Logo of envhperEnvironmental Health PerspectivesBrowse ArticlesAbout EHPGeneral InformationAuthorsMediaProgramsPartnerships
Environ Health Perspect. 2002 Oct; 110(10): 1003–1008.
PMCID: PMC1241026
Research Article

Mechanisms of the genotoxicity of crocidolite asbestos in mammalian cells: implication from mutation patterns induced by reactive oxygen species.


Asbestos is an important environmental carcinogen in the United States and remains the primary occupational concern in many developing countries; however, the underlying mechanisms of its genotoxicity are not known. We showed previously that asbestos is a potent gene and chromosomal mutagen in mammalian cells and that it induces mostly multilocus deletions. Furthermore, reactive oxygen species (ROS) are associated with the mutagenic process. To evaluate the contribution of ROS to the mutagenicity of asbestos, we examined their generation, particularly hydrogen peroxide, and compared the types of mutants induced by crocidolite fibers with those generated by H(2)O(2 )in human-hamster hybrid (A(L)) cells. Using confocal scanning microscopy together with the radical probe 5,6 -chloromethy-2,7 -dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), we found that asbestos induces a dose-dependent increase in the level of ROS among fiber-treated A(L) cells, which is suppressed by concurrent treatment with dimethyl sulfoxide. Using N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red reagent) together with horseradish peroxidase, we further demonstrated that there was a dose-dependent induction of H(2)O(2) in crocidolite-treated A(L) cells. The amount of H(2)O(2 )induced by asbestos reached a plateau at a dose of 6 microg/cm(2). Concurrent treatment with catalase (1,000 U/mL) inhibited this induction by 7- to 8-fold. Mutation spectrum analysis showed that the types of CD59(-) mutants induced by crocidolite fibers were similar to those induced by equitoxic doses of H(2)O(2). These results provide direct evidence that the mutagenicity of asbestos is mediated by ROS in mammalian cells.

Full Text

The Full Text of this article is available as a PDF (1.0M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Mossman BT. In vitro studies on the biologic effects of fibers: correlation with in vivo bioassays. Environ Health Perspect. 1990 Aug;88:319–322. [PMC free article] [PubMed]
  • Merchant JA. Human epidemiology: a review of fiber type and characteristics in the development of malignant and nonmalignant disease. Environ Health Perspect. 1990 Aug;88:287–293. [PMC free article] [PubMed]
  • Anderson HA, Lilis R, Daum SM, Fischbein AS, Selikoff IJ. Household-contact asbestos neoplastic risk. Ann N Y Acad Sci. 1976;271:311–323. [PubMed]
  • Oliver LC, Sprince NL, Greene R. Asbestos-related disease in public school custodians. Am J Ind Med. 1991;19(3):303–316. [PubMed]
  • Sebastien P, Bignon J, Martin M. Indoor airborne asbestos pollution: from the ceiling and the floor. Science. 1982 Jun 25;216(4553):1410–1412. [PubMed]
  • Stanton MF, Laynard M, Tegeris A, Miller E, May M, Kent E. Carcinogenicity of fibrous glass: pleural response in the rat in relation to fiber dimension. J Natl Cancer Inst. 1977 Mar;58(3):587–603. [PubMed]
  • Hei TK, He ZY, Suzuki K. Effects of antioxidants on fiber mutagenesis. Carcinogenesis. 1995 Jul;16(7):1573–1578. [PubMed]
  • Walker C, Everitt J, Barrett JC. Possible cellular and molecular mechanisms for asbestos carcinogenicity. Am J Ind Med. 1992;21(2):253–273. [PubMed]
  • Schapira RM, Ghio AJ, Effros RM, Morrisey J, Dawson CA, Hacker AD. Hydroxyl radicals are formed in the rat lung after asbestos instillation in vivo. Am J Respir Cell Mol Biol. 1994 May;10(5):573–579. [PubMed]
  • DeGraff WG, Krishna MC, Russo A, Mitchell JB. Antimutagenicity of a low molecular weight superoxide dismutase mimic against oxidative mutagens. Environ Mol Mutagen. 1992;19(1):21–26. [PubMed]
  • Korkina LG, Durnev AD, Suslova TB, Cheremisina ZP, Daugel-Dauge NO, Afanas'ev IB. Oxygen radical-mediated mutagenic effect of asbestos on human lymphocytes: suppression by oxygen radical scavengers. Mutat Res. 1992 Feb;265(2):245–253. [PubMed]
  • Turver CJ, Brown RC. The role of catalytic iron in asbestos induced lipid peroxidation and DNA-strand breakage in C3H10T1/2 cells. Br J Cancer. 1987 Aug;56(2):133–136. [PMC free article] [PubMed]
  • Huang SL, Saggioro D, Michelmann H, Malling HV. Genetic effects of crocidolite asbestos in Chinese hamster lung cells. Mutat Res. 1978 May;57(2):225–232. [PubMed]
  • Daniel FB. In vitro assessment of asbestos genotoxicity. Environ Health Perspect. 1983 Nov;53:163–167. [PMC free article] [PubMed]
  • Hei TK, Piao CQ, He ZY, Vannais D, Waldren CA. Chrysotile fiber is a strong mutagen in mammalian cells. Cancer Res. 1992 Nov 15;52(22):6305–6309. [PubMed]
  • Both K, Henderson DW, Turner DR. Asbestos and erionite fibres can induce mutations in human lymphocytes that result in loss of heterozygosity. Int J Cancer. 1994 Nov 15;59(4):538–542. [PubMed]
  • Park SH, Aust AE. Participation of iron and nitric oxide in the mutagenicity of asbestos in hgprt-, gpt+ Chinese hamster V79 cells. Cancer Res. 1998 Mar 15;58(6):1144–1148. [PubMed]
  • Lezon-Geyda K, Jaime CM, Godbold JH, Savransky EF, Hope A, Kheiri SA, Dzmura ZM, Uehara H, Johnson EM, Fasy TM. Chrysotile asbestos fibers mediate homologous recombination in Rat2 lambda fibroblasts: implications for carcinogenesis. Mutat Res. 1996 Dec 12;361(2-3):113–120. [PubMed]
  • Waldren C, Correll L, Sognier MA, Puck TT. Measurement of low levels of x-ray mutagenesis in relation to human disease. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4839–4843. [PMC free article] [PubMed]
  • Hei TK, Liu SX, Waldren C. Mutagenicity of arsenic in mammalian cells: role of reactive oxygen species. Proc Natl Acad Sci U S A. 1998 Jul 7;95(14):8103–8107. [PMC free article] [PubMed]
  • Long JF, Dutta PK, Hogg BD. Fluorescence imaging of reactive oxygen metabolites generated in single macrophage cells (NR8383) upon phagocytosis of natural zeolite (erionite) fibers. Environ Health Perspect. 1997 Jul;105(7):706–711. [PMC free article] [PubMed]
  • Weitzman SA, Graceffa P. Asbestos catalyzes hydroxyl and superoxide radical generation from hydrogen peroxide. Arch Biochem Biophys. 1984 Jan;228(1):373–376. [PubMed]
  • Liu SX, Athar M, Lippai I, Waldren C, Hei TK. Induction of oxyradicals by arsenic: implication for mechanism of genotoxicity. Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1643–1648. [PMC free article] [PubMed]
  • Mohanty JG, Jaffe JS, Schulman ES, Raible DG. A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine derivative. J Immunol Methods. 1997 Mar 28;202(2):133–141. [PubMed]
  • Xu A, Wu LJ, Santella RM, Hei TK. Role of oxyradicals in mutagenicity and DNA damage induced by crocidolite asbestos in mammalian cells. Cancer Res. 1999 Dec 1;59(23):5922–5926. [PubMed]
  • Fridovich I. The biology of oxygen radicals. Science. 1978 Sep 8;201(4359):875–880. [PubMed]
  • McGuinness SM, Shibuya ML, Ueno AM, Vannais DB, Waldren CA. Mutant quantity and quality in mammalian cells (AL) exposed to cesium-137 gamma radiation: effect of caffeine. Radiat Res. 1995 Jun;142(3):247–255. [PubMed]
  • Zhou H, Zhu LX, Li K, Hei TK. Radon, tobacco-specific nitrosamine and mutagenesis in mammalian cells. Mutat Res. 1999 Nov 29;430(1):145–153. [PubMed]
  • Jaurand MC. Mechanisms of fiber-induced genotoxicity. Environ Health Perspect. 1997 Sep;105 (Suppl 5):1073–1084. [PMC free article] [PubMed]
  • Shatos MA, Doherty JM, Marsh JP, Mossman BT. Prevention of asbestos-induced cell death in rat lung fibroblasts and alveolar macrophages by scavengers of active oxygen species. Environ Res. 1987 Oct;44(1):103–116. [PubMed]
  • Oya Y, Yamamoto K, Tonomura A. The biological activity of hydrogen peroxide. I. Induction of chromosome-type aberrations susceptible to inhibition by scavengers of hydroxyl radicals in human embryonic fibroblasts. Mutat Res. 1986 Dec;172(3):245–253. [PubMed]
  • Adachi S, Yoshida S, Kawamura K, Takahashi M, Uchida H, Odagiri Y, Takemoto K. Inductions of oxidative DNA damage and mesothelioma by crocidolite, with special reference to the presence of iron inside and outside of asbestos fiber. Carcinogenesis. 1994 Apr;15(4):753–758. [PubMed]
  • Maples KR, Johnson NF. Fiber-induced hydroxyl radical formation: correlation with mesothelioma induction in rats and humans. Carcinogenesis. 1992 Nov;13(11):2035–2039. [PubMed]
  • Hansen K, Mossman BT. Generation of superoxide (O2-.) from alveolar macrophages exposed to asbestiform and nonfibrous particles. Cancer Res. 1987 Mar 15;47(6):1681–1686. [PubMed]
  • Vilím V, Wilhelm J, Brzák P, Hurych J. Stimulation of alveolar macrophages by mineral dusts in vitro: luminol-dependent chemiluminescence study. Environ Res. 1987 Feb;42(1):246–256. [PubMed]
  • Root RK, Metcalf J, Oshino N, Chance B. H2O2 release from human granulocytes during phagocytosis. I. Documentation, quantitation, and some regulating factors. J Clin Invest. 1975 May;55(5):945–955. [PMC free article] [PubMed]
  • Gabrielson EW, Rosen GM, Grafstrom RC, Strauss KE, Harris CC. Studies on the role of oxygen radicals in asbestos-induced cytopathology of cultured human lung mesothelial cells. Carcinogenesis. 1986 Jul;7(7):1161–1164. [PubMed]
  • LeBel CP, Ischiropoulos H, Bondy SC. Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol. 1992 Mar-Apr;5(2):227–231. [PubMed]
  • Rothe G, Valet G. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2',7'-dichlorofluorescin. J Leukoc Biol. 1990 May;47(5):440–448. [PubMed]
  • Ambrosio G, Zweier JL, Duilio C, Kuppusamy P, Santoro G, Elia PP, Tritto I, Cirillo P, Condorelli M, Chiariello M, et al. Evidence that mitochondrial respiration is a source of potentially toxic oxygen free radicals in intact rabbit hearts subjected to ischemia and reflow. J Biol Chem. 1993 Sep 5;268(25):18532–18541. [PubMed]
  • Hancock JT, Jones OT. The inhibition by diphenyleneiodonium and its analogues of superoxide generation by macrophages. Biochem J. 1987 Feb 15;242(1):103–107. [PMC free article] [PubMed]
  • Freeman BA, Crapo JD. Biology of disease: free radicals and tissue injury. Lab Invest. 1982 Nov;47(5):412–426. [PubMed]
  • Howden PJ, Faux SP. Fibre-induced lipid peroxidation leads to DNA adduct formation in Salmonella typhimurium TA104 and rat lung fibroblasts. Carcinogenesis. 1996 Mar;17(3):413–419. [PubMed]
  • Turrens JF, Alexandre A, Lehninger AL. Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria. Arch Biochem Biophys. 1985 Mar;237(2):408–414. [PubMed]
  • Zhu S, Manuel M, Tanaka S, Choe N, Kagan E, Matalon S. Contribution of reactive oxygen and nitrogen species to particulate-induced lung injury. Environ Health Perspect. 1998 Oct;106 (Suppl 5):1157–1163. [PMC free article] [PubMed]
  • Park SH, Aust AE. Regulation of nitric oxide synthase induction by iron and glutathione in asbestos-treated human lung epithelial cells. Arch Biochem Biophys. 1998 Dec 1;360(1):47–52. [PubMed]
  • Juedes MJ, Wogan GN. Peroxynitrite-induced mutation spectra of pSP189 following replication in bacteria and in human cells. Mutat Res. 1996 Jan 17;349(1):51–61. [PubMed]
  • Termini J. Hydroperoxide-induced DNA damage and mutations. Mutat Res. 2000 May 30;450(1-2):107–124. [PubMed]
  • Dahm-Daphi J, Sass C, Alberti W. Comparison of biological effects of DNA damage induced by ionizing radiation and hydrogen peroxide in CHO cells. Int J Radiat Biol. 2000 Jan;76(1):67–75. [PubMed]
  • Gustafson DL, Franz HR, Ueno AM, Smith CJ, Doolittle DJ, Waldren CA. Vanillin (3-methoxy-4-hydroxybenzaldehyde) inhibits mutation induced by hydrogen peroxide, N-methyl-N-nitrosoguanidine and mitomycin C but not (137)Cs gamma-radiation at the CD59 locus in human-hamster hybrid A(L) cells. Mutagenesis. 2000 May;15(3):207–213. [PubMed]
  • Goodglick LA, Pietras LA, Kane AB. Evaluation of the causal relationship between crocidolite asbestos-induced lipid peroxidation and toxicity to macrophages. Am Rev Respir Dis. 1989 May;139(5):1265–1273. [PubMed]

Articles from Environmental Health Perspectives are provided here courtesy of National Institute of Environmental Health Science


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Compound
    PubChem chemical compound records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records. Multiple substance records may contribute to the PubChem compound record.
  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem chemical substance records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records.

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...