New genetically modified crops are being promoted for nutritional or health benefits. Health depends on a balance of macro and micronutrients, cofactors and vitamins, best achieved by organic agricultural practices. Overdose of many single nutritional factors can be toxic, and hence food crops genetically modified to overproduce single nutrients could be public health hazards.
Above all, genetic modification fails to address climate change and the depletion of energy, water, soil nutrients, and other agricultural resources that already threaten food security; it is a dangerous waste of time and dwindling resources. Prof. Joe Cummins and Dr. Mae-Wan Ho
The Codex Alimentarius Commission of the United Nations is deliberating Safety Assessment of Food Derived from Recombinant-DNA Plants Modified for Nutritional or Health Benefits. Governments and international organizations wishing to submit comments on the subject matters are invited to do so no later than 1 October 2006, preferably in electronic format, for the attention of Dr. FUJII Mitsuru, Fax No: +81 3 3503 7965; E-mail:codexj@mhlw.go.jp with a copy to the Secretary, Codex Alimentarius Commission, Joint FAO/WHO Food Standards Programme, Viale delle Terme di Caracalla, 00100 Rome, Italy (Fax +39.06.5705.4593; E-mail: Codex@fao.org ).
Our submission from the Institute of Science in Society consists of a review followed by specific comments.
The first genetically modified crop, Calgene's Flavr Savr tomato for prolonged shelf life, was approved for commercial release in 1992. It was not a commercial success. Since then, however, the area planted to GM crops has been increasing, and according to industry sources, reached 90 million ha in 2005 [1]. Two traits, herbicide-tolerance and insect-resistance, currently account for nearly all GM crops; but that will soon change.
New GM crops with other traits are poised to enter the market, in the guise of nutritional benefits and health foods. In 2004, Monsanto received approval for commercial release of high lysine corn modified with a bacterial gene to produce enhanced production of the amino acid lysine [2] ( Why Not Transgenic High Lysine Maize ). In the same year, Monsanto released ‘Vistive' soybeans with reduced linolenic acid fat, a trait selected using traditional breeding but bred into a glyphosate resistant soybean [3] ( Beware Monsanto's 'Vistive Soybeans' ) , and should not have been considered a crop modified only for food quality. In 2005, Syngenta applied for commercial release of a corn modified to produce a heat stable amylase enzyme as an aid in food and feed processing [4]. There are many more crop modifications for ‘food quality' in the pipelines to be petitioned for commercial release. But are they safe? And what criteria are used in their food safety assessment?
A number of reviews have appeared, one of the most comprehensive coming from the International Life Sciences Institute of Washington DC, whose Trustees include academics as well as representatives from Monsanto, Syngenta, Novartis, Unilever and other corporations [5]. “Metabolic engineering” is highlighted, which consists of up or down regulating metabolic pathways, or introducing entirely new pathways to enhance production of key nutrients in foods [6]. Genes for biosynthetic enzymes or regulatory genes are introduced, as well as anti-sense genes to block competing pathways to increase production of desired secondary metabolites [7]. Engineering soybeans has focused on altering functional properties of soy protein to improve processing and on improving the flavour of soy products [8].
Plant genomics is also put forward as an alternative to genetic modification. Using marker assisted selection in conventional breeding to improve flavour, health and nutritional value would obviate the need for genetic modification of the crops [9], and ultimately prove much more acceptable to consumers worldwide.
Nevertheless, a lot of work involving genetic modification is in progress, targeting every aspect of nutrition and health, from the improvement of storage proteins to increased mineral content.
Cassava is a staple food for over 500 million people in the tropics. Its root is rich in starch and contains proteins with a balanced amino acid composition, but at low concentrations. In order to add a storage protein with a balanced amino acid composition, a completely synthetic gene was inserted that had codons optimised for plants, and placed under the control of the CaMV 35S promoter and the nos terminator. A hygromycin antibiotic resistance gene served as the selectable marker. Nutritional improvement of the cassava root was reported [10, 11]. A synthetic gene expressing a synthetic protein is a novel approach, and requires extra careful scrutiny, especially with regard to allergenic and other immunogenic potentials.
The nutritional value of transgenic potato was increased using the seed albumin gene from Amaranthus hypochondriacus ( Prince-of-Wales feather). The transgene was driven by a tuber-specific promoter for high levels of protein production in the tuber. The transgenic protein was believed to be non-allergenic based on a single experiment with mouse pups injected with tuber, which were found to produce IgG antibodies but not the IgE antibodies of allergy [ 12]. IgG antibodies may be associated with inflammation, which could be quite severe, but that effect was not studied. We should be extremely wary of transgenic proteins, as even gene transfer between closely related species will alter the glycosylation patterns of proteins and hence its immunogenicity. A harmless bean protein turned into a potent immunogen when transferred to pea, eliciting serious inflammation reactions in mice [13, 1 4] ( Transgenic Pea that Made Mice Ill ) . Such considerations apply to all transgenic proteins discussed here.
Soybean is an important protein source in both food and feed. However it is deficient in sulphur amino-acids, particularly methionine. A methionine-rich maize delta-zein storage protein was used to transform soybean , but it failed to increase the methionine content of seed flour [15]. Storage protein was enhanced in rice, but only by introducing transgenes into low storage protein mutant strains that had “room” in the seed for the transgenic protein [16].
Glutenin is a major storage protein in barley. Barley is malted to make beer. During malting, glutenin is digested by a beta-glucanase enzyme. The heat stability of the enzyme can be problematic during industrial scale malting. A heat stable hybrid enzyme was made from genes of two bacillus bacteria species, with codon adjustments in the DNA sequence to enhance protein synthesis in barley. The synthetic hybrid gene is reported to have improved the malting characteristics of the transgenic barley [ 17, 18], but the fate and safety of the transgenic glucanase is unknown.
As in the enhancement of storage proteins, genetic engineering for improved food processing requires much more in-depth research. Marker assisted selection may be the best way forward, as has been used i n improving the carbohydrates of cereals [19].
There is a growing effort to enhance production of cancer fighting antioxidants and related plant products that reduce the risk of cancer and improve health in many ways. The evidence that organically grown crops are richer in cancer fighting antioxidants [20, 21] ( Organic Agriculture Helps Fight Cancer ; Organic Strawberries Stop Cancer Cells ) seems to have spurred biotechnologists on to create GM crops with enhanced levels of these compounds. Flavonoids are the plant antioxidants that have received the most attention a mong biotechnologists. In feats of metabolic engineering, novel structural or regulatory genes, antisense or sense suppressing genes, have all been introduced in efforts to enhance fl avonoid production [22].
Tomato has been the first target. Red wine is rich in the flavonoid stilbene, thought to be responsible its health benefit in preventing heart disease, and much effort has been devoted to metabolically engineering tomato to produce high levels of stilbene. Stilbene is provided by a gene from petunia flower, which was up regulated in tomato fruit by regulatory genes from maize. To further increase flavone production, genes from grape, alfalfa and the flower Gerbera were also incorporated into the tomato, resulting in the production of high levels of health related flavonoids [23]. But the safety of the transgenic tomato containing so many transgenes has not been addressed.
In another experiment, phenolic precursors of the health related flavonoids, lignans and phenols were enhanced in tomato by down regulating a competing metabolic pathway using RNAi to inhibit the gene for cinnanomyl-CoA reductase [24]. The use of RNAi in genetic engineering food crops and animals has been put into question by the recent observation that RNAi caused excessive fatality in mice due to the over-saturation of RNAi pathways [25, 26] ( Gene Therapy Nightmare for Mice ) .
The human C-reactive proteins are associated with cardiovascular risk, mice modified with human C-reactive proteins fed transgenic flavonoid tomato showed a greater decrease in C-reactive protein than mice fed conventional tomato [27].
Tomatoes transformed with the grape stilbene synthase gene driven by the CaMV promoter to provide constitutive synthesis showed in creased ascorbate and glutathione ; the soluble and total antioxidant activity was enhanced while lipid peroxidation was decreased [28].
Engineered polyamine accumulation in tomato enhanced phytonutrient content, juice quality and vine life [29, 30]. The problem with polyamine accumulation is the impact of the polyamines putresine and cadaverine, which are well known to impair human health [31] ( Drought Resistant GM Rice Toxic? ) ; and hence the promoters of genetically modified wine yeast claim that its greatest benefit is in decreasing polyamine content of the wine. Perhaps, eating high polyamine tomatoes will cause severe hangovers?
Tomatoes were also modified with the genes for enzymes that enhanced production of phytosterols by altering the isoprenoid pathway. The genes influencing isoprenoid formation were isolated from Arabidopsis and the bacterium E. coli . A chloroplast transit gene from tomato was attached to the isoprenoid genes, allowing both cytoplasmic and chloroplast isoprenoid pathways to be enhanced, and the modified tomatoes produced elevated phytosterols [32].
Tomatoes are not the only crop to be genetically modified for human health benefits, apple too, has been modified to enhance stilbene synthesis. A gene for stilbene synthase from grape, with the wound/pathogen inducible promoter also from grape, was introduced into apple along with the bar gene for herbicide tolerance driven by the nos promoter. The transgenic apple showed an increase in the stilbene reveratrol and in total flavonoids [ 33].
Seed phytosterol levels were enhanced in tobacco using a shortened gene for a rubber tree enzyme 3-hydroxy-3-methyl-CoA reductase (the gene was shortened to remove a cell membrane binding domain to increase activity in seeds). Phytosterol was increased more than 3 fold to 3.5 percent of the seed oil [34]. Presumably, such constructs will be transferred to food oil crops such as canola, soybean or maize. Metabolic engineering of proanthocyanidins using genes for anthocyanidin reductase and for the Myb protein transcription factor from Arabidopsis provided a way to enhance the (epi)-flavan-3-ol antioxidants. The antioxidants not only provide health benefits to humans, but also prevent bloating in ruminants. Tobacco was transformed to produce quantities of antioxidant capable of preventing bloating in ruminants. Anthocyanidin reductase alone enhanced antioxidant production in a forage legume annual alfalfa, Medicago truncatula , but the levels were not sufficient to prevent bloating in ruminants [35]. It is worth mentioning that the Myb family of transcription activators was first discovered as a viral oncogene (cancer-associated gene). Even though the factor is prevalent in plants, its amplified use in transgenic food and feed requires thorough risk assessment and safety testing.
A 2004 review of micronutrients in staple food crops and plant breeding for improvements [36] identified a crisis in the availability of certain micronutrients globally. Children (primarily the poor) may be dying from deficiencies of iron, zinc and vitamin A, in particular. The problem is best addressed through agriculture, and biotech proponents have put forward solutions; though they are certainly not the only solutions. Sustainable farming practices that emphasize internal organic inputs can address the problem of micronutrient deficiencies across the board, without the need for genetic modifications [21, 37] ( Dream Farm 2 - Story So Far ).
Carotenoid, the precursor to vitamin A, can be over-produced in plants by either direct insertion of transgenes or by altering the flux of metabolites to carotenoid synthesis [38]. “Golden rice” was promoted as the answer to vitamin A deficiency. The entire beta -carotene biosynthesis pathway had been engineered into rice endosperm in a single transformation step. The genes for phytoene synthase ( psy ) and lycopene beta-cyclase ( beta-lcy ) originated from the daffodil and the gene for phytoene desaturase ( crt1 ) from bacteria [39]. The daffodil gene psy was subsequently replaced with maize psy to enhance synthesis in rice. Recent versions of golden indica rice used an endosperm-specific promoter for psy and CaMV promoters for beta-icy and crt1 . Selection was done using a mannose medium in cell culture, based on co-transformation of the rice with a phosphomannose isomerase gene driven by a cestrum yellow leaf curling virus promoter. Mannose selection depends on the enzyme phosphomannose isomerase converting mannose-phosphate, which cannot be metabolised by the plant cell, to fructose- phosphate, which can be metabolised. Unmodified cells accumulate mannose-phosphate, causing them to die from starvation . Mannose selection avoids antibiotic resistance marker selection and the carry over of resistance genes into food. Antibiotic resistance markers have also been removed from golden indica rice by cross breeding and selection [40].
The Institute of Science in Society critically reviewed golden rice in 2000 [41]. Among the major concerns was that the rice produced too little beta-carotene to relieve the existing dietary deficiency. Since then, golden rice strains have been improved, but still fall short of relieving dietary deficiency. On the other hand, increasing the level of beta-carotene may cause vitamin A overdose for those consuming a normal balanced diet with multiple carotene sources. Vitamin A supplements taken during pregnancy can cause birth defect, and even moderate to small doses may induce birth defects (perhaps subtle) during early gestation [42]. In fact, both vitamin A deficiency and supplementation may cause birth defects [43], and it seems that the developers of golden rice are caught between a rock and a hard place. This is where labelling is absolutely necessary if golden rice is to be sold in the market, to alert sensitive people of its potential adverse impacts.
Vitamin E genetically engineering has begun. Plants are the source of vitamin E, a class of compounds called tocochromanols comprising four tocopherols and four tocotrienols. Corn seed oil, soybean seed oil and wheat germ oil are all rich in tocopherols. Vitamin E enhancement has been achieved by mutation and by genetic manipulation. Arabidopsis has been the main source of genes to enhance production of vitamin E by over-expression in Arabidopsis , canola and soybean. Further metabolic engineering of the tocochromanol pathways may lead to greater production of the most significant tocopherols [44, 45]. V itamin E supplementation has been promoted for preventing heart disease and cancer [46] and in treating cancer [47]. However, vitamin E supplementation caused significantly elevated ovarian cancer in one study [48], while another major study found that high dosage supplementation increased all-cause mortality and should be avoided [49]. Like vitamin A, vitamin E over-production in food crops is of dubious value, and may indeed be harmful.
Vitamin C is commercially synthesized from glucose. The vitamin has been produced in modified bacteria and yeast and these approaches may have some limited advantage over chemical synthesis [50]. Plants have been subject to metabolic engineering to over produce vitamin C, but the increases have been very modest [51].
Folate levels have been enhanced in Arabidopsis using the bacterial gene encoding the enzyme GTP cyclohydrolase [52], but with yet no success when transferred to crop plants [53]. Folate enhancement is a global public health issue. In Canada, USA and Chile, flour is fortified with folate, resulting is a striking decrease in neural tube birth defects while in Europe, where fortification is not mandatory, there has been no decline in neural tube defects [54]. In an area of China where neural tube defects appeared in 1.4 percent of births, a public information campaign was run to promote folate supplement for women of childbearing age, but the campaign failed. Folate fortified flour is inexpensive and would probably have prevented the birth defects [55]. In the immediate future, mandatory supplementation will prove more effective than genetic modification.
B vitamins include riboflavin (B2) and pantothenate (B5). Their metabolic pathways in crop plants are known, but there has been no success as yet to engineer over- production of the vitamins in food crops [56, 57].
The metabolic pathways of vitamin synthesis in food crops are well understood. This knowledge can be exploited for marker assisted breeding to enhance vitamin production much more profitably than genetic modification.
There has been extensive genetic manipulation to improve the mineral nutrition of plants for both macronutrients such as calcium and nitrogen, and micronutrients such as selenium.
A 3-fold enhancement of calcium in potato tubers was achieved using an Arabidopsis calcium exchanger and transporter gene. The exchanger had been shortened at the N terminus to eliminate an auto-inhibitory regulatory domain of the protein. Two different promoters were tested, the CaMV promoter and a cell division cycle gene promoter, both of which appeared to have equivalent effects on calcium uptake [58]. The modifications did not affect yield or quality of the potatoes. Milk is currently the main source of calcium in children's diet, but is that to be soon replaced by fries?
Inorganic nitrogen fertilizer is linked to a variety of problems including the pollution of drinking water, harming infants and causing eutrification of water and depletion of oxygen for aquatic animals. Improving utilization of nitrates by crops should lead to human health benefits in terms of cleaner drinking water, which could be offset by increased levels of nitrates in crops. The best way to improve nitrate utilization by crop plants is to phase out chemical fertilizers in favour of organic inputs, which decrease nitrate levels in the water as well as in the crop plants, where cancer-fighting antioxidants, mineral nutrients and micronutrients are also increased [21, 59] ( Organic Strawberries Stop Cancer Cells ; Organic Farms Make Healthy Plants Make Healthy People ) . So far, th ere has been little progress in developing transgenic crops that take up and utilize nitrogen more efficiently [60].
Iron deficiency in food crops plagues much of the globe, particularly Asia. About 40 percent of the world's women suffer some degree of iron deficiency. Pre-menopausal women are most severely affected by iron deficiency, while men tend to retain iron. Increased dietary iron is desirable and rice is the preferred crop for increasing iron content. Mugineic acid phytosiderophores (siderophores are compounds that bind metals in the soil and enhance their cellular uptake) iron transporters are re-adsorbed by the plant roots. In response to iron deficiency, the phytosiderophores are markedly increased. Transgenic rice modified with barley genes for the precursors of the phytosiderophore produce elevated levels of phytosiderophore and had enhanced tolerance to low iron soil, and had a greater yield than conventional rice on alkaline soil [61, 62]. Constitutive expression of a gene for soybean ferritin in wheat and rice resulted in increased levels of iron in the leaves and stems of transgenic rice but not the grains [63]. The soybean ferritin gene linked to an endosperm specific promoter was expressed in the seeds of maize resulting in elevated iron content [64] ( Rice in Asia: Too Little Iron, Too Much Arsenic ) . Food crops enhanced with elevated iron content must be labelled in the marketplace because iron overload is a significant problem in males, and may lead to haemochromatosis, a disorder of excessive absorption and storage of iron that could damage the liver and other organs, resulting in liver cancer or colorectal cancer. One percent of the population may carry a mutation (hereditary haemochromatosis) that makes them sensitive to iron overload at relatively modest iron intake levels; and there is an association between increasing iron stores and risk of cancer [65].
Selenium is essential for humans, but has a toxic side that makes it poisonous at relatively low levels. In Australia, selenium is generally inadequate for optimum human health, so agronomic bio-fortification of food g r ains has been proposed [66]. In western USA, on the other hand, high soil selenium levels are encountered. Seleniun toxicity is caused by replacement of protein sulphur with selenium forming selenocysteine, which is needed at low levels but toxic at higher levels. A mouse gene specifying an enzyme selenocysteine lyase was introduced into the Brassica juncea (canola) chloroplast genome to limit accumulation of selenocysteine to mitigate selenium toxicity. The transgenic canola had a reduced content of selenium in its proteins [67]. Like iron and other minerals, selenium is an essential nutrient, but becomes toxic at high levels.
It is clear that manipulating single genes to overproduce any mineral (or vitamin) is fraught with difficulties as these essential nutrients are often toxic at inappropriately high levels. This highlights the importance of getting a balanced mineral content in our food, which can only be achieved by moving away from unbalanced external inputs of chemical fertilisers in favour of organic fertilisers [21, 60].
Long chain fatty acids are the focus of a great deal of interest because they play an important role in health and nutrition. Long chain polyunsaturated acids are vital for human health. Fish and marine oils are the main sources of long chain polyunsaturated fatty acids, but efforts are being made to modify oil crop plants to produce the essential fish fats. The key fatty acids are eicosapentaenoic acid (20 carbon fatty acid with five unsaturated double bonds) and docosahexaenoic acid (22 carbon fatty acid with six double bonds). Genes from a marine microalga Isochrysis galbana , an oil-producing fungus Mortierella alpina and a green protozoan flagellate Euglena gracillus were used to transform Arabidopsis to modify the plant seed oils to fish fatty acids. The transforming genes were involved in elongating and desaturating the plant fatty acids [68-70]. The crucial long chain polyunsaturated fatty acids were synthesized in Arapidopsis , a tiny plant grown in small petri dishes, which is thus unlikely to produce commercial quantities of the essential fatty acids. The process will have to be transferred to oil crops; but why not grow fish instead? Fish and vegetables together make a much more satisfactory diet, and certainly a more enjoyable meal for most people.
Monsanto engineered canola seeds to accumulate stearidonic acid, another long chain polyunsaturated omega-3 fatty acid that has 18 carbons and 4 double bonds. The transgenic canola was modified with genes targeted to the seeds, and included genes for desaturase from the oil fungus Mortierella alpina and from canola [71].
USDA and the University of Nebraska created transgenic soybean to produce stearidonic acid. The soybean was modified with desaturase genes from borage (a tasty salad green) and Arabidopsis , driven by a seed-specific promoter from soybean. Stearidonic acid made up 60 percent of the seed oil in the modified soybean [72].
Sunflower seed oil has been modified with multiple copies of a desaturase gene from castor bean to act on stearic acid, which reduces the quality of sunflower seed oil. The transgenic oil had reduced levels of stearic acid and was superior to unmodified sunflower oil [73]. Antisense technology was used to down regulate a cottonseed desaturase gene resulting in enhanced production of desirable oleic acid and reduced the content of linoelic acid, which is highly undesirable [74] .
The modifications of plant fatty acids are impressive, but the impact of the transgenes and transgenic plants on human health remain unknown. As discussed earlier, the protein products of transgenes may have undesirable effects on the immune system. Metabolic engineering in general may create unintended toxins and immunogens. Another factor to take into account is that the essential polyunsaturated fatty acids are also known to be toxic at high levels. There is good evidence that n-3 and n-6 polyunsaturated fatty acids are therapeutic at moderate levels in the diet but they may be detrimental at high levels by causing oxidation stress and forming lipid peroxides which are toxic. Daily intake of the polyunsaturated fatty acids above 10 percent of energy intake is not recommended [75]. Furthermore, high intake of marine fat rich in n-3 polyunsaturated fatty acids may prolong gestation, producing high birth weight [76] and gestational exposure to methylmercury in fish. The n-3 fatty acids interact with the mercury pollutant found in fish and unmask its toxic effects [77].
Certain amino acids are essential for the human diet because mammals cannot synthesize the amino acids. The essential amino acids are histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. In addition, the amino acids arginine, cysteine, glycine and tyrosine are considered conditionally essential. Certain crops, such as maize, are not complete foods or feeds because they are deficient in an essential amino acid. Maize is normally deficient in lysine. As indicated earlier, high lysine transgenic maize was approved for commercial release in 2004. The transgenic lysine is modified with a bacterial gene while high lysine maze varieties are already available through conventional breeding. The transgenic maize differs from high lysine maize derived from conventional breeding, which contains storage proteins with elevated lysine. Transgenic maize, on the other hand, has lysine elevated in the metabolic pools, and while it may provide adequate lysine in fodder, the soluble lysine may be lost during processing of the grain [2]. High lysine cer e al crops other than maize are considered desirable [79].
Methionine is deficient in some food and feed crops, and there are efforts to enhance methionine levels in these crops. A sunflower seed albumin, rich in the sulphur amino acids methionine and cysteine, was used to modify lupine, a significant feed crop in many countries. The transgenic construct included a herbicide tolerant gene (Bar) and a gus reporter gene. The modified lupine seeds performed significantly better than unmodified lupine in feeding trials [79]. Lupine is not the only legume deficient in sulphur amino acids as all legume storage proteins have low levels of these amino acids, preventing them from being a complete diet. Methionine and cysteine were enhanced in alfalfa by over-expressing an Arabidopsis cystathionine gamma-synthase gene (the first enzyme in the metabolic pathway for methionine). The enzyme was driven by a pea chloroplast rubisco promoter, and directed to the chloroplast by adding a pea rubisco transit sequence. The modified plants were enhanced in both soluble and protein bound methionine and cysteine [80].
Maize has a high methionine-rich storage protein that is usually under-represented in the seeds. A cis -acting regulatory site giving limited messenger RNA stability was replaced by a sequence imparting greater messenger RNA stability, resulting in increased levels of methionine in transgenic seeds. This provided feed and food that did not require addition of synthetic methionine [81]. A bacterial ( E. coli ) serine acetyl-transferase gene , driven by the CaMV promoter, was transferred to potato to increase cysteine and glutathione. An Arabidopsis transit sequence was added to direct the transgenic protein to the chloroplast. The resulting transgenic potato plants had elevated levels of cysteine and glutathione. Metabolic engineering was used to enhance production of sulphur-containing compounds in potato [82].
Glutathione (GSH) is an antioxidant consisting of three amino acids that protects cells from free radicals and participates in metabolic reaction. GSH is the most abundant low molecular weight thiol (compounds with -SH group) in plants. It accumulates to high concentrations particularly in response to stress. A bacterial enzyme catalyzing glutathione synthesis and lacking feed back inhibition was used to enhance glutathione production in plants [83]. Glutathione is important in maintaining nutritional homeostasis and great caution must be exercised in manipulating glutathione levels in plants. Increasing glutathione levels in tobacco unexpectedly resulted in continuous oxidative stress in the plants [84].
Anti-microbial peptides provide the first line of defence against invading bacteria, fungi and viruses in both plants and animals and are part of the host's innate immunity, acting mainly at the cell membrane. They are 15 to 40 amino acids in length, most of them hydrophobic (water-hating) and cationic (positively charged), and are beginning to find applications in medicine and in crop protection.
A synthetic peptide D4E1 based on the cecropin B peptide toxin (obtained from the moth, Cecropia), consists of a linear sequence of 17 amino acids: FKLRAKIKVRLRAKIKL (F for phenylalanine, K for lysine, L for leucine, R for arginine, A for alanine, I for isoleucine, V for valine). The peptide protected against Aspergillus and Fusarium fungi. It act s by binding to ergosterol, a sterol present in fungal cell walls [85]. On further tests, D4E1 was found to have broad-spectrum anti-microbial action, and was active against fungi belonging to the orders Ascomycete, Basidiomycete, Deuteromycete and Oomycetes, as well as bacterial pathogens Psuedomonas and Xanthomonas [86]. The D4E1 toxin also proved effective in treating Chlamydia infection in humans [87]. Synthetic peptides of 11 amino acids proved effective against bacterial plant pathogens, with minimal cytotoxicity and protease degradation, offering improved crop protection as an external pesticide or incorporated into transgenic crops [88].
A DNA sequence encoding synthetic peptide 10 amino acids long replaced the active region of the tomato prostemin gene in order to enhance production and processing of the peptide. The hybrid gene facilitated the transfer and insertion of the peptide into tobacco plants where it proved active against microbial pests [89].
Researchers at the National Agricultural Research Center, Niigata, Japan, have created transgenic rice with the anti-microbial peptide defensin from Brassica . The transgenic rice plants were resistant to rice blast disease caused by the fungus Magnaporthe grisea . The researchers then systematically altered the genetic code for defensin to produce synthetic peptides that were far more toxic to the fungus than the natural peptides [90]. Rice with the synthetic genes and peptides are being proposed for field-testing prior to commercial release in Japan.
A potato virus X expression system was used to produce a killer peptide derived from a single chain anti-idiotype antibody of a broad spectrum microbiocidal yeast killer toxin with a strong activity against human pathog ens. The killer peptide was tested against both bacterial and fungal plant pathogens and proved very effective. The killer toxin was fused to the virus X coat protein in a system that allowed its rapid production [91]. The virus production system is capable of spreading the toxin to potato, for better or for worse.
There have been criticism and objections to open field-testing of crops modified with the synthetic peptides. The evolution of resistance to anti-microbial peptides will severely compromise both the natural defence of the human immune system against disease and the possibilities of effective therapies emerging in the wake of the disaster of widespread antibiotic resistance [92] ( No to Releases of Transgenic Plants with Antimicrobial Peptides ) . As versions of the peptides also provide defence against pathogens in other animals and plants, the ecological impact of resistant pathogens could be devastating. Another factor adding to the hazards to health and the environment is that the synthetic transgenes code for peptides that are significantly different from the natural versions. This may itself be responsible for toxic or other harmful effects that cannot be known unless thoroughly tested.
Probiotic microbes including Lactobacillus species, Bifidobacterium species and the yeast Saccharomyces boulardii , have been used as food supplement. The health benefits of probiotic microbes include antagonistic effects on gastroenteric pathogens, neutralisation of food mutagens produced in the colon, shifting the immune system to alleviate allergy and lowering serum cholesterol [93] ( Health-promoting Germs ) . Probiotic microbes are being developed as vectors for gene therapy and genetically modified to “improve” the quality of food.
The probiotic lactic acid bacteria have been extensively modified to serve the food industry and other purposes. Modifications included modulation of the proteolytic system to enhance cheese ripening, increasing the production of the Kreb's cycle enzyme alpha keto glutarate, using antisense RNA to silence lytic Lactoccocus phage, introducing a folate gene cluster, re-routing pyruvate to L-alanine, and over-expressing the riboflavin biosynthesis pathway. Further genetic modifications of lactic acid bacteria involved inactivation of glucose fermentation and introduction of lactose fermentation, introduction of alpha-galactosidase, of phytase, of alpha amylase and cellulose. Lactic acid bacteria have also been genetically modified with bacteriocin toxin to prevent dental carries; for increased activity of beta-galactosidase, for lacticin (a bacteriocin) production, for increased nicin production, and increased proteolytic and acidifying activity. The probiotic bacteria have been enhanced for glutathione production, and for oxidative stress tolerance. Lysostaphin, a glycylglycine endopeptidase that specifically cleaves the pentaglycine cross-bridges found in the staphylococcal peptidoglycan was inserted into lactic acid bacteria for use in destroying the pathogen. The safety assessment and regulation of these GM probiotic bacteria were discussed in recent reviews [94, 95].
The production of nutraceuticals (foods or components with health benefits) with genetically modified food grade microbes and their safety assessment were also reviewed [96]. GM probiotic bacteria have not received much public scrutiny mainly because the regulation of such microbes is separate from the regulation of GM crops. There is a strong likelihood that GM probiotic bacteria may be introduced to the market before serious safety concerns are addressed.
Using GM probiotic bacteria requires special caution. These bacteria are natural symbionts of the gastrointestinal tract, and have adapted to their human and animal hosts over millions if not billions of years of evolution. Genetically modifying them could easily turn them into pathogens pre-adapted to invade the human and animal gut [97]. Furthermore, the gastroinstestinal tract is an ideal environment for horizontal gene transfer and recombination, the major route to creating pathogens. For these reasons, we have proposed that any genetic modification of probiotic bacteria should be banned [98, 99] ( Ban GM Probiotics ; GM Probiotic Bacteria in Gene Therapy ).
Fowls grew faster when fed transgenic yeast Pichia pastoris modified with a pig growth hormone gene with an alcohol oxidase promoter and an alpha-factor signal peptide. The modified yeast mixed with the fowl diet made the fowl grow about 10 percent faster than controls [100] . Growth hormone food microbes may be attractive for chicken farmers, but their use may carry the microbes over into the human population; and not everyone would want to grow like pigs.
Microbial bio-control agents have been developed and the impact of such agents on foods requires careful consideration. A modified Trichoderma atroviride with a glucose oxidase gene from Aspergilus niger rapidly overgrew and lysed the plant pathogens Rhizoctonia solani and Pythium ultimum . The transgenic bio-control agent both defeated the pathogens and induced systemic resistance in treated plants [101], but they should be studied extensively for their impact on food safety and quality.
The modification of food microbes requires comprehensive public scrutiny especially as numerous modified strains are awaiting release into the commercial markets.
The pressure to use genetics to boost crop yield, first with the green revolution, and then with genetic modification, may have resulted in a crisis in nutrition. Too many of the crops have become depleted in mineral nutrients, vitamins and essential building blocks [102], particularly as soils become depleted and exhausted. Healthy soils are needed for the production of healthy crops [59].
But maximising yield does not necessarily sacrifice nutrition if the land is properly managed for maximum internal organic input. This can be achieved by turning otherwise polluting livestock and crops wastes into food and energy resources [37], thereby mitigating climate change and solving the global energy crisis while providing food security for all.
Genetic modification fails to address climate change and the depletion of energy, water, soil nutrients, and other agricultural resources that already threaten food security, and is a diversion of time and resources that the world can ill afford.
Much effort is being devoted to improving the nutritional quality of crops intended for food through genetic modification to enhance production of single nutrients, minerals or vitamins. We question this approach as a whole. Nutrition depends on a balance of macro and micronutrients as well as cofactors and vitamins, which is best achieved by adopting organic agricultural practices. Furthermore, overdose of any single nutritional factor is likely to be toxic; and hence these genetically modified ‘health-enhancing' food crops may well turn out to be serious public health hazards.
Codex's consultation document [103] states: “Working Group members recognized that safe upper intake levels should be determined for nutrients and related substances to prevent excessive intake by vulnerable populations. It was also recognized that there is a need to determine the safety of nutrients and related substances when upper limits have not been determined and to also consider the history of safe use of the nutrient when appropriate. However, it was also recognized that the issue was generic in nature.”
It is already clear that many of the nutrients being genetically manipulated have upper limits of toxicity, particularly iron and selenium among minerals, vitamin A and possible vitamin E.
It would be misleading and indeed dangerous to market foods enhanced in single nutritional factors as ‘health' foods. And it is imperative to label such products clearly in order to avoid toxic overdose.
Codex has not addressed the safety of synthetic genes that are being used, some of which produce proteins that are completely new to our food chain. Extreme examples are the synthetic storage protein genes to enhance amino acid content, and synthetic peptides for controlling pathogens. By definition, the products of synthetic genes are not “substantially equivalent” as they have no natural counterparts.
Apart from the completely synthetic genes, many genes are synthetic approximations of natural genes or hybrid genes made of synthetic approximations of two or more genes. These too, constitute novel proteins in our food chain. All transgenic proteins should be comprehensively tested for toxicity and immunogenicity, bearing in mind that gene transfer even between closely related species involves changes in glycosylation patterns that may transform a normally harmless protein into a potent immunogen.
It is stated in the Codex consultation document [103] that, “foods derived from rDNA plants that have undergone modification to intentionally alter nutritional quality or functionality should be subjected to additional nutritional assessment - beyond that conducted when modifications are for other purposes - to assess the consequences of the changes and whether the nutrient intakes are likely to be altered by the introduction of such foods into the food supply.”
Many of the genetic modifications involve massive alterations, such as the replacement of one or more metabolic pathways. Such changes increase the probability of creating unintended toxic by-products, and call for comprehensive comparisons of metabolic- transcriptional- and protein-profiles between GM varieties and the non-GM controls, as well as extensive safety trials.
RNAi is being used increasingly to up or down regulate genes and pathways. The need for adequately testing such constructs is clear; the massive deaths of mice subjected to RNAi ‘gene therapy' should serve as a warning. RNA interference is currently not included as genetic modification, and requires special attention.
The Codex consultation document [103] states: “…additional safety and nutritional considerations for the assessment of foods derived from rDNA plants modified for nutritional or health benefits include such aspects as bioavailability and physiological function of the intended modification. Particular focus will be given to staple crops of interest to populations in developing countries.”
Safety and nutritional testing of all of the modifications described in the accompanying literature is clearly essential for all the areas of the world. But we emphasize that the initial testing of the modified crops should not be done on children of developing countries under the guise of providing medical care, as has been the case with GM rice producing proteins found in milk [104] ( FDA in Third World Drug Trial Scandals ) , and those undertaking such unethical tests should be prosecuted.
The genetic modification of probiotic bacteria should be banned until and unless extensive studies and safety tests have been carried out. These bacteria have co-evolved with their animal and human hosts for millions and billions of years, with an intricate network of relationships that is only just beginning to be understood, and if thrown out of balance, could result in serious disease. Genetically modifying these bacteria runs the risk of creating pathogens that are preadapted to invade the gastrointestinal tracts of their hosts, where horizontal gene transfer and recombination are rife.
Finally, the concept “substantial equivalence” has no validity in risk assessment of GM food and food products, least of all in the area of metabolic engineering (see our comments for Genetically modified food animals [105] and should be rejected by Codex Alimentarius.
Article first published 24/09/06
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