Monolayer Cultures Of Rat Hepatocytes Research Articles

Kupffer cell-mediated differential down-regulation of cytochrome P450 metabolism in rat hepatocytes

Nonparenchymal cells, particularly Kupffer cells, might play an important role in the modulation of xenobiotic metabolism in liver and its pharmacological and toxicological consequences. This intercellular communication via the exchange of soluble factors was investigated in primary rat Kupffer cells and hepatocytes. Freshly isolated rat Kupffer cells were seeded onto cell culture inserts and cocultured with 5 day old serum-free rat hepatocyte monolayer cultures at a ratio of 1:1 for 2 days. Hepatocyte cultures, Kupffer cell cultures or cocultures were treated with 0.1 ng/ml–10 μg/ml lipopolysaccharide (LPS). Within this concentration range, no significant toxicity was observed in either cell type. In LPS-exposed cocultures, tumor necrosis factor α (TNFα) levels rose up to 5 ng/ml within 5 h; nitric oxide (NO) levels increased up to 70 μM within 48 h of treatment, both in a dose-dependent fashion. The release of negative (albumin) and positive (α1-acid-glycoprotein) acute phase proteins from the hepatocytes was strongly down- and up-regulated, respectively. The simultaneous treatment of the cocultures with phenobarbital and LPS (10 ng/ml) or 3-methylcholanthrene and LPS (10 ng/ml) resulted in a strong down-regulation (85%) of the phenobarbital-induced cytochrome P450 (CYP) isoform CYP2B1 in the hepatocytes whereas the 3-methylcholanthrene-induced isoform CYP1A1 was only weakly affected (15%). This specific down-regulation of CYP2B1 was mediated exclusively by TNFα, released from the Kupffer cells. It was not linked with NO release from or inducible NO synthase activity in the hepatocytes. The TNFα release was not affected by the two xenobiotics. Acetaminophen tested in these cocultures showed no direct interaction with the Kupffer cells. The use of liver cell cocultures is therefore a useful approach to investigate the influence of intercellular communication on xenobiotic metabolism in liver.

Effect of Kurozu(Brewed Rice Vinegar) on Maintenance of Primary Culture of Rat Hepatocytes.

Kurozu is a brewed rice vinegar prepared by a traditional method in Kagoshima prefecture, Japan, and is regarded as a healthful food. The effects of the non-volatile components obtained from Kurozu (the residue of Kurozu) on the primary monolayer culture of rat hepatocytes were examined. In serum-free L-15 medium supplemented with the residue of Kurozu, about 60% of the hepatocytes inoculated to a dish survived for 6 days of culture. In contrast, without the residue, the number of hepatocytes decreased to less than one-third of the inoculated cells during the culture. The amount of albumin secreted in the medium was monitored as an expression of liver-specific function. In the medium with the residue of Kurozu, the rat hepatocytes secreted a considerable amount of albumin in 4-day culture (2.5 μg/ml medium/24 h), and even at 6 days (1.3 μg/ml medium/24 h), while albumin secretion by the hepatocytes cultured without the residue decreased rapidly with time. These results suggest that some components in Kurozu residue are effective to maintain the primary culture of rat hepatocytes and thus for albumin production by the hepatocytes.

Increased P-glycoprotein messenger RNA stability in rat liver tumors in vivo.

P-glycoproteins (Pgp) are comprised of a small family of plasma membrane proteins whose abundance in cultured cells is often associated with the multidrug resistance phenotype. Overexpression of Pgp has been observed in many types of human cancers, but the molecular basis for this overexpression has not been established. We have used primary monolayer cultures of adult rat hepatocytes and a stepwise model of rat liver carcinogenesis to study the regulation of Pgp gene expression. We observed a marked overexpression of Pgp, specifically the class II Pgp, in both systems. In addition, we observed that a number of unrelated genes including alpha-tubulin, beta-actin, gamma-actin, cytokeratin 8, cytokeratin 18, and c-myc are overexpressed in cultured hepatocytes, and they are also overexpressed during liver carcinogenesis and in transplantable tumors. Nuclear run-on assays showed no increase in the transcriptional activity of Pgp genes in transplantable liver tumors compared to normal liver. Studies of in vivo mRNA stability, however, revealed that all three Pgp mRNAs were relatively stable in transplantable liver tumors (t(1/2) > 12 h), in contrast to what was found in normal liver (t(1/2) < 2 h). In addition, mRNA for several other genes, including alpha-tubulin, c-myc, and cyclin D1, all appear to be stabilized in the tumors. These findings suggest that the overexpression of Pgp genes in rat liver tumors may be the result of a mechanism involving stabilization of a diverse group of mRNAs.

Induction of megamitochondria by some chemicals inducing oxidative stress in primary cultured rat hepatocytes

Effects of hydrazine, hydrogen peroxide and bromobenzene, inducers of free radicals, and those of erythromycin and cycloheximide, inhibitors of protein synthesis on structural changes of mitochondria in primary monolayer culture of rat hepatocytes were examined using laser confocal microscope and electron microscope. After 22 h of incubation of hepatocytes with 0.2 mM hydrogen peroxide or 10 μg ml −1 of erythromycin, mitochondria became extremely enlarged. Mitochondria of hepatocytes isolated from control rats became slightly to moderately enlarged in the presence of 2 mM hydrazine, while those of hepatocytes isolated from phenobarbital-pretreated animals became extremely enlarged in the presence of 2 mM hydrazine. Cycloheximide (0.5–10.0 μg ml −1) and bromobenzene (0.1–1.0 mM) failed to induce structural changes of mitochondria. The level of cytochrome P-450 in freshly prepared hepatocytes from phenobarbital-treated rats was 2.5 times higher than that from the control rats, and remained about three times higher than the latter after 22 h of incubation with 2 mM hydrazine. The level of malondialdehyde was invariably elevated when megamitochondria were induced. These results may suggest that oxidative stress is intimately related to the mechanism of the formation of megamitochondria and that the inhibition of cytoplasmic protein synthesis seems not to contribute the phenomenon. However, the detailed mechanism by which free radicals may induce megamitochondria remains to be elucidated.

Effect of pyruvate on glutathione s-transferase expression in primary cultures of rat hepatocytes

Recently, primary rat hepatocyte cultures supplemented with 30 m m pyruvate and various hormones have been proposed as suitable long-term in vitro models for xenobiotic metabolism studies. In this study, the effect of 30 m m pyruvate on the cytosolic phase II biotransformation enzyme glutathione S-transferase (GST, EC 2.5.1.18) has been investigated. Adult male rat hepatocytes were brought into primary culture in serum free Williams' medium E with and without supplementation of 30 m m pyruvate. Total, Mu and Alpha class GST activities as well as GST subunit patterns were determined on the day of the isolation and as a function of culture time. Cell morphology and albumin secretion were also examined. After 7 days of culture, the morphology of pyruvate-treated cultures was still intact and bile canaliculi were clearly visible, whereas control cells without pyruvate had deteriorated and lost their normal morphology. The albumin secretion rate was higher in pyruvate-supplemented than in non-supplemented cultures. Total, Mu and Alpha class GST activities were well maintained in the presence of pyruvate; conversely, without pyruvate, all GST isoenzyme activities were significantly decreased. These findings were confirmed at the protein level. However, when hepatocytes received medium containing pyruvate for 7 days, the Pi class subunit 7, normally not present in adult liver cells, was expressed at a much higher level than was the case in untreated hepatocyte cultures. This study clearly shows that GST activities and GST proteins, as well as cell morphology and albumin secretion, are better maintained in primary monolayer cultures of adult rat hepatocytes supplemented with 30 m m pyruvate than in control cultures. However, it was also found that pyruvate is a very strong inducer of GST Pi. Consequently, the percentage GST subunit pattern of cultured rat hepatocytes is significantly affected by pyruvate supplementation, which may have consequences for in vitro xenobiotic metabolism studies.

Effect of tertiary butylhydroperoxide-induced oxidative stress on glutathione content and thiobarbituric acid reactive substances production in cultures and co-cultures of adult rat hepatocytes

The production of thiobarbituric acid reactive substances (TBARS) and the content of reduced glutathione (GSH) were determined in primary monolayer cultures of adult rat hepatocytes and in their co-cultures with rat epithelial cells, both exposed for 90 min to 2 mm tertiary butylhydroperoxide. Under these stress-induced conditions, TBARS production tended to be less increased in co-cultured cells than in conventionally cultured hepatocytes. A total depletion of GSH content was measured in conventional cultures from the third culture day, whereas in co-cultures a decrease in GSH content was observed until the third day of culture, followed by a low but constant GSH level that was maintained until the end of the culture period. Oxidative stress appears to be less pronounced in co-cultures than in conventional cultures; the former cope better with externally induced oxidative stress than do the latter.

Transforming growth factor β (TGF-β) expression in isolated and cultured rat hepatocytes

It is still a subject of debate whether hepatocytes have the ability to express TGF-beta. Therefore, we investigated in freshly isolated and in monolayer cultures of rat hepatocytes the expression of TGF-beta isoform s at the RNA and protein level applying RT-PCR, immunocytochemistry, immunoblotting, and functional assays of TGF-beta. TFG-beta 1, -beta 2, and -beta 3 transcripts were detected in cultured cells, and the level of m RNA increased up to 48/72 h, but TGF-beta 1 transcripts were absent in freshly isolated cells. Using APAAP stainings the proteins of all three TGF-beta isoforms were observed in hepatocyte cultures from 5-96 h, but in hepatocytes in the liver in situ and in freshly isolated cell suspensions TGF-beta staining was negative. SDS-PAGE under reducing conditions followed by Western blotting detected in cell lysates the subunit of mature TGF-beta at about 13 kd. Analysis of TGF-beta bioactivity with the mink cell (Mv1Lu) proliferation inhibition assay and competitive radioligand assay confirmed in activated (i.e., acidified and subsequently neutralized) hepatocyte-conditioned media the presence of TGF-beta, which, however, is almost entirely in the latent form. It is concluded that TGF-beta can be expressed in cultured hepatocytes and that the level of expression is quickly upregulated under abnormal, not yet known, microenvironmental conditions.

Effect of 350 kDa Glycoprotein in Royal Jelly on Primary Culture of Rat Hepatocytes.

The functions of the 350 kDa glycoprotein in royal jelly (RJ) on the primary monolayer culture of rat hepatocytes were examined. In the serum-free L-15 medium supplemented with the RJ protein, the monolayer culture of hepatocytes was maintained for 20 days. In contrast, without the RJ glycoprotein, the number of the hepatocytes which adhered to the dish decreased to less than one-half the number of inoculated cells during 20 days of culture. The amount of albumin secreted in the medium was monitored as an expression of liver-specific function. In the medium with the RJ glycoprotein, the hepatocytes maintained the secretion of albumin at a similar high level for 2 days compared to that in the starting culture, followed by diminishment to a low level. In contrast, the secretion of albumin by the hepatocytes cultured without RJ glycoprotein was decreased rapidly with time. As a control experiment, bovine serum albumin added to the medium did not show any effect both on the maintenance or albumin synthesis of rat hepatocytes.

An evaluation of the roles of metabolic denitrosation and alpha-hydroxylation in the hepatotoxicity of N-Nitrosodimethylamine.

N-Nitrosodimethylamine (NDMA) is a potent hepatotoxic agent in rats. NDMA causes cell death that does not correlate with known mechanisms of toxicity such as the production of oxidative stress or covalent binding to proteins. The following studies were designed to determine whether NDMA cytotoxicity is the result of metabolic denitrosation or alpha-hydroxylation of the nitrosamine. We determined the toxicity of various metabolites of NDMA in monolayer cultures of primary rat hepatocytes. NDMA was toxic at 0.1 mM in our cultures, but the metabolites formaldehyde, methanol, and methylamine were not toxic at this concentration. We used diazeniumdiolates that spontaneously release nitric oxide (NO) in aqueous media to deliver NO to hepatocytes in culture. The results show that, while NO released from diazeniumdiolates causes death in hepatocytes, the levels of NO produced during NDMA metabolism are insufficient to account for the toxicity of the nitrosamine. NDMA-d6, the fully deuteriated form of NDMA that undergoes approximately twice as much denitrosation in vivo as NDMA, was significantly less cytotoxic than NDMA. In contrast, N-nitroso(acetoxymethyl)methylamine (AcO-NDMA), a stable precursor of the methanediazonium ion, was found to cause toxicity equivalent of NDMA on a molar basis. Altogether, our results with methylamine, formaldehyde, methanol, the diazeniumdiolates, and NDMA-d6 indicate that NDMA toxicity is not the result of metabolic denitrosation, while the toxicity of AcO-NDMA provides strong strong evidence that the formation of the methanediazonium ion via alpha-hydroxylation leads to cell death.

Cryopreservation of rat hepatocyte monolayer cultures.

1. Most previous attempts to cryopreserve hepatocytes have used suspensions stored at either -70 degrees C or in liquid nitrogen, and the major problem is that these do not, on subsequent thawing, attach well in culture. This limits their use in studies of drug metabolism and xenobiotic-induced toxicity. In this manuscript we demonstrate successful cryopreservation of rat hepatocytes as monolayers attached to a collagen film. 2. Monolayers can be frozen and thawed without significant loss of cells, and although damage to the internal and plasma membranes is evident immediately post-thaw, a remarkable repair process takes place over 24-48 h post-thaw. Immediately post-thaw only 10% of the cells exclude Trypan Blue, but by 48 h 80-90% of the thawed cells are viable, indicating that repair of the plasma membranes has taken place. 3. The cells post-thaw retain aspects of liver-specific function including cytochrome P450 content and albumin synthesis. However, cytosolic proteins are lost through the damaged membranes and, probably because of this, urea synthesis from ammonia is retained at only 25% of pre-freeze values. 4. A cryopreservation method based on adherent hepatocytes on a collagen substrate overcomes the problems encountered with culture of cryopreserved hepatocyte suspensions, and may provide a practical means of establishing a 'bank' of hepatocytes from several donors and species.

Effect of glycine on valproate toxicity in rat hepatocytes.

The interaction between the amino acid glycine and valproate (VPA), an antiepileptic drug (AED) that occasionally causes hepatotoxicity, was studied in rat hepatocytes in monolayer culture. Valproate caused a dose-dependent increase in leakage of lactic acid dehydrogenase (LDH), and glycine prevented this toxic response. L-Carnitine, L-alanine, and L-cysteine did not protect hepatocytes from VPA. Glycine also partially antagonized inhibition of fatty acid beta-oxidation by VPA, as estimated by the generation of acid-soluble products from [14C]palmitic acid. These results are consistent with the hypothesis that glycine prevents VPA toxicity by removing acyl-CoA esters, which accumulate during VPA exposure and interfere with fatty acid beta-oxidation. Glycine, however, also antagonized the toxic effects of acetaminophen on hepatocytes, although at higher concentrations than required to protect hepatocytes from VPA. Because the mechanism of toxicity of acetaminophen probably is different from that of VPA, a nonspecific cytoprotective effect may contribute to glycine antagonism of valproate toxicity. Our results emphasize the importance of glycine in protecting hepatocytes from noxious insult in general as well as from VPA in particular.

Regulation of the acid-labile subunit of the insulin-like growth factor complex in cultured rat hepatocytes.

The acid-labile subunit (ALS) is a glycoprotein that forms a ternary complex in serum with insulin-like growth factors and insulin-like growth factor-binding protein-3. This study investigates the regulation of ALS production, measured by RIA, and messenger RNA (mRNA) content, measured by Northern analysis, in primary rat hepatocyte monolayer cultures. Hepatocytes produced ALS at a linear rate over 48 h. Exposure to human GH (30 ng/ml) caused a maximum 2.2-fold stimulation of ALS production compared to that in control cultures, giving a rate of 200 ng/10(6) cells.48 h. ALS mRNA appeared as a predominant 2-kilobase band and increased 4-fold by administration of 30 ng/ml GH. Both dexamethasone and epidermal growth factor (EGF) inhibited ALS production, with maximal effects at 100 nM dexamethasone and 50 ng/ml EGF (both approximately 50% inhibition). ALS mRNA levels measured 24 or 48 h after dexamethasone addition were decreased 75-80% compared to the control value. A similar decrease in ALS mRNA was observed 24 h after EGF addition, but a second addition of EGF at 24 h was required to maintain this decrease for 48 h. This study demonstrates that rat hepatocytes secrete immunoreactive ALS under GH regulation, and that EGF and corticosteroid inhibit ALS production and mRNA levels. The quantitative discrepancies between ALS production rates and mRNA levels suggest that posttranscriptional events may have a role in ALS regulation.

The expression of liver-specific genes within rat embryonic hepatocytes is a discontinuous process

Abstract The onset of transcription and mRNA accumulation of two liver-specific genes, carbamoylphosphate synthase (CPS) and phosphoenolpyruvate carboxykinase (PEPCK) in individual embryonic rat hepatocytes was investigated with in situ hybridization. In vitro CPS and PEPCK mRNAs can be induced prematurely in monolayer cultures of embryonic rat hepatocytes by glucocorticosteroids and cyclic AMP, i.e. the hormones that also regulate the expression of these genes in vivo. Upon exposure to hormones the cultures showed an interhepatocyte heterogeneity in CPS and PEPCK mRNA content. The pattern of accumulation of nuclear CPS mRNA-precursors indicates that this heterogeneity is generated by intercellular differences in the timing of the onset of transcription. However, under induced steady-state conditions the heterogeneity in the hepatocyte population persisted. The degree of heterogeneity is inversely related to the half life of the gene product (i.e. higher for PEPCK than for CPS and higher for mRNAs than for the respective proteins) and to the concentrations of inducing hormones. Accordingly, the interhepatocyte heterogeneity was most pronounced for the nuclear CPS mRNA-precursor. In contrast, no intercellular differences in the rate of degradation of the mRNAs were seen. These observations reveal that although all hepatocytes can and do express the genes, transcription of a gene in a particular cell is a discontinuous process.

Comparative cytotoxicity of non-steroidal anti-inflammatory drugs in primary cultures of rat hepatocytes

The cytotoxicities of 12 non-steroidal anti-inflammatory drugs (NSAIDs) in primary monolayer cultures of rat hepatocytes were compared. Toxicity was determined by measuring the release of lactate dehydrogenase into the culture medium after 20 hr of exposure. Diflunisal was the most cytotoxic, followed, in order, by mefenamic acid, diclofenac, indomethacin, flurbiprofen, piroxicam, sulindac and ibuprofen. Ketoprofen, naproxen, tolmetin and acetylsalicylic acid (ASA) were the least cytotoxic. Phenobarbital pretreatment in vivo potentiated the in vitro toxicity of diclofenac, ketoprofen and piroxicam, and SKF525-A addition to the medium reduced their toxicity. These results indicate that the cytocidal hepatotoxicity of diclofenac, ketoprofen and piroxicam may be mediated, at least partially, by cytochrome P-450 metabolism. The cytotoxicity of the other nine NSAIDs appears not to be significantly influenced by cytochrome P450 modulation.

Regulation of Asialoglycoprotein Receptor Expression in Rat Hepatocytes Cultured under Proliferative Conditions

The expression of many glycoproteins on the surface of hepatocytes has been related to the state of differentiation of the liver. In particular, the asialoglycoprotein receptor (ASGP-R) is modulated in many physiological and pathological conditions in which hepatocyte proliferative activity is modified. We studied ASGP-R expression in primary monolayer cultures of rat hepatocytes stimulated to proliferate by the addition of epidermal growth factor and insulin. In proliferating hepatocytes the receptor concentration on the cell surface was lowered, with no modifications in its distribution; similarly, both the binding activity and the amount of specific transcripts were decreased. The decreases were related to the peak of cellular growth, as judged by [ 3H]thymidine incorporation into DNA. The results suggest the presence of a cell cycle-dependent ASGP-R expression also in in vitro systems; this control could be exerted by a decreased availability of receptor-specific mRNA molecules or on the stability of transcripts.

Sphingomyelin transport to the cell surface occurs independently of protein secretion in rat hepatocytes.

The transport of sphingomyelin from its site of synthesis, in the cis/medial-Golgi, to the plasma membrane has been investigated in monolayer cultures of rat hepatocytes. Sphingomyelin was synthesized intracellularly from [methyl-3H]choline, and the arrival of sphingomyelin at the plasma membrane was monitored by treatment of the cells with sphingomyelinase, which hydrolyzed only sphingomyelin at the cell surface. When hepatocytes were maintained at temperatures from 5 to 37 degrees C, both protein secretion and sphingomyelin trafficking to the cell surface were inhibited at low temperatures, especially at temperatures below 25 degrees C. Treatment of cells with the energy poison potassium cyanide prevented protein secretion and reduced the incorporation of [3H]choline into sphingomyelin, but did not affect the percentage of newly synthesized sphingomyelin that was transported to the cell surface. Brefeldin A and monensin, both of which disrupt protein movement through the Golgi apparatus, were used to evaluate the mechanism of transport of sphingomyelin to the plasma membrane. Although brefeldin A and monensin each effectively blocked protein secretion, sphingomyelin movement to the cell surface was not inhibited, even though sphingomyelin synthesis normally occurs in the Golgi. We conclude that the most likely mechanism of sphingomyelin transport to the cell surface is a vesicle-mediated process (because of the known topology of sphingomyelin synthesis and distribution). However, the putative vesicles that are involved in the movement of sphingomyelin to the plasma membrane are distinct from those that transport proteins for secretion. In addition, the route of trafficking of sphingomyelin to the plasma membrane is not one that requires an obligatory passage through the Golgi apparatus.

Movement of apolipoprotein B into the lumen of microsomes from hepatocytes is disrupted in membranes enriched in phosphatidylmonomethylethanolamine.

When monolayer cultures of rat hepatocytes are incubated with the ethanolamine/choline analogue, monomethylethanolamine, the secretion of apolipoproteins B100 and B48, as well as the lipid constituents, of very low density lipoprotein (VLDL) is inhibited by approximately 50% (Vance, J. E. (1991) J. Lipid Res. 32, 1971-1982). In the present study we have investigated the mechanism by which monomethylethanolamine disrupts VLDL secretion. Hepatocytes were treated with 400 microM monomethylethanolamine overnight, which resulted in an increase in the cellular content of the derived phospholipid, phosphatidylmonomethylethanolamine, from 0.32 +/- 0.15 to 2.92 +/- 0.74 nmol/mg of cell protein. The biosynthesis of apoproteins B100 and B48 was not impaired by treatment of cells with monomethylethanolamine. However, monomethylethanolamine decreased by approximately 50% the amount of apoproteins B, but not of the typical secretory protein, albumin, present in the luminal content subfraction of microsomes. The intracellular degradation of apoproteins B was also increased in phosphatidylmonomethylethanolamine-enriched, compared with control, cells. Moreover, the pool of apoprotein B present in intact microsomes from hepatocytes incubated with monomethylethanolamine was more accessible to exogenously added trypsin, presumably because a larger pool of the apoprotein B was exposed on the cytosolic surface of these microsomes. The data strongly suggest that an increase in the microsomal content of phosphatidylmonomethylethanolamine diminishes the ability of apoprotein B to translocate across the endoplasmic reticulum membrane into the luminal compartment. Consequently, the association of apoprotein B with lipids and/or the normal assembly of mature VLDL particles is impaired.

Generation of trifluoroacetylated protein antigens in cultured rat hepatocytes exposed to halothane in vitro

Previous investigations have implicated an immune response to trifluoroacetylated proteins (TFA-proteins) in the pathogenesis of halothane hepatitis. The objective of this study was to establish conditions for generation of TFA-proteins in hepatocytes exposed to halothane in vitro. Monolayer cultures of rat hepatocytes were incubated in sealed flasks with or without added halothane, then subcellular fractions were prepared by differential centrifugation and analysed by immunoblotting for reactivity with anti-TFA antiserum. The specificity of the antiserum was verified by hapten inhibition with N-ϵ-TFA- l-lysine. TFA-proteins were generated when hepatocytes were cultured with halothane, but not when hepatocytes were cultured without halothane, and were concentrated in the microsomal fraction. Generation of TFA-proteins was greater when hepatocytes were exposed to an initial halothane concentration of about 0.17 m m-halothane than when hepatocytes were exposed to higher concentrations (0.6 m m and 1.4 m m). The molecular masses of the major TFA-proteins produced in vitro (100, 80 and 60 kDa) were very similar, if not identical, to the molecular masses of the major TFA-proteins produced in livers of rats treated ip with halothane in vivo, as were the kinetics of TFA-protein formation and turnover.

Differential feedback regulation of cholesterol 7 alpha-hydroxylase mRNA and transcriptional activity by rat bile acids in primary monolayer cultures of rat hepatocytes.

We have used primary monolayer cultures of rat hepatocytes to study the effects of physiological concentrations of various bile acids, commonly found in bile of normal rats, on the mechanism of regulation of cholesterol 7 alpha-hydroxylase and bile acid synthesis. Addition of taurocholic acid, the most predominant bile acid in rat bile, to the culture medium suppressed cholesterol 7 alpha-hydroxylase activity and mRNA time- and dose-dependently. The decrease in enzyme activity paralleled the changes in mRNA. Maximal suppression of cholesterol 7 alpha-hydroxylase mRNA (-91%) and enzyme activity (-89%) was observed after a 16 h incubation period with 50 microM taurocholic acid. The declines in mRNA and enzyme caused by taurocholic acid were tightly coupled and followed first-order kinetics with a half-life of 4 h. Transcriptional activity, as assessed with nuclear run-on assays, was decreased by 44% at 50 microM taurocholic acid. Mass production of bile acids (chenodeoxycholic acid and beta-muricholic acid) was inhibited to a similar extent as the cholesterol 7 alpha-hydroxylase when different concentrations of taurocholic acid were used, giving maximal inhibition (-81%) at 50 microM taurocholic acid. Glycocholic acid and unconjugated cholic acid were equally effective as taurocholic acid in suppressing cholesterol 7 alpha-hydroxylase mRNA. The more hydrophobic bile acids (chenodeoxycholic acid and deoxycholic acid) showed profound suppression of the cholesterol 7 alpha-hydroxylase mRNA by 85% and 75% respectively, whereas the other trihydroxy bile acids in rat bile, alpha- and beta-muricholic acid, were not or only marginally active. We conclude that rat bile acids, in particular the more hydrophobic ones, in concentrations commonly observed in portal blood, exert negative feedback control at the level of cholesterol 7 alpha-hydroxylase mRNA in cultured rat hepatocytes through a direct effect on the hepatocytes, and that down-regulation of transcription is only one of the mechanisms involved in this regulation.

Paradoxical transcriptional activation of rat liver cytochrome P-450 3A1 by dexamethasone and the antiglucocorticoid pregnenolone 16 alpha-carbonitrile: analysis by transient transfection into primary monolayer cultures of adult rat hepatocytes.

The family 3A cytochromes P-450, among the most abundant members of this supergene family of microsomal hemoproteins expressed in animal and human liver, are inducible by glucocorticoids but also by such antiglucocorticoids as pregnenolone 16 alpha-carbonitrile (PCN). To investigate the mechanism for this nonclassical glucocorticoid effect, we analyzed the ability of 1.5 kilobases of DNA or of its successive subsegments isolated from the 5' flanking region of the rat CYP3A1 structural gene to modulate transcription of a reporter gene consisting of a viral promoter coupled to the chloramphenicol acetyltransferase (CAT) structural gene (expression vector pBLCAT2) and transiently expressed in a homologous cell system consisting of primary monolayer cultures of adult rat hepatocytes in which CYP3A1 mRNA and protein are inducible. The CAT activity measured after chimeric gene constructions were transferred into the cultured rat hepatocytes by lipofection increased as much as 7.2-fold if the cells were treated with dexamethasone (DEX). One CYP3A1 fragment (positions -220 to -56; 164 base pairs), which does not contain a traditional glucocorticoid responsive element, conferred dose-dependent DEX responsiveness independent of its orientation but not its position in pBLCAT2. This construction was activated by addition of PCN to the cultures and was synergistically induced by PCN plus DEX. In contrast, induction of CAT activity in cultures containing MMTVCAT, a plasmid containing the CAT gene controlled by the mouse mammary tumor virus long terminal repeat, was unaffected by PCN treatment, required lower concentrations of DEX for a maximal response, and was inhibited by treatment with DEX plus PCN. We conclude that a primary mechanism for induction of CYP3A1 is stimulated transcription through a pathway activated by steroid hormones.