Antidiabetic Effect of Passiflora ligularis Leaves in High Fat-Diet/Streptozotocin-Induced Diabetic Mice.

Type 2 diabetes mellitus (T2DM) is a major global public health concern, prompting the ongoing search for new treatment options. Medicinal plants have emerged as one such alternative. Our objective was to evaluate the antidiabetic effect of an extract from the leaves of Passiflora ligularis (P. ligularis). For this purpose, T2DM was first induced in mice using a high-fat diet and low doses of streptozotocin. Subsequently, an aqueous extract or an ethanolic extract of P. ligularis leaves was administered for 21 days. The following relevant results were found: fasting blood glucose levels were reduced by up to 41%, and by 29% after an oral glucose overload. The homeostasis model assessment of insulin resistance (HOMA-IR) was reduced by 59%. Histopathologically, better preservation of pancreatic tissue was observed. Regarding oxidative stress parameters, there was an increase of up to 48% in superoxide dismutase (SOD), an increase in catalase (CAT) activity by 35% to 80%, and a decrease in lipid peroxidation (MDA) by 35% to 80% in the liver, kidney, or pancreas. Lastly, regarding the lipid profile, triglycerides (TG) were reduced by up to 30%, total cholesterol (TC) by 35%, and low-density lipoproteins (LDL) by up to 32%, while treatments increased high-density lipoproteins (HDL) by up to 35%. With all the above, we can conclude that P. ligularis leaves showed antihyperglycemic, hypolipidemic, and antioxidant effects, making this species promising for the treatment of T2DM.

Effects of pulse repetition rate in static electrochemotherapy models.

Electroporation alters cell membrane structure and tissue electrical properties by short and intense pulsed electric fields (PEF). Static mathematical models are often used to explain the change in electrical properties of tissues caused by electroporation. Electric pulse repetition rate may play an important role, as tissue dielectric dispersion, electroporation dynamics, and Joule heating may affect the electrical properties. In this work, we investigate the effects on the magnitude of the electric current when the repetition rate of the standard electrochemotherapy protocol is increased. Liver, oral mucosa, and muscle tissues were studied. Ex vivo animal experiments show that the magnitude of the electric current increases when the repetition rate is changed from 1 Hz to 5 kHz (10.8% for liver, 5.8% for oral mucosa, and 4.7% for muscle). Although a correction factor could reduce the error to less than 1%, dynamic models seem to be necessary to analyze different protocol signatures. Authors should be aware that static models and experimental results can only be compared if they use exactly the same PEF signature. The repetition rate is a key information to consider in the pretreatment computer study because the current at 1 Hz PEF differs from a 5 kHz PEF.

Synthesis of a novel glibenclamide-pioglitazone hybrid compound and its effects on glucose homeostasis in normal and insulin-resistant rats.

A new library of hybrid compounds that combine the functional parts of glibenclamide and pioglitazone was designed and developed. Compounds were screened for their antihyperglycemic effects on the glucose tolerance curve. This approach provided a single molecule that optimizes the pharmacological activities of two drugs used for the treatment of diabetes mellitus type 2 (DM2) and that have distinct biological activities, potentially minimizing the adverse effects of the original drugs. From a total of 15 compounds, 7 were evaluated in vivo; the compound 2; 4- [2- (2-phenyl-4-oxo-1,3-thiazolidin-3-yl) ethyl] benzene-1-sulfonamide (PTEBS) was selected to study its mechanism of action on glucose and lipid homeostasis in acute and chronic animal models related to DM2. PTEBS reduced glycemia and increased serum insulin in hyperglycemic rats, and elevated in vitro insulin production from isolated pancreatic islets. This compound increased the glycogen content in hepatic and muscular tissue. Moreover, PTEBS stimulated the uptake of glucose in soleus muscle through a signaling pathway similar to that of insulin, stimulating translocation and protein synthesis of glucose transporter 4 (GLUT4). PTEBS was effective in increasing insulin sensitivity in resistance rats by stimulating increased muscle glucose uptake, among other mechanisms. In addition, this compound reduced total triglycerides in a tolerance test to lipids and reduced advanced glycation end products (AGES), without altering lactate dehydrogenase (LDH) activity. Thus, we suggest that PTEBS may have similar effects to the respective prototypes, which may improve the therapeutic efficacy of these molecules and decrease adverse effects in the long-term.

Estradiol and 1α,25(OH)2 vitamin D3 share plasma membrane downstream signal transduction through calcium influx and genomic activation in immature rat testis.

The aim of this study was to investigate the rapid response pathway and gene and protein expression profiles of the rat testis in response to estradiol (E2) and 1α,25(OH)2 vitamin D3 (1,25-D3), to understand how they mediate their effects on the first spermatogenic wave. To do this, we compared the effects of 1,25-D3 and E2 on 45calcium(Ca2+) uptake and the involvement of estrogen receptors (ESR) in their rapid responses. Additionally, we studied the downstream signal transduction effects of 1,25-D3 and E2 on cyclin A1/B1 and cellular cycle protein expression. As previously observed for 1,25-D3, E2 also increased 45Ca2+ uptake in immature rat testes via voltage-dependent Ca2+ channels, Ca2+-dependent chloride channels and via the activation of protein kinase C, protein kinase A and mitogen-activated protein kinase kinase (MEK). Elevated aromatase expression by testes was observed in the presence of 1,25-D3 and both hormones decreased ESR mRNA expression. Furthermore, 1,25-D3 and E2 diminished cyclin A1 mRNA expression, but E2 did not affect cyclin B1 mRNA levels. Consistent with these findings, the immunocontent of cyclin A1 and B1 in the testes was also increased by 1,25-D3 and E2. 1,25-D3 increased expressions of the p16 and p53 proteins, supporting the anti-proliferative and pro-apoptotic properties of 1,25-D3, while E2 also augmented p16. Data indicate that both hormones trigger rapid responses at the plasma membrane that may control the expression of gene and proteins related to cell cycle regulation, and thereby modulate spermatogenesis.

Biological activity of 2α,3ß,23-trihydroxyolean-12-ene on glucose homeostasis.

We studied the effect and the mechanisms of action of 2α,3ß,23-trihydroxyolean-12-ene (THO), from Croton heterodoxus Baill. (Euphorbiaceae), in glucose uptake in hyperglycemic rats. The effect of in vivo pretreatment with THO in hyperglycemic rats was analyzed. The in vitro effects of THO were observed in adipocytes and in adipose tissue. THO reduced glycemia, in part by increasing serum insulin and augmenting the disposal of glucose as glycogen in hepatocytes but did not change the serum concentration of glucagon-like peptide-1. THO increased glucose uptake in adipocytes and in adipose tissue by a mechanism dependent on phosphatidylinositol 3-kinase vesicular traffic and on the process of vesicle fusion at the plasma membrane in regions containing cholesterol, indicating the involvement of glucose transporter-4 (GLUT4). This triterpene may act solely via the activation and translocation of GLUT4 (rather than via nuclear actions, such as upregulation of GLUT4 synthesis), since THO did not alter the amount of GLUT4 mRNA or the content of GLUT4. Consistent with these data, the stimulatory effect of this triterpene on the quantity of GLUT4 in the membrane fraction was dependent upon p38 phosphorylation. In this experimental model, orally administered 10 mg/kg THO did not modulate extracellular serum lactate dehydrogenase. In conclusion, THO decreases hyperglycemia by increasing serum insulin and hepatic glycogen content. The THO mechanism of action on adipose tissue for glucose uptake is suggested to be via GLUT4 translocation stimulation mediated by a p38-dependent mechanism. THO is a potential antihyperglycemic agent that acts in a target tissue for glucose homeostasis.

Pyriproxyfen induces intracellular calcium overload and alters antioxidant defenses in Danio rerio testis that may influence ongoing spermatogenesis.

We investigated the in vitro effects of pyriproxyfen on ionic balance in the testis of the zebrafish by measuring 45Ca2+ influx. In vivo pyriproxyfen treatment was carried out to study oxidative stress, and conduct morphological analysis of the testis and liver. Whole testes were incubated in vitro with/without pyriproxyfen (10-12, 10-9 or 10-6 M; 30 min) and 45Ca2+ influx determined. To study pyriproxyfen's mechanism of action, inhibitors/activators of ionic channels or pumps/exchangers, protein kinase inhibitors or a calcium chelator were added 15 min before the addition of 45Ca2+ and pyriproxyfen. We evaluated the in vivo effects of 7 day exposure to waterborne pyriproxyfen (10-9 M) on reactive oxygen species (ROS) formation, lipid peroxidation, and reduced glutathione content (GSH), glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) and γ-glutamyltransferase (GGT) activity. Morphological analyses of the testis and liver were carried out after in vivo exposure of D. rerio to pyriproxyfen. Pyriproxyfen increased 45Ca2+ influx by opening the voltage-dependent T-type channels (T-type VDCC), inhibiting sarco/endoplasmic reticulum 45Ca2+-ATPase (SERCA) and the NCX exchanger (forward mode) and by mobilizing calcium from stores. The involvement of potassium channels and protein kinase C (PKC) was also demonstrated in pyriproxyfen-induced intracellular calcium elevation. In vivo pyriproxyfen treatment of D. rerio increased lipid peroxidation, decreased GSH content and increased GST activity in testes, in addition to increasing the number and size of spermatogonia cysts and inducing hepatocyte basophilia and dilation of blood vessels in the liver. The toxicity of pyriproxyfen is mediated by calcium overload, increased lipid peroxidation, and a diminished antioxidant capacity in the testis, due to GSH depletion, and altered spermatogenesis. The development of high basophilia in the liver suggests that pyriproxyfen may have estrogenic activity, possibly acting as an endocrine-disruptor. These findings indicate that these alterations may contribute to pyriproxyfen toxicity and spermatogenesis disruption.

Dibutyl phthalate rapidly alters calcium homeostasis in the gills of Danio rerio.

This study investigates the impacts of exposure to an environment Ca2+ challenge and the mechanism of action of dibutyl phthalate (DBP) on Ca2+ influx in the gills of Danio rerio. In vitro profile of 45Ca2+ influx in gills was verified through the basal time-course. Fish were exposed to low, normal and high Ca2+ concentrations (0.02, 0.7 and 2 mM) for 12 h. So, gills were morphologically analysed and ex vivo45Ca2+ influx at 30 and 60 min was determined. For the in vitro studies, gills were treated for 60 min with DBP (1 pM, 1 nM and 1 µM) with/without blockers/activators of ionic channels, Ca2+ chelator, inhibitors of ATPases, ionic exchangers and protein kinase C to study the mechanism of DBP-induced 45Ca2+ influx. Exposure to high environmental Ca2+ augmented 45Ca2+ influx when compared to fish exposed to normal and low Ca2+ concentrations. Additionally, histopathological changes were observed in the gills of fish maintained for 12 h in low and high Ca2+. In vitro exposure of gills to DBP (1 pM) disturbed Ca2+ homeostasis. DBP stimulated 45Ca2+ influx in gills through the transitory receptor potential vanilloid 1 (TRPV1), and reverse-mode Na+/Ca2+ exchanger (NCX) activation, protein kinase C and K+ channels and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). These data suggest that in vivo short-term exposure of gills to low and high Ca2+ leads to 45Ca2+ influx and histopathological changes. Additionally, the DBP-induced rapid 45Ca2+ influx is mediated by TRPV1, NCX activation with the involvement of PKC, K+-channels and SERCA, thereby altering Ca2+ homeostasis.

Role of bisphenol A on calcium influx and its potential toxicity on the testis of Danio rerio.

This study investigated the acute in vitro effect of low-concentration bisphenol A (BPA) on calcium (45Ca2+) influx in zebrafish (Danio rerio) testis and examined whether intracellular Ca2+ was involved in the effects of BPA on testicular toxicity. In vitro studies on 45Ca2+ influx were performed in the testes after incubation with BPA for 30 min. Inhibitors were added 15 min before the addition of 45Ca2+ and BPA to testes to study the mechanism of action of BPA. The involvement of intracellular calcium from stores on lactate dehydrogenase (LDH) release and on triacylglycerol (TAG) content were carried out after in vitro incubation of testes with BPA for 1 h. Furthermore, gamma-glutamyl transpeptidase (GGT) and aspartate aminotransferase (AST) activities were analyzed in the liver at 1 h after in vitro BPA incubation of D. rerio. Our data show that the acute in vitro treatment of D. rerio testes with BPA at very low concentration activates plasma membrane ionic channels, such as voltage-dependent calcium channels and calcium-dependent chloride channels, and protein kinase C (PKC), which stimulates Ca2+ influx. In addition, BPA increased cytosolic Ca2+ by activating inositol triphosphate receptor (IP3R) and inhibiting sarco/endoplasmic reticulum calcium ATPase (SERCA) at the endoplasmic reticulum, contributing to intracellular Ca2+ overload. The protein kinases, PKC, MEK 1/2 and PI3K, are involved in the mechanism of action of BPA, which may indicate a crosstalk between the non-genomic initiation effects mediated by PLC/PKC/IP3R signaling and genomic responses of BPA mediated by the estrogen receptor (ESR). In vitro exposure to a higher concentration of BPA caused cell damage and plasma membrane injury with increased LDH release and TAG content; both effects were dependent on intracellular Ca2+ and mediated by IP3R. Furthermore, BPA potentially induced liver damage, as demonstrated by increased GGT activity. In conclusion, in vitro effect of BPA in a low concentration triggers cytosolic Ca2+ overload and activates downstream protein kinases pointing to a crosstalk between its non-genomic and genomic effects of BPA mediated by ESR. Moreover, in vitro exposure to a higher concentration of BPA caused intracellular Ca2+-dependent testicular cell damage and plasma membrane injury. This acute toxicity was reinforced by increased testicular LDH release and GGT activity in the liver.

Acute exposure to bis(2-ethylhexyl)phthalate disrupts calcium homeostasis, energy metabolism and induces oxidative stress in the testis of Danio rerio.

Bis(2-ethylhexyl)phthalate (BEHP) negatively affects testicular functions in different animal species, disturbing reproductive physiology and male fertility. The present study investigated the in vitro acute effect of BEHP on the mechanism of action of ionic calcium (Ca2+) homeostasis and energy metabolism. In addition, the effect of BEHP on oxidative stress was studied in vitro and in vivo in the testis of Danio rerio (D. rerio). Testes were treated in vitro for 30 min with 1 µM BEHP for 45Ca2+ influx measurements. Testes were also incubated with 1 µM BEHP for 1 h (in vitro) or 12 h (in vivo) for the measurements of lactate content, 14C-deoxy-d-glucose uptake, lactate dehydrogenase (LDH) and gamma-glutamyl transpeptidase (GGT) activity, total reactive oxygen species (ROS) production and lipid peroxidation. In addition, the effect of BEHP (1 µM) on GGT, glutamic oxaloacetic transferase (GOT) and glutamic pyruvic transferase (GPT) activity in the liver was evaluated after in vivo treatment for 12 h. BEHP disturbs the Ca2+ balance in the testis when given acutely in vitro. BEHP stimulated Ca2+ influx occurs through L-type voltage-dependent Ca2+ channels (L-VDCC), transitory receptor potential vaniloid (TRPV1) channels, reverse-mode Na+/Ca2+ exchanger (NCX) activation and inhibition of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). BEHP affected energy metabolism in the testis by decreasing the lactate content and LDH activity. In vitro and in vivo acute effects of BEHP promoted oxidative stress by increasing ROS production, lipid peroxidation and GGT activity in the testis. Additionally, BEHP caused liver damage by increasing GPT activity.

Astragalin augments basal calcium influx and insulin secretion in rat pancreatic islets.

Astragalin is a flavonol glycoside with several biological activities, including antidiabetic properties. The objective of this study was to investigate the effects of astragalin on glycaemia and insulin secretion, in vivo, and on calcium influx and insulin secretion in isolated rat pancreatic islets, ex vivo. Astragalin (1 and 10 mg / kg) was administered by oral gavage to fasted Wistar rats and serum glucose and plasma insulin were measured. Isolated pancreatic islets were used to measure basal insulin secretion and calcium influx. Astragalin (10 mg/ kg) decreased glycaemia and increased insulin secretion significantly at 15-180 min, respectively, in the glucose tolerance test. In isolated pancreatic cells, astragalin (100 µM) stimulated calcium influx through a mechanism involving ATP-dependent potassium channels, L-type voltage-dependent calcium channels, the sarcoendoplasmic reticulum calcium transport ATPase (SERCA), PKC and PKA. These findings highlight the dietary coadjuvant, astragalin, as a potential insulin secretagogue that may contribute to glucose homeostasis.

New ionic targets of 3,3',5'-triiodothyronine at the plasma membrane of rat Sertoli cells.

The functions of Sertoli cells, which structurally and functionally support ongoing spermatogenesis, are effectively modulated by thyroid hormones, amongst other molecules. We investigated the mechanism of action of rT3 on calcium (45Ca2+) uptake in Sertoli cells by means of in vitro acute incubation. In addition, we performed electrophysiological recordings of potassium efflux in order to understand the cell repolarization, coupled to the calcium uptake triggered by rT3. Our results indicate that rT3 induces nongenomic responses, as a rapid activation of whole-cell potassium currents in response to rT3 occurred in <5 min in Sertoli cells. In addition, the rT3 metabolite, T2, also exerted a rapid effect on calcium uptake in immature rat testis and in Sertoli cells. rT3 also modulated calcium uptake, which occurred within seconds via the action of selective ionic channels and the Na+/K+ ATPase pump. The rapid response of rT3 is essentially triggered by calcium uptake and cell repolarization, which appear to mediate the secretory functions of Sertoli cells.

Experimentally-induced maternal hypothyroidism alters enzyme activities and the sensorimotor cortex of the offspring rats.

In this study, we used an experimental model of congenital hypothyroidism to show that deficient thyroid hormones (TH) disrupt different neurochemical, morphological and functional aspects in the cerebral cortex of 15-day-old offspring. Our results showing decreased glutamine synthetase (GS) activity and Ca2+ overload in the cerebral cortex of hypothyroid pups suggest misregulated glutamate metabolism associated with developmentally induced TH deficiency. The 14C-MeAIB accumulation indicates upregulated System A activity and glutamine uptake by neurons. Energy metabolism in hypothyroid cortical slices was preserved, as demonstrated by unaltered glucose metabolism. We also found upregulated acetylcholinesterase activity, depleting acetylcholine from the synaptic cleft, pointing to disrupted cholinergic system. Increased reactive oxygen species (ROS) generation, lipid peroxidation, glutathione (GSH) depletion, which were associated with glutathione peroxidase, superoxide dismutase and gamma-glutamyltransferase downregulation suggest redox imbalance. Disrupted astrocyte cytoskeleton was evidenced by downregulated and hyperphosphorylated glial fibrillary acidic protein (GFAP). Morphological and structural characterization of the sensorimotor cerebral cortex (SCC) showed unaltered thickness of the SCC. However, decreased size of neurons on the layers II & III and IV in the right SCC and increased NeuN positive neurons in specific SCC layers, suggest that they are differently affected by the low TH levels during neurodevelopment. Hypothyroid pups presented increased number of foot-faults in the gridwalk test indicating affected motor functions. Taken together, our results show that congenital hypothyroidism disrupts glutamatergic and cholinergic neurotransmission, Ca2+ equilibrium, redox balance, cytoskeleton integrity, morphological and functional aspects in the cerebral cortex of young rats.

The potent insulin secretagogue effect of betulinic acid is mediated by potassium and chloride channels.

Betulinic acid (BA) has been described as an insulin secretagogue which may explain its potent antihyperglycemic effect; however, the exact role of BA as an insulinogenic agent is not clear. The aim of this study was to investigate the mechanism of BA on calcium influx and static insulin secretion in pancreatic islets isolated from euglycemic rats. We found that BA triggers calcium influx by a mechanism dependent on ATP-dependent potassium channels and L-type voltage-dependent calcium channels. Additionally, the voltage-dependent and calcium-dependent chloride channels are also involved in the mechanism of BA, probably due to an indirect stimulation of calcium entry and increased intracellular calcium. Additionally, the downstream activation of PKC, which is necessary for the effect of BA on calcium influx, is involved in the full stimulatory response of the triterpene. BA stimulated the static secretion of insulin in pancreatic islets, indicating that the abrupt calcium influx may be a key step in its secretagogue effect. As such, BA stimulates insulin secretion through the activation of electrophysiological mechanisms, such as the closure of potassium channels and opening of calcium and chloride channels, inducing cellular depolarization associated with metabolic-biochemical effects, in turn activating PKC and ensuring the secretion of insulin.

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.

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

Oestrogens 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.

In vivo inhibition of tumor progression by 5 hydroxy-1,4-naphthoquinone (juglone) and 2-(4-hydroxyanilino)-1,4-naphthoquinone (Q7) in combination with ascorbate.

The purpose of the study was to obtain further in vivo data of antitumor effects and mechanisms triggered by juglone and Q7 in combination with ascorbate. The study was done using Ehrlich ascites tumor-bearing mice. Treatments were intraperitoneal every 24 h for 9 days. Control group was treated with excipient. Previous tests selected the doses of juglone and Q7 plus ascorbate (1 and 100 mg/kg, respectively). Samples of ascitic fluid were collected to evaluate carbonyl proteins, GSH and activity of antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase. Hypoxia inducible factor HIF-1α, GLUT1, proteins driving cell cycle (p53, p16 and cyclin A) and apoptosis (poly-ADP-polymerase PARP, Bax and Bcl-xL) were assessed by western blot. Tumor cells were categorized by the phase of cell cycle using flow cytometry and type of cell death using acridine orange/ethidium bromide. A glucose uptake assessment was performed by liquid scintillation using Ehrlich tumor cells cultured with (14)C-deoxyglucose. Treatments caused increased protein carbonylation and activity of antioxidant enzymes and decreased levels of GSH, HIF-1α, GLUT1 and glucose uptake in tumor cells. They also caused increased number of tumor cells in G1, p53 and p16 activation and decreased cyclin A, but only when combined with ascorbate. Apoptosis was induced mostly when treatments were done with ascorbate, causing PARP and Bax cleavage, and increased Bax/Bcl-xL ratio. Juglone and Q7 in combination with ascorbate caused inhibition of tumor progress in vivo by triggering apoptosis and cell cycle arrest associated with oxidative stress, suppression of HIF-1 and uncoupling of glycolytic metabolism.

Novel sulfonyl(thio)urea derivatives act efficiently both as insulin secretagogues and as insulinomimetic compounds.

Glibenclamide is widely used in the management of non-insulin dependent diabetes mellitus, but numerous risks limit its use in therapy. In the search for novel structures that are safer and more efficient than glibenclamide, we obtained new chemical analogs based on bioisosterism, through the treatment of benzenesulfonamide derivatives with isothiocyanates and isocyanates, affording (thio)ureas with good yield. We also verified the hypoglycemic activity, through an in vivo approach. Most of these synthesized compounds improved glucose tolerance, and the mechanism of action of the best compound (7) suggests that its effect is mediated by insulin secretion, while its hypoglycemic action is triggered by glucose uptake involving GLUT4 expression and translocation through PI-3K and PKA activity and active de novo protein synthesis in skeletal muscle. Taking all these factors together, sulfonylthiourea 7 acts as an insulin secretagogue and insulinomimetic agent on glucose homeostasis, and does not exhibit toxicity in acute treatment.

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.