The role of tumor necrosis factor alpha in lipopolysaccharide/ranitidine-induced inflammatory liver injury.

Exposure to a nontoxic dose of bacterial lipopolysaccharide (LPS) increases the hepatotoxicity of the histamine-2 (H2) receptor antagonist, ranitidine (RAN). Because some of the pathophysiologic effects associated with LPS are mediated through the expression and release of inflammatory mediators such as tumor necrosis factor alpha (TNF), this study was designed to gain insights into the role of TNF in LPS/RAN hepatotoxicity. To determine whether RAN affects LPS-induced TNF release at a time near the onset of liver injury, male Sprague-Dawley rats were treated with 2.5 x 10(6) endotoxin units (EU)/kg LPS or its saline vehicle (iv) and 2 h later with either 30 mg/kg RAN or sterile phosphate-buffered saline vehicle (iv). LPS administration caused an increase in circulating TNF concentration. RAN cotreatment enhanced the LPS-induced TNF increase before the onset of hepatocellular injury, an effect that was not produced by famotidine, a H2-receptor antagonist without idiosyncrasy liability. Similar effects were observed for serum interleukin (IL)-1beta, IL-6, and IL-10. To determine if TNF plays a causal role in LPS/RAN-induced hepatotoxicity, rats were given either pentoxifylline (PTX; 100 mg/kg, iv) to inhibit the synthesis of TNF or etanercept (Etan; 8 mg/kg, sc) to impede the ability of TNF to reach cellular receptors, and then they were treated with LPS and RAN. Hepatocellular injury, the release of inflammatory mediators, hepatic neutrophil (PMN) accumulation, and biomarkers of coagulation and fibrinolysis were assessed. Pretreatment with either PTX or Etan resulted in the attenuation of liver injury and diminished circulating concentrations of TNF, IL-1beta, IL-6, macrophage inflammatory protein-2, and coagulation/fibrinolysis biomarkers in LPS/RAN-cotreated animals. Neither PTX nor Etan pretreatments altered hepatic PMN accumulation. These results suggest that TNF contributes to LPS/RAN-induced liver injury by enhancing inflammatory cytokine production and hemostasis.     

Enhancement of allyl alcohol hepatotoxicity by endotoxin requires extrahepatic factors.

Noninjurious doses of bacterial endotoxin (lipopolysaccharide; LPS) enhance allyl alcohol-induced liver damage in rats in a Kupffer cell (KC)-dependent fashion. To investigate the mechanism by which KCs contribute to liver injury in this model, isolated KCs and hepatocytes (HCs) were cocultured. Addition of LPS to the cocultured cells did not enhance allyl alcohol-induced cytotoxicity. In addition, recirculating perfusion of isolated livers from na?ve rats with LPS for 2 h did not significantly enhance allyl alcohol-induced toxicity as measured by release of alanine aminotransferase (ALT). These results suggest an extrahepatic factor is required for LPS potentiation of allyl alcohol hepatotoxicity. To examine whether the coagulation cascade contributes to injury in this model, rats were given either warfarin at 42 and 18 h before LPS, or heparin at 1 h before LPS, and were treated with allyl alcohol 2 h after LPS. Warfarin and heparin each significantly blocked the decrease in plasma fibrinogen levels and attenuated the increase in plasma ALT activity in rats treated with LPS and allyl alcohol. To assess the role of thrombin in this injury, isolated livers from rats pretreated with LPS were perfused with thrombin or vehicle and allyl alcohol. Though LPS pretreatment enhanced the toxicity of allyl alcohol compared with livers from na?ve rats, perfusion with thrombin did not increase sensitivity to allyl alcohol. In summary, LPS augments the hepatotoxicity of allyl alcohol through a mechanism involving extrahepatic factors, one of which may be a component of the coagulation cascade.     

Involvement of nonparenchymal cells in oxygen-dependent hepatic injury by allyl alcohol.

Allyl alcohol injury to hepatocytes in the perfused liver is oxygen-dependent. It is not known if this injury involves direct action of allyl alcohol on hepatocytes or requires participation of other cell types (e.g., Kupffer cells) present in the liver. Accordingly, the action of allyl alcohol (100-500 microM) on isolated hepatocytes was studied using cells maintained at either 95 or 21% O2. Allyl alcohol toxicity, as indexed by trypan blue uptake, lactate dehydrogenase release, and ATP content, did not differ in the two groups of cells, suggesting that O2 dependency of allyl alcohol toxicity involves other cell types. Administration of allyl alcohol (30 or 40 mg/kg, ip) to rats caused extensive hepatic necrosis localized primarily to periportal regions. To test the involvement of Kupffer cells in the genesis of this injury, male rats (200-350 g) were treated with gadolinium chloride (GdCl3, 10 mg/kg, iv) which diminishes Kupffer cell function and number. The extent of hepatic damage assessed by light microscopy and serum enzymes, aspartate aminotransferase and alanine aminotransferase, was markedly attenuated by pretreatment of rats with GdCl3 24 hr prior to allyl alcohol injection. Thus, O2-dependent hepatic necrosis caused by allyl alcohol involves the presence of Kupffer cells. Since GdCl3 did not prevent toxicity in the perfused liver, circulating blood elements may also contribute to injury of the liver by allyl alcohol in vivo.     

Neutrophils contribute to endotoxin enhancement of allyl alcohol hepatotoxicity

Nontoxic doses of endotoxin (lipopolysaccharide, LPS) enhance the hepatotoxicity of many xenobiotic agents, including allyl alcohol. Systemic LPS exposure induces an inflammatory response, including accumulation and activation of neutrophils (PMNs) in the liver. The hypothesis that PMNs play a causal role in LPS enhancement of allyl alcohol hepatotoxicity was tested. Rats were pretreated with an anti-neutrophil antibody (anti-PMN immunoglobulin [lg]) to deplete circulating PMNs. Subsequently, they were given LPS or its vehicle, and 2 h later allyl alcohol was administered. The numbers of circulating and hepatic PMNs were decreased in rats pretreated with anti-PMN lg, and liver toxicity induced by cotreatment with LPS and allyl alcohol was attenuated. Treatment with allyl alcohol diminishes the concentration of reduced glutathione (GSH) in liver, raising the possibility that antioxidant defense was compromised in these livers. Accordingly, the hypothesis was tested that allyl alcohol-induced reduction in GSH renders liver cells more sensitive to reactive oxygen species produced by activated PMNs. Isolated hepatocytes were incubated with allyl alcohol in the presence and absence of isolated PMNs stimulated to produce reactive oxygen species. Allyl alcohol produced a concentration-dependent increase in ALT release from hepatocytes. Activated PMNs produced a statistically significant increase in cell killing that was so small it is unlikely to explain the role of PMNs in liver injury in vivo. To test the hypothesis that proteases released from activated PMNs increase the sensitivity of liver cells to allyl alcohol, isolated hepatocytes were incubated with medium from PMNs activated to undergo degranulation. Protease-containing medium from PMNs did not affect allyl alcohol-induced release of ALT from hepatocytes. Taken together, these results indicate that PMNs play a role in the potentiation of allyl alcohol toxicity by LPS. It is unlikely that PMNs contribute to this injury through release of reactive oxygen species or proteases, and other mechanisms must be involved.     

The role of Kupffer cells and TNF-alpha in monocrotaline and bacterial lipopolysaccharide-induced liver injury.

Coexposure to small, noninjurious doses of the pyrrolizidine alkaloid phytotoxin monocrotaline (MCT) and bacterial lipopolysaccharide (LPS) results in synergistic hepatotoxicity. Both centrilobular and midzonal liver lesions occur and are similar to those seen from large, toxic doses of MCT and LPS, respectively. The nature of the lesions in vivo and results from studies in vitro suggest that injury is mediated indirectly rather than from a simple interaction of MCT and LPS with hepatic parenchymal cells. Accordingly, the role of inflammatory factors, such as Kupffer cells and TNF-alpha, in the development of MCT/LPS-induced liver injury was investigated. In Sprague-Dawley rats, MCT (100 mg/kg, i.p.) was administered 4 h before LPS (7.4 x 10(6) EU/kg, i.v.). Pretreatment of these animals with gadolinium chloride, an inhibitor of Kupffer cell function, attenuated liver injury 18 h after MCT administration. An increase in plasma TNF-alpha preceded the onset of hepatic parenchymal cell injury, raising the possibility that this inflammatory cytokine contributes to toxicity. Either pentoxifylline, an inhibitor of cellular TNF-alpha synthesis, or anti-TNF-alpha serum coadministered to MCT/LPS-treated animals significantly attenuated liver injury. These results suggest that Kupffer cells and TNF-alpha are important mediators in the synergistic hepatotoxicity resulting from MCT and LPS coexposure.     

Effect of aging on liver glutathione levels and hepatocellular injury from carbon tetrachloride, allyl alcohol or galactosamine

Severity of liver damage 24 hr after i.p. administration of carbon tetrachloride (0.2 ml/kg), allyl alcohol (0.036 ml/kg) or galactosamine (400 mg/kg) was evaluated in male rats at 4-5, 14-15 or 24-25 months of age. Allyl alcohol hepatotoxicity, as judged by light microscopy and serum alanine aminotransferase levels, increased markedly as a function of age. In contrast, carbon tetrachloride and galactosamine toxicities were unchanged or slightly diminished in old rats. Hepatic glutathione (GSH) concentrations were unaffected by aging; thus, the age-dependent increase in susceptibility to allyl alcohol toxicity was not a result of diminished GSH availability in old age. Hepatotoxicant-induced changes in GSH were observed in allyl alcohol-treated old rats (20% increase) and in galactosamine-treated young-adult and middle-aged rats (30% decrease).     

15-deoxy prostaglandin J2 enhances allyl alcohol-induced toxicity in rat hepatocytes.

Allyl alcohol causes hepatotoxicity that is potentiated by small doses of bacterial lipopolysaccharide (LPS) through a cyclooxygenase-2 (COX-2)-dependent mechanism. The COX-2 product prostaglandin D(2) (PGD(2)) increases hepatocyte killing by allyl alcohol in vitro. In the present study the ability of the nonenzymatic product of PGD(2), 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)), to increase the cytotoxicity of allyl alcohol was evaluated. In a concentration-dependent manner, 15d-PGJ(2) significantly augmented cell death caused by allyl alcohol in isolated rat hepatocytes. 15d-PGJ(2) also increased the cytotoxicity of acrolein, the active metabolite of allyl alcohol. An agonist for the PGD(2) receptor neither reproduced the increase in allyl alcohol-mediated cytotoxicity nor altered the response to 15d-PGJ(2). Similarly, these responses were not affected by either an agonist or an antagonist for the peroxisome proliferator-activated receptor-gamma. The enhancement by 15d-PGJ(2) of allyl alcohol-mediated cell killing was unaffected by augmentation or inhibition of cAMP. Protein synthesis was markedly decreased by 15d-PGJ(2), but inhibition of protein synthesis alone with cycloheximide did not increase allyl alcohol-mediated cell killing. Allyl alcohol at subtoxic concentrations increased translocation of nuclear factor kappa B (NF-kappaB), whereas at cytotoxic concentrations no translocation occurred. 15d-PGJ(2) inhibited translocation of NF-kappaB from the cytosol to the nucleus both in the presence and absence of allyl alcohol. Like 15d-PGJ(2), MG132, an inhibitor of NF-kappaB activation, enhanced allyl alcohol-induced hepatocyte death. Together these results indicate that 15d-PGJ(2) augments hepatocyte killing by allyl alcohol, and the mechanism may be related to the inhibition of NF-kappaB activation.     

Effects of phycocyanin extract on tumor necrosis factor-alpha and nitrite levels in serum of mice treated with endotoxin

Phycocyanin is a biliprotein which exerts antioxidative and anti-inflammatory effects in various in vivo and in vitro experimental models. In this study phycocyanin effects on tumor necrosis factor-alpha (TNF alpha) and nitrite levels in serum of mice treated with lipopolysaccharide (LPS) was examined. TNF alpha was measured by cytotoxicity on L-929 cells and nitrite by the Griess reaction, after reduction of all nitrates to nitrites by nitrate reductase, 1 h after LPS injection (0.5 mg/kg i.p.) there was a significant increase in TNF alpha levels in mouse serum. Phycocyanin (50-300 mg/kg p.o.), administered 1 h before LPS, reduced dose-dependently the TNF alpha concentration in serum. After 18 h, LPS (30 mg/kg i.p.) also induced a substantial increase in serum nitrite levels, which were reduced dose-dependently by phycocyanin pretreatment (100-300 mg/kg p.o.). The results indicate that phycocyanin exerts inhibitory effects on TNF alpha and NO production which might be ascribed to the antioxidative properties of the biliprotein.     

Kupffer cell-mediated differential down-regulation of cytochrome P450 metabolism in rat hepatocytes

Nonparenchymal cells, particularly Kupffer cells, might play an important role in the modulation of xenobiotic metabolism in liver and its pharmacological and toxicological consequences. This intercellular communication via the exchange of soluble factors was investigated in primary rat Kupffer cells and hepatocytes. Freshly isolated rat Kupffer cells were seeded onto cell culture inserts and cocultured with 5 day old serum-free rat hepatocyte monolayer cultures at a ratio of 1:1 for 2 days. Hepatocyte cultures, Kupffer cell cultures or cocultures were treated with 0.1 ng/ml-10 microg/ml lipopolysaccharide (LPS). Within this concentration range, no significant toxicity was observed in either cell type. In LPS-exposed cocultures, tumor necrosis factor alpha (TNFalpha) levels rose up to 5 ng/ml within 5 h; nitric oxide (NO) levels increased up to 70 microM within 48 h of treatment, both in a dose-dependent fashion. The release of negative (albumin) and positive (alpha1-acid-glycoprotein) acute phase proteins from the hepatocytes was strongly down- and up-regulated, respectively. The simultaneous treatment of the cocultures with phenobarbital and LPS (10 ng/ml) or 3-methylcholanthrene and LPS (10 ng/ml) resulted in a strong down-regulation (85%) of the phenobarbital-induced cytochrome P450 (CYP) isoform CYP2B1 in the hepatocytes whereas the 3-methylcholanthrene-induced isoform CYP1A1 was only weakly affected (15%). This specific down-regulation of CYP2B1 was mediated exclusively by TNFalpha, released from the Kupffer cells. It was not linked with NO release from or inducible NO synthase activity in the hepatocytes. The TNFalpha release was not affected by the two xenobiotics. Acetaminophen tested in these cocultures showed no direct interaction with the Kupffer cells. The use of liver cell cocultures is therefore a useful approach to investigate the influence of intercellular communication on xenobiotic metabolism in liver.     

Effect of allyl alcohol on hepatic transporter expression: Zonal patterns of expression and role of Kupffer cell function

During APAP toxicity, activation of Kupffer cells is critical for protection from hepatotoxicity and up-regulation of multidrug resistance-associated protein 4 (Mrp4) in centrilobular hepatocytes. The present study was performed to determine the expression profile of uptake and efflux transporters in mouse liver following treatment with allyl alcohol (AlOH), a periportal hepatotoxicant. This study also investigated the role of Kupffer cells in AlOH hepatotoxicity, and whether changes in transport protein expression by AlOH are dependent on the presence of Kupffer cells. C57BL/6J mice received 0.1 ml clodronate liposomes to deplete Kupffer cells or empty liposomes 48 h prior to dosing with 60 mg/kg AlOH, i.p. Hepatotoxicity was assessed by plasma ALT and histopathology. Hepatic transporter mRNA and protein expression were determined by branched DNA signal amplification assay and Western blotting, respectively. Depletion of Kupffer cells by liposomal clodronate treatment resulted in heightened susceptibility to AlOH toxicity. Exposure to AlOH increased mRNA levels of several Mrp genes, while decreasing organic anion transporting polypeptides (Oatps) mRNA expression. Protein analysis mirrored many of these mRNA changes. The presence of Kupffer cells was not required for the observed changes in uptake and efflux transporters induced by AlOH. Immunofluorescent analysis revealed enhanced Mrp4 staining exclusively in centrilobular hepatocytes of AlOH treated mice. These findings demonstrate that Kupffer cells are protective from AlOH toxicity and that induction of Mrp4 occurs in liver regions away from areas of AlOH damage independent of Kupffer cell function. These results suggest that Kupffer cell mediators do not play a role in mediating centrilobular Mrp4 induction in response to periportal damage by AlOH.     

Role of lipid peroxidation as a mechanism of liver injury after acetaminophen overdose in mice.

Mitochondrial oxidant stress and peroxynitrite formation have been implicated in the pathophysiology of acetaminophen-induced (AAP-induced) liver injury. Therefore, we tested the hypothesis that lipid peroxidation (LPO) might be involved in the injury mechanism. Male C3Heb/FeJ mice fed a diet high in vitamin E (1 g d-alpha-tocopheryl acetate/kg diet) for 1 week had 6.7-fold higher hepatic tocopherol levels than animals on the control diet (8.2 +/- 0.1 nmol/g liver). Treatment of fasted mice with 300 mg/kg AAP caused centrilobular necrosis with high plasma alanine aminotransferase (ALT) activities at 6 h (3280 +/- 570 U/l) but no evidence of LPO (hepatic malondialdehyde, 4-hydroxynonenal). Animals on the vitamin E diet had similar injury and LPO as mice on the control diet. To verify a potential effect of the vitamin E diet on drug-induced liver injury, animals were pretreated with a combination of phorone, FeSO4, and allyl alcohol. We observed, 2 h after allyl alcohol, massive LPO and liver cell injury in the livers of animals on the control diet, as indicated by a 32-fold increase in malondialdehyde levels, extensive staining for 4-hydroxynonenal, and ALT activities of 2310 +/- 340 U/l. Animals on the vitamin E diet had 40% lower hepatic malondialdehyde levels and 85% lower ALT values. Similar results were obtained when animals were treated for 3 days with alpha- or gamma-tocopherol (0.19 mmol/kg, ip). Both treatments reduced LPO and injury after allyl alcohol but had no effect on AAP hepatotoxicity. Thus, despite the previously shown mitochondrial oxidant stress and peroxynitrite formation, LPO does not appear to be a critical event in AAP-induced hepatotoxicity.     

Attenuation of bacterial lipopolysaccharide-induced hepatotoxicity by betaine or taurine in rats.

The effects of betaine or taurine on hepatotoxicity induced by lipopolysaccharide (LPS) were examined in adult male SD rats. Rats were provided with drinking water containing either 1% betaine or taurine for 2 weeks prior to challenge with LPS (5 mg/kg, iv). Supplementation with betaine or taurine protected the animals from induction of LPS hepatotoxicity as measured by changes in aspartate aminotransferase (AST), alanine aminotransferase (ALT) activities and total bilirubin levels in serum, and hepatic glutathione contents. LPS challenge increased serum TNF-alpha and nitrate/nitrite in rats, which were reduced by betaine or taurine intake. Taurine depletion induced by supply of drinking water containing 3% beta-alanine for 7 days did not enhance the LPS-induced hepatic damage or the decrease in hepatic glutathione level. The results indicate that intake of betaine or taurine attenuates the LPS-induced hepatotoxicity resulting from activation of Kupffer cells.     

Effect of perftoran on experimental hepatitis

The effect of perftoran on the course of experimental acute hepatitis in albino rats was studied on the hepatitis models induced by allyl alcohol or P. acnes culture with typhoid fever endotoxin. Perftoran (10 ml/kg) favored more rapid cytolytic syndrome elimination by affecting the lipid peroxidation in rat liver. The drug inhibits the activity of prooxidant enzymes (xanthine oxidase and myeloperoxidase of Kupffer cells) and induces the synthesis of factors accounting for the antiperoxidation protection in hepatocytes such as catalase, glucose-6-phosphate dehydrogenase, and reduced glutathione.     

Examination of the differential hepatotoxicity of diallyl phthalate in rats and mice

In this study we confirmed that diallyl phthalate (DAP) is more hepatotoxic to rats than to mice, and we demonstrated the same species difference in toxicity for allyl alcohol (AA). The data suggest that the toxicity of DAP probably results from AA cleaved from DAP. To determine if the species difference in susceptibility to hepatotoxicity resulted from differences in the disposition and metabolism of DAP, Fischer-344 rats and B6C3F1 mice were given [14C]DAP, 1, 10, or 100 mg/kg po or 10 mg/kg iv, and placed in metabolism cages for 24 hr. In rats, 25-30% of the DAP was excreted as CO2, and 50-70% appeared in the urine within 24 hr. In mice, 6-12% of the DAP was excreted as CO2, and 80-90% was excreted in the urine within 24 hr. Monoallyl phthalate (MAP), allyl alcohol, 3-hydroxypropylmercapturic acid (HPMA), and an unidentified polar metabolite (PM) were found in the urine of rats and mice dosed with DAP. The polar metabolite was present in the urine of rats dosed with DAP or AA, indicating that the compound is a metabolite of AA. There was no difference between the species in the quantity of AA excreted, but mice excreted more MAP (39 vs 33%), HPMA (28 vs 17%), and PM (20 vs 8%) than rats. Because DAP is metabolized to AA, a potent periportal hepatotoxicant, and because the mouse produced more HPMA than rats, we postulate that the differential hepatotoxicity of DAP is related to the extent of glutathione conjugation with allyl alcohol or acrolein (the active metabolite of AA).     

Methoxyflurane enhances allyl alcohol hepatotoxicity in rats. Possible involvement of increased acrolein formation

The effect of methoxyflurane anesthesia on allyl alcohol-induced hepatotoxicity and the metabolism of allyl alcohol was studied in male rats. Hepatotoxicity was assessed by the measurement of serum alanine aminotransferase activity and histopathological examination. Allyl alcohol-induced hepatotoxicity was enhanced when allyl alcohol (32 mg/kg) was administered 4 hr before or up to 8 days after a single 10-min exposure to methoxyflurane vapors. The possibility that methoxyflurane increases alcohol dehydrogenase-dependent oxidation of allyl alcohol to acrolein, the proposed toxic metabolite, was evaluated by measuring the rate of acrolein formation in the presence of allyl alcohol and liver cytosol. The effect of methoxyflurane on alcohol dehydrogenase activity in liver cytosol was also assessed by measuring the rate of NAD+ utilization in the presence of ethyl alcohol or allyl alcohol. Alcohol dehydrogenase activity and rate of acrolein formation were elevated in methoxyflurane-pretreated rats. The results suggest that a modest increase in alcohol dehydrogenase activity and rate of acrolein formation markedly enhances allyl alcohol-induced hepatotoxicity.     

Lipopolysaccharide activates nuclear factor kappaB in rat intestine: role of endogenous platelet-activating factor and tumour necrosis factor

1. We examined the effect of lipopolysaccharide (LPS), a cell wall constituent of Gram negative bacteria, on nuclear factor kappaB (NF-kappaB) activation in the intestine and the roles of endogenous platelet-activating factor (PAF), tumour necrosis factor-alpha (TNF) and neutrophils. We also compared the time course of NF-kappaB activation in response to PAF and LPS. 2. Ileal nuclear extracts from LPS (8 mg kg(-1), IV)-injected rats were assayed for NF-kappaB-DNA-binding activity and identification of the subunits. Some rats were pretreated with WEB2170 (a PAF receptor antagonist), anti-TNF antibody, or anti-neutrophil antiserum. NF-kappaB p65 was localized by immunohistochemistry. An additional group was challenged with PAF (2 microg kg(-1), IV) for comparison. 3. LPS activates intestinal NF-kappaB, both as p50-p50 and p50-p65 dimers within 15 min, and the effect peaks at 2 h. The effect is slower and more sustained than that of PAF, which peaks at 30 min. Activated NF-kappaB was immunolocalized within epithelial and lamina propria cells. LPS effect was reduced by 41, 37 and 44%, respectively, in animals pretreated with WEB2170, anti-TNF antibody, or anti-neutrophil antiserum (P<0.05). 4. LPS activates intestinal NF-kappaB in vivo and neutrophil activation is involved in its action. The LPS effect is mediated by both endogenous PAF and TNF.     

Alcohol-induced S-adenosylhomocysteine accumulation in the liver sensitizes to TNF hepatotoxicity: possible involvement of mitochondrial S-adenosylmethionine transport

Hepatocytes are resistant to tumor necrosis factor-alpha- (TNF) induced killing/apoptosis under normal circumstances, but primary hepatocytes from rats chronically fed alcohol have increased TNF cytotoxicity. Therefore, there must be mechanism(s) by which alcohol exposure "sensitizes" to TNF hepatotoxicity. Abnormal metabolism of methionine and S-adenosylmethionine (SAM) are well-documented acquired metabolic abnormalities in ALD. S-adenosylhomocysteine (SAH) is the product of SAM in hepatic transmethylation reactions, and SAH hydrolase (SAHH) is the only enzyme to metabolize SAH to homocysteine and adenosine. Our previous studies demonstrated that chronic intracellular accumulation of SAH sensitized hepatocytes to TNF cytotoxicity in vitro. In the current study, we extended our previous observations by further characterizing the effects of chronic alcohol intake on mitochondrial SAM levels in liver and examining its possible involvement in SAH sensitization to TNF hepatotoxicity. Chronic alcohol consumption in mice not only increased cytosolic SAH levels, but also decreased mitochondrial SAM concentration, leading to decreased mitochondrial SAM to SAH ratio. Moreover, accumulation of hepatic SAH induced by administration of 3-deaza-adenosine (DZA-a potent inhibitor of SAHH) enhanced lipopolysaccharide (LPS)/TNF hepatotoxicity in mice in vivo. Inhibition of SAHH by DZA resulted not only in accumulation of cytoplasmic SAH, but also in depletion of the mitochondrial SAM pool. Further studies using mitochondrial SAM transporter inhibitors showed that inhibition of SAM transport into mitochondria sensitized HepG2 cells to TNF cytotoxicity. In conclusion, our results demonstrate that depletion of the mitochondrial SAM pool by SAH, which is elevated during chronic alcohol consumption, plays a critical role in SAH induced sensitization to TNF hepatotoxicity.     

Analysis of strain difference in sensitivity to cadmium-induced hepatotoxicity in Fischer 344 and Sprague-Dawley rats.

Acute administration of cadmium (Cd) in rats results in hepatotoxicity that appears to involve the activation of Kupffer cells and the subsequent production of proinflammatory chemokines and cytokines. However, the importance of these endogenous mediators in Cd-induced hepatotoxicity is unknown. Therefore, this study was conducted to define and utilize a rat strain difference in sensitivity to Cd-induced hepatotoxicity to elucidate the role of cytokines and chemokines in Cd-induced hepatotoxicity. Doses were selected from a dose-response study of the effect of Cd on serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities. Hepatotoxic doses of 2.0 mg Cd/kg in Fischer 344 (F344) rats and 3.0 mg Cd/kg in Sprague-Dawley (SD) rats, as well as a relatively nontoxic dose of 2.0 mg Cd/kg in SD rats, were chosen for the time-course experiment. Blood and liver from F344 (saline or 2.0 mg Cd/kg iv) and SD rats (saline or 2.0 or 3.0 mg Cd/kg iv) were collected at 0, 1, 3, 6, 10, 18, 24, and 48 h after Cd administration. Cadmium treatment caused an increase in serum ALT and SDH by 3 h and peaked between 18 and 24 h in both strains. Hepatic Cd content, metallothionein (MT) induction, and nonprotein sulfhydryl (NPSH) content were quantified and determined to be consistent with dosing rather than strain differences. Total RNA samples isolated from liver samples were analyzed for chemokine (CINC-1 and MCP-1) and cytokine (TNF-alpha, IL-1beta, IL-6, and IL-10) mRNA levels by the Quantigene branched DNA signal amplification assay. Lipopolysaccharide treatment served as a positive control for chemokine and cytokine induction. After Cd administration, F344 rat livers did not contain higher levels or earlier induction of chemokine and cytokine mRNAs than SD rats. Therefore, this study demonstrates a strain difference in sensitivity to Cd-induced hepatotoxicity that appears to be unrelated to Cd, MT, NPSH, or cytokine expression.