Primary Monolayer Cultures Of Hepatocytes Research Articles

Fluorescence based cell counting in collagen monolayer cultures of primary hepatocytes.

Accurate determination of cell number is essential for the quantitative description of biological processes. The changes should be related to a measurable reference e.g. in the case of cell culture, the viable cell number is a very valuable reference parameter. Indirect methods of cell number/viability measurements may have up to 10% standard deviation. This can lead to undesirable large deviations in the analysis of "-omics" data as well as time course studies. Such data should be preferably normalized to the exact viable cell number at a given time to allow meaningful interpretation and understanding of the biological processes. Manual counting of cell number is very laborious and not possible in certain experimental setups. We therefore, developed a simple and reliable fluorescence based method with an accuracy of 95-98% for the determination of the viable cell number in situ. We optimized the seeding cell densities for primary rat hepatocytes for optimal cell adhesion. This will help in efficient use of primary cells which are usually limited in availability. The method will be very useful in the application of "-omics" techniques, especially metabolome analysis where the specific rates of uptake/production of metabolites can be reliably calculated.

Measurement of Apoptotic and Necrotic Cell Death in Primary Hepatocyte Cultures.

Hepatotoxicity, including drug-induced liver injury, is frequently accompanied by cell death. The latter is typically driven by apoptosis or necrosis, which substantially differ based upon biochemical and morphological criteria. This chapter describes two commonly used methods to probe apoptotic and necrotic activities in adherent monolayer cultures of primary hepatocytes. The apoptosis assay uses a prototypical substrate of caspase 3, the main executor of apoptotic cell death, which can be cleaved, yielding a product that can be measured fluorimetrically. The second assay relies on the disruption of the cell plasma membrane, which typically occurs in necrotic cell death and that results in the extracellular release of cytoplasmic enzymes, such as lactate dehydrogenase. The latter can be indirectly assessed by spectrophotometrically measuring the consumption of reduced nicotinamide adenine dinucleotide.

Long-term culture of primary hepatocytes: new matrices and microfluidic devices.

Prediction of in vivo drug-induced hepatotoxicity by in vitro cell culture systems is still one of the main challenges in drug development. To date, most in vitro approaches are based on monolayer cultures of primary hepatocytes, although it is known that they rapidly lose their morphology and liver-specific functions, such as activities of drug-metabolizing enzymes and transporters. Hepatocyte dedifferentiation can be delayed by culturing cells in a 3D environment. Combination with continuous medium flow, which creates a more physiological situation, further improves the maintenance of hepatic functions. Here, we present recently developed hydrogels and scaffolds for 3D culture of hepatocytes, which aim at preserving hepatic morphology and functionality for up to 4weeks in culture. Furthermore, major benefits and drawbacks of microfluidic devices for in vitro hepatotoxicity screening are discussed. Although promising advances have been made regarding the preservation of hepatic functions in 3D flow culture, major issues, such as expensive equipment, large cell numbers and low throughput, are still hampering their use in drug toxicity screening. For these devices to be applied and accepted in the drug-developing industry, it is necessary to combine easily accessible matrices that highly preserve the activities of drug-metabolizing enzymes with a user-friendly microfluidic platform, thereby finding the right balance between reflecting the in vivo situation and enabling satisfying throughput for drug candidate screening.

Characterization of spontaneous cell death in monolayer cultures of primary hepatocytes

Monolayer cultures of primary hepatocytes, isolated from freshly removed livers, represent widely used in vitro tools in the area of liver physiology and pathology, pharmacology and toxicology. However, a major shortcoming of these systems is that they cope with dedifferentiation, which is accompanied by spontaneous cell death. The goal of the present study was to elucidate the mechanisms that drive the process of self-generated cell demise in primary hepatocyte cultures. For this purpose, isolated rat hepatocytes were cultivated under conventional conditions, and the occurrence of apoptosis and necrosis was monitored during 4days by performing a set of acknowledged cell death assays. These included examination of cell morphology by light microscopy, quantification of apoptotic and necrotic cell populations by Hoechst 33342 and propidium iodide in situ staining, assessment of apoptotic and necrotic activities by measuring caspase 3-like activity and extracellular leakage of lactate dehydrogenase, and studying the expression of apoptosis regulators through immunoblot analysis. In essence, two cell death peaks were observed, namely shortly after cell seeding and in the final stages of the cultivation period, both involving apoptotic and necrotic actions. The outcome of this study not only sheds new light onto the molecular processes that underlie spontaneous cell death in primary hepatocyte cultures, but also opens perspectives for the establishment of strategies to increase cell survival in these popular in vitro systems.

Regulation of bile acid synthesis by estradiol and progesterone in primary cultures of rat hepatocytes

We have used primary monolayer cultures of hepatocytes from rats fed standard and cholestyramine-diet to study the effects of 17 beta-estradiol and progesterone on the activity of cholesterol 7 alpha-hydroxylase (EC 1.14.13.17) and bile acid synthesis. Cholesterol 7 alpha-hydroxylase activity in hepatocytes freshly isolated from rats fed either diet mentioned above declined gradually during attachment and the first day of culture. Exposure of cell monolayers to 1 or 10 microM estradiol or progesterone resulted in rapid and transient increases in cholesterol 7 alpha-hydroxylase activity, the maximal stimulation of enzyme activity being observed after a 6 h culture period. Bile acid synthesis in standard cells was markedly activated by both hormones, but in cholestyramine cells only the effect caused by 10 microM progesterone was significant. The cellular content of total bile acids was not significantly altered by the presence of the hormones, except by 10 microM progesterone, which provoked an initial cellular depletion of bile acids that was rapidly restored. Bile acid output was enhanced by treating primary cultures with 10 microM estradiol or progesterone, but whereas the increases caused by progesterone were marked and sustained, those caused by estradiol were minor and transient. We conclude that progesterone and 17 beta-estradiol, in this order of potency, enhance short-term bile acid synthesis in rat hepatocyte monolayers.

Hepatoprotective evaluation of Anogeissus latifolia: in vitro and in vivo studies.

To evaluate the hepatoprotective activity of a hydroalcoholic extract of the bark of Anogeissus latifolia; in vitro in primary rat hepatocyte monolayer culture and in vivo in the liver of Wistar rats intoxicated by carbon tetrachloride (CCl(4)). In the in vitro study, a primary hepatocyte monolayer culture was treated with CCl(4) and extract of Anogeissus latifolia. Hepatoprotective activity was demonstrated in the CCl(4) damaged primary monolayer culture. In the in vivo study, the hepatoprotective activity of a hydroalcoholic extract of Anogeissus latifolia was analyzed in liver injured CCl(4)-treated rats. Biochemical parameters including serum transaminases [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)] and alkaline phosphatase (ALP) in serum were analyzed. The biochemical findings were supplemented with histopathological examination of rat liver sections. In vitro: primary hepatocyte monolayer cultures were treated with CCl(4) and extract of Anogeissus latifolia. A protective activity could be demonstrated in the CCl(4) damaged primary monolayer culture. In vivo: Hydroalcoholic extract of Anogeissus latifolia (300 mg/kg) was found to have protective activity in rats with CCl(4)-induced liver damage as judged from serum marker enzyme activity. The above findings lead to the conclusion that the hydroalcoholic extract of Anogeissus latifolia is hepatoprotective. Hence, we suggest that the inclusion of this plant in the management of liver disorders is justified.

Characterization of the cellular damage induced by Aflatoxin B1 in sea bream (Sparus aurata Linnaeus, 1758) hepatocytes

Gilthead sea bream (Sparus aurata L.) is one of the most intensively farmed fish species in the Mediterranean, greatly studied for the relevant economic value, although its sensitivity to Aflatoxin B1 (AFB1) has to be investigated, yet. The aim of this study was to perform an in vitro evaluation of cytotoxic potential of AFB1 on S. aurata hepatocytes in order to grade the range of AFB1 toxicity, and the boundary between acute and long-term toxicity. Primary monolayer cultures of hepatocytes from S. aurata juveniles were treated with a wide range of concentrations from 5x103 ng/ml to 2x10-5 ng/ml of AFB1 for a different period of exposure (24, 48, 72 hours). The cytotoxic activity was characterized by MTT reduction assay. After each exposition hepatocytes were examined for morphologic alterations and apoptosis induction. AFB1 exposure significantly reduced cell viability in a dose- and time-dependent manner. Dose-response curves obtained after 24, 48 and 72 hrs revealed that prolonged exposure times lead to a significant increase of the toxic potency of AFB1. Our results demonstrate that S. aurata hepatocytes are highly sensitive to AFB1 exposure. Such scientific findings could provide new insights to investigate the real impact of aflatoxin on marine farmed fish.

Hepatoprotective activity of Sapindus mukorossi and Rheum emodi extracts: In vitro and in vivo studies

To study the hepatoprotective capacity of Sapindus mukorossi (S. mukorossi) and Rheum emodi (R. emodi) extracts in CCl(4) treated male rats. The dried powder of S. mukorossi and R. emodi was extracted successively with petroleum ether, benzene, chloroform, and ethanol and concentrated in vacuum. Primary rat hepatocyte monolayer cultures were used for in vitro studies. In vivo, the hepatoprotective capacity of the extract of the fruit pericarp of S. mukorossi and the rhizomes of R. emodi was analyzed in liver injured CCl(4)-treated male rats. In vitro: primary hepatocytes monolayer cultures were treated with CCl(4) and extracts of S. mukorossi & R. emodi. A protective activity could be demonstrated in the CCl(4) damaged primary monolayer culture. In vivo: extracts of the fruit pericarp of S. mukorossi (2.5 mg/mL) and rhizomes of R. emodi (3.0 mg/mL) were found to have protective properties in rats with CCl(4) induced liver damage as judged from serum marker enzyme activities. The extracts of S. mukorossi and R. emodi do have a protective capacity both in vitro on primary hepatocytes cultures and in in vivo in a rat model of CCl(4) mediated liver injury.

Characteristics of the carbohydrate moiety and the intermolecular associations of the high-molecular-weight forms of alpha 2u-globulin.

We have investigated some of the biochemical characteristics of the glycosylated forms of α2u-globulin, the major protein of adult male rat urine. Labeled α2u-globulin that was secreted into the culture medium of primary monolayer cultures of hepatocytes was immunoprecipitated from the medium with antibody specific for urinary α2u-globulin and the complex used for further study. α2u-Globulin existed in the medium as a high-molecular-weight aggregate that could be dissociated with reducing agents and detergents into two glycosylated molecules of Mr 75000 and 50000 and one nonglycosylated molecule of Mr 20000. The glycosylated forms of α2u-globulin were shown by kinetic studies not to be precursors of the molecule of Mr 20000. When the glycoproteins were synthesized in the presence of radioactive sugar and amino acid precursors, the ratio of sugar to amino acid suggested that the highest-molecular-weight glycoprotein had 1.5–2 times the carbohydrate content of the faster-migrating glycoprotein. The oligosaccharide moiety, isolated from an exhaustive pronase digest of the combined glycoprotein molecules, was of Mr 5000 as determined by gel nitration methods. Sequential glycosidase digestion with neuraminidase, β-galactosidase, and β-N-acetylglucosaminidase produced a core oligosaccharide of Mr 1200. Anion-exchange chromatography demonstrated that the charge microheterogeneity of the α2u-globulin glycopeptides could be abolished by treatment with neuraminidase. Our results indicate that the oligosaccharide moiety of α2u-globulin is similar to those oligosaccharides found on other serum proteins, and suggest that formation of the species of α2u-globulin of Mr 75000 and 50000 requires either the attachment of many oligosaccharide chains to a single polypeptide of Mr 20000 or the covalent linkage of poly-peptides containing one or more oligosaccharide chains to form dimers and trimers.

The use of liver spheroids as an in vitro model for studying induction of the stress response as a marker of chemical toxicity

Stress protein induction has been advocated as a sensitive indicator of compound-induced toxicity. In monolayer cultures of primary hepatocytes, however, the two stress proteins, Hsp25 and Hsp72/3 are up-regulated, probably due to the effect of the isolation procedure and adaptation of the cells to the culture conditions. The aim of the current studies was to determine whether liver spheroids would provide an improved experimental model for the study of heat shock protein induction in vitro. Primary rat hepatocytes were cultured as liver spheroids and the expression of Hsp25 and Hsp72/3 measured along with the levels of ATP, GSH and albumin secretion. Hsp72/3 was initially increased in spheroid culture but returned to in vivo levels after 3 days of culture. Hsp25 was maintained at in vivo levels until day 6 of culture, after which levels increased slightly. The effects of the two hepatotoxins, hydrazine and cadmium chloride (CdCl 2), were therefore measured on day 6 of spheroid culture. CdCl 2 had no effect on Hsp25 but increased Hsp72/3 at concentrations that affected other biochemical parameters. Hydrazine caused a rapid reduction in ATP levels and albumin secretion, but did not affect Hsp72/3. Hsp25 was slightly induced by hydrazine at later sampling times at concentrations, however, that affected other biochemical parameters. It can be concluded that liver spheroids provide a model for studying stress protein expression. However, the increase in stress proteins appears to be a relatively insensitive parameter compared to other more conventionally used toxicity endpoints and the response appears to vary with individual toxins under study.

Isolation and genomic analysis of the rat polymeric immunoglobulin receptor gene terminal domain and transcriptional control region.

The polymeric immunoglobulin receptor (pIgR) transports IgA and IgM across secretory epithelial cells and is essential in external immunity maintenance. We report here the structural characterization of the single-copy rat gene distributed over 30 kb of chromosomal DNA and analysis of its transcriptional control region. RNA sequencing and genomic analysis show a 5' terminal region originates at a major (+1) and a minor site producing an unusual 124-bp nontranslated exon I separated from a small 96-bp initiator ATG coding exon II by a 7.5-kb intron. The pIgR 5' region comprises a structured promoter with abundant helix-loop-helix (bHLH) cis elements positioned within an equivalent internal -70, -290, -528, and three centered at -745. The three latter bHLH elements each occur within 30-bp repeats at -690 to -780. Transient expression assays show a 1.3-kb 5' region is sufficient to drive expression in rat primary hepatocyte monolayer cultures, transformed human hepatic (HepG2) cells, and a mammary epithelial tumor cell line MCF-7, but is inactive in the rodent fibroblast 3T3 cell line. A minimal transcriptional promoter domain was deduced from sequentially deleted vectors revealing a +40 to -922 sequence to be sufficient for full activity. Further deletions within this region yield incremental losses in cis activity, indicating that multiple subregions comprise an extended transcriptional control region.

Retinoids stimulate ApoA-I synthesis by induction of gene transcription in primary hepatocyte cultures from cynomolgus monkey (Macaca fascicularis)

The influence of different retinoids on apolipoprotein A-I (apoA-I) synthesis and secretion was investigated in primary monolayer cultures of hepatocytes from cynomolgus monkeys. Addition of retinol (vitamin A) and retinoic acid to the culture medium resulted in a time- and dose-dependent increase in the secretion of apoA-I. No effect was observed during the first 24-hour incubation period; however, apoA-I secretion was enhanced 1.5-fold in the following 24-hour period in the presence of 10 mumol/L retinoic acid. Maximal stimulation (2.7-fold) was obtained at 10 mumol/L retinoic acid during a third 24-hour incubation. In these experiments apoB-100 secretion was unaffected. When [35S]methionine incorporation studies were performed de novo synthesis of apoA-I was increased, whereas total protein synthesis remained constant. These observations indicated that the induction of apoA-I synthesis is not part of a general effect of retinoic acid on hepatic protein synthesis. Among different natural and synthetic retinoids, retinoic acid and its 9-cis and 13-cis isomers were equally active and were the most potent inducers of apoA-I synthesis, whereas the maximal stimulation induced by retinol was lower (1.6-fold). ApoA-I mRNA abundance was increased threefold in hepatocytes exposed for 72 hours to 10 mumol/L retinoic acid, which was associated with a twofold increase in the transcriptional rate of the apoA-I gene. In contrast, no changes were found in the apoB-100 mRNA level and transcriptional activity of the apoB-100 gene. We conclude that retinoids enhance apoA-I synthesis in simian hepatocytes by transcriptional regulation.

Spectrophotometric measurement of flavin-containing monooxygenase activity in freshly isolated rat hepatocytes and their cultures

The determination of the mixed function flavin-containing monooxygenase activity in rat liver and in hepatocytes and their cultures by spectrophotometric measurement of the oxygenation of methimazole is complicated by an inhibition caused by some of the reagents used during this method. Optimal conditions were determined for measuring this enzyme activity in microsomal preparations of rat liver and its hepatocytes. Optimal flavin-containing monooxygenase activities were obtained for measurements performed in a 0.25 m N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine—EDTA buffer at pH 8.7 and at a methimazole concantration of 2 m m. Data are also presented which show that no interferences caused by either cytochrome P 450-dependent enzymes or by the reduction of methimazole disulfide by glutathione have to be taken into account when determining methimazole oxygenation. Finally, the above assay was also used to study flavincontaining monooxygenase activity in primary monolayer cultures of hepatocytes for 6 days.

Application of the neutral red assay (NR assay) to monolayer cultures of primary hepatocytes: rapid colorimetric viability determination for the unscheduled DNA synthesis test (UDS)

The neutral red (NR) absorption method was adapted for the determination of cell viability in the UDS assay with primary hepatocyte cultures of the rat. The NR method is rapid, easy to perform, and suitable for handling of large numbers of cultures simultaneously. It can be used for concentration range-finding pre-experiments. In addition, it can easily be integrated into a UDS test protocol for documentation of toxic effects if supplementary cultures for each concentration are established. The time schedule required for the NR assay makes it possible for one person to process the hepatocytes for autoradiography and at the same time determine the toxicity.

Actin Filament Alterations in Rat Hepatocytes Induced In Vivo and In Vitro by Microcystin-LR, a Hepatotoxin from the Blue-green Alga, Microcystis aeruginosa

The morphologic effects of microcystin-LR (MCLR) were examined in vitro and in vivo to identify the specific cell type(s) affected and to characterize the actin filament changes occurring in hepatocytes. Male Sprague Dawley rats were used for all studies. For in vitro studies, hepatic cells were isolated by collagenase perfusion of liver, while parenchymal cells (hepatocytes) and nonparenchymal cells were prepared by pronase digestion and metrimazide gradient centrifugation. Cell suspensions and and primary hepatocyte monolayer cultures were treated with MCLR at doses up to 10 micrograms/ml; cultured hepatocytes were also treated with phalloidin or cytochalasin B at a dose of 10 micrograms/ml; and rats were treated intraperitoneally with MCLR at 180 mg/kg. Cultured hepatocyte preparations and frozen liver sections were stained with rhodamine-labeled phalloidin for filamentous actin. In cell suspensions, MCLR did not affect nonparenchymal cells but caused rapid, progressive, blebbing of the plasma membrane in hepatocytes. In cultured hepatocytes, MCLR caused plasma membrane blebbing as well as marked reorganization of actin microfilaments. These alterations were dose and time dependent. Cultured hepatocytes treated with phalloidin or cytochalasin B also showed extensive plasma membrane blebbing and actin filament alterations; however, actin filament changes were morphologically distinct from those induced by MCLR. In vivo, MCLR-induced hepatocyte actin alterations occurred at the same time as, or slightly preceded, histologic changes that began 30 minutes after dosing. These studies suggest that early MCLR-induced morphologic changes occurring both in vivo and in vitro are due to alterations in hepatocyte actin filaments.

Induction of peroxisomal acyl CoA oxidase activity and lipid peroxidation in primary rat hepatocyte cultures

Peroxisome proliferators have been suggested to induce liver carcinogenesis as a result of increased peroxisomal hydrogen peroxide production and cellular oxidative stress. Primary monolayer cultures of hepatocytes isolated from male F344 rats were incubated in medium containing one of three different peroxisome proliferators and examined for the induction of peroxisomal CoA oxidase activity and lipid peroxidation. The latter parameter was determined by measuring levels of conjugated dienes in lipid fractions extracted from harvested cells. The peroxisome proliferators used in these studies were nafenopin and clofibric acid (two hypolipidemic drugs) and mono(2-ethylhexyl)phthalate (MEHP), the primary metabolite of the industrial plasticizer, di(2-ethylhexyl)phthalate (DEHP). The relative specific activity of peroxisomal acyl Coa oxidase was increased by about 300% after incubation for 44 h with 200 μM nafenopin; lower levels of induction were observed with clofibric acid or MEHP. Relative to controls, the level of conjugated dienes was increased approximately 2-fold after incubation with 200 μM nafenopin; there was no apparent increase in conjugated dienes after incubation with up to 200 μM MEHP or 400 μM clofibric acid. The increase in conjugated dienes with 200 μM nafenopin was inhibited by co-incubation with the antioxidant, N, N'-diphenyl- p-phenylenediamine. Thus, peroxisomal enzyme induction by nafenopin can result in membrane lipid peroxidation and monolayer cultures of rat hepatocytes may provide a useful model system for studying relationships between peroxisome proliferation, enhanced hydrogen peroxide production and cellular changes due to hepatic oxidative stress.

Epidermal growth factor and cyclic AMP stimulate Na+/H+ exchange in isolated rat hepatocytes

Na+/H+ exchange in acid-loaded isolated hepatocytes was measured using the intracellular pH indicator biscarboxyethyl-carboxyfluorescein (BCECF) to follow intracellular pH (pHi). The rate of amiloride-sensitive Na(+)-dependent recovery from cytoplasmic-acid-loading was found to be increased in cells treated with epidermal growth factor (EGF), 8-(4-chlorophenylthio)adenosine 3',5'-monophosphate (ClPhScAMP) or phorbol 12-myristate 13-acetate (PMA). These three agents increased the rate of Na+/H+ exchange to similar extents and their effects were not additive. The stimulation was shown in all three cases to be due an alkaline shift of 0.1 in the set point pH of the Na+/H+ exchanger. Experiments measuring the uptake of 22Na+ into acid-loaded primary hepatocyte monolayer cultures confirmed these results. EGF, ClPhScAMP and PMA significantly increased the amiloride-inhibitable accumulation of 22Na+, thus providing further evidence that Na+/H+ exchange is stimulated by these effectors.

Interaction of hypoxia and carbon tetrachloride toxicity in hepatocyte monolayers

The toxicity of carbon tetrachloride (CCl 4) 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% O 2. CCl 4 was administered in the vapor phase at concentrations that produce aqueous concentrations at 37°C of 0.4, 2.0, and 4.0 m M. 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% O 2. Only in the case of 0.5% O 2 and 4 m M CCl 4 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 CCl 4 and oxygen concentration was not evident until the 6-hr time point. The monolayers exposed to 4 m M CCl 4 and 1, 2, or 20% O 2 exhibited little immediate damage but were all 100% dead 6 hr later. The monolayers exposed to 2 m M CCl 4 and 0.5, 1, 2, or 20% O 2 were 53, 48, 40, and 22 ± 2% dead after 6 hr, respectively. These results suggest that effects of CCl 4 exposure, for example alterations in the function or synthesis of essential proteins, require several hours to affect cell viability.

Effects of Nucleosides and a Nucleotide on DNA and RNA Syntheses by the Salvage and De Novo Pathway in Primary Monolayer Cultures of Hepatocytes and Hepatoma Cells

Studies were made on the effects of inosine, guanosine 5'-monophosphate (GMP), cytidine, uridine, thymidine, and their mixture (4:4:4:3:1, OG-VI) on DNA and RNA syntheses in primary monolayer cultures of normal hepatocytes and cultures of hepatoma cells, AH130, to use these compounds for total parenteral nutrition. Addition of an appropriate amount of inosine, GMP, uridine, or thymidine to primary cultures of hepatocytes enhanced both DNA and RNA syntheses by the salvage and de novo pathways. Cytidine appeared to have lower optimal concentration for enhancing these pathways. The OG-VI mixture also enhanced the syntheses of DNA and RNA, but the composition of the mixture was not optimal. Additions of inosine, GMP, uridine, and thymidine to cultured hepatoma cells also enhanced their DNA and RNA syntheses, but the cells consumed more of the added nucleic acid compounds than hepatocytes did. Addition of cytidine had no effect on proliferation of the cells. The OG-VI mixture at relatively higher concentration inhibited the syntheses of DNA and RNA by hepatoma cells. Addition of high concentrations of nucleic acid compounds was found to suppress the proliferation of both hepatocytes and hepatoma cells. These results suggest that addition of optimal amounts of nucleic acid compounds such as nucleosides and nucleotides would enhance growth of hepatocytes, particularly during liver regeneration, but that they may also enhance proliferation of tumor cells in the liver.

Toxicity of 1,2-dibromoethane in primary hepatocyte monolayer cultures: Lack of dependence on oxygen concentration

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.