Abstract
BackgroundThe repeated-dose liver micronucleus (RDLMN) assay is an effective and important in vivo test for detecting genotoxic compounds, particularly for those that require metabolic activation to show genotoxicity. In a collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS) – Mammalian Mutagenicity Study Group (MMS), micronucleus induction of 22 chemicals with the RDLMN assay employing the collagenase digestion method was examined and reported on. Recently, we have developed a method which enables retrospective evaluation of micronucleus induction in formalin-fixed liver tissues (the formalin-fixed method) obtained in general toxicity studies completed in the past. Using this method, we were able to easily evaluate clastogenic potential of chemicals from the formalin-fixed tissues obtained in the general toxicity studies.In this study, to evaluate the usefulness of the formalin-fixed method, we have conducted a liver micronucleus assay using the formalin-fixed liver samples obtained from the above collaborative study (18 of 22 test chemicals) and carried out a comparison with the results obtained by the collagenase digestion method.ResultsComparison of the collagenase digestion and formalin-fixed methods was conducted using the results of the micronucleus assays with a total of 18 test chemicals which included 12 genotoxic hepatocarcinogens (Group A), 4 genotoxic carcinogens but not liver targeted (Group B), and 2 nongenotoxic hepatocarcinogens (Group C). The formalin-fixed method obtained the similar results as the collagenase digestion method in 10 out of the 12 chemicals of Group A, and all chemicals of Group B and Group C. Although the results were statistically contradictive due to different levels of concurrent negative control, the 2 other chemicals of Group A showed comparable responses between the two methods.ConclusionThe present study shows that the formalin-fixed method is capable of detecting liver carcinogens with sensitivity equal to or higher than that of the collagenase digestion method. We recommend use of the formalin-fixed method because of its capability of enabling retrospective evaluation of micronucleus induction in the formalin-fixed liver tissues obtained in general toxicity studies completed in the past.
Highlights
The liver is not targeted in the routine micronucleus assay, the liver is an important tissue in general toxicology studies and in carcinogenicity bioassays because test chemicals are metabolized and on occasion activated with toxicological significance in the liver
In a recently improved formalin-fixed method, procedures to prepare samples for liver micronucleus assays have been provided [19]. Because this method enables retrospective evaluation of micronucleus induction in the formalin fixed liver tissues obtained in general toxicity studies completed in the past, clastogenic potential of chemicals from the materials obtained in the general toxicity studies are able to be evaluated
When compared to the collagenase digestion method employed in the previous collaborative study, the formalin-fixed method induced almost the same levels of micronuclei in most of the chemicals as the collagenase digestion method
Summary
The liver is not targeted in the routine micronucleus assay, the liver is an important tissue in general toxicology studies and in carcinogenicity bioassays because test chemicals are metabolized and on occasion activated with toxicological significance in the liver. The micronucleus assay using the liver, which is the main organ for drug metabolism, has been known to be important but not widely used because hepatocyte (HEP) proliferation in adult rats is slow and micronuclei are difficult to produce To overcome this shortcoming, partial hepatectomy [6,7,8], mitogen treatment [9, 10], and the use of juvenile rats [11,12,13,14] have been introduced to the assays. We have developed a new method, the repeated-dose liver micronucleus (RDLMN) assay, to evaluate liver micronucleus through repeated administration of test chemicals, e.g., 14-day or 28-day repeated-dose treatments [18] This method is expected to produce an accumulation of micronucleated hepatocytes (MNHEPs) through long-term continuous treatment, HEP turnover is slow [18]. To evaluate the usefulness of the formalin-fixed method, we have conducted a liver micronucleus assay using the formalin-fixed liver samples obtained from the above collaborative study (18 of 22 test chemicals) and carried out a comparison with the results obtained by the collagenase digestion method
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