Why novel dsRNA molecules in GM food are of little to no concern

posted in: Science | 14

Recently, concerns were raised about the potential risks of dietary double stranded RNA (dsRNA) and microRNA (miRNA) molecules silencing human genes, after research by Zhang et al. showed the presence of plant miRNA in human blood plasma, as well as providing evidence that this plant miRNA enters the system by dietary uptake in mice. The group then demonstrated that this plant miRNA could silence genes in the mice, leading other researchers to separately raise concerns that diets consisting of genetically modified organisms could lead to the uptake of novel dsRNA molecules that could silence human genes.

Gene-silencing by RNA interference, or RNAi, typically occurs by way of short sequences of RNA which bind to a target messenger RNA sequence (mRNA) and inhibit it, either signaling the mRNA for deletion or inhibiting its expression. This occurs through perfect or near-perfect base pairing of the sequence to a short segment on the mRNA strand. Uptake of dsRNA or miRNA at levels that would lead to gene silencing would therefore be an important consideration in food safety, including the safety of GMOs.

Diagram detailing the creation of miRNA. From Wikimedia Commons.

However, recent studies have failed to reproduce these results and questioned the likelihood of dietary uptake of plant miRNA molecules in mammals. Witwer and other scientists from Johns-Hopkins showed that the plant miRNA discovered in human cells (miR160 and miR166) by Zhang et al. did not reflect the concentrations of miRNA in rice. In rice, miR166 levels were higher than miR160 levels, however, in humans, miR160 levels were substantially higher than miR166 levels. Witwer et al. showed that blood levels of these plant miRNAs were not appreciably changed after eating, and never resembled the levels present in the dietary sources. Reported levels of plant miRNA levels were also significantly lower and more variable than previously reported.

This raises questions of dose. Responses in biological systems vary dramatically with respect to dose, such as how drinking sea water can induce vomiting while a pinch of salt in a glass of water may be refreshing. Previous studies have established that levels of miRNA below 100 copies per human cell would have no impact on gene expression and therefore no regulatory capacity. Zhang et al. found 853 copies of miR160 per cell in mice, low compared to native miRNA levels but at levels with gene-silencing capability. The lower levels of miR160 reported by Witwer et al., along with the fact that plasma miRNA levels did not reflect the dietary sources and the high variation in observed plant miRNA levels makes it unlikely that dietary sources impact gene expression through miRNA uptake.

In addition, the amount of rice in the diet of the mice in the Zhang et al. study were abnormally high. The amount of rice fed daily to the mice would equate to a human eating 33 kg of rice/day, 50 times the daily amount of rice consumption in Brunei, which has the highest rice consumption per capita. Rice consumption per capita also sharply falls in other countries, making the diet in the study 70 times the daily consumption in Vietnam, the next country in terms of rice consumption. Since we can reasonably expect miRNA uptake levels to be proportional to the amount of rice consumed if diet is the primary source of these miRNA molecules, the reported 853 copies/cell would fall to an estimated 17 and 12 copies/cell in the average Bruneian and Vietnamese diets, respectively. The levels reported by Zhang et al. would there fall well outside the range of significant biological activity in a typical diet, and therefore should not impact gene expression under realistic circumstances.

franknrice
Frank is looking for elusive effects of dsRNA in rice

The lower concentrations of reported by other researchers compound these concerns, and make it highly unlikely that any dietary sources of dsRNA would have any impact on gene expression. Furthermore, the risks presented by dsRNA and other forms of RNAi are not exclusive to transgenic organisms, nor are they substantially changed by the presence of transgenes. Non-genetically modified plants, such as in the study by Zhang et al., also produce dsRNA. The addition of one or multiple transgenes and the resulting additional dsRNA would not significantly change the amount of dsRNA our system digests daily, nor would these transgenes be more likely to inhibit human gene expression than genes native to a dietary source.

Overall, these studies show that it is highly unlikely that dietary sources of RNA could have biologically relevant impacts on human gene expression. Further studies may elucidate whether RNA sequences are actually assimilated into blood plasma from dietary sources, however, it is improbable that these RNA molecules would be present in sufficient quantities to have any detrimental (or beneficial) effect on humans. Ultimately, although some researchers may maintain concerns about the physiological role of dietary sources of RNA in humans, these claims are not well-established and can currently be considered improbable.

References:

Zhang, L.; Hou, D.; Chen, X.; Li, D.; Zhu, L.; Zhang, Y.; Li, J.; Bian, Z.; Liang, X.; Cai, X.; Yin, Y.; Wang, C.; Zhang, T.; Zhu, D.; Zhang, D.; Xu, J.; Chen, Q.; Ba, Y.; Liu, J.; Wang, Q.; Chen, J.; Wang, J.; Wang, M.; Zhang, Q.; Zhang, J.; Zen, K.; Zhang, C.-Y. Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA. Cell research 2011, 22, 107–26.

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. Environment International 2013, 55.

Witwer, K. W.; McAlexander, M. A.; Queen, S. E.; Adams, R. J. Real-time quantitative PCR and droplet digital PCR for plant miRNAs in mammalian blood provide little evidence for general uptake of dietary miRNAs: Limited evidence for general uptake of dietary plant xenomiRs. RNA Biology 2013, 10.

AE, Isaacs SK, Baggish AL, Chan
SY. Ineffective delivery of diet-derived microRNAs to recipient animal organisms. RNA Biology
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Brown, B.; Gentner, B.; Cantore, A.; Colleoni, S.; Amendola, M.; Zingale, A.; Baccarini, A.; Lazzari, G.; Galli, C.; Naldini, L. Endogenous microRNA can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state. Nature biotechnology 2007, 25, 1457–67.

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Graduate student in protein structure function and proteomics and an absolute skeptic obsessed with learning more, helping clear misconceptions, and listening to terrible music.