In vitro effect of some mutagens/carcinogens on cultured fish cells

Fibroblast cells derived from juvenile bluegill sunfish were tested for their response the effects of known mutagens/carcinogens. The cells grew well and cloned in Eagle's minimal essential medium which contained fetal bovine serum. Growth rate was temperature dependent and increased steadily as temperature was raised from 15 to 33 degrees C. Direct mutagens and promutagens were toxic to the cells, and fluorescence spectroscopy revealed the capability of metabolizing at least one promutagen, benzo(a)pyrene, to water-soluble intermediates. Ouabain resistant mutants were produced by exposing the cells to N-methyl-N-nitro-nitrosoguanidine (MNNG) or to benzo(a)pyrene (B(a)P). The mutant clones were resistant to 100-fold increases in concentration of the selective agent ouabain, over that which was lethal to wild type cells. Spontaneous mutation frequency to ouabain in mass cultures averaged 1.2 X 10(-6) for all experiments. A fluctuation test confirmed an expected random occurrence of spontaneous mutations. Visible crystals formed within the cells when high concentrations of B(a)P (greater than 5 micrograms/ml were added to the tissue culture medium. Crystal formation resulted in no apparent cell damage but did reduce the growth rate of the cultures. The crystals were rhombic, resembling those described for pure native B(a)P, and gradually disappeared when the cells were exposed to mutagen-free medium which contained serum.