Hemolysis of human red blood cells induced by the combination of diethyldithiocarbamate (DDC) and divalent metals: Modulation by anaerobiosis, certain antioxidants and oxidants

The objective of the present communication is to describe the role played by combinations between diethydithiocarbamate (DDC) and divalent metals in hemolysis of human RBC. RBC which had been treated with DDC (10-50 μM) were moderately hemolyzed (about 50%) upon the addition of subtoxic amounts of Cu²⁺ (50 μM). However, a much stronger and a faster hemolysis occurred either it mixtures of RBC-DDC were immediately treated either by Co²⁺ (50 μM) or by a premixture of Cu²⁺ and Co²+ (Cu:Co) (50 μM). While Fe²⁺ and Ni²⁺, at 50 μM, initiated 30-50% hemolysis when combined with DDC (50 μM), on a molar basis, Cd²⁺ was at least 50 fold more efficient than any of the other metals in the initiation of hemolysis by DDC. On the other hand, neither Mn²⁺ nor Zn²⁺, had any hemolysis-initiating effects. Co²⁺ was the only metal which totally blocked hemolysis if added to DDC prior to the addition of the other metals. Hemolysis by mixtures of DDC + (Cu:Co) was strongly inhibited by anaerobiosis (flushing with nitrogen gas), by the reducing agents glutathione, N-acetyl cysteine, mercaptosuccinate, ascorbate, TEMPO, and α-tocopherol, by the PLA₂ inhibitor bromophenacylbromide (BrPACBr), by tetracycline as well as by phosphatidyl choline, cholesterol and by trypan blue. However, TEMPO, BrPACBr and PC were the only agents which inhibited hemolysis induced by DDC:Cd²⁺ complexes. On the other hand, none of the classical scavengers of reactive oxygen species (ROS) employed e.g dimethylthiourea, catalase, histidine, mannitol, sodium benzoate, nor the metal chelators desferal and phenanthroline, had any appreciable inhibitory effects on hemolysis induced by DDC + (Cu:Co). DDC oxidized by H₂O₂ lost its capacity to act in concert either with Cu²⁺ or with Cd²⁺ to hemolyze RBC. While either heating RBC to temperatures greater than 37°C or exposure of the cells to glucose-oxidase-generated peroxide diminished their susceptibility to hemolysis, exposure to the peroxyl radical from AAPH, enhanced hemolysis by DDC + (Cu:Co). The cyclovoltammetry patterns of DDC were drastically changed either by Cu²⁺, Co²⁺ or by Cd²⁺ suggesting a strong interaction of the metals with DDC. Also, while the absorbance spectrum of DDC at 280 nm was decreased by 50% either by Co²⁺, Cd²⁺ or by H₂O₂, a 90% reduction in absorbance occurred if DDC + H₂O₂ mixtures were treated either by Cu²⁺ or by Co²⁺, but not by Cd²⁺. Taken together, it is suggested that DDC-metal chelates can induce hemolysis by affecting the stability and the integrity of the RBC membrane, and possibly also of the cytoskeleton and the role played by reducing agents as inhibitors might be related to their ability to deplete oxygen which is also supported by the inhibitory effects of anaeobiosis.