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Manganese (Mn) plays an important role in many physiological processes. Nevertheless, Mn accumulation in the brain can cause a parkinsonian-like syndrome known as manganism. Unfortunately, the therapeutic options for this disease are scarce and of limited efficacy. For this reason, a great effort is being made to understand the cellular and molecular mechanisms involved in Mn toxicity in neuronal and glial cells. Even though evidence indicates that Mn activates autophagy in microglia, the consequences of this activation in cell death remain unknown. In this study, we demonstrated a key role of reactive oxygen species in Mn-induced damage in microglial cells. These species generated by Mn 2+ induce lysosomal alterations, LMP, cathepsins release and cell death. Besides, we described for the first time the kinetic of Mn 2+ -induced autophagy in BV-2 microglial cells and its relevance to cell fate. We found that Mn promotes a time-dependent increase in LC3-II and p62 expression levels, suggesting autophagy activation. Possibly, cells trigger autophagy to neutralize the risks associated with lysosomal rupture. In addition, pre-treatment with both Rapamycin and Melatonin enhanced autophagy and retarded Mn 2+ cytotoxicity. In summary, our results demonstrated that, despite the damage inflicted on a subset of lysosomes, the autophagic pathway plays a protective role in Mn-induced microglial cell death. We propose that 2 h Mn 2+ exposure will not induce disturbances in the autophagic flux. However, as time passes, the accumulated damage inside the cell could trigger a dysfunction of this mechanism. These findings may represent a valuable contribution to future research concerning manganism therapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the presence of misfolded proteins, amyloid-β (Aβ) aggregates, and neuroinflammation in the brain. Microglial cells are key players in the context of AD, being capable of releasing cytokines in response to Aβ and degrading aggregated proteins by mechanisms involving the ubiquitin-proteasome system and autophagy. Here, we present in vivo and in vitro evidence showing that microglial autophagy is affected during AD progression. PDAPPJ20 mice-murine model of AD-exhibited an accumulation of the autophagy receptor p62 and ubiquitin+ aggregates in Iba1+ microglial cells close to amyloid deposits in the hippocampus. Moreover, cultured microglial BV-2 cells showed an enhanced autophagic flux during a 2-h exposure to fibrillar Aβ, which was decreased if the exposure was prolonged to 24 h, a condition analogous to the chronic exposure to Aβ in the human pathology. The autophagic impairment was also associated with lysosomal damage, depicted by membrane permeabilization as shown by the presence of the acid hydrolase cathepsin-D in cytoplasm and altered LysoTracker staining. These results are compatible with microglial exhaustion caused by pro-inflammatory conditions and persistent exposure to aggregated Aβ peptides. In addition, we found LC3-positive autophagic vesicles accumulated in phagocytic CD68+ microglia in human AD brain samples, suggesting defective autophagy in microglia of AD brain. Our results indicate that the capacity of microglia to degrade Aβ and potentially other proteins through autophagy may be negatively affected as the disease progresses. Preserving autophagy in microglia thus emerges as a promising approach for treating AD. Graphical abstract.

Hydroxyapatite (Ca10(PO4)6(OH)2; HAP) is an essential component of the human bone inorganic phase. At the nanoscale level, nano-HAP (nHAP) presents marked emergent properties differing substantially from those of the bulk counterpart. Interestingly, these properties depend on nanoparticle characteristics. In this study, we investigated the cytotoxicity of rod-shaped crystalline nHAP (10-20 nm × 50-100 nm) in both normal (ARPE-19, BV-2) and tumoral (HepG2, HEp-2, A549 and C6) cells. We found that nHAP was cytotoxic in tumor HEp-2, A549, and C6 cells. Moreover, it induced an expansion of the lysosomal compartment at sublethal concentrations in different cell lines, while lysosomal membrane damage was not detected. In C6 glioma cells, the most sensitive cell line to nHAP, these nanoparticles increased reactive oxygen species (ROS) production and induced DNA damage measured by γ-H2AX phosphorylation. Interestingly, our data also show for the first time that nHAP affects both cell unlimited proliferative capacity and cell migration, two of the major pathways involved in cancer progression. The present results showed the cytotoxic and antiproliferative effects of nHAP and suggest its potential as an alternative agent for glioma therapy., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Age-related macular degeneration (AMD) is a multifactorial retinal disease characterized by a progressive loss of central vision. Retinal pigment epithelium (RPE) degeneration is a critical event in AMD. It has been associated to A2E accumulation, which sensitizes RPE to blue light photodamage. Mitochondrial quality control mechanisms have evolved to ensure mitochondrial integrity and preserve cellular homeostasis. Particularly, mitochondrial dynamics involve the regulation of mitochondrial fission and fusion to preserve a healthy mitochondrial network. The present study aims to clarify the cellular and molecular mechanisms underlying photodamage-induced RPE cell death with particular focus on the involvement of defective mitochondrial dynamics. Light-emitting diodes irradiation (445 ± 18 nm; 4.43 mW/cm 2 ) significantly reduced the viability of both unloaded and A2E-loaded human ARPE-19 cells and increased reactive oxygen species production. A2E along with blue light, triggered apoptosis measured by MC540/PI-flow cytometry and activated caspase-3. Blue light induced mitochondrial fusion/fission imbalance towards mitochondrial fragmentation in both non-loaded and A2E-loaded cells which correlated with the deregulation of mitochondria-shaping proteins level (OPA1, DRP1 and OMA1). To our knowledge, this is the first work reporting that photodamage causes mitochondrial dynamics deregulation in RPE cells. This process could possibly contribute to AMD pathology. Our findings suggest that the regulation of mitochondrial dynamics may be a valuable strategy for treating retinal degeneration diseases, such as AMD.

Aim: Amino functionalization is a first step modification aiming to achieve biomedical applications of silicon nanoparticles, for example, for photodynamic therapy or radiotherapy. Nevertheless, toxicity and low quantum yields due to the positive charge of amino groups emerge as a problem that could be solved with subsequent derivatizations., Materials & Methods: Folic and PEG-conjugated nanoparticles were obtained from amino-functionalized silicon nanoparticle (NH 2 SiNP). Cytotoxicity was determined on a tumor cell line at low and high concentrations. Four end points of in vivo toxicity were evaluated on zebrafish (Danio rerio)., Results: Folic acid functionalization reduced the cytotoxicity in comparison to amino and PEG-functionalized nanoparticles. In zebrafish, folic functionalization lowered toxicity in general while PEG increased it., Conclusion: Functionalization of NH 2 SiNP with folic acid reduced the toxic effects in vitro and in vivo. This could be useful for therapeutic applications. PEG functionalization did not lower the toxicity.

Apoptosis plays an important role in cellular processes such as development, differentiation, and homeostasis. Although the participation of angiotensin II (Ang II) AT 2 receptors (AT 2 R) in cellular apoptosis is well accepted, the signaling pathway involved in this process is not well established. We evaluated the participation of signaling proteins focal adhesion kinase (FAK), RhoA, and p38 mitogen-activated protein kinase (p38MAPK) in apoptosis induced by Ang II via AT 2 R overexpressed in HeLa cells. Following a short stimulation time (120 to 240 minutes) with Ang II, HeLa-AT 2 cells showed nuclear condensation, stress fibers disassembly and membrane blebbing. FAK, classically involved in cytoskeleton reorganization, has been postulated as an early marker of cellular apoptosis. Thus, we evaluated FAK cleavage, detected at early stimulation times (15 to 30 minutes). Apoptosis was confirmed by increased caspase-3 cleavage and enzymatic activity of caspase-3/7. Participation of RhoA was evaluated. HeLa-AT 2 cells overexpressing RhoA wild-type (WT) or their mutants, RhoA V14 (constitutively active form) or RhoA N19 (dominant-negative form) were used to explore RhoA participation. HeLa-AT 2 cells expressing the constitutively active variant RhoA V14 showed enhanced apoptotic features at earlier times as compared with cells expressing the WT variant. RhoA N19 expression prevented nuclear condensation/caspase activation. Inhibition of p38MAPK caused an increase in nuclear condensation and caspase-3/7 activation, suggesting a protective role of p38MAPK. Our results clearly demonstrated that stimulation of AT 2 R induce apoptosis with participation of FAK and RhoA while p38MAPK seems to play a prosurvival role., (© 2018 Wiley Periodicals, Inc.)

Microglia, the brain resident immune cells, play prominent roles in immune surveillance, tissue repair and neural regeneration. Despite these pro-survival actions, the relevance of these cells in the progression of several neuropathologies has been established. In the context of manganese (Mn) overexposure, it has been proposed that microglial activation contributes to enhance the neurotoxicity. However, the occurrence of a direct cytotoxic effect of Mn on microglial cells remains controversial. In the present work, we investigated the potential vulnerability of immortalized mouse microglial cells (BV-2) toward Mn 2+ , focusing on the signaling pathways involved in cell death. Evidence obtained showed that Mn 2+ induces a decrease in cell viability which is associated with reactive oxygen species (ROS) generation. In this report we demonstrated, for the first time, that Mn 2+ triggers regulated necrosis (RN) in BV-2 cells involving two central mechanisms: parthanatos and lysosomal disruption. The occurrence of parthanatos is supported by several cellular and molecular events: (i) DNA damage; (ii) AIF translocation from mitochondria to the nucleus; (iii) mitochondrial membrane permeabilization; and (iv) PARP1-dependent cell death. On the other hand, Mn 2+ induces lysosomal membrane permeabilization (LMP) and cathepsin D (CatD) release into the cytosol supporting the lysosomal disruption. Pre-incubation with CatB and D inhibitors partially prevented the Mn 2+ -induced cell viability decrease. Altogether these events point to lysosomes as players in the execution of RN. In summary, our results suggest that microglial cells could be direct targets of Mn 2+ damage. In this scenario, Mn 2+ triggers cell death involving RN pathways., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Heme Oxygenase-1 (HO-1), a stress- responsive enzyme which catalyzes heme degradation into iron, carbon monoxide, and biliverdin, exerts a neuroprotective role involving many different signaling pathways. In Parkinson disease patients, elevated HO-1 expression levels in astrocytes are involved in antioxidant defense. In the present work, employing an in vitro model of Mn 2+ -induced Parkinsonism in astroglial C6 cells, we investigated the role of HO-1 in both apoptosis and mitochondrial quality control (MQC). HO-1 exerted a protective effect against Mn 2+ injury. In fact, HO-1 decreased both intracellular and mitochondrial reactive oxygen species as well as the appearance of apoptotic features. Considering that Mn 2+ induces mitochondrial damage and a defective MQC has been implicated in neurodegenerative diseases, we hypothesized that HO-1 could mediate cytoprotection by regulating the MQC processes. Results obtained provide the first evidence that the beneficial effects of HO-1 in astroglial cells are mediated by the maintenance of both mitochondrial fusion/fission and biogenesis/mitophagy balances. Altogether, our data demonstrate a pro-survival function for HO-1 in Mn 2+ -induced apoptosis that involves the preservation of a proper MQC. These findings point to HO-1 as a new therapeutic target linked to mitochondrial pathophysiology in Manganism and probably Parkinson´s disease., (Copyright © 2018. Published by Elsevier B.V.)

Silicon blue-emitting nanoparticles (NPs) are promising effectors for photodynamic therapy and radiotherapy, because of their production of reactive oxygen species (ROS) upon irradiation., Results: Amino-functionalized silicon NPs (NH 2 SiNP) were intrinsically nontoxic below 100 μg/ml in vitro (on two tumor cell lines) and in vivo (zebrafish larvae and embryos). NH 2 SiNP showed a moderate effect as a photosensitizer for photodynamic therapy and reduced ROS generation in radiotherapy, which could be indicative of a ROS scavenging effect. Encapsulation of NH 2 SiNP into ultradeformable liposomes improved their skin penetration after topical application, reaching the viable epidermis where neoplastic events occur., Conclusion: Subsequent derivatizations after amino-functionalization and incorporation to nanodrug delivery systems could expand the spectrum of the biomedical application of these kind of silicon NPs.

Parkinson's disease is a neurodegenerative movement disorder caused by the loss of dopaminergic neurons from substantia nigra. It is characterized by the accumulation of aggregated α-synuclein as the major component of the Lewy bodies. Additional common features of this disease are the mitochondrial dysfunction and the activation/inhibition of autophagy both events associated to the intracellular accumulation of α-synuclein. The mechanism by which these events contribute to neural degeneration remains unknown. In the present work we investigated the effect of α-synuclein on mitochondrial dynamics and autophagy/mitophagy in SH-SY5Y cells, an in vitro model of Parkinson disease. We demonstrated that overexpression of wild type α-synuclein causes moderated toxicity, ROS generation and mitochondrial dysfunction. In addition, α-synuclein induces the mitochondrial fragmentation on a Drp-1-dependent fashion. Overexpression of the fusion protein Opa-1 prevented both mitochondrial fragmentation and cytotoxicity. On the other hand, cells expressing α-synuclein showed activated autophagy and particularly mitophagy. Employing a genetic strategy we demonstrated that autophagy is triggered in order to protect cells from α-synuclein-induced cell death. Our results clarify the role of Opa-1 and Drp-1 in mitochondrial dynamics and cell survival, a controversial α-synuclein research issue. The findings presented point to the relevance of mitochondrial homeostasis and autophagy in the pathogenesis of PD. Better understanding of the molecular interaction between these processes could give rise to novel therapeutic methods for PD prevention and amelioration., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Manganese (Mn) is an essential trace metal which plays a critical role in brain physiology by acting as a cofactor for several enzymes. However, upon overexposure, Mn preferentially accumulates within the basal ganglia leading to the development of a Parkinsonism known as Manganism. Data from our group have proved that Mn induces oxidative stress-mediated apoptosis in astrocytoma C6 cells. In the present study we described how cathepsins impact on different steps of each apoptotic cascade. Evidence obtained demonstrated that Mn generates lysosomal membrane permeabilization (LMP) and cathepsin release. Both cathepsins B (Ca-074 Me) and D (Pepstatin A) inhibitors as well as Bafilomycin A1 prevented caspases-3, -7, -8 and -9 activation, FasL upregulation, Bid cleavage, Δφm disruption and cytochrome c release. Results from in vivo studies showed that intrastriatal Mn injection increased cathepsin D levels from corpus striatum and substantia nigra pars compacta. Our results point to LMP and lysosomal cathepsins as key mediators in the apoptotic process triggered by Mn. These findings highlight the relevance of targeting the lysosomal pathway for Manganism therapy.

Two decades ago, Galectin-8 was described as a prostate carcinoma biomarker since it is only expressed in the neoplastic prostate, but not in the healthy tissue. To date, no biological function has been attributed to Galectin-8 that could explain this differential expression. In this study we silenced Galectin-8 in two human prostate cancer cell lines, PC3 and IGR-CaP1, and designed a pre-clinical experimental model that allows monitoring the pathology from its early steps to the long-term metastatic stages. We show for the first time that the natural and conserved expression of Gal-8 in tumour cells is responsible for the metastatic evolution of prostate cancer. In fact, Gal-8 controls the rearrangement of the cytoskeleton and E-Cadherin expression, with a major impact on anoikis and homotypic aggregation of tumour cells, both being essential processes for the survival of circulating tumour cells during metastasis. While localized prostate cancer can be cured, metastatic and advanced disease remains a significant therapeutic challenge, urging for the identification of prognostic markers of the metastatic process. Collectively, our results highlight Galectin-8 as a potential target for anti-metastatic therapy against prostate cancer.

A primary mode-of-action of all pyrethroid insecticides (PYRs) is the disruption of the voltage-gated sodium channel electrophysiology in neurons of target pests and nontarget species. The neurological actions of PYRs on non-neuronal cells of the nervous system remain poorly investigated. In the present work, we used C6 astrocytoma cells to study PYR actions (0.1-50 μM) under the hypothesis that glial cells may be targeted by and vulnerable to PYRs. To this end, we characterized the effects of bifenthrin (BF), tefluthrin (TF), α-cypermethrin (α-CYP), and deltamethrin (DM) on the integrity of nuclear, mitochondrial, and lysosomal compartments. In general, 24- to 48-h exposures produced concentration-related impairment of cell viability. In single-compound, 24-h exposure experiments, effective concentration (EC) 15 s 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT assay) were computed as follows (in μM): BF, 16.1; TF, 37.3; α-CYP, 7.8; DM, 5.0. We found concentration-related damage in several C6-cell subcellular compartments (mitochondria, nuclei, and lysosomes) at ≥ 10 -1 μM levels. Last, we examined a mixture of all PYRs (ie, Σ individual EC 15 ) using MTT assays and subcellular analyses. Our findings indicate that C6 cells are responsive to nM levels of PYRs, suggesting that astroglial susceptibility may contribute to the low-dose neurological effects caused by these insecticides. This research further suggests that C6 cells may provide relevant information as a screening platform for pesticide mixtures targeting nervous system cells by expected and unexpected toxicogenic pathways potentially contributing to clinical neurotoxicity., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Alzheimer's disease (AD) is a progressive neurodegenerative disease without effective therapy. Brain amyloid deposits are classical histopathological hallmarks that generate an inflammatory reaction affecting neuronal and glial function. The identification of early cell responses and of brain areas involved could help to design new successful treatments. Hence, we studied early alterations of hippocampal glia and their progression during the neuropathology in PDAPP-J20 transgenic mice, AD model, at 3, 9, and 15 months (m) of age. At 3 m, before deposits formation, microglial Iba1+ cells from transgenic mice already exhibited signs of activation and larger soma size in the hilus, alterations appearing later on stratum radiatum. Iba1 immunohistochemistry revealed increased cell density and immunoreactive area in PDAPP mice from 9 m onward selectively in the hilus, in coincidence with prominent amyloid Congo red + deposition. At pre-plaque stages, GFAP+ astroglia showed density alterations while, at an advanced age, the presence of deposits was associated with important glial volume changes and apparently being intimately involved in amyloid degradation. Astrocytes around plaques were strongly labeled for LC3 until 15 m in Tg mice, suggestive of increased autophagic flux. Moreover, β-Amyloid fibrils internalization by astrocytes in in vitro conditions was dependent on autophagy. Co-localization of Iba1 with ubiquitin or p62 was exclusively found in microglia contacting deposits from 9 m onward, suggesting torpid autophagy. Our work characterizes glial changes at early stages of the disease in PDAPP-J20 mice, focusing on the hilus as an especially susceptible hippocampal subfield, and provides evidence that glial autophagy could play a role in amyloid processing at advanced stages., (© 2015 Wiley Periodicals, Inc.)

Mitochondria are dynamic organelles that undergo fusion and fission processes. These events are regulated by mitochondria-shaping proteins. Changes in the expression and/or localization of these proteins lead to a mitochondrial dynamics impairment and may promote apoptosis. Increasing evidence correlates the mitochondrial dynamics disruption with the occurrence of neurodegenerative diseases. Therefore, we focused on this topic in Manganese (Mn)-induced Parkinsonism, a disorder associated with Mn accumulation preferentially in the basal ganglia where mitochondria from astrocytes represent an early target. Using MitoTracker Red staining we observed increased mitochondrial network fission in Mn-exposed rat astrocytoma C6 cells. Moreover, Mn induced a marked decrease in fusion protein Opa-1 levels as well as a dramatic increase in the expression of fission protein Drp-1. Additionally, Mn provoked a significant release of high MW Opa-1 isoforms from the mitochondria to the cytosol as well as an increased Drp-1 translocation to the mitochondria. Both Mdivi-1, a pharmacological Drp-1 inhibitor, and rat Drp-1 siRNA reduced the number of apoptotic nuclei, preserved the mitochondrial network integrity and prevented cell death. CsA, an MPTP opening inhibitor, prevented mitochondrial Δψm disruption, Opa-1 processing and Drp-1 translocation to the mitochondria therefore protecting Mn-exposed cells from mitochondrial disruption and apoptosis. The histological analysis and Hoechst 33258 staining of brain sections of Mn-injected rats in the striatum showed a decrease in cellular mass paralleled with an increase in the occurrence of apoptotic nuclei. Opa-1 and Drp-1 expression levels were also changed by Mn-treatment. Our results demonstrate for the first time that abnormal mitochondrial dynamics is implicated in both in vitro and in vivo Mn toxicity. In addition we show that the imbalance in fusion/fission equilibrium might be involved in Mn-induced apoptosis. This knowledge may provide new therapeutic tools for the treatment of Manganism and other neurodegenerative diseases.

Manganese (Mn) is an essential trace element due to its participation in many physiological processes. However, overexposure to this metal leads to a neurological disorder known as Manganism whose clinical manifestations and molecular mechanisms resemble Parkinson's disease. Several lines of evidence implicate astrocytes as an early target of Mn neurotoxicity being the mitochondria the most affected organelles. The aim of this study was to investigate the possible mitochondrial dynamics alterations in Mn-exposed human astrocytes. Therefore, we employed Gli36 cells which express the astrocytic markers GFAP and S100B. We demonstrated that Mn triggers the mitochondrial apoptotic pathway revealed by increased Bax/Bcl-2 ratio, by the loss of mitochondrial membrane potential and by caspase-9 activation. This apoptotic program may be in turn responsible of caspase-3/7 activation, PARP-1 cleavage, chromatin condensation and fragmentation. In addition, we determined that Mn induces deregulation in mitochondria-shaping proteins (Opa-1, Mfn-2 and Drp-1) expression levels in parallel with the disruption of the mitochondrial network toward to an exacerbated fragmentation. Since mitochondrial dynamics is altered in several neurodegenerative diseases, these proteins could become future targets to be considered in Manganism treatment., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a gradual loss of motoneurons. The majority of ALS cases are associated with a sporadic form whose etiology is unknown. Several pieces of evidence favor autoimmunity as a potential contributor to sporadic ALS pathology. To gain understanding concerning possible antigens interacting with IgGs from sporadic ALS patients (ALS-IgGs), we studied immunoreactivity against neuromuscular junction (NMJ), spinal cord and cerebellum of mice with and without the Ca(V) 2.1 pore-forming subunit of the P/Q-type voltage-gated calcium (Ca(2+)) channel. ALS-IgGs showed a strong reactivity against NMJs of wild-type diaphragms. ALS-IgGs also increased muscle miniature end-plate potential frequency, suggesting a functional role for ALS-IgGs on synaptic signaling. In support, in mice lacking the Ca(V) 2.1 subunit ALS-IgGs showed significantly reduced NMJ immunoreactivity and did not alter spontaneous acetylcholine release. This difference in reactivity was absent when comparing N-type Ca(2+) channel wild-type or null mice. These results are particularly relevant because motoneurons are known to be early pathogenic targets in ALS. Our findings add further evidence supporting autoimmunity as one of the possible mechanisms contributing to ALS pathology. They also suggest that serum autoantibodies in a subset of ALS patients would interact with NMJ proteins down-regulated when P/Q-type channels are absent., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Manganese (Mn) is a trace element known to be essential for maintaining the proper function and regulation of many biochemical and cellular reactions. However, chronic exposure to high levels of Mn in occupational or environmental settings can lead to its accumulation in the brain resulting in a degenerative brain disorder referred to as Manganism. Astrocytes are the main Mn store in the central nervous system and several lines of evidence implicate these cells as major players in the role of Manganism development. In the present study, we employed rat astrocytoma C6 cells as a sensitive experimental model for investigating molecular mechanisms involved in Mn neurotoxicity. Our results show that C6 cells undergo reactive oxygen species-mediated apoptotic cell death involving caspase-8 and mitochondrial-mediated pathways in response to Mn. Exposed cells exhibit typical apoptotic features, such as chromatin condensation, cell shrinkage, membrane blebbing, caspase-3 activation and caspase-specific cleavage of the endogenous substrate poly (ADP-ribose) polymerase. Participation of the caspase-8 dependent pathway was assessed by increased levels of FasL, caspase-8 activation and Bid cleavage. The involvement of the mitochondrial pathway was demonstrated by the disruption of the mitochondrial membrane potential, the opening of the mitochondrial permeability transition pore, cytochrome c release, caspase-9 activation and the increased mitochondrial levels of the pro-apoptotic Bcl-2 family proteins. In addition, our data also shows for the first time that mitochondrial fragmentation plays a relevant role in Mn-induced apoptosis. Taking together, these findings contribute to a deeper elucidation of the molecular signaling mechanisms underlying Mn-induced apoptosis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The effect of molecular oxygen and water on the blue photoluminescence of silicon nanoparticles synthesized by anodic oxidation of silicon wafers and surface functionalized with 2-methyl 2-propenoic acid methyl ester is investigated. The particles of 3 +/- 1 nm diameter and a surface composition of Si(3)O(6)(C(5)O(2)H(8)) exhibit room-temperature luminescence in the wavelength range 300-600 nm upon excitation with 300-400 nm light. The luminescence shows vibronic resolution and high quantum yields in toluene suspensions, while a vibronically unresolved spectrum and lower emission quantum yields are observed in aqueous suspensions. The luminescence intensity, though not the spectrum features, depends on the presence of dissolved O(2). Strikingly, the luminescence decay time on the order of 1 ns does not depend on the solvent or on the presence of O(2). To determine the mechanisms involved in these processes, time-resolved and steady-state experiments are performed. These include low-temperature luminescence, heavy atom effect, singlet molecular oxygen ((1)O(2)) phosphorescence detection, reaction of specific probes with (1)O(2), and determination of O(2) and N(2) adsorption isotherms at 77 K. The results obtained indicate that physisorbed O(2) is capable of quenching nondiffusively the particle luminescence at room temperature. The most probable mechanism for (1)O(2) generation involves the energy transfer from an exciton singlet state to O(2) to yield an exciton triplet of low energy (<0.98 eV) and (1)O(2). In aqueous solutions, excited silicon nanoparticles are able to reduce methylviologen on its surface.

Background: A comparison of the 21-mg NiQuitin patch with other marketed nicotine patches reported significant differences in pharmacokinetic profiles, even among patches of the identical labeled dose strength. The 25-mg Nicorette Invisi patch became available in the United Kingdom at the end of 2008. No published studies have directly compared the pharmacokinetic profile of this new patch with that of the 21-mg NiQuitin patch., Objectives: This study was conducted to compare the single-dose pharmacokinetics of the 21-mg/24-hour patch and the 25-mg/16-hour patch. To determine whether any pharmacokinetic differences might be related to differences in wear time, a post hoc exploratory analysis evaluated the nicotine delivery profiles of the patches under the assumption that the 21-mg patch was removed after 16 rather than 24 hours., Methods: This was a single-center, randomized, open-label, single-dose, 2-way crossover study in healthy adults who smoked >10 cigarettes per day in the 6 months before the study. Eligible subjects were housed at the study center for 2 baseline and 2 treatment sessions; no smoking was permitted during the baseline or treatment sessions. Subjects were allocated to receive either the 21-mg patch (removed after 24 hours) or the 25-mg patch (removed after 16 hours) during the first treatment session, after which they crossed over to the alternative sequence in the second treatment session. Blood samples were obtained at predetermined time points before and after patch application. The primary pharmacokinetic parameter was the AUC(0-infinity), an indication of total nicotine exposure. Secondary pharmacokinetic parameters included AUC(0-t), C(max), and T(max). Post hoc exploratory parameters were the AUC(0-16) and the AUC(0-infinity) assuming a 16-hour application time for the 21-mg patch. The differences in AUC(0-infinity), AUC(0-t), Cmax, AUC(0-16), and AUC(0-infinity) assuming a 16-hour application time for the 21-mg patch were considered significant if the lower limit of the 90% CI for the geometric mean ratio (21 mg:25 mg) was >100%. T(max) values were compared using a signed-rank test. Adverse events were elicited using a standard open-ended question on each day of confinement; spontaneously reported events were also captured. The topical effects of the patch (erythema; edema; extent of erythema/papules/pustules; self-reported pruritus) were assessed by study staff before patch application and 1 and 8 hours after patch application using a 4-point rating scale; any topical effects were recorded as adverse events., Results: Fifty otherwise healthy smokers (29 men, 21 women) were enrolled; 47 (94%) were white. Their mean (SD) age was 31.5 (9.57) years (range, 20-53 years), mean weight was 70.24 (9.56) kg (range, 51.0-95.9 kg), and mean height was 173.0 (8.02) cm (range, 156-194 cm). Subjects reported smoking between 11 and 40 cigarettes per day before the study. The AUC(0-infinity) was significantly higher for the 21-mg patch worn for 24 hours than for the 25-mg patch worn for 16 hours (382.36 vs 243.69 ng/mL . h, respectively; geometric mean ratio: 156.90%; 90% CI, 148.10%-166.23%; P < 0.001). T(max) was reached significantly sooner with the 21-mg patch than with the 25-mg patch (6.0 vs 12.0 hours; P < 0.001). C(max) was significantly higher for the 21-mg patch compared with the 25-mg patch (18.34 vs 16.56 ng/mL; geometric mean ratio: 110.72%; 90% CI, 104.82%-116.94%; P < 0.01). The exploratory analyses suggested that the 21-mg patch applied for 16 hours may provide greater total nicotine exposure than the 25-mg patch applied for 16 hours. Although most subjects reported adverse events (75.0% with the 21-mg patch, 89.8% with the 25-mg patch), the majority of these events were mild., Conclusions: In this single-dose study in adult smokers, the 21-mg patch was associated with significantly greater nicotine exposure compared with the 25-mg patch. The 21-mg patch provided a maximal nicotine concentration faster than did the 25-mg patch.

Manganese induces the central nervous system injury leading to manganism, by mechanisms not completely understood. Chronic exposure to manganese generates oxidative stress and induces the mitochondrial permeability transition. In the present study, we characterized apoptotic cell death mechanisms associated with manganese toxicity in rat cortical astrocytes and demonstrated that (i) Mn treatment targets the mitochondria and induces mitochondrial membrane depolarization followed by cytochrome c release to the cytoplasm, (ii) Mn induces both effector caspases 3/7 and 6 as well as PARP-1 cleavage and (iii) Mn shifts the balance of cell death/survival of Bcl-2 family proteins to favor the apoptotic demise of astrocytes. Our model system using cortical rat astrocytes treated with Mn would emerge as a good tool for investigations aimed to elucidate the role of apoptosis in manganism.

Objective: Controversy exists over the effect of tobacco deprivation in nicotine-dependent individuals and the efficacy of nicotine in reversing performance decrements. This study's aim was to assess the efficacy of nicotine (4-mg lozenge) versus placebo on aspects of cognitive and psychomotor performance, mood, and withdrawal symptoms in male and female established smokers., Methods: Male and female smokers (N = 22; mean age, 28.8 years), with a smoking history of more than 1 year and time to first cigarette of less than 30 minutes upon waking, were enrolled. Baseline measures were obtained at 17 hours of abstinence. At 18-hour abstinence, nicotine or placebo was administered every 2 hours over an 8-hour period. Cognitive and psychomotor performance measurements were taken 30 minutes after dose. Cognitive test battery included Rapid Visual Information Processing, Continuous Tracking Task, Critical Flicker Fusion, Choice Reaction Time, Stroop Test, and Sternberg's Short-term Memory Scanning Task. Withdrawal (Modified Minnesota Withdrawal Scale) and mood (Positive and Negative Affect Schedule) were also assessed. A mixed-models analysis of covariance was performed., Results: Compared with placebo nicotine (4 mg) significantly improved vigilance, divided attention, executive functioning, working memory, and sensorimotor performance in abstinent volunteers (P < or = 0.05). Withdrawal symptoms including craving, difficulty concentrating, irritability, and restlessness were also attenuated, and affective state was improved after nicotine 4 mg administration., Conclusions: Compared with placebo, nicotine (4 mg) improved measures of vigilance, memory, and attention; improved mood; and reduced withdrawal symptoms. These findings suggest that repeated nicotine replacement therapy over a period of 8 hours can improve cognitive deficits associated with nicotine withdrawal.

The 'at least as good as' criterion, introduced by Laster and Johnson for a continuous response variate, is developed here for applications with dichotomous data. This approach is adaptive in nature, as the margin of non-inferiority is not taken as a fixed difference; it varies as a function of the positive control response. When the non-inferiority margin is referenced as a high fraction of the positive control response, the procedure is seen to be uniformly more efficient than the fixed margin approach, yielding smaller sample sizes when sizing non-inferiority trials under identically specified conditions. Extending this method to proportions is straightforward, but highlights special considerations in the design of non-inferiority trials versus superiority trials, including potential trade-offs in statistical efficiency and interpretability., (Copyright 2005 John Wiley & Sons, Ltd.)

1 We have previously demonstrated that nitric oxide (NO) triggers CD34(+)-derived megakaryocyte apoptosis. We here show that prostacyclin (PGI(2)) inhibits PAPA/NO-induced megakaryocyte death detected by fluorescent microscopy and flow cytometry. 2 The cAMP-specific phosphodiesterase inhibitor, Ro 20-1724, and the permeable analog dibutyryl-cAMP also delayed apoptosis. PGI(2) effect was fully prevented when adenylyl cyclase activity was suppressed by SQ 22536, and partially reversed by the permeable protein kinase A inhibitor PKI 14-22 amide. ELISA showed that while both PGI(2) and NO alone or synergistically raised cAMP, only NO was able to increase intracellular cGMP levels. 3 Treatment of megakaryocytes with PGI(2) abolished both basal and NO-raised cGMP levels. Addition of 8-pCPT-cGMP or activation of soluble guanylyl cyclase by BAY 41-2272 induced cell death in a concentration-dependent manner, and ODQ, an inhibitor of guanylyl cyclase, prevented both PAPA/NO- or BAY 41-2272-induced apoptosis. Specific cGMP phosphodiesterase inhibition by Zaprinast or suppression of adenylyl cyclase by SQ 22536 enhanced the PAPA/NO proapoptotic effect. 4 PGI(2) completely inhibited NO-mediated generation and the increased activity of the cleaved form of caspase-3. 5 In conclusion, our results demonstrate that contrary to their well-known direct and synergistic inhibitory effects on platelets, PGI(2) and NO regulate opposite megakaryocyte survival responses through a delicate balance between intracellular cyclic nucleotide levels and caspase-3 activity control.

During oxidative stress, cell apoptosis is promoted through the mitochondrial death pathway. Increased reactive oxygen species (ROS) are linked to excess cell loss and mediate the induction of apoptosis in various cell types. However, the role of ROS in the apoptotic pathway has not been clearly established. The aims of this study were to investigate the biochemical and morphological responses of rat astrocytes to hydrogen peroxide-mediated cell death and to define the role that melatonin might play in the apoptotic cascade. Hydrogen peroxide (H2O2; 0.1-1.0 mM) significantly reduced cell viability. Astrocyte death was associated with enhanced ROS production in a dose-dependent manner, as measured by 2',7'-dichloro-fluorescein fluorescence. H2O2-induced cell death was found to be mediated through an apoptotic pathway as treated cells exhibited cell shrinkage, nuclear condensation and marked DNA fragmentation. H2O2 also triggered caspase-3 activation and Bax expression. The ability of different antioxidants to prevent H2O2-induced apoptosis was examined by pre-incubating rat astrocytes with N-acetylcysteine (10 mM), glutathione (0.5 mM) or melatonin (0.1 mM and 10 nM). Results showed that N-acetylcysteine and glutathion can protect astrocytes against ROS accumulation and caspase-3 activation, whereas 0.1 mM melatonin can inhibit H2O2-induced apoptosis by regulating Bax expression and by inhibiting caspase-3 activation. Antiapoptotic effect of 10 nM melatonin associated to inhibition of Bax expression, give rise to new therapeutic approaches.

Accumulation of delta-aminolevulinic acid (ALA), as it occurs in acute intermittent porphyria (AIP), is the origin of an endogenous source of reactive oxygen species (ROS), which can exert oxidative damage to cell structures. In the present work we examined the ability of different antioxidants to revert ALA-promoted damage, by incubating mouse astrocytes with 1.0 mM ALA for different times (1-4 hr) in the presence of melatonin (2.5 mM), superoxide dismutase (25 units/mL), catalase (200 units/mL) or glutathione (0.5 mM). The defined relative index [(malondialdehyde levels/accumulated ALA) x 100], decreases with incubation time, reaching values of 76% for melatonin and showing that the different antioxidants tested can protect astrocytes against ALA-promoted lipid peroxidation. Concerning porphyrin biosynthesis, no effect was observed with catalase and superoxide dismutase whereas increases of 57 and 87% were obtained with glutathione and melatonin, respectively, indicating that these antioxidants may prevent the oxidation of porphobilinogen deaminase, reactivating so that the AIP genetically reduced enzyme. Here we showed that ALA induces cell death displaying a pattern of necrosis. This pattern was revealed by loss of cell membrane integrity, marked nuclear swelling and double labeling with annexin V and propidium iodide. In addition, no caspase 3-like activity was detected. These findings provide the first experimental evidence of the involvement of ALA-promoted ROS in the damage of proteins related to porphyrin biosynthesis and the induction of necrotic cell death in astrocytes. Interestingly, melatonin decreases the number of enlarged nuclei and shows a protective effect on cellular morphology.

It is well known that porphyrins cause a toxic light-mediated effect due to their capability to generate free radicals. Several reports have proved that melatonin is a potent free radical scavenger. The aim of this work has been to study the ability of melatonin to prevent the cell damage caused by porphyrins in the Harderian gland of female Syrian hamsters. Cell injury was evaluated estimating the percentage of damaged cells found in the gland and analyzing the degree of this damage at ultrastructural level. To explain the mechanism by which this hormone could prevent the cell damage caused by porphyrins, its capability to both decrease porphyrin synthesis and increase the mRNA levels for antioxidant enzymes was evaluated. Our results demonstrate that melatonin administration decreases the percentage of damaged cells, porphyrin synthesis, and aminolevulinate synthase (ALA-S) mRNA levels and increases the mRNA levels for manganese superoxide-dismutase and copper-zinc superoxide dismutase. When observed under an electron microscope, the lesions in the clear cells of the treated females were much less severe than in the corresponding cells of the control animals. Melatonin exerts a cytoprotective effect by inhibiting the ALA-S gene expression (and so porphyrin synthesis) and by raising the mRNA levels for several antioxidant enzymes.

It is known that the Harderian gland of male Syrian hamster synthesizes a much smaller amount of porphyrins than the gland of the female and that castration greatly increases this synthesis. We have studied in this experimental model the behavior of the different classes of secretory cells and their role in the synthesis of porphyrins, attempting to clarify the participation of these compounds in the cell damage leading to the formation of clear cells previously described in the gland of females. We have also investigated the mechanism underlying the death of these secretory cells after porphyrin accumulation (necrosis vs apoptosis). To achieve this, we have utilized the following techniques: (a) morphometrical; (b) ultrastructural; (c) biochemical (fluorescence spectrophotometry); and (d) molecular (DNA nick-end labeling in methacrylate sections and dot blot analysis). The glands from male hamsters (serving as control) present a very low rate of damaged cells that progressively rises after castration. This rise runs parallel to that of porphyrin synthesis, porphyrin deposits, and the decrease of Type II secretory cells. The damage and subsequent death of the secretory cells in the gland is produced by the deposit of porphyrins in the mitochondrial membrane. This porphyrin accumulation leads to a complete mitochondrial destruction that finally results in cell death and its secretion into the lumen. We finally conclude that this event is not a physiological cell death (apoptosis) but the consequence of the toxic accumulation of porphyrins (necrosis).

The response of nerve cells to high exogenous aminolevulinic acid (ALA) concentrations was studied by examining the changes in its uptake and in porphyrin biosynthesis. ALA was shown to be taken up by cerebral cortex particles by a non-saturable process. As opposed to other previously described experimental systems, it was also observed that 84-87% of porphyrins formed was found within the cells. Exposure of cerebral cortex particles to high exogenous ALA (0.8-4.0 mM) showed that ALA can be accumulated in relatively high concentrations in brain cells (21.04 +/- 1.05 nmol/mg protein). Under these experimental conditions, porphyrin biosynthesis was found to be markedly inhibited (52%). 2.4 mM ALA caused an initial stimulation of glucose uptake after 1 hr incubation and a later fall to below control values, being consistent with the fact that acute porphyric crisis could be precipitated by the action of ALA on energy metabolism. ALA toxicity could be due both to its accumulation in the cells and to deficient heme concentrations, with an additional effect on glucose metabolism. These findings provide the basis for a useful brain tissue model to investigate the nature of the metabolic mechanisms occurring in acute intermittent porphyria (AIP) patients.

Background: The Harderian glands of female Syrian hamsters contain very high concentrations of protoporphyrin (in the range of micrograms per mg of tissue) which accumulate in the tubulo-alveoli of the gland. We have studied the process of synthesis, accumulation, and secretion of this cyclic compound by the secretory cells of the hamster Harderian glands., Methods: The animals used were female Syrian hamster of 15, 35, 75, 180, and 360 days of age. Items first examined were (1) percentage of the "clear cells," (2) area occupied by intraluminal porphyrins, and (3) histological characteristics of "clear cells" by light and transmission electron microscopy (TEM). In a second study the total content of porphyrins was determined. Finally, the levels of mRNA for the enzyme aminolevulinate synthase (ALV-S) were measured., Results: In the glands of female hamsters, both the tissue concentration and the intraluminal area occupied by protoporphyrin correlate with the appearance of a special type of cell (clear cells) which show signs of cell degeneration. In addition, the expression of the gene for ALV-S, which is the limiting enzyme in porphyrin production, also parallels the relative number of clear cells. Analyzed under TEM, these clear cells display dilated mitochondria and short and swollen endoplasmic reticulum cisternae. In a late phase of necrosis, the nuclear envelope appears disorganized with scarce chromatin. The mitochondria undergo complete destruction, resulting in electron-dense bacillary formations which progressively coalesce in large and dense areas of protoporphyrin. The cell dies after this accumulation, being secreted by a "cytogen" mechanism., Conclusions: In view of our results, the Harderian gland of female Syrian hamster may provide a useful model for the study of the mechanism by which the anomalous accumulation of protoporphyrin induces cell damage in human protoporphyria.

Porphobilinogen-deaminase (PBG-D) and PBG-D-isomerase complex (PBG-D-I) from pig liver were isolated and partially purified. Uroporphyrinogen I and III formation was found to be linear with time and protein concentration. Optimal pH was about 7.4 and 7.6-7.8 for PBG-D and PBG-D-I complex, respectively. Some properties of the isolated enzymes were studied. Molecular mass determination gave a value of 40,000 Da for PBG-D and 50,000 Da for the complex. Both enzymes exhibited classical Michaelis-Menten kinetics. Km and Vmax parameters were estimated. The effect of several divalent cations, ammonia and thiol reagents was also investigated. The differential action of some of these chemicals on PBG-D and PBG-D-I system would suggest that PBG-D and isomerase may not be only physically adjacent but actually associated.

The detection and accumulation of tetrapyrrole intermediates synthesized by the action of bovine liver porphobilinogen deaminase immobilized to Sepharose 4B is reported. Employing Sepharose-deaminase preparations, two phases in uroporphyrinogen I synthesis as a function of time were observed, suggesting the accumulation of free and enzyme-bound intermediates, the concentration and distribution of which were time dependent. The deaminase-bound intermediate behaves as a substrate in uroporphyrinogen I synthesis whereas the free intermediates produce enzyme inhibition. The tetrapyrrole intermediate bound to the Sepharose-enzyme is removed from the protein by the binding of porphobilinogen. Free as well as enzyme-bound intermediates are shown to be substrates for cosynthetase with formation of 80% uroporphyrinogen III.

Rhodopseudomonas palustris uroporphyrinogen I synthetase (URO-S) has been chemically attached to Sepharose 4B and some of its properties have been studied. When 7-8 mg protein/ml activated Sepharose was used, immobilized URO-S retained 45% of the activity of the original soluble preparation, with a coupling yield of 66% after a period of 15 h. Optimal incubation conditions for the activity of gel-enzyme were determined. Unlike the soluble enzyme, the Sepharose-bound URO-S showed a biphasic substrate saturation curve, indicating that a protein conformational change had occurred during the process of immobilization. Immobilized URO-S stored at 4 degrees C for 35 days retained 90% of activity and when repeatedly used, up to 5 times, retained 48% of the original activity. Attachment of URO-S to Sepharose led to an enhanced thermal stability.

A simpler method for purifying human red cell deaminase, using a mixture of n-butanol and chloroform, which denatures hemoglobin, followed by ammonium sulphate fractionation, heat treatment, Sephadex G-100 and DEAE-cellulose chromatography, yielding a 3400 fold purified enzyme is described. Some properties of purified deaminase were studied. The enzyme seems to have a strict requirement for oxygen, neither PBG consumption nor uroporphyrinogens formation were measured under anaerobiosis. Uroporphyrinogens formation was linear with both protein and time over a wide range of enzyme concentration and up to 2 h. The optimum pH was 7.4 and the mol. wt was 40,000 +/- 4000. The enzyme was heat-stable and increased its activity by heating. Ammonium and hydroxylamine ions inhibited the reaction. K+ and Na+ ions did not greatly affect activity, while most divalent cations tested significantly diminished uroporphyrinogen formation and to a lesser degree PBG consumption. Direct plots of velocity against PBG concentration were hyperbolic, however double-reciprocal plots were non-linear, Hill plots gave an n value of 2 and Eadie plots were bell-shaped, indicating the existence of weakly positive cooperative effect between 2 binding sites for PBG per molecule of deaminase.

1. Porphobilinogenase (PBGase) from Rp. palustris has been isolated and some properties of a partially purified fraction were studied. 2. PBGase has an optimum pH of 7.4 when activity was expressed in terms of porphyrins formed and two pH maxima at 7.4 and 8.5 when activity was based on the amount of PBG consumed. 3. Cyclotetramerization rate and distribution of reaction products were not affected either by the presence or absence of oxygen. 4. Two PBGase active species of mol. wt 115,000 and 50,000 were found, by means of gel filtration through a calibrated Sephadex G-100 column. 5. Kinetic data show the existence of positive cooperative effects for porphyrin formation, while a hyperbolic behaviour for PBG consumption was observed.