More Information on HGT from Biotech Crops to Intestinal Cells

Studies from Doerfler's laboratory at the University of Cologne in Germany have addressed the question of the ultimate fate of ingested DNA in mammals. In the first set of experiments the Doerfler team fed mice pure, circular DNA extracted from the phage virus M13, and, 1 to 7 hours after feeding, they identified small amounts of absorbed DNA fragments (200-500bp) in the blood, epithelial cells of the intestinal wall, peripheral leukocytes, spleen, and liver cells. In follow-up studies, they screened 109 clones of splenocyte DNA extracted from the mice and found one M13-positive 1.3 kilobase fragment clone. The M13 DNA fragment was covalently linked to an 80-nucleotide fragment with 70% homology for the IgE receptor gene (Schubbert et al., 1997). Additional chimeric molecules of M13 DNA fragments were found associated with bacterial DNA (the authors suggest from normal gut bacteria) and with rearranged lambda phage DNA. These later findings raise serious questions about the technical conduct of these studies and suggest that the results are possibly common cloning artifacts as cautioned when creating genomic libraries (Maniatis, 1982). That concern is independent of the question of why an 'altered' IgE sequence (or even how the IgE sequence was altered) was found in the same clone.

These studies have been reviewed and criticized (Beever and Kemp, 2000). In general, they raise the possibility of technical methodological errors given the infrequent occurrence of incorporated M13 DNA fragments identifiable only after powerful amplification techniques were used. More specific issues related to experimental design include the

• excessive amount of DNA fed to the mice compared to normal DNA intake, and
• use of circular DNA from the M13 virus

Unlike plant or animal DNA, M13 DNA has short specific sequences that are not methylated. Human and mouse white blood cells, specifically the macrophages, respond to M13 DNA as if it were an infectious agent.

While DNA fragments from digested food may move from the mammalian digestive tract into other cells of the organism, there is no conclusive evidence that intact genes from foods, whether biotech or non-biotech, integrate into the genome of animal cells (Schubbert et al. 1997, 1998; Hohlweg and Doerfler, 2001; Einspanier et al., 2001).

Khumnirdpetch et al. (2001) attempted to detect transgenic DNA in broiler chickens fed meal from glyphosate tolerant soybeans from birth to seven weeks of age. Samples (meat, skin, duodenum and liver) were isolated from the birds at 1, 3, 5 and 7 weeks. PCR results of all samples taken over the seven-week feeding period were negative. The authors speculated that the negative detection results suggest that the transgenic DNA in glyphosate tolerant soybean meal had been fully degraded in the digestive tract of the broilers.

Weber and Richert (2001) attempted to detect the Bt gene and an endogenous corn gene in DNA extracted from 24 pork loin samples (12 fed Bt corn and 12 fed a control conventional corn). PCR followed by Southern blot analysis for ~200 bp fragments of the Bt transgene and endogenous shrunken-2 (sh-2) gene were uniformly negative. By comparison, adding corn DNA to the sample of swine DNA produced positive results, indicating that the DNA quality and PCR conditions were both favorable for detection of DNA fragments, had they been present in the original samples.

Using PCR followed by Southern blot, Klotz and Einspanier (1998) could not detect the transgene from glyphosate tolerance soybeans in either the blood or milk of cows. However, this highly sensitive technique detected a small fragment of a soybean chloroplast gene in white blood cells but not milk.

Einspanier's laboratory has published data from a study in which dairy cows, beef steers and broiler chickens were fed either grain from biotech maize or grain from non-biotech maize (Einspanier et al., 2001; Flachowski et al., 2000). The investigators used two DNA detection technologies [standard Polymerase Chain Reaction (PCR) and Light Cycler "real time" PCR]. No portion of the Bt gene could be detected by either method in any samples from the cows, steers or chickens fed Bt corn. Similar to the previous Klotz and Einspanier (1998) report, however, they could detect a small fragment of a chloroplast gene (tRNAleu) in lymphocytes of dairy cows and in muscle, liver, spleen and kidneys cells of chickens, but not in dairy milk, or any tissue samples from steers.

In a similar study, where pigs were fed Bt maize, short chloroplast DNA fragments were detectable by PCR in intestinal juices, but not in any pig organ investigated. Bt maize specific transgenic DNA fragments were never detected in any pig sample (Klotz et al., 2002).

Therefore, in light of the Einspanier et al. (2001) and Schubbert et al. (1997, 1998) studies, a primary determinant in detecting fragments of ingested DNA in host animal tissues may be the copy number of the sequence ingested. The copy number of the chloroplast genome per cell ranges from ~500 to 10,000 copies in roots and leaves, respectively (Bendich, 1987). Therefore, the copy number of the detected chloroplast gene is orders of magnitude higher than a transgene in a transgenic crop, which typically has only one copy per haploid genome. In addition, chloroplast gene sequences are also present in high numbers in the plant's nuclear genome, with >100 copies of some sequences being recorded (Ayliffe et al., 1998).

Based on these studies, it would be expected that fragments of both native genes and transgenes, as a consequence of food ingestion, would be distributed in a variety of animal tissues, probably in amounts that cannot be detected with current methods. It is important to reiterate that if uptake of small amounts of transgenic DNA fragments does occur, the rate will be trivial when compared to the uptake of other DNA fragments from ingested food. In the gut or in the environment, the amount of non-transgenic DNA exceeds transgenic DNA by many orders of magnitude.

The World Health Organization (1993) and the U.S. Food and Drug Administration (1992) concluded there is no risk in consuming the DNA of biotech crops. The basis of this decision was that mammals have always ingested a large quantity of DNA from plants, animals, bacteria, parasites and viruses, and the proportion of transgenic DNA within native DNA of a biotech crop, for example, is less than 0.00042% (Beever and Kemp, 2000).

In a review on novel DNA in mammalian systems, Doerfler et al. (1998) conclude with the following perspective:

"Of course, we and our ancient history ancestors have all exposed our gastrointestinal surfaces to large amounts of foreign DNA of most variable origins for millions of years. Whatever the evolutionary consequences of this mass experiment in natural gene technology may have been, we just begin to understand in a modest way these consequences by applying concepts of gene technology ourselves. In the light of the magnitude and millenia duration of these natural processes, concerned scenarios about the application of gene technology and its consequences in our age belong to the realm of fairy tales."