Chem. Res. Toxicol. 1995,8, 1014-1019
1014
Characterization of DNA Adducts Formed by anti-Benzo[g]chrysene 11,12=Dihydrodiol13,14-Epoxide J a n Szeliga,f John E. Page,' Bruce D. Hilton,$ Alexander S. Kiselyov,§ Ronald G. Harvey,§ Yuriy M. Dunayevskiy,Il Paul Vouros,II and Anthony Dipple*>' Chemistry of Carcinogenesis Laboratory, ABL-Basic Research Program, and Chemical Synthesis and Analysis Laboratory, SAIC Frederick, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, Ben May Institute, University of Chicago, Chicago, Illinois 60637, and Department of Chemistry and Barnett Institute, Northeastern University, Boston, Massachusetts 02115 Received March 20, 1995@ The anti-11,12-dihydrodiol 13,14-epoxide of benzo[g]chrysene, a fjord-region-containing hydrocarbon, was found to react with DNA in vitro to yield, as the major product, a n adduct in which the epoxide of the llR,12S,13S,14R enantiomer was opened trans by the amino group of deoxyadenosine. The structures of this adduct and other deoxyadenosine and deoxyguanosine adducts were established by spectroscopic methods. In reactions with deoxyguanylic acid, a product tentatively identified as a 7-substituted guanine was also detected. The mutagenic properties of this dihydrodiol epoxide in shuttle vector pSP189 showed that mutation at AT pairs accounted for 39% of base change mutations whereas chemical findings indicated that about 60% of adducts formed in calf thymus DNA involved adenines. Since calf thymus DNA is 56% AT and the target supF gene is 41% AT, the findings represent a fairly close relationship between adduct formation and mutagenic response. Overall, the chemical and mutagenic selectivities for the two purine bases in DNA were similar, though not identical, to those for the only other fjord-region-containing hydrocarbon studied in depth, i.e., benzo[clphenanthrene. A major difference for these two hydrocarbon derivatives, however, is that benzo[clphenanthrene dihydrodiol epoxides react to much higher extents ( d - f o l d ) with DNA than did the benzoklchrysene derivative.
Introduction Polycyclic aromatic hydrocarbons, formed by incomplete combustion of organic matter, are known environmental carcinogens. Metabolism plays an important role in the carcinogenic action of most carcinogens ( I ) , and for the hydrocarbons, dihydrodiol epoxides in which the epoxide is located in a bay or fiord region are the metabolites most frequently associated with carcinogenic properties (2, 3). The DNA reactions of a number of hydrocarbon dihydrodiol epoxides have been studied, and in general, it has been found that the amino groups of the purine bases in DNA are the major sites of reaction ( 4 , 5 ) . It has also been noted that, whereas guanine residues are the principal site of reaction for dihydrodiol epoxides derived from planar hydrocarbons such as benzo[a]pyrene (6-8), adenine residues are targeted as, or more, effectively than guanine residues when the reactive metabolite is derived from a nonplanar hydrocarbon such as 7,12-dimethylbenz[a]anthracene(9, 10). Nonplanarity can derive from the presence of a fjord region or from the presence of a methyl substituent in a bay region, but DNA adducts for only a limited number of each of these structural types have been studied so far. Benzo[clphenanthrene represents the one fjord region hydrocarbon for which DNA adducts from both syn- and anti-dihydrodiol epoxides have been fully characterized (11,12). Recently, we have reported the DNA ABL-Basic Research Program. Cancer Research and Development Center. 5 University of Chicago. 11 Northeastern University. Abstract published in Advance ACS Abstracts, August 1, 1995.
* NCI-Frederick +
@
adducts derived from syn-benzo[g]chrysene 11,12-dihydrodiol13,14-epoxide (13), a structure very similar to that of benzo[c]phenanthrene dihydrodiol epoxide except that it carries an extra benzene ring. In the present work, we report the DNA adducts derived from the anti diastereomer of benzo[g]chrysene dihydrodiol epoxide so that there are now two examples of adduct characterization from both diastereomers of a fjord region dihydrodiol epoxide, Both of these diastereomeric dihydrodiol epoxides of benzo[g]chrysene have been found to be potent mutagens in bacterial and in mammalian cells (14),and both form adducts in mammalian cells, as determined by postlabeling (15). Benzo[g]chrysene is present in a standard reference of an air particulate sample (161, and it has been classified as a carcinogen with moderate carcinogenic activity (reviewed in ref 2).
Materials and Methods Materials. Racemic anti-benzo[glchrysene 11,12-dihydrodiol 13,14-epoxide ( 1 7 )was synthesized as described elsewhere (27). C-18 Sep-Pak cartridges were purchased from Waters (Milford, MA). All HPLC solvents, calf thymus DNA, deoxyribonucleotides, and enzymes were obtained commercially and used without further purification. Adduct Formation and Isolation. Reactions with calf thymus DNA and with each deoxyribonucleotide were done exactly as described previously for syn-benzoklchrysene dihydrodiol epoxide (13). Tetraol extraction, enzymatic conversion of reaction products to carcinogen-deoxyribonucleoside adducts, and isolation of these adducts were essentially as described previously (18). In DNA digestions, the ratio of venom phos-
0893-228x/95/2708-1014$09.00/00 1995 American Chemical Society
Benzo[g]chrysene -DNA Adducts
Chem. Res. Toxicol., Vol. 8, No. 8,1995 1016
Scheme 1. Structures of Adducts of Racemic anti-Benzo[glchrysene 11,12-Dihydrodiol 13,14-Epoxide and Purine Deoxyribonucleosides That Are Generated upon Reaction with DNA or Purine Deoxyribonucleotides
A. DNA
A
8. dGMP
C. dAMP
p /
HO
4 OH
OH
(S)Gc
I DNA OT Nucleotide 2 Enzymatic Hydrolysis
Ho)-4& HO
10
20
30
40
50
60
Time (min) Figure 1. HPLC separations in 24% acetonitrile of products of reaction of anti-benzolglchrysene dihydrodiol epoxide with DNA (A), deoxyguanylic acid (B), and deoxyadenylic acid (C).
phodiesterase to DNA was twice that previously found necessary to fully digest 7,12-dimethylbenz[alanthracene-modified DNA
(19).
Tetraol and Adduct Separation. All deoxyribonucleoside adducts from reactions with DNA or nucleotides were separated chromatographically on a Beckman Ultrasphere ODS column eluted with acetonitrile/water systems (5 pm, 4.6 x 250") using a Hewlett-Packard Model 1090 Series I1 high pressure liquid chromatograph equipped with a diode array detector. Tetraols were recovered from the organic extract and were separated isocratically with 30% acetonitrile in water to yield a major trans-opened tetraol a t 13 min and a minor cis-opened tetraol at 20 min. Initial comparisons of adducts derived from DNA with those from deoxyribonucleotides were run in 24% acetonitrile. For adduct identification, sufficient amounts of adduct were pooled from many separate HPLC runs. The four deoxyadenosine adducts were separated isocratically with 24% acetonitrile, and retention times were 26, 29, 41, and 47 min. The deoxyguanosine adducts were separated with 20% acetonitrile to yield four peaks a t 48,53,66, and 74 min. The pooled peak at t~ 66 min was separated into two components with 14% THF a t 40 "C ( t 47 ~ and 53 min). After acquiring CD and mass spectra, all adducts were acetylated (13)and then purified on a normal-phase DuPont Zorbax SIL column (5 pm, 4.6 x 250") using 5% ethyl acetate in dichloromethane for tetraol tetraacetates, using 90% methanovethyl acetate/dichloromethane (1: 15:84) for adducts from deoxyadenylic acid and (5:14:81) for adducts from deoxyguanylic acid. Adduct and Tetraol Characterization. All spectroscopic data were obtained with adducts from reactions with deoxyribonucleotides. Ultraviolet absorption spectra were recorded online with a diode array detector or off-line using a Milton Roy spectronic 3000 diode array spectrophotometer. Circular dichroism spectra were measured on a JASCO Model J500A spectropolarimeter and normalized t o 1.0 absorbance unit at A,, Le., 264 nm.
Proton NMR spectra of acetylated tetraol and acetylated adducts were obtained at 500 MHz in acetone-de with a Varian VXR-500s spectrometer using an indirect detection 2.5 mm probe (Nalorac, Inc., Martinez, CA). Proton spectra of two trans deoxyguanosine adducts were recorded at both 27 and -20 "C, whereas spectra for the four deoxyadenosine adducts and the trans-tetraol were recorded at -20 "C because resolution was considerably improved (13). Electrospray ionization mass spectra were obtained using a FisonsNG Instruments (VG Analytical, Altrincham, U.K.) QUATTRO triple quadrupole mass spectrometer. Collision induced dissociation (CID) mass spectra were acquired a t -40 eV collision energy, 1 mTorr Ar gas. Mutation Studies. The mutational specificity studies with the shuttle vector pSP189 were done exactly as described for the syn-benzoklchrysene dihydrodiol epoxide diastereomer (13).
Results Based on findings for other dihydrodiol epoxides, the reactions of racemic anti-benzo[glchrysene 11,12-dihydrodiol 13,14-epoxide with DNA and its four deoxyribonucleotide components were expected to yield primarily, after conversion to deoxyribonucleoside adducts, two cisand two trans-opened products from each purine nucleoside. These result from opening of the epoxide ring of each benzo[glchrysene dihydrodiol epoxide enantiomer at the benzylic CI4position by the purine exocyclic amino groups (Scheme 1). Chromatographic comparisons of the adducts generated in DNA with those from the four deoxyribonucleotides (all run in 24% acetonitrile in water) showed no detectable level of deoxycytidine and thymidine adducts in the nucleotide or DNA reactions, so that adducts derived from deoxyadenosine and deoxyguanosine accounted for all the DNA adducts found (Figure 1). Only two of the four possible deoxyadenosine
1016 Chem. Res. Toxicol., Vol. 8, No. 8, 1995
Szeliga et al. A. dGuo -trans cis
A.
- ..
3
c?
B
e
w
s
U
I 10
20
30
50
40
60
70
80
90
B. dAdo -trans cis
---
H
e s:
2
10
20
30
40
50
Time (min)
Figure 2. HPLC separation of products from reaction of antibenzo[g]chrysene dihydrodiol epoxide with deoxyguanylic acid eluted in 20% acetonitrile (A). The peak at 66 min was pooled and rechromatographed in 14% tetrahydrofuran (B).
adducts ((S)At and (R)At) were found to be present in substantial amounts in the DNA, and only three of the deoxyguanosine adducts ((RIGc, (R)Gt, and (S)Gt) represented substantial contributors to DNA adduct formation. The reaction with deoxyguanylic acid yielded a product eluting between the two major adducts that has been tentatively identified as a 7-substituted guanine (7G) adduct. In the figures and text, adducts have been labeled to indicate absolute stereochemistry at C14 (Ror S),the deoxyriboside attached through its amino group to C14 [i.e., deoxyguanosine (GIor deoxyadenosine (A)], and the relationship between the purine nucleoside and hydroxyl group on C13 [i.e., cis (c) or trans (t)l. The justification for these assignments is found later on in the text. In order to characterize these anti-benzo[glchrysene dihydrodiol epoxide adducts, it was necessary to separate substantial quantities of material to permit spectral analyses. The deoxyguanosine adduct separation shown in Figure 1presented difficulties in this regard. Changing elution solvent to 20% acetonitrile in water (Figure 2A) allowed a clean separation of three of the expected deoxyguanosine adducts, but two adducts were eluted together at t~ -66 min. This latter peak was resolved into two major components by further chromatographic purification using 14%tetrahydrofuran in water (Figure 2B). The U V absorption spectra of the deoxyribonucleoside adducts and tetraol obtained in these studies (Figure 3) allowed distinction to be made between the tetraol and the adducts. The spectra for the deoxyguanosine and deoxyadenosine adducts also differed slightly. The spectrum of 7-G (not shown) was similar to that of the deoxyguanosine adducts. CD spectra for deoxyribonucleoside adducts, normalized to 1 absorbance unit a t
250
230
290
270
330
310
Wavelength (nm) Figure 3. W absorption spectra of the amino-substituted deoxyribonucleoside adducts of anti-benzo[g]chrysene dihydrodiol epoxide for deoxyguanosine (A) and deoxyadenosine (B)and for the corresponding trans-tetraol (C). 20
-20
40
1
-
I
,
I
1
1
-40
230
260
290
320
350
230
260
290
320
350
Wavelength (nm)
Figure 4. Circular dichroism spectra in methanol for benzoIglchrysene-deoxyribonucleoside adducts. The corresponding structures are given in Scheme 1.
264 nm (Figure 41, could be paired on the basis of mirror image relationships to each other. For a given purine
Chem. Res. Toxicol., Vol. 8, No. 8, 1995 1017
Benzo[g]chrysene-DNA Adducts
Table 1.1HNMR Data for Acetates of DeoxyribonucleosideAdducts and Tetraols
trans-tetraol (R)Gta (S)Gta O1
6.41 (511~2= 6.9) 6.41 ( 5 1 1 = ~ 6.8) ~
7.54 (J14,NH = 9.1) 6.60 6.62 6.69 6.32 ( 5 1 4 , ~=~4.6) 6.38
5.87 (512~3= 2.9) 5.81 ( 5 1 2 ~ 3= 2.5)
7.01
6.85 7.15
Spectra were determined at 27 "C. All others were at -20 "C.
nucleoside, each pair of CD spectra is derived from two cis or from two trans adducts, consistent with the fact that the hydrocarbon moieties would have an enantiomeric relationship in such pairs. NMR spectra were recorded for peracetylated adducts after purification by normal-phase HPLC. The yields of the two cis adducts of deoxyguanosine and of the cistetraol were insufficient to allow useful NMR data to be obtained, but the data collected for the other six nucleoside adducts and the truns-tetraol are summarized in Table 1. The methine proton assignments were based on the proton spectra, on COSY experiments, on observations of Overhauser connectivities to distinguish between protons on CI and C14, and on deuterium oxide exchange experiments to identify the NH proton. Confirmation of the assignments was provided by observation of longrange proton J coupling connectivities. The NMR data for the benzo[glchrysene-deoxyribonucleoside adducts were very similar to those derived earlier for benzo[clphenanthrene-deoxyribonucleoside adducts (11). Cis and trans configurations were assigned on the basis of the empirical generalization that, in a cis1 trans pair of compounds, the benzylic proton is always found further downfield in the cis product than in the trans product. Assignments were obvious, therefore, when spectral data were available for both cis and trans isomers. Because the chemical shifts were so similar for these adducts and the benzo[clphenanthrene adducts, assignments were also obvious when data for only one isomer were obtained. As for the analogous benzo[clphenanthrene adducts, the coupling constants (Table 1) were similar for both cis and trans isomers. The NMR data for acetylated 7-G suggested the absence of deoxyribose protons, but signal to noise was too low for confident peak assignment. Full scan mass spectra using positive ion electrospray ionization of unacetylated WAC,(S)At, WGc, and (R)Gt gave the anticipated protonated ion peaks (M H)+ (mlz 580 for the deoxyadenosine adducts and mlz 596 for deoxyguanosine adducts). Structurally informative product ion spectra were obtained when the protonated molecule was subjected to CID (Figure 5). The product ion spectra did not distinguish between cis and trans isomers but did reveal fragments resulting from the loss of deoxyribose (loss of 116 daltons) at mlz 464, as well as fragments at m l z 265,293, and 311. The latter three ions are characteristic of benzolglchrysene and, therefore, were present in both the deoxyadenosine and deoxyguanosine adduct spectra. The fragment at mlz 252 in Figure 5 corresponds in mass to protonated deoxyadenosine. The spectrum of the peracetylated 7-G adduct, formed in reactions with deoxyguanylic acid, showed an (M H)+of mlz 648.5. The CID spectrum of this ion exhibited fragments whose masses were consistent with those
+
+
OH
Figure 5. CID mass spectrum of the parent ion m l z 580 of @)At.
expected for the tetraacetyl derivative of a 7-substituted guanine. For example, the fragment at mlz 194 corresponds in mass to a protonated N-acetylguanine, while major fragments at mlz 395, 353, 311, and 293 reflect the presence of the acetylated hydrocarbon residue. In line with this plausible assignment, no loss of a sugar group from mlz 648.5 was observed. For the deoxyribonucleoside adducts, absolute configuration at the site of nucleoside attachment to the hydrocarbon residue was established through comparison of CD spectra for these adducts and for benzo[c]phenanthrene dihydrodiol epoxide adducts (11). Thus, S configuration was assigned to adducts with a positive CD band in the 260-270 nm region and a negative band in the 280-290 nm region and R configuration to those for which the sign of these bands was reversed. Once assignments of structure and stereochemistry had been made, the relative amounts of individual adducts formed in reactions with DNA and nucleotides could be estimated from the areas under the HPLC peaks (Table 2). DNA trapped about 16%of the anti-benzolglchrysene dihydrodiol epoxide, and deoxyadenosine adducts accounted for 60% of total adducts. As found for other hydrocarbons, the (R,S)-dihydrodiol (S,R)-epoxide reacted more extensively with DNA (giving 70% of total products) than its enantiomer. The differences in extents of reaction of each enantiomer with DNA were more substantial than in reactions with nucleotides, presumably because of the chiral nature of the DNA molecule (Table 2). To determine to what extent the chemical findings would be reflected in a biological response, the mutagenic properties of unti-benzolglchrysene dihydrodiol epoxide in the supF gene of the shuttle vector pSP189 were examined. For vector treated at 10 ng of dihydrodiol epoxidelpg of DNA, mutation frequency was 26 x
1018 Chem. Res. Toxicol., Vol. 8, No. 8, 1995
Szeliga et al.
120 130 140 150 160 170 180 I I I I I I I I 6-GGTGGGGTTCCCGAGCGGCCAAAGGGAGCAGACTCTAAATCTGCCGTCATCGACTTCGAAGGTTCGAATCCTTCCCCCACCACCACG-3' CTTT T T T TCAATTTT T T C CA T T T A GAG C A GCG TT TGC A AA AG AG 100
110
I
G T
AT G
TTCA T
T A
A T T A T T G O
T O T
GA AA
A GCT A G T
AGT T
TA A A A
AG AG AT A G A A
T
Figure 6. Distribution of mutations induced by racemic anti-benzo[g]chrysene dihydrodiol epoxide through the supF gene of shuttle vector pSP189. The sequence of the wild-type gene is given, and below that, base change mutations are indicated. 0 indicates a deletion and A indicates a n insertion.
Table 2. Adduct Distributions (S)in Reactions of anti-Benzo[g]chrysenel1,12-Dihydrodiol 13,14-Epoxide with DNA and Nucleotidesa DNAb dihydrodiol epoxide S,R,R,S
R,S,S,R
dGuo cis trans G€--CG (12%) > AT-GC (10%) > GC-AT (7%) > AT-CG (5%). Although substantial numbers of mutations were generated at AT base pairs (39% of total), this was smaller than the fraction of total reaction with deoxyadenosine residues (60%) (see Table 2). The distribution of point mutations through the target gene (Figure 6) was nonrandom (determined using the Poisson distribution) but was relatively even. Only one site (site 176) was a hotspot for mutation (defined as a site where the observed number of mutations exceeded the expected number by a factor of 5 or more), but several other sites had almost as many mutations. No other hydrocarbon dihydrodiol epoxide examined so far has given a hotspot for base substitution at site 176.
Discussion While structural assignment should be considered tentative, the mass spectral data suggest the formation of a 7-substituted guanine adduct in reactions with deoxyguanylic acid. MacLeod et al. (20)have previously described such an adduct formed from a non-bay region dihydrodiol epoxide, and Cheh et al. characterized a 7-substituted guanine derived from benz[a]anthracene (4S,3R)-dihydrodiol(!LR,lS)-epoxide(211. Since our studies were undertaken with a racemic dihydrodiol epoxide, we cannot implicate one specific enantiomer in formation
of the supposed 7-substituted guanine, but it should be noted that DNA strand breaks, a known consequence of substitution on the 7-position of guanine residues, are preferentially generated by the (S,R)-dihydrodiol (R,S)epoxides of benzo[alpyrene and benzo[clphenanthrene (22).
The reaction of the anti-benzo[g]chrysene dihydrodiol epoxide to yield adducts on the amino groups of the purines was the main chemistry observed in reactions with DNA. In comparison with the structurally similar dihydrodiol epoxide of benzo[clphenanthrene, the larger aromatic system of benzolglchrysene led to less extensive reaction with DNA, i.e., 16% of the racemic benzo[g]chrysene dihydrodiol epoxide was trapped by DNA compared with 60% and 75% for the two enantiomeric antidihydrodiol epoxides of benzo[clphenanthrene (23). This finding is consistent with previous suggestions (5, 2 4 ) that increasing size of the aromatic system may favor ionization of the epoxide and preferentially facilitate hydrolysis over reaction with DNA. The fraction of hydrocarbon residues on adenine versus guanine residues in DNA was greater for the R,S,S,R enantiomer than for the S,R,R,S enantiomer (Table 2) as had been found previously for benzo[clphenanthrene dihydrodiol epoxides (12). However, the relative yields of individual adducts were notably different in some cases for these two closely related hydrocarbons. In particular, the trans deoxyguanosine adduct from the S,R,R,S enantiomer was about a 1.4-foldlarger fraction of total aminosubstituted adducts for benzo[glchrysene whereas the cis deoxyadenosine adduct for the same enantiomer represented 19% of these adducts for benzo[clphenanthrene and only 2.5% for benzo[glchrysene. The mutagenicity studies of the anti-benzo[glchrysene dihydrodiol epoxide revealed that mutations at AT pairs accounted for 39% of the base change mutations whereas the chemical findings indicated that about 60% of the adducts formed involved adenines. A similar result was obtained in studies of the syn diastereomer of benzoklchrysene dihydrodiol epoxide (13). Since the chemical studies have been undertaken with calf thymus DNA (56% AT) and the 85 bp target sequence for mutation of supF is 41% AT, there is a rough parallelism between extents of modification and mutation a t adenines. This is similar to previous findings for benzo[clphenanthrene dihydrodiol epoxides (25). A recent study with dihydrodiol epoxides from 5,6-dimethylchrysene indicated that mutation at adenines was more extensive than might be expected based on the extent of reaction at adenines (261, and these dihydrodiol epoxides were also unusual in that substantial fractions of mutations (26% and 28%) were AT-GC transitions. The latter transition accounted for only 10% of base changes in the present study. The distribution of mutations over the target sequence in the present study showed only one hotspot of mutation. Compared with other dihydrodiol epoxides examined in
Chem. Res. Toxicol., Vol. 8, No. 8, 1995 1019
Benzo[g]chrysene -DNA Adducts this system, the distribution of mutations through the supF structural gene was relatively even (Figure 6).
(10) Bigger, C. A. H., Sawicki, J. T., Blake, D. M., Raymond, L. G.,
and Dipple, A. (1983) Products of binding of 7J2-dimethylbenz[alanthracene to DNA in mouse skin. Cancer Res. 43,5647-5651. (11) Aganval, S. K., Sayer, J . M., Yeh, H. J. C., Pannell, L. K., Hilton, Overall, the additional benzene ring in the benzo[glB. D., Pigott, M. A., Dipple, A,, Yagi, H., and Jerina, D. M. (1987) chrysene, versus the benzo[c]phenanthrene structure, Chemical characterization of DNA adducts derived from the configurationally isomeric benzo[clphenanthrene-3,4-diol1,2resulted in a substantial reduction in the fraction of epoxides. J.Am. Chem. SOC. 109, 2497-2504. dihydrodiol epoxide trapped by DNA and a consequent (12) Dipple, A,, Pigott, M. A., Aganval, S. K., Yagi, H., Sayer, J . M., increase in conversion to tetraol. Although there were and Jerina, D. M. (1987) Optically active benzo[clphenanthrene diol epoxides bind extensively to adenine in DNA. Nature 327, some overall similarities in the distribution of reaction 535-536. products with DNA for the reactive derivatives of these (13) Szeliga, J., Lee, H., Harvey, R. G., Page, J. E., Ross, H. L., two hydrocarbons, there were also some substantial Routledge, M. N., Hilton, B. D., and Dipple, A. (1994) Reaction with DNA and mutagenic specificity of syn-benzolglchrysene differences, notably in the reactions of S,R,R,S enanti11,12-dihydrodiol13,14-epoxide. Chem. Res. Toxicol. 7,420-427. omers. In terms of the kinds of mutations generated, (14) Glatt, H., Piee, A., Pauly, K., Steinbrecher, T., Schrode, R., Oesch, GC-TA and AT--TA were the two most prominent kinds R., and Seidel, A. (1991) Fjord- and bay-region diol-epoxides of mutations formed in both the benzolglchrysene and investigated for stability, SOS induction in Escherichia coli, and mutaeenicitv in Salmonella tvDhimurium and mammalian cells. benzo[clphenanthrene cases, but the distribution of Cancer Res. "61, 1659- 1667. mutations through the supF gene was quite different in 15) PhilliDs, D. H., Hewer, A., Seidel, A,, Steinbrecher, T., Schrode, these two cases. Tumorigenicity studies for the benzoR., Oesch, F., and Glatt, H. (1991) Relationship between mutagenicity and DNA adduct formation in mammalian cells for fjord[glchrysene dihydrodiol epoxides have yet to be reported, and bay-region diol-epoxides of polycyclic aromatic hydrocarbons. so the relationship of the in vitro chemical and mutagenChem.-Biol. Interact. 80, 177-186. esis studies for these agents to their in vivo effects cannot 16) Wise, S. A., Benner, B. A., Cheder, S. N., Hilpert, L. R., Vogt, C. R., and May, W. E. (1986) Characterization of the polycyclic yet be evaluated. aromatic hydrocarbons from two standard reference material air particulate samples. Anal. Chem. 58, 3067-3077. (17) Bushman, D. R., Grossman, S. J., Jerina, D. M., and Lehr, R. E Acknowledgment. Research sponsored in part by (1989) Synthesis of optically active fjord-region ll,12-diol 13,14the National Cancer Institute, DHHS, under contracts epoxides and the K-region 9,lO-oxide of the carcinogen benzolglwith ABL, with SAIC Frederick, and by National Instichrysene. J. Org. Chem. 54, 3533-3544. (18) Cheng, S. C., Hilton, B. D., Roman, J. M., and Dipple, A. (1989) tute for Environmental Health Sciences Grant ES 04732 DNA adducts from carcinogenic and noncarcinogenic enantiomers and American Cancer Society CN-22 to R.G.H. and by of benzo[alpyrene dihydrodiol epoxide. Chem. Res. Toxicol. 2, U.S. E.P.A. Grant 820113-01-0 to P.V. The contents of 334-340. this publication do not necessarily reflect the views or (19) Dipple, A., and Pigott, M. A. (1987) Resistance of 7,la-dimethylbenz[alanthracene-deoxyadenosine adducts in DNA to hydrolysis policies of the Department of Health and Human Serby snake venom phosphodiesterase. Carcinogenesis 8,491-493. vices, nor does mention of trade names, commercial (20) MacLeod, M. C., Evans, F. E., Lay, J., Chiarelli, P., Geacintov, products, or organizations imply endorsement by the U.S. N. E., Powell, K. L., Daylong, A., Luna, E., and Harvey, R. G. Government. (1994) Identification of a novel, N7-deoxyguanosine adduct as the major DNA adduct formed by a non-bay-region diol epoxide of benzo[a]pyrene with low mutagenic potential. Biochemistry 33, References 2977-2987. (21) Cheh, A. M., Chadha, A., Sayer, J. M., Yeh, H. J. C., Yagi, H., (1) Gonzalez, F. J., and Gelboin, H. V. (1994) Role of human Pannell, L. K., and Jerina, D. M. (1993) Structures of covalent cytochromes P450 in the metabolic activation of chemical carnucleoside adducts formed from adenine, guanine, and cytosine cinogens and toxins. Drug Metab. Reu. 26, 165-183. bases of DNA and the optically active bay-region 3,4-diol 1,2(2) Dipple, A., Moschel, R. C., and Bigger, C. A. H. (1984)Polynuclear epoxides of benz[alanthracene. J. Org. Chem. 58, 4013-4022. aromatic carcinogens. In Chemical Carcinogens (Searle, C. E., Ed.) (22) Bigger, C. A. H., Cheh, A., Latif, F., Fishel, R., Canella, K. A,, pp 41-163, American Chemical Society, Washington. Stafford, G. A., Yagi, H., Jerina, D. M., and Dipple, A. (1994) DNA (3) Harvey, R. G. (1991) Polycyclic Aromatic Hydrocarbons: Chemstrand breaks induced by configurationally isomeric hydrocarbon istry and Carcinogenicity, Cambridge University Press, Camdiol epoxides. Drug Metab. Rev. 26, 287-299. bridge, England. (23) Jerina, D. M., Sayer, J . M., Aganval, S. R , Yagi, H., Levin, W., (4) Jerina, D. M., Chadha, A,, Cheh, A. M., Schurdak, M. E., Wood, Wood, A. W., Conney, A. H., Pruess-Schwartz, D., Baird, W. M., A. W., and Sayer, J. M. (1990) Covalent bonding of bay-region Pigott, M. A,, and Dipple, A. (1986)Reactivity and tumorigenicity diol epoxides to nucleic acids. Adu. Exp. Med. 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