Chem. Res. Toaicol. 1993,6, 364-371
364
Identification and Quantitation of 7,12-Dimethylbenz[alanthracene-DNA Adducts Formed in Mouse Skin Prabhakar D. Devanesan,? N. V. S. RamaKrishna,+N. S. Padmavathi,? Sheila Higginbotham,? Eleanor G. Rogan,? Ercole L. Cavalieri,*l+Glenn A. Marsch,t Ryszard Jankowiak,t and Gerald J. Smallt 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
Identification and quantitation of the depurination and stable DNA adducts of 7,12dimethylbenz[al anthracene (DMBA) formed by cytochrome P450 in rat liver microsomes previously established one-electron oxidation as the predominant mechanism of activation of DMBA to bind to DNA. In this paper we report the identification and quantitation of the depurination and stable DMBA-DNA adducts formed in mouse skin. The depurination adducts, which constitute 99 % of all the adducts detected, are DMBA bound at the 12-methyl group to the N-7 of adenine or guanine, namely, 7-methylbenz[alanthracene(MBA)-l2-CHz-N7Ade and 7-MBA-12-CH2-N7Gua. The depurination adducts were identified by HPLC and fluorescence line narrowing spectroscopy. The stable DNA adducts were analyzed by the 32P-postlabeling method. Almost 4 times as much of the depurination adduct 7-MBA-12-CHZ-N7Ade (79 % ) was formed compared to 7-MBA-12-CH2-N7Gua (20%). The stable adducts accounted for only 1% of all the adducts detected and 80% of these were formed from DMBA diolepoxide. The binding of DMBA to DNA specifically at the 12-CH3group is consistent with the results of carcinogenicity experiments in which this group plays a key role. When DMBA was bound to RNA or denatured DNA in reactions catalyzed by microsomes or by horseradish peroxidase (HRP), no depurination DNA adducts of DMBA were detected. The amount of stable DNA adducts observed with denatured DNA was 70% lower in the H R P system and 30% lower in the microsomal system compared to native DNA. In the microsomal system, just the two major adduct spots decreased, whereas the amounts of all of the adducts decreased with HRP. These results demonstrate that in mouse skin, DMBA-DNA adducts are predominantly formed via one-electron oxidation and that the adducts formed by the diolepoxide pathway are minor. The double helical structure of DNA is necessary for formation of adducts by one-electron oxidation but is not a critical factor in the diolepoxide pathway.
Introduction Knowledge of the mechanism of metabolic activation of polycyclic aromatic hydrocarbons (PAH) provides essential information for understanding the process of tumor initiation. Based on the metabolism of PAH and the structures of their DNA adducts, two main pathways for the activation of this class of compounds have been elucidated: one-electron oxidation that produces radical cation intermediates and monooxygenation that produces diolepoxide intermediates (1-4). Cavalieri and Rogan have suggested that individual PAH are predominantly activated by either one of these mechanisms or by both (2). Determination of the structure of biologically-formed PAH adducts can provide evidence on the mechanism of activation. Identification and quantitation of the DNA adducts formed by 3-methylcholanthrene (MC)-induced
* To whom correspondence should be addressed.
University of Nebraska Medical Center. Iowa State University. BP, benzo[alpyrene; DMBA, 7,12-dimethylbenz[alanthracene;FLN, fluorescence line narrowed FLNS, fluorescence line narrowing spectroscopy; HRP, horseradish peroxidase; MBA, methylbenz[a]anthracene; MC, 3-methylcholanthrene; PAH, polycyclic aromatic hydrocarbon(s); PDA, photodiode array detector. +
' Abbreviations:
rat liver microsomes demonstrated that benzo[alpyrene (5) and 7,12-dimethylbenz[alanthracene(DMBA) (6) are activated primarily by one-electron oxidation to form adducts that are lost by depurination. DMBA-3,4-dihydrodiol,precursor to the bay-regiondiolepoxide, has been shown to be a potent tumor initiator in mouse skin (7). DMBA-DNA adducts formed in mouse epidermis by enantiomersof tram-3,4-dihydrodiolvia their DMBA-3,Cdihydrodiol 1,2-epoxideshave been reported (8). Three stable DNA adducts arising from reaction of bay-region diolepoxides of DMBA with dA and dG have been previously identified (9,101. Four stable adducts in rat mammary gland have been identified as those formed by diolepoxides with dG or dA (11). Analysis by the 32Ppostlabeling technique, however, revealed several minor unidentified DMBA-DNA adducts formed in mouse skin in addition to those formed from the diolepoxides (12). One limitation of these studies is that only the stable DNA adducts were investigated, although the majority of DMBA adducts formed after microsomal activation are lost by depurination (6). Thus, to evaluate the mechanisms of activation of DMBA in vivo, we undertook a study to identify and quantitate both the stable and depurination adducts formed in mouse skin. Formation of DNA adducts
QS93-22Sx193/2706-Q364~Q4.QQiQ 0 1993 American Chemical Society
Chem. Res. Toxicol., Vol. 6, No. 3, 1993 365
DMBA-DNA Adducts Formed in Mouse Skin
7-MBA-12-CH2-N7Gua
7-MBA-12-CH2-N7Ade
Figure 1. Structures of DMBA-DNA adducts.
by DMBA activated by horseradish peroxidase (HRP) or microsomes was also studied to compare the adducts formed with native DNA, denatured DNA, and RNA. The results of this comparison demonstrate the importance of the native structure of DNA on the nature of the adducts formed. Synthetic deoxyribonucleoside adducts of DMBA (13) were used as standards in identification of the depurination adducts by HPLC and fluorescence line narrowing spectroscopy (FLNS) (6, 14,15). The stable adducts were quantitated by the 32P-postlabelingtechnique (16). We report here that depurination adducts (Figure 1) account for 99% of the total adducts detected in mouse skin and that 80% of the detected stable adducts are formed from DMBA-trans-3,4-dihydrodiol. We also report that the double helical structure of native DNA is necessary for the formation of depurination adducts obtained by one-electron oxidation.
Methods Chemicals. Authentic 7-methylbenz[a]anthracene (MBA)12-CH2-N7Gua,7-MBA-12-CHz-N7Ade (Figure l),12-MBA-7-
CHz-N7Gua, and 12-MBA-7-CHz-N3Adewere synthesized by anodic oxidation of DMBA in the presence of dG or dA (13). [3H]DMBA(sp act. 57 Ci/mmol) was purchased from Amersham Life Sciences (Arlington Heights, IL), and DMBA-trans-3,4dihydrodiol was purchased from the NCI Chemical Carcinogen Repository (Bethesda, MD). [3HlDMBA was used at a specific activity of 370 Ci/mol. Caution: DMBA and DMBA-3,4dihydrodiol are hazardous chemicals; they are handled in accordance with NIH guidelines (17). Binding of DMBA and DMBA-3,4-dihydrodiol to Mouse Skin DNA. In groups of eight mice (female 8-week-old Swiss, Eppley Colony), a shaved area of dorsal skin was treated with 200 nmol of [SHIDMBA in 50 pL of acetone and the mice were either allowed to live for 4 h or they were sacrificed immediately and the treated area of skin was maintained for 4 h in culture medium. In one type of experiment ("in vivo"), duplicate groups were killed after 4 h, and the treated area was excised. In the other ("in culture"), duplicate groups were killed immediately, and the treated area was excised and maintained for 4 h a t 37 "C in 95% air/5% CO2 atmosphere in RPMI-1640 medium with 10% fetal bovine serum and 15 mM N-(2-hydroxyethyl)piperazine-Nf-(2-ethanesulfonic acid) (HEPES). The culture medium was reserved for analysis of depurination adducts. Epidermis from each group was prepared (181, pooled, ground in liquid Nz, and split into two equal samples weighing approximately 1 g. One was used to purify DNA (16)and to analyzethe stable adducts by the 32P-postlabelingmethod (16). The other was Soxhlet extracted for 48 h with CHC13to recover the depurination adducts, and the adducts were analyzed by HPLC and FLNS (6). The amount of DNA in each sample of ground epidermis was determined by the diphenylamine reaction (19) to be 8.5 0.4 Wmol of DNA-P/g of epidermis. The level of binding in vivo was about 1 rmol of DMBA/mol of DNA-P. Two groups of four mice were treated with 200 nmol of DMBA3,4-dihydrodiol in 50 WLof acetone and the mice were sacrificed after 4 h. The treated skins were excised,epidermis was prepared (18) and ground in liquid Nz, and DNA was purified.
*
loa
P 0
151
f 500
250
TIME, MIN
Figure 2. Initial HPLC separation of depurination adducts. The YMC 5-rm ODs-AQ reverse-phase analytical column (6.0 X 250 mm) was eluted with 30% CH30H in HzO for 5 min, followed by a linear gradient to 100% CH30H in 75 min a t a flow rate of 1.0 mL/min.
Devanesan et al.
366 Chem. Res. Toxicol., Vol. 6, No. 3, 1993 Binding of DMBA 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 quickcooling in ice-water. Yeast RNA, type IIS (3.0 mM, Sigma Chemical Co., St. Louis, MO) was used. As previously described (16), [3H]DMBA (80 pM) was bound to native and denatured DNA in reactions catalyzed by MC-induced rat liver microsomes, and [3H]DMBAwas also bound to DNA and RNA by HRP. All of the DNA and RNA reactions were 15mL in volume; they were incubated for 30 min at 37 OC. At the end of the reaction, a 1-mL aliquot of the mixture was used to determine the level of DMBA binding to DNA or RNA and the amount of stable DNA adducts by the 32P-postlabeling P1 nuclease enrichment method (16). The DNA or RNA from the remaining 14-mLmixture 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 CHC13extract of the epidermis was evaporated to dryness under vacuum. The medium from the 4 h in culture was 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 CHC13 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 from the in vitro samples and the in vivo and in culture mouse skin samples were dissolved in dimethylsulfoxide/ CH30H (1:l). 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 (6). 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/H20and CH3CNiHzO 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/H20gradient 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 30 % ,with the larger variations in the minor adducts. Identification of the depurination adducts by FLNS was conducted as previously described (6, 13, 14). Calculationof Adduct Levels. The amount of stable adducts was calculated by the 32P-postlabeling method as previously described (16). For quantitation of the depurination adducts, each of the peaks eluting at 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/H20gradient and the percentage of the injected radioactivity eluting at 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 skin epidermal sample.
Results and Discussion Identification of Adducts Formed in Mouse Skin. Mouse skin was treated with DMBA either in vivo or in culture. The DNA depurination adducts observed in
t
200c
lam
m.00
s.00
ram
sam
60.00
70.33
aam
TIME, MIN
Figure 3. Rechromatographyof individual depurination adducts (A) 7-MBA-12-CHz-N7Adeand (B) 7-MBA-12-CHz-N7Gua.The analytical column was eluted with 20% CH&N in HzOfor 5 min, followed by a linear gradient to 100% CH3CN in 80 min a t a flow rate of 0.8 mL/min.
mouse skin 4 h after treatment with DMBA were identified by comparison of their HPLC elution times and FLN spectra with those of authentic standards. The stable adducts were partially identified by the 32P-postlabeling method. Two depurination adducts formed in mouse skin by oneelectron oxidation of DMBA were separated by HPLC in two consecutive solvent systems. The adducts were first separated by using a CH30H/H20 gradient (Figure 2) and peaks corresponding to the retention times of the two standard adducts were collected. Each peak was then rechromatographed with a CH3CN/H20 gradient (Figure 3) to obtain the individual adducts for identification by FLNS or quantitation (see Methods). Both adducts are formed by substitution at the 12-CH3 group of DMBA, namely, 7-MBA-12-CHz-N7Ade and 7-MBA-l2-CHz-N7Gua (Figure 1). The corresponding adducts substituted at the 7-CH3 group of DMBA were not detected. This selective formation of adducts at the 12-CH3was previously observed in the cytochrome P450catalyzed binding of DMBA to DNA in vitro (6). The structures of the depurination adducts were confirmed by comparing their fluorescence line narrowed (FLN) spectra with those of authentic standards (Figures 4 and 5). The standard FLN spectra of the electrochemically synthesized 7-MBA-l2-CHz-N7Ade adduct are shown as the upper spectra (a) in Figure 4. Excitation wavelengths of 392.8 nm (A) and 390 nm (B) provide the SI state vibronic excitations of the 7-MBA-12-CHzchromophore, and prominent zero-phonon origin bands due to -200 to -700 cm-' vibrations are observed. The middle (b) and bottom (c) spectra correspond to the
Chem. Res. Toxicol., Vol. 6, No. 3, 1993 367
DMBA-DNA Adducts Formed in Mouse Skin
.-
,-, I
c
-
396
,
,/ ,
,
,
!
ex=390 nm
,
I
396
400
398
r
398
400
Wavelength (nm)
Figure 4. Comparison of vibronically excited FLN spectra of the synthesized 7-MBA-12-CH2-N7Adestandard (a) with FLN spectra of depurination adducts from mouse skin in vivo (b) and in culture (c). Spectra were obtained for excitation wavelength A,, = 392.8 nm (A) and A,, = 390 nm (B). Bands are labeled according to excited-state vibrational frequencies (cm-1). T = 4.2 K. Gate delay 50 ns; gate width 50 ns.
396
398
400
Wavelength (nm) Figure 5. Comparison of a vibronically excited FLN spectrum of the synthesized 7-MBA-12-CH2-N7Guastandard (a)withFLN spectra of a second depurination adduct from mouse skin in vivo (b) and in culture (c). Spectra were obtained at T = 4.2 K in standard glass with A,, = 390 nm. Gate delay 50 ns; gate width 50 ns.
depurination adducts isolated from mouse skin in vivo and in culture, respectively. These spectra are identical to that of the synthesized 7-MBA-12-CHz-N7Ade adduct. The spectra in Figure 5 identify, in a similar manner, the second major isolated depurination adduct as 7-MBA12-CHz-N7Gua. Additional support for these assignments was provided by FLN spectra obtained with other excitation wavelengths (data not shown). We think it is highly improbable that the HPLC cuts analyzed by FLNS were significantly contaminated by unknown compounds. The high selectivity of FLNS, which is apparent in this and the accompanying paper (201,was demonstrated in several early papers (for a review see ref 21). For example, FLNS can distinguish between BPDE-10-N2dG in external, basestacked and quasi-intercalative helix configurations and cis vs trans conformations (22). In addition, FLNS can distinguish between the tetraol of BPDE and the BPDE-
Y
, 396
I
398
400
402
Wavelength (nm) Figure6. FLNspectraoftheDMBAmetabolite 7,12-(CH~0H)~BA, obtained at T = 4.2 K in standard glass with A,, = 392.8, 388.0, and 390.0 nm. Gate delay 25 ns; gate width 50 ns. 10-N2dG adduct (22, 23). The selectivity stems to a considerable extent from the ability to generate sharp FLN spectra with a series of excitation wavelengths, as well as gated detection. In fact, in the early stages of this research, FLNS was routinely used to determine whether or not HPLC cuts contained the adducts of interest. The FLN spectra of 7,12-(CHzOH)2-BA, obtained for excitation wavelengths of 392.8, 388.0, and 390.0 nm (Figure 61,illustrate the selectivity further. Comparison of these spectra with those of Figures 4 and 5 shows that this compound is easily distinguished from the two adducts. Based on our experience with polar metabolites whose parent fluorophore is the same as that of the adducts of interest, it is unlikely that the spectra of the synthesized standard and the isolated adducts would agree so well if contamination by related metabolites (or even unknown one-electron oxidation adducts) was greater than 10$7,. Contamination by metabolites and adducts in which one or more of the benzene rings of DMBA is saturated is not possible due to their different retention times on HPLC. Thus, comparison of the vibronically excited FLN spectra (Figures 4 and 5) clearly demonstrates that the depurination adducts 7-MBA-12-CHz-N7Ade and 7-MBA-12CHz-N7Gua are formed in mouse skin both in vivo and in culture. By using the 32P-postlabeling method, four stable adducts were detected in mouse skin DNA treated in vivo and three adducts in the in culture skin samples (Figures 7A,B). Two adducts were detected when the skin was treated in vivo with DMBA-truns-3,4-dihydrodiol (Figure 7C). These adducts have yet to be identified, but the mobilitiesof the adduct spots suggestthat the major adduct (1)formed by DMBA-truns-3,4-dihydrodiol corresponds to the major stable adduct (1)formed in the skin by DMBA. DMBA-trans-3,4-dihydrodiol has been shown to form DNA adducts via the bay-region diolepoxide, DMBA-3,4dihydrodiol 1,2-epoxide (8). The level of stable DMBA adducts observed by 32P-postlabelingcompared to that of BP (20)is about 20-fold lower, although the level of binding of both compounds to DNA in mouse epidermis is about the same. We think this is a problem of incomplete enzymic reactions in the 32P-postlabelingmethodology.
368 Chem. Res. Toxicol., Vol. 6, No. 3, 1993
Devanesan et al.
Figure 7. Autoradiogram of 32P-postlabeledDNA containing DMBA adducts formed in mouse skin treated with (A) DMBA in vivo, (B)DMBA in culture, or (C) DMBA-3,4-dihydrodiol in vivo. The film was exposed a t room temperature for 36 h (A and B) or 6 h
(0. The profile of the DMBA adducts in mouse skin (Figure 7) differs from that obtained with rat liver microsomes (Figure 8A,B and ref 6). A difference was first observed by Bigger, et al. (24-26). The diolepoxide adduct was the most abundant in mouse skin and mouse embryo cells, but with activation by rat liver microsomes, the most abundant appeared to be adducts formed from the K-region of DMBA and the diolepoxide adducts were just minor. In our experiments, this seems to be an intrinsic difference between mouse skin and rat liver microsomes, since little or no repair of DMBA-DNA adducts would be expected during the 4-h treatment period. Quantitation of Adducts Formed in Mouse Skin. The 32P-postlabelingmethod, with P1nuclease enrichment of the adducted nucleotides, was utilized for quantitation of the stable adducts. The efficiencyof the different steps in the method was not established and, hence, the values reported are minimum values. The amount of DNA in the skin samples was determined to be 8.5 f 0.4 pmol DNA-P/g of epidermis, based on numerous determinations by the diphenylamine reaction (19). Quantitation of the depurination adducts was achieved by HPLC with a radiation flow monitor. Four hours after treatment of mouse skin with DMBA, at least 99% of the DNA adducts observed were depurination adducts formed by one-electron oxidation (Table
I). Both types of skin samples, in vivo and in culture, contained 79 % of the adducts as 7-MBA-12-CHrN7Ade and 20 % of the adducts as 7-MBA-12-CH2-N7Gua (Table I). The stable DNA adducts constituted less than 1% of the DMBA adducts detected in mouse skin. The overall level of binding was probably lower in culture than in vivo, but the recovery of depurination adducts was higher in culture than in vivo, where adducts are lost from the tissue during the 4 h after treatment with DMBA. The major stable adduct (1)detected from DMBA in mouse skin, which constitutes approximately 80% of the stable adducts, appears to correspond to the major adduct (1) detected from DMBA-trans-3,4-dihydrodiol (Figure 7). Treatment of mouse skin with DMBA-trans-3,Cdihydrodiol produced two stable addu-, with the major adduct (1)accounting for 90% of the adducts detected (Figure 7C). DMBA Adducts Formed in Vitro with RNA and Denatured DNA. Activation by HRP or rat liver microsomes was used to compare the DMBA adducts formed in RNA or denatured DNA to those observed with native DNA (Table 11). This study was conducted to determine whether the double helical structure of DNA is required for adduct formation. In addition, information was obtained on the ability of RNA to form depurination adducts by one-electron oxidation. The depurination
Chem. Res. Toxicol., Vol. 6, No. 3, 1993 369
DMBA-DNA Adducts Formed in Mouse Skin
Table I. Quantitation of DMBA-DNA Adducts Formed in Mouse Skina stable DNA adducts, (mol of adduct/mol of DNA-P) X
incubation svstem
total adducts (fimol/mol DNA-P)
unidentified
4.19 1.22
0.036' 1.22d
7.53
0.005e
depurination DNA adducts, (mol of adduct/mol of DNA-P) X 106
lo6
stable adducts, % of total
7-MBA-12- 7-MBA-12CH2-N7Ade CH2-N7Gua
depurination ratio of adducts, depurinationl % of total stable
~~
4 h in vivo DMBA DMBA-3,4-dihydrodiol 4 h in culture DMBA
0.9
100
3.32 (79)b 0.83 (20) N.D., N.D.
99
5.99 (80)
99.9
0.07
1.53 (20)
115.3 1504
Values are the average of determinations from two preparations. The amount of each adduct varied between 10 and 30% in the two preparations, with larger variations with the minor adducts. Number in parentheses is percentage of total adducts. 4 adduct spots. 2 adduct spots. e 3 adduct spots. f N.D. = not determined. a
Table 11. Quantitation of DMBA-DNA Adducts Formed in Vitroa stable DNA adducts, (mol of adduct/mol of DNA-P)
incubation system MC-microsomes native DNA denatured DNA HRP native DNA denatured DNA RNA
total adducts (pmollmol DNA-P)
unidentified
X
lo6
depurination of DNA adducts, (mol of adduct/mol of DNA-P) X lo6
stable adducts, % oftotal
7-MBA-12CH*-N7Ade
7-MBA-12CHz-N7Gua
depurination adducts, % of total
9.8 0.77
1.1c 0.77d
11 100
7.2 (73)b
