Janaina Sena de Souza

Perinatal exposure to glyphosate-based herbicide alters the thyrotrophic axis and causes thyroid hormone homeostasis imbalance in male rats

Glyphosate-based herbicides (GBHs) are widely used in agriculture. Recently, several animal and epidemiological studies have been conducted to understand the effects of these chemicals as an endocrine disruptor for the gonadal system. The aim of the present study was to determine whether GBHs could also disrupt the hypothalamic-pituitary-thyroid (HPT) axis. Female pregnant Wistar rats were exposed to a solution containing GBH Roundup®Transorb (Monsanto). The animals were divided into three groups (control, 5mg/kg/day or 50mg/kg/day) and exposed from gestation day 18 (GD18) to post-natal day 5 (PND5). Male offspring were euthanized at PND 90, and blood and tissues samples from the hypothalamus, pituitary, liver and heart were collected for hormonal evaluation (TSH-Thyroid stimulating hormone, T3-triiodothyronine and T4-thyroxine), metabolomic and mRNA analyses of genes related to thyroid hormone metabolism and function. The hormonal profiles showed decreased concentrations of TSH in the exposed groups, with no variation in the levels of the thyroid hormones (THs) T3 and T4 between the groups. Hypothalamus gene expression analysis of the exposed groups revealed a reduction in the expression of genes encoding deiodinases 2 (Dio2) and 3 (Dio3) and TH transporters Slco1c1 (former Oatp1c1) and Slc16a2 (former Mct8). In the pituitary, Dio2, thyroid hormone receptor genes (Thra1 and Thrb1), and Slc16a2 showed higher expression levels in the exposed groups than in the control group. Interestingly, Tshb gene expression did not show any difference in expression profile between the control and exposed groups. Liver Thra1 and Thrb1 showed increased mRNA expression in both GBH-exposed groups, and in the heart, Dio2, Mb, Myh6 (former Mhca) and Slc2a4 (former Glut4) showed higher mRNA expression in the exposed groups. Additionally, correlation analysis between gene expression and metabolomic data showed similar alterations as detected in hypothyroid rats. Perinatal exposure to GBH in male rats modified the HPT set point, with lower levels of TSH likely reflecting post-translational events. Several genes regulated by TH or involved in TH metabolism and transport presented varying degrees of gene expression alteration that were probably programmed during intrauterine exposure to GBHs and reflects in peripheral metabolism. In conclusion, the role of GBH exposure in HPT axis disruption should be considered in populations exposed to this herbicide.

Evaluation of globins expression in brain, heart, and lung in rats exposed to side stream cigarette smoke

The side stream cigarette smoke (SSCS) is a contributing factor in the pathogenesis of cigarette smoking‐induced toxicity. Hemoglobin (Hb), myoglobin (Mb), neuroglobin (Ngb), and cytoglobin (Cygb) are globins with different distributions and functions in the tissues and have similar actions by providing O2 (oxygen) for respiratory chain, detoxification of ROS and nitric oxide (NO), and protect tissues against irreversible lesions. We aimed to investigate the effects of SSCS exposure on gene and protein expression of Ngb, Cygb, and Mb in different tissue. The Ngb and Cygb gene and protein expression in the cerebral cortex increased after 1 week of rat exposure to SSCS. In hippocampus, the Ngb gene and protein expression increased after 1 week or more of exposure and no change was observed in Cygb gene and protein expression. In myocardium, Mb and Cygb gene expression increased at 1 and 4 weeks of exposure, while protein expression of both increased at 1, 2, 3, and 4 weeks. In lung, observed an increase in Cygb gene and protein expression after 2, 3, and 4 weeks of exposure. The findings suggest that SSCS modulates Ngb, Cygb, and Mb in central and peripheral tissue

Down Syndrome iPSC-Derived Astrocytes Impair Neuronal Synaptogenesis and the mTOR Pathway In Vitro

Several methods have been used to study the neuropathogenesis of Down syndrome (DS), such as mouse aneuploidies, post mortem human brains, and in vitro cell culture of neural progenitor cells. More recently, induced pluripotent stem cell (iPSC) technology has offered new approaches in investigation, providing a valuable tool for studying specific cell types affected by DS, especially neurons and astrocytes. Here, we investigated the role of astrocytes in DS developmental disease and the impact of the astrocyte secretome in neuron mTOR signaling and synapse formation using iPSC derived from DS and wild-type (WT) subjects. We demonstrated for the first time that DS neurons derived from hiPSC recapitulate the hyperactivation of the Akt/mTOR axis observed in DS brains and that DS astrocytes may play a key role in this dysfunction. Our results bear out that 21 trisomy in astrocytes contributes to neuronal abnormalities in addition to cell autonomous dysfunctions caused by 21 trisomy in neurons. Further research in this direction will likely yield additional insights, thereby improving our understanding of DS and potentially facilitating the development of new therapeutic approaches.

Unravelling the intravenous and in situ vasopressin effects on the urinary bladder in anesthetized female rats: More than one vasopressin receptor subtype involved?

ABSTRACT Urinary bladder dysfunctions show high prevalence in women. We focused to investigate the intravenous and in situ (topic) vasopressin effects on the bladder and also to characterize the vasopressin receptor subtypes in the bladder. Adult female Wistar rats anesthetized with isoflurane underwent to the cannulation of the femoral artery and vein, and also urinary bladder for mean arterial pressure, heart rate and intravesical pressure (IP) recordings, respectively. Doppler flow probe was placed around the renal artery for blood flow measurement. After baseline recordings, intravenous injection of saline or vasopressin at different doses (0.25, 0.5, 1.0ng/ml/kg of b.w.); or 0.1ml of saline or 0.1ml of vasopressin at different doses (0.25, 0.5, 1.0ng/ml) was randomly dropped on the bladder. In another group of rats, the UB was harvest for gene expression by qPCR and also for protein expression by Western blotting of the vasopressin receptor subtypes. We observed that either intravenous or in situ vasopressin evoked a huge increase in the IP in a dose‐dependent manner compared to saline, whilst no differences were observed in the cardiovascular parameters. The genes and the protein expression of V1a, V1b and V2 vasopressin receptors subtypes were found in the bladder. Intravenous injection of V1a or V2 receptor antagonist evoked a huge fall in IP and 30min later, i.v or in situ vasopressin evoked responses on IP were significantly attenuated. Therefore, intravenous or in situ vasopressin increases the IP due to binding in V1a or V2 receptors localized in the bladder.

Antidepressant behavior in thyroidectomized Wistar rats is induced by hippocampal hypothyroidism

Thyroidectomy is a surgical procedure indicated in cases of several maligned or benign thyroid diseases, thus, the aim of our study was to verify how the hypothyroidism induced by thyroidectomy influences behavioral parameters and its relation to thyroid hormones metabolism and neurogenesis at hippocampus. For this purpose, Adult male Wistar rats underwent to thyroidectomy to induce hypothyroidism. Behavioral tests, the thyroid profile and hippocampal gene expression were evaluated in control and in thyroidectomized animals. It was observed that thyroidectomized group had a significant increasing in serum thyroid-stimulating hormone (TSH) and a decreasing in thyroxine (T4) levels as well as in triiodothyronine (T3) serum level. It was also observed reduction of the monocarboxylate transporter 8 (Mct8), thyroid hormone receptor alfa (Trα1), deiodinase type 2 (Dio2), ectonucleotide pyrophosphatase/phosphodiesterase 2 (Enpp2) and brain-derived neurotrophic factor (Bdnf) mRNA expression in hippocampus of thyroidectomized animals. In the forced swimming test, it was verified that thyroidectomy promotes a decrease in time of immobility and climbing when compared with the control group. In summary, we demonstrated that antidepressant behavior in thyroidectomized Wistar rats is induced by hippocampal hypothyroidism. This effect could be associated to an impaired neuronal activity in acute stress response as it is observed in forced swimming paradigm.

Erratum to: Anatomical specificity of the brain in the modulation of Neuroglobin and Cytoglobin genes after chronic bisphenol a exposure

The aim of this study was to investigate the influence of Bisphenol A (BPA) exposure on Neuroglobin (Ngb) and Cytoglobin (Cygb) as well as oxidative stress gene expression in the cerebellum, hippocampus, hypothalamus and cortex. Male Wistar rats were randomly divided into 3 groups: Control and two groups receiving 2 different daily BPA dosages, 5 or 25 mg/kg from postnatal day 50 (PND50) through PND90 and they were euthanized at PND105. In the cortex, we found an increase in Ngb gene expression and also in superoxide dismutase 1 and Catalase (Cat). In the cerebellum, we found an increase in Ngb and Cat, in the hypothalamus, there was a decrease in Cygb and an increase in glutathione peroxidase and Cat and in hypoxia-inducible factor 1 alpha (Hif1α) at the low dosage and a decrease in Hif1α at the high BPA dosage. Finally, in the hippocampus, we observed a decrease in Ngb and Cygb and an increase in Hif1α. In summary, BPA promotes the modulation of both Ngb and Cygb, but such changes occur by different mechanisms depending on the exposure dose and anatomical area.

Novel lincRNA Susceptibility Gene and Its Role in Etiopathogenesis of Thyrotoxic Periodic Paralysis

Thyrotoxic periodic paralysis (TPP) is a life-threatening neuromuscular complication of thyrotoxicosis characterized by muscle weakness and hypokalemia and with an unclear etiopathogenesis. However, the 17q24.3 locus had been genetically linked to TPP, in which the genetic variant rs312691 (TC genotype) in long intergenic noncoding RNA (lincRNA) CTD-2378E21.1 is located downstream of inward-rectifier potassium (Kir) channel genes [KCNJ2 and its antisense KCNJ2 (AS-KCNJ2)]. A TPP patient with a suppressed thyroid-stimulating hormone level, a high free thyroxine level of (5.8 ng/dL), and low serum potassium level of (2 mEq/L) was evaluated for Kir channel expression during and after recovery from thyrotoxicosis. We observed that circulating lincRNA and Kir expression varied in accordance with thyroid status and TC genotype. To endorse this association of a lincRNA-rs312691 variant with a genetic risk of TPP, an additional series of 37 patients with TPP and 32 patients with thyrotoxic without paralysis (TWP) were assessed. We verified that the risk of minor allele C was greater in TPP than in TWP (odds ratio, 5.289; P = 0.0062), and protective major allele T was more frequent than observed in the 1000 genome controls (odds ratio, 11.90; P < 0.0001). AS-KCNJ2 was downregulated during thyrotoxicosis in the TWP controls carrying allele T and were upregulated in those with TPP with risk allele C. Moreover, KCNJ2 (Kir2.1) expression was reduced during thyrotoxicosis and restored in euthyroid status. We further excluded any other coding variant by performing targeted exome sequencing mutational screening in 17q24.3. Our data suggest that high lincRNA AS-KCNJ2 and CDT-2378E21.1 expression, possibly driven by the triiodothyronine regulatory mechanism, reduces the Kir2.1 expression observed during thyrotoxicosis. This finding could contribute to the understanding of the reduced inward-rectifying current observed during muscle weakness in genetically susceptible TPP patients.

Novel immunoassay for TSH measurement in rats

Measuring thyroid hormones is an important aspect for the study of metabolism and for monitoring diseases in both human and animal models. The traditional method for hormone measurement in rats is the radioimmunoassay (RIA). However, the RIA is associated with some practical disadvantages, including the use of radioactive material, the need for specialized equipment and expert staff, the short shelf-life of kits according to the half-life of the radioisotope and high costs. The objective of this study was to develop a new cost-effective method for measuring TSH levels in rats that avoids the use of radioactive material. We developed an in-house competitive immunoassay using a reference standard, polyclonal antibody produced in rabbits and biotinylated antigen. This method was tested in 64 Wistar rats that were divided into a control group (n = 41) and a group with hypothyroidism (n = 23). Our assay demonstrated an analytical sensitivity of 0.24 ng/mL (n = 12) and an intra-assay coefficient of variation (CV) of 8.9% for sera with TSH levels of 1.5 ng/mL and 13.2% for sera with TSH levels of 17.5 ng/mL (n = 14). The inter-assay CV was 13.5% for sera with TSH levels of 1.4 ng/mL and 14.5% for TSH levels of 18.2 ng/mL (n = 5). The analysis of mean TSH levels in control rats (5.06 ± 0.5701) and hypothyroid rats (51.09 ± 5.136) revealed a statistically significant difference (p < 0.001) between the groups. This method showed good sensitivity, can be automated and is low-cost compared with RIA. Our method offers a viable alternative for TSH measurement in rats.

Parvalbumin expression and distribution in the hippocampal formation of Cebus apella.

New World primates play an important role in biomedical research. However, the literature still lacks information on many structural features of the brain in these species, particularly structures of the hippocampal formation that are related to long‐term memory storage. This study was designed to provide information, for the first time, about the distribution and number of neurons expressing parvalbumin‐immunoreactivity (PV‐I) in the subregions of the hippocampal formation in Cebus apella, a New World primate species commonly used in biomedical research. Our results revealed that for several morphometric variables, PV‐I cells differ significantly among the subregions CA1, CA2, CA3, and the hilus. Based upon our findings and those of other studies, we hypothesize that the proportional increase from monkeys to humans in PV‐I cell density within CA1 is a factor contributing to the evolution of increased memory formation and storage. Am. J. Primatol. 77:449–461, 2015.