Cadmium-promoted thyroid hormones disruption mediates ROS, inflammation, Aß and Tau proteins production, gliosis, spongiosis and neurodegeneration in rat basal forebrain.

Cadmium (Cd) produces cognition decline following single and repeated treatment, although the complete mechanisms are still unrevealed. Basal forebrain (BF) cholinergic neurons innervate the cortex and hippocampus, regulating cognition. Cd single and repeated exposure induced BF cholinergic neuronal loss, partly through thyroid hormones (THs) disruption, which may cause the cognition decline observed following Cd exposure. However, the mechanisms through which THs disruption mediate this effect remain unknown. To research the possible mechanisms through which Cd-induced THs deficiency may mediate BF neurodegeneration, Wistar male rats were treated with Cd for 1- (1 mg/kg) or 28-days (0.1 mg/kg) with or without triiodothyronine (T3, 40 µg/kg/day). Cd exposure promoted neurodegeneration, spongiosis, gliosis and several mechanisms related to these alterations (increased H202, malondialdehyde, TNF-α, IL-1ß, IL-6, BACE1, Aß and phosphorylated-Tau levels, and decreased phosphorylated-AKT and phosphorylated-GSK-3ß levels). T3 supplementation partially reversed the effects observed. Our results show that Cd induces several mechanisms that may be responsible for the neurodegeneration, spongiosis and gliosis observed in the rats' BF, which are partially mediated by a reduction in THs levels. These data may help to explain the mechanisms through which Cd induces BF neurodegeneration, possibly leading to the cognitive decline observed, providing new therapeutic tools to prevent and treat these damages.

Cadmium-induced neurotoxic effects on rat basal forebrain cholinergic system through thyroid hormones disruption.

Cadmium (Cd) single and repeated exposure produces cognitive dysfunctions. Basal forebrain cholinergic neurons (BFCN) regulate cognitive functions. BFCN loss or cholinergic neurotransmission dysfunction leads to cognitive disabilities. Thyroid hormones (THs) maintain BFCN viability and functions, and Cd disrupts their levels. However, Cd-induced BFCN damages and THs disruption involvement was not studied. To research this we treated male Wistar rats intraperitoneally with Cd once (1 mg/kg) or repetitively for 28 days (0.1 mg/kg) with/without triiodothyronine (T3, 40 µg/kg/day). Cd increased thyroid-stimulating-hormone (TSH) and decreased T3 and tetraiodothyronine (T4). Cd altered cholinergic transmission and induced a more pronounced neurodegeneration on BFCN, mediated partially by THs reduction. Additionally, Cd antagonized muscarinic 1 receptor (M1R), overexpressed acetylcholinesterase S variant (AChE-S), downregulated AChE-R, M2R, M3R and M4R, and reduced AChE and choline acetyltransferase activities through THs disruption. These results may assist to discover cadmium mechanisms that induce cognitive disabilities, revealing a new possible therapeutic tool.

Bisphenol A single and repeated treatment increases HDAC2, leading to cholinergic neurotransmission dysfunction and SN56 cholinergic apoptotic cell death through AChE variants overexpression and NGF/TrkA/P75NTR signaling disruption.

Bisphenol-A (BPA), a widely used plasticizer, induces cognitive dysfunctions following single and repeated exposure. Several studies, developed in hippocampus and cortex, tried to find the mechanisms that trigger and mediate these dysfunctions, but those are still not well known. Basal forebrain cholinergic neurons (BFCN) innervate hippocampus and cortex, regulating cognitive function, and their loss or the induction of cholinergic neurotransmission dysfunction leads to cognitive disabilities. However, no studies were performed in BFCN. We treated wild type or histone deacetylase (HDAC2), P75NTR or acetylcholinesterase (AChE) silenced SN56 cholinergic cells from BF with BPA (0.001 µM-100 µM) with or without recombinant nerve growth factor (NGF) and with or without acetylcholine (ACh) for one- and fourteen days in order to elucidate the mechanisms underlying these effects. BPA induced cholinergic neurotransmission disruption through reduction of ChAT activity, and produced apoptotic cell death, mediated partially through AChE-S overexpression and NGF/TrkA/P75NTR signaling dysfunction, independently of cholinergic neurotransmission disruption, following one- and fourteen days of treatment. BPA mediates these alterations, in part, through HDAC2 overexpression. These data are relevant since they may help to elucidate the neurotoxic mechanisms that trigger the cognitive disabilities induced by BPA exposure, providing a new therapeutic approach.

Aryl Hydrocarbon Receptor Activation Produces Heat Shock Protein 90 and 70 Overexpression, Prostaglandin E2/Wnt/ß-Catenin Signaling Disruption, and Cell Proliferation in MCF-7 and MDA-MB-231 Cells after 24 h and 14 Days of Chlorpyrifos Treatment.

The biocide chlorpyrifos (CPF) was described to increase breast cancer risk in humans, to produce breast cancer in animals, and to induce cell proliferation in MCF-7 and MDA-MB-231 cells after 1 and 14 days of treatment. The entire mechanisms related to these CPF actions remain unknown. CPF induced cell proliferation in MCF-7 and MDA-MB-231 cells after 1 and 14 days of treatment by AhR activation through the PGE2/Wnt/ß-catenin pathway and HSP90 and HSP70 overexpression. Our results reveal new information on CPF toxic mechanisms induced in human breast cancer cell lines, which could assist in elucidating its involvement in breast cancer.

Chlorpyrifos induces cell proliferation in MCF-7 and MDA-MB-231 cells, through cholinergic and Wnt/ß-catenin signaling disruption, AChE-R upregulation and oxidative stress generation after single and repeated treatment.

Chlorpyrifos (CPF) biocide, is associated with breast cancer. The processes underlying this association have not been elucidated to date. CPF increases MCF-7 and MDA-MB-231 cell proliferation after acute and long-term treatment, partially through KIAA1363 overexpression and aryl-hydrocarbon receptor activation but also through estrogen receptor-alpha activation after 24 h exposure in MCF-7 cells, suggesting other mechanisms may be involved. CPF induces reactive oxygen species (ROS) generation, acetylcholine accumulation, and overexpression of acetylcholinesterase-R/S (AChE-R/S) variants, while it also alters the Wnt/ß-catenin pathway, both in vitro and in vivo, in processes different from cancer. These latter mechanisms are also linked to cell proliferation and could mediate this effect induced by CPF. Our results show that CPF (0.01-100 µM), following one-day and fourteen-days treatment, respectively, induced ROS generation and lipid peroxidation, and acetylcholine accumulation due to AChE inhibition, Wnt/ß-catenin up- or downregulation depending on the CPF treatment concentration, and AChE-R and AChE-S overexpression, with the latter being mediated through GSK-3ß activity alteration. Finally, CPF promoted cell division through ACh and ROS accumulation, AChE-R overexpression, and Wnt/ß-catenin signaling disruption. Our results provide novel information on the effect of CPF on human breast cancer cell lines that may help to explain its involvement in breast cancer.

Manganese increases Aß and Tau protein levels through proteasome 20S and heat shock proteins 90 and 70 alteration, leading to SN56 cholinergic cell death following single and repeated treatment.

Manganese (Mn) produces cholinergic neuronal loss in basal forebrain (BF) region that was related to cognitive dysfunction induced after single and repeated Mn treatment. All processes that generate cholinergic neuronal loss in BF remain to be understood. Mn exposure may produce the reduction of BF cholinergic neurons by increasing amyloid beta (Aß) and phosphorylated Tau (pTau) protein levels, altering heat shock proteins' (HSPs) expression, disrupting proteasome P20S activity and generating oxidative stress. These mechanisms, described to be altered by Mn in regions different than BF, could lead to the memory and learning process alteration produced after Mn exposure. The research performed shows that single and repeated Mn treatment of SN56 cholinergic neurons from BF induces P20S inhibition, increases Aß and pTau protein levels, produces HSP90 and HSP70 proteins expression alteration, and oxidative stress generation, being the last two effects mediated by NRF2 pathway alteration. The increment of Aß and pTau protein levels was mediated by HSPs and proteasome dysfunction. All these mechanisms mediated the cell decline observed after Mn treatment. Our results are relevant because they may assist to reveal the processes leading to the neurotoxicity and cognitive alterations observed after Mn exposure.

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.

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.

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.

Time trends of human papillomavirus types in invasive cervical cancer, from 1940 to 2007.

Contribution over time of human papillomavirus (HPV) types in human cancers has been poorly documented. Such data is fundamental to measure current HPV vaccines impact in the years to come. We estimated the HPV type-specific distribution in a large international series of invasive cervical cancer (ICC) over 70 years prior to vaccination. Paraffin embedded ICC cases diagnosed between 1940 and 2007 were retrieved from eleven countries in Central-South America, Asia and Europe. Included countries reported to have low-medium cervical cancer screening uptake. Information on age at and year of diagnosis was collected from medical records. After histological confirmation, HPV DNA detection was performed by SPF-10/DEIA/LiPA25 (version1). Logistic regression models were used for estimating the adjusted relative contributions (RC) of HPV16 and of HPV18 over time. Among 4,771 HPV DNA positive ICC cases, HPV16 and HPV18 were the two most common HPVs in all the decades with no statistically significant variations of their adjusted-RC from 1940-59 to 2000-07 (HPV16-from 61.5 to 62.1%, and HPV18-from 6.9 to 7.2%). As well, the RC of other HPV types did not varied over time. In the stratified analysis by histology, HPV16 adjusted-RC significantly increased across decades in adenocarcinomas. Regarding age, cases associated to either HPV16, 18 or 45 were younger than those with other HPV types in all the evaluated decades. The observed stability on the HPV type distribution predicts a high and stable impact of HPV vaccination in reducing the cervical cancer burden in future vaccinated generations.

Human papillomavirus genotype distribution in cervical cancer cases in Spain. Implications for prevention.

OBJECTIVE: Human papillomavirus (HPV) genotype distribution in invasive cervical cancer (ICC) is critical to guide the introduction and to assess the impact of HPV prophylactic vaccines. This study aims to provide specific information for Spain. METHODS: 1043 histological confirmed ICC cases diagnosed from 1940 to 2007 from six Spanish regions were assembled. HPV DNA detection was performed by SPF(10) broad-spectrum PCR followed by deoxyribonucleic acid enzyme immunoassay and genotyping by reverse hybridization line probe assay (LiPA(25)) (version 1). RESULTS: Of 1043 ICC cases, 904 were HPV DNA positive (adjusted prevalence: 89.1%). The eight most common types, in decreasing order, were HPV 16, 18, 33, 31, 45, 35, 52 and 56, accounting for more than 90% of cases. HPV 16 and 18 contributed to 72.4% of all HPV positive ICC cases. In cervical adenocarcinomas, this contribution increased up to 94%. HPV 16 and 18 relative contributions showed a stable pattern over the 60 year study period. HPV 45, 18 and 16-positive ICC cases presented at younger ages than cases with other HPV types (adjusted mean age: 43.8, 45.2, 52.6 and 57.7 years, respectively). CONCLUSIONS: HPV 16 and 18 accounted together for a 72.4% of positive cases, with no statistically significant changes in their relative contributions over the last decades. In 94% of cervical adenocarcinomas we identified at least one of the two HPV types included in the current vaccines (HPV 16/18). Results suggest a major impact of HPV vaccines on reduction of ICC burden in Spain in the HPV vaccinated cohorts.

Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study.

BACKGROUND: Knowledge about the distribution of human papillomavirus (HPV) genotypes in invasive cervical cancer is crucial to guide the introduction of prophylactic vaccines. We aimed to provide novel and comprehensive data about the worldwide genotype distribution in patients with invasive cervical cancer. METHODS: Paraffin-embedded samples of histologically confirmed cases of invasive cervical cancer were collected from 38 countries in Europe, North America, central South America, Africa, Asia, and Oceania. Inclusion criteria were a pathological confirmation of a primary invasive cervical cancer of epithelial origin in the tissue sample selected for analysis of HPV DNA, and information about the year of diagnosis. HPV detection was done by use of PCR with SPF-10 broad-spectrum primers followed by DNA enzyme immunoassay and genotyping with a reverse hybridisation line probe assay. Sequence analysis was done to characterise HPV-positive samples with unknown HPV types. Data analyses included algorithms of multiple infections to estimate type-specific relative contributions. FINDINGS: 22,661 paraffin-embedded samples were obtained from 14,249 women. 10,575 cases of invasive cervical cancer were included in the study, and 8977 (85%) of these were positive for HPV DNA. The most common HPV types were 16, 18, 31, 33, 35, 45, 52, and 58 with a combined worldwide relative contribution of 8196 of 8977 (91%, 95% CI 90-92). HPV types 16 and 18 were detected in 6357 of 8977 of cases (71%, 70-72) of invasive cervical cancer. HPV types 16, 18, and 45 were detected in 443 of 470 cases (94%, 92-96) of cervical adenocarcinomas. Unknown HPV types that were identified with sequence analysis were 26, 30, 61, 67, 69, 82, and 91 in 103 (1%) of 8977 cases of invasive cervical cancer. Women with invasive cervical cancers related to HPV types 16, 18, or 45 presented at a younger mean age than did those with other HPV types (50·0 years [49·6-50·4], 48·2 years [47·3-49·2], 46·8 years [46·6-48·1], and 55·5 years [54·9-56·1], respectively). INTERPRETATION: To our knowledge, this study is the largest assessment of HPV genotypes to date. HPV types 16, 18, 31, 33, 35, 45, 52, and 58 should be given priority when the cross-protective effects of current vaccines are assessed, and for formulation of recommendations for the use of second-generation polyvalent HPV vaccines. Our results also suggest that type-specific high-risk HPV-DNA-based screening tests and protocols should focus on HPV types 16, 18, and 45.