New research confirms that DNA fragments derived from meals, large enough to carry complete genes, can escape degradation and enter the human circulatory system, and so can RNA, raising serious concerns over new nucleic acids introduced into the human food chain via genetically modified organisms.
by Dr Mae-Wan Ho
Food RNA gets into blood and so does DNA
We have alerted readers to research showing how tiny RNA molecules in food eaten can circulate in the bloodstream and turn genes off in the body , raising concerns over the safety of genetically modified organisms (GMOs), which introduce many novel and synthetic nucleic acids into the human food chain ( How Food Affects Genes, SiS 53). New research shows that pieces of DNA large enough to code for complete genes can also escape degradation in the gut and enter the human circulatory system, and the presence of circulating RNA from food is much more extensive and widespread.
DNA known to resist digestion and may form part of circulating cell free DNA
A study led by Sándor Spisák who holds a joint appointment at Hungarian Academy of Sciences in Budapest and Harvard Medical School Boston, Massachusetts in the USA analysed over 1 000 human adult samples from four independent studies, and found DNA fragments derived from food in all plasma samples, some large enough to code for complete genes .
Previous animal feeding studies have demonstrated that a minor proportion of fragmented dietary DNA may resist digestion , but the degradation of long chains of DNA and the possible uptake and transport into the bloodstream are not at all understood. Circulating cell free DNA (cfDNA) in the human bloodstream, first described in 1948, are mostly double-stranded molecules with a wide range of fragment sizes from 180 – 21 k bp.
Most people think cfDNA are from apoptotic cells (resulting from programmed cell-death), and in different diseases such as inflammation, autoimmune, trauma and cancer, necrotic cells (from non-programmed cell death) may increase the amount. In fact, both DNA and RNA are found circulating in the bloodstream, and there is good evidence that they are actively secreted from living cells as a nucleic acid intercommunication system (see  Intercommunication via Circulating Nucleic Acids, SiS 42).
Apart from the individual’s own cells, DNA of the foetus can be detected in maternal plasma. Viral DNA, bacterial DNA may also be found in various disease states. DNA from consumed food is not usually considered, although there are animal studies suggesting that small fragments of nucleic acids may pass into the bloodstream and even into various tissues. For example, foreign DNA fragments were detected by PCR in the digestive tract and leukocytes of rainbow trout  fed GM soybean, and other similar findings were reported in goats ., pigs [8, 9] and mice .
These recent discoveries were possible thanks to huge advances in nucleic acid sequencing technology, in particular, next generation deep sequencing (NGS) (see Box).
Next generation deep sequencing [10, 11]
Next generation sequencing (NGS) extends sequencing across millions of reactions taking place in parallel rather than being limited to a single or a few DNA fragments. This enables rapid sequencing of large stretches of DNA base pairs spanning entire genomes, with instruments capable of producing hundreds of gigabase (Gb) data in a single sequencing run. To sequence a single genome, the genome is first fragmented into a library of small segments that can be uniformly and accurately sequenced in millions of parallel reactions. The newly identified strings of bases, called reads (of a defined length) are then reassembled using a known reference genome as a scaffold (resequencing), or in the absence of a reference genome (de novo sequencing), assembled by overlaps. The full set of aligned reads reveals the entire sequence of each chromosome in the genome.
NGS data output has been rising steeply since its invention in 2007, when a single sequencing run could produce a maximum of about one Gb data. By 2011, the rate has reached nearly a terabase (Tb, 1012b), a thousand fold increase. By 2012, researchers can sequence more than 5 human genomes in a single run, producing data in roughly one week at a cost of less than $5 000 per genome. The $1 000 genome is now within our grasp .
NGS high throughput capacity has enabled ‘deep sequencing’ of genomes and transcriptomes to look for rare DNA variants or rare species of RNA transcripts. Deep sequencing means that the total number of reads is many times larger than the length of the sequence under study. ‘Depth’ (coverage) is the average number of times a nucleotide is read.
Surveys of existing next generation sequencing database
Spisák’s team did a first survey on samples from 200 human individuals pooled into four groups based on colonoscopy diagnosis as having inflammatory bowel disease, adenoma, colorectal cancer or negative . NGS gave 50 nt long reads, and a total of 86.6 G bases. On average 71 % of reads could be mapped to the human reference genome. The goal of the original study was to find human genetic differences between the four groups according to the stage of their disease, but there were relatively large amounts of unmapped reads, and the researchers wanted to find out where that DNA could have originated.
Before searching for foreign (non-human) genomes, the reads that matched the reference human genome were discarded. The remaining sequences were then aligned to foreign genomes using stringent sequence matching criteria. Chloroplast DNA sequences from tomatoes were identified, with hints of other food species, such as chicken, but larger samples would be needed to get convincing results for meat (because meat DNA is more similar to human DNA).
The number of aligning short reads shows large differences between samples. Most of the matches are for the longest intact DNA segments. This is surprising, in view of the current assumption that during digestion and absorption DNA is degraded down to nucleotide level. Instead, the results showed that not just some of the DNA can avoid complete degradation but fragments large enough to carry complete genes can pass from the digestive tract into the bloodstream.
To investigate further, they searched the publicly available NGS archives for circulating cell free DNA sequence data, as NGS technology is evolving so fast and sequences are produced at such a great rate that detailed understanding of the information cannot keep pace with the accumulation of data. The team found 909 samples from 907 individuals in three studies. The analysis of these independent data confirms their hypothesis that foreign DNA in human plasma is not unusual, though there is large variation from subject to subject. There was no trace of plant DNA in cord blood samples, which act as a good negative control, while more than 1 000 reads were detected in the maternal plasma. An independent sample from a subject with inflammation showed high plant DNA concentration, higher than human DNA.
There were alignments to dozens of plant species differing between individuals. The first three species, beans, are members of the Fabaceae family, the next eight species belong to the Brassicaceae family. There are four members from the Solanaceae famlly (potato, tobacco) and one from the Convolvulaceae (Ipomoea, Cuscuta) family, members of the Solanales order. The remaining eight species are from the Poaceae family of the Monocots clade. All 24 plants are often consumed by humans or are close relatives of frequently eaten species while many non-edible plants do not show up on the list. Inedible species can show up because they are genetically related to other species, and not all the frequently eaten plant species are part of the chloroplast genome collection. The only outlier, the non-edible Ipomoea purpurea (morning glory) shows up because it is similar to the genome of Ipomoea batatas (sweet potato) or Ipomoea aquatica (kangkong) a common vegetable eaten in Southeast Asia. Individuals can be grouped according to those with high Poaceae (grains), high Fabaceae and “high everything else except Poaceae”.
In general the DNA present in plasma reflected the diet of the individuals concerned, leaving little doubt that DNA from food ingested can resist digestion in the gut and pass into the circulatory system, potentially to be taken up by cells within the body with unknown effects.
Bacterial, fungi and food RNA in human plasma
In another study, researchers in Seattle Washington in the United States led by Kai Wang and David Galas at the Institute for Systems Biology and Paul Wiles at University of Luxembourg carried out a survey of human plasma for miRNA using NGS . They found a significant fraction originating from exogenous species, including bacteria and fungi as well as food species. Some of the RNAs are detected in intracellular complexes and may be able to influence cellular activities.
Initially, NGS was done on 9 plasma samples, 3 from healthy individuals, 3 from patients with colorectal cancer prior to any treatment and 3 from individuals suffering from ulcerative colitis. On first examination, the team noticed that less than 1.5 % of the processed reads (proportional to frequency) actually mapped to human miRNAs. About 11 % of the remaining reads mapped to human transcripts and human genome sequence when no sequence mismatch was allowed. With a higher tolerance of sequence mismatches (2 mismatches), the fraction of reads that could be mapped to known human transcripts rose to about 42 % and 15 % to other human genome sequences. This still left over 40 % of reads with an unknown origin. After carefully eliminating the human sequences, a significant number of the unmapped reads aligned with various bacterial and fungal sequences.
The reads (~7 %) covered all major bacteria phyla and two archaea phyla, Euryarchaeola and Crenarchaeota. The bacterial phylum Firmicules, typically the most abundant bacteria phylum in the human gut is the 3rd most abundant sequence populations in human plasma. The bacterium that accounts for the highest number of reads is an uncultured bacterium, followed by Pseudomonas fluorescens, an important beneficial bacterium in agriculture, followed by bacteria from the genus Ralstonia pp, then Achromobacter pechaudii, identified in some clinical blood samples.
Fungi represent the largest source of exogenous RNA, about 14 %, covering all major fungal phyla, Ascomycota the most abundant. Metarhizium anisopliae, a common fungus in soil had the most mapped reads and Thielavia terrestris, a thermophilic fungus is the next most abundant. A significant number of reads mapped to yeast Saccharomyces cerevisae commonly used in baking and brewing.
After carefully examining sequences mapped to species other than bacteria and fungi, they found a significant number of reads (~ 2-3 %) that mapped to common food species. The most abundant food RNA sequences are corn (Zea mays) followed by rice (Oryza sativa Japonica group), with corn reads 66 times higher on average than rice. The data from a Chinese individual gave sequence abundance between corn and rice reversed: rice was 55-fold the number from corn. Apart from common cereal grains, RNA from other food items included soybeans, tomato, and grape.
Like endogenous miRNA, the levels of specific exogenous miRNA and rRNA were reduced significantly after Triton X-100, protease, RNAse and protease followed by RNAse treatments, suggesting that some of the exogenous RNA molecules like the endogenous miRNAs are associated with protein and/or lipid complexes in circulation.
Some of the micro-RNA-like molecules from observed exogenous miRNA sequences and some highly abundant exogenous sequences (bacterial rRNAs) were synthesized and transfected into a mouse fibroblast cell line. The expression profiles of a number of genes in the cells were affected by some of the exogenous RNA sequences.
MiRNA in milk
Finally, researchers at Moringa Milk Industry, Zama Kanagawa, Japan, using more conventional microarray and quantitative PCR analyses identified 102 miRNA in bovine milk, 100 in colostrum and 53 in mature milk, and 51 were common to both. Among them, several immune- and development-related miRNAs including miR-15b, miR-27b, miR-34a, miR-106b, 130a, 155 and 223 were more highly expressed in colostrum than in mature milk. Some messenger(m)RNA was also found in bovine milk. While synthetic miRNA spiked in the raw milk whey were degraded, naturally existing miRNA and mRNA in raw milk were resistant to acidic conditions and RNase treatment; unexpectedly, miRNA and mRNA were also found in infant formulas purchased from Japanese market .
Nucleic acids (both DNA and RNA) from food can resist digestion in the human gut and enter the circulatory system, with the potential of being taken up by cells to influence gene expression and/or become incorporated into the cell’s genome. This underscores the hazards of GM and other unknown nucleic acids introduced into the human food chain by GMOs.