- Email: [email protected]
Transgenes for tea?
Opinion
TRENDS in Biotechnology
Vol.23 No.1 January 2005
Transgenes for tea? John Heritage School of Biochemistry and Microbiology, University of Leeds, Leeds, LS2 9JT, UK
So far, no compelling scientific evidence has been found to suggest that the consumption of transgenic or genetically modified (GM) plants by animals or humans is more likely to cause harm than is the consumption of their conventional counterparts. Despite this lack of scientific evidence, the economic prospects for GM plants are probably limited in the short term and there is public opposition to the technology. Now is a good time to address several issues concerning GM plants, including the potential for transgenes to migrate from GM plants to gut microbes or to animal or human tissues, the consequences of consuming GM crops, either as fresh plants or as silage, and the problems caused by current legislation on GM labelling and beyond. Despite qualified approval for the commercialization of GM crops for human food and animal feed purposes in the United Kingdom, granted in March of this year [1], largescale industrial research in this area has been severely curtailed. The reasons for this curtailment are the poor commercial prospects in Europe for the technology rather than scientifically based opposition. In the European Community, vocal opposition to GM technology in agriculture has been evident for years (A report to the EC Directorate General for Research from the project ‘Life Sciences in European Society’ QLG7-CT-1999-00286, http://europa.eu.int/comm/public_opinion/archives/eb/ ebs_177_en.pdf), as has been well illustrated by the National GM Public Debate [Genetic Modification (GM) Public Debate (2003) GM Nation? The findings of the national debate, http://www.gmnation.org.uk/docs/ gmnation_finalreport.pdf] which was held in the United Kingdom during 2003. There were three strands to the National GM Public Debate: a public consultation (http://www.gmnation.org. uk/docs/gmnation_finalreport.pdf), a review of the economic consequences of commercializing GM crops [UK Cabinet Office Strategy Unit (2003) Field work: weighing up the costs and benefits of GM crops, http://www.number-10. gov.uk/su/gm/downloads/gm_crop_report.pdf] and a review of the science underpinning the technology [GM Science Review Panel (2003) GM Science Review First Report: An open review of the science relevant to GM crops and food based on interests and concerns of the public, http://www. gmsciencedebate.org.uk/report/pdf/gmsci-report1-full.pdf]. The science review found no evidence for harm in the introduction of transgenic plants as food or feed. The economic review concluded that none of the situations examined was entirely good or bad: each required a trade-off Corresponding author: John Heritage ( [email protected]). Available online 25 November 2004
between costs in some areas and benefits in others, necessitating value judgements to be made weighing the pros and cons of introducing transgenic plants as food or feed. The public consultation found that people in the United Kingdom are generally uneasy about GM food and feed, and that the more that people engage with GM issues, the harder are their attitudes and the deeper are their concerns. Opposition to agricultural applications of GM plants is not universal. The growth of insect-resistant plants has seen the crystal toxin of Bacillus thuringiensis transformed from a foliar insecticide with limited practical applications to a toxin engineered into ‘Bt crops’ that, at the turn of the millennium, were being grown on 11.4 million hectares worldwide [2]. By 2003, the latest year for which figures were available at the time of writing, the area devoted to cultivation of these Bt crops had risen to 12.2 million hectares worldwide, and the total global area supporting the growth of transgenic plants for food or feed purposes had grown to 67.7 million hectares [Global status of commercialized transgenic crops (2003) ISAAA Briefs No. 30-2003l http://www.isaaa.org]. It has been argued that inserting the gene encoding the crystal toxin of B. thuringiensis into target plants where only pests will consume the toxin is more environmentally friendly than is spraying the toxin widely across fields and thereby exposing insects that do no damage to plants, as well as those that do, to the toxin. In this article, I focus on the potential for transgenes to migrate from GM plants to gut microbes and/or to animal or human tissues. In addition to exploring the consequences of consuming fresh GM plants, I discuss the issue of ensilage and the problems caused by current legislation on GM labelling and beyond. Cause for concern? Despite the absence of scientific evidence showing that potential harm to human or animal health is associated with the commercialization of GM plants (http://www. number-10.gov.uk/su/gm/downloads/gm_crop_report.pdf), the UK economic review revealed that any economic benefits from the application of this technology are likely to be limited, at least in the short term (http://www. number-10.gov.uk/su/gm/downloads/gm_crop_report.pdf). Furthermore, many people in Great Britain are uneasy about the use of GM plants for food or feed purposes, and the more these people engage with the topic, the more hardened become their attitudes (http://www.gmnation. org.uk/docs/gmnation_finalreport.pdf). But are there reasons for concern? One area of particular public concern is the potential for the inserted genes to migrate from GM plants to microbes in the guts of
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the animals or humans who consume the GM material; another is the possibility that these genes might associate with animal or human tissues. I consider these issues below. The potential for gene flow in the gut and beyond There has been considerable interest in the report that foreign DNA consumed in food is not degraded completely in the mouse intestinal tract [3]. When mice were fed on material containing DNA from bacteriophage M13mp18, DNA fragments of 976 bp were detected in the bloodstream for up to 8 h after feeding. By fluorescent in situ hybridization (FISH) analysis, about 1 in 1000 peripheral white blood cells were found to be carrying bacteriophage DNA in the test mice, whereas none was found in the controls. Bacteriophage DNA was observed in gut epithelial cells, in the Peyer’s patches, in liver cells, and in lymphocytes and macrophages found in the spleen [3]. More recent studies using various foreign DNA sources have confirmed that DNA sequences are taken up from the diet of humans, food animals and laboratory rodents, and that increasing the concentration of bulky dietary fibre material accelerates the passage of food through the gastrointestinal tract, speeding up the clearance of foreign DNA from the gut [4]. If foreign DNA is capable of transferring from a food source into the tissues of an animal that eats the test material, could transgenes incorporated into GM plants that are used for food or feed purposes do the same? GM foods and feeds are not a homogenous group and range from plant material that is consumed fresh and unprocessed to pure chemical compounds, such as sucrose, that are derived from GM plants. For pure compounds in which there is no trace of the GM event, there is clearly no potential for gene flow; however, the same might not be true for GM food or feed material that undergoes little or no processing. Both high temperature and low pH reduce the survival of DNA in food and feed, and these effects are additive [5,6]. The physical nature of DNA affects its chances of survival during its passage through the gut. In a study using gnotobiotic rats, plasmid DNA was recovered throughout the intestinal tract of the rats. Furthermore, the DNA recovered retained the ability to transform electrocompetent Escherichia coli cells [7], indicating that not only had it survived passage through the digestive tract but it had remained biologically active. This distinction between the detection of total DNA and DNA that retains biological activity is important because the ability to transform cells is a crucial step in potential gene flow and it is a property that is lost before the ability to amplify target DNA from samples is lost [8,9]. In vitro model studies have confirmed that transgenes in some GM foods can survive passage through the small intestine [10]. Gene transfer events to the microflora of the intestinal tracts of animals and humans eating GM plants involving transgenes are likely to be very rare events. Indeed, searching for them might be likened to seeking needles in haystacks. In this area of research, however, a recent publication stands out. Netherwood et al. [11] examined the fate of transgenic DNA in food given to human volunteers. Food containing significant amounts of GM www.sciencedirect.com
Vol.23 No.1 January 2005
soya was fed to people who had had an ileostomy, an operation in which the ileum is passed through the wall of the abdomen to form an artificial anus so that the contents of the gut at this point can be collected with ease. The fate of transgenic DNA in the ileostomy fluid of volunteers was examined. Although Netherwood et al. [11] concluded that gene transfer events did not occur during the feeding experiment, three of the seven volunteers showed evidence of low-frequency gene transfer events from GM soya into the small bowel microflora that had occurred before the experiment began. Transgenic DNA fragments could be amplified from bacteria cultivated in liquid culture from the ileostomy fluid at the outset of the experiment. Furthermore, bacteria containing the transgene template could be detected after six serial passages in broth culture. Intriguingly, these bacteria were not recoverable on conventional solid growth media, making further studies in this area challenging, although it is likely from the work reported so far that the bacteria are obligately symbiotic facultative Gram-positive bacteria [11]. Netherwood et al. concluded that these DNA transfer events from transgenic plants to gut microflora are highly unlikely to alter gastrointestinal function and thus do not pose a risk to human health. This is a view with which I agree completely [12]. “.these DNA transfer events from transgenic plants to gut microflora are highly unlikely to alter gastrointestinal function and thus do not pose a risk to human health.” The problem of antibiotic resistance marker genes It is not only the potential for transfer of transgenes that requires consideration when contemplating the commercialization of GM plants for food or feed purposes. What is encoded by the DNA might be also significant. In this context, DNA sequences that encode traits that have the potential to harm health require active deliberation. It is unlikely that DNA sequences that encode increased bacterial virulence will be incorporated into GM plants destined for food or feed usage; however, another area of potential concern is the inclusion of DNA sequences that encode antibiotic resistance. In Europe, the use of such markers is being phased out and so the associated issues will take on a lesser significance. Elsewhere in the world, the use of antibiotic resistance markers in GM plants has caused less concern on the basis that, first, there is already a very high prevalence of bacteria resistant to antibiotics in the gastrointestinal tracts of the humans and animals that are likely to consume GM plant material; and second, the very low probability of gene transfer from GM plant material to the gut microflora is unlikely to have any effect on the overall prevalence of resistance. This issue has also been the subject of recent reviews, which have concluded that the likelihood of antibiotic resistance genes escaping from GM plants and transforming gut bacteria with antibiotic resistance is slight and that the consequences are negligible given the level of resistance already present in bacteria associated with
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humans [13,14]. I have previously commented that the most significant threat to the continued use of antibiotics comes from their use and misuse in human medicine, but I also retain my concern that everyone must take responsibility to ensure that the spread of antibiotic resistance genes is minimized [15]. There is another aspect of research pertaining to the presence of antibiotic resistance genes in GM plants that has received scant attention. The administration of antibiotics might have a profound effect on the microflora of the gut [16] and provides a selective pressure for resistance to antibiotics among bacteria exposed to antimicrobial compounds. It is not known what effects the administration of antibiotics has on the potential for gene flow to humans or animals that consume GM plant material containing DNA that encodes antibiotic resistance. Does the administration of antibiotics to humans or animals feeding on transgenic plant material increase the chance that antibiotic resistance markers, or indeed other transgenic DNA, will transfer to the gut microflora? We do not know. Would silage research lead to pastures new? Much of the maize crop grown in northern Europe is used to make silage. If GM plants are used for this purpose, then there is the potential for gene flow from the GM plant material, either to the bacteria responsible for ensilage or to the microflora of animals fed on silage. Because the lactic acid bacteria responsible for silage production can act as opportunist pathogens, the potential problems caused by the ensilage of transgenic plant material do not come directly from the food chain but might arise if the bacteria from silage production were to cause infection in humans working with or animals fed on silage material. Most of the studies published on ensiled GM crops have focused on the nutritional qualities of this feed material, and none has shown a difference between GM crops and their conventional counterparts with respect to feed quality [17,18]. High molecular weight DNA survives the ensilage process and whole gene sequences are present in maize silage [6]. Large fragments of transgenic DNA are not recoverable by PCR in the rumen fluid of sheep fed on GM maize silage, although the same target can be amplified from the rumen fluid of animals fed on GM maize grains [8]. Shorter DNA sequences (211 bp) can be recovered from the rumen fluid of sheep fed on maize silage for up to 3 h after feeding. More recently, the persistence of large- or medium-sized fragments of plant DNA was found to be dependent not only on the time spent in the rumen but also on the copy number of the target gene [19]. The fate of transgenic DNA during silage production has received little research interest. Silage is a product of mixed microbial fermentation, in which lactic acid bacteria form the predominant flora [20]. Among these flora are enterococci, which might act as opportunistic human pathogens. Furthermore, these bacteria are naturally competent for genetic transformation. Consequently, ensilage provides an environment in which high molecular weight DNA from GM plants is in close proximity to a diverse population of bacteria, some of www.sciencedirect.com
Vol.23 No.1 January 2005
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which are both competent for natural transformation and opportunistic human pathogens. It is perhaps surprising that so little attention has been paid to the potential for gene flow during the ensilage of GM plant material. Wider issues: labelling what is and what is not GM The debate on using GM plants for food or feed purposes goes much wider than just scientific arguments. In Europe, strong lobbying by pressure groups is being manifested in an increase in legislation regarding the use of GM crops for food and feed purposes. In a recent article [21], Derek Burke, founder chairman of the UK Advisory Committee on Novel Foods and Processes, argues cogently against an extension of legislation with respect to GM food. A similar argument has been made with respect to the mandatory labelling of GM foods in Canada [22]. The European Commission are enacting legislation that requires all food made with products from GM plants to be so labelled. This applies to food ingredients that contain no traces of transgenic protein or DNA. The reason underpinning the legislation is that consumers should be offered informed choice. Those who wish to buy GM foods are free so to do; but people who wish to avoid consuming GM plants will also have this freedom. In my view, this legislation is unworkable because someone who fails to label Bourbon whiskey distilled from GM maize will have committed the same offence as someone who fails to label GM sweetcorn. In the latter crop, both transgenic DNA and protein will be readily detectable; in the former, no trace of the transgenic event will be found. In both cases, however, failure to label the product as ‘GM’ will mean that a fraudulent act will have been perpetrated. This situation is to be deprecated; it will be much easier to prove the latter case than the former. Another consequence of such labelling legislation is that producers who wish to avoid prosecution might well label any feed that could contain GM material as so doing to avoid prosecution, even if there is nothing of GM origin in the product. The precedent for the precautionary principle in labelling is the ‘may contain nuts’ label that warns people with peanut allergies. Where does this leave the consumer’s right to informed choice? “.this legislation is unworkable because someone who fails to label Bourbon whiskey distilled from GM maize will have committed the same offence as someone who fails to label GM sweetcorn.” Beyond labelling. The issue of labelling is even more complex than I have outlined above. So far, the products of animals fed on GM plants, including meat, milk and eggs, have been exempt from being labelled as containing GM material. The same exclusion is likely to apply also to honey from bees that have visited GM plants. Is this exclusion justified? This question is pertinent, particularly in light of the results obtained when mice were fed bacteriophage DNA (see above) [3]. Neither fragments of transgenic DNA nor the proteins encoded by transgenes have been found in poultry meat or eggs, although plant-specific DNA
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sequences have been found in the muscles, liver, spleen and kidneys of broiler chickens and laying hens [23]. Several studies using pigs or cattle have reported similar findings in animal tissues: a lack of transgenic DNA, but the presence of multicopy plant DNA sequences, particularly those derived from chloroplast genomes [24–30]. Notably, these studies have detected only short fragments of target DNA, which disappear from tissues on prolonged fasting, suggesting that these sequences are unlikely to integrate permanently into the host DNA [31]. Foreign DNA has typically not been found in eggs or milk, but there has been a report of faint signals in milk tested for the presence of chloroplast DNA [32]. When working with milk samples and when PCR is used to detect target DNA, care must be taken with the integrity of the samples to ensure, for example, that they are not contaminated with food components or with airborne feed particles that might carry the target DNA sequences [33]. The observation that small fragments of multicopy DNA sequences derived from food or feed sources might be found in the tissues of animals is not confined to plants, and studies have not been confined to animals. A recent publication reports the use of nested PCR to detect the transient presence in blood samples of rabbit mitochondrial DNA in two healthy male volunteers after the consumption of cooked rabbit [34]. Although these studies pose further questions relating to the control of DNA in our diets, they show that the uptake of DNA derived from food or feed material is a natural process that relates, at least in part, to the copy number of the DNA that is detected. It is not clear whether the failure to detect foodderived DNA that is typically present in low copy number is due to its absence in the tissues of the animals or humans who have consumed it or to the insensitivity of current assay systems. Whatever the answer to this question is, it has consequences for future trends in plant biotechnology. Although there are compelling reasons for exploiting plastid transformation systems relating to the expression of multiple copies of transgenes, there is an increased likelihood that DNA fragments associated with transgenic events will be detected in the tissues of animals or humans who eat such plants and this will have to be taken into account in any risk assessment process. This underlines the need for risk assessment to be made on a case-by-case basis, although – given that the size of DNA fragments typically reported in these studies is small – it is unlikely that a significant threat to human or animal health will be posed by such constructs if they are used for food or feed purposes. Concluding remarks Although many studies have examined the fate of transgenic DNA from GM plant material in its passage through the gastrointestinal tract, most have concentrated on the detection of DNA per se rather than on the detection of DNA that retains its biological activity. Although it is more challenging to study the latter, only when the fate of biologically active DNA is studied will we gain insight into gene flow as opposed to the passage of DNA. Much evidence indicates that DNA derived from the diet can www.sciencedirect.com
Vol.23 No.1 January 2005
be detected in animal and human tissues irrespective of whether the subjects consume GM plants. In general, however, the targets detected are too small to carry whole genes. Although the prospect of gene flow from GM plants during digestion is often cited as a cause for concern, there remains no scientific evidence to show that the presence of transgenes in our food and feedstuffs poses a threat to human or animal health. Herein I have identified areas in which knowledge could be extended and that deserve active research to further our understanding of issues that are of public concern. Even with the phasing out in Europe of the use of antibiotic resistance markers, the need to study the effects of antibiotic challenge on the potential for gene flow remains a challenge. The potential for ensilage of transgenic crops also warrants further study. The apparent transfer of plant transgenes to the microflora of the ileum, as reported by Netherwood et al. [11], is another area that is worthy of more attention. Gene flow seems to occur into bacteria that are not recoverable as discrete colonies on the solid media tested, but that are nevertheless capable of growth in liquid culture. Netherwood et al.’s study was limited in that no information was obtained regarding the nature of the DNA identified in the gut bacteria. Work has begun to address this issue and should be supported strongly. Acknowledgements I thank several colleagues who have commented critically on my manuscript.
References 1 Vogel, G. (2004) Transgenic crops – Britain opts for brave new GM world. Science 303, 1590 2 Shelton, A.M. et al. (2002) Economic, ecological, food safety, and social consequences of the deployment of Bt transgenic plants. Annu. Rev. Entomol. 47, 845–881 3 Schubbert, R. et al. (1997) Foreign (M13) DNA ingested by mice reaches peripheral leukocytes, spleen, and liver via the intestinal wall mucosa and can be covalently linked to mouse DNA. Proc. Natl. Acad. Sci. U. S. A. 94, 961–966 4 Palka-Santini, M. et al. (2003) The gastrointestinal tract as the portal of entry for foreign macromolecules: fate of DNA and proteins. Mol. Genet. Genomics 270, 201–215 5 Bauer, T. et al. (2003) The effect of processing parameters on DNA degradation in food. Eur. Food Res. Technol. 217, 338–343 6 Chiter, A. et al. (2000) DNA stability in plant tissues: implications for the possible transfer of genes from genetically modified food. FEBS Lett. 481, 164–168 7 Wilcks, A. et al. (2004) Persistence of DNA studied in different ex vivo and in vivo rat models simulating the human gut situation. Food Chem. Toxicol. 42, 493–502 8 Duggan, P.S. et al. (2003) Fate of genetically modified maize DNA in the oral cavity and rumen of sheep. Br. J. Nutr. 89, 159–166 9 Mercer, D.K. et al. (2001) Transformation of an oral bacterium via chromosomal integration of free DNA in the presence of human saliva. FEMS Microbiol. Lett. 200, 163–167 10 Martin-Orue, S.M. et al. (2002) Degradation of transgenic DNA from genetically modified soya and maize in human intestinal simulations. Br. J. Nutr. 87, 533–542 11 Netherwood, T. et al. (2004) Assessing the survival of transgenic plant DNA in the human gastrointestinal tract. Nat. Biotechnol. 22, 204–209 12 Heritage, J. (2004) The fate of transgenes in the human gut. Nat. Biotechnol. 22, 170–172 13 Bennett, P.M. et al. (2004) An assessment of the risks associated with
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the use of antibiotic resistance genes in genetically modified plants: report of the Working Party of the British Society for Antimicrobial Chemotherapy. J. Antimicrob. Chemother. 53, 418–431 Kuiper, H.A. and Kleter, G.A. (2003) The scientific basis for risk assessment and regulation of genetically modified foods. Trends Food Sci. Technol. 14, 277–293 Heritage, J. (1999) OGM alimentaires: une le´gitime re´sistance? Biofutur 192, 24 Sullivan, A. et al. (2001) Effect of antimicrobial agents on the ecological balance of human microflora. Lancet Infect. Dis. 1, 101–114 Flachowsky, G. and Aulrich, K. (2001) Nutritional assessment of feeds from genetically modified organism. J. Anim. Feed Sci. 10, 181–194 Flachowsky, G. and Aulrich, K. (2002) Food of animal origin after feeding of feeds from genetically modified plants (GMP). ErnahrungsUmschau 49, 84 Einspanier, R. et al. (2004) Tracing residual recombinant feed molecules during digestion and rumen bacterial diversity in cattle fed transgene maize. Eur. Food Res. Technol. 218, 269–273 Lin, C.J. et al. (1992) Epiphytic lactic-acid bacteria succession during the pre-ensiling and ensiling periods of alfalfa and maize. J. Appl. Bacteriol. 73, 375–387 Burke, D. (2004) The dead hand of regulation – who pays the cost of excessive regulation on GM food? Biologist 51, 63 Smyth, S. and Phillips, P.W.B. (2003) Labeling to manage marketing of GM foods. Trends Biotechnol. 21, 389–393 Chesson, A. and Flachowsky, G. (2003) Transgenic plants in poultry nutrition. World’s Poult. Sci. J. 59, 201–207 Jennings, J.C. et al. (2003) Attempts to detect transgenic and endogenous plant DNA and transgenic protein in muscle from broilers fed YieldGard Corn Borer Corn. Poult. Sci. 82, 371–380
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25 Klotz, A. et al. (2002) Degradation and possible carry over of feed DNA monitored in pigs and poultry. Eur. Food Res. Technol. 214, 271–275 26 Aumaitre, A. et al. (2002) New feeds from genetically modified plants: substantial equivalence, nutritional equivalence, digestibility, and safety for animals and the food chain. Livest. Prod. Sci. 74, 223–238 27 Reuter, T. and Aulrich, K. (2003) Investigations on genetically modified maize (Bt-maize) in pig nutrition: fate of feed-ingested foreign DNA in pig bodies. Eur. Food Res. Technol. 216, 185–192 28 Aulrich, K. et al. (2002) Novel feeds – a review of experiments at our institute. Food Res. Int. 35, 285–293 29 Chowdhury, E.H. et al. (2003) Detection of corn intrinsic and recombinant DNA fragments and Cry1Ab protein in the gastrointestinal contents of pigs fed genetically modified corn Bt11. J. Anim. Sci. 81, 2546–2551 30 Chowdhury, E.H. et al. (2004) Fate of maize intrinsic and recombinant genes in calves fed genetically modified maize Bt11. J. Food Prot. 67, 365–370 31 Tony, M.A. et al. (2003) Safety assessment of Bt 176 maize in broiler nutrition: degradation of maize DNA and its metabolic fate. Arch. Tierernahr. 57, 235–252 32 Einspanier, R. et al. (2001) The fate of forage plant DNA in farm animals: a collaborative case-study investigating cattle and chicken fed recombinant plant material. Eur. Food Res. Technol. 212, 129–134 33 Poms, R.E. et al. (2003) Model studies on the detectability of genetically modified feeds in milk. J. Food Prot. 66, 304–310 34 Forsman, A. et al. (2003) Uptake of amplifiable fragments of retrotransposon DNA from the human alimentary tract. Mol. Genet. Genomics 270, 362–368
Free journals for developing countries The WHO and six medical journal publishers have launched the Access to Research Initiative, which enables nearly 70 of the world’s poorest countries to gain free access to biomedical literature through the Internet. The science publishers, Blackwell, Elsevier, the Harcourt Worldwide STM group, Wolters Kluwer International Health and Science, Springer-Verlag and John Wiley, were approached by the WHO and the British Medical Journal in 2001. Initially, more than 1000 journals will be available for free or at significantly reduced prices to universities, medical schools, research and public institutions in developing countries. The second stage involves extending this initiative to institutions in other countries. Gro Harlem Brundtland, director-general for the WHO, said that this initiative was ’perhaps the biggest step ever taken towards reducing the health information gap between rich and poor countries’. See http://www.healthinternetwork.net for more information. www.sciencedirect.com
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Transgenes for tea? John Heritage School of Biochemistry and Microbiology, University of Leeds, Leeds, LS2 9JT, UK
So far, no compelling scientific evidence has been found to suggest that the consumption of transgenic or genetically modified (GM) plants by animals or humans is more likely to cause harm than is the consumption of their conventional counterparts. Despite this lack of scientific evidence, the economic prospects for GM plants are probably limited in the short term and there is public opposition to the technology. Now is a good time to address several issues concerning GM plants, including the potential for transgenes to migrate from GM plants to gut microbes or to animal or human tissues, the consequences of consuming GM crops, either as fresh plants or as silage, and the problems caused by current legislation on GM labelling and beyond. Despite qualified approval for the commercialization of GM crops for human food and animal feed purposes in the United Kingdom, granted in March of this year [1], largescale industrial research in this area has been severely curtailed. The reasons for this curtailment are the poor commercial prospects in Europe for the technology rather than scientifically based opposition. In the European Community, vocal opposition to GM technology in agriculture has been evident for years (A report to the EC Directorate General for Research from the project ‘Life Sciences in European Society’ QLG7-CT-1999-00286, http://europa.eu.int/comm/public_opinion/archives/eb/ ebs_177_en.pdf), as has been well illustrated by the National GM Public Debate [Genetic Modification (GM) Public Debate (2003) GM Nation? The findings of the national debate, http://www.gmnation.org.uk/docs/ gmnation_finalreport.pdf] which was held in the United Kingdom during 2003. There were three strands to the National GM Public Debate: a public consultation (http://www.gmnation.org. uk/docs/gmnation_finalreport.pdf), a review of the economic consequences of commercializing GM crops [UK Cabinet Office Strategy Unit (2003) Field work: weighing up the costs and benefits of GM crops, http://www.number-10. gov.uk/su/gm/downloads/gm_crop_report.pdf] and a review of the science underpinning the technology [GM Science Review Panel (2003) GM Science Review First Report: An open review of the science relevant to GM crops and food based on interests and concerns of the public, http://www. gmsciencedebate.org.uk/report/pdf/gmsci-report1-full.pdf]. The science review found no evidence for harm in the introduction of transgenic plants as food or feed. The economic review concluded that none of the situations examined was entirely good or bad: each required a trade-off Corresponding author: John Heritage ( [email protected]). Available online 25 November 2004
between costs in some areas and benefits in others, necessitating value judgements to be made weighing the pros and cons of introducing transgenic plants as food or feed. The public consultation found that people in the United Kingdom are generally uneasy about GM food and feed, and that the more that people engage with GM issues, the harder are their attitudes and the deeper are their concerns. Opposition to agricultural applications of GM plants is not universal. The growth of insect-resistant plants has seen the crystal toxin of Bacillus thuringiensis transformed from a foliar insecticide with limited practical applications to a toxin engineered into ‘Bt crops’ that, at the turn of the millennium, were being grown on 11.4 million hectares worldwide [2]. By 2003, the latest year for which figures were available at the time of writing, the area devoted to cultivation of these Bt crops had risen to 12.2 million hectares worldwide, and the total global area supporting the growth of transgenic plants for food or feed purposes had grown to 67.7 million hectares [Global status of commercialized transgenic crops (2003) ISAAA Briefs No. 30-2003l http://www.isaaa.org]. It has been argued that inserting the gene encoding the crystal toxin of B. thuringiensis into target plants where only pests will consume the toxin is more environmentally friendly than is spraying the toxin widely across fields and thereby exposing insects that do no damage to plants, as well as those that do, to the toxin. In this article, I focus on the potential for transgenes to migrate from GM plants to gut microbes and/or to animal or human tissues. In addition to exploring the consequences of consuming fresh GM plants, I discuss the issue of ensilage and the problems caused by current legislation on GM labelling and beyond. Cause for concern? Despite the absence of scientific evidence showing that potential harm to human or animal health is associated with the commercialization of GM plants (http://www. number-10.gov.uk/su/gm/downloads/gm_crop_report.pdf), the UK economic review revealed that any economic benefits from the application of this technology are likely to be limited, at least in the short term (http://www. number-10.gov.uk/su/gm/downloads/gm_crop_report.pdf). Furthermore, many people in Great Britain are uneasy about the use of GM plants for food or feed purposes, and the more these people engage with the topic, the more hardened become their attitudes (http://www.gmnation. org.uk/docs/gmnation_finalreport.pdf). But are there reasons for concern? One area of particular public concern is the potential for the inserted genes to migrate from GM plants to microbes in the guts of
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the animals or humans who consume the GM material; another is the possibility that these genes might associate with animal or human tissues. I consider these issues below. The potential for gene flow in the gut and beyond There has been considerable interest in the report that foreign DNA consumed in food is not degraded completely in the mouse intestinal tract [3]. When mice were fed on material containing DNA from bacteriophage M13mp18, DNA fragments of 976 bp were detected in the bloodstream for up to 8 h after feeding. By fluorescent in situ hybridization (FISH) analysis, about 1 in 1000 peripheral white blood cells were found to be carrying bacteriophage DNA in the test mice, whereas none was found in the controls. Bacteriophage DNA was observed in gut epithelial cells, in the Peyer’s patches, in liver cells, and in lymphocytes and macrophages found in the spleen [3]. More recent studies using various foreign DNA sources have confirmed that DNA sequences are taken up from the diet of humans, food animals and laboratory rodents, and that increasing the concentration of bulky dietary fibre material accelerates the passage of food through the gastrointestinal tract, speeding up the clearance of foreign DNA from the gut [4]. If foreign DNA is capable of transferring from a food source into the tissues of an animal that eats the test material, could transgenes incorporated into GM plants that are used for food or feed purposes do the same? GM foods and feeds are not a homogenous group and range from plant material that is consumed fresh and unprocessed to pure chemical compounds, such as sucrose, that are derived from GM plants. For pure compounds in which there is no trace of the GM event, there is clearly no potential for gene flow; however, the same might not be true for GM food or feed material that undergoes little or no processing. Both high temperature and low pH reduce the survival of DNA in food and feed, and these effects are additive [5,6]. The physical nature of DNA affects its chances of survival during its passage through the gut. In a study using gnotobiotic rats, plasmid DNA was recovered throughout the intestinal tract of the rats. Furthermore, the DNA recovered retained the ability to transform electrocompetent Escherichia coli cells [7], indicating that not only had it survived passage through the digestive tract but it had remained biologically active. This distinction between the detection of total DNA and DNA that retains biological activity is important because the ability to transform cells is a crucial step in potential gene flow and it is a property that is lost before the ability to amplify target DNA from samples is lost [8,9]. In vitro model studies have confirmed that transgenes in some GM foods can survive passage through the small intestine [10]. Gene transfer events to the microflora of the intestinal tracts of animals and humans eating GM plants involving transgenes are likely to be very rare events. Indeed, searching for them might be likened to seeking needles in haystacks. In this area of research, however, a recent publication stands out. Netherwood et al. [11] examined the fate of transgenic DNA in food given to human volunteers. Food containing significant amounts of GM www.sciencedirect.com
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soya was fed to people who had had an ileostomy, an operation in which the ileum is passed through the wall of the abdomen to form an artificial anus so that the contents of the gut at this point can be collected with ease. The fate of transgenic DNA in the ileostomy fluid of volunteers was examined. Although Netherwood et al. [11] concluded that gene transfer events did not occur during the feeding experiment, three of the seven volunteers showed evidence of low-frequency gene transfer events from GM soya into the small bowel microflora that had occurred before the experiment began. Transgenic DNA fragments could be amplified from bacteria cultivated in liquid culture from the ileostomy fluid at the outset of the experiment. Furthermore, bacteria containing the transgene template could be detected after six serial passages in broth culture. Intriguingly, these bacteria were not recoverable on conventional solid growth media, making further studies in this area challenging, although it is likely from the work reported so far that the bacteria are obligately symbiotic facultative Gram-positive bacteria [11]. Netherwood et al. concluded that these DNA transfer events from transgenic plants to gut microflora are highly unlikely to alter gastrointestinal function and thus do not pose a risk to human health. This is a view with which I agree completely [12]. “.these DNA transfer events from transgenic plants to gut microflora are highly unlikely to alter gastrointestinal function and thus do not pose a risk to human health.” The problem of antibiotic resistance marker genes It is not only the potential for transfer of transgenes that requires consideration when contemplating the commercialization of GM plants for food or feed purposes. What is encoded by the DNA might be also significant. In this context, DNA sequences that encode traits that have the potential to harm health require active deliberation. It is unlikely that DNA sequences that encode increased bacterial virulence will be incorporated into GM plants destined for food or feed usage; however, another area of potential concern is the inclusion of DNA sequences that encode antibiotic resistance. In Europe, the use of such markers is being phased out and so the associated issues will take on a lesser significance. Elsewhere in the world, the use of antibiotic resistance markers in GM plants has caused less concern on the basis that, first, there is already a very high prevalence of bacteria resistant to antibiotics in the gastrointestinal tracts of the humans and animals that are likely to consume GM plant material; and second, the very low probability of gene transfer from GM plant material to the gut microflora is unlikely to have any effect on the overall prevalence of resistance. This issue has also been the subject of recent reviews, which have concluded that the likelihood of antibiotic resistance genes escaping from GM plants and transforming gut bacteria with antibiotic resistance is slight and that the consequences are negligible given the level of resistance already present in bacteria associated with
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TRENDS in Biotechnology
humans [13,14]. I have previously commented that the most significant threat to the continued use of antibiotics comes from their use and misuse in human medicine, but I also retain my concern that everyone must take responsibility to ensure that the spread of antibiotic resistance genes is minimized [15]. There is another aspect of research pertaining to the presence of antibiotic resistance genes in GM plants that has received scant attention. The administration of antibiotics might have a profound effect on the microflora of the gut [16] and provides a selective pressure for resistance to antibiotics among bacteria exposed to antimicrobial compounds. It is not known what effects the administration of antibiotics has on the potential for gene flow to humans or animals that consume GM plant material containing DNA that encodes antibiotic resistance. Does the administration of antibiotics to humans or animals feeding on transgenic plant material increase the chance that antibiotic resistance markers, or indeed other transgenic DNA, will transfer to the gut microflora? We do not know. Would silage research lead to pastures new? Much of the maize crop grown in northern Europe is used to make silage. If GM plants are used for this purpose, then there is the potential for gene flow from the GM plant material, either to the bacteria responsible for ensilage or to the microflora of animals fed on silage. Because the lactic acid bacteria responsible for silage production can act as opportunist pathogens, the potential problems caused by the ensilage of transgenic plant material do not come directly from the food chain but might arise if the bacteria from silage production were to cause infection in humans working with or animals fed on silage material. Most of the studies published on ensiled GM crops have focused on the nutritional qualities of this feed material, and none has shown a difference between GM crops and their conventional counterparts with respect to feed quality [17,18]. High molecular weight DNA survives the ensilage process and whole gene sequences are present in maize silage [6]. Large fragments of transgenic DNA are not recoverable by PCR in the rumen fluid of sheep fed on GM maize silage, although the same target can be amplified from the rumen fluid of animals fed on GM maize grains [8]. Shorter DNA sequences (211 bp) can be recovered from the rumen fluid of sheep fed on maize silage for up to 3 h after feeding. More recently, the persistence of large- or medium-sized fragments of plant DNA was found to be dependent not only on the time spent in the rumen but also on the copy number of the target gene [19]. The fate of transgenic DNA during silage production has received little research interest. Silage is a product of mixed microbial fermentation, in which lactic acid bacteria form the predominant flora [20]. Among these flora are enterococci, which might act as opportunistic human pathogens. Furthermore, these bacteria are naturally competent for genetic transformation. Consequently, ensilage provides an environment in which high molecular weight DNA from GM plants is in close proximity to a diverse population of bacteria, some of www.sciencedirect.com
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which are both competent for natural transformation and opportunistic human pathogens. It is perhaps surprising that so little attention has been paid to the potential for gene flow during the ensilage of GM plant material. Wider issues: labelling what is and what is not GM The debate on using GM plants for food or feed purposes goes much wider than just scientific arguments. In Europe, strong lobbying by pressure groups is being manifested in an increase in legislation regarding the use of GM crops for food and feed purposes. In a recent article [21], Derek Burke, founder chairman of the UK Advisory Committee on Novel Foods and Processes, argues cogently against an extension of legislation with respect to GM food. A similar argument has been made with respect to the mandatory labelling of GM foods in Canada [22]. The European Commission are enacting legislation that requires all food made with products from GM plants to be so labelled. This applies to food ingredients that contain no traces of transgenic protein or DNA. The reason underpinning the legislation is that consumers should be offered informed choice. Those who wish to buy GM foods are free so to do; but people who wish to avoid consuming GM plants will also have this freedom. In my view, this legislation is unworkable because someone who fails to label Bourbon whiskey distilled from GM maize will have committed the same offence as someone who fails to label GM sweetcorn. In the latter crop, both transgenic DNA and protein will be readily detectable; in the former, no trace of the transgenic event will be found. In both cases, however, failure to label the product as ‘GM’ will mean that a fraudulent act will have been perpetrated. This situation is to be deprecated; it will be much easier to prove the latter case than the former. Another consequence of such labelling legislation is that producers who wish to avoid prosecution might well label any feed that could contain GM material as so doing to avoid prosecution, even if there is nothing of GM origin in the product. The precedent for the precautionary principle in labelling is the ‘may contain nuts’ label that warns people with peanut allergies. Where does this leave the consumer’s right to informed choice? “.this legislation is unworkable because someone who fails to label Bourbon whiskey distilled from GM maize will have committed the same offence as someone who fails to label GM sweetcorn.” Beyond labelling. The issue of labelling is even more complex than I have outlined above. So far, the products of animals fed on GM plants, including meat, milk and eggs, have been exempt from being labelled as containing GM material. The same exclusion is likely to apply also to honey from bees that have visited GM plants. Is this exclusion justified? This question is pertinent, particularly in light of the results obtained when mice were fed bacteriophage DNA (see above) [3]. Neither fragments of transgenic DNA nor the proteins encoded by transgenes have been found in poultry meat or eggs, although plant-specific DNA
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sequences have been found in the muscles, liver, spleen and kidneys of broiler chickens and laying hens [23]. Several studies using pigs or cattle have reported similar findings in animal tissues: a lack of transgenic DNA, but the presence of multicopy plant DNA sequences, particularly those derived from chloroplast genomes [24–30]. Notably, these studies have detected only short fragments of target DNA, which disappear from tissues on prolonged fasting, suggesting that these sequences are unlikely to integrate permanently into the host DNA [31]. Foreign DNA has typically not been found in eggs or milk, but there has been a report of faint signals in milk tested for the presence of chloroplast DNA [32]. When working with milk samples and when PCR is used to detect target DNA, care must be taken with the integrity of the samples to ensure, for example, that they are not contaminated with food components or with airborne feed particles that might carry the target DNA sequences [33]. The observation that small fragments of multicopy DNA sequences derived from food or feed sources might be found in the tissues of animals is not confined to plants, and studies have not been confined to animals. A recent publication reports the use of nested PCR to detect the transient presence in blood samples of rabbit mitochondrial DNA in two healthy male volunteers after the consumption of cooked rabbit [34]. Although these studies pose further questions relating to the control of DNA in our diets, they show that the uptake of DNA derived from food or feed material is a natural process that relates, at least in part, to the copy number of the DNA that is detected. It is not clear whether the failure to detect foodderived DNA that is typically present in low copy number is due to its absence in the tissues of the animals or humans who have consumed it or to the insensitivity of current assay systems. Whatever the answer to this question is, it has consequences for future trends in plant biotechnology. Although there are compelling reasons for exploiting plastid transformation systems relating to the expression of multiple copies of transgenes, there is an increased likelihood that DNA fragments associated with transgenic events will be detected in the tissues of animals or humans who eat such plants and this will have to be taken into account in any risk assessment process. This underlines the need for risk assessment to be made on a case-by-case basis, although – given that the size of DNA fragments typically reported in these studies is small – it is unlikely that a significant threat to human or animal health will be posed by such constructs if they are used for food or feed purposes. Concluding remarks Although many studies have examined the fate of transgenic DNA from GM plant material in its passage through the gastrointestinal tract, most have concentrated on the detection of DNA per se rather than on the detection of DNA that retains its biological activity. Although it is more challenging to study the latter, only when the fate of biologically active DNA is studied will we gain insight into gene flow as opposed to the passage of DNA. Much evidence indicates that DNA derived from the diet can www.sciencedirect.com
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be detected in animal and human tissues irrespective of whether the subjects consume GM plants. In general, however, the targets detected are too small to carry whole genes. Although the prospect of gene flow from GM plants during digestion is often cited as a cause for concern, there remains no scientific evidence to show that the presence of transgenes in our food and feedstuffs poses a threat to human or animal health. Herein I have identified areas in which knowledge could be extended and that deserve active research to further our understanding of issues that are of public concern. Even with the phasing out in Europe of the use of antibiotic resistance markers, the need to study the effects of antibiotic challenge on the potential for gene flow remains a challenge. The potential for ensilage of transgenic crops also warrants further study. The apparent transfer of plant transgenes to the microflora of the ileum, as reported by Netherwood et al. [11], is another area that is worthy of more attention. Gene flow seems to occur into bacteria that are not recoverable as discrete colonies on the solid media tested, but that are nevertheless capable of growth in liquid culture. Netherwood et al.’s study was limited in that no information was obtained regarding the nature of the DNA identified in the gut bacteria. Work has begun to address this issue and should be supported strongly. Acknowledgements I thank several colleagues who have commented critically on my manuscript.
References 1 Vogel, G. (2004) Transgenic crops – Britain opts for brave new GM world. Science 303, 1590 2 Shelton, A.M. et al. (2002) Economic, ecological, food safety, and social consequences of the deployment of Bt transgenic plants. Annu. Rev. Entomol. 47, 845–881 3 Schubbert, R. et al. (1997) Foreign (M13) DNA ingested by mice reaches peripheral leukocytes, spleen, and liver via the intestinal wall mucosa and can be covalently linked to mouse DNA. Proc. Natl. Acad. Sci. U. S. A. 94, 961–966 4 Palka-Santini, M. et al. (2003) The gastrointestinal tract as the portal of entry for foreign macromolecules: fate of DNA and proteins. Mol. Genet. Genomics 270, 201–215 5 Bauer, T. et al. (2003) The effect of processing parameters on DNA degradation in food. Eur. Food Res. Technol. 217, 338–343 6 Chiter, A. et al. (2000) DNA stability in plant tissues: implications for the possible transfer of genes from genetically modified food. FEBS Lett. 481, 164–168 7 Wilcks, A. et al. (2004) Persistence of DNA studied in different ex vivo and in vivo rat models simulating the human gut situation. Food Chem. Toxicol. 42, 493–502 8 Duggan, P.S. et al. (2003) Fate of genetically modified maize DNA in the oral cavity and rumen of sheep. Br. J. Nutr. 89, 159–166 9 Mercer, D.K. et al. (2001) Transformation of an oral bacterium via chromosomal integration of free DNA in the presence of human saliva. FEMS Microbiol. Lett. 200, 163–167 10 Martin-Orue, S.M. et al. (2002) Degradation of transgenic DNA from genetically modified soya and maize in human intestinal simulations. Br. J. Nutr. 87, 533–542 11 Netherwood, T. et al. (2004) Assessing the survival of transgenic plant DNA in the human gastrointestinal tract. Nat. Biotechnol. 22, 204–209 12 Heritage, J. (2004) The fate of transgenes in the human gut. Nat. Biotechnol. 22, 170–172 13 Bennett, P.M. et al. (2004) An assessment of the risks associated with
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the use of antibiotic resistance genes in genetically modified plants: report of the Working Party of the British Society for Antimicrobial Chemotherapy. J. Antimicrob. Chemother. 53, 418–431 Kuiper, H.A. and Kleter, G.A. (2003) The scientific basis for risk assessment and regulation of genetically modified foods. Trends Food Sci. Technol. 14, 277–293 Heritage, J. (1999) OGM alimentaires: une le´gitime re´sistance? Biofutur 192, 24 Sullivan, A. et al. (2001) Effect of antimicrobial agents on the ecological balance of human microflora. Lancet Infect. Dis. 1, 101–114 Flachowsky, G. and Aulrich, K. (2001) Nutritional assessment of feeds from genetically modified organism. J. Anim. Feed Sci. 10, 181–194 Flachowsky, G. and Aulrich, K. (2002) Food of animal origin after feeding of feeds from genetically modified plants (GMP). ErnahrungsUmschau 49, 84 Einspanier, R. et al. (2004) Tracing residual recombinant feed molecules during digestion and rumen bacterial diversity in cattle fed transgene maize. Eur. Food Res. Technol. 218, 269–273 Lin, C.J. et al. (1992) Epiphytic lactic-acid bacteria succession during the pre-ensiling and ensiling periods of alfalfa and maize. J. Appl. Bacteriol. 73, 375–387 Burke, D. (2004) The dead hand of regulation – who pays the cost of excessive regulation on GM food? Biologist 51, 63 Smyth, S. and Phillips, P.W.B. (2003) Labeling to manage marketing of GM foods. Trends Biotechnol. 21, 389–393 Chesson, A. and Flachowsky, G. (2003) Transgenic plants in poultry nutrition. World’s Poult. Sci. J. 59, 201–207 Jennings, J.C. et al. (2003) Attempts to detect transgenic and endogenous plant DNA and transgenic protein in muscle from broilers fed YieldGard Corn Borer Corn. Poult. Sci. 82, 371–380
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25 Klotz, A. et al. (2002) Degradation and possible carry over of feed DNA monitored in pigs and poultry. Eur. Food Res. Technol. 214, 271–275 26 Aumaitre, A. et al. (2002) New feeds from genetically modified plants: substantial equivalence, nutritional equivalence, digestibility, and safety for animals and the food chain. Livest. Prod. Sci. 74, 223–238 27 Reuter, T. and Aulrich, K. (2003) Investigations on genetically modified maize (Bt-maize) in pig nutrition: fate of feed-ingested foreign DNA in pig bodies. Eur. Food Res. Technol. 216, 185–192 28 Aulrich, K. et al. (2002) Novel feeds – a review of experiments at our institute. Food Res. Int. 35, 285–293 29 Chowdhury, E.H. et al. (2003) Detection of corn intrinsic and recombinant DNA fragments and Cry1Ab protein in the gastrointestinal contents of pigs fed genetically modified corn Bt11. J. Anim. Sci. 81, 2546–2551 30 Chowdhury, E.H. et al. (2004) Fate of maize intrinsic and recombinant genes in calves fed genetically modified maize Bt11. J. Food Prot. 67, 365–370 31 Tony, M.A. et al. (2003) Safety assessment of Bt 176 maize in broiler nutrition: degradation of maize DNA and its metabolic fate. Arch. Tierernahr. 57, 235–252 32 Einspanier, R. et al. (2001) The fate of forage plant DNA in farm animals: a collaborative case-study investigating cattle and chicken fed recombinant plant material. Eur. Food Res. Technol. 212, 129–134 33 Poms, R.E. et al. (2003) Model studies on the detectability of genetically modified feeds in milk. J. Food Prot. 66, 304–310 34 Forsman, A. et al. (2003) Uptake of amplifiable fragments of retrotransposon DNA from the human alimentary tract. Mol. Genet. Genomics 270, 362–368
Free journals for developing countries The WHO and six medical journal publishers have launched the Access to Research Initiative, which enables nearly 70 of the world’s poorest countries to gain free access to biomedical literature through the Internet. The science publishers, Blackwell, Elsevier, the Harcourt Worldwide STM group, Wolters Kluwer International Health and Science, Springer-Verlag and John Wiley, were approached by the WHO and the British Medical Journal in 2001. Initially, more than 1000 journals will be available for free or at significantly reduced prices to universities, medical schools, research and public institutions in developing countries. The second stage involves extending this initiative to institutions in other countries. Gro Harlem Brundtland, director-general for the WHO, said that this initiative was ’perhaps the biggest step ever taken towards reducing the health information gap between rich and poor countries’. See http://www.healthinternetwork.net for more information. www.sciencedirect.com
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