Proof of horizontal gene transfer
RNA molecules in GM food crops can enter the serum and organs of mammals
Date Added to website 19th June 2013
Well over a decade ago Prof Jack Heinemann took an interest in dsRNA-mediated gene silencing. In 2004 when he started actively engaging with regulators in Australia and New Zealand, he began advocating for testing for unintended dsRNA effects because of the possibility that intended and unintended dsRNAs might be transmissible through food. (This form of genetic manipulation was only discovered in 1998, and the work was recognized to be useful with a Nobel prize in 2006. However, the Codex Alimentarius safety assessment guidelines were written in 2003, and give no reference to this sort of genetic construct.)
Using assumption-based reasoning (that is, not using experiments to come to a conclusion), food safety regulators, including published scientists, dismissed the predictions, asserting that "the scientific evidence does not support the theory that RNA molecules in food can be transmitted" because they would be unstable during cooking, storage and digestion, unable to access mammalian cells or exert an effect on gene expression in mammalian cells (1). Prof Heinemann's repeated calls to test these assumptions, and those made by others to the Australian Office of the Gene Technology Regulator, were met with, effectively, silence.
In 2011, a landmark paper published in Cell Research by Zhang et al (4) proved these assumptions wrong (assumptions made, it should be noted, by the scientists serving both as regulators and as peer-reviewers for the regulator). Zhang and colleagues concluded that "these results indicate that exogenous plant [dsRNAs] are able to enter the serum and organs of mammals via food intake, and that plant [dsRNA] can bind to the nucleotide sequence located in exon 4 of mammalian LDLRAP1, leading to the inhibition of LDLRAP1 expression in vivo."
That dsRNA could cause silencing in humans was considered surprising to the science community just 12 years ago, because of assumptions that it should not work in us, and because initial attempts to make it work in human tissue culture cells failed (2). It turns out that these assumptions were wrong, just like the assumptions that dsRNA would not transmit through food, and the failure in this case was due to using dsRNA molecules that were too long. This suggests that we should not lead with assumption-based reasoning when it comes to risk assessment.
In such assessment, the first step is what is sometimes called a "hazards" analysis. This analysis attempts to identify anything new, or anything produced at new concentrations, that might be the cause of an adverse effect. For the effect to happen, there must be exposure to the hazard. If exposure is a possibility, then the developer can be asked to determine whether the adverse effect is possible (or even probable), and to determine as much as possible what the severity of the consequences might be. Then the regulator makes a determination of whether the potential adverse effect can be mitigated or eliminated. If it can, or if the regulator then determines that harm to be negligible, the product may be recommended for testing or use.
That makes early risk assessments by definition speculative. And that is a good thing. It would be best to have identified what should be tested while the adverse effect is speculative rather than after a product causes harm to human health or the environment.
It may be asked why, if people have been eating dsRNAs from plants (as shown by Zhang et al), should test be done on those created in GM wheat and other GM varieties. Prof Heinemann has already answered that question in a peer-reviewed and published article in the risk assessment literature. "Likewise, as GMOs modified by regulatory RNAs (e.g., dsRNA of the siRNA, antisense, and shRNA kind) grow in number, it will be critical to determine the on and off target effects of the novel RNA" (3).
The sequence and the structure of the dsRNA intended to be produced in the GM wheat has never been eaten by humans before. Prof Heinemann says: "We have no way of knowing if this molecule is benign, beneficial or harmful. We don't have a history of safe use of this wheat. "Neither overall amounts of small RNA molecules, nor the presence of benign small RNAs in conventional plants are sufficient as evidence that all novel small RNAs will be safe in the food chain or environment" (3). The potential effect is caused by the sequence and the sequence is unique to the GM wheat."
When Heinemann (5) and others recently questioned the Australian Office of the Gene Technology Regulator about what it required from the developer of a GM wheat variety using a new dsRNA molecule, the Australian and New Zealand versions of the Science Media Centre orchestrated a campaign of personal attack and deflection, using 'scientists' with no particular expertise or insight into this wheat to draw doubt on the need for such assessments.
Those 'science' commentators on this matter drew further, and more outrageous, risk CONCLUSIONS. Among other things, they claimed that the wheat was safe (in the absence of testing).
These conclusions from scientists should, of course, have been based on open, reviewed and accessible methodology. Indeed, to say that harm is unlikely is a claim that many would feel should have gone through anonymous peer-review and have been published in a journal. In reality, the "conclusion" is nothing more than an OPINION, lacking in support.
Interestingly, risk assessments are rarely if ever published in journals following an anonymous peer-review. For example, it is not FSANZ's policy to do so. Regulators use almost exclusively unpublished material from the developer at the time they make their decisions.
Heinemann retorted to the SMC and its chosen scientists: "Why are there no objections to the lack of anonymous peer review and journal publication here, especially as these assessments will actually determine what goes into the food supply?"
In contrast, Prof Heinemann's 2012 report on a risk assessment (5) met the same or higher criteria for peer review that regulators and the industry use routinely. Unlike their work, his report had no potential to put an unsafe product in front of the public, and was open to complete public review. Yet it was criticised for not appearing in a journal.
Worse than this, many commentators who addressed his report in the media chose ad hominem argumentation rather than discussing the outlined exposure pathways and the nature of the experiments that are needed to test legitimate hypotheses about what and how adverse effects could arise. They made mistakes, such as failing to check facts on where the target gene had come from. Importantly, not a single commentator offered any evidence that a risk assessment on the dsRNA molecules, or secondaries that might arise, was done by anyone. Much less did they offer even a single shred of data from any testing specific to this risk of the GM wheat being considered by OGTR.
Science routinely shows prevailing assumptions, such as those made earlier about dsRNA, to have been wrong. The proper response to challenges to assumptions is further research. This, not denunciation of the challengers, is the way to maintain public trust in the regulatory system, and in science.
For GM food crops (such as the GM wheat debated so hotly in Australia and New Zealand) there may be unintended off-target effects from RNAs hitting non-target genes in non-target organisms, following ingestion and transmission. (Note: The change intended to be introduced into the genetically modified (GM) wheat through genetic engineering was the production of novel RNA molecules that 'turn off' the expression of genes; these are called regulatory RNAs (a type of non-coding RNA). The vast majority of existing commercial GM plants (e.g., herbicide tolerant or insecticide traits) are not intended to make RNA molecules that are involved in gene regulation. This type of modification is therefore rare and has not benefited from extensive or validated safety testing procedures. )
What are the real implications of the recent research? It confirms -- not for the first time -- that horizontal TRANSFER OF GENETIC MATERIAL DOES actually happen, in spite of denials from GM developers and regulators. Secondly, it shows that unintended dsRNA molecules can be produced in any process of plant genetic modification, with unknown and unintended effects that might well impact on animal health. Thirdly, it shows that there is inadequate regulation in this field (6); this is now urgently required, if major detrimental effects are to be anticipated and avoided as a new generation of GM plants begins to emerge from laboratories and into field trials.
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NOTES.
1. FSANZ. 2006. Draft Assessment Report Application 549 Food Derived from High Lysine Corn LY038. Food Standards Australia New Zealand.
2. Gura, T. 2000. A silence that speaks volumes. Nature 404:804-808.
3. Heinemann, J. A., B. Kurenbach, and D. Quist. 2011. Molecular profiling — a tool for addressing emerging gaps in the comparative risk assessment of GMOs. Env. Int. 37:1285-1293.
4. Zhang, L., D. Hou, X. Chen, D. Li, L. Zhu, Y. Zhang, J. Li, Z. Bian, X. Liang, X. Cai, Y. Yin, C. H. Wang, T. Zhang, D. Zhu, D. Zhang, J. Xu, Q. Chen, Y. Ba, J.-J. Liu, Q. Wang, J. Chen, J. Wang, M. Wang, Q. Zhang, J. Zhang, K. Zen, and C.-Y. Zhang. 2012. Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA. Cell Res 22:107-126.
5. http://www.inbi.canterbury.ac.nz/Documents/Reports%20and%20others/Heinemann-Report-20120828.pdf
Evaluation of risks from creation of novel RNA molecules in genetically engineered wheat plants and recommendations for risk assessment An expert opinion of Professor Jack A. Heinemann, PhD, 28 August 2012 , for the Centre for Integrated Research in Biosafety
6. Comments from MADGE about the GM wheat line considered by FSANZ -- and the potential human risk.
http://www.madge.org.au/Docs/MR-110329-All-aboard-the-Hindenburg.pdf
http://www.madge.org.au/Docs/MR-110722-Bring-CSIRO-to-account.pdf
http://www.madge.org.au/madge-asks-csiro-gm-wheat-code
(Acknowledgements to Prof Jack Heinemann, from whose blog comments this note has been assembled.)