Low-concentration exposure to glyphosate-based herbicide modulates the complexes of the mitochondrial respiratory chain and induces mitochondrial hyperpolarization in the Danio rerio brain.

Glyphosate (N-phosphonomethyl-glycine) (GLY) is the active ingredient of the most used herbicides in the world. GLY is applied in formulated products known as glyphosate-based herbicides (GBH), which could induce effects that are not predicted by toxicity assays with pure GLY. This herbicide is classified as organophosphorus compound, which is known to induce neurotoxic effects. Although this compound is classified as non-neurotoxic by regulatory agencies, acute exposure to GBH causes neurological symptoms in humans. However, there is no consensus in relation to neurotoxic effects of GBH. Thus, the aim of this study was to investigate the neurotoxic effects of the GBH in the zebrafish Danio rerio, focusing on acute toxicity, the activity and transcript levels of mitochondrial respiratory chain complexes, mitochondrial membrane potential, reactive species (RS) formation, and behavioral repertoire. Adult zebrafish were exposed in vivo to three concentrations of GBH Scout®, which contained GLY in formulation (fGLY) (0.065, 1.0 and 10.0 mg L-1 fGLY) for 7 d, and an in vitro assay was performed using also pure GLY. Our results show that GBH induced in zebrafish brain a decrease in cell viability, inhibited mitochondrial complex enzymatic activity, modulated gene expression related to mitochondrial complexes, induced an increase in RS production, promoted hyperpolarization of mitochondrial membrane, and induced behavioral impairments. Together, our data contributes to the knowledge of the neurotoxic effects of GBH. Mitochondrial dysfunction has been recognized as a relevant cellular response that should not be disregarded. Moreover, this study pointed to the mitochondria as an important target of GBH.

Development of quantitative competitive PCR for determination of copy number and expression level of the synthetic glyphosate oxidoreductase gene in transgenic canola plants

Background: For successful in vitro plant regeneration, plant cell lines with multiple transgene integration and low transgene expression levels need to be ruled out. Although real-time polymerase chain reaction (RT-PCR) is a rapid way to accomplish this, it is also expensive and typically limits the size of the target sequence. Quantitative competitive PCR (QC-PCR) is proven to be a safe and accurate method for determination of both copy number and quantification of transcript levels of synthetic transgenes in transformed plants. Results: The glyphosate oxidoreductase genewas chemically synthesized and used to transform Brassica napus L. via Agrobactrium-mediated transformation. A construct containing the mutated form of a synthetic glyphosate oxidoreductase (gox) gene (internal standard) was prepared. Gene copy number was estimated in nine independent transgenic lines using QC-PCR as well as the standard method of Southern blot analysis. By quantitative RT-PCR, transcript levels were also determined in these lines. High (> 3), medium to high (2.2-3), medium to low (1-2.2), and low (< 1) levels of transcript were detected. Conclusions: No direct relationship was found between copy number and transgene expression levels. QC-PCR method could be implemented to screen putative transgenic plants and quickly select single T-DNA inserts. QC-PCR methods and the prepared competitor construct may be useful for future quantification of commercial transgenic food and feed.