Influence-of-Genetically-modified-Soya-on-the-Birth-Weight-and-Survival-of-Rat-Pups.doc

Influence of Genetically modified Soya on the Birth-Weight and Survival of Rat Pups 转基因大豆对幼鼠出生重量和生存状况的影响By Irina V.EmakovaAbstract Investigation of the influence of GM soya on the birthrate and survival of the offspring后代of Wistar rats2 were performed. A group of female rats were fed GM soya flour before mating 交配and pregnancy. The control group of females were fed traditional soya and the third group of females, the positive control group, received feed without any soya. The weight and the mortality死亡 rate of the newborn pups were analyzed. The study showed that there was a very high rate of pup mortality (55.6%) in the GM soya group in comparison with the control group and the positive control group (9% and 6.8% respectively. Moreover, death in the first group continued during lactation哺乳, and the weights of the survivors are lower those from the other two groups. It was revealed in these experiments, that GM soya could have a negative influence on the offspring of Wistar rats.摘要我们进行了转基因大豆对大白鼠后代出生率和生存状况的影响研究。一组母鼠在交配和怀孕前被喂食转基因大豆粉。母鼠的控制组被喂食传统大豆，第三组母鼠，即积极控制组，吃的食物没有任何大豆。我们分析了新生幼鼠的体重和死亡率。研究表明相比控制组合积极控制组的死亡率（分别是9%和6.8%），转基因大豆组幼鼠死亡率（55.6%）很高。另外，转基因大豆组的幼鼠死亡率在哺乳期还在继续，存活幼鼠的体重也比其他两组幼鼠要轻。这些实验表明，转基因大豆对大白鼠后代可能有消极的影响。IntroductionThe term genetically modified organism (GMO3s) 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. Four main sources of the hazards危险 of GMO are discussed by scientists worldwide: 1) those due to the new genes, and gene products introduced; 2) unintended effects inherent内在的 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异花授粉 as well as by horizontal gene transfer (World Scientists Statement 2000).介绍术语“转基因生物”指的是拥有从其他物种传递来的基因的植物、微生物和动物，转移基因是为了产生某些新的特点（比如抗病虫，或抗除草剂），并且是由重组DNA技术产生的。全世界的科学家讨论了四种转基因生物风险的主要来源：1）那些因为新的基因，以及引入的基因产品导致的风险；2）技术内在的无意间的影响；3）外来基因和宿主基因的相互作用；4）因为普通的异花传粉和水平基因传递导致的引入基因传播产生的风险（世界科学家声明，2000年）GM crops contain material, which is not present in them under natural conditions, 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 in 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 1997; Pusztai 1999 and 2001; 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 lectin4 (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 (Ermakova 2005).转基因作物包含一些材料，在自然条件下这些材料不会出现在作物中，转基因作物成为了我们日常食物的一部分。为了理解它们对我们和动物有什么影响，研究这些转基因植物在几代不同生物体中的影响是非常重要的。目前，这些研究还缺乏科学文献。同时，转基因作物对动物新陈代谢的几种不利影响已被揭示。在很多调查中转基因生物对动物和环境的危险也被揭示。（Traavik1995;）。早期有研究表明，动物食用转基因食品会导致有机体负面变化。A.Pustzai做的实验表明，嵌入雪花莲凝集素（一种杀虫剂蛋白质）基因之后的转基因土豆，阻碍了老鼠的生长，极大地影响了它们的一些重要器官，包括肾脏、胸腺、腓肠肌和其它器官（1998）并且损伤了它们的肠子和免疫系统（EwenandPustzai1999）。俄罗斯营养学院也得到了类似的转基因土豆对老鼠影响的结果。 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 plasmids5 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 Schubbert et al. (1998) the plasmid containing the gene for the green fluorescent protein6 (pEGFP-C1) or bacteriophage 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. At the same time Brake and Evenson (2004) analyzing the testis in mice as a sensitive biomonitor of potential toxic, didnt find negative effects of transgenic soybean diet on fetal, postnatal, puber talor adult testicular development. 在风险评估文件中提出已经转变的植物中转基因成分在人类和动物的消化道中被完全破坏，还有其它被发现的转基因材料也是如此。但是，外来DNA质粒比最初人们认为地更能抵御消化作用。在肠道和唾液微生物中发现了质粒DNA和转基因DNA(Merceretal1998;)。对老鼠的实验研究表明被吸收的外来DNA可以以片段的形式持续存在于胃肠道中，穿透肠壁，到达白细胞、脾细胞和肝细胞的细胞核。（Schubbertetal.1994）。在Schubbert等人做的另一项研究中（1998），包含绿色荧光蛋白（pEGFP-c1）基因或噬菌体M13DNA的质粒被喂给怀孕的老鼠。外来DNA被怀孕的老鼠口服后，在胚胎和新生幼鼠的血液（白细胞）、脾、肝、心、脑、睾丸和其它器官都有发现。作者们认为被雌性老鼠吸收的外来DNA可能是正在发育的胚胎的潜在诱变因素。同时Brake和Evenson(2004)分析了老鼠中的睾丸，它是敏感的潜在毒性物质的生物监控器，他们并未发现转基因大豆食物对胎儿、产后、青年和成年睾丸器官的发展有消极影响。 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 mother were fed diets supplemented with the Roundup- Ready soya, a kind of GM food.转基因作物对哺乳动物，尤其对他们的繁殖功能的影响还缺乏调查。因此，我们决定进行一项研究，以确认最常用的转基因作物对幼鼠的出生率、死亡率和体重增加的影响，而生这些幼鼠的母鼠被喂食的食物补充了“抗农达”转基因大豆，是一种转基因食物。MethodsAnimals Wistar rats were used in the experiment. The animals were brought up to sexual maturity on laboratory rat feed. When their weight reached about 180-200g, the female rats were divided into 3 groups, and housed in groups (3 rat/cage), and kept under normal laboratory conditions.方法在实验中被用到大白鼠。这些大白鼠被喂食实验室老鼠饲料，养育到性成熟阶段。当它们的重量达到180-200g,母鼠被分成三组，按组放在不同的笼子里（每笼三只老鼠），并且处于正常的实验室条件下。 The feeding scheme was as follows. Females in every cage daily received dry pellets from a special container placed on the top of their cage. 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.喂食计划如下：每个笼子里的母鼠每天从位于笼子顶部的特殊容器接受干的颗粒。那些接受大豆粉补充物的老鼠从放在笼子里的小容器中得到大豆粉（20g和40ml水），笼子里有三只老鼠，所以每天每只老鼠5-7克大豆粉。Experiment One group of female rats of 180-200g weight was allocated to the experimental group, and received 5-7 soy a flour/rat/day prepared from Roundup-Ready soya, added to the rat feed 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 the traditional soya in which only traces (0.08+ 0.04%) of the GM construct was present, most likely resulting from cross-contamination. 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 feed without any supplementation, although it is acknowledged that the energy and protein content of this diet was less than in the other two groups.实验一组重180-200g的母鼠被分配到实验组，每只老鼠每天接受5-7g来自“抗农达”的转基因大豆粉，这些大豆粉加到老鼠食物中，持续2周。另一组母鼠（3只）被分配到控制组，但是它们的食物补充的是同量的大豆粉，从传统的大豆磨制，其中只有微量的（0.08+0.04%）的转基因构成物出现，最有可能是因为交叉感染。我们还引入了一个积极控制组（两个笼子：每个笼子3只老鼠），这一组没有接触大豆粉。所以这些母鼠只有标准的实验室食物，没有任何补充物。尽管我们承认这种食物的能量和蛋白质含量比其它两组少。 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 feed and water was available for all animals during the experimental period. When rat pups opened their eyes and could feed themselves (from 13-14 days of age), the daily dose of soya supplement was increased till 2-3g for every pup, 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 level of mortality was analyzed by the one-way ANOVA7, using of Newman-Keuls test8 for share distribution. The pups weight and its distribution were checked by Mann-Whitney test9 and Chi-square10 in StatSoft Statistica v6.0 Multilingua (Russia).食用相应的食物两周后，所有各组的3只母鼠与两只同龄的健康公鼠交配，这两只公鼠从没接触过大豆粉补充物。首先是第一只，然后是另外一只被放进笼子三天。为了避免母鼠感染，精子的计数和质量没有被确定。在交配和怀孕期间，我们继续给母鼠喂食各自的食物。分娩之后，所有的母鼠被转移到单独的笼子。对于每只刚出生的幼鼠，大豆补充物的数量额外增加了一克。在实验期间对于所有动物实验室的食物和水都可得到。当幼鼠睁开眼睛并且可以自己进食（从13-14天开始），每只幼鼠每天大豆补充物的量增加到2-3g,尽管所有的老鼠都可以自由靠近大豆。所有老鼠都很好地食用了自己份内的大豆。有些小鼠的器官被取出来并且称重。利死亡率由单方方差分析进行，对于份额分布使用N-K测试。小鼠的重量和分布有曼-惠特尼测试进行检验，还有卡方检验，都由俄罗斯的Statsoft统计V6.0多语软件进行。Results 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 4 rats who became pregnant from 6 females on the positive control diet gave birth to 44 pups (an average of 11 pups/female), while the four females, from the six on GM soya flour supplemented groups gave birth to 45 (11.3 pups/female), and 3 from traditional soya group33 pups (11 pups/mother).结果到实验结束时为止，在实验包括的15只母鼠中，11只分娩，总共生132只小鼠。积极控制饮食组的6只母鼠中，4只怀孕，生44只小鼠（平均每只母鼠生11只小鼠），而补充转基因大豆粉的那组6只母鼠中，4只怀孕生45只小鼠（平均每只母鼠生11.3只小鼠），传统大豆组中3只母鼠怀孕，生33只小鼠（平均每只母鼠生11只小鼠） 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. High pup mortality was generally characteristic for females fed the GM soya flour (Table2). Among the pups from the females fed the positive control diet, 2 pups died during the first week, and 1 during the second week after delivery. All pups from females fed traditional soya flour died during the first week after birth. However, pups from females fed the GM soya flour supplemented diet kept dying during lactation period as it is evident from Table3.到哺乳第三周结束时，在母鼠饮食中补充转基因大豆导致45只出生的小鼠中25只死亡。而同时期补充传统大豆食物组33只小鼠中只有3只死亡。积极控制组中小鼠的死亡数也是3只，但是出生的小鼠总数更大，如表1所示。对于食用转基因大豆粉的母鼠，通常小鼠高死亡率是其特点（表2）。食用积极控制食物的母鼠生的小鼠中，出生后一周内有2只死亡，两周内有一只死亡。食用传统大豆粉的母鼠生的小鼠中，死亡的都是发生在出生后第一周内。但是，食用转基因大豆粉补充食物的母鼠生的小鼠，在整个哺乳期都有死亡，这在表3中可以证明。 In two weeks after their birth the weight of pups (with SE) from the GM soya supplemented group was less (23.95g 1.5g) than that of the pups of the positive control group (30.03g1.1g; p0.005), or from the traditional soya flour supplemented group (27.1g 0.9g; 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 g, in comparison with the 6% in the positive control group, and with the 6.7% found in the traditional soya supplemented diet group (Table 4). Study of pups 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 (Table 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. No mortality of females and survived young pups eating the GM soya flour supplemented diet was observed.来自转基因大豆补充食物组的小鼠（有标准误）出生两周后重量（23.95g+1.5g）不如积极控制组的小鼠（30.03+1.1g；p0.005）,也不如传统的大豆粉补充组（27.1g+0.9g;p0.1）。因为存活的小鼠数量非常不同，在表4中小鼠的重量分布被比较。从数据中可以明显看出，来自转基因大豆组的小鼠有36%重量不足20g,而在积极控制组中只有6%，在传统大豆补充食物组中有6.7%（表4）。对小鼠器官的质量研究表明，除了脑部质量外，相比其它组的相同器官，来自转基因组的小鼠器官都很小（表5）。这个事实表明来自转基因组的小鼠和其它组的小鼠年龄相同，但是随着内部器官的发展，变化就发生了。在接受传统大豆食物组中发现了轻微的负面影响，但这种影响不显著。食用转基因大豆粉补充食物的母鼠和存活幼鼠中没有观察到死亡现象。Discussion The reproductive behaviour of female rats fed on standard laboratory feed supplemented with soya flour prepared from either genetically modified soya or traditional soya 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 soybean contains a number of anti-nutrients, such as the lectins, trypsin inhibitors, etc. (Pusztai et al. 1998), and also female hormone-like substances, it was thought to be necessary to compare these data also with those from a positive control group when animals were not exposed to any soya flour supplementation.讨论母鼠的繁殖行为被研究，它们食用的是标准实验室食物，这些食物中补充了大豆粉，大豆粉来自转基因大豆或传统大豆。研究母鼠的行为是想了解食物对怀孕、哺乳和小鼠生长的影响。因为大家都认为天然状态的大豆包含很多反营养物质，比如植物血凝素，胰蛋白酶抑制剂等（Pusztaietal,1998）,还有雌性的类似荷尔蒙的物质，当动物没有接触任何大豆粉补充物时，人们认为有必要和积极控制组比较这些数据。 In order to understand the mechanism how this widely consumed GM crop exerts its influence on the reproductive performance of mammals and their offspring, it would be necessary to perform complex researches, including histological, genetic 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 the experiments of Brake and Evenson (2004), who started to feed pregnant mice, in our experiments the diets supplemented with GM or traditional soya flours were already given to the female rats 2 weeks before mating already, and we continued to treat them with their respective diet until the pups were weaned.为了理解这种被广泛食用的转基因作物是如何按照其机制对哺乳动物及其后代的繁殖行为施加影响的，有必要进行复杂的研究，包括毒理学的、基因的和胚胎组织学的研究。不过，我们得把实验限制在比较短的时间范围，并且在交配前两周开始喂养母鼠。但是，不像Brake和Evenson(2004)的实验，他们开始喂养怀孕的母鼠，而在我们的实验中，在母鼠交配前两周就已经给它们喂养补充了转基因的或传统的大豆粉，知道小鼠断奶我们一直继续给它们各自相应种类的食物。 Upon delivery, very unexpectedly a very high rate of pup mortality (55.6%) was observed in the the group of females whose diet was supplemented with the GM 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 occurred 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 the pups 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 were not affected by consuming the GM soya flour.在分娩后，出人意料的是，在食物中补充了转基因大豆粉的母鼠组中观察到了非常高的小鼠死亡率（55.6%），这相比积极控制组（6.8%）和传统大豆粉补充组（9%）都是很高的。并且在这组中小鼠在整个哺乳期还在继续死亡，这种现象只在转基因大豆喂养组出现。同时，这组中存活的小鼠体重也更轻。更令人惊讶的是，因为小鼠更小，大约是其它组的一半，对于每个小鼠可以得到的乳液应该更多。如果乳液的数量，还有/或者品质没有受到食用转基因大豆粉的影响，它们本应有更好的机会在最优条件下生长。 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 enter into the sexual/stem cells, or/and into cells of the fetus, as it was observed by Schubbert et al. (1998). Secondly, negative effect of GM soya could be mediated by the accumulation of Roundup residues in GM soya residues. 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 two first factors. (2,086 words)我们的数据让我们猜测并认定转基因大豆对新生小鼠的消极影响或许是被两种可能的因素引起。第一，可能是变化过程、外来基因的嵌入的结果，外来基因能进入性细胞/干细胞，或者/并且进入胚胎细胞，就像Schubbert等观察到的一样（1998）。第二，转基因大豆的消极影响可能是被转基因大豆残留物中的累积残留引起的。但是，在母鼠和存活幼鼠中没有观察到死亡现象，尽管幼鼠也开始食用转基因大豆，据推断这种效果可能是由第一条原因中的两个因素引起的。References Brake DG and Evenson DP (2004). A generational study of glyphosate-tolerant soybeans on mouse fetal, postnatal, pubertal and adult testicular development. Food Chemistry and Toxicology, 42: 29-36.Coghlan A (2002). GM crop DNA found in human gut bugs. New Scientist. 2002.Ermakova IV (2005). Conclusion to the report about feeding of rats by genetically modified potatoes Russet Burbank Agrarian Russia 2005. 62-64.Ewen SW, Pusztai A (1999). Effect of diets containing genetically modified potatoes expressing Galanthus nivalis lectin on rat small intestine. Lancet 354 (9187).Ho MW and Tappeser B (1997). Potential contributions of horizontal gene transfer to the transboundary movement of living modified organisms resulting from modern biotechnology. In Transboundary Movement of Living Modified Organisms Resulting from Modern Biotechnology: Issues and Opportunities for Policy-Makers (K. J. Mulongoy, Ed.) International Academy of the Environment, Switzerland:171-193.Kuznetcov W, Kulikov AM, Mitrohin IA and Cidendambaev VD (2004). Genetically modified organisms and biological safety. Ecos 2004: 3-64. Ultrastructural, morphometrical and immunocytochemical analysis of hepatocyte nuclei from mice fed on genetically modified soybean. Cell Struc