2--deoxyguanosine-5--phosphate and 5-5-dimethyl-1-pyrroline-1-oxide

Ultraviolet-A and melanin are implicated in melanoma, but whether melanin in vivo screens or acts as a UVA photosensitiser is debated. Here, we investigate the effect of UVA-irradiation on non-pigmented, lightly and darkly pigmented melanocytes and melanoma cells using electron spin resonance (ESR) spectroscopy. Using the spin trap 5,5 Dimethyl-1-pyrroline N-oxide (DMPO), carbon adducts were detected in all cells. However, higher levels of carbon adducts were detected in lightly pigmented cells than in non-pigmented or darkly pigmented cells. Nevertheless, when melanin levels were artificially increased in lightly pigmented cells by incubation with L-Tyrosine, the levels of carbon adducts decreased significantly. Carbon adducts were also detected in UVA-irradiated melanin-free cell nuclei, DNA-melanin systems, and the nucleoside 2'-deoxyguanosine combined with melanin, whereas they were only weakly detected in irradiated synthetic melanin and not at all in irradiated 2'-deoxyguanosine. The similarity of these carbon adducts suggests they may be derived from nucleic acid- guanine - radicals. These observations suggest that melanin is not consistently a UVA screen against free-radical formation in pigmented cells, but may also act as a photosensitizer for the formation of nucleic acid radicals in addition to superoxide. The findings are important for our understanding of the mechanism of damage caused by the UVA component of sunlight in non-melanoma and melanoma cells, and hence the causes of skin cancer.

Topics: Carbon; Cell Line, Tumor; Cell Nucleus; Cyclic N-Oxides; Deoxyguanine Nucleotides; DNA; DNA Damage; Electron Spin Resonance Spectroscopy; Free Radicals; Humans; Melanocytes; Melanoma; Ultraviolet Rays

Fecapentaene-12 (fec-12), excreted in human faeces, is genotoxic to human cells and a known animal carcinogen. The mechanism of its genotoxicity is unknown but may involve direct alkylation and/or free-radical generation. The formation of reactive species during fec-12 aerobic degradation was thus investigated by electron paramagnetic resonance (EPR) and NMR spectroscopic techniques. Oxy- and alkyl-radicals were detected as the 5,5'-dimethyl-1-pyrroline-N-oxide spin-trap adducts at fec-12 concentrations of between 0.1 and 2.0 mM. Under anaerobic conditions no free-radical generation was observed. NMR spectroscopy indicated that fec-12 degraded at least initially into three unsaturated aldehydes. The co-formation of free-radicals and unsaturated aldehydes suggests that fec-12 decomposed aerobically via a process analogous to lipid peroxidation. As both types of species, thus formed, may subsequently interact with DNA to form adducts, fec-12-induced DNA damage was investigated by 32P-postlabelling techniques. Using procedures that detect alkyl-type adducts, a number of putative adducts were detected in fec-12-treated DNA; two of similar mobility were observed in fec-12-treated 2'-deoxyguanosine-3'-monophosphate. Adducts with similar mobility have been detected in acrolein-treated DNA. One adduct with similar mobility was also observed in DNA obtained from normal human fibroblasts treated with fec-12. Using a C-18 ODS column, these putative adducts were eluted in 60-85% methanol, whereas 8-hydroxydeoxyguanosine-3'-monophosphate (8OHdGp) was eluted with 1% acetonitrile. Also unlike these putative adducts, the detection of 8OHdGp required HPLC fractionation prior to 32P-postlabelling. The formation of adducts, possibly aldehyde-related, and free-radical damage suggests that fec-12 genotoxicity may be the result of several different mechanisms, the relative importance of each is as yet unknown. Hydroxyl radicals were also detected during the aerobic decomposition of deca-2,4,6,8-tetraenal, a possible degradation product of fec-12 and a less potent mutagen, suggesting that free-radical generation may have only a minor role in fec-12-induced genotoxicity.

Topics: Autoradiography; Cyclic N-Oxides; Deoxyguanine Nucleotides; DNA; Humans; Mutagens; Phosphorus Radioisotopes; Polyenes; Spin Labels