Toxicity mouse bioassay

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. 

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.

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 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.)... 

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. 

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. 

Screening for toxicity Toxicity of the crude acetone soluble and insoluble products was determined using the mouse bioassay. Measured quantities of these products were administered via intraperitoneal injection into outbred Harlan Sprague Dawley ICR (BR) mice weighing approximately 20 g. The carrier was 1% Tween 60 in 0.15M sodium chloride and the injection volume was 0.5 ml per mouse. Extracts which caused death of mice within 48 hours were deemed toxic. However, it was apparent that some fractions were toxic even though death was not observed. 

The results of TLC indicated that concavum produced at least three and possibly as many as five separate toxins (Table V). The three relatively distinct toxic components were designated PC-1, PC-3, and PC-5. Results from TLC did not clearly distinguish PC-2 or PC-4 from PC-5. However, mouse bloassay of these fractions (from preparative TLC) revealed that PC-4 was a fast-acting toxin in contrast to the slow-acting PC-2 and PC-5. Of course, the variation in results of the mouse bioassays could have been a consequence of different amounts and levels of purity of the respective fractions. Regardless, preliminary results from TLC and bioassay suggest that PC-2 may be the same as PC-5, the former being a water soluble carryover in the initial ether fraction. 

Currently, all five toxic components from concavum are being subjected to column and high performance liquid chromatography. Products of these treatments will be evaluated and characterized using the mouse bioassay and isolated nerve-muscle preparations. 

Mouse Bioassay Procedures. For initial screening and LD50 determinations outbred female swiss mice (Harlan Sprague Dawley ICR BR ) weighing between 19 to 21 g were used. Doses of toxic extract were suspended in 0.5 ml of 0.1% Tween 60 in 0.15 M NaCl and administered by i. p. injection. Mice were observed for a period of 48 hours. 

Mouse Bioassay. When administered at 5 mg per mouse in 0.5 ml dose during the initial screening, the WSAP from G. toxicus caused death in all test mice within 120 minutes. The toxin had a latency period of approximately 30 minutes after which signs of toxicity were noticeable, and included in order of occurrance inactivity and piloerection followed by cyanosis of the tail and feet with concurrent hypothermia, vasodilation in the ears ( scarlet ears ), lacrimation, ptosis of the eye lid on the side of injection, ptosis of the abdomen (loss of muscular tone), asthenia, impairment of hind limb motor ability followed shortly by complete paralysis with the hind limbs extended posteriorly (complete prostration), and dyspnea (respiratory distress). Death occurred without convulsions and the eyes became cataracted just prior to or after death. 

A mouse bioassay method is useful for screening of the toxicity of various organisms from affected areas, although this method may not be sufficient for the identification of the toxin. Recently, distribution of tetrodotoxin in the marine ecosystem has expanded from puffer-fishes to some other animals. Rapid and accurate determination of the toxin occurring in those organisms is becoming increasingly important from the public health standpoint. 

On the other hand, some pufferfishes which have not been eaten before, are sometimes marketed in Japan. In our mouse bioassay test a high toxicity was detected in the tissues from Tetraodon alboreti-culatus, one of such pufferfishes 2870 MU/g ovary and 31 MU/g liver ( 5). Thus, re-examination seems to be necessary for toxic potency of the pufferfishes landed from Japanese and adjacent waters. 

Hanna PA, Jankovic J, Vincent A (1999) Comparison of mouse bioassay and immunoprecipitation assay for botulinum toxin antibodies. J Neurol Neurosurg Psychiatry 66 612-16 Hanson MA, Stevens RC (2000) Cocrystal structure of synaptobrevin-II bound to botulinum neurotoxin type B at 2.0 A resolution. Nat Struct Biol 7 687-92 Harlow ML, Ress D, Stoschek A, Marshall RM, McMahan UJ (2001) The architecture of active zone material at the frog s neuromuscular junction. Nature 409 479-84 Harris JB (1997) Toxic phospholipases in snake venom an introductory review. Symp. zool. Soc. Lond. 70 235-50... 

The MS profiles of McTx-A and McTx-B were both very similar to the respective profile of reference palytoxin, but the estimated molecular masses (between 2500-2535 Da) were lower than that of reference palytoxin (2680 Da) or other palytoxins and ostreocin-D. Nevertheless, the MS profile and fragmentation patterns of McTx-A and McTx-B together with mouse bioassay symptomatology and delayed haemolytic activity confirm the palytoxin-like character of these compounds. Quantitative differences in the hemolytic action and mouse lethality, as well as minor deviations in the MS spectra and retention times, could be attributed to structural variations between mascarenotoxins and the reference palytoxin (Lenoir et al. 2004). This is also supported by Usami et al. (1995), who showed that small changes in the structure of palytoxin analogues can have an impact on mouse toxicity, haemolytic potency, and cytotoxicity. 

In Scotland, during 1981-1982, cyanobacteria from several lakes were found to be toxic by mouse bioassay (i.p.) (Richard et al. 1983), but the first case of AN intoxication due to benthic cyanobacteria was reported 10 years later (Edwards et al. 1992). The neurotoxic bloom consisted mainly of Planktothrix species and was associated with three canine fatalities AN was identified in the stomach contents of one of the dogs (Gunn et al. 1992). In Finland, the first survey of cyanobacterial blooms during 1985-1987 revealed that 13 out of 30 bloom samples contained AN (Sivonen etal. 1989). Toxin content was in the range 12 360 pg AN/g lyophilized material. During this... 

Although there can be no doubt that some PTXs are toxic to mice by i.p. administration, the question of whether these compotmds are orally toxic, or cause diarrhoea when administered orally, is more controversial. There have been relatively few oral dosing studies, due to the scarcity of high-pruity PTXs, and the results have not been consistent. However, recent evidence suggests that PTXs are weakly diarrhetic at most and are of relatively low oral toxicity. Based on their toxicity by the i.p. route in the mouse bioassay, PTX-1 and PTX-2 are currently regulated in shellfish together with okadaic acid... 

The semipurified toxic fraction obtained from this alga and selected on the basis of a mouse bioassay was applied to LC-diode array detection (LC-DAD) and LC/electrospray-MS (LC/ESl-MS) analysis. The UV absorption at 259, 269, and 280 mn due to the conjugated triene of PA, PA2, and PA3, and X at 231 mn due to the conjugated diene of PB2 were used to detect them by... 

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