Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats.

Kupffer cells are involved in the pathogenesis of chemically mediated liver injury through release of biologically active mediators that promote the pathogenic process. The purpose of this study was to elucidate specific biochemical and molecular changes occurring in Kupffer cells throughout a time course of carbon tetrachloride (CCl(4))-mediated liver injury and fibrosis. Rats were administered 1 ml/kg of CCl(4) (10% v/v olive oil) twice weekly for up to 6 weeks. Plasma alanine aminotransferase values and hematoxylin-and-eosin- and trichrome-stained liver sections indicated minor liver damage at 2 weeks followed by increased damage and collagen deposition by 4 and 6 weeks. Additionally, mRNA levels in Kupffer cells isolated from CCl(4)-treated rats demonstrated significant increases in tumor necrosis factor alpha (TNF alpha); tumor growth factor beta; interleukin-6 (IL-6); interleukin 1 beta; cyclooxygenase 2; CD14, and I kappa B alpha transcripts after 2 and 4 weeks of treatment. However, the expression of these genes at 6 weeks was similar to that of controls. Increased gene expression of cytokines in Kupffer cells isolated from CCl(4)-treated rats was accompanied by increases in protein production of TNF alpha, IL-6, IL-1 beta, and interleukin 10 following lipopolysaccharide stimulation. Further, liver sections stained for ED2-positive cells demonstrated an increase in the number of resident macrophages at 2 and 4 weeks with a slight decrease in ED2-positive cells by week 6 but still significantly more than control. Analysis of reduced glutathione (GSH) and oxidized glutathione (GSSG) indicated that Kupffer cells from CCl(4)-treated animals exhibited a 50% decrease in GSH at 2 and 4 weeks, whereas no significant changes were observed for GSSG. In conclusion, these data implicate Kupffer cells as a critical mediator of the inflammatory and fibrogenic responses during CCl(4)-mediated liver damage and provide new insight into the temporal molecular and biochemical changes associated with the ability of these resident macrophages to modulate liver injury.

Metabolism of 4-hydroxynonenal by rat Kupffer cells.

Kupffer cells are known to participate in the early events of liver injury involving lipid peroxidation. 4-Hydroxy-2,3-(E)-nonenal (4-HNE), a major aldehydic product of lipid peroxidation, has been shown to modulate numerous cellular systems and is implicated in the pathogenesis of chemically induced liver damage. The purpose of this study was to characterize the metabolic ability of Kupffer cells to detoxify 4-HNE through oxidative (aldehyde dehydrogenase; ALDH), reductive (alcohol dehydrogenase; ADH), and conjugative (glutathione S-transferase; GST) pathways. Aldehyde dehydrogenase and GST activity was observed, while ADH activity was not detectable in isolated Kupffer cells. Additionally, immunoblots demonstrated that Kupffer cells contain ALDH 1 and ALDH 2 isoforms as well as GST A4-4, P1-1, Ya, and Yb. The cytotoxicity of 4-HNE on Kupffer cells was assessed and the TD50 value of 32.5+/-2.2 microM for 4-HNE was determined. HPLC measurement of 4-HNE metabolism using suspensions of Kupffer cells incubated with 25 microLM 4-HNE indicated a loss of 4-HNE over the 30-min time period. Subsequent production of 4-hydroxy-2-nonenoic acid (HNA) suggested the involvement of the ALDH enzyme system and formation of the 4-HNE-glutathione conjugate implicated GST-mediated catalysis. The basal level of glutathione in Kupffer cells (1.33+/-0.3 nmol of glutathione per 10(6) cells) decreased significantly during incubation with 4-HNE concurrent with formation of the 4-HNE-glutathione conjugate. These data demonstrate that oxidative and conjugative pathways are primarily responsible for the metabolism of 4-HNE in Kupffer cells. However, this cell type is characterized by a relatively low capacity to metabolize 4-HNE in comparison to other liver cell types. Collectively, these data suggest that Kupffer cells are potentially vulnerable to the increased concentrations of 4-HNE occurring during oxidative stress.

Characterization of 4-hydroxy-2-nonenal metabolism in stellate cell lines derived from normal and cirrhotic rat liver.

During oxidative stress, reactive aldehydes, including trans-4-hydroxy-2-nonenal (4-HNE), are generated by peroxidation of membrane lipids and purportedly stimulate hepatic stellate cells to produce excessive extracellular matrix, including type I collagen. An important question concerning the ability of 4-HNE to modulate collagen production by stellate cells is the potential of these specialized cells to detoxify 4-HNE. The objective of the present study was to characterize the ability of stellate cell lines, derived from normal (NFSC) and cirrhotic (CFSC) rat livers, to metabolize 4-HNE by oxidative, reductive and conjugative pathways. These two stellate cell lines were noted to have differing susceptibilities to the cytotoxic effect of 4-HNE. Treatment of both stellate cell lines with a range of 4-HNE doses demonstrated that the concentration which was cytotoxic to 50% of CFSC (TD(50)) was 25% greater than that for NFSC (967.57+/-9.26 nmol/10(6) cells vs. 769.90+/-5.32 nmol/10(6) cells respectively). The capacity of these cell lines to metabolizes 4-HNE was determined by incubating them in suspension with 50 microM 4-HNE (10 nmol/10(6) cell); 4-HNE elimination and metabolite formation were quantified over a 20 min time course. Both stellate cell lines rapidly metabolized 4-HNE, with the CFSC line eliminating 4-HNE at a rate that was approx. 2-fold greater than the NFSC line. The rate of 4-HNE metabolism attributable to glutathione S-transferase (GST) was similar in both cell lines, though differential cell specific expressions of GST isoforms GSTP1-1 and GSTA4-4 were observed. The greater rate of 4-HNE elimination by CFSC was attributable to its aldehyde dehydrogenase (ALDH) activity which accounted for approx. 50% of 4-HNE metabolism in CFSC but was insignificant in NFSC. Neither cell line had detectable alcohol dehydrogenase activity or protein levels. Measurement of cellular GSH concentrations revealed that NFSC contain approx. 2-fold greater concentrations of GSH when compared to CFSC and that following 4-HNE treatment, GSH levels were rapidly depleted from both cell lines. Concomitant with 4-HNE mediated GSH depletion, a corresponding increase in the 4-HNE-glutathione adduct formation was observed with the NFSC line forming greater amounts of the glutathione adduct than did the CFSC line. Taken together, these data demonstrate that both stellate cell lines have the capacity to metabolize 4-HNE but that CFSC have a greater rate of metabolism which is attributable to their greater ALDH activity, suggesting that the stellate cells isolated from cirrhotic liver may be differentially responsive to the biologic effects of 4-HNE.

Involvement of p65 in the regulation of NF-kappaB in rat hepatic stellate cells during cirrhosis.

We have examined the NF-kappaB binding and functional activities in two stellate cell lines derived from normal (NFSC) and cirrhotic (CFSC) rat liver. Gel mobility shift assays revealed two bands in NFSC nuclear extracts that correspond to p65/p50 heterodimers and p50/p50 homodimers. In contrast, a single and more intense band that migrates faster was detected in CFSC nuclear extracts. This band supershifts with either p65 or p50 antibody. The differential NF-kappaB binding observed in these two cell lines appears to depend on the phosphorylation of the p65 subunit rather than the expression levels of either p65 or p50. The nonphosphorylated NF-kappaB form, present in CFSC cells, possesses significantly lower transcriptional activity compared to phosphorylated NF-kappaB, found in NFSC cells. To our knowledge, this is the first report on the NF-kappaB regulation at the p65 protein in hepatic stellate cells. It is likely that this regulation affects IL-6 expression and may represent a mechanism regulating hepatocyte death during fibrogenesis.

Formation of malondialdehyde adducts in livers of rats exposed to ethanol: role in ethanol-mediated inhibition of cytochrome c oxidase.

BACKGROUND: Previous studies in our laboratory demonstrated that short-term ethanol consumption by maternal rats increased the hepatic levels of 4-hydroxynonenal (HNE) in both the adult and the fetus. Additionally, HNE inhibited cytochrome c oxidase (COX) by forming adducts with the enzyme subunits. The present study examined modification of COX by another major aldehydic lipid peroxidation product, malondialdehyde (MDA), and its role in COX inhibition by ethanol. METHODS AND RESULTS: It is demonstrated in vitro that MDA inhibits the activity of purified COX while forming adducts with the enzyme. Compared with HNE, MDA is a more potent inhibitor of COX. Overnight incubation at room temperature caused an 80% decrease in COX activity by MDA versus a 67% decrease by HNE. MDA produced marked inhibition of COX activity at physiologically relevant concentrations, e.g., 43% inhibition at 10 microM. Although our previous studies documented that HNE formed adducts primarily with subunit IV of COX via histidine residues, the current report showed that MDA forms adducts with both subunit IV and subunit V via lysine residues. Furthermore, both aldehydes induce carbonyl formation in subunit IV. The in vivo role of MDA in the impairment of COX by ethanol is assessed in both adult and fetal liver after maternal ethanol consumption. CONCLUSIONS: The results showed that: (1) there are significant increases in MDA levels in liver homogenate as well as mitochondria in both adult and fetal livers after ethanol exposure; (2) these MDA levels are in the nanomole/mg protein range, in contrast to picomole/mg protein range of HNE in identical setting; and (3) ethanol-induced production of MDA is accompanied by enhanced formation of MDA adducts with COX. These findings suggest that MDA may play at least as equally an important role as HNE in ethanol-induced inhibition of COX.

Characterization of hepatic iron overload following dietary administration of dicyclopentadienyl iron (Ferrocene) to mice: cellular, biochemical, and molecular aspects.

A unique organic form of iron (dicyclopentadienyl iron; ferrocene) has been used to further elucidate specific hepatic histopathologic, biochemical, and molecular parameters associated with dietary iron overload. Male C57BL/6Ibg mice fed a diet containing 0.04-0.2% w/w ferrocene for 115 days displayed severe hepatic siderosis of hepatocytes accompanied by a 15-fold induction of nonheme iron content compared to control mice receiving a diet with normal amounts of iron. The ferrocene treatment led to significant increases in hepatocellular necrosis as measured by plasma alanine aminotransferase activity. Histological assessment of hepatic fibrosis revealed mild increases in collagen deposition localized with accumulations of hemosiderin primarily in centrilobular hepatocytes. Hepatic fibrosis was confirmed by measurement of hepatic hydroxyproline content that was increased 4-fold in ferrocene-fed animals compared to control animals not ingesting ferrocene. Hepatic siderosis was accompanied by significant increases in hepatic malondialdehyde content suggesting the ferrocene-induced iron burden initiated lipid peroxidation in vivo. Expression of the heavy-chain isoform of ferritin mRNA and protein measured in liver after ferrocene feeding was increased approximately 8- and 2-fold, respectively, compared to the appropriate controls. These results, using an organic form of iron fed to genetically well-characterized inbred mice, provide new additional insight into the specific molecular and biochemical events that occur in association with histopathologic changes initiated by iron-induced liver injury. These data support the hypothesis that peroxidation of cellular membrane lipids is an important mechanism involved in the toxicity of excess hepatic iron and possibly the initiation of liver fibrogenesis. The results presented here also provide novel in vivo evidence documenting the cellular modulation of ferritin in response to the toxic effects of hepatic iron overloading and iron-mediated oxidative stress.

Role of aldehyde dehydrogenases in endogenous and xenobiotic metabolism.

Aldehydes are highly reactive molecules that are intermediates or products involved in a broad spectrum of physiologic, biologic and pharmacologic processes. Aldehydes are generated from chemically diverse endogenous and exogenous precursors and aldehyde-mediated effects vary from homeostatic and therapeutic to cytotoxic, and genotoxic. One of the most important pathways for aldehyde metabolism is their oxidation to carboxylic acids by aldehyde dehydrogenases (ALDHs). Oxidation of the carbonyl functional group is considered a general detoxification process in that polymorphisms of several human ALDHs are associated a disease phenotypes or pathophysiologies. However, a number of ALDH-mediated oxidation form products that are known to possess significant biologic, therapeutic and/or toxic activities. These include the retinoic acid, an important element for vertebrate development, gamma-aminobutyric acid (GABA), an important neurotransmitter, and trichloroacetic acid, a potential toxicant. This review summarizes the ALDHs with an emphasis on catalytic properties and xenobiotic substrates of these enzymes.

4-Hydroxynonenal and malondialdehyde hepatic protein adducts in rats treated with carbon tetrachloride: immunochemical detection and lobular localization.

The metabolism of CCl(4) initiates the peroxidation of polyunsaturated fatty acids producing alpha,beta-unsaturated aldehydes, such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). The facile reactivity of these electrophilic aldehydic products suggests they play a role in the toxicity of compounds like CCl(4). To determine the rate at which CCl(4)-initiated lipid peroxidation results in the formation of 4-HNE and/or MDA hepatic protein adducts, rats were given an intragastric dose of CCl(4) (1.0 ml/kg) and euthanized 0-72 h after administration. Rabbit polyclonal antisera directed toward 4-HNE- or MDA-protein epitopes were employed in immuno-histochemical and immuno-precipitation/Western analyses to detect 4-HNE and MDA-protein adducts in paraffin-embedded liver sections and liver homogenates. As early as 6 h post CCl(4) exposure, 4-HNE and MDA adducts were detected immuno-histochemically in hepatocytes localized to zone 2 of the hepatic acinus. Liver injury was progressive to 24 h as lipid peroxidation and hepatocellular necrosis increased. The hallmark of CCl(4) hepatotoxicity, zone 3 necrosis, was observed 24 h after CCl(4) administration and immuno-positive hepatocytes were observed in zone 2 as well as zone 3. Immuno-positive cells were no longer visible by 36 to 72 h post CCl(4) administration. From 6 to 48 h after CCl(4) administration, at least four adducted proteins were immuno-precipitated from liver homogenates with the anti-MDA or anti-4HNE serum, which corresponded to molecular weights of 80, 150, 205, and greater than 205 kDa. These results demonstrate that 4-HNE and MDA alkylate specific hepatic proteins in a time-dependent manner, which appears to be associated with hepatocellular injury following CCl(4) exposure.

Formation and export of the glutathione conjugate of 4-hydroxy-2, 3-E-nonenal (4-HNE) in hepatoma cells.

The cellular metabolism of 4-hydroxy-2-nonenal (4-HNE), a cytotoxic and genotoxic product of oxidative stress-induced lipid peroxidation, was investigated in rat H35 hepatoma cells. Previous studies from our laboratory (1) have characterized the degree to which oxidative, reductive, and conjugative metabolic pathways function simultaneously during hepatocellular metabolism of 4-HNE to rapidly eliminate the compound from suspensions of freshly isolated rat hepatocytes. In the current studies, we have extended the investigation of 4-HNE metabolism to examine the pharmacokinetic parameters of 4-HNE elimination and export in a hepatoma cell line and determined that the ensuing oxidative and conjugative metabolites of 4-HNE are rapidly and efficiently transported out the cell. Low concentrations of 4-HNE (25 microM) were used in an attempt to simulate physiologically relevant conditions. The H35 hepatoma cell line studied was first evaluated for enzymes known to play important roles in the metabolism of 4-HNE and were found to possess activities for glutathione S-transferase, aldehyde dehydrogenase (ALDH), and alcohol dehydrogenase of 24.00 +/- 1.12, 3. 45 +/- 0.17, and 6.44 +/- 0.29 nmol min-1 mg-1 protein, respectively. Hepatoma cells were incubated with 25 microM 4-HNE and metabolites in intra- and extracellular fractions were quantitated by reversed-phase HPLC over the time course of treatment. Reduced glutathione (GSH) and the GSH metabolites of 4-HNE were quantitated by reversed-phase HPLC as the dinitrobenzene derivatives. Uptake of 4-HNE from the extracellular medium occurred with an estimated rate of 0.398 +/- 0.181 min-1 10(6) hepatoma cells-1. The oxidative metabolite of 4-HNE, 4-hydroxy-2-nonenoic acid (HNA), produced by ALDH, appeared rapidly in the intracellular fraction achieving concentrations of 0.28 HNA nmol 10(6) hepatoma cells-1 and was efficiently eliminated with a first-order rate constant of 0.988 min-1. The GST-mediated conjugative metabolite, 3-glutathionyl-4-hydroxy-2-nonanal (4-HNE-SG), rapidly reached maximal intracellular concentrations of 1.88 +/- 0.44 nmol 10(6) hepatoma cells-1 and was eliminated at a rate of 0.101 +/- 0.033 min-1. Extracellular rates of formation, representing export, for HNA and 4-HNE-SG were 0.247 +/- 0.045 and 0.044 +/- 0.009 min-1 10(6) hepatoma cells-1, resulting in maximal extracellular concentrations for HNA and 4-HNE-SG of 0.70 +/- 0.10 and 3.03 +/- 0. 84 nmol 10(6) hepatoma cells-1. Approximately 75% of the administered concentration of 4-HNE was converted to measurable metabolites, with the 4-HNE-GSH conjugate accounting for 61% of total administered 4-HNE and HNA accounting for 14%. Collectively, these results demonstrate that oxidative and conjugative pathways are primarily responsible for elimination of 4-HNE at low concentrations in the hepatoma cell line evaluated and that the 4-HNE metabolites resulting from these pathways are rapidly and efficiently exported out of the cell.

Alpha,beta-unsaturated aldehydes increase glutathione S-transferase mRNA and protein: correlation with activation of the antioxidant response element.

A series of alpha,beta-unsaturated aldehydes was evaluated to determine if these compounds could mediate inducible expression of glutathione S-transferase (GST) through the 5'-flanking antioxidant response element (ARE). The ARE from rGST A1 was subcloned into a luciferase reporter construct and used to transiently transfect rat Clone 9 hepatoma cells. Transfected cells were treated with 4-hydroxy-trans-2-nonenal (4-HNE), trans-2-hexenal (t-2-HE), 2-propenal (acrolein, 2-PE), and ethacrynic acid (EA), a control compound also containing an alpha,beta-unsaturated carbonyl moiety. Each compound was evaluated for cytotoxicity to construct dosing regimens in transfection studies. IC50 values for growth inhibition were measured using 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide. IC50 values in Clone 9 cells were: 4-HNE, 6.3 +/- 0.7 microM; t-2-HE, 16.0 +/- 0.7 microM; 2-PE, 2.2 +/- 0.4 microM; and EA, 38.0 +/- 1.6 microM. A dose-dependent increase in luciferase activity was observed in transfected cells with all four compounds tested, indicating that alpha, beta-unsaturated aldehydes function as direct activators of the ARE. To determine whether or not the observed promoter activation led to increased transcriptional and translational induction of GST, cells were treated with the various compounds and assayed for increases in GST mRNA, protein, and enzyme activity. Studies in Clone 9 cells revealed increased steady-state message for GST A1 and A4, increased GST A4-4 protein by Western blotting, and increased GST activity toward 1-chloro-2,4-dinitrobenzene in response to treatment with all four compounds evaluated. Collectively, these studies demonstrate that EA and certain alpha,beta-unsaturated aldehydes produced as a result of cellular membrane lipid peroxidation are activators of the ARE and efficient inducers of GST A1-1 and A4-4.

Formation of liver microsomal MDA-protein adducts in mice with chronic dietary iron overload.

Lipid peroxidation has been proposed to be a major mechanism involved in the pathophysiology of hepatic iron overload. Hepatic microsomal lipid peroxidation has been demonstrated in animals with dietary iron overload, and major products of lipid peroxidation with known cytotoxicity, such as malondialdehyde (MDA), may be involved in iron-induced hepatocellular injury by covalent binding to microsomal proteins. This investigation examined whether DBA/2Ibg mice fed a diet enriched with ferrocene-iron for 16 weeks, results in hepatic lipid peroxidation, and if liver microsomes contain proteins adducted by MDA. Chronic iron feeding to mice resulted in a severe hepatic iron overload with hepatic stores of iron 12-fold greater than those measured in control mice and a three-fold increase in hepatic concentrations of MDA, indicating the occurrence of iron-induced lipid peroxidation in vivo. Hepatic collagen content was increased by over three-fold (p < 0.05) in iron-fed mice as compared to control animals, suggesting increased fibrogenesis. Using rabbit antiserum specific for MDA amine protein adducts and immunoprecipitation-Western blotting, we documented formation of 10 liver microsomal proteins adducted by MDA in iron overload mice (approximate molecular weights; 214, 140, 129, 121, 103, 83, 62, 60, 48, and 43-kD). Control mice did not exhibit positive immunostaining for these protein adducts. The incubation of synthetic MDA with liver microsomes isolated from untreated mice demonstrated formation of MDA-adducted proteins with molecular weights comparable to those detected following in vivo iron overload. The data from this animal study are the first to demonstrate that lipid-derived aldehydes produced from hepatic iron overload in vivo, covalently bind and hence, chemically modify numerous proteins in microsomes. These data suggest that MDA modified proteins in microsomes may play a role in a sequence of events that lead to cell injury during metal-induced liver damage.

Association of glutathione S-transferase isozyme-specific induction and lipid peroxidation in two inbred strains of mice subjected to chronic dietary iron overload.

The alpha-class glutathione S-transferases are proposed to play a prominent role in catalyzing the conjugation of glutathione with electrophilic aldehydic products of lipid peroxidation. The effect of iron-induced lipid peroxidation on induction of glutathione S-transferase (GST) isozymes A1 and A4 in the livers of male C57/BL6Ibg and DBA/J2Ibg mice was studied. C57 and DBA mice were fed for 4 months on a diet supplemented with iron as ferrocene and then were assessed for liver injury, hepatic iron loading, indices of lipid peroxidation, GST activity, and induction of GST isozymes A1 and A4. Iron-treated animals displayed a loss in body weight from pair-fed controls and had large increases in hepatic non-heme iron with concomitant liver injury, as measured by serum alanine aminotransferase. Hepatic lipid hydroperoxides, a direct measure of oxidized membrane lipids, were significantly increased only in C57 mice, but hepatic concentrations of reduced glutathione (GSH) were significantly increased in both inbred strains. Total GST activity toward 1-chloro-2,4-dinitrobenzene was significantly increased in C57 mice but not in DBA. Western blot studies using polyclonal antibodies specific for GST A1 and A4 revealed significant increases of 1.5-2.0-fold in these GST isoforms in both inbred strains. These results in a unique murine model for hepatic iron overload further support recent in vivo studies (Khan et al., Toxicol. Appl. Pharmacol., 131, 63-72, 1995) that have associated induction of GST A4 with protection against oxidative stress-induced lipid peroxidation. The observed increases in lipid hydroperoxides, hepatic GSH, GST activity, and GST A1 and A4 protein strongly support the hypothesis that induction of GST A1 and A4 represents an important protective event in the detoxification of electrophilic products of lipid peroxidation.

Prooxidant-initiated lipid peroxidation in isolated rat hepatocytes: detection of 4-hydroxynonenal- and malondialdehyde-protein adducts.

Toxicity associated with prooxidant-mediated hepatic lipid peroxidation is postulated to originate from the interaction of the aldehydic end products of lipid peroxidation with cellular constituents. The principal alpha,beta-unsaturated aldehydic products of lipid peroxidation, 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA), are known to modify proteins through covalent alkylation of lysine, histidine, and cysteine amino acid residues. To detect and characterize the formation of 4-HNE- and MDA-adducted proteins during prooxidant-initiated lipid peroxidation, rabbit polyclonal antibodies were raised to 4-HNE-sulfhydryl, dinitrophenylhydrazine (DNPH)-4-HNE-sulfhydryl, and MDA-amine conjugates of keyhole limpet hemocyanin (KLH). Each antiserum displayed high antibody titers to either 4-HNE-metallothionein, DNPH-albumin, or MDA-albumin adducts when measured by ELISA. To study the formation of 4-HNE- and MDA-protein adducts during prooxidant-initiated cellular injury, isolated hepatocytes were exposed to either carbon tetrachloride or iron/ascorbate for 2 h. Indices of hepatocellular oxidative stress (i.e., cell viability and glutathione status) and lipid peroxidation (i.e., formation of 4-HNE, protein carbonyls, and MDA) were monitored continuously. Hepatocellular viability was affected moderately by carbon tetrachloride, while cellular reduced glutathione status was moderately affected by both iron/ascorbate and carbon tetrachloride. Levels of MDA and protein carbonyls increased dramatically with both prooxidants, whereas 4-HNE levels did not change significantly over the time course studied. In addition, hepatocellular proteins were immunoprecipitated with each antiserum, and aldehyde-modified immunopositive proteins were detected by immunoblotting. Prooxidant-induced increases in MDA corresponded with increases in intensity and number of MDA-adducted proteins over the time course studied. A total of 13 MDA-modified proteins (20, 25, 28, 30, 33, 38, 41, 45, 80, 82, 85, 130, and 150 kDa) were detected with the MDA-amine antiserum. Additionally, both iron/ascorbate- and carbon tetrachloride-induced formation of DNPH-derivatizable protein carbonyls corresponded quantitatively with the ability to detect specific proteins (80, 100, 130, and 150 kDa) with the DNPH-4-HNE-cysteine antiserum. Neither CCl4 nor iron/ascorbate elicited changes in 4-HNE or induced the formation of 4-HNE-modified proteins when assessed by immunoprecipitation-immunoblot analysis with the 4-HNE-sulfhydryl antiserum. In all instances detection of aldehyde-modified proteins was not associated with cell death and may be related to the function of these proteins as aldehyde-binding proteins which sequester electrophilic molecules during oxidative liver injury.

Co-metabolism of ethanol, ethanol-derived acetaldehyde, and 4-hydroxynonenal in isolated rat hepatocytes.

Our laboratory has previously reported on the ability of 4-hydroxynonenal (4-HNE), a primary product of lipid peroxidation, to inhibit acetaldehyde metabolism in isolated mouse liver mitochondria. The purpose of the present study is to determine whether the co-metabolism of ethanol and 4-HNE compromises the elimination of either substrate in isolated rat hepatocytes. Hepatocytes were isolated and incubated with ethanol and 4-HNE. Ethanol elimination and acetaldehyde accumulation were monitored by gas chromatography, whereas 4-HNE elimination and metabolite accumulation were measured by UV detection and reversed-phase HPLC at 202 nm. In the absence of 4-HNE, hepatocytes metabolized ethanol at an initial rate of 9.4 nmol/min/million cells. Ethanol elimination was moderately inhibited by the presence of 4-HNE. Accumulation of ethanol-derived acetaldehyde was not apparent in incubations with only ethanol. In contrast, in incubations containing both substrates, ethanol-derived acetaldehyde accumulation exceeded that observed in hepatocytes exposed only to ethanol and was proportional to the 4-HNE concentration in the incubations. In all instances, the rate of 4-HNE elimination was not compromised by the presence of ethanol. Accordingly, ethanol metabolism did not alter the oxidative or conjugative metabolism of 4-HNE. However, the reductive metabolism of 4-HNE was affected by the presence of ethanol, wherein accumulation of 1,4-dihydroxy-2-nonene increased > 2-fold of that observed in incubations with only 4-HNE. To determine further if 4-HNE and ethanol are metabolized through the same metabolic pathways, cells were preincubated with either 4-methylpyrazole or cyanamide to inhibit alcohol dehydrogenase (E.C. 1.1.1.1.) and aldehyde dehydrogenase (E.C. 1.2.1.2.), respectively. Expectantly, 4-methylpyrazole blocked the formation of 1,4-dihydroxy-2-nonene, but had no effect on the rate of 4-HNE elimination. In contrast, cyanamide substantially inhibited the formation of 4-hydroxy-2-nonenoic acid, decreased the rate of 1,4-dihydroxy-2-nonene formation, but did not decrease the elimination rate of 4-HNE. Overall, these results support our previous observation that 4-HNE inhibits acetaldehyde metabolism and establish that ethanol and 4-HNE are metabolized through the same alcohol dehydrogenase- and aldehyde dehydrogenase-mediated pathways. These data continue to suggest that, as a consequence of enhanced lipid peroxidation resulting from chronic ethanol consumption, increased 4-HNE levels could compromise cellular elimination of ethanol-derived acetaldehyde and thus function in the potentiation of alcoholic liver fibrosis.

Alcohol mediates increases in hepatic and serum nonheme iron stores in a rat model for alcohol-induced liver injury.

The notion that prolonged ethanol consumption promotes hepatocellular damage through interactions with iron was evaluated in rats fed ethanol with or without supplemental dietary carbonyl iron. The individual and combined pro-oxidant potential of these agents was evaluated in terms of their ability to perturb iron homeostasis and initiate hepatocellular injury. Sprague-Dawely rats received a high fat liquid diet for 8 weeks supplemented with: 35% ethanol-derived calories (Alcohol group), 0.02 to 0.04% (w/v) carbonyl iron (Iron group), ethanol plus carbonyl iron (Alcohol + Iron group), or a diet containing carbohydrate-derived isocaloric calories (Control group). Hepatic and serum nonheme iron stores were significantly elevated (p < 0.05) in all treatment groups, compared with the Controls. Catalytically active low-molecular weight iron was detected in rats consuming alcohol and was markedly elevated (p < 0.05) in rats ingesting iron alone or iron in combination with alcohol. Elevations in serum ALT indicated significant hepatocellular injury in rats ingesting only alcohol, but was most prominent in the rats consuming ethanol in combination with iron (p < 0.05). Significant hepatic fatty infiltration, increased hydroxyproline content, and perturbations in reduced glutathione were also observed in the Alcohol and Iron treatment groups. Histochemical assessment of hepatic iron sequestration revealed that alcohol feeding resulted in deposition of ferric iron in the centrilobular area of the liver lobule. This unique alcohol-mediated iron deposition was histologically graded above Control group and was observed in both hepatocytes and Kupffer cells. Data presented herein suggest that alcohol alone or in combination with iron results in rather specific lobular patterns of hepatic iron deposition relevant to iron overload observed in human alcoholics. Furthermore, data suggest that alcohol- and iron-initiated prefibrotic events occur before extensive hepatocellular necrosis.

Iron-induced lipid peroxidation in rat liver is accompanied by preferential induction of glutathione S-transferase 8-8 isozyme.

Since previous studies from this laboratory have suggested that glutathione S-transferase (GST) 8-8 of rat belongs to a distinct subgroup of GST isozymes which may be involved in the detoxification of the products of lipid peroxidation (Zimniak et al., J. Biol. Chem. 269, 992-1000, 1994), during the present studies we examined the effect of iron-induced lipid peroxidation on the expression of GST 8-8 in rat liver. Rats treated with 100 mg/kg body wt iron showed a significant increase in lipid peroxidation in liver. This was accompanied by a concomitant increase in the expression of GST 8-8 in liver as observed in isoelectrophoretic analysis of rat liver GSTs, and an increase in GST activity toward 4-HNE, a toxic product of lipid peroxidation toward which GST 8-8 displays high specific activity. Western blot studies using polyclonal antibodies specifically recognizing GST 8-8 also indicated that, among the GST isozymes of rat liver, GST 8-8 was preferentially induced upon iron treatment. These findings were further confirmed by purifying and quantitating GST 8-8 protein from the controls and iron-treated rats. Significant differences in the specific activities of GST 8-8 purified from the controls and iron-treated rats were observed, indicating that more than one GST isozyme related to GST 8-8 may be present in rat liver. This observation is consistent with the observed heterogeneity in mouse mGSTA4-4 which is an ortholog of rat GST 8-8. Iron treatment also caused significant increase in GSH levels probably because of de novo synthesis as indicated by an increase in gamma-glutamyl cysteine synthetase activity. The results of these studies suggest that GST 8-8, and possibly other related GST isozymes, may play an important role in defense mechanisms against lipid peroxidation.