- Email: [email protected]
Ionizing Radiation and Hydrogen Peroxide Induced Oxidative DNA Base Damage in Two L5178Y Cell Lines
Free Radical Biology & Medicine, Vol. 24, Nos. 7/8, pp. 1250 –1255, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/98 $19.00 1 .00
PII S0891-5849(97)00452-8
Original Contribution IONIZING RADIATION AND HYDROGEN PEROXIDE INDUCED OXIDATIVE DNA BASE DAMAGE IN TWO L5178Y CELL LINES TOMASZ H. ZASTAWNY,* MARCIN KRUSZEWSKI,†
and
RYSZARD OLINSKI*
*Department of Clinical Biochemistry, University School of Medical Sciences, Karlowicza 24, 85-092 Bydgoszcz, Poland, † Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-195 Warsaw, Poland (Received 23 June 1997; Revised 28 October 1997; Accepted 17 November 1997)
Abstract—Seven oxidized DNA bases were quantified, by gas GC/MS-SIM, in chromatin from g-rays and H2O2 treated mouse lymphoma L5178Y (LY) cells, inversely cross-sensitive to these agents. In H2O2 treated cells (2 mM, 1 h, 37°C) we found more damage in LY-R cells than in LY-S cells. On the contrary, in gamma-rays (400 Gy) treated cells we found more damaged DNA bases in LY-S cells. The yield of damaged bases in control cells was similar in both cell lines, with the exception of 8OHAde and FapyGua that were found at a much higher level in LY-S cells. The yields of damaged bases were related to cellular sensitivity to damaging agent; this observation points to a relationship between DNA base damage induction, antioxidant defense system in the intracellular milieu and cell sensitivity. © 1998 Elsevier Science Inc. Keywords—Free radical, DNA damage, Oxidative base damage, Radiation sensitivity, H2O2 sensitivity
x-rays than LY-S subline.8 In contrast, LY-S cells are 3.6 times and 11 times more resistant to H2O2 in 37°C and 4°C, respectively.9 The high sensitivity of LY-S cells to ionizing radiation is related to a defect in DNA double strand break (DSB) rejoining,10,11 whereas the reasons of high sensitivity of LY-R cells to H2O2 are less clear and more complex. It has been suggested that LY sublines differ in transition metal ion content and/or their availability, 7,12 antioxidant defense system13 or in DNA higher order structure and these factors may be responsible for the differential H2O2 sensitivity of the sublines. To study oxidative chromatin damage in LY sublines we use the isotope dilution gas chromatography/mass spectrometry with selected-ion monitoring (GC/MS-SIM). This technique makes it possible to identify and quantify seven oxidation products of DNA bases in chromatin. This knowledge may be very important in understanding the molecular mechanism of the differential sensitivity of LY-R and LY-S murine lymphoma lines to H2O2 or ionizing radiation. The different initial DNA base damage in both cell lines after gamma-radiation and H2O2 treatment may play an important role in their cross-sensitivity to these agents.
INTRODUCTION
Reactive oxygen species (ROS) generated by ionizing radiation or oxidative metabolism and inflammatory reactions produce a variety of DNA damages.1–3 A number of reports have provided significant advances in the understanding of the mechanisms of ROS interaction with cellular DNA (for review see Ref. 4). However, the relations between molecular and cellular effects of ROS action need further exploration. An important DNA lesion type induced by ROS is oxidative base damage. Some of these lesions may lead to mutagenesis, carcinogenesis and reproductive cell death5,6 to the extent that largely depends on the cellular context. A good example of such dependencies is provided by the pair of L5178Y (LY) sublines. Although most of the damage inflicted both by H2O2 and x-rays is by ROS generated in Fenton-type reaction or by water radiolysis, these two sublines LY-R and LY-S are inversely cross-sensitive to X/g rays and H2O2.7 LY-R subline is about 2 times more resistant to killing by Address correspondence to: Dr. Tomasz H. Zastawny, Department of Clinical Biochemistry, University School of Medical Sciences, Karlowicza 24, 85-092 Bydgoszcz, Poland; Tel: (0-048) (52) 414916; Fax: (0-048) (52) 415933; E-Mail: [email protected]. 1250
Ionizing radiation and H2O2 DNA damage MATERIALS AND METHODS
Chemicals Triton X-100 was purchased from Sigma Chemical Company. Internal standards were gifts from Dr. M. Dizdaroglu from the National Institute of Standards and Technology (Gaithersburg MD, USA). Acetonitrile and bis(trimethylsilyl)-trifluoroacetamide (BSTFA) containing 1% trimethylchlorosiliane were obtained from Pierce Chemical Co. Formic acid was from Mallincrodt.
Cells cultures Murine leukaemic lymphoblast LY-R and LY-S were maintained in suspension cultures in Fischer’s medium supplemented with 8% bovine serum and antibiotics, as described by Szumiel.14 Asynchronous populations in exponential phase of growth were used in all experiments.
Irradiation of cells Cells were collected by centrifugation, and resuspended in cold Fisher’s medium containing 8% bovine fetal serum (4 3 106 cells/ml). Cells were exposed to g-rays in a 60Co g-rays source (MINEOLA, dose rate 39.2 Gy/min), while submerged in an ice bath. The irradiation dose was 200 or 400 Gy. Immediately after irradiation cells were frozen in liquid nitrogen and stored at 280°C until chromatin isolation.
Treatment with H2O2 Cells were collected by centrifugation, resuspended in phosphate-buffered saline (PBS; 2 3 105 cells/ml) and an appropriate amount of H2O2 was added for 1 h to the final concentration of 2 mM. After incubation at 37°C the cells were centrifuged, washed in PBS, frozen in liquid nitrogen and stored at 280°C until chromatin isolation.
1251
Hydrolysis, trimethylsiliation and GC/MS The chromatin samples containing 100 mg of DNA (as determined by spectrophotometry) were supplemented with the following internal standards: 2 nmol of thymine-a,a,a,6-2H4 and 0.5 mmol each of 5-hydroxyhydantoin-1,3-15N2-2,4-13C2, 5-hydroxycytosine1,3- 15 N 2 -2- 13 C, tymine- a , a , a ,6- 2 H 4 glycol, 5-hydroxymethyluracil-2,4-13C2-a,a-2H2, 5,6-dihydroxyuracil-1,3-15N2-2-13C (isodialuric acid-1,3-15N2-2-13C), 4,6-diamino-5-formamidopyrimidine-1,3-15N2-2-13C-(5formamido-15N,2H), 8-hydroxyadenine-1,3,7-15N3-2,813 C2, 8-hydroxyguanine-1,3-15N2-(2-amino-15N)-2-13C, 5-hydroxyuracil-1,3-15N2-2-13C, 5-(hydroxymethyl)uracil-2,4-13C2-a,a-2H2, 5,6-dihydroxyuracil-1,3-15N2-213 C, 8-hydroxyadenine-1,3,7-15 N 3 -2,8- 13 C 2 , 2,6-diamino-4-hydroxy-5-formamidopyrimidine-1,3-15N2-(5amino-15N)-2-13C. The samples were lyophilized and hydrolysed with 0.5 ml of 60% formic acid in evacuated and sealed tubes for 30 min at 140°C.19 The hydrolysates were lyophilized and then trimethylsilylated in polytetrafluorethylene-capped hypovials (Pierce Chemical Co.) with 100 ml mixture of BSTFA and acetonitrile (4/1, v/v) by heating for 30 min at 130°C under nitrogen. After hydrolysis and derivatization, the samples were analyzed by gas chromatography/isotope-dilution mass spectrometry with selected ion-monitoring according to the method described by Dizdaroglu.18 A Hewlett Packard Model 5890 Series II Model gas chromatograph interfaced to a Hewlett Packard Model 5972 mass selective detector was used. The injection port and GC/MS interface were both maintained at 250°C and the ion source at about 200°C. Separations were carried out using a fused-silica capillary column (Ultra 2, 12.5 m 3 0.2 mm, Hewlett Packard) coated with crosslinked 5% phenylmethyl silicone, film thickness 0.33 mm. An aliquot (4 ml) of each derivatized sample was injected without any further treatment into the injection port of the gas chromatograph by means of an autosampler. Thymine-a,a,a,6-2H4 was used as an internal standard for thymine to verify the amount of DNA in chromatin samples.19 RESULTS
Isolation of chromatin Isolation of chromatin was performed according to the modified procedure of Mee and Adelstein,15 as previously described.16 Chromatin characterization was carried out as described before.17 DNA content in chromatin preparations was determined by measurement of the absorbance at 258 nm using a molar coefficient of 6.6 3 103 M21; RNA content in chromatin was found to be 5% of the amount of DNA.
In this work the yields of 7 oxidized DNA bases (Fig. 1) were quantified in chromatin from g-irradiated or H2O2 treated LY cells. In H2O2 treated (2 mM, 1 h, 37°C) LY-R cells we found a marked increase in the yield of 5OHUra, 5OHMeUra, 8OHAde, FapyGua and 8OHGua—11, 19, 72, 39, 15-fold, respectively, whereas in H2O2 treated LY-S cells the yields of all products were increased between 0.8-fold and 2.7-fold (Fig. 2). In g-irradiated (400 Gy) cells we found a marked increase in 5OHUra, 5OHMeUra,
T. H. ZASTAWNY et al.
1252
Fig. 1. Chemical structures of DNA base products measured in the present work.
8OHAde, FapyGua and 8OHGua in LY-R cells—3, 7, 22, 9, 6-fold, respectively; in LY-S cells the increases were 11, 17, 9, 4, 4-fold in 5OHMeUra, 8OHAde, FapyGua, 8OHGua and FapyAde, respectively (Fig. 2). The yield of damaged bases in control cells was similar in both cell lines, with the exception of 8OHAde and FapyGua that were found to yield much higher levels in LY-S cells, 3.3 and 54-fold, respectively. In H2O2 treated cells we found more 5OHUra , 5OHMeUra, 5OHCyt and 8OHAde in LY-R cells than in LY-S cells (2.7, 8, 1.7, 28-fold, respectively), a similar yield of FapyAde, but 3.3-fold less FapyGua. On the contrary, in gamma irradiated cells we have found more damaged bases in LY-S cells—FapyAde, 8OHAde and FapyGua (3, 2.5, 50-fold, respectively) and a similar yield of 5OHUra, 5OHMeUra, 5OHCyt and 8OHGua. The major base product was FapyGua in all cases but the ratio FapyGua/ 8OHGua was much higher in LY-S cells. DISCUSSION
Both X/g radiation and H2O2 produce DNA damage through reactive oxygen intermediates, such as OH radical or superoxide anion.3 Generation of ROS resulting from radiation exposure has been considered the most important indirect mechanism of radiation injury, whereas transition metal ion-driven Fenton reaction was found to be the main cause of H2O2 genotoxicity.20 Oxidative DNA product formation has been shown to depend on the free radical producing system and the presence or absence of oxygen.
We characterized g-radiation- and H2O2-induced oxidative DNA products in mammalian cell chromatin from two closely related cell sublines that are inversely cross sensitive to these agents. The products identified in this work were those that have been found previously in the chromatin of X-irradiated or H2O2-treated other mammalian cell lines.21 However, after irradiation we found a more abundant formation of damaged purines in the radiation sensitive LY-S cell line than in the radiation resistant LY-R cell line. Our results are in agreement with the higher abundance of oxidized DNA bases that was for the first time reported by Mori and Dizdaroglu22 in the radiation sensitive mouse lymphoma M10 mutant, as compared with parental L5178Y cell line. Similar to our results for the DNA base damage, a higher induction of DSBs was observed by Olive et al. in LY-S cells10,23 and by Kelland et al. in radiation sensitive cervix carcinoma cell line.24 On the other hand, no difference in the induction of DNA DSBs has been found between other radiation sensitive mutants and radiation resistant parent cell lines.10,26 –31 Also, no difference in induction of single strand breaks (SSBs) has been found in irradiated L5178Y cell sublines.9,10,31,32 In contrast, in H2O2-treated cells we found more oxidized bases in LY-R cells than in LY-S cells, with the exception of formamidopyrimidines. In LY-R cells there was also a more marked increase of the yield of H2O2damaged bases as compared to control cells than in LY-S cells. High induction of initial DNA damage expressed as an overall DNA breakage or increased DNA unwinding in LY-R cells treated with hydrogen peroxide has been also previously reported, as compared to LY-S cells.7,9,12 The most abundant product in H2O2 treated cells was FapyGua in both cell sublines. Although, the identified products were typical for H2O2 treated chromatin, our results did not comply with the results of other authors, as 8OH-purines were previously reported to be the most abundant oxidative DNA lesions after H2O2 treatment.33 This discrepancy may result from the differences in cell types used in our and previous studies (lymphoma cells vs. hybridoma cells), however, the abnormally high yield of FapyGua found in the course of this study in LY-S cells suggests that the intranuclear environment of this cell subline promotes the induction of FapyGua over 8OHGua or adenine damage. Since hydroxyl radical adducts of guanine exhibit “redox ambivalence”, the reduction or oxidation of C-2 and C-4 resonance by the constituents of the intranuclear milieu may lead to different DNA products, FapyGua or 8OH-Gua, respectively.34 Higher content of reduced glutathione (and possibly other monobromobimane reactive thiols) found in LY-S cells than in LY-R,13 supports this idea; on the other hand, there is evidence that GSH is excluded from
Fig. 2. Yields of modified DNA bases in LY-R and LY-S mouse lymphoma L5178Y (LY) cells after gamma-radiation and H2O2 treatment. 1 nmol of a modified DNA base/mg DNA equals to 32 modified bases/105 bp DNA. Each data point represents the mean 7 SEM in three independent experiments. 1253
1254
T. H. ZASTAWNY et al.
the immediate vicinity of DNA in the cell nucleus.35,36 Nevertheless, G-Px induction by Se gives a considerable protection from H2O2-induced DNA damage in LY sublines, as detected by the comet assay.13 Although proneness to apoptosis may be a factor influencing the cellular sensitivity to DNA-damaging agents, this factor seems to be unimportant in the case of DNA damage estimation in hydrogen peroxide-treated LY cells: H2O2 (50 mM, 1 h, 37°C) does not induce intranucleosomal DNA degradation in LY sublines (4 h or 48 h after treatment); apoptosis can be observed after such treatment only after inactivating p21ras with a farnesylation inhibitor.37 Moreover, apoptotic processes do not affect base damage, as can be judged from the papers published on this subject. Although the doses of damaging agents applied in this work were supralethal and there is always a danger in extrapolating to low dose levels, the relation between base damage and the cell’s susceptibility to the given damaging agent is striking. This observation points to the role of initial DNA base damage in the intrinsic sensitivities of the LY-S and LY-R cells to the X/g-rays and H2O2. Preliminary results of experiments with the use of lower doses of the damaging agents and the comet assay combined with specific endonuclease treatment support the above conclusion. Acknowledgements—We are grateful to Dr. Miral Dizdaroglu from National Institute of Standards and Technology (Gaithersburg, MD, USA) for the stable isotope labeled internal standards used in this work. We appreciate the expert assistance of Dr. Teresa Wronska in the MINEOLA g-source operation. We are also indebted to Prof. Irena Szumiel for critical reading of the manuscript and helpful discussion. Financial support was from Regional Environmental Protection and Water Conservation Fund in Bydgoszcz. This work was also supported by KBN Grant 6P04A.047.14.
ABBREVIATIONS
5OHHyd—5-hydroxyhydantoin 5OHCyt—5-hydroxycytosine ThyGlycol—thymine glycol 5OHMeUra—5-hydroxymethyluracil 5,6-diOHUra—5,6-dihydroxyuracil FapyAde— 4,6-diamino-5-formamidopyrimidine FapyGua—2,6-diamino-4-hydroxy-5-formamidopyrimidine 8OHAde— 8-hydroxyadenine 8OHGua— 8-hydroxyguanine GC/MS— gas chromatography/mass spectrometry REFERENCES 1. Te´oule, R. Radiation-induced DNA damage and its repair. Int. J. Radiat. Biol. 51:573–589; 1987. 2. Dizdaroglu, M. Oxidative damage to DNA in mammalian chromatin. Mutat. Res. 275:331–342; 1992.
3. von Sonntag, C. eds. The chemical basis of radiation biology. New York: Taylor & Francis; 1987. 4. Steenken, S. Purine bases nucleosides and nucleotides: Aqueous solution redox chemistry and transformation reactions of their radical cations end e- and OH adducts. Chem. Rev. 89:503–520; 1989. 5. Painter, R.B. The role of DNA damage and repair in cell killing induced by ionizing radiation. In: Meyn, R. E.; Withers, H. R., eds. Radiation biology in cancer research. New York: Raven Press; 1980; 59 – 68. 6. Breimer, L.H. Molecular mechanisms of ferric nitrilotriacetate: a model of experimental hemochromatosis Amer. J. Patho. 95:663– 674; 1990. 7. Szumiel, I.; Kapiszewska, M.; Kruszewski, M.; Iwanienko, T.; Lange, C. S. Content of iron in the nuclei and induction of pH 9-labile lesions in L5178Y sublines inversely cross-sensitive to H2O2. Radiat. Environ. Biophys. 34:113–119; 1995. 8. Beer, J. Z.; Jakobson, E. D.; Evans, H. H.; Szumiel, I. X-ray and UV mutagenesis in two L5178Y cells strains differing in tumorogenicity, radiosensitivity and DNA repair. Br. J. Cancer 49:107– 111; 1983. 9. Kruszewski, M.; Grean, M. H.; Lowe, J. E.; Szumiel, I. DNA strand breakage, cytotoxicity and mutagenicity of hydrogen peroxide treatment at 4°C and 37°C in L5178Y sublines. Mutat. Res. 308:233–241; 1994. 10. Wlodek, D.; Hittelman, W. N. The repair of double-strand DNA breaks correlates with radiosensitivity of L5178Y-S and L5178Y-R cells. Radiat. Res. 112:146 –155; 1987. 11. Evans, H. H.; Ricanati, M.; Horng, M. F. Deficiency in DNA repair in mouse lymphoma strain L5178Y-S. Proc. Natl. Acad. Sci. USA 84:7562–7566; 1987. 12. Kruszewski, M.; Grean, M. H.; Lowe, J. E.; Szumiel, I. Comparision of effects of iron and calcium chelators on the response of L5178Y sublines to x-rays and H2O2. Mutat. Res. 326:155–163; 1995. 13. Bouzyk, E.; Iwanenko, T.; Jarocewicz, N.; Kruszewski, M.; Sochanowicz, B.; Szumiel, I. Antioxidant defence system in differentially hydrogen peroxide sensitive L5178Y sublines. Free Rad. Biol. Med. 22: 697–704; 1997. 14. Szumiel, I. Response of two strains of L5178Y cells to cisdichlorobis(cyclopentylamine) platinum II. I. Cross-sensitivity to cis-PAD and UV light. Chem. Biol. Interact. 24:51–72; 1979. 15. Mee, L. K.; Adelstein, S. J. Predominance of core histones in formation of DNA-protein crosslinks in g-irradiated chromatin. Proc. Natl. Acad. Sci. USA 78:2194 –2198; 1981. 16. Gajewski, E.; Dizdaroglu, M. Hydroxyl radical-induced crosslinking of cytosine and tyrosine in nucleohistone. Biochemistry 29:977–980; 1990. 17. Berezney, R.; Coffey, D. S. Identification of a nuclear protein matrix. Biochem. Biophys. Res. Commun. 60:1410 –1417; 1974. 18. Dizdaroglu, M. Chemical determination of oxidative DNA damage by gas chromatography-mass spectrometry. Meth. Enzymol. 234: 3–16; 1994. 19. Djuric, Z.; Heilbrun, L. K.; Reading, B. A.; Broomer, A.; Veleriote, F. A.; Martino, S. Effects of a low-fat diet on levels of oxidative damage to DNA in human peripherial nucleated blood cells. J. Natl. Cancer Inst. 83:766 –769; 1991. 20. Halliwell, B.; Gutteridge, J. M. C., eds. Free radicals in biology and medicine. Oxford: Clarendon Press; 1989. 21. Dizdaroglu, M.; Oxidative damage to DNA in mammalian chromatin. Mutat.Res. 275:331–342; 1992. 22. Mori, T.; Dizdaroglu, M. Ionizing radiation causes greater DNA base damage in radiation-sensitive mutant M10 cells than in parent mouse lymphoma L5178Y cells. Radiat. Res. 140:85–90; 1994. 23. Olive, P. L; Wlodek, D.; Banath, J. P.; DNA double-strand breaks measured in individual cells subjected to gel electrophoresis. Cancer Res. 51:4671– 4676; 1991. 24. Kelland, L. R.; Edwards, S. M.; Steel, G. G. Induction and rejoining of DNA double-strand breaks in human cervix carci-
Ionizing radiation and H2O2 DNA damage
25. 26. 27. 28. 29.
30.
noma cell lines of differing radiosensitivity. Radiat Res. 116: 526 –538; 1988. Eguchi-Kasai, K.; Kosaka, T.; Sato, K.; Kaneko, I. Repairability of DNA double-strand breaks and radiation sensitivity in five mamalian cell lines. Int. J. Radiat. Biol. 59:97–104; 1991. Kemp, L. M.; Sedvick, S. G.; Jeggo, P. A. X-ray sensitive mutants of Chinese hamster ovary cells defective in double-strand break rejoning. Mutat. Res. 132:189 –196; 1984. Giaccia, A.; Weistein, R.; Hu, J.; Stamato, T. D. Cell-dependent repair of double-strand DNA breaks in a g-ray sensitive Chinese hamster cell. Somat. Cell Genet. 11:485– 491; 1985. Weibezahn, K. F.; Lohrer, H.; Herrlich, P. Double-strand break repair and G2 block in Chinese hamster ovary cells and their radiosensitive mutants. Mutat. Res. 145:177–183; 1985. Schwartz, J. L.; Rotmensch, J.; Giovanazzi, S.; Cohen, M. B.; Weichselbaum, R. R. Faster repair of DNA double-strand breaks in radioresistant human tumor cells. Int. J. Radiat. Oncol. Biol. Phys. 15:907–912; 1988. Iliakis, G.; Okayasu, R.; Seaner, R.. Radiosensitive xrs-5 and parental CHO cell show identical DNA neutral filter elution doseresponse: implication for a relationship between cell radiosensitivity and induction of DNA double-strand breaks. Int. J. Radiat. Biol. 54:55– 62; 1988.
1255
31. Kapiszewska, M.; Wright, W. D.; Lange, C. S.; RotiRoti, J. L. DNA supercoiling changes in nucleoids from irradiated L5178Y-S and -R cells. Radiat. Res. 119:569 –575; 1989. 32. Dizdaroglu, M.; Nackerdien, Z.; Chao, B.-C.; Gajewski, E.; Rao, G. Damage to DNA bases in mammalian chromatin by hydrogen peroxide in the presence of ferric and cupric ions. Arch. Biochem. Biophys. 285:317–324; 1991. 33. Breen, A.; Murphy, J.A. Reactions of oxyl radicals with DNA. Free Radical Biol. Med. 18:1033–1077; 1995. 34. Ward, J. F. The yield of double-strand breaks produced intracellulary by ionizing radiation: a review. Int. J. Radiat. Biol. 57: 1141–1150; 1990. 35. Smoluk, G. D.; Fahey, R. C.; Ward, J. F. Interaction of glutathione and other low-molecular-weight thiols with DNA: evidence of counterion condensation and coion depletion near DNA. Radiat. Res. 114:2–10; 1988. 36. Zeng, S.; Newton, G. L.; Gonick, G.; Fahey, R. C.; Ward, J. F. Radioprotection of DNA by thiols: relationship between the net charge on a thiol and its ability to protect DNA. Radiat. Res. 114:11–27; 1988. 37. Sochanowicz, B.; Szumiel, I.; Gradzka, I.; Jaworska, A. (in preparation).
PII S0891-5849(97)00452-8
Original Contribution IONIZING RADIATION AND HYDROGEN PEROXIDE INDUCED OXIDATIVE DNA BASE DAMAGE IN TWO L5178Y CELL LINES TOMASZ H. ZASTAWNY,* MARCIN KRUSZEWSKI,†
and
RYSZARD OLINSKI*
*Department of Clinical Biochemistry, University School of Medical Sciences, Karlowicza 24, 85-092 Bydgoszcz, Poland, † Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-195 Warsaw, Poland (Received 23 June 1997; Revised 28 October 1997; Accepted 17 November 1997)
Abstract—Seven oxidized DNA bases were quantified, by gas GC/MS-SIM, in chromatin from g-rays and H2O2 treated mouse lymphoma L5178Y (LY) cells, inversely cross-sensitive to these agents. In H2O2 treated cells (2 mM, 1 h, 37°C) we found more damage in LY-R cells than in LY-S cells. On the contrary, in gamma-rays (400 Gy) treated cells we found more damaged DNA bases in LY-S cells. The yield of damaged bases in control cells was similar in both cell lines, with the exception of 8OHAde and FapyGua that were found at a much higher level in LY-S cells. The yields of damaged bases were related to cellular sensitivity to damaging agent; this observation points to a relationship between DNA base damage induction, antioxidant defense system in the intracellular milieu and cell sensitivity. © 1998 Elsevier Science Inc. Keywords—Free radical, DNA damage, Oxidative base damage, Radiation sensitivity, H2O2 sensitivity
x-rays than LY-S subline.8 In contrast, LY-S cells are 3.6 times and 11 times more resistant to H2O2 in 37°C and 4°C, respectively.9 The high sensitivity of LY-S cells to ionizing radiation is related to a defect in DNA double strand break (DSB) rejoining,10,11 whereas the reasons of high sensitivity of LY-R cells to H2O2 are less clear and more complex. It has been suggested that LY sublines differ in transition metal ion content and/or their availability, 7,12 antioxidant defense system13 or in DNA higher order structure and these factors may be responsible for the differential H2O2 sensitivity of the sublines. To study oxidative chromatin damage in LY sublines we use the isotope dilution gas chromatography/mass spectrometry with selected-ion monitoring (GC/MS-SIM). This technique makes it possible to identify and quantify seven oxidation products of DNA bases in chromatin. This knowledge may be very important in understanding the molecular mechanism of the differential sensitivity of LY-R and LY-S murine lymphoma lines to H2O2 or ionizing radiation. The different initial DNA base damage in both cell lines after gamma-radiation and H2O2 treatment may play an important role in their cross-sensitivity to these agents.
INTRODUCTION
Reactive oxygen species (ROS) generated by ionizing radiation or oxidative metabolism and inflammatory reactions produce a variety of DNA damages.1–3 A number of reports have provided significant advances in the understanding of the mechanisms of ROS interaction with cellular DNA (for review see Ref. 4). However, the relations between molecular and cellular effects of ROS action need further exploration. An important DNA lesion type induced by ROS is oxidative base damage. Some of these lesions may lead to mutagenesis, carcinogenesis and reproductive cell death5,6 to the extent that largely depends on the cellular context. A good example of such dependencies is provided by the pair of L5178Y (LY) sublines. Although most of the damage inflicted both by H2O2 and x-rays is by ROS generated in Fenton-type reaction or by water radiolysis, these two sublines LY-R and LY-S are inversely cross-sensitive to X/g rays and H2O2.7 LY-R subline is about 2 times more resistant to killing by Address correspondence to: Dr. Tomasz H. Zastawny, Department of Clinical Biochemistry, University School of Medical Sciences, Karlowicza 24, 85-092 Bydgoszcz, Poland; Tel: (0-048) (52) 414916; Fax: (0-048) (52) 415933; E-Mail: [email protected]. 1250
Ionizing radiation and H2O2 DNA damage MATERIALS AND METHODS
Chemicals Triton X-100 was purchased from Sigma Chemical Company. Internal standards were gifts from Dr. M. Dizdaroglu from the National Institute of Standards and Technology (Gaithersburg MD, USA). Acetonitrile and bis(trimethylsilyl)-trifluoroacetamide (BSTFA) containing 1% trimethylchlorosiliane were obtained from Pierce Chemical Co. Formic acid was from Mallincrodt.
Cells cultures Murine leukaemic lymphoblast LY-R and LY-S were maintained in suspension cultures in Fischer’s medium supplemented with 8% bovine serum and antibiotics, as described by Szumiel.14 Asynchronous populations in exponential phase of growth were used in all experiments.
Irradiation of cells Cells were collected by centrifugation, and resuspended in cold Fisher’s medium containing 8% bovine fetal serum (4 3 106 cells/ml). Cells were exposed to g-rays in a 60Co g-rays source (MINEOLA, dose rate 39.2 Gy/min), while submerged in an ice bath. The irradiation dose was 200 or 400 Gy. Immediately after irradiation cells were frozen in liquid nitrogen and stored at 280°C until chromatin isolation.
Treatment with H2O2 Cells were collected by centrifugation, resuspended in phosphate-buffered saline (PBS; 2 3 105 cells/ml) and an appropriate amount of H2O2 was added for 1 h to the final concentration of 2 mM. After incubation at 37°C the cells were centrifuged, washed in PBS, frozen in liquid nitrogen and stored at 280°C until chromatin isolation.
1251
Hydrolysis, trimethylsiliation and GC/MS The chromatin samples containing 100 mg of DNA (as determined by spectrophotometry) were supplemented with the following internal standards: 2 nmol of thymine-a,a,a,6-2H4 and 0.5 mmol each of 5-hydroxyhydantoin-1,3-15N2-2,4-13C2, 5-hydroxycytosine1,3- 15 N 2 -2- 13 C, tymine- a , a , a ,6- 2 H 4 glycol, 5-hydroxymethyluracil-2,4-13C2-a,a-2H2, 5,6-dihydroxyuracil-1,3-15N2-2-13C (isodialuric acid-1,3-15N2-2-13C), 4,6-diamino-5-formamidopyrimidine-1,3-15N2-2-13C-(5formamido-15N,2H), 8-hydroxyadenine-1,3,7-15N3-2,813 C2, 8-hydroxyguanine-1,3-15N2-(2-amino-15N)-2-13C, 5-hydroxyuracil-1,3-15N2-2-13C, 5-(hydroxymethyl)uracil-2,4-13C2-a,a-2H2, 5,6-dihydroxyuracil-1,3-15N2-213 C, 8-hydroxyadenine-1,3,7-15 N 3 -2,8- 13 C 2 , 2,6-diamino-4-hydroxy-5-formamidopyrimidine-1,3-15N2-(5amino-15N)-2-13C. The samples were lyophilized and hydrolysed with 0.5 ml of 60% formic acid in evacuated and sealed tubes for 30 min at 140°C.19 The hydrolysates were lyophilized and then trimethylsilylated in polytetrafluorethylene-capped hypovials (Pierce Chemical Co.) with 100 ml mixture of BSTFA and acetonitrile (4/1, v/v) by heating for 30 min at 130°C under nitrogen. After hydrolysis and derivatization, the samples were analyzed by gas chromatography/isotope-dilution mass spectrometry with selected ion-monitoring according to the method described by Dizdaroglu.18 A Hewlett Packard Model 5890 Series II Model gas chromatograph interfaced to a Hewlett Packard Model 5972 mass selective detector was used. The injection port and GC/MS interface were both maintained at 250°C and the ion source at about 200°C. Separations were carried out using a fused-silica capillary column (Ultra 2, 12.5 m 3 0.2 mm, Hewlett Packard) coated with crosslinked 5% phenylmethyl silicone, film thickness 0.33 mm. An aliquot (4 ml) of each derivatized sample was injected without any further treatment into the injection port of the gas chromatograph by means of an autosampler. Thymine-a,a,a,6-2H4 was used as an internal standard for thymine to verify the amount of DNA in chromatin samples.19 RESULTS
Isolation of chromatin Isolation of chromatin was performed according to the modified procedure of Mee and Adelstein,15 as previously described.16 Chromatin characterization was carried out as described before.17 DNA content in chromatin preparations was determined by measurement of the absorbance at 258 nm using a molar coefficient of 6.6 3 103 M21; RNA content in chromatin was found to be 5% of the amount of DNA.
In this work the yields of 7 oxidized DNA bases (Fig. 1) were quantified in chromatin from g-irradiated or H2O2 treated LY cells. In H2O2 treated (2 mM, 1 h, 37°C) LY-R cells we found a marked increase in the yield of 5OHUra, 5OHMeUra, 8OHAde, FapyGua and 8OHGua—11, 19, 72, 39, 15-fold, respectively, whereas in H2O2 treated LY-S cells the yields of all products were increased between 0.8-fold and 2.7-fold (Fig. 2). In g-irradiated (400 Gy) cells we found a marked increase in 5OHUra, 5OHMeUra,
T. H. ZASTAWNY et al.
1252
Fig. 1. Chemical structures of DNA base products measured in the present work.
8OHAde, FapyGua and 8OHGua in LY-R cells—3, 7, 22, 9, 6-fold, respectively; in LY-S cells the increases were 11, 17, 9, 4, 4-fold in 5OHMeUra, 8OHAde, FapyGua, 8OHGua and FapyAde, respectively (Fig. 2). The yield of damaged bases in control cells was similar in both cell lines, with the exception of 8OHAde and FapyGua that were found to yield much higher levels in LY-S cells, 3.3 and 54-fold, respectively. In H2O2 treated cells we found more 5OHUra , 5OHMeUra, 5OHCyt and 8OHAde in LY-R cells than in LY-S cells (2.7, 8, 1.7, 28-fold, respectively), a similar yield of FapyAde, but 3.3-fold less FapyGua. On the contrary, in gamma irradiated cells we have found more damaged bases in LY-S cells—FapyAde, 8OHAde and FapyGua (3, 2.5, 50-fold, respectively) and a similar yield of 5OHUra, 5OHMeUra, 5OHCyt and 8OHGua. The major base product was FapyGua in all cases but the ratio FapyGua/ 8OHGua was much higher in LY-S cells. DISCUSSION
Both X/g radiation and H2O2 produce DNA damage through reactive oxygen intermediates, such as OH radical or superoxide anion.3 Generation of ROS resulting from radiation exposure has been considered the most important indirect mechanism of radiation injury, whereas transition metal ion-driven Fenton reaction was found to be the main cause of H2O2 genotoxicity.20 Oxidative DNA product formation has been shown to depend on the free radical producing system and the presence or absence of oxygen.
We characterized g-radiation- and H2O2-induced oxidative DNA products in mammalian cell chromatin from two closely related cell sublines that are inversely cross sensitive to these agents. The products identified in this work were those that have been found previously in the chromatin of X-irradiated or H2O2-treated other mammalian cell lines.21 However, after irradiation we found a more abundant formation of damaged purines in the radiation sensitive LY-S cell line than in the radiation resistant LY-R cell line. Our results are in agreement with the higher abundance of oxidized DNA bases that was for the first time reported by Mori and Dizdaroglu22 in the radiation sensitive mouse lymphoma M10 mutant, as compared with parental L5178Y cell line. Similar to our results for the DNA base damage, a higher induction of DSBs was observed by Olive et al. in LY-S cells10,23 and by Kelland et al. in radiation sensitive cervix carcinoma cell line.24 On the other hand, no difference in the induction of DNA DSBs has been found between other radiation sensitive mutants and radiation resistant parent cell lines.10,26 –31 Also, no difference in induction of single strand breaks (SSBs) has been found in irradiated L5178Y cell sublines.9,10,31,32 In contrast, in H2O2-treated cells we found more oxidized bases in LY-R cells than in LY-S cells, with the exception of formamidopyrimidines. In LY-R cells there was also a more marked increase of the yield of H2O2damaged bases as compared to control cells than in LY-S cells. High induction of initial DNA damage expressed as an overall DNA breakage or increased DNA unwinding in LY-R cells treated with hydrogen peroxide has been also previously reported, as compared to LY-S cells.7,9,12 The most abundant product in H2O2 treated cells was FapyGua in both cell sublines. Although, the identified products were typical for H2O2 treated chromatin, our results did not comply with the results of other authors, as 8OH-purines were previously reported to be the most abundant oxidative DNA lesions after H2O2 treatment.33 This discrepancy may result from the differences in cell types used in our and previous studies (lymphoma cells vs. hybridoma cells), however, the abnormally high yield of FapyGua found in the course of this study in LY-S cells suggests that the intranuclear environment of this cell subline promotes the induction of FapyGua over 8OHGua or adenine damage. Since hydroxyl radical adducts of guanine exhibit “redox ambivalence”, the reduction or oxidation of C-2 and C-4 resonance by the constituents of the intranuclear milieu may lead to different DNA products, FapyGua or 8OH-Gua, respectively.34 Higher content of reduced glutathione (and possibly other monobromobimane reactive thiols) found in LY-S cells than in LY-R,13 supports this idea; on the other hand, there is evidence that GSH is excluded from
Fig. 2. Yields of modified DNA bases in LY-R and LY-S mouse lymphoma L5178Y (LY) cells after gamma-radiation and H2O2 treatment. 1 nmol of a modified DNA base/mg DNA equals to 32 modified bases/105 bp DNA. Each data point represents the mean 7 SEM in three independent experiments. 1253
1254
T. H. ZASTAWNY et al.
the immediate vicinity of DNA in the cell nucleus.35,36 Nevertheless, G-Px induction by Se gives a considerable protection from H2O2-induced DNA damage in LY sublines, as detected by the comet assay.13 Although proneness to apoptosis may be a factor influencing the cellular sensitivity to DNA-damaging agents, this factor seems to be unimportant in the case of DNA damage estimation in hydrogen peroxide-treated LY cells: H2O2 (50 mM, 1 h, 37°C) does not induce intranucleosomal DNA degradation in LY sublines (4 h or 48 h after treatment); apoptosis can be observed after such treatment only after inactivating p21ras with a farnesylation inhibitor.37 Moreover, apoptotic processes do not affect base damage, as can be judged from the papers published on this subject. Although the doses of damaging agents applied in this work were supralethal and there is always a danger in extrapolating to low dose levels, the relation between base damage and the cell’s susceptibility to the given damaging agent is striking. This observation points to the role of initial DNA base damage in the intrinsic sensitivities of the LY-S and LY-R cells to the X/g-rays and H2O2. Preliminary results of experiments with the use of lower doses of the damaging agents and the comet assay combined with specific endonuclease treatment support the above conclusion. Acknowledgements—We are grateful to Dr. Miral Dizdaroglu from National Institute of Standards and Technology (Gaithersburg, MD, USA) for the stable isotope labeled internal standards used in this work. We appreciate the expert assistance of Dr. Teresa Wronska in the MINEOLA g-source operation. We are also indebted to Prof. Irena Szumiel for critical reading of the manuscript and helpful discussion. Financial support was from Regional Environmental Protection and Water Conservation Fund in Bydgoszcz. This work was also supported by KBN Grant 6P04A.047.14.
ABBREVIATIONS
5OHHyd—5-hydroxyhydantoin 5OHCyt—5-hydroxycytosine ThyGlycol—thymine glycol 5OHMeUra—5-hydroxymethyluracil 5,6-diOHUra—5,6-dihydroxyuracil FapyAde— 4,6-diamino-5-formamidopyrimidine FapyGua—2,6-diamino-4-hydroxy-5-formamidopyrimidine 8OHAde— 8-hydroxyadenine 8OHGua— 8-hydroxyguanine GC/MS— gas chromatography/mass spectrometry REFERENCES 1. Te´oule, R. Radiation-induced DNA damage and its repair. Int. J. Radiat. Biol. 51:573–589; 1987. 2. Dizdaroglu, M. Oxidative damage to DNA in mammalian chromatin. Mutat. Res. 275:331–342; 1992.
3. von Sonntag, C. eds. The chemical basis of radiation biology. New York: Taylor & Francis; 1987. 4. Steenken, S. Purine bases nucleosides and nucleotides: Aqueous solution redox chemistry and transformation reactions of their radical cations end e- and OH adducts. Chem. Rev. 89:503–520; 1989. 5. Painter, R.B. The role of DNA damage and repair in cell killing induced by ionizing radiation. In: Meyn, R. E.; Withers, H. R., eds. Radiation biology in cancer research. New York: Raven Press; 1980; 59 – 68. 6. Breimer, L.H. Molecular mechanisms of ferric nitrilotriacetate: a model of experimental hemochromatosis Amer. J. Patho. 95:663– 674; 1990. 7. Szumiel, I.; Kapiszewska, M.; Kruszewski, M.; Iwanienko, T.; Lange, C. S. Content of iron in the nuclei and induction of pH 9-labile lesions in L5178Y sublines inversely cross-sensitive to H2O2. Radiat. Environ. Biophys. 34:113–119; 1995. 8. Beer, J. Z.; Jakobson, E. D.; Evans, H. H.; Szumiel, I. X-ray and UV mutagenesis in two L5178Y cells strains differing in tumorogenicity, radiosensitivity and DNA repair. Br. J. Cancer 49:107– 111; 1983. 9. Kruszewski, M.; Grean, M. H.; Lowe, J. E.; Szumiel, I. DNA strand breakage, cytotoxicity and mutagenicity of hydrogen peroxide treatment at 4°C and 37°C in L5178Y sublines. Mutat. Res. 308:233–241; 1994. 10. Wlodek, D.; Hittelman, W. N. The repair of double-strand DNA breaks correlates with radiosensitivity of L5178Y-S and L5178Y-R cells. Radiat. Res. 112:146 –155; 1987. 11. Evans, H. H.; Ricanati, M.; Horng, M. F. Deficiency in DNA repair in mouse lymphoma strain L5178Y-S. Proc. Natl. Acad. Sci. USA 84:7562–7566; 1987. 12. Kruszewski, M.; Grean, M. H.; Lowe, J. E.; Szumiel, I. Comparision of effects of iron and calcium chelators on the response of L5178Y sublines to x-rays and H2O2. Mutat. Res. 326:155–163; 1995. 13. Bouzyk, E.; Iwanenko, T.; Jarocewicz, N.; Kruszewski, M.; Sochanowicz, B.; Szumiel, I. Antioxidant defence system in differentially hydrogen peroxide sensitive L5178Y sublines. Free Rad. Biol. Med. 22: 697–704; 1997. 14. Szumiel, I. Response of two strains of L5178Y cells to cisdichlorobis(cyclopentylamine) platinum II. I. Cross-sensitivity to cis-PAD and UV light. Chem. Biol. Interact. 24:51–72; 1979. 15. Mee, L. K.; Adelstein, S. J. Predominance of core histones in formation of DNA-protein crosslinks in g-irradiated chromatin. Proc. Natl. Acad. Sci. USA 78:2194 –2198; 1981. 16. Gajewski, E.; Dizdaroglu, M. Hydroxyl radical-induced crosslinking of cytosine and tyrosine in nucleohistone. Biochemistry 29:977–980; 1990. 17. Berezney, R.; Coffey, D. S. Identification of a nuclear protein matrix. Biochem. Biophys. Res. Commun. 60:1410 –1417; 1974. 18. Dizdaroglu, M. Chemical determination of oxidative DNA damage by gas chromatography-mass spectrometry. Meth. Enzymol. 234: 3–16; 1994. 19. Djuric, Z.; Heilbrun, L. K.; Reading, B. A.; Broomer, A.; Veleriote, F. A.; Martino, S. Effects of a low-fat diet on levels of oxidative damage to DNA in human peripherial nucleated blood cells. J. Natl. Cancer Inst. 83:766 –769; 1991. 20. Halliwell, B.; Gutteridge, J. M. C., eds. Free radicals in biology and medicine. Oxford: Clarendon Press; 1989. 21. Dizdaroglu, M.; Oxidative damage to DNA in mammalian chromatin. Mutat.Res. 275:331–342; 1992. 22. Mori, T.; Dizdaroglu, M. Ionizing radiation causes greater DNA base damage in radiation-sensitive mutant M10 cells than in parent mouse lymphoma L5178Y cells. Radiat. Res. 140:85–90; 1994. 23. Olive, P. L; Wlodek, D.; Banath, J. P.; DNA double-strand breaks measured in individual cells subjected to gel electrophoresis. Cancer Res. 51:4671– 4676; 1991. 24. Kelland, L. R.; Edwards, S. M.; Steel, G. G. Induction and rejoining of DNA double-strand breaks in human cervix carci-
Ionizing radiation and H2O2 DNA damage
25. 26. 27. 28. 29.
30.
noma cell lines of differing radiosensitivity. Radiat Res. 116: 526 –538; 1988. Eguchi-Kasai, K.; Kosaka, T.; Sato, K.; Kaneko, I. Repairability of DNA double-strand breaks and radiation sensitivity in five mamalian cell lines. Int. J. Radiat. Biol. 59:97–104; 1991. Kemp, L. M.; Sedvick, S. G.; Jeggo, P. A. X-ray sensitive mutants of Chinese hamster ovary cells defective in double-strand break rejoning. Mutat. Res. 132:189 –196; 1984. Giaccia, A.; Weistein, R.; Hu, J.; Stamato, T. D. Cell-dependent repair of double-strand DNA breaks in a g-ray sensitive Chinese hamster cell. Somat. Cell Genet. 11:485– 491; 1985. Weibezahn, K. F.; Lohrer, H.; Herrlich, P. Double-strand break repair and G2 block in Chinese hamster ovary cells and their radiosensitive mutants. Mutat. Res. 145:177–183; 1985. Schwartz, J. L.; Rotmensch, J.; Giovanazzi, S.; Cohen, M. B.; Weichselbaum, R. R. Faster repair of DNA double-strand breaks in radioresistant human tumor cells. Int. J. Radiat. Oncol. Biol. Phys. 15:907–912; 1988. Iliakis, G.; Okayasu, R.; Seaner, R.. Radiosensitive xrs-5 and parental CHO cell show identical DNA neutral filter elution doseresponse: implication for a relationship between cell radiosensitivity and induction of DNA double-strand breaks. Int. J. Radiat. Biol. 54:55– 62; 1988.
1255
31. Kapiszewska, M.; Wright, W. D.; Lange, C. S.; RotiRoti, J. L. DNA supercoiling changes in nucleoids from irradiated L5178Y-S and -R cells. Radiat. Res. 119:569 –575; 1989. 32. Dizdaroglu, M.; Nackerdien, Z.; Chao, B.-C.; Gajewski, E.; Rao, G. Damage to DNA bases in mammalian chromatin by hydrogen peroxide in the presence of ferric and cupric ions. Arch. Biochem. Biophys. 285:317–324; 1991. 33. Breen, A.; Murphy, J.A. Reactions of oxyl radicals with DNA. Free Radical Biol. Med. 18:1033–1077; 1995. 34. Ward, J. F. The yield of double-strand breaks produced intracellulary by ionizing radiation: a review. Int. J. Radiat. Biol. 57: 1141–1150; 1990. 35. Smoluk, G. D.; Fahey, R. C.; Ward, J. F. Interaction of glutathione and other low-molecular-weight thiols with DNA: evidence of counterion condensation and coion depletion near DNA. Radiat. Res. 114:2–10; 1988. 36. Zeng, S.; Newton, G. L.; Gonick, G.; Fahey, R. C.; Ward, J. F. Radioprotection of DNA by thiols: relationship between the net charge on a thiol and its ability to protect DNA. Radiat. Res. 114:11–27; 1988. 37. Sochanowicz, B.; Szumiel, I.; Gradzka, I.; Jaworska, A. (in preparation).