An integrated approach to the evaluation of GMPs as industrial products

Marcello Buiatti, Department of animal Biology and Genetics

The University of Florence, Italy

Premise

The debate on genetically engineered organisms ( the terms more frequently used such as “genetically modified” or “biotechnological” are misleading) has unfortunately been centred only on the effects on human and animal health of Genetically Modified Plants and particularly only on the four engineered crops now in the market ( Maize, Soybeans, Canola and Cotton). This approach has opened a never ending discussion between two opposite factions, discussing often on the adoption or not of the GMPs in general, by unfortunately avoiding a specific integrated product by product evaluation as it should happen in any evaluation procedure.

Now, GMPs can be and should be considered as industrial products as they are being developed and released by multifunctional industries of agriculture related products which include seeds of cultivated varieties of a large number of crops, but also fertilizers, pesticides etc. For this reason, evaluation procedures should be case by case and include several aspects such as the economic, and social ones as well as those related to bio-safety and human and animal health issues.

In our opinion this approach is the only one which could be leading to a discussion on scientifically proven facts thus avoiding the useless waving of opposite flags. We shall therefore in this short review discuss the different mentioned items with reference not only to the present day products but to the technology itself.

a) The technology: failure or success?

Strangely enough opinion polls almost always show that the vast majority of people think that a vast number of edible crops has been successfully modified and released into the market. In the development of such a wide-spread feeling a major role has been played by both the opponents and the “lovers” of GMPs, the first probably reflecting the feeling of being under siege by the scientists envisaged as all powerful-magicians, the second for badly conducted propaganda of Genetic engineering. The reality of the facts gives us a completely different picture. Genetically engineered plants now on the market have been developed in the late eighties of last century and are modified only for two characters, namely resistance to herbicides and to insects. Only four crops have been transformed with the aforementioned genes, only one of which ( maize) being an important carbohydrate source, particularly in South America. Thousands of other transgenic plants have been produced in private and public laboratories and a number of them have reached the last phase of field trials. Some of them have also been introduced into the market but have been withdrawn from it. The first example of this kind has been the famous Flavr Savr tomato, transgenic inhibited in the galacturonase activity and therefore resistant to rotting by saprophytes during conservation. This tomato was withdrawn for its bad texture and taste and its fate was followed by a high oleic acid content soybean, and, recently also by the “Golden rice”, a provitamin A producer developed by Syngenta, advertised as the solution for the lack of vitamins in developing Countries. This particular product had to be withdrawn because of a too low expression level of the inserted transgene. The very low amount of successful products in more than twenty years of intense and expensive research has led the most important companies such as Monsanto, Syngenta and Dupont to slowly divert their interest from edible crops to the use of plants for the production of compounds of industrial and pharmaceutical interest. A couple of pharmaceutical products are already in the market and a new interest in transgenic crops is growing at the moment in relation to the area of possible materials for bio-fuels although in this case the crops proposed are mainly soybean and maize, also in this case transgenic for resistance to herbicides and insects. In other words, with the exception of a few cases in the pharmaceutical area , where single extracted and purified compounds are used instead of the whole plant, in the last twenty years plant biotechnology research has not been able to release in the market more than two types of successful products. One may ask now what may be the reason for this result, very poor if confronted with the expectations of the early years of this research area. The answer comes from the new conception of the organisation of living systems emerging from the experimental data obtained in the third millennium with the new powerful tools of Molecular Biology. It should be recalled here that the idea behind the use of recombinant DNA techniques, (not by chance the new technology was called genetic engineering) has been that the DNA of an organism is its only “program” possible and acts in a perfectly deterministic way during the whole life cycle. A consequence of this conception is that a gene coming from one species, could be integrated into the genome of another not inter-fertile with the first, without unpredictable effects, just as it happens with the modification of an algorithm in a computing program. As discussed more thoroughly in an earlier paper ( M.Buiatti, 2004), this mechanistic view has been challenged by the demonstration of the functional relevance of multi-level non additive interactions in living systems and of the partially unpredictable character of the different steps involved in the creation of a transgenic plant. In fact all researchers in the field know very well that present day techniques do not allow the exact prediction of the location of trans-gene insertions, the number of copies to be integrated, the final structure of the insert, the interaction of gene products with plant metabolism, of the PGM with the ecosystem, not to mention the social and economic contexts, that is the final “judges” of the product released in the market. The literature of the last few years gives us a vast number of reports confirming the presence in the first generations of genetically modified plants of re-arrangements of the inserted constructs, mutational events deriving from trans-gene integration, modifications in gene expression levels and inactivation through methylation, changes in the metabolome etc. ( See for recent reviews on the subject, J.Latham et al., 2006, H.Rischer and K.M.Oksman-Caldentey, 2006; M.Filipecki and S.Malepszy,2006 ). From the point of view of the final result of the transformation events and of their commercial exploitation the most interesting data come from the analysis of physiological unpredicted effects and of the metabolome of transgenic plants. The data on transgenic experiments which never led to products released in the market, fully confirm the scientific explanation of the low number of successful PGM. In many cases where the integrated gene interferes with the pre-existent metabolism unpredicted effects are present generally affecting specific areas of the metabolic network. Just to give an example, in our laboratory we obtained Nicotiana spp. plants transgenic for the rat gluco-corticoid receptor gene which was supposed not to interfere with the plant hormonal system very different from the animal one and , on the contrary, we observed gross changes in plant physiology and even morphology leading to full sterility in the third generation. It should be stressed here that not by chance , the two PGM types successful in the market did not show any negative effect at this level, bacterial genes not interfering in any ways with plant metabolism. However we should also recall that the sequence of Bt genes had to be artificially changed in the early years of product development because of the discrepancy between the bacterial and plant t-RNA populations and codon usage. The data just discussed all suggest that the present day low rate of success of plant genetic engineering is due to a lack of basic knowledge of the structures and dynamics of plant genetic and metabolic networks and, consequently to the lack of efficient tools for the control of transgene insertion, its possible modification by the plant, interactions with the receiving genome and the the metabolome. The answer to the question posed at the beginning of this paragraph would then be that genetic engineering is by no means fully unsuccessful but it may well become so if research is limited to the development of plants other than maize, canola, soybean, cotton, transformed with the same two groups of genes which have been used until now, without giving priority to basic research in the new fields of genome and metabolome dynamics leading to a much better control of the effects of transformation.

b) GMP Biosafety

Rather unfortunately, the term “unintended effects” is taken by both the anti and pro GMP groups as a synonym of “undesirable” or eve “dangerous”. It should not be so because only a part of the unpredicted events which may be following plant transformation are liable to really affect bio-safety. However the data just reported certainly do mean that biotechnologists are confronted with a fairly high level of intrinsic unpredictability of genetic transformation. Therefore, according to the precautionary principle great care should be taken when dealing with GMPs and all facets of possible risks should be analysed. That such an approach is unavoidable is confirmed by the fact that even several products already on the market, when re-analysed with updated techniques often reveal unknown unpredicted changes. Just to take some examples, Windels et al., 2001 found a series of previously unknown modifications in Roundup Ready soybean insert and flanking regions. In maize Hernandez et al, (2005) showed a deletion of the termination sequence in ma MON 810, and Rang et al., (2005) observed a similar change in Round up Ready soybean leading to the transcription of “fusion RNAs deriving from the deletion of a termination site. It should be noted that only in a few cases long term analyses on the possible deleterious effects on animal or human health at the histological and biochemical level have been carried out and no macroscopic damages have been shown to occur. However, recently evidence to be taken into consideration has been obtained by the group of Malatesta for histological modifications induced in rats ( see for instance Vecchio et al, 2004) and by the laboratory of Seralini for negative effects on human cell viability ( ……..) of feeds containing genetically modified soybean. These data, for the precautionary principle suggest that more attention should be given in the future to the possible presence of risks. Moreover the recent data reported and many others proposing the need of further research have been obtained generally by independent researchers through the use of new tools for molecular, biochemical, physiological analyses and therefore suggest that present day bio-safety rules should be updated along with the methods and techniques of analysis of transgenic materials. Moreover, safety testing should be carried out by public, independent research groups committed by the public authority and not by privates coherently with the rules in other areas of bio-safety related risk assessments. Innovation is needed also in the planning of experimental protocols to be followed by testing groups. Just to take a few examples of possible innovations in present days rules, experiments should be planned with the appropriate controls as required by the referees of all international journals. By appropriate control we mean isogenic non transformed genotypes or at least lines whose pedigree is known as well as the distance from the PGM to be tested. Unfortunately this almost never occurs in the present practice where isogenic lines are never utilised and the comparison is often made between the PGM and a number of not transformed cultivars “representative of natural variation” a term often used which of course does not mean anything to a geneticist. The use of this term in assessment reports suggests that the test is not aimed at establishing the risks of a specific GMP but has been planned to understand whether the eventually observed variability is “natural” or not, this being an obviously unscientific and irrelevant statement. Protocols should also be established for the planning of experiments in view of a statistical analysis of the data with a high resolution power. This means that numbers should be high, the use of randomised repeats in different environments and cultural conditions should be recommended etc. as they normally are in all research laboratories. Also the interpretation of the results, as suggested by Seralini et al. (2007) should be considered, particularly in the testing of possible nutritional problems where otherwise the final statement may be too subjective and different depending on who is carrying out the tests and particularly between independent laboratories or by PGM producers. As far as the molecular analysis is concerned protocols included in risk assessment only require the demonstration of the presence of the inserted trans-gene, its expression, and the estimate of its copy number. Now, it is known, as mentioned before, that DNA sequences other than those belonging to the trans-gene have been often found in transgenic materials as well as fragments of the construct used. (see for instance the very accurate work carried out by the group of Svitashev on transgenic oats). Moreover, re-arrangements of the original sequence have been shown to occur in transgenic materials but also spontaneously (Brunner et al., 2004) potentially leading to the displacement of genes, whose expression levels could be altered by possible position effects. Therefore it seems to us critical the introduction into the required bio-safety tests, insertion site detection and sequencing assessing the possible disruption of a pre- existing gene of the receiver plant eventually leading to its inhibition and to the possible formation of fusion RNA sequences potentially translated into novel proteins. Fragments search should moreover be carried out and again fragment insertion sites determined and sequenced. Moreover, a genome wide analysis for novel mutations can be carried out through the use of such techniques as for instance AFLP or other molecular methods leading to estimates of genome wide mutations as suggested by the work of Labra et al. 2001. A specific attention should then be given to the proteome for the eventual identification of new proteins deriving from fusion materials or from complex formation between different polypeptides as it has been shown to occur in the case of maize-made human tripsin produced by Prodigene . Finally, changes in the metabolome carried out utilizing up to date techniques allowing the contemporary analysis of a wide range of key metabolic components should be compulsory as these data are the needed background for the full understanding of the meaning of results from experiments aiming at assessing the potential nutritional risks of the tested PGM. A final critical point should be introduced here, very often source of discussion in all risk assessment debates: acute toxicity analysis is not sufficient but long term studies are required as it is known since the very beginning of toxicity assessments of pharmaceutical of other industrial products.

All the putative rules here suggested for introduction in EFSA required protocols come from a long lasting experience in the evaluation procedures of the two types of products at the moment in the market. Although unfortunately up to now all the discussion has been centred on PGM resistant to herbicides or to insects, however, this problem is going to be much more relevant in the future. The reason for this statement is that while, at least in our opinion, the risks of present day PGM are very low if not absent ( the only real risk is due to herbicides given immediately before harvest), plants producing pharmaceuticals and other industrial products should be carefully monitored in long term experiments. It should be recalled that American Bio-safety procedures do allow open air cultivation of GM crops without labels once they have been authorized for experimental purposes. Just to take one example, the aforementioned Prodigene products are already in the market and they derive from transgenic maize plants, liable to cross- pollinate other non GM or GM cultures. Europe therefore may be in danger in the near future of importing maize processed products containing trypsin or anti-trypsin or any other dangerous pharmaceutical and industrial product liable to have medium and long term effects.

c) Environmental, social, economic effects

When GMPs environmental effects are taken into consideration the discussion generally concerns the possible direct actions of GMP products released in the surrounding ecosystems and/or affecting specific microbial, plant or animal components. We shall introduce here a different and broader point of view taking into account also indirect effects deriving from agricultural practices and involving relevant social problems. In our opinion GMPs are a sort of emblem of the general trend in world agricultural policies towards the “optimisation” and homogenization of the agro-ecosystems following the systemic analogue of the concept of “ideotype” proposed by Donald long time ago in the early years of the “green revolution”. This concept does not take in principle into account the value of variability “per se” for the adaptation of living systems, suggesting that natural plasticity could be complemented by chemical and energy inputs through sound agriculture practices thus obtaining good production levels with the same or similar genotypes in almost any environment. As mentioned in the introduction genetic engineering also started as the most modern tool for the realisation of the wanted optimisation and, since the Cartagena protocol on Biosafety, GMPs introduction is the key for the penetration of the model supported by industrial agricultures as they are the only plant products which may offer a basis for closing national barriers to seed import on the ground of their supposedly related dangers for human or animal health or for social problems. Therefore industrial agriculture model penetration goes together with the increase of PGMs cultivation in developing countries. It should be stressed here that such a model has been developed in industrialised Countries and still is in competition with two other cultivation systems, namely subsistence agriculture and what has been called “craftsman-like” agriculture. The first is typical of poor areas where the farmers live directly on the products of their fields. These farmers, as was shown by an opinion poll in the region of Oaxaka ( Mexico) in 1998 by FAO, do not necessarily need a very high production level but they do require some harvest every year to support their families and this cannot obviously be achieved with genetically homogeneous cultivars as those produced in the North of the world, costs of chemical and energy inputs being too high for the poorest areas. “Craftsmanlike” agriculture is on the other hand also typical of several European Countries where the tendency is towards high quality–high prices products competitive in the market. Now, both agricultures need a high level of variability although for different reasons and are severely disturbed by the homogenisation process in general and particularly by GMPs in Europe due to the low acceptance by consumers of GMP containing products. Different economical reasons suggest therefore that genetically engineered crops with their relatively low number of homogeneous cultivars and the low acceptance by consumers have some serious drawbacks. Moreover, USDA official data show that maize and soybean production/acre, from the eighties, when no GMPs were in the market, to 2007 when a large majority of the two crops is genetically modified, have been increasing at the same pace, with no evidence of improvement given by the transformation. These data, of course limited to the products now in the market, do not show any significant positive effect on the production of food of present day GMPs, which, therefore for the moment do not seem to be useful in the fight against famine. On the contrary, as shown by data from Argentina, the growth of soybean cultivated areas is negatively correlated with those of rice, wheat and maize. Particularly, in the period 1996-2002 the production of soybean increased (74%) , while the other crops decreased (maize –26,2; rice –44,0%; wheat –3,5%).

Finally , at the same time, the increase in soybean production was parallel to a decrease in crop diversity as shown by the Simpson index, an indicator of variability, increased from 0,179 to 0,305 in the period 1997-2005 ( Simpson index = 1 means total homogeneity). What really is happening is that large soybean plantations are destroying local production of subsistence agriculture by small farmers and on the other hand soybean cultivation does not increase food availability as most of soybean produced is exported to be used elsewhere as animal feed and, lately as a source of vegetable oil to be used as bio-fuel. Broadly speaking anyway , GMPs being limited to maize, soybean, canola and cotton , their value for a significant impact on the famine problem is necessarily very low as only maize is a staple food in a limited area of South America.

From the environmental point of view one should consider on one hand the possible direct effects of GMP cultivation on the agro-ecosystem and on the other the impact of agriculture homogenisation on natural diversity particularly in areas critical for climate change like for instance Amazon forests. Direct effects of GMPs on biodiversity can be present at the level of soil communities both as far as microbial communities are concerned ( see M.Giovannetti et al. (2005) for a comprehensive review) and invertebrates (D.A Bohan et al.,2005). In both cases the cause of the observed damage is not necessarily the GMP itself, most problems being related to herbicide treatment for the whole duration of plant life cycles. Moreover, there are several although controversial, data on negative effects on insect populations particularly affecting bees or other pollinators but also in this case it is sometimes difficult to distinguish the direct effect of Bt toxin from chemicals used in transgenic fields ( see S.Kilner and P.Peduzzi for a recent summary of the literature). However particularly soybean cultivation is strongly correlated with deforestation for instance in Brasil and recently several commitments have been taken by the Brasilian government for economic reasons with USA ad Europe, which will certainly increase the pace of Amazon destruction in favour of massive sowing of oil producing crops to be used as bio-fuels.

d) Some conclusions

The main conclusions that can be taken from the present short survey of the state of the art of GMP Technology may be the following:

n From the mere technological point of view, present day GMPs, very limited in number of species and characters, are by far not at the level of expectations from this new way of improving plants for food. This possibly temporary situation is mainly due to the high level of unpredictability and control of the effect of the integration of genes not co-evolved with the receiving genotype and of several relevant parameters like the number of copies inserted, the “active reaction” of the receiver etc. These problems hopefully will be solved in the future but only if a much stronger attention is given to basic science leading to a better understanding and control of genome and metabolome dynamics.

n The intrinsic unpredictability of transgenosis leads obviously to several problems related to bio-safety and requires therefore a drastic change in present evaluation procedures needing an update of methods , techniques and approach also in view of the entrance into the market of potentially dangerous industrial and pharmaceutical products

n Present day GMPs , although cultivated in very large areas of the world do not seem to be at the level of expectations from the point of view of small farmers and consumers although a part of industrial agriculture and the producing industry, mainly controlled by a very small number of companies is profiting from them. This is not specifically due to the molecular techniques used nor to the very nature of GMP but to the fact that they are the emblem of industrial agriculture, a model with serious drawbacks particularly in developing countries and in relation to the problem of bio-diversity conservation both at the crop and natural resources levels

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-Morgante

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– Vecchio L., B.Cisterna, M.Malatesta, T.E. Martin, M.Biggioggera (2004) Ultrastructural analysis of testes from mice fed on genetically modified soybean. Eur.J. Histochem. 2004: 449-452

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