REVIEWS

UDC 604.6 doi: 10.15407/biotech9.01.007 PROBLEM OF GENETICALLY MODIFIED FOODS SAFETY: A TOXICOLOGIST’S VIEW

E. L. LEVITSKY

Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, Kiyv

E-mail: [email protected] Received 20.10.2015

This study aimed to analyze the published literature regarding the problem of safety of consuming food products containing genetically modified organisms. Genetically modified food products are given a brief definition, purpose and methods of their production are described, and the pro- and contra- arguments for their consumption are presented. The discussion is mostly focused on results of evaluating possible toxicity of such foods and their safety for macroorganism using traditional methods of toxicological analysis. Test results for long-term toxic effects, namely allergenicity, carcinogenicity, reproductive toxicity, and the possibility of mutagenic effects of these food products on the human body and the intestinal microflora are discussed separately. These data are based on the current understanding of the laws of the penetration and functioning of foreign genetic material outside the body, its entry and the possibility of integration into the during intake of foods manufactured by . The basic principles of the toxicological and hygienic regulation of these food products are also considered. An analysis of published experimental results allowed to draw a general conclusion about the absence of reliable scientific information indicating the presence of the toxic properties of genetically modified foods, and therefore of credible evidence of the dangers of consuming for humans and pets.

Key words: genetically modified foods, toxicity, safety.

In one article it is impossible to fully Over the past decades, as a illuminate the problem associated with the synthesis of molecular , microbiology, consumption of foods containing components , botany, zoology and emerging of genetically modified organisms (GMOs). technologies, including nano-, reached Unfortunately, its solution affects both the indisputable success. It is connected, first interests of manufacturers of traditional of all, with the achievements of genetic food, produced using pesticides and other engineering that allow getting new high- toxic chemicals and thus toxic to animals and yielding, pest-resistant crop varieties, breeds humans, as well as of the producers of modern of domestic animals, effective and popular genetically modified food (GMO foods) [1–9]. pharmaceuticals, as well as coming close to Therefore, only the most common questions the introduction of methods of treating the relating to the scientific evidence of possible most dangerous diseases through toxicity and safety of consuming foods that transplantation and therapy. Also on contain genetically modified ingredients are the agenda is the creation of artificial organs discussed here. to replace the damaged ones via integration The present century is rightly called the of special microchips developed with modern century of biology. Specifically, the hopes of computer technologies [13–15]. this science solving the urgent problems in the One of the most important biotechnological fields of industry, agriculture, pharmacy, and achievements was the creation of GMOs, medicine are pinned on the rapid development successfully implemented in agriculture, in of one of the most promising of its practical biomedicine, and to create high-performance sectors, namely biotechnology [10–12]. biofuels [16].

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There are objective reasons that cause The aim of this review was to analyze, in the rapid development of biotechnology. the terms of available scientific information, Thoughtless use of toxic substances in food whether concerns related to the consumption and agriculture is a growing concern of the of GMO foods are consistent with modern world community. Environmental pollution by ideas about the laws of functioning of foreign harmful products of industry and agricultural in vitro and their possible penetration chemistry results in an extremely adverse (integration) into of humans and pets effect on the health of all living things, global if their food contained components with genes warming, deterioration of soil, food, water of organisms obtained by genetic engineering quality. And these are only some of the effects techniques, with possible consequences of of environmental and agricultural crisis. The toxic effect and occurrence of mutations (up to development of traditional medicine is at a lethal) in the organism. standstill because of ignorance of the basic molecular mechanisms of diseases and the Determination of GMO foods and the absence of effective methods of treatment purpose of their production [13–16]. In modern world, the development of Against this background, the most plant genetics and industrial agriculture lead promising for solving the problems is the to completely new varieties of exceedingly use of the approaches and achievements of high-yield crops, amazing in size and notably biotechnology. adaptive to climatic conditions, and with fruits The use of GMOs — , bacteria, that bear long-term storage while maintaining yeasts, fungi, plants and animals — is the the form of smell and taste. reality of the modern biotechnological world, Extended genetic engineering experiments the world of the third millennium, nuclear substantiated the idea of replacing some energy, the Internet, microchips, hardware, parts of DNA strand in order to increase the space exploration and genetic engineering. productivity of various crops. The genetic Regardless of our opinion on GMOs, their material of animals served as the hereditary development and creation is one of the factors information introduced into the genotype of of human progress. And like any other product cultivated plants. Thus, scientists have been of scientific and technological development, able to raise unique species different from GMOs can be an unquestinable boon, but their parents in a number of features [1–12]. can also be seriously dangerous. The GMOs GMO food products are produced from are constantly and heatedly debated over, GMO plants (as a rule) or animals. If the food sometimes passing from the area of pseudo- is produced using GMO and it includes at least scientific discussion and information exchange one of the GMO-derived components, the food into the political and emotional fields, may also be considered genetically modified complicating the already difficult situation depending on the national legislation. GMOs even more. have some new properties due to the transfer Recently, the media heavily rumors about of separate genes theoretically from any the alleged unsafe use of human food and pet organism (in case of trans-genesis) or from the food containing GMO genes. The authors of genome of closely related species (cis-genesis) these publications suppose that the danger into a chosen genome [12]. An organism lies primarily in the possibility of “harmful” is referred to as genetically modified if it mutations due to incorporation of GMO genes possesses an intentionally altered genotype, in the DNA of either macroorganisms or and the changes are purposeful and carried microorganisms that form intestinal flora. out with the help of genetic engineering The problem under consideration is methods. Genetic engineering allows to work too extensive to be analyzed in one article. not only with the normal genetic material of an Therefore, the emphasis here will be placed organism, but also to introduce foreign genes solely on the analysis of the evaluation or a synthetic nucleotide sequence (so-called results of possible toxicity and safety “”), previously not typical of the of GMO food products for humans and recipient. animals. Determination and methods of The aim of such operations is to obtain GMO their production will be examined here very with predetermined and desirable properties briefly. The range of issues associated with (in the case of edible GMO plants, these the development and practical use of GMOs properties would be, for example, drought and is in more detail covered in other scientific pest resistance, higher yield compared with publications, for example see [17, 18]. conventional plants, etc.).

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Methods of production From the viewpoint of toxicology based It should be noted that the production of on classical analysis of toxicity and safety GMO foods (here the primary subject will be of various objects and substances, the study eating GMO plants that are most widely used of acute and subacute toxicity of GMO foods as a food source) has a long history. Classical does not make sense, because their toxic selection experiments also were based on the concentrations are very high and do not transfer of necessary genes, however, unlike differ from such ones for conventional foods the genetic engineering techniques, entire gene (although such studies have been done and will clusters were transferred in them. Selection be discussed). Regarding their possible real driven by genetic engineering approaches danger, only chronic toxicity and long-term allows purposefully obtaining products with toxic effects can be argued. preset properties by transferring one or more For an objective answer to these and genes of interest. similar questions related to the safety of the GMO plants are produced by trans- GMO products, it is necessary, first of all, to formation using one of the following introduce a common procedure for testing of methods: agrobacterial-mediated transfer, the presence (or absence) of harmful (toxic ballistic transformation, and other listed above) properties. As it was or viral transformation [12]. A lot of already noted, in toxicology acute, subacute, commercial transgenic plants are generated chronic, and specific toxicity (reproductive using agrobacterial transfer or ballistic toxicity, mutagenicity, allergenisity, etc.) transformation. Normally, the transfer is are typically determined. All substances carried out with a plasmid containing a gene or products that pose a potential danger to whose activity imparts the desired properties, human health (pesticides, pharmaceuticals, the regulating the activation of and so on) are subjected to such mandatory this gene and the transcription terminator testing procedure. Upon detection of such cassette which comprises a selective antibiotic properties the matter will be settled. In their resistance gene for kanamycin antibiotic or absence, given the possibility of insufficient herbicides. Creating new plant varieties and resolving power of the applied testing methods breeding animals with new technologies is we should, at least theoretically, consider much faster and less expensive than traditional possible mechanisms of potential toxic and breeding techniques. Furthermore, desired mutagenic properties of GMO foods, associated changes can be achieved in fewer generations. with the possible penetration and insertion of Increased resistance to pests, drought and soil their genes into the genome of a person or of salinity makes it possible to grow a lot of grain intestinal microflora, followed by induction of crops cultures in places where previously it deleterious mutations. This possibility is the could not be implemented [16, 17]. very foundation of fears of the general public Genetic modification can impart to the about the dangers of eating these foods. plant and its alimentary produce a veritable Theoretically, this situation may be number of essential features. Most cultivated provoked by either DNA, RNA fragments or GMOs are resistant to the pathogens (viruses foreign originated from the GMO food and fungi), insect pests or herbicides. This [20, 21]. Most of recently created transgenic greatly facilitates the cultivation, and also plants are different from the parent varieties reduces the costs of pesticide treatments. by the presence of a that determines a new character, and the gene that codes Evaluation of the toxicity of GMO food the synthesis of this protein (recombinant products for humans and animals DNA). Therefore the safety evaluation is Typically, to assess the danger of a focused on studying these carriers of genetic compound to the body, its toxicological modification. The presence of the recombinant profile is determined in animal studies DNA itself in foods and feeds does not pose a according to the following parameters: risk to human and animal health, as compared determination of the target (or targets) of with conventional products, since any DNA possible toxic effect and the critical effect (s) consists of nucleotide bases and a genetic value; dose-response; NOAEL (level at which modification leaves their chemical structure there are no side effects — the “threshold” unchanged and does not increase the overall concept); safety factor, an acceptable genetic material. level of consumption (ADI, mg per kg As for the possibility of penetration of body mass), the minimum level into the organism, the food DNA arrives of safety [19]. in the gastrointestinal tract and is almost

9 BIOTECHNOLOGIA ACTA, V. 9, No 1, 2016 completely decomposed into nucleotides whose of examined organs of the cows. However, chemical composition is the same for all living shorter amplified fragment of the gene of organisms. Hence, in this case the risk of chloroplast DNA were revealed in tissues of inserting foreign genes into the host genome examined organs of chickens. In the eggs, is minimum. the foreign DNA was not detected. Bt-gene- Aspects concerning the safety of GMO specific constructs derived from Bt-corn were foods have been comprehensively analyzed in not found in any of the examined bird organs. [22]. They include: the possibility of acquiring However, it was found that small fragments of foreign DNA from food products fragments of the foreign DNA still remain containing transgenic sequences; horizontal in the gastrointestinal tract after the gene transfer caused by these sequences; food is digested, and can be absorbed from integration of transgenic DNA fragments the intestinal mucosa of the host. More into the genomes of the host and microbial information on this subject can be found intestinal microflora. Approaches and in the monograph [22]. Also, in [24] there guidelines on applying methods of modern is a list of studies relating to digestion molecular genetics to assess the safety of these and incorporation of transgenic DNA and foods were generalized. Also, based on the proteins into mammalian cells. Kuiper noted results of published experimental researches, [22] that “in the process of transgenic DNA the following conclusions indicate a lack of digestion in the gastrointestinal tract out of toxicity and existence of the safety of the the corresponding food products, it quickly consumption of GMO foods. Firstly, it is stated becomes unavailable for transformation, but that small fragments of bacterial and plant theoretically such transformation of bacteria DNA (prior to 100 genes) can be detected after can not be excluded, especially if the presence ingestion of food in human gastrointestinal of homologous sequences is considered. tract (GIT). However, since they degrade very Although the presence of small fragments of fast, horizontal gene transfer from bacteria transgenic DNA in gastrointestinal tract cells and plants to man could not be detected of mammals have been demonstrated, there [22]. Moreover, these fragments were not is no evidence of its presence in the germ-line detected in germ-line cells. It was found that cells. The transfer of antibiotic resistance the transfer of marker genes for antibiotics genes from GMO plant food into the bacterial resistance from GMO plants into the genome cells of the human intestinal microflora and of human intestinal microflora and expression their subsequent expression are very rare of such genes are extremely rare events. The events, given the small amounts of undigested grounds for this are specific conditions in plant DNA as a result of the environment in the gastrointestinal tract that contribute the gastrointestinal tract that promotes its to rapid degradation of the fragments of digestion”. Further in the same article it is foreign DNA (acidic environment, presence stated that given the existing conditions in of DNases, temperature conditions, etc.). the gastrointestinal tract that contribute to In such conditions, degradation of plasmid the degradation of the foreign DNA, as well DNA of GMO food products by DNAase I was as the presence of a “competing” bacterial demonstrated. In addition, the foreign DNA is population, transformation and horizontal degraded by enzymes of intestinal microflora. gene transfer are very rare events. Acidic No toxic effect of the consumption of environment in the gastrointestinal tract and vegetable feed containing either normal or high temperature promote rapid degradation of recombinant corn possessing recombinant foreign DNA. Acidic environment catalyzes its plant DNA, Bacillus thuringiensis toxin- depurination. However, fragments of foreign maize (Bt-maize), for domestic animals (cattle DNA can be detected in the gastrointestinal tract and chickens) was found in [23]. Only the even at 1 hour after consumption of GMO food probability of PCR detection of chloroplast- products. In the chyme in the small intestine of specific gene fragments of different lengths rats and pigs, the DNA is rapidly degraded to (from 199 to 532 base pairs) and of Bt-maize- concentrations that can not be detected by PCR. specific fragment has been shown. It was However, despite the rapid degradation of DNA found that short fragments of DNA (less than in the small intestine, small transiently existing 200 base pairs) from plant chloroplasts can be DNA fragments were detected in the intestines detected in blood lymphocytes of cattle (bulls). of rats even in case of consumption of free DNA. In all other organs of these animals (muscle, Apparently, there are mechanisms by which liver, spleen, kidneys), plant DNA was absent, they can avoid nuclease degradation even in the moreover, it also has not been found in any absence of the membrane or cell wall.

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The authors of a review published in 2012 the human blood is rigidly separated from the [25] studied the possibility of transforming inner (GIT) and the external environment, in DNA in rats with the DNA of food and DNA accordance with standard paradigm large food of GIT microbiota. They pointed out the length macromolecules can not pass directly into the of such DNA sequences, insufficient for bloodstream. In the process of digestion, food both these species to presence of homologous proteins and DNA are degraded to smaller recombination (the result of which could be fragments, amino acids and nucleic acids the transfer of antibiotic resistance genes). respectively, which are than absorbed in a The DNA of the GIT bacteria was injected complex active process, and then the blood with plasmid DNA constructed with two circulation system distributes them in various resistance genes (nptI and aadA), homologous body compartments. Based on analysis of to DNA present in the digestive tract, with more than 1000 samples of human blood, the genes 16S rRNA and 23S rRNA. The the authors identified food-originated DNA resulting bacteria were fed to rats. Six fragments, large enough to contain entire rats with normal microflora were fed daily genes, which may avoid degradation and for four days with food containing this by unknown mechanism penetrate into the constructed DNA. Then the microbiota from human bloodstream. In one of the studied different parts of the GIT (stomach, small, blood samples, the concentration of plant GMO large intestine and cecum) was analyzed. DNA was even higher a person’s DNA. The Two rats were used as negative control. exact log-normal distribution of plant DNA in Screening for recombination of introduced the plasma was determined, while outside the DNA with antibiotic-resistant colonies on plasma (in cord blood) of the control samples, selective medium using PCR was performed. plant DNA was not detected. In [30] the No transformants were found among the 441 authors found transgenic DNA in milk of cows tested isolates. Based on these studies, the fed GMO foods. authors concluded that extensive digestion of Convincing experimental evidence, the DNA (100 μg of plasmid per day) did not testifying in favor of the safety of GMO food increase the proportion of kanamycin-resistent products, is given in [31]. In this study, the bacteria and transformants detected in aerobic fate of orally and intramuscularly injected microbiota in six rats. The findings coincide DNA fragments of M13 and with the results of similar studies and indicate the cloned gene of green fluorescent protein no detectable bacterial transformation in (GFP) in the organs of mice was analysed. mammalian GIT. Using RT-PCR method, absence of horizontal In [26] it is noted that the existing evidence transfer of foreign genes, as well as of the indicates the equivalence of GMO food and foreign DNA fragments in the intestine and normal food on indicators such as composition, muscle cells of experimental animals was nutritional value and digestibility: “In established. Their removal is likely to occur hundreds of scientific studies such equivalence through the mechanism of “liver-bile-gut”. has been established, and the presence of GMO In this case, “as indeed it was expected, the DNA and proteins in the tissues of domestic entire DNA was eliminated, and there was no animals (meat, milk and eggs) that consumed case of its insertion into the genome of mice, GMO food, was not detected”. either as a result of oral consumption or as a Previously (2000), such evidence has been result of injection. Hence, even if the foreign analyzed in the review of Beever and Kemp DNA pervades the blood in the form of large [27]. The authors came to the conclusion that fragments, germline transfer is not observed”. there is full equivalence of DNA behavior Recently the term “resistome” was of normal and transgenic food. The same introduced to indicate the resistance to emphasis is in [28]: “Based on available data, insertion of foreign DNA into the genome of we do not believe that there is gene transfer the macroorganism host [32]. The authors of DNA of GMO foods of plant origin into the emphasize that in recent decades the topic of tissues of animals that consume this food, antibiotic resistance of bacterial pathogens in and if this process occurs its frequency is not connection with the consumption of GMO food different from that of traditional foods”. has become particularly relevant. The human However, in some studies, the foreign intestine contains microbial population, DNA has been detected in the tissues. In 2013, the so-called intestinal microbiota, which presence of foreign DNA fragments (up to may theoretically serve as a target for the whole genes) in food was established even in horizontal transfer of genetic material, human blood [29]. The authors claim that as including antibiotic resistance genes. Recent

11 BIOTECHNOLOGIA ACTA, V. 9, No 1, 2016 advances in the development of appropriate This pattern was also confirmed in the research methods allowed to study the study of the possible toxicity of the proteins dynamics of the distribution and stability of of GMO foods. Toxicological evaluation of the genes of the microbiota (corresponding proteins introduced into the organism in term: “the gut resistome”). Based on analysis corn, soybean, rice, canola foods revealed of available data, the authors conclude that that changes in the amino acid composition of the genes responsible for antibiotic resistance GMO proteins do not cause the development are ubiquitous among human intestinal of toxic properties. Various effects faced by microbiota, and the majority of these genes these proteins when introduced into the body are masked by strictly anaerobic intestinal (mechanical, changing pH, temperature, commensals. The horizontal transfer of denaturation) likewise do not occasion such genetic material, including conjugation properties, known for other marker proteins and , is a fairly frequent event toxic to mammals [35, 36]. for intestinal microbiota. But in most There is also other evidence of the absence cases this is determined by nonpathogenic of toxicity introduced with GMO food proteins. intestinal commensals which dominate into For example, Lutz et al. [37] using the method the intestine of a healthy individual. The of immunoblotting showed degradation of transfer of these genes from the commensals Cry1Ab-protein of GM maize in the GIT of the to opportunistic pathogens is relatively rare, bull. but may contribute in a way to the emergence However, some authors still allow for of multidrug-resistant strains, as illustrated the possibility that toxic effects of GM food for the vancomycin-resistant determinants, proteins occur in the mammal macroorganism. common for aerobic intestinal commensals and According to the author of [38], there are nosocomial pathogen Enterococcus faecium. problems associated with the production of The research on of GMO food is in transgenic food and its possible negative a similar situation. In [33] the safety of GMO impact on the body. In the light of data foods was evaluated based on the analysis on molecular mechanisms of formation of of mediated non-coding RNAs (ncRNAs), the protein structure and of sustenance of involved in gene regulation. Aside from the interprotein complementarity there is a today widely used analysis of small interfering hypothesis about the complex nature of the RNA (siRNA), suggestions were made to functioning of the structure-supporting, include other RNA variants in this analysis depleting and eliminating systems. The of GMO plants: artificial miRNA (amiRNA), author considers it is possible that the use miRNA mimics and artificial transacting of GMO products leads to development of siRNAs (tasiRNAs). This approach was applied certain disorders of interprotein coordination in [33], and evaluation of the possible toxicity mechanisms likely with consequences for the of GMO plant foods due to the presence of organism. However, given the fact that GMO foreign RNA was conducted. It was based on foods are exposed to the abovementioned the analysis of low molecular weight RNA heavy impacts in the GIT (pH change, in conventional and GMO-containing food denaturation, thermal treatment, reducing products. In that study, the authors compared agents, mechanical stress, etc.) that alter the the genetic suppression mechanisms by structural profile of GM-proteins, resulting determining mediators of RNA interference in their denaturation and loss of functional (RNAi): extended double-stranded RNA activity, consumption of this food can be (dsRNA), small interfering RNA (siRNA) considered safe [36]. and micro RNA (miRNA) in conventional and Without going into detail on the highly GMO foods. It was found that the systematic publicized researches of Ermakova, Pusztai consumption of both types of products by and Seralini [39–41], in which the authors higher organisms is accompanied by intense allegedly discovered the presence of toxic, degradation of digested nucleic acids, and that and in carcinogenic and particular allergenic there are biological barriers for such dietary properties of the GMO foods, we only note ingredients. A small amount of short RNA can that when other researchers tried to revise be absorbed in the intestine consuming GMO these experiments, they failed to reproduce food products. However it was found that, the results due to the wrong setting and despite the possibility of activation of RNA- interpretation of original ones. In 2013 an mediated gene regulation, the GMO foods are article was published in “Nature” [42], in as safe for consumption as conventional plant which the author analyzes in detail the reasons food [34]. for withdrawal of Seralini’s publication.

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Publications of aforementioned authors have protein, albumin and globulin, and activities caused an outcry in the international scientific of aspartate and alanine aminotransferase, community including such prestigious alkaline phosphatase, gamma-glutamyl organizations as the EFSA and Germany’s transferase, amylase and creatine kinase and Federal Institute for Risk Assessment (Berlin), also urea volume, urine nitrogen, creatinine, which also did not support the conclusions of uric acid. The results revealed strong evidence their researches. of absence of significant differences between Recently, in connection with the ongoing the experimental and control groups of rats debate over the safety of GMO foods and on indicators such as the relative weight of inconsistent results obtained in some studies organs, blood creatinine, total protein and [43], there are more and more reports globulin. Only minimal histopathological demanding thorough evaluation studies of changes were found in the liver and kidneys. GMO safety and more open discussion on the In another recent paper regarding this scientific problem. For example, Devos et al. aspect [48], the background of the issue was [44] call for more open debates, more thorough illuminated with description of results of the data analysis, discussion of conflicting results relevant experiments, and with appropriate of some researches, as well as the relation conclusions. The authors note that there between the factors of “benefit-risk” using is 50-year history of safe use of microbial GMO plants in human and pets’ nutrition. pesticides based on Bacillus thuringiensis (Bt) As highlighted in that publication, the in agriculture. These pesticides include such inconsistency of data on the safety of GMO active insecticidal ingredients as Cry proteins. products is most often caused by political Their coding genes have been introduced motives. In particular, in developing countries into the corresponding GMO products using that are still strongly influenced by pesticides modern biotechnological approaches. Often, producing companies, and where frightening these genes are modified to prohibit expression propaganda regarding the consumption of in plant cells, and a few Cry proteins were GMO foods is widely used, people are not changed to increase biological activity. Also, ready to perceive them as an alternative to by combining the respective domains, these their familiar food. For example, the author proteins have been structurally converted of [43] from Turkey and most of the authors with increased insecticidal activity. This cited by him that testify in favor of the was done by extensive research involving alleged evidence of toxicity in GMO foods such subjects as invertebrates, mammals and also come from developing countries. He birds. Mammals were used for consumption considers foods produced through genetic and evaluation of the safety of the GMO food modification to be able to cause undesirable products. The results of these experiments mutations and determine the development allowed the authors to confirm their safety for of their toxic to man properties. In this case, man and studied animal species. he refers to another research of Turkish Thus the author of [43] quotes very early, authors [45], in non-scientific publication, outdated work from the 1960–1980s, with as evidence. He further claims that this toxic results testifying about the alleged toxic material penetrates into the soil and water, effect of GMO foods on the human body that causing ecological pollution. These toxins can have been refuted by later studies [49, 50]. possibly enter the food chain formed by other The authors of two monographs [51, 52] organisms. The author cites an old study of once again sum up and summarize the results 1998 [46] when the possibility of getting Bt- of studies of GMO foods safety, concluding toxin gene into the human body through the that in recent years the assessment involves soil in which these genes are supposedly stored the latest methods and high-precision for a long time was widely discussed. Based on technologies. The general conclusion to be the more recent works, for example [47], that drawn from these works is that there are no possibility was discarded later. In this paper, signs of the genetic modification, as well as of the authors studied the effects of transgenic unforeseen events, even when using traditional and normal food on three rat generations. Rat methods of crossing and selection of plants. In stomach, duodenum, liver and kidneys were addition, it is emphasized that “unexpected investigated histopathologically. The volume effect” will not necessarily be harmful for and average diameter of the renal tubule, as humans and pets. well as thickness of the adrenal cortex were A recent review [53] presented an counted. The biochemical parameters in the analysis of the safety studies of GMO foods. blood serum that were analysed included, total It was stated that at the time of writing,

13 BIOTECHNOLOGIA ACTA, V. 9, No 1, 2016 the production technology of genetically was no evidence of any biologically significant engineered plants has been applied for 30 differences in the studied parameters between years, and one of its main achievements was control and experimental animals”. Further the creation of GMO food products. The food’s information regarding the safety of GMO foods safety has over the years been the subject can be found in [57–61]. of intensive research, the results of which Thus, on the basis of the information are often ignored by the general public. The provided we can draw a general conclusion authors extensively reviewed the scientific about the absence of serious researches literature on this subject over the last 10 indicating the presence of toxic properties of years. They collected and processed surveys, the GMO food compared with conventional experimental articles, reports and modern food. opinions on this issue; given its importance (it is sufficient to note that at the time of Test results of long-term effects of GMO publication, GMO vegetable products have food products been widely used all over the world). The main Research of the researches on possible conclusion drawn by the authors is that the long-term toxic effects from GMO foods will results of researches carried out so far indicate be considered on the example of the usually the absence of any danger of the use of these evaluated allergenicity, mutagenicity and products. However, discussions on this issue reproductive toxicity. are continuing. Creating a scientific research base will help all professionals engaged Allergenicity in the industry, as well as a wide circle of A lot of people are allergic to certain non-scientific public to obtain reliable and foods (non-GMO). In particular, the soybean impartial information regarding the safe use allergen is particularly problematic because of GMO products. In the end, the authors note soy products are finding increasing use in food that 5% of the cited papers present negative production due to the high nutritional value of results. soy proteins. This means that people allergic A 90-days trial in rodents, described in to soy are finding it increasingly difficult to [54], aimed to identify possible toxic effects obtain non-allergenic foods. In addition, pigs of GMO foods based on corn, soybeans and and calves consuming soy food can also have cotton that differed from the usual plants by allergic reactions. Food allergens are almost increased content of some biologically active always natural proteins. One highly-allergenic substances. Foods containing no GMOs were soybean seed protein is Gly-m-Pd-30-K, which used as a control. A number of parameters is about 1% of total seed protein. This protein relating to possible sub-acute toxicity were causes more than 65% of allergy sufferers determined: the expression of GM-foreign to react. Using genetic engineering it is proteins, the presence of altered metabolites possible to lock the gene of this protein and with known toxicity, arising from the protein to develop soybean lines that do not contain degradation. It has been found that the the allergen [62]. margins of safety for GMO foods reach 100- Cotton yield per kilogram of fiber produces fold and do not differ from those for ordinary approximately 1.6 kg of seeds, which contain food. The same applies to the frequency of about 20% oil. After soybean, cotton is the possible side effects, which also did not differ second most rich oil source, with limited usage for GMO- and traditional foods. Based on the in food due to high amounts of gossypol and studies the authors report the absence of any other terpenoids. Gossypol is toxic to the heart, toxic effects in of the GMO food compared with liver, reproductive system. Theoretically, conventional food. 44 megatons of cottonseed each year could The authors of the review [55] conclude satisfy the need for oil to 500 mln people. that there is sufficient evidence of absence of There are conventional methods to produce acquired toxic properties of GMO food, and gossypol-less cotton, but in this case the plant that it is not expedient to resume coincident is left unprotected from insect pests. Genetic experiments on animals. In another survey engineering techniques enable purposeful [56] it is stated that: “The results of testing interruption of one of the first steps of GMO foods in rodents suggest the existence of the biochemical synthesis of gossypol in of extended safety margins (at least 100- seeds. Gossypol content in seeds is reduced fold) of the food consumption without having by 99%, while the remaining organs of plants observed adverse effects (of recalculated daily continue to produce it protecting the plant consumption of this food by humans). There from insects [63].

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Reducing allergencity and detoxification were observed. Reiner et al. [68] evaluated the of foods by genetic engineering methods are ability of GMO food induce allergic reactions in process of scientific development. Possible in mice after feeding them GM maize GM allergenicity of GMO food is also a concern of Bacillus thuringiensis (Bt)-maize (MON810). its opponents. Food allergies are an adverse No noticeable allergic reactions in mice in reaction to food that affects the immune the model of allergic asthma were induced. system, it affects about 8–10% of children and In a similar study, Andreassen et al. [69] 1–2% of adults. In theory, each protein may investigated the possible activity of plant- act as an allergen. The most common allergens originated Cry1Ab expressed in transgenic are milk, eggs, fish, soy, peanuts, nuts and corn MON810 as adjuvant against allergen wheat. As evidence of allergenicity of GMO ovalbumin via aerosol administration to foods opponents of GM plants usually refer to mice. No systemic adjuvant effect under the problems associated with the use of transgenic experimental conditions was detected. soybean and corn. Thus, on the basis of the available However, there is strong evidence of experimental material it can be argued that absence of allergenic properties of the GMO food products possess no more allergenic GMO food proteins. For example, based on activity compared to normal diet. significant experimental data it was concluded that GM proteins are no more allergenic than Reproductive toxicity similar conventional food proteins [64, 65]. Considering the above material evidencing In truth, the genetic modification alters the the lack of significant toxic potential of the protein structure of the plant, introducing main components of GMO food products (DNA, new proteins, modifies or alters their existing RNA and proteins), it is difficult to assume amount, and so plant’s allergenicity after toxic effects on the reproductive system and modification can also vary. That’s the reason the presence of mutagenic properties. why GM plants are carefully and mandatory Nevertheless, studies have been conducted tested for allergenicity. in the vein of evaluating reproductive toxicity Most scientists believe that the risk of of the consumption of food products containing inducing allergy is much more from the new GMOs. For example, in [54] the effect of GM rarely checked for allergenicity food, than maize in the pre- and postnatal development from comprehensively studied GM products. of the rat offspring was evaluated. Corn One or two new proteins are consumed with was included in the diet as much as possible GMO foods while a new product can carry without upsetting the balance of main hundreds of new proteins (the same applies nutrients. Analysis of the data did not reveal to using traditional selection methods). Par any impact of GMO maize on the development example, the broad sell of kiwis caused the and emergence of the rat offspring. development of allergies to this fruit (similarly In the 90-days trial on rodents that to soy). It was later found that fruits of this included histopathological evaluation and plant contain several allergenic proteins. If the measurement of the mass of reproductive kiwi first came on the market today, under the organs of adults, no reproductive or current rules it would be considered as a new developmental toxicity in the use of GMO product, tested for allergenicity, and perhaps foods was shown [53, 54]. Another already it never would be on sale [66]. mentioned research [47] evaluated the effects Here are results of several researches of GMO maize on some histopathological and that proved the absence of any allergenic biochemical indicators in three generations of properties of GMO foods. For example, in rats. Samples of rat stomach, duodenum, liver [67] mice were injected with purified Cry1Ab and kidneys were used in histopathological protein from GMMON810 maize, and its evaluation. The volume and average diameter effect on metabolism and immune status of of the renal tubule, as well as thickness of the mouse organism was evaluated. The results adrenal cortex were counted. The biochemical confirmed the presence of immunogenic parameters in and urine that were additionally potential of this protein in absence of allergic analyzed included urea volume, urine nitrogen, reactions. Immunological and metabolic creatinine, uric acid, total protein, albumin tests have revealed slight differences in the and globulin, and in the blood serum activities metabolic profile of the experimental rats of aspartate and alanine aminotransferases, compared with controls at introduction of the alkaline phosphatase, gamma-glutamyl protein, but no reliable unforeseen effects of transferase, amylase and creatine kinase. No genetic modification on the immune response statistically significant differences in the

15 BIOTECHNOLOGIA ACTA, V. 9, No 1, 2016 relative mass of the organs within the groups resistant to glufosinate ammonium on the were found. Insignificant changes were generative function of rats generations P0–P1 detected in levels of creatinine, total protein was experimental proved, as well as the lack of and globulin. influence of GM corn resistant to glufosinate Tyshko et al. [70] evaluated the effect of ammonium on the prenatal development of GM maize Liberty Link® on pre- and postnatal the offspring of rats generations P1–P2. development of the offspring of three Comparative analysis of indicators generations of Wistar rats. In the experiment, characterizing the prenatal development of the 630 adult animals and 2837 immature rats offspring revealed no significant difference were used. The animals were divided into 5 between the control and experimental groups. groups that received corn-enriched diets: GM Evidence of absence of influence of GM maize maize was given to the experimental group, on the postnatal development of the offspring the traditional analog of GM maize in such of rat generations P1–P2 was thus proved. investigations was fed to the control group, Comparison of indicators characterizing and 3 traditional varieties of corn, ROSS 144 the postnatal development of the offspring MW, ROSS 197 MW and Dokuchaevsk 250 showed no significant differences between MB were given to 1st, 2nd and 3rd reference the control and experimental groups: physical groups respectively. Corn was included in the development of the offspring and the dynamics diet as much as possible without upsetting of zoometric parameters correspond to the the balance of main nutrients. Analysis of the values of physiological characteristics of the data did not reveal any impact of GM maize animals of that species and age”. on the development of the rat offspring: the Thus, on the basis of published data, study of reproductive toxicity of GM maize the absence of GMO food toxic effects on Liberty Link® on three generations of rats the reproductive system and offspring, i.e. found no negative impact of GM maize on the absence of reproductive toxicity, can be the reproductive function in experimental considered proven. animals. Parallel studies of the reproductive toxicity of traditional maize varieties showed Mutagenicity the absence of specific varietal effects on Batista et al. [71] compared the effects reproductive function, pre- and postnatal on gene expression of rice obtained by a development of the offspring, and, at the conventional method of breeding (mutation same time, a fairly wide range of fluctuations breeding, in this case the gamma-irradiation) of the studied parameters, consistent with and transgenic rice. The authors used a the literature. The results of the research method of oligonucleotide microarrays for can be regarded as direct evidence of absence transcriptome modification assessment. As of any negative effect of GM maize on the a result, the researchers found that plants reproductive function in experimental animals obtained by conventional breeding, as and on the development of their offspring. compared with the control, caused a far more In her doctoral thesis, Utembayeva [8] significant changes in gene expression of notes: “... an algorithm was developed for non-specific genes (through by abiotic stress evaluation of the reproductive toxicity induction) than GM plants (ratio 10: 3). of GMO of plant origin, including a study Previously nucleic acids and proteins were of the generative function, prenatal and shown to have mutagenic activity [72–75]. postnatal development of the offspring of However, as already mentioned above, these three generations of rats; defined a set of substances upon consumption are broken down methods to assess the reproductive toxicity to small organic molecules by the digestive of GMOs, including a study of the generative enzymes. The ordered information stored in function by fertility, hormonal status and the product’s DNA is entirely destroyed, that level of gametogenesis in the gonads of males is, the food is eaten but it does not change our and females; prenatal development of the DNA. GMO food differs from conventional in offspring by the pre- and post-implantation that it has a few extra genes. At the same time, mortality by zoometric parameters of state if GMO product is consumed, these genes are of internal organs and skeletal system of the digested in the same way as conventional food. fetus; postnatal development of the offspring Similar findings were made in [76]: “The by the dynamics of zoometric indicators, likelihood of unintended mutations is much parameters of physical development, viability greater with using for sustenance plants from 0 to 5th and 6th to 25th days of life. The obtained by means of conventional breeding, lack of impact of genetically modified corn as compared with GMO foods. In addition, the

16 Reviews latter, in contrast to the traditional food, are in one or even several identical genes does subject to rigorous testing in rats and cattle not lead to catastrophic consequences for the before entering the distribution chain”. And cell. The unchanged copies are sufficient to further: “It is unlikely that consumption ensure cell’s normal functioning [82, 83]. Of of foods containing transgenic DNA, and considerable importance is also the functional approving such food products can have any nonequivalence of amino acid substitutions in significant harm to human health”. the polypeptide. If the new and the replaced Apart from the above arguments amino acids are of similar physical and supporting the safety of GMO food products, chemical properties, changes in the tertiary it should be noted that there are special structure and biological properties of the mechanisms in the organism that reduce the protein are insignificant. The occurrence adverse effect of harmful genetic mutations. of mutations and the impermanence of As a result of their appearance, the meaning the genome are an essential mechanism of of biological information changes. The variation and the driving force of evolution consequences of this are twofold. With [77, 82, 84, 85]. habitat conditions varying only slightly, new Hence, these mechanisms contribute to information usually reduces the survival rate. the preservation of selected genes during If there is a rapid change in living conditions, evolution, simultaneously accumulating in case of settling in a new ecological niche different alleles in the gene pool of a it is useful to have variable information. population, forming a reserve of genetic Thus, the intensity of the mutation process variation. The latter determines high in nature is maintained at a level not causing evolutionary plasticity of the population, a dramatic drop in viability of the species. i.e. the ability to survive in different An important role in limiting the adverse conditions. As already mentioned, there are effects of mutations belongs to anti-mutation no scientifically sound evidences of the GMO mechanisms arising in the course of evolution. food exhibiting more pronounced mutagenic First of all, these are specifics of the properties compared to conventional food. functioning of DNA polymerase alpha that Thus, the National Academy of Sciences of selects the required nucleotides during DNA the United States of America considers it replication, and ensures self-correction appropriate to carry out regular testing of during the formation of a new strand of DNA possible mutagenic activity of GMO food along with endonuclease. Various repair products instead of having on the market the mechanisms of DNA structure and the role foods derived through mutation breeding of the degeneracy of the genetic code, etc. [86]. This is supported by mentioned above are studied in detail [77–81]. Realization frequency of mutations that occur when using of this task can be the triplet genetic code, genetic engineering methods much rarer than which allows for a minimum number of with the methods of plant mutagenesis [71]. substitutions within the triplets, leading to distortion of information.For example, 64% The basic principles of toxicological and of substitutions in the third nucleotide of a hygienic regulation of GMO foods triplet do not change their meaning. However, All existing evaluating systems of GMO replacements of the second nucleotide distorts food products’ safety involve as the primary the meaning of the triplet in 100%. Another phase the analysis of information about the factor of protection against the adverse effects plant to be modified, about the donor organism of gene mutations is the paired of new genes, and on the nature of the genetic in diploid karyotypes of eukaryotic somatic modification [84]. cells. Pairing alleles prevents the phenotypic In the early 1990, the Organization for expression of mutations if they are recessive Economic Cooperation and Development in nature. Some contribution to the reduction (OECD) has developed the concept of of harmful consequences of gene mutations is substantial equivalency, currently shared by contributed by phenomenon of extra-replicated the majority of experts in the countries of genes encoding vital macromolecules, present the world community, including the World in the genotype in a few tens and sometimes Health Organization (WHO). This concept hundreds of identical copies of such genes. is based on a comparison of the GMO with its Examples include genes of rRNA, tRNA, traditional analog source, in respect of which histone proteins, without which vital there is a long history of safe use as a food or functions of cells are impossible. If there are food product, according to their appearance, extra-replicated copies, mutational changes key substances’ (protein and amino acid

17 BIOTECHNOLOGIA ACTA, V. 9, No 1, 2016 composition, fat and fatty acid composition, implements Directive 90/219/EEC and governs carbohydrates, vitamins, minerals) content, all of the GMO contained uses including the toxins that are standardized in food and production of nutritional supplements or forage, allergens and biologically active other purposes. All programs must thoroughly substances, typical for this type of product assess the risks with special emphasis on the [85, 86]. possible organism changes resulting from the In the absence of sufficient equivalence of consumption of GMO foods. GMO food product to its traditional analog, It should be noted that none other new further safety assessment comprises of the technology has been the object of as much following steps: the study of nutritional value attention of scientists around the world as of the product; quotas in the diets of humans the technology of production of GMO foods. and animals; methods of use in nutrition, This is due to the fact that the scientists and during breast-feeding; digestibility, have differing opinions about the safety of evaluation of intake of individual components genetically modified food sources [34, 89, (if the expected intake is more than 15% of the 90]. There is no scientific evidence against the daily requirement); impact on the intestinal use of transgenic products. At the same time, microflora (if GM product contains live some experts believe that there is a risk of microorganisms). Then, such characteristics release of unstable species of plants, transfer of GM product are analyzed: the toxicokinetics of the specified properties to weeds, the of the chemicals present only in the test GM impact on biodiversity of the planet, and, most product, and not in traditional products; importantly, the potential threat to biological DNA-damaging activity of GM product or and human health due to the transfer of the its individual components that distinguish it inserted gene in the intestinal microflora, or from the traditional product; allergenicity; the formation of the modified proteins due if the product contains live microorganisms, to exposure of normal enzymes, and so-called including genetically modified, potential minor components that can have a negative gastrointestinal colonization and impact [6, 91]. pathogenicity are evaluated. If test product Most of the presently developed transgenic exhibits DNA-damaging activity, long-term plants differ from parental varieties by studies for carcinogenicity are carried out [27, presence of protein that determines a new 87, 88]. character, and of gene that encodes the When new biotechnology products come to synthesis of this protein (recombinant DNA). the market, the consumer must be confident Therefore safety evaluation is focused on of their quality and safety. Therefore, there studying these carriers of genetic modification. must be toxicological approaches for the As noted above, the presence of recombinant development of new food products and their DNA itself in the food and forage does not pose components, to assess any potential risks of a risk to human and animal health, as compared biotech products. Safety assessment of new with conventional products, since any DNA foods and food ingredients must meet the consists of nucleotide bases and a genetic needs of producers, regulators and consumers. modification leaves unchanged their chemical It is essential that this approach is consistent structure and does not increase the overall with accepted scientific theories, the results content of the genetic material. An individual of the safety assessment could be reproduced human daily ingests (with food) DNA and and are acceptable to the health authorities, RNA in an amount of from 0.1 to 1.0 g and the result must satisfy and convince the depending on the type of food consumed and consumer. the extent of their processing. Furthermore, it Currently, the EU has a regulating was found that the percentage of recombinant (controlling) structure established to protect DNA into the genome of a genetically modified human health and the environment. Adopted crop is negligible. For example, in the lines by the Directive, which involves software of pest-resistant maize, the percentage of horizontal control, control unnecessary use recombinant DNA is 0.00022, in pesticide- and development of GMOs. Control over the resistant soybean lines it is 0.00018, in pest- use of GMOs is regulated by the regulation resistant potato varieties it’s 0.00075. Food “Genetically modified organisms (Contained processing significantly reduces the amount of Use)”, published by Health and Safety DNA in the product. Highly refined products Executive (HSE) in the UK. HSE receives the such as sugar produced from sugar beet or recommendations of the Advisory Committee soybean oil contain trace amounts or no DNA. on Genetic Modification. This regulation The experts fear possible transfer of antibiotic

18 Reviews resistance genes used in creating transgenic herbicides). In addition, biotechnological plants into the genome of the bacteria of the approach allows to manufacture products gastrointestinal tract. However, the bulk with predetermined useful properties. of food DNA would be destroyed in GIT and, However, this food, obtained with the help therefore, survival of the entire gene with of gene technologies poorly understood by appropriate regulatory sequences is unlikely. ordinary consumers (the history of this In addition, the transfer of recombinant DNA misunderstanding goes back to the days of into bacterial genome is virtually impossible, “Lysenkoism”), has at first caused a flurry as it requires a sequence of certain stages. of rejection and criticism. Imaginary threats These are: penetration of the DNA through the that supposedly may result from its use are cell wall and membrane of the microorganism, “horror stories” that intimidated and continue and withstanding the bacterial mechanism of to intimidate the commoners, unfamiliar with the destruction of foreign DNA; incorporation possible mechanisms of these unjustified fears. and stable integration in a specific area of The spread of these delusions is owed mainly the host’s DNA; expression of the gene in the to representatives of pesticide manufacturers microorganism. Despite the extremely low that bear huge financial losses as a result of the probability of introducing marker genes into ever-increasing introduction of GMO foods on the genome of microorganisms, methods of the market. Risking total bankruptcy, they removing these genes from the plant genome exaggerate and revive long-forgotten myths are currently intensively being developed. and legends, as a result of which our biological In particular, the resistant to glyphosate sciences altogether and the emergent from soybean line 40-3-2, and most others recently them biotechnology in particular fared so created transgenic plants contain no antibiotic poorly. resistance genes. The discussion and analysis Of course, one cannot categorically claim of the problem of safety of food DNA allowed that GMO food is potentially completely safe. the world scientific community to conclude Some authors consider the data and evidence that the DNA from genetically modified of safety of currently produced GMO foods to organisms is as safe as any other DNA in be insufficient [43, 49, 50, 55]. According to the food product. These findings can also be the author of the latest review, GMO foods are attributed to forage [49, 50, 92]. now widely available in the markets of most The GMO safety assessment system countries, and approved for use by the relevant focuses on the study of proteins bearing national legislative bodies. According to the new characters [18, 35, 36, 93]. Amino acid estimates given in the legislations, there is composition of such protein is compared no risk associated with the toxicity of these to known structures of protein toxins and foods. However, according to the author, who allergens in genetic databases [GenBank, used the information published in Medline EMBL, PIR and Swiss Prot], and based on the database, there are not enough reviews analysis, conclusions of degree of similarity relating to toxicological studies of GMO food are made. Further evaluation of the protein products. This applies to researches, conducted includes determination of acute toxicity in on GM plant food (tomatoes, potatoes, corn, laboratory animals, destruction speed in rice, peppers, peas and canola) regarding its gastric and intestinal juices on models in vitro potential toxicity to humans and animals. and in animals, decay during cooking and In addition, they were performed mostly in potential allergenicity. If it is shown that the research laboratories of biotech companies protein is slowly broken down during digestion that produce these products, the assessment of and its amino acid composition has a structure which could have been biased. Thus, the author similar to known protein toxins or allergens, is right to question: is there any scientific then chronic toxicity of the protein in question proofs of the toxicological safety of plant GMO is studied. In the absence of toxicity of the foods? protein, GMO products are deemed as safe as But no similar categorical statements conventional. exist about the safety of traditional foods All in all, we can say that today there is produced using pesticides. From the point of no evidence suggesting the presence of toxic view of toxicologist, GMO foods even are much properties of GMO foods. better studied than ordinary traditional food Thus, the establishment of vegetable (which among other things contains GMO foods was caused by objective reasons, pesticide residues [18, 60]), and are at least primarily higher yields due to pest resistance no more dangerous than products obtained and the lack of need for chemicals (pesticides, by conventional techniques. Indeed, short

19 BIOTECHNOLOGIA ACTA, V. 9, No 1, 2016 stretches of transgenic DNA and proteins will gradually replace its traditional toxic, can penetrate the cells of the gastrointestinal allergenic “pesticidal” analog. mucosa. However, no specific and well-proved Analysis of the published literature results of their future potential toxic effect indicates that the production process of GMO were published. A lot of testing of these foods foods does not cause any toxic properties other on animals did not identify their direct or than known for ordinary food. The safety of indirect toxic effect. Delayed toxic effects these products can be reliably tested using (mutagenicity, allergenicity, reproductive conventional methods of analytical chemistry, toxicity, etc.) have not been found either. toxicology and nutraceuticals. Substantial According to all these indicators, GMO food limitations may occur in the future, if the use was not different from the usual. This is of transgenic technologies will lead to more natural, given the existence of the currently significant and complex changes in food. known powerful extra- and intracellular For now, there are no methods for complete biochemical detoxification mechanisms. evaluation of whole foods (as compared with These include the degradation of the foreign a singular chemical component) regarding nucleotide and protein sequences, repair of the safety of GMO food products. Progress damaged DNA regions, and the existence can also be achieved with the development of multiple copies of duplicate genes, and of conclusive methods for identification and finally, the inconstancy of the genome characterization of protein allergens, and now [18, 72, 87], which allows to avoid long- it is the main focus of relevant studies. Another term mutagenic effects of relevant factors. important objective is the improvement of Moreover, potentially toxic DNA fragments methods of analysis of metabolites of plants and proteins completely degrade from and microorganisms, as well as proteins in case exposure to both the environment (mechanical of their gene expression. This might be useful stresses, temperature, denaturation, pH in detecting sudden changes in the GMO and in etc.) and enzymes (nucleases and proteases, establishing the level of sufficient equivalence acting against the “unknown” chemical and of GMO food products. biological targets) before their entry into Security level of modern GMO products is target cells. for consumers equivalent to that of traditional Biotechnology does not stand still. The foods. There is no scientifically proven development of new genetic engineering information on the adverse health effects techniques that allow for better targeted of these products. However, this conclusion control over the fate of inserted genes and cannot guarantee that all subsequent genetic their safe disappearance during consumption modifications will have the same positive of GMO food, gives reason to believe that in and predictable results. Further development the near future, this food will no longer be of toxicological methods and management the object of violent attacks of opponents of strategies is a prerequisite for maintaining the its introduction in the consumer market, and level of safety of GMO foods [85, 86].

REFERENCES 5. Rudishin S. D. Transgenic plants and problems 1. Biological Engineering. http://www.be.usu. of biosafety. Proceedings of scientific articles edu. 2011. ІІІ Ukrainian meeting of ecologists with 2. Blum Ya., Borlaug N., Sugik L., Sivolap Yu. international specialists. Vinnytsia. 2011, V. 1, Modern biotechnology. Calling time. Kyiv: PA P. 250–253. (In Ukrainian). http://eco.com.ua/. NOVA. 2002, 102 p. (In Russian). 6. The Safety of Genetically Modified Foods 3. Blum Ya., Novogilov O. The transgenic plant Produced through Biotechnology. Oxford J. organisms: economic impact and risks to Med. Health Toxicol. Sci. 2003, V. 71, Issue 1, biota. International Symposium “Problems P. 2–8. of biological safety of genetically modified 7. Modern food biotechnology, human health and organisms, new scientific approaches, development: an evidence-based study. Food regulation and public perception” (May 10–14 safety department. World health organization. 2006, Yalta). Visnyk Natsionalnoi Akademii 2005. nauk Ukrainy. 2006, N 9, P. 56–59. (In 8. Utembayeva N. T. Estimate of influence of Ukrainian). genetically modificated food products on 4. Glazko V. I. Genetically modificated organisms: rat reproduction system and their posterity. from bacteria to human. Kyiv: КVITS. 2002, Avtoreferat dis. kand. med. nauk. Moskva. 210 p. (In Russian). 2011, 86 p. (In Russian).

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9. Aksyuk I. N., Anisimova O. V., Kirpatovsky N. A., from genetically modified organisms to the Kravchenko L. V., Kirpichnikov M. P., Mazo V. K., microflora of the food chain and human gut. Onishchenko G. G., Rogov I. A., Semenov B. F., Conclusions. P. 138–141. Skryabin K. G., Sorokin E., Tutelian V. A., 23. Einspanier R., Klotz A., Kraft J., Aulrich K., Tyshko N. V., Chernysheva O. (eds. Tutelian V. A.). Poser R., Schwägele F., Jahreis G., Genetically modified food: evaluation of safety Flachowsky G. The fate of forage plant and control. Moskva: Izd-vo RAMN. 2007, DNA in farm animals: a collaborative case- 440 p. (In Russian). study investigating cattle and chicken fed 10. Sasson A. Biotechnology: Accomplishments recombinant plant material. Europ. Food Res. and Hopes. Moskva: Mir. 1987, 412 p. (In Technol. 2001, 212 (1), 129–134. Russian). 24. Federation of Animal Science Societies. 11. Glick B., Pasternak Ch. Molecular 2005. References pertaining to transgenic Biotechnology. Principles and Applications. DNA and protein and livestock products Moskva: Mir. 2002, 590 p. (In Russian). (meat, milk, eggs). http://www.fass.org/ 12. Biotechnology. Wikipedia. 2015.https:// references/Transgentic_DNA.htm. uk.wikipedia.org/wiki. (In Russian). 25. Rizzi A., Raddadi N., Sorlini C., Nordgrd L., 13. Ellingsen J. E., Lyngstadaas S. P. Bio-Implant Nielsen K. M., Daffonchio D. Review. The Interface: Improving Biomaterials and Tissue stability and degradation of dietary DNA Reactions. CRC Press. 2003, 464 p. in the gastrointestinal tract of mammals: 14. Bronzino J. D. The Biomedical Engineering implications for horizontal gene transfer and Handbook, Third Edition. CRC Press. the biosafety of GMOs. Crit. Rev. Food Sci. 2006, 3800 p. ISBN 978-0-8493-2124-5. Nutr. 2012, 52 (2), 142–161. http://crcpress.com/product/isbn/ 26. Van Eenennaam A. Does genetically 9780849321245. engineered DNA or protein get into milk, 15. Wood A. Physiology, Biophysics and Biomedical meat or eggs? Genet. Engineer. Animal Feed. Engineering. CRC Press Textbook. 2012, 782 p. 2014, V. 8, P. 41–48. 16. Ermichine A. P., Podpisskih V. E., Voron- 27. Beever, D. E., Kemp C. F. Safety issues kov V. E., Anoshenko B. Yu., Zarkov V. M. associated with the DNA in animal feed Biotechnology, Biosafety, . Minsk: derived from genetically modified crops. Tekhnalogiya. 2005, 430 p. (In Russian). A review of scientific and regulatory 17. Kozub N. A., Pilipenko L. A., Sozіnov І. O., procedures. Nutrition Abstracts Reviews, Blume Y. B., Sozіnov O. O. Genetically series B Livestock Feeds and Feeding. 2000, modified plants and plant protection 70 (3), 175–182. problems: achievements and assessment of 28. Mazza R., Soave M., Morlacchini M., Piva G., potential risks. Tsytolohiia i henetyka. 2012, Marocco A. Assessing the transfer of 46 (4), 73–78. (In Ukrainian). genetically modified DNA from feed to 18. Tutelyan V. A. (edit.). Genetically Modified animal tissues. Transg. Res. 2005, 14 (5), Food Sources. Academic Press. 2013, 333 p. 775–784. (In Russian). 29. Spisák S., Solymosi N., Ittzés P., Bodor A., 19. Kurlandskiy B. A., Filov V. A. (ed.) General Kondor D., Vattay G., Barták B. K., Sipos F., Toxicology. Moskva: Medicina. 2002, 608 p. Galamb O., Tulassay Z., Szállási Z., (In Russian). Rasmussen S., Sicheritz-Ponten T., Brunak S., 20. Global Status of Commercialized Biotech. Molnár B., Csabai I. Complete Genes May GM Crops. 2014, N 49. Pass from Food to Human Blood. PLoS One. 21. Ladics G. S., Bartholomaeus A., Bregitzer P., 2013, 8 (7), e69805. Doerrer N. G., Gray A., Holzhauser T., Jordan M., 30. Phipps R. H., Beever D. E., Humphries D. J. Keese P., Kok E., Macdonald P., Parrott W., Detection of transgenic DNA in milk from Privalle L., Raybould A., Rhee S. Y., Rice E., cows receiving herbicide tolerant (CP4EPSPS) Romeis J., Vaughn J., Wal J. M., Glenn K. soyabean meal. Livesock Sci. 2002, V. 74, Issue Genetic basis and detection of unintended 3, P. 269–273. doi: http://dx.doi.org/10.1016 effects in genetically modified crop plants. /S0301-6226(02)00038-6. Transg. Res. 2015, 24 (4), 587–603. doi: 31. Hohlweg U., Doerfler W. On the fate of plant 10.1007/s11248-015-9867-7. or other foreign genes upon the uptake in 22. Fergal O’Gara. Biosafety research directed at food or after intramuscular injection in more sustainable food production. A decade mice. Mol. Genet. Genomics. 2001, 265 (2), of EU-funded GMO research. European 225–233. Commission. 2001–2010, P. 44–47; 32. Van Schaik W. The human gut resistome. Kuiper H. A. Chapter 2. GMO and food safety. Philos. Trans. R Soc. Lond. B Biol. Sci. P. 128–133; Van der Vossen J. M. B. M. 2015, 370 (1670), 20140087. doi: 10.1098/ Safety evaluation of horizontal gene transfer rstb.2014.0087.

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33. Ramesh S. V. Non-coding RNAs in crop biotechnology. Transg. Res. 2014, genetic modification: considerations and 23 (6), 933–943. predictable environmental risk assessments 45. Ozdogan S., Ekmen Z. I. What is genetic (ERA). Mol. Biotechnol. 2013, 55 (1), 87– engineering? http://www.yunus. hacettepe. 100. doi: 10.1007/s12033-013-9648-6. edu.tr/aacorner/GEN/02/genetik. 34. Petrick J. S., Brower-Toland B., Jackson A. L., htm/2002. Access to Date: 20. 12. 2002. Kier L. D. Safety assessment of food and feed 46. Tapp H., Stotzky G. Persistence of from biotechnology-derived crops employing the insecticidal toxins from Bacillus RNA-mediated gene regulation to achieve thurigiensissusp, kurstaki in soil. Soil Biol. desired traits: a scientific review. Regul. Biochem. 1998, V. 30, P. 471–476. Toxicol. Pharmacol. 2013, 66 (2), 167–176. 47. Kiliç A., Akay M. T. A three generation doi: 10.1016/j.yrtph.2013.03.008. study with genetically modified Bt corn 35. Hammond B. G., Jez J. M. Impact of food in rats: Biochemical and histopathological processing on the safety assessment for investigation. Food. Chem. Toxicol. 2008, 46 (3), proteins introduced into biotechnology- 1164–1170. doi: 10.1016/j.fct.2007.11.016. derived soybean and corn crops. Food. Chem. 48. Koch M. S., Ward J. M., Levine S. L., Baum J. A., Toxicol. 2011, 49 (4), 711–721. doi: 10.1016/j. Vicini J. L., Hammond B. G. The food and fct.2010.12.009. Epub 2010 Dec 16. environmental safety of Bt-crops. Front. 36. Hammond B. G., Kough J., Herouet-Guiche- Plant. Sci. 2015, V. 6, P. 283. doi: 10.3389/ ney C., Jez J. M. Toxicological evaluation fpls.2015.00283. of proteins introduced into food crops. Crit. 49. Domingo J. L. Toxicity studies of Rev. Toxicol. 2013, Suppl. 2, P. 25–42. doi: genetically modified plants: a review of 10.3109/10408444.2013.842956. the published literature. Crit. Rev. Food 37. Lutz B., Wiedemann S., Einspanier R., Sci. Nutr. 2007, 47 (8), 721–733. doi: Mayer J., Albrecht C. Degradation of Protein 10.1080/10408390601177670. from Genetically Modified Maize in the 50. Domingo J. L., Giné B. J. A literature review on Bovine Gastrointestinal Tract. J. Agric. the safety assessment of genetically modified Food Chem. 2005, 53 (5), 1453–1456. doi: plants. Environ Int. 2011, 37 (4), 734–742. 10.1021/jf049222x. doi: 10.1016/j.envint.2011.01.003. 38. Veriovka S. V. Peculiarities of the structure 51. Bronzino J. D. The Biomedical Engineering of the transgenic protein and the resulting Handbook, Third Edition. CRC Press. 2006, risks. Bіotekhnologіya. 2008, N 2, P. 13–23. 3800 p. ISBN 978-0-8493-2124-5. http:// (In Russian). crcpress.com/ product/isbn/ 9780849321245. 39. Ermakova I. V. Effect of soybean EPSPS 52. Wood A. Physiology, Biophysics, and CP4 gene on the physiological condition Biomedical Engineering. CRC Press Textbook. and the reproductive function of rats in the 2012, 782 p. first two generations. Sovr. probl. nauki 53. Nicolia A., Manzo A., Veronesi F., Rosellini D. i obrazovaniya. 2009, N 5, P. 15–21. (In An overview of the last 10 years of genetically Russian). engineered crop safety research. Crit. 40. Ewen S. W., Pusztai A. Effect of diets Rev. Biotechnol. 2014, 34 (1), 77–88. doi: containing genetically modified potatoes 10.3109/07388551.2013.823595. expressing Galanthus nivalis lectin on rat 54. Zhu Y., He X., Luo Y., Zou S., Zhou X., small intestine. Lancet. 1999, 354 (9187), Huang K., Xu W. A 90-day feeding study 1353–1354. of glyphosate-tolerant maize with the G2- 41. Seralini G. E., Clair E., Mesnage R., Gress S., aroA gene in Sprague-Dawley rats. Food. Defarge N., Malatesta M. Hennequin D., Chem. Toxicol. 2013, 51 (2), 280–287. doi: de Vendomois J. S. Long term toxicity of a 10.1016/j.fct.2012. 9.008. Roundup herbicide and a Roundup-tolerant 55. Bartholomaeus A., Parrott W., Bondy G., genetically modified maize. Food. Chem. Walker K. The use of whole food animal Toxicol. 2012, 50 (11), 4221–4231. studies in the safety assessment of 42. Reddit B. C. Study linking GM maize to rat genetically modified crops: limitations tumours is retracted. Nature. 2013, V. 28, P. and recommendations. Crit. Rev. Toxicol. 14268. doi:10.1038/nature.2013. 2013, 43 (1), Suppl. 2, P. 1–24. doi: 43. Ozkok G. A. Genetically Modified Foods and 10.3109/10408444.2013.842955. the Probable Risks on Human Health. Int. 56. Alink G., Barlow S., Cockburn A., Flachowsky J. Nutr. Food Sci. 2015, 4 (3), 356–363. doi: G. Safety and nutritional assessment of GM 10.11648/j.ijnfs. 20150403.23. plants and derived food and feed: the role of 44. Devos Y., Sanvido O., Tait J., Raybould A. animal feeding trials. Food. Chem. Toxicol. Towards a more open debate about 2008, Suppl. 1, P. 2–70. doi: 10.1016/j. values in decision-making on agricultural fct.2008. 02.008.

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57. Kuiper H. A., Kleter G. A., H. P Noteborn. J. M., 70. Tyshko N. V., Zhminchenko V. M., Pashorina V. A., Kok E. J. Assessment of the food safety issues Selyaskin K. E., Saprykin V. P., Utembayeva related to genetically modified foods. Plant N. T., Tutelian V. A. Assessment of GM plant J. 2001, V. 27, Is. 6, P. 503–528. on development of the offspring of rats in 58. Tzotzos G. T., Head G. P., Hull R. Genetically three generations. Voprosy pitaniya. 2011, modified plants. Assessing safety and 80 (1), 14–28. (In Russian). managing risk. Acad. Press. 2009, 240 p. 71. Batista R., Saibo N., Lourenco T., Olivei- 59. U. S. General Accounting Office (2002). ra M. M. Microarray analyses reveal Genetically Modified Foods: Experts view that plant mutagenesis may induce more regimen of safety tests as adequate, but transcriptomic changes than FDA’s evaluation process could be enhanced. insertion. Proc. Natl. Acad. Sci. USA. Document GAO-02–566. 2008, 4 (105), 3640–3645. doi: 10.1073/ 60. Chemical and Biological Safety. 2004, 3–4 pnas.0707881105. (15–16), 3–7. 72. Gershenzon S. M. Mutagenic effects of DNA 61. Doerfler W. (2000). Foreign DNA in and the problem of directed mutations. Mammalian Systems. Wiley-VCH, Genetika. 1966, N 5, P. 3–13. (In Russian). Weinheim. 73. Gershenzon S. M. Mutagenic action of some 62. Herman E. M., Helm R. M., Jung R., biopolymers in Drosophila. Jap. J. Genet. Kinney A. J. Genetic Modification Removes 1969, V. 44, Suppl. 1, P. 114–119. an Immunodominant Allergen from Soybean. 74. Gershenzon S. M., Alexandrov Yu. N. Plant Physiol. 2003, V. 132, P. 36–43. Mutagenic effects of natural and synthetic 63. Sunilkumar G., Campbell L. M., Puckhaber L., nucleotides and the problem of directed Rathore K. S. Engineering cottonseed for mutations. Zh. obshchey biologii. 1982, 43 (6), use in `12human nutrition by tissue-specific 747–763. (In Russian). reduction of toxic gossypol. Proc. Natl. Acad. 75. Piven O. A., Lukash L. L. Effect of Sci. USA. 2006, 103 (48), 18054–18059. exogenous proteins in the mutation process. doi:10.1073/pnas.0605389103. Citologiya i genetika. 2011, 45 (1), 68–79. 64. Taylor S. L., Hefle S. L. Will genetically (In Russian). modified foods be allergenic? J. Allergy Clin. 76. Val Giddings L. A National Framework for Immunol. 2001, 107 (5), 765–771. the Review and Labeling of Biotechnology in 65. Helm R. M. Food biotechnology: is this good Food. U. S. House of Representatives Energy or bad? Implications to allergic diseases. & Commerce Subcommittee on Health. Ann. Allergy Asthma Immunol. 2003, 90 (6), 18 June 2015. Suppl 3, 90–98. 77. Kunakh V. A. Mobile genetic elements and 66. NRC (2000). Genetically Modified Pest- plasticity of the genome of plants. Kyiv: Protected Plants: Science and Regulation. Logos. 2013, 299 p. (In Ukrainian). Committee on Genetically Modified Pest- 78. Levitsky E. L. Mechanisms and age Protected Plants, National Research characteristics of the replication of nuclear Council. National Academy Press, DNA. Ukr. biokhim. zh. 1984, 56 (4), 460–472. Washington, DC. (In Russian). 67. Adel-Patient K., Guimaraes V. D., Paris A., 79. Levitsky E. L. DNA polymerase activity of Drumare M. F., Ah-Leung S., Lamourette P., adult and old rats livers. Ukr. biokhim. zh. Nevers M.C., Canlet C., Molina J., Bernard H., 1986, 58 (3), 72–74. (In Russian). Créminon C., Wal J. M. Immunological and 80. Ichas M. Genetic code. Moskva: Mir. 1971, metabolomic impacts of administration 351 p. (In Russian). of Cry1Ab protein and MON 810 maize in 81. Ratner V. A. The genetic code as the system. mouse. PLoS One. 2011, 6 (1), e16346. doi: Soros obrazovatelniy zh. 2000, 6 (3), 17–22. 10.1371/journal.pone.0016346. (In Russian). 68. Reiner D., Lee R. Y., Dekan G., Epstein M. M. 82. Hesin R. B. Variability of the genome. No adjuvant effect of Bacillus thuringiensis- Moskva: Nauka. 1984, 482 p. (In Russian). maize on allergic responses in mice. PLoS 83. GMOs vs. mutagenesis vs. conventional One. 2014, 9 (8), e103979. doi: 10.1371/ breeding: Which wins? December 3, journal.pone.0103979. eCollection 2014. 2013. FAO publication: Induced Plant 69. Andreassen M., Bøhn T., Wikmark O. G., van Mutations in the Genomics Era, Food and den Berg J., Løvik M., Traavik T., Nygaard U. Agriculture Organization of the United C. Cry1Ab protein from Bacillus thuringiensis Nations. http://www.geneticliteracyproject. and MON810 cry1Ab-transgenic maize exerts org/2013/12/03/gmos-vs-mutagenesis-vs- no adjuvant effect after airway exposure. conventional-breeding-which-wins/ Scand. J. Immunol. 2015, 81 (3), 192–200. 84. Knudsen I., Poulsen M. Comparative doi: 10.1111/sji.12269. safety testing of genetically modified foods

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in a 90-day rat feeding study design allowing 89. Nielsen K. M. Biosafety Data as Confidential the distinction between primary and Business Information. PLoS Biol. 2013, secondary effects of the new genetic event. 11 (3), e1001499. doi: 10.1371/journal. Regul. Toxicol. Pharmacol. 2007, 49 (1), pbio.1001499. PMCID: PMC3589341. 53–62. 90. Dona A., Arvanitoyannis I. S. Health Risks 85. Royal Society (2002). Genetically Modified of Genetically Modified Foods. Crit. Rev. Plants for Food Use and Human Health – An Food Sci. Nutrition. 2009, 49 (2), 164–175. Update. Policy Document 4/02. The Royal doi:10.1080/ 10408390701855993. Society, London. 91. Bonnefoi M. S., Belanger S. E., Devlin D. J., 86. FDA/CFSAN/OFAS. 2004 (update). Redbook Doerrer N. G. Human and environmental 2000, Toxicological principles for the safety health challenges for the next decade (2010– assessment of food ingredients. Food and 2020). Crit. Rev. Toxicol. 2010, 40 (10), Drug Admin/Center for Food Safety and 893–911. Applied Nutrition. Office of Food Additive 92. Doerfler W. Foreign DNA in Mammalian Safety, Washington, DC. Systems. Wiley-VCH, Weinheim. Chapter 87. Kuriakov I. A., Gayduchenko Yu. S., Ischak E. P. 11. Uptake of foreign DNA from the Genetically modified foods are harmful or environment: the gastrointestinal tract helpful? Sibirskiy torgovo-ekonomicheskiy and the placenta as portals of entry. 2000, zh. 2012, N 16. http://cyberleninka. P. 147–158. ru/journal/n/sibirskiy-torgovo- 93. Val Giddings L. A National Framework for ekonomicheskiy-zhurnal. (In Russian). the Review and Labeling of Biotechnology in 88. Herman R. A., Ekmay R. Do whole-food Food. U. S. House of Representatives Energy animal feeding studies have any value in & Commerce Subcommittee on Health. 2015, the safety assessment of GM crops? Regul. 1599 p. Toxicol. Pharmacol. 2014, 68 (1), 171–174. doi: 10.1016/j.yrtph.2013.07.003. Epub 2013 Jul 11.

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ПРОБЛЕМА БЕЗПЕКИ ПРОБЛЕМА БЕЗОПАСНОСТИ ГЕНЕТИЧНО МОДИФІКОВАНИХ ГЕНЕТИЧЕСКИ МОДИФИЦИРОВАННЫХ ПРОДУКТІВ ХАРЧУВАННЯ: ПРОДУКТОВ ПИТАНИЯ: ПОГЛЯД ТОКСИКОЛОГА ВЗГЛЯД ТОКСИКОЛОГА

Є. Л. Левицький Е. Л. Левицкий

Інститут біохімії ім. О. В. Палладіна Институт биохимии им. А. В. Палладина НАН України, Київ НАН Украины, Киев

E-mail: [email protected] E-mail: [email protected]

Метою роботи був аналіз даних літера- Целью работы был анализ данных лите- тури стосовно проблеми безпеки вживання ратуры, касающейся проблемы безопасности продуктів харчування, що містять генетич- употребления продуктов питания, содержащих но модифіковані організми. Подано стисле генетически модифицированные организмы. визначення генетично модифікованих про- Дано краткое определение генетически моди- фицированных продуктов питания, описаны дуктів харчування, описано мету і методи цель и методы получения, приведены мнения отримання, наведено думки «за» і «проти» «за» и «против» их употребления. Основное їх вживання. Основну увагу приділено об- внимание уделено обсуждению результатов говоренню результатів оцінки можливої оценки их возможной токсичности и безопас- токсичності і безпеки їх для макроорганізму ности для макроорганизма с использованием з використанням традиційних методів токси- традиционных методов токсикологического кологічного аналізу. Окремо обговорюються анализа. Отдельно обсуждаются результаты те- результати тестування віддалених ефектів стирования отдаленных эффектов этих пище- цих харчових продуктів, а саме: алерген- вых продуктов, а именно: аллергенности, кан- ності, канцерогенності, репродуктивної церогенности, репродуктивной токсичности, а токсичності, а також можливості мутаген- также возможности мутагенного влияния на ного впливу на організм людини і мікрофло- организм человека и микрофлору кишечника. ру кишечника. Ця інформація базується Эта информация базируется на современных на сучасних уявленнях про закономірності представлениях о закономерностях проник- проникнення і функціонування чужорідно- новения и функционирования чужеродного го генетичного матеріалу поза організмом і генетического материала вне организма и воз- можливості його потрапляння (вбудовуван- можности его попадания (встраивания) в геном ня) в геном у разі споживання продуктів при потреблении продуктов питания, получен- харчування, отриманих методами генної ін- ных методами генной инженерии. Приведены женерії. Наведено основні принципи токси- основные принципы токсиколого-гигиениче- ского регламентирования таких продуктов колого-гігієнічного регламентування таких питания. продуктів харчування. Анализ опубликованных эксперименталь- Аналіз опублікованих експерименталь- ных результатов позволил сделать общий вы- них результатів дав змогу зробити загальний вод об отсутствии научной информации, свиде- висновок про відсутність наукової інформа- тельствующей о наличии токсических свойств ції, яка свідчить про наявність токсичних у генетически модифицированных продуктов властивостей у генетично модифікованих питания, и, следовательно, достоверных дока- продуктів харчування, і, отже, вірогідних зательств опасности их употребления человеком доказів небезпеки їх вживання людиною и домашними животными. і домашніми тваринами.

Ключові слова: генетично модифіковані про- Ключевые слова: генетически модифициро- дукти харчування, токсичність, безпека. ванные продукты питания, токсичность, безо- пасность.

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