Novel Dimeric hHv1 Model and Structural Bioinformatic Analysis Reveal an ATP-Binding Site Resulting in a Channel Activating Effect.

The human voltage-gated proton channel (hHv1) is a highly selective ion channel codified by the HVCN1 gene. It plays a fundamental role in several physiological processes such as innate and adaptive immunity, insulin secretion, and sperm capacitation. Moreover, in humans, a higher hHv1 expression/function has been reported in several types of cancer cells. Here we report a multitemplate homology model of the hHv1 channel, built and refined as a dimer in Rosetta. The model was then subjected to extensive Gaussian accelerated molecular dynamics (GaMD) for enhanced conformational sampling, and representative snapshots were extracted by clustering analysis. Combining different structure- and sequence-based methodologies, we predicted a putative ATP-binding site located on the intracellular portion of the channel. Furthermore, GaMD simulations of the ATP-bound dimeric hHv1 model showed that ATP interacts with a cluster of positively charged residues from the cytoplasmic N and C terminal segments. According to the in silico predictions, we found that 3 mM intracellular ATP significantly increases the H+ current mediated by the hHv1 channel expressed in HEK293 cells and measured by the patch-clamp technique in an inside-out configuration (2.86 ± 0.63 fold over control at +40 mV). When ATP was added on the extracellular side, it was not able to activate the channel supporting the idea that the ATP-binding site resides in the intracellular face of the hHV1 channel. In a physiological and pathophysiological context, this ATP-mediated modulation could integrate the cell metabolic state with the H+ efflux, especially in cells where hHv1 channels are relevant for pH regulation, such as pancreatic ß-cells, immune cells, and cancer cells.

Chronic exposure to low concentrations of chlorpyrifos affects normal cyclicity and histology of the uterus in female rats.

Chlorpyrifos (CPF), the most used insecticide in Argentina, can act as an endocrine disruptor at low doses. We previously demonstrated that chronic exposure to CPF induces hormonal imbalance in vivo. The aim of this work was to study the effects of low concentrations of CPF (0.01 and 1 mg/kg/day) on the reproductive system of virgin adult rats. In the ovary, we studied the effects of CPF on steroidogenesis by determining steroid hormone content by RIA and CYP11 and CYP19 enzyme expression by qRT-PCR. The estrous cycle was evaluated by microscopic observation of vaginal smear, as well as by changes in uterine histology. In endometrium, we determined the fractal dimension and expression of PCNA, ERα and PR by IHC. Our results showed that chronic exposure to CPF affects ovarian steroid synthesis, causing alterations in the normal cyclicity of animals. In addition, CPF induced proliferative changes in the uterus, suggesting that it could affect reproduction or act as a risk factor in the development of uterine proliferative pathologies.

Differential expression of the long and truncated Hv1 isoforms in breast-cancer cells.

Metabolic reprogramming of cancer cells results in a high production of acidic substances that must be extruded to maintain tumor-cell viability. The voltage-gated proton channel (Hv1) mediates highly selective effluxes of hydronium-ion (H+ ) that prevent deleterious cytoplasmic acidification. In the work described here, we demonstrated for the first time that the amino-terminal-truncated isoform of Hv1 is more highly expressed in tumorigenic breast-cancer-cell lines than in nontumorigenic breast cells. With respect to Hv1 function, we observed that pharmacologic inhibition of that channel, mediated by the specific blocker 5-chloro-2-guanidinobenzimidazole, produced a drop in intracellular pH and a decrease in cell viability, both in monolayer and in three-dimensional cultures, and adversely affected the cell-cycle in tumorigenic breast cells without altering the cycling of nontumorigenic cells. In conclusion, our results demonstrated that the Hv1 channel could be a potential tool both as a biomarker and as a therapeutic target in breast-cancer disease.

El hexaclorobenceno como factor de riesgo en el cáncer de mama / Hexachlorobenzene as a risk factor in breast cancer

Hexachlorobenzene (HCB) is a widespread environmental pollutant and an endocrine disruptor. Chronic exposure of humans to HCB elicits porphyria, neurologic symptoms, immune disorders and thyroid dysfunctions. It is a dioxin-like compound and a weak ligand of the AhR (aryl hydrocarbon receptor), a transcription factor that modulates genes related to detoxification, proliferation, migration and invasion. This study was carried out to revise the results of HCB action on mammary gland and breast cancer, summarizing the main ideas of its mechanism of action. HCB increases tumor development and active c-Src/EGFR (epidermal growth factor receptor) signaling pathways, while reducing tyrosine537-ER-alpha (estrogen receptor-alpha) phosphorylation, and promoting a phenotype with enhanced malignancy and lung metastasis in different animal models. In a rat mammary gland, HCB promotes an estrogenic microenvironment by activation of ER-alpha and Insulin/IGFs (insulin growth factors) pathways. HCB induces cell proliferation, promoting cell cycle progression and enhancing cyclin D1 expression and c-Src/p27 interaction in (ER-alpha) MCF-7 human breast cancer cell line. In (ER-alpha)(-) MDA-MB-231 breast cancer cells, the pesticide enhances cell migration and invasion as well as metalloproteases and TGF-beta1 (transformig growth factor-beta1) expression. In conclusion our current study suggests that alterations in the estrogenic microenvironment may influence the biological behavior of mammary gland or breast tumors, leading to preneoplastic lesions or enhanced malignancy, respectively. Our findings suggest that HCB may be a risk factor for human breast cancer progression.

El hexaclorobenceno (HCB) es un contaminante ambiental ampliamente distribuido y un desorganizador endocrino. Su exposición crónica en seres humanos produce porfiria, síntomas neurológicos, trastornos inmunitarios y disfunciones tiroideas. Es un agonista débil del receptor de hidrocarburos aromáticos (AhR), un factor de transcripción que modula genes relacionados con el metabolismo de xenobióticos, la proliferación, la migración y la invasión. Nuestro objetivo es revisar los efectos del HCB en la glándula mamaria y el cáncer mamario, resumiendo los principales mecanismos de acción. El HCB aumenta el desarrollo tumoral y activa vías de señalización de c-Src/receptor del factor de crecimiento epidérmico (EGFR), mientras que disminuye la fosforilación de tirosina 537/receptor de estrógenos alfa (RE-alfa), promoviendo un fenotipo de mayor malignidad y metástasis pulmonar en diferentes modelos con animales. En la glándula mamaria de rata genera un microambiente estrogénico por activación del RE-alfa y las vías de insulina/factores de crecimiento similares a la insulina (IGF). En células de cáncer mamario humanas MCF-7 (RE-alfa) induce proliferación celular, promoviendo la progresión del ciclo, aumentando la ciclina D1 y la interacción p27/c-Src. En MDA-MB-231 (-RE-alfa) estimula la migración e invasión, así como la expresión de metaloproteasas y factor de crecimiento transformante beta 1 (TGF-beta 1). Estos estudios indican que las alteraciones en el microambiente estrogénico podrían influir el comportamiento biológico de la glándula mamaria y los tumores, lo que provoca lesiones preneoplásicas o aumento en la malignidad tumoral mamaria. Nuestros hallazgos sugieren que el HCB podría ser un factor de riesgo para la progresión del cáncer de mama humano.

Evaluation of sodium arsenite exposure on reproductive competence in pregnant and postlactational dams and their offspring.

We investigated arsenite exposure on the reproductive axis of dams (during pregnancy and at cyclicity resumption) and their offspring. Pregnant rats were exposed to 5 (A5) or 50ppm (A50) of sodium arsenite in drinking water from gestational day 1 (GD1) until sacrifice at GD18 or two months postpartum. Offspring were exposed to the same treatment as their mothers from weaning to adulthood. A50-pregnant rats gained less weight, showed increased testosterone and estradiol but pregnancy was unaffected. After lactation, arsenic-exposed dams presented compromised cyclicity, decreased estradiol, increased follicle-stimulating hormone (FSH), less preovulatory follicles and presence of ovarian cysts, suggesting impaired reproduction. A50-offspring presented lower body weight; A50-female-offspring showed elevated gonadotropin releasing hormone (GnRH), FSH and testosterone, while A50-males showed diminished GnRH/FSH, but normal testosterone. We conclude that arsenite at the present exposure levels did not compromise pregnancy outcome while it negatively affected reproductive physiology in postpartum dams and their offspring.

Arsenite in drinking water produces glucose intolerance in pregnant rats and their female offspring.

Drinking water is the main source of arsenic exposure. Chronic exposure has been associated with metabolic disorders. Here we studied the effects of arsenic on glucose metabolism, in pregnant and post-partum of dams and their offspring. We administered 5 (A5) or 50 (A50) mg/L of sodium arsenite in drinking water to rats from gestational day 1 (GD1) until two months postpartum (2MPP), and to their offspring from weaning until 8 weeks old. Liver arsenic dose-dependently increased in arsenite-treated rats to levels similar to exposed population. Pregnant A50 rats gained less weight than controls and recovered normal weight at 2MPP. Arsenite-treated pregnant animals showed glucose intolerance on GD16-17, with impaired insulin secretion but normal insulin sensitivity; they showed dose-dependent increased pancreas insulin on GD18. All alterations reverted at 2MPP. Offspring from A50-treated mothers showed lower body weight at birth, 4 and 8 weeks of age, and glucose intolerance in adult females, probably due to insulin secretion and sensitivity alterations. Arsenic alters glucose homeostasis during pregnancy by altering beta-cell function, increasing risk of developing gestational diabetes. In pups, it induces low body weight from birth to 8 weeks of age, and glucose intolerance in females, demonstrating a sex specific response.

Hexachlorobenzene modulates the crosstalk between the aryl hydrocarbon receptor and transforming growth factor-ß1 signaling, enhancing human breast cancer cell migration and invasion.

Given the number of women affected by breast cancer, considerable interest has been raised in understanding the relationships between environmental chemicals and disease onset. Hexachlorobenzene (HCB) is a dioxin-like compound that is widely distributed in the environment and is a weak ligand of the aryl hydrocarbon receptor (AhR). We previously demonstrated that HCB acts as an endocrine disruptor capable of stimulating cell proliferation, migration, invasion, and metastasis in different breast cancer models. In addition, increasing evidence indicates that transforming growth factor-ß1 (TGF-ß1) can contribute to tumor maintenance and progression. In this context, this work investigated the effect of HCB (0.005, 0.05, 0.5, and 5µM) on TGF-ß1 signaling and AhR/TGF-ß1 crosstalk in the human breast cancer cell line MDA-MB-231 and analyzed whether TGF-ß1 pathways are involved in HCB-induced cell migration and invasion. RT-qPCR results indicated that HCB reduces AhR mRNA expression through TGF-ß1 signaling but enhances TGF-ß1 mRNA levels involving AhR signaling. Western blot analysis demonstrated that HCB could increase TGF-ß1 protein levels and activation, as well as Smad3, JNK, and p38 phosphorylation. In addition, low and high doses of HCB were determined to exert differential effects on AhR protein levels, localization, and activation, with a high dose (5µM) inducing AhR nuclear translocation and AhR-dependent CYP1A1 expression. These findings also revealed that c-Src and AhR are involved in HCB-mediated activation of Smad3. HCB enhances cell migration (scratch motility assay) and invasion (Transwell assay) through the Smad, JNK, and p38 pathways, while ERK1/2 is only involved in HCB-induced cell migration. These results demonstrate that HCB modulates the crosstalk between AhR and TGF-ß1 and consequently exacerbates a pro-migratory phenotype in MDA-MB-231 cells, which contributes to a high degree of malignancy. Taken together, our findings help to characterize the molecular mechanism underlying the effects of HCB on breast cancer progression.

Pesticide chlorpyrifos acts as an endocrine disruptor in adult rats causing changes in mammary gland and hormonal balance.

Endocrine disruptors (EDs) are compounds that interfere with hormone regulation and influence mammary carcinogenesis. We have previously demonstrated that the pesticide chlorpyrifos (CPF) acts as an ED in vitro, since it induces human breast cancer cells proliferation through estrogen receptor alpha (ERα) pathway. In this work, we studied the effects of CPF at environmental doses (0.01 and 1mg/kg/day) on mammary gland, steroid hormone receptors expression and serum steroid hormone levels. It was carried out using female Sprague-Dawley 40-days-old rats exposed to the pesticide during 100 days. We observed a proliferating ductal network with a higher number of ducts and alveolar structures. We also found an increased number of benign breast diseases, such as hyperplasia and adenosis. CPF enhanced progesterone receptor (PgR) along with the proliferating cell nuclear antigen (PCNA) in epithelial ductal cells. On the other hand, the pesticide reduced the expression of co-repressors of estrogen receptor activity REA and SMRT and it decreased serum estradiol (E2), progesterone (Pg) and luteinizing hormone (LH) levels. Finally, we found a persistent decrease in LH levels among ovariectomized rats exposed to CPF. Therefore, CPF alters the endocrine balance acting as an ED in vivo. These findings warn about the harmful effects that CPF exerts on mammary gland, suggesting that this compound may act as a risk factor for breast cancer.

El organofosforado clorpirifos como disruptor estrogénico y factor de riesgo para el cáncer de mama / The organophosphorus chlorpyrifos as endocrine disruptor and breast cancer factor risk

El clorpirifos (CPF) es un insecticida de amplio espectro que se utiliza en Argentina y en otros países de Latinoamérica. Se emplea para el control de plagas en la producción de frutas, hortalizas, cereales y plantas ornamentales. El principal mecanismo de acción descripto para este insecticida es la inhibición de la acetilcolinesterasa. Sin embargo, reportes más recientes sugieren múltiples efectos del plaguicida independientes de la inhibición de esa enzima. El objetivo de este trabajo es transmitir a la comunidad los resultados de nuestras investigaciones obtenidos utilizando diferentes dosis de CPF en distintos modelos experimentales, tanto in vitro como in vivo. En relación a esto, hemos evidenciado una acción del CPF sobre el sistema redox celular que conduce al incremento de especies reactivas del oxígeno y consecuentemente a la activación de diferentes vías de señalización. Además, hemos determinado que el insecticida CPF puede comportarse como un disruptor endócrino modulando la acción de los estrógenos y alterando la normal estructura del tejido mamario. Nuestros resultados alertan sobre el impacto que este compuesto podría tener sobre la salud, sugiriendo la necesidad de revisar su uso dado que manifiesta acciones a dosis encontradas en el ambiente.

Chlorpyrifos (CPF) is a broad spectrum insecticide used in Argentina and other Latin American countries. It is commonly used for pest control in the production of fruits, vegetables, cereals and ornamental plants. The main mechanism of action described for this insecticide is the inhibition of acetylcholinesterase activity. However, more recent reports suggest multiple effects for this pesticide in an independent way from the inhibition of this enzyme. The objective of this work is to convey to the community the results of our investigations obtained using different doses of CPF in various experimental models, both in vitro and in vivo. In this connection, we have shown a CPF action on the cellular redox system which leads to increased reactive oxygen species and the consequent activation of different signaling pathways. In addition, we have determined that the insecticide CPF acts as an endocrine disruptor modulating the action of estrogen and altering the normal structure of breast tissue. Our findings warn about the impact that this compound might have on health, suggesting the need to review its use since adverse actions were found at environmentally relevant doses.

Chlorpyrifos inhibits cell proliferation through ERK1/2 phosphorylation in breast cancer cell lines.

It is well known the participation of oxidative stress in the induction and development of different pathologies including cancer, diabetes, neurodegeneration and respiratory disorders among others. It has been reported that oxidative stress may be induced by pesticides and it could be the cause of health alteration mediated by pollutants exposure. Large number of registered products containing chlorpyrifos (CPF) is used to control pest worldwide. We have previously reported that 50 µM CPF induces ROS generation and produces cell cycle arrest followed by cell death. The present investigation was designed to identify the pathway involved in CPF-inhibited cell proliferation in MCF-7 and MDA-MB-231 breast cancer cell lines. In addition, we determined if CPF-induced oxidative stress is related to alterations in antioxidant defense system. Finally we studied the molecular mechanisms underlying in the cell proliferation inhibition produced by the pesticide. In this study we demonstrate that CPF (50 µM) induces redox imbalance altering the antioxidant defense system in breast cancer cells. Furthermore, we found that the main mechanism involved in the inhibition of cell proliferation induced by CPF is an increment of p-ERK1/2 levels mediated by H2O2 in breast cancer cells. As PD98059 could not abolish the increment of ROS induced by CPF, we concluded that ERK1/2 phosphorylation is subsequent to ROS production induced by CPF but not the inverse.

Enhancement of ionizing radiation response by histamine in vitro and in vivo in human breast cancer.

The radioprotective potential of histamine on healthy tissue has been previously demonstrated. The aims of this work were to investigate the combinatorial effect of histamine or its receptor ligands and gamma radiation in vitro on the radiobiological response of 2 breast cancer cell lines (MDA-MB-231 and MCF-7), to explore the potential molecular mechanisms of the radiosensitizing action and to evaluate the histamine-induced radiosensitization in vivo in a triple negative breast cancer model. Results indicate that histamine significantly increased the radiosensitivity of MDA-MB-231 and MCF-7 cells. This effect was mimicked by the H1R agonist 2-(3-(trifluoromethyl)phenyl)histamine and the H4R agonists (Clobenpropit and VUF8430) in MDA-MB-231 and MCF-7 cells, respectively. Histamine and its agonists enhanced radiation-induced oxidative DNA damage, DNA double-strand breaks, apoptosis and senescence. These effects were associated with increased production of reactive oxygen species, which correlated with the inhibition of catalase, glutathione peroxidase and superoxide dismutase activities in MDA-MB-231 cells. Histamine was able also to potentiate in vivo the anti-tumoral effect of radiation, increasing the exponential tumor doubling time. We conclude that histamine increased radiation response of breast cancer cells, suggesting that it could be used as a potential adjuvant to enhance the efficacy of radiotherapy.

Antitumor activity of histamine and clozapine in a mouse experimental model of human melanoma.

BACKGROUND: Functional presence of histamine H4 receptor (H4R) was demonstrated in human melanoma cell lines and biopsies. OBJECTIVE: The purposes of this work were to investigate signal transduction pathways and biological responses triggered by the activation of H4R in human primary (WM35) and metastatic (M1/15) melanoma cell lines and to evaluate the in vivo antitumor activity of histamine (HA) and clozapine (CLZ) on human M1/15 melanoma xenografts. METHODS: Clonogenic assay, incorporation of BrdU, cell cycle distribution, phosphorylation levels of ERK1/2 and cAMP production were evaluated in vitro. An experimental human melanoma model was developed into athymic nude mice. Tumor growth, survival and histochemical studies were performed in order to investigate the expression levels of H4R, HA, PCNA, mitotic index (MI), and angiogenesis. RESULTS: The results indicate that H4R agonists inhibited forskolin-induced cAMP levels only in M1/15 cells while increased phosphorylation levels of ERK1/2 and decreased proliferation in both cell types. In vivo studies show that HA and CLZ (1mgkg(-1), sc) significantly increased median survival and decreased tumor volume. These effects were associated to a reduction in MI, in the expression of proliferation marker and in intratumoral neovascularization. CONCLUSIONS: We conclude that HA and CLZ exhibit an antitumoral effect in vitro and in vivo on human melanoma, suggesting the therapeutic potential of these compounds for the treatment of malignant melanoma.

Differential mechanisms of action are involved in chlorpyrifos effects in estrogen-dependent or -independent breast cancer cells exposed to low or high concentrations of the pesticide.

It has reported that many environmental compounds may display estrogenic actions and these findings led to researchers to associate breast cancer risk with the use of some pesticides. The aim of this work was to investigate the effect of chlorpyrifos (CPF) on cell proliferation and the ERα-dependence of this action employing MCF-7 and MDA-MB-231 breast cancer cell lines. We have also analyzed CPF action on the cell cycle distribution and the cyclins that are implicated in G1-S and intra-S checkpoints. Finally, the action on cell death and ROS production were studied. We demonstrated the ability of CPF 0.05µM to induce cell proliferation through ERα in hormone-dependent breast cancer cells. In contrast, CPF 50µM induces intra-S arrest modifying checkpoints proteins, through a mechanism that may involve changes in redox balance in MCF-7. In MDA-MB-231, we have found that CPF 50µM produces an arrest in G2/M phase which could be related to the capacity of the pesticide for binding to tubulin sites altering microtubules polymerization. Altogether, our results provide new evidences on the action of the pesticide CPF as an environmental breast cancer risk factor due to the effects that causes on the mechanisms that modulate breast cell proliferation.

TRPC6 mutational analysis in a large cohort of patients with focal segmental glomerulosclerosis.

BACKGROUND: Mutations in the TRPC6 gene have been reported in six families with adult-onset (17-57 years) autosomal dominant focal segmental glomerulosclerosis (FSGS). Electrophysiology studies confirmed augmented calcium influx only in three of these six TRPC6 mutations. To date, the role of TRPC6 in childhood and adulthood non-familial forms is unknown. METHODS: TRPC6 mutation analysis was performed by direct sequencing in 130 Spanish patients from 115 unrelated families with FSGS. An in silico scoring matrix was developed to evaluate the pathogenicity of amino acid substitutions, by using the bio-physical and bio-chemical differences between wild-type and mutant amino acid, the evolutionary conservation of the amino acid residue in orthologues, homologues and defined domains, with the addition of contextual information. RESULTS: Three new missense substitutions were identified in two clinically non-familial cases and in one familial case. The analysis by means of this scoring system allowed us to classify these variants as likely pathogenic mutations. One of them was detected in a female patient with unusual clinical features: mesangial proliferative FSGS in childhood (7 years) and partial response to immunosupressive therapy (CsA + MMF). Asymptomatic carriers of this likely mutation were found within her family. CONCLUSIONS: We describe for the first time TRPC6 mutations in children and adults with non-familial FSGS. It seems that TRPC6 is a gene with a very variable penetrance that may contribute to glomerular diseases in a multi-hit setting.