Cultures Of Adult Rat Hepatocytes Research Articles

Urea production in long-term cultures of adult rat hepatocytes

To study the functionality of the urea cycle in long-term cultures of adult rat hepatocytes, urea production and the activity of two urea cycle enzymes were measured in hepatocytes cultured on 3T3 cells for 15 days. Urea production was also measured in cultures maintained with medium containing either 0.4 m m arginine or 0.4 m m ornithine and in cultures exposed to different concentrations of NH 4Cl, an in vivo inducer of urea production. In hepatocytes seeded on 3T3 cells, urea production decreased gradually to 50% of the initial value after 15 days. Urea production was similar in 3T3-hepatocyte cultures maintained for 11 days with medium containing ornithine or arginine. Hepatocytes exposed for 24 hr to 1, 3 and 5 m m NH 4Cl showed an average increase in urea production of 25, 50 and 69%, respectively, above that of unexposed cultures over 15 days. Ornithine transcarbamylase (OTC) activity decreased by 84% after 5 days in culture and remained constant thereafter, while arginase activity remained constant over 15 days. In contrast, in hepatocytes seeded on plastic substratum, urea production decreased to 24% of the initial value after 8 days in culture. OTC and arginase activities also decreased to 13 and 10% of their initial values after 8 days in culture. These results show that 3T3-hepatocyte cultures from adult rats produce urea from ornithine and/or arginine for at least 15 days and respond to an inducer of urea production as in vivo. They also show that these cultures have decreasing and constant levels of OTC and arginase activities, respectively, owing probably to an adaptative response dependent on substrate concentrations and hormonal regulation. These findings also suggest that 3T3-hepatocyte cultures are a suitable in vitro system to study urea production, its regulation by substrates and hormones and its alteration by drugs and toxic chemicals.

Regulation of cytochrome P450 2B1/2 mRNAs by Kepone ® (chlordecone) and potent estrogens in primary cultures of adult rat hepatocytes on Matrigel

We previously reported that when primary cultures of rat hepatocytes were treated with phenobarbital (PB) or one of several organochlorine pesticides, including Mirex ®, there was co-induction of cytochrome P450 2B1 and 2B2 mRNAs and immunoreactive proteins, whereas Kepone ® selectively induced 2B2 (Kocarek et al. (1991) Mol. Pharmacol. 40, 203–210). Indeed, Kepone treatment actively suppressed induction of 2B1 and 2B2 mRNAs in hepatocytes cotreated with phenobarbital. Because Kepone differs chemically from Mirex only in the replacement of 2 chlorine atoms with a ketone group, which exists in aqueous solution as a gem-diol and appears to confer weak estrogenic properties, we treated hepatocyte cultures with one of 3 potent estrogens, β-estradiol, 17α-ethinylestradiol or diethylstilbestrol. Treatment with each of these estrogens induced 2B1 and 2B2 mRNA only at very high doses (10 −4 M). β-Estradiol (10 −4 M) treatment also induced 2B1/2 mRNA in hepatocyte cultures prepared from a prepubescent female rat. The anti-estrogen tamoxifen failed to reverse 2B1/2 mRNA induction following β-estradiol or Kepone treatment of adult hepatocyte cultures. High doses of β-estradiol or 17α-ethinylestradiol failed to induce 2B1/2 mRNA in treated rats. We also examined the effects of chloral hydrate, a simple gem-diol, on 2B1/2 mRNA induction in the hepatocyte cultures. Treatment with chloral hydrate (3 × 10 −3 M), like Kepone (10 −5 M), suppressed 2B1/2 mRNA induction following phenobarbital (10 −4 M) treatment, while Kepone alcohol (10 −5 M), which is not a gem-diol, produced less suppression. Our results suggest that selective induction by Kepone of 2B2 is unlikely related to its effects as a weak classical estrogen, while the ability of Kepone to suppress induction of 2B1 and 2B2 by PB may be related to its properties as a gem-diol.

Stimulation of hepatocyte DNA synthesis by neurotensin.

Epidermal growth factor and transforming growth factor alpha stimulated DNA synthesis in primary cultures of adult rat hepatocytes. Neurotensin amplified epidermal growth factor-stimulated or transforming growth factor alpha-stimulated DNA synthesis by three- to eightfold. Neurotensin by itself did not stimulate DNA synthesis. Amplification of DNA synthesis by neurotensin was observed as low as 10(-10) M, and it was increased in a dose-dependent manner with maximal effects at 10(-8) M. These results were obtained when hepatocytes were cultured in Williams' medium E, but not in Leibovitz L-15 medium, suggesting that a minor component(s) in the medium is required for hepatocytes to fully respond to neurotensin. Neurotensin effect on DNA synthesis was observed not only in normal rat hepatocytes but also in partially hepatectomized rat hepatocytes, although its effect was stronger in normal hepatocytes. Amplified DNA synthesis was inhibited by transforming growth factor beta. Secondary mitogens (co-mitogens) such as insulin, vasopressin, or angiotensin II interacted additively with low concentrations of epidermal growth factor as well as with neurotensin. Neurotensin-related peptides such as kinetensin or neuromedin-N, which was released from blood plasma by pepsin digestion, did not have this amplifying effect on DNA synthesis at any concentrations tested. Neurotensin mRNA was found in several organs including brain and intestine, but not liver. These results suggest that neurotensin can be regarded as a new secondary mitogen and that it may be involved in cell proliferation, including regenerating liver as a gastrointestinal hormone and/or a neurotransmitter.

Anin vitro model of ethanol-dependent liver cell injury

Primary cultures of adult rat hepatocytes were incubated (6 to 96 hr) with 50 to 150 mmol/L ethanol, 0.5 mmol/L linoleate, 0.5 mmol/L palmitate, 0.5 mmol/L 4-methylpyrazole, 0 to 25 mumol/L vitamin E phosphate or selected combinations of these agents. Agent-dependent changes in liver cell viability (AST release and reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and function (phospholipid peroxidation, hydrolysis, biosynthesis and triacylglycerol biosynthesis) were determined. The influence of ethanol on liver cell function and viability was dose and incubation time dependent. Short periods (24 hr or less) of exposure to 100 mmol/L ethanol increased liver cell triacylglycerol biosynthesis and phospholipid hydrolysis, peroxidation and biosynthesis without altering cell viability. However, longer periods (72 hr or more) of exposure to 100 or 150 mmol/L ethanol resulted in significant reductions (30% to 50%) in cell viability, function and phosphatidylcholine biosynthesis and content. The ethanol-dependent decreases in cell function and viability were potentiated by linoleate and reduced by vitamin E phosphate, palmitate and 4-methylpyrazole. These results suggest that ethanol-induced liver cell injury in vitro is not a result of ethanol per se, but factors such as acetaldehyde or oxyradicals produced as a consequence of ethanol metabolism. Therefore the incubation of cultured hepatocytes with ethanol may be an appropriate model in vitro for determining the mechanisms by which ethanol intake disrupts liver cell function in vivo.

An in vitro model of ethanol-dependent liver cell injury

Primary cultures of adult rat hepatocytes were incubated with 100 mM ethanol, 0.5 mM linoleate, 0.5 mM 4-methylpyrazole (4-MP), 0.1 mM vitamin E and selected combinations of these agents. Agent-dependent alterations in cell viability and function were determined. Short periods of ethanol exposure stimulated the incorporation of labeled glycerol and choline into cellular lipids but did not alter cell viability. Longer periods of ethanol exposure; however, produced significant reductions in cell viability and cell function. The ethanol-dependent reductions in cell viability and function were potentiated by linoleate and reduced by 4-MP or Vitamin E. These results suggest that ethanol metabolism may induce liver cell injury in vitro by some oxidative event such as lipid peroxidation. In summary, these studies suggest that: (1) the long-term incubation of cultured hepatocytes with ethanol may be an appropriate in vitro model to determine the mechanisms by which ethanol induces liver cell injury; (2) vitamin E may prevent or reduce ethanol-dependent liver cell injury in vitro.

Genotoxic properties of 4-hydroxyalkenals and analogous aldehydes

4-Hydroxynonenal (HNE), one of the major products of lipid peroxidation, has been demonstrated to induce genotoxic effects in the micromolar range. HNE has too structural domains, a lipophilic tail and a polar head with three functional groups: the aldehyde and hydroxy groups and the trans CC double bond. To evaluate their relative importance, the genotoxic effects of HNE were compared with those of the homologous aldehydes 4-hydroxyhexenal and 4-hydroxyundecenal (different lengths of the lipophilic tail), and the analogous aldehydes 2- trans-nonenal (lacking the OH group) and nonanal (lacking the OH group and the trans CC double bond). This investigation was carried out on primary cultures of adult rat hepatocytes in order to further determine the influence of biotransformation- and/or detoxification reactions. A 3-h treatment with HNE induces statistically significant levels of SCE at concentrations ≥0.1 μM, micronuclei at concentrations ≥ 1 μM and chromosomal aberrations at a concentration of 10 μM. Compared to HNE the homologous aldehydes induced a significant genotoxic effect at higher concentrations. Statistically significant increases in SCE frequency were obtained at concentrations ≥ 1 μM for 4-hydroxyundecenal and at a concentration of 10 μM for 4-hydroxyhexenal. The induction of chromosomal aberrations was significantly elevated at concentrations of ≥ 10 μM and 10 μM for 4-hydroxyhexenal and 4-hydroxyundecenal, respectively. Except for a 4-hydroxyhexenal concentration of 1 μM, both aldehydes did not induce statistically significant levels of micronucleis. The HNE analogous aldehydes 2- trans-nonenal and nonanal induced statistically significant frequencies of SCE at concentrations of ≥ 1 μM (nonanal) and ≥ 10 μM (2- trans-nonenal). No significant induction of chromosomal aberrations or micronuclei could be demonstrated. The structure of the aldehydes investigated appears to influence the cyto- and genotoxic potential in the following ways. (1) The lenght of the lipophilic tail has no influence on chromosomal aberration induction, but appears to determine the yield of SCE and micronuclei, and the cytotoxic potential. (2) The lack of the OH group (2- trans-nonenal) reduces the SCE-inducing potential of the aldehyde shifting the dose-effect curve to higher concentrations. The similar shape compared to SCE induction by HNE indicates that possibly the same active metabolite is formed. (3) The lack of both the OH group and the CC double bond (nonanal) does not result in a complete loss of the SCE-inducing activity. The different shape of the dose-response curve suggests a different metabolism and/or a different mode of interaction with DNA.

Glutathione dependent detoxication in adult rat hepatocytes under various culture conditions.

In order to obtain more information concerning the effects of culture and medium conditions on the glutathione dependent detoxication system in hepatocyte cultures, glutathione reductase (GR) and glutathione peroxidase (GPx) activities were studied in both pure cultures of adult rat hepatocytes and their co-cultures with rat epithelial cells. Cells were isolated either with an oxygen saturated Krebs Henseleit buffer (KHB) or with a non-gassed Hepes buffer. As medium conditions, additions of 10% fetal calf serum (FCS), 25 mM nicotinamide, 0.1 microM selenium and 2% dimethylsulphoxide, respectively, to the culture medium were examined. It was found that co-cultures of rat hepatocytes can cope better with oxidative stress than pure cultures do. This conclusion was reached from the following observations. When oxygenated KHB was used as isolating buffer, GR and GPx activities increased during the first days of pure culture and then slowly decreased. This was observed for all the medium conditions studied and no significant differences between the different media could be observed. For co-cultures, however, after some initial variations GR and GPx activities reached stabilized levels which were not only significantly lower than those observed for pure cultures, but were also maintained throughout the whole culture period. Supplementation of the medium had no effect on these findings with the exception of high GPx activities when Se was added to the co-culture medium. When Hepes buffer with a low oxygen content was used in cell isolation, pure cultures showed significantly lower GR and GPx activities than those first mentioned.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of mitogens and co‐mitogens on the formation of small‐cell colonies in primary cultures of rat hepatocytes

Colonies of small hepatocytes appeared after the culture of primary adult rat hepatocytes for 4 days in serum-free Dulbecco's modified Eagle's medium containing 10 mM nicotinamide and 10 ng/ml of epidermal growth factor (EGF), acidic and basic fibroblast growth factors (FGF), hepatocyte growth factor (HGF), or transforming growth factor-alpha (TGF-alpha). Every colony consisted of cells that each had a single nucleus and a higher nucleus/cytoplasm ratio than surrounding hepatocytes, and immunocytochemically the cells induced by any mitogen were stained with albumin, transferrin, cytokeratin-8 and -18. But these cells expressed neither cytokeratin-7 nor -19. When 6 x 10(5) cells were plated on 35-mm dishes, about 15 colonies per 1,000 attached cells were observed in the cultures treated with EGF, HGF, and TGF-alpha. Although FGFs could also induce colonies, their number was less than half of the number induced by EGF. Furthermore, the numbers of colonies induced by the combinations of EGF+HGF, EGF+TGF-alpha, and HGF+TGF-alpha were not different from those of the colonies induced by each mitogen alone. To examine the ability of co-mitogenic factors to induce small-cell colonies, angiotensin-II, insulin-like growth factor-I, norepinephrine, tumor necrosis factor, and vasopressin were used. In the cells cultured without EGF, these co-mitogens neither stimulated DNA synthesis nor induced colonies. On the other hand, in cells cultured with both EGF and each co-mitogen, although the DNA synthesis of the hepatocytes was enhanced, the number of colonies detected was not significantly different from the number which EGF alone could induce. These results showed that the small-cell colonies in primary cultures of rat hepatocytes were inducible by EGF, HGF, TGF-alpha, or FGFs and that the co-mitogens did not influence the formation of the small-cell colonies.

Cell Density-Dependent DNA Fragmentation and Its Suppression by Heparin in Primary Culture of Adult Rat Hepatocytes

We show here that the internucleosomal DNA fragmentation, which is a biochemical hallmark of apoptosis, was induced in a cell density-dependent manner in primary culture of adult rat hepatocytes. This DNA fragmentation could be suppressed by a gene expression inhibitor, indicating the active nature of this process. Moreover, the viability changes in high and low cell density cultures showed a tendency corresponding with the incidence of the DNA fragmentation in them. These results suggest that in hepatocytes there may be a cell density-dependent apoptosis mechanism. In this report, we also show that heparin could suppress this DNA fragmentation with high specificity, and the cell death to some extent.

Aquatic genotoxicity testing with rat hepatocytes in primary culture. I. SCE induction

Primary cultures of adult rat hepatocytes were tested for their suitability to assess the genotoxic potential of effluent water samples by means of sister chromatid exchange induction. The results obtained with a variety of industrial effluents indicate a high sensitivity of this system since significantly elevated levels of SCE could be demonstrated in the undiluted sample and dilutions down to concentrations of 0.01%. However, the results also showed that the sole examination of an undiluted sample can lead to misinterpretations due to the shape of the dose response curves; in the worst case no genotoxic effect will be found although the sample has both toxic and genotoxic activity. Therefore, to assess the potential genotoxicity, it will be necessary to determine SCE induction with several dilutions of the effluents under investigation.

Measurement of malondialdehyde in cultures of adult rat hepatocytes

Peroxidation of unsaturated fatty acids is commonly measured by quantification of malondialdehyde production using the thiobarbituric acid assay. This technique has been applied to cultures of rat hepatocytes and the following observations were made. Optimal pigment production occurred when homogenates of cultured cells were heated at 90°C for 15–20 min. Linearity was observed in homogenates containing 0.5–2.5 mg protein/ml (originating from 0.5–2.5 × 10 6 cultured hepatocytes). Samples have to be stored for at least 24 hr at 0–4°C in the dark in order to precipitate the cloudiness that is due to the presence of lipids. Storage for up to 7 days is possible without any alteration. When peroxide production was provoked by tertiary butylhydroperoxide (tBHP), malondialdehyde measurements remained linear at least within the range 0 to 2 m m-tBHP, and 30 min was the optimal reaction time. The reproducibility of the whole technique applied to hepatocyte cultures was satisfactory (mean coefficient variation of four cultures was 3.5% ± 1.4%), and the mean inter-culture variation coefficient was 37 ± 21% (n = 16). Using this technique, it was found that the peroxidation rate was significantly lower in co-cultures than in pure cultures. These findings are in agreement with earlier experiments, which indicated that co-cultures suffer less from oxidative stress than do pure cultures.

Regulation of Phenobarbital-Inducible Cytochrome-P450 2B1/2 mRNA by Lovastatin and Oxysterols in Primary Cultures of Adult Rat Hepatocytes

Lovastatin (LOVA) is a potent inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase widely used in clinical practice. We treated primary cultures of adult rat hepatocytes, maintained in a minimal, serum-free medium on Matrigel, a reconstituted basement membrane, with this drug, and found that the amounts of P450 2B2 mRNA detected on Northern blots were increased at the same doses (10−5 to 3 × 10−5 M) required for induction of HMG-CoA reductase mRNA, a gene known to be under oxysterol regulatory control. LOVA treatment produced selective effects increasing also the mRNA levels for P450s 2C6, 2C7, 3A1, and 4A1 but not for 1A1, 2A1/2, or NADPH-cytochrome P450 oxidoreductase. LOVA treatment increased the induction of 2B1/2 mRNA in cells cotreated with either phenobarbital (PB; 10−4 M) or clotrimazole (CTZ; 10−5 M), or of 3A1 mRNA in cells cotreated with PB (2 × 10−3 M), but not dexamethasone (10−5 M). LOVA treatment did not potentiate the induction of 1A1 or 4A1 mRNA in cells cotreated with β-naphthoflavone (10−5 M) or ciprofibrate (10−4 M), respectively. In contrast to the potentiation of 2B1/2 mRNA induction produced by treatments with LOVA in combination with PB or CTZ, cotreatment of hepatocytes with PB and CTZ did not result in increased induction relative to that seen in cells treated with either agent alone. Treatment of hepatocyte cultures with either mevalonate (3 × 10−4 to 3 × 10−3 M), the immediate product of HMG-CoA reductase, or 25-hydroxycholesterol (10−6 to 10−5 M), a model oxysterol, resulted in dose-dependent suppression of 2B1/2 mRNA induction in cells treated with PB-like inducers. Taken together, our results demonstrate that LOVA is a unique inducer of P450 mRNA in cultured rat hepatocytes and implicate oxysterols as potential intracellular modulators of 2B1/2 induction. We conclude that endogenous metabolic factors including those related to cholesterol biosynthesis are critical in induction of liver cytochromes P450 2B1 and 2B2 by PB and "PB-like" agents.

Genotoxic effects of selected peroxisome proliferators

Peroxisome proliferators, a class of structurally dissimilar chemicals including hypolipidemic drugs and industrial plasticizers, have been shown to be associated with hepatocarcinogenesis although an initiating effect could not yet be demonstrated in the cell systems utilized. For this reason the genotoxic potential of the peroxisome proliferators nafenopin, ciprofibrate and di(2-ethylhexyl)adipate (DEHA) was determined in primary cultures of adult rat hepatocytes. The further test if these compounds are genotoxic per se or the genotoxic effect is due to peroxisome proliferation, the cultures were exposed for 3 and 51 h. Treatment for 3 h with the hypolipedemic drugs nafenopin abd ciprofibrate induced statistically significant increases of SCE at concentrations ≥30 and 100 μM respectively. At higher concentrations statistically significant increases of chromosomal aberrations (nafenopin: 100 μM; ciprfibrate: ≥ 100 μM μM) and micronuclei (ciprodibrate: ≥250 μM) were also found. The presence of peroxisome proliferators in the media until harvesting (51 h) did not significantly alter the dose response of SCE, micronuclei and chromosomal aberration induction by ciprofibrate, while long-term exposure to nafenopin resulted in statistically significant increase of chromosomal aberrations and micronuclei at concentrations The differences were statistically significant at 30 and 100 μM for micronuclei, and at 30 μM for ≥3 chromosomal aberrations. Neither short- nor long-term exposure to DEHA produced a significant genotoxic effect up to 200 μM. The peroxisome proliferators tested were not cytotoxic at any concentration, as determined by mitotic index. These results clearly demonstrate that the peroxisome proliferators nafenopin and ciprofibrate can cause genotoxic effect in primary cultures of adult rat hepatocytes. The comparison of short- and long-term exposure does not suggest a strong correlation between the induction of peroxisome proliferation and genotoxicity, since long-term exposure did not significantly alter the dose response and - expect for nafenopin - extent of the genotoxic effects.

α-Asarone Toxicity in Long-Term Cultures of Adult Rat Hepatocytes

In this work we studied the toxic effects of alpha-asarone, a hypolipidemic active principle of Guatteria gaumeri Greenman, on long-term cultures of adult rat hepatocytes cultivated on a feeder layer of 3T3 cells. The exposure for one and two weeks to alpha-asarone (1-50 micrograms/ml) produced intracytoplasmic lipid droplets and at higher concentrations (25-50 micrograms/ml) retraction of the hepatocyte cords and cell detachment. Ultrastructurally, the treated cultures (10 micrograms/ml) showed enlargement and vacuolization of the mitochondria in addition to lipid droplets. The triacylglycerol content increased up to 2.3-fold in the cultures treated for one week with 50 micrograms/ml, whereas the protein content per culture, a rough estimate of cell number and viability, decreased by up to 53% in the cultures treated for two weeks with 50 micrograms/ml. The synthesis and secretion of proteins, measured by the incorporation of [3H]-leucine into cellular and secreted macromolecules, decreased also in the cultures exposed. After one and two week exposure to 50 micrograms/ml of alpha-asarone, the secretion of labeled proteins decreased by 53 and 67%, respectively, whereas the synthesis of cellular and total proteins decreased by 48-67%, respectively. The secretion of proteins was the most sensitive parameter of alpha-asarone toxicity. The mean inhibitory dose (ID50), i.e, that producing 50% inhibition in the incorporation of the labeled precursor, was 22.12 and 5.04 micrograms/ml after one and two weeks exposure, respectively. Our results show that long-term exposure to micromolar concentrations of alpha-asarone produces morphologic and ultrastructural alterations, triacylglycerol accumulation (fatty liver), and inhibition of protein synthesis and secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of Ca2+ in stimulation of DNA synthesis by epidermal growth factor and tumor promoters in cultured rat hepatocytes.

This study examines the effects of extracellular Ca2+ concentrations, [Ca2+]o, and of treatments known to modulate intracellular Ca2+ levels on the extent and timing of DNA synthesis in primary cultures of adult rat hepatocytes. In cultures exposed to insulin and EGF, the extent of DNA synthesis between 40 h and 70 h in culture was independent of [Ca2+]o in the range 25-1,800 microM, although the peak of DNA synthesis occurred 5-10 h earlier with 1.2 mM Ca2+ than with 25 microM Ca2+. Complete removal of extracellular Ca2+ using EGTA blocked DNA synthesis if Ca2+ was removed on the second day after EGF addition but not if Ca2+ was absent only on day 1. Treatment of cultures in 1.2 mM Ca(2+)-containing media with Ca(2+)-ionophore A23187 or with thapsigargin, agents expected to raise cytosolic [Ca2+], failed to augment the stimulation of DNA synthesis by EGF. These observations suggest that hepatocytes may have a permissive requirement for [Ca2+]o > 0 at least late in the sequence of events leading from growth factor stimulation to DNA synthesis. However, sustained elevation of cytosolic [Ca2+] does not appear to be important as an early signalling event either in mediating or augmenting EGF action in hepatocytes. The ability of liver tumor promoters alpha-hexachlorocyclohexane or DDT to stimulate DNA synthesis in combination with EGF was independent of [Ca2+]o. By contrast, the skin tumor-promoting phorbol ester, TPA, or liver tumor promoter, phenobarbital, were without effect or inhibitory at low [Ca2+]o but in combination with EGF, stimulated DNA synthesis at [Ca2+]o > 0.4 mM, suggesting that Ca2+ may have some role in mediating or modulating the stimulatory effects of these agents.

Inhibition of Lipid Synthesis and Secretion in Long-Term Cultures of Adult Rat Hepatocytes by α-Asarone

In this work we studied the effect of alpha-asarone, a hypolipidemic active principle of Guatteria gaumeri Greenman, on hepatic lipid metabolism using adult rat hepatocytes cultured on a feeder layer of 3T3 cells. These cultures synthesize and secrete for at least two weeks various lipids from [14C]-acetic and [14C]-oleic acid. Exposure for one or two weeks to 10 micrograms/ml of alpha-asarone decreased the secretion of various lipids to the culture medium; triacylglycerol secretion was inhibited by 80-97%, phospholipid secretion by 70-87%, cholesterol by 64-70%, and cholesterol esters by 50-92%. The incorporation of [14C]-acetic acid into cellular lipids decreased by 30-81% and that of [14C]-oleic acid into phospholipids by 25-47% whereas the incorporation of [14C]-oleic acid into triglycerides and cholesterol esters increased 3.2 fold and by 28-36%, respectively. Similarly, the activities of glycerol-3-phosphate dehydrogenase and malic enzyme, marker enzymes of glycerolipid and fatty acid synthesis, decreased by 22-50% and 30-76%, respectively. Our results show that the exposure of the 3T3-hepatocyte cultures to micromolar concentrations of alpha-asarone significantly inhibits lipid secretion and probably lipid synthesis. They also suggest that at least part of the hypolipidemic effect could be due to a decrease in the secretion of lipids (i.e., lipoproteins) by the hepatocytes.

Occurrence and possible consequences of multipolar mitoses in primary cultures of adult rat hepatocytes

Proliferating primary cultures of adult rat hepatocytes are characterized by the occurrence of multipolar mitoses, and chromosome loss resulting in the formation of micronuclei at telophase. The percentage of multipolar mitotic figures was determined to be 12.76 +/- 7.9%, 80% of which were tripolar. Multipolar mitotic stages showed a high incidence of chromosome loss, increasing from meta- (61.7 +/- 16.6%) to telophase (72.1 +/- 19.3%). Regular bipolar mitotic figures on the other hand also showed chromosome loss, however, to a lesser degree and decreasing from meta- (49.5 +/- 10.4%) to telophase (34.9 +/- 7.9%). The incidence of chromosome loss even in regular mitotic figures is very high compared to other cells and appears to depend on another special feature of hepatocytes: they remain flat and well attached during mitosis, so that shearing forces could be responsible for the separation of chromosomes from the mitotic spindle. Additionally this morphology creates a situation allowing for a maximal interaction of mitotic spindles of binucleated cells, leading to the high rate of multipolar mitoses observed. Both multipolar mitoses and chromosome loss could also explain the consecutive detachment of hepatocytes reported for proliferating primary cultures, since the aneuploid daughter cells generated can be expected to be non-viable in most cases and eventually detach.

Pharmacodynamics of cytochrome P450 2B induction by phenobarbital, 5-ethyl-5-phenylhydantoin, and 5-ethyl-5-phenyloxazolidinedione in the male rat liver or in cultured rat hepatocytes.

The pharmacodynamics of rat hepatic cytochrome P450 2B (P450 2B) induction by phenobarbital (PB) and two structural congeners, dl-5-ethyl-5-phenylhydantoin (EPH) and dl-5-ethyl-5-phenyloxazolidinedione (EPO), were investigated. The in vivo induction of P450 2B was probed in F344/NCr rats by measuring immunoreactive hepatic P450 2B1 protein and by assaying the hepatic 16 beta-hydroxylation of testosterone and O-dealkylation of (benzyloxy)- and pentoxyresorufin. The induction of (benzyloxy)resorufin O-dealkylation activity was also measured in adult rat hepatocyte cultures exposed to the three xenobiotics. The concentration of xenobiotic at the putative active site in the in vivo studies was approximated by measuring serum total xenobiotic levels, while in the hepatocyte culture studies, the nominal xenobiotic concentration in the culture medium was used. Concentration-dependent induction of P450 2B activities was observed in the in vivo and hepatocyte culture studies. The in vivo ED50 values for P450 2B induction were approximately 110, approximately 100, and approximately 3000 dietary ppm (14 days administration) for PB, EPH, and EPO, respectively. The in vivo EC50 values for P450 2B induction were approximately 9, approximately 6, and approximately 130 microM (total serum) for PB, EPH, and EPO, respectively. In cultured rat hepatocytes, the ED50 values for induction of (benzyloxy)resorufin O-dealkylation activity were 14.5, 14.2, and 108 microM for PB, EPH, and EPO, respectively. These data indicate that pharmacodynamic results obtained with cultured hepatocytes represent a good qualitative and quantitative approximation of the in vivo hepatic responses in male rats caused by PB-type inducers.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced proliferation of fetal rat hepatocytes in primary culture induced by ritodrine

Although ritodrine crosses the placenta, its direct effect on fetal cell proliferation has not been reported. We hypothesized that beta 2-adrenergic receptor stimulation could promote fetal liver growth. Ritodrine was added to serum- and hormone-free primary cultures of fetal, neonatal, or adult rat hepatocytes. We measured both tritiated thymidine incorporation into deoxyribonucleic acid and nucleus number. The effect of ritodrine on cell cycle was also analyzed with flow cytometry. Ritodrine enhanced the proliferation of fetal rat hepatocytes. Ritodrine remarkably stimulated deoxyribonucleic acid synthesis of fetal and neonatal but not adult hepatocytes. The effect was dose dependent and was antagonized by propranolol. Analysis of the nuclear deoxyribonucleic acid content derived from flow cytometry revealed that cells stimulated by ritodrine entered S phase. These results indicate that ritodrine may promote the proliferation of fetal hepatocytes through the stimulation of beta 2-adrenergic receptors, followed by induction of deoxyribonucleic acid synthesis.

Insulin-mimetic actions of phorbol ester in cultured adult rat hepatocytes. Lack of phorbol-ester-elicited inhibition of the insulin signal

The actions of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on glucose metabolism, amino acid transport and enzyme inductions were studied in primary cultures of adult-rat hepatocytes and compared with the effects of insulin. PMA and insulin stimulated glycolysis 5- and 7-fold respectively. The half-maximal effective dose of PMA was 60 nM. Stimulation of glycolysis was accompanied by an insulin- or PMA-dependent and okadaic acid-sensitive activation of 6-phosphofructo-2-kinase and pyruvate kinase, as well as by an increase in fructose 2,6-bisphosphate. Glucose production from glycogen was decreased to 50% by PMA and to 15% by insulin, whereas glycogen synthesis was stimulated 2- and 7-fold respectively. PMA also increased aminoisobutyrate uptake, induced ornithine decarboxylase and counteracted the glucagon-dependent induction of phosphoenolpyruvate carboxykinase. PMA strongly antagonized the hormonal activation of glycogen synthesis, but all other insulin actions assayed were not decreased by the phorbol ester. Whereas additive effects of PMA and insulin were not detected, PMA and a simultaneous increase in the glucose concentration had additive effects on glycolysis and glycogen metabolism. Cell exposure to insulin resulted in receptor autophosphorylation and a more than 10-fold activation of the receptor tyrosine kinase. PMA did not alter these effects, and also had no effect on the receptor phosphorylation status in the absence of insulin. Long-term (15 h) pretreatment of the cells with PMA abolished all PMA effects, but not the insulin effects. It is concluded that PMA does not generally antagonize the action of insulin in differentiated adult hepatocytes, and that insulin and PMA may use related signal-transduction pathways.