NOTE: This is a very slightly edited version of an affidavit provided in evidence by Dr. Arpad Pusztai to India's Supreme Court. It usefully summarises research done worldwide on the toxicity of Bt crops. Dr. Pusztai, incidentally, is widely acknowledged as being the world's foremost nutritional expert on lectins, and the Bt toxin is a form of lectin.

EXTRACT: The MON 863 study revealed that feeding rats on transgenically expressed Bt toxin in maize caused kidney and liver problems in addition to interfering with the normal growth of young rats (Seralini et al. 2007). Bt toxin expressed in potatoes caused major changes in the small intestine of mice (Fares, N.H. and El-Sayed, A.K. (1998). Fine structural changes in the ileum of mice fed on delta-endotoxin-treated potatoes and transgenic potatoes (Natural Toxins 6, 219-233). The evidence for the survival of the Bt toxins in the digestive tract and internal organs is clear-cut.

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Dr Arpad Pusztai, FRSE

AFFIDAVIT

I am afraid, we come back to the same old problem: the absence of data in the submission for allowing the large scale field trial of Bt brinjal (eggplant) makes it impossible to formulate a critical evaluation of the proposal. Unless there is now access to the GEAC* arguments (in favour of the Bt brinjal large-scale field trials) based on actual results, hard data and SCIENCE and not PR, one can only exchange opinions and insults with the GEAC. Their assurances that they have these results but will not disclose them to us for evaluation are worthless. (*the GEAC is India's apex GM regulatory body)

In contrast, there is now plenty of evidence that some Bt toxins are harmful for insects by binding to surface receptors in the digestive system. Evidence that lectin binding to the digestive system of insects is the main reaction mechanism for the insecticidal effect of lectins in transgenic plants is now generally accepted. The evidence for binding of the various Bt toxins to the digestive system of insects is particularly strong (I. Gomez, D.H. Dean, A. Bravo and M. Soberon: Molecular basis for Bacillus thuringiensis Cry1Ab specificity: Two structural determinants in the Manduca sexta Bt-R1 receptor interact with loops -8 and 2 in domain II of Cry1Ab toxin. Biochemistry 42, 10482-10489 (2003); J.L. Jurat-Fuentes, L.J. Gahan, F.L. Gould, D.G. Hechel and M.J.Adang: The HevCaLP protein mediates binding specificity of the Cry1A class of Bacillus thuringiensis toxins in Heliothis virescens. Biochemistry 43, 14299-14305 (2004); P.J.K. Knight, J. Carroll and D.J. Ellar: Analysis of glycan structures on the 120kDa aminopeptidase N of Manduca sexta and their interactions with Bacillus thuringiensis Cry1Ac toxin. Insect Biochemistry and Molecular Biology 34, 101-112 (2004); X. Zhang, M. Candas, N.B. Griko, L. Rose-Young and L.A. Bulla, Jr: Cytotoxicity of Bacillus thuringiensis Cry1Ab toxin depends on specific binding of the toxin to the cadherin receptor BT-R1 expressed in insect cells. Cell Death and Differentiation 12, 1407-1416 (2005); X. Zhang, M. Candas, N.B. Griko, R. Taussig and L.A. Bulla, Jr: A mechanism of cell death involving adenylyl cyclase/PKA signalling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis. Proceedings of the National Academy of Sciences US 103, 9897-9902 (2006); H. Barros Moreira Beltrao,de and M.H.Neves Lobo Solva-Filha: Interaction of Bacillus thuringiensis svar. israelensis Cry toxins with binding sites from Aedes aegypti (Diptera: Culicidae) larvae midgut. FEMS Microbiology Letters 266, 163-169 (2007); L. Pardo-Lopez, I. Gomez, C. Rausell, J. Sanchez, M. Soberon and A. Bravo: Structural changes of the Cry1Ac oligomeric pre-pore from Bacillus thuringiensis induced by N-acetylgalactosamine facilitates toxin membrane insertion. Biochemistry 2006, 10329-10336 (2006).) The Bt toxin directly and specifically binds glycolipids and this binding is carbohydrate-dependent and relevant for toxin action in vivo (J.S. Griffitts, S.M. Haslam, T. Yang, S.F. Garczynski, B. Mulloy, H. Morris, P.S. Cremer, A. Dell, M.J. Adang and R.V. Aroian: Glycolipids as receptors for Bacillus thuringiensis crystal toxin. Science 307, 922-925 (2005)).

With the work of Vazquez-Padron and others, however, it has been demonstrated that Bt toxins bind not only to the insect gut but also to the mammalian gut, leading to various immunity problems. (Bernstein, I.L., Bernstein, J.A., Miller, M., Tierzieva, S., Bernstein, D.I., Lummus, Z., Selgrade, M.K., Doerfler, D.L. and Seligy, V.L. (1999). Immune responses in farm workers after exposure to Bacillus thuringiensis pesticides (Environmental Health Perspectives 107, 575-582) The claimed exclusiveness of the specificity of Bt toxin-binding to the insect gut can therefore no longer be maintained, as there is credible scientific evidence that some Bt toxins will also bind to the gut of mammalian species (A. Pusztai and S.Bardocz: GMO in animal nutrition: potential benefits and risks. In: 'Biology of Nutrition in Growing Animals' (ed. Mosenthin, R. Zentek, J.and Zebrowska, T.) 2006 Elsevier Limited, pp. 513-540).

The capacity of various A-B toxin-lectins, including Bacillus thuringiensis (Bt) Cry1Ac protoxin to stimulate and modulate both the systemic and mucosal immune systems is now firmly established (RI. Vázquez, L. Moreno-Fierros, L. Neri-Bazán, G.A. De la Riva and R. López-Revilla: Bacillus thuringiensis Cry1Ac protoxin is a potent systemic and mucosal adjuvant. Scandinavian Journal of Immunology 49, 578-584 (1999); Vazquez Padron, R.I., Moreno Fierros, L., Neri Bazan, L., De la Riva, G.A. and Lopez Revilla, R. Intragastric and intraperitoneal administration of Cry1Ac protoxin from Bacillus thuringiensis induces systemic and mucosal antibody responses in mice. Life Sciences 64, 1897-1912. (1999); Vazquez-Padron, R.I., Moreno-Fierros, L., Neri-Bazan, L., Martinez-Gil, A.F., de la Riva, G.A. and Lopez-Revilla, R. Characterization of the mucosal and sytemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice. Brazilian Journal of Medical and Biological Research 33, 147-155 (2000); Vazquez Padron, R.I., Gonzalez Cabrera, J., Garcia Tovar, C., Neri Bazan, L., Lopez Revilla, R., Hernandez, M., Morena Fierros, L. and De la Riva, G.A. Cry1Ac protoxin from Bacillus thuringiensis sp. kurstaki HD73 binds to surface proteins in the mouse small intestine. Biochemical and Biophysical Research Communications 271, 54-58 (2000)). In a more recent study the cellular immune response induced by Cry1Ac and its mutants in mice has been analysed (G.G. Guerrero, W.M. Russel and L. Moreno-Fierros: Analysis of the cellular immune response induced by Bacillus thuringiensis Cry1Ac toxins in mice: Effect of the hydrophobic motif from diphtheria toxin. Molecular Immunology 44, 1209-1217 (2007)). It was shown that the production of Th1 and Th2 type cytokines by Cry1Ac toxins was inhibited by N-acetylgalactosamine, in accordance with the lectinic properties of this Bt toxin.

Effect of Bt toxin on human cells: Tayabali AF and Seligy VL. Human cell exposure assays of Bacillus thuringiensis commercial insecticides: production of Bacillus cereus-like cytolytic effects from outgrowth of spores. Environ Health Perspect 108: 919-930, (2000).

The MON 863 study revealed that feeding rats on transgenically expressed Bt toxin in maize caused kidney and liver problems in addition to interfering with the normal growth of young rats (Seralini et al. 2007). Bt toxin expressed in potatoes caused major changes in the small intestine of mice (Fares, N.H. and El-Sayed, A.K. (1998). Fine structural changes in the ileum of mice fed on delta-endotoxin-treated potatoes and transgenic potatoes (Natural Toxins 6, 219-233). The evidence for the survival of the Bt toxins in the digestive tract and internal organs is clear-cut. Thus, it is expected that the situation with Bt brinjal will not be different. Accordingly, all the already described potentially harmful effects on consumers of the Bt toxins can also be expected to occur with Bt brinjal. As their release into the environment is an irreversible act, sanctioning such large scale field trials would be highly irresponsible.

Dr Arpad Pusztai
30 August 2007