Document QkQjgpdoKyqMpB65r6pD2Rq85

Carcinogenesis vol 7 no.11 pp.1849-1851, 1986 -__ . Formation of 8-hydroxyguanine moiety in cellular DNA by agents producing oxygen radicals and evidence for its repair H.KSai', P.F.Crain', Y.Kuchinol, S.Nishimural, A.Ootsuyama* and H.Tanooka2 IBiology and 'Radiobiology Divisions, National Cancer Center Research bstinrte, 5-1-1 Tsukiji, Chuo-ku. Tokyo 104, Japan &Hydroxydeoxyguanosine (8-OH-dG) was detected in DNA isolated from HeLa cells after the cells in tissue culture had been irradiated with X-rays and from the liver of mice after the whole animals had been irradiated with y-rays. The amounts of &OH-dG in DNA after in vivo irradiation were three orders of magnitude lower than those after in vitro ir- radiation (0.008 -0.032 8-OH-dG residue/lV dG/krad). The 8-OH-dG produced in liver DNA by irradiation of mice decreased with time, suggesting the presence of a repair en- zyme($ acting on &OH-dG in mouse liver. Treatment of Salmonella Ophimurium cefk with hydrogen peroldde also caused increase in the 8-OH-dG content. These results indicate that 8-OH-dG is formed in vivo in cellular DNA on treatment with various oxygen radical-producing agents and that it is repairable. Introduction We previously found that the C-8 position of deoxyguanosine residues in DNA is hydroxylated to produce S-hydroxydeoxyguanosine (8-OH-dG*) in vitro by various oxygen radicalproducing agents, such as reducing agents ( l ) , X-rays ( 2 ) , asbestos plus hydrogen peroxide (H20(23)), and polyphenol with H,O, and ferric ion (4).Since many mutagens, tumor promoters and carcinogens are known to generate oxygen radicals ( 5 ) , and generation of oxygen radicals in vivo is thought to be relevant to carcinogenesis (6),8-OHdG formation in DNA may be related to tumorigenesis. The questio-n arises as to whether 8-OH-dG is formed in cellular DNA in vivo when intact cells are treated 'with oxygen radical-producing agents. In the present study we demonstrate that 8-OH-dG is in fact produced in cellular DNA in vivo by ionizing radiation or H202. Materials and methods Irrudiarion of H e h cells und whole b d i r s of mice HeLa cells ( I x IO9 cells) were cultured in Eagle's minimum essential medium Supplemented with 5% calf serum, collected and suspended in IO ml of DulbecCo's phosphate buffered saline (PBS).The cell suspension was divided into three pans: one as a control and the other two for irradiation. Cells were irradiated with 250 kVp X-rays generated by a Manitron 300 (General Electric CO.)at a dose rate of 390 ndlmin at 4C with a 2 m m Cu filter. After irradiation. the cells were collected by centrifugation and quickly frozen at -80C until isolation of DNA. Female mice (C3HIHe)were i d i a t e d with y-rays from a "CO source (1000 ci. Toshiba Electric Co.. Tokyo) at a dose rate of 130 nd/min and a total dose of 60 k r d or 173 krad. In repair experiments. mice were irradiated with y-rays from a T o Cammacell (12000 Ci. Atomic Energy of Canada) at a dose rate 01 14 kradhin. After irradiation. the mice were killed by cervical dislocation snd their livers were quickly isolated and frozen at - 80C. b 8-OH-dG 1 EC c A290 0 10 20min 0 10 20min Retention time Fig. 1. Detection of 8-OH-dG with the h.p.1.c.-EC sysizii. The detector response of EC and AI,,, were recorded. (a) Control m o w liver DNA; (b) liver DNA from a mouse irradiated with 173 krad of y-rays. X 4 a y Dose (Krad) Fig. 2. Formation of 8-OH-dG in DNA of HeLa cells by X-ray irradiation. 8-OH-dG level in control DNA was subtracted. Points and bars represent mean values and standard deviations for thrce independent analyses. 7-Ray Dose ( Krad1 'Ahhreviations: 8-OHIIG. 8-hydroxydeoxygwnosine: dG, deoxyguanosine; ctT, thymidine; EC. electrochemical. 3 IRL Press Limited. Oxford. England Fig. 3. Formation of 8-OHdG in mouse liver DNA on %hole body irradintion with y-rays. I 849 P H.Kasai ef ai. b Trearrrtertr of Sdrrtonella yphitnuriurn cells wirh HzOz Salmonella iyltt'murt'um strain TALCQ was grown in I I of nutrient broth (Difco) medium. A sample of 2 g of wet cclls collected by centrlfugatton was suspended in I 1 of 100 mM sodium phosphate buffer (pH 7.4) and divided into four pans. One part was used as a control without treatment, and the other three were treated with different concentrations of H,O, as specified at 4C for 30 min. The cells were collected by centrifugation, suspended in 40 ml of the above buffer, and collected again by centrifugation. This washing procedure was repeated twice more and then the cells were frozen at -80C. Analysis of 8-OHdG with an h.p.1. c. -electrochemical derecror system DNA was isolated from cells or tissues by Marmur's method (8,except that cells were lysed by 2% sodium dodecyl sulfate at 37C for 30 min. Portions -of DNA samples (4 5 units) were dissolved in 200 pi of 20 mM sodium acetate buffer (pH 4.8). digested with 20 pg of nuclease PI at 37C for 30 min, and then treated with 1.3 units of E. coli alkaline phosphornonesterase in 0.1 M Tris-HCI buffer (pH 7.5) for 1 h. The resultingdeoxynucleoside mixture was injected into an h.p.1.c. apparatus coupled with an electrochemical [EC) detector (7): apparatus, Toyo Soda HLC-803D; column, Beckman Ultrasphere ODS (0.46 x 25 cm); eluent, 10% aqueous mthanol containing 12.5 mM citnc acid, 25 mM scdium acetate, 30 mM NaOH, 10 mtvl acetic acid; flow rate, 1 ml/min; U.V. detector. Toyo Soda W-8, 290 nm; EC detector, Toyo Soda EC-8000.600mV (oxidation).The molar ratio of 8-0HdG to deoxyguanosine (dG) in each DNA sample was determined based on the peak height of authentic 8-OHdC with the EC detector and the U.V. absorbance at AZg0of dG. On prolonged incubation of the DNA sample with E. coli alkaline phosphomonoesterase under the conditions described above. the contentof W H d G increased at the rate of 0.3 h 0.1 residue/l@ d G h Therefore this blank value for 8-OHdG was subtracted from the observed data. In Figures 2-5. background 8-OHdC level in control DNA (0.6- 1.4 residue/lOSdG) was subtracted. Results HeLa cells and whole bodies of mice were irradiated with different doses of X-rays and y-rays, respectively. DNA was then isolated from HeLa cells and the livers of the mice, and the quantity of 8-OH-dG was determined as described in Materials and t methods. Typical chromatographic profiles of 8-OH-dG found in liver DNA of control mice and irradiated mice are shown in Figure 1. The irradiated DNA sample clearly contained more 8-OHdG, but it is noteworthy that control liver also contained some 8-OH-dG. As shown in Figure 2 and 3 , the content of 8-OH-dG increased with increase in the dose of ionizing radia. tion. The formation of 8-OH-dG in cellular DNA on radiation was estimated to be 0.008 -0.018 residue/105 dG/krad. To determine whether 8-OH-dG in DNA is repaired in vivo, mice were killed at various times afier irradiation with ?-rays (80 had), and their liver DNA was analyzed. The 8-OHdG content in samples obtained 90 min after irradiation was considerably less than that in those obtained immediately after irradiation (Figure 4).This result suggests that mouse liver cells have a repair enzyme(s) for 8-OH-dG in DNA. In addition to the effect of ionizing radiation, we also examin- ed the effect of H,O, on formation of 8-OH-dG in DNA in vivo. H202is known to have mutagenic (9), clastogenic (10) and carcinogenic (11) activities. It induced DNA damage in vivo such as strand scissions and thymine glycoI formation (12). We previously observed the formation of 8-OH-dG on treatment of calf thymus DNA with H202in vitro (3). In the present experiment, cells of S. ~phirnrrriurnstrain TAlOO were treated with various concentrations of H202.As shown in Figure 5, the content of 8-OH-dG in their DNA increased with increase in the concentration of H202. Discussion The sensitive assay procedure recently developed by Floyd n al., using an EC detector coupled with an h.p.1.c. apparatus (7) was found to be very useful for measurement of small amounts of 8-OH-dG in DNA. On treatment of cells or mice with oxygen radical producing-agents, considerable portions of the cells may be killed or become non-functional. However, repair of 8-OH-dG in liver DNA after y-ray irradiation was observed mice, indicating that 8-OH-dC is formed in intact cells. The results shown in Figure 3 were obtained by irradiation for several hours at a low dose rate of I30 rad/min. Therefore, some repair may have occurred during irradiation, and the true yield of 8 4 H dG may have been higher, as indicated from the results at a high dose rate shown in Figure 4 (yield of 8-OHdG: 0.032 residuell@ 20 In X (3 0 4 (3 P 0 10 a Y Time ( m i d Fig. 4. Repair of 8-OHdC residues in liver DNA after whole body irradiation of mice with y-rays. Mice were irradiated with 80 knd of y-rays (14 kradlmin). Livers were isolated at various rimes aRer irradidtion and their DNA was analyzed as described. in the experimental section. 1850 ( Concentration of HlO, (mM) Fig. 5. Formation of 8-OH-dG in DNA of S. fyhinnihurm TA100 cells treated with H20p 3 3 found hown in :d mort lntaina ntent of g radiaadiation d. 'in vivo, y-nys .dG con,iderab\y -adiation :a rep& examin- . in vivo. and car,ivosuch 12). We tment of t experiited with the conje in the Floyd et tratus (7) amounts vith oxy' the cells repair of served in ells. The )r several ne repair of 8-OHI at a high :sidue/lp A 3 0 cells Formation of 8-hydroxyguanine moiety in cellulnr DNA dG/krad). Repair of 8-OHdG may be related to unscheduled DNA synthesis, which was observed in mouse skin after ionizing irradiation (13). A small amount of 8-OH-dG (0.6 - I .4 residue/105 dG) was also detected in control DNA isolated from HeLa cells, mouse liver and S. vphimuriurn that had not been treated with oxygen ndicdl-produCing agents. These results suggested that in normal conditions cellular DNA is damaged with formation of 8-OHdG residues by oxygen radicals that are generated by normal ,-ellular metabolism. This is compatible with the fact that another product of DNA damage, thymine glycol, is excreted in the urine of normal rats and humans (14). Mutations were also observed in Salmonella strains TA2662 and TA103 recovered from the liver and spleen of mice, suggesting that oxygen radicals act as mutagens in these normal tissues (15). It should be mentioned that on isolation of DNA with phenol that has not been freshly distilled the 8-OH-dG content of the DNA is Substantially increased. In this case 8-OH-dG is presumably produced during isolation of DNA by oxidizing components present in the phenol. However, on DNA extraction with freshly distilled phenol, the 8-OH-dG content was the same as that obtained using Marmur's method employed in this work. Although we speculate that 8-OHdG is actually present in cellular DNA, the possibility cannot be completely excluded even with Marmur's method that it is an artifact produced in DNA. For example, peroxides are reported to be formed on incubation of homogenized mammalian tissues (16), and we observed that the 8-OH-dG content increased on prolonged homogenization of mouse liver. Formation of 8-OH-dG in DNA is three orders of magnitude less on ionizing irradiation in vivo than on irradiation in vitro (2). Thus in intact cells there must be some mechanism for preventing formation of 8-OH-dG in DNA by oxygen radicalforming agents. The conformation of DNA in the nucleosome form may be more resistant to the agents, or some cellular components may inhibit the action of oxygen radicals. Another possibility is that there is an active repair mechanism for converting 8-OH-dG to dG in the cells. Further work is needed on this problem. The extent of formation of 8-OH-dG in cellular DNA by ionizing radiation (0.008 -0.032. residuellp dGlkrad) observed in this study seems to be of roughly the same order as that of thymine glycol formation (0.034 residue/ 10s dT/krad, calculated from data in ref. 17). Therefore, formation of 8-OH-dG in cellular DNA should be considered as a possible mechanism a f involvement of oxygen radical-forming agents in mutation and carcinogenesis. Acknowledgements This work was wpported in part by a Grant-in-Aid from the Ministry of Health and Welfare for a Comprehensive IO-Year Strategy for Cancer Control and by a grant from the Ministry of Wucation. Science and Culture. P.F.C. was the holder of a Foreign Research Fellowship from the Foundation for the Promotion of Cancer Research. Tokyo. Her present address is, Department of Medicinal Chemistry and Biochemistry. University of Utah. References 1 . Kasai.H'. and Nishimun.S. (1984) Hydroxylation of deoxyguanosine at the C-8 position by ascorbic acid and other reducing agents. Nucleic Acids Res., 12. 2137-3145. 2 . K&sai,H.. Tanooka.H. and NishimuraS. (1984) Formation of S-hydroxyguanine redues in DNA by X-irradiation. Gunn. 75. 1037- 1039. 3. Kasai.H. and Nishimura.S. (1984) DNA damage induced by asbestos in the prexnce 0 1 hydrogen peroxide. Gum. 75, 841 -844. 4. 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(1983)Clastogenicactivity ofcaffeic acid and its relationship to hydrogen peroxide generated during autooxidation. Murar. Res., 116, 333-339. 11. Ito,A., Naito,M.. Nait0.Y. and Watanabe,H. (1982) Induction and characterization of gastroduodenal lesions in mice given continuous oral administration of hydrogen peroxide. Gann. 73. 315-322. 12. Lewis.J.G. and Adams,D.O. (1985) Induction of 5.6-ring-saturated thymine base in NIH-3T3 cells by phorbol ester-stimulatedmacrophages: role of reactive oxygen intermediates. Cancer Res., 45, 1270-1275. 13.Ootsuyama.A. and Tanooka,H. (1986) Unscheduled DNA synthesis after beta-irradiation of mouse skin in siru. Murar. Res., 116, 183-185. 14. Cathcart.R., Schwiers,E., Sau1,R:L. and Ames.B.N. (1984) Thymine glycol and thymidine glycol in human and rat urine: a possible assay for oxidative DNA damage. Proc. Narl. Acad. Sei. USA, 81, 5633-5637. 15. Wright.A.E. and R0senberg.L.T. (1986) Mutation in Safmonelh fyhimuriwn recovered from livers and spleens of mice. Murar. Res., 159, 1-11. 16. Cut1e.R.C. (1985) Peroxide-producing potential of tissues: inverse correlation with longevity of mammalian species. h o c . Narl. Acad. Sci. USA, 82, 4798-4802. 17. Lead0n.S.A. and Hanawa1t.P.C. (1983) Monoclonal antibody to DNA containing chymine glycol. Mum. Res., 112, 191-200. Received on I2 Ma! 1986; accepted on 3 Seprember 1986 1851