Gene mutation assays limitations

Species. In vivo genotoxicity assays mainly use rodents (rats and mice), because they are small (standard animal facilities and low amount of test article needed) and because abundant toxicologic and metabolic data are available on these species. Most assays are also theoretically applicable to noiu odent animals, but limited data have been reported in the literature. Most of the assays are applicable to wild-type and readily available animal strains. It should nevertheless be noted that a few models require specific strains (e.g., some gene mutation assays). 

Principle, Interpretation, Limitations, and Strengths. Tables 12.3 and 12.4 summarize the main characteristics of the different gene mutation assays that are further compared below. 

Metals are probably one of the most susceptible forms in the B. subtilis rec-assay system. Among 121 metal compounds rec-assayed with cold incubation, 44 are positive (Table 8B). These were then examined as to their mutagenicity by the Ames assay, and some of them were found to be positive. The frequency of the mutagenic samples among the rec-assay-positive cases is rather limited compared to the other categories such as food additives and pesticides. It seems that further improvements are required in bacterial reversion assays for metal compounds. Most rec-assay-positive metals either are carcinogens or cause chromosome aberrations or gene mutations in cultured cells. 

The use of yeast cells as a eukaryotic complement to the Ames test led to the development of several protocols for the detection of mutation, gene conversion and recombination. The formal introduction of methods [23] followed by much development work from Zimmermarm s laboratory led to large systematic studies [24, 25] and OECD guidelines for the test battery (OECD 480, 481). However the assays are now rarely used, at least in part because of concerns over low sensitivity, thought to reflect limited permeability of the cell wall. 

In order to define the limits of the ARE and the bases critical for binding of the protein and for transcriptional activity, the ARE sequence was subjected to a mutational analysis. Mutations were introduced into the ARE using mutant oligonucleotides and these mutant sequences were multimerised to give a four copy ARE promoter. These 4x mutant ARE constructs were assayed for their ability to bind the protein factor or to compete with binding of the wild-type ARE element to the protein. The 4x mutant ARE elements were also linked to the GUS reporter gene and introduced into protoplasts by electroporation to determine their activity as transcriptional effectors under aerobic and anaerobic conditions. 

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