Salsolinol-Like Compounds As Biomarkers of Human Alcohol Consumption, Disease and Toxicity

Home , Metanephrines, Norsalsolinol, Tetrahydroisoquinoline

Salsolinol-like compounds as biomarkers of human alcohol consumption, disease and toxicity

______

Thesis Presented

Sean Edward Kocur

The Bouvè Graduate School of Health Sciences in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Pharmaceutical Sciences with an Interdisciplinary Specialization

NORTHEASTERN UNIVERSITY

BOSTON, MASSACHUSETTS

December, 2016

Signature page 1 (Signed by Dean and Library)

Northeastern University

Bouve Graduate School of Health Sciences

Thesis title: Salsolinol-like compounds as biomarkers of human alcohol consumption, disease and toxicity

Author: Sean Edward Kocur

Program: Pharmaceutical Science with an Interdisciplinary Specialization

Approval for thesis requirements of the Doctor of Philosophy in Pharmaceutical Science

Thesis Committee (Chairman) ______Date:______

______Date:______

Director of the Graduate School ______Date:______

Dean ______Date:______

Copy Deposited in Library Date: ______Date:______iii

Signature page 2 (Not Signed by Dean and Library)

Northeastern University

Bouve Graduate School of Health Sciences

Thesis title: Salsolinol-like compounds as biomarkers of human alcohol consumption, disease and toxicity

Author: Sean Edward Kocur

Program: Pharmaceutical Science with an Interdisciplinary Specialization

Approval for thesis requirements of the Doctor of Philosophy in Pharmaceutical Science

Chairman: ______Date:______

Thesis Committee Member ______Date:______

Director of the Graduate School ______Date:______

Table of Contents

Page #

LIST OF TABLES …………………………………………...……………………….………. ..vii

LIST OF FIGURES ……………………………………………..……………………….. ……viii

LIST OF ABBREVIATIONS …………………………………………....………………….……x

ABSTRACT ………………………………………………………………………………….…xii

ACKNOWLEDGEMENTS …………………………………………….………………...... xv

OBJECTIVE ……………………………………………………………….………………...…xvi

SPECIFIC AIMS ……………………………………………...... …………….…xvii

CHAPTER 1. THE BIOLOGY OF ETHANOL CONSUMPTION AND ALCOHOLISM

1. Alcoholism and Alcohol Use Disorders……………………………………………………....1 1.1. History of ethanol use…………………………………………………………………….2 1.2. Epidemiology………………………………………………………………………..……4 1.3. Clinical Presentation……………………………………………………………….…..…7 1.4. Metabolism……………………………………………………………………...………10 1.5. Health Effects………………………………………………………………….…….….14 1.6. Treatments, Interactions, and Disulfiram……………………………………….………21 1.7. Significance of Study…………………………………………………………………....24

CHAPTER 2. METABOLIC DISORDERS AND DISEASES ASSOCIATED WITH ALCOHOL CONSUMPTION

2. Metabolic Disorders…………………………………………………………………………25 2.1. Introduction………………………………………………………………………….….26 2.2. Metabolic syndrome…………………………………………………………….………27 2.3. Glucose Metabolism………………………………………………………………...…..28 2.4. Lysosomal Storage Diseases…………………………………………………………….29 2.5. Alzheimer’s Disease…………………………………………………………………….30

CHAPTER 3. EFFECTS OF TETRAHYDROISOQUINOLINES ASSOCIATED WITH ALCOHOL CONSUMPTION

3. Tetrahydroisoquinolines (TIQs)………………………………………………………….…..32 3.1. Introduction……………………………………………………………………………...33 3.2. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)……………………………..….33 3.3. Salsolinol…………………………………………………………………………..……35 3.4. Carboxysalsolinol……………………………………………………………………….38 3.4.1. 1-Carboxysalsolinol……………………………………………………………..39 3.4.2. 3-Carboxysalsolinol……………………………………………………………..41 3.5. N-Methyl-Salsolinol………………………………………………………………….....42 3.6. 1-acetyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (ADTIQ)…..………………….42

CHAPTER 4. SIMULTANEOUS QUANTITATION OF CATECHOLAMINES AND SALSOLINOL-LIKE TETRAHYDROISOQUINOLINES BY LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY/MASS SPECTROMETRY

4. Quantitation Method 4.1. Introduction………………………………………………………………………...……46 4.2. Methods & Materials……………………………………………………………………46 4.3. Results…………………………………………………………………………………...58 4.4. Conclusions……………………………………………………………………………...60

CHAPTER 5. DETERMINATION OF ENDOGENOUS LEVELS OF SALSOLINOL-LIKE TETRAHYDROISOQUINOLINES IN MOUSE BRAINS

5. Endogenous Level 5.1. Introduction………………………………………………………………………...……62 5.2. Results………………………………………………………………………………..….65 5.3. Conclusions……………………………………………………………………………...67

CHAPTER 6. REGIONAL DISTRIBUTION OF 3-CARBOXYSALSOLINOL IN MOUSE BRAINS FOLLOWING IV INJECTION

6. Distribution with IV dosing 6.1. Introduction………………………………………………………………………..…….69 6.2. Results………………………………………………………………………………..….71 6.3. Conclusions…………………………………………………………………………..….72

CHAPTER 7. REGIONAL DISTRIBUTION OF 3-CARBOXYSALSOLINOL & 6-METHOXY-3- CARBOXYSALSOLINOL FOLLOWING IV DOSING OF 3-CARBOXYSALSOLINOL IN MOUSE BRAINS AT TIME POINTS 1,6, AND 10 MINUTES

7. Ethanol and L-DOPA 7.1. Introduction……………………………………………………………………………...73 7.2. Results………………………………………………………………………………..….76 7.3. Conclusions…………………………………………………………………………...…79

CHAPTER 8. EVALUATION OF THE EFFECTS OF COMT, MAO, AND AADC INHIBITORS ON THE FORMATION OF CATECHOLAMINES, METABOLITES AND SALSOLINOL-LIKE TETRAHYDROISOQUINOLINES

8. MAO & COMT Inhibitors 8.1. Introduction………………………………………………………………………….…..81 8.2. Results………………………………………………………………………………..….85 8.3. Conclusions……………………………………………………………………..……….88

CHAPTER 9. EVALUATION OF THE EFFECTS OF ACUTE AND CHRONIC ALCOHOL ADMINISTRATION ON CATECHOLAMINE AND SALSOLINOL-LIKE TETRAHYDROISOQUINOLINES LEVELS IN THE MOUSE BRAIN

9. Acute & Chronic Alcohol 9.1. Introduction……………………………………………………………………….....….90 9.2. Results……………………………………………………………………………….….93 9.3. Conclusions……………………………………………………………………………..99

CHAPTER 10. CONCLUSIONS 10. Conclusions 10.1. Conclusions……………………………………………………………………..100 10.2. Future Considerations………………………………………………………..…102

REFERENCES ...... …………...……104

APPENDICES……………………………………………………………………………….…124

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LIST OF TABLES

1. MRM parameters of catecholamines, tetrahydroisoquinolines, & metabolites…….57

2. Method validation data for CA’s and TIQs in mouse brain…………………………59

3. Enzymes, substrates, and inhibitors…………………………………………………...83

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LIST OF FIGURES

1. Global incidence rates for alcohol……………………………………………………....5 2. Historical trends of ethanol consumption 1850-1997.……………………………..…..6 3. Ethanol as the most harmful drug in the world…………………………………..……7 4. Blood alcohol concentrations and associated effects…………………...... 8 5. Oxidative pathways for ethanol metabolism………………………………………….11 6. Genetic mutations that affect ethanol consumption……….…………………………13 7. Non-oxidative consequences of ethanol consumption………………………………...14 8. Ethanol allosteric binding site on the GABA receptor……………………………….16 9. Ethanol targets receptors, signaling systems, and neuromodulators………………..17 10. Possible long term effects of ethanol……………………………………………….….18 11. Lipid metabolism in diabetes mellitus………………………………………………...22 12. Common therapeutic classes with drug-alcohol interactions………………………..23 13. Bioenergetic homeostasis link between diabetes & neurodegeneration………….…26 14. MPTP like structures of tetrahydroisoquinolines…………………………………….35 15. Pathways for the formation of salsolinol and N-methylsalsolinol…………………...38 16. Formation of 1-carboxysalsolinol, 3-carboxysalsolinol and salsolinol……………....41 17. Salsolinol and 1-carboxysalsolinol in Alcoholics and Non-Alcoholics……………....41 18. Formation of ADTIQ from Dopamine and Methylglyoxal……………………..……44 19. Alternative Products of Glycolysis & Formation of ADTIQ…………………...……45 20. Interplay between glucose, catecholamine, and ethanol metabolism………………..49 21. Pictet-Spengler reaction for the formation of 3-carboxysalsolinol…………………..51 22. Methylation of 3-carboxysalsolinol to 6,7-methoxy-3-carboxysalsolinol……………52 23. Chromatogram of catecholamines and TIQs……….…………………………….…..55 24. Mass spectrum for 3-carboxysalsolinol………………………………………………..56 25. Enzymatic & non-enzymatic metabolism of CA’s and the formation of TIQs……..63 26. Endogenous levels of 3-carboxysalsolinol in mouse striatum…………………….….65 27. Endogenous levels of salsolinol in mouse brains………………………………...……66 28. Endogenous TIQs in Mouse Brain…………………………………………………….68 29. 3-carboxysalsolinol in mouse brain after IV injection………………………………..72 30. Methylation of 3-carboxysalsolinol by COMT………………………………………..74 ix

31. 3-CS and 6M-3-CS in mouse brain at 1 minute……………………………………....76 32. 3-CS and 6M-3-CS at 6 minutes in mouse brain……………………………………...77 33. 3-CS and 6M-3-CS at 10 minutes in mouse brain……………………………...……..78 34. Time course for 3-CS in mouse striatum………………………..…………………….80 35. Metabolism pathway for TIQs, catecholamines, and their metabolites………….....82 36. MAO, COMT, and the benserazide inhibitors…………………………………….….84 37. 3-carboxysalsolinol in mouse striatum……………………………………………...…85 38. In vivo formation of HVA in mouse striatum …………………...……………..…….86 39. In vivo formation of salsolinol in mouse striatum……………………………...…….87 40. Levels of TIQs & metabolites in mouse brain…………………………………..…….89 41. Catecholamine and TIQs pathways with ethanol inhibitors………………...………91 42. Catecholamines in mouse striatum with ethanol and treatment dosing………….…93 43. Catecholamines in mouse cerebellum with ethanol and treatment dosing………….94 44. TIQs in mouse striatum after acute and chronic treatments…………………...……95 45. TIQs in mouse cerebellum after acute and chronic treatments………………….….96 46. Salsolinol & N-methyl-salsolinol in mouse striatum………………………………….97 47. Salsolinol & N-methyl-salsolinol in mouse cerebellum………………………….……98

LIST OF ABBREVIATIONS

1-CS…………1-carboxysalsolinol 3-CS…………3-carboxysalsolinol 5-HT3………. 5-hydroxytryptamine 6M-3CS…….. 6-methoxy-3-carboxysalsolinol AADC……… amino acid decarboxylase inhibitor AcH………… acetaldehyde AD………….. Alzheimer’s disease ADH…………alcohol dehydrogenase ADHD……… attention deficit hyperactivity disorder ADME……… absorption, distribution, metabolism, and elimination ADTIQ………1-acetyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline AGEs……….. Advanced glycation end products ALDH………. aldehyde dehydrogenase AMA……….. American medical association AMPA……… α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid APP………… amyloid precursor protein ATP………… Adenosine triphosphate ATPase………adenosine 5'-triphosphatase hydrolase AUD…………alcohol use disorder BAC………….blood alcohol concentrations BBB………… blood brain barrier CA……………catecholamines CB……………cerebellum CAGE………..cut down, agitate, guilty, eye-opener CHD……….…coronary heart disease CNS………….central nervous system COMT……….catechol-O-methyl transferase CSF………….cerebrospinal fluid CVD…..……..cardiovascular disease DA…..…….…dopamine DAT…………dopamine transporter DHBA……….3,4-Dihydroxybenzylamine DOPAC……...3,4-dihydroxyphenylacetic acid DOPAL……...3,4-dihydroxyphenylacetaldehyde ED……………emergency room ER……………endoplasmic reticulum ETC…………..electron transport chain FAEEs………..fatty acid ethyl esters Fortified………standards spiked into analyte free matrix GABA……….. gamma -Aminobutyric acid GC-MS……….gas chromatography-mass spectrometry GI…………….gastrointestinal GIRK…………G protein-coupled inwardly-rectifying potassium channels GSH…………..glutathione xi

H2O2 ………….hydrogen peroxide HIAA…………5-Hydroxyindole-3-acetic acid HP…………….hippocampus HPLC-ECD…...high-performance liquid chromatography-electrochemical detection HVA…………..homovanillic acid IGF…………….insulin growth factors IV……………...intravenous LB……………..Lewy body LC-MS/MS……liquid chromatography-tandem mass spectrometry LOD…………...limit of detection LOQ…………...limit of quantitation MAST…………Michigan Alcoholism Screening Test MAO…………..monoamine oxidase Methylglyoxal…MG or MGO MPP+………….1-methyl-4-phenylpyridinium ion MPDP…………1-methyl-4-phenyl-2, 3-dihydropyridium MPTP…………1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MRM………….multiple reaction monitoring NAC…………..N-acetyl-L-cysteine NAD+…………nicotinamide adenine dinucleotide oxidized NADH…………nicotinamide adenine dinucleotide reduced nFKb…………..nuclear factor kappa B NMDA………..N-Methyl-D-aspartic acid PD……………..Parkinson’s disease PLD……………phospholipase D ROS……………reactive oxygen species RB……………..rest of brain RSD……………relative standard deviation SAL……………salsolinol SOD……………superoxide dismutase SPE…………….solid phase extraction ST……………...striatum TH……………..tyrosine hydroxylase THP……………tetrahydropapaveroline TIQ…………….1,2,3,4-tetrahydroisoquinoline ULOL………….upper limits of quantitation UPS……………ubiquitin-proteasome system VMA…………..4-Hydroxy-3-methoxymandelic Acid

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ABSTRACT

Introduction. The use, misuse, and abuse of ethanol, man’s oldest drug, has constituted a major public health concern. Ethanol use is a contributing factor for more than 200 diseases, making it perhaps man’s most dangerous drug. Paradoxically, moderate alcohol consumption has beneficial effects and may prevent heart disease, the leading cause of death in the United States.

There is much debate on what constitutes a “safe” dose of alcohol. The human body has evolved to be able to handle relatively high doses of alcohol and it is often considered a food rather than a drug. Thus, the pharmacological effects of a single recreational dose of ethanol wear off relatively quickly, yet the physiological consequences of chronic dosing may last a lot longer, or even be permanent. Alcohol’s proximal metabolite, acetaldehyde, is a highly toxic compound that reacts with many different biomolecules and may be a direct causative agent for dementia and cancer. Acetaldehyde and brain catecholamines, which are important neurotransmitters, may react to form tetrahydroisoquinoline compounds, which are toxic chemicals that have been linked to neurodegeneration and the disruption of glucose metabolism. The objective of this research was to further elucidate our understanding of the formation and behavior of tetrahydroisoquinoline compounds. To this end, we have developed a combined method for detection of catecholamines and tetrahydroisoquinolines by LC-MS/MS.

Methods. LC-MS/MS has become the gold standard for measuring catecholamines due to its sensitivity and selectivity. [177-180] It is used clinically for diagnosing rare tumors called pheochromocytomas and paragangliomas. Simultaneous quantitative determination of catecholamines and tetrahydroisoquinolines is attainable in a single method using solid phase extraction for sample preparation. In this method, we achieved linearity to four orders of xiii magnitude, as well as reach the sensitivity of 1 ng/ml for all analytes, with the one exception being DOPAC, which had a sensitivity of 2 ng/ml. In comparison with immunoassays, GC-MS, and HPLC-ECD, the LC-MS/MS technology allows for a simpler sample prep, requires less sample volume, does not need to be hydrolyzed, derivatized, evaporated, or ion-paired. [180-186]

Experimentally, we administered ethanol to mice and observed acute and chronic changes in levels of catecholamines and tetrahydroisoquinoline in the brain. Specifically, we looked at levels in the dopamine enriched striatum, since it condenses with acetaldehyde to form the previously established neurotoxin, salsolinol.

Results: The major findings of this research are the following: Acute dosing of ethanol results in an increase of L-DOPA and dopamine in the striatum, which are potentiated using MAO and

COMT inhibitors. Chronic dosing of ethanol results in the formation of salsolinol, 3- carboxysalsolinol, 1-carboxysalsolinol, and ADTIQ in the striatum, which are potentiated by a pretreatment with disulfiram, an inhibitor of acetaldehyde metabolism. The inhibition of L-

DOPA metabolism resulted in the increase in formation of 3-carboxysalsolinol, but prevented the formation of dopamine metabolites, salsolinol and n-methylsalsolinol. Lastly, we found that 3- carboxysalsolinol localizes in the striatum and is metabolized within 10 minutes after administration.

Discussion:

Using LC-MS/MS, we could accurately quantitate both catecholamines and tetrahydroisoquinolines in one combined method. In this research, we found that acute effects of alcohol consumption increased catecholamine levels in the brain. This has previously been xiv documented elsewhere. Chronically, ethanol administration increased the formation of tetrahydroisoquinolines which have been reported to be neurotoxic in the brain, alter glucose metabolism, and are associated with alcoholism, ADHD, diabetes, Parkinson’s disease, and psychiatric disorders.

We also found that administration of L-DOPA, which is used to treat Parkinson’s disease, increased the levels of 3-carboxysalsolinol and salsolinol in the striatum. Moreover, inhibition of

L-DOPA metabolism potentiates the formation of these TIQs. While L-DOPA treatments alleviate the symptoms of Parkinson’s disease in the short term, it also increases the exposure to

TIQs specifically in dopaminergic cells in the striatum, and may ultimately be neurotoxic. The formation of TIQs as a result of chronic ethanol administration was potentiated by the administration of disulfiram, which is used to treat alcoholics. In addition to exposing the alcoholic to elevated levels of acetaldehyde, these treatments also subject the patient to potentially neurotoxic TIQs. Taken together, patients being treated for Parkinson’s disease are advised against consuming alcohol in any amount.

ACKNOWLEDGEMENTS

This work was the culmination of many hours (years), mentors, students, colleagues, and family providing so much patience, love, support and time to help one person realize their lifelong dream. I am humbled to tears that their generosity and belief in me kept me going long after I have given up on myself.

I would like to thank my advisor Dr. Gatley for accepting me into his lab, letting me work full time, and not giving up on me when he had every right to do so. His kindness and anecdotes will live with me forever. I would also like to thank my committee members. Thank you, Dr. Ralph Loring, for all your help and sticking with me all these years. I was deeply saddened by the loss of Dr. Louis Traficante who was like a father to me, and the more recent loss of Dr. Torbjorn Jarbe who was such a nice guy and gentle soul. With the retirement of Dr.

Bob Schatz, I was faced with having to replace all three committee members. What would seem like an impossible task, I was blessed with the offer of four noble men that stepped in at nearly the last minute to save the day. Thank you, Dr. Adam Hall, Dr. Carl Selavka, Dr. Jonghan Kim, and Dr. Jong O Lee. for stepping in so late in the game. I am honored to have such high character individuals be on my committee.

I would like to thank all the Professors in the Pharmaceutical Sciences department for their support in my education and this research.

Lastly, and most importantly, I would like to thank my family and four children that have sacrificed so much time with their father so that he could pursue his dreams. I love you all dearly.

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OBJECTIVE

Reactions of acetaldehyde and brain catecholamines (important neurotransmitters) condense to form tetrahydroisoquinolines, which are toxic chemicals, and have been linked to neurodegeneration and the disruption of glucose metabolism. Chronic alcohol consumption has been shown to be a risk factor for developing diabetes and Alzheimer’s disease. The biological objective of this research is to further elucidate our understanding about the connection between the effects of acute and chronic alcohol on the disruption of catecholamines and formation of tetrahydroisoquinolines.

To address this, we have developed an efficient and robust sample preparation that was applied to a LC-MS/MS platform for detection of catecholamines and tetrahydroisoquinolines in a single analytical method. In addition, we synthesized 3-carboxysalsolinol, 3-carboxysalsolinol- d2, and 6-methoxy-3-carboxysalsolinol to achieve the most accurate and reliable quantitation of

3-CS, 1-CS and its metabolites. This is the first method of its kind to use deuterated internal standard analogs to quantify these two carboxysalsolinols.

Additionally, we wanted to compare regional distributions for TIQs, thus we performed these experiments looking at mouse striatum, cerebellum, and hippocampus, since many of the

TIQs are formed in dopamine enriched areas where there is ample supply of L-DOPA and dopamine (striatum), but not in in the cerebellum.

Lastly, we wanted to determine if there is a difference between a heavy dose of alcohol consumed acutely versus heavy doses consumed chronically over four days (binge drinking). To accomplish these, we proposed the following specific aims:

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SPECIFIC AIMS

Aim 1: Determine endogenous levels of 3-carboxysalsolinol in striatum, cerebellum, and hippocampus using untreated mice.

Hypothesis :

Endogenous levels of 3-CS will be detectable in mouse brain with highest levels in dopamine enriched areas where there are the highest levels of L-DOPA.

Aim 2: Repeat the measurements of Aim (1) at after intravenous administration of 3-carboxysalsolinol (1 mg/kg).

Hypothesis:

IV injection of a 1 mg/kg dose of 3-CS will result in detectable drug in the brain

Aim 3: Repeat the measurements of Aim (1) at 1, 6, and 10 mins after intravenous administration of 3- carboxysalsolinol (1 mg/kg).

Hypothesis: IV injection of a 1 mg/kg dose of 3-CS will result in detectable levels of 3-CS and 6- methoxy-3-CS in the brain.

Aim 4: Repeat the measurements of Aim (1) at 30 minutes after IP administration of alcohol (50 mg/kg), L-DOPA (50 mg/Kg), pargyline (50 mg/Kg), benserazide (50 mg/Kg), and tolcapone (30 mg/kg)

Hypothesis: The inhibition of catecholamine and TIQ metabolism will result in elevated levels of their metabolites.

Aim 5:

Repeat the measurements of Aim (1) after an acute dose of 1g/kg given IP, or a chronic dose on 1g/kg given daily IP for 4 consecutive days prior to treatment. Challenge with inhibitors for alcohol dehydrogenase (4-methylpyrazole (50 mg/kg)) and aldehyde dehydrogenase (disulfiram (30 mg/Kg)).

Hypothesis: Acute and chronic ethanol consumption results will result in different effects on levels of catecholamines and tetrahydroisoquinolines in the brain.

CHAPTER 1.

THE BIOLOGY OF ETHANOL CONSUMPTION AND ALCOHOLISM

1. Alcoholism and Alcohol Use Disorders 1.1. History of ethanol use 1.2. Epidemiology 1.3. Clinical Presentation 1.3.1. Amount and pattern of use 1.3.2. Diagnosis 1.4. Metabolism 1.4.1. Non-oxidative 1.4.2. Oxidative 1.5. Health Effects 1.5.1. Short term acute effects 1.5.1.1. Central Nervous System 1.5.1.2. Cardiovascular System 1.5.2. Long term chronic effects 1.5.2.1. Liver 1.5.2.2. Nervous System 1.5.2.3. Cardiovascular System 1.5.2.4. Immune System 1.6. Treatments, Interactions, and Disulfiram 1.7. Significance of Study

1.1 History of ethanol use

Human ethanol consumption may date back as far as 12,000 years ago, and is thought to be mankind’s oldest drug. Evidence exists that the consumption of ethanol may be a naturally selected behavior in primates. This evidence comes from evolutionary biology that links the development of primate olfactory sense organs to ethanol fermentation, with the gathering and consuming of fruits that contain ethanol. [1] When fruit ripens, and has its highest sugar content, yeast converts these sugars to ethanol, attracting herbivores for consumption. It is not known if the fruit’s production of ethanol is evolutionarily conserved for its ability to attract, or if it is a defense mechanism to preserve the fruit from microbial degradation. Either way, herbivores, including primates, can sense when a piece of fruit is ripe or rotten based upon how much fermentation has occurred. The consumption of a ripe piece of fruit has the reinforcing effects of caloric nutritional gain as well as the reinforcing effects from the ethanol present. Taken together, the historical origins of exposure to ethanol and the subsequent drug seeking behaviors, precede the ability of humans to produce alcohol itself, and may have inspired them to do so. [1]

The levels of ethanol in fermented fruit sugars are relatively low. The primary motivation for consumption of fruit, and by extension ethanol, is thought to be for calories and nutrition. The juices from these fruits were the primary liquids consumed. This is also a result of the fact that water quality was so poor and known to be associated with diseases.

The earliest evidence of fermented beverages dates to the stone age (10,000 BC). Jars that have been recovered from this period, have residue traces of ethanol. [2] In fact, this technique of determining residue traces of ethanol, found that fermented grapes, berries, honey, and rice were stored in pottery recovered from China dating back to 7000 BC. [3] Over the subsequent

3 centuries, many nations began practicing this behavior of intentionally fermenting sugars from all sorts of fruits and plants. References to ethanol consumption began showing up in the writings of Babylonians, Sumerians, and Egyptians. Religions began incorporating the use of ethanol in the rituals to the extent of naming deities for the existence of wine. [4] Around 2000

BC, humans began using ethanol for medicinal purposes. Indications include sickness, depression, and death and are prescribed by the Hebrews in the bible. Later, ethanol was commonly used as a medicinal diluent, disinfectant, and for mouthwashes. It is still used today in pharmacies as a diluent and is available in many over the counter cold medications, analgesics and hand sanitizers.

It wasn’t until the 1800’s, when water purification procedures were developed, that water replaced beers and wines as the primary beverage for humans. [5] This is around the same time that methods for the distillation of ethanol lead to the production of much higher concentrations of alcohol by volume, called distilled spirits. A shift from consuming alcohol for its nutritional benefits and mild euphoria to its use as a part of social recreation occurred.

In general, the amount of ethanol in the beers and wines that were produced in these historical times, were not very high. In addition, ethanol’s pharmacological potency is quite low.

Thus, ethanol is one of the only, if not the only, drug of abuse that requires gram doses to have an effect. To achieve these effects of alcohol, people then and now need to consume large quantities of alcohol. Thus, ethanol is often considered to be more of a food than a drug.

Consumption of large quantities and drunkenness was viewed by society as a character issue and to a lesser extent a medical issue. The major difference between then and now is the significantly higher concentrations by volume of ethanol per drink. This allows drinkers to consume higher

4 doses in a shorter amount of time, and the health effects become more apparent to the individual and society.

Heavy, chronic consumption of ethanol causes a constellation of adverse effects on health and is associated with a withdrawal syndrome that promotes tolerance, dependence and subsequent abuse and addiction. Also, motor vehicle accidents and other events associated with ethanol intoxication are major problems. On the other hand, many people consume ethanol in moderate doses on an occasional basis with no apparent ill effects. They appreciate its mild euphorigenic and anxiolytic properties, and evidence supports the view that a glass of red wine a day benefits the cardiovascular system. Partaking of moderate doses of ethanol (especially red wine) more closely resembles the patterns of consumption that humans were fermenting in clay pots, thousands of years earlier. The differences between moderate/heavy or acute/chronic ethanol consumption, resulting in either its beneficial or detrimental effects are specific to the individual drinker. Moreover, the mechanisms to explain these differences (behavioral and physiological) are not completely understood. The direct actions of ethanol on targets such as glutamate and GABA receptors are probably responsible for much of the drug's psychoactivity, but there is still much research to be done. In this research, we are looking to answer some of these questions, by measuring the effects of ethanol directly, and on the formation of metabolites that form in vivo and have been previously shown to be neurotoxic.

1.2 Epidemiology

Epidemiology for the field of alcohol consumption focuses on patterns, development, and risks associated with ethanol use, abuse, and dependence. [6] Ethyl alcohol is likely the most abused drug in the world. Adult use is legal in almost every civilized nation, and its use is not

5 specific to any age, gender, or nationality. However, incidence rates for consumption are higher in drinkers between the ages of 15-25, males, and those in the northern hemisphere countries

(Figure 1).

Figure 1. Global incidence rates for alcohol https://commons.wikimedia.org/wiki/File:Alcohol_by_Country.png

Ethanol use and abuse is one of the costliest health care issues of our time. This price tag is not only in currency but also in the damaging effects that ethanol causes to the body and quality of life. The economic cost to Americans is more than $225 billion dollars annually. It is estimated that alcoholics have an average decrease in life expectance of between 12-15 years. [7]

In the United States, nearly 14 million people abuse ethanol, and between 5-10% of the total population are dependent on ethanol.[8] This abuse is three times more likely in men than woman.

Alcoholism results in the loss of as many as 200,000 lives per year in the US and 3 million worldwide, and is the number one cause of death for persons between the ages of 15-45, as well as the number one cause of preventable death. Alcoholism is the leading risk factor for increased rates of mortality and morbidity. [9]

The overall consumption of alcohol in terms of gallons consumed per year, over the past

160 years, has not significantly changed (~2 gallons/year) (Figure 2). The consumption of distilled spirits has declined since 1975 while beer and wine drinking have remained steady.

Historical Trends: 1850-1997

d Figure 2. Historical trends of ethanol consumption 1850-1997 http://pubs.niaaa.nih.gov/publications/Social/Module1Epidemiology/Module1.html

Studies show that these trends are partly due to the increase in adolescent drinking and the increase in social drinking among women. Despite restrictions and penalties for underage drinking, use is quite common in this age group. During this developmental period adolescents are more susceptible to the adverse effects of ethanol, and are more likely to develop tolerance and dependence. Among adults, as many as 75% consume ethanol at some point during one year.

Of those that consumed ethanol within that year, 20% of these individuals consumed the equivalent of five or more drinks in a sitting at least once. [10]

There is a 50% increased risk for traffic fatalities, as well as deaths from accidents due to fire, when ethanol is involved. There is also a two-thirds increased risk for homicides and

7 drowning when ethanol is used. [11] Nearly 3 million victims of violent crimes per year are assaulted by perpetrators who were under the influence of ethanol. [12] Statistics that compare the harm of alcohol to that of other drugs, show that ethanol is the most harmful drug in the world

(Figure 3). Ethanol consumption has been identified as a contributing factor for more than 200 diseases. [13]

Figure 3. Ethanol as the most harmful drug in the world David J Nutt, Leslie A King, Lawrence D Phillips, on behalf of the Independent Scientific Committee on Drugs. Lancet 2010; 376: 1558–65

1.3 Clinical Presentation

Ethanol consumption can have a wide range of clinical effects that are dependent upon the following factors; amount of alcohol consumed, the quality of the alcohol (congener impurities), pattern of drinking, what food or other drugs were also taken, the history of use, health, age, gender, and the genetic predisposition of the user. [13]

The standard dose of alcohol is about 14 grams of pure ethanol. This is also about the amount that the average adult can metabolize in one hour. This is equal to about one beer, shot of a spirit, or glass of wine. According to the Dietary Guidelines for Americans, [14] moderate alcohol consumption is defined as one dose per day for women, and two doses for men.

Moderate doses relieve anxiety and gives an overall sense of well-being. Consumption of more than 8 drinks per week for women, and 15 drinks per week for men is considered heavy drinking.

In addition, binge drinking is a pattern of alcohol consumption that is usually defined as 4 drinks for women and 5 drinks for men in a two-hour period, per the National Institute on Alcohol

Abuse and Alcoholism or about 60 grams as defined by the World Health Organization. This is also approximately the amount that is considered the legal limit for operating a motor vehicle in the United States and is equivalent to 80 mg/dL or 0.08%. At this level, an individual has slower reaction times, impaired motor function, slurred speech, and ataxia (Figure 4).

Figure 4. Blood alcohol concentrations and associated effects www.cdc.gov/motorvehiclesafety/pdf/BAC-a.pdf

The effects that are consistent with higher doses including vomiting, sedation, loss of protective reflexes, seizures, coma, and death. The signs of intoxication can also include flushing, tachycardia, hypotension, hypothermia, hypoventilation, and hypoglycemia. Medical staff in emergency rooms should suspect alcohol if the patient has unexplained trauma or seizures. The seizures may be due to the hypoglycemia. Consuming food is a key factor in mitigating some of the harmful effects for ethanol. Food delays gastric emptying into the small intestine, and thus delays the absorption of ethanol. One of the primary consequences of higher doses is the development of metabolic tolerance. This can occur in a few hours in a single binge episode or in chronic patterns of consumption. Tolerance is defined as the reduced sensitivity to a drug (ethanol), such that increased doses are required to achieve the same level of effects. For alcohol, this is particularly problematic, since higher doses are a risk factor for the development of physical and psychological dependence, and ethanol has a small therapeutic index for toxicity.

The quality of the alcohol is also an important consideration. Most alcoholic beverages have “residue congeners” that are left in for flavor and aroma. These congeners can include toxic methanol, acetone, and aldehydes. Congeners are thought to contribute to the occurrence of hangovers. In addition, microbrews and home brews have become more and more popular, and lack the tight controls applied to commercial alcoholic beverages. Methanol is a particularly dangerous contaminant, as it metabolizes into toxic formaldehyde and formic acid, and its presence has been associated with blindness, coma and death.

Nearly 10% of the population in the US have an alcohol use disorder (AUD) and are considered alcoholics. Alcoholism is a disease that can be defined as a chronic and progressive disorder in which tolerance and dependence are apparent. It is considered a disease of the brain and was established as such by the American Medical Association (AMA) in 1956. The AMA

10 now classifies the disease as being both a psychiatric and a medical disorder. Alcoholism may also include dysfunctional changes and irreversible damage to various other organ systems. Early detection is very important to mitigate these pathological changes that may occur. Risk factors for developing alcoholism include but are not limited to anxiety, stress, social isolation, depression, environment and genetic predispositions. Some researchers have shown that with early detection, followed by abstinence, tissues including the brain can recover. [15] Laboratory tests for alcohol consumption are available, but there is still a need for tests that can identify the extent of the progression of the disease. The two most prevalent diagnostic screening systems that have been created to help with early detection of alcoholism are; the Michigan Alcoholism

Screening Test (MAST) and the CAGE questionnaire. These questionnaires are quick, can be performed in the ER, and are good indicators of heavy alcohol consumption and by extension alcoholism.

1.4 Metabolism

The primary route of administration for ethanol is oral consumption. The oral cavity does not contribute significantly to absorption. The stomach is the first site for ethanol absorption, and accounts for nearly 20% of the amount consumed. [16] The stomach resident time is influenced by the presence of food, alcohol content, and other co-administered drugs. First pass metabolism occurs through the portal vein, and contributes to some of the immediate effects of alcohol consumption. The stomach then empties its content into the small intestine, where the remaining alcohol is absorbed. In the small intestine, absorption can take about an hour in the absence of food, but can take as much as 6 hours when food and other drugs are also co-administered. [17]

Absorption is influenced by an individual’s GI health and genetic factors.

Metabolism starts as early as the oral cavity, continues in the stomach, and then in the small intestine. In the stomach, alcohol dehydrogenase (ADH) enzymes in the gastric mucosa convert ethanol to acetaldehyde. Metabolism in the GI tract reduces absorption and subsequent toxicity, thus is considered protective. This is especially evident in men, and allows larger doses to be consumed. In contrast, about 30% of Japanese are deficient in gastric ADH, and this results in higher systemic doses, increased adverse effects and toxicities, and greater susceptibility to tolerance and dependence. [18]

Ethanol that is not metabolized in the stomach or the small intestine is absorbed and and transferred via the portal vein to the liver, where nearly 90% of an alcohol dose is ultimately metabolized. There are also smaller extra-hepatic metabolic contributions in other parts of the body including the lungs and brain. ADH represents one of the oxidative pathways for ethanol metabolism. In addition, the cytochrome P450 system 2E1, and peroxisome-catalase pathways also metabolize ethanol oxidatively (Figure 5).

Figure 5. Oxidative pathways for ethanol metabolism Samir Zakhari, 2006, vol. 29, no.4, Alcohol Research & Health [19]

The cytosolic ADH enzymes of the liver have been divided into five different classes, and at least ten isozymes. These enzymes use zinc as a cofactor. ADH1, ADH2*1, ADH3*1, ADH4, and ADH6 are all are present in the liver, with ADH2*1 having the highest activity. [19] Ethanol is thought to be the only CNS depressant that is primarily metabolized by cytosolic enzymes

(ADH), as opposed to the microsomal P450 enzymes. However, saturation of ADH occurs at low doses, and becomes rate limiting for ethanol metabolism. Clearance of alcohol then exhibits zero order kinetics. Metabolism of ethanol with ADH generates acetaldehyde and reduces the dehydrogenase cofactor NAD + to NADH. In 90% of Asians, genetic polymorphisms result in the gain of function ADH2*2 which make them rapid metabolizers of ethanol to acetaldehyde. This causes severe reactions that lead to reduced consumption in this population. [20] As ethanol doses are increased, the NAD +/NADH ratio is decreased, resulting in a reduced cytosolic environment.

This reduced redox potential may contribute to the metabolic disorders, alcoholic ketoacidosis, altered fatty acid metabolism, and reduced gluconeogenesis seen in chronic alcoholism.

A second consequence of saturation of ADH activity is that there is an increased metabolic flux through the microsomal P450 2E1 pathway at higher doses. Ethanol metabolism through this pathway generate reactive oxygen species (ROS), free radicals, and hydrogen

[19] peroxide (H 2O2) all of which can cause tissue damage. Moreover, 2E1 is an inducible enzyme, thus increased activity due to saturation of ADH results in increases in 2E1 enzyme synthesis and the capacity for additional tissue damage.

Acetaldehyde is a highly toxic, highly reactive metabolite and usually maintained in very low

µM concentrations in the liver cytosol by the NADH/NAD + couple which favors a high ethanol/acetaldehyde ratio, and by oxidation to acetate. In binge episodes, or with chronic exposure, the levels of acetaldehyde can be elevated significantly and the metabolite can enter

13 the circulation. Concentrations of acetaldehyde as high as 50 µmol/L have been measured in the blood after an intoxicating dose of alcohol. NAD + -linked aldehyde dehydrogenase (ALDH) rapidly metabolizes acetaldehyde into acetate. Acetaldehyde can form adducts with many cellular components; including proteins in the endoplasmic reticulum thought to disrupt protein folding, microtubules which may prevent transportation of proteins to the cell surface, the neurotransmitter dopamine to form salsolinol which may contribute to alcohol reinforcing behaviors, and DNA whose adducts may be involved in certain cancers. Acetaldehyde may also mediate some of the rewarding properties of alcohol. One group of researchers has claimed that the reinforcing effects of acetaldehyde, on a molar basis, may be as high as 1000 times greater than that of ethanol. [21] ALDH also has genetic variants and polymorphisms where ALDH2*1 and ALDH2*2 are inactive. Nearly 50% of Asians have the ALDH2*2 gene and phenotype that prevents acetaldehyde from being metabolized. ALDH polymorphisms of the ALDH2*2 allelic variant results in severe flushing, vomiting, nausea, and reduced alcohol consumption (Figure 6).

Figure 6. Genetic mutations that affects ethanol consumption

Acetaldehyde metabolism can also be inhibited pharmacologically, by disulfiram, metronidazole, cefotetan, and trimethoprim. [22] Disulfiram (Antabuse) has been used for the treatment for alcohol use disorders and causes severe acetaldehyde toxicity from the treatment.

Patients are required to be informed about the adverse effects that are associated with the treatment.

Non-oxidative metabolism of ethanol occurs to a lesser extent than oxidative metabolism, but may have physiological and pathological significance. Ethanol can react with fatty acids to produce fatty acid ethyl esters (FAEEs). A second non-oxidative metabolic pathway for ethanol is the reaction with the enzyme phospholipase D (PLD) (Figure 7).

Figure 7. Non-oxidative consequences of ethanol consumption Samir Zakhari, 2006, vol. 29, no.4, Alcohol Research & Health [19]

1.5 Health Effects

1.5.1 Acute Consumption

Ethanol is a colorless liquid that is both water and lipid soluble. It readily diffuses across cell and organelle membranes, and partitions into the total body water of the user. Therefore, it influences all cells, tissues, and organ systems, and at high doses has properties somewhat like those of the volatile anesthetics. Ethanol has a high systemic bioavailability limited to a small extent by ADH metabolism in the upper gastrointestinal tract and by first pass liver metabolism.

It is introduced orally and its effects on the esophagus, stomach, and intestines are significant.

Esophageal reflux, tears, ruptures, and cancer can occur. In the stomach, there can be increases in gastric secretions leading to increases in histamine and gastrin release. [23] This condition is exacerbated by the consumption of food, since the presence of food decreases absorption, and delays the emptying of ethanol into the intestine, thereby prolonging ethanol’s direct contact with the esophagus and stomach tissue and cells. [24]

The amount and quality of ethanol consumed, patterns of usage such as binge drinking, and an individual’s history of consumption are important factors that contribute to alcohol’s effects on the body. Repeated short-term heavy exposures are not equivalent to exposures from chronic low-to-moderate drinking, even if the total amount of alcohol consumed is the same if averaged over a period of a week or a month. Binge drinking (4-5 drinks in 2 hours), is not heavy chronic use, but both patterns can lead to the development of tolerance and dependence. Chronic binge drinking is associated with the most pronounced pathological effects and the poorest outcomes.

Small to moderate doses are defined as consumption of between 1-2 drinks (32 g) of alcohol at a sitting. This is the amount that is considered safe and even by some authorities beneficial, especially in alcoholic wines. Ethanol in small acute doses is associated with relaxation, relief of anxiety, enhancement of mood, warming sensation, release of inhibitions, and an overall sense of well-being. At the receptor level, ethanol increases neuronal activity by enhancing GABA A receptor chloride ion channels. Ethanol does not bind as an agonist, but as a positive allosteric modulator on the delta subunit of the pentamer (Figure 8). GABA is an inhibitory neurotransmitter, thus enhancing the inhibitory effect may be linked to the sedation seen in alcohol consumption. This enhancement of an inhibitory function is sometimes referred

to as disinhibition. Agonists for the GABA A receptor result in ethanol-like behaviors, and antagonists attenuate the effects. Ethanol also inhibits the excitatory neurotransmitter action of glutamate at NMDA receptors as a negative allosteric modulator, resulting in the relaxation and release of anxiety. The sense of a warming feeling may be related to the vasodilation effect that ethanol has in the periphery.

Figure 8. Ethanol allosteric binding site on the GABA receptor https://en.wikipedia.org/wiki/Alcohol_intoxication

Ethanol also has a direct effect on signaling receptors, channels, transporters and neuromodulators that include; amino acid release, dopamine release, endogenous opioid release,

AMPA and Kainate allosteric modulators, NAC and 5-HT3 agonists, glycine, sodium/potassium

ATPase, adenynyl cyclase, phospholipase C, adenosine reuptake inhibitors, voltage-gated Ca2+ channel blockers, and GIRK channel openers (Figure 9). [25]

Figure 9. Ethanol targets receptors, signaling systems, and neuromodulators Fuller, RK et al. JAMA 1986;256:1449

Heavier doses are associated with impairments of learning, reasoning, memory, self- control, motor dysfunctions, ataxia, slurred speech, and vomiting; collectively these are generally referred to as intoxication. The degree of intoxication is also dependent upon the drinker’s history of use. Heavier doses more quickly result in tolerance and dependence. Thus, the drinker needs to consume more, to get the same effects. Higher doses to an already low therapeutic index drug, can be dangerous and result in respiratory depression, coma and in some cases death.

Chronic users can adapt to the signs of intoxication and can remain surprisingly functional, even with large doses. Heavy consumption also result in increasing enhancement of GABA A receptors and the inhibition of NMDA subtype of glutamate receptors. The NMDA receptor activated by the excitatory neurotransmitter, glutamate, resulting in positively charged ions to flow. This ligand-gated ion-channel also possesses a binding site for glycine, and has been shown to be associated with learning and memory. [25] It is thought that with an intoxicating dose of alcohol, the extent of NMDA inhibition may be linked to the loss of memory (blackout) during the episode.

1.5.2 Chronic Consumption

As with acute consumption, the effects of chronic consumption depend upon many factors, but the most important factor to consider is the amount ethanol consumed, and the genetic disposition of the drinker. Ethanol rapidly diffuses to all body tissues and has a pervasive effect on all organ systems. Ethanol’s most profound effects are on the GI, liver, nervous, immune, and cardiovascular system. Small to moderate doses taken chronically can result in beneficial effects. However, heavy chronic alcohol consumption adversely affects all organ systems (Figure 10).

Figure 10. Possible long term effects of ethanol https://en.wikipedia.org/wiki/Long-term_effects_of_alcohol_consumption

Liver

Heavy, chronic ethanol consumption is associated with liver disease in about 30% of such drinkers. This disease starts with the metabolism of ethanol to acetaldehyde then to acetate which

19 results in the increase in NADH:NAD + ratio and thus a reduced cytosolic environment in the liver. Activation of CYP 2E1 metabolism results in an increase in reactive oxygens species

(ROSs) (Figure 5). One of the consequences of this reduced and toxic cytosolic environment, is altered glucose metabolism, since elevated NADH levels in the cell favors the conversion of pyruvate to lactate. The cellular response to this lack of energy is to increase gluconeogenesis, amino acid metabolism, and fatty acid mobilization from adipose tissue. Fatty acid accumulation is the first hallmark of this disease. The liver can proliferate new hepatocytes and reverse this fatty acid infiltration. [26] Unchecked, fatty liver becomes inflamed and can progress into hepatitis. Co-occurring non-alcohol related hepatitis (hepatitis B or C) exacerbates the liver’s ability to regenerate, and contributes to the progression of alcohol related hepatitis to cirrhosis, an irreversible scarring of the liver. [27]

Nervous System

Heavy, chronic use of ethanol results in the development of tolerance, physical, and psychological dependence. Heavier doses also lead to higher receptor occupancy. This results in an upregulation of receptor density on the cell membrane. The downregulation of GABA A receptor ion channels and upregulation of NMDA receptors and voltage-gated calcium channels may be involved in tolerance, and result in withdrawal. Moreover, these changes may result in seizures associated with withdrawal syndrome. Ethanol also regulates neuronal activity in the mesolimbic dopaminergic pathways and can release the neurotransmitter dopamine into the nucleus accumens. In addition to dopamine, ethanol affects serotonin, cannabinoid, and opioids in the reward pathway, and may involve the signaling pathways of these neurotransmitters. The appetite-regulating system and the stress response systems may also be involved in ethanol’s rewarding behavior. These include leptin, ghrelin, and neuropeptide Y, and corticotrophin

20 releasing factors respectively. Chronic, heavy alcohol consumption leads to neuropathy in the hands and feet which can include gait dysfunction and ataxia. [28-30]

Cardiovascular System

Heavy, chronic alcohol consumption disrupts heart cell membranes, disrupts mitochondrial function, and increases fatty acid accumulation in the sarcoplasmic reticulum. It also upregulates voltage-gated calcium channels. The result of these effects is cardiomyopathy with inflammation progressing to fibrosis. Alcoholic hearts are also smaller in size with reduced functional capacity. The seizures associated with withdrawal syndrome may also include heart arrhythmias. This may be connected to the increases in catecholamine release and the disruption of potassium and magnesium metabolism. Arrhythmias are more prevalent in chronic, heavy use, but are also seen in binge patterns of alcohol consumption. High blood pressure is also associated with use of alcohol, and results in nearly 5% of cases of hypertension. [30]

In contrast to heavy drinking regular consumption of low doses of alcohol is thought to be beneficial for cardiovascular disease (CVD) and may be able to prevent coronary heart disease (CHD). This effect is sometimes referred to as the “French Paradox”. The French consume a high fat diet, yet have very low incidence of CHD. One explanation for this is that the

French consume moderate levels of red wine. The grapes that go into making red wine have many antioxidant components including the phenolic compound resveratrol. Whether this effect is due to the grapes or the Mediterranean diet in general is still a debate for discussion. [31-32] We do know that ethanol from all origins, has the beneficial effect of increasing high-density lipoproteins into the blood, dilation of blood vessels, and reducing inflammation. These beneficial effects are out-weighed by the harmful effects of heavy doses, especially with chronic consumption. Heavier consumption inhibits the proliferation of cells from the bone marrow

21 resulting in deficiencies of blood elements. Red blood cells are most affected, causing anemia from iron and folic acid deficiencies. Heavy chronic alcohol also results in hyperlipidemia, negating the beneficial effects of moderate consumption. It should be noted that moderate consumption levels that may be beneficial in healthy individuals do not ameliorate CHD once this is present. These effects considered, The World Health Organization does not recommend the consumption of even moderate doses, saying that abstinence is the healthiest choice. [13]

Immune System

The effects of ethanol on the immune system vary from one organ system to another. The antioxidant effects from resveratrol and other berries may only be related to wines and moderate consumption. Alcohol suppresses macrophages and T-cells (immune response cells) in the lungs, resulting in an anti-inflammatory effect. Thus, with ethanol consumption, the lungs become susceptible to infections and are at a higher risk for pneumonia and death. [33-34]

The liver, being the primary site of ethanol metabolism, is the organ most affected by the immunological response. Many inflammatory cytokines are elevated in the liver after a heavy dose of ethanol.

1.6 Treatments, Interactions, and Disulfiram

Treatment for an intoxicated patient in the ER usually includes restoring fluids and electrolytes, dextrose, vitamins (especially thiamine), and sometimes potassium and magnesium.

Signs including seizures and unconsciousness can result in a misdiagnosis of hyperglycemia to which metformin is administered. Metformin is the first line of defense for treating hyperglycemia in diabetes. Unlike type II diabetics, alcoholics often have a low caloric intake and poor nutritional habits. As many as 22% of patients admitted to the ER for alcohol

22 intoxication are hypoglycemic. [35-36] Thus, treatment with metformin that can further lower glucose levels can cause a stroke or even worse death.

Dextrose (glucose) ensures a restoration and increase in insulin release, decrease in glucagon release, and a reduction of fatty acid oxidation (Figure 11). Dextrose restores ATP energy to cells, and reverses the pyruvate to lactate shift and the low NAD+/NADH ratio, ameliorating the reduced cellular environment.

Figure 11. Lipid metabolism in diabetes mellitus http://slideplayer.com/slide/3010248/

Emergency room staff must also consider drug-drug interactions, genetic history, and patient history of consumption when treating a patient. This can be difficult if the patient is unconscious or incapable of understandable speech. Patients with a history of altered ethanol metabolism as seen with genetic variants of ADH and ALDH present with increased risk of fatal consequences. Alcoholics may also have higher levels of CYP 2E1, which can have severe

23 adverse effects if combined with drugs that act on the cardiovascular system, antibiotics or analgesics (Figure 12). Diagnosis for an alcohol use disorder, can be performed quickly in a clinical setting with the use of two well-established assessment tests. These clinical assessment tests are called the C.A.G.E. (cut down, annoyed, guilty, eye-opener) questionnaire, and the

M.A.S.T. (Michigan Alcohol Screening Test). Examples of these questionnaires can be found in the appendix.

Figure 12. Common therapeutic classes with drug-alcohol interactions

The most severe adverse effects may come from the administration of disulfiram (Antabuse) which makes people feel ill if they drink alcohol. Disulfiram inhibits the metabolism of acetaldehyde resulting is severe nausea, vomiting, abdominal pain, depression, and movement

24 disorders. Patients become sick if they drink, and thus are dissuaded and negatively reinforced to prevent relapse. Disulfiram is rarely used today, however, several commonly prescribed drugs have disulfiram-like side effects in combination with alcohol (Figure 12).

1.7 Significance of Study and Need for Research

There are many remaining questions concerning ethanol consumption that merit systematic research. There is currently great interest in alcohol’s effects on neurotransmitter and neuromodulators systems, and on neural and metabolic their pathways. Indirect as well as direct effects of ethanol itself and its metabolites on these pathways may be involved in the pathological and psychological changes associated with prolonged ethanol exposure. Some researchers in this field postulate that most of the effects of ethanol come from exposure to acetaldehyde, and that the disease should be called acetaldehydism instead of alcoholism. Much of the focus has been on the direct pathological effects of ethanol, and not enough on the metabolic consequences of exposures to high levels of acetaldehyde or of acetate. It is possible also that some effects of ethanol may come from salsolinol, an alkaloid-like tetrahydroisoquinoline compound formed by reaction of acetaldehyde with the neurotransmitter dopamine. [37] Other reaction products of acetaldehyde and biological amines could also be involved in the pharmacology and toxicology of ethanol. The present research evaluates the formation and localization of some of the metabolites of acetaldehyde associated with ethanol consumption that are formed in vivo in mice.

CHAPTER 2.

METABOLIC DISORDERS AND DISEASES ASSOCIATED WITH ALCOHOL

CONSUMPTION

1. Introduction

2. Metabolic Disorders

2.1. Metabolic syndrome

2.1.1. Impaired insulin signaling

2.1.2. Methylglyoxal

2.1.2.1. AGEs

2.1.2.2. ADTIQ

2.1.3. Diabetes

3. Glucose Metabolism

3.1.1. Acetate

3.1.2. Lipid Membrane Composition

3.1.3. Dopamine transporters (DAT)

4. Lysosomal Dysfunction

5. Alzheimer’s Disease

Introduction:

The convergence of evidence around the progressive disorders such as alcoholism, diabetes,

Alzheimer’s disease, and Parkinson’s disease, supports the theory that these diseases may be initiated by unique pathways, but they all ultimately have the same end-point of progressive cell death. A unifying biological mechanism for this progression could be impaired insulin signaling because of altered glucose metabolism and disruption of bioenergetic homeostasis. [38] (Figure

13).

Figure 13. Bioenergetic homeostasis link between diabetes and neurodegeneration M. Stroh et al. / Biochemical Pharmacology 88: 573-583, 2014.

To better understand how alcohol’s ability to impair glucose metabolism as it relates to catecholamine regulation and the formation of tetrahydroisoquinolines, we have measured brain tissue concentrations of 1-carboxysalsolinol and of ADTIQ. Both are derived from the catecholamine, dopamine, by reaction with pyruvate and methylglyoxal, respectively.

Metabolic Disorders:

Metabolic Syndrome

Metabolic syndrome is a disorder that may occur because of disrupted metabolism of glucose, and the ability to store and use energy. There does not appear to be one specific pathognomonic indicator for this disorder, and thus proper diagnosis has been challenging for the medical industry. General hallmarks of the disorder include hypertension, hyperglycemia, and hyperlipidemia, and is considered a risk factor for the development of diabetes and CVD which increases with age.

The connection with glucose metabolism suggests that insulin regulation and signaling may be involved. Impaired insulin signaling may be a connection between alcoholism, diabetes, and Alzheimer’s disease. Brain specific insulin, insulin growth factors (IGF), and insulin signaling may be involved as they regulate glucose metabolism, oxidative stress, protein folding, and inflammation. [39-40]

Methylglyoxal is a highly toxic aldehyde metabolite of glucose metabolism that can induce the formation of advanced glycation end products (AGEs), and is associated with inflammation, hypertension, diabetes, aging, and induces apoptosis. [41] AGEs are formed because of high levels of sugar, where glucose and other sugars, react irreversibly with proteins and lipids. Methylglyoxal and AGEs accumulate in diabetic patients and may play a role in the cytotoxicity of endothelial cell damage resulting in stroke and other degenerative diseases like alcoholism, diabetes, and Alzheimer’s disease. [42] Methylglyoxal increases expression of caspase 3, caspase 8, FAS, and activation of nuclear factor kappa B (nFKb) translocation to the nucleus. All of these are associated with the apoptotic pathway. [42] Interestingly, consumption of the mushroom Agaricus bisporus, for which health benefits are claimed, is associated with

28 decreased serum concentrations of methylglyoxal, as well as other biomarkers of oxidative stress. [43]

Diabetes is a risk factor for Parkinson’s disease, and since diabetes induces the formation of methylglyoxal, this compound could provide a link between the two conditions.

Methylglyoxal reacts with dopamine to form a tetrahydroisoquinoline called ADTIQ that occurs in vivo under physiological conditions, and has been discovered in the brains of Parkinson’s patients’ post-mortem. In animal studies, ADTIQ was significantly elevated in mice with dysfunctional α-synuclein proteins, and diminished cell viability and induced apoptosis in SH-

SY5Y cell cultures. Moreover, in rats with diabetes, methylglyoxal and ADTIQ levels were significantly elevated, and this may be due to hyperglycemia and altered glucose metabolism. [44]

Nearly 25.8 million Americans have been diagnosed with diabetes. Diabetes is associated with hyperglycemia, hyperinsulinemia, CVD, and neurodegeneration, and has implicated in obesity and aging.

Glucose metabolism

Heavy alcohol consumption and intoxication decreases metabolism of glucose as an energy substrate. Decreased glucose metabolism also occurs in starvation and eating disorders. The initial physiological response is to convert glycogen into glucose for energy. The glycogen energy stores are quickly depleted in both heavy alcohol consumption and in starvation.

Moreover, alcoholics often have poor nutrition in combination with alcohol consumption. Once the glycogen stores are depleted, the body progresses to use proteins and lipids as carbon sources for production of glucose.

In the case of alcohol consumption, acetate, the oxidative metabolite of acetaldehyde, can also be used to some extent as an energy source to replace glucose. In addition, non-oxidative metabolism to fatty acid ethyl esters also occurs. In chronic exposures, the use acetate in the brain for the energy to perform basic functions becomes more prevalent. In withdrawal, when the acetate is removed, the brain is particularly vulnerable to dysfunction including hypoglycemic seizures unless glucose is reintroduced into the recovery diet. [45]

Heavy alcohol consumption results in significant changes in membrane lipid composition, specifically sphingolipids, fatty acids, lysophosphatidylcholines, and glycerophospholipids. [46] Chronic alcohol, more so than acute, reduced coupling between behavior and a parameter termed “functional connectivity density” in MRI studies. [47] Alcohol craving in dependent individuals, manifests itself as intense physiological and psychological desire to consume it. This condition is exacerbated in those individuals that are genetical predisposed to consume and in those that are surrounded by environmental cues that have been associated or linked to their drinking behavior. Relapse is often the outcome unless these cues are avoided. The neurobiology of cravings, cued behavior, and depression in alcoholism has been linked to activation in the amygdala, nucleus accumbens, and may involve the dopamine transporter (DAT), a target that has been implicated in effects of many addictive substances.

Recent studies have indicated that the methylation state of the promoter regions of the dopamine transporter gene is elevated in the leukocytes of alcoholics and may be involved in the cravings and depression associated with chronic alcohol consumption. [48]

Lysosomal dysfuntion:

Lysosomal dysfunction has been associated with diabetes, aging, and neurodegenerative disorders. Diabetic hyperglycemia, hypercholesterolemia, and reduced insulin sensitivity may be

30 involved in the disruption of the lysosomal membranes. [49] The lysozymes are the organelles that process all the cellular and extracellular waste with hydrolytic enzymes namely; lipases, carbohydrases, proteases, and nucleases. More than 60 lysosomal enzymes have been identified.

Genetic variations and mutations of DNA that code for these enzymes have resulted in the identification of more than 50 metabolic diseases. The central issue in most of these diseases, is the cells’ inability to remove and degrade waste, which can therefore accumulate in the cell.

These genetic diseases clinically present and are related to neurodegenerative diseases, CVD, cancer, and age related disorders. [50] The two main processes that regulated the identification, transportation, and disposal of this cellular waste are autophagy for cytosolic waste, and endocytosis for extracellular waste. Thus, the lysosomes are important for removing the accumulation of cellular and extracellular debris, protein folding, transportation, and secretion, plasma membrane function, signaling, energy metabolism and cell death. [51] Biomarkers of lysosomal damage can be detected prior to indications of memory loss and neurodegeneration, and may be important indicators of the progression of the aging, age-related diseases, alcoholism, diabetes, cancer, CVD, and Alzheimer’s disease.

Alzheimer’s Disease:

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder involving progressive memory loss and dementia, that may be linked to hypertension, alcoholism and major depressive disorder. Significant memory loss and other symptoms of dementia generally does not occur until after the age of 65. Strict diagnosis of AD depends on post mortem histological imaging of amyloid plaque and neurofibrillary tangles, though it is now possible to use PET imaging to visualize plaque. The cause of AD may involve defects in the folding and degradation of

31 proteins, but there is not 100% consensus on this issue. Amyloid precursor protein (APP) is an extra cellular membrane protein that is important for the growth and repair of neurons. [52] In AD, proteolytic secretases responsible for recycling and degrading APP fail, thus amyloid beta peptide fragments of APP accumulate on the outside of the cell, and some APP protein is internalized and degraded by cytosolic secretases that form amyloid beta peptides inside the cell.

This results in formation of insoluble plaques of amyloid beta peptides. Additionally, in AD, tau proteins, responsible for securing microtubules in place for protein and nutrient transport in the cell cytosol, become hyper-phosphorylated and form tangles that are disruptive to cell function.

Tau tangles are associated with Parkinson’s disease (PD), as well as AD-type dementia. [53] The pancreas secretes hormonal amylin and insulin from the beta-cells. Amylin can accumulate forming aggregates and adducts in islet cells and in brain neurons, and has been associated with increases in pro-inflammatory cytokines, oxidative stress, and membrane disruption consistent with diabetes and Alzheimer’s disease and may be a link between the two diseases. [54]

CHAPTER 3.

EFFECTS OF TETRAHYDROISOQUINOLINES ASSOCIATED WITH ALCOHOL

CONSUMPTION

1. Tetrahydroisoquinolines (TIQs)

1.1. Introduction

1.2. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

1.3. Salsolinol

1.3.1. Synthesis

1.3.2. Behavioral effects

1.3.3. Inhibitory effects

1.3.4. Neurotransmitter dysregulation

1.4. N-Methyl-Salsolinol

1.5. 1-acetyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (ADTIQ)

Introduction:

Tetrahydroisoquinolines (TIQ) have been an area of great research in the past four decades, since it was reported by Sandler et al., 1973 [55] that TIQ alkaloids, salsolinol and tetrahydropapaveroline, were detected in the urine of Parkinson’s patients being treated with L-

DOPA. TIQ’s are of interest since they are found and formed in vivo via a non-enzymatic condensation reaction between acetaldehyde and biogenic amines. Endogenous levels have been seen in all areas of the brain with elevated levels in dopamine rich areas, especially the substantia nigra. [56]

TIQs are found in nature and are part of our diet. [57] Some have also been shown to readily cross the blood-brain barrier. [58] They affect metabolism, motor activity, and neurotransmission. [59] Moreover, they directly modulate signaling of dopamine and norepinephrine [60] by dysregulating the metabolism of these neurotransmitters. [61]

They have been associated with ADHD, [62] Parkinson’s disease, [56] and alcoholism. [63]

They are elevated in the CSF of patients with psychiatric disorders [64] and in low levels in the brain of patients without psychiatric disorders postmortem. [65] DeCuypere et al., performed an extensive review of the distribution of the TIQs in the brains of mice, rats, and human, and compared endogenous levels with levels see in Parkinson’s disease.

One of the goals of the present research was to evaluate changes in catecholamine and

TIQ levels that occur in the brain as a result of the consumption of alcohol.

MPTP

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a piperidine derivative that was first discovered in a clandestine laboratory environment in which designer drug derivatives of

34 meperidine were being synthesized for their morphine-like effects on the opioid system. MPTP was a contaminant in batches of the illicit drugs. Individuals who used these substances very quickly developed parkinsonian symptoms, causing an epidemic of apparent PD in people far younger than is normal for this disease. MPTP has since been used as a chemical inducer and model for idiopathic Parkinson’s disease. Rotenone and paraquat are pesticide that have some structural similarity to MPTP, and have also been shown to result in parkinsonism in animal models. Other structurally similar compounds that are neurotoxic include the tetrahydroisoquinolines (Figure 14).

MPTP is not considered toxic on its own, and requires activation to MPP+ to elicit its neurotoxic effects. The activation occurs in glial cells enzymatically with monoamine oxidase B

(MAO B). Once formed, MPP+ can readily move out of glial cells and into neurons. MPP+ can act as a substrate for the dopamine transporter, inhibits its own metabolism at MAO-B, and inhibits tyrosine hydroxylase the rate limiting step for catecholamine synthesis. Most importantly, MPP+ inhibits complex I of the mitochondrial respiratory chain, which results in apoptotic cell death. [66-70] TIQs have been detected in urine, CSF, and brain tissue from alcoholics and Parkinson’s brain. Endogenous low levels of many TIQs have been detected in disease free patients, and may be associated with the low-grade degeneration associated with progressive, age-related pathologies of aging, diabetes, Alzheimer’s, alcoholism, and Parkinson’s diseases.

Figure 14. MPTP like structures of tetrahydroisoquinolines

Salsolinol

Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) is a catecholamine derived neurotoxic tetrahydroisoquinoline that can be found in nature as well as in the body.

Salsolinol is in many of the foods that we consume (cheese, chocolate, bananas, mushrooms) and all alcoholic drinks. [71-73] Salsolinol can also be formed in the body by two main pathways and crosses the blood brain barrier. [74] The enzymatic pathway uses salsolinol synthase to combine dopamine and acetaldehyde in a stereospecific manner to form (R)-salsolinol. The non- enzymatic pathway occurs as dopamine and acetaldehyde readily condense in vivo via the Pictet-

Spengler reaction to form both (R) and (S) enantiomers of salsolinol (Figure 15). Hereafter, the use of the word salsolinol will refer to the (R) confirmation. Salsolinol is considered toxic, displaying MPTP-like effects.

Interest in salsolinol as an alcohol metabolite began in 1973, when Sander and colleagues first reported significant levels in urine from Parkinson patients on L-DOPA treatment.

Salsolinol has also been linked to many of the neurochemical and behavioral effects of alcoholics, and is present in urine after alcohol consumption, [75] and significantly elevated in the brains of alcoholics, post mortem. [76] In chronic ethanol consumption, salsolinol levels are significantly higher in the hippocampus [77] and the compound is present in urine for days even in

36 withdrawal. [78] Salsolinol and O-methylated salsolinol metabolites are elevated in alcoholics proportional to blood acetaldehyde levels for 2-3 days. [79-80] Females show higher salsolinol levels in urine than men. [81]

The literature on salsolinol’s effects includes many findings for which replication by other authors has not been reported, but which are, for the most part, plausible. Moderate drinkers have significantly higher levels of salsolinol than light drinkers, [81] but binge consumption has been reported to lower urinary salsolinol levels than chronic moderate drinkers.

Salsolinol has been detected in the cerebral spinal fluid (CSF) of healthy volunteers and alcoholic patients. [82-83]. Levels in alcoholics have a strong correlation with age and level of dementia. [84] Salsolinol levels are higher in the striatum with chronic consumption. [85] Heavy drinkers have less salsolinol derived from their diet than light drinkers. [86] . This may be due to heavier drinkers having poorer diets. Urinary levels of salsolinol increase during the first four days of withdrawal. [87] PD patients who have received transplanted dopaminergic neurons have significantly increased concentrations of salsolinol in CSF. [88] High concentrations of salsolinol have been found in amniotic fluid but not in umbilical plasma. [89]

Behaviorally, salsolinol has been found to increase preference for alcohol and addiction, regulate drinking and dependence and is self-administered; the reinforcing effects involve the nucleus accumbens and ventral tegmental area and receptors for serotonin and dopamine have been implicated in these effects. [90-93] In a study of antisocial alcoholics, salsolinol levels were found to be inversely related to scores on a rating scale for antisocial tendencies. [94] In another study, patients with hallucinations associated with psychiatric disorders had three times higher levels of salsolinol than non-hallucinatory patients. [95]

The literature on salsolinol’s molecular interactions is similarly complex. Salsolinol is metabolized by catecholamine O-methyl transferase (COMT). [96] It is also a competitive inhibitor of COMT and of monoamine oxidases MAOs. [97-98] It has been reported to be a selective inhibitor of MAO A [99], but a reversible inhibitor for MAO B. [100] Inhibitors of MAOs are known to increase nicotine self-administration and maintain sensitization. [101] Moreover, nicotine inhibits salsolinol toxicity, via anti-apoptotic properties that are mediated by nicotine receptors. [102-103]

Salsolinol is a potent competitive inhibitor of tyrosine hydroxylase with biopterin, a cofactor that binds directly to the tyrosine hydroxylase enzyme. [104-107] Tyrosine hydroxylase is the rate limiting step in catecholamine synthesis, thus its inhibition results in a depletion of L-

DOPA and the catecholamine neurotransmitters (dopamine, norepinephrine and epinephrine) that are derived from this amino acid. Salsolinol has no effect on dopa decarboxylase. [107] Infusion of salsolinol into the brain results in dopamine and serotonin release from synaptic vesicles, [108-

109] resulting in decreased brain dopaminergic and serotonergic activity. [110] Salsolinol acts as an agonist for alpha adrenergic receptors, [111] and as an antagonist for beta adrenergic receptors.

[112] This results in enhancement of norepinephrine, which in turn inhibits adenylate cyclase resulting in inhibition of cAMP and blood vessel constriction.

Salsolinol is an agonist for D2 and D3 dopamine receptors, and the most potent of the known TIQ’s for inhibiting the binding of apomorphine on dopaminergic receptors. Salsolinol also shows opiate like activity upon stimulation. Affinity is higher for dopamine receptors than opiate receptors, especially D3. Salsolinol acts like an endogenous opioid, encephalin. Its toxicity may be due to its role in inhibiting complex I of the electron transport chain and inhibition of ATP in the mitochondria like MPTP. [113-114]

Figure 15. Pathways for the formation of salsolinol and N-methylsalsolinol DeCuypere, M et al. Journal of Neurochemistry, 2008, (107) 1398-1413

Carboxysalsolinol

Carboxysalsolinols are tetrahydroisoquinolines that have a carboxylic acid group on one of the carbons adjacent to the N-atom of the piperidine ring. (Figure 16). 1-carboxysalsolinol is formed as a condensation product of dopamine and pyruvic acid, whereas 3-carboxysalsolinol is formed as a product of L-DOPA and acetaldehyde. Very little research has been performed with these carboxy-TIQs. 1-CS is commercially available, and 3-CS can be readily synthesized in the laboratory following the method by Brossi et al., 1971. Given the ease to procure these TIQs, it is surprising that more work has not been done in this area. 1-CS and 3-CS share many of the same toxic characteristics as the rest of the TIQ family, but they do have a few unique traits. 3-

CS is thought to be the only TIQ that readily crosses the blood brain barrier (BBB) which may be

39 from its amino acid moieties like L-DOPA. Thus, 3-CS may use the amino acid transport system across the BBB like L-DOPA. Moreover, 3-CS is thought to be the only TIQ that can induce alcohol consumption with peripheral (IP) injections. Other TIQs require central administration to affect alcohol consumption and place preference. [115] 3-CS treatments enhance the voluntary consumption of alcohol, [116] and generalize as a discriminative stimulus. [117-118]

Carboxysalsolinol’s were determined to be significantly elevated in the urines of alcoholics and in brains of Parkinson’s disease. The carboxysalsolinol’s were evaluated at for these mechanistic links, yet inconsistent and inconclusive data resulted in researchers in this area to focus on other TIQs. There were also analytical challenges at detecting some of these TIQs due to sample preparation techniques and instrument sensitivity. It has been nearly forty years since the first experiments were performed in this area.

Quantitative ADME studies for the carboxysalsolinols, have not been reported. Acute and chronic ethanol consumption does not significantly alter endogenous levels of 1- carboxysalsolinol (Figure, 17). [119] Moreover, 1-carboxysalsolinol levels in the striatum of alcoholic and non-alcoholics were not different [120] In another study, 1-CS was detected in alcoholic’s post mortem brains, CSF, and urine. In addition, these levels were significantly elevated when alcohol was still present in the system. [121] Design study, sampling, and analytical detection appear to be key factors in these varying results. It is not surprising that 1-CS is not a direct biomarker of consumption since it is formed from dopamine and pyruvate and does not have a direct link to alcohol consumption. Both 1-CS and 3-CS are readily metabolized by catechol-O-methyl transferase (COMT), and excreted in urine predominantly as the mono- methylated form, and non-enzymatically, oxidative decarboxylated in the liver to dehydrosalsolinol. [122] Neither 1-CS nor 3-CS are converted to salsolinol. [123] Carbidopa

40 inhibits metabolism and enhances 3-CS ethanol induced prolonged sleep times [124], and depressant effects [125] , and enhances apomorphine. These effects may be related to the high binding affinity that both carboxysalsolinols have for opiate receptors. [90] Moreover, carboxysalsolinol displays analgesia like effects that are enhanced with carbidopa and blocked by naloxone. [125]

Both carboxysalsolinols can competitively bind to tyrosine hydroxylase and inhibit catecholamine synthesis. ] Yet, neither has any inhibitory effect on dopa decarboxylase. [105]

Acute consumption of ethanol increases 1-CS and interferes with glucose metabolism by 1-CS reacting with pyruvate and acetaldehyde inhibition of pyruvate dehydrogenase complex. [126]

Acute administration of 3-CS increased serotonin in the striatum by 70-90%, and chronic

[127] administration reduced striatal serotonin by 52%.

1-CS is significantly increased in ethanol consumption that is accompanied by poor nutrition and thiamine deficiency [128] 3-CS inhibits angiotensin converting enzyme (ACE), the enzyme that regulates blood pressure, [129-130] and is a selective antagonist vasopressin 2, an antidiuretic hormone. [131]

Figure 16. Formation of 1-carboxysalsolinol, 3-carboxysalsolinol and salsolinol

Figure 17. Salsolinol and 1-carboxysalsolinol in Alcoholics and Non-Alcoholics MA. Collins, Parkinsonism and Related Disorders, 8 (2002) 417-422

N-methyl-salsolinol

N-methyl-(R)-salsolinol is a dopamine derived alkaloid that is an endogenous neurotoxin that has been implicated in many disorders. It is derived from salsolinol and the enzyme N-methyl- transferase (Figure 18). Highest concentrations are found in dopamine-rich areas and it is reported as being selective for the nigrostriatal pathway [132] . Moreover, N-methyl-salsolinol has a direct effect on the catecholamines by lowering dopamine levels. Of all the tetrahydroisoquinoline derivatives, its toxic effects were most closely related to the neurotoxic

Parkinsonian drug MPTP [133] and its mechanism of toxicity is thought to be similar to MPTP as it reduces mitochondrial potential, [134] decreases GSH, increases ROS and therefore oxidative stress [135] and activates an apoptotic cascade. [136] Additionally, N-methyl-salsolinol has been shown to be significantly elevated in CSF of Parkinson patients [137] and is significantly elevated in urine of children with ADHD. [59] N-methyl-salsolinol inhibits mitochondrial monoamine oxidases, complex I, and α-ketoglutarate dehydrogenase [138] and Rasagiline a MAO inhibitor protects neurodegeneration [139] . Protection from MAO inhibition implies that N-methyl- salsolinol like MPTP requires activation to take effect. Deactivation and metabolism of N- methylsalsolinol occurs enzymatically with catechol-O-methyl transferase (COMT).

ADTIQ

1-acetyl-6,7- dihydroxyl-1, 2, 3, 4- tetrahydroisoquinoline (ADTIQ) is an endogenous MPTP- like neurotoxin that is formed from a condensation (Pictet-Spengler) reaction between dopamine and methylglyoxal under normal physiological conditions (Figure 18). Methylglyoxal is a pyruvate-aldehyde that is mainly produced during the glycolytic pathway. Methylglyoxal is a toxic metabolite formed from an enzymatic reaction between glyceraldehyde 3-phosphate and dihydroxyacetone with the recovery of phosphate (Figure 19). Higher glucose levels result in a

43 nearly 6-fold increase in methylglyoxal levels in the blood. Methylglyoxal readily reacts with amino acids (lysine, arginine, and cysteine), proteins, lipids, and catecholamines. Hyperglycemic individuals have elevated levels of methylglyoxal derived metabolites, including advanced glycation end products (AGEs).

AGEs are proteins that react with sugars due to elevated blood glucose, and have been associated with hypertension, diabetes, renal failure and neurodegenerative diseases

(Alzheimer’s, Parkinson’s). Moreover, there is evidence that implicates AGEs in chronic inflammatory disorders associated with aging and neuropathy. [140] Diabetes, Alzheimer’s, and

Parkinson’s are progressive degenerative disorders that may be related to the chronic inflammation because of elevated AGEs. Diabetes is also a risk factor for Alzheimer’s and

Parkinson’s. More recently, the reaction of methylglyoxal with dopamine has become of interest because significant levels of ADTIQ have been determined in the human brains of Parkinson patients’ post-mortem. This may be the connection between diabetes, glucose metabolism, metabolic syndrome, and Parkinson’s disease. [141] The glyoxalase system is a detoxification pathway for the degradation and removal of methylglyoxal. This pathway uses glutathione and two glyoxalase enzymes with lactate as an end-product.

In animal models of hypertension and diabetes, methylglyoxal, ADTIQ, and metabolic enzymes were significantly elevated in rat brains. [142] Additionally, adenosine triphosphate

(ATP) and superoxide dismutase (SOD) levels were significantly decreased in the diabetic rat model. These results indicate that glucose metabolism has been altered and neurotoxins have been formed. ADTIQ has been determined in all brain regions of Parkinson patients post mortem, including the substantia nigra, putamen, frontal cortex, and the cerebellum. [143]

Excessive glucose results in excessive amounts of methylglyoxal and ADTIQ, resulting in

44 induced oxidative stress, mitochondrial damage, neuronal damage, and apoptosis. [144] ADTIQ is associated with mitochondria apoptosis as evident by key protein markers, Bax, Bcl-2, caspase-3.

[145] . Bax and caspase-3 are pro-apoptosis, and Bcl-2 is an anti-apoptotic protein that helps release cytochrome c from mitochondria.

In addition, methylglyoxal induces tyrosine hydroxylase (TH), and dopamine transporter

(DAT) expression levels, dopamine and salsolinol. [146] ADTIQ levels are significantly elevated in mice with mutations of alpha-synuclein, are associated with oxidative stress, and may be responsible for the dysregulation of dopamine seen in diabetics. Alpha-synuclein is also associated with familial Parkinson’s disease, Alzheimer’s disease, and alcoholism, and is thought to form aggregates that can disrupt the membranes of the mitochondria. In alcoholism, glucose metabolism is disrupted, in response, the cell uses acetate for energy. This creates a crisis in the cell for energy sources, during alcohol withdrawal.

Figure 18. Formation of ADTIQ from Dopamine and Methylglyoxal

Figure 19. Alternative Products of Glycolysis & Formation of ADTIQ

CHAPTER 4.

SIMULTANEOUS QUANTITATION OF CATECHOLAMINES AND SALSOLINOL-

LIKE TETRAHYDROISOQUINOLINES BY LIQUID CHROMATOGRAPHY-MASS

SPECTROMETRY/MASS SPECTROMETRY

ABSTRACT

Rationale: Chronic alcohol consumption has been shown to be a risk factor for developing diabetes. This association may be due to the interactions of alcohol itself or through alcohol metabolites on glucose utilization and metabolism. In order to further elucidate the connection between alcohol and glucose metabolism, the development of an analytical method for the determination of these metabolites was necessary.

Methods: LC-MS/MS was used to determine quantitative levels of catecholamines (CAs) and tetrahydroisoquinolines (TIQs) associated with alcohol and glucose metabolism in mice.

Simultaneous determination was achieved with a Waters HLB solid phase extraction column with is an all-purpose hydrophilic and lipophilic reverse phase sorbent, chromatographic separation, quantification with standard curves and analyte-matched deuterated internal standards.

Results: CAs and TIQs were validated in a single method with sensitivity of less than 1 ng/ml in many cases. Matrix matched standard curves gave linearity with more than four orders of magnitude ranging from 0.25 to 1000 ng/ml.

Conclusions: Elevated levels of TIQs have been implicated in many diseases including alcoholism, diabetes, Parkinson’s and Alzheimer’s. A quantitative method for CAs and TIQs was validated for brain, liver, urine and blood. One or more of these biomarkers may help in the determination and assessment of the progression of a disease or disorder and therefore, may have both clinical and forensic relevance.

INTRODUCTION:

Heavy, chronic consumption of alcohol (ethanol) causes a constellation of adverse effects on health and is associated with a withdrawal syndrome that promotes addiction. Conversely, many people consume alcohol in moderate doses on an occasional basis with no apparent ill effects. In spite of decades of research on what is surely mankind's oldest drug, the exact ways in which alcoholic beverages induce all of their many effects are not completely understood. The direct actions of ethanol on targets such as glutamate and GABA receptors are probably responsible for much of the drug's psychoactivity. However, it is possible that metabolites of alcohol are responsible for some of the effects, and alcohol metabolites also have utility as clinical and forensic biomarkers of alcohol consumption.

There are indications that moderate alcohol consumption may be protective against the development of diabetes. [147] However, chronic alcohol exposure is considered a risk factor for the occurrence of diabetes. [148] The direct impact of alcohol on bio-energetic homeostasis is not clear, and warrants further investigation. It has been proposed that diseases like diabetes,

Parkinson’s, and Alzheimer’s may be associated with the effects of patient specific altered energy metabolism and mitochondrial dysfunction. [149] Moreover, many neurodegenerative symptoms are also associated with diabetes. [149] Alzheimer’s disease has been directly linked to

48 disrupted metabolic processes and synaptic transmission similar to the way that alcohol disrupts transmission of GABA and acetylcholine in beta and alpha-cells in the pancreas respectively. [150-

152] Beta cells in the pancreas use similar machinery as neurons in the brain for the purposes of storing chemical messengers, responding to stimuli, and transmitting a signal. [153] A direct connection between alcohol consumption and glucose metabolism has been well established. [154]

Recent studies have also made connections between glucose metabolism and catecholamine neurotransmitter homeostasis. [155]

The connection between glucose and catecholamine metabolism introduces a newly discovered tetrahydroisoquinoline (TIQ) named 1-acetyl-6,7-dihydroxy-1,2,3,4- tetrahydroisoquinoline (ADTIQ). [156] TIQs have been extensively studied as they have been shown to be neurotoxic. The TIQs that have received the most attention are salsolinol and N- methylsalsolinol (Figure 14).

Salsolinol is formed endogenously and enzymatically in the body from the condensation of dopamine and the reactive ethanol metabolite, acetaldehyde. [157] Salsolinol is converted enzymatically to, N-methylsalsolinol, which is structurally similar to MPTP (a chemical that provides a model for Parkinson’s disease) and which is neurotoxic in SH-SY5Y cell viability studies. [158-160] The dopamine precursor L-DOPA can also react with acetaldehyde in vivo to give in this case 3-carboxysalsolinol (3-CS). [161] 3-CS has been given special attention because it is known to readily crosses the blood brain barrier. [162] It is presumed to be a substrate for the amino acid transport system that can carry L-DOPA into the brain. This highlights the urgency and need for diagnostic methods that tests for these metabolites.

Levels of TIQs have been shown to be elevated in alcoholism, [163] attention deficit hyperactivity disorder, [164] Parkinson’s, [165] and psychiatric disorders. [166] The transient nature of

CAs and their rapid oxidation and metabolism make them difficult to test and interpret.

However, TIQs are relatively more stable biologically than CAs. [165] As such, they offer potential advantages as compared to CAs for physiological and pathological assessments in clinical and forensic investigations. More studies on the quantification of TIQs as biomarkers for disease progression and toxicity are clearly warranted.

Alcohol is readily metabolized to acetaldehyde by alcohol dehydrogenase, and this occurs predominantly in the liver. Acetaldehyde is a volatile and reactive compound, but is rapidly converted to acetate by aldehyde dehydrogenase. Acetate may provide a link between alcohol consumption and disorders of glucose metabolism, since acetate can be used metabolically for fuel. [167] It competes with pyruvate for the citric acid cycle elevating pyruvate to accumulate

(Figure 21). [168]

Figure 21. Interplay between glucose, catecholamine, and ethanol metabolism

Elevated pyruvate concentrations can lead to pyruvate being converted to lactate or condensing with dopamine to form 1-carboxysalsolinol (1-CS), which has been shown to be

50 elevated in saliva, urine and blood after chronic alcohol consumption. [169-171] Lastly, elevated pyruvate caused by inhibiting glycolysis may lead to substrates being consumed by the glyoxylate cycle. [172] Methylglyoxal, a byproduct of the glyoxylate system, condenses with dopamine in vivo , to form another TIQ, 1-acetyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline

(ADTIQ; Figure 2). [173] ADTIQ levels have been shown to be elevated in patients with diabetes and Parkinson’s disease. [174]

In this paper, we report simultaneous quantification of CAs and TIQs in biological tissues and fluids. This method will be used to assess the effects of alcohol consumption on catechol and glucose metabolism by monitoring the formation of these TIQs under different treatment conditions in experimental animals treated with ethanol.

EXPERIMENTAL:

Chemicals and reagents

3,4-Dihydroxy-L-phenylalanine (L-DOPA), 3-(3,4-Dihydroxyphenyl-2,5,6-d3)-L-alanine (L-

DOPA-d3), 3,4-Dihydroxyphenylacetic acid (DOPAC), Homovanillic acid (HVA), DL-4-

Hydroxy-3-methoxymandelic acid (VMA), Dopamine hydrochloride, DL-Norepinephrine hydrochloride, (±)-Epinephrine hydrochloride, L-Tyrosine, 3,4-Dihydroxybenzylamine hydrobromide (DHBA), Serotonin hydrochloride, 5-Hydroxyindole-3-acetic acid (5-HIAA),

Salsolinol hydrobromide, were all purchased from Sigma-Aldrich. Dopamine-d4 HCL, (±)-

Epinephrine-D6, (±)-4-Hydroxy-3-methoxymandelic Acid-D3 (ring-D3) (VMA-D3), 4-

Hydroxy-3-methoxyphenyl-D3-acetic-D2 Acid (HVA-D5), 5-Hydroxyindole-4,6,7-D3-3-acetic-

D2 Acid (5-HIAA-D5) and (±)-Norepinephrine-D6 were purchased from Cerilliant. Salsolinol-1- carboxylic acid was purchased from Chemdea. (R)-Salsolinol-d4 HBR was purchased from

Cambridge Isotope Laboratories, Inc. 3-O-Methyl-L-DOPA was purchased from Santa Cruz

Biotechnology. HPLC-grade water, methanol, and acetonitrile as well as formic acid and ammonium formate were purchased from Fisher Scientific.

Synthesis of 3-carboxysalsolinol, 3-carboxysalsolinol-d2, and 6-methoxy-3- carboxysalsolinol.

The carboxylated tetrahydroisoquinolines, 3-carboxysalsolinol and 3-carboxysalsolinol- d2 were synthesized following the method as described by Brossi et al. [175] This synthesis condenses L-DOPA with acetaldehyde to yielded a 95:5 ratio of cis:trans forms of 3- carboxysalsolinol (Figure 21). Since L-DOPA was used instead of D-DOPA for this reaction, and L-DOPA is almost exclusively seen endogenously, it is likely that the 3-cis- carboxysalsolinol isoform is more prevalent in the body than the 3-trans-carboxysalsolinol isoform. Therefore, the levels determined in this study for 3-carboxysalsolinol (3-CS), represent the 3-cis-carboxysalsolinol isoform fraction. 6-methoxy-3-carboxysalsolinol was synthesized by using 3-methyl-L-DOPA and acetaldehyde. In vivo, 3-carboxysalsolinol is metabolized with methyltransferases (Figure 22) to 6- and 7-methoxy-3-carboxysalsolinol.

Figure 21. Pictet-Spengler reaction for the formation of 3-carboxysalsolinol

Figure 22. Methylation of 3-carboxysalsolinol to 6,7-methoxy-3-carboxysalsolinol

Animals used for all experiments

Male Swiss-Webster mice were purchased from Taconic Farms. These mice were maintained in the animal care center at the university. Experiments performed were in accordance with protocols approved by the institutional animal care and use committee (IACUC). Sample sizes were N=5 for both control and treatment groups.

Standards preparation and calibration

The stock standard solutions of 1 mg/ml and 100 µg/ml for drug and internal standards respectively were either purchased or prepared from powder, and stored at -80 ˚C until analysis.

At the time of analysis, a 10-point calibration curve with points ranging from 0.25 – 1000 ng/ml was fortified in matrix, where standards are spiked directly into an analyte-free (blank) matrix matched to the matrix being analyzed. This is important because different matrices have different

53 effects on the quantitation of the analyte. This method was used to analyze catecholamines and

TIQs in mouse brains, blood, urine and liver. Additionally, quality control standards were made fresh each day that this method was used. Four levels of controls were made to reflect the four orders of magnitude achieved with this method. Namely, 1, 10, 100, and 1000 ng/ml.

Quantitation was achieved by preparing a standard curve with fresh calibrators (1, 2.5, 5, 10, 25,

50, 100, 250, 500, 1000 ng/ml) and quality controls with every batch of samples. Calibrators were injected in duplicate and quality controls were injected before and after tissue samples to verify method ruggedness, column integrity, and instrument sensitivity.

Sample preparation for mouse brain tissues

Tissue samples were prepared as described by DeCuypere et al. [56] Briefly, mice were anesthetized and brain samples from the striatum, cerebellum, and hippocampus were collected on ice. These tissues were then weighed, and placed into vials for homogenization. The homogenization buffer was 0.1M perchloric acid with sodium bisulfite and EDTA to prevent catecholamine oxidation. The volume of buffer was 100 µl per 10 mg of wet tissue. These samples were then homogenized on ice with a Tissuemiser® (Fisher Scientific) until there were no visible signs of any tissue matter. Samples were then centrifuged at 14,000g for 30 mins at

4˚C. The supernatants were then filtered with a 0.22 µm membrane filter to remove high molecular weight compounds, and then applied to a Waters HLB® solid phase extraction column directly. This column has an all-purpose hydrophilic-lipophilic sorbent that allows for the additional cleanup of the matrix, while providing excellent recovery of catecholamines and tetrahydroisoquinolines. The eluent was evaporated and reconstituted in mobile phase A (DIH 20 with 10 mM ammonium formate), then transferred to an autosampler vial for analysis.

Instrumentation

Liquid Chromatography method development

Liquid chromatography was performed with an Agilent 1260 system with an Infinity refrigerated autosampler. The column compartment was set at 4˚C for the entire method. Mobile phase A was

HPLC grade DIH 20 with 10 mM ammonium formate, and mobile phase B was HPLC grade acetonitrile with 0.1% formic acid. The LC column was a Discovery® HS F5-3 (5 µm x 150 mm x 4.6 mm) from Supelco Analytical with a 10 µl injection. The gradient was as follows: 95% A at t=0-2 mins, 95%-70% A for 2 to 20 mins, 70%-40% A for 20 to 24 mins, then 40%-95% A for

24-26 mins to re-equilibrate the column. A post-run time of 5 mins was also applied to prevent any carryover and to equilibrate the column. Figure 24 shows baseline resolution of all compounds.

It is important to note that 1-CS and 3-CS are isobaric compounds. Therefore, a shallow gradient was utilized at the beginning of this run to ensure the separation of these compounds at

5.9 min (1-CS) and 6.5 min (3-CS). Chiral separation for several compounds was also achieved with this method, but was not the focus of this research. The (R)-isomer of the tetrahydroisoquinolines was predominantly seen in biological samples, and thus was used for quantitation. For example, in Figure 23 the peaks have been selected for (R)-salsolinol at a RT of

16.834 minutes and (S)-salsolinol at RT of 21.203 minutes. Chromatographic separation of 1- carboxysalsolinol and 3-carboxysalsolinol was achieved with the use of the Discovery HS F5-3

LC column.

Figure 23. Full chromatographic resolution and quantification of all analytes tested in a single 30-minute run. The x-axis in time in minutes, and the y-axis is relative abundance. The individual peaks have the following retention times.

1 1-carboxysalsolinol 5.90 2 3-carboxysalsolinol d2 6.52 3 3-carboxysalsolinol 6.52 4 acetylcholine 6.90 5 L-dopa-d3 7.47 6 L-DOPA 7.51 7 ADTIQ 7.90 8 glutamate 8.40 9 norepinephine 9.21 10 L-tyrosine 10.17 11 DHBA 11.89 12 6-methoxy 3-carboxy 11.96 13 epinerphine 12.05 14 3-methoxydopa 12.57 15 DOPAC 13.38 16 dopamine d4 14.90 17 dopamine 14.95 18 VMA 15.91 19 salsolinol 16.83 20 N-methylsalsolinol 19.63 21 HVA 19.75 22 Serotonin 22.01

Mass spectrometry method development and validation

Tandem mass spectrometry was performed with an Agilent 6490 system. All standards and their deuterated internal standards were infused with a syringe pump for optimization and the two most prevalent transitions were used for analysis. The most abundant transition was used in

MRM mode for quantitation. Quantitation was performed with standard curves ranging from 1 to

1000 ng/ml. Samples were normalized with their deuterated internal standard when available.

Analytes that did not have a paired deuterated internal standard, 3,4-Dihydroxybenzylamine

(DHBA) was used. DHBA is not found endogenously, elutes chromatographically near the middle of the run, and was not found to have significant ion suppression or enhancement.

ADTIQ transitions and parameters were added based upon previously published work. [173]

Ionization was achieved with an electrospray ionization probe with the following source parameters: Gas temperature 325 ˚C, gas flow 10 L/min, nebulizer 40 psi, capillary 4000V.

Positive ionization mode was used for all compounds except, 5-HIAA, DOPAC, HVA, and

VMA, in which the negative ionization mode was employed. 1-CS and 3-CS share the same quantitative 224.1-178.1 MRM transition, so chromatographic resolution is essential for proper quantitation. Qualitative distinction between 1-CS and 3-CS is possible by using the second transition of 137.1 and 161.1 respectively. Figure 24 shows the mass spectrum for 3-CS. The remaining MS parameters can be found in Table 1.

Figure 24. Mass spectrum for 3-carboxysalsolinol.

Table 1.

Method validation for CAs and TIQs in mouse brain

Validation for catecholamines, tetrahydroisoquinolines, and their metabolites was performed for all matrices analyzed (brain, blood, urine, and liver). Here report validation data for mouse brain only. This validation included precision, accuracy, limits of detection (LOD), limits of quantitation (LOQ), and upper limits of quantitation (ULOL), carryover, interferences, ion suppression/enhancement, stability, and recovery from filtration as well as from solid phase extraction (SPE).

Statistical analysis for method validation and determination of significance

Some of the validation data was performed within the Agilent MassHunter software. This program provided the R 2, LOD, LOQ, and regression equations. Relative standard deviation

(RSD) values were calculated as such: RSD = (SD/observed mean) x 100. In addition, this method was used experimentally on mice exposed to alcohol (ethanol). Statistical data for these experiments (reported elsewhere), between control and treated groups, were compared using a T- test analysis. P values less than 0.001 were considered statistically significant. In addition, a two- way ANOVA was performed using GraphPad Prism 7 software.

RESULTS AND DISCUSSION:

Linearity of four orders of magnitude

Optimization and quantitative determination of both catecholamines and tetrahydroisoquinolines were made possible by chromatographic baseline resolution of all analytes, isomers, and separation of isobars. With this method development, we could achieve linearity to four orders of magnitude, as well reach the sensitivity of 0.25 ng for some analytes in fortified tissue samples (Table 2). A weighting factor of 1/x was used in all cases. The highest calibrator used was 1000 ng/ml with no observed detector saturation resulting in a non-linear response. The limits of quantitation were deemed acceptable for the brain tissue matrix. The high organic content of the brain matrix were accounted for in the sample prep, and any additional matrix effects were accounted for with internal standards.

Precision and accuracy of calibrators and quality control standards

Inter and Intra-day precision was determined by running two replicates of each calibration point over five days (n=10). Each point was integrated with an independent calibration standard curve for each analyte, and values are expressed as relative standard

59 deviations (Table 2). Accuracy was determined as the percent difference the theoretical concentration and the calculated concentrations.

Table 2. Method validation data for CA's and TIQ's in mouse brain

LOD (ng/ml) LOQ ng/ml Linear Range Precision Analyte R2 Accuracy (%) Recovery (%) S/N >3 S/N >10 (ng/ml) (n=10 RSD) 1-carboxysalsolinol 0.996 0.5 3 0.5 - 500 8.7 94.8% 87.2% 3-carboxysalsolinol 0.991 0.5 3 0.5 - 500 11.2 96.3% 85.6% acetylcholine 0.994 1 5 1.0 - 1000 2.8 98.1% 92.4% L-DOPA 0.992 1 5 1.0 - 1000 4.1 94.2% 91.7% glutamate 0.995 1 5 1.0 - 1000 6.3 97.6% 95.0% norepinephine 0.993 0.5 3 0.5 - 500 7.7 96.6% 101.1% L-tyrosine 0.992 1 5 1.0 - 1000 5.5 95.6% 97.4% 6-methoxy 3-carboxy 0.990 1 5 1.0 - 1000 12.2 89.6% 86.6% epinerphine 0.998 0.25 1 0.25 - 500 3.8 98.6% 100.9% 3-methoxydopa 0.991 1 5 1.0 - 1000 9.4 93.5% 85.3% DOPAC 0.992 2 5 2 - 1000 14.3 98.5% 82.8% dopamine 0.997 0.25 1 0.25 - 1000 2.7 97.7% 102.4% salsolinol 0.999 0.25 1 0.25 - 1000 2.9 97.6% 95.1% N-methylsalsolinol 0.997 0.5 3 0.5 - 500 5.8 92.9% 94.6% HVA 0.992 1 5 1.0 - 1000 11.7 86.3% 84.4% Serotonin 0.996 0.5 3 0.5 - 500 2.1 99.2% 98.7% 5-HIAA 0.990 1 5 1.0 - 1000 6.9 97.4% 81.0% Table 2.

Extraction recovery from sample preparation and matrix effects from mouse brain.

CA and TIQ recovery from tissue is essential for accurate quantitative determination.

Standards were spiked (fortified) into blank matrix and compared with neat extracted samples.

Preliminary results showed matrix effects, and issues of recovery from sample prep. Thus, we added a filtration step with a 0.22 µm pore size membrane filter. The acceptance criteria of greater than 80% recovery from sample preparation and less than 30% ion suppression from matrix was achieved with the addition of this membrane filter.

CONCLUSIONS:

Simultaneous quantitative determination of catecholamines and salsolinol-like tetrahydroisoquinolines is attainable in a single method utilizing sample filtration preparatory steps, solid phase extraction, liquid chromatographic separation, and the sensitivity and specificity of mass spectrometry. LC-MS/MS is considered the gold standard and method of choice for analyzing catecholamines. [176-179] In comparison with immunoassays, GC-MS, and

HPLC-ECD, the LC-MS/MS technology allows for a simpler sample prep, requires less sample volume, does not need to be hydrolyzed, derivatized, evaporated, or ion-paired. [180-186]

Analytically, LC-MS/MS is faster, cheaper, more accurate, sensitive, selective, robust, and can be automated for high-throughput than GC-MS or HPLC-ECD. [187-199] It also has fewer interferences, with a larger dynamic range, and a greater breath of analytes that can be analyzed with a single injection. [200-210] TIQs share many of these advantages, but have not been as extensively studied as the catecholamines.

We have validated our methodology for liver, urine, blood and plasma, as well as for brain. Catecholamines are currently being used diagnostically as tumor markers for pheochromocytoma, paraganglioma, and neuroblastoma. [211-212] TIQs have been previously shown to be elevated in urine of patients with ADHD, alcoholism, melanoma, and Parkinson’s disease. [213-216]

Standardization of methods are important if these biomarkers are to be used to determine the status and progression of a disease or disorder. Previous methods have had challenges with the analytical sensitivity to meet a diagnostically appropriate cutoff and as such have under-reported the presence of the cancers mentioned above. [217-218] Moreover, the interferences that occur with these prior methods have led to the reporting of false positive results. LC-MS/MS reduces the

61 gaps between the false negatives and false positives, as diagnostic instrument leading to better clinical outcomes. Catecholamine derived-TIQs may also be important biomarkers to help early detection and proactive assessment for increases in risks associated with related diseases. Here we reported an LC-MS/MS method that determines both catecholamines and salsolinol-like tetrahydroisoquinolines.

CHAPTER 5.

DETERMINATION OF ENDOGENOUS LEVELS OF 3-CARBOXYSALSOLINOL,

(R/S)-SALSOLINOL, N-METHYL-(R/S)-SALSOLINOL, AND DOPAMINE IN MOUSE

STRIATUM, HIPPOCAMPUS, AND CEREBELLUM

Introduction:

Salsolinol can also be formed in the body by two main pathways and crosses the blood brain barrier. The enzymatic pathway uses salsolinol synthase to combine dopamine and acetaldehyde in a stereospecific manner to form (R)-salsolinol. The non-enzymatic pathway occurs as dopamine and acetaldehyde readily condense in vivo via the Pictet-Spengler reaction to form both (R) and (S) enantiomers of salsolinol (Figure 15). An elevated levels of (R)-salsolinol implies enzymatic formation of salsolinol. Moreover, food and beverage sources of salsolinol are predominantly the (S)-enantiomer, and are not considered to be toxic. Endogenous levels of salsolinol and N-methylsalsolinol have been reported in the brains of human, rats, and mice, and are elevated in Parkinson brains. N-methyl-salsolinol is a metabolite of salsolinol, and is thought to be even more toxic, given its greater structural similarity to MPTP.

Carboxysalsolinols are tetrahydroisoquinolines that have a carboxylic acid group on one of the carbon atoms adjacent to the indole ring nitrogen (Figure 25). 3-carboxysalsolinol is formed as a condensation product of L-DOPA and acetaldehyde. Very little research has been performed pertinent to this TIQ. In fact, 3-CS has not been reported in the literature in nearly 15 years, and prior to that, only twenty relevant papers are listed in pubmed (see appendix). 3-CS can be readily synthesized in the laboratory following the method by Brossi et al., 1971. [175] 3-

CS is thought to be the only TIQ that readily crosses the blood brain barrier (BBB) which may be

63 because of the amino acid-like structure retained from its precursor L-DOPA. Thus, 3-CS may use the aromatic amino acid transport system to cross the BBB like L-DOPA, and allow 3-CS formed peripherally to access the central nervous system.

Figure 25. Enzymatic & non-enzymatic metabolism of CA’s and the formation of TIQs

Hypothesis:

Endogenous levels of 3-CS will be detectable in mouse brain with highest levels in dopamine enriched areas where there are the highest levels of L-DOPA.

Materials, animals, sample preparation, and analytical method:

Materials, animals, sample preparation, and analytical method were as described in Chapter 4.

Treatments:

Reagents:

1. No treatments were necessary, as this experiment is to examine the endogenous levels of

catecholamines and TIQs.

Method:

1. Weigh the mouse on a balance accurate for grams

2. Euthanize the mouse and collected the required samples on ice including plasma, urine,

liver, striatum, cerebellum, hippocampus and the rest of brain.

3. After sample collection and weighing, samples were stored in -80 ℃ freezer.

Results:

Endogenous levels of 3-carboxysalsolinol were not detected in mouse brains

3-CS levels in mouse striatum, hippocampus, and cerebellum were not detected at the limit of sensitivity of our methodology, and endogenous levels may not be present in mouse brains

(Figure 26). In contrast, salsolinol and N-methylsalsolinol were detected, with the (R)- enantiomer present at significantly higher concentrations than the (S) form.

Fig 26: Quantification of mouse brain homogenates by LC-MS/MS to determine endogenous levels of tetrahydroisoquinolines. Data are expressed as an average of five experiments in terms of ng/g wet weight of tissue. Error bars represent SEM. Legend (from left to right): 3-carboxysalsolinol, (R)-salsolinol, (S)-salsolinol, N-methyl-(R)-salsolinol, N-methyl-(S)-salsolinol. *p<0.05 (R) vs (S) #p<0.05 (R)-salsolinol vs (R) N-methyl-(R)-salsolinol

Dopamine-derived salsolinol highest in the striatum

The highest levels of salsolinol and N-methylsalsolinol (not shown) were detected in the dopamine rich striatum in mouse brains, as compared to the hippocampus and the cerebellum

(Figure 27).

Fig 27: Quantification of mouse brain homogenates by LC-MS/MS to determine endogenous levels of (R)-salsolinol in different brain regions. Data is expressed as an average of five experiments in terms of ng/g wet weight of tissue. Error bars represent SEM. Legend (from left to right): striatum, hippocampus, cerebellum, and rest of brain. *p<0.05 striatum vs hippocampus

Conclusions:

Endogenous levels of 3-carboxysalsolinol were not detected in mouse brains. In this method, we used 3-carboxysalsolinol-d2 and a multi-level calibration curve utilizing mass spectrometry for detection. This is the first method to report quantitative results for 3-CS using a drug specific deuterated internal standard. The sensitivity of this method is such that we can detect less than 1 ng/ml for 3-CS. Thus, it does not appear that 3-CS occurs endogenously in the mouse brain. In contrast, both salsolinol and N-methyl-salsolinol were detected in all three brain areas (striatum, hippocampus, and cerebellum). If 3-CS is formed in the brain at the very low acetaldehyde concentration pertaining in the absence of alcohol ingestion, it may be rapidly metabolized and/or cleared, since 3-carboxysalsolinol readily crosses the BBB.

Salsolinol and N-methylsalsolinol may have lower BBB permeability than 3-CS, although recent evidence indicates that in elevated doses, salsolinol can penetrate the CNS to some extent. Consequently, centrally formed salsolinol and N-methylsalsolinol may persist in the brain for sufficient periods to potentially cause cell damage. Post-mortem samples from alcoholic and Parkinson human brains show significantly elevated levels of salsolinol and N- methylsalsolinol in all brain regions, with the highest levels at the site of endogenous formation in dopaminergic areas (Figure 28). Moreover, higher levels of (R)-salsolinol suggests in vivo formation, and the participation of the stereo-selective salsolinol synthase. In this experiment, we detected salsolinol and N-methylsalsolinol in mouse brains with higher levels of the (R)- enantiomer (Figure 28).

Fig 28: Quantification of mouse brain homogenates by LC-MS/MS to determine endogenous levels of tetrahydroisoquinolines. Data is expressed as an average of five experiments in terms of ng/g wet weight of tissue. Error bars represent SEM. Legend (from left to right): 3-carboxysalsolinol, (R)-salsolinol, (S)-salsolinol, N-methyl-(R)-salsolinol, N-methyl-(S)-salsolinol. *p<0.05 striatum versus hippocampus

CHAPTER 6.

REGIONAL DISTRIBUTION OF 3-CARBOXYSALSOLINOL IN MOUSE BRAINS

FOLLOWING IV INJECTION

Introduction:

Carboxysalsolinol are tetrahydroisoquinolines that have a carboxylic acid group on nitrogen containing ring. 3-carboxysalsolinol is formed as a product of L-DOPA and acetaldehyde condensation (Figure 22). 3-CS is thought to be the only TIQ that readily crosses the blood brain barrier (BBB) which may be because of its amino acid-like structure from its precursor L-DOPA

(Figure 22). Thus, 3-CS may use the amino acid transport system across the BBB like L-DOPA, and allow peripherally formed or administered concentrations access to central nervous system.

In this experiment, we injected 1 mg/kg 3-CS into the tail-veins of mice. A dose of 1 mg/kg was chosen as approximating the amount formed from an intoxicating dose of alcohol.

Hypothesis:

IV injection of a 1 mg/kg dose of 3-CS will result in detectable drug in the brain

Materials, animals, sample preparation, and analytical method:

Materials, animals, sample preparation, and analytical method were as described in Chapter 4.

Treatments:

Reagent:

1. 3-carboxysalsolinol was dissolved in 1.2N HCL, and 0.8% NaH 2PO 4

Method:

1. Weigh the mouse on a balance accurate for grams

2. Each mouse was injected IV with 0.2ml of 3-CS in solution

3. Euthanize the mouse and collected the required samples on ice including plasma, urine, liver,

striatum, cerebellum, hippocampus and the rest of brain.

4. After sample collection and weighing, samples were stored in -80 ℃ freezer.

Results:

3-carboxysalsolinol detected in all brain regions

3-carboxysalsolinol penetrated all brain regions in the mouse brain after IV injection (Figure 29).

3-CS localized in the striatum at significantly higher levels versus the hippocampus and the

cerebellum.

Fig 29: Quantification of mouse brain homogenates by LC-MS/MS to determine levels of 3- carboxysalsolinol in different brain regions after an IV dose of 1 mg/kg was given. Data is expressed as an average of five experiments in terms of ng/g wet weight of tissue. Error bars represent SEM. Legend (from left to right): striatum, hippocampus, cerebellum, and rest of brain. *p<0.05 3-carboxysalsolinol vs control #p<0.05 striatum vs hippocampus

Conclusions:

3-carboxysalsolinol readily crosses the blood brain barrier. Minutes after a 1 mg/kg dose of 3-CS, detectable levels were seen in all brain regions (Figure 29). Highest levels were detected in the brain fraction labeled “rest of brain”. Regarding specific brain regions, 3-CS was elevated in the striatum.

In summary, the lack of endogenous levels of 3-CS in mouse brains was verified in this experiment, penetration of 3-CS into the brain was rapid (under 5 mins), and 3-CS was well distributed to all brain regions after IV dosing with 1 mg/kg.

CHAPTER 7.

REGIONAL DISTRIBUTION OF 3-CARBOXYSALSOLINOL & 6-METHOXY-3-

CARBOXYSALSOLINOL FOLLOWING IV DOSING OF 3-CARBOXYSALSOLINOL IN

MOUSE BRAINS AT TIME POINTS 1,6, AND 10 MINUTES

Introduction:

3-CS is thought to be the only TIQ that readily crosses the blood brain barrier (BBB) which may be because of its amino acid-like structure from its precursor L-DOPA. Thus, 3-CS may use the amino acid transport system across the BBB like L-DOPA, and allow peripherally formed concentrations to have free access the central nervous system. 3-CS being catecholamine derived, has two ring hydroxyl groups that can undergo methylation enzymatically with catecholamine O-methyl transferase (COMT) (Figure 30). Collins & Origitano determined that the 6-methyoxy (60%) metabolite is preferred versus 7-methyoxy (40%), and that this may be due to steric hindrance with the COMT enzyme. In addition, formation of the di-methoxy metabolite is negligible.

Previously, our lab determined that 3-carboxysalsolinol was not present in the brain in control mice (i.e. animals not treated with alcohol. We also determined that following IV injection of 3-CS this compound was detectable in the brain 2 minutes after the injection, but not after 60 minutes (Data not shown). The experiment described in the present chapter was performed to determine how quickly 3-CS gets into the brain, and how quickly it can be degraded by COMT to 6-methoxy-3-carboxysalsolinol (6M-3CS).

Figure 30. In vivo Methylation of 3-carboxysalsolinol by COMT

Hypothesis:

IV injection of a 1 mg/kg dose of 3-CS will result in detectable levels of 3-CS and 6M-3CS in the brain.

Materials, animals, sample preparation, and analytical method:

Materials, animals, sample preparation, and analytical method were as described in Chapter 4.

Treatments:

Reagent:

1. 3-carboxysalsolinol was dissolved in 2 ml of DIH 2O, with 9% saline solution added

Method:

1. Weigh the mouse on a balance accurate for grams

2. The half the mice were pre-treated 30 minutes prior to the 3-CS treatment

3. All mice were injected IV with 0.2ml of 3-CS in solution

4. Euthanize the mouse and collected the required samples on ice including plasma, urine, liver,

striatum, cerebellum, hippocampus and the rest of brain.

5. After sample collection and weighing, samples were stored in -80 ℃ freezer.

Results:

3-CS in mouse brain at 1 minute

3-CS given IV reached the mouse brain in under one minute (Figure 31). The principle

metabolite of 3-CS, 6M-3CS was not detected above the limits of quantification for this assay.

Levels of 3-CS in the striatum were significantly higher than levels in the hippocampus or the

cerebellum.

Fig 31: Quantification of mouse brain homogenates by LC-MS/MS to determine levels of 3- carboxysalsolinol and 6-methoxy-3-carboxysalsolinol in different brain regions after an IV dose of 1 mg/kg was given. Samples were taken 1 minute after dosing. Data is expressed as an average of five experiments in terms of ng/g wet weight of tissue. Error bars represent SEM. Legend (from left to right): striatum, hippocampus, cerebellum *p<0.05 striatum vs hippocampus

6M-3CS predominates at 6 minutes

At 6 minutes after 3-CS IV dosing, the 3-CS metabolite, 6M-3CS, was present at higher concentrations than the parent compound. Levels of 3-CS and 6M-3CS were significantly higher in the striatum than the hippocampus or the cerebellum (Figure 32). The presence of 6M-3CS in non-dopaminergic areas like the cerebellum, suggests that this metabolite might be formed peripherally and enter the brain from the blood in these regions.

Fig 32: Quantification of mouse brain homogenates by LC-MS/MS to determine levels of 3- carboxysalsolinol and 6-methoxy-3-carboxysalsolinol in different brain regions after an IV dose of 1 mg/kg was given. Samples were taken 6 minutes after dosing. Data is expressed as an average of five experiments in terms of ng/g wet weight of tissue. Error bars represent SEM. Legend (from left to right): striatum, hippocampus, cerebellum *p<0.05 striatum vs hippocampus, #p<0.05 6- methoxy-3-carboxysalsolinol vs 3-carboxysalsolinol

6M-3CS predominates at 10 minutes

At 10 minutes after 3-CS IV dosing, the 3-CS metabolite, 6M-3CS was still present in all brain regions of the mouse. However, the levels of 3-CS itself were significantly decreased. 6M-3CS levels were higher than at 6-minutes. Levels 6M-3CS are significantly higher in the striatum as compared to the hippocampus and the cerebellum at 10 mins (Figure 33).

Fig 33: Quantification of mouse brain homogenates by LC-MS/MS to determine levels of 3- carboxysalsolinol and 6-methoxy-3-carboxysalsolinol in different brain regions after an IV dose of 1 mg/kg was given. Samples were taken 10 minutes after dosing. Data is expressed as an average of five experiments in terms of ng/g wet weight of tissue. Error bars represent SEM. Legend (from left to right): striatum, hippocampus, cerebellum *p<0.05 striatum vs hippocampus

Conclusions:

3-CS given IV reached the mouse brain in under one minute (Figure 31). These findings are supported by the two-minute study that was perform in Chapter 6. In addition to verifying this work previously reported by our lab, a 10-minute time course for the occurrence and metabolism of 3-CS and its principle metabolite 6M-3CS was performed at 1, 6, and 10 minutes. At the 1- minute time point, 3-CS penetrated all brain regions tested, with higher levels in the striatum.

The 6-minute time point appears to be close to the steady state for 3-CS and 6M-3CS. In all regions of the brain, the 6M-3CS is slightly higher than 3-CS at 6-minutes.

3-CS is primarily formed in the striatum of mice since the striatum is rich in dopaminergic neurons that contain higher levels of L-DOPA. Previous studies have reported that the highest levels of TIQs in the brain are in the dopamine rich areas. Consumption of alcohol

(acetaldehyde exposure) and patients that are being treated for Parkinson’s disease (L-DOPA) have also been associated with elevated levels of TIQs in dopamine rich cells. Yet, it is interesting to note that neither acetaldehyde nor L-DOPA were administered in the present experiment, and still the highest levels of 3-CS were determined to be in the striatum. One possible explanation for this is that 3-CS may be more stable in the striatum, or it may be that transport of 3-CS from the blood is greater in dopaminergic areas.

Taken together, these results suggest that exogenously administered 3-CS moves freely into the brain and is extensively metabolized by 10-minutes by brain catecholamine O-methyl transferases (COMT). It is not known if the 10-minute brain exposure to 3-CS causes any physiological, behavioral, or pathological consequences. Nonetheless, there are significantly higher levels of 3-CS in the striatum.

In summary, 3-CS exposure has the greatest effects on the striatum, in which 3-CS is metabolized in under 10-minutes to the 6M-3CS mouse brains (Figure 34).

Fig 34: Quantification of mouse brain homogenates by LC-MS/MS to determine levels of 3- carboxysalsolinol in mouse striatum IV dose of 1 mg/kg was given. Samples were taken at 1,6, and 10 minutes after dosing. Data is expressed as an average of five experiments in terms of ng/g wet weight of tissue. Error bars represent SEM. Legend (from left to right): 3-carboxysalsolinol, 6- methyoxy-3-carboxysalsolinol *p<0.05 1 minute to 6 minutes **p<0.05 6 minutes to 10 minutes

CHAPTER 8.

EVALUATION OF THE EFFECTS OF COMT, MAO, AND AADC INHIBITORS ON

THE FORMATION OF CATECHOLAMINES, METABOLITES AND SALSOLINOL-

LIKE TETRAHYDROISOQUINOLINES

Introduction:

Ethanol metabolism can cause a constellation of neurochemical and behavioral effects (see chapter 1 for review). Briefly, the consumption of mM concentrations of ethanol, results in the formation of µM concentrations of acetaldehyde, and mM concentrations of acetate, as well as a reduced cellular environment. As mentioned, moderate doses of ethanol may be beneficial, and are often used to address anxiety and depression. Yet, heavy doses are associated with several psychiatric disorders including depression. Treatment for depression, especially when it is associated with Parkinson’s, often includes the use on monoamine oxidase inhibitors.

Monoamines, including catecholamines are essential for cellular communication and signaling pathways.

Catecholamines, including; L-DOPA, dopamine, norepinephrine and epinephrine are present in the central and peripheral nervous systems. As endogenous compounds, they are metabolized as DOPAC, VMA and HVA through catalytic reaction with COMT and/or MAO

(Figure 35). Under abnormal conditions, for example, high levels of alcohol consumption or drug-drug interactions, these endogenous compounds, especially L-DOPA and dopamine, are readily available to react with acetaldehyde to form 3-carboxysalsolinol, salsolinol, N- methylsalsolinol, 1-carboxysalsolinol, and 6-methoxy-3-carboxysalsolinol (Figure 35) which are all tetrahydroisoquinoline (TIQs) and may be neurotoxic.

Figure 35. Metabolism pathway for TIQs, catecholamines, and their metabolites

MAO and COMT inhibition results in elevated levels of catecholamines necessary for maintaining sufficient dopaminergic tone and loss of coordination seen in alcoholics and in

Parkinson’s patients alike. However, it also results it the formation, elevation, and persistence of these TIQ neurotoxins.

This study will measure levels of 3-carboxysalsolinol in striatum, cerebellum, hippocampus, and the rest-of-the-brain, using control (untreated) mice and treated mice after different combination administrations of MAO (pargyline), COMT (tolcapone), and amino acid decarboxylase inhibitor, (AADC) benserazide (Table 3).

Enzyme Substrate Inhibitors

Catalase ethanol aminotriazole

cimetidine, ethanol, acetominophen, CYP2E1 diethyldithiocarbamate, dapsone, theophylline, disulfiram

4-methyl pyrazole, ADH methanol, ethanol ranitidine

formaldehyde, ALDH disulfiram acetaldehyde

AADC L-dopa, 5-HTP, histadine benserazide , carbidopa

serotnin, epinephrine, and MAO type A resveratrol, rasagiline norepinephrine

dopamine, pargyline , Selegiline, MAO type B phenylethylamine rasagiline

dopamine,epinephrine, and COMT tolcapone norepineprine

Table 3.

Hypothesis:

The inhibition of aromatic aminoacid decarboxylase, monoamine oxidase and catecholamine O- methyl transferase will result in altered levels of metabolites of catecholamines and TIQ’s.

Materials, animals, sample preparation, and analytical method:

Materials, animals, sample preparation, and analytical method were as described in Chapter 4.

Treatments:

Reagent:

1. Heparin (1 mg/ml), 9% Saline solution, D.I. water, 5% Arabic gum (dissolved in 9% Saline

solution), 20% Cyclodextrin solution.

2. Mixture solution of drugs: Added the ethanol, tolcapone, benserazide and pargyline into the

5% Arabic gum completely and mixed them together by vortex. The final amount of each drug

in 200 µl of this mixture solution was 50 mg/kg benserazide, 50 mg/kg ethanol and 50 mg/kg

pargyline, and 30 mg/kg tolcapone.

3. L-DOPA solution: L-DOPA was dissolved in the 20% Cyclodextrin so that the final amount

of L-DOPA in 200 µl solution was 1 mg/kg for a mouse.

Method:

1. Weigh the mouse on a balance accurate for grams

2. The mouse was injected drugs mixture solution IP

3. At 15 minutes after administration of drugs mixture solution, inject the same mouse with same

volume of L-DOPA solution IV or not treatment for control.

4. The half the mice were pre-treated 30 minutes prior to the 3-CS treatment

5. All mice were injected IV with 0.2ml of 3-CS in solution

6. Euthanize the mouse and collected the required samples on ice including plasma, urine, liver,

striatum, cerebellum, hippocampus and the rest of brain.

7. After sample collection and weighing, samples were stored in -80 ℃ freezer.

Figure 36. MAO, COMT, and the benserazide inhibitors

Results:

In vivo formation of 3-carboxysalsolinol in mouse striatum

The administration of an IP dose of a 50 mg/kg ethanol and 1 mg/kg L-DOPA resulted in the in

vivo formation of 3-carboxysalsolinol. The co-administration of pargyline and tolcapone potentiated the increase in the formation of 3-CS. Treatment group three received a co-

administration of benserazide and tolcapone. Group three also had a significant elevation in the

formation of 3-CS. The group that received all three inhibitors resulted in the highest detectable

levels of 3-CS (Figure 37).

Fig 37: Quantification of mouse brain homogenates by LC-MS/MS to determine levels of 3- carboxysalsolinol in mouse striatum after an IP dose of 50 mg/kg of ethanol was given. Samples were taken at 30 minutes after dosing. Data is expressed as an average of five experiments in terms of ng/g wet weight of tissue. Error bars represent SEM. Legend (from left to right): ethanol plus L-DOPA; ethanol plus L-DOPA,pargyline,tolcapone; ethanol plus L- DOPA,benserazide,tolcapone; ethanol plus L-DOPA,pargyline,benserazide; ethanol plus L- DOPA,pargyline,tolcapone,benserazide; L-DOPA/pargyline/tolcapone *p<0.05 treatment doses versus ethanol plus L-DOPA control.

In vivo formation of HVA in mouse striatum.

The administration of an IP dose of a 50 mg/kg ethanol and 1 mg/kg L-DOPA resulted in the in vivo formation of HVA. The co-administration of pargyline and tolcapone resulting in a significant decrease in the formation of HVA. Treatment group three received a co- administration of benserazide and tolcapone. Group three also had significantly reduced levels of

HVA in the striatum. The group that received all three inhibitors, resulted in nearly total depletion of HVA in the striatum of mice (Figure 38).

Fig 38: Quantification of mouse brain homogenates by LC-MS/MS to determine levels of homovanillic acid (HVA) in mouse striatum after an IP dose of 50 mg/kg of ethanol was given. Samples were taken at 30 minutes after dosing. Data is expressed as an average of five experiments in terms of ng/g wet weight of tissue. Error bars represent SEM. Legend (from left to right): ethanol plus L-DOPA; ethanol plus L-DOPA,pargyline,tolcapone; ethanol plus L- DOPA,benserazide,tolcapone; ethanol plus L-DOPA,pargyline,benserazide; ethanol plus L- DOPA,pargyline,tolcapone,benserazide; L-DOPA/pargyline/tolcapone *p<0.05 treatment doses versus ethanol plus L-DOPA control.

In vivo formation of salsolinol in mouse striatum

The administration of an IP dose of a 50 mg/kg ethanol and 1 mg/kg L-DOPA resulted in the in vivo formation of salsolinol. The co-administration of pargyline and tolcapone resulting in a significant increase in the formation of salsolinol. Treatment group three received a co- administration of benserazide and tolcapone. Group three had attenuated levels of salsolinol in the striatum, and the group that received all three inhibitors resulted in the most significant decreases. (Figure 39).

Fig 39: Quantification of mouse brain homogenates by LC-MS/MS to determine levels of salsolinol in mouse striatum after an IP dose of 50 mg/kg of ethanol was given. Samples were taken at 30 minutes after dosing. Data is expressed as an average of five experiments in terms of ng/g wet weight of tissue. Error bars represent SEM. Legend (from left to right): ethanol plus L-DOPA; ethanol plus L-DOPA,pargyline,tolcapone; ethanol plus L-DOPA,benserazide,tolcapone; ethanol plus L-DOPA,pargyline,benserazide; ethanol plus L-DOPA,pargyline,tolcapone,benserazide; L- DOPA/pargyline/tolcapone *p<0.05 treatment of ethanol plus L-DOPA,pargyline,tolcapone versus ethanol plus L-DOPA control.

Conclusions:

The importance of maintaining proper homeostatic balance of catecholamine neurotransmitters for cellular function cannot be overstated. The catecholamine system in both the periphery as well as the central nervous system act as first responders to environmental stimuli, whether it is in the form of exposure to a drug or to a cue from our senses. Dysregulation of this system is associated with the pathology of many diseases including alcoholism, Alzheimer’s, ADHD, diabetes, and Parkinson’s disease.

In this experiment, our lab established that exposures to ethanol and L-DOPA result in the formation of 3-carboxysalsolinol. Moreover, the amount of 3-CS formed is directly associated with the presence of L-DOPA. Inhibitors affecting L-DOPA metabolism, directly and proportionally resulted in a significant increase in the formation of the tetrahydroisoquinoline, 3-

CS in the striatum of mice. These treatment schedules also resulted in the significant changes in the levels of dopamine, as is indicated by the significant decreases in metabolism of dopamine to

HVA. Treatments with L-DOPA are used clinically to treat Parkinson’s disease. While inhibiting the metabolism of L-DOPA and dopamine assures restoration of a dopaminergic tone, and mitigates some of the movement disorders, the consequences of elevated levels of neurotoxic

TIQs may be a contributing factor to the progression of the disease. 3-CS has a very low half-life in the brain (10-minutes), as shown by previous work performed in our lab. However, salsolinol, thought to be the primary neurotoxic TIQ associated with Parkinson’s, is not as readily metabolized, and may persist in dopaminergic neurons for a physiologically and perhaps pathologically relevant amount of time. In this study, the highest levels of salsolinol were the result of the combination of the inhibitors pargyline (MAO) and tolcapone (COMT) (Figure 40).

Salsolinol has been shown to be elevated in post mortem brain samples from alcoholics and well

89 as Parkinson brains. Caution is warranted in taking these inhibitors in combination with alcohol consumption.

Fig 40: Quantification of mouse brain homogenates by LC-MS/MS to determine levels of tetrahydroisoquinoline and dopamine metabolites in mouse striatum after an IP dose of 50 mg/kg of ethanol was given. Samples were taken at 30 minutes after dosing. Data is expressed as an average of five experiments in terms of ng/g wet weight of tissue. Error bars represent SEM. Legend (from left to right): 3-carboxysalsolinol, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), salsolinol, and N-methyl-salsolinol. *p<0.05 ethanol plus L-DOPA control versus ethanol plus L-DOPA,pargyline,tolcapone treatment.

CHAPTER 9.

EVALUATION OF THE EFFECTS OF ACUTE AND CHRONIC ALCOHOL

ADMINISTRATION ON CATECHOLAMINE AND SALSOLINOL-LIKE

TETRAHYDROISOQUINOLINE LEVELS IN THE MOUSE BRAIN

Introduction:

The amount of ethanol consumed, pattern (binge) of usage, and history of consumption are important factors that contribute to alcohol’s effects on the body. Small to moderate doses are defined as consuming between 1-2 drinks (32 mgs) of alcohol at a sitting. This is the amount that is considered safe and even beneficial, especially in alcoholic wines. Ethanol in small acute doses is associated with relaxation, relief of anxiety, enhancement of mood, warming sensation, release of inhibitions, and an overall sense of well-being. At the receptor level, ethanol increases neuronal activity by enhancing GABA A receptor for chloride ion channels. GABA is an inhibitory neurotransmitter, thus enhancing the inhibitory effect may be linked to the sedation seen in alcohol consumption. Enhancement of an inhibitory function is sometimes referred to disinhibition. Agonists for the GABA A receptor result in ethanol like behaviors, and antagonists attenuate the effects. Ethanol also inhibits the excitatory neurotransmitter action of glutamate at

NMDA receptors as a negative allosteric modulator, resulting in the relaxation and release of anxiety. The sense of a warming feeling may be related to the vasodilation effect that ethanol has in the periphery.

Acute alcohol has been shown to increase dopamine levels in the brain. In addition, opioid receptors have been identified as playing a role in the rewarding and reinforcing effects of ethanol, yet alcohol does not bind to the opioid receptors directly. Tetrahydroisoquinolines

(TIQs), have been shown to produce alcohol like effects, and can substitute for alcohol in

91 conditioned place preference tests. TIQs have also been shown to bind to both dopamine and opioid receptors in the brain. Acetaldehyde, and one of its byproducts, salsolinol, have been shown to have the reinforcing potential of 100 to 1000 times that of ethanol itself respectively.

[93] Moreover, some researchers have implicated salsolinol as being the key initial factor that results in an increasing voluntary consumption and the progression of an alcohol use disorder.

Salsolinol is thought to be the “first-hit” that reinforces consumption. [37] Chronic alcohol consumption results in an overall decrease in catecholamine levels in the brain.

In this experiment, we will be measuring the effects of acute (immediate) and chronic

(sub-acute) alcohol consumption on catecholamine levels, and the subsequent formation of TIQs in mouse brains. We will also explore the effects that alcohol dehydrogenase (ADH) inhibitor (4- methylpyrazole) and aldehyde dehydrogenase (ALDH) inhibitor (disulfiram), have on the catecholamine system and TIQ formation (Figure 41).

Figure 41. Catecholamine and tetrahydroisoquinoline pathways with ethanol inhibitors

Hypothesis:

Acute and chronic ethanol consumption results in disturbances in and at times the opposite effect on the catecholamine neurotransmitter system, and there is a link between these effects and the formation and accumulation of tetrahydroisoquinolines.

Treatments:

Reagent:

1. 4-methylpyrazole (50 mg/kg) in water or disulfiram (100 ng/kg) in 5% Arabic gum (dissolved

in 9% Saline solution) were given orally as a pre-treatment 1 hour prior to ethanol dosing.

2. 1 g/kg of ethanol was given orally as a 0.2 ml in a 12.5% solution

Method:

1. Weigh the mouse on a balance accurate for grams

2. At 1 hour prior to ethanol dosing, mice were given either 4-methylpyrazole or disulfiram

3. 1 hour after pretreatment, 1 g/kg of ethanol or saline control was given, for chronic

consumption, 1 g/kg was given for four consecutive days prior to testing.

4. At 30 minutes, mice were euthanized, and collection of the required samples were performed

on ice for plasma, urine, liver, striatum, cerebellum, hippocampus and the rest of brain.

5. After sample collection and weighing, samples were stored in -80 ℃ freezer.

Results:

Acute alcohol increases L-DOPA, dopamine, and DOPAC in the striatum

Acute dosing of 1 g/kg of ethanol under all treatment conditions, resulted in an increase in the

levels of L-DOPA,dopamine, and DOPAC (Figure 42). Chronic ethanol dosing showed a

reduction in striatal dopamine.

Fig 42: Quantification of mouse brain homogenates by LC-MS/MS to determine levels of catecholamines in mouse striatum after an oral dose of 1g/kg ethanol was given with and without pretreatment of 4-methylpyrazole (50 mg/kg) or disulfiram (100 mg/kg). Data is expressed as an average of five experiments in terms of ng/g wet weight of tissue. Error bars represent SEM. Legend (from left to right): L-DOPA, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA). *p<0.05 treatment versus control

Acute and chronic alcohol decreases L-DOPA in the cerebellum

Acute and chronic dosing of 1 g/kg of ethanol under all treatment conditions, resulted in a significant decreases in the levels of L-DOPA (Figure 43). Chronic, but not acute dosing, showed decreases in dopamine in the cerebellum. Interestingly, the disulfiram pre-treatment resulted in decreases in the dopamine metabolite, DOPAC, but not dopamine or HVA, a secondary metabolite of dopamine.

Fig 43: Quantification of mouse brain homogenates by LC-MS/MS to determine levels of catecholamines in mouse cerebellum after an oral dose of 1g/kg ethanol was given with and without pretreatment of 4-methylpyrazole (50 mg/kg) or disulfiram (100 mg/kg). Data is expressed as an average of five experiments in terms of ng/g wet weight of tissue. Error bars represent SEM. Legend (from left to right): L-DOPA, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA). *p<0.05 treatment versus control

Chronic alcohol increases 1-CS, 3-CS ,and ADTIQ in the Striatum

Chronic alcohol dosing of 1 g/kg for 4 days resulted in significant increases in the levels of 1-CS,

3-CS, and ADTIQ in the striatum of mice (Figure 44). Acute ethanol resulted in signficant increases in 3-CS and ADTIQ with all treatment protocols.

Levels of TIQs in mouse striatum

60 Control ** ** ** Acute Ethanol 1g/kg 40 ** * * Acute Ethanol 1g/kg w/MP ** * Acute Ethanol 1g/kg w/Disulfiram ** 20 Chronic Ethanol 1g/kg/day

0 l lino ADTIQ

xysalso bo -car 1-carboxysalsolinol 3

Fig 44: Quantification of mouse brain homogenates by LC-MS/MS to determine levels of tetrahydroisoquinolines in mouse striatum after an oral dose of 1g/kg ethanol was given with and without pretreatment of 4-methylpyrazole (50 mg/kg) or disulfiram (100 mg/kg). Data is expressed as an average of five experiments in terms of ng/g wet weight of tissue. Error bars represent SEM. Legend (from left to right): 1-carboxysalsolinol, 3-carboxysalsolinol, and 1-acetyl-6,7-dihydroxy- 1,2,3,4-tetrahydroisoquinoline (ADTIQ) *p<0.05 treatment versus control, **p<0.05 treatment versus control

Chronic alcohol increases 1-CS, 3-CS, and ADTIQ in the cerebellum

Chronic dosing of 1 g/kg of ethanol increased levels of 1-CS, 3-CS, and ADTIQ in the cerebellum (Figure 45). Acute ethanol decreases the levels of 1-CS in all treatment conditions.

Pre-treatment with disulfiram increased levels of 3-CS in the cerebellum.

Fig 45: Quantification of mouse brain homogenates by LC-MS/MS to determine levels of tetrahydroisoquinolines in mouse cerebellum after an oral dose of 1g/kg ethanol was given with and without pretreatment of 4-methylpyrazole (50 mg/kg) or disulfiram (100 mg/kg). Data is expressed as an average of five experiments in terms of ng/g wet weight of tissue. Error bars represent SEM. Legend (from left to right): 1-carboxysalsolinol, 3-carboxysalsolinol, and 1-acetyl- 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (ADTIQ) *p<0.05 treatment versus control, **p<0.05 treatment versus control

Disulfiram pre-treatment results in increases in salsolinol and N-Methyl-salsolinol in the striatum

Acute ethanol dosing with disulfiram pre-treatment resulted in significant increases in salsolinol and N-methyl-salsolinol (Figure 46). Acute alcohol without pre-treatments, resulted in increases in salsolinol but not N-methyl-salsolinol. Chronic alcohol dosing also resulted in increases of salsolinol but not N-methyl-salsolinol in the striatum.

Fig 46: Quantification of mouse brain homogenates by LC-MS/MS to determine levels of salsolinol and N-methyl-salsolinol in mouse striatum after an oral dose of 1g/kg ethanol was given with and without pretreatment of 4-methylpyrazole (50 mg/kg) or disulfiram (100 mg/kg). Data is expressed as an average of five experiments in terms of ng/g wet weight of tissue. Error bars represent SEM. Legend (from left to right): salsolinol, N-methyl-salsolinol *p<0.05 treatment versus control, *p<0.05 acute treatment versus acute treatment with disulfiram, **p<0.01 treatment versus control

Disulfiram pretreatment results in increases in salsolinol & N-Methyl-salsolinol in the cerebellum

Acute ethanol dosing with disulfiram pretreatment resulted in significant increases in salsolinol and N-methyl-salsolinol (Figure 47). Acute alcohol without pretreatments, resulted in decreases in salsolinol and N-methyl-salsolinol in the striatum. Chronic alcohol dosing resulted in the attentuation of salsolinol but not N-methyl-salsolinol in the cerebellum.

l ino linol lso a Salsol thyl-s e -m N

Fig 47: Quantification of mouse brain homogenates by LC-MS/MS to determine levels of salsolinol and N-methylsalsolinol in mouse cerebellum after an oral dose of 1g/kg ethanol was given with and without pretreatment of 4-methylpyrazole (50 mg/kg) or disulfiram (100 mg/kg). Data is expressed as an average of five experiments in terms of ng/g wet weight of tissue. Error bars represent SEM. Legend (from left to right): salsolinol, N-methyl-salsolinol *p<0.05 treatment versus control, **p<0.05 treatment versus control

Conclusions:

Brain concentrations of catecholamines and tetrahydroisoquinolines were altered after administration of ethanol. In this experiment, an acute heavy dose of 1 g/kg was given to mice orally. The catecholamine response was an increase in L-DOPA, dopamine and DOPAC in the striatum. Similar responses were not seen in the cerebellum, where there is very little dopamine in this brain region. Acute and repeated (4 daily doses) alcohol dosing also had pronounced effects on the formation of TIQs. Acute doses resulted in elevated levels of 3-CS, ADTIQ, salsolinol, and N- methyl-salsolinol. The repeated doses had a similar effect, elevating all TIQs tested (1-CS, 3-CS, ADTIQ, and salsolinol) except for N-methyl-salsolinol. 1-CS levels were elevated after repeated dosing, but not with an acute dose. 1-CS is formed from the reaction of dopamine and pyruvate. It is reasonable to assume that the elevated dopamine levels were counterbalanced with depleted pyruvate levels in the acute exposure. Yet, with repeated exposure, levels of 1-CS were elevated. This is consistent with human data from alcoholics that have elevated levels of 1-CS with chronic consumption. Disulfiram pretreatment most profoundly affected the formation of TIQs in both the striatum and the cerebellum. Disulfiram inhibits the oxidative metabolism of acetaldehyde to acetate. It is also known to be a promiscuous and non-selective inhibitor with pathological consequences of its own. Taken together, it is not surprising that with the burden of elevated levels of acetaldehyde and disulfiram, pathological consequences will ensue. For the purposes of this discussion, some of those pathological consequences may be associated with the dramatic elevation of TIQs in the central nervous system as evidenced in this experiment. In summary, the neurochemical response to an acute versus a chronic exposure to ethanol can be assessed at the cellular level by measuring catecholamines and tetrahydroisoquinolines. Individuals who are treated with drugs that have a disulfiram-like effect, and persons with a

100 genetic disability to metabolize acetaldehyde, as well as persons that consume too much alcohol are particularly at risk to exposures to neurotoxic TIQs in the brain. CHAPTER 10

Concluding remarks:

Some compounds taken for medicinal or recreational reasons exert their effects, or some of their effects, via “active metabolites”. Classical examples are the analgesic codeine, which is a prodrug for its O-desmethyl metabolite, morphine, and the psychodelic psilocybin, which is dephosphorylated to the active compound psilocin. The discovery that the tetrahydroisoquinoline compound, salsolinol, could be formed in vivo by condensation of ethanol-derived acetaldehyde with the neurotransmitter dopamine raised the question of whether some effects of alcoholic beverages could be due to salsolinol. This was clearly a notion worth exploring for two reasons. First, salsolinol’s structure is reminiscent of “vegetable alkaloids”, many of which (e.g. morphine, cocaine, physostigmine, atropine, nicotine….) have pronounced pharmacological and/or toxicological effects. Secondly, although only a small fraction of ingested alcohol might be converted to salsolinol, the amounts of ethanol consumed by an individual in a single drinking session is enormous compared to that of other recreational drugs.

A liter of a strong beer might contain 46g of ethanol, or one mole. In contrast, other recreational drugs are consumed in millimolar quantities or less. Thus, conversion of 0.1% or less of a dose of alcohol to a potent active metabolite might indeed be a factor in explaining some of alcohol’s many psychoactive actions, which include mild euphoria and anxiolytics in the early stages of a drinking session, and the severe dysphoria of a hangover several hours after a heavy drinking session, which can also be associated with disturbances of sleep architecture, antisocial behaviors, loss of motor coordination and loss of consciousness. Chronic heavy drinking can lead to dependence and addiction, and to withdrawal-associated hallucinations, as well as

101 neurodegeneration. During the 40+ years since the discovery of the in vivo production of salsolinol, increasing evidence suggests that many of the effects of alcohol consumption maybe from salsolinol directly. Nevertheless, it remains possible that metabolites of alcohol are involved in some aspects of its psychoactivity and/or toxicity. Such metabolites include the proximal oxidative product acetaldehyde, and its oxidative product, acetate, which may disrupt cerebral energy metabolism by acting as a metabolic fuel bypassing controls on glucose utilization. Non-oxidative metabolites, such as ethyl esters of long chain fatty acids, may also be important.

The present studies began with the idea of a re-evaluation of in vivo formation of 3- carboxy salsolinol, formed from acetaldehyde plus L-DOPA, since several early papers had indicated that this compound had behavioral and reinforcing activity. This work required the development and validation of liquid chromatography-mass spectrometric method to detect to simultaneously determine brain concentrations of catecholamines, tetrahydroisoquinolines and their metabolites (see Chapter 4). Improvements to analytical instrumentation as well as separation chemistry has been tremendous in the past several decades since researchers became interested in TIQs. LC-MS/MS has revolutionized several fields of study, including the measurement of catecholamines to diagnosis certain cancers.

We used our LC-MS/MS method to show that while 3-carboxy salsolinol was undetectable in brains of control (untreated) animals, it was present in brains of mice given ethanol plus L-DOPA. Unlike the case for 3-CS, levels of salsolinol and N-methyl-salsolinol were detected in the striatum of mice. Salsolinol levels were higher in the striatum than in the hippocampus or cerebellum. Acute dosing of ethanol results in an increase of L-DOPA and dopamine in the striatum, which is potentiated using MAO and COMT inhibitors. Chronic

102 dosing of ethanol results in the formation of salsolinol, 3-carboxysalsolinol, 1-carboxysalsolinol, and ADTIQ in the striatum, which are potentiated by a pretreatment with disulfiram, an inhibitor of acetaldehyde metabolism. The inhibition of L-DOPA metabolism resulted in the increase in formation of 3-carboxysalsolinol, but prevented the formation of dopamine metabolites, salsolinol and n-methylsalsolinol. Lastly, we found that 3-carboxysalsolinol localizes in the striatum and is metabolized within 10 minutes after administration. Pharmacological inhibition of L-DOPA metabolism resulted in an increased brain content of 3-carboxysalsolinol, but prevented the increase in the concentrations of the dopamine metabolites, salsolinol and N- methylsalsolinol.

While L-DOPA treatments alleviate the symptoms of Parkinson’s disease, this drug tends to lose its effectiveness with time as the number of dopamine secreting cells in the midbrain continues to decrease. It is possible that this decline is in part due to TIQ-related neurotoxicity.

Since acetaldehyde is known to be a toxic compound, alcohol consumption by individuals with

Parkinson’s disease should probably be discouraged as it increases exposure to both TIQ’s and acetaldehyde. Consistent with expectations, we found that the formation of TIQs as a result of chronic ethanol administration was potentiated by the administration of the acetaldehyde dehydrogenase inhibitor, disulfiram, which is sometimes used as “aversion therapy” to discourage alcohol consumption. Thus, in addition to exposure to alcohol and elevated levels of acetaldehyde, L-DOPA treatment may also subject individuals to potentially neurotoxic TIQs.

Future studies:

Many questions remain unanswered, including:

-1. The exposure of brain regions to the several known TIQ’s over a range of alcohol doses, and

103 whether some TIQ’s are unable to cross cell membranes and so accumulate in brain cells over long periods, causing progressive toxicity. Thorough animal PK studies are warranted.

-2. Whether concentrations of TIQ’s achieved in the brain after alcohol ingestion are toxic to brain cells. Performing toxicological tissue-culture studies using immortalized cell lines and primary mid-brain cell cultures would be one approach to investigating this area.

104

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