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Departments of Pharmacology and Toxicology and * Pathology, The University of Texas Medical Branch, Galveston, TX 77555–1031
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • methionine • methylation • propargylglycine • glutathione • hamsters
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This is
because it participates in many fundamental biological processes, e.g.,
in numerous S-adenosylmethionine
(SAM)3-dependent transmethylation reactions, as a precursor
for cysteine and glutathione (GSH), in protein synthesis. Thus, the
deficiency of Met will affect a variety of metabolic housekeeping
processes.
The presence of cystine in diets decreases Met loss via the
cystathionine pathway (Fig. 1
)(Finkelstein and Mudd 1967
, Womack and Rose 1941
). GSH, which is present in many foods including meats,
fresh fruits and vegetables (Jones et al. 1992
), is
capable of delivering 100% of its cysteine content to the animal as
bioavailable cysteine (Harter and Baker 1977
). Thus,
GSH-derived cysteine would decrease Met loss in the same manner as that
found with dietary cystine. The Met-cycle allows Met to be conserved
via homocysteine methylation resulting in the increased availability of
methyl groups to fulfill the biological requirements for methylation
(Finkelstein 1990
). However, a rapid and extensive loss
of GSH after an exposure to a GSH-depleting agent depletes Met and
impairs methylation (Lertratanangkoon et al. 1996
). This
is because mammalian liver cells are capable of resynthesizing GSH
after GSH is depleted, and GSH turnover occurs at the expense of Met
(Reed and Orrenius 1977
). An increase of Met catabolism
via the cystathionine pathway decreases Met recycling. This, in turn,
limits the availability of SAM for methylation.
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). Necrosis was
prevented by the administration of an intraperitoneal (ip) dose of
N-acetylmethionine (NAM) 5 h after BB treatment. A
decrease in toxicity was accompanied by a striking increase in urinary
excretion of the O- and S-methylated
bromothiocatechols and S-methylated
bromodihydrobenzenethiolols. On the basis of these findings, we
postulate that methyl deficiency occurring in response to GSH
depletion/turnover plays a role in BB toxicity. We showed in a recent
study that GSH depletion-induced impairment in methylation has a
significant effect on DNA methylation; a progressive impairment in
genomic DNA methylation was found in BB-treated hamsters
(Lertratanangkoon et al. 1997b
). The metabolic pathways
shown in Figure 1
are common in mammalian metabolism, and agents that
deplete GSH will deplete Met and impair methylation. We further
demonstrated that GSH depletion-induced impairment in DNA methylation
is a common event and that this molecular alteration is a general one
that is followed by another GSH-depleting agent, acetaminophen
(Lertratanangkoon and Savaraj 1997
). The administration
of acetaminophen to Syrian hamsters also progressively impaired genomic
DNA methylation. We also showed (Lertratanangkoon et al. 1997a
) that an increased utilization of Met for the resynthesis
of GSH diverts folate from thymidylate biosynthesis to the resynthesis
of Met (Fig. 1)
. This event, which causes thymidylate insufficiency for
DNA repair synthesis, plays a role in BB toxicity
(Lertratanangkoon et al. 1997a
).
The observation that NAM protection was accompanied by a striking
increase in the methylation capability raises questions concerning the
mechanism of NAM protection and also the role of methyl deficiency in
BB toxicity. If replacement of the methyl donor is the essential
process, then blocking the resynthesis of GSH in the methyl-repleted
animals should not decrease NAM protection. This hypothesis was
examined in this study. Propargylglycine (PPG), an irreversible
inhibitor of cystathionase (Beatty and Reed 1980
,
Cho et al. 1991
, Reed 1995
), was used to
inhibit the metabolic conversion of cystathionine to cysteine and GSH
(Fig. 1)
. This inhibition should also block sulfate formation; the
formation of sulfate is a cysteine-dependent reaction (Kim et al. 1995
, Rao et al. 1990
, Stipanuk et al. 1992
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reference compounds and other materials used in this study have been
described (Lertratanangkoon and Scimeca 1993
,
Lertratanangkoon et al. 1996
).
DL-Propargylglycine (2-amino-4-pentynoic acid) was
purchased from Sigma Chemical (St. Louis, MO). All other reagents were
of the highest grade commercially available.
Animal treatments.
Young adult male Golden Syrian hamsters were obtained from Charles River Laboratories (Wilmington, MA). The hamsters were 32–35 d of age and weighed between 80 and 100 g. They were housed in groups of four on a bedding of hardwood shavings in shoe-box polycarbonate cages. They were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Animal protocols received prior approval from the Animal Care and Use Committee of the University of Texas Medical Branch at Galveston. Hamsters were allowed to acclimate in the animal facility for 4–5 d before use. They had free access to food (Purina Lab Diet 5008; Purina, St. Louis, MO) and water.
After the acclimation period and 24 h before BB administration, hamsters were pretreated with either PPG or saline. One group received an ip dose of PPG [30 mg/kg (265 µmol/kg)] in 0.2 mL normal saline solution; the other group received normal saline ip. During the pretreatment period, all hamsters had free access to food and water. At 24 h after PPG treatment, hamsters were divided into the following three groups; PPG + BB, PPG + BB + NAM, and PPG. The saline-treated hamsters were divided into six groups as follows: BB, BB + NAM, NAM, saline, BB + Met, and Met.
PPG + BB hamsters.
Each of these hamsters received an ip dose of BB [800 mg/kg (5.12
mmol/kg)] in 0.2 mL corn oil. After dosing, they received no food, but
had free access to water. At various time points after BB treatment (1,
3, 5, 7, 9, 15 or 24 h), hamsters (n = 4) were
anesthetized with ether. Blood was drawn by cardiac puncture into a
heparin-treated syringe, and plasma was separated after centrifugation.
Plasma glutamate pyruvate transaminase (GPT) activities were determined
using Sigma kit 505. Due to the inhibitory effect of the transaminase
by PPG (Burnett et al. 1980
, Cornell et al. 1984
, Marcotte and Walsh 1975
, Tanase and Morino 1976
), GPT activity was not used as a biochemical index
for liver injury in this study. Livers were excised and gallbladders
were removed. The livers were rinsed with ice-cold saline, patted dry
and quickly weighed. The same lobe from each liver was sliced and fixed
in 10% buffered neutral formalin solution for histopathological
examinations. The rest of the liver was frozen in liquid nitrogen and
stored at -70°C for GSH determination.
For urinary collection, each of the 24-h time point hamsters was housed in a metabolism cage after BB, and a 24-h urine sample was collected. Urine samples were stored at -20°C until analyzed. Other hamsters were housed as described above.
PPG + BB + NAM hamsters.
Each hamster (n = 4) received an ip dose of BB as
described, followed 5 h later with an ip dose of NAM [1200 mg/kg
(6.28 mmol/kg)] in 1 mL distilled water (pH of the NAM solution was
carefully adjusted to 7.4 with NaOH). The selected dosage and the time
at which NAM was administered were to ensure consistency with our
previous experiments (Lertratanangkoon and Scimeca 1993
). Each of these hamsters was housed in a metabolism cage
after BB treatment, and a 24-h urine sample was collected. At 24 h
after BB administration, they were anesthetized with ether and treated
as described.
PPG hamsters.
These hamsters (n = 4) received no further injections, but they were anesthetized with ether and livers were excised and treated as described.
BB hamsters.
Each hamster received an ip dose of BB as described. At various time points after BB administration (1, 3, 5, 11, 15 or 24 h), hamsters (n = 4) were anesthetized with ether, and livers were excised and treated as described. Urine samples were collected from each of the 24-h time point hamsters, and stored at -20°C until analyzed.
BB + NAM hamsters.
These hamsters (n = 4) received BB and NAM as described. Each hamster was housed in a metabolism cage and a 24-h urine sample was collected and stored at -20°C until analyzed. They were anesthetized with ether and livers were excised and treated as described.
NAM hamsters.
Twenty-four hours after saline treatment, these hamsters (n = 4) received corn oil and NAM as described. They were housed in groups of four on a bedding of hardwood shavings in shoe-box polycarbonate cages. At 24 h after corn oil, they were anesthetized with ether and livers were excised and treated as described.
Saline hamsters.
These hamsters (n = 4) received no further injections, but they were anesthetized with ether and livers were excised and treated as described.
BB + Met and Met hamsters.
The efficiency of the Syrian hamster to utilize N-acetylated
Met as a precursor for GSH was examined. In this experiment, a small
dose of Met was substituted for NAM, and the effect of Met on GSH
resynthesis was examined and compared with that obtained from NAM. Two
groups of hamsters, BB + Met and Met, were used (n = 4 per group). In the BB + Met group, an ip dose of BB was given
as described, followed 5.5 h later with an ip dose of Met [450
mg/kg (3 mmol/kg)] in 1 mL distilled water. The selected dosage and
the time at which Met was administered were to ensure consistency with
our recent experiments (Lertratanangkoon et al. 1996
).
Met hamsters received corn oil and Met as described. All hamsters were
anesthetized 24 h after BB or corn oil treatment and treated as
described.
Histopathological evaluation.
Liver slices were embedded in paraffin, sectioned at 2–4 µm, stained with hematoxylin and eosin and examined by light microscopy. Lesion severities were scored as follows: 0 = absent; 1 = mild; 2 = moderate; 3 = marked; 4 = severe. Scoring was performed without knowledge of treatment.
Glutathione assay.
A slight modification of Ellman's reagent method (1959)
was used for the determination of liver GSH. This method was described
recently (Lertratanangkoon et al. 1996
). Briefly, frozen
livers were crushed between two polystyrene weighing dishes over a
large piece of dry-ice. A 500 mg sample of the crushed frozen liver was
then homogenized with a Tissumizer (Tekmar Company, Cincinnati, OH) at
4°C in 4 volumes of ice-cold 100 mmol/L potassium phosphate buffer
(pH 7.4). A known aliquot of the homogenate was deproteinized with an
equal volume of 157 mmol/L sulfosalicylate. After centrifugation, a
known aliquot of the supernatant was diluted with potassium phosphate
buffer (100 mmol/L, pH 8.0), and an aliquot of 10 mmol/L
5,5'-dithio-bis(2-nitrobenzoate) (pH 8.0) was added.
Reduced GSH was proportional to the absorbency at 412 nm. A reduced GSH
reference standard was used to prepare the calibration curve.
Analyses of urinary metabolites.
An aliquot of the 24-h urine samples (usually one fifth of total
volume) was hydrolyzed with Glusulase (9000 units ß-glucuronidase and
1000 units sulfatase). Neutral and phenolic metabolites were extracted
by the ammonium carbonate-ethyl acetate procedure (Horning et al. 1984
). Analytical studies were conducted with the neutral
and phenolic extract that contained the metabolites of interest.
Neutral and phenolic metabolites were converted to trimethylsilyl ether (TMS) derivatives. An aliquot of the ethyl acetate extract (usually one fifth of the total extract) was transferred to a 1-mL Reacti-vial and carefully dried under nitrogen. The residue was dissolved in 10 µL pyridine and silylated with 10–15 µLbis(trimethylsilyl)acetamide (Pierce Chemical, Rockford, IL). The reaction mixture was heated at 60°C for 1 h. Gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) studies were carried out with 0.2–0.6 µL samples.
GC analyses were carried out with a Hewlett-Packard 5890 Gas Chromatograph equipped with a flame-ionization detector. A 30-m (0.32 mm i.d., 0.25 µm film thickness) fused silica DB-5 capillary column (J & W Scientific, Folsom, CA) was used. Helium was used as the carrier gas. All GC analyses were temperature programmed from 60 to 300°C at the rate of 2°C/min.
BB metabolites were confirmed by GC-MS analyses. GC and GC-MS
properties (TMS derivatives) of the neutral and phenolic metabolites of
BB have been described (Lertratanangkoon and Horning 1987
, Lertratanangkoon 1993
). GC-MS analyses
were conducted with a Nermag R10–10C (originally Delsi Nermag
Instrument, Houston, TX) mass spectrometer coupled to a Varian 3400 gas
chromatograph (Varian Instrument, Walnut Creek, CA). A PDP 11/73 data
system (Digital Equipment, Bedford, MA) was used. The mass spectrometry
analyses were conducted in an electron impact ionization mode as
recently described (Lertratanangkoon et al. 1996
).
A known amount of n-eicosane, in isooctane, was added to the ethyl acetate extracts before the derivatization step to serve as internal standard. Inasmuch as the methylated thiol–containing metabolites are not available as reference standards, a response factor of unity was assumed for all GC separations. Quantitative determination of the methylated thiol–containing metabolites was based on peak height analyses of the TMS derivatives.
Statistical analysis.
Data were subjected to computer analyses and are presented as means ± SEM. Data were examined by one-way ANOVA, followed by post-hoc t tests (Instat Graph Pad Software, San Diego, CA) comparing the PPG- with the non-PPG-treated groups and each treatment group with controls. Differences with a P-value of < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At 24 h after BB treatment, all hamsters showed severe liver
necrosis with massive intrahepatic hemorrhage (Fig. 2
A and Table 1
).BB caused a rapid and extensive depletion of liver GSH during the first
5 h (Fig. 3
).After this initial depletion, all hamsters could resynthesize GSH. At
24 h after BB treatment, liver GSH rebounded to 40% of the
initial pretreatment value.
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The administration of a high dose of NAM (6.28 mmol/kg) at 5 h
after BB treatment protected the liver from necrosis (Fig. 2
B and Table 1
). The protection, however, was not
accompanied by a marked GSH resynthesis in the liver. At 24 h
after BB administration, liver GSH in the BB + NAM group was
13.7 ± 1.2 µmol compared with 12.6 ± 2.3 µmol in the
nonprotected BB-treated hamsters (Fig. 3)
. When a small dose of Met (3
mmol/kg) was substituted for NAM at 5.5 h after BB administration,
liver GSH rebounded to a much higher level (24.5 ± 8.2 µmol)
than that seen in the BB + NAM group (P < 0.05).
Although Met enhanced GSH resynthesis, the protection by Met was not
significantly better (data not shown) than that observed in the BB + NAM group. Histological examination indicated that centrilobular
degeneration of hepatocytes occurred in both groups; however, the
extent of cellular swelling was higher in the BB + NAM group than
in the BB + Met group. The protection by Met found in this study
is comparable to that found in our recent study in which the deuterated
L-Met-methyl-d3 was used as an
antidote against BB toxicity (Lertratanangkoon et al. 1996
).
NAM hamsters.
The high dosage of NAM employed caused a mild degree of hepatocyte
degeneration (Fig. 2
C and Table 1
). NAM did not alter liver
GSH significantly. When the experiments were terminated, liver GSH in
the NAM group was 32.2 ± 3.9 µmol compared with 31.2 ±
1.4 µmol in saline-treated hamsters.
PPG hamsters.
PPG (30 mg/kg) produced no detectable effects on the liver (Fig. 2
F), consistent with results of an earlier study (Cho et al. 1991
). At 24 h after PPG treatment, which was just
before BB administration, histopathological examination indicated
essentially normal livers (Table 1)
.
The inhibition of cystathionase by PPG (Fig. 1)
decreased the content
of liver GSH in the PPG-treated hamsters. At 24 h after PPG
treatment, which was just before BB administration, liver GSH was
28.1 ± 1.9 µmol in the PPG-treated group (Fig. 4
)compared with 31.1 ± 1.2 µmol in saline-treated hamsters (Fig. 3)
.
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The administration of BB to the PPG-pretreated hamsters resulted in
massive liver necrosis. Intrahepatic hemorrhage, which is a typical
characteristic of BB toxicity in Syrian hamsters, was more pronounced
in the PPG + BB group (Fig. 2
D) than in the group
treated with BB alone (Fig. 2
A). In the BB group, some
viable hepatocytes surrounding the portal triad areas were still noted,
whereas most hepatocytes in the PPG + BB–treated hamsters showed
extensive degeneration, necrosis and hemorrhage (Table 1)
.
Pretreatment with PPG had no significant effect on the metabolism of
BB. All of the neutral and phenolic metabolites of BB that were found
in our previous study of BB hamsters (Lertratanangkoon and Scimeca 1993
) were also found in the PPG + BB group.Figure 5(upper panel) shows
a typical GC separation of urinary neutral and phenolic metabolites of
BB (as TMS derivatives) obtained from a PPG + BB hamster. This
metabolite profile is comparable to that found for the BB-treated
hamsters (data not shown). The PPG + BB and the BB groups also had
similar time-response curves of GSH depletion during the first 5 h
(Figs. 4 and 3
, respectively). These results suggested that PPG had no
effect on the conjugation of BB metabolite(s) with GSH. PPG, however,
significantly inhibited (P < 0.05) the ability of the
liver cells to regenerate GSH after GSH was depleted. After the initial
depletion, liver GSH in the PPG + BB group continued to decline,
and only a negligible level could be detected at 24 h (Fig. 4)
.
This result is quite different from that found for the BB group (Fig. 3)
. In the BB group, all of the hamsters were able to resynthesize GSH;
liver GSH rebounded to ~40% of the initial pretreatment value at
24 h. The results indicate that an ip dose of PPG (30 mg/kg), as
employed in this study, is sufficient to inhibit GSH resynthesis in
vivo. The results also provide evidence that the cystathionine pathway
is a major route for GSH synthesis in mammals. Our result is consistent
with an early report using isolated rat hepatocytes (Beatty and Reed 1980
).
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Despite the massive necrosis found in the PPG + BB group, the
administration of an ip dose of NAM at 5 h after BB treatment
resulted in marked protection of the liver against necrosis (Table 1
;
P < 0.05). Light microscopic examinations indicated
that NAM provided better protection in the PPG + BB + NAM
hamsters (Fig. 2
E) than in the BB + NAM group (Fig. 2
B). Although NAM has proven to be an excellent antidote for
BB, light microscopic examinations indicated that diffuse
hepatocellular swelling with mild centrilobular degeneration of
hepatocytes was still present in the BB + NAM group (Table 1)
. In
the PPG + BB + NAM–treated hamsters, only mild diffuse
hepatocellular swelling, with focal areas of degeneration surrounding
central veins, was present.
If the mechanism of protection by NAM is mediated through an increase
of GSH resynthesis, the marked protection by NAM in the PPG +
BB + NAM group should be accompanied by a marked increase in the
GSH level, and this level should be significantly higher than that
found for the BB + NAM group. When GSH was determined at 24 h
after BB treatment, the GSH level in the PPG + BB + NAM group
was not significantly greater than that observed in the PPG + BB
group (Fig. 4)
, and this level was lower than that observed in the
BB + NAM group (Fig. 3)
. Although NAM did not significantly
enhance GSH resynthesis, the availability of an external source of NAM
prevented a further decline of GSH in the PPG + BB + NAM
hamsters compared with the PPG + BB group (Fig. 4)
.
Effect of PPG on the methylation of bromothiocatechols.
The administration of NAM to BB-treated hamsters strikingly increased
urinary excretion of the four isomeric O- andS-methylated bromothiocatechols (Table 2
)(Lertratanangkoon and Scimeca 1993
). These methylated
bromothiocatechols were minor metabolites in the hamsters treated with
BB alone. Pretreatment with PPG had no effect on their excretions (Fig. 5
, upper panel); similar amounts were excreted in
the PPG + BB and the BB groups (Table 2)
. However, the
administration of NAM to the PPG + BB hamsters markedly
(P < 0.05) enhanced the excretion of two of these
methylated thiol–containing metabolites (44.4 and 46.1 min GC
retention times; Fig. 5
, lower panel) compared
with the BB + NAM group. Increases of ~46 and 39%,
respectively, were found (Table 2)
. The excretions of the third and
fourth isomeric methylated bromothiocatechols (43.1 and 48.6 min GC
retention times), however, were not significantly different. The
remaining urinary neutral and phenolic metabolites from the PPG +
BB + NAM group were comparable to those found previously for the
BB + NAM-treated hamsters (Lertratanangkoon and Scimeca 1993
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The time point of NAM administration, both in our previous
(Lertratanangkoon and Scimeca 1993
) and present studies,
was after liver GSH had been extensively depleted (Fig. 3)
. Our
experimental design was different from that of an earlier study
(Jollow et al. 1974
) in which multiple doses of
cysteine, an immediate precursor of GSH, were administered before and
shortly after BB treatment. The availability of cysteine at the time of
BB administration could very well have prevented extensive depletion of
endogenous GSH. This, in turn, could have prevented an excessive
utilization of Met for the resynthesis of GSH (Fig. 1)
. Although
cyst(e)ine cannot replace Met, the availability of cyst(e)ine would
lower Met requirements (Finkelstein and Mudd 1967
,
Womack and Rose 1941
). If the mechanism of protection by
NAM is mediated through an increase of GSH resynthesis, the
administration of a large dose of NAM after GSH has already been
extensively depleted should result in an enhancement of GSH
resynthesis. When GSH was determined at 24 h after BB
administration, the level was not different in the BB + NAM group
than in the BB-treated group (Fig. 3)
, indicating that NAM is not a
good precursor for GSH in Syrian hamsters. An inefficiency of
N-deacetylase could account for this effect. Our suggestion
is supported by the observation that a much higher level of GSH was
found when a small dose of Met was substituted for NAM (Fig. 3)
. The
limited amounts of Met that were generated in the BB + NAM group
would enhance Met conservation via homocysteine methylation rather than
Met catabolism via the cystathionine pathway to generate GSH (Fig. 1)
.
The percentage of homocysteine that is transulfurated is related
directly to the availability of the methyl groups in diets (Mudd and Poole 1975
, Mudd et al. 1980
). This explains
why NAM is a good source of methyl groups (Lertratanangkoon and Scimeca 1993
), but a poor source of GSH resynthesis in Syrian
hamsters (Fig. 3)
.
Pretreatment with PPG further inhibited the ability of liver cells to
utilize NAM as a precursor for GSH. At 24 h after BB treatment,
GSH levels in the PPG + BB group (Fig. 4)
were significantly
(P < 0.05) lower than those found in the BB group
(Fig. 3)
. All of the PPG + BB–treated hamsters also had massive
liver necrosis and more pronounced intrahepatic hemorrhage than did the
BB hamsters that were not treated with PPG. These data alone suggested
that an increase in toxicity in the PPG + BB hamsters was due to
the insufficiency of GSH for detoxification. If this assumption were
correct, the administration of NAM to these hamsters, which have
limited capabilities to utilize NAM for GSH resynthesis, should result
in little or no protection. Histological examinations (Fig. 2
E and Table 1
) indicated that NAM protected the PPG +
BB + NAM group. Interestingly, the protection by NAM was better in
this group than in the BB + NAM group (Fig. 2
B and
Table 1
). Furthermore, the levels of GSH were lower in the PPG + BB + NAM group (Fig. 4)
than in the BB + NAM group (Fig. 3)
.
The results demonstrate that the protection by NAM is not correlated
with the degree of GSH resynthesis and thus provide evidence that the
insufficiency of GSH for conjugation may not be the direct cause of BB
toxicity in Syrian hamsters.
Liver cells respond to GSH depletion by a prompt and rapid increase of
Met synthesis (Lertratanangkoon et al. 1996
). This
initial increase is followed by a rapid and extensive Met catabolism.
Under a general condition such as that in the BB-treated hamsters, the
end product of Met catabolism is GSH (Fig. 1)
. However, when the
cystathionine pathway is blocked by PPG, the end product is
cystathionine (Beatty and Reed 1980
, Cho et al. 1991
). The inability of liver cells to replace the depleted GSH
in PPG-treated hamsters continues to signal for more Met catabolism.
Under conditions in which an external source of Met is not available,
an increased Met requirement is fulfilled by a further increase in
homocysteine methylation. This Met-exhausted metabolic condition would
further divert folates from the biosyntheses of purines and the
pyrimidine, thymidylate, resulting in a further increase of
deoxynucleotide imbalance (Lertratanangkoon et al. 1997a
). The continuation of the exhausted-Met cycle produces a
small amount of methyl groups for methylation. This explains why there
was a slightly higher (P < 0.05) urinary excretion of
the methylated bromothiocatechols in the PPG + BB–treated
hamsters compared with the BB-treated group (Table 2)
. The severity of
this metabolic imbalance together with an increased formation of the
potentially toxic bromothiocatechols (discussed below) could account
for more toxic effects in the PPG + BB group than in the BB group.
The enhanced protection by NAM in the PPG + BB + NAM group
was accompanied by a further increase in urinary excretion of
specific O- and S-methylated bromothiocatechols.
The amounts that were excreted were significantly (P <
0.05) higher in the PPG + BB + NAM group than in the BB + NAM group (Table 2)
. This may be due to an increased availability of
the methyl donor and also to an increased bromothiocatechol formation.
These bromothiocatchols are the 3,4-series thiol-containing metabolites
of BB (Lertratanangkoon 1993
). They are the end products
of a long sequence of metabolic reactions that involve the extension of
the GSH conjugates of BB 3,4-oxide. The formation of bromothiocatechols
requires a cleavage action of a C-S ß-lyase in which the cysteine
conjugates of BB 3,4-oxide serve as substrates (Lertratanangkoon and Denney 1993
, Lertratanangkoon et al. 1993
). Specific C-S ß-lyase and transaminase are known to be
related (Lertratanangkoon and Denney 1993
,
Stevens et al. 1986
). Inhibition of transaminases by PPG
could lead to an increase in C-S ß-lyase products. The formation of
bromothiocatechols could be increased when the transamination is
inhibited by PPG. Although the toxicological importance of
bromothiocatechols is not currently known, results from our previous
and present studies show a strong relationship between BB toxicity and
the impairment in their methylation, and that a decrease in BB toxicity
by NAM or Met is accompanied by a striking increase in urinary
excretion of their methylated counterparts. If bromothiocatechols are
indeed toxic, an increase in their formation would lead to an increase
in toxicity. In this instance, the extent of liver necrosis found in
the PPG + BB–treated hamsters was far more pronounced than that
in the BB-treated group.
GSH depletion/regeneration has long been a subject of interest for
toxicologists and also for clinicians involved in drug-resistant
chemotherapy. This is because GSH depletion is generally associated
with an increase in toxicity or an increased sensitization of tumor
cells to chemotherapy and radiation treatment (Arrick et al. 1982
, Vistica and Ahmad 1989
). However, this
does not necessarily indicate that the insufficiency of GSH for
conjugation alone is solely responsible for such effects. Extensive GSH
depletion/turnover provokes a cascade of biological events (those
currently known are Met insufficiency, impairment inO-, S- and DNA-methylation, and deoxynucleotide
imbalance), which perturb many essential metabolic processes. The
consecutive events of GSH depletion, Met insufficiency and impairment
in methylation have made it rather difficult to distinguish these
individual effects. However, the observation in this study that NAM
provided better protection in the hamsters that have limited
capabilities to resynthesize GSH provides strong evidence that methyl
deficiency in response to GSH depletion/turnover plays a role in BB
toxicity. Met metabolism and transmethylation have long been recognized
as central to mammalian metabolism (Cantoni 1975
,
Chiang et al. 1996
, Finkelstein 1990
,
Mato et al. 1997
). Perturbation of such essential
processes would profoundly affect the integrity of all cellular
functions. The mechanism through which methyl deficiency mediates
toxicity by the model GSH-depleting hepatotoxin, BB, is currently under
investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
3 Abbreviations used: BB, bromobenzene; GC, gas
chromatography; GC-MS, gas chromatography-mass spectrometry; GPT,
glutamate pyruvate transaminase; GSH, glutathione; ip, intraperitoneal;
NAM, N-acetylmethionine; PPG, propargylglycine; SAM,
S-adenosylmethionine; TMS, trimethylsilyl ether.
Manuscript received June 25, 1998. Initial review completed September 10, 1998. Revision accepted December 7, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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methionin 
