Article

# Oxidation Process of Adrenaline in Freshly Isolated Rat Cardiomyocytes: Formation of Adrenochrome, Quinoproteins, and GSH Adduct

Authors:
• University of Porto, Faculty of Pharmacy, Porto, Portugal
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... ROS production is involved in this process since superoxide dismutase/catalase-mimetics or catalase overexpression prevent 1mediated JNK activation and apoptosis [125]. The sources of ROS are still under debate but are most likely multifactorial, as they can result from mitochondrial uncoupling caused by continuous 1adrenoceptor stimulation and from the catecholamine enzymatic and non-enzymatic degradation processes [128,129]. ...
... The cardiotoxicity induced by isoproterenol, NA and ADR has been studied for many years [351][352][353][354][355][356][357][358][359][360][361]. Several mechanisms have been postulated to explain their toxic effects, including relative hypoxia, increased sarcolemma permeability, calcium overload, cAMP elevation, activation of -and -adrenergic receptors (Fig. 2), and the formation of oxidative catecholamine metabolites [119,128,129,219,356,[362][363][364][365]. At the cellular level the effects of NA, ADR, and isoproterenol are qualitatively identical, namely the disruptive effects upon contractile myofilaments, however, glycogen depletion and fat deposition are more extensive with ADR [366,367]. ...
... When the enzymes dealing with the catabolism of catecholamines are unable to cope efficiently, their levels rise and catecholamines can undergo oxidation. The oxidation rate is faster under enzymatic or metal catalysis [428][429][430], in the presence of the O 2 •or higher pH [129,431]. Although at physiological pH, the catecholamines' oxidation seems to occur very slowly, it has been found to occur in in vivo situations, namely in septic shock [432]. ...
Article
Pathologic heart conditions, particularly heart failure (HF) and ischemia-reperfusion (I/R) injury, are characterized by sustained elevation of plasma and interstitial catecholamine levels, as well as by the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Despite the continuous and extensive research on catecholamines since the early years of the XX(th) century, the mechanisms underlying catecholamine-induced cardiotoxicity are still not fully elucidated. The role of catecholamines in HF, stress cardiomyopathy, I/R injury, ageing, stress, and pheochromocytoma will be thoroughly discussed. Furthermore and although the noxious effects resulting from catecholamine excess have traditionally been linked to adrenoceptors, in fact, several evidences indicate that oxidative stress and the oxidation of catecholamines can have important roles in catecholamine-induced cardiotoxicity. Accordingly, the reactive intermediates formed during catecholamine oxidation have been associated with cardiac toxicity, both in in vitro and in vivo studies. An insight into the influence of ROS, RNS, and catecholamine oxidation products on several heart diseases and their clinical course will be provided. In addition, the source and type of oxidant species formed in some heart pathologies will be referred. In this review a special focus will be given to the research of cardiac pathologies where catecholamines and oxidative stress are involved. An integrated vision of these matters is required and will be provided along this review, namely how the concomitant surge of catecholamines and ROS occurs and how they can be interconnected. The concomitant presence of these factors can elicit peculiar and not fully characterized responses on the heart. We will approach the existing data with new perspectives as they can help explaining several controversial results regarding cardiovascular diseases and the redox ability of catecholamines.
... The observed cytotoxicity of MTX was time and concentration dependent in both lactate dehydrogenase leakage assay and MTT reduction assay. Two therapeutic concentrations (100 nM and 1 lM) and three time points were selected (24,48, and 96 h) for further studies. Both MTX concentrations caused a significant increase in caspase-3 activity, which was not prevented by inhibiting MTX CYP450-metabolism. ...
... Conversely, the cells were scrapped and treated with the same protocol described for the evaluation of glutathione levels (detailed above). ATP intracellular levels were measured as previously described [24]. ...
... No significant changes were observed in the malondialdehyde levels after incubation with MTX (100 nM and 1 lM) at any time point tested (24,48, or 96 h) in comparison with control levels (data not shown). The expression of ATP-synthase was evaluated through western immunoblotting in all time points (24,48, and 96 h), after incubation with MTX in both working concentrations. ...
Article
Mitoxantrone (MTX) is a chemotherapeutic agent that emerged as an alternative to anthracycline therapy. However, MTX also causes late cardiotoxicity, being oxidative stress and mitochondrial-impaired function proposed as possible mechanisms. This work aimed to investigate the relevance of these mechanisms to the MTX toxicity in H9c2 cells, using therapeutic concentrations. The observed cytotoxicity of MTX was time and concentration dependent in both lactate dehydrogenase leakage assay and MTT reduction assay. Two therapeutic concentrations (100 nM and 1 μM) and three time points were selected (24, 48, and 96 h) for further studies. Both MTX concentrations caused a significant increase in caspase-3 activity, which was not prevented by inhibiting MTX CYP450-metabolism. Significant decreases were observed in the total and reduced glutathione levels only in MTX 100 nM at 96 h; however, neither alterations in oxidized glutathione nor increases in the malondialdehyde levels were observed at any time or concentrations tested. On the other hand, changes in the intracellular ATP levels, mitochondrial membrane potential, and intracellular calcium levels were observed in both concentrations and all time tested. Noteworthy, decreased levels of ATP-synthase expression and activity and increases in the reactive species generation were observed at 96 h in both working concentrations. However, the radical scavenger N-acetylcysteine or the mitochondrial function enhancer L-carnitine did not prevent MTX cytotoxicity. Thus, this work evidenced the early MTX-induced energetic crisis as a possible key factor in the cell injury.
... Glutathione is a strong antioxidant well known as cells protector which prevents against damages caused by radicals, peroxides, lipid peroxides and heavy metals presence. However, more recently its detoxification properties which help to eliminate oxidation products of EP and others catecholamines has been discredited, and even a generation of the toxic adducts was reported [5,44,45]. In light of this fact, we decided also to study a formation of EP-GHS adducts and their stability. ...
... Intensity of these peaks decreases with increasing potential. In the same time, a peak from adrenochrom (Fig. 8a), a final product of EP oxidation, occurring at m/z 180, increases [45]. At the final applied potential its intensity is much higher than that at m/z 184 at the beginning when no potential was applied, what suggest that oxidation of EP is related with diminishing of both: m/z 184 and m/z 166, accordingly. ...
... A closer inspection of the MS spectrum in Fig. 7a reveals the presence of peak at m/z 181, which corresponds to leucoadrenochrome-o-semiquinone radical [45] -one of inter-mediate oxidation products. It suggests that the process of electrochemical oxidation of EP proceeds through a clearly separate stage of the formation of leucoadrenochrome-o-semiquinone radical [35], intermediator in leucoadrenochrome oxidation to adrenochrome (Fig. 8a). ...
Article
Epinephrine (EP) is a neurotransmitter and hormone involved in an enormous number of processes in living organisms, however its highly active oxidation metabolites can cause hard damages and lead to serious health consequences. This paper described a relatively simple method of fabrication of Au nanotube array electrodes for electrochemical determination of EP, which could be easily transferred to non-laboratory conditions. On the basis of Tafel plot, some parameters of EP oxidation process like number of electrons involved in the rate-determining step (nα = 2) and electron transfer coefficient (α = 0.45) were evaluated for the studied electrode. The linear sweep voltammetry (LSV) revealed a very good linear response in the EP concentration range of 60–600 μM EP, however to 1000 μM its linearity differs insignificantly. Using differential pulse voltammetry (DPV), the calibration curve for EP determination is close to linear in the range of 10–150 μM, but the detection limit of 2.8 μM was evaluated for a lower concentration range 10–60 μM. The proposed DPV method was successfully applied to the determination of EP in a pure, ascorbic acid containing and real samples. The DPV results were evaluated and compared with those obtained by the high-performance liquid chromatography (HPLC) method. Combining the ROXY™ EC System with mass spectrometry creates an analytical tool for oxidative products of EP and conjugation of EP reactive potential metabolites characterized for I and II stage of biotransformation. The presence of leucoadrenochrome-o-semiquinone radicals confirmed by mass spectrometry implies that the ECE (electron transfer-chemical reaction-electron transfer) mechanism of EP oxidation at the Au electrode is more feasible.
... When the enzymes dealing with the catabolism of catecholamines are unable to cope efficiently, their levels rise and catecholamines can undergo oxidation. The oxidation rate is faster under enzymatic or metal catalysis (Heacock, 1959, Bindoli et al., 1992, Foppoli et al., 1997), in the presence of the superoxide anion (O 2 @BULLET – ) or high pH (West, 1947, Spencer et al., 1995, Costa et al., 2007). Although at physiological pH, the oxidation of catecholamines seems to occur very slowly, it has been found to occur in vivo namely in the septic shock (Macarthur et al., 2000). ...
... For ADR, at physiological pH, partial deprotonation of the amine group of the side chain leads to an irreversible 1,4-intramolecular cyclization, a reaction that occurs through nucleophilic attack of the nitrogen atom at position 6 of the quinone ring, to form " leucoadrenochrome " ; leucoadrenochrome is subsequently oxidized to form adrenochrome (Heacock and Mahon, 1958, Bindoli et al., 1992, Bindoli et al., 1999). This indole is often represented as a zwitterionic structure in aqueous solutions (Heacock and Mahon, 1958, Costa et al., 2007) (Figure 5). In summary, the oxidation of catecholamines occurs through two-stages whereby a total of four electrons is removed and an indole is formed by cyclization (Costa et al., 2011).Fig. 5. Postulated pathway for the oxidation of catecholamines. ...
... Adrenochrome is the most studied aminochrome due to its stability (Heacock, 1959, Rupp et al., 1994). When adrenochrome is formed and ADR still exists in solution, adrenochrome accelerates the oxidation process of the remaining ADR (Bindoli et al., 1999, Costa et al., 2007). Furthermore, the yield of adrenochrome increases if ADR semi-quinone reacts with O 2 to form O 2 @BULLET – (Costa et al., 2007). ...
... Determination of intracellular dopamine in both undifferentiated and differentiated cells by high-performance liquid chromatography linked with electrochemical detection (HPLC-ECD). Dopamine intracellular quantification was based on previously described assays ( Costa et al., 2007;Silva et al., 2007). After the 6 days-differentiation protocol, both DC and UNC cultured in petri dishes were washed with PBS and the culture medium was replaced by fresh medium alone (control) or containing dopamine (100 M) for 1 h. ...
... The protein pellet was dissolved in 0.3 M NaOH and the protein content was measured by the Lowry method, using bovine serum albumin (BSA) as the standard. The mobile phase used in the present study was adapted from a previously described method already used in our lab ( Costa et al., 2007;Silva et al., 2007). In order to maximize resolution and definition of dopamine peaks, a mobile phase containing 10% methanol was used (10% methanol in 50 mM citric acid, 0.46 mM octanessulfonic acid, adjusted to pH 3.0). ...
... Each cellular sample in 5% perchloric acid was injected into the HPLC system by an auto sampler. The dopamine levels were assessed from calibration curves constructed with at least six concentrations of dopamine and performed in the same day that samples were to be injected, as described previously ( Costa et al., 2007;Silva et al., 2007). Dopamine intracellular levels were normalized to the total protein content. ...
Article
"Ecstasy" (3,4-methylenedioxymethamphetamine or MDMA) is a widely abused recreational drug, reported to produce neurotoxic effects, both in laboratory animals and humans. MDMA metabolites can be major contributors for MDMA neurotoxicity. This work studied the neurotoxicity of MDMA and its catechol metabolites, α-methyldopamine (α-MeDA) and N-methyl-α-methyldopamine (N-Me-α-MeDA) in human dopaminergic SH-SY5Y cells differentiated with retinoic acid and 12-O-tetradecanoyl-phorbol-13-acetate. Differentiation led to SH-SY5Y neurons with higher ability to accumulate dopamine and higher resistance towards dopamine neurotoxicity. MDMA catechol metabolites were neurotoxic to SH-SY5Y neurons, leading to caspase 3-independent cell death in a concentration- and time-dependent manner. MDMA did not show a concentration- and time-dependent death. Pre-treatment with the antioxidant and glutathione precursor, N-acetylcysteine (NAC), resulted in strong protection against the MDMA metabolites' neurotoxicity. Neither the superoxide radical scavenger, tiron, nor the inhibitor of the dopamine (DA) transporter, GBR 12909, prevented the metabolites' toxicity. Cells exposed to α-MeDA showed an increase in intracellular glutathione (GSH) levels, which, at the 48h time-point, was not dependent in the activity increase of γ-glutamylcysteine synthetase (γ-GCS), revealing a possible transient effect. Importantly, pre-treatment with buthionine sulfoximine (BSO), an inhibitor of γ-GCS, prevented α-MeDA induced increase in GSH levels, but did not augmented this metabolite cytotoxicity. Even so, BSO pre-treatment abolished NAC protective effects against α-MeDA neurotoxicity, which were, at least partially, due to GSH de novo synthesis. Inversely, pre-treatment of cells with BSO augmented N-Me-α-MeDA-induced neurotoxicity, but only slightly affected NAC neuroprotection. In conclusion, MDMA catechol metabolites promote differential toxic effects to differentiated dopaminergic human SH-SY5Y cells.
... Experimental research into the cardiotoxic effects of catecholamines has focused almost exclusively on norepinephrine and epinephrine, and multiple adverse cardiac effects have been ascribed to excess levels of these two catecholamines, including microvascular dysfunction, inflammation, fibrosis, and cardiac hypertrophy (13). Moreover, excess levels of these two catecholamines were shown to directly induce cardiomyocyte damage and death in vitro (12,14) as well as in animals in vivo (45). In contrast, although cardiac and circulating dopamine levels are also increased in patients with stress cardiomyopathy, knowledge regarding putative adverse effects of excess dopamine levels on the heart and on cardiomyocytes in particular is very scarce. ...
... Important factors in catecholamine-induced cardiomyocyte damage and death appear to be oxidative stress and lipotoxicity, i.e., the intracellular accumulation of lipids. Both epinephrine (500 M) and norepinephrine (2 and 100 M) induced reactive oxygen species (ROS) production in H9c2 rat cardiomyoblasts and in isolated adult rat cardiomyocytes in vitro (14,20). Moreover, increased ROS levels were found in cardiomyocytes in patients with Takotsubo cardiomyopathy (40). ...
... Next to lipotoxicity, catecholamine-induced cardiotoxicity has also been attributed to excess ROS formation. Indeed, increased ROS levels were detected in cardiomyocytes in patients with Takotsubo cardiomyopathy (41), and both epinephrine (500 M) and norepinephrine (2 and 100 M) were found to increase ROS levels in H9c2 cells and isolated adult rat cardiomyocytes (14,20). We now observed that also dopamine (200 M) significantly increased intracellular nitrotyrosine levels indicative of ROS production. ...
Article
Aims: Excess catecholamine levels are suggested to be cardiotoxic and to underlie stress-induced heart failure. The cardiotoxic effects of noradrenaline and adrenaline are well recognized. However, although cardiac and circulating dopamine levels are also increased in stress-cardiomyopathy patients, knowledge regarding putative toxic effects of excess dopamine levels on cardiomyocytes is scarce. We now studied the effects of elevated dopamine levels in H9C2 cardiomyoblasts. Methods and results: H9C2 cells were cultured and treated with dopamine (200 μM) for 6, 24 and 48 hours. Subsequently, the effects on lipid accumulation, cell viability, flippase activity, reactive oxygen species (ROS) production, subcellular NOX protein expression and ATP/ADP and GTP/GDP levels were analysed. Dopamine did not result in cytotoxic effects after 6 hours. However, after 24 and 48 hours dopamine treatment induced a significant increase in lipid accumulation, nitrotyrosine levels, indicative of ROS production, and cell death. In addition, dopamine significantly reduced flippase activity and ATP/GTP levels, coinciding with phosphatidylserine exposure on the outer plasma membrane. Furthermore, dopamine induced a transient increase in cytoplasmic and (peri)nucleus NOX1 and NOX4 expression after 24 hours, which subsided after 48 hours. Moreover, while dopamine induced a similar transient increase in cytoplasmic NOX2 and p47(phox) expression, in the (peri)nucleus this increased expression persisted for 48 hours, where it co-localized with ROS. Conclusion(s): Exposure of H9C2 cells to elevated dopamine levels induced lipid accumulation, oxidative stress and a pro-inflammatory status of the plasma membrane. This can, in part, explain the inflammatory response in patients with stress-induced heart failure.
... Formation of superoxide anion O 2 •− (due to an oxidation pathway that involves the formation of highly reactive intermediaries o-semiquinones and o-quinones) [74] has been observed in both in vivo [75] and in vitro models [74,76]. The superoxide anion O 2 •− can cause the oxidation of epinephrine [52,77,78] and of metal ions copper [79] and iron [77], enhancing the oxidation of catecholamines. ...
... Formation of superoxide anion O 2 •− (due to an oxidation pathway that involves the formation of highly reactive intermediaries o-semiquinones and o-quinones) [74] has been observed in both in vivo [75] and in vitro models [74,76]. The superoxide anion O 2 •− can cause the oxidation of epinephrine [52,77,78] and of metal ions copper [79] and iron [77], enhancing the oxidation of catecholamines. ...
... Catecholamines metabolites can also reduce antioxidant defences by decreasing the levels of reduced glutathione (GSH) and increasing the oxidized glutathione (GSSG) content as observed in adrenaline-treated isolated rat cardiomyocytes [74,79]. ...
Article
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Oxidative stress (OS) is thought to play an important role in the pharmacological and toxic effects of various drugs of abuse. Herein we review the literature on the mechanisms responsible for the cardiovascular and hepatic toxicity of cocaine with special focus on OS-related mechanisms. We also review the preclinical and clinical literature concerning the putative therapeutic effects of OS modulators (such as N-acetylcysteine, superoxide dismutase mimetics, nitroxides and nitrones, NADPH oxidase inhibitors, xanthine oxidase inhibitors, and mitochondriotropic antioxidants) for the treatment of cocaine toxicity. We conclude that available OS modulators do not appear to have clinical efficacy.
... The fundamental role of oxidative stress (OS) in COCinduced cardiovascular toxicity is well established [8,11]. Moreover, formation and accumulation of reactive oxygen species (ROS) as a consequence of αand β-adrenergic receptors stimulation [12,13], as well as of enzymatic or nonenzymatic catabolism of catecholamines [14,15], have been demonstrated in cardiac and vascular cells. Mitochondrial dysfunction leading to the production of ROS is implicated in cardiovascular toxicity [16,17]. ...
... As noted, a crucial role in COC-induced toxicity is played by transformation of catecholamines into aminochromes, that is, the oxidative catecholamine metabolites [93]. Indeed, when the level of catecholamines rises and the enzymes responsible for their catabolism become less efficient, as it might likely occur during COC abuse, catecholamines can undergo oxidation [14,94] with formation of aminochromes (adrenochrome, dopachrome, and noradrenochrome); these molecules are very active from the redox cycling point of view. In bovine heart, it has been demonstrated that adrenochrome is reduced into semiquinone by mitochondrial complex I [95] inducing in cardiomyocyte mitochondria the formation of O 2 −· [9,94,96]. ...
... Genova and coworkers [97] had shown that mitocondrial complex I is involved both in initial generation of superoxide and in the reduction of adrenochrome to its semiquinone form. Furthermore, the superoxide anion O 2 −· in turn increases the adrenaline oxidation rate [14,97]. Thus, the mitochondria, on one side, are largely responsible for cardiomyocyte oxidative stress, while on the other side, they are themselves targets of the stress. ...
Article
Full-text available
Cocaine abuse has long been known to cause morbidity and mortality due to its cardiovascular toxic effects. The pathogenesis of the cardiovascular toxicity of cocaine use has been largely reviewed, and the most recent data indicate a fundamental role of oxidative stress in cocaine-induced cardiovascular toxicity, indicating that mitochondrial dysfunction is involved in the mechanisms of oxidative stress. The comprehension of the mechanisms involving mitochondrial dysfunction could help in selecting the most appropriate mitochondria injury biological marker, such as superoxide dismutase-2 activity and glutathionylated hemoglobin. The potential use of modulators of oxidative stress (mitoubiquinone, the short-chain quinone idebenone, and allopurinol) in the treatment of cocaine cardiotoxic effects is also suggested to promote further investigations on these potential mitochondria-targeted antioxidant strategies.
... The GSHt and GSSG levels of tissue homogenates were determined by the 5,5 0 -dithiobis(2-nitrobenzoic acid) DTNB-GSSG reductase recycling assay, as previously described (Costa et al., 2009(Costa et al., , 2007. For the quantification of GSSG, 10 mL of 2-vinylpyridine was added to the acidic supernatant and shaken for 1 h in ice to block reduced glutathione (GSH). ...
... In protocol 2, ATP levels in the tissues were determined through the formation of bioluminescence using the firefly luciferinluciferase system, as previously described (Costa et al., 2007). ...
... In protocol 2, the levels of noradrenaline and adrenaline in the heart were determined by HPLC, as previously described (Costa et al., 2007). The HPLC mentioned in the previous section was coupled to electrochemical detection, namely a Colouchem II (ESA, Chelmsford, USA) equipped with a guard cell (ESA 5020) and a analytical cell (ESA 5011A). ...
... Representative electrochemical chromatograms of (D) blank 5% perchloric acid and 5% perchloric acid spiked to contain 5 g mL −1 of ␣-amanitin; (E) blank liver, and liver sample spiked to contain 5 g mL −1 of ␣amanitin and (F) blank kidney, and kidney sample spiked to contain 5 g mL −1 of ␣amanitin. used in a wide range of pH (2.1 < pH < 6.4) and provides low background noise [24,27]. It was observed that the ratio of citric acid and methanol (80:20) was key to a good separation and resolution of the peaks. ...
... As an alternative, we used citrate as a buffer of the mobile phase. Citrate can be used in a wide range of pH (2.1 < pH < 6.4) and provides low background noise, which is very important in electrochemical analysis [24,27]. The ratio of citrate: methanol (80:20): 0.46 mM octanessulfonic acid and the pH 5.5 were key to obtain good separation and resolution of the ␣-amanitin peak. ...
... ATP levels in the heart, kidneys, liver and brain areas were assessed by a bioluminescent assay based on the firefly reaction of luciferin-luciferase system, as previously described (Costa et al. 2007). For each measurement, an ATP standard curve was done (ATP ≥ 99% purity according to Sigma-Aldrich). ...
... The determination of total glutathione (total GSH) and GSSG levels was done by the 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)-GSSG reductase recycling assay according to previous works (Costa et al. 2007;Teixeira-Gomes et al. 2016). The levels of GSH were determined considering the following ratio: GSH = total GSH − 2×GSSG. ...
Article
Full-text available
3,4-Methylenedioxymethamphetamine (MDMA or “ecstasy”) is a widespread drug of abuse with known neurotoxic properties. The present study aimed to evaluate the differential toxic effects of MDMA in adolescent and aged Wistar rats, using doses pharmacologically comparable to humans. Adolescent (post-natal day 40) (3 × 5 mg/kg, 2 h apart) and aged (mean 20 months old) (2 × 5 mg/kg, 2 h apart) rats received MDMA intraperitoneally. Animals were killed 7 days later, and the frontal cortex, hippocampus, striatum and cerebellum brain areas were dissected, and heart, liver and kidneys were collected. MDMA caused hyperthermia in both treated groups, but aged rats had a more dramatic temperature elevation. MDMA promoted serotonergic neurotoxicity only in the hippocampus of aged, but not in the adolescents’ brain, and did not change the levels of dopamine or serotonin metabolite in the striatum of both groups. Differential responses according to age were also seen regarding brain p-Tau levels, a hallmark of a degenerative brain, since only aged animals had significant increases. MDMA evoked brain oxidative stress in the hippocampus and striatum of aged, and in the hippocampus, frontal cortex, and striatum brain areas of adolescents according to protein carbonylation, but only decreased GSH levels in the hippocampus of aged animals. The brain maturational stage seems crucial for MDMA-evoked serotonergic neurotoxicity. Aged animals were more susceptible to MDMA-induced tissue damage in the heart and kidneys, and both ages had an increase in liver fibrotic tissue content. In conclusion, age is a determinant factor for the toxic events promoted by “ecstasy”. This work demonstrated special susceptibility of aged hippocampus to MDMA neurotoxicity, as well as impressive damage to the heart and kidney tissue following “ecstasy”.
... ATP levels were quantified by a bioluminescent assay using the luciferin-luciferase system, as described in detail in previous works [26,32]. The results are presented in nmol of ATP per mg of protein. ...
... The GSHt or GSSG levels were evaluated by the 5,5′dithiobis(2-nitrobenzoic acid)-GSSG reductase recycling assay, as previously we described in detail [26,32]. GSH levels were calculated by the equation: GSH = GSHt -2 × GSSG. ...
Article
Full-text available
Background 3,4-Methylenedioxymethamphetamine (MDMA or “ecstasy”) is a worldwide drug of abuse commonly used by adolescents. Most reports focus on MDMA’s neurotoxicity and use high doses in adult animals, meanwhile studies in adolescents are scarce. We aimed to assess in rats the acute MDMA toxicity to the brain and peripheral organs using a binge dose scheme that tries to simulate human adolescent abuse. Methods Adolescent rats (postnatal day 40) received three 5 mg/kg doses of MDMA (estimated equivalent to two/three pills in a 50 kg adolescent), intraperitoneally, every 2 h, while controls received saline. After 24 h animal sacrifice took place and collection of brain areas (cerebellum, hippocampus, frontal cortex and striatum) and peripheral organs (liver, heart and kidneys) occurred. Results Significant hyperthermia was observed after the second and third MDMA doses, with mean increases of 1 °C as it occurs in the human scenario. MDMA promoted ATP levels fall in the frontal cortex. No brain oxidative stress-related changes were observed after MDMA. MDMA-treated rat organs revealed significant histological tissue alterations including vascular congestion, but no signs of apoptosis or necrosis were found, which was corroborated by the lack of changes in plasma biomarkers and tissue caspases. In peripheral organs, MDMA did not affect significantly protein carbonylation, glutathione, or ATP levels, but liver presented a higher vulnerability as MDMA promoted an increase in quinoprotein levels. Conclusions Adolescent rats exposed to a moderate MDMA dose, presented hyperthermia and acute tissue damage to peripheral organs without signs of brain oxidative stress.
... Conversely, the cells were scrapped and treated with the same protocol described for the evaluation of glutathione levels (detailed above). ATP intracellular levels were measured as previously described [24]. ...
... After 96 h incubation with MTX (100 nM and 1 lM), the ATP intracellular levels were about 1.31 ± 0.42 nmol/mg protein and 1.33 ± 0.78 nmol/mg protein, respectively, compared to 2.20 ± 0.83 nmol/mg protein for the control group. The expression of ATP-synthase was evaluated through western immunoblotting in all time points (24,48, and 96 h), after incubation with MTX in both working concentrations. Results were normalized to the actin content and were expressed as the optic density ratio between ATPsynthase/actin (% relative to the control). ...
... The ATP levels of tissue homogenates were determined by bioluminescence, which is based on the generation of light using the luciferin-luciferase system (Costa et al., 2007;Rossato et al., 2014a). The method uses the enzyme luciferase that catalyzes the oxidation reaction of the luciferin reagent, consuming ATP. ...
... The tGSH and GSSG levels of tissue homogenates were determined by the DTNB-GSSG reductase recycling assay (Costa et al., 2007;Teixeira-Gomes et al., 2016). The method involves oxidation of GSH by the sulfhydryl reagent DTNB to form the yellow derivative 5'-thio-2-nitrobenzoic acid (TNB), measurable at 415 nm. ...
Article
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P-glycoprotein (P-gp) is an ATP-binding cassette transporter involved in the efflux of numerous compounds that influences the pharmacokinetics of xenobiotics. It reduces intestinal absorption and exposure of target cells to toxicity. Thioxanthones are compounds able to induce and/or activate P-gp in vitro. Particularly, 1-(propan-2-ylamino)-4-propoxy-9H-thioxanthen-9-one (TX5) behaves as a P-gp inducer and activator in vitro. The aims of this study were: i) to perform a histological characterization, by testing a single high dose of TX5 [30 mg/kg, body weight (b.w.), gavage], administered to Wistar Han rats, 24 hours after administration; and ii) to perform both a complete histological characterization and a preliminary safety evaluation, in distinct target organs, 24 hours after administration of a single lower dose of TX5 (10 mg/kg, b.w., gavage) to Wistar Han rats. The results showed a relevant histological toxicity for the higher dose of TX5 administered (30 mg/kg, b.w.), manifested by extensive hepatic necrosis and splenic toxicity (parenchyma with hyperemia, increased volume of both white and red pulp, increased follicles marginal zone). Moreover, in the kidneys, a slight hyperemia and tubular edema were observed in TX5-treated animals, as well as an inflammation of the small intestine. On the contrary, for the lower tested dose (10 mg/kg, b.w.), we did not observe any relevant histological toxicity in the evaluated organs. Additionally, no significant differences were found in the ATP levels between TX5-exposed and control animals in any of the evaluated organs, with the exception of the intestine, where ATP levels were significantly higher in TX5-treated rats. Similarly, TX5 caused a significant increase in the ratio GSH/GSSG only in the lungs. TX5 (10 mg/kg, b.w.) did not induce any change in any of the hematological and biochemical circulating evaluated parameters. However, TX5 was able to significantly reduce the activated partial thromboplastin time, without affecting the prothrombin time. The urine biochemical analysis revealed a TX5-mediated increase in both creatinine and sodium. Taken together, our results show that TX5, at a dose of 10 mg/kg, does not induce considerable toxicity in the biological matrices studied. Given this adequate safety profile, TX5 becomes a particularly interesting compound for ex vivo and in vivo studies, regarding the potential for induction and activation of P-gp at the intestinal barrier.
... Administration of norepinephrine in ratspromotes lipid peroxidation, alters the activities of antioxidant enzymes, reduces GSH/GSSG ratio, as well as evokes a pro-inflammatory and apoptotic response in the cardiac tissue (162). Several studies substantiated the cardio-toxic role of adrenaline and pointed out to the fact that oxidative metabolites of epinephrine such as adrenochrome, adrenolutin and quinoproteins are the initiators of the biochemical pathway leading to cardiac damage (162,164,165). Melatonin protects against adrenaline induced cardio-toxicity (165). ...
... Athletes in competitive sports often experience anticipatory stress and anxiety which lead to an increase in circulatory epinephrine and other catecholamines (45). These molecules exert cardio-toxic effects (162,164). Melatonin administration provides protection against myocardial injury and cardiac arrhythmia caused by epinephrine (165,184) or other β-adrenergic receptor agonists viz., isoproterenol (182,185). Exercise in moderate-intensity also exacerbates the pathological state of patients suffering from CHF (52). ...
Article
Exercise conducted at an optimum training load is usually beneficial for the overall health of an individual. However, an unaccustomed intense exercise carried out by untrained individuals or elite athletes during over-training and/or competition-related stress often bear inevitable cardiovascular risks. Although many alterations occurring in the cardiovascular system during exercise are the results of training adaptations, sudden cardiovascular deaths reported in competitive athletes is a matter of grave concern. Several oxidative biomarkers that depict the underlying structural and functional impairment of the myocardial tissue have been identified in the individuals subjected to extensive exercise. The exercise-mediated cardiomyopathy is free radical related and also associated with pro-inflammatory response. In this review we will highlight the possible role of melatonin in obviating irrevocable oxidative cardiovascular injury triggered by extensive exercise stress. Melatonin effectively reduces exercise-induced lipid peroxidation, restores natural cellular antioxidant pool and supresses the innate immune cascade reaction that, otherwise, jeopardize cardiovascular integrity. Melatonin blocks the IKK/IκB/NFκB signaling as well as suppress iNOS and COX-2 mediated inflammation in cardiac tissue. In addition, melatonin reduces blood lactate accumulation and accelerates glucose utilization, thereby, promoting energy metabolism in athletes during their training and competition. Physical exertion associated overheating and the resultant sympathetic outflow impede cardiovascular homeostasis. Melatonin not only attenuates the sympathomedullary stimulation but also protects the cardiac cells from the cytotoxic effect of catecholamines. The available information regarding the efficacy of melatonin in amelioration of exercise-driven oxidative insult in cardiac tissue has been discussed and summarized.
... Catecholamines excess can conceivably result in cardiomyopathy through recurrent coronary vasospasm, tachycardia, hypertension, accelerated atherosclerosis, and/or direct myocardial toxicity (Costa et al. 2011). Importantly, the surge of catecholamines in the presence of MAO inhibition and/or oxidative stress favors the autoxidation of catecholamines, which potentiates heart injury (Costa et al. 2007(Costa et al. , 2011. ...
... In particular, the mitochondrial dysfunction, the oxidative and nitrosative stress (evaluated by tyrosine nitration) (Lord et al. 2010), and inflammation (Islam et al. 2009;Varner et al. 2002) caused by the METH-induced catecholamine surge is crucial to impair heart function, which is particularly redox sensitive (Costa et al. 2011). It is well known that the phagocytic response can exacerbate the catecholamine-induced oxidative stress and leads to catecholamine auto-oxidation (Costa et al. 2011), which, on the other hand, causes alterations in cardiac proteins and energetic metabolism (Costa et al. 2007(Costa et al. , 2009. ...
Article
Amphetamines represent a class of psychotropic compounds, widely abused for their stimulant, euphoric, anorectic, and, in some cases, emphathogenic, entactogenic, and hallucinogenic properties. These compounds derive from the β-phenylethylamine core structure and are kinetically and dynamically characterized by easily crossing the blood-brain barrier, to resist brain biotransformation and to release monoamine neurotransmitters from nerve endings. Although amphetamines are widely acknowledged as synthetic drugs, of which amphetamine, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) are well-known examples, humans have used natural amphetamines for several millenniums, through the consumption of amphetamines produced in plants, namely cathinone (khat), obtained from the plant Catha edulis and ephedrine, obtained from various plants in the genus Ephedra. More recently, a wave of new amphetamines has emerged in the market, mainly constituted of cathinone derivatives, including mephedrone, methylone, methedrone, and buthylone, among others. Although intoxications by amphetamines continue to be common causes of emergency department and hospital admissions, it is frequent to find the sophism that amphetamine derivatives, namely those appearing more recently, are relatively safe. However, human intoxications by these drugs are increasingly being reported, with similar patterns compared to those previously seen with classical amphetamines. That is not surprising, considering the similar structures and mechanisms of action among the different amphetamines, conferring similar toxicokinetic and toxicological profiles to these compounds. The aim of the present review is to give an insight into the pharmacokinetics, general mechanisms of biological and toxicological actions, and the main target organs for the toxicity of amphetamines. Although there is still scarce knowledge from novel amphetamines to draw mechanistic insights, the long-studied classical amphetamines-amphetamine itself, as well as methamphetamine and MDMA, provide plenty of data that may be useful to predict toxicological outcome to improvident abusers and are for that reason the main focus of this review.
... The levels of whole brain ATP were determined through the firefly luciferin-luciferase bioluminescent assay as previously described (Costa et al. 2007). The ATP levels were normalized to the total protein content and expressed as % control. ...
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Mitoxantrone (MTX) is a topoisomerase II inhibitor used to treat a wide range of tumors and multiple sclerosis but associated with potential neurotoxic effects mediated by hitherto poorly understood mechanisms. In adult male CD-1 mice, the underlying neurotoxic pathways of a clinically relevant cumulative dose of 6 mg/kg MTX was evaluated after biweekly administration for 3 weeks and sacrifice 1 week after the last administration was undertaken. Oxidative stress, neuronal damage, apoptosis, and autophagy were analyzed in whole brain, while coronal brain sections were used for a closer look in the hippocampal formation (HF) and the prefrontal cortex (PFC), as these areas have been signaled out as the most affected in ‘chemobrain’. In the whole brain, MTX-induced redox imbalance shown as increased endothelial nitric oxide synthase and reduced manganese superoxide dismutase expression, as well as a tendency to a decrease in glutathione levels. MTX also caused diminished ATP synthase β expression, increased autophagic protein LC3 II and tended to decrease p62 expression. Postsynaptic density protein 95 expression decreased in the whole brain, while hyperphosphorylation of Tau was seen in PFC. A reduction in volume was observed in the dentate gyrus (DG) and CA1 region of the HF, while GFAP-ir astrocytes increased in all regions of the HF except in the DG. Apoptotic marker Bax increased in the PFC and in the CA3 region, whereas p53 decreased in all brain areas evaluated. MTX causes damage in the brain of adult CD-1 mice in a clinically relevant cumulative dose in areas involved in memory and cognition.
... Ischemia/reperfusion insult promotes the release of large amounts of hormones from cells, including adrenaline (Kanda and Watanabe, 2007), glucagon (Thorens, 2011) and glucocorticoid, such as hydrocortisone (Kainuma et al., 2009), all of which can raise the blood glucose levels or influence glucose metabolism (Costa et al., 2007;Godinez-Rubi et al., 2013). We thus investigated if these hormones would regulate the expression of TIGAR during reperfusion. ...
... 77 In isolated cardiomyocytes, exposure to high concentrations of adrenaline demonstrated its redox ability, reflected by GSH depletion, with formation of GSSG, adrenaline-o-quinones, adrenochrome, and several other highly reactive species, namely, HO • and ONOO − . 56 detect, especially in biological matrixes such as plasma or blood. 71,79 Many studies underestimate adrenaline-redox ability, while other experiments use supplemented medium with antioxidants to avoid catecholamine oxidation (i.e., ascorbic acid) since authors do not exclude the contribution of catecholamine oxidative metabolites to the observed effects. ...
Article
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The heart is a target organ for oxidative stress-related injuries. Due to its very high energetic metabolic rate, the heart has the highest rate of production of reactive oxygen species, namely hydrogen peroxide (H2O2), per gram of tissue. Additionally, the heart has lower levels of antioxidants and of total activity of antioxidant enzymes when compared to other organs. Furthermore, drugs that have relevant antioxidant activity and that are used in the treatment of oxidative stress related cardiac diseases demonstrate better clinical cardiac outcomes than other drugs with similar receptor affinity but with no antioxidant activity. Several xenobiotics particularly target the heart and promote toxicity. Anticancer drugs, like anthracyclines, cyclophosphamide, mitoxantrone, and more recently tyrosine kinase targeting drugs are well known cardiac toxicants whose therapeutic application has been associated to a high prevalence of heart failure. High levels of catecholamines or drugs of abuse, namely amphetamines, cocaine, and even the consumption of alcohol for long periods of time are linked to cardiovascular abnormalities. Oxidative stress may be one common link for the cardiac toxicity elicited by these compounds. We aim to revise the mechanisms involved in cardiac lesions caused by the above mentioned substances specially focusing in oxidative stress related pathways. Oxidative stress biomarkers can be useful in the early recognition of cardiotoxicity in patients treated with these drugs and aid to minimize the setting of cardiac irreversible events.
... It is known that the toxic effects induced by catecholamines oxidation processes is mainly related to production of aminochromes [15]. Aminochromes are catecholamines oxidation products that can induce oxidative stress and were described to be cytotoxic [16,17]. However, it seems that aminochromes, namely adrenochrome, rapidly disappears from blood by tissue uptake or by rearrangement and metabolism to adrenolutin and 5,6-dihydroxy-N-methylindone (DHMI) [18]. ...
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... Although catecholamines play a vital role under stressful conditions, excess circulating levels of adrenaline may induce detrimental effects in cells. Cardiotoxicity of adrenaline is well documented [7,8]. It was found that adrenaline stimulates proliferation of esophageal cell carcinoma via β-adrenoreceptor activation [9]. ...
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Adrenaline is a neurotransmitter and hormone that plays an important role in physiological regulatory mechanisms. The objective of this study was to assess primary DNA damage in isolated human lymphocytes exposed to adrenaline using the in vitro comet assay. Dose-response of human lymphocytes was determined at concentration range of adrenaline from 0.01 μM to 300 μM for various treatment times (1h, 2h, 4h and 24h). The obtained results showed that adrenaline induced DNA damage at concentration range from 5 μM to 300 μM after 1h, 2h and 4h of treatment. The slightest DNA damage was observed after 24 h of adrenaline treatment - only the highest concentrations of adrenaline (150 μM and 300 μM) caused increased level of DNA damage. In order to evaluate the potential contribution of reactive oxygen species (ROS) in adrenaline-induced DNA damage we used antioxidants catalase (100 IU/mL and 500 IU/mL) and quercetin (100 μM and 500 μM). Co-treatment of lymphocytes with adrenaline (300 μM) and antioxidants for 1 h, signifi cantly reduced the quantity of DNA in the comet tails. Therefore, it can be concluded that adrenaline exhibits genotoxic effects mainly through induction of reactive oxygen species and that some of the DNA damage is repaired during the fi rst four hours following the treatment with adrenaline.
... The protocol used for the LDH leakage assay in Caco-2 cells was performed as already described [29]. In the cardiomyocyte suspensions, the percentage of rod-shaped cells and the LDH leakage assay were determined as previously described [30][31][32]. In order to confirm the sampling homogeneity, the protein levels were determined by the method described by Lowry [33]. ...
... Oxidative stress was analysed by evaluation of total (GSHt), reduced (GSH) and oxidised (GSSG) glutathione levels. The intracellular levels of GSH and GSSG in Sh-SEA-treated HCV29 cells were evaluated by the DTNB-GSSG reductase recycling assay, as previously described (Costa et al., 2007). After exposure to Sh-SEA, cells were lysed and proteins were precipitated with 5% HClO 4 . ...
Article
Chronic infection with the blood fluke, Schistosoma haematobium, is associated with squamous cell carcinoma of the bladder. Previously, it has been shown that soluble extracts of mixed sex adult S. haematobium worms (SWAP) are tumourigenic, both in vitro and in vivo. In addition, oestrogen-related molecules in SWAP of S. haematobium down-regulate oestrogen receptors (ERs) alpha and beta in oestrogen responsive cells. Moreover, schistosome oestrogens occur in sera of persons with schistosomiasis haematobia and repress transcription of ERs in urothelial cells. Given that eggs of S. haematobium are the developmental stage directly responsible for urogenital disease during schistosomiasis haematobia, we suspected that soluble antigens from S. haematobium eggs exhibit similar or more potent tumorigenic capacity. Here we investigated the tumorigenic potential of soluble egg antigens (Sh-SEA) of S. haematobium and the endocrine system in favouring parasitism by schistosomes. The findings confirmed that 6.25μg/ml of Sh-SEA was enough to stimulate cell proliferation, reduce apoptosis and increase oxidative stress of Sh-SEA-exposed urothelial cells. In addition, genotoxic effects of Sh-SEA on these cells were determined by using alkaline single-cell gel electrophoresis (Comet). Furthermore, Liquid Chromatography Diode Array Detection Electron Spray Ionisation Mass Spectrometry indicated the presence of catechol-oestrogens in S. haematobium SEA. A prospective oestrogen-DNA adduct mediated pathway in S. haematobium egg induced bladder cancer is also discussed.
... This reaction occurs at a modest rate, but oxidation of catecholamine in the presence of reactive oxygen species is thought to occur much faster [170]. The oxidation of catecholamines by ROS was initially believed to be protective, in that it resulted in the removal of the radical oxygen species, but recent evidence is emerging to suggest that the reduced adrenaline products result in cellular toxicity as well. ...
... 60,61 Interestingly, the inability of ISO to induce detectable formation of ROS was in accordance with our previous studies. 62,63 This phenomenon has also been described by Costa et al.,64 where no changes were shown in lipid peroxidation, protein carbonylation, or activity of various antioxidant enzymes after up to 3 hours of incubation of isolated adult rat cardiomyocytes with 500 μM adrenaline. Moreover, direct antioxidant activity of catecholamines under pro-oxidant conditions has been described. ...
Article
Objectives: Rutin, quercetin-3-O-rutinoside, a natural flavonol glycoside, has shown various in vitro benefits with potential use treating human diseases, especially cardiovascular system disorders. Antioxidant properties are assumed to underlie the majority of these benefits. Yet rutin pro-oxidant properties have been reported as well. Our research group has recently shown aggravating effects on isoprenaline (ISO)-induced cardiotoxicity in Wistar:Han rats after 24 hours. Methods: This study was designed to examine in more detail the reasons for the negative effects of rutin (11.5 and 46 mg/kg, i.v.) after administration of ISO (100 mg/kg, s.c.) in rats within 2 hours of continuous experiment and in the H9c2 cardiomyoblast-derived cell line. Results: Like our previous findings, rutin did not (11.5 or 46 mg/kg, i.v.) reduce the ISO-induced mortality within 2 hours although the lower dose significantly reduced cardiac troponin T (cTnT) and partly improved the histological findings. In contrast, the higher dose increased the mortality in comparison with solvent (1.26% w/v sodium bicarbonate). This was not caused by any specific haemodynamic disturbances. It appears to be associated with oxidative stress as rutin enhanced intracellular reactive oxygen species formation in vitro and had the tendency to increase it in vivo. Conclusions: Rutin, likely due to its pro-oxidative effects, can exacerbate catecholamine cardiotoxicity depending on the dose used.
... Hsp70 can inhibit the main producer of ROS (NADPH oxidase) in human neutrophils, and thereby suppress ROS production [6]. The chemical oxidation of adrenaline can possible explain observed decrease in ROS production [7]. The negative correlation between intracellular Hsp70 levels and ROS production was discovered in the previous work [8]. ...
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We have developed a simple and reliable method to measure the sensitivity of individuals to oxidative stress. This method utilizes luminol-amplified chemiluminescence to quantify production of reactive oxygen species (ROS) by opsonized zymosan-stimulated neutrophils that have been subjected to short-term stress via heat shock. In this study, the chemiluminescence reaction was used to monitor the dynamics of ROS production in neutrophils derived from 17 patients of different ages and genders before and after these neutrophils were subjected to heat shock. In addition, we determined expression of Toll-like receptors using fluorescent-labeled antibody. The effects of adrenaline, dexamethasone, aspirin, and indomethacin, as well as different doses of exogenous heat shock protein 70 (Hsp70), on the production of ROS by stimulated neutrophils was also investigated. Our data showed that adrenaline and exogenous Hsp70 both suppressed ROS production by stimulated neutrophils. Furthermore, TLR4 expression was upregulated upon heat stress. Thus, adrenaline, HSPs, and TLRs may all play a role in regulating stress responses in phagocytes.
... The pellet was stored at −20 °C and used for protein evaluation. ATP levels were assessed by a bioluminescence reaction with the firefly luciferin-luciferase system, as previously described (Costa et al. 2007). The levels of ATP were expressed as nmol of ATP per amount of protein or as % of control after protein normalization. ...
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Mitoxantrone (MTX) is an antineoplastic agent used to treat several types of cancers and on multiple sclerosis, which shows a high incidence of cardiotoxicity. Still, the underlying mechanisms of MTX cardiotoxicity are poorly understood and the potential toxicity of its metabolites scarcely investigated. Therefore, this work aimed to synthesize the MTX-naphthoquinoxaline metabolite (NAPHT) and to compare its cytotoxicity to the parent compound in 7-day differentiated H9c2 cells using pharmacological relevant concentrations (0.01–5 µM). MTX was more toxic in equivalent concentrations in all cytotoxicity tests performed [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction, neutral red uptake, and lactate dehydrogenase release assays] and times tested (24 and 48 h). Both MTX and NAPHT significantly decreased mitochondrial membrane potential in 7-day differentiated H9c2 cells after a 12-h incubation. However, energetic pathways were affected in a different manner after MTX or NAPHT incubation. ATP increased and lactate levels decreased after a 24-h incubation with MTX, whereas for the same incubation time and concentrations, NAPHT did not cause any significant effect. The increased activity of ATP synthase seems responsible for MTX-induced increases in ATP levels, as oligomycin (an inhibitor of ATP synthase) abrogated this effect on 5 µM MTX-incubated cells. 3-Methyladenine (an autophagy inhibitor) was the only molecule to give a partial protection against the cytotoxicity produced by MTX or NAPHT. To the best of our knowledge, this was the first broad study on NAPHT cardiotoxicity, and it revealed that the parent drug, MTX, caused a higher disruption in the energetic pathways in a cardiac model in vitro, whereas autophagy is involved in the toxicity of both compounds. In conclusion, NAPHT is claimed to largely contribute to MTX-anticancer properties; therefore, this metabolite should be regarded as a good option for a safer anticancer therapy since it is less cardiotoxic than MTX.
... Also, the production of reactive oxygen species (ROS), either via adrenoceptors or via autoxidation cannot be overlooked in the cardiotoxicity inflicted by catecholamines, namely adrenaline. 88,89 Similar findings were also observed after noradrenaline administration, where mixed -and -blockade did not fully counteract oxidative stress. 90 ...
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Cardiovascular diseases are a leading cause of morbidity and mortality in most developed countries of the world. Pharmaceuticals, illicit drugs, and toxins can significantly contribute to the overall cardiovascular burden and thus deserve attention. The present article is a systematic overview of drugs that may induce distinct cardiovascular toxicity. The compounds are classified into agents that have significant effects on the heart, blood vessels, or both. The mechanism(s) of toxic action are discussed and treatment modalities are briefly mentioned in relevant cases. Due to the large number of clinically relevant compounds discussed, this article could be of interest to a broad audience including pharmacologists and toxicologists, pharmacists, physicians, and medicinal chemists. Particular emphasis is given to clinically relevant topics including the cardiovascular toxicity of illicit sympathomimetic drugs (e.g., cocaine, amphetamines, cathinones), drugs that prolong the QT interval, antidysrhythmic drugs, digoxin and other cardioactive steroids, beta-blockers, calcium channel blockers, female hormones, nonsteroidal anti-inflammatory, and anticancer compounds encompassing anthracyclines and novel targeted therapy interfering with the HER2 or the vascular endothelial growth factor pathway.
... Since the rates of cyclization reactions of dopamine o-quinone and its subsequent oxidation significantly exceed the rate of aminochrome rearrangement, the latter can temporarily accumulate in the cytosol [42,88] and, being a substrate of NQO2, contribute to the intensification of oxidative stress [26,27,32,42] (Figure 1). Similar patterns have been demonstrated for norepinephrine [19] and adrenaline [89]. In addition, the contribution of NQO2 to cellular damage may also be associated with the oxidation of the co-substrate NRH to NR+ with subsequent formation of 4-pyridone-3-carboxamide riboside in the presence of O 2 ...
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The ability of NQO2 to increase the production of free radicals under enhanced generation of quinone derivatives of catecholamines is considered to be a component of neurodegenerative disease pathogenesis. The present study aimed to investigate the neuroprotective mechanisms of original NQO2 inhibitor M-11 (2-[2-(3-oxomorpholin-4-il)-ethylthio]-5-ethoxybenzimidazole hydrochloride) in a cellular damage model using NQO2 endogenous substrate adrenochrome (125 µM) and co-substrate BNAH (100 µM). The effects of M-11 (10–100 µM) on the reactive oxygen species (ROS) generation, apoptosis and lesion of nuclear DNA were evaluated using flow cytometry and single-cell gel electrophoresis assay (comet assay). Results were compared with S29434, the reference inhibitor of NQO2. It was found that treatment of HT-22 cells with M-11 results in a decline of ROS production triggered by incubation of cells with NQO2 substrate and co-substrate. Pre-incubation of HT-22 cells with compounds M-11 or S29434 results in a decrease of DNA damage and late apoptotic cell percentage reduction. The obtained results provide a rationale for further development of the M-11 compound as a potential neuroprotective agent.
... Briefly, the primary function of GSH is to recycle oxidized ascorbic acid (dehydroascorbic acid or DHA) back into reduced ascorbic acid (ASC), thereby mediating cellular oxidative stress [136]. GSH also recycles epinephrine, norepinephrine, dopamine, and related adrenergic amines, which oxidize to form toxic adrenochrome-like compounds or forms adducts with them that are then excreted [137][138][139][140][141][142][143][144][145]. Similarly, opioids such as morphine and the enkephalins bind to GSH antagonizing its antioxidant function and forming inactive adducts [146][147][148][149][150][151]. ...
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Cross-talk between opioid and adrenergic receptors is well-characterized and involves second messenger systems, the formation of receptor heterodimers, and the presence of extracellular allosteric binding regions for the complementary ligand; however, the evolutionary origins of these interactions have not been investigated. We propose that opioid and adrenergic ligands and receptors co-evolved from a common set of modular precursors so that they share binding functions. We demonstrate the plausibility of this hypothesis through a review of experimental evidence for molecularly complementary modules and report unexpected homologies between the two receptor types. Briefly, opioids form homodimers also bind adrenergic compounds; opioids bind to conserved extracellular regions of adrenergic receptors while adrenergic compounds bind to conserved extracellular regions of opioid receptors; opioid-like modules appear in both sets of receptors within key ligand-binding regions. Transmembrane regions associated with homodimerization of each class of receptors are also highly conserved across receptor types and implicated in heterodimerization. This conservation of multiple functional modules suggests opioid–adrenergic ligand and receptor co-evolution and provides mechanisms for explaining the evolution of their crosstalk. These modules also suggest the structure of a primordial receptor, providing clues for engineering receptor functions.
... However, adrenaline at other catecholamine at doses exceeding physiological levels may cause toxic effects [4,5]. There are studies indicating toxic effects of adrenaline via signal transduction pathways [6,7]. ...
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This study is aimed at analysing biochemical and genetic endpoints of toxic effects after administration of adrenaline. For this purpose, the study was carried out on Wistar rats and three doses of adrenaline were used: 0.75 mg/kg, 1.5 mg/kg, and 3 mg/kg body weight. To achieve these aims, we investigated the effects of adrenaline on catalase (CAT), Cu, Zn-superoxide dismutase (SOD), malondialdehyde (MDA), nitrite (NO 2 −), carbonyl groups (PCC), and nitrotyrosine (3-NT). Total activity of lactate dehydrogenase (LDH), its relative distribution (LDH 1 –LDH 5 ) activity, level of acute phase proteins (APPs), and genotoxic effect were also evaluated. The obtained results revealed that all doses of adrenaline induced a significant rise in CAT activity, MDA level, PCC, NO 2⁻ , and 3-NT and a significant decrease in SOD activity compared to control. Adrenaline exerted an increase in total activity of LDH, LDH 1 , and LDH 2 isoenzymes. Further study showed that adrenaline significantly decreased serum albumin level and albumin-globulin ratio, while the level of APPs ( α1 -acid glycoprotein and haptoglobulin) is increased. The micronucleus test revealed a genotoxic effect of adrenaline at higher concentrations (1.5 mg/kg and 3 mg/kg body weight) compared to untreated rats. It can be concluded that adrenaline exerts oxidative and nitrative stress in rats, increased damage to lipids and proteins, and damage of cardiomyocytes and cytogenetic damage. Obtained results may contribute to better understanding of the toxicity of adrenaline with aims to preventing its harmful effects.
... Yet, in the presence of uric acid (end product of hypoxanthine oxidation) with/without XO, no reaction occurs; this indicates the importance of ROS (98). In a similar experiment on isolated rat cardiomyocytes, there was a concomitant decrease in GSH (99). A membrane system with NADH/NADPH on separated bovine cardiac sarcolemma also induces adrenochrome formation (100,101). ...
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Indirect evidences in reviews and case reports on Takotsubo syndrome (TTS) support the fact that the existence of oxidative stress (OS) might be its common feature in the pre-acute stage. The sources of OS are exogenous (environmental factors including pharmacological and toxic influences) and endogenous, the combination of both may be present, and they are being discussed in detail. OS is associated with several pathological conditions representing TTS comorbidities and triggers. The dominant source of OS electrones are mitochondria. Our analysis of drug therapy related to acute TTS shows many interactions, e.g., cytostatics and glucocorticoids with mitochondrial cytochrome P450 and other enzymes important for OS. One of the most frequently discussed mechanisms in TTS is the effect of catecholamines on myocardium. Yet, their metabolic influence is neglected. OS is associated with the oxidation of catecholamines leading to the synthesis of their oxidized forms – aminochromes. Under pathological conditions, this pathway may dominate. There are evidences of interference between OS, catecholamine/aminochrome effects, their metabolism and antioxidant protection. The OS offensive may cause fast depletion of antioxidant protection including the homocystein-methionine system, whose activity decreases with age. The alteration of effector subcellular structures (mitochondria, sarco/endoplasmic reticulum) and subsequent changes in cellular energetics and calcium turnover may also occur and lead to the disruption of cellular function, including neurons and cardiomyocytes. On the organ level (nervous system and heart), neurocardiogenic stunning may occur. The effects of OS correspond to the effect of high doses of catecholamines in the experiment. Intensive OS might represent “conditio sine qua non” for this acute clinical condition. TTS might be significantly more complex pathology than currently perceived so far.
Article
Alzheimer's disease (AD), the most common cause of dementia, is thought to be a progressive neurodegenerative disease that is clinically characterised by a decline of memory and other cognitive functions. Mild cognitive impairment (MCI) is considered to be the prodromal stage of AD. However, the relationship between AD and MCI and the development process remains unclear. The amygdala is one of the most vulnerable structures in the early stages of AD. To our knowledge, this is the first report on the alteration of the functional connectivity of the amygdala in AD and MCI subjects. We hypothesised that the amygdala-cortical loop is impaired in AD and that these alterations relate to the disease severity. In our study, we used resting-state functional MRIs to investigate the altered amygdala connectivity patterns in 35 AD patients, 27 MCI patients and 27 age- and gender-matched normal controls (NC). Compared with the NC, the decreased functional connectivity found in the AD patients was mainly located between the amygdala and the regions that are included in the default mode, context conditioning and extinction networks. Importantly, the decreased functional connectivity between the amygdala and some of the identified regions was positively correlated with MMSE, which indicated that the cognitive function impairment is related to an altered functional connectivity pattern.
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The reaction of adrenaline autooxidation in an alkaline carbonate buffer followed by formation of superoxide radicals and the product of its oxidation, adrenochrome, which models the quinone pathway of adrenaline metabolism in the body, is accompanied by oxygen consumption. A study of this process by the polarographic method enabled one to apply this reaction to determine the activity of superoxide dismutase and antioxidant properties of biological and chemical compounds; it is based on evaluation of a latent period and the rate of oxygen consumption, which are measured in the presence of examined compounds. It was suggested that known neuroand cardiotoxicity of the quinone products of adrenaline oxidation may be associated not only to their intrinsic properties and reactive oxygen species formed but also local hypoxia of those regions of the cell and tissue where the quinone oxidation of adrenaline occurs. Keywordsadrenaline–adrenochrome–superoxide–oxygen–polarography–superoxide dismutase
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Eye is the official journal of the Royal College of Ophthalmologists. It aims to provide the practising ophthalmologist with information on the latest clinical and laboratory-based research.
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The reaction of adrenaline autoxidation in an alkaline buffer with the formation of superoxide radicals and the product of its oxidation, adrenochrome, which models the quinoid pathway of adrenaline conversion in the body, is accompanied by oxygen consumption. This reaction is applicable for polarographic determination of the activity of superoxide dismutase and the antioxidant properties of biological and chemical compounds, it is based on evaluation of the latent period and the rate of oxygen consumption, which are measured in the presence of the compounds examined. It was assumed that the neuro- and cardiotoxicity of quinone products of adrenaline oxidation is related not only to their "own" properties and reactive oxygen species formed but also the hypoxia of those regions of the cell and tissue where the quinoid oxidation of adrenaline occurs.
Article
This review is focused on literature data and our own research of the nontrivial quinoid pathway for the oxidation of adrenaline. All catecholamines can be oxidized similarly with formation of corresponding aminochromes. This process is simulated in vitro in an alkaline medium and is known as the adrenaline autoxidation chain reaction, whose products are adrenochrome and radical compounds, superoxide anions ($${\text{O}}_{2}^{{ - {\kern 1pt} \centerdot }}$$), and other. This reaction was previously used to determine the activity of superoxide dismutase as a model of superoxide generation. We have proposed various new methodical approaches that allow the determination of the enzyme activity and reveal the anti/prooxidant properties of various compounds and materials. This pathway of conversion of one of the catecholamines (dopamine) is currently described as a “preclinical model of Parkinson’s disease.” In this regard, we have proposed the reaction of adrenaline autoxidation to be used in search for substances that can inhibit the process of quinoid oxidation, that is, to identify potential neuroprotective agents. Experimental and theoretical studies of this reaction expand the understanding of the mechanisms of free radical processes that occur in the body.
Article
Over forty years ago, biochemist Lauro Galzigna conducted an in-vitro experiment showing that the antipsychotic chlorpromazine reacted with the putative psychotogen adrenochrome to form a polymer resembling melanin. The field of psychopharmacology has essentially ignored that simple but illustrative experiment in the intervening time. The present study reproduces principle elements of Galzigna's experiment and expands the scope to include the antipsychotic medications olanzapine and minocycline. The rate of reaction was slow, with maximal yield of black polymer being achieved by 4, 10 and 7 days with chlorpromazine, olanzapine and minocycline, respectively. Changing the pH was most informative for chlorpromazine and minocycline reactions, where yield increased sharply between pH 6.1 and 6.9, and decreased slightly between pH 6.9 and 7.8, consistent with reaction profiles expected for aromatic substitution. Pre-incubation of olanzapine with iodine doubled the polymer yield, facilitated by the addition of iodine to the aromatic ring and presumably followed by its departure as a "leaving group". Increasing the salt concentration 1.5-fold depressed yields for all three drugs, most likely via ionic shielding of charged functional groups, diminishing reactivity. The results are discussed in regards to the mechanism of action of antipsychotic medications, casting doubt on commonly held theories. The time course of the chemical reactions presented here and the concentrations required, are much more consistent with clinical results than are models concerning receptor-mediated mechanisms. Furthermore, minocycline was effective in this model, but does not appear to have affinity for the primary receptor families thought by many to mediate antipsychotic efficacy.
Article
The physiologically active metal ions with fixed valence Ca2+ and Mg2+ were shown to accelerate epinephrine autoxidation at an alkaline pH, which proceeds via the known quinoid pathway and is accompanied by the generation of reactive oxygen species. A higher efficiency was observed for Ca2+ ions compared with Mg2+ ions. The activation of epinephrine autoxidation was evident from a decrease in the time of the initiation of the chain reaction to begin (i.e., the reaction lag) and an increase in the rate of both oxygen uptake and the formation of adrenochrome. Based on the observed effects, Ca2+ and Mg2+ cations were assumed to have the potential to play a role in the free radical processes that are associated with redox reactions in the cell and can also modulate the effect of epinephrine in the organism its oxidation via the quinoid pathway.
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Adrenaline at high pharmacological doses may lead to oxidative damages in diverse organs including gut. In this study, we attempt to elucidate the potentially protective effects of melatonin on gastrointestinal (GI) tissue damages induced by adrenaline. Rats were injected (s.c.) with different doses (0.125, 0.25 and 0.50 mg/kg) of adrenaline bitartrate (AD) for 15 days with or without melatonin (2.5, 5 and 10 mg/kg; orally). The results showed that adrenaline caused massive histological and ultra-structural GI injuries and melatonin (20 mg/kg) effectively protected these injuries. The protective mechanisms are related to the antioxidant and anti-inflammatory activities of melatonin indicated by increased glutathione levels and antioxidant enzymes as well as decreased oxidative stress markers and pro-inflammatory cytokines in GI tissues. The signal pathways of melatonin include up-regulating expression of Nrf2, SIRT1 and Bcl2, while down-regulating NFκB, TNFα and Bax. Melatonin also targeted mitochondrial energy homeostasis and biogenesis by up-regulating expression of PGC1α, AMPKα and SOD2 and reduced leakage of cytochrome c. The SIRT1-NFκB and PGC1α-AMPKα signal transduction pathways seem to play the central roles involving in melatonin's protective effects on gastric damages induced by the high doses of adrenaline.
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Ochratoxin A (OTA) is a mycotoxin produced by several species of fungi from the Aspergillus and Penicillium genera that frequently grow in improperly stored food products. OTA has carcinogenic, teratogenic and nephrotoxic potential and sustains a high half-life in human blood. Despite the recently efforts to decontaminate OTA through its conversion into its metabolite ochratoxin alpha (OTα), there are just a few reports in literature comparing the toxic effects of these toxins. Thus, herein we studied and compared the proinflammatory and toxicological effects of OTA and its metabolite OTα in human neutrophils in vitro. The effect of OTA and OTα on human neutrophils viability was evaluated by trypan blue, annexin-V and propidium iodide methods as well as by the analysis of the cytomorphological alterations. The ATP assay was performed using the luciferin-luciferase bioluminescence assay. The alteration on mitochondrial potential was assessed by a mitoscreen flow cytometry mitochondrial membrane potential detection kit and the intracellular calcium levels through the probe FLUO-4/AM. To study the human neutrophils' oxidative burst, the fluorescent probe dichlorodihydrofluorescein diacetate was used. OTA induces an increase on the intracellular calcium, human neutrophils' oxidative burst followed by depletion of ATP levels and alterations on mitochondrial potential leading to cell death by necrosis, while OTα did not induce significant toxic effects. Our results strongly suggest that the toxicity of human neutrophils induced by OTA started with the release of calcium(+) from internal stores triggering several neutrophils' activities that culminate in cell death by necrosis.
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The spontaneous oxidation of adrenaline in an alkaline medium was investigated by electronic spectroscopy. It was shown that the pH of the medium has an effect on the position of the maxima in the absorption spectrum of adrenaline. Analysis of the absorption spectra of the reaction medium during the oxidation process revealed the dynamics of change in the optical density of the products. The spectra of adrenaline, adrenolutine, and adrenochrome were calculated theoretically by quantum chemical methods within the scope of density functional theory. A procedure was developed for quantitative analysis of the products from autoxidation of adrenaline.
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The addition of nitro blue tetrazolium (NBT) into the reaction of adrenaline autooxidation allows direct identification of superoxide anion formation (O 2−⊙) as well as demonstration of kinetics of their accumulation in this superoxide-generating system. The kinetics of adrenochrome and O 2−⊙ formation has been compared under the same conditions. Three possible approaches to the use of the adrenaline autooxidation reaction for the determination of superoxide dismutase activity (SOD) and revealing antioxidant properties of various compounds are discussed. Two of these approaches have been described previously: the spectro-photometric method of registration of adrenochrome, an end product of adrenaline autooxidation, at 347 nm (Sirota, 1999) and the polarographic method, which measures oxygen consumption used for O 2−⊙ formation (Sirota, 2011). Here, a novel approach to this problem is presented; it is based on spectrophotometric determination of O 2−⊙ using NBT. The employment of this approach results in a significant decrease of the pH value of carbonate buffer from 10.5 to 9.7 and a 4-fold decrease in the amounts of added adrenaline, thus creating milder conditions for the revealing and investigation of antioxidant properties of materials being examined.
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The current study aimed to demonstrate the potentially adverse effects of adrenaline, an endogenous stressor, on the melatonergic system, oxidative status, antioxidative responses and inflammatory markers in different parts of gastrointestinal tract of Wistar rat. These included stomach, duodenum and colon and they were incubated with different concentrations (2.5, 5.0 and 10.0 µg/mL) of adrenaline for 1h, respectively. The levels of melatonin, gene expressions of arylalkylamine N-acetyltransferase (AANAT) and melatonin receptor 1 (MT1) as well as other stress-induced parameters including NF-kB expression, levels of cAMP, calcium, malondialdehyde, protein carbonyl content, reduced glutathione, nitrate, superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferase, tumour necrosis factor-α, IL-1β, IL6 and IL10 were systemically measured in these tissues. An adrenaline dose-dependent decrease in level of melatonin, AANAT, MT1 and NF-kB in these tissues were observed. In contrast, the profound increases in the levels of cAMP, calcium and all oxidative stress markers, inflammatory cytokines (except IL10), and activities of antioxidant enzymes (except superoxide dismutase) were observed after adrenaline treatment. A maximum effect was found in tissues treated with 5 µg/mL of adrenaline. The Correlation studies between melatonin level and other parameters (any two at a time) indicated a potentially physiological interplay between adrenaline stress and melatonin tissue levels. Collectively, the results provided the novel data on the adverse effects of adrenaline on the endogenous melatonergic system, antioxidant and inflammatory responses in the gastrointestinal tissues of rats.
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Nucleic acid (NA)‐based therapy is proposed to address serious diseases such as cardiovascular diseases (CVDs). Powerful NA delivery vehicles are essential for effective gene therapy. Herein, a novel type of delivery vehicle, an unlockable core–shell nanocomplex (Hep@PGEA) with self‐accelerating NA release, is structurally designed. Hep@PGEA is composed of disulfide‐bridged heparin nanoparticle (HepNP) core and low‐toxicity PGEA cationic shell. In comparison with NA, heparin, a negatively charged polysaccharide macromolecule, exhibits stronger interactions with cationic species. Upon the breakdown of redox‐responsive HepNP cores, unlocked heparin would interact with the outer cationic shells and replace the condensed NA to facilitate NA release. Such unique Hep@PGEA is successfully explored for effective miRNA–pDNA staged gene therapy of myocardial infarction (MI), one of the most serious CVDs. With the progression of MI, glutathione amounts in heart tissues increase. MiR‐499 (for the inhibition of cardiomyocyte apoptosis) and plasmid encoding vascular endothelial growth factor (for the promotion of angiogenesis) are sequentially delivered for systemic treatment of MI. Such treatment produces impressive results in restoring heart function and suppressing cardiac hypertrophy. Due to the wide existence of redox agents in cells, the proposed unlockable delivery nanovehicle and staged therapy strategy can provide new methods to effectively treat different serious diseases. Unlockable core–shell nanocomplexes with self‐accelerating nucleic acid release are structurally designed as a novel type of delivery vehicle. The newly designed unlockable vector is successfully utilized for effective miRNA–pDNA staged gene therapy of serious cardiovascular diseases.
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Methylphenidate (MPH) is a first-line stimulant drug to treat attention deficit hyperactivity disorder (ADHD). Overdiagnosis of ADHD and MPH abuse lead to serious concerns about the possible long-term adverse consequences of MPH in healthy children and adolescents. We aimed to evaluate MPH effects in adolescent male Wistar rats (postnatal day 40) using an oral dose scheme (2 daily MPH doses 5 mg/kg in a 5% sucrose solution, 5 h apart, for 7 days) that mimics the therapeutic doses given to human adolescents. Twenty-four hours after the last MPH administration, rats were sacrificed and brain areas [cerebellum, prefrontal cortex (PFC), hippocampus, and striatum], peripheral organs (liver, heart, and kidneys), and blood were collected for biochemical and histological analysis. MPH treatment did not alter rats’ body temperature or weight, neither food or water intake throughout the experiment. The ratio of reduced glutathione/oxidized glutathione (GSH/GSSG) significantly increased in the PFC and hippocampus of MPH-treated rats, meanwhile protein carbonylation remained unchanged in the brain. In the heart, the GSH/GSSG ratio and GSH levels were significantly increased, with decreased GSSG, while histology revealed significant damage, namely interstitial edema, vascular congestion, and presence of a fibrin-like material in the interstitial space. In the kidneys, MPH treatment resulted in extensive necrotic areas with cellular disorganization and cell infiltration, and immunohistochemistry analysis revealed a marked activation of nuclear factor-ĸB. This study showed that clinically relevant oral MPH doses improve the GSH redox status in the brain and heart, but evoke heart and kidney tissue damage to adolescent rats.
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The rate of autoxidation of epinephrine and the sensitivity of this autoxidation to inhibition by superoxide dismutase were both augmented, as the pH was raised from 7.8 → 10.2. O2⁻, generated by the xanthine oxidase reaction, caused the oxidation of epinephrine to adrenochrome and the yield of adrenochrome produced per O2⁻ introduced, increased with increasing pH in the range 7.8 → 10.2 and also increased with increasing concentration of epinephrine. These results, in conjunction with complexities in the kinetics of adrenochrome accumulation, lead to the proposal that the autoxidation of epinephrine proceeds by at least two distinct pathways, only one of which is a free radical chain reaction involving O2⁻ and hence inhibitable by superoxide dismutase. This chain reaction accounted for a progressively greater fraction of the total oxidation as the pH was raised. The ability of superoxide dismutase to inhibit the autoxidation of epinephrine at pH 10.2 has been used as the basis of a convenient and sensitive assay for this enzyme.
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Although assays for the most popular markers of exercise-induced oxidative stress may experience methodological flaws, there is sufficient credible evidence to suggest that exercise is accompanied by an increased generation of free radicals, resulting in a measurable degree of oxidative modifications to various molecules. However, the mechanisms responsible are unclear. A common assumption that increased mitochondrial oxygen consumption leads per se to increased reactive oxygen species (ROS) production is not supported by in vitro and in vivo data. The specific contributions of other systems (xanthine oxidase, inflammation, haem protein auto-oxidation) are poorly characterised. It has been demonstrated that ROS have the capacity to contribute to the development of muscle fatigue in situ, but there is still a lack of convincing direct evidence that ROS impair exercise performance in vivo in humans. It remains unclear whether exercise-induced oxidative modifications have little significance, induce harmful oxidative damage, or are an integral part of redox regulation. It is clear that ROS play important roles in numerous physiological processes at rest; however, the detailed physiological functions of ROS in exercise remain to be elucidated.
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3.4-Methylenedioxymethamphetamine (MDMA or "Ecstasy") is a widely abused, psychoactive recreational drug. There are growing evidences that the MDMA neurotoxic profile may be highly dependent on its hepatic metabolism. MDMA metabolism leads to the production of highly reactive derivates, namely catechols, catechol thioethers, and quinones. In this study the electrochemical oxidation-reduction processes of MDMA human metabolites, obtained by chemical synthesis, were evaluated by cyclic voltammetry based on an electrochemical cell with a glassy carbon working electrode. The toxicity of alpha-methyldopamine (alpha-MeDA), N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA) and 5-(glutathion-S-yl)-alpha-methyldopamine [5-(GSH)-alpha-MeDA] to rat cortical neurons was then correlated with their redox potential. The obtained data demonstrated that the lower oxidation potential observed for the catecholic thioether of alpha-MeDA correlated with the higher toxicity of this adduct. This accounts for the use of voltammetry data in predicting the toxicity of MDMA metabolites.
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Quinones and related quinonoid substances catalyze redox cycling at an alkaline pH in the presence of excess glycine as reductant. With nitroblue tetrazolium and oxygen present there is concomitant reduction of the tetrazolium to formazan. This property of quinonoid compounds is used for the specific staining of quinoproteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. The dopa-containing vitelline proteins and the 6-hydroxydopa-containing bovine serum amine oxidase are stained with the nitroblue tetrazolium/glycinate reagent. Also, the mammalian quinoproteins, diamine oxidase and lysyl oxidase, purported to contain pyrroloquinoline quinone, tested positive in this procedure. No quinonoid components were detected in three putative pyrroloquinoline quinone-containing quinoproteins, dopamine beta-hydroxylase, lipoxygenase, and peptidylglycine-amidating monoxygenase. Redox-cycling staining therefore confirms the presence of covalently bound quinones in the copper-dependent amine oxidases, but not in two putative quinoprotein oxygenases. Clarification of the biological significance of quinolation should be facilitated by identification of quinoproteins using this approach.
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The contribution of lipid peroxidation to myocardial injury by free radicals (FR) is still unclear. Consequently, we examined the functional damages inflicted on cultured rat cardiomyocytes (CM) during FR stress provoked by the xanthine/xanthine oxidase system (X/XO) or by a hydroperoxidized fatty acid ((9 Z, 11 E, 13 (S), 15 Z)-13-hydroperoxyocta-decatrienoic acid; 13-HpOTrE), in order to simulate in vitro the initial phase and the propagation phase of the FR attack, respectively. Transmembrane potentials were recorded with glass microelectrodes and contractions were monitored photometrically. The EPR spectroscopy showed that X/XO produced superoxide and hydroxyl radicals during 10 min. The X/XO system altered sharply and irreversibly the spontaneous electrical and mechanical activities of the CM. However, the gas chromatographic analysis showed that these drastic functional damages were associated with comparatively moderate membrane PUFA degradation. Moreover, the EPR analysis did not reveal the production of lipid-derived FR. 13-HpOTrE induced a moderate and reversible decrease in electrical parameters, with no change in CM contractions. These results indicate that the functional consequences of FR attack are dependent on the radical species present and do not support the idea that the membrane lipid breakdown is a major factor of myocardial oxidant dysfunction.
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Quinones represent a class of toxicological intermediates which can create a variety of hazardous effects in vivo, including acute cytotoxicity, immunotoxicity, and carcinogenesis. The mechanisms by which quinones cause these effects can be quite complex. Quinones are Michael acceptors, and cellular damage can occur through alkylation of crucial cellular proteins and/or DNA. Alternatively, quinones are highly redox active molecules which can redox cycle with their semiquinone radicals, leading to formation of reactive oxygen species (ROS), including superoxide, hydrogen peroxide, and ultimately the hydroxyl radical. Production of ROS can cause severe oxidative stress within cells through the formation of oxidized cellular macromolecules, including lipids, proteins, and DNA. Formation of oxidatively damaged bases such as 8-oxodeoxyguanosine has been associated with aging and carcinogenesis. Furthermore, ROS can activate a number of signaling pathways, including protein kinase C and RAS. This review explores the varied cytotoxic effects of quinones using specific examples, including quinones produced from benzene, polycyclic aromatic hydrocarbons, estrogens, and catecholamines. The evidence strongly suggests that the numerous mechanisms of quinone toxicity (i.e., alkylation vs oxidative stress) can be correlated with the known pathology of the parent compound(s).
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A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
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This toxicology update reviews the oxidative stress metabolites of catecholamines, postulated to be the biochemical initiators of cardiotoxicity. A brief overview of catecholamine metabolism is provided with several noteworthy historical observations relating to the autoxidation and rearrangement of epinephrine. The basic chemical and physical properties of adrenochrome and adrenolutin are discussed. The autoxidative, enzymatic and cellular basis for the transformation of catecholamines to oxidative metabolites is reviewed. Mechanisms seeking to account for the observed cardiotoxic changes in isolated heart perfusion studies and in vivo models are described. Copyright © 2001 John Wiley & Sons, Ltd.
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The mechanisms of thiol metabolism and chemistry have particular relevance to both cellular defenses against toxicant exposure and to redox signaling. Here, we will focus on glutathione (GSH), the major endogenous low- molecular-weight nonprotein thiol synthesized de novo in mammalian cells. The major pathways for GSH metabolism in defense of the cell are reduction of hydroperoxides by glutathione peroxidases (GSHPx) and some peroxiredoxins, which yield glutathione disulfide (GSSG), and conjugation reactions catalyzed by glutathione-S-transferases. GSSG can be reduced to GSH by glutathione reductase, but glutathione conjugates are excreted from cells. The exoenzyme γ-glutamyltranspeptidase (GGT) removes the glutamate from extracellular GSH, producing cysteinyl-glycine from which a dipeptidase then generates cysteine, an amino acid often limiting for de novo GSH synthesis. Synthesis of GSH from the constituent amino acids occurs in two regulated, enzymatically catalyzed steps. The signaling pathways leading to activation of the transcription factors that regulate these genes are a current area of intense investigation. The elucidation of the signaling for GSH biosynthesis in human bronchial epithelial cells in response to 4-hydroxynonenal (4HNE), an end product of lipid peroxidation, will be used as an example. GSH also participates in redox signaling through the removal of H2O2, which has the properties of a second messenger, and by reversing the formation of sulfenic acid, a moiety formed by reaction of critical cysteine residues in signaling proteins with H2O2. Disruption of GSH metabolism will therefore have major a impact upon function of cells in terms of both defense and normal physiology.
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Oxidation of l-3,4-dihydroxyphenylalanine (l-DOPA) and dopamine (DA) to generate semiquinones/quinones, oxygen radicals, and other reactive oxygen species may play a role in neuronal cell death in Parkinson's disease (PD). In particular, semiquinones/quinones can form conjugates with thiol compounds such as GSH and cysteine. Exposure of l-DOPA, DA, and other catecholamines to a system generating O2•− radical led to O2•−-dependent depletion of added GSH (or cysteine), accompanied by the formation of thiol-DA or -DOPA adducts as detected by HPLC. Superoxide could additionally cause destruction of these adducts. Iron or copper ions could also promote conjugate formation between GSH or cysteine and DA and l-DOPA, especially if H2O2 was present. We applied HPLC to measure glutathionyl and cysteinyl conjugates of l-DOPA, DA, and 3,4-dihydroxyphenylacetic acid (DOPAC) in postmortem brain samples from PD patients and normal control subjects. Conjugates were detected in most brain areas examined, but levels were highest in the substantia nigra and putamen. In most regions, adduct levels were lower in PD, but there were significant increases in cysteinyl adducts of l-DOPA, DA, and DOPAC in PD substantia nigra, suggesting that acceleration of l-DOPA/DA oxidation occurs in PD, although we cannot say if this is a primary feature of the disease or if it is related to therapy with l-DOPA. In vitro, conjugate formation could be inhibited by the dithiol dihydrolipoate but not by its oxidised form, lipoic acid.
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The purpose of this study was to evaluate rat tissue antioxidant status after repeated administration of d-amphetamine. Three groups of four rats each were used: control, d-amphetamine sulphate dosed (s.c., 20 mg/kg per day), and pair-fed. After 14 days of d-amphetamine daily administration, superoxide dismutase (CuZnSOD and MnSOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GRed), glutathione-S-transferase (GST), glutathione (GSH), cysteine and thiobarbituric acid reactive substances (TBARS) were measured in liver, kidney, and heart. Various serum and urine parameters were also analysed. d-Amphetamine treatment induced an increase of liver GSH, as well as a decrease of cysteine and MnSOD levels in this organ. A small increase in serum transaminases was also observed in comparison to the pair-fed group. Hepatic levels of TBARS, GPx, GRed and CuZnSOD were found to be similar among the three groups of rats. d-Amphetamine treatment induced an increase of kidney GST, GRed and catalase levels, and an elevation of N-acetyl-β-d-glucosaminidase efflux to the urine, accompanied by a decrease in urinary creatinine, compared to the pair-fed group. In d-amphetamine treated animals, heart cysteine levels were significantly depleted when compared to the pair-fed group, but all three groups of rats were found to have similar heart antioxidant enzyme levels. These results indicate that repeated administration of d-amphetamine caused a certain degree of stress in liver and kidney, which was followed by adaptations of antioxidant defences. The mechanisms involved in d-amphetamine-induced toxicity may explain the different adaptations observed for the studied organs.
The adrenaline release from the adrenal medulla increases during exercise, but at a given absolute work intensity the magnitude of this response is less pronounced in endurance trained vs sedentary individuals most likely due to a lower sympathetic stimulation of the adrenal medulla. However, when trained and untrained subjects are compared at identical relative work loads as well as in response to numerous non-exercise stimuli. endurance trained athletes have a higher epinephrine secretion capacity compared to sedentary individuals. This indicates a development of a so-called "sports adrenal medulla" as a result of a long term adaptation of an endocrine gland to physical training. Such an adaptation is parallel to adaptations taking place in other tissues like skeletal muscle and the heart. and can be advantageous in relation to both exercise performance in the competing athlete and cause a biological rejuvenation in relation to aging.
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Adult rat heart muscle cells were isolated after simultaneous perfusion of multiple (two to eight) hearts with buffered salt solutions containing collagenase and hyaluronidase. Yields (35 to 50% of ventricular weight with approximately 70% viability) are quantitatively suitable for metabolic studies. Viability has been determined by the ability of intact cells to exclude trypan blue and the inability of intact cells to oxidize exogenous succinate. Micrographs show that the fine structure of the isolated cells is well ordered. Cell concentrations of glycogen, glucose 6-phosphate, citrate, and various enzymes were similar to those of intact heart. ATP and creatine phosphate concentrations were lower than in whole hearts. Adenosine 3′,5′-monophosphate concentrations were somewhat elevated. Deoxyribonucleic acid was lower than in whole tissue. The isolated cells retain certain metabolic control mechanisms. The uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, increased oxygen consumption severalfold, whereas exogenous ADP had no effect on respiration. Under anaerobic conditions the rates of glucose utilization and lactate production were faster than in the presence of oxygen, indicating retention of the Pasteur effect. The addition of glucose and insulin caused a decrease in oxygen uptake or the Crabtree effect. Exogenously added pyruvate decreased glycolytic flux and produced a pronounced increase in intracellular citrate and glucose 6-phosphate. Isoproterenol stimulated adenylate cyclase activity of the isolated cells at the same concentrations effective with intact heart preparations. Isoproterenol and glucagon caused the activation of phosphorylase. The cells deteriorated as a function of incubation time, as indicated by a decrease in ATP content and a loss of lactate dehydrogenase into the medium. Cell deterioration was greatly accelerated by Ca2+ at concentrations greater than 10−5m.
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A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Article
The normal catabolism of catecholamines proceeds through enzymatic pathways (monoaminooxidase, catechol-o-methyltranserase, and phenolsulphotransferase). In addition, nonenzymatic oxidative pathways might take place since catechols are readily oxidized. In this review article, the pathways of formation of the oxidation products of catecholamines and their reactions are described. The interactions of these products with different biological systems and their toxicity are examined. Among the reactions known to occur is that with sulfhydryls, which results in either a covalently linked adduct or disulfide production. Another interesting pathway to toxicity involves the oxidation of these catecholamine products by oxygen, with the formation of damaging oxygen-derived species. The action of the oxidation products of catecholamines is outlined, with special attention to the nervous and cardiac systems.
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Cytotoxicity associated with exposure to quinones has generally been attributed to either redox cycling, and the subsequent development of "oxidative stress," and/or to their interaction with cellular nucleophiles, such as protein and non-protein sulfhydryls. Glutathione (GSH) is the major non-protein sulfhydryl present in cells, and conjugation of potentially toxic electrophiles with GSH is usually associated with detoxication and excretion. However, this review discusses the biological (re)activity of quinone-thioethers. For example, quinone-thioethers are (1) capable of redox cycling (2) substrates for, and inhibitors of, a variety of enzymes (3) methemoglobinemic (4) potent nephrotoxicants (5) DNA reactive and (6) may contribute to quinone-mediated carcinogenicity and neurotoxicity. The ubiquitous nature of quinones, and the high intracellular concentrations of GSH, ensures that cells and tissues will be exposed to quinone-thioethers. The toxicological importance of quinone-thioethers in quinone-mediated toxicities therefore deserves further attention.
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Reperfusion, without doubt, is the most effective way to treat the ischaemic myocardium. Late reperfusion may however cause further damage. Myocardial production of oxygen free radicals above the neutralizing capacity of the myocytes is an important cause of this reperfusion damage. There is evidence that prolonged ischaemia reduces the naturally occurring defence mechanisms of the heart against oxygen free radicals, particularly mitochondrial manganese superoxide dismutase, and intracellular pool of reduced glutathione. Consequently, reperfusion results in a severe oxidative damage, as evidenced by tissue accumulation and release of oxidized glutathione. An oxygen free radical-mediated impairment of mechanical function also occurs during reperfusion of human heart. In fact we observed during surgical reperfusion of coronary artery disease (CAD) patients, a prolonged and sustained release of oxidized glutathione;the degree of oxidative stress was inversely correlated with recovery of mechanical and haemodynamic function. These findings represent the rationale for therapeutic interventions which increase the cellular antioxidant capacities and improve the efficacy of myocardial reperfusion.
Article
Increasing evidence suggests that oxygen free radicals play a major role in the pathogenesis of reperfusion injury. Initial indirect evidence was based on beneficial effects of free radical scavengers administered exogenously at the time of postischemic reperfusion. Recent electron paramagnetic resonance (EPR) spectroscopy studies show a burst of oxygen-centered free radical generation during the first 60 seconds of reflow and administration of either a free radical scavenger, such as superoxide dismutase (SOD), or an iron chelator, such as deferoxamine, prevents this burst. The in vitro data obtained in a perfused rabbit heart model and the impressive reduction in infarct size, shown in an intact canine model, suggest that well-designed, randomized, placebo-controlled clinical trials of free radical scavengers and/or antioxidants should be performed to determine if postischemic reperfusion injury can be shown and/or prevented in humans.
Article
Publisher Summary This chapter discusses methods to determine carbonyl content in oxidatively modified proteins. The methods described are (1) reduction of the carbonyl group to an alcohol with tritiated borohydride; (2) reaction of the carbonyl group with 2,4-dinitrophenylhydrazine to form the 2,4-dinitrophenylhydrazone; (3) reaction of the carbonyl with fluorescein thiosemicarbazide to form the thiosemicarbazone; and (4) reaction of the carbonyl group with fluorescein amine to form a Schiff base followed by reduction to the secondary amine with cyanoborohydride. Van Poelje and Snell have also quantitated protein-bound pyruvoyl groups through formation of a Schiff base with p-aminobenzoic acid followed by reduction with cyanoborohydride. Although a systematic investigation has not appeared, this method should also be useful in detecting other protein-bound carbonyl groups. Carbonyl content of proteins is expressed as moles carbonyl/mole subunit for purified proteins of known molecular weight. For extracts, the results may be given as nanomoles carbonyl/milligram protein. For a protein having a molecular weight of 50,000, a carbonyl content of 1 mol carbonyl/mol protein corresponds to 20 nmol carbonyl/mg proteins.
Article
The preparation and properties of isolated adult cardiac myocytes are reviewed, with the goal being to evaluate their usefulness as a model system for measuring cardiotoxicity. Some important factors in cell isolation methodology which impact on the quality of the preparation are identified, along with criteria for assessing the quality of cells after isolation. By all criteria, myocytes isolated by good procedures appear to largely retain their original properties. Moreover, the distinctive behavior of adult myocytes under metabolic stress endows them with a particular usefulness as monitors of toxicity. Overall, we conclude that the art of adult heart cell isolation and culture is now sufficiently advanced for either freshly isolated cells in suspension or cells in culture to be a useful model system for toxicity studies.
Article
Adrenochrome is reduced by ascorbate in a reaction accompanied by a large and rapid oxygen uptake. The rates of adrenochrome reduction and the concomitant oxygen uptake are decreased in the presence of superoxide dismutase or catalase. The species formed on the one-electron reduction of adrenochrome (i.e., the semiquinone) was shown by pulse radiolysis to rapidly react with oxygen (9.10(8) M-1.s-1), indicating the occurrence of a redox cycling in a system formed by adrenochrome, a reducing agent, and oxygen. Adrenochrome is also reduced to the corresponding semiquinone by complex I of beef heart submitochondrial particles supplemented with NADH, while succinate is unable to support this reduction. The o-semiquinone is the intermediate species in the superoxide-generating cycle resulting from both non-enzymatic and enzymatic reduction. The toxic effects of adrenochrome and its pathophysiological role can be explained, at least in part, on the basis of the demonstrated cycle.
Article
The first part of the present review deals with the chemical and enzymatic synthesis of adrenochrome and other aminochromes from the corresponding catecholamines. A description of the most significant pathways of formation and the reactivity of the aminochromes is presented. In the second part of the toxicity of aminochromes, mainly at the cardiac and CNS level, is described and some of the molecular mechanisms of the toxic action are outlined. The toxicity of the aminochromes appears to depend mainly on the production of reduced oxygen species through redox cycling. The interaction of aminochromes with sulfhydryl groups and the induced depletion of oxygen, ascorbate and glutathione are additional mechanisms resulting in noxious effects at a cellular level.
Article
Oxygen radical production by polymorphonuclear leucocytes stimulated the oxidation of adrenalin through the adrenochrome pathway. This was detected either spectrophotometrically at 480 nm or separated by hplc and detected radiochemically. The oxidation was detectable within 5 min and continued for at least 4 h. Over the adrenalin concentration range 0.3 microM to 10 mM more than 80% of the oxidation that occurred was through the adrenochrome pathway, the remainder being through the amine oxidase, catechol methyl transferase pathway. Medium isolated after stimulation of the polymorphonuclear leucocytes was also able to oxidise adrenalin to adrenochrome. The results provide a cellular mechanism for the formation of adrenochrome and the other metabolites on this pathway of adrenalin metabolism, in inflammatory conditions where polymorphonuclear leucocyte infiltration occurs.
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