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Expression of cytochrome P450 in rat pleural mesothelial cells in secondary cultures
A Buard1, P H Beaune, A Renier
1Laboratoire de Pathologie Cellulaire et Moleculaire de l'Environnement, INSERM U. 139, C.H.U. Henri Mondor, Creteil, France.
Abstract:
Cultured rat pleural mesothelial cells (RPMC) isolated from male Sprague-Dawley rats have been shown to metabolize polycyclic aromatic hydrocarbons to more oxygenated metabolites. This capacity, which is maintained with passages, suggested the presence of monooxygenase enzymes. In order to clarify the enzymatic pathway, we investigated the expression of cytochromes P450 (CYP) in cultured RPMC by Western and Northern blot analyses. Cells were cultured in Ham's F10 medium supplemented with 10% fetal calf serum. The CYP expression was studied from passage 9 to 16 on different cell strains treated for 48 hours with P450 inducers. CYP1A1 apoprotein expression was very low in untreated cells, but was markedly induced after treatment with 1 microM 3-methylcholanthrene or 22 microM beta-naphthoflavone. CYP1A1 mRNA was not detected in untreated cells and appeared after 3-methylcholanthrene treatment. CYP2E1 apoprotein was constitutively expressed in cultured RPMC, and markedly increased by 170 mM ethanol, and 0.1 microM or 1 microM dexamethasone treatments. Unexpectedly, whereas the amount CYP2E1 mRNA was not modified by ethanol treatment, dexamethasone has a marked inductive effect on CYP2E1 mRNA level. The CYP expression pattern was found similar in RPMC issued from different rats, and not dependent on passage number. The CYP expression and the detection of NADPH-P450 reductase, and of epoxide hydrolase, ascertained that RPMC contain the overall enzymatic pathway required for the biotransformation and activation of procarcinogen compounds, such as polycyclic aromatic hydrocarbons and nitrosamines. Both expression and regulation properties are maintained in long-term cultures of RPMC.
Insights
Cultured rat pleural mesothelial cells (RPMC) metabolize polycyclic aromatic hydrocarbons via cytochrome P450 enzymes. This study identifies specific CYP enzymes and confirms RPMC
Area of Science:
- Cell Biology
- Biochemistry
- Toxicology
Background:
- Cultured rat pleural mesothelial cells (RPMC) metabolize polycyclic aromatic hydrocarbons (PAHs).
- This metabolic capacity suggests the presence of monooxygenase enzymes, specifically cytochromes P450 (CYP).
- Understanding the enzymatic pathway is crucial for assessing the biotransformation of procarcinogens.
Purpose of the Study:
- To investigate the expression of cytochromes P450 (CYP) in cultured RPMC.
- To clarify the enzymatic pathway involved in PAH metabolism by RPMC.
- To determine the regulation of CYP expression in RPMC under various treatments.
Main Methods:
- Western and Northern blot analyses were used to study CYP expression.
- RPMC were cultured and treated with specific CYP inducers (3-methylcholanthrene, beta-naphthoflavone, ethanol, dexamethasone).
- Expression levels of CYP1A1 and CYP2E1 were analyzed at different passages and after inducer treatments.
Main Results:
- CYP1A1 apoprotein and mRNA were induced by 3-methylcholanthrene and beta-naphthoflavone.
- CYP2E1 apoprotein was constitutively expressed and induced by ethanol and dexamethasone.
- Dexamethasone significantly increased CYP2E1 mRNA levels, while ethanol did not.
- CYP expression patterns were consistent across different cell strains and passages.
Conclusions:
- Cultured RPMC express functional CYP enzymes (CYP1A1, CYP2E1) involved in procarcinogen metabolism.
- RPMC possess the complete enzymatic machinery, including NADPH-P450 reductase and epoxide hydrolase, for biotransformation.
- These metabolic capabilities and regulatory properties are maintained in long-term RPMC cultures.