Dichloroacetate and Trichloroacetate Toxicity in AML12 Cells: Role of Oxidative Stress

The toxicity of the drinking water disinfection by products dichloroacetate (DCA) and trichloroacetate (TCA) was studied in the alpha mouse liver (AML12) cells at concentrations ranging between 770 and 4100 ppm and at incubation times ranging from 24 to 72 h. Cellular viability, superoxide anion (SA) and lipid peroxidation (LP) production, as well as superoxide dismutase (SOD) activity were determined. DCA and TCA resulted in time‐ and concentration‐dependent decreases in cellular viability, and also in significant increases in SA and LP production, and in SOD activity at specific concentrations and time points. The effective toxic concentrations of the compounds in these cells were found to be 10‐fold higher than those producing similar effects in the mouse liver. It has been concluded that the AML12 is a good screening system to identify toxic concentrations of the halaocetates present in the drinking water that may need further in vivo testing.     

The induction of oxidative stress and cellular death by the drinking water disinfection by-products,dichloroacetate and trichloroacetate in J774.A1 cells

The in vitro toxicity of the drinking water disinfection by products dichloroacetate (DCA) and trichloroacetate (TCA) were studied using the J774A.1 macrophage cell line. DCA and TCA were added to cell cultures at concentrations ranging between 8-32 mM and incubated for 24, 36 and 60 h. DCA and TCA effects on cellular viability, lactate dehydrogenase (LDH) release and superoxide anion (SA) production by the cells, as well as superoxide dismutase (SOD) activities of the cells were determined. DCA and TCA caused time- and concentration-dependent increases in cellular death, in LDH release and production of SA by the cells. The compounds also caused modulations in SOD activities of the cells, with increases observed at the lower concentrations and/or shorter periods of incubations and suppression with the higher concentrations and/or longer periods of incubation. The results of the study indicate that DCA and TCA induce macrophage activation and that the activation is associated with cellular toxicity. Also, DCA and TCA are found to be equitoxic to J774.A1 cells.     

The induction of tumor necrosis factor‐alpha,superoxide anion,myeloperoxidase, and superoxide dismutase in the peritoneal lavage cells of mice after prolonged exposure to dichloroacetate and trichloroacetate

The induction of phagocytic activation in response to prolonged treatment with different doses of dichloroacetate (DCA) and trichloroacetate (TCA) has been investigated in mice. Groups of B6C3F1 male mice were administered 7.7, 77, 154, and 410 mg of DCA or TCA/kg/day, postorally, for 4‐ and 13‐weeks. Peritoneal lavage cells (PLCs) were isolated and assayed for the different biomarkers of phagocytyic activation, including superoxide anion (SA), tumor necrosis factor‐alpha (TNF‐α), and myeloperoxidase (MPO). In addition, the role of superoxide dismutase (SOD) in the SA production was also assessed. DCA and TCA produced significant and dose‐dependent increases in SA and TNF‐α production and in MPO activity, but the increases in response to the high doses of the compounds (>77 mg/kg/day) in the 13‐week treatment period were less significant than those produced in the 4‐week treatment period. Also, dose‐dependent increases in SOD activity were observed in both periods of treatments. In general, the results demonstrate significant induction of the biomarkers of phagocytic activation by doses of DCA and TCA that were previously shown to be noncarcinogenic, with significantly greater increases observed at the earlier period of exposure, as compared with later period. These findings may argue against the contribution of those mechanisms to the hepatotoxicity/hepatocarcinogenicity of the compounds and suggest them to be early adaptive/ protective mechanisms against their long‐term effects. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:136–144, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20322     

The Induction of Phagocytic Activation by Mixtures of the Water Chlorination By‐Products,Dichloroacetate‐ and Trichloroacetate,in Mice after Subchronic Exposure

In this study, groups of B6C3F1 male mice were treated with dichloroacetate (DCA), trichloroacetate (TCA), and mixtures of the compounds (Mix I, II, and III) daily by gavage, for 13 weeks. The tested doses were 7.5, 15, and 30 mg DCA/kg/day and 12.5, 25, and 50 mg TCA/kg/day. The DCA: TCA ratios in Mix I, II, and III were 7.5:12.5, 15:25, and 30:50 mg/kg/day, respectively. Peritoneal lavage cells were collected at the end of the treatment period and assayed for the biomarkers of phagocytic activation, including superoxide anion and tumor necrosis factor‐alpha production, and myeloperoxidase activity. The mixtures produced nonlinear effects on the biomarkers of phagocytic activation, with Mix I and II effects were found to be additive, but Mix III effects were found to be less than additive. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:237‐242, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21476     

Effects of superoxide dismutase and polyclonal tumor necrosis factor-alpha antibodies on chloroacetate-induced cellular death and superoxide anion production by J774.A1 macrophages

Dichloroacetate (DCA) and trichloroacetate (TCA) are by-products that are formed during the process of water chlorination and have been previously shown to induce superoxide anion (SA) production and cellular death when added to J774.A1 macrophage cultures. In this study, the effects of superoxide dismutase (SOD) and polyclonal tumor necrosis factor-alpha (TNF-alpha) antibodies on DCA- and TCA-induced SA production and cellular death have been tested on the J774.A1 macrophage cultures. TCA and DCA were added to different cultures either alone, each at a concentration of 16 mM, or in combination with SOD (2-12 units/ml), or with TNF-alpha antibodies (10 and 25 units/ml). Cells were incubated for 48 h, after which cellular death/viability, lactate dehydrognase (LDH) leakage by the cells, and SA production by the cells were determined. While TCA and DCA caused significant cellular toxicity, indicated by reduction in cellular viability and increases in LDH leakage and SA production, SOD addition resulted in significant reduction of the effects induced by the compounds. On the other hand, addition of TNF-alpha antibodies to the DCA- and TCA-treated cultures resulted in significant reduction of DCA- but not TCA-induced cellular death and SA production by the cells. Although these results suggest a significant role for SA in DCA- and TCA-induced cellular death, they may also suggest two different mechanisms for the chloroacetate-induced SA production by the cells.     

Dichloroacetate- and trichloroacetate-induced phagocytic activation and production of oxidative stress in the hepatic tissues of mice after acute exposure

Dichoroacetate (DCA) and trichloroacetate (TCA) are by-products formed during chlorination of the drinking water and were found to be hepatotoxic and hepatocarcinogenic in rodents. In this study, the abilities of the compounds to induce oxidative stress and phagocytic activation have been studied in B6C3F1 mice. Groups of mice were administered 300 mg/kg of either DCA or TCA, p.o, and were sacrificed after 6 or 12 h. Peritoneal lavage cells (PLCs) were isolated and assayed for superoxide anion (SA) production, and hepatic tissues were assayed for the production of SA, lipid peroxidation (LP), and DNA-single strand breaks (SSBs). TCA resulted in significant production of SA in the PLCs, and in the production of SA, LP, and DNA-SSBs in the hepatic tissues, 12 h after dosing, as compared with the control. DCA administration, on the other hand, resulted in significant increases in the productions of LP and DNA-SSBs in the hepatic tissues at both time points, and in SA production in PLCs and hepatic tissues, 6 h after dosing. However, DCA-induced increases in SA production in PLC and hepatic tissues declined at the 12-h time point, reaching control level in the hepatic tissues. These results may implicate the contribution of phagocytic activation to the induction of oxidative stress in the hepatic tissues and also the role of SA production in the induction of LP and/or DNA damage in those tissues, in response to the compounds. The results also suggest studying the involvement of these mechanisms in the long-term hepatotoxicity/hepatocarcinogencity of the compounds.     

Mutation of PEB4 alters the outer membrane protein profile of Campylobacter jejuni

Although anaerobic bioremediation of chlorinated organic contaminants in the environment often requires exogenous supply of hydrogen as an electron donor, little is known about the ability of hydrogen-producing bacteria to grow in the presence of chlorinated solvents. In this study, 18 Clostridium strains including nine uncharacterized isolates originating from chlorinated solvent contaminated groundwater were tested to determine their ability to fermentatively produce hydrogen in the presence of three common chlorinated aliphatic groundwater contaminants: 1,2-dichloroethane (DCA), 1,1,2-trichloroethane (TCA), and tetrachloroethene (PCE). All strains produced hydrogen in the presence of at least 7.4 mM DCA, 2.4 mM TCA, and 0.31 mM PCE. Some strains produced hydrogen in media containing concentrations as high as 29.7 mM DCA, 9.8 mM TCA, and 1.1 mM PCE. None of the strains biotransformed chlorinated solvents under the conditions tested. Results demonstrate that many Clostridium species are chlorinated solvent tolerant, producing hydrogen even in the presence of high concentrations of DCA, TCA, and PCE. These findings have important implications for bioremediation of contaminated soil and groundwater.     

Developmental effects of trichloroacetate in Zebrafish embryos: Association with the production of superoxide anion

To assess the developmental toxicity of trichloroacetate (TCA), zebrafish embryos were exposed to 8 to 48 mM of TCA and evaluated for developmental milestones from 8‐ to 144‐hour postfertilization (hpf). All developmental toxicities are reported in this paper. Embryos were found to have developed edema in response to 16 to 48 mM of TCA exposure at 32‐ to 80‐hpf, experienced delay in hatching success in response to 24 to 48 mM at 80‐hpf. Lordosis was observed in developing embryos exposed to 40 to 48 mM at 55‐ to 144‐hpf. The observed toxic effects of TCA exposure were found to be concentration and exposure period independent. Effects were found to be associated with increases in superoxide anion production, but these increases were also found to be concentration and time independent. TCA resulted in concentration‐dependent increases in embryonic lethality at 144‐hpf, with an LC₅₀ determined to be 29.7 mM.     

Chromium-induced production of reactive oxygen species,DNA single-strand breaks,nitric oxide production,and lactate dehydrogenase leakage in J774A.1 cell cultures

The involvement of oxidative stress in the toxicity of chromium (VI) and chromium (III) has been proposed. We have therefore examined the effects of these cations on the production of superoxide anion, nitric oxide (NO), and DNA single strand breaks (SSB) in J774A.1 macrophage cells in culture as well as the effects on lactate dehydrogenase (LDH) leakage and cell viability. Following a 48 hour incubation, over twofold increases in superoxide anion and NO production were observed at concentrations of approximately 0.30 and 50 μM for Cr (VI) and Cr (III), respectively. The patterns of cell viability and LDH leakage paralleled superoxide anion and NO production for Cr (VI) and Cr (III). A 50% decrease in viability was observed at approximately the concentrations that produced a twofold increase in superoxide and NO production. Concentration-dependent increases in DNA-SSB were observed after incubation with Cr (III) with maximum increases occurring at a concentration of approximately 60 μM. Cr (VI) had no effect on the incidence of DNA-SSB at any of the tested concentrations. The results indicate that Cr (VI) and Cr (III) are toxic to the J774A.1 cell line, and the toxicity may be due at least in part to an oxidative stress induced by the production of reactive oxygen species. © 1996 John Wiley & Sons, Inc.     

Improving culture performance and antibody production in CHO cell culture processes by reducing the Warburg effect

Lactate is one of the key waste metabolites of mammalian cell culture. High lactate levels are caused by high aerobic glycolysis, also known as the Warburg effect, and are usually associated with adverse culture performance. Therefore, reducing lactate accumulation has been an ongoing challenge in the cell culture development to improve growth, productivity, and process robustness. The pyruvate dehydrogenase complex (PDC) plays a crucial role for the fate of pyruvate, as it converts pyruvate to acetyl coenzyme A (acetyl‐CoA). The PDC activity can be indirectly increased by inhibiting the PDC inhibitor, pyruvate dehydrogenase kinase, using dichloroacetate (DCA), resulting in less pyruvate being available for lactate formation. Here, Chinese hamster ovary cells were cultivated either with 5 mM DCA or without DCA in various batch and fed‐batch bioreactor processes. In all cultures, DCA increased peak viable cell density (VCD), culture length and final antibody titer. The strongest effect was observed in a fed batch with media and glucose feeding in which peak VCD was increased by more than 50%, culture length was extended by more than 3 days, and the final antibody titer increased by more than twofold. In cultures with DCA, lactate production and glucose consumption during exponential growth were on average reduced by approximately 40% and 35%, respectively. Metabolic flux analysis showed reduced glycolytic fluxes, whereas fluxes in the tricarboxylic acid (TCA) cycle were not affected, suggesting that cultures with DCA use glucose more efficiently. In a proteomics analysis, only few proteins were identified as being differentially expressed, indicating that DCA acts on a posttranslational level. Antibody quality in terms of aggregation, charge variant, and glycosylation pattern was unaffected. Subsequent bioreactor experiments with sodium lactate and sodium chloride feeding indicated that lower osmolality, rather than lower lactate concentration itself, improved culture performance in DCA cultures. In conclusion, the addition of DCA to the cell culture improved culture performance and increased antibody titers without any disadvantages for cell‐specific productivity or antibody quality.     

Anaerobic transformation of 1,1,1-trichloroethane by municipal digester sludge

Anaerobic transformations of 1,1,1-trichloroethane (TCA), 1,1-dichloroethane (DCA), and chloroethane (CA) were studied with sludge from a lab-scale, municipal wastewater sludge digester. TCA was biologically transformed to DCA and CA and further to ethane by reductive dechlorination. TCA was also converted to acetic acid and 1,1-dichloroethene (11DCE) by cell-free extract. 11DCE was further biologically converted to ethene. This pathway was confirmed by transformation tests of TCA, DCA and CA, by tests with cell-free extract, and by chloride release during TCA degradation. With cell-free extract, acetic acid accounted for approximately 90% of the TCA transformed; tests with live cells indicate that the fraction of TCA transformed by this pathway decreased with lower biomass. The dechlorination of DCA to CA and CA to ethane was not stoichiometric. A high rate of TCA removal was observed under the experimental conditions. The results indicate that removal of TCA in anaerobic digestion should be complete, but DCA and CA could persist in a normally operating digester.     

Oestrogenic compounds and oxidative stress (in human sperm and lymphocytes in the Comet assay)

Reactive oxygen species (ROS) are produced by a wide variety of chemicals and physiological processes in which enzymes catalyse the transfer of electrons from a substrate to molecular oxygen. The immediate products of such reactions, superoxide anion radicals and hydrogen peroxide can be metabolised by enzymes such as superoxide dismutase (SOD) and catalase (CAT), respectively, and depending on its concentration by Vitamin C (Vit C). Under certain circumstances the ROS form highly reactive hydroxyl radicals. We examined human sperm and lymphocytes after treatment with six oestrogenic compounds in the Comet assay, which measures DNA damage, and observed that all caused damage in both cell types. The damage was diminished in nearly all cases by catalase, and in some instances by SOD and Vit C. This response pattern was also seen with hydrogen peroxide. This similarity suggests that the oestrogen-mediated effects could be acting via the production of hydrogen peroxide since catalase always markedly reduced the response. The variable responses with SOD indicate a lesser involvement of superoxide anion radicals due to SOD-mediated conversion of superoxide to hydrogen peroxide generally causing a lower level of DNA damage than other ROS. The variable Vit C responses are explained by a reduction of hydrogen peroxide at low Vit C concentrations and a pro-oxidant activity at higher concentrations. Together these data provide evidence that inappropriate exposure to oestrogenic compounds could lead to free-radical mediated damage. It is believed that the observed activities were not generated by cell free cell culture conditions because increased responses were observed over and above control values when the compounds were added, and also increasing dose-response relationships have been found after treatment with such oestrogenic compounds in previously reported studies.     

Neutrophil superoxide anion production,and CAT and GSSGR activity in patients with tuberculosis

Tuberculosis (TB) is a chronic infection disease caused by Mycobacterium tuberculosis (MTB), as an intracellular pathogen. Various cytokines (TNF-alpha, IL's, GSF etc.) and other factors play important preventing roles and are secreted during the infection. It may cause changes in the metabolism of neutrophils. Production of superoxide anion and antioxidative enzymes activities, such as glutathione reductase (GSSGR) and catalase (CAT) may be changed during MTB infection in the host. In this study, the control group consisted of ten healthy subjects and ten patients with TB were studied before anti-TB treatment. Level of superoxide anion production, activity of CAT and activity of GSSGR were studied from peripheral neutrophils of healthy subjects and patients with TB. Catalase activities of the neutrophils were significantly lower in patients with TB than normal subjects (p < 0.01). Glutathione reductase activities of the neutrophils were also significantly lower in patients with TB than normal subjects (p < 0.05). Superoxide anion production in the neutrophils did not show any significant difference between TB and normal subjects (p > 0.05). As a result, the activities of CAT and GSSGR were lower in the peripheral neutrophils of patients with TB than normal subjects, whereas superoxide anion production in the neutrophils did not differ between in TB patients than normal subjects.     

Differential modulation of glucose,lactate, and pyruvate oxidation by insulin and dichloroacetate in the rat heart

Despite the fact that lactate and pyruvate are potential substrates for energy production in vivo, our understanding of the control and regulation of carbohydrate metabolism is based principally on studies where glucose is the only available carbohydrate. Therefore, the purpose of this study was to determine the contributions of lactate, pyruvate, and glucose to energy production in the isolated, perfused rat heart over a range of insulin concentrations and after activation of pyruvate dehydrogenase with dichloroacetate (DCA). Hearts were perfused with physiological concentrations of [1-13C]glucose, [U-13C]lactate, [2-13C]pyruvate, and unlabeled palmitate for 45 min. Hearts were freeze clamped, and 13C NMR glutamate isotopomer analysis was performed on tissue extracts. Glucose, lactate, and pyruvate all contributed significantly to myocardial energy production; however, in the absence of insulin, glucose contributed only 25-30% of total pyruvate oxidation. Even under conditions where carbohydrates represented >95% of substrate entering the tricarboxylic acid (TCA) cycle, we found that glucose contributed at most 50-60% of total carbohydrate oxidation. Despite being present at only 0.1 mM, pyruvate contributed between approximately 10% and 30% of total acetyl-CoA entry into the TCA cycle. We also found that insulin and DCA not only increased glucose oxidation but also exogenous pyruvate oxidation; however, lactate oxidation was not increased. The differential effects of insulin and DCA on pyruvate and lactate oxidation provide further evidence for compartmentation of cardiac carbohydrate metabolism. These results may have important implications for understanding the mechanisms underlying the beneficial effects of increasing cardiac carbohydrate metabolism.     

Dichloroacetate-induced developmental toxicity and production of reactive oxygen species in zebrafish embryos

Dichloroacetate (DCA) is one of the toxic by products that are formed during the chlorine disinfection process of drinking water. In this study, the developmental toxicity of DCA has been determined in zebrafish (Danio rerio) embryos. Embryos were exposed to different concentrations (4, 8, 16, and 32 mM) of the compound at the 4 h postfertilization (hpf) stage of development, and were observed for different developmental toxic effects at 8, 24, 32, 55, 80, and 144 hpf. Exposure of embryos to 8-32 mM of DCA resulted in significant increases in the heart rate and blood flow of the 55 and 80 hpf embryos that turned into significant decreases at the 144 hpf time point. At 144 hpf, malformations of mouth structure, notochord bending, yolk sac edema and behavioral effects including perturbed swimming and feeding behaviors were also observed. DCA was also found to produce time- and concentration-dependent increases in embryonic levels of superoxide anion (O2*-) and nitric oxide (NO), at various stages of development. The results of the study suggest that DCA-induced developmental toxic effects in zebrafish embryos are associated with production of reactive oxygen species in those embryos.     

The effect of the trichloroethylene metabolites trichloroacetate and dichloroacetate on peroxisome proliferation and DNA synthesis in cultured human hepatocytes

Dichloroacetate (DCA) and trichloroacetate (TCA) are metabolites of the environmental contaminant trichloroethylene (TCE) that are thought to be responsible for its hepatocarcinogenicity in B6C3F₁ mice. TCA and DCA induce peroxisomal proliferation and are mitogenic in rodent liver. The susceptibility of humans to TCA- and DCA-induced hepatocarcinogenesis is unknown. The current studies were aimed at using both primary and long-term human hepatocyte cultures to study the effects of TCA, DCA, and a potent peroxisome proliferator, WY-14,643, on peroxisomal activity and DNA synthesis in human hepatocytes. Peroxisome proliferation, as assessed by palmitoyl-CoA oxidation activity, was below the limit of detection in all human cell lines tested. However, the human cell lines did display small but significant increases in CYP450 4A11 levels following treatment with WY-14,643 (0.1 mmol/L), indicting that the CYP 4A11 gene may be regulated by peroxisome proliferator-activated receptor α in humans. Similarly to their effect in rodent hepatocyte cultures, TCA and DCA were not complete mitogens in human hepatocyte cultures. In fact, DNA synthesis tended to be significantly decreased following treatment of the cells with WY-14,643, TCA, or DCA. In contrast to rodent hepatocyte responses, TCA and DCA did not increase palmitoyl-CoA oxidation and caused a decrease in DNA synthesis in human hepatocyte cultures, suggesting that humans may not be susceptible to TCA- and DCA-induced hepatocarcinogenesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Antioxidant activity of liver growth factor,a bilirubin covalently bound to albumin

We previously reported that treatment of spontaneously hypertensive rats (SHR) with liver growth factor (LGF), an albumin–bilirubin complex with a covalent bond, reduces blood pressure, improves nitric oxide (NO)-dependent vasodilatation, and exerts vascular antifibrotic actions. Because bilirubin, albumin, and albumin-bound bilirubins have antioxidant properties, we hypothesize that LGF might exert its cardiovascular actions through an antioxidant mechanism. We have tested in vitro the capacity of LGF to scavenge ABTS cation and peroxyl and hydroxyl radicals and to protect vascular NO from degradation by superoxide anion. We have also compared the antioxidant capacity of LGF with that of its molecular components albumin and bilirubin and the reference antioxidant trolox. LGF exhibited antioxidant capacity against all free radicals tested at lower concentrations than albumin, bilirubin, and trolox. LGF, bilirubin, and albumin were also able to protect endothelial NO from superoxide anion degradation in a fashion similar to that of superoxide dismutase or tiron, but at much lower concentrations. These data, together with our previous results in SHR, suggest that LGF might exert its cardiovascular regenerative actions, at least in part, through an antioxidant mechanism and that LGF could be a relevant circulating antioxidant in situations of oxidative stress.     

Inhibition of macrophage activation by isoquinolinesulfonamides, phenothiazines, and a napthalenesulfonamide

The influence of isoquinolinesulfonamides (H-7 and H-8), phenothiazines(trifluoperazine and fluphenazine), and a naphthalenesulfonamide (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on stimulated superoxide anion production and phosphatidyl inositol (PI) cycle activity was investigated in the guinea pig alveolar macrophage. All five drugs were able to inhibit superoxide anion production stimulated by n-formyl-nel-leu-phe (FNLP), leukotriene B4 (LTB4), and phorbol-12,13-dibutyrate (PDB). The order of potency was trifluoperazine greater than or equal to fluphenazine greater than H-7 = W-7 greater than H-8. The dose response curves could be shifted to less efficacy by increasing extracellular calcium. By itself, W-7 markedly stimulated 45Ca+2 efflux, fluphenazine and trifluoperazine slightly stimulated 45Ca+2 efflux, while H-7 and H-8 had no effect on 45Ca+2 efflux from macrophages preloaded with 45Ca+2. Consistent with these results, W-7 markedly stimulated PI cycle activity, fluphenazine and trifluoperazine slightly stimulated PI cycle activity, while H-7 and H-8 had no significant effects on PI cycle activity. In addition, W-7 by itself was able to stimulate a weak and short-lived "burst" of superoxide anion production. In order to evaluate whether a site of action of the inhibitors was at protein kinase C and whether protein kinase C was involved in terminating the normally short-lived FNLP- and LTB4-stimulated macrophage activation, fluphenazine and H-7 were used to evaluate the duration of FNLP- and LTB4-stimulated PI cycle activity, at concentrations of the inhibitors that significantly blocked stimulated superoxide anion production. In all cases, FNLP and LTB4 still stimulated PI cycle activity, which still terminated even though protein kinase C was inhibited. These results suggest that all five drugs block protein kinase C, but H-7 was the most specific in its action at protein kinase C, while the phenothiazines and W-7 have multiple sites of action. In addition, these results suggest that protein kinase C may not function to cause the termination of FNLP- and LTB4-stimulated PI cycle activity and subsequent superoxide anion production.     

Formation of mixed disulfide of cystatin-beta in cultured macrophages treated with various oxidants

Macrophage cell cultures were treated with menadione, zymosan, or phorbol myristate acetate (PMA), and changes in productions of superoxide anion and hydroperoxide, and in glutathione oxidation and S-thiolation of cystatin-beta (formation of a mixed disulfide of cystatin-beta and glutathione) were examined. All three compounds promoted production of superoxide anion and hydroperoxide, but only menadione caused extensive oxidation of glutathione. Menadione caused S-thiolation of cystatin-beta in a dose-dependent fashion, but the other two compounds did not. Removal of menadione promptly reduced the oxidation of glutathione and S-thiolation of cystatin-beta induced by menadione. Inhibition of catalase by aminotriazol caused slight increase in the GSSG content in both menadione- and zymosan-treated cells, but not in S-thiolation of cystatin-beta in zymosan-treated cells. None of the three compounds influenced appreciably the activity of glutathione peroxidase, glutathione reductase, or superoxide dismutase in cultured cells. These results indicate that S-thiolation of cystatin-beta occurs in cells in response to oxidative challenge by menadione but not by zymosan or by the tumor promoter PMA. Dethiolation of cystatin-beta by purified thiol transferase and protein disulfide isomerase in the presence of different concentrations of GSH was examined in vitro. Both enzymes catalyzed dethiolation of cystatin-beta at a much lower level of GSH than that required for the non-enzymatic reaction, suggesting the importance of enzymatic catalysis of S-thiolation and dethiolation of cystatin-beta in cells.     

Modulation by ellagic acid of DCA-induced developmental toxicity in the zebrafish (Danio rerio)

The ability of ellagic acid (EA) to modulate dichloroacetic acid (DCA)-induced developmental toxicity and oxidative damage was examined in zebrafish embryos. Embryos were exposed to 20 mM EA administered concomitantly with 32 mM DCA at 4 hours postfertilization (hpf) and 20 h later. Embryos were observed through 144 hpf for developmental malformations, and production of superoxide anion (SA) and nitric oxide (NO) was determined in embryonic homogenates. DCA was shown to produce developmental abnormalities and significant levels of SA and NO in zebrafish embryos. EA exposure alleviated the developmental malformations observed in treated embryos and decreased the levels of SA and NO in those same embryos. Less than 10% of DCA + EA exposed embryos showed developmental malformations compared to 100% of embryos treated with DCA alone. Animals in this group that developed malformations were shown to have fewer defects than those treated with DCA only. Taken together, the results confirm the involvement of oxidative stress in the developmental toxicity of DCA in zebrafish embryos, and suggest possible protection against those effects with the use of antioxidants.