The Role of Microsomes and Nuclear Envelope in the Metabolic Activation of Benzo(A)Pyrene Leading to Binding with Nuclear Macromolecules1

[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.

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Alexandrovef al. (1) and JernstrÃ¶m 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 A45â€ž_,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 37Â°with microsomes, [3H]BP, and 1 /Â¿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 Â¡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. Prior to use, the rats were given a daily i.p. olites bound per mg of DNA, RNA, or protein. All proce injection of MC (25 mg/kg, in corn oil), for 4 days or no dures were performed in dim light. All binding studies were treatment. For each study, between 3 and 10 rats were used repeated at least 1 time, and the results were qualitatively in each group. The animals were given a commercial labo similar. ratory chow and water ad libitum and kept in air-condi Gross Binding of [3H]BP to Nuclei and Microsomes. To tioned quarters with a 12-hr light-dark cycle. determine the overall distribution of [3H]BP under our assay Isolation of Microsomes and Nuclei. The rats were sacri conditions, nuclei and microsomes were incubated at 37Â° ficed by decapitation, and the livers were excised, placed in for 30 min with [3H]BP. The reaction mixture was then ice cold 0.14 M NaCI, and rinsed 4 to 6 times. All subsequent procedures were performed at 0-4Â°unless otherwise indi placed on ice, and 2 ml of cold Buffer A were added. Following homogenization, the nuclei were sedimented by cated. Microsomes were isolated by differential centrifuga- centrifugation at 800 x g for 10 min, washed with 4 ml of tion as previously described (40) and stored frozen in small portions at -90Â°. Metabolically inactive (denatured) micro Buffer A, and digested with 1 ml of Protosol (New England Nuclear, Boston, Mass.). Microsomes were recovered from somes were prepared as follows. The microsomal suspen the supernatant by centrifugation at 105,000 x g for 1 hr. sion was brought to pH 12 with 1 N NaOH and left at room The microsomal pellet was homogenized in 4 ml of Buffer A, temperature for 10 min. The pH was then adjusted to neu recentrifuged, and digested with Protosol. trality with 1 N HCI, followed by the addition of buffer (40 rriM Tris-HCI, pH 7.4; 0.25 M sucrose; 6 mM MgCI2; and 2 mM EDTA) and sonic disruption (25). RESULTS Nuclei were isolated by the method of Kasper (18). This entails homogenizing the minced liver in 2 volumes (v/w) of In considering the role of nuclei or microsomes in carcin 0.25 M sucrose-TKM. Following filtration through 2 and 4 ogen activation, the problem of possible microsomal con-

3428 CANCER RESEARCH VOL. 37

lamination in the nuclear preparation is of great impor Table 1 tance. There is no biochemical marker for checking micro- Cytochrome P-450 content and AHH activity of nuclei and somal contamination in a nuclear preparation, and the pres microsomes AHH activity and cytochrome P-450 were determined as de ence of disrupted nuclei in the sample may complicate the scribed in "Materials and Methods." The values given for control interpretation of electron micrographs. We have examined and MC nuclei are an average of 2 and 6 preparations, respectively. several methods of nuclear isolation. The method of Kasper The microsomal data are from 1 preparation, and they are within (18) was found to be most suitable and was adopted in this the range of values obtained with other microsomal preparations in study. The yield of this method is lower than that of our our laboratory. previous method (25), but the nuclei appear to be more pure AHH (pmoles product/ as judged by the lack of a contaminating 420 nm peak as min) well as the lower cytochrome P-450 content and AHH activ P- pmole ity (data not shown). In order to test the purity of the nuclear 450 (pmoles/ mg cytochrome preparation, a dilute sample of isolated nuclei was subject SampleControl mg protein)36 protein11 P-4500.306 to a 2nd discontinuous sucrose gradient centrifugation; the nuclei cytochrome P-450 content and AHH activity of the nuclei MC nuclei 84 92 1.10 were not decreased. The nuclear preparation is similar to Control microsomes 850 470 0.553 that of Berezneyef al. (3) in terms of yield, AHH activity, and MC microsomesCytochrome1,730Per 2,090Per 1.21 cytochrome P-450 content (data not shown). The carbon monoxide difference spectra of nuclei and microsomes are shown in Chart 1. Control nuclei and control microsomes The cytochrome P-450 contents and the AHH activities of both have absorption peaks at 450 nm, whereas MC nuclei and MC microsomes have peaks at 448 nm. This is in agree the samples are shown in Table 1. In agreement with pre ment with previous reports (15, 30) that treatment of the vious reports (1, 15, 19, 30) MC treatment resulted in an 8.4-fold increase in activity when expressed on the basis of animals with MC induces nuclear and microsomal cyto chrome P-448, a form of cytochrome P-450 that has higher protein. On the same basis, microsomal activity was in AHH activity than does the cytochrome P-450 of control duced only 4.4-fold. Comparison of the turnover numbers animals. The extent of induction for microsomes and nuclei (pmoles of product formed per min per pmole cytochrome was 2.0- and 2.3-fold, respectively. The spectra of NaOH- P-450), however, indicates that the hydroxylation of BP by treated MC microsomes is also shown; the peak at 420 nm microsomes and nuclei is similar. and the absence of a 450 nm peak indicating complete The effect of BP concentration on the binding to the denaturation of cytochrome P-450. macromolecules of MC nuclei is shown in Table 2. A 30-min incubation time was used in this experiment. In agreement with the results of Vaught and Bresnick (37), the addition of microsomes reduced the levels of BP binding when 1 or 2 /UMBP were used. This inhibition was not observed with 30 fj.M BP, consistent with our previous observation (25). Fur Ã›A = 004 thermore, when equal amounts of denatured microsomes were added to the incubation systems, the levels of binding were reduced in all the experiments except the DMA and histone samples in Experiment 4. These reduced levels of

NciOH Denatured binding were also reflected in control values (no NADPH MC Microtomes added). These results suggest that due to the physical bind ing of BP to microsomes, the quantity of BP available for metabolism by the nuclei was reduced. This resulted in a MC Microsomes lower covalent binding of BP to nuclear macromolecules with the addition of denatured microsomes or with the addition of active microsomes when lower BP concentra tions were used. A comparison between the levels of bind Control Microsomes ing observed with denatured and active microsomes indi

MC Nuclei cates that the metabolism of BP by active microsomes con tributed to the total BP binding to nuclear components. Such an effect is not apparent with low BP concentrations but became clear as the concentration of BP increased. Control Nuclei Table 3 shows the binding of BP metabolites to the mac romolecules of control nuclei, again with a 30-min incuba tion time. At all the BP concentrations tested, control mi 450 crosomes were able to increase the binding. Addition of MC Nanometers microsomes resulted in much higher levels of binding, com Chart 1. The carbon monoxide difference spectra of nuclei and micro parable to those obtained with MC nuclei. It is therefore somes. The respective protein concentrations (mg/ml) were: control nuclei, 3.8; MC nuclei, 4.0; control and MC microsomes, both 0.40; and NaOH apparent that metabolism by microsomes can increase the denatured MC microsomes, 0.28. level of BP metabolites bound to nuclear components, even

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Table 2 Effect of BP concentration on the binding to MC nuclear macromolecules Each incubation contained MC nuclei (5 mg protein) or MC nuclei plus native or denatured MC microsomes (4 mg protein). [3H]BP was added in 0.02 ml of acetone, and the incubation was at 37Â°for 30 min. Binding is expressed as pmoles of [3H]BP bound per mg of macromolecules. Unless indicated, the incubation contained NADPH. Experi mentno.1234BPMicrosomes(/UM)None added to nuclei tone63.124.126.569.232.644.7106.937.6107.823.77.1113.858.9220.7 1DenaturedMC

inducedNone

2DenaturedMC

inducedNone

4DenaturedMC

inducedNone

30MC (-NADPH) (-NADPH)NoneDenaturedMCinduced

inducedDMA25.110.79.944.517.417.662.425.433.915.99.862.564.6152.0RNA249.874.4101.8345.9104.9187.0309.9141.4355.0102.455.9310.9217.81090.2Histone23.07.49.235.512.518.037.117.231.68.82.830.229.1109.6Nonhis-

Table 3 Effect of BP concentration on the binding to control nuclear macromolecules Control nuclei (5 mg protein) were incubated at 37Â°for 30 min with microsomes (4 mg protein). Control microsomes were denatured for some studies. Other conditions were the same as those in Table 2. Experi mentno.1234BPMicrosomes(/

inducedDenatured

2ControlMC

inducedDenatured

4ControlMC

inducedControl

30DenaturedControlMC(-NADPH)

inducedDMA1.52.56.62.12.912.62.13.625.84.67.910.9122.7RNA12.123.287.015.826.6157.021.841.6348.146.181.7100.3984.1Histone0.51.56.60.81.713.31.02.426.01.73.67.983.1Nonhis- when low concentrations of BP are used in the incubation the presence of NADPH, the fraction of BP physically asso system. ciated with microsomes decreased to 34% and that with The effect of incubation time on the binding of [3H]BP nuclei decreased to 14%. About half of the BP molecules metabolites to nuclear macromolecules is shown in Table 4. were found in the supernatant. This is probably due to the MC nuclei, MC or denatured microsomes, 2 Â¿IM[3H]BP,and production of hydroxylated BP metabolites that are more NADPH were incubated for the indicated time at 37Â°.With soluble in the aqueous media. Similar results were also short incubation times (2 to 8 min) the level of binding was observed with 30 /nM [3H]BP. greater when native MC microsomes were included. The difference diminished with an incubation time of 12 min and disappeared with 30 min of incubation. Microsomes also DISCUSSION increased the binding of BP to nuclear components with short incubation times when the experiment was repeated The existence of cytochrome P-450 and AHH activity in with 1 H.MBP. the nuclear envelope has been observed in several laborato The distribution of [3H]BP physically bound to the compo ries (1,15,17-19, 25, 29-31). Removal of components of the nents in the incubation system is shown in Table 5. With 2 nuclear envelope by detergent treatment (9) abolishes the /nM BP, a large portion of BP (68%) bound to microsomes, enzyme-dependent binding of BP to nuclear DNA (30). The 22% bound to nuclei, and 10% was in the supernatant. In proximity of the enzyme system to possible carcinogen

3430 CANCER RESEARCH VOL. 37

Table 4 Effect of incubation time on the binding of [3H]BP metabolites to nuclear macromolecules MC nuclei (5 mg protein) were incubated with native or denatured MC microsomes (4 mg protein) at 37Â°for the indicated time. The concentration of [3H]BP was 2 pM. The zero time samples were kept on ice. Other conditions were the same as those in Table 2.

Experiment added of incuba no.123456MicrosomestonucleiDenatured tion02481230DMA1.5 tone1.8

MCinducedDenatured 1.42.0 11.818.6 0.30.9 2.14.1

MCinducedDenatured 6.03.4 63.131.4 5.31.7 12.06.2

MCinducedDenatured 7.26.3 105.454.8 8.74.5 19.010.6

MCinducedDenatured 8.712.0 163.6118.0 11.29.9 17.621.3

MCinducedDenatured 10.813.0 117.1180.8 14.716.6 22.434.2

MC inducedMinutes 12.2RNA11.2210.2Histone0.415.9Nonhis- 32.7

Table 5 all, it appears that the mixed-function oxidase system in the Distribution of [3H]BP in incubations containing nuclei and nuclear envelope is similar to that in microsomes. microsomes The present study confirmed our previous results (25) MC nuclei (5 mg protein) were incubated with MC microsomes (4 mg protein) at 37Â°for 30 min with 2 or 30 /Â¿MBP in duplicate. that, in an incubation system containing isolated nuclei, Averaged values are given as the percentage of the total [3H]BP [3H]BP, and NADPH, the addition of microsomes increases added to the incubation. Unbound is that which was recovered in the binding of BP to nuclear macromolecules. This is appar the supernatant after centrifuging at 105,000 x g for 60 min. Wash ent in experiments with 30 /U.MBP,and with 15 to 112 /nMBP ing the nuclei or microsomes with Buffer A removed 2 to 3% of the as shown previously (25). With a BP concentration of about radioactivity. 3 fj.M, Vaught and Bresnick (37) observed that the added BP (MM)2 Nuclei22+ microsomes decreased the binding. We now report that with BP concentrations as low as 1 /AM, the addition of 2 14 34 52 microsomes to MC nuclei does not cause a reduction in 30 26 6335Unbound101146 binding when compared with an appropriate control, the 30NADPH + 19Microsomes68 addition of denatured microsomes (Table 2). The apparent reduction of carcinogen bound to the macromolecules of MC nuclei is probably due to physical binding of BP by the binding sites in the nucleus has led to the suggestion that microsomes. This interpretation is consistent with the ob the nuclear enzyme may play an important role in carcino served high affinity of BP for microsomes (Table 5). The gen activation (29). It has been reported that MC treatment microsomes, therefore, have 2 opposite effects on the me preferentially induces the AHH activity in nuclei (19, 30), in tabolism of BP: Effect A, the metabolic activation of BP by contrast to phÃ©nobarbital, which induces microsomal but microsomes; and Effect B, the physical binding of BP to not nuclear cytochrome P-450(17,19). These results further microsomes causing a decrease in the effective concentra suggest the uniqueness of the nuclear envelope mixed- tion of BP to nuclear enzymes. The Kmof the MC-induced function oxidase system. In the present study, MC treatment mixed-function oxidase system has been reported to have caused a 2.3-fold induction in nuclear cytochrome P-450 values ranging from 0.3 to 2.4 /*M BP (2, 21, 28). With 30 ^M that is slightly higher than the induction of microsomal BP added to incubations containing microsomes, the de cytochrome P-450, and an 8-fold increase in nuclear AHH creased BP concentration should still be much higher than activity that is significantly higher than the increase in mi the Km; therefore, Effect A outweighs Effect B, and an crosomes. The latter difference may be a reflection of the enhanced level of binding is seen. With lower BP concen low AHH activity of the control nuclei, rather than any differ trations, when the metabolism is substrate limiting, e.g., 2 ence between the cytochrome P-448 in MC nuclei and MC fj,M, Effect B outweighs Effect A, and decreased binding is microsomes, since both have a similar turnover number in seen. Under these conditions, the enzymes in the micro catalyzing the AHH reaction. The pattern of BP metabolites somes should contribute to the binding. This is demon produced with the nuclear enzyme also resembles that ob strated by the experiments with shorter incubations in Table tained with microsomes (1, 15). Moreover, treatment of the 4. Such an effect is less apparent with longer incubations, animals with phÃ©nobarbital results in approximately a 2.5- due possibly to the exhaustion of the substrate, BP (Tables and 2-fold increase in cytochrome P-450 contents of micro 2 and 4). With control nuclei, which have very low AHH somes and nuclei, respectively (unpublished results). Over activity, Effect A outweighs Effect B even at low BP concen-

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1977 American Association for Cancer Research. J. M. Pezzuto et al. trations when either control or MC microsomes are used W. Jones (eds.), Carcinogenesis-A Comprehensive Survey, Vol. 1. pp. (Table 3). 283-297. New York: Raven Press, 1976. 8. Daudel, P., Duquesne, M., Vigney, P., Grover, P. L., and Sims, P. JernstrÃ¶met al. (15) have suggested that the microsomes Fluorescence Spectral Evidence that Benzo(a)pyrene-DNA Products in metabolize BP to a form that the nuclei can convert more Mouse Skin Arise from Diol-Epoxides. Federation European Biochem. efficiently than BP to a DNA-binding species. Our results Soc. Letters. 57: 250-253, 1975. 9. Franke, W. W. Structure, Biochemistry, and Functions of the Nuclear however, suggest that such a 2-step process may not be Envelope, Intern. Rev. Cytol., 4 (Suppl.): 71-236, 1974. needed. The metabolism-dependent binding of BP to the 10. Gelboin, H. V. A Microsome-Dependent Binding of Benzo(a)pyrene to DMA. Cancer Res., 29: 1272-1276, 1969. components of control nuclei (which contain little AHH 11. Goshman, L. M., and Heidelberger, C. Binding of Tritium-labeled Poly- activity) is very low in the absence of microsomes but is cyclic Hydrocarbons to DNA of Mouse Skin. Cancer Res., 27. 1678-1688, 1967. greatly enhanced when microsomes are added. The ob 12. Grover, P. L., and Sims, P. Enzyme-catalyzed Reactions of Polycyclic served levels of binding are similar to those attained when Hydrocarbons with Deoxyribonucleic Acid and Protein in Vitro. Bio MC nuclei are incubated with microsomes. Further investi chem. J., 110: 159-160, 1968. 13. Heidelberger, C. Chemical Carcinogenesis. Ann. Rev. Biochem., 44: 79- gation of this aspect of carcinogen activation is needed. 121,1975. The fact remains that the AHH system of isolated nuclei 14. Jerina, D. M., and Daly, J. W. Arene Oxides: A New Aspect of Drug can activate BP and cause binding to nuclear components. Metabolism. Science, 785: 573-582, 1974. 15. JernstrÃ¶m,B., Vadi, H., and Orrenius, S. Formation in Isolated Rat Liver The active metabolites of BP produced by microsomes can Microsomes and Nuclei of Benzo(a)pyrene Metabolites that Bind to DNA. also enter into the nuclei and react with nuclear macromol- Cancer Res., 36: 4107-4113, 1976. 16. Jungmann, R. A., and Schweppe, J. S. Binding of Chemical Carcinogens ecules. Since most of the AHH activity is associated with the to Nuclear Proteins of Rat Liver. Cancer Res., 32: 952-959, 1972. endoplasmic reticulum, it appears that the endoplasmic 17. Kasper, C. Biochemical Distinctions between the Nuclear and Micro- reticulum may play a more important role than nuclei in the somal Membranes from Rat Hepatocytes. J. Biol. Chem., 246: 577-581, 1971. activation of BP when the carcinogen is present in high 18. Kasper, C. B. Isolation and Properties of the Nuclear Envelope. Methods concentrations, e.g., 15 to 30 Â¿Â¿M.Whenthe cellular con Enzymol., 37: 279-292, 1974. 19. Khandwala, A. S., and Kasper, C. B. Preferential Induction of Aryl Hydro centration of BP is 2 fj.Mor lower, a condition that may more carbon Hydroxylase Activity in Rat Liver Nuclear Envelope by 3-Methyl- closely approximate the situation in carcinogenesis, the cholanthrene. Biochem. Biophys. Res. Commun., 54: 1241-1246, 1973. endoplasmic reticulum should contribute to the metabolic 20. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem., 793: 265- activation of BP, although it would also physically bind BP 275, 1951. and decrease the effective carcinogen concentration 21. Lu, A. Y. H., and West, S. Reconstituted Liver Microsomal Enzyme around the nucleus. The latter effect should also be mani System that Hydroxylates Drugs, Other Foreign Compounds, and En dogenous Substrates. III. Properties of the Reconstituted 3,4-Benzpyr fested by other organelles and cellular membranes. The ene Hydroxylase System. Mol. Pharmacol., 8: 490-500, 1972. relative importance of the endoplasmic reticulum and the 22. Miller, E. C.. and Miller, J. A. Biochemical Mechanisms of Chemical Carcinogenesis. In: H. Busch (ed.), The Molecular Biology of Cancer, nuclear envelope in carcinogen activation remains to be pp. 377-402. New York: Academic Press, Inc., 1974. unequivocally defined, since it may also depend upon cyto- 23. Nebert, D. W., and Gelboin, H. V. Substrate-inducible Microsomal Aryl plasmic detoxification reactions (35) and the mechanisms Hydroxylase in Mammalian Cell Culture. I. Assay and Properties of In duced Enzyme. J. Biol. Chem., 243: 6242-6249, 1968. of carcinogen transport. 24. Omura, T., and Sato, R. 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SEPTEMBER 1977 3433

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1977 American Association for Cancer Research. The Role of Microsomes and Nuclear Envelope in the Metabolic Activation of Benzo( a)pyrene Leading to Binding with Nuclear Macromolecules

John M. Pezzuto, Michael A. Lea and Chung S. Yang

Cancer Res 1977;37:3427-3433.

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