Screening test for insecticides interfering with cuticular sclerotization

Article abstract of DOI:10.1002/(SICI)1096-9063(199612)48:4<315::AID-PS481>3.0.CO;2-C

The potential of known and new insecticides to interfere with cuticle sclerotization was investigated using assays for key enzymes such as phenoloxidase, quinone methide isomerase and DOPA decarboxylase. Homogenates from the blowfly Lucilia cuprina and from the epithelial cell line from Chironomus tentans were used to examine the compounds under investigation. Phenoloxidases are known to oxidize DOPA, the substrate for DOPA decarboxylase. Since phenoloxidases were not detectable in C. tentans cell homogenates, inhibitor and kinetic studies were done for comparison with DOPA decarboxylase of this insect cell line. DOPA decarboxylase and phenoloxidase of L. cuprina exerted highest specific activities at early pupal stages (day 7). The apparent K(m) values for the two enzymes were 0.47(±0-21) mM and 0.71(±0.16) mM, respectively, using L-DOPA as substrate. DOPA decarboxylase from C. tentans had a K(m) value of 0.42(±0.18) mM. Quinone methide isomerase was most active in young pupae. In terms of substrate specificity for enzymic (mushroom-tyrosinase) production of different quinones from their corresponding catechols, that with dopamine quinone proved to be the most efficient. Synthesis of derivatives of L-DOPA and L-tyrosine led to a compound which inhibited both phenoloxidase and quinone methide isomerase. DOPA decarboxylase from L. cuprina and from cells of C. tentans was inhibited by carbidopa (IC₅₀ values of 0.021(±0.011) μM and 0.031(±0.019) μM, respectively) and indomethacine (IC₅₀ values of 22.6(±7.1) μM and 18.8(±9.7) μM). Both compounds exerted a competitive type of inhibition and were able to interfere with development of L. cuprina.