Abstract
The isolation and purification of two major phenobarbitone-induced hepatic cytochromes P-450 from male Wistar rats is described. These forms (designated cytochromes P-450 B1 and P-450 B2) were extensively characterised and structurally and functionally compared to two other homogeneous isoenzymic forms, cytochromes P-452 (clofibrate-induced) and cytochrome P-447 (BNF-induced). These characterisation studies (including catalytic, immunological, physical and spectral analysis) indicated that all four isoenzymes were distinct, unique hemoproteins. The above characterisation was extended towards the induction profiles of these hemoproteins in hepatic and renal microsomes, following single dose xenobiotic pretreatment. Of primary interest was the immunoquantitation data which revealed the presence of cytochrome P-452 as a major constitutive isoenzyme. This data in conjunction with the metabolic data also indicated that xenobiotic induction of cytochromes P-450 was both a specific and precise event. In order to determine the induction profile of these hemoproteins following xenobiotic pretreatment, analysis of the mRNA species was also undertaken. In vitro rabbit reticulocyte translation systems in conjunction with immunoprecipitation analysis (using monospecific antibodies against the specific cytochrome P-450) revealed that following phenobarbitone and beta-napthoflavone pretreatment the induction of the specific translatable mRNA species was due to a difference in the amount of specific mRNA and not to a change in its translatability. This induction profile corresponded to that found on analysis of the holoenzyme cytochromes P-450 present within hepatic microsomes. The temporal aspects of the induction of the mRNA species coding for cytochrome P-452 was not in agreement with the metabolic and immunochemical data for this isoenzyme in hepatic microsomes. This discrepancy is possibly believed to relate to the activation/repression of genes coding for similar and related cytochrome P-452 isoenzymes. A preliminary analysis of renal mRNA species coding for the cytochromes P-450 in conjunction with metabolic data was strongly suggestive for there being different modes of induction in the liver and kidney.