Glutathione depletion in human erythrocytes and rat liver: a study on the interplay between bioactivation and inactivation functions of liver and blood.

The interplay between bioactivation and inactivation functions of human erythrocytes and rat liver was studied. Glutathione depletion was used as a measure of the amount of reduced glutathione (GSH)-reactive compound. Iodoacetamide (IAcA), N-ethylmaleimide (NEM) and diethyl maleate (DEM), which are electrophiles that need no metabolic activation, were able to deplete GSH in incubations with either aqueous GSH solution or erythrocytes. These results indicate that these compounds can pass the erythrocyte membrane. Cyclophosphamide (CP), 3-hydroxyacetanilide (3-HAA) and 2-methylfurane (2-MF) needed metabolic activation by rat liver microsomes to deplete glutathione in incubations with aqueous GSH solution or erythrocytes. By measuring the sum of both reduced and oxidized glutathione [ = total glutathione (GT)] it became clear that GSH-reactive metabolites are generated out of CP, 3-HAA and 2-MF by the action of microsomes and that these metabolites can pass through the erythrocyte membrane. As GT depletion was higher when microsomes of phenobarbital-pretreated rats were used, the metabolites were (are expected to be) generated by phenobarbital-inducible enzymes. GT was also depleted in incubations with haemolysate and 3-HAA or 2-MF but not in incubations with aqueous GSH solution, which indicates that erythrocyte cytosol can metabolize 3-HAA and 2-MF into GSH-reactive compounds. The pesticides monuron and monulinuron did not affect GT concentrations when aqueous GSH solution, haemolysate or erythrocytes with or without microsomal activating system were tested. When hepatocytes were incubated with 3-HAA or CP (2 mm), about 2 mm of internal GT concentration was depleted. The hepatocytes excreted GSH-reactive metabolites generated from 3-HAA and CP (about 20% of the metabolites formed for 3-HAA). Erythrocyte GT was not depleted in co-incubations of hepatocytes and erythrocytes with 3-HAA. This can be explained by the amounts of GSH-reactive metabolites excreted by the hepatocytes, which would require very effective uptake by the erythrocytes in order to be detectable.