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Content archived on 2024-04-19

Genotoxicity of environmental alkylating agents; role of DNA damage and DNA repair systems studied in mammalian cells expressing cloned DNA repair genes


Most genotoxic agents exert mutagenic and/or carcinogenic effects because they introduce lesions in DNA which can not be handled properly by the DNA replication machinery of the cell. Therefore, several types of biological effects can be expected, including the induction of gene mutations, chromosomal aberrations and death of the cell. However, most genotoxic agents introduce a mixture of DNA adducts, not all of which are equally efficient in the induction of genetic and/or toxic effects. The reason for this can be that there are intrinsic differences in the mutagenic effectiveness between types of DNA adducts, but also DNA repair processes can preferentially remove some of the DNA damage in time, thereby preventing deleterious consequences. The aim of the project is to investigate the contribution of defined types of DNA adducts to mutation induction in mammalian cells. This will be achieved by modulating the frequency of DNA adducts by introducing cloned DNA repair genes in mammalian cells having different specificity towards the various adducts. These experiments will be performed under such conditions that these genes will produce specific DNA repair proteins which will otherwise be present in these cell at a much lower concentration. Furthermore, repair proteins produced by the transgene can have a different specificity compared to the endogenous repair proteins. Subsequently, the response of these cells towards treatment with genotoxic agents will be investigated.

The chemicals studied in this project will be alkylating agents because they are environmental and industrial carcinogens and they cause DNA damage from which it is known that it can be handled by the DNA repair proteins produced by the genetically engineered cells.

First mammalian expression vectors will be constructed which carry genes from E. coli or from the rat coding for glycosylases which specifically remove defined alkylation products from DNA. Chinese hamster cells will be transfected with these constructs and clones will be isolated which express the DNA glycosylase. Subsequently, mutation induction experiments will be performed with the cell lines carrying the cloned glycosylase genes using the alkylating agents MMS, MNU, MNNG, EMS and ENU. The frequency of mutations at the hprt gene will be compared with that found in the parental celle line. Similar comparisons will be performed concerning the toxic effects of these alkylating agents. Hprt deficient mutants will be isolated and the molecular nature of the mutations will be analyzed by DNA sequencing. The mutation spectra thus obtained will be compared with those found in the parental cell lines.

The repair kinetics of the DNA lesions in the gene in which also the genetic effects is monitored i.e. the hprt gene, will be determined in order to interpret the molecular nature of the mutations induced. The technology to perform such gene specific measurements will be further developed for the types of DNA damage investigated in this project using purified DNA glycosylases from E. coli which are specific for the different DNA adducts of interest.

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Rijksuniversiteit Leiden
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2300 RA Leiden

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