Elsevier

Toxicology

Volume 313, Issues 2–3, 16 November 2013, Pages 122-128
Toxicology

Ethoxylated adjuvants of glyphosate-based herbicides are active principles of human cell toxicity

https://doi.org/10.1016/j.tox.2012.09.006Get rights and content

Abstract

Pesticides are always used in formulations as mixtures of an active principle with adjuvants. Glyphosate, the active ingredient of the major pesticide in the world, is an herbicide supposed to be specific on plant metabolism. Its adjuvants are generally considered as inert diluents. Since side effects for all these compounds have been claimed, we studied potential active principles for toxicity on human cells for 9 glyphosate-based formulations. For this we detailed their compositions and toxicities, and as controls we used a major adjuvant (the polyethoxylated tallowamine POE-15), glyphosate alone, and a total formulation without glyphosate. This was performed after 24 h exposures on hepatic (HepG2), embryonic (HEK293) and placental (JEG3) cell lines. We measured mitochondrial activities, membrane degradations, and caspases 3/7 activities. The compositions in adjuvants were analyzed by mass spectrometry. Here we demonstrate that all formulations are more toxic than glyphosate, and we separated experimentally three groups of formulations differentially toxic according to their concentrations in ethoxylated adjuvants. Among them, POE-15 clearly appears to be the most toxic principle against human cells, even if others are not excluded. It begins to be active with negative dose-dependent effects on cellular respiration and membrane integrity between 1 and 3 ppm, at environmental/occupational doses. We demonstrate in addition that POE-15 induces necrosis when its first micellization process occurs, by contrast to glyphosate which is known to promote endocrine disrupting effects after entering cells. Altogether, these results challenge the establishment of guidance values such as the acceptable daily intake of glyphosate, when these are mostly based on a long term in vivo test of glyphosate alone. Since pesticides are always used with adjuvants that could change their toxicity, the necessity to assess their whole formulations as mixtures becomes obvious. This challenges the concept of active principle of pesticides for non-target species.

Introduction

Pesticide formulations are mixtures of adjuvants and so-called “active principles” on plants for herbicides, and insects for insecticides, etc. The supposed specificity of active principles on their targets does not mean a priori that they are the most toxic compounds of the formulations on human cells. Numerous mammalian (Colborn et al., 1993) and other animal studies (Hawthorne and Dively, 2011) evidenced side effects for pesticides. The toxicology of mixtures cannot be fully understood without knowing the differential toxicity of the various compounds of the formulations and their combined effects. Surprisingly, to measure their side effects, the active principles of pesticides are generally tested alone at a regulatory level in long-term mammalian trials, although their adjuvants are developed at least to enhance their stability and penetration into cells. However, most of the adjuvants are classified as inert.

Here we tested the differential and combined cytotoxicity of the major pesticides in the world which are glyphosate-based herbicides (GBH), and analyzed their composition and mechanisms of action. The residues of the GBH such as Roundup (R) are also among the first contaminants of ground and surface waters (IFEN, 2006), and of some food and feed because they are present since more than 15 years in around two third of genetically modified (GM) cultivated edible plants, because they are designed at least to tolerate R (James, 2011). Glyphosate (G) is toxic in plant cells by inhibition of 5-enolpyruvylshikimate-3-phosphate synthase used as a first step in aromatic amino acid synthesis (Boocock and Coggins, 1983). Adjuvants considered as inert include, according to the formulations, surfactants like POEAs (polyethoxylated alkylamines, Fig. 1), isobutane, light petroleum distillate, etc. that may induce among other DNA damages (Cox, 2004). However G is still generally hypothesized to be the active ingredient for non-target side effects. Unexpected side effects of G-based formulations were evidenced on non-target species, among other endocrine disruptions during spermatogenesis or pregnancy (Beuret et al., 2005, Clair et al., 2012, Dallegrave et al., 2007, Daruich et al., 2001, Oliveira et al., 2007, Romano et al., 2011, Savitz et al., 1997, Yousef et al., 1995). This may be related to adjuvants in formulation. They are indeed more and more considered as responsible for GBH toxicity (Mesnage et al., 2010, Williams et al., 2012), but the mechanistic and the nature of the cytotoxic agent(s) on human cells are still unknown. This is a general question that can arise for all pesticides.

The detailed known composition indicate that major adjuvants are ethoxylated, such as POEAs which are themselves mixtures of di-ethoxylates of tallowamines characterized by their oxide/tallowamine ratio. POEA commonly used in GBH is the POE (15) tallowamine (POE-15). We thus compared the toxicity and the composition of 9 formulations varying in adjuvants contents: Roundup Ultra, Roundup GT, Roundup GT+, Roundup Bioforce, Roundup 3plus, Glyphogan, Topglypho 360, Clinic E.V., and Bayer GC. For controls, we tested a formulation containing POE-15 without G (Genamin T200), and POE-15 alone. The compositional analysis of these products was performed by a non-quantitative mass spectrometry (MALDI-TOF MS/MS), considered as the best way to analyze pesticides formulations (Corbera et al., 2010, Cserháti and Forgács, 1997). Physico-chemical properties of POE-15 were approached by the measurements of its critical micelle concentration (CMC), determined by absorption changes in its presence of Coomassie blue CBB R-250.

We used HEK293, JEG3 and HepG2 cell lines, three models where unexpected effects of GBH have already been demonstrated (Benachour and Seralini, 2009, Gasnier et al., 2009). JEG3 cells are a useful model for examining placental toxicity (Letcher et al., 1999), and HepG2 for hepatic toxicity (Urani et al., 1998). HEK293 were chosen because of the sensitivity of embryonic cells, Roundup causing pregnancy outcomes (Savitz et al., 1997). Moreover, we have demonstrated that these cell lines are even less sensitive than primary cells (Benachour and Seralini, 2009, L’Azou et al., 2005), and therefore are possibly representative of a real cellular toxicity. For cytotoxicity measurements, we assayed mitochondrial succinate dehydrogenase (SD) activity (MTT assay), G and its formulations are indeed known to target mitochondria (Astiz et al., 2009, Peixoto, 2005). Cytotoxicity was also characterized by the measurement of apoptosis and necrosis, respectively by caspases 3/7 activation (Liu et al., 2005) and adenylate kinase leakage after membrane alterations (Crouch et al., 1993).

Overall, we questioned if an active toxic principle in a target species may be always generalized as such in a non target one, and thus if the regulatory toxicological tests on active principles alone are relevant.

Section snippets

Chemicals

Glyphosate (N-phosphonomethyl glycine, G, CAS: 1071-83-6) was purchased from Sigma–Aldrich (Saint Quentin Fallavier, France). GBH formulations available on the market were by alphabetical order: Bayer GC (12.5% of G, 1–5% of POE-15, homologation 05873567), Clinic EV (42% of G, 11% of POE-15, homologation 9900039), Genamin T200 (60–80% of POE-15, homologation 8500170), Glyphogan (39–43% of G, 13–18% of POE-15, homologation 9100537), Roundup Grand Travaux (400 g/L of G, R GT, homologation

Results

Here we studied for the first time the precise involvement of the adjuvants and G in GBH induced toxicity, on three human cell lines from different embryonic origins (kidney, liver, and placenta) in order to test their specificities. We first compared mitochondrial respiration (SD activity) in presence of 9 formulated mixtures of G and adjuvants, G alone, formulating agents without G (Genamin), and a major adjuvant of some formulations, POE-15 (Fig. 2). All chemicals are cytotoxic, inducing

Discussion

This study unravels the differential nature and cytotoxicity of the main compounds from the major herbicide formulations in the world. These formulations are conceived to enhance the pesticide activity through mixtures of adjuvants and G. The latter is the active principle toxic in plants; in this study we checked how this active principle is differentially toxic on non-target organisms in comparison to the so-called inert adjuvants in numerous formulations.

Here we demonstrate that all

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

We gratefully thank Angelo San Filippo for technical support for adjuvants isolation. We acknowledge the Regional Council of Low Normandy for R.M. fellowship, but also the Charles Leopold Mayer (FPH) and Denis Guichard Foundations, together with CRIIGEN, for structural supports.

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