Triterpene betulin may be involved in the acute effects of pulp and paper mill effluent on testis physiology in zebrafish.

Pulp and paper mill effluent can cause changes in the morphology and energy metabolism in the zebrafish (Danio rerio) testis. Betulin, a naturally occurring triterpene is commonly present in this type of effluent and is suspected of being involved in these effects. The aim of this study was to compare the effects pulp and paper mill effluent and betulin on various aspects of testicular physiology in the zebrafish. This included the in vitro effects of effluent and betulin on testicular lactate content and lactate dehydrogenase (LDH) activity. In addition, the effects of betulin on glucose uptake, glycogen, alanine aminotransferase (ALT), reactive oxygen and nitrogen species formation and oxidative damage in the testes were determined. Furthermore, we compared the effects and mechanism of action of betulin and effluent on calcium homeostasis in testes. In vitro exposure to both effluent and betulin decreased lactate and calcium influx, possibly due to the activation of the sodium­calcium exchanger (NCX) pump. Additionally, betulin-treated testes had higher reactive oxygen species (ROS) and reduced glutathione (GSH) content, as well as increased glutathione transferase (GST) activity and a tendency towards decreased catalase (CAT) activity. Thus, this study shows that alterations in testis physiology caused by the pulp and paper mill effluent in the testis may be due in part to the actions of betulin.

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.

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.

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.

Pulsed ultrasound and dimethylsulfoxide gel treatment reduces the expression of pro-inflammatory molecules in an animal model of muscle injury.

Studies have shown an exacerbated increase in proinflammatory markers during and after muscle injury. In this way, interventions that reduce inflammatory activation appear to be of great interest in muscle injury therapy. Thus, the preset study evaluated the effect of low-intensity pulsed ultrasound (LIPUS) and dimethylsulfoxide (DMSO) on the proinflammatory molecules in an animal model of traumatic muscle injury. Forty-eight 3-month old male Wistar rats were divided into six groups (n = 8/group): sham; muscle injury without treatment; muscle injury and gel-saline (0.9%); muscle injury and gel-DMSO (15 mg/kg); muscle injury and LIPUS plus gel-saline; and muscle injury and LIPUS plus gel-DMSO. Two, 12, 24 and 48 h after trauma, four groups received one of the treatments described. One hour after, Western blot was performed to quantify proinflammatory protein levels. We observed greater protein levels of TNFα (3.9 times), IL-1ß (3.6 times), JNK phosphorylation (4.2 times) and NFκB (3.8 times) in muscle injury group. However, the combined LIPUS with DMSO resulted in significantly lower levels of TNFα (2.2 times), IL-1ß (2.1 times), JNK phosphorylation (2.4 times), and NFκB (2.1 times). The results demonstrate that LIPUS associated with DMSO gel can attenuate TNFα, IL-1ß, NFκB protein levels and JNK phosphorylation in traumatic muscle injury.

Sodium butyrate decreases the activation of NF-κB reducing inflammation and oxidative damage in the kidney of rats subjected to contrast-induced nephropathy.

BACKGROUND: Contrast-induced nephropathy (CIN) is associated with a combination of hypoxic and toxic renal tubular damage, renal endothelial dysfunction and altered intra-renal microcirculation. Recently, sodium butyrate (SB) has been focused on since it possesses anti-inflammatory activities. Thus, based on the lack of information on the effects of SB in acute kidney injury (AKI), we investigated the possible effects of SB after CIN in rats. METHODS: Wistar rats were divided into three groups: (1 sham) control, (2 MI) AKI treated with contrast medium and (3 MI + SB) AKI plus SB. Six days after contrast administration, blood and kidney were removed for the determination of creatinine, interleukin (IL)-6 levels, oxidative damage parameters and histologic analyses. Nuclear factor kappa B (NF-κB), pIκBα and vasodilator-stimulated phosphoprotein (VASP) protein content were determined by immunoblotting. RESULTS: After 6 days, the levels of creatinine increased significantly in the MI group, and this was attenuated using SB. SB treatment was associated with a decrease on the levels of lipid peroxidation, but not the protein oxidation, and IL-6 levels, as well as tubular damage. These effects are probably mediated, in part, by a decrease on the activation of NF-κB in the kidney, but not alteration in pVASP content. CONCLUSIONS: The current experiment suggests that NF-κB induced an inflammatory response after CIN and SB could inhibit NF-κB expression protecting against CIN in rats.

Short-term inhibition of SREBP-1c expression reverses diet-induced non-alcoholic fatty liver disease in mice.

OBJECTIVE: The present study investigates the level of Sterol-regulatory element-binding proteins (SREBP-1c) and related proteins in obese mice (DIO) treated with SREBP-1c antisense oligonucleotide (ASO) to observe a reversal of steatosis. MATERIALS AND METHODS: Swiss mice were fed on chow containing 61 kJ% saturated fat for 8 weeks to develop obesity. After this period, one group of animals was used to assess the molecular effects of SREBP-1c antisense oligonucleotide treatment by immunoblot analysis in a dose-response curve (0; 1.0; 2.0; 3.0; 4.0 nmol/day). After the dose (3.0 nmol/day) was determined, another group was treated for 14 days. After a period of 24 h following the last injection mice were killed and plasma and hepatic tissue were obtained to evaluate plasma triglycerides and total liver fat. Western blot was performed to evaluate SREBP-1c, FAS, SCD-1, PPARγ and CPT1 expression and AMPK[Thr172] and ACC[Ser79] phosphorylation. Livers were stained using the hematoxylin and eosin method for histological analysis. RESULTS: Body weight, epididymal fat and glucose levels were not affected by one daily dose of ASO. However, total plasma triglycerides and total liver fat were significantly reduced. Also, this treatment inhibited SREBP-1c and reduced protein levels of a series of proteins involved in lipogenesis, including ACC, FAS and SCD-1. Moreover, mice treated with ASO presented a significant reduction in macroscopic and microscopic features of hepatic steatosis. CONCLUSION: Our results demonstrate that the inhibition of SREBP-1c decreased the expression of lipogenic enzymes, reducing the accumulation of triglycerides and, finally, reversing hepatic steatosis in mice.

Ketamine plus imipramine treatment induces antidepressant-like behavior and increases CREB and BDNF protein levels and PKA and PKC phosphorylation in rat brain.

A growing body of evidence has pointed to the N-methyl-d-aspartate (NMDA) receptor antagonists as a potential therapeutic target for the treatment of major depression. The present study investigated the possibility of synergistic interactions between antidepressant imipramine with the uncompetitive NMDA receptor antagonist ketamine. Wistar rats were acutely treated with ketamine (5 and 10mg/kg) and imipramine (10 and 20mg/kg) and then subjected to forced swimming tests. The cAMP response element bindig (CREB) and brain-derived neurotrophic factor (BDNF) protein levels and protein kinase C (PKC) and protein kinase A (PKA) phosphorylation were assessed in the prefrontal cortex, hippocampus and amygdala by imunoblot. Imipramine at the dose of 10mg/kg and ketamine at the dose of 5mg/kg did not have effect on the immobility time; however, the effect of imipramine (10 and 20mg/kg) was enhanced by both doses of ketamine. Ketamine and imipramine alone or in combination at all doses tested did not modify locomotor activity. Combined treatment with ketamine and imipramine produced stronger increases of CREB and BDNF protein levels in the prefrontal cortex, hippocampus and amygdala, and PKA phosphorylation in the hippocampus and amygdala and PKC phosphorylation in prefrontal cortex. The results described indicate that co-administration of antidepressant imipramine with ketamine may induce a more pronounced antidepressant activity than treatment with each antidepressant alone. This finding may be of particular importance in the case of drug-resistant patients and could suggest a method of obtaining significant antidepressant actions whilst limiting side effects.

Endurance exercise training ameliorates insulin resistance and reticulum stress in adipose and hepatic tissue in obese rats.

Obesity-induced endoplasmatic reticulum (ER) stress has been demonstrated to underlie the induction of obesity-induced JNK and NF-κB activation inflammatory responses, and generation of peripheral insulin resistance. On the other hand, exercise has been used as a crucial tool in obese and diabetic patients, and may reduce inflammatory pathway stimulation. However, the ability of exercise training to reverse endoplasmatic reticulum stress in adipose and hepatic tissue in obesity has not been investigated in the literature. Here, we demonstrate that exercise training ameliorates ER stress and insulin resistance in DIO-induced rats. Rats were fed with standard rodent chow (3,948 kcal kg(-1)) or high-fat diet (5,358 kcal kg(-1)) for 2 months. After that rats were submitted to swimming training (1 h per day, 5 days for week with 5% overload of the body weight for 8 weeks). Samples from epididymal fat and liver were obtained and western blot analysis was performed. Our results showed that swimming protocol reduces pro-inflammatory molecules (JNK, IκB and NF-κB) in adipose and hepatic tissues. In addition, exercise leads to reduction in ER stress, by reducing PERK and eIF2α phosphorylation in these tissues. In parallel, an increase in insulin pathway signaling was observed, as confirmed by increases in IR, IRSs and Akt phosphorylation following exercise training in DIO rats. Thus, results suggest that exercise can reduce ER stress, improving insulin resistance in adipose and hepatic tissue.