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

Our plain-English summary of Mechanisms of the genotoxicity of crocidolite asbestos in mammalian cells: implication from mutation patterns induced by reactive oxygen species, by A. Xu, H. Zhou, D.Z. Yu, et al (Environmental Health Perspectives, 2002).

The study

• Authors: A. Xu, H. Zhou, D.Z. Yu, T.K. Hei
• Journal: Environmental Health Perspectives
• Published: 2002;110(10):1003-8
• Cited by: 66 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

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.

In plain English

This laboratory study used cells grown in dishes, not people. The authors exposed human–hamster hybrid cells (called AL cells) to crocidolite asbestos fibers and, for comparison, to hydrogen peroxide. Hydrogen peroxide is a chemical that produces reactive oxygen species, highly reactive forms of oxygen that can damage DNA. A dose of 6 micrograms of crocidolite per square centimeter raised reactive oxygen levels in the cells 5-fold compared with untreated cells, and the scavenger chemical DMSO lowered this signal. Crocidolite also made the cells release hydrogen peroxide, and the enzyme catalase reduced this 7- to 8-fold. Crocidolite caused mutations in a gene on human chromosome 11, reaching about 4 times the background level at 4 micrograms per square centimeter, and DMSO or catalase reduced these mutations. Doses of hydrogen peroxide that killed similar shares of cells produced similar types of mutations. These were mainly losses of DNA spanning several genes, which increased with dose. The authors concluded that this was direct evidence that the genotoxicity of asbestos fibers is mediated by reactive oxygen species. They noted that their experiments did not identify where these molecules came from.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) experiment in cultured cells. Cells were exposed to crocidolite or hydrogen peroxide, with or without the antioxidants catalase or DMSO, and then tested for reactive oxygen production, survival, mutation rate and mutation type.
• Who was studied: A human–hamster hybrid cell line (AL cells) containing a standard set of Chinese hamster ovary (CHO-K1) chromosomes and a single copy of human chromosome 11. The work was done at Columbia University, New York. No people or animals were studied.
• Exposure: International Union Against Cancer standard reference crocidolite fibers (average length 3.2 plus or minus 1.0 µm; average diameter 0.22 plus or minus 0.01 µm), given for 24 hours at graded doses (for example 2, 4 and 6 µg/cm2). For comparison, cells were given hydrogen peroxide (for example 4.4 mM and 13.2 mM) for 15 minutes. Catalase (1,000 U/mL) and DMSO (0.5%) were used as antioxidants. Titanium dioxide was used as a negative control.
• Main results:
  • In AL cells, a 6 µg/cm2 dose of crocidolite produced a 5-fold increase in reactive oxygen species compared with control cells (p less than 0.05).
  • With DMSO present, the relative fluorescence (a measure of reactive oxygen) in cells given 6 µg/cm2 crocidolite fell from 59.01 plus or minus 6.74 to 19.30 plus or minus 0.85 (p less than 0.05).
  • Hydrogen peroxide release from crocidolite-treated AL cells rose with dose and peaked at 0.32 plus or minus 0.055 µM at 6 µg/cm2.
  • In cells given 6 µg/cm2 crocidolite, catalase (1,000 U/mL) suppressed hydrogen peroxide induction 7- to 8-fold (p less than 0.05).
  • Survival of AL cells was 62 plus or minus 4% after 2 µg/cm2 crocidolite and 26 plus or minus 5% after 4 µg/cm2. The article reports survival of 56 plus or minus 10% and 24 plus or minus 5% after 4.4 mM and 13.2 mM hydrogen peroxide.
  • The mutant fraction at the CD59 gene rose with crocidolite dose. At 4 µg/cm2 it was about 4-fold higher than background. Background averaged 52 plus or minus 15 mutants per 100,000 surviving cells.
  • At equally toxic doses, the mutant fraction was slightly higher with hydrogen peroxide than with crocidolite, but the difference was not statistically significant.
  • Mutation yields from 4 µg/cm2 crocidolite or 13.2 mM hydrogen peroxide were suppressed by 0.5% DMSO or 1,000 U/mL catalase (p less than 0.05).
  • Among spontaneous (untreated) mutants, 22 of 30 (73%) showed no detectable changes in any of the five marker genes tested on human chromosome 11.
  • Only 9 of 27 mutants (33%) from cells given 2 µg/cm2 crocidolite kept all marker genes. None of the 29 mutants from 4 µg/cm2 crocidolite kept all five markers.
  • Mutation patterns after hydrogen peroxide were similar. 9 of 23 mutants (39%) from 4.4 mM kept all five markers, and none of the mutants from 13.2 mM did.
  • Titanium dioxide, the negative control, was neither toxic nor mutagenic to AL cells at doses up to 12 µg/cm2. The data for this were not shown.
• The authors' conclusion: The authors concluded that their results provide direct evidence that the genotoxicity of asbestos fibers is mediated by reactive oxygen species. They also said that the similar mutation patterns from crocidolite and hydrogen peroxide gave strong circumstantial evidence that similar mutagenic mechanisms are involved.
• Limitations the authors state:
  • The experiments did not identify the source of the reactive oxygen species inside the cells.
  • Because most reactive oxygen species are short-lived, the authors stated it is still not clear how they reach the cell nucleus to cause mutations.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Crocidolite.

Medical terms in this study, explained in our Lexicon: Genotoxicity, Crocidolite, Reactive Oxygen Species (ROS).

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