A Reliable, Efficient, Microinjection Apparatus and Methodology for the In Vivo Exposure of Rainbow Trout and Salmon Embryos to Chemical Carcinogens23

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Abstract
A modular apparatus and technique for the injection of salmonid fish embryos with chemical carcinogens are described. A key feature of the methodology is the relative ease of routine through-the-eggshell injection, into the yolk sac of living salmonid fish embryos, inside the “eyed-stage” egg. The procedure is sufficiently rapid that 2 persons working as a team can give injections to 200 embryos per hour. The injection per se induces low mortality, i.e., optimal net survival rates (controls given an injection of dimethyl sulfoxide vs. those not given an injection) in the range of 70–90%. Because only small amounts of chemical are handled in relatively dilute form, the exposure method poses low risks to both the experimentalist and the environment. Preliminary results in a test of 4 carcinogens that differed widely in their structures and requirements for metabolic activation indicated that hepatocellular neoplasms were induced in rainbow trout (Salmo gairdneri) in response to 100 ng aflatoxin B1 (CAS: 1162-65-8)/egg, 1 μg N- methyl-N′-nitro-N- nitrosoguanidine (CAS: 70-25-7)/egg, and 10 μg benzo[a]pyrene (CAS: 50-32-8)/egg. Nine months after exposure, liver neoplasms were observed in 25, 21, and 9%, respectively, of the rainbow trout, but no neoplasms were observed in rainbow trout exposed to 100 μg dimethylnitrosamine (CAS: 62-75-9)/egg. Liver neoplasms were also induced in 17% of coho salmon (Oncorhynchus kisutch) given an injection as embryos of 90 ng aflatoxin B1/egg or 5 μg N- methyl-N′-nitro-N- nitrosoguanidine/egg.