Chem. Res. Towicol. 1990, 3, 580-586
580
Metabolism and Mutagenicity of Dibenzo[ a ,e Ipyrene and the Very Potent Environmental Carcinogen Dibenzo[ a ,/]pyrene Prabhakar D. Devanesan, Paolo Cremonesi, Janet E. Nunnally, 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 June 11, 1990
Dibenzo[a,l]pyrene (DB[a,l]P) is one of the most potent carcinogens ever tested in mouse skin and rat mammary gland. DB[a,l]P is present in cigarette smoke and, presumably, in other environmental pollutants. Metabolism and mutagenicity studies of this compound compared to the weak carcinogen dibenzo[a,e]pyrene (DB[a,e]P) can provide preliminary evidence on its mechanism of carcinogenesis. The mutagenicity of DB[a,l]P, DB[a,e]P, and benzo[a]pyrene (BP) was compared in the Ames assay with Aroclor-induced rat liver S-9. B P was the strongest mutagen. In strain TA100, DB[a,l]P and DB[a,e]P were marginally mutagenic. In strain TA98 both compounds were mutagenic, and DB[a,l]P induced more than twice as many revertants as DB[a,e]P. T h e mutagenicity of DB[a,l]P does not correlate with its carcinogenicity, since DB[a,l]P is a much stronger carcinogen, but a much weaker mutagen, than BP. T h e NADPH-supported metabolism of DB[a,e]P and DB[a,l]P was conducted with uninduced and 3-methylcholanthrene-induced rat liver microsomes. Metabolites were analyzed by reverse-phase HPLC and identified by NMR, UV, and mass spectrometry. Uninduced microsomes produced only traces of metabolites with either compound. The major metabolites of DB[a,l]P with induced microsomes were DB[a,l]P 8,9-dihydrodiol, DB[a,l]P 11,12-dihydrodiol, 7-hydroxyDB[a,l]P, and a DB[a,l]P dione. The metabolites of DB[a,e]P with induced microsomes were DB[a,e]P 3,4dihydrodiol, 3-hydroxyDB [ a,e]P, 7-hydroxyDB [a,e]P, and 9-hydroxyDB[a,e]P. Some of these metabolites are very useful in assessing possible pathways of activation in the initiation of cancer.
Introduction Dibenzo[a,l]pyrene (DB[a,l]P,' Figure 1) was recently found to be one of the most potent carcinogenic polycyclic aromatic hydrocarbons (PAH) ( I ) . Although toxic to both mice and rats at the single dose tested, it exhibited potent tumor-initiating activity in mouse skin and potent carcinogenicity by direct application in rat mammary gland. In the mouse skin experiment at an initiating dose of 800 nmol, papillomas began appearing 5 weeks after initiation with DB[a,l]P and only 2 weeks after promotion began (promotion was delayed because of a DB[a,l]P-induced erythema). In the mammary experiment, rats were treated by intramammillary injection at eight glands with 4 Mmol of DB[a,l]P per gland. Tumors began appearing 5 weeks after treatment, and by 10 experimental weeks there were seven tumors per rat. In a previous experiment by Masuda and Kagawa (2),which has been largely ignored due to lack of experimental detail, DB[a,l]P was reported to be 200-400 times more carcinogenic than benzo[a]pyrene (BPI, although the data for BP were taken from another, similar experiment ( 3 ) . In contrast, dibenzo[a,e]pyrene (DB[a,c]P)is a very weak tumor initiator in mouse skin and is inactive in rat mammary gland ( 1 ) . DB[a,l]P has been identified in a biologically active fraction of cigarette smoke condensate ( 4 ) . It is also thought to be a component of other environmental pollutants (5-7), but positive identification has not been pursued and analytical data obtained prior to 1968 are incorrect. This compound has not been considered a very * To whom correspondence should be addressed. 0893-228xj90j2703-0580$02.50j o
significant environmental carcinogen for two reasons. First, tumorigenicity tests prior to 1968 incorrectly used the weakly active dibenzo[a,e]fluoranthene instead of DB[a,l]P itself (8,9). Second, analytical data quantitating its presence in cigarette smoke and other environmental hazards have not been pursued. Therefore, the very potent carcinogenic activity of this compound suggests that it may pose a significant risk in cigarette smoke and other environmental pollutants, even if present in very small amounts. One of the major research objectives concerning DB[a,l]P is to elucidate its mechanism of carcinogenssis. As part of this investigation, mutagenicity and metabolism studies have been conducted comparing DB[a,l]P with the isomeric borderline carcinogen DB[a,e]P.
Materials and Methods BP and DB[a,e[P were available in our laboratory and were purified by column chromatography on silica gel eluted with hexane/benzene (82). BP was recrystallized from benzene/hexane (mp 178-179 "C). DB[a,e]P was recrystallized from xylenes (mp 238-239 "C). DB[a,l]P was obtained from the National Cancer Institute Chemical Carcinogen Repository, Bethesda, MD (mp 161-162 "C). The compounds were analyzed by reverse-phase high-pressure liquid chromatography (HPLC) on a Spectra Physics 8100 system with an Altex Ultrashpere ODS 5-pm column and a 60-min linear gradient of 6C-lOO% methanol in water. They Abbreviations: BP, benzo[a]pyrene; DB[a,e]P, dibenzo[a,e]pyrene; DB[a,l]P,dibenzo[a,l]pyrene; HPLC, high-pressure liquid chromatography; MC, 3-methylcholanthrene; MS, mass spectrometry; NMR, nuclear magnetic resonance; PAH, polycyclic aromatic hydrocarbons; 2-D COSY, two-dimensional chemical shift correlation spectroscopy.
0 1990 American Chemical Society
DB[a,e]P and DB[a,l]P:
Metabolism a n d Mutagenicity 3
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F i g u r e 1. Structures of compounds tested. were >99% pure. [3H]DB[a,e]P(sp act. 1860 mCi/mmol) and [3H]DB[a,l]P(sp act. 1340 mCi/mmol) were obtained by general tritiation a t Chemsyn Science Laboratories (Lenexa, KS) and purified by HPLC to >99% purity. Mutagenicity. The compounds were dissolved in DMSO (50.1 mL/plate). Mutagenicity tests were performed in Salmonella typhimurium strains TA98 and TAlOO by using the liquid preincubation method described by Maron and Ames (10). Metabolic activation was supplied by S-9 (9OOOg) preparations from 8-week-old male Sprague-Dawley rats (Sasco, Omaha), either uninduced or induced with Aroclor 1254 (10). The concentration of protein, 1 mg/plate, was selected by assaying 0.5-5 mg of protein/plate with various levels of compound. At levels of 51 mg of protein, the mutagenicity of DB[a,e]P was less, and the mutagenicity of BP and DB[a,l]P was about the same with 1 or 2.5 mg of protein/plate. Positive and negative controls were included in each experiment. Revertant colonies were scored after 48 h a t 37 “C. Data points represent a t least two separate experiments, each run in triplicate. Metabolism. Liver microsomes were prepared from uninduced and 3-methylcholanthrene (MC) induced (100 pmol/kg body weight in olive oil on 2 consecutive days, killed 24 h later) 8week-old male Wistar rats (Eppley Colony) as previously described (11). Reaction mixtures of 1-mL volume containing 1 mg of microsomal protein, NADPH-generating system (0.43 mM NADP, 1.29 mM glucose 6-phosphate, 0.43 units/mL glucose-6-phosphate dehydrogenase), 150 mM KC1, and 5 mM MgCl, in 50 mM Tris-HC1, pH 7.5, were preincubated for 5 min a t 37 “C, and the incubations were continued for 30 min after addition of 80 nmol of [3H]DB[a,e]Por [3H]DB[a,l]P.The reactions were terminated by addition of 1 mL of acetone, and the metabolites were extracted three times with 2 mL of ethyl acetate. Analysis of Metabolites by HPLC. The ethyl acetate extracts were evaporated to dryness under argon and dissolved in methanol/DMSO (1:l). Samples were analyzed by HPLC using a Spectra Physics 8700 system and a YMC-Pack 5-pm ODS-AQ313 column (YMC, Morris Plains, NJ). The column was eluted with 60% methanol in water for 10 min followed by a linear gradient to 100% methanol in 60 min a t a flow rate of 1.2 mL/min. The eluant was monitored for UV absorbance a t 254 nm (Kratos Spectroflow monitor) and for radioactivity with a continuous-flow system using a RAMONA radioactivity detector (IN/US, Fairfield, NJ) with a 2-mL liquid cell. Radioactivity data were processed with an automatic data integration system. Isolation a n d Characterization of Metabolites. Metabolites were isolated from 250-mL incubation mixtures by HPLC as described above, and these pure compounds were used for analysis by UV spectroscopy, mass spectrometry (MS), and nuclear magnetic resonance (NMR). The UV spectra were obtained during HPLC with the methanol/water gradient described above by using a Waters 990 diode array detector (Millipore Corp., Waters Division, Milford, MA). Proton and homonuclear two-dimensional chemical shift correlation spectroscopy (2-D COSY) NMR spectra were recorded on a Varian XTAOO at 299.938 MHz as solutions in DMSO-d,. Chemical shifts (6)are reported relative to tetamethylsilane, which was employed either as a primary internal reference or as a secondary reference relative to residual DMSO at 2.50 ppm. Mass spectra were recorded on a Kratos MS-50 instrument at the Midwest Center for Mass Spectrometry, Department of Chemistry, University of Nebraska-Lincoln. DB[a,e]P a n d Metabolites. DB[a,e]P. U V A,, (nm) 272, 290,302,340,356,372. NMR: d 7.79-7.84 (m, 2 H, 11-H, 12-H), 7.86-7.90 (m, 2 H, 2-H, 3-H), 8.10 (t, 1 H, J = 7.8 Hz, 8-H), 8.39
Chem. Res. Toxicol., Vol. 3, No. 6, 1990 581 (d, 1 H, J = 7.8 Hz, 7-H), 8.44 (d, 1 H, J = 9.3 Hz, 6-H), 8.45-8.49 (m, 1 H, I-H), 8.92-8.95 (m, 1H, lO-H), 9.03 (d, 1 H, J = 7.7 Hz, 9-H), 9.09-9.12 (m, 1 H, 13-H), 9.17-9.20 (m, 1H, 4-H), 9.23 (d, 1 H, J = 9.3 Hz, 5-H), 9.56 (s, 1 H, 14-H). D B [ a , e ] P 3,4-Dihydrodiol. UV: A, (nm) 292, 328, 342. N M R 6 4.33 (d, 1 H, J = 5.5 Hz, 3-H), 5.44 (s, 1H, 4-H), 6.32-6.37 (m, 1 H, 2-H); 7.09 (d, 1 H, J = 8.5 Hz, 1-H), 7.81-7.84 (m, 2 H, 11-H, 12-H),8.09 (t, 1 H, J = 7.6 Hz, 8-H), 8.23 (d, 1 H, J = 8.6 Hz, 6-H), 8.31 (d, 1 H, J = 7.6 Hz, 7-H), 8.48 (d, 1 H, J = 8.6 Hz, 5-H), 8.98 (s, 1 H, 14-H), 8.98-9.09 (m, 3 H, 9-H, 10-H, 13-H). MS: m / z (relative intensity) 336.1139 (6.9, M), in agreement with an elemental formula of C24H1602;318.1037 (100, M - H2O); 289.1022 (47.8, M - CH302). 3-OH-DB[a,e]P. UV: A, (nm) 278,294,306,376,394,416. NMR: b 7.43 (bd, 1 H, J = 8.7 Hz, 2-H), 7.7C-7.79 (m, 2 H, 11-H, 12-H),8.04 (t, 1 H, J = 7.8 Hz, 8-H), 8.30-8.35 (m, 4 H, 1-H, 4-H, 6-H, 7-H), 8.87 (bd, 1 H, J = 8.1 Hz, 10-H), 8.92-8.96 (m, 2 H, 5-H, 9-H), 9.01 (bd, 1 H, J = 7.7 Hz, 13-H), 9.42 (s, 1 H, 14-H). MS: m/z (relative intensity) 318.1039 (100, M), in agreement with an elemental formula of C24H&; 289.1008 (37.5, M - CHO). (nm) 256,276,296,308, 7- a n d 9-OH-DB[a,e]P. UV: A, 360, 380, 406. NMR of 7-OH-DB[a,e]P: b 7.53 (d, 1 H, J = 8.5 Hz, 8-H), 7.64-7.73 (m, 2 H, 11-H, 12-H),7.82-7.86 (m, 2 H, 2-H, 3-H), 8.40-8.43 (m, 1 H, 1-H), 8.63 (d, 1 H, J = 9.3 Hz, 6-H), 8.72-8.75 (m, 1 H, 10-H),8.81 (d,. 1 H, J = 8.5 Hz, 9-H), 8.97-9.00 (m, 1 H, 13-H), 9.10 (d, 1 H, J = 8.5 Hz, 5-H), 9.10-9.13 (m, 1 H, 4-H), 9.41 (s, 1H, 14-H). NMR of 9-OH-DB[a,e]P: b 7.64-7.67 (m, 1 H, 8’-H), 7.77, 7.80 (m, 2 H, 2’-H, 3’-H), 8.15 (d, 1 H, J = 8.7 Hz, 7’-H), 8.22 (d, 1 H, J = 9.1 Hz, 6’-H), 8.36-8.39 (m, 1 H, 1’-H), 8.90 (d, 1 H, J = 9.1 Hz, 5’-H), 9.04-9.07 (m, 1 H, 4’-H), 9.30 (s, 1 H, 14’-H); the chemical shifts of the four additional protons are buried under the chemical shifts of the more abundant isomer, 7-OH-DB[a,e]P. MS: m / z (relative intensity) 318.1041 (100, M), in agreement with an elemental formula of C24H14O; 289.1018 (58.8, M - CHO). DB[a,l]P a n d Metabolites. DB[a,I]P. UV: A, (nm) 238, 270,302,316,336,354,370,388. NMR: b 7.80-7.88 (m, 4 H, 2-H, 3-H, 12-H, 13-H), 8.03 (d, 1 H, J = 9.1 Hz, 8-H), 8.08-8.15 (m, 2 H, 6-H, 9-H), 8.25 (d, 1 H, J = 7.5 Hz, 7-H), 8.41-8.44 (m, 1 H, I l - H ) , 8.76 (s, 1 H, 10-H), 9.06-9.13 (m, 3 H, 1-H, 4-H, 5-H), 9.17-9.20 (m, 1 H, 14-H). DB[a,I]P 8,9-Dihydrodiol. UV: A, (nm) 270 (s), 278,288, 310, 324, 336. NMR: d 4.81 (d, 1 H, J = 8.5 Hz, 8-H), 4.93 (d, 1 H, J = 8.5 Hz, 9-H), 7.63-7.77 (m, 5 H, 2-H, 3-H, 6-H, 12-H, 13-H), 7.86 (d, 1 H, J = 7.2 Hz, 7-H), 8.11-8.14 (m, 1 H, 11-H), 8.17 (s, 1 H, 10-H), 8.73 (d, 1 H, J = 8.3 Hz, 5-H), 8.84-8.90 (m, 3 H, 1-H, 4-H, 14-H). MS: m / z (relative intensity) 336.1140 (38.0, M), in agreement with an elemental formula of CUHl6O2;318.1047 (100, M - H2O); 289.1007 (51.6, M - CH302). DB[a,I]P 11,12-Dihydrodiol. UV: A, (nm) 252,266, 292, 304, 335 (s), 344, 360. NMR: b 4.58-4.63 (m, 1 H, 12-H), 4.72 (d, 1 H, J = 10.5 Hz, 11-H), 6.30 (dd, 1 H, J = 10.1 Hz, J ’ = 2.2 Hz, 13-H), 7.27 (dd, 1H , J = 10.1 Hz, J’= 2.1 Hz, 14-H),7.71-7.80 (m, 2 H, 2-H, 3-H), 8.01-8.17 (m, 3 H, 6-H, 8-H, 9-H), 8.26 (d, 1 H, J = 7.5 Hz, 7-H), 8.43 (s, 1 H, 10-H), 8.53 (d, 1 H, J = 9.1 Hz, 1-H), 8.95-9.01 (m, 2 H, 4-H, 5-H). MS: m/z (relative intensity) 336.1146 (5.2, M), in agreement with an elemental formula of C24H1602;318.1047 (100, M - H@); 289.1015 (43.9, M - CH302). 7-OH-DB[a,I]P. UV: A, (nm) 240,264,306,318,332,376, 392, 414,440. N M R 6 7.60 (d, 1 H, J = 8.6 Hz, 6-H), 7.65-7.77 (m, 4 H, 2-H, 3-H, 12-H, 13-H), 7.96 (d, 1 H, J = 9.3 Hz, 9-H), 8.17 (d, 1 H, J = 9.3 Hz, 8-H), 8.32-8.36 (m, 1 H, 11-H), 8.61 (s, 1 H, IO-H), 8.88-8.95 (m, 3 H, 1-H, 4-H, 5-H), 9.06-9.10 (m, 1 H, 14-H). MS: m / z (relative intensity) 318.1039 (100 M), in agreement with an elemental formula of C&&; 289.1030 (23.1, M - CHO). DB[a,l]P Dione. UV: A, (nm) 244,294,342,426,580. MS: m/z (relative intensity) 332.0830 (100, M), in agreement with an elemental formula of CZ4H12O2;304.0874 (19.2, M - CO); 276.0924 (25.3, M - 2CO).
Results Mutagenicity. The mutagenic activity of BP, DB[a,l]P, and DB[a,e]P in S. typhimurium strains TA98 and TAlOO
582 Chem. Res. Toxicol., Vol. 3, No. 6, 1990
Devanesan et al.
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was found to require metabolic activation. Using Aroclor-induced S-9 preparations for activation and strain TA98, BP at 1 nmol/plate was well above background, and it reached a maximum of almost 20 times background by 12 nmol/plate (Figure 2A). DB[a,l]P was less mutagenic than BP. The low level of activity observed at 1 nmol/ plate increased to a maximum of about 10 times background at 10 nmol/plate. In contrast, the activity of DB[a,e]P slowly increased to about 4-5 times background at 100 nmol/plate, the highest dose tested. When the compounds were assayed in strain TA100, BP showed significant activity at 1 nmol/plate and was 7 times background at 3 nmol/plate (Figure 2B). In this strain, DB[a,l]P and DB[a,e]P exhibited similar activity, rising only to about twice background at 100 nmol/plate. When uninduced S-9 was used for activation, only BP showed slight mutagenicity in strain TA98 and none showed activity in TAlOO (data not shown), although they were tested at levels which gave activity with Aroclor-induced S-9: 1 and 3 nmol/plate for BP, 6 and 30 nmol/ plate for DB[a,l]P, and 20 and 100 nmol/plate for DB[a,e]P. The Aroclor-induced S-9 had 0.48 nmol of cytochrome P-45O/mg of protein, whereas the uninduced S-9 had 0.038 nmol/mg of protein. When the levels of mutagenicity of BP at 3 nmol/plate obtained with 0.5-5 mg
of Aroclor-induced S-S/plate were compared to the same amounts of uninduced S-9, the number of revertants/nmol of cytochrome P-450 was about the same with induced and uninduced S-9. Metabolism. The NADPH-supported metabolism of DB[a,e]P and DB[a,l]P was conducted with uninduced and MC-induced rat liver microsomes. Uninduced microsomes produced only traces of metabolites with either compound (data not shown). With MC-induced microsomes, several metabolites were purified and analyzed by reverse-phase HPLC (Figure 3). Elucidation of the structures was obtained by a combination of UV (Figures 4 and 51, NMR (Figures 6-8), and MS (see Materials and Methods). The major metabolites of DB[a,e]P with induced microsomes (Figure 3A) were the colorless DB[a,e]P 3,4-dihydrodiol (8% yield), the yellow 3-OH-DB[a,e]P (5% yield), and the yellow mixture of 7- and 9-OH-DB[a,e]P (ratio 7525) (5% yield). The structure of the dihydrodiol was deduced from its UV spectrum (Figure 4A), which showed a blue shift with respect to its parent compound, and the mass spectrum (see Materials and Methods), in which the molecular ion peak and its fragmentation are characteristic of an aromatic dihydrodiol. However, unequivocal elucidation of structure was obtained from its NMR spectrum (Figure 6), in which assignments of the various protons were made by applying empirical rules that designate different chemical shifts for protons bound to certain carbon atoms (e.g., a- or 0-naphthalenic, mesoanthracenic, etc.) (12). Support for the assignments was obtained by spin-spin decoupling and 2-D COSY. With saturation of a double bond in the 1,2,3,4-ring, the aromatic moiety of DB[a,e]P 3,4-dihydridiol should display an aromatic NMR spectrum similar to that of benzo[e]pyrene. The angular protons 9,10, 13, and 14 are the ones shifted downfield the most, whereas 0 protons 11 and 12 are shifted the most to the high field. Protons 5 and 6 are easily identified by their coupling constant and 2-D COSY. Proton 8-H is recognized as the characteristic triplet at 8.12
DB[a,e]P and DB[a,l]P:
Chem. Res. Tonicol., Vol. 3, No. 6, 1990 583
Metabolism and Mutagenicity
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ppm and is coupled with 7-H. This metabolic product is identified as DB[a,e]P 3,4-dihydrodiol by the very small coupling constant of 3-H and 4-H, which indicates that these protons are diequatorial. This implies that the two hydroxyl groups are diaxial. The only two hydroxyl groups that assume the axial configuration are the 3 and 4, due to the steric constraint of the angular proton 5-H. The remaining two major metabolites of DB[a,e]P were DB[a,e]P phenols, as determined from the molecular ion and typical fragmentation in the mass spectrum, as well as the bathochromic effect observed in their UV spectra (Figure 4B). The phenol eluting first (Figure 3A) is 3OH-DB[a,e]P. The structure has been clearly elucidated by its NMR spectrum (Figure 7B). The characteristic feature of this spectrum is the lack of the 2-H and 3-H protons at 7.9 ppm seen in the parent compound (Figure 7A). Proton 2-H is now shifted upfield to 7.4 ppm and is coupled with one of the protons in the multiplet a t 8.35 ppm. The proton 4-H is no longer a t 9.2 ppm but is part of the multiplet at 8.35 ppm, where 1-H, 6-H, and 7-H are found. The latter phenol peak in Figure 3A contains a mixture of the two phenols 7- and 9-OH-DB[a,e]P, in a ratio of 75:25, respectively. This is apparent from the two singlets in the lower field corresponding the meso-anthracenic angular 14-H. Assignment of the major phenol as 7-OHDB[a,e]P is based on the absence of the typical triplet corresponding to 8-H in the parent compound and the presence of this proton as a doublet a t 7.50 ppm. This doublet is coupled with 9-H a t 8.83 ppm and exhibits the same coupling constant. With DB[a,l]P the major metabolites obtained with induced microsomes (Figure 3B) were DB[a,l]P 8,9-di-
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Discussion The relative mutagenicity of BP, DB[a,l]P, and DB[a,e]P does not correlate with their carcinogenic potency. In both mouse skin and rat mammary gland, DB[a,l]P is an exceptionally potent carcinogen, whereas DB[a,e]P is almost inactive (ref 1 and unpublished results). DB[a,l]P is only a moderately strong mutagen in strain TA98, although it is clearly more mutagenic than DB[a,e]P (Figure 2A). Both dibenzo[a]pyrenes are weak mutagens in strain TAlOO (Figure 2B). The results for DB[a,e]P are very similar to those reported by LaVoie et ai. (13). Furthermore, BP is a much stronger mutagen than DB[a,l]P in the Ames assay (Figure 2), although the carcinogenic potency of DB[a,l]P far exceeds that of B P (refs. 1-3 and unpublished results). In conclusion, study of the
DB[a,e]P and DB[a,l]P:
Chem. Res. Toxicol., Vol. 3, No. 6,1990 585
Metabolism and Mutagenicity +-I
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of both. One-electron oxidation of DB[a,l]P and DB[a,e]P produces radical cations which specifically react with acetate ions at positions 10 and 14, respectively, as determined by manganic oxidation of the compounds (unpublished results). However, the reaction of DB[a,l]P is quantitative and very efficient, whereas the reaction of DB[a,e]P is slow and incomplete. In terms of the monooxygenation pathway catalyzed by cytychrome P-450, DB[a,l]P produces the potential proximate carcinogen DB[a,l]P 11,12-dihydrodiol, which
corresponds to BP 7,8-dihydrodiol. In contrast, DB[a,e]P does not yield the corresponding proximate dihydridiol, namely, DB[a,e]P 1,2-dihydrodiol. Thus, the results of the metabolism studies reported here for DB[a,e]P and DB[a,l]P and the chemical properties of their radical cations provide critical preliminary data for elucidating the mechanisam of tumor initiation of DB[a,l]P. Studies of the binding of DB[a,l]P to DNA and determination of the structure of DB[a,l]P-DNA adducts formed in vitro and in vivo are anticipated to provide the evidence needed
586
Chem. Res. Toxicol., Vol. 3, No. 6, 1990
to elucidate the mechanism of activation of this very potent carcinogen.
Acknowledgment. We thank Dr. R. Todorovic and Dr. N. V. S. RamaKrishna for their contributions to this research. This research was supported by Public Health Service Grants CA25176 and CA44686 from the National Cancer Institute and by Grants CA36727 from the National Cancer Institute and ACS SIG-16 from the American Cancer Society. Mass spectral determinations were performed at the Midwest Center for Mass Spectrometry at the University of Nebraska in Lincoln, an NSF Regional Instrumentation Facility (Grant CHE-8620177).
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