Departments of 1 Internal Medicine and 3 Pathology, College of Medicine, University of Kentucky, and the 2 Lexington Veterans Affairs Medical Center, Lexington, Kentucky 40536
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ABSTRACT |
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S-adenosylmethionine
(Adomet) is a substrate for de novo synthesis of choline. Adomet
deficiency occurs in certain types of liver injury, and the injury is
attenuated by exogenous Adomet. Tumor necrosis factor- (TNF-
) is
also a mediator of these models of hepatotoxicity. We investigated the
role of Adomet in lipopolysaccharide (LPS)-induced liver injury in rats
made deficient in both Adomet and choline. Rats were maintained on
either a methionine-restricted and choline-deficient (MCD) diet or a
diet containing sufficient amounts of all nutrients [methionine
and choline sufficient (MCS)] and then administered either LPS or
saline. MCS-LPS rats had normal liver histology and no change in serum
transaminases compared with the MCS-saline control group. MCD-saline
rats had hepatosteatosis but no necrosis, and a five- to sevenfold
increase in transaminases vs. the MCS-saline group. MCD-LPS rats
additionally had hepatonecrosis and a 30- to 50-fold increase in
transaminases. Exogenous Adomet administration to MCD-LPS rats
corrected the hepatic deficiency of Adomet but not of choline,
prevented necrosis but not steatosis, and attenuated transaminases.
Serum TNF-
was sixfold higher in MCD rats even without LPS challenge
and 300-fold higher with LPS challenge. Exogenous Adomet attenuated
increased serum TNF-
in MCD-LPS rats.
tumor necrosis factor-; choline deficiency; liver injury
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INTRODUCTION |
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S-adenosylmethionine (Adomet) is an
important metabolic intermediate in the transsulfuration pathway and is
formed from methionine and ATP in a reaction catalyzed by methionine
adenosyltransferase (MAT) (6, 23). The pathway is predominantly
localized in liver, although MAT has been identified in most tissues
examined (20, 23). There is increasing evidence to indicate that Adomet concentrations play an important role in the development of liver injury caused by several toxins. For example, administration of galactosamine, alcohol, or carbon tetrachloride results in hepatic Adomet deficiency, and exogenous Adomet alleviates the liver injury (9,
10, 13, 26). Adomet deficiency is probably a result of subnormal
synthesis. Although this deficiency has not been clearly established in
animals administered acetaminophen, exogenous Adomet decreases the
liver injury resulting from this hepatotoxin (4, 21). Tumor necrosis
factor- (TNF-
), a pleiotropic proinflammatory cytokine, is
postulated to be one mediator for these models of liver injury, and
antibodies to TNF-
or soluble TNF-
receptors attenuate the
hepatic injury in these models (3, 11, 15, 27). TNF-
is synthesized
by many cell types, including monocytes, macrophages, and hepatic
Kupffer cells, and its synthesis is stimulated by multiple factors such
as viral and fungal antigens, immune complexes, and bacterial
lipopolysaccharide (LPS) (25). Potential interactions between Adomet
and TNF-
in the development of hepatotoxicity have not been
addressed.
Adomet is a precursor for the biosynthesis of glutathione (GSH) and is
a vital biological methylating agent for a variety of molecules,
including macromolecules such as nucleic acids and proteins and low
molecular weight molecules such as biogenic amines (6, 23).
Furthermore, Adomet is required as a substrate for the de novo
synthesis of the lipotrope choline by phosphatidylethanolamine N-methyltransferase (5, 23). Limited
availability of Adomet can result in choline deficiency, especially if
the dietary intake of choline is also curtailed (28). Thus rats
maintained on diets with limited amounts of methionine and deficient in
choline (MCD) have a systemic deficiency of choline and develop hepatic
steatosis, abnormalities in lipoprotein and bile secretion, and
subnormal acetylcholine in the brain (22, 28). We previously observed that rats on MCD diets exhibited enhanced liver injury when challenged with LPS and that antibody to TNF- attenuated the liver injury (12).
The objective of this study was to determine whether administration of
exogenous Adomet to MCD rats 1)
prevents hepatic deficiencies of Adomet and choline,
2) attenuates LPS-induced hepatic
injury, and 3) attenuates the
LPS-stimulated increase in serum TNF-
concentrations.
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MATERIALS AND METHODS |
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All animal studies were approved by the Animal Studies
Subcommittee of the Lexington Veterans Affairs Medical
Center. Male Sprague-Dawley rats (100-120 g, ~4 wk old), were
obtained from Harlan Sprague Dawley (Indianapolis, IN). Both the MCD
and the methionine- and choline-sufficient (MCS) diets were the same as those used in our previous study (12) and were obtained from ICN
Biochemicals (Cleveland, OH). Both diets contained casein and -soy
protein as sources of protein, and both diets were stored at 4°C
when not in use. The MCD diet contained a limited amount of methionine,
was deficient in choline, and contained sufficient amounts of all other
nutrients. The MCS diet contained sufficient amounts of all nutrients.
Adomet 1,4-butanedisulfonate salt and the sodium salt of its anion were
provided by Dr. Robert O'Brian of Knoll Pharmaceuticals (Piscataway,
NJ). LPS (E. coli 0111:B4 endotoxin)
was purchased from Difco (Detroit, MI). All reagents were of the
highest grade of purity available and were obtained from Sigma Chemical
(St. Louis, MO), unless otherwise indicated.
Animal studies.
Animals were housed in cages with wire-mesh bottoms and had ad libitum
access to food and water. After an initial 24-h acclimation period,
rats were divided into four groups (shown in Table
1; n = 8 animals/group) and administered MCD or MCS diets for 16 days. One group
of MCD rats was administered Adomet (170 µmol · kg1 · day
1
im) for 16 days while another MCD group received an equimolar amount of
placebo 1,4-butanedisulfonate. Blood for TNF-
analysis was obtained
90 min after intravenous injection of 2 mg/kg of LPS, and 24 h after
LPS injection rats were anesthetized to obtain blood for analyses of
the liver transaminases alanine aminotransferase (ALT) and aspartate
aminotransferase (AST). At that time, animals were exsanguinated and
livers were removed and stored in 10% formaldehyde for hematoxylin and
eosin staining and in liquid nitrogen for biochemical analyses.
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Statistical methods. Comparison of group means was done using a two-tail, two-sample t-test for unequal variances, with degrees of freedom approximated by Satterthwaite's method. To assure an overall type I error rate of 0.05, P values for individual t-tests were adjusted by Bonferroni's procedure. With six t-tests per end point, significance was declared on an individual t-test only if P < 0.0083.
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RESULTS |
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The control group in all experiments was MCS rats injected with saline. Data on the hepatic Adomet concentrations in the various groups are summarized in Fig. 1. Adomet concentrations in the control MCS rats were in the normal range reported in the literature (24). As expected, hepatic Adomet was significantly (P < 0.001) lower in MCD rats. Exogenous Adomet corrected the deficiency (68.1 ± 12.6 nmol/g tissue) to the same range as the levels observed in the MCS-saline group (56.2 ± 8.8 nmol/g tissue; P = 0.062). Administration of LPS to the MCS rats (MCS-LPS) increased their hepatic Adomet concentrations significantly. The ratio of Adomet to S-adenosylhomocysteine remained 2 or higher in all groups.
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Hepatic choline levels in the MCD group were ~50% of those in the MCS group (212.0 ± 44.3 vs. 412.8 ± 110.0 µmol/g tissue; P < 0.001). Exogenous Adomet supplementation raised hepatic choline concentrations in MCD rats to 266.7 ± 51.0 µmol/g tissue, but the values did not approach normal levels. Hepatic GSH concentrations were not significantly lowered in saline-injected MCD rats (Fig. 2). LPS treatment significantly elevated hepatic GSH concentrations in MCD rats compared with their saline-treated counterparts (P < 0.001).
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Changes in liver histology in the various groups are summarized in Table 1 and shown in Fig. 3. No steatosis or necrosis was noted in MCS-saline rats (Fig. 3A), whereas massive macrovesicular fatty changes, but no necrosis, were observed in the MCD-saline group (Fig. 3B). In MCD-LPS rats, large areas of punched out necrosis and extensive steatosis were evident (Fig. 3C) in ~10% of the areas examined. Administration of exogenous Adomet to MCD-LPS rats resulted in marked attenuation of necrosis with little change in steatosis, whereas administration of the Adomet placebo altered neither steatosis nor necrosis in MCD-LPS rats (data not shown).
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Changes in the serum transaminases AST and ALT are summarized in Table 2. In the control MCS group, transaminases did not change significantly on LPS injection. However, transaminases in the MCD-saline group were five to sevenfold higher compared with the MCS-saline control group. Injection of LPS caused a 30- to 50-fold increase in transaminases in MCD rats, indicating an exacerbation of the injury. Administration of exogenous Adomet to MCD-LPS rats lowered serum transaminases; this attenuation paralleled the improvements noted in liver histology. Administration of the Adomet placebo to MCD-LPS rats did not lower serum transaminases (data not shown).
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Serum TNF- concentrations are also shown in Table 2. LPS
challenge resulted in a ~30-fold increase in serum TNF-
in MCS rats. Interestingly, serum TNF-
concentrations in the MCD rats without any LPS administration showed an approximately sixfold increase
over the levels in the control MCS group. After LPS challenge serum
TNF-
concentration in MCD rats increased ~300-fold, suggesting a
synergy of Adomet deficiency and LPS challenge in increasing TNF-
levels. Moreover, exogenous Adomet supplementation in these rats
markedly lowered serum TNF-
levels, just as it attenuated changes in
serum transaminases and hepatic necrosis.
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DISCUSSION |
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Rats administered MCD diets for 16 days had significantly lower hepatic
concentrations of both Adomet and choline. These changes are consistent
with results reported by other investigators (29). Daily administration
of exogenous Adomet to these rats normalized concentrations of hepatic
Adomet. Interestingly, LPS treatment increased hepatic Adomet
concentrations in rats fed either the MCS or MCD diet. The mechanisms
for increased Adomet after LPS treatment are not clear but may include
increased MAT activity, impaired utilization of hepatic Adomet by
various transmethylation reactions, or both. Avila et al. (2) reported
a downregulation of MAT activity after a 6-h exposure to LPS, but the
hepatic Adomet concentrations showed a modest increase. MAT activity
normalized after 12-h exposure to LPS (personal communication from J. M. Mato). Therefore, our findings of increased Adomet after 24-h treatment with LPS are consistent with these observations. In contrast
to its effects on hepatic Adomet, exogenous Adomet did not normalize
hepatic choline levels. The dose of Adomet administered (170 µmol · kg1 · day
1
im) was in the high range compared with that used in other
investigations (30-170
µmol · kg
1 · day
1)
to examine the beneficial effects of Adomet in chemically induced liver
injury (4, 10, 26). Under our experimental conditions, it appears that
exogenous Adomet cannot be used to synthesize optimal amounts of
choline via phosphatidylethanolamine
N-methyltransferase in MCD rats. As a
result, a dietary supplement of choline may be necessary to normalize
hepatic choline concentrations in MCD rats to levels observed in MCS
rats.
This study suggests an important interaction between Adomet and LPS in the development of liver injury. As reported in other studies, MCD rats in our study with no LPS challenge had hepatic steatosis and elevated serum ALT and AST (22, 28). However, LPS challenge significantly exacerbated the hepatic injury to these rats, as evidenced by the appearance of hepatic necrosis in addition to steatosis and a further increase in concentrations of serum transaminases (Fig. 3 and Table 2). Exogenous Adomet supplements normalized hepatic Adomet concentrations, alleviated hepatic necrosis, and lowered serum transaminases but did not markedly affect hepatic steatosis. On the basis of these observations, it may be postulated that Adomet deficiency is necessary for necrosis in this injury model and that choline deficiency alone results only in hepatic steatosis. These observations are similar to our findings in hypoxic rats that have a hepatic deficiency of Adomet but not choline (8). When these rats were challenged with LPS, they showed no steatosis but had marked necrosis (Watson and Chawla, unpublished observations).
The present study demonstrates for the first time an effect of
Adomet/choline deficiency on in vivo TNF- metabolism, as determined by serum TNF-
concentrations. The serum TNF-
concentrations in
MCD rats treated with saline were approximately sixfold higher than
those in similarly treated MCS rats. In the MCS group injected with LPS, the mean serum TNF-
concentration increased ~30-fold compared with the baseline concentration. However, the increase in
serum TNF-
in the LPS-injected MCD rats was ~300-fold higher compared with baseline. Moreover, administration of exogenous Adomet to
MCD-LPS rats blunted the serum TNF-
increase. Thus hepatic Adomet
appears to significantly modulate TNF-
metabolism while
concomitantly affecting the severity of TNF-
hepatotoxicity. This
observation is strengthened by a recent report in which exogenous Adomet suppressed the release of TNF-
by LPS-stimulated pulmonary macrophages in vitro (1). Our study does not examine the issue of
whether the observed increase in serum TNF-
concentrations reflects
increased synthesis, decreased clearance, or both. Also, this study
does not examine the type of cells responding to Adomet deficiency and
repletion (e.g., hepatic Kupffer cells or blood monocytes). It is
relevant to note that TNF-
gene expression is sensitive to the
methylation status of the CpG island in its promoter
region (17, 24) and that hypomethylation of this region due to a
limited availability of Adomet could conceivably affect its expression
in TNF-
-producing cells.
Because Adomet is a precursor for hepatic GSH and hepatic Adomet deficiency is often associated with GSH deficiency (13, 23), it was relevant to examine the role of GSH in this model of liver injury. Our data showed no significant decline in hepatic GSH in any of the treatment groups. Instead, we noted that LPS injection caused an increase in hepatic GSH concentrations in MCD rats compared with the corresponding saline-treated group. However, hepatic GSH was measured only 24 h after LPS administration, and it is possible that transient oxidation of GSH induced by LPS, as reported by others (14, 19), may somehow be modulated by Adomet. Moreover, we measured only total hepatic GSH and did not measure mitochondrial GSH. Depleted mitochondrial GSH has been postulated to play a role in alcoholic liver disease, and Adomet therapy has been used to increase mitochondrial GSH and to attenuate liver injury in rodents fed alcohol (14, 19).
In summary, hepatic Adomet deficiency is associated with increased
susceptibility to LPS-induced hepatotoxicity and increased serum
TNF- concentrations, and administration of exogenous Adomet attenuates these effects. Hepatic injury caused by several chemical toxins is also associated with hepatic Adomet deficiency and increased serum TNF-
concentrations. We postulate that Adomet administration may be beneficial in certain toxin-induced liver injuries (e.g., alcohol-induced liver injury) not only because of its previously observed beneficial effects on mitochondrial GSH concentrations but
also because of its effects on TNF-
metabolism. Because TNF-
cytotoxicity is mediated, at least in part, through mitochondrial dysfunction and oxidative stress, Adomet may also attenuate TNF-
cytotoxicity by correcting these mitochondrial abnormalities. These
data provide an additional rationale for exploring the use of Adomet
therapy in certain types of liver injury.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the help of Dr. Richard Kryscio, Dept. of Biostatistics, University of Kentucky, for statistical analysis of the data.
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FOOTNOTES |
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This study was supported by merit awards (R. K. Chawla and C. J. McClain) from the Dept. of Veterans Affairs and by National Institutes of Health Grants AA-08565 and AA-10496 (to R. K. Chawla), MO1-RR2602, AA-010762, and 1PO1NG 331220 (to C. J. McClain), and ES-07266 (to W. H. Watson).
Address for reprint requests: R. K. Chawla, Division of Digestive Diseases and Nutrition, Dept. of Internal Medicine, MN 650, Univ. of Kentucky Medical Center, Lexington, KY 40536.
Received 14 August 1997; accepted in final form 30 March 1998.
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