Doctoral Dissertations

Date of Award

5-1994

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Lee R. Shugart

Abstract

In order to interpret genetic toxicological data in an ecological setting, studies were conducted to determine the relationship between laboratory and field research, and between individual and population-level responses. studies are as follows: These 1) The temporal expression of various genotoxic responses was determined in bluegill sunfish (Lepomis macrochirus) exposed under controlled laboratory conditions to sediment contaminated with genotoxic compounds (polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and heavy metals). In addition, non-genotoxic responses were also examined over a chronic (up to 40 weeks) exposure. Genotoxic responses included DNA strand breakage, DNA adduct formation, cell-to-cell variation in DNA content (determined by flow cytometry), and changes in chromosomal protein profiles. Nongenotoxic responses included mixed function oxidase, acetylchol inesterase, and Na+/K+ ATPase activities, histopathology, stress protein induction, and hematological parameters. Biomarkers in the laboratory-exposed fish were similar to those of indigenous sunfish sampled from the site of origin of the contaminated sediment. Several patterns of development of biomarkers over time were evident, depending upon specific biomarkers and the tissue in which they were examined. 2) DNA, isolated from the blood cells of the same bluegill sunfish as above, was examined for strand breakage by agarose gel electrophoresis under either neutral (pH 7) or alkaline (pH 12) conditions. The median molecular length (MML) of the DNA distributed in the gel was determined. These quantitative measures were used to estimate the difference in the number of double strand and single breaks between DNA preparations. Both types of strand breakage were found to be greater in fish exposed to sediment contaminated with genotoxic compounds as compared to non-exposed fish. 3) Mosquitofish (Gambusia affinis) were collected from Pond 3513, a small pond that is contaminated with 137cs, 90Sr and other radionuclides, from White Oak Lake, a settling basin contaminated with radionuclides and chemical genotoxins, and from non-contaminated populations. DNA was isolated from both liver and blood cells and examined for DNA total- (both single and double) and double-strand breaks alone by gel electrophoresis. In general, total- and double-strand DNA breaks were slightly more prevalent in fish from radionuclide-contaminated sites than from uncontaminated sites. In addition, there were differences in the number of strand breaks between liver and blood cells. Also, fecundity and number of malformed embryos were determined in fish from all sites. It was found that, for fish from the contaminated sites, the number of DNA strand breaks was negatively correlated with fecundity, and that females with malformed embryos in their broods had slightly more DNA strand breaks than did females with no malformed embryos. 4) DNA polymorphisms, using the RAPD (Randomly Amplified Polymorphic DNA) technique, and allozyme polymorphisms were examined in mosquitofish (Gambusia affinis) from the same populations as above. The RAPD technique uses the polymerase chain reaction with a short oligonucleotide primer to produce DNA fragments which form banding patterns similar to DNA fingerprints. Using the RAPD technique, the genetic diversity of the contaminated populations were found to be greater than the non-contaminated populations. Furthermore, heterozygosity, both at the nucleoside phosphorylase locus and at all other loci, was found to be elevated in contaminated populations relative to control sites. Additionally, RAPD bands found at high frequency in Pond 3513 and White Oak Lake populations were present at a lower frequency or absent in the non-contaminated populations. For some primers, the contaminated populations showed more DNA bands per individual than the non-contaminated populations. 5) DNA polymorphisms revealed by RAPD and allozyme analysis were compared to relative amounts of strand breakage as determined by gel electrophoresis in mosquitofish (Gambusia affinis). These fish were exposed to radionuclide contamination in the field and to X-rays in the laboratory. It was found in a Part 5 that in some instances the average number of bands and the frequency of certain bands were elevated in radionuclide-contaminated relative to reference populations. Therefore the median molecular length (MML) in each individual was compared to RAPD genotype in terms of the two metrics mentioned above. In several cases, the median molecular length (MML) of the DNA (which is inversely proportional to the amount of strand breakage) was correlated to the number of bands per individual. Also, for bands which had a higher population frequency in contaminated sites, the MML in many cases was higher for fish with than without these bands. Results from laboratory X-ray exposures paralleled those from the field. The results of these studies will be discussed in terms of application to ecotoxicology and ecological risk assessment.

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