|
||
|
THIRD WORLD NETWORK BIOSAFETY INFORMATION SERVICE 8 June 2005
RE: BT10 IS NOT THE SAME AS BT11 In mid-May, Food Standards Australia New Zealand (FSANZ), responding to increasing public pressure, released several documents that purport to support the FSANZ view that Bt10 is virtually identical to Bt11, according to Greenpeace Australia. Bt10, an unapproved and experimental GM corn, was inadvertently mistaken for Bt11, approved in some countries, and released by Syngenta from 2001-2004. FSANZ has argued that "the two varieties have been modified in the same way and produce the same novel proteins. The presence of a non-functional antibiotic resistance marker gene (BLA) in Bt-10 corn, that is not present in Bt-11, has no impact on the safety of food produced from Bt-10 corn." FSANZ has argued that because Bt10 is for all intents and purposes the same as Bt11 and Bt11 has been deemed safe for human consumption, then FSANZ is justified in taking no steps to remove potential Bt10 products from Australian supermarket shelves or to prevent possible continuing imports of Bt10 corn products. However, as the Syngenta documents released by FSANZ and the critique by Dr Jack Heineman of the NZ Institute of Gene Ecology at the University of Canterbury in New Zealand (Item 1) as well as the leaked documents received by the Institute for Science in Society (ISIS) (Item 2) show, these claims do not hold water.
"The Syngenta documents you have provided indicate that there are additional and possibly substantial differences between BT10 and BT11." Further, claims relating to the similarity of Bt10 and Bt11 cannot be ascertained from the materials released. "The report SSB-112-05 indicates that there were differences in the profiles of PAT and Cry1Ab proteins and thus there may be other undetected differences." The Syngenta documents are now available on the Greenpeace Australia website at http://www.greenpeace.org.au/features/features_details.html?site_id=45&news_id=1672 In a separate analysis, ISIS came to a similar conclusion after studying leaked documents that Syngenta sent to the US Environment Protection Agency earlier this year. The data suggested that "Bt10 is completely different from Bt11".
With best wishes, Chee Yoke Heong
Critique of Syngenta's documents
- Dr Jack Heinemann, New Zealand Claire Bleakley Re: Syngenta Biotechnology Report SSB-104-05 and SSB-112-05 on BT10 Dear Claire Thank you for forwarding these two reports to us. I have read the reports and will attempt to answer the question you presented in your email of 13 May, 2005. By way of background and outside the context of the documents you sent, it has been reported that the BT10 line differs from the BT11 in that BT10 retains the gene that confers resistance to the antibiotic ampicillin on some bacteria [Nature Biotechnology v23:514 (2005)]. I can find no reference to the resistance gene in the reports you sent. Since the insert in BT10 was sequenced for purposes of establishing its similarity to BT11, I assume that the antibiotic resistance gene has integrated elsewhere, or at least outside of the sequenced region. It is impossible for me to determine the importance of that integration event without knowing where it is, what genes it may have integrated into or what novel hybrid (or fusion) proteins might emerge from what is called a read-through into the ampicillin-resistance gene. In addition, it is a common phenomenon for transgene constructs to integrate in multiple places in the genome, and for very small parts of the construct to integrate independently of full sized versions (references for this statement can be found in our submission on A549 which is available on our website). Without knowing if, or how well, the genome of BT10 was inspected for potential secondary insertions of full or partial composition, I also cannot determine the relative equivalence of BT10 and BT11. Since the DNA for these studies was sourced from plants that were derived from a series of crosses to the original BT10 line, it is impossible to say how their genomes would represent the genomes of the plants (if indeed those lines are different) that have mixed with commercial BT11 lines, and whether by chance they have retained or lost the chromosomes that may carry other insertions. Impacts arising from uncharacterised insertions cannot be predicted from characterised insertions. Impacts from other insertions might be seen if BT10 had been subjected to a "substantial equivalence" test. While such tests do not prove equivalence, they can provide evidence of alterations in genes or genome function that result in changes to the overall normal composition of the organism. The documents you provided do not include such an analysis, nor do they discuss other "product-oriented" tests, such as the results of feeding studies. Again, without such information, I could not comment on the equivalence of BT10 and BT11. The reports do make clear several other differences between BT10 and BT11. First, the characterised insert in BT10 resides in chromosome 1 whereas in BT11 the insert is in chromosome 8. The context of the insert would obviously be different, as would be the genes that may be affected directly by the insertion. Second, there are three recorded basechanges in the sequenced part of the insert in BT10 that is common to BT11. These three nucleotide changes were the essence of your question to me, but my answer will include other context issues too. The context of the insertion is relevant for several reasons. As indicated above, the integration may have effects on adjacent genes by, for example, introducing a change in chromatin structure or inducing a change in methylation (which often is initiated de novo from duplicated DNA sequences that may arise from secondary but unknown inserts). While it is possible that the insert may reside in a region of no known function, (cis-acting) regulatory DNA sequences, for example those with weak promoter or enhancer activity, are still difficult to identify and we cannot exclude the possibility that one or more such sequences were changed as a result of the insertion(s) in BT10. Furthermore, read-through transcription-initiated somewhere in the insert and ending outside it, or initiated in adjacent regions and ending in the insert-may be the source of novel RNA and proteins. Abortive transcription from read-through might, for example, produce novel short and double-stranded (ds)RNA molecules. A risk factor emerging from the production of novel dsRNA is the potential to induce gene silencing either locally or on other genes (again, a reference list is provided in our submission on A549). The collection of DNA elements that have been used for the construction of the BT10 event have both known functions and probably unknown functions; the latter can only be identified by analyses that go well beyond DNA sequencing. It has recently been demonstrated, for example, that DNA sequences of the nos terminator, used in two places in the BT10 event (and perhaps distributed elsewhere in the genome), may be cis-acting alternative splice signals (see references to submission on A549). As you are aware, the DNA sequence is a template for the transcription of an RNA molecule. Less commonly appreciated is that the RNA molecule may undergo a large series of transformations, including splicing to remove introns, but also alternative splicing, that results in families of different RNA molecules all derived from the same original source. These families do not necessarily give rise to the same proteins or proteins with similar functions. While the nos terminator is not derived from an organism like corn, it may be processed by the enzymes that mediate splicing and generate novel RNA molecules in the process. It is in my view reasonable to query the effect of this cryptic splicing activity on the transcriptome of the BT10 lines; it is even more important given the nucleotide change reported in nos in BT10. The other two reported DNA sequence changes are adjacent to the 35S promoter used to initiate transcription of the cry1Ab(syn) gene. This is in a region called an "intervening sequence", presumably so-called because it has not been demonstrated to be transcriptionally active. I also presume that the assignment was made in the donor organism, not in the corn plant. The level of demonstration is not made clear in the report so it remains formally possible that the assignment of "intervening sequence" is based on a bioinformatic analysis and not empirical testing. I would have low confidence in this assignment if the conclusion were drawn only from bioinformatic analysis. Regardless of how it was determined that the DNA adjacent to the promoter was an "intervening sequence", since the DNA was sequenced it is reasonable to assume that it is part of the construct and new to that position in chromosome 1. Its potential to contribute novel cis-acting regulatory influences has not been tested in the set of experiments described in these reports and should not be dismissed without test, both because it is adjacent to presumably different genes in BT10 than in BT11, and because it is adjacent to presumably very different DNA sequences in chromosome 1 than in chromosome 8. It is clearly documented that introns can influence promoter activity and RNA processing reactions such as splicing and RNA editing, making premature the conclusion that this "intervening sequence" is functionally inert without a test. The Syngenta documents you have provided indicate that there are additional and possibly substantial differences between BT10 and BT11. If the position of the insert (chromosome 1 vs. 8) and effects of other contextual elements (such as the altered nos and intervening sequence) were of no consequence, I would expect that the profile of purified Cry1Ab and PAT proteins from BT11 and BT10 would be identical. If they are not, then we should, in my opinion, rule out other changes. The report SSB-112-05 indicates that there were differences in the profiles of PAT and Cry1Ab proteins and thus there may be other undetected differences. Interestingly, three bands are visible in the Western blot of Cry1Ab beyond the band that migrates at an apparent molecular weight of 68,900 Da. Two of these are acknowledged in the conclusion of the report, those migrating at an apparent molecular weight of 46 and the 52 kDa. Another unacknowledged band appears at approximately 67 kDa. The two identified bands of lower molecular weight are said to be "minor breakdown fragments", presumably of Cry1Ab protein. Nothing in the report confirms this identification and it is well within the abilities of protein science to make such a confirmation. It is also possible that they are proteins with a common epitope to Cry1Ab but derived from a different mRNA (due to alternative splicing or read-through). It is a concern that the relative concentration of these alleged breakdown products is very different in BT11 and BT10. The concentration of Cry1AB (69 kDa species) and the ~67 kDa species is nearly the same in both plants, but these bands are barely visible at the 46 and 52 kDa region of the blot for protein preparations isolated from BT10. (In fact, when I printed the report for my use I could not see these bands. They became only just visible when they were printed using a different printer!) I would expect these profiles to be identical unless the proteins detected were not breakdown products at all, but legitimately synthesised proteins of different mRNA transcripts. I also do not know whether their characterisation as breakdown products was ever challenged by regulatory authorities evaluating BT11. This new work by Syngenta, along with the very recent observation of transcription across the nos terminator and processing of transcripts that include the nos region, may provide reason to reconsider the safety analysis of BT11. Different inconsistencies are apparent in the PAT Western blot. The PAT levels in BT10 appear to be much higher than in BT11. This is not readily explained by the overall DNA sequence similarity between the BT10 and BT11 events. Moreover, the differences in concentration could indicate a higher overall stability of the PAT protein, and possibly Cry1AB protein (if it is true that the lower molecular weight bands are indeed breakdown products in BT11), produced by BT10. If indeed these proteins are inherently more stable, or delivered to consumers at higher concentrations, their potential to be allergens may be underestimated by studies using BT11 as the source. In summary, it is not possible for me to assess the importance of the three reported base changes in the characterised BT10 event, because the importance of DNA sequences is in part derived from their context and contribution to higher-order events on the RNA and protein level. While a difference of three nucleotides may seem small, we know that it may not be unimportant. Clearly the events are in different contexts (by being on different chromosomes) and the Western blots suggest, but do not prove (since they were semi-quantitative), that there are significant differences in expression profiles between BT10 and BT11. The New Zealand Institute of Gene Ecology is a public research centre that emphasises responsiveness to the research questions from the public, particularly those who do not have direct access to the resources to conduct their own research. We have conducted this analysis free-of-charge as part of the University's role as critic and conscience of society. Sincerely, Cry1Ab Syngenta recombinant
allele of a gene sourced from Bacillus
ISIS Press Release 18/05/05 Bt10 Detection Method Unacceptable The detection method for Syngenta's illegal GM maize is flawed; there must now be a full disclosure of information and access to reference material for retrospective risk assessment and risk management. Dr. Mae-Wan Ho and Prof. Joe Cummins Concerted move to reassure the European public Swiss biotech firm Syngenta had accidentally sold illegal GM maize Bt10 in the US for the past four years, resulting in about 133 million kilograms of the maize making its way into food and feed. The news broke on 22 March 2005 in the science journal Nature ("Syngenta's GM maize scandals", SiS 26), although Syngenta had entered into talks with the US government since December 2004. Under pressure from public protests across the world, the US government fined Syngenta a derisory US$375 000 (euro 270 000) for the mishap. And on 18 April, the European Commission imposed an emergency measure to ban certain GM maize imports from the US unless they are accompanied by an original analytical report issued by an accredited laboratory demonstrating that the product does not contain Bt10 ("Europe acts swiftly to keep out unapproved GM maize", SiS26). Scarcely a week later, the EU authorities announced that Syngenta had presented a detection test for Bt10, which was already validated by the EU authorities. The validation report [2] from the Joint Research Centre, also Europe's Community Reference Laboratory (CRL) for GM Food and Feed, said it carried out an in-house validation of the event-specific detection method "proposed by GeneScan on Bt10 maize developed by Syngenta Crop Protection AG." Syngenta provided the DNA samples (genomic DNA extracted from the Bt10 maize line and from a control maize line), and GeneScan provided the event-specific detection method based on a qualitative polymerase chain reaction (PCR) assay. Monopoly on detection method declared So who, or what is GeneScan? GeneScan advertises itself on its website as "the world market leader in the field of molecular biological testing for Genetically Modified Organisms (GMOs) in food, feed and agricultural raw materials." The GeneScan website has a link to a page on Syngenta's website, which advertises the "European Union Bt10 Detection Method" [3] as a "validated detection methodology that has been thoroughly tested for accuracy, reliability and sensitivity" using authentic samples to ensure actual targeted material is detected reliably when present. The method is designed, it says, to exclude "false positives" in the hands of "highly qualified scientific personnel with specific experience with the protocol", working under "exemplary laboratory practice and standard operation procedures (SOPS) from an ...accredited lab", with "provisions for retesting false positives". The same Syngenta page advises us that GeneScan is "the only private service laboratory that fulfils the elements listed above for Bt10 testing", and the fact that the EU Joint Research Centre has certified the GeneScan method on April 22, 2005 as "the only EU official method for Bt10 detection." Following that, yet again, the admonition to guard against "false positives" is repeated. In contrast, there's not a word said about false negatives, which as every molecular geneticist knows, is also a problem with the PCR detection method, particularly if the GM insert is unstable, and prone to deletions and rearrangements, as revealed in recent analyses by European government laboratories ("Transgenic lines proven unstable", SiS20; "Unstable transgenic lines illegal", SiS21). This three-way mutual reinforcement between Europe's Joint Research Centre (the European Commission's official laboratory), Syngenta and GeneScan seems just a bit too cosy to be reassuring. What's more, they have jointly declared a monopoly on the detection method, ruling out all others that could give "false positives". It is a case of the poacher turned gamekeeper with the help of the governor. The validation report issued by the Joint Research Centre (JRC) goes on to state [2], "The results of the JRC validation demonstrated that the method reliably detects an amplification product specific for Bt10 maize, and therefore allows discriminating event Bt10 from other GM-events in maize lines. The sensitivity of the method is below 0.1%..... "The method is therefore considered by the CRL as fit for the purpose of Bt10 detection and it is the only accepted to certify the presence of Bt10 in maize commodities in accordance with the Commission Decision 1005/317/EC). (emphasis added) When is a positive false? In fact, the method amplifies and detects a small 130base pair fragment of Bt10 DNA, said to be specific for Bt10. It is not stated which gene fragment from Bt10 is being amplified. A strict protocol is laid out in detail. The Bt10 and wild type DNA supplied by Syngenta were analysed along with other reference and non-reference material contained in the JRC's Community Reference Laboratory. The 130 bp band was indeed specifically amplified only in Bt10. But unfortunately, bigger bands were amplified and detected in other GM maize lines, and even in the wild-type maize DNA supplied by Syngenta. Strangely enough, these higher molecular weight bands were absent from the Bt10 DNA from Syngenta. The origins of the "unspecific amplicons" (amplified DNA) were not investigated further, but effectively dismissed with the remark, "This suggests that the method can be further optimised." Consequently, only the 130bp amplicon is regarded as a definite positive. The conclusion of the validation report states that the method is "fit for its intended purpose", with the qualification [3], "However, at this stage of testing, the method produces a higher molecular- weight multi-band pattern in GM and non-GM maize which requires additional efforts in its optimisation." Still further qualifications are contained in a later report [4] on the detection method: "The analyst shall be aware that other validation experiments indicated that the method might perform less reliably at annealing temperatures higher than specified in the protocol. Moreover, in some incidents unspecific amplification was observed with PCR profiles that used high numbers of cycles than specified in the protocol. Time constraints did not permit to rectify these concerns..." As mentioned earlier, fragmentation or rearrangements of the GM insert can change the size of the amplicon, or otherwise fail to give the specific amplicon. Consequently, unless fragmentation or rearrangement of the Bt10 GM insert can be ruled out, it is not legitimate to conclude that amplicons of other sizes are "false positives". Further data, further confusion Syngenta's reports sent to the US Environment Protection Agency earlier this year have been leaked to ISIS. The first report dated 28 January 2005 [5] is intended to present the DNA sequence of Bt10 compared with Bt11, the GM maize line that Bt10 had contaminated by accident. The Bt10 insert was mapped to chromosome 1 of the maize genome, while Bt11 insert had been mapped to chromosome 8. This alone will indicate that Bt10 is completely different from Bt11. In addition, there were three nucleotide changes in Bt10 compared with Bt11: two in an unspecified sequence contained within the Bt10 insert (unspecified sequence 1 in Figure 1 below), and one located in the nos terminator associated with the crylAb gene. No nucleotide changes were identified in any of the coding sequences and promoters within the Bt10 insert. However, the map of the Bt10 insert presented can only be partial, as it did not include the ampicillin antibiotic resistance marker gene, unless that marker gene has inserted elsewhere in the genome. The map presented also contained at least three unspecified, unknown sequences (Fig. 1). Unspecified sequence 1 (>1000 bp)-p35S (516pb)-IVS6 maize adh1S (477bp)-crylAb(syn) (1848bp)-tnos (267bp)-Unspecified sequence 2 (~400bp)- p35S(422bp)-IVS2 maize adh1S (180bp)- pat (522bp)-tnos (259 bp)-unspecified sequence 3 (~160bp) Figure 1. Map of Bt11 from Syngenta's report to US EPA The second report from Syngenta to the EPA is of a study comparing the transgenic proteins expressed in Bt10 compared with those in Bt11 [6]. The proteins were extracted from leaves of the plants, and subjected to western blot analyses, a technique dependent on staining the protein bands with specific antibodies after separating them by migration in an electric field through a gel matrix. This report claims that the analyses "revealed similar dominant immunoreactive bands" in both Bt11 and Bt10 corresponding to the predicted Cry1Ab protein (for insect resistance) and phosphinothricin acetyltransferase (PAT) (for tolerance to the herbicide glufosinate ammonium) of about 69 000 and 22 000 daltons respectively. However, the photographs of the western blots contained in the report tell a different story. Bt11 showed a series of bands at 46 000, 63 000 and 52 000 daltons (in order of strength of staining) besides the dominant 63 000 daltons band, whereas Bt 10 only had the 63 000 daltons fragment besides the main predicted band. The PAT protein bands in Bt10 and Bt 11 were also different from each other and from the purified standard, with many high molecular weight bands reacting to the antibody. Neither report contains information on the breeding history of the GM maize lines analysed, such as the number of generations since the transformation event; nor data from appropriate reference material. These are sure signs of sloppy science. Full disclosure of molecular data and access to reference material required The detection method for Bt10 is flawed by the admission of the European authorities. The identity of the 130 bp amplicon, supposed to be specific for Bt10, is not made explicit. The molecular data supplied to the US EPA are incomplete. It is impossible to judge if the detection method is adequate in the absence of full molecular data including those from reference material proving that Bt10 had remained genetically stable since it was first unintentionally released. Bt11 had already been exposed to be unstable, and to be contaminated with another Syngenta maize Bt176, implicated in the death of dairy cows in Hesse Germany ("Cows ate GM maize and died", SiS 21). Syngenta has admitted that Bt10, as distinct from Bt11, contains an ampicillin resistance marker gene, which, according to an Opinion issued by the Scientific Panel on Genetically Modified Organisms of the European Food Safety Authority in 2004, "should not be present in GM plants to be placed on the market". No official information has been forthcoming regarding the ampicillin resistance marker gene in Bt10, nor any attempt to ascertain whether the marker gene has contaminated other maize varieties, GM or otherwise. As Bt10 has already entered the market and the human food chain, it must go through retrospectively the risk assessment process that would have been applied to a GM product approved for market. This is also essential for effective post-release risk management. At the very least, Syngenta must be required to provide the following:
Furthermore, regulatory authorities on both sides of the Atlantic must make public all information on Bt10 that they have received from Syngenta or other sources. Please circulate this report widely and send it to your elected representatives.
|
||
