[CANCER RESEARCH 37, 3427-3433, September 1977] The Role of Microsomes and Nuclear Envelope in the Metabolic Activation of Benzo(a)pyrene Leading to Binding with Nuclear Macromolecules1 John M. Pezzuto, Michael A. Lea, and Chung S. Yang2 Department of Biochemistry, College of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07013 SUMMARY spread atmospheric pollutant that has also been shown to be a component of cigarette smoke (7). Like many chemical In an attempt to resolve existing conflicting reports and carcinogens, unmetabolized BP does not covalently react further substantiate the roles of microsomes and the with macromolecules (10, 12). Metabolic activation results nuclear envelope in the metabolic activation of in binding with several cellular nucleophilic centers (13,14, benzo(a)pyrene (BP), factors affecting the binding of BP to 22, 33), and such binding will presumably lead to carcino- the DNA, RNA, histone, and nonhistone proteins of isolated genesis. Recent evidence suggests that metabolism by the nuclei were investigated. Examination of the spectra and cytochrome P-450-containing mixed-function oxidase catalytic properties of the mixed-function oxidase systems (AHH) in conjunction with epoxide hydrase results in the of nuclei and microsomes indicated that they are similar. formation of a diol-epoxide that is probably the ultimate Regard less of the BP concentration used, microsomes from carcinogen (4, 8, 34, 36, 38, 41). The relatively short lifetime control or 3-methylcholanthrene-treated rats increased the of this species (36, 38) and the existence of cytoplasmic binding of BP to the components of control nuclei. With 30 detoxification mechanisms (35) raise the question of IJLMBP, microsomes enhanced the binding to the nuclei whether metabolic activation by the endoplasmic reticulum from 3-methylcholanthrene-treated rats. With lower BP con can lead to covalent reactions with macromolecules within centrations (1 to 2 /J.M),addition of microsomes reduced the the cell nucleus. The presence and inducibility of AHH in the binding. A reduction was also observed when denatured nuclear envelope have been well documented (17,19, 31). It microsomes were added. It was shown that the reduction has been suggested that carcinogen metabolism at this site was due to physical binding rather than the metabolism of might more readily lead to binding with nuclear compo BP by microsomes, and in fact the latter contributed to the nents and therefore is critical in the process of carcinogen- binding of BP to nuclear components. With incubation sys esis (29). Carcinogens have been shown to bind to nuclear tems containing microsomes and nuclei, the results indi macromolecules with experiments in vivo (5, 6, 8, 11, 16, cated that microsomes can (a) activate BP leading to bind 27), but the subcellular site of carcinogen activation cannot ing with nuclear macromolecules; and (o) physically bind be determined in such experiments. BP and reduce the effective BP concentration around the We have recently described conditions in which metabol- nuclei. Both the microsomes and nuclear envelope are po ically activated BP covalently binds to the DMA, RNA, his tentially important in the activation of carcinogens. The tone, and nonhistone proteins of nuclei isolated from rat endoplasmic reticulum may play a more important role than liver or lung (25). Incubation of isolated nuclei with NADPH the nuclei in the activation of BP when the carcinogen is in the presence of molecular oxygen resulted in binding. present in high concentrations. When the concentration of Treatment of the animals with MC increased the level of the carcinogen is low, the endoplasmic reticulum should nuclear AHH and the binding of BP to nuclear components. still contribute to the metabolic activation of BP, although it Similar results have also been reported by several other would also physically bind BP and lower the concentration laboratories (1, 15, 29, 30, 37). The addition of liver micro of BP available for nuclear metabolism. somes to the incubation system greatly enhanced the level of BP bound to the components of liver or lung nuclei. The nuclei were isolated from either control or MC-treated rats, INTRODUCTION and the maximal levels of bound carcinogen were similar. On the basis of this analysis, it was concluded that both the It is generally agreed that chemicals are a major causative factor in the initiation of cancer (13, 22). BP3 is a wide- endoplasmic reticulum and nuclear envelope were poten tially important sites of carcinogen activation. Consistent with this concept, increased binding of BP to nuclear DMA Received March 8, 1977; accepted June 10, 1977. 1 This work was supported by Grants CA-16788, CA-12933, and CA-16274 in the presence of microsomes has also been reported by from the National Cancer Institute and Grant 472 from the Nutrition Founda tion. Some preliminary results have appeared in an abstract (26). bon hydroxylase; MC, 3-methylcholanthrene; TKM, 50 mM Tris-HCI buffer, 2 Recipient of Faculty Research Award PRA-93 from the American Cancer ph 7.5, containing 25 mM KCI, and 5 mM MgCI..; MC microsomes or MC Society. To whom requests for reprints should be addressed. nuclei, respectively, isolated from animals that were pretreated with 3- 3 The abbreviations used are: BP, benzo (a) pyrene; AHH, aryl hydrocar- methylcholanthrene. SEPTEMBER 1977 3427 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1977 American Association for Cancer Research. J. M. Pezzato et al. Alexandrovef al. (1) and Jernström et al. (15). On the other layers of cheesecloth, 20 ml of 2.3 M sucrose-TKM were hand, Rogan and Cavalieri (29) and, more recently, Vaught added to 10 ml of the homogenate in a 40-ml cellulose and Bresnick (37) have observed that upon addition of nitrate tube and were thoroughly mixed. This was underlay- microsomes to isolated nuclei, the level of bound BP was ered with 5 ml of 2.3 M sucrose-TKM and centrifuged at reduced. It may be inferred from the last 2 reports that the 24,000 rpm for 1 hr in a Beckman SW-27 rotor. The nuclear metabolic role of microsomes is one of detoxification. pellets were washed with 1.0 M sucrose-TKM, 0.25 M su In order to resolve the existing conflict and further sub crose-TKM, and finally suspended in 20 mw Tris-HCI buffer, stantiate the roles of microsomes and the nuclear envelope pH 7.4, containing 0.25 M sucrose, 3 mw MgCU, and 1 mw in carcinogen activation, we have undertaken the present EDTA (Buffer A). Total nuclear protein was measured by the investigation. We have examined the mixed-function oxi method of Lowry ef al. (20). When binding studies were to dase system of the nuclear envelope and studied the effect be performed, the preparation was used immediately. of added microsomes on BP binding to nuclear macromole- Assay of AHH. This was done by the fluorimetrie method cules under a variety of conditions. The results of these of Nebert and Gelboin (23) with some modifications (39, 40). studies and a discussion of the cellular sites of carcinogen Duplicate determinations were made with either 0.2 to 0.4 activation are included in this report. mg of nuclear protein or 0.05 to 0.1 mg of microsomal protein and an incubation time of 5 to 10 min. The fluores cence of the phenolic products was measured with a Far- MATERIALS AND METHODS rand spectrofluorometer, and the amount of the prod uct was quantitated by comparison with a 3-hydroxy- Chemicals and Biochemicals. BP, NADPH, DL-isocitric benzo(a)pyrene standard. The activity was expressed as acid, isocitric dehydrogenase, Triton X-100, and protease pmoles of product formed per min. (Streptomyces griseus, type VI) were obtained from Sigma Measurement of Cytochrome P-450. The carbon monox Chemical Co., St. Louis, Mo. MC was from Mann Research ide difference spectra of reduced microsomes or nuclei Laboratories, New York, N. Y. Bovine pancreatic RNase and were recorded with a Gary Model 17 spectrophotometer. An DNase were obtained from Worthington Biochemical Corp., extinction coefficient of 91 mivrVcrrr1 for A45„_,90nmwas Freehold, N. J. 3-Hydroxybenzo(a)pyrene was supplied by used for cytochrome P-450 (24). the National Cancer Institute, Bethesda, Md. All other Binding of [3H]BP to Nuclear Components. The method chemicals were of reagent grade and were used as supplied utilized has been described in detail (25). The nuclei were by commercial sources. incubated at 37°with microsomes, [3H]BP, and 1 /¿moleof Radiochemical. Generally labeled [3H]BP was obtained NADPH in a total volume of 2.0 ml. After 2 washes with 1% from Amersham/Searle, Arlington Heights, III., with a spe Triton X-100 in Buffer A, h ¡stoneswere extracted with 0.24 N cific activity of 8.3 Ci/mmole. Prior to use, the benzene HCI and repeatedly precipitated with acetone. RNA, DMA, solvent was removed by a stream of nitrogen, and unlabeled and nonhistone proteins were then selectively extracted by BP was added in acetone to a specific activity of 0.5 Ci/ enzymic digestions and treated with ether to remove non- mmole. During the course of these studies, the radiochemi- covalently bound BP. In some experiments, the microsomes cal purity was not less than 96% as analyzed by thin-layer were omitted or replaced with denatured microsomes. chromatography with benzene as the solvent (32). Background levels of radioactivity associated with the mac- Treatment of Animals. Male Long-Evans rats with a body romolecules were determined by omitting NADPH from the weight of about 100 g were obtained from Marland Farms, incubation. The data are expressed as pmoles of BP metab Hewitt, N. J.