Sub-chronic toxicological evaluation of genetically modified AVP1 sugarcane in albino rabbits

Paper Details

Research Paper 01/08/2018
Views (285) Download (9)

Sub-chronic toxicological evaluation of genetically modified AVP1 sugarcane in albino rabbits

Farheen Bhatti, Shaheen Asad, Qaiser Mahmood Khan, Ameena Mobeen, Muhammad Asif
Int. J. Biosci.13( 2), 276-290, August 2018.
Certificate: IJB 2018 [Generate Certificate]


Genetically modified crops require biosafety evaluation for approval from the regulatory competent authorities prior to commercialization. Genetically modified (GM) sugarcane expressing the Arabidopsis Vacuolar H+-pyrophosphatase gene (AVP1) which confers drought tolerance has been developed at NIBGE. The risks associated with GM sugarcane containing the AVP1 gene were evaluated in a sub-chronic feeding study on rabbits. Various parameters including body weight, food consumption, serum biochemistry, haematology, absolute and relative organ weight were compared between rabbits fed on GM and non-GM sugarcane after consumption for 90-day. In addition, genotoxicity was conducted among treatment groups and mutagenic potential was determined with AVP1 purified protein in an Ames assay. No adverse effects related to GM and normal diet were detected. The results obtained did not reveal any statistically significant differences in the micronuclei frequency or any DNA damage in rabbit’s peripheral blood cells. Furthermore, AVP1 purified protein showed no mutagenic activity at the concentration of 512µg/plate. This is the first report on biosafety of AVP1 sugarcane in Pakistan and scientific data generated through this research will be helpful for the commercial release of AVP1 GM sugarcane.


Allmann M, Candrian U, Höfelein C, Lüthy J. 1993. Polymerase chain reaction (PCR): a possible alternative to immunochemical methods assuring safety and quality of food detection of wheat contamination in non-wheat food products. Zeitschrift für Lebensmitteluntersuchung und-forschung A 196, 248-251.

Ames BN, McCann J, Yamasaki E. 1975. Methods for detecting carcinogens and mutagens with the Salmonella mammalian-microsome mutagenicity test. Mutation Research/Environmental Mutagenesis and Related Subjects 31, 347-363.

Appenzeller LM, Munley SM, Hoban D, Sykes GP, Malley LA, Delaney B. 2008. Subchronic feeding study of herbicide-tolerant soybean DP-356Ø43-5 in Sprague–Dawley rats. Food and Chemical Toxicology 46, 2201-2213.

Arjó G, Capell T, MatiasGuiu X, Zhu C, Christou P, Piñol C. 2012. Mice fed on a diet enriched with genetically engineered multivitamin corn show no sub‐acute toxic effects and no sub‐chronic toxicity. Plant Biotechnology Journal 10, 1026-1034.

Auffan M, Decome L, Rose J, Orsiere T, De Meo M, Briois V, Chaneac C, Olivi L, Berge-Lefranc J-L, Botta A. 2006. In vitro interactions between DMSA-coated maghemite nanoparticles and human fibroblasts: a physicochemical and cyto-genotoxical study. Environmental Science & Technology 40, 4367-4373.

Bhilwade HN, Chaubey RC, Chauhan PS. 2004. Gamma ray induced bone marrow micronucleated erythrocytes in seven strains of mouse. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 560, 19-26.

Bohnert HJ, Jensen RG. 1996. Strategies for engineering water-stress tolerance in plants. Trends in Biotechnology 14, 89-97.

Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254.

Chen ZL, Gu H, Li Y, Su Y, Wu P, Jiang Z, Ming X, Tian J, Pan N, Qu L-J. 2003. Safety assessment for genetically modified sweet pepper and tomato. Toxicology 188, 297-307.

Collins AR. 2002. The comet assay: principles, applications, and limitations. In Situ Detection of DNA Damage: Methods and Protocols 203, 163-177.

Colognato R, Bonelli A, Ponti J, Farina M, Bergamaschi E, Sabbioni E, Migliore L. 2008. Comparative genotoxicity of cobalt nanoparticles and ions on human peripheral leukocytes in vitro. Mutagenesis 23, 377-382.

Craig W, Tepfer M, Degrassi G, Ripandelli D. 2008. An overview of general features of risk assessments of genetically modified crops. Euphytica 164, 853-880.

Delaney B, Appenzeller LM, Roper JM, Mukerji P, Hoban D, Sykes GP. 2014. Thirteen-week rodent feeding study with processed fractions from herbicide tolerant (DP-Ø73496-4) canola. Food and Chemical Toxicology 66, 173-184.

Delaney B, Goodman RE, Ladics GS. 2017. Food and Feed Safety of Genetically Engineered Food Crops. Toxicological Sciences 162, 361-371.

Domingo JL. 2016. Safety assessment of GM plants: An updated review of the scientific literature. Food and Chemical Toxicology 95, 12-18.

Dryzga M, Yano B, Andrus A, Mattsson J. 2007. Evaluation of the safety and nutritional equivalence of a genetically modified cottonseed meal in a 90-day dietary toxicity study in rats. Food and Chemical Toxicology 45, 1994-2004.

EFSA. 2008. Safety and nutritional assessment of GM plants and derived food and feed: the role of animal feeding trials. Report of the EFSA GMO Panel Working Group on animal feeding trials 52.

FAO. 2008. GM food safety assessment. Tools for trainers.

FAO/WHO. 2000. Safety aspects of genetically modified foods of plant origin [computer file]; 2000. World Health Organization.

FAO/WHO. 2003. Codex Alimentarius: Food hygiene, basic texts: Food & Agriculture Organization.

Fenech M. 2008. The micronucleus assay determination of chromosomal level DNA damage. Environmental Genomics 185-216.

Garcia-Alonso M. 2013. Safety assessment of food and feed derived from GM crops: using problem formulation to ensure “fit for purpose” risk assessments. Collection of Biosafety Reviews 8, 72-101.

Gaxiola RA, Li J, Undurraga S, Dang LM, Allen GJ, Alper SL, Fink GR. 2001. Drought-and salt-tolerant plants result from overexpression of the AVP1 H+-pump. Proceedings of the National Academy of Sciences 98, 11444-11449.

Ghumman M. 2017. Meeting on sugar export today. Business Recorder Islamabad (Pakistan), March 16, 2017. 5250

Hamada S, Yamasaki K-i, Nakanishi S, Omori T, Serikawa T, Hayashi M. 2001. Evaluation of the general suitability of the rat for the micronucleus assay: the effect of cyclophosphamide in 14 strains. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 495, 127-134.

Hammond B, Dudek R, Lemen J, Nemeth M. 2006. Results of a 90-day safety assurance study with rats fed grain from corn borer-protected corn. Food and Chemical Toxicology 44, 1092-1099.

He X, Huang K, Li X, Qin W, Delaney B, Luo Y. 2008. Comparison of grain from corn rootworm resistant transgenic DAS-59122-7 maize with non-transgenic maize grain in a 90-day feeding study in Sprague-Dawley rats. Food and Chemical Toxicology 46, 1994-2002.

Healy C, Hammond B, Kirkpatrick J. 2008. Results of a 13-week safety assurance study with rats fed grain from corn rootworm-protected, glyphosate-tolerant MON 88017 corn. Food and Chemical Toxicology 46, 2517-2524.

Heddle JA. 1990. Micronuclei in vivo. Progress in Clinical Biological Research 340B, 185-194.

ISAAA. 2017. Brazil approves GM sugarcane for commercial use. Crop Biotech Updates.

ISAAA G. 2015. Approval Database. Updated

James C. 2013. ISAAA Brief 46-2013: Executive Summary. Global Status of Commercialized Biotech/GM Crops:

James C. 2016. Global Status of Commercialized Biotech/GM Crops: 2014. ISAAA Brief No. 49. ISAAA: Ithaca (NY).

James C, Teng P, Arujanan M, Aldemita RR, Flavell RB, Brookes G, Qaim M. 2015. Invitational Essays to Celebrate the 20th Anniversary of the Commercialization of Biotech Crops (1996 to 2015): Progress and Promise.

Jaszczak K, Kruszewski M, Baranowski A, Parada R, Bartlomiejczyk T, Zimny J, Rosochacki S. 2008. Micronucleus test and comet assay on mice fed over five generations a diet containing genetically modified triticale. Journal of Animal and Feed Sciences 17, 100.

Kiaie S, Abtahi Hs, Alikhani M, Mosayebi G. 2012. Expression, purification and antigenic evaluation of toxin-coregulated pilus B protein of Vibrio cholera. African Journal of Microbiology Research 6, 5188-5192.

Kinkema M, Geijskes J, Palupe A, Shand K, Coleman HD, Brinin A, Williams B, Sainz M, Dale JL. 2014. Improved molecular tools for sugarcane biotechnology. Plant Molecular Biology 84, 497-508.

Klaude M, Eriksson S, Nygren J, Ahnström G. 1996. The comet assay: mechanisms and technical considerations. Mutation Research/DNA Repair 363, 89-96.

Klümper W, Qaim M. 2014. A meta-analysis of the impacts of genetically modified crops. PloS One 9, e111629.

Konopacka M, Widel M, Rzeszowska-Wolny J. 1998. Modifying effect of vitamins C, E and beta-carotene against gamma-ray-induced DNA damage in mouse cells. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 417, 85-94.

Köppel E, Stadler M, Lüthy J, HuÈbner P. 1998. Detection of wheat contamination in oats by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Zeitschrift für Lebensmitteluntersuchung und-Forschung A 206(6), 399-403.

Krishna G, Kropko M, Ciaravino V, Theiss J. 1991. Simultaneous micronucleus and chromosome aberration assessment in the rat. Mutation Research Letters 264, 29-35.

Lucht JM. 2015. Public acceptance of plant biotechnology and GM crops. Viruses 7, 4254-4281.

MacKenzie SA, Lamb I, Schmidt J, Deege L, Morrisey MJ, Harper M, Layton RJ, Prochaska LM, Sanders C, Locke M. 2007. Thirteen-week feeding study with transgenic maize grain containing event DAS-Ø15Ø7-1 in Sprague–Dawley rats. Food and Chemical Toxicology 45, 551-562.

Malley LA, Everds NE, Reynolds J, Mann PC, Lamb I, Rood T, Schmidt J, Layton RJ, Prochaska LM, Hinds M. 2007. Subchronic feeding study of DAS-59122-7 maize grain in Sprague-Dawley rats. Food and Chemical Toxicology 45, 1277-1292.

Maluszynska J, Juchiniuk J. 2005. Plant genotoxicity. Archives of Industrial Hygiene and Toxicology 56, 177-184.

Maron DM, Ames BN. 1983. Revised methods for the Salmonella mutagenicity test. Mutation Research/ Environmental Mutagenesis and Related Subjects 113, 173-215.

McCleary BV, Harrington J. 1988. Purification of β-D-glucosidase from Aspergillus niger. Methods in Enzymology 160, 575-583.

Mejia LA, Korgaonkar CK, Schweizer M, Chengelis C, Marit G, Ziemer E, Grabiel R, Empie M. 2009. A 13-week sub-chronic dietary toxicity study of a cruciferin-rich canola protein isolate in rats. Food and Chemical Toxicology 47, 2645-2654.

Noteborn H, Bienenmann-Ploum M, Van den Berg J, Alink G, Zolla L, Reynaerts A, Pensa M, Kuiper H. 1995. Safety assessment of the Bacillus thuringiensis insecticidal crystal protein CRYIA (b) expressed in transgenic tomatoes. ACS Publications 134-167.

OECD. 2010. Consensus Document on Molecular Characterization of Plants Derived from Modern Biotechnology. Organization for Economics-operation and Development (OECD) Paris, France.

Pasapula V, Shen G, Kuppu S, PaezValencia J, Mendoza M, Hou P, Chen J, Qiu X, Zhu L, Zhang X. 2011. Expression of an Arabidopsis vacuolar H+pyrophosphatase gene (AVP1) in cotton improves drought and salt tolerance and increases fibre yield in the field conditions. Plant Biotechnology Journal 9, 88-99.

Raza G, Ali K, Ashraf MY, Mansoor S, Javid M, Asad S. 2016. Overexpression of an H+-PPase gene from Arabidopsis in sugarcane improves drought tolerance, plant growth, and photosynthetic responses. Turkish Journal of Biology 40, 109-119.

Schrøder M, Poulsen M, Wilcks A, Kroghsbo S, Miller A, Frenzel T, Danier J, Rychlik M, Emami K, Gatehouse A. 2007. A 90-day safety study of genetically modified rice expressing Cry1Ab protein (Bacillus thuringiensis toxin) in Wistar rats. Food and Chemical Toxicology 45, 339-349.

Séralini GE, Cellier D, de Vendomois JS. 2007. New analysis of a rat feeding study with a genetically modified maize reveals signs of hepatorenal toxicity. Archives of Environmental Contamination and Toxicology 52, 596-602.

Singh NP, McCoy MT, Tice RR, Schneider EL. 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research 175, 184-191.

Singh R, Kumar P, Tiwari N, Rastogi J, Singh S. 2013. Current status of sugarcane transgenic: an overview. Advances in Genetic Engineering 2, 2169-0111.1000112.

Smart RD, Blum M, Wesseler J. 2017. Trends in approval times for genetically engineered crops in the United States and the European Union. Journal of Agricultural Economics 68, 182-198.

Tukey JW. 1993. The problem of multiple comparisons. Multiple Comparisons: 1948-1983, H I Braun, Ed, VIII (The Collected Works of John W. Tukey, Chapman Hall), 1-300.

Venkataramana S, Rao PG, Naidu K. 1986. The effects of water stress during the formative phase on stomatal resistance and leaf water potential and its relationship with yield in ten sugarcane varieties. Field Crops Research 13, 345-353.

WHO. 1995. Application of the principles of substantial equivalence to the safety evaluation of foods or food components from plants derived by modern biotechnology: report of a WHO workshop WHO/FNU/FOS/95.1. World Health Organization, Geneva, Switzerland.