phosphorus-radioisotopes and Xeroderma-Pigmentosum

Tamoxifen, a breast cancer drug, has recently been approved for the chemoprevention of this disease. However, tamoxifen causes hepatic carcinomas in rats through a genotoxic mechanism and increases the risk of endometrial tumors in women. DNA adducts have been detected at low levels in human endometrium, and there is much interest in determining whether DNA damage plays a role in tamoxifen-induced endometrial carcinogenesis. This study investigates the mutagenicity of tamoxifen DNA adducts formed by alpha-acetoxytamoxifen, a reactive ester producing the major DNA adduct formed in livers of tamoxifen-treated rats. pSP189 plasmid DNA containing the supF gene was treated with alpha-acetoxytamoxifen and adduct levels (0.5-8.0 adducts per plasmid) determined by (32)P-postlabeling. Adducted plasmids were transfected into nucleotide excision repair proficient (GM00637) or deficient (GM04429, XPA) human fibroblasts. After replication, plasmids were recovered and screened in indicator bacteria. Relative mutation frequencies increased with the adduct level, with 1.3-3.6-fold higher numbers of mutations in the XP cells compared to the GM00637 cells, indicating that NER plays a significant role in the removal of these particular tamoxifen DNA adducts. The majority of sequence alterations (91-96%) occurred at GC base pairs, as did mutation hotspots, although the type and position of mutations was cell-specific. In both cell lines, as the adduct level increased, the proportion of GC --> AT transitions decreased and GC --> TA transversions, mutations known to arise from the major tamoxifen adducts, increased. Given the high mutagenicity of dG-N(2)-tamoxifen adducts, if not excised, they may potentially contribute to the initiation of endometrial cancer in women.

Topics: Base Sequence; Cell Line, Transformed; DNA Adducts; DNA Mutational Analysis; DNA Repair; Genes, Suppressor; Genetic Complementation Test; Genetic Predisposition to Disease; Humans; Molecular Sequence Data; Mutagenesis; Mutagenicity Tests; Phosphorus Radioisotopes; Plasmids; RNA, Transfer; Tamoxifen; Transfection; Xeroderma Pigmentosum

The DNA synthesized shortly after ultraviolet (UV) irradiation of Potorous tridactylis (PtK) cells sediments more slowly in alkali than that made by nonirradiated cells. The size of the single-strand segments is approximately equal to the average distance between 1 or 2 cyclobutyl pyrimidine dimers in the parental DNA. These data support the notion that dimers are the photoproducts which interrupt normal DNA replication. Upon incubation of irradiated cells the small segments are enlarged to form high molecular weight DNA as in nonirradiated cells. DNA synthesized at long times ( approximately 24 h) after irradiation is made in segments approximately equal to those synthesized by nonirradiated cells, although only 10-15% of the dimers have been removed by excision repair. These data imply that dimers are not the lesions which initially interrupt normal DNA replication in irradiated cells. In an attempt to resolve these conflicting interpretations, PtK cells were exposed to photoreactivating light after irradiation and before pulse-labeling, since photoreactivation repair is specific for only one type of UV lesion. After 1 h of exposure approximately 35% of the pyrimidine dimers have been monomerized, and the reduction in the percentage of dimers correlates with an increased size for the DNA synthesized by irradiated cells. Therefore, we conclude that the dimers are the lesions which initially interrupt DNA replication in irradiated PtK cells. The monomerization of pyrimidine dimers correlates with a disappearance of repair endonuclease-sensitive sites, as measured in vivo immediately after 1 h of photoreactivation, indicating that some of the sites sensitive to the repair endonuclease (from Micrococcus luteus) are pyrimidine dimers. However, at 24 h after irradiation and 1 h of photoreactivation there are no endonuclease-sensitive sites, even though approximately 50% of the pyrimidine dimers remain in the DNA. These data indicate that not all pyrimidine dimers are accessible to the repair endonuclease. The observation that at long times after irradiation DNA is made in segments equal to those synthesized by nonirradiated cells although only a small percentage of the dimers have been removed suggests that an additional repair system alters dimers so that they no longer interrupt DNA replication.

Topics: Animals; Bromodeoxyuridine; Carbon Radioisotopes; Cell Line; Centrifugation, Density Gradient; DNA; DNA Repair; DNA Replication; Endonucleases; Humans; Hydrogen-Ion Concentration; Hydroxyurea; Isotope Labeling; Kidney; Light; Marsupialia; Phosphorus Radioisotopes; Radiation Effects; Skin; Thymidine; Time Factors; Tritium; Ultraviolet Rays; Xeroderma Pigmentosum

Topics: Humans; Phosphorus; Phosphorus Radioisotopes; Phosphorus, Dietary; Radioactivity; Xeroderma Pigmentosum