Metabolism of Benzo(a)pyrene by Subcellular Fractions of Rat Liver: Evidence for Similar Patterns of Cytochrome P-450 in Rough and Smooth Endoplasmic Reticulum but not in Nuclei and Plasma Membrane

Since our earlier work (P. Stasiecki, F. Oesch, G. Bruder, E. D. Jarasch, and W. W. Franke, Eur. J. Cell Biol., 21: 79-92, 1980) had shown that carcinogen-metabolizing monooxygenase activ ity was present in almost all investigated cellular membranes, the possibility of differential control of the various metabolic pathways in the individual cellular membranes arose. Using high pressure liquid chromatography we have now studied the benzo(a)pyrene metabolites formed by rough and smooth endoplasmic reticulum, nuclei, and plasma membrane as well as mitochondrial fractions and investigated the metabolic coopera tion between the monooxygenases and epoxide hydrolase in these fractions. Since various cytochrome P-450 isozymes cat alyze the oxidative attack on the benzo(a)pyrene molecule at defined preferential sites, this analysis also provides an indirect trace of potential differences in the pattern of cytochrome P-450 isozymes present in the individual membranes. The metabolic profiles produced by the two most active frac tions, smooth and rough endoplasmic reticulum, were very sim ilar to each other but different from those produced by the other three preparations. The metabolite pattern produced by incuba tions containing nuclear fractions differed slightly from that pro duced by the fractions of endoplasmic reticulum, but plasma membrane and mitochondria produced markedly different pat terns. Since the similarity of the benzo(a)pyrene metabolite pattern produced by the smooth and rough endoplasmic reticulum sug gested similar cytochrome P-450 isozyme patterns in these two subfractions, they were further investigated by the use of selec tive Âinducersas well as a broad spectrum substrate, 7-ethoxycoumarin, in the absence and presence of selective inhibitors. Treatment of animals with frans-stilbene oxide or phA©nobarbital (a) increased the total amount of metabolites per protein mass and time, (b) changed the pattern of metabolites, but (c) induced a pattern of metabolites which was again very similar in rough and smooth endoplasmic reticulum. Even more distinct changes were found following treatment with 3-methylcholanthrene or AŸnaphthoflavone. Both of these compounds (a) preferentially in duced the activity of rough endoplasmic reticulum, (b) changed the profile of metabolites, but (c) again did not disturb the similarities of the benzo(a)pyrene metabolite pattern between both fractions. Selective inhibitors (metyrapone, a-naphthoflavone, tetrahydrofuran) had characteristically different effects on 7-ethoxycoumarin 0-deethylase activity in preparations from un treated versus frans-stilbene oxide (or phenobarbital)-treated versus /3-naphthoflavone (or 3-methylcholanthrene)-treated rats, but in any of these situations the effects of the selective inhibitors Received 6/28/83; revised 12/5/84, accepted 6/3/85. were similar in the smooth and rough endoplasmic reticulum fraction. These findings indicate that the composition of cytochrome P450 isozymes is different in nuclei, plasma membrane, mitochon drial fractions, and the endoplasmic reticulum but that the isoenzyme pattern of smooth and rough endoplasmic reticulum is similar even after enzyme induction. The total amount of benzo(a)pyrene metabolites produced by smooth and rough endoplasmic reticulum fractions from controls and from the animals treated with the various inducers correlated well with 7-ethoxycoumarin O-deethylase activities in absence of inhibitors and in presence of metyrapone or tetrahydrofuran (r = +0.983, +0.995, +0.990) but not in the presence of anaphthoflavone (r = -0.225), indicating that the monooxygenase forms inhibited by a-naphthoflavone were major contributors to benzo(a)pyrene metabolism in all these fractions. No good cor relation was found between dihydrodiol formation and epoxide hydrolase activity. In line with the relatively high ratio of epoxide hydrolase to monooxygenase in rat liver microsomes, this indi cates that in all these fractions epoxide hydration is not the ratedetermining step in the formation of the dihydrodiols studied.