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 P450 (PB1 and PB2) have been isolated from the livers of rats treated with phenobarbital. PB2 (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. PB1 and PB2 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 PB1 to be localized in a large area and PB2 in a narrow range of cells around the central vein. This demonstrates the heterogeneity of hepatocytes 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 MC1 (mol. wt. 54 500) and MC2 (mol. wt. 57 000) were present at very low levels, MC2 mostly in the periportal region but also diffusely distributed throughout the lobule including some centrilobular cells, MC1 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 P450 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: (i) PB2, MC1, EH and GST 'C' type proteins were concentrated in a narrow area around the central vein; (ii) PB1 and GST 'B' type proteins occupied a large centrilobular area; (iii) MC2 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 beta-naphthoflavone led to a shift of MC1 to the periportal 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple forms of cytochrome P-450 related to forms induced marginally by phenobarbital. Differences in structure and in the metabolism of alkoxyresorufins

“…PB2b (PB21) on the basis of yield from the purification, appears to be present in rat microsomal fractions in low concentration. From previous studies PB2, was shown to be phenobarbital-inducible and highly localized in the centrilobular region of the liver [12], in contrast with the PB1 proteins, which are more diffusely distributed in this area [12]. We have also shown that both PB1-and PB2-related proteins are expressed in significant concentrations in human liver, but differences in the level of expression are observed between individuals.…”

“…In a previous report [12] we have described the isolation of two distinguishable forms of rat liver cytochrome P-450 (PB1 and PB2), which are only marginally induced by phenobarbital relative to certain other forms, namely PB35 and PB3b [5,6,8]. We have now isolated a series of proteins that appear structurally related to PB1 and PB2.…”

“…Although they appear to have identical N-terminal sequences, PBia and PBlb can be clearly distinguished from each other by their different Mr values, spectra and substrate specificities. Although PB1, appears to be another member of this group, it was isolated from a different microsomal preparation by a different purification procedure [12]. The N-terminal sequence of PBia and PBlb are identical with that of the enzyme described by Waxman & Walsh [11] as PB1.…”

“…Members of this family have been isolated from rat liver and termed 'PB1' by Waxman & Walsh [11] and 'PB-C' by Guengerich et al [6]. We have previously described the isolation and purification of two cytochrome P-450 forms (PB11I and PB2) marginally inducible by phenobarbital and a variety of other reagents such as trans-stilbene oxide and Aroclor 1254 [12]. Interestingly, PB1 also appears to be elevated at early time points in preneoplastic foci in rat liver [13].…”

Immunohistochemical demonstration of β-naphthoflavone-inducible cytochrome P450 1A1/1A2 in rat intrahepatic biliary epithelial cells

“…[6][7][8] Indeed, a significant body of evidence has been presented showing that the anatomical distributions of xenobiotic-metabolizing enzymes within tissues can greatly influence the cell specificity of chemically induced toxicities resulting from the biotransformation of relatively inert xenobiotics into electrophilically reactive metabolites. 6,7,9,10 While the localizations, distributions, and inductions of various phase I and phase II xenobiotic-metabolizing enzymes within parenchymal cells (i.e., hepatocytes) throughout the liver have received considerable attention during the past two decades, [6][7][8][9][11][12][13][14] little is currently known regarding the presence and inducibility of xenobiotic-metabolizing enzymes, particularly phase I enzymes, within intrahepatic biliary epithelial cells and other nonparenchymal liver cells. [15][16][17] Intrahepatic biliary epithelial cells have recently received considerable attention because of their ability to proliferate under certain pathophysiological conditions and because of the possibility that they might serve as the progenitor cell in the liver.…”