Bioassay mouse

The brine shrimp (Anemia salina) has been evaluated as an alternative to the mouse bioassay for use in cyanobacterial toxicity screening assays." " " As in the... 

Not all cyanobacterial blooms and scums contain detectable levels of toxins. Indeed, the incidence of toxicity detection by mouse bioassay, and toxin detection by HPLC among environmental samples, ranges from about 40% to However, in view of this high occurrence, it is the policy of regulatory authorities and water supply operators in some countries to assume that blooms of cyanobacteria are toxic until tested and found to be otherwise. In the absence of available analytical facilities or expertise or for logistical reasons, this precautionary principle should be regarded as sensible and prudent. 

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. 

It is important that the employment of the mouse bioassay and the interpretation of its results take these factors into consideration to ensure that the data produced are as reliable as possible and that the eventual conclusions are well founded. 

Figure 10. Calculated changes in the mouse bioassay toxicity of a sample initially containing 1 xmol of toxin C2. The horizontal axis represents the percentage of 11- -hydroxysulfate. The lower line indicates the toxicity of the sample with the 21-sulfo group intact, but with varying degrees of epimerization. The upper line indicates the toxicity of the corresponding carbamates, formed by hydrolysis of the 21-sulfo group.

Pharmacological Methods and Results. The data upon which the following discussion is based were accumulated using three techniques mouse bioassay, displacement of radiolabelled saxitoxin from rabbit brain membranes, and blockage of sodium conductance through rat sarcolemmal sodium channels incorporated into planar lipid bilayers. The results are summarized in Figures 11 and 12. 

The mouse bioassay for PSP, described in its original form by Sommer in 1937 (29), involves i.p. injection of a test solution, typically 1 mL, into a mouse weighing 17-23 g, and observing the time from injection to death. From the death time and mouse weight, the number of mouse units is obtained by reference to a standard table 1 mouse unit is defined as the amount of toxin that will kill a 20-g mouse in 15 min (77). The sensitivity of the mouse population used is calibrated using reference standard saxitoxin (70). In practice, the concentration of the test solution is adjusted to result in death times of approximately 6 min. Once the correct dilution has been established, 5 mice will generally provide a result differing by less than 20% from the true value at the 95% confidence level. The use of this method for the various saxitoxins and indeterminate mixtures of them would appear... 

N-1. In the mouse bioassay (Figure 11), 7 is slightly more potent than 1 in the... 

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... 

It is emphasized that some investigations show that bacterial cultures contain TTX-like substances which are not detected by mouse bioassay and are "difficult to detect" by HPLC and GC-MS analyses. Structural analyses of these substances with other techniques was not reported. 

Experimental evidence indicates that many marine bacteria produce TTXs. However, TTX production by some bacteria has not been validated since TTX and anhydro-like TTX are described as "difficult to detect" by using HPLC and GC-MS methods, and show no activity in the mouse bioassay. 

Deteimination of palytoxin. A mouse bioassay method proposed by Teh and Gardiner (25) for determination of L. pictor toxin (=palytoxin) was useful in our studies on the palytoxin-containing animals. The toxin amount in mouse units (MU,... 

Due to false positives, zinc may confound interpretation of the paralytic shellfish poisoning (PSP) mouse bioassay, one of the routine tests used to measure shellfish safety for human consumption. For example, mice injected intraperitoneally with extracts of healthy oyster tissues showed extreme weakness, a drop in body temperature, cyanosis, and some deaths (McCulloch et al. 1989). The threshold for a toxic PSP response corresponds to a drained tissue zinc level >900 mg/kg FW, and this overlaps the zinc concentration range of 230 to 1650 mg/kg FW (1900 to 9400 mg/kg DW) recorded in healthy oyster soft tissues (McCulloch et al. 1989). 

The relevance (and return on investment) for the bioassays preformed in mice have been questioned for some time. In 1997, ICH opened the possibility for the substitution of some form of short- or medium-term mouse test as an alternative for the traditional lifetime mouse bioassay. FDA has subsequently stated that it would accept validated forms of a set of medium-term mouse studies based on transgenic models, and significant effort has since gone into such validation. 

As performance data has become available on these strains, ICH (1997) has incorporated their use into pharmaceutical testing guidelines in lieu of the second rodent species tests (that is, to replace the long-term mouse bioassay when the traditional rat study has been performed). FDA has stated that they would accept such studies when performed in a validated model. In fact, CBER has accepted such studies as a sole carcinogenicity bioassay in some cases where there was negative traditional genotoxicity data and strong evidence of a lack of a mechanistic basis for concern. 

How wide and unconditional will FDA (and other regulatory bodies) acceptance be of these models in lieu of the traditional two-year mouse bioassay ... 

Figure 2.2 Three piperidine alkaloid teratogens from Conium maculatum (poison-hemlock) (a) coniine, (b) y-coniceine, and (c) A-methyl coniine, with accompanying LD50 as determined in a mouse bioassay.

The mouse bioassay, an indirect assay, historically has been used to evaluate shellfish toxicity (especially for PSP). Other bioassay procedures have been developed but not generally applied for regulatory purposes (Schantz et al., 1958). The mouse bioassay involves intraperitoneal (i.p.)... 

Indirect measurement (bioassay) Mouse bioassay Mouse bioassay Neuroblastoma assay (Truman et at, 2002) Mouse bioassay Protein-phosphatase inhibition assay (Mountfort et at, 2001) Mouse bioassay Cytotoxicity assay (Flanagan et at, 2001) Mouse bioassay Neuroblastoma assay (Matta et at, 2002)... 

Bioassay detection limit Mouse bioassay 20 pg SIX eq/100 g Mouse bioassay 20 pg STX eq/lOOg Neuroblastoma assay 2 pg STX eq/ 100 g Mouse bioassay 20 pg STX eq/100 g Protein-phosphatase inhibition assay 20 pg STX eq/100 g Unknown Unknown Neuroblastoma assay 10 ng/mL... 

Jellett, J.F., et al.. Detection of paralytic shellfish poisoning (PSP) toxins in shellfish tissue using MIST Alert, a new rapid test, in parallel with the regulatory AOAC mouse bioassay, Toxicon, 40, 10, 1407, 2002. 

The more classical approach to assess the presence of marine biotoxins in seafood is the in vivo mouse bioassay. It is based on the administration of suspicious extracted shellfish samples to mice, the evaluation of the lethal dose and the toxicity calculation according to reference dose response curves, established with reference material. It provides an indication about the overall toxicity of the sample, as it is not able to differentiate among individual toxins. This is a laborious and time-consuming procedure the accuracy is poor, it is nonspecific and generally not acceptably robust. Moreover, the mouse bioassay suffers from ethical implications and it is in conflict with the EU Directive 86/609 on the Protection of Laboratory Animals. Despite the drawbacks, this bioassay is still the method of reference for almost all types of marine toxins, and is the official method for PSP toxins. 

Despite the uncertainties in HOP for the toxins, there is reason to suspect that their mouse intraperitoneal potencies (MIP), the ri for the standard mouse bioassay system, do not bear a uniform relationship to them. Early pharmacological work ( ) on the paralytic shellfish toxins was conducted with shellfish extracts. 

The sulfamate saxitoxins have very low potencies relative to their carbamate hydrolysis products. This relationship has been observed in every assay system tried, including the standard mouse bioassay (Figure 3), squid giant axon (1 ), frog sciatic nerve (16), mammalian brain (1 ), and single rat sarcolemma sodium channels incorporated into lipid bilayers (15). It seems unlikely that human oral potencies are an exception to this trend. 

In an assay that offers acceptable HOPi/ri ratios for the carbamates, potential HOP can be estimated by preparing the sample under conditions that insure hydrolysis of the sulfamates. Unfortunately, the conditions specified for sample preparation in the standard mouse bioassay are not sufficiently acidic to insure complete hydrolysis ( ). As currently employed in state monitoring laboratories, the mouse assay may substantially underestimate the potential HOP of samples containing the sulfamate toxins. 

Bioassay. Toxicity of the materials was measured by the standard mouse bioassay for paralytic shellfish toxins and expressed by mouse unit (MU) as defined by the method (16). For testing the low toxin levels of algal specimens, extracts were treated with a charcoal column prior to injection into mice. 

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