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Characterization of the genetic toxicity profile of chemical carcinogens in mammals : relation to heritable and somatic mutations

Objective

The objective is to analyze the behaviour of a selected group of chemicals in animal model systems in which the induction of genetic effects will be determined and characterized, in parallel to measurements of DNA damage formation, persistence of DNA damage and levels of enzymatic activities of selected DNA repair enzymes. The genetic effects are analyzed in transgenic mice and the results are calibrated against observations in endogenous genes of the same animals or in parallel experiments using other
strains of mice or rats.

The various assay systems necessary to investigate the genetic and biochemical characteristics of the test chemicals which are all methylating or ethylating agents has been established, and a first characterization of a number of chemicals has been performed. This includes: establishment of the conditions under which mutation frequencies can be determined in T-lymphocytes obtained from the spleen of rats treated with genotoxic agent;
the dose response relationship for mutation induction by ethyl nitrosourea, hydroxy-ethyl nitrosourea and ethyl-methane sulphonate at the hprt gene in T-lymphocytes has been determined;
the spectrum of changes at the deoxyribonucleic acid (DNA) sequence level in the induced mutations has been determined in the case of the chemical mentioned;
DNA adduct measurements in different organs (small intestine, liver, lung, bone marrow, kidney and T-lymphocytes) of rats and mice exposed to the test chemicals has been performed. A comparison has been made between the different organs in order to see whether the frequency observed in T-lymphocytes is a good parameter for adduct frequencies in internal organs;
the effects of administering ethyl nitrosourea in a split dose protocol was investigated in the mouse by determining mutation induction at the Dlb-I locus of the small intestine and in male germ cells using the specific locus assay;
Lambda gt10.lacZ mice were crossed with C57B1/6J mice in order to obtain animals heterozygous for Dlb-I which will be used to score mutations at the Dlb-I locus as well as on the LacZ gene;
pilot experiments to determine the frequency of LacZ mutations induced by ethyl nitrosourea in germ cells of the mouse were performed.
The genetic toxicity profile of each model chemical is determined by measuring the induction of gene mutations in a number of endogenous genes in somatic cells of various organs of mice and rats, in male germ cells of the mouse and in transgenic mice, in which a transgene has been inserted in the genome. This transgene will be recovered from different organs or tissues and then analyzed for the presence of mutations upon introduction in the bacterium Escherichia coli.
The genes which are used in this analysis are:

1. The dbl-I locus in intestinal epithelium of the mouse.

2. The hprt locus in lymphocytes of the rat.

3. Seven loci measured simultaneously in male germ cells of the mouse (specific locus assay).

4. The lacZ gene of E. coli which serves as transgene and which can be analyzed in any organ of the mouse.

Genotoxic agents are injected intra-peritoneally in the animals and the appearance of mutations are analyzed in the same animals following different periods of time (dlb-l, phrt, LacZ). In the mouse specific locus assay the induction of mutations is determined in offspring of treated males and untreated female mice.

The chemicals used are all methylating or ethylating agents, some of which are activated by mammalian enzyme systems in specific organs. The organ specificity of the chemicals is investigated by the measurement of DNA damage formation in various organs, the persistence of the DNA damage with time and the modulation of a number of enzymes involved in the repair of DNA alkylation damage.

The work will result in calibration of the transgenic animal model system and provide insight into the mechanisms underlying organ specificity of genotoxic agents.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Rijksuniversiteit Leiden
Address
72,Wassenaarseweg
2300 RA Leiden
Netherlands

Participants (3)

Centre International de Recherche sur le Cancer
France
Address
150 Cours Albert Thomas
69372 Lyon
GSF-RESEARCH CENTER FOR ENVIRONMENT AND HEALTH
Germany
Address
Ingolstaedter Landstrasse 1
85764 Neuherberg
Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek
Netherlands
Address
137-139,Lange Kleiweg
2280 HV Rijswijk