Abstract
We developed a novel method for the simultaneous extraction and analysis of total tissue RNA and DNA to quantify the RNA and DNA oxidation products 8-oxo-7,8-dihydroguanosine and 8-oxo-7,8-dihydro-2′-deoxyguanosine using HPLC coupled to electrochemical detection (HPLC-ECD). The protein denaturing agents guanidine thiocyanate and phenol/chloroform at neutral pH were found to be very efficient for the isolation of RNA and DNA from rat brain, liver and muscle. The method is very fast, allows extraction at 0°C, gives high yields of pure RNA and DNA with low background oxidation levels, and also determines the RNA/DNA ratio. Experiments with isolated RNA and DNA exposed to the Fenton reagents H2O2/ascorbate/Fe3+ (or Cu2+) resulted in significantly greater RNA oxidation. The RNase inhibitor 2-mercaptoethanol, commonly used for RNA extraction, acted as a pro-oxidant during nucleic acid extraction, an effect attenuated by the inclusion of the metal chelator deferoxamine mesylate. In vivo, administration of doxorubicin (an oxidant generator) to Fisher-344 rats resulted in a significant increase in liver RNA oxidation, but no significantly increased DNA oxidation. This new method could be useful to assess oxidatively damaged RNA and DNA simultaneously, and our data show that RNA is more susceptible to oxidative stress than DNA in vivo and in vitro.
References
Aas, P.A., Otterlei, M., Falnes, P.O., Vagbo, C.B., Skorpen, F., Akbari, M., Sundheim, O., Bjoras, M., Slupphaug, G., Seeberg, E., and Krokan, H.E. (2003). Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA. Nature421, 859–863.10.1038/nature01363Search in Google Scholar
Abe, T., Tohgi, H., Isobe, C., Murata, T., and Sato, C. (2002). Remarkable increase in the concentration of 8-hydroxyguanosine in cerebrospinal fluid from patients with Alzheimer's disease. J. Neurosci. Res.70, 447–450.10.1002/jnr.10349Search in Google Scholar
Abe, T., Isobe, C., Murata, T., Sato, C., and Tohgi, H. (2003). Alteration of 8-hydroxyguanosine concentrations in the cerebrospinal fluid and serum from patients with Parkinson's disease. Neurosci. Lett.336, 105–108.10.1016/S0304-3940(02)01259-4Search in Google Scholar
Barja, G. (2004). Aging in vertebrates, and the effect of caloric restriction: a mitochondrial free radical production-DNA damage mechanism? Biol. Rev. Camb. Philos. Soc.79, 235–251.10.1017/S1464793103006213Search in Google Scholar
Byrne, E., Trounce, I., and Dennett, X. (1991). Mitochondrial theory of senescence: respiratory chain protein studies in human skeletal muscle. Mech. Ageing Dev.60, 295–302.10.1016/0047-6374(91)90042-XSearch in Google Scholar
Cheng, K.C., Cahill, D.S., Kasai, H., Nishimura, S., and Loeb, L.A. (1992). 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G→T and A→C substitutions. J. Biol. Chem.267, 166–172.10.1016/S0021-9258(18)48474-8Search in Google Scholar
Childs, A.C., Phaneuf, S.L., Dirks, A.J., Phillips, T., and Leeuwenburgh, C. (2002). Doxorubicin treatment in vivo causes cytochrome c release and cardiomyocyte apoptosis, as well as increased mitochondrial efficiency, superoxide dismutase activity, and Bcl-2:Bax ratio. Cancer Res.62, 4592–4598.Search in Google Scholar
Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., and Rutter, W.J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry18, 5294–5299.10.1021/bi00591a005Search in Google Scholar
Chomczynski, P. and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem.162, 156–159.10.1016/0003-2697(87)90021-2Search in Google Scholar
Conaway, C.C., Nie, G., Hussain, N.S., and Fiala, E.S. (1991). Comparison of oxidative damage to rat liver DNA and RNA by primary nitroalkanes, secondary nitroalkanes, cyclopentanone oxime, and related compounds. Cancer Res.51, 3143–3147.Search in Google Scholar
Cox, R.A. (1968). The use of guanidinium chloride in the isolation of nucleic acids. Methods Enzymol.12B, 120–129.10.1016/0076-6879(67)12123-XSearch in Google Scholar
Doroshow, J.H. (1983). Effect of anthracycline antibiotics on oxygen radical formation in rat heart. Cancer Res.43, 460–472.Search in Google Scholar
Doroshow, J.H., Locker, G.Y., Baldinger, J., and Myers, C.E. (1979). The effect of doxorubicin on hepatic and cardiac glutathione. Res. Commun. Chem. Pathol. Pharmacol.26, 285–295.Search in Google Scholar
Fiala, E.S., Conaway, C.C., and Mathis, J.E. (1989). Oxidative DNA and RNA damage in the livers of Sprague-Dawley rats treated with the hepatocarcinogen 2-nitropropane. Cancer Res.49, 5518–5522.Search in Google Scholar
Finkel, T. and Holbrook, N.J. (2000). Oxidants, oxidative stress and the biology of ageing. Nature408, 239–247.10.1038/35041687Search in Google Scholar PubMed
Frelon, S., Douki, T., Favier, A., and Cadet, J. (2003). Hydroxyl radical is not the main reactive species involved in the degradation of DNA bases by copper in the presence of hydrogen peroxide. Chem. Res. Toxicol.16, 191–197.10.1021/tx025650qSearch in Google Scholar
Gedik, C.M., Collins, A., and ESCODD (2005). Establishing the background level of base oxidation in human lymphocyte DNA: results of an interlaboratory validation study. FASEB J.19, 82–84.Search in Google Scholar
Giorgiev, G.P., Mantieva, V.L., and Zbarsky, I.B. (1960). RNA fractions in cell nuclei isolated by phenol and by sucrose-glycerophosphate. Biochim. Biophys. Acta37, 373–374.10.1016/0006-3002(60)90257-2Search in Google Scholar
Green, P.S. and Leeuwenburgh, C. (2002). Mitochondrial dysfunction is an early indicator of doxorubicin-induced apoptosis. Biochim. Biophys. Acta1588, 94–101.10.1016/S0925-4439(02)00144-8Search in Google Scholar
Halliwell, B. and Gutteridge, J.M.C. (1999). Free Radicals in Biology and Medicine, 3rd ed. (Oxford, UK: Oxford University Press).Search in Google Scholar
Harman, D. (1972). The biologic clock: the mitochondria? J. Am. Geriatr. Soc.20, 145–147.10.1111/j.1532-5415.1972.tb00787.xSearch in Google Scholar PubMed
Helbock, H.J., Beckman, K.B., Shigenaga, M.K., Walter, P.B., Woodall, A.A., Yeo, H.C., and Ames, B.N. (1998). DNA oxidation matters: the HPLC-electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine. Proc. Natl. Acad. Sci. USA95, 288–293.10.1073/pnas.95.1.288Search in Google Scholar PubMed PubMed Central
Hofer, T. (2001). Oxidation of 2′-deoxyguanosine by H2O2-ascorbate: evidence against free OH• and thermodynamic support for two-electron reduction of H2O2. J. Chem. Soc. Perkin Trans.2, 210–213.10.1039/b006394kSearch in Google Scholar
Hofer, T. and Moller, L. (1998). Reduction of oxidation during the preparation of DNA and analysis of 8-hydroxy-2′-deoxyguanosine. Chem. Res. Toxicol.11, 882–887.10.1021/tx980041xSearch in Google Scholar PubMed
Hofer, T. and Moller, L. (2002). Optimization of the workup procedure for the analysis of 8-oxo-7,8-dihydro-2′-deoxyguanosine with electrochemical detection. Chem. Res. Toxicol.15, 426–432.10.1021/tx015573jSearch in Google Scholar PubMed
Hofer, T., Badouard, C., Bajak, E., Ravanat, J.L., Mattsson, A., and Cotgreave, I.A. (2005). Hydrogen peroxide causes greater oxidation in cellular RNA than in DNA. Biol. Chem.386, 333–337.10.1515/BC.2005.040Search in Google Scholar PubMed
Honda, K., Smith, M.A., Zhu, X., Baus, D., Merrick, W.C., Tartakoff, A.M., Hattier, T., Harris, P.L., Siedlak, S.L., Fujioka, H., et al. (2005). Ribosomal RNA in Alzheimer's disease is oxidized by bound redox-active iron. J. Biol. Chem.280, 20978–20986.10.1074/jbc.M500526200Search in Google Scholar PubMed
Jang, Y.M., Kendaiah, S., Drew, B., Phillips, T., Selman, C., Julian, D., and Leeuwenburgh, C. (2004). Doxorubicin treatment in vivo activates caspase-12 mediated cardiac apoptosis in both male and female rats. FEBS Lett.577, 483–490.10.1016/j.febslet.2004.10.053Search in Google Scholar
Kang, Y.J., Chen, Y., and Epstein, P.N. (1996). Suppression of doxorubicin cardiotoxicity by overexpression of catalase in the heart of transgenic mice. J. Biol. Chem.271, 12610–12616.10.1074/jbc.271.21.12610Search in Google Scholar
Kang, Y.J., Chen, Y., Yu, A., Voss-McCowan, M., and Epstein, P.N. (1997). Overexpression of metallothionein in the heart of transgenic mice suppresses doxorubicin cardiotoxicity. J. Clin. Invest.100, 1501–1506.10.1172/JCI119672Search in Google Scholar
Kawanishi, S., Inoue, S., Sano, S., and Aiba, H. (1986). Photodynamic guanine modification by hematoporphyrin is specific for single-stranded DNA with singlet oxygen as a mediator. J. Biol. Chem.261, 6090–6095.10.1016/S0021-9258(17)38496-XSearch in Google Scholar
Kotamraju, S., Konorev, E.A., Joseph, J., and Kalyanaraman, B. (2000). Doxorubicin-induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone spin traps and ebselen. Role of reactive oxygen and nitrogen species. J. Biol. Chem.275, 33585–33592.10.1074/jbc.M003890200Search in Google Scholar PubMed
Kuchino, Y., Mori, F., Kasai, H., Inoue, H., Iwai, S., Miura, K., Ohtsuka, E., and Nishimura, S. (1987). Misreading of DNA templates containing 8-hydroxydeoxyguanosine at the modified base and at adjacent residues. Nature327, 77–79.10.1038/327077a0Search in Google Scholar PubMed
Leeuwenburgh, C. and Heinecke, J.W. (2001). Oxidative stress and antioxidants in exercise. Curr. Med. Chem.8, 829–838.10.2174/0929867013372896Search in Google Scholar PubMed
Lin, H.S., Jenner, A.M., Ong, C.N., Huang, S.H., Whiteman, M., and Halliwell, B. (2004). A high-throughput and sensitive methodology for the quantification of urinary 8-hydroxy-2′-deoxyguanosine: measurement with gas chromatography-mass spectrometry after single solid-phase extraction. Biochem. J.380, 541–548.10.1042/bj20040004Search in Google Scholar
Liu, J., Head, E., Gharib, A.M., Yuan, W., Ingersoll, R.T., Hagen, T.M., Cotman, C.W., and Ames, B.N. (2002). Memory loss in old rats is associated with brain mitochondrial decay and RNA/DNA oxidation: partial reversal by feeding acetyl-l-carnitine and/or R-α lipoic acid. Proc. Natl. Acad. Sci. USA99, 2356–2361.10.1073/pnas.261709299Search in Google Scholar PubMed PubMed Central
Ma, W.J., Cao, E.H., and Qin, J.F. (1999). The involvement of singlet oxygen in copper-phenanthroline/H2O2-induced DNA base damage: a chemiluminescent study. Redox Rep.4, 271–276.10.1179/135100099101535115Search in Google Scholar PubMed
Maquat, L.E. and Carmichael, G.G. (2001). Quality control of mRNA function. Cell104, 173–176.10.1016/S0092-8674(01)00202-1Search in Google Scholar
Martinet, W., de Meyer, G.R., Herman, A.G., and Kockx, M.M. (2004). Reactive oxygen species induce RNA damage in human atherosclerosis. Eur. J. Clin. Invest.34, 323–327.10.1111/j.1365-2362.2004.01343.xSearch in Google Scholar
Metzler, D.E. (1977). Biochemistry: The Chemical Reactions of Living Cells, 1st ed. (New York, USA: Academic Press).Search in Google Scholar
Miquel, J., de Juan, E., and Sevila, I. (1992). Oxygen-induced mitochondrial damage and aging. EXS62, 47–57.10.1007/978-3-0348-7460-1_5Search in Google Scholar
Nakae, D., Mizumoto, Y., Kobayashi, E., Noguchi, O., and Konishi, Y. (1995). Improved genomic/nuclear DNA extraction for 8-hydroxydeoxyguanosine analysis of small amounts of rat liver tissue. Cancer Lett.97, 233–239.10.1016/0304-3835(95)03980-BSearch in Google Scholar
Nohl, H. (1987). A novel superoxide radical generator in heart mitochondria. FEBS Lett.214, 269–273.10.1016/0014-5793(87)80068-6Search in Google Scholar
Nohl, H., Gille, L., and Staniek, K. (1998). The exogenous NADH dehydrogenase of heart mitochondria is the key enzyme responsible for selective cardiotoxicity of anthracyclines. Z. Naturforsch. (C)53, 279–285.10.1515/znc-1998-3-419Search in Google Scholar PubMed
Nunomura, A., Perry, G., Pappolla, M.A., Wade, R., Hirai, K., Chiba, S., and Smith, M.A. (1999). RNA oxidation is a prominent feature of vulnerable neurons in Alzheimer's disease. J. Neurosci.19, 1959–1964.10.1523/JNEUROSCI.19-06-01959.1999Search in Google Scholar
Park, E.M., Shigenaga, M.K., Degan, P., Korn, T.S., Kitzler, J.W., Wehr, C.M., Kolachana, P., and Ames, B.N. (1992). Assay of excised oxidative DNA lesions: isolation of 8-oxoguanine and its nucleoside derivatives from biological fluids with a monoclonal antibody column. Proc. Natl. Acad. Sci. USA89, 3375–3379.10.1073/pnas.89.8.3375Search in Google Scholar PubMed PubMed Central
Ravanat, J.L., Douki, T., Duez, P., Gremaud, E., Herbert, K., Hofer, T., Lasserre, L., Saint-Pierre, C., Favier, A., and Cadet, J. (2002). Cellular background level of 8-oxo-7,8-dihydro-2′-deoxyguanosine: an isotope based method to evaluate artefactual oxidation of DNA during its extraction and subsequent work-up. Carcinogenesis23, 1911–1918.10.1093/carcin/23.11.1911Search in Google Scholar PubMed
Rhee, Y., Valentine, M.R., and Termini, J. (1995). Oxidative base damage in RNA detected by reverse transcriptase. Nucleic Acids Res.23, 3275–3282.10.1093/nar/23.16.3275Search in Google Scholar PubMed PubMed Central
Shan, K., Lincoff, A.M., and Young, J.B. (1996). Anthracycline-induced cardiotoxicity. Ann. Intern. Med.125, 47–58.10.7326/0003-4819-125-1-199607010-00008Search in Google Scholar
Shen, Z., Wu, W., and Hazen, S.L. (2000). Activated leukocytes oxidatively damage DNA, RNA, and the nucleotide pool through halide-dependent formation of hydroxyl radical. Biochemistry39, 5474–5482.10.1021/bi992809ySearch in Google Scholar
Shigenaga, M.K. and Ames, B.N. (1991). Assays for 8-hydroxy-2′-deoxyguanosine: a biomarker of in vivo oxidative DNA damage. Free Radic. Biol. Med.10, 211–216.10.1016/0891-5849(91)90078-HSearch in Google Scholar
Sies, H. and Menck, C.F. (1992). Singlet oxygen induced DNA damage. Mutat Res.275, 367–375.10.1016/0921-8734(92)90039-RSearch in Google Scholar
Van Remmen, H., Ikeno, Y., Hamilton, M., Pahlavani, M., Wolf, N., Thorpe, S.R., Alderson, N.L., Baynes, J.W., Epstein, C.J., Huang, T.T., et al. (2003). Life-long reduction in MnSOD activity results in increased DNA damage and higher incidence of cancer but does not accelerate aging. Physiol. Genomics16, 29–37.10.1152/physiolgenomics.00122.2003Search in Google Scholar
Wacker, W.E. and Vallee, B.L. (1959). Nucleic acids and metals. J. Biol. Chem.234, 3257–3262.10.1016/S0021-9258(18)69661-9Search in Google Scholar
Wardman, P. and Candeias, L.P. (1996). Fenton chemistry: an introduction. Radiat. Res.145, 523–531.10.2307/3579270Search in Google Scholar
Weimann, A., Belling, D., and Poulsen, H.E. (2002). Quantification of 8-oxo-guanine and guanine as the nucleobase, nucleoside and deoxynucleoside forms in human urine by high-performance liquid chromatography-electrospray tandem mass spectrometry. Nucleic Acids Res.30, E7.10.1093/nar/30.2.e7Search in Google Scholar
Yamamoto, K. and Kawanishi, S. (1989). Hydroxyl free radical is not the main active species in site-specific DNA damage induced by copper(II) ion and hydrogen peroxide. J. Biol. Chem.264, 15435–15440.10.1016/S0021-9258(19)84847-0Search in Google Scholar
Yen, H.C., Oberley, T.D., Vichitbandha, S., Ho, Y.S., and St Clair, D.K. (1996). The protective role of manganese superoxide dismutase against adriamycin-induced acute cardiac toxicity in transgenic mice. J. Clin. Invest.98, 1253–1260.10.1172/JCI118909Search in Google Scholar PubMed PubMed Central
Zhang, J., Perry, G., Smith, M.A., Robertson, D., Olson, S.J., Graham, D.G., and Montine, T.J. (1999). Parkinson's disease is associated with oxidative damage to cytoplasmic DNA and RNA in substantia nigra neurons. Am. J. Pathol.154, 1423–1429.10.1016/S0002-9440(10)65396-5Search in Google Scholar
©2006 by Walter de Gruyter Berlin New York