Development of a Novel In Vitro System for Potential Application in Marine Toxicological Analysis
IAAAM 1991
Michael J. Carvani
Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station, TX

The long-term effects of toxic chemicals on the oceans, one of our most fragile and productive ecosystems, are unknown. Marine mammals and man are at the top of the oceanic food chain, where the bioaccumulation of toxin is more immediate and of greater magnitude. Toxicological assessment of smaller, more abundant, marine organisms is feasible in a laboratory situation, but the size of marine mammals and their status as protected and endangered species make traditional methodologies impossible. There is a serious need for development of new in vitro model systems to conduct analyses of the effects of both naturally occurring and polluting chemicals on marine mammals, especially cetaceans. Cetaceans are excellent indicators of long-term environmental health and stability. Their sensitivity to environmental stress is evident in the recent die-offs: the summer of 1987 (east coast, U.S.), winter of 1990 (Gulf of Mexico coast, U.S.), and summer of 1991 (western Mediterranean Sea).

Complex tissues and organs can be investigated utilizing cells in culture. Beluga whale (Delphinapterus leucas) and spotted dolphin (Stenella attenuate) epidermal cells were cultured with modifications to the techniques for serial culture of human epidermal keratinocytes. Skin samples were collected from live or recently dead specimens. Cells in culture were identified using histochemical and immunocytochemical techniques to verify their identity. Variations in metabolic rate, growth, proliferation and substrate utilization, with changes in the culture environment (temperature, pH, media osmolarity, nutrient availability, fibroblast co-culture and dissolved gas content) were used to characterize the cells in culture, optimize the system, and establish baseline data for use in toxicological assays.

The epidermis is well suited for research in toxicology and cell/molecular biology due to its dynamic nature, availability as living tissue, extensive cytochrome P-450 system, environmental barrier function, and its metabolic and spatial association with the blubber, where large amounts of various toxins are stored. There are currently no good experimental data available on the cytotoxic effects of natural and pollutant chemicals on any cetacean tissues.

This system can be utilized to study cetacean cytotoxic response to contaminants and their capacity to detoxify harmful compounds. Unfortunately, the metabolic intermediates from such biotransformations are highly electrophilic and extremely toxic, binding to the nucleophilic regions of macromolecules (DNA, RNA and proteins) altering their information, content and expression. Hence, these metabolic intermediates may cause cellular damage leading to immunological injury, tissue necrosis, oxygen toxicity and malignancy. By utilizing such model systems in conjunction with the tools of cellular and molecular biology, we have the potential to determine cetacean-specific cytotoxicity and its relationship to oceanic processes including analysis of both natural and human-induced flux upon the quality of that environment, from biotoxins to pesticide, petroleum and heavy metal contamination.

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Michael J. Carvani, III

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