7,12-Dimethylbenz[a]anthracene-DNA adduct formation in Wistar rat and Syrian hamster embryo cell cultures

Chem.-Biol. Interactions, 56 (1985) 1-12

1

Elsevier Scientific Publishers Ireland Ltd.

7 , 1 2 - D I M E T H Y L B E N Z [ a ] A N T H R A C E N E - D N A A D D U C T FORMATION IN WISTAR RAT AND SYRIAN H A M S T E R EMBRYO CELL C U L T U R E S

SAID M. SEBTI*, T E R E S A A. S M O L A R E K , BAIRD**

C Y N T H I A P. S A L M O N

and W I L L I A M M.

Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, IN47907 (U.S.A.)

(Received May 10th, 1985) (Revision received July 21st, 1985) (Accepted July 23rd, 1985)

SUMMARY

The binding of 7,12-dimethylbenz[a]anthracene (DMBA) to DNA was examined in Syr i a n h a m s t e r and Wistar r a t em bryo cell cultures exposed to DMBA for 5, 24, 48 and 72 h. The level of binding of DMBA to DNA was ab o u t twice as great in the h a m s t e r em b ryo cells as in the r a t e m b r y o cells at all times. Analysis of the DMBA-deoxyribonucleoside adducts by immobilized boronate c h r o m a t o g r a p h y d e m o n s t r a t e d t h a t the ratio of adducts with no cis vicinal h y d r o x y l groups to t hose containing cis vicinal h y d r o x y l groups was m u c h gr eat er in the r a t e m b r y o cells (from 2 . 2 : 1 to 2.9: 1) t h a n in the h a m s t e r e m b r y o cells (from 1.3: I to 1.6: 1). The h a m s t e r e m b r y o cells contained t hr e e m a j o r DMBADE-DNA adducts: based upon their c h r o m a t o g r a p h i c behavior and comparison with the t hree m a j o r DMBA-DNA adducts described by Dipple et al. in mouse e m b r y o cell cultures (Biochemistry, 24 (1985) 2291), two were tentatively identified as resulting f r o m the r eact i on of a n t i - D M B A D E (the isomer of 1,2-epoxy-3,4-dihydroxy1,2,3,4-tetrahydro-DMBA with the epoxide and benzylic h y d r o x y l on the opposite faces of t he molecule) with d e o x y g u a n o s i n e and deoxyadenosi ne and one adduct resulted f r o m r e a c t i on of syn-DMBADE (epoxide and benzylic

*Present address: Department of Pharmacology, Yale University School of Medicine, New Haven, CN, U.S.A. **To whom correspondence should be addressed. Abbreviations: BaP, benzo[a]pyrene; BaPDE, 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroBaP; dA, deoxyadenosine;dG, deoxyguanosine; DMBA, 7,12-dimethylbenz[a]anthracene;antiDMBADE, the isomer of 1,2-epoxy-3,4-dihydroxy-l,2,3,4-tetrahydro-DMBAwith the epoxide and benzylic hydroxyl on opposite faces of the molecule; syn-DMBADE, the isomer of 1,2-epoxy-3,4-dihydroxy-l,2,3,4-tetrahydro-DMBAwith the epoxide and benzylic hydroxyl on the same face of the molecule. 0009-2797/85/$03.30

O 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

2 hydroxyl on the same face of the molecule) with deoxyadenosine. The anti-DMBADE-deoxyguanosine, syn-DMBADE-deoxyadenosine, and antiDMBADE-deoxyadenosine adducts were present in h a m s t e r embryo cell DNA in a ratio of 1.2 : 2 : 1. The Wistar rat embryo cell DNA contained a much larger proportion of the syn-DMBADE-deoxyadenosine adduct. The relative proportions of the three major DMBA-DNA adducts in Syrian hamster embryo cells were similar at all times, but the proportion of syn-DMBADEdeoxyadenosine adduct decreased slightly with time in the rat embryo cells. These results indicate t h a t there are species specific differences in the stereospecificity of activation of DMBA to DNA-binding diol epoxides which parallel those observed for benzo[a]pyrene (BaP). The high proportion of deoxyadenosine adducts suggests t h a t they may have an important role in the induction of biological effects by DMBA.

Key words: 7,12-Dimethylbenz[a]anthracene - 7,12-Dimethylbenz[a]anthracene diol epoxide - 7,12-Dimethylbenz[a]anthracene-DNA adduct 7,12-Dimethylbenz[a]anthracene-deoxyadenosine adduct - Rodent embryo cell cultures - Immobilized boronate chromatography INTRODUCTION Several lines of evidence suggest t h a t the interaction of carcinogenic polycyclic aromatic hydrocarbons such as BaP and DMBA with DNA is a critical step in tumor induction by these compounds [1-3]. The m a j o r BaP-DNA adduct formed in cells and tissues in culture and in vivo after t r e a t m e n t with BaP results from the interaction of the (+)anti isomer (the isomer with the 7-hydroxyl and epoxide on opposite faces of the molecule) of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaPDE) with deoxyguanosine (dG) (reviewed in Ref. 4). Smaller amounts of other adducts such as syn(7-hydroxyl and epoxide on the same face of the molecule)BaPDE-dG and syn- and anti-BaPDE-deoxyadenosine (dA) are also formed in some cells and tissues [4]. The DNA interaction products of DMBA, a more potent carcinogen t h a n BaP, are not as well characterized, for due to the difficulty of synthesizing DMBA diol epoxide, synthetic markers of DMBA-DNA adducts have not yet been reported. Several lines of evidence [5-12] suggest t h a t the 'bay-region' diol epoxide, 3,4-dihydroxy-l,2-epoxy-l,2,3,4-tetrahydro DMBA (DMBADE), is involved in the binding of DMBA to DNA. Recently Dipple et al. [8] found t h a t DMBA was bound to both dA and dG in DNA from DMBA-treated NIH Swiss mouse embryo cell cultures. Sawicki et al. [11] demonstrated by the use of immobilized boronate chromatography and fluorescence spectroscopy t h a t both anti and syn isomers of DMBADE were involved in the binding of DMBA to DNA in NIH Swiss mouse embryo cell cultures. To characterize the binding of DMBA to DNA in rodent embryo cell cultures from other species, we analyzed the DMBA-DNA adducts formed in Wistar rat and Syrian

3 hamster embryo cell cultures after various lengths of time of exposure to DMBA by immobilized boronate chromatography and HPLC [13]. MATERIALS AND METHODS

Treatment o f cells with [3H]DMBA Embryo cell cultures were prepared from Syrian hamsters (13th day of gestation, Engle Laboratory Animals, Inc., Farmersburg, IN), and Wistar rats (17th day of gestation, Harlan Sprague-Dawley, Inc., Indianapolis, IN) as described previously [14]. Cultures were grown to confluence in 175 cm 2 culture flasks (Falcon, Oxnard, CA) containing 50 ml of medium and refed with fresh medium 2 A. A.8h prior to treatment with DMBA. Cultures from hamster embryo cells were grown in minimum Eagle's medium (MEM, Gibco Laboratories, Grand Island, NY): rat embryo cell cultures were grown in basal medium Eagle's (BME, M.A. Bioproducts, Walkersville, MD) [13]. The medium was supplemented with 10% fetal calf serum (Reheis Chemical Co., Phoenix, AZ). Tertiary cultures were treated with [G-SH]DMBA (Amersham, Arlington Heights, IL; final concentration of 0.1 ~g/ml medium and spec. act., 10 Ci/mmol) for the lengths of time stated in Results. Media samples were removed and analyzed by organic solvent extraction and HPLC to determine the amount of DMBA metabolized [15]. Typical values were: hamster embryo cells 5h, 46% metabolized; 24h, 92%; 48h, 99%; 72 h, 100%: rat embryo cells 5h, 44%; 24h, 90%; 48h, 98%; 72h, 100%. The cells were harvested with 0.025% trypsin/0.01% EDTA, washed twice with phosphatebuffered saline and the nuclei isolated as described previously [13,16]. DNA isolation and analysis o f D M B A - D N A adducts The DNA was isolated by proteinase K and RNase A treatment, chloroform/isoamyl alcohol (24: 1) extractions and ethanol precipitations as described previously [17]. The purified DNA was degraded to deoxyribonucleosides with DNase I from bovine pancreas, phosphodiesterase from Crotalus atrox venom and alkaline phosphatase from E. coli [9,13]. The amount of DMBA bound per mg DNA was determined by measuring the amount of DNA by A2so and radioactivity by liquid scintillation counting. The DMBA-modified deoxyribonucleoside adducts were isolated by chromatography on Quik-Sep Sephadex LH-20 columns and analyzed either by HPLC or by immobilized boronate chromatography followed by HPLC [13]. HPLC analyses were carried out on a 25 cm × 4.6 mm Ultrasphere octyl reverse phase column (Beckman Instruments, Inc., St. Louis, MO). Samples were eluted with methanol/water (50 : 50) for 35 min, then for 10 min with a linear gradient of methanol/H20 (50:50 to 55: 45) followed by 24 min at 55: 45 methanol/H20 at a flow rate of 1 ml/min. Twenty 1-min fractions and 160 0.3-min fractions were collected in scintillation vials and counted by liquid scintillation counting. The DMBA-DNA adducts that contained cis vicinal hydroxyl groups were separated from the other DMBA-DNA adducts by chromatography on a column of [N-[N'-[m-(dihydroxyboryl)phenyl]-

succinamyl]amino]ethyl cellulose [ 11] as described by Pruess-Schwartz et al. [ 13]. The DMBA-deoxyribonucleoside adducts that did not contain cis vicinal hydroxyl groups were eluted with 1 M morpholine buffer at pH 9. Those with cis vicinal hydroxyl groups were then eluted in 1 M morpholine buffer at pH 9 containing 10% sorbitol. The adducts in the morpholine buffer fractions and in the sorbitol-morpholine buffer fractions were pooled, concentrated by Quick-Sep Sephadex LH-20 chromatography and the individual D M B A - D N A adducts were analyzed by reverse-phase HPLC as described above. RESULTS

Effect o f the length o f time o f exposure on D M B A - D N A binding levels Cultures of Wistar rat embryo and Syrian hamster embryo cells were exposed to [aH]DMBA for 5, 24, 48 and 72 h. The DNA was then isolated, degraded to deoxyribonucleosides and the amount of DMBA bound per mg of DNA was determined as described in Materials and Methods. In rat embryo cells the level of binding increased between 5 and 2 4 h from 12.4 to 3 4 . 4 p m o l / m g D N A , remained at 36.1pmol/mg at 4 8 h and decreased to 23.6 pmol/mg at 72 h. The levels of binding in hamster embryo cells were higher than those in rat embryo cells. After 5 h of exposure, there were 37.6 pmol of DMBA bound per mg of hamster embryo cell DNA, a value similar to that of the highest level of binding observed in rat embryo cells. After 24 h of exposure to DMBA, the level of binding in hamster embryo cells reached a maximum value of 74.4 pmol/mg DNA and decreased to 60 pmol/mg after 4 8 h and 56pmol/mg at 72h. This difference between the rat and hamster embryo cells was not due to the a m o u n t of DMBA metabolized, for both cells metabolized more than 90% by 24 h. Thus, the level of binding of DMBA to DNA was dependent upon the species of origin of the cells as well as the length of time of exposure to DMBA. Analysis o f the D M B A - D N A adducts in rat and hamster embryo cells To determine the individual D M B A - D N A adducts present in these cells, the DNA samples from the cultures exposed for 24 h were degraded enzymatically to deoxyribonucleosides. The DMBA-deoxyribonucleoside adducts were separated from unreacted deoxyribonucleosides by chromatography on Sephadex LH-20 and an aliquot was analyzed by reverse-phase HPLC as described in Materials and Methods. HPLC column elution profiles of the D M B A - D N A adducts isolated from rat and hamster embryo cells are shown in Fig. 1 (A and B, respectively). In rat embryo cells (Fig. 1A) the HPLC profile demonstrated the presence of one m a j o r and several minor adduct peaks. In contrast, the elution profile from hamster embryo cells (Fig. 1B) was more complex and there were at least 3 maj or and several minor adduct peaks. To characterize these adducts the remainder of the DMBAdeoxyribonucleosides were separated into adducts that contained cis vicinal hydroxyl groups (e.g. anti-DMBADE-DNA adducts) and adducts that did not contain cis vicinal hydroxyls (e.g. syn-DMBADE-DNA adducts) by immobil-

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FRACTION NUMBER Fig. 1. HPLC column elution profiles of DMBA-DNA adducts isolated from Wistar rat (A,C,E) and Syrian hamster (B,D,F) embryo cells. Cultures were exposed to [aH]DMBA for 24 h, the DNA was then isolated and degraded to deoxyribonucleosides. The DMBA-deoxyribonucleosides were isolated, by Sephadex LH-20 chromatography and an aliquot was analyzed by reverse-phase HPLC (A,B). The remainder was separated by immobilized boronate chromatography into DMBA-deoxyribonucleoside adducts that did contain cis vicinal hydroxyls and those that did not contain cis vicinal hydroxyls as described in Materials and Methods. Insets show immobilized boronate chromatograms of the DMBA-DNA adducts isolated from Wistar rat (A) and Syrian hamster (B) embryo cell cultures. The morpholine (fractions 1--40 in insets) and the sorbitolmorpholine (fractions 41-80 in insets) buffer fractions were analyzed by reverse-phase HPLC [(C,D) and (E,F) respectively]. The arrow indicates the elution position of a [14C](+)anti-BaPDEdG adduct marker added to each sample.

ized boronate chromatography (Figs. 1A and 1B, insets). The morpholine buffer fractions (1-40) contained D M B A - D N A adducts with no cis vicinal hydroxyls whereas the sorbitol-morpholine buffer fractions (41-80) contained those adducts with c/s vicinal hydroxyls. The ratio of the radioactive material eluted in the morpholine buffer fractions to that eluted in the sorbitol-morpholine buffer fractions was 2.2 : 1 for rat and 1.5 : 1 for h a m s t e r embryo cells.

The DMBA-deoxyribonucleoside adducts in the morpholine and the sorbitol-morpholine buffer fractions (Figs. 1A and 1B, insets) were concentrated by Sephadex LH-20 chromatography and analyzed by reverse phase HPLC. In both rat (Fig. 1C) and hamster (Fig. 1D) embryo cell DNA samples, the morpholine buffer fractions contained one major adduct peak (M4). This peak eluted in the same relative position as the m a j o r morpholine buffer fraction peak isolated from NIH Swiss mouse embryo cell cultures that was tentatively identified by Dipple and coworkers [8,11] as a syn-DMBADE-dA adduct. The other adducts M 1, M2, M3 and M5 are unidentified. (The identity of the morpholine buffer-eluted adducts from DMBA-treated mouse embryo cell cultures is discussed in detail by Dipple et al. [18]). Although the relative proportion of the total adducts eluted in the morpholine buffer fraction was larger in rat than in hamster embryo cells, the HPLC profiles of the morpholine buffer-eluted material from both cell types were similar and both contained one m a j o r adduct, syn-DMBADE-dA (M4). HPLC analysis of the sorbitol-morpholine buffer fractions resolved 4 m a j o r adduct peaks in the rat embryo cell DNA sample (Fig. 1E) and 2 m a j o r adduct peaks in the hamster embryo cell DNA sample (Fig. 1F). Adducts $2 and $4 eluted in the same relative positions as those tentatively identified as anti-DMBADE-dG and anti-DMBADE-dA, respectively in the mouse embryo cell culture s y s t e m of Dipple and coworkers [8,11]. Adducts $3 and S1 (in the hamster S1 was resolved into 2 peaks Sla and Slb) are unidentified. (The identity of the sorbitol buffer-eluted adducts from DMBA-treated mouse embryo cell cultures is described in detail by Dipple et al. [18]).

D M B A - D N A adducts in rat and hamster embryo cells after various lengths of time o f exposure to DMBA The effect of the length of time of exposure to DMBA on the D M B A - D N A adducts was determined by treating cultures of rat and hamster embryo cells with [3H]DMBA for 5, 24, 48 and 72 h. The HPLC column elution profiles of the D M B A - D N A adducts isolated from rat embryo cells (Fig. 2) contained one m a j o r peak of syn-DMBADE-dA adduct (M4) and several smaller adduct peaks. Although the profiles appeared similar at all times, the relative proportion of the syn-DMBA-dA adduct (M4) decreased slightly with the length of time of exposure to DMBA and the relative proportion of antiDMBADE-dG ($2) and anti-DMBADE-dA ($4) increased. Exposure of hamster embryo cells to [3H]DMBA resulted in a more complex D M B A - D N A adduct profile (Fig. 3). Three large and several small adduct peaks were eluted. The relative proportion of the adducts in each peak was similar at all times. The actual amounts of the 3 major adducts, $2, M4 and $4, present in both cell types at the 4 times of exposure are shown in Fig. 4. In both cells all adducts reached a maximum value at 24-48 h and decreased by 72 h. The relative proportion of the three maj or adducts remained almost constant in hamster: embryo cells (Fig. 4B). The only change in the rat embryo cell cultures was the slight relative decrease in the syn-DMBADE-dA adduct

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Fig. 2. HPLC column elution profiles of D M B A - D N A adducts isolated from Wistar r a t embryo cells. Cultures were exposed to [3H]DMBA for A(5 h), B(24 h), C(48 h) and D(72 h). The DNA from each sample was t h e n isolated and the DMBA-deoxyribonucleosides were analyzed by reverse-phase HPLC as described in Materials and Methods. The arrow indicates the elution position of a [14C](+)-anti-BaPDE-dG adduct m a r k e r added to each sample. Fig. 3. HPLC column elution profiles of D M B A - D N A adducts isolated from Syrian h a m s t e r embryo cells. Cultures were exposed to [3H]DMBA for A(5 h), B(24 h), C(48 h) and D(72 h). The DNA from each sample was t h e n isolated a n d the DM BA-deoxyribonucleosides were analyzed by reverse-phase HPLC as described in Materials a n d Methods. The arrow indicates the elution position of a [14C](+)anti-BaPDE-dG adduct m a r k e r added to each sample.

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Fig. 4. A m o u n t s of the three m a j o r D M B A - D N A adducts present in W i s t a r r a t (A) and Syrian h a m s t e r (B) embryo cells after various lengths of time of exposure to DMBA. The adducts were analyzed as described in Figs. 2 and 3. $2 anti-DMBADE-dG adduct, M4 syn-DMBADE-dA adduct, $4 anti-DMBADE-dA adduct.

(M4). The presence of the larger proportion of the syn-DMBADE-dA adduct is also the only m a j o r difference between the adducts in the rat and hamster embryo cells, for the ratio of the anti-DMBADE-dG adduct to the antiDMBADE-dA adduct was similar in both cells at all times. DISCUSSION

The use of immobilized boronate chromatography as described by Sawicki et al. [11] has permitted the resolution of D M B A - D N A adducts formed from the syn- and anti-isomers of DMBADE and allowed the tentative identification of three of the m a j o r adducts formed from DMBA in mouse embryo cells. Previous studies of D M B A - D N A adducts using LH-20 chromatography or HPLC had provided some information about the D M B A DNA adducts formed in cells and tissues from various species [3,5,6,19,20], but assignment of the diol epoxide isomer responsible for the formation of the adducts was usually not possible. Thus, the role of specific DMBADE isomers in the binding of DMBA to DNA in cells from species other than mice has not been established. M o u s e embryo cell cultures from N I H Swiss mice contained approximately similar amounts of three m a j o r adducts; anti-DMBADE-dG, syn-DMBADE-

9

dA and anti-DMBADE-dA [8,11]. The relative proportions of the three adducts remained similar after various lengths of time of exposure to DMBA. Epidermis from mice treated with DMBA in vivo also contained similar relative amounts of these three adducts [21,22] as did mouse epidermal cell cultures [22]. In all of these studies the proportion of anti-DMBADE-DNA adducts appeared greater than that of the syn-DMBADE-DNA adducts based upon the relative amounts of material in these three m a j o r adduct peaks. Based upon immobilized boronate chromatography of the D M B A - D N A adducts from N I H Swiss mouse embryo cell cultures after 24 h of exposure to 0.125 t~gDMBA/ml medium, Sawicki et al. [11; Table I] reported that the syn-DMBADE-DNA adducts (those not retained by the column) represented only 40% of the total D M B A - D N A adducts. In the Syrian hamster embryo cell cultures, 58% of the D M B A - D N A adducts were not retained on the immobilized boronate column and 72% of the D M B A - D N A adducts from Wistar rat embryo cells were not retained (did not contain cis vicinal hydroxyl groups). Thus the proportion of adducts that presumably resulted from the syn isomer of DMBADE was greater in rat embryo cells than in h a m s t e r embryo cells and greater in hamster cells than in mouse embryo cells [11]. The same relationship between the proportion of syn and anti diol epoxide isomer adducts was also observed for benzo[a]pyrene in embryo cell cultures from mice, rats and hamsters [13,17]. After 4 8 h of exposure to benzo[a]pyrene the proportions of benzo[a]pyrene-DNA adducts that did not contain cis vicinal hydroxyl groups (syn-benzo[a]pyrene-7,8-diol-9,10epoxide adducts) were: Baib/c mouse embryo cell cultures, 14%; Sencar mouse embryo cell cultures, 18%; Syrian hamster embryo cell cultures, 3()'~ ; Fischer rat embryo cell cultures 32%; Wistar rat embryo cell cultures, 47(Z [17]. In all five types of embryo cell cultures there was an increase in the proportion of anti diol epoxide isomer DNA adducts formed with increased length of time of exposure to BaP [17]. In contrast, proportions of the individual D M B A - D N A adducts in the hamster and rat embryo cell cultures were relatively independent of the length of time of exposure to DMBA. There were essentially no time-dependent changes in the D M B A - D N A adduct profile from hamster embryo cells and only a slight decrease in the proportion of the syn-DMBADE-dA adduct in rat embryo cells. The three m a j o r D M B A - D N A adducts observed in the hamster embryo cell cultures were similar to those reported in mouse embryo cell cultures and mouse skin [8,11,22]. In the hamster embryo cells the relative amount of the syn-DMBADE-dA peak was greater than that observed in the mouse embryo cells [8]. In the Wistar rat embryo cell cultures the syn-DMBADE-dA peak is much larger than either the anti-DMBADE-dG or the anti-DMBADE-dA peak. Previous studies of D M B A - D N A adducts in liver of rats exposed in vivo [3,19,20] have also demonstrated that in the area of the HPLC gradient in which D M B A D E - D N A adducts were expected to elute, only one m a j o r adduct peak was present. The actual identity of the adducts and the possibility that both syn- and anti-DMBA adducts eluted in that peak could

10 not be established by HPLC alone [20]. However, the elution profiles and the presence of smaller peaks on each side of the m a j o r adduct peak suggest that the adduct reported by Daniel and coworkers [20] may also be the syn-DMBADE-dA adduct. Both the syn and anti isomers of benzo[a]pyrene-7,8-diol-9,10-epoxide bind almost exclusively with deoxyguanosine residues in DNA in solution and in cells exposed to benzo[a]pyrene (reviewed in Ref. 4). The anti isomer of benz[a]anthracene-3,4-diol-l,2 epoxide also binds mainly to deoxyguanosine [23]. In contrast, the anti isomer of DMBADE bound to deoxyguanosine to only a slightly greater extent than to deoxyadenosine in DNA of DMBA treated cells from mice, rats and hamsters. The syn isomer of DMBADE bound almost exclusively to deoxyadenosine in all of the above cells. The relative roles of the syn and a n t i - D M B A D E - D N A adducts in the induction of biological effects has not yet been established. Sawicki and Dipple [10] found that the antioxidant butylated hydroxyanisole, an inhibitor of tumor induction by DMBA [24], selectively decreased the relative proportion of syn-DMBADE-dA adduct in DMBA-treated mouse embryo cell cultures. DiGiovanni et al. [22] compared the binding of DMBA and 10-FDMBA, a more potent initiator of mouse skin carcinogenesis than DMBA, to DNA in mouse epidermis. They found that the proportion of the syn-10FDMBADE-dA adduct was decreased in the 10-F-DMBA-DNA samples, although the total level of binding of 10-F-DMBA was greater than for DMBA [22]. The outstanding difference between DMBA and benzo[a]pyrene in all of the biological s y s t e m s examined is the relatively high proportion of deoxyadenosine adducts formed from DMBA. Dipple et al. [25] have pointed out that the relative carcinogenic potencies of DMBA and benzo[alpyrene in mouse skin differ by roughly the same factor as the amount of hydrocarbon-deoxyadenosine residues formed in mouse skin. The formation of high proportions of DMBA deoxyadenosine adducts in rat and hamster embryo cell cultures suggests that such adducts may also be involved in the high carcinogenic potency of DMBA in those species. The finding that rodent embryo cell cultures vary greatly in the proportion of syn- and antiD M B A D E - D N A adducts formed suggests that they will be useful for determining the role of specific DMBADE isomers and adducts in the induction of biological effects. ACKNOWLEDGMENTS The authors thank Constance G. Moynihan for valuable technical assistance and Marilyn Hines for typing this manuscript. The [14C]-antibenzo[a]pyrene diol epoxide was purchased from the Radiochemical Repository, Division of Cancer Etiology, National Cancer Institute. This work was supported by grants CA 28825 and CA 30234 from the National Cancer Institute, DHHS.

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