[33] Induction protocols for cytochromes P450IIIA in Vivo and in primary cultures of animal and human hepatocytes

doi:10.1016/0076-6879(91)06104-B

Methods in Enzymology

Volume 206, 1991, Pages 345-353

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This chapter discusses the induction protocols for cytochromes P450IIIA in vivo and in primary cultures of animal and human hepatocytes. Although induction of cytochromes P450IA and P450IIB by the prototypic inducers 3-methylcholanthrene and phenobarbital, respectively, was well established in the mid 1970s, the existence of a “third class” of inducers was only realized in the early 1980s. The protocols described here have been used successfully in the laboratory to induce cytochromes P450IIIA both in vivo and in primary cultures of animal and human hepatocytes. In both rabbit and human untreated hepatocyte cultures, P450IIIA gene transcription, messenger RNA (mRNA) and protein accumulation, and related monooxygenase activities rapidly decline after plating to levels hardly detectable after 48 hr. These genes retain their capacity to be activated by their specific inducers in young and up to 3-week-old cultures from rabbits.

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Cytochrome P450 (CYP) 3A1 was detected in the cytoplasm of giant cells in the trophoblastic region of rat placenta through pregnancy (Ejiri et al. 2001). In the present study, changes in the expression of CYP3A1 protein as well as in the histology of pregnant rat and fetal livers and placenta after treatment with dexamethasone (DEX) or pregnenolone-16α-carbonitrile (PCN), well-known CYP3A1 inducers, at 16 day of gestation (DG) (DEX₁ and PCN₁ groups: 100 mg/kg) or from 13 to 16 DG (DEX₄ group: 25 mg/kg/day; PCN₄ group: 50 mg/kg/day). All animals were killed at 17 DG, and Western blot and immunohistochemical analyses on CYP3A1 expression and histological examination were done. Western blot analysis revealed that PCN induced CYP3A1 more clearly than DEX and that the induction was most prominent in the fetal liver and lowest in the placenta. Except for the placenta, changes in the immunohistochemical stainability for CYP3A1 almost corresponded to those in the expression of CYP3A1 by Western blot analysis. In addition, swelling and and/or vacuolization of hepatocytes were generally observed in the mother and fetal livers showing increased expression of CYP3A1. These results suggest that DEX and PCN might pass through the placenta with no prominent induction of CYP3A1 at the placenta and be distributed to the fetal liver rapidly, resulting in high induction of CYP3A1 in the fetal liver.
Alteration of cellular phosphorylation state affects vitamin D receptor-mediated CYP3A4 mRNA induction in Caco-2 cells
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Expression of cytochrome P450 3A4 (CYP3A4) is induced by 1,25-dihydroxyvitamin $D_{3} (1, 25 {(OH)}_{2} D_{3})$ in Caco-2 cells. However, since a typical vitamin D responsive element has not been found in the 5^′-flanking region of the CYP3A4 gene, the mechanism of 1,25(OH)₂D₃-induced CYP3A4 mRNA expression is poorly understood. In the present study, we demonstrated that vitamin D receptor (VDR) is a critical factor for the induction using the antisense oligonucleotide technique. In addition, we found that treatment of Caco-2 cells with the protein kinase C (PKC) inhibitors, staurosporine and GF109203X, and the tyrosine kinase inhibitor, genistein, but not with the protein kinase A inhibitor, H-89, suppressed CYP3A4 mRNA induction by 1,25(OH)₂D₃. The depletion of PKC by prolonged treatment with phorbol ester abolished the induction. On the other hand, protein kinase inhibitors used had no effects on the constitutive expression of VDR mRNA. Therefore, these observations suggest that 1,25(OH)₂D₃-induced CYP3A4 mRNA expression might be involved in phosphorylation events in addition to transcriptional regulation via VDR. However, 1,25(OH)₂D₃ did not rapidly activate PKC in the Caco-2 cells used, while the treatment with staurosporine and GF109203X, but not genistein, decreased basal PKC activity by ∼30% of the controls. Taken together, these findings suggest that the change in the phosphorylation state via PKC and tyrosine kinase might, at least in part, modulate 1,25(OH)₂D₃-induced CYP3A4 mRNA expression via VDR.
Modulation of CYP3A4 expression by ceramide in human colon carcinoma HT-29 cells
2002, Biochemical and Biophysical Research Communications
Cytochrome P450 3A4 (CYP3A4) enzyme is responsible for the metabolic activation and inactivation of the majority of clinically used drugs in human liver and intestines. Recent studies have increasingly implicated various inflammatory stimuli to cause changes in the activities and expression levels of CYPs. However, the underlying mechanisms are largely unknown. In the present study, our studies investigated the effects of ceramide on CYP3A4 expression in human colon carcinoma HT-29 cells. Treatment with the cell-permeable ceramide analog C₆-ceramide to the cells significantly decreased the expression of CYP3A4. By contrast, C₆-dihydroceramide, a biologically inactive analog of C₆-ceramide, did not affect CYP3A4 expression. We found that bacterial sphingomyelinase (SMase) and tumor necrosis factor-α (TNF), which are known to increase intracellular ceramide levels, also markedly suppressed the synthesis of CYP3A4. To elucidate whether nitric oxide (NO) participates in suppression of CYP3A4 expression by ceramide, the effects of NO modulators were determined. Treatment with N^G-monomethyl-l-arginine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), was able to protect ceramide-dependent CYP3A4 suppression. In contrast, the addition of S-nitroso-N-acetylpenicillamine, a NO donor, to HT-29 cells reduced CYP3A4 expression. The addition of iNOS antisense oligonucleotide prevented ceramide-mediated induction of iNOS expression and restored CYP3A4 expression. Wortmannin which is known to inhibit phosphatidylinositol 3-kinase (PI3-K) blocked CYP3A4 suppression by ceramide. Taken together, our results demonstrate that ceramide-mediated suppression of CYP3A4 is due to production of NO, which might result from activation of PI3-K.
Nuclear Receptor Response Elements Mediate Induction of Intestinal MDR1 by Rifampin
2001, Journal of Biological Chemistry
Citation Excerpt :
The reported co-induction of CYP3A4 andMDR1 (11, 12) and the recently identified involvement of PXR in xenobiotic induction of CYP3A genes (18) prompted us to investigate whether PXR is also involved in induction ofMDR1. Treatment of LS174T cells with the knownCYP3A4 inducers reserpine, clotrimazole, RU486, carbamazepine, dexamethasone, and pregnenolone-16α-carbonitrile (12,25, 26) and the known activators for PXR, nifedipine, corticosterone, 5β-pregnane-3,20-dione, and 6,16α-dimethylpregnenolone (14-17), induced MDR1. Only the PXR activator coumestrol did not induce MDR1.
Intestinal P-glycoprotein, which is encoded by the MDR1 gene, plays an important role in the absorption and presystemic elimination of many xenobiotics. Hence, an understanding of the factors regulating its expression and function is of substantial interest. In addition to genetic factors, exposure to drugs such as rifampin can profoundly affect its expression. So far, the mechanisms by which rifampin induces MDR1 expression are poorly understood. Recent studies demonstrate that the nuclear receptor PXR (pregnane X receptor) is involved in xenobiotic induction of CYP3A4. Because CYP3A4 and MDR1are often co-induced, we investigated whether a similar mechanism is also involved in MDR1 induction. The human colon carcinoma cell line LS174T was used as an intestinal model to study induction because in these cells the endogenous MDR1 gene is highly inducible by rifampin. The 5′-upstream region of human MDR1was examined for the presence of potential PXR response elements. Several binding sites were identified that form a complex regulatory cluster at about −8 kilobase pairs. Only one DR4 motif within this cluster is necessary for induction by rifampin. We conclude that induction of MDR1 is mediated by a DR4 motif in the upstream enhancer at about −8 kilobase pairs, to which PXR binds.
Expression and DNA-binding activity of C/EBPα and C/EBPβ in human liver and differentiated primary hepatocytes
2001, Journal of Hepatology
Background/Aims: Limited information is available on the expression and role of C/EBP factors in human liver and hepatocytes. We investigated the expression and DNA-binding activity of C/EBPα and C/EBPβ in human liver needle biopsies, surgical lobectomies and differentiated cultured hepatocytes derived from lobectomies.
Methods: RNA and protein extracts were analyzed by RNAse protection, immunoblot and gel shift assays.
Results: C/EBP mRNAs, isoforms and DNA-binding activities were low/undetectable in lobectomies. In contrast, several C/EBPα (47, 45, 35 and 33 kDa) and C/EBPβ isoforms (47, 43, 40, 35 and 21 kDa) were observed in needle biopsies. In cultured hepatocytes, the C/EBP expression pattern dramatically changed with time. C/EBPα mRNA and the 45 kDa isoform increased in parallel, reaching a maximum after 3–4 weeks coincident with weak DNA-binding activity. C/EBPβ mRNA and isoform expression increased rapidly reaching a plateau within 1–2 weeks; all C/EBPβ isoforms were phosphorylated. C/EBPβ exhibited greater DNA-binding activity than C/EBPα, and this activity paralleled C/EBPβ isoform expression.
Conclusions: C/EBP isoforms exhibit markedly different expression patterns in lobectomies, needle biopsies and cultured hepatocytes. Stress stimuli during and/or after surgery for lobectomy resections may account for this difference. The pattern of C/EBP isoform expression in long-term highly differentiated cultured hepatocytes is close to that observed in needle biopsies.
Interleukin-6 negatively regulates the expression of pregnane X receptor and constitutively activated receptor in primary human hepatocytes
2000, Biochemical and Biophysical Research Communications
The marked impairment of hepatic drug metabolism during inflammation and infections has been known for many years and shown to result from down-regulation of cytochrome P450s (CYP) by cytokines. However, the mechanism of this repression is unknown. Using primary cultures of human hepatocytes, we show here that interleukin-6 (IL-6) rapidly and markedly decreases the expression of PXR (pregnane X receptor) and CAR (constitutively activated receptor) mRNAs, but does not affect the levels of dioxin receptor and glucocorticoid receptor mRNA. In parallel, IL-6 decreases both rifampicin- and phenobarbital-mediated induction of CYP2B6, CYP2C8, CYP2C9, and CYP3A4. As the transcriptional activity of PXR and CAR is not affected by IL-6 in cell-based reporter assays, our data suggest that the loss of CYP2 and CYP3 inducibility results from the negative regulation of PXR and CAR gene expression by this cytokine.

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[33] Induction protocols for cytochromes P450IIIA in Vivo and in primary cultures of animal and human hepatocytes

Publisher Summary

J. Biol. Chem.

Biochem. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochem. Pharmacol.

Biochem. Biophys. Res. Commun.

Gastroenterology

Biochem. Biophys. Res. Commun.

Biochemistry