MODULATION OF THE METABOLISM AND

CYTOTOXICITY OF FORMALDEHYDE

IN ISOLATED RAT HEPATOCYTES

Shirley Hsueh Li Teng

A thesis submitted in confomiity with the requirements For the degree of Master of Science

Graduate Department of Pharmaceutical Sciences University of Toronto

0 Copyright by Shirley Hsueh Li Teng 2001

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ABSTRACT --

Modulation of the Metabolism and Cytotoxicity of Formaldehyde in Isolated Rat Hepatoc ytes

Shirley Hsueh Li Teng MSc, August 2001 Department of Phannaceutical Sciences Faculty of Pharmacy, University of Toronto

HCHO was toxic to isolated rat hepatocytes in a dose- and time-dependent manner by

targeting mitochondria and causing oxidative stress (GSH depletion, ROS formation,

lipid peroxidation). A decrease in HCHO metabolisrn caused by inhibiting the

metabolising enzymes alcohol dehydrogenase, aldehyde dehydrogenase or formaldehyde

dehydrogenase correlated with increased cytotoxicity. C W 2E l -catalysed HCHO

metabolism was greater in CYP 2E 1 -induced rat liver microsomes venus non-induced or

CYP 2E1 inhibited microsomes. Inhibition of CYP 2E1 prevented HCHO metabolism

and formate production by hepatocytes and increased cytotoxicity. Removal of HCHO by

increasing enzymatic rnetabolism with NADH generators or oxidisers or by trapping with

amines or thiols decreased cytotoxicity. However trapping HCHO with catecholamines

resulted in Uicreased cytotoxicity. This stuây suggests that persons who are deficient in

HCHO-metabolishg enzymes may be more susceptible to HCHO toxicity. Amines/thiols

may be used as antidotes for HCHO poisoning, however the mechanism of toxicity of the

reaction with catecholamines needs to be fiuther examined.

ACKNOWLEDGEMENTS

"nere hr no such thing as an unossisted goal. "

Here 1 go, talking about hockey again. Seriously, though, that was something 1 heard

in a ment commercial and it really made me think about al1 of those who contributed in one

way or another in helping me reach my goal: an MSc. What 1 have done towards

accomplishing this goal huly could not have been possible without the assistance of so many.

1 am grateful to you dl. So here goes.. .

Dr. Peter O'Brien. Thank you so much for so many things. For putting me on this

project in the first place. For keeping an open mind, which cnabled this research to expand in

so many directions. For providing invaluable guidance thughout. The weight that you

c k e d in seeing this project through cannot be stressed enougb.

Dr. Peter Pennefather and Dr. Manuela Neuman. Thank you both for your input, your

criticisms, your comments. You helped me focus and get on the right track. You helped me

strengthen the weahesses of my work and pointed me in the right direction. Your expertise

helped me to better understand my work.

Dr. Raymond Pwn. Thank you for initiating this project and for bringing in the

finances h m Heaith Canada needed to cany it through. Your assistance with the formate

d y s i s is also greatly appreciated, as is your input on interpretation of the data. Dealing

with the govemment was quite palliless with you as the link!

Mom, Dad, Pat. Thanks for being there. Th& for feigning interest while 1 babbled

endlessly about work (blah, blah, blah), for listening while 1 complained, for teaching me to

face my problems h a n and not to back d o m when things got rough.

iii

The members of the O'Brien lab. So many of you have corne and gone but your

contributions remain. Thanks for helping me with techniques and theories, especially Kristin

for assisting with HPLC and Majid for synthesising compounds and for assisting me with

mass spec. Thanks to everyone for making me laugh (although the Mooseheads were also a

big heip) and for helping me keep my (in)sanity. 1 am honoured to share a spot on the Wall of

Shame with you!

And finally, Tom. Thank you for everyihing. 1 honestly don't h o w how 1 would've

gotten through any of this without you.

ACKNOWLEDGEMENT OF FINANCIAL SUPPORT

This study was conducted as a joint effort with the Bureau of Chemical Hazards at

Healtb Canada in Ottawa, and financial support was provided by Health Canada. The

majority of the experimentai work was donc at the Faculty of Pharmacy, University of

Toronto, 19 Russell Street, Toronto, Ontario, Canada The formic acid analysis was done at

Health Canada, Tunney's Pasture, Ottawa, Ontario, Canada.

Shirley Teng was supported by a University of Toronto Open Fellowship and the Ben

Cohen Bursary Fund.

LIST OF PUBLICATIONS AND ABSTRACTS

Publication:

Teng, S., Beard, K., Pourahmad, J., Moridani, M., Easson, E., Poon, R. and O'Brien, P.J. (201). The formaldehyde metabolic detoxifîcation enzyme systmis and molecular c ytotoxic mechanism in isolated rat hepatoc ytes. Chem. Biol. intetac. 1 30- 1 32,285-96.

Reprinted with permission h m Elsevier Science.

Abstracts:

Teng, S., Beard, K., Pourahrnad, J., Mondani, M., Easson, E., Poon, R. and O'Brien, P.J. The fomaldehyde molecular cytotoxic mechanisms: modulation by inhibiting metabolizing enzymes. Presented at the Enzymology and Molecular Biology of Carbonyl Metabolism loth International Meeting. Taos, NM, July 14,2000.

Teng, S., Poon, R., Easson, E. and O'Brien, P.J. Involvement of CYP 2E1 in fomaldehyde/methanol metabolism and cytotoxicity. Presented at the American College of Toxicology Meeting. San Diego, CA, November 12- 15,2000.

Table 3.1

Table 3.2

Table 3.3

Table 3.4

Table 4.1

Table 4.2

Table 4.3

Table 4.4

Table 5.1

Table 5.2

Table 5.3

Table 5.4

Table 6.1

Modulation of HCHO-induced lipid peroxidation and cytotoxicity by inhibitors of the HCHO metabolising enzyme systems

Effect of aldehyde or alcohol metabotising enzyme inhibitors on HCHO metabolism in isolated hepatocytes versus HCHO cytotoxicity

Effect of HCHO on mitochondnal respiration and membrane potential in isolated hepatocytes

Oxidative stress induced by HCHO as indicated by ROS formation and GSH depletion in isolated hepatocytes

HCHO metabolism by insect ce11 control SUPERSOMES~, rat liver microsomes, CYP 2El (pyrazo1e)-induced rat liver microsomes and human CYP 2E1 + P450 reductase + cytochrome b5 SUPERSOMES"

HCHO metabolism is inhibited in CYP 2E1- and P450 reductase- inhibited hepatocytes, which correlates with the inhibition of formate production

HCHO-induced cytotoxicity increases when CYP 2E1 or P450 reductase are inhibited in isolat4 rat hepatocytes

ROS generation fiom microsomes and NADPH with HCHO versus ethanol as substrates

The effect of NADH generators or NADH oxidisers on HCHO-induced cytotoxiciy and lipid peroxidation and HCHO metabolism

HCHO production from methanol with glycolate and its modulation

Effect of replacing the hepatocyte incubation buffer on HCHO cyto- toxiciiy

The disappearance of HCHO upon the addition of L-cysteine, D-penicill- amine, aminoguanidine, metforniin, hydroxylamine or L-cystine to hepatocytes and the associated cytotoxic effects

The metabolism of HCHO by catecholarnines and the associated

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82 cytotoxic effects

vii

Figure 1.1

Figure 1.2

Figure 1.3

Figure 1.4

Figure 3.1

Figure 5.1

Figure 5.2

Figure 5.3

Figure 6.1

Figure 6.2

Figure 6.3a

Figure 6.3b

Figure 6.4

Figure 7.1

Figure 7.2

The enzymatic pathways of formaldehyde metabolism

The non-enzymatic metabolism of formaldehyde

The metabolism of formate to CO2 and the mle of folate in formate and formaldeh yde metabolism

The role of mitochondria in ce11 death

HCHO cytotoxicity in isolated rat hepatocytes is dose- and time- dependent

Spectrophotometric cornparison of thiazolidine-4-carboxylic acid (TC) versus HCHO + L-cysteine

The disappearance of HCHO in the presence of amino and thiol compounds

Structures of the amines and thiols used and the proposed products formed by their reactions with HCHO

The chemical metabolism of HCHO by catecholamines

Spectrophotometric cornparison of NMNS versus HCHO + deoxyepi- nephrine

MSMS profile of NMNS

MSMS profile of the reaction of HCHO and deoxyepinephrine with hepatoc ytes after incubation for 15'

MS/MS profile of the reaction betwecn HCHO and deoxyepinephrine in 0.1M phosphate buffer, pH 7.4 d e r incubation for 15'

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73

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84

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88

Stnictures of the catecholamines and THIQs used in this study and MPTP 92

The pmposed c ytoioxic mechanism of formaldehyde 102

Ovdl scheme of the metabolic pathways of HCHO and their effects 107 on HCHO-induced cytotoxicity

viii

SUMMARY OF APPENDICES

Page

Appendix 3.1 HCHO inhibits hepatocytes respiration which is reversai by 45 the addition of L-cysteine

Appendix 3.2 HCHO metabolism in isolated rat hepatocytes 46

Appendix 3.3 Inactivation of glutathione reductase by HCHO + NADPH 47

Appendix 6.1 MS of hepatocyte incubation buffer treated with HCHO and 95 deoxyepinephrine for 15 minutes

ADH1 ADH3 ALDH2 ATP BDL BHT CO2 COMT CYP 1A2 CYP 2E1 CYP 4A2 CYP 4A11 DCF DCPP DEDC DMSO DNA DNFB EDTA GR GSH GSSG Hz02 HCHO HEPES HPLC IL- la a - i p IL-6 m i.p. LD50 MAO MeDEDC MPT MPTP MPP+ MS MS/MS NAD+ NADH NADP+

Alcohol dehydrogenase Formaldehyde dehydrogenase Aldehyde dehydrogenase Adenosine 5 '-triphosphate Beyond detaction lirnit (of the assay) Butylated hydroxy toluene Carbon dioxide Catechol-O-methyl transferase Cytochrome P450 1A2 Cytochrome P450 2E1 Cytochrome P450 4A2 Cytochrome P450 4A11 2', 7'-Dichlorofiuorescin diacetate 2.6-Dichlorophenolindophenol Diethyldithiocarbamate Dimethylsul foxide Deoxyribonucleic acid 2,4-Dinitrofluorobaene Ethylene-diaminetetraacetic acid Glutathione reductase Glutathione (reduced fom) Glutathione (oxidised fomi) Hydrogen peroxide Formaldehyde N-(2-hydroxyethyl)piperazine-N'-(2-ethane~ulfonic acid) High performance liquid chromatography Interleukin- 1 alpha Interleukin- 1 beta Interleukin-6 p-Iodonitrotetrazolimn viola htraperitoneal Lethal dose 50% Monoamine oxidase Methyl diethyldithiocarbamate Mitochondnal pemeability transition 1-Methyl4phenyl- l,2,3,6-tetrahydropyridine 1 -Methyb4-p yridinium ion Mass spectra W u c t ion mass spectra Nicotinamide adenine dinucleotide (oxidised fom) Nicotinamide ad& dinuckotide (reduced fom) Nicotinamide adenine dinucleotide phosphate (oxidised fom)

NADPH NLrKB NMNS 02*- OH' P45qs) PMS ROS S.E. SSAO TBA TBARS TC TCA THF THIQ(s) TNF-a 'hi

Nicotinamide adenine dinucleotide phosphate (reduced form) Nuciear factor kappa B N-methylnorsalsolinol Superoxide radical Hydroxyl radical Cytochrome P450(s) Phenazine methosulfate Reactive oxygen species Standard Error Semicarbazide-sensitive amine oxidase 2-Thiobarbituric acid Thiobarbihiric acid reactive substances Thiazolidine~arboxylic acid Trichioroacetic acid Tetrahydrofolic acid Teüahydroisoquinoline(s) Turnor necrosis factor alpha Mitochondrial membrane potential

Abstract

Acknowledgements

Acknowledgement of financial support

List of publications

Summary of tables

Summary of figures

Summary of appendices

List of abbreviations used

Table of contents

Chapter 1 - General Introduction

Chapter 2 - Materials and Methods

Chapter 3 - The Formaldehyde Metabolic Detoxification Enzyme Systems and Molecular Cytotoxic Mechanism in Isolated Rat Hepatocytes

Chapter 4 - The Metabolisrn of Formaldehyde by CYP 2E1 and the Associated Cytotoxic Consequences in Isolated Rat Hepatocytes

Chapter 5 - Antidotes to Formaldehyde Cytotoxicity: Amino-Thiols or Modulating Celluiar Redox f otential

Chapter 6 - The Effect of Catecholamines on Formaldehyde Metabolism and Cyto- toxicity in Isolated Rat Hepatocytes

Chapter 7 - Summary, Final Conclusions and Future Perspectives

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GENERAL INTRODUCTION

1.1 Human exposure to formaldehyde

Formaldehyde (HCHO) is a compound that is found throughout the environment. In

the 1980s. as much as 9 billion pounds of HCHO were manufachued in the United States per

year (Ma and Harris, 1988). It is present in many building materials including paint,

plywood, foam insulation and particle board, and is also a component of leather, adhesives,

cosmetics, and disinfectants to name only a few commonly-used household items (Conaway

et al., 1996). The out-gasing of HCHO from construction materials has been suggested to be

associatcd with the onset of the symptoms of "sick building syndrome", which includes skin,

eye and upper respiratory tract imtation and headaches (Letz, 1990). HCHO is also

comonly used as a preservation agent for biological tissues; thus, exposure levels are

increased for histologists and pathologists. HCHO is a component of detergents and many

cleaning agents because of its antimicrobial action, thus members of cleaning staffs

fiequently come into contact with HCHO. Finally, HCHO is a component of root canal

filling sealen, thus exposure to HCHO occurs for dentists and their assistants as well as their

patients. The workers most exposed to HCHO are those employed in the manufacture of

plywood and particle board (Conaway et ai., 1996 ). Serious health pmblems have been

linked to the use of ma-HCHO foam insulation in U.S. houses, resultiag in the banning of

its use in the 1970s (Ma and Harris, 1988). HCHO is dso a component of cigarette smoke

and photochernical smog and is produced during the combustion of hydrocarbons such as

gasohe, rendering it to be a prevaient environmental poliutant (Conaway et al., 1996).

Finally, HCHO is found naturally in some foods including meat, fish, fhits, sofl drinks and

beer (Boeniger, 1 987; Restani and Galli, 1 99 1 ; Trezl et al.. 1 998).

Human exposure to HCHO stems not only directly h m environmental sources, but

also h m the metabolism of xenobiotics. For instance, HCHO is produced during N-

dealkylation reactions catalysed by cytochrome P450s (Waydhas et al., 1 W8), the hydiol ysis

of aspartame (Trocho et al., 1998) and the oxidation of methanol. With the possible

introduction of methanol as a component of gasoline in the future, the general population

would be exposed regularly to a HCHO-generating source in addition to present HCHO

emissions in the environment. Thus researching the health risks associated with HCHO

exposure is important. HCHO may also contribute to the cytotoxic mechanism and the DNA

cross-linking induced by the antitumor drup daunorubich and doxorubicin. The metabolism

of these dmgs leads to the production of HCHO, which then re-associates with the dnig to

fonn a complex capable of cross-linking DNA (Kato et al., 2000). As well, the

carcinogenicity of dichloromethane requires its dehalogenation to HCHO through a pathway

involving glutathione-S-ûansferase (Casanova et al., 1997). HCHO is also produced

physiologically h m the metabolism of epinephrine, creatinine and sarcosine via their

comrnon metabolite methylamine (Yu and Zuo, 1993). It is important physiologically as a

carbon source in contributhg to biological methylation by folic acid for the synthesis and

repair of DNA. Heck et al (1985) found 2.61 pg igd 2.24 HCHO per gram of venous

blood in unexposed humaas and rats respectively, hirther emphasising the importaace of

endogenous HCHO as a contributor to physiological metaboiism.

HCHO is a very volatile compound; thus, in the majority of cases, c h m ~ c or acute

exposure to HCHO occurs through respiration. However, because it is such a small

compound, HCHO is also readily absorbeà through the skin. In addition, cases of HCHO

ingestion bave also been documenteci.

1.2 The metabolism of formaldehyde

HCHO is metabolised enzymatically by three prirnary pathways, as shown in Figure

1.1. HCHO cm be reduced to methanol by the cytosolic alcohol dehyhgenase (ADHI) or

oxidised to formic acid by mitochondnal aldehyde dehydrogenase (ALDH2) or the cytosolic

GSH-dependent formaldehyde dehydrogenase (ADH3). These abbreviations are based on the

suggested nomenclature for their respective genetic enzyme classes.

Other routes of enzymatic HCHO metabolism such as by catalase or aldehyde

reductase have also been suggested to occur, although their involvement in mammalian

HCHO metabolism have not been detemllned. A fomaldehyde dismutase was found in the

bactena Pseudomonas pufida and StuphyIocococncr aureus (Mason and Sanders, 1989; Kato et

al., 1986). Abeles and Lee (1960) showed HCHO dismutation to formic acid by home liver

alcohol dehydrogenase, whereas Svensson et al. (1996) showed that butanal is dismutated by

human liver alcohol dehydrogenase to butanol and butyric acid. As of yet, this aldehyde

dismutase activity of alcohol dehydrogenase has not been dernonstrated in vivo or in cells.

ADH1, ALDH2 and ADH3 are al1 present in abundance in the liver, although their

distribution is wide and includes the kidney, stomach, brain and erythrocytes. ADH3 in

particula. is present in high levels in crythrocytes which may explain why a plasma half-life

of only l .S minutes was found upon intravenous injection of HCHO into monkeys

(McMartin et al., 1979). The reduction of HCHO by ADHI is dependent on NADH, and

HCHO oxidation by ALDH2 and ADH3 are both dependent on NAD' as a coenzyme. Thus

the iedox state of the cell may have an effect on the pathway of metabolism and the extent of

HCHO metabolism by these pathways. The activity of ADH3 is also dependent on the

presmce of GSH although GSH is not consumed during the reaction; instead, GSH is

regenerated at the end of the catalytic mechanism. HCHO condenses with GSH to form S-

hyâroxymethylglutathione, which can ie~ an alcohol functional group. S-

hydroxyrnethylglutahione is in fact the true substrate for oxidation by ADH3 to S-

fonnylglutathione, which may explain why ADH3 was genetically classified as an alcohol

dehydrogenase (Koivusalo et al., 1989). S-Fomylglutathione is hydrolysed by S-

formylglutathione hydrolase to fom fomic acid with the release of GSH. ADH 1 catalyses

bodi the oxidation of methanol to HCHO and the reduction of HCHO back to rnethanol.

Pocker and Hong (1 990) showed that kinetically, ADHl favoun the reduction of HCHO over

the oxidation of methanol. Thus, the reduction of HCHO is an important route of

metabolism.

--A

Figure 1.1 The enzymatic pathways of formaldehyde metabolism.

FORMALDEHYDE

H3C-OH METHANOL

OH

S-G FORME AClD Shydroxymethyîglutathione

Fdate-dependent NAD* pathway

GSH

NADH

hydrdase

H S-G

HCHO is the simplest of aldehydes in t m s of its structure. The carbon atom of its

carbonyl group is electrophilic due to the electronegative nature of the carbonyl's oxygen

atom. nius, HCHO is very reactive towards nucleophilic campounds including thiols and

amines such as the sulfhydryl amino acid c ysteine, GSH and the nucleic acids of DNA.

HCHO is also lmown to cause cross-linking in nucleohistones (Bnitlag et al., 1969). Its

reactivity with cellular macromolecules can cause immense protein, DNA or protein-DNA

cross-linking. The use of HCHO as a biological preservative is based on its ability to cross-

link proteins to prevent h m h m breaking dom. The reactivity of HCHO with DNA leads

to the formation of DNA-HCHO adducts which results in carcinogenesis.

In studies of the fate of injected radiolabeled HCHO in rats, some of the radioactivity

was detected in the urine. The radioactivity was incorporated in metabolites other than

formate, indicating that other pathways of HCHO metabolism exist. Two of the most

prevalent metabolites were that of a HCHO-cysteine condensation product, chiazolidine-4-

carboxylic acid (Hemminki, 1 984) and hydrox ymethyl adducts of urea (Mashford and Jones,

1982). This reinforces the very reactive nature of HCHO with endogenous amines and thiols.

It has been suggested that the importance of ADH3 lies not only in its role as a HCHO-

detoxification enzyme, but that it also prevents the depletion of GSH following the formation

of S-hydroxyrnethylglutathione îiom its reaction with HCHO (Uotila and Koivusalo, 1989)

since S-hydroxymethyiglutathione eflluxes h m hepatocytes, thereby depleting the cell of

GSH (Ku and Billings, 1984). The electrophilic nature of HCHO also extends to its reactions

with hydrazine or hydrazine denvatives. Exposure to hydrazine results in the formation of

formaldehyde hydrazone, which when activateci by catalase becomes carcinogenic as it

methylates guanine in DNA (Lambert and Shanlc, 1988). HCHO is also known to react in

vitro with endogenous amines such as catecholarnines to fonn isoquinoline denvatives such d- -

as tetrahydroisoquino1ines (THIQs). THIQs have been suggested to be involved in the

etiology of neurological disorders such as Parkinson's Disease because of their structural

similady to 1-methyl-4-phenyl- l,2,3,6-tetrahydropyridine (MPTP), a well-known inducer of

Parkinsonism (McNaught et al., 1998) (Figure 1.2).

Figure 1.2 The non-enzymatic metabolism of fonnaldehyde.

GSH s

HCHO

H K H

O II H

A, ,N. thiazolidine-4-carbox ylic acid HO C CH, H 1 H ~ C - ~

isoquinolines e - ~ H"JJ'"'J HO CH3

K O N-h ydrox ymethylurea, N,N'-bis(hydroxymeth yi)urea HO-N NH2 HO H

H H H

5,10-methylene tetrahydrofolate

protein-NH-CH,OH ,

1.3 The toxicity of formaldehyde

13.1 The systemic toxicity of fornaldehyde and its invoivewnt in diseases

HCHO is known to be a very reactive and toxic compound. Acute exposure to HCHO

often causes irritation primarily near the area of exposure such as the respiratory tract or the

skin. Its toxicity has been mainly attribut4 to the electrophilic nature of its carbonyl group

leading to adduct formation with proteins and DNA. This results in protein denaturation or

inactivation, the onset of genetic mutations or immune responses.

The carcinogenicity of HCHO is well-known, especially in the nasal passage and

respiratory tract where most initial contact with HCHO occurs. It was first reported by

Swenberg et al. in 1980 that exposure to 14.3 ppm HCHO by inhalation over 24 months

induced squamous ceIl carcinomas in the nasal cavity of 60% of treated rats.

However, Heck et al. (1983) found that 22% of inhaled radiolabeled HCHO in rats

was deposited in the unne and feces. This suggests that HCHO that is inhaleà can reach

tissues and organs distant fiom the site of exposure. Indeed, HCHO has also been suggesteà

to be involved in systemic toxicity. Strubelt et al. (1990) showed that HCHO is a cardiotoxin

in that it caused a decrease in blood pressure, heart rate and cardiac output once infbsed in

rats.

The teratogenicity of HCHO has also been studied. Katakura et al. (1993)

demonstrated that exposure to HCHO by injection into pregnant mice resulted in HCHO

accumulation in fetal liver, brain and DNA. Saillenfait et al. (1989) showed that matemal

inhalation of HCHO in rats caused a reduction in fetal body weight, but other teratogenic

effects (e.g. physical malfonnations) were not observecl.

In ment years, some attention has been paid to the possible d e of HCHO in the

development of neurological disorders. A mal1 amount of inhaleci radioactive HCHO was

detected in the brains of rats, suggesting that HCHO can cross the blood brin barrier (Heck

et al., 1983). Kilburn (1994) linked several cases of neurobehavioural impairnent with

chronic occupational exposure to HCHO or acute, high dose exposure fkom industrial

accidents. In each case, subjects had decreased motor ability, impaired cognitive hct ions

and changes in behaviour (e.g. increased irritability). In another study, rats exposed to HCHO

by inhalation were less able to perfoxm behavioural exercises than their control counterparts

(Pitten et al., 2000). Many cases of methanol poisoning have resulted in motor dysfunction

(Guggenheim et al., 197 1 ; McLean et al., 1980; Anderson et al., 1989) including one

documented case of Parkinsonism (Oliveras-Ley and Gali, 1983) although these studies did

not determine whether the effects were the result of methanol itself or its metabolites such as

HCHO or formate. Despite these many indications that exposure to HCHO is linked to

neurological impairment, the mechanism of neurotoxicity has not been elucidated.

Finally, hepatotoxicity has also been associated with exposure to HCHO. It was

reported that hepatic GSH levels decreased in guinea pigs following the inhalation of HCHO

(Mecler, 1978). Inhalation of HCHO in rats was also reported to cause morphological

changes and ce11 death in the liver (Feldman and Bonashevskaya, 197 1) and increased

alkaline phosphatase activity (Murphy et al., 1964). HCHO-induced GSH depletion was also

reported in the liver, kidney, lung and brain of rats injected with a sub-lethal dose of HCHO

(Faroqui et al., 1986) and in isolated/perfused rat liver and lung (Ayres et al., 1 985).

However, the toxicity of low levels of HCHO has been largely overlooked because

HCHO is rapidly metabolised and removed h m the body. Because of the many routes

through which HCHO can be detoxified as discussed previously, it is generally believed that

low levels of HCHO are not present long enough to exert sigificant toxic effects. Instead,

the toxicity of methanol, for exarnple, has been pnmarily attributed to the formation and

accumulation of fonnic acid (Tephly, 1991). Fonnic acid causes metabolic acidosis and

targets the optic nerve, therefore causing blindness in humans and other primates. These

symptoms are used as clinical indicators of methanol poisoning and formate levels have been

suggested to be a good indicator of exposure to HCHO (Boeniger, 1987). Some studies have

shown, though, that exposure of rats to methanol by ingestion or i.p. results in decreased

antioxidant potential in the liver (Skrzydlewska and Farbiszewski, 1998) and increased

activity of aspartate amino transferase and sorbitol dehydrogenase in serum, which is

indicative of liver damage (Kadiiska and Mason, 2000).

The end product of methanol metabolism is CO2, which is generated fiom formate

through a folk acid-dependent pathway (Figure 1.3). Catalase is also involved in the

metabolism of formate to CO2, however the lack of physiological H202 may render this

pathway to be a minor one at best in humans. Humans and monkeys exhibit the symptoms of

methanol poisoning as descnbed because of an inefficiency in formate metabolism.

Compared to rats, which do not suffer from methanol poisoning to the sarne extent, primates

have lower tetrahyârofolate levels and the activity of 10-fomyltetrahydrofolate

dehydrogenase is lower (Johîin et al., 1987).

In diabetes, the levels of aldehydes such as methylglyoxal, glycolaldehyde and evm

HCHO are increased. The increased presence of these aldehydes likely contributes to

diabetic complications such as cardiovascular damage which has been attributed to advanced

glycation (Yu, 1998b). It war suggested that the generation of HCHO fkom methylamine may

be a cause of diabetic complications (Yu, 1998a). Methylamine produced h m the

deamination of epinephrine, nicotine and choline is furth= deaminated to fom HCHO as

catalysed by semicarbazide-sensitive amine oxidase (SSAO) in senun. The activity of SSAO

has been shown to increase in diabetes, thus increasing the generation of HCHO and the risk

for toxicity. In fact, it was demonstrateci that methylamine toxicity can be prevented by the

addition of SSAO inhibitors such as arninoguanidine and MDL-72974A (Deng et al., 1998).

This irnplicated HCHO as the toxic metabolite of methylarnine and suggests its importance

as a toxin in diabetes. The toxicity associated with smoking has also been suggested to

involve the production of HCHO fkom nicotine via methylamine (Yu, 1998a).

However despite the known toxicity of HCHO, i t remains important as a source of

carbon for biological methylation and DNA synthesis and repair. Humans reportedly have

2.61 pig/g HCHO physiologically in blwd (Heck et al., 1985). HCHO reacts with

tetrahydrofolic acid (TKF) to fom 5.10-methylenetetrahydrofolate which is actively

involved in the synthesis of serine, methionine or thymidine (Figure 1.3). Thus although

HCHO is indeed a very toxic compound, it is a necessary one for maintainhg biological

processes.

Figure 1.3. The metabolism of fonnate to C a and the role of folate in formate and --- - fomatdehyde metabolism.

METHANOL

FORMALDEHYDE

10-FORMYL THF rn CO2

I \ 1 O-Formyl THF dehydrogenase

.) Purines 5,l O-methylidyne TM;

J 1 CH3-purines

THF i

Fomaldehyde h 5 , l O-methylene THF

homocysteine dTMP glycine

methionine THF

DNA synthesis, npair f Proteins Dietary Folk Ac id

1.3.2 The mechanism of HCHO toxkity

The toxicity of HCHO has been mainly attributed to its carcinogenicity through its

reactivity towards proteins and DNA in the cells of tissues localisecl to the area of HCHO

exposure. This is important when considering the toxicity of meihanol in that methanol is

metabolised to HCHO pnmarily in the liver. Thus, the liver would be the first tissue of

contact for HCHO.

It would be interesting to compare the mechanism of HCHO toxicity, especially in the

liver, to that of acetaldehyde, as HCHO and acetaldehyde are sirnilar compounds in ternis of

their chernical nature and their metabolism. Acetaldehyde is formed by the oxidation of

ethanol by ADHl or CYP 2E 1 and is considered to be the toxic metabolite of ethanol,

leading to the development of alcoholic liver disease. It is known to be carcinogenic and it

causes oxidative stress, Oxidative stress occurs when the oxidative state of the cell has

overcome its antioxidant defences, such as when ROS are formed or when antioxidant

enzymes such as glutathione peroxidase become exhausted. This condition is often

rnanifested by the onset of lipid peroxidation or the oxidation of cellular macromolecules.

Oxidative stress is thought to be involved in the onset of aging (Finkel and Holbrook, 2000)

and in the development of many diseases including Parkinson's Disease (Offen et al., 1999),

atherosclerosis (Young and Woodside, 2001) and alcoholic liver disease (Lieber, 1997) to

narne only a few. Oxidative stress caused by acetaldehyde is associateci with the depletion of

GSH (Vendemaile et al., 1998). Like HCHO, acetaldehyde is highly electrophilic and can

condense with GSH itself or its precursor, cysteine (Lieber, 1988). The depletion of GSH

favours the onset of lipid pemxidation ( K w s e et al., 1997) and the decrease in cellular

protein sulfhydryl content (Vendemaile et al., 1998) that were reported ta be caused by

ethanol in culhired rat hepatocytes and liver, respectively. These oxidative effects were

prevmted by the ADHl inhibitor 4-methylpyrazole and were enhanced with ALDH2

inhibitors such as cyanamide, suggesting that acetaldehyde was responsible for these effects.

Furthemore, acetaldeh yde toxicity may be associated with mitochondrial darnage, as

acetaldehyde was reported to be an inhibitor of complex I of the mitochondrial electron

transport chain (Cederbaum et al., 1974) and it decreases the mitochondrial membrane

potential (vm) (Kurose et al., 1 997).

The toxic mechanisms of HCHO have been poorly studied compared to acetaldehyde

possibly because the similarities in the metabolism and chemistry of HCHO and

acetaldehyde would lead one to predict that their toxic mechanisms would be similar. Studies

have s h o w that HCHO depletes GSH and protein sulfhydryls, induces lipid peroxidation

and inhibits respiration in isolated rat hepatocytes or whole livers (Ku and Billings, 1986;

Strubelt et al., 1989). Al1 of this indicates an oxidative stress mechanism of HCHO toxicity,

possibly initiated by damage to mitochonâria since inhibition of respiration results in the

generation of ROS (Tunens, 1997). Similady, studies using isolated rat hepatocytes have

show that acetaldehyde induces GSH depletion, lipid peroxidation and cytotoxicity (Stege,

1982; Vina et al., 1980) in addition to other effects such as protein kinase C inactivation

(hmenicotti et al., 1996). However, huther investigation is needed to elucidate the toxic

mechanism of HCHO.

13.3 The role of mitochondria in ce11 death

Mitochondria are the organelles responsible for producing 90% of a cell's energy

requirements and thus play an important role in maintaining a cell's viability. Damage to

mitochondna cm severely undennine cellular integrity. The majority of activity in

mitochondria occurs in the matrix or in close proximity to the inner membrane. This includes

the proteins involved in oxidative phosphorylation.

Mitochondnal defects often result fiom conditions of oxidative stress. Mitochondria

themselves generate ROS through the rnitochondrial electron transport chain. It has been

estimated that 98999% of the oxygen taken up is reduced to H20 whereas only 1.2% of the

oxygen is reduced to ROS, and mitochonâria readily detoxify the ROS formed with

antioxidant enzymes including glutathione peroxidase and superoxide dismutase. Respiratory

inhibitors however can readily increase ROS formation to the point of saturating the

mitochondrial detoxification system and creating a harmful oxidative environment in the

mitochondria (Kowaltowski and Veresci, 1999). Proteins can become oxidised by ROS,

especially at cysteine residues to create disulfide bonds and protein thiol cross-links which

can contribute to the opening of the mitochondrial pemeability transition pore (MPT)

(Castiho et al., 1996). Clearly, the integrity of the mitochondria is important in maintaining

the overall integrity of the ceIl and imbalances in the oxidative state of the ceIl cm have

detrimental results.

It is interesting that in addition to the role of mitochondna in maintaining the health

of the cell, mitochondria are also responsible for triggering a cascade of events leading to ce11

death. A loss of mitochondrial membrane integrity or the opening of the MPT can allow the

leakage of proteins such as cytochrome c into the cytosol. The release of cytochrome c

results in the activation of a senes of caspases that lead to ce11 death by apoptosis (Cai et al., -- - >.-

1998). Howeva, apoptosis (programmed ce11 death) is an ATP-dependent process and under

conditions in which cellular ATP levels are insacient to enable apoptosis to occur, ce11

death occurs by necrosis (accidental ce11 death) (Tsujimoto, 1997) (Figure 1.4).

Figure 1.4 The mle of mitochonâria in ce11 death.

1.4 Purpose of the study

This study originally began as a joint effort with the Bureau of Chernical Hazards at

Health Canada to help detemine the health nsks associateâ with the use of methanol as a

component of gasoline. The incorporation of rnethanol into gasoline may be economically

favowable because methanol is produced fkom the hydrolysis of wheat (Bunce, 1994). This

risk assessrnent posed the question of whether or not HCHO, the first oxidative metabolite of

methanol, is toxic towards isolated rat hepatocytes and also examined what conditions can

modify its toxicity.

However as the study progressed, it became clear that HCHO metabolism plays a

major role in its toxicity. There have been many studies on the metabolism or toxicity of

HCHO, but none have correlated them. HCHO is a very toxic compound and its removal is

important in preventing toxicity. However, HCHO metabolism may be hindered if the

enzymes responsible for HCHO metabolism are inactive or are deficient. It is therefore

important to detemine the cytotoxic effects of the inhibition of HCHO-metabolising

enzymes. HCHO metabolism can also occur through its reaction with endogenous

compounds such as GSH, amino acids and catecholamines. The effects of these reactions on

toxicity must also be evaluated. The main objective is therefore to relate HCHO metabolism

to its cytotoxicity towatds isolated rat hepatocytes. The mechanism of HCHO cytotoxicity

was also investigated since exposure to HCHO was found to cause hepatotoxicity in rats as

discussed above. By elucidating the toxic mechanism of HCHO, methods for treating its

toxicity may be discovered.

The isolated hepatocyte system is ideal for this study for several reasons. Firstly,

hepatocytes express high levels of al1 of the p ~ c i p l e HCHO-metabolising enzymes (ADH 1,

ALDH2, ADH3). Thus metabolism studies will not be hindered by a lack of these enzymes.

Secondly, the liver is the organ responsible for the buk of xenobiotic metabolism. Thirdly,

the target organ for acetaldehyde is the liver and HCHO has also been shown to exert toxic

effects on the liver (Feldman and Bonashevskaya, 1971; Mecler, 1978; Faroqui et al., 1986),

thus the cytotoxicity studies are physiologically relevant. Finally, the isolated hepatocyte

system is easy to manipulate. That is, desired conditions (e.g. the addition of toxins or

antidotes, oxygen levels, etc.) are easily achieved and endpoints are easily assayed. This is

extremely important in this study, in which the modification of HCHO metabolism and

monitoring its cytotoxic effects are key.

The modulation of HCHO metabolism will be done using inhibitors of

ADHl/ALDHZ or ADH3 or NADH generators/oxidisers to inhibit or enhance the enzymatic

metabolism of HCHO, respectively. Increasing the non-enzymatic metabolism of HCHO will

be done with trapping agents such as various amines and thiols. The mechanism of HCHO

cytotoxicity will be determined by examining the effect of HCHO on mitochondrial function

(respiration, membrane potential) and by measuring oxidative stress indicators (ROS, lipid

peroxidation, GSH).

f .S Hypothesk

1) HCHO-induced cytotoxicity occurs through its toxic effects on mitochondria and

the induction of oxidative stress,

2) The inhibition of HCHO metabolisrn results in increased HCHO-induced

c ytotoxicity.

2.1 Chemicals

Al1 chemicals, biochemicals and enzymes used were purchased From either Sigma

Chemical Co. (St. Louis, MO) or Aldrich Chemical Co. (Milwaukee, W1) with the exception

of a few, as described below.

For the preparation and use of rat hepatocytes, collagenase (fiom Closh'dium

histoljticum) was obtained fiom Worthington Biochemical Corp. (Lakewood, NJ), sodium

pentobarbital h m M.T.C. Pharmaceuticals (Cambridge, ON) and sodium heparin h m

Organon Teknika (Toronto, ON). Calcium chloride (anhydrous) was fiom Fisher Scienti fic

(Fair Lawn, NJ), potassium chloride and sodium chloride were h m BDH hc. (Toronto,

ON), sodium bicarbonate was from ACP Chernicals Inc. (Montreal, PQ) and magnesuium

sulfate (heptahydrate) was purchased fiom Bioshop Canada h c . (Burlington, ON). Carôogen

gas (95% 026% CO2) was obtained fiom BOC Gases (Mississauga, ON).

For the microsome expenments, human CYP 2E1 + P450 reductase + cytochmme b5

SUPERSOMES@, human CYP 4A11+ P450 reductase + cytochrome bs SUPERSOMES'

(derived from human cDNA-expressing baculovirus (Autognpha cali/mica)-infected Hi5

insect cells) and insect ce11 control SUPERSOMES~ (Hi5 insect cells infected with wild type

baculovirus) wen purchased h m GenTest Corp. (Wobum, MA).

In ternis of other chemicals used, methanol (HPLC grade) and acetic acid (glacial)

were pwhased h m Fisher Scientific (Fair Lawn, NJ), ethanol(95%) was from Commercial

Alcohols Inc. (Brampton, ON), DMSO and dithiothreitol were h m Caledon Laboratones

Ltd. (Georgetown, ON) and hyhxylamine was h m Fisher Scientific (Fair Lawn, NJ). 2'.

7'dichlorofluorescin diacetate was pwhased h m Eastman (Rochester, NY). Unless

otherwise indicated, al1 chernicals useâ were of the highest grade available.

Methyl-diethyldithiocarbamate (MeDEDC) was prepand from

diethyldithiocarbamate @EDC) according to the method of Faiman et al. (1983) and

propiolaldehyde was synthesised h m propargyl alcohol according to the method of Veliev

and Gnseinov (1 980). N-methyl-6'7-dihydroxy- l,2,3,4-tetrahydraisoquinoline (N-

methylnorsalsolinol) hydrobromide was synthesised according to the method of Srnissman et

al. (1 976).

2.2 Animals

Adult male Sprague-Dawley rats (280-300g) obtained from Charles River (St.

Constant, PQ) were used in al1 experiments. The animals were given water ad libitum and

were fed a standard chow diet of Purina Rodent Chow from Woodlyn Laboratones Ltd.

(Guelph, ON). The animals were kept in a controlled environment of 12 hours light/dark

cycle.

2.3 Isolation and incubation of nt hepatocytes

The isolation of rat hepatocytes was done according to the collagenase perhsion

methoâ of Moldeus et al. (1978). The cells were suspendeci in Krebs-Henseleit buffer with

HEPES at pH 7.4. 10 mL suspensions of hepatocytes (106 cells/mL) were kept in rotating 50

mL round-bottomed flasks in a water bath maintainecl at 37°C with an atmosphen of 95% O2

and 5% Ca. The cells were incubated under these conditions for 30 minutes prior to the

addition of chemicals in order to allow for the cellular suspension to equilibrate with the

atmosphere. Only cells with an initial viability of at least 85% were used.

In experhents in which the incubation medium was replaced after various tirne

points, the cells were centrifbged and then resuspended in fnsh incubation buffer. h

expenments in which HCHO was added to the cells, the flasks were sealed with parafilm and

were incubated in a stationary position in the 37OC water bath for 30 minutes before being

rotated and exposed to the atmosphere again. This was done to prevent the vapourisation of

HCHO.

2.4 Determination of cell viabilitylcytotoxicity '

The viability of hepatocytes was determined by measuring the intactness of the

plasma membrane by its exclusion of trypan blue (final concentration 0.1% w/v) as describeci

by Moldeus et al. (1978). Trypan blue exclusion was viewed under a light microscope and

the percentage of cells which excluded trypan blue was calculated as the extent of viability.

2.5 Preparation of GSH-depleted and ADHl-inhibited hepatocytes

Hepatocyte GSH was depleted by adding 200 pM 1-bromoheptane to the ce11

suspension and incubating for 30 minutes prior to the addition of other chernicals (Khan and

O'Brien, 1991). Hepatocyte ADHl was inhibited by preincubating the cells with 1 pM 4-

methylpyrazole for 30 minutes prior to the addition of other chemicals.

2.6 Preprration and use of microsoma =-A.

Microsornes w m prepared by centrifugation of the rat liver homogenate, according

to the method of Lake (1 987). CYP 2El-induced mimsomes were prepared by injecting rats

i.p. with pyrazole (200 mgkg) for 2 days (Krikum and Cederbaurn, 1984). The microsornes

were suspendeci in O. IM potassium phosphate buffer, pH 7.4 in plastic tubes. Incubations

were done at 37"C in a gently shaking water bath. The P450 inhibitors used (e.g.

benzylimidauile, phenylimidazole, methylpyrazole or diphenyliodonium) were preincubated

in the suspensions for 30 minutes prior to the addition of 2 mM NADPH followed

immediately by 250 pM HCHO. The tubes were then capped to prevent the escape of the

highly volatile HCHO. 500 PL aliquot suspensions were removed at various time points and

reactions were stop@ with a final concentration of 4.5% trichlomacetic acid before HCHO

levels were measured as described below.

For experiments with purifid human CYP 2El+ P450 reductase + cytochrome bs

SUPERSOMES@, 50 pmol CYP 2E1 was incubated and analysed for HCHO levels in the

same conditions and manner as described above. Insect control SUPERSOMES' of an equal

amount of protein as the human CYP 2El + P450 reductase + cytochrome bs

SUPERSOMES~ was used to control for metabolism by enzymes endogenous to the host

insect cells used to express CYP 2E1 h m hurnan CYP 2El cDNA-infected baculovinis.

2.7 Measunment of formaldebyde metabobm by rat hepatocyta or microsornes

HCHO levels were determined according to the coloMmetric method of Nash (1953).

This method measures the amount of diacevldihydrolutidine formed h m the reaction

between HCHO aud a solution of ammonium acetate and acetylacetone. To measure

hepatocyte HCHO uptake or microsomal HCHO metabolism. 500 pi, ceIl suspensions or -- -

microsorne aliquots were taken at various times and 4.5% tnchiomacetic acid was added to

precipitate pmteins and to therefore stop metabolic reactions h m f i d e r o c c d g . The

cells or aliquots were then centrifigeci to separate the cells or microsornes fiom the

incubation buffer. The supematants were removed and 500 pi, of reagent (2 M ammonium

acetate, 0.05 M acetic acid and 0.02 M acetylacetone) was added to each sample of

supernatant. The samples were then incubated at 37"C for 40 minutes to allow for the

derivatisation of HCHO with the reagent. The absorbance (indicating the amount of

diacetyldihydrolutidine) at 412 nm was then measwed on a Shirnaâzu W 240

spectrophotometer (Kyoto, Japan).

2.8 Measurement of formic acid leveh

Hepatocyte fomic acid levels were detemined using the enzymatic assay of Grady

and Osterloh (1986). Following separation of the cells h m the incubation medium by

cmhifiigation, 100pL aliquots of the supematants were added 10 3 mL of 0.1 M phosphate

buffer pH 6.0 containhg 1 5 mM NAD*, 0.8 U/mL diaphorase and 2.1 mM p-

iodonitrotetrazoüum violet (INT). O. 1 U/mL formate dehydrogenase was then added to

oxidise formate ushg the NAD+, producing NADH. The INT was reduced by NADH and

diaphorne, and the level of INT metabolite was useà as the end point. The total solution

containhg the sample, NAD+, INT, diaphorase and fomate dehydrogenase was left at room

temperature for 10 minutes following the addition of formate dehydrogenase. Absorbantes

were th- read on a Cobas Fara II analps (Roche Diagnostics, Mississauga, ON) high-

throughput spectrophotometer at 500 nm.

2.9 Meamremeut of lipid peroxidation A- -

Hepatocyte lipid peroxidation was measured according to the method of Smith et al.

(1982). The amount of the lipid peroxidation product malondialdehyde produced was

detennined by measuring the amount of product (TBARS) fomed fiom its reaction with 2-

thiobarbitwic acid. 1 rnL ceIl suspensions were removed at various time points and reactions

were stopped by the addition of 70% trichloroacetic acid. 0.8% 2-thiobarbituric acid was then

added and the suspensions were incubated for 15 minutes in a boiling water bath to allow the

reaction betwea malondialdehyde and 2-thiobarbituric acid to occur. Following removd of

the cells by centrihigation, TBARS levels were meastueci spectrophotometrically at 532 m.

2.10 Measurement of GSH and GSSG levets

Intracellular GSH and GSSG levels were measured according to the HPLC method of

Reed et al. (1980), in which GSH was denvatised with iodoacetic acid and 2,4-

dinitrofluorobenzene (DNFB). 800 pL cell suspensions of each sample were removed at

various time points and the cells were separated fiom the incubation buffer by centrifugation.

Following mnoval of the supematants, the cells were deproteinised with 25%

metaphosphoric acid and centrihged again to remove proteins. The subsequent supematants

were treated with excess sodium bicarbonate and 1.5% iodoacetic acid and were allowed to

stand in the dark over night. 1.5% DNFB was then added and the mixture allowed to stand

for at least 4 hours. HPLC analysis was done using a Waters HPLC system (Mode15 10

pumps, WISP 710B autoinjector, Mode1 410 W i s detector) with a Waters p Bondapaka

NH2 3.9 x 300 rnM cotumn.

Measurement of cellular respiration

Oxygen uptake by hepatocytes was measureà using a Clark-type oxygen electrode

(Yellow Springs Instrument Co., Yellow Springs, IL). 2 mL ce11 suspensioas were placed in

the incubation chamber which was kept at 37°C. Chmicals were aâded to the chamber a few

minutes later, when the normal rate of O2 uptake was determined. The percentage of

hepatocyte respiration as compared to the control was measured 10 minutes f ier the addition

of compounds (cg. HCHO).

2.12 Measurement of reactlve oxygen species in hepatocytes and microsornes

Reactive oxygen species (ROS) formation in hepatocytes was measured based on the

method of LeBel et al (1 992). 3 rnL ceil suspensions were removed at various time points

and centrifuged to separate the cells fiom the incubation buffer. Following removal of the

supematant, the cells were resuspended in 3 rnL of 50 mM Tris-HC1 buffer with Hanks

incubation medium, containing 16 pM of 2'. 7'-dichlorofîuorescin diacetate @CF). During a

10 minute incubation at 37OC, 2', 7'-dichlorofluorescin diacetate was hydrolysecl in the cells

to dichlorofluorescin, which was oxidised by ROS to the fluorescent product

dichlorofluorescein. Dichlorofluorescein effluxed the ce11 and following centrifugation, was

measured in the supematant at 500 nm and 520 nm excitation and emission wavelengths,

respectively, on a Shimadni RF5000 spectrofluorometer (Kyoto, Japan).

In micmsomes, ROS levels were detennined using a slightly modified method h m

that with hepatocytes. Based in part on the method of Puntarulo and Cederbaurn (1 998). cYP

2E1 or CYP 4A11 + P450 reductase + cytochrome bs SUPERSOMES~ (50 pmol) were pre-

incubated in 40 mM potassium phosphate buffa (pH 7.4.37OC) with 1 mM sodium azide for

28

15 minutes to inactivate any catalase that may ôe present which may remove some of the - --

ROS fonneû. 0.5 mM NADPH was th- added followed by the substrate. The ~actions were

allowed to pioceed for 1.5 hours at 37°C before the addition of 5 pM DCF. DCF was

incubated with the sample for 10 minutes before being placeâ on ice and then nad on a

spectmfluorimeter at 488 nm and 552 nrn excitation and emission wavelengths, respectively.

2.13 Measorement of mitochoidrial membrane potenaal

The integrity of mitochondrial membrane potential (Ayi,,,) was measureâ based on the

method of Eamus et al. (1 986), in which the membrane potential-dependent mitochondnal

uptake of the cationic fluorescent dye rhodamine 123 was measured. 500 pL ce11 suspensions

were removeci at various time points and the cells were separateâ h m the incubation

medium by centrifugation. The supematants were then removed and the cells were

resuspended in fresh incubation buffer containing 1.5 pM rhodamine 123. The suspension

was incubated for 10 minutes with gentle shaking in a 37°C water bath and then centrifugeci

to remove the cells. The fluorescence of the supernatant was analysed at 490 nm and 520 nm

excitation and emission wavelengths, respectively, for the amount of remaining rhodamine

123.

2.14 Identükrtion of tetrahydroisoquioolincs (TA1Qs) in bepatocytes

The formation of N-methylnorsaisolino1 (NMNS) in the hepatocyte incubation buffer

was dctennined by h t l y sepatating the cells h m the buffer by centrifugation. Extraction of

NMNS firom the buffei was done with ethylacetate followed by washing with 50 mM

hydrochlonc acid. The acidic fiaction was extracted with ethylacetate, which was then

evaporated. The left-over solid was redissolved in methano1 for malysis by mas 7 -

spectrometry.

The mass spectra of the extracteci pmduct was done and a peak at 180 m/z was found,

which matches the mass of the target compound, NMNS. The product ion mass specûa

(MSiMS) was then acquired for NMNS.

One- and two-array analysis of vafiance (ANOVA) followed by the Scheffe's test for

significant difference were used for cornparison of the multiple-heated hepatocyte or

microsomal incubates and the relative controls. The data represent the mean f S.E. of at least

3 separate experiments.

3. - -L -

THE FORMGLDEBYDE METABOLIC DETOXIFICATION ENZYME SYSTEMS AND MOLECULAR CYTOTOXIC MECHANISM IN ISOLATED RAT HEPATOCYTES

3.1 Introduction

Humans are exposed to fomaldehyde (HCHO) h m both direct environmental

sources as well as h m the metabolism of xenobiotics. HCHO is commonly used as a tissue

pnsenation agent and is produced in large quantities industnally. Everyday exposure to

HCHO inchides building materials (e. g. paint, plywood), cosmetics, cigarette smoke,

photochernical smog and even various h i t s (Conaway et al., 1996; Trezl et al., 1998).

HCHO is also formed during P45O-catal ysed dealkylation reac tions (e.g. of nitrosamines),

aspartame hydrolysis and the oxidatioa of methanol (Conaway et al., 19%; Trezl et al., 1998;

Trocho et al., 1998).

HCHO has been implicated as a cause of carcinomas, especially in the nasal passage,

due to its highly reactive nature with proteins and DNA. The carcinogenicity of hydrazine

has also been atûibuted to DNA reactive formaldehyde hydrazone formation h m

physiological HCHO (Lambert and Shank, 1988). In addition, some studies have linked

chmnic HCHO exposure in humans to neumdcgenerative disorders (Kilbum, 1994).

Teratogenicity induced in mice by HCHO has been attributed to folic acid deficiency in the

embryo (Sakanashi et al., 19%).

However, the toxicity of HCHO as a consequence of chronic exposun to HCHO-

generating xenobiotics has been largely overlded kause HCHO is rapidly metabolised

and removed h m the organism. HCHO could be duced to methanol by cytosolic alcohol

dehydrogenase (ADH 1) or oxidised to formate by mitochondrial aldehyde dehydrogenase

(ALDE) or cytosolic GSH-dependent formaldehyde dehydrogenase (ADH3) (Pocker and

Hong, 1990; Uotila and Koivusalo, 1989). It has also been suggested that HCHO is a

substrate for CYP 2E1 and can thus be oxidised by the endoplasmic reticulum (BellParikh

and Guengeric h, 1 999).

The kinetics of HCHO metabolism by ADH1, ALDH2 and ADH3 have been studied

as have the effects of various enzyme inhibitors on its rate of metabolism (Pocker and Hong,

1990; Uotila and Koiwsalo, 1989; Dicker and Cederbaurn, l984a,b) but few metabolism

studies have been correlated with HCHO-induced cytotoxicity. Since methanol or HCHO

given orally to rodents causes hepatotoxicity and decreases antioxidant enzyme levels

(Slaydlewska and Farbiszewski, 1996), we have used isolated hepatocytes to compare the

cytoprotective activity of these metabolising enzymes and detemine the molecular

mec hanisms involved in HCHO-induced ce11 lysis.

3.2 Hypothesis

Inhibition of the HCHO-metabolishg enzymes ADHI, ALDM or ADH3 increases

hepatocyte susceptibility to HCHO as HCHO is much more toxic to mitochondria than other

metabolites such as methanol or formic acid.

3.3 Results

As shown in Figure 3.1, HCHO was found to cause hepatocyte lysis in a dose- and

tirnedependent manner with approximately 4 rnM HCHO causing 50% ce11 tysis in 2 hours

(LDS0). Toxicity was greatly hcreased when either of the enzymes responsible for the

- metabolism of HCHO were inhibited. As show in Table 3.1, HCHO-induced üpid

peroxidation and cytotoxicity were markedly incmed by the ADHl inhibitor 4-

methylpyrazole or by the ALDH2 inhibitors cyanamide or chlord hydrate. The

concentrations of the ADH 1 and ALDH2 inhibitors used did not affect hepatocyte viability

or increase lipid pemxidation over 3 hours. Furthemore HCHO-induced lipid peroxidation

and cytotoxicity were ais0 markedly increased if hepatocyte GSH was depleted so as to

inhibit ADH3 before the addition of HCHO. The ALDHZ inhibitor disulfirarn used in

aversion therapy for alcoholism or its active metabolites diethyldithiocarbamate (DEDC) and

methyl-diechyldithiwafiamate (MeDEDC), however, inhibited HCHO-induced lipid

peroxidation and cytotoxicity presumably because of their antioxidant activity.

As shown in Table 3.1, the NADH generators xylitol, fnctose and lactate (used at

concentrations which did not affect hepatocyte viability over 3 hours) also markedly

decreased HCHO-induced hepatocyte cytotoxicity in control hepatocytes but did not prevent

HCHO cytotoxicity in ADH 1 -inhibitecl hepatocytes.

Figure 3.1 HCHO cytotoxicity in isolated rat hepatocytes is dose- and time-dependent.

Control hepatocytes; . 2 mM HCHO; A 4 mM HCHO; X 10 mM HCHO.

Hepatocytes (106 cellJ/mL) were incubated in Krebs-Henseleit buffer (pH 7.4) at 37°C. The detedation of cytotoxicity w u camed out as described in Materials and Methods.

Values are expressad as the means of at least 3 separate experimmts S.E.

Table 3.1 Modulation of HCHO-induced lipid peroxidation and cytotoxicity by inhibitors of -- L tht HCHOmctabolisingenyme systems.

Treaûnent

Lipid peroxidation

Cytotoxicity absorbance at % Trypan blue uptake 532 nrn

60' 120' 180' 1 80' Control hepatocytes + 1 m M HCHO + 2.5 mM HCHO

+ 1 mM chloral hydrate + 200 pM cyanamide + 20 pM disulhm + 50 pM DEDC + 50 pM MeDEDC

+ 4 mM HCHO + 10 mM xylitol + 10 mM lactate + 10 rnM fkuctose

GSH-depleted hepatoc ytes + 2.5 mM HCHO ADH 1 -inhibited hepatocytes + 2.5 mM HCHO

+ 10 rnM xylitol + 10 mM lactate + 10 mM hctose

0.024 * 0.004 0.057 k 0.03 0.190 *0.07' 0.234 * 0.09~ 0.271 * 0.09~ 0.025 * 0.002~ 0.026 * 0.003~ 0.028 * 0.003~ 0.343 +0.08' O. 156 A 0.07' O. 1 16 0.05' 0.128 * 0.03' 0.028 0.004 0.42 1 IO. 1 zd 0.025 0.003 0.26 t ~0.08~ 0.202 =t 0.06' O. 1 8 10.07~ 0. t 9 ~0.07'

Hepatocytes (106 celldml) were incubated in Krebs-Henseleit buffer (pH 7.4) at 37'C. The determination of cytotoxicity and lipid peroxidation were carried out as described in Materials and Methods.

Values are expressed as the means of at least 3 separate expenrnents f S.E.

'Signi ficantl y different fkom contml hepatocytes (P < 0.05) b~ignificantly different fiom control hepatocytes + 2.5 mM HCHO (P < 0.05). 'Signincantly different fiom control hepatocytes + 4 mM HCHO (P < 0.05). d~ignificantly different from GSH-depleted hepatocytes or control hepatocytes + 2.5 m M HCHO (P c 0.05). 'Significantly different fiom ADH 1 -inhibited hepatocytes or control hepatocytes + 2.5 mM HCHO (P < 0.05).

A cornparisonof the effectivenesmfvarious ALDH2inhibibrs at inhibithg HCHO

metabolism in hepatocytes was studied by following the disappeanmce of HCHO added to

GSH-depleted/ADHl-inhibitd hepatocytes. This was then correlated with the effect of the

ALDH2 inhibitors on HCHO cytotoxicity. Inhibition of ALDH2 was found to correspond to

an increase in hepatocyte susceptibility to HCHO. As shown in Table 3.2, 100 @id

glycolalâehyde or 10 mM acetaldehyde were the most effective at inhibiting ALDH2 and

increasing HCHO cytotoxicity. Methylglyoxal, cihal, crotonaldehyde and propiolaldehyde,

d l cornpetitive inhibitors, were also effective. MeDEDC was more effective than DEDC, (its

metabolic precursor) at inhibiting HCHO metabolism but only slightly increased cytotoxicity

presumably because of theu antioxidant activity (Table 3.1). The CYP 2El inhibitor

isoniazid was also effective at inhibiting HCHO rnetabolism and increasing HCHO

cytotoxicity.

A ---. Table 3.2 Enéct of alâehyde or aicohol metabolising enzyme inhibitors on HCHO metabolism in isolated hepatocytes venus HCHO cytotoxicity.

% Treatment % HCHO metabolised c yîotoxicity

60' 120' 1 80' 1 80' GSH-depletdADH 1 hhibited Hepatocytes + 1 mM HCHO + 10 m M acetalâehyde + 300 pM crotonaldehyde + 5 0 piid methylgiyoxal + 100 phd propiolaldehyde + 200 pM cyanamide + 1 m M chloral hydrate + 50 pM D E N + 50 pM MeDEDC + 100 phf glycolaldehyde + 25 )iM citral + 2 niM isoniazid + 1 m M penicillamine + 1 mM cysteine

Hepatocytes (lo6 celldrnL) were incubated in Krebs-Henseleit buffer (pH 7.4) at 37°C. Detemination of HCHO metabolism and cytotoxicity were carrieû out as describeci in Materials and Methods.

Values are expmsed as the means of at least 3 separate experirnents f S.E.

'Significantly different from GSH depletedIADH 1 -inhibited hepatocytes + 1 mM HCHO (P c 0.05).

As shown in Table 3.3, isolated hepatocytes treated with HCHO resuited in a dose-

dependent inactivation of mitochondrial respiration with more than 50% inactivation at 1

mM HCHO. Interestingly respiration was restored by the subsequent addition of

dithiothreitol, penicillamine or cysteine. The HCHO-inducd inhibition of hepatocyte

respiration was accompanied by a marked dose-dependent decrease in the mitochondrial

membrane potential (y,,,) (Table 3.3). Both respiration and W, were compromised M e r in

GSH-depleted cells. Further evidence indicating that HCHO causes opening of the

mitochondrial pmeability transition pore was the finding that cytotoxicity induced by

HCHO (4 m M caused 70 6% cytotoxicity in 3 hours) was prevented by 2 pM cyclosporin

A (46 4% cytotoxicity in 3 hours) or 2 mM camitine (49 * 4% cytotoxicity in 3 hours).

Dithiothreitol also prevented cytotoxicity when added 30 minutes afier the HCHO (Table

3.3).

The thiols @cillamine or cysteine prevented cytotoxicity when added 30 minutes

after the HCHO (Table 3.3). Addition of the antioxidant BHT prevented cytotoxicity as did

the addition of the iron chelator desferoxarnine. Because antioxidants prevented HCHO

cytotoxicity, the role of oxidative stress in the cytotoxic mechanism of HCHO was also

investigated. As shown in Tables 3.1 and 3.4, the addition of HCHO to hepatocytes resulted

in the generation of ROS and induction of lipid peroxidation in a dose- and tirne-dependent

manner. Both were fiirther increased in GSH-depleted cells, coiresponding with an increase

in HCHO-induced cytotoxicity. HCHO also induced hepatocyte GSH depletion in a dose-

and time-dependent manner. A non-toxic dose of 1 mM HCHO was able to deplete

hepatocyte GSH by 50% in only 30 minutes whereas 4 mM HCHO was requind to deplete

Lk-- . over 90% of the GSH in 3 hours. This madred depletionof hepatocyte GSH comlated with

the high cytotoxicity induceà by 4 rnM HCHO.

Tabk 3.3 Effwt of HCHO on mitochondnal respiration and membrane potential in isolated hepatocytes.

Treatment

Cytotoxicity % Hepatocyte % Hepatocyte % Trypan blue

respiration Wm up take 1 0' 120' 120'

Control hqatocytes 100 94* 3 21f3 + 1 m M HCHO 44 6' 33 * Sa 2 5 f 3 + 2 rnM HCHO 35 * 6' 26 * 4a 39 f 3' + 4 mM HCHO 151 2a 18*Z0 52 f 5'

+ 50 pIi4 BHT 24I5 80 * 3' 31 f 3b + 250 pM desfmxamine 20 f 4 82 I 4b 32 f 4' + 4 rnM D-penicillamine (30') 97. zb 85 f 3b 34 î 3b + 4 mM Ltysteine (30') 98 i 2' 86 f sb 32 f 3b + 4 rnM dithiothreitol(30') 97 i 2b 89 * 4b 35 f 3' GSH depleted hepatocytes 98*2 93*3 23 * 4 + 2 mM HCHO 17 * 5' 14* 2' 68 f SC

Hepatocytes (106 ce1ldm.L) were incubated in Kxbs-Henseleit buffer (pH 7.4) at 37OC. Hepatocyte respiration (oxygen uptake) was measured on an oxygen electrode as described in Materials and Methods. Mitochoncùial membrane potential (y,) was measured as described in Materials and Methods.

Values are expressed as the means of at least 3 separate expenments A S.E.

aSignificantly di fferent âom control hepatocytes (P < 0.05). b~ignificantly different fiom conhol hepatocytes + 4 mM HCHO (P c 0.05). CSignificantly different fiom control hepatocytes + 2 mM HCHO or GSH-depleted hepatoc ytes (P c 0.05).

Tabk 3.4 ûxidative stress induced by HCHû as indicated by RQS formation and GSH depletion in imlated hepatocytes.

Measwement Time (min) 30' 60' 120'

(1) HCHOIinduced ROS formation (fluorescence units) Control hepatocytes + 1 mM HCHO + 2 mM HCHO GSH-depleted hepatocytes + 1 mM HCHO

(2) HCHO-induced GSH depletion (GSH levels as % of contml) Control hepatocytes 100 95f 9 92 f 8 + 1 mM HCHO 53 I 4 4 5 I 4 35 1: 3' + 2 mM HCHO 39f4 3 2 I 3 10 12' + 4 mM HCHO 35f 4 2 7 I 3 4.6 f 2'

Hepatocytes (10' ce l l im~) were incubateci in Krebs-Hmseleit buffér (pH 7.4) at 37'C. ROS and GSH levels were measured as describeci in Materials and Methods.

Values are expressed as the means of at least 3 separate experiments * S.E.

'Significantly diffkrent from control hepatocytes (P < 0.05). b~ignificantly different h m control hepatocytes + 1 mM HCHO or GSH-depleted hepatocytes (P < 0.05).

3.4 Discussion

The toxicity of methanol has usually been atûibuted to its metabolite formate

(McMartin et al., 1979) and little consideration has been given to the toxic effects of HCHO,

mainly because HCHO is rapidly metabolised. HCHO is believed to undergo metabolism in

isolated hepatocytes by cytosolic GSH-dependent fonnaldehyde dehydrogenase (K, HCHO

< 1 piid (Uotila and Koivusalo, 1989)) identified as ADH3 and by mitochonârial ALDH2

(M, HCHO 380 phd (Siew and Deitrich, 1976)). However HCHO-induced c ytotoxicity was

increased by the ADHl inhibitor Cmethylpyrazole which suggests that ADHl catalyses the

reduction of HCHO to methanol. Furthemore the NADH generators xylitol, lactate or

hctose prevent HCHO cytotoxicity towards control isolated hepatocytes but not in ADHI-

inhibited hepatocytes. This suggests that increasing the cytosolic NADH:NAD+ ratio

increases HCHO reduction to methanol catalysed by NADWADH1 which decreases HCHO-

induced cytotoxicity.

Hepatocyte susceptibility to HCHO was also increased if hepatocyte GSH was

depleted beforehand so as to inhibit the GSH-dependent ADH3 which catalyses HCHO

oxidation to formate. Various inhibitors of ALDH2 were shown to inhibit HCHO metabolism

in GSH depleted/ADH 1 inhibited hepatoc ytes whic h also markedl y increased HCHO-

induced cytotoxicity. Acetaldehyde (10 mM) was particularly effective at inhibithg ALDHZ

and increasing HCHO-induced cytotoxicity, which was not surprising considering that the

K, of ALJ)H2 for acetaldehyde is less than 5 pIb4 as compared to 380 phi for HCHO (Siew

and Deitrich, 1976). Glycolddehyde (100 IiM) and methylglyoxal(500 @id), aldehydes that

are increased in diabetes, were also potent ALDH2 inhibitors (with Kms for ALDHZ of 46

pM and 8.6 pM, respectively (Pietruszko et al., 1999)) which ais0 increased HCHO-induced

cytotoxiciîy. DisUtnrani, curtently wed as an ALDE inhibitar in avmion aicrapy fbr

alcoholism is believed to be bioactivated by reduction to DEDC, thiomethylated to MeDEM:

and oxidised to a sulfoxide (Lipsky et al, 2001). hterestingly MeDEDC was more effective

than DEDC at inhibithg hepatocyte ALDH2 but did not increase HCHO cytotoxicity. This is

likely because disulfiram and its metabolites are antioxidants (Liu et al., 1996). The CYP

2El inhibitor isoniazid (Qum et al., 1992) also inhibited HCHO metabolism and increased

HCHO cytotoxicity.

The increased cytotoxicity seen in GSH-depleted cells was likely due to three factors.

Firstly, the lack of GSH inhibited the GSH-dependent ADH3 activity, thus removing one

major pathway for HCHO metabolism. Secondly GSH when coupled with GSH rductase is

the major antioxidant defence system of the ceIl and HCHO-induced lipid peroxidation was

particularly increased in GSH-depleted hepatwytes. Thirdly GSH may also play a role in

preventing enzyme inactivation by HCHO e.g. the antioxidant enzyme GSH rediictase is

readily inactivated by HCHO if NADPH is present (see appendix 3.3).

The molecular cytotoxic mechanism seems to involve lipid peroxidation as

antioxidants decrûise cytotoxicity. Mitochonârial enzymes are also HCHO targets, as HCHO

rapidly and rnarkedly inhibited mitochondrial respiration, possibly targeting complex 1,

which is the case for acetddehyde (Cederbaum et al., 1974). The inhibition of respiration

caused a decrease in ATP production and caused membrane blebbing. HCHO also induced

openhg of the mitochondnal permeability transition pore as cyclosporin A or cmitine

pieventai HCHO-induced cytotoxicity. Cyclosporin A or camitine are used to protect the

hepatocyte mitochondrial penneability transition pore (Pastorino et al., 1993). Furthemore

the cytoprotection by the reductive dithi01 dithiothreitol against HCHO cytotoxicity likely

-=L- reoultcd the ability of dithiohmit01 temitore the protein dithiois that arc mponsibte fbr

closing the mitochondrial transition pore (Kushnareva and Sokolove, 2000).

By inhibithg respiration, ROS were fùrther generated, thus contributing to oxidative

stress. Lipid peroxidation likely contributed to the openhg of the mitochondrial pemeability

transition pore which caused the collapse of yr, and possibly the leakage of ca2+ and

cytochrome c h m the mitochondna. The HCHO-induced oxidative stress found therefore

anses h m the impairment of cellular enzymatic antioxidant defences and an increase in

mitochondnd ROS formation. Mitochondrial respiration was restored and cytotoxicity

prevented by the addition of penicillarnine or cysteine which rapidly fomed non-toxic

thiazolidines with HCHO (Kallen, 197 1). Cysteine and penicillamine are therefore effective

antidotes for HCHO cytotoxicity.

The cytotoxicity of HCHO was clearly related to its metatmlism. inhibition of ADHI,

ALDH2 and ADH3 were found to inhibit the removal of HCHO by the hepatocytes, which

resulted in increased cytotoxicity through oxidative stress mechanisms. It is reasonable to

hypothesise that individuals with deficiencies in any of the above enzymes as well as those

who have lower levels of GSH will be more susceptible to HCHO toxicity. Such individuais

are likely to include approxirnately 50% of ûrientals, who possess a mutant, inactive ALDH.2

(Ooode et al., 1992). as well as diabetics who already have carbonyl glycoxidative stress as a

mult of ddehyde accumulation. In addition, it has been show that the activity of ALDHZ is

partially hormonally regulated in that high levels of f a a l e honnones such as estmgen and

progesterone c m downregulate ALDH2 (Jeavons and Zeiner, 1984). Thus, women who are

pregnant or are taking oral contraceptives may be more susceptible to HCHO. Although

HCHO ia indcedra~iciiy rrmoved in Whesrlthy individual, extra caution must be tskea by

those who lack any part of the HCHO cellular defaice system.

- Appendix 3.1 4 m M HCHO inhibits hepatocyte respiration, which is reversai by the addition of L-cysteine.

A - conmi hepatocytes

100% O2 Oxygen level 0% 0 2

B - 4 mM HCHO

4 mM HCHO 2 C - 4 m M HCHO + 4 rnM L-cvsteine at 10' A

Appeodix 3.2 HCHO metabolisrn in isolated rat hepatocytes.

O 30 60 90 120 150 1 80

Time (minutes)

l niM HCHO; . 2 mM HCHO; A 3 mM HCHO; 4 rnM HCHO

Broken line (-) indicates estimated metabotisrn as HCHO levets were too high at 30 minutes and could not be d e t d n e d .

Hepatocytes (lob cells/m~) were incubated in Krebs-Henseleit buffer (pH 7.4) at 37°C. Determination of HCHO levels was carried out as described in Materials and Methods.

Values are expresseci as the means of at least 3 separate experiments * S.E.

Appenâix 3 3 Inactivation of giutathione reductase by HCHO + NADPH.

O ! I 1 I

O 30 60 90 120 Time (minutes)

- control; -- - -- + 1 mM HCHO; + 1 m M HCHO + O. 1 mM NADPH;

Values are expresscd as the means of at least 3 separate experiments. *Significantly différent as compared to control (P c 0.05).

Dctamination of giutathione reductasc (GR) activity was based on the method of Vander Jagt et al. (1997). 5 pg/mL GR was incubated at 37°C in 0.1 M potassium phosphate buffer (pH 7.0) containing 2 mM EDTA and 1 mM GSSG. with 0.1 mM NADPH in indicatcd samples. NADPH was generated with 2.5 unitdmL glucose-6-phosphate dehydrogenase. 7.5 mM glucosed-phosphate, 0.5 mM NADP' and 5 mM MgCl*. At the t h e points indicated, an aliquot of the sample was taken and GR activity was detennined by following the rate of change in absorbwce at 340 nm for 2 minutes after the addition of 0.1 mM NADPH.

THE METABOLISM OF FORMALDEHYDE BY CYP 2El AND THE ASSOCIATED CYTOTOXlC CONSEOUENCES IN ISOLATED RAT HEPATOCYTES

4.1 Introduction

The metabolism of fonnaldehyde via dehydrogenation by its three primary

metabolising enzymes - alcohol dehydrogenase (ADH 1), aldehyde dehydrogenase (ALDH2)

and fomaldehyde dehydrogenase (ADH3) - has been studied extensively with in vitro

enzyme systems. However, other enzymatic routes of fonnaldehyde metabolism have also

been suggested to exist. These w thought to involve oxidation by catalase (Sies, 1974) and

dismutation by alcohol dehydtogenase (Abeles and Lee, 1 960). Their involvement in

mammalian HCHO metabolism has remained large1 y unstudied.

In ment years, much attention has been paid to the involvement of microsomal

enzymes in the oxidative metabolism of xenobiotics. Microsomal ce11 hctions are the source

of the cytochrome WSOs, an extensive group of enzymes involved in the metabolism of

many xenobiotics. The P450s are NADPH-dependent membrane-bound hemeproteins

located in the endoplasmic reticulurn. They are involved in over 40 oxidative reactions with

over 10) hown substrates (Hasler et al., 1999). Moreover, new substrates are continuously

being discovered.

Many aldehydes have been pmriously shown to be substrates for P450s. These

include retinal (Roberts et al., 1992) and phenolic aldehydes (Waianabe et al., 1990). Terelius

et al. (1991) demonsûated that CYP 2E1 is able to oxidise acetaldehyde to its corresponding

acid, acetic acid, using both micmsomal enzymes and reconstituted vesicles from s t d ,

acetone-treated rats. Kunitoh et al. (1997) reporteci similar results using human liver

microsomes. Except for the GSH-dependent ADH3 pathway, the other pathways of HCHO

metabolism that have been established appear to be p d l e l to the pathways of acetaldehyde

metabolism. It was therefore hypothesised that HCHO is also a substrate for oxidation

catalysed by CYP 2El. In the following experhents, the oxidation of HCHO to formic acid

catalysed by CYP 2El has been demonstrateû using isolated rat hepatocytes, rat liver

microsomes and human CYP 2E1 + P450 reductase + cytochrome bs SUPERSOMES? The

cytotoxic consequences associated with deficiencies in this metabolic pathway were also

studied in hepatocytes.

4.2 Hypotbesis

HCHO is oxidised to formic acid by CYP 2El and inhibition of CYP 2EI results in

an increase in HCHO-induced cytotoxicity.

4.3 Results

HCHO metabolism by the cytochrome P450s was detemiined in vitro using a

microsomal system by measuring the disappearance of HCHO over time. As shown in Table

4.1, HCHO was metabolised by normal, non-induced rat liver microsomes in the presence of

2 mM NADPH. Less HCHO loss occurred in the absence of NADPH and may be attributed

to the loss of HCHO by either vaporisation or binding to microsumal protein. To confimi that

the metabolism of HCHO observed was due to the involvement of P4SOs and not other

microsoma1 enzymes, the P450 reductase inhibitor âiphenylidonium was added 30 minutes

pnor to the addition of HCHO and NADPH. Diphenyliodonium, which inhibits the activity

of al1 P450 isoforms, prevented HCHO metabolism by the microsomes. To determine if CYP

2E1 specifically was involved in HCHO metabolism, the CYP 2E1 inhibitor phenyiimidazole

-- was added. In contrast to the uninhibiteci microsomes, HCHO metabolism occmed to a

lesser extent in the presence of phenylimidazole.

HCHO was metabolised to a rnuch greater extent using CYP 2El-induced

microsomes as compareci to non-induced microsomes (Table 4.1). Using these microsomes,

HCHO metabolism decreaseâ when CYP 2E1 was inhibited by benzylimidazole or

phenylimidazole or when P450 reductase was inhibited by diphenyliodonium. Furthexmore,

HCHO metabolism did not occur when NADH, NAD' or NADP* were used as cofactors

instead of NADPH.

The metabolism studies were carried out M e r using a system of hurnan CYP SE1 +

P4SO reductase + c ytochrome br SUPERSOMES? HCHO metabolism occuned with this

enzyme system, which gives M e r evidence for the involvement of CYP 2E1 in the

metabolism of HCHO. Metabolism with this system was much greater than with the insect

ce11 contml SUPERSOMES', which were used to control for any metabolism by native

insect ce11 proteins.

Tabk 4.1 HCHO metabolism by insect ce11 control SUPERSOMES@, rat liver microsomes, CYP 2E1 @yrazole)-hduced rat Iiver microsames and human CYP 2E1 + P450 reductase + cytochrome bs SUPERSOMES?

Microsorne treatrnent pM HCHO metabolised 60' 120' 180'

Control (no NADPH) Non-induced rat liver microsomes + 50 pM diphenyliodonium + 300 pM phenylimidazole

CYP 2El -inducd rat liver microsomes + 100 benzylimidazole + 300 phenylimidazole + 50 piid diphenyliodonium + 2 mM NADH + 2 rnM NAD' + 2 r n ~ NADP+

Insect control SUPERSOMES" Human CYP 2E1+ P4SO reductase + cytochrome b5 SUPERSOMES~

Rat liver (0.5 m m ) or insect cell control SUPERSOMES~ (0.25 mg/mL) or human CYP 2E1 + P450 reductase + cytochrome b SUPERSOMES" (50 pmol) were incubated in 0.1M phosphate buffer, pH 7.4 at 3PC with 250 pM HCHO and 2 m M NADPH (except for the control, in which NADPH was omitted, and the samples in which NADPH was replaced with NADH, NAD' or NADP?. Inhibitors were added to the microsomes 30 minutes pnor to the addition of HCHO and NADPH. HCHO levels were detennined by the method of Nash (1953) as described in Materials and Methods.

Values are expressed as the means of at least 3 separate experiments A S.E.

'Significantly different (P c 0.05) as compareci ta control (no NADPH). b~ignificantly di ffmnt (P < 0.05) as compared to non-induced rat liver microsomes. CSignificmtly âiffmnt (P < 0.05) as cornparrd to non-induced rat liver microsomes. d~ignificantly differeat (P c 0.05) as compared to CYP 2El-induced rat liver microsomes. 'Significantiy differeat (P c 0.05) as compared to insect control SUPERSOMES@.

HCHO metabolism by the P450 systern was aiso investigated using isolated rat

hepatocytes. As shown in Table 4.2, HCHO metabolism occurred to a lesser extent when the

cells were treated with the CYP 2El inhibitors benzylimidazole, phenylimidazole or

methylpyrazole. The P450 reductase inhibitor diphenyliodonium also inhibited HCHO

metabolism. However isosafble, a CYP 1A2 inhibitor, did not affect HCHO metabolism.

F o d c acid formation by HCHO metabolism was lower in hepatocytes treated with

benzylimidazole, phenylimidazole, methylpyrazole or diphenyliodonium (Table 4.2). The

inhibition of formic acid production when CYP 2E1 or P450 reductase were inhibited

correlated with the inhibition of HCHO metabolism. This suggests that CYP 2El catalyses

HCHO oxidation to fomiic acid. CYP 1A2 inhibition by isosafiole, however, did not affect

fonnic acid fornation.

Table 4.2. HCHO metabolism is inhibited in CYP 2El- and f450 reductase-inhibited hepatocytes, which correlates with the inhibition of formic acid production.

Hepatocyte treatment % HCHO metabolised F o d c acid

Control hepatocytes + 1 mM HCHO 65r2 85*2 95*4 613A18 + 100 pM benzylimidazole 57 * 2 73 *3 80* 3' 426 123~ + 300 pM phenylimidazole 54* 3 76*3 83 2' 437*20a + 1 m M methylpyrazole 53 I2 65f3 74f4a 3 8 3 ~ 1 7 ~ + 50 pM diphenyliodonium 57h3 70*2 78*3' 387*11a + 100 @id isosahle 62* 1 84*2 94*3 600*27

Hepatocytes (106 cells/ml) were incubated in Krebs-Henseleit buffer (pH 7.4) at 37OC. HCHO metabolism and fonic acid levels were determineci by the methods of Nash (1953) and Grady and Osterloh (1986). respectively, as described in Matenals and Methods.

Values are expresseci as the means of at least 3 different experiments * S.E. 'Significantly different as compared to control hepatocytes + 1 mM HCHO (P < 0.05).

nie effect of inhibiting the P450-catalysed metabolism of HCHO on hcpatocyte

viability was also investigated. As show h Table 4.3, HCHO-induced cytotoxicity increased

when non-toxic concentrations of CYP 2E 1 or P450 reductase inhibitors were used whereas

isosahle had no effect on cytotoxicity. These results suggest that CYP 2E1 plays a role in

the metabolism and detoxification of HCHO.

CYP 2E1 is involved in the oxidation of ethanol to acetaldehyde, however this

reaction has toxic consequmces in that reactive oxygen species (ROS) are fomeû as by-

products during the P450 catalytic cycle. It was therefore of interest to examine the potmtial

for ROS generation by CYP 2El-catalysed HCHO metabolism. The incubation of CYP 2El

+ P450 reductase + cytochrome bs SUPERSOMES~ with ethanol and NADPH caused a

marked increase in ROS generation compared to the control (microsomes + NADPH only) as

indicated by dichlorofluorescin oxidation (Table 4.4). Incubation of the microsornes with

HCHO and NADPH also generated ROS, but the ROS levels were markedly less than that

produced with ethanol as a substrate. ROS production h m CYP 4A11 + P45O reductase +

cytochrome bJ SUPERSOMES~ was also examined. ROS production by these microsomes

with NADPH was much greater than that by the CYP 2El microsornes with NADPH and the

addition of HCHO or ethanol had no significant effect on ROS levels with NADPH.

However the CYP 4A11 substrate laurate decreased ROS formation by the CYP 4A11

SUPERSOME~ and NADPH system.

- Table 4.3. HCHO-induced cytotoxicity increases when CYP 2E1 or P450 reductase arc inhibited in isolated rat hepatocytes.

Treatment

Control hepatocytes + 2.5 m M HCHO

+ 100 pM benzylimidazole + 300 pM phenylimidazole + 1 mM methylpyrazole + 50 pM diphenyliodonium + 100 pM isosafrole

Cytotoxicity % trypan blue uptake

60' 120' 180'

Hepatocytes (106 cells/mL) were incubated in Krebs-Henseleit buffer (pH 7.4) at 37OC. Cytotoxicity was detennined as described in Materials and Methods.

Values are expressed as the means of at least 3 different experiments I S.E.

'Significantly different fiom control hepatocytes + 2.5 mM HCHO (P < 0.05).

Table 4.4 ROS generation h m microsornes and NADPH with HCHO versus ethanol or Iaurate as substrates.

ROS (fluorescence units)

1.5 h CYP 2E1 + P450 reductase + cytochrome b5 SUPERSOMES" + 0.5 mM NADPH 95 î 7

+ 1 rnM ethanol 159* 13' + 1 rnM HCHO 117 * 9'>

CYP 4A11+ P450 reductase + cytochrome bs SUPEROMES@+ 0.5 m M NADPH 164k 17

+ 1 rnM laurate 117* 12' + 1 mM HCHO 151 10 + t rnM ethanol 154k 13

Human CYP 2El or CYP 4A11+ P450 reductase + cytochrome bs SWERSOMES~ (50 pmol) wen incubated in 40 mM potassium phosphate bufEer, pH 7.4 at 37OC with 1 mM sodium azide and 0.5 m M NADPH. ROS was detemineci by dichlorofluorescin oxidation as described in Materials and Methods.

Values are expressed as the means of at least 3 separate experiments * S.E. 'Significantly different (P < 0.05) as compared to CYP 2E1 SUPERSOMES? b~ignificantly different (P c 0.05) as compared to incubation with ethanol as substrate. 'Significantly different (P < 0.05) as compared to CYP 4A11 SUPERSOMES?

- L 4.4 Discussion

These results suggested that CYP 2El catalyses HCHO oxidation. This investigation

also showed that inhibition of this HCHO metabolic pathway had cytotoxic consequences for

isolated rat hepatocytes. HCHO metaboiism occurred to a mucb greater extent in CYP 2E1-

induced rat liver microsomes as compared to normal, non-inducd rnicrosomes, which

demonstrated the importance of this particular isoform in HCHO metabolism. HCHO

metabolism wes also catalysed by human CYP 2E1 + P450 reductase + cytochrome bs

SUPERSOMES? Moteover, the inhibition of CYP 2E1 in both microsomes and hepatocytes

inhibited HCHO metabolism. CYP 2E1 inhibition also inhibiteci hepatocyte-catalysed

formate formation fiom HCHO.

P450s have also been shown to catalyse the oxidation of retinal and phenolic

aldehydes such as perilaldehyde and veraûum aldehyde (Roberts et al., 1992; Watanabe et

al., 1990). Acetaldehyde is a compound that is stnicturally very similar to HCHO and these

two aldehydes follow many of the sarne metabolic pathways. Acetaldehyde was shown to be

metabolised Co acetate by liver microsomes fiom starved/acetone-treated rats (Terelius et al.,

1991) as well as human hepatic rnicrosomes (Kunitoh et al., 1997). The substrates of CYP

2E1 Vary greatly in terms of structure, however most are low molecular weight compounds

(Tanaka et ai., 20ûû), hiriber indicating that HCHO was also a possible substrate for this

particular isoform. However, the possibility remained open that HCHO may be metaboüsed

by additional isofonns other than CYP 2E1. Kunitoh et al. (1997) report4 that acetaldehyde

is also metabolised by other P450 isoforms such as CYP 1A2 and CYP 4A2, although these

reactions were not as catalyticdly active as that for CYP 2El. However our studies suggest

that CYP 1A2 was not involved in HCHO metabolisrn in that the CYP 1A2 inhibitor

isosaErole did not affect HCHO metabolism, fonnic acid formation or HCHO-induced

cytotoxicity. Many compounds undago enzyme multiplicity in that several enzymes catalyse

the same reaction with a particular subsmite. The involvement of other isozymes in HCHO

rnetabolism should be investigated in the future.

The possible involvement of other microsomal enzymes in the metabolism of HCHO

observed was also investigated as a control. HCHO metabolism did not occur when NADH,

NAD' or NADP+ were used as cofacton instead of NADPH. This confirms firstly that

NADPH is indeed the cofactor required for HCHO metabolism catalysed by CYP 2E1.

Secondly, the observed metabolism of HCHO was not due to reactions catalysed by other

microsomal enzymes such as microsomal aldehyde dehydrogenase, which requires NAD' in

order to be catalytically active (Martini and Murray, 1996).

Fomic acid production was inhibiteci in CYP 2E1- and P450 reductase-inhibited

hepatocytes, suggesting that CYP 2El oxidised HCHO to formic acid. Unfomuiately, fonnic

acid production could not be m e a s d in the microsomal systems due to interference by the

high level of NADPH (which has an absorption peak at 340 nm) in measuring the reduction

of iodonitrotetrazoliwn violet at 500 nm.

The catalytic mechanism of PMOs requires the oxidation of NADPH and the

reduction of molecular oxygen, thus giving rise to the tenn ''mixed function oxidase." nie

reduction of molecular oxygm, however, may lead to the formation of reactive oxygen

species (ROS) such as Hz&, qP and 'OH during the P450 catalytic cycle. ROS generation

is ofien the result of instability of the enzyme-substrate-oxygen complex, which can

dissociate and release ROS. In the case of CYP ZEl, the redox potential of the enzyrne is

quite low, making it tess able to be reduced by NADPH and P450 reductase in order to

continue with the catalytic cycle, and more capable than other P450s for autoxidising and

forming ROS (Hu et ai., 1999). Indeed, the toxicity of ethanol and the onset of alcoholic liver

disease has been parily attributed to the generation of ROS during the oxidation of ethanol by

CYP 2El (Lieber, 1997). In the case of HCHO though, cytotoxicity was increased whm

CYP 2El was inhibited; this was possibly due to two mechanisms. Firstly, the removal of

HCHO by CYP 2E1 was a detoxification mechanism in that metabolites that were less toxic

than HCHO were produced. Second1 y, the generation of ROS during HCHO metabolism by

CYP 2E1 was minimal so that the potential of any toxic threat by ROS was overcome by the

cellular antioxidant defences.

This second possibility is supported by our results with microsornes which showed

that ROS was generated during CYP 2El-catalysed HCHO metabolism, but ROS levels were

much less than that generated with ethanol as a substrate. On the other hand, laurate

prevented ROS formation with CYP 4A11 + P450 reâuctase + cytochrome bs

SUPERSOMES'@ and NADPH because it is a stable substrate for CYP 4A11 and uncoupling

of the enzyme-substrate-oxygen complex did not occur. HCHO or ethanol incubated with

these CYP 4A11 SUPERSOMES@ and NADPH did not affect ROS generation because

HCHO and ethanol are not substrates for CYP 4A11. Future studies should examine ROS

generation by CYP 2E1 with HCHO in hepatocytes in order to better evaluate the cytotoxic

potential of this reaction.

The hding that CYP 2E1 may also catalyse a HCHO detoxification pathway lads to

many potential toxic clinical implications in ternis of dmg-HCHO interactions. For example,

coincidental expsure to ethanol and HCHO may result in toxicity due to two reasons.

Fhtly, etbanol cm likely out-compte HCHO for oxidation by CYP 2E 1, leaving the ce11

vuluerable to a build-up of HCHO. Secondly, the oxidation of ethanol by both CYP 2E1 and

alcohol dehydrogenase pmduces acetaldehyde, which may be able to m e r out-compete

HCHO for oxidation by both aldehyde dehydrogenase and CYP 2E 1. This would result in a

M e r build-up of HCHO levels. We had found that the inhibition of HCHO metabolism in

hepatocytes, especiaily by acetaldehyde, increaseà their susceptibility to HCHO cytotoxicity

(Teng et al., 2001).

CYP 2El is also a polymorphic enzyme, with several restriction fragment length

variations having been found among the general population (Hasler et al., 1999). Studies

have found that there is no iùnction for the variations since they appear to afîéct only non-

coding ngions of the CYP 2E1 gene (Hu et al., 1997), yet it has also been shown that mutant

versions of the enzyme possess increased hydroxylation activity with chlonoxazone as a

substrate (McCarver et al., 1998). Despite the controversy, it is interesting to consider that if

a functional variation does exist which results in decreased HCHO metabolising activity,

such a variation may increase the susceptibility of some individuals to HCHO toxicity.

Several enzymatic metabolic pathways of HCHO, which detoxify this reactive and

toxic compound, have already been f k l y established. This present investigation has shown

that CYP 2E1 is anotha metabolic route for HCHO. The relative importance of this

metabolic route in tenns of contribution to the overall metabolisrn of HCHO was not

detedned, however this is a difficult cietennination to make since the activity of CYP 2El

can vary greatly.

The potential implications of the findings of this investigation are numemus. CYP

2E 1 oxidises many compounds, thus drug-HCHO interactions are Wtely to arise. CYP 2El is

also induced or inhibited by many compounds, thus the extent of the involvement of CYP

2El in HCHO metabolism may Vary depending on the presence of inducers or inhibitors,

competing substrates and any functional polymorphisms of the enzyme. Ia fact, HCHO itself

was reported to induce rat lung P450 levels following chronic, repeated exposures to inhaled

HCHO (Dallas et al., 1 989). This may have a fiirther effect on HCHO metabolism by P450s

and thus cytotoxicity. Clearly, the involvement of P4SOs in HCHO metabolism is an ana that

should be exploreci m e r as it has the potential to have significant consequences on HCHO

toxicity.

ANTIDOTES TO FORMALDEHYDE CYTOTOXICI'IX AMINOITHIOLS OR MODULATING CELLULAR REDOX POTENTIAL

5.1 Introduction

The buk of formaldehyde (HCHO) metabolism is catalysed by three enzymes:

alcohol dehyhgenase (ADH 1 ), aldehyde dehydrogenase (ALDH2) and fomaldehyde

dehydrogenase (ADH3). ADHl is cytosolic and reduces HCHO to methanol using NADH as

a coenzyme. ALDH2 and ADH3, which are mitochondrial and cytosolic, respec tively, both

oxidise HCHO to fonnic acid using NAD' as a coenzyme, although ADH3 also requues

GSH in order to be catalytically active.

HCHO is a very toxic compound, thus its metabolism and removai fiom the ce11 is

essential for its detoxification. Therefore, the relative activities of ADHI, ALDH2 and

ADH3 are important in determining the toxicity of HCHO towards cells. We showed that

inhibiting HCHO-metabolising enzymes results in a build-up of HCHO, which subsequently

increases HCHO-induced cytotoxicity towards rat hepatocytes, whereas NADH generators

decreased c ytotoxicity (Teng et al., 200 1). The dependence of HCHO metabolism on

enzymes requiring NAD+ or NADH suggest that NAD' and NADH levels may also be

important in determining the rate or extent of HCHO metabolism by ADH 1, ALDH2 or

ADH3 and consequently the cytotoxicity of HCHO.

Considering that increasing the enzymatic metabolism of HCHO may serve as a

detoxification mechanism, it is aiso possible that increasing the metabolism of HCHO by

adàing HCHO trapping agents should also protect hepatocytes h m HCHO cytotoxicity.

HCHO is very reactive towards nucleophiles such as amines and thiols. Thus it readily

attacks proteins, DNA and cellular thiols such as GSH, leading to toxicity. This reactivity

with amines and thiols, however, may be used instead to prevent or treat HCHO toxicity.

Studies have already shown that some amines and thiols can prevent aldehyde toxicity both

in vivo and in vitro. For example, metformin is an amino drug which reacts with and removes

dicaibonyls such as methylglyoxal and glycolaldehyde, which are present in excess levels

under diabetic conditions (Ruggiero-Lopez et al., 1999). One of the causes of alcoholic liver

disease is the production of the toxic metabolite acetaldehyde fiom ethanol. Investigators

have found that the administration of L-cysteine by oral intubation to rats 30-45 minutes

prior to exposure to a lethal dose of acetaldehyde prevents acetaldehyde-induced death

(Spnnce et al., 1974).

in the following, the effect of NADH generators or NADH oxidisers on HCHO

metabolism and cytotoxicity is examined. The chemical metabolism of HCHO by reaction

with amines and thiols is also examined for its effect on HCHO-induced cytotoxicity.

5.2 Hypothesis

HCHO-induced cytotoxicity may be prevented by increasing its metabolisrn with

NADH generators or NADH oxidisers or by trapping with some amines or thiols.

5.3 Results

HCHO-induced cytotoxicity and lipid peroxidation were markedly decreasd in

hepatocytes treated with the NADH generators xylitol, hctose and lactate (used at

concentrations which did not affect hepatocyte viability over 3 hours) (Table 5.1). The

addition of the NADH oxidisers phenazine methosulfate (PMS), 2,6-dichlorophenolindo-

phenol (DCPIP) or pyruvate also decreased HCHO-induced cytotoxicity and lipid

pemxidation.

As shown in Table 5.1, the cytoprotective effects of the NADH grnerators xylitol,

lactate and hctose was accompanied by increased HCHO metabolism by hepatocytes and

suggests that HCHO metabolism results in its detoxification. The NADH oxidisers pymvate,

PMS or DCPIP also prevented HCHO c ytotoxicity and increased HCHO metabolism.

The possible involvement of peroxisomal catalase in methanol metabolism was

investigated by the addition of glycolate, which produces H202 in peroxisomes. Glycolate

increased HCHO production h m methanol (Table 5.2) which suggests that catalase

catalyses the oxidation by H 2 q of methanol to HCHO. HCHO production was inhibited

when catalase was inhibited by non-toxic concentrations of azide, 3-amino- 1,2,4-tnazole or

cyanide, but cytotoxicity did not occur, indicating that the HCHO concentration reached was

not sufficient to cause ce11 death. HCHO levels were decreased in the presence of pyruvate,

xylitol and lactate. On the other hand, the effact of xylitol at decreasing HCHO levels was

prevented by methylpyrazole, an alcohol dehydrogenase inhibitor. Interestingly, cytotoxicity

produced by glycolate in GSH-depleted hepatocytes (74 6% at 3 hours) was prevented by

methanol(37 * 4 at 3 hours) and was accompanied by HCHO production (429 * 13 ph4 at 3

hours).

To detemine the reversibility of HCHO-induced cytotoxicity, the effect of nmoving

HCHO h m the ce11 incubation medium by discarding the buffa and replacing it with h h

bWa was examined. As shown in Table 5.3, resuspending the hepatocytes in HCHO-fke

buffer after 30 minutes pmented ce11 blebbing and cytotoxicity h m tùrther occurring.

However resuspension after 60 minutes was less cytoprotective and resuspension after 120 --A -

minutes was not cytoprotective.

Table 5.1 The efféct of NADH generators or NADH oxidisers on HCHO-induceû cytotoxicity and lipid peroxidation and HCHO metabolism.

Treatment

Lipid Cytotoxiciîy peroxidation % Trypan absorbance at % HCHO

blue uptake 532 nm metabolised 180' 180' 60'

Control hepatoc ytes 24* 2 0.024 0.004 - + ~ ~ M H C H O - 70 a 6' 0.343 0.08' 59*4

+ 10 mM xylitolc 4814~ 0.156*0.07~ 77 * 3b + 10 mM lactateC 40 i 4b 0.1 16 k 0.05~ 75 * 4b + 10 mM fhctosec 4 5 1 9 0.128*0.03~ 71 1 4 ~ + 10pMPMS 38*5b 0.12110.04~ 81 1 4 ~ + 10 pM DCPIP 39h4b 0.119*0.05~ 86 *4b + 10 mM pyruvate 50 * 6b O. 182 * 0.07~ 74. 3b

Hepatocytes (106 cellslmL) were incubated in Krebs-Henseleit buffer (pH 7.4) at 3TC. The detemination of cytotoxicity, lipid peroxidation and HCHO levels were carried out as described in Matenals and Methods.

Values are expressed as the means of at lest 3 separate expenments S.E.

'Signi ficantl y di fferent from control hepatoc ytes (P < 0.05). b~ipificantly different fiom control hepatocytes + 4 mM HCHO (P < 0.05). 'Results taken from Teng et al., 2001.

-= -A - Tabk 5.2 HCHO production h m methanol with glycolate and its modulation.

Treatment pM HCHO produced 60' 120' 180'

Control hepatocytes + 75 mM methml 26f5 33f6 37f 5 + S m M glycolate Control hepatocytes + 150 rnM methanol + 2 mM glycolate + 5 m M glycolate

+ 500 pibi aide + 15 mM 3-amino-1,2,4-triazole + 1 m M cyanide + 10 m M xylitol + 10 mM lactate + 10 mM pyruvate + 10 mM xylitol+ 200 pM

methylpyrazole

Hepatocytes (106 cells/mL) were incubated in Krebs-Henseleit buffer (pH 7.4) at 37OC. The determination of HCHO levels was carried out as described in Materials and Methods.

Values are expressed as the means of at least 3 separate expenments * S.E.

'Sipificantly different fiom control hepatocytes + 150 m M methanol (P c 0.05). b~ignifi~antly different fiom control hepaioc ytes + 150 mM methmol+ 5 rnM glycolate (P < 0.05).

Table 5.3 Effect of replacing the hepatocyte incubation buffer on HCHO cytotoxicity.

Treatment % Cytotoxicity 30' 60' 120' 180'

Control hepatocytes 1 8 f 2 2 0 I 2 2 1 f 3 2 3 f 3 + 4 m M HCHO 3 9 f 3 4 5 I 4 5 4 f 4 6 8 f 4

+ replaced buffer at 30 min. 3 6 f 2 3 8 f 3 a 3 9 f 3 ' 4 1 f 3 ' + replaced buffer at 60 min. 4 0 f 3 4 6 f 4 4 9 i 4 5213' + replaceà buffer at 120 min. 3 8 f 4 4 7 f 4 5 3 f 4 7 3 f 3

Hepatocytes (106 celIs/mL) were incubated in Krebs-Henseleit buffer (pH 7.4) at 37OC. The cells were cenûifbged and then resuspended in HCHO-fiee buffer at the indicated tirne points. The determination of cytotoxicity was c h e d oui as describeci in Matenals and Methods.

Values are expressed as the means of at least 3 separate expenments f S.E.

'Significantly different from control hepatocytes + 4 mM HCHO (P < 0.05).

As show in Table 5.4, the addition of non-toxic concentrations of the amino-thiols

L-cysteine or D-penicillamine or the amine hydroxylamine to hepatocytes treatad with an

equMolar dose of HCHO resulted in a rapid disappearance of HCHO and protection h m

HCHO-induced c ytotoxicity . L-c ysteine, D-penici Ilamine and hydrox ylarnine were still

cytoprutective even if administered 30 minutes after the addition of HCHO. The amine

aminoguanidine also protected hepatocytes from HCHO-induced cytotoxicity and increased

the disappearance of HCHO, although it was less effective than the other compounds tested.

An equimolar concentration of metformin, an antidotal h g similar in structure to

aminopanidine, had no effect on HCHO removal or cytotoxicity. However a higher dose of

metformin increased cytotoxicity markedly. Cystine, the disulfide dimer of cysteine, had no

effect on HCHO metabolism or cytotoxicity, suggesting the importance of the thiol group of

cysteine in its reaction with HCHO. Interestingly, when hydroxylamine, but not the other

compounds, was added to GSH-depleteâ hepatocytes treated with HCHO, c yto toxicity

increased despite a rapid disappearance of HCHO. This suggested that a product formed fiom

HCHO and hydroxylamine was toxic possibly through an oxidative stress-rnediated

mec hanisrn.

- Table 5.4 The disappearance of HCHO upon the addition of lcysteine, D-peni~illarnine~ aminoguaoidinev metformh, hydroxyïamhe or L-cystine to hepatocytes and the associafed cytotoxic effects.

Treatment

Control hepatocytes + 4 mM HCHO

+ 4 mM L-cysteine + 4 mM L-cysteine (30') + 4 mM penicillamine + 4 mM penicillarnine (30') + 4 mM hydroxylamhe + 4 rnM hydmxylamim (30') + 4 mM aminoguanidine + 4 mM metfonnin + 10 m M metfonnin + 4 mM cystine

GSH-depleted hepatoc ytes +2 mM HCHO

+ 2 mM L-cysteine + 2 mM D-penicillarnine + 2 rnM hydroxylamine + 2 mM aminoguanidine

% HCHO removed % cytotox. 30' 60' 120' 180' 180' --

BDL 87 f 3' BDL

83 f 4' BDL 90 * 3' BDL BDL BDL BDL BDL -- 27* 3 84 * 4b 86 4b 90 sb BDL

C

63 I S 90 * 4a 89 * 4' 88 * 4' 83 * 3' 92 k 3' 84 I 3' 69 i 4 BDL BDL BDL -- 43 * 3 88 * sb 87 * 4b 91 * 4b 62 * 3b

Hepatocytes (106 celWmL) were incubated in Krebs-Henseleit buffer at pH 7.4.37OC. Cytotoxicty and HCHO metabolism were determined as described in Materials and Methods.

Values are expressed as the means of at least 3 separate experiments * S.E. 'Significant difference as compared to control hepatocytes + 4 mM HCHO (P < 0.05). b~ignificant difference as cornparrd to GSH-depleted hepatocytes + 2 mM HCHO (P < 0.05)- BDL: beyond the detection limit of the assay

To determine if the rapid disappearance of HCHO with Gcysteine, D-penicillamine,

aminoguanidine and hydroxylamine was due to the chernical trapping of HCHO by these

compounds and not by an effect on the HCHO-metabolishg enzymes or co-enzymes, the

reactivity of these compounds with HCHO was examhed. Figure 5.1 shows that HCHO

reacted with Ltysteine to fonn thiazolidine4~arboxylic acid (TC) as suggesteà by their

identical W spectra. In addition, HCHO rapidly reacted with L-cysteine, D-peniciliamine,

hydroxylamine and aminoguanidine in the absence of hepatocytes, as demonstrated by

kinetic scans at various wavelengths in phosphate buffer (Figure 5.2). The reactions took

place within 5 minutes, suggesting that even if the compounds were able to increase the

enzymatic metabolism of HCHO, the trapping of HCHO would occur before the enzymes

were affected. Cystine, the disulfide dimer of cysteine, did not react chemically with HCHO

and thus had no effmt on HCHO trapping or cytotoxicity in hepatocytes.

Figure 5.1 Spectmphotometric cornparison of thiazolidine-4-carboxylic acid (TC) versus HCHO + Gcysfeine.

200 210 220 230 240 250 260

Wsvdength (nm)

Legend: - (hiazolidine-4-carbox ylic acid . . . L-c ysteine on1 y -- Ltysteine + HCHO at 10 min.

250 ph4 thiazolidine-4-carboxylic acid (TC) was compared to 250 pM HCHO + 250 pM L-cysteine in 0.1 M phosphate buffer (pH 7.4). The HCHO + Lcysteine reaction was incubated at 37OC for 10 minutes before scanning.

Figure 5.2 The disappearance of HCHO in the presence of amino and thiol compounds.

500 phd HCHO was reacted with 500 pM amino or thiol compounds in O. 1 M phosphate buffer, pH 7.4. Reaction rates were measured by following the absorbance at specific wavelengths for each individual amine or thiol. These wavelengths were determineci by firstly scanning the spectrum of the amine or thiol and then comparing it with the spectrum after the addition of HCHO. The wavelength of a newly-formed peak was useci to detemine the reaction rate in a kinetic scan. The following wavelengths were used: cysteine, 201 nm; penicillamine, 200 nrn; hydroxylamine, 215 nm; aminoguanidine, 225 nm; cystine, 206 m.

Values are express4 as the means of 3 separate experimmts * S.E. * Significantly different as compared to HCHO + L-cysteine (P < 0.05).

5.4 Discussion

It was shown previously that inhibiting the HCHO metabolising enzymes ADHI,

ALDH2 and ADH3 resulted in an increase in HCHO-induccd cytotoxicity in isolated rat

hepatocytes (Teng et al., 2001). Modulating the redox state of the cell (NAD'/ NADH levels)

however decreased cytotoxicity due to an increase in enzyme activity. The NADH generators

xylitol, fhctose or lactate decreased HCHO-induced cytotoxicity and lipid peroxidation. This

was accompanied by an increase in HCHO metabolism, presumably due to an increase in

HCHO reduction to methanol as catalysed by ADHI. Similarly, the oxidation of NADH to

NAD+ by PMS, DCPLP or pynivate resulted in an increase in the ALDHZ- and ADH3-

catalysed oxidation of HCHO to formate, and decreased HCHO-induced cytotoxicity. In a

study by Waydhas et al. (1978), the NAD' reâuctants 3-hydroxybutyrate, lactate or xylitol

inhibited the in hi de pendent oxidation of HCHO to COz but not fomate to CO2 in

perfused rat liver, which M e r emphasises the importance of the NADH:NAD+ ratio in

detemining the route and the extent of HCHO metabolism.

Peroxisomal catalase was found to be involved in the oxidation of methanol to

HCHO, since glycolate, which produces HCHO during its oxidation to glyoxylate by

glycolate oxidase in peroxisomes, increased HCHO production k m methanol. HCHO

production was prevented when catalase was inhibited by azide, 3-amino- 1,2,4-tnazole or

cyanide. The metabolism of the HCHO produced h m methanol was aiso af'fécted by cellular

levels of NAD+ or W H , as there was less HCHO when NAD+ or NADH levels were raised

by pynivate, xylitol or lactate. These results demonstrate that increasing NAD+ or NADH

levels may prevent methanol toxicity due to HCHO by increasing HCHO metabolism.

- -

Cytotoxicity could alm be prevented if the remaining HCHO was removeci 30

minutes later following the addition of HCHO by resuspending the hepatocytes in fresh

buffer. However removing HCHO at 60 minutes was less effective at preventing cytotoxicity

and removing HCHO at 120 minutes did not a e c t the cytotoxicity suggesting that there is an

apparent ''point of no renun" for the omet of HCHO cytotoxicity. This suggests that HCHO

poisoning could not be treated by removing HCHO with slower methods such as dialysis.

Trapping HCHO with amines or thiols was found to be another route of

detoxification. The prevention of HCHO-induced cytotoxicity by the addition of L-cysteine

(Figure 5.3a) even 30 minutes following the administration of HCHO was attributed to the

reaction of L-cysteine with HCHO to fom thiazolidine-4-carboxylic acid (TC) (Figure 5.3b).

This supports the work of Guem et al. (1976), who found that death induced by i.p.

administration of lethal doses of HCHO in rats was attmuated by the injection of thiol agents

including cysteine several hours after HCHO exposw. ûther studies showed that

pretreatment of rats with cysteine either orally (Spnnce et al., 1979) or i.p. (Strubelt et al.,

1990) prevented HCHO-induced death and cardiovascular failure, respectively. This

protective effect of L-cysteine has been attributed to the trapping of HCHO to form TC,

which is non-toxic and is excreteâ in the urine in vivo (Hernrninki, 1984). The reaction

between HCHO and L-cysteine is known to involve firstly a nucleophilic attack by the amino

group of L-cysteine on the HCHO caibon to subsequently fom a Schiff-base intermediate

which then cyclises to fom TC, a closed ring (Kallen, 1971).

Despite the obvious thetapeutic effocts of L-cysteine in treating and preventing

HCHO toxicity both in vivo and in vitro with isolateci hepatocytes, the clinical use of L-

cysteine may be limited due to the rapid metabolism of L-cysteine when administemxi in

vivo. High doses of cysteine would be more effective but could be toxic in vivo (Harper et

al., 1970). Cystine (Figure 5.3e). the disulfide dimer of cysteine, was not very reactive

towards HCHO and was thus not effective at preventilig HCHO-induced cytotoxicity likely

because of the absence of a free sulfhydryl group which prevented thiazolidine ring

formation.

n ie limitations of using L-cysteine as a HCHO antidote may make D-penicillarnine a

more attractive alternative. Nagasawa et al. (1 977; 1 980) demonstrated that D-penicillarnine

(Figure 5.3~) is an effective sequestering agent for acetaldehyde through the formation of a

thiazolidine ring (Figure 5.3d) and proposed the use of D-penicillamine as a means for

preventing acetaldehyde-induced toxicity in alcoholics or in ALDH2-deficient individuals.

As shown in our study, the ability of D-penicillamine to trap HCHO and prevent HCHO-

induced cytotoxicity in isolated hepatocytes was equal to that of L-cysteine. D-penicillamine

is cunently used clinically to treat rheumatoid arthntis and to chelate copper ions in the

treatment of Wilson's disease (Howard-Lock et al., 1986), however its use could be extended

to include the treatment of HCHO poisoning.

Metformin (Figure 5.3f) is a dmg in clinical use for the treatment of diabetes by

acting as a gluconeogenesis inhibitor and scavenger of a-oxoaldehydes (Beisswenger et al.,

1999; Ruggiero-Lopez et al., 1999). It was not able to scavenge HCHO, however cytotoxicity

ensued with the higher dose of metformin likely because of the inhibitos, effect of metformin

on cellular respiration (El-Mir et al., 2000; Owen et al., 2000). Aminoguanidine (Figure 53g)

is another dnig that scavenges a-oxoaldehydes (Thomalley et al., 2000) and is being

suggested as a treaûnent for âiabetic complications. However it was found to have oniy a

slight cytoprotective effect in hepatocytes as a HCHO scavenger. Its cytoprotective eEect

was markedîy less than that of Lcysteine or D-penicillamine, perhaps because the product

formed from HCHO and aminoguanidine was unstable or dissociated quickly. The

thiazolidines formed fiom HCHO and Lcysteine or D-pencillamine, on the otha hand, are

stable because of their closed ring structure. Another possible reason for the lesser

effectiveness of atninoguanidine is that the b u h e s s of the substituent on the amine of

aminoguanidine rendered the compound to be less nucleophilic due to stenc hindrance. Thus,

its ability to react with HCHO was compromised.

The marked increase in cytotoxicity observed when hydroxylamine (Figure 5.2h) was

added to HCHO-treated hepatocytes that were depleted of GSH @ut not in hepatocytes with

normal levels of GSH) was suspected to be due to the fornation of an oxime (Figure 5.2i).

Previous studies have shown that oximes have oxidative effects in erythrocytes (e.g.

hemoglobin oxidation, lipid peroxidation, GSH depletion), but interestingly oxime-induced

lipid peroxidation and GSH depletion were not seen in hepatocytes (Palmen and Evelo,

1998). This contrasts our result which suggests that the oxime was toxic by an oxidative

stress-mediateci mechanism. Thus the toxic mechanism of the oxime remains to be stuàied.

In conclusion, the cytotoxicity of HCHO can clearly be manipulateci by modulating

its elimination. Whereas our previous studies showed that inhibiting HCHO metabolism

increases its cytotoxicity (Teng et al., 2001), the present investigation shows that increasing

HCHO metabolism either enzymatically (with NADH generators or NADH oxidisers) or

chemically (by trapping HCHO with amines or thiols) results in detoxification. These results

may be used towards the development of more effective methods of treating HCHO

poisonhg by removing it more quickly h m the body before irreversible toxicity ensues.

Figure 5 3 Structures of the amines and thiols used and the proposed proâucts f o d by their reactions with HCHO.

I H2 H2 I CH-C-S-S-C-CH

(a) L-cysteine; (b) thiazolidine-4-carboxyüc acid (TC); (c) D-peniciliamine; (d) 5,s-dimethylthiazolidine-eCarboxylic acid; (e) cystine; (f) metfocmin (dimethyi biguanide); (g) aminoguanidine; (h) hydroxylamine; (i) oxime

THE E M C T OF CATECHOLAMINES ON FORMALDEHYDE METABOLISM AND CYTOTOXICITY IN ISOLATED RAT HEPATOCYTES~

6.1 Introduction

Fomaldehyde (HCHO) readily reacts with nucleophilic compounds such as amines

and thiols including DNA, cysteine residues in proteins and GSH. We showed previously

that the reaction of HCHO with amines such as L-cysteine, D-penicillamine, aminoguanidine

or hydroxylamine al1 resulted in the trapping of HCHO and subsequently decreased HCHO-

induced cytotoxicity in hepatocytes (Chapter 5 of this thesis). The reactivity of HCHO with

amines is well-known, however a more ment area of shidy has focused specifically on the

reactions between aldehydes and catecholarnines.

Aldehydes are able ta react with catecholamines to form isoquinoline derivatives such

as tetrahydroisoguinolines (THIQs). Although the reactions between HCHO and the amines

studied previously resulted in the detoxification of HCHO by the formation of non-toxic

conjugates with HCHO, there is growing concem over the possible toxicity of THlQs. Moura

et al. (1977) showed that the THIQ of acetaldehyde and epinephrine (1 2-dimethyl-4,6,7-

trihyâroxy- 1,2,3,4-tetrahydroisoquinolinc) caused extensive necrosis in rat liver in vivo. This

may lead to speculation on the role of THIQs in the hepatotoxicity of ethanol. However

interest in THIQ toxicity is greatest with respect to the etiology of neurological disorclers as

THIQs have been suggested to be involveà in the idiosyncratic development of Parkinson's

discase because of their structural similarity to 1 -methyl-4-phenyl- l,2,3,6-teûahydrop yridine

(MPTP), a well-known inducer of Parkinson-like symptoms in mammals. Cases of

idiosyncratic Parkinson's disease may occur as a result of chronic exposure to either THIQs

themselves or their parent compounâs such as aldehydes, which may result in the

endogenous formation of THIQs. Cases of neurobehaviouraî impairment in humans and rats

have resulted h m chronic or acutc exposure to HCHO or methanol, although the mechanisrn

of this impairment has not been detennined (Guggenheim et al., 1971; Kallen, 1971; McLean

et al., 1980; Oliveras-Ley and Gali, 1983; Boja et al., 1985; Anderson et al., 1989; Pitten et

al., 2000). Thus the effect of catecholamines on HCHO cytotoxicity would be of great

interest to study because of the possible association between THIQs and both neurotoxicity

and hepatotoxicity. in the following, we used isolated rat hepatocytes as a ceIl mode1 for

detennining the effect of catecholamines on HCHO metabolism and cytotoxicity.

6.2 Hypotbesis

HCHO-induced c ytotoxici ty is incna

form tetrahydroisoquinolines.

, trapping HCHO with catecholamines to

6.3 Results

HCHO rapidly disappeared in the presence of equimolar non-toxic doses of the

catecholamines dopamine, epinephrine, norephephrine and deoxyepinephrine (Table 6.1).

Although HCHO was almost entirely removed by the catecholamines within 30 minutes,

extensive cytotoxicity did not occur until3 hours. N-acetyldoparnine, which lacks the fret

amino group of dopamine. did not remove HCHO and had no efféct on HCHO cytotoxicity.

Interestingly, the addition of 6,7-dihydroxy-Nmethy1-1,2,3,4-tetrahyd.isoquinoline

(N-methyInofSallsolino1 (NMNS)), which is formed by the condensation of HCHO with

deoxyephephrine, was less toxic than the addition of stoichiometric doses of its individuai

parent compounds. 2 rnM NMNS was needed to achieve the same cytotoxicity as 1 mM L L -

HCHO and 1 m M deoxyepinephrine added separately. Similarly, a pre-mixed solution of

HCHO and deoxyepinephrine (1 mM each, pre-incubated for 15 minutes at room

temperature) was also not toxic. However, a solution that was pre-incubated for only 5

minutes was cytotoxic.

To determine if the lack of toxicity seen with pre-fomed NMNS was possibly due to

a difficulty in THIQ transport into the cell, NMNS was added to cells treated with

imipramine, an inhibitor of catecholamine transport into cells (Westwood et al., 1 996).

However, cytotoxicity was not prevented, suggesting that NMNS is taken up by another

transporter that is not affected by imipramine, or that transporters are not involved at al1 in

NMNS uptake. The efléct of pre-loading the cells with deoxyepinephrine for 30 minutes

prior to the addition of HCHO was also briefly investigated. Cytotoxicity still ensued, even if

the deoxyepinephrine was washed nom the incubation buffer afler 30 minutes (100%

cytotoxicity at 3 hours in both cases). These results suggest that NMNS can fonn inside the

ce11 when deoxyepinephrine and HCHO were addeô to the cells separately, and that pre-

fonned NMNS may have a difficulty in penetrating the ce11 membrane. On the other hand

imiprarnine, which blocks norepinephrine uptake into the cell, did not pnvent the toxicity

induced by norepinephrine and HCHO. This suggests that unlike NMNS, the product of

norepinephrine condensation with HCHQ 4,6,7-bihydroxy-1,2,3,4-tetrahydmisoquin01ine, is

better able to enter the ce11 after forming in the extracellular medium.

Table 6.1 The metabolism of HCHO by catecholamines and îhe associated cytotoxic effects.

Treat men t

Control hepatocytes + 1 m M HCHO + 1 rnM epinephrine + 1 mM dopamine + 1 mM deoxyepinephnne + 1 m M norepinephrine + 1 mM N-acetyldopamine

1 m M N M N S 2 mM NMNS 1 mM HCHOI1 mM deoxyepi. pre-incubated solution (5') 1 mM HCHO/l mM deoxyepi. pre-incubateci solution (1 5')

% HCHO metabolised 30' 60' 120' 180'

Hepatocytes (1 o6 cellshn~) were incubated in Krebs-Henseleit buffer (pH 7.4) at 37°C. Detemination of cytotoxicity and HCHO metabolism were carried out as described in Materials and Methods.

Values are expressed as the means of at least 3 separate experiments * S.E.

'Significantly different from control hepatocytes + 1 rnM HCHO (P < 0.05).

To c o ~ m that a chernical ~ a c t i o n does take place betwem HCHO and the

catecholamines, the nactions were monitored for the rate of THIQ formation. As shown in

Figure 6.1, the reactions occurred quickly except for N-acetyldopamine, which suggests the

amino p u p of the catecholamines participates in the reaction with HCHO. The increase in

HCHO metabolism observeci with catecholamines was not due to an enhancing effact of the

catecholamines on the activity of the HCHO-metabolising enzymes such as alcohol

dehydrogenase, aldehyde dehydrogenase or formaldehyde dehydrogenase since the reactions

took place very quickly (completion before 15 minutes).

A cornparison between the spectrophotometic scans of the HCHO-deoxyepinephrine

reaction and that of NMNS shows that the scans are identical, suggesting that NMNS is

indeed formed h m HCHO and deoxyepinephrine (Figure 6.2). Moreover, NMNS was

formed with hepatocytes as shown by mass spectrometry. The MSlMS profiles of NMNS

(Figure 6.3a), the reaction product of HCHO and deoxyepinephrine formed afler 15 minutes

incubation with hepatocytes (Figure 6.3b) and the reaction product fomed after 15 minutes

in phosphate buffer (Figure 6 . 3 ~ ) are identical. The presence of NMNS in hepatocytes

indicates that NMNS was responsible for the toxicity seen with HCHO and deoxyepinephnne

in hepatocytes. However, the mechanism of toxicity remains to be determined.

Figure 6.1 The chernical metabolism of HCHO by catecholarnines.

500 pM HCHO was reacted with 500 pM catecholamine in 0.1 M phosphate buffer, pH 7.4. Reaction rates were measureâ by following the absorbance at specific wavelengths for each individual catecholamine. niese wavelengths were detennined by firstly scanning the spectnun of the catecholamine and then comparing it with the spectrum af?er the addition of HCHO. The wavelength of a newly-formed peak was used to determine the reaction rate in a kinetic scan. The wavelengths were the following: deoxyepinephrine, 484 nm; dopamine, 482 imi; cpmcphrint, 482 mn; nomphcphrint, 483 mn; N-acttytdopamint, 482 mn.

Values are expnssed as the means of 3 separate experirnents * S.E.

* Significantly different as cornparrd to HCHO + deoxyepinephnne (P < 0.05).

230 250 270 290 310 330 350 Wavtltngth (nm)

Legend: - NMNS . . . deoxyepinephrine only .. deoxyepinephrine + HCHO at 10 minutes. - -

500 pM NMNS was compared to 500 pM HCHO + 500 ph4 deoxyepinephrine in 0.1 M phosphate buffer (pH 7.4). The HCHO + deoxyepinephrine reaction was incubated at 37OC for 1 O minutes befort scanning.

Figure 6.3b MS/MS profile of the reaotion of HCHO and deoxyepinephrine with hepatocytes after incubation for 15'.

- 6 . 4 D b h

In assessing the cytotoxicity of HCHO, it is important to consider the reactivity of

HCHO towards endogenous amines or thiols. HCHO is known to react with sulfhydryl amino

acids in proteins, the amino groups of DNA nucleotides and GSH in addition to

catecholamines. Cstecholarnines are known to form THIQs upon condensation with

aldehydes. For example, alcoholics have been found to have high levels of acetaldehyde-

deriveci THIQs, the most noted of which is 1 -rnethyl6,7=dihydroxy- 1,2,3,4-

tetrahydroisoquinoline (salsolinol) (Figure 6.4a), fonned by the condensation of acetaldehyde

with dopamine (Collins et al., 1979).

HCHO was found to be rapidly removed by hepatocytes in the presence of

catecholamines which resulted in a marked increase in cytotoxicity. A spectrophotometric

scan and MS/MS analysis showed that HCHO and deoxyepinephrine (Figure 6.4b) react to

form NMNS (Figure 6.4~) in a cell-free system. suggesting this to be the product formed by

trapping HCHO with deoxyepinephrine in hepatocytes. Interestingly, NMNS itself was also

show to be cytotoxic which firther suggests that NMNS was formed with hepatocytes and

was the compound that was mponsible for the cytotoxicity observed. n ie fonnation of

NMNS with hepatocytes was confirrned by MS/MS analysis. However the lack of toxicity

seen with pre-formed NMNS relative to the addition of HCHO and deoxyepinephrine

separately to cells suggests that NMNS may have difficulty in cmssing the ceIl membrane

whereas HCHO and deoxyepinephrine can enter the cells separately before fomiing NMNS

inside the cell. The onset of toxicity with pre-loaded deoxyepinephrine, even following the

washing of deoxyepinephrine h m the incubation mddium, supports this hypothesis. On the

other hand, 4,6,7-trihydroxy- 1,2,3,4-tetrahydoisoquinole (Figure 6 4 ) formed h m the

- condensation of HCHO with norepinephrine may be more membrane permeable as

inhibiting norepinephrine uptake with imipramine (Limer et al., 1999) did not prevent

cytotoxicity. This demonstrates that 4,6,7-trihyroxy-l,2,~,~-teüahydroisoquinoline fomed

outside of the cell, but was able to peneûate the ce11 membrane in order to cause toxicity.

Re-mixing the HCHO-deoxyepinephrine stock solution for 5 minutes was more cytotoxic

than pre-mixing for 15 minutes. This may have been due to incomplete NMNS formation in

the solution at 5 minutes, therefore allowing the remaining free HCHO and deoxyepinephrine

to enter the ceIl before forming NMNS. On the other hand, by 15 minutes, al1 of the HCHO

and deoxyepinephnne had reacted and the NMNS formed was not toxic because of a lack of

membrane permeability. Altematively, pre-formed NMNS may have been metabolised either

in the stock solution or in the extracetlular medium, and this metabolite was either less

membrane permeable or less toxic. Clearly, further investigation is needed to determine the

reason for the differences in cytotoxicity seen based on the method of THIQ dosing to

hepatocytes.

The mechanism of THIQ cytotoxicity also remains unclear, however it may be

similar to that of MPTP (Figure 6.4e), a known inducer of Parkinsonism and a structural

analog to the THIQs. MPTP is metabolised in astrocytes to 1-methyl-4-pyridinium ion

(MPP~ , which is taken into dopamimrgic neurons by dopamine transporten. MPP+ (Figure

6.49 is then able to inhibit complex 1 of the mitochondrial electron transport chah, thereby

pmenting the production of ATP, leading to an energy crisis followed by ce11 death (Cleeta

et al., 1992). McNaught et al. (1995) reportecl that various isoquinoline derivatives an able to

inhibit mitochondrial respiration at complex 1, suggesting tbat this effcct may be involved in

the toxic mechanisn of isoquinolines. In contrast, Storch et al. (2000) reporteci that salsolinol

.-A. .- - . . . . . is-toxic to.neuroblastoma SH-SYSY celloby inhibithg complex H. However, the

characteristic delay followed by a sudden extensive increase in cytotoxicity observed in our

study is suggestive of an autoxidation-mediated mechanism of cytotoxicity. 1,2-dimethyl-

6,7-dihydroxy-l,2,3,4-tetrahydroisoquinoline (N-methyl-(R)-salsolinol) has been

demonstrated to promote hydroxyl radical production in vivo in rat striatum (Maruyama et

al., 1995a) and in vitro (Maruyarna et al., 1995b) during its nonenzymatic oxidation to 1,2-

dimethyl-6.7-dihydroxyisoquinolinium ion. It is therefore possible that the toxic mechanian

of THIQs may involve firstly the inhibition of complex 1, which results in an increased

kakage of electrons from the electron transport chah. These electrons can then reduce

molecular oxygen to form supemxide radicals and other toxic reactive oxygen species. The

inhibition of complex 1 could also nsult in increased intracellular oxygen levels which would

autoxidise the tetrahydroisoquinolines to quinones, while generating reactive oxygen species

at the sarne time. N-methylated isoquinolinium ions were show to be more potent

respiratory inhibitors than unrnethylated THIQs (McNaught et al., 1995). This may explain

why deoxyepinephrine and epinephrine, which f m e d N-methylated T H Q upon

condensation with HCHO, were more cytotoxic with HCHO than other catecholarnines.

4,6,7-trihydmxy-1,2,3,4-tetrahydroisoquinoline f o d h m norepinephrine and HCHO, for

example, would require mon extensive metabolism before finally forming an isoquinolinium

cation that inhibits complex 1.

a - (R)-salsolinol b - deoxyepinephnne c - W S

C d - 4,6,7-trihydroxy-l,2,3,4 tetrah ydroisoquinolim

HO CH3

e - MPTP f - MF'P+

Mo= et al. (1977) rrportedtbat the fondeafatioaproduct of acetaldehyde d

epinephrine, 1 , 2 ~ e t h y l - 4 , 6 , 7 - t r i h y d r o x y - l , 2 , 3 , 4 - t e ~ , caused hepatic

necrosis following i.p. injection in rats. Our study rnay lead to speculation that hepatotoxicity

h m THIQ formation may be a consequence of exposure to HCHO or methanol. However,

the consequences of the fornation and cytotoxicity of these HCHO-based THIQs in isolated

hepatocytes may be more clinically relevant in tenns of their potmtial neurotoxicity. THIQs

formed fiom the condensation of HCHO with phenylalanine or dopamine induced abnomal

behaviour in rats (Makowski et al., 1981) and NMNS inhibited tyrosine hydroxylase activity

in rat brain (Scholz et al., 1997). Furthemore, several studies have shown that exposure to

HCHO induces neurobehavioural damage. Neurotoxicity including a case of Parkinson's

disease has been linked to chronic or acute occupational exposure to HCHO (Kilbum, 1994).

Furthemore rats inhaling HCHO have suffered neurobehavioural disabilities (Boja et al.,

1985; Pitten et al., 2000). and rnany cases of methanol poisoning have msulted in impaired

motor huiction and other neurological disorders in humans (Guggenheim et al., 1971 ;

McLean et al., 1980; Oliveras-Ley and Gali, 1983; Anderson et al., 1989).

The cytotoxicity results of this study may serve as a mode1 for HCHO-induced

neurotoxicity. These results should also bnng forth consideration of the dangers of exposure

of the general public to chronic doses of HCHO or methanol through cleaning agents,

environmental air pollution and perhaps even methanol-based gasolines in the fiiture.

Monova, it was suggested that foods contaidg isoquinoline derivatives such as wine,

cheese, cocoa, bananas and milk may lead to the onset of parkinsonism (Makino et al., 1988;

Niwa et al., 1989). This was supported by reports that THIQs are able to cross the blood-

brain-barris (Makino et al., 1988; Niwa et al., 1988).

Ovd1, ~te~~1tssuggestthatthcEzteofHCHU isimportmtindetermining ito

cytotoxicity. Those who are exposed to HCHO either acutely or on a chronic bais should be

aware of the interactions betweai HCHO and endogenous compounds such as

catecholarnines that can lead to enhanced HCHO cytotoxicity.

SUMMARY. GENERAL, DISCUSSION AND FüTURE PERSPECTIVES

7.1 Summary of researeb resulb

nie aim of this study was to relate the metabolism of formaldehyde (HCHO) to its

cytotoxicity in isolated rat hepatocytes. The metabolism of HCHO was investigated h m

both an enzymatically-catalysed standpoint as well as h m a chernical standpoint through

reactions with nucleophilic compounds.

We firstly showed that HCHO induced isolated rat hepatocyte death in a dose- and

the-dependent manner with an appmximate (2 houn) of 4 m . . HCHO was much

more effective than the metabolites methanol or formic acid for the effects leading to

cytotoxicity including inhibition of mitochondnal respiration, collapse of membrane potential

(y,,,), GSH depletion, generation of reactive oxygen species (ROS) and lipid peroxidation.

These effects and thus cytotoxicity were prevented by inhibitors of the mitochondrial

penneability transition pore (MPT), antioxidants, the disulfide reductant dithiothreitol and

iron chelators, suggesting that HCHO is a mitochondrial toxin and exerts its toxic effect by

an oxidative stress mechanism. Inhibithg the oxidation of HCHO to formic acid by aldehyde

dehydrogenase (ALDH2) or formaldehyde dehydrogenase (ADH3) or inhibithg the

reduction of HCHO to methanol by alcohol dehyhgenase (ADHI) resulted in increased

cytotoxicity.

Following up on these results, we next investigated the effect of increasing HCHO

metabolism on its cytotoxicity. Using NADH grnerators or NADH oxidisers, HCHO

metabolism increased and cytotoxicity subsequently decreaseâ due to an hcrease in HCHO

redwioo and oxidation, respectively. MetbaaoC wagshowa tebe a substrate for oatalspt, as

addition of the H202 generator glycolate increased HCHO production h m methanol.

Inhibition of catalase by azide, 3-amino-l,2,rl-triazole or cyanide prevented HCHO

formation, and NAD+ or NADH generators decreased HCHO levels likely by increasing

HCHO mymatic metabolism. Moreover, HCHO is known to react with nucleophilic

compounds such as amines and thiols. We sought to determine the cytotoxic effect of HCHO

by trapping HCHO with amines or thiols. L-cysteine, D-penicillamine, hydroxylamine and

aminoguanidine were al1 effective at trapping HCHO and preventing cytotoxicity. L-cysteine,

D-penicillamine and hydroxylamine had antidotal properties as they were even effective

when added 30 minutes after the addition of HCHO to hepatocytes. However, trapping

HCHO with hydroxylamine resulted in increased cytotoxicity under GSH-depleted

conditions, suggesting that the product formecl between hydroxylamine and HCHO was

toxic.

The trapping of HCHO with catecholarnines was also found to increase cytotoxicity.

Catecholamines and HCHO reacted with each other to fom tetrahydn,isoquinolines

(THIQs), and THIQ formation was confinned with hepatocytes by mass spectrometry. The

observed cytotoxicity was thought to be due to the formation of THIQs, which are

stmchually similar to the hown neurotoxin MPTP. However the mechanism of cytotoxicity

is unclear. To begin with, the mechanism of THIQ uptake into hepatocytes has not been

established, as the addition of HCHO and the catecholamine deoxyepinephrine separately to

hepatocytes caused cytotoxicity whereas the addition of an equimolar amount of the reaction

product, N-methylnorsalsolinol (NMNS), was less toxic. It is possible that NMNS had

--- -= - difficulty i~penetreting the cell memhm. Fuithei work is- acedcd to etttcidate the

mechanism of THIQ-induced hepatotoxicity.

The role of the cytochrome P450s in HCHO metabolisrn and cytotoxicity was also

investigated. HCHO was metaboüsed by a rat liver microsoma1 system, and metabolism was

greater in CYP 2El-induced microsomes. Furthemore, HCHO was metabolised by human

CYP 2E1 + P450 reductase + cytochmme bs SUPERSOMES? in hepatocytes, HCHO

metaboüsm and fonnic acid production were inhibited when CYP 2El or P450 reductase

were inhibited but not when CYP 1A2 was inhibited. In addition, HCHO-induced

cytotoxicity was enhanced in CYP 2E 1 - or P450 ductase-inhibited hepatocytes but not in

CYP 1 A2-inhibiteci hepatocytes. ROS fornation was lower using CYP 2E1 SUPERSOMES~

with HCHO as a substrate than with ethanol. Together, these results suggested HCHO was a

substrate for CYP 2E1 and that HCHO metabolism by this pathway resulted in

detoxification. More importantly, it is Iikely that this alcohol inducible P450 contributes to

HCHO and acetaldehyde metabolism in humans. This could compensate for the lower GSH

levels found in alcoholics which would be expected to increase hepatocyte susceptibility to

HCHO and acetaldehyde.

7.2 General dlscussiodimplications of research

Although the metabolism of HCHO by ADHI, ALDH2 and ADH3 has been well-

studied in in vitro systems, the relationship between HCHO metabolism and cytotoxicity has

not been established. It was shown in this study for the first time that there was a direct

correlation between inhibition of the metaboüsm of HCHO and its cytotoxicity.

It is gemmIly thought that HCHO is toxic piiiRarily te tissues local to the m a of

exposure (e.g. the respiratory tract or skin) and HCHO produced from the oxidation of

methanol is detoxified rapidly enougb so that its toxicity is minimal. In fact, methano1

toxicity is mainly attributed to the formation of formic acid and subsequent acidosis rather

than the etrects of HCHO (McMartin et al., 1979). However the results from this study

suggested that individuals who are deficient in any of the HCHO detoxification enzymes may

be more susceptible to HCHO toxicity due to an inability to metabolise HCHO. This includes

those who have less active enzymes due to polymorphisms, those who are taking enzyme-

inhibiting dnigs (e.g. d i s u l f m for alcohol aversion îherapy) or those who have decreased

GSH levels due to disease conditions such as diabetes. ADH3-nul1 mice were found to be

more susceptible to HCHO-induced death than control mice (Deltour et al., 1999) and

alcoholics who are ALDHZ-deficient were more susceptible to the development of cancers of

the gastrointestinal tract due to acetaidehyde (Yokoyama et al., 1998). In addition, ALDH2

and ADHl are subjected to inhibition by many compounds including hormones (Jeavons and

Zeiner, 1984) and competing substrates (Dicker and Cederbaurn, 1984a and 1984b; Teng et

al., 2001). In addition to having lower GSH levels penons with diabetes may also be at

greater risk for HCHO toxicity due to their higher methylglyoxal and glycolaldehyde levels

(Yu, 1993). Clearly, exposure to HCHO may have more severe toxic consequences for some

individuals over others.

Acetaldehyde and HCHO share many of the same metabolic pathways, thus it was not

surprishg to find that HCHO was a substrate for oxidation by CYP 2E1, as previous reports

showed that acetaldehyde was also a CYP 2E1 substrate. However many clinical

implications may arise due to the involvement of CYP 2E1 in HCHO metabolism and

dctoxihcation. CYP 2E1 metabolises mmy xenobiotics imcl&g Qugs, and toxic HCHO-

drug interactions rnay occur. Our work suggests that the contribution of CYP 2E1 in the

overail metabolisrn of HCHO is less than that of ADHI, ALDH2 or ADH3, however its

involvement rnay become more pronounced if CYP 2E1 is Uiduced. Many drugs are known

to cause the induction of CYP 2E1, thus exposure to CYP 2E1 inducers pnor to HCHO

exposure rnay affect not only the extent of the involvement of CYP 2El in HCHO

metabolism relative to other enzymes, but rnay also affect HCHO cytotoxicity in that

increased ROS rnay be generated due to the increased levels of CYP 2E1. This is especially

important considering that the low redox potential of CYP 2E1 renders the enzyme to be

more capable than other P450s of generating ROS by-products during its catalytic cycle.

ROS are involved in oxidative stress-mediated cytotoxicity, and increased ROS

generation rnay be responsible for the cytotoxic effects of HCHO. Our studies contïnn the

hdings of others who reporteci that HCHO depletes hepatocyte GSH (Jones et al., 1978;

Ayres et al., 1985; Ku and Billings, 1986) and inhibits mitochondrial respiration (Stmbelt et

al., 1989). However we have shown for the first time that the integrity of y, is also affected

by HCHO and that antioxidants, iron chelators, disulfide reductants and protectors of the

mitochondrial permeability transition pore (MPT) can prevent HCHO-induced c ytotoxicity

and Iipid peroxidation. Thus, the mechanism of HCHO-induced cytotoxicity was linked to

mitochondrial toxicity. Previously, Ku and Billings (1986) suggested that hepatocyte GSH

depletion made hepatocytes more vulnnable to protein sulfhydryl oxidation by HCHO in that

GSH acts as the fht defence mechanism against HCHO by reacting with it to fonn S-

hydroxyn~ethylglutathione which is subsequently eflluxed h m the cell. They found that

HCHO causes protein sulfhyâryl oxidation, however the afExted sdfhydryl protein(s) that

W t e d l de& w a e not detemined. Givea thet prdectorsof the MPT end dithiothroitd, 0

disulfide reductant, prevented HCHO-induced cytotoxicity in our study, it is possible that

HCHO caused oxidation of sulfhydryl proteins associated with the pore, and that the

oxidation of these proteins is responsible for pore opening and hence c ytotoxicity. Castilho et

al. (1996) reported that prooxidant-induced membrane permeability is due to protein thiol

cross-linking and protein aggregation. In this case, GSH depletion pnor to HCHO exponire

was more detrimentai to hepatocytes than controls not only because it would allow for the

oxidation of protein sulfhydryls by HCHO, but also because GSH-dependent antioxidant

enzymes such as glutathione peroxidase would not be able to function. Moreover, our

preliminary results suggest that HCHO inactivates GSH reductase, an enzyme that is

dependent on sulfhydryl groups in its active site for its enzymatic activity. These oxidative

effects of HCHO would lead to a cascade of events eventually resulting in ce11 death (Figure

7.1).

-- . Figure 7.1 The propofed cytotoxic mechariism of forrmldehyde.

The toxK mhenisnt of HCHO was f d te be compsrable to that of acdddebyds.

Shidies have s h o w that ethanol induces MPT opening and decreases v, in culhued rat

hepatocytes and perhised liver (Kurose et al., 1997), and depletes cellular GSH, oxidises

protein sulfhydryls and induces lipid peroxidation in the livers of rats afier oral

administration of ethanol (Vendemaile et al., 1998). These effects were prevented with 4-

methylpyrazole and enhanced with cyanamide, suggesting that they were dependent on the

metabolite acetaldehyde. Furthemore, acetaldehyde isdministered orally to rats fsted

ovemight caused a depletion of liver GSH and induced lipid peroxidation (Videla et al.,

1982). Ail of these effects are involved in the oxidative stress theory of the development of

alcoholic liver disease (Lieber, 1997). Acetaldehyde, like HCHO, cm react with GSH

directly or with its precwsor cysteine. However the effect of acetaldehyde on MPT has not

been reported in the Merature. Pastorino et al. (1999) showed that mitochondria h m rats that

were chronically fed ethanol were more susceptible to MPT opening than controls. Hirokawa

et al. (1998) also showed that MPT opening preceded the death of gastric mucosal cells of

rats fed ethanol. However, the specific role of acetaldehyde in inducing these effects was not

established. It is possible that ROS pmduced h m the P450-catalysed oxidation of ethanol

may also induce MPT opening.

Just as Uihibiting HCHO metabolism Unreases HCHO-induceà cytotoxicity, we have

s h o w that the cytotoxicity of HCHO can be prevented through the removal of HCHO by

increasing emymatic metaboüsm or by chernical trapping. The detoxification seen with L-

cysteine, D-penicillamine or aminoguanidine suggests that these compounds may be

candidate antidotes for HCHO poisoning. Our results support the work of others who showed

that cysteine administered i.p. or orally to rats before or after HCHO exposure was protective

- -- agpinst HCHO-inducd death (Guecri et al., 1976; S-e et al., t979). However due te the

rapid metabolism of L-cysteine in vivo, its eflectiveness may be minimal. D-penicillamine

and aminoguanidine, on the other hand, are drugs that are already marketed as treatments for

rheumatoid arihntis and diabetes, respectively, thus their fùnctions may be extended to

include HCHO scavenging and detoxification. D-penicillamine scavenges acetaldehyde and

has been suggested as a means of preventing acetaldehyde toxicity for those who are

deficient in ALDH2 (Nagasawa and Goon, L 977). The use of aminoguanidine in the

treatment of diabetes is based on its ability to detoxiQ dicarbonyls that are involved in the

formation of advanced glycation endproducts (Thomailey et al., 2000). Standard treatment

for methanol poisoning cunently involves dosing with ethanol to prevent the oxidation of

methanol to HCHO. along with dialysis to remove HCHO that is already produced. Since this

is a rather slow process, aldehyde trapping agents such as D-penicillarnine or

aminoguanidine may increase the efféctiveness of the current treatment. The importance of

removing HCHO as quickly as possible to prewent toxicity was indicated by the lack of

c ytoprotec tion sem w hen the hepatoc yte incubation bu ffer was replaced wi th a HCHO- fiee

buffer at later t h e points. Clearly, HCHO cytotoxicity can be reversed, however its toxic

mechanism includes a step in which a cornmitment to ceIl death is made. This step of

cormitment m a i n s unknown, however its identification would be valuable towards finding

other means of treathg or preventing HCHO toxicity.

Although the majority of HCHO metabolic pathways resulted in the detoxification of

HCHO, an increase in HCHO-induced hepatocyte cytotoxicity was seen with

catecholamines Moura et al. (1977) showed that the condensation product of acetaldehyde

and epinephrine caused hepatic necrosis in vivo in rats. Catecholarnines circulate b u g h the

liver, where they are metaboliseci by MAO or COMT (Kopin, 19- Howeva the p r e ~ c e

of aldehydes can result in the formation of tetrahydroisoquinolincs (THIQs) upon their

condensation with catecholamines. These findings suggest that increased aldehyde levels in

the liver may be toxic due to THIQ formation. For instance, alcohol consumption raises liver

acetaldehyde levels and as show previously, acetaldehyde is hepatotoxic with epinephrine.

Its toxicity with other catecholamines remains to be detemined. Reactions with

catecholamines may also conhibute to methanol toxicity following HCHO production.

However despite the toxicity towards hepatocytes, research into THIQ toxicity has focused

primarily on its neuralogical effects instead.

HCHO and its precursor methanol have been report4 to cause neurological

impairnent and have even been implicated in a case of Parkinson's Disease (Guggenheim et

al., 1971; Anderson et al., 1989; Kilbum, 1994; Pitten et al., 2000). n i e mechanism of

HCHO-induced neurological damage has not been detennineà but our studies suggest a

possible mechanism. HCHO and catecholamines undergo a Pictet-Spengler condensation to

fonn THIQ derivatives, which are similar in structure to the known neurotoxin MPTP. It is

possible that HCHO enters the brin and reacts with catecholarnines to fonn THIQs that

induce neurotoxicity through a mechanism similar to that of MPTP. Indeed, high levels of

various denvatives of THIQs have been found in the brains of Parkinson's patients (Niwa et

al., 1987; Moser and Kompf. 1992) and THIQs have been show to be toxic to various

cultureci neuronal ce11 lines (McNaught et al., 1996; Mamyarna et al., 1997; Storch et al.,

2000). However as our study used hepatocytes, the actual neurotoxicity of HCHO-derived

THIQs has not been determineci. These findings may serve as a starting point, though, for

investigating the bais of HCHO-induced nemtoxicity.

O v d I , our shidy brings together the mIationshipbetwan HCHO metabolism snd its

cytotoxicity. It is clear that in most cases, the metabolism of HCHO leads to its

detoxification, however the reaction of HCHO with nucleophilic compounds can be antidotal

or it cm lead to the formation of mon toxic products (sumrnarised in Figure 7.2). This study

also provides evidence towards putting together a possible mechanism for HCHO-induced

hepatotoxicity. Clearly, the toxicity of HCHO goes beyond its carcinogmic effects and the

extent of its toxicity may be dependent on an individual's ability for HCHO metabolism.

Figure 7.2 Overall scheme of the metablic pathways of HCHO and their effécts on HCHO-induced cytotoxicity.

thiazulidines

HCHO-aminopnidine tetrahydroiquinolines complex -- a-*

œ œ . * catecholamiaes

FORMALDEHYDÉ

(nonenzymatic) Forrnic acid

Methanol S-hydmxpe th yiGSH

GSH 1 CO2

S-formylGSH hydrolase

S-formylGSH

Legend: - detoxification pathway " " - * toxic pathway

7.3 Lindtatiaas of rcstarcb

The buk of this research was done using isolated rat hepatocytes. The use of this

system has its advantages. Firstly, the isolated ce11 systern is easily manipulateci in order to

achieve the desired conditions (e.g. atmosphere, doses, etc.). Cytotoxicity and metabolism

snidies are easily conducted, and biochemical endpoints are also easily assayed. The system

allows one to study the effects of compounds on the cells directly without a need to consider

the pharmacokinetics of the compound. The interaction of the compound with the tissues

directly smunding the cells in the intact liver also does not need to be considered. Another

advantage is that al1 the toxic effécts of HCHO reported by other investigators using

subcellular fractions or enzymes, proteins or nucleic acids can be repeated with intact cells to

detemine whether these effects contribute to cytotoxicity.

However, the isolated ce11 system is limited to the study of acute toxicity, as the

system is only viable for a few hours. In the case of HCHO toxicity, however, exposure for

most people occurs over a chronic period rather than acutely. The effects of HCHO over a

chronic pend (with lower doses) may differ h m those of acute, hi& dose toxicity.

The use of rats as the species of study poses limitations as to the extent to which the

results cm be extrapolated to a human situation. It is likely that humans may be even more

susceptible to HCHO toxicity than rats because humans and other primates have lower levels

of GSH (Siegen et al., 1982; Ketterer et al., 1983; Woodhouse et al., 1983) and folate (Johlin

et d., 1987) as compand to rats. Humans and monkeys, but not rats, arr susceptible to

methand-induced blindness and acidosis, which has been attributed to the accwnulation of

for& acid due to deficimt formic acid metabolism (Tephly, 1991). GSH, as discussed,

plays a major role in the detoxification of HCHO, and folate is important in both the

oxid8tbn of f& acid podwed fiom se well as h the reaction of 4th H m

to incorporate HCHO Uito the folic acid cycle. Thus with lower GSH and folate Ievels,

HCHO toxicity may be potentiated in humans as compared to the rat.

7.4 Future perspectives

The metabolism and cytotoxicity of HCHO remains a vastly undersnidid topic in

cornparison to the metabolism and toxicity of acetaidehyde. The popularity of acetaldehyde

study stems primarily h m its relationship to ethanol and alcoholic liver disease. Expsure to

methanol and HCHO, on the other hand, occurs less fiequently and thus there is generally

less interest. However endogenous HCHO formation is likely much greater than

physiological acetaldehyde formation.

As discussed above, the isolated hepatocyte system allows only for the study of acute

toxicity. Cultureâ hepatocytes can be used instead with lower HCHO concentrations for

longer pends of t h e in order to better simulate the effects of chronic exposure to HCHO.

On the other hand, in order to m e r investigate the mitochondrial toxicity of HCHO,

experiments with isolated rat liver mitochondria can be used rather than the intact cell.

Mitochonârial swelling due to MPT opening can be examinai spectmphotometrically and

respiration cm be studied diiectly. The mechanism of mitochondnal respiration inhibition by

HCHO should be determineci (Le. the electron transport chah protein tatget of HCHO). tn

addition, it would be appropriate to examine ca2+ leveis, as mitochondrial ca2+ plays a major

d e MPT openhg. Ce11 membrane blebbing is considmd to be a morphological

characteristic of apoptosis. Since blebbing was clearly obsaved in HCHO-treated

hepatocytes, the importance of apoptosis as a mode of HCHO-induced ce11 death should be

- - -- investigated. In psiticular, considerhg the invdvement of the MPT in HCHO-induoed

cytotoxicity, the release of cytochrome c from mitochondria should be determineâ in

conjunction with the effects of caspase inhibitors on cytotoxicity. The best way to detennine

apoptosis, though, would be to examine the morphology of the ce11 using electron

microscopy, as cells undergoing apoptosis have prominent morphological features as

cornparad to necrotic cells.

The role of oxidative stress in HCHO-induced cytotoxicity can be m e r studied by

exarnining the efiect of HCHO on the activity of antioxidant enzymes such as glutathione

reductase, glutathione peroxidase or superoxide dismutase. Vander Jagt et al. (1 997) showed

that various aldehydes are able to inactivate glutathione reductase, and Slaydlewska and

Farbiszewski (1997) showed that methanol decreased the activity of glutathione reductase

and glutathione peroxidase, although the specific d e of HCHO in inactivation was not

discloseâ. Our preliminary studies also showed that glutathione reductase is inactivated by

HCHO, however this needs to be demonstrated in cells.

The involvernent of cytokines in the development of many pathologicaî conditions

has been well-snidied. Cytokines have been implicated in the pathology of alcoholic liver

disease (McClain et al., 1999) and moreover, ROS cm modulate the activation of NFKB, a

cytokine transcription factor (Shreck et al., 1992). Neuman et al. (1998) showed that ethanol

treatment for 24 hours increaseâ the release and expression of IL-la, IL6 and TNF-a in

HepG2 cells Similady, 24 hour treatment of HepG2 cells with acetaldehyde resulted in the

release of IGl fl and increased the expression of TNFa (Gutiem-Ruiz et al., 1999). GSH

depletion also occumd in both of these studies, suggesting that cytokine release and

expression were involved in a response to oxidative stress induceû by ethanol or

clcBtiildehyde. W e showed that oxidative stress, iacludiag the depletion of GSH, is invoIved

in the cytotoxic meçbanism of HCHO. It would be of interest to Uivestigate the possible role

of cytokines in HCHO cytotoxicity, as modulation of cytokine expression or release may be

therapeutic against HCHO toxicity.

The metabolisrn of HCHO by other enzymes should also be studied m e r . This

includes 0 t h P450 isofoms, catalase and aldehyde dismutases. Sies (1 974) suggested that

catalase is involved in HCHO metabolism, however, this needs to be M e r elucidated. The

NAD*-dependent aldehyde dismutase activity of alcohol dehyhgenases has been reported

for some enzymes and aldehyde substrates, however it has not been demonstrated for HCHO

in intact cells. In the case of P450s. acetaldehyde was reported to be a substrate for CYPs

1 A2 and 4A2 in addition to CYP 2E1 although the oxidation activities of CYPs 1 A2 and 4A2

w m less than that of CYP 2E1 (Kunitoh et al., 1997). The role of additional P450 isofoms

in HCHO metabolism should be investigated in order to create a more complete pichire of

enzymatic HCHO metabolism. Based on out study, it appears that HCHO is not metablised

by CYP 1A2 since the inhibition of CYP 1A2 had no effect on HCHO metabolism. formic

acid production or HCHO-induced cytotoxicity. Nevertheless, (his needs to be M e r

confirmed through metabolism stuâies with CYP 1A2 microsomes. The CYP 2E1 studies

pmented hem should be expanded to include the determination of the kinetics of the

oxidation of HCHO, e.g. km tb etc. Knowledge of these values would aid in detennining

the dative contribution of CYP 2E1 in the overall metabolism of HCHO. It would also be of

interest to shidy the metabolism and cytotoxicity of HCHO in CYP 2El-induced hepatocytes

versus normal, non-induced hepatocytes. The potential for CYP 2El-catalysed HCHO

metaboiism to induce drug-HCHO interactions can also ôe investigated using microsomes to

-.- - - d e t d n e the Wty of HCHO to inhibit the metabolism of t h w n CYP 2E1 subetratesuch

as pnitrophenol. Moreover, using hepatocytes, the potential toxicity of these interactions

can ais0 be investigated.

The toxic consequences of CYP 2El-catalysed HCHO metabolism can be m e r

shidied by determining ROS generation during HCHO metabolism by CYP 2El in

hepatocytes. We have shown using microsornes that ROS is produced during this reaction,

however ROS levels were Iess than that pduced during CYP 2El-catalysed ethanol

oxidation. The potential for ROS generation in CYP 2E l -induceci hepatocytes should be

studied, since it would be expected that CM? 2El would play more of a role in HCHO

metabolism in this case. Thus the potential for ROS generation would be greater and the

toxic consequences for HCHO metabolism by this pathway may be enhanced.

Our research presents THIQs as a possible cause of HCHO-induced hepatotoxici ty

and neurotoxicity. There have been many reports of neurological impairment in humans and

rats induced by acute or chronic exposure to HCHO or methanol, although the mechuiisrn of

toxicity has not been detennjned. Resemh in THIQs has been pursued in only the past

couple of decades ever since the discovery that MfTP, a compound similar in structure to

THIQs, is a potent inducer of parkinsanism (Langston et al., 1983). Since then, much work

has gone into resolving the mechanism of MPTP toxicity and in looking for other compounds

with similar effects. In particular, endogenous MPTP-like compounds have been of major

interest.

Our hepatocyte studies on THIQ cytotoxicity can be taken huther. The toxicity of

MPTP is known to involve the inhibition of complex I of the electron transport chah, whicb

results in balted ATP production and then ce11 death. Therefore the effect of THIQs on

mitochandrid rcspiratim and ATP pmducth sbuH beexammed dong with cpm as these

are al1 indicators of mitochondrial integrity. MPTP toxicity has also k e n associated with

oxidative stress, since the inhibition of respiration results in superoxide production. Thus

indicators of oxidative stress such as GSH depletion, ROS fonnation and lipid peroxidation

should be measund. Finally, THIQs likely require bioactivation to fonn N-methylated

cations in order to exert toxicity, and that cation formation requires catalysis by MAO

(McNaught et al., 1998). This role of THIQ metabolites in toxicity can be determined by

looking at the cytotoxicity associateâ with MAO-inhibited hepatocytes, as well as by

identifjing the presence of metabolites by m a s spectrometry.

It has been suggested that (R)-salsolinol, the THIQ fomed fiom dopamine and

acetaldehyde, is synthesised enzymatically in the human brain (Naoi et al., 1996). Although

THIQ formation is known to procead non-enzymatically, the possibility of the involvement

of an enzyme may have implications in the rate of THIQ formation and may bring forth

ways to prevent or decrease THIQ formation so as to prevent or decrease toxicity. The

enzymatic formation of THIQs should be m e r investigateâ.

Despite the demonstrated toxicity of THIQs towards hepatocytes, the toxicity of

THIQs towards neurons should be examinad in order to confirm the neurotoxicity of THIQs

and that THIQ formation is the cause of reported cases of HCHO-induced neurotoxicity in

humans and rats.

Finally, it would be interesthg to test in vivo whether HCHO, rather than one of its

metabolites, is responsible for the onset of neuiological disorciers seen in rats exposed

chronically to HCHO. Rats exposed to methanol with or without the ADHl inhibitor 4-

methylpyrazole or rats exposcd to HCHO with or without ALDH2 inhibitors should be

---- exclaiinsd for whether or not they presmt symptom8 ofneurobgkd dieordem. ShouId

HCHO be the toxic responsible for the effects, 4methylpyiszole would be expected to

prevent HCHO production h m methanol, thus preventing nemtoxicity. ALDH2 inhibitors

should potentiate HCHO toxicity in vivo.

Overdl, studies on HCHO toxicity have focused rnainly on its carcinogenicity or

mutagenicity. Few studies have looked at its systemic toxicity or the biochemical changes

induced by HCHO. Future research should address the issue of WHO-induced liver damage

as well as toxicity to other tissues or organs.

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