ABSTRACT
Dicamba tolerant (DT) soybean, cotton and maize were developed through constitutive expression of dicamba mono-oxygenase (DMO) in chloroplasts. DMO expressed in three DT crops exhibit 91.6-97.1% amino acid sequence identity to wild type DMO. All DMO forms maintain the characteristics of Rieske oxygenases that have a history of safe use. Additionally, they are all functionally similar in vivo since the three DT crops are all tolerant to dicamba treatment. None of these DMO sequences were found to have similarity to any known allergens or toxins. Herein, to further understand the safety of these DMO variants, a weight of evidence approach was employed. Each purified DMO protein was found to be completely deactivated in vitro by heating at temperatures 55 °C and above, and all were completely digested within 30 s or 5 min by pepsin and pancreatin, respectively. Mice orally dosed with each of these DMO proteins showed no adverse effects as evidenced by analysis of body weight gain, food consumption and clinical observations. Therefore, the weight of evidence from all these protein safety studies support the conclusion that the various forms of DMO proteins introduced into DT soybean, cotton and maize are safe for food and feed consumption, and the small amino acid sequence differences outside the active site of DMO do not raise any additional safety concerns.
Subject(s)
Crops, Agricultural/toxicity , Dicamba/pharmacology , Drug Resistance , Food, Genetically Modified/toxicity , Glycine max/toxicity , Gossypium/toxicity , Herbicides/pharmacology , Mixed Function Oxygenases/toxicity , Oxidoreductases, O-Demethylating/toxicity , Plants, Genetically Modified/toxicity , Zea mays/toxicity , Administration, Oral , Amino Acid Sequence , Animals , Computational Biology , Consumer Product Safety , Crops, Agricultural/enzymology , Crops, Agricultural/genetics , Databases, Protein , Drug Resistance/genetics , Enzyme Stability , Female , Food Safety , Food, Genetically Modified/parasitology , Gene Expression Regulation, Plant , Gossypium/enzymology , Gossypium/genetics , Humans , Male , Mice , Mixed Function Oxygenases/administration & dosage , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Pancreatin/metabolism , Pepsin A/metabolism , Plants, Genetically Modified/enzymology , Plants, Genetically Modified/genetics , Protein Denaturation , Proteolysis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/toxicity , Risk Assessment , Glycine max/enzymology , Glycine max/genetics , Stenotrophomonas maltophilia/enzymology , Stenotrophomonas maltophilia/genetics , Temperature , Toxicity Tests, Acute , Zea mays/enzymology , Zea mays/geneticsABSTRACT
In order to assess the role of oxidative stress in the cytotoxicity of natural hydroxyanthraquinones, we compared rhein, emodin, danthron, chrysophanol, and carminic acid, and a series of model quinones with available values of single-electron reduction midpoint potential at pH 7.0 (E(1)7), with respect to their reactivity in the single-electron enzymatic reduction, and their mammalian cell toxicity. The toxicity of model quinones to the bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK), and HL-60, a human promyelocytic leukemia cell line, increased with an increase in their E(1)7. A close parallelism was found between the reactivity of hydroxyanthraquinones and model quinones with single-electron transferring flavoenzymes ferredoxin: NADP+ reductase and NADPH:cytochrome P450 reductase, and their cytotoxicity. This points to the importance of oxidative stress in the toxicity of hydroxyanthraquinones in these cell lines, which was further evidenced by the protective effects of desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine, by the potentiating effects of 1,3-bis-(2-chloroethyl)-1-nitrosourea, and an increase in lipid peroxidation.