John C. Harshbarger

Tumor prevalence and biomarkers of exposure and response in brown bullhead (Ameiurus nebulosus) from the Anacostia River, Washington, DC and Tuckahoe River, Maryland, USA

We evaluated liver and skin tumor prevalence and biomarkers of exposure and response in brown bullhead (Ameiurus nebulosus) from three locations in the Anacostia River (Washington, DC, USA), a Chesapeake Bay region of concern. The Tuckahoe River (Maryland, USA) served as a reference. Each river was sampled in fall 2000 and spring 2001. In the Anacostia, prevalence of liver tumors was 50 to 68%, and prevalence of skin tumors was 13 to 23% in large (> or = 260 mm, age > or = 3 years) bullheads. Liver and skin tumor prevalence was 10 to 17% and 0%, respectively, in small (150-225 mm, age 1-2 years) bullheads. Tuckahoe bullhead liver tumor prevalence was 0 to 3% (large) and 0% (small); none had skin tumors. Biliary polynuclear aromatic hydrocarbon (PAH)-like fluorescent metabolites and liver DNA adduct concentrations were elevated in large and small Anacostia bullheads. Mean adduct concentrations were 16 to 28 times higher than those in Tuckahoe fish. Chromatograms revealed a diagonal radioactive zone, indicating polycyclic aromatic compound (PAC)-DNA adducts. The biomarker data and the 10 to 17% liver tumor prevalence at ages 1 to 2 suggest that these year classes are likely to have a high prevalence as they reach age 3 and older. This study provides the strongest evidence to date of the role of PAHs in tumor development in Anacostia bullheads.

A Proposed Classification Scheme for Xiphophorus Melanomas Based on Histopathologic Analyses

Abstract: 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.

Histological Assessment of Goiters in Elasmobranch Fishes

Abstract Twelve thyroid glands from free-living (n = 3) and captive (n = 9) elasmobranch fishes were investigated. Histological observation of the thyroids revealed two euthyroid glands, one case of mild chronic thyroiditis, three diffuse hyperplastic goiters, three diffuse colloid goiters, and three multinodular colloid goiters. The term goiter best describes the thyroid enlargement that results from hyperplasia and hypertrophy. Although elasmobranch goiters are typically linked to low aquatic iodine concentrations, iodine deficiency alone does not explain all of the observed thyroid conditions. Diffuse hyperplastic goiters typically result from either iodine deficiency or goitrogenic release of thyroglobulin from the follicle. Colloid goiters result from fluctuating iodine concentrations or goitrogenic agents that block the release of iodine from the thyroid gland. Although nitrate is a potential goitrogenic agent in aquaria, research indicates that nitrate interferes with the uptake and retention of io...

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.

Cutaneous mastocytomas in the neotenic caudate amphibians Ambystoma mexicanum (axolotl) and Ambystoma tigrinum (tiger salamander)

Abstract Spontaneous mastocytomas studied in 18 axolotls (Ambystoma mexicanum) and six tiger salamanders (Ambystoma tigrinum) were gray-white, uni- to multilobular cutaneous protrusions from 2 mm to 2 cm in diameter. Tumors were moderately cellular unencapsulated masses that usually infiltrated the dermis and hypodermis with the destruction of intervening tissues. Some tumors were invading superficial bundles of the underlying skeletal muscle. Tumors consisted of mitotically active cells derived from a single lineage but showing a range of differentiation. Immature cells had nearly smooth to lightly cleft or folded basophilic nuclei bordered by a band of cytoplasm with few cytoplasmic processes and containing a few small uniform eccentric granules. Mature cells had basophilic nuclei with deep clefts or folds and abundant eosinophilic cytoplasm with multiple long intertwining cytoplasmic extensions packed with metachromatic granules. The axolotls were old individuals from an inbred laboratory colony. The tiger salamanders were wild animals from a single polluted pond. They could have been old and inbred. Both groups were neotenic. These are the first mastocytomas discovered in cold-blooded animals.

Renal Adenocarcinoma in Mozambique Tilapia, Neurofibroma in Goldfish, and Osteosarcoma in Channel Catfish from a Puerto Rican Hatchery

Abstract A renal adenocarcinoma in a Mozambique tilapia Tilapia mossambica, neurofibromas in several goldfish Carassius auratus, and an osteosarcoma in a channel catfish Ictalurus punctatus were found at a fish hatchery in Puerto Rico. Both osteosarcomas and renal adenocarcinomas are rare in fish and have not been previously reported in these species. No carcinogenic pollutants are known to be contaminating the water or feed used by this hatchery, and no relationships between these tumors were found except for their occurrence at the same hatchery.

Tumor Prevalence in Mummichogs from the Delaware Estuary Watershed

Mummichogs Fundulus heteroclitus were collected in 2002-2004 from six locations within the Delaware Estuary watershed and examined histopathologically. The objective was to compare the prevalence of skin and liver tumors in fish from locations with a range of sediment polynuclear aromatic hydrocarbons (PAHs) based on available data. Fish were collected from (1) three locations within the industrialized Christina River subwatershed (Hershey Run, Newport Marsh (upriver and downriver)); (2) one location in the Motiva Enterprises refinery discharge canal to the Delaware River; (3) one location in the St. Jones River; and (4) one location in Blackbird Creek. Adult mummichogs (≥70 mm; n = 21-30 fish per location) were seined and held for necropsy. No neoplastic skin lesions were diagnosed in any fish. No liver tumors were diagnosed in the St. Jones, Motiva, Newport Marsh upriver, or Newport Marsh downriver collections. One of 30 Blackbird Creek mummichogs had foci of hepatocellular alteration (a putative preneoplastic lesion) and one had hepatocellular carcinoma. There was a significant difference in hepatocellular carcinoma prevalence in Hershey Run mummichogs in 2002 (9 of 21 fish, or 43%) and in 2003 (3 of 29 fish, or 10%) compared with all other locations pooled (1 of 145 fish, or 0.7%). Hershey Run is contaminated with creosote from an adjacent Superfund site, where sediments were frequently reported to have total PAH concentrations greater than 100 mg of total PAH/kg and a maximum of 13,300 mg/kg. No other locations had reports of total PAHs higher than 11 mg/kg. In conclusion, liver neoplasia was associated with exposure to sediment containing high concentrations of PAHs derived from creosote. Studies that include tumor prevalence, biomarkers (e.g., DNA adducts and PAH metabolites in bile), and sediment and/or tissue chemistry are recommended to develop a weight of evidence for specific chemical classes.

Suprasellar germinoma in three lake whitefish (Coregonus clupeaformis)

Abstract Suprasellar germinomas were identified in three wild-caught lake whitefish (Coregonus clupeaformis) from the St. Lawrence River, Quebec, Canada. Histologically, the three tumors expanded the subarachnoid space of the ventral surface of the brain immediately adjacent to the pituitary gland and, in one case, infiltrated the adjacent neuropil. These tumors were characterized by nests and sheets of round cells with a high mitotic rate, separated by a scant amount of loose fibrovascular stroma. The stroma was infiltrated by a moderate number of small mononuclear cells, including rare CD3-immunoreactive lymphocytes. This is the first report of intracranial germinoma in a fish species.

Granulosa Cell Tumor in a Farm-Reared Channel Catfish

Abstract Multiple white-to-tan, solid, nodular masses were present in the abdominal cavity of a subadult female farm-reared channel catfish Ictalurus punctatus. The masses ranged in size from pinpoint to approximately 1 cm in diameter and were present on the ovaries, liver, spleen, posterior kidney, serosal surface of the intestine, and peritoneal adipose tissue. Histologically, the masses consisted of sheets and packets of large (approximately 12.5–10 × 10–7.5 μm) polyhedral cells with abundant foamy cytoplasm arranged in cords and short interweaving bundles and separated by fine fibrovascular tissue. The nuclei were small to medium in size, round, and pale, often having a distinct nucleolus and rare mitotic figures. The widespread ovarian involvement together with cytological and pattern features is most consistent but not definitive for a gonadal stromal cell tumor (granulosa cell tumor).