Localization and identification of cytochrome P-450 in differentiating rat embryo cells in vitro and in foetal tissue in vivo by immunocytochemistry

Abstract

The presence of the isoenzymes b, e and c of cytochrome P-450 in foetal rat limb-bud and mid-brain tissue has been investigated in vivo and in micromass cell cultures of limb-bud and mid-brain cells derived from rat embryos by a sensitive immunocytochemical technique. The cytochromes could not be detected by antibody staining at the start of the culture nor in 13-day-old embryos from which cultures were prepared. Two different antibodies directed against cytochrome P-450 revealed the ontogenic profile of the phenobarbitone-inducible b and e forms, which appeared at an earlier stage of development, day 1 of culture (equivalent to day 14 of gestation), than did the 3-methylcholanthrene-inducible c form, which appeared on day 3 of culture (equivalent to day 16 of gestation). These isoenzymes were not tissue specific. Comparison of the localization and intensity of staining of cells cultured in vitro for 5 days with tissue from the equivalent foetal developmental stage (day 18) in vivo revealed the presence of cytochrome P-450 in corresponding areas. In day 18 limb sections, cytochrome P-450 was localized in the perichondrial and myogenic tissue, which corresponded to the cells in the periphery of the chondrogenic foci in vitro. In mid-brain whole tissue, the enzyme was located in connective tissue and neurofibrils, corresponding to cells in the periphery of the foci of neurones in vitro. The correlation between in vitro and in vivo observations from time course, location and quantitative aspects, illustrated that the micromass culture technique is a valid model for metabolism studies with these specific isoenzymes.