Use of Two-Compartment Teflon Flow-Through Diffusion Cells to Study Cutaneous Absorption Kinetics of Malathion in the Bullfrog (Rana catesbeiana) and Marine Toad (Bufo marinus)
There is increased concern about the sublethal effects of OP compounds and their impact on the global decline of amphibian populations.5 Malathion is one of the most widely used organophosphorus (OP) pesticides.3 For environmentally applied pesticides to exert a toxicological effect, they must be systemically absorbed. Cutaneous absorption is a significant route of environmental exposure for amphibians.5 Thus, to conduct risk assessments, it is important to research and develop components for a model of the absorption kinetics of these compounds through the skin.
An in vitro system commonly used to determine the absorption kinetics of xenobiotics and toxins across the skin is the two-compartment Teflon flow-through diffusion cell system.1 Porcine skin diffusion cells have been used to extrapolate in vivo cutaneous exposure and metabolism of OP compounds in humans.2 This system provides a morphologically complete and intact skin barrier. This model also eliminates confounding factors in the analysis of in vivo cutaneous absorption, including absorption, metabolism, or elimination by other routes. We hypothesize that the two-compartment Teflon flow-through diffusion cell system will adequately model cutaneous absorption in bullfrogs (Rana catesbeiana) and marine toads (Bufo marinus). We also hypothesize that cutaneous absorption will differ between dorsal and ventral sites and between species due to differential skin thickness and gland distribution.
To establish cutaneous absorption kinetics of malathion, six dorsal and six ventral full thickness circular sections (0.64 cm2) taken from each of three bullfrogs and marine toads were run in two-compartment Teflon flow-through diffusion cells. The perfusate was a modified amphibian Ringer's solution maintained at pH 7.2-7.4, 70°F, and flow rate of 4 ml/h.6 Malathion-2,3-14C was diluted in 100% ethanol to achieve a 10 µl dose of 40 µg/cm2 applied to each sample. Perfusate was collected at 0, 15, 30, 45, 60, 75, 90, 105, 120, 150, 180, 210, 240, 300, and 360 minutes of malathion exposure and analyzed in a scintillation counter. Kinetic parameters, including venous efflux profile (% dose/min), were calculated from these data. At the end of six hours, surface swabs, tape strips, biopsy punches of the dosed area of skin, and peripheral samples were oxidized and analyzed for partitioning effects. Morphometric measurements of skin were obtained using light microscopy. Malathion absorption was significantly greater in the ventral skin of bullfrogs than in dorsal skin. However, more malathion was retained on the surface of frog skin and in the stratum externum of dorsal samples due to a thickened stratum externum. Also, significantly more malathion was retained in the dosed area of dorsal skin in frogs compared with ventral skin.
The authors are grateful to Mr. Joseph Faulk and the Laboratory Animal Resources staff at NCSU for their assistance in animal care. We also thank the Center for Cutaneous Toxicology and Residue Pharmacology and the Laboratory for Advanced Electron and Light Optical Methods (LAELOM) at NCSU. This research was made possible by a grant from the United States Environmental Protection Agency.
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