Dismutase Inhibitor Diethyldithiocarbamate Research Articles

Cloning, Purification, and Characterization of Recombinant Human Extracellular Superoxide Dismutase in SF9 Insect Cells.

A balance between production and degradation of reactive oxygen species (ROS) is critical for maintaining cellular homeostasis. Increased levels of ROS during oxidative stress are associated with disease conditions. Antioxidant enzymes, such as extracellular superoxide dismutase (EC-SOD), in the extracellular matrix (ECM) neutralize the toxicity of superoxide. Recent studies have emphasized the importance of EC-SOD in protecting the brain, lungs, and other tissues from oxidative stress. Therefore, EC-SOD would be an excellent therapeutic drug for treatment of diseases caused by oxidative stress. We cloned both the full length (residues 1–240) and truncated (residues 19–240) forms of human EC-SOD (hEC-SOD) into the donor plasmid pFastBacHTb. After transposition, the bacmid was transfected into the Sf9-baculovirus expression system and the expressed hEC-SOD purified using FLAG-tag. Western blot analysis revealed that hEC-SOD is present both as a monomer (33 kDa) and a dimer (66 kDa), as detected by the FLAG antibody. A water-soluble tetrazolium (WST-1) assay showed that both full length and truncated hEC-SOD proteins were enzymatically active. We showed that a potent superoxide dismutase inhibitor, diethyldithiocarbamate (DDC), inhibits hEC-SOD activity.

Disulfiram may mediate erythrocyte hemolysis induced by diethyldithiocarbamate and 1,4-naphthoquinone-2-sulfonate

The increase in 1,4-naphthoquinone-2-sulfonate (NQS)-induced hemolysis by the superoxide dismutase inhibitor diethyldithiocarbamate (DEDC) was formerly attributed to increased superoxide anion levels in the erythrocyte. Our results show that removal of DEDC after preincubation and prior to the addition of NQS did not produce a significant increase in hemolysis, which suggests that hemolysis is primarily caused by the reaction products of DEDC with NQS and not to the inactivation of superoxide dismutase. Disulfiram, the oxidized product of DEDC, was found to be the main product formed when excess DEDC was reacted with NQS. Oxygen uptake also occurred and hydrogen peroxide was formed. The latter caused the oxidation of DEDC to disulfiram as catalase prevented disulfiram formation. Disulfiram was found to readily hemolyze erythrocytes at low concentrations as well as to crosslink the proteins in the erythrocyte membrane. Furthermore, disulfiram-induced hemolysis was markedly enhanced in glutathione-depleted erythrocytes. Disulfiram was subsequently found to readily oxidize glutathione in red blood cells. When equimolar concentrations of DEDC and NQS were reacted, the major product formed was the diethyldithiocarbamate:1,4-naphthoquinone (DEDC:NQS) conjugate. However, the principal mediator of erythrocyte hemolysis when excess DEDC is reacted with 1,4-naphthoquinone-2-sulfonate is disulfiram, whose mode of action may be to modify membrane protein sulfhydryls.

H 2O 2 production, modification of the glutathione status and methemoglobin formation in red blood cells exposed to diethyldithiocarbamate in vitro

Human red blood cells treated with the CuZn superoxide dismutase inhibitor diethyldithio-carbamate (DDC) undergo metabolic modifications in addition to the superoxide dismutase inhibition: oxidation of the reduced glutathione (GSH) to oxidized glutathione (GSSG), methemoglobin formation, and increased hexose monophosphate shunt activity were observed. The magnitudes of these changes are dependent on the DDC concentration. Under nitrogen, only superoxide dismutase inhibition occurs. After removal of the GSH with N-ethylmaleimide, production of H 2O 2 can be detected by measuring the red cell catalase inhibition in the presence of 3-amino-1,2,4-triazole. H 2O 2 production is not altered by conversion of oxyhemoglobin to methemoglobin by sodium nitrite prior to incubation. GSH oxidation and methemoglobin formation are stopped when DDC is eliminated from the incubation medium after completion of the superoxide dismutase inhibition. These data indicate that methemoglobin formation and modification of the GSH status in red cells treated by DDC are not a direct consequence of the CuZn Superoxide dismutase inhibition but are due rather to a DDC-dependent production of H 2O 2.

Superoxide anion as a mediator of drug-induced oxidative hemolysis.

The superoxide dismutase inhibitor diethyldithiocarbamate (DDC) was utilized to study the toxic effect of 1,4-naphthoquinone 2-sulfonate (NQ), a structural analog of the hemolytic drug, menadione, on red cells. NQ was shown to react with hemoglobin and result in the generation of superoxide anion (O2-). Red cells treated with NQ were found to undergo a gradual disappearance of their oxyhemoglobin and also hemolyze. Red cells pretreated with DDC to inhibit cellular superoxide dismutase were found to be markedly sensitive to oxyhemoglobin destruction and hemolysis in the presence of NQ. Superoxide dismutase-inhibited red cells were also found to undergo a slow autohemolysis in the absence of NQ. No evidence for lipid peroxidation was obtained for red cells treated with NQ either in the presence or the absence of DDC. Ghosts prepared from superoxide dismutase-inhibited red cells exposed to NQ were found to retain a green hemoglobin-derived pigment.