Male Rat Hepatocytes Research Articles

Expression of p53 mutant protein(s) in diethylnitrosamine-induced foci of enzyme-altered hepatocytes in male Fischer-344 rats

Several types of human and animal tumors have been shown to carry mutations in the p53 gene. While the translation product of the wild type gene has tumor suppressor properties, mutant alleles of the gene produce proteins that can cooperate with other oncogene products in transforming cells. In this paper, evidence is presented indicating that a p53 gene mutation(s) occurs in foci of enzyme-altered hepatocytes induced by diethylnitrosamine in male Fisher-344 rats. The evidence was obtained by means of immunohistochemical and immunoblotting techniques, using antibodies directed against mutant forms of the p53 protein.

Examination of potential mechanisms of carcinogenicity of 1,4-dioxane in rat nasal epithelial cells and hepatocytes

Several long-term studies with 1,4-dioxane (dioxane) have shown it to induce liver tumors in mice, and nasal and liver tumors in rats when administered in amounts from 0.5 to 1.8% in the drinking water (Argus et al. 1965; Kociba et al. 1974; National Cancer Institute, 1978). In order to examine potential mechanisms of action, chemically-induced DNA repair (as an indicator of DNA reactivity) and cell proliferation (as an indicator of promotional activity) were examined in nasal turbinate epithelial cells and hepatocytes of male Fischer-344 rats treated with dioxane. Neither dioxane nor 1,4-dioxane-2-one, one of the proposed metabolites, exhibited activity in the in vitro primary rat hepatocyte DNA repair assay, even from cells that had been isolated from animals given either 1 or 2% dioxane in the drinking water for 1 week to induce enzymes that might be responsible for producing genotoxic metabolites. No activity was seen in the in vivo hepatocyte DNA repair assay in animals given a single dose of up to 1000 mg/kg dioxane or up to 2% dioxane in the drinking water for 1 week. Treatment of rats with 1.0% dioxane in the drinking water for 5 days yielded no increase in liver/body weight nor induction of palmitoyl CoA oxidase, indicating that dioxane does not fit into the class of peroxisomal proliferating carcinogens. The percentage of cells in DNA synthesis phase (S-phase) was determined by administration of 3H-thymidine and subsequent quantitative histoautoradiography. The hepatic labeling index (LI) did not increase at either 24 or 48 h following a single dose of 1000 mg/kg dioxane.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo and in vitro estimations of the direct effect of estrogen on rat hepatocytes tested by the changes in the unusual estrogen-binding protein content

The direct effect of estradiol (E 2) on the hepatocytes of mature male rats has been examined by measuring the changes in the unusual estrogen-binding protein (UEBP) content and parallel measuring the level of liver estrogen receptors (ER). The content of UEBP ( N UEBP) and ER ( N ER) in the liver were determined using the quantitative methods for differential specific determination of the E 2-binding sites of these proteins. It has been shown that the administration of E 2 in vivo induced a considerable decrease in hepatic N UEBP not only in intact males, but also in hypophysectomized males during the initial period after the operation (when the content of hepatic ER was still high) and produced no effect in hypohysectomized males during the later period (when liver ER were depleted). Repeated administration of human growth hormone (hGH) (twice a day) resulted in a considerable increase in n er in hypophysectomized males and restored the sensitivity to the subsequent inhibitory effect of E 2 on UEBP. We also used rat hepatocytes after a 4-day primary culturing. These cells had a stable morpho-functional status, high ER level, and sex-differentiated UEBP content. Culturing of mature male rat hepatocytes in the medium containing E 2 at concentrations close to physiological levels (10 −10−10 −7) decreased N UEBP in a dose-dependent manner. Hexestrol (10 −7M) but not cholesterol (10 −5M) also exhibited a direct effect on N UEBP in cultured rat hepatocytes. The effect of E 2 was reversible: statistically significant increase in N UEBP was observed 3 days after 10 −9 M E; had been removed from the culturing medium. It was concluded that hepatocytes may be a primary target for E 2 under physiological conditions and that GH may modulate the direct effect of E 2 at the hepatic level by modifying the content of liver ER.

Androgen regulated expression of the alpha 2u-globulin gene in pancreatic hepatocytes of rat.

Under a copper-deficient regimen, pancreatic cells in the adult rat can be found to undergo differentiation into hepatocytes. Pancreatic hepatocytes induced in male and female rats were examined for the expression of the androgen-inducible hepatic protein, alpha 2u-globulin. Alpha 2u-Globulin protein was demonstrable by immunoperoxidase method in all the pancreatic hepatocytes of male rats. Northern blot analysis confirmed the presence of 1.3 kb alpha 2u-globulin mRNA transcript in the pancreas of male rats with hepatocytes. Orchiectomy resulted in marked decrease of alpha 2u-globulin protein and its mRNA. Administration of dihydrotestosterone to castrated rats resulted in increased levels of alpha 2u-globulin mRNA and the amount of alpha 2u-globulin protein in the pancreatic hepatocytes. Unlike normal males, in intact and ovariectomized females alpha 2u-globulin was not detectable in pancreatic hepatocytes. These results indicate that similar to hepatic parenchymal cells pancreatic hepatocytes synthesize alpha 2u-globulin under androgenic regulation. Furthermore, unlike in liver where it is expressed predominantly in perivenular and midlobular hepatocytes, there is no localized difference in the expression of this gene in the transdifferentiated pancreatic hepatocytes.

Inhibition of hepatocyte gap junctional intercellular communication by endosulfan, chlordane and heptachlor

The cyclodiene pesticides endosulfan, chlordane and heptachlor have been reported to be non-genotoxic rodent hepatocarcinogens. These three compounds and several metabolites of endosulfan (endosulfan sulfate, endosulfan ether and endosulfan lactone) were examined for their effects on gap junctional intercellular communication (GJIC) in primary cultured male F344 rat hepatocytes and B6C3F1 mouse hepatocytes. GJIC was evaluated by Lucifer Yellow CH dye-coupling. Endosulfan and endosulfan sulfate inhibited rat and mouse hepatocyte GJIC in a dose-responsive manner (50-200 microM) after 4 h treatment. Endosulfan ether inhibited rat hepatocyte GJIC only at 200 microM and had no effect on mouse hepatocytes. Endosulfan lactone did not affect rat or mouse hepatocyte GJIC. Chlordane and heptachlor inhibited both mouse and rat hepatocyte GJIC at concentrations of 50-200 microM. The inhibition of GJIC by the cyclodienes showed similar dose-response relationships and kinetics of onset of inhibition and reversibility for both mouse and rat hepatocytes. Concomitant treatment of the cells with inhibitors of cytochrome P450 monooxygenases (SKF-525A, piperonyl butoxide or carbon monoxide) did not alter the inhibition of GJIC by the cyclodienes, suggesting that cytochrome P450 metabolism was not involved in the inhibitory mechanism. Dibutyryl cyclic AMP (0.5 mM), however, decreased the inhibition of GJIC by the cyclodienes and may indicate that these compounds inhibit intercellular communication through a cAMP-dependent process. The inhibition of mouse and rat hepatocyte GJIC by the cyclodienes correlated with previous reports indicating that these compounds are non-genotoxic rodent liver carcinogens.

Oxygen dependence of glutathione synthesis in hepatocytes

The O 2 dependence of glutathione (GSH) synthesis was studied in freshly isolated hepatocytes of white male rats. The rate of synthesis with methionine as the sulfur-containing amino acid precursor was decreased at hypoxic O 2 concentrations and was half-maximal at 5 μ m O 2. ATP-dependent formation of S-adenosylmethionine was the rate-limiting step in GSH synthesis under these hypoxic conditions as shown by studies of S-adenosylmethionine concentrations and effects of compounds that inhibit mitochondrial ATP production. GSH synthesis with cysteine as the sulfur-containing precursor amino acid was relatively resistant to O 2 deficiency. The rate under anoxia was 48% of the aerobic rate and the O 2-dependent rate was half-maximal at 0.9 μ m O 2. These results indicate that GSH synthesis from methionine is likely to be impaired under physiological and pathological conditions involving hypoxia, but synthesis from cysteine is not likely to be greatly affected except during anoxia. In addition, the sensitivity of the cystathionine pathway to hypoxia suggests that other products of the pathway, such as choline, creatine, epinephrine, and methylated tRNA's, may also be decreased by hypoxia.

In-vivo uptake of human growth hormone in male rat liver.

125I-Labelled human GH (hGH) was injected i.v. to male rats and its subcellular distribution in the hepatocyte was examined using fractionation techniques. Uptake into liver homogenates was maximal by 15 min after injection and represented 24% of the injected radioactivity; it was markedly inhibited by coinjection of native hGH. 125I-Labelled hGH taken up by the liver underwent a time-dependent translocation process. The peak of specific labelling of plasma membranes occurred at 3 min whereas later on the radioactivity was concentrated in low-density structures present in Golgi-endosome fractions. To characterize the ligand-associated structures better, endosome-enriched fractions were prepared from a microsomal fraction by isopycnic centrifugation in a sucrose gradient and a Nycodenz gradient. The radioactivity was in one peak with a median density of 1.096 g/cm3 in the Nycodenz gradient fractions. The peak of radioactivity was distinct from that of galactosyltransferase activity which appeared at a median density of 1.114 g/cm3. The labelled material eluted from the various subcellular fractions appeared as intact hGH. Upon in-vivo interaction with male rat hepatocytes, 125I-Labelled hGH was internalized with a sequential association with plasma membranes and endocytic structures distinct from Golgi elements.

Carbonic anhydrase localization to perivenous hepatocytes

It was shown by immunohistochemical methods, using antisera against highly purified carbonic anhydrases CA I, II and III, that CA II and CA III are specifically located in hepatocytes around central veins of adult rat livers. CA II was found in these perivenous hepatocytes of both male and female rats. Its physiological role is probably to facilitate the secretion of bicarbonate ions by these cells into canalicular bile. CA III and CA I were found in the same hepatocytes but only in the livers of male rats. The function of CA I and CA II exclusively in these cells of male rats cannot be simply explained.

Effect of benzyl chloride on rat hepatocytes.

Benzyl chloride (BCl) is used in the manufacture of basic and acidic dyes, pharmaceutical products, resins, and synthetic tannins. BCl is known to have caused liver malfunctions in some workers exposed to 2 ppm BCl vapors. This study was conducted to investigate the effect of BCl on isolated male rat hepatocytes using several toxicity parameters. The hepatocytes were isolated by a collagenase perfusion technique and were incubated in airtight tubes with 1.8 and 3.6 mM BCl in a shaking water bath at 37 degrees C for 10, 30, 60, and 120 min. Throughout the incubation period the cell viability was determined by trypan blue exclusion and leakage of cytosolic enzymes such as lactate dehydrogenase (LDH), aspartate transaminase (AST), and alanine transaminase (ALT). Exposure to BCl resulted in a significant decrease in cell viability as assessed by trypan blue and significant increase in leakage of these enzymes compared to the controls.

In vitro binding of aflatoxin B1 and 2-acetylaminofluorene to rat, mouse and human hepatocyte DNA: the relationship of DNA binding to carcinogenicity.

DNA binding levels were determined and compared in cultured hepatocytes from male and female rats as well as other animal species following exposure to aflatoxin B1 (AFB1) or 2-acetylaminofluorene (2-AAF). When human, rat (both male and female) and mouse hepatocytes in primary culture were exposed to 2.0 X 10(-7) M [3H]AFB1 (sp. act. 2.63 microCi/nmol) for 24 h, male rat hepatocytes had the highest degree of [3H]AFB1-DNA binding (203 pmol/mg DNA) and human hepatocytes contained the next highest binding level (42 pmol/mg DNA). Hepatocytes from female rats contained 38 pmol/mg DNA while cultured mouse hepatocytes contained only 1.4 pmol/mg DNA. When the same dose of [3H]AFB1 was administered to the cultured male rat hepatocytes at 24 h, 48 h, 72 h and 1 week after seeding, and incubated for 24 h, the DNA binding levels were 189, 175, 76, 75 pmol/mg DNA respectively. In parallel experiments to the cultured male rat hepatocytes above, the AFB1-DNA binding levels in the cultured female hepatocytes were 42, 41, 37 and 34 pmol/mg DNA respectively. Human, male and female rat hepatocytes in primary culture were exposed to 5.2 X 10(-5) M 2-acetylamino[9-14C]fluorene (sp. act. 0.0094 microCi/nmol) for 24 h. It was determined that male rat hepatocytes contained the highest amount of radioactively labeled 2-AAF bound to their DNA (1.57 nmol/mg DNA), female rat hepatocytes contained 0.62 nmol/mg DNA and human hepatocytes contained 0.29 nmol/mg DNA. Results from our in vitro hepatocyte culture system correlate well with in vivo animal studies dealing with species and sex differences in DNA binding and carcinogenic susceptibility. This indicates that hepatocytes in vitro maintain many of the biological properties necessary for carcinogen response similar to liver cells in vivo. In addition, comparison of genotoxic effect in cultured hepatocytes from animals as well as humans may be useful in evaluating carcinogenic potential of xenobiotics in human liver.

Metabolism of alfentanil by isolated hepatocytes of rat and dog.

1. The biotransformation of 3H-alfentanil was studied using suspension cultures of isolated hepatocytes of male and female rats and of dogs. 2. In hepatocytes of the male rat, alfentanil was readily metabolized, following linear Michaelis-Menten kinetics over the concentration range 5-400 microM. The metabolism was strongly inhibited by the cytochrome P-450 inhibitors metyrapone, alpha-naphthoflavone and piperonyl butoxide. 3. The major metabolites of alfentanil, which were formed in suspension cultures of male rat hepatocytes, were identified by h.p.l.c. co-chromatography and by mass spectrometry and included N-[4-(hydroxymethyl)-4-piperidinyl]-N-phenylpropanamide, N-[4-(methoxymethyl)-4-piperidinyl]-N-phenylpropanamide or noralfentanil and N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1-H-tetrazol-1-yl)ethyl]- 4-(hydroxymethyl)-4-piperidinyl]-N-phenylpropanamide or desmethylalfentanil. 4. The major in-vitro metabolic pathways of alfentanil in hepatocytes of the three sources were oxidative N-dealkylation at the piperidine nitrogen and oxidative O-demethylation at the methoxymethyl moiety.

Chemical reactivity, cytotoxicity, and mutagenicity of chloropropanones

Studies were conducted to assess the in vitro toxicity of three chloropropanones: monochloropropanone (MCP), 1,1-dichloropropanone (1,1-DCP), and 1,3-dichloropropanone (1,3-DCP). Chloropropanones reacted directly with reduced glutathione (GSH) in sodium phosphate buffer at pH 7.4. All chloropropanones were cytotoxic to suspensions of male rat hepatocytes in a concentration range of 0.5–10 m M. Cytotoxicity was preceded by rapid decline in cellular GSH levels. Mutagenic potencies among the chloropropanones in Salmonella typhimurium bacteria differed greatly. 1,3-DCP was mutagenic in the nanomole range, 1,1-DCP was weakly mutagenic in the micromole range, and MCP was not mutagenic. Mutagenicity of the dichloropropanones was evident without metabolic activation. These results suggest that the three chloropropanones may, in part, be directly cytotoxic but only 1,3-DCP and 1,1-DCP are directly mutagenic.

Metabolism and covalent binding of hexachlorobenzene by isolated male and female rat hepatocytes

Metabolism and covalent binding of hexachlorobenzene by isolated male and female rat hepatocytes

Cytoplasmic androgen binding protein of rat liver: molecular characterization after photoaffinity labeling and functional correlation with the age-dependent synthesis of .alpha.2u-globulin

The liver of the mature male rat contains a moderate affinity (Kd = 10(-8)M), low-capacity, cytoplasmic androgen binding protein (CAB) whose appearance during puberty and disappearance during senescence correlate with the androgen-dependent synthesis of alpha 2u-globulin. Molecular properties of CAB were examined by photoaffinity labeling with tritiated methyltrienolone (R-1881), a synthetic androgen, and by its localization within the hepatocytes which are competent to produce alpha 2u-globulin. Photoaffinity labeling of the liver cytosol derived from postpubertal male rats, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, showed a predominant androgen binding band corresponding to Mr 31,000. This 31-kilodalton (kDa) binding component was conspicuously absent in the liver of androgen-insensitive prepubertal and senescent male rats and in adult male rats treated with estradiol-17 beta. In addition, unlike the cytoplasmic extract, the nuclear lysate of the male rat hepatocytes did not contain the 31-kDa androgen binder. Disappearance of the 31-kDa androgen binding band from the cytosolic fraction of androgen-insensitive animals was associated with a concomitant appearance of a minor androgen binding component of apparent Mr 29,000. The livers of postpubertal male rats normally contain two subpopulations of hepatocytes, only one of which is highly active (competent) in alpha 2u-globulin synthesis. Separation of these two subpopulations through a fluorescence-activated cell sorter followed by whole cell labeling showed more than a 2-fold higher uptake of R-1881 by the competent cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of the covalent binding potential of 2,2,4-trimethylpentane to rat α2u-globulin

Subchronic exposure of male rats to the nephrotoxin 2,2,4-trimethylpentane (TMP) causes an accumulation of protein droplets in the epithelial cells of the renal cortex. Experimental evidence suggests that these droplets contain α 2u-globulin, a low-molecular-weight protein found specifically in the urine of male rats. It has been proposed that aldehyde metabolites of TMP form Schiff base adducts with the lysine groups of α 2u-globulin and thereby inhibit renal lysosomal processing of the protein. Accordingly, the ability of TMP and its metabolites to covalently bind to α 2u-globulin was examined. As a model, a [ 14C]formaldehyde -α 2u - globulin Schiff base was formed. This protein adduct was stabilized by reduction with cyanoborohydride and could be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein analysis by SDS-PAGE demonstrated that hepatocytes isolated from male Fischer-344 rats produced significant quantities of α 2u-globulin in culture, whereas hepatocytes from female rats did not. A 15-hr exposure of metabolically competent, primary cultures of male rat hepatocytes to [ 14C]TMP (0.1 and 0.5%, v/v), followed by reduction with cyanoborohydride, dialysis, and analysis with SDS-PAGE, revealed no evidence of radiolabeled α 2u-globulin. When [ 14C]TMP was administered to an adult male Fischer-344 rat (300 mg/kg, ig) 22, 16, and 10 hr before sacrifice, 16% of the administered radioactivity was eliminated in the urine as TMP metabolites. Analysis as above showed no TMP-derived radioactivity in fractions containing α 2u-globulin from liver, blood, kidney cortex, or urine. The absence of a detectable covalent interaction between TMP and α 2u-globulin following in vitro or in vivo exposure suggests that a TMP- α 2u-globulin adduct is not responsible for the excessive formation of protein droplets in the renal cortex of exposed male rats.

Potential significance of phenotypic heterogeneity of focal lesions at different stages in hepatocarcinogenesis.

In this study we used morphometric methods to investigate the number, size and phenotype of foci of altered hepatocytes in male rats after limited (7 weeks) oral administration of N-nitrosomorpholine (stop-model). We found a chronological sequence from clear and eosinophilic cell foci (CCF and ECF) of early appearance followed by intermediate and mixed (MCF) and basophilic cell foci (BCF). Eventually, neoplastic nodules (NN) and hepatocellular carcinomas (HC) developed. The animals killed first (7 weeks) showed a large number of CCF and ECF and virtually no MCF and BCF. During the following weeks, we observed a temporary increase in the number of MCF and a progressive decrease in the number of CCF and ECF. A few BCF appeared for the first time 5 weeks after cessation of treatment. Subsequently there was an increase in the number of BCF and a decrease in the number of MCF, the latter starting 20 weeks after withdrawal of the carcinogen. MCF were found most frequently in animals with a high incidence of CCF. BCF and tumours were found most frequently in animals with a high incidence of MCF. The increase in the number of CCF was due to the appearance of small foci of this phenotype. However, the increase in the number of MCF and BCF was not related to an increase in number of small foci of these phenotypes. On the contrary, while the total number of MCF and BCF increased, there was a decrease in the incidence of small foci, but an increase in the incidence of large foci of these phenotypes. From these results, we concluded that phenotypically different foci essentially reflect different stages in the process of hepatocarcinogenesis. Moreover, the lack of small MCF and BCF suggests that the transition from CCF and ECF to MCF and finally to BCF is the result of a conversion of large cell populations within foci from 'early' to 'late' phenotypes rather than the consequence of a repeated clonal selection.

Effect of volatile anesthetics on protein synthesis and secretion by rat hepatocyte suspensions.

The effect of volatile anesthetics on protein synthesis and secretion by isolated rat hepatocytes in suspension was investigated. Halothane and enflurane inhibited protein synthesis in a dose-dependent manner. Diethyl ether had little effect on protein synthesis while isoflurane caused a mild inhibition. This effect was more pronounced in hepatocytes from phenobarbital treated male rats when compared to hepatocytes from control rats. Protein synthesis in hepatocytes from phenobarbital treated female rats was inhibited similar to that seen with control male rat hepatocytes. Isoflurane, enflurane, and halothane also caused a dose-dependent inhibition of protein secretion, while diethyl ether was only mildly inhibitory. From these studies it appears that inhibition of protein synthesis and secretion might be an early and sensitive indicator of cellular injury by volatile anesthetics.

Inhibition of gluconeogenesis and urea synthesis in isolated rat hepatocytes by acetazolamide

Citrulline generation in isolated rat liver mitochondria is markedly inhibited by the carbonic anhydrase inhibitor acetazolamide (Dodgson et al., 1983). This finding suggests that hepatic mitochondrial-matrix carbonic anhydrase may exert an important controlling effect on ureogenesis by supplying bicarbonate for carbamoyl phosphate synthesis. Three different carbonic anhydrase isoenzymes have been identified in mammals and designated CAI, CAII and CAIII. In liver CAII is uniquely sensitive to inhibition by acetazolamide and is present in a 4-fold higher concentration in the hepatocyte cytosol of female rats than in males (Jeffrey et al., 1984). with higher cytosol concentration of CAIII being found in males (Shiels et al., 1984). Sex differences in mitochondrial carbonic anhydrase have not been reported. We have extended these findings by examining the effect of acetazolamide on rates or ureogenesis and gluconeogenesis in isolated rat hepatocytes from male and female rats. Isolated rat hepatocytes were prepared from 48 h-starved male and female SpragueDawley rats by collagenase perfusion and suspended in Krebs bicarbonate buffer (Krebs & Henseleit, 1932) gassed with O-,/CO, (19 : 1). Samples (2ml) of cell suspension (30-60mg wet wt.) were incubated with I .8mM-NH4CI. SmM-L-( +)-lactate and 0.2mM-ornithine in the presence or absence of different concentrations of acetazolamide. Incubations were terminated after either 10 or 20min by the additions of 4.5ml of 0.6M-HCI0,. Urea and glucose were determined in the deproteinized supernatant by standard spectrophotometric methods. Rates of ureogenesis and gluconeogenesis were determined between the 10 and 20min time points. Ureogenesis and gluconeogenesis in male rat hepatocytes were markedly inhibited by acetazolamide (Fig. 1 ) . Maximum inhibition of ureogenesis (7506) occurred at acetazolamide concentrations between 0.5 and I .OmM, with 25% inhibition evident at 0.005 mM. Gluconeogenesis was inhibited by a maximum of 400; at I.0rnM-acetazolamide. with l2:$ inhibition at 0.05mM. An identical degree of inhibition of ureogenesis was obtained with hepatocytes from female rats. These data suggest that inhibition of ciirbonic anhydrase by acetazolamide may decrease the supply of intramitochondrial bicarbonate for both the carbamoyl-phosphate synthetase and pyruvate carboxylase reactions. I t is thus possible that bicarbonate availability may be a limiting factor in both urea and glucose production. This conclusion is supported by the demonstrations of inhibition of ureogenesis by metabolic acidosis at a site proximal to citrulline synthesis (Monson r t ul.. 1984). The observed similarity between acetarolamide inhibition of urea production in hepatocytes from mule and female rats suggests the presence of similar mitochondria1 concentrations in males and females of one or more susceptible carbonic anhydrase isozymes. The possibility of mechanisms other than carbonic anhydrase inhibition being responsible for the effects of acetazolc 20

Unscheduled DNA synthesis caused by norethindrone and related contraceptive steroids in short-term male rat hepatocyte cultures.

The genotoxic potential of oral contraceptive steroids such as norethindrone was investigated in short-term rat hepatocyte cultures by measurement of unscheduled DNA synthesis. Norethindrone caused a small dose-dependent increase in unscheduled DNA synthesis in male rat hepatocytes as judged by the incorporation of [methyl-3H]thymidine into DNA. This was assessed either by liquid scintillation counting following isolation of DNA or by autoradiography. No increase in unscheduled DNA synthesis could be detected in female rat hepatocytes treated with norethindrone. Pre-treatment of male rats with phenobarbitone prior to hepatocyte preparation decreased the norethindrone mediated unscheduled DNA synthesis relative to control hepatocyte cultures while 3-methylcholanthrene pre-treatment had little effect. Unscheduled DNA synthesis in norethindrone treated control male rat hepatocytes was reduced by the mixed function oxidase inhibitors SKF 525A or metyrapone. In 24- or 52-hour-old hepatocyte cultures in which the cytochrome P-450 content was lower than in freshly prepared cells, or in a hepatocyte-derived cell line lacking cytochrome P-450, unscheduled DNA synthesis due to norethindrone was either decreased or abolished. Structure activity studies showed that only steroids containing a 17 alpha-ethynyl substituent caused an increase in unscheduled DNA synthesis.

Cytosolic Ca2+ variations induced by specific receptor-mediated stimulation in male and female rat hepatocytes

Cytosolic Ca2+ variations induced by specific receptor-mediated stimulation in male and female rat hepatocytes