Biochemical and Molecular Action of Nutrients

Inhibition of Glutathione Synthesis with Propargylglycine Enhances

N-Acetylmethionine Protection and Methylation in
Bromobenzene-Treated Syrian Hamsters
1
Khingkan Lertratanangkoon,
2
Joseph M. Scimeca* and Jing-na Wei
Departments of Pharmacology and Toxicology and *Pathology, The University of Texas Medical Branch,
Galveston, TX 775551031
ABSTRACT The nding that liver necrosis caused by the environmental glutathione (GSH)-depleting chemical,
bromobenzene (BB) is associated with marked impairment in O- and S-methylation of BB metabolites in Syrian
hamsters raises questions concerning the role of methyl deciency in BB toxicity. N-Acetylmethionine (NAM) has
proven to be an effective antidote against BB toxicity when given after liver GSH has been depleted extensively.
The mechanism of protection by NAM may occur via a replacement of methyl donor and/or via an increase of GSH
synthesis. If replacement of the methyl donor is an important process, then blocking the resynthesis of GSH in the
methyl-repleted hamsters should not decrease NAM protection. This hypothesis was examined in this study.
Propargylglycine (PPG), an irreversible inhibitor of cystathionase, was used to inhibit the utilization of NAM for GSH
resynthesis. Two groups of hamsters were pretreated with an intraperitoneal (ip) dose of PPG (30 mg/kg) or saline
24 h before BB administration (800 mg/kg, ip). At 5 h after BB treatment, an ip dose of NAM (1200 mg/kg) was
given. Light microscopic examinations of liver sections obtained 24 h after BB treatment indicated that NAM
provided better protection (P 0.05) in the PPG BB NAM group than in the BB NAM group. Liver GSH
content, however, was lower in the PPG BB NAM group than in the BB NAM group. The Syrian hamster
has a limited capability to N-deacetylated NAM. The substitution of NAM with methionine (Met; 450 mg/kg) resulted
in a higher level of GSH in the BB Met group than in the BB NAM group (P 0.05). The enhanced protection
by PPG in the PPG BB NAM group was accompanied by higher (P 0.05) urinary excretions of specic O-
and S-methylated bromothiocatechols than in the BB NAM group. The results suggest that NAM protection
occurs primarily via a replacement of the methyl donor and that methyl deciency occurring in response to GSH
repletion plays a potential role in BB toxicity. J. Nutr. 129: 649656, 1999.
KEY WORDS: methionine methylation propargylglycine glutathione hamsters
The sulfur-containing amino acid, methionine (Met), has
long been recognized as an essential nutrient for the normal
growth and development of mammals (Finkelstein 1990). This
is because it participates in many fundamental biological pro-
cesses, e.g., in numerous S-adenosylmethionine (SAM)
3
-de-
pendent transmethylation reactions, as a precursor for cysteine
and glutathione (GSH), in protein synthesis. Thus, the de-
ciency 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 methyl-
ation resulting in the increased availability of methyl groups to
fulll 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.
We have previously shown that liver necrosis caused by a
single high dose of a model GSH-depleting hepatotoxin, bro-
mobenzene (BB), is associated with signicant impairment in
O- and S-methylation of 4- and 5-bromo-2-hydroxythiophe-
nols (bromothiocatechols) and S-methylated 4- and 5-bromo-
2-hydroxy-1,2-dihydrobenzenethiols (bromodihydrobenzene-
thiolols) in Syrian hamsters (Lertratanangkoon and Scimeca
1993). Necrosis was prevented by the administration of an
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.
2
To whom correspondence should be addressed.
3
Abbreviations used: BB, bromobenzene; GC, gas chromatography; GC-MS,
gas chromatography-mass spectrometry; GPT, glutamate pyruvate transami-
nase; GSH, glutathione; ip, intraperitoneal; NAM, N-acetylmethionine; PPG,
propargylglycine; SAM, S-adenosylmethionine; TMS, trimethylsilyl ether.
0022-3166/99 $3.00 1999 American Society for Nutritional Sciences.
Manuscript received 25 June 1998. Initial review completed 10 September 1998. Revision accepted 7 December 1998.
649

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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 bromodihy-
drobenzenethiolols. On the basis of these ndings, we postu-
late that methyl deciency 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 signicant 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 methyl-
ation 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 insufciency for
DNA repair synthesis, plays a role in BB toxicity (Lertratan-
angkoon et al. 1997a).
The observation that NAM protection was accompanied by
a striking increase in the methylation capability raises ques-
tions concerning the mechanism of NAM protection and also
the role of methyl deciency 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 cysta-
thionine 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).
MATERIALS AND METHODS
Materials. 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-pen-
tynoic 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 3235 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 45 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 mol/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 cen-
trifugation. 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 xed in 10% buffered neutral
formalin solution for histopathological examinations. The rest of the
liver was frozen in liquid nitrogen and stored at 70C 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 20C 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 (Lertratanangk-
oon 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 de-
scribed. 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 20C until
analyzed.
FIGURE 1 Metabolic pathways leading from N-acetylmethionine
3 methionin 3 glutathione. Propargylglycine (PPG), an irreversible
inhibitor of cystathionase, inhibits the conversion of cystathionine to
cysteine. This inhibits the utilization of N-acetylmethionine as precursor
for glutathione synthesis. [Modied from Lertratanangkoon et al. (1996);
reprinted with permission.]
LERTRATANANGKOON ET AL. 650

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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 20C 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 de-
scribed.
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 efciency 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 adminis-
tered were to ensure consistency with our recent experiments (Ler-
tratanangkoon 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
parafn, sectioned at 24 m, 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 modication of Ellmans reagent
method (1959) was used for the determination of liver GSH. This
method was described recently (Lertratanangkoon et al. 1996).
Briey, 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 Com-
pany, Cincinnati, OH) at 4C in 4 volumes of ice-cold 100 mmol/L
potassium phosphate buffer (pH 7.4). A known aliquot of the ho-
mogenate was deproteinized with an equal volume of 157 mmol/L
sulfosalicylate. After centrifugation, a known aliquot of the superna-
tant 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 absor-
bency 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 fth of total volume) was hydrolyzed with
Glusulase (9000 units -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 fth of the total extract) was transferred to a 1-mL
Reacti-vial and carefully dried under nitrogen. The residue was dis-
solved in 10 L pyridine and silylated with 1015 L bis(trimethyl-
silyl)acetamide (Pierce Chemical, Rockford, IL). The reaction mix-
ture was heated at 60C for 1 h. Gas chromatography (GC) and gas
chromatography-mass spectrometry (GC-MS) studies were carried
out with 0.20.6 L samples.
GC analyses were carried out with a Hewlett-Packard 5890 Gas
Chromatograph equipped with a ame-ionization detector. A 30-m
(0.32 mm i.d., 0.25 m lm thickness) fused silica DB-5 capillary
column (J & W Scientic, Folsom, CA) was used. Helium was used
as the carrier gas. All GC analyses were temperature programmed
from 60 to 300C at the rate of 2C/min.
BB metabolites were conrmed by GC-MS analyses. GC and
GC-MS properties (TMS derivatives) of the neutral and phenolic
metabolites of BB have been described (Lertratanangkoon and Horn-
ing 1987, Lertratanangkoon 1993). GC-MS analyses were conducted
with a Nermag R1010C (originally Delsi Nermag Instrument, Hous-
ton, TX) mass spectrometer coupled to a Varian 3400 gas chromato-
graph (Varian Instrument, Walnut Creek, CA). A PDP 11/73 data
system (Digital Equipment, Bedford, MA) was used. The mass spec-
trometry 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 inter-
nal standard. Inasmuch as the methylated thiolcontaining metabo-
lites are not available as reference standards, a response factor of unity
was assumed for all GC separations. Quantitative determination of
the methylated thiolcontaining 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 signicant.
RESULTS
BB hamsters. At 24 h after BB treatment, all hamsters
showed severe liver necrosis with massive intrahepatic hem-
orrhage (Fig. 2A and Table 1). BB caused a rapid and exten-
sive depletion of liver GSH during the rst 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.
BB NAM hamsters. The administration of a high dose
of NAM (6.28 mmol/kg) at 5 h after BB treatment protected
the liver from necrosis (Fig. 2B 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 mol compared with
12.6 2.3 mol in the nonprotected BB-treated hamsters
(Fig. 3). When a small dose of Met (3 mmol/kg) was substi-
tuted for NAM at 5.5 h after BB administration, liver GSH
rebounded to a much higher level (24.5 8.2 mol) than that
seen in the BB NAM group (P 0.05). Although Met
enhanced GSH resynthesis, the protection by Met was not
signicantly 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-d
3
was used as an antidote against BB toxicity (Lertra-
tanangkoon et al. 1996).
NAM hamsters. The high dosage of NAM employed
caused a mild degree of hepatocyte degeneration (Fig. 2C and
Table 1). NAM did not alter liver GSH signicantly. When
the experiments were terminated, liver GSH in the NAM
group was 32.2 3.9 mol compared with 31.2 1.4 mol
in saline-treated hamsters.
PPG hamsters. PPG (30 mg/kg) produced no detectable
effects on the liver (Fig. 2F), 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 adminis-
tration, liver GSH was 28.1 1.9 mol in the PPG-treated
group (Fig. 4) compared with 31.1 1.2 mol in saline-
treated hamsters (Fig. 3).
METHYL DEFICIENCY IN LIVER INJURY 651

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PPG BB hamsters. 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. 2D) than in the group treated with BB
alone (Fig. 2A). In the BB group, some viable hepatocytes
surrounding the portal triad areas were still noted, whereas
most hepatocytes in the PPG BBtreated hamsters showed
extensive degeneration, necrosis and hemorrhage (Table 1).
Pretreatment with PPG had no signicant 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
FIGURE 2 Light microscopic comparisons of liver sections (hematoxylin and eosin stained) obtained from hamster treated with bromobenzene
(BB; panel A), bromobenzene N-acetylmethionine (BB NAM; panel B), N-acetylmethionine (NAM; panel C), propargylglycine bromobenzene
(PPG BB; panel D), propargylglycine bromobenzene N-acetylmethionine (PPG BB NAM; panel E), and propargylglycine (PPG; panel F).
Magnication X200.
TABLE 1
Histopathological evaluations of liver sections obtained 24 h
after treatment with bromobenzene (BB) show enhanced
N-acetylmethionine (NAM) protection by propargylglycine
(PPG) in Syrian hamsters
1,2
Treatment
Lesion severity
Degeneration Necrosis Hemorrhage
BB 3.5 0.3 3.0 0.0 3.5 0.3
BB NAM 3.0 0.0 1.0 0.0 0.3 0.3
PPG BB
3
4.0 0.0 4.0 0.0 4.0 0.0
PPG BB NAM
4
2.3 0.3 0.8 0.3 0
NAM
5
1.0 0.0 0 0
PPG
5
0.3 0.0 0 0
Saline
5
0 0 0
1
The extent of lesions was graded as followed: 0 absent; 1
mild; 2 moderate; 3 marked; 4 severe.
2
Values are means SEM; n 4.
3
Extent of degeneration, necrosis and hemorrhage was more pro-
nounced in the PPG BB hamsters than in the BB-treated group;
P 0.05.
4
NAM provided better protection (degeneration and necrosis) in the
PPG BB NAM group than in the BB NAM group; P 0.05.
5
Liver sections from NAM, PPG and saline groups were obtained
24 h after corn oil, PPG and saline treatments, respectively.
FIGURE 3 Efciencies of the liver cells from bromobenzene (BB)-
treated hamsters to utilize N-acetylmethionine (NAM) and methionine
(Met) as precursors for glutathione (GSH) resynthesis. An intraperito-
neal dose of NAM or Met was given to BB-treated hamsters at 5 or
5.5 h, respectively, after BB administration. GSH concentrations were
determined 24 h after BB treatment. Values are expressed as means
SEM, n 4. GSH level in the BB NAM group was not signicantly
different from that in the group treated with BB alone. GSH level in the
BB Met group, however, was signicantly (P 0.05) higher than the
BB- and the BB NAM-treated groups.
LERTRATANANGKOON ET AL. 652

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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 prole 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 rst 5 h (Figs. 4 and 3, respectively).
These results suggested that PPG had no effect on the conju-
gation of BB metabolite(s) with GSH. PPG, however, signif-
icantly 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
sufcient 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).
PPG BB NAM hamsters. 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 ham-
sters (Fig. 2E) than in the BB NAM group (Fig. 2B).
Although NAM has proven to be an excellent antidote for
BB, light microscopic examinations indicated that diffuse hep-
atocellular swelling with mild centrilobular degeneration of
hepatocytes was still present in the BB NAM group (Table
1). In the PPG BB NAMtreated hamsters, only mild
diffuse hepatocellular swelling, with focal areas of degenera-
tion surrounding central veins, was present.
If the mechanism of protection by NAM is mediated
through an increase of GSH resynthesis, the marked protec-
tion by NAM in the PPG BB NAM group should be
accompanied by a marked increase in the GSH level, and this
level should be signicantly higher than that found for the
BB NAM group. When GSH was determined at 24 h after
BB treatment, the GSH level in the PPGBB NAM group
was not signicantly 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 signicantly 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).
FIGURE 5 Typical gas chromatographic separation of urinary neu-
tral and phenolic metabolites of bromobenzene (trimethylsilyl ether
derivatives) obtained from hamsters treated with propargylglycine
bromobenzene (PPG BB; upper panel) and propargylglycine
bromobenzene N-acetylmethionine (PPGBB NAM; lower panel).
NAM signicantly (P 0.05) enhanced the urinary excretion of the O-
and S-methylated bromothiocatechols in the PPG BB NAM group
compared with the hamsters treated with PPG BB.
FIGURE 4 Effect of propargylglycine (PPG), an irreversible inhibi-
tor of cystathionase (Fig. 1), on glutathione (GSH) resynthesis in the
livers of bromobenzene (BB)-treated hamsters. Twenty-four hours be-
fore BB administration, hamsters were pretreated with PPG as de-
scribed in Materials and Methods. In the PPG BB N-acetylmethi-
onine (NAM) group, an intraperitoneal dose of NAM was given 5 h after
BB administration. GSH concentrations were determined 24 h after BB
administration, and the results were compared with those in the ham-
sters not treated with PPG (Fig. 3). Values are expressed as means
SEM, n 4. PPG signicantly (P 0.05) inhibited GSH turnover in the
PPG BB and the PPG BB NAM groups compared with the
respective groups of hamsters not treated with PPG.
METHYL DEFICIENCY IN LIVER INJURY 653

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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- and S-
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 adminis-
tration of NAM to the PPG BB hamsters markedly (P
0.05) enhanced the excretion of two of these methylated
thiolcontaining metabolites (44.4 and 46.1 min GC reten-
tion 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 re-
tention times), however, were not signicantly 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 (Lertratan-
angkoon and Scimeca 1993).
DISCUSSION
The results of this study demonstrate that the protection by
NAM against BB toxicity was enhanced when the metabolic
conversion of cystathionine to cysteine was inhibited by PPG.
An increase in the protection by NAM in the PPG BB
NAM group was accompanied by a further increase in
urinary excretion of the methylated thiolcontaining metab-
olites of BB. The results provide evidence that NAM protec-
tion may occur primarily via a replacement of the methyl
donor. This, in turn, suggests that methyl deciency may be
involved in BB toxicity.
The time point of NAM administration, both in our pre-
vious (Lertratanangkoon and Scimeca 1993) and present stud-
ies, 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 resyn-
thesis of GSH (Fig. 1). Although cyst(e)ine cannot replace
Met, the availability of cyst(e)ine would lower Met require-
ments (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 inefciency 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 percent-
age 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 ham-
sters (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
signicantly (P 0.05) lower than those found in the BB
group (Fig. 3). All of the PPG BBtreated hamsters also had
massive liver necrosis and more pronounced intrahepatic hem-
orrhage 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 insufciency of
GSH for detoxication. 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. 2E
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. 2B 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 insufciency 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
TABLE 2
Effects of propargylglycine (PPG) and N-acetylmethionine (NAM) on urinary excretion of the O- and S-methylated
bromothiocatechol metabolites of bromobenzene (BB) in Syrian hamsters
1
BB BB NAM PPG BB PPG BB NAM
GC retention time, min mol/24 h
43.1 0.14 0.03 2.93 0.86 0.17 0.03 3.10 1.52
44.4 3.62 3.38 50.14 5.52 4.59 2.62 73.07 3.38
2
46.1 0.76 0.52 10.55 1.66 0.79 0.28 14.62 2.93
2
48.6 1.41 0.28 9.59 1.35 2.10 0.69 8.97 3.17
1
Values are means SEM; n 4 urine samples for each value.
2
Signicantly different from the BB NAM group (P 0.05). GC, gas chromatography.
LERTRATANANGKOON ET AL. 654

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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 fullled 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 BBtreated
hamsters compared with the BB-treated group (Table 2). The
severity of this metabolic imbalance together with an in-
creased 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 specic O- and S-methylated bromothio-
catechols. The amounts that were excreted were signicantly
(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 bromothiocat-
chols 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 bro-
mothiocatechols requires a cleavage action of a C-S -lyase in
which the cysteine conjugates of BB 3,4-oxide serve as sub-
strates (Lertratanangkoon and Denney 1993, Lertratanang-
koon et al. 1993). Specic C-S -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 -lyase products. The formation of
bromothiocatechols could be increased when the transamina-
tion is inhibited by PPG. Although the toxicological impor-
tance of bromothiocatechols is not currently known, results
from our previous and present studies show a strong relation-
ship between BB toxicity and the impairment in their meth-
ylation, 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 in-
deed toxic, an increase in their formation would lead to an
increase in toxicity. In this instance, the extent of liver ne-
crosis found in the PPG BBtreated 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 in-
creased sensitization of tumor cells to chemotherapy and ra-
diation treatment (Arrick et al. 1982, Vistica and Ahmad
1989). However, this does not necessarily indicate that the
insufciency of GSH for conjugation alone is solely responsi-
ble for such effects. Extensive GSH depletion/turnover pro-
vokes a cascade of biological events (those currently known
are Met insufciency, impairment in O-, S- and DNA-meth-
ylation, and deoxynucleotide imbalance), which perturb many
essential metabolic processes. The consecutive events of GSH
depletion, Met insufciency and impairment in methylation
have made it rather difcult 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 deciency in response to GSH depletion/turnover
plays a role in BB toxicity. Met metabolism and transmethyl-
ation 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 pro-
cesses would profoundly affect the integrity of all cellular
functions. The mechanism through which methyl deciency
mediates toxicity by the model GSH-depleting hepatotoxin,
BB, is currently under investigation.
ACKNOWLEDGMENTS
We thank Mary F. Kanz and Patricia K. Seitz for their critical
reading of the manuscript and helpful suggestions.
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