Comet assay evaluation of six chemicals of known genotoxic potential in rats

Abstract

As a part of an International validation of the in vivo rat alkaline comet assay (comet assay) initiated by the Japanese Center for the Validation of Alternative Methods (JaCVAM) we examined six chemicals for potential to induce DNA damage: 2-acetylaminofluorene (2-AAF), N-nitrosodimethylamine (DMN), o-anisidine, 1,2-dimethylhydrazine dihydrochloride (1,2-DMH), sodium chloride, and sodium arsenite. DNA damage was evaluated in the liver and stomach of 7- to 9-week-old male Sprague Dawley rats. Of the five genotoxic carcinogens tested in our laboratory, DMN and 1,2-DMH were positive in the liver and negative in the stomach, 2-AAF and o-anisidine produced an equivocal result in liver and negative results in stomach, and sodium arsenite was negative in both liver and stomach. 1,2-DMH and DMN induced dose-related increases in hedgehogs in the same tissue (liver) that exhibited increased DNA migration. However, no cytotoxicity was indicated by the neutral diffusion assay (assessment of highly fragmented DNA) or histopathology in response to treatment with any of the tested chemicals. Therefore, the increased DNA damage resulting from exposure to DMN and 1,2-DMH was considered to represent a genotoxic response. Sodium chloride, a non-genotoxic non-carcinogen, was negative in both tissues as would be predicted. Although only two (1,2-DMH and DMN) out of five genotoxic carcinogens produced clearly positive results in the comet assay, the results obtained for o-anisidine and sodium arsenite in liver and stomach cells are consistent with the known mode of genotoxicity and tissue specificity exhibited by these carcinogens. In contrast, given the known genotoxic mode-of-action and target organ carcinogenicity of 2-AAF, it is unclear why this chemical failed to convincingly increase DNA migration in the liver. Thus, the results of the comet assay validation studies conducted in our laboratory were considered appropriate for five out of the six test chemicals.

Genotoxic effects of N-nitrosodimethylamine in somatic and generative cells of mice

“…Такие исследования ранее были проведены в условиях in vivo с использованием метода ДНК-комет. НДМА вызывал повреждения ДНК в печени крыс, а также печени и же-лудка мышей [16]. Целью наших исследований было определение органной специфичности генотоксического действия НДМА, и поэтому анализ разрывов ДНК про-водили параллельно в четырех органах мышей: печени, почках, легких и селезенке.…”

“…Ранее было установлено, что НДМА индуцирует мута-ции, выявляемые в тестах Эймса, по учету генных и хро-мосомных мутаций, сестринских хроматидных обменов, незапланированного синтеза ДНК [15][16][17][18][19]. Одна ко ис-следования in vivo на млекопитающих немногочисленны и носят противоречивый характер [20][21][22][23].…”

Re-analysis results using medians of the data from the JaCVAM-organized international validation study of the in vivo rat alkaline comet assay

“…Data re-analysis was conducted by two types of investigations, i.e., one used each laboratory analysis report [4][5][6][7][8][9][10][11][12][13][14][15][16] and the other was our unified analysis. For both analyses, based on TG 489 [3], the median % tail DNA for each slide was determined, the mean of the 2 median values was calculated for each animal, and then the mean of the individual animal means was determined to give a group mean.…”

“…In laboratory analysis, statistical significance between a negative control group and chemical treatment groups except for the positive control group was analyzed with a one-way analysis of variance (ANOVA) using log-transformed values (3 labs; Lab B: one-sided Simple meanapproach Significant 18 3 3 24 Significant in either statistic 0 5 3 8 Non-significant 2 4 47 53 Number of data 20 12 53 85 Significant: statistically significant in Dunnett's and linear trend test, non-significant: no statistically significant in both statistical analysis methods, significant in either statistic: statistically significant in either but not both of the two statistical analysis methods. and p < 0.03 [4], Lab D: p < 0.05 [6], Lab G: p < 0.001 [9]) followed by a multiple-comparison such as Dunnett's test (3 labs; Lab C: p < 0.05 [5], Lab L: two-sided and p < 0.05 [14], Lab M: two-sided and p < 0.05 [15]), a multiple-comparison with Dunnett's test for nonlog-transformation values (5 labs; Lab E: two-sided and p < 0.05 [7], Lab F: two-sided and p<0.05 [8], Lab I: two-sided and p < 0.05 [11], Lab J: p < 0.05 [12], Lab N: two-sided and p < 0.05 [16]), or a post-hoc right one-sided pairwise comparison using log-transformed values (1 lab; Lab K: one-sided and p < 0.05 [9]). One laboratory (Lab H) did not use statistics because the values in all treatment groups were lower than the respective control values [10].…”

“…Here, we refer to the former method of analysis as the simple mean approach and the latter as the median based approach. Due to TG 489, the individual laboratory reports of the main validation study (Phase 4-2) from most of the participating laboratories were revised and described using their own statistical approach based on using medians (and means) [4][5][6][7][8][9][10][11][12][13][14][15][16]. Also, we separately re-analyzed the Phase 4-2 data based on the median based approach in the same manner as the original statistical analysis of the JaCVAM-organized validation study as reported by Uno et al [2] in order to compare the sensitivity/specificity using the simple mean approach with the median based approach.…”