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Helicoverpa armigera is an insect that causes important economic losses in crops. To reduce this loss, pyrethroids have been commonly used against H. armigera in farming areas. However, excess and continuous usage of pyrethroids cause resistance in H. armigera. Therefore, expressions of midgut proteins of two H. armigera field populations were compared to those of a susceptible strain by 2-D PAGE and MALDI-ToF-MS. Our results indicate that H. armigera reacts to pyrethroid-induced stress mainly by increasing the expression of energy metabolism-related proteins, such as ATP synthase and arginine kinase. NADPH cytochrome P450 reductase, also up-regulated, could play a role in detoxification of toxic pyrethroid metabolites, such as 3-phenoxybenzaldehyde. Interestingly, while GSTs were not found up-regulated in the comparative proteome analysis, biochemical assays showed significant increases of enzyme activities in both field populations as compared to the susceptible strain. Similarly, although esterases were not found differentially expressed, biochemical assays showed significant increases of esterase activities in both field populations. Thus, esterases are also proposed to be involved in metabolic responses towards pyrethroid insecticide-induced stress. In conclusion, we suggest increased energy metabolism in the midgut tissue of H. armigera as a general prerequisite for compensating the costs of energy-consuming detoxification processes. (C) 2013 Elsevier Inc. All rights reserved.

Gundelia tournefortii L. is an important food source and a well-known medicinal plant in Eastern Anatolia. Therapeutic effects of medicinal plants are known to be closely related to their antioxidant capacities. Antioxidant activities of G. tournefortii, both for the aerial parts and seeds, were investigated by using both 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and lipid peroxidation inhibition methods. The seeds were found to have higher antioxidant potential than the aerial, with IC50 values of 0.073 mg/mL for DPPH scavenging and 0.146 mg/mL for lipid peroxidation inhibition capacities. In addition, total phenolic contents of the Gundelia tournefortii L. extracts, especially the seed extracts correlates to its high antioxidant activity with 105.1 +/- 8.7 mu g gallic acid equivalents (GAEs) per mg of seed extract. Plant extracts with high phenolics content are known to have important effects on various enzymes, as well as glutathione-S-transferases, which are important detoxification enzymes in phase 11 systems with an important role in developing multi-drug resistance to chemotherapy in tumour cells. Consequently, the effects of G. tournefortii extracts on crude cytosolic glutathione-S-transferase was also studied and the seed extracts have shown effective inhibition of cytosolic GST activity, with an IC50 of 97.51 mu g/mL. (c) 2005 Elsevier Ltd. All rights reserved.

1. Nitrofurantoin reductase which catalyzes the bioactivation of nitrofurantoin was purified to electrophoretic homogenity from sheep liver and lung microsomes, with a yield of 15% and 35%, respectively. The specific activity of both reductases was found to be similar (140 nmol/min/mg protein). 2. The effects of nitrofurantoin and NADPH concentrations, pH, ionic strength, amount of enzyme and reaction period, on the enzyme activity were studied and the optimum conditions for maximum activity of purified liver and lung nitrofurantoin reductases were determined. 3. The enzyme concentration was found proportional with the square root of the rate of nitrofuratoin reduction up to approximately 15 micrograms protein/ml and 25 micrograms protein/ml incubation mixture for liver and lung nitrofurantoin reductases, respectively. 4. The plots of inverse of the nitrofurantoin concentration against the inverse of the square root of the velocity for the reduction of nitrofurantoin by liver and lung enzymes gave Km values as 27.78 microM and 32.25 microM, respectively. 5. The purified liver and lung enzymes were also saturated by NADPH at similar concentrations and the Km values were calculated as 29.4 microM and 35.5 microM, respectively. 6. The effects of magnesium, nickel, cadmium and copper ions on the nitrofurantoin reductase activity were examined. Magnesium ion was found to have almost no effect, whereas the other ions inhibited the activity of both liver and lung reductases.

Treatment of rabbits with benzene (880 mg/kg/day), s.c. for 3 consecutive days, caused 3.8- and 5.7-fold increases in aniline 4-hydroxylation rates of liver and kidney microsomes, respectively. Benzene treatment markedly enhanced hydroxylation rats of p-nitrophenol by liver and kidney by 7.2- and 4.2-fold, respectively. Both of these enzymes are associated with cytochrome P-450 LM3a. In contrast, the activity of benzphetamine N-demethylase, associated with P-450 LM2, was not altered significantly in either liver or kidney microsomes. Although the total cytochrome P-450 contents of liver and kidney microsomes were not altered significantly by the benzene treatment, in the case of liver microsomes, formation of a new cytochrome P-450 with an apparent Mr of 51,400 was observed on SDS-PAGE. On the other hand, in the kidney microsomes, the intensity of the bands corresponding to approximate Mr of 50,000 and 51,400 was markedly increased. The results of the present work, in combination with those of the previous work (Arinç et al. 1988), indicate the existence of tissue specificity in the induction of rabbit P-450 isozyme by benzene.

Rheum species are medicinally important plants due to the presence of anthracene derivatives. This study was designed to determine the antioxidative, cytotoxic and apoptotic properties of Rheum ribes shoot and root ethyl acetate extracts using human promyelocytic leukemia (HL-60) cell line as a model system. R. ribes shoot and root dry powder samples were prepared and extracted with ethyl acetate. The extracts were revealed to be a potential scavenger of DPPH radicals (IC50 value of 206.28 μg/ml for shoot and 10.92 μg/ml for root) and the chemical composition of the extracts was quantified by colorimetric determination of total phenol (GAE) and flavonoid (CAE) contents. HL–60 cells were cultured in the presence of various concentrations of extracts up to 72 h. R. ribes inhibited the survival of HL-60 cells in a concentration- and time-dependent manner, shown by XTT assay. R. ribes caused HL-60 cells apoptosis via formation of phosphatidylserine externalization, as evidenced by flow cytometry. Exposure of HL-60 cells to higher concentrations of extracts for 72 h resulted in a shift of 87% of the cell population from normal to the early/late apoptotic stage. These findings suggest that Rheum ribes ethyl acetate root extracts exhibits potential antioxidant and cytotoxic properties against HL-60 cells better than shoot extracts and exert their toxicity via induction of apoptosis.

The sulphation of bile acids is an important pathway for the detoxification and elimination of bile acids during cholestatic liver disease. A dehydroepiandrosterone (DHEA) sulphotransferase has been purified from male and female human liver cytosol using DEAE-Sepharose CL-6B and adenosine 3′,5′-diphosphate-agarose affinity chromatography [Falany, Vazquez & Kalb (1989) Biochem. J. 260, 641-646]. Results in the present paper show that the DHEA sulphotransferase, purified to homogeneity, is also reactive towards bile acids, including lithocholic acid and 6-hydroxylated bile acids, as well as 3-hydroxylated short-chain bile acids. The highest activity towards bile acids was observed with lithocholic acid (54.3 +/- 3.6 nmol/min per mg of protein); of the substrates tested, the lowest activity was detected with hyodeoxycholic acid (4.2 +/- 0.01 nmol/min per mg of protein). The apparent Km values for the enzyme are 1.5 +/- 0.31 microM for lithocholic acid and 4.2 +/- 0.73 microM for taurolithocholic acid. Lithocholic acid also competitively inhibits DHEA sulphation by the purified sulphotransferase (Ki 1.4 microM). No evidence was found for the formation of bile acid sulphates by sulphotransferases different from the DHEA sulphotransferase during purification work. The above results suggest that a single steroid sulphotransferase with broad specificity encompassing neutral steroids and bile acids exists in human liver.

1. 1. Reductase was purified to electrophoretic homogeneity from sheep liver and lung microsomes. The specific activity of both enzymes ranged from 55 to 66 μ mol cytochrome c reduced/min/mg protein. 2. 2. Liver and lung reductases appeared to have similar kinetic and spectral properties. K m , (NADPH) and K m (cytochrome c ) values were calculated to be 14.3 ± 1.23 μ M and 22.2 ± 2.78 μ M for liver and 11.1 ±0.70 μ M and 20.0 ± 2.15 μ M for lung reductase, respectively. Kinetic studies showed that cytoehrome c can bind the oxidized form of the enzyme as well as its reduced form and both reductases operated through a ping-pong type mechanism. 3. 3. These reductases cannot be distinguished on the basis of monomer molecular weights (M r 78,000) except that the liver reductase was found to be more susceptible to proteolytic attack. 4. 4. Both reductases supported aniline 4-hydroxylation and ethylmorphine N -demethylation reactions to the same extent in the reconstituted systems. However, sheep lung reductase appeared only 36.5 and 14.8% as effective in catalyzing benzo[ a ]pyrene reaction as an equivalent amount of reductase from liver in the presence of liver cytochrome P -450 and 3MC-treated rat liver cytochrome P -448, respectively.

1. 1. NADPH-cytochrome c reductase was purified to electrophoretic homogeneity from detergent solubilized sheep lung microsomes. 2. 2. The specific activity of the purified enzyme ranged from 56 to 67 μmol cytochrome c reduced/min/mg protein and the yield was 48–52% of the initial activity in lung microsomes. 3. 3. The reductase had Mr of 78,000 and contained 1 mol each of FAD and FMN. 4. 4. Km values obtained in 0.3 M phosphate buffer, pH 7.8 at 37°C for NADPH and cytochrome c were 11.1±0.70 μM and 20.0±2.15 μM. Lung reductase was inhibited by its substrate, cytochrome c when its concentration was above 160 μM. 5. 5. The lung reductase exhibited a ping-pong type kinetic mechanism for NADPH mediated cytochrome c reduction. 6. 6. Purified lung reductase was biocatalytically active in supporting benzo(a)pyrene hydroxylation reaction when coupled with lung cytochrome P-450 and lipid.

1. The specific activity of the aniline 4-hydroxylase which catalyses hydroxylation of aniline to p-aminophenol was found to be 0.65 (N = 10) and 0.15 (N = 13) nmol p-aminophenol formed/mg protein, min. in sheep liver and lung microsomes, respectively. 2. The effects of aniline concentration. pH. cofactors. amount of enzyme and incubation period, on enzyme activity were studied, and the optimum conditions for maximum activity of liver and lung microsomes were determined. 3. Liver and lung microsomal aniline 4-hydroxylase activity was found to be completely dependent on the presence of cofactor NADPH. 4. The Lineweaver-Burk and Eadie-Hofstee plots of the liver enzyme were found to be curvilinear, suggesting that the enzyme did not follow the Michaelis-Menten kinetics. From these graphs, two different Km values were calculated for the liver enzyme as 3.21 and 0.072 mM aniline. Km of the lung enzyme was calculated to be 1.43 mM aniline from its Lineweaver-Burk graph. 5. The effects of magnesium, nickel and cadmium ions on the liver and lung aniline 4-hydroxylase activity were examined. Magnesium ion was found to have stimulatory effect, whereas nickel and cadmium ions inhibited the activity of the both liver and lung enzyme.