Cutaneous Chromatophoromas in Four Species of Australian Elapid Snake.

This report documents the clinicopathological features of cutaneous chromatophoromas in four wild-caught, captive Australian elapid snakes: a strap-snouted brown snake (Pseudonaja aspidoryncha), a tiger snake (Notechis scutatus), an Eastern brown snake (Pseudonaja textilis) and a Mengden's brown snake (Pseudonaja mengdeni). All tumours were subclassified as melanophoromas, with three assessed as malignant on the basis of invasive growth or presence of intracoelomic metastases. The chromatophoromas were single or multiple, black or orange pigmented, cutaneous, sometimes ulcerated, plaques or nodules. Microscopically, the neoplastic cells were often spindle shaped with low or variable pigmentation. Neoplastic cells in one tumour were notable for their pleomorphic round cell morphology and high mitotic rate. One snake with late-stage metastasis survived for over 5 years. There are few reports of chromatophoromas in elapid snakes and, to our knowledge, this is the first report of these tumours in Australian elapid snakes.

Immunohistochemical analysis of pigment cell tumors in two cyprinid species.

Pigment cell tumors, also known as chromatophoromas, are cutaneous spindle cell neoplasms originating from pigment cells (chromatophores) in the dermis of teleosts, amphibians, and reptiles. Chromatophoromas share similar histologic morphology to other spindle cell tumors and are not always pigmented. Therefore, immunohistochemical analysis may be useful in distinguishing these neoplasms from tumors of other cellular origin when poorly pigmented. We performed 3 immunohistochemistry assays (PNL-2, melan A, and SOX10) on 8 cutaneous neoplasms from 8 teleosts diagnosed as chromatophoromas based on histologic morphology. Semiquantitative analysis of immunoreactivity was evaluated on each immunohistochemical assay using a 0-3 scale. PNL-2 exhibited mild-to-moderate (1 or 2) immunoreactivity in 7 of the cases, and resident chromatophores (internal control) were also immunoreactive in these cases. Melan A exhibited mild-to-moderate (1 or 2) immunoreactivity in 4 cases (and with resident chromatophores in these cases); SOX10 was not immunoreactive in any cases. Our results indicate that PNL-2 may be a useful marker in teleosts to distinguish tumors of chromatophore origin. Melan A could also be useful, but appears to be less sensitive, and SOX10 is likely not a useful marker for these neoplasms in teleosts.

Cutaneous Chromatophoromas in Captive Snakes.

Chromatophoromas are neoplasms arising from pigment-bearing cells (chromatophores) of the dermis. While isolated cases have been reported in the literature, the prevalence and biological behavior of chromatophoromas in snakes are unknown. Forty-two chromatophoromas were identified among 4663 submissions (0.9%) to a private diagnostic laboratory in a 16-year period. The most commonly affected snakes were colubrids (23 cases, 55%) and vipers (8 cases, 19%). The San Francisco garter snake was the most commonly affected species (6 cases; 14% of all affected snake species and 3.7% of all garter snake submissions). No sex predilection was found. The age of 28 snakes ranged from 5 to 27 years. Single cutaneous chromatophoromas were most commonly observed and presented as pigmented cutaneous masses or plaques along any body segment. Euthanasia or death due to progressive neoplastic disease or metastasis was reported in 8 (19%) and 4 (10%) cases, respectively. The survival time of 4 animals ranged from 4 to 36 months. Microscopically, xanthophoromas, iridophoromas, melanocytic neoplasms, and mixed chromatophoromas were identified, with melanocytic neoplasms being most common. Microscopic examination alone was generally sufficient for the diagnosis of chromatophoroma, but immunohistochemistry for S-100 and PNL-2 may be helpful for diagnosing poorly pigmented cases. Moderate to marked nuclear atypia appears to be consistently present in cutaneous chromatophoromas with a high risk of metastasis, while mitotic count, lymphatic invasion, the level of infiltration, and the degree of pigmentation or ulceration were not reliable predictors of metastasis.

HISTOLOGIC, IMMUNOHISTOCHEMICAL, AND ELECTRON MICROSCOPIC CHARACTERIZATION OF A MALIGNANT IRIDOPHOROMA IN A DWARF BEARDED DRAGON (POGONA HENRYLAWSONI).

A dwarf bearded dragon (Pogona henrylawsoni) was presented with a white subcutaneous mandibular mass and multiple nodules in the oral mucosa, heart, liver, kidney, intestine, and visceral fat. Histologically, the tumor consisted of densely packed spindle-shaped cells with brow intracytoplasmic pigment that exhibited white-blue birefringence with polarized light. Immunohistochemical staining was negative for S-100 and weakly positive with melan A. Electron microscopic examination revealed cytoplasmic irregular and oblong empty spaces, laminated and often arranged into short stacks, compatible with reflecting platelet profiles typically seen in iridophores. However, in unstained ultrathin sections, electron-dense crystalline material was present, which filled the empty spaces described for stained sections before. Based on histology, immunohistochemistry, and biologic behavior, a malignant iridophoroma was diagnosed. To the authors' knowledge, iridophoromas in lizards have rarely been characterized by using electronic microscopy. Moreover, this is the first description of an iridophoroma in a dwarf bearded dragon.

Melanophoromas and iridophoromas in reptiles.

Chromatophoromas are tumours of pigment-producing cells of the skin and are rarely reported in reptiles. These tumours are subclassified on the basis of the type of pigment. The present study characterizes chromatophoromas arising in 26 reptiles, including six snakes, 19 lizards and a tortoise. These include the first reports of melanophoromas in a yellow anaconda (Eunectes notaeus), pigmy rattlesnake (Sistrurus spp.), southern water snake (Nerodia fasciata), veiled chameleon (Chamaeleo calyptratus) and leopard gecko (Eublepharis macularius); the first reports of benign iridophoromas in a savannah monitor (Varanus exanthematicus), veiled chameleon and bearded dragon (Pogona vitticeps); and the first description of a malignant iridophoroma in a bearded dragon. Additionally, in three bearded dragons a 'mucinous' type of melanophoroma is described for the first time. Chromatophoromas generally arose from the skin of the body and head and ranged in size from 0.2 to 2.0cm in diameter. In six cases the animals were humanely destroyed immediately after diagnosis. Three further animals were humanely destroyed following recurrence of their tumour. Six of these nine reptiles had visceral metastases. Grossly, melanophoromas (n=20) were grey or black, while iridophoromas (n=6) were white in colour. Microscopically, most of the tumours were composed of spindle cells with varying pigmentation and 0-2 mitoses per 10 high power fields. Six of the 20 melanophoromas were classified as malignant due to the presence of intravascular tumour cells, visceral metastases, high pleomorphism and/or mitotic figures. Five of the six iridophoromas were classified as benign and the one malignant tumour was defined by the presence of intravascular tumour cells and visceral metastases. Immunohistochemically, melan A and S100 were coexpressed by all of the chromatophoromas.

Chromatophoroma in a crevice kelpfish (Gibbonsia montereyensis).

A captive adult crevice kelpfish, Gibbonsia montereyensis, developed a cutaneous mass, approximately 9 × 7 mm on the right side of the head in an area of nonscaled skin. Following surgical debulking, examination of both impression smears and histologic sections of the tumor revealed a predominant population of round to spindloid to polygonal cells with a moderate amount of lightly basophilic cytoplasm. The cytoplasm was filled with round, variably-sized reddish-brown granules that often obscured the nucleus. Nuclei were round to ovoid with coarsely granular chromatin. There was minimal anisocytosis and anisokaryosis. The cytoplasmic granules in histologic sections were weakly positive by the Fontana-Masson method, and staining was eliminated with melanin bleach. Immunohistochemical staining was strongly positive with a murine monoclonal antibody for melan A. As the specificity of melan A for melanophores is not clearly defined in nonmammalian species, the tumor was examined by transmission electron microscopy. Melanophores were not detected. Instead, neoplastic cells were filled with numerous intracytoplasmic organelles with triple-limiting membranes composed of concentric lamellae; these structures were most compatible with pterinosomes, which are the pigment-containing organelles of cells called xanthophores and erythrophores. As both of these organelles are ultrastructurally indistinguishable and as kelpfish skin is known to contain both xanthophores and erythrophores, a diagnosis of a mixed pigment cell tumor or chromatophoroma was made. As the tumor was grossly reddish-brown, the possibility of a neoplastic population of only erythrophores could not be excluded. Pigment cell tumors, arising from cells of the embryonic neural crest, are common in reptiles and bony fish.

Diagnosis and clinical management of multiple chromatophoromas in an eastern yellowbelly racer (Coluber constrictor flaviventris).

A wild-caught subadult eastern yellowbelly racer (Coluber constrictor flaviventris) presented with three raised, circumscribed, freely moveable subcutaneous masses over the dorsal and right epaxial musculature. Microscopic examination and special staining of surgically excised masses were diagnostic for chromatophoroma. No metastases were apparent on whole-body plain film radiographs and computed tomography. Additional excisional biopsies of previously affected sites performed 13 wk later demonstrated normal skin and subcutis. The snake recovered uneventfully and continued to do well 18 mo later. Surgical excision of chromatophoromas remains the treatment of choice in snakes. Subcutaneous masses in snakes should be thoroughly investigated.

The yellow mutation in the frog Rana rugosa: pigment organelle deformities in the three types of chromatophore.

Crossing experiments revealed that a single recessive gene mutation (yellow) gives rise to the yellow phenotype of Rana rugosa in Japan. Ultrastructural observation of dermal chromatophores showed that the pigment organelles; melanosomes, pterinosomes, and reflecting platelets, all had structural deformities. This suggests that the yellow gene acts at the level of a primordial pigment organelle common to the three types of chromatophore.

Effect of water extract of the bark of Buchanania lanzan linn. on behaviour and chromatophores of a fresh water fish, Labeo rohita.

Fish exposed to 150 ppm and 250 ppm doses saparately, showed eratic swimming with hyper-excitability, spiralling, convulsion and mortality. However at 350 ppm dose fish became lethargic and steady at the bottom of aquarium. This behaviour appears to be due to sedative effects of fish implicating the phychoactive nature of the plant B. Ianzan. At 150 ppm to 350 ppm doses, fish scale changed from reticulate to punctate chromatophores; and treated fish when transferred to fresh water, were slowly regained to normal behaviour after 96 h exposure. Hence doses regaining from 150 ppm to 350 ppm is considered to be psychoative in nature. In higher doses 450 ppm and 550 ppm fish could survive only 76 h and 4.30 h respectively and then died indicating toxicity of plant for the said doses. It is found that scale present on its body is most suitable test system for the study of psychoactivity and toxicity of plant extracts on fish, Labeo rohita.

Zebrafish pigmentation mutations and the processes of neural crest development.

Neural crest development involves cell-fate specification, proliferation, patterned cell migration, survival and differentiation. Zebrafish neural crest derivatives include three distinct chromatophores, which are well-suited to genetic analysis of their development. As part of a large-scale mutagenesis screen for embryonic/early larval mutations, we have isolated 285 mutations affecting all aspects of zebrafish larval pigmentation. By complementation analysis, we define 94 genes. We show here that comparison of their phenotypes permits classification of these mutations according to the types of defects they cause, and these suggest which process of neural crest development is probably affected. Mutations in eight genes affect the number of chromatophores: these include strong candidates for genes necessary for the processes of pigment cell specification and proliferation. Mutations in five genes remove part of the wild-type pigment pattern, and suggest a role in larval pigment pattern formation. Mutations in five genes show ectopic chromatophores in distinct sites, and may have implications for chromatophore patterning and proliferation. 76 genes affect pigment or morphology of one or more chromatophore types: these mutations include strong candidates for genes important in various aspects of chromatophore differentiation and survival. In combination with the embryological advantages of zebrafish, these mutations should permit cellular and molecular dissection of many aspects of neural crest development.

Chromatophoromas and chromatophore hyperplasia in Pacific rockfish (Sebastes spp.).

Pacific rockfish from Cordell Bank, off central California (United States), were collected and histologically examined from 1985 to 1990. Hyperplastic and neoplastic cutaneous lesions, involving dermal chromatophores, were observed in five species; yellowtail rockfish (Sebastes flavidus), bocaccio (S. paucispinis), olive rockfish (S. serranoides), widow rockfish (S. entomelas), and chilipepper rockfish (S. goodei). Yearly prevalences were highest in S. paucispinis (29-38%). Prevalence was initially low in S. flavidus, but increased more than 3-fold from 1985 (7.5%) to 1990 (25%). The majority of lesions were black, but white, yellow, orange, red, and mixed-color variants were also seen. Lesions were found in skin, fins, lips, gingiva, tongue, urogenital papilla, conjunctiva, and cornea of the eye. Flat lesions were consistent with melanophore (black), xanthophore (yellow or orange), and erythrophore (red) hyperplasia. Neoplastic lesions included melanophoromas, amelanotic melanophoromas, xanthophoromas, erythrophoromas, and mixed chromatophoromas. Although etiology has not been determined, interest is currently focused on potential exposure to chemical and radioactive carcinogens from the Farallon Island Radioactive Waste Dump, 30 km to the south.

Appearance of tumorous phenotypes in goldfish erythrophores transfected with ras, src, and myc oncogenes and spontaneous differentiation of the transformants in vitro.

When goldfish erythrophores isolated from the skin by tissue digestion and centrifugation in a Percoll density gradient were transfected in a monolayer-culture with v-Ha-ras or v-src oncogene either singly or in combination with v-myc by means of calcium phosphate-DNA co-precipitation, there appeared a certain number of transformants manifesting a chromatoblast-like profile and tumorous phenotypes as seen in the capability for unlimited growth, and piling-up in a monolayer-culture or colony formation in semi-solid soft agar. After successive growth in vitro for longer than one month which was scarcely observed with the erythrophores, the vast majority of such transformants began to differentiate into erythrophores and ceased proliferation spontaneously. The onset of their differentiation was ascertained by the deposition of marker pteridine pigments. None of the transformants differentiated into melanophores or iridophores or other neural crest derivatives as seen in goldfish erythrophoroma cells. Little difference was observed in their transforming efficiency (0.2-0.3 transformants/micrograms DNA) between the combinations of oncogenes applied but a tendency was noted that cells transfected with ras or src in combination with myc developed the capacity to grow for a longer period and differentiated at a later stage than those transfected solely with ras or src. One cell line (ESM-1) derived from the erythrophores transfected with src/myc grew successively over nine months, indicating its acquisition of immortality. The expression of the transfected oncogenes in this cell line was examined in comparison with the erythrophoroma cells by Western and Northern blot analyses.

Pigment cells and pigment cell tumors in fish.

The three basic pigment cell types found in poikilothermic vertebrates, melanocytes (melanin-producing cells), erythrophores (red or yellow pigment cells), and iridophores (iridescence-producing cells), are derived from neural crest. Neoplasms of pigment cells in fish are also of three phenotypes, melanomas (melanophoromas), erythrophoromas, and iridophoromas, showing the phenotypes of their corresponding normal pigment cells. These pigment cell tumors are among the most common types in bony fish and seem to be more common in fish than in mammals, including humans. Moreover, there are no mammalian neoplasms corresponding to erythrophoromas and iridophoromas in fish. The complexities in the nature and classification of pigment cell tumors in fish will be discussed on the basis of a survey of our collection of these tumors at the Cancer Institute. The etiology of pigment cell tumors in fish is obscure. In order to know whether activated oncogene is involved in the genesis of erythrophoromas in goldfish, the ras genes from normal and erythrophoroma cells were cloned and their nucleotide sequences were compared. The goldfish ras gene and human ras genes showed striking homology. However, no point mutation at the 12th codon was observed in ras genes isolated from erythrophoromas. Besides pigment cell tumors in fish, abnormal pigmentation or depigmentation in flounders associated with diseased conditions is also described.

Environment: peculiar pigment cell neoplasm in fish.

Chromatophoroma in the croaker (Nibea mitsukurii) showed a unique geographic distribution. The contribution of environmental chemicals to the cause of chromatophoroma in the feral croaker is considered likely on the basis of the following results in our studies. 1) Chromatophoroma was induced in tank-reared N. mitsukurii by administration of certain kinds of known carcinogens such as 7,12-dimethyl-benz(a)anthracene, N-methyl-N'-nitro-N-nitrosoguanidine, and nifurpirinol. 2) Local accumulation of pigment-cell hyperplasia in the catfish (Protosus anguillaris) showed similar tendencies to those of chromatophoroma in N. mitsukurii. 3) Removal of contaminated sediment from the harbor and the river appeared to reduce the incidence from 47% in 1973-1983 to about 20% in 1985-1987. 4) Waste water from a factory located at the station where the incidence of the neoplasm was the highest contained mutagenic substances such as chloroacetones and glyoxals [5]. Exposure of catfish to the waste water induced pigment-cell hyperplasia on the skin.

Morphologic and biochemical characterization of erythrophoromas in goldfish (Carassius auratus).

The properties of spontaneous cutaneous erythrophoromas that occurred in 17 adult goldfish (Carassius auratus) were described. This study was based on combined histology, cytochemistry, electron microscopy, and biochemical analysis. Tumors that varied from 0.4 to 2.7 cm in maximum diameter were located on various parts of the skin. As shown by light microscopy, tumors were composed of red- to orange-pigmented, dendritic, stellate, or spindle-shaped cells, which coalesced into an excrescence in the dermal layer. No metastasis was observed. Evidence of invasiveness in five examples and local recurrence in three examples indicated that some of these tumors were malignant. Electron microscopy revealed numerous round cytoplasmic organelles (pterinosomes), which measured 0.4--0.7 mu, and well-developed tubular, smooth endoplasmic reticulum in the tumor cells. Biochemical analysis showed that red pigmentation of tumor cells was imparted by pteridines and carotenoids, most of which were detectable in normal adult erythrophores. These data indicate that tumors were comprised of neoplastic erythrophores.