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Chem. Res. Toricol. 1989, 2, 12-14
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Communications Sequence Selectivity in the Reaction of Optically Active Hydrocarbon Dihydrodiol Epoxides with Rat H-ras DNA Sir: Several structurally diverse chemical carcinogens activate the ras protooncogene by mutation in the codon for amino acid 12 or 61 of the p21 ras protein (1-8). Extensive studies show that the alkylating agent N methyl-N-nitrosourea (NMU) consistently activates the rat H-ras gene by mutation at guanine in the codon 12 GGA sequence ( 9 ) , whereas the hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA) activates mouse and rat H-ras by mutation at adenine residues in the codon 61 CAA sequence (5, 7, 9, 10). These findings are consistent with the chemical preference of NMU for reaction with guanine residues (II), and of DMBA for extensive reaction with adenine, as well as guanine, residues in DNA (12-I4), and have prompted us to investigate the extent to which chemical properties of reactive carcinogens determine sequence selectivity in reactions with DNA. Here, we report that both optical isomers and stereoisomers of the highly carcinogenic benzo[c]phenanthrene (BcPh) dihydrodiol epoxide (15) (Figure 1)exhibit subtle differences in sequence selectivity in their reactions with rat ras DNA in vitro and that major differences in sequence selectivity exist for BcPh and the extensively studied benzo[a]pyrene (BaP) dihydrodiol epoxides. The former were reactive toward both codon 12 and codon 61 sequences whereas the latter exhibited a preferential reaction with codon 12 sequences. A plasmid containing rat c-H-ras sequences (16) was reacted to similar extents with each optically active hydrocarbon dihydrodiol epoxide described in Figure 1. The sites of reaction were then detected by denaturing the double-stranded DNA in alkali, annealing with a 32Pend-labeled primer complementary to a sequence adjacent to codon 12 or codon 61, incubation with nucleoside triphosphates and Sequenase [a modified T7 DNA polymerase (17)], and determination of the lengths of newly synthesized DNA by electrophoresis in 6% polyacrylamide denaturing gels (Figure 2). This follows the general approach used by Strauss and his colleagues (18),wherein bulky adducts or apurinic sites lead to termination of DNA synthesis. It is clear that treatment of PAL-7 with solvent alone did not lead to early arrest of the polymerase in any of our experiments (Figure 2), and in this regard, Sequenase was superior to the Klenow fragment of Escherichia coli polymerase I and AMV reverse transcriptase in our hands. Although the overall appearances of the bands from each isomeric BcPh dihydrodiol epoxide are similar, close inspection of Figure 2A,B indicates that each isomer is unique in its sequence specificity. For example, at the guanine labeled 1,the (-)-1isomer clearly gives the most intense band, whereas at the adenine labeled 2, the (+)-2 isomer provides the most effective inhibition of the polymerase. There are other sites where all isomers are similarly effective, i.e., the bands labeled 3, and there are sites, e.g., the bands labeled 4, where the (-)-isomers are more effective than the (+)-isomers. Many other differences among the isomers can also be seen. Another point of interest is that the BcPh derivatives generate arrest sites associated with the purines in the template strand, but
(+)-BCPhDE-1
(-)-BCPhDE-1
( +)-BcPhDE-P
(-)-BcPhDE-2
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(-)-BaPDE-2
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(+)-BaPDE-2
Figure 1. Structures of dihydrodiol epoxides (DE) of BcPh and
BaP. Diastereomers wherein the epoxide oxygen and benzylic hydroxyl group are cis are identified by the suffix 1 and those wherein these groups are trans are identified by the suffix 2. where the template contains runs of pyrimidines (e.g., a t regions marked 5 and 6), polymerase arrest is not seen. This is consistent with previous chemical studies with these agents indicating that adenine and guanine residues in DNA are the principal sites for reaction (19,20). In contrast, the arrest sites formed with BaP dihydrodiol epoxides follow the guanine residues in the sequence fairly closely, with fewer sites being associated with adenine residues. This is, again, generally consistent with the chemistry established for the BaP derivatives (21). Although some subtle differences between the (+)- and (-)-enantiomers for BaP dihydrodiol epoxides can be seen (indicated by 7 and 8), both isomers show a preference for reaction at guanines, as indicated by the absence of bands in the region labeled 9, which contains cytosines, adenines, and thymines but no guanines. This contrasts with expectations from earlier studies suggesting that the (-)enantiomer preferentially reacts with adenines (22). With respect to the selectivity of these hydrocarbon derivatives for reaction at codon 61, it is clear that all four BcPh derivatives lead to polymerase arrest at the two adenines of the CAA sequence in the sense strand, although the bands at this codon are not the most intense bands found. In contrast, bands at this codon were very weak for the BaP derivatives (Figure 2C), in concert with a limited chemical reactivity toward deoxyadenosine residues. In the antisense strand where the complement of 8 1989 American Chemical Society
Communications
Chem. Res. Toxicol., Vol. 2, No. 1, 1989 13
Codon 61 Sense BcPh DES
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-_ Figure 2. Autoradiographs showing sites of arrest of DNA polymerase (Sequenase) caused by chemical modification of ras DNA sequences by the optically active dihydrodiol epoxides described in Figure 1. Panels A-D show sequences adjacent to codon 61 in the sense and antisense strands, while panels E and F show the sequence adjacent to codon 12 in the sense strand. T h e hydrocarbon derivatives used to treat the DNA are indicated on the figure as are the locations of codon 61 and codon 12. T h e symbols C, G, T, and A indicate the sequence of the template strand of unmodified PAL-7. Aliquots of PAL-7 [this pBR322 derivative, which contains a 2.9-kb Hind111 DNA fragment encompassing all the coding sequences of the normal rat H - ~ Qgene s except the first 12 nucleotides (16), was a gift from Dr. Mariano Barbacid, NCI-Frederick Cancer Research Facility] (0.8 or 0.2 mg/mL in 0.01 M Tris-0.001 M EDTA buffer, pH 7.4, for BcPh and BaP derivatives in solvent, respectively) were treated with BcPhDEs (25) (Figure 1)a t 7.5,7.2, 11.7, and 6.6 ng/pg of DNA for the (+)-l, (-)-l,(+)-2, and (-1-2 isomers, respectively, so that approximately 1 in every 200 nucleotides was modified and the final concentration of acetonitrile was 22% in each case. Similar levels of DNA modification by the BaP derivatives were obtained by using 68 and 208 ng/pg of DNA of the (+)-2 and (-)-2 enantiomers (NCI Chemical Carcinogen Repository, NCI, Bethesda, MD). The BaP derivatives were in tetrahydrofuran/triethylamine (19:1), and the final concentration of organic solvent in buffer was 22%. Reactions were for 3 h a t 37 "C in the dark. As a control, some DNA was treated with the solvents alone. Treated p A L 7 was denatured with 0.1 volume of 2 M NaOH-0.002 M EDTA, and after 5 min, an equimolar amount of =P-labeled primer was added prior to neutralization with 0.1 volume of 2 M ammonium acetate, pH 4.5. After precipitation with ethanol and drying, the DNA pellet was resuspended in 0.03 M Tris-HC1, pH 7.5,0.015 M MgC12, 0.037 M NaCl, and 0.0075 M dithiothreitol(7 pL/pg of DNA). T o 7 pL of DNA solution was added nucleotide solution, 80 pM in each of dATP, dGTP, dCTP, and TTP (5 pL), and 6.25 units of Sequenase (United States Biochemical Corp., Cleveland, OH) (4 pL). After 5 min a t 37 "C, 4 pL of stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyano1 FF) was added. Aliquots with equal amounts of radioactivity were heated to 90 "C for 3 min and electrophoresed in 6% denaturing polyacrylamide gels. Sequencing of unmodified DNA followed the general approach of Zagursky e t al. (26) and was essentially as described above except that the termination reactions followed the Sequenase protocol.
codon 61 is "TG, no evidence for any substantial reaction was seen with the BaP derivatives (Figure 2B) or the BcPh derivatives (Figure 2D), though weak arrest sites could be seen immediately adjacent to the guanine in both cases. The sequence specificity of reactions in the region of the codon 12 GGA sequence was also explored (Figure 2E,F), but since the complement contains all pyrimidines, which are not reactive toward these derivatives, the antisense strand was not examined. All six dihydrodiol epoxides reacted a t the GGA of codon 12. The intensities of the bands formed with the BcPh derivatives were similar to those of many other bands elsewhere, indicating little se-
lectivity. The BaP-induced bands a t this codon were not the most intense bands on the gel, but they were more intense than bands a t many other guanines, suggesting some degree of selectivity in reaction a t this codon. Our findings indicate that the BaP metabolites exhibit some preference for reaction a t codon 12 rather than codon 61 of the rat rus sequences and that the BcPh derivatives clearly are reactive toward both of these codons. Vousden et al. (23)have found preferential in vitro mutation of the human ras sequences a t codon 61 by the racemic mixture of the BaP derivatives examined here, but the sequence for human ras is GGC a t codon 12 and CAG a t codon 61.
14 Chem. Res. Toxicol., Vol. 2, No. 1, 1989
In contrast, You et al. (6) have recently found activating mutations at codon 12 of K-ras in lung tumors of strain A mice exposed to BaP in vivo. Codon 12 in c K-ras from mouse is GGT (24). The findings described herein indicate that optical isomers of the same compound, e.g. (+)-land (-)-lfrom BcPh, can react with subtly different sequence selectivities, that agents with the same absolute stereochemistry but with a different hydrocarbon residue, e.g., (+)-2 from BaP and (-)-2 from BcPh, exhibit widely divergent sequence selectivities, and that different agents can exhibit different selectivities for codons 12 and 61 of the c-H-ras oncogene. Although clearly not the only factor involved, these studies indicate that the different chemical properties of carcinogen metabolites are important in determining sites of reaction on biologically important macromolecules and, therefore, in determining biological effects.
Acknowledgment. We thank Dr. Michael M. Seidman and Dr. J. M. Sayer for helpful advice. This research was sponsored in part by the NCI, DHHS, under Contract N01-CO-74101 with Bionetics Research, Inc. Registry No. (+)-BcPhDE-1, 82510-59-6; (-)-BcPhDE-1, 82510-56-3; (+)-BcPhDE-2,82510-57-4;(-)-BcPhDE-2,82510-5&5; (-)-BaPDE-2, 63323-30-8; (+)-BaPDE-2, 63323-31-9.
References (1) Barbacid, M. (1987) ras Genes.
Annu. Reo. Biochem. 56, 779-827. (2) Guerrero, I., Villasante, A., Corces, V., and Pellicer, A. (1985) Loss of the normal N-ras allele in a mouse thymic lymphoma induced by a chemical carcinogen. Proc. Natl. Acad, Sci. U.S.A. 82, 7810-7814. (3) Bargman, C. I., Hung, M.-C., and Weinberg, R. A. (1986) Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185. Cell 45, 649-657. (4) Wiseman, R. W., Stowers, S. J., Miller, E. C., Anderson, M. W., and Miller, J. A. (1986) Activating mutations of the c-Ha-ras protooncogene in chemically induced hepatomas of the male B6C3 F, mouse. Proc. Natl. Acad. Sci. U S A . 83, 5825-5829. (5) Bizub, D., Wood, A. W., and Skalka, A. M. (1986) Mutagenesis of the Ha-ras oncogene in mouse skin tumors induced by polycyclic aromatic hydrocarbons. Proc. Natl. Acad. Sci. U.S.A.83, 6048-6052. (6) You, M., Candrian, V., Widegren, B., Maronpot, R., Stoner, G . D., and Anderson, M. W. (1988) Activation of the K-ras oncogene in spontaneously occurring and chemically-induced lung tumors of strain A mice. Proc. Am. Assoc. Cancer Res. 29, 143. (7) Quintanilla, M., Brown, K., Ramsden, M., and Balmain, A. (1986) Carcinogen-specific mutation and amplification of H a m s during mouse skin carcinogenesis. Nature 322, 78-80. (8) Topal, M. D. (1988) DNA repair, oncogenes and carcinogenesis. Carcinogenesis 9, 691-696. (9) Zarbl, H., Sukumar, S., Arthur, A. V., Martin-Zanca, D., and Barbacid, M. (1985) Direct mutagenesis of Ha-ras-1 oncogenes by N-nitroso-N-methylurea during initiation of mammary carcinogenesis in rats. Nature 315, 382-385. (10) Dandekar, S., Sukumar, S., Zarbl, H., Young, L. J. T., and Cardiff, R. D. (1986) Specific activation of the cellular Harvey-ras oncogene in dimethylbenzanthracene-inducedmouse mammary tumors. Mol. Cell. Biol. 6, 4104-4108. (11)Swann, P. F., and Magee, P. N. (1968) The alkylation of nucleic acids of the rat by N-methyl-N-nitrosourea, dimethylnitrosamine, dimethyl sulphate and methyl methanesulphonate. Biochem. J. 110, 39-47. (12) Dipple, A., Pigott, M., Moschel, R. C., and Costantino, N. (1983) Evidence that binding of 7,12-dimethylbenz[a]anthraceneto DNA in mouse embryo cell cultures results in extensive substitution of both adenine and guanine residues. Cancer Res. 43,4132-4135.
Communications (13) Cheng, S. C., Prakash, A. S., Pigott, M. A,, Hilton, B. D., Lee, H., Harvey, R. G., and Dipple, A. (1988) A metabolite of the carcinogen 7,12-dimethylbenz[a]anthracenethat reacts predominantly with adenine residues in DNA. Carcinogenesis 9, 1721-1723. (14) Dipple, A., Sawicki, J. T., Moschel, R. C., and Bigger, C. A. H. (1983) 7,12-Dimethylbenz[a]anthracene-DNAinteractions in mouse embryo cell cultures and mouse skin. In Extrahepatic Drug Metabolism and Chemical Carcinogenesis (Rydstrom, J., Montelius, J., and Bengtsson, M., Eds.) pp 439-448, Elsevier, Amsterdam. (15) Levin, W., Chang, R. L., Wood, A. W., Thakker, D. R., Yagi, H., Jerina, D. M., and Conney, A. H. (1986) Tumorigenicity of optical isomers of the diastereomeric bay-region 3,4-diol-1,2-epoxides of benzo[c]phenanthrene in murine tumor models. Cancer Res. 46, 2257-2261. (16) Sukumar, S., Notario, V., Martin-Zanca, D., and Barbacid, M. (1983) Induction of mammary carcinomas in rats by nitroso-methylurea involves malignant activation of H-ras-1 locus by single point mutations. Nature 306, 658-661. (17) Tabor, S., and Richardson, C. C. (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 84, 4767-4771. (18) Sagher, D., and Straws, B. (1985) Abasic sites from cytosine as termination signals for DNA synthesis. Nucleic Acids Res. 13, 4285-4298. (19) Dipple, A., Pigott, M. A., Agarwal, S. K., Yagi, H., Sayer, J. M., and Jerina, D. M. (1987) Optically active benzo[c]phenanthrene diol epoxides bind extensively to adenine in DNA. Nature 327, 535-536. (20) Agarwal, S. K., Sayer, J. M., Yeh, H. J. C., Pannell, L. K., Hilton, B. D., Pigott, M. A., Dipple, A. Yagi, H., and Jerina, D. M. (1987) Chemical characterization of DNA adducts derived from the configurationally isomeric benzo[c]phenanthrene-3,4-diol1,2-epoxides. J . Am. Chem. SOC. 109, 2497-2504. (21) Cooper, C. S., Grover, P. L., and Sims, P. (1983) The metabolism and activation of benzo[a]pyrene. Prog. Drug Metab. 7, 295-396. (22) Meehan, T., and Straub, K. (1979) Double-stranded DNA stereoselectively binds benzo[a]pyrene diol epoxides. Nature 277, 410-412. (23) Vousden, K. H., Bos, J. L., Marshall, C. J., and Phillips, D. H. (1986) Mutations activating human c-Ha-rasl protooncogene (HRAS1) induced by chemical carcinogens and depurination. Proc. Natl. Acad. Sci. U.S.A. 83, 1222-1226. (24) Guerrero, I., Villasante, A., Corces, V., and Pellicer, A. (1984) Activation of a c-K-ras oncogene by somatic mutation in mouse lymphomas induced by y radiation. Science 225, 1159-1162. (25) Yagi, H., Thakker, D. R., Ittah, Y., Croisy-Delcey, M., and Jerina, D. M. (1983) Synthesis and assignment of absolute configuration to the trans 3,4-dihydrodiol and 3,4-diol-1,2-epoxides of benzo[c]phenanthrene. Tetrahedron Lett. 24, 1349-1352. (26) Zagursky, R. J., Baumeister, K., Lomax, N., and Berman, M. L. (1985) Rapid and easy sequencing of large linear double-stranded DNA and supercoiled plasmid DNA. Gene Anal. Tech. 2,89-94.
Dean B. Reardon,' C. Anita H. Bigger' Judy Strandberg,' Haruhiko Yagi2 Donald M. Jerina; Anthony Dipple*,' BRI-Basic Research Program NCI-Frederick Cancer Research Facility Frederick, Maryland 21 701, and Laboratory of Bioorganic Chemistry N a t i o n a l Institute of D i a b e t e s and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892 Received December 1, 1988
* To whom correspondence should be addressed. NCI-Frederick Cancer Research Facility.
* National Institutes of Health.
