The objective is to develop and validate techniques for identification of germ cell specific mutagens
The project objectives are to develop and validate techniques for identification of germ cell mutagens and for quantification of genetic risk. It consists of three integral parts: detection and characterization of germ cell mutagens;
standardization and validation of new germ cell tests;
development of a data base on germ cell mutagenicity.
Detection and characterization of germ cell mutagens. Acrylamide induced dominant lethal mutations, heritable translocations and specific locus mutations in mouse spermatids. It is most important for genetic risk evaluation that specific locus mutations were also induced in stem cell spermatogonia of mice. 1,3-Butadiene was mutagenic in a variety of assay systems after subacute exposure. It presents a serious genetic risk to the human population. Trophosphamide, a trifunctional alkylating agent, induced dominant lethal mutations, heritable translocations and specific locus mutations in mouse spermatozoa and spermatids. Trophosphamide also induced micronuclei in mousebone marrowerythrocytes. Urethane did not induce dominant lethal mutations or congenital malformations after acute or chronic exposure in drinking water.
Standardization and validation of new germ cell tests. The flow cytometric analysis of sperm deoxyribonucleic acid (DNA) content and the cytogenetic analysis of first cleavage division chromosomes showed that primary spermatocytes are the most sensitive stage for aberration induction by chlorambucil in male germ cells. Validation of the spermatid micronucleus assay showed that S-dependent clastogens such as mitomycin C are readily recognized. Acrylamide, which is specifically clastogenic in late spermatids, showed only weak effects in the spermatid micronucleus assay after repeated treatments of primary spermatids during the pre-leptotene S-phase. Urethane showed no effect. Development of a data base on germ cell mutagenicity. The mutagenicity and carcinogenicity data base consists of entries of 300 chemicals of in vitro mutagenicity test data, toxicity data and rodent carcinogenicity data. It has been complemented with dominant lethal data from the literature on 21 compounds. The computer analysis of test responses showed that the dominant lethal assay has a position not related to any of the other tests which indicates that it responds to chemicals in its own particular way.
The question whether chemicals exist that cause mutations in germ cells but not in somatic cells is of utmost importance in view of the present national and international legislative efforts to correctly label chemicals as mutagens and/or carcinogens. The base set of mutagenecity testing in the EC for industrial chemicals requires 2 short term tests. One test detects gene mutations in bacteria (Ames test) and the other identifies structural chromosome damage in somatic cells in vitro or in vivo. If some chemicals are not genotoxic in somatic cells but are mutagenic in germ cells this could have serious consequences for their recognition. With the commonly required short term tests, a specific germ cell mutagen would not be detected.
The project consists of 3 integral parts as follows.
A data base on germ cell mutagenecity and the analysis of structure and activity correlations using artificial intelligence. The development of this data base for germ cell mutagens is of high priority. Currently, for the induction of mutations in mammalian germ cells the data base is too small to be useful for predictions. Experimental data from germ cell mutagenecity tests, including the dominant lethal and the specific locus assay, are obtained in the project to extend the data base.
The second part is standardization and validation of the new methods of spermatid micronucleus test (SPTD-MNT) and sperm deoxyribonucleic acid(DNA) flow cytometry test (SPM-FLOW test). To be able to evaluate a larger number of chemicals for germ cell effects alternative methods have to be developed that are not as time consuming and animal consuming as the classical germ cell mutagenicity tests. The SPTD-MNT determines indirectly the clastogenic activity of chemicals during meiosis by scoring micronuclei in derived early round spermatids. The SPM-FLOW test also determines indirectly clastogenic activity by changes in the coefficient of varience of the DNA content of mature sperm measured by flow cytometry and covers the entire process of male germ cell development.
The third part is a search for germ cell specific mutagens will be systematically undertaken by simultaneous performance of in vivo somatic cell and germ cell mutagenicity tests. The mouse bone marrow micronucleus test and the mouse spot test are included in the project to determine mutagenic effects in somatic cells.
The results will allow predictions from rapid germ cell tests and/or somatic mutagenicity tests for heritable germ cell effects. The main goal is to facilitate the recognition of germ cell mutagens and to regulate their presence in the environment.
Funding SchemeCSC - Cost-sharing contracts
SM5 4DS Carshalton
00060 Santa Maria Di Galeria (Roma)
2333 AL Leiden