Mammals assay techniques

Our preliminary results with fish brain preparations suggest that ion flux techniques may be valuable in studies of target site differences between species. We have demonstrated veratridine-stimulated, tetrodotoxin-sensitive sodium uptake in a vesicular preparation from fish brain, thus confirming the presence of functional sodium channels in this preparation. Our results with , -DDT in this system also agree well with the action of DDT analogs and pyrethroids in mouse brain assays. Further studies wih both preparations should allow the exploration of target site differences between mammals and fish that have been inferred from whole animal toxicity studies. 

Finally, it is possible that these techniques can be extended successfully to insect CNS preparations. Methods now exist to prepare functional insect synaptosomes from insect ganglia (1 ), and these preparations have been used to demonstrate veratridine-dependent neurotransmitter release and enhancement of this release by deltamethrin in a superfusion assay (2j)). Further refinement of these methods should allow direct measurement of sodium channel-mediated sodium fluxes in insect CNS preparations, thus allowing the investigation of target site differences not only between mammals and insects but also between susceptible and resistant insect strains. 

The host-mediated assay is a simple technique that attempts to bridge the gap between in vitro microbial studies and definitive tests in mammals. The numerous advantages of microorganisms as determinants of hereditary change are utilized in an experimental mammal after it has had an opportunity to metabolize the compound under investigation. This assay was developed to determine the ability of laboratory animals to either potentiate or detoxify compounds in regard to their mutagenic activity. In addition to the studies in mammals, it is essential to conduct the in vitro assay as a control for the host-mediated assay. 

The enzymes which catalyze the formation of GSH have been found in the livers of pigeons (1, 2) as well as of various mammals (3), and may be obtained in soluble form from acetone-dried tissue preparations. The presence of the GSH synthesizing system in extracts of E. coli has recently been demonstrated by Samuels (4). Synthesis of GSH can be followed either by the conventional glyoxalase assay (5), or, as in our experience, more conveniently by measuring the incorporation of radioactive amino acids into the tripeptide which is isolated by the carrier technique (1, 2). 

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