Characterization of cytochromes P450 in liver and kidney of rats treated with di-(2-ethylhexyl)phthalate.
A polyclonal antibody was made to a liver cytochrome P450 purified from di-(2-ethyl-hexyl)phthalate (DEHP)-treated Sprague-Dawley rats and was used to identify the CYP4A forms in liver and kidney cortex microsomes of control rats and rats treated with this peroxisome proliferator. Three clearly separated major protein bands were recognized on western blots in liver microsomes of control male rats or male rats treated with a single dose of DEHP, which, based on the description of relative mobility, tissue specificity, and sex dependent expression of CYP4A forms (Sundseth and Waxman (1992). J. Biol. Chem., 267, 801-810), correspond to the migration pattern of forms 4A1, 4A2, and 4A3 in clofibrate-treated rats. The administration of DEHP for 2 or 3 days caused a loss of resolution of two of the protein bands. The protein band corresponding to 4A2 was absent in liver or kidney cortex microsomes of DEHP-treated or control female rats and was not always visible in the livers of control male rats. The purified P450DEHP supported the hydroxylation of arachidonic acid at both the 19- and 20-carbon atoms with turnover rates of 1.4 +/- 0.2 and 22.7 +/- 2.5 nmoles per minute per nmol P450, respectively. No measurable amounts of hydroxylated products were obtained when prostaglandin E1, leukotriene B4, or testosterone were used as substrates. Another member of the CYP4 family, 4B1 from rabbit lung microsomes, was also recognized by this antibody on western blot analysis; however, rabbit lung form 4A4 showed only minimal cross-reactivity with this antibody.
Okita
J R
JR
Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman 99164-6510.
Castle
P J
PJ
Okita
R T
RT
D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D002994:Clofibrate / Q000494:pharmacology; D003429:Cross Reactions; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000378:metabolism*; D004051:Diethylhexyl Phthalate / Q000494:pharmacology*; D015777:Eicosanoids / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D005260:Female; D006900:Hydroxylation; D066298:In Vitro Techniques; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012727:Sex Characteristics
D015777:Eicosanoids; D005227:Fatty Acids; D003577:Cytochrome P-450 Enzyme System; D004051:Diethylhexyl Phthalate; D002994:Clofibrate