Heinemann has previously provided evidence concerning the risk assessment and hazard identification of GM crops and in particular the appraisal of Monsanto’s bio-safety dossier of Bt brinjal (molecular analyses) for the Supreme Court. He was also one of the independent academic scientists who responded to Shri Jairam Ramesh in his review process of Bt brinjal in 2009-2010. Heinemann’s credentials are well known. This document is his further analyses of the molecular characterisation of Bt brinjal and follows from a request that I (AR) made last year (ref. the Preamble). It seemed appropriate to undertake a deeper investigation of Bt brinjal Event EE 1 given the disquiet generated as a result of several separate appraisals of Monsanto’s dossier by leading international scientific experts, which pointed to a shoddy environmental risk assessment; of studies claimed to have been done, but were not done or poorly done and significant health safety issues which raise concern. All were comprehensively denied by the regulators and the EC II (Expert Committee II appointed by the apex regulator the GEAC to deal with various analyses of the Dossier and objection received to it). The clear need for long term testing for chronic toxicity (cancers and other problems) was also turned down, but which Shri Jairam Ramesh required when he imposed an indefinite moratorium on Bt brinjal pending independent scientific and long term studies. This is that further report of Prof H.
I am astounded at Heinemann’s findings and their serious implications. At the outset, Heinemann says that a “critical and fundamental characterisation of the event was not completed, usually because of assumption-based reasoning. When such fundamental misunderstandings of the basic tools of the procedure were demonstrated by the developer, (which) seemingly went unchallenged by the regulator, it was very difficult to accept assurances that the other procedures in the evaluation of Bt brinjal could be trusted”. In other words if we may draw a very crude analogy, this stage of the risk assessment of a GMO (the molecular characterisation) is akin to the first steps of learning to read as in CAT= cat and MAT= mat. There is also a curious divergence between the Monsanto reference work of 1997 and their Bt brinjal dossier. Did the regulators not bother to even check Monsanto 1997? Without going into undue detail (please see pages 6-9), I draw your attention to the following key points of Heinemann’s analyses along with my comments.
- At this very first stage of the risk assessment and testing of a GM crop, (the molecular testing and characterisation) it is now apparent that there has been no regulatory oversight at all by either the RCGM nor the apex regulator the GEAC. The EC II report was as unreliable for safety oversight.
“The commercial trait in Bt brinjal is conferred by the Cry1Ac-like derived protein. This protein is not Cry1Ac isolated from natural plasmids of B. thuringiensis v. kurstaki, but a protein made from a series of in vitro modifications. —The GE “plasmid was the vector PVLEBK04, also called pMON10518 (Monsanto, 1997). Taken together, PV-LEBK04 has at least 10 different DNA elements that have been taken from different species, including soybeans, viruses, plasmids isolated from different species of bacteria, and many of which have also been extensively and separately subject to in vitro modification after being taken from their natural sources (see Table 2 of Monsanto, 1997)”.
- Chimeric or fusion gene: The Bt B gene is claimed as a fusion gene of Cry1Ac and Cry1Ab. Monsanto and the EC II report claimed that Bt B event EE1 was 99.4 % identical in amino acids to the natural Cry1Ac protein (p. 33 of Mahyco, 2008). There is no mention in the Dossier or the EC II report of the multiple origins that form the fusion (see above). They further went on to say this corresponded to only a difference in 1 amino acid. This is astonishing for other reasons than there can be no safety comfort derived from ‘only. However, Dr Bhargava very quickly corrected this fundamental error to say that on this basis, the difference was in fact 7 amino acids. Prof Michael Antoniou confirmed this as easily and as quickly. This is how basic such a calculation is to a molecular biologist. So what does this say of the GEAC, the apex regulator? (The RCGM/DBT have become the official vending machine dispensing GM crops and are currently embarked openly, on aggressively pushing formal public-private partnership agreements to provide the vehicle to promote GE crops, thereby, formalising corruption.
Heinemann says: ( EC II described the construct in Bt brinjal as composed of (the first) 466 amino acids from Cry1Ab and 712 amino acids from Cry1Ac (amino acids 1178 to 467). The regulator said in EC II: “This difference of 0.6% is attributed to the difference in presence of one amino acid at position 766 i.e. serine in place of leucine.”
However, as noted earlier, currently registered sequences for these proteins do not support that they were this similar. Moreover, a single amino acid difference in a sequence of 1178 amino acids would have been 0.1% rather than 0.6% as the scale of difference between the fusion and nature reported by the developer and accepted by EC II. Neither the original Monsanto dossier nor the EC II numbers support the EC II conclusion that the fusion had a single amino acid difference from what is found in nature (JH).
“At 99.4% identity, there would be approximately 7 amino acid differences between the chimera fusion and natural Cry1Ac (consistent withoriginal description by Monsanto, 1997)”.
“However, when I construct the fusion using sequences published in Genbank, I find that the fusion is a maximum of 94% identical to Cry1Ac (GenBank: ABD37053.1) and only 95% identical to Cry1Ab (GenBank: ABV91087.1). Based on these matches, it is also not clear why the developers have historically called their fusion construct after cry1Ac rather than after cry1Ab, or more precisely, cry1Ac-like to accurately identify it as a product of modern biotechnology formed as a chimera of multiple origins. Thefirst 40% of the amino acids found in Cry1Ac2 were replaced with 466 amino acids from Cry1Ab, another insecticidal protein.
- Furthermore, the developer reports that a leucine residue at position 766 has been replaced by a serine residue in planta. What appear to be small differences can be physiologically and immunogenically important. The change from leucine to serine at position 766 is of interest to the biosafety investigator because only the latter can be O-linked glycosylated by the addition of N-acetylgalactosamine through a side chain hydroxyl group (Mitra et al., 2006). “O-linked glycosylation has a profound effect on the antigenic properties of peptides—-“
- The implications of this are that the fundamental affinity-based tools used by developers to list and characterise all forms of the protein produced in the plant (e.g., western blots) may be compromised if they were not trained on the glycosylated forms that the plant may produce. All subsequent studies that are based on isolated proteins (e.g., digestibility, toxicity, and cooking studies) may then be invalid because they only have available to them a subset of the relevant protein isoforms that may be in the plant.“
- · At 94% identity (consistent with current GenBank comparison), there could be up to 70 different amino acids. To conclude that a novel protein is likely to be of no safety concern because of even as few differences as 7 amino acids is not a research-based conclusion. Changes of single amino acids can significantly alter the characteristics of proteins (a fact that underpins the field of directed evolution) —–. One of the characteristics that can be changed is immunogenicity. For example, several groups reported significant decreases of IgE binding to a major peanut allergen after mutating single nucleotides. Even more surprising, in some cases even a synonymous (i.e., differences in the nucleotide sequence of a gene that do not alter the resulting amino acid sequence) coding change can alter the characteristics of a transcript or protein, or levels of expression A single nucleotide polymorphism that results in a synonymous change can change the substrate specificity of the resulting protein, potentially by affecting its folding patterns during translation. Furthermore, sequence identical proteins with differing tertiary structures can turn benign proteins into toxins –or agents that cause pathogenesis as demonstrated for the Prp proteins causing Creutzfeld-Jacob disease and mad cow disease
These are startling findings. They mean that Monsanto’s dossier is fundamentally flawed at its starting point. This is to put in politely. Without overstating the case, the more correct description is, fraudulent. There also appears to have been a deliberate cover-up of differences with their original doc of Monsanto 1997 and their Bt brinjal dossier. However, fraudulent dossiers are nothing new for Monsanto. There have been similar cases with other products in the EU and New Zealand. This kind of behaviour is to be expected from a company that has been formally indicted for bribes and cover-up, hounding farmers into bankruptcy, false advertising both in France where they had to retract and more recently in Vidharba for paid advertising and lies. Monsanto has also been indicted for some of the worst crimes against humanity. This is part of the formal record.
But what of our regulators and in particular the GEAC? The charges against Monsanto in India for illegal field trials, illegal plantings, are serious enough to have pushed even the GEAC into the action of serving a show-cause notice, seemingly impotent. The recent case of the supposedly Desi BN cotton gene that has a Monsanto gene discovered in it, merely underscores the case that we have no regulation of any value that serves to protect India, her farmers and the public interest. As seriously, it betrays either a significant lack of competence or a conflict of interest so deep that there is a great facilitation to promote GM crops and Monsanto especially, mortgaging the public interest. This means that we have had 15 years of unsafe field trials and accelerating in the last 2-3 years to encompass numerous Bt genes in virtually all our food crops and including the failed technology in herbicide resistant crops. These greatly risk our bio-safety and indigenous seed stock because of irreversible contamination, which is the main reason why untested open field trials are not acceptable.
Finally, the performance of Bt cotton is being hyped in order to prepare the way for other Bt crops. But without reliable statistics and a PMM(post market monitoring) of Bt cotton which should have been instituted by a conscionable regulator years ago, there is no evidence for the claims being made by Monsanto and the government regulator and other public institutions. Dr Kranthi ‘s analyses of the performance of Bt cotton shows the onset of ‘resistance’ and the experience of other countries where insect resistance to Cry proteins is proven (ref the recent US EPA report on Bt corn 863) underscores the fact that this was a predicted outcome.