THIRD WORLD NETWORK BIOSAFETY INFORMATION SERVICE 

Dear Friends and Colleagues 

Gene Silencing Products Set to Enter the Food and Feed Chain 

A new wave of genetically engineered (GE) crops is set to enter the food and feed supply chain using an emerging technology called RNA interference or RNAi. RNAi induces a plant to silence the expression of its own genes, such as those responsible for natural processes like browning or lignin production. However, RNA manipulations may end up turning down or shutting off other genes in the plant, as well as affecting a cascade of genes including up-regulating some genes. Current testing requirements are unable to reliably detect such effects.

The Center for Food Safety (CFS) has alerted American consumers of the approval by the US Department of Agriculture (USDA) of two new products using RNAi technology (Item 1). The first is the Simplot GE potato, genetically engineered by the J.R. Simplot Co. to reduce browning and the production of acrylamide during frying and baking. It will soon be sold to unsuspecting customers as the US does not require labelling of GE food. The second is a low-lignin GE alfalfa from Monsanto, which would be used as forage for animals. The CFS questions the USDA’s failure to conduct the legally required rigorous and overarching analysis of the impacts of these new GE crops before approval.

Dr. Jack Heinemann from the Centre for Integrated Research in Biosafety (INBI), New Zealand raises concerns about the potential of double stranded (ds)RNA, which induces silencing effects such as RNAi, to cause human health effects and unintended environmental effects (Item 2). He cites the recent evaluation commissioned by the US Environmental Protection Agency (EPA) of dsRNA-based pesticide products, which raised many significant uncertainties about potential risks, and found that existing risk assessment frameworks were insufficient to evaluate them for safety.

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Third World Network

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A NEW FORM OF GENETIC ENGINEERING WILL SOON BE SOLD TO UNSUSPECTING CONSUMERS 

November 7, 2014 (Washington, DC) — Center for Food Safety (CFS) is today warning consumers about a new genetically engineered (GE) potato that may soon enter the food supply. Because GE foods are not required to be labeled, the new GE potato will be sold to consumers without their knowledge. The GE potato was one of two new crops approved today by the U.S. Department of Agriculture (USDA) that uses a new, little understood form of genetic engineering called RNA interference (RNAi). The other is a new low-lignin alfalfa from Monsanto. Despite the unprecedented nature of these approvals, USDA has inexplicably failed to undertake the legally required rigorous and overarching analysis of the GE crops’ impacts or reasonably foreseeable consequences.

“We simply don’t know enough about RNA interference technology to determine whether GE crops developed with it are safe for people and the environment.  If this is an attempt to give crop biotechnology a more benign face, all it has really done is expose the inadequacies of the U.S. regulation of GE crops. These approvals are riddled with holes and are extremely worrisome,” said Doug Gurian-Sherman, Ph.D., CFS director of sustainable agriculture and senior scientist.

Analysis of RNAi by a panel of independent scientists requested by the Environmental Protection Agency concluded that there were many significant uncertainties about potential risks from this technology, and that current risk assessment procedures were not adequate. Despite such cautions USDA is rushing the technology forward.

Unlike earlier genetic engineering techniques that splice in segments of DNA, the new technique used in the Simplot potato and Monsanto’s low-lignin alfalfa is based on the manipulation of the plant’s RNA-based control mechanisms. RNA interference (RNAi) induces the plant to silence or dial back expression of the plant’s own genes, such as those responsible for natural processes like browning or lignin production. However, RNA manipulations may end up turning down, or off, genes other than those that were targeted because many genes contain similar, or even identical, stretches of DNA. Current testing requirements do not reliably detect such effects on other important crop genes.

Developed by the J.R. Simplot Company, the potato would be the only GE potato variety on the U.S. commercial market. The Simplot potato has been genetically engineered with RNAi technology to reduce browning by silencing the expression of one of five polyphenol oxidase genes, which is normally highly expressed in potato tubers. This is attractive to the potato processing industry because bruised potatoes are culled for cosmetic reasons. However, bruised potatoes have not been associated with health risks.

These potatoes are also silenced for genes affecting sugar production and the amino acid asparagine, which during frying and baking lead to the production of acrylamide, a probable carcinogen. However, it is unclear whether the observed reductions will lead to positive health outcomes, given that acrylamide is found in many other foods. In addition, fried potato products have other serious negative health effects.

“In light of the obesity crisis, there has been an important national push to discourage children and adults from eating large quantities of fried foods like french fries or chips. In creating the false illusion that fried potatoes are now healthy, the Simplot potato sends the absolute opposite message,” said Elizabeth Kucinich, policy director at CFS. “Claims of health benefits by USDA and Simplot are short sighted, misleading, and in the light of the science, could actually be potentially dangerous.”

The asparagine gene has also been shown in recent research to be important in plant defenses against pathogens. The Simplot potato was not adequately tested for a possible weakening of its ability to defend itself against disease.  If this occurs in the field, it could lead to increased fungicide use, greater farmer expense, and possibly reduced productivity. The latter effect was seen in several tests of these potatoes.

“We need answers to these questions before these potatoes are commercialized,” said Gurian-Sherman.

Read CFS’s full comments to USDA here.

Concerns with Monsanto’s Low-Lignin Alfalfa:

Monsanto and Forage Genetics International (FGI) have genetically engineered alfalfa for reduced levels of lignin through the suppression of a key enzyme in the lignin biosynthetic pathway. It represents the first non-regulated GE crop with reduced lignin levels.  Lignin and its building blocks perform many functions in plants, including structural stability and plant defense.  Lowering lignin levels could make the alfalfa more prone to attack by insects or diseases, and potentially increase pesticide use.  Moreover, there are still many unknowns about how plants make lignin, making it premature to manipulate this important pathway. Additionally, alfalfa is a perennial crop and can cross-pollinate at great distances, allowing it to interbreed with other types of alfalfa. Any adverse impacts of the new variety will therefore be spread rapidly through much or all of the alfalfa seed supply.

Regulatory Failures:

By Dr. Jack Heinemann

The first intentionally pesticidal GM crop trait based on RNA is before food safety regulators for approval to release as a food for humans or feed for animals. Will regulators substitute their assumptions, seemingly validated by the bold statements of some scientists, for data on its safety?

This new trait is based on the expression of what are called double-stranded (ds)RNA molecules. These molecules influence the expression of genes. The types of dsRNA molecules relevant to this essay are also called, among other things, siRNA, miRNA and microRNA, and cause ‘silencing’ effects such as RNAi (interference), PTGS (post-transcriptional gene silencing), and DNA methylation. While we are most familiar with the ability of dsRNA molecules to either ramp down or prevent translation of a messenger RNA (mRNA) into a protein, dsRNA can also up-regulate some genes and can interfere with RNA that is not mRNA. For a technical review, see 1, 2, or 3.

The use of dsRNA molecules as gene regulators appears to be nearly universal in biology. Nevertheless, our knowledge of the biochemistry is still incomplete. Our ability to predict the cascade of genes affected by any particular dsRNA molecule is poor. Therefore, I hold the view that novel dsRNA molecules created through genetic engineering techniques or applied to the surface of crops in the form of pesticides should be tested for the potential to cause human health effects and unintended environmental effects ⁽¹⁾.

However, at least one food safety regulator, Food Standards Australia New Zealand (FSANZ), disagrees that novel dsRNAs in food could have human health effects. I find that regulator’s arguments for not undertaking a risk assessment unconvincing, but I do agree that there has been a lack of proof that ingested dsRNAs from plants have a biological effect on people (or mammals in general).

A key plank in the argument made by the food regulator and some scientists is that ingested dsRNAs are too fragile to survive digestion and therefore we cannot be ‘exposed’ to them in an active form. A second plank in the argument is that the concentration of dsRNAs in food would be too low to have an effect even if they were taken up. These arguments are rapidly losing their power to convince as a growing body of scientific evidence suggests that nature doesn’t share their assumptions.

Furthermore, risk assessment by food regulators does not take into account environmental effects. Therefore, their opinions are not relevant to the potential for adverse effects to arise in the environment and are not the final word when it comes to generating the full picture of GMO safety, for people or the environment.

The US Environmental Protection Agency (EPA) recently concluded a large-scale evaluation of dsRNA-based pesticide products and whether or not existing risk assessment frameworks are sufficient to evaluate them for safety. The answer, succinctly, was ‘no’.

Overall, the Panel agreed with the concerns raised by the EPA regarding the inadequacies of the current environmental fate and non-target effects testing frameworks for dsRNA PIPs [plant incorporated protectant] and exogenously applied dsRNA products. Uncertainties in the potential modes of action in non-target species, potential for chronic and sublethal effects, and potential unintended consequences in the various life stages of non-target organisms are sufficient justification to question whether the current Agency framework for ecological effects testing is applicable to dsRNA PIPs or exogenously applied non-PIP end-use products. Due to the modes of action of RNAi, no one set of test species will serve as an adequate representation of non-target species for all pesticidal products using RNAi technology. The classic approach of developing and assembling effects data for a standard set of test species will likely not work well for this technology.

The EPA evaluation involved a standing group of scientists taking both written and oral submissions from scientists and other interested persons from around the world. Despite the lack of definitive proof of biological activity from dsRNA molecules ingested by mammals, considerable uncertainty remains. This is because it is unlikely that dsRNA in actual food will be pure, the form used in laboratory studies, but will instead be protected by other kinds of molecules, like those that form ‘exosomes’. EPA therefore said that degradation of dietary dsRNA cannot be assumed. They “recommended experimental testing of the mammalian blood and exposed tissues be done to ensure that the siRNAs processed from the PIP dsRNAs are not present,” to confirm that they have been degraded “since these could have off-target effects after human consumption” (emphasis added).

Importantly, ingestion is not the only exposure pathway. The EPA also wants other exposure pathways such as via the lungs through inhalation, or through contact with skin or mucosa, to be tested. These pathways could produce very different exposure potentials. The EPA highlighted that other exposure pathways remain unexplored and may at times be more relevant than ingestion.

Moreover, the argument that RNA won’t survive digestion is hypothetical because there are few studies on dsRNA stability through digestion, and none that prove complete removal of dsRNA at the stomach acidity levels typical of different kinds of consumers. Consequently, the EPA Panel “recommended that the stability of dsRNA in individuals that manifest diseases, immune compromised, elderly, or children be investigated.”

The EPA also noted that bioinformatics and use of long dsRNAs does not guarantee absence of risk. “While ‘long’ dsRNA” which may be produced by the genetic engineering of the plant “may have no similarity to mammalian genes, processing of dsRNA into shorter siRNAs may present additional issues if these siRNAs have a high degree of similarity to sequences in non-target species including mammals.” The short active form may have many more targets than predicted from the intended longer form. “Chances of off-target binding increase as the siRNA becomes shorter and if sequences mismatches between target and off-target sites occur,” the EPA Panel said. Off-target effects can result in unintended silencing of other genes in animals or humans, potentially causing unanticipated adverse effects.

Thus EPA has come substantially to the same conclusions we did in our previous two publications, 1 and 2. This will be disappointing news for the likes of Profs. Rick Roush and Peter Langridge, who have publicly stated precisely the opposite without providing any evidence, via the Science Media Centre and other media outlets.

Probably more troubling to the scientists and regulators who have attempted to paint a one-dimensional picture of dsRNA risk assessment will be two new publications. The first demonstrates that dsRNAs (miRNAs) found in cow milk are biologically active in humans. The research, published in the Journal of Nutrition, found that the miRNA in the cow milk survived digestion and could alter gene expression. The authors said: “We conclude that miRNAs in milk are bioactive food compounds that regulate human genes” ⁽²⁾.

These are the very things that prominent regulators such as FSANZ, some scientists and the editor of Nature Biotechnology said couldn’t happen. The focus of their previous dismissals was a paper that reported that dsRNA of plant origin could be detected in human blood, alter gene expression in human cells in culture, and alter genes in the liver of live mice fed these plants (³⁾. The editor of Nature Biotechnology said that the detection of plant dsRNAs in human blood and mice organs “went against a large body of research in which the systemic administration of double-stranded RNAs was shown incapable of triggering the RNA interference pathway in humans (and mice).” However, the editor failed to acknowledge the full range of studies that report detections of exogenous dsRNA in humans and animals, e.g., Wang et al. (2012) ⁽⁴⁾ and Lam (2012) ⁽⁵⁾. This new study should raise more questions about the editorial objectivity of Nature Biotechnology, and the subjective views of some food safety regulators.

The first study showed that dsRNA in breast milk could be transferred via ingestion, survive digestion and cause changes in gene expression, invalidating claims that it could not. This next study provides the latest evidence that dsRNAs derived from plants are found in human and pig breast milk, packaged in exosomes ⁽⁶⁾. “Our study shows that plant miRNA molecules are abundant in human and porcine breast milk exosomes,” the authors said. The obvious recipients of these dsRNAs would be babies. This study establishes that novel dsRNAs introduced into plants or animals by genetic engineering, or sprayed onto plants as a pesticide, may very well survive digestion and accumulate in some tissues.

Our diets are full of dsRNA. It is a natural component of all life. Our diets are also full of proteins. However, no credible dietitian or toxicologist would suggest that if all proteins in a carrot are safe, then all proteins a carrot can be made to make would be safe. That is why risk assessments consider novel proteins in genetically modified food products. Yet this same argument is used to pre-determine the safety of dsRNA. For example, FSANZ says: “There is no scientific basis for suggesting that small dsRNAs present in some GM foods have different properties or pose a greater risk than those already naturally abundant in conventional foods.”

This kind of argument might have had some validity when we thought RNA was biologically inert except when used as a template for the production of proteins. But now we know that RNA has, when in double-stranded form, activities that transcend its informational content as a template. That activity is revealing surprises on a nearly daily basis. It is being pressed into service as a form of biotechnology product (sometimes even unwittingly, see: ⁷), and those products are meant for our food and environment. As my colleague, toxicology Prof. Ian Shaw, said on the Science Media Centre site: “currently data relating to dsRNAs and their effects are not required as part of the dossier supplied to a regulatory authority (e.g. FSANZ) as part of the regulatory process for assessing GM foods. Therefore, dsRNAs are not considered during the risk assessment. I agree with Heinemann et al; THEY SHOULD BE.”

Denying that dsRNAs deserve to pass a risk assessment based on valid measures of their potential to cause an adverse effect is only creating more unnecessary distrust in a technology that has some promise.

References

1.     Heinemann, J.A., Agapito-Tenfen, S.Z. & Carman, J.A. A comparative evaluation of the regulation of GM crops or products containing dsRNA and suggested improvements to risk assessments. Environ Int 55, 43-55 (2013).

2.     Baier, S.R., Nguyen, C., Xie, F., Wood, J.R. & Zempleni, J. MicroRNAs Are Absorbed in Biologically Meaningful Amounts from Nutritionally Relevant Doses of Cow Milk and Affect Gene Expression in Peripheral Blood Mononuclear Cells, HEK-293 Kidney Cell Cultures, and Mouse Livers. J. Nutr. 144, 1495-1500 (2014).

3.     Zhang, L. et al. Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA. Cell Res 22, 107-126 (2012).

4.     Wang, K. et al. The complex exogenous RNA spectra in human plasma: an interface with human gut biota? PLoS ONE 7, e51009 (2012).

5.     Lam, E.  (2012). WO 2012135820 A2. http://www.google.com/patents/WO2012135820A2

6.     Lukaski, A. & Zielenkiewicz, P. In silico identification of plant miRNAs in mammalian breast milk exosomes – a small step forward? PLoS ONE 9, e99963 (2014).

7.     Sanders, R.A. & Hiatt, W. Tomato transgene structure and silencing. Nat. Biotechnol. 23, 287-289 (2005).