It is generally agreed that sunlight exposure is one of the etiologic agents in malignant melanoma of fairskinned individuals. However, the wavelengths responsible for tumorigenesis are not known, although DNA is assumed to be the target because individuals defective in the repair of UV damage to DNA are several thousandfold more prone to the disease than the average population. Heavily pigmented backcross hybrids of the genus Xiphophorus (platyfish and swordtails) are very sensitive to melanoma induction by single exposures to UV. We irradiated groups of five 6-day-old fish with narrow wavelength bands at 302, 313, 365, 405, and 436 nm and scored the irradiated animals for melanomas 4 months later. We used several exposures at each wavelength to obtain estimates of the sensitivity for melanoma induction as a function of exposure and wavelength. The action spectrum (sensitivity per incident photon as a function of wavelength) for melanoma induction shows appreciable sensitivity at 365, 405, and probably 436 nm, suggesting that wavelengths not absorbed directly in DNA are effective in induction. We interpret the results as indicating that light energy absorbed in melanin is effective in inducing melanomas in this animal model and that, in natural sunlight, 90-95% of melanoma induction may be attributed to wavelengths > 320 nm-the UV-A and visible spectral regions.The incidence of malignant melanoma has been increasing for several years at a rate of -5% per year among fair-skinned individuals in North America and Europe, probably due to changes in lifestyle. The disease has a complex etiology. Although sunlight exposure is implicated, melanoma is not associated with chronic exposure nor is it located primarily on highly exposed areas of the body (1, 2). Because individuals with the DNA repair-deficient disease xeroderma pigmentosum are several thousandfold more susceptible than unaffected individuals (3), sunlight damages to DNA are thought to be initiating carcinogenic events. However, the wavelengths effective in melanoma induction are not known. The wavelengths in sunlight between 280 and 320 nm (UV-B) are more strongly absorbed by DNA than are the longer UV-A wavelengths. On the other hand, the melanin in melanocytes absorbs UV at all wavelengths and energy absorbed by this pigment might affect DNA by energy or free-radical transfer to DNA (4). Useful animal models for determining melanoma induction as a function of wavelength-the action spectrumare the hybrid offspring from intra-and interspecific crosses between pigmented and nonpigmented fishes of the genus Xiphophorus (5

Sunlight exposure is strongly indicated as one of the important etiologic agents in human cutaneous malignant melanoma. However, because of the absence of good animal models, it has not been possible to estimate the wavelengths or wavelength regions involved. We have developed a useful animal model from crosses and backcrosses of platyfish (Xiphophorus maculatus) and swordtails (Xiphophorus heUeri).Two strains of these fish are susceptible to invasive melanoma induction by exposure to filtered radiation from sunlamps in the wavelength ranges A > 290 nm and A > 304 nm. Multiple exposures on 5-20 consecutive days beginning on day 5 after birth or a single exposure of =200 J/(m2 day) of A > 304 nm result in a tumor prevalence of 20% to 40% at 4 months of age compared with a background rate of 12% in one strain and 2% in another. Exposure of the fish to visible light after UV exposure reduces the prevalence to background. The melanomas are similar in many respects to mammalian melanomas, as judged by light and electron microscopy. The genetics of the crosses determined by others and the high sensitivity of the hybrids to melanoma induction indicate that the UV radiation probably inactivates the one tumor repressor gene (or a small number of tumor repressor genes) in the hybrid fish. The small size of the animals and their high susceptibility to melanoma induction make them ideal for action spectroscopy.Agents that cause a decrease in stratospheric ozone would cause an increase in UV-B (A = 280-320 nm) intensities at the earth's surface without appreciably changing the longer UV or visible intensities of light. Melanoma among the white population of the United States and Europe is increasing dramatically as a function of time, probably as a result of changing lifestyles (1). The relation between latitude and melanoma mortality suggests that there is a correlation between the average solar radiation and mortality from malignant melanoma. However, it is not known which parts of the solar spectrum can plausibly be related to the increasing mortality because, although sunlight exposure seems to be an essential component in melanoma incidence, it is not the only one. Body areas most exposed to light are not the primary locations of melanomas as they are for basal and squamous cell carcinomas. There is good evidence that UV-B is tumorigenic in animals (2) and can cause neoplastic transformation in vitro (3). Four types of experiment indicate that light energy absorbed in DNA can cause cellular damage leading to tumors: (i) the tumorigenicity of fish cells as a result of UV-irradiation in vitro can be photoreactivated (4)-a process that monomerizes UV-induced cyclobutane pyrimidine dimers in DNA; (ii) the wavelengths effective in neoplastic transformation in vitro are those absorbed by DNA (5); (iii) transformation in vitro by UV is photoreactivable (6); and (iv) xeroderma pigmentosum individuals are defective in the ability to repair UV damages in their DNA and are extraordinarily sensitive to cancer induction, inc...

ABSTRACr The Amazon molly, Poecilia formosa, is a small fish that grows in clones. Hence, cells from one animal may be transplanted to another without danger of rejection. Cells from thyroid and adjacent tissue were irradiated with UV light in vitro and injected into the abdominal cavity of isogeneic recipients. At appropriate UV doses and numbers of cells injected, all recipients showed exuberant thyroid proliferation. We give arguments and data indicating that the proliferation is a tumor, not a goitrogenic response. If the UV irradiation is followed, but not preceded, by photoreactivating illumination, the yield of thyroid growths is markedly decreased. Because other investigtions have shown that photoreactivation monomerizes UV-induced cyclobutylpyimiine dimers in DNA and does not affect other photoproducts, our data indicate that pyrimidine dimers in DNA can give rise to tumors. A number of physical and chemical environmental agents damage DNA in vivo. Many of these agents are carcinogenic (1-3),-and various mutagen test systems show that there is an excellent correlation between carcinogenicity and DNA damage as measured by mutagenesis (4). Nevertheless, because treatment of DNA with carcinogens results in the formation of many products, it has not been possible to identify particular molecular changes in DNA as initiating carcinogenic events. To make such an identification, one needs an experimental trick that will change the relative proportions of the various products and permit observation of how tumor induction depends on these proportions. UV radiation makes many types of photoproducts in DNA. One of these-cyclobutylpyrimidine dimers (pyrimidine dimers)-has been shown to have lethal and mutagenic effects in microorganisms by virtue of the fact that such dimers can be monomerized by photoreactivating (PR) enzyme plus light of wavelength >320 nm (5, 6). Because, to the best of our knowledge, enzymic photoreactivation works only on pyrimidine dimers and not on other products, our finding of a PR effect strongly implicates pyrimidine dimers as the important initial DNA damage in tumor induction.There is excellent evidence that most human skin cancer arises from solar UV radiation (7) and it is a good inference that DNA is the target molecule (8 (12), cooked spinach (6), marine fish meal (6), high-protein baby cereal (4), wheat germ (2), and iodized salt (1).Cell Isolation and Injections. Donor animals (2-3 months old, 2 cm long) were placed on ice for 5 min and then given 3-min washes in each of 2.6% sodium hypochlorite, cold 95% ethanol, and sterile fish Ringer's solution. Scales were removed and tissue was excised from along the ventral aorta. The excised region contained thyroid, muscle, and cardiac tissue (from the thyroid gland, urohyal, and heart, respectively). Tissues were pooled and homogenized in fish Ringer's solution without Ca2+ or Mg2+ and containing isoniazid (0.7 mg/ml), streptomycin (1 ,sg/ml), and penicillin (1000 units/ml). Homogenization yielded primarily small clumps of two to five ...

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Genetic hybrids of Xiphophorus fishes have been used for decades to study heritable melanoma formation. In these models, overexpression of pigmentation patterns from melanin-producing pigment cells can lead to genetically regulated melanoma formation in backcross hybrids. In the best studied of these models, the Gordon-Kosswig hybrid melanoma, tumors form spontaneously in all individuals of a subset of backcross hybrids between the platyfish Xiphophorus maculatus Jp 163 A and the swordtail species Xiphophorus helleri. Backcross hybrids susceptible to melanoma formation inherit a sex-linked oncogene, Xmrk, associated with the spotted dorsal (Sd) pigment pattern and have lost both copies of an autosomal gene, DIFF, from the X. maculatus parent. Spontaneous melanoma formation conforms to simple, two-gene Mendelian inheritance in which DIFF behaves as a recessive tumor suppressor gene. Recently, Xiphophorus hybrids in which melanomas can be induced by UV and near-UV visible light exposure have been described. We report here results of genetic linkage analysis of one of these Xiphophorus light-inducible hybrid melanoma models, in backcross hybrids between the two platyfish species X. maculatus Jp 163 B and Xiphophorus couchianus. Our linkage results provide the first estimate of recombination between the tumor suppressor locus, DIFF, and glycerate-2-dehydrogenase (GLYDH) in Xiphophorus linkage group V. Also, they demonstrate that DIFF regulates hyperplasia of spotted side (Sp) pigment cells in this hybrid model, analogous to its regulation of hyperplasia of Sd pigment cells in the "classical" Gordon-Kosswig hybrid. Joint segregation analyses of melanoma-bearing fish indicate that segregation of DIFF is genetically linked to melanoma induction by 405 nm light in this model but that induction of melanomas by UV wavelengths apparently does not depend on segregation of the DIFF locus.

The Xiphophorus hybrid melanoma model represents one of the earliest reported cases of genetically regulated tumor susceptibility. Melanoma formation in Xiphophorus hybrids may be explained by the inheritance of two genes: a sex-linked oncogene, Xmrk, and a putative tumor suppressor locus, termed DIFF, located in Linkage Group V (LG V). Several genetic mapping procedures were used to produce a new Xiphophorus LG V map with 20 loci. All markers, particularly a recently cloned Xiphophorus CDKN2 gene family member, called CDKN2X, were tested for associations of genotype with degree of macromelanophore pigment pattern modification and susceptibility to melanoma formation in backcross hybrids of seven genetic types, involving 1,110 fish and three pigment patterns. Highly significant associations of CDKN2X genotypes with such phenotypic effects suggests that this gene is a strong candidate for the classically defined DIFF tumor suppressor gene. Because published results have documented the involvement of the CDKN2A (p16, MTS1, and INK4A) tumor suppressor gene in human melanoma formation, the possibility of CDKN2 genes acting as tumor suppressors in both man and Xiphophorus is likely.

The unisexual fish Poecilia formosa (the Amazon molly) reproduces by gynogenesis, a process in which sperm from the males of the host bisexual species activates development of its eggs. Unisexuals live with one of the host species in nature and compete with bisexual females for the males. It was long thought that male discrimination and mate selection established a balance between the unisexual and bisexual populations. Thus, hierarchies of males were set up in which dominant males mated with their conspecific females and subordinates mated with the Amazon molly. Recent evidence suggested, however, that male fish do not discriminate between their own females and the Amazon molly, and that there always are more males available than sexually receptive females of both species.Our findings indicate that male behaviour may be more complex than suggested by either hypothesis. Mate discrimination and courtship behaviour appear to increase with age, so that large males show almost complete preference for their conspecific females, but smaller males will mate with the Amazon molly. In complex groups, small males often dart in and mate with their own females whilst the large males are engaged in courtship activities and defence of territory.