INDUCTION AND LOCALIZATION OF CYTOCHROME P4501B1 (CYP1B1) PROTEIN IN THE LIVERS OF TCDD-TREATED RATS - DETECTION USING POLYCLONAL ANTIBODIES RAISED TO HISTIDINE-TAGGED FUSION PROTEINS PRODUCED AND PURIFIED FROM BACTERIA

Knowledge of the response of cytochrome P450 1B1 (CYP1B1) to exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in both humans and roden ts is limited. To improve the analysis of CYP1 proteins, specific CYP1 B1 and CYP1A1 polypeptides were expressed as hexahistidine-tagged fusi on proteins in Escherichia coli, purified to homogeneity and used to p roduce polyclonal antibodies in rabbits. Immunoblot analyses showed th at these antibodies were specific and sensitive, detecting both the hu man and rat forms of the respective isozymes and exhibiting negligible cross-reactivity between the two known CYP1 subfamilies. We show that CYP1B1, CYP1A1 and CYP1A2 protein levels were induced in the livers o f female Sprague-Dawley rats following either acute (single dose of 25 mu g TCDD/kg) or chronic (125 ng TCDD/kg/day for 30 weeks) exposure t o TCDD, CYP1B1 protein exhibited a dose-response to TCDD that was diff erent from those of CYP1A1 and CYP1A2, CYP1B1 induction appeared to be less sensitive to TCDD exposure, with induction occurring at higher d oses of TCDD than that required for induction of CYP1A1 or CYP1A2. Imm unohistochemical analysis showed that in animals chronically exposed t o TCDD (35 ng/kg/day for 30 weeks), CYP1B1 was induced only in centril obular hepatocytes, a pattern of expression similar to that of CYP1A1 and CYP1A2. These observations of cellular co-localization of the CYP1 cytochromes in livers of TCDD-treated rats and apparent differences i n both protein amounts and dose-response are indicative of both common and unique regulation of CYP1 induction.