Treatment with pentylenetetrazole (PTZ) and 4-aminopyridine (4-AP) differently affects survival, locomotor activity, and biochemical markers in Drosophila melanogaster.

PTZ is a convulsive agent that acts via selective blockage of GABAA receptor channels, whereas 4-AP leads to a convulsive episode via blockage of K+ channels. However, the mechanism(s) by which pentylenetetrazole (PTZ) and 4-aminopyridine (4-AP) cause toxicity to Drosophila melanogaster needs to be properly explored, once it will help in establishing an alternative model for development of proper therapeutic strategies and also to counteract the changes associated with exposure to both epileptic drugs. For the purpose, we investigated the effects of exposure (48 h) to PTZ (60 mM) and/or 4-AP (20 mM) on survival, locomotor performance, and biochemical markers in the body and/or head of flies. 4-AP-fed flies presented a higher incidence of mortality and a worse performance in the open field test as compared to non-treated flies. 4-AP also caused a significant increase in the reactive species (RS) and protein carbonyl (PC) content in the body and head. Also a significant increase in catalase and acetylcholinesterase (AChE) activities was observed in the body. In the same vein, PTZ exposure resulted in a significant increase in RS, thiobarbituric acid reactive substances (TBARS), PC content, and catalase activity in the body. PTZ exposure also caused a significant increase in AChE activity both in body and head. It is important to note that PTZ-treated flies also down-regulated the NRF2 expression. Moreover, both 4AP- and PTZ-fed flies presented a significant decrease in MTT reduction, down-regulation, and inhibition of SOD in body. However, SOD was significantly more active in the head of both 4-AP and PTZ-treated flies. Our findings provide evidence regarding the toxicological potential of both PTZ and/or 4-AP to flies. This model will help in decoding the underlying toxicological mechanisms of the stated drugs. It will also help to properly investigate the therapeutic strategies and to counteract the drastic changes associated with both epileptogenic drugs.

Continuous liquid feeding: New method to study pesticides toxicity in Drosophila melanogaster.

Fly fruit Drosophila melanogaster (DM) has been extensively employed as an in vivo model system to study pesticides toxicity. Pesticide administration to the fly traditionally involves feeding in an agar-gelled feed fly's medium (AM). However, AM method has several limitations such as uncertainty regarding the bioavailability and amount of pesticides ingested. And also high manipulation of the treated flies. We developed a new method of exposure the flies to pesticides, called Continuous Liquid Feeding (CLF). This method successfully delivers food to the flies at much higher concentrations than the AM method, and requires little manipulation of flies under treatment.

Mentha pulegium crude extracts induce thiol oxidation and potentiate hemolysis when associated to t-butyl hydroperoxide in human's erythrocytes.

Mentha pulegium (Lamiaceae) tea has been used as a traditional medicine; however, the modulatory effect of M. pulegium extracts on damage to human erythrocytes associated to t-butyl hydroperoxide (t-BHP) exposure remains to be investigated. Accordingly, we perform this study in order to test the hypothesis that aqueous and ethanolic extracts of M. pulegium could modulate the hemolysis associated to t-BHP exposure, non-protein thiol (NPSH) oxidation and lipid peroxidation (measured as thiobarbituric acid reactive substances - TBARS) in human erythrocytes. Samples were co-incubated with t-BHP (4 mmol/L) and/or aqueous or ethanolic extracts (10-1000 mg/mL) during 120 min to further analysis. We found that both extracts, when associated to t-BHP, potentiate NPSH oxidation and hemolysis. Moreover, both extracts significantly prevents against t-BHP-induced TBARS production. A significant correlation among hemolysis and NPSH levels was found. Taking together, our data points that the association of M. pulegium extracts with t-BHP culminates in toxic effect to exposed erythrocytes, besides its protective effect against t-BHP-induced TBARS production. So, we infer that the use of this extract may exert negative effect during painful crisis in sickle cell anemia. However, more studies are still necessary to better investigate/understand the mechanism(s) involved in the toxic effect resultant from this association.

Ilex paraguariensis crude extract acts on protection and reversion from damage induced by t-butyl hydroperoxide in human erythrocytes: a comparative study with isolated caffeic and/or chlorogenic acids.

BACKGROUND: Studies comparing the effects of phytochemicals under different regimens of exposure are necessary to give a better indication about their mechanism(s) of protection. Hence, in the present study, we investigated the preventive (pre-incubation), protective (co-incubation) and/or remediative (post-incubation) activity of chlorogenic acid and caffeic acids, in comparison with Ilex paraguariensis crude extract, against t-butyl hydroperoxide (t-BHP)-induced damage to human erythrocytes. RESULTS: We found that both caffeic and chlorogenic acids were able to prevent and revert the hemolysis associated with t-BHP exposure. By contrast, isolated compounds (alone or in combination) presented no effect on basal and/or t-BHP-induced non-protein thiol (NPSH) oxidation or production of thiobarbituric acid reactive substances (TBBARS). In turn, I. paraguariensis extract was effective to prevent, protect and revert the hemolysis associated with t-BHP exposure. Moreover, I. paraguariensis significantly protects and reverts t-BHP-induced NPSH oxidation and TBARS production. CONCLUSIONS: We have found that I. paraguariensis extract acts better with respect to the protection and reversion of t-BHP-associated changes, whereas isolated compounds are more active in preventing and reverting t-BHP pro-hemolytic action. Moreover, our data suggest that the pro-hemolytic activity of t-BHP may occur via mechanism(s) other(s) than lipid peroxidation and/or NPSH oxidation. © 2016 Society of Chemical Industry.

Peumus boldus (Boldo) Aqueous Extract Present Better Protective Effect than Boldine Against Manganese-Induced Toxicity in D. melanogaster.

The cellular, intracellular and molecular mechanism(s) underlying the toxicity of Mn are still incompletely understood, although several points concerning Mn neurotoxicity have been addressed. Importantly, oxidative changes have been reported to be involved in Mn-induced toxicity. As a consequence, antioxidants are expected to offer protection in Drosophila melanogaster exposed to this metal. So, in this study we evaluated the hypothesis that the aqueous extract of boldo (Peumus boldus), and its alkaloids boldine, could prevent/ameliorate behavioral and oxidative alterations induced by Mn in a D. melanogaster intoxication model. Adult wild-type flies were concomitantly exposed to Mn (3 mM) and boldo aqueous extract (5 mg/mL) or boldine (327.37 µg/mL) in the food during 9 days. Mn-fed flies had a worse performance in the negative geotaxis assay and in the open-field test, as well as a higher incidence of mortality and TBARS levels in head and body, when compared to control group. Boldo aqueous extract was found to reduce the mortality rate of the flies exposed to Mn. In turn, boldine was ineffective against Mn-induced mortality and significantly increases mortality per se. Additionally, Mn-induced locomotors dysfunction were fully ameliorated by boldo crude extract and only partially ameliorated by boldine. Likewise, boldo completely normalize head and body TBARS levels, whereas boldine only partially normalize in body. Finally, we found that flies treated with Mn presented significantly decrease in dopamine levels. Our results suggest that boldo crude extract can exert protective effect against Mn-induced toxicity in D. melanogaster, whereas boldine do not. Moreover, our data confirm the utility of this model to investigate potential therapeutic strategies on movement disorders, such as that caused by Mn.

Cryotherapy reduces skeletal muscle damage after ischemia/reperfusion in rats.

The aim of this study was to analyze the effects of cryotherapy on the biochemical and morphological changes in ischemic and reperfused (I/R) gastrocnemius muscle of rats. Forty male Wistar rats were divided into control and I/R groups, and divided based on whether or not the rats were submitted to cryotherapy. Following the reperfusion period, biochemical and morphological analyses were performed. Following cryotherapy, a reduction in thiobarbituric acid-reactive substances and dichlorofluorescein oxidation levels were observed in I/R muscle. Cryotherapy in I/R muscle also minimized effects such as decreased cellular viability, levels of non-protein thiols and calcium ATPase activity as well as increased catalase activity. Cryotherapy also limited mitochondrial dysfunction and decreased the presence of neutrophils in I/R muscle, an effect that was corroborated by reduced myeloperoxidase activity in I/R muscle treated with cryotherapy. The effects of cryotherapy are associated with a reduction in the intensity of the inflammatory response and also with a decrease in mitochondrial dysfunction.

MeHg exposure impairs both the catecholaminergic and cholinergic systems resulting in motor and non-motor behavioral changes in Drosophila melanogaster.

Human exposure to the natural environmental contaminant methylmercury (MeHg) has been associated to adverse health effects. Importantly, the mechanisms by which this organomercurial exerts its neurotoxicity have yet to be fully clarified. Therefore, the aim of this study was to evaluate whether exposure to MeHg alters dopamine (DA) and octopamine (OA) levels, acetylcholinesterase (AChE) activity and impacts both motor and non-motor behaviours. We studied the effect of MeHg by feeding 1-2 d old flies (male and females) with 25 and 50 µM MeHg for 4 d and determined effects on survival, motor and non-motor behaviours, oxidative stress, AChE and tyrosine hydroxylase (TH) activities, as well as DA and OA levels. We found that Drosophila melanogaster (D. melanogaster) exposed to MeHg showed a reduction in survival rate, associated with the inhibition of AChE and TH activities in head of flies and decreased DA and OA levels. These changes were accompanied by behavioural alterations, such as locomotor deficit and increased grooming behaviour, in addition to an increase in oxidative stress markers both in head and in body of flies, and an increase in glutathione-S-transferase (GST) activity in head of flies. Collectively, our data support the hypothesis that MeHg neurotoxicity is associated with altered OA and DA levels, AChE inhibition, which may serve, at least in part, as the underpinnings of both motor and non-motor behavioural changes.

Guanosine and synthetic organoselenium compounds modulate methylmercury-induced oxidative stress in rat brain cortical slices: involvement of oxidative stress and glutamatergic system.

Excessive formation of reactive oxygen species (ROS) and disruption of glutamate uptake have been pointed as two key mechanisms in methylmercury-toxicity. Thus, here we investigate the involvement of glutamatergic system in methylmercury (MeHg) neurotoxicity and whether diphenyl diselenide, ebselen and guanosine could protect cortical rat brain slices from MeHg-induced ROS generation. MeHg (100 and 200 microM) increased 2',7'-dichlorodihydrofluorescin (DCFH) oxidation after 2h of exposure. At 50 microM, MeHg increased DCFH oxidation only after 5h of exposure. Guanosine (1 and 5 microM) did not caused any effect per se; however, it blocked the increase in DCFH caused by 200 or 50 microM MeHg. Ebselen (5 and 10 microM) decreased significantly the DCFH oxidation after 2 and 5h of exposure to MeHg. Diphenyl diselenide (5 microM) did not change the basal DCFH oxidation, but abolished the pro-oxidant effect of MeHg. MK-801 also abolished the pro-oxidant effect of MeHg. These results demonstrate for the first time the potential antioxidant properties of organoseleniun compounds and guanosine against MeHg-induced ROS generation after short-term exposure in a simple in vitro model. In conclusion, endogenous purine (guanosine) and two synthetic organoselenium compounds can modulate the pro-oxidant effect of MeHg in cortical brain slices.

Thimerosal inhibits Drosophila melanogaster tyrosine hydroxylase (DmTyrH) leading to changes in dopamine levels and impaired motor behavior: implications for neurotoxicity.

Thimerosal (THIM) is a well-established antifungal and antiseptic agent widely used as a preservative in vaccines. Recent studies identified the neurotoxic effects of THIM, including malfunction of the monoaminergic system. However, the underlying cytotoxic mechanisms are not well understood. Here we used the fruit fly Drosophila melanogaster to investigate the mechanisms of THIM-induced neurotoxicity. We focused on the dopaminergic system, and the rate-limiting enzyme tyrosine hydroxylase (DmTyrH), to test the hypothesis that THIM can impair dopamine (DA) homeostasis and subsequently cause dysfunction. We studied the effect of THIM by feeding 1-2 day old flies (both sexes) food supplemented with 25 µM THIM for 4 days and determined THIM-induced effects on survival, oxidative stress, and metabolic activity based on MTT assay and acetylcholinesterase (AChE) activity. Our results demonstrate that D. melanogaster exposed to THIM present changes in DmTyrH expression and activity, together with altered DA levels that led to impaired motor behavior. These phenotypes were accompanied by an increase in oxidative stress, with a decrease in MTT reduction, in AChE activity, and also in survival rate. These findings suggest an initiating and primary role for THIM-mediated DmTyrH dysfunction that leads to impaired DA function and behavioral abnormalities, ultimately causing oxidative stress-related neurotoxicity.

Cellular Responses in Drosophila melanogaster Following Teratogen Exposure.

Studies focusing on the teratogenicity of a series of new chemicals that are produced in a daily basis represent an important focus in toxicological/pharmaceutical research, particularly due to the risks arising from occupational exposure of the subjects. However, the complex mating procedures, scheduling of treatments, requirements for trained personnel, and elevated costs of traditional teratological assays with mammals hamper this type of assessments. Accordingly, the use of Drosophila melanogaster as a model for teratological studies has received considerable attention. Here some general protocols about Drosophila exposure-at different stages of their life cycle-to any chemical with putative teratological activity are presented. Importantly, some details about D. melanogaster embryonic, larval, pupal, or adult endpoints, that can be used to assess teratogenicity using flies as a model organism, are presented.

Diphenyl diselenide decreases the prevalence of vacuous chewing movements induced by fluphenazine in rats.

RATIONALE: Chronic treatment with neuroleptics causes, as a side effect, tardive dyskinesia in humans; however, the mechanisms involved in its pathophysiology remain unclear. OBJECTIVES: The purpose of this study was to examine the effects of diphenyl diselenide, an organoselenium compound with antioxidant properties, in an animal model of vacuous chewing movements (VCMs) induced by long-term treatment with fluphenazine. RESULTS: Adult male rats were treated during 24 weeks with fluphenazine (25 mg/kg, intramuscularly [i.m.], once every 21 days) and diphenyl diselenide (1 mg/kg, subcutaneously, three times a week). VCMs and body weight gain were quantified every 3 weeks. The fluphenazine treatment produced VCMs in the majority of the treated rats (87% after 24 weeks). Concomitant treatment with diphenyl diselenide decreased the prevalence of VCMs to 50%. Additionally, we separated the rats that developed or did not develop VCMs. We did not find any statistical differences among the groups when oxidative stress parameters were evaluated. Chronic fluphenazine treatment significantly decreased [(3)H]-dopamine uptake. Concomitant treatment with diphenyl diselenide was not able to prevent this decrease in those rats that developed VCMs. CONCLUSIONS: Our data suggest that the reduction in dopamine transport can be a possible mechanism related to the maintenance of VCMs in rats. Moreover, diphenyl diselenide seems to be a promising pharmacological agent in the reduction in the prevalence of VCMs in rats.

Paraquat exposure-induced Parkinson's disease-like symptoms and oxidative stress in Drosophila melanogaster: Neuroprotective effect of Bougainvillea glabra Choisy.

Extracts from the leaves of Bougainvillea glabra Choisy are used in traditional medicines, but their actions on the central nervous system have not been studied. In the present study, we investigated the potential neuroprotective effects of Bougainvillea glabra Choisy leaf extract (BG extract) against paraquat (PQ)-induced neurotoxicity. Male adult wild-type flies (1- 4days old) were exposed to PQ (3.5mM) and/or BG extract (120µg/mL) through food for 4days. PQ-fed flies had decreased locomotor capacity in negative geotaxis and crossing number assays and had a higher incidence of mortality than the control group. PQ neurotoxicity was also associated with a marked decrease in dopamine levels and increase in acetylcholinesterase (AChE) activity, reactive oxygen species (ROS) production and lipid peroxidation. Co-exposure to BG extract prevented mortality, and dopamine depletion, improved locomotor performance and decreased AChE activity, ROS production and lipid peroxidation. GC-MS and HPLC analyses of BG extract revealed the presence of many antioxidant compounds such as phytol, α,γ-tocopherol, squalene, stigmasterol, geranylgeraniol, quercetin, and caffeic, vanillic, coumaric, ferulic acids. Our results showed neuroprotective effects of BG extract, reflecting the presence of antioxidant compounds. Thus, we suggested that B. glabra leaves could be considered an effective agent in the prevention of neurological disorders, where dopamine depletion and/or oxidative stress are involved, as in Parkinson's disease (PD).

Mitochondrial electron transfer chain complexes inhibition by different organochalcogens.

Mitochondrial dysfunction plays a pivotal role in the cell toxicology and death decision. The aim of the present study was to investigate the effect of three organocompounds (ebselen [Ebs], diphenyl diselenide [(PhSe)(2)] and diphenyl ditelluride [(PhTe)(2)]) on mitochondrial complexes (I, II, I-III, II-III and IV) activity from rat liver and kidney to determine their potential role as molecular targets of organochalcogens. All studied organochalcogens caused a statistically significant inhibition of the mitochondrial complex I activity. Ebs and (PhTe)(2) caused a statistically significant inhibition of the mitochondrial complex II activity in both hepatic and renal membranes. Hepatic mitochondrial complex II activity was practically unchanged by (PhSe)(2), whereas it significantly inhibited renal complex II activity. Mitochondrial complex IV activity was practically unchanged by the organochalcogens. Furthermore, organochalcogens inhibited the mitochondrial respiration supported by complex I or complex II substrates. The inhibitory effect of Ebs, (PhSe)(2) and (PhTe)(2) on mitochondrial complex I was prevented by NADH, but it was not prevented by catalase (CAT) and/or superoxide dismutase (SOD). Additionally, the organochalcogens-induced inhibition of complex I and II was completely reversed by reduced glutathione (GSH). In conclusion, Ebs, (PhSe)(2) and (PhTe)(2) were more effective inhibitors of renal and hepatic mitochondrial complex I than complex II, whereas complexes III and IV were little modified by these compounds. Taking into account the presented results, we suggest that organochalcogen-induced mitochondrial complexes I and II inhibition can be mediated by their thiol oxidation activity, i.e., Ebs, (PhSe)(2) and (PhTe)(2) can oxidize critical thiol groups from mitochondrial complexes I and II. So, mitochondrial dysfunction can be considered an important factor in the toxicity of Ebs, (PhSe)(2) and (PhTe)(2).

The influence of Bauhinia forficata Link subsp. pruinosa tea on lipid peroxidation and non-protein SH groups in human erythrocytes exposed to high glucose concentrations.

ETHNOPHARMACOLOGICAL RELEVANCE: Bauhinia forficata (BF) has been traditionally used as tea in folk medicine of Brazil for treatment of Diabetes mellitus (DM). AIM OF THE STUDY: To evaluate the effects of BF leaf tea on markers of oxidative damage and antioxidant levels in an experimental model of hyperglycemia in human erythrocytes in vitro. MATERIALS AND METHODS: Human erythrocytes were incubated with high glucose concentrations or glucose and BF tea for 24h and 48h. After incubation lipid peroxidation and non-protein SH levels were analyzed. Moreover, quantification of polyphenols and flavonoids, iron chelating property, scavenging of DPPH, and prevention of lipid peroxidation in isolated lipids were also assessed. RESULTS: A significant amount of polyphenols and flavonoids was observed. The main components found by LC-MS analysis were quercetin-3-O-(2-rhamnosyl) rutinoside, kaempferol-3-O-(2-rhamnosyl) rutinoside, quercetin-3-O-rutinoside and kaempferol-3-O-rutinoside. BF tea presents important antioxidant and chelating properties. Moreover, BF tea was effective to increase non-protein SH levels and reduce lipid peroxidation induced by high glucose concentrations in human erythrocytes. CONCLUSION: The antioxidant effects of BF tea could be related to the presence of different phenolic and flavonoids components. We believe that these components can be responsible to protect human erythrocytes exposed to high glucose concentrations against oxidative damage.

Apocynin prevents vascular effects caused by chronic exposure to low concentrations of mercury.

UNLABELLED: Mercury increases the risk of cardiovascular disease and oxidative stress and alters vascular reactivity. This metal elicits endothelial dysfunction causing decreased NO bioavailability via increased oxidative stress and contractile prostanoid production. NADPH oxidase is the major source of reactive oxygen species (ROS) in the vasculature. Our aim was to investigate whether treatment with apocynin, an NADPH oxidase inhibitor, prevents the vascular effects caused by chronic intoxication with low concentrations of mercury. Three-month-old male Wistar rats were treated for 30 days with a) intramuscular injections (i.m.) of saline; b) HgCl(2) (i.m. 1(st) dose: 4.6 µg/kg, subsequent doses: 0.07 µg/kg/day); c) Apocynin (1.5 mM in drinking water plus saline i.m.); and d) Apocynin plus HgCl(2). The mercury treatment resulted in 1) an increased aortic vasoconstrictor response to phenylephrine and reduced endothelium-dependent responses to acetylcholine; 2) the increased involvement of ROS and vasoconstrictor prostanoids in response to phenylephrine, whereas the endothelial NO modulation of such responses was reduced; and 3) the reduced activity of aortic superoxide dismutase (SOD) and glutathione peroxidase (GPx) and increased plasma malondialdehyde (MDA) levels. Treatment with apocynin partially prevented the increased phenylephrine responses and reduced the endothelial dysfunction elicited by mercury treatment. In addition, apocynin treatment increased the NO modulation of vasoconstrictor responses and aortic SOD activity and reduced plasma MDA levels without affecting the increased participation of vasoconstrictor prostanoids observed in aortic segments from mercury-treated rats. CONCLUSIONS: Mercury increases the vasoconstrictor response to phenylephrine by reducing NO bioavailability and increasing the involvement of ROS and constrictor prostanoids. Apocynin protects the vessel from the deleterious effects caused by NADPH oxidase, but not from those caused by prostanoids, thus demonstrating a two-way action.

Mentha pulegium crude extracts induce thiol oxidation and potentiate hemolysis when associated to t-butyl hydroperoxide in human's erythrocytes

ABSTRACT Mentha pulegium (Lamiaceae) tea has been used as a traditional medicine; however, the modulatory effect of M. pulegium extracts on damage to human erythrocytes associated to t-butyl hydroperoxide (t-BHP) exposure remains to be investigated. Accordingly, we perform this study in order to test the hypothesis that aqueous and ethanolic extracts of M. pulegium could modulate the hemolysis associated to t-BHP exposure, non-protein thiol (NPSH) oxidation and lipid peroxidation (measured as thiobarbituric acid reactive substances - TBARS) in human erythrocytes. Samples were co-incubated with t-BHP (4 mmol/L) and/or aqueous or ethanolic extracts (10-1000 mg/mL) during 120 min to further analysis. We found that both extracts, when associated to t-BHP, potentiate NPSH oxidation and hemolysis. Moreover, both extracts significantly prevents against t-BHP-induced TBARS production. A significant correlation among hemolysis and NPSH levels was found. Taking together, our data points that the association of M. pulegium extracts with t-BHP culminates in toxic effect to exposed erythrocytes, besides its protective effect against t-BHP-induced TBARS production. So, we infer that the use of this extract may exert negative effect during painful crisis in sickle cell anemia. However, more studies are still necessary to better investigate/understand the mechanism(s) involved in the toxic effect resultant from this association.

Mitochondrial dysfunction induced by different organochalchogens is mediated by thiol oxidation and is not dependent of the classical mitochondrial permeability transition pore opening.

Ebselen (Ebs) and diphenyl diselenide [(PhSe)(2)] readily oxidize thiol groups. Here we studied mitochondrial swelling changes in mitochondrial potential (Deltapsim), NAD(P)H oxidation, reactive oxygen species production, protein aggregate formation, and oxygen consumption as ending points of their in vitro toxicity. Specifically, we tested the hypothesis that organochalchogens toxicity could be associated with mitochondrial dysfunction via oxidation of vicinal thiol groups that are known to be involved in the regulation of mitochondrial permeability (Petronilli et al. J. Biol. Chem., 269; 16638; 1994). Furthermore, we investigated the possible mechanism(s) by which these organochalchogens could disrupt liver mitochondrial function. Ebs and (PhSe)(2) caused mitochondrial depolarization and swelling in a concentration-dependent manner. Furthermore, both organochalchogens caused rapid oxidation of the mitochondrial pyridine nucleotides (NAD(P)H) pool, likely reflecting the consequence and not the cause of increased mitochondrial permeability (Costantini, P., Chernyak, B. V., Petronilli, V., and Bernardi, P. (1996). Modulation of the mitochondrial permeability transition pore (PTP) by pyridine nucleotides and dithiol oxidation at two separate sites. J. Biol. Chem. 271, 6746-6751). The organochalchogens-induced mitochondrial dysfunction was prevented by the reducing agent dithiothreitol (DTT). Ebs- and (PhSe)(2)-induced mitochondrial depolarization and swelling were unchanged by ruthenium red (4microM), butylated hydroxytoluene (2.5microM), or cyclosporine A (1microM). N-ethylmaleimide enhanced the organochalchogens-induced mitochondrial depolarization, without affecting the magnitude of the swelling response. In contrast, iodoacetic acid did not modify the effects of Ebs or (PhSe)(2) on the mitochondria. Additionally, Ebs and (PhSe)(2) decreased the basal 2' 7' dichlorofluorescin diacetate (H(2)-DCFDA) oxidation and oxygen consumption rate in state 3 and increased it during the state 4 of oxidative phosphorylation and induced the formation of protein aggregates, which were prevented by DTT. However, DTT failed to reverse the formation of protein aggregates, when it was added after a preincubation of liver mitochondria with Ebs or (PhSe)(2). Similarly, DTT did not reverse the Ebs- or (PhSe)(2)-induced Deltapsim collapse or swelling, when it was added after a preincubation period of mitochondria with chalcogenides. These results show that Ebs and (PhSe)(2) can effectively induce mitochondrial dysfunction and suggest that effects of these compounds are associated with mitochondrial thiol groups oxidation. The inability of cyclosporine A to reverse the Ebs- and (PhSe)(2)-induced mitochondrial effects suggests that the redox-regulated mitochondrial permeability transition (MPT) pore was mechanistically regulated in a manner that is distinct from the classical MPT pore.

Krebs cycle intermediates modulate thiobarbituric acid reactive species (TBARS) production in rat brain in vitro.

The aim of this study was to investigate the effect of Krebs cycle intermediates on basal and quinolinic acid (QA)- or iron-induced TBARS production in brain membranes. Oxaloacetate, citrate, succinate and malate reduced significantly the basal and QA-induced TBARS production. The potency for basal TBARS inhibition was in the order (IC50 is given in parenthesis as mM) citrate (0.37) > oxaloacetate (1.33) = succinate (1.91) > > malate (12.74). alpha-Ketoglutarate caused an increase in TBARS production without modifying the QA-induced TBARS production. Cyanide (CN-) did not modify the basal or QA-induced TBARS production; however, CN- abolished the antioxidant effects of succinate. QA-induced TBARS production was enhanced by iron ions, and abolished by desferrioxamine (DFO). The intermediates used in this study, except for alpha-ketoglutarate, prevented iron-induced TBARS production. Oxaloacetate, citrate, alpha-ketoglutarate and malate, but no succinate and QA, exhibited significantly iron-chelating properties. Only alpha-ketoglutarate and oxaloacetate protected against hydrogen peroxide-induced deoxyribose degradation, while succinate and malate showed a modest effect against Fe2+/H2O2-induced deoxyribose degradation. Using heat-treated preparations citrate, malate and oxaloacetate protected against basal or QA-induced TBARS production, whereas alpha-ketoglutarate induced TBARS production. Succinate did not offer protection against basal or QA-induced TBARS production. These results suggest that oxaloacetate, malate, succinate, and citrate are effective antioxidants against basal and iron or QA-induced TBARS production, while alpha-ketoglutarate stimulates TBARS production. The mechanism through which Krebs cycle intermediates offer protection against TBARS production is distinct depending on the intermediate used. Thus, under pathological conditions such as ischemia, where citrate concentrations vary it can assume an important role as a modulator of oxidative stress associated with such situations.