Differential interactions of ethacrynic acid and diethyl maleate with glutathione S-transferases and their glutathione co-factor in the house fly

Glutathione S-transferases (GSTs) are an important class of enzymes that facilitate the conjugation of reduced glutathione (GSH) with electrophilic substrates, including some insecticides. Two inhibitors of GSTs, ethacrynic acid (EA) and diethyl maleate (DEM), are often used as diagnostic tools to implicate GST involvement in insecticide resistance, but their modes of action against insect GSTs are largely assumed based on mammalian studies. In mammalian studies, there are two proposed mechanisms of inhibition of GST function by EA and DEM: 1) scavenging or “depleting” cytosolic GSH through non-enzymatic conjugation, and 2) inhibition of GST activity directly by the inhibitor-GSH conjugate (EA-SG and DEM-SG).

The objective of this study was to characterize putative inhibitory mechanisms of EA and DEM against insect (house fly) GSTs and the co-factor GSH. Both EA and DEM synergized topical applications of naled and propoxur but not permethrin. As a GSH scavenger, EA was ∼10-fold more potent compared to DEM. Conditions such as pH, GSH concentration, and incubation time significantly affected the ability of both inhibitors to scavenge GSH. EA demonstrated scavenging at a wider pH range than DEM and scavenged GSH at a faster rate than DEM. Whereas EA peak scavenging was observed almost instantly, there was a 54.4 % increase in scavenged GSH for DEM between 0 and 30 min of incubation. Increasing concentration of GSH diminished the effect of scavenging at the highest tested concentrations of both inhibitors. In the presence of both GSH and GSTs in crude homogenate, EA was 300-fold more potent as a GST inhibitor compared to DEM at pH 7.5. No comparison was made at pH 6.5 because the tested concentrations of DEM did not produce enough inhibition to derive an IC₅₀ value while EA concentrations did. With purified GSTs, EA-SG was 205-fold more potent as an inhibitor compared to DEM-SG, while EA alone was 7.6-fold more potent than EA-SG and 1565-fold more potent than DEM-SG. These findings establish in insects that the insecticide synergists EA and DEM function mainly by scavenging the GST co-factor GSH, with some inhibition due to interactions with GSTs and the inhibitor-GSH conjugates, rather than through interaction between the inhibitors and the GST protein itself. These resulting impacts are two-fold, whereby (i) GSH bioavailability is limited and (ii) the GSH-inhibitor complex attenuates GST-based xenobiotic metabolism.