ISIS Report 13/03/04

Exposed: More Shoddy Science in GM Maize Approval

Dr. Mae-Wan Ho The Food Standards Agency appears to be selectively promoting and suppressing research results in projects it funds

Scientists should be afraid, very afraid of the extent to which the academic-industrial-military complex is bending science to suit its purpose. No, I am not only speaking about the United States, but the United Kingdom here.

Horizontal gene transfer – a major uncertainty in GM safety

In the current issue of Science in Parliament [1], I found no less than three mentions of horizontal gene transfer as a major uncertainty in the safety of GM crops: in an article by Michael Meacher MP, "GM: the politics of uncertainty", in a Parliamentary debate on biotechnology by Joan Ruddock, and in the article " Nanotechnology: friend or foe?" by Prof. George Smith of Oxford University.

Horizontal gene transfer is the process whereby genetic material jumps into foreign genomes, or put the other way round, whereby foreign genetic material jumps into genomes. That is exactly what genetic modification involves: artificially constructed DNA (called GM DNA or transgenic DNA) cobbled together from a wide variety of sources or simply made in the laboratory, is inserted into the genomes plants, animals and livestock.

But horizontal gene transfer can also happen unintentionally and without our knowledge. It is the main process creating new viruses and bacteria that cause disease epidemics and spreads antibiotic and drug resistance besides, making the diseases more difficult to treat. Foreign genes jumping into genomes, as many investigations on the genetic modification process itself reveal, causes extensive genetic damage, scrambling and rearranging genomes, resulting in inappropriate gene expression that can trigger cancer.

There’s lots of evidence that transgenic DNA may be more unstable and more mobile than natural DNA [2], and molecular analyses of commercially approved GM crops, carried out belatedly last year, found that practically all the inserts have rearranged since characterised by the company [3,4].

One big question is: what happens to the transgenic DNA that’s in GM food and feed? I have raised this question on numerous occasions with our government over the past ten years, one of the more recent occasions during the public hearing on Chardon LL T25 transgenic maize organised by the Advisory Committee on Releases to the Environment (ACRE) in 2002 [5].

At the same hearing, it was revealed that twice as many broiler chickens died eating GM maize as non-GM maize. But because the experimental design was so flawed, statistical analysis failed to detect a significant difference between the two groups. Prof. Orskov of the Macaulay Institute raised the issue of whether milk from dairy cows was safe to drink, and spokespersons from Friends of the Earth also pressed for feeding tests in the appropriate species – cattle - instead of rats and broiler chickens.

ACRE’s written response to the Chardon LL Hearing (December 2002) stated [6]: "The Company - Bayer - has commissioned a dairy cattle feeding study with T25 maize and will present the data to the French competent authorities when completed. As is the standard practice, ACRE will review new information generated in this trial and update the risk assessment accordingly."

Mystery of the missing study

But the result of this study has yet to see the light even though the GM maize has now been approved for commercial growing as cattle feed.

Dr. Brian John of GM-Free Cymru wrote to ACRE 24 February expressing his concern that there are no published or peer-reviewed ruminant feeding studies on the effects of T25 maize [7] and asked to see the study mentioned by ACRE, especially in view of the recent report that twelve dairy cows in Hesse Germany died after eating Syngenta’s GM maize 176 [8]. ACRE has not replied, despite a reminder sent 5 March.

The study was supposed to have been done by Professor Richard Phipps in the Centre for Dairy Research (CEDAR) at Reading University.

"There is no mention of the study on the CEDAR website." Brian John said, "If the Chardon LL used in the study came from the FSE trial sites, that would have contravened the FSE rules, and a separate consent must have been signed by somebody."

Bayer was reportedly given a report of the study late in 2002, and one of the researchers involved, David Beever, claimed the report had gone to ACRE.

On 9 March, Brian John finally received, via the Welsh Assembly, a "Background Information" paper from Richard Phipps, stating that the study was conducted at the University of Reading for Bayer Crop Science. The objective of the study was to determine the effect of silage derived from T25 maize on feed intake and milk production in lactating dairy cows compared with a near isogenic counterpart and silage of two further commercial maize hybrids.

Furthermore, it stated that the study has been completed and presented to the company, and they are in the process of preparing their data for publication in an international scientific journal once the peer review process has been completed, the same process followed in the study they conducted for the UK Food Standards Agency, published in the Journal of Dairy Science [9], a copy of which was enclosed.

They then went on to state,

"While it is not our policy to release details of studies prior to peer review we feel able to say that the compositional, fermentation characteristics and nutritional values of all four silages were comparable and that there were no significant differences in milk yield, milk composition and yield of milk constituents, when comparing the four rations. In addition GM DNA was not detected in any of the milk samples analysed by Polymerase Chain Reaction methodology. Cows remained in good health throughout the study period."

Notice that the study focussed on silage, not on maize grain, which is also widely fed to cattle. It concentrated on detecting GM DNA in milk, but not the mouth, rumen or intestinal contents, blood, meat or other animal tissues, where positive results have already been reported (see below).

The FSA-sponsored study is presumably the reason our government has given the go-ahead for approving Chardon LL T25 maize to be grown for cattle feed, as the T25 study has yet to be published and there is a dearth of published studies on feeding ruminants with GM feed. So, how does that study stand up to scrutiny?

Study that found no results worth reporting is worthless

The study failed to find significant survival of GM DNA, or indeed any single copy DNA in most of the tissue and tissue contents examined; but it is deeply flawed.

First of all, the FSA-sponsored study has nothing to do with Chardon LL maize. It was work carried out with a mixture of both Monsanto’s Roundup Ready soya GTS 40-3-2 (as soya meal) and Mon 810 maize (as maize grain) at the same time, comprising only 13% and 18.5% respectively of the total diet. This inevitably decreases the chance of detecting the GM DNA belonging to the varieties.

Second, only six cows were used, three fed the GM diet and the other non-GM. But a peculiar "single reversal design with three 4-wk periods" was used, which I believe, meant that the groups of three cows alternated between GM and non-GM diets. Thus one group would spend the first four weeks on GM, the next four weeks on non-GM and then four weeks back on GM; while the feeding regime for the other group would be non-GM, GM, and non-GM. This design generates in effect 9 data points each for the GM diet and non-GM diet. But, it also guarantees to balance out the effects of GM versus non-GM diet and hence is utterly worthless as far as detecting difference in weight gain or any other developmental or physiological indicators between the diets.

Third, the researchers made a big blunder. Two of the cows in the non-GM group were inadvertently fed on the GM-diet, so they ended up with 13 data points in the GM diet group and only 5 data points in the control non-GM diet group.

Fourth, even though they had taken apparently carefully timed samples from individual animals in each four week period, they pooled all the samples from the same animal together, thus losing potentially valuable information regarding the time course of the clearing of GM DNA from the gut to the tissues and out of the body.

Fifth, and most serious of all, their PCR method for detecting GM DNA is neither validated nor standardized. Its sensitivity varied over 1000 fold between different tissues and tissue-contents. The limits of detection is such that in some samples, I calculate that more than 4 000 copies of the soya genome or 900 copies of the maize genome must be present in the sample before a positive result is obtained. The usual detection limit of PCR is 10 copies or less. Thus, given the minute amounts of tissues and tissue contents used in a PCR test, as for example, 0.3 millilitres of milk, it is no wonder that the only DNA that can be detected at all reliably is the chloroplast gene, which exists in 10 000 copies per plant cell. And no wonder there is a rather large number of neither positives nor negatives, but "inconclusives" in the data.

Poor PCR amplification is one of the most common causes of failing to detect GM DNA

The "Background Information" on the study on Chardon LL claims that their as yet unpublished results "support the 30 other peer-reviewed papers in international scientific journals, which have failed to detect GM DNA in milk, meat and eggs derived from animals fed diets containing GM feeds."

In fact, poor PCR amplification is probably one of the most common causes of having "failed to detect GM DNA". A Japanese research team, which has documented the survival of both GM DNA and Bt toxin protein in the digestive tract of mice, pigs and cattle [10], nevertheless reported a failure to detect GM DNA in blood because as they stated, the PCR did not work in blood. There are many unknown PCR inhibitors in different tissues that can give false negatives. Phipps and coworkers also failed to detect single-copy DNA in blood, GM or otherwise, they failed to detect even the abundant chloroplast gene in the vast majority of samples.

GM DNA found to survive when PCR is adequate

One recent study documenting the survival of GM DNA in the mouth and rumen of sheep was also funded by the Food Standards Agency [11]. This research group from Leeds University found that DNA fragments containing the entire coding region of the synthetic cry1Ab gene was still amplifiable from rumen fluid 5 hours after feeding maize grains, though not from rumen fluid sampled from sheep fed silage prepared from the genetically modified maize line. But PCR amplification of a shorter (211-bp) sequence was possible with rumen fluid sampled up to 3 and 24 h after feeding silage and maize grains, respectively.

It is clear that GM DNA in maize grains persists, and "may, therefore, provide a source of transforming DNA in the rumen".

But the authors are wrong to claim that the 211-bp sequence is "very unlikely to transmit genetic information". For such sequences could be promoters or enhancers containing hundreds of binding motifs for transcription factors, and capable of boosting the expression of genes inappropriately.

The researchers also found that plasmid DNA introduced into the mouth of sheep and extracted from saliva sampled after 8min was still capable of transforming Escherichia coli bacteria to kanamycin resistance, "implying that DNA released from the diet within the mouth may retain sufficient biological activity for the transformation of competent oral bacteria".

They conclude: "The use of GM crops harbouring antibiotic resistance genes, in particular the use of unprocessed grains in animal feed, possibly deserves further evaluation."

A great deal of uncertainty remains over the fate of GM DNA. Further research must be carried out with properly validated quantitative PCR methods.

ISIS has discovered that information on this research is not easily found on the FSA website, although the FSA has clearly funded the research (the research grant is G01010). The FSA website does have information on the research they fund (there is a Research Project List for its Safety of Novel Foods Research Programme), some with the papers themselves, others with just short information on the projects; this is available for G01007-G01021, with the exception of G01010 and G01014. A search of the FSA website with the term ‘G01010’ eventually turned up some information on the project, although not the papers arising from the research.

References

1. Science in Parliament 2004, Spring, The Parliamentary and Scientific Committee.
2. Ho MW. Living with the Fluid Genome, Chapter 11, ISIS & TWN, London and Penang, 2003.
3. Ho MW. Trangenic Lines Proven Unstable. Science in Society 2003, 20, 35-36.
4. Ho MW. Unstable transgenic lines illegal. Science in Society 2004, 21, 23.
5. Ho MW. GM maize approved on bad science in the UK. Science in Society 2002, 15, 10.
6. ACRE’s Response to the Chardon LL Hearing, December 2002 http://www.defra.gov.uk/environment/acre/advice.advice20d.htm
7. Letter to Dr. Clare Pitcher, ACRE Secretariat, DEFRA, from Brian John, GM-Free Cymru, 24 February 2004.
8. Ho MW and Burcher S. Cows ate GM maize and died. Science in Society 2004, 21, 4-6.
9. Phipps RH, Deaville ER, Maddison BC. Detection of transgenic and endogenous plant DNA in rumen fluid, duodenal digesta, milk, blood and feces of lactating dairy cows. J. Dairy Sci. 2003, 86:JDS 3275 Take H502.
10. Chowdhury EH, Kuribara H, Hino A, Sultana P, Mikami O, Shimada N, Guruge KS, Saito M, Nakajima Y. Detection of corn intrinsic and recombinant DNA fragments and CrylAb protein in the gastrointestinal contents of pigs fed genetically modified corn Bt11. J Anim Sci 2003, 81, 2546-51, and references therein; reviewed by Ho MW. Transgenic DNA and Bt toxin survive digestion. Science in Society 2004, 21, 11.
11. Duggan PS, Chambers PA, Heritage J and Forbes JM. Fate of genetically modified maize DNA in the oral cavity and rumen of sheep. Journal of Nutrition 2003, 89, 159-166.

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