Phenobarbital-inducible Cytochrome P-450 Research Articles

Attenuation of phenobarbital-induced cytochrome P450 expression in carbon tetrachloride-induced hepatitis in mice models.

Certain pathological conditions, such as inflammation, are known to affect basal cytochrome P450 (CYP) expression by modulating transcriptional regulation, and the pharmacokinetics of drugs can vary among patients. However, changes in drug-induced CYP expression under pathological conditions have not been elucidated in detail. Here, we investigated the effects of hepatic inflammation and injury on phenobarbital-induced expression of CYP isoforms in mice. Phenobarbital was administered once as a CYP inducer in the carbon tetrachloride-induced hepatitis model mice. The mRNA expression levels of Cyp3a11 and Cyp2b10 in the liver and small intestine were measured using reverse transcription polymerase chain reaction. The enzymatic activity of CYP3A in liver S9 was evaluated using midazolam as the substrate. Phenobarbital increased the mRNA expression of Cyp3a11 and Cyp2b10 in the liver of healthy mice, but not in the small intestine. Increased mRNA expression of hepatic Cyp3a11 and Cyp2b10 by phenobarbital was significantly suppressed in the hepatitis model mice. Hepatitis also suppressed the increased CYP3A enzymatic activity induced by phenobarbital in liver S9, consistent with the results of Cyp3a11 mRNA expression. These results suggest that the inducibility of CYP by phenobarbital may vary in patients with hepatitis, indicating that pharmacokinetic drug-drug interactions can be altered under certain pathological conditions.

Proteome studies on liver tissue in a phenobarbital-induced rat model

Many drugs may affect the activity of cytochrome P450 (CYP), which is a major source of adverse drug interactions (ADR). Phenobarbital (PB) is the typical inducer of cytochrome P450. The aim of our study was to determine the changes in the cytosolic proteins expression in rat liver at a protein level following induction of cytochrome P450. Firstly, we made a phenobarbital-induced cytochrome P450 rat model. The total cytosolic proteins were then extracted from rat liver tissue and separated by 2-D gel electrophoresis (2-DE). Differentially expressed spots were identified by MALDI-TOF/TOF tandem mass spectrometry followed by database searching. Eight differentially expressed proteins were identified and these proteins were found to be involved in protein degradation, oxidative stress, energy metabolism, biotransformation, and the synthesis of quinolinic acid (QUIN). These findings should provide useful information for research into the regulation of cytochrome P450 gene expression, drug metabolism and drug interaction.

Hormone receptor-like in 96 and Broad-Complex modulate phenobarbital induced transcription of cytochrome P450 CYP6D1 in Drosophila S2 cells

Phenobarbital (PB) is a prototypical inducer for studies of xenobiotic responses in animals. In mammals, the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) have been identified as key transcription factors regulating PB induced transcription of xenobiotic responsive genes. In insects, much less is known about the transcription factors involved in regulating PB induced transcription, although CAR and PXR have a single orthologue hormone receptor-like in 96 (HR96) in Drosophila melanogaster. Using dual luciferase reporter assays in Drosophila Schneider (S2) cells, constructs containing variable lengths of the promoter of the PB inducible cytochrome P450 CYP6D1 were evaluated in the presence and absence of PB. The promoter region between -330 and -280 (relative to the position of transcription start site, +1) was found to be critical for PB induction. Putative binding sites for Drosophila Broad-Complex (BR-C) and deformed (Dfd) were identified within this promoter region using TFsearch. RNA interference (RNAi) treatment of S2 cells in conjunction with CYP6D1 promoter assays showed that suppression of Drosophila HR96 and BR-C transcription in S2 cells resulted in a significant decrease and increase, respectively, of PB induction. Effects of HR96 and BR-C in mediating PB induction were PB specific and PB dependent. This represents new functional evidence that Drosophila HR96 and BR-C can act as an activator and repressor, respectively, in regulating PB induced transcription in insects.

Xanthohumol, a prenylated chalcone from hops, modulates hepatic expression of genes involved in thyroid hormone distribution and metabolism

In the present study, we analyzed the influence of xanthohumol (XN) on thyroid hormone (TH) distribution and metabolism in rats. A potent and selective competition of XN for thyroxine (T4) binding to transthyretin (IC(50)=1 microM at 1.7 nM [(125)I]T4) was found in human and rat sera in vitro. Female rats treated orally with XN showed increased hepatic expression of T4-binding globulin and decreased transthyretin and albumin. Thyrotropin levels and hepatic type 1 deiodinase activity were moderately increased. Northern blot analysis revealed diminished expression of liver sulfotransferase (Sult1a1) and uridine-diphosphate glucuronosyltransferase (Ugt1a1) after XN treatment. The transcript levels of constitutive androstane receptor (CAR), known to be involved in regulation of enzymes metabolizing hormones, drugs and xenobiotics, was lower in rats treated with >10 mg XN/kg body weight per day. Immunoblot analysis indicates reduced amounts of CAR protein. The phenobarbital-inducible cytochrome P450 mRNA level was decreased in rats treated with >10 mg XN/kg/day, in agreement with reduced CAR protein. Although only moderate changes in TH serum levels were observed, the XN-dependent altered expression of components involved in TH homeostasis might be important not only for hormone metabolism, but also for hepatic phase I and II elimination of drug metabolites and xenobiotics.

Docosahexaenoic acid down-regulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes via the sphingomyelinase/ceramide pathway

Docosahexaenoic acid (DHA) regulates the expression of cytochrome P450 2B1 (CYP 2B1) in rat primary hepatocytes in response to xenobiotics. Ceramide, a lipid signaling molecule, is involved in various physiological processes and can be generated by the hydrolysis of sphingomyelin via sphingomyelinase (SMase). DHA activates SMase and increases ceramide formation in vitro. Ceramides differentially enhance adenylyl cyclase activity in vitro depending on the chain length of their fatty acids. In addition, the cAMP-dependent PKA pathway down-regulates CYP 2B1 expression induced by phenobarbital (PB). In the present study, we determined the effect of DHA on SMase transactivation and the downstream pathway in CYP 2B1 expression induced by PB. SMase was activated by DHA 2 h after treatment, and D609 (an SMase inhibitor) attenuated the inhibition of PB-induced CYP 2B1 expression by DHA. Ceramide formation reached a maximum 3 h after DHA administration. C2-ceramide dose-dependently inhibited PB-induced CYP 2B1 expression and increased intracellular cAMP concentrations. SQ22536 (an adenylyl cyclase inhibitor) and H89 (a PKA-specific inhibitor) partially reversed the inhibition of PB-induced CYP 2B1 expression by C2-ceramide. These results suggest that stimulation of SMase, generation of ceramide and activation of the cAMP-dependent PKA pathway are involved in the inhibition exerted by DHA.

Stem Cell Therapy for Liver Disease: Parameters Governing the Success of Using Bone Marrow Mesenchymal Stem Cells

Liver transplantation is the primary treatment for various end-stage hepatic diseases but is hindered by the lack of donor organs and by complications associated with rejection and immunosuppression. There is increasing evidence to suggest the bone marrow is a transplantable source of hepatic progenitors. We previously reported that multipotent bone marrow-derived mesenchymal stem cells differentiate into functional hepatocyte-like cells with almost 100% induction frequency under defined conditions, suggesting the potential for clinical applications. The aim of this study was to critically analyze the various parameters governing the success of bone marrow-derived mesenchymal stem cell-based therapy for treatment of liver diseases. Lethal fulminant hepatic failure in nonobese diabetic severe combined immunodeficient mice was induced by carbon tetrachloride gavage. Mesenchymal stem cell-derived hepatocytes and mesenchymal stem cells were then intrasplenically or intravenously transplanted at different doses. Both mesenchymal stem cell-derived hepatocytes and mesenchymal stem cells, transplanted by either intrasplenic or intravenous route, engrafted recipient liver, differentiated into functional hepatocytes, and rescued liver failure. Intravenous transplantation was more effective in rescuing liver failure than intrasplenic transplantation. Moreover, mesenchymal stem cells were more resistant to reactive oxygen species in vitro, reduced oxidative stress in recipient mice, and accelerated repopulation of hepatocytes after liver damage, suggesting a possible role for paracrine effects. Bone marrow-derived mesenchymal stem cells can effectively rescue experimental liver failure and contribute to liver regeneration and offer a potentially alternative therapy to organ transplantation for treatment of liver diseases.

DHA down-regulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes by attenuating CAR translocation

The constitutive androstane receptor (CAR) plays an important role in regulating the expression of detoxifying enzymes, including cytochrome P450 2B (CYP 2B). Phenobarbital (PB) induction of human CYP 2B6 and mouse CYP 2b10 has been shown to be mediated by CAR. Our previous study showed that PB-induced CYP 2B1 expression in rat primary hepatocytes is down-regulated by both n-6 and n-3 polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA); however, the mechanism for this down-regulation by DHA was previously unknown. The objective of the present study was to determine whether change in CAR translocation is involved in the down-regulation by n-6 and n-3 PUFAs of PB-induced CYP 2B1 expression in rat primary hepatocytes. We used 100 μM arachidonic acid, linoleic acid, eicosapentaenoic acid, and DHA to test this hypothesis. PB triggered the translocation of CAR from the cytosol into the nucleus in a dose-dependent and time-dependent manner in our hepatocyte system, and the CAR distribution in rat primary hepatocytes was significantly affected by DHA. DHA treatment decreased PB-inducible accumulation of CAR in the nuclear fraction and increased it in the cytosolic fraction in a dose-dependent manner. The down-regulation of CYP 2B1 expression by DHA occurred in a dose-dependent manner, and a similar pattern was found for the nuclear accumulation of CAR. The results of immunoprecipitation showed a CAR/RXR heterodimer bound to nuclear receptor binding site 1 (NR-1) of the PB-responsive enhancer module (PBREM) of the CYP 2B1gene. The EMSA results showed that PB-induced CAR binding to NR-1 was attenuated by DHA. Taken together, these results suggest that attenuation of CAR translocation and decreased subsequent binding to NR-1 are involved in DHA's down-regulation of PB-induced CYP 2B1 expression.

Defined Conditions for Development of Functional Hepatic Cells from Human Embryonic Stem Cells

Human embryonic stem (hES) cells provide an important means to evaluate specific soluble and cell-bound stimuli that regulate development of specific cell lineages. Here, we examined specific cytokines and extracellular matrix (ECM) proteins that support differentiation of hES cells to hepatocytes. Tests of several different conditions determined that addition of fibroblast growth factor (FGF)-4 and hepatocyte growth factor in completely serum-free cultures of hES cell-derived embryoid bodies subsequently allowed to attach to type I collagen-coated dishes led to maximal differentiation into cells, not only with the morphologic and phenotypic characteristics of hepatocytes but also the functional characteristics. Expression of common hepatic transcription factors including HNF-3beta, HNF-1, and GATA-4 were all significantly induced under these conditions. Hepatocyte function was demonstrated by multiple complementary criteria: production of urea and albumin, phenobarbital-induced cytochrome P450 expression, and uptake of indocyanine green. These hES cell-derived hepatocytes will serve as a resource to understand normal human hepatocyte development and for applications such as cell replacement therapies and screening of pharmacologic drugs.

In vitro metabolism of 2,2′,3,4′,5,5′,6-heptachlorobiphenyl (CB187) by liver microsomes from rats, hamsters and guinea pigs

The metabolism of 2,2′,3,4′,5,5′,6-heptachlorobiphenyl (heptaCB) (CB187) was studied using liver microsomes of rats, hamsters and guinea pigs, and the effect of cytochrome P450 (CYP) inducers, phenobarbital (PB) and 3-methylcholanthrene (MC), was also investigated. In untreated animals, guinea pig liver microsomes formed three metabolites which were deduced to be 4′-hydroxy-2,2′,3,5,5′,6-hexachlorobiphenyl (M-1), 4′-hydroxy-2,2′,3,3′,5,5′,6-heptaCB (M-2) and 4-OH-CB187 (M-3) from the comparison of GC/MS data with some synthetic authentic samples. The formation rate of M-1, M-2 and M-3 was 18.1, 36.6, 14.7 pmol h−1 mg protein−1, respectively. Liver microsomes of untreated rats and hamsters did not form CB187 metabolites. In guinea pigs, PB-treatment increased M-1 and M-2 significantly to 1.9- and 3.4-fold of untreated animals but did not affect the formation of M-3. In rats, PB-treatment resulted in the appearance of M-2 and M-3 with formation rates of 87.1 and 13.7 pmol h−1 mg protein−1, respectively, but M-1 was not observed. In hamsters, PB-treatment formed only M-2 at a rate of 29.4 pmol h−1 mg protein−1. On the other hand, MC-treatment of guinea pigs decreased the formation of M-1 and M-2 to less than 50% of untreated animals. MC-microsomes of rats and hamsters produced no metabolites. Preincubation of antiserum (300 µl) against guinea pig CYP2B18 with liver microsomes of PB-treated guinea pigs produced 80% inhibition of M-1 and the complete inhibition of M-2 and M-3. These results suggest that PB-inducible CYP forms, especially guinea pig CYP2B18, rat CYP2B1 and hamster CYP2B, are important in CB187 metabolism and that CB187 metabolism in guinea pigs may proceed via the formation of 3,4- or 3′,4′-oxide and subsequent NIH-shift or dechlorination.

Proteasome-dependent degradation of cytochromes P450 2E1 and 2B1 expressed in tetracycline-regulated HeLa cells

The degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible cytochrome P450 2B1 (CYP2B1) expressed in tetracycline (Tc)-inducible HeLa cell lines was characterized. A steady-state pulse-chase analysis was used to determine a half-life of 3.8 h for CYP2E1 while the half-life of CYP2B1 was 2.3-fold greater in the same cell line. In contrast, NADPH cytochrome P450 reductase which is constitutively expressed in Tc-HeLa cells had a half-life of about 30 h. Lactacystin and other selective proteasome inhibitors including N-benzyloxycarbonyl-leucyl-leucyl-leucinal (MG132) and N-benzyloxycarbonyl- l-leucyl- l-leucyl- l-norvalinal (MG115) significantly inhibited both CYP2E1 and CYP2B1 degradation. The turnover of CYP2E1 was slightly inhibited by calpain inhibitors while CYP2B1 turnover was not altered. Inhibitors of lysosomal proteolysis had no effect on the degradation of either protein. Treatment of cells with brefeldin A did not alter the degradation of either P450 which suggested the degradation occurred in the endoplasmic reticulum (ER). Even in the presence of proteasome inhibitors high molecular weight ubiquitin conjugates were not observed. Mutagenesis of two putative ubiquitination sites (Lys 317 and 324) did not alter the degradation of CYP2E1. The role of ubiquitination in the degradation of CYP2E1 was also examined in a Chinese hamster mutant cell line E36ts20 that contains a thermolabile ubiquitin-activating enzyme (E1). The turnover of CYP2E1 was not significantly different at the nonpermissive temperature in the ts20 when compared to the control E36 cells. Furthermore, the addition of the hsp90 inhibitors geldanamycin, herbimycin, and radicicol had no effect on the turnover of CYP2E1, differentiating the degradation of CYP2E1 from other substrates for proteasome-dependent degradation.

In vitro Hepatic Differentiation of Human Mesenchymal Stem Cells

This study examined whether mesenchymal stem cells (MSCs), which are stem cells originated from embryonic mesoderm, are able to differentiate into functional hepatocyte-like cells in vitro. MSCs were isolated from human bone marrow and umbilical cord blood, and the surface phenotype and the mesodermal multilineage differentiation potentials of these cells were characterized and tested. To effectively induce hepatic differentiation, we designed a novel 2-step protocol with the use of hepatocyte growth factor and oncostatin M. After 4 weeks of induction, cuboidal morphology, which is characteristic of hepatocytes, was observed, and cells also expressed marker genes specific of liver cells in a time-dependent manner. Differentiated cells further demonstrated in vitro functions characteristic of liver cells, including albumin production, glycogen storage, urea secretion, uptake of low-density lipoprotein, and phenobarbital-inducible cytochrome P450 activity. In conclusion, human MSCs from different sources are able to differentiate into functional hepatocyte-like cells and, hence, may serve as a cell source for tissue engineering and cell therapy of hepatic tissues. Furthermore, the broad differentiation potential of MSCs indicates that a revision of the definition may be required.

Stereoselective hydroxylation of 4-methyl-2-cyclohexenone in rats: its relevance to R-(+)-pulegone-mediated hepatotoxicity

R-(+)-Pulegone, a monoterpene ketone, is a potent hepatotoxin. One of the major metabolites of pulegone has been shown to be p-cresol, a glutathione depletor and a known toxin. Allylic hydroxylation of 4-methyl-2-cyclohexenone results in the formation of p-cresol. The present study documents for the first time the involvement of cytochrome P-450 system and the stereochemical preference in this hydroxylation reaction. Incubation of PB-induced rat liver microsomes as well as reconstituted PB-induced cytochrome P-450 system with ±4-methyl-2-cyclohexenone in the presence of NADPH and O 2 resulted in the formation of 4-hydroxy-4-methyl-2-cyclohexenone and p-cresol. From the assay mixture, the unreacted substrate, viz., 4-methyl-2-cyclohexenone was isolated and purified and its optical rotation was found to be 2.2 (in CHCl 3). The observed enantiomeric excess in the recovered substrate was further confirmed by circular dichroism (CD) studies. The CD spectrum has a peak at 292 nm and a trough at 270 nm. The enantiomeric excess in the recovered substrate indicates that the hydroxylation at C-4 position is stereoselective. The significance of these results with respect to pulegone-mediated hepatotoxicity is discussed.

Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells.

We have derived from normal human, mouse, and rat postnatal bone marrow primitive, multipotent adult progenitor cells (MAPCs) that can differentiate into most mesodermal cells and neuroectodermal cells in vitro and into all embryonic lineages in vivo. Here, we show that MAPCs can also differentiate into hepatocyte-like cells in vitro. Human, mouse, and rat MAPCs, cultured on Matrigel with FGF-4 and HGF, differentiated into epithelioid cells that expressed hepatocyte nuclear factor-3beta (HNF-3beta), GATA4, cytokeratin 19 (CK19), transthyretin, and alpha-fetoprotein by day 7, and expressed CK18, HNF-4, and HNF-1alpha on days 14-28. Virtually all human, as well as a majority of rodent cells stained positive for albumin and CK18 on day 21; 5% (rodent) to 25% (human) cells were binucleated by day 21. These cells also acquired functional characteristics of hepatocytes: they secreted urea and albumin, had phenobarbital-inducible cytochrome p450, could take up LDL, and stored glycogen. MAPCs, which can be expanded in vitro and maintained in an undifferentiated state for more than 100 population doublings, can thus differentiate into cells with morphological, phenotypic, and functional characteristics of hepatocytes. MAPCs may therefore be an ideal cell for in vivo therapies for liver disorders or for use in bioartificial liver devices.

Long-term maintenance of cytochrome P450 activities by rat hepatocyte/3T3 cell co-cultures in heparinized human plasma.

Little information on the effect of plasma on hepatocyte cytochrome P450 (CYP) activities is currently available. We characterized the effect of plasma on CYPs of hepatocyte-mesenchymal cell co-cultures, which exhibit stable liver specific functions and may be potentially useful for bioartificial liver design. Rat hepatocyte-mouse 3T3-J2 cell co-cultures were maintained for 6 days in medium, and then switched to heparinized human plasma containing 3-methylcholanthrene (3MC; 2 microM), phenobarbital (PB; 1 mM), or no inducer for up to 7 days. CYP activities were measured in situ based on the o-dealkylation of ethoxy- (EROD), methoxy- (MROD), pentoxy- (PROD), or benzyloxy- (BROD) resorufin. Plasma alone increased PROD/BROD but not EROD/MROD. The endogenous inducer was in the high molecular weight fraction (>5 kD) of plasma and inhibited by >5 nM okadaic acid and >10 microM dibutyryl cyclic AMP, two inhibitors of PB-inducible CYPs. Furthermore, plasma increased CYP1A1 and CYP2B1/2 mRNA levels. In plasma, 3MC induced EROD/MROD to about 60% of the level induced in culture medium while PB induced PROD/BROD that were three- to 10-fold above levels induced in medium. CYP activities decreased between days 2 and 7 of plasma exposure, but were enhanced by plasma supplementation with amino acids, insulin, glucagon, and hydrocortisone.

Identification of estrogen-responsive genes in chick liver.

Identification of targets of estrogen is an important step in understanding the mechanisms of estrogen action. A two-step strategy was developed to identify estrogen-responsive genes (ERGs) in chick liver. Initially, differential-display, reverse transcription polymerase chain reaction (DDRT-PCR) was introduced to isolate ERGs. Isolated ERGs were then analyzed using Northern blot hybridization. A number of differentially expressed cDNA fragments were isolated following estrogen exposure. Four cDNA fragments that displayed dramatic change after estrogen administration were identified as liver adenylosuccinate lyase (ADL), phenobarbital-inducible cytochrome P-450 (CYP450), ovoinhibitor, and glutathione-dependent prostaglandin D2 synthase (PGDS). Time sequence analysis showed that estrogen-induced alteration occurred as early as 0.5 h and peaked between 1 and 4 h after estrogen exposure. Nuclear runoff assay indicated that estrogen significantly increased the transcription rate of these genes. To determine whether the observed alteration was due to the direct effect of estrogen, protein synthesis was inhibited by cycloheximide (CHX) during stimulation by estradiol, Estrogen-mediated upregulation of PGDS was completely abolished by a concurrent treatment with CHX, suggesting that its activation requires the participation of some newly synthesized factor(s). In contrast, CHX did not affect the expression of other genes, indicating the alteration is a direct response to estrogen. In conclusion, ADL, CYP450, ovoinhibitor, and PGDS represent the novel targets of estrogen, which regulates the transcriptional activity of these genes.

Apolipoprotein A-I gene expression is upregulated by polychlorinated biphenyls in rat liver

Xenobiotics such as polychlorinated biphenyls (PCB) increase serum cholesterol level (especially high density lipoprotein cholesterol) and apolipoprotein A-I (apo A-I) level in rats. The effect of PCB on serum apo A-I and hepatic apo A-I gene expression and the relationship between apo A-I and drug-metabolizing enzymes in rats were investigated. Serum levels of cholesterol and apo A-I were increased by dietary addition of PCB in a dose-dependent manner (0–500 mg/kg diet). Hepatic apo A-I mRNA level was also elevated by PCB in a similar fashion. Serum level of cholesterol gradually increased during feeding period of PCB (200 mg/kg diet, 105 days) and reached a two-fold higher level in PCB group than in controls. The levels of serum apo A-I and hepatic apo A-I mRNA linearly elevated during feeding period of PCB and were increased 3- or 4-fold, respectively, compared to controls. Although acute administration (16 hr) of PCB, 3-methylcholanthrene, and phenobarbital induced cytochrome P-450 gene expression in the liver, hepatic apo A-I gene expression was not increased by these xenobiotics. These results indicated that the serum levels of cholesterol and apo A-I had positive correlation with hepatic level of apo A-I mRNA in rats fed PCB, and that hepatic apo A-I gene expression was dependent upon intake of PCB but was not directly related to the induction of drug-metabolizing enzymes. This study demonstrated that xenobiotic-induced hyper-alpha-cholesterolemia would be caused by the increased apo A-I gene expression and cholesterol synthesis in the liver, coordinately.

Effect of phenobarbital on intralobular expression of CYP2B1/2 in livers of rats: Difference in the expression between single and repetitive administrations

Phenobarbital (PB) was shown to induce the major PB-inducible cytochrome P450 (CYP) isoforms, CYP2B1/2, in perivenular hepatocytes by a single injection, and in midzonal and periportal hepatocytes in addition to perivenular hepatocytes by injections of the same dosage once a day for 3 days in rat livers. The present study was undertaken to determine whether the spread of enzyme induction to midzonal and periportal hepatocytes is caused by the increase in total dose of the drug by repetitive injections or by the repetitive injections of the drug themselves. Male adult rats were administered PB by a single injection (80 mg/kg) or repetitive injections (20 mg/kg once a day for 4 days; a total dose of 80 mg/kg), and the molar content of CYP2B1/2 was measured by quantitative immunohistochemistry in the cytoplasm of perivenular, midzonal, and periportal hepatocytes. In addition, the molar content of total CYP in the cytoplasm was measured by microphotometry, and the expression of CYP2B2 mRNA was examined by in situ hybridization. When animals received the single injection, the isoforms and CYP2B2 mRNA increased markedly in perivenular hepatocytes, increased somewhat in midzonal hepatocytes, and remained unchanged in periportal hepatocytes. If animals received the repetitive injections, however, although the isoforms and the mRNA increased markedly in perivenular hepatocytes, they also increased markedly in midzonal hepatocytes and somewhat in periportal hepatocytes. These findings demonstrated that the enlargement of the sublobular area in which induction of the isoforms occurred was caused by the repetitive injections of PB themselves.

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.

Glutaredoxin is a direct target of oncogenic jun.

We have analysed differential gene expression in v-jun-transformed chicken embryo fibroblasts (CEF) compared to normal CEF by using the directional tag PCR subtraction method. From a first generation of putative Jun targets four clones were selected for study; they are upregulated in jun-transformed cells. Three of these clones showed homology to known genes: glutaredoxin, growth associated protein (GAP)-43/neuromodulin, and phenobarbital-induced cytochrome P450. The expression of these genes was analysed in fibroblasts transformed by various oncogenes. Expression of the glutaredoxin mRNA could be induced by a Jun-estrogen receptor chimaera in the absence of de novo protein biosynthesis. Based on this observation we conclude that glutaredoxin is a direct target of v-Jun.

Molecular approaches for production of pravastatin, a HMG-CoA reductase inhibitor: transcriptional regulation of the cytochrome P450 sca gene from Streptomyces carbophilus by ML-236B sodium salt and phenobarbital

We have characterized the transcriptional regulation of ML-236B·Na and phenobarbital-inducible cytochrome P450 sca-2 (Cyt P450 sca-2) from Streptomyces carbophilus, an industrial pravastatin-producing strain. ML-236B·Na and phenobarbital enhanced the expression of the cytP450 sca-2 gene in S. carbophilus. The cytP450 sca-2 gene was also ML-236B·Na-inductive in S. lividans. Analysis of various deletion and mutation of the 5′-flanking region of the cytP450 sca-2 gene revealed that the 1-kb region was required for ML-236B·Na-dependent Cyt P450 sca-2 induction. We have found a putative ORF in the 5′-flanking region that encodes a protein of 174 amino acid residues containing a helix–turn–helix DNA-binding motif. A gel mobility shift assay showed that the protein was bound by an imperfect palindromic sequence between −46 bp and −24 bp in the 5′-flanking region, and ML-236B·Na was found to inhibit its binding. These findings suggest that induction of Cyt P450 sca-2 is negatively regulated at the transcriptional level and that the protein encoded by the putative ORF is possibly functional as a repressor of the cytP450 sca-2 gene.