Chlorpyrifos-induced cell proliferation in human breast cancer cell lines differentially mediated by estrogen and aryl hydrocarbon receptors and KIAA1363 enzyme after 24 h and 14 days exposure.

Organophosphate biocide chlorpyrifos (CPF) is involved with breast cancer. However, the mechanisms remain unknown. CPF increases cell division in MCF-7 cells, by estrogen receptor alpha (ERα) activation, although it is a weak ERα agonist, suggesting other mechanisms should be involved. Aromatic hydrocarbon receptor (AhR) activation increases cell division in human breast cancer cells, and CPF strongly activates it. Finally, the KIAA1363 enzyme, which is regulated by CPF, is overexpressed in cancer cells. Accordingly, we hypothesized that CPF or its metabolite chlorpyrifos-oxon (CPFO) could induce cell viability promotion in MCF-7 and MDA-MB-231 cell lines, through mechanisms related to ERα, AhR, and KIAA1363, after 24 h and 14 days treatment. Results show that, after acute and long-term treatment, CPF and CPFO alter differently KIAA1363, AhR, ER and cytochrome P450 isoenzyme 1A1 (CYP1A1) expression. In addition, they induced cell proliferation through ERα activation after 24 h exposure in MCF-7 cells and through KIAA1363 overexpression and AhR activation in MCF-7 and MDA-MB-231 cells after acute and long-term treatment. The results obtained in this work provide new information relative to the mechanisms involved in the CPF toxic effects that could lead to breast cancer disease.

Primary hippocampal estrogenic dysfunction induces synaptic proteins alteration and neuronal cell death after single and repeated paraquat exposure.

The extensively utilized herbicide Paraquat (PQ) was reported to generate cognitive disorders and hippocampal neuronal cell death after unique and extended exposure. Although, most of the mechanisms that mediate these actions remain unknown. We researched whether PQ induces synaptic protein disruption, Tau and amyloid beta protein formation, oxidative stress generation, and hippocampal neuronal cell loss through anti-estrogen action in primary hippocampal neurons, after day and two weeks PQ treatment, as a probable mechanism of such learning and memory impairment. Our results reveal that PQ did not alter estrogen receptors (ERα and ERß) gene expression, yet it decreased ER activation, which led to synaptic proteins disruption and amyloid beta proteins generation and Tau proteins hyperphosphorylation. Estrogenic signaling disruption induced by PQ also downregulated the NRF2 pathway leading to oxidative stress generation. Finally, PQ exposure induced cell death mediated by ER dysfunction partially through oxidative stress and amyloid beta proteins generation and Tau proteins hyperphosphorylation. The results presented provide a therapeutic strategy to protect against PQ toxic effects, possibly giving an explanation for the learning and memory impairment generated following PQ exposure.

Proteasome 20S and Rab5 Alteration after 24 h and 14 Days Chlorpyrifos Exposure Lead to ß-Amyloid and Tau Protein Level Increases and SN56 Neuronal Cell Death.

The biocide chlorpyrifos (CPF) was shown to produce cognition impairment following single and long-term exposure. The complete mechanisms that lead to the CPF induced cognitive disorders remain to be discovered. Aß and tau proteins production was induced in basal forebrain SN56 cholinergic cells, by CPF, through proteasome 20S inhibition and Rab5 overexpression, leading to cell death both after acute and repeated administration, which was related with cognitive disorders induction. The results obtained in our study procure novel information related to the mechanisms involved in CPF neurodegeneration, which could be responsible for cognitive dysfunction and may lead to a promising alternative treatment of these effects.

Oxidative stress and cell death induction by amitraz and its metabolite BTS-27271 mediated through cytochrome P450 and NRF2 pathway alteration in primary hippocampal cell.

Amitraz is a neurotoxic formamidine pesticide that induces cell death in hippocampal neurons, although its mechanisms are unknown. Amitraz produces reactive oxygen species (ROS), which could lead to cell death. Amitraz was shown to induce different cytochrome P450 (CYP) isoenzymes involved with ROS and apoptotic cell death induction. Finally, amitraz was described to decrease the activity of antioxidant enzymes regulated through KEAP1/NRF2 pathway, thus likely leading to a reduction of ROS elimination and to cell death induction. We evaluated the effect of amitraz or BTS-27271 co-treatment with or without the antioxidant N-acetylcysteine and/or the unspecific CYP inhibitor 1-aminobenzotriazole on cell viability and its related mechanisms in wild type and silenced primary hippocampal neurons after 24 h treatment. We observed that amitraz produced oxidative stress and CYPs induction leading to apoptotic cell death. ROS generation was partially mediated by CYPs induction and downregulation of NRF2-pathway through KEAP1 overexpression. These data could help explain the mechanism by which amitraz induces cell death and oxidative stress and provide a therapeutic strategy to protect against this effect in case of poisoning.

Manganese induced ROS and AChE variants alteration leads to SN56 basal forebrain cholinergic neuronal loss after acute and long-term treatment.

Manganese (Mn) induces cognitive disorders and basal forebrain (BF) cholinergic neuronal loss, involved on learning and memory regulation, which could be the cause of such cognitive disorders. However, the mechanisms through which it induces these effects are unknown. We hypothesized that Mn could induce BF cholinergic neuronal loss through oxidative stress generation, cholinergic transmission and AChE variants alteration that could explain Mn cognitive disorders. This study shows that Mn impaired cholinergic transmission in SN56 cholinergic neurons from BF through alteration of AChE and ChAT activity and CHT expression. Moreover, Mn induces, after acute and long-term exposure, AChE variants alteration and oxidative stress generation that leaded to lipid peroxidation and protein oxidation. Finally, Mn induces cell death on SN56 cholinergic neurons and this effect is independent of cholinergic transmission alteration, but was mediated partially by oxidative stress generation and AChE variants alteration. Our results provide new understanding of the mechanisms contributing to the harmful effects of Mn on cholinergic neurons and their possible involvement in cognitive disorders induced by Mn.

Cadmium alters heat shock protein pathways in SN56 cholinergic neurons, leading to Aß and phosphorylated Tau protein generation and cell death.

Cadmium, a neurotoxic environmental compound, produces cognitive disorders, although the mechanism remains unknown. Cadmium induces a more pronounced cell death on cholinergic neurons from basal forebrain (BF), mediated, in part, by increase in Aß and total and phosphorylated Tau protein levels, which may explain cadmium effects on learning and memory processes. Cadmium downregulates the expression of heat shock proteins (HSPs) HSP 90, HSP70 and HSP27, and of HSF1, the master regulator of the HSP pathway. HSPs proteins reduce the production of Aß and phosphorylated Tau proteins and avoid cell death pathways induction. Thus, we hypothesized that cadmium induced the production of Aß and Tau proteins by HSP pathway disruption through HSF1 expression alteration, leading to BF cholinergic neurons cell death. Our results show that cadmium downregulates HSF1, leading to HSP90, HSP70 and HSP27 gene expression downregulation in BF SN56 cholinergic neurons. In addition, cadmium induced Aß and total and phosphorylated Tau proteins generation, mediated partially by HSP90, HSP70 and HSP27 disruption, leading to cell death. These results provide new understanding of the mechanisms contributing to cadmium harmful effects on cholinergic neurons.

SN56 neuronal cell death after 24 h and 14 days chlorpyrifos exposure through glutamate transmission dysfunction, increase of GSK-3ß enzyme, ß-amyloid and tau protein levels.

Chlorpyrifos (CPF) is an organophosphate insecticide described to induce cognitive disorders, both after acute and repeated administration. However, the mechanisms through which it induces these effects are unknown. CPF has been reported to produce basal forebrain cholinergic neuronal cell death, involved on learning and memory regulation, which could be the cause of such cognitive disorders. Neuronal cell death was partially mediated by oxidative stress generation, P75NTR and α7-nAChRs gene expression alteration triggered through acetylcholinesterase (AChE) variants disruption, suggesting other mechanisms are involved. In this regard, CPF induces Aß and tau proteins production and activation of GSK3ß enzyme and alters glutamatergic transmission, which have been related with basal forebrain cholinergic neuronal cell death and development of cognitive disorders. According to these data, we hypothesized that CPF induces basal forebrain cholinergic neuronal cell death through induction of Aß and tau proteins production, activation of GSK-3ß enzyme and disruption of glutamatergic transmission. We evaluated this hypothesis in septal SN56 basal forebrain cholinergic neurons, after 24 h and 14 days CPF exposure. This study shows that CPF increases glutamate levels, upregulates GSK-3ß gene expression, and increases the production of Aß and phosphorylated tau proteins and all these effects reduced cell viability. CPF increases glutaminase activity and upregulates the VGLUT1 gene expression, which could mediate the disruption of glutamatergic transmission. Our present results provide new understanding of the mechanisms contributing to the harmful effects of CPF, and its possible relevance in the pathogenesis of neurodegenerative diseases.

Cadmium induced ROS alters M1 and M3 receptors, leading to SN56 cholinergic neuronal loss, through AChE variants disruption.

Cadmium, an environmental neurotoxic compound, produces cognitive disorders, although the mechanism remains unknown. Previously, we described that cadmium induces a more pronounced cell death on cholinergic neurons from basal forebrain (BF). This effect, partially mediated by M1 receptor blockade, triggering it through AChE splices variants alteration, may explain cadmium effects on learning and memory processes. Cadmium has been also reported to induce oxidative stress generation leading to M2 and M4 muscarinic receptors alteration, in hippocampus and frontal cortex, which are necessary to maintain cell viability and cognitive regulation, so their alteration in BF could also mediate this effect. Moreover, it has been reported that antioxidant treatment could reverse cognitive disorders, muscarinic receptor and AChE variants alterations induced by cadmium. Thus, we hypothesized that cadmium induced cell death of BF cholinergic neurons is mediated by oxidative stress generation and this mechanism could produce this effect, in part, through AChE variants altered by muscarinic receptors disruption. To prove this, we evaluated in BF SN56 cholinergic neurons, whether cadmium induces oxidative stress and alters muscarinic receptors, and their involvement in the induction of cell death through alteration of AChE variants. Our results show that cadmium induces oxidative stress, which mediates partially the alteration of AChE variants and M2 to M4 muscarinic receptors expression and blockage of M1 receptor. In addition, cadmium induced oxidative stress generation by M1 and M3 receptors alteration through AChE variants disruption, leading to cell death. These results provide new understanding of the mechanisms contributing to cadmium harmful effects on cholinergic neurons.

Primary hippocampal neuronal cell death induction after acute and repeated paraquat exposures mediated by AChE variants alteration and cholinergic and glutamatergic transmission disruption.

Paraquat (PQ) is a widely used non-selective contact herbicide shown to produce memory and learning deficits after acute and repeated exposure similar to those induced in Alzheimer's disease (AD). However, the complete mechanisms through which it induces these effects are unknown. On the other hand, cholinergic and glutamatergic systems, mainly in the hippocampus, are involved on learning, memory and cell viability regulation. An alteration of hippocampal cholinergic or glutamatergic transmissions or neuronal cell loss may induce these effects. In this regard, it has been suggested that PQ may induce cell death and affect cholinergic and glutamatergic transmission, which alteration could produce neuronal loss. According to these data, we hypothesized that PQ could induce hippocampal neuronal loss through cholinergic and glutamatergic transmissions alteration. To prove this hypothesis, we evaluated in hippocampal primary cell culture, the PQ toxic effects after 24h and 14 consecutive days exposure on neuronal viability and the cholinergic and glutamatergic mechanisms related to it. This study shows that PQ impaired acetylcholine levels and induced AChE inhibition and increased CHT expression only after 14days exposure, which suggests that acetylcholine levels alteration could be mediated by these actions. PQ also disrupted glutamate levels through induction of glutaminase activity. In addition, PQ induced, after 24h and 14days exposure, cell death on hippocampal neurons that was partially mediated by AChE variants alteration and cholinergic and gultamatergic transmissions disruption. Our present results provide new view of the mechanisms contributing to PQ neurotoxicity and may explain cognitive dysfunctions observed after PQ exposure.

Muscarinic M1 receptor partially modulates higher sensitivity to cadmium-induced cell death in primary basal forebrain cholinergic neurons: A cholinesterase variants dependent mechanism.

Cadmium is a toxic compound reported to produce cognitive dysfunctions, though the mechanisms involved are unknown. In a previous work we described how cadmium blocks cholinergic transmission and induces greater cell death in primary cholinergic neurons from the basal forebrain. It also induces cell death in SN56 cholinergic neurons from the basal forebrain through M1R blockage, alterations in the expression of AChE variants and GSK-3ß, and an increase in Aß and total and phosphorylated Tau protein levels. It was observed that the silencing or blockage of M1R altered ChAT activity, GSK-3ß, AChE splice variants gene expression, and Aß and Tau protein formation. Furthermore, AChE-S variants were associated with the same actions modulated by M1R. Accordingly, we hypothesized that cholinergic transmission blockage and higher sensitivity to cadmium-induced cell death of primary basal forebrain cholinergic neurons is mediated by M1R blockage, which triggers this effect through alteration of the expression of AChE variants. To prove this hypothesis, we evaluated, in primary culture from the basal forebrain region, whether M1R silencing induces greater cell death in cholinergic neurons than cadmium does, and whether in SN56 cells M1R mediates the mechanisms described so as to play a part in the cadmium induction of cholinergic transmission blockage and cell death in this cell line through alteration of the expression of AChE variants. Our results prove that M1R silencing by cadmium partially mediates the greater cell death observed on basal forebrain cholinergic neurons. Moreover, all previously described mechanisms for blocking cholinergic transmission and inducing cell death on SN56 cells after cadmium exposure are partially mediated by M1R through the alteration of AChE expression. Thus, our results may explain cognitive dysfunctions observed in cadmium toxicity.

Molecular mechanisms of amitraz mammalian toxicity: a comprehensive review of existing data.

Amitraz is a formamidine pesticide widely used as an insecticide and acaricide. Amitraz poisoning cases in humans and animals are still being described to date, which is a cause of concern for health authorities. Amitraz was reported not to pose unreasonable risks or adverse effects to humans or the environment unlike the other commercialized member of the formamidine family, chlordimeform, which was removed from the market because of carcinogenic effects in animal studies. Amitraz was classified as a nonquantifiable "Suggestive Evidence of Carcinogenicity" and not genotoxic, but recently, it has been reported that it could induce genotoxic effects. Moreover, ever since the previously published evaluations made by the Environmental Protection Agency (EPA) and the Joint Meeting of Pesticide Residues (JMPR) there have been new reported data on amitraz toxicity related to genotoxicity, oxidative stress, cell death, immunotoxicty, endocrine disruption, and developmental toxicity which indicate that the risk of this compound could be underestimated. Furthermore, there is missing information about the dose-response relationship for some mechanisms and toxic effects described for amitraz and its metabolites, the mechanism of action by which several toxic effects are produced, and amitraz pharmacokinetics on different species. According to this, the new information reported should be taken into account, and more studies should be performed to fill in the gaps of missing information for a complete hazard identification and therefore an exhaustive risk assessment of amitraz. This review is aimed at updating the current knowledge on molecular mechanisms of amitraz mammalian toxicity, pointing out the missing information, providing some possible explanation of the mechanism by which some toxic effects observed are produced, and suggesting future direction of its research. To our knowledge, this is the first review on the molecular mechanisms of amitraz toxicity.

Liver and kidney damage induced by 4-aminopyridine in a repeated dose (28 days) oral toxicity study in rats: gene expression profile of hybrid cell death.

4-Aminopyridine (4-AP) is an orphan drug indicated for the treatment of neuromuscular disorders. There is a great controversy around the use of this drug because of its narrow safety index and because a large number of adverse effects have been reported. Moreover, it was shown to induce cell death in different cell lines, being reported mainly apoptosis and necrosis as the principal pathways of cell death mediated by blockage of K channels or the Na, K-ATPase, but until now it was not described in vivo cell death induced by 4-aminipyridine. To provide new subchronic toxicity data and specifically, evaluate if 4-AP is able to induce in vivo cell death process and the main pathways related to it, a repeated dose (28 days) oral toxicity study, at therapeutic range of doses, was conducted in rats. The anatomical pathology, the biochemical and hematological parameters were analyzed and a real-time PCR array analysis was developed with an Ingenuity Pathway Analysis (IPA). The leucocytes number, the lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) enzymatic activity were increased at all dose but the erythrocytes number, the hemoglobin concentration, the alkaline phosphatase (FAL) and alanine aminotransferase (ALT) enzymatic activity were increased only at highest dose studied. However, glucose levels decreased at all doses. The biochemical results are indicative of hepatic damage. The anatomy pathology studies showed cell death only on liver and kidney, and the real-time PCR array on liver tissue expressed a gene expression profile of necrotic and apoptotic induced cell death. The present work shows for the first time in vivo cell death on liver and kidney with features of apoptosis and necrosis induced by 4-AP and the gene expression profile shows that the cell death is mediated by necrotic and apoptotic pathways that support this finding.