Ultraviolet A does not induce melanomas in a Xiphophorus hybrid fish model David L. Mitchella,b,1, André A. Fernandeza, Rodney S. Nairna,b, Rachel Garciaa, Lakshmi Panikera, David Tronoa, Howard D. Thamesb,c, and Irma Gimenez-Contia,b aDepartment of Carcinogenesis, University of Texas MD Anderson Cancer Center, Smithville, TX 78957; bGraduate School of Biomedical Sciences, University of Texas, Houston TX 77225; and cDepartment of Biostatistics,University of Texas, Houston, TX 77024 Edited* by James E. Cleaver, University of California, San Francisco, CA, and approved April 13, 2010 (received for review January 8, 2010) We examined the wavelength dependence of ultraviolet (UV) ra- domestica) and several genetically modified mouse models, dem- diation (UVR)-induced melanoma in a Xiphophorus backcross hy- onstrate that UVA does not induce melanomas (5–7). In contrast, brid model previously reported to be susceptible to melanoma in- evidence from all of these models, including Xiphophorus, sup- duction by ultraviolet A (UVA) and visible light. Whereas ultraviolet ports a role for UVB in the etiology of melanoma. Unlike placental B (UVB) irradiation of neonates yielded high frequencies of mela- mammals, fish are very efficient at removing UVB-induced direct nomas in pigmented fish, UVA irradiation resulted in melanoma damage in DNA using photoenzymatic repair (PER) (8, 9) and this frequencies that were not significantly different from unirradiated mechanism greatly decreases melanoma formation in the Xipho- fish. Spontaneous and UV-induced melanoma frequencies corre- phorus melanoma model (1, 3). The most abundant damage in- lated with the degree of pigmentation as expected from previous duced by UVB irradiation results from the direct absorption of studies, and the histopathology phenotypes of the melanomas photons by DNA and includes the formation of cyclobutane py- were not found in significantly different proportions in UV-treated rimidine dimers (CPD) and (6-4) pyrimidine dimers [(6-4)PD] (10, and -untreated tumor-bearing fish. Our results support the conclu- 11). Collectively, the results from the vast majority of animal sion that a brief early-life exposure to UVB radiation causes mela- studies strongly suggest that one (or both) of these lesions is re- noma formation in this animal model. These data are consistent quired for melanoma formation. with an essential role for direct DNA damage, including cyclobutane Xiphophorus offers some significant advantages over other ani- dimers and (6-4) photoproducts, in the etiology of melanoma. mal models for investigating melanoma. The classical, spontaneous Xiphophorus hybrid melanoma model has been studied for over ultraviolet B | DNA damage | cyclobutane dimer | reactive oxygen 80 years and offers powerful genetic and biochemical approaches species | melanin for revealing determinants of melanomagenesis (12–14). The Xi- phophorus melanoma receptor kinase gene (XMRK)behavesas n the late 1980s Setlow and coworkers used genetic hybrids a dominant oncogene in this fish model and is a mutated derivative Ifrom interspecific crosses involving several species of the fish of the fish ortholog for the human epidermal growth factor receptor genus Xiphophorus to investigate the effects of UVR on the in- (EGFR/ErbB-1). Consistent with the activity of mammalian EGFR duction of cutaneous malignant melanoma (CMM) (1). These in melanocytes (15–17), activation of the XMRK oncoprotein leads pioneering studies demonstrated that ultraviolet B (UVB) irra- to numerous downstream signaling cascades including, but not li- diation of backcross hybrids generated from a specific genetic mited to, the RAS/RAF/MAPK and PI3-K/AKT signaling path- crossing scheme induced melanomas at significant frequencies ways (12). In addition to these signaling cascades, the transformed above spontaneous levels. These results were later confirmed, phenotype in Xiphophorus also involves participation of transcrip- and the genetic basis of UVB-induced melanoma susceptibility tion factors (e.g., STAT5) and glycoproteins (e.g., osteopontin, in this cross was recognized to be the same as in the well-studied OPN) that are intimately involved in cellular proliferation and spontaneous Xiphophorus hybrid melanoma model (2). In 1993, antiapoptotic responses that characterize numerous human can- Setlow used a different Xiphophorus interspecies cross (desig- cers. Hence, Xiphophorus hybrid models offer ideal experimental nated as Sp-couchianus; Fig. 1) to study the wavelength de- platforms to further elucidate the biochemistry underlying mela- pendence of melanoma induction and reported that wavelengths nomagenesis within the context of a controlled genetic background. in the ultraviolet A (UVA) and visible ranges were effective in A recent publication (18) exploited the same Xiphophorus Sp- fi inducing melanomas in rst-generation backcross (BC1) hybrids couchianus hybrid melanoma model used by Setlow for UVA in- generated from this particular cross (3). An action spectrum for duction studies (3) to investigate photosensitization of melanin as melanoma induction was proposed with maxima in the UVB a possible mechanism for melanoma formation. In this report, fl (302/313 nm) and UVA (365 nm) ranges. Because UVA uence electron paramagnetic resonance was used to describe the UVR MEDICAL SCIENCES is quantitatively much greater than UVB in sunlight incident to wavelength dependence of reactive melanin radical formation in ’ ∼ the earth s surface ( 10-fold), Setlow suggested that, on the basis pigmented fish skin. The action spectrum for melanin-sensitized of this action spectrum, UVA was more effective than UVB in generation of reactive radicals derived from these experiments causing melanomas in the human population (2, 4). This report positively correlated with the action spectrum for melanoma for- fi had signi cant public health consequences; it suggested that the mation reported by Setlow (3). These results are consistent with use of commercially available sunscreens that effectively blocked a role for UVB and UVA in generating melanin-derived reactive UVB but not UVA encouraged more lengthy recreational sun- light exposure and thereby increased the exposure to UVA and its associated risks. Over the past 20 years, these data have be- Author contributions: D.L.M. and R.S.N. designed research; D.L.M., R.G., L.P., and D.T. come central to the debate on the role of UVA in melanoma and performed research; D.L.M., A.A.F., R.S.N., L.P., H.D.T., and I.G.-C. analyzed data; and the risks associated with recreational and artificial exposures to D.L.M., A.A.F., R.S.N., H.D.T., and I.G.-C. wrote the paper. UVA wavelengths. The authors declare no conflict of interest. Debate over the action spectrum for melanoma has only in- *This Direct Submission article had a prearranged editor. tensified because subsequent research using a variety of animal 1To whom correspondence should be addressed. E-mail: [email protected]. models has not corroborated these results. Studies in mammalian This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. models, including the South American opossum (Monodelphis 1073/pnas.1000324107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1000324107 PNAS | May 18, 2010 | vol. 107 | no. 20 | 9329–9334 Downloaded by guest on October 1, 2021 melanoma frequency = 44.3%; control: 40 tumor bearing fish out of 216 individuals, melanoma frequency = 18.5%; χ2 = 31.99, P < 0.0001). However, there was no difference in melanoma incidence between the UVA-irradiated fish and that of the control fish (UVA: 35 tumor-bearing fish out of 282 individuals, melanoma frequency = 12.4%; control: 40 tumor-bearing fish out of 216 individuals, melanoma frequency = 18.5%; χ2 = 3.57, P > 0.05) Therefore, our results contradict the report of Setlow and col- leagues (3) and indicate that UVB but not UVA significantly induces melanomas in this Xiphophorus melanoma model (Fig. 2). We used doses of 6.4 and 80 kJ/m2 UVB and UVA, respectively, for each of the five UV treatments; Setlow and colleagues used a dose range of 0.35–3.0 and 0.5–11.75 kJ/m2 313 (UVB) and 365 (UVA), respectively, for their treatments. Because of differences in emission spectra and dosimetry, these doses are not directly comparable. However, on the basis of our toxicity and DNA Fig. 1. The Sp-couchianus backcross hybrid breeding scheme. F1 hybrids are damage determinations, our incident doses are equal to or exceed produced by mating a macromelanophore pigmented “spotted side” (Sp/ those of Setlow et al. (3). Sp) X. maculatus female from strain Jp 163 B (Upper Left)toaX. couchianus (Xc) male not carrying the Sp allele (+/+) (Upper Right), therefore not Exposure of Neonates to UVA or UVB Did Not Affect the Degree of exhibiting any macromelanophore pigmentation. F1 hybrids are back- Adult Pigmentation. The degree of pigmentation was visually es- crossedtoeithermaleorfemaleX. couchianus individuals and produce BC1 fi i ≈ timated in each sh by two observers (L.P. and D.T.) as ( ) light progeny of which 50% exhibit the Sp (spotted side) (Sp/+) and 50% the – ii – wild-type (+/+) pigment phenotypes. with 50 70% coverage, ( ) intermediate with 70 90% coverage, and (iii) heavy with >90% coverage. The distribution of pig- mentation in the different experimental groups was determined oxygen species (ROS) in melanoma causation. However, the ob- at 4 months and at the time of sacrifice at 12–14 months (Table servation that virtually all other animal models developed to study 1). From Table 1, it is clear that the amount of pigmentation an melanoma are refractory to UVA-induced melanoma calls into individual possessed increased with their age in all treatment question the role of melanin-derived ROS in melanoma induction. groups. However, it should be noted that there did not appear to With these considerations in mind, we used the Xiphophorus Sp- be any differences in the distribution of pigmentation among the couchianus model in experiments designed to replicate the results treatment groups at the time of sacrifice.