Chem. Res. Toxicol. 1995, 8, 143-147
143
Mutational Spectra for 5,6-Dimethylchrysene 1,2-Dihydrodiol3,4-Epoxidesin the supF Gene of pSPl89 John E. Page,? Jan Szeliga,? Shantu Amin,$ Stephen S. Hecht,#and Anthony Dipple*9? Chemistry of Carcinogenesis Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, P.O. Box B, Frederick, Maryland 21 702,and American Health Foundation, Dana Road, Valhalla, New York 10950 Received July 27, 1994@ Dihydrodiol epoxides from 5,6-dimethylchrysene exhibit properties similar to those of fjord region-containing hydrocarbon derivatives in t h a t they react extensively with deoxyadenosine residues in DNA and consequently generate substantial numbers of mutations a t AT pairs a s well as GC pairs. The syn-dihydrodiol epoxide favors reaction with deoxyadenosine (68% of adducts) to a greater extent t h a n does the anti-dihydrodiol epoxide (52% of adducts), and point mutations at AT pairs (72% for syn- and 45% for anti-dihydrodiol epoxide) follow the same trend. A novel feature of the mutagenicity of the 5,6-dimethylchrysene derivatives is t h a t they exhibit a higher fraction of AT GC transitions (28% and 26% for syn and anti, respectively) t h a n has been seen for other hydrocarbon derivatives to date.
-
Introduction A substantial amount of evidence suggests that bay region dihydrodiol epoxide metabolites of polycyclic aromatic hydrocarbons may be responsible for the carcinogenic properties associated with these environmental carcinogens (reviewed in refs 1-3). For this reason, the chemical (4, 5 ) and biological properties of these dihydrodiol epoxides (6-8) have been the subject of fairly intense investigation. The tumor initiating activities of dihydrodiol epoxides derived from hydrocarbons with hindered bay regions or fjord regions have been found to be exceptionally high (7), and chemical studies have indicated that these dihydrodiol epoxides in particular [e.g., those from 7,12-dimethylbenz[alanthracene (91, benzoklphenanthrene (10,11),5,6-dimethylchrysene (12, 13),and benzo[g]chrysene (14)lreact extensively with deoxyadenosine residues in DNA as well as with deoxyguanosine residues. To clarify the relationship between the established chemical specificities of these agents and their biological effects, we have examined mutational spectra in the supF gene for dihydrodiol epoxides from two hydrocarbons containing fjord regions, i.e., benzo[clphenanthrene (15) and benzolglchrysene (14). Each of these metabolites induced many mutations at AT pairs, as well as a t GC pairs, in line with expectations from their chemistry. The 5,6-dimethylchrysene 1,2-dihydrodiol 3,bepoxides (Figure 1) do not contain fjord regions, but do have a sterically hindered bay region resulting from the methyl groups at the 5- and 6-positions. Consequently, their chemistry is similar to that for compounds containing fjord regions (12,13). In the present work, we have extended chemical analyses of DNA adduct formation by 5,6-dimethylchrysene dihydrodiol epoxides and determined their mutagenic properties. With respect to reactivity toward deoxyadenosine residues and the distribution of mutations over AT and GC base pairs, these + ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center. *American Health Foundation. Abstract published in Advance ACS Abstracts, December 1,1994. @
syn 1,Zdihydrodiol 3,4-epoxide of 5,8dimrthylchrysene
ant/ I , ~ d i h y d ~ d i o l W - e ~ o x i d e of 6,6dimethylchrysene
Figure 1. Relative stereochemistries of syn- and anti-dihydrodiol epoxides. Racemates were used in the present work, but only one enantiomer is shown in the figure.
agents behave like fjord region-containing hydrocarbons.
Experimental Section The anti- and syn-5,6-dimethylchrysene1,2-dihydrodio13,4epoxides were synthesized as described previously (12). Mutation Assay. The mutation assay utilizing the pSP189 shuttle vector of Parris and Seidman (16)was essentially as described earlier (15,17).Briefly, the dihydrodiol epoxideswere dissolvedin tetrahydrofuran, and aliquots of pSP189 DNA (100 pg in 0.05 mL Tris/EDTA buffer, pH 7) were treated with dihydrodiol epoxide solution such that the dihydrodiol epoxide: DNA ratios were 50,25,10, and 1ng/,ug of DNA. Mer leaving overnight at 37 "C,the solutions were extracted with ethyl acetate (3x ) and with ether, and the DNA was then precipitated with ethanol, washed with 70%ethanol, and dried. The DNA samples were resuspended in 55 pL of TridEDTA buffer (pH 7) and used to transfect Ad293 cells by the calcium phosphate coprecipitation technique, all as described (18).After 48 h, the vector DNA was recovered, was digested with DpnI to remove unreplicated DNA, and was used to transform Escherichia coli MBM 7070 cells by electroporation (17). Transformed cells were grown on LB plates with ampicillin (50pg/plate) overlain with 5-bromo-4-chloro-3-indolylj3-&galactoside (X-Gal,' 2 mg/plate) and isopropyl B-D-thiogalactoside (IPTG,l 5 mdplate). White and pale blue mutant colonies were selected, restreaked, and grown up in overnight cultures. Vector DNA was isolated using the Wizard Miniprep plasmid purification kit (Promega, MadiAbbreviations: X-Gal, 5-bromo-4-chloro-3-indolyl B-&galactoside; IPTG, isopropyl B-D-thiogalactoside,
0893-228x/95/2708-0143$09.00/00 1995 American Chemical Society
144 Chem. Res. Toxicol., Vol. 8, No. 1, 1995
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Page et al. ~
3
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1 03 04
A2
L A
1
,
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B T
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7
10
30
50
A 70
90
Time (min) Fi2. HPLC separation of deoxyribonucleoside adducts obtained from reaction^ of the syn- (panel A) and anti- (panel B) dihydrotliol epoxides of 5,tdimethylchrysene with calf thymue DNA in vitro. Reaction and separation conditions are given under Experimental Section. Deoxyguanosine adducts (Gl-G4) and deoxyadenosineadducts (Al-A4) were identified by comparison of retention times and W absorption spectra with thow of adduets derived by reaction of the dihydrodiol epoxides with deoxyguanosine and deoxyadenosine 5'-phosphaw, respectively. T denotes a tetraol that was incompletely extracted from the reaction mixture prior to chromatography.
son, WI)and sequenced through the supF gene and signature sequence using a Sequenase I1 dideoxy sequencing kit (USB, Cleveland, OH). A mutation hotspot was defined as a site at which the number of mutations found exceeded those expected from a Poisson distribution by a factor of 5 or more. Chemical Studies. Calf thymus DNA (1mg/mL) in 0.1 M Tris-HC1buffer, pH 7, was treated with 0.05 volume of a solution of the dihydrodiol epoxide (2 mg/mL) in tetrahydrofuran. ARer 20 h, the solutions were extracted (4x) with water-saturated 1-butanol and with ether (3x1. Thereafter, 0.1 volume of 0.01 M MgC12, 0.01 M Tris-HC1buffer, pH 7, and 0.1 mL of DNase (1mg/mL) were added, and the solution was incubated at 37 "C for 1.5 h. The pH was then raised by the addition of 0.2 M Tris-HC1buffer, pH 9, and snake venom phosphodiesterase (1 unit) was added, followed by incubation for 48 h at 37 "C. Alkaline phosphatase (20 units) was then added, and after a further 24 h incubation, the solution was passed through a SepPak that was then washed with water and 5%methanol before elution of the adducts in methanol. Individual adducts were then separated by HPLC using a Beckman Ultrasphere ODS column (0.46 x 25 cm) and elution with 40% methanol for 10 min followed by a linear increase to 56%methanol over 80 min and isocratic elution for a further 10 min (for the antzdihydrodiolepoxide adducts) or with 23%acetonitrile for 10 min followed by a gradient that decreased acetonitrile t o 0% and increased methanol to 50% over the next 50 min, followed by a further increase to 65% methanol over the next 40 min (for the syn-dihydrodiol epoxide adducts).
Results Figure 2 shows a comparison of deoxyribonucleoside adducts formed by reaction of calf thymus DNA with either the syn- (panel A) or anti- (panel B) dihydrodiol epoxides of 5,6-dimethylchrysene (Figure 1). Comparison of the retention times and absorbance spectra of the adducts obtained with those formed in separate reactions with deoxyadenylic and deoxyguanylic acids for each diastereomer allowed the adducts to be identified as deoxyguanosine adducts (G1-G4) or as deoxyadenosine adducts (Al-A4). In two separate experiments, some
tetraol (T) was present in the adducts from the antidihydrodiol epoxide but not in those from the syndihydrodiol epoxide. A slightly larger fraction of the anti(32%) than the syn- (24%) dihydrodiol epoxide reacted with calf thymus DNA, and whereas reaction with deoxyadenosine residues in DNA was prevalent for the syn-dihydrodiol epoxide (dAdo:dGuo adducts = 2.1/1), each nucleoside was similarly susceptible to reaction with the anti-dihydrodiol epoxide (dAdo:dGuo adducts = 1.11 1). The findings for the anti-dihydrodiol epoxide were consistent with previous studies (12,131. Detailed studies of the syn-dihydrodiol epoxide have not been reported before. When shuttle vector DNA was exposed to a range of epoxide doses, i.e., 50,25,10, and 1ng/pg of vector DNA, it was found, after plating out 1mL of transformation reaction solution, that no transformants were recovered a t the two highest doses for both the anti and syn isomers, indicating that these compounds were toxic at these levels. At the 10 ng/pg dose, 10 mutant phenotypes were recovered from 2080 transformants and 100 were recovered from 23040 transformants for the anti and syn derivatives, respectively. At the lowest dose used, 181 29120 and 6/22880 transformants were mutant phenotype from the syn- and anti-dihydrodiol epoxides. Although mutants were not fully characterized in this preliminary study, the findings indicated that the two agents could most readily be compared by collecting mutants from DNA exposed to the 10 ng/pg of DNA dose for both compounds. Further transformations for both agents were undertaken, and approximately equal numbers of mutant phenotypes were collected in each case. After retransformation, to detect wild types that had been selected because of a mutation in the bacterial @-galactosidase gene, 95 and 82 mutants, corresponding to mutation frequencies of 23 x and 35 x low4for the syn- and anti-dihydrodiol epoxides, respectively, remained for analysis. Thus, mutation frequency was considerably elevated from background levels (0.1 x in both cases, and the anti isomer was somewhat more mutagenic than the syn isomer in concert with the relative extents of reaction with DNA found in the chemical studies. Analysis of these mutants by sequencing indicated that all of the mutants arising from treatment with the antidihydrodiol epoxide resulted from point mutations and that four of the mutants contained point mutations a t two sites such that 86 point mutations were detected overall. Of the 95 mutants derived from treatment with the syn-dihydrodiol epoxide, 91 were found to be the result of point mutation (four were deletions of two or more base pairs) and 3 of these were double mutations (one of these three was a tandem mutation), leading to a total of 94 point mutations. The types of point mutations found for each diastereomeric dihydrodiol epoxide are summarized as percentages of total mutations in Table 1. It is clear that these dihydrodiol epoxides yielded substantial numbers of mutations at both AT as well as GC pairs. Thus, 72% of the syn diastereomer-induced mutants were at AT pairs, and 45% of the anti diastereomer-induced mutants were a t AT pairs. The latter number contrasts sharply with a 7% value obtained previously for the anti-dihydrodiol epoxide of 5-methylchrysene (191, but the mutation data are consistent with the increased reactivity of the 5,6dimethylchrysene derivatives toward deoxyadenosine residues in DNA (12, Figure 2) compared with the
Mutational Spectra for 5,6-Dimethylchrysene
Chem. Res. Toxicol., Vol. 8, No. 1, 1995 145
anti 5,B-Dimethylchrysene Dihydrodiol Epoxide 110
100
120
130
140
150
170
160
180
I I I I I I I I I S-GGTGGGGTTCCCGAGCGGCCAAAGGGAGCAGACTCTAAATCTGCCGTCATCGACTTCGAAGGTTCGAATCCTTCCCCCACCACCACG-3' A TT ATG G T T T AG G A G AA AGTAAG G T AATTCAA G A AGAT G AG T C G C T G O A G TG T A A A G A A G A G A T G G G G T AG T G A G T G A G G G T T C
syn 5,B-Dlmethylchrysene Dihydrodlol Epoxide 100 110 120 130 140 150 I60 170 180 I I I I I I I I I S-GGTGGGGTTCCCGAGCGGCCAAAGGGAGCAGACTCTAAATCTGCCGTCATCGACTTCG~AGGTTCG~ATCCT~CCCCCACCACC~CG-3' TAT G T T GTCT G CT A A A G A TTT A C T G A T T G A T G GCT G T A A A T A C T A T G A T T o G A A 0 T A T G A G T A T G T G A T A T T G A T C T G C C
X(
0
G G
A
G
Figure 3. Mutational spectra for 5,6-dimethylchrysenedihydrodiol epoxidesin shuttle vector pSP189. indicates a tandem mutation. an insertion, and Table 1. Analysis of Point Mutations (Percentage) Induced in supF Gene by 6,B-Dimethylchrysene Dihydrodiol Epoxides ant9
CG
14 6 41
CG
3
35 14 16 3
2 28 2 3
6 26 0 0
-AT - TA AT transition -AT AT -
transversion GC TA GC
GC
GC
insertion (+1)
deletion (-1) a 94 total mutations. 86 total mutations.
Discussion
predominant reaction with deoxyguanosine reported for the 5-methyl derivative (20,211. Similarly, the greater yield of mutations at AT pairs for the syn- versus the anti-dihydrodiol epoxide parallels the greater propensity of the syn isomer for reaction with deoxyadenosine (Figure 2). Other remarkable features of the data in Table 1 are that each diastereomer induced a large fraction of AT GC transitions. Using the pSP189 and the pS189 shuttle vector [from which pSP189 was derived (2611 with other dihydrodiol epoxides, AT GC changes have always been less than 10% of the total base change mutations in earlier work. Additionally, whereas AT TA transversions were the most prominent single kind of base change for the syn diastereomer, GC TA transversions were the single most frequent mutagenic change encountered for the anti diastereomer. Similar differences between syn and anti diastereomers have been reported for one pair of benzo[clphenanthrene dihydrodiol epoxide diastereomers (15)and for syn- (14) and anti2-benzo[glchrysene dihydrodiol epoxides. The distribution of the mutations through the supF sequence is summarized in Figure 3. For the anti
-
-
-
-
indicates
diastereomer, only three sites (122, 128, and 163) were identified as mutation hotspots (siteswith 2 5 times more mutations than expected from a Poisson distribution), and only 21 out of 86 (24%) of the point mutations were localized in these hotspots. Five hotspots (101,128,140, 180, and 183), accounting for 41 of 94 (44%) of point mutations, were detected for the syn diastereomer, and the mutations for this compound seemed to be less widely distributed through the target gene, therefore. Only one hotspot at 128 was common to both diastereomeric dihydrodiol epoxides.
dihydrodiol epoxide s.yna
base change
G El indicates deletion:
The chemical properties of the 5,6-dimethylchrysene dihydrodiol epoxides are similar to those of fjord regioncontaining hydrocarbons (refs 12 and 13 and Figure 2) in that extensive modification of both adenine and guanine residues in DNA was observed, as reported earlier for the benzo[clphenanthrene derivatives (10 , l l ) and for the syn diastereomer of benzo[glchrysene (14). In contrast, these chemical properties differ from those of the diastereomeric 5-methylchrysene dihydrodiol epoxides for which reaction with deoxyguanosine residues is more predominant (20-22). Although the structures of 5-methyl- and 5,6-dimethylchrysene are obviously very similar, the former accommodates steric crowding in the bay region mainly by in-plane distortions, whereas the buttressing effect of the 6-methyl group forces the dimethyl derivative to accommodate the crowding in the bay region by out-of-plane distortions (23). Thus, it appears that the out-of-plane distortion, which is exhibited also by the fjord region-containing hydrocarbons, is the property that is primarily associated with the increased preference for reaction with deoxyadenosine residues in DNA. The mutational spectra for the diastereomeric syn- and anti-dihydrodiol epoxides of 5,6-dimethylchrysene indi-
* J. Szeliga, J. Page, H. Lee, R. G. Harvey, and A. Dipple, unpublished results.
146 Chem. Res. Toxicol., Vol. 8, No. 1, 1995
Page et al.
cate that the biological effects of these agents parallel their chemical properties in producing mutations a t both AT pairs and GC pairs in DNA. Moreover, the greater mutagenic activity of the anti diastereomer and the greater preference for mutation a t AT pairs for the syn diastereomer follow the chemical findings fairly closely. Overall, the chemical and mutagenic properties of the 5,6-dimethylchrysene dihydrodiol epoxides show that these agents behave much more like dihydrodiol epoxides derived from hydrocarbons containing fjord regions, such as benzo[c]phenanthrene (15)and benzolglchrysene (141, than like those from bay region-containing hydrocarbons, such as benzo[a]pyrene (24, 25) or 7-methylbenz[alanthracene (26,271. In one respect, the substantial fraction of AT GC transition mutations observed in this study (28% and 26% for the syn- and anti-dihydrodiol epoxides, respectively), the 5,6-dimethylchrysene dihydrodiolepoxides are unusual. Under the same conditions as this present study, syn-benzo[g]chrysene dihydrodiol epoxide gave only 10% AT GC transitions (14) and the anti diastereomer gave only 4%.2 Our previous work with benzo[clphenanthrene derivatives (15) used optically active derivatives, which does not prevent direct comparison (because the racemates contain both enantiomers in equal amounts), but also this work was done in the related but not identical plasmid, pS189. Recent studies have shown that the positions of mutational hotspots are sensitive to changes in sequence at sites remote from the hotspots (28,29) although effects on the types of mutation seen were not large (28). The yields of AT GC transitions for the four benzo[clphenanthrene configurational isomers were 2%) 3%, 8%,and 9%. The mutation data for the 5,6-dimethylchrysene derivatives in the supF gene clearly distinguish them from anti-dihydrodiol epoxides derived from planar hydrocarbons with no steric hindrance in the bay region (no data are available for syn diastereomers) because these generate base change mutations almost exclusively a t GC pairs, Examples of these latter agents would be the dihydrodiol epoxides from benzo[a]pyrene [90% mutations a t GC pairs (24)], 5-methylchrysene [93%mutations a t GC pairs (19)], and 7-methylbenz[a]anthracene 195% mutations a t GC pairs (26)l. The present data (24% and 57% of mutations a t GC pairs for the syn- and antidihydrodiol epoxides, respectively) are close to the range of findings for dihydrodiol epoxides derived from hydrocarbons that contain fjord regions and are, therefore, distorted from planarity. These agents are more targeted toward adenine residues, and therefore, the fraction of base change mutations arising from changes a t GC pairs is lower and ranges from 32% to 73% for the four optically active benzo[c]phenanthrene dihydrodiol epoxides (15) and is 51% for the racemic syn-benzo[g]chrysene dihydrodiol epoxide (14). Overall, the current findings suggest that the 5,6dimethylchrysene derivatives can be categorized with the dihgdrodiol epoxides from fjord region-containing hydrocarbons with respect to their mutational targeting of both AT and GC pairs in DNA. This targeting is associated, therefore, with the distortions from planarity common to both fjord region (30)and certain bay region methylsubstituted hydrocarbons (23)rather than with a structural feature, such as a bay region methyl group. Earlier, deformation from planarity has been identified as the key factor favoring reaction with deoxyadenosine in DNA (10,
-
-
-
12,21), and the present findings are consistent with this view.
Acknowledgment. Research sponsored in part by the National Cancer Institute, DHHS, under Contracts N01-CP-21115 with the American Health Foundation and N01-CO-46000 with Al3L. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
References (1) Dipple, A., Moschel, R. C., and Bigger, C. A. H. (1984) Polynuclear aromatic carcinogens. In Chemical Carcinogens (Searle, C. E., Ed.) pp 41-163, American Chemical Society, Washington. (2) Thakker, D. R., Yagi, H., Levin, W., Wood, A. W., Conney, A. H., and Jerina, D. M. (1985) Polycyclic aromatic hydrocarbons: metabolic activation to ultimate carcinogens. Bioact. Foreign Compd. 7,177-242. (3) Guengerich, F. P. (1992) Metabolic activation of carcinogens. Pharmacol. Ther. 54, 17-61. (4) Jerina, D. M., Chadha, A,, Cheh, A. M., Schurdak, M. E., Wood, A. W., and Sayer, J. M. (1990) Covalent bonding of bay-region diol epoxides to nucleic acids. Adu. Exp. Med. Biol. 283,533553. (5) Dipple, A. (1994) Reactions of polycyclic aromatic hydrocarbons with DNA. In DNA Adducts: Identification and Biological Significance (Hemminki, K, Dipple, A., Segerback, D., Kadlubar, F. F., Shuker, D., and Bartach, H., Eds.) IARC Scientific Publications, Lyon. (6) Jerina, D. M., Sayer, J. M., Agarwal, S. K., Yagi, H., Levin, W., Wood, A. W., Conney, A. H., Pruess-Schwartz, D., Baird, W. M., Pigott, M. A., and Dipple, A. (1986)Reactivity and tumorigenicity of bay-region diol epoxides derived from polycyclic aromatic hydrocarbons. In Biological Reactive Intermediates IZZ (Kocsis, J. J., Jollow, D. J., Witmer, C. M., Nelson, J. O., and Snyder, R., Eds.) pp 11-30, Plenum Press, New York. (7) Amin, S., Desai, D., and Hecht, S. S. (1993) Tumor-initiating activity on mouse skin of bay region diol-epoxides of 5,6-dimethylchrysene and benzo[clphenanthrene. Carcinogenesis 14,20332037. (8) Dipple, A., Peltonen, K., Cheng, S. C . , Ross, H., and Bigger, C. A. H. (1994) Chemical and mutagenic specificities of polycyclic aromatic hydrocarbon carcinogens. In Diet and Cancer: Markers, Prevention, and Treatment (Jacobs, M. M., Ed.) pp 101-112, Plenum Press, New York. (9) 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 cultwes results in extensive substitution of both adenine and guanine residues. Cancer Res. 43, 41324135. (10)Dipple, A., Pigott, M. A., Aganval, S. K., Yagi, H., Sayer, J. M., and Jerina, D. M. (1987) Optically active benzo[clphenanthrene diol epoxides bind extensively to adenine in DNA. Nature 327, 535-536. (11) Agarwal, S. K., Sayer, J . M., Yeh, H. J. C., Pannell, L. IC,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-diol1,2epoxides. J. Am. Chem. SOC.109, 2497-2504. (12) Misra, B., Amin, S., and Hecht, S. S. (1992) Dimethylchrysene diol epoxides: Mutagenicity in Salmonella typhimurium, tumorigenicity in newborn mice, and reactivity with deoxyadenosine in DNA. Chem. Res. Toxicol. 6, 248-254. (13) Misra, B., Amin, S., and Hecht, S. S. (1992)Metabolism and DNA binding of 5,6-dimethyIchrysene in mouse skin. Chem. Res. Toxicol. 5, 242-247. (14) Szeliga, J., Lee, H., Harvey, R. G., Page, J . E., Ross, H. L., Routledge, M. N., Hilton, B. D., and Dipple, A. (1994) Reaction with DNA and mutagenic specificity of syn-benzoLg1chrysene ll,l2-dihydrodiol13,14-epoxide.Chem. Res. Toxicol. 7,420-427. (15) Bigger, C. A. H., St.John, J., Yagi, H., Jerina, D. M., and Dipple, A. (1992) Mutagenic specificities of four stereoisomeric benzo[clphenanthrene dihydrodiol epoxides. Proc. Natl. Acad. Sci. U S A . 89, 368-372. (16)Parris, C. N., and Seidman, M. M. (1992) A signature element distinguishes sibling and independent mutations in a shuttle vector plasmid. Gene 117,1-5.
Mutational Spectra for 5,6-Dimethylchrysene (17) Routledge, M. N., Wink,D. A., Keefer, L. K., and Dipple, A. (1993) Mutations induced by saturated aqueous nitric oxide in the pSP189 supF gene in human Ad293 and E. coli MBM7070 cells. Carcinogenesis 14, 1251-1254. (18) Bigger, C. A. H., Strandberg, J., Yagi, H., Jerina, D. M., and Dipple, A. (1989) Mutagenic specificity of a potent carcinogen, benzo[cluhenanthrene (4R,3S)-dihvdrodio1(2S.lR)e~oxide, which reacts 4 t h adenine and guanine in DNA. PAC.Ndtl. Acad. Sci. U S A . 86,2291-2295. (19) Bieeer. C. A. H.. Flickineer. D. J.. Strandbere. J.. Pataki. J.. Harvey, R. G., and Dipple~A.'(1990jMutational-apecificityof the anti 1,2-dihydrodiol 3,4-epoxide of 5-methylchrysene. Carcinogenesis 11, 2263-2265. (20) Melikian, A. A., Amin, S., Hecht, S.S., Hoffmann, D., Pataki, J., and Harvey, R. G. (1984) Identification of the major adducts formed by reaction of 5-methylchrysene anti-dihydrodiol-epoxides with DNA in vitro. Cancer Res. 44,2524-2529. (21) Reardon, D. B., Prakash, A. S., Hilton, B. D., Roman, J. M., Pataki, J., Harvey, R. G., and Dipple, A. (1987) Characterization of 5-methvlchrvsene-l.2-dihvdrodiol-3,4-e~oxide-DNA adducts. . Carcinogekesis"8, 1317-1322. (22) Peltonen, IC, Hilton, B. D., Pataki, J., Lee, H., Harvey, R. G., and Dipple, A. (1991) Spectroscopic characterization of syn-5methylchrysene 1,2-dihydrodi013,4-epoxid~-deoxyribonucleoside adducts. Chem. Res. Toxicol. 4, 305-310. (23) Zacharias, D. E., Kashino, S.,Glusker, J. P., Harvey, R. G., Amin, S., and Hecht, S. S. (1984) The bay-region geometry of some 5-methylchrysenes: steric effects in 5,6- and 5J2-dimethylchrysenes. Carcinogenesis 6, 1421-1430. (24) Yang, J.-L., Maher, V. M., and McCormick, J. J. (1987) Kinds of mutation formed when a shuttle vector containing adducts of
Chem. Res. Toxicol., Vol. 8, No. 1, 1995 147 (+/-)-7~,8a-dihydroxy-9u,10u-epoxy-7,8,9,10-tetrahydrobenzor a 1 pyrene replicates in human cells. Proc. Natl. Acad. Sci. U S A . 84,3787-3791. (25) Cheng, S. C., Hilton, B. D., Roman, J. M., and Dipple, A. (1989) DNA adduds from carcinogenic and noncarcinogenic enantiomers of benzo[alpyrene dihydrodiol epoxide. Chem. Res. Toxicol. 2, 334-340. (26) Bigger, C. A. H., Flickinger, D. J., St. John, J., Harvey, R. G., and Dipple, A. (1991)Preferential mutagenesis at GGC base pairs by the anti 3,4-dihydrodiol 1,a-epoxide of 'I-methylbenz[a]anthracene. Mol. Carcinog. 4, 176-179. (27) Peltonen, K., Cheng, S. C., Hilton, B. D., Lee, H., Cortez, C., Harvey, R. G., and Dipple, A. (1991) Effect of bay region methyl group on reactions of anti-benz[alanthracene 3,4-dihydrodiol1,2epoxides with DNA. J. Org. Chem. 56, 4181-4188. (28) Courtemanche, C., and Anderson, A. (1994) Shuttle-vector mutagenesis by aflatoxin B1 in human cells: Effects of sequence context on the supF mutational spectrum. Mutat. Res. Fundam. Mol. Mech. Mutagen. 306,143-151. (29) Parris, C. N., Levy, D. D., Jessee, J., and Seidman, M. M. (1994) Proximal and distal effects of sequence context on ultraviolet mutational hotspots in a shuttle vector replicated in xeroderma cells. J. Mol. BWl. 236,491-502. (30) Hirshfeld, F. L. (1963) The structure of overcrowded aromatic compounds. Part VII. Out-of-plane deformation in benzo[clphenanthrene and 1,12-dimethylbenzo[clphenanthrene.J.Chem. SOC., 2126-2135.
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