Dear Sir, I read the research findings of Martino-Roth et al. (2003) with interest. They studied occupational genotoxicity risk in storage battery renovation workers and car painters using the comet assay and micronucleus (MN) test. Highly significant effects of exposure were found with both assays. MN-inducing effect of paints in exfoliated buccal cells was shown recently (6-fold increase compared with control; Pinto et al., 2000), and the authors confirmed strong mutagenic effect of paints (3-fold increase of cells with MN). The difference in mutagenic effect in two papers could be due to the different paints used, and also to various levels of cells with MN in control subjects (0.03 and 0.11%, respectively). Discussions concerning MN level in exfoliated cells of healthy humans published recently (Nersesyan, 2003, 2005a,b) raise the importance of this problem. The number of cells with MN in control subjects is about 0.1% (Martino-Roth et al., 2003), and it is in accordance with other researches carried out in Brazil. They are in the middle of the range between 0.03-0.05% (Gattas et al., 2001; Ramirez and Saldanha, 2002; Cerqueira et al., 2004) and 0.2% (Agostini et al., 1996). The same genetic endpoints (the study of MN frequency in buccal cells and the comet assay), used by Martino-Roth et al. (2003), were applied to workers exposed to chromium and nickel recently (Danadevi et al., 2004), and both biomarkers showed significant increase compared with non-exposed subjects. Hence, these two endpoints are valuable biomarkers of genotoxic exposure. However, together with the presentation of very interesting results, the paper has some shortcomings which should be clarified. In the abstract the authors wrote that ‘The micronucleus (MN) test and the alkaline single cell gel or comet assay were applied to exfoliated cells of the buccal mucous in order to evaluate the genotoxic risk associated with occupational exposure ….’. But the MN assay was applied only to exfoliated cells, and the comet assay to lymphocytes. Hence, the right version of this sentence should be: ‘The micronucleus (MN) test in exfoliated cells of the buccal mucous and the alkaline single cell gel electrophoresis or the comet assay in lymphocytes were applied, in order to evaluate the genotoxic risk associated with occupational exposure ….’. The sentence ‘The comet assay was found to be rapid, of simple visualization, and it is a sensitive technique for measuring and analyzing DNA damage in human cells.’ ought to be omitted because before this paper this statement was shown in numerous other papers many years ago. In ‘Material and Methods’ the authors indicated that they recorded cells undergoing degenerative processes, but there is no word about them in further text of the article. They wrote that both negative and positive controls were used to test the efficiency and electrophoresis conditions, but again there is no other mention in the text about both controls. Moreover, the sentence concerning positive control is not clear, especially about two (!) final concentrations: ‘In the positive control, 200 µl of whole blood was incubated for 2 h at 37°C with 50 µl methyl methanesulfonate (MMS; final concentrations of 8 x 10-5 M and 4 x 10-5 M).’ It is really unclear for readers the following: ‘Comet tail lengths (nuclear region + tail) were measured in arbitrary units. One unit was approximately 5 µm at 200X magnification.’. But why they indicated size of the comet tails in µM, and not in arbitrary units (Table 3, page 414)? Real ‘arbitrary units’ used in the comet assay studies are indicated in the paper of Collins et al. (1995) cited and quoted by authors: ‘Cells were also scored visually into five classes, according to tail size (from undamaged - 0, to maximally damaged - 4) and a value was assigned to each comet according to its class. The final overall rating for 100 cells, DNA damage score between 0 (completely undamaged) and 400 (maximum damage), was obtained by summation (Collins et al., 1995).’ In 4 control subjects damage index was equal to 0 (Table 4). I doubt if it is possible - no one damaged cell among 400! The authors presented information about smoking and alcohol consuming habits of study participants. But they never mentioned if habits influence genotoxic endpoints, unlike Danadevi et al. (2004), who showed the influence of smoking on both biomarkers. They wrote that the car painters were exposed to lead paint, solvents and benzene, but no data were presented concerning agents to which the storage battery renovation workers were exposed. This is a really important question for other investigations on these agents. In Table 3 the authors presented the individual data concerning sizes of the comet tails as mean ± SD, but the mean of these means was presented with SE. Three papers should not be cited in the context of the aim and results obtained in this research - Wojewodska et al., Baltaci et al. and Stavreva et al., concerning workers exposed to radiation, women with habitual abortions, and tobacco cells (?) exposed to mutagen, respectively. ‘We found large differences in the damage index in the exposed groups, compared to their controls, approximately 11 times greater in the battery renovator group and 7 times in the car painter group, which was similar to what was found by Collins et al. (1995).’ This quotation is wrong because Collins et al. studied DNA damage only in HeLa cells and in human lymphocytes exposed in vitro to H2O2. Moreover, the data concerning this paper and Goethen (1997) are wrong. Correct version of citation of both papers are presented in my reference list (find the last paper as Van Goethem et al., 1997). In conclusion, the authors obtained very interesting results but presented them in very confusing manner with a lot of errors and shortcomings. REFERENCES Agostini, J.M.S., Otto, P.A. and Wajntal, A. (1996). Chromosome damage in underground coal miners: detection by conventional cytogenetic techniques and by submitting lymphocytes of unexposed individuals to plasma form at-risk groups. Braz. J. Genet. 19: 641-646. Cerqueira, E.M., Gomes-Filho, I.S., Trindade, S., Lopes, M.A., Passos, J.S. and Machado-Santelli, G.M. (2004). Genetic damage in exfoliated cells from oral mucosa of individuals exposed to X-rays during panoramic dental radiographies. Mutat. Res. 562: 111-117. Collins, A.R., Ma, A.G. and Duthie, S.J. (1995). The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells. Mutat. Res. 336: 69-77. Danadevi, K., Rozati, R., Banu, B.S. and Grover, P. (2004). Genotoxic evaluation of welders occupationally exposed to chromium and nickel using the Comet and micronucleus assays. Mutagenesis 19: 35-41. Gattas, G.J., Cardoso Lde, A., Medrado-Faria Mde, A. and Saldanha, P.H. (2001). Frequency of oral mucosa micronuclei in gas station operators after introducing methanol. Occup. Med. 51: 107-113. Martino-Roth, M.G., Viegas, J. and Roth, D.M. (2003). Occupational genotoxicity risk evaluation through the comet assay and the micronucleus test. Genet. Mol. Res. 2: 410-417. Nersesyan, A.K. (2003). Letter to Editor. Mutat. Res. 538: 181-182. Nersesyan, A.K. (2005a). Arsenic and drinking water in West Bengal. Cancer Epidemiol. Biomarkers Prev. 14: 757-759. Nersesyan, A.K. (2005b). Letter to Editor. Mutat. Res. 582: 163-164. Pinto, D., Ceballos, J.M., Garcia, G., Guzman, P., Del Razo, L.M., Vera, E., Gomez, H., Garcia, A. and Gonsebatt, M.E. (2000). Increased cytogenetic damage in outdoor painters. Mutat. Res. 467: 105-111. Ramirez, A. and Saldanha, P.H. (2002). Micronucleus investigation of alcoholic patients with oral carcinomas. Genet. Mol. Res. 1: 246-260. Van Goethem, F., Lison, D. and Kirsch-Volders, M. (1997). Comparative evaluation of the in vitro micronucleus test and the alkaline single cell gel electrophoresis assay for the detection of DNA damaging agents: genotoxic effects of cobalt powder, tungsten carbide and cobalt-tungsten carbide. Mutat. Res. 392: 31-43. |
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