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32P-postlabeling of 1,3-butadiene and 4-vinyl-1-cyclohexene metabolite-DNA adducts: in vitro and in vivo applications
ELSEVIER
Toxicology 113 (1996) 341-344
32P-postlabeling of 1,3-butadiene and 4-vinyl-1-cyclohexene metabolite-DNA adducts: in vitro and in vivo applications Nathalie Mabon, Kurt Randerath* Division of Toxicology, Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030, USA
Abstract
Epoxides of 1,3-butadiene, i.e. 1,2-epoxy-3-butene and 1,2:3,4diepoxybutane are DNA-reactive metabolites. No methods have been reported for detecting DNA adducts of these compounds in exposed animals or humans. The purpose of this study has been to develop a 32P-postlabeling assay for adducts of 1,3-butadiene and its dimer, Cvinyl-1-cyclohexene. The assay was applied to skin DNA of mice exposed topically to diepoxides of these dienes. A dose-dependent increase in in vivo adduct formation was observed for both compounds. The newly developed assay will find applications in mechanistic studies on these and related compounds and in human biomonitoring. Keywords:
1,3-Butadiene; rl_Vinyl-1-cyclohexene;
Diene diepoxides; 32P-postlabeling
DNA adducts; Mouse skin
1. Introduction
The diepoxides vinyl-1-cyclohexene
of 1,3-butadiene (BD) and 4(VCH) (Fig. 1) are rodent
carcinogens (van Duuren et al., 1963; IARC, 1976; Chhabra, 1989). As aliphatic epoxides, they represent ultimate electrophiles acting through covalent binding to informational macromolecules (DNA, RNA, and protein). Both BD and VCH are carcinogens to which humans are exposed occupationally. The diepoxide of BD, 1,2:3,4-diepoxybutane (BDE), is considerably more mutagenic than the corresponding monoepoxide (de Meester, 1988) and, therefore, is viewed as the ultimate carcinogenic metabolite of BD (reviewed by Melnick and Kohn, 1995; Bond
*Corresponding author, Tel: + 1 713 798 4465; fax: + 1 713 798 3 145.
et al., 1995), with mice being more susceptible than rats to the carcinogenic action of BD. The human carcinogenicity of these compounds remains an unresolved issue, however (Bond et al., 1995; Melnick and Kohn, 1995). DNA adduct formation has been reported for BD epoxides in vitro, with guanine and adenine being targets (Citti et al., 1984; Leuratti et al., 1994). No data are available on DNA adduct formation for VCD in vitro or in vivo. Existing methods for the detection of DNA adducts formed from these diepoxides and their monoepoxide analogues are not sufficiently sensitive for in vivo applications in exposed animals or humans. We have modified the 32P-postlabeling assay for DNA adducts (Randerath et al., 1981, 1989) in order to make it applicable to BD- and 4-vinyl-l-cyclohexenemetabolite adducts.
0300-483X/96/$15.00 0 1996 Elsevier Ireland Ltd. All rights reserved PI1 SO300-483X(96)03470-1
N. Mabon, K Randerathl Toxicology 113 (19%) 341-344
342
‘“\“-c”-cH
Ii& -
\,/
1,3-b&dime
=
diepoxide
LA”
0
2
O
4-wnyl-l-cyclohexene
diepoxide
(VW
WE)
Fig. 1. Structures of BDE and VCD.
2. Materials and methods 2.1. ModiJication of DNA
derath et al., 1989) under modified chromatographic conditions. Thus, adducts from 5 pg DNA were resolved by 2-dimensional PEI-cellulose TLC. The solvents were 0.3 M LiCl (first dimension, Dl), 0.2 M NaCl, 0.02 M H,BO,, pH 7.6 (D2) for BDE adducts; and 0.3 M LiCl (Dl), 0.4 M Tris chloride, pH 8.2 (D2) for VCD adducts. Adducts were located by screen-intensified autoradiography and quantified by scintillation counting. Data were expressed as relative adduct labeling (RAL x 10’) values, representing minimum estimates of the number of adducts in 10’ DNA nucleotides (Randerath et al., 1989).
Calf-thymus DNA (1.6 mM DNA-P) was incubated with 40 mM BDE or VCD in 10 mM Tris buffer, pH 7.4, for 16 h at 37°C. After ether extraction, DNA was precipitated with ethanol. For in vivo modification of female ICR mouse DNA, topical applications of acetone (150 ~1) or different doses of BDE or VCD (17, 51, and 153 pmol/mouse in 150 ~1 acetone) were performed for 3 days. Skin DNA was isolated from five individual mice/group 5 h after the third treatment.
3. Results Chromatographic adduct patterns were similar in vitro and in vivo (Fig. 2), but differed for the two carcinogens. Re-chromatography of isolated individual in vitro and in vivo adducts showed identity of corresponding adducts except for BDE spot 5 (data not shown). A significant (r > 0.99) linear dose-response was documented for BDE in vivo (Table l), with a total RAL x lo8 value of 26.6 at the lowest dose (17 pmol/mouse). Preliminary in vivo studies also showed a dose-dependent response for VCD, with total RAL x 10’ values being lower than for BDE (Table 1).
2.2. 32P-postlabeling Skin DNA adducts were measured by the 32P-postlabeling assay (Ranmonophosphate CON
BDE
VCD
BDE
VCD
IN VITRO . CON
Fig. 2. Representative autoradiograms of TLC maps from control (CON) and BDE- and VCD-modified DNA. Animal treatments were with 1.53 pmol of each compound. BDE-DNA adduct levels were determined after subtraction of the corresponding background spots (nos. 2’ and 53, which were also detected in the in vivo control samples.
N. Mabon, K Ranakrathl Toxicology 113 (19%)
343
341-344
Table 1 Dose.-dependent formation of major DNA adducts in mouse skin treated with BDE or VCD Dose. (ymol/mouse)
Adduct levels (RAL x 10” + S.E.M.) 1
2
3
Total
rb
5.2 f 0.2 10.6 + 0.5 32.6 + 1.0 > 0.99
10.6 + 0.9 23.3 k 0.4 70.2 f 0.5 > 0.99
10.8 + 0.7 25.2 f 1.8 82.8 k 4.9 > 0.99
26.6 If: 1.8 59.1 + 2.5 185.6 + 5.3 > 0.99
17’ 51’ 153’
NDd 19.5 f 2.3 69.9 + 2.4
ND 6.0 + 1.3 28.9 + 4.8
ND 4.4 f 0.3 14.4 + 1.8
ND 29.9 f. 3.4 113.2 f 14.9
17” 51” 153
“Treatment with BDE. bLinear correlation coefficient. ‘Treatment with VCD. dAdducts not consistently detected.
4. Discussion and conclusions
BDE- and VCD-DNA adducts in tissue DNA are measured by a newly developed 32P-pastlabeling assay. Low levels of adducts are detected in mouse skin in a dose-dependent manner. As shown for BDE adducts, the assay correlates linearly with the carcinogen dose. The assay is currently being developed further to increase its sensitivity (which is at present N 1 adduct in lo* DNA nucleotides) so as to make it applicable to human biomonitoring as well as to mechanistic studies in model systems. BDE (IARC, 1976) and VCD (Chhabra, 1989) are comparatively weak skin carcinogens in mice. In accord with these observations, the adduct levels measured herein were at least lOO-fold below those formed with comparable doses of potent carcinogens, such as benzo[a]pyrene and other polynuclear aromatics (Schurdak and Randerath, 1989). The new methodology, applied to human biomonitoring and mechanistic studies, is expected to answer important questions relating to the possible human carcinogenicity of BD and 4-vinyl-l-cyclohexene, such as: (i) do human tissues, like mouse but not rat tissues, have the capacity to metabolize BD to the diepoxide? (ii) are diepoxide adducts detectable in human, especially target tissue? (iii) do adducts from both olefins persist in target cells or are they readily repaired? and (iv) what is the relationship between adduct for-
mation/repair and carcinogenicity of the compounds? Thus, assays for the diepoxide adducts, and their further refinement, will provide biomarkers of internal dose and contribute to the evaluation of human risk posed by industrially produced dienes and their derivatives.
Acknowledgements
We thank Drs. E. Randerath and B. Moorthy for advice and discussion. This research was supported in part by USPHS grants CA32157 awarded by the National Cancer Institute and ES04917 awarded by the National Institute of Environmental Health Sciences, and by grant CR817863 awarded by the U.S. Environmental Protection Agency.
References Bond, J.A., Recio, L. and Andjelkovich, D. (1995) Epidemiological and mechanistic data suggest that 1,3-butadiene will not be carcinogenic to humans at exposures likely to be encountered in the environment or workplace. Carcinogenesis 16, 165-171. Chhabra, R. (1989) Toxicology and carcinogenesis of CvinylI-cyclohexene diepoxide (CAS No. 106-87-6) in F344/N rats and B6C3Fl mice (dermal studies). NTP Technical Report No. 362. U.S. Department of Health and Human Services.
344
N. Mabon. K RandwathIToxicology 113 (19%) 341-344
Citti, L., Gervasi, PG., Turchi, G., Bellucci, G. and Bianchini, R. (1984) The reaction of 3,4-epoxy-1-butene with deoxyguanosine and DNA in vitro: synthesis and characterization of the main adducts. Carcinogenesis $47-52. De Meester, C. (1988) Genotoxic properties of 1,3-butadiene. Mutat. Res. 195,273-281. International Agency for Research on Cancer (1976) Diepoxybutane. IARC Monograph, Vol. 11, IARC, Lyon, France, pp. 115-123. Leuratti, C., Jones, N.J., Ma&ante, E., Kostiainen, R., Peltonen, K. and Waters, R. (1994) DNA damage induced by the environmental carcinogen butadiene: identification of a diepoxybutane-adenine adduct and its detection by 32P-postlabeling. Carcinogenesis 15, 1903-1910. Melnick, R.L. and Kohn, MC. (1995) Mechanistic data indicate that 1,3-butadiene is a human carcinogen. Carcinogenesis 16, 157- 163.
Randerath, K., Reddy, M.V. and Gupta, R.C. (1981) a2Plabeling test for DNA damage. Proc. Nat]. Acad. Sci. USA 78,6126-6129. Randerath, K., Randerath, E., Danna, T.F., van Golen, K.L. and Putman, K. L. (1989) A new sensitive 32P-postlabeling assay based on the specific enzymatic conversion of bulky DNA lesions to radiolabeled dinucleotides and nucleoside S-monophosphates. Carcinogenesis 10, 123l1239. Schurdak, M.E. and Randerath, K. (1989) Effects of routes of administration on tissue distribution of DNA adducts in mice: comparison of 7H-dibenzo[c,g]carbazole, benzo[a]pyrene, and 2-acetylaminofluorene. Cancer Res. 49,26332638. Van Duuren, B.L., Nelson, N., Orris, L., Palmes, E.D. and Schmitt, F.L. (1963) Carcinogenicity of epoxides, lactones, and peroxy compounds. J. Nat]. Cancer Inst. 31,41-55.
Toxicology 113 (1996) 341-344
32P-postlabeling of 1,3-butadiene and 4-vinyl-1-cyclohexene metabolite-DNA adducts: in vitro and in vivo applications Nathalie Mabon, Kurt Randerath* Division of Toxicology, Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030, USA
Abstract
Epoxides of 1,3-butadiene, i.e. 1,2-epoxy-3-butene and 1,2:3,4diepoxybutane are DNA-reactive metabolites. No methods have been reported for detecting DNA adducts of these compounds in exposed animals or humans. The purpose of this study has been to develop a 32P-postlabeling assay for adducts of 1,3-butadiene and its dimer, Cvinyl-1-cyclohexene. The assay was applied to skin DNA of mice exposed topically to diepoxides of these dienes. A dose-dependent increase in in vivo adduct formation was observed for both compounds. The newly developed assay will find applications in mechanistic studies on these and related compounds and in human biomonitoring. Keywords:
1,3-Butadiene; rl_Vinyl-1-cyclohexene;
Diene diepoxides; 32P-postlabeling
DNA adducts; Mouse skin
1. Introduction
The diepoxides vinyl-1-cyclohexene
of 1,3-butadiene (BD) and 4(VCH) (Fig. 1) are rodent
carcinogens (van Duuren et al., 1963; IARC, 1976; Chhabra, 1989). As aliphatic epoxides, they represent ultimate electrophiles acting through covalent binding to informational macromolecules (DNA, RNA, and protein). Both BD and VCH are carcinogens to which humans are exposed occupationally. The diepoxide of BD, 1,2:3,4-diepoxybutane (BDE), is considerably more mutagenic than the corresponding monoepoxide (de Meester, 1988) and, therefore, is viewed as the ultimate carcinogenic metabolite of BD (reviewed by Melnick and Kohn, 1995; Bond
*Corresponding author, Tel: + 1 713 798 4465; fax: + 1 713 798 3 145.
et al., 1995), with mice being more susceptible than rats to the carcinogenic action of BD. The human carcinogenicity of these compounds remains an unresolved issue, however (Bond et al., 1995; Melnick and Kohn, 1995). DNA adduct formation has been reported for BD epoxides in vitro, with guanine and adenine being targets (Citti et al., 1984; Leuratti et al., 1994). No data are available on DNA adduct formation for VCD in vitro or in vivo. Existing methods for the detection of DNA adducts formed from these diepoxides and their monoepoxide analogues are not sufficiently sensitive for in vivo applications in exposed animals or humans. We have modified the 32P-postlabeling assay for DNA adducts (Randerath et al., 1981, 1989) in order to make it applicable to BD- and 4-vinyl-l-cyclohexenemetabolite adducts.
0300-483X/96/$15.00 0 1996 Elsevier Ireland Ltd. All rights reserved PI1 SO300-483X(96)03470-1
N. Mabon, K Randerathl Toxicology 113 (19%) 341-344
342
‘“\“-c”-cH
Ii& -
\,/
1,3-b&dime
=
diepoxide
LA”
0
2
O
4-wnyl-l-cyclohexene
diepoxide
(VW
WE)
Fig. 1. Structures of BDE and VCD.
2. Materials and methods 2.1. ModiJication of DNA
derath et al., 1989) under modified chromatographic conditions. Thus, adducts from 5 pg DNA were resolved by 2-dimensional PEI-cellulose TLC. The solvents were 0.3 M LiCl (first dimension, Dl), 0.2 M NaCl, 0.02 M H,BO,, pH 7.6 (D2) for BDE adducts; and 0.3 M LiCl (Dl), 0.4 M Tris chloride, pH 8.2 (D2) for VCD adducts. Adducts were located by screen-intensified autoradiography and quantified by scintillation counting. Data were expressed as relative adduct labeling (RAL x 10’) values, representing minimum estimates of the number of adducts in 10’ DNA nucleotides (Randerath et al., 1989).
Calf-thymus DNA (1.6 mM DNA-P) was incubated with 40 mM BDE or VCD in 10 mM Tris buffer, pH 7.4, for 16 h at 37°C. After ether extraction, DNA was precipitated with ethanol. For in vivo modification of female ICR mouse DNA, topical applications of acetone (150 ~1) or different doses of BDE or VCD (17, 51, and 153 pmol/mouse in 150 ~1 acetone) were performed for 3 days. Skin DNA was isolated from five individual mice/group 5 h after the third treatment.
3. Results Chromatographic adduct patterns were similar in vitro and in vivo (Fig. 2), but differed for the two carcinogens. Re-chromatography of isolated individual in vitro and in vivo adducts showed identity of corresponding adducts except for BDE spot 5 (data not shown). A significant (r > 0.99) linear dose-response was documented for BDE in vivo (Table l), with a total RAL x lo8 value of 26.6 at the lowest dose (17 pmol/mouse). Preliminary in vivo studies also showed a dose-dependent response for VCD, with total RAL x 10’ values being lower than for BDE (Table 1).
2.2. 32P-postlabeling Skin DNA adducts were measured by the 32P-postlabeling assay (Ranmonophosphate CON
BDE
VCD
BDE
VCD
IN VITRO . CON
Fig. 2. Representative autoradiograms of TLC maps from control (CON) and BDE- and VCD-modified DNA. Animal treatments were with 1.53 pmol of each compound. BDE-DNA adduct levels were determined after subtraction of the corresponding background spots (nos. 2’ and 53, which were also detected in the in vivo control samples.
N. Mabon, K Ranakrathl Toxicology 113 (19%)
343
341-344
Table 1 Dose.-dependent formation of major DNA adducts in mouse skin treated with BDE or VCD Dose. (ymol/mouse)
Adduct levels (RAL x 10” + S.E.M.) 1
2
3
Total
rb
5.2 f 0.2 10.6 + 0.5 32.6 + 1.0 > 0.99
10.6 + 0.9 23.3 k 0.4 70.2 f 0.5 > 0.99
10.8 + 0.7 25.2 f 1.8 82.8 k 4.9 > 0.99
26.6 If: 1.8 59.1 + 2.5 185.6 + 5.3 > 0.99
17’ 51’ 153’
NDd 19.5 f 2.3 69.9 + 2.4
ND 6.0 + 1.3 28.9 + 4.8
ND 4.4 f 0.3 14.4 + 1.8
ND 29.9 f. 3.4 113.2 f 14.9
17” 51” 153
“Treatment with BDE. bLinear correlation coefficient. ‘Treatment with VCD. dAdducts not consistently detected.
4. Discussion and conclusions
BDE- and VCD-DNA adducts in tissue DNA are measured by a newly developed 32P-pastlabeling assay. Low levels of adducts are detected in mouse skin in a dose-dependent manner. As shown for BDE adducts, the assay correlates linearly with the carcinogen dose. The assay is currently being developed further to increase its sensitivity (which is at present N 1 adduct in lo* DNA nucleotides) so as to make it applicable to human biomonitoring as well as to mechanistic studies in model systems. BDE (IARC, 1976) and VCD (Chhabra, 1989) are comparatively weak skin carcinogens in mice. In accord with these observations, the adduct levels measured herein were at least lOO-fold below those formed with comparable doses of potent carcinogens, such as benzo[a]pyrene and other polynuclear aromatics (Schurdak and Randerath, 1989). The new methodology, applied to human biomonitoring and mechanistic studies, is expected to answer important questions relating to the possible human carcinogenicity of BD and 4-vinyl-l-cyclohexene, such as: (i) do human tissues, like mouse but not rat tissues, have the capacity to metabolize BD to the diepoxide? (ii) are diepoxide adducts detectable in human, especially target tissue? (iii) do adducts from both olefins persist in target cells or are they readily repaired? and (iv) what is the relationship between adduct for-
mation/repair and carcinogenicity of the compounds? Thus, assays for the diepoxide adducts, and their further refinement, will provide biomarkers of internal dose and contribute to the evaluation of human risk posed by industrially produced dienes and their derivatives.
Acknowledgements
We thank Drs. E. Randerath and B. Moorthy for advice and discussion. This research was supported in part by USPHS grants CA32157 awarded by the National Cancer Institute and ES04917 awarded by the National Institute of Environmental Health Sciences, and by grant CR817863 awarded by the U.S. Environmental Protection Agency.
References Bond, J.A., Recio, L. and Andjelkovich, D. (1995) Epidemiological and mechanistic data suggest that 1,3-butadiene will not be carcinogenic to humans at exposures likely to be encountered in the environment or workplace. Carcinogenesis 16, 165-171. Chhabra, R. (1989) Toxicology and carcinogenesis of CvinylI-cyclohexene diepoxide (CAS No. 106-87-6) in F344/N rats and B6C3Fl mice (dermal studies). NTP Technical Report No. 362. U.S. Department of Health and Human Services.
344
N. Mabon. K RandwathIToxicology 113 (19%) 341-344
Citti, L., Gervasi, PG., Turchi, G., Bellucci, G. and Bianchini, R. (1984) The reaction of 3,4-epoxy-1-butene with deoxyguanosine and DNA in vitro: synthesis and characterization of the main adducts. Carcinogenesis $47-52. De Meester, C. (1988) Genotoxic properties of 1,3-butadiene. Mutat. Res. 195,273-281. International Agency for Research on Cancer (1976) Diepoxybutane. IARC Monograph, Vol. 11, IARC, Lyon, France, pp. 115-123. Leuratti, C., Jones, N.J., Ma&ante, E., Kostiainen, R., Peltonen, K. and Waters, R. (1994) DNA damage induced by the environmental carcinogen butadiene: identification of a diepoxybutane-adenine adduct and its detection by 32P-postlabeling. Carcinogenesis 15, 1903-1910. Melnick, R.L. and Kohn, MC. (1995) Mechanistic data indicate that 1,3-butadiene is a human carcinogen. Carcinogenesis 16, 157- 163.
Randerath, K., Reddy, M.V. and Gupta, R.C. (1981) a2Plabeling test for DNA damage. Proc. Nat]. Acad. Sci. USA 78,6126-6129. Randerath, K., Randerath, E., Danna, T.F., van Golen, K.L. and Putman, K. L. (1989) A new sensitive 32P-postlabeling assay based on the specific enzymatic conversion of bulky DNA lesions to radiolabeled dinucleotides and nucleoside S-monophosphates. Carcinogenesis 10, 123l1239. Schurdak, M.E. and Randerath, K. (1989) Effects of routes of administration on tissue distribution of DNA adducts in mice: comparison of 7H-dibenzo[c,g]carbazole, benzo[a]pyrene, and 2-acetylaminofluorene. Cancer Res. 49,26332638. Van Duuren, B.L., Nelson, N., Orris, L., Palmes, E.D. and Schmitt, F.L. (1963) Carcinogenicity of epoxides, lactones, and peroxy compounds. J. Nat]. Cancer Inst. 31,41-55.