Subacute exposure to Roundup® changes steroidogenesis and gene expression of the glutathione-glutaredoxin system in rat ovaries: Implications for ovarian toxicity of this glyphosate-based herbicide.

Studies have indicated that glyphosate induces endocrine disruption and may adversely affect the male reproductive system. However, evidence of its effects on ovarian function is poorly understood so far, making further studies necessary on the mechanisms of the glyphosate toxicity in the female reproductive system. The aim of this work was to evaluate the effect of a subacute exposure (28 days) to the glyphosate-based formulation Roundup® (1.05, 10.5 and 105 µg/kg b.w. of glyphosate) on steroidogenesis, oxidative stress, systems involved in cell redox control and histopathological parameters in rat ovaries. Hence we quantify plasma estradiol and progesterone by chemiluminescence; non-protein thiol levels, TBARS, superoxide dismutase and catalase activity by spectrophotometry; gene expression of steroidogenic enzymes and redox systems by real-time PCR; and ovarian follicles by optical microscopy. Our results demonstrated that oral exposure increased progesterone levels and the mRNA expression of 3ß-hydroxysteroid dehydrogenase. Histopathological analysis revealed a decrease in the number of primary follicles and an increase in the number of corpus luteum in rats exposed to Roundup®. An imbalance of the oxidative status was also evidenced by decreasing the catalase activity at all groups exposed to the herbicide. Increased lipid peroxidation and gene expression of glutarredoxin and decreased of glutathione reductase were also observed. Our results indicate that Roundup® causes endocrine disruption of hormones related to female fertility and reproduction and changes the oxidative status by altering antioxidant activity, inducing lipid peroxidation, as well as changing the gene expression of the glutathione-glutarredoxin system in rat ovaries.

The neurotoxic mechanism of Jack Bean Urease in insects involves the interplay between octopaminergic and dopaminergic pathways.

In the last decades, the entomotoxicity of JBU and its derived peptides became an object of study, due mainly to the ubiquitous interaction of these compounds with different species of insects and their potential as natural insecticides. In this work, we investigated the neurotoxic effects of JBU in Nauphoeta cinerea cockroaches by dissecting pharmacologically the monoaminergic pathways involved. Selective pharmacological modulators for monoaminergic pathways in in vivo and ex vivo experimental models were employed. Thus, the analysis of N. cinerea neurolocomotory behavior demonstrated that JBU (1.5 and 3 µg/g) induces a significant decrease in the exploratory activity. In these assays, pretreatment of animals with phentolamine, SCH23390 or reserpine, interfered significantly with the response of JBU. Using in vivo abductor metathoracic preparations JBU (1.5 µg/g) induced progressive neuromuscular blockade, in 120 min recordings. In this set of experiments, the previous treatment of the animals with phentolamine, SCH23390 or reserpine, completely inhibited JBU-induced neuromuscular blockade. The recordings of spontaneous compound neural action potentials in N. cinerea legs showed that JBU, only in the smallest dose, significantly decreased the number of potentials in 60 min recordings. When the animals were pretreated with phentolamine, SCH23390, or reserpine, but not with mianserin, there was a significant prevention of the JBU-inhibitory responses on the action potentials firing. Meanwhile, the treatment of the animals with mianserin did not affect JBU's inhibitory activity. The data presented in this work strongly suggest that the neurotoxic response of JBU in N. cinerea involves a cross talking between OCTOPAMIN-ergic and DOPAMIN-ergic nerve systems, but not the SEROTONIN-ergic neurotransmission. Further molecular biology studies with expression of insect receptors associated with voltage clamp techniques will help to discriminate the selectivity of JBU over the monoaminergic transmission.

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.

Entomotoxic Activity of Prasiola crispa (Antarctic Algae) in Nauphoeta cinerea Cockroaches: Identification of Main Steroidal Compounds.

Prasiola crispa is a macroscopic green algae found in abundance in Antarctica ice free areas. Prasiola crispan-hexaneextract (HPC) induced insecticidal activity in Nauphoeta cinerea cockroaches after 24 h of exposure. The chemical analysis of HPC revealed the presence of the followingphytosterols: ß-sitosterol, campesterol and stigmasterol. The incubation of cockroach semi-isolated heart preparations with HPC caused a significant negative chronotropic activity in the heartbeats. HPC affected the insect neuromuscular function by inducing a complete inhibition of the cockroach leg-muscle twitch tension. When the isolated phytosterols were injected at in vivo cockroach neuromuscular preparations, there was a progressive inhibition of muscle twitches on the following order of potency: ß-sitosterol > campesterol > stigmasterol. HPC also provoked significant behavioral alterations, characterized by the increase or decrease of cockroach grooming activity, depending on the dose assayed. Altogether, the results presented here corroborate the insecticide potential of Prasiola crispa Antarctic algae. They also revealed the presence of phytosterols and the involvement of these steroidal compounds in the entomotoxic activity of the algae, potentially by modulating octopaminergic-cholinergic pathways. Further phytochemical-combined bioguided analysis of the HPC will unveil novel bioactive compounds that might be an accessory to the insecticide activity of the algae.

Iron and manganese present in underground water promote biochemical, genotoxic, and behavioral alterations in zebrafish (Danio rerio).

Iron (Fe) and manganese (Mn) are metals commonly found at high concentrations in underground water. These metals are essential for the good functioning of living organisms, but high concentrations lead to imbalance, potentiating the appearance of pathologies. This study aimed to evaluate the effect of exposure to naturally occurring metals in groundwater, using zebrafish (Danio rerio) as an experimental model. Thus, zebrafish were exposed to Fe (0.8 and 1.3 mg/L), Mn (0.2 and 0.4 mg/L), and groundwater collected from deep tube wells with Fe and Mn (Fe 0.8/Mn 0.2 mg/L and Fe 1.3/Mn 0.4 mg/L) for 30 days. Bioaccumulation of these metals has been demonstrated in the livers and muscles of zebrafish. Acetylcholinesterase activity changed only in zebrafish muscles in all groups. Sulfhydryl levels changed mainly in the group Mn 0.4. SOD/CAT ratio decreased in the groups Fe 0.8 and 1.3, Mn 0.4, and Fe 0.8/Mn 0.4. An increase in the frequency of micronucleus in all groups was shown as a consequence of these changes. Behavioral parameters (time and distance traveled, mean speed, turn angle, latency, and number of crossings between compartments) have also changed, mainly in the groups Fe 1.3, Mn 0.4, and Fe 1.3/Mn 0.4. Therefore, long-term exposure to Fe and Mn, even at not so high concentrations, may cause biochemical, genotoxic, and behavioral changes in zebrafish.

Jaburetox, a natural insecticide derived from Jack Bean Urease, activates voltage-gated sodium channels to modulate insect behavior.

Jaburetox (Jbtx) is an insecticidal peptide derived from Canavalia ensiformis urease, whose mechanism of action is not completely elucidated. We employed behavioral, electromyographical and electrophysiological protocols to identify the cellular and molecular targets involved in the Jbtx entomotoxicity in cockroaches and locusts. In Nauphoeta cinerea, Jbtx (32 µg/g) altered the locomotory behaviour inducing a significative decrease in the distance travelled followed by a significant increase in stopped time (52 ±â€¯85 cm and 2573 ±â€¯89 s, p < .05, n = 40). Jbtx (8 to 32 µg/g body weight, respectively) also increased the leg and antennae grooming activities (p < .05, n = 40, respectively). Jbtx (8 to 16 µg/g) induced a maximum neuromuscular blockade of 80.72% (n = 6, p < .05) and was cardiotoxic, decreasing the cockroach heart rate. The electrophysiological profiles of both muscle and nerve of L. migratoria showed that Jbtx (2.5 × 10-7 and 2.5 × 10-3 µg/ body weight) induced a significant increase in the amplitude of nerve action potentials (n = 5, p < .05). Voltage clamp analysis of Jbtx (200 nM) applied in Xenopus laevis oocytes heterologously expressed with Nav 1.1 channels showed a significant increase in the sodium currents. In conclusion, this work revealed that the entomotoxic activity of Jbtx involves complex behavioral alterations that begins with an initial activation of voltage-gated sodium channels.

Neurotoxic effects of sublethal concentrations of cyanobacterial extract containing anatoxin-a(s) on Nauphoeta cinerea cockroaches.

The detection of cyanotoxins, such as the anatoxin-a(s), is essential to ensure the biological safety of water environments. Here, we propose the use of Nauphoeta cinerea cockroaches as an alternative biological model for the biomonitoring of the activity of anatoxin-a(s) in aquatic systems. In order to validate our proposed model, we compared the effects of a cyanobacterial extract containing anatoxin-a(s) (CECA) with those of the organophosphate trichlorfon (Tn) on biochemical and physiological parameters of the nervous system of Nauphoeta cinerea cockroaches. In brain homogenates from cockroaches, CECA (5 and 50 µg/g) inhibited acetylcholinesterase (AChE) activity by 53 ±â€¯2% and 51 ±â€¯7%, respectively, while Tn (5 and 50 µg/g) inhibited AChE activity by 35 ±â€¯4% and 80 ±â€¯9%, respectively (p < 0.05; n = 6). Moreover, CECA at concentrations of 5, 25, and 50 µg/g decreased the locomotor activity of the cockroaches, diminishing the distance travelled and increasing the frequency and duration of immobile episodes similarly to Tn (0.3 µg/g) (p < 0.05, n = 40, respectively). CECA (5, 25 and 50 µg/g) induced an increase in the leg grooming behavior, but not in the movement of antennae, similarly to the effect of Tn (0.3 µg/g). In addition, both CECA (50 µg/200 µl) and Tn (0.3 µg/200 µl) induced a negative chronotropism in the insect heart (37 ±â€¯1 and 47 ±â€¯8 beats/min in 30 min, respectively) (n = 9, p > 0.05). Finally, CECA (50 µg/g), Tn (0.3 µg/g) and neostigmine (50 µg/g) caused significant neuromuscular failure, as indicated by the monitoring of the in vivo neuromuscular function of the cockroaches, during 100 min (n = 6, p < 0.05, respectively). In conclusion, sublethal doses of CECA provoked entomotoxicity. The Tn-like effects of CECA on Nauphoeta cinerea cockroaches encompass both the central and peripheral nervous systems in our insect model. The inhibitory activity of CECA on AChE boosts a cascade of signaling events involving octopaminergic/dopaminergic neurotransmission. Therefore, this study indicates that this insect model could potentially be used as a powerful, practical, and inexpensive tool to understand the impacts of eutrophication and for orientating decontamination processes.

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.

Acute effect of bisphenol A: Signaling pathways on calcium influx in immature rat testes.

We investigated the acute effect of low concentrations of BPA on calcium influx and the mechanism of action of BPA in this rapid response in the rat testis. BPA increased calcium influx at 1 pM and 1 nM at 300 s of incubation, in a similar manner to that of estradiol. At 1 pM, BPA stimulated calcium influx independently of classical estrogen receptors, consistent with a G-protein coupled receptor. This effect also involves the modulation of ionic channels, such as K+, TRPV1 and Cl- channels. Furthermore, BPA is able to modulate calcium from intracellular storages by inhibiting SERCA and activating IP3 receptor/Ca2+ channels at the endoplasmic reticulum and activate kinase proteins, such as PKA and PKC. The rapid responses of BPA on calcium influx could, in turn, trigger a cross talk by MEK and p38MAPK activation and also mediate genomic responses.

Central and peripheral neurotoxicity induced by the Jack Bean Urease (JBU) in Nauphoeta cinerea cockroaches.

BACKGROUND: Ureases of Canavalia ensiformis are natural insecticides with a still elusive entomotoxic mode of action. We have investigated the mechanisms involved in the neurotoxicity induced by Jack Bean Urease (JBU) in Nauphoeta cinerea (Olivier). METHODS: To carry out this study we have employed biochemical and neurophysiological analysis of different cockroach organ systems. RESULTS AND CONCLUSIONS: The injection of the insects with JBU (0.75-6µg/g animal), although not lethal within 24h, caused significant inhibition of the brain acetylcholinesterase activity (60±5%, p<0.05, n=6). JBU (1.5µg/200µL), acetylcholine (0.3µg/200µL) or neostigmine (0.22µg/200µL), induced a positive cardiac chronotropism (∼25%) in the cockroaches (p<0.05, n=9). JBU (6µg/g) increased the insects' grooming activity (137±7%), similarly to octopamine (15µg/g) (p<0.05, n=30, respectively). Pretreating the insects with phentolamine (0.1µg/g) prevented the JBU- or octopamine-induced increase of grooming activity. JBU (6µg/g) caused 65±9% neuromuscular blockade in the cockroaches, an effect prevented by bicuculline (5µg/g) (p<0.05, n=6). JBU (6µg/g) decreased the frequency whilst increasing the amplitude of the spontaneous neural compound action potentials (1425±52.60min-1, controls 1.102±0.032mV, p<0.05, n=6, respectively). Altogether the results indicate that JBU induces behavioral alterations in Nauphoeta cinerea cockroaches probably by interfering with the cholinergic neurotransmission. The neuromuscular blocking activity of JBU suggests an interplay between acetylcholine and GABA signaling. GENERAL SIGNIFICANCE: The search for novel natural molecules with insecticide potential has become a necessity more than an alternative. Understanding the mode of action of candidate molecules is a crucial step towards the development of new bioinsecticides. The present study focused on the neurotoxicity of Canavalia ensiformis urease, a natural insecticide, in cockroaches and revealed interferences on the cholinergic, octopaminergic and GABA-ergic pathways as part of its entomotoxic mode of action.

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 αvß3. 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.

Pesquisa das medidas do crescimento da sensaçäo do loudness numa populaçäo de ouvintes e deficientes auditivos / Research on the loudness growth function in a population of hearing and hearing impaired individuals

Considerando que a näo linearidade do sistema auditivo comprometido exige uma maior atençäo no trabalho com a amplificaçäo, o presente estudo foi realizado no módulo - O Homem por trás da surdez -, no curso de fonoaudiologia na PUC-SP - DERDIC, com o objetivo de fazer um levantamento das medidas do crescimento da sensaçäo do loudness numa populaçäo de indivíduos ouvintes e em deficientes auditivos; verificar quais as características específicas à amplificaçäo deveria apresentar cada paciente, a partir das medidas do loudness dos deficientes auditivos. Os resultados mostraram que as medidas do loudness representam uma contribuiçäo significativa para o processo de adaptaçäo de AASI