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The relationship between the induction of DNA damage, genetic alterations and tumors in the skin of hairless mice by UV-B radiation. A model for skin cancer induction in man

Objective

The objective is to make comparisons and eventually to establish qualitative and quantitative correlations between types and frequencies of DNA damage in the skin following UV-B radiation, the repair of DNA damage and the manifestation of genetic alterations in skin and in skin tumours.

The increased level of ultraviolet B (UVB) irradiation that will result from the depletion of stratospheric ozone is likely to give rise to a variety of impacts of human health such as increased risk of skin cancer. For ultraviolet (UV) carcinogenesis it is evident that the wavelengths responsible for cancer induction are predominantly in the UVB range.

Tumour induction experiments have been carried out by daily exposure of hairless mice to UVB light or the combination of UVB and ultraviolet A (UVA) light. The total carcinogenic dose appeared to be the sum of the effective carcinogenic UVA and UVB dose. The hypothesis was tested whether the tumorigenic response expressed as the median induction time of skin tumours (T50) following the different radiation regimes, correlated with the accumulation of UV induced cyclobutane pyrimidine dimers (CPD). A relationship between the CPD load in the epidermis and the T50 values was found for UVB irradiation. However the results also suggest the involvement of another type of deoxyribonucleic acid (DNA) lesion in UVA carcinogenicity. Molecular analysis of 13 tumours from the skin of UVB/UVA irradiated mice revealed the absence of mutations in the P53 gene and only one single modification of the Ha-ras gene. DNA damage induction and repair was investigated in skin cells of hairless mice exposed to UVB. DNA damage levels in the epidermis measured as antibody binding sites, initially accumulated in daily exposed mice, but then dropped due to an increase of thickness of the skin. The results suggest an important role of cell proliferation in shielding of basal cells from DNA damaging effects of UV. Efficient repair of CPD was observed both in suprabasal and basal cells employing the thymine dimer specific antibody. However biochemical analysis revealed a considerable heterogeneity of repair strongly resembling the repair kinetics of CPD in cultured rodent cells. The observed difference between the immunological and biochemical assay might be explained by a modification of a subset of lesions causing the loss of antibody binding sites. The project is continuing.
The increased level of UV-B irradiation that will result from the depletion of stratospheric ozone is likely to give rise to a variety of impacts of human health such as increased risk of skin cancer. For UV-carcinogenesis it is evident that the wavelengths responsible for cancer induction are predominantly in the UV-B range. The project will focus on the mechanisms underlying the induction of skin cancer by UV-B irradiation employing an animal model system. It is generally believed that damage induced by UV in chromosomal DNA is at the origin of genetic alterations in the skin and that induction of certain types of genetic alterations plays a crucial role in the carcinogenic process.

Albino hairless Skh mice will be treated with single and multiple exposures of UV-B or UV-B combined with UV-A. The combined treatment with UV-B and UV-A light is included since UV-A can alter the effects of pure UV-B irradiation and since it is abundantly present in sunlight. Frequencies of DNA damage and genetic alterations in skin and skin tumours will be assessed at various time-intervals after irradiation. DNA damage will be analysed in skin sections and isolated skin cells employing specific antibodies against DNA photolesions, and at the molecular level of transcriptionally active and inactive genes. Analysis will focus on two major photolesions i.e. the cyclobutane pyrimidine dimer and the pyrimidine 6-4 pyrimidone photoproduct. Epidermal cell suspensions will be used for cytogenetic analysis and analysis of HPRT mutation frequencies. The frequency of skin tumours in UV-irradiated mice will be determined. Skin tumour tissue will be analyzed for oncogene activation and the molecular nature of changes in these tumours will be defined.

The work will allow a detailed study, at the cellular and molecular level, of the effects of UV-B irradiation starting from the first step (exposure) up to the final stage (induction of tumours). It will provide an expansion of our understanding of UV-induced carcinogenesis and enable comparison of the genotoxic effects in mice with those in man.

Coordinator

Rijksuniversiteit Leiden
Address
72,Wassenaarseweg
2300 RA Leiden
Netherlands

Participants (3)

Centre National de la Recherche Scientifique (CNRS)
France
Address
7 Rue Guy Môquet
94801 Villejuif
NETHERLANDS ORGANISATION FOR APPLIED SCIENTIFIC RESEARCH - TNO
Netherlands
Address
9,Zernikedreef 9 Gaubius
2301 CE Leiden
Rijksuniversiteit Utrecht
Netherlands
Address
100,Heidelberglaan
3508 GA Utrecht