Research in mutagenesis has almost exclusively dealt with nuclear DNA and therefore there is an obvious lack of knowledge concerning the process of mutagenesis, repair and function of mitochondrial DNA (mtDNA). There is also little information on the effects of mutagenic chemicals and other agents on mtDNA.
The present project aims at filling some of these gaps in our knowledge. This will be acquired through experimental studies on spontaneous and induced induction of mitochondrial mutations and their repair, as well as their consequences on the properties and function of mitochondria.
In recent years it has been established that alterations in mtDNA can have severe deleterious effects on the organism and such alterations may be involved in tumorigenicity. The research in this project can be expected to have some practical relevance in that context.
The project involves four levels of investigations : analysis of mitochondrial morphology and function, measurements of DNA adduct formation, establishment of repair processes and characterization of mutations induced.
Mitochondrial Morphology and Function
A group of widely different chemicals cause proliferation of peroxisomes and some have been demonstrated to be efficient rodent carcinogens by a mechanism, which is not understood. It was observed that treatment with peroxisome proliferators causes a change in the morphology of hepatic mitochondria, resulting in a subpopulation of small mitochondria. This effect on mitochondria will be investigated by measuring fatty acid beta-oxidation and the formation of reactive oxygen species. In this context hydroxylation of guanine from hydroxyradical formation will be studied. These parameters may be of relevance for the carcinogenic properties of peroxisome proliferators.
Induction and Repair of DNA Lesions
The frequency of DNA adducts and lesions, induced by UV-radiation or treatment with alkylating agents will be assessed in restriction fragments of mtDNA either immediately after exposure or after incubation to allow for repair. Two UV-induced lesions will be analysed quantitatively, cyclobutane pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproduct. Oxidative damage or DNA lesions induced by alkylating agents will be assessed by generating AP-sites through the use of glycosylases/AP endonucleases. In a subsequent step AP sites will be converted into DNA strand breaks by endonuclease S1. Thymine glycols and hydrates, 8-hydroxyguanine and 3-methyladenine will be measured. The frequency of breaks is determined by neutral gel electrophoresis and Southern blotting. For some lesions specific antibodies are available and they will be used to detect and quantify lesions. Slotblot or PCR techniques will be employed to analyse lesion frequencies in specific sequences.
Experiments will be carried out to investigate, whether DNA repair mechanisms of nuclear DNA are operating on mtDNA. For that purpose studies will be performed with normal and excision repair defective xeroderma pigmentosum fibroblasts. Also rodent cell lines and mutants sensitive to UV-radiation or alkylating agents will be studied.
The binding of peroxisome proliferators to mtDNA will be studied as well as the formation of 8-hydroxyguanine in mtDNA as compared to nuclear DNA by these compounds.
Induced mutations in mitochondria will be measured in different ways. The Swansea group has sequenced a large portion of mtDNA in the mussel Mytilus and primers have been prepared for amplification of specific regions. This will be used to identify and characterize point mutations and deletions in germ cells as well as in somatic cells in different tissues. The possible involvement of mutations in mtDNA in ageing will be studied in Mytilus by analysis of somatic cells of different age. The mutagenic effects of environmental chemicals will be investigated both by monitoring in the field and by laboratory experiments.
To study mutagenic effects on mitochondria by peroxisome proliferators restriction site analysis of mtDNA will be carried out in rodents.
In human cells mitochondrial mutations will be analysed after exposure to different DNA damageing agents - MNU, UV-radiation and ionizing radiation. Non phenotypic mutations will be assayed by means of RFLP and PCR of selected endonuclease sites.
Funding SchemeCSC - Cost-sharing contracts
2300 RA Leiden
SA2 8PP Swansea