be petrochemicals. As men tioned previously, according to the laboratory testing that was conducted under the direction of the Campaign for Safe Cosmetics and was analyzed later by the Environmental Working Group, 38 hidden chemicals were uncovered in 17 name‐brand fragrance products [41]. Topping the list was American Eagle’s Seventy Seven, which contained 24 undisclosed chemicals, followed by Chanel’s Coco with 18, and Britney Spears’ Curious and Giorgio Armani’s Acqua Di Gio with 17 secret chemicals each (as shown in Chapter 1). Among those are chemicals, such as musk ketone and diethyl phthalate (DEP), that are responsible for allergic reactions and hormone disruption [7,40]. Although these chemicals have been found to accumulate in human tissues, they have not yet been adequately analyzed for safety in products. As a result of an exemption for cosmetics and fragrances in the Federal Fair Packaging and Labeling Act of 1973 [42], the producers of cosmetics and fragrances are not required to disclose the chemical nature of their products, nor do they have to test the safety of flavors present in innumerable chemicals that may cause health problems [41,42]. It is a common practice for businesses to list some chemicals simply as “fragrance,” which may mean that many ingredients are never revealed to buyers. Even worse, people who use cologne, fragrances, body spray, and other scented cosmetics may unknowingly expose themselves to dangerous chemicals since the Food and Drug Administration lacks author ity to mandate testing of all fragrances for safety before being released to the public. The worst of all, there are now companies that synthetize chemicals that test, smell, and feel like the “real” thing. One of these companies was fea tured in a CBS program entitled “Tweaking tastes and creating cravings” [43]. These chemicals are designed to enter the human body and if an unsuspecting fertile woman is pregnant, these chemicals can get into the brain of the fetus, and we do not know what might then happen. Fragrances are everywhere, and when applied on the skin or sprayed, many chemicals from these fragrances are absorbed or inhaled, and some of these chemicals damage the body from before birth to until the end of life. Research by the Environmental Working Group (EWG) found tonalide and galaxolide, two manmade musks, in the cord blood of newborns [41]. Although toxic to the endocrine system, musks were discovered in most of the 17 fragrances Contribution of Fragrances to ASD 179 tested [41]. Also, during pregnancy, the use of fragrances and other cosmetics may expose the developing fetus to DEP, a common fragrance solvent that can cause abnormal development of reproductive organs in infant males, attention deficit disorder in children, and sperm damage in adults [7,29–33]. Compared with genetic studies of ASD, where DNA or RNA can be easily extracted, sequenced and analyzed, studies of environmental risk factors are very difficult to carry out. The primary reason is the lack of a suitable in vitro model that can mimic the in vivo condition. Although animal models have been used extensively, one major drawback is that rodent and other mamma lian models (i.e., wolves, meerkats, and small primates) lack the ability to com municate and convey to the observers what is happening in their brains. Furthermore, most of them have very small brain size or lack the complexity of the Homo sapiens brain to discern qualitative information. The industrial test ing services that some of the chemical industries employ generally utilize mouse models and look for toxicity by determining if the animals injected with a particular chemical(s) have died or have specific adverse effects. These are giant companies that have vast profit margins and work for the EPA or the chemical manufacturers. Some of the testing companies have been criticized and some of their evaluation methods and results are questionable, according to published data available for public review [44]. The Ames test, originally described by Ames et al. in 1973 [45], is a well‐ established and widely applied biological assay to assess the mutagenic poten tial of chemical compounds. A positive test indicates that the chemical is mutagenic; therefore, it may act as a carcinogen, since cancer is often linked to mutations [3]. The test serves as a convenient screening assay to evaluate the carcinogenic potential of a compound; standard carcinogen assays on mice or in vitro tests are time‐consuming and expensive. The test utilizes specific strains of Salmonella typhimurium that carry mutations in genes involved in histidine synthesis. This strain is called an auxotrophic mutant and requires histidine for growth and is unable to produce histidine. Histidine is an amino acid. The method tests the capability of mutagenic agents to create mutations that can result in a reversion back to a nonauxotrophic state, which permits cells to grow in a histidine‐free medium (meaning that this strain now can make its own histadine). In order to study the mutagenic mutagenic/carcino genic potential of perfumes, one can mix 1 μl of the bacteria in an agar tube containing 1.5 ml of agar at 46 °C with 1 μl of histidine (final dilution 1:15,000) in duplicates. This mixture is pipetted on a plate and allowed to culture at 37 °C for 48 h. The small amount of histidine added in the growth medium allows the bacteria to grow for an initial time, and have the opportunity to mutate. With histidine depletion, only those bacteria that have an “induced gain‐of‐ function mutation” that allows them to produce their own histidine will survive. The mutagenicity of a substance is proportional to the number of colo nies produced on an agar plate. 180 Autism and Exposure to Environmental Chemicals

We have searched for an affordable and realistic technology that would be as valid as the Ames test that can detect mutagenic and carcinogenic chemicals with a reasonable degree of certainty [45]. As shown below, we have developed an assay that can estimate the neuromodifying effects of a chemical(s) that can damage a developing fetus. We have evaluated neuroblastoma cells that, in our opinion, represent vari ous stages of developing fetal brain progenitor neurons and react to various environmental insults very similar to what one would expect for a fetal brain exposed to an exogenous chemical. As mentioned previously, we are able to identify a large array of neuronal progenitor cells that are present in different NBCs. For example, we carried out single‐cell cloning of two NBCs – one from each gender – that we screened to determine if each of these cell lines carried large repertoires of human fetal brain progenitor cells. In the single‐cell clon ing method, each cell from the cell line was isolated and then allowed to grow into an individual cell line (Figure 7.1). By utilizing this single‐cell cloning method, we were able to grow and char acterize numerous cell lines to determine if each of them represents a unique type of fetal brain progenitor cell. An example is shown below where three different single‐cell clones were characterized by immunological markers. In addition, the cell lines were exposed to retinoic acid to induce differentiation. As shown in Figure 7.1, each of the cell lines developed by single‐cell cloning represents a unique progenitor cell with a specific array of immunological markers and differentiation. In addition, this in vitro model may also be able to assist us in determining gen der bias. Just as the Ames test can be used to screen for mutagenic/carcinogenic agents, we believe that this model can be used to screen for neuromodifying chemicals. This technology could also be adapted for high throughput screening of neurotoxic agents. We believe that this is an excellent in vitro model to study fetal progenitor cells. By utilizing this model, one can test the precise amount of a chemical by exposing the neuronal cells to even a fentomolar concentration of putative neurotoxic agent, thereby exposing them to similar amounts of synthetic or natural chemicals that would be found in a fetus’s amniotic fluid at various stages of gestation. Our approach is highly appropriate to study gender bias, pre cise exposure measurement, and reliable timing of exposure in relation to critical developmental periods. It should also take into account the dynamic interplay between genes and the environment by using genetic analyses, receptor expression dynamics, and gene and environmental factor interactions [1–6,18,20] (Figure 7.2). In addition to neuroblastoma cells having a similar morphology to those in an ASD brain when exposed to synthetic chemicals, they also appear to exhibit a similar immunological profile, where oxytocin‐ and arginine vasopressin (AVP)‐receptor positive neurons are reduced and axons are significantly affected. Such an outcome was observed in our recent study where all five male NBCs exposed to fentomolar concentrations of fragrances exhibited Contribution of Fragrances to ASD 181

(a)

(b) 1 234 5 6 7 8 9 A B C D E Each of the single-cell clones NBC cell line containing Single cells were seeded into were expanded and the unknown numbers of each well of 96-well plate and clones were characterized progenitor cells single cells were allowed to grow with monoclonal antibodies until each single-cell clone reach to brain specific antigens confluency. The single-cell clones were then transferred into larger flask and allowed to grow until 90% confluency Screened with monoclonal antibodies

Growth pattern of a single-cell clone from a single cell into a larger cluster of cells from day 0 to day 8

Figure 7.1 (a) Various kinds of neuronal progenitor cells. Each color represents a unique progenitor cell in weeks 4–8 fetal brain, destined to become one of the compartments of an adult brain. Elimination of one of the cell types may result in complete or partial absence of a future compartment of a brain. Depletion of a single cell at an early developmental stage can result in a cognitive deficit. (b) The process of single‐cell cloning. The neuroblastoma cell line is cultured in a specialized cell culture flask, and the cells are then trypsinized to separate each of the cells (each representing a unique fetal brain progenitor cell). The individual cells are seeded into a 96‐well plate until they grow in a cellular cluster. Each of the clusters is then transferred into a new culture flask. Each clone is then characterized by immunostaining (c). (c) The visual microscopic presentation of a single-cell clone that grows into a large cluster-from day 0 to day 8. (See insert for color representation of this figure.) 182 Autism and Exposure to Environmental Chemicals

D3 D6 F11 D3 retinoic D6 retinoic F11 retinoic control acid control acid control acid

Synapto- physin

CD56

Oxytocin receptor

Chromo -granin A

Figure 7.2 An example of three clones developed by the single‐cell cloning method from a female neuroblastoma cell line (i.e., CRL‐2266). Each of the clones was characterized by using an array of antibody markers (four of which are shown here) that showed that each single cell clone represents a unique progenitor neuron. In addition, each single‐cell clone responded differently to retinoic acid.

significant down‐regulation in oxytocin‐receptor positive neurons, reducing the ability of the hormone oxytocin to bind effectively, when a child is born, to certain parts of the brain that are essential for social interaction in humans. It should be noted that, in fentomolar concentrations, when a pregnant woman is exposed to a particular perfume, the perfume chemicals penetrate the blood circulation via the lung (breathing) or through the woman’s skin (absorption). These chemicals are diluted in about 6 liters of the woman’s blood/fluid vol ume and subsequently make their way to the tiny fetal brain. We believe that only a few molecules make their way to the brain of the fetus and kill only a few brain progenitor cells (hence the fentomolar concentrations used). However, the impact of these few molecules could be enough to produce autism or ASD (see below). One might wonder how only a few molecules can damage a fetal brain. We all have encountered or know someone who has encountered an allergic reaction to a bee, hornet, or certain perfumes. In each of these clinical situations, only a few molecules trigger a cascade of events that can cause a victim to break into hives or experience obstructive swelling of the lips, tongue, and/or throat, trouble swallowing, shortness of breath or wheezing, turning blue, a drop in blood pressure (feeling faint, confused, weak, passing out), loss of consciousness, chest pain, or even death. All of these takes place due to very few molecules of bee venom or perfume. We will take up the subject of allergic reaction to fragrances later in this chapter. As we covered in Chapter 4, oxytocin is involved in various social pro cesses that appear to be absent in ASD children, suggesting low binding Contribution of Fragrances to ASD 183 efficiency of oxytocin to its respective receptor [5,6]. Interestingly, the three female fetal brain cell lines that we tested did not show substantial reduction in oxytocin‐receptor positive neurons, shedding light on the extreme male bias for ASD. Increased susceptibility of male fetal brains to synthetic chemicals that can reduce the numbers of oxytocin‐receptor positive neurons, combined with the mutagenic ability of fragrances (and perhaps other synthetic chemicals), may be the major contributors to ASD and male bias [46]. It should be noted that when scientists are investigating causes of autism and when they are looking at the etiology of ASD, they are essentially looking for mutations found in the ASD child, particularly de novo mutations, and associate these mutations with ASD symptoms. We contend that the hundreds or even thousands of mutations reported so far likely represent the secondary effects of synthetic chemicals since they are highly mutagenic and not the primary cause of ASD. As we have shown, it may be the depletion of specific progenitor neurons (i.e., oxytocin‐ and AVP‐receptor positive neurons) that are most likely responsible for the observed symptoms of ASD. Since, currently, there is no method for assess ing the number of oxytocin‐receptor positive neurons present in the ASD child, an alternate solution would be to examine other social mammals (i.e., meerkats, wolves, rabbits, gerbils, guinea pigs, etc.) exposed to fragrances and other synthetic chemicals at equivalent doses during various stages of their pregnancies. Support for the unusual vulnerability of oxytocin‐receptor positive neurons came indirectly from numerous studies that revealed that children with autism have significantly lower levels of plasma oxytocin and AVP than their typical peers [47–52]. A recent study also showed that mothers of ASD children have low levels of oxytocin and AVP, but high levels of testosterone [48]. In addition, in children without ASD, lower concentrations of oxytocin in plasma are associated with lower social and cognitive functioning [47–57]. Furthermore, AVP, oxytocin, and their respective receptors, share similar sequences in animals, allowing these peptides to activate each other’s receptors [54,58–63]. Animal and human studies have revealed sex differences in AVP and oxytocin, such that males appear to have enhanced AVP and oxytocin functioning compared with females; these differences, however, vary by brain region and species [62–64]. We hypothesize that fragrances, which are complex chemical mixtures, may have differential effects on human neural development and differentiation. To investigate these effects male and female origin NBCs have been used for an in vitro developing fetal brain model. As mentioned previously, these cell lines represent a suitable model since they possess biochemical properties of human neurons in vivo [2–6,44]. Furthermore, these cell lines are acquired from human tumors, which encourage continuous division and provide the required quantities of cells for investigations without significant changes in variability [2–5]. 184 Autism and Exposure to Environmental Chemicals

Effects of Fragrances on Male Oxytocin‐Receptor Positive Neurons

Here we find that male, but not female, fetal progenitor brain neurons exposed to femtomolar concentrations of certain fragrances exhibit signifi cant reductions in both oxytocin‐ and AVP‐receptor positive neurons (Figures 7.3 and 7.4). Interestingly, neuromodifications are observed by light microscopy in both male and female NBCs following exposure to femtomolar

Control

Fragrance A

Fragrance B

Fragrance C

Figure 7.3 Effects of fragrances on male oxytocin receptor neurons. The top row shows control cells immunostained for oxytocin male neurons at 400× magnification, and hematoxylin and eosin (H & E) stained cells at 100× and 400× magnifications (respectively, left to right). Similarly, the second, third and fourth rows show male NBCs exposed to fragrances A, B, and C at 1:1 million dilutions, respectively. As shown in the immunostained 400× pictures, exposure to all three fragrances causes significant depletions of oxytocin neurons. The H & E stained cells clearly show significant modulation of neurons in cellular proliferation (increased numbers of cell clusters), axon shape, size, length and thickness, as well as profound chromatolysis of the neurons exposed to these fragrances. Source: Adapted from Ref. [4]. Effects of Fragrances on Male Oxytocin‐Receptor Positive Neurons 185

Control

Fragrance A

Fragrance B

Fragrance C

Figure 7.4 Effects of fragrances on female oxytocin‐receptor positive neurons: female NBC control neurons showed oval‐shaped neurons with axons that normally extended ~2× the length of the average neuron size in this cell line. The immunostaining showed that ~35% of the female NBCs were oxytocin‐receptor positive neurons. Female NBCs exposed to fragrances exhibited increased expression of oxytocin‐receptor positive neurons, notable modulation of neurons, increased areas of cell clustering, increases in cell size to 2× or more, and increases in axonal length to 3–6×, as compared with the controls. In addition, the immunostaining showed stippling, suggesting membrane antigen redistribution and modulation. Source: Adapted from Ref. [4].

concentrations of certain fragrances, but this is more pronounced in male fetal progenitor brain neurons. Our studies are the first to reveal a direct connection between fragrance exposure and neuromodifications that may contribute to autism and provide insight into gender bias. As shown in Figure 7.3, male NBCs exposed to extremely small concentrations of fra grances (fragrances A, B, and C) have profound neuromodifying effects. Most interesting among these findings was the depletion or significant reduction in oxytocin‐ and AVP‐receptor positive neurons in male, but not female, cell lines. 186 Autism and Exposure to Environmental Chemicals

Effects of Fragrances on Female Oxytocin‐Receptor Positive Neurons

Female NBC exposed to the same amounts of fragrances as male NBCs did not show depletion of oxytocin‐ or AVP‐receptor positive neurons. In the previous chapter, we explained in detail the importance of oxytocin‐ and AVP‐receptor positive neurons and their interaction with the oxytocin and AVP neuropep tides and their central role in social communications and interaction.

How Synthetic Chemicals in Fragrances Affect Fetal Brain Development

Over 4,000 synthetic chemicals are found in some fragrances [21]. This is one of the most rapidly developing industries and is highly competitive [65] and the current market value, according to Statistica, is estimated to be US$40.1 billion and will continue to increase [65]. Organic chemists around the world are con stantly vying to bring new fragrances to the consumer market. The way fra grances are being synthetized, marketed and then mass produced is vividly depicted in Luca Turin’s autobiographic account entitled The Secret of Scent: Adventures in Perfume and the Science of Smell [66] (Table 7.2). It would take an army of neuroscientists to investigate the potential adverse effects of each of the chemicals found in the most commonly sold perfumes [21,65,66]. Here we present preliminary evidence of potential adverse effects from just a few chemicals. We selected eight chemicals to evaluate what kind of effects they may have on fetal brain neurons if a fetus is exposed to even a minuscule amount. Keep in mind that we have exposed the fetal brain neurons to each of these chemicals separately. Imagine what would happen to the devel oping brain when many neurotoxic chemicals reach the brain simultaneously. The chemicals we used for our preliminary test were: DEP, tonalide (musk ketone), octinoxate, d‐limonene, eugenol, benzyl benzoate, benzyl salicylate, and (+)‐α‐pinene.

Synthetic Musks

Synthetic musks are common chemicals used in fragrances and other cosmet ics. Synthetic musks are also referred to as white musks in the perfume industry. To the best of our knowledge, the first artificial musk was synthetized by Albert Baur in 1888 by condensing toluene and isobutyl bromide in the pres ence of aluminum chloride [21]. This was an accidental discovery when Baur was trying to make a TNT explosive bomb. Of note, all of these chemicals are Table 7.2 Chemical structures and adverse effects of the eight chemicals selected.

Chemical name Structure Function (effect)

Tonalide H3C CH3 Affects estrogen, H C 3 CH3 androgen receptors

CH3

H3C CH3 O

Octinoxate (octyl O Estrogen endocrine methoxycinnamate disruptor such that O CH3 H it binds to estrogen CH3 receptors in human H3CO cells; linked to being toxic and disruptive to the thyroid endocrine pathway in animals such as rats

Eugenol H3CO Antiestrogenic activity; has been HO deemed a causative allergen in fragrances Diethyl phthalate O Affects hormonal balance; cancer O CH3 causing agent (e.g., O CH3 breast cancer)

d‐limonene CH3 Can affect those with asthma; linked to being a contact allergen C H3C CH2

Benzyl benzoate O Estrogenic activity in a study with O human breast cancer cells Benzyl salicylate O Estrogenic activity in a study with O human breast OH cancer cells

(+)‐α‐Pinene Major respiratory irritant 188 Autism and Exposure to Environmental Chemicals

toxic to humans and other biological life forms. They emulate the scent of deer musk or ox musk, but chemically they are not like the natural musk. Synthetic musks have a clean, smooth, and sweet scent lacking the animalic notes of natural musks. These compounds are an essential part of modern perfumery and form the base note foundations of most perfume formulas. Currently, almost all musk fragrances used in perfumery today are synthetic. There are three different types of synthetic musks: aromatic (a nitro musk with a similar structure to TNT explosive), polycyclic, and macrocyclic rings – all chemicals known to be highly toxic, especially to fetal brain neurons. Musk xylene and musk ketone are nitro musks. Nitro musks were a main ingredient in many perfumes and other consumer products, however, concerns raised regarding their bioaccumulation, toxicity to various life forms, and persistence in the environment has led to reduced production in EU nations [67]. For example, between 1995 and 2000, the total use of nitro musks amounted to 300 t (tons), but then declined to 200 t [68]. On the other hand, the production of the poly cyclic musks galaxolide and tonalide has increased significantly and they are the mostly widely used today [69,70]. In fact, over 4,000 t of polycyclic musk was sold in 2005 [71]. Synthetic musks are rarely listed on the label of products or tested for safety, including testing for effects on unborn fetuses or on rapidly developing off spring, since they are exempt according to the Federal Fair Packaging and Labelling Act of 1973 [42]. Synthetic musks are found in many common house hold detergents, soaps, perfumes, shampoos, air fresheners, and cosmetics (reviewed in Refs [67, 68]). Originally introduced as cheap substitutes for natu ral musk fragrances obtained from the musk deer and musk ox, synthetic musk fragrance consumption leapt in the 20th century and was estimated to be 8,000 t/year in 1996 [67]. In Europe in 1995 the combined usage rate of the two most commonly used synthetic musk compounds, HHCB (1,3,4,6,7,8‐ hexahydro‐4,6,6,7,8,8‐hexamethylcyclopenta‐γ‐2‐benzopyran) and AHTN (7‐acetyl‐1,1,3,4,4,6‐hexamethyl‐1,2,3,4‐tetrahydronaphthalene), was about 15.5 mg/day [69]. Additionally, the US EPA lists HHCB as a high production volume chemical (more than 1 million pounds produced or imported into the USA each year). Synthetic musks accumulate in the environment (i.e., in waste water), fish, shrimp, and bioaccumulate inside humans (e.g. in body fat, blood, human breast milk, and umbilical cords), similar to PCBs and organochlorine pesticides [72–76]. It is well documented that synthetic musks can disrupt human endocrine systems (hormonal functions and other physiological pro cesses) [72–76]. They are highly ubiquitous in our daily life and are commonly found in perfumes, colognes, soaps, body wash, sprays, lotions, hair products, detergents, softeners, air fresheners, and in the environment. It is likely that they may be present in some children’s medicines to make them more palata ble. They are listed as “fragrance,” phantolide, celestolide, transeolide, cash meran, musk ketone, musk xylene, galaxolide, tonalide, and so on. Synthetic Musks 189

In recent years, bioaccumulation of various synthetic musks has increased to the point that they are now considered to be major emerging pollutants by environmental biologists around the globe, due to their long‐term persistence in the environment and hazardous potential to ecosystems, even at fentomolar concentrations. This is consistent with increased US use of fragrance chemi cals in consumer products [73]. The synthetic musks are widespread environ mental contaminants, particularly in freshwater and marine ecosystems [72–76]. They have been measured in rivers, lakes, sediment, soil, sewage sludge, and effluent from wastewater treatment plants in Canada, the USA, the UK, and elsewhere in Europe [67]. Synthetic musk compounds have also been detected in outdoor air [67] and indoor air due to their use in numerous con sumer products. Studies in Europe, North America, and Japan have reported bioaccumulation of synthetic musks in a wide range of aquatic wildlife species including cetaceans, sharks, fish, crustaceans, and shellfish [69]. Nitromusks and polycylic musks have also been found in human blood, adipose (fat) tissue, and breast milk [67–70]. Many of the polycyclic and nitro musks are listed on the US EPA’s Toxic Substances Control Act (TSCA) Inventory (TSCA, 2003). The use of musk xylene and musk ketone in cosmetics is now restricted under the EU Cosmetics Directive, due to their tendency to build up in the environment.

How Do Synthetic Musks Get Into the Food Chain?

Widespread use of synthetic musks in consumer products results in large amounts of these chemicals being washed down the drain after being applied to skin, hair, and clothing. These chemicals enter water drainage, sewage, water treatment plants, and wastewater systems, and then find their way into the aquatic environment [67–72}. Once in the aquatic environment, synthetic musks can enter the food chain, being taken up by wildlife such as fish and shellfish. Following wastewater treatment, sewage sludge is digested via a reme diation process, dewatered, and then the resulting solid waste is commonly disseminated onto agricultural land. Synthetic musks in the sludge that have not been degraded during digestion are therefore introduced to the terrestrial eco system via the soil [75]. Most alarming is that because of their use in so many consumer products, musks can also escape into air and dust, potentially enter ing a pregnant woman’s system or a young child’s body during critical times of brain development [2,3,75]. A simple wastewater system is shown in Figure 7.5.

How Are People Exposed to Synthetic Musks?

The main exposure route for most of the synthetic chemicals found in per fumes, including musks, is through the skin (dermal absorption) as they are applied directly onto the skin [67,76]. Absorption through the skin is also Industry trade Domestic sewage

TIMEX micro drum filter Percolating water

TIMEX SVF Sand automatic filter Preliminary Activating Final Rake catcher sedimentation basin sedimentation

Oversized Return activated sludge material Sand

Sludge treatment TIMEX KMF filter Disposal

Effluent To draining canal

Figure 7.5 A schematic illustration of a wastewater treatment system. Source: Adapted from https://in.pinterest.com/ pin/527484175084049726/. Diethyl Phthalate 191 important route from fragrances left on clothes from laundry detergents. Another major route is inhalation. Fragrances detected in air, which by their very nature are volatile chemicals that can evaporate and reach the nose so that the olfactory system can recognize them, easily enter through the lungs to the blood stream [76].

Musks in Food

Exposure to synthetic musks can also occur through flavored drinks and food. Synthetic flavors are created by highly specialized food flavor experts and are increasingly being introduced into our daily diet and drinks. Flavored drinks have gained enormous popularity among fast food chains, including various so called fruit drinks in the form of water, ice, shakes, and soft drinks. No real information is available on the nature of these chemicals. Most information regarding synthetic musk residues is on fish and shellfish species as the fresh water and marine ecosystems receive the greatest inputs of these compounds (from sewage and wasterwater discharges) [75]. Synthetic musks have endocrine disrupting properties – that is, they are capable of interacting with the normal functioning of human hormonal sys tems. Both tonalide and galaxolide can bind to ERs (estrogen receptors) in cells and can be both estrogenic and anti‐estrogenic, depending on the type of cell and the type of ER involved [76]. Several of the musks have been shown to mimic the action of the hormone estrogen in experiments with human cell lines [67]. In in vitro tests, musk xylene, musk ketone, a major metabolite of musk xylene (p‐amino‐musk xylene), and the polycyclic musk fragrance tonalide have been shown to increase the proliferation rate of human MCF‐7 breast cancer cells, demonstrating their ability to mimic estrogen [67,76]. Although a causal relationship has not been established, higher levels of musk xylene and musk ketone concentrations in women’s blood have been correlated to higher rates of miscarriage [67,68]. Exposure to tonalide also has been shown to cause acute liver damage in rodents. As we have shown in Chapter 1, many perfumes are highly mutagenic [75,76].

Diethyl Phthalate

Phthalates and phthalate esters are a large group of compounds used as liquid plasticizers and are found in a wide range of products of household items including plastics, coatings, perfumes, personal care products, makeup, cos metics, and medical tubing. These compounds have been long‐standing ingre dients in plastic industry products and were first introduced as additives in the1920s and were in widespread use in poly(vinyl chloride) (PVC) in the 1930s and later (reviewed in Ref. [7]). Phthalates are not chemically bound to the 192 Autism and Exposure to Environmental Chemicals

plastic and can readily leach into the environment. Furthermore, a large number of consumer products use various phthalates, and in fact, phthalate contamination has been found in a large variety of foods, including sports drinks, fruit beverages, tea drinks, fruit jam or jelly, and health foods or supple ments in powder or tablet form, ice cream, frozen food, and cake mixes [77]. Phthalates are detectable in human urine, serum, and breast milk [78–80], and the estimated daily exposure to one major phthalate, di(2‐ethylhexyl) phthalate (DEHP), ranges from 3 to 30 μg/kg/day [81]. When metabolized by the liver, DEP is a phthalate ester and is a solvent for many organic compounds. It is most commonly found in fragrance, cos metics, and perfumes. Numerous well‐documented studies suggest that DEP can cause damage to the nervous system as well as to the reproductive organs in males and females [82–84]. DEP is reduced and excreted in the urine as monoethyl phthalate (MEP; Figure 7.6). DEP is hydrolyzed to the monoester, monoethyl phthalate, and ethanol after oral administration in the lumen of the gastrointestinal tract or in the intestinal mucosal cells. Hydrolysis of DEP also takes place in the kidney and liver after systemic absorption. After tissue distribution throughout the body, DEP accumulates in the liver and kidney. The metabolites are excreted in the urine. DEP is metabolized by carboxyl esterase, which is synthesized in the human liver. In vitro studies show that DEP reduces the glucuronyl transferase activity. It was also observed that the activity of peroxisomal enzyme carnitine acetyl transferase is increased in cultures of rat liver cells [82–84]. Furthermore, DEP induces the enzyme activity of catalase, which leads to hepatic peroxi some proliferation and possibly causes hyperplasia. Teratogenic effects of DEP have also been reported [85].

O H O O R R group O OH hydroxylation Phase II COOH O O OH O O R H O Phase I O Phase II O OH O O R Biotransformation R Biotransformation O OH R O O O Diester phthalate Monoester phthalate Phthalate glucuronide R group Phase II oxylation O H O O R O O

Figure 7.6 Metabolic pathways of phthalates. Octinoxate 193

MEP can be detected in urine and can be used as a biomarker for exposure to DEP [79]. Major concerns of the damaging effects of being exposed to DEP are mainly geared towards pregnant women and their unborn fetus that were exposed to harmful phthalates such as DEP during the gestation period. Studies have shown that the chemical DEP acts as an endocrine disruptor and mimics the role of estrogen and androgen hormones in females and males by binding to estrogen and androgen receptors in the exposed fetuses [84–90]. This dis turbs the fine hormonal balance in a developing fetal body. Furthermore, DEP is mutagenic and has been linked to breast cancer in women. Our study shows that DEP can be linked to autism by inducing neuromodifications in fetal brain cells. Furthermore, children that have a prolonged exposure to phthalates exhibit social communication impairments that are a hallmark symptom that children with autism express. Consumers come into contact with various chemicals through air, food, water, plasticizers, synthetic musks (i.e., tonalide) and other various modes of inadvertent exposures. In a study conducted in Paris, France, an analytical model was developed to detect various amounts of endocrine disrupting chemicals (EDCs) in indoor air environments. The model had the ability to detect EDCs in two atmospheric phases, including gas and particulate phases. The results from the model showed that phthalates, because of their high volatile characteristics, were one of the most abundant indoor contaminants and were mostly found in the gaseous phase at a varying concen tration of 8.39–1.246 ng/mg3. In addition, musk ketones were the second most detected EDCs in the indoor air environment, which had a concentration of 30 ng/m3. The results showed that these chemicals are found abundantly in the indoor air environment of homes. If the indoor air quality is contaminated with these chemicals, the inhabitants of these homes (especially young chil dren and pregnant women with developing fetuses) may be affected by these chemicals [85–90].

Octinoxate

Octinoxates are colorless to light yellow liquids and a main chemical ingredient in sunscreen, shampoos, and skin creams, nail polishes, and so on. Octinoxate has been linked to being a possible endocrine disruptor [88]. It has also been linked to breast cancer and affects the development of reproductive organs [7,21]. For example, studies carried out at The Technical University of Denmark exposed octinoxate to unborn fetuses of pregnant rats at various concentra tions during the gestation and lactation period [91]. The offspring of rats that were exposed to the various levels of octinoxate were observed for effects during pre‐ and postnatal stages, up to 8 months of age [91]. The results of the study revealed reduced weight of the testes and prostate glands in high‐dose‐ exposed male rats. Decreased motor abilities were observed in female rats 194 Autism and Exposure to Environmental Chemicals

exposed to octinoxate. In addition, in the age range up to 8 months, the male offspring showed a drastic decrease in sperm count and weight of the testes. Of note, exposure to octinoxate yielded different effects in males and females [91] (Table 7.2).

Benzyl Benzoate and Benzyl Salicylate

Benzyl benzoate and benzyl salicylate are both chemical ingredients that are found in various personal care products such as fragrances, cosmetics, after shave, hair color, air fresheners, moisturizers, and so on. Benzyl benzoate, also known as ascabiol or ascabin, is a benzyl ester (Figure 7.6). The physical state of benzyl benzoate is usually a clear colorless liquid. The odor of benzyl benzo ate is usually a faint yet sweet balsamic‐like odor. Benzyl salicylate, also known as salicylic acid, is a benzyl ester. Salicylic acid has a clear liquid appearance. The odor of benzyl salicylic acid is mildly sweet and slightly balsamic. Certain studies have investigated the effects of benzyl benzoate and benzyl salicylate to find the chemicals that may be estrogenic endocrine disruptors and possibly induce breast cancer in women [92]. Studies conducted at the Perinatal Centre, Institute for the Health of Women and Children, Sahlgrenska Academy, Gothenburg, Sweden found the extent of cell proliferation in MCF7 breast cancer cell lines was correlated with certain levels of exposure to benzyl benzoate or benzyl salicylate over a period of 7–35 days [92] (Table 7.2).

d‐Limonene

d‐limonene is a chemical that is predominantly used in fragrances and is used as an additive for a lemon‐like flavor. The d‐limonene chemical is derived from citrus peels. Common consumer goods that have synthetic d‐Limonene as an additive include fragrances, soaps, automobile tire cleaner, cleaning products (degreasers), shampoo, beverages (such as orange juice), and chewing gum. The chemical structure of d‐limonene is that of a monocyclic monoterpene. Limonene is a colorless liquid hydrocarbon classified as a cyclic terpene. The more common D‐isomer possesses a strong smell of oranges. Limonene takes its name from the lemon, since the rind of the lemon, like other citrus fruits, contains considerable amounts of this compound, which contributes to its distinct odor. Currently, widespread use of limonene is due to its ease of bio synthesis. These are not natural chemicals, but are synthesized chemically on a mass scale (Table 7.2). Dietary intake from food ingredients is an alternative route of exposure; thus, limonene is found as a flavoring compound in tea resulting in both dietary and inhalative exposure [93]. Also, secondary reactions during construction produce many odors. Monoterpenes with α‐pinene are a primary example emitted from pine and Synthetic EDCs 195 spruce construction products and can also be inhaled during forest walking (e.g., from pine wood trees), and accumulated in plasma [94]. Limonene belongs to the terpene family. Terpenes are one of the largest groups of volatile organic compounds (VOCs) that are widely used to make us feel good in our work environments. Terpenes are also used as solvents and in paints. Terpenes constitute ~13% of the total VOC concentration in pine wood flooring material. These chemicals are highly reactive with air and create ozone. Some components in air fresheners combine with ozone and produce strong toxins such as oxidative products, secondary aerosols, and formalde hyde, all of which can damage a developing fetal brain when a pregnant woman is breathing these chemicals. Limonene causes respiratory disease as well as damage to the liver, kidney, and nervous system [95].

α‐Pinene

α‐Pinene, an alkene that has a reactive four‐membered ring, is a chemical that was originally derived from natural oil from pine trees. Like limonene, it is also a terpene, and is chemically synthesized. A clear liquid with a sweet piney odor, α‐pinene is the main ingredient and starting material in making aroma‐like chemicals such as terpineol. In addition, α‐pinene is also an ingredient in various consumer goods such as fragrances, soaps, and disin fectants. Presence of α‐pinene creates a reaction in the air and the combina tion of ozone and α‐pinene produces formaldehyde, acetone, and picric acid, three air pollutants. Serious pulmonary effects of α‐pinene byproducts have been reported [96]. There are no reports on the potential adverse effects of α‐pinene on fetal brain cells.

Synthetic EDCs

Both male and female hormone disruptive chemicals are known to impart adverse health effects through interaction with the endocrine and central nerv ous systems (summarized in Table 7.1). The US EPA defines EDCs as exoge nous agents that interfere with normal physiological or biochemical functions of human beings. These include (but are not limited to) to synthesis, secretion, metabolism, transport, binding action, detoxification of natural blood‐borne hormones that are present in the body and are responsible for reproduction, and development and homeostasis of human bodily functions. These chemi cals can accumulate and persist in various parts of the human body and may or may not cause clinically observable effects (reviewed in Refs [7,96]). We will concentrate on synthetic chemicals that may change the natural levels of sex hormones in the fetal amniotic fluid during gestation. Exposure of 196 Autism and Exposure to Environmental Chemicals

the fetuses to any abnormal amount of sex hormones may interfere with normal development. As an example, we show below the normal parameters of testos terone or estrogens in a developing male and female from developing fetus to childhood. There is a long history of research investigating the effects of EDCs in male reproductive health in humans and other lifeforms [7]. One of the best‐studied endocrine based examples is exposure to a potent synthetic nonsteroidal estro gen, diethylstilbestrol (DES), taken during pregnancy by women from the 1940s to 1975 to prevent miscarriage and other complications. DES is an estro genic chemical that binds with high affinity to the ERs, ERα and ERβ, which play an important role in adiposity regulation as well as central and peripheral energy balance [97]. DES was prescribed at doses from <100 up to 47,000 mg, depending on the stage of pregnancy (the higher the pregnancy stage, the higher the dose). Its use was discontinued when a subset of exposed daughters presented with early‐onset vaginal clear‐cell adenocarcinoma [98]. A 40‐fold increase in risk was found compared with unexposed individuals [99]. Eventually, it was determined that the DES‐exposed offspring of both sexes had increased risk for multiple reproductive disorders, including cryptorchidism in male offspring, and numerous other diseases [100]. Furthermore, emerging data suggest that there is significantly increased disease risk in grandchildren [101]. Of note, the DES scenario first suggested the possibility that synthetic estrogens or other potentially environmental estrogenic synthetic chemicals can affect not only female but also male reproductive health. This discovery prompted a broad evaluation on how hormone exposure affected human health, eventually leading to a better understanding of how EDCs, especially those that are anti‐androgenic chemicals, may play key roles in the develop ment and maintenance of male health. Therefore, not surprisingly, a plethora of examples is emerging for increased disease susceptibility later in life as a function of developmental exposures to EDCs that include bisphenol A (BPA), phthalates, PCBs, pesticides, dioxins, and tributyltin (TBT), and thousands of other synthetic chemicals that are being introduced almost every day and whose effects on the human body are still unexplored. Among the most serious ones are found in fragrances that can reach human fetuses without anyone suspecting their adverse effects (reviewed in Refs [7,53]).

Why Is It Important To Look at EDCs and Their Potential Effects on Our Next Generations?

The endocrine glands are distributed throughout the body and produce the hormones that act as signaling molecules after release into the circulatory system. As shown in Figures 7.7 and 7.8, development, physiological processes, and homeostatic functions are regulated and maintained by hormones. The hormonal regulation and maintenance is conducted like a giant orchestra Fine brittle hair Bulging eyes Healthy Hyperthyroidism and hair loss

Increased Enlarged thyroid perspiration Bulging eyes

Enlarged liver Abnormal heart rhythms Goiter

Nausea Increased appetite vomiting diarrhea Enlarged Loss of libido Normal thyroid thyroid Hand tremors amenorrhea

Irritability Excessive production of thyroid hormones Hyperactivity High blood sugar

T3 T4 Intolerance to heat Low serum cholesterol

Figure 7.7 Symptoms of hyperthyroidism. Reproduced with permission of Top 10 Home Remedies, Natural Therapeutics Health Centre Inc. 198 Autism and Exposure to Environmental Chemicals

Thinning hair Loss of eyebrow hair hair loss

Enlarged thyroid Puffy face

Dry and Slow heartbeat coarse skin

Poor appetite

Constipation Infertility heavy menstruation Cool extremities and Carpal tunnel swelling of the limbs syndrome

Weight gain The thyroid gland does not produce Poor memory enough thyroid Intolerance to cold hormone Feeling of tiredness

Figure 7.8 Symptoms of hypothyroidism. Source: https://www.massagemag.com/ massage‐therapy‐thyroid‐health‐34099/. Reproduced with permission of Natural Therapeutics Health Centre Inc.

where a finite number of hormonal interactions creates an infinite music of life. The bindings of natural hormones to their respective receptors are critical for both health and disease. Interference by EDCs can disturb a fine balance of processes of life and if a fetus is exposed to EDCs they may cause problems in brain development, cellular differentiation, organogenesis and perturb numer ous molecular and physiological processes, including regulation of gene and protein expression, exocytosis of vesicles containing peptide or protein hor mones, metabolism and steroidogenesis of lipophilic hormones, transport through circulation (often in association with binding partners), actions at Where in Fetal Life Are Androgen Receptors Expressed? 199 target receptors, metabolism, degradation, excretion, and many others. The natural homeostasis of hormonal levels during a fetal life involves the ability of the specific glands throughout prenatal development to precisely regulate the hormonal levels by interactions with their natural targets. These interactions, most often by negative feedback of a hormone through its receptor in the original target brain and the rest of the fetal body, determine the best outcome for a normotypic offspring. The finely tuned interplay keeps the levels of any hormone within a physiologically relevant range to be most effective. Excursions outside of that range to elevated or depressed levels for any extended period can result in dysfunction in brain development (along with other organs and the rest of the fetal body). A prime example of an unbalanced hormone and its adverse outcome is the thyroid system: normal levels of the thyroid hormones are needed for appropriate metabolic health. Hyperthyroidism is associated with a range of symptoms due to elevated metabolism, whereas hypothyroidism, with a very different disease phenotype, results from depressed hormone levels (Figures 7.7 and 7.8).

Where in Fetal Life Are Androgen Receptors Expressed?

Androgen receptors (ARs) belong to the super family of ligand‐responsive transcription regulators, which includes retinoic acid receptors, thyroid hor mone receptors, and several steroid hormone receptors. Androgens have a great variety of effects on many target tissues [102,103]. They induce the devel opment and physiological function of male accessory sex organs, such as the prostate and seminal vesicles, and later in life they control the functional activ ity of target tissues. Androgen action in these organs and tissues is believed to be mediated by ARs. Using immunohistochemical techniques, ARs have been clearly demonstrated in nearly all human adult tissues [3], and in fetal tissues from second and third trimester gestation, suggesting that ARs are involved in early development [4], Figure 5.2. Analyses of AR expression in a variety of extra‐ genital tissues during weeks 8–12 of gestation revealed expression in the thymus gland, bronchial epithelium of the lungs, and in cardiac valves. AR expression was absent in the kidneys, adrenal glands, liver, and bowel. The study suggested an important role of AR in organogenesis. Of note, the thymus is a primary immune organ and an essential part of the adaptive immune defense system. In human fetuses, the first lymphocytes in the human thymus primordium appear around 8 weeks of gestation. In the thymus, lymphocytes begin to proliferate in the epithelial stroma, under the influence of various thymic hormones that induce stimulation, proliferation, and differentiation, and become competent T lymphocytes by weeks 14–15 of gestation. This is the period during which thymocytes go through a process called “clonal selection” and any thymocyte that can recognize self‐antigen is destroyed. A major implication of these 200 Autism and Exposure to Environmental Chemicals

observations is that androgenic steroids might be capable of exerting immuno‐ modulatory effects on the maturing lymphocytes within the thymic environ ment and, upon maturing, the cells might no longer express the ARs. It also suggests that if an EDC interferes with this special process, it may contribute to development of autoimmunity, such as rheumatoid arthritis, lupus, celiac disease, Sjogren’s syndrome, multiple sclerosis, type 1 diabetes, vasculitis, and many other diseases [104].

Why Testosterone is Essential for Engineering a Male Brain

As shown in Figures 7.9 and 5.2, testosterone is an essential part of the normal development of male gonads that begins early in embryogenesis with the differentiation of the testis from the bipotential gonad. The internal sex organs arise as a consequence of the maintenance of the mesonephric ducts (or Wolffian ducts) and the regression of the paramesonephric (or Müllerian ducts) due to the testicular hormones testosterone and anti‐Müllerian hor mone, respectively. The development of external genitalia, the scrotum, and testicular descent are under the control of several genetic and hormonal path ways. Testosterone and its metabolite dihydrotestosterone drive the masculini zation of the external genitalia. Testicular insulin‐like peptide 3 is also necessary for testicular descent. Timing of the hormone action is important. In humans, normal androgen (testosterone) action during gestational weeks 8–15 is criti cal for normal development of the genitalia (penile malformations arise during this period), whereas development of sperm production capacity occurs over a much broader time window that continues to puberty (Figure 7.10). Interference of endocrine disruptors on the timing of male fetal develop ment, especially brain development, can wreak havoc on the engineering of the fetal male brain. The female reproductive system, even though much complex and requires a proper structure and function for many organs, includ ing the ovary, uterus, vagina, and anterior pituitary, does not require testoster one during the early fetal life as shown in Figure 5.2. But, still, EDCs have the potential to interfere with female reproduction by adversely affecting the structure and/or function of female reproductive organs. The male brain is engineered entirely differently than its female counterpart and high levels of testosterone during weeks 8–24 of fetal development attests to this unique engineering of the male brain. The majority of our information regarding the differences between male and female brain structures has been obtained from rodent animal models of fetal development. New information is emerging from sheep and other larger size mammals. So far, the research shows that exposure of the vertebrate brain to testosterone during a critical period of fetal development establishes perma nent sex differences in reproductive physiology as well as a broad spectrum of Bodily systems affected by testosterone

Brain/Focus Testosterone levels can affect mood, mental focus, memory and libido. Hypothalamus

Muscle mass Helps build muscle GnRH strength and endurance.

Kidney Anterior pituitary Stimulation of erythropoietin.

(essential hormone for red cell production) k

ACTH c a

Lean body mass b Adrenal glands d Testosterone improves body e e Gn f

composition. e

v i t

a g e Bone N Low levels of testosterone can affect Testes bone density and can increase risk of osteoporosis.

Testosterone

*Source: “Testosterone: A Man’s Guide” Second Edition by Nelson Vergel (2011)

Figure 7.9 Wide‐ranging effects of testosterone on the human male body. Reproduced with permission of Nelson Vergel. 202 Autism and Exposure to Environmental Chemicals

Testosterone action

Bioavailable Bound

Free Albumin SHBG (0.5 – 3.0%) (50 – 70%) (30 – 45%)

Excretion (90%) Testosterone (5 mg/d)

5α-Reductase Aromatase (6–8%) (0.3%)

Dihydrotestosterone(DHT) Testosterone Estradiol

External genitalia ∙ ∙ Wolffian duct ∙ Hypothalamic/ ∙ Prostate growth ∙ Bone formation pituitary feedback ∙ Acne ∙ Muscle mass ∙ Bone resorption ∙ Facial/body hair ∙ Spermatogenesis ∙ Epiphyseal closure ∙ Scalp hair loss ∙ Gynecomastia ∙ Some vascular and behavioral effects

Figure 7.10 The target organs and action sites of testosterone. Source: Adapted from https://www.slideshare.net/KAPILVASANTH/hypogonadism‐final.

behaviors and cognitive functions [104,105]. Testosterone and estrogen are also responsible for sexual dimorphisms in brain regions that regulate these functions [106,107]. For example, sexual dimorphisms in the volume of the medial preoptic area (MPOA) occur in several species, in particular the central region of the medial preoptic nucleus, which has been named the sexually dimorphic nucleus of the preoptic area in rats where it was first described [108,109]. The dimorphic nucleus of the preoptic area of males is larger in Why Testosterone is Essential for Engineering a Male Brain 203

Mullerian duct Genital ridge Mesonephros Wolffian duct

AMH

Male Female Ovary Epididymis Oviduct Testis

Uterus Vas deferens

Seminal vesicle Vagina

Figure 7.11 Differentiation of the male and female reproductive systems does not occur until the fetal period of development. Source: Adapted from https://upload.wikimedia.org/ wikipedia/commons/thumb/e/e3/2915_Sexual_Differentation‐02.jpg/300px‐2915_Sexual_ Differentation‐02.jpg volume than that of females (Figures 7.11 and 7.12). This correlates with quan titatively greater sexual intercourse behavior exhibited by males [110,111]. This difference in nuclear volume has been attributed to the presence of more neurons in the dimorphic nucleus of the preoptic area of males than of females [112]. Sex hormones determine neuron number in part by controlling the occurrence of cell death during brain sexual differentiation in rodents [113]. In addition, gonadal steroids have direct effects on the size and the dendritic growth of a particular part of the brain region, which also contribute to volume differences in these regions. In a study carried out by Reddy et al. [114] in sheep, a cluster of neurons was found to exist bilaterally in the central portion of the medial preoptic area. This structure is called the ovine sexually dimorphic nucleus (oSDN) because it is approximately two times larger in heterosexual males and contains more neu rons than in rams that are homosexual or in ewes. The oSDN develops prior to birth and is enlarged in females by exposure to exogenous testosterone during a prenatal critical period that occurs after the external genitalia have differentiated. Exposure of adult sheep to exogenous testosterone does not affect oSDN volume, confirming that nuclear size in the male is pre‐engineered Corpus callosum Paraventricular Intermediate mass nucleus of thalamus Lateral preoptic nucleus Dorsomedial nucleus Medial preoptic nucleus Sagittal Posterior plane hypothalamic Anterior nucleus hypothalamic nucleus Arcuate Ventromedial Suprachiasmatic Key: nucleus nucleus nucleus Mammillary region Mammillary Infundibulum Supraoptic Tuberal region body nucleus Optic chiasm Optic (II) nerve Supraoptic region Pituitary gland Preoptic region

Figure 7.12 Important areas of the hypothalamus that are different in size and neuronal numbers between male and females. Source: http://what‐when‐how.com/wp‐content/uploads/2012/04/tmp15F20_thumb2.jpg. Why Testosterone is Essential for Engineering a Male Brain 205 prenatally. They also cultured the primary oSDN in vitro and showed that testosterone stimulates morphological differentiation of cultured fetal lamb neurons derived from the cerebral cortex and hypothalamus by increasing neurite outgrowth and size. These data suggest that testosterone acts as a morphogenic signal for developing sheep neurons and by so doing contributes to the maturation of a fully masculine brain. The result establishes that sexual differentiation during a long gestation (as in humans) occurs prior to birth. Another important study, carried out by Zuloaga et al. [115], showed that exposure to testosterone early in fetal life imparts a monumental effect on vari ous parts of the brain that are designed to engineer key features of the male brain. The study demonstrated that the effects of testosterone are mediated primarily by binding of testosterone (androgen) to its specific receptors (ARs). ARs found on various parts of the developing fetal brain become relatively larger and have more neuron and neuronal connects in males compared with female counterparts in all mammalian species. Of course, it is established that in the male brain, activation of AR normally mediates masculinization of the nervous system and behavior. Some of the first evidence for this idea came from an analysis of motoneurons in the spinal nucleus of the bulbocavernosus (SNB; Figure 7.12), which mediates penile reflexes during intercourse. There is a sex difference in the number of SNB motoneurons that are dependent on AR activation, with males having greater numbers than females. Morphology of the posterodorsal medial amygdala (MePD; Figure 7.12), which receives olfactory and pheromonal information and is important for certain male sexual behavior, is highly dependent on testosterone. The MePD volume is 1.5 times greater in male rats than in females. Structural plasticity in the adult MePD appears to be mediated through activation of both ARs and ERs. Testosterone treatment increased MePD soma size, but did not affect volume. Interestingly, MePD volumes are greater in the right hemisphere than the left. This laterality in MePD volume is not found in females, suggesting that its presence may depend on some masculinizing factor [116]. The male brain engineering is not only related to the male’s ability to have high sexual drive and right hemispheric dimorphism, but it also affects the brain’s biological clock! The vasoactive intestinal polypeptide (VIP)‐containing subnucleus of the suprachiasmatic (SCN) is twice as large in men as in women. In addition, the vasopressin‐containing subnucleus of the SCN has a different shape in men than in women. Sexual orientation has also been correlated with the size of the human SCN. The vasopressin subnucleus of the SCN in homo sexual men has been found to be 1.7 times larger and contain twice as many cells compared with heterosexual men. The ventromedial hypothalamus (VMH) is another sexually differentiated region that is involved in sexual and parental behaviors. VMH volume and number of neurons are greater in males than in females, and males also have a 206 Autism and Exposure to Environmental Chemicals

higher concentration of ARs in the VMH than do females. The VMH contains four morphologically distinct regions, including the anterior (VMHa), dorso medial (VMHdm), ventrolateral (VMHvl), and central (VMHc) subdivisions, which have distinct connectivity patterns to other brain regions and may affect a variety of functions (Figure 7.12). In a recent study, volume and neuronal numbers of the entire VMH and its four subdivisions were compared between males and females, and, confirming earlier findings, the overall VMH volume was greater in males than in females. The bed nucleus of the stria terminalis (BNST) is a center of integration for limbic information and balance monitoring. The BNST, sometimes referred to as the extended amygdala, is located in the basal forebrain and is a sexually dimorphic structure comprised of between 12 and 18 subnuclei. These subnu clei are rich with distinct neuronal subpopulations of receptors, neurotrans mitters, transporters and proteins. The BNST is important in a range of behaviors such as the stress response, extended duration fear states, and social behavior, which are all crucial determinants of dysfunction in human psychiat ric diseases [117]. A portion of this region, the posteromedial nucleus of the bed nucleus of the stria terminalis (BSTMPM) exhibits several sexual dimorphisms, including greater AR density and regional volume in males compared with females. Interestingly, there was a sex difference in BSTMPM volume in both the left and right hemisphere (males > females), where volume was increased in males compared with females only in the left hemisphere. These results suggest that, as in the MePD, ARs normally contribute to the full masculinization of the BSTMPM in males [117]. AVP innervation of the septal area (including the BST), which plays a role in pair bonding, parental and aggressive behavior [118], is also sexually differenti ated. AVP innervation of this area is greater in males than in females and is dependent upon androgens in both early development and adulthood. The locus coeruleus (LC), a brainstem nucleus implicated in the physiological response to stress and panic, is an example of a sexual dimorphism in which females show a larger volume and a greater number of neurons than do males. This dimorphism appears to be under the control of both testicular and ovar ian hormones, with testicular steroids decreasing and ovarian steroids increas ing growth and survival of LC cells. The hippocampus is another sexually dimorphic area of the brain in which males show a greater overall volume than females. One area of the hippocampus, the dentate gyrus, is sexually dimor phic in some strains of mice in which males show a greater number, size, and density of cells within this region [119]. As we have explained above, many parts of male brain are larger and have more compact neuronal mass than females, identifying specific regions that have a unique role in male behavior. Therefore, along with the emerging role of high levels of testosterone in weeks 8–24 of gestation and expression of the AR Why Testosterone is Essential for Engineering a Male Brain 207 in shaping brain morphology, evidence also suggests that fetal testosterone and the AR are important in shaping male behavior, both during its early organization and its later activation in adulthood. Although aromatization may account for many sex differences in behavior, research suggests that tes tosterone and ARs play a critical role in a variety of behaviors ranging from sexual to cognitive. These include spatial memory and anxiety‐related behavior.

Spatial Memory

In general, human males outperform females in spatial memory performance. These gender differences have been ascribed to the organizational effects of testosterone, since neonatal castration of males or administration of testoster one to newborn females eliminates this observed sex difference. This also fits into the “extreme male brain” hypothesis we have detailed in Chapter 5. The biochemical conversion of testosterone to estrogens (called aromatization) may be particularly important in the development of the sex difference, since neonatal administration of estrogen masculinizes spatial ability in female rats. However, this may not be the only mechanism. In fact, other evidence suggests that perinatal AR expression during early stages of gestation activation may also play a role in enhancing male spatial memory performance. Isgor and Sengelaub [120] found that prenatal estrogen treatment did not masculinize performance, whereas treatment with testosterone did. In addition, in experi mental animals, males administered AR antagonists prenatally showed poorer performance than control males, eliminating the sex difference in this behav ior, play fighting and aggression, and sexual/social behavior.

Anxiety‐Related Behavior

Anxiety is a psychological, physiological, and behavioral state induced in animals and humans by a threat to well‐being or survival, either actual or potential. It is characterized by increased arousal, expectancy, autonomic and neuroendocrine activation, and specific behavior patterns. The function of anxiety‐related behavior is to facilitate coping with an adverse or unexpected situation. Pathological anxiety interferes with the ability to cope successfully with normal life tasks. Anxiety‐related behavior appears to be a result of predisposing factors, including gene–environment interactions during the early developmental period as well as life events. In humans, anxiety disorders are estimated to affect about 1 in 5 people and there are striking sex differences in the prevalence of these disorders. Social anxiety disorders are more prevalent in women than men and women tend to receive the majority of diagnoses for specific phobias (e.g., panic disorder, post‐traumatic stress disorder, fear of spiders, fear of heights, etc.). Differences in circulating sex hormones, estrogen and testosterone, are likely 208 Autism and Exposure to Environmental Chemicals

involved in the occurrence of gender differences in anxiety. Estrogen replace ment has repeatedly been shown to increase mood and decrease anxiety in postmenopausal women. Other studies indicate that testosterone also plays a critical role in the regulation of anxiety in humans. Chemical castration through androgen blockade therapy in men with prostate cancer was corre lated with an increase in anxiety. When treatment ceased, anxiety levels decreased. Androgens play a critical role in the regulation of the hypothalamic– pituitary–adrenal (HPA) axis, with administration of testosterone reducing the rise of stress hormones [adrenocorticotropic hormone (ACTH) and corti costerone] following exposure to a stressful situation (Figures 7.7–7.9). Increased HPA axis activation is often correlated with increased anxiety, which suggests that androgens may regulate the display of anxiety‐related behavior by depressing HPA axis activation.

Play Fighting and Aggression

Play fighting includes two major components (playful attack and playful defense) and is a common juvenile behavior in many mammalian species that appears to be regulated by sex hormones. In rodents, play fighting peaks 30–40 days after birth, and occurs more frequently in males than females. Play fighting is decreased in male rodents castrated at birth and its frequency begins to decline at the onset of puberty. In summary, it is reasonable to conclude that testosterone and its receptors, ARs, normally contribute to masculinization and engineering of male brain morphology and behavior.

Adverse Effects of EDCs and Their Mechanisms of Action

It will require many books to provide analyses of the adverse effects of EDCs. However, in Figure 7.13 we have summarized some of the adverse effects of EDCs. In Table 7.1 we have further depicted the known mode of action of EDCs. The effects are varied and not entirely clear. Recent research indicates that cer tain EDCs may cause epigenetic changes (heritable changes in gene expression that are not due to mutations in the genes); we have already discussed (Chapter 6) the definition and the mechanisms of epigenetic changes that may last for gen erations (transgenerational effects) across numerous organ systems [121–125]. Studies consistently show that some EDCs impair key processes in ovarian development in animal models, adversely affect the structure and function of the postnatal ovary by inhibiting follicle growth and/or increasing programmed cell death (apoptosis) in animal models, and disrupt steroid hormone levels in animals and women. Adverse Effects of EDCs and Their Mechanisms of Action 209

Synthetic chemicals that disrupt testosterone homeostatic

Bioavailable Bound

Free Albumin SHBG (0.5 – 3.0%) (50 – 70%) (30 – 45%)

Excretion (90%) Testosterone (5 mg/d)

DEP DES 5α-Reductase PCBs Aromatase (6–8%) (0.3%) PCBs BPA

Dioxins

Dihydrotestosterone(DHT) Testosterone Estradiol

∙ External genitalia ∙ Wolffian duct ∙ Hypothalamic/ ∙ Prostate growth ∙ Bone formation pituitary feedback ∙ Acne ∙ Muscle mass ∙ Bone resorption ∙ Facial/body hair ∙ Spermatogenesis ∙ Epiphyseal closure ∙ Scalp hair loss ∙ Gynecomastia ∙ Some vascular and behavioral effects

Figure 7.13 Adverse effects of EDCs on testosterone metabolism. 210 Autism and Exposure to Environmental Chemicals

Although the data are not always consistent between experimental and epidemiological studies, they suggest that some EDCs may adversely affect the structure and/or function of the uterus, vagina, and anterior pituitary. Some EDCs are also associated with abnormal puberty, irregular monthly cycle, reduced fertility, infertility, endometriosis, fibroids, preterm birth, and adverse birth outcomes [126]. Limited information is available to explain the inconsistencies in findings among studies and to explain the mechanisms by which EDCs adversely affect female reproduction. Thus, more research is needed in this area [127]. Many potential EDCs have not been examined at all in experimental or epidemiological studies. There are two major reasons for this paucity of infor mation. First, there are so many synthetic chemicals being made as fragrance and food flavors that it is not possible to keep up with this rapidly expanding market [65,66]. Secondly, cosmetic and fragrance laws currently do not require the industries who are marketing the products to test cosmetics, fragrances, and food fragrances (i.e., flavors) for their safety. Even if some products are tested, adult rodents are often used in the limited safety tests, with no assess ment of potential adverse effects on fetuses, as detailed by Bioscience, and backed up with 20,000 pages of documents [44]. Thus, there is a real and urgent need for future studies to focus on the effects and mechanisms by which fra grances, food flavors, and EDCs affect fetal development and reproductive systems in both experimental and epidemiological studies. Natural mutations or hormonal abnormalities have been extremely informa tive in helping us better understand the consequences of disturbances in the developmental processes described above, including their consequences on male brain and reproductive organs (shown in Figure 7.13 and Table 7.1). Genetic mutations affecting testosterone levels or AR expression or function cause testicular dysgenesis syndrome (TDS), including cryptorchidism, hypo spadias, impaired semen quality, and markedly increased risk of testicular cancer [126]. The mutations encompass a large number of genes, including those that encode ARs, key steroidogenic enzymes, and regulatory transcrip tion factors. We already are aware that ASD has a multifactorial etiology, and environmental factors play an important role. EDCs that can mimic testoster one or bind ARs can have detrimental effects on the developing fetal brain and may contribute towards the development of the gender bias of ASD observed in male children [127]. Considering the importance of normal androgen activity in male reproduc tive development and function, it is not surprising that chemical compounds that disrupt androgen production or activity can cause ASD more often in males than females. Although there are no equivalent animal models for human ASD, since our brains are so large and unique, it is still meaningful to test the EDCs that disturb testosterone balance in human fetuses on developing primordial neurons for potential adverse effects. Animal models show that Effects of Testosterone or AR Mimicking EDCs 211 anti‐androgens can act in a dose‐additive manner, which has challenged the current no adverse effect levels because adverse outcomes have appeared when animals have been exposed to a combination of chemicals far below their individual levels. In addition to anti‐androgens, estrogens and dioxins cause similar effects, via their equivalent ERs or ARs, respectively. The balance between these hormone‐receptor pathways is also important, beyond the indi vidual roles of each hormonal system. Because the bulk of studies have focused on anti‐androgens, these are currently the main focus of EDC research in male reproduction.

Effects of Testosterone or AR Mimicking EDCs

Here we would like to briefly discuss what sort of physical and anatomic effects one can observe when humans are exposed to EDCs. Some EDCs like phtha late esters (Table 7.1 and Figure 7.13), inhibit testosterone production, whereas others block the AR (e.g., the pesticide DDE, and fungicides vinclozolin and procymidone). Despite their dissimilar mechanisms of action, these chemicals act in a dose‐dependent manner, with increased likelihood of adverse effects when low doses of individual chemicals are combined in a mixture [128]. Flame retardants, PCBs, DDT, and dioxin have been assessed for their rela tionships with cryptorchidism. Cryptorchidism is the absence of one or both testes from the scrotum. It is the most common birth defect of the male genital and about 3% of full‐term and 30% of premature infant boys are born with at least one undescended testis. However, about 80% of undescended testes descend by the first year of life, usually within 3 months of birth. Undescended testes are associated with reduced fertility and increased risk of testicular cancer. In addition, boys may suffer psychological problems. Without intervention, an undescended testicle will usually descend during the first year of life, but to reduce these risks, undescended testes can be brought into the scrotum in infancy by a surgical procedure called an orchiopexy. Figure 7.14 summarizes the data. It is interesting to note that incidence of cryptorchidism at birth is increasing in many developed nations [129]. The list of EDCs that have an anti‐testosterone (anti‐androgenic) effect is growing (Table 7.1). The compounds that act at the ARs (i.e., antagonists or partial agonists) include widely spread pesticide congeners such as dichlorodi phenyldichloroethylene (DDE) and fungicides such as vinclozolin and procy midone. The most alarming problem is that a much larger group of EDCs and other synthetic chemicals disturb testosterone synthesis. Phthalates are well‐ documented examples of these chemicals. Interestingly, phthalates show vari ation in effects on different species, whereas the effects of receptor antagonists are similar over these species. Since testing is performed with rodent models, 212 Autism and Exposure to Environmental Chemicals

Denmark 10.0 United kingdom Finland 8.5

7. 5 chidism (%)

or 5.0 5.0 ypt 3.5

2.7 2.5 2.1

Incidence of cr 1. 8

0.0

1960 1980 2000 Year

Figure 7.14 Incidence of cryptorchidism at birth on the basis of prospective clinical studies from the 1950s to the 2000s in Denmark, Finland, and the UK. The data points are marked on the year of the publication of the study which represents the preceding incidence rate. Source: Adapted from Ref. [129].

the harmful effects on human health of some anti‐androgens, such as testos terone and estrogen, that might disturb human sex hormones without affecting rodents may go unnoticed. Dioxin levels in breast milk were associated with an increased risk of cryp torchidism in Danish boys [130,131]. Dibutyltin concentrations in placenta were associated with an increased incidence of cryptorchidism in Danish boys. A French case‐control study on cryptorchidism measured concentrations of DDE, PCBs, di‐n‐butyl phthalate, and metabolite monobutyl phthalate (MBP) in colostrum and cord blood (Figure 7.6, [132]). The boys in the high‐exposure group (based on combined exposure to DDE and PCBs) had a higher risk of cryptorchidism than boys in the lower exposure group [133]. A similar ten dency was found for MBP. A US study that examined testicular position at the mean age of 12.8 months reported an association between levels of maternal urinary mono‐2‐ethyhexyl phthalate and incomplete testes descent [134]. Maternal urinary levels of five phthalate metabolites were also associ ated negatively with anogenital distance (AGD; [135]), suggesting an anti‐ androgenic effect [134]. The Finnish–Danish study reported an association between phthalate levels in mother’s milk and serum testosterone and lutein izing hormone (LH) concentrations at 3 months of age in boys, although there was no direct association between cryptorchidism and milk phthalate Effects of Testosterone or AR Mimicking EDCs 213 levels [136]. High phthalate exposure was associated with a decline in free testosterone and an increase in LH concentration, again suggesting an anti‐ androgenic effect (Figure 7.13). The key question to ask is: is human exposure to one or a mixture of many of EDCs enough to introduce disruptions of male programming and brain engineering? In experimental animals, when a mixture of the chemicals is introduced (to mimic the real life daily exposure to these agents in humans), these chemicals can induce the double or triple jeopardy manner (additive effect) and render harmful effects. The experimental results from exposing animals to mixtures of chemicals can impart dose–response curves of the indi vidual chemical. Estrogenic chemicals and dioxins can also cause cryptorchidism. In humans, exposure to the synthetic estrogen DES was linked to increased rate of cryptorchidism, but environmental estrogens are typically much less potent. However, together with anti‐testosterone they might act in a synergistic manner and deliver a double effect [128,137]. Epidemiological studies that can link exposures to endocrine disruptors and cryptorchidism have analyzed single chemicals or chemical groups, and only a few have attempted to integrate these data (Figure 7.15). Exposures have been measured in blood, urine, placenta, and breast milk that serve as a biomarker to a woman’s levels of chemicals during pregnancy. In some cohort studies, careful ascertainment of the diagnosis has been combined with exposure measurements. The results vary according to the experimental design. The breast milk level of polybrominated flame retardants was associated with an increased risk of cryptorchidism. Similarly, dioxin levels in breast milk of Danish women were associated with an increased risk of cryptorchidism [129]. The effects of exposure to multiple chemicals have been analyzed in only a limited number of studies. After combining data from several pesticide

Abdominal (15%)

Inguinal canal (25%) High scrotal (60%)

Normal Subcoronal MidshaftPenoscrotal

Figure 7.15 cryptorchidism and hypospadias in newborn boys. Source: http://img.tfd.com/ mk/C/X2604‐C‐73.png. 214 Autism and Exposure to Environmental Chemicals

exposures by permutation analysis, an association between the level of chlorin ated pesticides in breast milk and risk of cryptorchidism in offspring was found. Greenhouse workers exposed to pesticides during pregnancy were also shown to have an increased risk of cryptorchidism. Phthalate levels in breast milk were not associated with cryptorchidism risk, but they were linked to an increased LH‐to‐free testosterone ratio in the son at the age of 3 months, sug gesting testicular impairment during lactation. It is clear that there is a need for more research as there are large gaps in our understanding of this problem. In addition to clinical experience in humans, experimental animal studies show that exposure to anti‐androgenic EDCs lead to cryptorchidism, similar to the effect of these compounds on hypospadias (Figures 7.14 and 7.15). Hypospadias is a birth defect of the urethra where the urinary opening is not at the usual location on the head of the penis. It is the second most common birth abnormality found in male newborns, and this affects 1:250 males at birth. In the majority of cases, the opening is on or near the head of the penis (Figures 7.14 and 7.15), while in rare cases hypospadias will be located near or within the scrotum. In addition to anti‐testosterone chemicals, compounds that act like estrogen (estrogenic) can also lead to cryptorchidism. Thus, a mixture effects of EDCs may be due to their various estrogenic and anti‐androgenic proper ties. There is no reason why the same mechanisms do not apply to humans, who share the same biological mechanisms of testicular descent as rodents.

Early Puberty in Males

Skakkebaek et al. [129] also have clearly shown that there is another alarming trend – males as well as females are reaching puberty earlier. Early puberty in females is well documented but this trend appears to be present in males also. Several well documented studies suggest a significant downward trend in male pubertal timing. For example, a European study of 21,612 boys born in 1935–1969 showed a downward trend in age at peak height velocity (PHV), although other European studies did not find such changes. Male puberty marks the transitional period during which the infantile boy achieves reproductive capacity and develops into a matured man. Puberty usually starts at 11.5 years of age, although with large inter‐individual variability. A boy generally reaches puberty between 9 years and 14 years of age and any one falling outside of this range is considered pathological and requires clinical evaluation to exclude underlying pathologies. The timing of puberty is deter mined by genetic as well as environmental factors such as body composition, physical fitness, nutritional and socioeconomic status, ethnicity, residence, foreign adoption, and exposure to endocrine disrupters. Testosterone pro duced by the Leydig cells in the testes is the major male sex steroid that deter mines the timing of male puberty. Production of testosterone is stimulated by Effects of EDCs on Neurodevelopmental and Neuroendocrine Systems 215

LH secreted by the pituitary, which itself is stimulated by gonadotropin releas ing hormone (GnRH) from the hypothalamus. On the other hand, circulating testosterone has, together with estrogen, an inhibitory effect on both GnRH and LH secretion. In the adult male, a balance between LH and testosterone level is thus achieved through a finely regulated hormonal negative‐feedback loop, which is part of the hypothalamo–pituitary–testis hormone axis. About 98–989 of circulating testosterone and estrogen is bound to serum proteins called sex steroid binding protein (SHBG) testosterone and only 1–2% of circu lating testosterone occurs free in the bloodstream. The target tissues of the body, including the hypothalamus and pituitary, only sense the free‐unbound sex steroids, and SHBG serum level is therefore an important regulator in the GnRH–LH–testosterone hormonal feedback loop. In several studies, data indicate that both SHBG and free‐unbound testosterone levels are declining significantly in males. In addition, sperm count is also declining in males from developed nations [129].

Change in Sex Ratio

Furthermore, the sex ratio of offspring is also changing in a significant manner. Generally, there are 105 male births to 100 female births, leading to 51.5% rate of male birth. However, recent data suggest a decline in the sex ratio in many developed nations [129].

Effects of EDCs on Neurodevelopmental and Neuroendocrine Systems

Parts of the brain function as an endocrine gland. In Chapter 4, we covered certain aspects of the human brain’s endocrine function and role of oxytocin and AVP and their respective receptors in ASD. Specialized kinds of neurons release neurohormones, and oxytocin and AVP are neuropeptides that func tion like hormones in human brains. These are evolutionarily conserved neuropeptides and their origin can be tracked back to many early vertebrate life forms. The central neuroendocrine region is located at the base of the brain, the hypothalamus, and is integrally involved in controlling how the body adapts to the environment and in the regulation of several peripheral endocrine systems. Different groups of hypothalamic neurons control reproduction, growth, metabolism, lactation, stress responsiveness, uterine contractions at parturi tion, energy balance, circadian rhythms, temperature regulation, and electro lyte balance, among other functions. We will focus primarily on the hypothalamic–pituitary–gonadal (HPG) and neuroendocrine axes controlling reproduction and stress, respectively (Figure 7.16). Importantly, the steroid Hypothalamus

Selective estrogen receptor modulators

GnRH GnIH Estradiol

Anterior pituitary

Aromatase inhibitors

Aromatase

FSH Inhibion B LH Testosterone (serum~500 ng/dl) Activin

Testes

Leydig cells

Testosterone (~500 ng/dl)

Sertoli cells

Figure 7.16 Hypothalamic–pituitary–gonadal network. Source: Adapted from http://www. maleinfertility.org/sites/default/files/styles/panopoly_image_original/public/new_hpg. jpg?itok=J3X4BcOu and Pinterest: hypothalamic‐pituitary‐adrenal. EDC Effects on Steroid Hormone Receptors in the Brain 217 hormones not only act on sex organs but also act upon the brain via steroid hormone receptors that are not only expressed in the hypothalamus but are also widely and heterogeneously expressed throughout the nervous system and the rest of the human body. EDCs target the human brain in two basic ways: by directly disturbing neuroendocrine balance that originates in the hypo thalamus; and by binding the steroid hormone receptors and other signaling pathways that occur much more widely throughout the brain and the body. In humans, uncovering the effects of EDCs on the brain is difficult since the analyses require postmortem tissues for which no studies are available. Neuroendocrine neurons cannot be directly evaluated because hypothalamic releasing hormones cannot be measured in peripheral blood samples, urine, or other tissues. Therefore, human data are scarce and most of the findings are based on behavioral consequences. However, it is not very difficult to surmise the effects of EDCs on testosterone (ARs) and estrogen (ERs). As we discussed earlier, the brain’s dense and widespread distribution of hormone receptors, its high hormone sensitivity, and its ability to synthesize steroids through the expression of steroidogenic enzymes, among other characteristics, make it particularly vulnerable to hormonal‐like EDCs that disturb its fine‐tuned bal ance. This is central to the idea that EDCs disturb the highly organized process of brain development and contribute towards ASD, since there are critical periods during which even minuscule fluctuations in hormone exposures can affect a neurobiological outcome. This aspect of sensitive life periods is pivotal in our understanding of the effects of EDCs on brain development.

EDC Effects on Steroid Hormone Receptors in the Brain

There is ample evidence that EDCs alter the degree of expression and distribu tion of ARs and ERs in the developing central nervous system. This results in interference in normal mRNA levels, protein expression, and neuroanatomical changes to hormone receptors studied to date, as well as functional conse quences of altered receptor action. In this book we cannot describe all the adverse events from thousands of EDCs, but as an example we would like to briefly mention two chemicals that have been shown to impart significant alterations in a developing brain. Because these types of experiments cannot be carried out on humans, we would like to present a small sample of research carried out in zebrafish that were exposed to BPA for which there is the most evidence for EDC activity on hormone receptors in the brain. BPA is a compound used in the production of diverse consumer products, ranging from baby bottles to thermal paper used for credit card receipts. Even though adults can experience adverse health following continued exposure to BPA, the fetal brain is especially vulnerable because of an immature xenobi otic‐metabolizing system and the blood–brain barrier. A recent examination 218 Autism and Exposure to Environmental Chemicals

of urine samples in the USA and Asia confirmed previous work showing that 93% of people had detectable levels of BPA, but surprisingly also showed that 81% had detectable levels of bisphenol S (BPS), illustrating the widespread use of this poorly known bisphenol analogue in consumer products. The physiological effects of BPA on human adults are well documented, but the mode of BPA action on the developing brain has yet to be examined in primates, especially in the hypothalamus, which plays an important role in the neuroendocrine system, neurodevelopmental disorders, and directly impacts on human health (e.g., obesity, precocious puberty, anxiety, and hyperactivity). BPA is commonly thought to exert its effects by acting as a weak ER competitor that binds to ER receptors instead of the natural estrogen. However, interfer ence at ARs and thyroid receptors (ThRs) has been shown also. Proliferating cells in the developing hypothalamus express ERs and ARs, and the roles of estrogen and testosterone in regulating neurogenesis are rapidly being researched. In the hypothalamus of rodents, as well as in sheep, exposures to BPA during fetal development affect expression of the nuclear ER genes and proteins. For example, several studies have shown that hypothalamic ERα pro tein and mRNA expression was increased by prenatal and/or early postnatal BPA. In female sheep, ERs in the MPOA were increased by BPA (50 μg/kg/day or 5 mg/kg/day, from E15 to P21) in both sexes. An elegant study carried out by Kinch et al. [138] demonstrated that BPA exposure during a time point parallel to the second trimester in humans has real and measurable effects on brain development and behavior of the zebrafish brain. The study showed that BPS, a replacement used in BPA‐free products, equally affects neurodevelopment. These findings suggest that BPA‐free prod ucts are not necessarily safe and support a societal push to remove all structur ally similar bisphenol analogues and other compounds with endocrine‐disruptive activity from consumer goods. Their study results combined with numerous reported physiological and behavioral studies carried out in humans begin to point to the prenatal period as a BPA window of vulnerability and suggest that pregnant women should limit exposure to plastics and receipts. Numerous other animal studies have also demonstrated that a number of chemicals can reduce circulating levels of thyroid hormone, including, but not limited to, PCBs, PBDEs, some phthalates, and perchlorate. Some chemicals that affect the thyroid system in animals have been shown to be associated with cognitive deficits in humans. Besides the hypothalamus, the hippocampus, developmental exposure of male rats to BPA changed expression of the ERα gene and protein. This observed effect was dependent upon the age at which ERα was measured, and this effect was blocked by the ER antagonist. Nearly all of the steroidogenic chemicals are detectable in the brain, with each agent having unique developmental and brain region‐specific profiles. Beginning with those enzymes necessary for cholesterol transport and side Are the EDCs and other Synthetic Chemicals Depopulating the Human Race? 219 chain cleavage, and continuing through the pathways leadings to glucocorticoids, mineralocorticoids, estrogens, progestins, and androgens, these hormones are capable of interconverting, metabolizing, and inactivating steroid hormones in the brain. This is still an unexplored field of science and connection of these pathway disturbances to development of ASD is still in its infancy. In summary, the experimental animal studies and observations in humans exposed to EDC consistently show that the structure and function of the brain’s neuroendocrine systems can be altered by developmental exposures to EDCs.

Are the EDCs and other Synthetic Chemicals Depopulating the Human Race?

After looking at the evidence, is it possible that synthetic chemicals that have entered our lives may be actually responsible for depopulating the human race? If synthetic chemicals are interfering with fetal development and neurodevel opment of our next generations, then there must be some evidence for this. In our view, exposure to synthetic chemicals, in the forms of fragrances, herbi cides, EDCs, and any other forms, are affecting our unborn at a very early stage of pregnancy. If this is true, then the spontaneous abortions rate must have increased significantly in the industrialized regions of the world where the use of synthetic chemicals is the highest, as compared with developing nations. There is evidence supporting such an outcome. For example, since the intro duction of synthetic chemicals during the last 50–100 years, populations of industrialized countries all over the world have experienced a decline in total fertility rates (average number of live births per woman) far below 2.1, which is the rate considered necessary to sustain a population size at current numbers. In addition, other male reproductive problems, several of which are linked to testicular cancer, including disorders of spermatogenesis, are widespread and have recently been the focus of many basic and clinical research projects (Figure 7.17). Is the incidence of spontaneous abortions are increasing? We have to clarify certain facts about pregnancy: 50% of all conceptions are lost and remain unrecognized. About 15% of recognized pregnancies are lost, generally around weeks 12–14 of gestation. About 10–20% of women suffer one sporadic spon taneous abortion. About 2% of women suffer two spontaneous abortions and about 0.4% suffer three spontaneous abortions. Therefore, if a larger percent age of women are suffering from unrecognized loss of pregnancies, it would be difficult to determine. The only barometer one can use is to determine if total fertility rates have been declining in the nations that use the largest amounts of synthetic chemicals in the forms of fragrances, EDCs, herbicides and synthetic food flavors, drinks and other forms of consumables. Let us see if this notion is supported. <10% 10–<15% ≥15% Data not available 01250 2500 5000 km Not applicable

Figure 7.17 Global birth rates. Source: http://www.thelancet.com/journals/lancet/article/PIIS0140‐6736(12)60820‐4/ fulltext. Are the EDCs and other Synthetic Chemicals Depopulating the Human Race? 221

A study by Skakkebaek et al. [129] compiled many fascinating facts. It is predicted that Japan and the European Union will soon experience appreciable decreases in their populations due to persistently low total fertility rates below replacement level (2.1 children per woman). In the USA, where the total fertility rate has also declined, there are ethnic differences. Caucasians have rates below replacement, while total fertility rates among African‐Americans and Hispanics are higher. As shown in Figure 7.18a, the total fertility rate has dramatically decreased below replacement level to between 1.0 and 1.5 in many nations of the European Union, Japan, the USA. It is difficult to state with some degree of certainty, but environmental factors are greatly contributing to low fertility rates in these nations, along with other social and economic factors, including contracep tives and access to induced abortions. Of note, total fertility rates have been declining decades before the contraceptive pill and legal abortion were intro duced (Figure 7.18b). Skakkebaek et al. [129] have summarized some surprising aspects of current trends in male reproductive disorders and fertility. They have described possible links between environmental agents and the rising incidence of testicular cancer, disorders of sex development, cryptorchidism, hypospadias, low testosterone levels, poor semen quality, childlessness and changed sex ratio [138]. Based on their review of genetic and environmental factors, we conclude that environmental exposures arising from modern lifestyle, rather than genetics, are the most important factors in the observed trends. These environmental factors might act either directly or via epigenetic mechanisms. In the latter case, the effects of exposures might have an impact for several generations post exposure. As we hypothesized earlier, exposure to environmental agents that specifi cally deplete or reduce the numbers of certain progenitor neurons may con tribute to the development of ASD. They have taken this one step further and have hypothesized that the increasing incidence of testicular germ cell cancer is the result of interference in spermatogenesis process mainly due to environ mental agents. The key pathogenetic event is insufficient masculinization and impaired function of the testicular somatic cell niche, which in fetal life is mainly composed of Sertoli and Leydig cells. The insufficient stimulation of developing germ cells causes arrest of gonocyte differentiation to spermatogonia and prolonged expression of pluripotency genes (Figure 7.19). The delayed gonocytes (pre‐germ cell neoplasia in situ or pre‐GCNIS cells) then gradually acquire secondary genetic defects, while adapting to the changing niche, espe cially during and after pubertal hormonal stimulation of the testis. Increased proliferation results in malignant transformation of GCNIS cells into an invasive tumor, either a seminoma or nonseminoma (the latter through the reprogrammed pluripotent stage of embryonal carcinoma). Normal germ cell development is shown in the top part of Figure 7.19. 222 Autism and Exposure to Environmental Chemicals

(a) 4 European union Japan 3.5 USA Replacement level

2.5 (per women)

te 2 y ra ilit rt 1. 5 fe tal To 1

0.5

10 960 965 970 975 980 985 990 995 1 1 1 1 1 1 1 1 2000 2005 20

(b) 4.5

3.5

(per women) 2.5 te 2 y ra

ilit 1. 5 rt fe 1 tal

To 0.5

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010

Figure 7.18 (a) Total fertility rates of the European Union, Japan and the USA, 1960–2013. The dotted line represents a fertility rate of 2.1, below which a population cannot be sustained Source: Adapted from http://databank.worldbank.org/data/views/ variableselection/selectvariables.aspx?source=world‐development‐indicators. (b) Total fertility rate of Denmark, 1901–2014. The dotted line represents a fertility rate of 2.1. Note that a downwards trend in total fertility rate started long before introduction of the contraceptive pill in the 1960s. Apparently the trend was interrupted by the First and Second World Wars. Source: Adapted from Statistics Denmark. http://www.statistikbanken. dk/statbank5a/default.asp?w=1600. Summary 223

BirthPuberty

Spermatids i and leydig tol c r el se l l fu a n c m t Normal r i o o

n development N

Gonocyte Spermatogonia Spermatocytes Migration

Fetal testis Prepubertal testis Adult testis PGC

or leyd toli ig Delayed er ce Migration s ll d fu gonocyte SEMINOMA re n i c “pre-GCNIS” Proliferation a t i p o

n m Impaired I “A daptation” GCNIS with development Arrest Genomic invasive capacity aberrations Gonocyle Re-programming NONSEMINOMAS EC Somatic Environmental insults differentiation gene mutations

Figure 7.19 Model for the pathogenesis of testicular germ cell tumors of young adults. Reproduced with permission of University of South Carolina.

Summary

In this chapter we have covered a wide range of issues that relates to environ mental contaminations, some visible and others invisible, have major impact on our daily lives and have a devastating impact on our offspring that could potentially last for generations to come. We have documented that synthetic chemicals, fragrance, EDCs, and other chemicals such as pesticides and herbi cides, can have a major role in causing ASD. We have presented data that synthetic chemicals in our tasty foods, flavored drinks, and environment are responsible for causing a plethora of illnesses and the adverse effects of synthetic chemicals is not limited to the alarming rise of autism but many diverse disorders, syndromes, and spec trums. We are aware that ASD is four to five times more common in males than females but it appears that current exposure to industrial synthetic chemicals also imparts much greater adverse effects on males compared with females. This is mostly due to anti‐androgenic and estrogenic properties of the chemicals. Is the relative high incidence of ASD also related to potential androgenic effects of fragrances? We have shown that many common chemi cals found in fragrances have anti‐androgenic or estrogenic quality. For example, tonalide, octinoxate, DEP, benzyl benzoate, and benzyl salicylate have androgenic or estrogenic properties. These chemicals are not only linked to causing cancer but also are neuromodifying agents and contribute in the etiologies of ASD. 224 Autism and Exposure to Environmental Chemicals

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235

Maternal Antibodies to Fetal Brain Neurons and Autism

We can believe in the future and work to achieve it and preserve it, or we can whirl blindly on, behaving as if one day there will be no children to inherit our legacy. The choice is ours; the earth is in balance. Al Gore, 1992, Earth in the Balance: Ecology and the Human Spirit

Link between Damage to the Fetal Brain and Maternal Antibodies: A Double Jeopardy

In the previous chapters, we have detailed the role of synthetic chemicals, particularly those that are found commonly in fragrances, detergents, soap, air fresheners, car wash, food flavors, drinks, air, water and so many other places. Many are endocrine disrupting chemicals that can perturb the body’s homeostasis. Industrial chemicals can harm a developing fetal brain in ways that cannot be perceived easily by currently available technology. However, as is shown in well‐documented studies, many of the synthetic chemicals would be neurotoxic to rapidly replicating human fetal brain cells. General neurotoxicity or “‘highly selective cytotoxicity” to specific types of neurons [i.e., oxytocin or arginine vasopressin (AVP) receptors, olfactory neurons] would cause neuronal death to a larger extent than what occurs as part of the well‐orchestrated brain development, where pro grammed cell death is part of fetal brain proliferation and differentiation processes. During normal embryogenesis, fetal development and all through life many cell types go through programmed cell death (called apoptosis) and antigens that are released are either digested by specialized cells (as in the fetus) or never cross the amniotic membrane and reach the

Autism and Environmental Factors, First Edition. Omar Bagasra and Cherilyn Heggen. © 2018 John Wiley & Sons, Inc. Published 2018 by John Wiley & Sons, Inc. 236 Maternal Antibodies to Fetal Brain Neurons and Autism

maternal circulation [1–3]. The programmed cell death is an essential part of embryogenesis and normal functioning of life. This is a huge subject by itself and beyond the scope of this book. Of note, cellular death is akin to carving a statue by a sculptor, where a chisel is used to remove the unwanted pieces of a stone, and apoptosis is similar, where unwanted cells are removed by a highly orchestrated system and at the end there is a “perfect offspring”. In this process there is no antigenic residue left behind to confuse the immune system. However, in the case of the extraordinary killing of brain cells by harmful chemicals or sometimes in utero infection, the apoptosis mechanism is saturated and reaches a threshold, and the cells are not com pletely processed. This results in a larger than normal amount of neuroan tigens that would spill out into the maternal blood circulation (if the fetal antigens cross the placenta and reach the maternal circulation). In such a situation, the mother’s adaptive immune system would produce antibodies in response to those antigens. These antibodies can enter the fetal circula tion and can damage the fetus and sometimee even kill the fetus.

Are there Examples of Such an Immune Mechanism?

A prime example is a disease called “erythroblastosis fetalis”. There are two main causes of erythroblastosis fetalis: Rh incompatibility; and ABO incom patibility. Human red blood cells have specific chemicals that determine our blood group: A, B, AB, and O. In addition, blood can be either Rh positive (Rh+) or Rh negative (Rh−). If a person is type A and Rh+, then he or she is A+. Similarly, if a person is AB−, then he or she has both A and B antigens on the surface of the red blood cells but lacks the Rh antigen [4,5].

Rh Incompatibility

Rh incompatibility occurs when an Rh− mother conceives from an Rh+ father. The result can be an Rh+ baby. In such an event, the baby’s Rh antigens will leak into the maternal blood circulation during the birth process and the mother’s adaptive immune system recognizes the Rh antigen as foreign antigen, similar to the way pathogenic viruses or bacteria are recognized by the immune system. If the mother gets pregnant with another baby who is also Rh+ then antibodies to the Rh antigen would enter the fetal blood circulation and begin to destroy the fetal red blood cells. This second Rh+ fetus is in danger of developing fetal anemia and may be stillborn. Therefore, Rh incom patibility is not as much of a concern for the first baby but may cause fetal anemia in subsequent Rh+ babies. Figure 8.1 illustrates this concept and the immune mechanism that can be applied in the case of fetal antigen release into the maternal blood [6]. Are there Examples of Such an Immune Mechanism? 237

Release of fetal brain antigens

Early stage fetus

1 Environmental factors enter 2 Neuronal antigens from the damaged 3 Mother’s lgG enters the pregnant mother’s circulatory neurons leak out into mother’s fetal brain and interferes systems at early stages of circulation and adaptive in the normal development gestation and damage immune system develop of fetal brain, resulting in certain brain progenitor antibodies to the fetal ASD. neurons. neuronal antigens. +

Figure 8.1 Illustration of neuroantigens released from the fetal brain and role of maternal neuroantibodies that can damage a developing fetal brain and may cause autism spectral disorder (ASD). (See insert for color representation of this figure.)

ABO Incompatibility

In this type of hemolytic disease of newborns, the ABO blood type is incom patible. This occurs when the mother’s blood type of A, B, or O carries a unique antigen similar to the Rh antigen (i.e., Kell, Duffy, Kidd, Lutheran, Diego, Xg, P, Ee, Cc, MNSs) that is not found on the mother’s red blood cell surfaces but is on the fetus’s red blood cells. This condition is considerably less harmful, very uncommon, and generally not life threatening [4–6]. It is not difficult to imagine that if a fetus is exposed to neurotoxic chemical(s) that have killed a significant number of neurons, the “unique antigens” that have leaked into the maternal circulation could lead the mother to produce antibodies to these antigens which could enter the fetus’s system and further damage the fetus’s brain development. Here, there is a perpetual double jeop ardy for the future offspring! The neural antibodies produced during the first (or previous) pregnancy can harm the subsequent fetuses of these “exposed” mothers. In Figure 8.1, we have illustrated the mechanism of this terrible double whammy – the synthetic chemicals (or occasionally an infection) could kill certain progenitor neurons that lead to specific types of antigens released from 238 Maternal Antibodies to Fetal Brain Neurons and Autism

these killed neurons. These antigens could enter the maternal immune system and the adaptive immune system of the mother could form antibodies to these antigens that enter the fetus’s brain and further damage the fetal brain develop ment. Here, we hypothesize that the five criteria must be met and kept in mind: 1) Only selective types of progenitor neurons are destroyed initially due to synthetic chemicals. 2) The mothers only produce antibodies to specific neuronal antigens [i.e., lactose dehydrogenase (LDH) A, LDH B, STIP1, CRMP1, or perhaps against oxytocin and AVP receptors; see below]. 3) The release of neuroantigens may be more relevant during the early stages of fetal brain development (i.e., weeks 4–8 of gestation) with regards to development of autism and other neurodevelopmental disorders. The rationale for this theory is that in the early stages, any elimination of brain progenitor cells would have a much broader impact on the overall brain construction and neural damage, as compared with later exposure, since at later stages the blood–brain barrier (BBB) and a much thicker placental/ amniotic barrier may filter out many of the neurotoxic chemicals compared with earlier stages of pregnancy. 4) After exposure to a neuroantigens(s) in the maternal circulation, it would take several weeks to form antibodies to those early expressed neuroantigens. 5) The anti‐neuronal antibodies formed during the first trimester may not always be harmful to the fetal brain during the later stages of brain develop ment (second and third trimester fetal brain) due to development of the BBB and/or due to lack of expressions of the particular neuroantigens (due to enormous and rapid brain differentiation).

Why are Fetal Neuroantigens Immunogenic to the Maternal Immune System?

In an earlier paragraph we used the term “unique antigens,” which warrants scientific clarification. In immunological terms, the human body does not form antibodies to its own antigens. This is due to self‐tolerance of antigens. However, there are few sites in human organs where this “self‐tolerance” does not apply. These sites are called immunologically “privileged” sites and include the brain, the retina of the eye, and male testes. Therefore, if any antigens leak from these sites, the body will form antibodies to the leaked antigens. This is the primary reason why in infection by the mumps virus of only one testis that results in leakage of testicular antigens, the antibodies made against the testis antigens can destroy the other testis. This would be due to antibodies not the virus! Furthermore, sperm are also considered a “foreign” antigen by the body and in some cases immune response Why are Fetal Neuroantigens Immunogenic to the Maternal Immune System? 239 to sperm antigen may result in low sperm count and infertility [7]. Unfortunately, the mump vaccine has been reported to induce autoimmune orchitis or infer tility. We will return to this topic later in the chapter [8]. Similarly, multiple sclerosis (MS) is considered to be due to leakage of certain specific types of neuroantigens (myelin) into the patient’s blood circulation. MS is a disease of the central nervous system (CNS). The CNS consists of the brain, optic nerves, and spinal cord. In MS patients, the immune system responds by causing inflammation of the CNS and damaging the myelin sheath (insulated covering) that protects and covers the axons of the neurons. The damage to the myelin disrupts the smooth flow of nerve impulses. As a result, the speed of information from the central location in the brain and spinal cord to other parts of the body is delayed causing the symptoms observed in MS [9,10]. Figure 8.2 illustrates the autoimmune response to myelin antigen and development of MS. The good thing about the “immunological privileged” sites is that a “foreign graft” can be accepeted from other humans without matching the human leuko cyte antigens or carrying out “tissue typing”. The antigens inside the privileged

Normal myelin in MS

Normal

Cell body Myelin Damaged myelin (demyelination) Nerve fibre (axon)

Distorted messages

Damaged myelin in MS

Figure 8.2 The immunological mechanism that leads to MS. (See insert for color representation of this figure.) 240 Maternal Antibodies to Fetal Brain Neurons and Autism

sites are protected from the immune system. Hence, we regularly implant cornea from the eye. Many of us agree to allow our corneas to be donated; this is often done when the driver’s license is obtained. Similarly, human testis can be transplanted with minimum rejection. The main reason for the unusual nature of immunological privileged sites is that the antigens in the brain, testes, and eyes appear after the constitution of self‐tolerance (i.e., germ cells) or are sealed during the fetal development by specialized barriers (e.g., BBB, Sertoli‐blood barrier and vitreous‐humor barrier, for the testis, brain and eye, respectively). These so called neo‐antigens are sequestered from the early stages of immune development where the “self‐recognition” process takes place. Of course, this is the simplest way this highly complex concept can be described; it is beyond the scope of this book for us to describe this concept further. Readers are invited to refer to the literature to explore this topic in more detail [11,12] (Figure 8.3).

Neuroantigens/Abs

odegeneration LDHA/B Neur STIPl

CRMPl Protective OTR (destructive)

AVPR

Immune system Migration to the CNS

Immune response to Dendritic released antigens from cells fetal brain

Figure 8.3 An autoimmune hypothetical model of the immunopathogenesis of ASD: The role of the immune system in a primary neurodegenerative scenario. The figure illustrates the molecular and immunological mechanism by which autoimmunity can play a role in development of ASD. The middle section shows the leakage of selective brain antigens passing through the placental barrier (green). Top left shows antibodies to the leaked fetal neroantigens (i.e., LDH, YBX1, cypin, STIP1, CRMP1 and CRMP2) from the brain after an environmental injury. The maternal adaptive immune system develop antibodies to these neuroantigens and maternal autoantibodies cross the feto‐amniotic barrier (shown in green) and enters the brain, causing damage to the fetal brain only in selected areas. (See insert for color representation of this figure.) The Detection of Fetal Brain Neuroantigens in the Maternal Blood 241

Is There Any Evidence of a Link Between Synthetic Chemicals Exposure, Neurotoxicity, and Autoimmunity?

There is a large body of evidence that supports the notion that there is direct link between neuromodifying, androgenic or androgen receptor (AR) mimicking chemicals, the immune system of pregnant women and anti‐brain antibodies with autism spectrum disorders (reviewed in Refs [13] [14]). In the following section we will summarize some the most prominent exam ples of such scientific evidence.

The Relationship between Autoimmunity and ASD

As we have hypothesized, there are five points of interest that relate to ASD, maternal antibodies, and neural antigens released from the developing fetal brain. Autism shares many of the commonly recognized characteristics of autoimmune disorders. There is a strong family tendency of ASD, suggesting a link between potential immune disorders and children with autistic traits. It should be emphasized that every family has a specific environment the family clusters around and environmental factors can increase or contribute to the risk of developing a neurodevelopmental disorder. Most importantly, one should look at the 1:5 male gender bias in ASD. Interestingly, endocrine disrupting chemicals (EDCs) described in detail in Chapter 7 can shed a light on this gender bias. A large percentage of EDCs affect male hormones more than the female hormones, and exposure to those EDCs may be the main cause of gender disparity. The epidemiological links between autoimmunity and ASD are persuasive. In the following section, we will detail some of the links between ASD and maternal antibodies to the neuroantigens, as well as autoimmunity to numerous other neurodevelopmental disorders that are considered genetic in origin.

The Detection of Fetal Brain Neuroantigens in the Maternal Blood

As we have already pointed out, development of anti‐brain antibodies would be unusual and unlikely in normal circumstances and would defy the evolu tionary role of placental life forms to protect their fetuses from any kind of damage. However, we link the rising pandemic of autism to the invasion of chemicals, especially EDCs, into our daily life. These chemicals are responsible for the brain injuries that perturb normal brain development and most likely are responsible for leakage of neuroantigens into the maternal circulation. 242 Maternal Antibodies to Fetal Brain Neurons and Autism

Since the encroachment of the neo‐chemicals, many investigators also have been noticing the negative role of the maternal immune system and potential presence of anti‐brain antibodies [13,14]. In 1990, Warren et al. [15] noticed a connection between ASD and maternal autoantibodies that reacted to proteins displayed on the fathers’ immune cells, found predominantly in women with a history of miscarriage. These antibodies were suspiciously more common in mothers with children with ASD. This seminal study aroused further investigation and Dalton et al. [16] were able to show that a single sample from the serum of a mother with a child with ASD reacted to mouse neurons resulting in deficits in the exploratory behaviors in the resulting offspring when injected into a dam during gestation. Here we would like to define a type of antibody called IgG. In humans and other mammals, antibodies come in different forms (i.e., IgD, IgM, IgA, IgE, and IgG). IgG is the most abundant in human blood, plasma, and serum. IgG maternal antibodies readily cross the placenta to the fetus. These antibodies are highly beneficial to the newborns and serve as a protection against numer ous possible infectious agents [10,11]. These maternal IgG antibodies persist in newborns for up to 6 months after birth [11]. However, along with protective IgG antibodies, neuroantibodies can react to fetal “neuroantigens,” which can cross the placenta and cause damage to a developing fetal brain, if the mother has developed neuroantibodies to the fetal brain. Some of the most outstanding and elegant research studies were carried out by Braunschweig et al. [17,18] who analyzed 61 children with autism and 62 normotypic (normal) children, along with their mothers, and uncovered potential neuroantibodies in autistic children. Maternal antibodies that were present in 10% of the pregnant women were found to recognize certain anti gens in the developing fetal brain cells. An important aspect of the study was the understanding of the role of the BBB during fetal development. Western blot analysis revealed that maternal antibodies (IgG) reacted with fetal proteins (i.e., LDH, YBX1, cypin, STIP1, CRMP1, and CRMP2) and thus could adversely affected fetal neurodevelopment, since the BBB is permeable to the maternal IgG throughout the gestational period [11,18–20]. This study showed a signifi cant correlation between the paired maternal antibody reactive to fetal brain neuroantigens located within the 37‐ and 73‐kDa molecular‐weight bands on a human fetal brain immunoblot and a diagnosis of ASD in the child. Further, this reactivity was observed only in plasma samples obtained from families affected with ASD and not in families that had normal children or children diagnosed with developmental delay. This appears to play an important role in certain cases of ASD. The research suggests that over 50% of the mothers of children with ASD showed reactivity in relation to neuroantigens [17,18]. This hypothesis was further validated by utilizing the Rhesus monkey model [19,20]. Infusion of these neuroantibodies into the pregnant Rhesus monkeys showed that various combinations of neuroantibodies infused into the pregnant What are the Functions of the Neuroantigens that are Being Destroyed by the Maternal Antibodies? 243 monkeys can contribute to a higher percentage of ASD and that, when it comes to assessing behavior, combinations of these neuroantigens play a key role in behavioral activity [18–20]. Gender differences were also evident. Within vari ous male groups, a significant difference in growth rate in fetal brain develop ment was observed as compared with females. The unanswered question is why do certain mothers develop the antibodies to fetal brain antigens in the first place? We believe that this may be the result of leakage of developing fetal brain neuroantigens into the maternal blood circulation; initiating humoral immune responses to these antigens, after a synthetic or other type of insult (see below) that a fetal brain suffers. As mentioned above, since the brain is a privileged site in mammals, the leaked fetal brain antigens would be recog nized as foreign by the maternal immune system and subsequently affect fetal brain development. We would like to point out that our studies, shown throughout this book, have shown that oxytocin and AVP receptors are also severely damaged and have molecular weights that are close to fetal brain neu roantigens located within the 37‐ and 73‐kDa molecular weight bands. For example, the oxytocin receptor is composed of 389 amino acids and has molec ular weight of 43 kDa. Similarly, several of the AVP receptors in the human fetal brain range from 34, 41 and 47 kDa. The potential presence of neuroanti bodies to these antigens is under investigation in our research laboratory. Further studies by Braunschweig et al. [18], where they analyzed the banked mid‐pregnancy blood samples also observed that maternal autoantibody binding to neuroantigens near 37 and 73 kDa was only found in women whose children later received a diagnosis of ASD. Surprisingly, all the aforemen tioned reports were unable to find a correlation between a family history of autoimmunity and the presence of maternal neuroantibodies.

What are the Functions of the Neuroantigens that are Being Destroyed by the Maternal Antibodies?

As mentioned above, Braunschweig et al. [18–20] uncovered several neuroan tigens against which the mothers with ASD children formed neuroantibodies (i.e., LDH, YBX1, cypin, STIP1, CRMP1, and CRMP2). So, what are these anti gens and what do they really do? How would a fetal brain be harmed by these antibodies? Do they affect the behavior of children exposed to these neuroan tibodies during their fetal development? In one of their research studies, they were able to link the neuroantibodies against LDH, YBX1, cypin, STIP1, CRMP1, and CRMP2 with lower expressive language scores in the children whose mothers had antibodies to these neuroantigens. Furthermore, the absence of these antibodies correlated with development of normal children (in the control mothers). Most importantly, it was noted in a follow up study that children born to mothers with this antibody‐binding pattern also 244 Maternal Antibodies to Fetal Brain Neurons and Autism

exhibited abnormal brain enlargement when compared with both children with ASD born to mothers who did not harbor neuroantibodies and normally developing control children. Further, reactivity to a band near 39 kDa was later discovered, and paired reactivity to neuroantigens at 39 and 73 kDa correlated with a broader diagnosis of ASD (which was distinct from full autism at that time), as well as increased irritability on the Aberrant Behavioral Checklist scale [19]. We would like to remind readers here that the molecular weight of one of the most important communication molecules (i.e., oxytocin and AVP receptors) falls near this range. Of course, in order to comprehend how these neuroantibodies could poten tially contribute to the development of ASD one needs to figure out what their functions are during fetal brain development. Studies by Edmiston et al. (13), which utilized highly sophisticated analytical tools, determined that the mater nal neuroantibodies recognize seven developmentally regulated proteins in the fetal brain that include LDH A and LDH B, stress‐induced phosphoprotein 1 (STIP1), guanine deaminase, collapsin response mediator proteins 1 (CRMP1) and 2 (CRMP2), and Y‐box binding protein 1 (Figure 8.1). These proteins are critical for normal brain development. They are necessary for a fetal brain’s development and play a critical role in neuronal migration and neural network construction. For example, guanine deaminase has an integral role in dendritic branching of hippocampal neurons. The hippocampus is an essential part of the human limbic system and regulates emotions and long‐term memory. It is good to keep in mind that some ASD children lack both of these normal func tions. STIP1, in combination with the cellular prion protein, is responsible for neuritogenesis. Neuritogenesis is a key process in fetal brain growth. This pro cess is where neurons develop and, more precisely, the individual neurons sprout out axons from their bodies. These are the main events in neurodevel opment that create connections between neurons. The sprouting of neurites (axons and dendrites), which mature into axons and dendrites, is an important event in fetal brain neuronal differentiation. Studies in living neurons indicate that neuritogenesis begins immediately after neuronal commitment, with the activation of membrane receptors by extracellular cues. These receptors acti vate intracellular cascades that trigger changes in the actin cytoskeleton, which promote the initial breakdown of symmetry. Then, through the regulation of gene transcription, and of microtubule and membrane dynamics, the newly formed neurite becomes stabilized. Any interruption in the molecular machin ery that regulates the neuritogenesis during initial neurite sprouting can dis rupt the balance of neurodevelopment. These two proteins also increase neuronal survival. CRMPs 1–5 are necessary for proper growth of fetal neu rons and are required for proper cell migration and axon‐dendrite specifica tion. Y‐box binding protein 1 is involved in almost all DNA‐ and messenger RNA‐dependent processes and LDH is essential for energy metabolism. Animal Models and Neuroantibodies to Autism 245

Further, studies by Fox‐Edmiston et al. [14] described behavioral outcomes in the children of neuroantibody‐positive mothers. These mothers who had ASD children also produced antibodies to LDH, STIP1, and CRMP1 antigens. About 7% of mothers who produced these combined antibodies also had chil dren with ASD with an increase in stereotypic behaviors in the child. Maternal neuroantibodies have been associated with the behavioral and mental deficits that characterize ASD, but because these studies have been conducted in human participants, it is difficult to determine the mechanism by which these neuroantibodies lead to ASD. Experimental animal models were used to decipher if these neuroantibodies play a pivotal role in ASD.

Animal Models and Neuroantibodies to Autism

In order to determine whether maternal neuroantibodies are important in the development of neurodevelopmental disorders, researchers have utilized several animal models. The most significant findings are described below.

Rhesus Macaques Model

Rhesus macaques are a very useful animal model to study human neurodevel opment because of their social repertoire and established battery of social tests that can objectively measure their behavior. In a study carried out by Martin et al. [19] in which neuroantibodies were infused into to pregnant rhesus macaques, the group of monkeys that was exposed through passive transfer to purified IgG neuroantibodies from mothers of children with ASD had signifi cantly more ASD stereotypic behaviors and higher levels of motor activity when placed in an unfamiliar social setting than monkeys treated with IgG from mothers with normally developing children [20]. Furthermore, these findings were replicated in a larger study using monkeys who were adminis tered IgG from mothers of children with ASD specific for the dominant 37‐ and 73‐kDa band pattern (the three combined neuroantibodies that correspond to LDH, CRMP1, and STIP1). In the macaques model, it was observed that offspring treated with IgG from mothers of children with ASD exhibited abnormal social behaviors and, most surprisingly, enlarged brain volume com pared with control IgG‐treated animals (which were infused with IgG from normal mothers). It was also found that the pregnant macaques receiving the ASD‐associated IgG displayed heightened maternal protectiveness toward their progeny [21]. These studies enhance our insight into the potential mecha nism by which the maternal neuroantibodies may impart neuromodulation in developing fetal brains and contribute to ASD. We will come back to the obser vation of brain enlargement. 246 Maternal Antibodies to Fetal Brain Neurons and Autism

Studies Using Rodent Models

Parallel to the studies described above that were carried out in rhesus macaques, scientists duplicated the studies in mice in which pregnant mice were infused with a single intravenous injection of human maternal plasma. The plasma contained neuroantibodies that were reactive to the 37‐ and 73‐kDa banding pattern. The offspring from the mice exhibited increased anxiety and response to stress, along with impaired motor and sensory development [20]. The investigators concluded that the offspring born to pregnant mice injected with IgG from mothers with autistic children, and not from mothers with normally developing children, showed some of the symptoms seen in ASD, including alterations in sociability and increased activity and anxiety [22].

Why Do Some Autistic Children Have Bigger Brains?

The normal human offspring is born with limited processing and behavioral abilities since at the time of birth, the neural circuitry is still limited in many brain regions, most especially in the compartments representing higher order reasoning, language, social, emotional, and self‐awareness functions. Throughout the first years of a newborn, the rise of functional ability depends heavily on the establishment, generation and improvement of innu merable eruptions of neural circuitry. At this stage of neurogenesis, syn apses are being formed between near and distal neurons at lightning speed. These processes are integrated with high speed experience based learning to very high neuronal proliferation, and differentiation, and myelination. This is one of the most important periods of a newborn’s learning processes that permanently stabilize adaptive connections. These essential growth and selection processes follow regionally ordered and precisely timed sequences, and as a result neurobehavioral functions emerge and become refined in an orderly hierarchical fashion during the first year of life and continue in a similar manner for a few more years. During the first year and subsequent two years, the rate of brain growth and the regions where the brain growth is critical appear to differ significantly in ASD babies as compared with nor mal babies [23] (Figure 8.4). It is during the first few years of brain growth combined with the experiential learning period that an ASD baby is differentiated from a normal one. During the first year it is not possible to observe any clear‐cut difference between the two babies but innumerable retrospective studies and parental observations based on subtle motor, sensory, attention and social behavioral abnormalities show differences may be present as early as the first or second year of life [23]. However, around 2–3 years, ASD babies fail to reach normal language and Why Do Some Autistic Children Have Bigger Brains? 247

Autistic child Cerebral hemisphere Spinal cord

Hind brain Mid brain Mid brain 40 days Fore brain 35 days 50 days Cerebellum

Pons

25 days Medulla 100 days

5 months

9 months

Figure 8.4 The potential mechanism by which an autistic brain is differentiated from a typical growing fetal brain. We propose that the actual injury occurs during the first trimester, predominantly in the future forebrain. Certain progenitor cells are destroyed by chemicals or other environmental insult, which allows other progenitor neurons to fill the empty space created by the death of the target neurons. These new progenitor neurons grow at a faster rate than the original progenitor neurons (i.e., oxytocin‐ or AVP‐receptor positive neurons) and, subsequently, result in a larger than average brain size. social developmental milestones. Usually, at around 3 years old there is enough evidence to suggest to parents and physicians that a child might have autism. Interestingly, magnetic resonance imaging studies of babies that utilized head circumference as a potential guide of brain size during the first few years of life have identified a remarkable observation in ASD. The children with ASD experience an unusual period of disproportionate brain growth during the first few years of life, a period of time that coincides with the commencement of ASD symptoms. Importantly, the rapid increase in brain size is followed by an atypically reduced rate of brain growth. Several studies have shown that 248 Maternal Antibodies to Fetal Brain Neurons and Autism

increased brain growth may be more prominent in frontal lobes, which is the largest region of the human brain and is the largest among all life forms. The frontal lobes go through growth and selection processes later and longer than other areas of the brain. We believe that this region of the brain is most affected from exposure to synthetic chemicals during the early stages of fetal brain development. Selective destruction of fetal brain progenitor cells subsequently leads to the abnormal growth pattern during the first few years of the postnatal period in ASD children. Dysregulation of frontal lobe progenitor neurons would result in dysfunction in higher order social communication, emotional processing, language, and cognition, while regions mediating more low‐level functions would be relatively spared (Figure 8.4). We have hypothesized that early rapid growth in ASD is the result of abnormal proliferations of types of neurons that have replaced the oxytocin‐ and AVP‐ receptor positive neurons at the early stage of fetal brain development. These replacement neurons infil trate into the empty regions (small holes) left after the normal neurons have been killed and they have a higher proliferation rate than the normal neurons that were killed by neurotoxic chemicals. We are currently working on an in vitro model to establish this process. This concept is depicted in Figure 8.4. Some evidence from the most recent studies carried out in mice elucidated a potential mechanism by which maternal neuroantibodies modify fetal brain development. In one study, investigators injected specific maternal neuroanti bodies into the cerebral ventricles of embryonic mice. These injections resulted in increased cellular proliferation in the subventricular zone, increased size of adult cortical neurons, and increased adult brain size and weight compared with animals exposed to antibody from normal mothers with normal children [13,14]. It should be noted that these studies are in mice and do not perfectly represent a huge human brain, but they do give us a glimpse into the bigger brain phenomenon. In another parallel study, the investigators focused on the behavioral outcomes of the exposed offspring. The offspring injected with neuroantibodies exhibited increased stereotypic behaviors and altered social phenotypes observed in the children born to mothers with this brain pattern reactivity [24]. Of note, it is well documented that maternal antibodies, espe cially IgG, are able to reach the fetal brain, at least during the early stages of gestation when the fetal BBB is still under construction [25].

Is There a Link between Autoimmunity and Other Forms of Neurodevelopmental or Neurodegenerative Disorders?

In logical and pure immunological terms, if one attempts to find an association between autoimmunity and ASD it would be like looking for a needle in a hay stack. Simply, the phenomenon we described above, where fetal neuroantigens would leak into the maternal blood and the maternal neuroantibodies would Is There a Link between Autoimmunity and Other Forms of Neurodevelopmental 249 contribute towards ASD, is a highly specific phenomenon and cannot be gen eralized to other autoimmune diseases where a mother is suffering from an autoimmune disorder(s). In the case of any autoimmune disease, a mother suffers from a breakdown of the normal immune system and develops anti bodies to a “specific” antigen or antigens of her own tissue. These disorders originate from the mother’s own body and not from a fetus (which we described above in detail). For example, in the case of autoimmune hemolytic anemia, a mother forms antibodies to Rh I antigen found on her own red blood cells. This should not be confused with erythroblastosis fetalis, where the problem arises from the incompatible fetus (i.e., Rh+ red blood cells). It would be ridiculous to claim that all mothers who were exposed to Rh+ red blood cells from their fetuses would have ASD children! Similarly, in another example, in the case of acute rheumatoid arthritis, the mother forms antibod ies to a common bacterial agent, Streptococcus. Antibodies formed to this pathogen can result in arthritis (inflammation of joints), myocarditis (inflam mation of heart tissues), and sometimes inflammation of heart valves. In the majority of cases, the autoantibodies a mother forms are directed to her own body and may not have any effect on the developing fetus in her womb. However, we believe that in some cases and in some forms of autoimmune disorders, the mother can form antibodies to a wide range of antigens in her own body that have the potential to cross the placenta and reach a developing fetus. A prime example of such an unusual illness is systemic lupus erythema tosus (SLE). In this case, a SLE patient develops antibodies to DNA, histones, ribosomes, small nuclear ribosomal nuclear proteins, and a large array of other tissue antigens. If the maternal IgG autoantibodies cross the placenta, they may harm the fetus. Keeping the above limitations in mind and that all mothers suffering from autoimmune disorders would not give birth to ASD offspring, a large study by Vinet et al. [25] evaluated whether children born to mothers with SLE would have an increased risk of ASD compared with children born to mothers with out SLE. Their study population included 719 children born to 509 SLE women and 8,493 children born to 5,824 control women. Children born to women with SLE more frequently had a diagnosis of ASD compared with con trols. The difference between the SLE and control groups was 0.8%. In conclu sion, compared with children from the general population, children born to SLE women had an increased risk of ASD, although, in real life, it represents a rare event. If should also be noted that SLE individuals are clinically different than typical individuals. A study by Tiosano et al. (26) analyzed 5,018 indi viduals with SLE and 25,090 matching controls for schizophrenia in Israel. They found a positive association between SLE and schizophrenia across patients of different age, gender, and social economic status. This association may provide additional insight into our understanding of the pathophysiology of the two disorders. 250 Maternal Antibodies to Fetal Brain Neurons and Autism

A Chicken and Egg Conundrum

Several investigators have been looking at the possibility that some mothers with autoimmune disorders may possess antibodies to fetal brain antigens. Therefore, with regard to antibodies to different neuroantigens that we described in the preceding section, there is evidence that there may be maternal antibodies that target different fetal brain antigens than LDH, YBX1, cypin, STIP1, CRMP1, and CRMP2 that are in the 39‐ and 73‐kDa molecular weight bands. An association between the presence of neuroantibodies and the inci dence of numerous behavioral disorders has been described [27–29]. Antibodies reactive to neuronal tissues have been identified in schizophrenia [27–30].

Tourette Syndrome [30–32] and Obsessive Compulsive Disorder [33]

Unlike the neuroantibodies identified and well‐characterized in mothers of ASD children, the neuroantibodies observed in children, and in other neurologic disor ders, bind to adult, rather than fetal, brain tissue, suggesting that they are targeting adult or older children rather than the fetal brain. In neurologic illnesses that arise later in life, such as schizophrenia, these different kinds of neuroantibodies may be crossing the BBB after the child is born, when the BBB is sealed! It is hypothesized that there must be another event that increases barrier permeability, allowing anti bodies to traverse the BBB and alter neuronal processes that cause episodic schizo phrenia, Tourette syndrome, obsessivecompulsive disorder, and many similar psychiatric disorders. We have been working on the potential role of synthetic chemicals that activate Interleukin‐6 (IL‐6; [34]). IL‐6 has been shown to make the BBB transiently leaky (see Chapter 9). The question is, if maternal neuroantibodies are playing a role in many neurodevelopmental disorders, then how are the key neuroantigens entering the maternal blood circulation? We hypothesize that the leakage of brain antigens is the result of synthetic chemicals that reach the adult brains as much as they are reaching the fetal brains. Environmental factors and tim ing of exposure during different stages of fetal development and after birth are the major contributors to the differing symptoms and manifestations of ASD as well as numerous neuropsychiatric disorders, which are all rising at an alarming rate. We believe that the real culprits are the synthetic chemicals that first damage certain fetal brain neurons, release the neuroantigens, and enter the maternal circulation, and the neuroantibodies produced by the mothers, cause the damage [34].

Other Contributing Factors in Neuropsychiatric Disorders

Infectious Flu Virus Influenza Vaccine and Narcolepsy In recent years there has been a special interest in vaccination with Pandemrix–a pandemic influenza A vaccine – and the development of narcolepsy. The Other Contributing Factors in Neuropsychiatric Disorders 251

concern emerged from reports that increased numbers of cases of narcolepsy were apparent in nonvaccinated subjects infected with wild Influenza A (H1N1) pandemic influenza virus. This suggested a role for the viral antigen(s) in disease development [35–37]. Ahmed et al. (35) have found that the peptide in influenza nucleopeptide A (one of the antigenic compo nents in the Pandemrix influenza vaccine) shares an uncanny similarity with human brain molecules (hypocretin receptor 2), which have been linked to narcolepsy. Due to the enormous fear that emerged after reports that a global influenza A (H1N1) pandemic would kill billions of humans in June 2009, mass vaccination campaigns were carried out using newly developed monovalent influenza A (H1N1) pandemic vaccines. A number of coun tries, using a range of vaccine protocols, carried out large scale immuniza tions. An estimated 31 million doses of European AS03‐adjuvanted A (H1N1) pandemic vaccine were used in more than 47 countries, and the Canadian AS03‐adjuvanted A (H1N1) pandemic vaccine was given to 12 million people. As no similar narcolepsy association has been reported to date with the AS03‐adjuvanted A (H1N1) pandemic vaccine made using the Canadian inactivation/purification protocol, it appeared that the AS03 adjuvant alone may not be responsible for the narcolepsy association. To date, no narcolepsy association has been reported with the MF59®‐adju vanted A (H1N1) pandemic vaccine. The connection between narcolepsy and the Pandemrix vaccine may be a factor in other cases where cross reac tivity occurs. Looking back on the percentage of Americans vaccinated for influenza, focusing on child‐bearing women, if this does contribute to autism, then the rate of occurrence would be significantly higher. Furthermore, a blanket association with influenza vaccine and development of ASD may not be scientifically valid, since each year a new vaccine is pro duced directed against the predicted new strain of influenza virus. Therefore, it is unlikely that all different strains of anti‐influenza with variations in antigenic molecules would have adverse effects on fetal brain development. In addition, it appears that from the global H1N1 vaccination we have learned that certain adjuvants may be better in prevention of potential nar colepsy [38,39]. Most recently, Mahic et al. [38] have addressed the issue of influenza infec tion during pregnancy. These investigators obtained information from ques tionnaires and samples from the Autism Birth Cohort, a prospective birth cohort comprising mothers, fathers, and offspring recruited in Norway from 1999 to 2008. Through questionnaires, referrals, and linkages to the Norwegian National Patient Registry, they identified 338 mothers of children with ASD and 348 frequency‐matched controls for whom plasma samples had been col lected mid‐pregnancy and after delivery. They analyzed the influenza virus sera from both groups and found that neither influenza A nor influenza B virus infection was associated with increased ASD risk. Integration of reports of symptoms of influenza‐like illness with serology revealed an increase in risk 252 Maternal Antibodies to Fetal Brain Neurons and Autism

for women who tested positive for influenza infection with symptoms, but this increase was not statistically significant (P<0.05) in comparison with women who tested negative for influenza infection and who were without symptoms. The authors proposed that even though evidence of gestational influenza virus infection alone (by laboratory test) was not associated with risk, positive labo ratory tests and symptoms of influenza‐like illness cannot yet be definitively ruled out as a risk factor for ASD. This means that there may be a slight risk for ASD, according to the authors. We believe that this is like looking for a needle in a haystack, and the risk may be minuscule and may not be related to the influenza infection during pregnancy and there may be other factors. A comprehensive report by Persson et al. [39] who analyzed over 3.3 million individuals vaccinated with Pandemrix and compared them with 2.5 million control individuals for any neurological and immunological diseases, including narcolepsy, concluded that for a large number of selected neurological and immune‐related diseases, there was neither confirmation or any causal asso ciation with Pandemrix nor did it refute entirely a small excess risk. They confirmed an increased risk for a diagnosis of narcolepsy in individuals below 20 years of age and observed a trend towards an increased risk amongst young adults between 21 years old and 30 years old [39]. Here, we would remind the readers that environmental factors, which were not considered in the large‐ scale analyses, may be contributing to some of these unusual issues. Flavored drinks that are so prevalent in fast food, and flavored food that contain EDCs and plastic bottles that could leach out bisphenol A and other neurodamaging chemicals must be kept in mind.

Other Viral and Nonviral Infections

As we have described in detail, there is a great body of research that suggests maternal infections during pregnancy are associated with the risk of neurode velopmental disorders, including ASD [37–40]. The major reason for such an association is that there are numerous pathogenic agents that infect a growing fetus, particularly during early fetal development when the BBB is still weak and infectious agents can penetrate the fetal brain. We will describe a detailed account of BBB development during various stages of fetal development shortly, but at this juncture it is sufficient to state that the fetal brain is not equally susceptible to infectious agents at all stages of development and protective mechanisms keep the fetus free from most of the infections at later stages of development, as opposed to early stages of fetal development. A prime example of the development of a stronger BBB is congenital Rubella infection [41]. In the case of maternal infection with Rubella, the virus crosses the placenta and infects the fetus prior to week 18 of gestation, but after weeks 18–20 of gestation there are unlikely to be any con genital defects in the fetus [42,43]. Similarly, in the current pandemic of the Zika Other Contributing Factors in Neuropsychiatric Disorders 253 virus, it is well established that there are significantly fewer congenital anomalies in fetuses infected after the first trimester [42]. Mahic et al. [38] have recently evaluated possible associations between maternal infection with Toxoplasma gondii, rubella virus, cytomegalovirus (CMV), herpes simplex virus 1 (HSV‐1), and HSV‐2. They analyzed the serum samples collected during mid‐pregnancy from 442 mothers of children with ASD and compared them with 464 frequency‐matched control IgG antibodies to the infectious agents mentioned above (from Norway). They uncovered that high levels of HSV‐2 IgG antibodies in maternal mid‐pregnancy plasma were associated with increased risk of ASD in male offspring when adjusted for parity and child’s birth year. No association was found between ASD and the presence of IgG antibodies to Toxoplasma gondii, rubella virus, CMV, or HSV‐1. Their study has created controversy and awaits replication from other scientists. More recently, epidemiological studies have reported increased risk of ASD associated with maternal infection during pregnancy. However, as we stated above, that high risk may only be associated during the first trimester of the maternal infection, since we believe that the fetal BBB is much firmer after the first trimester. Generally, the results are negative with regards to maternal infection and having offspring that develop ASD. For example, a population based study of all children born in Denmark from 1980 to 2005 found no overall association between maternal infection diagnosis and ASD over the course of the entire pregnancy, but did report a nearly threefold increased risk for ASD following hospitalization for viral infection in the first trimester as well as an increased risk following hospitalization for bacterial infections in the second trimester [44]. Similarly, in another Danish population based study there was no association between common cold during pregnancy and an increased risk of ASD [45]. The only caveat was influenza infection that was associated with a nearly twofold risk of ASD and fever for >1 week was associ ated with a nearly threefold increase. The fever appears to be an issue during early pregnancy and a study from California (Kaiser Permanente) found that fever during pregnancy, particularly fever experienced without taking anti‐ fever medication, was associated with an increased risk of ASD, though over all experiences of maternal influenza were not associated with an increased risk in this study [46]. A subsequent study found that severe maternal infec tions that required hospitalization were associated with an increased risk of ASD, whereas diagnosed infections that did not need hospitalization were not associated with ASD. Here, we would like to point out that the potential conundrum of influenza infection and the potential ability of influenza anti bodies to react with neuroantigens needs further investigation, as pointed out by many investigators. We hypothesize that the leakage of the fetal antigens into the maternal blood may be the result of certain environmental factors that are cytopathic to 254 Maternal Antibodies to Fetal Brain Neurons and Autism

specific progenitor cells in the developing fetal brains, resulting in develop ment of maternal autoantibodies. This hypothesis can be examined by inject ing fetal brain antigens into pregnant Rhesus monkeys at early gestation. Our hypothesis may explain why a small percentage of mothers give birth to multi ple ASD children. However, we do not discount the possibility that other anti gens, after an infection with viral agents or microorganisms that cross react with fetal brain antigens, may play a role in the development of ASD.

Blood–Brain Barrier

What is the BBB?

In humans the CNS is protected by a complex vascularized and dense capil lary connection that serves as a barrier against any insult from the blood circulation. This highly sophisticated system allows the measured transport of oxygen and essential nutrients and removes carbon dioxide and harmful waste products. The BBB is the result of a specialized construct of blood vessels. These blood vessels are distinguished from vasculature in other tis sues, enabling CNS vessels to strictly regulate the delivery of cells (i.e., micro glial cells, monocytes), molecules and ions between the circulatory system blood and the CNS tissue (Figure 8.5). Therefore, a better understanding of the true nature of the BBB in a developing fetus is essential in understanding the ways a fetal brain may sustain injury from synthetic chemicals, infections, and mechanical trauma of the woman carrying the fetus. As shown in Figure 8.5, during fetal development many of the properties of the BBB are possessed by the endothelial cells that form the walls of the blood vessels but are assimilated through a series of complex cellular interactions with the microenvironment. The BBB is not an organ but is a “neurovascular unit” and it comprises the endothelial cells, pericytes, microglia, astrocytes, and basement membrane that are characteristic of the cerebral vasculature (Figure 8.5). It is a widely believed among neuroscientists that the BBB in the embryo, fetus, and newborn is “immature” and poorly formed. This unfortunate con cept of a so called “‘leaky” BBB is incorrect [47] and without any scientific evi dence; it was thoroughly examined and rebuffed by Saunders et al. [47] in their outstanding and comprehensive review of this subject. One historical reason for the belief in barrier immaturity comes from an early paradigm that was established in the 1930s that the fetal brain would not need a barrier, because the fetus is protected by a placenta (Figure 8.5). The developing brain is neces sarily immature compared with that of the adult, but the real question should be about the functional status of the BBB mechanisms in embryos, fetuses, and infants, compared with adults. Blood–Brain Barrier 255

Abluminal membrane Tight junction

Astrocyte

Luminal membrane Astrocyte Brain capillary Blood Hg

Endothelial cell

Neuron

Pericyte Basement membrane

Figure 8.5 Cross section of a cerebral capillary. This is the structure of the blood–brain barrier. The blood–brain barrier is not a distinct organ, but rather a functional concept. Source: http://captain‐nitrogen.tumblr.com/post/4927485305/a‐fortress‐of‐intelligence.

In two of the examples mentioned earlier, Rubella infection and Zika virus infection of the fetus, the congenital anomalies are rarely observed after 20 weeks of gestation even though maternal viremia can be documented [41]. These observations point towards the presence of a functional BBB in a fetus after 20 weeks of gestation. Recent evidence confirms that the fetal brain develops tight junctions and many of the transport mechanisms between the blood, brain, and cerebrospinal fluid (CSF) during the very early stages of development. Some properties of these barrier mechanisms and their susceptibility to disruption may lead to brain damage and later neurological disorders. Understanding the role of BBB mechanisms in normal brain development and possible deleterious effects should these mechanisms be dysfunctional is important from the clinical perspective of whether or not drugs or toxins, once they cross the placenta, may have access to the vulnerable developing brain. A reason given by regulatory bodies in the USA and the European Union for caution in giving drugs to pregnant women or infants is the “imma turity” of the BBB [38]. There is no doubt that blood vessels in the developing fetal brain are signifi cantly more fragile than in an adult brain. These concepts were established from experiments in which excessive volumes of fluid were injected into fetuses [47]. A specific example of susceptibility of cerebral blood vessels in 256 Maternal Antibodies to Fetal Brain Neurons and Autism

the developing brain is illustrated by the effect of lipopolysaccharides on the permeability of blood vessels in brain tissue at a critical stage of brain develop ment, carried out in rats and opossums equivalent to weeks 22–28 gestation in humans. In these experiments these blood vessels showed a leakage of plasma proteins that were confirmed in postnatal animals. In a clinical setting if a mother develops an infection, the fetus may be born prematurely and in some cases brain injury has been detected with the devel opment of cerebral palsy. Leakage of proteins from plasma into white matter in the presence of maternal infection has been suggested to be part of the etiol ogy. As elegantly reviewed by Saunders et al. [47], many other possibilities of functionally important changes in influx or efflux mechanisms following a pathological insult to the developing brain have rarely been considered. In evolutionary terms, it is counterintuitive to believe that placental life (viviparous) forms that give live birth and carry the placenta would be so suc cessful if they did not co‐evolve parallel tools to protect their fetuses against pathogens. In many circumstances, viviparity of various forms offers excellent protection from microbial and viral infections and predators and permits flexibility in adapting to unreliable weather and adverse circumstances. The evidence presented would attest to the fact that very few adverse natural circumstances can perturb the balance of life between a pregnant woman and her offspring. It is certain that our ancestors and early humans were exposed to a much greater number and variety of infections agents than we are today. So, why are ASD and the other myriad of neurodevelopmental and neuropsy chiatric disorders increasing at such a high rate? We believe that manmade chemicals are hurting human life on earth.

Summary and Conclusions

Maternal antibodies directed against fetal brain antigens are detected in ASD by numerous researchers in a subset of mothers of children with ASD. Further, there is now an abundance of evidence supporting their deleterious role in neurodevelopment. For the most part, these studies have described similar experimental outcomes, and given the clinical and biological heterogeneity of ASD, there likely exists a complex relationship between the presence of mater nal neuroantibodies and the developmental course of exposed offspring. It is still unclear how and when these maternal neuroantibodies arise in the mater nal blood and what factors permit the leakage of neuroantigens from a fetal brain into the maternal circulation. We have provided detailed analyses of the mechanism underlying the origins of fetal neuroantigens and the maternal origins of neuroantibodies– autoantibodies, which cannot be generalized as causes of other neuro developmental and neuropsychiatric disorders. However, we believe that References 257 further characterization of neuroantigens may provide appropriate therapeutic interventions that may block the production of those specific neuroantigens, and neutralize them by blocking antibodies (i.e., anti‐Rh antibodies given to mothers with Rh‐incompatibility to protect against development of erythro blastosis fetalis), thus raising the optimistic prospect that some future cases of ASD or related neurodevelopmental and psychiatric disorders could be prevented.

References

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44 Vogel N, Kirisits M, Michael E, et al. (1996). Congenital toxoplasmosis transmitted from an immunologically competent mother infected before conception. Clin. Infect. Dis., 23:1055–60. 45 Landais I, Nelson JA (2013). Functional genomics approaches to understand cytomegalovirus replication, latency and pathogenesis. Curr. Opin. Virol., 3:408–15. 46 Woo EJ, Winiecki SK, Arya D, Beeler J (2016). Adverse events after MMR or MMRV vaccine in infants under nine months old. Pediatr. Infect. Dis. J., 35(8):e253–7. 47 Saunders NR, Liddelow SA, Dziegielewska KM (2012). Barrier mechanisms in the developing brain. Front. Pharmacol., 3:46. 261

Vaccines and Autism

There are over 165 studies that have focused on Thimerosal, an organic‐ mercury (Hg) based compound, used as a preservative in many childhood vaccines, and found it to be harmful. Of these, 16 were conducted to specifically examine the effects of Thimerosal on human infants or children with reported outcomes of death; acrodynia; poisoning; allergic reaction; malformations; auto‐immune reaction; Well’s syndrome; developmental delay; and neurodevelopmental disorders, including tics, speech delay, language delay, attention deficit disorder, and autism. Brian Hooker, Janet Kern, David Geier, et al., 2014, Methodological issues and evidence of malfeasance in research purporting to show thimerosal in vaccines is safe. Biomed. Res. Int., 2014:247218.

The risk of serious complications associated with the development of diseases in unvaccinated individuals far outweighs the potential risk of adverse consequences associated with immunization with thiomersal‐ containing vaccines. Aleksandra Goloś and Anna Lutyńska, 2015, Thiomersal‐containing vaccines – a review of the current state of knowledge. Przegl. Epidemiol., 69(1):59–64, 157–61.

Childhood Vaccines and Regressive Autism

Childhood immunization is a factor that has received much scrutiny as a potential environmental cause of autism. Claims of a link between vaccines and autism first arose in the late 1990s in England, the USA, and other

industrialized countries and were triggered by clinical observation of the onset of autism in the days immediately following vaccination [1–7]. In England, these claims focused on the measles–mumps–rubella (MMR) vaccine [2,3]. In the USA, the focus was on thimerosal, a preservative that contains ethyl mercury, which was added to multi‐dose vials of many vaccines as a preservative to prevent bacterial and fungal growth. In multi‐dose vaccine vials, needles are inserted into the vaccine bottle many times, which can introduce contamination into the vial [1–4]. In the 1990s, thimerosal was used in most pediatric vaccines. Although all of the vaccines on the currently recommended Centers for Disease Control and Prevention (CDC) early childhood vaccination schedule are available as thimerosal‐free, parental perceptions that vaccines pose safety concerns are affecting vaccination rates, particularly in light of the much expanded and more complex schedule in place today. Table 9.1 depicts the recommended childhood and adolescent immunization schedules approved by the American Academy of Pediatrics, the Advisory Committee on Immunization Practices of the CDC, the American Academy of Family Physicians, and the American College of Obstetricians and Gynecologists in 2017 (https://www.cdc.gov/vaccines/schedules/hcp/imz/ child‐adolescent.html). In addition to the childhood vaccinations, adults also receive numerous vaccines at different stages in their life. In Table 9.2 we list these highly recommended vaccines that are administered regularly to adults in industrialized nations. Many of these vaccines contain weakened live viruses (attenuated viruses). Live virus based vaccines should be avoided in children and adults with compromised immune systems. We will return to this topic later in the chapter.

Politics Versus Science in the Vaccination Era

Currently, vaccination is a prominent topic on the political stage. On one side are the autism deniers, who generally believe that vaccination has no side effects. On the other side are vaccine safety advocates, who contend that many vaccines are unsafe and are responsible for untold illnesses, particularly MMR, which some believe is responsible for regressive autism (more later). However, key errors and conflicts of interest continue to be discovered in papers describing studies trying to link vaccines to autism, leading to the papers being with- drawn from publication in scientific journals [1–8]. It should be realized, however, that neither side could be correct. It should be stated without any doubt that is always a small degree of risk associated with any kind of medicine used today, including something as seemingly benign as aspirin, and vaccines are prophylactic treatments to fend off more serious and T able 9.1 Recommended immunization schedule for children and adolescents aged 18 years or younger (USA, 2017).

19–23 Vaccine Birth 1 mo 2 mos4 mos6 mos9 mos 12 mos15 mos18 mos 2–3 yrs 4–6 yrs 7–10 yrs 11–12 yrs 13–15 yrs 16 yrs 17–18 yrs mos

Hepatitis B1 (HepB) 1st dose 2nd dose 3rd dose

2 Rotavirus (RV) RV1 (2-dose st nd See series); RV5 (3-dose series) 1 dose 2 dose footnote 2

Diphtheria, tetanus, & acellular 1stdose 2nd dose 3rd dose 4th dose 5th dose pertussis3 (DTaP: <7 yrs)

Haemophilus influenzae type b4 See 3rd or 4th dose, 1stdose 2nd dose (Hib) footnote 4 See footnote 4

Pneumococcal conjugate5 1stdose 2nd dose 3rd dose 4th dose (PCV13)

Inactivated poliovirus6 1stdose 2nd dose 3rd dose 4th dose (IPV: <18 yrs)

Annual vaccination (IIV) 7 Annual vaccination (IIV) 1 or 2 doses Influenza (IIV) 1 dose only

Measles, mumps, rubella8 (MMR) See footnote 8 1stdose 2nd dose

Varicella9 (VAR) 1stdose 2nd dose

Hepatitis A10 (HepA) 2-dose series, See footnote10

Meningococcal11 (Hib-MenCY nd ≥6 weeks; MenACWY-D ≥9 mos; See footnote 11 1stdose 2 dose MenACWY-CRM ≥2 mos)

Tetanus, diphtheria, & acellular Tdap pertussis12 (Tdap: ≥7 yrs)

13 See footnote Human papillomavirus (HPV) 13

See footnote 11 Meningococcal B11

5 Pneumococcal polysaccharide See footnote 5 (PPSV23)

Range of recommended Range of recommended ages Range of recommended ages Range of recommended ages fornon-high-risk No recommendation ages for all children for catch-up immunization for certain high-risk groups groups that may receive vaccine, subject to individual clinical decision making

Source: http://pediatrics.aappublications.org/content/133/2/357. 264 Vaccines and Autism

Table 9.2 Adult immunization schedule (age based recommendations).

19 – 26 27 – 49 50 – 59 60 – 64 Vaccine/Age group ≥ 65 Years Years Years Years Years

Tetanus, Diptheria, Pertussis Substitute one time dose of Tdap with Td, then Td booster every (Tdap) booster with Td every 10 years 10 years

Human pappiloma vaccine 3 doses

Varicella 2 doses

Zoster 1 dose

Measles, Mumps, Rubella 1 or 2 doses 1 dose

Influenza 1 dose annually

Pnemococcal (polysaccharide) 1 or 2 doses 1 dose

Hepatitis A 2 doses

Hepatitis B 3 doses

Meninngicoccal 1 or more doses

Recommended if some risk factor is present

All persons who meet the age criteria

No recommendation

Source: https://www.slideshare.net/abhaydhanorkar5/seminar‐adult‐immunization‐16550441.

deadly illness. Most importantly, the risk should be not denied and should be spelled out clearly and managed appropriately to prevent adverse events. In addition, the known risk factors need to be addressed and eliminated completely or minimized as far as possible. In the following section, we will describe factors underlying potential risk factors and suggest possible remedies. We also describe in detail the possible contributing chemicals and factors that could contribute to regressive autism.

A Short Glimpse of the History of Vaccines: Justification for Using Vaccines

In the pre‐vaccination era, the world was plagued with a plethora of illnesses that claimed the lives of millions of people annually. Diseases such as smallpox, diphtheria, anthrax, and pertussis killed millions of children before they reached puberty. Respiratory diseases like whooping cough obstructed airways, and polio crippled an untold number of children and adults. Even one of the US presidents, Franklin D. Roosevelt, was paralyzed from polio viral infection. What is in the Vaccines? 265

The idea of vaccination originated with an English doctor, Edward Jenner, in 1796. Jenner, located in a rural area, performed the first vaccination by inocu- lating the pus from a cowpox lesion from a milkmaid’s hand into an 8‐year‐old boy [9,10]. Vaccines as we know then today can be credited to the renowned microbiologist and chemist, Louis Pasteur, who created the first “vaccine” for rabies in 1885. He developed an antitoxin that was used as a post infection antidote via passive immunization. He is recognized for introducing attenuated and inactivated microorganisms into the human body to elicit an immunological response thereby preventing infectious disease (prophylactic vaccination) [9,10]. By the 1900s, after better understanding of the immune system, especially the adaptive arm of the immune system and the antigen– antibody relationship, individuals began receiving “vaccinations” to prevent the future occurrence of certain illnesses. It was not until the 1940s that the USA began recommending that all children be vaccinated. At the time, only diphtheria, pertussis, and tetanus (DPT), and smallpox were targeted with vaccines. Today more than 25 doses of vaccine are administered within 24 months of birth (https://www.cdc.gov/vaccines/schedules/hcp/imz/child‐adolescent. html; Tables 9.1 and 9.2). To this day, vaccines remain one of the leading factors that contribute to the welfare of children and their long, healthy life span. Therefore, it is reasonable to suggest that our modern societies cannot remain healthy and robust without vaccination. The major point of dispute is that we make sure that vaccines are safe and do not cause any unreasonable degree of risk to the recipients of the vaccines, both children and adults.

What is in the Vaccines?

The development of vaccines predates the discovery of the immune system [9] and our understanding of germ prevention and therefore it was not uncommon for individuals to acquire other diseases when receiving a vaccine. The very first vaccine was the smallpox vaccine that was derived from a live cowpox virus. Although it was similar to the deadly smallpox virus, it only caused localized ulcers at the site of inoculation and prevented infection from the highly pathogenic smallpox [11–14]. At this point, it would be pertinent to briefly mention that smallpox, like several other human pathogens, has been used as a biological weapon. Smallpox has deliberately and unwittingly been used as a weapon of biological warfare. Its arrival via an infected slave from the Narváez 1520 expedition is believed to be responsible for the massive post‐Columbian depopulation of Central and South America, which played an influential role in the conquest of Mexico and Peru [14]. The British are known to have employed smallpox as an agent of death during the French and Indian Wars (1754–1763) [14]. In Australia, it has been suggested that the British used smallpox pur- posefully in the 1789 defense of fragile military positions [14]. Throughout the 266 Vaccines and Autism

last century, American, Japanese, Soviet, and British researchers examined the potential for weaponizing smallpox but this was thwarted mainly due to the efficacy of vaccines. Individuals who received early versions of Jenner’s vaccine were immune to smallpox. Even before Jenner introduced his vaccine, it was a common folk remedy in many parts of Asia to scratch a dried scab from a smallpox ulcer on to an individual’s skin. Most of these “scratched” individuals experienced a less severe form of smallpox. These observations led to the practice of variolation where people were inoculated artificially using pus from a pock or dried scab. This practice, which likely originated in China during the 10th century, spread at first to India and, by the 13th century, appeared in Egypt [14]. By the 17th century, variolation was used widely, being introduced to England from the Ottoman territories by Lady Mary Wortley Montagu in 1721. Interestingly, Edward Jenner was also variolized prior to his discovery of vaccination in 1796. After the advent of heat‐killed and formalin‐fixed bacterial vaccines, most notably DPT, it was recognized that some sort of action needed to be taken to ensure that vaccines were effective, safe, and free from other microbial agents. This included the prevention of contamination and potential accidental transfer of other diseases or infections. Traditionally, it has been difficult to add antimicrobial agent(s) to live‐attenuated (weakened form of live virus) vaccines, since these agents can also harm the vaccine agents. Thus, preservatives began to be added. Today, vaccines contain several components that aid in the effectiveness and safety of the doses administered, in the form of adjuvants and preservatives. Adjuvants are agents that make vaccines more powerful, enhance their effects, and allow the immune systems to respond to the vaccine antigens more robustly [15,16]. Preservatives are designed to prevent contamination of unwanted microbes or viruses that can creep into a vaccine. Here, the addition of thimerosal comes in. Thimerosal is an organomercury compound known for its antiseptic and antifungal properties and has been used as an antibacterial agent in pharmaceutical products, including vaccines and other injectable biological products. Aside from thimerosal, some other chemicals being used in vaccines can have known harmful side effects when introduced into the body [16–22]. β‐Propiolactone is an organic compound commonly used since the 1950s as a sterilizing agent for blood plasma, vaccines, bone and tissue grafts, surgical instruments and enzymes. It is a colorless liquid with a slightly sweet odor and is highly soluble in water. Controversially, it is considered to be a potential carcinogenic agent, and has been shown to cause cancer when introduced to mice, hamsters, guinea pigs, and rats [23]. Another additive, formalin, a 40% solution of formaldehyde in water, is also used in vaccines to preserve the freshness and effectiveness of vaccines. As mentioned earlier, some of the bacterial agents in DPT vaccine were treated with formalin to render them inactive. When ingested, formalin can affect the What is in the Vaccines? 267 central nervous system (CNS), heart (cardiovascular system), and liver and kidney (hepato‐renal system), potentially leading to blood loss from the gut (gastrointestinal hemorrhage), cardiovascular collapse and unconsciousness or convulsions, and severe metabolic and acute respiratory distress syndrome [24–28]. This is a major chemical used by morticians to embalm a dead body. Aluminum is not a preservative but an immune stimulating agent and is present in a large majority of vaccines in an effort to induce a greater response to the agents in the vaccine. Not all vaccines require adjuvants. Live weakened vaccines (e.g., smallpox, cholera, typhoid, rabies, and some other vaccines) do not need adjuvants as they cause a milder form of the actual infection and so are effective without any adjuvants. Generally, adjuvants are substances added to vaccines to enhance the immunogenicity of the selected antigens that have insufficient immune‐stimulating capabilities, and have been used in human vaccines for almost a hundred years. Over the years, numerous adjuvants have been used and are still being used in many vaccines, including aluminum salt, and oil‐in‐water emulsions (sequalene, AS03). The choice and use of a combination of adjuvants in a given vaccine is considered a proprietary (similar to disclosure of synthetic chemicals in a perfume) and is generally not disclosed on the label of a vaccine vial. The experts who prepare and test the vaccines for vaccine manufacturers have been testing a large number of adjuvants to determine which ones work best for a particular vaccine to elicit a protective immune response. These vaccines are rigorously tested, including for safety and efficacy, typically in experimental animals (generally mice or rats) before they go to human use, but may not be tested as extensively for their potential effects on the developing brain. Some of the most difficult vaccines to test for potential adverse effects on humans are influenza vaccines since investigators have to predict which strains of influenza are going to be dominant in the next year and have to prepare millions of doses of vaccine before the influenza season arrives. That only leaves a small window to test the vaccine for its potential effects in real time. In the previous chapter, we provided a detailed account of various influenza vaccines that have been implicated in numerous neurological illnesses including narcolepsy and others associated with the H1N1 influenza vaccine. However, the CDC found no indication of any association between any US Food and Drug Administration licensed H1N1 or seasonal influenza vaccine and narcolepsy when they examined the US Vaccine Adverse Event Reporting System (VAERS) and the Vaccine Safety Datalink (VSD) [29]. Similarly, no increased risk for narcolepsy was found when the CDC analyzed 650,000 recipients of the 2009 pandemic influenza vaccine and 870,000 recipients of the 2010/2011 seasonal influenza vaccine. It is also important to note that a recent study found that influenza shot administration during pregnancy did not statistically increase the risk of autism diagnosis in the child [29]. However, it should be understood that adjuvants are still an evolving component of vaccination and their addition to a vaccine is a double‐edged sword. 268 Vaccines and Autism

On the one hand, the right combination of antigens and adjuvants can benefit a recipient’s adaptive immune response, enabling the development of new efficacious vaccines. On the other hand, some adjuvants may have unknown adverse effects. Some infectious diseases of global importance are not currently preventable by vaccination. Adjuvants are the most advanced new technology in the search for improved or novel vaccines against elusive pathogens and for vulnerable populations that respond poorly to traditional vaccines. Aluminum adjuvants are neurotoxic when injected directly into the spine of mice [30]. There is minimal knowledge that explains the actual mechanism behind this action. Aluminum has been linked to brain, bone and renal disease when exposed in excessive amounts [30,31]. Despite the notion that aluminum in vaccines is safe, experimental evidence has clearly refuted this claim by the vaccine makers. As a matter of fact, aluminum adjuvants have a potential to induce serious immunological disorders in humans. In particular, aluminum in adjuvant form carries a risk for autoimmunity, long‐term brain inflammation, and associated neurological complications and may thus have intense and widespread adverse health consequences. One of the most fascinating investigations examining potential effects of aluminum adjuvant took place in France in 1998 [32]. Safety concerns about the use of aluminum in vaccines arose in France as a result of deltoid muscle (the muscle that forms the rounded contour of the shoulder) biopsies in patients with a constellation of symptoms including muscle pain and fatigue. Close scrutiny of microscopic histological lesions revealed aluminum salts, which were shown to persist for up to 10 years. Because these lesions occurred at the traditional vaccination site in the deltoid, the illness was linked with the administration of aluminum‐containing vaccines [33]. However, to date, there is no reliable scientific evidence that absolutely links vaccination sites to long‐ term pain at the deltoid site [34,35]. In 2008 the World Health Organization (WHO) Global Advisory Committee on Vaccine Safety (GACVS) issued a statement concluding that: “From the most recent evidence available, there is no reason to conclude that a health risk exists as a result of administration of aluminium containing vaccines, nor is there any good reason for changing current vaccination practice. The GACVS will continue to review the evidence that might emerge from on‐going studies” [35]. Phenol and phenoxyethanol are also added to vaccines as a form of disin- fectant. Phenol, chemically known as carbolic acid, is produced both naturally and synthetically. Naturally, phenol can be found in coal tar, as well as in human and animal waste, indicating that it is present in foods that are consumed [36–38]. The CDC has reported that exposure to phenol by any route is capable of causing systemic poisoning, which includes transient CNS stimulation, followed very quickly by CNS depression. Other possible symptoms following exposure include nausea, vomiting, hypotension, arrhythmia What is in the Vaccines? 269

(irregular heart beat), pulmonary edema, tachycardia, and methemoglobine- mia. Phenoxyethanol is a very common personal care preservative, which is used as a bactericide. Studies have shown phenoxyethanol to be toxic to the human body, even in minute doses. Phenoxyethanol affects the nervous system, including the brain at moderate doses, the endocrine system, the bladder, and has also been seen to cause acute pulmonary edema in animals [38]. Polysorbate 80 is a frequently used surfactant in the cosmetic, pharmaceutical and food industries. The purpose of a surfactant is to reduce the surface tension and to act as an emulsifier, to increase the solubility between two liquids that are typically unable to dissolve together [36,39]. In vaccines, polysorbate 80’s purpose is to help disperse all other ingredients of the vaccine equally throughout the liquid. The use of this chemical generates concerns as it is used in medications to assist in the delivery of certain drugs or chemotherapeutic agents across the blood–brain barrier (BBB). Human research is limited, but based on animal research, we are aware of the potential for polysorbate 80 to affect the reproductive system and act as a possible carcinogen. Large amounts of this chemical can cause abdominal spasms and diarrhea. More alarming, animal studies have shown polysorbate 80 to cause cardiac abnormalities, changes in behavior, and weight loss. Studies by Coors et al. [39] have shown that polysorbate 80 can also cause severe nonimmunologic anaphy- lactic reactions. Glutaraldehyde is a sterilizing agent that is mostly used to disinfect equipment that cannot be heat sterilized. Glutaraldehyde has several applications in a number of different areas; it is commonly used as a preservative in cosmetics, a cold sterilant in the healthcare industry, a tissue fixative in histology and pathology laboratories, a hardening agent in the development of X‐rays, and also it is used in embalming solutions, to name just a few uses. The CDC notes this chemical to be harmful through inhalation and skin contact; however, no research has yet been completed evaluating the effect intravenously. Possible side effects include throat and lung irritation, asthma and difficulty breathing, contact and/or allergic dermatitis, nasal irritation, sneezing, burning eyes, and conjunctivitis [40–43]. Monosodium glutamate (MSG), which is a commonly used food spice, is believed to cause allergic reactions such as headaches, nausea, dizziness, skin rash, lethargy, numbness, rapid or irregular heartbeat, and excessive flushing. There is no definitive research to support this theory, but it has been acknowledged that a small percentage of people may experience these allergic reactions after consuming products which contain MSG [44–46]. Other components included in vaccines include tributyl phosphate, antibiotics, and genetically modified DNA and RNA. Tributyl phosphate is a liquid typically used as a plasticizer for cellulose esters, lacquers, plastics, vinyl resins and also in fire‐resistant aircraft hydraulic fluid [47]. The inclusion of antibiotics 270 Vaccines and Autism

increases the chances of developing an antibiotic resistance and the introduction of genetically modified DNA and RNA is risky in the sense that it may take years to fully understand the pros and cons of inserting foreign genes into our genome.

Thimerosal

Thimerosal is an organomercury compound which contains 50% mercury. It is commonly used as an antiseptic agent, a preservative in vaccines, in skin tests for allergies, eye drops, nasal sprays, tattoo inks, immunoglobulin preparations, and many other products. It was patented in 1927 by a University of Maryland chemist, Morris Kharasch, and was marketed by Eli Lilly as Merthiolate [47]. Thimerosal was first added into vaccines in 1940 as a preservative to prevent contamination of multi‐dose vials by inhibiting the growth of harmful microbes and fungus. Whenever a new needle is inserted into a vial it increases the chances of that vial being contaminated by bacteria, which may cause serious infection. This became apparent after two reported cases. One of the cases occurred in 1916 where four children died, and 60 others suffered from severe symptoms brought on due to contamination of a typhoid vaccine with Staphylococcus aureus. The other case occurred in Australia and claimed the lives of 12 out of 21 children who received a diphtheria vaccine containing the same bacterium [47]. It is also used in the preparation stages of some vaccines, for a similar purpose. Thimerosal via vaccines became a major contributor of mercury exposure to children in the first 36 months of life, exposing these children to an amount of mercury beyond that to be considered safe by guidelines [47,48]. Once metabolized by the body, thimerosal forms ethylmercury, which is bound to thiosalicylate [48]. We know that the half‐life of ethylmercury is shorter than that of methylmercury, but that does not discredit the fact that all forms of mercury can be harmful to the human body. Organic mercury has a raised potential of harm compared with inorganic mercury because organic mercury is a bio‐accumulator, meaning it has a tendency to accumulate in the human body as it is absorbed by tissue and fat. Thimerosal‐preserved drugs have been proven to be a contributing factor in the long‐term accumulation of the mercury‐body burden and can cause potentially harmful levels of mercury in the human brain [49]. Methylmercury and ethylmercury distribute to all body tissues, crossing the BBB and the placental barrier, and ethylmercury also moves freely throughout the body [49–51]. Concerns based on extrapolations from methylmercury caused thimerosal to be removed from US childhood vaccines, starting in 1999. Since then, it has been found that ethylmercury is eliminated from the body and the brain significantly faster than methylmercury, so the late 1990s risk assessments turned out to be overly conservative. Though inorganic mercury metabolized from ethylmercury has a much longer half‐life in the brain, at least 120 days, it appears to be much less toxic than the inorganic mercury Thimerosal 271 produced from mercury vapor, for reasons not yet understood. It is well documented in the scientific literature that exposure to mercury can elicit immune, neurological, gastrointestinal, motor, sensory, and even behavioral dysfunctions that mimic the symptoms of those seen in autistic individuals [52–55]. The use of organomercury in vaccines is a very contentious issue and it is claimed by vaccine makers that thimerosal is no longer used in vaccines, especially in children’s vaccines [55]. However, there are always exceptions! In the USA, it is claimed that the only exceptions among vaccines routinely recommended for children are some formulations of the inactivated influenza vaccine for children older than 2 years of age [52–55]. Several other children’s vaccines also contain thimerosal, including DT, Td (tetanus and diphtheria), and TT (tetanus toxoid). Some other vaccines contain supposedly traces of thimerosal, according to the CDC [52–56]. The multi‐dose versions of the influenza vaccines Fluvirin and Fluzone also contain a large amount of thimerosal (i.e., 25 μg of mercury per dose of thiemerosal) [53,54]. Also, several antisnake venom vaccines (i.e., pit viper, coral snake, and black widow venom) contain a large amount of thimerosal [55]. Outside North America and Europe, in the poorer nations, the majority of the vaccines contain thimerosal [56,57]. The WHO has concluded that there is no apparent toxicity from thimerosal in vaccines and no reason on safety grounds to change to more expensive single‐dose administration [57–59]. This means that the children of poorer nations are exposed to factors potentially contributing to regressive autism and to the neurotoxicity of organic mercury. Ironically, due to the lack of medical facilities, the adverse effects are generally not noticeable or recorded and the WHO has backed away from an earlier proposal of adding thimerosal in vaccines to the list of banned compounds in a treaty aimed at reducing exposure to mercury worldwide. The WHO has cited medical and scientific consensus that thimerosal in vaccines posed no safety issues, but that eliminating the preservative in multi‐ dose vaccines, primarily used in developing countries, would lead to high cost and a requirement for refrigeration which the developing countries can ill afford [60]. It is possible that the WHO decision has been influenced by the “Big Pharma”. Exposure to mercury in food or other ingestible forms has different consequences and may not be as harmful, but direct injection of the compound can have several serious consequences. The presence of thimerosal in numerous vaccines has greatly eroded public confidence in vaccine safety and efficacy, and trust in companies and agencies that manufacture or recommend vaccines, and trust in the CDC which has reported safety of this chemical in vaccines, particularly MMR vaccines. This has adversely changed some perceptions around the need for vaccination, and has had significant adverse impacts on vaccine uptake in specific populations. Recently, the documentary “Vaxxed” has been widely viewed, and this has increased fears that both the US government and the vaccine manufacturers are withholding the truth about potential adverse effects of the MMR vaccine [61]. In our own research we 272 Vaccines and Autism

have discovered significant neurotoxicity of vaccines containing thimerosal and shown serious neuromodifying effects of many live vaccines on fetal brain‐ like neurons in vitro (unpublished data). As it is evident from the quotations shown at the start of this chapter, the vaccination debate has taken a political tone and it is difficult for individual parents to decide if exposure of their children to an organomercury containing vaccine would lead to a higher risk of regressive autism [62]. At this juncture, we would like to explain that there is a huge difference between classical autism and the kind of autism that children acquire after receiving a vaccine (either containing thimerosal or not containing thimerosal). In the case of regressive autism, children are born healthy, have apparently normal development, and have been reaching their developmental milestones (as clearly documented in their medical records) and then suddenly develop autism‐like symptoms shortly after receiving a schedule vaccine. In some circles, regressive autism is called atypical autism and referred to as a condition “where young children lose early language and social skills.” This does not clarify the fact that in some cases this happens just after a child has received a vaccine [61,62]. Regressive autism is twice as common in African‐American children as compared with Caucasian children, and 50% higher in Hispanic/ Latino children as compared with Caucasian children [63]. We will discuss in detail the underlying scientific evidence and why we think this happens. In order to provide the basic evidence and reasons for regressive autism,” we first present the latest evidence in support of increased risks for regressive autism. Then we will discuss that out of millions of children who have received the same vaccines, why we believe only a small number of children suffer from regressive autism, and how it can be prevented with a reasonable degree of certainty if specific, easy to carry out screening can be implemented a few minutes before delivering a vaccine to a child. This latter idea will be presented as a hypothesis based on our ongoing research and related testing of the BBB.

What is the Evidence That Organomercurial Vaccines Pose a Higher Risk of Regressive Autism?

Recently, Geier et al. [64] published an interesting research report that explored potential risks of thimerosal‐containing hepatitis B vaccine (or HepB). Three doses of this vaccine are given to children before the age of 18 months. They evaluated a large number of individuals (i.e., 15,216) who received HepB vaccine and concluded that [64]: “Cases diagnosed with atypical autism were statistically significantly more likely to have received greater overall and dose‐ dependent exposures to thimerosal‐containing mercury from TM‐HepB vaccines administered within the first month of life, first two months of life, and first six months of life than the controls. Similar phenomena were observed when cases and controls were separated by gender.” Thimerosal 273

Geier et al. [64] evaluated the mercury exposure among the cases and controls by utilizing the vaccination database and included individuals who received no doses of HepB vaccine. A comparison was made between the children who received HepB that contained mercury (12.5 μg of mercury per dose is present in that particular vaccine) to those receiving 0 μg mercury per dose for those receiving either combined haemophilus influenza type b (Hib)‐hepatitis B vaccine (a thimerosal‐free hepatitis B vaccine) or neither of the aforementioned vaccines. The dosing of thimerosal‐containing mercury cases and controls during the first 6 months of life ranged from a minimum of 0 μg thimerosal‐containing mercury (for children receiving no doses of hepatitis B vaccine and/or Hib‐ hepatitis B vaccine) to a maximum of 37.5 μg thimerosal‐containing mercury (from children who received three doses of Hepatitis B vaccine). Analyses showed that children who were diagnosed with regressive (atypical) autism were significantly more likely to have received increased doses of mercury from thimerosal‐containing hepatitis B vaccines than controls within the first month, within the first 2 months, and within the first 6 months of life. They also observed, by using the logistic regression statistical test, that cases diagnosed with regressive autism in comparison with controls were in a dose‐dependent fashion more likely to have received increased mercury doses from thimerosal‐ containing hepatitis B vaccines administered within the first 6 months of life. In this chapter, we will not be able to discuss in detail all the pro‐vaccine versus anti‐vaccine groups and their views. However, we would like to summarize the support of the hypothesis that thimerosal‐containing vaccines may deliver a toxic amount of mercury to neonates, toddlers and young children that may increase risks for regressive autism in susceptible individuals.

Why Thimerosal‐Containing Vaccines are Harmful: A Scientific Narrative

In a simple scientific manner, we can state that thimerosal‐mercury causes oxidative stress [65,66]. Studies find higher levels of oxidative stress in autism spectrum disorder (ASD) and the degree of oxidative stress levels are observed to be higher in individuals with more severe symptoms of ASD [67]. There have been numerous reports of immune dysfunction associated with ASD. We have discussed this topic in the previous chapter. The study of Geier et al. [64] confirms previous studies and validates the studies that examined the medical diagnoses of ASD, autism spectrum, pervasive developmental disorders or regressive autism and found a significant association between thimerosal‐containing mercury exposure and regressive autism. For example, an ecological study of the vaccine safety datalink database for the birth cohort prevalence of ASD in comparison with the average thimerosal‐containing mercury dose infants received by birth cohort revealed a significant dose‐dependent increased risk of diagnosed ASD that correlated with the increasing microgram thimerosal‐ containing mercury exposure within the first 7 months and the first 13 months of 274 Vaccines and Autism

life [68]. A subsequent case‐control study in the vaccine safety datalink revealed that cases diagnosed with ASD were significantly more likely than controls to have received increased thimerosal‐containing mercury from thimerosal‐containing vaccines administered within the first month, the first 2 months, and the first 6 months of life [69]. Other case‐control studies in the vaccine safety datalink revealed dose‐dependent correlation of higher thimerosal‐containing mercury exposures at specific periods of development within the first 15 months of life [70,71]. Other investigators examined data from the National Health Interview Survey (NHIS) of the CDC on the relationship between thimerosal‐containing HepB vaccination of male neonates and ASD diagnosis. A threefold significantly increased male gender bias for ASD was observed among boys vaccinated as neonates compared with boys never vaccinated or vaccinated after the first month of life [72]. Additional investigators recently analyzed data from the Vaccine Adverse Event Reporting System (VAERS) database of the CDC. It was revealed in a cohort study [69], and in a case‐control study [73] that administration of additional doses of thimerosal‐containing mercury vaccine was associated with an increased frequency of autism adverse events reported to the VAERS database. Finally, a meta‐ analysis of environmental epidemiological studies examining the relationship between prenatal and early infancy thimerosal‐containing mercury exposure and autism risk found a significant overall relationship [73]. It should be pointed out that not all the studies that have looked at the effects of thimerosal‐containing vaccines have found the same outcome as described above, and other epidemiological studies of thimerosal‐containing vaccines and the risk of ASD have found no correlation or no significant association between thimerosal‐containing mercury vaccine and ASD [74–79]. Critical analyses of these reports have found methodological errors and in some cases lack of transparency [64].

How Much Mercury is Given to Children Before the Age of 3 years?

It must be realized that a child receives a large amount of thimerosal‐containing vaccine exceeding the guidelines according to the US Environmental Protection Agency or CDC or any governmental agency in the world [59]. How much? Table 9.3 shows the mercury‐containing influenza vaccines.

Why Do Only Small Numbers of Children Develop Regressive Autism After Vaccination?

Here, we would like to present a hypothesis based on scientific data and confirmed by large epidemiologic studies that post vaccine regressive autism is due to a specific immunological phenomenon. We believe that this type of ASD can be prevented by a simple, inexpensive test that can be administered just before vaccination with thimerosal‐containing vaccine. Thimerosal 275

Table 9.3 Mercury‐containing influenza vaccines.

MAX ALLOWABLE FACTOR AVG WT MERCURY EXPOSURE MERCURY OVER EPA AGE FOR AGE (for avg weight at age) IN FLU SHOT LIMITS

6 Months 7.7 kg 0.77 mcg 12.5 mcg 16.2 x 12 Months 10.5 kg 1.05 mcg 12.5 mcg 11.9 x 24 Months 12.3 kg 1.23 mcg 12.5 mcg 10.2 x 36 Months 14.5 kg 1.45 mcg 25 mcg 17.2 x 4 Years 16.3 kg 1.63 mcg 25 mcg 15.3 x 6 Years 20.5 kg 2.05 mcg 25 mcg 12.2 x Fetus 1.0 kg 0.01 mcg 25 mcg 2,500 x Adult 70 kg 7.0 mcg 25 mcg 3.5 x

Mercury‐containing influenza vaccinations in early childhood will exceed EPA guidelines for maximum daily mercury exposure [based on EPA reference dose (0.1 mcg/kg) example for 28‐week gestation].

We have described the role of BBB and how it prevents entry of any unwanted chemicals into the brain. However, there are events that can make a young child’s BBB transiently leaky [80–82]. In our own research, we have discovered that there are cytokines that, if present, can make a child’s BBB leaky. The BBB is a filtering mechanism of the capillaries that carry blood to the brain and spinal cord tissue, blocking the passage of certain substances. Therefore, in any normal circumstances the BBB can prevent the entry of any neurotoxic agent(s) including thimerosal or mercury into the brain. However, there are certain immunologic conditions that would make a BBB slightly leaky. The transient leakiness of the BBB can now be deciphered through various methods [81]. Many epidemiological studies have shown inflammatory cytokines can make a neonatal BBB leaky [82–86]. Inflammatory cytokines are types of immune signaling molecules that are released from T‐lymphocytes, macrophages and mast cells that increase inflammation. Inflammation sometimes causes fever but also can make the BBB transiently leaky. Figure 9.1 shows a hypothetical model of a leaky BBB that contributes to the entry of mercury into a neonatal or a young child’s brain. Is there any evidence that the ASD child suffers from increased amounts of inflammatory cytokine levels and, if so, that they have a leaky BBB? A number of inflammatory molecules have been shown to be increased in the brain and cerebrospinal fluid (CSF) of many ASD individuals including 276 Vaccines and Autism

(a) Abluminal membrane Tight junction

Astrocyte

Luminal membrane Astrocyte Brain capillary Blood Hg

Endothelial cell

Neuron

Pericyte Basement membrane

(b) Abluminal membrane Tight junction

Astrocyte

Luminal membrane Astrocyte Brain capillary Inflammatory cytokines Blood Hg Mercury in vaccines/ Environmental chemicals Endothelial cell Leaky BBB Neuron

Pericyte Basement membrane

Figure 9.1 The blood–brain barrier (BBB) is a highly selective semipermeable membrane barrier that separates the circulating blood from the brain extracellular fluid in the CNS. The BBB is formed by brain endothelial cells, which are connected by tight junctions. (a) A normal BBB. (b) A leaky BBB, induced by inflammatory cytokines, particularly interleukin 6 (IL‐6). (See insert for color representation of this figure.) Thimerosal 277

IL‐1β, IL‐6, tumor necrosis factor (TNF), monocyte chemotactic protein (MCP)‐1 and CCL8 (IL‐8) [87–90]. Plasma levels of IL‐1β, IL‐6 and IL‐8 were increased in children with ASD and correlated with regressive autism, as well as impaired communication and aberrant behavior [90,91]. Abnormal immune responses in individuals with ASD have been reported for nearly 40 years including the presence of self‐reactive antibodies to neuroantigens that we have described in Chapter 8. The evidence of increased neuroinflammation in brain specimens obtained from subjects with ASD has shown increased infil- tration of lymphocytes and macrophages, validating a leaky BBB. For example, Vargas et al. analyzed autopsied brain tissues from cerebellum, midfrontal, and cingulate gyrus from 11 patients with autism and fresh‐frozen tissues from seven patients and CSF from six living autistic patients for cytokine protein profiling [92,93]. They showed an active neuroinflammatory process in the cerebral cortex, white matter, and in the cerebellum of autistic patients. Immunocytochemical studies showed marked activation of microglia and astroglia, and cytokine profiling of the CSF indicated that MCP‐1 and TNF‐β1, derived from neuroglia, were the most prevalent cytokines in brain tissues. CSF showed a unique proinflammatory profile of cytokines, including a marked increase in MCP‐1. Their findings, which have been verified by a large group of other investigators, indicate that innate neuroimmune reactions play a pathogenic role in an undefined proportion of autistic patients, suggesting that future therapies might involve modifying neuroglial responses in the brain [88,94]. Several studies have shown immune abnormalities in patients with ASD, specifically in their blood circulation including abnormal or skewed T helper cell cytokine profiles [95–99], decreased lymphocyte numbers [98], abnormal T lymphocyte responses [97–102], and disparity of serum immunoglobulin levels [100]. In summary, these findings support modifications in the immune responses in a significant proportion of children with ASD. Masi et al. (103) have reported that serum concentrations of IL‐1β, IL‐6, IL‐8, interferon‐gamma (IFN‐γ), and MCP‐1 were significantly higher in individuals with ASD than in controls. Xu et al. (104) carried out a series of studies that strongly suggested that IL‐6, TNF‐α, and IFN‐γ are significantly elevated in different tissues in ASD patients. Importantly, these aforementioned biomarkers can be manifestations of neurointoxication or neuroinflammation, and mercury‐containing vaccines in particular induce neuroinflammation [103–106]. For example, oxidative stress is commonly known as one of the main effects of mercury present in thimerosal‐containing vaccines that induces neuroinflammation. In humans, exposure to metallic mercury is known to cause elevated levels of IL‐1β, IL‐2, IL‐6, IL‐8, IFN‐γ, and TNF‐α [106]. Mercury‐containing vaccines can also cause autoimmune dysfunction. Studies completed on human workers exposed to metallic mercury vapors show auto‐ antibodies to S‐100 proteins, myelin basic protein (MBP), and myelin‐associated glycoprotein (MAG) [106]. 278 Vaccines and Autism

Can Measurements of Abnormal Cytokines Identify Children Who Are at Increased Risk for Regressive ASD?

Few studies have addressed whether cytokine patterns differ in the sera or plasma of ASD [84,87,90,106,107]. Three investigating groups have evaluated whether certain specific cytokine levels are associated with core symptoms of ASD or whether behavior impairments are associated with the onset patterns of ASD. A subset of these studies has demonstrated increased levels of cytokines that can induce inflammation in ASD, such as IFN‐γ or IL‐12 [106], or a decreased production of cytokines that negatively regulate inflammation, such as TGF‐β1 [98]. The role of numerous environmental factors in neuroinflammation cannot be ignored [106–108]. In a large, comprehensive study, cytokine levels were assessed in ASD children from 24 to 60 months and compared with age‐matched typically developing children and children with developmental disabilities other than ASD. In addition, cytokine profiles in children with ASD were explored for any associations with clinical behavioral outcomes. Ashwood et al. [98,99] concluded that levels of IL‐6 and IL‐12p40 were significantly higher in children with ASD compared with typically developing and children with developmental disabilities other than autism controls. IL‐6 appears to be a common denominator among hundreds of reports and seems to play a pivotal role in making the BBB leakiness and is associated with regressive autism. We propose that a simple test can be used to identify children with increased risk for regressive autism. This test can be carried out by nursing staff before administration of any thimerosal‐containing vaccine. We believe this will significantly prevent the incidence of post‐vaccination associated regressive autism. We feel this is a scientific solution to the political debate of regressive autism. Avoiding vaccination is not the solution, since vaccines save lives. However, scientifically validated studies that suggest an increased risk of regressive ASD in children who have received thimerosal‐containing vaccine should not be ignored [109–111]

Summary of Contributing Immunological Factors to ASD

An emerging focus of research into the etiology of ASD has suggested neuroinflammation as one of the major candidates underlying the biological model. There is no doubt that organomercury significantly contributes to inducing regressive ASD and this environmental component can be eliminated. The study of inflammation in ASD provides a great opportunity to examine potential cytokines that can be identified via a simple test before administration of any vaccine that contains mercury or other metallic or nonmetallic agent that can make a child’s BBB leaky. Here, we have highlighted some of the common References 279 indications of immunological dysregulation as potential contributing pathogenic processes for, at least in certain subgroups of individuals, particularly regressive ASD. Nevertheless, it is unclear whether the role of inflammation in ASD induces epigenetic change, via the activation of signaling cascades, or whether it is a direct result of mercury that causes upregulation of inflammatory cytokines, which makes the recipient’s BBB leaky. While, there is a growing body of work to support the role of inflammatory cytokines in ASD, future work is needed to demonstrate how we can prevent regressive ASD associated with mercury‐containing vaccines. Furthermore, we strongly recommend that the use of mercury‐containing vaccines should continue to be curtailed and, if this is not feasible, the amount of organomercury used must be reduced. In addition, we recommend that children under 18 months of age should be monitored for the presence of inflammatory cytokine levels that may harm the child upon receiving any type of thimerosal based vaccine. Despite potential risks, vaccines save lives and have significantly reduced the risk of, and even in some cases eliminated, many serious diseases. Decreased vaccination rates have led to some focused outbreaks in recent years, including a recent outbreak of measles in Minnesota, USA. Thus, the importance of vaccination cannot be overstated. All the known and potential neurotoxic agents must be eliminated from all vaccines.

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Epilogue

“Anecdotal” is often used as a pejorative term in scientific circles, meaning unreliable. In practice it often means isolated, and therefore hard to assess. Think of a new field of science as a large jigsaw puzzle. Pieces are discovered one by one, and at first they are unlikely to fit together to make a picture. Things can look distinctly unpromising, sometimes for decades. But if you can bear the pain of feeling stupid and humiliation of being wrong, anecdotal evidence is the call of the wild, the sunset sign of the undiscovered. Columbus set sail on the basis of anecdotal evidence. The Mayan hieroglyphs were deciphered using anecdotal evidence. Life‐saving remedies based on plants, such as aspirin and digitalis, were found by scientists who paid attention to anecdotal evidence. Luca Turin, 2007, The Secret of Scent

Why do we have so many endocrine disturbing chemicals (EDCs) in our environment? Why is autism spectrum disorder (ASD) rising? Why is legal protection lacking to shield our offspring from synthetic chemicals? Special interest groups and the viral topics of the day, whether based on accurate scientific evidence or not, often dominate public discussion. The recent documentary, “Vaxxed” [1], illustrates how certain agendas may be pushed forward shifting attention from where it needs to be focused to better understand the mechanisms underlying autism – examining the scientific evidence [1]. Misinformation on autism has had detrimental effects, as has been seen with recent outbreaks of diseases easily prevented by vaccines in communities that have under‐ vaccinated their children, such as the recent outbreaks of measles in Minnesota in the spring of 2017. However, the situation is not hopeless. If we want to make a difference to our future generations, we have to act.

In this book we have done our best to present scientific data that were originally anecdotal observations. Many may think that the whole of ASD is essentially an anecdotal field of science that combines biology, psychology, social science, humanities, neurology, neuroscience, neuroimmunology, neuropsychoimmunology, immunology, developmental biology, embryol- ogy, endocrinology, toxicology, organic chemistry, physics, music therapy, special education, radiology, computation medicine, and so on, and so many areas of research and science and medicine that it is a giant jigsaw puzzle. This jigsaw has millions of pieces and it is yet to be fully assembled. We have attempted to bring some of these pieces together so that we can at least bring a picture that is still way out of focus into focus. We do not claim to have all the answers or even part of the answers to the autism conundrum. We hope that readers will forgive us for any errors and occasional mono- lithic concepts. Our goal is to try to gather as many pieces together as possible in this book. We hope to add new pieces so that the blurred picture of autism may become more focused. We request that readers point out any errors and help us make us make this puzzle more solvable. A Facebook Page (https://www.facebook.com/autisman denvironmentalfactors/?notif_id=1512833578907283¬if_t=page_admin) has been created for this book and we hope to hear from readers so that the underlying mechanisms of autism may become visible and to help stem the alarming rise of autism cases. Each of those affected by autism, those silent offspring, represents a piece of the puzzle – a shining star that never got the chance to glow! Let us find a way together to quash the “invisible smells” and prevent autism. The next time you add fragrance crystals to your laundry, remember Chapter 1 and the section “Smell of Autism”. The next time you wear perfume, reread Chapter 7’s section “Contribution of Fragrances to ASD”. The next time you spray herbicide in your yard, look again at Figure 1.4 which shows the correlation between autism prevalence and glyphosphate applied to crops. We believe no one has presented the predicament of human life more accurately than Rachel Carson in her book Silent Spring when she stated [2]:

As crude a weapon as the cave man’s club, the chemical barrage has been hurled against the fabric of life – a fabric on the one hand delicate and destructible, on the other miraculously tough and resilient, and capable of striking back in unexpected ways. These extraordinary capacities of life have been ignored by the practitioners of chemical control who have brought to their task no "high‐minded orientation," no humility before the vast forces with which they tamper.

We hope that the next generation – our offspring – are born without the threat of synthetic chemicals that affect their brains while they are still in the womb and EDCs that change their DNA, epigenetics, and hormones. Epilogue 289

References

1 Wakefield A (Director) (2016). Vaxxed: from Cover‐Up to Catastrophe. https:// www.amazon.com/Vaxxed‐Cover‐Up‐Catastrophe‐Del‐Bigtree/dp/ B01IHSJO6M/ref= sr_1_2?s=music&ie=UTF8&qid= 1501950017&sr= 8‐2&keywords=Vaxxed. Accessed November 4, 2017. 2 Carson R (1962). Silent Spring. Houghton Mifflin Company, Boston.

291

Index

Note: Page numbers followed by ‘f’ refer to Figures; those followed by ‘t’ refer to Tables

(+)‐ α‐pinene 100, 186, 187t, 195 aldrin 172t β‐propiolactone 266 allethrin 173t 1,2‐dibromoethane 172t aluminum 149 11‐deoxycortisol 126 adjuvants 43, 267, 268 17‐hydroxyprogesterone 126 Alzheimer’s disease 84, 144 17α‐hydroxyprogesterone 11–12, 125 damage to dentate gyrus and 21‐hydroxylase deficiency 126 olfactory bulb neurons in 7 2D:4D digit ratio 10–11, 10f, 12, 12f, olfaction loss in 14, 84, 86 124, 125, 129 oxytocin binding site density in the brain 98 a projected prevalence 146f Aberrant Behavioral Checklist scale 244 American Eagle: Seventy Seven 178 ABO Incompatibility 237–8 Ames, Bruce 65 acetochlor 172t Ames test 158–9, 179–80 acetyl ethyl tetramethyl tetraline amitrol 173t (AETT) 13, 131, 171 amyotrophic lateral sclerosis adjuvants, vaccine 43, 149, 267–8 (ALS) 84 age androgen insensitivity syndrome 11 maternal, and autism 161–3, 162f androgen receptors (ARs) 199–200 and olfactory function 84 androgens 171 AHTN (7‐acetyl‐1,1,3,4,4,6‐ exposure in utero 124 hexamethyl‐1,2,3,4‐ androstenedione 11–12, 125 tetrahydronaphthalene) 188 Angelman syndrome 52t, 72, 143 alachlor 172t animal models alcohol 66 genetic mutations 34–5 abuse 92 limitations of 179 olfactory damage and 91–2 and neuroantibodies to autism 245

anthrax 264 b antibiotics 269–70 Bacteroides fragilis 9 antisnake venom vaccines 271 bad mothering 67, 146 anxiety‐related behavior 207–8 Baur, Albert 4, 186 apoptosis 208, 235–6 bed nucleus of the stria terminalis Arabidopsis thaliana 74 (BNST) 206 arginine vasopressin (AVP) 36–7, 76 benzene hexachloride 172t neurons producing 103f benzene ring products 29 arginine vasopressin receptor benzo[a]pyrene 176t (AVPR) 29, 40, 235 benzophenone 176t AVPR1α and/or AVPR1β 106 benzophenone‐1 13, 37, 76, 131, Armani, Giorgio: Acqua Di Gio 178 170, 171 aromatase enzyme 130, 132, 133 benzophenone‐2 13, 37, 76, 131, arsenic 170, 177t 170, 171 artificial vanilla 4 benzyl benzoate 13, 37, 76, 100, 131, ascabin see benzyl benzoate 170, 186, 194, 223 ascabiol see benzyl benzoate chemical structure and adverse Asperger, Hans 23, 53–5, 56 effects of 187t Asperger syndrome 20, 39, 40, 53, 54, benzyl salicylate 13, 37, 76, 100, 131, 61, 63, 67 170, 186, 194, 223 brain size 41 chemical structure and adverse male gender bias in 9, 40 effects of 187t assisted reproductive technologies 170 Bettelheim, Bruno 53, 57, 169 atrazine 173t birth rates, global 220f attention‐deficit/hyperactivity disorder bisphenol A (BPA) 151, 176t, 196, (ADHD) 67, 68 217–18, 252 atypical autism see regressive autism bisphenol F 176t autism spectrum disorder (ASD) 55f bisphenol S (BPS) 218 definition 62 blood–brain barrier (BBB) 42, 254–6, diagnosis of 51, 12 274–5 genesis 158–9 ‘leaky’ 42, 250, 254, 275–8, 276f, 279 persistent deficits 61, 62–3 normal 276f “spectrum” 20–2, 39–41, 155f brain severity 67–8 areas that process olfactory data 84 with and without intellectual ASD genesis ans 31–2 disability 69–70 areas affected by neuromodifying autistic disorder 63, 64 agents during fetal autoimmune hemolytic anemia 249 development 29f autoimmune hypothetical model 240f development 15, 17, 22f, 32–4, 33f, autoimmune orchitis 239 36–9, 99 autoimmunity 241, 248–9 gender and 132–4 avian flu 2 human, vs mouse 72 AZT 26 human, vs nonhuman primate 35 Index 293

neocortex 17 Childhood Autism Spectrum Test neural network 19f (CAST) 126 neuroantigens in maternal blood childhood disintegrative disorder 241–3 (CDD) 63, 64–5 structural connectivity in chlofentezine 173t twins 150 chlordane 14, 171, 173t structure, adult 15, 15f chloroform 172t see also brain size chromatin remodeling genes 69 brain size 20, 36 cigarette smoke 65 in ASD 16, 28, 246–8, 247f clonal selection 199 in classical autism 41 Clostridium difficile 9 human vs other mammals 18f Cohen syndromes 72 importance of 74–5 Cohen, Baron 11 breast cancer 15 collapsin response mediator proteins Britney Spears’ Curious 178 CRMP1 243, 244, 245 butyl benzyl phthalate 176t CRMP2 243, 244 butylated hydroxyanisole 172t common cold during pregnancy 253 butylated hydroxytoluene 13, 37, 76, communication skills 39 131, 171 complete androgen insensitivity butylphenyl methylpropional 13, 37, syndrome 125 76, 131, 170 congenital adrenal hyperplasia 11, 125, 126 c congenital rubella infection 252 cadmium 177t connectome dysregulation 132 Caenorhabditis elegans 74 copy number variations (CNVs) 23, Campaign for Safe Cosmetics 178 69, 156–7 carbaryl 173t corpus callosum 15–16, 125 carbendazim 177t Courchesne, Eric 40 carbolic acid 268 cowpox virus 265 Carcinogenic Potency Database 65 creatine kinase, brain‐type (CKB) 151 Carson, Rachel: Silent Spring 1, 4, 51, cryptorchidism 210–14, 212f, 221 83, 97, 123, 143, 169, 288 CYP19A1 (aromatase) gene 130, cashmeran 188 132–3 caustic fibrosis 169 cypermethrin 173t Cavendish, Henry 53 cypin 243 celestolide 188 cytokines, abnormal, regressive ASD celiac disease 200 and 278 cerebral capillary 255f cytomegalovirus (CMV) 253 cerebral palsy 256 ch22q11 deletion syndrome d 52t, 69 daidzein 177t ch22q11 duplication syndrome 52t Darwin, Charles 19 Chanel: Coco 178 DDT 52, 170, 173t, 211 294 Index

de novo mutations 23–4, 68, 69, e 156–7 Ebola virus 2 deaf community 59 Einstein, Albert 19, 39 DEHP 172, 176t, 177t electromagnetic radiation 34 dentate gyrus 7, 88 Eli Lilly 270 depression, olfaction in 86 emotional word‐face task (using di(2‐ethylhexyl) phthalate see DEHP functional MRI) 150 diabetes empathizing theory 125 gestational 66 encephalization quotient (EQ) 35, type 1 200 35t, 74 Diagnostic and Statistical Manual of human vs animals 74–5, 75t Mental Disorders endocrine disrupting chemicals (EDCs) DSM‐4 63 12, 14–15, 130, 146, 193, 195–9 DSM‐5 40, 60–2, 84, 110 adverse effects and mechanisms of dibutyltin 212 action 208–11, 209f dichlorodiphenyldichloroethylene effects of testosterone (DDE) 211 mimicking 211–14 dichlorodiphenyltrichloroethane see effects on neurodevelopmental and DDT neuroendocrine systems dichorionic diamniotic twins 148f 215–17 dicofol (Kelthane) 173t effects on steroid hormone receptors dieldrin 173t in brain 217–19 diethyl phthalate (DEP) 6, 52, 100, and gender bias 241 176t, 178, 179, 186, 191–3, 223 and infertility 219–23 chemical structure and adverse endogenous transposons 34 effects of 187t endosulfan 173t diethylstilbestrol (DES) 196, 213 entorhinal cortex 90, 91 DiGeorge syndrome see ch22q11 Environmental Protection Agency (US deletion syndrome EPA) 14, 171 di‐n‐butyl phthalate 176t Toxic Substances Control Act dioxins 172t, 196, 211–13 (TSCA) Inventory 189 diphtheria 264 epigenetic change 160–1 vaccine 270 epilepsy 25, 67 diphtheria, pertussis, and tetanus erythroblastosis fetalis 236, 249 (DPT) vaccine 265, 266 ethane dimethane sulfonate 177t D‐limonene 100, 186, 194–5 ethyl alcohol 170 chemical structure and adverse ethyl mercury 262 effects of 187t ethylene thiourea 173t DNA mutation 73 ethylmercury 270 dosage, chemical and toxicity 65–6 EUAIMS Longitudinal European Down syndrome 52t, 70f, 71, 143 Autism Project (LEAP) 67 Drosophila 74 eugenics 53 Duesberg, Professor Peter 25 eugenol 100, 186, 187t Index 295

European Union Cosmetics in fetal neuroblastoma cells 6, 6f Directive 189 mutagenic/carcinogenic effects exome sequencing analysis 23 of 5–6, 5f extreme male brain (EMB) popular, and secret ingredients 7f theory 10–12, 124, 125, 126, Freud, Sigmund 57 130, 132, 207 Frozen (Disney movie) 60 fungicides 211 f furans 172t famine, prenatal, and schizophrenia 161 FYN Tyrosine Kinase Protooncogene Federal Fair Packaging and Labeling (FYN) 151 Act (1973) (US) 6, 178, 188 femtomolar concentrations 37, 76–7, g 177, 184 galaxolide 13, 37, 76, 131, 170, 171, fenarimol 173t 178, 188, 191 fenbuconazole 174t Gates, Bill 19 fenitrothion 174t gender bias in ASD 9, 171 fenvalerate 174t for ASD 171 fertility rates 219, 221, 222f in Asperger syndrome 123 fetal alcohol spectrum disorders in classical autism 123 (FASDs) 38, 38f, 91–2 male 123, 131 fetal alcohol syndrome 66 molecular basis of 134–7 feto‐fetal transfusion syndrome (FFTS) genetic deletions 3–4 147–8, 147f genetic drift 52 fipronil 174t genetic mutations 3–4, 23, 34–5, 52f, flame retardants 211 72–3 Fleishmann, Carly 16–17 genetic variation 68 fluvirin 271 genetics and ASD 2, 51–2, 68, 69 fluzone 271 vs environment 75–7 folic acid deficiency 66 family 69 Food and Drug Administration as myth 67 (US) 6, 163 genistein 177t food antioxidant 172t genome formalin 266–7 analyses of ASD 68 Fragile X syndrome 61, 70–1, 70f, architecture of ASD 69 72, 107 human vs mouse 72–3 fragrance abuse 92 genomic imprinting 134 fragrances, synthetic 5–7, 12–13, 31, gestational diabetes 66 52, 100, 170, 178–83 Gillberg, Christopher 1 effects on female oxytocin‐receptor glucose 66 positive neurons 185f, 186 glutaraldehyde 269 effects on male oxytocin receptor glycolytic pathway 66 neurons 184–5, 184f glyphosate 7–9, 8f, 43, 149, 288 fetal exposure to 37 gonocytes, delayed 221 296 Index

Gore, Al 235 hypothalamus, gender and 204f Grandin, Professor Temple 53, hypothyroidism 198f, 199 59–60 i guanine deaminase 244 gustation 83 immunization schedule gustatory diagnostic assessment 85 adult (age based recommendations 264t h for children and adolescents (USA) H1N1 influenza vaccine 251, 267 263t Haarmann, Wilhelm 4 immunological factors, ASD and handedness 149–50 278–9 head size 40–1 Industrial Bio‐Test Laboratories heavy metals 149, 151, 177t (IBT) 170 hemoglobin S (HbS) gene 3 infertility, male 239 hemolytic disease of newborns 237 influenza vaccines 267, 271, 275t hemophilia‐AIDS 25 H1N1 251, 267 hepatitis B vaccine (HepB) 42, and narcolepsy 250–2, 267 272–3, 274 influenza virus 250–4 heptachlor 174t insecticides 4 heptachlor‐epoxide 174t intellectual disability 67 herbicides 4, 149, 288 interleukin‐6 (IL‐6) 250 herd immunity 56 International Classification of Diseases, herpes simplex virus 10th Revision (ICD‐10) 11 HSV‐1 253 Internet, information dissemination HSV‐2 253 and 60 hexachlorocyclohexane 174t intestinal disorders 67 HHCB (1,3,4,6,7,8‐ hexahydro‐4,6,6, Iprodione 174t 7,8,8‐hexamethylcyclopenta‐γ‐ IQ 68 2‐benzopyran) 188 irritable bowel syndrome 109 high angular resolution diffusion j imaging (HARDI) 150 Jackson, Michael 19 hippocampus, neuronal projections Jefferson, Thomas 19 in 101f Jenner, Edward 265, 266 history of autism 53–60, 151–2 Hitler, Adolf 53, 54 k HIV/AIDS 25, 26, 57–8, 65 Kanner, Chaskel Leib (Leo) 23, 53, HIV‐1 26 55–6, 67, 69 Hoffman, Dustin 53, 60 Karate 174t Huntington’s chorea 57, 84, 169 kepone (chlordecone) 174t hyperthyroidism 197f, 199 ketoconazole 174t hypospadias 210, 213, 214, 221 Kharasch, Morris 270 hypothalamic–pituitary–gonadal Krebs cycle 66 (HPG) axis 215, 216f Kuhn, Thomas 1 Index 297 l methoxychlor 174t lactose tolerance 3, 4 methylmercury 170, 270 LDH 243, 245 metribuzin 174t LDH A 244 mirex 175t LDH B 244 Mohawk Homeobox (MKX) 151 lead 170, 177t mono‐2‐ethyhexyl phthalate 212 leaky gut syndrome 9 monochorionic monoamniotic leukemia 15 twins 148f Life 60 monoethyl phthalate (MEP) 192, 193 Lincoln, Abraham 19 monosodium glutamate (MSG) 269 lindane 174t monozygotic twins, discordant linuron 174t ASD discordance 146–9 locus coeruleus (LC) 206 disease discordance 152 long‐distance cortical “under‐ environment and 149–51 connectivity” theory 133–4 types 146–7 Lou Gehrig’s disease 84 Montagu, Lady Mary Wortley 266 lupus 200 Mozart, Wolfgang Amadeus 19 lymphoma 15 Mullis, Dr Kary B. 26 multiple sclerosis (MS) 200, 239, 239f m mump vaccine 239 malaria 2–3 mumps virus 238 malathion 174t muscular dystrophy 169 mancozeb 174t musk ambrette 14, 131, 171 maneb 174t musk ketone 4, 13, 37, 76, 100, 131, manganese 170 170, 178, 186, 188, 189, 191 maternal immune system musk xylene 188, 189, 191 238–40 mutation rate in human genome 74 maternal twins see monozygotic mutations of coding genes that 30 twins myocarditis 249 measles 41, 279 measles–mumps–rubella (MMR) n vaccine 262, 271 narcolepsy, influenza vaccine and mechanistic target of rapamycin 250–2, 267 (mTOR) signaling pathway 72 nasal passages 86, 87f media and public perceptions of National Society for Autistic autism 60 Children 58 medial preoptic area (MPOA) 202 Nazism 53–4 mercury 42, 177t, 270–1 neo‐antigens 240 in food 271 nested lineage trees 34 in immunization schedule 52 neural plate folding 17 in vaccines 4–5, 274 neural tube defect 66 merthiolate see thimerosal neuritogenesis 244 methomyl 174t neuroantigens 243–5 298 Index

neuroblastoma cell line (NBC) 76, olfactory epithelium 86, 87, 88 132, 171 olfactory odor neurons 88 neuroblastomas 159–60 olfactory receptor cells 86 neuroendocrine axes 215 olfactory sensory neurons (OSNs) 86 neurofibromatosis 169 open reading frames (ORFs) 18–9 neurofibromatosis 1 (NF1) 152–3 orbitofrontal cortex 88, 90 neuronal progenitor cells 181f orchiopexy 211 neuropeptide arginine organochlorine pesticides 188 vasopressin 102–3 organohalogens, persistent 172t Newton, Isaac 19 organomercury in vaccines 42, 149, Nicksic‐Springer, Professor Taryn 59 271, 272–3 nitro musks 188, 189 organophosphate insecticides 170 nitrofen 175t orthonasal olfaction process 87, 88 nonachlor 175t ovine sexually dimorphic nucleus nonseminoma 221 (oSDN) 203–5 nonviral Infections 252–4 oxybenzone 13, 37, 76, 131, 170, 171 nonylphenol 177t oxychlordane 175t oxygen free radicals 34 o oxytocin 37, 76, 89, 97–9 obsessive compulsive disorder 250 abnormalities of 112–13 octachlorostyrene 172t in ASD 99–103 octinoxate 13, 37, 76, 100, 131, 170, AVP neurons and 103–4 171, 186, 193–4, 223 and communication abilities 160 chemical structure and adverse developmental neurological effects of 187t disorders and 106–7 octyl methoxycinnamate see octinoxate effects on human physiological octylphenol 177t responses 105f odor perception 84, 86 efficiency to its respective odorant molecules 86 receptor 182–3 odorant neurons, fetal, synthetic exogenous treatments 108–9 chemicals and 88–9 functions 112 odorant receptors (ORs) 83 gender and 98 olfaction and autism 83 intranasal and intravenous, in diagnostic assessment 85 ASD 109–10, 109f, 111 neural pathways of 85f low plasma 36–7 paucity of 16 in mice 106–7 process of 87–92 neurons producing 103f olfactory bulb neurons 7, 98 social cognition and 98–9 olfactory bulbs 28, 86 social experience in development olfactory cells, regeneration of 86 and 104–5 olfactory cortex 86, 88 synthetic 98 olfactory dysfunction 84, 86 trials 110–12 alcohol and 91–2 oxytocin receptor genes in mice 106–7 Index 299 oxytocin receptor depleted (knockout) polybrominated biphenyls (PBBs) 172t mice 107 polybrominated diphenyl ethers oxytocin receptors 105, 106t (PBDEs) 172t, 218 developmental trajectories in rat polychlorinated biphenyls (PCBs) brain 100f 43, 52, 149, 170, 172t, 188, 196, 211, 218 p polycyclic musks 188, 189 p,p′‐DDE 173t polymerase chain reaction (PCR) p‐amino‐musk xylene 191 method 26, 27f pandemrix 250–2 polysorbate 80 269 Parkinson’s disease 84, 98, 169 posterodorsal medial amygdala olfactory dysfunction 84, 86 (MePD) 205 Pasteur, Louis 265 posteronedial nucleus of bed nucleus p‐cresol 9 of the stria terminalis PDD‐NOS 61 (BSTMPM) 206 pendimethalin 175t postpartum depression 109 pentachloronitrobenzene 175t post‐traumatic stress disorder pentachlorophenol 172t (PTSD) 109 perchlorate 218 Potocki–Lupski syndrome 52t perfluorooctanesulfonate 177t Prader–Willi syndrome 52t, permethrin 175t 105, 143 persistent organic pollutants 151 pre‐germ cell neoplasia in situ pertussis 264 (pre‐GCNIS cells) 221 Pervasive Developmental Disorder Not prevalence of ASD 43 Otherwise Specified In developed countries 43 (PDD‐NOD) 63–4 rise in 2, 3–4, 12, 170 pesticides 43, 149, 172–6t, 196 in USA 43 cryptorchidism and 214 primary sensing 86 phantolide 188 primate brain enlargement 35 Phelan–McDermid syndrome 52t procymidone 175t, 211 phenol in vaccines 268 prodiamine 175t phenotypic heterogeneity 62, 67–8 progesterone 11–12, 125 phenoxyethanol in vaccines 268, 269 prostate cancer 15 phenylketonuria 169 puberty, male 214–15 phthalate esters 191, 211 pyrimethanil 175t phthalates 43, 149, 151, 176t, 191–3, 196, 211–12, 213, 218 q metabolic pathways 192f Q‐CHAT (Quantitative Checklist for phytoestrogens 177t Autism in Toddlers) 125–6 piriform cortex 90, 91 polio 264 r pollutants with endocrine‐disrupting rabies 265 effects 172t Rain Man (movie) 60 300 Index

Reading the Mind in the Eyes (RMET) Seneff, Dr 8, 9 test 108 serum sulfate deficiency 9 refrigerator mother theory 57, 67, sex hormones, normal ranges of 130f 146, 169 sex steroid binding protein (SHBG) regressive autism 42, 63, 65, 272, testosterone 215 274–7 Shikimate pathway 8, 9 abnormal cytokines and 278 sickle cell anemia 2–3, 4, 10, 57, 169 ‘Regressive Autism and Vaccines’ Silberman, Steve: NeuroTribes 53 (documentary) 287 silent offspring 2, 144, 159, 288 reproductive systems Simons Simplex Collection 68 differentiation 203f single‐cell cloning method 182f resorcinol 177t Sjogren’s syndrome 200 restricted and repetitive behaviors skin pigmentation 4 (RRBs) 110 smallpox 264 retinoic acid‐related orphan receptor vaccine 265–6 alpha (RORA) 132 as weapon 265–6 retronasal olfaction process 87, 88 Smith–Magenis syndrome Rett syndrome 61, 63, 64, 72, 143, 52t, 143 150–1 smoking, maternal 151, 170 Rh Incompatibility 236 sniff test 89 Rhesus macaques model 245 Sniffin’ Sticks 85 rheumatoid arthritis, acute 200, 249 SNPs, and degree of autism 25 Rimland, Bernard 53 social communication disorder (SCD) Infantile Autism 58 61, 62 risk genes 69 spatial memory 207 Rodent Models 246 spermatogenesis, disorders of 219 Roosevelt, Franklin D. 264 spinal nucleus of the bulbocavernosus RORA gene 132–3 (SNB) 205 RoundupTM 7–9 RoundupTM spontaneous abortions 219 rubella virus 253, 255 Staphylococcus aureus 270 Streptococcus 249 s stress‐induced phosphoprotein 1 salicylic acid see benzyl salicylate (STIP1) 243, 244, 245 Salmonella typhimurium 179 stuck twin 148 schizophrenia 55–6, 67, 108, 109, styrene dimers and trimers 177t 144, 146, 249 sumithrin 175t antibodies reactive to neuronal suppress arginine vasopressin tissues in 250 (AVP) 98 olfactory dysfunction in 84 suprachiasmatic nucleus (SCN) 205 prenatal famine and 161 sympathizing theory 125 self‐recognition process 240 symptom severity, gender and 68 semen, impaired quality of 210 synthetic chemicals 12–13, 13f, 66, seminoma 221 170, 241 Index 301 synthetic musks 13, 37, 76, 131, 170, thiazopyr 175t 178–9, 186–91 thimerosal 41–2, 56–7, 170, 261, 262, exposure to 189–91 266, 270–7 in fish and shellfish 191 thiram 175t in food 191 thyroid hormone disruption 131 in food chain 189 Tiemann, Ferdinand 4 types of 188 tonalide 13, 37, 76, 100, 131, 170, 171, synthetic oxytocin 98 178, 186, 188, 191, 223 Syntocinon 111 chemical structure and adverse systemic lupus erythematosus effects of 187t (SLE) 249 tonalide oxybenzone 13 Tourette syndrome 250 t toxaphene 175t Tarstar 175t toxic parenting 57 technologically based activities 59 Toxoplasma gondii 253 teratogens 20, 21, 27f transduction 87, 87f fetal developmental progression and transeolide 188 susceptibility to 28f, 29f transfusion‐AIDS, 25 teratology, principles of 20 triadimefon 175t terpenes 195 triadimenol 175t terpineol 195 tributyl phosphate 269 testicular cancer 210, 211, 219, 221 tributyltin (TBT) 175t, 196 testicular dysgenesis syndrome trifluralin 176t (TDS) 210 tris‐BP 65 testicular germ cell cancer 221, 223f TSc2 (hamartin) 72 testosterone 76, 123, 125, 211–14 TSC2 (tuberin) 72 in amniotic fluid 11–12, 125 tuberous sclerosis 71–2, 71f conversion into estrogen 130 Turin, Luca: Secret of Scent, The 186 dosage 123 Turner syndrome 135, 136 during pregnancy 124 twin‐to‐twin transfusion syndrome fetal 11, 14 (TTTS) 147–8, 147f fetal brain and 128 typhoid vaccine 270 male brain and 200–7 male gender bias and 9–12 u neuronal morphology and 133–4 undescended testicle 211 prenatal, and cognitive unique antigens 237, 238 development 124 sex differences in autistic traits 124 v target organs and action sites of 202f vaccinations 56, 170 timing of 123 prophylactic 265 wide‐ranging effects of 201f Vaccine Adverse Event Reporting thalidomide 27f, 32 System (VAERS) 267, 274 theory of mind 39 Vaccine Safety Datalink (VSD) 267 302 Index

vaccines 41–2 w contents 265–70 wastewater treatment system 190f live weakened 267 Waterhouse, Lynn 1 organomercurial 4–5, 42, 149, 271, whooping cough 264 272–3 Williams syndrome 52t politics versus science 262–4 Wilson, James 66 preservatives 266 Wing, Lorna 53, 56 regressive autism 261–2 Wing, Susie 56 valporic acid 26, 169 World Health Organization (WHO): variolation 266 Global Advisory Committee on vasculitis 200 Vaccine Safety (GACVS) 268 vasoactive intestinal polypeptide (VIP) 205 y ‘Vaxxed’ (documentary) 271 Y‐box binding protein 1 velocardiofacial syndrome see ch22q11 (YBX1) 243, 244 deletion syndrome ventromedial hypothalamus z (VMH) 205–6 Zika virus 2, 52, 252–3, 255 vinclozolin 176t, 211 Zineb 176t viral infections 252–54 Ziram 176t Cerebrum Corpus callosum

Hypothalamus

Thalamus

Pons

Amygdala Cerebellum

Medulla oblongata

Spinal cord

Figure 1.8 An illustration of a typical adult human brain structure. Source: Adapted from https://www.khemcorp.com/autism‐and‐social‐anxiety‐research‐summary‐and‐science/.

Autism and Environmental Factors, First Edition. Omar Bagasra and Cherilyn Heggen. © 2018 John Wiley & Sons, Inc. Published 2018 by John Wiley & Sons, Inc. Short-tailed Star-nosed Smoky Hamster Rat shrew Mouse mole Eastern mole shrew

1.802g 0.176g 0.347g 0.416g 1.020g 0.802g 0.999g 36 M52M 71 M90M 131M 200M 204M

Guinea pig Marmoset Agouti Galago Owl monkey

1cm 1cm 1cm 3.759g 7.78 g 10.15g 18.365 g 15.73g 240M 634M 936M 857M 1468 M

Capybara Squirrel monkey Capuchin monkey

1cm

30.22g 76.036 g 3246 M 53.21g 1600 M 3690 M Human

Macaque monkey

1cm

87.35g 6376 M 1508 g 86000 M

Figure 1.9 The size of the human brain compared with that of other mammals. Source: https://www.google.com/search?newwindow=1&hl=en&site=imghp&tbm=isch& source=hp&biw=1264&bih=576&q=relative+size+of+mammalian+brains&oq=relative+ size+of+mammalian+brains&gs_l=img.3...1668.11635.0.13335.34.34.0.0.0.0.66.1418.33.33.0.... 0...1.1.64.img..1.19.861.0..0j35i39k1j0i30k1j0i24k1.isfvXm_tTjU#imgrc=BGRS6TbiE0ByLM. (a)(b)

Figure 1.10 (a) A normal brain neural network versus (b) ASD brain neural network. Typical child

Spinal cord Cerebral hemisphere

Hind brain Mid brain Mid brain 40 days Fore brain 35 days 50 days Cerebellum Pons

25 days Medulla 100 days

5 months

9 months

Autistic child

Spinal cord Cerebral hemisphere

Hind brain Mid brain Mid brain 40 days Fore brain 35 days 50 days Cerebellum Pons 25 days Medulla 100 days

5 months

9 months

Figure 1.12 Development of a typical human and an autistic human fetal brain from 25 days to 5 months of gestation. In the majority of autism cases, the problems arise during the very early stages of the fetal brain development. A few progenitor neurons, destined to mature into a specialized faculty are killed or mutated, resulting in ASD. An autistic child’s brain is larger but has parts missing (see text for details). Figure 1.13 Illustration showing how the Exome sequencing exomes are assembled and exome sequencing is carried out. Only ~1% of human genomes contain exons. In exome sequencing all the exons are joined together into one large assembled format and then sequenced. The exome represents all the genes that direct the production of thousands of proteins. Source: https:// d2gne97vdumgn3.cloudfront.net/api/file/ AuHulxYbQ46mPuuqdJ6H. Exons Transcription, elimination of intron transcript segments, and splicing of exons

Exome

Figure 1.14 Various malformations resulting from teratogenic agents, including unique malformations that are organ specific. Among these are malformations caused by exposure to thalidomide. One does not see any of these types of mutations in ASD children. Source: Adapted from http://www.thalidomide.ca/recognition‐of‐thalidomide‐defects/; http://www.merckmanuals.com/professional/pediatrics/congenital‐craniofacial‐and‐ musculoskeletal‐abnormalities/common‐congenital‐limb‐defects; http://www.dailymail. co.uk/news/article‐2198015/Paralympic‐swimmers‐incredible‐journey‐Iraqi‐orphanage‐ London‐2012‐Australia.html; and https://www.researchgate.net/figure/26760442_fig6_ FIGURE‐6‐Mirror‐hand‐attributed‐to‐ZPA‐cells‐in‐the‐anterior‐limb‐margin‐and. http:// keywordsuggest.org/gallery/573175.html 123 45 678916 20–36 38 Period of dividing zygote, Embryonic period, weeks Fetal period, weeks implantation and embryo, Brain Palate weeks Eye Ear Eye Ear Heart Eye Heart

External genitalia

Central nervous system

Heart

Upper limbs

Eyes

Lower limbs

Teeth

Palate

External genitalia

Not susceptible to teratogens Ear

Prenatal death Major congenital anomalies (red)Functional defects and minor congenital anomalies

Olfactory bulb Hippocampus Odor/smell stimulation Raphe nucleus signaling Locus ceruleus Olfactory epithelium Pituitary gland Hypothalamus

(a) Oxytocin receptor developmental expression

First appearance of Progressive appearance of OXR First transition to the Second transition to OXR in the brain in discrete brain regime adult pattern the adult pattern

Embryonic Embryonic Prenatal day 10 Prenatal Gestational Prenatal day 10 Age Gestational day 14 day 18 Prenatal day 13 day 19 week 5 week 8 Prenatal day 13

Infant pattern Adult pattern (b) Dorsal

Caudate nucleus PN-Postnatal

Hypothalamus ventro- Olfactory medial nucleus Periform Bed nucleus of the cortex Amygdala Nucleus accumens Lateral septum stria terminalis Anterior Transient OXR expression Permanent OXR expression

Growth pattern of a single-cell clone from a single cell into a larger cluster of cells from day 0 to day 8

Early stage fetus

Normal

Cell body Myelin Damaged myelin (demyelination) Nerve fibre (axon)

Distorted messages

Damaged myelin in MS

Figure 8.2 The immunological mechanism that leads to MS. Neuroantigens/Abs

odegeneration LDHA/B Neur STIPl

CRMPl Protective OTR (destructive)

AVPR

Immune system Migration to the CNS

Immune response to Dendritic released antigens from cells fetal brain

Astrocyte

Luminal membrane Astrocyte Brain capillary Blood Hg

Endothelial cell

Neuron

Pericyte Basement membrane

(b) Abluminal membrane Tight junction

Astrocyte

Luminal membrane Astrocyte Brain capillary In ammatory cytokines Blood Hg Mercury in vaccines/ Environmental chemicals Endothelial cell Leaky BBB Neuron

Pericyte Basement membrane

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