Overexpression of human OGG1 in mammalian cells decreases ultraviolet A induced mutagenesis.

7,8-dihydro-8-oxo-guanine (8-oxoG) is a mutagenic DNA lesion that is induced by ultraviolet A (UVA) radiation. 8-oxoG results in increased frequency of GC-->TA transversion mutations. UVA-induced mutant frequency was measured in the guanine phosphoribosyl transferase (gpt) gene of Chinese hamster ovary cells (AS52) that were stably transfected to overexpress the hOGG1 protein, the human DNA repair glycosylase for 8-oxoG. This mutant frequency was compared with ultraviolet A-induced mutant frequency in AS52 cells stably transfected with the same vector without the hOGG1 gene. The mutant frequency was significantly decreased in the hOGG1 overexpressing cells irradiated with 300 and 400 kJ/m2 ultraviolet A radiation, corresponding to 25% and 10% cell survival, respectively. The hOGG1 overexpressing cells repaired oxidative DNA lesions three times faster than the vector only cells as measured by a semi-automated alkaline elution assay using FPG enzyme, the bacterial OGG1 analogue, to cut DNA at oxidative base modifications. Thus, the lower mutation frequency in UVA-induced mutations of the hOGG1 overexpressing cells may be related to the increased repair of 8-oxoG. No GC-->TA mutations were detected in the UVA-irradiated hOGG1 overexpressing cells. The results suggest a link between the 8-oxoG lesion and UVA-induced mutagenesis. We propose that hOGG1 has a role in maintaining genomic stability in mammalian cells after oxidative stress.

Bystander effects in UV-induced genomic instability: antioxidants inhibit delayed mutagenesis induced by ultraviolet A and B radiation.

BACKGROUND: Genomic instability is characteristic of many types of human cancer. Recently, we reported that ultraviolet radiation induced elevated mutation rates and chromosomal instability for many cell generations after ultraviolet irradiation. The increased mutation rates of unstable cells may allow them to accumulate aberrations that subsequently lead to cancer. Ultraviolet A radiation, which primarily acts by oxidative stress, and ultraviolet B radiation, which initially acts by absorption in DNA and direct damage to DNA, both produced genomically unstable cell clones. In this study, we have determined the effect of antioxidants on induction of delayed mutations by ultraviolet radiation. Delayed mutations are indicative of genomic instability. METHODS: Delayed mutations in the hypoxanthine phosphoribosyl transferase (hprt) gene were detected by incubating the cells in medium selectively killing hprt mutants for 8 days after irradiation, followed by a 5 day period in normal medium before determining mutation frequencies. RESULTS: The UVB-induced delayed hprt mutations were strongly inhibited by the antioxidants catalase, reduced glutathione and superoxide dismutase, while only reduced glutathione had a significant effect on UVA-induced delayed mutations. Treatment with antioxidants had only minor effects on early mutation frequencies, except that reduced glutathione decreased the UVB-induced early mutation frequency by 24%. Incubation with reduced glutathione was shown to significantly increase the intracellular amount of reduced glutathione. CONCLUSION: The strong effects of these antioxidants indicate that genomic instability, which is induced by the fundamentally different ultraviolet A and ultraviolet B radiation, is mediated by reactive oxygen species, including hydrogen peroxide and downstream products. However, cells take up neither catalase nor SOD, while incubation with glutathione resulted in increased intracellular levels of glutathione. Previously, we have shown that ultraviolet induced delayed mutations may be induced via a bystander effect and that this effect is 5-fold higher for UVB radiation than for UVA radiation. Therefore, we propose that the antioxidants inhibit an ultraviolet radiation-induced bystander effect and that the effect is transmitted via the medium and via an internal transfer between cells, like gap junctional intercellular communication, for UVB radiation and only by the latter mechanism for UVA radiation.

The pheomelanin precursor 5-S-cysteinyldopa protects melanocytes from membrane damage induced by ultraviolet A radiation.

Pheomelanin and pheomelanin precursors have been implicated as risk factors for induction of melanoma by ultraviolet radiation. The pheomelanin precursor, 5-S-cysteinyldopa, has been shown to sensitise DNA to oxidative damage by ultraviolet radiation. We here show that 5-S-cysteinyldopa significantly protects melanocytes from membrane damage (permeability) induced by ultraviolet A radiation. Thus, 5-S-cysteinyldopa, may at the same time sensitise DNA and protect membranes from damage induced by ultraviolet radiation.

Bystander effects may modulate ultraviolet A and B radiation-induced delayed mutagenesis.

Ultraviolet irradiation of cells can induce a state of genomic instability that can persist for several cell generations after irradiation. However, questions regarding the time course of formation, relative abundance for different types of ultraviolet radiation, and mechanism of induction of delayed mutations remain to be answered. In this paper, we have tried to address these questions using the hypoxanthine phosphoribosyl transferase (HPRT) mutation assay in V79 Chinese hamster cells irradiated with ultraviolet A or B radiation. Delayed HPRT(-) mutations, which are indications of genomic instability, were detected by incubating the cells in medium containing aminopterin, selectively killing HPRT(-) mutants, and then treating the cells with medium containing 6-thioguanine, which selectively killed non-mutant cells. Remarkably, the delayed mutation frequencies found here were much higher than reported previously using a cloning method. Cloning of cells immediately after irradiation prevents contact between individual cell clones. In contrast, with the present method, the cells are in contact and are mixed several times during the experiment. Thus the higher delayed mutation frequency measured by the present method may be explained by a bystander effect. This hypothesis is supported by an experiment with an inhibitor of gap junctional intercellular communication, which reduced the delayed mutation frequency. In conclusion, the results suggest that a bystander effect is involved in ultraviolet-radiation-induced genomic instability and that it may be mediated in part by gap junctional intercellular communication.

Multiplex polymerase chain reaction analysis of UV-A- and UV-B-induced delayed and early mutations in V79 Chinese hamster cells.

We previously reported that approximately 10% of V79 Chinese hamster fibroblast populations clonally derived from single cells immediately after irradiation with either ultraviolet B (UV-B, 290-320 nm, mainly 311 nm) or ultraviolet A (UV-A, 320-400 nm, mainly 350-390 nm) radiation exhibit genomic instability. The instability is revealed by relatively high mutation frequencies in the hypoxanthine phosphoribosyl transferase (hprt) gene up to 23 cell generations after irradiation. These delayed mutant clones exhibited higher levels of oxidative stress than normal cells. Therefore, persistently increased oxidative stress has been proposed as a mechanism for UV-induced genomic instability. This study investigates whether this mechanism is reflected in the deletion spectrum of delayed mutant clones. Eighty-eight percent of the delayed mutant clones derived from UV-A-irradiated populations were found to have total deletion of the hprt gene. Correspondingly, 81% of UV-A-induced early mutations (i.e. detected shortly after irradiation) also had total deletions. Among delayed UV-B-induced mutant clones, 23% had total deletions and 8% had deletion of one exon, whereas all early UV-B events were either point mutations or small deletions or insertions. In conclusion, the multiplex polymerase chain reaction deletion screen showed that there were explicit differences in the occurrence of large gene alterations between early and delayed mutations induced by UV-B radiation. For UV-A radiation the deletion spectra were similar for delayed and early mutations. UV-A radiation is, in contrast to UV-B radiation, only weakly absorbed by DNA and probably induces mutation almost solely via production of reactive oxygen species. Therefore, the present results support the hypothesis that persistent increase in oxidative stress is involved in the mechanism of UV-induced genomic instability.

Increased level of oxidative stress in genomically unstable cell clones.

Recently, we reported that ultraviolet radiation induces delayed mutations in mammalian cells. At the same level of cell death the oxidative component of sunlight (ultraviolet A radiation) was as potent in inducing this kind of genomic instability as ultraviolet B radiation. Ultraviolet B radiation predominantly harms cells by direct damage to DNA and thus is much more mutagenic than ultraviolet A radiation. From that study, clones with a significantly increased mutation rate in the hypoxanthine phosphoribosyl transferase gene were obtained. These genomically unstable clones were also found to have a higher variance in the number of chromosomes than the unirradiated control cells, indicating chromosomal instability. The mechanisms for induction and maintenance of radiation induced genomic instability are not known, but some studies suggest that reactive oxygen species might be involved. In the present study, we have measured the level of potentially mutagenic peroxides in the genomically unstable clones. The levels of intracellular peroxides and lipid peroxides were measured using the probes dihydrorhodamine 123 and diphenyl-1-pyrenyl-phosphine, respectively. The unstable clones had elevated levels of oxidants, supporting the hypothesis that intermediate reactive oxygen species might have a role in the maintenance of genomic instability induced by ultraviolet radiation.

Pigmented melanocytes are protected against ultraviolet-A-induced membrane damage.

The dominant skin pigment melanin is believed to protect human skin against several harmful effects of ultraviolet radiation. It is not clear, however, how melanin located inside melanin-producing melanocytes modulates the effect of ultraviolet radiation on melanocytes themselves. We have determined membrane damage in pigmented and unpigmented albino mouse melanocytes after ultraviolet A radiation, which is suspected to induce melanoma. Unpigmented cells were much more susceptible to ultraviolet-A-induced membrane permeability than pigmented cells. Unpigmented cells were also more susceptible to ultraviolet-A-induced lipid peroxidation than strongly pigmented cells. Furthermore, unpigmented cells were much more susceptible to ultraviolet-A-induced depletion of glutathione than pigmented cells. Reduced glutathione is known to be a major antioxidant of unpigmented skin cells such as fibroblasts and keratinocytes. To examine whether or not glutathione is also a major antioxidant in melanocytes, melanocytes were depleted of glutathione by means of buthionine sulfoximine. We found that depletion of glutathione in pigmented melanocytes did not change lipid damage induced by ultraviolet A radiation. In unpigmented melanocytes, however, depletion of glutathione significantly increased lipid damage induced by ultraviolet A radiation. Thus, pigmented melanocytes apparently contain antioxidants more potent than glutathione, protecting them from ultraviolet-A-induced membrane damage.

Induction of delayed mutations and chromosomal instability in fibroblasts after UVA-, UVB-, and X-radiation.

Mutations in critical genes are believed to be a necessary part of cancer induction. The conventional view of radiation mutagenesis is that radiation induces most mutations in cells shortly after irradiation, because of false repair or lack of repair of DNA damage before or during DNA replication. In contrast, we here show that delayed mutations in the hypoxanthine phosphoribosyltransferase locus of Chinese hamster fibroblasts (V79) arise many cell generations after three types of carcinogenic irradiation: (a). UVA-; (b). UVB-; or (c). X-radiation. The frequency of mutations at the hypoxanthine phosphoribosyltransferase locus was measured in clones 14 days after irradiation with doses killing 80% of the cells. The proportion of unstable clones, as indicated by mutant fractions 10-7500-fold above background, was higher for the cells treated with UVA (13.2%) than for cells treated with UVB (9.2%) and X-radiation (9.6%). In contrast, UVA produces few immediate mutations compared with UVB and X-radiation. Thus, UVA-radiation, which is suspected to cause melanomas, produces few immediate mutations but more delayed mutations than UVB or X-radiation. Clones of cells that developed delayed mutations were examined for markers of chromosome instability, such as increased numbers of centrosomes, DNA content, and variability in the number of chromosomes. All radiation types increased the variability in the number of chromosomes in unstable clones. Although UVB and X-radiation, which damages DNA by direct interaction, resulted in an increased number of centrosomes in cell clones, the oxidative UVA-radiation did not. Thus, the mechanism of UVA-induced chromosomal instability is apparently different from that of UVB and X-radiation.