**8. Conclusions**

146 Soybean – Genetics and Novel Techniques for Yield Enhancement

herbicide on human/animal health, particularly as it is used in large amounts. Indeed, there are a number of recently published papers that all indicate possible damaging effects of glyphosate on health and reproduction which need to be taken seriously. On previous work the findings of Marc et al. (2005) have confirmed and extended their previous results by showing that the main ingredient of commercial Roundup formulations, glyphosate, in a mM concentration range, particularly when used together with the obligatory polyoxyethylene amine surfactant, inhibited the transcription of one of the enzymes involved in hatching of sea urchin embryos and therefore significantly delayed their hatching. When it is considered that farm workers inhale commercial herbicide sprays in which the active ingredient concentration exceeds by about 25 times of that used in the transcription inhibition studies by Marc et al. (2005), health concerns due to the use of glyphosate must be acute. In another study it was shown that in the oral treatment of Wistar rats with increasing concentrations of the herbicide Glyphosate-Biocarb, a formulation used in many countries such as Brazil, the number of Kupffer cells in hepatic sinusoids increased, followed by large deposition of reticulin fibres and the leakage of hepatic aspartate aminotransferase and alanine aminotransferase into the circulation, indicating hepatic damage in these animals (Benedetti et al., 2004). Successively, Richard et al. (2005) and Benachour et al. (2007) showed that glyphosate, particularly as used together with polyoxyethylene amine surfactant in Roundup Ready formulations, was toxic to human placental JEG3 cells at concentrations lower than that used in agricultural practices. Even at subtoxic concentrations RR was an endocrine disruptor on aromatase activity and its mRNA level as glyphosate interacted with the active site of the purified enzyme. It is possible that the pregnancy problems in agricultural workers using Roundup may be traced back to the

Recently, Gasnier et al. (2009) exposed human liver HepG2 cells, a well-known model to study xenobiotic toxicity, to four different formulations and to glyphosate, which is usually tested alone in chronic *in vivo* regulatory studies. They measured cytotoxicity with three assays (Alamar Blue®, MTT, ToxiLight®), plus genotoxicity (comet assay), anti-estrogenic (on ER\_, ER\_) and anti-androgenic effects (on AR) using gene reporter tests. They also checked androgen to estrogen conversion by aromatase activity and mRNA. All parameters were disrupted at sub-agricultural doses with all formulations within 24 h. These effects were more dependent on the formulation than on the glyphosate concentration. First, the observed a human cell endocrine disruption from 0.5ppm on the androgen receptor in MDA-MB453-kb2 cells for the most active formulation (R400), then from 2ppm the transcriptional activities on both estrogen receptors were also inhibited on HepG2. Aromatase transcription and activity were disrupted from 10 ppm. Cytotoxic effects started at 10ppm with Alamar Blue assay (the most sensitive), and DNA damages at 5 ppm. According to the authors, the direct G action is most probably amplified by vesicles formed by adjuvants or detergent-like substances that allow cell penetration, stability, and probably change its bioavailability and thus metabolism. These detergents can also be present in rivers as polluting contaminants. The type of formulation should then be identified precisely in epidemiological studies of G-based herbicides effects. Of course to drive hypotheses on *in vivo* effects, not only dilution in the body, elimination, metabolism, but also bioaccumulation and time-amplified effects should be taken into account. These herbicides mixtures also present endocrine effects on human cells, at doses far below agricultural dilutions and toxic

exposure to this herbicide (Savitz et al., 2000).

levels on mitochondrial activities and membrane integrity.

The debate on the safety of genetically modified organisms (GMOs) used for food and feed is still very lively throughout the world, more than 15 years after their first commercial release. Huge social, economical, and political issues have been raised. Unfortunately, although some stakeholders claim that a history of safe use of GMOs can be upheld, there are no human or animal epidemiological studies to support such a claim as yet, in particular because of the lack of labeling and traceability in GMO-producing countries. As a matter of fact, 97% of edible GMOs among cultivated GMOs (soy, corn and oilseed rape or canola, excluding cotton) are grown in South and North America, where GMOs are not labeled. All these plants have been modified to tolerate and/or produce one or more pesticides, and contain therefore such residues at various levels. Most are Roundup residues (it is a major herbicide used worldwide and tolerated by about 80% of GMOs).

As stated by the EFSA (2008), several aspects have to be investigated when considering whether or not recombinant DNA from GM plants, or the derived proteins can end up in animal tissues and products. These include (i) the fate of the recombinant DNA and protein during feed processing and ensiling; (ii) the fate of the recombinant DNA and protein in the gastrointestinal tract of animals fed with GM feed; (iii) the potential absorption of the digested pieces of DNA or protein into animal tissues/products and (iv) the potential of biological functionality of absorbed DNA and protein fragments.

The mere detection of recombinant DNA fragments in animal organs and tissues could not justify, by itself, public concerns regarding human consumption of products from farm animals fed transgenic crops. However, the persistence of DNA after dietary exposure is one aspect of risk assessment for novel food. Indeed, as concerns the hypothetical horizontal gene transfer of recombinant DNA from GM crop-derived feeds to animal and human gut microflora, Netherwood et al. (2004), found that a small proportion of feed DNA survives passage through the human upper gastrointestinal tract and a very small proportion of the small intestinal microflora containing transgenic feed. According to the authors, even if this result does not indicate a complete transgenic transfer to the prokaryotes, the survival of transgenic DNA during the passage through the small intestine should be considered in future safety assessments of GM foods. In addition, any alteration in cell metabolism should be taken into account in this field. For instance, the modification in LDH synthesis suggests an increase in cell metabolism. Therefore, possible long-term effects of such an alteration need to be elucidated.

In conclusion, taking into account the potential risks related to GMP impact, further researches are needed in this area, including studies to determine DNA transport or entry

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