Formulants of glyphosate-based herbicides have more deleterious impact than glyphosate on TM4 Sertoli cells.

Roundup and Glyphogan are glyphosate-based herbicides containing the same concentration of glyphosate and confidential formulants. Formulants are declared as inert diluents but some are more toxic than glyphosate, such as the family of polyethoxylated alkylamines (POEA). We tested glyphosate alone, glyphosate-based herbicide formulations and POEA on the immature mouse Sertoli cell line (TM4), at concentrations ranging from environmental to agricultural-use levels. Our results show that formulations of glyphosate-based herbicides induce TM4 mitochondrial dysfunction (like glyphosate, but to a lesser extent), disruption of cell detoxification systems, lipid droplet accumulation and mortality at sub-agricultural doses. Formulants, especially those present in Glyphogan, are more deleterious than glyphosate and thus should be considered as active principles of these pesticides. Lipid droplet accumulation after acute exposure to POEA suggests the rapid penetration and accumulation of formulants, leading to mortality after 24 h. As Sertoli cells are essential for testicular development and normal onset of spermatogenesis, disturbance of their function by glyphosate-based herbicides could contribute to disruption of reproductive function demonstrated in mammals exposed to these pesticides at a prepubertal stage of development.

Dig1 protects against locomotor and biochemical dysfunctions provoked by Roundup.

BACKGROUND: Plant medicinal extracts may be claimed to prevent or cure chemical intoxications. Few of these are tested for their mechanisms of actions in vivo and for their cellular impacts. In 2011, we demonstrated that hepatic cell mortality induced by environmentally realistic levels of the widely used herbicide Roundup (R) in vitro can be almost entirely prevented by plant extracts called Dig1 (D, Digeodren). METHODS: We tested the in vivo effects of D alone (1.2 ml/kg bw/d), but also prior to and during 8 days of R intoxication (at 135 mg/kg bw/d) in a total of 4 groups of 40 adult Sprague-Dawley male rats each. After treatments, horizontal and vertical locomotor activities of the animals were measured by use of actimeters. Brain, liver, kidneys, heart and testes were collected and weighted. Body weights as well as feed and water consumption were recorded. Proteins, creatinine, urea, phosphate, potassium, sodium, calcium, chloride ions, testosterone, estradiol, AST and ALT were measured in serum. In liver S9 fractions, GST, GGT, and CYP450 (1A2, 2C9, 2C19, 2D6, 3A4) were assessed. RESULTS: D did not have any physiological or biochemical observable impact alone at 2 %. Out of a total of 29 measured parameters, 8 were significantly affected by R absorption within only 8 days. On these 8 parameters, only 2 were not restored by D (GGT activity and plasmatic phosphate), 5 were totally restored (horizontal and vertical locomotor activities, CYP2D6 activity, plasmatic Na + and estradiol), and the 6th was almost restored (plasmatic K+). The specificities of the toxic effects of R and of the therapeutic effects of D treatment were thus demonstrated, both at the behavioural and biochemical levels. CONCLUSIONS: D, without any side effect observable in these conditions, presented strong preventive and therapeutic properties in vivo after a short-term intoxication by the widely used pesticide Roundup.

Effects of Roundup(®) and glyphosate on three food microorganisms: Geotrichum candidum, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus.

Use of many pesticide products poses the problem of their effects on environment and health. Amongst them, the effects of glyphosate with its adjuvants and its by-products are regularly discussed. The aim of the present study was to shed light on the real impact on biodiversity and ecosystems of Roundup(®), a major herbicide used worldwide, and the glyphosate it contains, by the study of their effects on growth and viability of microbial models, namely, on three food microorganisms (Geotrichum candidum, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus) widely used as starters in traditional and industrial dairy technologies. The presented results evidence that Roundup(®) has an inhibitory effect on microbial growth and a microbicide effect at lower concentrations than those recommended in agriculture. Interestingly, glyphosate at these levels has no significant effect on the three studied microorganisms. Our work is consistent with previous studies which demonstrated that the toxic effect of glyphosate was amplified by its formulation adjuvants on different human cells and other eukaryotic models. Moreover, these results should be considered in the understanding of the loss of microbiodiversity and microbial concentration observed in raw milk for many years.

A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cells in vitro, and testosterone decrease at lower levels.

The major herbicide used worldwide, Roundup, is a glyphosate-based pesticide with adjuvants. Glyphosate, its active ingredient in plants and its main metabolite (AMPA) are among the first contaminants of surface waters. Roundup is being used increasingly in particular on genetically modified plants grown for food and feed that contain its residues. Here we tested glyphosate and its formulation on mature rat fresh testicular cells from 1 to 10000ppm, thus from the range in some human urine and in environment to agricultural levels. We show that from 1 to 48h of Roundup exposure Leydig cells are damaged. Within 24-48h this formulation is also toxic on the other cells, mainly by necrosis, by contrast to glyphosate alone which is essentially toxic on Sertoli cells. Later, it also induces apoptosis at higher doses in germ cells and in Sertoli/germ cells co-cultures. At lower non toxic concentrations of Roundup and glyphosate (1ppm), the main endocrine disruption is a testosterone decrease by 35%. The pesticide has thus an endocrine impact at very low environmental doses, but only a high contamination appears to provoke an acute rat testicular toxicity. This does not anticipate the chronic toxicity which is insufficiently tested, and only with glyphosate in regulatory tests.

Effects of the methanol extract of Basella alba L (Basellaceae) on steroid production in Leydig cells.

In this study, Leydig cells were purified from 70 day-old Sprague Dawley male rats and incubated with 10 and 100 µg/mL of methanol extract of Basella alba (MEBa) for 4 hours followed by the evaluation of cell viability, steroid (testosterone and estradiol) production, and the level of aromatase mRNA. Results showed that MEBa did not affect Leydig cell viability. At the concentration of 10 µg/mL, MEBa significantly stimulated testosterone and estradiol production (p < 0.01 and p < 0.03, respectively), and enhanced aromatase mRNA level (p < 0.04). These observations suggest that MEBa directly stimulated testosterone, estradiol and aromatase mRNA levels in isolated Leydig cells.

Defined plant extracts can protect human cells against combined xenobiotic effects.

BACKGROUND: Pollutants representative of common environmental contaminants induce intracellular toxicity in human cells, which is generally amplified in combinations. We wanted to test the common pathways of intoxication and detoxification in human embryonic and liver cell lines. We used various pollutants such as Roundup residues, Bisphenol-A and Atrazine, and five precise medicinal plant extracts called Circ1, Dig1, Dig2, Sp1, and Uro1 in order to understand whether specific molecular actions took place or not. METHODS: Kidney and liver are major detoxification organs. We have studied embryonic kidney and hepatic human cell lines E293 and HepG2. The intoxication was induced on the one hand by a formulation of one of the most common herbicides worldwide, Roundup 450 GT+ (glyphosate and specific adjuvants), and on the other hand by a mixture of Bisphenol-A and Atrazine, all found in surface waters, feed and food. The prevention and curative effects of plant extracts were also measured on mitochondrial succinate dehydrogenase activity, on the entry of radiolabelled glyphosate (in Roundup) in cells, and on cytochromes P450 1A2 and 3A4 as well as glutathione-S-transferase. RESULTS: Clear toxicities of pollutants were observed on both cell lines at very low sub-agricultural dilutions. The prevention of such phenomena took place within 48 h with the plant extracts tested, with success rates ranging between 25-34% for the E293 intoxicated by Roundup, and surprisingly up to 71% for the HepG2. By contrast, after intoxication, no plant extract was capable of restoring E293 viability within 48 h, however, two medicinal plant combinations did restore the Bisphenol-A/Atrazine intoxicated HepG2 up to 24-28%. The analysis of underlying mechanisms revealed that plant extracts were not capable of preventing radiolabelled glyphosate from entering cells; however Dig2 did restore the CYP1A2 activity disrupted by Roundup, and had only a mild preventive effect on the CYP3A4, and no effect on the glutathione S-transferase. CONCLUSIONS: Environmental pollutants have intracellular effects that can be prevented, or cured in part, by precise medicinal plant extracts in two human cell lines. This appears to be mediated at least in part by the cytochromes P450 modulation.

Dig1 protects against cell death provoked by glyphosate-based herbicides in human liver cell lines.

BACKGROUND: Worldwide used pesticides containing different adjuvants like Roundup formulations, which are glyphosate-based herbicides, can provoke some in vivo toxicity and in human cells. These pesticides are commonly found in the environment, surface waters and as food residues of Roundup tolerant genetically modified plants. In order to know their effects on cells from liver, a major detoxification organ, we have studied their mechanism of action and possible protection by precise medicinal plant extracts called Dig1. METHODS: The cytotoxicity pathways of four formulations of glyphosate-based herbicides were studied using human hepatic cell lines HepG2 and Hep3B, known models to study xenobiotic effects. We monitored mitochondrial succinate dehydrogenase activity and caspases 3/7 for cell mortality and protection by Dig1, as well as cytochromes P450 1A1, 1A2, 3A4 and 2C9 and glutathione-S-transferase to approach the mechanism of actions. RESULTS: All the four Roundup formulations provoke liver cell death, with adjuvants having stronger effects than glyphosate alone. Hep3B are 3-5 times more sensitive over 48 h. Caspases 3/7 are greatly activated in HepG2 by Roundup at non-cytotoxic levels, and some apoptosis induction by Roundup is possible together with necrosis. CYP3A4 is specifically enhanced by Roundup at doses 400 times less than used in agriculture (2%). CYP1A2 is increased to a lesser extent together with glutathione-S-transferase (GST) down-regulation. Dig 1, non cytotoxic and not inducing caspases by itself, is able to prevent Roundup-induced cell death in a time-dependant manner with an important efficiency of up to 89%, within 48 h. In addition, we evidenced that it prevents Caspases 3/7 activation and CYP3A4 enhancement, and not GST reduction, but in turn it slightly inhibited CYP2C9 when added before Roundup. CONCLUSION: Roundup is able to provoke intracellular disruption in hepatic cell lines at different levels, but a mixture of medicinal plant extracts Dig1 can protect to some extent human cell lines against this pollutants. All this system constitutes a tool for studying liver intoxication and detoxification.

First synthesis of 7alpha- and 7beta-amino-DHEA, dehydroepiandrosterone (DHEA) analogues and preliminary evaluation of their cytotoxicity on Leydig cells and TM4 Sertoli cells.

Efficient syntheses of new DHEA analogues, and their apoptotic and necrotic effects on Leydig cells and TM4 Sertoli cells are described. The key step in the synthetic strategy of 7-amino-DHEA derivatives involves a bromination on C-7 position to give an epimeric mixture of bromides which were substituted by azides and reduced to give 7alpha- and 7beta-amino-3beta-hydroxyandrost-5-en-17-ones. No cytotoxic effect induced by apoptosis mechanism was observed on Leydig and TM4 Sertoli cells by treatment with these amino-DHEA analogues. A necrotic effect was induced only in TM4 Sertoli cells. The best activity was obtained with 7alpha,beta-amino-androst-5-en-3beta-ol and 7beta-amino-3beta-hydroxy-androst-5-en-17-one.

Induction of apoptosis by 25-hydroxycholesterol in adult rat Leydig cells: protective effect of 17beta-estradiol.

Testicular macrophages can convert cholesterol into 25-hydroxycholesterol which strongly stimulates Leydig cell testosterone production. We demonstrated that 25-hydroxycholesterol reduced cholesterol biosynthesis in adult rat Leydig cells. This oxysterol can also be cytotoxic. As hydroxylated cholesterol can induce apoptosis in various cells, we investigated cell death produced by 25-hydroxycholesterol. Apoptosis was characterized by TUNEL assay and by DAPI test. Addition of 25-hydroxycholesterol, during 24h, induced a dose dependent increase of apoptosis. This effect was reduced by a treatment with a caspase-3 inhibitor (Ac-DEVD-CHO). 25-Hydroxycholesterol is known to stimulate testosterone production, but an increase of intracellular or culture medium testosterone level does not modify significantly the percentage of apoptotic cells. In contrast, addition of 17beta-estradiol (2 nM) induced a decrease of apoptotic cells. These data suggested that this oxysterol can be used by rat Leydig cells in culture for sterol metabolism, but also induces apoptosis which could be inhibited by 17beta-estradiol.