ISIS Report 17/06/04
DNA in GM Food & Feed
The government’s scientific advisory committees have repeatedly
tried to reassure the public that there is nothing to fear from genetically
modified (GM) DNA, but critics disagree. Dr. Mae-Wan Ho offers a quick guide
for the perplexed
A fully referenced version of this article is posted
on ISIS Members’ website. Details here.
Is GM DNA different from natural DNA?
"DNA is DNA is DNA," said a proponent in a public debate in trying to
convince the audience that there is no difference between genetically modified
(GM) DNA and natural DNA, "DNA is taken up by cells because it is very
nutritious!"
"GM can happen in nature," said another proponent. "Mother Nature got
there first."
So, why worry about GM contamination? Why bother setting contamination
thresholds for food and feed? Why award patents for the GM DNA on grounds that
it is an innovation? Why don’t biotech companies accept liabilities if
there’s nothing to worry about?
As for GM happening in nature, so does death, but that doesn’t
justify murder. Radioactive decay happens in nature too, but concentrated and
speeded up, it becomes an atom bomb.
GMDNA and natural DNA are indistinguishable according to the most
mundane chemistry, i.e., they have the same chemical formula or atomic
composition. Apart from that, they are as different as night and day. Natural
DNA is made in living organisms; GMDNA is made in the laboratory. Natural DNA
has the signature of the species to which it belongs; GMDNA contains bits
copied from the DNA of a wide variety of organisms, or simply synthesized in
the laboratory. Natural DNA has billions of years of evolution behind it; GMDNA
contains genetic material and combinations of genetic material that have never
existed.
Furthermore, GMDNA is designed – albeit crudely - to cross species
barriers and to jump into genomes. Design features include changes in the
genetic code and special ends that enhance recombination, i.e., breaking into
genomes and rejoining. GMDNA often contains antibiotic resistance marker genes
needed in the process of making GM organisms, but serves no useful function in
the GM organism.
The GM process clearly isn’t what nature does (see "Puncturing the
GM myths", SiS22). It bypasses reproduction, short circuits and greatly
accelerates evolution. Natural evolution created new combinations of genetic
material at a predominantly slow and steady pace over billions of years. There
is a natural limit, not only to the rate but also to the scope of gene
shuffling in evolution. That’s because each species comes onto the
evolutionary stage in its own space and time, and only those species that
overlap in space and time could ever exchange genes at all in nature. With GM,
however, there’s no limit whatsoever: even DNA from organisms buried and
extinct for hundreds of thousands of years could be dug up, copied and
recombined with DNA from organisms that exist today.
GM greatly increases the scope and speed of horizontal gene
transfer
Horizontal gene transfer happens when foreign genetic material jumps
into genomes, creating new combinations (recombination) of genes, or new
genomes. Horizontal gene transfer and recombination go hand in hand. In nature,
that’s how, once in a while, new viruses and bacteria that cause disease
epidemics are generated, and how antibiotic and drug resistance spread to the
disease agents, making infections much more difficult to treat.
Genetic modification is essentially horizontal gene transfer and
recombination, speeded up enormously, and totally unlimited in the source of
genetic material recombined to make the GMDNA that’s inserted into the
genomes plants, animals and livestock to create genetically modified organisms
(GMOs).
By enhancing both the rate and scope of horizontal gene transfer and
recombination, GM has also increased the chance of generating new
disease-causing viruses and bacteria. (It is like increasing the odds of
getting the right combination of numbers to win a lottery by betting on many
different combinations at the same time.) That’s not all. Studies on the
GM process have shown that the foreign gene inserts invariably damages the
genome, scrambling and rearranging DNA sequences, resulting in inappropriate
gene expression that can trigger cancer.
The problem with the GM inserts is that they could transfer again into
other genomes with all the attendant risks mentioned. There are reasons to
believe GM inserts are more likely to undergo horizontal transfer and
recombination than natural DNA, chief among which is that the GM inserts (and
the GM varieties resulting from them) are structurally unstable, and often
contain recombination hotspots (such as the borders of the inserts).
After years of denial, some European countries began to carry out
‘event-specific’ molecular analyses of the GM inserts in commercially
approved GM varieties as required by the new European directives for deliberate
release, novel foods and traceability and labelling. These analyses reveal that
practically all the GM inserts have fragmented and rearranged since
characterised by the company. This makes all the GM varieties already
commercialised illegal under the new regime, and also invalidates any safety
assessment that has been done on them (see "Transgenic lines proven unstable",
SiS 20 and
"Unstable transgenic lines illegal",
SiS 21). As
everyone knows, the properties of the GM variety, and hence its identity,
depend absolutely on the precise form and position of the GM insert(s). There
is no sense in which a GM variety is "substantially equivalent" to non-GM
varieties.
GMDNA in food & feed
In view of the strict environmental safety assessment required for
growing GM crops in Europe, biotech companies are bypassing that by applying to
import GM produce for food and processing only. Is GM food safe? There are both
scientific and anecdotal evidence indicating it may not be: many species of
animals were adversely affected after being fed different species of GM plants
with a variety of GM inserts (see "GM food safe?" series,
SiS 21),
suggesting that the common hazard may reside in the GM process itself, or the
GMDNA.
How reliably can GMDNA be detected?
DNA can readily be isolated and quantified in bulk. But the method
routinely used for detecting small or trace amounts of GMDNA is the polymerase
chain reaction (PCR). This copies and amplifies a specific DNA sequence based
on short ‘primers strings’ of DNA that match the two ends of the
sequence to be amplified, and can therefore bind to the ends to
‘prime’ the replication of the sequence through typically 30 or more
cycles, until it can be identified after staining with a fluorescent dye.
There are many technical difficulties associated with PCR amplification.
Because of the small amount of the sample routinely used for analysis, it may
not be representative of the sample, especially if the sample is inhomogeneous,
such as the intestinal contents of a large animal. The primers may fail to
hybridise to the correct sequence; the PCR itself may fail because inhibitors
are present. Usually, the sequence amplified is a small fraction of the length
of the entire GM insert, and will therefore not detect any other GM fragment
present. If the target sequence itself is fragmented or rearranged, the PCR
will also fail. For all those reasons, PCR will almost always underestimate the
amount of GMDNA present, and a negative finding cannot be taken as evidence
that GMDNA is absent.
A new review on monitoring GM food casts considerable doubt over the
reliability of PCR methods. Mistakes can arise if the sample is not large
enough to give a reliable measure, or if the batch of grain sampled is
inhomogeneous, or the PCR reaction not sensitive enough, or the data presented
to the regulatory authorities simply not good enough. Consequently, the level
of contamination is almost invariably underestimated.
There is an urgent need to develop sensitive, standardized and validated
quantitative PCR techniques to study the fate of GMDNA in food and feed.
Regulatory authorities in Europe are already developing such techniques for
determining GM contamination. One such technique has brought the limit of
detection down to 10 copies of the transgene (the GM insert or a specific
fragment of it).
In contrast, the limit of PCR detection in investigations on the fate of
GMDNA in food and feed is extremely variable. In one study commissioned by the
UK Food Standards Agency, the limit of detection varied over a thousand fold
between samples, with some samples requiring more than 40 000 copies of the GM
insert before a positive signal is registered. Such studies are highly
misleading if taken at face value, given all the other limitations of the PCR
technique.
Despite that, however, we already have answers to a number of key
questions regarding the fate of DNA in food and feed.
1. Does DNA break down sufficiently during food processing?
The answer is no, not for most commercial processing. DNA was found to
survive intact through grinding, milling or dry heating, and incompletely
degraded in silage. High temperatures (above 95 deg. C) or steam under pressure
were required to degrade the DNA completely.
"The results imply that stringent conditions are needed in the
processing of GM plant tissues for feedstuffs to eliminate the possibility of
transmission of transgenes." The researchers warned.
They pointed out for example, that the gene aad, conferring
resistance to the antibiotics streptomycin and spectinomycin, is present in GM
cottonseed approved for growth in US and elsewhere (Monsanto’s Bollgard
(insect-protected) and Roundup Ready (herbicide tolerant)). Streptomycin is
mainly used as a second-line drug for tuberculosis. But it is in the treatment
of gonorrhoea that spectinomycin is most important. It is the drug of choice
for treating strains of Neisseria gonorrhoeae already resistant to
penicillin and third generation cephalosporins, especially during pregnancy.
The release of GM crops with the blaTEM gene for ampicillin
resistance is also relevant here, because that’s where resistance to
cephalosporins has evolved.
Another study found large DNA fragments in raw soymilk of about 2 000bp
(base pairs, unit of measurement for the length of DNA), which degraded
somewhat after boiling, but large fragments were still present in tofu and
highly processed soy protein. Heating in water under acid conditions was more
effective in degrading DNA, but again, the breakdown was incomplete (fragments
larger than 900bp remaining).
It is generally assumed, incorrectly, that DNA fragments less than
200bp pose no risk, because they are well below the size of genes. But
that’s a mistake, as these fragments may be promoters (signals
needed by genes to become expressed), and sequences of less than 10bp can be
binding sites for proteins that boost transcription. The CaMV 35S promoter, for
example, is known to contain a recombination hotspot, and is implicated in the
instability of GM inserts.
2. Does DNA break down sufficiently rapidly in the
gastrointestinal tract?
Although free DNA breaks down rapidly in the mouth of sheep and humans,
it was not sufficiently rapid to prevent gene-transfer to bacteria inhabiting
the mouth. DNA in GM food and feed will survive far longer. The researchers
conclude: "DNA released from feed material within the mouth has potential to
transform naturally competent oral bacteria."
Several studies have now documented the survival of DNA in food
throughout the gastrointestinal tract in pig and mice, in the rumen of sheep
and in the rumen and duodenum of cattle. The studies were variable in quality,
depending especially on the sensitivity of the PCR methodology used to amplify
specific sequences for detection. Nevertheless they suggest that GMDNA can
transfer to bacteria within the rumen and in the small intestine. In neither
sheep nor cattle was feed DNA detected in the faeces, suggesting that DNA
breakdown may be complete by then.
The only feeding trial in human volunteers was perhaps the most
informative. After a single meal containing GM soya containing some
3x1012 copies of
the soya genome, the complete 2 266 bp epsps transgene was recovered
from the colostomy bag in six out of seven ileostomy subjects (who had their
lower bowel surgically removed). The levels were highly variable among
individuals as quantified by a small 180bp PCR product overlapping the end of
cauliflower mosaic virus (CaMV) 35S promoter and the beginning of the gene:
ranging from 1011
copies (3.7%) in one subject to only 105 copies in another. This is
a strong indication that DNA in food is not sufficiently rapidly broken down in
transit through the gastrointestinal tract, confirming the results of an
earlier experiment by the same research group.
No GMDNA was found in the faeces of any of 12 healthy volunteers tested,
suggesting that DNA has completely broken down, or all detectable fragments
have passed into the bloodstream (see later) by the time food has passed
through the body. This finding is in agreement with the results from ruminants.
In general, the studies report that GMDNA degrades to about the same
extent and at about the same rate as natural plant DNA. However, no
quantitative measurements have been made, and GMDNA was often compared with the
much more abundant chloroplast DNA, which outnumbers the transgene by 10 000 to
one.
3. Does GMDNA get taken up by bacteria and other micro-organisms?
The answer is yes. The evidence was reported in the human feeding trial
mentioned. The transgene was not detected in the content of the colostomy bag
from any subject before the GM meal. But after culturing the bacteria,
low levels were detected in three subjects out of seven: calculated to be
between 1 and 3 copies of the transgene per million bacteria.
According to the researchers, the three subjects already had the
transgene transferred from GM soya before the feeding trial, probably by having
eaten GM soya products unknowingly. No further transfer of GM DNA was detected
from the single meal taken in the trial.
The researchers were unable to isolate the specific strain(s) of
bacteria that had taken up the transgene, which was not surprising, as
"molecular evidence indicates that 90% of microorganisms in the intestinal
microflora remain uncultured. …they can only grow in mixed culture, a
phenomenon seen with other microorganisms."
Actually, GMDNA can already transfer to bacteria during food processing
and storage. A plasmid was able to transform Escherichia coli in all 12
foods tested under conditions commonly found in processing and storage, with
frequencies depending on the food and on temperature. Surprisingly, E.
coli became transformed at temperatures below 5 degrees C, i.e. under
conditions of storage of perishable foods. In soy drink this condition resulted
in frequencies higher than those at 37 degrees C.
4. Do cells lining the gastrointestinal tract take up DNA?
The answer is yes. Food material can reach lymphocytes (certain white
blood cells) entering the intestinal wall directly, through Peyer’s
patches. And fragments of plant DNA were indeed detected in cows’
peripheral blood lymphocytes.
It is notable that in the human feeding trial, a human colon carcinoma
cell line CaCo2 was directly transformed at a high frequency of 1 in 3 000
cells by an antibiotic resistance marker gene in a plasmid. This shows how
readily mammalian cells can take up foreign DNA, as we have pointed out some
years ago (see also below).
5. Does DNA pass through the gastrointestinal tract into the blood
stream?
The answer is yes, as mentioned above, fragments of plant DNA was
detected in cow’s peripheral blood lymphocytes. However, attempts to
amplify plant DNA fragments from blood have failed, most likely on account of
the presence of inhibitors of the PCR amplification.
6. Does DNA get taken up by tissue cells?
The answer is yes, and this has been known since the mid 1990s. GMDNA
and viral DNA fed to mice ended up in cells of several tissues, and when fed to
pregnant mice, the DNA was able to cross the placenta, and enter the cells of
the foetus and the newborn. These results were confirmed in 2001, when soya
DNA, too, was found taken into the tissue cells of a few animals.
In general, abundant chloroplast sequences have been detected in the
tissues of pig and chicken but not single gene DNA nor GMDNA. But rare events
are most likely to go undetected, on account of the limitations of the PCR
technique.
Recently, "spontaneous transgenesis" – the process of spontaneous
uptake of foreign DNA resulting in gene expression - has been rediscovered by a
team of researchers looking for new possibilities in gene therapy. They
documented the phenomenon in several human B lymphocyte cell lines as well as
peripheral blood B lymphocytes. The transgene in a plasmid was readily taken up
and was found in many cell compartments including the nucleus, where gene
transcription took place. The plasmid was not integrated into the genome, but
the researchers say that its eventual integration cannot be ruled out.
7. Is GM DNA more likely to insert into genomes?
This is perhaps the most important question. There are reasons to
believe GMDNA is more likely to insert into genomes after it is taken up into
cells, chief among which, its sequence similarities (homologies) to a wide
variety of genomes, especially those of viruses and bacteria. Such homologies
are known to enhance horizontal gene transfer to bacteria up to a billion fold.
More significantly, the integration of non-homologous genetic material
can occur at high frequencies when flanked by homologous sequences. A recent
report highlights the importance of this "homology-facilitated illegitimate
recombination", which increases the integration of foreign (non-homologou) DNA
at least 105 fold
when it was flanked on one side by a piece of DNA homologous to the recipient
genome.
No experiment has yet been done to assess whether GMDNA is more
likely to transfer horizontally than natural DNA. However, in the human
feeding trial, where three ileostomy volunteers tested positive for the soya
transgene in the bacteria cultured from their colostomy bag, the soya lectin
gene Le was not detected in the bacterial cultures from any of
the subjects.
The researchers found it necessary to remark, "Although the plant
lectin gene was not detected in the microbial population…it is premature
to conclude that the epsps transgene is more likely than endogenous
plant genes to transfer into the microbial population."
But until this possibility has been adequately addressed, it cannot be
ruled out.
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