Date of Award

1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Co-H. Wayne Taylor

Second Advisor

Jay C. Means

Abstract

A need exists for fish sentinel species for monitoring environmental changes. These studies were performed to examine the suitability of western mosquitofish (Gambusia affinis) as a sentinel species and to explore basic mechanisms of hepatocarcinogenesis. Neoplastic lesions were induced in laboratory-reared mosquitofish fry and adults using single pulse exposures to 1 or 10 ppm methylazoxymethanol acetate. Early changes in the liver of exposed fish included severe cytotoxicity, megalocytosis, and widespread apoptosis. Neoplastic changes were found in the liver in approximately 20% of the exposed fish by 8 weeks after exposure and increased to approximately 70% by 41 weeks. These included foci of cellular alteration, hepatocellular carcinomas and cholangiocellular carcinomas. There was also significant incidence of a spindle cell tumor, believed to be of hepatobiliary stem cell origin. Tumors were found in less than 1% of controls. Immunohistochemistry was performed on histologic sections of the fish from the histopathology study. Specimens were stained for proliferating cell nuclear antigen (PCNA, PC10). Significantly increased cell proliferation detected in liver cell populations of the high dose groups at early time points was attributed primarily to regenerative hyperplasia, and at later time points mainly to preneoplastic and/or neoplastic cell proliferation. Additional sections of the same fish specimens were stained for p53 protein. Overexpression of p53 was detected as early as 48 hours after exposure and in approximately 38% of the tumors. However, at the early sampling points, p53 immunohistochemistry did not appear to have good predictive value of later tumor formation. Levels of the DNA adducts O$\sp6$-methylguanine and 7-methylguanine were measured in the liver in additional alkylating agent exposures using stable isotope dilution-GC/mass spectrometry. Adapted for small fish species, this assay gave detection limits of approximately 1.6 femtomoles per microgram of DNA, or about 1 adduct per 2 million DNA bases. Levels of O$\sp6$-methylguanine ranging from approximately 55-185 picograms per microgram DNA, measured in the first 72 hours after exposure, were correlated with a 33% liver tumor incidence after 25 weeks in parallel histopathology studies. The carcinogen sensitivity, ease of laboratory culture, biology, and wide range of Gambusia affinis appear to indicate excellent potential for its use as a warmwater sentinel species.

Pages

183

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