Abstract
Hexavalent chromium (Cr (VI)) is reduced intracellularly to Cr (V), Cr (IV) and Cr (III) by ascorbate (Asc), cysteine and glutathione (GSH). These metabolites induce a spectrum of genomic DNA damage resulting in the inhibition of DNA replication. Our previous studies have shown that treatment of DNA with Cr (III) or Cr (VI) plus Asc results in the formation of DNA‐Cr‐DNA crosslinks (Cr‐DDC) and guanine‐specific arrests of both prokaryotic and mammalian DNA polymerases. GSH not only acts as a reductant of Cr (VI) but also becomes crosslinked to DNA by Cr, thus, the focus of the present study was to examine the role of GSH in Cr‐induced DNA damage and polymerase arrests. Co‐incubation of Cr (III) with plasmid DNA in the presence of GSH led to the crosslinking of GSH to DNA. GSH co‐treatment with Cr (III) also led to a decrease in the degree of Cr‐induced DNA interstrand crosslinks relative to Cr (III) alone, without affecting total Cr DNA binding. DNA polymerase arrests were observed following treatment of DNA with Cr (III) alone, but were markedly reduced when GSH was added to the reaction mixture. Pre‐formed polymerase‐arresting lesions (Cr‐DDC) were not removed by subsequent addition of GSH. Treatment of DNA with Cr (VI), in the presence of GSH, resulted in crosslinking of GSH to DNA, but failed to produce detectable DNA interstrand crosslinks or polymerase arrests. The inhibitory effect of GSH on Cr‐induced polymerase arrest was further confirmed in human genomic DNA using quantitative PCR (QPCR) analysis. Treatment of genomic DNA with Cr (III) resulted in a marked inhibition of the amplification of a 1.6 kb target fragment of the p53 gene by Taq polymerase. This was almost completely prevented by co‐treatment with GSH and Cr (III). These results indicate that Cr‐induced DNA interstrand crosslinks, and not DNA‐Cr‐GSH crosslinks, are the principal lesions responsible for blocking DNA replication. Moreover, the formation of DNA‐Cr‐GSH crosslinks may actually preclude the formation of the polymerase arresting lesions.
Similar content being viewed by others
References
IARC: Chromium, nickel, and welding. In: International Agency for Research on Cancer Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Human, vol. 49. IARC, Lyon, France, 1990, pp 1–214
Clarkson TW: Molecular ionic mimicry of toxic metals. Annu Rev Pharmacol Toxicol 32: 545–571, 1993
DeFlora S, Wetterhahn KE: Mechanisms of chromium metabolism and genotoxicity. Life Chem Rep 7: 169–244, 1989
DeFlora S, Bagnasco M, Serra D, Zanacchi P: Genotoxicity of chromium compounds. A review. Mutat Res 238: 99–172, 1990
Jones P, Kortenkamp A, O'Brien P, Wang G, Yang G: Evidence for the generation of hydroxyl radicals from a chromium (V) intermediate isolated from the reaction of chromate with glutathione. Arch Biochem Biophys 286: 652–655, 1991
Kasprzak KS: The role of oxidative damage in metal carcinogenicity. Chem Res Toxicol 4: 604–615, 1991
Kortenkamp A, O'Brien P, Beyersmann D: The reduction of chromate is a prerequisite of chromium binding to cell nuclei. Carcinogenesis 12: 1143–1144, 1991
Léonard A: Mechanisms in metal genotoxicity: The significance of in vitro approaches. Mutat Res 198: 321–326, 1988
Levis AG, Majone F: Cytotoxic and clastogenic effects of soluble and insoluble compounds containing hexavalent and trivalent chromium. Br J Cancer 44: 219–235, 1981
Manning FCR, Xu J, Patierno SR: Transcription inhibition by carcinogenic chromate: Relationship to DNA damage. Mol Carcinogen 6: 270–279, 1992
Sugiyama M, Ando A, Nakao K, Ueta H, Hidaka T, Ogura R: Influence of vitamin B2 on formation of chromium (V), alkali-labile sites, and lethality of sodium chromate (VI) in Chinese hamster V-79 cells. Cancer Res 49: 6180–6184, 1989
Xu J, Wise JP, Patierno SR: DNA damage induced by carcinogenic lead chromate particles in cultured mammalian cells. Mutat Res 280: 129–136, 1992
Xu J, Manning FCR, Patierno SR: Preferential formation and repair of chromium-induced DNA damage in nuclear matrix DNA. Carcinogenesis 15: 1443–1450, 1994
Bennicelli C, Camoirano A, Petruzzeli S, Zanacchi P, DeFlora S: High sensitivity of Salmonella TA 102 in detecting hexavalent chromium mutagenicity and its reversal by liver and lung preparations. Mutat Res 122: 1–5, 1983
Chen J, Thilly WG: Mutation spectrum of chromium (VI) in human cells. Mutat Res 323: 21–27, 1994
Cheng L, Liu S, Dixon K: Analysis and repair and mutagenesis of chromium-induced DNA damage in yeast, mammalian cells, and transgenic mice. Environ Health Perspect 106(suppl 4): 1027–1032, 1998
Liu S, Medvedovic M, Dixon K: Mutational specificity in a shuttle vector replication in chromium (VI)-treated mammalian cells. Environ Mol Mutagen 33: 313–319, 1999
Liu S, Dixon K: Induction of mutagenic DNA damage by chromium (VI) and glutathione. Environ Mol Mutagen 28: 71–79, 1996
Yang JL, Hsieh YC, Wu CW, Lee TC: Mutational specificity of chromium (VI) compounds at the hprt locus of Chinese hamster ovary-K1 cells. Carcinogenesis 13: 2053–2057, 1992
Bakke O, Jakobsen K, Eik-Nes KB: Concentration-dependent effects of potassium dichromate on the cell cycle. Cytometry 5: 482–486, 1984
Bridgewater LC, Manning FC, Patierno SR: Arrest of replication by mammalian DNA polymerases alpha and beta caused by chromium-DNA lesions. Mol Carcinogen 23: 201–6, 1998
Bridgewater LC, Manning FCR, Patierno SR: Base-specific arrest of in vitro DNA replication by carcinogenic chromium: Relationship to DNA intrastrand crosslinks. Carcinogenesis 15: 2421–2427, 1994
Bridgewater LC, Manning FCR, Patierno SR: DNA polymerase arrest by adducted trivalent chromium. Mol Carcinogen 9: 122–133, 1994
Xu J, Bubley GJ, Detrick B, Blankenship LJ, Patierno SR: Chromium (VI) treatment of normal human lung cells results in guanine-specific DNA polymerase arrest and blockade of cell cycle and DNA synthesis. Carcinogenesis 17: 1511–1517, 1996
Bianchi V, Dal Toso R, Debetto P, Levis AG, Luciani S, Majone F, Tamino G: Mechanisms of chromium toxicity in mammalian cell cultures. Toxicology 17: 219–224, 1980
Levis AG, Buttignol M, Bianchi V, Sponza G: Effects of potassium dichromate on nucleic acid and protein syntheses and on precursor uptake in BHK fibroblasts. Cancer Res 38: 110–116, 1978
Hamilton JW, Wetterhahn KE: Differential effects of chromium (VI) in constitutive and inducible gene expression in chick embryo liver in vivo and correlation with chromium (VI)-induced DNA damage. Mol Carcinogen 2: 274–286, 1989
Jakobsen K, Bakke O, Østgaard K, White LR, Eik-Nes KB: Effects of potassium dichromate on the cell cycle of an established human cell line (NHIK 3025). Toxicology 24: 281–292, 1982
Blankenship LJ, Carlisle DL, Wise JP, Orenstein JM, Dye LE III, Patierno SR: Induction of apoptotic cell death by particulate lead chromate: Differential effects of vitamins C and E on genotoxicity and survival. Toxicol Appl Pharmacol 146: 270–280, 1997
Blankenship LJ, Manning FCR, Orenstein JM, Patierno SR: Apoptosis is the mode of cell death caused by carcinogenic chromium. Toxicol Appl Pharmacol 126: 75–83, 1994
Singh J, Pritchard DE, Carlisle DL, Mclean JA, Montaser A, Orenstein J, Patierno SR: Internalization of carcinogenic lead chromate particles by cultured normal human lung epithelial cells: formation of lead-inclusion bodies and induction of apoptosis. Toxicol Appl Pharmacol 161: 240–248, 1999
Singh J, Carlisle DL, Pritchard DE, Patierno SR: Chromium-induced apoptosis: relationship to chromium carcinogenesis. Oncology Rep 5: 1307–1318(review), 1998
Carlisle DL, Pritchard DE, Singh J, Owens BM, Blankenship LJ, Orenstein JM, Patierno SR: Apoptosis and p53 induction in human fibroblasts exposed to chromium (VI): Effects of ascorbate and tocopherol. Toxicol Sci 55: 60–68, 2000
Carlisle DL, Pritchard DE, Singh J, Patierno SR: Chromium (VI) induces p53 dependent apoptosis in diploid human lung and mouse dermal fibroblasts. Mol Carcinogen 28: 111–118, 2000
Arslan P, Beltrame M, Tomasi A: Intracellular chromium reduction. Biochim Biophys Acta 931: 10–15, 1987
Standeven AM, Wetterhahn KE: Ascorbate is the principal reductant of chromium (VI) in rat liver and kidney ultrafiltrates. Carcinogenesis 12: 1733–1737, 1991
Wiegand HJ, Ottenwalder H, Bolt HM: The reduction of chromium (VI) to chromium (III) by glutathione: An intracellular redox pathway in the metabolism of the carcinogen chromate. Toxicology 33: 341–348, 1984
Liu KJ, Shi X, Dalal NS: Synthesis of Cr (IV)-GSH, its identification and its free hydroxyl radical generation: A model compound for Cr (VI) carcinogenicity. Biochem Biophys Res Commun 235: 54–58, 1997
Aiyar J, Berkovits HJ, Floyd RA, Wetterhahn KE: Reaction of chromium (VI) with glutathione or with hydrogen peroxide: Identification of reactive intermediates and their role in chromium (VI)-induced DNA damage. Environ Health Perspect 92: 53–62, 1991
Borges KM, Boswell JS, Liebross RH, Wetterhahn KE: Activation of chromium (VI) by thiols results in chromium (V) formation, chromium binding to DNA and altered DNA conformation. Carcinogenesis 12: 551–561, 1991
O'Brien P, Kortenkamp A: Chemical models important in understanding the ways in which chromate can damage DNA. Environ Health Perspect 102(suppl 3): 3–10, 1994
Shi XL, Dalal NS: On the mechanism of the chromate reduction by glutathione: Evidence for the glutathionyl radical and an insoluble Cr (V) intermediate. Biochem Biophys Res Commun 156: 137–142, 1988
Borges KM, Wetterhahn KE: Chromium crosslinks glutathione and cysteine to DNA. Carcinogenesis 10: 2165–2168, 1989
Zhitkovich A, Voitkun V, Costa M: Glutathione and free amino acids form stable complexes with DNA following exposure of intact mammalian cells to chromate. Carcinogenesis 16: 907–913, 1995
Capellmann M, Mikalsen A, Hindrum M, Alexander J: Influence of reducing compounds on the formation of DNA-protein crosslinks in HL-60 cells induced by hexavalent chromium. Carcinogenesis 16: 1135–1139, 1995
Lin X, Sugiyama M, Costa M: Differences in the effect of vitamin E on nickel sulfoxide or nickel chloride-induced chromosomal aberrations in mammalian cells. Mutat Res 260: 159–164, 1991
Casadevall M, Kortenkamp A: The generation of apurinic/apyrimidinic sites in isolated DNA during the reduction of chromate by glutathione. Carcinogenesis 15: 407–409, 1994
Casadevall M, Kortenkamp A: The formation of both apurinic/apyrimidinic sites and single-strand breaks by chromate and glutathione arises from attack by the same single reactive species and is dependent on molecular oxygen. Carcinogenesis 16: 805–809, 1995
Kawanishi S, Inoue S, Sano S: Mechanism of DNA cleavage induced by sodium chromate (VI) in the presence of hydrogen peroxide. J Biol Chem 261: 5952–5958, 1986
Shi XL, Dalal NS: One-electron reduction of chromate by NADPH-dependent glutathione reductase. J Inorg Biochem 40: 1–12, 1990
Cupo DY, Wetterhahn KE: Modification of chromium (VI)-induced DNA damage by glutathione and cytochrome P-450 in chicken embryo hepatocytes. Proc Natl Acad Sci USA 82: 6755–6759, 1985
Sugiyama M, Tsuzuki K: Effects of glutathione depletion on formation of paramagnetic chromium in Chinese hamster V-79 cells. FEBS Lett 341: 273–276, 1994
Misra M, Alcedo JA, Wetterhahn KE: Two pathways for chromium (VI)-induced DNA damage in 14 day chick embryos: Cr-DNA binding in liver and 8-oxo-2′-deoxyguanosine in red blood cells. Carcinogenesis 15: 2911–2917, 1994
Voitkun V, Zhitkovich A, Costa M: Cr (III)-mediated crosslinks of glutathione or amino acids to the DNA phosphate backbone are mutagenic in human cells. Nucleic Acids Res 26: 2024–2030, 1998
Sambrook J, Fritsch EF, Maniatis T: In: Molecular Cloning, A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989
Feinberg A, Vogelstein B: Addendum to ‘A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.’ Anal Biochem 137: 266–267, 1984
Tornaletti S, Rozek D, Pfeifer GP: The distribution of UV photoproducts along the human p53 gene and its relation to mutations in skin cancer. Oncogene 8: 2051–2057, 1993
Denissenko MF, Cahill J, Koudriakova TB, Gerber N, Pfeifer GP: Quantitation and mapping of aflatoxin B1-induced DNA damage in genomic DNA using aflatoxin B1-8,9-epoxide and microsomal activation systems. Mutat Res 425: 205–211, 1999
Wise JP, Orenstein JM, Patierno SR: Inhibition of lead chromate clastogenesis by ascorbate: Relationship to particle dissolution and uptake. Carcinogenesis 14: 429–434, 1993
McCarthy MJ, Rosenblatt JI, Lloyd RS: A modified quantitative polymerase chain reaction assay for measuring gene-specific repair of UV photoproducts in human cells. Mutat Res 363: 57–66, 1996
Grimaldi KA, Bingham JP, Souhami RL, Hartley JA: DNA damage by anticancer agents and its repair: Mapping in cells at the subgene level with quantitative polymerase chain reaction. Anal Biochem 222: 236–242, 1994
Oshita F, Saijo N: Rapid polymerase chain reaction assay to detect variation in the extent of gene-specific damage between cisplatin-or VP16-resistant and sensitive lung cancer cell lines. Jpn J Cancer Res 85: 669–673, 1994
Kalinowski DP, Illenye S, Van Houten B: Analysis of DNA damage and repair in murine leukemia L1210 cells using a quantitative polymerase chain reaction assay. Nucleic Acids Res 20: 3485–3494, 1992
Jennerwein MM, Eastman A: A polymerase chain reaction-based method to detect cisplatin adducts in specific genes. Nucleic Acids Res 19: 6209–6214, 1991
Govan HL, Valles-Ayoub Y, Braun J: Fine-mapping of DNA damage and repair in specific genomic segments. Nucleic Acids Res 18: 3823–3830, 1990
Jenkins GJ, Burlinson B, Parry JM: The polymerase inhibition assay: A methodology for the identification of DNA-damaging agents. Mol Carcinogen 27: 289–297, 2000
Standeven AM, Wetterhahn KE: Ascorbate is the principal reductant of chromium (VI) in rat lung ultrafiltrates and cytosol, and mediates chromium-DNA binding in vitro. Carcinogenesis 13: 1319–1324, 1992
Larkworthy LF, Nolan KB, O'Brien EC: Chromium. In: G. Wilkinson, R.D. Gillard, J.A. McCleverty (eds). Comprehensive Co-ordination Chemistry. Pergamon Press, Oxford, 1988, pp 699–969
Hneihen AS, Standeven AM, Wetterhahn KE: Differential binding of chromium (VI) and chromium (III) complexes to salmon sperm nuclei and nuclear DNA and isolated calf thymus DNA. Carcinogenesis 14: 1795–1803, 1993
Abdullah N, Barrett J, O'Brien P: Synthesis and characterization of some chromium (III) complexes with glutathione. J Chem Soc Dalton Trans 5: 2085–2089, 1985
Fiol JJ, Terron A, Moreno V: Chromium (III) interactions with nucleotides. Inorg Chim Acta 83: 69–73, 1984
Campromar JA, Fiol JJ, Terron A: Chromium (III) interactions with nucleotides. II. Inorg Chim Acta 124: 75–81, 1986
Krishnamoorthy CR, Harris GM: An equilibrium and rate study of the interaction of aqueous chromium (III) ion with adenine. J Coord Chem 10: 69–73, 1980
Mikulski CM, Mattucci L, Smith Y, Tran TB, Karayannis NM: Guanine complexes with first transition metal perchlorates. Inorg Chim Acta 80: 127–133, 1983
Suzuki Y, Fukuda K: Reduction of hexavalent chromium by ascorbic acid and glutathione with special reference to the rat lung. Arch Toxicol 64: 169–176, 1990
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
O'Brien, T., Xu, J. & Patierno, S.R. Effects of glutathione on chromium‐induced DNA. Crosslinking and DNA polymerase arrest. Mol Cell Biochem 222, 173–182 (2001). https://doi.org/10.1023/A:1017918330073
Issue Date:
DOI: https://doi.org/10.1023/A:1017918330073