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.

Nurses in action--an introduction to Action Research in nursing.

Action Research is one of the new generation of qualitative research methods in the social sciences which has special significance for nurses in South Africa. The collaborative, participative and reflective qualities of Action Research appeal to practitioners, and lend themselves to joint problem solving activities in local contexts. This paper sets out a rationale for Action Research, then describes its features, strengths, and limitations, Ways of overcoming the latter are suggested. The paper concludes that Action Research has potential applications in the field of nursing, not only for the purposes of practical problem solving, but also for improving the personal and professional practice of nurses, and for emancipating nurses from their subordinate position in the hierarchy of health science.

Human liver lauric acid hydroxylase activities.

Nine male and five female human liver microsomal sample were examined for laurate 11- and 12-hydroxylase activities. The mean specific activities for the 11- and 12-hydroxylation reactions were 0.78 +/- 0.33 and 1.07 +/- 0.12 nmol/min/mg protein, respectively. Antibody inhibition experiments, using a polyclonal antibody to a cytochrome P450 (P450) isolated from diethylhexyl phthalate-treated rats, which recognizes forms P4504A1, P4504A2, and P4504A3 of the rate, inhibited the 12-hydroxylase activity by 65%, but did not affect 11-hydroxylase activity. Western-blot analyses of the 14 human liver microsomal samples identified one major protein band at 52 kDa that comigrated with human form 4A11. A correlation coefficient of only 0.19 was calculated when comparing laurate 12-hydroxylase activities and the densitometric values of the immunochemically reactive protein bands in the human liver microsomal samples, which strongly suggests that additional P450 forms also support the 12-hydroxylation of lauric acid. Laurate 11-hydroxylase activity was inhibited by diethyldithiocarbamate, an inhibitor of P4502E1-mediated reactions, and by chlorzoxazone, a P4502E1 substrate. A comparison of laurate 11-hydroxylase activities with densitometric values of the P4502E1 protein bands indicated a strong correlation existed (0.82). An analysis of microsomal samples containing expressed human forms P4501A2, P4502A6, P4502C8, P4502C9, P4502D6, P4502E1, and P4503A4 showed that only form P4502E1 supported the 11-hydroxylation reaction.

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.