Effect of arsenite on induction of CYP1A, CYP2B, and CYP3A in primary cultures of rat hepatocytes.

In earlier studies, sodium arsenite treatment was shown to decrease induction of enzymatic activities associated with hepatic CYPs in rats. Here we investigated the effect of sodium arsenite on induction of CYP2B, CYP1A, and CYP3A in primary cultures of rat hepatocytes. Arsenite decreased the induction of all three families of CYP, as measured enzymatically and immunochemically. These decreases in CYPs occurred at concentrations of arsenite (2.5-10 microM) at which no toxicity was observed; however, toxicity was observed at 25 microM arsenite. With 3-methylcholanthrene as inducer, 5 microM arsenite caused a 55% decrease in CYP1A1 immunoreactive protein and enzyme activity, but only a 25% decrease in CYP1A1 mRNA. With phenobarbital (PB) as the inducer, 2.5 microM arsenite decreased CYP2B enzyme activity and immunoreactive protein 50%, with only a 25% decrease in CYP2B1 mRNA. 5 microM Arsenite decreased CYP2B enzyme activity and immunoreactive protein 80%, but decreased CYP2B1 mRNA only 50%, while CYP3A protein was decreased greater than 75% with no decrease in CYP3A23 mRNA. With dexamethasone (DEX) as inducer, 5 microM sodium arsenite caused a 50% decrease in immunoreactive CYP3A and a 30% decrease in CYP3A23 mRNA. Although arsenite-mediated increases in heme oxygenase (HO) inversely correlated with decreases in CYP2B or CYP1A activity, inclusion of heme in cultures treated with inducers of CYP1A or CYP2B did not prevent the arsenite-mediated decreases in these CYPs. Even though added heme induced HO to similar levels with and without arsenite, decreases in CYPs were only observed in the presence of arsenite. These results suggest that, in rat hepatocytes, elevated levels of HO alone are not responsible for arsenite-mediated decreases in CYP.     

Negative regulation by dexamethasone of fluvastatin-inducible CYP2B expression in primary cultures of rat hepatocytes: role of CYP3A

Fluvastatin (Fluva), a synthetic inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, induces CYP2B1/2 in rat liver and primary cultured rat hepatocytes. However, the overall profile of CYP induction, which includes induction of CYP4A, suggests that Fluva is not a typical "phenobarbital (PB)-like" inducer. Several treatments affecting diverse cell signaling pathways have been reported to modify PB-inducible CYP2B expression in primary cultured rat hepatocytes. We examined the effects of selected treatments on the ability of Fluva to induce CYP2B1/2 mRNA. Only dexamethasone (Dex) produced effects on Fluva-inducible CYP2B1/2 mRNA expression that differed from those produced on PB-inducible CYP2B1/2 mRNA expression. Dex concentrations up to 10(-7) M of potentiated PB (10(-4) M)-mediated CYP2B1/2 mRNA induction, while higher Dex concentrations produced a progressive reduction in PB-induced CYP2B1/2 mRNA levels. By contrast, Dex concentrations up to 10(-8) M had no effect on Fluva (3 x 10(-5) M)-induced CYP2B1/2 mRNA levels, while Dex concentrations of 10(-7) M and higher markedly suppressed Fluva-mediated CYP2B1/2 mRNA induction. The concentrations of several glucocorticoids that produced suppression of Fluva-induced CYP2B1/2 mRNA levels were the same concentrations that induced CYP3A mRNA. Treatment with pregnenolone 16 alpha-carbonitrile also produced a concentration-dependent suppression of Fluva-induced CYP2B1/2 mRNA levels. Dex-mediated suppression of Fluva-induced CYP2B1/2 mRNA was concentration-dependently reversed when hepatocytes were cotreated with troleandomycin, a selective CYP3A inhibitor. The amounts of Fluva detected in culture medium and cells were reduced significantly when hepatocytes were incubated with Dex. However, Dex-mediated suppression of Fluva-induced CYP2B1/2 mRNA expression was not overcome when hepatocytes were incubated with Fluva concentrations greater than 3 x 10(-5) M, suggesting that mechanisms other than CYP3A-catalyzed metabolism may contribute to Dex-mediated suppression of Fluva-induced CYP2B1/2 expression.     

Expression and induction of CYP1A1/1A2, CYP2A6 and CYP3A4 in primary cultures of human hepatocytes: a 10-year follow-up

1. The aims were to refine experimental conditions (using 76 human hepatocyte preparations) in terms of the selection of enzyme inducers and their optimal concentration, the treatment duration with inducers and the choice of specific cytochrome P450 isoform(s) probes to optimize the use of primary hepatocytes for predicting the potential induction by new chemical entities of cytochrome P450 isoforms in vivo in man. 2. In the absence of any inducer, basal cytochrome P450 isoform(s)-mediated activities decreased to 20% of their initial activity (end of the seeding period) by 72-96 h. In contrast, UGT-dependent enzyme activities remained at a constant level (+/- 20%) up to the fifth day of culture. 3. Beta-naphthoflavone, at an optimal concentration of 50 microM and after a 3-day treatment, specifically and potently induced 7-ethoxyresorufin (10.4 +/- 10.4-fold, n = 74) and phenacetin (6.6 +/- 6.4-fold, n = 60) O-deethylation processes, markers for CYP1A1 and CYP1A2 isoforms respectively. Only a 2-fold increase was noted following treatment with 2 mM phenobarbitone, whereas dexamethasone and rifampicin had no effect at all. 4. A 3-day treatment of human hepatocytes with 50 microM dexamethasone was associated with a major induction of both coumarin 7-hydroxylation (9.4 +/- 11.4-fold, n = 49) and nifedipine dehydrogenation (4.7 +/- 3.8-fold, n = 61), markers for CYP2A6 and CYP3A4 respectively. Phenobarbitone, however, exhibited a broad but moderate inducing effect on 7-ethoxyresorufin (2.2 +/- 1.5-fold, n = 55) and phenacetin (1.7 +/- 0.9-fold, n = 54) O-deethylation, coumarin 7-hydroxylation (3.9 +/- 9.2-fold, n = 50) and nifedipine dehydrogenation (2.1 +/- 2.0-fold, n = 47). 5. Km obtained for the different cytochrome P450 isoform substrates in untreated hepatocytes were in the same range of magnitude that those determined on human hepatic microsomal fractions. Enzyme induction processes were characterized by a large increase in apparent Vmax whereas apparent Km were not affected. 6. These studies demonstrate that human hepatocytes in primary culture can respond specifically and quantitatively to model inducers. This in vitro system offers a useful approach to study the regulation of human hepatic biotransformation activities and should facilitate the demand for a reproducible method for addressing cytochrome P450 induction.     

Expression and induction of CYP1A1/1A2, CYP2A6 and CYP3A4 in primary cultures of human hepatocytes: a 10-year follow-up

1. The aims were to refine experimental conditions (using 76 human hepatocyte preparations) in terms of the selection of enzyme inducers and their optimal concentration, the treatment duration with inducers and the choice of specific cytochrome P450 isoform(s) probes to optimize the use of primary hepatocytes for predicting the potential induction by new chemical entities of cytochrome P450 isoforms in vivo     

Lack of association between CYP2A5 induction and apoptosis in mouse primary hepatocytes

Upregulation of mouse hepatic cytochrome P450 2A5 (CYP2A5) is a process closely associated with hepatocellular damage and formation of liver tumours. 2-Aminopurine, a protein kinase inhibitor modulating cell cycle control, was recently shown to strongly induce CYP2A5 in mouse hepatocytes. The objective of this study was to determine the association between CYP2A5 induction and apoptosis in mouse primary hepatocytes. Five well-characterised CYP2A5 inducers were tested for their ability to affect apoptosis rate, determined by immunohistochemical in situ 3'-end-labelling technique, in a primary mouse hepatocyte model. Transforming growth factor beta (TGFbeta) was used as a positive (proapoptotic) control. Phenobarbital, pyrazole and the mitogen-activated protein kinase inhibitor PD98059 did not significantly affect apoptosis rate in hepatocytes. Norcocaine induced apoptosis at 6 hr (1.8-fold) and 2-aminopurine 12 hr (1.4-fold) after treatment, which is considerably earlier than peaks in the amount of CYP2A5 mRNA. TGFbeta reduced CYP2A5 marker activity, coumarin 7-hydroxylase by 74%. These results indicate that in a primary hepatocyte model (a) there is no systematic correlation between apoptosis and CYP2A5 induction; (b) phenobarbital does not significantly affect the rate of apoptosis; and (c) the induction of apoptosis caused by the chemicals tested occurs considerable earlier than elevation of CYP2A5 expression. Thus, no causal link appears to exist between induction of CYP2A5 and apoptotic rate.     

Distinct role of bilobalide and ginkgolide A in the modulation of rat CYP2B1 and CYP3A23 gene expression by Ginkgo biloba extract in cultured hepatocytes.

In the present study, primary cultures of rat hepatocytes were treated for 48 h with one of several extracts of Ginkgo biloba (10, 100, or 1000 microg/ml). Maximal increase in CYP2B1 and CYP3A23 mRNA levels was obtained at 100 microg/ml. This concentration of G. biloba extract also increased CYP3A2 and CYP3A18 mRNA expression in addition to CYP2B-mediated 7-benzyloxyresorufin O-dealkylation (BROD) and CYP3A-mediated testosterone 6beta-hydroxylation. In other experiments, cultured hepatocytes were treated for 48 h with bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, kaempferol, quercetin, isorhamnetin, or a flavonol diglycoside at a concentration that represented the level present in a 100 microg/ml concentration of an extract. Only bilobalide (2.8 microg/ml) increased CYP2B1 mRNA expression, and the -fold increase (7.9 +/- 0.5; mean +/- S.E.M.) was similar to that (8.3 +/- 1.7) by the extract. By comparison, only ginkgolide A (1.1 microg/ml) increased CYP3A23 mRNA expression, but the extent (2.6 +/- 0.5-fold) was less than the 5.3 +/- 1.7-fold increase by the extract. A greater concentration (5 microg/ml) of ginkgolide A was required to elevate CYP3A2 and CYP3A18 mRNA expression. Over the range of 1 to 5 microg/ml, bilobalide increased CYP2B1 mRNA and BROD, but not CYP3A23 mRNA or testosterone 6beta-hydroxylation, whereas ginkgolide A increased CYP3A23 mRNA and testosterone 6beta-hydroxylation, but not CYP2B1 mRNA or BROD. Overall, our novel results indicate a distinct role of bilobalide and ginkgolide A in the modulation of CYP2B1 and CYP3A23 gene expression and enzyme activities by G. biloba extract in primary cultures of rat hepatocytes.     

Possible differential induction of phase 2 enzyme and antioxidant pathways by american ginseng, Panax quinquefolius

Human immunodeficiency virus (HIV)-infected patients often take herbal medicines, which may interact with antiretrovirals. American ginseng induces phase 2 and antioxidant enzymes in vitro and might increase the clearance of zidovudine and/or enhance antioxidant activity. Ten healthy volunteers received 300 mg of zidovudine orally before and after 2 weeks of treatment with a ginsenoside-enriched American ginseng extract 200 mg twice daily. This ginseng extract induced the phase 2 enzyme quinone reductase with an average concentration of doubling of enzyme activity of 190 microg/mL. Total ginsenoside content was 8.5 +/- 0.5%. Pharmacokinetic profiles of zidovudine and oxidative stress marker concentrations were measured post-zidovudine dose. American ginseng does not significantly affect the formation clearance of zidovudine to its glucuronide (ratio post- to pre-American ginseng = 1.17; 90% confidence interval: 0.95-1.45; P = .21), total clearance (ratio = 0.97; 0.82-1.14; P = .70), or plasma zidovudine AUC0-8 (ratio = 1.03; 0.87-1.21; P = .77). Oxidative stress biomarkers are reduced post-American ginseng (F2-isoprostane ratio = 0.79; 0.72-0.86; P < .001; 8-hydroxy-deoxyguanosine ratio = 0.74; 0.59-0.92; P = .02). Two weeks of American ginseng does not alter zidovudine pharmacokinetics but reduces oxidative stress markers.     

Cell shape, cell-cell contact, cell-extracellular matrix contact and cell polarity are all required for the maximum induction of CYP2B1 and CYP2B2 gene expression by phenobarbital in adult rat cultured hepatocytes

The effect of cell shape, cell density, contact with extracellular matrix and cell polarity on the phenobarbital (PB)-induced gene expression of CYP2B1 and CYP2B2 (CYP2B1/2B2) in adult rat hepatocytes was investigated. High cell density enhanced the induction of CYP2B1/2B2 gene expression by PB. Hepatocytes cultured on EHS gel showed a spherical cell shape and highly enhanced the induction of CYP2B1/2B2 gene expression by PB. Although monolayer hepatocytes cultured on type I collagen (TIC) and type IV collagen exhibited poor induction of CYP2B1/2B2 gene expression by PB, monolayer cells on laminin showed substantial induction. The addition of soluble laminin to media did not show any effect on induction in monolayer hepatocytes cultured on TIC. Dishes coated with different concentrations of immovable laminin demonstrated complicated effects. Coating with higher concentrations of laminin resulted in greater induction of CYP2B1/2B2 gene expression by PB. On the other hand, when hepatocytes were cultured on dishes coated with lower concentrations of laminin, they became round and greater induction of CYP2B1/2B2 gene expression by PB was observed. Spherical hepatocytes cultured on low concentrations of TIC also showed highly enhanced induction of CYP2B1/2B2 gene expression by PB. EHS gel overlay to hepatocytes cultured on TIC and collagen sandwich configurations that are known to induce cell polarity enhanced the induction by PB. The induction of CYP2B1/2B2 gene expression needed cytoskeleton organization, such as actin filament, microtubule filament and intermediate filament. These results demonstrate that cell shape, cell density, contact with extracellular matrix and cell polarity all play critical roles in the induction of CYP2B1/2B2 gene expression by PB.     

Inhibition of heme oxygenase-1 partially reverses the arsenite-mediated decrease of CYP1A1, CYP1A2, CYP3A23, and CYP3A2 catalytic activity in isolated rat hepatocytes

Heme oxygenase (HO-1), the rate-limiting enzyme in the physiological breakdown of heme, is ubiquitous, and its expression can be increased by arsenite [As(III)], and similar other stimuli that induce cellular oxidative stress. Interestingly, it has been shown that the As(III)-induced HO-1 is inversely correlated with a decrease in cytochromes P450 (P450s) activity; however, the direct role for HO-1 in the inhibition of P450 enzymes remains unknown. Our results showed that As(III) at a concentration of 5 μM decreased the constitutive and inducible expression of CYP1A1, CYP1A2, CYP3A23, and CYP3A2 at the mRNA, protein, and catalytic activity levels. Moreover, As(III) decreased the nuclear accumulation of aryl hydrocarbon receptor (AhR) and pregnane X receptor without increasing their degradation. As(III) also increased the binding of cytosolic AhR to heat shock protein 90 and hepatitis B virus X-associated protein 2. In the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin as an inducer for CYP1A and rifampin as an inducer for CYP3A, As(III) decreased the enzymatic activity of the four P450s more than it decreased their mRNA or protein expression levels. It is noteworthy that treatment with the competitive HO-1 inhibitor, tin-mesoporphyrin, or supplementing external heme partially reversed the As(III)-mediated decrease in activities of the four P450s. In conclusion, the current study provides the first evidence that As(III) decreases CYP1A1, CYP1A2, CYP3A23, and CYP3A2 expression in freshly isolated rat primary hepatocytes. Furthermore, inhibiting the As(III)-mediated induction of HO-1 partially restores the enzymatic activity of these P450s that was initially decreased by As(III), confirming the direct role of HO-1 in the inhibition of P450s.     

The induction of CYP1A2, CYP2D6 and CYP3A4 by six trade herbal products in cultured primary human hepatocytes

The aim of this study was to evaluate the in vitro inductive potential of six commonly used trade herbal products on CYP1A2, CYP2D6 and CYP3A4 metabolic activities. Herbal components were extracted from the trade products in a way that ensured a composition equal to that present in the original product. Primary human hepatocytes and specific CYP substrates were used. Classic inducers were used as positive controls and herbal extracts were added in in vivo-relevant concentrations. Metabolites were determined by high performance liquid chromatography (HPLC). St. John's wort and common valerian were the strongest inducing herbs. In addition to induction of CYP3A4 by St. John's wort, common valerian and Ginkgo biloba increased the activity of CYP3A4 and 2D6 and CYP1A2 and 2D6, respectively. A general inhibitory potential was observed for horse chestnut, Echinacea purpurea and common sage. St. John's wort inhibited CYP3A4 metabolism at the highest applied concentration. Horse chestnut might be a herb with high inhibition potentials in vivo and should be explored further at lower concentrations. We show for the first time that G. biloba may exert opposite and biphasic effects on CYP1A2 and CYP2D6 metabolism. Induction of CYP1A2 and inhibition of CYP2D6 were found at low concentrations; the opposite was observed at high concentrations. CYP2D6 activity, regarded generally as non-inducible, was increased by exposure to common valerian (linear to dose) and G. biloba (highest concentration). An allosteric activation is suggested. From the data obtained, G. biloba, common valerian and St. John's wort are suggested as candidates for clinically significant CYP interactions in vivo.     

Comparison of inducibility of CYP1A and CYP3A mRNAs by prototypical inducers in primary cultures of human, cynomolgus monkey, and rat hepatocytes

This study was conducted to investigate the effects of treatment with the prototypical inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) on the mRNA levels of drug-metabolizing enzymes in primary cultures of cryopreserved human, cynomolgus monkey, and rat hepatocytes. Analysis was performed by quantitative real-time RT-PCR using primers and TaqMan probes. Treatment with Ome substantially increased the mRNA levels of both CYP1A1 and CYP1A2 in human hepatocytes, but increased only the mRNA level of CYP1A1 in monkey hepatocytes, whereas it had no marked effect on the mRNA levels of CYP1A1 or CYP1A2 in rat hepatocytes. Treatment with Rif or Dex did not markedly affect the mRNA level of CYP1A in any of the hepatocyte cultures under the conditions used. All three inducers increased the mRNA level of CYP3A8 in monkey hepatocytes (in the order Rif>Dex>or=Ome), and a similar profile was observed for the mRNA level of CYP3A4 in human hepatocytes, but the potency of induction was markedly attenuated. In contrast, only Dex substantially increased the mRNA level of CYP3A1 in rat hepatocytes, with Rif and Ome showing no effects. These results indicate that the molecular mechanisms responsible for the regulation of CYP1A2 genes differ between humans and cynomolgus monkeys, although the regulatory mechanisms for CYP1A1 and CYP3A genes are similar.     

Activation of CYP1A1 gene expression during primary culture of mouse hepatocytes

Expression of CYP1A1 mRNA in mouse hepatocytes in primary culture was investigated. The expression was obvious on day 3 of culture without addition of any known ligands of the aryl hydrocarbon receptor and increased with culture period. Removal of insulin from and addition of hydrogen peroxide to the medium enhanced and suppressed the expression, respectively. The CYP1A1 mRNA expression was also enhanced in the presence of anti-oxidant, t-butylhydroquinone, in the medium. Several kinds of kinase inhibitors markedly increased the CYP1A1 mRNA expression. In contrast, the inhibitory expression was prolonged in the presence of okadaic acid, a potent inhibitor of serine/threonine phosphatase PP1 and PP2. These observations suggest that there might be a repressive pathway in the regulation of CYP1A1 mRNA expression and that the presently observed expression pathway differs at several points from those previously reported, such as ligand-activated aryl hydrocarbon receptor- or omeprazole-mediated expression. Modulation of CYP1A2 mRNA expression after exposing hepatocytes to agents affecting phosphorylation pathways differed from that of CYP1A1 mRNA. This implies that regulatory pathways for CYP1A1 and CYP1A2 expression may differ.     

Role of nitric oxide in down-regulation of CYP2B1 protein, but not RNA, in primary cultures of rat hepatocytes.

There are conflicting reports about the role of nitric oxide in the down-regulation of cytochrome P450 that occurs when animals or cultured hepatocytes are exposed to inflammatory stimuli. Here, we investigated the participation of NO in the down-regulation of CYP2B1 by bacterial endotoxin (LPS) in rat hepatocytes cultured on Matrigel. LPS caused the down-regulation of CYP2B1 mRNA to 20% of control values within 12 h of treatment, and this was not reversed by concentrations of NO synthase inhibitors that completely blocked NO production. LPS was several orders of magnitude more potent in the down-regulation of CYP2B1 mRNA than in induction of NO production. In contrast, concentrations of LPS in the 1 to 100 ng/ml range induced NO production and produced a rapid down-regulation of CYP2B1 protein to 30% and <5% of control at 6 and 24 h, respectively, that could be completely prevented both by inhibitors of NO synthase and by LY83583, which prevents NO synthase-2 induction. The blockade of CYP2B1 down-regulation by NO synthase inhibitors was reversed by arginine, and the NO donors S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine mimicked CYP2B1 protein suppression. Taken together, these experiments demonstrate two independent mechanisms of CYP2B1 down-regulation by LPS: a rapid, NO-dependent suppression of the protein occurring at high concentrations of LPS and a slower, NO-independent pretranslational suppression occurring at low concentrations of LPS.     

Regulation of CYP2B6 and CYP3A expression by hydroxymethylglutaryl coenzyme A inhibitors in primary cultured human hepatocytes.

The effects of treatment with the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitors lovastatin, simvastatin, pravastatin, fluvastatin, and atorvastatin on the contents of cytochrome p450 mRNAs were examined in primary cultures of human hepatocytes prepared from three different livers. Treatment of 2- to 3-day-old human hepatocyte cultures with 3 x 10(-5) M lovastatin, simvastatin, fluvastatin, or atorvastatin for 24 h increased the amounts of CYP2B6 and CYP3A mRNA by an average of 3.8- to 9.2-fold and 24- to 36-fold, respectively. In contrast, pravastatin treatment had no effect on the mRNA level of either CYP2B6 or CYP3A, although treatment with pravastatin did produce the expected compensatory increase in HMG-CoA reductase mRNA content, indicating effective inhibition of cholesterol biosynthesis. Although treatment with the active (+), but not the inactive (-), enantiomer of atorvastatin increased the amount of HMG-CoA reductase mRNA, treatment with each enantiomer significantly induced both CYP2B6 and CYP3A mRNA levels. Treatment of primary cultured rat hepatocytes with the atorvastatin enantiomers effectively increased the amount of CYP3A mRNA, but had no effect on CYP2B or CYP4A mRNA levels, in contrast to fluvastatin, which increased both. Findings for p450 proteins by Western blotting were consistent with the mRNA results. These findings indicate that the ability of a drug to inhibit HMG-CoA reductase activity does not predict its ability to produce p450 induction in primary cultured human hepatocytes, and demonstrate that some, but not all, of the effects of these drugs that occur in primary cultured rat hepatocytes are conserved in human hepatocyte cultures.