A Comparative Study of GSH-S-Aryltransferase and DDT-Dehydrochlorinase in Insects

Abstract

A comparative study of the GSH binding-sites of housefly and grass-grub GSH-S-aryltransferase, and of housefly DDT-dehydrochlorinase, was undertaken. A variety of triphenylmethane compounds isosteric to GSH were tested for inhibition of both enzymes. Amongst the acidic triphenylmethanes the coloured as well as the leuco-forms were found to be effective inhibitors of both enzymes. With the basic triphenylmethanes, only the coloured forms inhibited GSH-S-aryltransferase while the leuco-forms did not. However, DDT-dehydrochlorinase was inhibited by both the coloured as well as the leuco-forms of basic triphenylmethanes. The dicarboxylic acids were found to be poor inhibitors of both DDT-dehydrochlorinase and GSH-S-aryltransferase. However it was found with both enzymes that dicarboxylic acids with their carboxyl groups spaced at distances greater than those on GSH were better inhibitors than in which these distances were smaller than in GSH. Studies on the minimal structural requirements for maximum inhibition amongst the triphenylmethanes were carried out with respect to both DDT-dehydrochlorinase and GSH-S-aryltransferase. With the acidic triphenylmethanes, it was found that a molecule such as p, p’-dihydroxydiphenylmethane with halogen substituents ortho to the phenolic group was just as effective an inhibitor as the best of the brominated phthaleins or sulphonphthaleins. Amongst the basic triphenylmethanes, a molecule such as p p’-diaminodiphenylmethane with dialkyl substituents on the amino group was just as effective as the coloured as well as the leuco-forms of the basic triphenylmethanes towards DDT-dehydrochlorinase. However this type of structure did not inhibit GSH-S-aryltransferase unless it carried a positive charge on the molecule. All the diphenylmethane and triphenylmethane compounds as well as the dicarboxylic acids, acted competitively towards the GSH-binding-site of both GSH-S-aryltransferase and DDT-dehydrochlorinase. DDT toxicity studies were conducted on two strains of houseflies, namely the A-strain and the V-strain. The LD50 for DDT with the A-strain was 50 μg per fly (mixed sexes) while that for the V-strain was 0.5 μg per fly (mixed sexes). Some of the best DDT-dehydrochlorinase inhibitors were tested as potential synergists against DDT-resistant flies in combination with DDT. Among them tetram ethyldiaminodiphenylmethane (TDM), a GSH competitor, was found to synergise DDT moderately in the highly DDT-resistant A-strain although it was not comparable to WARF-antiresistant, a DDT competitor, on the same strain. However, both TDM and WARF-antiresistant have been shown by other workers to synergise DDT to the same extent in the V-strain. Further, with the A-strain, the methylenedioxyphenyl compounds, which inhibit microsomal enzymes, did not synergise DDT toxicity, thus implying that microsomal enzymes if present did not contribute significantly to DDT-resistance in this strain. The rate of in vivo- metabolism of DDT was studied in the A-strain flies. It was found to be similar to the rate of DDT metabolism in six other strains including the V-strain, studied by other workers. The rate of in vitro metabolism of DDT in the A-strain was found to be almost similar to that of the V-strain and VD-strain. An attempt was made to study the distribution, properties and molecular weights of both GSH-S-aryltransferase and DDT-dehydro-chlorinase in a variety of animal species. Although GSH-S-aryltransferase was present in both aquatic and terrestrial vertebrates and invertebrates, the occurrence of DDT-dehydrochlorinase was detected in measureable amounts in houseflies only. Detectable amounts of DDT-dehydrochlorinase could not be found in the other species studied in spite of the sensitivity of the technique used. Both enzymes were found to have a single activity peak with a molecular weight of 37,000 ± 3000 on gel-filtration (sephadex-G-100 and Bio-gels P-100 and P-150). No association or dissociation of either enzyme was noted in the strains studied, although such phenomena with respect to DDT-dehydrochlorinase have been observed by other workers for other strains of houseflies.