Vacuolation, proliferation and neoplasia in the liver of Boston Harbor winter flounder (Pseudopleuronectes americanus)

Abstract

Neoplasia has been found in the livers of bottom-feeding fish taken from heavily contaminated freshwater and marine habitats. This study examined the progressive development and ultimate diversity of liver neoplasia in winter flounder (Pseudopleuronectes americanus) from Deer Island Flats, Boston Harbor, MA., U.S.A., and encompassed histopathology, ultrastructural pathology, immunohistochemistry and experimental toxicology. It was found that liver neoplasia was most prevalent adjacent to a major sewage outfall, and that the predominant neoplastic cell type was cholangiocellular. Cholangiocellular neoplasms ranged from non-invasive tubular cholangiomas to invasive anaplastic cholangiocellular carcinomas. The latter were solid, tubular, cystic and scirrhous in form. Hepatocellular adenomas and carcinomas were also present, but only infrequently. Abnormally vacuolated hepatic epithelia were intimately associated with neoplastic lesions of all types. These vacuolated cells were first seen in the center of the hepatic tubule, as vacuolated preductular biliary epithelial cells. Later, cells of the entire hepatic tubule were vacuolated. Foci of vacuolated cells were visible grossly, and often contained or were adjacent to neoplastic lesions. Vacuolation, biliary hyperplasia, aggregation of macrophages and necrosis were first seen in two year old fish. These lesions then appeared to progress, becoming more severe and prevalent as the fish grew. Of the fish for which age data were available, the youngest fish to contain a liver neoplasm was 5 years old. Prevalence of neoplasia did not differ between gender of fish. Liver neoplasia and vacuolation persisted in fish that were maintained in clean water on clean food for five months. However, the prevalence of vacuolation decreased with increasing distance from urban contamination, being absent in fish from Georges Bank. Ultrastructural examination of winter flounder liver from clean and contaminated sites revealed a loss of hepatic glycogen and lipid stores with increasing environmental contamination, with a concomitant increase of abnormal proliferated endoplasmic reticulum (ER). Fluid accumulation in the cisternal space of the ER, and the perinuclear space and mitochondra led to vesicle formation. These vesicles coalesced, to form large cellular vacuoles that compressed the nucleus and residual cytoplasm to the margins of the cell. Vacuolation appeared to be a process that affected preductular cells, hepatocytes, cholangiocytes, neoplastic cells, and exocrine pancreatic cells. To assess the role of vacuolated cells in the progression to neoplasia, evidence for replicative nuclear DNA synthesis was sought by assaying for the nuclear incorporation of a nucleotide analog, bromodeoxyuridine (BrdU). Tissue sections from fish labeled with BrdU were stained imunohistochemically using an anti-BrdU monoclonal antibody. Constitutive DNA synthesis was observed in basal gill and intestinal epithelia, and renal hemopoietic cells. Increased levels of DNA synthesis were observed in…