Environmental Engineering Reference
In-Depth Information
5.1
Bioassay Methods
5.1.1
In Vivo Mouse Bioassays
Mouse bioassay (MBA) is the only assay that measures the biological response of
whole animals, thus allowing some correlation with human toxic effects. This method
was fi rst applied by Sommer and Meyer in
1937
. The MBA is an internationally
recognized analysis method for PST, and has been standardized by the Association
of Offi cial Analytical Chemists to offer quick and accurate measurements (AOAC
1995
). The advantages of this assay are that it is cheap, simple and accurate. However,
it does not provide information on the specifi c toxin composition. Moreover, the use
of live animals in this procedure is required and therefore is a growing challenge
vis-a-vis animal ethics. The results of this mouse bioassay can be confounded by
the presence of high salt content, the presence of metals and gender of the mouse.
A high salt concentration suppresses the toxic effects by 20-50% (Schantz et al.
1958
),
while the presence of zinc results in overestimates of toxicity (Aune et al.
1998
).
Aune et al. (
2008
) found that the neurotoxin concentration required to kill a female
mouse was sevenfold higher than that for a male mouse of the same weight.
5.1.2
In Vitro Cell Assays
The basic principle of
in vitro
cell assays is to lace two biologically active drugs
(veratridine and ouabine), in combination, into an established neuroblastoma cell
line culture, then add the toxin that is to be tested. Veratridine is a sodium channel
activator that causes infl ux of sodium, leading to cell swelling and eventually cell
lysis. Ouabine blocks the action of Na/K-ATPases. The presence of the toxin,
e.g., tetrodotoxin, acts as a sodium channel blocker, preventing the infl ux of sodium
ions and thus protecting the mouse neuroblastoma cells. Vital staining is used to
determine cell viability, and the intensity of color is proportional to the amount of
toxin present (Jellett et al.
1992
). The commercially available and shippable kit
(MIST: Maritie
In vitro
Shellfi sh Test) available for performing this analysis has a
3 week shelf life, and was developed by Jellet Biotek Ltd. (Dartmouth, Nova Scotia;
Canada) from a neuroblastoma cell bioassay (Jellett et al.
1998
). Three versions of
the MIST kit are available, and they vary by having different aims, detection accu-
racy and requirements for detection equipment. The Mini-MIST™ kit can only be
used for pre-screening for the presence or absence of saxitoxin. The semi-
quantitative version is designed to detect whether toxins in a sample exceed the
regulatory limit. The fully quantitative version of MIST has a sensitivity of 2
ʼ
g
STXeq/100 g for shellfi sh tissue, which is 20-fold more sensitive than the standard
mouse bioassay (Jellett et al.
1992
). This method, however, does not provide infor-
mation on the specifi c toxin composition and has a short shelf life of only about
2-3 weeks.
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