Photochemistry and Photobiology, 2005, 81: zyxwvuts 114-1 zyxwvutsr 19 Multiplex Polymerase Chain Reaction Analysis of UV-A- and UV-6-induced Delayed zyxw and Early Mutations in V79 Chinese Hamster Cells? Jostein Dahle*, Paul Noordhuis, Trond Stokke, Debbie Hege Svendsrud and Egil Kvam Department of Radiation Biology, The Norwegian Radium Hospital, Montebello, Oslo, Norway Received 19 May 2004; accepted 20 September 2004 ABSTRACT zyxwvutsrq We previously reported that approximately 10% of V79 Chinese hamster fibroblast populations clonally derived from single cells immediately after irradiation with either ultravi- olet 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 pop- ulations were found to have total deletion of the hprt gene. Correspondingly, 81 % of UV-A-induced early mutations zyxwvu (Le. 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. f/Posted on the website on 28 September 2004. *To whom correspondence should be addressed: Department of Radiation Biology, The Norwegian Radium Hospital, Montebello, 03 10 Oslo, Norway. e-mail: zyxwvutsrqp jostein.dahle@labmed.uio.no zyxwvutsrqpon Abbreviations: cDNA, complementary DNA, CHO, Chinese hamster ovary; EF2, elongation factor 2; hprt, hypoxanthine phosphoribosyl transferase; mRNA, messenger RNA, PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction; 6TG, 6- thioguanine; UV-A, ultraviolet A (320400 nm); UV-B, ultraviolet B (290-320 nm). zyxwvutsrqp 0 2005 American Society for Photobiology 003 1 zyxwvutsrqponmlk -8655/05 INTRODUCTION The increased incidence of human skin cancer has been attributed to an increase in environmental levels of ultraviolet B radiation (UV-B, 290-320 nm), caused by depletion of stratospheric ozone, and changes in recreational suntanning habits (1-3). Ultraviolet A radiation (UV-A, 3 2 0 4 0 0 nm) has previously been found to be around 10 times less mutagenic than UV-B radiation when compared at the same level of cell survival (4). However, the fluence of UV-A radiation in sunlight is up to 100 times higher than the fluence of UV-B radiation (3, and UV-A radiation also penetrates deeper into skin (6). UV-B radiation not only acts initially by absorption in DNA and production of direct DNA damage but also generates oxidative stress, especially in the form of hydrogen peroxide and lipid peroxides (7). In contrast, UV-A radiation acts primarily by absorption of radiation in endogeneous photosensitizers that generate reactive oxygen species such as singlet oxygen in exposed cells (7,8). Thus, UV-A radiation induces different spectrum and time course of reactive oxygen species than UV-B radiation (7,9,10). The relative roles of UV-A radiation and UV-B radiation in the induction of human skin cancers, particularly malignant melanoma, are not well established. UV radiation-induced mutations have been assumed to be the result of DNA damage inflicted during the irradiation. This damage is converted to a mutation either during DNA replication or as the result of errors during DNA repair, and mutations should therefore occur shortly after irradiation. However, recent evidence suggests that mutations can occur up to 23 cell generations after UV radiation (11). Remarkably, UV-A radiation was as effective as UV-B radiation in inducing mutations 23 cell generations after radiation. Both types of radiation induced significantly increased mutation frequencies in around 10% of the clones. Delayed mutations were measured by culturing cells for 14 days after irradiation (approximately 23 cell generations) before measuring mutation frequency by the hypoxanthine phosphoribosyl trans- ferase (hprt) mutation assay. One hprt mutant colony from each unstable clone was picked and used in this study. The molecular characteristics of UV-induced delayed mutations are unknown, but the mechanism of induction of UV-induced delayed mutations might be related to oxidative stress (12-14). In a study of some of the clones analyzed in this article, we found a trend that UV-induced delayed mutant clones had higher levels of oxidative stress than normal cells (13). Furthermore, the antioxidant reduced glutathione can inhibit both UV-A- and UV-B-induced delayed mutagenesis (J. Dahle and E. Kvam, unpublished). 114