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     

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.     

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.     

Comparative toxicity studies on bromochloroacetate,dibromoacetate, and bromodichloroacetate in J774A.1 macrophages: Roles of superoxide anion and protein carbonyl compounds

The brominated and mixed bromo‐chloro‐haloacetates, such as dibromoacetate (DBA), bromochloroacetate (BCA), and bromodichloroacetate (BDCA), are by‐products of water chlorination and are found at lower levels than the fully chlorinated acetates in the drinking water. The toxicities of the compounds were assessed in J774A.1 cells and were found to induce concentration‐dependent increases in cell death and superoxide anion and protein carbonyl compounds production. Compared to the previously tested concentrations of dichoroacetate (DCA) and trichloroacetate (TCA) in the same cell line, the tested haloacetates induced similar effects on cellular viability and superoxide anion production but at DBA and BCA concentrations that were approximately 40–160 times lower than those of DCA and TCA, and at BDCA concentrations that were 4–16 times lower than those of DCA and TCA. Also, production of super oxide anion, protein carbonyl compounds, and induction of phagocytic activation are suggested to play a role in their toxicity.     

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.     

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.     

Salvianolic acid B protects against acute lung injury by decreasing TRPM6 and TRPM7 expressions in a rat model of sepsis

This study aimed to investigate the protective effects of salvianolic acid B (SA‐B) on acute lung injury (ALI) through decreasing the expressions of channel kinase's TRPM6 and TRPM7. Wistar Septic rat models were established by lipopolysaccharide (LPS), which were separated into the control, lipopolysaccharide (LPS), SA‐B, SA‐B + si‐TRPM6, SA‐B + si‐TRPM7, si‐TRPM6, and si‐TRPM7 groups. Arterial blood gas, protein content, total white blood cell (WBC) count and the percentage of polymorphonuclear neutrophils (PMN%) were measured. Levels of TNF‐α and IL‐6 levels in bronchoalveolar lavage fluid (BALF) were monitored. Lung coefficient, myeloperoxidase (MPO) and superoxide dismutase (SOD) activity were conducted by MPO and SOD kit. The mRNA expressions of TRPM6 and TRPM7 were detected by qRT‐PCR. Compared with the control group, the PaO₂ and PaO₂/FiO₂ values exhibited decreases in other group, while the PaCO₂ value_, protein content, total WBC, PMN%, TNF‐α, IL‐6 levels and lung coefficient values all increased. MPO activity in lung tissue increased, while SOD activity decreased. TRPM6 and TRPM7 expressions increased significantly. Compared with the LPS group, the SA‐B, SA‐B + si‐TRPM6, SA‐B + si‐TRPM7, si‐TRPM6, and si‐TRPM7 groups had increased PaO₂ and the PaO₂/FiO₂, while decreased PaCO_2, protein content, total WBC, PMN%, TNF‐α, IL‐6 levels, and lung coefficient. MPO activity in lung tissue decreased while SOD activity increased. Decreased mRNA expressions of TRPM6 and TRPM7 in the SA‐B, SA‐B + si‐TRPM6, and SA‐B + si‐TRPM6 groups were observed. Through decreasing the expressions of the channel kinase TRPM6 and TRPM7, SA‐B protects against ALI in septic rats.     

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.     

The role of antioxidant enzymes in TCDD-induced oxidative stress in various brain regions of rats after subchronic exposure

The induction of oxidative stress by TCDD in various brain regions of rats has been investigated after subchronic exposure. TCDD was administered by gavage to female Sprague-Dawley rats at daily doses of 0, 10, 22, and 46 ng/kg for 13 weeks. The brains were dissected to cerebral cortex (Cc), hippocampus (H), cerebellum (C), and brain stem (Bs); the production of superoxide anion (SA) and lipid peroxides and the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px) were determined in those regions. TCDD caused dose-dependent increases in the production of SA and lipid peroxidation in Cc and H and those were associated with dose-dependent suppressions of SOD. While a TCDD dose of 10 ng/kg/d resulted in significant increases in catalase and GSH-Px activities in Cc and H, doses of 22 and 46 ng/kg/d resulted in dose-dependent suppressions of these two enzymes in the same regions. In the C and Bs, TCDD treatment did not result in significant production of SA and lipid peroxidation but it resulted in dose-dependent increases in the activities of various antioxidant enzymes. These results suggest that Cc and H are vulnerable to TCDD-induced oxidative stress after subchronic exposure, and that C and Bs are protected against that effect.     

Myeloperoxidase, xanthine oxidase and superoxide dismutase in the gastric mucosa of helicobacter pylori positive and negative pediatric patients

The aim of this study was determination and comparison of the levels of myeloperoxidase (MPO), xanthine oxidase (XO), and superoxide dismutase (SOD) in gastric mucosa of children who were infected and noninfected with Helicobacter pylori (HP). The MPO, and XO enzyme activities were detected via kinetic measurement, and the MPO, XO and SOD enzyme protein levels were detected via Western blot, in antral mucosa specimens of 43 patients who underwent upper gastrointestinal endoscopy with various indications. The diagnosis of HP infection was made with a positive rapid urease test and histopathologic detection. MPO activity and enzyme protein levels were measured in 14 [8 HP (+) and 6 HP (−)], and in 9 [5 HP (+) and 4 HP (−)] while XO activity and enzyme protein levels were measured in 16 [10 HP (+) and 6 HP (−)] and in 9 [5 HP (+) and 4 HP (−)] patients, respectively. SOD protein level was detected in 13 [7 HP (+) and 6 HP (−)] patients. Of 43 patients 25 were HP (+) and 18 were HP (−). MPO activities were 75.6 ± 40.5 and 98.8 ± 44.1 U/g. protein (p = 0.302) while XO activities were 0.5 ± 0.3 and 0.4 ± 0.2 U/g. protein in HP (+) and HP (−) patients, respectively (p = 0.625). Measured enzyme protein levels of MPO, XO and SOD were found statistically indifferent in HP (+) and HP (−) patients (p = 0.327, p = 0.086, and p = 0.775, respectively). The results of this study revealed that, MPO, XO and SOD conditions in gastric mucosa alone were not affected from HP presence. That's why MPO, XO, and SOD may not have important roles in the pathogenesis of HP related gastric disease in children.     

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O₂^- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O₂^- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.

Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O₂^- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O₂^- production and MPO release from stimulated neutrophils.     

Induction of manganese superoxide dismutase by tumor necrosis factor in human breast cancer MCF-7 cell line and its TNF-resistant variant

Tumor necrosis factor (TNF)-resistant variant of human mammary cancer MCF-7 cell line was isolated by stepwise selection. The final TNF-resistant variant Tnf-1000 showed more than 100-fold higher resistance than the parental MCF-7 cell. Saturation kinetics for 125I-TNF binding showed that TNF-1000 cells had similar TNF receptor numbers as MCF-7 cells, but of a lower affinity. Induction of superoxide dismutase (SOD) was compared between MCF-7 and Tnf-1000 cells treated with TNF: SOD scavenges potentially toxic superoxide radicals. TNF induced more mitochondrial manganese SOD (SODm) in MCF-7 than in Tnf-1000 whereas there appeared to be no significant induction of cytosolic copper/zinc SOD (SODc) by TNF in both MCF-7 and Tnf-1000 cell lines. Acquirement of TNF-resistance in MCF-7 cells might be correlated with expression of SODm.     

The possible neurobehavioral protective effects of natural antioxidant against phototoxicity attenuation of antimicrobial quinolone group in rats

The fluoroquinolones absorb light in the 320 to 330 nm ultraviolet A (UV‐A) wavelength and produce reactive oxygen species (ROS) such as superoxide anion, hydroxyl radical, and hydrogen peroxide; thus, the photodynamic generation of ROS may be the basis of phototoxicity of quinolones in human beings and animals. This study aimed to evaluate the damaging effects of UV‐A radiation at different periods of exposure on rats' brains administered with ciprofloxacin. Ciprofloxacin administration in UV‐A exposed animals exaggerated the brain‐oxidative stress biomarkers and decreased the locomotor activity. Exposure of rats to UV‐A for 60 minutes induced a significant increase of malondialdehyde (MDA), myeloperoxidase (MPO), and a decrease in the values of superoxide dismutase (SOD), glutathione (GSH) compared to a normal one; these changes were UV‐A exposure time–dependent. However, the administration of vitamin C to the UV‐60‐treated group decreased the values of MDA, MPO, and shifted the values of SOD, GSH toward the normal values. Vitamin C, probably due to its strong antioxidant properties, could improve and partially counteract the toxic effect of UV‐A on oxidative stress parameters and prevent the damage in rat's brain tissues.     

Effects of niacin on bleomycin-induced increases in myeloperoxidase,prolyl hydroxylase,and superoxide dismutase activities and collagen accumulation in the lungs of hamsters

It has been shown that lung nicotinamide adenine dinucleotide (NAD) depletion accompanies bleomycin (BL)-induced lung fibrosis in the hamster and that treatment with niacin (NA), a precursor of NAD, was found to attenuate lung fibrosis caused by this agent. Niacin was used in the present study to investigate changes in some biochemical parameters and enzymes involved in the development of BL-induced lung fibrosis in the hamster. Niacin (500 mg/kg, IP), or an equivalent volume of saline (SA, IP), was given daily 2 days prior to intratracheal instillation of BL (7.5 U/5 mL/ kg) or SA and everyday thereafter throughout the study. Hamsters were killed at 1, 4, 7, 10, and 14 days after the BL or SA instillation and their lungs processed for various biochemical assays. Hydroxyproline content and superoxide dismutase (SOD) activity in SABL treated animals were significantly (P ± 0.05) elevated at 7 and 10 days, peaking at 14 days to 161 ± 11% and 159 ± 11% of the SASA treated animals, respectively. Although the hydroxyproline level of NABL treated animals was significantly elevated at 7 and 10 days and peaked at 14 days to 123 ± 8% of the NASA control, these values were significantly lower than the SABL treated animals at the corresponding times. The lung SOD activity of NABL groups was significantly higher at 4 days but significantly lower at 10 and 14 days than the SABL groups at the corresponding times. Prolyl hydroxylase (PH) activity and total lung calcium in SABL treated groups were significantly elevated compared to SASA treated groups starting at 4 days, with PH peaking at 10 days to 163 ± 13% and calcium peaking at 7 days to 148 ± 8% of SASA treated groups. The NABL treated animals displayed a significant elevation in PH activity at 4 days only (132 ± 15%), while the calcium content in this group was significantly increased at 4 and 14 days compared to NASA treated animals. However, the activity of PH in the NABL treated animals was significantly lower than the SABL treated animals at 7, 10, and 14 days. The calcium content of the NABL group was significantly lower than the SABL group at 7 and 10 days. The thiobarbituric acid reactive substance equivalents (TBARS) content and myeloperoxidase (MPO) activity were significantly elevated at all time points in SABL groups as compared to SASA groups, with peak elevation of TBARS to 160 ± 9% at 4 days and MPO to 268 ± 40% at 1 day. The TBARS content of NABL groups was significantly elevated above NASA groups at 1, 4, 7, and 10 days. The MPO activity in NABL groups was significantly elevated above NASA groups at 1, 4, 7, and 14 days, but the activity was significantly lower than SABL groups at 4 and 14 days. The data suggest that NA inhibits the extent of oxidative damage or enhances processes involved in the repair of BL-induced alveolar epithelial damage.     

Effect of pre-treatment with dichloroacetic or trichloroacetic acid in drinking water on the pharmacokinetics of a subsequent challenge dose in B6C3F1 mice

Dichloroacetate (DCA) and trichloroacetate (TCA) are prominent by-products of chlorination of drinking water. Both chemicals have been shown to be hepatic carcinogens in mice. Prior work has demonstrated that DCA inhibits its own metabolism in rats and humans. This study focuses on the effect of prior administration of DCA or TCA in drinking water on the pharmacokinetics of a subsequent challenge dose of DCA or TCA in male B6C3F1 mice. Mice were provided with DCA or TCA in their drinking water at 2 g/l for 14 days and then challenged with a 100 mg/kg i.v. (non-labeled) or gavage (14C-labeled) dose of DCA or TCA. The challenge dose was administered after 16 h fasting and removal of the haloacetate pre-treatment. The haloacetate blood concentration-time profile and the disposition of 14C were characterized and compared with controls. The effect of pre-treatment on the in vitro metabolism of DCA in hepatic S9 was also evaluated. Pre-treatment with DCA caused a significant increase in the blood concentration-time profiles of the challenge dose of DCA. No effect on the blood concentration-time profile of DCA was observed after pre-treatment with TCA. Pre-treatment with TCA had no effect on subsequent doses of DCA. Pre-treatment with DCA did not have a significant effect on the formation of 14CO2 from radiolabeled DCA. In vitro experiments with liver S9 from DCA-pre-treated mice demonstrated that DCA inhibits it own metabolism. These results indicate that DCA metabolism in mice is also susceptible to inhibition by prior treatment with DCA, however the impact on clearance is less marked in mice than in F344 rats. In contrast, the metabolism and pharmacokinetics of TCA is not affected by pre-treatment with either DCA or TCA.     

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O₂^- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O₂^- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.

Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O₂^- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O₂^- production and MPO release from stimulated neutrophils.     

Hypobromous acid and bromamine production by neutrophils and modulation by superoxide

MPO (myeloperoxidase) catalyses the oxidation of chloride, bromide and thiocyanate to their respective hypohalous acids. We have investigated the generation of HOBr by human neutrophils in the presence of physiological concentrations of chloride and bromide. HOBr was trapped with taurine and detected by monitoring the bromination of 4-HPAA (4-hydroxyphenylacetic acid). With 100 microM bromide and 140 mM chloride, neutrophils generated HOBr and it accounted for approx. 13% of the hypohalous acids they produced. Addition of SOD (superoxide dismutase) doubled the amount of HOBr detected. Therefore we investigated the reaction of superoxide radicals with a range of bromamines and bromamides and found that superoxide radicals stimulated the decomposition of these species, with this occurring in a time- and dose-dependent manner. The protection afforded by SOD against such decay demonstrates that these processes are superoxide-radical-dependent. These data are consistent with neutrophils generating HOBr at sites of infection and inflammation. Both HOBr and bromamines/bromamides have the potential to react with superoxide radicals to form additional radicals that may contribute to inflammatory tissue damage.     

Induction of resistance to TNF cytotoxicity and mitochondrial superoxide dismutase on U-937 cells by 1,25-dihydroxyvitamin D3

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) dose-dependently inhibited the cytotoxicity of tumor necrosis factor (TNF) in a human monoblastic leukemic cell line, U-937. Combination of TNF and 1,25(OH)2D3 remarkably increased mitochondrial superoxide dismutase (mSOD) of U-937 cells, TNF alone increased it only slightly and 1,25(OH)2D3 alone did not. The cytosolic SOD (cSOD) activity was not changed by TNF or/and 1,25(OH)2D3. The mSOD activity was not inhibited by 2 mM KCN, suggesting that mSOD should be a manganese SOD (MnSOD). These results suggest that 1,25(OH)2D3 may reduce the susceptibility to TNF cytotoxicity of U-937 cells by enhancing the ability of inducing MnSOD by TNF.     

Innate Immune Responses and Antioxidant/Oxidant Imbalance Are Major Determinants of Human Chagas Disease

Background

We investigated the pathological and diagnostic role of selected markers of inflammation, oxidant/antioxidant status, and cellular injury in human Chagas disease.

Methods

Seropositive/chagasic subjects characterized as clinically-symptomatic or clinically-asymptomatic (n = 116), seronegative/cardiac subjects (n = 102), and seronegative/healthy subjects (n = 45) were analyzed for peripheral blood biomarkers.

Results

Seropositive/chagasic subjects exhibited an increase in sera or plasma levels of myeloperoxidase (MPO, 2.8-fold), advanced oxidation protein products (AOPP, 56%), nitrite (5.7-fold), lipid peroxides (LPO, 12–17-fold) and malondialdehyde (MDA, 4–6-fold); and a decline in superoxide dismutase (SOD, 52%) and glutathione (GSH, 75%) contents. Correlation analysis identified a significant (p<0.001) linear relationship between inflammatory markers (AOPP/nitrite: r = 0.877), inflammation and antioxidant/oxidant status (AOPP/glutathione peroxidase (GPX): r = 0.902, AOPP/GSH: r = 0.806, Nitrite/GPX: 0.773, Nitrite/LPO: 0.805, MDA/MPO: 0.718), and antioxidant/oxidant levels (GPX/MDA: r = 0.768) in chagasic subjects. Of these, MPO, LPO and nitrite biomarkers were highly specific and sensitive for distinguishing seropositive/chagasic subjects from seronegative/healthy controls (p<0.001, training and fitting AUC/ROC >0.95). The MPO (r = 0.664) and LPO (r = 0.841) levels were also correlated with clinical disease state in chagasic subjects (p<0.001). Seronegative/cardiac subjects exhibited up to 77% decline in SOD, 3–5-fold increase in LPO and glutamate pyruvate transaminase (GPT) levels, and statistically insignificant change in MPO, AOPP, MDA, GPX, GSH, and creatine kinase (CK) levels.

Conclusions

The interlinked effects of innate immune responses and antioxidant/oxidant imbalance are major determinants of human Chagas disease. The MPO, LPO and nitrite are excellent biomarkers for diagnosing seropositive/chagasic subjects, and MPO and LPO levels have potential utility in identifying clinical severity of Chagas disease.     

The effect of dichloroacetic acid and trichloroacetic acid on DNA methylation and cell proliferation in B6C3F1 mice

The chlorine disinfection by-products, dichloroacetic acid (DCA) and trichloroacetic acid (TCA), are carcinogenic in mouse liver. We have previously reported that DCA and TCA induced DNA hypomethylation in mouse liver. In the present study, we determined the temporal association for DNA hypomethylation and cell proliferation. Female B6C3F1 mice were administered daily doses of 500 mg/kg DCA or TCA by gavage and sacrificed at 24, 36, 48, 72, and 96 hours after the first dose. The proliferating cell nuclear antigen-labeling index in the liver was increased at 72 and 96 hours by both DCA and TCA, that is, at 72 hours the index was 1.00 +/- 0.21, 0.51 +/- 0.11, and 0.095 +/- 0.016 for DCA, TCA, and the vehicle control, respectively. The mitotic index was also significantly increased at 96 hours. The promoter region for the c-myc gene was hypomethylated only at 72 and 96 hours and not at the earlier sacrifices. Similarly, the methylation of the c-myc gene in the kidney and urinary bladder was decreased only at 72 and 96 hours. In summary, enhancement of cell proliferation and decreased methylation of the c-myc gene were first observed simultaneously at 72 hours after the start of exposure. Thus, the results support the hypothesis that DCA and TCA induce DNA hypomethylation by inducing DNA replication and preventing the methylation of the newly synthesized strands of DNA.