Determination of genotoxic stress in feral populations of bullheads and carp using the alkaline single cell gel assay.
Monitoring genotoxicity of the environment using endemic organisms as sentinels requires the development of sensitive assays. Towards this end, I explored the feasibility of applying the ALKALINE SINGLE CELL GEL (SCG) or "COMET" ASSAY. This approach involves detection, under alkaline conditions, of cell DNA fragments which on electrophoresis migrate from the nuclear core resulting in a 'comet with tail' formation. The length of the tail has been correlated with the level of genotoxicant exposure in a number of organisms. The fish used in this study were benthic feeding bullheads (Ameiurus nebulosus) and carp (Cyprinus carpio). In preliminary studies, on electrophoresis of erythrocyte DNA under alkaline conditions I found a linear increase in the DNA length:width ratio over a broad range of cyclophosphamide (a clastogen) doses. Subsequent examination of two direct acting mutagenic agents (methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (NMU)), two indirect acting mutagens (benzo(a)pyrene (B(a)P)) and 7,12-dimethylbenz(a)anthracene (DMBA) and two cell cycle specific agents (methotrexate (MTX) and colchicine) over a range of doses showed that all, except the lower methotrexate concentration, resulted in significant (p $<$ 0.001) DNA damage in fish erythrocytes. Freshly caught bullheads from seven different sites showed a wide range of DNA damage. Bullheads from Big Creek (western Lake Erie), Hamilton Harbour (western Lake Ontario) and a number of sites along the Detroit River gave ratios v ranging from 3.36 to 4.65. Samples from Lake St. Clair, Thames River, Lake Huron and Port Franks gave ratios ranging from 1.30 to 2.19. Fish from a hatchery in Brockport, New York, gave ratios between 1.35 and 1.40. Based on polycyclic aromatic hydrocarbons (PAH) and polychlorinated biphenyls (PCB) levels, the sediment at the Detroit River sites is considered to be heavily polluted. Bullheads from Big Creek, maintained in the laboratory for three months, gave ratios which dropped from 4.78 to 1.41. The latter are similar to those obtained in hatchery-bred fish. Carp from Big Creek gave ratios of about 4.50, while carp from Lake Huron and laboratory-maintained carp gave values of 1.23 and 1.36, respectively. Bullheads placed in cages at three different sites in the Detroit River, one near Lake St. Clair (Peche Island), and the other two midway between the source and the mouth (near Turkey Island and in the Trenton channel) showed significant increases (p $<$ 0.05) at two sites (Turkey and Trenton) after two days of exposure. The level of damage increased with the duration of exposure. Bullheads caged at two other sites (Big Creek and LaSalle), for eight days also showed significantly higher ratios than controls (p $<$ 0.01). Finally, DNA length:width ratios for bullheads exposed to sediments from Lake St. Clair (relatively clean) did not differ from the control group ratios. In contrast, animals exposed to Big Creek sediment (highly polluted) showed an increase in DNA ratios. The results, from the bullhead and carp systems, indicate that the SCG assay is extremely sensitive and should be useful in detecting DNA damage caused by environmental contaminants. Moreover, the results from freshly caught fish, fish placed in cages and fish placed on sediment were consistent suggesting that any of these approaches could be used in monitoring a particular habitat.Dept. of Biological Sciences. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1997 .P36. Source: Dissertation Abstracts International, Volume: 59-08, Section: B, page: 3911. Adviser: M. Petras. Thesis (Ph.D.)--University of Windsor (Canada), 1997.
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