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The accumulation of two metals (copper and zinc) and the specific activities of two enzyme systems glutathione S-transferases (GSTs) and cytochrome P450-dependent monooxygenases (P450-MO) were measured in four tissues (liver, kidney, small and large intestine) of the frog Rana ridibunda, collected from a wetland near the town of Lagadas, Northern Greece. Very high concentrations of the two metals were found in all studied tissues. The specific activities of both enzyme systems showed a significant decrease in frogs collected from the Lagada area compared to the controls. In the liver, both activities were reduced by about 88–90%. GST activity decreased by about 73% and 87% in the kidneys and small intestine, respectively, whereas the reduction in P450-MO activity was somewhat lower (about 59% in the kidney and 40% in the small intestine). Total protein correlated negatively with GST in the liver and kidney. Negative correlation was also found between copper and GST activity in the kidney. It is concluded ...

The effect of two heavy metals, namely chromium and cadmium, on GST and P450-MO activities in the liver, kidney and gut of the frog Rana ridibunda, and the use of these detoxification enzyme systems as possible markers of heavy metal contamination of rivers in Northern Greece was investigated. In control animals, the highest P450-MO activity was observed in the liver and the lowest activity in the gut. As regards GST, the liver and kidneys showed the highest activity. Exposure of Rana to either Cr or a mixture of Cr and Cd caused a decrease in liver GST and P450-MO and renal GST activities. An important finding was that both GST and P450-MO activities were negatively correlated with concentrations of both metals in the liver of mixture-exposed animals. The results suggest that the determination of GST and P450-MO activities, in combination, could serve to indicate heavy metal contamination in rivers of Northern Greece.

The correlation between the natural levels of GST and the tolerance to the insecticide decamethrin (dMT), as well as the interaction between the molecules of affinity purified enzyme and the insecticide were investigated in order to collect further information on the obscure role of the Glutathione S-transferase system (GST) as a mechanism of defence against pyrethroids. The studies were carried out, comparatively, on the larvae and pupae developmental stages of the coleopteran Tenebrio molitor, which exhibit varying natural levels of GST activity. No stage dependent susceptibility of the insect against pyrethroid insecticides was found during the first 24 h, however 48 h after treatment, the KD50 dose increased significantly due to the recovery of some individuals from the larvae stage. Simultaneous injection of decamethrin with compounds which inhibit GST activity in vitro, resulted in an increased tolerance, which was more pronounced in the pupae stage. Inhibition studies combined with competitive fluorescence spectroscopy and high pressure liquid chromatography (HPLC) showed that the insecticide binds probably to the active site of the enzyme inhibiting its activity towards CDNB in a competitive manner, but is not conjugated with GSH. According to this, GST offers a passive protection towards pyrethroid insecticides by binding to their molecule in a sequestering mechanism.

The correlation between the natural levels of glutathione S-transferase (GST) and the tolerance to the organophosphorus insecticides parathion-methyl and paraoxon-methyl, as well as the interaction of affinity-purified enzyme and the insecticides were investigated in order to collect further information on the role of the glutathione S-transferase system as a mechanism of defence against insecticides in insects. The studies were carried out on the larvae and pupae of the coleopteran Tenebrio molitor L, which exhibit varying natural levels of GST activity. Stage-dependent susceptibility of the insect against insecticides was observed during the first 24 h. However, 48 h after treatment, the KD50 value increased significantly due to the recovery of some individuals. Simultaneous injection of insecticide with compounds which inhibit GST activity in vitro caused an alteration in susceptibility of insects 24 or 48 h post-treatment, depending on stage and insecticide used. Inhibition studies combined with competitive fluorescence spectroscopy revealed that the insecticides probably bind to the active site of the enzyme, thus inhibiting its activity towards 1-chloro-2,4-dinitrobenzene in a competitive manner. High-performance liquid chromatography and gas chromatography revealed that T molitor GST catalyses the conjugation of the insecticides studied to a reduced form of glutathione (GSH). From the above experimental results, it is considered that GST offers a protection against the organophosphorus insecticides studied by active site binding and subsequent conjugation with GSH.

The isoenzymes of glutathione S-transferase expressed in the three developmental stages [larvae( L ), pupae( P ), adults( A )] of Tenebrio molitor , an insect of economic significance, were studied. Four major isoenzymes [A (L,P,A) , B (L,P,A) , C (L,P,A) and D (L,P,A) ] were present throughout development as judged by chromatofocusing at pH ranges 7–4 and 9–6. GSTs were purified by means of S-hexyl-GSH-Epoxy activated Sepharose 6B affinity chromatography and subsequent chromatofocusing at pH range 7–4. The molecular weight of the native isoenzymes was estimated to be 50.5 kDa, after gel filtration chromatography, while that of the subunits was 27 kDa, indicating that the enzyme was dimeric. With the exception of the molecular weights which remained constant, the isoelectric points, substrate specificities, sensitivities to inhibitors, pH optima of CDNB-conjugating activity and kinetic parameters of the isoenzymes altered in the three developmental stages.

Cytosolic glutathione S-transferase activity was studied in the three developmental stages of Tenebrio molitor . The highest activity towards both CDNB and DCNB was observed in the newly emerged pupae, while towards ethacrynic acid was in the young larvae. The pH optimum of GST activity towards CDNB was 8.5 in larvae and pupae and 8.25 in adults. GSTs operate at a high rate even at pH values 1 or 2 units from their pH optimum. A second lower pH optimum apparently existed in pupae (pH 8). The kinetic parameters (K m and V max ) for both GSH and CDNB were determined in the three developmental stages and showed the total amount of GSTs to be elevated in pupae. Three fractions ( not bound, low affinity and high affinity ) were obtained after GSH-Epoxy activated Sepharose 6B affinity chromatography of larval homogenate. Chromatofocusing of the fractions, in the pH range 7-4, revealed the presence of four isoenzymes. Changes in kinetic properties and substrate specificities of GSTs during development suggest a differential isoenzymic expression between larval and pupal stages, or alternatively, expression of the same isoenzymes at a different percentage. They also indicate a potentially lower importance of GST detoxification system in the adult stage.