Javier del Pino

A review of metal-catalyzed molecular damage: Protection by melatonin

Metal exposure is associated with several toxic effects; herein, we review the toxicity mechanisms of cadmium, mercury, arsenic, lead, aluminum, chromium, iron, copper, nickel, cobalt, vanadium, and molybdenum as these processes relate to free radical generation. Free radicals can be generated in cells due to a wide variety of exogenous and endogenous processes, causing modifications in DNA bases, enhancing lipid peroxidation, and altering calcium and sulfhydryl homeostasis. Melatonin, an ubiquitous and pleiotropic molecule, exerts efficient protection against oxidative stress and ameliorates oxidative/nitrosative damage by a variety of mechanisms. Also, melatonin has a chelating property which may contribute in reducing metal‐induced toxicity as we postulate here. The aim of this review was to highlight the protective role of melatonin in counteracting metal‐induced free radical generation. Understanding the physicochemical insights of melatonin related to the free radical scavenging activity and the stimulation of antioxidative enzymes is of critical importance for the development of novel therapeutic strategies against the toxic action of these metals.

Higher sensitivity to cadmium induced cell death of basal forebrain cholinergic neurons: a cholinesterase dependent mechanism.

Cadmium is an environmental pollutant, which is a cause of concern because it can be greatly concentrated in the organism causing severe damage to a variety of organs including the nervous system which is one of the most affected. Cadmium has been reported to produce learning and memory dysfunctions and Alzheimer like symptoms, though the mechanism is unknown. On the other hand, cholinergic system in central nervous system (CNS) is implicated on learning and memory regulation, and it has been reported that cadmium can affect cholinergic transmission and it can also induce selective toxicity on cholinergic system at peripheral level, producing cholinergic neurons loss, which may explain cadmium effects on learning and memory processes if produced on central level. The present study is aimed at researching the selective neurotoxicity induced by cadmium on cholinergic system in CNS. For this purpose we evaluated, in basal forebrain region, the cadmium toxic effects on neuronal viability and the cholinergic mechanisms related to it on NS56 cholinergic mourine septal cell line. This study proves that cadmium induces a more pronounced, but not selective, cell death on acetylcholinesterase (AChE) on cholinergic neurons. Moreover, MTT and LDH assays showed a dose dependent decrease of cell viability in NS56 cells. The ACh treatment of SN56 cells did not revert cell viability reduction induced by cadmium, but siRNA transfection against AChE partially reduced it. Our present results provide new understanding of the mechanisms contributing to the harmful effects of cadmium on the function and viability of neurons, and the possible relevance of cadmium in the pathogenesis of neurodegenerative diseases.

Cadmium-induced cell death of basal forebrain cholinergic neurons mediated by muscarinic M1 receptor blockade, increase in GSK-3β enzyme, β-amyloid and tau protein levels

Abstract Cadmium is a neurotoxic compound which induces cognitive alterations similar to those produced by Alzheimer’s disease (AD). However, the mechanism through which cadmium induces this effect remains unknown. In this regard, we described in a previous work that cadmium blocks cholinergic transmission and induces a more pronounced cell death on cholinergic neurons from basal forebrain which is partially mediated by AChE overexpression. Degeneration of basal forebrain cholinergic neurons, as happens in AD, results in memory deficits attributable to the loss of cholinergic modulation of hippocampal synaptic circuits. Moreover, cadmium has been described to activate GSK-3β, induce Aβ protein production and tau filament formation, which have been related to a selective loss of basal forebrain cholinergic neurons and development of AD. The present study is aimed at researching the mechanisms of cell death induced by cadmium on basal forebrain cholinergic neurons. For this purpose, we evaluated, in SN56 cholinergic mourine septal cell line from basal forebrain region, the cadmium toxic effects on neuronal viability through muscarinic M1 receptor, AChE splice variants, GSK-3β enzyme, Aβ and tau proteins. This study proves that cadmium induces cell death on cholinergic neurons through blockade of M1 receptor, overexpression of AChE-S and GSK-3β, down-regulation of AChE-R and increase in Aβ and total and phosphorylated tau protein levels. Our present results provide new understanding of the mechanisms contributing to the harmful effects of cadmium on cholinergic neurons and suggest that cadmium could mediate these mechanisms by M1R blockade through AChE splices altered expression.

Wnt Signaling Pathway, a Potential Target for Alzheimer's Disease Treatment, is Activated by a Novel Multitarget Compound ASS234

Figure 1 (A) Represents chemical structure of ASS234. (B–G) Shows results from real-time PCR arrays targeting select genes after ASS234 (5 lM) treatment. (H) Representative table of data, in which specific gene expression was compared with controls [cells treated with DMSO (0.1%) were the negative control]. Each bar represents mean SEM of six independent experiments. ACTB was used as an internal control. ***P < 0.001, significantly different from controls.

Acute and long-term exposure to chlorpyrifos induces cell death of basal forebrain cholinergic neurons through AChE variants alteration

Chlorpyrifos (CPF) is one of the most widely used organophosphates insecticides that has been reported to induce cognitive disorders both after acute and repeated administration similar to those induced in Alzheimers disease (AD). However, the mechanisms through which it induces these effects are unknown. On the other hand, the cholinergic system, mainly basal forebrain cholinergic neurons, is involved in learning and memory regulation, and an alteration of cholinergic transmission or/and cholinergic cell loss could induce these effects. In this regard, it has been reported that CPF can affect cholinergic transmission, and alter AChE variants, which have been shown to be related with basal forebrain cholinergic neuronal loss. According to these data, we hypothesized that CPF could induce basal forebrain cholinergic neuronal loss through cholinergic transmission and AChE variants alteration. To prove this hypothesis, we evaluated in septal SN56 basal forebrain cholinergic neurons, the CPF toxic effects after 24h and 14 days exposure on neuronal viability and the cholinergic mechanisms related to it. This study shows that CPF impaired cholinergic transmission, induced AChE inhibition and, only after long-term exposure, increased CHT expression, which suggests that acetylcholine levels alteration could be mediated by these actions. Moreover, CPF induces, after acute and long-term exposure, cell death in cholinergic neurons in the basal forebrain and this effect is independent of AChE inhibition and acetylcholine alteration, but was mediated partially by AChE variants alteration. Our present results provide a new understanding of the mechanisms contributing to the harmful effects of CPF on neuronal function and viability, and the possible relevance of CPF in the pathogenesis of neurodegenerative diseases.

Toxicological and pharmacological evaluation, antioxidant, ADMET and molecular modeling of selected racemic chromenotacrines {11-amino-12-aryl-8,9,10,12-tetrahydro-7H-chromeno[2,3-b]quinolin-3-ols} for the potential prevention and treatment of Alzheimer’s disease

The pharmacological analysis of racemic chromenotacrines (CT) 1-7, bearing the 11-amino-12-aryl-8,9,10,12-tetrahydro-7H-chromeno[2,3-b]quinolin-3-ol ring skeleton, in a series of experiments targeted to explore their potential use for the treatment of Alzheimers disease (AD), is reported. The toxicological evaluation showed that among all these chromenotacrines, CT6 is much less hepatotoxic than tacrine in a range of concentrations from 1 to 300 μM, measured as cell viability in HepG2 cells. Moreover, CT6 did not significantly increase lactate dehydrogenase, aspartate transaminase, and alanine transaminase release in HepG2 cells. Besides, CT6 treatment exerts a high protective effect against the lipid peroxidation induced after H₂O₂-treated SH-SY5Y cells, in a concentration-dependent manner. CT6 showed an excellent antioxidant profile in the AAPH test, and protects against the decrease in cell viability induced by respiratory chain inhibitors (Oligomicyn A/Rotenone) and NO donors in neuronal cultures. This effect could be due to a mixed antiapoptotic and antinecrotic neuroprotective effect at low and intermediate CT6 concentrations, respectively. CT1-7 are potent and selective inhibitors of EeAChE in the submicromolar range. CT3 [IC₅₀ (EeAChE) = 0.007 ± 0.003 μM], and CT6 [IC₅₀ (EeAChE) = 0.041 ± 0.001 μM] are the most potent AChE inhibitors. Kinetic studies on the non-toxic chromenotacrine CT6 showed that this compound behaves as a non-competitive inhibitor (Ki = 0.047 ± 0.003 μM), indicating that CT6 binds at the peripheral anionic site, a fact confirmed by molecular modeling analysis. In silico ADMET analysis showed also that CT6 should have a moderate BBB permeability. Consequently, non-toxic chromenotacrine CT6 can be considered as an attractive multipotent molecule for the potential treatment of AD.

Neuroprotective or neurotoxic effects of 4-aminopyridine mediated by KChIP1 regulation through adjustment of Kv 4.3 potassium channels expression and GABA-mediated transmission in primary hippocampal cells.

4-Aminopyridine (4-AP) is a potassium channel blocker used for the treatment of neuromuscular disorders. Otherwise, it has been described to produce a large number of adverse effects among them cell death mediated mainly by blockage of K(+) channels. However, a protective effect against cell death has also been described. On the other hand, Kv channel interacting protein 1 (KChIP1) is a neuronal calcium sensor protein that is predominantly expressed at GABAergic synapses and it has been related with modulation of K(+) channels, GABAergic transmission and cell death. According to this KChIP1 could play a key role in the protective or toxic effects induced by 4-AP. We evaluated, in wild type and KChIP1 silenced primary hippocampal neurons, the effect of 4-AP (0.25μM to 2mM) with or without semicarbazide (0.3M) co-treatment after 24h and after 14 days 4-AP alone exposure on cell viability, the effect of 4-AP (0.25μM to 2mM) on KChIP1 and Kv 4.3 potassium channels gene expression and GABAergic transmission after 24h treatment or after 14 days exposure to 4-AP (0.25μM to1μM). 4-AP induced cell death after 24h (from 1mM) and after 14 days treatment. We observed that 4-AP modulates KChIP1 which regulate Kv 4.3 channels expression and GABAergic transmission. Our study suggests that KChIP1 is a key gene that has a protective effect up to certain concentration after short-term treatment with 4-AP against induced cell injury; but this protection is erased after long term exposure, due to KChIP1 down-regulation predisposing cell to 4-AP induced damages. These data might help to explain protective and toxic effects observed after overdose and long term exposure.

Toxicogenomic profile of apoptotic and necrotic SN56 basal forebrain cholinergic neuronal loss after acute and long-term chlorpyrifos exposure

Chlorpyrifos (CPF) is an organophosphate insecticide reported to induce, both after acute and repeated exposure, learning and memory dysfunctions, although the mechanism is not completely known. CPF produces basal forebrain cholinergic neuronal loss, involved on learning and memory regulation, which could be the cause of such cognitive disorders. This effect was reported to be induced through apoptotic process, partially mediated by AChE overexpression, although neuronal necrosis was also described after CPF exposure. Accordingly, we hypothesized that CPF induces apoptotic and necrotic basal forebrain cholinergic cell death. We evaluated, in septal SN56 basal forebrain cholinergic neurons, the CPF effect after 24h and 14days exposure on apoptosis and necrosis induction and the apoptotic and necrotic gene expression pathways. This study shows that CPF induces, after acute and long-term exposure, apoptosis and necrosis, partially mediated through AChE overexpression. Evaluation of cell death pathways supports the necrosis and apoptosis data and revealed that some genes are altered at lower concentrations than those at which the effects observed are produce and below the No Observed Adverse Effect Level (NOAEL). The present finding suggests that the use of gene expression profile could be a more sensitive and accurate way to determine the CPFs NOAEL.

Upregulation of Antioxidant Enzymes by ASS234, a Multitarget Directed Propargylamine for Alzheimer's Disease Therapy

Alzheimer’s disease (AD), a major neurodegenerative disorder, is a matter of health concern because of the lack of effective drugs to treat it. Thus, our group has recently identified N-((5-(3-(1-benzylpiperidin-4-yl) propoxy)-1-methyl-1H-indol-2-yl)methyl)-Nmethylprop-2-yn-1-amine (ASS234, Figure 1), a new multitarget propargylamine for the potential treatment and prevention of AD, and described that this compound is able to act simultaneously both as a reversible inhibitor of human acetylcholinesterase (AChE)/butyrylcholinesterase (BuChE) and as an irreversible inhibitor of human monoamine oxidase A/B [1]. In addition, ASS234 has been reported to inhibit in vitro b-amyloid (Αb)1–42 aggregation induced by AChE and Αb1–40 self-aggregation, thus limiting the formation of fibrillar and oligomeric species and reducing Αb1–42-mediated toxicity [2]. However, the mechanism underlying its neuroprotective effect remains unclear still. On the other hand, oxidative stress has been implicated as a possible mechanism in the etiology and progression of AD [3]. In this regard, our previous studies showed that ASS234 is a potent antioxidant due to its free radical scavenging capacity, preventing, also, the Ab1–42-induced depletion of antioxidant enzymes such as catalase and superoxide dismutase-1 (SOD-1) [2], which are mainly regulated by the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway [4]. Using Ingenuity pathways analysis (IPA) (Ingenuity H Systems, Redwood City, CA, USA) to evaluate the Nrf2 pathway, we found that this molecule regulates many antioxidant enzymes (Figure 2), such as NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione reductase (GSR), thioredoxin reductase 1 (TRXR1), sequestosome 1 (SQSTM1), which have been described to be affected in AD disease [3,5–8]. According to this information, we tested whether ASS234 induced the gene expression of the Nrf2related antioxidant enzymes CAT, SOD-1, NQO1, GCLC, GCLM, GSR, TRXR1 and SQSTM1 in SH-SY5Y cells as a mechanism of neuroprotection. Relative changes in gene expression were calculated using the Ct (cycle threshold) method. The expression data are shown as actual change multiples. At least six replicates for each experimental condition were carried out, and the presented results were representative of these replicates. Data are represented as means standard error of the mean (SEM). Comparisons between experimental and control groups were performed by Student’s t-test. Statistical difference was accepted when P ≤ 0.05. Statistical analysis of data was carried out by computer using GraphPad Prism software v. 5.02 (La Jolla, CA, USA). The analysis performed after incubating ASS234 (5 lM) in SHSY5Y cells during a 24-h period showed that this molecule was able to induce the gene expression of CAT, SOD1, GCLC, GCLM, GSR, QSTM1, TXNRD1 and NQO1 (Figure 1) in a significant manner. The target genes studied have been involved in the development of AD. In this context, a decrease in GSH levels and GSR expression and subsequent GSSG production has been linked to neuronal loss in AD [3]. The first and rate-limiting step in GSH synthesis is catalyzed by glutamate cysteine ligase (GCL), which is a heterodimeric protein composed of catalytic (GCLC) and modifier (GCLM) subunits that are expressed from different genes [9]. Moreover, immunohistochemical analysis of frontal cortex samples from patients with AD revealed an overall decrease in TXNRD1 levels in neurons, but an increase in TXNRD1 immunoreactivity in astrocytes when compared with age-matched control brains [5]. Exogenous addition of thioredoxin or TXNRD1 has been shown to protect in vitro cell cultures from Ab toxicity [5]. Furthermore, it was also reported that NQO1, a flavoenzyme that is important in maintaining the

Melatonin as potential candidate to prevent the toxicity induced by chemical warfare agents

crosses easily cell membranes, including blood brain barrier (Costa et al. 1995), reaching all subcellular compartments and allowing it to be administered either orally or intravenously. Taking into account melatonin’s low toxicity and that patients treated with high doses of melatonin do not experience any harmful side effects (Seabra et al. 2000), its potential spectrum for improving medical treatment against CWAs seems to be wide. The toxic effects of organophosphates (OPs) compounds such as the nerve agents are not limited to acetylcholinesterase inhibition. Oxidative stress is a major mechanism in the pathophysiology of several toxins and diseases. In both acute and chronic OPs toxicity, changes in antioxidant enzymes occur, and lipid peroxidation increases in many organs, especially in the brain. Moreover, an important neuro-inflammation process occurs after OPs exposure (Collombet 2011). Recent insights and new therapeutic perspectives about melatonin’s anti-inflammatory properties and molecular aspects have been recently reviewed (Mauriz et al. 2012). However, it would be important to determine if the neuroprotective efficacy of melatonin against OPs could be effective as prophylactic and/or as post-exposure treatment, because some drugs exert higher protective activity when given under one set of conditions versus the other. Oxidative stress is a key element in the pathogenesis of blister agent toxicity. Some studies have suggested that oxidative stress due to reactive oxygen species (ROS) play an important role in the toxic mechanism of action of mustard gas action (Jafari 2007). However, the powerful nitrosating agent ONOO is involved in the initial detrimental effects of all mustards (Korkmaz et al. 2006). Nowadays, both melatonin and its metabolites have important advantages when compared to “classical antioxidants” including iNOS inhibition and ONOO scavenging properties against mustard’s induced acute toxicity (Sadir et al. 2007). In a recent Chemical Warfare Agents (CWAs) are substances that can kill, injure or incapacitate people because of its pathophysiological effects. Many CWAs are able to generate free radicals and derived reactants, excitotoxicity and inflammatory processes; as consequence, they can produce neurological symptoms and damage in different organs. Nowadays, total immediate decontamination after CWAs exposure is difficult to achieve, and there are no completely effective antidotes or treatments against these agents. In this complex scenario, we think that a broadspectrum multipotent molecule, such as melatonin, would be an interesting antidote and its use could provide a good strategy to counteract CWAs-induced injury. Melatonin (N-acetyl-5-methoxytryptamine), a versatile and pleiotropic molecule that modulates and controls oxidative stress by several mechanisms (Hengstler and Bolt 2007), is a well-known antioxidant and free radical scavenger (Tan et al. 1993; Reiter et al. 2001). Melatonin is also involved in several and important functions such as vasomotor control and adrenal function, possesses antiexcitatory actions, regulates immune function and energy metabolism, including anti-inflammatory properties (Hardeland et al. 2011). Melatonin is highly lipophilic and consequently