[CANCER RESEARCH 51. 3882-3885. August 1. I991|

Elevated 8-Hydroxydeoxyguanosine Levels in DNA of Diethylstilbestrol-treated

Syrian Hamsters: Covalent DNA Damage by Free Radicals Generated byRedox Cycling of Diethylstilbestrol1

Deodutta Roy, Robert A. Floyd, and Joachim G. I iclirDepartment of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77550-2782 ¡D.R., J. 6". /../. and Molecular Toxicology Research

Program, Oklahoma Medical Research Foundation, Oklahoma City. Oklahoma 73104 [R. A. F.J

ABSTRACT

The generation of free radicals by microsome-mediated redox cyclingbetween catechol estrogens or diethylstilbestrol and their correspondingquiñoneshas previously been demonstrated in vitro. However, the reaction of free radicals with DNA has not yet been detected in animalstreated with estrogen and is the subject of this investigation. The reactionof guanine bases of DNA with hydroxyl radicals to form 8-hydroxydeox-yguanosine has been used as a monitor of free radical generation in kidneyand liver of Syrian hamsters, a species prone to estrogen-induced carci-nogenesis. Prior to in vivo measurements, the in vitro hydroxylation ofguanine bases of DNA under conditions of redox cycling of estrogen wasinvestigated. In incubations of DNA or deoxyguanosine with hamsterkidney microsomes, NADPH, and diethylstilbestrol 4',4"-quinone, the

hydroxylation of guanine bases of free deoxyguanosine or of DNA was50 to 100% higher than in controls. When incubations were carried outin the presence of iront III ) chloride, the hydroxylation of guanine baseswas 2.5- or 10-fold higher than control values. There was a 65% increasefrom control values in levels of 8-hydroxydeoxyguanosine in liver DNAof hamsters treated with 20 mg/kg/day diethylstilbestrol for 3 days and100 mg/kg on the 4th day. In hamsters treated chronically with diethylstilbestrol implants for 15 days, 8-hydroxydeoxyguanosine levels morethan doubled from control values in kidney but not liver DNA. Treatmentof hamsters with estradiol for various time periods did not induce anychanges in levels of hydroxylated guanine in either kidney or liver. It wasconcluded that in vitro and in vivo redox cycling of diethylstilbestrolhydroxylated guanine bases in DNA.

INTRODUCTION

Microsomal enzymes mediate the redox cycling of DES1 or

of catechol estrogens [reviewed by Liehr and Roy (1)]. Forinstance, DES or 2-hydroxyestradiol are oxidized by cyto-chrome P-450 to DES Q or 2,3-estradiol quinone, respectively.NADPH-dependent cytochrome P-450 reducíasecatalyzes thereduction of these quiñonesto corresponding hydroquinones,i.e., DES or 2-hydroxyestradiol (2). This redox cycling of estrogens is a mechanism to generate free radicals. For instance,Superoxide radicals are formed by NADPH-dependent cytochrome P-450 reductase-catalyzed reduction of DES Q presumably by reaction of molecular oxygen with DES semiquinone,the postulated intermediate in this reduction (3).

The generation of free radicals in vivo by redox cycling ofestrogens has not yet been demonstrated and is the subject ofthis study. The direct measurement of free radicals in vivo isvery difficult if not impossible because of the low concentrations

Received 12/5/90; accepted 5/17/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by Grants CA 43233 and CA42854 from the

National Cancer Institute, National Institutes of Health.2To whom requests for reprints should be addressed, at Department of

Pharmacology and Toxicology. University of Texas Medical Branch, Galveston.TX 77550-2782.

3The abbreviations used are: DES, diethylstilbestrol; DES Q. diethylstilbes-trol-4',4"-quinone: 8-OH-dGuo. 8-hydroxy-2'-deoxyguanosine: dGuo, 2'-

deoxyguanosine.

and short half-lives of these reactive intermediates. However, itis possible to obtain evidence of their existence by monitoringthe products of their reactions with cellular components. Several assays have been developed to measure the products generated by the free radical reactions with cellular protein orDNA. The reaction of guanine bases of DNA with hydroxylradicals to form 8-OH-dGuo has been quantitated and utilizedas evidence of free radical generation in vivo (4). Also, theoxidation of amino groups of proteins to carbonyl by freeradicals has been measured by a 'H-labeled sodium borohydride

reduction assay (5, 6). In the present study, 8-OH-dGuo hasfirst been quantitated in vitro to examine the validity of theassay under conditions of redox cycling of estrogen. Secondly,8-OH-dGuo concentrations have been measured in DNA hy-drolysates of hamsters treated with DES or 17/i-estradiol toassess the extent of free radical generation by estrogen in vivo.Hamsters have been selected for these studies because chronictreatment with DES or 17/í-estradiolfor 6 to 8 months induceskidney tumors in almost all of these animals (7). The 8-OH-dGuo content has been measured in DNA hydrolysate of kidney, a target of estrogen-induced cancer, and compared withvalues of liver, which does not develop neoplasms under thetreatment conditions.

MATERIALS AND METHODS

Materials. 17/i-Estradiol, DES, cholesterol, catatase from bovineliver, and NADPH were purchased from Sigma Chemical Co., St.Louis, MO. DNase I was obtained from Calbiochem Corp., San Diego.CA; endonuclease, phosphodiesterase I, and alkaline phosphatase werefrom Boehringer Mannheim Biochemicals, Indianapolis, IN. DES Qand 8-OH-dGuo were synthesized as described previously (8, 9). Allsolvents and common chemicals used were of analytical grade or ofhighest grade available.

Estrogen Treatment of Animals. Male Syrian hamsters (purchasedfrom Harlan/Sprague-Dawley, Houston, TX, at 8 weeks of age) wereused for all experiments.

Short-Term Treatment. A first group of hamsters received 20 mg/kgDES i.p. for 3 days and 100 mg/kg DES on the 4th day. A secondgroup of hamsters received only a single injection of DES (100 mg/kgi.p.). Control hamsters were given injections of corn oil. After 4 h, thehamsters were killed and their livers and kidneys were excised.

Chronic Treatment. Each animal was treated with one s.c. implant of17/3-estradiol or DES (25 mg of estrogen containing 10% cholesterol).Control hamsters remained untreated. After 8, 15, 90, and 150 days ofestrogen treatment, hamsters were killed by decapitation. Their liversand kidneys were excised. Tissues were homogenized in 0.25 M sucrosecontaining 10 m.MEDTA. DNA was prepared by the method of Gupta(10). Microsomes needed for the in vitro experiments were preparedfrom kidney cortex homogenate of untreated hamsters by differentialcentrifugation as described previously (11).

Incubation of DNA or dGuo with Microsomes and DES Q. Micro-some-mediated reductions of DES Q were carried out as describedpreviously to generate Superoxide radicals (3). The incubation mixturesconsisted of 4 mg kidney cortex microsomal protein, 200 ^M DES Q,50 i!M iron(III) chloride, 5 miMNADPH, and 1 mg liver DNA or dGuo

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ELEVATED 8-OH-dGuo LEVELS IN DNA OF DES-TREATED HAMSTERS

in a final volume of 1 ml 0.01 M phosphate buffer, pH 7.5. Some ofthe reactions were carried out in the presence of catalase (250 n% =1250 units). The reactions were carried out for 60 min at 37°C.DNA

or dGuo was extracted by the method of Gupta (10). Free DESmetabolites were extracted by organic solvents as described previously(2, 3).

Assay of 8-OH-dGuo. DNA was digested by incubation with DNaseI, endonuclease, phosphodiesterase I, and alkaline phosphatase according to the procedures of Beland et al. (12). The amount of 8-OH-dGuoin the DNA digest was measured by high performance liquid chroma-tography with electrochemical detection as described previously (13).In some cases, the amount of dGuo in the DNA digests was determinedby UV detection. The UV detector was placed in line between thecolumn and the electrochemical detector.

RESULTS

Hydroxylation of Guanine Bases by Redox Cycling of Estrogens. In incubations of dGuo with microsomes and DES Q, aredox cycling system known to produce free radicals (3), levelsof 8-OH-dGuo were double the values in controls incubated inthe absence of substrate (Table 1). When reactions were carriedout in the presence of iron(III) chloride, levels of 8-OH-dGuowere 10-fold higher than those in the absence of substrate. Theaddition of catalase inhibited the hydroxylation of dGuo by50%. These results confirm previously published data of hydroxylation of dGuo in incubations with DES, horseradishperoxidase, and hydrogen peroxide (14). In this latter system,DES semiquinone has been presumed to be an intermediate inthe oxidation of DES to DES Q (14).

In DNA incubated with DES Q, microsomes, and NADPH,8-OH-dGuo levels were increased by 50 or 120% from valuesof controls carried out in the absence of substrate or cofactor,respectively (Table 2). In the presence of iron(III) chloride, thehydroxylation of guanine bases was enhanced even further. Theaddition of catalase inhibited the hydroxylation of guanine inDNA by 22%.

In summary, the data demonstrate that microsome-mediatedredox cycling of stilbene estrogen stimulates the hydroxylationof guanine bases of free dGuo or within DNA by hydroxylradicals. Iron(III) chloride, which catalyzes the conversion ofhydrogen peroxide to hydroxyl radicals, enhances further thehydroxylation of guanine bases, and this reaction is inhibitedby addition of catalase.

8-OH-dGuo Levels in DNA of Estrogen-treated Hamsters.The effect of estrogen treatment of hamsters on the levels of 8-OH-dGuo was tested at two doses, a large acute dose administered by injection and an intermediate chronic dose by implantation of estrogen pellets, which are known to release approximately 60 fig/hamster/day (15). When hamsters were primed

Table 1 Formation of 8-OH-dGuo by incubation of dGuo, DES Q, and hamsterkidney microsomes

The incubation mixture consisted of 4 mg kidney cortex microsomal protein,200 MMDES Q. 5 mM NADPH. and 1 mg dGuo in the presence or absence ofnomili i chloride in a final volume of 1 ml 0.01 M phosphate buffer, pH 7.5. Insome of the incubations catalase (250 >jg= 1250 units) was added before startingthe reaction. Reactions were carried out for 1 h. Each reaction was carried out induplicate, and each experiment was analyzed twice. Thus, each value representsa mean of four measurements.

Conditions 8-OH-dGuo/dGuo x 10s

No NADPHNADPHNADPH + Fed,NADPH + DES QNADPH + FeClj + DES QNADPH + Fed, + DES Q + catalase

0.180.150.351.50.7

Table 2 Hydroxylation of guanine bases of DNA by hydroxyl radicals formed byDES Q and hamster kidney microsomes

The incubation conditions were the same as described in the legend to Table Iexcept that DNA was used instead of dGuo. Each reaction was carried out induplicate, and each experiment was analyzed twice. Thus, each value representsa mean of four measurements.

Conditions 8-OH-dGuo/dGuo x IO5

No DES QDESQDES Q + FeCljNADPHDES Q +NADPHDES Q + FeClj + NADPHDES Q + FeCI, + NADPH + catalase

8.69.4

12.513.621.028.822.5

12.&T

(•- p < 0.01. min«Student1•t-tMt)

CO

(.-pCO.OS. udng Student1•t-tMt)

Fig. 1. Levels of 8-OH-dGuo in liver (top) or kidney (bottom) DNA of hamsterstreated with a large acute dose of DES. Animals received a single injection of 100mg/kg DES (Q) or 20 mg/kg DES for 3 days and 100 mg/kg DES on the 4thday (•).Control hamsters received corn oil (•).Columns, means (n = 5). Bars,SD. dG, dGuo.

with 20 mg/kg DES for 3 days and treated with 100 mg/kgDES on the 4th day, their liver DNA contained significantlyhigher levels of 8-OH-dGuo (65% increase) than did that ofuntreated controls (Fig. 1). The corresponding values in liverDNA of hamsters treated with a single 100-mg/kg injection ofDES were unchanged from controls. The 8-OH-dGuo contentof renal DNA was unchanged after a single large injection ofDES and significantly decreased from control levels after priming and injection of 100 mg/kg DES on the fourth day.

The chronic intermediate dose of DES, achieved by implantation of hamsters with stilbene for 8 or 15 days, was selectedbecause such treatment for 6-8 months induces kidney tumors(7). The levels of 8-OH-dGuo were more than double the controlvalues specifically in renal DNA treated with DES for 15 days(133% increase) (Fig. 2). There were no changes in 8-OH-dGuoconcentrations after 8 days of treatment. 8-OH-dGuo levels inhepatic DNA of hamsters treated for 8 or 15 days were significantly decreased from control values.

The treatment of hamsters for 8 or 15 days with 17/3-estra-diol, the natural hormone, which also induces kidney tumorsin this species, did not significantly change the levels of 8-OH-dGuo from control values in either kidney or liver (Table 3).After chronic treatment for 3 or 5 months, 8-OH-dGuo levels

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ELEVATED 8-OH-dGuo LEVELS IN DNA OF DBS-TREATED HAMSTERS

0

40i

8 DATS 15 DAYS(•-p 0.03. u*>g Studwfi t-U*)

8 DAYS 15 DAYS(~ - p <0.01. Mkig Staxtenfi t-t~t)

Fig. 2. Levels of 8-OH-dGuo in liver (top) or kidney (bottom) DNA of hamsterstreated with DES implants for 8 or 15 days. Animals received implants (25-mgpellets consisting of 90% DES and 10% cholesterol) on day 0. Their kidney orliver DNAs (•)or control DNAs from age-matched untreated hamsters (•)wereanalyzed as described in "Materials and Methods." Columns, means (n = 5).

Bars, SD. <IG.dGuo.

Table 3 8-OH-dCuo levels in hamsters treated chronically with 17>1-estradiolMale Syrian hamsters received 17/<-estradiol implants at time 0 and were killed

after various time periods. Kidney DNA was isolated, and Ihe ratios of 8-OH-dGuo/dGuo x 10* were determined as described previously (13). Values areexpressed as mean ±SD (n = 5-8).

8-OH-dGuo/dGuo x10*Period

oftreatmentKidney

8 days15 days3 mo5moLiver

8 days15 days3 moControl18.4

±4.412.6 ±1.67.4 ±2.15.7 ±2.929.3

±5.721.0 ±2.2

3.1 ±1.317fi-Estradiol-

treared15.2

±5.515.6 ±4.04.2 ±1.83.0 ±1.521.6

±2.719.4 + 2.45.0 ±1.9

were not significantly different from control values. 17,0-Estra-diol treatment of hamsters for 3 months did not induce anychanges in levels of hydroxylated guanine in hepatic DNA.

In summary, acute treatment of hamsters with a large doseof DES increased 8-OH-dGuo levels in liver DNA. The chronicexposure of animals for 15 days to DES pellets doubles thelevels of 8-OH-dGuo content over control values specifically inthe kidney, the target of estrogen-induced cancer. 17/i-Estradioltreatment did not significantly change 8-OH-dGuo levels fromcontrol values.

DISCUSSION

Mechanism of 8-OH-dGuo Formation by Estrogen. The administration of DES to male Syrian hamsters at various doselevels induces the hydroxylation at C-8 of guanine bases inDNA. Moreover, microsome-mediated redox cycling of stilbeneestrogen in vitro increases 8-OH-dGuo formation by hydroxylation of either dGuo or DNA. The generation of 8-OH-dGuofrom dGuo or DNA by redox cycling of estrogen supports amechanism of hydroxylation as shown in Fig. 3. Redox cyclingbetween DES and DES Q is catalyzed by cytochrome P-450

(oxidation) or cytochrome P-450 reducíase(reduction) (2) andthus generates low steady state concentrations of DES semi-quinone. This semiquinone free radical may react with molecular oxygen to form Superoxide radicals, which in turn may beconverted to hydrogen peroxide either spontaneously or catalyzed by Superoxide dismutase. The generation of Superoxideradicals by redox cycling of stilbene estrogen has been describedpreviously (3). Quinone reducíase(DT-diaphorase), which reduces DES Q to DES in a two-electron reduction bypassing Ihesemiquinone inlermediale, inhibils this generation of free radicals (3). Hydrogen peroxide may be reduced by metal ions(iron, copper) in a Fenton-type reaction to yield hydroxylradicals, if H:O2 is not detoxified by a catalase-mediated reaction (16). The decrease in 8-OH-dGuo formation caused byaddition of catatase (Tables 1 and 2) supports Ihe mechanismproposed above.

Sleroid estrogens such as 17/i-estradiol are metabolicallyoxidized to catechol estrogens, which also are capable of redoxcycling (2). Although free radical generation by redox cyclingbetween 2- or 4-hydroxyeslradiol and iheir corresponding quiñoneshas not yet been demonstrated, the increase in carbonylcontent in proteins and the increase in fluorescent products oflipid peroxidation specifically in kidney of hamsters treatedwith 17ß-estradiol(6, 17) have been taken as evidence of freeradicals generated by redox cycling of catechol estrogens. 8-OH-dGuo levels did not rise in response to 17^-estradiol treatment of hamsters. However, the time course of guanine hydroxylation in response to 17/tf-estradiol treatment may bedifferent from that following DES treatment. Furthermore, therepair mechanisms involved may occur at a rate such that thesteady state levels of 8-OH-dGuo may not be greater thanbackground levels. Free radical generation by redox cycling ofcatechol estrogens in vivo will also be dependent on the optimalconversion of 17/i-estradiol to catechol estrogens. Activities ofestrogen 2- or 4-hydroxylases are known to decrease in response

to estrogen treatment (18).Conditions of 8-OH-dGuo Formation. Elevated 8-OH-dGuo

levels require an increase in metabolic events which supportredox cycling and free radical generation and/or a decrease inspecific DNA repair activity. For instance, a very large dose ofDES may result in high levels of free radicals only in liverbecause of high hepatic metabolic capacity. Consequently, highhepatic 8-OH-dGuo formation may exceed DNA repair in thisorgan. In contrast, under chronic treatment conditions with

. DES

HjO.Oj

Fig. 3. Proposed mechanism of hydroxylation of guanine bases by redox cyclingof DES. Redox cycling between DES and DES Q is mediated by microsomalenzymes, i.e., cytochrome P-450. 1, and cytochrome P-450 reducíase,2. Theintermediate in this cycling. DES semiquinonc, may react with molecular oxygenand form Superoxide radicals (3). Superoxide is converted to hydrogen peroxideby Superoxide dismutase. 3. Hydrogen peroxide may be reduced to hydroxylradicals which in turn may hydroxylate guanine at C-8. Detoxifying enzymes arecatalasc, 4, which converts H2O; to water and molecular oxygen, and quinonereducíase,5. which reduces DES Q to DES bypassing the DES semiquinoneintermediate, thus decreasing the Superoxide yield (3). d(j, dGuo.

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ELEVATED 8-OH-dGuo LEVELS IN DNA OF DES-TREATED HAMSTERS

intermediate doses of DES (estrogen implants), the liver detoxification and DNA repair mechanisms may be able to cope withintermediate levels of free radical generation and DNA damage.However, the renal detoxification and DNA repair systems maybe inadequate for the continuous challenge posed by redoxcycling of DES. Activities of cytochrome P-450 oxidase andcytochrome P-450 reducíase, the enzymes mediating redoxcycling, have been shown to increase in kidneys of hamsterstreated chronically with estrogen (18). In contrast, the activitiesof catatase which detoxifies H2O2 and of quinone reducíasewhich lowers concentrations of DES Q, the substrate for redoxcycling, decrease substantially in kidney of hamsters Irealedchronically with stilbene or sleroid estrogens compared to control values (3, 17, 19). Thus, the increase in 8-OH-dGuo levelsspecifically in kidney of hamsters treated chronically with DES(Fig. 2) coincides with the target-specific increase in activitiesof enzymes supporting redox cycling and decrease in detoxifying enzyme activities such as catalase and quinone reducíase.

8-OH-dGuo Formation and Carcinogenesis. Covalent damagelo cellular macromolecules by reaclive free radicals has beenpostulated to initiale and/or promole the development of lu-mors (20). Likewise, Ihe largel-specific increase in 8-OH-dGuolevels in renal DNA of hamslers Irealed chronically wilh DESprior lo kidney malignancy supports a role of free radicaldamage in estrogen-induced carcinogenesis. Other lypes ofcovalenl DNA damage such as DES-DNA adduci formalion(21) or Ihe kidney-specific increase in endogenous DNA adduclsin response lo eslrogen ireatment of hamslers (22) have alsobeen postulated lo be carcinogenic evenls in DES-induced carcinogenesis. The exacl role of each of ihese processes in Iheinduclion of malignancy by DES requires addilional sludies.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Mark L. Winter for his help withanimal experiments, for calculation of data, and for the preparation ofgraphs; and Melinda West and Kent Eneff for analyses of 8-OH-dGuo

content. The typing of the manuscript by Rosalba Ortiz is gratefullyacknowledged.

REFERENCES

1. Liehr, J. G., and Roy, D. Free radical generation by redox cycling ofestrogens. Free Radical Biol. Med., 8: 415-423. 1990.

2. Liehr. J. G., Ulubelen, A. A., and Strobel. H. W. Cytochrome P-450-mediatedredox cycling of estrogens. J. Biol. Chem., 261: 16865-16870, 1986.

3. Roy, D., and Liehr, J. G. Temporary decrease in renal quinone reducíaseactivity induced by chronic administration of estradiol to male Syrian hamsters: increased Superoxide formation by redox cycling of estrogen. J. Biol.Chem., 263:3646-3651, 1988.

4. Kasai, H., Crain, P. F., Kuchino, Y., Nishimura, S., Ootsuyama, A., andTanooka, H. Formation of 8-hydroxy guanine moiety in cellular DNA byagents producing oxygen radicals and evidence for its repair. Carcinogenesis(Lond.), 7: 1849-1851, 1986.

5. Levine, R. L., Garland, D., Oliver, C. N., Amici, A., Climent, I„Lenz, A.G., Ahn, B., Shamuel, S., and Stadtman, E. R. Determination of carbonylcontent in oxidatively modified proteins. Methods Enzymol.. 186: 464-478,1990.

6. Winter, M. L., and Liehr. J. G. Quantification of oxidative damage to proteinin estrogen-induced hamster kidney. Proc. Am. Assoc. Cancer Res., 30:129,1989.

7. Kirkman, H. Estrogen-induced tumors of the kidney in the Syrian hamster.Nati. Cancer Inst. Monogr. No. /, 1-58, 1959.

8. Kasai. H.. and Nishimura, S. Hydroxylation of deoxyguanosine at the C-8position by ascorbic acid and other reducing agents. Nucleic Acids Res., 12:2137-2145, 1984.

9. Liehr. J. G., DaGue, B. B., Ballatore, A. M., and Henkin. J. Diethylstilbestrol(DES) quinone: a reactive intermediate in DES metabolism. Biochem. Phar-macol., Õ2:3711-3718, 1983.

10. Gupta, R. C. Non-random binding of the carcinogen yV-hydroxy-2-acetyla-minofluorene to repetitive sequences of rat liver DNA in vivo. Proc. Nati.Acad. Sci. USA, 81: 6943-6947, 1984.

11. Dignam, J. D., and Strobel. H. W. NADPH-cytochrome P-450 reducíasefrom rat liver: purification by affinity chromatography and characterization.Biochemistry, 16: 1116-1123. 1977.

12. Beland, F. A., Dooley. K. L.. and Casciano, D. A. Rapid isolation ofcarcinogen-bound DNA and RNA by hydroxy-apatite chromatography. J.Chromatogr., / 74: 177-186, 1979.

13. Floyd. R. A.. Watson, J. J., Wong, P. K.. Altmiller, D. H., and Rickard, R.C. Hydroxyl free radical adduci of deoxyguanosine: sensitive detection andmechanisms of formation. Free Radical Res. Commun., /: 163-172. 1986.

14. Rosier, J. A., and Van Peteghem, C. H. Peroxidative in vitro metabolism ofdiethylstilbestrol induces formation of 8-hydroxy-2'-deoxyguanosine. Carcinogenesis (Lond.). 70:405-406, 1989.

15. Liehr, J. G., Stance!, G. M., Chorich, L. P., Bousfield, G. R., and Ulubelen,A. A. Hormonal carcinogenesis: separation of estrogenicity from carcinogen-icity. Chem.-Biol. Interact., 59: 173-184, 1986.

16. Floyd, R. A., West, M. S.. Eneff, K. L., Hogsett, W. E., and Tingey, D. T.Hydroxyl free radical-mediated formation of 8-hydroxyguanine in isolatedDNA. Arch. Biochem. Biophys., 262: 266-272, 1988.

17. Roy, D., and Liehr. J. G. Changes in activities of free radical detoxifyingenzymes in kidneys of male Syrian hamsters treated with estradiol. CancerRes., 49: 1475-1480, 1989.

18. Liehr. J. G., Roy, D., Bui, Q., and Weisz, J. Effect of chronic estrogentreatment of Syrian hamsters on microsomal enzymes mediating formationof catecholestrogens and their redox cycling: implications for carcinogenesis.J. Steroid Biochem.. 35: 555-560, 1990.

19. Segura-Aguilar, J., Cortes-Vizcaino V., Llombart-Bosch. A., Ernster, L.,Monsalve, E.. and Romero. F. J. The levels of quinone reductases. Superoxidedismutase and glutathione related enzymatic activities in diethylstilbestrol-induced carcinogenesis in the kidney of male Syrian golden hamsters. Carcinogenesis (Lond.), //: 1727-1732. 1990.

20. Floyd, R. A. The role of 8-hydroxyguanine in carcinogenesis. Carcinogenesis(Lond.), ;/: 1447-1450, 1990.

21. Gladek, A., and Liehr, J. G. Mechanism of genotoxicity of diethylstilbestrolin vivo. ]. Biol. Chem., 264: 16847-16852. 1989.

22. Liehr. J. G.. Avitts, T. A.. Randerath. E.. and Randerath. K. Estrogen-induced endogenous DNA adduction: possible mechanism of hormonal cancer. Proc. Nati. Acad. Sci. USA. 83: 5301-5305, 1986.

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1991;51:3882-3885. Cancer Res   Deodutta Roy, Robert A. Floyd and Joachim G. Liehr  DiethylstilbestrolDamage by Free Radicals Generated by Redox Cycling ofDiethylstilbestrol-treated Syrian Hamsters: Covalent DNA Elevated 8-Hydroxydeoxyguanosine Levels in DNA of

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