Effects of different maternal diets on adipose tissue inflammation and liver tissue oxidative stress in dams and their female offspring.

Pregnancy and lactation are important stages of fetal development. Therefore, this study investigated how different maternal diets offered during gestation and lactation periods affect adipose tissue inflammation and liver tissue oxidative stress of dams and their female offspring. Female BALB/c albino mice (60 days old) were randomized into three groups receiving a standard (CONT), hypercaloric (HD), or restricted (RD) diet during the pregnancy. After birth, female offspring weaned at 21 days were divided into two groups that received a standard or restricted diet (CONT/CONT, CONT/RD, RD/CONT, RD/RD, HD/CONT, and HD/RD) until 100 days old. Histological, oxidative parameters and inflammatory infiltrate of dams' and offspring's liver and adipose tissue were evaluated. HD dams presented non-alcoholic steatohepatitis (NASH) diagnosis and an increase in tumor necrosis factor-alpha (TNF-α) concentrations when compared to the RD and CONT dams, indicating a pro-inflammatory state. High concentrations of malondialdehyde (MDA) formation and catalase (CAT) activity in HD when compared to the CONT in the liver. SOD activity decreased in RD mice compared to CONT, and the SOD/CAT ratio was decreased in the RD and HD in comparison to the CONT. The maternal diet leads to an increase in SOD in RD/RD compared to HD/RD. RD-fed dams showed an increase in inflammatory infiltrates compared to CONT, evidencing changes caused by a restrictive diet. In the HD/CONT offspring, we verified an increase in inflammatory infiltrates in relation to the offspring fed a standard diet. In conclusion, HD, and RD, during pregnancy and lactation, altered the liver and adipose tissues of mothers. Furthermore, the maternal diet negatively impacts the offspring's adipose tissue but does not cause liver damage in these animals in adult life.

Taurine counteracts the neurotoxic effects of streptozotocin-induced diabetes in rats.

Diabetes is a chronic metabolic disease associated with oxidative stress, damage to biomolecules such as DNA, and neuroinflammation. Taurine, a sulfur-containing amino acid widespread in the brain, has neuroprotective properties that might prevent tissue injury and DNA damage induced by chronic hyperglycemia. We evaluated the effects of chronic taurine treatment on oxidative stress parameters, DNA damage and inflammatory markers in the frontal cortex, and hippocampus of streptozotocin-induced diabetic rats. Diabetic rats displayed increased levels of reactive oxygen species (ROS) and DNA damage in both areas, evidencing the pro-oxidant effects of diabetes in the brain. Moreover, this condition increased levels of several inflammatory mediators, such as IL-6, IL-12, TNF-γ, and IFN-α, more pronouncedly in the hippocampus. Supporting our hypothesis, taurine treatment reduced ROS, DNA damage, and inflammatory cytokine levels, providing evidence of its beneficial effects against genotoxicity and neuroinflammation associated with diabetes. Our data endorse the necessary clinical trials to evaluate the efficacy and safety of taurine supplementation in the prevention and treatment of neurochemical and metabolic alterations related to diabetes.

Proton-Transfer-Based Azides with Fluorescence Off-On Response for Detection of Hydrogen Sulfide: An Experimental, Theoretical, and Bioimaging Study.

This work describes the synthesis of photoactive proton transfer compounds based on the benzazolic core containing the azide group. The compounds present absorption in the UV region and fluorescence emission in the visible region of the spectra with large Stokes shift due to a phototautomerism in the excited state (ESIPT). The azide location on the benzazolic structure presented a noteworthy role on their photophysics, leading to fluorescence quenching. A photophysical study was performed in the presence of NaHS to evaluate their application as an H2S sensor. The methodology employed was the reduction of azides to amines using NaHS to mimic H2S, resulting in an off-on response fluorescence mechanism. The observed photophysical features were successfully used to explore the azides as fluorescent probes in biological media. In addition, DFT and TD-DFT calculations with the CAM-B3LYP/cc-pVDZ and CAM-B3LYP/jun-cc-pVTZ level, respectively, were performed in order to understand the photophysics features of azide derivatives, where the main interest was to investigate the fluorescence quenching experimentally observed in the azide derivatives.

DNA damage and oxidative stress induced by seizures are decreased by anticonvulsant and neuroprotective effects of lobeline, a candidate to treat alcoholism.

The alkaloid lobeline (Lob) has been studied due to its potential use in treatment of drug abuse. This study evaluates the possible anticonvulsant and neuroprotective activities of Lob to obtain new information on its properties that could confirm it as a candidate in the treatment of alcohol addiction. The anticonvulsant effect of Lob was evaluated using a pilocarpine-induced seizure model. In addition, possible neuroprotective effects were investigated measuring DNA damage using the comet assay, assessing free radical levels by dichlorofluorescein diacetate (DCF) oxidation, and measuring the antioxidant potential using the α, α-diphenyl-ß-picrylhydrazyl (DPPH) scavenging assay, besides measuring superoxide dismutase (SOD) and catalase (CAT) enzyme activities in brain tissues. Lobeline increased the latency to the first seizure and decreased the percentage of seizures in a similar way as diazepam, used as control. DNA damage induced by Pil and hydrogen peroxide were decreased in hippocampus and cerebral cortex from mice treated with Lob. The levels of free radicals and CAT activity increased in cortex and hippocampus, respectively, in mice treated with Pil. Lobeline decreased CAT in hippocampus, leading to similar values as in the saline negative control. In conclusion, Lob has anticonvulsant and neuroprotective actions that may be mediated by antioxidant-like mechanisms, indicating its potential as candidate drug in alcoholism therapy.

Globotriaosylsphingosine induces oxidative DNA damage in cultured kidney cells.

Fabry disease (FD) is a lysosomal disorder caused by mutations leading to a deficient activity α-galactosidase A with progressive and systemic accumulation of its substrates. Substrates deposition is related to tissue damage in FD, but the underlying molecular mechanisms remain not completely understood. DNA damage has been associated with disease progression in chronic diseases and was recently described in high levels in Fabry patients. Once renal complications are major morbidity causes in FD, we investigated the effects of the latest biomarker for FD - globotriaosylsphingosine (lyso-Gb3) in a cultured renal lineage - human embryonic kidney cells (HEK-293 T) - on DNA damage. In concentrations found in Fabry patients, lyso-Gb3 induced DNA damage (by alkaline comet assay) with oxidative origin in purines and pyrimidines (by comet assay with endonucleases). These data provide new information about a deleterious effect of lyso-Gb3 and could be useful to studies looking for new therapeutic strategies to FD.

Development and characterization of a temozolomide-loaded nanoemulsion and the effect of ferrocene pre and co-treatments in glioblastoma cell models.

BACKGROUND: Glioblastoma is a severe brain tumor that requires aggressive treatment involving surgery, radiotherapy, and chemotherapy, offering a survival rate of only 15 months. Fortunately, recent nanotechnology progress has enabled novel approaches and, alongside ferrocenes' unique properties of cytotoxicity, sensitization, and interaction with reactive oxygen species, have brought new possibilities to complement chemotherapy in nanocarrier systems, enhancing treatment results. METHODS: In this work, we developed and characterized a temozolomide-loaded nanoemulsion and evaluated its cytotoxic potential in combination with ferrocene in the temozolomide-resistant T98G and temozolomide-sensitive U87 cell lines. The effects of the treatments were assessed through acute assays of cell viability, cell death, mitochondrial alterations, and a treatment protocol simulation based on different two-cycle regimens. RESULTS: Temozolomide nanoemulsion showed a z-average diameter of 173.37 ± 0.86 nm and a zeta potential of - 6.53 ± 1.13 mV. Physicochemical characterization revealed that temozolomide is probably associated with nanoemulsion droplets instead of being entrapped within the nanostructure, allowing a rapid drug release. In combination with ferrocene, temozolomide nanoemulsion reduced glioblastoma cell viability in both acute and two-cycle regimen assays. The combined treatment approach also reversed T98G's temozolomide-resistant profile by altering the mitochondrial membrane potential of the cells, thus increasing reactive oxygen species generation, and ultimately inducing cell death. CONCLUSIONS: Altogether, our results indicate that using nanoemulsion containing temozolomide in combination with ferrocene is an effective approach to improve glioblastoma therapy outcomes.

Dietary interventions in mice affect oxidative stress and gene expression of the Prlr and Esr1 in the adipose tissue and hypothalamus of dams and their offspring.

Maternal diet is key to the progeny's health since it may impact on the offspring's adult life. In this study, mice dams received standard (CONT), restrictive (RD), or hypercaloric (HD) diets during mating, pregnancy, and lactation. Male offspring of each group of dams also received these diets: CONT, RD, HD. Aiming to evaluate the oxidative stress in the adipose tissue, reactive oxygen species (ROS) production, catalase (CAT), and superoxide dismutase (SOD) activities were analyzed in dams and offspring. In the adipose tissue and hypothalamus, gene expression of prolactin (Prlr) and estrogen alpha (Esr1) receptors was performed in dams and offspring. Protein expression of Stat5 was evaluated in the adipose tissue of the offspring from RD-fed dams. HD-fed dams increased triglycerides and leptin serum concentrations, and decreased SOD activity in the adipose tissue. In the offspring's adipose tissue, we observed a maternal diet effect caused by HD, with increased ROS production and SOD and CAT activities. Gene expression of Prlr and Esr1 in the offspring's adipose tissue was decreased due to maternal RD. Mice from HD-fed dams showed higher Stat5 expression compared to the offspring from CONT and RD dams in the adipose tissue. In the hypothalamus, we found decreased expression of Prlr in RD and HD dams, compared to CONT; and a maternal diet effect on Prlr and Esr1 gene expression in the offspring. In conclusion, we can affirm that maternal nutrition impacts the redox state and influences the gene expression of Prlr and Esr1, which are involved in energy metabolism, both peripherally and centrally in the adult life of the female offspring.

Nek1-inhibitor and temozolomide-loaded microfibers as a co-therapy strategy for glioblastoma treatment.

Malignant glioblastoma (GB) is the predominant primary brain tumour in adults, but despite the efforts towards novel therapies, the median survival of GB patients has not significantly improved in the last decades. Therefore, localised approaches that treat GB straight into the tumour site provide an alternative to enhance chemotherapy bioavailability and efficacy, reducing systemic toxicity. Likewise, the discovery of protein targets, such as the NIMA-related kinase 1 (Nek1), which was previously shown to be associated with temozolomide (TMZ) resistance in GB, has stimulated the clinical development of target therapy approaches to treat GB patients. In this study, we report an electrospun polyvinyl alcohol (PVA) microfiber (MF) brain-implant prepared for the controlled release of Nek1 protein inhibitor (iNek1) and TMZ or TMZ-loaded nanoparticles. The formulations revealed adequate stability and drug loading, which prolonged the drugs' release allowing a sustained exposure of the GB cells to the treatment and enhancing the drugs' therapeutic effects. TMZ-loaded MF provided the highest concentration of TMZ within the brain of tumour-bearing rats, and it was statistically significant when compared to TMZ via intraperitoneal (IP). All animals treated with either co-therapy formulation (TMZ + iNek1 MF or TMZ nanoparticles + iNek1 MF) survived until the endpoint (60 days), whereas the Blank MF (drug-unloaded), TMZ MF and TMZ IP-treated rats' median survival was found to be 16, 31 and 25 days, respectively. The tumour/brain area ratio of the rats implanted with either MF co-therapy was found to be reduced by 5-fold when compared to Blank MF-implanted rats. Taken together, our results strongly suggest that Nek1 is an important GB oncotarget and the inhibition of Nek1's activity significantly decreases GB cells' viability and tumour size when combined with TMZ treatment.

Gap junctions and expression of Cx36, Cx43 and Cx45 in the posterodorsal medial amygdala of adult rats.

The posterodorsal medial amygdala (MePD) has an adapted synaptic organization that dynamically modulates reproduction and other social behaviors in rats. Discrete gap junctions between glial cells were previously reported in the MePD neuropil. Connexins (Cx) are components of gap junctions and indicative of cellular electrical coupling. Here, we report the ultrastructural occurrence of gap junctions between neurons in the MePD and demonstrate the expression and immunofluorescent labeling of Cx36, Cx43 and Cx45 in this subcortical area of adult male rats. Few neuronal gap junctions were found in the MePD and, when identified, occurred between dendrites. On the other hand, there is a diffuse presence and distribution of punctate labelling for the tested Cxs. Puncta were visualized isolated or forming clusters in the same focal plane of cell bodies or along the MePD neuropil. The Cx36 puncta were found in neurons, Cx43 in astrocytes and Cx45 in both neurons and astrocytes. Our data indicate the presence of few gap junctions and different Cxs composition in the MePD. Because Cxs can assemble, form hemichannel units and/or serve as transcriptional regulator, it is likely that additional modulation of intercellular communication can occur besides the chemical transmission in the MePD of adult rats.

Neurotrophic factors in the posterodorsal medial amygdala of male and cycling female rats.

The posterodorsal medial amygdala (MePD) has a high concentration of receptors for gonadal hormones, is a sexually dimorphic region and dynamically controls the reproductive behavior of both males and females. Neurotrophic factors can promote dendritic spine remodeling and change synaptic input strength in a region-specific manner. Here, we analyzed the gene and protein expression of brain-derived neurotrophic factor (BDNF), insulin-like growth factor-I (IGF-1), polysialylated neural cell adhesion molecule (PSA-NCAM) and Ephrin-A4 in the MePD of adult males and females in diestrus, proestrus and estrus using real-time qPCR and fluorescent immunohistochemistry. The first approach showed their amplification except for Igf1 and the latter revealed that BDNF, IGF-1, PSA-NCAM and Ephrin-A4 are expressed in the MePD of the adult rats. Protein expression of these neurotrophic factors showed no differences between groups. However, proestrus females displayed a higher number of labelled puncta than males for BDNF expression and diestrus females for IGF-1 expression. In conjunction, results indicate that IGF-1 might be released rather than synthetized in the MePD, and the expression of specific neurotrophic factors varies specifically during proestrus. The dynamic modulation of BDNF and IGF-1 during this cyclic phase is coincident with synaptic changes and spine density remodeling in the MePD, the disinhibition of gonadotrophin secretion for ovulation and the display of sexual behavior.

Electrospun PVA-Dacarbazine nanofibers as a novel nano brain-implant for treatment of glioblastoma: in silico and in vitro characterization.

Malignant glioblastoma (GB) treatment consists of resection surgery followed by radiotherapy and chemotherapy (CT). Despite several implications, such as systemic toxicity and low efficacy, CT continues to be used for GB therapy. Aiming to overcome the blood-brain barrier (BBB) limitations, one of the most promising approaches is the use of drug delivery systems (DDS) to treat the cancer cells in situ. Dacarbazine (DTIC) is an antitumor agent that has limited application given its high toxicity to healthy cells. However, it is effective against GB recurrent cells. In this study, DTIC polymeric nanofibers (NF) were successfully prepared, characterized and its in vitro anticancer efficacy was determined. This system demonstrated high drug loading of 83.9 ± 6.5%, good stability and mechanical properties and sustained drug release, improved in tumor pH (6.8). This controlled release prolonged the uptake of GB improving DTIC antitumor effects such as DNA damage and cell death by apoptosis. Molecular dynamics simulations revealed that DTIC interacts with PVA, possibly explaining the controlled release of the drug. Therefore, DTIC NF brain-implants show great potential as a promising drug delivery system for GB therapy.

Rosmarinic acid improves oxidative stress parameters and mitochondrial respiratory chain activity following 4-aminopyridine and picrotoxin-induced seizure in mice.

Studies have indicated that epilepsy, an important neurological disease, can generate oxidative stress and mitochondrial dysfunction, among other damages to the brain. In this context, the use of antioxidant compounds could provide neuroprotection and help to reduce the damage caused by epileptic seizures and thereby the use of anticonvulsant drugs. Rosmarinic acid (RA) is an ester of caffeic acid and 3,4-dihydroxyphenylactic acid that prevents cell damage caused by free radicals, acting as an antioxidant. It also presents anti-inflammatory, antimutagenic, and antiapoptotic properties. In this work, we used two models of acute seizure, 4-aminopyridine (4-AP) and picrotoxin (PTX)-induced seizures in mice, to investigate the anticonvulsant, antioxidant, and neuroprotective profile of RA. Diazepam and valproic acid, antiepileptic drugs already used in the treatment of epilepsy, were used as positive controls. Although RA could not prevent seizures in the models used in this study, neither enhance the latency time to first seizure at the tested doses, it exhibited an antioxidant and neuroprotective effect. RA (8 and 16 mg/kg) decreased reactive oxygen species production, superoxide dismutase activity, and DNA damage, measured in hippocampus, after seizures induced by PTX and 4-AP. Catalase activity was decreased by RA only after seizures induced by 4-AP. The activity of the mitochondrial complex II was increased by RA in hippocampus samples after both seizure models. The results obtained in this study suggest that RA is able to reduce cell damage generated by the 4-AP and PTX seizures and therefore could represent a potential candidate in reducing pathophysiological processes involved in epilepsy.

In vitro model to study cocaine and its contaminants.

Cocaine is one of the most popular illicit drug worldwide. Due its great addictive potential, which leads to euphoria and hyperactivity, it is considered a public health concern. At the central nervous system, the drug acts inhibiting catecholamine re-uptake. It is now known that in addition to the toxicity of the drug itself, the contaminants present in the street drug have raised concern about the harmful effects on health. Toxicological in vivo and in vitro studies have demonstrated the toxic effects of cocaine correlated with the generation of reactive oxygen species (ROS), which in turn lead to oxidative damage to the cells. Therefore the aim of this work was to propose an in vitro model that reunites the main parameters of toxicity of the cocaine already observed in the literature so far, and we tested this model using cocaine and seizure cocaine sample (SCS), kindly provided by Federal Police of Brazil. For that, we used a C6 glioblastoma cells and evaluated cell death, oxygen reactive species induction, oxidation of macromolecules as membrane lipids and DNA and loss of mitochondrial membrane potential after cocaine exposure. The results showed that cocaine can decrease cellular viability in a dose-dependent way in the C6 cell immortalized and astrocytes primary culture. Cocaine also induced cellular death by apoptosis. However, in the seizure cocaine sample (SCS), the predominant cell death was due to necrosis. Using dichlorofluorescein (DCF) assay, we confirmed ROS production after cocaine exposition. In agreement with these findings, occurred an increasing in MDA production, as well as increased superoxide dismutase (SOD) and catalase (CAT) activity. The induction of DNA damage was observed after cocaine. Our results demonstrate the occurrence of mitochondrial dysfunction by depolarization of mitochondrial membrane as a consequence of cocaine treatment. In summary, these results demonstrated that cocaine can induce reactive oxygen species formation, leading to oxidative stress. As a consequence of this unbalance, DNA damage, lipidic peroxidation and loss of mitochondrial membrane occurred, which could be an answer to cell death observed.

Influence of PARP-1 inhibition in the cardiotoxicity of the topoisomerase 2 inhibitors doxorubicin and mitoxantrone.

Doxorubicin (DOX) and Mitoxantrone (MTX) are very effective drugs for a range of tumors despite being highly cardiotoxic. DNA topoisomerase 2 beta (Top2ß) was revealed as key mediator of DOX-induced cardiotoxicity, although ROS generation is also an important mechanism. Oxidative stress is also an important issue in MTX-induced cardiotoxicity that is manifested by mitochondrial dysfunction. Studies have demonstrated the relationship between PARP-1 overactivation and cell viability in DOX-treated cardiomyocytes. In reference of MTX, data regarding PARP-1 overactivation as the mechanism responsible for cardiotoxicity is difficult to find. The aim of this study was to evaluate the influence of PARP-1 inhibitor DPQ on DOX- and MTX-mediated cardiotoxicity. Cells were exposed for 24 h to DOX or MTX in the presence or absence of DPQ. Viability, apoptosis, and genotoxicity assays were carried out. Immunofluorescence of phosphorylated histone H2AX was analyzed in H9c2 cells and cardiomyocytes from neonatal rats. Results demonstrated that DPQ co-treatment increases DOX-induced apoptosis in H9c2 cells. DPQ also prevents DOX and MTX-ROS generation in part by increasing SOD and CAT activities. Furthermore, DPQ co-treatment increased the generation of DNA strand breaks by DOX and MTX whilst also inducing phosphorylation of H2AX, MRE11, and ATM in H9c2 cells. Our results demonstrated that as well as increasing DNA damage and inducing apoptotic cell death, DPQ enhances DOX- and MTX-mediated cytotoxicity in H9c2.

Dietary interventions in mice affect oxidative stress and gene expression of the Prlr and Esr1 in the adipose tissue and hypothalamus of dams and their offspring

Maternal diet is key to the progeny’s health since it may impact on the offspring’s adult life. In this study, mice dams received standard (CONT), restrictive (RD), or hypercaloric (HD) diets during mating, pregnancy, and lactation. Male offspring of each group of dams also received these diets: CONT, RD, HD. Aiming to evaluate the oxidative stress in the adipose tissue, reactive oxygen species (ROS) production, catalase (CAT), and superoxide dismutase (SOD) activities were analyzed in dams and offspring. In the adipose tissue and hypothalamus, gene expression of prolactin (Prlr) and estrogen alpha (Esr1) receptors was performed in dams and offspring. Protein expression of Stat5 was evaluated in the adipose tissue of the offspring from RD-fed dams. HD-fed dams increased triglycerides and leptin serum concentrations, and decreased SOD activity in the adipose tissue. In the offspring’s adipose tissue, we observed a maternal diet effect caused by HD, with increased ROS production and SOD and CAT activities. Gene expression of Prlr and Esr1 in the offspring’s adipose tissue was decreased due to maternal RD. Mice from HD-fed dams showed higher Stat5 expression compared to the offspring from CONT and RD dams in the adipose tissue. In the hypothalamus, we found decreased expression of Prlr in RD and HD dams, compared to CONT; and a maternal diet effect on Prlr and Esr1 gene expression in the offspring. In conclusion, we can affirm that maternal nutrition impacts the redox state and influences the gene expression of Prlr and Esr1, which are involved in energy metabolism, both peripherally and centrally in the adult life of the female offspring. (AU)