Characterization, localization and regulation of a novel phenobarbital-inducible form of cytochrome P450, compared with three further P450-isoenzymes, NADPH P450-reductase, glutathione transferases and microsomal epoxide hydrolase

Abstract

Two cytochromes P ₄₅₀ (PB ₁ and PB ₂ ) have been isolated from the livers of rats treated with phenobarbital. PB ₂ (mol. wt. 53 500) is novel and is the first example of a phenobarbital-inducible enzyme with a Soret peak at 447 nm. Using an enzyme-linked immunosorbent assay, some immunochemical and structural similarities were observed between these cytochromes. PB ₁ and PB ₂ were induced by phenobarbital, Aroclor 1254, trans-stilbene oxide and to a lesser extent by isosafrole. Immunohistochemical localization of these proteins in the liver of untreated rats showed PB ₁ to be localized in a large area and PB ₂ in a narrow range of cells around the central vein. This demonstrates the heterogeneity of hepato-cytes even within the centrilobular area and indicates that the synthesis of these two proteins is regulated differently although both are induced by the same agent, phenobarbital. Two 3-methylcholanthrene inducible cytochromes MC ₁ (mol. wt. 54 500) and MC ₂ (mol. wt. 57 000) were present at very low levels, MC ₂ mostly in the periportal region but also diffusely distributed throughout the lobule including some centrilobular cells, MC ₁ concentrated in the centrilobular region. The localization of two major groups of glutathione transferases (GST's) was also different. ‘C’ type proteins (Yb Yb') and microsomal epoxide hydrolase (EH), were concentrated around the central vein, whereas the ‘B’ type proteins (Ya Yc) and cytochrome P ₄₅₀ reductase were distributed in a larger area of this region. Thus, the localization was different for some members of the same enzyme family, whilst similarities in the localization existed across the border of the families: (1) PB ₂ , MC ₁ , EH and GST ‘C’ type proteins were concentrated in a narrow area around the central vein; (ii) PB ₁ and GST ‘B’ type proteins occupied a large centrilobular area; (iii) MC ₂ levels were very low, predominantly periportal but also diffusely distributed throughout the lobule. Treatment of the animals with inducers increased the staining intensity and in several cases extended the areas of cells containing these proteins over the adjacent zone without fundamentally altering their distributions. However, treatment with β-naphthoflavone led to a shift of MC ₁ to the peri-portal area. This suggests that the expression of these proteins in certain cells is not an irreversible quality of differentiation but depends on the degree of suppression and derepression of regulatory components. The differences in the localization between the predominantly detoxifying enzymes EH and GST's and the cytochromes P ₄₅₀ which are frequently involved in the activation of carcinogens in all likelihood represent an important factor in the susceptibility of certain regions to chemical carcinogens, although it must be kept in mind that the method allows detection of immunoreactive protein but not that of enzyme activity.