nitrophenols and diethyl-maleate

The mosquito vector Culex quinquefasciatus is known to be resistant to insecticides worldwide, including Thailand. This study was the first investigation of the insecticide resistance mechanisms, involving metabolic detoxification and target site insensitivity in C. quinquefasciatus from Thailand. Adult females reared from field-caught larvae from six provinces of northern Thailand were determined for resistant status by exposing to 0.05% deltamethrin, 0.75% permethrin and 5% malathion papers using the standard WHO susceptibility test. The overall mortality rates were 45.8%, 11.4% and 80.2%, respectively. A fragment of voltage-gated sodium channel gene was amplified and sequenced to identify the knock down resistance (kdr) mutation. The ace-1 gene mutation was determined by using PCR-RFLP. The L1014F kdr mutation was observed in all populations, but the homozygous mutant F/F1014 genotype was found only in two of the six provinces where the kdr mutation was significantly correlated with deltamethrin resistance. However, none of mosquitoes had the G119S mutation in the ace-1 gene. A laboratory deltamethrin resistant strain, Cq_CM_R, has been established showing a highly resistant level after selection for a few generations. The mutant F1014 allele frequency was significantly increased after one generation of selection. A synergist assay was performed to assess the metabolic detoxifying enzymes. Addition of bis(4-nitrophenyl)-phosphate (BNPP) and diethyl maleate (DEM), inhibitors of esterases and glutathione S-transferases (GST), respectively, into the larval bioassay of the Cq_CM strain with deltamethrin showed no significant reduction. By contrast, addition of piperonyl butoxide (PBO), an inhibitor of cytochrome P450 monooxygenases, showed a 9-fold reduction of resistance. Resistance to pyrethroids in C. quinquefasciatus is widely distributed in northern Thailand. This study reports for the first time for the detection of the L1014F kdr mutation in wild populations of C. quinquefasciatus in Thailand. At least two major mechanisms, kdr and cytochrome P450 monooxygenases, confer resistance to deltamethrin in Thai C. quinquefasciatus populations.

Topics: Animals; Culex; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Female; Insecticide Resistance; Insecticides; Larva; Malathion; Maleates; Mosquito Control; Nitriles; Nitrophenols; Permethrin; Piperonyl Butoxide; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Pyrethrins; Thailand; Voltage-Gated Sodium Channels

Glutathione plays an important role in the protection of the liver against several hepatotoxins. The hepatocarcinogen N-hydroxy-2-acetylaminofluorene is converted in the rat in vivo to reactive metabolites that bind covalently to cellular macromolecules. These metabolites may also react with glutathione, resulting in the formation of glutathione conjugates and in the detoxification of reactive metabolites. The role of glutathione in detoxification was investigated by depletion of glutathione in the rat in vivo with diethyl maleate. When rats were pretreated with diethyl maleate, 45 min before the administration of N-hydroxy-2-acetylaminofluorene, excretion of 2-acetylaminofluorene:glutathione conjugates in bile was decreased by 60% as compared to controls. However, total covalent binding to rat liver protein was not increased, and total binding to DNA was even decreased (p less than 0.1), apparently at the expense of the acetylated carcinogen-DNA adducts. Formation of deacetylated, 2-aminofluorene adducts to DNA was not affected by diethyl maleate. Pretreatment with diethyl maleate had no major effect on the acute hepatotoxic effects of N-hydroxy-2-acetylaminofluorene. The results indicate that glutathione does not play a vital role in the detoxification of reactive metabolites generated from the carcinogen N-hydroxy-2-acetylaminofluorene, since glutathione is not very effective in competing with macromolecules for trapping of reactive metabolites of N-hydroxy-2-acetylaminofluorene. Thus, 1 mM glutathione did not decrease the covalent binding of 2-acetylaminofluorene-N-sulfate (one of the main reactive metabolites that is formed in vivo) to DNA in vitro, while 10 mM glutathione decreased the covalent binding to RNA by only 20% and to DNA by only 40%.

Topics: 2-Acetylaminofluorene; Animals; Cysteine; DNA; Glutathione; Hydroxyacetylaminofluorene; In Vitro Techniques; Kidney; Liver; Male; Maleates; Nitrophenols; Protein Binding; Rats; Rats, Inbred Strains; RNA; Sulfuric Acids