Human protein assay developed as an alternative to the mouse LD50 assay for shellfish toxin

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Human protein assay developed as an alternative to the mouse LD50 assay for shellfish toxin

Published: August 20, 2008
The testing of biologicals represents a large segment of the animals used in toxicity testing. Much of this testing involves the mouse bioassay where the endpoint, LD50, is the dose that kills 50% of the test animals.

New Zealand scientists have developed a method for drug and toxin testing using a recombinant human skeletal muscle protein that provides a potential alternative to the mouse LD50 assay. Scientists involved in this project received the 2008 National Animal Ethics Advisory Committee (NAEAC) Three Rs Award for using this new in vitro method to develop an assay to detect algal neurotoxins. Seafood or water suspected of contamination by algal toxins is currently assessed for safety using the mouse LD50 assay. According to John Martin of the NAEAC, “this prototype has the potential to contribute significantly to the reduction of the use of animals in testing.”

Monitoring programs for algal toxins are conducted worldwide to screen shellfish and water for a variety of biotoxin types to protect public health. A World Health Organization (WHO) report concluded that: a) the mouse bioassays “have severe technical and ethical limitations and generally lack adequate validation for Codex [international food standards] purposes,” and b) “functional assays based on the common biochemical activity of a group of toxins are attractive alternatives to multi-toxin methods but few thoroughly validated assays are widely available” (WHO, 2004). The WHO panel recommended that Member states improve and validate shellfish toxin detection methods. The report from the 2005 European Centre for the Validation of Alternative Methods (ECVAM) Workshop, Three Rs Approaches in Marine Biotoxin Testing, noted the concerns of various stakeholders over the use of the animal bioassay for toxin detection, which include: a) animal welfare, b) lack of detection of all classes of toxins, and c) lack of the state-of-the-art methods in current practices (Hess, et al., 2006).

The new in vitro assay is based on the binding of the algal neurotoxins to voltage-gated sodium ion channel human skeletal muscle proteins. “Human voltage-gated sodium ion channels are major sites of action for drugs and toxins that modulate cellular excitability, and are therefore key molecular targets for…high throughput screening for new drugs and toxin detection” (Zhang, et al., 2007). This new test method is, therefore, based on a biologically relevant effect of the toxins in the human body. The in vitro assay is also much faster than the mouse bioassay, with results obtained in hours rather than several days.

The ion channel proteins must retain their biological function for use in this assay. The recombinant human voltage-gated skeletal muscle sodium channels are produced in insect cell cultures, and are then purified by affinity chromatography. The ion channel proteins retained their pharmacological function when incorporated into planar bilayer lipid membranes, and tetrodotoxin and saxitoxin inhibited the activity of the protein (Zhang, et al., 2007).

The researchers have proposed the use of this protein in the development of biosensors and high throughput screening assays for biotoxin detection. Dr. Julie Dalziel recently received a grant from the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) to continue research and development on the shellfish toxin assay, which she named the mini-mouse toxin detection assay. The grant will be used to “investigate the ability of this assay to determine the concentration of brevetoxins (activators) and saxitoxins (inhibitors).” The researcher’s goal is to develop this assay into a method capable of detecting a range of shellfish biotoxins by incorporating different types of ion channel receptor proteins into the membranes used for the assay.

Hess, P., Grune, B., Anderson, D.B., et al. (2006). Three Rs Approaches in Marine Biotoxin Testing. ECVAM Workshop Report 55. Altern. Lab. Anim. 34, 193-224.

Intouch. (2008). New toxicity test could replace animal testing. Intouch (March 2008) 36, 7. Available here.

WHO (2004). Report of the Joint FAO/IOC/WHO ad hoc Expert
Consultation on Biotoxins in Bivalve Molluscs. Oslo, Norway, Sept. 26-30, 2004. Available here.

Zhang, Y.L., Dunlop, J. & Dalziel, J.E. (2007). Recombinant human voltage-gated skeletal muscle sodium channels are pharmacologically functional in planar lipid bilayers. Biosens. Bioelectron. 22, 1006-1012.