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Chem. Res. Toxicol. 1989, 2, 312-315
32P-Postlabeling Analysis of Benzo[ a Ipyrene-DNA Adducts Formed in Vitro and in Vivo W. J. Bodell,? P. D. Devanesan,l E. G. Rogan,*J and E. L. Cavalieril Brain Tumor Research Center of the Department of Neurological Surgery, School of Medicine, H S W -783, University of California, San Francisco, California 94143, and Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42nd Street and Dewey Avenue, Omaha, Nebraska 68105 Received May 1 , 1989
Benzo[a]pyrene (BP) was bound to DNA by horseradish peroxidase, rat liver microsomes, and rat liver nuclei in vitro and in mouse skin in vivo. The BP-DNA adducts formed were analyzed by the 32P-postlabeling technique. Activation by microsomes and nuclei resulted in the detection of five adducts, including a major adduct (55%) which cochromatographed with (BPDEthe adduct (f)-10~-deoxyguanosin-~-y1-7~,8a,9a-trihydroxy-7,8,9,10-tetrahydro-BP N2dG) formed by reaction of (f)-7~,8a-dihydroxy-9a,lOa-epoxy-7,8,9,lO-tetr~ydr~BP (BPDE) with DNA or by microsomal activation of B P 7,8-dihydrodiol. Activation by horseradish peroxidase, which catalyzes one-electron oxidation, produced seven adducts, including a major one (30%) that coeluted with an adduct observed with microsomal (2%) and nuclear (14%) activation. The pattern of adducts formed in mouse skin treated with B P in vivo for 4 or 24 h contained four of the same adducts observed with nuclei or microsomes in vitro, and the predominant adduct detected (86%) was BPDE-N2dG. The adduct common to horseradish peroxidase, microsomes, and nuclei was also detected in mouse skin DNA (2%). These results demonstrate that multiple BP-DNA adducts are formed in these in vitro and in vivo systems and suggest that a t least one adduct is formed in common in all of the systems. Thus, it appears that stable B P adducts can be formed in mouse skin DNA by both monooxygenation and one-electron oxidation.
Introduction Benzo[a]pyrene (BP)’ is metabolized to reactive intermediates which, after binding to DNA, presumably initiate carcinogenesis. Metabolic activation of BP proceeds by two major mechanisms, one-electron oxidation ( 1 , 2 ) and monooxygenation ( 3 , 4 ) . The predominant DNA adduct obtained by monooxygenation arises from formation of a covalent bond between the C-10 of (f)-70,8a-dihydroxy9a,lOa-epoxy-7,8,9,10-tetrahydro-BP (BPDE) and the 2-exocyclicamino group of deoxyguanosine (BPDE-PdG) ( 4 ) . Two adducts have been identified from one-electron oxidation of BP. The major adduct contains BP bound at C-6 to the N-7 of guanine (BP-N7Gua) (5). The second adduct contains a bond between the C-6 of BP and the C-8 of deoxyguanosine (BP-C8dG) ( 5 ) . The BP-N7Gua adduct is rapidly lost from DNA by depurination, but the BP-C8dG adduct is more stable ( 5 ) . The structures of these different adducts reflect their mechanisms of activation. Binding of BP to DNA is mediated by several in vitro systems, including horseradish peroxidase (HRP), rat liver microsomes, and rat liver nuclei. HRP binds BP to DNA by one-electron oxidation, and our studies have demonstrated formation of BP-N7Gua and BP-C8dG in DNA by this mechanism (5). In addition, we have found that the BP-N7Gua adduct is formed by rat liver microsomes (ref 6 and manuscript submitted for publication) and nuclei (7). These results suggest that, in addition to the wellstudied BPDE-N2dG, DNA adducts formed by one-electron oxidation may contribute to the biological effects of BP. *To whom correspondence should be addressed. ‘University of California. University of Nebraska Medical Center.
32P-Postlabeling, developed by Randerath and coworkers, is a very sensitive method for the detection and quantitation of aromatic adducts that are stable in DNA ( 8 ) . Previous investigations have used postlabeling to detect the formation and repair of BP-DNA adducts in mouse skin and other organs. BPDE-WdG was found, but additional adducts were also detected (8-10). Some of them may correspond to adducts formed by one-electron oxidation of BP. The purpose of these investigations was to use 32Ppostlabeling to compare the stable DNA adducts formed by HRP, microsomal, and nuclear activation of BP in vitro and by mouse skin in vivo. These results provide information on the presence and relative abundance of various stable adducts formed in these biological systems.
Experimental Procedures Binding of BP to DNA. HRP (type VI), bovine pancreas ribonuclease (type l-AS),and proteinase K were purchased from Sigma Chemical Co. (St. Louis, MO), and calf thymus (CT)DNA was from Pharmacia (Piscataway, NJ). [3H]BPwas purchased from Amersham (Arlington Heights, IL), whereas [I4CC]BP7,8dihydrodiol (sp act. 52 mCi/mmol) and [3H]BPDE (1380 mCi/ mmol) were from Chemsyn Science Laboratories (Lenexa, KS). Rat liver microsomes were prepared from 3-methylcholanthrene-induced (100 pmollkg body weight on two consecutive days) 8-week-old male Wistar rats (Eppley Colony) as previously described ( 1 1 ) . Rat liver nuclei were isolated from
’ Abbreviations: BP, benzo[a]pyrene;BPDE, (+)-7,9,8a-dihydroxy-
9a,lOa-epoxy-7,8,9,1O-tetrahydro-BP; BPDE-NedG, (+)-lO@-deoxyguanosin-N2-yl-78,8a,9a-trihydroxy-7,8,9,lO-tetrahydr~BPBP-CMG, g b e n z o [ a ] p y r e n - 6 - y l d e o x y ~ ~BP-N7Gua, ~e; 7-benzo[a]pyren-6-y1-
guanine; CT, calf thymus; 3’dNp, 2’-deoxyribonucleoside 3’-mOnOphosphate; 3’,5’dpNp,2’-deoxyribonucleoside3’,5’-bisphosphate,HRP, horseradish peroxidase;PEI, poly(ethy1enimine);SSC, standard saline citrate, 0.15 M NaC1-0.015 M sodium citrate.
0893-228~/89/2702-0312$01.50/0 0 1989 American Chemical Society
32P-Postlabeling Analysis of BP-DNA Adducts 3-methylcholanthrene-induced(100 pmol/kg body weight on two consecutive days) 3-week-old male Wistar rats as previously described (12). [3H]BPDE[0.1 mg in 0.2 mL of THF/TEA (191)] was reacted with 1 mg of DNA in 1mL of 50 mM Tris-HC1,150 mM KC1, and 5 mM MgC12, pH 7.5, for 30 min. The DNA was purified by extraction with phenol and chloroform, followed by ethanol precipitation,and the level of binding was determined by counting 3H (5).DNA was estimated by absorbance at 260 nm. The level of binding measured €or the purified DNA was 107 f 15adducts per lo6 nucleotides. [3H]BP (140 mCi/mmol) was bound to DNA in the HRPcatalyzed reaction (5). After purification of the DNA, the level of binding was determined to be 75 i 5 adducts per lo6 nucleotides. [3H]BPand [14C]BP7,8-dihydrodiolwere bound to DNA by using microsomes under the reaction conditions described for BP, and the DNA was purified and analyzed (11). Binding of BP was 45 f 3 adducts per lo6 nucleotides, whereas the binding of BP 7,8-dihydrodiolwas much higher, 992 f 225 adducts per lo6 nucleotides. BP was bound to endogenous DNA in nuclei as previously described (12),and the DNA was purified and analyzed for bound BP. The level of binding was 9.8 f 2.3 adducts per lo6 nucleotides. Groups of five female Swiss mice (6 weeks old, Eppley Colony) were treated for 4 or 24 h with [3H]BP (200 nmol per mouse, 316 mCi/mmol) on a shaved area of dorsal skin (13).The skin was excised, and epidermiswas prepared and ground in liquid nitrogen, as previously described (13).The powdered epidermis was suspended in 10 mL of 0.1 M Tris, 0.1 M NaCl, 50 mM EDTA, pH 8.0, and 1%sodium dodecyl sulfate. It was shaken 10 min. Solid NaCl was added to give a 1M solution, which was shaken 10 min. The solution was extracted twice with equal volumes of chloroform/isoamyl alcohol (24:1), and the DNA was further purified as previously described (14).The DNA was dissolved in 0.5 mL of 0.1 X SSC. At 4 h there were 1.3 f 0.3 adducts per lo6 nucleotides, whereas at 24 h, 2.1 f 0.3 adducts per lo6 nucleotides were bound. P1-Nuclease-Modified 32P-Postlabeling. The 32P-postlabeling method used was as described by Reddy and Randerath (15).Purified DNA (4 pg) was enzymaticallydigested to 3'dNp's at 37 "C for 2 h with micrococcal nuclease (Worthington Biochemicals, Freehold, NJ) and spleen phosphodiesterase (Worthington Biochemicals). PI-nuclease (Pharmacia) was added to the digest, and incubation was continued at 37 "C for 60 min. The modified nucleotides were converted into 3P-labeled 3',5'dpNp's by incubation of the modified nucleotides with 150 pCi of [-]ATP (6000 Ci/mmol) and T4polynucleotide kinase (Bethesda Research Laboratories, Gaithersburg, MD) for 60 min at 37 "C. Potato apyrase (Sigma) was added and the mixture was incubated for 30 min at 37 "C. Chromatography. The postlabeled mixtures were applied to 10 cm X 10 cm poly(ethy1enimine) (PE1)-cellulose plates (Brinkman, Des Plaines, IL). A paper wick was attached to the top of each TLC plate. The TLC plates were developed overnight in 1M NaH2P04,pH 6. The plates were subsequently washed twice with H20, followed by a wash with 0.15 M ammonium formate, pH 3.5. The plates were dried and developed in 3.0 M lithium formate and 7.0 M urea, pH 3.5, from the bottom to the top of the plate. The plates were washed twice in H20, air-dried, and then developed at a right angle to the previous direction of development in 0.72 M NaH2P04,0.45 M Tris-HC1, and 7.6 M urea, pH 8.7, to approximately 4-5 cm onto the paper wick. They were washed twice with H 2 0and then, with new wicks attached, developed in the same direction to approximately 4-5 cm onto the wick with 1.7 M NaH2P04,pH 6.0. The plates were washed for 5 min in H20. The adducts were located by autoradiography using Kodak XAR-5 film and a Du Pont Chronex Lightning Plus intensifying screen. When control samples (no activation of BP) were analyzed, no radioactive spots were observed in the areas where adducts travel. Calculation of Adduct Frequency. The radioactive spots on the PEI-cellulose sheets detected by autoradiography were scraped into liquid scintillation vials containing 5 mL of scintillation cocktail (Safety Solve, Research Products, Inc.), and radioactivity was determined by liquid scintillation counting. Regions adjacent to the radioactive spots were also scraped, placed
Chem. Res. Toxicol., Vol. 2, No. 5, 1989 313 A
B
9
C
D
Figure 1. (A) Autoradiogram of 32P-postlabeledCT-DNA containing BP adducts formed after microsomal activation. The f h was exposed a t room temperature for 2 h. (B) Autoradiogram of 32P-postlabeled CT-DNA containing BP adducts formed after HRP activation. The film was exposed at room temperature for 1h. (C) Autoradiogram of 32P-postlabeledDNA containing BP adducts formed after nuclear activation. The film was exposed a t room temperature for 18 h. (D) Autoradiogram of 32P-postlabeled DNA containing BP adducts formed in mouse skin DNA. The film was exposed at room temperature for 18 h. into scintillationvials, and counted for background.determination. We have previously determined the counting efficiency for 32P to be 0.760 with Safety Solve as the scintillation cocktail (16). The background counts were subtracted from those of the BP adducts. The level of BP modifcation was calculated as described by Reddy and Randerath (15). Assuming that 4 pg of DNA is 1.21 X lo4 pmol of 3'dNp and that the specific activity of the [32P]ATPwas 9.36 X lo6cpm/pmol, adduct levels (per specified number of nucleotides) were calculated as follows: adduct level = (cpm in adducts)/[(l.21 X lo4 pmo1)(9.36 X lo6 cpm/pmol)] The values calculated by using% or '.I "c are generally expressed to two significant figures, whereas those calculated by using 32P contain three significant figures.
Results DNA adducts formed by reaction of CT-DNA with microsomally activated BP were detected by 32P-postlabeling (Figure 1A). The extent of DNA modification was 11.8 f 2.1 adducts per lo6 nucleotides, based on 11 determinations. The extent of binding measured by 3H radioactivity was 45 f 3 adducts per lo6 nucleotides. With postlabeling, five adducts were detected. Adducts 1and 3 were the principal adducts and accounted for 55.2 f 5.0% and 39.7 f 5.3% of the detected modification. Adducts 2, 4, and 5 each accounted for 2.2 f 0.9%, 1.2 f 0.9%, and 1.4 f 1.1%of the detected modification. 32PPostlabeling of CT-DNA reacted with either BPDE or with
314 Chem. Res. Toxicol., Vol. 2, No. 5, 1989
microsomally activated BP 7,8-dihydrodiol gave a single adduct, BPDE-N2dG, which had the same migration on PEI plates as microsomal adduct 1. Reaction of CT-DNA with HRP-activated BP produced seven adducts detected by 32P-postlabeling(Figure 1B). The extent of DNA modification was 16.1 f 4.3 adducts per lo6 nucleotides, based on 18 determinations. The extent of DNA modification determined by [3H]BP binding was 75 f 5 adducts per lo6 nucleotides. Adducts 2 and 6 were the principal adducts detected and accounted for 29.2 f 7.0% and 19.8 f 3.8% of the detected modification. Better resolution of adducts 2 and 6 was not obtained with varying the ionic strength or the pH of the second dimension solvent. However, we found that adducts 7 and 8, which comigrated in the second dimension at pH 8.2, were well-resolved at pH 8.7. Adducts 7 and 8 represented 11.0 f 2.4% and 12.7 f 5.4% of the detected modification. The remaining adducts 9, 10, and 11 each accounted for 11.7 f 1.9%, 10.9 f 1.9%,and 2.5 f 0.5%, respectively, of the detected modification. Comparisons of the HRP adducts with those of the microsomally activated BP were made by running postlabeled DNA mixtures from the two activation systems at the same time so that the position of adducts could be directly compared. The adducts formed were generally distinct. However, adduct 2 of the HRP reaction and adduct 2 of the microsomal reaction appeared to be a common adduct formed by both systems. The autoradiograms presented for microsomal, HRP, and mouse skin (panels A, B and D of Figure 1)were run simultaneously on separate 10 X 10 cm TLC plates derived from the same 20 X 20 cm plate. Binding of BP to the DNA of rat liver nuclei (samples) isolated from MC-treated rats was determined. The average extent of DNA modification detected by 32P-postlabeling was 0.76 f 0.19 adducts per lo6 nucleotides, compared to 9.8 f 2.3 adducts per lo6 nucleotides based on measurement of 3H radioactivity. Five adducts were detected (Figure lC), with adduct 1, BPDE-N2dG, being the principal adduct and accounting for 56.1 f 5.3% of the detected modification. Adducts 2 and 3 each accounted for 13.9 f 5.0% and 21.6 f 5.8% of the detected modification, whereas adducts 4 and 5 were minor and represented 3.6 f 1.1%and 1.3 f 0.4% of the detected modification. Comparison of the BP adducts formed in the DNA of nuclei with those in CT-DNA after either microsomal or HRP activation shows that most of the adducts are in common with one or both of the activation systems. Nuclear adduct l appears to correspond to microsomal adduct 1, BPDE-N2dG. Nuclear adduct 2 corresponds to adduct 2 of both the microsomal and HRP systems. Nuclear adducts 3, 4, and 5 correspond to microsomal adducts 3, 4, and 5, respectively. The extent of BP binding to the DNA of mouse skin at 4 and 24 h after application of BP was determined. The average extent of DNA modification of two samples was 0.65 f 0.08 adducts per lo6 nucleotides at 4 h and 1.07 f 0.09 adducts per lo6 nucleotides at 24 h, as determined by 32P-postlabeling. Measurement of 3H radioactivity gave values of 1.3 f 0.3 adducts per lo6 nucleotides and 2.1 f 0.3 adducts per 106 nucleotides for 4 and 24 h. Comparison of the extent of DNA binding at 4 and 24 h indicates that most of the binding occurs in the first four hours. The BP-DNA adducts in mouse skin 4 h after treatment with BP, as detected by 32P-postlabeling,are shown in Figure 1D. Four adducts were detected. Adduct 1,BPDE-N2dG, was the principal one, representing 86.4 f 2.9% of the
Bodell et al.
detected modification. Adducts 2, 3, and 4 represented 1.8 f 0.7%, 8.5 f 1.8%, and 4.4 f 1.4% of the detected modification. A similar adduct pattern was found for the 24-h sample, with adduct 1being the principal one (86.5 f 3.0%) and adducts 2,3, and 4 representing 1.3 f 1.1%, 8.9 f 2.1 % , and 3.6 f 0.5% of the detected modification. Comparison of the BP-DNA adduct pattern detected in the skin with that in the nuclei shows that they are very similar. The major difference between the two samples is the level of adduct 1. It was higher in the DNA of mouse skin compared to the liver nuclei. Comparison of the DNA adducts detected in mouse skin with those of the microsomdy activated BP shows that they are also very similar. Skin adducts 1,3, and 4 correspond to microsomal adducts 1,3, and 4. Skin adduct 2 appears to correspond to adduct 2 from HRP, microsomes, and nuclei.
Discussion Covalent binding of BP to nucleic acid bases produces two main kinds of adducts, labile ones, which are lost by depurination or depyrimidination, and stable ones (5,17). Previous studies have shown that the abundant BP-N7Gua observed with activation by HRP, microsomes, or nuclei is rapidly depurinated (refs 5-7 and 18 and manuscript submitted for publication) and thus is not detectable by 32P-postlabeling. The amount of N7Gua adduct isolated is 1.4-9 times greater than the total amount of stable BP-DNA adducts in these in vitro systems (refs 5-7 and 18 and manuscript submitted for publication). Comparison of the extent of DNA binding estimated by 3?P-postlabelingand by measurement of tritium indicates that the postlabeling method measures a fraction of the total DNA adducts. The level of binding estimated from 32P-postlabelingranged from 8% of the level determined by tritium with the nuclear DNA to 21% with HRP activation, 26% with microsomes, and 50% in the mouse skin samples. While we cannot explain this discrepancy at this point, there are several possible reasons, including the following: (1)measurement of bound [3H]BPmay include some noncovalently bound [3H]BP,whereas 32Pmeasures only adducts, (2) some of the adducts may be resistant to enzymatic digestion to 3’dNp’s (this is particularly significant for bulky compounds like BP), (3) some adducts may be lost by slow depurination or depyrimidination during the digestion, postlabeling, or chromatography procedures, or (4) some adducts may be dephosphorylated by PI-nuclease. The 50% ratio we obtained in the mouse skin samples, in which BPDE-WdG was the predominant stable adduct, is similar to the 40-50% recovery of the BPDE I-DNA adducts reported by Gupta et al. (8). Since almost all 32P-postlabeling analyses are conducted on samples exposed to nonradioactive carcinogens, it is impossible to determine whether our apparent recovery of 10-50% of the adducts in the postlabeling procedure is typical or not. In previous investigations we have shown that HRP activation produces BP adducts at the N-7 and C-8 positions of guanine in DNA (5). The studies reported here indicate that HRP activation of BP produces multiple stable adducts that can be detected by 32P-postlabeling (Figure 1B). Studies are in progress to determine which of the adducts detected by postlabeling corresponds to the BP-C8dG adduct and to identify the other unknown adducts formed in this system, with particular emphasis on adduct 2. This adduct appears to be formed in HRP, microsomes, nuclei, and mouse skin, as evidenced by the same migration when three of the samples were run simultaneously (Figure lA,B,D) and in other experiments
92P-Postlabeling Analysis of BP-DNA Adducts
in which the various samples were chromatographed simultaneously. Microsomal activation of BP produced multiple stable DNA adducts detected by postlabeling (Figure 1A). The principal adduct detected was BPDE-N2dG. A second major adduct accounts for 41?% of the total modification, but its structure is not known. In general, the adducts formed by HRP and microsomal activation of BP were distinct. However, microsomal adduct 2 is of particular interest because adduct 2 of the HRP system had the same migration on PEI-cellulose plates (see above). This suggests that at least one adduct is formed by both enzymes. The stable DNA adducts of nuclei (Figure IC) were very similar to microsomally activated BP-DNA adducts. Nuclear adduct 2 had the same migration as adduct 2 from microsomes and HRP, but it was more prevalent in nuclei than with microsomes. Multiple stable DNA adducts were detected by postlabeling in the DNA of mouse skin at 4 h (Figure 1D) and 24 h after treatment with BP, and the adduct patterns were similar at both times. The levels of binding indicate that most of the binding occurred in the first 4 h and that rapid repair of the adducts does not occur. The principal adduct detected by postlabeling corresponds to BPDEN2dG. However, skin adduct 2 appears to correspond to HRP adduct 2, suggesting that in skin BP can be activated by both the one-electron oxidation and monooxygenation mechanisms. Our investigations are very similar to those of Randerath et al. (9), who also found BPDE-N2dG to be the principal stable adduct in the skin. However, other investigations in human and animal tissues by Lu et al. (10) and Seidman et al. (29) have shown that multiple stable BP-DNA adducts are detected by the postlabeling method at various relative levels of abundance. Comprehensive study of BP-DNA adducts will include identification of not only the stable adducts resolved by the 32P-postlabelingtechnique but also the labile adducts which can be separated from DNA in vitro and recovered as excretion products in vivo.
Acknowledgment. This work was supported by USPHS Grants R01-CA25176 and R01-CA44686 from the National Cancer Institute and Grant P42-ES04705 from the National Institute of Environmental Health Sciences. Core support was provided by USPHS Grant P30CA36727 from the National Cancer Institute.
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Chem. Res. Toricol., Vol. 2, No. 5, 1989 315 (3) Sims, P., and Grover, P. L. (1981) Involvement of dihydrodiols and diol epoxides in the metabolic activation of polycyclic hydrocarbons other than benzo[a]pyrene. In Polycyclic Hydrocarbons and Cancer (Gelboin, H. V., and Ts’o, P. 0. P., Eds.) Vol. 3, pp 117-181, Academic Press, New York. (4) Conney, A. H. (1982) Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons: G. H. A. Clowes Memorial Lecture. Cancer Res. 42, 4875-4917. (5) Rogan, E. G., Cavalieri, E. L., Tibbels, S. R., Cremonesi, P., Warner, C. D., Nagel, D. L., Tomer, K. B., Cerny, R. L., and Groas, M. L. (1988) Synthesis and identification of benzo[a]pyrene guanine nucleoside adducts formed by electrochemical oxidation and horseradish peroxidase-catalyzed reaction of benzo[a]pyrene 110,4023-4029. with DNA. J. Am. Chem. SOC. (6) Cavalieri, E., Devanesan, P., and Rogan, E. (1988) Cytochrome P-450-catalyzed binding of benzo[a]pyrene to DNA by one-electron oxidation. Proc. Am. Assoc. Cancer Res. 28, 94. (7) Zamzow, D., Jankowiak, R., Cooper, R. S., Small, G. J., Tibbels, S. R., Cremonesi, P., Devanesan, P., Rogan, E. G., and Cavalieri, E. L. (1989) Fluorescence line narrowing spectrometric analysis of benzo[a]pyrene-DNA adducts formed by one-electron oxidation. Chem. Res. Toxicol. 2, 29-34. (8) Gupta, R. C., Reddy, M. V., and Randerath, K. (1982) 32Ppostlabeling analysis of non-radioactive aromatic carcinogen-DNA adducts. Carcinogenesis 3, 1081-1092. (9) Randerath, E., Agrawal, H. P., Reddy, M. V., and Randerath, K. (1983) Highly persistent polycyclic aromatic hydrocarbonDNA adducts in mouse skin: Detection by a2P-postlabeling analysis. Cancer Lett. 20, 109-114. (10) Lu, L.-J. W., Disher, R. M., Reddy, M. V., and Randerath, K. (1986) 32P-Postlabeling assay in mice of transplacental DNA damage induced by the environmental carcinogens safrole, 4aminobiphenyl and benzo[a]pyrene. Cancer Res. 46,3046-3054. (11) Wong, A. K. L., Cavalieri, E. L., and Rogan, E. G. (1986) Dependence of benzo[a]pyrene metabolic profile on the concentration of cumene hydroperoxide with uninduced and induced rat liver microsomes. Biochem. Pharmacal. 35, 1583-1588. (12) Rogan, E. G., and Cavalieri, E. (1974) 3-Methylcholanthreneinducible binding of aromatic hydrocarbons to DNA in purified rat liver nuclei. Biochem. Biophys. Res. Commun. 58,1119-1126. (13) Rogan, E., Roth, R., Katomski, P., Benderson, J., and Cavalieri, E. (1978) Binding of benzo[a]pyrene at the 1,3,6 positions to nucleic acids in vivo on mouse skin and in uitro with rat liver microsomes and nuclei. Chem.-Biol. Interact. 22, 35-51. (14) Bodell, W. J., and Banerjee, M. R. (1976) Reduced DNA repair in mouse satellite DNA after treatment with methyl methanesulfonate and N-methyl-N-nitrosourea. Nucleic Acids Res. 3, 1689-1701. (15) Reddy, M. V., and Randerath, K. (1986)Nuclease Pl-mediated enhancement of sensitivityof s2p-postlabeliig test for structurally diverse DNA adducts. Carcinogenesis 7, 1543-1551. (16) Liu, S. F., Rappaport, S. M., Rasmussen, J., and Bodell, W. J. (1988) Detection of styrene oxide DNA adducts by 32P-postlabeling. Carcinogenesis 9, 1401-1404. (17) Osborne, M., and Merrifield, K. (1985) Depurination of benzo[a]pyrene-diolepoxidetreated DNA. Chem.-Biol. Interact. 53, 183-195. f>8) Cavalieri, E., Devanesan, P., Cremonesi, P., Higginbotham, S., and Rogan, E. (1989) Binding of benzo[a]pyrene to DNA by one-electron oxidation catalyzed by cytochrome P-450 in rat liver nuclei. Proc. Am. Assoc. Cancer Res. 29, 117. (19) Seidman, L. A., Moore, C. J., and Gould, M. N. (1988) Postlabeling analysis of DNA adducts in human and rat mammary epithelial cells. Carcinogenesis 9, 1071-1077.
