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 Dr Mae-Wan Ho
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
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 .
discoveries were possible thanks to huge advances in nucleic acid sequencing
technology, in particular, next generation deep sequencing (NGS) (see Box).
generation deep sequencing [10, 11]
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.
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 .
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.
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.
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
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
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
sativaJaponica 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,
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.