25th Annual Meeting and Symposium of the
Desert Tortoise Council, April 21-24, 2000
Abstracts

Desert Tortoises As Sentinels of Environmental Toxicants

Bruce L. Homer¹, Lisa M. Domico¹, J. Elliot Williams¹, Bert Heaton-Jones¹, and Kristin H. Berry^2
1Department of Pathobiology, University of Florida, PO Box 110880, Gainesville, FL 32611  ²U.S. Geological Survey, Biological Resources Division, 6221 Box Springs Blvd., Riverside, CA 92507

Animals exposed to environmental toxicants often suffer health effects similar to those in humans. Data collected from studies of these animals may play a vital role in the risk assessments of the toxicants on the environment and of the environment on human health. For instance, because of their exquisite sensitivity to carbon monoxide, canaries were carried into mines to monitor air quality. Recognition of other animal sentinels has been more fortuitous. Epidemiologic studies of epizootic liver cancer in fish have shown a strong correlation with the presence of carcinogens in the aquatic environment. For an animal species to be a suitable sentinel for a hazardous environmental agent, members of the species must exhibit the following traits: 1) a long life span; 2) a large enough size to yield adequate tissue samples for analysis; 3) non-migratory behavior; 4) territorial behavior; 5) easy apprehension; 6) a sufficient population size and density to be enumerated; 7) inhabit the area to be monitored; 8) accumulate the toxicant without being killed; 9) a correlation between the concentration of the toxicant in tissues with that in the environment; 10) express a measurable response to the toxicant; and 11) respond to the toxicant analogously to humans. For the most part, the desert tortoise fits the profile (traits 1-7) of a desirable sentinel species. As part of a study of 49 ill, dying or dead desert tortoises from the Mojave and Colorado deserts of California, the concentrations of up to 22 trace elements were determined in fresh sections of liver, kidney and scute. Toxic or potentially toxic metals assayed included cadmium, lead, mercury, nickel, chromium, selenium, zinc, copper, and molybdenum, the concentrations of which appeared to be elevated in liver, kidney and scute of one or more tortoises from multiple study sites (trait 8). Haxel, Knight, Chaffee and Berry are investigating uptake of metals in plants and biogeochemical pathways of metals as means of correlating concentrations of metals in tissues with that in the environment (trait 9).

We have not detected a consistently measurable response of tortoises to exposure to toxic metals, although we suspect that elevated concentrations of toxic metals have contributed to tortoise morbidity and mortality (trait 10). Thus we have initiated a study to determine if the presence of the metal-binding protein metallothionein (MT) in tissues and fluid samples in free-ranging desert tortoises can be used as an indicator of environmental toxic metal exposure. Certain metals, including cadmium, nickel and mercury, can induce synthesis of MT in vertebrates. We propose to induce MT synthesis in juvenile desert tortoises by administering multiple injections of cadmium chloride. Low molecular weight proteins, including metallothionein isoforms, will be isolated from liver of these tortoises by molecular weight gel-filtration column chromatography. Metallothioneins will be further purified by anion exchange high performance liquid chromatography (HPLC). Purified metallothioneins will be injected into rabbits to induce anti-MT antibody formation. Rabbit polyclonal MT antibodies will be utilized to develop a competitive enzyme-linked immunosorbent assay (ELISA) for desert tortoise MT in tissues (particularly liver and kidney), serum and urine. The MT-ELISA test will ultimately serve as a simple and sensitive assay for MT content in desert tortoise tissue and fluid specimens, and should provide correlative information about which metals induce synthesis of MT in desert tortoises, and whether elevated concentrations of MT correlate with elevated concentrations of toxic metals in desert tortoises. If we determine that MT occurs in plasma or urine of metal-exposed tortoises, then it may be possible to develop a non-invasive laboratory and/or field test to assess exposure to heavy metals in live desert tortoises. Polyclonal antibodies will also be utilized for immunohistochemical (IHC) detection and localization of MT in paraffin-embedded liver, kidney, and other tissues of tortoises that were submitted for complete necropsy and for which metal analyses have been conducted. Like the ELISA test, the IHC studies should help address which metals induce MT in desert tortoises and whether the presence and sites of localization of MT in tissue are useful indicators of metal exposure.