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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