**5. Fate of transgenic DNA and new protein in animal organs and products**

One of the most important questions about the use of GM products in animal nutrition is the possibility that modified DNA could be transferred from plants to animal products or to bacteria, with harmful consequences (FAO, 2004). Other problems regard the ability of transgenic proteins to provoke food intolerance or allergic reaction in susceptible people. Hence, it is necessary to consider the destiny of these molecules within the animal organism (Alexander et al., 2007).

The gastrointestinal tract is constantly exposed to DNA that is released from partially or completely digested food, ingested microbes, and DNA from intestinal microflora. Ingested food is mechanically disrupted and the released DNA, although poorly digested, is cleaved through acid hydrolysis and enzymatic digestion into small DNA fragments. Eventually some of these fragments are converted to single nucleotides. Acid hydrolysis in the gastrointestinal tract is expected to depurinate most adensine and guanine nucleotides of the food DNA (Klinedinst & Drinkwater, 1992). The presence of various phosphatases and deaminases continue to destroy the structural integrity of any free DNA. The breakdown products of DNA are absorbed for using at the cellular level for synthetic processes as they may be found in blood and tissues (McAllan, 1982). All though there were conflicting reports on the fate of GM DNA in the biological system it was observed that DNA could pass through the gut wall into the blood stream and taken up by cells in the blood, liver, spleen and passed through the placenta to the cells of the feoetus and the newborn one (Doerfler & Schubert, 1998).

In ruminants, experimental evidence suggests that more than 80% of DNA is completely disrupted after 2 hours (Wiedemann et al., 2006). However, this degradation is not complete and not immediate (van den Eede et al., 2004).

In animal tissues some fragments of chloroplast DNA have been found. The reason why chloroplast DNA is more frequently detected in animal products is the number of the genes involved and the sensitivity of the PCR method. In transgenic plants, every cell contains hundreds chloroplast genes, but only one transgenic gene (Aumaitre et al., 2002).

Genetically Modified Soybean in Animal Nutrition 139

et al., 1994). The fate of ingested GM soybean DNA fragments (120 and 195 bp) and a 180 bp fragment of the lectin gene of soybean in Atlantic salmon and their survival through the gastrointestinal tract (GIT) were investigated and the DNA was traced in a variety of fish tissues (liver, muscle and brain) (Sanden et al., 2004). Only the smaller GM DNA fragment (120 bp) was amplified from the content of the stomach, pyloric region, mid intestine and distal intestine, while no transgenic or conventional soybean DNA fragments were detected. The uptake of dietary DNA into blood, kidney and liver of salmon was investigated also by other authors (Nielsen et al., 2005) which determined the DNA fragment size if dietary DNA was detected. Most of the feed (partially digested) was found in the pyloric region, mid intestine, and distal intestine at 4, 8, and 16 h after force-feeding, while the highest concentrations of dietary DNA in liver and kidney were found 8 h after force-feeding, and blood up to 64 h. Finally, the cauliflower mosaic virus 35S promoter fragment (220 bp) of the GM defatted soybean meal was detected in the muscle of rainbow trout receiving both levels of GM soybean (approximately 15 and 30%) diet by nested PCR, but the frequency of detection was greater at the higher inclusion level (Chainark et al., 2006). Additionally, the promoter fragment was not detected by the fifth day after changing the diet to non-GM soybean. Conversely, the promoter fragment was not detected from fish fed with the non-GM SBM diet. Successively, Chainark et al. (2008) traced foreign DNA fragments from genetically modified defatted soybean meal (GM SEM) in rainbow trout by nested polymerase chain reaction (PCR) and located by in situ hybridization. Either a GM or non-GM SBM formulated diet (42% protein) was fed to fish (average weight 50.5 g) for 2 weeks. The degradation results showed that the cauliflower mosaic virus 35S promoter (220 bp) fragment was detected in the contents of digestive system only in fish fed the GM SBM diet, and it was not detected on the third day after changing the diet to the non-GM SBM diet. For the possible transferral results, the promoter fragment was detected in the leukocyte, head kidney and muscle only of fish fed the GM SBM diet; it was not detected on the fifth day after changing the diet to the non-GM SBM diet. These results suggest that a foreign DNA fragment was not completely degraded and might be taken up into organs through the gastrointestinal tract. However, foreign DNA was not detected after the withdrawal period. Thus, the data show that uptake of DNA from GM SBM might not remain in the tissues of fish fed GM SBM diet. Similarly, Ran et al. (2009) found DNA fragments from RR soybean in

different tissues and organs of tilapias (*Oreochromis niloticus*, GIFT strain).

fragments gave undetectable results.

Tudisco et al. (2006) in order to evaluate the presence of plant DNA fragments in tissues to follow the fate of plant fed, carried out a research on twenty weaned 30-day-old New Zealand rabbits (10 males and 10 females), individually caged, which were equally assigned to control (C) and treated (T) groups. The animals were given a diet containing soybean meal (solved extracted) which was from conventional or Roundup Ready beans, for group C and T, respectively. The presence of chloroplast DNA was found in tissues and blood from both control and treated groups. The percentage of positive samples were: 50% (blood), 70% (muscle), 80% (heart), 70% (liver) and 80% (kidney). By contrast specific fragments of soybean were not detected in all samples but only in the plant samples. Similarly transgenic

Subsequently, the same authors (Tudisco et al. 2010) investigated the presence of DNA fragments in blood and milk from goats fed conventional (control) or Roundup Ready soybean and in blood, skeletal muscle and organs from their offspring. Transgenic target DNA sequences (35S and CP4 EPSPS) were detected in blood and milk from goats that received a diet containing transgenic soybean as well as from some samples of their

Research on the fate of foreign DNA in the mammalian organism showed that PCR products specific to foreign DNA could be detected therein. It was concluded that DNA fragments from the gastrointestinal tract could reach the bloodstream and be transported through the epithelium of the gut and the cells of the Peyer's patches to spleen and liver cells. Such DNA fragments are probably retained for a short while and then digested (Schubbert et al., 1994, 1997, 1998). While the intestinal tract does not seem an absolute barrier against the uptake of macromolecules or even of microorganisms, the mechanism of foreign DNA uptake by the intestinal wall epithelia is unknown. In addition, not much is known about the degradation and integration of the DNA. There is some evidence that fragments of foreign DNA are not digested in the gut and might enter the organism or become incorporated into cells lining the gut wall (Doerfler, 2000; Tony et al., 2003).

Transfer of the plant DNA to bacteria needs several steps, and the expectation seems to be extremely low (Kuiper et al., 2003; Sharma e al., 2004). Bacterial resistance to antibiotics is not a specific problem of genetic engineering. According to Directive 2001/18/EC, use of GMOs containing antibiotic-resistant genes will be forbidden starting from 01/01/2009 (European Commission, 2001).

Data are also available on the fate of recombinant plant DNA in the gastrointestinal tract of humans. By in vitro simulation of human digestion, 80% of the transgene in naked GM soybean DNA was degraded in the gastric simulations, while no degradation of the transgenes contained within GM soybean and corn was observed in these acidic conditions (Martin-Orúe et al., 2002). In the small intestinal simulations, transgenes in naked soybean DNA were degraded. In contrast, the corn nucleic acid was hydrolysed in the small intestinal simulations in a biphasic process in which approximately 85% was rapidly degraded, while the rest of the DNA was cleaved at a low rate of degradation.

The number of transgene copies passing to the small intestine of human ileostomists consuming GM soya were successful quantified, and up to 3.7% of the transgene could survive passage (Neterwood et., 2004).

Finally, another factor that will directly affect gene persistence throughout the digestive tract and therefore indirectly affect the chance of passage across the GIT epithelium is the digestibility of the ingested plant species. Feedstuffs with relatively greater digestibility, such as soybean meal, are likely to have their DNA degraded more rapidly, decreasing the chance of absorption.

A low copy endogenous (soybean lectin) and recombinant (CP4 epsps) gene in longissimus dorsi muscle samples from pigs fed herbicide-tolerant soybean meal, in the grower and finisher phases, respectively were attempted to detect (Jennings et al., 2003). The same results about the fate of the CP4 epsps gene in other species were reported (Klotz & Einspanier, 1998).

The CP4 epsps transgenic gene was not found in muscle and liver of chicken fed herbicidetolerant soybean up to 7 weeks after ingestion (Khumnirdpetch et al., 2001). According to the authors, GM soybean fragment were degraded in the gastrointestinal tract.

As regards ruminants, chloroplast gene fragments were found in the leucocytes of dairy cows fed small quantities of transgenic soybean meal, while no fragments of the transgenic DNA were found in any tissue examined and in milk (Klotz & Einspanier, 1998). Similarly, high copy chloroplast "rubisco" gene fragments were found in the blood of cattle fed GM and soybean meal, but transgenic sequences were never detected (Phipps et al., 2003).

In fish, soybean meal is used as dietary source of protein in their diet, however, because of the presence of anti-nutritional factors (ANFs) their inclusion levels should be kept low (Olli

Research on the fate of foreign DNA in the mammalian organism showed that PCR products specific to foreign DNA could be detected therein. It was concluded that DNA fragments from the gastrointestinal tract could reach the bloodstream and be transported through the epithelium of the gut and the cells of the Peyer's patches to spleen and liver cells. Such DNA fragments are probably retained for a short while and then digested (Schubbert et al., 1994, 1997, 1998). While the intestinal tract does not seem an absolute barrier against the uptake of macromolecules or even of microorganisms, the mechanism of foreign DNA uptake by the intestinal wall epithelia is unknown. In addition, not much is known about the degradation and integration of the DNA. There is some evidence that fragments of foreign DNA are not digested in the gut and might enter the organism or

become incorporated into cells lining the gut wall (Doerfler, 2000; Tony et al., 2003).

degraded, while the rest of the DNA was cleaved at a low rate of degradation.

(European Commission, 2001).

survive passage (Neterwood et., 2004).

chance of absorption.

Einspanier, 1998).

Transfer of the plant DNA to bacteria needs several steps, and the expectation seems to be extremely low (Kuiper et al., 2003; Sharma e al., 2004). Bacterial resistance to antibiotics is not a specific problem of genetic engineering. According to Directive 2001/18/EC, use of GMOs containing antibiotic-resistant genes will be forbidden starting from 01/01/2009

Data are also available on the fate of recombinant plant DNA in the gastrointestinal tract of humans. By in vitro simulation of human digestion, 80% of the transgene in naked GM soybean DNA was degraded in the gastric simulations, while no degradation of the transgenes contained within GM soybean and corn was observed in these acidic conditions (Martin-Orúe et al., 2002). In the small intestinal simulations, transgenes in naked soybean DNA were degraded. In contrast, the corn nucleic acid was hydrolysed in the small intestinal simulations in a biphasic process in which approximately 85% was rapidly

The number of transgene copies passing to the small intestine of human ileostomists consuming GM soya were successful quantified, and up to 3.7% of the transgene could

Finally, another factor that will directly affect gene persistence throughout the digestive tract and therefore indirectly affect the chance of passage across the GIT epithelium is the digestibility of the ingested plant species. Feedstuffs with relatively greater digestibility, such as soybean meal, are likely to have their DNA degraded more rapidly, decreasing the

A low copy endogenous (soybean lectin) and recombinant (CP4 epsps) gene in longissimus dorsi muscle samples from pigs fed herbicide-tolerant soybean meal, in the grower and finisher phases, respectively were attempted to detect (Jennings et al., 2003). The same results about the fate of the CP4 epsps gene in other species were reported (Klotz &

The CP4 epsps transgenic gene was not found in muscle and liver of chicken fed herbicidetolerant soybean up to 7 weeks after ingestion (Khumnirdpetch et al., 2001). According to

As regards ruminants, chloroplast gene fragments were found in the leucocytes of dairy cows fed small quantities of transgenic soybean meal, while no fragments of the transgenic DNA were found in any tissue examined and in milk (Klotz & Einspanier, 1998). Similarly, high copy chloroplast "rubisco" gene fragments were found in the blood of cattle fed GM and soybean meal, but transgenic sequences were never detected (Phipps et al., 2003). In fish, soybean meal is used as dietary source of protein in their diet, however, because of the presence of anti-nutritional factors (ANFs) their inclusion levels should be kept low (Olli

the authors, GM soybean fragment were degraded in the gastrointestinal tract.

et al., 1994). The fate of ingested GM soybean DNA fragments (120 and 195 bp) and a 180 bp fragment of the lectin gene of soybean in Atlantic salmon and their survival through the gastrointestinal tract (GIT) were investigated and the DNA was traced in a variety of fish tissues (liver, muscle and brain) (Sanden et al., 2004). Only the smaller GM DNA fragment (120 bp) was amplified from the content of the stomach, pyloric region, mid intestine and distal intestine, while no transgenic or conventional soybean DNA fragments were detected. The uptake of dietary DNA into blood, kidney and liver of salmon was investigated also by other authors (Nielsen et al., 2005) which determined the DNA fragment size if dietary DNA was detected. Most of the feed (partially digested) was found in the pyloric region, mid intestine, and distal intestine at 4, 8, and 16 h after force-feeding, while the highest concentrations of dietary DNA in liver and kidney were found 8 h after force-feeding, and blood up to 64 h. Finally, the cauliflower mosaic virus 35S promoter fragment (220 bp) of the GM defatted soybean meal was detected in the muscle of rainbow trout receiving both levels of GM soybean (approximately 15 and 30%) diet by nested PCR, but the frequency of detection was greater at the higher inclusion level (Chainark et al., 2006). Additionally, the promoter fragment was not detected by the fifth day after changing the diet to non-GM soybean. Conversely, the promoter fragment was not detected from fish fed with the non-GM SBM diet. Successively, Chainark et al. (2008) traced foreign DNA fragments from genetically modified defatted soybean meal (GM SEM) in rainbow trout by nested polymerase chain reaction (PCR) and located by in situ hybridization. Either a GM or non-GM SBM formulated diet (42% protein) was fed to fish (average weight 50.5 g) for 2 weeks. The degradation results showed that the cauliflower mosaic virus 35S promoter (220 bp) fragment was detected in the contents of digestive system only in fish fed the GM SBM diet, and it was not detected on the third day after changing the diet to the non-GM SBM diet. For the possible transferral results, the promoter fragment was detected in the leukocyte, head kidney and muscle only of fish fed the GM SBM diet; it was not detected on the fifth day after changing the diet to the non-GM SBM diet. These results suggest that a foreign DNA fragment was not completely degraded and might be taken up into organs through the gastrointestinal tract. However, foreign DNA was not detected after the withdrawal period. Thus, the data show that uptake of DNA from GM SBM might not remain in the tissues of fish fed GM SBM diet. Similarly, Ran et al. (2009) found DNA fragments from RR soybean in different tissues and organs of tilapias (*Oreochromis niloticus*, GIFT strain).

Tudisco et al. (2006) in order to evaluate the presence of plant DNA fragments in tissues to follow the fate of plant fed, carried out a research on twenty weaned 30-day-old New Zealand rabbits (10 males and 10 females), individually caged, which were equally assigned to control (C) and treated (T) groups. The animals were given a diet containing soybean meal (solved extracted) which was from conventional or Roundup Ready beans, for group C and T, respectively. The presence of chloroplast DNA was found in tissues and blood from both control and treated groups. The percentage of positive samples were: 50% (blood), 70% (muscle), 80% (heart), 70% (liver) and 80% (kidney). By contrast specific fragments of soybean were not detected in all samples but only in the plant samples. Similarly transgenic fragments gave undetectable results.

Subsequently, the same authors (Tudisco et al. 2010) investigated the presence of DNA fragments in blood and milk from goats fed conventional (control) or Roundup Ready soybean and in blood, skeletal muscle and organs from their offspring. Transgenic target DNA sequences (35S and CP4 EPSPS) were detected in blood and milk from goats that received a diet containing transgenic soybean as well as from some samples of their

Genetically Modified Soybean in Animal Nutrition 141

soybeans. The authors did not find adverse effects in mice following acute oral exposure to GM-HRA at a dose of at least 436 mg/kg of body weight, or in a 28-day repeated dose dietary toxicity study at doses up to 1247 mg/kg of body weight/day. It was concluded that

In contrast to the above results, in a long-term study on female mice fed with a GM modified soybean (insertion of the bacterial CP4 EPSPS gene to confer a high level of tolerance to glyphosate), focused on assessing the effects of this diet on liver of old animals (until 24 months of age) and to elucidate possible interference with aging, Malatesta et al. (2008a) found that GM soybean intake could influence the liver morpho-functional features during the physiological process of aging. Several proteins belonging to hepatocyte metabolism, stress response, calcium signaling and mitochondria were differentially expressed in GM-fed mice, indicating a more marked expression of senescence markers in comparison to controls. Moreover, hepatocytes of GM-fed mice showed mitochondrial and nuclear modifications indicative of reduced metabolic rate. In previous studies on hepatocytes from young and adult (2–8 months of age) female mice fed GM soybeans, nuclear modifications involving structural constituents of the transcription and splicing properties pathways were seen (Malatesta et al., 2002a). Although the cause(s) of the observed alterations could not be conclusively established, it was noted that these modifications disappeared when GM soybean was replaced by a non-GM one in the diet (Malatesta et al., 2005). Since the GM soybean used was tolerant to glyphosate and was treated with the glyphosate-containing herbicide Roundup, the effects observed might be due to herbicide residues. Accordingly, and aiming to verify this hypothesis, Malatesta et al. (2008b) treated rat hepatoma tissue culture (HTC) cells with 1–10 mM Roundup and analyzed cellular features by flow cytometry, fluorescence, and electron microscopy. Under these experimental conditions, the death rate and the general morphology of HTC cells were not affected, as well as most of the cytoplasmic organelles. However, in HTC-treated cells, lysosome density increased and mitochondrial membranes were modified indicating a decline in the respiratory activity. In addition to the above, nuclei underwent morphofunctional modifications suggesting a decreased transcriptional/splicing activity. The authors did not exclude that factors other than the presence of the herbicide residues could be responsible for the cellular modifications described in GM-fed mice. However, they indicated that the concordance of the effects induced by low concentrations of Roundup on HTC cells suggested that the presence of Roundup residues could be one of the factors

Cisterna et al. (2008) investigated the ultrastructural and immunocytochemical features of pre-implantation embryos from mice fed either GM or non-GM soybean in order to verify whether the parental diet could affect the morpho-functional development of the embryonic ribonucleoprotein structural constituents involved in pre-mRNA pathways. Morphological observations revealed that the general aspect of embryo nuclear components were similar in the GM and non-GM soybean-exposed groups. However, immunocytochemical and in situ hybridization results suggested a temporary decrease of pre-mRNA transcription and splicing in 2-cell embryos and a resumption in 4–8-cell embryos from mice fed GM soybean. In addition, pre-mRNA maturation seemed to be less efficient in both 2-cell and 4–8-cell

Battistelli et al. (2010) investigated the duodenum and colon of mice fed on genetically modified (GM) soybean during their whole life span (1-24 months) by focusing their attention on the histological and ultrastructural characteristics of the epithelium, the

GM-HRA protein is safe when used in agricultural biotechnology.

interfering with multiple metabolic pathways.

embryos from GM-fed mice than in non-GM-fed animals.

offspring, not in the control group. Those findings show plant DNA fragments are likely to survive digestive processes to some extent (Duggan et al., 2003; Einspanier et al., 2004), as well as their transfer to blood and milk. In addition, the detection of plant DNA in tissues and organs of nursed kids could support the hypothesis of a gene transfer through milk.
