Mesude İşcan

The bioaccumulation of nonyphenol and its adverse effect on the liver of rainbow trout (Onchorynchus mykiss).

Alkylphenol polyethoxylates (APEs) are widely used as nonionic surfactants. Nonylphenol (NP), one of the derivatives of APEs, has been found in the aquatic environment in ranges from nanograms per liter to milligrams per liter. In this study, juvenile rainbow trout were exposed to 0 (control), 66, 220, or 660 micro g NP/L for up to 28 days. Fish remained healthy under NP exposures of 0, 66, and 220 micro g/L for the length of the experiment. All fish died after 4 days of exposure to 660 micro g NP/L. Time-dependent NP bioaccumulation was detected in the tissues of fish exposed to 220 micro g NP/L (P<0.05) and histopathological changes were observed in the livers of fish exposed to 220 micro g NP/L. Furthermore, an increase in the activity of glutathione-S-transferase (GST) was found in the liver of fish exposed to 220 micro g NP/L for 1 week (P<0.05). There was an increase in GST activity in the liver of fish exposed to 66 micro g NP/L but it did not occur before 2 weeks of exposure to NP. The GST activity then decreased in a time-dependent manner in treatment groups, and this decrease was lower in the livers of fish treated with 66 and 220 micro g NP/L than in control fish after 3 weeks of exposure (P<0.05). These results indicated that sublethal doses of NP were accumulating in the bodies of the fish and causing histopathological and biochemical changes in the livers of rainbow trout.

DNA single-strand breakage in rat lung, liver and kidney after single and combined treatments of nickel and cadmium

Single-strand breaks were observed in rat lung and kidney after acute treatment of animals with CdCl2 (4 mg/kg body weight) injected intraperitoneally and NiCl2, (44.4 mg/kg body weight) injected subcutaneously. In the rat liver, no single-strand breakage was evident with those doses in single and combined metal treatments. The most susceptible tissue in rats to cadmium or nickel chloride treatment was the lung tissue. The single-strand breaks were higher in cadmium treatment than in nickel treatment in the rat lung. Also the response to cadmium treatment was obtained earlier than nickel. Rat kidney was also responsive to cadmium treatment. However, the response, although statistically significant, was much lower than the one obtained in rat lung. The combined treatment, which was done by administrating cadmium prior to nickel administration, reduced the number of single-strand breaks significantly and reversed them to control values in rat lung and kidney. This study confirms that cadmium and nickel create single-strand breaks when administered alone in the rat lung. This effect, which was seen in the single metal treatments, was reduced in the combined treatments.

Sister chromatid exchanges in human lymphocytes treated in vitro with cadmium in Go and S phase of their cell cycles

Sister chromatid exchanges (SCEs) were analyzed in human phytohemagglutinin-activated peripheral lymphocyte cultures exposed to varying concentrations (10(-7)-10(-3) M) of cadmium chloride in vitro at two different stages of the cell cycle, G(o) and early S phase. When cadmium chloride was administered at the G(o) phase, no increase in the SCEs were observed for the doses 10(-6) and 10(-5) M. Concentrations equal to or larger than 10(4) M cadmium chloride were lethal to human lymphocytes in our experimental conditions. A highly statistically significant increase was observed in the SCE frequency with increasing cadmium chloride concentration (10(-7)-10(-4)) when cadmium was administered at the early S phase, which was 24 h after culture initiation. The increase in SCE frequency was higher when the cultures were terminated at 54 h, compared to termination at 72 h. In order to examine the effects of cadmium administered at the S phase on SCE frequency in different individuals, 10(-5) M concentration was used and the cultures were terminated at 54 h after culture initiation. A 2- to 3-fold increase in the SCE frequency was observed in all six individuals examined. A progressive decrease in the proliferative index was also observed by increasing cadmium chloride concentration. These results demonstrate that the genotoxicity of cadmium chloride may be changed depending on the stage of the cell cycle in human lymphocytes. This may be one of the reasons of contradictory findings in the literature.

Comparative studies of sheep liver and lung microsomal aniline 4-hydroxylase

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.

In vivo effects of 3-methylcholanthrene, phenobarbital, pyrethrum and 2,4,5-t isooctylester on liver, lung and kidney microsomal mixed-function oxidase system of guinea-pig: A comparative study

The optimum conditions (pH, microsomal protein amount and substrate concentration) of guinea-pig liver, lung and kidney microsomal aniline 4-hydroxylase, ethylmorphine N-demethylase and benzo[a]pyrene hydroxylase activities were determined. Male guinea-pigs weighing 500-700 g were administered 3-methylcholanthrene (25 mg/kg, i.p. 3 days), phenobarbital (75 mg/kg, i.p. 3 days), pyrethrum (120 mg/kg, i.p. 2 days) and 2,4,5-T isooctylester (200 mg/kg, i.p. 3 days). 3-Methylcholanthrene treatment caused significant increases in liver microsomal benzo[a]pyrene hydroxylase and kidney microsomal aniline 4-hydroxylase activities. However, with phenobarbital treatment the only significant increase was observed in liver microsomal ethylmorphine N-demethylase activity. Pyrethrum treatment decreased kidney microsomal ethylmorphine N-demethylase activity significantly. 2,4,5-T isooctylester treatment increased liver microsomal aniline 4-hydroxylase and lung microsomal ethylmorphine N-demethylase activities significantly. Liver microsomal NADPH-cytochrome c reductase activity was increased significantly by phenobarbital and pyrethrum treatment. The other treatments did not cause any significant changes in microsomal NADPH-cytochrome c reductase activities of liver, lung and kidney. Cytochrome P-450 content of guinea-pig liver microsomes were increased significantly about 2.5-fold and 2-fold by treatment with 3-methylcholanthrene and phenobarbital, respectively. 3-Methylcholanthrene also caused 1 nm spectral shift in the absorption maxima of CO difference spectrum of the dithionite-reduced liver microsomal cytochrome P-450, forming P-449.

Kinetic and structural properties of biocatalytically active sheep lung microsomal NADPH-cytochrome c recutase

Abstract 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.

Alkylphenol concentrations in two rivers of Turkey.

Alkylphenol polyethoxylates (APEs) called environmental endocrine disruptors has been shown to accumulate in water around the world. In this study, the pollution level of alkylphenolic compounds was measured and quantified in water, sediment, and the tissues of fishes collected in two rivers, Sakarya and Degirmendere Rivers, Turkey. Butylphenol (BP) were detected in sediment samples at one sampling stations of both rivers with 1.68 and 3.15 μg/g sediment, while nonylphenol (NP) were detected with the amount of 4.46 μg/g sediment in one sampling station in Degirmendere river. Fish samples also showed the presence of alkylphenolic compounds in both rivers. The level of alkylphenol pollution in two rivers of Turkey was determined to be in the range of alkylphenol level reported in Europe but lower than that of in the USA.

Stimulatory effects of benzene on rabbit liver and kidney microsomal cytochrome P-450 dependent drug metabolizing enzymes

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 rates ofp-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 50000 and 51400 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.

Molecular adaptations of Helicoverpa armigera midgut tissue under pyrethroid insecticide stress characterized by differential proteome analysis and enzyme activity assays.

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.

Combined effect of cadmium and nickel on rat hepatic monooxygenases: possible stimulation of certain cytochrome P-450 isozymes

When male rats were given either a single dose of cadmium (3.58 mg CdCl2.6H2O/kg, i.p.) 72 h prior to sacrifice or a single dose of nickel (59.5 mg NiCl2.6H2O/kg, s.c.) 16 h prior to sacrifice, the activities of ethylmorphine N-demethylase, aminopyrine N-demethylase and aniline 4-hydroxylase, and the levels of cytochrome P-450 and microsomal heme were significantly decreased. Cadmium decreased the cytochrome b5 level significantly, whereas it did not alter the NADPH-cytochrome c reductase activity significantly. In contrast, Ni did not alter the cytochrome b5 level significantly but decreased the NADPH-cytochrome c reductase activity significantly. For the combined treatment, animals received the single dose of nickel 56 h after the single dose of cadmium and then they were killed 16 h later. In these animals ethylmorphine N-demethylase, aminopyrine N-demethylase and NADPH-cytochrome c reductase activities and cytochromes P-450 and b5 levels increased significantly as compared to those of controls, whereas aniline 4-hydroxylase activity and microsomal heme level remained unaltered. In concordance with the increase in the enzyme activities, certain P-450 protein bands were observed to be elevated when studied on SDS-polyacrylamide gel electrophoresis. Furthermore, when the monooxygenase activities and SDS-polyacrylamide gel electrophoresis profiles of combined metal-treated animals were compared with those of the animals treated with classic inducers such as phenobarbital (75 mg/kg i.p., 72, 48 and 24 h prior to sacrifice) and 3-methylcholanthrene (20 mg/kg i.p., 72, 48 and 24 h prior to sacrifice), the combination of metals seemed to have tendency to stimulate certain phenobarbital and 3-methylcholanthrene inducible cytochrome P-450 isozymes.