OGM :15 ANNI DOPO Il limite del paradigma biotecnologico e le implicazioni per la salute umana

1. OGM :15 ANNI DOPOIl limite del paradigma biotecnologico e le implicazioni per la salute umana Mariano Bizzarri Dept. Experimental Medicine, University La Sapienza SystemsBiology Group Lab

2. The SAFETY “ QUESTION“

4. THE ORIGINAL SIN • THE EXPERIMENTAL MODEL • CONFLICT of INTEREST • GENE TRANSFER • UNEXPECTED EFFECTS • HEALTH EFFECTS • TRACEABILITY MAIN CHALLENGES

5. THE ORIGINAL SIN

6. the CENTRAL DOGMA of the BIOLOGY Crick FHC (1970) The central dogma ofmolecularbiology. Nature 227:561–563 one gene one protein one function --AAA--GAG--TCC-- P H E N O T Y P E all the biological informations are stored into dna sequences GENOTYPE

7. SIGN SIGNIFICANCE MESSAGE I II III IV V Digital Information GENOTYPE decodification P H E N O T Y P E D N A TRANSLATION SPLICING SIGNAL PROTEIN TRANSDUCTION FOLDING INTRA and EXTRA-CELLULAR MODULATION E.T.Wang, et al. Nature 27, 456 (2008).

8. ONE GENE, THOUSAND of PROTEINS The sameproteinswith the same amino acid sequences can, in differentenvironments, beviewedastotallydifferentmoleculeswithdifferentphysical and chemicalproperties. S. RothmanLessonfrom the living cell: the limitsofReductionism N. York, McGraw Hill, 2002. ∼30.000 GENES PROMOTER HESONS TERMINATOR ALTERNATIVE PROCESSING 100.000 PROTEINS

9. The DNA-MANIA Genes are notworking in isolation. Their “success” doesnotlie in its code. Itlies in howthat code isinterpreted and in how the resultofthatinterpretationfitinto the overalllogicof the system. Therefore, the question “whatis a gene” needtobere-addressed A. Pichot, Histoire de la notion de gène, Paris, 1999. I discovered the gene that makesusbelievethateverythingis in the genes

10. Bothbiologicalfunctions and phenotypes are linearlydeterminedonlybygenes and gene products (proteins) Gene expressionisunrelatedtoboth the intra and the extracellular milieu Genes and genomes are stable and immutable (the onlyexceptionisrepresentedbymutation) Genesdontmovefromtheirchromosomal position Gene functionmustbeunderstoodas a part ofanintegrated network. Causualityshouldbeconsideredas a multidimensionalloop, notasmerelylinear Genes and genomes are subjectto a complex feedback regulatory network, regulatedbyboth the intra and the extra-cellular milieu Genes and genomes are “fluid” and dynamicssystems. Genes and segmentsfromgenes are moving and ableto “jump” from a side toanother (transposomes) Gens can flow horizontallybetweendifferentspecies. Recombinanteffects are likelytooccur The GENOME: opposite PARADIGMS

11. THE EXPERIMENTAL MODEL HOW TO ASK PROPERLY

12. METHODOLOGICAL INADEQUACIES LABORATORY EXPERIMENTS havebeenverylimited. There are no long-termtoxicological, oncological, neurological or reproductivestudies on GM foods. SAFETY DATA PRESENTATION IS NOT MANDATORY. Unsatisfactoryhandlingbyregulatoryagencies. NO SAFETY ASSESSMENT METHODS are fullyreliable. Inadequacyof the SubstantialEquivalencePrinciple. GENETIC CONTROL of CELL FUNCTION is VERY POOR UNDERSTOOD HUMAN EXPERIENCE with GM hasbeen VERY LIMITED no post-market surveillancestudieswererealizeduntilnow !

13. none of these previous investigations have included a detailed follow-up of the animals, including multiple (up to 11) blood and urine sampling over 2 years, and none has investigated either the GM NK603 R-tolerant maize or Roundup • Furthermore, evaluation of long-term toxicity of herbicides is generally performed on mammalian physiology employing only their active principle, rather than the complete formulations as used in agriculture • The rodent feeding studies routinely performed by industry to support regulatory authorization of their products generally last for a maximum of 90 days, a subchronic period that is too short to reveal long-term effects. • Long-term animal studies should be followed by systematic post-commercialization monitoring, in which products containing the GMO are labelled as such and the market monitored for human health impacts such as allergies, chronic toxicity, carcinogenicity, reproductive and developmental toxicity, and teratogenicity. These impacts cannot, by definition, be studied in short 90-day toxicity tests in rodents. HOW LONG SHOULD BE THE FOLLOW-UP ?

14. Indeed, it has been demonstrated that R concentrations in the range of 103 times below the MRL can induce endocrine disturbances in human cells and toxic effects thereafter • This may explain toxic effects seen in experiments in rats in vivo as well as in farm animals IS THERE ANY THRESHOLD VALUE ?

15. the conceptof “substantialequivalence” hasneverbeenproperlydefined. itisexactlythisvaguenessthatmakes the conceptusefultoindustrybutunacceptablefor the consumer

16. PLS-DA model manage to discriminate between the two kind of samples: Representation of seeds belonging to the groups maize3 and maize4 in the PCA space spanned by the score of the first and the second principal components. all the transgenic ones (maize4) have positive score values all the unmodified samples (maize3) have negative score values. Transgenic samples (maize4) Unmodified samples (maize3)

17. The study found that GM soybeans, claimed to be substantially equivalent to non-GM comparators by industry and regulators, could, with 100% accuracy, be differentiated from non-GM using only 5 of the parameters measured in the study. It was also reported that the GM soybeans contained high levels of glyphosate, whereas no such residues were present in the non-GM or organic soybeans. Also the nutritional profile of organic soybeans was superior a commercialized GM maize, MON810, had a markedly different profile of proteins compared with the isogenic non-GM counterpart when grown under the same conditions. Such differences can result in unexpected toxicity or allergenicity.

18. A studyof GM VicaFabaanincrease in estrogen levelshasbeenfound. Thatfindingraiseshealth issues – especially in infantsoyformulas. Milk fromcowswithrBHGcontainssubstantiallyhigher Levelsof pus, bacteria and fat. Monsanto’s analysisofglyphosate-resistantsoyashowed the GM-linecontained 28% more Kunitz-trypsin inhibitor, a knownanti-nutrient and allergen. Toooften the toxicityof GM foodsis untested and the potentialhazards thatthey pose tochildrenhave notbeenexamined. Nobodyhasevaluatedwhetherintra- uterine and infantexposuretoGMFs mayhaveprofoundpermanent and irreversibleconsequenceseven in adult life. GeneticallyModifiedFoods and ChildrenPotentialhealthRisks Cantani A., Micera M. Un. La Sapienza, Roma Eur RevMedPharmacol Sci 2001, 5: 25-29 CHILDREN’S HAZARD

19. CONFLICT of INTEREST CAN WE RELY ON MONSANTO DATA ?

20. NEED FOR INDEPENDENT INQUIRY

21. In an attempt to deal with this persistent criticism, EFSA instituted a new independence policy. But that did not appear to solve EFSA’s problems. A 2013 report by Corporate Europe Observatory found that over half of the agency’s experts who give opinions on the safety of GMOs, food contaminants, and additives had conflicts of interest with industry.

22. GENE TRANSFER “There is nothing known to science today to reverse the contamination already spread over two-thirds of arable US farmland and heading everywhere unless checked”.

23. GM GENES FOUND IN HUMAN GUT Netherwood T., et al. Assessing the survival of transgenic plant DNA in the human gastrointestinal tract Nat. Biotechnol. 2004, 22: 204-209. Thesefindings indicate that DNA releasedfrombacteria or foodsources (GMO) within the mouthhas the potentialtotrasformnaturallycompetentbacteria "Tomyknowledgethisstudydemonstratedclearlythatyou can get GM plant DNA in the gutbacteria. Everyoneusedtodenythatthiswasimpossible. Resultssuggeststhatyou can getantibioticmarkergenesspreadingaround the stomachwhichwould compromise antibioticresistance. This can happeneven at very low levelsafter just onemeal." M. Antonio (senior lecturer in moleculargenetics at King's College MedicalSchool, London)The GuardianJuly 17, 2004

24. we report the surprising finding that exogenous plant miRNAs are present in the sera and tissues of various animals and that these exogenous plant miRNAs are primarily acquired orally, through food intake. MIR168a is abundant in rice and is one of the most highly enriched exogenous plant miRNAs in the sera of Chinese subjects. Functional studies in vitro and in vivo demonstrated that MIR168a could bind to the human/mouse low-density lipoprotein receptor adapter protein 1 (LDLRAP1) mRNA, inhibit LDLRAP1 expression in liver, and consequently decrease LDL removal from mouse plasma. These findings demonstrate that exogenous plant miRNAs in food can regulate the expression of target genes in mammals GMOs are being engineered to make novel microRNA molecules that have never before been in the food supply, such as molecules that can kill insects or silence genes. These RNA molecules emphatically do not have a “history of safe use”.

25. UNEXPECTED EFFECTS not everything is written in the genes, after all

26. A BRIEF SURVEY

27. ROUNDUP’S ENZYMATIC-ENDOCRINE EFFECTS Roundup is always more toxic than its active ingredient. We tested the effects of glyphosate and Roundup at lower nontoxic concentrations on aromatase,the enzyme responsible for estrogen synthesis. The glyphosate-based herbicide disrupts aromatase activity and mRNA levels and interacts with the active site of the purified enzyme, but the effects of glyphosate are facilitated by the Roundup formulation in microsomes or in cell culture. We conclude that endocrine and toxic effects of Roundup, not just glyphosate, can be observed in mammals. We suggest that the presence of Roundup adjuvants enhances glyphosate bioavailability and/or bioaccumulation.

28. TOXIC SECONDARY METABOLITES In conclusion, the results of this study clearly demonstrate that the introduction of anti-invertase and maize RIP genes into potato can result in significant changes in the levels of glycoalkaloids and sesquiterpenes in tuber tissue. An observation from this study, which may be of some concern, is the high levels of total glycoalkaloids found in GM samples. Mean levels frequently reached twice the accepted threshold of 20 mg/100 g, with some individual samples exceeding 72 mg/100 g; clearly, tubers destined for human consumption containing these levels of glycoalkaloids could represent a significant health concern. Transgenic potato plants showed the expected changes in anthocyanins synthesis level. A significant decrease in anthocyanin level was observed when the plant was transformed with a corresponding antisense construct. The transformation of potato plants was also accompanied by significant changes in steroid alkaloid glycosides (SAG) level in transgenic potato tuber.

29. UNEXPECTED THREATS Terminator technology that renders seed infertile to guarantee seed corporations’ yearly sales may eventually be abused for economic warfare. If terminator crops become widespread, it would be easy for a transnational company that controls the technique to stop sales to a specific country or region for political or economic purposes. After some years of planting such seeds, only limited quantities of other seed would be available, thus agriculture could be paralyzed, leading to serious economic crisis and/or famine The Sunshine Project. An introduction to biological weapons, their prohibition, and the relationship to biosafety. April 2002. The American Society for Microbiology coded a long series of rules to identify and label agents and substances that could be used by terrorists. Among these, GMOs are in category F: GMOs are generally first and second class agents of infection (Category A [UN2814 or UN2900], Category B [UN3373]) or else substances or organisms in class 9 (miscellaneous dangerous goods) American Society of Microbiology. Sentinel Laboratory: Guidelines for suspected agents of bioterrorism, in: Biological Safety: Principles and Practice, 4th ed., Fleming D.O. & Hunt D.L. (Eds.), ASm Press, Washington DC, 2006. Just a few bushels of “pharmacorn” producing a swine vaccine could, if strategically planted by terrorists, contaminate virtually the entire US corn supply and close international markets to us for years Gilmore R. US food safety under siege? Nat Biotechnol. 2004, 22: 1503-1505.

30. HEALTH EFFECTS ACUTE and CHRONIC TOXICITIES

31. IMMUNOLOGICAL ALTERATIONS Weaning and oldmicewerefed a dietcontaining MON810 or itsparentalmaizefor 30 and 90 days. MON810 maizeinducedalterations in the percentageof T and B cells and of CD4+, CD8+, γδT, and αβT subpopulationsofrats, at the gut and peropheralsites. An increaseofserum IL-6, IL-13, IL-12p70, and MIP-1βafter MON810 feedingwasalsofound. “As both PHA-E lectin and Cry1Ab protein were capable of inducing an antigen-specific antibody response it is important to make careful considerations when designing future animal studies to avoid intake of proteins from the other groups by inhalation as well as to examine the sensitization and elicitation potential of ‘foreign’ proteins before introduction to the world market”.

32. HEPATO-RENAL TOXICITY Health risk assessment of genetically modified organisms (GMOs) cultivated for food or feed is under debate throughout the world, and very little data have been published on mid- or long-term toxicological studies with mammals. One of these studies performed under the responsibility of Monsanto Company with a transgenic corn MON863 has been subjected to questions from regulatory reviewers in Europe, where it was finally approved in 2005. We observed that after the consumption of MON863, rats showed slight but dose-related significant variations in growth for both sexes, resulting in 3.3% decrease in weight for males and 3.7% increase for females. Chemistry measurements reveal signs of hepatorenal toxicity, marked also by differential sensitivities in males and females. Triglycerides increased by 24-40% in females (either at week 14, dose 11% or at week 5, dose 33%, respectively); urine phosphorus and sodium excretions diminished in males by 31-35% (week 14, dose 33%) for the most important results significantly linked to the treatment in comparison to seven diets tested. With the present data it cannot be concluded that GM corn MON863 is a safe product.

33. TUMORS

34. Our findings show that the differences in multiple organ functional parameters seen from the consumption of NK603 GM maize for 90 days escalated over 2 years into severe organ damage in all types of test diets. This included the lowest dose of R administered (0.1 ppb,50 ng/L G equivalent) of R formulation administered, which is well below permitted MRLs in both the USA (0.7 mg/L) and European Union (100 ng/L) • Overall, our study is the first in-depth life-long toxicology study on the full commercial Roundup formulation and NK603 GM maize, with observations on 34 organs and measurement of 56 parameters analyzed at 11 time points for most organs, and utilizing 3 doses. • Surprisingly, there was also a clear trend in increased tumor incidence, especially mammary tumors in female animals, in a number of the treatment groups • Our data highlight the inadequacy of 90-day feeding studies and the need to conduct long-term (2 years) investigations to evaluate the life-long impact of GM food MAIN RESULTS

35. The major criticisms of the Seralini manuscript were that the proper strain of rats was not used and their numbers were too small. Neither criticism is valid. The strain of rat is that required by the FDA for drug toxicology, and the toxic effects were unambiguously significant. In fact, Monsanto published a similar study in the same journal eight years before using the same number and strain of rats. Their study was for 90 days and claimed no harm. In contrast, the Seralini study was for two years and did not see any tumors until after nine months. Therefore, it is clear that the short 90-day feeding paradigm is not sufficiently long to detect the carcinogenic effects of GM products. It takes a long time before low-level exposure to environmental toxins affect health. For example, a recent Associated Press report documented the dramatic increase in birth defects and cancer in areas of Argentina that have grown GM soy for a decade. Given these facts, what was the justification for the editorial decision to retract the Seralini manuscript? • The editors claim the reason was that “no definitive conclusions can be reached.” As a scientist, I can assure you that if this were a valid reason for retracting a publication, a large fraction of the scientific literature would not exist • However, before the retraction of the Seralini paper, a former Monsanto scientist was brought into the journal as biotechnology editor. Therefore, a combination of intense pressure from scientists and a new pro-industry editor led to the elimination of an exceptionally important study. • I am convinced that there is significant evidence, like that presented by Seralini, that some GM foods are hazardous to human health. In order for data supporting this possibility to enter public discourse, scientists must place their ethical responsibilities above corporate profits and cease their continual assault on the science relating to GM safety. The protection of scientists’ right to publish their findings without censorship or retribution must be preserved. Schubert, Ph.D., is a professor with the Salk Institute for Biological Studies.

36. ....silence like cancer grows

37. TRACEABILITY WHY THE US CONTRASTS TRACEABILITY?

38. “ I want to know — quite technically, in all the detail available — how my food is produced, and I’m far from alone. We’d be able to make saner choices, and those choices would greatly affect Big Food’s ability to freely use genetically manipulated materials, an almost unlimited assortment of drugs and inhumane and environmentally destructive animal-production methods “ “ I WANT TO KNOW “

39. The Transatlantic Trade and Investment Partnership (TTIP) is being negotiated among European governments, the US and Canada, with the active participation of dozens of large businesses. It has already attracted strong criticism from democracy campaigners, who say it could mean the UK could have to open the National Health Service further to private companies, and complaints against large companies could be treated in secret without proper legal recourse. • The European commission has strongly denied that the partnership would allow North American companies to circumvent EU food standards, particularly with regard to genetic modification • However, documents from various US and Canadian government agencies and business trade bodies suggest strong pressure is being brought to bear from US industries to allow GM products and other foods into EU markets that would violate the EU’s current standards, in the name of free trade A NEW THREAT: the TTIP

40. DELEGITTIMAREL’AVVERSARIO Profeti inascoltati

41. The economic benefits of BtG. hirsutum over non-BtG. hirsutum are well documented and include increase in yields, increase in farmers' net revenue • However, it is unclear to what extent irrigation influences the performance of BtG. hirsutum on smallholder farming in India, and if, in the absence of irrigation, growing BtG. hirsutum provides greater economic benefits. • we compare the economic impact of growing BtG. hirsutum with growing G. arboreum under rainfed conditions in the Indian state of Maharashtra, and show that G. arboreum can generate similar net revenue, and thus similar economic benefits for smallholder farmers compared with growing BtG. hirsutum. • even though BtG. hirsutum yields increase with irrigation, the net revenue does not significantly increase because farmers using irrigation spend significantly more than farmers growing BtG. hirsutum without irrigation MAINSTREAM HYPOCRISY

42. A vast scientific literature has raised specific questions that governments, companies and scientists have to answer. Indignant tirades and partisan speculations are unacceptable. Transgenic supporters’ accusations of unscientific arguments should be returned to the sender, and the discussion resumed on an equal footing of mutual attention and respect, without prejudice or ideological preconceptions CONCLUSIONS

43. DISTRUGGERE L’IDENTITÀ “In parole povere Bruxelles decide che per adeguarci alle schifezze in uso negli altri Paesi europei dobbiamo permettere anche noi la produzione del formaggio «zero latte». E per raggiungere questo bel risultato la Commissione Ue si è presa il disturbo e la fatica di mandarci una lettera ufficiale di messa in mora per infrazione. Del resto Bruxelles ha già dato via libera al cioccolato senza cacao, al vino senza uva e alla carne annacquata Non è così che si aumenta il prestigio e la legittimità delle istituzioni europee”. La Stampa 27 Giugno 2015