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

AbstractPrevious 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 45Ca2+ influx and cell viability. Moreover, we investigated the pesticide effects on oxidative stress parameters, 14C-α-methyl-amino-isobutyric acid (14C-MeAIB) accumulation, as well as glutamate uptake, release and metabolism. Results showed that acute exposure to Roundup® (30min) increases 45Ca2+ influx by activating NMDA receptors and voltage-dependent Ca2+ 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 3H-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 3H-glutamate uptake and metabolism, while induced 45Ca2+ uptake and 14C-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

Rapid responses to reverse T₃ hormone in immature rat Sertoli cells: calcium uptake and exocytosis mediated by integrin.

There is increasing experimental evidence of the nongenomic action of thyroid hormones mediated by receptors located in the plasma membrane or inside cells. The aim of this work was to characterize the reverse T₃ (rT₃) action on calcium uptake and its involvement in immature rat Sertoli cell secretion. The results presented herein show that very low concentrations of rT₃ are able to increase calcium uptake after 1 min of exposure. The implication of T-type voltage-dependent calcium channels and chloride channels in the effect of rT₃ was evidenced using flunarizine and 9-anthracene, respectively. Also, the rT₃-induced calcium uptake was blocked in the presence of the RGD peptide (an inhibitor of integrin-ligand interactions). Therefore, our findings suggest that calcium uptake stimulated by rT₃ may be mediated by integrin αvβ₃. In addition, it was demonstrated that calcium uptake stimulated by rT₃ is PKC and ERK-dependent. Furthermore, the outcomes indicate that rT₃ also stimulates cellular secretion since the cells manifested a loss of fluorescence after 4 min incubation, indicating an exocytic quinacrine release that seems to be mediated by the integrin receptor. These findings indicate that rT₃ modulates the calcium entry and cellular secretion, which might play a role in the regulation of a plethora of intracellular processes involved in male reproductive physiology

1α,25(OH)2-vitamin D3 stimulates rapid plasma membrane calcium influx via MAPK activation in immature rat Sertoli cells

International audienceIt was characterized that the rapid response to 1α,25(OH)(2)-vitamin D(3) (1,25D(3)) on (45)Ca(2+) influx in rat Sertoli cells was mediated by voltage-dependent Ca(2+) channels (VDCCs), PKC, ERK1/2 and p38 MAPK pathways. In primary culture of 10 day-old rat Sertoli cells as well as in the whole testis, the time-course of (45)Ca(2+) influx did not change significantly in basal conditions. However, 1,25D(3) showed stimulatory effect on (45)Ca(2+) influx from 10(-15) to 10(-8) M after 60 s of incubation. The maximum effect was around 140% at 10(-12) M on purified Sertoli cells showing a steady state on (45)Ca(2+) influx between 10(-11) and 10(-9) M. Under this experimental condition, 1,25D(3) stimulated (45)Ca(2+) influx from 73% to 106% and no effect was observed at 10(-16), 10(-8) and 10(-7) M in whole testis. VDCC activities are mandatory for a full and complete stimulatory effect of 1,25D(3) in these approaches. K(+) and Cl(-) channels also are strongly involved in this rapid response coordinated by 1,25D(3). The participation of some selected kinases, points to PKC and ERK1/2 upstream activity to p38 MAPK activation suggesting an intracellular cross-talk between rapid (45)Ca(2+) influx and nuclear events. In addition, the comparative effect of microtubule disassembles and ClC-3 channel blocker on (45)Ca(2+) influx provides evidence of secretory activity of Sertoli cells triggered by 1,25D(3). Our results suggest that 1,25D(3) activates p38 MAPK and reorganizes microtubules, involving Ca(2+), PKC and ERK1/2 as upstream regulators and that extracellular Ca(2+) have a central role to rapidly start hormone-induced gene transcription and/or the secretory activity of Sertoli cell

Natural Products as Outstanding Alternatives in Diabetes Mellitus: A Patent Review

Diabetes mellitus (DM) is a metabolic syndrome that can be considered a growing health problem in the world. High blood glucose levels are one of the most notable clinical signs. Currently, new therapeutic alternatives have been tackled from clinicians’ and scientists’ points of view. Natural products are considered a promising source, due to the huge diversity of metabolites with pharmaceutical applications. Therefore, this review aimed to uncover the latest advances in this field as a potential alternative to the current therapeutic strategies for the treatment of DM. This purpose is achieved after a patent review, using the Espacenet database of the European Patent Office (EPO) (2016–2022). Final screening allowed us to investigate 19 patents, their components, and several technology strategies in DM. Plants, seaweeds, fungi, and minerals were used as raw materials in the patents. Additionally, metabolites such as tannins, organic acids, polyphenols, terpenes, and flavonoids were found to be related to the potential activity in DM. Moreover, the cellular transportation of active ingredients and solid forms with special drug delivery profiles is also considered a pharmaceutical technology strategy that can improve their safety and efficacy. From this perspective, natural products can be a promissory source to obtain new drugs for DM therapy

Interactions between oestrogen and 1α,25(OH)2-vitamin D3 signalling and their roles in spermatogenesis and spermatozoa functions

International audienceOestrogens and 1α,25(OH)2-vitamin D3 (1,25-D3) are steroids that can provide effects by binding to their receptors localised in the cytoplasm and in the nucleus or the plasma membrane respectively inducing genomic and non-genomic effects. As confirmed notably by invalidation of the genes, coding for their receptors as tested with mice with in vivo and in vitro treatments, oestrogens and 1,25-D3 are regulators of spermatogenesis. Moreover, some functions of ejaculated spermatozoa as viability, DNA integrity, motility, capacitation, acrosome reaction and fertilizing ability are targets for these hormones. The studies conducted on their mechanisms of action, even though not completely elicited, have allowed the demonstration of putative interactions between their signalling pathways that are worth examining more closely. The present review focuses on the elements regulated by oestrogens and 1,25-D3 in the testis and spermatozoa as well as the interactions between the signalling pathways of both hormones.L’œstradiol et la 1α,25(OH)2-vitamin D3 (1,25-D3 ou calcitriol) sont respectivement la forme la plus active des œstrogènes et la forme hormonalement active de la vitamine D. Ces stéroïdes peuvent exercer leurs effets biologiques après fixation à des récepteurs localisés dans le cytoplasme et le noyau (récepteurs dit nucléaires) ou par fixation à des récepteurs localisés à la membrane plasmique (récepteurs membranaires) à l’origine d’effets appelés génomiques et non génomiques respectivement. Bien que les œstrogènes aient longtemps été considérés comme uniquement des hormones féminines, de nombreux travaux ont permis de montrer leur importance dans le bon déroulement de la spermatogenèse et la qualité des gamètes. De même, la 1,25-D3 est capable de réguler les fonctions testiculaires suggérant son importance dans la fertilité. Les études réalisées sur leurs mécanismes d’action, bien qu’ils ne soient pas complètement élucidés, ont permis de mettre en évidence des interactions entre les voies de signalisation de ces deux hormones. Cette revue est centrée sur les évènements régulés par les œstrogènes et la 1,25-D3 dans les testicules et les spermatozoïdes et les interactions entre leurs voies de signalisation

1α,25-Dihydroxyvitamin D 3 Signaling Pathways on Calcium Uptake in 30-Day-Old Rat Sertoli Cells

International audience1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is the active metabolite of vitamin D(3) and the major calcium regulatory hormone in tissues. The aim of this work was to investigate the mechanism of action of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells from 30-day-old rats. Results showed that 10(-9) and 10(-12) M 1,25D(3) increased the rate of (45)Ca(2+) uptake 5 and 15 min after hormone exposure and that 1α,25(OH)(2) lumisterol(3) (JN) produced a similar effect suggesting that 1,25D(3) action occurs via a putative membrane receptor. The involvement of voltage-dependent calcium channels (VDCC) in 1,25D(3) action was evidenced by using nifedipine, while the use of Bapta-AM demonstrated that intracellular calcium was not implicated. Moreover, the incubation with ouabain and digoxin increased the rate of (45)Ca(2+) uptake, indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, we demonstrated that the mechanism underlying the hormone action involved extracellular signal-regulated kinase (ERK) and protein kinase C (PKC) activation in a phospholipase C-independent way. Furthermore, a local elevation of the level of cAMP, as demonstrated by incubating cells with dibutyryl cAMP or a phosphodiesterase inhibitor, produced an effect similar to that of 1,25D(3), and the inhibition of protein kinase A (PKA) nullified the hormone action. In conclusion, the stimulatory effect of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells occurs via VDCC, as well as PKA, PKC, and ERK activation. These protein kinases seem to act by inhibiting Na(+)/K(+)-ATPase or directly phosphorylating calcium channels. The Na(+)/K(+)-ATPase inhibition may result in Na(+)/Ca(2+) exchanger activation in reverse mode and consequently induce the uptake of calcium into the cells

Involvement of ionic channels and intracellular calcium on stimulatory effect of rT₃ on ⁴⁵Ca²⁺ uptake.

<p>(A) Influence of flunarizine, (B) BAPTA-AM and (C) 9-AC on stimulatory effect of rT₃ on ⁴⁵Ca²⁺ uptake in Sertoli cells. Pre-incubation: 15 min in KRb, additional pre-incubation: 60 min with 0.1 µCi/mL of ⁴⁵Ca²⁺ and incubation time: 60 s with 0.1 µCi/mL of 45Ca2+ in the presence or absence of flunarizine (1 µM), BAPTA-AM (50 µM) and 9-AC (1 µM) with/without rT3 (10-17 M). Means ± S.E.M. For control, n=10; rT3, n=7; flunarizine, n=8; rT3 + flunarizine, n=8; BAPTA-AM, n=8; rT3 + BAPTA-AM, n=6; 9-AC, n=6; rT3 + 9-AC, n=6. ***P < 0.001 and **p < 0.01 compared with control group; ###p < 0.001; ##p < 0.01 and #p < 0.05 compared with rT3 group.</p

Involvement of kinases proteins on stimulatory effect of rT₃ on ⁴⁵Ca²⁺ uptake in Sertoli cells.

<p>(A) Influence of stearoylcarnitine and (B) PD 98059. Pre-incubation: 15 min in KRb, additional pre-incubation: 60 min with 0.1 µCi/mL of ⁴⁵Ca²⁺ and incubation time: 60 s with 0.1 µCi/mL of ⁴⁵Ca²⁺ in the presence or absence of stearoylcarnitine (1 µM) and PD 98059 (30 µM) with/without rT₃ (10^-17 M). Means ± S.E.M. For control, n=9; rT₃, n=6; stearoylcarnitine, n=8; rT₃ + stearoylcarnitine, n=9; PD 98059, n=8; rT₃ + PD 98059, n=8. ***<i>p</i> < 0.001 and *<i>p</i> < 0.05 compared with control group; ##<i>p</i> < 0.01 and #<i>p</i> < 0.05 compared with rT₃ group.</p