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The overall objective of this research was the development of closed systems previously devised in our laboratory to assay the effects of chemical carcinogens on marine teleosts. The results include the following: The LC-50 (96 hrs) for benzidine dihydrochloride (BEN) with respect to Cyprinodon variegatus (sheepshead minnow) was determined to be 64 ppm. Exposure of C. variegatus weekly to contaminations of 1 ppm BEN resulted in some individuals developing liver lesions at 25-29 weeks. The livers of these individuals contained large fibrotic regions within which a proliferation of various types of tubular profiles can be observed. Exposure of early C. variegatus embryos to BEN at various concentrations produced abnormalities at concentrations of 50 ppm and above. Anomalies in the order of frequency of occurrence were tubed heart syndrome with distended pericardia, poor circulation, sparse distribution of melanophores around yolk, inability to hatch, abnormal head morphology, scoliosis, and faint RBC pigmentation. Acute toxicity concentrations were established for benzo(a)pyrene (BaP), BEN, and diethylnitrosamine with respect to a cell line from Archosargus probatocephalus (the sheepshead). Long-term exposures provided evidence that BaP and BEN have mutagenic effects on this cell line. A dechorionation technique was developed to better observe detailed cellular and subcellular activities during early embryonic development of C. variegatus. Employment of this technique to observe inverted blastoderms provided evidence that the ectodermal cells that cover the yolk travel from the superficial blastoderm via a pathway along the blastoderm floor. Detailed studies of the gross and histologic structure of the digestive tract and histologic studies of the peripheral blood cells of C. variegatus were conducted. Three novel techniques were developed to study the effects of carcinogens on C. variegatus at the cellular level: an aseptic embryo technique that provides the opportunity to study embryos in a sterile environment; an embryo-primary cell culture technique that incorporates, in one system, characteristics of both intact embryos and primary cell cultures; and a primary hepatocyte cell culture technique that will be employed to study the effects of carcinogens on teleost hepatocytes. In order to study the immune system of C. variegatus. standard immunological techniques were miniaturized. Serum electrophoresis disclosed considerable variation between BEN-exposed and unexposed fish, and the presence of antibody-forming cells in spleen suspensions from C. variegatus immunized with human type O erythrocytes was demonstrated by a modified immune rosette procedure. |
