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Formation of covalent deoxyribonucleic acid benzo[a]pyrene 4,5-epoxide adduct in mouse and rat skin
125
Cancer Letters, 14 (1981) 125-129 Elsevier/North-Holland Scientific Publishers Ltd.
FORMATION OF COVALENT DEOXYRIBONUCLEIC ACID BENZO[a]PYRENE 4,5-EPOXIDE ADDUCT IN MOUSE AND RAT SKIN*
W. BAER-DUBOWSKA**, Znstitut de Recherches (France)
Ch. FRAYSSINET
Scientifiques
and K. ALEXANDROV***
sur le Cancer, B.P. No. 8, 94802
Villejuif
Cedex
(Received 27 July 1981) (Accepted 6 August 1981)
SUMMARY
The covalently bound products of [ 3H]benzo [alpyrene (BP) were determined in the DNA from the skin of mice and rats. The quality and the distribution of bound products was similar. The formation of products corresponding to 4,5-dihydro-4,5-epoxy benzo [a] pyrene (BPE) and to a further metabolite of 9-hydroxybenzo[a]pyrene bound to DNA were found in the 2 species at the 24 h end point.
INTRODUCTION
The covalent binding to DNA of BP metabolites is believed to be one of the critical biological events causing tumors [ 131. The studies of Brookes and Lawley [ 71 and more recently of Phillips et al. [ 151 showed a positive correlation between the levels of binding to DNA and the carcinogenic potencies of polycyclic aromatic hydrocarbons. Subsequent work has shown *This research was partially supported by the DGRST (No. 80-70449). **Present address: Department of Biochemistry, Institute of Bioanalysis and Environmental Protection, Academy of Medicine, GrunwaIdzka 6, 60-780 Poznan, Poland. ***TO whom reprint requests should be addressed. Abbreviations: BP, benzo[a]pyrene; (+) BPDE I, (~)-(7~,8a)dihydroxy-(9a,lOa)epoxy7,8,9,10-tetrahydrobenzo[a]pyrene; (*)BPDE II, (~)-(7~,8a)-dihydroxy-(S~,lO~)epoxy7,8,9,10-tetrahydrobenzo[a]pyrene; (7R)BPDE IdA, (7R)-N’-[10-(7p,8a,9ar_trihydroxy7,8,9,10-tetrahydrobenzo[a]pyrene)yl]deoxyadenosine; (7R) or (7S)BPDE ldG, (7R)or (7S)-N1-[10-(7p,~,9~-trihydroxy-7,8,9,lO-tetra[a]pyrene)yl]deoxyguanosine; BPDE II-dG, N2-[ 10-(78,8~,9p-trihydroxy-7,8,9,lO-tetrahbenz[a]pyrene)yl] deoxyguanosine; BPE, 4,5dihydroxy-4,5epoxybenzo[a]pyrene; BPE-DNA, covalent adduct of BPE to DNA; HPLC, high-pressure liquid chromatography; 90HBP-4,5epoxide, 9-hydroxybenzo[a]pyrene_4,5epoxide; g-OH BPEdG, covalent adduct of 9-OH BPE to deoxyguanosine. 0304-3835/81/000~000/$02.50 o 1981 Elsevier/North-Holland
Scientific Publishers Ltd.
126
that the BP diol epoxide (BPDE)-DNA is the major adduct formed in mouse skin [8] and many diverse biological systems (for review see Ref. 2) with the preferential formation of (7R)BPDEdG and minor contribution of BPDE IIdG and (7S)BPDEdG. (7R)BPDE I is considered the ultimate carcinogenic metabolite [ 2,6,15]. Although BPDE has received much attention, it has been shown to be less active than BP as a complete carcinogen or skin tumor initiator (for review see Ref. 6). The possibility of involvement of other metabolites has been suggested [lo]. Using LH20 chromatography, Pelkonen et al. [14] found the formation in mouse skin of benzo[a]pyrene4,5-oxide-DNA while others identified it as 9-OH-BP-4,5-oxide-DNA [ 171 adduct. Benzo [al pyrene-4,5-oxide and 9-OH-benzo [a]pyrene (90H BP) are weak and poor carcinogens, respectively. It is known that rat skin is resistant to BP carcinogenesis [4]. In our previous study we found lower binding levels of BP to rat skin DNA and the formation of (7R)BPDE IdA. In this study we report the formation of BP4,5-oxide-DNA adducts in skin susceptible (mouse) and resistant (rat) to BP carcinogenesis. MATERIALS
AND METHODS
Chemicals T’ritium labeled BP (40 Ci/mmol) was purchased from the Radiochemical Centre, Amersham, Bucks, England and was diluted with cold BP prior to use to give a material of specific activity 1.9 Ci/mmol. (*)BPDE I, (+)BPDE II, tritium labeled (+ )BPDE I, g-OH BP and BP-4,5-epoxide were kindly supplied by the NC1 Standard Chemical Carcinogen Repository and Midwest Research Institute, Kansas City (U.S.A.). All other chemicals were of analytical grade. Mouse and rat skin treatment Groups of 10 female Swiss mice weighing 18-22 g and 5 male Wistar rats (150-200 g) were used in each experiment. [ 3H]BP was applied topically on the backs of animals and the epidermis removed as described previously
r31. Isolation of DNA and high-pressure liquid chromatography (HPLC) of modified deoxyribonucleosides The epidermal DNA was isolated and enzymatically degraded to deoxyribonucleosides as previously described [ 11. HPLC of modified deoxyribonucleosides was performed according to the method of Meehan et al. [12]. (*)BPDE I, (&)BPDE II, [3H](f)BPDE I, [3H](*)BPE reacted DNA and poly(dG) and [3H]9-OH BP microsome mediated binding to DNA and poly(dG) were used as fluorescent and radioactive markers for the formed BP-metabolites DNA adducts. RESULTS
AND DISCUSSION
The enzymic hydrolysates of DNA obtained from mouse and rat skins
3i
I 7 0 ; : z g
1.5
n ,II! I1 I ; I I 1.
\
,-a 69
d 2 @z2-
,-
I
! :j_ : : i n/k
-
/Y
40
;o
60
FRACTION
80
100
NUMBER
Fig. 1. HPLC profile of BP-modified nucleosides isolated from mouse and rat skin DNA pretreated with [ 3H]BP. Identification of the major and small DNA bound products was achieved by co-chromatography with deoxyribonucleoside products from (t’ )BPDE I and (k ) BPDE II reacted DNA as fluorescent markers [ 12 1. The radioactive peaks of nucleoside obtained from [3H] (*)BPDE I, [3H] (*)BPE and [‘H]9-OH BP (microsome mediated) reacted calf thymus DNA and poly(dG) were also used to identify the position of alkylated adducts. (A) Unknown; (B) g-OH BPEdG + (7S)BPDE IdG; (C) (7R)BPDE IdG; (D) (7R)BPDE II-dG; (E) (7R)BPDE I-dA; (F) (+)BPE-DNA, BPE-dG. (a) Mouse skin; (b) rat skin; fractions O-80 were eluted with 50% MeOH; fractions 80-110 were eluted with 50-100% H,O : MeOH gradient.
treated with [3H]BP were analysed by HPLC as described in Materials and Methods. Several distinct BPdeoxyribonucleoside adducts were clearly present (Fig. 1). The data presented show the resolution of 4 3H-labeled components in 50% MeOH and 1 3H-labeled component in a 50-100% MeOH gradient. The major product (peak C) co-chromatographed with the TABLE 1 BP ADDUCT DISTRIBUTION Sample
Mouse Rat
IN DNA FROM MOUSE AND RAT SKIN
% of total BP adducts’ A
B
C+D
E
F
3.2 4.1
7.4 8.6
82.4 80.2
3.8 3.5
3.2 3.6
Epidermal DNA was isolated 24 h after application of BP and BP-modified nucleosides were separated by HPLC [ 121. The relative adduct distribution was calculated from the radioactivity data of the products. Three separate experiments were performed. a (A) Unknown; (B) 9-OH BPE-dG + (7S)BPDEdG; (C + D) (7R)BPDE IdG + (7R)BPDE IIdG; (E) (7R)BPDE IdA; (F) BPE-DNA, BPE-dG.
128
BP deoxyribonucleoside adduct which is formed between (7R)BPDE I, (7R)BPDE II and deoxyguanosine. This adduct made up about 80% of the HPLC adducts (Table 1). Previous analysis of these deoxyguanosine adducts demonstrates the major contribution of (7R)BPDE I and minor presence of (7R)BPDE II [ 11,121. As previously, we observed the formation of (7R)BPDE I-dA (peak E) adducts [3]. The majority of product B is probably due to further metabolism of 9-OH BP. A similar adduct was observed with hydrolysate from calf thymus DNA or poly(dG) reacted with [3H]9-OH BP in the presence of liver microsomes (Fig. lb, dashed line). There is fluorescence spectral evidence that this product is 9-OH-4,5-oxide [17]. (7S)BPDE I has the same retention time as the product derived from 9-OH BP but its contribution to peak B seems to be minor [ 121. The product eluted early (peak A) is unknown. There is some evidence that this is reaction product between BPDE I and deoxycytidine [ 121. Our data from a 50-100% MeOH gradient show the presence of 3H-labeled products from both mouse and rat skin DNA which co-chromatographed with the product formed from BP-4,5-oxide and exogenous DNA or poly(dG). The reactions of epoxy-derivatives of polycyclic aromatic hydrocarbons with guanosine or deoxyguanosine appears to be a general property [2,9,12]. This product accounts for about 3-4% of the bound products on the HPLC profile. Its relatively small formation was similar (Table 1) in mouse and rat skin. Rat skin shows lower binding capacity [ 31. To the best of our knowledge, this study provides the first evidence that BPE-DNA adducts are formed in vivo in rat skin non-susceptible to BP carcinogenesis. The adducts are also found in mouse skin susceptible to BP carcinogenesis (Fig. 1, Ref. 14). Ashurt and Cohen found a late running product on HPLC from DNA hydrolysates of mouse skin but did not mention the nature of this adduct [2]. BP-4,5-oxide-DNA product was observed in rat lung DNA after i.v. injection of [3H]BP at the l-h end point. The major adduct appeared to result from further metabolism of BP phenols while a relatively small amount was observed with BPDE I [ 51. Lung cells A 549 show low efficiency of formation and high excisability though 20% of BPE-dG resist the excision [9]. The rate of persistence of the minor adducts and their biological role in skin carcinogenesis has still to be determined.
ACKNOWLEDGEMENTS
We wish to acknowledge the skilled technical assistance of Roland Guerry and Mrs. Agnes Clolus for assistance in preparing the manuscript. The investigation has been supported in part by grant from DGRST. No. 8070449. The authors are indebted to the Cancer Research Program of the National Cancer Institute, Division of Cancer Cause and Prevention, Bethesda, MD, U.S.A., for the supply of labeled and non-labeled benzo[a]pyrene derivatives. Dr. W.B.D. is the recipient of a French Fellowship.
129 REFERENCES 1 Alexandrov, K. and Thompson, M.H. (1977) Influence of inducers inhibitors of mixed-function oxidases on benzo(a)pyrene binding to the DNA of rat liver nuclei. Cancer Res., 37, 1443-1449. 2 Ashner, S.W. and Cohen, G.M. (1981) In uiuo formation of benzo(a) pyrene diol epoxide-deoxyadenosine adducts in the skin of mice susceptible to benzo(a)pyreneinduced carcinogenesis. Int. J. Cancer, 27, 357-364. 3 Baer-Dubowska, W. and Alexandrov, K. (1981) The binding of benzo [alpyrene to mouse and rat skin DNA. Cancer Letters, 13,47-52. 4 Berenblum, I. (1974) Carcinogenesis as a biological problem. North-Holland Publishing Company. 5 Boronjerdi, M., Kung, H., Wilson, A.G.E. and Anderson, M.W. (1981) Metabolism and DNA binding of benzo(a)pyrene in vivo in the rat. Cancer Res., 41, 951-957. 6 Brookes, P. (1979) The binding of mouse skin DNA of benzo[a]pyrene, its 7,8diol and 7,8diol9,10epoxides in relation to the tumourigenicity of these compounds. Cancer Letters, 6, 285-289. 7 Brookes, P. and Lawley, P.D. (1964) Evidence for the binding of polynuclear aromatic hydrocarbons to the nucleic acids of mouse skin: relation between carcinogenic power of hydrocarbons and their binding to DNA. Nature (London), 202, 781.-784. 8 Daudel, P., Duquesne, M., Vigny, P., Grover, P.L. and Sims, P. (1975) Fluorescence spectral evidence that benzo(a)pyrene-DNA products in mouse skin arise from diolepoxides. Fed. Eur. Biochem. Sot. Meet., 57,250-253. 9 Feldman, G., Remsen, J., Wang, T.V. and Cerrutti, P. (1980) Formation and excision of covalent deoxyribonucleic acid adducts of benzo(a)pyrene 4,5-epoxide and benzo(a)pyrene diol epoxide 1 in human lung cells A 540. Biochemistry, 19, 1095-1101. 10 Kodama, M., Ioni, Y. and Nagata, C. (1980) Dose-dependent effect of trichloropropene oxide on benzo(a)pyrene carcinogenesis. J. Cancer Res. Clin. Oncol., 98, 105.5107. 11 Koreeda, M., Moore, P.D., Wislocki, P.G., Levin, W., Conney, A.H., Yagi, H. and Jerina, D.W. (1978) Binding of benzo(a)pyrene 7,8-diol-9,lO epoxides to DNA, RNA, and protein of mouse skin occurs with high stereoselectivity. Science, 199, 778 780. 12 Meehan, T., Straub, K. and Calvin, M. (1977) Benzo(a)pyrene diol epoxide covalently binds to deoxyguanosine and deoxyadenosine in DNA. Nature, 269, 725-727. 13 Miller, E.C. (1978) Some current perspectives on chemical carcinogenesis in humans and experimental animals. Cancer Res., 38, 1479.. -1496. 14 Pelkonen, O., Boobis, A.R., Levitt, R.C., Kouri, R.E. and Nebert, D.W. (1979) Genetic differences in the metabolic activation of benzo(a)pyrene in mice. Pharmacology, 18, 281~-293. 15 Phillips, D.H., Grover, P.L. and Sims, P. (1978) The covalent binding of polycyclic hydrocarbons to DNA in the skin of mice of different strains. Int. J. Cancer, 22, 487-497. 16 Sims, P., Grover, P.L., Swaisland, A., Pal, K. and Hewer, A. (1974) Metabolic activation of benzo(a)pyrene proceeds by a diol-epoxide. Nature, 252, 326-328. 17 Vigny, P., Ginot, Y.M., Kinds, M., Cooper, C.S., Grover, P.L. and Sims, P. (1980) Fluorescence spectral evidence that benzo(a)pyrene is activated by metabolism in mouse skin to a diol-epoxide and a phenol-epoxide. Carcinogenesis, 1, 9455950.
Cancer Letters, 14 (1981) 125-129 Elsevier/North-Holland Scientific Publishers Ltd.
FORMATION OF COVALENT DEOXYRIBONUCLEIC ACID BENZO[a]PYRENE 4,5-EPOXIDE ADDUCT IN MOUSE AND RAT SKIN*
W. BAER-DUBOWSKA**, Znstitut de Recherches (France)
Ch. FRAYSSINET
Scientifiques
and K. ALEXANDROV***
sur le Cancer, B.P. No. 8, 94802
Villejuif
Cedex
(Received 27 July 1981) (Accepted 6 August 1981)
SUMMARY
The covalently bound products of [ 3H]benzo [alpyrene (BP) were determined in the DNA from the skin of mice and rats. The quality and the distribution of bound products was similar. The formation of products corresponding to 4,5-dihydro-4,5-epoxy benzo [a] pyrene (BPE) and to a further metabolite of 9-hydroxybenzo[a]pyrene bound to DNA were found in the 2 species at the 24 h end point.
INTRODUCTION
The covalent binding to DNA of BP metabolites is believed to be one of the critical biological events causing tumors [ 131. The studies of Brookes and Lawley [ 71 and more recently of Phillips et al. [ 151 showed a positive correlation between the levels of binding to DNA and the carcinogenic potencies of polycyclic aromatic hydrocarbons. Subsequent work has shown *This research was partially supported by the DGRST (No. 80-70449). **Present address: Department of Biochemistry, Institute of Bioanalysis and Environmental Protection, Academy of Medicine, GrunwaIdzka 6, 60-780 Poznan, Poland. ***TO whom reprint requests should be addressed. Abbreviations: BP, benzo[a]pyrene; (+) BPDE I, (~)-(7~,8a)dihydroxy-(9a,lOa)epoxy7,8,9,10-tetrahydrobenzo[a]pyrene; (*)BPDE II, (~)-(7~,8a)-dihydroxy-(S~,lO~)epoxy7,8,9,10-tetrahydrobenzo[a]pyrene; (7R)BPDE IdA, (7R)-N’-[10-(7p,8a,9ar_trihydroxy7,8,9,10-tetrahydrobenzo[a]pyrene)yl]deoxyadenosine; (7R) or (7S)BPDE ldG, (7R)or (7S)-N1-[10-(7p,~,9~-trihydroxy-7,8,9,lO-tetra[a]pyrene)yl]deoxyguanosine; BPDE II-dG, N2-[ 10-(78,8~,9p-trihydroxy-7,8,9,lO-tetrahbenz[a]pyrene)yl] deoxyguanosine; BPE, 4,5dihydroxy-4,5epoxybenzo[a]pyrene; BPE-DNA, covalent adduct of BPE to DNA; HPLC, high-pressure liquid chromatography; 90HBP-4,5epoxide, 9-hydroxybenzo[a]pyrene_4,5epoxide; g-OH BPEdG, covalent adduct of 9-OH BPE to deoxyguanosine. 0304-3835/81/000~000/$02.50 o 1981 Elsevier/North-Holland
Scientific Publishers Ltd.
126
that the BP diol epoxide (BPDE)-DNA is the major adduct formed in mouse skin [8] and many diverse biological systems (for review see Ref. 2) with the preferential formation of (7R)BPDEdG and minor contribution of BPDE IIdG and (7S)BPDEdG. (7R)BPDE I is considered the ultimate carcinogenic metabolite [ 2,6,15]. Although BPDE has received much attention, it has been shown to be less active than BP as a complete carcinogen or skin tumor initiator (for review see Ref. 6). The possibility of involvement of other metabolites has been suggested [lo]. Using LH20 chromatography, Pelkonen et al. [14] found the formation in mouse skin of benzo[a]pyrene4,5-oxide-DNA while others identified it as 9-OH-BP-4,5-oxide-DNA [ 171 adduct. Benzo [al pyrene-4,5-oxide and 9-OH-benzo [a]pyrene (90H BP) are weak and poor carcinogens, respectively. It is known that rat skin is resistant to BP carcinogenesis [4]. In our previous study we found lower binding levels of BP to rat skin DNA and the formation of (7R)BPDE IdA. In this study we report the formation of BP4,5-oxide-DNA adducts in skin susceptible (mouse) and resistant (rat) to BP carcinogenesis. MATERIALS
AND METHODS
Chemicals T’ritium labeled BP (40 Ci/mmol) was purchased from the Radiochemical Centre, Amersham, Bucks, England and was diluted with cold BP prior to use to give a material of specific activity 1.9 Ci/mmol. (*)BPDE I, (+)BPDE II, tritium labeled (+ )BPDE I, g-OH BP and BP-4,5-epoxide were kindly supplied by the NC1 Standard Chemical Carcinogen Repository and Midwest Research Institute, Kansas City (U.S.A.). All other chemicals were of analytical grade. Mouse and rat skin treatment Groups of 10 female Swiss mice weighing 18-22 g and 5 male Wistar rats (150-200 g) were used in each experiment. [ 3H]BP was applied topically on the backs of animals and the epidermis removed as described previously
r31. Isolation of DNA and high-pressure liquid chromatography (HPLC) of modified deoxyribonucleosides The epidermal DNA was isolated and enzymatically degraded to deoxyribonucleosides as previously described [ 11. HPLC of modified deoxyribonucleosides was performed according to the method of Meehan et al. [12]. (*)BPDE I, (&)BPDE II, [3H](f)BPDE I, [3H](*)BPE reacted DNA and poly(dG) and [3H]9-OH BP microsome mediated binding to DNA and poly(dG) were used as fluorescent and radioactive markers for the formed BP-metabolites DNA adducts. RESULTS
AND DISCUSSION
The enzymic hydrolysates of DNA obtained from mouse and rat skins
3i
I 7 0 ; : z g
1.5
n ,II! I1 I ; I I 1.
\
,-a 69
d 2 @z2-
,-
I
! :j_ : : i n/k
-
/Y
40
;o
60
FRACTION
80
100
NUMBER
Fig. 1. HPLC profile of BP-modified nucleosides isolated from mouse and rat skin DNA pretreated with [ 3H]BP. Identification of the major and small DNA bound products was achieved by co-chromatography with deoxyribonucleoside products from (t’ )BPDE I and (k ) BPDE II reacted DNA as fluorescent markers [ 12 1. The radioactive peaks of nucleoside obtained from [3H] (*)BPDE I, [3H] (*)BPE and [‘H]9-OH BP (microsome mediated) reacted calf thymus DNA and poly(dG) were also used to identify the position of alkylated adducts. (A) Unknown; (B) g-OH BPEdG + (7S)BPDE IdG; (C) (7R)BPDE IdG; (D) (7R)BPDE II-dG; (E) (7R)BPDE I-dA; (F) (+)BPE-DNA, BPE-dG. (a) Mouse skin; (b) rat skin; fractions O-80 were eluted with 50% MeOH; fractions 80-110 were eluted with 50-100% H,O : MeOH gradient.
treated with [3H]BP were analysed by HPLC as described in Materials and Methods. Several distinct BPdeoxyribonucleoside adducts were clearly present (Fig. 1). The data presented show the resolution of 4 3H-labeled components in 50% MeOH and 1 3H-labeled component in a 50-100% MeOH gradient. The major product (peak C) co-chromatographed with the TABLE 1 BP ADDUCT DISTRIBUTION Sample
Mouse Rat
IN DNA FROM MOUSE AND RAT SKIN
% of total BP adducts’ A
B
C+D
E
F
3.2 4.1
7.4 8.6
82.4 80.2
3.8 3.5
3.2 3.6
Epidermal DNA was isolated 24 h after application of BP and BP-modified nucleosides were separated by HPLC [ 121. The relative adduct distribution was calculated from the radioactivity data of the products. Three separate experiments were performed. a (A) Unknown; (B) 9-OH BPE-dG + (7S)BPDEdG; (C + D) (7R)BPDE IdG + (7R)BPDE IIdG; (E) (7R)BPDE IdA; (F) BPE-DNA, BPE-dG.
128
BP deoxyribonucleoside adduct which is formed between (7R)BPDE I, (7R)BPDE II and deoxyguanosine. This adduct made up about 80% of the HPLC adducts (Table 1). Previous analysis of these deoxyguanosine adducts demonstrates the major contribution of (7R)BPDE I and minor presence of (7R)BPDE II [ 11,121. As previously, we observed the formation of (7R)BPDE I-dA (peak E) adducts [3]. The majority of product B is probably due to further metabolism of 9-OH BP. A similar adduct was observed with hydrolysate from calf thymus DNA or poly(dG) reacted with [3H]9-OH BP in the presence of liver microsomes (Fig. lb, dashed line). There is fluorescence spectral evidence that this product is 9-OH-4,5-oxide [17]. (7S)BPDE I has the same retention time as the product derived from 9-OH BP but its contribution to peak B seems to be minor [ 121. The product eluted early (peak A) is unknown. There is some evidence that this is reaction product between BPDE I and deoxycytidine [ 121. Our data from a 50-100% MeOH gradient show the presence of 3H-labeled products from both mouse and rat skin DNA which co-chromatographed with the product formed from BP-4,5-oxide and exogenous DNA or poly(dG). The reactions of epoxy-derivatives of polycyclic aromatic hydrocarbons with guanosine or deoxyguanosine appears to be a general property [2,9,12]. This product accounts for about 3-4% of the bound products on the HPLC profile. Its relatively small formation was similar (Table 1) in mouse and rat skin. Rat skin shows lower binding capacity [ 31. To the best of our knowledge, this study provides the first evidence that BPE-DNA adducts are formed in vivo in rat skin non-susceptible to BP carcinogenesis. The adducts are also found in mouse skin susceptible to BP carcinogenesis (Fig. 1, Ref. 14). Ashurt and Cohen found a late running product on HPLC from DNA hydrolysates of mouse skin but did not mention the nature of this adduct [2]. BP-4,5-oxide-DNA product was observed in rat lung DNA after i.v. injection of [3H]BP at the l-h end point. The major adduct appeared to result from further metabolism of BP phenols while a relatively small amount was observed with BPDE I [ 51. Lung cells A 549 show low efficiency of formation and high excisability though 20% of BPE-dG resist the excision [9]. The rate of persistence of the minor adducts and their biological role in skin carcinogenesis has still to be determined.
ACKNOWLEDGEMENTS
We wish to acknowledge the skilled technical assistance of Roland Guerry and Mrs. Agnes Clolus for assistance in preparing the manuscript. The investigation has been supported in part by grant from DGRST. No. 8070449. The authors are indebted to the Cancer Research Program of the National Cancer Institute, Division of Cancer Cause and Prevention, Bethesda, MD, U.S.A., for the supply of labeled and non-labeled benzo[a]pyrene derivatives. Dr. W.B.D. is the recipient of a French Fellowship.
129 REFERENCES 1 Alexandrov, K. and Thompson, M.H. (1977) Influence of inducers inhibitors of mixed-function oxidases on benzo(a)pyrene binding to the DNA of rat liver nuclei. Cancer Res., 37, 1443-1449. 2 Ashner, S.W. and Cohen, G.M. (1981) In uiuo formation of benzo(a) pyrene diol epoxide-deoxyadenosine adducts in the skin of mice susceptible to benzo(a)pyreneinduced carcinogenesis. Int. J. Cancer, 27, 357-364. 3 Baer-Dubowska, W. and Alexandrov, K. (1981) The binding of benzo [alpyrene to mouse and rat skin DNA. Cancer Letters, 13,47-52. 4 Berenblum, I. (1974) Carcinogenesis as a biological problem. North-Holland Publishing Company. 5 Boronjerdi, M., Kung, H., Wilson, A.G.E. and Anderson, M.W. (1981) Metabolism and DNA binding of benzo(a)pyrene in vivo in the rat. Cancer Res., 41, 951-957. 6 Brookes, P. (1979) The binding of mouse skin DNA of benzo[a]pyrene, its 7,8diol and 7,8diol9,10epoxides in relation to the tumourigenicity of these compounds. Cancer Letters, 6, 285-289. 7 Brookes, P. and Lawley, P.D. (1964) Evidence for the binding of polynuclear aromatic hydrocarbons to the nucleic acids of mouse skin: relation between carcinogenic power of hydrocarbons and their binding to DNA. Nature (London), 202, 781.-784. 8 Daudel, P., Duquesne, M., Vigny, P., Grover, P.L. and Sims, P. (1975) Fluorescence spectral evidence that benzo(a)pyrene-DNA products in mouse skin arise from diolepoxides. Fed. Eur. Biochem. Sot. Meet., 57,250-253. 9 Feldman, G., Remsen, J., Wang, T.V. and Cerrutti, P. (1980) Formation and excision of covalent deoxyribonucleic acid adducts of benzo(a)pyrene 4,5-epoxide and benzo(a)pyrene diol epoxide 1 in human lung cells A 540. Biochemistry, 19, 1095-1101. 10 Kodama, M., Ioni, Y. and Nagata, C. (1980) Dose-dependent effect of trichloropropene oxide on benzo(a)pyrene carcinogenesis. J. Cancer Res. Clin. Oncol., 98, 105.5107. 11 Koreeda, M., Moore, P.D., Wislocki, P.G., Levin, W., Conney, A.H., Yagi, H. and Jerina, D.W. (1978) Binding of benzo(a)pyrene 7,8-diol-9,lO epoxides to DNA, RNA, and protein of mouse skin occurs with high stereoselectivity. Science, 199, 778 780. 12 Meehan, T., Straub, K. and Calvin, M. (1977) Benzo(a)pyrene diol epoxide covalently binds to deoxyguanosine and deoxyadenosine in DNA. Nature, 269, 725-727. 13 Miller, E.C. (1978) Some current perspectives on chemical carcinogenesis in humans and experimental animals. Cancer Res., 38, 1479.. -1496. 14 Pelkonen, O., Boobis, A.R., Levitt, R.C., Kouri, R.E. and Nebert, D.W. (1979) Genetic differences in the metabolic activation of benzo(a)pyrene in mice. Pharmacology, 18, 281~-293. 15 Phillips, D.H., Grover, P.L. and Sims, P. (1978) The covalent binding of polycyclic hydrocarbons to DNA in the skin of mice of different strains. Int. J. Cancer, 22, 487-497. 16 Sims, P., Grover, P.L., Swaisland, A., Pal, K. and Hewer, A. (1974) Metabolic activation of benzo(a)pyrene proceeds by a diol-epoxide. Nature, 252, 326-328. 17 Vigny, P., Ginot, Y.M., Kinds, M., Cooper, C.S., Grover, P.L. and Sims, P. (1980) Fluorescence spectral evidence that benzo(a)pyrene is activated by metabolism in mouse skin to a diol-epoxide and a phenol-epoxide. Carcinogenesis, 1, 9455950.