Pyruvate remediation of cell stress and genotoxicity induced by haloacetic acid drinking water disinfection by‐products

Monohaloacetic acids (monoHAAs) are a major class of drinking water disinfection by‐products (DBPs) and are cytotoxic, genotoxic, mutagenic, and teratogenic. We propose a model of toxic action based on monoHAA‐mediated inhibition of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as a target cytosolic enzyme. This model predicts that GAPDH inhibition by the monoHAAs will lead to a severe reduction of cellular ATP levels and repress the generation of pyruvate. A loss of pyruvate will lead to mitochondrial stress and genomic DNA damage. We found a concentration‐dependent reduction of ATP in Chinese hamster ovary cells after monoHAA treatment. ATP reduction per pmol monoHAA followed the pattern of iodoacetic acid (IAA) > bromoacetic acid (BAA) >> chloroacetic acid (CAA), which is the pattern of potency observed with many toxicological endpoints. Exogenous supplementation with pyruvate enhanced ATP levels and attenuated monoHAA‐induced genomic DNA damage as measured with single cell gel electrophoresis. These data were highly correlated with the S_N2 alkylating potentials of the monoHAAs and with the induction of toxicity. The results from this study strongly support the hypothesis that GAPDH inhibition and the possible subsequent generation of reactive oxygen species is linked with the cytotoxicity, genotoxicity, teratogenicity, and neurotoxicity of these DBPs. Environ. Mol. Mutagen. 54:629–637, 2013. © 2013 Wiley Periodicals, Inc.     

Evaluation of the protective efficacy of four newly identified surface proteins of Erysipelothrix rhusiopathiae

Erysipelothrix rhusiopathiae is the causative agent of animal erysipelas and human erysipeloid. Bacterial surface proteins are promising vaccine candidates. We recently identified 3 E. rhusiopathiae surface proteins (GAPDH, HP0728, and HP1472) and characterized their roles as virulence factors. However, their efficacy as protective antigens is still unknown. The N-terminal region of a previously identified surface protein, CbpB (CbpB-N), is speculated to be a protective antigen, but this needs to be verified. The aim of this study was to evaluate the protective efficacy of GAPDH, HP0728, HP1472, and CbpB-N. Immunization with recombinant GAPDH provided complete protection in a mouse model, recombinant CbpB-N provided partial protection, while recombinant HP0728 and HP1472 provided no protection. Recombinant GAPDH also provided good protection in a pig model. GAPDH antiserum exhibited significant blood bactericidal activity against E. rhusiopathiae. In conclusion, GAPDH and CbpB-N were found to be protective antigens of E. rhusiopathiae, and GAPDH is a promising vaccine candidate.     

Surface-displayed glyceraldehyde 3-phosphate dehydrogenase and galectin from Dirofilaria immitis enhance the activation of the fibrinolytic system of the host

Cardiopulmonary dirofilariosis is a cosmopolitan disease caused by Dirofilaria immitis, a filaroid parasite whose adult worms live for years in the vascular system of its host. Previous studies have shown that D. immitis can use their excretory/secretory (ES) and surface antigens to enhance fibrinolysis, which could limit the formation of clots in its surrounding environment. Moreover, several isoforms of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and galectin (GAL) were identified in both antigenic extracts as plasminogen-binding proteins. The aim of this work is to study the interaction of the GAPDH and GAL of D. immitis with the fibrinolytic system of the host. This study includes the cloning, sequencing and expression of the recombinant forms of the GAPDH and GAL of D. immitis (rDiGAPDH and rDiGAL) and the analysis of their capacity as plasminogen-binding proteins. The results indicate that rDiGAPDH and rDiGAL are able to bind plasminogen and stimulate plasmin generation by tissue plasminogen activator (tPA). This interaction needs the involvement of lysine residues, many of which are located externally in both proteins as have been shown by the molecular modeling of their secondary structures. In addition, we show that rDiGAPDH and rDiGAL enhance the expression of the urokinase-type plasminogen activator (uPA) on canine endothelial cells in culture and that both proteins are expressed on the surface of D. immitis in close contact with the blood of the host. These data suggest that D. immitis could use the associated surface GAPDH and GAL as physiological plasminogen receptors to shift the fibrinolytic balance towards the generation of plasmin, which might constitute a survival mechanism to avoid the clot formation in its intravascular habitat.     

Glutathione-S-transferase P1-1 protects aberrant crypt foci from apoptosis induced by deoxycholic acid

BACKGROUND & AIMS: Aberrant crypt foci, precursors of colonic adenoma, are frequently positive for glutathione-S-transferase P1-1. Because deoxycholic acid is an apoptosis-inducing xenobiotic in the colon, we examined the possibility that aberrant crypt foci, through the cytoprotecting function of glutathione-S-transferase P1-1, resist deoxycholic acid-induced apoptosis, thereby surviving to become adenomas and subsequently cancer. METHODS: Glutathione-S-transferase P1-1 or cyclooxygenase-2 expression and the percentage of apoptotic cells in aberrant crypt foci were examined by immunohistochemistry and by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, respectively. Glutathione-S-transferase P1-1 was transfected into colon cancer cells (M7609) and human lung fibroblasts, and deoxycholic acid-induced apoptosis was evaluated by a dye-uptake assay and flow cytometry. Binding of deoxycholic acid to glutathione-S-transferase P1-1 was analyzed by circular dichroism and immunoprecipitation. Caspase activities were determined by colorimetric protease assay, and sulindac binding to glutathione-S-transferase P1-1 was determined by inhibition assay of glutathione-S-transferase P1-1 activity. RESULTS: Aberrant crypt foci showed positive immunostaining for glutathione-S-transferase P1-1 but negative staining for cyclooxygenase-2. The percentage of apoptotic cells in aberrant crypt foci was significantly lower than in healthy epithelium, and the difference became more apparent with deoxycholic acid treatment. The impaired sensitivity of aberrant crypt foci to deoxycholic acid was restored by the glutathione-S-transferase P1-1-specific inhibitor gamma-glutamyl-S-(benzyl)cysteinyl-R-phenylglycine diethylester. By transfection of glutathione-S-transferase P1-1, M7609 cells became more resistant to deoxycholic acid-induced apoptosis than mock transfectants. Direct binding of glutathione-S-transferase P1-1 to deoxycholic acid was proven by circular dichroism and by immunoprecipitation. The aberrant crypt foci in adenoma patients treated with sulindac, which was shown to bind to glutathione-S-transferase P1-1, underwent apoptosis in 4 days and mostly regressed in 2-3 months. CONCLUSIONS: Glutathione-S-transferase P1-1 protects aberrant crypt foci from deoxycholic acid-induced apoptosis and may play a pivotal role in early colon carcinogenesis.     

Salvianolic acid B protects human endothelial progenitor cells against oxidative stress-mediated dysfunction by modulating Akt/mTOR/4EBP1, p38 MAPK/ATF2, and ERK1/2 signaling pathways

The vascular endothelium is specifically sensitive to oxidative stress, and this is one of the mechanisms that causes widespread endothelial dysfunction in most cardiovascular diseases and disorders. Protection against reactive oxygen species (ROS)-mediated oxidative damage via antioxidant mechanisms is essential for tissue maintenance and shows therapeutic potential for patients suffering from cardiovascular and metabolic disorders. Salvianolic acid B (SalB), a natural bioactive component known from Traditional Chinese Medicine, has been reported to exert cellular protection in various types of cells. However, the underlying mechanisms involved are not fully understood. Here, we showed that SalB significantly promoted the migratory and tube formation abilities of human bone marrow derived-endothelial progenitor cells (BM-EPCs) in vitro, and substantially abrogated hydrogen peroxide (H₂O₂)-induced cell damage. SalB down-regulated Nox4 and eNOS, as well as nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase expression upon H₂O₂ induction that in turn prevents oxidative-induced endothelial dysfunction. Moreover, SalB suppressed the Bax/Bcl-xL ratio and caspase-3 activation after H₂O₂ induction. Furthermore, our results provide mechanistic evidence that activation of the mTOR/p70S6K/4EBP1 pathways is required for both SalB-mediated angiogenic and protective effects against oxidative stress-induced cell injury in BM-EPCs. Suppression of MKK3/6-p38 MAPK-ATF2 and ERK1/2 signaling pathways by SalB significantly protected BM-EPCs against cell injury caused by oxidative stress via reduction of intracellular ROS levels and apoptosis. Taken together, by providing a mechanistic insight into the modulation of redox states in BM-EPCs by SalB, we suggest that SalB has a strong potential of being a new proangiogenic and cytoprotective therapeutic agent with applications in the field of endothelial injury-mediated vascular diseases.