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Content archived on 2024-04-19

Molecular dosimetry of chemical mutagens. Relationship between DNA adduct formation and genetic changes analyzedat the molecular level


The objective of the project is to analyze the relationship between the exposure to genotoxic chemicals and the induction of genetic effects in several genotoxicity assays as they are used under laboratory conditions. A series of model compounds which include methylating and ethylating agents were used to determine the nature and the frequency of different types of deoxyribonucleic acid (DNA) damage caused by the chemicals. To this end several assay systems were first developed to measure DNA adduct frequencies. These include immunological assays, high performance liquid chromatography (HPLC) procedures using an electrochemical detector and an enzymatic assay.

Parallel to the measurements of DNA damage the frequency of mutations was determined in different organisms which include cultured mammalian cells, mouse germ cells, Drosophila, yeast, Escherichia coli and Salmonella typhimurium.

The results to date include that measurements of DNA adduct frequencies can give information concerning the mutagenic potency of a genotoxic agent, but these determinations should be accompanied by measurements of the effects of DNA repair mechanisms on the persistence of the DNA lesions. The expected nature of the induced genetic changes can be confirmed by molecular analysis of the DNA sequence changes. Such a combined approach will generate a genotoxicity profile of a chemical or a group of related chemicals, which can be used in risk assessment procedures.
The project described in this research proposal is focused on the relationship between the exposure to genotoxic chemicals and the induction of genetic effects in several genotoxicity assays as they are used under laboratory conditions for regulatory purposes. The work proposed can be divided into the following two
1. Investigations concerning the correlation between exposure to chemicals and formation of DNA adducts. This will be investigated in organisms and under conditions which are also used in the test assays for genotoxicity, e.g. bacteria, yeast, mammalian cells in culture, Drosophila, mice and rats. This part of the project can be divided into two parts:
a. Further development of methodologies for the detection and quantification of DNA adducts in the genome overall, which will involve: (i) The use of radioactively labelled model compounds which allow the quantification of labelled DNA adducts at low frequency. (ii) Development of HPLC separation systems using an electrochemical detector, which would allow the detection of certain unlabelled adducts. (iii) Isolation and characterization of monoclonal antibodies against DNA containing specific DNA adducts or against a purified DNA adduct itself. (iv) Further development of various types of immunological assays using the newly characterized antibodies. (v) Further development of immuno histochemical methods for the detection of DNA adducts in various organs and cell types of mammals. (vi) Development of a sensitive assay for the measurement of apurinic sites in DNA. (vii) Further development of post-labelling methods for other than bulky DNA adducts.
b. Development of methodologies for the detection of DNA
adducts in specific DNA sequences. There is evidence that the distribution of DNA adducts over the genome immediately after exposure is not random, but that certain DNA sequences are damaged to a higher extent. Furthermore, removal of certain type of DNA damage from actively transcribed genes is likely to be faster than from the genome overall.
2. Correlation between DNA adduct formation and biological
effect. Most chemicals induce more than one type of DNA adduct. The mutagenic properties of each of these type of adducts can vary significantly. Moreover most organisms are equipped with DNA repair mechanisms which remove specific DNA adducts from their genome with varying efficiency. This part of the project can be divided in the following parts:
a.Determination of DNA adduct frequencies as a function of time in: Different organs of mice, hamsterand rat, including mouse and rat germ cells. In eukaryotic systems with different DNA repair characteristics. In E. coli and Salmonella typhimurium.
b.Determination of molecular mutation spectra of base pair
changes under normal and repair deficient conditions. This will allow a direct correlation between the fate of certain DNA adducts and the type of base pair changes induced. Especially the
determination of DNA adducts in the same gene to be used for mutation analysis will allow a direct correlation between DNA adduct formation and the type of induced mutations.

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Rijksuniversiteit Leiden
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2300 RA Leiden

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Participants (15)