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Ethanol-induced fatty liver in rats was attenuated by repeated running exercise, and the protective effect of exercise was associated with the synergistic expression of heat shock proteins (HSP72). Rats were placed in four groups of six. The two ethanol-fed groups of rats received a liquid diet (Lieber-DeCarli formulation) in which 36% of the calories were derived from ethanol. One group remained sedentary (S/E), whereas the other was trained to run on a rodent treadmill at a speed of 27 m/min, 1 hr/day, 5 days/week, for 7 weeks (R/E). Two other groups--one exercised as previously mentioned (R/C) and one sedentary (S/C)--received control-liquid diets in which the ethanol was isocalorically substituted with a dextran/maltose mixture. The degree of fatty infiltration in liver sections stained with hematoxylin and eosin was graded on a 0-4 scale and the data analyzed by ANOVA on ranks. Ethanol significantly induced fatty infiltration in the S/E group, whereas fatty infiltration in the livers of the R/E group was not different from the S/C group. Electrophoresis and Western blotting of liver homogenates demonstrated that HSP72 was not expressed in either the S/C or S/E groups and was only slightly expressed in the R/C group. The combination of exercise and ethanol, however, resulted in an elevated expression of HSP72 in the R/E group. The content of HSP73 was unaffected by any treatment.

Alcohol dramatically reduced loss of heat shock proteins (HSP73) and prevented morphological damage in monolayers of hepatocytes prepared from alcohol-fed rats. The monolayers were treated with 0, 5, 25, or 100 mM alcohol and triplicate samples were assayed at 24, 48, 72, and 96 hr after exposure. The content of HSP73 was measured by PAGE electrophoresis and Western blotting with a mouse monoclonal anti-HSP70 IgG antibody. HSP72 is not expressed under these conditions. Damage to the hepatocytes, quantified by leakage of lactate dehydrogenase (LDH), was also decreased by 100 mM alcohol. Although the initial 100 mM alcohol concentration decreased logarithmically to 1.7 mM over the first 24 hr, the effect of alcohol on HSP73 loss, LDH leakage, and morphological damage was most pronounced at 96 hr.

Previous studies have shown that exposure of phenobarbital-pretreated rats to halothane in 10% O2 causes centrilobular necrosis, induces expression of the 72-kDa heat shock protein (HSP72), and produces several trifluoroacetylated adducts. In the present study the time course of development of the centrilobular lesion, as measured by histochemistry, was compared with the time course of appearance of both trifluoroacetylated adducts and HSP72, as measured by Western blotting. One group of 20 rats was pretreated with phenobarbital for 5 days, whereas a second group of two rats was left as untreated controls. Ten phenobarbital-pretreated rats were exposed for 2 hr to 1% halothane in 10% O2 and 10 were exposed to 1% halothane in 20% O2. At either 2, 4, 6, or 24 hr after exposure, livers were excised and frozen without fixation. Thin sections stained with hematoxylin and eosin demonstrated that centrilobular lesions occurred at 6 hr and became extensive at 24 hr in rats pretreated with phenobarbital and exposed to 1% halothane in 10% O2. The time course of appearance of both trifluoroacetylated adducts and HSP72 was determined by Western blotting. Trifluoroacetylated adducts appeared in all rats exposed to halothane by 2 hr, lasted until 6 hr, and then diminished by 24 hr. In contrast, HSP72 was induced only in the rats pretreated with phenobarbital and exposed to 1% halothane in 10% O2. HSP72 appeared in both the nuclear and supernatant fractions at 6 hr after exposure and was intense 24 hr after exposure.

These experiments were designed to observe specific binding of fluorescein-conjugated FAB'2 secondary antibodies to epitopes on the surface of isolated hepatocytes. The hepatocytes were attached as monolayers on microscope cover slips and an antigenic adduct known to be formed during metabolism of halothane, -trifluoroacetyl-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, was exchanged into their surface. Then the monolayers of hepatocytes were incubated with primary rabbit antibodies specific for the trifluoroacetyl group. Each coverslip was mounted in a perfusion chamber on a fluorescence microscope and a set of digital fluorescence images was made. Then fluorescein-conjugated goat-anti-rabbit FAB'2 secondary antibodies were flowed over the monolayer, the perfusion chamber was washed with buffer, and a second set of digital fluorescence images was made. The difference of these two sets of images demonstrated intense fluorescence superimposed on the outline of the cells. This intense fluorescence was not observed in control experiments in which the primary antibodies were omitted.

Immunocytochemical studies have revealed that one of the major heat shock proteins, HSP72, is induced in livers of rats that have been pretreated with phenobarbital and then administered halothane in a hypoxic gas mixture of 10% oxygen. To determine the sub-cellular localization of HSP72 in the livers of these rats 24 hr after halothane administration, cytoplasmic and nuclear fractions were prepared and separated by PAGE electrophoresis. Western blotting with a mouse monoclonal anti-HSP70 IgG antibody, which recognizes both the constitutive (HSP73) and inducible (HSP72) forms, revealed that HSP72 was induced and translocated into the nucleus in only those rats exposed to halothane under hypoxia following phenobarbital pretreatment. Nuclear translocation of HSP72 under the latter conditions was confirmed by immunocytochemical staining using gold-conjugated secondary antibodies followed by digital laser microscopy with Nomarski optics. Neither phenobarbital pretreatment alone nor phenobarbital plus hypoxia treatment induced HSP72. No alteration in amount of HSP73 was observed under any of these conditions.

We have previously shown that antibodies raised against acetaldehyde adducts of protein cross-react with an acetaldehyde adduct of dioleoylphosphatidylethanolamine, N-ethyl-dioleoylphosphatidylethanolamine, when the latter is incorporated into hexagonal phase phospholipid micelles. In the present study we demonstrate that these same IgG antibodies cross-react with N-ethyl-dioleoylphosphatidylethanolamine when this adduct is incorporated into the surface of hepatocytes. Hapten-specific IgG antibodies were purified from the sera of rabbits sensitized to an albumin-acetaldehyde conjugate that had been reduced with sodium cyanoborohydride (N-ethyl-RSA). The N-ethyl-RSA was coupled to an Affi-Gel-10 column to affinity purify the IgG. Liposomes containing N-ethyl-dioleoylphosphatidylethanolamine were fused with isolated hepatocytes, the affinity purified primary IgG antibodies were added, then fluorescein-conjugated second antibodies were added, and antibody binding to hepatocytes was measured by flow cytometry. The fluorescence of these hepatocytes was significantly greater (p less than 0.01) than control hepatocytes prepared with (1) pre-immune primary IgG antibodies with fluorescein-conjugated second antibodies, (2) no primary antibody but with fluorescein-conjugated second antibodies, and (3) no fluorescein-conjugated second antibodies.

The toxicity of carbon tetrachloride (CCl4) in monolayer cultures of primary hepatocytes was investigated at oxygen concentrations that prevail in the liver under conditions that range from normoxia to hypoxia: 0.5, 1, 2, and 20% O2. CCl4 was administered in the vapor phase at concentrations that produce aqueous concentrations at 37 degrees C of 0.4, 2.0, and 4.0 mM. Damage was assayed by leakage of aspartate transaminase and the inclusion of Trypan Blue immediately after the 2-hr incubation and after an additional 6-hr incubation in 20% O2. Only in the case of 0.5% O2 and 4 mM CCl4 were the monolayers damaged (18%) immediately after the 2-hr exposure; all other exposed cells were undamaged at that time point and the dose response of cell death as a function of CCl4 and oxygen concentration was not evident until the 6-hr time point. The monolayers exposed to 4 mM CCl4 and 1, 2, or 20% O2 exhibited little immediate damage but were all 100% dead 6 hr later. The monolayers exposed to 2 mM CCl4 and 0.5, 1, 2, or 20% O2 were 53, 48, 40, and 22 +/- 2% dead after 6 hr, respectively. These results suggest that effects of CCl4 exposure, for example alterations in the function or synthesis of essential proteins, require several hours to affect cell viability.

Rat hepatocyte homogenates convert 5-hydroperoxyeicosatetraenoic acid (5-HPETE) into biologically active leukotriene B4 (LTB4) as well as less active all-trans-LTB4 (i.e., 6-trans-LTB4 and 6-trans-12-epi-LTB4). Here, we present a hypothesis of the reaction mechanism and the minimal structural requirements of the active enzyme based on the following experimental evidence: The ED50 of the inhibitors 5,8,11,14-eicosatetraynoic acid (ETYA) and 5,6-dehydro-eicosatetraenoic acid was approximately 100-fold higher than for 5-lipoxygenase. Propanethiol and O2 were strong inhibitors of LTB4 formation, whereas butylated hydroxytoluene, nordihydroguaiaretic acid, metyrapone, Desferal and CO had no effect. Cytochrome c, catalase, hematin, and a Fe3+/Fe2+ couple, but not iron-free protoporphyrin IX, catalyzed the formation of only all-trans-LTB4. LTB4 formation in hepatocyte homogenates was heat- and trypsin-sensitive whereas all-trans-LTB4 formation was not. We propose that a ferric heme iron forms a ferryl-hydroxo complex upon homolytic scission of the oxygen-oxygen bond in 5-HPETE and the resulting 5,6-trans-epoxide radical is oxidized by the ferryl-hydroxo complex to yield LTA4. A mechanism for hydrolysis of LTA4 is described that results in formation of LTB4 (less than 1% yield) rather than all-trans-LTB4.

Inasmuch as it is known that the toxicity of anesthetic agents is potentiated by hypoxia and that the reductive metabolism of these agents results in the formation of lipid hydroperoxides, we investigated the toxicity of hydroperoxides under low-oxygen concentrations. We found that hypoxia exacerbates the toxicity of t-butyl hydroperoxide, shifting the dose-response curve of t-butyl hydroperoxide vs. lysis of hepatocytes approximately an order of magnitude to the left. Furthermore, although at the end of a 4-h exposure to 0.5% O2 hepatocyte monolayers seemed normal by three indices (release of 51Cr and serum glutamate transaminase or exclusion of trypan blue), they were completely lysed after an additional 20 h reoxygenation at 20% O2. In contrast, monolayers exposed to 2% O2 for 4 h seemed normal after 20 h reoxygenation. However, cells exposed to both a subtoxic dose of hydroperoxide and 4 h of 2% O2, although seeming healthy at the end of the hypoxic period, were completely lysed within 20 h after reoxygenation.

Binding of halothane metabolites to rat liver histones was investigated after in vivo administration of 14C-halothane. Animals were injected with either a mixture of triiodothyronine, glucagon and heparin (TGH) to stimulate liver growth or with saline as a control. Twenty-four hours later, animals were administered 14C-halothane and maintained at 8--10 per cent O2 for 6 hours. Detergent washed nuclei from liver homogenates were subfractionated to allow quantitative measurements of 14C-halothane binding to histones. Although our studies suggest that much of the previously reported binding of halothane metabolites to major cell fractions was a result of redistribution of endoplasmic reticulum components during isolation procedures, carefully controlled experiments demonstrated that the radioactivity associated with histones could not be due to residual microsomal lipid. Of the initial 132 mumol of 14C-halothane administered, 1.1 mumol remained as nonvolatile metabolites in the liver homogenate and 25 pmol were associated with purified histones. This corresponds to approximately one halothane moiety per 15,000 histone molecules. No significant binding to liver cell RNA or DNA was observed. With this low level of histone modification and lack of convincing evidence of halothane metabolite binding to hepatic DNA or RNA, it is unlikely that significant alteration of the genome occurs after exposure to halothane.

Leukotriene B4 was found to be metabolized by rat hepatocyte monolayers at a rate that was linear with increasing substrate concentration from 74 to 740 nM leukotriene B4. The rates of metabolism were dependent on the O2 concentration and were 315, 213, 80, and 36 pmol leukotriene B4 per min per nmol cytochrome P-450 at 20% (212 microM), 4% (42.5 microM), 2% (21.2 microM), and 1% (10.6 microM) O2, respectively. The metabolic rate was not linear with respect to O2 concentration; however, half maximal rate occurred at 4% O2, and O2 concentration found in the pericentral region of normally oxygenated liver. These results suggest that in vivo conditions of hypoxia or ischemia that lead to blood O2 concentrations less than 4% may drastically decrease hepatic clearance of leukotriene B4.

Potentiation of chemical toxicity by hypoxia was studied in confluent hepatocyte monolayers. Addition of either hydroperoxyarachidonic acid (50 micrograms), leukotriene C4 (10 micrograms), or calcium ionophore A23187 (1.8 micrograms) to hepatocyte monolayers followed by incubation in 2% oxygen for 24 h killed 95% of the hypoxic cells, but was without effect on the normoxic cells. The greater than 10-fold increase in toxicity of A23187 suggests that hypoxic cells are less able to regulate intracellular calcium. The increased toxicity of hydroperoxyarachidonic acid and leukotriene C4 may be due to a related reduction in activity of protective enzymes.

SR 4233 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is presently undergoing investigation as an antitumor agent because of its high selective toxicity for hypoxic cells in vitro and in vivo. It has been found to be 15 to 200 times more toxic to hypoxic rodent and human cell lines than their normoxic counterparts. We investigated the toxicity of SR 4233 in primary cultures of hepatocytes under various oxygen tensions, ranging from 1% to 20% oxygen. The 50% lethal dose of SR 4233 was found to be 50 times lower in hepatocyte monolayers at 1% O2 versus 20% O2. Even at 4% O2, a concentration that prevails in the pericentral area of the liver under conditions of normal blood flow, SR 4233 was an order of magnitude more toxic than at 20% O2. All samples were analyzed for metabolites, and metabolism was found to be dependent on both the SR 4233 concentration and the oxygen tension. Formation of the major metabolite SR 4317 occurred to the greatest extent at the lowest oxygen concentration and the highest SR 4233 concentration. Very little metabolism occurred at 10 to 20% O2, which is in agreement with data in Chinese hamster ovary cells under aerobic conditions.

Hypoxia, phenobarbital induction, and halothane anesthesia have been implicated in the pathogenesis of hepatotoxicity in the rat model. However, a controversy exists over the role of halothane in liver injury; does it act by reducing hepatic blood flow, thereby inducing hypoxia, or do its metabolites initiate the injury? These variables are difficult to separate during in vivo halothane exposure. In the present experiments, effects of halothane on hepatic perfusion were eliminated by exposing confluent monolayers of hepatocytes isolated from Fisher 344 rats livers, both with and without phenobarbital pretreatment, to 1.5% halothane or 2.0% isoflurane in 1%, 2%, or 4% (control) oxygen. Isoflurane exposure was included for a control of anesthetic effects on hepatocytes, because it is known to be metabolized minimally and probably is not associated with hepatic dysfunction. Oxygen levels were chosen to approximate those that may occur in the liver in vivo. Cell death was assayed via aspartate aminotransferase (AST) release, both immediately following a 2-h oxygen +/- anesthetic exposure and 6 h post-exposure. Per cent cell death data were analyzed using multiple regression techniques. Results obtained immediately, and 6 h after, exposure demonstrate that low oxygen levels, halothane, and phenobarbital were each highly significant factors (P less than .001) in relation to cell death, in agreement with the halothane-phenobarbital-hypoxia rat model. A toxic effect of isoflurane was not observed under identical experimental conditions. The results of the study clearly indicate that the origin of cell death in hepatocyte monolayers is multi-factorial; hypoxia, phenobarbital induction, and halothane exposure each contribute to the hepatocyte damage observed in our in vitro model.

Increased cytoplasmic calcium has been implicated in hepatic necrosis induced by cytochrome P-450-mediated halocarbon metabolism; hepatic necrosis is exacerbated hypoxia. It is known that addition of the calcium ionophore A23187 to a cell can mimic the metabolic causes of increased cytoplasmic calcium. In the present experiments, confluent monolayers of rat hepatocytes were exposed to A23187 (0.5-50 microM) under conditions of normoxia and 18-fold during a 4-h incubation at 0.5% (5 microM) O2 compared with 20% (212 microM) O2. The ED50 values of A23187 were 0.92, 1.84, 10.98, and 16.81 microM at 0.5, 1, 2, and 20% O2, respectively. The results demonstrate that small reductions in the oxygen concentrations normally encountered by pericentral hepatocytes (i.e., below 28 microM) may severely decrease the ability of these already compromised hepatocytes to manage perturbations in calcium homeostasis.

Rat hepatocyte homogenates converted 5-hydroperoxyeicosatetraenoic acid into leukotriene B4 (LTB4). The reaction was dependent on time and protein and substrate concentration, did not require NADPH or oxygen, and was not supported by heat-inactivated hepatocyte homogenates. The authenticity of the biologically generated LTB4 that eluted at the position of synthetic LTB4 during high performance liquid chromatography was established by UV spectrophotometry, mass spectral analysis, radioimmunoassay, and a LTB4 receptor displacement assay. In addition, a leukotriene bioassay is described in which transient increases in cytosolic Ca2+ within human neutrophils are measured by means of fura-2 fluorescence. Biologically generated LTB4 was 40, 40, and 33% as active as synthetic LTB4 in the radioimmunoassay, receptor displacement assay, and cytosolic calcium bioassay, respectively. This activity is consistent with the biologically derived LTB4 being an epimeric mixture of (5S),(12R)-LTB4 and the much less active (5S),(12S)-LTB4. The formation of LTB4 was inhibited by 5,8,11,14-eicosatetraynoic acid (1 mM), 5,6-dehydro-arachidonic acid (50 microM), propanethiol (1 mM), and O2 (100%) to the extent of 53, 42, 48, and 66%, respectively. No inhibition was observed in the presence of diethylcarbamazine (1 mM) and desferal (1 mM). A possible contribution towards LTB4 formation by contaminating Kupffer cells was excluded (less than 0.2%). These results suggest that hepatocytes can convert lipid peroxides into potent chemoattractants that may alter the homeostasis of immunomediators within the liver.

Hepatic 1,2-dibromoethane (DBE) metabolism proceeds via two pathways: oxidation by cytochrome P-450 and direct conjugation with the ubiquitous tripeptide glutathione (GSH) via the GSH S-transferases. The toxicity of DBE in monolayers of hepatocytes was assessed to establish whether the toxicity of this compound is increased under conditions of reductive metabolism at low oxygen concentrations. Our previous studies with t-butyl hydroperoxide and the calcium ionophore A23187 suggested that hypoxia would exacerbate toxicity that was mediated through lipid peroxidation or loss of calcium homeostasis. Monolayers of hepatocytes were exposed for 2 hr to 0, 14, 140, 1400, or 14,000 ppm of DBE in an atmosphere of either 1, 2, or 20% oxygen. Toxicity was measured by leakage of aspartate aminotransferase (AST) and trypan blue exclusion. The time course of the development of cytotoxicity was examined by assaying cell death both immediately following a 2-hr exposure and 24 hr later. The LC50 of DBE vapor was found to be approximately 14,000 ppm when assayed immediately after exposure but only 140 ppm when assayed 24 hr after exposure. The similarity of the percentages of DBE-induced cell death after incubations at 1, 2, and 20% oxygen demonstrates that the toxicity of DBE is oxygen-independent. We conclude that while DBE is highly toxic to rat hepatocytes, hypoxia does not appear to contribute to the toxicity of DBE, even under conditions of low oxygen concentrations. This result is in direct contrast to a previous report where we showed that the toxicity of halothane is potentiated under hypoxic conditions.