Bioassay drawback

Although bioassays directly assess product potency (i.e. activity), they suffer from a number of drawbacks, including ... 

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. 

This chapter is concerned with the sampling, isolation, separation, and measurement of toxicants, including bioassay methods. Bioassay does not measure toxic effects rather, it is the quantitation of the relative effect of a substance on a test organism as compared with the effect of a standard preparation of a basic toxicant. Although bioassay has many drawbacks, particularly lack of specificity, it can provide a rapid analysis of the relative potency of toxicants in environmental samples. 

The major drawback of bioassay approaches is that they do not provide much information on the identity of the chemicals responsible for the toxicity of the sample tested. 

Important aspects bioassays as well as their types are discussed below. One of the main drawbacks with the bioassays is that some of them have long durations, which can delay the results. Hence, researches on new and faster bioassays are carried out. An interesting new possible bioassay involves the use of olfactory j3-waves that is, bursts of c.20 Hz fast waves that are elicited in the olfactory bulb and pyriform cortex in rats are observed.13 These fast waves are also observed in voles.14 These studies indicate that these /3-waves may provide an easy means of identifying new antifeedants in small herbivores. 

Mouse bioassays have been used in most laboratories to analyse microcystins in samples. The main drawbacks associated with these bioassays are related with the lack of sensitivity and specificity as well as a high variability on the results (Falconer 1993). Mouse bioassay has been used primarily to... 

Bioassay is the quantitative method in which the endpoint is an observable effect on a biological system or an organism. The classical approach to microbial detection involves the use of differential metabolic assays (monitored colormetrically) to determine species type in the case of most bacteria, or the use of cell culture and electron microscopy to diagnose viruses and some bacteria that are intracellular parasites. Samples taken from the environment, such as soil and water, and most clinical samples must be cultured in order to obtain sufficient numbers of various cell types for reliable identification. The time required for microbial outgrowth is typically 4-48 hours (or two weeks for certain cases, such as Mycobacterium tuberculosis). Furthermore, bacterial culture suffers from an inherent drawback cells that are viable may not be culturable, because they possess unanticipated nutritional requirements as a result of genetic mutation. 

On the other hand, a number of factors hamper the development and validation of analytical methodologies alternative to the biological bioassays. A serious problem is that accurate and readily available calibration standards are required for each of the various toxins. The lack of pure analytical standards and reference materials has always been—and still is—the major drawback both in shellfish toxin research and monitoring. Moreover, only limited progress has been achieved in the synthesis of these compounds. 

Owing to the difficulties in distinguishing between DSP toxins and pectenotoxins by the traditional mouse bioassay, several chemical analyses have been developed. These methods are based on liquid chromatography (LC) with detection by ultraviolet (UV) absorption [4-7,20,25,26], fluorescence (FL) [6,7,9-11,25-27], andmass spectrometry (MS) [5-8,10-21,23,24,28-36]. Amajor drawback of these LC techniques is the lack of analytical standards. In the present chapter, chemistry, metabolism, and chemical detection methods of pectenotoxins are described. Pharmacology and toxicology of pectenotoxins will be discussed in other chapters. 

However, the use of these transgenic plants does have several drawbacks. First, the rate-limiting step for this bioassay is the absorption and translocation of the POPs into plants. POPs with high liposolubihty are not easily absorbed and translocated into plants. In particular, absorption of POPs from weathered soil and soil with a high organic content is difficult. Cucurbitaceae species, which are known to actively absorb some POPs, may be useful for this bioassay [25]. Second, climate-independent assay is required for consistent results. Of course, assay results will have to be fuUy validated by comparison with the results of the... 

The Association for Official Analytical Methods (AOAC) has approved the mouse bioassay [183] as a standard method for the screening of PSTs in seafood. It involves the intraperitoneal injection of 1 ml acid extract into a 20 g mouse, and the recording of symptoms and time to death. One mouse unit (MU) is defined as the toxin amount that kills a 20 g mouse in 15 min, which is equivalent to 0.18 mg saxitoxin. An obvious drawback of this method is animal cruelty. In addition, this method is not specific for PSTs, and it is prone to interference from substances that are imrelated to PSTs. Also the dose-response relationship is not always linear. 

Currently, in the EU and worldwide, the monitoring system of choice for most of the toxins is the mouse bioassay [4r-6]. Either for lypophilic or hydrophilic toxins, an extract is injected into three mice, for which the dead of two out of three mice is considered a positive result. In that case, extraction and industrial processing is stopped. The main advantage of the mouse bioassay is, by far, its ability to detect anything toxic. On the other hand, this advantage also serves as its main drawback, and has created legal complications (as is the case of YTX), for compounds that are not toxic but show only interferences that cause false positives. 

Important drawbacks of the bioassay are the interferences of high-salt concentrations, low sensitivity, high variability with high concentrations of toxins, an inherent variability of +20%, and, more important, the necessity of large amounts of mice can make it very expensive. Large number of animals are sacrificed which results in an ethical problem. 

Bioassays have been used to confirm that Lact. reuteri CRL 1098 produces Cbl in the order of 50 ig/l, which is a modest amount compared to the values obtained by the strains used in industrial production that surpass 200 mg/1 (Martens et al. 2002). However, these values could be considered adequate when the daily requirement of vitamin is taken into account (2-3 pg for an adult) because they could be sufficient for the development of Bj bio-enriched foods with. A possible drawback in the technological development is that the major form of the corrinoid produced in anaerobiosis by Lact. reuteri is pseudocobalamin (Santos et al. 2007) which, although present as a co-enzyme in many anaerobic and facultative anaerobic bacteria, is not active in animals and humans (Brandt et al. 1979 Rucker et al. 2001). Our studies indicate that in microaerophilic conditions Lact. reuteri CRL1098 not only produces pseudo-B, but also a small amount of other corrinoids including co-enzyme B (Vera 2007). 

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