A Thesis Entitled the Effects of Benzyl Alcohol on the Developing

A Thesis

entitled

The Effects of Benzyl Alcohol on the Developing Zebrafish (Danio rerio)

Alaina M. Schnapp

Submitted to the Graduate Faculty as partial fulfillment of the requirements for

The Master of Science Degree in Pharmaceutical Sciences.

______

Committee: Dr. Frederick E. Williams

______

Committee Member: Dr. Ming-Cheh Liu

______

Committee Member: Dr. Steven M. Peseckis

______

Dr. Patricia R. Komuniecki, Dean

College of Graduate Studies

University of Toledo

May 2013

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author.

An Abstract of

The Effects of Benzyl Alcohol on the Developing Zebrafish (Danio rerio)

Alaina M. Schnapp

Submitted to the Graduate Faculty in partial fulfillment of the requirements for

The Master of Science Degree in Pharmaceutical Sciences

University of Toledo

May 2013

Benzyl alcohol (C6H5CH2OH) is a preservative used in IV preparations, cosmetics, hair dyes, perfumes, and soaps. It is also used as a solvent for many organic processes and in industrial applications. It is an organic compound whose polarity and low vapor pressure make it a useful industrial solvent. We designed a series of experiments to study the developmental effects of benzyl alcohol exposure on zebrafish

(Danio rerio), a well-established model for development. Zebrafish were exposed starting at high blastula stage of development (4 hours post-fertilization, hpf) and finishing at free swimming larvae stage (144 hpf). Observations of critical developmental endpoints were conducted at 8 hpf, 24 hpf, 32 hpf, 55 hpf, 80 hpf, and 144 hpf. The exposure of zebrafish embryos to varying concentrations of benzyl alcohol at 4 hpf in development resulted in developmental defects including delayed and/or abnormal eye development, cardiac/yolk sac edema, and cardiovascular abnormalities. The chemical’s effects on neuronal outgrowth as a measured by acetylated alpha-tubulin staining were inconclusive for the concentrations studied.Cell death was observed using an acridine orange assay, showing a dramatic increase in cell death in a dose-dependent

i manner. While more work is needed to elucidate the effects of benzyl alcohol, these preliminary data suggest that there is a strong link between exposure to benzyl alcohol and the developmental defects observed.

I would like to dedicate this thesis to my sister, Victoria.

iii

Acknowledgements

I would like to thank everyone in the Department of Pharmacology for their advice, support, and encouragement throughout my time at the University of Toledo. I would like to give special thanks to my advisor, Dr. Williams, for his guidance and optimism throughout the project as well as for giving me the opportunity to attend the

International Meeting on Zebrafish Genetics and Development in Madison, Wisconsin. I would also like to thank Dr. Liu, Dr. Peseckis, and Dr. Sari for agreeing to serve on my thesis committee, for their flexibility, and their patience. My heartfelt thanks go out to

Dr. Steinmiller for her inestimable assistance in the writing and critiquing of this thesis.

Thank you to my fellow graduate students Shay Riegsecker, Jessica Ciesler, and

Valerie Desmond for their support and humor throughout our time in graduate school.

Finally, I would like to thank my family and friends for their inspirational comments, steadfast confidence in me and patience with me. Whenever I have needed anything, they have been there for me. My family in particular has played a key role in making this journey a positive experience, and I will never stop being thankful that they are a part of my life. When I was unsure of myself, they never failed to offer me inspiration and to cheer me on. I would also like to thank Josh Coleman for his

iv invaluable assistance with Microsoft Office products (as deadlines approached) and for his endless stream of encouragement.

Table of Contents

Abstract……………………………………………………………………………….....…i

Dedication………………………………………………………………………………...iii

Acknowledgements…………………………………………………………………….....iv

Table of Contents……………………………………………………………………...….vi

List of Figures…………………………………………………………………………...viii

1 Introduction

1.1 Preservatives and Disinfectants in Developmental Toxicology……...…….....1

1.2 Benzyl Alcohol………………………………………………………..………2

1.3 Zebrafish as a model for developmental toxicology studies………..…………6

1.4 Zebrafish developmental stages…………..………………………………...... 7

1.5 Oxidative Stress……..……………………………………………………...... 8

1.6 Cell Death………………..……………………………………………………8

1.7 Microtubule Formation…………..……………………………………………8

2 Objectives……………………………………………………………………………...10

3 Methods and Materials

3.1 Materials..……………………………………………………………………11

3.2 Preparation of Zebrafish Embryos for Exposure to Benzyl Alcohol…..…….11

3.3 Exposure of Zebrafish Embryos to Benzyl Alcohol……..…………………..12

3.4 ZETA Assay…………..……………………………………………………...13

3.5 Acridine Orange Assay…………………..…………………………………..13

3.6 Immunohistochemistry Assay……………………………..…………………14

3.7 Superoxide Anion Assay………..……………………………………………15

4 Results…………….……………………………………………………………………16

5 Discussion….…………………………………………………………………………..26

6 Future Investigations………………….………………………………………………..31

References………………………………………………………………………………..32

vii

List of Figures

Figure 1 Percent survival data……………………………………………………..17

Figure 2 The gross morphological effects of benzyl alcohol on developing

zebrafish (Danio rerio)…………………………………………………..19

Figure 3 Average heart rate data…………………………………………………..20

Figure 4 The effects of benzyl alcohol exposure on acetylated alpha-

tubulin staining in zebrafish……………………………………………...22

Figure 5 The effects of benzyl alcohol exposure on Acridine Orange

staining in zebrafish larvae………………………………………………24

Figure 6 Superoxide anion production in embryos exposed to benzyl alcohol……25

viii

Chapter 1

Introduction

1.1 Preservatives and Disinfectants in Developmental Toxicology

Preservatives and disinfectants are present in many of the products we encounter every day. From lotions to foods to medications, they make up a soup of chemicals that contact our skin and are absorbed through our lungs and gastrointestinal tract extending from in utero exposure until death. Preservatives and disinfectants have been implicated in a wide range of neurological and developmental disorders, such as ADHD and autism, and as a result, the field of developmental toxicology has been steadily growing.

Disinfectants and preservatives both achieve similar goals: they prevent the growth of bacteria in products and on surfaces. They are particularly used in cosmetic products to prevent microbial contamination by killing bacteria (Besito, 2012). These products can contain levels of preservatives that can be cytotoxic, depending on the type of product, the concentration of preservative in the product, the duration of use, location of use, and the extent to which the chemical can penetrate into the system (de Carvalho, et al, 2011).

There is strong case study evidence that suggests that chemicals present in safe levels for adults can cause serious birth defects, developmental disorders, and death to developing

1 organisms; therefore, it is important to verify the safety of the preservatives that are in use, as well as those being developed for future use, to determine fetal risk.

1.2 Benzyl Alcohol

Benzyl alcohol, an aromatic alcohol, is a valuable solvent due to its low vapor pressure, polarity, and its low toxicity to adults; however, its volatility and nature as a solvent means that precautions need to be taken during storage and use. It is soluble in water at low concentrations, which makes it ideal for study with zebrafish.

There are four main categories of use for this chemical: flavor industries (oral consumables), medical preservative, industrial solvent, and over-the-counter (OTC) products. In the flavoring industry, it has a wide range of applications: as an adhesive, a flavoring agent, a color diluent, a resin/polymer coating, and to seal gaskets for food storage. The acceptable daily intake according to the FDA is 0-5 mg/kg body weight in this form. As a medical preservative, it is found in IV medical preparations and in some topical products. It is the active ingredient in an oral medication to treat dental pain, though the FDA does not recommend its use in children under two years of age.

Chemical industry uses it as a solvent in inks, paints, epoxy resin coatings and lacquers.

It is also present in a plethora of OTC products, such as soap, perfume/cologne, cosmetics, lotions, shampoos, conditioners, and body washes. Its role in the cosmetic industry is as a fragrance component, a preservative (to prevent bacterial growth), a solvent, and as a viscosity-reducing agent. The FDA has established that benzyl alcohol can only be used in cosmetics in concentrations up to 25%.

The chemical, by its nature and uses, has many pathways of exposure: it can be consumed orally, absorbed dermally, injected intravenously or inhaled. It is rapidly absorbed in the gastrointestinal tract (GIT). Up to 60.5% of the dose was absorbed dermally when applied with petrolatum. Once absorbed, benzyl alcohol is metabolized via simple oxidation to benzoic acid (Flavor and Extract Manufacturers’ Association,

1984). Benzoic acid is then conjugated with glycine (which is the rate-limiter in the conversion to its metabolite) to form hippuric acid in the liver. Hippuric acid is excreted rapidly in the urine. The total dose is usually eliminated in 2 to 3 days, though more than

75% of the dose is excreted within the first 6 hours Informatics, 1972). Lipoic acid and valproic acid reduce the clearance of benzoic acid in rats (despite previous “loading” with glycine) by reducing the availability of hepatic coenzyme A, which is needed for the

ATP-dependent conjugation with glycine (Gregus et al., 1993 and Gregus et al., 1996).

Benzyl alcohol is also a membrane “fluidizer” that affects lipid bilayer structure

(Ebihara et al., 1979). It is a well-documented nondenaturant that has been shown to act on the membranes of erythrocytes (Burgen et al., 1970; Basse et al., 1992) and hepatocytes (Gordon et al., 1980). A variety of cellular effects can be ascribed to benzyl alcohol: it increases the activity of membrane-bound Ca2+-dependent enzymes such as adenylate cyclase ( Voorheis and Martin 1982; Martin, McConkey, and Stokes 1985;

Needham and Houslay 1988) as well as inhibiting the activity of various glycosyltransferases in the rat liver Golgi membrane (Mitranic, Boggs and Moscarello,

1982). Short-term intake of 2% benzyl alcohol in normal drinking water results in inhibition of hepatic alcohol dehydrogenase and mitochondrial aldehyde dehydrogenase isoenzyme activities in female rats, but not in males (Messiha, 1991). Benzyl alcohol

3 non-competitively inhibits the activity of hepatic alcohol dehydrogenase in rats maintained for a short time on ethanol (5%) (Messiha et al 1992).

The following oral LD-50 values have been cited for benzyl alcohol: mouse 1580 mg/kg; rat 1230 to 3200 mg/kg; and rabbit 1040 mg/kg. This chemical has not been studied in zebrafish, either as adults or as embryos. The oral LD-50 for benzoic acid was

1996 mg/kg in mice, 2000-2500 mg/kg in rats. The oral LD-100s for benzoic acid are

1520-2000 mg/kg in rabbits, 2000 mg/kg in cats, and 2000 mg/kg in dogs. The chronic oral toxicity of benzyl alcohol has been established in rats, and extrapolated by the EPA to a human reference dose for chronic oral exposure of 0.3 mg/kg/day in 1989. Acute parenteral exposure of mice to benzyl alcohol resulted in convulsions, dyspnea, and reduced mobility within the first 24 hours, when mice were dosed at concentrations lower than the established LD-50. Blood samples from benzyl alcohol-treated mice had a potential for hemolysis and precipitation. Along with dimethyl sulfoxide, polyethylene glycol 400, dimethylformamide and absolute ethanol, undiluted benzyl alcohol was ranked the most toxic. When a 10% solution of benzyl alcohol was applied to the skin of male nude mice for 24 hours, the resultant skin samples had severe compact hyperkeratosis, acanthosis, spongiosis, intracellular edema, and some areas of ulceration

(FASEB 1973; Flavor and Extract Manufacturers’ Association 1984).

Benzyl alcohol’s developmental and reproductive effects have been previously studied in mouse and rat models. The primary effects seen were maternal toxicities

(including death) as well as toxicity in the pups of the mothers who survived. The fetal effects were described as low birth weight, decreased litter size, and decreased weight gain. There does not appear to be any further data as to the specific toxicities

4 experienced by the developing organisms, though the delay in weight gain and low birth weight indicate that a developmental delay is occurring (Hardin et al 1987). Results have been inconsistent in the nearly three decades that this chemical has been studied. Oral- dose teratogenicity studies (using mice) gave negative results in one study, questionable results in another, and was considered a suspected reproductive hazard in the third (Nair,

2001). Benzyl alcohol’s developmental effects have never been evaluated in the zebrafish (Danio rerio).

In the early 1980s, benzyl alcohol in isotonic saline (used to flush catheters) was suspected of causing several neonatal deaths (Brown et al., 1982; Gershanik et al., 1982).

The observed effects of benzyl alcohol exposure in these neonates were metabolic acidosis, central neural depression, respiratory distress progressing to gasping respiration, hypotension, renal failure and sometimes seizures and intracranial hemorrhages.

Biochemical evidence of benzyl alcohol was found in the blood and urine samples of these neonates, including high concentrations of the parent compound (benzyl alcohol) and its metabolites (benzoic acid and hippuric acid). The 1983 revision of the US

Pharmacopeia contained warnings that the two most common classes of products that were implicated in benzyl alcohol exposure (bacteriostatic water for injection and bacteriostatic sodium chloride injection) stating that they were “not for use in newborns.”

Further regulation was deemed unnecessary due to the reduced number of cases of benzyl alcohol toxicity after the revision of the US Pharmacopeia and the manufacturers of bacteriostatic water and bacteriostatic sodium chloride voluntarily added warning labels to their products, cautioning against their use in newborns (Nair, 2001).

1.3 Zebrafish as a model for developmental toxicology studies

The zebrafish (Danio rerio) is a small freshwater fish measuring approximately

1.5 inches long in adulthood and is a common aquarium fish. It has grown in popularity as an animal model for various toxicological applications and as such has become respected as one of the most practical models for biological studies and chemical toxicity work (Tsang, 2010; Oberemm, 2000; and Powers, 1989).

The zebrafish (Danio rerio) has distinct advantages in the field of developmental toxicology. In recent years, this animal model has grown in popularity due to its ease of use and genetic clarity; however, in terms of developmental toxicology, its usefulness is centered around the transparency of the zebrafish embryo and chorion during development (Ton, 2006). It is possible to watch development taking place with the use of a light microscope. In addition to this obvious advantage over other models (mouse, rat, etc.), the availability of a relatively large number of eggs laid weekly and the rapidity with which these animals develop makes it possible to collect a large amount of data in a short period of time. The fish are small in size, they have a short generation time, and husbandry is a simple process. While there are distinct morphological and physiological differences between zebrafish and humans, many of the toxicological processes are conserved (Carvan, et al., 2000 and Carvan, et al., 2004). Responses to chemicals that cause reproductive toxicity, behavioral defects, teratogenesis, cardiotoxicity, ototoxicity, etc. are largely identical. The high-throughput nature of the animal model and the conservation of toxicological pathways taken together demonstrate the usefulness of zebrafish in developmental toxicological screening (Blader, 2000). When bred in proper ratios, the abundance of eggs regularly produced minimizes the inconsistencies stemming

6 from small sample sizes. They are more biologically relevant for correlation with expected human outcomes than smaller models (such as C. elegans or Drosophila) due to their vertebrate nature. Early life stages are highly conserved among vertebrates (Laale,

1977).

1.4 Zebrafish developmental stages

There are 8 stages of development in zebrafish that are broken down into embryonic periods which include: zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching period. The zygote period, from 0 to ¾ hpf, extends from fertilization until cleavage occurs. The cleavage period (3/4 – 2 ¼ hpf) includes the period of time during which the blastomeres divide at a rate of once every 15 minutes until the 32 cell stage. Next is the blastula period (2 ¼ - 5 1/4) hpf), during which the blastodisc begins to look ball-like and the embryo enters epiboly. The following phase, the gastrula phase (5 ¼ - 10 hpf), continues the epiboly process and forms the germ ring.

Then, the segmentation period begins (10-24 hpf) during which the primary organs become visible. It is during this phase that the somites develop. The pharyngula stage

(24-48 hpf) refers to the time when the classic vertebrate body plan is now visible in the embryo. The final embryonic stage is the hatching period (48 – 72 hpf) when the fish typically hatches from its chorion. After the 3rd day, it is considered to be a larvae regardless of whether or not it has hatched. Hatching times can also vary within the same clutch of eggs. Once in the larval stage, it will continue to grow and inflate its swim bladder. External feedings must begin 5 days post fertilization, at which point the yolk sac will have been consumed. Zebrafish reach sexual maturity 100 days post fertilization

(Hill et al., 2003 and Kimmel et al., 1995).

1.5 Oxidative stress

Superoxide anion (SA) is formed endogenously by normal aerobic metabolic reactions and plays a role in reactions which yield free radicals (e.g. hydrogen peroxide and peroxynitrite) which can lead to DNA damage and lipid peroxidation. SA is converted to hydrogen peroxide, which is then converted into water; however, during oxidative stress situations, SA is generated in excess. To deal with this, superoxide dismutase (SOD) is induced to counterbalance the increased free radical formation. SOD works by removing SA (Hassoun, et al., 2005).

1.6 Cell death

Apoptosis is a form of programmed cell death that is involved in embryogenesis, metamorphosis, cellular homeostasis and as a defense mechanism (Makani, 2002). It constitutes one of many end results of oxidative stress. Acridine orange exclusively binds to exposed DNA in cells undergoing apoptosis. Through this method, cells undergoing apoptosis may be visualized.

1.7 Microtubule formation

Microtubules are cytoskeletal complexes comprising of alpha and beta tubulin and microtubule associated protein. Microtubule arrays function in intra-cellular trafficking of nutrients and other cellular products from the center of the cell to the periphery.

Microtubules undergo dynamic instability by addition of tubulin subunits at the beta tubulin (positive) side and loss of subunits at the alpha (negative) side (Lewis et al, 1997,

Tian et al 1999). If the subunits forming the microtubule array cannot associate and

8 disassociate, cellular trafficking breaks down (Chretien et al., 1995). Axons involved in neurotransmission in the brain rely on microtubules for cytoplasmic transport.

Our objective was a systematic study of the developmental toxicity of benzyl alcohol in zebrafish embryos. Embryos were exposed at 4 hpf and remained in the exposure solution until 144 hpf, at which point the experiment was concluded. The following concentrations of benzyl alcohol were selected for study: 1 mM, 300 uM, 100 uM, 30 uM, 10 uM, 3 uM, 1 uM. Morphological changes, mortality, hatching rates, and heart rates were observed and analyzed. Neuronal outgrowth was observed via an immunohistochemical assay (Novak, 2003). Cell death was observed through the use of an acridine orange assay. Superoxide anion production was also measured. Further work is needed to define a clear connection between benzyl alcohol exposure and developmental defects, but the current data suggest that a connection does exist and hints at what specific developmental defects result from this form of chemical exposure.

Chapter 2

Objectives

Preservatives and disinfectants have become increasingly popular areas of toxicological research in recent years due to public concern over the effects these chemicals exhibit in the body. While the effects of these sorts of chemicals in adult organisms are well known, the effects on developing organisms have not been well- studied. Preservatives are present in many products and have been implicated in a wide range of neurological and developmental disorders, such as ADHD and autism, leading to growth in the field of developmental toxicology. There is strong case study evidence that chemicals present in safe levels for adults can cause serious birth defects, developmental disorders, and death to developing organisms; therefore, it is important to verify the safety of the preservatives that are in use, as well as those being developed for future use, to determine fetal risk. Benzyl alcohol’s wide-spread use in a variety of industrial and clinical applications, as well as its water-solubility, makes it an attractive target for research. The objective of this study was to elucidate the developmental effects of benzyl alcohol on zebrafish embryos.

Chapter 3

Methods and Materials

3.1 Materials

Benzyl Alcohol was obtained from Fisher Scientific (Fair Lawn, New Jersey,

USA). Brine shrimp were acquired from Brine Shrimp Direct (Ogden, UT). All conical tubes and other disposable sterile materials were obtained from Corning through Fisher

Scientific. Monoclonal Anti-tubulin antibody from mouse was obtained from Sigma-

Aldrich. Fluorescent Avidin Kit and Vectastain ABC kit were acquired from Vector

Laboratories, Inc (Burlingame, CA). All other chemicals were obtained from Fisher

Scientific (Chicago, IL, USA) and were analytical grade or the highest grades commercially available.

3.2 Preparation of Zebrafish Embryos for Exposure to Benzyl Alcohol

Adult zebrafish were obtained from ZIRC (University of Oregon) and maintained in a system of buffered water (pH 7.2) at 28° Celcius and fed brine shrimp and Tetramin dried flakes (Tetra, Blacksburg, VA, USA). The light-dark cycle was maintained at

14:10h. Two males and four females were placed in a 10 gallon breeding tank with

11 marbles as a substrate. Breeding is dependent on the transition from dark to light and the presence of the marble substrate, so breeding occurred at first light. Eggs were collected by siphoning, and the fertilized eggs were sorted from the rest using a light microscope.

Eggs were then washed in a 0.5% bleach solution, rinsed several times with egg water

(30 mg Instant Ocean in 500 mL distilled H2O, autoclaved), before being divided into

Petri dishes for exposure and subsequent experiments. Eggs were collected approximately an hour post fertilization (hpf) and divided among different exposures.

3.3 Exposure of Zebrafish Embryos to Benzyl Alcohol

A 0.1 M stock solution of benzyl alcohol in distilled water was prepared, parafilmed, and stored in the fridge. Dilutions for exposure were made using this stock solution and egg water. Depending on the experiment being done, between 20 and 60 eggs were placed in each Petri dish, with the number of exposure groups needed determining the number of groups. Once dilutions were prepared, they were warmed in a hot-air incubator to temperature (~28° C). All egg water in the dish with the embryos was aspirated, then replaced with the warmed exposure solutions to ensure that the concentrations were accurate.

Exposure to benzyl alcohol was conducted at 4 hpf, and regular observations were taken at the following developmental timepoints: 8 hpf (75% epiboly of gastrulation), 24 hpf (prim-6), 32 hpf (prim-16), 55 hpf (pec fin), 80 hpf (protruding mouth), and 144 hpf

(early larva). Exposure was performed by placing eggs in an egg water solution (30mg

Instant Ocean/500mL distilled H20), with the following concentrations of benzyl alcohol added: 1 μM , 3 µM, 10 μM, 30 µM, 100 μM, 300 µM, 1 mM, and 3 mM. To expose the

12 eggs, first the water was aspirated off the eggs until a minimal amount of water remained in the Petri dish. The solution for exposure was then added to the Petri dish, and the eggs were incubated at 28.5° Celsius. Since benzyl alcohol is volatile, the plates were parafilmed to prevent cross-contamination and fluctuations in chemical concentration.

Observations were conducted with use of a microscope. Gross development, mortality, and heart rate were observed and recorded.

3.4 ZETA Assay

Embryos were observed at specific developmental timepoints: 8 hpf, 24 hpf, 32 hpf, 55 hpf, 80 hpf, and 144 hpf. Heart rates were measured at the 32 hpf, 55 hpf, and

80 hpf time points and recorded. Any morphological changes or aberrations in development were recorded, and examples of these were photographed.

3.5 Acridine Orange Assay

Cell death in benzyl alcohol-exposed zebrafish embryos was documented by

Acridine Orange (AO) staining (2mg/mL) at 70-80 hours post-fertilization (hpf) for 1 hour (Carvan, et al., 2004). AO is not cell-permeable and binds to DNA in cells that have undergone apoptosis (Carvan, et al., 2004). The embryos were washed with PBS four times for 20 minutes on an orbital shaker. Embryos were examined using a FITC filter and Zeiss Axiovert 25 Fluorescent Microscope (Thornwood, NY) and documented with a

Sony Cyber Shot Carl Zeiss Vario Sonnar Digital Camera (Thornwood, NY).

3.6 Immunohistochemistry Assay

Exposed embryos (80hpf) were soaked in cytofix (BD biosciences, San Diego,

CA) for two hours at room temperature with low stirring on an orbital shaker. The fish were washed two times for five minutes with 1X phospho-buffered saline (PBS) and then once for five minutes in water. All washes were combined with gentle stirring on the orbital shaker.

Next, the fish were soaked in glass beakers in cold acetone for ten minutes in the refrigerator. The specimens were then washed one time for five minutes with water and two times with 1 X PBS for five minutes each and washed once with BDP for five minutes. BDP was a mixture of 1g BSA plus 1 mL DMSO brought up to 100 mL with

1X PBS.

Embryos were then exposed to horse serum. The ratio made was 100 µl serum for every 5 ml of 1X PBS. The zebrafish were put in the horse serum mixture for 30 minutes at room temperature with gentle stirring, followed by two washes for five minutes each with BDP and incubation overnight with antibody at 4° Celsius with gentle stirring on the orbital shaker. The monoclonal anti-acetylated alpha-tubulin clone antibody (Sigma-Aldrich, St. Louis, MO) was diluted with BDP in a ratio of 1:2000.

Embryos were washed three times for 30 minutes with BDP followed by incubation with the anti-mouse secondary antibody from the Vectastain kit (Vector Laboratories,

Burlingame, CA) for one hour at 37° Celsius. The mixture contained one drop concentrated stock to five ml of PBS containing 1.5% normal serum (Vector

Laboratories, Burlingame, CA).

After incubation with the secondary antibody, the embryos were washed three times for 30 minutes with BDP and incubated 1:1 with diluted ABC solution (Vector

Laboratories, Burlingame, CA) for 30 minutes at room temperature. Embryos were washed three times for 30 minutes with BDP. The last step was the staining procedure.

The stain was mixed according to instructions, with fluorescent dye (Fluorescent Avidin

Kit, Vector Laboratories, Burlingame, CA) and BDP. The zebrafish were placed in a 12- well plate for staining. The fish were stained for two minutes. Cytofix was added to halt the staining.

3.7 Superoxide Anion Assay

Embryos exposed as described above were homogenized in Tris-KCl buffer

(1.15% KCl solution in 0.05 M Trizma-HCl pH 7.4) at 70 hpf, then stored on ice. A small sample of the homogenate (50 uL) was then added to a test tube, followed by the addition of 1.5 mL cytochrome C stock solution (8.3 mg cytochrome c from horse heart obtained from Sigma dissolved in 45 mL 1 X PBS). A blank consisting of Tris-KCl buffer and cytochrome c was also prepared. All samples (including blank) were then incubated for 20 minutes in a 37°C air incubator. Absorbance was measured at 550 nm.

Chapter 4

Results

Mortality Rate

Embryos were exposed to concentrations of benzyl alcohol in egg water ranging from 1 uM to 10 mM, and survival through key time points was observed and recorded.

Embryos were observed at regular developmental time points (8, 24, 32, 55, 80, and 144 hpf) for each survival experiment. Death was determined either by lack of heart beat (if heart was developed and visible) or by sufficient evidence of cell death (blebbing, opacity in embryos that had not yet developed a distinct heart). Survival rates were recorded and plotted in Figure 1. The data series were averaged and plotted as percentages. None of the embryos exposed to 10 mM benzyl alcohol survived past 8 hpf. All of the animals in the control group survived through 80 hpf.

This assay was run in triplicate alongside the heart rate assay and the observation of the gross morphological effects, with a minimum of 15 embryos per experimental group and control. Results shown below were obtained from one experimental exposure and are indicative of the trends seen overall.

Survival Data 120

100

80 control 1 uM 60 10 uM 100 uM 40 1 mM

Number survivingNumber of embryos 20 10 mM

0 4hpf 8 hpf 24 hpf 32 hpf 55 hpf 80 hpf Developmental timepoint

Figure 1: Percent survival data.

Gross Morphological Effects

Gross morphological observations were taken at critical time points: 8, 24, 32, 55,

80 and 144 hpf. All gross morphological changes between the exposure groups and the control group were photographed for comparison at the same time point.

Starting at 32 hpf, the 10 uM and higher exposure groups started to exhibit mild cardiac edema. At this time point, the highest concentration (1 mM) had notably smaller heads, more prevalent and severe cardiac edema, small/slightly misplaced eyes which were less developed and less regularly-shaped, and shortened snouts.

At 55 hpf, the prevalence of mild cardiac edema in the groups exposed to 10 uM and higher concentrations of benzyl alcohol increased. All of the embryos in the 1 mM exposure group had some level of cardiac edema (moderate to severe) and eye development abnormalities.

Embryos in the 10 and 100 uM groups began to exhibit light avoidance behavior at 80 hpf. All of the embryos in the 1 mM exposure group showed severe cardiac edema, some form of craniofacial deformation, and eye irregularities. One embryo at this concentration exhibited pigmentation abnormalities in the eye, snout malformation, severe cardiac edema with blood pool, and a dehydrated yolk sac.

For all time points, the embryos exposed to the 1 uM did not exhibit the dramatic edema and eye abnormalities of the higher concentrations; however, their survival rate was less than that of the control embryos.

Figure 2: The gross morphological effects of benzyl alcohol on developing zebrafish (Danio rerio).

Heart rate

Heart rates were observed and recorded at 32, 55, and 80 hpf. Surprisingly, embryos exposed to the 1 uM concentration of benzyl alcohol showed an aberrant drop in heart rate at 55 hpf. Heart rate was determined by visually counting heart beats for 10 seconds and estimating heart rates by multiplying the number of beats by 6.

Heart Rate Data 250.0000

200.0000

150.0000 control 1 microM 100.0000 10 microM

100 microM Heart beats/minute Heart 50.0000 1 mM

0.0000 32 hpf 55 hpf 80 hpf Developmental timepoint

Figure 3: Average heart rate data.

Immunohistochemical Assay

An immunohistochemistry assay showed the extent of neuronal outgrowth as measured by detection of an epitope on acetylated alpha tubulin. Zebrafish were exposed to benzyl alcohol as described above, and treated according to the immunohistochemical protocol described in the Methods section. Stained embryos were visualized by using a confocal microscope with a 10X focal length and a FITC filter in order to assess any damage caused by the presence of benzyl alcohol to neuronal outgrowth. This assay was run in triplicate. The pictures shown were indicative of results seen overall. The green fluorescence indicates where neuronal outgrowth was occurring. The neuronal outgrowth exhibited by the exposed larvae is less organized and less prevalent than that of the control larvae. Higher exposure concentrations of benzyl alcohol increased the presence of other abnormalities such as edema and craniofacial anomalies, but did not show any further effect on anti-acetylated alpha tubulin staining.

Figure 4: The effects of benzyl alcohol exposure on acetylated alpha-tubulin staining in zebrafish are shown. As compared with the control fish, the other fish have less staining,

indicating poor polymerization of acetylated alpha-tubulin in axons.

Acridine Orange Assay

Acridine orange is used to characterize cell death in whole embryos (Carvan, et al. 2004). Following exposure to benzyl alcohol as described above, embryos were treated with acridine orange. After multiple washes, pictures were taken and are shown below. There were distinct differences between the exposure groups and the control group. The control animal showed organized and distinct cell death occurring in expected locations such as in the brain and along the notochord as well as in the GI tract.

The exposed animals showed levels of disorganized cell death that increased in a dose- dependent manner.

This assay was run in triplicate. At 70 hpf, five to ten surviving embryos were selected and placed in a six-well plate for exposure. Photos were taken of embryos that were indicative of the whole exposure or control group once the assay was completed.

These photos are shown below.

Figure 5: The effects of benzyl alcohol exposure on Acridine Orange staining of zebrafish larvae. The yellow and orange staining increases in a dose-dependent manner.

Superoxide Anion Assay

Superoxide anion detection by cytochrome C is used to determine the production of superoxide anions as an indicator of oxidative stress. Exposure to benzyl alcohol was carried out as described above, and at 70 hpf embryos were homogenized and treated as described in the methods. An increase in superoxide anion production was seen in concentrations up to 100 uM. At concentrations higher than 100 uM, there is a drop in superoxide anion production. This assay was run in triplicate, and all results showed the same trend. Results shown in the graph below are representative of this trend. The number of surviving embryos available for each trial varied from 15 embryos up to 40, so the specific concentration of superoxide anion produced varied based on how many embryos were included. The increase in superoxide anion concentration up to 100 uM was seen in all trials.

Superoxide Anion Assay

0.000007

0.000006

0.000005

0.000004

0.000003 [SA] 0.000002

0.000001

Superoxide Superoxide AnionConcentration 0 control 1 uM 3 uM 10 uM 30 uM 100 uM 300 uM 1 mM Benzyl Alcohol Concentration

Figure 6: Superoxide anion production in embryos exposed to benzyl alcohol.

Chapter 5

Discussion

The overall goal of this project was to characterize and quantify the developmental toxicity of benzyl alcohol in the zebrafish (Danio rerio). The results indicate that benzyl alcohol causes a variety of morphological defects in developing zebrafish embryos, ranging from edema to fluctuations in heart rate to neurological problems. The literature implies that depressed heart rate was likely related to the cardiotoxic potential of this chemical in adult organisms, which the data stated previously supports.

Cell death related to benzyl alcohol exposure was primarily localized to the head and notochord, though there was some peripheral cell death (not shown) in the tail end.

This was attributed to lack of blood perfusion to the distal end of the tail. The cell death seen in the control animal is attributable to neuronal plasticity and normal cell death that occurs during development (Purves, 2007). The added observed cell death in treated embryos could either be attributed to direct toxicity of the chemical on nervous system cells or to lack of perfusion caused by cardiotoxicity of the chemical.

The immunohistochemistry results indicate that motor neurons are affected by benzyl alcohol exposure, and the effect is seen in high concentrations (approaching the

LD-100). Disorganization of neuronal outgrowth is the primary dysfunction rather than lack of outgrowth overall as seen in Figure 5. Taken together, these results indicate that the nervous system is negatively affected by benzyl alcohol exposure during development; however, it is unclear if this is due to cardiotoxicity and perfusion problems caused by the chemical or if this is a direct result of neuronal exposure to benzyl alcohol.

Future investigations into the direct effect of benzyl alcohol on specific cell types in culture would clarify if direct toxicity to neuronal cells causes the cell death seen in the central nervous system, or if this phenomenon is a down-stream result of benzyl alcohol’s cardiotoxic and perfusion effects.

The production of superoxide anions in benzyl alcohol-exposed zebrafish embryos increases in a dose-dependent manner up to the 100 uM exposure concentration.

Above this concentration, the production of superoxide anions drops off. Cell death is occurring at a much higher rate at the higher benzyl alcohol concentrations, and this is the likely culprit behind the drop in superoxide anion concentration. Again, it is unclear if the oxidative stress seen is due to stress caused by lack of perfusion or if it is due to direct toxicity of benzyl alcohol. Further clarification of benzyl-alcohol induced oxidative stress is needed, such as examination of glutathione levels and nitric oxide levels.

Studying benzyl alcohol’s effects in cell culture would clarify if the above noted oxidative stress is caused by perfusion problems or is toxic to the specific cell type.

The known toxicokinetics of benzyl alcohol in zebrafish mirror human exposure.

The modes of exposure available in the zebrafish model are gastrointestinal, through the gills, and dermal exposure. Initial human in utero exposure would likely occur through maternal circulation (which is already subject to some level of metabolism, both through

27 the maternal hepatic system and through the placenta which is mildly metabolic in its own right); however, once in the fetus, the parent compound and metabolites would be excreted into the amniotic fluid, followed by fetal re-exposure through respiration (the fetus practices breathing with the amniotic fluid), swallowing of the amniotic fluid, and dermal absorption. Part of the extreme toxicity benzyl alcohol causes in neonates is due to the reduced metabolic capacity of newborns. Neonates lack sufficient enzyme levels to clear benzyl alcohol until several weeks after birth (LeBel et al., 1988). Clarification of which enzymes are involved in metabolizing benzyl alcohol would be topic for future work. Benzyl alcohol’s effects on clutch size was not studied in zebrafish, though previous mammalian work suggests that exposure would cause fertility problems

(Inveresk Research International Ltd, 1983).

At the molecular level, benzyl alcohol’s toxicity may be due to its nature as a membrane “fluidizer.” The pharmacodynamics of this chemical have not been well- studied. It seems to follow similar toxicological pathways as ethanol, though the amount of cell death seen in the benzyl alcohol-exposed embryos suggests that there are other additive effects happening. Benzene is known to intercalate with DNA and cause single- and double-strand breaks in DNA leading to cell death, but it is unclear if a similar phenomenon is occurring during exposure to benzyl alcohol.

The literature suggests that benzyl alcohol causes developmental delay in mice

(Informatics, 1972). The data presented here agrees with that, and further clarifies that the developmental delay is probably due to cardiotoxicity of the chemical, as well as having neurological effects, though not specific to motor neurons. Clarification of which cell types in the CNS are affected by this chemical would be a provocative topic for

28 further work. The oxidative stress observed in the zebrafish may play an additional role in the developmental delay seen, though this also could be studied in greater depth.

Currently there are minimal regulations in place regarding benzyl alcohol’s use in non-medical products (Nair, 2001). Multiple routes of exposure are likely, and no warnings exist regarding maternal exposure to the chemical. The data presented previously suggests that benzyl alcohol presents significant fetal risk at low concentrations. It is probable that these toxic concentrations could be reached easily through the multiple routes of exposure available. Moreover, any small amount of the parent compound or its metabolites that reach the fetus despite maternal metabolism and placental protection would be re-absorbed and cause fetal toxicities until the compounds left the amniotic fluid in a concentration-dependent manner. Therefore, it would be beneficial to the public if this chemical was more closely regulated and pregnant women were cautioned to avoid exposure to it.

These data have offered new insight into the toxicity of benzyl alcohol on developing organisms. The available literature failed to offer understanding of the localization of toxicity in the developing organism and whether or not oxidative stress plays a role in the developmental delay associated with in utero exposure to benzyl alcohol. It is now known that exposure to this chemical to zebrafish embryos causes a wide array of developmental defects, from craniofacial abnormalities to the production of superoxide anion, an indicator of oxidative stress. This information will lead to new research directions, as well as being clinically useful when counseling pregnant women.

The growing field of developmental toxicology will also benefit from the delineation of

29 this compound, which will be a useful addition to the library of toxic chemicals due to its complexity and its ubiquitous nature.

Chapter 6

Future Investigations

1.) Elucidate the type of oxidative stress being experienced by the developing organism through evaluation of glutathione and nitric oxide levels.

2.) Determine the cell type being affected, with particular attention paid to glial cells.

3.) Determine the mechanism of action and evaluate the toxicokinetics of this chemical.

4.) Correlate developmental dysfunctions seen with human disorders, such as ADHD and autism.

5.) Determine which enzymes are lacking in neonates that are involved in benzyl alcohol metabolism. Re-evaluation of the developmental defects seen in zebrafish could be accomplished with morpholino knock-down experiments.

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