MNU induction of neoplasia in a platyfish model.

Interspecific hybrid crosses between members of the fish genus Xiphophorus have been used for over 70 years to study the genetic aspects of melanoma formation. In the well-established "Gordon-Kosswig" cross, the platyfish X. maculatus is outcrossed to the swordtail X. helleri, and the resulting backcross segregants spontaneously develop melanoma. We recently produced a distinct cross between X. maculatus and another platyfish species, X. couchianus. X. maculatus strain Jp 163 A is homozygous for several X-linked pigment pattern genes, including the Spotted dorsal (Sd), Dorsal red (Dr), and Anal fin spot (Af). Af is a sex-limited trait, coding exclusively for melanophores distributed on the modified anal fin or "gonopodium" in the adult male fish. Within F1 and BC1 hybrids (to X. couchianus), the Sd pigment pattern is phenotypically suppressed, whereas Dr and Af are enhanced. We exposed BC1 hybrids to the direct-acting carcinogen N-methyl-N-nitrosourea (MNU). Treatment led to the development of schwannomas, fibrosarcomas, and retinoblastomas. In addition, numerous MNU-treated males that inherited Af developed a pronounced melanotic phenotype, with melanin-containing cells oftentimes totally covering the gonopodium and extending further to grow within the ventral regions of the fish. Genetic linkage analysis of the BC1 hybrids revealed a significant (p < 0.01) association between CDKN2X genotype and the phenotypic degree of melanization. Such an association is consistent with a locus within linkage group V playing a role in the development of melanosis and delineates three genetic preconditions and a carcinogenic scheme resulting in melanosis of the ventral regions of hybrid fish. The overall study further alludes to the potential of using Xiphophorus fish to study carcinogenic mechanisms for tumors other than melanoma (schwannoma, fibrosarcoma, and retinoblastoma) and should enable extensive pathologic and molecular genetic studies of derived neoplastic abnormalities.

Three unique experimental fish stories: Poecilia (the Past), Xiphophorus (the Present), and Medaka (the Future).

These three stories exemplify the uniqueness of fish models in their abilities to answer important biological questions. The first one identifies the major UV-induced damage (pyrimidine dimers) that is responsible for tumor induction. Thyroid cells from isogenic fish ( Poecilia formosa) were exposed to UV in vitro, then either exposed or not to photoreactivating light that monomerizes dimers, and the cells were injected into isogenic recipients. In the absence of photoreactivating light, the recipients developed tumors; in its presence, there were very few tumors. The second story describes our use of backcross hybrids of Xiphophorus as a model for melanoma induction by several UV and visible wavelengths. All the wavelengths were effective. (Squamous cell carcinomas in mice are induced preferentially by wavelengths <320 nm.) The data strongly suggest that light absorbed by the black pigment melanin damages DNA. The third story is designed to determine the mutagenic effects on sperm of the high atomic number, high-energy (HZE) nuclei present in cosmic rays by measuring mutations in progeny of exposed male medaka.

A proposed classification scheme for Xiphophorus melanomas based on histopathologic analyses.

We studied the histopathologic characteristics of melanomas induced in the Xiphophorus model. This fish model has been used for several decades to study the molecular and genetic mechanisms underlying its susceptibility to melanoma induction. Numerous distinct interspecific hybrid crosses currently are being used in research on carcinogenesis. We previously reported that tumors were induced in such hybrid crosses after treatment with N-methyl-N-nitrosourea or UV radiation. In this report, we describe the histopathologic features of Xiphophorus melanomas and propose a new classification system. We suggest that melanomas in these fishes can be classified as follows: melanocytic melanomas; melanophorous-macromelanophorous polymorphic melanomas; spindle cell type melanomas; epithelioid cell melanomas; and amelanotic melanomas. The new classification of Xiphophorus melanomas should allow correlations between histopathologic characteristics and carcinogen treatment, and between histopathologic characteristics and the genetic background of the hybrid fish.

Environmental factors in nonmelanoma and melanoma skin cancer.

We discuss the role of sunlight, mostly ultraviolet light (UV), in the induction of nonmelanoma and melanoma skin cancer. Whilst the former seems to be correlated with accumulated exposure, the causation of melanoma is more complex, and may also involve the pattern of, and age at, exposure. The efficacy of sunscreens is debatable; while they protect against UVB wavelengths (290-320 nm), and so extend the time that may be spent in the sun before becoming sunburnt, their use may subject wearers to excessive exposure to UVA (320-400 nm) and visible light. Both epidemiological surveys and experiments with animal models suggest that UVA, and perhaps the visible, may induce melanomas. Although Japanese have a much lower incidence of skin cancer than Caucasians, the dramatic rise in skin cancer in Japanese-Americans in Hawaii exposed to high-intensity irradiation raises concerns. If the Japanese people adopt sun-seeking behavior, or should the levels of UV irradiation rise significantly through depletion of the ozone layer, then this could become an important health problem in future.

Localization of a CDKN2 gene in linkage group V of Xiphophorus fishes defines it as a candidate for the DIFF tumor suppressor.

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.

Aging, the fishy side: an appreciation of Alex Comfort's studies.

Many years ago, Alex Comfort experimentally refuted Bidder's hypothesis that fish potentially were immortal. Later morphological and physiological studies, together with observations from fish populations in the wild, revealed that fish age in a way similar to that in other vertebrates. More recently, assessments of the age of fish have been revised, and have shown that some species live much longer than was estimated. These findings, together with the difficulties of demonstrating any increase in the rate of mortality with age in the long-lived, heavily exploited populations of fish, revived Bidder's ideas. I briefly review some of the more recent literature, and conclude that there is no evidence to suggest that fish are exceptional; like other vertebrates, sooner or later they grow old and die.

Nonmammalian models for sunlight carcinogenesis: genetic analysis of melanoma formation in Xiphophorus hybrid fish.

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.

Temporal changes in the incidence of malignant melanoma: explanation from action spectra.

The incidence of malignant cutaneous melanoma has been increasing for more than 50 years, and is now rising more rapidly than that of any other cancer. This increase is not explicable by changes in the physical environment, particularly by any observed increase in UVB radiation (290-320 nm). The distribution of melanomas on the body differs from the site distribution of nonmelanoma skin cancer (relatively many more melanomas occur on areas of the body not chronically exposed to sunlight, such as the back of the trunk in males, and the legs in females). This localization of melanoma, together with its epidemiology, suggest that a change in lifestyle has contributed to the fast-rising incidence in many countries. There is no convenient mammalian animal model for malignant melanoma. However, certain inter- and intra-specific hybrids of fish of the genus Xiphophorus are very sensitive to light-induced melanomas; we have used them to determine the wavelengths effective in melanoma induction. The action spectrum has a relatively very large component in the UVA region (320-400 nm) compared to human erythema. Hence, if the human and fish spectra were similar, the use of sunscreens that minimize erythema would have little effect in preventing the induction of melanoma, and if people using sunscreens expose themselves to sunlight for longer periods, they will be increasing dramatically their exposure to these melanoma-inducing wavelengths. Such considerations are sufficient to explain the rising incidence of malignant melanoma and its distribution on the body.

Wavelengths effective in induction of malignant melanoma.

It is generally agreed that sunlight exposure is one of the etiologic agents in malignant melanoma of fair-skinned 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.

Animal model for ultraviolet radiation-induced melanoma: platyfish-swordtail hybrid.

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 helleri). Two strains of these fish are susceptible to invasive melanoma induction by exposure to filtered radiation from sunlamps in the wavelength ranges lambda greater than 290 nm and lambda greater than 304 nm. Multiple exposures on 5-20 consecutive days beginning on day 5 after birth or a single exposure of approximately 200 J/(m2.day) of lambda greater than 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.

Established cell lines from nonmammalian vertebrates: models for DNA repair studies.

Several established cell lines from different classes of vertebrates were assayed for the presence of O6-methylguanine acceptor protein. This protein is instrumental in removing adducts from DNA caused by exposure to alkylating agents. Cultured cells had levels of acceptor protein activity within the range found in fresh tissues from animals in the same class. We suggest that cells from lower vertebrates are satisfactory in vitro models for studies of this DNA repair function.

Presence of O6-methylguanine acceptor protein in the tissues of different classes of vertebrates and invertebrates.

We have measured the ability of extracts of tissues from several species of mammals, birds, reptiles, amphibia and fish to demethylate adducts of O6-methylguanine in exogenous DNA by transfer of the methyl group to an acceptor protein. Our study also encompassed tissues from a smaller number of invertebrates, from arthropods, molluscs and annelids. The vertebrate tissues used were liver, brain, spleen and kidney. In the case of the invertebrates we sampled liver, neural tissue, gonads, digestive tract and hepatopancreas. There was no consistent change in the amount of acceptor activity per unit of protein or DNA going from cold-blooded to warm-blooded vertebrates. Liver invariably had the highest amount; this finding was not unexpected since metabolic processes in the liver are high, and good cellular protective mechanism important. Inter-class comparisons within the vertebrates are highly speculative, and hindered by the fact that there is little information on carcinogenesis in animals other than rodents and humans. O6-methylguanine acceptor activity was found in all the invertebrate tissues tested. The amounts were variable, 0.003-0.0051 fmol/micrograms cellular DNA, but the values fell within the range of those found in the tissues of vertebrates.

Levels of O6-methylguanine acceptor protein in tissues of rats and their relationship to carcinogenicity and aging.

N-nitroso compounds react with cellular DNA to produce various damaging adducts, one of the more important being O6-alkylguanine. DNA restoration is accomplished by transfer of the alkyl group to a cysteine residue of an acceptor protein. The levels of acceptor activity were compared in several tissues from well-fed and dietary-restricted inbred SD rats 30-1,194 days of age. Striking and consistent differences were found in the levels of acceptor activity in different tissues from both groups; these levels corresponded to their sensitivity to tumorigenesis by alkylating agents. Acceptor activity levels were highest in the liver and somewhat less in the spleen; there were significantly lower levels in brain and kidney. The random loss with time in the integrity of DNA may cause alterations in cellular function or limit cellular proliferation, thus leading to senescence and death. DNA repair processes may alter the rate of accumulation of damage, thereby affecting potential longevity. There were no significant age-associated changes in the ability of cells from either dietary group to remove DNA adducts and there was no evidence of alterations in the acceptor protein with age that would compromise its functional activity.

The Amazon molly, Poecilia formosa, as a test animal in carcinogenicity studies: chronic exposures to physical agents.

The Amazon molly, Poecilia formosa, a small, live-bearing fish native to Texas, has an unusual mode of reproduction that makes it highly suitable for carcinogenicity studies. The species is easy to rear and breed, and withstands handling well. Large broods of young are born, numbering up to 90 when the parent fish are fully grown. The Amazon molly reproduces gynogenetically; eggs are activated after mating with males of closely related species. The male makes no genetic contribution; sperm provides only the stimulus for egg development. The offspring of a female comprise a clone with genotypes identical to that of the mother's so that cell and tissue transplants made between members of the clone are not rejected. We used the Amazon molly as an animal model to show that damage to DNA caused by UV and ionizing radiation and by certain chemicals results in tumor development. Thyroid cells were taken from donor mollies, treated precisely in vitro with the agents, and then injected into homologous recipients. Eight months later, recipient fish had developed large thyroid tumors. The genetic homogeneity of the clone also makes the Amazon molly invaluable in acute or chronic bioassays because variability in the responses of the individual fishes is minimal. Therefore, a limited number will suffice to give statistically significant results. The findings from a study of chronic exposure to asbestos are also described.

Aging changes in the optic tectum of the guppy Poecilia (Lebistes) reticulatus.

Histological surveys of the brains of guppies throughout their lifespan showed no overall loss of tissue with advancing age. Brain and body increased in size at a similar rate throughout adult life in male fish. In old females after the age of two years, brain growth apparently ceased, although body growth continued at a low rate. In both sexes there was a loss of neurones from the stratum griseum periventriculare in the midbrain roof in old age; the decrease in size was significant. The midbrain is a major correlative center concerned with spatial orientation. How far neuronal loss may contribute to functional behavioral disorder in old age is unknown, but a loss of orientation could render the older fish more susceptible to predation.

Aging changes in the heart of a poeciliid fish, the guppy Poecilia reticulatus.

Histological surveys of the hearts of guppies Poecilia reticulatus throughout their lifespan showed no aging changes until the fish were three years old. In younger fishes the hearts were healthy, even in three individuals with "wasting" disease, where there was considerable loss of muscle throughout the body. There was a loss of muscle fibers in the ventricle in old fish, and deposition of collagen in the bulbus arteriosus. Guppies of three years and older also had a marked accumulation of melanomacrophages in the atrium. It seems unlikely that degeneration of the heart is involved in mortality of guppies in the wild.

Aging changes in the kidneys of two poeciliid fishes, the guppy Poecilia reticulatus and the Amazon molly P. formosa.

Histological surveys of the kidneys of two poeciliid fishes, the Amazon molly Poecilia formosa and the guppy P. reticulatus, throughout their lifespans showed no regular aging trends. Few kidney lesions were found in the Amazon molly until very late in life, 60 months or more, when obsolescent glomeruli and dilated renal tubules occurred. Guppies showed involutional changes of the renal system earlier, and the lesions became more severe with age, particularly in male fish. Hemopoietic tissue was reduced in amount in older fish of both species. Guppies of a year and older had marked accumulation of melanin in the melanomacrophage centers of the kidney, and the amount present increased with age. By contrast, there was little melanin deposition in mollies until almost the end of the lifespan. Hyaline droplets were consistently seen in the renal tubules of the mollies, but were rare in guppies. The degenerative changes in the kidneys of these two teleosts are similar to those seen in the kidneys of aging mammals. Despite the loss of normal structure in older fish, it seemed unlikely that degeneration of the kidney was directly involved in aging and death.

Photoreactivating enzyme activity in the rat tapeworm, Hymenolepis diminuta.

There has been considerable speculation about the occurrence of photoreactivating enzyme in different organisms and about its biological purpose. We have developed a simple, sensitive assay for estimating pyrimidine dimers in DNA which is useful in making a rapid survey for the presence of the enzyme. Using this method, we have found photoreactivating enzyme activity in the tissues of the rat tapeworm Hymenolepis diminuta. This parasite spends the majority of its life span in the bodies of its definitive or intermediate hosts, but a period is spent externally. We suggest that photoreactivating enzyme may be important in preserving the integrity of embryonic DNA during this free-living stage.