Abstract
In the present study, we describe the effects of medium composition in primary cultures of rat hepatocytes on the expression of two major constituent female-dependent CYP isoforms, CYP2C12 and CYP2A1. When female rat hepatocytes were cultured with the serum-free medium HepatoZYME, currently used to attain long-term maintenance of hepatocyte phenotypic expression, CYP2C12 mRNA and protein levels were markedly suppressed, despite the constant presence of growth hormone, the essential regulator of liver CYP2C12. Conversely, rat hepatocytes cultured in the serum-free medium Dulbecco's modified Eagle's medium-F12K, also supplemented with growth hormone, sustained near normal expression levels of CYP2C12 mRNA and protein for the 7 days of observations. Although media composition had no significant effect on mRNA expression of CYP2A1, protein content decreased dramatically in hepatocytes cultured with HepatoZYME medium. We were able to demonstrate the plasticity of the cells by restoring/suppressing the expression of CYP2C12 and CYP2A1 mRNA by reverting the culture conditions. Addition of the mitogen epidermal growth factor present in the HepatoZYME formulation to the Dulbecco's modified Eagle's medium-F12K culture media appreciably decreased expression of both CYP2C12 and CYP2A1 in female hepatocytes, while briefly sustaining levels of the cyclin inhibitor p21. Lastly, reduced CYP protein content observed in hepatocytes cultured with epidermal growth factor was not the result of an absence or reduction in the CCAAT/enhancer-binding protein α, a requisite transcription factor for CYP2C12 expression.
Footnotes
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Send reprint requests to: Bernard Shapiro, Ph.D., Laboratories of Biochemistry, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce St., Philadelphia, PA 19104-6048. E-mail:shapirob{at}vet.upenn.edu
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This work was supported by National Institutes of Health Grants GM45758 and HD16358.
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↵1 The terms sex-dependent, sex-predominant or -dominant, and sex-specific are often used indiscriminately. We use sex-dependent to imply that expression levels are dependent on the existence of sex; sex-predominant indicates that expression levels, regardless of magnitude, are consistently greater in one sex; and sex-specific implies that expression is basically restricted to only one sex.
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↵3 In our efforts to detect CYP2C12 mRNA in hepatocytes cultured without growth hormone, we prolonged autoradiographic exposure time, which unavoidably resulted in overexposure of those samples expressing the isoform (Fig. 1).
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↵4 An uncoupling of transcription from translation of hepatic P450s is not unique and has been reported in phenobarbital induction of CYP2B1 (Agrawal and Shapiro, 1996) and growth hormone induction of CYP2C11 (Pampori and Shapiro, 1994).
- Abbreviations used are::
- CYP
- cytochrome P450
- EGF
- epidermal growth factor
- DMEM
- Dulbecco's modified Eagle's medium
- DMEM-F12 and -F12K
- DMEM-Ham's F12 and F12K, respectively
- C/EBPα
- CCAAT/enhancer-binding protein α
- MTS
- CellTiter 96 Aqueous One solution
- HepatoZYME
- HepatoZYME-serum-free medium
- Received August 14, 2000.
- Accepted October 20, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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