Control Rat Hepatocytes Research Articles

Dynamics of proglycogen and macroglycogen in hepatocytes of normal and cirrhotic rat liver at various stages of glycogenesis

The content and structure of glycogen in hepatocytes of normal and cirrhotic rat liver were examined at different time intervals after glucose administration to starving animals. We used an original cytofluorimetric method for detection and quantification of proglycogen (PG) and macroglycogen (MG) of isolated hepatocytes. The method is based on the use of reagents of the Schiff type with different spectral characteristics. The content of MG in hepatocytes of control rats was increased by 52% (p < 0.01) as early as after 10 min. The MG content in the cirrhotic liver cells was increased by 43% (p < 0.05) only 20 min after glucose administration to the starving animals. The correlation coefficient between MG content and the total glycogen content at various stages of glycogenesis in rats of both groups was from 0.90 to 0.99 (p < 0.001). Increase in the PG content in hepatocytes of control rats was observed in intervals of 10–30 and 45–75 min. The PG content in cirrhosis was increased only in 60 min after the beginning of glycogenesis, but in 120 min it was 1.5 times higher than the control values (p < 0.001). The correlation coefficients between PG and the total glycogen content in the cells were on average 0.86 (p < 0.001) and 0.77 (p < 0.001) in the control and experimental groups, respectively. Thus, the change in the total glycogen content in hepatocytes of normal and cirrhotic liver are associated mainly with changes in the MG level. The contribution of PG was most significant in normal liver at the beginning of glycogenesis (10–30 min); in cirrhotic liver, at later stages (75–120 min).

Chronic EtOH administration alters liver Mg2+ homeostasis.

Ethanol (EtOH) administration to rats for 4 wk markedly decreased Mg(2+) content in several tissues, including liver. Total cellular Mg(2+) accounted for 26.8 +/- 2.4 vs. 36.0 +/- 1.4 nmol Mg(2+)/mg protein in hepatocytes from EtOH-fed and control rats, respectively, and paralleled a 13% decrease in cellular ATP content. Stimulation of alpha(1)- or beta-adrenergic receptor or acute EtOH administration did not elicit an extrusion of Mg(2+) from liver cells of EtOH-fed rats while releasing 5% of total tissue Mg(2+) content from hepatocytes of control rats. Despite the 25% decrease in Mg(2+) content, hepatocytes from EtOH-fed rats did not accumulate Mg(2+) following stimulation of protein kinase C signaling pathway, whereas control hepatocytes accumulated approximately 2 nmol Mg(2+). mg protein(-1). 4 min(-1). Together, these data indicate that Mg(2+) homeostasis and transport are markedly impaired in liver cells after prolonged exposure to alcohol. The inability of liver cells, and possibly other tissues, to accumulate Mg(2+) can help explain the reduction in tissue Mg(2+) content following chronic alcohol consumption.

Increased expression of multidrug resistance-associated protein 1 (mrp1) in hepatocyte basolateral membrane and renal tubular epithelia after bile duct ligation in rats

Components of the multidrug resistance-associated protein (mrp) family mediate the adenosine triphosphate (ATP)-dependent transport of conjugated organic anions in the liver. Of these, mrp1 and mrp2 have been shown to have similar substrate specificity and nucleotide sequence. The intracellular localization and distribution of mrp1 under normal condition and cholestasis have not been as yet completely elucidated. To clarify this point, in the present study we evaluated the intracellular localization of mrp1 in rat liver and kidney after bile duct ligation (BDL). Bile duct was ligated in Wistar rats. Sequential staining of mrp1 by immunofluorescence was carried out in rat liver and kidneys 1, 3, and 5 days after bile duct ligation using confocal laser scanning microscopy. Weak granular staining of mrp1 was observed in cytoplasm of control rat hepatocytes. In addition to increased cytoplasm staining of mrp1, belt-and granule-like staining of mrp1 in basolateral membrane of hepatocytes was also shown after BDL. Furthermore, mrp1 immunofluorescence increased over time after BDL. No specific immunoflurescence of mrp1 was detected in control rat kidney. However, mrp1-positive staining was observed in epithelia of some renal tubules after BDL. This study showed that mrp1 immunofluorescence increased in hepatocyte basolateral membrane and cytoplasm and epithelia of some renal tubules after BDL. This increased mrp1 expression may be an adaptive response to impairment of hepato-biliary organic anion transport during obstructive cholestasis.

Immunohistochemical demonstration of beta-naphthoflavone-inducible cytochrome P450 1A1/1A2 in rat intrahepatic biliary epithelial cells.

Although intrahepatic biliary epithelial cells are targets for certain hepatotoxic chemicals, including some procarcinogens, their ability to monooxygenate, and thereby bioactivate and inactivate xenobiotics, remains to be established. Thus, the present study was undertaken to immunohistochemically determine if cytochrome P450 (CYP) 1A1/1A2 is present and can be induced within these nonparenchymal liver cells. Immunoperoxidase and immunofluorescent staining for CYP1A1/1A2 was detected within intrahepatic biliary epithelial cells as well as hepatocytes of control rats and was markedly enhanced in both cell types by beta-naphthoflavone (BNF). Color confocal laser microscopic analyses of dual immunofluorescent staining for CYP1A1/1A2 and cytokeratins 6 and 9 (56 and 64 kd, respectively) provided unequivocal evidence for the presence and induction of CYP1A1/1A2 within intrahepatic bile duct epithelia. Moreover, microdensitometric analyses of immunoperoxidase staining intensities for CYP1A1/1A2 revealed that intrahepatic biliary epithelial cells of control rats contain 44%, 56%, and 58% as much CYP1A1/1A2 as do centrilobular, midzonal, and periportal hepatocytes, respectively. These analyses further revealed that BNF increased the content of CYP1A1/1A2 in biliary epithelial cells by approximately 120%, while CYP1A1/1A2 levels in centrilobular, midzonal, and periportal hepatocytes were increased by 82%, 159%, and 160%, respectively. The results of this study represent the first in situ demonstration that mammalian intrahepatic biliary epithelial cells contain a CYP isoform, and further that CYP1A1/1A2 can be induced in these cells by BNF. These findings therefore indicate that intrahepatic biliary epithelial cells can oxidatively metabolize xenobiotics in situ and that their ability to bioactivate and inactivate xenobiotics can be significantly enhanced by CYP1A1/1A2 induction.

Bile acid synthesis in primary cultures of rat and human hepatocytes.

The regulation of hepatic bile acid formation is incompletely understood. Primary cultures of mammalian hepatocytes offer an opportunity to examine putative regulatory factors in relative isolation. Using rat and human hepatocytes in primary culture, we examined bile acid composition and the expression of the rate-limiting enzyme of formation, cholesterol 7alpha-hydroxylase. Control rat hepatocytes showed a declining bile acid production over 4 days, from 156 +/- 24 ng/mL (67% cholic acid) on day 1 to 55 +/- 11 ng/mL (55% cholic acid) on day 4. In addition to cholic acid, chenodeoxycholic acid, alpha-muricholic acid, and beta-muricholic acid were formed. Treatment with triidothyronine (T3) or dexamethasone alone had no significant effect on bile acid production. A combination of T3 and dexamethasone significantly increased the total bile acid production on day 4 (224 +/- 54 ng/mL) and resulted in a marked change in composition to 23% cholic acid and 77% non-12alpha-hydroxylated bile acids. Control rat hepatocytes had a cholesterol 7alpha-hydroxylase activity of 3.3 +/- 0.6 pmol/mg protein/min after 4 days in culture. Cells treated with the combination of dexamethasone and T3 had an activity of 16.4 +/- 3.6 pmol/mg protein/min. The cholesterol 7alpha-hydroxylase messenger RNA (mRNA) levels, determined by solution hybridization after 4 days of culture, showed results similar to those for the activity data; control cells had 5.3 +/- 0.9 cpm/microg total nucleic acids (tNAs). T3 or dexamethasone-treated cells did not differ from control cells, whereas the combination of T3 and dexamethasone increased the mRNA levels to 20.6 +/- 2.8 cpm/microg tNAs. In human hepatocytes, isolated from donor liver, bile acid formation increased from 206 +/- 79 ng/mL on day 2 to 1490 +/- 594 ng/mL on day 6 and then declined slightly. Cholic acid and chenodeoxycholic acid were formed, constituting about 80% and 20%, respectively. The combined addition of T3 and dexamethasone had a tendency to decrease rather than increase bile acid formation. Also, mRNA levels of the cholesterol 7alpha-hydroxylase increased severalfold in the human hepatocytes from day 2 to day 4 and then declined. The addition of T3 or dexamethasone did not effect the mRNA levels in any consistent way. It is noteworthy that the capacity of the cultured human hepatocytes to produce bile acids was higher than that of cultured rat hepatocytes, in spite of the fact that the production of bile acids in rat liver is 3- to 5-fold higher than that in human liver in vivo. It is also evident that while hormonal factors appear to regulate bile acid synthesis in the rat, no evidence for this was found in human hepatocytes. As the composition of bile acids secreted by human hepatocytes in primary culture closely resembles that found in vivo, this represents a useful model for further studies of the synthesis and regulation of bile acids.

Effects of phenobarbital and 3-methylcholanthrene induction on the formation of three glucuronide metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a tobacco specific carcinogen believed to be a causative agent for human lung cancer. To exert its carcinogenic potential, NNK must be metabolically activated, by α-hydroxylation, at either the methyl or methylene carbons adjacent to the N-nitroso group. We recently reported the presence of a glucuronide conjugate of 4-(hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (α-hydroxymethylNNK-Gluc) in the urine of Phenobarbital (PB) treated rats, and in the media of PB induced hepatocytes incubated with NNK. PB induces the α-hydroxylation of NNK, which generates the aglycon, as well as several UDP-glucuronosyl transferases. In the study presented here, we compared the metabolism of NNK to α-hydroxymethylNNK-Gluc by PB induced, 3-methylcholanthrene (3-MC) induced and control rat hepatocytes. Media was analyzed for the products of α-hydroxylation, N-oxidation and glucuronidation by radioflow HPLC. PB induced both N-oxidation and α-hydroxylation of NNK. 3-MC did not induce N-oxidation but induced α-hydroxylation more than 10-fold. α-HydroxymethylNNK-Gluc was not detected (< 0.05% total metabolites) when control hepatocytes were incubated with 1 to 100 μM NNK. When 3-MC and PB induced hepatocytes were incubated with 1–100 μM NNK α-hydroxymethylNNK-Gluc, expressed as the average percent of metabolites, accounted for 0.725 ± 0.27 and 1.35 ± 0.24% (± S.D.) of the NNK metabolites, respectively. The percent of NNK metabolized to α-hydroxymethylNNK-Gluc is small. But this glucuronide is potentially important in NNK carcinogenesis, since its formation results in the direct conjugation of an active metabolite responsible for DNA adduct formation. When PB induced rats were injected with NNK the level of NNK hemoglobin adducts, which can serve as surrogates for DNA adducts, decreased 50% compared to control rats administered NNK. Hepatic microsomal metabolism increased 2-fold and urinary α-hydroxymethylNNK-Gluc increased more than 10-fold in PB treated rats. One explanation for the decrease in NNK hemoglobin adducts may be a PB induced increase in the glucuronidation of α-hydroxymethylNNK, the metabolite responsible for adduct formation.

Hypocalcemia decreases the early and late responses to epidermal growth factor in rat hepatocytes

Extreme variations in extracellular Ca2+ concentrations ([Ca2+]e) modify the signaling generated by many hormones and growth factors. However, the influence of physiological changes in [Ca2+]e on the response to hepatic mitogens remains largely unknown. To study the influence of [Ca2+]e on the response to epidermal growth factor (EGF), hepatocytes from normal rat livers were equilibrated in vitro at [Ca2+]e similar to those observed in normocalcemia or hypocalcemia. To further investigate the effect of hypocalcemia in vivo, hepatocytes were obtained from chronically hypocalcemic rats and kept in vitro at the [Ca2+]e prevailing in vivo. Intracellular Ca2+ concentrations ([Ca2+]i) and DNA synthesis were evaluated after increasing doses of EGF. [Ca2+]e strongly influenced the [Ca2+]i response to EGF with significantly smaller [Ca2+]i increases in hepatocytes of normal rats kept in low [Ca2+]e compared with those kept in normal [Ca2+]e. In hypocalcemic rat hepatocytes, the response was further decreased and found to be significantly lower than that obtained in control cells kept in vitro at either 1.25 mmol/L or 0.8 mmol/L [Ca2+]e. In normal [Ca2+]e, the EGF-induced increases in [Ca2+]i were abolished by inhibiting EGF receptor autophosphorylation and by blocking calcium channels. Low in vitro [Ca2+]e significantly dampened the EGF-mediated DNA synthesis in normal rat hepatocytes but hypocalcemia in vivo further reduced the proliferative response compared with that obtained in control rat hepatocytes maintained in normal, or low [Ca2+]e. Furthermore, the blunted responses in [Ca2+]i mobilization and DNA synthesis associated with hypocalcemia could not be overcome by increasing concentrations of EGF nor by normalization of [Ca2+]e in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypocalcemia decreases the early and late responses to epidermal growth factor in rat hepatocytes

Extreme variations in extracellular Ca 2+ concentrations ([Ca 2+] e) modify the signaling generated by many hormones and growth factors. However, the influence of physiological changes in [Ca 2+] e on the response to hepatic mitogens remains largely unknown. To study the influence of [Ca 2+] e on the response to epidermal growth factor (EGF), hepatocytes from normal rat livers were equilibrated in vitro at [Ca 2+] e similar to those observed in normocalcemia or hypocalcemia. To further investigate the effect of hypocalcemia in vivo, hepatocytes were obtained from chronically hypocalcemic rats and kept in vitro at the [Ca 2+] e prevailing in vivo. Intracellular Ca 2+ concentrations ([Ca 2+] i) and DNA synthesis were evaluated after increasing doses of EGF. [Ca 2+] e strongly influenced the [Ca 2+] i response to EGF with significantly smaller [Ca 2+] i increases in hepatocytes of normal rats kept in low [Ca 2+] e compared with those kept in normal [Ca 2+] e. In hypocalcemic rat hepatocytes, the response was further decreased and found to be significantly lower than that obtained in control cells kept in vitro at either 1.25 mmol/L or 0.8 mmol/L [Ca 2+] e. In normal [Ca 2+] e, the EGF-induced increases in [Ca 2+] i were abolished by inhibiting EGF receptor autophosphorylation and by blocking calcium channels. Low in vitro [Ca 2+] e significantly dampened the EGF-mediated DNA synthesis in normal rat hepatocytes but hypocalcemia in vivo further reduced the proliferative response compared with that obtained in control rat hepatocytes maintained in normal, or low [Ca 2+] e. Furthermore, the blunted responses in [Ca 2+] i mobilization and DNA synthesis associated with hypocalcemia could not be over-come by increasing concentrations of EGF nor by normalization of [Ca 2+] e in vitro. These data demonstrate that [Ca 2+] e within the physiological concentration range can strongly influence the hepatocyte response to EGF. At [Ca 2+] e comparable to that of hypocalcemia in vivo, our data point to the appearance of a phenomenon of hepatocellular resistance to the early (increases in [Ca 2+] i) and late (DNA synthesis) cellular responses to EGF.

Heterogeneous expression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase genes in the rat liver lobulus.

We investigated the lobular localization and molecular level of expression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase, two key enzymes in bile acid synthesis, in isolated periportal and pericentral hepatocytes and by in situ hybridization of rat liver. Enzyme activity, mRNA, and gene transcription of cholesterol 7 alpha-hydroxylase were predominant in pericentral hepatocytes of control rats, being 7.9-, 9.9-, and 4.4-fold higher than in periportal hepatocytes, respectively. Similar localization was found for sterol 27-hydroxylase: 2.9-, 2.5-, and 1.7-fold higher enzyme activity, mRNA, and gene transcription, respectively, was found in pericentral hepatocytes. Interruption of the enterohepatic circulation with colestid resulted in upregulation of these parameters for both enzymes, as a consequence of stimulated gene expression mainly in the periportal zone. In contrast, mRNA levels and gene transcription of 3-hydroxy-3-methylglutaryl CoA reductase showed opposite lobular distribution. Selective periportal expression for the latter was enhanced, but remained local, after colestid treatment. In situ hybridization showed unambiguously that cholesterol 7 alpha-hydroxylase mRNA is localized exclusively in the pericentral zone and that sterol 27-hydroxylase mRNA is expressed preferentially in the pericentral region, though less pronounced. Administration of colestid led to expression of both genes within a larger area of the liver lobulus. In conclusion, we suggest that cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase are coordinately regulated by the bile acid gradient over the lobulus, resulting in predominant expression in the pericentral zone. Opposite lobular localization of cholesterol and bile acid synthesis provides an alternative view to interregulation of these metabolic pathways.

A microtiter plate assay for total glutathione and glutathione disulfide contents in cultured/isolated cells: performance study of a new miniaturized protocol.

The microtiter plate technique reported by Baker and colleagues for the glutathione reductase-DTNB recycling assay of total glutathione (GSx) and glutathione disulfide (GSSG) has been modified according to Anderson's recommendations, in order to improve the reliability and accuracy of this miniaturized method for the measurement of glutathione status in cultured/isolated cells. Dilute HCl (10 mmol/L) has been used to lyse cells, before protein removal by centrifugation in the presence of 1.3% sulfosalicylic acid. The final DTNB, GSSG-reductase and NADPH concentrations in the reaction mixture have been increased to 0.7 mmol/L, 1.2 IU/ml and 0.24 mmol/L, respectively. The procedure specificity has been tested by spiking and dilution assays, showing that about 90% of the expected GSx amounts could actually be recovered, while no changes of GSSG concentrations were caused in the cells. Accuracy has been assessed by analysis of within-series precision as well as of intra- and interassay reproducibility, showing coefficient variation of < 10%. Glutathione changes measured either in control rat hepatocytes or in primary cultures treated with paracetamol or menadione were in good agreement with well-known literature data. These data suggest that the experimental conditions reported in this paper are suitable for the analysis of total glutathione and glutathione disulfide concentrations in cultured/isolated cells.

Modulation of KC/gro Protein (Interleukin-8 Related Protein in Rodents) Release from Hepatocytes by Biologically-Active Mediators

Modulation of the release of KC/gro protein (a chemoattractant for neutrophils; IL-8 related protein in rodents) from isolated hepatocytes after stimulation with biologically active mediators was investigated. The release of KC/gro protein from hepatocytes of control rats was enhanced by stimulation with lipopolysaccharide, IL-1beta or TNF-alpha in a dose dependent manner, but was not enhanced by IL-6. In contrast, although spontaneous release of KC/gro protein from the hepatocytes of chronically ethanol-fed rats was markedly enhanced as compared with control rats, the relative increase by stimulation with lipopolysaccharide, IL-1beta or TNF-alpha was significantly smaller than in controls. These findings suggest that the regulation of hepatocyte KC/gro protein production might be disturbed in chronically ethanol-fed rats.

Cellular distribution of nuclear factor kappa B binding activity in rat liver.

The cellular localization of nuclear factor kappa B (NF-kappa B) binding activity in rat liver has been investigated using electrophoretic mobility shift assay on extracts of highly purified hepatocytes and Kupffer cells obtained from liver perfused in vivo with collagenase. Constitutive NF-kappa B binding activity was demonstrated in nuclear extracts of control Kupffer cells, and this was not apparently influenced by injection of lipopolysaccharide (LPS) into rats 24 h before perfusion. In contrast, little nuclear NF-kappa B binding activity was present in hepatocytes from control animals, although there was detectable inactive, inhibitor-bound, NF-kappa B in the cytoplasm. However, nuclear NF-kappa B binding activity was increased in hepatocytes from LPS-treated animals and after in vitro culture of control rat hepatocytes. Thus NF-kappa B binding activity has been demonstrated in highly purified hepatocytes and appears to be inducible both in vivo and in vitro. These findings support a role for NF-kappa B in hepatocyte gene regulation which may be important in the modulation of the hepatic acute phase response.

Immunolocalization, quantitation and cellular heterogeneity of apolipoprotein B in rat hepatocytes.

Hepatocyte autofluorescence represents a major problem in immunofluorescence studies with fluorescein conjugates because of significant spectral overlap. We describe a method for immunostaining hepatocytes with R-phycoerythrin (a fluorochrome with minimal overlap with autofluorescence) with paraformaldehyde fixation and Triton X-100 permeabilization for better antibody penetration. This method produced both perinuclear (presumed Golgi apparatus) and dispersed, reticular staining (presumed endoplasmic reticulum) in rat hepatocytes in culture stained with a monoclonal antibody to rat apolipoprotein B. Treatment with brefeldin A resulted in loss of apolipoprotein B perinuclear staining and increased reticular immunofluorescence consistent with known properties of brefeldin A (inhibition of protein transport within the secretory pathway by dissolution of Golgi bodies). This suggests that apolipoprotein B epitopes are present in both Golgi bodies and endoplasmic reticulum. To demonstrate the utility of the technique for quantitative studies, static cell cytofluorometry of brefeldin A-treated cells was performed, demonstrating increases in specific immunofluorescence of apolipoprotein B corresponding closely to results estimated by monoclonal antibody radioimmunoassays of cellular homogenates. The technique was then used with flow cytometry of single-cell suspensions of control rat hepatocytes derived from immunostained primary cultures to reveal cell-to-cell heterogeneity of apolipoprotein B epitope expression manifested as apolipoprotein B-negative and positive populations. Results for brefeldin A-treated cells revealed even clearer delineation of heterogeneity as indicated by frank bimodality of the populations, along with not only higher mean apolipoprotein B levels but also a significantly higher proportion of apolipoprotein B-positive cells than in the control.

Secretion of high and low molecular weight phosphorylated apolipoprotein B by hepatocytes from control and diabetic rats. Phosphorylation of APO BH and APO BL.

Apolipoprotein B (apo B) phosphorylation was examined in primary cultures of hepatocytes from control and nonketotic streptozotocin diabetic rats. Following a 5-h incubation with ortho[32P]phosphate, media lipoproteins (d less than 1.063 g/ml) were isolated, and delipidated apolipoproteins were separated by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis (SDS-PAGGE), and gels were heat fixed. Autoradiographic bands corresponding to high (apo BH) and low molecular weight apo B (apo BL) were observed in media lipoproteins isolated from control rats, and these bands were more prominent in media lipoproteins isolated from diabetic rats. Apo B-specific activity was estimated from aqueous alcohol-precipitated radioactivity and apo B monoclonal immunoassay of isolated media lipoproteins. In lipoproteins secreted by hepatocytes of diabetic rats, the calculated apo B specific activity was between 18- and 31-fold greater than that secreted by hepatocytes of control rats, consistent with the SDS-PAGGE gel data. The increase in secretory 32P-labeled apo B from hepatocytes of diabetic rats was due, at least in part, to an increase in labeled phospho-tyrosine as determined by phosphoamino acid analysis. These data demonstrate that apo BH may be secreted as a phosphorylated protein and that apo B phosphorylation occurs on tyrosine as well as serine residues.

Acute and chronic effects of ethanol on secretion of alpha-tocopherol from primary cultures of rat hepatocytes

Primary cultures of rat hepatocytes were used to study the effect of acute and chronic ethanol exposure on α-tocopherol content in cells and media. Cells treated acutely with 60 mM ethanol secreted 74.5 ± 18.0% ( P < 0.05), and their cellular α-tocopherol content was 85.7 ± 15.4% (not significant) of controls after 20 h incubation. At this time total recovery of α-tocopherol was significantly reduced in ethanol-exposed cells (43.1 ±8.4%) as compared to control cells (52.8 ±5.0%, P < 0.05). Hepatocytes isolated from chronic ethanol-fed rats (35% of total energy intake as ethanol for 5 weeks) secreted 41.9 ± 12.7% less α-tocopherol than did cells of pair-fed controls during 20 h incubation ( P < 0.05). The amount of α-tocopherol secreted was then 15.6 ± 4.2 and 19.8 ± 3.8% of cell-associated α-tocopherol at start of incubation for chronic ethanol-fed and control rats, respectively ( P < 0.05). When 60 mM ethanol was added to the incubation medium, hepatocytes of control rats secreted significantly less α-tocopherol (about 30%, P < 0.05), whereas α-tocopherol secretion was not significantly reduced in hepatocytes of chronic ethanol-fed rats. We conclude that both acute and chronic ethanol exposure reduce α-tocopherol secretion from rat hepatocytes.

Inflammation-induced synthesis of proteoheparan sulfate: A novel acute-phase reactant in rat hepatocytes

The synthesis of proteoheparan sulfate in hepatocytes is positively regulated under acute-phase conditions produced either by turpentine or deep back incision. In both cases the incorporation of [35S]sulfate and [14C]glucosamine is doubled during a 4-h incubation period if compared with control rat hepatocytes. Neither the fractional secretion rate of heparan sulfate into the medium (less than 0.1 of cell-associated glycosaminoglycans) nor the composition of newly formed proteoglycans in hepatocytes are affected during acute phase reaction.

Endocytosis and binding of asialoorosomucoid by hepatocytes from rats with jejunoileal bypass.

Rats with jejunoileal bypass were used to study the biological activity of the hepatic binding protein. Hepatocytes were prepared 11 weeks after surgical procedure, and presence of asialoorosomucoid in serum has been determined. Compared to control rat hepatocytes, endocytosis of [3H]asialoorosomucoid by bypassed rat hepatocytes was decreased by about 60%. This was due to a decreased number of total and surface receptors. In most sera, an accumulation of asialoorosomucoid was found. A reduction of protein synthesis or turnover could be considered.

Aroclor 1254 pretreatment effects on DNA repair in rat hepatocytes elicited by in vivo or in vitro exposure to various chemicals.

Inducers of liver mixed function oxidase (MFO) activities have profound effects on the genotoxicity of substances that undergo metabolic activation by the MFO system. The polychlorinated biphenyl mixture Aroclor 1254 is a broad-spectrum inducer of liver MFO activities that has been employed as a pretreatment to augment the metabolic activation capabilities of rat liver fractions used in a number of short-term tests for genotoxicity, including the Ames Salmonella/bacterial mutagenicity assay. The present study was designed to characterize the effects of Aroclor pretreatment of rats on the DNA repair responses elicited by various chemicals in the in vitro hepatocyte primary culture/DNA repair (HPC/DR) assay as well as the in vivo/in vitro HPC/DR assay. The amount of DNA repair produced in vitro by diethylnitrosamine (DEN), benzo(a)pyrene (B(a)P), 3-methylcholanthrene (3-MC), 2-acetylaminofluorene (2-AAF), o-aminoazotoluene (o-AT), and aflatoxin B1 (AFB1) was significantly greater in hepatocytes derived from Aroclor-pretreated rats than in control rat hepatocytes; in vitro responses to dimethylnitrosamine (DMN), 7,12-dimethylbenzanthracene (DMBA), benzidine (BZ), and 2-naphthylamine (2-NA) were not significantly affected by Aroclor pretreatment. DNA repair elicited by the direct-acting alkylating agents methyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine was also not increased by Aroclor pretreatment, which indicated that Aroclor does not exert a general stimulatory effect on the hepatocellular DNA repair capacity. Therefore, the pretreatment-related potentiation of DNA repair observed for 6 out of 12 compounds tested in vitro was considered to be due to enhanced metabolic activation. These results suggested that pretreatment with Aroclor may increase the sensitivity of the in vitro HPC/DR assay to certain compounds. In contrast, Aroclor pretreatment had little effect on the amount of hepatocellular DNA repair elicited by in vivo administration of DMN, DEN, o-AT, 2-AAF, 3-MC, or AFB1, which indicated that this pretreatment regimen may have little utility for improving the sensitivity of the in vivo/in vitro HPC/DR assay.

Bile acids and lipids in isolated rat hepatocytes. II. Source of cholesterol used for bile acid formation, estimated by incorporation of tritium from tritiated water, and by the effect of ML-236B

After incubation in the presence of tritiated water, incorporation of tritium into cholesterol and into different bile acids was several-fold higher using hepatocytes of cholestyramine-fed rats than that found using hepatocytes of control rats. Labeling of the trihydroxylated cholic and beta-muricholic acids was markedly greater than that of dihydroxycholanoic acid. The total amount of label in all bile acids was 30% or less of that in free cholesterol, in both types of hepatocytes. In combination with the data on bile acids mass production we could calculate the average number (N(a)) of tritium atoms incorporated per molecule of newly-formed bile acid. The experimental values of N(a) for cholic and beta-muricholic acid were compared with values of N(n) or N(o), theoretically predicted if these bile acids were derived entirely from newly made or pre-existent cholesterol, respectively. It was deduced for hepatocytes of cholestyramine-fed rats that the bile acids produced in the first hour of incubation should be totally derived from pre-existent cholesterol, whereas 50% and 25% of the cholic and beta-muricholic acid, respectively, produced during the second and third hours of incubation should be derived from newly synthesized cholesterol. The contribution of newly made cholesterol as substrate for bile acid production was also estimated by using ML-236B. In a concentration of 12 micro M, it depressed cholesterol synthesis by 90% during 1 or 3 hours of incubation of hepatocytes of cholestyramine-fed rats. Mass production of cholic acid was depressed by 25% and that of beta-muricholic acid was not inhibited at all by ML-236B during the first hour of incubation, while they were depressed by 71 and 52%, respectively, during the second plus third hours of incubation. It is concluded that 1) in isolated hepatocytes newly made cholesterol can be a significant substrate for bile acid formation; 2) there are separate cholesterol substrate pools for the productions of cholic or beta-muricholic acid; 3) of the total carbon flux directed into cholesterol synthesis, the major part ends up as free cholesterol and only a minor part as bile acids, even in hepatocytes with a derepressed bile acid production.-Kempen, H. J., M. Vos-van Holstein, and J. de Lange. Bile acids and lipids in isolated rat hepatocytes. II. Source of cholesterol used for bile acid formation, estimated by incorporation of tritium from tritiated water, and by the effect of ML-236B.

Immunohistochemical localization of metallothionein in cell nucleus and cytoplasm of rat liver and kidney

Metallothionein (Mt) is a low molecular weight metalloprotein and its synthesis is induced by various divalent metals (Cd, Zn, Hg and Cu). The Intracellular distribution of Mt in hepatic and renal cells from control and CdCl 2 injected rats was investigated by immunohistochemical methods. Antiserum to purified rat-liver Mt was prepared in rabbits after partial polymerization of the protein. The unlabelled peroxidase-antiperoxidase method using the specific rabbit anti-rat liver Mt provided a sensitive technique to localize Mt in tissue sections. In control rats, Mt or thionein (metal-free protein) was mainly localized in the cytoplasm of hepatocytes, renal collecting duct epithelium and distal convoluted tubular epithelium. In rats, injected intraperitoneally (i.p.) with CdCl 2 (0.6 mg/kg) for 2 weeks, Mt was present mainly in the nuclei which were largely negative in control rats. Repeated injection with CdCl 2 for 4–8 weeks resulted in the appearance of Mt in both the nucleus and cytoplasm. Intraluminal staining was also noted in proximal convoluted tubules along with marked vacuolation in the cytoplasm at 6 and 8 weeks. High intensity staining was observed in proximal convoluted tubules and collecting duct epithelium of the kidneys in CdCl 2 injected rats. The bile duct epithelium in liver samples, renal glomerular mesangial cells, glomerular visceral epithelial cells and vascular smooth muscle cells showed weak to moderately intense staining. No staining was seen in vascular endothelial cells, fibroblasts or leukocytes. The staining for Mt in this technique was abolished when the antibody was absorbed with rat-liver Mt in vitro or by substitution of the antiserum with normal rabbit serum, demonstrating the specificity of the staining reaction for Mt. The results showed the presence of small amounts of Mt predominantly in the cytoplasm of control rat hepatocytes and renal tubular epithelium and its appearance in both the nucleus and cytoplasm after its synthesis induced by CdCl 2 injections.