Mouse assays detection

Some Chemical Considerations Relevant to the Mouse Bioassay. Net toxicity, determined by mouse bioassay, has served as a traditional measure of toxin quantity and, despite the development of HPLC and other detection methods for the saxi-toxins, continues to be used. In this assay, as in most others, the molar specific potencies of the various saxitoxins differ, thus, net toxicity of a toxin sample with an undefined mixture of the saxitoxins can provide only a rough approximation of the net molar concentration. Still, to the extent that limits can be placed on variation in toxin composition, the mouse assay can in principle provide useful data on trends in net toxin concentration. However, the somewhat protean chemistry of the saxitoxins makes it difficult to define conditions under which the composition of a mixture of toxins will remain constant thus, attaining a reproducible level of mouse bioassay toxicity is difficult. It is therefore useful to review briefly some of the chemical factors that should be considered when employing the mouse bioassay for the saxitoxins or when interpreting results. Similar concepts will apply to other assays. 

A recent area of major concern has been the development of a practical and specific assay for the detection of ciguatoxin directly from fish tissues. Earlier assays, some of which are still used, relied on whole tissues, crude extracts and partially purified ciguatoxin (8, 9, 20). For the mongoose and cat assays, large amounts (10-15% of body weight) of fresh tissues were required, while the mouse assay required concentrated lipid extracts of the fish tissue. 

The 35 mm dish assay detects compounds that are cytotoxic to or alter proliferation of the multipotent stem cells, but the main limitation of the 35 mm dish CFU assay is the need to microscopically identify and count the colonies to determine the compound concentration that decreased the number of colonies by 50% (IC50) or 90% (IC90). The European Community for Validation of Alternative Methods (ECVAM) supported the recommendation of using the mouse and human CFU-GM IC90 with... 

Unfortunately, the dose-survival times for the DSP toxins in the mouse assay fluctuate considerably and fatty acids interfere with the assay, giving false-positive results consequently, a suckling mouse assay that has been developed and used for control of DSP measures fluid accumulation after injection of the shellfish extract. Considerable effort has been applied recently to development of chemical assays to replace these bioassays. As a result a good high performance liquid chromatography (HPLC) procedure has been developed to identify individual PSP toxins (detection limit for saxitoxin = 20 fg per 100 g of meats 0.2 ppm), an excellent HPLC procedure (detection limit for okadaic acid = 400 ngg 0.4 ppm), a commercially available immunoassay (detection limit for okadaic acid=lfg per 100 g of meats 0.01 ppm) for DSP, and a totally satisfactory HPLC procedure for ASP (detection limit for domoic acid = 750 ngg 0.75 ppm). 

Wang J, Sawyer JR, Chen L, Chen T, Honma M et al (2009) The mouse lymphoma assay detects recombination, deletion, and aneu-ploidy. Toxicol Sci 109 96-105... 

These studies may provide a mechanism for the toxicity demonstrated in the epidemiological studies to date. This in vitro model has the potential to be developed as a functional assay for detection of AZA and other marine toxins. The assay may well be more suitable than the mouse assay. The assay has high sensitivity with very significant effects being demonstrated at 5 nM AZA. 

It is obvious from the provisional risk assessment values for microcystins, and, being of the same order of magnitude of mammalian toxicity, similar values may be calculated for the cyanobacterial neurotoxins, that sensitive detection methods are required to detect these low concentrations of toxins. Of the biological methods of detection discussed earlier, the mouse and invertebrate bioassays are not sensitive enough without concentration of water samples, in that they are only able to detect mg of microcystins per litre. Only the immunoassays (ng-/rg 1 and the protein phosphatase inhibition assays (ng O... 

Mouse Bioassay. The mouse is the traditional animal of choice for detecting biological activity due to STX and TTX. Mice receive an intraperitoneal injection of sample and are observed for symptoms of intoxication, i.e., dypsnea, convulsions, and death. This method is effective for detecting biological activity of STX and TTX in numerous samples. For the standard STX assay, one mouse unit is defined as that quantity of STX injected i.p. in 1 ml solution that will... 

Tissue Culture Assay. Kogure et al. (48) report a novel tissue culture assay for detecting several types of sodium channel blockers. The mouse neuroblastoma cell line ATCC CCL 131 is grown in RPMI 1640 supplemented with 13.5% fetal bovine serum and 100 pg/ml gentamycin, in an atmosphere of 5% C0 95% air at 37 C. Ninety-six well plates are seeded with 1 x 10 cells in 200 pi of medium containing 1 mM ouabain and 0.075 mM veratridine. Veratridine and ouabain cause neuroblastoma cells to round-up and die. In the presence of sodium channel blockers (e.g., TTXs or STXs), the lethal action of veratridine is obviated and cells retain normal morphology and viability. An important feature of this assay is that a positive test for sodium channel blockers results in normal cell viability. Since bacterial extracts can contain cytotoxic components, this assay offers an advantage over tests that use cell death as an endpoint. The minimum detectable level of TTX is approximately 3 nM, or approximately 1/1000 mouse unit. 

Ponzetto, C., Wadewitz, A. G., Pendergast, A. M., Witte, O. N., and Wolgemuth, D. J. (1989). P I50c ai,/is detected in the mouse male germ line by an in vitro kinase assay and is associated in haploid cells with a stage-specific phosphoprotein. Oncogene 4 685-690. 

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