Effect of angiotensin II on energetics,glucose metabolism and cytosolic NADH/NAD and NADPH/NADP redox in vascular smooth muscle

We investigated the potential of chronic administration of an oral daily dose (10 mg/kg) of the dietary flavonoid quercetin to prevent hypertension and oxidative stress induced by deoxycorticosterone acetate (DOCA)-salt in rats. We have compared its effects to those produced by the well-known anti-hypertensive drug verapamil, administered orally (20 mg/kg/day). Quercetin and verapamil treatments reduced systolic blood pressure of DOCA-salt rats in approximately 67.6 and 63.3% respectively, producing no effect in control animals. Both drugs reduced significantly hepatic and renal hypertrophy induced by DOCA-salt administration, while only quercetin prevented cardiac hypertrophy. Decreased endothelium-dependent relaxation to acetylcholine of aortic rings from DOCA-salt-treated rats was improved by quercetin, but verapamil only enhanced it in the presence of superoxide dismutase (SOD) plus catalase. Increased plasma and heart thiobarbituric acid reactive substances (TBARS) and total glutathione (GSH) levels in liver and heart, decreased liver glutathione peroxidase (GPX) and liver and kidney glutathione transferase (GST) activities were observed in DOCA-salt-treated rats compared to the control animals. The antihypertensive effect of quercetin was accompanied by normalisation of plasma TBARS values, improvement of the antioxidant defences system in heart and liver, restoring total GSH levels in both organs and altered liver GST and GPX activities, and improving kidney GST activity. Verapamil treatment only restored GSH levels in heart, having no effect on other alterations induced by DOCA-salt chronic administration in the antioxidant defences analysed. In conclusion, quercetin shows both antihypertensive and antioxidant properties in this model of mineralocorticoid hypertension, while verapamil exhibits only antihypertensive effects.     

Reduced glutathione increases quercetin stimulatory effects on HDL- or apoA1-mediated cholesterol efflux from J774A.1 macrophages

Abstract

In our in vitro study, we analyzed the effects of incubation of J774A.1 macrophages with reduced glutathione (GSH) and quercetin on the extent of cellular cholesterol efflux by high-density lipoprotein (HDL) or apolipoprotein A1 (apoA1). This combination was the most potent one among other exogenous and endogenous antioxidant combinations, since it significantly increased the extent of HDL-mediated cholesterol efflux from macrophages by 47% versus control cells, whereas quercetin (20 μM) or GSH (200 μM) alone increased it by only 37% or 17%, respectively. Similarly, apoA1-mediated cholesterol efflux was increased by 11% or 22% in quercetin or quercetin + GSH-treated cells, respectively, versus control cells. These stimulatory effects were noted only after 20 h of cell incubation. The combination of quercetin + GSH demonstrated high scavenging capacity of free radicals versus quercetin or GSH alone. In addition, quercetin + GSH significantly decreased macrophage oxidative stress as measured by the scavenging capacity of free radicals in the cells, the formation of reactive oxygen species, and the levels of cellular glutathione and lipid peroxides. There was no significant effect of quercetin + GSH on cellular HDL binding, on ATP-binding cassette A1 (ABCA1) activity, or on ABCG1 messenger RNA (mRNA) levels.

In contrast, mRNA levels for ABCA1 and peroxisome proliferator-activated receptor alpha (PPARα) were both significantly increased by 89% and 93%, respectively, in quercetin + GSH-treated cells versus control cells. Quercetin alone increased the mRNA levels for ABCA1 or PPARα by 42% or 77%, respectively, whereas GSH alone increased it by 22% or 28%, respectively. Mass spectra analysis revealed that oxidized quercetin reacts with GSH to form a new adduct product. We thus conclude that the stimulatory effects of quercetin + GSH on apoA1- or HDL-mediated macrophage cholesterol efflux are related to the ability of GSH to preserve quercetin in its reduced form.     

Reversal of Aging and Chronic Ethanol-induced Cognitive Dysfunction by Quercetin a Bioflavonoid

Cognitive dysfunction, one of the most striking age-related impairments seen in human beings, has been correlated to the vulnerability of the brain to increased oxidative stress during aging process. Quercetin is a bioflavonoid with strong antioxidant properties. Experiments were performed to study the possible effects of quercetin on cognitive performance of young, aged or ethanol-intoxicated mice (an animal model for cognition dysfunction) using one trail step down type of passive avoidance and elevated plus maze tasks, respectively. Aged or chronic ethanol-treated mice showed poor retention of memory in step-down passive avoidance and in elevated plus-maze task. Chronic administration of quercetin (10, 25 and 50 mg/kg) for 30 days or its co-administration with ethanol (15% w/v, 2 g/kg per orally) for 24 days significantly reversed the age-related or chronic ethanol-induced retention deficits in both the test paradigms. However, in both memory paradigms chronic administration of quercetin failed to modulate the retention performance of young mice. Chronic quercetin administration for 30 days also reversed age associated increase in TBARS levels and decline in forebrain total glutathione (GSH), SOD and catalase levels. Chronic ethanol administration to young mice produced an increase in lipid peroxidation, and a decline in forebrain total glutathione (GSH), SOD and catalase levels, which was significantly reversed by the co-administration of quercetin (10, 25 and 50 mg/kg). The results of the present study showed that chronic quercetin treatment reverses cognitive deficits in aged and ethanol-intoxicated mice, which is associated with its antioxidant property.     

Reversal of aging and chronic ethanol-induced cognitive dysfunction by quercetin a bioflavonoid

Cognitive dysfunction, one of the most striking age-related impairments seen in human beings, has been correlated to the vulnerability of the brain to increased oxidative stress during aging process. Quercetin is a bioflavonoid with strong antioxidant properties. Experiments were performed to study the possible effects of quercetin on cognitive performance of young, aged or ethanol-intoxicated mice (an animal model for cognition dysfunction) using one trail step down type of passive avoidance and elevated plus maze tasks, respectively. Aged or chronic ethanol-treated mice showed poor retention of memory in step-down passive avoidance and in elevated plus-maze task. Chronic administration of quercetin (10, 25 and 50 mg/kg) for 30 days or its co-administration with ethanol (15% w/v, 2 g/kg per orally) for 24 days significantly reversed the age-related or chronic ethanol-induced retention deficits in both the test paradigms. However, in both memory paradigms chronic administration of quercetin failed to modulate the retention performance of young mice. Chronic quercetin administration for 30 days also reversed age associated increase in TBARS levels and decline in forebrain total glutathione (GSH), SOD and catalase levels. Chronic ethanol administration to young mice produced an increase in lipid peroxidation, and a decline in forebrain total glutathione (GSH), SOD and catalase levels, which was significantly reversed by the co-administration of quercetin (10, 25 and 50 mg/kg). The results of the present study showed that chronic quercetin treatment reverses cognitive deficits in aged and ethanol-intoxicated mice, which is associated with its antioxidant property.     

Modulation of Paraoxonase 2 (PON2) in Mouse Brain by the Polyphenol Quercetin: A Mechanism of Neuroprotection?

Quercetin is a common flavonoid polyphenol which has been shown to exert neuroprotective actions in vitro and in vivo. Though quercetin has antioxidant properties, it has been suggested that neuroprotection may be ascribed to its ability of inducing the cell’s own defense mechanisms. The present study investigated whether quercetin could increase the levels of paraoxonase 2 (PON2), a mitochondrial enzyme expressed in brain cells, which has been shown to have potent antioxidant properties. PON2 protein, mRNA, and lactonase activity were highest in mouse striatal astrocytes. Quercetin increased PON2 levels, possibly by activating the JNK/AP-1 pathway. The increased PON2 levels induced by quercetin resulted in decreased oxidative stress and ensuing toxicity induced by two oxidants. The neuroprotective effect of quercetin was significantly diminished in cells from PON2 knockout mice. These findings suggest that induction of PON2 by quercetin represents an important mechanism by which this polyphenol may exert its neuroprotective action.     

Effect of Solanum trilobatum on the antioxidant status during diethyl nitrosamine induced and phenobarbital promoted hepatocarcinogenesis in rat

AIM: The methanolic extract of Solanum trilobatum (ST) is cytotoxic and exerts an inhibitory effect on tumor growth and in the present study, its role on the antioxidant status of N-diethylnitrosamine (DEN) induced and phenobarbital (PB) promoted hepatocarcinogenesis was assessed. METHODS: The protective role of ST on DEN induced and PB promoted hepatocarcinogenesis in Wistar rats was assessed from total nodular incidence, nodule multiplicity and volume of persistent nodules after an experimental period of 3 and 6 months following co-administration. The levels of thiobarbituric reactive substances (TBARS), glutathione (GSH) and activities of antioxidant enzymes were assessed in the haemolysate and liver of experimental animals to evaluate the antioxidant status. RESULTS: In DEN+PB+ST animals, the nodular incidence, multiplicity and volume reduced significantly compared to DEN+PB treated animals. In DEN+PB animals, the levels of TBARS increased significantly, whereas the levels of GSH and the activities of antioxidant enzymes-superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and glucose 6 phosphate dehydrogenase showed significant alterations compared to control both in the haemolysate and liver. However, in DEN+PB+ST animals, the levels of TBARS decreased significantly and the levels of GSH increased with favorable alterations in the activities of antioxidant enzymes in both the haemolysate and liver. CONCLUSION: The present results suggest that ST exerts its chemopreventive effects by modulating the antioxidant status during DEN induced hepatocarcinogenesis.     

Effects of ciprofloxacin on fetal rat liver during pregnancy and protective effects of quercetin

Urinary tract infections are common in pregnant women and ciprofloxacin frequently is used as a broad spectrum antibiotic. It has been suggested that ciprofloxacin causes liver damage in fetuses. Quercetin is a flavonoid with antioxidant properties. We investigated the efficacy of quercetin treatment for preventing fetal liver damage caused by ciprofloxacin. Pregnant rats were divided into four groups: untreated control group (C), 20 mg/kg quercetin for 21 days group (Q), 20 mg/kg twice/day ciprofloxacin for 10 days group (CP), and 20 mg/kg, ciprofloxacin + quercetin for 21 days group (CP + Q). Fetal livers were removed on day 21 of gestation to measure antioxidants and for histological observation. Malondialdehyde (MDA) and glutathione (GSH) levels, and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities were measured in tissue samples. GSH-Px, SOD and CAT activities were significantly lower in the CP group compared to group C. A significant increase in MDA was observed in the CP group compared to group C. There was no significant difference in GSH levels in any group. MDA levels were lower and CAT, SOD and GSH-Px enzyme activities were higher in the CP + Q group compared to group CP. Liver samples of the CP group exhibited central vein dilation, portal vein congestion, pyknotic nuclei and cytoplasmic vacuolization in some hepatocytes. Histological changes were less prominent in the rats treated with quercetin. Use of ciprofloxacin during pregnancy caused oxidative damage in fetal liver tissue. Oxidative stress was ameliorated by quercetin. Quercetin supports the antioxidant defense mechanism and it is beneficial for treating fetal liver damage caused by ciprofloxacin.     

Quercetin attenuates lindane induced oxidative stress in wistar rats

A wide number of pesticides, including highly persistent organochlorine compounds, such as lindane (γ-Hexachlorocyclohexane), have deteriorative effect on fauna and flora by inducing oxidative stress. Lindane induces cell damage by producing free radicals and reactive oxygen species. Quercetin, a dietary flavonoid, is ubiquitous in fruits and vegetables and plays an important role in human health by virtue of its antioxidant function. In this study the flavonoid quercetin was used to investigate its antioxidative effect against lindane induced oxidative stress in rats. The level of lipid peroxidation, reduced glutathione (GSH) were analysed in addition to the antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD) and glutathione-s-transferase (GST) activities in the liver and kidney tissue. Levels of hepatic marker enzymes in serum like Aspartate transaminase (AST), Alanine transaminase (ALT), Alkaline phosphatase (ALP) and Lactate dehydrogenase (LDH) and renal markers like serum creatinine and serum urea were estimated. Administration of Lindane induced histopathological alterations and increased levels of serum hepatic and renal markers and malondialdehyde (MDA) with a significant decrease in GSH content and CAT, SOD, GPx and GST activities. Cotreatment of quercetin along with lindane significantly decreased the lindane induced alteration in histology, serum hepatic and renal markers and MDA and also improved the cellular antioxidant status. The results show that Quercetin ameliorates Lindane induced oxidative stress in liver and kidney. The quercetin exhibited chemopreventive effect when administered along with lindane.     

Effects of quercetin on antioxidant defense in streptozotocin-induced diabetic rats.

In light of evidence that some complications of diabetes mellitus may be caused or exacerbated by oxidative damage, we investigated the effects of subacute treatment with the antioxidant quercetin on tissue antioxidant defense systems in streptozotocin-induced diabetic Sprague-Dawley rats (30 days after streptozotocin induction). Quercetin, 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one, was administered at a dose of 10mg/kg/day, ip for 14 days, after which liver, kidney, brain, and heart were assayed for degree of lipid peroxidation, reduced and oxidized glutathione content, and activities of the free-radical detoxifying enzymes catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase. Treatment of normal rats with quercetin increased serum AST and increased hepatic concentration of oxidized glutathione. All tissues from diabetic animals exhibited disturbances in antioxidant defense when compared with normal controls. Quercetin treatment of diabetic rats reversed only the diabetic effects on brain oxidized glutathione concentration and on hepatic glutathione peroxidase activity. By contrast, a 20% increase in hepatic lipid peroxidation, a 40% decline in hepatic glutathione concentration, an increase in renal (23%) and cardiac (40%) glutathione peroxidase activities, and a 65% increase in cardiac catalase activity reflect intensified diabetic effects after treatment with quercetin. These results call into question the ability of therapy with the antioxidant quercetin to reverse diabetic oxidative stress in an overall sense.     

Flavonoids increase the intracellular glutathione level by transactivation of the gamma-glutamylcysteine synthetase catalytical subunit promoter

Fruits and vegetables protect against cancer by so far not well-characterized mechanisms. One likely explanation for this effect is that dietary plants contain substances able to control basic cellular processes such as the endogenous defense against oxidative stress. Oxidative stress is pivotal in many pathological processes and reduced oxidative stress is implicated in prevention of disease. Our results demonstrate that extract from onion and various flavonoids induce the cellular antioxidant system. Onion extract and quercetin were able to increase the intracellular concentration of glutathione by approximately 50%. Using a reporter construct where reporter expression is driven by the gamma-glutamylcysteine synthetase (GCS) heavy subunit (GCS(h)) promoter we show that onion extract, quercetin, kaempferol, and apigenin increased reporter gene activity, while a fourth flavonoid, myricetin and sugar conjugates of quercetin were unable to increase reporter expression. Quercetin was also able to induce a distal part of the GCS(h) promoter containing only two antioxidant-response/electrophile-response elements (ARE/EpRE). Our data strongly suggest that flavonoids are important in the regulation of the intracellular glutathione levels. This effect may be exerted in part through GCS gene regulation, and may also contribute to the disease-preventing effect of fruits and vegetables.     

Reactive oxygen species production and mitochondrial dysfunction contribute to quercetin induced death in Leishmania amazonensis

Background

Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects more than 12 million people worldwide. Quercetin has generated considerable interest as a pharmaceutical compound with a wide range of therapeutic activities. One such activity is exhibited against the bloodstream parasite Trypanosoma brucei and amastigotes of Leishmania donovani. However, the mechanism of protozoan action of quercetin has not been studied.

Methodology/Principal Findings

In the present study, we report here the mechanism for the antileishmanial activity of quercetin against Leishmania amazonensis promastigotes. Quercetin inhibited L. amazonensis promastigote growth in a dose- and time- dependent manner beginning at 48 hours of treatment and with maximum growth inhibition observed at 96 hours. The IC₅₀ for quercetin at 48 hours was 31.4 µM. Quercetin increased ROS generation in a dose-dependent manner after 48 hours of treatment. The antioxidant GSH and NAC each significantly reduced quercetin-induced cell death. In addition, quercetin caused mitochondrial dysfunction due to collapse of mitochondrial membrane potential.

Conclusions/Significance

The effects of several drugs that interfere directly with mitochondrial physiology in parasites such as Leishmania have been described. The unique mitochondrial features of Leishmania make this organelle an ideal drug target while minimizing toxicity. Quercetin has been described as a pro-oxidant, generating ROS which are responsible for cell death in some cancer cells. Mitochondrial membrane potential loss can be brought about by ROS added directly in vitro or induced by chemical agents. Taken together, our results demonstrate that quercetin eventually exerts its antileishmanial effect on L. amazonensis promastigotes due to the generation of ROS and disrupted parasite mitochondrial function.     

Protective effect of quercetin in primary neurons against Abeta(1-42): relevance to Alzheimer's disease

Quercetin, a flavonoid found in various foodstuffs, has antioxidant properties and increases glutathione (GSH) levels and antioxidant enzyme function. Considerable attention has been focused on increasing the intracellular GSH levels in many diseases, including Alzheimer's disease (AD). Amyloid beta-peptide [Abeta(1-42)], elevated in AD brain, is associated with oxidative stress and neurotoxicity. We aimed to investigate the protective effects of quercetin on Abeta(1-42)-induced oxidative cell toxicity in cultured neurons in the present study. Decreased cell survival in neuronal cultures treated with Abeta(1-42) correlated with increased free radical production measured by dichlorofluorescein fluorescence and an increase in protein oxidation (protein carbonyl, 3-nitrotyrosine) and lipid peroxidation (protein-bound 4-hydroxy-2-nonenal). Pretreatment of primary hippocampal cultures with quercetin significantly attenuated Abeta(1-42)-induced cytotoxicity, protein oxidation, lipid peroxidation and apoptosis. A dose-response study suggested that quercetin showed protective effects against Abeta(1-42) toxicity by modulating oxidative stress at lower doses, but higher doses were not only non-neuroprotective but also toxic. These findings provide motivation to test the hypothesis that quercetin may provide a promising approach for the treatment of AD and other oxidative-stress-related neurodegenerative diseases.     

Dietary Antioxidant,Quercetin, Protects Sertoli‐Germ Cell Coculture from Atrazine‐Induced Oxidative Damage

Quercetin (QT), a dietary‐derived flavonoid, is ubiquitous in fruits and vegetables and plays an important role in human health by virtue of its antioxidant function. The present study was designed to examine the effects of QT on oxidative damage that was induced by the herbicide, atrazine (ATZ), in mixed cultures of Sertoli‐germ cells. Results showed that treatment with QT increased cell viability and decreased catalase activity, malondialdehyde, and reactive oxygen species (ROS) levels. QT treatment also increased the mRNA expression of glutathione peroxidase (GSH‐Px), glutathione reductase (GR), glutathione‐S‐transferase, and superoxide dismutase‐1 and could not reversed to the control levels ATZ‐induced steady‐state mRNA levels of these antioxidant genes as well as the level of glutathione and activities of GSH‐Px and GR. QT has protective effect against ATZ‐induced oxidative stress through a reduction in ROS levels and lipid peroxidation. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:477‐485, 2012; View this article online at wileyonlinelibrary.com . DOI 10:1002/jbt.21449     

The effect of quercetin and galangin on glutathione reductase

Quercetin and galangin can change the activity of glutathione reductase. Quercetin (a catechol structure in the B-ring) and galangin (any hydroxyl group in the B-ring) have different biological activities but, both possess high antioxidant abilities. Quercetin during the antioxidative action, is converted into an oxidized products (o-semiquinone and o-quinone), and subsequently glutathionyl adducts may be formed or SH-enzyme can be inhibited. We have tried to see whether inhibition of glutathione reductase (GR) can be influenced by preincubation of enzyme with NADPH (a creation of reduced form of enzyme, GRH(2)) and whether diaphorase activity of the enzyme is decreased by these flavonoids. The results confirmed that quercetin inhibits GRH(2) and inhibition is reduced by addition of EDTA or N-acetylcysteine. Both of flavonoids have no effect on diaphorase activity of glutathione reductase and this enzyme could increase the production of free radicals by catalysis of reduction of o-quinone during action of quercetin in vivo.     

Antioxidative and prooxidative effects of quercetin on A549 cells

Quercetin, a common plant polyphenol, has been reported to show both antioxidant and prooxidant properties. We studied the effects of quercetin on A549 cells in in vitro culture. We found that low concentrations of the flavonoid stimulated cell proliferation and increased total antioxidant capacity (TAC) of the cells; while higher concentrations of the flavonoid decreased cell survival and viability, thiol content, TAC and activities of superoxide dismutase, catalase and glutathione S-transferase. Quercetin decreased production of reactive oxygen species in the cells but produced peroxides in the medium. The cellular effects of quercetin are therefore complex and include both antioxidant effects and induction of oxidative stress due to formation of reactive oxygen species in the extracellular medium.     

Oxidized quercetin reacts with thiols rather than with ascorbate: implication for quercetin supplementation

When an antioxidant scavenges a reactive species, i.e., when it exerts its antioxidant activity, the antioxidant is converted into potentially harmful oxidation products. In this way, the antioxidant quercetin might yield an ortho-quinone, denoted as QQ, which has four tautomeric forms, i.e., the ortho-quinone and three quinonmethides. We evaluated the interaction of QQ with ascorbate or glutathione (GSH). Ascorbate recycles QQ to the parent compound quercetin, while GSH forms two adducts with QQ, i.e., 6-GSQ and 8-GSQ. When both GSH and ascorbate are present, QQ is converted exclusively into GSQ. In the absence of GSH, protein thiols will be arylated by QQ. This protein arylation is not prevented by ascorbate. Thiol arylation by quinones and quinonmethides can impair several vital enzymes. This implies that the product formed when quercetin displays its antioxidant scavenging effect is toxic in the absence of GSH. Therefore, an adequate GSH level should be maintained when quercetin is supplemented.     

The reversibility of the glutathionyl-quercetin adduct spreads oxidized quercetin-induced toxicity

Quercetin is one of the most prominent dietary antioxidants. During its antioxidant activity, quercetin becomes oxidized into its o-quinone/quinone methide QQ. QQ is toxic since it instantaneously reacts with thiols of, e.g., proteins. In cells, QQ will initially form an adduct with glutathione (GSH), giving GSQ. We have found that GSQ is not stable; it dissociates continuously into GSH and QQ with a half life of 2min. Surprisingly, GSQ incubated with 2-mercapto-ethanol (MSH), a far less reactive thiol, results in the conversion of GSQ into the MSH-adduct MSQ. A similar conversion of GSQ into relatively stable protein thiol-quercetin adducts is expected. With the dithiol dihydrolipoic acid (L(SH)(2)), quercetin is formed out of GSQ. These results indicate that GSQ acts as transport and storage of QQ. In that way, the initially highly focussed toxicity of QQ is dispersed by the formation of GSQ that finally spreads QQ-induced toxicity, probably even over cells.     

Targeted activation of transcription in vivo through hairpin-triplex forming oligonucleotide in Saccharomyces cerevisiae

The aims of the present study were to analyse the effects of an oral daily dose (10 mg/kg) of the dietary flavonoid quercetin for five weeks in two-kidney, one-clip (2K1C) Goldblatt (GB) hypertensive rats. The evolution of systolic blood pressure was followed by weekly measurements, and morphological variables, proteinuria, plasma nitrates plus nitrites (NO_x) and thiobarbituric acid reactive substances (TBARS), liver oxidative stress markers and endothelial function were determined at the end of the experimental period. Quercetin treatment reduced systolic blood pressure of GB rats, producing no effect in control animals. It also reduced cardiac hypertrophy and proteinuria developed in GB hypertensive rats. Decreased endothelium-dependent relaxation to acetylcholine of aortic rings from GB rats was improved by chronic quercetin treatment, as well as increased endothelium-dependent vasoconstrictor response to acetylcholine and overproduction of TXB₂ by aortic vessels of GB rats, being without effect in normotensive animals. Increased plasma NO_x and TBARS, and decreased liver total glutathione (GSH) levels and glutathione peroxidase (GPX) activity were observed in GB hypertensive rats compared to the control animals. Normalisation of plasma NO_x and TBARS concentrations and improvement of the antioxidant defences system in liver accompanied the antihypertensive effect of quercetin. We conclude that chronic oral treatment with quercetin shows both antihypertensive and antioxidant effects in this model of renovascular hypertension. (Mol Cell Biochem 270: 147–155, 2005)M.F. García-Saura and M. Galisteo are equal contributors to this work