Influence of Genetically
Modified-SOYA on the
Birth-Weight and Survival of Rat Pups:
IRINA V. ERMAKOVA
Institute of Higher Nervous Activity and Neurophysiology
Russian Academy of Sciences (RAS)
Mindfully.org note: This paper was originally presented on 10 Oct 2005 to the symposium on genetic modification in Russia, which was organized by the National Association for Genetic Security (NAGS).
IrinaV.Ermakova Institute of Higher Nervous Activity and Neurophysiology RAS, Butlerov str., 5a, Moscow, Russia, I_Ermakova@mail.ru, (+7 095 334-43-13).
Investigations of the influence of genetically modified (GM)-plant, the Roundup Ready (RR)-soy on the birthrate and survival of the offspring of Wistar rats were performed. A group of female rats received additionally to the stock laboratory chow 5-7g/rat/day soya flour prepared from certified RR GM-soya for two weeks before mating, during mating, pregnancy, and an increased daily amount for every pup during lactation. The control group was fed in addition to the rat chow the same amount of traditional (Trad) soya. The third group of rats received only the chow, and was considered to be a positive control group. The behavior, physiological state, the weight and the mortality rate of the pups from the first mating were analyzed. High level of mortality (~ 55,6%) was observed with pups whose mothers received the GM-soya supplemented diets, and 36% of these pups weighed less than 20 grammas by the end of two weeks after the birth, in comparison with the Trad. soya supplemented group and with the positive control group (6.7% and 6% respectively). It was revealed in these experiments, that RR-soya could have a negative influence on the offsprings of Wistar rats, causing high level of mortality and decreased weight gain in some of the pups.
The term genetically modified organisms (GMOs) refers to plants, microbes and animals with genes transferred from other species in order to produce certain novel characteristics (for example resistance to pests, or herbicides), and are produced by recombinant DNA technology. There are two standard methods, which are generally used to introduce new DNA (genes) into a plant cell, which is going to be modified: 1) the particle acceleratio, or "shot-gun" technique, and 2) infecting the cells with a modified pathogen, with the help of Agrobacter tumefaciensis. Neither methods are perfect and do not guarantee that the rest of the plant genom remains unchanged. Therefore the safety of the GM-crops created with the help of these methods cannot be garanteed neither for human and animal health, nor forth the Environment (Ho and Tappeser, 1997; Kuznetcov et al., 2004; Wilson et al., 2004; Ermakova, 2005). Four main sources of the hazards of GMO are accepted by scientists worldwide: 1) those due to the new genes, and gene products introduced; 2) unintended effects inherent to the technology; 3) interactions between foreign genes and host genes; and 4) those arising from the spread of the introduced genes by ordinary cross-pollination as well as by horizontal gene transfer (World Scientists' Statement 2000).
GM-crops contain material, which under natural conditions is not present in them in nature, and they form a part of our daily diet. To understand what effect they can have on us and on our animals it is vitally important to study the influence of these GM-plants on different organisms for several generations. At the present, these studies are lacking from the scientific literature. Also, several detrimental effects of GM-crops had been showed on the metabolism of animals. The hazard of genetically modified organisms (GMO) was shown for animals and the environment in many investigations (Traavik, 1995; Ho and Tappeser B., 1997; Pusztai, 1999; 2001; Chirkov, 2002; Kuznetcov et al., 2004 and others). Earlier it was shown that consumption of GM-food by animals led to the negative changes in their organisms. Experiments, conducted by A. Pusztai showed that potatoes modified by the insertion of the gene of the snowdrop lectin (an insecticidal proteins), stunted the growth of rats, significantly affected some of their vital organs, including the kidneys, thymus, gastrocnemius muscle and others (1998) and damaged their intestines and their immune system (Ewen and Pusztai, 1999). Similar effect of GM-potatoes on rats was obtained at Institute of Nutrition in Russia (Report of Institute of Nutrition, 1998; Ermakova, 2005, Comments). In the investigations of Manuela Malatesta and coauthors the significant modifications in the cells of liver, exocrine pancreas and testis of mice, fed by diet containing GM-soybean (Roundup Ready), were shown (Malatesta et al., 2002, 2003; Vecchio et al., 2004).
Arpad Pusztai in his article "Genetically Modified Foods: Are They a Risk to Human/Animal Health" (2001) asked: "How can the public make informed decisions about GM foods when there is so little information about its safety?"
It is put forward in the risk assessment documents that the GM-components of transformed plants are completely destroyed in the digestive tract of humans and animals, together with the other genetic material found in them. However foreign DNA plasmids are steadier against the digestion, than it was originally believed. Plasmid DNA and GM-DNA were found in microorganisms of the intestine and in saliva (Mercer et al., 1998; Coghlan, 2002). Experimental researches in mice showed that ingested foreign DNA can persist in fragmented form in the gastrointestinal tract, penetrate the intestinal wall, and reach the nuclei of leukocytes, spleen and liver cells (Schubbert et al., 1994). In another research of Shubbert et al., (1998) the plasmid containing the gene for the green fluorescent protein (pEGFP-C1) or bacteriofaphage M13 DNA were fed to pregnant mice. Foreign DNA, orally ingested by pregnant mice, was discovered in blood (leukocytes), spleen, liver, heart, brain, testes and other organs of foetuses and newborn animals. The authors considered that maternally ingested foreign DNA could be potential mutagens for the developing fetus. However, Brake and Evenson (2004) analyzing the testis in mice as a sensitive biomonitor of potential toxic, didn’t find negative effects of transgenic soybean diet on fetal, postnatal, pubertal or adult testicular development.
There is a lack of investigations on the influence of GM/crops on mammals, especially on their reproductive function. Therefore, it was decided that we undertake a study to see the effect of the most commonly used GM crop on the birth rate, mortality and weight gain of rat pups, whose mothers were fed diets supplemented with Roundup-Ready soya.
MATERIALS AND METHODS
Diets and dietary components:
Roundup-Ready (RR) soya, genetically modified with the transgene CP4 EPSPS (40.3.2 line, Monsanto).
Since we had no access to the exact parent line, we bought a traditional (Trad) soya variety (Arcon SJ 91-330, ADM), which had a similar composition and nutritional value to the RR-soya, was bought from the Netherlands.
The soya flour was prepared from these varieties by grinding the raw whole soya bean seeds mix with water (40 ml) to form a paste.
Standard laboratory chow was obtained from Moscow, Russia (ПК-120-1).
Wistar rats from Moscow, Russia (Stolbovay) were used in the experiment. The animals were brought up to sexual maturity on laboratory rat chow. When their weight reached about 180-200 g, the female rats were divided into 3 groups, and housed in groups (3 rat/cage), and kept under normal laboratory conditions.
The feeding scheme was as follows. Females in every cage received dry pellets from a special container placed on the top of their cage, daily. They were also provided with 200 ml of drinking water/rat/day. Those rats receiving soya flour supplement, were given the soya flour in a small container placed inside their cage (20g x 40 ml water) for three rats and, so 5-7g flour for each rat every day.
One group of female rats of 180-200 g weight (in two cages: 3x3) was allocated to the experimental group, and received 5-7g soy a flour/rat/day prepared from Roundup-Ready soya, added to the rat chow for two weeks. Another group females (3) were allocated to the control group, but their diet was supplemented with the same amount of soya flour, prepared from a traditional soya Arcon SJ 91-330. We also introduced a positive control group (in two cages: 3x3), which had not been exposed to soya flour, therefore females have only got the standard laboratory chow, without any supplementation, although it is acknowledged, that the energy and protein content of this diet was less, than in the other two groups. After two weeks on the diets all groups of 3 females were mated with two healthy males of the same age, who have never been exposed to soya flour supplements. First the one, then the other male was put into the cage for 3 days. In order to avoid infection of females, the sperm count and quality has not been determined. We carried on with feeding the respective diets to all females during mating and pregnancy. Upon delivery, all females were transferred to individual cages, and the amount of soya supplement was increased by an additional g for every pup born. Lab chow and water was available ad libitum during the experimental period, for all animals. When rat pups open their eyes and could feed themselves (from 13-14 days of age), the dose of soya supplement was increased till 2-3g for every pup, daily, although all rats had free approach to the soya. All rats ate their soya portions well. Organs of some pups were taken out and weighed.
The weight of the rat pups were analyzed by the nonparametric Mann-Whitney test, and mortality rate and weight distribution - by the Newman-Keuls test, using the StatSoft Statistica v6.0 Multilingua (Russia).
Quantitative analysis of RR-soya by using the "CP4-LEC-RT-PCR" construct confirmed the presence of genetic modification in 100% of the flour. In the traditional, non GM-soya flour only traces (0.08+ 0.04%) of the same construct was present, most likely resulting from cross-contamination ((Altieri and et al., 2005).
By the end of the experiment, from the 15 females included in the experiment, 11 gave birth and produced a total of 132 rat pups. The four who became pregnant from six females on the positive control diet gave birth to 44 pups (an average of 11), while the four females, from the six on GM-soya flour supplemented groups gave birth to 45 (11.3 pups/female), and three from traditional soya-group - 33 pups (10 per mother).
Supplementation of the diet of the females with GM-soya led to the death of 25 pups, out of the 45 born by the end of the third week of lactation, while during the same period on the traditional soya supplemented diets only 3 pups died from 33. The mortality in the positive control group was also 3, but from the larger number of pups born, as it seen in Table 1 and Pict.1.
High pup mortality was characteristic of every litter from mothers fed the GM-soya flour (Table 2.).
From the litter of mothers fed the positive control diet 2 pups died on week first, and 1 on week second after delivery. All those pups died from the litters of mothers fed traditional soya flour, did so on the first week following birth. However, pups from mothers fed the GM-soya flour supplemented diet kept dying during lactation period as it is evident from Table 3.
In two weeks after their birth the weight of pups from the GM-soya supplemented group (23.95g +7.3 g) was less than that of the pups of the positive control group (30.03g +6.2 g; p<0.005), or from the traditional soya flour supplemented group (27.1 g + 3.3 g; p<0.1). Since the number of surviving pups were so different, the weigh distribution of the pups were compared in Table 4. From the data it is evident, that 36% of the pups from the GM-soya group weighed less than 20 grammas, in comparison with the 6% in the positive control group, and with the 6.7% found in the traditional soya supplemented diet group (see Figure 2, and also Table 4). Study of pup’s organs mass showed that the organs of small pups from GM-group were tiny in comparison with the same of other groups, except the brain mass (tabl.5). This fact indicated that the pups from the GM-group were the same age as others, but changes were occurred with the development of internal organs. Slight negative effect was found in the group, which received the traditional soya, but this effect was not significant, perhaps of number rat amount (tabl.1, 4). No lethality of females and survived young pups eating the GM-soya flour supplemented diet was observed.
The reproductive behavior of female rats fed on standard laboratory chow supplemented with soya flour prepared from either genetically modified (RR) or traditional soyabean seeds was studied to see the effect of the diet on pregnancy, lactation and the growth of the rat pups. Since it is well established, that raw soyabean contains a number of antinutrients (such as the lectins, trypsin inhibitors, etc. (Pusztai et al, 1998), and also female hormon-like substances, it was thought to be necessary to compare these data also with those from a positive control group, from animals not exposed to any soya flour supplementation.
In order to understand the mechanism, how this widely consumed GM-crop, which is present in about 50% of all foodstuff from bread to chocolate, exerts its influence on the reproductive performance of mammals and their offspring, it would be necessary to perform complex researches, including histological, genetical, and embryo-toxicological investigations. However, we had to restrict our experiments only for a short time-span, and starting to feed the female rats two weeks before mating. However, unlike in the experiments of Shubbert et al. (1998), or Brake and Evenson (2004), who started to feed already pregnant mice, in our experiments the diets supplemented with GM-, or traditional soya flours were given to the female rats 2 weeks before mating already, and we continued to treat them with their respective diet until the litters were weaned.
Upon delivery, very unexpectedly a very high rate of pup mortality (~ 55,6%) was observed in the litter of mothers, whose diet was supplemented with the genetically modified, Rundup-Ready soya flour, in comparison with the pups of both the positive control (6,8%), and the traditional soya flour supplemented (9%) groups. Also, in this group the pups continued to die over the period of lactation, which occured only in the GM-soya fed group. At the same time, the weights of the surviving rat pups were also lower. It is the more surprising, since litter numbers were smaller, about half, therefore more milk should have been available for the individual pups. They should have a better chance to grow optimally, unless the amount, and/or the quality of the milk was not affected by consuming the GM-soya flour.
Our data allow us to speculate and presume that the negative effect of GM-soya on the newborn pups could be mediated by two possible factors. Firstly, it can be the result of transformation, and insertion of the foreign genes, which could penetrate into the sexual/stem cells, or/and into cells of the fetus, as it was observed by Schubbert and collegues (1998). In their experiments the plasmid containing the green fluorescent protein (pEGFP-C1) gene, or the bacteriofaphage M13 DNA was fed to pregnant mice. The presence of the foreign DNA was detected in the cells of the mice in both cases. Also, the instability of gene constructs was described for GM-soya (Windels et al., 2001) and rice (Yang et al., 2005). Secondly, the negative effect of GM-soya could be mediated by the accumulation of Roundup residues in GM-soya. However, no mortality was observed with female rats, nor with the young pups survived, although they also began to eat the GM-soya, it is supposed that the effect could be mediated by the first factor.
I am very thankful to Dr. Susan Pusztai and Dr. Arpad Pusztai for valuable remarks and comments.
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Table 1.Mortality of rat pups by the end of the 3rd week of lactation
Number of Number of Dead pups/total Groups pups born dead pups born ( %) Positive control 44 3 6.8% (p=0,000118)* Trad. Soya 33 3 9% (p=0,000103)* GM-soya 45 25 55.6% *compared to the GM-soya flour supplemented group
Table 2. Number rat pups died from the litter of individual mothers on the GM-soya flour supplemented diet
Number of Number of Number of Females newborn rats cpups died dead pups/born (%) Female No. 1 11 7 64% Female No. 2 8 4 50% Female No. 3 13 6 46% Female No. 4 13 8 62%
Table 3.The number of dead pups (number and as %) from the treatment groups at different times after birth
Groups 1st week 2nd week 3rd week Positive control 4.5% (2) 2.3% (1) 0 Trad. Soya 9% (3) 0 0 GM-soya 31,1% (14) 13,4% (6) 11,1% (5)
Table 4. Weigh distribution of rat pups by 2 weeks of age on different diets
Group: 50-40 g 40-30 g 30-20 g 20-10 g Positive control 12.5% 37.5% 44% 6% Trad. soya 0% 20% 73.3% 6.7% GM-soya 0% 23% 41% 36%* * highly significant, p<0,001
Table 5. Examples of absolute values of organ mass in pups in three weeks after their birth. Fixation in formaldehyde 0.1M PBS, pH7.2.
NN Body Liver Lungs Heart Kidneys Spleen Testes Brain N26 normal 69 3.80 1.20 0.37 0.44/0.44 0.52 0.34/0.34 1.67 N27 normal 72 4.63 1.55 0.38 0.52/0.42 0.81 0.3/0.3 1.6 N28 GM-soya 35 1.83 0.6 0.19 0.28/0.28 0.21 0.13/0.14 1.60 N29 GM-soya 30 1.68 0.5 0.20 0.19/0.20 0.19 0.14/0.18 1.54 N30 Trad-soya 62 4.28 0.95 0.36 0.38/0.38 0.24 0.22/0.26 1.76 N31 Trad-soya 63 4.35 0.94 0.39 0.42/0.42 0.32 0.22/0.23 1.66
Figure1.Mortality of rat pups, whose mothers were fed with GM-, or traditional soya flour, or no soya at all ( %)
Figure2. Comparison of the size of the 19 days old rat pups from mothers fed the positive control diet and the GM-soya flour supplemented diet. The rats weights are 40 and 15 g, respectively