Tamara Lazarević-Pašti

Acetylcholinesterase Inhibitors: Pharmacology and Toxicology

Acetylcholinesterase is involved in the termination of impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine in numerous cholinergic pathways in the central and peripheral nervous systems. The enzyme inactivation, induced by various inhibitors, leads to acetylcholine accumulation, hyperstimulation of nicotinic and muscarinic receptors, and disrupted neurotransmission. Hence, acetylcholinesterase inhibitors, interacting with the enzyme as their primary target, are applied as relevant drugs and toxins. This review presents an overview of toxicology and pharmacology of reversible and irreversible acetylcholinesterase inactivating compounds. In the case of reversible inhibitors being commonly applied in neurodegenerative disorders treatment, special attention is paid to currently approved drugs (donepezil, rivastigmine and galantamine) in the pharmacotherapy of Alzheimer’s disease, and toxic carbamates used as pesticides. Subsequently, mechanism of irreversible acetylcholinesterase inhibition induced by organophosphorus compounds (insecticides and nerve agents), and their specific and nonspecific toxic effects are described, as well as irreversible inhibitors having pharmacological implementation. In addition, the pharmacological treatment of intoxication caused by organophosphates is presented, with emphasis on oxime reactivators of the inhibited enzyme activity administering as causal drugs after the poisoning. Besides, organophosphorus and carbamate insecticides can be detoxified in mammals through enzymatic hydrolysis before they reach targets in the nervous system. Carboxylesterases most effectively decompose carbamates, whereas the most successful route of organophosphates detoxification is their degradation by corresponding phosphotriesterases.

Inhibition of myeloperoxidase and antioxidative activity of Gentiana lutea extracts

The aim of this study was to investigate the inhibitory activity of Gentiana lutea extracts on the enzyme myeloperoxidase (MPO), as well as the antioxidant activity of these extracts and their correlation with the total polyphenol content. Extracts were prepared using methanol (100%), water and ethanol aqueous solutions (96, 75, 50 and 25%v/v) as solvents for extraction. Also, isovitexin, amarogentin and gentiopicroside, pharmacologically active constituents of G. lutea were tested as potential inhibitors of MPO. Antioxidant activity of extracts was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging test and also using cyclic voltammetry (CV). Among all extracts, the antioxidant capacity of 50% ethanol aqueous extract was the highest, both when measured using the DPPH test, with IC(50)=20.6 μg/ml, and when using CV. Also, 50% ethanol extract, showed the best inhibition of MPO activity in comparison with other extracts. In the group of the selected G. lutea constituents, gentiopicroside has proved to be the strongest inhibitor of MPO, with IC(50)=0.8 μg/ml. Also, the concentration of G. lutea constituents were determined in all extracts, using Ultra Performance Liquid Chromatography (UPLC).

Chronic administration of fluoxetine or clozapine induces oxidative stress in rat liver: a histopathological study.

Chronic exposure to stress contributes to the etiology of mood disorders, and the liver as a target organ of antidepressant and antipsychotic drug metabolism is vulnerable to drug-induced toxicity. We investigated the effects of chronic administration of fluoxetine (15mg/kg/day) or clozapine (20mg/kg/day) on liver injury via the measurement of liver enzymes, oxidative stress and histopathology in rats exposed to chronic social isolation (21days), an animal model of depression, and controls. The activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the liver content of carbonyl groups, malonyldialdehyde (MDA), reduced glutathione (GSH), cytosolic glutathione S-transferase (GST) and nitric oxide (NO) metabolites were determined. We also characterized nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2) and CuZn-superoxide dismutase (CuZnSOD) protein expression as well as histopathological changes. Increased serum ALT activity in chronically-isolated and control animals treated with both drugs was found while increased AST activity was observed only in fluoxetine-treated rats (chronically-isolated and controls). Increased carbonyl content, MDA, GST activity and decreased GSH levels in drug-treated controls/chronically-isolated animals suggest a link between drugs and hepatic oxidative stress. Increased NO levels associated with NF-κB activation and the concomitant increased COX-2 expression together with compromised CuZnSOD expression in clozapine-treated chronically-isolated rats likely reinforce oxidative stress, observed by increased lipid peroxidation and GSH depletion. In contrast, fluoxetine reduced NO levels in chronically-isolated rats. Isolation induced oxidative stress but histological changes were similar to those observed in vehicle-treated controls. Chronic administration of fluoxetine in both chronically-isolated and control animals resulted in more or less normal hepatic architecture, while clozapine in both groups resulted in liver injury. These data suggest that clozapine appears to have a higher potential to induce liver toxicity than fluoxetine.

Myeloperoxidase Inhibitors as Potential Drugs.

Myeloperoxidase (MPO) is an important member of the haem peroxidase - cyclooxygenase superfamily. This enzyme is physiologically expressed in circulating neutrophils, monocytes and some tissue macrophages including microglia. MPO plays an essential role in the antimicrobial and antiviral system of humans. The microbicidal activity of MPO exists due to its capability to oxidize halide and pseudohalide ions (CI(-), Br(-), I(-) and SCN(-)) by H2O2, thereby producing respective hypohalous acids (HOX). During the phagocytosis of pathogens, azurophilic granules release their content together with MPO into phagolysosomes. On the other hand, MPO can be discharged outside the phagocytes. Due to this, tissue damage during inflammation is greatly promoted by MPO-derived oxidants. Regarding its activity, MPO is a key factor in a great number of conditions within the group of cardiovascular diseases, inflammatory diseases, neurodegenerative diseases, kidney diseases and immune-mediated diseases. Therefore, MPO and its downstream inflammatory pathways might be attractive targets for both prognostic and therapeutic intervention in the prophylaxis of all mentioned illnesses. Nowadays, structure and reaction mechanism of MPO are known, which enable rational strategy in the development of specific MPO inhibitors that still preserve MPO activity during host defense from bacteria, but hinder pathophysiologically persistent activation of MPO. Various methods for MPO activity inhibition and unfavorable effects of MPO-derived oxidants remodeling will be discussed. Emphasis will be put on various known inhibitors, as well as on newly investigated natural products, which can also inhibit MPO activity.

Investigation of reaction between quercetin and Au(III) in acidic media: mechanism and identification of reaction products

The aim of the present paper was to investigate the reaction of quercetin, the flavonol very often used as a dietary supplement, with [AuCl4]− ions. The reaction was studied spectrophotometrically using the equimolar solutions in 1 : 1 water–methanol at pH ∼ 2. The spectrophotometric data indicated the formation of the products with an absorption maximum at 295 nm in all cases, characteristic of the oxidized forms of quercetin. HPLC coupled with DAD and LC-MS analysis of the reaction products suggested that the oxidation of quercetin resulted in the generation of similar metabolites including quinone and various oxidized quercetin–solvent adducts. In addition, cyclic voltammetric measurements confirmed that under applied experimental conditions, the reduction of Au(III) to Au(0) took place. The reduction species in the reaction mixture were Au(III) ions, while Au(I) disproportionates back to Au(III) and Au(0). The newly generated Au(III) ions further oxidized 3′,4′-dihydroxy groups of quercetin adducts obtained after first 2e− oxidation, giving the final reaction products. Based on the identification of reaction products, the reaction mechanism for the oxidation of quercetin in the presence of Au(III) which involves two 2e− transfer processes was proposed.

Modulators of Acetylcholinesterase Activity: from Alzheimer's Disease to Anti-cancer Drugs.

BACKGROUND Acetylcholinesterase (AChE) is involved in the termination of impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine in numerous cholinergic pathways in the central and peripheral nervous systems. The enzyme inactivation leads to acetylcholine accumulation, hyperstimulation of nicotinic and muscarinic receptors, and disrupted neurotransmission. Hence, acetylcholinesterase inhibitors, interacting with the enzyme as their primary target, are applied as relevant drugs for different neurodegenerative diseases (such as Alzheimers and Parkinsons) as well as toxins. At the same time, there are increasing evidence that in non-neuronal context, AChE is involved in the regulation of cell proliferation, differentiation, apoptosis and cell-cell interaction. An irregular expression of AChE has been found in different types of tumors, suggesting the involvement of AChE in the regulation of tumor development. Having all this in mind, there is a possibility that some AChE inhibitors could be used as anti-cancer agents. OBJECTIVE This contribution will discuss a broad range of possible application of different AChE inhibitors as drugs, from well-known anti-Alzheimers disease drugs to their use in cancer treatment in future. Emphasis will be put on various known AChE inhibitors classes, whose application as drugs could be controversy, as well as on newly investigated natural products, which can also modulate AChE activity. CONCLUSION It is not clear a patient treated for neurodegenerative condition prone to increased risk for some types of cancer and vice versa. This is necessary to keep in mind during rational drug design process for all therapies, which are based on AChE as a target molecule.

Influence of organophosphorus pesticides on peroxidase and chlorination activity of human myeloperoxidase

Inhibitory effects of five organophosphorus pesticides (diazinon, malathion, chlorpyrifos, azinphos-methyl and phorate) and their oxo-analogs on human myeloperoxidase (MPO) activity were investigated. While inspecting separately peroxidase and chlorination activity, it was observed that investigated OPs affect peroxidase activity, but not chlorination activity. Among investigated pesticides, malathion and malaoxon have showed the highest power to inhibit MPO peroxidase activity with IC50 values of the order of 3×10(-7) and 5×10(-9) M, respectively. It was proposed that inhibition trend is rendered by molecular structure which invokes steric hindrance for OPs interaction with MPO active center responsible for peroxidase activity. In addition, it was concluded that physiological function of MPO is not affected by any of the investigated OPs.

Heteroatom-doped mesoporous carbons as efficient adsorbents for removal of dimethoate and omethoate from water

Extensive use of organophosphate pesticides (OPs) invokes development of efficient procedures for their removal from the environment. By introducing low levels (<1 at%) of B, N or P into the structure of mesoporous carbons, we have produced a series of materials with different surface chemical composition, textural properties and level of structural disorder. These adsorbents were applied for removal of dimethoate and its oxo-analogue omethoate from aqueous solutions under batch adsorption conditions and by filtration using modified nylon membrane filters. Adsorption capacities up to 164 mg g−1 were measured, with OPs uptake typically above 80% for dimethoate concentration as high as 5 × 10−3 mol dm−3. After the adsorption, neurotoxic effects of OP-containing water samples were significantly reduced or completely removed. The level of structural disorder was identified as a key parameter for efficient removal of dimethoate and omethoate while in the filtration experiments surface area of adsorbents also played an important role. While the presented research appeals to new fundamental studies of OP–carbon surface interactions, it also indicates a possible strategy in designing new efficient adsorbents for OPs removal from water.

The impact of the structure of graphene-based materials on the removal of organophosphorus pesticides from water

The wide use of pesticides in modern agriculture and other areas results in an urgent need for their efficient removal from the environment. Adsorption of pesticides is one of the most commonly used strategies for this task. Here we analyze the adsorption of two organophosphorus pesticides, dimethoate (DMT) and chlorpyrifos (CPF), on graphene-based materials. The adsorption was found to be very sensitive to the structure of the adsorbents used. In particular, aliphatic DMT was found to prefer hydrophilic oxidized graphene surfaces. The CPF molecule, which contains an aromatic moiety, prefers adsorption on the surface of a graphene basal plane with high structural order and preserved π electron system. The toxicity of pesticide solutions is reduced after adsorption, suggesting that there is no oxidation of DMT and CPF to more toxic oxo forms. We emphasize that the combination of structural properties of adsorbents and adsorbates defines the adsorption of organophosphorus pesticides on graphene-based materials, while the specific surface area of adsorbents is not the major factor.

Ruthenium(II)-N-alkyl phenothiazine complexes as potential anticancer agents

In recent years, the search for effective anticancer compounds based on transition metal complexes has been the focus of medical investigations. The synergy between the ruthenium(II) and N-alkylphenothiazine counter-ions (chlorpromazine hydrochloride, thioridazine hydrochloride and trifluoperazine dihydrochloride, respectively) through the formation of three different complexes (1–3) was investigated. We explored whether the selected counter-ions and complexes might affect redox homeostasis and genome integrity of normal human blood cells, and induce an inhibition of Na+/K+-ATPase and AChE at pharmacologically relevant doses. Our results have shown that counter-ions and complexes did not affect the activity of Na+/K+-ATPase, while AChE activity was inhibited in a dose-dependent manner. All investigated compounds disturbed the viability and redox homeostasis of lymphocytes. Complexes 1 and 2 displayed potent cytotoxic and prooxidant action while complex 3 behaved as a weaker genotoxic inducer. Still, the tested complexes appeared to be less genotoxic and more cytostatic than the corresponding counter-ions. The effects of selected complexes were also tested in PC12 and U2OS cancer cells with special attention being given to the ability of phenothiazines to affect dopamine D2 receptors. Using the confocal laser scanning microscopy, we observed that all the complexes reduced cell viability. Although all investigated complexes have been bound to the dopamine receptor D2-eGFP, only complex 3 reduced its surface density and increased its lateral mobility in investigated cell lines. Albeit the role of alternative targets for complex 3 cannot be ruled out, its effects should be further examined as potential treatment strategy against cancer cells that overexpress D2.