Pigment Cells and Pigment Cell Tumors in Fish

Prince Masahito, Takatoshi Ishikawa, M.D., and Haruo Sugano, M.D. Departments of Experimental Pathology (PM,TI) and Pathology (HS), Cancer Institute, Tokyo, Japan.

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 nu­<;:leotide 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.] Invest Dermatol92:266S - 270S, 1989

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From a survey made on accumulated information con­cerning spontaneous fish tumors [1- 4], pigment cell tumors seem to be more common compared with those of mammals, including humans. According to the RTLA report (Registry of Tumors in Lower Animals at the

Smithsonian Institution, Washington, D.C.) [4), 30 (6.7%) pig­ment cell tumors were diagnosed from 447 tumors of bony fish collected and were most frequent next to 57 (12.8%) neurogenic tumors. These incidences were comparable to those for liver tumors (11.2%), hematopoietic tumors (8.7%), and kidney tumors (8.1 %). The underlying reason for the frequent occurrence of pigment cell tumors in fish is not yet known, but the fact that there are peculiar pigment cell neoplasms, that is, erythrophoromas and iridophor­omas, in fish that do not exist in mammals may be relevant in this context. The pigment cell tumors in fish include three main pheno­types: melanomas, erythrophoromas, and iridophoromas.

In Japan, goldfish and colored carp have been raised commer­cially in great numbers. These fish have been developed through repeated artificial matings for more than 200 years for their beauty. Japanese comparative oncologists are in a unique position to study tumors in such artificially selected fish species because of the numer­ous aquarium fish hobbyists in this country. Fishery is regarded as one of the most important food industries in Japan, and a large number of modern aquariums and hatcheries have been constructed.

This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science, and Culture, Japan.

Reprint requests to: Prince Masahito, Department of Experimental Pa­thology, Cancer Institute, 1-37-1, Kami-Ikebukuro, Toshima-ku, Tokyo, Japan.

Abbreviations: dopa: 3,4-dihydroxyphenylalanine H&E: hematoxylin and eosin RTLA: Registry of Tumors in Lower Animals at the Smithsonian Insti­

tution (Washington, D.C.)

Therefore, the information concerning the occurrence of neo­plasms in fish is easy to obtain.

In this review, the pathomorphology of pigment cell tumors will be described with the possible etiology of these tumors. Abnormal pigmentation in fish will also be mentioned in relation to some diseased conditions.

MATERIALS AND METHODS

The authors depended primarily on the following materials col­lected at the Cancer Institute, Tokyo, from various sources. These materials included 52 erythrophoromas in 38 goldfish (Carassius auratus) and 14 multicolored carp (CyprirlUs carpio); and 40 mela­nomas in 27 nibe croaker (Nibea mitsukurii), seven swordtail (Xi­phophorus helleri), four red medaka (Oryzias latipes), one bitterling (Aceilogllathus lallceolatus), and one chum salmon (Ollcorhynchus keta). Since there are no typical iridophoromas in our collections, the iridophoromas in fish are from the collection of R TLA at the Smithsonian Institution. However, the authors had an opportunity to study an iridophoroma in a nibe croaker having relation to multi­ple melanomas by electron microscopy. Some specimens, as mela­nomas in the nibe croaker, were obtained from natural habitats. Other specimens, as erythrophoromas in the goldfish and multicol­ored carp and melanomas in the red medaka and bitterling, were obtained from man-made habitats such as ponds, aquariums, or hatcheries.

RESULTS AND DISCUSSION

Pathomorphology of Pigment Cell Tumors in Fish

Erythrophoromas Among the fish pigment cell tumors, erythro­phoromas are the next most common, after melanomas. The eryth­rophoromas are neoplastic growths of erythrophores (red pigment cells), located in the dermis of fish, amphibians, and reptiles. When these cells include yellow pigments instead of red pigments, the term xanthophoromas is often used [5,6). However, here the term erythrophoromas will be used to conveniently describe such tumors

0022-202X/89/S03.50 Copyright ©1989 by The Society for Investigative Dermatology, Inc.

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Figure 1. Goldfish with a large erythrophoroma on the red part of the body. The tumor was orange to yellow (bar: 30 mm).

because there is no essential biologic difference between erythro­phoromas and xanthophoromas [5,6).

Single or multiple spontaneous erythrophoromas were found in the various parts of the body including eyes, head, operculum, flank, fins, and tail in 38 goldfish and 1 4 multicolored carp. The erythro­phoromas were found only in the red parts of the body of these fish (Fig 1 ). Tumors varied from 4 - 50 mm in maximum diameter; most protruded outward and lifted up the covering epidermis. They were deep red to orange in color when observed through the covering epidermis.

Histologically, dendric or spindle-shaped erythrophoroma cells were arranged as sheets or clusters with interlacing connective tissue bundles varying in amount from place to place. However, in con­ventional hematoxylin and eosin (H&E) section details of the pig­ment granules were not clear. The erythrophoroma cells often showed local invasion into muscle or adipose tissue. However, no distant metastases were observed in any fish examined. In some areas, tumor cells were often arranged in parallel rows with palisad­ing nuclei like human schwannomas (Fig 2); therefore, the present authors think that an accurate diagnosis of erythrophoroma requires the use of electron microscopy or a biochemical assay of cell pig­ments.

Ultrastructural observations revealed that the erythrophoroma cells were characterized by numerous large spherical cytoplasmic particles (0.4 - 0.7 11m in diameter) (Fig 3) [5,6)' which were identi­cal to the pterinosomes seen in normal erythrophores and regarded

Figure 2. Section of erythrophoroma in a goldfish, showing parallel rows with palisading nuclei like human schwannoma (bar: 200 )lm).

PIGMENT CELL TUMORS IN FISH 267S

Figure 3. Electron micrograph of erythrophoroma in a goldfish. Most cells are characterized by numerous spherical cytoplasmic particles (pterino­somes) (arrows) (bar: 5 flm).

as specific pigment granules [7). In addition to pterinosomes, many well-developed, tubular, smooth endoplasmic reticulum in the erythrophoroma cells were found. No basal laminae surrounding the tumor cells were observed.

Biochemical analysis using paper chromatography showed that the erythrophoroma cells contain several kinds of pteridines, such as n-hydroxybiopterin, isoxanthopterin, biopterin, sepiapterin, and 6-carboxypteridine [6). Their relative contents varied according to tumors. However, pteridines species in the erythrophoroma were almost identical to normal goldfish skin.

Mela/lOmas Melanomas are the most common pigment cell tumors and occur frequently in a wide range of fish species. The melanomas are considered to be melanocyte-derived neoplasms. In fish, mela­nocytes are located in the dermis [8). A few teleosts also have mela­nocytes in the epidermis [9). In fish, melanocytes are commonly found in viscera such as the intestine, kidney, and peritoneum and mesentery. Two melanomas of unusual body sites were recorded in the kidney and intestine of lungfish [4,10). The first record of pig­ment cell tumors in fish were two melanomas in a haddock (Melano­grammus aeglefinus) and an Atlantic cod (Gadus macrocephalus) by Prince and Steven [1 1 ) in 1 892.

The pathomorphology of melanomas is discussed by Dr. Kimura elsewhere in this volume. He studied melanomas extensively in nibe croaker living in shallow sea waters inJapan [12). As the pathology of fish melanomas has been dealt with in detail elsewhere [2,5,10-14) we will mention it only briefly here. Forty melanomas in five kinds of fish tumors were melanotic, but a few were amelanotic. Some were mixed-type melanotic melanoma and amelanotic mela­noma (Fig 4). Histologically, tumors were composed of dendritic or spindle-shaped tumorous melanocytes (Fig 5). The ultrastructure of fish melanoma has been described in detail [5,12,1 3). Many melan­osomes were densely aggregated in the main body of the cytoplasm and were also evident in the dendritic processes.

Iridophoromas The iridophoromas are rather rare, as compared with melanomas and erythrophoromas [4), and are neoplastic growths of iridophores. The iridophores are located in the dermis and are re­sponsible for the silver or blue iridescence so characteristic of fish coloration [15). These cells contain crystals of purines, mostly

268S MASAHITO, ISHIKAWA, AND SUGANO

Figure 4, Bitterling with a melanoma. Note pigmented and nonpigmented parts of tumors (bar: 20 mm).

guanine, which constitute the internal components of reflecting platelets [1 5J.

The first case of iridophoroma in fish was recorded in a greenling (Hexagrammos otakii) [16J. This tumor had a silver-white sheen. In all cases of iridophoromas, they were composed of spindle-shaped cells, as well as other kinds of pigment cell tumors (Fig 6A). The reflecting platelets could be found by due reflection in dark field under incident illumination on good preparation (Fig 6B). In the pigment cell tumors in nibe croaker, iridophoromas were often found associated with multiple melanomas [12J. Electron micro­scopic examination of the tumors in nibe croaker showed the exis­tence of rod-shaped reflecting platelets in the cytoplasm, which were 0.1- 0.2 fJ.m wide and 0.5 - 2.0 fJ.m long [5,12J.

The Etiology of Pigment Cell Tumors Generally speaking, the etiology of pigment cell tumors in fish is obscure, similar to mammalian melanomas. The pigment tumors may occur ill combi­nation with hereditary, carcinogenic, and aging factors. First, as Reed and Gordon [17J and other researchers [1 8, 19J reported, mela­nomas in platyfish/swordtail hybrids are strongly determined ge­netically. From a series of systematical studies, melanomas have been considered to develop as follows. The acti vity of the color gene located on the X chromosome is controlled by a group of modifying genes. Repeated backcrossing of the swordtail/platyfish hybrids with the swordtail successively reduces the number of modifying genes, which permits unlimited proliferation of melanocytes. The color gene has been thought to be an oncogene and was designated

Figure 5. Section of melanoma in a bitterling. The tumor was composed of spindle-shaped amelanotic cells interspersed with melanotic cell foci (bar: 10 Jim).

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Figure 6, A: Section of an iridophoroma in a freshwater drum, showing irregularly shaped cell foci bearing cytoplasmic accumulation of reflecting platelets (H&E, bar: 200 .um). B: The same field as A taken under incident illumination (bar: 200 .um).

as a tumor gene by Anders and his co-workers [19J. The ras gene of goldfish was first cloned from normal liver DNA, and the nucleo­tide sequences were analyzed by Nemoto of our group [20J. Com­parison of nucleotide and amino-acid sequences between the ras gene from normal goldfish liver DNA and H-ras, K-ras, and N-ras gene from human tissue revealed surprising homology. This was especially true in the goldfish ras gene and human K-ras gene, where 96% homology in amino-acid sequence was seen. The homology between the goldfish ras gene and human H-ras and N-ras gene in amino-acid sequences was 87% and 88%, respectively [20J. In mammalian ras gene from cancerous tissue, point mutation at the 1 2th or 61 st codon was often observed. Then the point mutations of ras gene from erythrophoromas were examined using the perma­nent cell lines [21 J from goldfish erythrophoromas established at our laboratory. There was no point mutation at the 1 2th codon of the erythrophoromas ras gene [22J. However, our examination was directed at only the first exon, and thus it is not clear at present whether activated oncogene is involved in the development of erythrophoromas in goldfish.

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Figure 7. Right-eye flounder with a jet-black pseudotumor in the depig­mented side (bar: 50 mm).

Second, several researchers were able to induce melanomas in fish by treatment with chemical carcinogens [12,23]. Experimental in­duction of melanomas by carcinogens using nibe croaker was ac­complished by Kimura [12]. Hyodo-Taguchi and Matsudaira [23] reported the induction of transplantable melanomas using inbred strains of medaka. These medaka were exposed in aquarium water containing N-methyl-N'-nitro-N-nitrosoguanidine for only 2 h. The tumors were amelanotic melanomas with positive dopa reac­tion.

From a study on the relatively large numbers of erythrophoromas in goldfish [5,6], the present authors suspected that erythrophor­OIllJS might predominantly affect fish more than three years old. This problem was investigated more specifically by Etoh and co-

Figure 9. Electron micrograph of pseudo­tumor in a right-eye flounder showing X cells with round pale nuclei and distinct nu­cleoli (har: 5 pm).

PIGMENT CELL TUMORS IN FISH 269S

Figure 8. Section of pseudotumor in a right-eye flounder. Tumor consisted of proliferation of so-called X cells within the epithelium. The X cells had poorl\' stained large, round nuclei. Note numerous melanocytes in the stroma (bar: 50 pm).

workers [24]. They examined large numbers of goldfish stocks, which were separated chronologically in ponds from the first to the twelfth year of life. Tumors developed at five years or older, and the incidence increased with age and finally attained a level of about 60% in the oldest stock in combination with possible environmental factors, such as chemical contaminants in the water or ultraviolet

270S MASAHITO, ISHIKAWA, AND SUGANO

light. However, no one succeeded in the induction of pigment cell tumors in fish by ultraviolet irradiation.

Abnormal Pigmentation in Fish Besides pigment cell tumors, abnormal pigmentation or depigmentation was often observed in right-eye flounders and bastard halibut.

Many right-eye flounders with single or multiple pseudo tumors are widely distributed between the coast of Japan and California through the Bering Sea [25]. The features of these pseudotumors will be described, based on our collections including two kinds of right-eye flounders (Pleuronectes schrenki and P. obscura). Grossly, pseudotumors showed mostly-black papilloma-like lesions in the skin, which were even present in the depigmented side (Fig 7). Histologically, abnormal pigmentation was due to an increased number of melanocytes which were observed only in the stroma of the pseudotumors. However, the main component of pseudotumors in the right-eye flounders consisted of proliferation of so-called X cells [26] within the epithelium (Fig 8). X cells had poorly stained, large, round nuclei. Round nuclei having distinct nucleoli were found in the X cells by electron microscopy (Fig 9). In several respects, X cells were different from host epithelial cells in the fish [26,27]. From the unique cell-division pattern, Dawe [27] reported that this pseudotumor might be caused by the parasitic infection of amoebas.

Abnormal depigmentation was found in many young bastard hal­ibut (Paralichthys olivaceus) raised in Japanese hatcheries [28]. The abnormal irregular depigmentation was considered to be caused by an imbalance of nutrition under artificial feeding.

We are gratqul to Dr, John c. Harshbarger and his staff, Registry of Tumors in Lower Animals (RTLA), to the Smithsonian Institution, Washington, D,C., for allowing us to use the iridophoroma specimen, and to Miss Tomoko Toyama and Mr, Hironori Murayama for their assistance in the preparation of this manuscript,

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