Improved separation and immunodetection of rat cytochrome P450 4A forms in liver and kidney

In the study of tissues that contain several forms of one cytochrome P450 subfamily, it is useful to develop immunoblotting techniques so that the various individual members of the family can be distinguished. This paper describes improvements in the immunoblotting technique to distinguish members of the rat cytochrome P450 4A subfamily, 4A1, 4A2, and 4A3, as they are present in Sprague-Dawley rat liver microsomes. This procedure was used to investigate differences in the cytochrome P450 4A forms observed under various conditions such as: untreated versus peroxisome proliferator treated rats, Sprague-Dawley versus Fischer 344 male versus female rats, and liver versus kidney microsomes. In liver microsomes of male Sprague-Dawley rats, forms 4A1, 4A2, and 4A3 were induced by the peroxisome proliferators, clofibrate, di-(2-ethylhexyl) phthalate, dehydroepiandrosterone, aspirin, and ibuprofen. Expression of the 4A forms shows strain specificity. A comparison of the cytochrome P450 4A forms in male Sprague-Dawley and Fischer 344 rats treated with peroxisome proliferators demonstrated that three distinct protein bands are visible on immunoblots of liver microsomes of Sprague-Dawley rats, whereas only two distinct protein bands are detectable in liver microsomes of Fischer 344 rats. The two protein bands in liver microsomes of male Fischer 344 rats migrate in positions corresponding to the 4A2 and 4A3 bands in male Sprague-Dawley rats. There did not appear to be a protein band corresponding to the 4A1 band of Sprague-Dawley rats. Expression of the 4A forms also shows gender specificity. In liver microsomes of female Sprague-Dawley rats, expression of the P450 4A2 form was not observed after treatment with a peroxisome proliferator. Expression of the 4A forms also shows tissue specificity. In kidney, 4A2 is the major protein band in male Sprague-Dawley rats with minor amounts of the 4A3 protein, whereas two prominent protein bands (4A2 and 4A3) are seen in male Fischer 344 rats.