Chem. Res. Toxicol. 1993,6, 530-534
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32P-PostlabelingAnalysis of the DNA Adducts of 6-Fluorobenzo[a]pyrene and 6-Methylbenzo[alpyrene Formed in Vitro Rosa Todorovic, Prabhakar D. Devanesan, Eleanor G. Rogan,' and Ercole L. Cavalieri Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-6805 Received January 21,1993
Studies of benzo[alpyrene (BP) and selected derivatives are part of the strategy to elucidate mechanisms of tumor initiation by polycyclic aromatic hydrocarbons. Substitution of B P a t C-6 with fluorine to form 6-fluorobenzo[alpyrene (6-FBP) or a methyl group to form 6-methylbenzo[a]pyrene (6-CHsBP) decreases tumorigenicity compared to BP. BP, 6-FBP, and 6-CH3BP formed adducts with DNA when (1)they were activated by 3-methylcholanthreneinduced rat liver microsomes, (2) they were activated by horseradish peroxidase (HRP), (3) their 7,8-dihydrodiols were activated by microsomes, or (4) the radical cation of BP, 6-FBP, or 6-CH3B P was directly reacted with DNA. With microsomes, 6.5 pmol of r3H16-FBP/mol of DNA-P and 10pmol of [14C16-CHJ3P/molof DNA-P were bound us 15pmol of i3H1BP. With microsomes, two major 6-FBP adducts and some minor adducts were obtained. One major adduct coincided with that from 6-FBP-7,8-dihydrodiol. With microsomes, the minor 6-FBP adducts coincided with the adducts obtained from 6-FBP radical cation plus DNA and the major adduct of HRPactivated 6-FBP. With microsomes, 6-CHsBP showed adducts similar to some formed with HRP and one from 6-CH3BP radical cation. 6-CH3BP-7,8dihydrodiol produced a small amount of one adduct that did not coincide with any from 6-CH3BP. The adducts of 6-FBP appear to be formed mostly through the diolepoxide pathway, whereas those of 6-CH3BP appear to arise mostly uia one-electron oxidation.
Introduction Elucidation of the mechanisms of activation of polycyclic aromatic hydrocarbons (PAH)' is central to understanding the process of tumor initiation. Studies of PAH-DNA adducts point thus far to two major mechanisms of activation, one-electron oxidation to form intermediate radical cations and monooxygenation to produce bayregion diolepoxides ( I ) . With one-electron oxidation, benzo[a]pyrene (BP) is oxidized to its radical cation (BP'+), which binds at C-6 (2-6) (Figure 1). Such BP adducts, constituting 80% of the total adducts formed in vitro and in vivo (5, 61,are rapidly lost from DNA by depurination. The major adduct [lo-(deoxyguanosin-Nyl)-7,8,9-trihydroxy-7,8,9,lO-tetrahydrobenzo[al pyrene (BPDE-lO-N2dG)Iformed by monooxygenation contains a bond between the 2-amino group of guanine and the C-10 of BP after the 7,8,9,10-ring is saturated and hydroxylated (5-8). This adduct is stable in DNA, constituting 15-2096 of the total adducts (5,6). Studies of BP and selected derivatives, including 6-fluorobenzo[a]pyrene (6-FBP) and 6-methylbenzo[alpyrene (6-CHsBP) (Figure l),are part of the strategy to elucidate mechanisms of tumor initiation by PAH. Over the years we have studied the tumorigenicity (9-11),radical cation chemistry (12), binding to DNA (2-6,13-16), and
* To whom correspondence should be addressed.
Abbreviations: BP, benzo[a]pyrene; BP'+,benzo[alpyrene radical cation;BPDElO-NadG,10-(deoxyguanoein-Na-yl)-7,8,9-trihydrory-7,8,9,lO-tetrahydrobenzo[alpyrene;6-BrBP,6-bromobenzo[alpyrene;6-CHsBP, (i-methylbenzo[a]pyrene; 6-ClBP,6-chlorobenzo[alpyrene; DMBA, 7,12-dimethylbenz[a]anthracene;6-FBP,6-fluorobenzo[alpyrene;HRP, horseradish peroxidme; MC, 3-methylcholanthrene; PAH, polycyclic aromatic hydrocarbon(s). 1
12
1
R
R = H , BP R = F, 6-FBP R = CH,, 6-CH3BP
Figure 1. Structure of BP, 6-FBP, and 6-CH8BP. metabolism (17-21) of BP, 6-halogenatedBPs, and 6-CH3BP. Tumorigenicity studies of BP us 6-FBP, 6-chlorobenzo[alpyrene (6-C1BP), and 6-bromobenzo[alpyrene (6BrBP) show that 6-FBP is active, although less potent than the parent compound (10, 11,22,23),whereas 6-ClBP and 6-BrBP are virtually inactive (9, 10). 6-FBP contains the fluoro substituent at the critical C-6, that is, the position of binding of BP'+ to DNA nucleophiles. At first glance, the one-electron oxidation pathway seems to be blocked by the 6-fluor0 substituent. Oxidation of 6-FBP by manganic acetate, however, shows that nucleophilic attack of the acetate ion in the 6-FBP'+ occurs at (2-6, with formation of 6-acetoxy-BP and displacementof the fluoro substituent (12). Displacement of fluorine ala0 occurs in the formation of BP 1,6-, 3,6-, and 6,la-dione from 6-FBP during metabolism catalyzed by cytochrome P450 (19,23)and by horseradish peroxidase
0S93-22S~/93/2706-053Q~Q4.O0/0 0 1993 American Chemical Society
DNA Adducts of 6-FBP and 6-CHaP
(HRP) and prostaglandin H synthase (20). Electrochemical one-electron oxidation of 6-FBP in the presence of deoxyguanosine produces a mixture of adducts a t C-1 and C-3 (24),which are the positions of highest charge density after C-6 in the 6-FBP'+ (12,25). In this case the fluoro substituent is not displaced. The noncarcinogenic 6-C1BP and 6-BrBP are not metabolized by cytochrome P450 (19) and do not form adducts by electrochemical oxidation in the preence of deoxyribonucleosides (24). Formation of 6-FBP-7,8-dihydrodiol, the proximate metabolite in the diolepoxide pathway, is catalyzed by cytochrome P450 (19,23), but the fluoro substituent at C-6 in the position peri to the hydroxyl group at C-7 forces the conformation of the dihydrodiol to be diaxial (23). Therefore, the lesser carcinogenic activity of 6-FBP compared to BP can be preliminarily attributed to either the modified structure of the diolepoxide or decreased reactivity of 6-FBP'+, which involves displacement of the fluoro substituent or, more likely, reaction a t C-1 and (2-3, as suggested by the electrochemical oxidation of 6-FBP in the presence of deoxyribonucleosides (24). The noncarcinogenicity of 6-C1BP and 6-BrBP can be explained by their resistance to metabolism (19) or formation of adducts by one-electron oxidation (25). 6-CH3BP, like 6-FBP, exhibits a lesser tumorigenicity than BP (9, 11). This model compound is particularly relevant to BP because the methyl group at C-6 can be activated and react with nucleophiles after the 6-CH3BP'+ has been metabolically formed. This has also been observed with oxidation of 6-CH3BPby manganic acetate, in which acetoxylation at the methyl group in the 6-CH3BP'+ competes with that at C-1 and C-3 (12). This suggests that the reactivity of the methyl group in 6-CH3BP0+is less than that of C-6 in BP'+, in which no products are formed at C-1 and C-3 (12). Electrochemical oxidation of 6-CH3BP in the presence of deoxyguanosine produces a mixture of adducts at the methyl group and at C-1 and C-3 (24). The lesser tumorigenicity of 6-CH3BP us BP could be due to cytochrome P45O-catalyzed formation of the diaxial6-CH3BP-7,8-dihydrodiol, which is similar to that of the diaxial6-FBP-7,8-dihydrodiol.If activated by one-electron oxidation, 6-CH3BP could resemble 6-FBP and react at C-1 and C-3, or react at the 6-methyl group, resembling the meso-anthracenic-methylated compound 7,1Qdimethylbenz[al anthracene (DMBA), in which binding to DNA by one-electron oxidation occurs specifically at the 12-methyl group (26,27). In this paper we report comparison of the profiles of stable DNA adducts of BP, 6-FBP, and 6-CH3BPdetected by the 32P-postlabelingtechnique. In this procedure, DNA containing adducts is enzymically hydrolyzed to 3'nucleotides, which are labeled with 32P to form 3',5'bisphosphates and are separated by two-dimensional thinlayer chromatography. Autoradiography provides quantitation of the adduct spots, and preliminary identification is obtained by comparison of their mobilities (16). Binding of the PAH to DNA was effected by different methods: (1) the PAH or their 7,8-dihydrodiols were incubated with 3-methylcholanthrene (MC)-induced rat liver microsomes and DNA, (2) the PAH were incubated with HRP and DNA, or (3) the PAH radical cation perchlorates were directly reacted with DNA. These different methods enabled us to compare profiles of adducts formed by one-electron oxidation and the diolepoxide pathway. The stable adducts of 6-CH3BP appear
Chem. Res. Toxicol., Vol. 6, No. 4, 1993 531 to be formed by the one-electron mechanism, whereas the stable adducts of 6-FBP resemble those of BP and are presumably formed predominantly by the diolepoxide pathway.
Materials and Methods Chemicals. The radical cation perchlorates of BP, 6-FBP, and 6-CHaBPadsorbed on AgI were synthesized in our laboratory (28). [3H]BP was purchased from Amersham Corp. (Arlington Heights,IL; sp act. >550 Ci/mol), [3H16-FBPwas obtained from Moravek Biochemicals (Brea,CA; sp act. 11Ci/mol),and [l4C16CHsBP was synthesized by Midwest Research Institute (now Chemsyn Science Laboratories, Lenexa, KS; sp act. 54 Ci/mol) according to published methods (29). The three PAH were used at specific activities of 255, 11, and 40 Ci/mol, respectively. Caution: BP, 6-FBP, and 6-CHSBPare hazardous chemicals; they are handled in accordancewith NZHguidelines(30). Calf thymus DNA was purchased from Pharmacia (Piscataway,NJ). Isolation of BP-'l,d-dihydrodiol, 6-FBP-7,8-dihydrodiol, and 6-CHsBP-7,8-dihydrodiol. The 7,Sdihydrodiols were isolated from metabolic reactions of their respective parent compounds catalyzed by MC-induced rat liver microsomes. Reaction mixtures containing 150 mM Tris-HC1 (pH 7.5), 150 mM KC1,5 mM MgClz, 0.6 mM NADPH, and 80 pM PAH in a total volume of 30 mL were incubated for 30 min at 37 "C. The reactions were terminated by addition of 30 mL of acetone,and the metaboliteswere extracted twice with 60 mL of ethyl acetate. After evaporation of the solvent, the residue was dissolved in dimethylsulfoxide/methanol(1:l) and separated by HPLC. The YMC ODS-AQ 5-pm, 120-Acolumn(6 X 25Omm, YMC, Overland Park, KS) was eluted with 60% methanol in water for 10 min, and the metaboliteswere then eluted with a linear gradient from 60% methanol in water to 100% methanol in 60 min at a flow rate of 1mL/min. The 7,8-dihydrodiolpeaks were identified by their retention times and UV spectra and were then collected. Binding of PAH to DNA. I3H1BP,r3H16-FBP,and [l4C16CH3BP (80pM each) were bound to DNA in reactions catalyzed by microsomes, as well as in reactions catalyzed by HRP (16). BP-7,8-dihydrodiol,6-FBP-7,8-dihydrodiol,and 6-CHaP-7,8dihydrodiol (40, 19, and 5.5 pM, respectively) were bound to DNA in microsomal reaction mixtures containing 2.6 mM calf thymus DNA in 150mM Tris-HC1 (pH 7.5),150mM KC1,5 mM MgC12, and NADPH. The concentration of each dihydrodiol was 3 times the concentration normally produced in metabolic reactions starting with 80 pM PAH. All reaction mixtures were incubated for 30 min at 37 O C , and the DNA was purified as previously described (16). The radical cations BP'+, 6-FBP'+, and 6-CH3BP0+were directly reacted with DNA. About 2 mg of the radical cation perchlorate adsorbed on AgI was suspended in 0.5 mL of dry CHCl3. It was immediately filtered (0.45-pm filter) to trap the solid particles and added to 1mL of DNA (2 mg) in 0.0067 M sodium-potassium phosphate (pH 7.0). The mixture was immediately agitated on a vortex mixer, and the DNA was purifiedas previously described (16). With microsomes, the level of binding of BP was 15pmoVmo1of DNA-P; of 6-FBP, 6.5 pmol; and of 6-CH3BP, 10 pmol. With HRP, the level of binding of BP was 65 pmol/mol of DNA-P; of 6-FBP, 29 pmol; and of 6-CH3BP, 14 pmol. Analysis of Stable Adducts by the *2P-Postlabeling Method. The stable DNA adducts were detected by the "Ppostlabeling method utilizing both butanol extraction and P1 nuclease enhancementsof the adducts, as previously described (16,31). No qualitative differences were observed in the adduct profiles with the twomethods, butthe amount of adductsobtained was higher with P1-nuclease enhancement. Chromatographic steps were performed simultaneously with all the samples to minimize the chances for any variance. Calculation of the level of adducts was performed as previously described (16).
532 Chem. Res. Toxicol., Vol. 6, No. 4, 1993
Todorovic et al.
Table I. Stable PAH-DNA Adducts Detected by the 32P-PostlabelingMethod (mol of adduct/mol of DNA-P) X 1Og
(mol/mol of DNA-P) X 106 incubation system microsomes BP BP-7,8dihydrodiol 6-FBP 6-FBP-78-dihydrodiol 6-CHaP 6-CH3BP-7,8-dihydrodiol HRP BP 6-FBP 6-CH3BP radical cation BP 6-FBP 6-CH&P
total adducts
BPDE derived
radical cation derived
other
5.6 26.9 0.9 0.5 5.8 0.01
5.0 (89)O 26.2 (97) 0.6 (63) 0.5b(100)
0.05 (0.9) 0.05b(5)
0.5b(10.1) 0.7b(3) 0.3c (32)
3.6 (62)
2.2b(38)
9.3 9.7 3.3
7.9 (85) 5.9 (61) 1.6 (49)
1.4b(15) 3.8b (39) 1.7c (51)
26.2 0.3 1.1
26.2 (100) 0.3 (100) 1.1 (100)
0.01 (100)
Number in parentheses is percentage of total adducts. 3 adduct spots. 4 adduct spots.
Results and Discussion Analysis of Adducts by 32P-Postlabeling.BP-7,8dihydrodiol, 6-FBP-7,8-dihydrodiol, and 6-CH3BP-7,8dihydrodiol were synthesized from their parent compounds in metabolic reactions catalyzed by microsomes and were isolated by HPLC. They were then used for binding to DNA in microsomal reactions. Direct reaction of the BP, 6-FBP, and 6-CH3BP radical cations to DNA was also conducted. Because the radical cations are unstable in solution, they were directly reacted with DNA within about 20 s after they were suspended in dry CHCl3. Analysis of the stable DNA adducts by the 32Ppostlabeling technique afforded comparison of the adduct spots and their mechanism of formation. No qualitative differences were observed in the adduct profiles using the P1 nuclease or the butanol extraction methods for enrichment of the adducts. The amount of adducts recovered, however, was higher with the P1 nuclease method of enrichment. Therefore, this method was subsequently used. The noncarcinogenic 6-C1BP and 6-BrBP did not form any adducts with activation by either microsomes or HRP. This result is in agreement with the lack of formation of metabolites when 6-C1BP and 6-BrBPwere incubated with microsomes (I9). In the microsomal system, both BP and 6-CH3BP produced about the same amount of stable DNA adducts, while 6-FBP formed approximately 5 times less adducts detected by the 32P-postlabelingmethod (Table I). On the other hand, in the HRP-activated system, BP and 6-FBP produced approximately the same amount of adducts detected by 32P-postlabeling, while 6-CH3BP formed about one-third the amount of detected adducts. Analysis of the stable BP-DNA adducts obtained with activation by MC-induced rat liver microsomes or HRP has previously been reported (5). Adduct spots were identified on the autoradiograms by their mobility and given numerical designations. Comparison of the adduct profiles (Figure 2) reveals that the major diolepoxide adduct (adduct 1)formed by BP-7,8-dihydrodiol (Figure 2B) coincides with the major adduct obtained from BP (Figure 2A). This adduct has been previously been identified as BPDE-10-N2dG (16). Adduct spot 2, which accounts for about 1%of the adducts in the microsomal profile, coincides with an adduct produced by BP*+(Figure 2D). This adduct spot also coincides with the major adduct spot (85%of the total) formed by BP in the HRP system
Figure 2. Autoradiograms of 32P-postlabeled DNA containing adducts formed after (A) activation of BP by microsomes, (B) activation of BP-78-dihydrodiol by microsomes, (C) activation of BP by HRP, and (D) direct reaction of BP*+with DNA. The film was exposed at room temperaturefor (A) 90 min, (B) 45 min, (C) 10 min, and (D) 5 min.
(Figure 2C). The identities of the other adduct spots have not yet been elucidated. From the profiles of stable 6-FBP-DNA adducts (Figure 3), it is apparent that 6-FBP is more readily activated by the HRP system than by the microsomal system. With microsomal activation (Figure 3A), two major adducts of 6-FBP, along with several minor adducts, were observed. Adduct 1,comprising 62 % of the total, coincides with the major adduct obtained from 6-FBP-7,8-dihydrodiol activated by microsomes (Figure 3B). Some of the minor adducts formed with microsomal activation (area 3) coincide with the adduct obtained by the reaction of 6-FBPo+ with DNA (Figure 3D). The major adduct (adduct 3,64%) obtained from 6-FBP activated by HRP (Figure 3C) also coincides with this radical cation adduct. Thus, the profiles of the stable adducts of 6-FBP resemble those of the parent compound, BP (Figure 2), in all four activation systems. The profiles of 6-CH3BP-DNA adducts (Figure 4) indicate that the adducts obtained from 6-CH3BP activated by microsomes are similar to some of the adducts obtained with HRP. Adduct 1in both the microsomal (Figure 4A) and HRP (Figure 4C) systems appears to coincide with the adduct formed by 6-CH3BPo+(Figure 4D). This adduct accounts for more than 60% of all the adducts formed in the microsomal system and about 50 %
DNA Adducts of 6-FBP and 6 - C H a P
Figure 3. Autoradiograms of 32P-postlabeledDNA containing adducts formed after (A) activation of 6-FBP by microsomes, (B) activation of 6-FBP-7,8-dihydrodiol by microsomes, (C) activation of 6-FBP by HRP, and (D) direct reaction of 6-FBP*+ with DNA. The film was exposed at room temperature for (A) 2 h, (B) 2 h, (C) 20 min, and (D) 5 min.
Chem. Res. Toxicol., Vol. 6, No. 4, 1993 533
shown to react with DNA to produce adducts. These adducts can be useful in identification of those obtained by one-electron oxidation in biological systems. By observing the profiles of adducts in the various preparations, the following observations can be made: (1)The profile of stable adducts of 6-FBP activated by microsomes (Figure 3A) resembles that of BP (Figure 2A) because the major adduct of both PAH arises via the 7,8dihydrodiol (Figures 2B and 3B). (2) The profile of stable 6-CH3BP adducts formed by microsomes (Figure 4A) does not resemble that obtained by activation of its 7,8-dihydrodiol (Figure 4B). Instead, this profile resembles that obtained by activation of 6-CH3BP with HRP (Figure 4C) or by direct reaction of 6-CH3BP*+ (Figure 4D). (3) Similar adduct profiles are seen for all three PAH with activation by HRP and direct reaction of the radical cation (Figures 2C,D, 3C,D, and 4C,D). (4) Stable adducts of 6-CH3BPseem to be predominantly formed by cytochrome P450-catalyzed one-electron oxidation, whereas those of BP and 6-FBP are predominantly formed by monooxygenation. These results coupled with the synthesis of some standard adducts (24) constitute the first steps in identifying the stable and depurination adducts formed biologically by these two PAH and their mechanisms of activation. On the basis of these results, we expect to find that most 6-CH3BP adducts are formed by one-electron oxidation, with a covalent bond a t the 6-methyl group and/or C-1 and C-3. For 6-FBP, we expect to identify adducts arising from the diolepoxide and also adducts at C-1 and C-3 formed from the radical cation. This information should provide further insight into the relative carcinogenicity of BP, 6-FBP, and 6-CH3BP.
Acknowledgment. This research was supported by Figure 4. Autoradiograms of 32P-postlabeled DNA containing adducts formed after (A) activation of 6-CH3BP by microsomes, (B) activation of 6-CH3BP-7,8-dihydrodiolby microsomes, (C) activation of 6-CH3BP by HRP, and (D)direct reaction of 6-CH3BP*+with DNA. The film was exposed at room temperature for (A) 90 min, (B) 90 min, (C) 45 min, and (D) 5 min.
in the HRP system. The reaction of 6-CH3BP-7,8dihydrodiolactivated by microsomes (Figure 4B) produced only one adduct in small amount, but this adduct does not coincide with any obtained with 6-CH3BP activated by microsomes. Thus, the profiles of 6-CH3BP adducts differ from those of both 6-FBP and BP. 6-CH3BP more closely resembles DMBA, in which the adduct formed by the 3,4dihydrodiol coincides with only a minor adduct formed from the parent compound activated by microsomes (25). With both 6-CH3BP and DMBA, the major adducts formed with microsomes coincide with those formed with HRP.
Conclusions The profiles of stable adducts obtained by activation of BP, 6-FBP, or 6-CH3BP with microsomes were compared to those formed by activation of the respective 7,8dihydrodiols with microsomes, those obtained with activation of the parent PAH by HRP, and those produced by direct reaction of the three radical cations with DNA. This is the first time that PAH radical cations solvated from the radical cation perchlorates adsorbed on AgI were
U.S.Public Health Service Grants R01-CA25176, R01CA44686, and Pol-CA49210. Core support to the Eppley Institute was from the National Cancer Institute (P30CA36727).
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