Abstract
The Western population of Steller sea lions is endangered and the cause is unknown. We hypothesize that exposure to metals may be a factor in this decline. This study characterizes cellular and sub-cellular effects of various heavy metals on cell lines derived by our laboratory from Steller Sea Lion organ explants as model systems. By comparing intracellular metal ion levels achieved in these in vitro assays with actual tissue levels of metals in free ranging animals, hypotheses about in vivo effects of these metals can be developed, helping to determine relative risks and priorities for intervention. We have cultured cells from testes, ovary, skin, liver, kidney, and lung. Our goal is to determine cytotoxic and genotoxic effects of a variety of soluble metal compounds on these cell types.
Cytotoxicity is determined using a clonogenic assay.1 After a 24h exposure to metal ions, cells are grown for two weeks. Cells surviving with sufficient cellular machinery/processes intact will reproduce daughter clones forming countable colonies. Cytotoxicity is reported as relative survival, the percentage of colonies relative to untreated controls. We find metals are cytotoxic to Steller sea lion cells in an organ and metal specific manner. Chromium induced concentration-dependent cytotoxicity with hepatic, testicular, bronchial, and ovarian cells more sensitive than renal or dermal cells. Cadmium was less toxic than chromium at all concentrations. Interestingly, cadmium was generally non-toxic to dermal cells but induced concentration-dependent toxicity in other cells. Testicular cells were the most sensitive to both metals which has transgenerational implications. Finally, the organ specific potency of toxicants suggests that a toxicant concentration that may be harmless to one organ may be detrimental to another.
DNA damage is determined using single cell gel electrophoresis or 'Comet' assay. After a 24h exposure to metal ions, cells are embedded in agarose on microscope slides. Slides are treated to lyse cellular membranes, expose chromatin, and relax chromosomes. An electrophoretic field causes liberated genetic material to migrate based on the size of DNA fragments. Genetic damage in the form of single and double strand breaks will increase the number of fragments increasing length and density of migration away from the nucleus forming a comet like tail. Preliminary data indicate sodium chromate has genotoxic effects at concentrations where little to no cytotoxicity is observed.
Metals exert their effect intracellularly. Inductively coupled plasma (ICP) emission spectrometry was used to determine intracellular concentrations achieved in laboratory assays. Testicular cells take up more chromium than bronchial or dermal cells, possibly explaining differences in cytotoxicity. Metal concentration in tissues from free-ranging animals will be determined using ICP mass spectrometry. Concentrations analyzed to date are below those that cause cytotoxicity in vitro. However, so far only tissue from pre-weaned pups has been available for tissue analysis.
Further research is aimed at testing additional tissues from multiple Steller sea lions, testing additional metals in all cell lines, and extending the lifespan of these cells in culture with telomerase.
Acknowledgments
Tissue collection was done by several individuals from the following organizations covered by the listed permits: Alaska Department of Fish and Game (NMFS Permit No. 158-364); Alaska SeaLife Center (NMFS Permit #881-1443-04); Mystic Aquarium (NMFS LOA 23 Dec 1980, USDA Research Permit #16-R-031); and The Marine Mammal Center (NMFS LOA #1514-10, California Department of Fish and Game, Live Marine Mammal Permit # 76-6). In particular, thanks go to Jen Burns, Tom Gelatt, Millie Gray, Kendal Mashburn, Jo-Ann Mellish, Natalie Noll, Lorrie Rea, Julie Richmond, Carol Stephens, Pam Tuomi, Jason Waite, Denise Greig, Amie Holmes, Andy Morin, Nishad Jayasundara, and Jennifer Little. Marine mammal cell line development and use by the Wise Laboratory for Environmental and Genetic Toxicology is performed under NMFS Permit 1008-1637-00. The analysis of tissues for metal content was performed by Robert Taylor at the Trace Element Research Laboratory, Texas A&M. Support for this research was provided in part by NOAA grant, NA16FX1412, and the Bioscience Research Institute of Southern Maine.
References
1. Wise JP, Wise SS, JE Little. 2002. The cytotoxicity and genotoxicity of particulate and soluble hexavalent chromium in human lung cell, Mutation Research, 517:221-229.