Induction of cytochrome P-4502B1-related mouse cytochrome P-450 and regulation of its expression by epidermal growth factor/transforming growth factor α in primary hepatocyte culture

doi:10.1016/0006-2952(95)00200-J

Biochemical Pharmacology

Volume 50, Issue 6, 7 September 1995, Pages 781-785

https://doi.org/10.1016/0006-2952(95)00200-J Get rights and content

Abstract

Phenobarbital-dependent induction of mouse cytochrome P-450 (Cyp) orthologous to rat CYP2B1 and its modulation by hepatotrophic growth factors were examined in primary hepatocyte cultures. Compared to rat hepatocytes, induction in mouse hepatocytes was more rapid and effective. Ligands of the EGF receptor, epidermal growth factor, and transforming growth factor α inhibited induction on the basis of protein expression and CYP2B-associated 7-pentoxyresorufin-O-depentylase activity. Furthermore, EGF led to repression of accumulation of corresponding mRNA under phenobarbital, an effect not blocked by inhibition of protein synthesis under cycloheximide. Ligands of the EGF receptor may contribute towards the decrease in hepatic CYP expression observed during (pre)neoplastic development and regeneration.

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Multidrug resistance type 1 P-glycoproteins (P-gp) and multidrug resistance associated proteins (MRP) were studied in differentiated primary human lung cells in culture, in comparison with permanent human lung cell lines and primary alveolar type II cells from rat lung. AII cells exhibited low basal levels of mdr1b mRNA, that increased over time and after oxygen radical production induced by paraquat. mRNAs coding for antioxidative enzymes catalase (CAT), maganese superoxide dismutase (Mn-SOD) and copper/zinc superoxide dismutase (Cu/Zn-SOD) were not changed. H358, A549, H322 cells expressed low levels of MDR1 mRNA, but the mdr1 substrate rhodamine 123 (Rh 123) was transported out of H358 and H322 cells in a non-invasive, single cell fluorescence assay. The dye efflux could be inhibited by the chemosensitizer, verapamil. Normal human bronchial epithelial cells (NHBEC) expressed immuno-reactive MDR1 P-gp and the MPR protein that was active in the fluorescence assay using the MRP substrate carboxy-dichlorofluorescein (CDF) and MK-571 as an inhibitor. We did observe inter-individual variation of MRP in both the mRNA and the immunoreactive protein in NHBEC culture. Over time (12 weeks) the protein was relatively stable in NHBEC and epithelial cells from peripheral lung (PLC), but the mRNA level was drastically increased when explant cultures were continued (18 weeks).
Induction of cytochrome P450 2B1 by pyrethroids in primary rat hepatocyte cultures
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Citation Excerpt :
The induction followed a kinetic profile similar to that of CYP2B1 induction by PB in primary rat hepatocyte cultures, where maximal induction was observed within 3 days of culture [13–15]. As demonstrated in Fig. 6A, CYP2B1 mRNA induction by 50 μM permethrin exceeded CYP2B1 induction by PB at 1.5 mM, the PB concentration shown to elicit maximal CYP2B1 mRNA induction in this culture system [13]. In the present study, the induction of CYP2B1 mRNA by pyrethroids was dose-dependent in a range of 25–100 μM, while CYP2B protein as well as CYP2B-associated enzyme activity were not dose-dependent in the examined concentration range.
Numerous xenobiotics are capable of inducing their own metabolism and by enzyme induction can also lead to enhanced biotransformation of other xenobiotics. In this project, we examined the influence of pyrethroids (permethrin, cypermethrin, and fenvalerate) on the expression and activity of the phenobarbital (PB)-inducible cytochrome P450 2B1 isoform (CYP2B1) in primary rat hepatocyte cultures. Incubation of hepatocyte cultures with pyrethroids resulted in a marked CYP2B1 induction. Among the tested pyrethroids, permethrin elicited the most pronounced induction of CYP2B1 mRNA, which exceeded maximal induction achieved by PB at concentrations approximately 10-fold higher. Furthermore, permethrin induced CYP3A1 mRNA expression, while the expression of the CYP1A1 isoform, which in vivo is not responsive to PB treatment, was not significantly affected by pyrethroids. Permethrin-dependent enhancement of CYP2B1 and CYP3A1 mRNA expression was repressed by the hepatotrophic cytokine epidermal growth factor, which is known to also inhibit PB-dependent induction of CYP2B1. Several metabolites of permethrin formed by hepatocytes (3-(2′,2′-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 3-phenoxybenzyl alcohol, and 3-phenoxybenzoic acid) were ineffective in inducing CYP2B1 mRNA. Furthermore, permethrin stimulated the expression of the luciferase reporter gene under control of the CYP2B1 promoter (comprising the PB-responsive enhancer module) in transiently transfected primary hepatocyte cultures. Thus, permethrin-stimulated gene expression occurred on the transcriptional level. Taken together, these results indicate that the pyrethroid permethrin is a PB-like inducer. Due to its superior potency in induction, permethrin appears as a useful substance for mechanistic studies to elucidate the mechanism of enzyme induction by phenobarbital.
Effects of epidermal growth factor on CYP inducibility by xenobiotics, DNA replication, and caspase activations in collagen I gel sandwich cultures of rat hepatocytes
2001, Biochemical Pharmacology
Citation Excerpt :
Here, we report that EGF, significantly decreases the increase of CYP1A and CYP2B1/2 activities by PB but not the 3-MC and β-NF induction of CYP1A1. However, the decrease of PB-mediated induction of CYP by EGF in the collagen I gel cultures is moderate compared to the complete inhibition of CYP1A1, 2B1/2 and 3A4 by EGF in primary conventional cultures treated with 3-MC, PB and rifampicine, respectively [6,27,28]. These results suggest that part of the inhibitory effect of EGF on CYP expressions in conventional primary cultures may be due to combined effects of EGF and phenotypic changes occurring in this model, especially, the cell cycle progression and the decrease of liver specific functions.
In this study, we investigated the combined effects of EGF and collagen I gel on the phenotype of cultured rat hepatocytes and we focussed our investigations on the regulation of xenobiotic-mediated induction of CYP, cell cycle progression and activation of capases 8 and 3. We found that EGF, added to basal culture medium or phenobarbital (3.2 mM) containing medium, provoked a moderate decrease of CYP1A1 and CYP2B1/2 activities. However, EGF did not exert any inhibitory effect on 3-methylcholantrene (5 μM) and β-naphtoflavone (25 μM) induction of CYP1A1 activities. In collagen gel sandwich cultures, hepatocytes remained arrested in mid-G1 phase of the cell cycle, even in the presence of EGF. In conventional primary cultures, caspases 8 and 3 were activated at 3 and 5 days after plating respectively. In collagen gel sandwich cultures, we found that neither collagen I nor EGF prevented activation of caspase 8 while collagen I gel inhibited activation of caspase 3, preventing spontaneous apoptosis of cultured rat hepatocytes. In contrast, EGF transiently increased caspase 3 activity at day 1 after plating. Altogether, our data demonstrate that collagen I gel triggers intracellular signals which strongly affect cultured hepatocyte phenotype, leading to a cell cycle arrest in G1 phase and long-term survival through the inhibition of caspase 3 activation and that EGF-free medium improves survival and liver-specific gene expression in hepatocytes maintained in collagen I gel sandwich cultures.
Phosphorylation/dephosphorylation steps are crucial for the induction of CYP2B1 and CYP2B2 gene expression by phenobarbital
1999, Biochemical and Biophysical Research Communications
The effects of several protein kinase activators and protein phosphatase inhibitors on the phenobarbital (PB)-induced gene expression of CYP2B1 and CYP2B2 (CYP2B1/2B2) in adult rat hepatocytes were investigated. Insulin, epidermal growth factor, interleukin 6, cAMP, phorbol 12-myristate 13-acetate, tumor necrosis factor α, vanadate, and okadaic acid were found to suppress the induction of CYP2B1/2B2 at mRNA and protein levels in hepatocytes. cAMP and vanadate completely suppressed the induction of CYP2B1/2B2 gene expression in both rat hepatocytes and liver. The addition of genistein to vanadate-treated hepatocytes partially recovered the induction of CYP2B1/2B1 gene expression by PB. These results of the present study demonstrate that phosphorylation/dephosphorylation steps are crucial for the induction of CYP2B1/2B2 gene expression by PB.
Effect of epidermal growth factor in collagen gel cultures of rat hepatocytes
1999, Toxicology in Vitro
Collagen gel cultures of hepatocytes represent a promising in vitro model in pharmaco-toxicology. Epidermal growth factor (EGF) is usually added to the culture medium, although one could question its value in a culture model aiming at maintaining a maximum of differentiated functional capacities. In this study, the effects of EGF (20 ng/ml) on albumin secretion, morphology and pentoxyresorufin O-depentylase (PROD), ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities have been examined in both collagen gel sandwich and immobilization gel cultures of adult rat hepatocytes. Transmission electron microscopy did not show an obvious influence of epidermal growth factor (EGF) on the intracellular organization of organelles of the rat hepatocytes. It was found that EGF addition had no effect on albumin secretion in both culture models. On the contrary, the presence of EGF in the culture medium provoked in collagen gel sandwich cultures, after 7 days, significant decreases of 66% and 25% in EROD and PROD activities, respectively. On GST activities, no effect of EGF could be observed in both collagen gel cultures. Removal of EGF from the culture medium seemed to have a positive effect on the maintenance of the phase 1 biotransformation capacity of rat hepatocytes. Its addition should therefore be avoided in collagen gel cultures used in pharmaco-toxicology.
Interactions of gender, growth hormone, and phenobarbital induction on murine Cyp2b expression
1998, Biochemical Pharmacology
The interactions of gender, growth hormone, and phenobarbital induction on Cyp2b expression were examined in phenotypically normal (lit/+) and growth-hormone deficient “little” (lit/lit) mice. Using an immunocrossreactive monoclonal antibody designed to identify rat CYP2B1 and 2B2 proteins, we observed three hepatic Cyp2b proteins in control (lit/+) females, but only two proteins, one at trace levels, in control males. Phenobarbital administration to lit/+ mice increased the expression of the two Cyp2b isoforms in the males by 3- to 4-fold, but produced an approximately 75% reduction in the female-expressed proteins. Whereas growth hormone depletion (lit/lit) had no effect on the expression profile of Cyp2b proteins in females, it had a de-repressive effect in males, resulting in the expression of three proteins at concentrations now comparable to those observed in female liver. Generally, phenobarbital had no inductive effects in the lit/lit mice of both sexes. In all groups, transcript levels measured by a CYP2B1 probe were in agreement with the protein findings. In contrast, Cyp2b mRNA identified by an oligonucleotide probe for CYP2B2 were repressed completely by growth hormone in both sexes, and was expressed as a female-predominant transcript in the lit/lit mice. In spite of an apparent high degree of sequence homology between the rat CYP2B and murine Cyp2b gene families, the present findings highlight fundamental differences in their constitutive and gender-dependent expression, growth hormone regulation, and phenobarbital inducibility.

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^†: Present address: Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, MA, U.S.A.

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Research paperInduction of cytochrome P-4502B1-related mouse cytochrome P-450 and regulation of its expression by epidermal growth factor/transforming growth factor α in primary hepatocyte culture

Abstract

Biochim Biophys Acta

Exp Pathol

Biochem Pharmacol

FEBS Lett

Arch Biochem Biophys

Biochem Biophys Res Commun

Meth Enzymol

J Biol Chem

J Biochem Biophys Methods

Arch Biochem Biophys

J Biol Chem

Anal Biochem

J Biol Chem

Biochem Pharmacol

Research paper
Induction of cytochrome P-4502B1-related mouse cytochrome P-450 and regulation of its expression by epidermal growth factor/transforming growth factor α in primary hepatocyte culture