Identification and quantitation of benzo[a]pyrene-DNA adducts formed in mouse skin

356 Chem. Res. Toxicol. 1993,6, 356-363

Identification and Quantitation of Benzo[a]pyrene-DNA Adducts Formed in Mouse Skin

Eleanor G. Rogan,*>t Prabhakar D. Devanesan,t N. V. S. RamaKrishna,t Sheila Higginbotham,t N. S. Padmavathi,t Kimberly Chapman,t

Ercole L. Cavalieri,t Hyuk Jeong,$ Ryszard Jankowiak,$ and Gerald J. Small$ Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical

Center, 600 South 42nd Street, Omaha, Nebraska 68198-6805, and Department of Chemistry and Ames Laboratory-USDOE, Iowa State University, Ames, Iowa 50011

Received October 26, 1992

The DNA adducts of benzo[alpyrene (BP) formed in vitro were previously identified and quantitated. In this paper, we report the identification and quantitation of the depurination adducts of BP, 8-(benzo[a]pyren-6-yl)guanine (BP-6-C8Gua), BP-6-N7Gua, and BP-6-N7Ade, formed in mouse skin by one-electron oxidation, as well as the major stable adduct formed via the diolepoxide pathway, BP diolepoxide bound a t C-10 to the 2-amino of dG (BPDE-10-N2dG). Identification of the depurination adducts was achieved by HPLC and fluorescence line narrowing spectroscopy. The depurination adducts, BP-6-C8Gua (34% ), BP-6-N7Gua (10% ), and BP- 6-N7Ade (30%), constituted 74% of the adducts found in mouse skin 4 h after treatment with BP. The stable adduct BPDE-10-N2dG accounted for 22 % of the adducts. Treatment of the skin with BP-7,8-dihydrodiol or BP diolepoxide yielded almost exclusively the stable adduct BPDE-10-NZdG. When BP or BP-7,8-dihydrodiol was bound to RNA or denatured DNA in reactions catalyzed by rat liver microsomes, no depurination adducts were detected. The profiles of stable adducts were similar both qualitatively and quantitatively with native or denatured DNA. With activation of BP by horseradish peroxidase, the profiles of stable adducts differed with native and denatured DNA. The total amount of adducts with denatured DNA was only 25 % of the amount detected with native DNA. No depurination adducts were detected with denatured DNA or RNA in the peroxidase system. The results reported here demonstrate that in mouse skin BP-DNA adducts are predominantly formed by one-electron oxidation and that this mechanism of activation requires double helical DNA for formation of adducts.

Introduction

Covalent binding of chemical carcinogens, including polycyclic aromatic hydrocarbons (PAH),l to target cell DNA is thought to be the first critical step in the process of tumor initiation (1,2). Two major mechanisms of PAH activation leading to carcinogenesis are one-electron oxidation with formation of radical cations (3, 4 ) and monooxygenation to yield bay-region diolepoxides (5-7). Some PAH are thought to be activated by the diolepoxide pathway alone, others by one-electron oxidation alone, and some by a combination of both pathways (4 ) . We have developed this overall view of PAH activation based on the physicochemical properties of PAH and the catalytic properties of the cytochrome P450s involved, as well as on tumorigenicity studies and the structures of DNA adducts ( 3 , 4 , 8).

The structure of PAH-DNA adducts formed biologically can indicate the mechanism of metabolic activation of the

* To whom correspondence should be addressed. + University of Nebraska Medical Center. 1 Iowa State University. 1 Abbreviations: BP, benzo[alpyrene; BPDE, (f)-r-7,t-8-dihydroxy-

t-9,10-epoxy-7,8,9,1O-tetrahydrobenzo[alpyrene (anti); BP-6-N7Ade, 7-(benzo[a]pyren-6-yl)adenine; BP-6-N7Gua, 7-(benzo[alpyren-6-yl)guanine; BP-6-C8Gua, 8-(benzo[alpyren-6-yl)guanine; BP-6-C8dG, 84ben- zo[a]pyren-6-yl)deoxyguanosine; BPDE-10-N2dG, 10-(deoxyguanosin- N~-yl)-7,8,9-trihydroxy-7,8,9,lO-tetrahydrobenzo[a]p~ene; BPDE-10- N7Ade, 10-(adenin-7-yl)-7,8,9-trihydroxy-7,8,9,lO-tetrahydrobenzo[a]pyrene; FLNS, fluorescence line narrowing spectroscopy; HRP, horseradish peroxidase; MC, 3-methylcholanthrene; PAH, polycyclic aromatic hydrocarbon(s).

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BP-6-CBGUa BPbN7Gua

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BPDE-1 0-N2dG Figure 1. Structures o f BP-DNA adducts.

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PAH. Identification and quantitation of benzo[alpyrene (BPI-DNA adducts has shown that the majority of the adducts obtained by activation with 3-methylcholanthrene (MC)-induced rat liver microsomes, are formed by one- electron oxidation (9). The depurination adducts generated by one-electron oxidation, 8-(benzo[alpyren-6- y1)guanine (BP-6-C8Gua), 7-(benzo[alpyrenS-yl)guanine (BP-6-N7Gua) and 7- (benzo [a] pyren-6-yl) adenine (BP- 6-N7Ade) (Figure 11, constitute 80% of all the adducts, whereas the depurination adduct formed by the BP diolepoxide, (f)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-

0 1993 American Chemical Society

BP-DNA Adducts Formed in Mouse Skin Chem. Res. Toxicol., Vol. 6, No. 3, 1993 357

200 nmol of tritiated BP, BP-7,8-dihydrodiol, or BPDE in 50 pL of acetone, and the mice were either allowed to live for 4 h (“in vivo”) or were sacrificed immediately and the treated area of skin was maintained for 4 h in culture medium (“in culture”). In one type of experiment, duplicate groups were killed after 4 h, and the treated area was excised. In the other, duplicate groups were killed immediately, and the treated area was excised and maintained for 4 h at 37 “C in 95% air/5% C02 atmosphere in RPMI-1640 medium with 10% fetal bovine serum and 15 mM N-(2-hydroxyethyl)piperazine-N’-2-ethanes~lfo~c acid (HEPES). The culture medium was reserved for analysis of depurination adducts. Epidermis from each group was prepared (22), pooled, ground in liquid N2, and split into two equal samples weighing approximately 1 g. One was used to purify DNA (23) and to analyze the stable adducts by the 32P-postlabeling method (23). The other was Soxhlet-extracted for 48 h with CHC13 to recover the depurination adducts, and the adducts were analyzed by HPLC and FLNS (9). The amount of DNA in each sample of ground epidermis was determined by the diphenylamine reaction (24) to be 8.5 i 0.4 pmol of DNA-P/g of epidermis. The level of binding in vivo was about 1.5 pmol of BP/mol of DNA-P.

Binding of PAH to DNA or RNA in Vitro. Calf thymus DNA (2.6 mM, Pharmacia, Piscataway, NJ) was denatured by heating in a boiling water bath for 5 min, followed by quick- cooling in ice-water. Yeast RNA, type IIS (3.0 mM, Sigma Chemical Co., St. Louis, MO) was used. As previously described (23), [3H]BP (80 pM) or [3H]BP-7,8-dihydrodiol (15 pM) was bound to RNA and native and denatured DNA in reactions catalyzed by MC-induced rat liver microsomes and I3H1BP was also bound to DNA and RNA by HRP. All of the DNA and RNA reactions were 15 mL in volume; they were incubated for 30 min a t 37 “C.

At the end of the reaction, a 1-mL aliquot of the mixture was used to determine the level of PAH binding to DNA or RNA and the amount of stable DNA adducts by the 32P-postlabeling method, by using P1 nuclease enrichment, as previously described (23). The DNA or RNA from the remaining 14-mL mixture was precipitated with two volumes of absolute ethanol (or cold ethanol for RNA) and the supernatant was used to identify and quantify the depurination adducts by HPLC and FLNS.

Analysis of Depurination Adducts by HPLC and FLNS. For the “in vivo” experiments, the Soxhlet CHC13 extract of the epidermis, which contained depurination adducts and metabolites, was evaporated to dryness under vacuum. For the in culture experiments, the Soxhlet CHCl3 extract of the epidermis was evaporated to dryness under vacuum. The medium from the 4 h in culture was also evaporated to dryness under vacuum, and the residue was extracted 4 times with ethanol/CHC13/acetone (2:l:l) and filtered through a 0.45-pm filter. This extract was then added to the CHCl3 extract of the corresponding epidermis before evaporation of the solvent. For the in vitro binding reactions, the ethanol-aqueous supernatant was evaporated to dryness under vacuum.

The residues were dissolved in dimethyl sulfoxide/CH3OH (1: 1). After sonication to enhance solubilization, the undissolved residue in each sample was removed by centrifugation.

The depurination adducts were analyzed by HPLC as previously described (9). For each type of experiment, the depurination adducts were analyzed in four separate samples. In the first experiment, the depurination adducts were identified by HPLC in both CH30H/H20 and CH3CN/H20 gradients in the presence of added authentic adducts. In the second experiment, the adducts were chromatographed in both solvent systems without added authentic adducts, and the material eluting at the respective retention times of the authentic adducts on the CH3CN/H20 gradient was collected for identification by FLNS. In the third and fourth experiments, quantitative data were collected for calculation of the amounts of both the depurination and stable adducts. The amounts of each adduct varied in the two experiments by 10 to 25%, with the larger variations in the minor adducts.

tetrahydrobenzo[a]pyrene (anti) (BPDE), 10-(adenin-7- yl)-7,8,9-trihydroxy-7,8,9,lO-tetrahydrobenzo[~lp~ene (BPDE-lO-N7Ade) (Figure l), accountsfor only0.5 % .The major stable DNA adduct observed by the 32P-postlabeling technique is the previously identified (10, 11) 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,1O-tetrahydrobenzo[a]pyrene (BPDE-10-N2dG) (Figure l), constituting 15% of the adducts. All of the stable adducts account for less than 20 % of the total adducts formed. Research using other methods has previously suggested that the major stable adduct formed with microsomal activation arises by binding of the 9-hydroxyBP 4,5-oxide (12-14). We do not know whether this adduct is present in the adduct spots generated by the 32P-postlabeling technique.

The only depurination adduct of BP previously detected in vivo is BP-6-N7Gua, which was found in rat urine and feces (15). Recent published studies by Ross et al. (16) report stable adducts of BP metabolites in rat lung, liver, and lymphocytes. Reddy et al. (17) identified DNA adducts formed by BP-7,&dihydrodiol in mouse skin. There have been, however, no published reports identifying both depurination and stable adducts formed in vivo. This information is necessary to understand the biological significance of DNA adducts in the initiation of carcinogenesis by BP.

In this paper, we report identification and quantitation of both stable and depurination adducts formed after topical application of BP to mouse skin. The depurination adducts formed by one-electron oxidation, BP-6-C8Gua, BP-6-N7Gua, and BP-6-N7Ade, were identified and quantitated, as well as the stable BPDE-10-N2dG adduct. The depurination adducts were identified by comparison of HPLC elution times with those of authentic standards synthesized chemically or electrochemically (18,191 and by fluorescence line narrowing spectroscopy (FLNS) (9, 20). The BPDE-10-N2dG adduct was identified in the 32P-postlabeling method by comparison with the primary DNA adduct formed by BP-7,8-dihydrodiol or BPDE.

In addition, the results of in vitro binding studies with RNA or denatured DNA indicate that the double helical structure of native DNA is a prerequisite for the formation of BP-DNA adducts by one-electron oxidation. In fact, with activation of BP by either microsomes or horseradish peroxidase (HRP), depurination adducts were detected only with native DNA.

Methods Chemicals. BP-6-C8Gua, BP-6-N7Gua, and BP-6-N7Ade

(Figure 1) were synthesized by anodic oxidation of BP in the presence of dG or dA (18,19). BPDE-lO-N7Ade (Figure 1) was prepared by chemical reaction of BPDE with dA (19).

[3H]BP (sp act. 550 Ci/mol) was purchased from Amersham (Arlington Heights, IL), whereas [3H]-(*)-trans-BP-7,8-dihydrodiol (sp act. 767 Ci/mol) and [3H]-(i)r-7,t-8-dihydroxy-t- 9,10-epoxy-7,8,9,10-tetrahydro-BP (anti) (sp act. 1380 Ci/mol) were purchased from the NCI Chemical Carcinogen Repository (Bethesda, MD). The nonradiolabeled (i)-trans-BP-7,8-dihydrodiol and (i) -r-7,t-8-dihydroxy- t-9,10-epoxy-7,8,9,1O-tetrahydro-BP (anti) were also purchased from the NCI Chemical Carcinogen Repository. [3H]BP was used at a specific activity of 400 Ci/mol; [3H]BP-7,8-dihydrodiol at 330 Ci/mol; and [3H]BPDE at 210 Ciimol. Caution: BP, BP- 7,8-dihydrodiol and BPDE are hazardous chemicals; they are handled in accordance with NIH guidelines (21).

Binding of BP, BP-7,8-dihydrodiol, and BPDE to Mouse Skin DNA. In groups of eight female Swiss mice (8-weeks-old, Eppley Colony), an area of shaved dorsal skin was treated with

358 Chem. Res. Toxicol., Vol. 6, No. 3, 1993 Rogan et al.

TIME, MIN

Figure 2. Initial HPLC separation of depurination adducts. The YMC 5-pm ODS-AQ reverse-phase analytical column (6.0 X 250 mm) was eluted with 30% CH30H in H20 for 5 min, followed by an 80-min convex (CV5) gradient to 100% CH30H a t a flow rate of 1 mlimin.

Table I. Quantitation of BP-DNA Adducts Formed in Mouse Skin" stable DNA adducts,

(mol of adductimol of DNA-P) X lo6 depurination DNA adducts,

(mol of adductimol of DNA-P) X lofi adducts stable depurination ratio of

incubation system DNA-P) 10-NZdG tified 7% of total 10-N7Ade C8Gua N7Gua N7Ade 7% of total stable (pmol/mol BPDE- uniden- adducts, BPDE- BP-6- BP-6- BP-6- adducts, depurinationi

4 h in vivo BP 3.56 0.79 (22) 0.15' (4) 26 <0.05 1.20 (34) 0.36 (10) 1.06 (30) 74 2.8 BP-7,8-dihydrodiol 0.77 0.77 (100) 0 100 <0.05 BPDE 5.62 5.58 (99) 0.04d (0.7) 100 <0.05

BP 1.96 0.01 (0.5) 0.001[ (0.07) 0.6 <0.05 0.47 (24) 0.37 (19) 1.11 (57) 99 166

a Values are the average of determinations from two preparations. The amount of each adduct varied between 10 and 30% in the two Number in parentheses is percentage oftotal adducts. 2 adduct spots. d 3 adduct

4 h in culture

preparations, with larger variations with the minor adducts. Spots.

Identification of the depurination adducts by FLNS was conducted as previously described (9,20).

Calculation of Adduct Levels. The amount of stable adducts was calculated by the 32P-postlabeling method as previously described (23). For quantitation of the depurination adducts, each of the peaks eluting a t the same time as an authentic adduct in HPLC using the CH30H/H20 gradient was collected and counted. These peaks were then reinjected individually in an CH3CN/H20 gradient and the percentage of the injected radio- activity eluting a t the same time as the authentic adduct was measured and calculated using the radiation flow monitor. The total amount of each of the adducts was calculated from the specific activity of the PAH and normalized to the amount of DNA used in the reaction or calculated from the weight of the mouse epidermis sample.

Results and Discussion Identification of Adducts. A strategy similar to the

one employed for the identification and quantitation of BP-DNA adducts formed in vitro was utilized for identification of the DNA adducts formed in vivo. The

depurination adducts from mouse skin were identified by comparison of their HPLC elution times with those of the authentic standards and by FLNS (9, 20). The stable BPDE-10-N2dG adduct was confirmed in the 32P-postlabeling method by comparison with the adduct formed with either BP-7,8-dihydrodiol or BPDE.

In mouse skin, the three depurination adducts formed by one-electron oxidation, namely, BP-6-C8Gua, BP-6- N7Gua, and BP-6-N7Ade (Table I), were separated by HPLC in consecutive solvent systems. The adducts were first separated by using a CH~OH/HPO gradient (Figure 2) and peaks corresponding to the retention times of the three standard adducts were collected. Each peak was then rechromatographed with a CH&N/H20 gradient (Figure 3) to obtain the individual adducts for identification by FLNS or quantitation (see Methods).

These three adducts were previously identified in reactions catalyzed by cytochrome P450 in MC-induced rat liver microsomes (9). The depurination adduct formed


laser light from the fluoresence and/or to resolve tempo- rally the fluorescence of components (e.g., contaminants) having different fluorescence lifetimes. All spectra in Figure 4 were obtained for a 50-ns gate delay of the observation window. No significant differences were observed for delay times between 20 and 100 ns for BP- 6-C8Gua (frame A, Figure 4) and for BP-6-N7Gua (frame B). Based on our experience, this is consistent with HPLC data indicating that possible contamination by metabolites or related compounds containing the BP moiety must be smaller than about 10%. Other FLN spectra, obtained for different excitation wavelengths (data not shown), further support the conclusion that we have analyzed single components for BP-6-C8Gua and BP-6-N7Gua.

For BP-6-N7Ade, however, FLN spectra were found to depend on the delay time of the gate. Contribution from contamination (due to some related compound) was observed for a delay time of 25 ns. Assuming that the quantum yields of the adduct and contaminant are equal, the contaminant is estimated to be less than 20%. An impurity is also visible in the corresponding HPLC profile (Figure 3B). The gated FLN spectra indicate that the lifetime of this unknown contaminant was approximately 40 ns and of the adduct about 100 ns. FLN spectra for BP-6-N7Ade (frame C, Figure 41, obtained for a gate delay of about 50 ns, reveal only the contribution of the major component, namely BP-6-N7Ade.

Comparison of the standard spectra a in Figure 4 with the spectra b of the adducts isolated from mouse skin in vivo shows that the spectra are identical, with the same vibronic mode intensity, distribution, and vibronic mode structure. Although we cannot exclude contamination by unknown compounds (110% for BP-6-C8Gua and BP- 6-N7Gua and 520% for BP-6-N7Ade), the spectra show that the depurination adducts produced in the in vivo binding of BP to DNA in mouse skin are BP-6-C8Gua, BP-6-N7Gua, and BP-6-N7Ade.

The major DNA adduct detected by the 32P-postlabeliig method in mouse skin appears to be BPDE-10-N2dG, both in vivo and in culture (Table I). This same adduct is detected when mouse skin is treated with either BP-7,8- dihydrodiol or BPDE. This adduct spot may also contain other adducts having the same mobility. The structures of the other minor stable adducts are still to be established.

Quantitation of DNA Adducts. Quantitation of the stable adducts was achieved by the 32P-postlabeling method, with P1 nuclease enrichment of the adducted nucleotides (23). The efficiency of the various steps involved was not established and so the values reported are minimum values. The average amount of DNA in the skin samples was determined to be 8.5 f 0.4 pmol of DNA- P/g of epidermis, based on numerous determinations by the diphenylamine reaction (24). Quantitation of the depurination adducts was accomplished by HPLC with a radiation flow monitor.

Four hours after treatment of mouse skin with BP in vivo, the major stable adduct observed was BPDE-10- N2dG (22 % of total), whereas the other minor, unidentified stable adducts constituted only 4% of the total (Table I, Figure 5A). These values are comparable, except for slightly higher amounts of BPDE-10-N2dG in the mouse skin, to the values reported earlier with microsomal activation (9). In contrast, BP-treated skin maintained 4 h in culture contained less than 1% of the adducts as stable adducts (Table I, Figure 5B). It appears that the

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TIME, MIN

Figure 3. Rechromatography of individual depurination adducts (A) BP-6-C8Gua, (B) BP-6-N7Ade, and (C) BP-6-N7Gua. The analytical column was eluted with 30% CH3CN in HzO for 5 min followed by a convex (CV5) gradient to 100 % CH&N in 75 min at a flow rate of 1 mL/min (A and B) or 20% CH3CN in HzO for 5 min, followed by a linear (CV6) gradient to 100% CH3CN in 80 min at a flow rate of 1 mL/min (C).

by monooxygenation, BPDE-lO-N7Ade, that was identified in vitro, was not detected in either in vivo or in culture skin samples (Table I). Presumably, the amount of this adduct formed was below 0.05 pmol/mol DNA-P, the calculated limit of detection for our HPLC studies.

The structures of these adducts were confirmed by comparing their FLN spectra with those of authentic standards. The FLN spectra not only enable identification of the adduct structures but also provide evidence on their relative purity. Identification, using FLNS (9, 201, was performed for several excitations chosen to reveal mode structure in the 400-1500 cm-1 range. As an example, in Figure 4 two excitations are shown, A,, = 395.7 nm for BP-6-C8Gua (A) and BP-6-N7Gua (B) and A,, = 386.5 nm for BP-6-N7Ade (C). Spectra a in Figure 4 were obtained for the standard adducts BP-6-C8Gua, BP-6-N7Gua, and BP-6-N7Ade, which were previously characterized in detail (9), and spectra b were the adducts isolated from mouse skin in vivo. Gated fluorescence detection, which allowed time discrimination, was employed to eliminate scattered

360 Chem. Res. Toxicol., Vol. 6, No. 3, 1993

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Rogan et al.

404 405 406 407 408

c v) z W c z w V z W 0

CL 0

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ex=395.7 nm

! 403 404 405 406 4 0 7 ,

WAVELENGTH (nm)

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ex=386.5 nm

Figure 4. Vibronically excited FLN spectra of (A) BP-6-C8Gua, (B) BP-6:N7Gua, and (C) BP-6-N7Ade. Spectra a correspond to the synthesized reference adducts and spectra b correspond to the adducts isolated from mouse skin in vivo. Bands are labeled according to excited-state vibrational frequencies (cm-'1. T = 4.2 K. Gate delay 50 ns; gate width 50 ns.

Table 11. Quantitation of BP-DNA Adducts Formed in Vitros

stable DNA adducts, (mol of adductimol of DNA-P) X lo6

depurination DNA adducts, (mol of adductimol of DNA-P) X lo6

total adducts depurination (Mmol/mol BPDE- uniden- stable adducts, BPDE- BP-6- BP-6- BP-6- adducts,

incubation system DNA-P) 10-N2dG tified % oftotal 10-N7Ade C8Gua N7Gua N7Ade % oftotal MC-microsomes + BP

native DNA 19.2 3.3 (17)b 1.2c (6) 23 0.1 (0.5) 2.2 (11) 1.8 (9) 10.6 (55) 77 denatured DNA 4.8 3.8 (79) LOd (21) 100 <0.05 <0.05 <0.05 <0.05 0 RNA 3.7 N.D.8 N.D. <0.05 <0.05 <0.05 <0.05 0

MC-microsomes + BP-7,3-dihydrodiol

native DNA 48.2 40.3 (83) 0.4e (0.8) 84 7.5 (16) 16 denatured DNA 33.3 32.5 0.P (2) 100 <0.05 0 RNA 106 N.D. N.D. <0.05 0

native DNA 24.8 10.2e (41) 41 2.3 (9) 1.9 (8) 10.4 (42) 59 denatured DNA 2.6 2.u (100) 100 <0.05 <0.05 <0.05 0 RNA 53 N.D. N.D. <0.05 C0.05 <0.05 0

HRP

Values are the average of determinations from two preparations. The amount of each adduct varied between 5 and 30% in the two 5 adduct DreDarations. with larger variations with the minor adducts. * Number in parentheses is percentage of total adducts. 3 adduct spots.

spots. e 4 adduct spots. f 9 adduct spots. 8 N.D. = not determined.

overall level of binding was lower in culture than in vivo, but the recovery of depurination adducts was much higher in culture than in vivo, where adducts are lost from the tissue during the 4 h after treatment with BP.

The depurination adducts BP-6-C8Gua, BP-6-N7Gua, and BP-6-N7Ade were predominant in both types of samples. Mouse skin in vivo contained these one-electron oxidation adducts in an approximate molar ratio of 3:1:3, respectively, accounting for 74% of the total adducts detected. Skin in culture for 4 h contained BP-6-C8Gua, BP-6-N7Gua, and BP-6-N7Ade in approximate molar ratio of 1:1:3, constituting more than 99% of the total adducts detected. The relative amount of BP-6-N7Ade in the skin in vivo and in culture was about 3 times the amotlnt of each of the Gua adducts (Table I). This differed from the profile of adducts obtained in vitro with microsomes or HRP, in which the relative amount of BP-6-N7Ade was about 5 times that of each Gua adduct (Table I1 and ref 9). In addition, in mouse skin in vivo the relative amount of BP-6-C8Gua was 3 times greater than that of BP-6- N7Gua (Table I). This result is consistent with the slower hydrolysis of the glycosidic bond in the BP-6-C8dG adduct (19). In vivo, as a result of this slower hydrolysis, BP-6- C8Gua is released from DNA more slowly and is still

present in mouse skin epidermal cells at 4 h, whereas the more rapidly depurinated BP-6-N7Gua and BP-6-N7Ade are lost from the epidermis more quickly.

The depurination adduct BPDE-lO-N7Ade, observed in microsomal reactions, was not detected in any of the skin samples. If present, this adduct was below the detection limit of 0.05 pmol/mol DNA-P for the methods employed in these studies.

When mouse skin was treated with BP-'l,&dihydrodiol, only the stable adduct, BPDE-10-N2dG, was detected and the amount observed was about that seen with BP (Table I, Figure 5C). Treatment with BPDE produced this adduct at 7 times the level seen with either BP or BP-7,8- dihydrodiol and three other minor adducts accounting for less than 1% of the total (Figure 5D). No depurination adducts were detected after treatment with either BPDE or BP-7,8-dihydrodiol.

BP Adducts Formed in Vitro with RNA or Dena- tured DNA. Activation by HRP or rat liver microsomes was used to compare formation of BP adducts with RNA or denatured DNA to those observed with native DNA (Table 11). This study was conducted to determine the effect of the double helical structure of DNA on adduct formation. In addition, information was obtained on the


Figure 5. Autoradiogram of 32P-postlabeled DNA containing BP adducts formed in mouse skin treated with (A) BP in vivo, (B) BP in culture, (C) BP-7,8-dihydrodiol in vivo, or (D) BPDE in vivo. The film was exposed at room temperature for 3.5 h (A), 18 h (B), 6 h (C), or 3 h (D).

ability of RNA to form depurination adducts by one- electron oxidation.

With microsomal activation, the profiles of stable adducts seen by 32P-postlabeling were similar qualitatively and quantitatively with denatured (Figure 6A) and native DNA (Figure 6B). With activation of BP by HRP, the 32P-postlabeled profiles of stable adducts for native and denatured DNA were different, and the amount of stable adducts observed with denatured DNA (Figure 6D) was approximately 75% less than that obtained with native DNA (Figure 6C).

Although depurination adducts produced by one- electron oxidation constitute the majority of the BP adducts with native DNA, these adducts were not detected with denatured DNA or RNA (Table 11). If any of the depurination adducts were produced, they were below the detection limit.

BP-7,8-dihydrodiol was also bound to native or denatured DNA, or RNA in the microsomal system. In this case, the stable adduct profiles and the amounts of the individual adducts were similar with either native or denatured DNA (Table 11). The depurination adduct BPDE-lO-N7Ade constituted 16% of the adducts when BP-7,8-dihydrodiol was bound to native DNA (Table 11), similarly to previous results (9). This adduct was not detected with either RNA or denatured DNA.

Conclusions In mouse skin, the predominant mechanism of activation

of BP to bind to DNA is one-electron oxidation, in agreement with previously published results for microsomal activation (9). The BP-DNA adducts formed by this mechanism at the N-7 of Gua or Ade and the C-8 of Gua are lost from the DNA by depurination. These depurination adducts account for more than 70% of all the adducts detected in mouse skin in vivo and more than 99% of the adducts detected in mouse skin in culture 4 h after topical treatment of the skin (Table I). In contrast, the stable adduct BPDE10-N2dG accounts for about 22 % of total adducts in vivo and less than 1 % in culture. When mouse skin is treated with BP-7,8-dihydrodiol or BPDE, only stable BPDE adducts are formed (Table I). The depurination adduct BPDE-lO-N7Ade, which was observed in small quantities in microsomal reactions (9), was not detected in any of the mouse skin samples and could only be present a t less than 0.05 pmollmol DNA-P, the limit of detection in our studies.

The profile of BP-DNA adducts formed in mouse skin closely resembles that obtained with microsomal activation, both qualitatively and quantitatively. These results differ from previous studies suggesting that the diolepoxide adduct is the major stable adduct in intact cells, but not with microsomal activation (12-14). Our studies of stable

362 Chem. Res. Toxicol., Vol. 6, No. 3, 1993 Rogan et al.

Figure 6. Autoradiogram of 32P-postlabeled DNA containing BP adducts formed by microsomal activation in the presence of (A) native DNA or (B) denatured DNA or by HRP in the presence of (C) native DNA or (D) denatured DNA. The film was exposed at room temperature for 1 h (A and B), 20 min (C), or 45 min (D).

adducts differ from previous ones in many respects. A major difference is the 32P-postlabeling method, which includes digestion to 3’-nucleotides postlabeled and separated by TLC, compared to the previously used digestion to 5’-nucleotides and then nucleosides separated by HPLC. The 32P-postlabeling method has two generally observed limitations that result in adducts not being detected (23). One is incomplete enzymic digestion to 3’-nucleotides and the other is adduct spots coinciding on the TLC plates. For BP, only 10 to 50 % of the stable adducts are detected.

Completion of these studies would include identification of the as yet unidentified stable adducts observed by the 32P-postlabeling technique, which account for 4 % of the total adducts formed in the mouse skin, as well as other stable adducts that may not be detected by this technique. For example, the BP-6-C8dG adduct is relatively stable (19) and is only partially lost from the DNA as BP-6- C8Gua. We are certain that BP-6-C8dG is present in our DNA samples, but we have never been able to detect it by the 32P-postlabeling method. Therefore, identification of stable adducts not detected by 32P-postlabeling will require alternative, complementary methods of analysis.

Identification of depurination adducts requires the use of reference adducts. With present technology, it is not yet possible to identify adducts without their synthesized standards. The quantitative analysis used in these experiments includes two consecutive HPLC separations that

result in loss of adduct material. Quantitation of the depurination adducts provides an estimated minimum amount because we have no way to determine the relative proportion of each adduct lost in the extraction and chromatographic steps.

To summarize, our future objective of complete identification and quantitation of DNA adducts requires the technology to identify adducts without authentic standards. Four-sector mass spectrometry (18, 19) has the potential to achieve this goal, a t least for in vitro experiments. For stable adducts, the major problem is incomplete enzymic hydrolysis to either the 3’- or 5’- nucleotide level. For example, in 32P-postlabeling analysis, a t most 50% of the BP adducts are detected, and for DMBA the maximum seems to be about 10% (see accompanying paper). The degree of hydrolysis depends on the structure of the adducts formed. Complementary chemical methods of hydrolysis that do not disrupt adduct structures might offer substantial improvement in this type of analysis.

With binding of BP to denatured DNA by rat liver microsomes, none of the depurination adducts formed by one-electron oxidation was produced (Table 11). The profile of stable adducts, however, was similar with both native and denatured DNA. The profile of stable adducts formed with BP-7,8-dihydrodiol was similar with either type of DNA, but the depurination adduct BPDE-10-

BP-DNA Adducts Formed in Mouse Skin

N7Ade was not formed with RNA or denatured DNA. In HRP-catalyzed one-electron oxidation of BP, no depurination adducts were detected with denatured DNA (Table 11). The amount of stable adducts decreased by 75%, but a number of new adducts were detected. No depurination adducts were detected with RNA.

These results indicate that the double helical structure of DNA is a necessary prerequisite for the formation of depurination adducts of BP (Table 11). This is especially significant since at least 70% of all the adducts formed in mouse skin arise by one-electron oxidation (Table I). These results indicate that the depurination adducts observed after treatment of mouse skin with BP are formed in DNA, not RNA.

The studies of BP-DNA adducts formed in mouse skin reported here demonstrate that one-electron oxidation is the predominant mechanism of activation and binding of BP to DNA in this target tissue. The roles in carcinogenesis of stable adducts and apurinic sites, formed when the adducts are lost from DNA by depurination, need to be investigated.

Chem. Res. Toxicol., Vol. 6, No. 3, 1993 363

Acknowledgment. This research was supported by U.S. Public Health Service Grants Pol-CA49210, awarded to both research groups, R01-CA44686 andR01-CA25176. Core support at the Eppley Institute was from the National Cancer Institute (P30-CA36727). R.J. at Ames Laboratory was partially supported by the Office of Health and Environmental Research. Ames Laboratory is operated for the U.S. Department of Energy at ISU under Contract W-7405-Eng-82.

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Identification and quantitation of benzo[a]pyrene-DNA adducts formed in mouse skin

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