Chem. Res. Toxicol. 1993,6, 356-363
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Identification and Quantitation of Benzo[a]pyrene-DNA Adducts Formed in Mouse Skin Eleanor G. Rogan,*>tPrabhakar 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 BP6-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
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1 Abbreviations: BP, benzo[alpyrene; BPDE, (f)-r-7,t-8-dihydroxyt-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, 84benzo[a]pyren-6-yl)deoxyguanosine; BPDE-10-N2dG, 10-(deoxyguanosinN~-yl)-7,8,9-trihydroxy-7,8,9,lO-tetrahydrobenzo[a]p~ene; BPDE-10N7Ade, 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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Figure 1. Structures o f BP-DNA adducts.
* To whom correspondence + University of Nebraska 1 Iowa State University.
BPbN7Gua
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 oneelectron oxidation (9). The depurination adducts generated by one-electron oxidation, 8-(benzo[alpyren-6y1)guanine (BP-6-C8Gua),7-(benzo[alpyrenS-yl)guanine (BP-6-N7Gua)and 7-(benzo[a]pyren-6-yl)adenine (BP6-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 tetrahydrobenzo[a]pyrene (anti) (BPDE), 10-(adenin-7yl)-7,8,9-trihydroxy-7,8,9,lO-tetrahydrobenzo[~lp~ene of acetone, and the mice were either allowed to live for 4 h (“in vivo”) or were sacrificed immediately and the treated area of (BPDE-lO-N7Ade) (Figure l),accountsfor only0.5 % .The skin was maintained for 4 h in culture medium (“in culture”). In major stable DNA adduct observed by the 32P-postlabeling one type of experiment, duplicate groups were killed after 4 h, technique is the previously identified (10, 11) 10-(deoxand the treated area was excised. In the other, duplicate groups yguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,1O-tetrahydroben- were killed immediately, and the treated area was excised and zo[a]pyrene (BPDE-10-N2dG) (Figure l), constituting maintained for 4 h at 37 “C in 95% air/5% C02 atmosphere in 15% of the adducts. All of the stable adducts account for RPMI-1640 medium with 10% fetal bovine serum and 15 mM N-(2-hydroxyethyl)piperazine-N’-2-ethanes~lfo~c acid (HEPES). less than 20 % of the total adducts formed. Research using The culture medium was reserved for analysis of depurination other methods has previously suggested that the major adducts. Epidermis from each group was prepared (22),pooled, stable adduct formed with microsomal activation arises ground in liquid N2, and split into two equal samples weighing by binding of the 9-hydroxyBP 4,5-oxide (12-14). We do approximately 1 g. One was used to purify DNA (23) and to not know whether this adduct is present in the adduct analyze the stable adducts by the 32P-postlabelingmethod (23). spots generated by the 32P-postlabelingtechnique. The other was Soxhlet-extracted for 48 h with CHC13 to recover The only depurination adduct of BP previouslydetected the depurination adducts, and the adducts were analyzed by in vivo is BP-6-N7Gua, which was found in rat urine and HPLC and FLNS (9). The amount of DNA in each sample of feces (15). Recent published studies by Ross et al. (16) ground epidermis was determined by the diphenylamine reaction report stable adducts of BP metabolites in rat lung, liver, (24)to be 8.5 i 0.4 pmol of DNA-P/g of epidermis. The level and lymphocytes. Reddy et al. (17) identified DNA of binding in vivo was about 1.5 pmol of BP/mol of DNA-P. adducts formed by BP-7,&dihydrodiol in mouse skin. Binding of PAH to DNA or RNA in Vitro. Calf thymus There have been, however, no published reports identifying DNA (2.6 mM, Pharmacia, Piscataway, NJ) was denatured by heating in a boiling water bath for 5 min, followed by quickboth depurination and stable adducts formed in vivo. This cooling in ice-water. Yeast RNA, type IIS (3.0 mM, Sigma information is necessary to understand the biological Chemical Co., St. Louis, MO) was used. As previously described significance of DNA adducts in the initiation of carcino(23),[3H]BP (80 pM) or [3H]BP-7,8-dihydrodiol (15 pM) was genesis by BP. bound to RNA and native and denatured DNA in reactions In this paper, we report identification and quantitation catalyzed by MC-induced rat liver microsomes and I3H1BPwas of both stable and depurination adducts formed after also bound to DNA and RNA by HRP. All of the DNA and RNA topical application of BP to mouse skin. The depurination reactions were 15 mL in volume; they were incubated for 30 min adducts formed by one-electron oxidation, BP-6-C8Gua, a t 37 “C. BP-6-N7Gua, and BP-6-N7Ade, were identified and At the end of the reaction, a 1-mL aliquot of the mixture was quantitated, as well as the stable BPDE-10-N2dGadduct. used to determine the level of PAH binding to DNA or RNA and The depurination adducts were identified by comparison the amount of stable DNA adducts by the 32P-postlabeling of HPLC elution times with those of authentic standards method, by using P1 nucleaseenrichment, as previouslydescribed synthesized chemically or electrochemically (18,191 and (23). The DNA or RNA from the remaining 14-mL mixture was precipitated with two volumes of absolute ethanol (or cold ethanol by fluorescence line narrowing spectroscopy (FLNS) (9, for RNA) and the supernatant was used to identify and quantify 20). The BPDE-10-N2dG adduct was identified in the the depurination adducts by HPLC and FLNS. 32P-postlabelingmethod by comparison with the primary Analysis of Depurination Adducts by HPLC and FLNS. DNA adduct formed by BP-7,8-dihydrodiol or BPDE. For the “in vivo” experiments, the Soxhlet CHC13 extract of the In addition, the results of in vitro binding studies with epidermis, which contained depurination adducts and metabRNA or denatured DNA indicate that the double helical olites, was evaporated to dryness under vacuum. For the in structure of native DNA is a prerequisite for the formation culture experiments, the Soxhlet CHCl3 extract of the epidermis of BP-DNA adducts by one-electron oxidation. In fact, was evaporated to dryness under vacuum. The medium from with activation of BP by either microsomes or horseradish the 4 h in culture was also evaporated to dryness under vacuum, peroxidase (HRP), depurination adducts were detected and the residue was extracted 4 times with ethanol/CHC13/acetone only with native DNA. (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 Chemicals. BP-6-C8Gua, BP-6-N7Gua, and BP-6-N7Ade dryness under vacuum. (Figure 1) were synthesized by anodic oxidation of BP in the The residues were dissolved in dimethyl sulfoxide/CH3OH(1: presence of dG or dA (18,19). BPDE-lO-N7Ade (Figure 1)was 1). After sonication to enhance solubilization, the undissolved prepared by chemical reaction of BPDE with dA (19). residue in each sample was removed by centrifugation. [3H]BP (sp act. 550 Ci/mol) was purchased from Amersham The depurination adducts were analyzed by HPLC as pre(Arlington Heights, IL), whereas [3H]-(*)-trans-BP-7,8-dihyviously described (9). For each type of experiment, the depudrodiol (sp act. 767 Ci/mol) and [3H]-(i)r-7,t-8-dihydroxy-trination adducts were analyzed in four separate samples. In the 9,10-epoxy-7,8,9,10-tetrahydro-BP (anti) (sp act. 1380 Ci/mol) first experiment, the depurination adducts were identified by were purchased from the NCI Chemical Carcinogen Repository HPLC in both CH30H/H20 and CH3CN/H20 gradients in the (Bethesda, MD). The nonradiolabeled (i)-trans-BP-7,8-dihy-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,1O-tetrahy- presence of added authentic adducts. In the second experiment, drodiol and (i) the adducts were chromatographed in both solvent systems dro-BP (anti) were also purchased from the NCI Chemical without added authentic adducts, and the material eluting at the Carcinogen Repository. [3H]BP was used at a specific activity respective retention times of the authentic adducts on the of 400 Ci/mol; [3H]BP-7,8-dihydrodiol at 330 Ci/mol; and CH3CN/H20gradient was collected for identification by FLNS. [3H]BPDE at 210 Ciimol. Caution: BP, BP- 7,8-dihydrodiol In the third and fourth experiments, quantitative data were and BPDE are hazardous chemicals; they are handled in collected for calculation of the amounts of both the depurination accordance with NIH guidelines (21). and stable adducts. The amounts of each adduct varied in the Binding of BP, BP-7,8-dihydrodiol,and BPDE to Mouse two experiments by 10 to 25%, with the larger variations in the Skin DNA. In groups of eight female Swiss mice (8-weeks-old, minor adducts. Eppley Colony), an area of shaved dorsal skin was treated with
Methods
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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"
incubation system
stable DNA adducts, depurination DNA adducts, (mol of adductimol of DNA-P)X lofi (mol of adductimol of DNA-P) X lo6 adducts stable depurination ratio of (pmol/mol BPDEunidenadducts, BPDEBP-6- BP-6- BP-6adducts, depurinationi DNA-P) 10-NZdG tified 7% of total 10-N7Ade C8Gua N7Gua N7Ade 7% of total stable
4 h in vivo
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