Utilization of the specific-locus assay in the ad-3 region of two-component heterokaryons of Neurospora for risk assessment of environmental chemicals

Abstract

The utilization of the specific-locus assay in the ad-3 region of two-component heterokaryons of Neurospora crassa is compared with that of other eukaryotic assay systems for the evaluation of the mutagenic effects of environmental chemicals. In contrast to other in vitro specific-locus assays, the Neurospora assay can detect mutations not only at the ad-3A and ad-3B loci but also recessive lethal mutations elsewhere in the genome. Mutational damage in this system can be characterized readily by means of classical genetic techniques involving heterokaryon tests to determine genotype, and allelic complementation among ad-3BR mutations. The percentages of ad-3BR mutations showing allelic complementation with polarized or nonpolarized complementation patterns provide a presumptive identification of the genetic alterations at the molecular level in individual mutants. Dikaryon and trikaryon tests (using 3 strains carrying multilocus deletion mutations as tester strains) distinguish ad-3 mutations resulting from gene/point mutation, multilocus deletion mutation, and various types of multiple-locus mutation. The array of ad-3 mutations recovered from forward-mutation experiments can be expressed in terms of Mutational Spectra, which make it possible to make comparisons of mutational types between different doses of the same mutagen, different mutagens, or the effects of the same mutagen on different strains. Another important feature of this specific-locus assay system is that the effects of mutagens can be studied in both DNA excision repair-proficient (H-12) and -deficient (H-59) two-component heterokaryons to evaluate both quantitative and qualitative differences between the spectra of induced ad-3 mutations. The utilization of this assay on large numbers of environmental chemicals has shown that some chemicals produce predominantly, or exclusively, gene/point mutations, whereas other agents produce both gene/point mutations and multilocus deletion mutations in H-12. When the mutagenic effects of the same chemicals were compared in H-12 and H-59, marked differences between forward-mutation frequencies and Mutational Spectra of ad-3 mutations were detected