Rat Hepatocyte Culture System Research Articles

Elevated sensitivity of macrosteatotic hepatocytes to hypoxia/reoxygenation stress is reversed by a novel defatting protocol.

Macrosteatotic livers exhibit elevated intrahepatic triglyceride (TG) levels in the form of large lipid droplets (LDs), reduced adenosine triphosphate (ATP) levels, and elevated reactive oxygen species (ROS) levels, and this contributes to their elevated sensitivity to ischemia/reperfusion injury during transplantation. Reducing macrosteatosis in living donors through dieting has been shown to improve transplant outcomes. Accomplishing the same feat for deceased donor grafts would require ex vivo exposure to potent defatting agents. Here we used a rat hepatocyte culture system exhibiting a macrosteatotic LD morphology, elevated TG levels, and an elevated sensitivity to hypoxia/reoxygenation (H/R) to test for such agents and ameliorate H/R sensitivity. Macrosteatotic hepatocyte preconditioning for 48 hours with a defatting cocktail that was previously developed to promote TG catabolism reduced the number of macrosteatotic LDs and intracellular TG levels by 82% and 27%, respectively, but it did not ameliorate sensitivity to H/R. Supplementation of this cocktail with l-carnitine, together with hyperoxic exposure, yielded a similar reduction in the number of macrosteatotic LDs and a 57% reduction in intrahepatic TG storage, likely by increasing the supply of acetyl coenzyme A to mitochondria, as indicated by a 70% increase in ketone body secretion. Furthermore, this treatment reduced ROS levels by 32%, increased ATP levels by 27% (to levels near those of lean controls), and completely abolished H/R sensitivity as indicated by approximately 85% viability after H/R and the reduction of cytosolic lactate dehydrogenase release to levels seen in lean controls. Cultures maintained for 48 hours after H/R were approximately 83% viable and exhibited superior urea secretion and bile canalicular transport in comparison with untreated macrosteatotic cultures. In conclusion, these findings show that the elevated sensitivity of macrosteatotic hepatocytes to H/R can be overcome by defatting agents, and they suggest a possible route for the recovery of discarded macrosteatotic grafts.

Anticancer drug cis-4-hydroxy-L-proline: Correlation of preclinical toxicology with clinical parameters of liver function

cis-4-Hydroxy-L-proline (CHP) is being clinically evaluated as an anticancer drug. Since this compound targets the production of L-proline-rich proteins and critical L-proline residues, its impact on long-term cultures of human hepatocytes and toxicity in rats was studied to investigate possible effects on hepatic function, previously reported in rat hepatocytes. In the HEPAC2 human hepatocyte culture system, concentrations of CHP below 3.2 mg/ml had no significant effects on the release of lactate dehydrogenase (LDH), albumin, and urea. In rats, continuous administration of three different doses of CHP were tested for 28 days and resulted in signs of liver damage, as indicated by elevations of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) at a dose of 903 mg/kg, corresponding to a plasma concentration of approximately 200 µg/ml. Data from a clinical study of CHP in bladder and prostate cancer patients showed no adverse effects of administration of 8 g CHP/day, 4 days/week for 3 weeks in liver parameters ALAT, ASAT, γ-glutamyltransferase (γ-GT) and alkaline phosphatase (AP). In conclusion, the HEPAC2 human hepatocyte culture system correlates well with clinical results of a Phase II study of CHP, whereas a previous rat hepatocyte culture system predicted the compound would have toxic effects. The HEPAC2 system therefore constitutes a valuable tool for the preclinical screening of the hepatotoxicity of chemotherapeutic and other drugs, thereby reducing the need for experimental animals.

BENEFICIAL EFFECT OF CARBON MONOXIDE-INHALATION AGAINST ISCHEMIA/REPERFUSION INJURY AFTER RAT LIVER TRANSPLANTATION: POSSIBLE MECHANISM OF iNOS/NO PATHWAY BLOCKADE

P84 Background: Heme oxygenase (HO)-1 is a pleiotropic molecule that has been shown to provide potent cytoprotection in numerous models of cellular stress. Carbon monoxide (CO), product of heme degradation by HO-1, has been shown to induce similar cytoprotection as HO-1 against ischemia/reperfusion (I/R) injury. On the contrary, roles of inducible nitric oxide synthase (iNOS), another stress inducible gene product, during I/R injury remain controversial. In this study, we examined whether CO would protect the liver graft against cold I/R injury associating with transplantation. Particular attention was paid to the influence of CO treatment on hepatic iNOS/NO pathway. Methods: Orthotopic syngeneic LEW rat liver transplantation (OLT) was performed with 18 hrs preservation in cold UW solution. Recipients were exposed to air or two different doses of CO (20 or 100 ppm) for 1 hr before and 24 hrs after OLT. Recipients were sacrificed 3-48 hrs for serum AST and NO levels, histopathology, and hepatic iNOS protein expression. Efficacy of CO was further analyzed in vitro primary rat hepatocyte culture system after stimulation with cytokine mixture (CM) (TNF-α 500U/ml, IL-1 200U/ml, IF-γ 100U/ml). iNOS protein, NO production and hepatocyte viability after CM stimulation were analyzed. Results: After OLT, CO-inhalation at 100 ppm effectively improved hepatic I/R injury. Serum AST levels and tissue necrosis were significantly reduced with 100 ppm CO, but not with 20 ppm, compared to air-treated controls. Hepatic iNOS protein expression was significantly decreased in CO-treated group as compared with air-treated controls at 3 and 48 hrs, associating with lower serum NO levels (Table, mean ± SE, n=3-6 animals per group, *P < 0.05). When rat hepatocytes were cultured in the presence of CO 100 ppm, CM-induced iNOS protein expression was markedly inhibited with significantly reduced NO levels at 24 hrs (20.3 ± 0.6 vs. 2.8 ± 1.8 μM in CO-group, p < 0.05). Hepatocyte viability also was increased with CO treatment at 24 hrs after CM stimulation (56.9 ± 0.02 vs. 89.9 ± 0.03 % in CO-group, p < 0.02). Conclusion: The results demonstrate that exogenous CO treatment efficiently ameliorates hepatic I/R injury. The possible mechanism, by which CO protects the liver against cold I/R, may include the down-regulation of iNOS/NO pathway. Thus, CO-inhalation treatment would be a novel therapeutic strategy to combat hepatic cold I/R injury.Figure

Repression of phenobarbital-dependent CYP2B1 mRNA induction by reactive oxygen species in primary rat hepatocyte cultures.

Xenobiotic-metabolizing cytochrome P-450 (P-450) enzymes not only play a pivotal role in elimination of foreign compounds but also contribute to generation of toxic intermediates, including reactive oxygen species, that may elicit cellular damage if produced excessively. Expression of several xenobiotic-metabolizing P-450 enzymes is induced by phenobarbital (PB). Pronounced induction is observed for the rat CYP2B1 isoform. A primary rat hepatocyte culture system was used to investigate whether reactive oxygen species might modulate PB-dependent CYP2B1 induction. In cells cultivated for 3 days with 1.5 mM PB, substantial CYP2B1 mRNA induction was observed (100%). Addition of H(2)O(2) or of the catalase inhibitor 3-amino-1,2,4-triazole (AT) to the medium repressed induction to approximately 30% (at 1 mM H(2)O(2) and 2 mM AT, respectively). Accordingly, treatment of hepatocytes with PB and the glutathione precursor N-acetylcysteine (NAC) led to enhanced PB-dependent induction (to over 1000% at 10 mM NAC). In primary hepatocyte cultures transfected with a CYP2B1 promoter-luciferase construct containing approximately 2.7 kilobase pairs of the native CYP2B1 promoter sequence, PB-dependent reporter gene activation was repressed by AT and stimulated by N-acetylcysteine. Furthermore, a 263-base pair CYP2B1 promoter fragment encompassing the phenobarbital-responsive enhancer module conferred suppression of PB-dependent luciferase expression by AT and activation by NAC in a heterologous SV40-promoter construct. In summary, these data demonstrate a regulatory mechanism that is dependent on the cellular redox status, which modulates CYP2B1 mRNA induction by PB on the transcriptional level, thus representing a feedback mechanism preventing further P-450-dependent production of reactive oxygen intermediates under oxidative stress.

Hepatotoxicity of Camptothecin Derivatives in a Primary Culture System of Rat Hepatocytes

The topoisomerase I inhibitors are a new class of antineoplastic agents currently under clinical development. Among these compounds there are some camptothecin (CPT) derivatives with improved toxicity profiles and antitumor activity: irinotecan (CPT-11) and topotecan (TPT), particularly active against colon, lung and ovarian cancer.The aim of this study was to evaluate the cytotoxicity of CPT, CPT-11, its metabolite SN38 and TPT in a primary culture system of rat hepatocytes. Cytotoxicity was evaluated by measuring the leakage of lactate dehydrogenase (LDH) into the medium and by assessing cell viability in terms of tetrazolium salts (MTT) reduction by mitochondrial dehydrogenase activity. Our results showed that cytotoxicity was limited in the case of short drug exposure. There was a significant and time-dependent increase in LDH leakage and a significant time- and dose-dependent decrease in MTT reduction after 3h of incubation (p<0.01). In the treatments with doses related to peak plasma levels, CPT-11 was less responsible for the observed in vitro hepatotoxicity than its metabolite SN38; TPT had lower LDH leakage compared to SN38 and CPT-11 but showed significant and early (3h) decrease in MTT reduction: this may mean a different mechanism of cellular damage. These results demonstrate that CPT derivatives are directly toxic to liver cells in a distinct time- and dose-related response.

Differential induction of cytochrome P450 gene expression by 4n-alkyl-methylenedioxybenzenes in primary rat hepatocyte cultures.

A well-characterized primary rat hepatocyte culture system was used to examine induction patterns of cytochrome 450 gene expression by a series of 4-n-alkyl-methylenedioxybenzene (MDBs) derivatives. Hepatocytes were treated for 24, 48, or 72 hours with 0-500 microM of the MDB compounds, and total cellular RNA and protein from each treatment was evaluated by hybridization and immunochemical techniques. Exposure to MDB congeners possessing increasing 4-n-alkyl side-chain length (C0-C8) resulted in dose- and structure-dependent activation of CYP2B1, 2B2, 3A1, 1A1, and 1A2 gene expression. At equivalent 100 microM concentrations, the C6 and C8 MDB congeners were more effective than the prototypical inducer phenobarbital (PB) with respect to induction potency of CYP2B1, CYP2B2, and CYP3A1 gene expression. In contrast to PB, longer side-chain-substituted MDBs effectively induced CYP1A1 and CYP1A2 gene expression, in addition to the CYP2B and CYP3A genes. At equivalent molar concentrations, the catechol derivative of C6-MDB was ineffective in its ability to induce CYP gene expression, indicating the importance of the intact methylenedioxy bridge in the induction mechanism. Levels of MDB-inducible CYP2B1 and CYP2B2 mRNA were highly correlated with CYP2B1/2 apoprotein levels, ascertained by immunoblot analysis of cultured hepatocyte S9 fractions. Compared with results from previous in vivo analysis (12), the current data indicate that pharmacodynamic factors may influence MDB induction profiles and that differences in MDB effects on CYP gene expression result depending on distinct structure-activity relationships.

N-Deacetyl ketoconazole-induced hepatotoxicity in a primary culture system of rat hepatocytes

Ketoconazole (KT) is an azole antifungal agent that has been associated with hepatotoxicity. The mechanism of its hepatotoxicity has not yet been resolved. It has been suggested that a reactive metabolite may be the cause of toxicity because the hepatic injury does not appear to be mediated through an immunoallergic mechanism. Several metabolites of KT have been reported in the literature of which the deacetylated metabolite, N-deacetyl ketoconazole (DAK), is the major metabolite which undergoes further metabolism by the flavin-containing monooxygenases (FMO) to form a potentially toxic dialdehyde. The objective of this study was to evaluate DAK's cytotoxicity and the role of FMO in a primary culture system of rat hepatocytes. Cytotoxicity was evaluated by measuring the leakage of the cytosolic enzyme, lactate dehydrogenase (LDH), into the medium and by assessing mitochondrial reduction of 3-(4,5-dimethythiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT). The cultures were exposed to various concentrations of DAK (20–160 μM) for 0.5–4 h. There was a significant increase ( P < 0.05) in LDH leakage and an immediate decrease in MTT reduction ( P < 0.05) as early as 0.5 h. The MTT reduction assay appeared to be more sensitive than the LDH assay in that lower concentrations were needed to observe a 50% reduction of MTT (107, 90, 75, 58 μM DAK at 0.5, 1.0, 2.0 and 4.0 h, respectively). The concentrations to observe 50% LDH leakage from the hepatocytes were 155, 133, 100, 70 μM DAK at 0.5, 1.0, 2.0 and 4.0 h, respectively. Moreover, co-treatment with methimazole, a competitive substrate for FMO, produced a significant decrease ( P < 0.05) in % LDH leakage as early as 0.5 h, when compared to cells treated solely with DAK. Also, the toxicity was significantly ( P < 0.05) enhanced as early as 0.5 h by n-octylamine, a known positive effector for FMO. These results demonstrate that DAK is a more potent cytotoxicant than its parent compound, KT, as reported previously by our laboratory (Rodriguez and Acosta, Toxicology, 96: 83–92, 1995) and its toxicity was expressed in a dose- and time-dependent manner. Furthermore, DAK's cytotoxicity was enhanced with n-octylamine and suppressed with methimazole, suggesting a role for FMO in the toxicity of the metabolite.

Regulation of cholesterol 7 alpha-hydroxylase expression by sterols in primary rat hepatocyte cultures.

The importance of cholesterol and "oxysterols" in the regulation of cholesterol 7 alpha-hydroxylase is not clear. Previous in vivo studies suggest that cholesterol may up-regulate cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis, but these studies are open to question as they were carried out in whole animals. Therefore, we used primary rat hepatocytes, cultured in serum-free medium, to determine the effects of cholesterol on the regulation of cholesterol 7 alpha-hydroxylase. Squalestatin, a specific squalene synthase inhibitor, was used to block sterol but not isoprenoid biosynthesis in this system. Squalestatin (1 microM) decreased cholesterol 7 alpha-hydroxylase specific activity to undetectable levels and decreased steady-state mRNA and transcriptional activity to 13% and 47% of controls, respectively. Mevalonolactone (2 mM) failed to restore cholesterol 7 alpha-hydroxylase specific activity or steady-state mRNA levels in squalestatin-treated cells. Addition of cholesterol, delivered in beta-cyclodextrin, to squalestatin-treated cells restored cholesterol 7 alpha-hydroxylase specific activity and steady-state mRNA to control levels in a concentration (25 microM to 200 microM) -dependent manner. In contrast, the individual addition of selected "oxysterols" (5-cholesten-3 beta, 7 alpha-diol; 5 alpha-cholestan-3 beta, 6 alpha-diol; cholestan-3 beta, 5 alpha,6 beta-triol; 5-(25R)-cholesten-3 beta,26-diol, all at 50 microM) failed to restore cholesterol 7 alpha-hydroxylase mRNA levels in squalestatin-treated cells. These experiments provide evidence that cholesterol rather than "oxysterols" regulate cholesterol 7 alpha-hydroxylase gene expression. Squalestatin (1 microM) treatment increased HMG-CoA reductase specific activity by 229% of controls. Addition of cholesterol (200 microM), but not mevalonolactone (2 mM), to squalestatin-treated cells decreased HMG-CoA reductase specific activity to 19% of control. The primary rat hepatocyte culture system in conjunction with a specific squalene synthetase inhibitor should be a useful model for elucidating the mechanism of regulation of cholesterol 7 alpha-hydroxylase gene expression by sterols.

Comparison of ketoconazole- and fluconazole-induced hepatotoxicity in a primary culture system of rat hepatocytes

Ketoconazole (KT) and fluconazole (FLU) are azole antifungal agents with a broad spectrum of activity against both superficial and systemic mycoses. KT is also an anticancer agent in the treatment of advanced prostate cancer. In many clinical and retrospective studies, KT has been reported to cause liver damage, i.e. chemical hepatitis. Histologie analysis of KT induced hepatotoxicity shows massive centrilobular necrosis in which the hepatotoxicity was not thought to be mediated through an immunoallergic mechanism. According to the medical literature, the pattern of hepatic injury appears to be primarily of the hepatocellular type. Because of the documented reports of KT and FLU hepatotoxicity, a cytotoxicity comparison of KT and FLU was implemented. The objective of this comparison was to evaluate the cytotoxicity of these azoles such that future mechanistic investigations of hepatotoxicity could be performed. The relative hepatotoxicity of KT and FLU was evaluated using primary cultures of postnatal rat hepatocytes. Cytotoxicity was evaluated by measuring the leakage of the cytosolic enzyme, lactate dehydrogenase (LDH), into the medium; by assessing mitochondrial reduction of 3-(4,5-dimethythiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT); by assessing lysosomal uptake of neutral red (NR); and by gross morphology (phase contrast microscopy). The cultures were exposed to various concentrations of KT (56–188 μM) for 0.5–4 h and to various concentrations of FLU (50 μM to 1.0 mM) for 0.5–6 h. There was a significant increase ( P < 0.05) in LDH leakage and a large decrease in MTT reduction and lysosomal uptake of NR at 4 h for KT. One millimolar FLU had minimal effects on the LDH leakage and MTT reduction. These results demonstrate that KT is a more potent cytotoxicant than FLU; and its toxicity was expressed in a dose- and time-dependent manner.

INTERACTION OF PROPRANOLOL WITH CYTOCHROME P450 -METABOLIC ACTIVATION, ENZYME INHIBITION AND INDUCTION-

We examined the mechanisims of inhibition andelelvation of CYP enzyme activities in liver microsomes (Ms) from propranolol (PL)-pretreated rats. In the studies on the inhibition, we measured radioactivities derived from side chain-[14C]-labeled and aromatic ring-[14C]-labeled 4-hydroxypropranolols (4-OH-PL) after incubation with Ms and NADPH. Radioactivities were significantly higher in aromatic ring-labeled 4-OH-PL than in side chain-labeled one. A strain difference was observed in radioactivities derived from the aromatic ring-labeled 4-OH-PL(Wistar>DA), but not in those from the side chain-labeled one. We speculated that CYP2D enzymes converted PL to 4-OH-PL, which was then converted to 1, 4-naphthoquinone (1, 4-NO), causing the enzyme inhibition by covalently binding to apoprotein. However, 1, 4-NQ or 1, 4-dihydroxynaphthalene, when added to the incubation medium containing Ms and NADPH, did not inhibit CYP2D activities. The results suggest that some other metabolite(s) together with 1, 4-NQ could contribute to the enzyme inhibition. In the studies on the elevation of enzyme activities, we observed that elevated PL N-desisopropylase activity highly correlated with O-deethylase activities of phenacetin (PA) and ethoxyresorufin (ER) in liver microsomes from PL-pretreated rats. After repeated administrations of PLrecemate and enantiomers to rats, the potencies in elevation of CYP1A1 protein and activities (ER oxidation and PL N-desalkylation) were RS>S>R, whereas those in elevation of CYPIA2 protein and activities (PA oxidation and PLN-desalkylation) were RS>R>S. In the primary culture system of rat hepatocytes, potencies in elevation of CYP1A1 protein and the activities were R>RS>S, and no effect was observed from CYP1A2. These results indicate that elevation of PLN-desisopropylase activities could be caused by induction of CYPIA enzyme by repeated administration of PL.

Dehydroepiandrosterone 3β-sulphate is an endogenous activator of the peroxisome-proliferation pathway: induction of cytochrome P-450 4A and acyl-CoA oxidase mRNAs in primary rat hepatocyte culture and inhibitory effects of Ca2+-channel blockers

The role of steroids related to the adrenal androgen dehydroepiandrosterone (5-androstene-3 beta-ol-17-one; DHEA) in regulating the expression of peroxisomal and cytochrome P-450 4A (CYP4A) enzymes active in fatty acid metabolism was assessed using a primary rat hepatocyte culture system. Exposure of hepatocytes to the peroxisome proliferator, clofibric acid (10-250 microM), for 48-96 h led to substantial increases in CYP4A protein, CYP4A1, CYP4A2 and CYP4A3 mRNAs, and the mRNAs encoding both forms of peroxisomal acyl-CoA oxidase (ACOX-I and ACOX-II), as judged by Northern-blot analysis using gene-specific oligonucleotide probes. Although DHEA treatment in vivo is effective in inducing these mRNAs in rat liver, it had no effect in the cultured hepatocytes. In contrast, treatment of the cells with DHEA 3 beta-sulphate (DHEA-S; 10-250 microM) stimulated major increases in CYP4A and ACOX mRNA levels. Examination of several analogues indicated a preference for 3 beta-sulphate over 17 beta-sulphated steroids and the inactivity of a 3 alpha-hydroxy-17 beta-sulphate derivative (DHEA-S > 5-androstene-3 beta,17 beta-diol 3-sulphate approximately 5 alpha-androstene-3 beta-ol-17-one 3-sulphate > 5-androstene-3 beta, 17 beta,17 beta-diol 17-sulphate approximately 5 beta-androstane-3 alpha-ol-17-one 3-sulphate >> 5 alpha-androstane-3 alpha, 17 beta-diol 17-sulphate). Induction of CYP4A mRNAs by either DHEA-S or clofibric acid was partially blocked by structurally diverse Ca(2+)-channel antagonists (nicardipine, nifedipine and diltiazem; 50 microM), suggesting that both the steroidal and fibrate classes of CYP4A inducers stimulate peroxisomal-proliferative responses via a Ca(2+)-dependent pathway. Retinoic acid alone slightly induced CYP4A mRNAs but did not enhance the induction by clofibrate or DHEA-S. As DHEA-S corresponds to a physiologically important major circulating androgen, these findings suggest that it may serve as an endogenous regulator of hepatic peroxisome enzyme levels. They further suggest that Ca(2+)-channel blockers may be useful pharmacological tools for the further study of the underlying cellular mechanism whereby endogenous steroids and fibrate drugs induce peroxisome proliferation, and the relationship of these events to activation of the peroxisome proliferator-activated receptor.

Differential modulation of complement factor H and C3 expression by TNF-α in the rat. In vitro and in vivo studies

The biosynthesis of alternative regulatory complement protein factor H was investigated using both an in vivo rat model and an in vitro rat hepatocyte culture system, and compared to that of C3 component. Subcutaneous injection of a single dose of 20 μg of recombinant murine tumor necrosis factor-α (rmTNF-α) had no effect on factor H liver mRNA levels, while it increased C3 mRNA levels. In correlation with this, serum factor H levels remained unchanged after rmTNF-α injection, whereas C3 levels were increased. In contrast in vitro studies showed that rmTNF-a had no effect on factor H and C3 expression by rat hepatocytes. Recombinant human interleukin-1α (rhIL-Iα) did not alter the expression of factor H, whereas it increased C3 expression, and recombinant human interleukin-6 (rhIL-6) stimulated expression of both proteins. This study shows that TNF-α is not directly responsible for the increased levels of factor H observed in vivo during induced inflammation in the rat. Its in vivo effect on C3 secretion might be secondary to the TNF-α-induced release of IL-l and/or IL-6.

Hepatocyte differentiation in vitro: initiation of tyrosine aminotransferase expression in cultured fetal rat hepatocytes.

A fetal rat hepatocyte culture system has been used to study the molecular mechanisms of tyrosine aminotransferase (TAT) gene expression during development. It has previously been shown that TAT activity can be detected in 19-d, but not 15-d, gestation hepatocytes on the first day of culture (Yeoh, G. C. T., F. A. Bennett, and I. T. Oliver. 1979. Biochem. J. 180:153-160). In this study enzyme activity, synthesis, and mRNA levels were determined in hepatocytes isolated from 13-, 15-, and 19-d gestation rats maintained in culture for 1, 2, or 3 d and exposed to dexamethasone. TAT expression is barely detectable in 13-d gestation hepatocytes even after 3 d in culture. Hepatocytes isolated from 15-d gestation fetuses have undetectable levels of enzyme activity and synthesis on the first day of culture; both can be assayed by days 2 and 3. TAT mRNA levels in these hepatocytes, measured by hybridization with a specific cDNA, increase substantially during culture. TAT activity, synthesis, and mRNA are evident on the first and subsequent days of culture in 19-d gestation hepatocytes. Transcription measurements in isolated nuclei indicate that the increase in TAT mRNA in 15- and 19-d gestation hepatocytes is associated with an increase in transcription of the gene. Immunocytochemical studies demonstrated that the increase in TAT expression correlated with an increase in the proportion of hepatocytes expressing the enzyme, rather than a simultaneous increase in all hepatocytes. These results support the proposal that a subpopulation of 15-d fetal hepatocytes undergo differentiation in culture with respect to TAT.

Ethanol-induced inhibition of liver cell function: I. Effect of ethanol on hormone stimulated hepatocyte DNA synthesis and the role of ethanol metabolism.

We have studied a short term effect (96 hr) of ethanol on hormone-stimulated DNA synthesis in a primary rat hepatocyte culture system. Studies were also performed with respect to total RNA and protein synthesis as well as albumin secretion measured by a solid-phase radioimmunoassay. We found that ethanol, when added to cultured hepatocytes, resulted in a substantial reduction in hormone-stimulated hepatocyte DNA synthesis and this effect was concentration dependent and occurred in serum-free medium. Ethanol also had an inhibitory effect on total RNA synthesis but protein synthesis and albumin secretion remained essentially unchanged. We determined that hepatocytes exposed to ethanol during the first 24 hr of culture were the most susceptible to inhibition of DNA synthesis. During the first 24 hr, alcohol dehydrogenase (ADH) activity was present in the cells at higher levels than at 48 and 72 hr. In human hepatoma cell lines and differentiated primary and secondary chick fibroblasts, no ADH activity was demonstrable; such cells were not inhibited by 100 mM ethanol additions and DNA synthesis rates were similar to untreated cultures. Other alcohols found to be metabolized by hepatocyte ADH were inhibitory towards hormone-stimulated DNA synthesis whereas those with less metabolism had little effect. Hepatocytes treated with 4-methylpyrazole, an inhibitor of ADH, were partially protected from ethanol effects. Taken together our results are consistent with the hypothesis that a major physiological effect of ethanol on the hepatocyte is a direct impairment of DNA synthesis and that alcohol metabolism is required.

Role of glutathione depletion in the cytotoxicity of acetaminophen in a primary culture system of rat hepatocytes

A primary culture system of postnatal rat hepatocytes was utilized to study the cytotoxicity of acetaminophen and the toxicological significance of glutathione (GSH) depletion. The relative time of onset and magnitude of GSH depletion, lipid peroxidation and cytotoxicity were contrasted in order to gain insight into their interrelationships. Exposure of the hepatocytes to acetaminophen resulted in time- and dose-dependent depletion of cellular GSH. The acetaminophen-induced GSH depletion and ensuing lactate dehydrogenase (LDH) leakage were quite modest and delayed in onset, in contrast to that caused by iodoacetamine (IAA) and by diethylmaleate (DEM), 2 well-known depletors of GSH. There was comparable LDH leakage, irrespective of drug treatment, when GSH levels decreased to about 20% of normal. Reduction of GSH levels below the 20% threshold by IAA treatment reseulted in marked LDH leakage and loss of viability. Maximal LDH leakage in response to IAA and acetaminophen preceded maximal malondialdehyde (MDA) formation, suggesting that lipid peroxidation may be a consequence of cell damage as well as GSH depletion. IAA and DEM produced a comparable, modest accumulation of MDA, yet IAA was much more cytotoxic. These findings indicate that lipid peroxidation does not play a central role in hepatocellular injury by compounds which deplete GSH, although it may contribute to degeneration of the cell. As events in the cultured postnatal hepatocytes paralleled those reported in vivo, the system can be a useful and valid model with which to study mechanisms of chemical toxicity.

Identification of the proximate peroxisome proliferator(s) derived from Di(2-ethylhexyl) phthalate

A primary rat hepatocyte culture system was utilized to determine the proximate peroxisome proliferator(s) derived from di(2-ethylhexyl) phthalate (DEHP). DEHP was administered to rats and the urinary metabolites were identified and isolated. The major metabolites were those resulting from initial ω- or ω — 1-carbon oxidation of the mono(2-ethylhexyl) phthalate (MEHP) moiety. These metabolites, together with MEHP and 2-ethylhexanol, were added to primary rat hepatocyte cultures and the effect on peroxisomal enzyme activity was determined. The ω-carbon oxidation products [mono(3-carboxy-2-ethylpropyl) phthalate (I) and mono(5-carboxy-2-ethylpentyl) phthalate (V)] and 2-ethylhexanol produced little or no effect on CN −-insensitive palmitoyl-CoA oxidation (a peroxisomal marker). MEHP and the ω — 1-carbon oxidation products [mono-(2-ethyl-5-oxohexy) phthalate (VI) and mono(2-ethyl-5-hydroxyhexyl) phthalate (IX)] produced a large (7-to 11-fold) induction of peroxisomal enzyme activity. Similar structure-activity relationships were observed for the induction of cytochrome P-450-mediated lauric acid hydroxylase and increase in cellular coenzyme A content. This identification of the proximate proliferators will aid in the elucidation of the mechanism by which DEHP causes proliferation of peroxisomes in the rodent liver. Oral administration of MEHP (150 or 250 mg/kg) to male guinea pigs did not produce hepatic peroxisome proliferation. Addition of MEHP (0 to 0.5 m m) or one of the “active” proliferators in the rat (metabolite IX, 0 to 0.5 m m) to primary guinea pig hepatocyte cultures also failed to produce an induction of peroxisomal β-oxidation. Possible reasons for this species difference are discussed.

Factors influencing peroxisome proliferation in cultured rat hepatocytes.

A primary rat hepatocyte culture system has been developed for the study of peroxisome proliferation. Maximal induction of peroxisomal activity requires supplementation of the culture medium with hydrocortisone. The addition of clofibric acid (0.01-1 mM), mono-(2-ethylhexyl)phthalate (0.01-0.5 mM) and trichloroacetic acid (0.1-5 mM) to cultured rat hepatocytes resulted in a time- and dose-related increase in CN- insensitive palmitoyl CoA oxidation (maximal increases: 27-, 15.5-, and 5-fold respectively) and mitochondrial alpha-glycerophosphate dehydrogenase activity (maximal increases: 7.3-, 5.8-, and 1.6-fold respectively). Electron microscopic examination revealed smooth endoplasmic reticulum proliferation and morphometric analysis indicated an increase in fractional peroxisomal volume of X 8 and X 4 for clofibric acid (1 mM) and trichloroacetic acid (2.5 mM), respectively. SDS-PAGE of cell homogenates revealed an intensified protein band of mol. wt. 76-78,000. The induction of peroxisomal beta-oxidation by clofibric acid was elevated from 9- to 12-fold by supplementation of the medium with L-carnitine (2 mM).

U.v.-enhanced reactivation of u.v.-irradiated herpes virus by primary cultures of rat hepatocytes.

Carcinogen treatment of cultured mammalian cells prior to infection with u.v.-irradiated virus results in enhanced virus survival and mutagenesis suggesting the induction of SOS-type processes. In this paper, we report the development of a primary rat hepatocyte culture system to investigate cellular responses to DNA damage which may be relevant to hepatocarcinogenesis in vivo. We have obtained data demonstrating that enhanced reactivation of u.v.-irradiated Herpes simplex virus type 1 (HSV-1) occurs in hepatocytes irradiated with u.v. Cultured hepatocytes were pretreated with u.v. at the time of enhanced DNA synthesis. These treatments caused an inhibition followed by a recovery of DNA synthesis. At various times after pretreatment, the hepatocytes were infected with control or u.v.-irradiated HSV-1 at low multiplicity, and virus survival was measured by direct plaque assay. U.v.-irradiated HSV-1 exhibited the expected two-component survival curve in control or u.v. pretreated hepatocytes. The magnitude of enhanced reactivation of HSV-1 was dependent on the u.v. dose to the hepatocytes, the time of infection following u.v. pretreatment, and the level of DNA synthesis at the time of pretreatment. These results suggest that u.v. treatment of rat hepatocytes causes the induction of SOS-type functions that may have a role in the initiation of hepatocarcinogenesis.