1 Principles and methods of evaluation
Principle of substantive equality
1993, oecd (Organization for Economic Cooperation and Development) put forward the principle of food safety evaluation-the principle of substantial equivalence [4]. That is, if a genetically modified food is similar to the existing traditional food of the same kind in characteristics, chemical composition, nutritional composition, toxins contained, feeding and feeding conditions of people and animals, then they are essentially equivalent. The determination of basic equivalence shows that this new food may be similar to non-transgenic varieties in health benefits [5].
Just because a new genetically modified food is not exactly the same as the existing similar food does not mean that it is unsafe. If the safety test is carried out, it must be based on the characteristics of this new food. According to its chemical composition and familiarity with it, it can be inferred whether animal experiments and in vitro research are needed. Not all genetically modified foods need animal experiments, and the evaluation must be based on the principle of individual cases. If it is necessary to carry out animal experiments with genetically modified foods or their components, the purpose of the experiments must be clear and the design must be rigorous.
[5].
1.2 evaluation method
1.2. 1 Monsanto's evaluation method
In the United States, there are many gene companies, Monsanto is the largest one, and its capital has penetrated into many companies that commercialize genetically modified foods. The company's experience in food safety evaluation is worth learning. Monsanto Company divides the evaluation content into three aspects according to the principle of substantive equality [6]:
Safety evaluation of (1) inserted gene expression protein;
(2) The accidental (pleiotropic) effect should be selectively and concretely analyzed. The important nutritional components of genetically modified food should be compared with the corresponding non-transgenic strains and their parents, and the analysis results should be compared with the existing data to ensure that the nutritional level is within the normal range. The anti-nutritional components should also be compared with the existing data and their controls to confirm that the endogenous toxins have not changed significantly. It is also necessary to analyze various components of food processing products to ensure that the measured parameters are within the normal range.
(3) Selective application of health test. Usually, in order to simulate commercial feeding practice, these feeding experiments are carried out with domestic animals and poultry. As for the food eaten by human beings, mice were fed with new food 25 times the maximum estimated intake of human beings, and experiments were carried out with mice of each sex for more than 4 weeks. In the whole food feeding experiment, animals are not sensitive to small changes in food. The parameters of health display test include daily health observation, weekly weight, food consumption and so on. Four weeks later, a full autopsy will be conducted. If any abnormality is found in the autopsy, these tissues will be examined through a microscope. This 28-day acute toxicology study is usually used to evaluate whether the food to be tested has any adverse effects during feeding. At autopsy, changes in organ weight, hematology, clinical chemistry and histopathology should be observed [7].
The application of 1.2.2 database
The database can provide basic information of food ingredients to evaluate whether the main nutrients and toxins in genetically modified foods have changed significantly. Of course, we should also consider that these main nutrients and toxic components have a certain range of changes, and we must ensure the quality of data and formulate effective methods to quantify these main components [8].
1.2.3 animal model in vitro and in vivo.
Animal models in vivo or in vitro can be used to evaluate the safety of genetically modified microbial food. The Institute of Toxicology of the Danish Ministry of Food has established several digestive tract models of mammals [9]. Among them, the rat model is the most complete, probably because the rat model is used more in the study. Sterile rat model can not only study the survival and migration of bacteria, but also study the transfer of genetic material between microorganisms. If the donor strain and the recipient strain carrying the plasmid pamβ 1 are fed to infertile rats, the products of dna conjugation and transfer will be found in the excreta of rats in a few days.
1.2.4 evaluation method for allergenicity of genetically modified foods [10]
If the genetically modified food contains protein from a known source, we should do some immune reactions with sds-page, and then do the rast inhibition test. If the result is positive, we can conclude that the genetically modified food is allergic to human beings, and further tests are not needed. If the result is negative, skin prick test and double-blind placebo-controlled food provocation test (DBP CFC, placing bo control food provocation test) were carried out. If the result is still negative, it can be concluded that this genetically modified food has no risk to consumers. If the protein contained in genetically modified foods comes from non-food sources or unusual foods, it cannot be detected by allergic individuals or their sera. In this case, the characteristics of this protein are compared with known food allergens.
1.2.5 pcr detection method
At present, the 35s promoter of cauliflower mosaic virus (camv) is often used to construct transgenic plants. Pcr detection of 35s promoter is the basis of new food management in EU. Recently, the European Union revised its food management strategy, and foods with gmo (genetically decorative organisms) content greater than 5% must be labeled, so qualitative pcr turned to quantitative pcr, and the method of quantitative competitive PCR (QC-PCR) was particularly useful [1 1].
2 Results and analysis
2. 1 potato
Ewen and pusztai officially published their research results in 1999 [12] after it was publicly announced on TV that transgenic potatoes with the gene of snowdrop lectin (gna) were harmful to the internal organs of rats. They studied the effects of transgenic insect-resistant potato with gna gene on different parts of gastrointestinal tract in rats. The experimental rats were fed with three kinds of feed containing transgenic potato, non-transgenic potato parents and non-transgenic potato plus gna. The results showed that the length of gastric mucosa, intestinal villi and intestinal recess changed in different degrees. After comparative discussion, they concluded that the thickening of gastric mucosa is mainly the result of gna gene expression, while the changes of small intestine and cecum are mainly caused by genetic manipulation or/and transgenic components, and the influence of gna gene expression is only a small part. Fenton et al [13] found that gna can bind to human leukocytes. Although this does not explain anything, it increases the risk of affecting human health. Experiments have proved that bt toxic protein is safe for humans and animals, and a transgenic potato is essentially equivalent to its corresponding non-transgenic variety [14]. For transgenic herbicide-resistant potato, it is very close to the corresponding non-transgenic parent strain [15].
2.2 tomatoes
The results showed that the main nutritional components (such as va and vc) of delayed ripening tomatoes produced by calgene Company did not change, and the natural toxins such as lycopene did not change significantly, and no harmful pleiotropic effect was found. No adverse reaction was found in the 7-day feeding experiment of rats [16]. In a food safety study of transgenic insect-resistant tomatoes [17 ~ 18], male rats, mice and rabbits in brown Norway were fed with bt protein equivalent to 2000kgbt tomatoes per day, and no systemic adverse reactions occurred. In the food safety evaluation of a transgenic tomato resistant to tobacco mosaic virus, compared with non-transgenic tomato, sugar, alkali, organic acid and polybasic acid have no obvious changes and no difference in mutagenicity.
2.3 soybeans
Nordlee et al. [3] evaluated the food safety of transgenic soybean with 2s albumin gene of Brazil nut. In the study, they extracted the sera of nine people who had a history of allergy to Brazil nuts. Rast research shows that the transgenic soybean extract can effectively compete with the protein extract from raw Brazil nuts to bind igec (immunoglobulin) in the serum of allergic people, while the inhibition phenomenon is observed in the protein extract of genetically corresponding non-transgenic soybean. Sds-page showed that a new protein band (about 9kd) appeared in transgenic soybeans, and Brazil nuts also had this band, which had the same mobility as partially purified 2s albumin, but there was no such band in non-transgenic soybeans. Immunoblot analysis showed that 8 out of 9 sera could recognize partially purified 2s albumin and react with 9kd protein in Brazil nut extract, and 7 out of 9 sera could recognize 9kd protein with the same mobility as 2s albumin in Brazil nut. In non-transgenic soybeans, no serum reacted with low molecular weight protein, and the serum from the control did not react with any soybean or Brazil nut protein. Therefore, it can be confirmed that Brazil nuts exist in this transgenic soybean.
According to the research results of fuchs et al [19,20], Monsanto's herbicide-resistant transgenic soybeans are essentially equivalent to the corresponding non-transgenic soybeans on the market.
2.4 cotton
Bt toxin protein expressed by Monsanto's Bt cotton can be quickly digested by mammals. Compared with non-transgenic cotton, the quality and anti-nutritional components of cottonseed and cottonseed oil have not changed significantly. Rats, quails and catfish were fed with 5% ~ 10%, 10% and 20% cottonseed respectively for 28 days, 8 days and 65438+.
Chen Song et al [22] used cottonseed powder to feed rats for 28 days and quails for 8 days. The results showed that there was no significant difference in the weight and food utilization rate of transgenic animals compared with the control group, and the tested animals grew normally and did not die. The liver, kidney, stomach, cecum, colon, small intestine and testis of rats were examined by tissue section, and no pathological changes were found. The weight ratio of liver, kidney and testis, the activity of alanine aminotransferase and the level of blood urea nitrogen in rats are all within the normal range, and the transgenic cotton has no obvious change compared with the control group, so this bt cotton is basically equivalent to conventional cotton.
2.5 Others
There is no significant difference between virus-resistant transgenic rice [23], virus-resistant transgenic zucchini strain zw20[24], virus-resistant transgenic tobacco [25] and transgenic asparagus [26] and their corresponding non-transgenic parents [26].
3 Discussion and conclusion
3. 1 Concept limitation of substantive equality
The concept of basic equivalence embodies the idea that organisms currently used as food or food sources can be used as the basis for comparison to evaluate the safety of food or food ingredients from biotechnology. However, the concept of substantial equivalence also has its limitations. For example, although a new food is 99% identical to a known food, that is, they are essentially equivalent except for a new toxin, this food may need further detailed inspection; Even if a new food is only 70% the same as its corresponding existing food, especially when different nutrients can be replaced by complex diet, this new food may only need a small degree of inspection and testing [27]. Therefore, the concept of substantial equivalence can not completely predict whether new foods need to be tested for substantial toxicity to animals.
Moreover, scientists can't predict the biochemical or toxicological effects of genetically modified foods convincingly by using the known chemical components of genetically modified foods. Genetically modified food is similar to natural food in chemical composition, which does not mean that it is safe for human consumption [28]. Some scientists believe that transgenic herbicide-resistant soybeans are essentially equivalent to their corresponding non-transgenic soybeans. Millstone et al. [28] believe that this conclusion is based on the assumption that there is no significant toxicological difference between known genetic and biochemical differences. In fact, even without herbicide, the chemical composition of this transgenic herbicide-resistant soybean is different from all its corresponding varieties. When this herbicide-resistant soybean is actually used in agricultural production, it is bound to be treated with herbicides frequently, and the chemical composition of the soybean treated with herbicides will inevitably change significantly, which is enough to prove. However, in the experiment that draws the conclusion of substantial equality, the transgenic soybeans used are not often treated with herbicides, but only some chemical components are compared, so it has no practical significance.
3.2 Limitations of Whole Food Feeding Research
As mentioned above, many safety evaluation work of genetically modified food is to use the whole genetically modified food to carry out acute experimental animal toxicology experiments. However, the experiment of feeding animals with whole food has certain limitations, and it is difficult to detect small changes in food. Food and nutritionists oppose the use of whole food feeding research methods in the safety evaluation of genetically modified foods. The International Food Biotechnology Committee (ifbc) pointed out that this method is generally not recommended to evaluate the safety of genetically modified foods. If it is really necessary, the experimental design of whole food feeding research should be very cautious and cautious, and the duration should not be too long to avoid adverse effects on animals due to factors such as unbalanced nutrition, thus concealing the safety of genetically modified foods [7].
In fact, the above experience and conclusions come from the experimental results of safety evaluation of irradiated food. At that time, I didn't know that overfeeding certain food to experimental animals would lead to unbalanced nutrition and adversely affect the health of animals. Hammond et al. [6] summarized some such examples, which may have high reference value for the evaluation of genetically modified foods.
3.3 Debate on the Safety of gna Transgenic Potato
Pusztai's experimental design and results were immediately criticized by many parties after he and his colleagues published the research results that gna transgenic potatoes would damage the digestive system and immune system of rats [1]. As mentioned earlier. After the corresponding research paper was published in the internationally famous magazine The Lancet, it also caused a lot of controversy. The editorial department of The Lancet thinks that although pusztai and his colleagues have some shortcomings in this research, this paper provides a research result, which is the beginning of a debate. It is necessary to let the public know the facts in order to enhance the public's trust in science. So The Lancet published this paper in 1999 [29]. Kuiper et al [30] think that there are several problems in this paper: ① The composition of different diets fed to rats is not introduced in this paper. Although pusztai et al. published some details on the Internet, the starch, polyglucose, lectin, trypsin and chymotrypsin in transgenic potatoes are different from other parental strains, but it is unknown whether this difference is caused by transgene or different strains. ② The diet of rats only contains 60% protein, which is easy to cause hunger reaction, thus leading to other adverse effects; ③ The experimental design is not rigorous, the number of rats in each feeding group is too small, and there is a lack of diet control, such as the standard rodent diet containing 15% protein and "blank".