Perinatal exposure to a glyphosate pesticide formulation induces offspring liver damage.

The present study investigated the effects of perinatal exposure to glyphosate-based herbicide (GBH) in offspring's liver. Pregnant Wistar rats were exposed to GBH (70 mg glyphosate/Kg body weight/day) in drinking water from gestation day 5 to postnatal day 15. The perinatal exposure to GBH increased 45Ca2+ influx in offspring's liver. Pharmacological tools indicated a role played by oxidative stress, phospholipase C (PLC) and Akt pathways, as well as voltage-dependent Ca2+ channel modulation on GBH-induced Ca2+ influx in offspring's liver. In addition, changes in the enzymatic antioxidant defense system, decreased GSH content, lipid peroxidation and protein carbonylation suggest a connection between GBH-induced hepatotoxic mechanism and redox imbalance. The perinatal exposure to GBH also increased the enzymatic activities of transaminases and gamma-glutamyl transferase in offspring's liver and blood, suggesting a pesticide-induced liver injury. Moreover, we detected increased iron levels in liver, blood and bone marrow of GBH-exposed rats, which were accompanied by increased transferrin saturation and decreased transferrin levels in blood. The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were increased in the liver of rats perinatally exposed to GBH, which were associated with. Increased phospho-p65NFκB immunocontent. Therefore, we propose that excessive amounts of iron in offspring's liver, blood and bone marrow induced by perinatal exposure to GBH may account for iron-driven hepatotoxicity, which was associated with Ca2+ influx, oxidative damage and inflammation. Further studies will clarify whether these events can ultimately impact on liver function.

Paraquat induces redox imbalance and disrupts glutamate and energy metabolism in the hippocampus of prepubertal rats.

Paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride; PQ) is a widely used herbicide in Brazilian crops, despite its banishment in many other countries. The present study investigated the effects of repeated dose of PQ on glutamate system, energy metabolism and redox parameters in the hippocampus of prepubertal rats. Twenty-two-day-old rats received daily intraperitoneal injections of PQ (10 mg/Kg) during 5 consecutive days and the effects of the pesticide were assessed 24 h after the last injection. The PQ exposure provoked cytotoxicity associated to decreased cell viability and increased glutamate excitotoxicity, as demonstrated by decreased 14C-glutamate uptake and increased 45Ca2+ uptake. Downregulated glutamine synthetase (GS) activity, further supports disrupted glutamate metabolism compromising the glutamate-glutamine cycle. Downregulated 14C-2-Deoxy-D-glucose indicates energy failure and upregulated lactate dehydrogenase (LDH) suggests the relevance of lactate as energy fuel. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) upregulation suggest Krebs cycle replenishment and piruvate production. In addition, PQ disturbed the redox status inducing lipid peroxidation, evaluated by increased TBARS and imbalanced antioxidant system. Downregulated glutathione reductase (GR), gamma-glutamyltransferase (GGT), glutathione-S-transferase (GST) and glucose-6-P-dehydrogenase (G6PD) activities together with upregulated superoxide dismutase (SOD) and catalase activities corroborate the oxidative imbalance. The mechanisms underlying PQ-induced neurotoxicity involves the modulation of GSK-3ß, NF-κB and NMDA receptors. These neurochemical and oxidative events observed may contribute to neuroinflammation and neurotoxic effects of PQ on hippocampal cells.

Reverse T3 interacts with αvß3 integrin receptor and restores enzyme activities in the hippocampus of hypothyroid developing rats: Insight on signaling mechanisms.

In the present study we provide evidence that 3,3',5'-triiodothyronine (reverse T3, rT3) restores neurochemical parameters induced by congenital hypothyroidism in rat hippocampus. Congenital hypothyroidism was induced by adding 0.05% propylthiouracil in the drinking water from gestation day 8 and continually up to lactation day 15. In the in vivo rT3 exposure, hypothyroid 12-day old pups were daily injected with rT3 (50 ng/kg body weight) or saline until day 14. In the ex vivo rT3 treatment, hippocampal slices from 15-day-old hypothyroid pups were incubated for 30 min with or without rT3 (1 nM). We found that ex vivo and/or in vivo exposure to rT3 failed in restoring the decreased 14C-glutamate uptake; however, restored the phosphorylation of glial fibrillary acidic protein (GFAP), 45Ca2+ influx, aspartate transaminase (AST), glutamine synthetase (GS) and gamma-glutamate transferase (GGT) activities, as well as glutathione (GSH) levels in hypothyroid hippocampus. In addition, rT3 improved 14C-2-deoxy-D-glucose uptake and lactate dehydrogenase (LDH) activity. Receptor agonists/antagonists (RGD peptide and AP-5), kinase inhibitors of p38MAPK, ERK1/2, CaMKII, PKA (SB239063, PD98059, KN93 and H89, respectively), L-type voltage-dependent calcium channel blocker (nifedipine) and intracellular calcium chelator (BAPTA-AM) were used to determine the mechanisms of the nongenomic rT3 action on GGT activity. Using molecular docking analysis, we found rT3 interaction with αvß3 integrin receptors, nongenomically activating signaling pathways (PKA, CaMKII, p38MAPK) that restored GGT activity. We provide evidence that rT3 is an active TH metabolite and our results represent an important contribution to elucidate the nonclassical mechanism of action of this metabolite in hypothyroidism.

Developmental exposure to glyphosate-based herbicide and depressive-like behavior in adult offspring: Implication of glutamate excitotoxicity and oxidative stress.

We have previously demonstrated that maternal exposure to glyphosate-based herbicide (GBH) leads to glutamate excitotoxicity in 15-day-old rat hippocampus. The present study was conducted in order to investigate the effects of subchronic exposure to GBH on some neurochemical and behavioral parameters in immature and adult offspring. Rats were exposed to 1% GBH in drinking water (corresponding to 0.36% of glyphosate) from gestational day 5 until postnatal day (PND)-15 or PND60. Results showed that GBH exposure during both prenatal and postnatal periods causes oxidative stress, affects cholinergic and glutamatergic neurotransmission in offspring hippocampus from immature and adult rats. The subchronic exposure to the pesticide decreased L-[14C]-glutamate uptake and increased 45Ca2+ influx in 60-day-old rat hippocampus, suggesting a persistent glutamate excitotoxicity from developmental period (PND15) to adulthood (PND60). Moreover, GBH exposure alters the serum levels of the astrocytic protein S100B. The effects of GBH exposure were associated with oxidative stress and depressive-like behavior in offspring on PND60, as demonstrated by the prolonged immobility time and decreased time of climbing observed in forced swimming test. The mechanisms underlying the GBH-induced neurotoxicity involve the NMDA receptor activation, impairment of cholinergic transmission, astrocyte dysfunction, ERK1/2 overactivation, decreased p65 NF-κB phosphorylation, which are associated with oxidative stress and glutamate excitotoxicity. These neurochemical events may contribute, at least in part, to the depressive-like behavior observed in adult offspring.

Maternal Exposure to Ethanol During Pregnancy and Lactation Affects Glutamatergic System and Induces Oxidative Stress in Offspring Hippocampus.

BACKGROUND: Alcohol abuse during pregnancy leads to intellectual disability and morphological defects in the offspring. The aim of this study was to determine the effect of chronic maternal ethanol (EtOH) consumption during pregnancy and lactation on glutamatergic transmission regulation, energy deficit, and oxidative stress in the hippocampus of the offspring. METHODS: EtOH was administered to dams in drinking water at increasing doses (2 to 20%) from the gestation day 5 to lactation day 21. EtOH and tap water intake by treated and control groups, respectively, were measured daily. RESULTS: Results showed that EtOH exposure does not affect fluid intake over the course of pregnancy and lactation. The toxicity of maternal exposure to EtOH was demonstrated by decreased offspring body weight at experimental age, on postnatal day 21. Moreover, maternal EtOH exposure decreased (45) Ca(2+) influx in the offspring's hippocampus. Corroborating this finding, EtOH increased both Na(+) -dependent and Na(+) -independent glial [(14) C]-glutamate uptake in hippocampus of immature rats. Also, maternal EtOH exposure decreased glutamine synthetase activity and induced aspartate aminotransferase enzymatic activity, suggesting that in EtOH-exposed offspring hippocampus, glutamate is preferentially used as a fuel in tricarboxylic acid cycle instead of being converted into glutamine. In addition, EtOH exposure decreased [U-14C]-2-deoxy-D-glucose uptake in offspring hippocampus. CONCLUSIONS: The decline in glucose transport coincided with increased lactate dehydrogenase activity, suggesting an adaptative response in EtOH-exposed offspring hippocampus, using lactate as an alternative fuel. These events were associated with oxidative damage, as demonstrated by changes in the enzymatic antioxidant defense system and lipid peroxidation. Taken together, the results demonstrate that maternal exposure to EtOH during pregnancy and lactation impairs glutamatergic transmission, as well as inducing oxidative stress and energy deficit in immature rat hippocampus.

Mechanisms underlying the neurotoxicity induced by glyphosate-based herbicide in immature rat hippocampus: involvement of glutamate excitotoxicity.

Previous studies demonstrate that glyphosate exposure is associated with oxidative damage and neurotoxicity. Therefore, the mechanism of glyphosate-induced neurotoxic effects needs to be determined. The aim of this study was to investigate whether Roundup(®) (a glyphosate-based herbicide) leads to neurotoxicity in hippocampus of immature rats following acute (30min) and chronic (pregnancy and lactation) pesticide exposure. Maternal exposure to pesticide was undertaken by treating dams orally with 1% Roundup(®) (0.38% glyphosate) during pregnancy and lactation (till 15-day-old). Hippocampal slices from 15 day old rats were acutely exposed to Roundup(®) (0.00005-0.1%) during 30min and experiments were carried out to determine whether glyphosate affects (45)Ca(2+) influx and cell viability. Moreover, we investigated the pesticide effects on oxidative stress parameters, (14)C-α-methyl-amino-isobutyric acid ((14)C-MeAIB) accumulation, as well as glutamate uptake, release and metabolism. Results showed that acute exposure to Roundup(®) (30min) increases (45)Ca(2+) influx by activating NMDA receptors and voltage-dependent Ca(2+) channels, leading to oxidative stress and neural cell death. The mechanisms underlying Roundup(®)-induced neurotoxicity also involve the activation of CaMKII and ERK. Moreover, acute exposure to Roundup(®) increased (3)H-glutamate released into the synaptic cleft, decreased GSH content and increased the lipoperoxidation, characterizing excitotoxicity and oxidative damage. We also observed that both acute and chronic exposure to Roundup(®) decreased (3)H-glutamate uptake and metabolism, while induced (45)Ca(2+) uptake and (14)C-MeAIB accumulation in immature rat hippocampus. Taken together, these results demonstrated that Roundup(®) might lead to excessive extracellular glutamate levels and consequently to glutamate excitotoxicity and oxidative stress in rat hippocampus.

Roundup disrupts male reproductive functions by triggering calcium-mediated cell death in rat testis and Sertoli cells.

Glyphosate is the primary active constituent of the commercial pesticide Roundup. The present results show that acute Roundup exposure at low doses (36 ppm, 0.036 g/L) for 30 min induces oxidative stress and activates multiple stress-response pathways leading to Sertoli cell death in prepubertal rat testis. The pesticide increased intracellular Ca(2+) concentration by opening L-type voltage-dependent Ca(2+) channels as well as endoplasmic reticulum IP3 and ryanodine receptors, leading to Ca(2+) overload within the cells, which set off oxidative stress and necrotic cell death. Similarly, 30 min incubation of testis with glyphosate alone (36 ppm) also increased (45)Ca(2+) uptake. These events were prevented by the antioxidants Trolox and ascorbic acid. Activated protein kinase C, phosphatidylinositol 3-kinase, and the mitogen-activated protein kinases such as ERK1/2 and p38MAPK play a role in eliciting Ca(2+) influx and cell death. Roundup decreased the levels of reduced glutathione (GSH) and increased the amounts of thiobarbituric acid-reactive species (TBARS) and protein carbonyls. Also, exposure to glyphosate-Roundup stimulated the activity of glutathione peroxidase, glutathione reductase, glutathione S-transferase, γ-glutamyltransferase, catalase, superoxide dismutase, and glucose-6-phosphate dehydrogenase, supporting downregulated GSH levels. Glyphosate has been described as an endocrine disruptor affecting the male reproductive system; however, the molecular basis of its toxicity remains to be clarified. We propose that Roundup toxicity, implicated in Ca(2+) overload, cell signaling misregulation, stress response of the endoplasmic reticulum, and/or depleted antioxidant defenses, could contribute to Sertoli cell disruption in spermatogenesis that could have an impact on male fertility.

Congenital hypothyroidism alters the oxidative status, enzyme activities and morphological parameters in the hippocampus of developing rats.

Congenital hypothyroidism is associated with delay in cell migration and proliferation in brain tissue, impairment of synapse formation, misregulation of neurotransmitters, hypomyelination and mental retardation. However, the mechanisms underlying the neuropsychological deficits observed in congenital hypothyroidism are not completely understood. In the present study we proposed a mechanism by which hypothyroidism leads to hippocampal neurotoxicity. Congenital hypothyroidism induces c-Jun-N-terminal kinase (JNK) pathway activation leading to hyperphosphorylation of the glial fibrillary acidic protein (GFAP), vimentin and neurofilament subunits from hippocampal astrocytes and neurons, respectively. Moreover, hyperphosphorylation of the cytoskeletal proteins was not reversed by T3 and poorly reversed by T4. In addition, congenital hypothyroidism is associated with downregulation of astrocyte glutamate transporters (GLAST and GLT-1) leading to decreased glutamate uptake and subsequent influx of Ca(2+) through N-methyl-D-aspartate (NMDA) receptors. The Na(+)-coupled (14)C-α-methyl-amino-isobutyric acid ((14)C-MeAIB) accumulation into hippocampal cells also might cause an increase in the intracellular Ca(2+) concentration by opening voltage-dependent calcium channels (VDCC). The excessive influx of Ca(2+) through NMDA receptors and VDCCs might lead to an overload of Ca(2+) within the cells, which set off glutamate excitotoxicity and oxidative stress. The inhibited acetylcholinesterase (AChE) activity might also induce Ca(2+) influx. The inhibited glucose-6-phosphate dehydrogenase (G6PD) and gamma-glutamyl transferase (GGT) activities, associated with altered glutamate and neutral amino acids uptake could somehow affect the GSH turnover, the antioxidant defense system, as well as the glutamate-glutamine cycle. Reduced levels of S100B and glial fibrillary acidic protein (GFAP) take part of the hypothyroid condition, suggesting a compromised astroglial/neuronal neurometabolic coupling which is probably related to the neurotoxic damage in hypothyroid brain.