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 vacuolated cells, hyperplastic biliary epithelia, and most particularly in neoplastic cells, some of which were vacuolated. These observations were taken to suggest that vacuolated cells were capable of DNA synthesis, and that this, along with their intimate spatial relationship with neoplastic cells implied that they may be involved in the progression to neoplasia. To further investigate these observations, attempts were made to recreate the feral disease in the laboratory. Methods were developed for atraumatic capture, transport and year round maintenance of winter flounder. Long term colonies were established and experiments designed to reproduce the situation in wild-caught fish. The long latency between first exposure of larvae to genotoxic carcinogens in the native fish from Boston, and the actual appearance of neoplasia many years later, leads to the assumption that chronic exposure to epigenetic carcinogens was the rate limiting step in this neoplastic progression. Technical grade chlordane was chosen as representative of the hepatotoxic epigenetic carcinogens present in Boston Harbor sediments. Acute and subacute exposures were conducted, to establish the toxicity of the fish to chlordane, and to examine the resultant histopathology. A chronic feeding study was then conducted for one year, using chlordane and benzo(a)pyrene. Histological alterations induced in treated fish included elevated levels of macrophage aggregations, perisinusoidal edema, necrosis, and a proliferative reaction that involved the formation of structures that were apparently primitive biliary tubules. These studies have shown that winter flounder exposed to chemical contaminants appear to undergo a set of histopathological changes that precede neoplastic change. Cellular vacuolation is a significant change that may be directly involved in the progression to neoplasia. It is a relatively common lesion and is an excellent marker in winter flounder for the detection of the chronic biological effects of the particular chemical contaminants in the Boston Harbor environment, at a stage long before overt neoplasia is evident.