ANTIDOTAL MECHANISMS FOR

HYDROGEN SULFIDE TOXICITY

ALEKSANDRA MIHAJLOVIC

A thesis submitted in the conformity with the requirements

for the degree of Master of Science

Department of Phannaceutical Sciences

Faculty of Phannacy

University of Toronto

O Copyright by Aleksandra Mihajlovie 1999

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DEDICATION

This thesis is dedicated to:

M y Father who taught me how to enjoy life,

My Mother who taught me how to fight Me,

My sister Maja who was there thmugh thick and thin,

My friends for being there.

Thank you al1 for al1 your unending love, help and support!

Tata znam da bi bio ponoson na mene!

Andjelki i Maji posveèqjern ovu tezu jer su verovale u mene., a baki i deki

Sto su ih &vali.

V i a Saga

ANTIDOTAL MECHAIIQTSMS FOR HVlbROGEN SULFIDE TOXICITY

M. Sc., 1999

Aleksandra Mihajlovic

Graduate Department of Pharmaceutical Sciences

Faculty of Pharmacy

University of Toronto

Hydrogen sulfide (H2S) toxicity mechanisms are still not well understood

and currently used antidotes are ineffective. H2S was found to be not

only toxic per se but was metabolically activated to reactive metabolites

which can react with GSH or protein thiols to form persulfides. The

activation was probably catalyzed by cytochrome P450 CYP 3A and 2B as

cytotoxicity and GSH de pletion was prevented by metyrapone or SKF-

525A and markedly increased by hydroperoxides. Cytochrome c

prevented H2S cytotoxicity, a process in which cytochrome c was

reduced. Methemoglobin prevented H2S cytotoxicity by trapping H2S to

fom sulfmethemoglobin which catalyzed H2S autoxidation. Molybdenum,

barium and lead prevented cytotoxicity by fonning sulfide complexes

without H2S autoxidation while copper, cobalt, nickel and iron fomed

active complexes that catalyzed H2S autoxidation. Hydroxocobalatnin

was highly effective as an antidote in preventing H2S cytotoxicity in Mtro

and in vivo, where it was more potent and effective than currently used

nitrite.

1 sincerely thank my research s u p e ~ s o r , Dr. Peter O'Brien for

facilitating the completion of my research project by providing much

guidance and encouragement and for sharing his enthusiasm for science.

1 would also like to express my gratitude to my advisory cornmittee

member, Dr. Peter Pennefather, for his thoughtful suggestions and

criticai reading of rny thesis as well as my examiners, Dr. Rebecca

Prokipcak and Dr. Rena Bendayan for their suggestions and insightful

comments.

A special thanks goes to Dr. Sumsullah Khan for all his scientific,

technical and moral support through the rainy days of the research.

Endless gratitude to often forgotten help through Scyllas and

Charybds of administration to Merrylee Greenan, Graduate Secretary.

1 shail dearly remember the camaraderie and help of my peers - Sylvia, Sophia, Reza, Jalal and Bin.

The investigations described in this thesis were financially

supported by a research grant from the Natural Science and Engineering

Research Council of Canada. The investigations were carrieci out in

Professor P. J. O'Brien's laboratory in the Faculty of Pharmacy,

University of Toronto, 19 Russell St., Toronto, Ontario, Canada, M5S

2S2.

Aleksandra Mihajlovic was fmancialiy supported by the University

of Toronto Open Fellowship (1997/98) and Ontario College of Pharmacists Bursary (1996). Attendance of the 37a Annual Meeting of

Society of Toxicology (1998), Seattle, WA, USA, was partially supported

by the Faculty of Pharmacy, University of Toronto.

TABLE OF CONTEWTS

Title

Abstract

Dedications

Acknowledgements

Acknowledgement of Financial Support

Table of Contents

Summary of Abbreviations

Summary of Tables

Summary of Figures

Chapter 1 Introduction

Chapter 2 Materials and Methods

Chapter 3 Hemoproteins as Antidotes and Metabolic

Activators of Hydrogen Sulfide Cytotoxicity

Chapter 4

Chapter 5

References

Metals as Antidotes and Activators

Summary and Conclusions

1

II

III

IV

v VI

VI1

Ix X

B 12

B 12a

CHP

CYP

Qt P450

Cyt. a, b or c

GSH

GSSG

Hb

HEPES

HPLC

Met-Hb

NADH

vitamin B 12 (cyanocobalarnin)

vitamin B 12a (hydroxocobaiamin)

cumene hydroperoxide

cytochrome P450

cytochrome P450

cytochrome a, b or c

reduced glutathione

oxidized glu tathione

hemoglobin

4-(2-hydroxyethy1)- 1 -piperazine-

ethanesulfonic acid

high performance liquid

chrornatography

intraperitoneal

lethal dose that U s 60 or 95%

animals respectively

methemoglobin

nicotinamide adenine dinucleotide

(reduced)

nicoharnide adenine dinucleotide

TRIS-HCI

U V

phosphate (reduced)

hydroxoco balamin

standard deviation

2-dimethylaminoe thyl-S,2-diphenyl-

-n-pentanoate

Trizma base (Tris [hydroxyme thyl] -

-amino methane)

ultra violet

Chaptef 3

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 4-5:

Hemoprotehs as antidotes and rnetaboîic

activators of hydrogen s d d e cytotoxicity

Antidotal effect of hemoproteins towards H2S

cytotoxicity

H2S modulated respiration in isolated hepatocytes

H2S autoxidation catalyzed by hemoproteins

Effect of P4SO inhibitors and activators on H2S

induced cytotoxicty

Metaïs as antidotes and activatorr

Antidotal effect of metal salts/complexes towards H2S

cyto toxicity

Modulation of HB autoxidation by various metal sdts

Effect of Fe, Cu and Ca chelating agents on H2S

induced cytotoxicity

Modulation of H2S autoxidation by metal chelating

agents

Antidotal properties of vitamin Bi2 versus vitamin Bila

against H2S cytotoxicity

Chapter 1 Introduction

Figure 1-1: Chernical structure of hydroxocobalamin

Chapter 3 Hemoproteims as antidotes and metaboiic

activators of hydrogen s a d e cytotdcity

ne 3-1: The influence of H2S on the absorption spectn

met-Hb

Figure 3-2: Reduction of cytochrome c by H2S

Figure 3-3: Hydrogen sulfide induced GSH depletion in isolated

hepatocytes

Figure 3-4: In vitro GSH depletion by H2S is catalyzed by

Hemoproteins

Chapte 4 M e t a b as antidotes and activators

Figure 4- 1: OH-cobalarnin complex formation with H2S

Figure 4-2: Antidotal eflectiveness of OH-cobalarnin against

Figure 4-3:

Figure 4-4:

Chapter 5

Figure 5- 1 :

lethality induced by different NaHS concentrations

A cornparison of the antidotal effectiveness of NaNO2

and OH-cobalamin against NaHS induced lethality

H2S does not react with cyanocobalamin

S u m m y and conclusions

Mechanisms of H2S activation and detoxifkation

Hydrogen sulfide (H2S) is a gas that is as toxic as cyanide and four

times more toxic than carbon monoxide. Its toxicity has never provoked

as much interest as the other two noxious gases probably because

poisoning by H2S was restricted to infrequent cases of occupational

exposure and has never been a major hazard for the general public.

However, it has been shown that H2S could play an etiological role in

diseases such as ulcerative colitis (Roediger, 1982; Curnmings et al.,

1987) as weli as periodontal disease (Carlsson et ai., 1993; Granlund-

Edstedt et al., 1993; Persson, 1992) e.g. halitosis (Shimura et al., 1996).

A physiological role for H2S formation in Yivo was suggested by the

discoveries that it may act as a neuromodulator in the brain, or as a

smooth muscle relaxant in synergy with nitric oxide in the thoracic aorta

(Abe and Kimura, 1996; Hosoki et al., 1997). H2S producing enzymes are

dso expressed in the ileum and portal vein (Hosoki et al., 1997). This

demonstrates the need for a better understanding of the role and

metabolic pathways involved in H2S production and detoxification in

humans.

The research presented in this thesis is aimed at elucidating the

cytotoxic mechanisms of H2S and its detoxifïcation mechanisms. A s a

result of these studies a new antidote, hydroxocobalamin, has been

discovered that is much more effective and safe than currently available

nitrite.

Hydrogen sultide (H2S) is best known for its characteristic odor of

rotten eggs. This inorganic sulfur compound is a colorless inflammable

gas that under chemically defiied normal conditions is heavier than air

(d= 1.19). The molecular weight is 34.08 and it is the sulfur analog of

water (Budavari et ai., The Mer& Index, Burnett et al., 1977).

The chernical properües of H2S are as follows: - H2S is water soluble and its aqueous solution has an acidic pH

H2S ++ H+ + HS- t, 2H+ + S2-

At a physiological pH 113 of the H2S is in the undissociated form and

213 exists as the hydrosulfide anion (HS-) (Reiffenstein et al., 1992). I t

has two acidic dissociation constants (pKa 7.04 and 1 1.96) (Claesson et

ai., 1989). - H2S also has reductive properties and in redox reactions with weak

oxidizing agents it is oxidized to elemental sulfur

e.g. 2Fe3+ + H2S(g) + 2Fe2+ + So(s) + 2H+

or 0 2 + 2 H2S + 2S(s) + 2 H 2 0

It is also lipid soluble and is thus membrane penneable.

H2S is an inevitable constituent of fossil fuel and is almost always

fonned when organic materiai biodegrades. It is present in deposits of

natural (sour) gas, sewer gas and as a by-product or necessary ingredient

in many industries e.g. paper and pulp, tanning, rubber vulcanizing,

heavy water production, pelt processing, metal renning and oil and gas

processing.

H2S is also produced endogenously in mammalian tissues mainly

by two pyridoxal-5'-phosphate-dependent enzymes, cystathionine P- synthetase and cystathionine y-lyase, from L-cysteine.

1.2. HYDROGEN SULFIDE POISONING

1.2.1. Symptoms and s e of hydrogen aulfide potonliq

H2S is a broad-spectnim toxicant - the brain and respiratory

system being its primary targets, but it also affects the eye, olfactory,

gastrointestinal, hematopoetic and immune systems to various degrees.

The syrnptoms of intoxication v q in severity depending on the level of

exposure but it is difficult to determine the dose received in non-lethal

cases of H2S poisoning. Increased urinary thiosulfate levels is the only

clinical indicator to prove H2S exposure in non-fatal cases, whilst in fatal

cases, increased sulfide and thiosulfate levels in the blood can be

detected (Kage et al., 1997).

The following describes the human physiological responses to

exposure by increasing concentrations of HB:

Concentration of H2S Phvsioloaical resnon ses

PP=' m g b 3 0.003-0 .O2 0.0042-0.028 Odor threshold

3- 10 4- 14 O bvious unpleasant odor

20-30 28-42 Strong offensive odor ("rotten eggs")

30 42 Sickening sweet odor

50 70 Conjuctival irritation

50- 100 70-140 Irritation of respiratory tract

100-200 140-280 Loss of smeli (olfactory fatigue)

150-200 2 10-280 Olfactory paralysis

Pulmonary edema

Anxiety, headache, ataxia,

dizziness, stimulation of respiration,

amnesia, unconsciousness

Respiratory paralysis leading to

death, immediate collapse, neural

paralysis, cardiac arrhythmias,

death

(Reif'fenstein et ai., 1992).

1.2.2. Molecular mech.nirms of hydrogen ruifide toxicity

The first data showing the binding of H2S to cytochrome c oxidase

were reported by Keilin in 1929 and was further confmed by Hill (Hill et

ai., 1984). Sulfide reduced and then formed a complex with CUB of

cytochrome a3 and femcytochrome aiî that resulted in cytochrome

oxidase inactivation (Hill et al., 1984; Nichoils and Kim, 1982).

Another mechanism that may be involved in H2S toxicity includes

hydrogen peroxide production and oxygen depletion during H2S oxidation

by oxy- and methemoglobin (Beck et al., 198 1).

0 2 + H2S -+ S0 + Hz02

H2$ also slightly inhibited bovine erythrocyte superoxide dismutase

(SOD) and inhibited catalase, which suggests that cellular damage could

be due to increased levels of reactive oxygen species (Khan et al., 1987).

Iron sulfide was also more efficient than ferrous iron in converting

hydrogen peroxide into hydroxyl radicals (Berglin and Carlsson, 1985),

which when coupled with the catalase inhibition (Carlsson et ai., 1988)

could contribute to toxicity.

HB may react with the essentiai disulfide bonds of proteins, as

sulfide reacted with protein disulfide bridges to fonn protein persulfides

(in 0.0 1 N NaOH) (Cavallini et al., 1970). I t has also been shown that

dithiothreitrol (DTT) liberates sulfide believed to be bound to proteins as

protein persulfides (Warenycia et al., 1990).

R-SS-R + H2S H R-SSH + R-SH

R-SSH + DTT(red) 4 RSH + H2S + DTT(ox)

A similar reaction of H2S with the disulfide bridge of oxidized

glutathione (GSSG) explains the in viuo protection by GSSG against H2S

toxicity (Smith and Abbanat, 1966). This conclusion, however, remained

otherwise unsubstantiated.

1.2.3. Currentîy rued treatments for hyârogen s a d e poisoning

I t is generaîly assumed that the mechanism of H2S poisoning has

similarities with cyanide poisoning. Therefore current therapies for H2S

intoxication include treatment with hyperbaric oxygen and the

intravenous administration of sodium nitrite or inhalation of amyl nitrite

(Smith et al., 1976, Whitcraft et al., 1984, Smilkstein et al., 1985).

Hyperbaric oxygen used for treating cyanide intoxication is believed to

act by displacing cyanide from cytochrome oxidase whereas nitrite is

used to induce methemoglobinemia so as to trap cyanide. The benefits of

these methods as therapy for sulfide poisoning are controversial. Several

authors propose it as the only treatrnent available (Beck et al., 1981;

Smilkstein et al., 1985), whilst some reported no beneficid effect of

oxygen for the management of H2S poisoning (Smith et al., 1976) and

others reported no beneficial effects of nitrite (Beck et al., 1981; Burnett

et al., 1977).

The main treatment for H2S poisoning is nitrite using the cyanide

antidotal kit avaiIable in North Amencan Poison Control Centers. Sulfide

poisoning rapidly inhibits respiratory enzymes and treatment has to be

quick to counteract this inhibition. However, nitrite administration and

consequent met-Hb formaüon takes time, and furthemore the

impairment of oxygen transport as a result of methemoglobinemia will

exacerbate the already hypoxic celis and decrease the oxygen available

for hydrogen sulfide cietoxifcation by autoxidation (Beck et al., 198 1).

Sodium thiosulfate was even proposed as a possible H2S antidote

as with the help of rhodanese it converts cyanide to thiocyanate (Smith et

al., 1976). This therapy though, can hardly be justified for H2S poisoning

(Stine et al., 1976).

1.3.1. Chemicd properties of hydraocobalamin

Hydroxocobaiamin (OH-cobabalmin; vitamin B 1 4 (Figure 1 - 1) is a

derivative of cobalamin, which is a cofactor of cobalamine enzymes.

Cobalamin enzymes act as catalysts in three types of reactions - (1)

intramolecular rearrangements of amino and hydroxyl groups or

substituted carbons, (2) methylations and (3) reduction of

ribonucleotides to deoxyribonucleotides (Stryer, 1995).

The core of cobalamin consists of a corrin ring with centrai cobalt

atom and its three-dimensional structure was elucidated by Dorothy

Hodgkin in 1956. The conin ring has four pyrrole units. Like porphyrin

iron, the cobalt atom in cobalamin is bonded to four pyrrole nitrogens.

The fiNi substituent, below the comn plane is a derivative of

dimethylbenzimidamle, which contains ribose 3-phosphate and

aminoisopropanol. The sixth substituent, above the corrin plane, can be

OH- (hydroxocobalamin, vitamin Bis$, -CH3 or the 5'-deoxyadenosyl unit.

The cobalt atom can exists in the +1, +2 or +3 oxidation state.

Hydroxocobalamin has the cobalt in a +3 oxidation state which can be

reduced to a divalent state, Bi2r(Co2+) by a flavoprotein reductase. The

B12r form is further reduced by a second flavoprotein reductase to

BI~~(CO+). NADH is the reductant in both reactions.

Bila (Co3+) + B12r(Co2+) + B 12s(C0+)

In the BI^^ form, Co+ displaces the triphosphate group at the 5' atom of

ATP and forms 5'-deoxyadenosylcobalamin (coenzyme B 12).

OH. -

1.3.2. H y d r o x o c o b ~ i n as .n antidote fez @de poiroaing

Hydroxocobalamin is currently used in Europe for the treatment of

cyanide poisoning but has not been approved by governmental agencies

in Canada and USA even though it has a better safety profile than nitrite

(Bowden and Krenzelok, 1997). The usual recommended dose of

hydmxocobalamin is 50 mg/kg and a single dose of 5g may be sufficient

for most patients (Bowden and Krenzelok, 1997).

Hydroxocobalamin has been shown to reverse the effects of cyanide

poisoning on respiratory distress and convulsions in mice (Mushett et al.,

1952) and has been recommended for the treatment and prevention of

nitroprusside-induced cyanide toxicity (Zerbe et al., 1993). The

mechanism of action is believed to involve displacement of the -OH group

of hydroxocobalamin with cyanide anion to form cyanocobalamin (Astier

and Baud, 1995; Rion et al., 1990).

In antidotal studies against cyanide poisoning no toxicity of

hydroxocobalamin has been observed with doses up to 400 pM/kg given

intravenously (Astier and Baud, 1995). The usual recommended dose of

hydroxocobalamin as a dietary suplement is 5- 10 mg/day.

Interestingly enough, hydroxocobalarnin has never been tested for

hydrogen sulfide poisoning even though al1 other cyanide antidotes have.

1.3. RESEARCH O B J E C T I ' AND HPPOTHEûES

The aim of this study is to obtain a better understanding of the

mechanism of hydrogen sulfde toxicity and in that regard to propose a

new and better antidote.

Our hypothesis is that hydrogen suîfide is not ody t d c pes se

but that upon entering the c d it gets metaboUc~y ac th ted to

motive metaboiites, which then react with protein thiol. and

glutathione to form pendfider.

Our results suggest that cytochrome P450 is involved in hydrogen

sulfide oxidation, an action that has never been suggested before in

respect to H2S toxicity. We also present evidence suggesting that HÎS

does not simply form an inactive complex with cytochrome oxidase and

hemoproteins but is rather oxidized to reactive metabolites. In this light,

the use of nitrite as an antidote that induces methemoglobinernia has

very little justification, as even though H2S forms a complex with

methemoglobin, it may actively oxidize H2S to toxic SO species. Therefore,

it is more reasonable to use another metal-containing compound as a

H2S trap such as hydroxocobalamin which does not catalyze H2S

oxidation to generate toxic metabolites.

Bovine hemoglobin (approximately 75% methemoglobin) , cytochrome c, myoglobin, hydroxocobalamin, cyanocobalamin, antimycin

A, cumene hydroperoxide and m a n blue were obtained from Sigma

Chemical Co. (St. Louis, MO).

Sodium hydrosulfide hydrate (NaHS x &O), SKF-525A (2-

dimethylarninoethyl-2,2-diphenyl-n-pentanoate) , metyrapone and batho-

phenanthroline disulfonate were obtained from Aldrich Chemical

Company Inc. (Milwaukee, WI).

Desferoxarnine was a gift from Ciba Geigy Canada Ltd. (Toronto,

ON). Collagenase (from Clostridium histolytz8cum), 4-(2-hydroxyrnethy1)- 1 - piperazine e thanesulfonic acid (Hepes) and bovine semm albumin (BSA)

were obtained from Boehringer-Manheirn (Montreal, PQ) . HPLC grade

solvents were obtained from Caledon (Georgetown, ON).

Mi other chernicals were of the highest grade commerciaiiy

available.

2.2. Animal treiitment and hepatocyte prepuition

Hepatocytes were isolated from adult male Sprague-Dawley rats,

250-300g, that had been obtained from Charles River Canada

Laboratories (Montreal, PQ). Animals were fed od libitum and were aliowed to acclimatize for one week on clay chip bedding. Freshly isolated

hepatocytes were chosen as an intact cell model in this study as their

high cytochrome P450 levels make them suitable for toxin metabolism

studies (Moldeus et al., 1978.). Hepatocytes were prepared by liver

perfusion with collagenase as described by Moldeus (Moldeus et ai.,

1978). The hepatocytes were incubated in Krebs-Hensleit bicarbonate

buffer and Hepes at ph 7.4, 37OC for 30 min before the addition of

chemicals. The incubations were c d e d out in continuously rotaüng 50

ml round bottom flasks under 10% 0215% C02/85% N2 atmosphere.

Stock solution of chemicds were made either in distilled water

(maximum of 1% of the incubation volume) or methanol (maximum of

0.196, which had no significant effect on ceil viability or the assays).

Unless otherwise noted aii chemicals were added at the same time as

NaHS. After the addition of NaHS ail flasks were sealed with Parafilm@ for

30 min, kept in the incubator and regularly rotated. This practice did not

affect cell viability and was carried out because of the gaseous nature of

forrned HaS.

Ce11 viability was measured by a Trypan blue (0.2% w/v) exclusion

method, which is a good indicator of plasma membrane disruption

(Moldeus et al., 1978). The viability of cells at the beginning of the

experirnent was 80-90% and was further deterniined after incubation for

60, 120 and 180 minutes at 37OC.

2.8. HPLC anaiysis of hepatocyte C3SH leveh

The total amount of GSH and GSSG in isolated hepatocytes (5x106

cells/ ml) was measured in deproteinized samples (25% metaphosphoric

acid) which was reacted with iodoacetic acid, followed by a derivatization

with 1-fluoro-2,4-dinitrobenzene (Reed et al., 1980) using a Waters HPLC

system (model 510 pump, WISP 710B autoinjector and model 410

W / V I S detector) equipped with Waters p BondapakB NH2 (10 PM) 3.9 x

300 mm column. This method is sensitive enough that it can be used for

the determination of nanomole concentrations of glutathione, glutathione

disulfide, cysteine glutathione-mixed disulfde and several other sulfur-

containing amino acids or derivatives.

2.4. Oxygen consumption

Oxygen consumption in a sealed 2.1 ml chamber was monitored

with a Clark oxygen electrode (Yellow Springs Instrument Co. Inc., Mode1

5300)

Animals used for in uiuo antidotal and toxicity studies were adult

male CD1 mice, 25-30 g body weight obtained from Charles River

Canada Laboratories (Montreal, PQ) that were aliowed to acclimatize for

at least 7 days prior to experirnent on standard chip bedding. AU anirnals

were fed ad libitum and were not fasted before experiments. Ali chemicals

used were dissolved in 0.9% sterile saline solution and were

administered by intraperitoneal injections (O. lml/25g volume per weight

ratio of single injection). The suwivai of animals was recorded 24 h after

the treatment.

The spectral analyses of reactions between hydrogen sulfide and

hemoproteins or hydroxocobalamin were carried out using a Shimadzu

W-visible recording spectrophotometer mode1 W-240 "Graphicord,

Shimadni Corporation, Kyoto, Japan.

Statistical cornparisons were canied out by a Student's t-test as a

Test of Significance with a probability level of pc 0.05 unless otherwise

noted. Results represent mean f standard deviation.

Chapter 3

HEMOPROTEïNS AS ANTIDOTE8 ABlD METABOLIC

ACTIVATORS OF HYDROOEn SULFïDE CYTOTOXICITY

Hemoproteins are proteins with heme as a prosthetic group. Herne

consists of an iron atom that can exist in either an Fe(I1) or Fe(II1)

oxidative state and a protoporphyrin IX made of four pyrrole groups. The

functions of the hemoprotein are the transport of oxygen/carbon dioxide

(hemoglobin) or electrons in redox reactions (cytochromes) . Oxygen (02) can unly bind to the heme femus (Fe2+) form of

deoxyhemoglobin. The heme fernec (Fe3+) form of hemoglobin is known as

methemoglobin. Myoglobin is hemoglobin's counterpart in muscles that

c m also exist in two forms, oxidized and reduced. The difference between

the two hemoproteins is in the apoprotein part of the molecule, which in

myoglobin contains one heme containing single polypeptide chah

whereas in hemoglobin it contains four heme con taining polypeptide

chahs. Hemoglobin can therefore c a q four oxygen molecules whereas

myoglobin stores one oxygen molecule.

Cytochrome c (cyt. c) is a part of the electron transport chah in

mitochondria that has heme iron as a one electron-carrier which can

exist as the oxidized - ferric (Fe3+) or reduced - ferrous (Fe?+) form.

Catalase is also a hemoprotein with one heme in each subunit of

the enzyme. This homotetrarneric protein (in mammals) functions as a

special kind of peroxidase, which converts two molecules of hydrogen

peroxide into water and oxygen. The native oxidative state of heme iron

in catalase is femc (Fe3+) which is oxidized to a higher oxidation state

(Compound 1) by H202.

Catalase (Fe3+) + H202 Catalase Compound I + Hz0

Catalase Compound 1 + H g 0 2 + Catalase (Fe3+) + O2 + H20

It has been known for some time that H2S bhds to the cytochrome

a2+a33+ heme iron (Fe3+) and CU^+) of the cytochrome c oxidase system

and inhibits mitochondrial respiration (Nicholls and Kim, 1982).

Rotenone, antimycin A and cyanide are inhibitors of mitochondrial

electron transport. Rotenone is an inhibitor of complex I

(NADH:ubiquinone oxidoreductase and u biquinone) (Singer, 1979))

antimycin is an inhibitor of complex III (ubiquinol-cytochrome c

oxidoreductase) (Slater, 1973) between Cyt b and CI and cyanide is an

inhibitor of cytochrome c oxidase (Nicholls et ai., 1972).

Rotenone Antimycin 1 5.

NADH + Nu4DH-Q -, Q (ubiquinone) + QHrcytOchtiomc c --, reductase mductuse

(adapted from Stryer, 1995)

The cytochrome P450 dependent mixed function oxidases are

responsible for the detoxification and bioactivation of many xenobiotics

and are located mostiy on the endoplasmatic reticular membrane

(Guengrich, 1991). Some of them are also present in the mitochondria of

steroid producing tissues. These enzymes are monooxygenases as they

insert one oxygen atom into the substrate and reduce the other one to

water. They also have heme as a prosthetic group. Currently there are

over 300 different cytochrome P450 isoenzymes characterized (Josephy,

1997), however it is believed that three P450 gene families (CYPI, CYP2

and CYP3) are hepatic catalysts for the hydroxylation of xenobiotics

(Wrighton and Stevens, 1992). No data has been reported on the

involvement of Cyt P450 in H2S activation/ detoxifcation although the

hepatotoxin CS2 is metabolically activated by Cyt P450 (Dalvi et al.,

1975). Our preliminary results suggested that H2S is also metabolically

activated by cytochrome P450. Furthemore when cumene hydroperoxide

was used to substitute for NADPH/cytochrome P450 reductaselO2 in the

monooxygenase function of cytochrome P450 (Anari et al., 1996), H2S

induced cytotoxicity was markedly increased and the increase was

prevented by Cyt P450 inhibitors.

3.2.1. Addotal effect of hemoproteins on hyûrogen suifide

cytotdcitp

The results shown in Table 3-1. compare the ability of

hemoproteins, namely methemoglobin (met-Hb) , cytochrome c and

catalase to protect isolated rat hepatocytes against H2S toxicity.

The EDSO of NaHS (0.5 mM) used was established experimentally

with 0.2 mM being the dose at which no toxicity was observed and 0.7

mM being the EDw at 3 hrs (data not shown).

Met-Hb showed a dose-dependent protection that was effective over

a dose range from 10-50 pM when added to the ce11 culture at the same

tirne as 500 pM H2S. Met-Hb (10 pM) decreased the H B cytotoxicity at 3

hours from 65% cytotoxicity to 18% cytotoxicity. Moreover, when added

30 and 60 minutes after the H2S challenge, met-Hb decreased the H2S

cytotoxicity from 65% cytotoxicity a t 3 hours to 24% cytotoxicity and

38% cyto toxicity respectively.

The absorption spectra of Figure 3-1. shows that a met-Hb - H2S

complex was fonned when H2S was added to the met-Hb solution. On the

other hand protection by such low doses as 10 pM met-Hb against 500

FM NaHS suggests that the cytoprotective mechanism is not sirnply due

to the complexation of H2S by met-Hb. A s shown in Table 3-3., met-Hb

also readily catalyzed H2S autoxidation.

Catalase inhibited met-Hb stimulated oxygen consurnption by 50%

presumably by releasing oxygen from the H202 formed by H2S

autoxidation (Table 3-3). This suggests that most of the oxygen

consumption results in H202 formation.

Qtochrome c was also highly effective at protecting hepatocytes

from NaHS (Table 3-1). A s shown in Table 3-3. cytochrome c also readily

catalyzed H2S autoxidation, and caused a 6-fold increase in O2

consumption. Catalase also inhibited cytochrome c stimulated oxygen

consumption by 50%.

Catalase, which also complexes H2S however did not catalyze H2S

autoxidation. Furthemore, as shown in Table 3-l., catalase or

myoglobin was also much less effective than met-Hb or cytochrome c at

protecting hepatocytes from NaHS.

Myoglobin was 50% less potent than methemoglobin in catalyzing

H2S autoxidation.

The oxygen electrode data shown in Table 3-2. indicates that NaHS

markedly increased hepatocyte respiration. However this was prevented

by the respiratory inhibitors antimycin and cyanide, and the inhibited

respiration was similar to the level found in the absence of NaHS.

Rotenone inhibited normal hepatocyte respiration but did not affect

NaHS induced hepatocyte respiration.

Table 3-1.

Antidotaî efScct of hemoproteins tonudr Ha8 cytotdcity

Cytotarçfdirar ( % of t ypan blue uptake ut time, min)

f I + cytochrome c (100 PM) 35 f 4 37t2 39 t 3" II + catalase (100U/ml) 46f4 57f 4 60 f 5*

It + myoglobin (50 PM) 4 2 I 3 73f 7 80I7

NaHS (0.5 mM) II + met-Hb (50 PM) II + met-Hb (50pM) at 60'

Control (no treatment) 1 9 I 2 2 1 f 2 23 t 1

Hepatocytes (106 ceils/rnl) were incubated in Krebs-Hensleit buffer, pH

7.4, at 37°C under 10% 0215% C02/85% NZ atmosphere. The viability of

cells was assessed by detennining the percentage of cells that exclude

Trypan blue. Values are expressed as the means of three separate

experiments f SD.

48f3

26f 1

50f 4

* represents significantiy different from NaHS treated group (p< 0.05)

78 i 5

2 9 I 3

58 k 5

88k5

35 i 3*

61 i3*

Met-Hb (10 IrM) and NaHS (30 wen diss01vtd in TRIS-HCl buffer (pH 7.4; 0.1 M). The W-spectrum of met-Hb was opet a wave1cngth

range of 450-650 nm. 1- Met-Hb; 2- Met-Hb + NaHS at 0'; 3- Met-Hb + NafS at 15'; 4 Met-Hb + NaHS at 30' V i s d è - ab~oribana; Horiu,ntal s& - W C U / C ~ (~n)

I

Cytochrome c (20 pM) and NaXS (20 ph41 werc dissolved in TRIS-HCI

buffer (pH 7.4; 0.1 M) and the W absorption spectrum was s c a ~ c d over a wavc1ength range of 300600 am.

1- Cytochrome c + N W ; 2- CJrtochrome c

Table 3-2.

Ha8 moddated respiration in isolated hepatocytes

Rate of -en consumption ( nM 02/ min/ 1 06 cells)

None

NaHS (0.2 mM)

+ Cyanide

+ Antimycin

+ Rotenone

+ Metyrapone

+ Metyrapone + Rotenone

Cyanide

An timycin

Rotenone

Metyrapone

Rotenone + Metyrapone

The rate of oxygen consumption was determined with a Clark-type

oxygen electrode. Isolated hepatocytes ( 106 ceus/ ml) were incubated at

37°C in Krebs-Hensleit buffer, pH = 7.4, under 10Y0 02/5% C02/85% N2

atmosphere. Potassium cyanide (100 FM), antimycin A (10 PM),

aotenone (20 PM) or metyrapone (1 mM) were added at the beguining of

the experiment and NaHS was added -5 min after the start of the

experiment. Values are expressed as the means of three separate

experiments f SD.

a Significantly different from control (non-treated) group (pc 0.05)

Significantly different from NaHS treated group (pc 0.05)

Table 3-3.

H2S autddation cataiyzed by hemoproteins

+ catalase (20 U) 3.9 * 0.4a + cytochrome c oxidase (7.8 U) 3.8 I 0.3*

+ cytochrome c (50 FM)

+ myoglobin (50 PM)

+ cytochrome c + catalase

+ met-Hb + catalase 25.2 f 2 2

The rate of oxygen consumption was determined with a Clark-type

oxygen electrode. The experiments were conducted in Tris-HC1 (0.1 M)

buffer, pH 7.4, at 37OC. AU compounds were dissolved in distilled water.

Values are expressed as the means of three separate experiments * SD.

a Significantly different from NaHS treated group (pc 0 .OS)

3.2.2. Activation amd detdcation by cytochrome P450

hemoproteins

As shown in Table 3-4. Qt P450 seems to contribute to H2S

activation as the Cyt P450 inhibitors, metyrapone and SKF 525-A (2-

diethylaminoethyl-2,2-diphenylvalerate), markedly protected hepatocytes

from NaHS induced cytotoxicity. Metyrapone also increased NaHS

induced respiration (Table 3-2). This suggests that some Cyt P450

isoenzymes are involved in H2S activation. Another result to support this

belief is that SKI? 525-A was unable to prevent H2S toxicity when added

at 60 min, presumably because H2S had already been metabolized. The

CYP 2Cll inhibitor cimetidine and the CYP 2E 1 inhibitor, piperonyl

butoxide had no effect whereas the CYP 2E1 inhibitors benzylirnidazole

and phenylirnidazole, slightly increased H2S cytotoxicity implying that

CYP 2E 1 may be involved in H2S detoxifcation.

The marked increase in H2S cytotoxicity with a non-toxic dose of

cumene hydroperoxide also suggests that Cyt P450 catalyzes the

oxygenation of H2S to form "reactive sulfur species" e.g. singlet sulfur

atorns.

Table 3-4.

meet of P4SO inhibitoxa amd activators on Ha8 imduced cytotoalcty

carto-ciar (% of mpan blue uptake at tirne, min)

" + metyrapone (1 mM)* 1 26f3 1 29f3 1 31i3* " + SKF 525-A (50 PM)* 1 34f 3 1 SOI5 1 54f 5'

" + SKF 525-A (50pM) at 60'* 1 39f4 / 5 9 I 5 1 70i7 1

" + cimetidine ( 1 mM)b 34k4 64I4 7 5 2 7

" + piperonyl butoxide ( 1 mM)c 40i4 61 1 6 7 7 I 8

" + phenylimidazole (100 pM)c 1 50 f 5 1 84 1: 8 1 100 1 r

" + CHP (100 P M ) ~ 62*4 100 100

" +CHP+-metyrapone 37f 4 45f4 53f S*

" + CHP + SKF 525-A 41 I 4 5 6 k 5 62I5"

Control (no treatrnent) 1 19f2 1 21f2 1 2 3 I 1

" + CHP (100 PM)

CHP - cumene hydroperoxide

a non-specific Cyt P450 inhibitors CYP 2C11 inhibitor CYP 2E 1 inhibitors substrate for P-450 peroxygenase/peroxidase

Hepatocytes (106 cells/ml) were incubated in Krebs-Hensleit buffer, pH

7.4, at 37°C under 10% 02/5% C02/85% N2 atmosphere. The viability of

cells was assessed by determinhg the percentage of celis that exclude

Trypan blue. Values are expressed as the means of three separate

experiments f SD.

* Signifcantly different from NaHS treated group @< 0.05)

3.2.3. Effect of Ha8 on hepatocpte GSH leveïs

Figure 3-3. shows that an E D ~ o concentration of NaHS depleted

70% of hepatocyte GSH in 60 min before cytotoxicity ensued. By 2 hrs

50% cytotoxicty occurred at which time 94% of the GSH had been

depleted.

Metyrapone prevented hepatocyte GSH depletion (Figure 3-3) and

by 2 hrs only 53% of total GSH was depIeted versus 94% GSH depleted

in the NaHS treated group.

As shown in Figure 3-4., H2S in vitro did not react with GSH.

However a marked depletion of GSH without oxidation occurred in the

presence of catalytic concentrations of met-hemoglobin even though GSH

levels were not affected by met-hemoglobin alone.

-8 induced GSH depletion in isolated hepatocytes

30 60 120 Tinre (min)

Rat hepatocytes (1x106 cells/ml) were incubated under a

constant flow of lO%O2/8S%N2/ 5% CO2 gas. GSH levels

were detennined by the method of Reed et al. (1980) as

described in Chapter 2. BCS(200 PM) and metyrapone (1

mM) were added to the corresponding groups at the same

time as NaHS (500 FM). Values are expressed as the means

of three separate experiments f SD.

In vitro GSH depletion by &8 is catalyzed by

hemoproteins

4- GSH (O. 1 mM)

+ + NaSH (1 mM)

+ + NaSH (0.25 mM) + hemoglobin (0.025mM)

-O- + NaSH (0.5 mM) + hemoglo bin (0.025mM)

+ + NaSH (1mM) +hemoglobin(O. 02 5mM)

O 30 60

Thne (min)

Experiments were carried out in 0.1M Tris-HC1 buffer and

GSH concentrations were determined by the HPLC method

described in Chapter 2. Results courtesy of Bîn W u (Bin

Wu, 1998, unpublished results).

3.3. DISCUSSION

HQS has a greater affmity for heme Fe3+ (oxidized) than Fe2+

(reduced) (Nicholls and Kim, 1982) which explains the ability of

methemoglobin to prevent its toxicity by fonning a sulfhemoglobin

complex (Fe3+-SH). The formation of this complex is supported by the fact

that a W spectral scan of met-Hb following the addition of sulfide, shows

increased absorption at 550 nm. (Figure 3-l), which has been attributed

to a sulfmethemoglobin complex formation (Smith et al., 1976). However

much less hemoglobin was required to prevent H2S cytotoxicity than

would be expected from that required for 1: l heme-HS- complex

formation. Oxygen consumption was also observed in a met-Hb+NaHS

solution in agreement with previously published results, which were

attributed to met-Hb catalyzing the autoxidation of NaHS (Beck et al.,

1981). The ability of metMb to protect even when given 30 to 60 minutes

following NaHS addition suggests that met-Hb theoretically could act as

a promising antidote.

Since myoglobin possesses only one heme oxygen binding site and

is a single polypeptide chain molecule, it therefore carries only one H2S

catalytic site. This could explain its inability to protect hepatocytes

against NaHS toxicity (Table 3-1) even though it was able to increase 0 2

consumption three-fold (Table 3-3) suggesting that it also catalyses H B

au toxidation.

Previously it was shown that NaHS reduces the heme (Fe3+) of

cytochrome c to f om femcytachrome c without forming a complex but

still increased NaHS autoxidation (Nicholls and Kim, 1982). This was

confmed in Table 33. and Figure 3-2. These results suggest that H2S

autoxidation is activated/catalyzed by cytochrome c, and could partially

prevent H2S from binding to mitochondrial cytochrome oxidase.

A s shown in Table 3-3., catalase unlike other hemoproteins

inhibited H2S autoxidation. This suggests that catalase does not catalyze

H2S autoxidation and furthermore that H2S foms Hz02 on autoxidation.

Catalase therefore inhibits oxygen consumption by releasing 0 2 from

H202. At higher H2S concentrations, catalase is inactivated by forming

heme Fe3+:SH complex (Carlsson et al., 1988). Added catalase was only

partially effective at protecting hepatocytes from H2S (Table 3-1) even

though catalase formed a complex with H2S.

The addition of H2S to hepatocytes surprisingly increased oxygen

consumption (Table 3-2) even though H2S inactivated cytochrome

oxidase. Sanide is a specific inhibitor of cytochrome oxidase, which

prevented 0 2 consumption induced by NaHS by blocking electron

transport from Cyt c to 0 2 . Antimycin A is a specific inhibitor of

mitochondrial electron transport at a site between Cyt b and CI and also

inhibited the increased 0 2 consumption induced by H2S. Rotenone is a

specific inhibitor of the NADH dehydrogenase moiety of mitochondrial

electron transport but did not inhibit sulfide-induced respiration. This

result may seem contradictov as H2S inactivates cytochrome oxidase

and we would expect that the rate of 0 2 consumption, which corresponds

to respiration, would be decreased. It can also be concluded that

hepatocytes do not catalyze H2S autoxidation as the increased 0 2

consumption was inhibited by cyanide or antimycin. This suggests that

H2S increases hepatocyte respiration by reducing cytochrome b and

ubiquinone and thereby feedhg electrons into the respiratory chah.

Other investigators have shown that sulnde oxidation by the

lugworm Arenicola marina, the clam Solemya reidi and the killifish

h<ndulus pampinnis is localized in the rnitochondria and is also

inhibited by antimycin but not rotenone. Sulfide oxidation in these

species is coupled to the synthesis of ATP and thiosulfate is the product

formed. However high sulnde concentrations inhibited clam and killifish

sulfide oxidation by inactivating cytochrome oxidase whereas lugworm

sulfide oxidation was resistant (V6lkel and Grieshaber, 1996). Sulfide

induced respiration was also less sensitive to cyanide than normal

respiration. This was attributed to an alternative terminal oxidase that

enables lugworm to detoxifl sulfide even at high tissue levels of sulfide.

High su!fide concentrations however inhibited liver mitochondna

catalyzed respiration and sulfide oxidation. Sulfde oxidation by isolated

rat liver mitochondria however has been reported not to be coupled to

ATP synthesis (Powell and Somero, 1986) but the sulfide concentrations

used in these studies are likely to have inactivated cytochrome oxidase.

A s shown in Table 3-4., the Cyt P450 inhibitors, metyrapone and

SKF 525-A protected against H2S induced cytotoxicity, which suggests

that CYP 3A and 2B are probably responsible for hydrogen sulfide

activation as they are for CS2 metabolic activation (Chengelis and Neal,

1987). The P450 dependent metabolic activation of carbon disulfide

leading to hepatotoxicity is beiieved to involve the following reactions:

0 2 O2 S=C=S + S + [S=C=S+-O-] --+ CO2 + HS-

monothiocarbonate

The final products are CO2 and H2S. The released sulfur atoms readily

react with protein cysteine on the microsomal membranes to presumably

form protein cysteine persulfide. Interestingly, the monothiocarbonate

intermediate is converted to carbonyl sulfde by carbonic anhydrase and

carbonyl sulfide has been actuaîiy found to be toxic to rats as a result of

H2S formation catalyzed by carbonic anhydrase (Chengelis and Neal,

1980). Cyt P450 may catalyze H2S oxidation to form 'reactive sulfur

species" e.g. SH' radicais that react with oxygen causing a subsequent

formation of reactive oxygen species and elemental sulfur, which oxidize

protein-SH groups or form protein persullides respectively. The inability

of SKF-525A to protect against H2S toxicity if added at 30-60 min was

like1y because the H2S had already been metabolized by the hepatocytes.

Metyrapone also inhibited the increased 0 2 consumption in the presence

of rotenone (Table 3-2) also suggesting that H2S autoxidation is catalyzed

by Cyt P450.

In the hepatocyte the following mitochondrial enzymes are likely

involved in the metabolic oxidation and detoxification of HS-:

0 2 0 2 0 2

HS- ,-> ---3 S032-- S0q2- cytochrome c GSH/ thiosu lfae suifite

or sulfide oxidase (2) reductase om'duse

On the other hand, CYP 2E1 inhibitors, cirnetidine, piperonyl

butoxide, benzylirnidazole and phenylimidazole were not able to prevent

H2S toxicity, which suggests that CYP 2E1 does not modulate the

oxidative metabolism of H2S to form the toxic SH' and Sa species.

Alternatively, the CYP 2E1 may be inactivated by H2S as was previously

shown for CS2 (Lauriault, 1992; Snyderwine et al., 1988) and would

explain the lack of effect of CYP 2E1 inhibitors on H2S toxicity. Indeed,

H2S may like CS2 prove to be a highly specific inhibitor for the CYP 2E1

isoenzyme.

The marked Hicrease in hydrogen sulfide cytotoxicity on addition of

a non-toxic dose of cumene hydroperoxide, a cofactor for Cyt P450

peroxygenase function, also suggests that H2S is oxidatively activated by

Cyt P450 to form cytotoxic 'reactive sulfur species*. In support of this the

CYP 3A/2B inhibitors metyrapone or SKF 525A also prevented the

cumene hydroperoxide enhanced HB cytotoxicity.

mirther proof that Cyt P450 can metabolically oxidize H2S to

'reactive sulfur species" was the fmding that hepatocyte GSH is readily

depleted by H2S (Figure 3-3) even though H2S does not react with GSH

(Figure 3-4). H2S did however deplete GSH in the presence of

methemoglobin (Figure 3-4). Furthemore hepatocyte GSH depletion by

H2S was inhibited by metyrapone, a Cyt P450 inhibitor (Figure 3-3).

Glutathione persulfide formation by the following set of reactions could

explain these results:

0 2 0 2

HS- -r HS'- SO hemoprotein (P450)

SO + GS- t, GSS-

GSH depletion was decreased in the presence of copper chelator

BCS (bathocuproine disulfonate), a result which will be discussed in the

next chapter of this thesis.

Chapter 4

METAL8 A8 ANTIDOTES AND ACTIVATORS

4 1 INTRODUCTION

A s discussed in Chapter 1 of this thesis, current treatments used

for treating H2S intoxication include exposure to hyperbaric oxygen and

the intravenous administration of thiosulfate and nitrite (Smith et al.,

1976, Whitcraft et al., 1984, Smilkstein et al., 1985) even though many

authors are in disagreement with the beneficial effects of these therapies.

Although the results presented in Chapter 3 speak in favor of met-

Hb induction as a promising antidote therapy, we believe that it is still

difiicult to safely achieve sumcient in vivo concentrations with nitrite so

as to be able to catalyze H2S detoxification. It could also be lethal if the

victim had also been exposed to carbon monoxide which converts

oxyhemoglobin to carbon monoxyhemoglobin. Therefore we have

searched for new antidotes that may be more rapid and effcient in the

management of H2S poisoning when time is crucial.

The naturally occurring ore of molybdenum and suifur - MoSl led

us to the idea that sodium molybdate may be able to complex H2S. There

was no data in the scientifc literature as to whether molybdate can

prevent hydrogen sulfide toxicity, although inorganic chemists have

known for some üme that H2S reacts in acidic solution with Mo022+ ion

to form MoS2.

H2S has also been used for a long time as a histochemical "trap"

for Zn2+ ions and has also been shown to react with the Cu?+ and Zn2+

ions of superoxide dismutase (Searcy et al., 1995). A paper that proposed

the use of Zn-acetate for H2S elidnation from the intestine of the

patients suffering from ulcerative colitis appeared recently but the

results of such a clinical trial have not been pubiished yet (Suarez et ai.,

1998).

Cobalt compounds - cobalt chloride (CoCh), hydroxo- (vitamin Biza)

and cyanocobalamin (vitamin Bi4 were also tested for their antidotal

properties. Smith rejected the use of cobalt chloride as an antidote as it

was considered too toxic and did not induce methemoglobinemia unlike

cobalt nitrite (Smith RP, 1969). This is to be expected, as it is the nitrite

moiety that oxidizes oxyhemoglobin. Ironically, Smith did not test the

antidotal effect of cobalt chloride. Hydroxocobalamin has never been

tested as an antidote for H2S toxicity, even though it has been used

cfinically in experimental cyanide poisoning (Muschett et al., 1952) and

is now the major antidote for cyanide poisoning in Europe.

In this Chapter the research results described suggest that metals

in non-toxic doses can act as antidotes for managing H2S poisoning but

that some of them may activate H2S. We have also discovered a much

more potent and safe antidote for treating H2S intoxication,

hydroxocobalamin (OH-cobalamin), than the currently used nitrite

compounds. Although our new antidote, hydroxocobalamin, has only

been tested in mice, its natural presence in the human body and its

powerful antidotal properties in preventing H2S toxicity show potential

for a promising treatrnent of hydrogen sulfide intoxication.

4.2. RESULTS

4.2.1. Inactivation of Ha8 by c o m p k formation with Pb, Mo and Ba

ions

A s shown in Table 4-1. molybdenum, barium and lead, in non-

toxic concentrations, were able to form a complex with H2S and protect

hepatocytes against its toxicity. Na-molybdate also prevents H2S

autoxidation (Table 4-1 and 4-2) which suggests that molybdate traps

H2S to form tetrathiomolybdate. Evidence of complex formation between lead and sulfide is the

formation of a gray-brown precipitate when a buffered solution of lead

acetate (Pb(CH00)2) is mixed with NaHS. Furthemore lead had no effect

on oxygen consumption (Table 4-2), which suggests that lead does not

catalyze H2S au toxidation. Barium hydroxide (Ba(OH)2) formed a white-

yellow precipitate and prevented NaHS induced hepatocyte toxicity.

4.2.2. Co, Cu, Ni and Fe catdyze Ha8 autoxidation

The protection against H2S induced cytotoxicity offered by non-

toxic concentration of cobalt chloride (CoC12) (Table 4- 1) was

accompanied by a marked increase in oxygen consumption (Table 4-2).

This suggested that the mechanism of detomcation by cobalt involves

the formation of oxygenated cobaltic sulfide complexes. The oxygen

consumption can be attributed to the formation of oxygenated cobaltous

and cobaltic sulfide products (Middleton and Ward, l933), a process that

involves the formation of transient Co(SH)3, Co(SH0)a species, S2032- and

S042- as end products.

Nickel, in non-toxic concentration, also caused a marked increase

in oxygen consumption (Table 4-2) likely because of the formation of

oxygenated sulfide nickelous complexes (Middleton and Wmd, 1935).

Protection by copper sulfate (CuSOq), given at non-toxic

concentration, probably results from cuprous sulfide formation, which

catalyzed H2S autoxidation. Thanks to the ability of copper to redox-cycle

between CU^+ and Cu+, H2S can be autoxidized as follows:

Cu(I1) + H2S + Cu(1) + HS'

Cu(1) + 0 2 + Cu(I1) + 02-

HS' + O2 + S0 + 0 2 -

Ferrous sulfate (FeSOq), at concentration that was not toxic to

hepatocytes, was not as potent as previously mentioned metals in

protecting against H2S toxicity most likely due to the cytotoxicity of

ferrous sulfide particularly if it penneates the ceil. Femus suifide reacts

rapidly with oxygen and can be used as a reducing agent for the culture

of anaerobes (Brock and O'Dea, 1977). The overd1 reaction is as follows:

FeS + 2.25 0 2 + 2.5 H 2 0 - t Fe(OH)3 + S0$- + 2H+

4.2.3. Cdcium increased Ha8 toxicity

As shown in Table 4-l., calcium chloride (CaCh) increased the

susceptibility of hepatocyte to hydrogen sulfide toxicity. Furthemore

Ca?+ chelators - EGTA [ethylene glycol bis(beta-aminoethylether-N, N, Nt,

Nt-tetraacetic acid] and BAPTA [ 1,2-bis(o-aminophenoxy)ethane-N, N, Nt,

N-tetraacetic acid] showed significant protection (pe0.05) against NaHS

toxicity (Table-4-3). These results suggest that extraceliular calcium ions

increased the susceptibility of hepatocytes to H2S toxicity. The decreased

H2S cytotoxicity caused by these chelators suggests that Ca2+ ions are

involved in H2S cytotoxicity.

Table 4-1.

Cyto-cftsr ( 5% of trypan blue uptake ut tinte, min)

Control (no treatment) 21f 1 23I2 26*2

Hepatocytes (106 ceUs/ml) were incubated in Krebs-Hensleit buffer, pH

7.4, at 37OC under 10% &/5% C02/85% N2 atmosphere. The viability of

ceîls was assessed by the percentage of cells excluding Trypan blue.

Values are expressed as the means of three separate experiments f SD.

* Signifîcantly different from NaHS treated group (pç 0.05)

Table 4-2.

Moduiation of Ha8 autorddation by various metd salts

None

NaHS (0.5 mM)

+ Na-molybdate (1 mM)

+ Pb(CH&00)2

+ FeCL (1 mM)

+ CuS04 (200 PM)

+ NiCh (1 mM)

+ CoCh ( ImM)

+ OH-cobalamin (0.1 mM)

+ cyanocobalamin (0.1 mM)

The rate of oxygen consumption was determined with a Clark-type

oxygen electrode. The experiments were conducted in Tris-HCl(0.1 M)

buffer, pH 7.4, at 37OC. All compounds were dissolved in distilled water.

Modulating agents were added after 3 min.

Values are expressed as the means of three separate experiments f SD.

* Arbitrarily established value for immediate reaction that consumes ail

oxygen within -1 min.

4.2.4. Effect of metd cheiators on Ha8 toxicity

BCS (bathocuproine disulfonate) , a Cu+ specific, non-cell-

permeable metal chelator protected against hydrogen sulfide toxicity

(Table 4-3) probably by chelating extracellular membrane Cu ions that

could be responsible for HB in activation by extraceliular autoxidation.

This was confmed as BCS also inhibited CuS04 stimulated oxygen

consumption in NaHS solution(Tab1e 4-4). As shown in Figure 3-3., the

Cu chelator BCS slightly delayed by 90 minutes the H2S induced GSH

depletion in isolated hepatocytes, thus suggesting that extracellular

copper ions are involved in H2S autoxidation to a metabolite that depletes

GSH.

On the other hand, BPS (bathophenathroline sulfonate), a Fez+

specific, non-ce11 permeable metal chelator was ineffective ai preventing

hydrogen sulfide toxicity.

Neocuproine (NC) (2,9-dimethyl- 1,lO-phenanthroline) , a cell-

permeable Cu+ chelator increased H2S toxicity (Table 4-3) probably

because the redox inert but ce11 penneable Cu:NC complex is toxic

(Quah, 1997). The inhibition of CuS04 cataiyzed H2S autoxidation by NC

(Table 4-4) suggests that copper ions are involved in H2S autoxidation.

Desferoxamine, an Fe3+ ion chelator (Halliwell, 1989) showed a

slight protection against H2S cytotoxicty, which could indicate that

intraceliularly released iron contributes to cytotoxicity.

Table 4-3.

Effect of Fe, Cu amd Ca chelating agents on Ha8 hduced

CytotoaioiQV

Cytotaxiwl (% of typan blue uptake ut tirne, min)

60' 120' 180' NaHS (0.5 mM)

1

48 f 5 57&6 6 4 f 5

" + EGTA (2 mM) 3 0 I 3 3 4 I 4 40 * 4*

" + BAPTA (100 PM) 1 32k2 1 38k3 1 4 3 f 4 *

" + BCS (200 PM) 1 3 5 f 3 1 3 9 I 4 1 44f 4*

" + BCS (100 PM) 1 4 5 f 3 151I4 1 5 7 f 4

" + desferoxamine (500 PM) -60' 41f4 4 9 I 4 5435

" + BPS (200 PM) 1 48f5 1 55*5 1 60f6

" + neocuproine (100 PM) 1 4 7 f 4 1 6 3 f 6 1 8 0 f 8

Control (no treatment) 21I1 23f2 2 6 f 2

Hepatocytes (106 cells/ml) were incubated in Krebs-Hensleit buffer, pH

7.4, at 37OC under 10% 02/5% C02/85% N2 atmosphere. The viability of

celis was assessed by measuring the percentage of cells that excluded

Trypan blue. Values are expressed as the means of three separate

experiments f SD.

* Significantly different from NaHS treated group (p< 0.05)

Table 4-4.

Modulation of HaS autddation by metai chehting agents

+ CuS04 (200 PM) + BCS (100 PM)

+ CuS04 (200 FM) + BCS (200 PM)

+ CuSO4 (200 PM) + neocuproine (100 PM)

+ CuS04 (200 FM) + neocuproine (200 PM)

The rate of oxygen consumption was deterrnined with a Clark-type

oxygen electrode. The experiments were conducted in Tris-HCl(0.1 M)

buffer, pH 7.4, at 37°C. Al1 compounds were dissolved in distiiled water.

Modulating agents were added after 3 min.

Values are expressed as the rneans of three separate experiments f SD.

4.2.6. Aatidotrl properties of hy&oxocobalamin in vitm and in u b

The results given in Table 4-1. and 4-5. show a dose-dependant

ability of hydroxocobalamin (OH-cobalamin) (50 and 100 PM) to prevent

H2S toxicity towards isolated hepatocytes. Antidotal properties were

observed even 60 min after the challenge with H2S.

As shown in Table 4-2., an immediate increase in oxygen

consumption, occurred on addition of OH-cobalarnin (Co3+) to a buffered

solution of NaHS. Furthemore, as shown in the spectral changes

described in Figure 4-l., two equivaients of NaHS added to OH-

cobalamin formed a sulfide complex in 5 minutes which caused a

decrease of the absorbance maxima at 361 nm and the appearance of a

peak at 370-372 nm characteristic of a methylcobalamin-like y-peak.

Such a spectral complex has not been reported before but a similar

spectra (but '~ithout the 420 absorbance maxima) is formed when GSH is

incubated with NaHS. Nuclear magnetic resonance and X-ray absorption

spectroscopy studies have shown that glutathione is coordinated to the

cobalt atom (Co3+) via the cysteine sulfur atom (Scheuring et al., 1994;

Brown et al., 1993).

Although cyanocobalamin (Co2+) offered the same rate of

hepatocyte protection as OH-cobalamin (Table 4-5) it did not affect H B

autoxidation to the sarne extent as OH-cobalamin (Table 4-2)

Studies in vivo (Figure 4-2) showed that OH-cobalamin prevented

NaHS-dose dependant-lethality in rnice. Animals were treated with i.p.

injections of OH-cobalamin within 2 min following a NaHS challenge.

Protection by OH-cobalamin was complete (100%) w&h an LD~o (25

mg/kg NaHS) dose while 53% of the animals s u ~ v e d treatment with an

LD95 (32 mg/kg NaHS) dose.

Figure 43. shows that NaHS induced lethality was also prevented

if the mice were pretreated (-20 min) with sodium nitrite so as to Muce

methemoglobinemia. However when nitrite was given 2 min after the

NaHS (LDgs) dose only 33% of the animals survived. The difference

between these two treatments was significantly different with a confidence level of p4.O 1. Thus we can conclude that OH-cobalamin is a more effective and desirable treatment for acute H2S poisoning.

The pretreatment of animals with OH-cobalamin (1 and 2 mM/kg)

was not protective probably because most of the OH-cobalamin had

metabolized by the üme of the NaHS challenge. Increased concentrations

of OH-cobalarnin (2 mM / kg] did not show statistically different protection

when given as an antidote (at 2 min) suggesting that 1 mM/kg of OH-

cobalamin is the optimal concentration for acute poisoning in mice.

Table 4-5.

b

IMwttment C y t o u a ~ (% of typan &lue uptake at time, min)

A 60' 120' 180' NaHS (0.5 mM) 5 2 f 4 70î5 87f 7

L

Control (no treatrnent) 21I2 23 f 1 2 6 I 2

Hepatocytes (106 cells/ml) were incubated in Krebs-Hensleit buffer, pH

7.4, at 37OC undei a 10% 0215% C01/85% N2 atmosphere. The viability

of cells was assessed by the percentage of cells excluding Trypan blue.

Values are expressed as the means of three separate experiments f SD.

* Significantly different from NaHS treated group (p< 0.05)

The reaction mixture containcd TRIS-HC1 buffer (pH 7.4,O. 1 Ml.

OH-cobalamin (10 pM] and N&S (20 pM) were dissolved in distilled

water. The W-spectrum of OH-cobalamin was scmned over a

wavelength range of 200-700 nm.

1- OH-cobalamin; 2- OH-cobaiamin + NaHS at timc 0'; 3- OH-c0bakm.h

+ NaHS at time 25'

Vertical scalc - absorbante; Ho&tantol d e - wavelength (m)

Antidotal effectimeu of OH-cobdarnin rigainst lethdity induced by different HaHS concentrations

NaHS (25 mg/kg) NaHS (30 mg/ks) NaHS (32 mg/kg)

Experiments were carried on male CD1 mice (25-30 g body weight). Each

experimental group consisted of 15 animals. The chart represents the

survival rate observed 24 hrs after NaHS challenge. Animals were treated

with i.p. injections of OH-cobalamin at 2 min after the NaHS.

The sumival of animals in al1 three groups were significantly different

(p<O.O 1).

A cornparison of the antidotal effectiveness of NaNOa and OH-cobalamiii against NoW8 induced

lethality

i NaHS (32 mg/kgj

OH-

I OH-

I OH- Y

Experiments were carried out with CD 1 male rnice (25-30 g body weight).

Each experimental group consisted of 15 anirnals. AU groups received

one single i.p. injection of NaHS (32 mg/kg). Three groups (- 20') received

a pretreatment with NaNO2 or OH-Cbl 20 min before NaHS, while the

other three groups received their antidotes within 2' of NaHS challenge.

The Survival rate was observed for 24 hrs after treatment.

Qanocobalamin (10 PM) and NaHS (20 pM and SOpM) were dissolved in

distiiled water and the rcaction mixture contained TRIS-HCI bmer (pH 7.4, 0.1 M). The W-spectrum of OH-cobalamin was scanned over a

wavelength range of 200-700 nm. 1- Cyanocobalamin; 2- Cyanocobalamin + NaHS (20 PM) at tirne 0';

3- Cyanocobalamin + NaHS (20 PM) at tirne 25'; 4- Qanocobalamin +

NaHS (50 PM) at time 2 5

Vertid s d e - absorbanœ; Horiu,ntd sccrlc - wavehgth (nm)

4.3. DISCUSSION

In the previous Chapter we have discussed how hemoproteins are

able to oxidize and/or trap hydrogen sulfide and thus participate in the

activation and detoxification of &S. In this Chapter we are showing how

various metals modulate H2S toxicity and are providing evidence for,

what we believe to be, the fiist effective antidote for Hd toxicity - hydroxycobalamin.

The inorganic salt of molybdenum was able to prevent H2S toxicity

probably by forming a metal:sulfide complex as there was a change in

the color of the hepatocyte suspension (from off-white to MoS2 blue-gray)

and a precipitate was fonned. Furthemore the H2S induced respiration

was prevented. The ability of molybdate to protect against HzS toxicity

was so great that it even acted as an antidote when administered 60 min

after the H2S had been added to the hepatocytes. The ingestion of

molybdate by ruminants has been shown to react with sulfide (generated

by the reductiun of dietary sulfate) to form tetrathiomolybdate

M004~* + 4 H2S + MoS$* + 4 H 2 0

which can be harmful by causing copper deficiency as a result of

complexing copper (Mius and El-Gallad, 1981) to form copper

tetrathiomolybdate. However even though molybdenum is present as a

cofactor of xanthine and aldehyde oxidase in the human body, Iittle is

known about molybdate toxicity, which would prevent us from saying

that it can be a new, safe antidote, although tetrathiomolybdate is

currently being used clinically as chelation therapy (instead of

peniciîîamine) for Wilson's disease (a copper hepatic overload disease)

(Brewer, 1995).

The protection by barium hydroxide and lead acetate against H2S

induced cytotoxicity is based on the sarne mechanism as molybdate Le.

by fonning PbS and BaS complexes. In both cases there were changes in

the color of the hepatocyte suspension and a precipitate was fonned.

However due to the high toxicity of these metals and poor elimination

they represent unsuitable antidotes.

Ferous sulfate (FeS04) was not as effective as other metals in

protecting against H2S cytotoxicity and could be due to the toxicity of

ferrous sulfide complexes.

Bathocuproine disulfonate, a membrane impermeable copper

chelator, prevented H2S cytotoxicity and partly delayed GSH depletion

which suggests that H2S is activated by copper ions on the cell

membrane and/or in the ce11 media. However bathophenantroline

sulfonate was ineffective and suggests that membrane-bound iron ions

did not affect H2S activation. The cytoprotection by desferoxamine, a

ferric chelator, suggests H2S causes the release of the intacelluiar Fe-

ions that contribute to H2S cytotoxicity. We also concluded that calcium

ions contribute to H2S cytotoxicity as a result of calcium influx following

the inhibition of rnitochondrial respiration by H2S.

Cobalt nitrite was first proposed as a treatment for H2S toxicity by

Smith (Smith, 1969). It was believed that the best treatment for hydrogen

sulfide toxicity was to induce methemoglobinernia so as to trap the H2S.

Currently sodium nitrite is used by North American Poison Control

Centers to treat cyanide poisoning. Cobalt nitrite was much more

effective than cobalt chloride at preventing H2S lethality in rnice which

was attributed to the relative effectiveness of nitrite at inducing

methemoglobinemia. Several authors have questioned the effectiveness of

inducing methemoglobin formation to prevent sulfide poisoning (Burnett

et al., 1977; Ravizza et al., 1982; Beck et al., 1981) and have pointed out

the possible contra-effects (Beck et al., 1981). Because of a need for a

new, safer and faster antidote, various in uitro and in vivo experiments

were carried out to test the effectiveness of OH-cobalamui as an antidote

against sulfide poisoning.

Our results suggest that OH-cobalamin can be used as an antidote

which is more effective and safer than the currently used nitrite.

Experiments with OH-cobablarnin in vitro revealed potent antidotal

properties that were further supported with results obtained from in vivo

experiments. Nitrite only prevented H2S leth* in mice if used

prophylactically. On the other hand, OH-cobalamin, when given 2 min

after a NaHS challenge showed significant protection (Fig. 4-2 and 4-3).

Its mechanism of protection is likely based on trapping the H2S as a

sulfide complex which then catalyzed extracellular Ha$ autoxidation

(Table 4-2). This mechanism would also suggest that concomitant

treatment with oxygen could be useful. OH-cobalamin was also

previously shown to catalyze the aerobic oxidation of thiols e.g. 2-

mercaptoethanol and dithioerythritol (Jacobsen et al., 1984). OH-cobalamin

2RSH + 0 2 - RSSR + Hz02

The proposed mechanism is as follows:

RS RS & 0 2 RS-

RSH + Co(1II) + Co(I1I) -+ Co(II1) + RSSR +H202 + Co(I1I) 7'

OH-cobalamin has never been tested as an antidote for H2S

poisoning, although various experimental and other clinical studies

indicate that it is a safe, rapid and effective cyanide antidote (Favier et

al., 1993; Rion et al., 1990; Vogel et al.,1981) and can remove cyanide

from the cyanide-cytochrome c oxidase complex (Lopes and Campello,

1976). A 5g infusion of OH-cobalamin in acute cyanide poisoning in

humans leads to a dramatic improvement in their clinical status

{Bowden and Krenzelok, 1997). Recently OH-cobalamin, an essential

physiological vitamin, has been shown to penneate cyanide loaded cells

and complex cyanide to form the non-toxic cyanocobalamin (vit. Bi4 an

essential dietary vitamin (Astier and Baud, 1996).

Cyanocobalamin was tested as an antidote for cyanide but was

ineffective probably because it is much less effective than OH-cobalarnine

at binding cyanide (Zerbe and Wagner, 1993). It was also ineEective at

binding sulfide (Figure 4-4) and explains the inability of cyanocobalamin

to catalyze H2S autoxidation (Table 4-2). On the other hand, OH-

cobalarnin formed a sulfide complex, catalyzed H2S autoxidation and

fuliy protected against H2S (Table 4-51, This was also supported by the

UV spectral scan of OH-cobalamin and NaHS (Figure 4- 1) , which shows a

decrease of the 361 nm absorption peak of OH-cobalarnin and a shift of

the 530 nm peak to 540 nm as was observed when the sulfur of GSH

complexes with the Co of OH-cobalamin (Scheuring et al., 1994; Brown

et al., 1993). It is dmcult to explain the cytoprotection observed with

cyanocobalamin but could be explained as cyanocobalamin is converted

by the hepatocytes to OH-cobalamin or methylcobalamin. Recently it has

been shown that cyanocobalamin is converted to glutathionylcobalamin

with a liver cytosolic fraction, NADPH and glutathione (Pezacka, 1993).

The enzyme involved has been named cyanocobalamin P-ligand

transferase. Methylcobalamin may aiso methylate H2S to form the less

toxic methanethiol (CH3SH) and dimethylsulfide (CH3SCH3). Such a

detoxifcation methylation reaction has been proposed by Weisiger et al.

(Weisiger et al., 1980) in which H2S methylation was catalyzed by

rnicrosomal thiol S-methyltransferase and S-adenosyl-L-methionine

(SAM) transferase (Weisiger et al., 1980) . SAM SAM

H2S - CHSH CH3SCH3 tramferase transferase

Hydrogen sulfide cytotoxicity seems to be partly mediated through

reactive metabolites, which rnay then react with cell constituents and

ultimately impair its metabolism.

In C m + 3, we showed that hydrogen sulfide reduced the iron

of cytochrome c and complexed the iron of methemoglobin. At the same

time these hemoproteins catalyzed hydrogen sulfide autoxidation to form

hydrogen peroxide and sulf'ur metabolites. Hydrogen sulfde also

increased hepatocyte respiration even though others have shown that it

inactivated cytochrome oxidase in vitro. Furthemore, hydrogen sulfide

also reduces cytochrome b and ubiquinone in some organisms, which

then feed electrons into the respiratory chah and causes ATP formation.

Evidence is provided suggesting that CYP 3 A and 2B catalyses

hydrogen sulfide metabolic activation to form hepatotoxic sulfur

metabolites as metyrapone prevented H2S induced cytotoxicity and

hepatocyte GSH depletion.

In Clmpter 4, we showed how various metals modulate hydrogen

sulfide toxicity. Some of them were able to form stable complexes that

usually form a precipitate in in Mtro studies while others were actively

involved in hydrogen sulfide autoxidation. Hydroxocobalamin was highly

effective at preventing hydrogen sulfide toxicity in hepatocytes by forming

what appears to be a sulfcobalamin complex which then catalyzed

extracellular hydrogen suhide autoxidation.

We have also Oiscmered that hydroxocobalamin was an effective

antidote for hydrogen sulfde lethaüty in vivo. In an in vivo cornparison of

two antidotes, the hydroxocobalamin treated mice survived better than

the nitnte treated mice. These results suggest that hydroxocobalamin

can be successfully used as an adjuvant therapy in diseases that may

result from endogenously elevated levels of hydrogen sulfide.

Future research should be aimed at further elucidating the role of

cytochrome P450 isoenzymes in hydrogen sulfide toxicity as well as

idenüfjdng the reactive intermediates in the oxidation pathway.

Hydroxocobalamin as a promising antidote in rnice needs further

chical evaluation. The best approach would be to treat healthy subjects

with non-toxic concentrations of hydrogen sulfide (similar to the levels

found in health spasl) following the administration of hydroxocobalarnin

and determine whether hydroxocobalamin prevented the increase of

u r i n q thiosulfate induced by hydrogen suüide.

A better understanding of the molecular mechanisms of hydrogen

sulfide toxicity could aid in designing better therapeutic treatments not

only for acute poisoning by hydrogen sulfide, but also for diseases such

as ulcerative colitis and periodontal disease.

Reactions in bold Spe represent the pathways of H2S detoxification

while others represent the pathways that lead to hydrogen sulfide

toxicity.

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