Co-Formulants in Glyphosate-Based Herbicides Disrupt Aromatase Activity in Human Cells below Toxic Levels

Abstract

Pesticide formulations contain declared active ingredients and co-formulants presented as inert and confidential compounds. We tested the endocrine disruption of co-formulants in six glyphosate-based herbicides (GBH), the most used pesticides worldwide. All co-formulants and formulations were comparably cytotoxic well below the agricultural dilution of 1% (18–2000 times for co-formulants, 8–141 times for formulations), and not the declared active ingredient glyphosate (G) alone. The endocrine-disrupting effects of all these compounds were measured on aromatase activity, a key enzyme in the balance of sex hormones, below the toxicity threshold. Aromatase activity was decreased both by the co-formulants alone (polyethoxylated tallow amine—POEA and alkyl polyglucoside—APG) and by the formulations, from concentrations 800 times lower than the agricultural dilutions; while G exerted an effect only at 1/3 of the agricultural dilution. It was demonstrated for the first time that endocrine disruption by GBH could not only be due to the declared active ingredient but also to co-formulants. These results could explain numerous in vivo results with GBHs not seen with G alone; moreover, they challenge the relevance of the acceptable daily intake (ADI) value for GBHs exposures, currently calculated from toxicity tests of the declared active ingredient alone.

Re-registration Challenges of Glyphosate in the European Union

“…In contrast, cell types with the lowest apparent sensitivity were human choriocarcinoma cells (JEG3) (Benachour et al, 2007;Benachour and Séralini, 2009;Gasnier et al, 2009;Romano et al, 2010;Mesnage et al, 2013a), human chorioplacental cells (JAr) (Young et al, 2015), human hepatoma cells (HepG2) (Benachour et al, 2007;Benachour and Séralini, 2009;Gasnier et al, 2009Gasnier et al, , 2010, murine osteoblast precursor cells (MC3T3-E1) (Farkas et al, 2018), human embryonic kidney cells (HEK293) (Benachour et al, 2007;Benachour and Séralini, 2009;Gasnier et al, 2009;Mesnage et al, 2013a), and human primary neonate umbilical vein endothelial cells (HUVEC) (Benachour et al, 2007;Benachour and Séralini, 2009;Gasnier et al, 2009). Cytotoxicity has also been detected by other biochemical markers e.g., mitochondrial functions, release of lactate dehydrogenase, cell proliferation determined by the use of sulforhodamine B, or membrane integrity and lysosomal activities indicated by the uptake of neutral red dye (Koller et al, 2012;Defarge et al, 2016). The interaction of between glyphosate and mitochondrial succinate dehydrogenase has been verified by molecular modeling (Ugarte, 2014).…”

“…Correlations were less apparent for other biochemical end points e.g., endocrine disrupting effects. Glyphosate was found to inhibit aromatases in JEG3 cells with EC 50 values of 7 mg/ml (Richard et al, 2005), but causing ∼10% inhibition only at 0.024 mg/ml (Defarge et al, 2016). It has not been reported to exert estrogen agonist effects in the estrogen receptor activation-reporter assay on JEG3 cells, but was proven to be anti-androgenic at subagricultural and non-cytotoxic dilutions (Gasnier et al, 2009).…”

“…Although reviews of genotoxicity studies deemed DNA damage by glyphosate and glyphosate-based formulations secondary to cytotoxic effects (Kier and Kirkland, 2013;Kier, 2015), DNA-damaging effects and genotoxicity of glyphosate and particularly of its formulations (Roundup R , Glyfos R , Glyphogan R , Glyphosate-Biocarb R , etc.) on vertebrates (murine and human cells) (Bolognesi et al, 1997;Koller et al, 2012;Young et al, 2015;Townsend et al, 2017), cytotoxic effects of glyphosate-based herbicides on human embryonic and placental cells (Benachour et al, 2007;Benachour and Séralini, 2009;Gasnier et al, 2009Gasnier et al, , 2010Mesnage et al, 2013a,b), indication of endocrine disrupting effects by showing activity on estrogen receptors in human hormone-dependent breast cancer cells (Thongprakaisang et al, 2013;Mesnage et al, 2017a), inhibition of the biosynthesis of testosterone and estradiol (Romano et al, 2010) and progesterone (Young et al, 2015) or inhibitory effects on aromatase, a key enzyme in steroid hormone biosynthesis (Cassault-Meyer et al, 2014;Defarge et al, 2016), teratogenic effects on vertebrates by inhibiting the retinoic acid signaling pathway (Lajmanovich et al, 2003;Paganelli et al, 2010;Carrasco, 2013), birth defects in rats , and nephrotoxic and hepatotoxic effects of Roundup R have been demonstrated in rats in connection to RR GM maize (originally published in the journal Food and Chemical Toxicology in September 2012, but retracted by the journal in November 2013 following an alleged intervention from the industry stakeholder (Foucart, 2016), and subsequently republished in another journal a year later) . The analysis of kidney and liver tissues from the same rats by molecular profiling (transcriptomics, proteomics, metabolomics) confirmed pathology of these organs in the lowest dose Roundup R treatment group culminating in non-alcoholic fatty liver disease (Mesnage et al, 2015a(Mesnage et al, ,b, 2017a.…”

“…Such mitigation in the requirements is consonant with the increase proposed also in the ADI value for glyphosate, on the one hand, yet appears to reflect as if regulatory rigor yielded to technology, as limits previously considered necessary and accepted by industry are proposed to be replaced with more permissive ones, on the other hand. As for compliance of MRL, it is worth mentioning that negative health outcomes have been observed in laboratory animal studies showing toxicity at levels of exposure below regulatory set safety limits (Mesnage et al, 2015b(Mesnage et al, , 2017bDefarge et al, 2016).…”

On glyphosate

“…Consequently, those adjuvants and their glyphosate formulations have gone under scrutiny (Mesnage et al 2013a), also in conjunction with parallel exposure to Bacillus thuringiensis toxins (Mesnage et al 2013b). As regards to glyphosate-based herbicides, it is still not clear how many or what percentage of its side effects described in the scientific literature are due to those "inert" components components (Szekacs et al, 2014;Defarge et al, 2016). However, a certain group of adjuvants, the frequently used tallow amines, have been banned recently (AgriLand 2016).…”