Elucidating the effects of pure glyphosate and a commercial formulation on early life stages of zebrafish using a complete biomarker approach: All-or-nothing!

The search for new methods in the toxicology field has increased the use of early life stages of zebrafish (Danio rerio) as a versatile organism model. Here, we use early stages of zebrafish to evaluate glyphosate as pure active ingredient and within a commercial formulation in terms of oxidative stress. Biomarkers involved in the oxidative status were evaluated along with other markers of neurotoxicity, genotoxicity, cytotoxicity, energy balance and motor performance, and the selected tools were evaluated by its sensitivity in determining early-warning events. Zebrafish embryos exposed to glyphosate active ingredient and glyphosate-based formulation were under oxidative stress, but only the commercial formulation delayed the embryogenesis, affected the cholinergic neurotransmission and induced DNA damage. Both altered the motor performance of larvae at very low concentrations, becoming larvae hypoactive. The energy balance was also impaired, as embryos under oxidative stress had lower lipids reserves. Although data suggest that glyphosate-based formulation has higher toxicity than the active ingredient itself, the most sensitive biomarkers detected early-warning effects at very low concentrations of the active ingredient. Biochemical biomarkers of defense system and oxidative damage were the most sensitive tools, detecting pro-oxidant responses at very low concentrations, along with markers of motor performance that showed high sensitivity and high throughput, suitable for detecting early effects linked to neurotoxicity. Alterations on morphology during embryogenesis showed the lowest sensitivity, thus morphological alterations appeared after several alterations at biochemical levels. Tools evaluating DNA damage and cell proliferation showed mid-sensitivity, but low throughput, thus they could be used as complementary markers.

Evaluation of neurological behavior alterations and metabolic changes in mice under chronic glyphosate exposure.

Glyphosate is a widely used active ingredient in agricultural herbicides, inhibiting the biosynthesis of aromatic amino acids in plants by targeting their shikimate pathway. Our gut microbiota also facilitates the shikimate pathway, making it a vulnerable target when encountering glyphosate. Dysbiosis in the gut microbiota may impair the gut-brain axis, bringing neurological outcomes. To evaluate the neurotoxicity and biochemical changes attributed to glyphosate, we exposed mice with the reference dose (RfD) set by the U.S. EPA (1.75 mg/Kg-BW/day) and its hundred-time-equivalence (175 mg/Kg-BW/day) chronically via drinking water, then compared a series of neurobehaviors and their fecal/serum metabolomic profile against the non-exposed vehicles (n = 10/dosing group). There was little alteration in the neurobehavior, including motor activities, social approach, and conditioned fear, under glyphosate exposure. Metabolomic differences attributed to glyphosate were observed in the feces, corresponding to 68 and 29 identified metabolites with dysregulation in the higher and lower dose groups, respectively, compared to the vehicle-control. There were less alterations observed in the serum metabolome. Under 175 mg/Kg-BW/day of glyphosate exposure, the aromatic amino acids (phenylalanine, tryptophan, and tyrosine) were reduced in the feces but not in the serum of mice. We further focused on how tryptophan metabolism was dysregulated based on the pathway analysis, and identified the indole-derivatives were more altered compared to the serotonin and kynurenine derivatives. Together, we obtained a three-dimensional data set that records neurobehavioral, fecal metabolic, and serum biomolecular dynamics caused by glyphosate exposure at two different doses. Our data showed that even under the high dose of glyphosate irrelevant to human exposure, there were little evidence that supported the impairment of the gut-brain axis.

Hormesis of glyphosate on ferulic acid metabolism and antifungal volatile production in rice root biocontrol endophyte Burkholderia cepacia LS-044.

Glyphosate (GP, N-phosphonomethyl glycine) is one of the most popular organophosphate herbicides widely used in agricultural practices worldwide. There have been extensive reports on the biohazard attributes and hormetic impacts of GP on plant and animal systems. However, the effects of GP on plant growth-promoting microbes and its ecological relevance remain unknown. Here, we show that GP does exert a hormetic impact on Burkholderia cepacia LS-044, a rice (Oryza sativa ssp. japonica cv. Tainung 71) root endophytic isolate. We used increasing doses of ferulic acid (FA, 1-25 mM) and GP (0.5-5 mM) to test the growth and antifungal volatile production in LS-044 by electrochemical, liquid chromatographic, gas chromatographic and spectrophotometric means. GP treatment at a low dose (0.5 mM) increased FA utilization and significantly (P < 0.0001) enhanced antifungal volatile activity in LS-044. Although FA (1 mM) was rapidly utilized by LS-044, no chromatographically detectable utilization of GP was observed at tested doses (0.5-5 mM). LS-044 emitted predominant amounts of tropone in addition to moderate-to-minor amounts of diverse ketones and/or their derivatives (acetone, acetophenone, 2-butanone, 1-propanone, 1-(2-furanyl-ethanone, 1-phenyl-1-propanone and 1-(3-pyridinyl)-1-propanone), d-menthol, 2-methoxy-3-(1-methylethyl)-pyrazine, dimethyl disulfide, pyridine and ammonium carbamate when grown under GP supplement. GP hormesis on LS-044 induced phenotypic variations in O. sativa ssp. japonica cv. Tainan 11 as evident through seed germination assay. Genes involved in the transformation of FA, and a key gene encoding 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) with Gly-94 and Tyr-95 residues localized at active site most likely rendering EPSPS sensitivity to GP, were detected in LS-044. This is the first report on the GP hormesis influencing morphological and metabolic aspects including volatile emission in a biocontrol bacterium that could modulate rice plant phenotype.

Melatonin ameliorates renal dysfunction in glyphosate- and hard water-treated mice.

Chronic interstitial nephritis in agricultural communities (CINAC) is a severe and widespread disease that has been associated with environmental and occupational exposure to glyphosate and hard water. However, the potential underlying mechanisms remain incompletely understood. Melatonin is reported to exert protective effects on the kidney, but whether melatonin can attenuate renal tubular injury in mice exposed to glyphosate combined with hard water is unclear. Here, mice were treated with high doses and environmentally relevant doses of glyphosate (100 mg/kg·bw and 0.7 mg/L, respectively) and/or hard water (2500 mg/L CaCO3 and 250 mg/L Ca2+, respectively) via their drinking water for 12 weeks. We found that high-dose glyphosate or hard water treatment significantly increased the levels of biomarkers of renal damage, including ß2-microglobulin, neutrophil gelatinase-associated lipid carrier protein, and/or albumin, in the urine; these increased biomarker levels were correlated with obvious morphological changes, and all of these changes were also observed in animals exposed to environmentally relevant doses of glyphosate and/or high Ca2+ water. Melatonin (10 mg/kg·bw, intraperitoneal injection, daily for 12 weeks) administered concomitantly with high doses of glyphosate and hard water inhibited the glyphosate- and hard water-induced increases in the levels of kidney injury biomarkers and changes in morphology; this result was intriguing. Additionally, glyphosate combined with hard water at both high and environmentally relevant doses significantly upregulated the expression of the endoplasmic reticulum (ER) stress marker proteins Bip, ATF6, and PERK as well as the pyroptosis-related proteins (NLRP3 and caspase 1 signaling proteins) in renal tissues. Similarly, melatonin significantly attenuated the increased ER stress and pyroptosis induced by high doses of glyphosate and hard water. In summary, we conclude that exposure to glyphosate and hard water at both high doses and environmentally relevant doses causes renal dysfunction in mice, and this dysfunction can be attenuated by melatonin, possibly through the inhibition of ER stress and pyroptosis. Our results support the notion that melatonin may have therapeutic potential for the treatment of chronic kidney diseases.

Glyphosate and neurological outcomes: A systematic literature review of animal studies.

Studies of nervous system effects of glyphosate, a widely used herbicide, have not been critically examined. The aim of this paper was to systematically review glyphosate-induced neurotoxicity literature to determine its usefulness in regulatory decision-making. The review was restricted to mammalian studies of behavior, neuropathology, and neuropharmacology; in vitro and other biochemical studies were considered supplementary information. Glyphosate formulation studies were also considered, despite uncertainties regarding toxicities of the formulated products; no studies used a formulation vehicle as the control. Inclusion criteria were developed a priori to ensure consistent evaluation of studies, and in vivo investigations were also ranked using ToxRTool software to determine reliability. There were 27 in vivo studies (open literature and available regulatory reports), but 11 studies were considered unreliable (mostly due to critical methodological deficiencies). There were only seven acceptable investigations on glyphosate alone. Studies differed in terms of dosing scenarios, experimental designs, test species, and commercial product. Limitations included using only one dose and/or one test time, small sample sizes, limited data presentation, and/or overtly toxic doses. While motor activity was the most consistently affected endpoint (10 of 12 studies), there were considerable differences in outcomes. In six investigations, there were no marked neuropathological changes in the central or peripheral nervous system. Other neurological effects were less consistent, and some outcomes were less convincing due to influences including high variability and small effect sizes. Taken together, these studies do not demonstrate a consistent impact of glyphosate on the structure or function of the mammalian nervous system.

Response of the nuclear xenobiotic receptors to alleviate glyphosate-based herbicide-induced nephrotoxicity in weaned piglets.

Glyphosate-based herbicides (GBHs) are widely used worldwide. Glyphosate (GLP) is the main active component of GBHs. The presence of GBH residues in the environment has led to the exposure of animals to GBHs, but the mechanisms of GBH-induced nephrotoxicity are not clear. This study investigated the effects of GBHs on piglet kidneys. Twenty-eight healthy female hybrid weaned piglets (Duroc × Landrace × Yorkshire) with an average weight of 12.24 ± 0.61 kg were randomly divided into four treatment groups (n=7 piglets/group) that were supplemented with Roundup® (equivalent to GLP concentrations of 0, 10, 20, and 40 mg/kg) for a 35-day feeding trial. The results showed that the kidneys in the 40-mg/kg GLP group suffered slight damage. Roundup® significantly decreased the activity of catalase (CAT) (P=0.005) and increased the activity of superoxide dismutase (SOD) (P=0.029). Roundup® increased the level of cystatin-C (Cys-C) in the plasma (linear, P=0.002 and quadratic, P=0.015). The levels of neutrophil gelatinase-associated lipocalin (NGAL) in plasma increased linearly (P=0.007) and quadratically (P=0.003) as the dose of GLP increased. The mRNA expression of intercellular cell adhesion molecule-1 (ICAM-1) in the 20-mg/kg GLP group was increased significantly (P<0.05). There was a significant increase in the mRNA levels of pregnenolone X receptor (PXR), constitutive androstane receptor (CAR), and uridine diphosphate glucuronosyltransferase 1A3 (UGT1A3) (P<0.05). Our findings found that kidney nuclear xenobiotic receptors (NXRs) may play an important role in defense against GBHs.

A subchronic low-dose exposure of a glyphosate-based herbicide induces depressive and anxious-like behavior in mice: quercetin therapeutic approach.

In this study, we investigated the possible role of pesticide exposure in contributing to neurological diseases such as depression. Here, we evaluated whether a subchronic low dose of a glyphosate-based herbicide (GBH) could induce alterations in the central nervous system, using the flavonoid quercetin as a therapeutic strategy. Forty mice were divided into four treatment groups: control, GBH, quercetin, and GBH+Quer groups and received 50 mg/kg of GBH solution, 30 mg/kg of quercetin, and/or vehicles for 30 days via gavage. After performing behavioral tests, such as the open field (OF), elevated plus maze (EPM), forced swim test (FST), and sucrose preference test (SPT), the mice were euthanized and their hippocampal tissues were collected to measure the levels of oxidative stress markers such as reactive species (RS), total antioxidant capacity (FRAP), reduced glutathione (GSH), and acetylcholinesterase activity (AChE), as well as for histological evaluation. The GBH group showed anxious and depressive-like behavior in the EPM and FST tests, as well as increased levels of RS and decreased GSH levels in the hippocampus. Quercetin treatment in the GBH+Quer group allowed partial or total improvement in behavioral tests (EPM and FST) and in the levels of oxidative stress markers (RS and GSH). However, the quercetin group showed similar behavior to the GBH group after treatment. The results revealed that oral exposure to a subchronic low dose of GBH was capable of promoting effects on behavior and oxidative stress in the hippocampus of mice. In addition, despite quercetin having a neuroprotective role, caution is needed when considering the possible per se effects of its continuous supplementation.

Growth regulation of bermudagrass (Cynodon dactylon) and zoysiagrass (Zoysia japonica) with glyphosate.

Glyphosate can generate positive effects on turfgrass maintenance as a form of growth control by decreasing the expenses associated with mowing. However, there is little information about the effects of this herbicide on turfgrasses. This study aimed to evaluate the response of bermudagrass and zoysiagrass to the herbicide glyphosate as a growth regulator. Two studies were performed in a greenhouse and repeated at different times. The treatments involved application of glyphosate at 10 different rates (0, 5.625, 11.25, 22.5, 45, 90, 180, 360, 720, and 1.440 g ae ha-1) with four replicates. Evaluations of green cover by digital analysis, injury, and plant height were performed at 7, 14, 21, and 28 days after application, and shoot dry matter of clippings was determined for the last evaluation period. Bermudagrass and zoysiagrass presented variedtolerance to glyphosate toxicity. Overall, the digital analysis showed that green content was negatively influenced by the increase in visual injury caused by glyphosate application. Moreover, increasing the glyphosate rate decreased plant height and shoot dry matter in both turfgrasses. Glyphosate application rates up to 45 g ae ha-1 for bermudagrass and 90 g ae ha-1 for zoysiagrass decreased plant growth without affecting the factors analyzed in this study.

The use of combined high-fructose diet and glyphosate to model rats type 2 diabetes symptomatology.

An ideal food-chemical combination that will promote insulin resistance and its consequent development of pancreatic beta-cell dysfunction may open a new vista for Type 2 diabetes (T2D) research. Thus, we investigated the modulatory effects of a high-fructose diet (FRC) combined with glyphosate (GP). Male albino Wistar rats were randomly divided into five groups of eight/group and received distilled water, FRC, GP, and their combinations orally for eight consecutive weeks. We assessed the changes in fasting blood glucose levels (FBGLs), biochemical indices, oxidative stress parameters, and organ histopathology. From the results obtained, FBGLs and serum insulin levels were increased in the FRC-GP (2.3-3.1 and 1.9-2.2 folds) treated rats compared with the control baseline group. Also, the FRC-GP high dose increased FBGLs (1.9 folds), insulin (1.4 folds), triglycerides (1.5 folds), and uric acid (2 folds) levels compared with the FRC group. Malondialdehyde levels increased in the pancreas (54% and 78%) and liver (31.3% and 56.6%) of the FRC-GP treated rats. The FRC-GP treatments reduced serum high-density lipoprotein (57%), total protein (47%), and antioxidant parameters (non-enzymatic and enzymatic, 1.6-1.9 folds) respectively in the treated animals. The weight of the pancreas relative to the body increased (2-3 folds) while we observed mild inflammation and vascular congestion in vital organs in the treated rats. Overall, these results demonstrate the potential of FRC-GP-diet to induce conditions of rats T2D. Also, this novel finding suggests a cost-effective GP as an alternative in this model type and provides further insight into understanding FRC-GP interactions.

Protective effects of black seed (Nigella sativa) diet supplementation in common carp (Cyprinus carpio) against immune depression, oxidative stress and metabolism dysfunction induced by glyphosate.

Sustainable aquaculture arises as key to increase food production in the coming years. However, the sector still faces many challenges such as the exposure of the cultured animals to pesticide-contaminated water. Pesticides used in agriculture can reach aquaculture systems either directly (integrated-agriculture aquaculture practices) or indirectly (soil leakage) and cause a broad range of ecotoxicological effects on cultured fish and shellfish. Here, we studied how glyphosate affects several haematological, biochemical, and immune parameters in common carp (Cyprinus carpio) fingerlings, the fourth most important cultured fish species worldwide. We also evaluated the potential of dietary supplementation with black seed (Nigella sativa, 0.25, 0.5 and 1%) to lower glyphosate-associated toxicity. Our results showed that 14-day sub-lethal exposure of common carp fingerlings to glyphosate increases oxidative stress, decreases antioxidant defences, affects several metabolic pathways, and induced immune depression. Furthermore, we showed that fish fed with N. sativa-enriched diets at 0.25, 0.5 and 1% for 60 days coped better with glyphosate exposure than control fish and displayed more stable levels of biochemical serum parameters (total protein, albumin, triglycerides, low-density lipoprotein LDL), cholesterol and high-density lipoprotein HDL), higher levels of immune defences (lysozyme and immunoglobulin) and higher antioxidant enzymes (superoxide dismutase SOD, glutathione peroxidase GPx) than control fish. Fish fed with all enriched diets also displayed lower lipid peroxidation (malondialdehyde MDA), lower metabolic enzymes (alanine aminotransferase ALT, aspartate aminotransferase AST and alkaline phosphatase ALP) levels in blood serum and lower cortisol levels than control fish. Altogether, our results show that dietary inclusion of black seed can be used as a sustainable bio-remediation strategy, mitigating many of the negative effects of glyphosate exposure in fish.

Gene expression response of the alga Fucus virsoides (Fucales, Ochrophyta) to glyphosate solution exposure.

Fucus virsoides is an ecologically important canopy-forming brown algae endemic to the Adriatic Sea. Once widespread in marine coastal areas, this species underwent a rapid population decline and is now confined to small residual areas. Although the reasons behind this progressive disappearance are still a matter of debate, F. virsoides may suffer, like other macroalgae, from the potential toxic effects of glyphosate-based herbicides. Here, through a transcriptomic approach, we investigate the molecular basis of the high susceptibility of this species to glyphosate solution, previously observed at the morphological and eco-physiological levels. By simulating runoff event in a factorial experiment, we exposed F. virsoides to glyphosate (Roundup® 2.0), either alone or in association with nutrient enrichment, highlighting significant alterations of gene expression profiles that were already visible after three days of exposure. In particular, glyphosate exposure determined the near-complete expression shutdown of several genes involved in photosynthesis, protein synthesis and stress response molecular pathways. Curiously, these detrimental effects were partially mitigated by nutrient supplementation, which may explain the survival of relict population in confined areas with high nutrient inputs.

Discovery of Clinical Candidate (5-(3-(4-Chlorophenoxy)prop-1-yn-1-yl)-3-hydroxypicolinoyl)glycine, an Orally Bioavailable Prolyl Hydroxylase Inhibitor for the Treatment of Anemia.

The design and discovery of a new series of (5-alkynyl-3-hydroxypicolinoyl)glycine inhibitors of prolyl hydroxylase (PHD) are described. These compounds showed potent in vitro inhibitory activity toward PHD2 in a fluorescence polarization-based assay. Remarkably, oral administration of 17, with an IC50 of 64.2 nM toward PHD2, was found to stabilize HIF-α, elevate erythropoietin (EPO), and alleviate anemia in a cisplatin-induced anemia mouse model with an oral dose of 25 mg/kg. Rat and dog studies showed that 17 has good pharmacokinetic properties, with oral bioavailabilities of 55.7 and 54.0%, respectively, and shows excellent safety profiles even at a high dose of 200 mg/kg in these animals. Based on these results, 17 is currently being evaluated in a phase I clinical trial for anemia.

Glyphosate-based herbicide has soil-mediated effects on potato glycoalkaloids and oxidative status of a potato pest.

Glyphosate is the most used herbicide worldwide, targeting physiological pathways in plants. Recent studies have shown that glyphosate can also cause toxic effects in animals. We investigated the glyphosate-based herbicide (GBH)-induced changes in potato (Solanum tuberosum) plant chemistry and the effects of a GBH on the survival rate and oxidative status of the Colorado potato beetle (Leptinotarsa decemlineata). The beetles were reared on potato plants grown in pots containing soil treated with a GBH (Roundup Gold, 450 g/l) or untreated soil (water control). The 2nd instar larvae were introduced to the potato plants and then collected in 2 phases: as 4th instar larvae and as adults. The main glycoalkaloids of the potato plants, α-solanine and α-chaconine, were measured twice during the experiment. The α-solanine was reduced in potato plants grown in GBH-treated soil, which can be detrimental to plant defenses against herbivores. GBH treatment had no effect on the survival rate or body mass of the larvae or the adult beetles. In the larvae, total glutathione (tGSH) concentration and the enzyme activity of catalase (CAT), superoxide dismutase, and glutathione-S-transferase were increased in the GBH treatment group. In the adult beetles, CAT activity and tGSH levels were affected by the interactive effect of GBH treatment and the body mass. To conclude, environmentally relevant concentrations of a GBH can affect the potato plant's glycoalkaloid concentrations, but are not likely to directly affect the survival rate of the Colorado potato beetle, but instead, modify the antioxidant defense of the beetles via diet.

Roundup-induced biochemical and histopathological changes in the liver and kidney of rats: the ameliorative effects of Linum usitatissimum oil.

The present study was undertaken to evaluate the protective effects of Linum usitatissimum oil (LuO) against sub-chronic Roundup (RDP)-induced toxicity and oxidative stress in rats. Rats were divided into four groups: control group (no treatment), RDP group (Roundup at 269.9 mg/kg b.w.), LuO group (0.5 g/kg b.w. of LuO) and RDP+LuO group (RDP and LuO simultaneously). LuO decreased the ferric reducing antioxidant power (FRAP) (IC50=10.36 µg/ml) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (IC50=22.85 mg/ml) in the tested tissues. The 30-day exposure of rats to RDP caused an increase in serum hepatic and renal markers: AST, ALT, ALP, LDH, γGT, bilirubin, urea, and creatinine. In addition, SOD, CAT and GST activities decreased by 43%, 61%, and 61%, respectively, while GPx activity, MDA and PCOs levels increased by 80%, 46%, 25%, respectively. LuO treatment alleviated hepatotoxicity in RDP-treated rats, showing improved levels of oxidative stress biomarkers and plasma biochemical parameters. The histological examination of the liver and kidney confirmed the changes in Roundup-treated rats and demonstrated the protective role of LuO.

Multiparametric analysis of the effectiveness of cisplatin on cutaneous squamous carcinoma cells using two different types of adjuvants.

The objective of this study was to regulate the cytotoxicity of cisplatin (cisPt) minimizing its adverse effects. For this purpose, the lowest cisPt concentration needed to obtain a significant positive response in cutaneous squamous cell carcinoma (cSCC) was explored. Two adjuvant agents as gold nanoparticles (AuNP) and chelating tricine were tested as enhancers in cisPt treatment. Effectiveness of all treatments was assessed by means of biochemical techniques, which offer quantitative data, as well as two microscopy-based techniques that provided qualitative cell imaging. The present work confirms the effectiveness of free cisplatin at very low concentrations. In order to enhance its effectiveness while the side effects were probably diminished, cisPt 3.5 µM was administered with AuNP 2.5 mM, showing an effectiveness practically equal to that observed with free cisPt. However, the second treatment investigated, based on cisPt 3.5 µM combined with tricine 50 mM, enhanced drug effectiveness, increasing the percentage of cells dying by apoptosis. This treatment was even better in terms of cell damage than free cisPt at 15 µM. Images obtained by TEM and cryo-SXT confirmed these results, since a notable number of apoptotic bodies were detected when cisPt was combined with tricine. Thus, tricine was clearly a better adjuvant for cisPt treatments.

Adjuvant contributes Roundup's unexpected effects on A549 cells.

Roundup® (RDP) is one of the most representative glyphosate-based herbicides (GBHs), which extensive use increases pressure on environmental safety and potential human health risk. The aim of this study was to investigate whether the adjuvant polyethoxylated tallow amine (POEA) or the herbicidal active ingredient glyphosate isopropylamine salt (GP) in formulation confers RDP cytotoxicity. We demonstrated that RDP and POEA could inhibit the proliferation of human lung A549 cells. Intracellular biochemical assay indicated that collapse of mitochondrial membrane, release of cytochrome c into cytosol, activation of caspase-9/-3, cleavage of poly (ADP-ribose) polymerase (PARP), oxidative DNA damage, DNA single-strand breaks and double-strand breaks are occurred in RDP and POEA treated A549 cells, not occurred in GP treated A549 cells. We conclude that the RDP's effect of apoptosis and DNA damage on human A549 cells is related to the presence of adjuvant POEA in formulation, independent of the herbicidal active ingredient GP. This study would enrich the theoretical basis of the RDP toxicity effects and attract attention on potential human health and environmental safety threat caused by adjuvant.

Assessment of oxidative balance in hydrophilic cellular environment in Chlorella vulgaris exposed to glyphosate.

The studied hypothesis is that glyphosate (GLY) can affect the oxidative balance in the hydrophilic intracellular medium in non-target Chlorella vulgaris cells. Analytical GLY (5 µM) and a commercial product (RUP) (5 µM) supplementation, did not affect the growth profile. Neither in latent (Lag) nor in exponential (Exp) phase of development, there were significant differences in the cellular abundance, evaluated as cell number, after the supplementation with GLY or RUP. The ascorbyl (A•) content was significantly increased in the presence of GLY or RUP, in Lag and Exp phase of growth. No changes were observed in stationary (St) phase after supplementation with either GLY or RUP. Ascorbate (AH-) content was decreased by 30% in Exp phase of development the presence of RUP. In St phase of the development both, the administration of either GLY or RUP decreased the antioxidant content by 34 and 37%, respectively. The supplementation with GLY and RUP lead to a significant 5- and 10-fold increase in Exp phase, respectively in the A•/AH-content ratio, assessed as a damage/protection ratio in the hydrophilic fraction of the cells, as compared to controls. Neither GLY nor RUP affected the ratio in cells in St phase of development. The data presented here showed experimental evidence that suggested that oxidative balance in the hydrophilic environment is affected by GLY, even at the low to medium concentrations currently used. The effect seems as reversible either because of the magnitude of the herbicide-dependent damage or the antioxidant activity activated.

Toxic effects of glyphosate on intestinal morphology, antioxidant capacity and barrier function in weaned piglets.

At present, the public is paying more attention to the adverse effects of pesticides on human and animal health and the environment. Glyphosate is a broad-spectrum pesticide that is widely used in agricultural production. In this manuscript, the effects of diets containing glyphosate on intestinal morphology, intestinal immune factors, intestinal antioxidant capacity and the mRNA expression associated with the Nrf2 signaling pathway were investigated in weaned piglets. Twenty-eight healthy female hybrid weaned piglets (Duroc × Landrace × Yorkshire) were randomly selected with an average weight of 12.24 ±â€¯0.61 kg. Weaned piglets were randomly assigned into 4 treatment groups and fed a basal diet supplemented with 0, 10, 20, and 40 mg/kg glyphosate for a 35-day feeding trial. We found that glyphosate had no effect on intestinal morphology. In the duodenum, glyphosate increased the activities of CAT and SOD (linear, P < 0.05) and increased the levels of MDA (linear and quadratic, P < 0.05). In the duodenum, glyphosate remarkably increased the relative mRNA expression levels of Nrf2 (linear and quadratic, P < 0.05) and NQO1 (linear and quadratic, P < 0.05) and reduced the relative mRNA expression levels of GPx1, HO-1 and GCLM (linear and quadratic, P < 0.05). In the jejunum, glyphosate remarkably increased the relative mRNA expression levels of Nrf2 (linear and quadratic, P < 0.05) and decreased the relative mRNA expression levels of GCLM (linear and quadratic, P < 0.05). Glyphosate increased the mRNA expression levels of IL-6 in the duodenum (linear and quadratic, P < 0.05) and the mRNA expression levels of IL-6 in the jejunum (linear, P < 0.05). Glyphosate increased the mRNA expression of NF-κB in the jejunum (linear, P = 0.05). Additionally, the results demonstrated that glyphosate linearly decreased the ZO-1 mRNA expression levels in the jejunum and the mRNA expression of claudin-1 in the duodenum (P < 0.05). In the duodenum, glyphosate increased the protein expression levels of Nrf2 (linear, P = 0.025). Overall, glyphosate exposure may result in oxidative stress in the intestines of piglets, which can be alleviated by enhancing the activities of antioxidant enzymes and self-detoxification.

Modulation of antioxidant gene expressions by Roundup® exposure in the decapod Macrobrachium potiuna.

Glyphosate-based herbicides (GBH), including Roundup®, are the most used herbicides in agricultural and non-agricultural areas, which can reach aquatic environments through drift during application or surface runoff. Some studies, mostly in fish, demonstrated that GBH caused oxidative stress in non-target animals. However, only few information is available on the GBH effects in the antioxidant and stress proteins of many other organisms, such as freshwater crustaceans. Thus, we aimed to investigate the effects of environmentally relevant GBH concentrations on the relative transcript expression (RTE) of the superoxide dismutase (sod1), catalase (cat), selenium-dependent glutathione peroxidase (gpx), glutathione-S-transferase (gst), thioredoxin (txn), heat shock protein (hsp70 and hsp90) in the hepatopancreas of the ecologically important freshwater prawn Macrobrachium potiuna. Moreover, this study aimed to assess the gender-differences responses to GBH exposure. Male and female prawns were exposed to three Roundup WG® concentrations (0.0065, 0.065 and 0.28 mg of glyphosate/L) and a control group (0.0 mg/L) for 7 and 14 days. In general, males had an under-expression of the studied genes, indicating an oxidative stress and possible accumulation of ROS in the hepatopancreas. In the opposite, females had an overexpression of the same genes, indicating a more robust antioxidant system, in order to cope with the possible ROS increase after Roundup WG® exposure. Therefore, results confirmed that gender could be a confounding factor in ecotoxicological assessment of GBH effects. Additionally, this work highlights that sod1, cat, gpx, gst, txn, hsp70 and hsp90 gene expressions seem to be useful biomarkers to investigate the oxidative stress caused by Roundup WG® in Macrobrachium sp.

The surfactant polyethoxylated tallowamine (POEA) reduces lifespan and inhibits fecundity in Drosophila melanogaster- In vivo and in vitro study.

In order to develop an understanding of the role of adjuvants in a popular glyphosate-based herbicide - Roundup® Concentrate Plus (RCP), on non-target organisms, the effects of pure glyphosate [N-(phosphonomethyl)-glycine], RCP and a non-ionic surfactant - polyethoxylated tallowamine (POEA) were studied in the fruit fly Drosophila melanogaster. Acute exposure to sub-lethal concentrations of RCP (15 µg/mL) and POEA (45 µg/mL) reduced (p < 0.001) lifespan of female flies compared to untreated controls or glyphosate (100 µg/mL). Negative geotaxis responses in female flies were reduced (p < 0.05) following acute exposure to sub-lethal concentrations of RCP and POEA whereas glyphosate did not significantly affect this response compared to untreated flies. Acute exposure to sub-lethal concentrations of RCP and POEA elevated (p < 0.05) protein carbonyl levels while markedly (p < 0.01) inhibiting carbonyl reductase activity whereas glyphosate treatment did not significantly affect protein carbonyl levels or carbonyl reductase activity. Fecundity was reduced (p < 0.05) following exposure to sub-lethal concentrations of RCP and POEA whereas glyphosate did not affect fecundity. In vitro treatment of ovarian stem sheath (OSS) cells with sub-lethal concentrations of RCP and POEA revealed decreased cell viability and enhanced caspase activity indicative of pro-apoptotic processes after 48 h compared to untreated controls. Glyphosate however was non-toxic at the concentration used. The results suggest that RCP and the surfactant POEA are more toxic than pure glyphosate and inhibit fecundity in Drosophila by impairing cell viability through enhanced apoptosis.