Increased glutathione S-transferase activity and glutathione content in an insecticide-resistant strain of Tribolium castaneum (Herbst)

Abstract

Cyfluthrin resistance in Tribolium castaneum (Herbst) was associated with increases in both glutathione (GSH) concentration and glutathione S-transferase (GST) activity. Cytosolic GSH content was increased twofold in resistant beetles. Similarly, conjugation of the GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) were elevated four- to sixfold in cytosols isolated from resistant beetles. However, no difference in GST activity was noted when p-nitrobenzylchloride (PNBC) was used as a substrate. Kinetic analysis of cytosolic CDNB activity revealed a four- to fivefold increase in apparent V max in resistant cytosols. Apparent K m , however, was similar in both resistant and susceptible cytosols. Partial purification of beetle cytosols revealed increased CDNB activity in resistant cytosols eluting as the unbound fraction following DE-52 chromatography when compared with cytosols isolated from susceptible insects. When electrophoresed on an equal protein basis, SDS-PAGE analysis revealed increased staining of a single protein band in the GST molecular weight region (24–30,000 Da) in cytosols isolated from resistant beetles. Taken together, these results are consistent with the increased expression of a common GST in resistant beetles. Microsomes isolated from resistant beetles exhibited a type I binding spectrum when incubated with piperonyl butoxide. In contrast, no spectral interaction was noted when piperonyl butoxide was added to microsomes isolated from susceptible insects. Similarly, metabolic intermediate (MI) complex formation with piperonyl butoxide was only observed in microsomes from resistant beetles. Addition of GSH to the incubation mixture prior to the initiation of piperonyl butoxide metabolism resulted in a dose-related decrease in complex formation (as measured by 427 nm peak height), 5 m M GSH being associated with a 50% decrease in complexation. This result suggests that increased GSH levels may influence MI complex formation in vivo and hence affect insecticide synergism.