PRINCIPLES OF TOXICOLOGY - Biology East Borneo

254 MUTAGENESIS AND GENETIC TOXICOLOGYGerm Cell AssaysA basic test used to detect specific gene mutations induced in germ cells of mammals is themouse-specific locus assay. This test involves treatment of wild-type mice, either male or female, witha test compound before mating them to a strain homozygous for a number of recessive genes that areexpressed visibly in phenotype. If no mutations occur, then all offspring will be of the wild type. If amutation has occurred at one of the test loci in the treated mice, then the recessive phenotype will bevisibly detectable in the offspring. The mouse-specific locus test is of special significance in humanmodeling because it is the only standardized assay that directly measures heritable germ cell genemutations in the mammal. A major drawback of the mouse-specific locus test is that extensive physicalplant facilities are required to execute this assay, and it has been estimated that one scientist and threetechnicians could execute 10 single-dose mouse-specific locus tests in one year, provided there arefacilities for 5000 cages.New and promising test procedures have been described for detecting germ cell mutations by usingalterations in selected enzyme activity as the phenotypic endpoint. A large group of somatic cellenzymes can be monitored for changes in activity and kinetics in the F 1generation. These changesindicate changes in the parental genome.A similar biochemical approach has been proposed for identifying germ cell mutations in humansthrough the monitoring of placental cord blood samples. The activity of several erythrocyte enzymes,such as glutathione reductase, can be monitored because the enzyme proteins are the products of asingle locus and because heterozygosity of a mutant allele for the chosen enzymes will result inabnormal levels of enzyme activity. Likewise, it has been proposed that gene mutations be directlymonitored in mammalian germ cells by searching for phenotypic variants with biochemical markerssuch as lactic acid dehydrogenase-X (LDH-X), an isozyme of lactic acid dehydrogenase found onlyin testes and sperm. The test is based on the fact that a monospecific antibody for rabbit LDH-X reactswith rat but not mouse LDH-X in sperm. The rat sperm fluoresce as a result of the reaction but themouse sperm do not fluoresce unless a phenotypic variant is present. If adapted to humans, this testhas potential use as a noninvasive screening test of germ cell mutations in males.It has been proposed that the induction of behavioral effects in the offspring of male rats exposedto a mutagenic agent may represent a genotoxic endpoint. For example, studies have demonstrated thatthe mutagen cyclophosphamide can induce genotoxic behavioral effects in the progeny of male ratsand that these effects correspond to observed genetic damage caused in the spermatozoa followingmeiosis. A similar effect has been attributed to vinyl chloride in at least one instance of occupationalexposures.Mammalian germ cells can be monitored for chromosomal aberrations, and normally the testes areused as the cell source. Mammalian male germ cells are protected by a biological barrier comparablein function to the barrier which retards the penetration of chemicals to the brain. The blood–testesbarrier is a complex system composed of membranes surrounding the seminiferous tubules and theseveral layers of spermatogenic cells organized within the tubules. This barrier restricts the permeabilityof high-molecular-weight compounds to the developing male germ cell. An advantage of in vivomammalian germ cell mutagenicity testing is that the protective contribution of this barrier isautomatically taken into account. Conventional procedures for harvesting mammalian male germ celltissue for metaphase-spread analysis involve mincing or teasing the seminiferous tubules to liberatemeiotic germ cells in suspension. This homogenate is centrifuged, the centrifuged pellet is discarded,and the suspended cells are collected and analyzed. However, it was found that the tissue fragmentsdiscarded during this conventional procedure contained more spermatogonial cells and meioticmetaphases than did the suspension. Thus, the method has been refined by using tissue fragments andadding collagenase to dissociate them. After collagenase treatment, the tissue fragments are gentlyhomogenized and centrifuged, and the pellet containing meiotic cells is resuspended and prepared formicroscopic analysis.