S-adenosylmethionine deficiency and TNF-alpha in lipopolysaccharide-induced hepatic injury

Rajender K. Chawla1,2, Walter H. Watson1,2, Charles E. Eastin1,2, Eun Y. Lee3, Jack Schmidt1,2, and Craig J. McClain1,2

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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-alpha (TNF-alpha ) 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-alpha was sixfold higher in MCD rats even without LPS challenge and 300-fold higher with LPS challenge. Exogenous Adomet attenuated increased serum TNF-alpha in MCD-LPS rats.

tumor necrosis factor-alpha ; choline deficiency; liver injury

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-alpha (TNF-alpha ), a pleiotropic proinflammatory cytokine, is postulated to be one mediator for these models of liver injury, and antibodies to TNF-alpha or soluble TNF-alpha receptors attenuate the hepatic injury in these models (3, 11, 15, 27). TNF-alpha 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-alpha 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-alpha 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-alpha concentrations.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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 · kg-1 · day-1 im) for 16 days while another MCD group received an equimolar amount of placebo 1,4-butanedisulfonate. Blood for TNF-alpha 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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Table 1.   Animal groups used and changes in their hepatic histology

Serum AST and ALT enzymes were assayed in the Clinical Laboratory of the University of Kentucky Medical Center. Serum TNF-alpha was assayed by an in vitro cytotoxicity bioassay, using L-M cells from mouse connective tissue (16). Tissue levels of Adomet were measured using the cation exchange high-performance liquid chromatography (HPLC) method as previously reported by Chawla et al. (8). Hepatic choline levels were assayed radioenzymatically according to the procedure in use in our laboratory (7). Hepatic GSH concentrations were assayed by HPLC analysis of dansylated protein-free hepatic extracts (18).

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.

    RESULTS
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Materials & Methods
Results
Discussion
References

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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Fig. 1.   Hepatic concentrations (nmol/g tissue) of S-adenosylmethionine (Adomet) and S-adenosylhomocysteine in different experimental groups. 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) diet] and then administered either saline or lipopolysaccharide (LPS), with or without exogenous Adomet. * Adomet concentration significantly different from control (MCS/saline) group. All values are means ± SD.

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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Fig. 2.   Hepatic concentrations of glutathione (GSH; nmol/mg protein) in different experimental groups. * Significantly different from MCS-saline and MCD-saline groups. All values are means ± SD.

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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Fig. 3.   Hematoxylin and eosin staining of MCS-saline (A), MCD-saline (B), and MCD-LPS (C) livers. A: no steatosis or necrosis. B: massive steatosis but no necrosis. C: both steatosis and necrosis. See text for details.

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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Table 2.   Profile of serum liver enzymes and TNF-alpha of rats maintained on various dietary regimens

Serum TNF-alpha concentrations are also shown in Table 2. LPS challenge resulted in a ~30-fold increase in serum TNF-alpha in MCS rats. Interestingly, serum TNF-alpha 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-alpha concentration in MCD rats increased ~300-fold, suggesting a synergy of Adomet deficiency and LPS challenge in increasing TNF-alpha levels. Moreover, exogenous Adomet supplementation in these rats markedly lowered serum TNF-alpha levels, just as it attenuated changes in serum transaminases and hepatic necrosis.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 · kg-1 · 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-alpha metabolism, as determined by serum TNF-alpha concentrations. The serum TNF-alpha 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-alpha concentration increased ~30-fold compared with the baseline concentration. However, the increase in serum TNF-alpha 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-alpha increase. Thus hepatic Adomet appears to significantly modulate TNF-alpha metabolism while concomitantly affecting the severity of TNF-alpha hepatotoxicity. This observation is strengthened by a recent report in which exogenous Adomet suppressed the release of TNF-alpha by LPS-stimulated pulmonary macrophages in vitro (1). Our study does not examine the issue of whether the observed increase in serum TNF-alpha 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-alpha 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-alpha -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-alpha 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-alpha 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-alpha metabolism. Because TNF-alpha cytotoxicity is mediated, at least in part, through mitochondrial dysfunction and oxidative stress, Adomet may also attenuate TNF-alpha 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.

    ACKNOWLEDGEMENTS

We gratefully acknowledge the help of Dr. Richard Kryscio, Dept. of Biostatistics, University of Kentucky, for statistical analysis of the data.

    FOOTNOTES

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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Discussion
References

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Am J Physiol Gastroint Liver Physiol 275(1):G125-G129
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