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In Vitro Cellular & Developmental Biology - Animal : Volume 35, Issue 2, February 1999

In Vitro Cellular & Developmental Biology - Animal : Volume 35, Issue 1, January 1999

In Vitro Cellular & Developmental Biology - Animal : Volume 34

In Vitro Cellular & Developmental Biology - Animal : Volume 33

Analysis of cytochrome P450 and phase II conjugating enzyme expression in adult male rat hepatocytes

Content Provider	Springer Nature Link
Author	Davila, Julio C. Morris, Dale L.
Copyright Year	1999
Abstract	The induction of cytochrome P450 (CYP450) and Phase II conjugating enzymes by prototypical hepatic enzyme inducers was studied in adult male rat hepatocytes. Hepatocytes were suspended and cultured in diluted Matrigel® in a basal serum-free Dulbecco’s modified Eagle medium and exposed to the prototypical liver enzyme inducers, 3-methylcholanthrene, phenobarbital, hydrocortisone, and clofibrate for 48 h. Total RNA and microsomes were isolated and prepared, respectively, at 72 h. The expression of CYP1A1, CYP1A2, CYP2B1, CYP2C11, CYP2E1, CYP3A1, CYP3A2, CYP4A1, fatty acyl-CoA oxidase, uridine diphosphate-glucuronosyltransferase, glutathione-S-transferase, and sulfotransferase was determined at the mRNA level with reverse transcriptase polymerase chain reaction (RT-PCR). The expression of CYP1A1, CYP2B1, CYP2C11, CYP2E1, and CYP4A1 was also measured at the apoprotein level by Western immunoblotting. Using these culture and expression analysis techniques, we have found that the expression of these metabolic enzymes can be maintained in culture for up to 7 d at the mRNA and apoprotein levels. In addition, hepatocytes were found to respond to chemical enzyme inducers with marked increases in enzyme expression at either the mRNA or protein level and in a concentration-related fashion. Cells were responsive to enzyme induction as early as 24 h after initial plating. The results obtained from this investigation indicate that the presence of diluted Matrigel® (at a concentration of 0.35 mg/ml), the use of low concentrations of insulin (1 µM), hydrocortisone (0.1 µM), and serum-free culture medium can maintain the differentiated phenotype and responsiveness of cultured hepatocytes to chemical-induced metabolic enzyme expression. Under the conditions used in this study, enzyme induction in adult male rat hepatocytes shows close agreement with enzyme induction observed in the livers of rats exposed to these or similar prototypical enzyme inducers. Rat hepatocytes cultured in the presence of diluted Matrigel® coupled with enzyme mRNA expression analysis with RT-PCR are proven to be a valuable and important in vitro toxicological approach to assess the chemical-induced changes in expression of liver CYP450 and Phase II conjugating enzymes.
Starting Page	120
Ending Page	130
Page Count	11
File Format	PDF
ISSN	10712690
Journal	In Vitro Cellular & Developmental Biology - Animal
Volume Number	35
Issue Number	3
e-ISSN	1543706X
Language	English
Publisher	Springer-Verlag
Publisher Date	1999-01-01
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	rat hepatocytes Matrigel® P450 RT-PCR immunoblotting Cell Biology Developmental Biology Animal Genetics and Genomics Animal Biochemistry Animal Physiology
Content Type	Text
Resource Type	Article
Subject	Cell Biology Developmental Biology Medicine

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