Characterization of distinct forms of cytochromes P-450, epoxide metabolizing enzymes and UDP-glucuronosyltransferases in rat skin

Mai Anh Pham, Jacques Magdalou, Muriel Totis, Sylvie Fournel-Glgleux, Gérard Siest, Bruce D. Hammock

Research output: Contribution to journalArticle

45 Scopus citations

Abstract

Study of drug metabolizing enzyme activity was undertaken in skin microsomal and cytosolic fractions of male and female rats. The presence of several isoforms was revealed from their activities towards selected substrates and from their cross immunoreactivity using antibodies raised against purified hepatic or renal cytochromes P-450, epoxide hydrolase and UDP-glucuronosyltransferases. Cytochrome P-450 content was precisely quantified by second derivative spectrophotometry, 23.1 and 16.5 pmol/mg protein in males and females, respectively. The monooxygenase activity associated to cytochromes P-450IIB1 and P-450IA1 was determined through O-dealkylation of ethoxy-; pentoxy- and benzoxy- resorufin. The activity ranged between 4 and 2 nmol/min/mg protein for male and female rats, respectively. These results and Western blot analysis indicated that rat skin microsomes contain both monooxygenase systems associated with cytochromes P-450IIB1 and P-450IA1. By contrast lauric acid hydroxylation, supported by cytochrome P-450IVA1, was not detectable. Activities of epoxide metabolizing enzymes (microsomal and cytosolic epoxide hydrolases; glutathione S-transferase) were also characterized in skin. Microsomes catalysed the hydratation of benzo(a)pyrene-4,5-oxide and cis-stilbene oxide at the same extent, whatever the sex, although the specific activity was 10 times lower than in liver. The hydratation of transstilbene oxide by soluble epoxide hydrolase was four times lower than in the liver. Conjugation of cis-stilbene oxide with glutathione in skin and liver proceeded at essentially similar rates, as the specific activity of glutathione S-transferase in skin was only two times less than that measured in hepatic cytosol. Glucuronidation of 1-naphthol, bilirubin but not of testosterone could be followed in the microsomal fraction. Revelation by Western blot indicated that both the isoforms involved in conjugation of phenols and bilirubin were present in skin microsomes. By contrast, the isoform catalysing the conjugation of testosterone was apparently missing. When immunoblotting was carried out using specific antibodies raised against the renal isoforms, the same result was obtained. In addition, an intense staining corresponding to a 57 kD-protein was observed.

Original languageEnglish (US)
Pages (from-to)2187-2194
Number of pages8
JournalBiochemical Pharmacology
Volume38
Issue number13
DOIs
StatePublished - Jul 1 1989

ASJC Scopus subject areas

  • Pharmacology

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