Document pBOXNx03JbnkKYXje3pkqRKjD

E n i w o n m e ~ t a lHealth Perspectives Vol 82. p p 253-257. 1989 32PAnalysis of DNA Adducts in Tissues of Benzene-Treated Rats by M. Vijayaraj Reddy,* Gary R. Blackburn,* Ceinwen A. Schreiner,* Myron A. Mehlman,* and Carl R. Mackerer* Solid tumors have been reported in the Zymbal gland, oral and nasal cavities, liver, and mammary gland of Sprague-Dawley rats following chronic, high-dose administration of benzene. The carcinogenic activity of benzene is thought to be caused by activation to toxic metabolites that can interact with DNA, forming covalent adducts. A nuclease Pl-enhanced '2P-postlabeling assay, having a sensitivity limit of 1 adduct in lo9- lo DNA nucleotides, was found suitable for measuring aromatic DNA adducts derived in vitro from catechol, benzenetriol (BT), phenol, hydroquinone (HQ),and benzoquinone ( B O , potential metabolites of benzene. When DNA specimens isolated from tissues of female Sprague-Dawley rats at 24 hr after an oral gavage dose of 200 to 500 mglkg, 5 dayslweek, in olive oil (3m u g ) for 1day, 1 week, 5 weeks, and 10 weeks were analyzed by the '*P-postlabeling procedure, no aromatic adducts were detected unequivocally with DNA samples of liver, kidney, bone marrow, and mammary gland. With Zymbal gland DNA, three weak spots at levels totaling four lesions per IO9DNA nucleotides were seen only after 10 weeks of treatment, and these adducts did not correspond chromatographically to mqjor adducts in uifm from the above specifiedcompounds. Consequently, this finding requires confirmatory experiments. This distinct adduct pattern may relate to tumor induction in this organ following benzene administration. Our results also indicate that DNA adducts derived from catechol, BT, phenol, HQ. and BQ are either not formed in viw with benzene or formed at levels below the detection limit of 1 adduct per lo9- lo DNA nucleotides. Introduction Benzene is a chemical intermediate used for the synthesis of pesticides, dyes, and plastic resins, a component of commercial gasoline, and is present in air, food, feed, tobacco, and pyrolysis products ( I ) . According to the investigations of Maltoni et al. (2)and the National Toxicology Program (S),the chronic, high-dose administration of benzene to rats produces a variety of tumors, including Zymbal gland carcinomas, oral cavity carcinomas, hepatocarcinomas, and possibly, mammary carcinomas. Studies on the mechanisms of carcinogenic action of benzene have focused on its conversion to toxic metabolites and its ability to damage DNA. Evidence suggests that benzene is hydroxylated in vivo to phenol and catechol (4,5).Phenol, which is the predominant product, can be further converted via a hydroquinone (HQ)intermediate to semiquinone and benzoquinone (BQ).Catechol may form either 1,2,Cbenzenetriol (BT),which can be oxidized to its semiquinone and quinone species, or to the ring-opened product, tmns,truns-muconaldehyde (4-6). *Environmental and Health Science Laboratory, Mobil Oil Corpordtion, Princeton, N J 08540. Address reprint requests to M. V. Reddy. Environmental and Health Science Laboratory, Mobil Oil Corporation, Princeton, NJ 08540. Semiquinones and quinones are generally considered to be toxic metabolites capable of interacting with DNA (7,8),but the genotoxicity of muconaldehyde, a hemato- toxin (9), has yet to be elucidated. BQ and HQ have been shown, by the application of HPLC and physical methods ( 7 ) ,to form adducts in vitro with calf thymus DSA, deoxyguanosine (dG),and deoxy- adenosine (dA).The major adduct formed by the reaction of dG with BQ has been characterized as (3'OH) benzetheno(N1,N')deosyquanosine ( 7 ) .In addition to BQ and HQ, phenol, catechol, BT, and 14C-benzene, have been shown to form adducts in vitro with DNA in the liver and in bone marrow mitoplasts prelabeled with 'H-dGTP or 3H-dATP, a s determined by t h e profiling of DNA hydrolysate on a Sephadex LH-20 column (8).Seven p a nine and two adenine adducts have been detected with benzene in this system (8).The question as to whether adducts similar to the in vitro products are generated in vivo has not been addressed, although administration of I4C-or 3H-benzeneto rats and mice has been shown to cause incorporation of low-level radioactivity into DNA of liver, kidney, spleen, and bone marrow ( I 0 , I I ) . In the present studies, we have addressed this question using a highly sensitive nuclease PI-enhanced "P-postlabeling assay (12,13),which allows the detection of aromatic and 2-54 REDDY ET .4L. bulky nonaromatic adducts at a frequency as low as 1 modification per lo9- lo DNA nucleotides. Our results show that in vitro adducts generated with the above aromatic benzene metabolites are not formed in vivo a t detectable levels in DNA samples isolated from the liver, kidney, and mammary gland of rats given benzene for 10 weeks. DNA from the Zymbal gland, however, showed three adducts, which appear not to correspond to the in vitro adducts from aromatic metabolites. Materials and Methods The sources of chemicals and biochemicals have been given in the accompanyingpaper (14),except where specified below. To validate the suitability of the 3ZP-postlabelingassay for benzene-type adducts, BQ-modified DNA (BQ-DNA) was prepared by incubation of rat kidney DNA (3 mM DNA-P) with BQ (9 mM, Aldrich Chemical Co.) a t 37OC for 12hr, followed by precipitation with ethanol. Control was carried out without the addition of BQ or DNA. To reduce the extent of adduction, BQ-DNA was mixed with unadducted DNA at the ratio of 1to 300. Female Sprague-Dawley rats, 4 months of age and weighing about 280 g (Charles River Laboratories, Raleigh, NC), were maintained on standard laboratory diet and water ad libitum.To prepare in vivo benzene-DNA samples, groups of rats (4 per group) were given, by oral gavage, 200 or 500 mg/kg benzene in olive oil (3mL/kg) every day, 5 dayslweek for 1day, 1week, 5 weeks, or 10 weeks; the dose was switched from 500 to 200 m g k g a t week 4 to reduce rat mortality from benzene toxicity. The rats were sacrificed at 24 hr after dosing, and the following tissues were collected liver, kidney, mammary gland, bone marrow (femur), and Zymbal gland. DNA was isolated from the tissues by a modified digestion and extraction procedure as described previously (15),except that the volumes of reagents were reduced 3-fold in the case of Zymbal glands. DNA modified in vivo with '2-acetylaminofluorene (AAF),used as a positive control, was prepared from the Zymbal glands of rats at '24 hr after a daily oral dosing of 40mglkg AAF for 5 days (14). The detection and quantitation of adducts in DNA samples (15 each) were accohplished by a nuclease PI-enhanced '$-postlabeling assay (12,181coupled with TLC performed according to method A in the accompanying paper (14)."P-Labeling of adducts without exposure of DNA digests to nuclease PI, i.e., the standard procedure, was according to previous conditions (12,16). nucleotides are, however, dephosphorylated with nuclease PI,resulting in their low recovery or loss (12,131, it was necessary to validate the nuclease PI-enhanced32ppostlabeling assay for adducts derived from aromatic henzene metabolites. The validation of the 3ZP-postlabeledadduct assay wag Rsceavrreireadl mouitnoerm Plo-ypionsgtlainbevlietdroadaddduucct tfsr.acOtinoenms wajeorreaded. tected with in vitro BQ-DNA (Fig. 1B)but not with con- trol DNA (Fig. IA).These adducted nucleotides were found to be virtually resistant to 3'-dephosphorylationby nuclease PI,since the adduct patterns, as well as adduct recoveries (data not shown), were similar with and without exposure (i.e., standard procedure) (Fig. 1Band ID) to the enzyme. The nuclease PI-mediated assay was, how- ever, about three orders of magnitude more sensitive than the standard procedure, as evident from the observed enhancement of 32Pincorporation into adducts (data not shown)and the approximately 500 times shorter film exposure required for adduct detection. Adduct 1 composed approximately82% of totalDNA modifications, which corresponded to 400 adducts per lo7nucleotides. Reducing DNA modification about 300 times by dilution A NU"CL"EcAS,E pl i STANDARD C0 2 w-11 b 1' CanbdDNA !Vlbo- Results Validation of Nuclease PIVersion of the "P-Postlabeling Assay Detection of aromatic DNA adducts by 32P-postlabeling can be enhanced three orders of magnitude by the pretreatment Of DNA digests with 9 which dephosphorylates normal nucleotides but not most ar0- matic adducted nucleotides (12,18).Since some adducted FIGI'R1E. Comparison of nuclease PIand standard assays for BQ-DNA adducts. Autoradiopams of "P-maps of labeled digests prepared from the indicated DNA samples with (A. B) and without (C,0)exposure to nuclease PI.DNA specimens (15 pg) were digested and labeled under standardand nuclease PIconditions(12).Labeled adducts were purified, transferredin situ (2.9, and resolved by PEI-cellulose TLC accordingto method A (11),except that the development in D3 solvent (bottom to top) was to 12.5 cm. Autoradiography was pP'- w&formed at 23C for 3min (A,@ and at -80% for 2 days(C,D). have been circled. OR, origin. The sensitivity of X-ray film is 4 to 5 times higher at -8ooCthan at 23C. "P-POSTLABELING OF BENZENE-DNA ADDC'CTS IN RATS 255 with unadducted kidney DNA still permitted the detection of BQ adducts, but an increase in background radioactivity was noticed both in control and treated samples (Fig.2). The background spots, some of which correspond to endogenous adducts (I-compounds) (17),reduced the sensitivity of detection for adducts of interest. The limit of detection of BQ-specific adducts varied from 1to 10adducts per 10" DNA nucleotides, depending on the position of an adduct in relation to endogenous adducts. For example, adduct 3 could be detected a t a higher sensitivity than adduct 1(Fig. 2), which in turn was detected at a greater sensitivity than adduct 2. Using the "P-postlabeling assay, we detected a major product chromatographically identical to adduct 1(Fig. L4 and 1B)when deoxyguanosine 3'-monophosphate was reacted with BQ (data not shown), showing that adduct 1is a guanine derivative. The reaction of BQ with dG has been shown to generate (3'OH)benzetheno(N1,N2)dGas the major derivative by HPLC and NMR analyses (7). These results, as well as the finding that DNA adducts are readily detectable in a dose-related fashion in Zymbal glands treated in culture with phenol, HQ, BQ, catechol, and BT (14),indicated that the nuclease PI version of "P-postlabeling assay is applicable for the sensitive measurement of adducts derived from these benzene metabolites. Analysis of In Vivo Adducts The validated nuclease PI-mediated 32P-postlabeling procedure was applied to the detection of adducts in DNA samples isolated from tissues at 24 hr after a daily oral gavage dose of 200 to 500 mglkg benzene for 1day, 1week, 5 weeks, or 10weeks. No aromatic adducts were detected unequivocally in the liver, kidney, and mam- mary gland a t any of the time points, while the Zymbal / 7- 5* # -6 03 4* ControlDNA h vitro BO-DNA (1:300 d&tjon) FIGUR2E. Effect of lowering of DNA modifications on the "P-postlabeling assay's sensitivity. In vitro BQ-DNA was diluted 300-fold with unadductedkidney DN.4 and was analyzed by the nuclease PI method. See the legend of Fig. 1 for additional details. Note that chromatographic development in the vertical direction was longer (20 cm) than that performed for maps shown in Fig. 1. Autoriadiography was at - H O T for 15 hr. A, B, and C indicate reference back- ground spots. glands showed adducts only after 10weeks of treatment. These findings are illustrated in Figure 3 with "P-maps derived from 5-week and 10-week DNA samples. For liver, kidney, and mammary glands, 32Pfingerprints of &week treated DNA specimens were both qualitatively and quantitatively very similar to those obtained with the corresponding control samples, indicating that benzene failed to elicit aromatic adducts a t detectable levels of 1 adduct in lo9nucleotides. Certain extra spots marked by arrows were seen inconsistently in treated samples and were not detectable after 10 weeks of treatment (maps not shown). The "P-map of Zymbal gland DNA after 5 weeks was also similar to that of control DNA, while the 10-week sample showed three extra spots indicative of adducts. Adduct levels corresponded to 4 lesions per lo9 DNA nucleotides. Some spots indicated by arrows, presumably endogenous adducts, were reduced after 10 weeks of benzene treatment. Bone marrow DNA isolated from rats treated with benzene for 1 day and 1week failed to show adducts (maps not shown). Adducts were readily detectable with in vitro BQ-DNA and in vivo AAF-DNA from Zymbal glands, both being used in this study as positive controls. Discussion Nuclease PI-enhanced 32P-postlabelinganalysis revealed no DNA adducts in the liver, kidney, bone marrow, and mammary gland of rats given an oral gavage dose of benzene for 1day, 1week, 5 weeks or 10weeks. Since the 3ZP-postlabelingassay is suitable for detection of DNA adducts de&ved in vitro from phenol, HQ, BQ, catechol,and BT (Fig. 1)(14),it can be concluded that adducts from these aromatic metabolites are not formed in vivo following benzene administration or are formed below detectable levels of l adduct in lo9DNA nucleotides, i.e., 0.003 fmolelpg DNA. It is conceivable that the aromatic metabolites formed from the metabolism of benzene in vivo (4,s)either undergo conjugation or bind to proteins in tissues-both are processes that can inhibit DNA adduct formation. The postlabeling assay results are not in agreement with the direct labeling findings that show a low level of radioactivity inco orated into the tissue DNA samples following 'H- or%-benzene administration to rats (10,Il).This discrepancy can,however, be explained if the in vivo adducts are predominantly nonaromatic and polar, as are those probably derived from the putative openring derivative, muconaldehyde (6). In contrast to direct labeling, the "P-postlabeling assay, under the conditions described, does not allow the recovery of such adducts. The most likely steps in the procedure at which adduct loss occurs are nuclease PI treatment of DNA digests (161,which can cause 3'-dephosphorylation of small adducts (16), and the purification of adducts by PEIcellulose TLC. However, no adducts were detected when several variations of postlabeling techniques were tried (data not shown). These included: a) labeling of adducts by standard (16,17)and intensification conditions (181, 256 REDDY ET AL. neither of which involves the exposure of DNA digests to nuclease PI;b) the resolution of adducts by a combination of reverse-phase TLC and PEI-cellulose TLC (f 5,f9,20), which allows the recovery of nonaromatic, bulky adducts (14); and c) the separation of adducts by two-dimensional PEI-cellulose TLC alone (16,21,22), which facilitates the recovery of simple alkylated prod. ucts. These variations of postlabeling are,however, three to five orders of magnitude less sensitive than the en- hanced procedure using nuclease PI (12-14). Therefore, the enhancement of the 32P-postlabelingassay's sensitiv. ity for small adducts by removal of unadducted nucltm. .a 1 i i# ' - Benzene Liver Control Kidney Benzene Kidney Control Mammary Gland Control Zymbal Gland, 5 wk Benzene Zymbal Gland, Control Zymbal Gland, 10 wk Benzene Zymbal Gland, 10 wk In Vitro BQ-DNA (1:300 dilution) AAF Zymbal Gland FIGUR3E. Autoradiograms of "P-maps obtained with DNA samples isolated from the indicated control and benzene-treated tissues. Autoradiography was performed at -80C for 48 hr (mammary glands) and 17 hr (others). A, B, and C indicate reference background spots. Adduct 1 seen with AAF-DNA has been identified previously as dpGpC8-(Nz-AF)and adduct 2 as dpGp-Nz-(C3-AAF()17). 3ZP-POSTLABELlNGOF BENZENE-DNA ADDUCTS IN RATS tides in DNA digeats by HPLC prior to 3LP-labeling would probably assist in the detection of ring-opened derivatives presumably formed with 3H-or "C-benzene (10,ll). The detection of aromatic DNA adducts in Zymbal glands after 10weeks of benzene treatment (Fig. 3)may datteo tumor formation in this Organ (2,5).Thesead- ducts apparently did not correspond chromatographically to the major adduct of BQ (Figs. 1and 2) or to the major adducts generated by the interaction of phenol, HQ,BQ, catechol, and BT with DNA in Zymbal glands maintained in culture (14). Adduct quantities from the in vivo Z P - bal gland DNA are too low (Q 0.01 fmole) to perform structural analysis by physicochemical methods, and thus, it is notpossible to identify the benzene metabo- litds) present in the 32P-postlabeledadducts. More data is needed to support the detection Of in vivo adducts, since this result is from a single time point, i.e., 10weeks (Fig. 3).Experiments indicating either the accumulation and persistence Of the upon benzene for a period longer than 10 weeks or the formation of identical adducts in other target organs should provide such supportive data. We thank Lisa A. Carmody for skillful assistance inpreparing the manuscript. This work was supportedby a Joint-Industry Research Pro- gram sponsored by Arnoco Ashland oil 1% DoW Chemical CO., Mobil Oil Corp., Standard Oil Co., and Sun Co., Inc. REFERENCES 1. Marcus, W. L. Chemical of current interest: Benzene.Toxicol. Ind. Health 3: 205-266 (1987). 2. Maltoni, C., Conti, B., Cotti, G., and Belpoggi,F. Experimental studies on benzene carcinogenicity at the Bologna Institute of On- colagy: Current results and ongoing research. Am. J. Ind. Med. 7 415-446 (1985). 3. Huff,J. E. Toucology and Carcinogenesis Studies of Benzene (CAS No. 71-43-2)in F344/N Rats and B6C3FI Mice (Gavage Studies).Technical Report No. 289.National Toxicology Program, Research Triangle, NC, 1985. 4. Cooper,K.R., and and the molecular Snyder, aspects R. of Benzene benzene metabolism: toxicity. In: TBoexnizceonltehcetaicrs- cinogenicity (M. Aksoy, Ed.), CRC Press, Boca Raton. Florida, 1988,pp. 33-58. 5. KaK, G. F. Recent advances in the metabolism and toxicity of ben- zene. Crit. Rev. Toxieol. 18:141-159 (1987). 6. Latrlano, L., Goldstein, B. D., and Witz, G. Formation of muconaldehyde and open-ring metabolite of benzene in mouse liver microsomes: an additional pathway for toxic metabolites. Proc. Natl. Acad. Sci. (US.)83:8356-8360(1986). s.,7. Jowa, L., Winkle, Kalf, G. F., WitZ, G., and Snyder, R. k X Y - guanosirieadducts from benzoquinone and hydroquinone. In: Ad- vances in Experimental Medicine and Biology: Biological Inter- mediates 111(J.Kocsis, D. J. Jollow, C. M. Witmer, J. 0. Nelson, and R. Snyder, Eds.), Plenum Publishing, 1986,pp. 825-832. 8. Rushmore, T., Snyder, R., and Kalf, G. Covalent binding of ben- zene and its metabolites to DNA in rabbit bone marrow mitochon- dria ui tvtm Chem.-Bid. Interact. 49: 133-154(1984). 9. Witz, G., Rae, G., and Goldstein, B. D.Short-term toxicity of trans,lrans-muconaldehyde.Toxicol. Appl. Pharmacal. 80:511-516 (1985). 10. Lutz, W.K..and Schlatter. C. H. Mechanism of the carcinogenic action of benzene: irre\ erslhle binding to rat liver DNA. Chem.- Biol. Interact. 18:241-245 (1977). 11. ArfeHini, G., Grilli, S., CoIacci, A., Mazzullo, M., and Prodi, G. In vivo and in vitro binding of benzene to nucleic acids and proteins of various rat and mouse organs. Cancer Lett. 28:159-168(1985). 12. Reddy, M. v.,and Randerath, K.h'uclease P,-mediated enhance- ment of sensitivity of '2P-postlabeIingtest for structurally diverse DNA adducts. Carcinogenesis 7 1543-1551(1986). 13. Reddy, M. v., and Randerath, K. "P-postlabeling assay for carcinogen-DNA adducts: nuclease PI-mediatedenhancement of its sensitivityand applications.Enviinn. Health Perspect. 76: 41-47 (1987). 14. Reddy, M. V., Blackburn, G. R., Irwin, S. E., Kommineni, C., Mackerer, C. R., and Mehlman, M. A. A method for zn vitro cul- ture of rat Zymbal gland use in mechanistic studies of benzene car- cinogenesis in combination with '2P-postlabeling.Environ. Health Perspect. 82: 239-247 (1989). 15. Reddy, M.V.,and Randerath, K. 32P-analysisof DNA adducts rn somatic and reproductive tissues of rats treated with the an- 16. tiecdandcye,rMa.nvti.b,icotuicP, mbRit,o. mc.y, chin kC. wMut,aEt..,Res. 179:75-88 (1987). K. 3zp- postlabeling test for covalent binding of chemicals in vivo:Appli- cations to a variety of aromatic carcinogens and methylating agents. Carcinogenesis 5: 231-243 (1984). 17. Gupta, R. C., Reddy, M. V., and Randerath, K. 32P-postlabeling analysisof non-radioactive mmatic carcinogen-DNA adducts.Car- cinogenesis 3: 1081-1092(1982). 18. Randerath, E., Agrawal, H.P., Weaver,J. A., Bordelon, C. B., and Randerath, K.32P-postlabelinganalysis of DNA adducts persist- ing for up to 42 weeks in the skin of epidermis and dermis of mice treated topically with 7,12-dimethylbenz(a)anthra~eneC.arcinogen- esis 6 1117-1126(1985). 19. Randerath, K.,Haglund, R. E., Phillips,D. H., and Reddy, M. V. 32P-postlabelinganalysis of DNA adducts formed in the h e r s of animals treated with safrole, estragole and other naturally occurring alkenylbenzenes. I. Adult female CD-1 mice. Carcino- genesis 5 1613-1622 (1984). 20. Reddy, M.V., Irvin,T. R., and Randerath, K. Formation and per- sistence of sterigmatocystin-DNAadducts in rat liver determined via 32P-postlabeliiganalysis. Mutat. Res. 152 85-96 (1985). 21. Randerath, K.,Reddy, M. V., and Gupta, R. C. '*P-labeling test for DNA damage.Proc. Natl. A d . Sei.(U.S.7)8: 6126-6129(1!%1). 22. Reddy, M. V., Gupta, R. C., and Randerath, K. "P-base analysis of DNA. Anal. Biochem. 117: 271-279 (1981). 23. Lu, L.-J. W., Disher, R. M., Reddy, M. V.,and Randerath, K. "P- postlabelingassay of transplacental DNA damage by the environ- mental carcinogenssafrole, 4-aminobipheny1,and benzo(a)pyrene. Cancer. Res. 4 6 3046-3054(1986).