26th Annual Meeting and Symposium of the
Desert Tortoise Council, March 16-18, 2001
Abstracts

STUDENT PAPER

Purification of Desert Tortoise (Gopherus agassizii) Metallothionein, A Potential Biomarker of Heavy Metal Exposure

L. Domico¹, B. Homer¹, K. Berry², R. Cousins³, E. Jacobson⁴, P. Klein¹, and E. Williams^1
1Department of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611
²U.S. Geological Survey, Riverside, CA 92507
³Food Science and Human Nutrition, University of Florida, Gainesville, FL 32611
⁴Department of Small Animal Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611

Identified causes of desert tortoise morbidity and mortality include upper respiratory tract disease, cutaneous dyskeratosis, shell necrosis, liver and kidney degeneration, and urolithiasis (Homer et al., 1998). Post-mortem examinations of several tortoises have revealed suspected elevated concentrations of heavy metals, including cadmium, mercury, lead, molybdenum, arsenic, selenium, chromium, and nickel, in the liver and kidney of ill tortoises (Homer et al., 1996; unpublished data). Free-ranging tortoises that showed evidence of liver and kidney degeneration have contained elevated mercury, cadmium, or lead concentrations (Homer et al., 1996). Elevated concentrations of these metals may contribute to tortoise morbidity and mortality.

The overall objective of this research is to determine if the presence of tissue, plasma, or urine metallothionein (MT), a metal-binding protein with a molecular weight of approximately 7000 daltons, is a useful indicator of environmental toxic metal exposure in free-ranging desert tortoises. We hypothesized that accumulation of toxic metals correlates with elevated concentrations, and hence, increased deposition of MT in tissues. Fourteen juvenile desert tortoises were injected daily with 1 mg/kg BW cadmium chloride intra chemically for 5 days to induce MT synthesis. Low molecular weight proteins, including Cd-MT complexes, were isolated from tortoise liver cytosol by molecular weight gel-filtration on a Sephadex G-75 column. Atomic absorption spectrophotometry (AAS) confirmed low molecular weight fractions containing Cd. Gel electrophoresis (SDS-PAGE) of gel-filtration fractions confirmed the presence of doublet protein bands at approximately 6500 and 7000 daltons, typically indicative of the isoforms MT-1 and MT-2, and at 14,000 daltons, a possible polymer or an additional MT protein. Metallothionein isoforms were further purified by anion exchange column chromatography. Two distinct Cd peaks were detected via AAS. Gel electrophoresis of purified peaks confirmed, thus far, the presence of three protein bands that possibly correspond to MT isoforms. Protein fractions from peak 1 (MT-1) and peak 2 (MT-2) displayed bands at approximately 6500 and 7000 daltons, respectively. A band at 14,000 daltons was present in fractions from peak 1. Thus far, amino acid sequencing has identified the presence of ubiquitin, a 7,000 dalton protein involved in nonlysosomal degradation of damaged intracellular proteins during stressful conditions. Results from further amino acid composition analyses and sequencing of the other bands are forthcoming. Liver samples from treated and control tortoises were analyzed for Cd content on a dry weight basis. The mean Cd concentration in livers from treated tortoises was 44.8 ppm; the median was 27.7 ppm. In control livers, the mean Cd concentration was 0.26 ppm; the median was 0.25 ppm. All livers and kidneys were submitted for histologic staining. Microscopic changes, if any, were minimal and included mild renal tubular dilatation and proteinosis.

Reverse-phase high performance liquid chromatography will further confirm the presence of MT isoforms. Purified MTs will be injected into rabbits to induce anti-MT antibody formation. A sandwich enzyme-linked immunosorbent assay (ELISA) will be developed utilizing the MT antibodies. The ELISA will be useful in determining MT concentrations in tissues and body fluids, including plasma and urine, of the desert tortoise. If it is determined that MT occurs in plasma or urine of metal-exposed tortoises, then it may be possible to develop a laboratory and/or field test to determine exposure to heavy metals in live desert tortoises. Polyclonal antibodies will also be utilized for immunohistochemical detection of MT in paraffin-embedded tissues of archived specimens. This test will be valuable in investigating the localization of MT in the liver, kidneys, and other tissues. A non-invasive technique for determining exposure to metals would be valuable when determining causes of illness in tortoises, or if tortoises were to serve as sentinels of environmental metal references.

REFERENCES

Homer, B. L., K. H. Berry, F. Ross, C. Reggiardo, and E. R. Jacobson. 1996. Potentially toxic metals in liver and kidney of desert tortoises in California (abstract). Pages 34-35 in B. Bartholomew (ed.), Proceedings of 1996 Desert Tortoise Council Symposium, Las Vegas, Nevada.

Homer, B. L., K. H. Berry, M. B. Brown, G. Ellis, and E. R. Jacobson. 1998. Pathology of diseases in wild desert tortoises from California. Journal of Wildlife Disease 34(3):508-523.