Final Report

The final outcome for our research is to develop a reliable and sensitive suite of genetic biomarkers for the rapid assessment of contaminant exposure in the Grand Calumet River and at the Lake Michigan interface.  Contaminant related genetic damage and damage related disease outcomes may provide information on potential human health risks associated with direct (environmental) or indirect (fish consumption) contaminant exposures. The research results produced from this work will be useful for those agencies involved in the remediation process of the river and any future remediations that may occur in the lake. Currently Ron Gregg, a student under my supervision is quantifying a new agarose gel based procedure for the quantification of single and double stranded DNA breaks in brown bullhead catfish. He has worked through as series of experiments which have successfully validated the DNA strand break analysis method in populations held for a short time period held at the Baker Aquaculture Facility and at experimental sites located in the Grand Calumet Lagoons. We have analyzed data from a collaborative project with Dr. R. Gillespie and have presented the results at two scientific meetings. R. Gregg is currently working on the protocol for alkaline DNA unwinding procedures for the brown bullhead catfish.

Major Goals and Objectives:

1. To determine if exposure to environmental PAHs will increase DNA damage of caged brown bullhead catfish

2. To determine the amount of time of exposure to PAH contaminated sediments required to show increased levels of DNA damage in brown bullheads

3. To determine the potential link between DNA damage and the formation of visible neoplasia and tumors

4. To determine if agarose gel electrophoresis is a more economical and more rapid method of assessing DNA damage than the conventional alkaline unwinding method

5. To determine if DNA damage in caged brown bullheads is a useful indicator of DNA damage in wild fish

6. To determine differential response to PAH exposure among individual brown bullheads

7. To determine if caged yellow bullheads and bluegill sunfish respond differently than brown bullheads to environmental exposure to PAHs

Summary of Goals and Objectives:

1. Brown bullheads in cages at locations with PAH contaminated sediments show more DNA damage than caged brown bullheads at locations with non-contaminated sediments or reference fish kept at the Purdue Aquaculture facility.

2. Brown bullheads show increased DNA damage after only 4 weeks of exposure to PAH contaminated sediments.

3. Visible neoplasia or tumors were not present in caged brown bullheads after 2 years of exposure to PAH contaminated sediments.

4. The agarose gel electrophoresis method of detecting DNA damage potentially offers a more economical and more rapid means to assess DNA damage, however, problems in quantifying the amount of DNA damage may hinder its application.

5. In Progress

6. In Progress

7. In Progress

Accomplishments:

• Brown bullheads maintained in cages for two years and sampled on four occasions.

• Caged brown bullheads have been analyzed for neoplasia and tumors.

• Alkaline unwinding of brown bullhead DNA has been optimized.

• Extensive testing to improve the agarose gel method of detecting DNA damage has been performed.

• Laboratory experiments with brown bullheads to examine individual differences in response to PAH exposure have been conducted.

• Yellow bullheads from GCL and uncontaminated location have been collected for analysis of DNA damage and histopathology.

• Field experiments have been conducted to compare DNA damage of brown bullheads to other species.

Narrative Report:  We maintained caged brown bullheads in the PAH contaminated Grand Calumet Lagoons (GCL) near Gary, Indiana for two years. Initial stocking of these cages in the fall of 1997 was unsuccessful in that nearly all of fish died or escaped over the winter when the cages were inaccessible due to ice. The cages were stocked again in the summer of 1998. Four samples of fish from these cages were collected (fall 1998, spring 1999, fall 1999, spring 2000) and compared to reference fish housed at the Purdue Aquaculture Facility. Fieldwork has also been completed in short-term experiments with yellow bullheads and bluegill sunfish.

In addition to stocking the cages in the fall of 1997, we released ~7,000 brown bullheads into the West GCL. Attempts to recapture these fish were unsuccessful until the spring of 2001, when six brown bullheads were captured. We think that the fish were too small to capture due to slow growth in this unproductive, contaminated environment.

Since the brown bullheads that we released into GCL cannot be captured in large enough numbers in a reasonable time frame, we decided to use a different approach in determining whether caged fish are good indicators of wild fish. We will examine both the histopathology (in collaboration with Dr. Randy White of ADDL) and DNA damage of yellow bullheads native to the GCL and native to an uncontaminated location (Crooked Lake near Columbia City, Indiana). We will be able to tell at what age native fish show signs of increased DNA damage, neoplasia, and lesions in PAH contaminated environments.

In collaboration with Dr. Robert Gillespie of IPFW, we have completed a short-term study of caged brown bullheads. Dr. Gillespie and his graduate students have determined that brown bullheads caged near contaminated sediments show high levels of metabolite and CYP1A enzyme production, which is characteristic of fish in PAH contaminated environments. We have found that these same brown bullheads show increased DNA damage when compared to reference fish. However this increase is not statistically significant. One reason for the lack of significance is the large variance around these sample points. This inflated variance potentially arises from two major sources, individual brown bullheads responding differently to PAH contaminants and inadequate standardization between agarose gels. We are addressing both of these problems.

We designed laboratory experiments to further elucidate the response of brown bullheads to PAH contamination. These experiments will verify if the DNA damage observed in brown bullheads collected from the GCL is due to PAH exposure since known amounts of PAHs are added to the aquaria in the laboratory. In addition, these experiments will indicate the response of individual fish, since blood will be repeatedly sampled from the same individuals multiple times and examined for DNA damage.

We are collaborating with Dr. James Novak and others at University of Georgia’s Savannah River Ecology Laboratory to determine the method that best quantifies DNA damage with minimal variation among agarose gels. Several analytical approaches and statistical analyses may be employed and we are currently designing experiments to determine the most effective method. Once this method is determined it will be compared to the alkaline unwinding method that we have optimized for DNA from brown bullheads.

In order to accomplish many of our major goals, we have had to design and perform additional experiments. This has resulted in more time spent doing fieldwork, laboratory tests with brown bullheads, laboratory tests with DNA, and statistical analyses. By carefully working through these analyses we can ensure that our results will be reliable and repeatable. This attention to detail now, will allow future researches to more easily quantify DNA damage of fish in other locations.

Brief Summary:  Brown bullheads were maintained in cages in the Grand Calumet Lagoons (GCL) near Gary, Indiana for two years. Fish from locations with high concentrations of PAHs in the sediments show more DNA damage than fish from non-contaminated locations. Elevated levels of DNA damage were detected after only 4 weeks of exposure to contaminants. DNA damage is harmful to the fish and may be an early indicator of tumor formation. However, none of the caged brown bullheads showed evidence of tumors or pre-cancerous lesion after two years of exposure to PAH contaminated sediments. We will examine yellow bullheads native to GCL to determine the age at which DNA damage and pre-cancerous lesions may be detected. A new method of quantifying DNA damage caused by exposure to PAH is being explored. This method is more rapid and more economical than conventional methods, however, standardization techniques must be worked out in order to make this method more reliable. Once this problem is solved this technique has applications for fish, wildlife, and humans to determine levels of DNA damage. This may be used to identify areas in which environmental contaminants pose cancer risk.