Genetically Modified Organisms in Food: Production, Safety, Regulation and Public Health

Genetically Modified Organisms in Food

Production, Safety, Regulation and Public Health

Ronald Ross Watson

Mel and Enid Zuckerman College of Public Health, Health Promotion Sciences Division, University of Arizona, Tucson, AZ, USA

Victor R. Preedy

Department of Nutrition and Dietetics, King’s College London, London, UK

Table of Contents

Cover image

Title page

Copyright

Contributors

Preface

Acknowledgments

Section I. Development, Testing and Safety of Plant and Animal GMO foods

Chapter 1. Soybean as a Food Source: Comparative Studies Focusing on Transgenic and Nontransgenic Soybean

Introduction

Some Comments About Transgenic Soybeans

Comparative Studies Involving Transgenic and Nontransgenic Soybean Seeds or Plants

Trends

Conclusions

Chapter 2. Genetically Modified Crops: Biosafety Regulations and Detection Strategies

Introduction

Biosafety Measurement

Labeling Issues

Bt Gene and Stacked Traits

Detection Strategies

PCR and Real-Time PCR

Biosensors

Protein-Based Detection

Immunoassays

Immunostrip

Immuno-PCR

Future Prospects

Chapter 3. Genetically Modified Food Animals: An Overview

Introduction

Genetically Modified Organisms

Applications of Transgenic Animals

Concerns

Future Directions

Chapter 4. Genetically Modified Aubergine (Also Called Brinjal or Solanum melongena)

General Description of Brinjal

Biochemical and Nutritional Properties

Insect Pests of Brinjal

Development of Insect-Resistant Bt-Brinjal

Fruit and Shoot Borer Management in Bt-Brinjal

Fungal-Resistant Dm-AMP1-Aubergine Plants

Detection of Bt-Brinjal

Current Regulatory Framework of India for Recombinant DNA Technology

Food Safety Assessment of Bt-Brinjal

Environmental Risk Assessment of Bt-Brinjal

Commercialization of Bt-Brinjal

Chapter 5. Nutritional Assessment of Genetically Modified Crops Using Animal Models

Crop Composition, Nutritional Context, and the Suitability of Animal Studies

History of Animal Studies for Nutritional Assessment of Genetically Modified Crops

Regulatory Assessments

Conclusion

Chapter 6. Noncoding RNA-Based Genetically Modified Crops: Concepts and Challenges

Introduction

Various ncRNA-Based Silencing Platforms

Apprehensions of Noncoding RNA-Based Genetically Modified Crops

Persistence of ncRNAs

Predictive Environmental Risk Assessment

Impact on Plant Protection Measures

Food and Feed Safety

Nutritional Composition and Equivalency

Conclusions and Future Directions

Chapter 7. Agrobacterium-Mediated Alien Gene Transfer Biofabricates Designer Plants

The Biology of Agrobacterium

Agrobacterium-Mediated T-DNA Transfer Process

Comparison of T-DNA Transfer to Conjugative DNA Transfer

Factors Influencing Agrobacterium-Mediated Transformation

Advances in Agrobacterium-Mediated Transformation

Applications

Biosafety Aspects

Summary and Future Prospects

Chapter 8. Understanding the Factors Influencing Attitudes toward Genetically Modified Rice

Introduction

Theoretical Framework and Hypotheses Development

Research Methodology

Results

Conclusion

Chapter 9. Qualitative and Quantitative Diagnostics for EE1 Event of Bt Eggplant

Introduction

Screening Strategies for Bt Eggplant

Qualitative Analysis of EE1 Event

Diagnostics for EE1 Event: To Ensure GM-Free Conservation of Germplasm

Conclusion

Chapter 10. Biosensors for Detection of Genetically Modified Organisms in Food and Feed

Introduction

Standard Approaches for Detection of GMO

GMO Biosensors

Chapter 11. Genetically Modified Organism Analysis as Affected by DNA Degradation

Introduction

DNA Quality and Purity: From Extraction to PCR Analysis

Effect of Food Processing on GMO Detection

Final Remarks

Chapter 12. Novel Strategies for Genetically Modified Organism Detection

Introduction

Biosensors

Microarrays

Alternative DNA Amplification Methods

Final Remarks

Chapter 13. Targeted Genetic Modification in Crops Using Site-Directed Nucleases

Introduction

Different Types of SDNs: From Meganucleases to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

The Different Uses of SDNs

What Has Already Been Done in Plants?

How the Nuclease is Delivered in Plant Cells

Comparison of SDN-Based Approaches to Currently Used Techniques in Plant Breeding

SDNs to Obtain New Plants for Commercial Uses: Legal Framework

Conclusions

Section II. Social and Economic Context of GMO Foods

Chapter 14. Agricultural Biotechnology and Public Attitudes: An Attempt to Explain the Mismatch between Experience and Perception

Introduction

Overview on Perception, Policies, and Politics on GM Crops in Europe, USA, and the Rest of the World

Public Attitudes and Trust in Institutions

The Framing of the Public Debate

Methodologies to Assess Public and Consumer Attitudes

Why Concrete Experience with the Technology Matters

Concluding Remarks

Chapter 15. Fishy Business: Genetic Engineering and Salmon Aquaculture

Introduction

Salmon Decline: From Wild to Farmed Fish

The Social and Economic Context of Genetically Modified Salmon

Genetically Modified Salmon: A Smaller Ecological Footprint?

Conclusion

Chapter 16. Consumer Behavior Regarding Genetically Modified Foods: A Mediator Model

Introduction

Theoretical Framework: A Mediator Model

Research Methods

Results: The Mediator Model

Conclusions

Chapter 17. Are Ready for Market Genetically Modified, Conventional and Organic Soybeans Substantially Equivalent as Food and Feed?

Introduction

Materials and Methods

Results

Discussion

Conclusion

Section III. Government Regulation and Litigation for GMO foods

Chapter 18. Consumer Acceptance and Willingness-to-Pay for Genetically Modified Foods with Enhanced Vitamin Levels

Introduction

Methods

Results

Case Study: Information Effects on WTP for Folate Biofortified Rice

Conclusions

Chapter 19. Detection of Genetically Modified Organisms in Feed

Introduction

GMO Feeds: General Characteristics, Regulations, and Health Issues

Detection Methods of Genetically Modified Organisms in Feed

Molecular Analysis

Conclusions

Chapter 20. Development of Molecular Strategies for Gene Containment and Marker-Free Genetically Modified Organisms

The Current Market Situation of Genetically Modified Organisms

Concerns about the Potential Problems of GMOs

Gene Containment

SMG and SMG Protein Free in GMOs

Conclusion

Chapter 21. Negative Regulators of Messenger RNA and the Role of microRNA for Plant Genetic Engineering

Introduction

Biogenesis and Mechanisms of Negative Regulators of mRNA

Roles of miRNA in Plant Genetic Modification

MiRNA-Based Gene Silencing Strategies in Plant Genetic Modification

Concluding Remarks

Chapter 22. Genetically Modified Organisms and European Journalism

Introduction

The European Context and GMOs

GMOs and Competing Journalistic Traditions of Journalistic Objectivity and Interpretation

The Challenge of Reporting on GMOs: Facts and Scientific Disputes

Conclusion

Chapter 23. Agricultural Biotechnology Regulation and Litigation: Preventing Contamination

Regulatory Background

Litigation Over Biotech Crops

Chapter 24. The European Union Reference Methods Database and Decision Supporting Tool for the Analysis of Genetically Modified Organisms: GMOMETHODS and JRC GMO-Matrix

The From Farm to Fork European Approach

Harmonized Analytical Approaches for GMO Detection: Role of EU-RL GMFF and the European Network of GMO Laboratories (ENGL)

GMOMETHODS Database: Data Source and Selection Criteria

The GMOMETHODS Database Content

Joint Research Center (JRC) GMO-Matrix

The Central Core Sequence Information System

The in silico Pipeline Supporting the JRC GMO-Matrix

Practical Considerations

Next Generation Sequencing

Challenges

Future Prospective for GMO Analysis

NGS

Conclusions

Section IV. Role of GMC (Genetically Modified Crops) in Increasing the Food Supply in the Developing and Developed Countries

Chapter 25. Genetically Modified Crops: An Alternative Source of Livestock Feeding

Introduction

Historical Background of GM Crops

What is a GM Crop?

Risks and Concerns of GM Crops

Feeding GM Crops in Livestock

Conclusions and Future Prospectives

Chapter 26. Transgenic Food: Uncertainty, Trust, and Responsibility

Introduction

Conclusion

Chapter 27. Engineering Stress Tolerance in Peanut (Arachis hypogaea L.)

Introduction

Factors Affecting Peanut Production

Approaches to Studying Stress Tolerance in Peanut

Enhanced Stress Tolerance in Peanut: A Case Study

Issues with the Transgenic/GMO

Conclusion and Future Prospective

Chapter 28. Why India Needs Biotechnology to Ensure Food and Nutrition Security?

Introduction

Population versus Agricultural Productivity

Nutrition Security

Cotton

Rice

Hybrid Rice

Marker Assisted Selection

Transgenic Rice

Rice Genomics

Horticulture

Enhancement of Nutrition Content

Oilseeds

Pulses

Conclusions

Chapter 29. Wheat Storage Proteins in Transgenic Rice Endosperm

Introduction

Section V. Potential Health Benefits, Acceptance and Risks due to Incorporation of Novel Plant Gene Products into the Food Supply

Chapter 30. Event-Specific Identification Technology of Genetically Modified Organisms

Introduction

The Traditional Methods of Genome Walking

The Novel Methods: A-T Linker PCR

Loop-Linker PCR

Randomly Broken Fragment PCR with 5′ End-Directed Adaptor for Genome Walking

Next-Generation Sequencing in Obtaining the Molecular Characterization of Genetically Modified Organisms

Prospect

Chapter 31. The Detection Techniques of Genetically Modified Organisms

Introduction

Qualitative PCR Detection Techniques

Quantitative PCR

Enzyme-Linked Immunosorbent Assay Detection Technique

High-Throughput Detection Techniques

Loop-Mediated Isothermal Amplification

Prospect

Chapter 32. Carotenoids, Genetically Modified Foods, and Vitamin A Nutrition

Introduction

Strategies for Metabolic Engineering in the Carotenoid Pathway

Assessing the Impact of Vitamin A Biofortification on Human Nutrition

Genetic Engineering of Carotenoid Accumulation in Cereal Grains

Genetic Engineering of Carotenoid Accumulation in Oilseeds and Legumes

Genetic Engineering of Carotenoid Accumulation in Stem and Tuber Crops

Genetic Engineering of Carotenoid Accumulation in Vegetables and Fruits

Future Perspectives

Chapter 33. Food from Genetically Engineered Plants: Tomato with Increased β-Carotene, Lutein, and Xanthophylls Contents

Introduction

Genetically Engineered Food from Tomato

Carotenoids Metabolic Engineering in Tomato

Final Comments

Chapter 34. Plant Defensins for the Development of Fungal Pathogen Resistance in Transgenic Crops

Introduction

Plant Defensins: Origin and Distribution in Plants

Plant Defensins: Structure

Plant Defensins: Mechanism of Action

Plant Defensins: Role in Normal Growth and Development

Engineering Fungal Resistance in Transgenic Plants Using Plant-Derived Defensins

Combinatorial Strategy Approach Using Plant Defensins

Tissue Specific Overexpression of Plant Defensins

Targeted Modification of Native Defensins for Improved Antimicrobial Activity

Biosafety Issues Regarding the Use of Plant Defensins

Future Prospects

Chapter 35. Bioinformatics Application in Regulatory Assessment for Potential Allergenicity of Transgenic Proteins in Food Crops

Introduction

Allergen Databases

Bioinformatics Methods to Predict Protein Allergenicity

Bioinformatics Methods Used in Regulatory Assessment of Protein Allergenicity

Conclusion

Chapter 36. Attitudes of Polish Adolescents toward Genetically Modified Organisms and Genetically Modified Food

Introduction

Objective

Materials and Method

Results

Discussion

Conclusion

Chapter 37. Consequences of Gene Flow between Transgenic, Insect-Resistant Crops and Their Wild Relatives

Introduction

Introgression

Evolutionary and Agricultural Consequences of Introgression of Insect-Resistance Genes

Conclusion

Section VI. Safety of Genetically Modified Foods for Humans and Animals

Chapter 38. Labeling of Genetically Modified Food in the United States

Introduction

The Current Biotechnology Debate

The Regulatory Environment of GM Food Labeling Policies in the United States

Conclusions

Chapter 39. The Food Safety Assessment of Bt Crops

Introduction

History of Safe Use of Bt Microbial Pesticides

Insecticidal Activity of Bt Cry Proteins

History of Safe Use and Human Dietary Exposure Assessment of Cry Proteins

Regulatory Guidance for the Safety Assessment of Cry Proteins Introduced into GM Crops

Digestibility of Cry Proteins When Consumed in the Diet

Assessment of Food Processing on Cry Protein Insecticidal Activity

Allergy and Immunogenicity Assessment of Cry Proteins

Toxicology Testing of Cry Proteins

Toxicology Feeding Studies in Rodents Fed Bt Crops

Food and Feed Safety Benefits of Bt Crops

Conclusions

Chapter 40. Allergen Analysis in Plants and Use in the Assessment of Genetically Modified Plants

Introduction

Molecular Properties of Food Allergens

Protein Families of Food Allergens and Allergen Databases

Applications of Allergenomics for Assessment of Allergenicity in GM Plants

Conclusions

Section VII. Demand and Uses of Non-Genetically Modified Foods, and GMO’s for Humans and Animals

Chapter 41. Usage of Genetically Modified Foods: The Extent of Genetically Modified Rice, Maize, and Soy Consumption in Saudi Arabia

Introduction

Materials and Methods

Results and Discussion

Conclusion

Chapter 42. Reasons Analysis of Chinese Urban Consumers Opposing Genetically Modified Food—An Overview

Introduction

Reason Analysis

Discussion

Recommendations

Index

Copyright

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Contributors

Philipp Aerni,     Center for Corporate Responsibility and Sustainability (CCRS), University of Zurich, Switzerland

A.I. Al-Turki,     Al-Qassim University, Department of Plant Production and Protection, Qassim, Kingdom of Saudi Arabia

Joana S. Amaral

REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal

ESTiG, Instituto Politécnico de Bragança, Bragança, Portugal

Latifah Amin,     Pusat Citra Universiti, Universiti Kebangsaan Malaysia, Selangor, Malaysia

Alexandre Angers-Loustau,     Molecular Biology and Genomics Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Ispra, Italy

Şule Arı

Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, Istanbul, Turkey

Research and Application Center for Biotechnology and Genetic Engineering, Istanbul, Turkey

M.A.Z. Arruda

University of Campinas–Unicamp, Institute of Chemistry, National Institute of Science and Technology for Bioanalytics, Campinas, Brazil

University of Campinas–Unicamp, Department of Analytical Chemistry, Campinas, Brazil

S.C.C. Arruda,     University of São Paulo, Department of Genetics, Laboratory of Genetics Biochemistry of Plants, ESALQ, Piracicaba, Brazil

Mary A. Arugula,     Auburn University, Department of Materials Engineering, Auburn, AL, USA

Chenna R. Aswath,     Division of Biotechnology, Indian Institute of Horticultural Research, Bangalore, India

R.A. Azevedo,     University of São Paulo, Department of Genetics, Laboratory of Genetics Biochemistry of Plants, ESALQ, Piracicaba, Brazil

Ferenc Békés,     FBFD Pty. Ltd., Beecroft, NSW, Australia

Dieter Blancquaert,     Ghent University, Laboratory of Functional Plant Biology, Department of Physiology, Ghent, Belgium

T. Bøhn

GenØk – Centre for Biosafety, Tromsø, Norway

UiT The Arctic University of Norway, Faculty of Health Sciences, Tromsø, Norway

Laura Bonfini,     Molecular Biology and Genomics Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Ispra, Italy

Jeroen Buysse,     Ghent University, Department of Agricultural Economics, Faculty of Bioscience Engineering, Ghent, Belgium

Özgür Çakır,     Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, Istanbul, Turkey

Josep M. Casacuberta,     Center for Research in Agricultural Genomics, CRAG (CSIC-IRTA-UAB-UB), Campus UAB, Cerdanyola del Vallès Barcelona, Spain

Sandip Chakraborty,     Animal Resources Development Department, Agartala, Tripura, India

Amit Kumar Chaturvedi,     Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar, Gujarat, India

Ilaria Ciabatti,     Biotechnologies Unit, DG SANTE, European Commission, Bruxelles, Belgium

Brett Clark,     University of Utah, Department of Sociology, Salt Lake City, UT, USA

Rebecca Clausen,     Fort Lewis College, Department of Sociology, Durango, CO, USA

Steve Cogill,     Clemson University, Department of Genetics and Biochemistry, Clemson, SC, USA

Cécile Collonnier,     INRA AgroParisTech, IJPB, UMR 1318, INRA Centre de Versailles, Versailles, France

Joana Costa,     REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal

M. Cuhra

GenØk – Centre for Biosafety, Tromsø, Norway

UiT The Arctic University of Norway, Faculty of Health Sciences, Tromsø, Norway

Caterina D’Ambrosio,     Centro Ricerche Metapontum Agrobios, ALSIA, Metaponto (MT), Italy

Henri Darmency,     Institut National de la Recherche Agronomique, UMR1347 Agroécologie, Dijon, France

B.K. de Campos

University of Campinas–Unicamp, Institute of Chemistry, National Institute of Science and Technology for Bioanalytics, Campinas, Brazil

University of Campinas–Unicamp, Department of Analytical Chemistry, Campinas, Brazil

Hans De Steur,     Ghent University, Department of Agricultural Economics, Faculty of Bioscience Engineering, Ghent, Belgium

Rajib Deb,     ICAR-Central Institute for Research on Cattle, Meerut, Uttar Pradesh, India

Cristina Delerue-Matos,     REQUIMTE, Instituto Superior de Engenharia do Porto, Instituto Politécnico do Porto, Porto, Portugal

Meenakshi Dwivedi,     Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India

R.D. Ekmay,     Dow AgroSciences LLC., Indianapolis, IN, USA

Rafaat M. Elsanhoty,     El-Sadat City University, Institute of Genetic Engineering and Biotechnology, Department of Industrial Biotechnology, Branch of Food and Dairy Biotechnology, Sadat City, Egypt

J. Fagan,     Earth Open Source Institute, Fairfield, IA, USA

Theodore A. Feitshans,     North Carolina State University, Raleigh, NC, USA

Shuyi Feng,     Nanjing Agricultural University, College of Public Administration, Weigang, Nanjing, China

Telmo J.R. Fernandes,     REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal

R.M. Galazzi

University of Campinas–Unicamp, Institute of Chemistry, National Institute of Science and Technology for Bioanalytics, Campinas, Brazil

University of Campinas–Unicamp, Department of Analytical Chemistry, Campinas, Brazil

Megan R. Galey,     Husch Blackwell LLP, St. Louis, MO, USA

Thumballi R. Ganapathi,     Plant Cell Culture Technology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India

Shaoping Gan,     Philosophy Institution of Chinese Academy of Social Science, Beijing, China

Francesco Gatto,     Molecular Biology and Genomics Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Ispra, Italy

Xavier Gellynck,     Ghent University, Department of Agricultural Economics, Faculty of Bioscience Engineering, Ghent, Belgium

Siddhesh B. Ghag,     Plant Cell Culture Technology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India

Giovanni Giorio,     Centro Ricerche Metapontum Agrobios, ALSIA, Metaponto (MT), Italy

Lalitha R. Gowda,     Chief Scientist (Former), CSIR-Central Food Technological Research Institute, Department of Protein Chemistry and Technology, Mysore, Karnataka, India

Vinod Goyal,     University of Delhi, Delhi, India

Liliana Grazina,     REQUIMTE, Departamento de Ciências Químicas, Universidade do Porto, Porto, Portugal

Shishir K. Gupta,     Indian Veterinary Research Institute, Izatnagar, Bareilley, Uttar Pradesh, India

Shishir Kumar Gupta

Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India

Indian Veterinary Research Institute, Izatnagar, Bareilley, Uttar Pradesh, India

Bruce Hammond,     Private Consultant, St Charles, MO, USA

Hasrizul Hashim,     Pusat Citra Universiti, Universiti Kebangsaan Malaysia, Selangor, Malaysia

R.A. Herman,     Dow AgroSciences LLC., Indianapolis, IN, USA

M.A. Herrera-Agudelo

University of Campinas–Unicamp, Institute of Chemistry, National Institute of Science and Technology for Bioanalytics, Campinas, Brazil

University of Campinas–Unicamp, Department of Analytical Chemistry, Campinas, Brazil

Anita Howarth,     Brunel University London, Kingston Lane, Uxbridge, UK

Bhavanath Jha,     Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar, Gujarat, India

Anna Jurkiewicz,     Institute of Rural Health, Department of Public Health, Lublin, Poland

Suchitra Kamle,     Shanghai Jiao Tong University, School of Pharmacy, Shanghai, China

Małgorzata Karbarz,     University of Rzeszów, Institute of Applied Biotechnology and Basic Sciences, Kolbuszowa, Poland

Joachim Kreysa,     Molecular Biology and Genomics Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Ispra, Italy

P.U. Krisnaraj,     College of Agriculture, Department of Agricultural Microbiology, Bijapur, India

Willy Lambert,     Ghent University, Laboratory of Toxicology, Department of Bioanalysis, Ghent, Belgium

Dawei Li,     Shanghai Jiao Tong University, School of Pharmacy, Shanghai, China

Antoon Lievens,     Molecular Biology and Genomics Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Ispra, Italy

Stefano B. Longo,     North Carolina State University, Department of Sociology and Anthropology, Raleigh, NC, USA

Hong Luo,     Clemson University, Department of Genetics and Biochemistry, Clemson, SC, USA

Isabel Mafra,     REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal

Lucia Martinelli,     MUSE – Science Museum, Trento, Italy

Yue Ma,     Graduate School of Chinese Academy of Social Science, Beijing, China

Shweta Mehrotra,     University of Delhi, Delhi, India

Liliana Meira,     REQUIMTE, Departamento de Ciências Químicas, Universidade do Porto, Porto, Portugal

Sinan Meriç

Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, Istanbul, Turkey

Kultur University, Faculty of Science and Letters, Department of Molecular Biology and Genetics, Istanbul, Turkey

Avinash Mishra,     Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar, Gujarat, India

Fabien Nogué,     INRA AgroParisTech, IJPB, UMR 1318, INRA Centre de Versailles, Versailles, France

Maria Beatriz P.P. Oliveira,     REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal

Maria Oszvald,     Eotvos Lorand University, Department of Plant Physiology and Molecular Plant Biology, Budapest, Hungary

Govindarajan Padmanaban,     Indian Institute of Science, Department of Biochemistry, Bangalore, India

Renu Pandey,     Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India

S. Papineni,     Dow AgroSciences LLC., Indianapolis, IN, USA

Vincenzo Pavone,     Institute of Public Goods and Policies (IPP-CSIC), Madrid, Spain

Mauro Petrillo,     Molecular Biology and Genomics Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Ispra, Italy

Alexandra Plácido

REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal

REQUIMTE, Instituto Superior de Engenharia do Porto, Instituto Politécnico do Porto, Porto, Portugal

Shelly Praveen,     Indian Agricultural Research Institute, Advanced Centre for Plant Virology, Division of Plant Pathology, New Delhi, India

T.V. Raja,     ICAR-Central Institute for Research on Cattle, Meerut, Uttar Pradesh, India

Mohamed Fawzy Ramadan

Zagazig University, Agricultural Biochemistry Department, Zagazig, Egypt

Umm Al-Qura University, Institute of Scientific Research and Revival of Islamic Heritage, Makkah, Kingdom of Saudi Arabia

S.V. Ramesh,     Indian Council of Agricultural Research-ICAR, Directorate of Soybean Research, Indore, India

Gurinder J. Randhawa,     Division of Genomic Resources, Indian Council of Agricultural Research (ICAR)-National Bureau of Plant Genetic Resources, New Delhi, India

Thomas P. Redick,     Global Environmental Ethics Counsel LLC, Clayton, MO, USA

Macario Rodríguez-Entrena,     IFAPA-Institute of Agricultural Research and Training, Department of Agricultural Economics and Rural Studies, Alameda del Obispo Center, Córdoba, Spain

Sabrina Rosa,     Molecular Biology and Genomics Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Ispra, Italy

Melania Salazar-Ordóñez,     Universidad Loyola Andalucía, Department of Economics, Córdoba, Spain

Daman Saluja,     Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India

Rie Satoh,     Analytical Science Division, National Food Research Institute, National Agriculture and Food Research Organization Tsukuba, Ibaraki, Japan

Ying Shang

China Agricultural University, Laboratory of Food Safety, College of Food Science and Nutritional Engineering, Beijing, China

The Supervision, Inspection & Testing Center of Genetically Modified Food Safety, Ministry of Agriculture, Beijing, China

Alex L. Simonian,     Auburn University, Department of Materials Engineering, Auburn, AL, USA

Upendra K. Singh Shekhawat,     Plant Cell Culture Technology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India

Monika Singh,     Division of Genomic Resources, Indian Council of Agricultural Research (ICAR)-National Bureau of Plant Genetic Resources, New Delhi, India

Umesh Singh,     ICAR-Central Institute for Research on Cattle, Meerut, Uttar Pradesh, India

Ping Song,     Dow AgroSciences LLC, Indianapolis, IN, USA

Adriana L. Stigliani,     Centro Ricerche Metapontum Agrobios, ALSIA, Metaponto (MT), Italy

Christophe Stove,     Ghent University, Laboratory of Toxicology, Department of Bioanalysis, Ghent, Belgium

Simon Strobbe,     Ghent University, Laboratory of Functional Plant Biology, Department of Physiology, Ghent, Belgium

László Tamás,     Eotvos Lorand University, Department of Plant Physiology and Molecular Plant Biology, Budapest, Hungary

Reiko Teshima,     Division of Foods, National Institute of Health Sciences, Setagaya, Tokyo, Japan

Li Tian,     University of California, Department of Plant Sciences, Davis, CA, USA

T. Traavik

GenØk – Centre for Biosafety, Tromsø, Norway

UiT The Arctic University of Norway, Faculty of Health Sciences, Tromsø, Norway

Dominique Van Der Straeten,     Ghent University, Laboratory of Functional Plant Biology, Department of Physiology, Ghent, Belgium

Caterina Villa,     REQUIMTE, Departamento de Ciências Químicas, Universidade do Porto, Porto, Portugal

Anton E. Wohlers,     Cameron University, Academic Enrichment, Lawton, OK, USA

Wentao Xu

China Agricultural University, Laboratory of Food Safety, College of Food Science and Nutritional Engineering, Beijing, China

The Supervision, Inspection & Testing Center of Genetically Modified Food Safety, Ministry of Agriculture, Beijing, China

Ning Yuan,     Clemson University, Department of Genetics and Biochemistry, Clemson, SC, USA

Shuangrong Yuan,     Clemson University, Department of Genetics and Biochemistry, Clemson, SC, USA

Preface

Recent media reports suggest that 70% of production in USA is by GMO (genetically modified organisms) plants and animals, principally corn.

Section I: Development, Testing and Safety of Plant and Animal genetically modified crops as foods

This book focuses on scientific evaluation of published research relating to general responses by public health agencies to new GMO food products to assert their safety as well as potential health risks. The initial section discusses production and safety of GMO. Therefore comparative studies on transgenic and standard soybeans are evaluated followed by discussions of biosafety regulations and detection strategies. GMO plants as food have been evaluated for nutrition using animal models. GMO plants based upon noncoding RNAs help understand the concepts and challenges of GMO. It is clear that some aspects of GMO affect people’s use so the factors influencing use of genetically modified rice are discussed. New and improved methods to detect GMO in food and feed are needed including biosensors, DNA degradation, and other novel strategies in three chapters.

Section II: Social and Economic Context of GMO Foods

GMO technology has doubled the yields of some crops which is huge in its effects on food availability and practice of agriculture with public health implications for areas with many inadequately fed peoples. However concerns about newly introduced nonfood genes into foods have resulted in General Mills, a major breakfast cereal food producer in the USA announcing in 2014 that its classic Cheerios breakfast cereal will be GMO free. The market could be the 20% of Americans with concerns about GMO foods, double what it was a decade ago. Half of the American states had bills introduced to require GMO labeling of foods, but all failed. The non-GMO breakfast cereal is possible because most of Cheerios is oats and noncorn sources will be used for sweetening. Yet the rest of General Mills other foods will contain GMO products due to economic and availability reason. Therefore what are the economic, nutrition, food availability, and risks/benefits of using these GMO crops as foods? Therefore Phillip Aerni tests agricultural biotechnology to explain mismatch between public attitudes and perceptions about GMO foods. Food animals, fish are appearing and affecting the salmon aquaculture whose sociological effects are described by Rebecca Clausen and others. A mediator model is used by Macario Rodriguea-Entrena to understand consumer behavior with GMO. A group in Norway asks and answers the important question whether GMO soybeans are equivalent as food and feed to organic ones.

Section III: Government Regulation and Litigation for GMO foods

The United States Agriculture Department proposed eliminating restrictions on the use of corn and soybean seeds genetically engineered to resist common weed killers including Roundup recently. This move was welcomed by many farmers but concerning to some scientists and environmentalists who worry it could invite growers to use more chemicals. The herbicide, known as 2, 4-D, has had limited use in corn and soybean farming because it is toxic to the plants early in their growth. While the American farmers and public have been generally welcoming of GMO crops European governments and public has been stronger in concerns about safety and health. To help understand the different views a group in Belgium review consumer acceptance and pay for high nutrient GMO in Europe. Ozgur Caku reviews detection of GMO in animal feeds. Then gene containment and marker free GMO are described with Shuangrong Yuan also discussing microRNA for plant engineering. A British author, A. Howard, discusses the effects of European journalism on GMO use. This is timely as in 2014 an insect-resistant corn is on the verge of being approved by the European Union. The majority of EU nations opposed this action but failed to block it. In the USA litigation is frequent for many issues. An attorney, Thomas Redick, reviews agricultural litigation and regulation. Finally in this section the European Union Reference methods database and decision supporting tool for the analysis of GMO is reviewed by a large group of authors in Italy.

Section IV: Role of genetically modified crops in increasing the food supply in the developing and developed countries

Supporters of GMO crops argue that they offer an unrivaled opportunity to increase yields, but opponents say they pose unknown health and environmental risks. American companies pushing this product note a legal obligation to [DuPont Pioneer], to their farmers and scientists and its trade partners. It has taken 13 years after submission to reach approval, suggesting strongly the need for compilation of reviews on many aspects of GMO preparation and public health effects. A group in India reviews the benefits of GMO in livestock feeding. Then Lucia Martinelle describes the role of transgenic food’s uncertainty as well as the producers’ responsibilities. Groups in developing countries review stress tolerance in peanuts to make it easier to grow under challenging environmental conditions while another describes why India needs biotechnology to ensure food and nutrition security. Then Maria Oszvald discusses an example in detail where key genes for protein in wheat are transferred to rice.

Section V: Potential Health benefits, acceptance and risks due to incorporation of novel plant gene products into the food supply

Acceptance of genetically modified foods is their identification and especially safety. Wentao Xu discusses event-specific identification technology as well as detection techniques. Several groups discuss putting into GMO different genes for precursors to nutrients like carotenoids for increased vitamin A, important particularly in developing countries where childhood vitamin A levels are often low. It is possible to reduce allergenicity of some crops as reviewed by Siddhesh B Ghag while plant defensins for development of fungal pathogen resistance can improve yield. Finally Henri Darmency identifies gene flow from GMC to wild or original crops.

Section VI: Safety of Genetically Modified Foods for Humans and Animals

Safety concerns persist while frequently scientific studies overwhelmingly show safety of GMO foods. Therefore Anton E. Wohlers discussed labeling of GMO foods in USA as part of safety concerns. Bruce Hammond also reviews aspects of GMO food safety. Rie Satoh has a chapter on allergen analysis in assessment of GMO foods.

Section VII: Demand and Uses of Non-Genetically Modified Foods, and GMO’s for Humans and Animals

Mohamed Fawzy Ramadan genetically modified rice, maize, and soy consumption in Saudi Arabia. Yue Ma concludes the book with reasons for Chinese urban consumers opposing GMO.

Acknowledgments

The work of Dr Ronald Watson’s assistant, Bethany L. Stevens, in communicating and working with authors on the manuscripts was critical to the successful completion of this book. It is very much appreciated. Support for Ms Stevens’ and Dr Watson’s work was graciously provided by the Natural Health Research Institute (www.naturalhealthresearch.org) and Southwest Scientific Editing & Consulting, LLC. Appreciation for suggesting the topic by Dr Michael Lelah is noted. Finally, the work of the librarian at the Arizona Health Sciences Library, Mari Stoddard, was vital and very helpful in identifying the key researchers who participated in the book.

Section I

Development, Testing and Safety of Plant and Animal GMO foods

Outline

Chapter 1. Soybean as a Food Source: Comparative Studies Focusing on Transgenic and Nontransgenic Soybean

Chapter 2. Genetically Modified Crops: Biosafety Regulations and Detection Strategies

Chapter 3. Genetically Modified Food Animals: An Overview

Chapter 4. Genetically Modified Aubergine (Also Called Brinjal or Solanum melongena)

Chapter 5. Nutritional Assessment of Genetically Modified Crops Using Animal Models

Chapter 6. Noncoding RNA-Based Genetically Modified Crops: Concepts and Challenges

Chapter 7. Agrobacterium-Mediated Alien Gene Transfer Biofabricates Designer Plants

Chapter 8. Understanding the Factors Influencing Attitudes toward Genetically Modified Rice

Chapter 9. Qualitative and Quantitative Diagnostics for EE1 Event of Bt Eggplant

Chapter 10. Biosensors for Detection of Genetically Modified Organisms in Food and Feed

Chapter 11. Genetically Modified Organism Analysis as Affected by DNA Degradation

Chapter 12. Novel Strategies for Genetically Modified Organism Detection

Chapter 13. Targeted Genetic Modification in Crops Using Site-Directed Nucleases

Chapter 1

Soybean as a Food Source

Comparative Studies Focusing on Transgenic and Nontransgenic Soybean

M.A.Z. Arruda¹,², R.M. Galazzi¹,², B.K. de Campos¹,², M.A. Herrera-Agudelo¹,², S.C.C. Arruda³,  and R.A. Azevedo³     ¹University of Campinas–Unicamp, Institute of Chemistry, National Institute of Science and Technology for Bioanalytics, Campinas, Brazil     ²University of Campinas–Unicamp, Department of Analytical Chemistry, Campinas, Brazil     ³University of São Paulo, Department of Genetics, Laboratory of Genetics Biochemistry of Plants, ESALQ, Piracicaba, Brazil

Abstract

For a very long time, humanity has been searching for more productive cultivars, employing techniques such as grafting, cutting, or artificial pollination. However, with the increasing demand for food, genetic modification has become of utmost importance. The insertion of the cp4-EPSPS gene for producing a soybean tolerant to herbicides was one the pioneers in terms of genetically modified organisms. This chapter briefly comments about this history as well as emphasizes comparative studies involving transgenic and nontransgenic soybeans (seeds and plants), focusing on (metallo)proteins, metabolites, some enzymes involved in the reactive oxygen species combat, and ions, which are evaluated in different studies. Additionally, research trends are also presented.

Keywords

Bioaccessibility; Enzymes; EPSPS; Glyphosate; Ionomics; Metabolomics; Metallomics; Proteomics; Stress

Introduction

Food, especially food derived from plant sources, has always played a vital role in human nutrition and development. Besides supplying nutrients, its importance is justified by its functional properties and capacity to protect human organisms against a diversity of diseases (Suliburska and Krejpcio, 2014). Hunger and malnutrition are among the most devastating problems affecting a large part of the world’s population. In this perspective, soybean (Glycine max (L.) Merril) is one of the most important crops because it is an inexpensive source of protein and oil in the human and animal diet. Typically, soybean seeds contain about 40% protein and 20% oil (Natarajan et al., 2013; Yamada et al., 2012), and they are rich in several elements important to body functions (Mataveli et al., 2010). In addition, soybean has physiologically active metabolites such as isoflavones, lecithins, tocopherols, and saponins, which are important for maintaining good health (Yamada et al., 2012). According to some studies, the regular consumption of soybean and soy productions reduces the risks of cancer and others illnesses (Natarajan et al., 2013; Cassidy and Faughnan, 2000). However, soy foods may exhibit allergenic symptoms due to antigenic proteins present in soybeans, causing allergic reactions in sensitive consumers (Natarajan et al., 2013; Berneder et al., 2013). Moreover, it exhibits a low content of methionine, an important and essential amino acid for animal nutrition (Azevedo et al., 1997).

Considering the significance of soy production in different countries and areas, its high production and productivity are imperative. One alternative to attain these necessities is the production of genetically modified organisms (GMOs). Over the last 20 years, transgenic crops have accounted for much of the world’s seed production. Soybean is one of the most important crops due to its commercial importance for a diversity of countries, including Brazil and the USA.

The genetic modification (GM) is carried out by the insertion, elimination, altered expression, and/or the replacement of exogenous genes in order to (1) synthesize new substances, (2) promote the absence of proteins, which were currently synthesized before the GM, or (3) enhance the synthesis of substances already present in the organism. In addition, GM improves the production and the nutritional quality of crops, and is therefore a possible solution for reducing or eliminating some problems associated with allergens and antinutrients present in nontransgenic (NT) soybeans.

In 2013, the global planted area of GMOs in 27 countries was more than 175  M  ha, corresponding to an increase of 3% of cultivated area when compared to 2012. Since 1996, the total cultivated area of transgenic crops increased over 100 times (James, 2013). Soybean is one of the most cultivated crops in the world. The USA is the largest producer, with c. 90  M tons in 2013 (USDA, 2014), and Brazil is the second largest producer, with c. 87  M tons in 2013/2014 (CONAB, 2014; EMBRAPA, 2014a).

As noted, the production of GMOs has intensified. This, due to a set of nonconventional tools, in rapid development, enables the transfer of genetic information from one organism, being a microorganism, a plant, or an animal, to another. Additionally, multiple genes or large amounts of transfers have also been reported in the literature, such as the application of rice, canola, and corn as well as genes conferring the production of polyunsaturated fatty acids and vitamin E in soybean and Arabidopsis (Shetty et al., 2007; Porfirova et al., 2002).

In this context, plants are the main targets of the studies. The advancement of techniques for the genetic improvement of these organisms allows for biotechnological goals such as increasing productivity, reducing postharvest losses, obtaining crops that are more tolerant to environmental stress, and/or obtaining crops that use nitrogen and phosphorus more efficiently, increasing the nutritional value of the food, resistance to herbicides, pests, and/or diseases, and the development of alternatives for industries such as the fuel and pharmaceutical enterprises (Herman et al., 2003; Kliebenstein et al., 2001).

Since the first report as a GMO, the importance of soybeans to different countries, including Brazil, exponentially increased. Different aspects of this culture, focusing on comparative studies involving transgenic (T) and NT soybean seeds and plants, will be emphasized in this chapter. These studies involve aspects such as bioaccessibility of metals and evaluation of differential enzymes and their activities as well as metabolites, proteins, and metalloproteins.

Some Comments About Transgenic Soybeans

The Necessity of Using an Herbicide

Soybean, like many others crops, is subject to the action of several stress factors, such as drought and temperature, actions of pathogens, and fungi and pests that currently reduce the production (Ghosh et al., 2013). Thus, to avoid the action of pests on crops some types of herbicides are used. Glyphosate [N-(phosphomethyl) glycine] is the highest selling herbicide in the world. It is employed to control pests in the cultivars because of its systemic and postemergent action. This herbicide is continually translocated from roots, accumulating in distinct compartments of plants, inhibiting the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) (Arruda et al., 2013a; Arango et al., 2014; Zobiole et al., 2010), which participates in the synthesis of aromatic amino acids, whose absence results in plant death (Arruda et al., 2013a; Nakatani et al., 2014; Gomez et al., 2009).

The impact of glyphosate in the soil microbiota and rhizosphere communities is still not well understood, and the literature reports antagonisms (Arango et al., 2014; Nakatani et al., 2014). For example, some studies showed that glyphosate application can modify the biomass content in soil microbiota as well as in microbial respiration (Nakatani et al., 2014). On the other hand, no significant effects on the soil microbiota biomass, respiration, and metabolism and CO2 production were observed in other studies after the application of the glyphosate (Arango et al., 2014; Nakatani et al., 2014).

Genetic Modification of Soybean–Glyphosate Resistance

In most cases, the GM is done by the introduction of a DNA fragment, usually from a bacterium that is tolerant or resistant to the action of herbicides, pests, and insects, among others (Natarajan et al., 2013; Xue et al., 2012; Li et al., 2013).

The first reported GM of the soybean was carried out for its improvement to herbicide tolerance. Although the glyphosate, as the main component of the herbicide, presents excellent characteristics, as already mentioned, it is nonselective. In other words, all plants could die, including those of commercial and nutritional value, such as the soybean.

For solving this problem, the insertion of the cp4-EPSPS exogenous gene into the soybean, from a soil bacterium Agrobacterium sp, was carried out, so that its expression resulted in a glyphosate tolerance phenotype. The EPSPS enzyme is now continuously produced, even in the presence of the herbicide, allowing the use of glyphosate on T soybean cultivation. Due to the production of T crops presenting tolerance to the glyphosate, the use of this herbicide as well as the GM crops has been increasing (Arango et al., 2014; Nakatani et al., 2014).

The first variety of T soybean was called Roundup Ready® (RR) (Natarajan et al., 2013; Li et al., 2013), and it was the first genetic crop in the world produced by the Monsanto Company. Considering all GM crops, those presenting tolerance to herbicides represent the most planted area, with 25.9  M  ha. In addition to GM, which confers tolerance to herbicides, another modification involving the insertion of Bt cry1Ac gene, which confers resistance to insects, corresponds to 16.2% of the total planted area (Céleres, 2013; Kim et al., 2012). More recently, another variety of the T soybean (Intacta RR2®), combining the resistance to pests with herbicide tolerance, was produced, which currently accounts for 7.8  M  ha (19.3% of the total cultivated area) (Céleres, 2013; MONSANTO, 2014).

A Brief History of the Transgenic Soybean in Brazil

The USA introduced soybeans to Brazil in 1882, and after some tests, the first cultivars were registered in distinct areas of Brazil between 1900 and 1901. In the mid-1950s, the soy crop was encouraged due to its properties and appropriated climatological conditions, especially in the south of Brazil. Since then, soybean production and its importance to the economy are increasing (EMBRAPA, 2014b).

Regarding the T soybean in Brazil, the first GM crop was obtained in 1998 (Embrapa Soja, 2003). GM soybean varieties account for 27  M  ha (91.1% of the total soybean crop area in 2013/2014), which represents 67.2% of the total GM crop area in Brazil. In terms of production, soybeans also represent the highest crop production in Brazil, with c. 87  M tons, including GM, such as maize and cotton, and non-GM crops (CONAB, 2014; Céleres, 2013). Considering the importance of soybean crops in different areas such as economics, food, and the environment, comparative studies involving T and NT soybeans are necessary to evaluate both varieties and their specificities.

Comparative Studies Involving Transgenic and Nontransgenic Soybean Seeds or Plants

Bioaccessibility Studies

Soybean seeds are known to contain elements such as Ca, Cu, Fe, Mg, Mn, P, and Zn, which serve several functions in the human body, such as bone and teeth formation. They are also constituents of a diversity of enzymes responsible for cell metabolism. An accurate determination of these elements in soybeans can provide access to their nutritional quality (Mataveli et al., 2010).

A comparative study regarding total element concentrations (Mataveli et al., 2010) revealed that Fe, Co, and Cu are present at a higher concentration in T (variety MSOY 7575 RR) rather than NT soybean seeds (variety MSOY 7501). As a result, the T seeds seem to have the ability to take up and store higher amounts of these elements. Differences in Cu and Fe concentrations between T and NT soybean seeds were also previously established by Sussulini et al., 2007.

One typical and valuable use would be the control of iron deficiency anemia in certain populations around the world through the consumption/use of iron-rich transgenic soybeans in food production. Considering other soybean plant compartments (roots, stems, and leaves) as well as other Roundup Ready transgenic varieties (MSOY 7211 RR–T and MSOY 8200–NT soybeans), Oliveira et al. (2014) carried out a tracer experiment to evaluate if GM can influence the uptake and translocation of Fe. This study concluded that T and NT plants present similar profiles on Fe distribution between plant compartments; however, NT plants were able to accumulate higher amounts of Fe than T plants, even when different sources of Fe were provided.

For estimating the nutritional soybean contribution, it is of utmost importance to know the amount of nutrients that are delivered (bioaccessible fraction) for the absorption in the organism during human digestion (Fernández-García et al., 2009), since not all of the total content is efficiently absorbed and used. In this way, in vivo and in vitro methods are currently used to determine the bioaccessible fraction encountered in the gastrointestinal tract during the digestion, that is, the fraction really available to enter the bloodstream (Hur et al., 2011).

In vivo methods allow the determination of the amount of nutrients absorbed, bioactive compounds, or their metabolites through studies of mass balance and tissue concentration (Fernández-García et al., 2009). The studies of mass balance determine the nutrient amount absorbed by measurements of the amount of ingested and excreted nutrients. In studies of tissue concentrations, the increase of the nutrient concentration into the blood plasma is monitored. Some examples in the literature report the application of these methods in animals or humans, with the aim of determining the absorption of carbohydrates, minerals, and vitamins, among others (Vaisberg et al., 2006; Weber et al., 2006; Scalbert and Williamson, 2000).

Most traditional in vitro methods are those considered to be statics, which are proposed to make a sequential exposure of the sample in order to simulate its digestion inside the mouth, stomach, and intestine, simulating human physiological conditions, such as pH, gastric, intestinal, and hepatic enzymes as well as residence time. These conditions have been currently modified/adapted for performing bioaccessibility methods. On the other hand, temperature is the only physiological condition that usually remains at 37  °C (Hur et al., 2011; Intawongse and Dean, 2006; Le et al., 2012). After the simulation, the remaining solution is used for quantifying the nutrient of interest, allowing the evaluation of its bioaccessibility.

A wide variety of in vitro methods have been developed with the aim of establishing the best representation of the human physiological system, such as the Relative Bioaccesibility Leaching Procedure method, recommended by the American Agency EPA, In Vitro Gastrointestinal, Physiologically Based Extraction Test, Dutch National Institute for Public Health and the Environment method, and Unified Bioaccessibility Method (Le et al., 2012).

In vitro models are an alternative to objectively establish an analysis of the matrix composition in the digestive process. For functional foods, in vitro methods provide a first insight into the behavior of the bioactive ingredient, transport, absorption, and bioaccessibility (Fernández-García et al., 2009). Several studies have been published, with the main objective to assess the bioavailability of selected chemicals in certain foods.

Studies of the metal composition of genetically modified food are often performed, but most of them are limited to the determination of total content (Xin et al., 2008; Gayen et al., 2013), providing little information about the amount available to be absorbed and utilized efficiently by the organism.

The work carried out by our research team (Mataveli et al., 2010) was, up until now, unique where the effects of GM in the bioaccessiblity of some elements by comparing T and NT soybean seeds were investigated. The analysis was carried out by an inductively couple plasma (ICP)-sector field-mass spectrometry (MS). The bioaccessible fractions obtained through a simulation of gastric and intestinal conditions revealed that the contributions of bioaccessible fractions of Cu, Fe, and other elements (Mn, S, Zn) for T soybean seeds appear to be larger than those found in NT soybean seeds. Therefore, the comparative studies involving bioaccessibility between nutritional species of T and NT soybeans are of utmost importance, due to the increase in demand for food worldwide and its quality as a functional food. Research with T soybeans is scarce, since there few studies available allow for the collection of unpublished and important data, which may drive the availability of nutritional information for society.

Enzymes Involved in the Oxidative Stress

Earth is the only planet able to support aerobic life as the way that we understand its meaning, and the presence of oxygen is derived from the photosynthetic activities of cyanobacterias and plants (Arruda et al., 2013b). Its presence in its atmosphere led to a diversity of oxygen-based reactions. Under some situations, such as normal metabolic activity or when under environmental disturbance, the oxygen can be switched to an excited state, with the production of free radicals and similar forms, such as H2O2; O2 ·; HO2−; and OH− (Gratão et al., 2005), which can act in different ways in the cellular environment (Foyer and Noctor, 2000). Positive and negative aspects regarding the oxygen-based reactions can occur, and this process is known as oxidative stress. Oxidative stress can be described as a central factor in abiotic and biotic stress that occurs due to imbalances in any cell compartment between the production of reactive oxygen species (ROS) and antioxidant defense (Gratão et al., 2005).

Despite the importance of evaluating ROS and the enzymes involved in its combat (i.e., superoxide dismutase–SOD (Fridovich, 1995), catalase–CAT (Scandalios et al., 1997), glutathione reductase–GR, among others), the few examples found in the literature for soybeans (Arruda et al., 2013a) are not focused in comparative studies involving T and NT soybeans. In fact, there are only two examples that were carried out by our research team and will be briefly reviewed in this section.

Barbosa et al. (2012) reported the evaluation of enzyme activity and the abundance of some proteins from T (MSOY 7575 RR) and NT (MSOY 7501) soybean seeds. The analysis of enzymes such as ascorbate peroxidase (APX), GR, and CAT revealed higher levels (30.6, 71.4, and 35.3%, respectively) in T than in NT seeds. Additionally, lipid peroxidation was also evaluated through the malondialdehyde (MDA) concentration, which is an indicator of lipid peroxidation and oxidative stress, showing 29.8% higher concentration in T than in NT seeds. Such results suggest that the T genotype naturally exhibited a level of stress higher than that of the NT genotype. Nevertheless, such higher levels also suggest that this genotype is possibly better prepared than NT when facing adverse conditions.

In the referred work, four proteins were differentially found by using 2-D DIGE, as glycine-rich RNA-binding protein, cytosolic glutamine synthetase, actin, and glycinin G1 subunit.

Even though there is no information in the literature regarding ROS production and GM, particularly when seeds are concerned, it is well known that plants are highly adapted to respond quickly to biotic or abiotic stresses by altering gene expression and metabolism as a result of cell signaling, which may be mediated by ROS (Schmidt et al., 2010). In the same report, the authors commented that there was a previous GM, conferring on the plant a resistance to glyphosate. In plants, posttranscriptional control mechanisms are growing in importance, and proteins involved in RNA processing and alternative splicing, RNA transport, messenger RNA (mRNA) translation, mRNA stability, and mRNA silencing mechanisms have been shown to be required for normal plant development and the responses of plants to altered environments (Kazan, 2003; Cheng and Chen, 2004; Wang and Li, 2007). In the work by Barbosa et al. (2012) only the glycine-rich RNA-binding protein was differentially found after DIGE analysis, a protein shown to be correlated with ROS production (Schmidt et al., 2010; Kim et al., 2007).

The cytosolic glutamine synthetase (another differentially abundant protein found through DIGE analysis) is involved in nitrogen fixation and is a target of herbicides. Pageau et al. (2006) also demonstrated that oxidative stress can control the expression of nitrogen-metabolism genes. It is easy to show that cytosolic glutamine synthetase can be altered because of the oxidative stress observed in the T soybean line, mainly when higher levels of H2O2 are present, which is correlated to higher activities of CAT.

There is no report in the literature concerning actin or glycinin G1 subunit and ROS production, and the authors commented that this fact might open up a new possibility for future studies (Barbosa et al., 2012).

Barbosa et al. (2012) also evaluated protein species and enzymes, but in T (MSOY 7575) and NT (MSOY 7501) soybean leaves, collected from plants obtained from the seeds used. The enzymes involved in ROS combat, such as APX, CAT, GR, and SOD exhibited higher activities (21, 70, 14, and 31%, respectively) in T leaves when compared to NT leaves. Such a relationship was also observed for MDA and H2O2 concentrations, which increased (c. 44 and 69%, respectively) in T when compared to NT leaves. These results indicated that the GM itself might be a stressful factor to the plant, possibly due to the need for a new equilibrium in its metabolism, since the stress condition is being maintained within the levels that the plant can deal with.

Metabolites and (Metallo)proteins

Techniques based on modern biotechnology known as omics sciences, studying DNA, RNA, proteins, metabolites, and metals, among others, have been used in an attempt to answer some questions about the soybean culture and for evaluating the effects of the T soybean species at the molecular level (Arruda et al., 2013a; Barbosa et al., 2012; Clark et al., 2013). Despite more than a decade of use, no case of T food human consumption was reported as malicious. However, there are some results about food safety in the literature obtained from rats showing some antagonisms, since the total absence of effects to changes in organic functions, such as increase in cholesterol and triglycerides (Brasil et al., 2009; Daleprane et al., 2009a,b, 2010; Battistelli et al., 2010). Additionally, several studies in the literature raise some criticism with regard to side effects that plants have suffered in its metabolism (Arruda et al., 2013b).

Recently, the metabolic profile between GM and non-GM soybean was performed by Clark et al., 2013 in order to obtain data on biosafety. For this task, they investigated the soybean metabolic profile from 29 different cultivars cultivated in different seasons. A total of 169 metabolites have been identified, 159 of them corresponding to primary metabolism and 15 to secondary metabolism. The primary metabolites are essential to plants for functions such as growth and fertilization, while the secondary metabolites perform other functions, such as protection and defense (Lin et al., 2014).

Most of the literature concerning the content or metabolite profile in biological samples reports the use of the mass spectrometry technique, usually coupled to a separation technique such as liquid or gas chromatography. The metabolites extraction for chemical analysis is well established and it is easy to handle. For example, the method proposed by De Voz et al. (2007) uses 100  mg of a macerated sample with N2 and 1  mL of methanol (75% v/v), acidified with 0.1% formic acid (v/v) for 15  min sonication.

Studies involving comparative metabolomics between T and NT soybeans are still scarce in the literature; much more comprehensive research should be on the agenda if a better understanding of the metabolism of the plant and the effects that can cause these genetically modified organisms is to be better understood. For instance, some studies suggest that metabolite levels may have been dramatically altered by combining genetic or environmental impacts (Clark et al., 2013). It is also known that the effects of T soybean plants are becoming evident when it comes to proteomic studies. To the naked eye, the T and NT plants developments are similar, although the T plant has increased productivity and presented a shorter cultivation period; however, it has not yet been possible to correlate these changes with the changes observed in their proteomic profile.

Proteomics is defined as the area of science that studies proteins, including not only identification but also quantification, determination, modifications, interactions, activities, and functions thereof. It is known that from the total protein content in soybean seeds, 70–80% is glycinin and β-conglycinin and its subunits, presenting storage function and being responsible for nutritional and physicochemical properties. Much of the studies found in the literature are focused on these proteins (Natarajan et al., 2009). However, other studies involve a more thorough proteomic investigation of soybean proteins (Arruda et al., 2013a; Barbosa et al., 2012; Garcia et al., 2006; Natarajan et al., 2007, 2009) as well as studies involving food safety and the possible effects T organisms (Daleprane et al., 2009; Krzyzowska et al., 2010).

Krzyzowska et al. (2010) evaluated the possible effects of T triticale, which is a hybrid between wheat and rye, on animal health in five successive generations of rats. The results indicated that the use of T triticale in rodent diets has increased the concentration of white blood cells and also the expansion of B-cells in secondary lymphoid organs. Similarly, Daleprane et al., 2009 studied the use of GM soybean in the diet of rats. The study was carried out by separating three groups of rats, the first being fed with feed organic soybean, the second with feed T soybean, and third using casein base. The results showed a significant difference in the weight of the rats in both groups (experimental and control) and also that the organic soybean has a better protein utilization in relation to the transgenic soybean line. However, as far as we are aware, there are no case studies yet available on the consequences of a feeding base of transgenic organisms in humans.

In order to display differential proteins between two different cultures, for example T and NT soybean, polyacrylamide gel electrophoresis (2D-DIGE) is clearly a good choice. In this context, Barbosa et al. (2012) studied, by means of proteomic comparison, T and NT soybeans. The image of the gels showed four spots with different intensities, indicating that the proteins had different abundances between samples due to GM suffered by T soybean seeds. This fact was confirmed by the identification of the protein cp4-EPSPS, characterizing the GM. Additionally, 192 proteins were able to be identified by 2D-PAGE separation and identification by MALDI-TOF.

Another area that studies omics content is metallomics, which is defined as the totality of metal and metalloid species in a cell or tissue, its identity, and location. Most metal biological organisms are bound to proteins, which are called metalloproteins (Arruda and Azevedo, 2009; Mataveli et al., 2012). Our group performed a comparative study of the content of metals present in T and NT soybeans by ICP-MS. It was observed that the T and NT soybeans have statistically different concentrations of some metals such as Cu and Fe (higher in T soybeans). Using a three-dimensional separation technique—the first chromatographic separation by size exclusion (SEC), the second using an anion exchange (AEX), and, finally, one dimension by electrophoresis (SDS-PAGE)—fractions containing different metals were identified. These fractions were taken for identification by mass spectrometry with electrospray ionization source (ESI-MS). This approach identified 33 proteins, some of them being metalloproteins such as seed lipoxygenase-1, β-conglycinin, the chain lipoxygenase-3, and beta-amylase, among others (Mataveli et al., 2010, 2012).

In this context, the genes encoding genetically modified organisms in different cultivars of plants, added to the plant with the aim of agronomic improvement as herbicide tolerance and insect resistance, should be used in a way that does not affect the performance of the culture and does not produce changes unsafe to food derived from these cultivars. The omics sciences in contribution with the analytical techniques have presented data suggesting an improvement regarding the safety of GM crops; however, more studies are needed for a biochemical vision and extensive vegetable physiology for conclusive results.

Trends

Several regulatory agencies, such as Food and Agricultural Organization of the United Nations (FAO), The European Food Information Council, Brazilian Health Surveillance Agency (ANVISA), and others regulate the safety levels of determined nutrients and metals in feed that can be consumed by humans. In this way, many studies evaluate the elemental composition of some foods as well as if a GM changes the levels of nutrients and if the transgenic food would be safe to the consumer. In addition to the elements determination in seeds, studies that evaluate the bioaccessibility of the main elements contained in T and NT seeds are still scarce, and more attention should be given to this type of research, such as the one published by Mataveli et al. (2010). In addition to the elemental composition and bioaccessibility studies evaluating the nutrients, an approach focusing on carotenoids, wherein the total concentration as well as the carotenoids composition changes according to different kinds of GM (Rivera et al., 2013), was also carried out. Nonetheless, despite the bioaccessibility studies already performed, which provide preliminary and important information on how each bioaccessible compound could be absorbed by the human body, more elaborate tests, named bioavailability studies, must be carried out because in such studies it is possible to evaluate whether a bioaccessible element is able to pass through membranes that simulate the digestive wall and can thus be absorbed. More than assessing what kind of changes the GM may cause in terms of bioavailability, a chemical speciation study to know what kind of given element/compound would be bioavailable and if these species are toxic to humans would also add interesting new information that is missing from current studies.

When GM foods are concerned, a study of the generation of seeds is important to assess whether the effects caused by T intensify or diminish with successive plantings of seeds obtained. Such studies can be performed using various approaches, such as comparing the proteomic profile of generations, studies evaluating the metabolites of each generation, bioaccessibility and bioavailability tests, and also enriched isotope tracer studies to assess the biochemical absorption by the plant, such as the unique study published by Oliveira et al. (2014).

Another study which would be interesting is related to the use of glyphosate in T crop planting to assess whether this herbicide changes the composition of seeds obtained as well as if glyphosate is incorporated into the plant and if it has the absorption of some potentially toxic metals of the association with glyphosate and the possible consequences of consumption, by humans, of these contaminated seeds.

Conclusions

Besides a diversity of byproducts (i.e., milk, cakes, flour), there is no doubt that soybean is a major source of proteins and oil, placing this legume species on a special list as a key plant species to be used as a food source. Additionally, the necessity for obtaining more productive cultivars opened some new possibilities in terms of using molecular genetics, creating a generation of GMOs, including T soybean lines. As an organism led by thousands of years in terms of evolutionary adaption, it is easy to realize that the modified species may follow the same path. In this way, some questions may appear. Is the behavior always the same when these species are compared with natural ones? What is, in fact, changed when both are compared? What are the consequences of these changes? Is the nutritional quality of these GMOs similar when it is compared to that of natural organisms? This chapter presented some comparative results involving T and NT soybeans, hoping to answer some of these questions but leaving a door open to future research on T soybean as well as T crops plants as a whole. We can say that there are some differences, indicating that the GMO, that is, the soybean, is searching for a new equilibrium as a living organism in nature. What is needed from now on is the understanding of each difference observed and perhaps making possible the demystification of the GM. Besides being of great opportunity to research, we also will be contributing to making our food safer.

References

Arango L, Buddrus-Schiemann K, Opelt K, Lueders T, Haesler F, Schmid M, Ernst D, Hartmann A. Effects of