Metabolic activation of benzo(a)pyrene by human amniotic fluid

The in vitro activation of benzo(a)pyrene was studied in amniotic fluid from ten 4-month pregnant women. Benzo(a)pyrene monooxygenase and epoxide hydrolase activities were in the same range in amniotic fluid as in human liver. Glutathione epoxide transferase activity was markedly lower than in hepatocytes. Human amniotic fluid also catalyzed the formation of hydrocarbon metabolites mutagenic to Salmonella typhimurium TA98 (Ames system). Profiles of amniotic fluid aromatic hydrocarbons from non smokers exhibited low benzo(a)pyrene concentration (less than 0.1 ng/ml).     

Comparative effects of the polychlorinated biphenyl mixture,aroclor 1242, on porphyrin and xenobiotic metabolism in kidney of japanese quail and rat

1. Aroclor 1242 (500 mg/kg, p.o.) produced a marked increase in porphyrin content of quail kidney (1800-fold), and of rat kidney but to a lesser extent (6-fold).2. δ-Aminolevulinic acid synthetase activity was increased 12-fold in quail kidney but was unchanged in rat kidney folowing Aroclor 1242 treatment.3. Uroporphyrinogen decarboxylase activity was significantly inhibited in quail kidney but not in rat kidney.4. Renal ethoxyresorufin O-deethylase activity was induced in rat and quail whereas renal ethoxycoumarin O-deethylase and glutathione S-transferase activities were induced only in rats by Aroclor 1242.     

Metabolism of xenobiotic compounds by enzymes in cell extracts of the fungus Cunninghamella elegans.

Cell extracts of the filamentous fungus Cunninghamella elegans contain epoxide hydrolase (EC 3.3.2.3), glutathione S-transferase (EC 2.5.1.18) and UDP-glucuronosyltransferase (EC 2.4.1.17) activities. Epoxide hydrolase activity was determined with p-nitrostyrene oxide as substrate and was shown to be associated with the 100 000 g pellet obtained from disrupted mycelia. Glutathione S-transferase activity was demonstrated with 1-chloro-2,4-dinitrobenzene and p-nitrobenzyl chloride as substrates. The presence of two or more glutathione S-transferase activities was indicated by different activity ratios for the two substrates in different extracts, and by distinct thermal denaturation curves. UDP-glucuronosyltransferase activity with 3-hydroxybenzo[a]pyrene as substrate was found only with the non-sedimentable fraction prepared from ruptured mycelia.     

Benzo(a)pyrene pretreatment of Drosophila simulans mutant strain results in the induction of aberrant isoform of cytochrome P-450 with increased capacity to metabolize benzo(a)pyrene]

The basal level of benzo(a)pyrene monooxygenase, epoxide hydrolase and glutathione S-transferase activity as well as the content of cytochrome P-450 were found the same in both compared benzo(a)pyrene (BP) sensitive D. simulans strain 364yv and BP-resistant wild one (Turku). Phenobarbital pretreatment resulted in the same increase level of these enzyme activities in both strains. BP-pretreatment of 364yv flies decreased the amount of the cytochrome P-450 but raised up the turnover of BP per molecule of cytochrome P-450. SDS-polyacrylamide gel electrophoresis of the microsomal proteins from BP-pretreated 364yv flies (but not from Turku) showed an increased hemoprotein content in the 56000 band. The relationship between BP-sensitivity of the strain 364yv and BP-induced aberrant isoform of the cytochrome P-450 has been discussed.     

Changes in liver glutathione S-transferase activities in Coturnix quail fed municipal sludge-grown cabbage with reduced levels of glucosinolates

Cabbage, green beans, or seeds from sunflowers grown either on municipal sewage sludge-amended soil or soil alone were fed to male and female Coturnix quail, as 50% of a complete diet, for 5 weeks. Specific activities of liver glutathione S-transferase (GST) were similar in all quail fed the latter two plant diets and also similar to quail fed a nonplant, control diet. Sludge-grown cabbage-treated quail exhibited liver GST activities significantly higher (P less than 0.05) than levels of liver GST in birds fed the other plants, with a further twofold activity increase in quail fed the soil-grown cabbage. This response seems to be correlated with the levels of glucosinolates present in the cabbage, i.e., 3040 and 9253 ppm (dry basis) in the sludge-grown and soil-grown cabbage, respectively.     

Involvement of an NAD(P)H oxidase as a pO2 sensor protein in the rat carotid body.

Selenium (Se) deficiency for 5 weeks in rats produced changes in the activity of a number of hepatic, renal and plasma enzymes. In animals whose food intake was restricted to 75% of normal for 2 weeks, Se deficiency produced significant increases in the activity of hepatic cytosolic 'malic' enzyme and mitochondrial alpha-glycerophosphate dehydrogenase (GPD), two enzymes that are particular sensitive to the thyroid-hormone concentrations in tissue. Propylthiouracil-induced hypothyroidism produced significant decreases in 'malic' enzyme and GPD activities. The effect of hypothyroidism on the activity of 'malic' enzyme, GPD and other enzymes studied in liver and plasma was often opposite to that seen in Se deficiency. Glutathione S-transferase (GST) activity was increased by both Se deficiency and hypothyroidism, but in hypothyroid animals further significant increases in GST were produced by Se deficiency. These data suggest that the changes in enzyme expression observed in Se deficiency are not caused by decreased tissue exposure to thyroid hormones.     

Evaluation of the relationship among gene expressions and enzyme activities with antioxidant role and presenilin 1 expression in Alzheimer's disease

It is known that oxidative stress originating from reactive oxygen species plays a role in the pathogenesis of Alzheimer's disease. In this study, the role of antioxidant status associated with oxidative stress in Alzheimer's disease was investigated. Peripheral blood samples were obtained from 28 healthy individuals (as control) and 28 Alzheimer's patients who met the National Institute of Neurological and Communicative Diseases and Stroke/Alzheimer's Disease and Related Disorders Association criteria. Catalase, glutathione S-transferase and paraoxonase 1 enzyme activities in blood plasma and glutathione S-transferase enzyme activities in erythrocytes were determined by spectrophotometer. Catalase, glutathione S-transferase and presenilin 1 gene expressions in leukocytes were determined using qRT-PCR. Data were analysed with SPSS one-way anova , a LSD post hoc test at p < 0.05. The activity of each enzyme was significantly reduced in Alzheimer's patients compared to control. The catalase gene expression level did not change compared to the control. Glutathione S-transferase and presenilin 1 gene expression levels were increased compared to the control.     

Studies on the glutathione S-transferase activity associated with rat liver mitochondria.

The major proportion of rat liver glutathione S-transferase is cytosolic. Carefully washed mitochondria contain 0.25-0.47% of the cytosolic activity. Subfractionation of washed mitochondria using digitonin treatment revealed that glutathione S-transferase release did not parallel that of any of the mitochondrial marker enzymes. Glutathione S-transferase release paralleled that of lactate dehydrogenase, suggesting that these 'mitochondrial' activities are due to loosely bound cytoplasmic forms.     

Enzymatic antioxidant response of a labrid fish (Coris julis) liver to environmental caulerpenyne

Exposure of marine animals to certain toxic compounds can enhance reactive oxygen species production with subsequent damage to macromolecules and alterations in oxidant defenses levels. Caulerpenyne is the major metabolite synthesized by Caulerpa species, used as chemical defense affecting several cellular and molecular targets. We assessed the changes produced by the presence of Caulerpa spp. in the activities of antioxidant enzymes as well as lipid peroxidation levels in liver of the teleost Coris julis. Fish were captured at two stations with Caulerpa species-Caulerpa taxifolia and Caulerpa prolifera-and at a region with the seagrass Posidonia oceanica as negative control. Caulerpenyne concentration was significantly higher in C. prolifera than in C. taxifolia (p<0.05). Glutathione S-transferase, glutathione peroxidase and glutathione reductase activities were significantly higher in both Caulerpa stations compared to the P. oceanica (p<0.05). No statistical difference (p>0.05) existed in catalase activity between groups. Glutathione reductase activity is significantly higher in C. prolifera station than in C. taxifolia (p<0.05). Despite the variations in the antioxidant enzyme activities, there was no significant difference in malondialdehyde concentration. In conclusion, the production of caulerpenyne by Caulerpa species could induce an antioxidant adaptation in the liver of C. julis in order to prevent oxidative damage.     

棉铃虫抗药性的生理生化机制研究

本文报道了棉铃虫Helicoverpa armigera田间抗性种群对杀虫剂抗药性的生理生化机制。抗性种群(HJ-R)5龄幼虫羧酸酯酶、谷胱甘肽转移酶、多功能氧化酶活力均明显高于相对敏感种群(HD-S)。两种群乙酰胆碱酯酶对杀虫剂敏感性没有显著差异。HJ-R种群的腹神经索对氰戊菊酯表现了2-3倍的神经不敏感性。HJ-R种群对氨基甲酸酯类杀虫剂的抗性主要是由代谢机制引起,其中多功能氧化酶可能起主导作用;对菊酯的抗性是由多功能氧化酶、酯酶、以及神经不敏感性几个因子综合作用的结果。     

Organic nitrate reductase: reassessment of its subcellular localization and tissue distribution and its relationship to the glutathione transferases

1. Using a specific and sensitive GLC method for the determination of glyceryl trinitrate (GTN), its subcellular and tissue distribution were reassessed. Liver was the most active tissue, but activity was also detected in the heart, kidney and gut. In all tissues activity was localized in the soluble fraction. The activity of soluble glutathione S-transferase followed the same pattern, liver exhibiting the highest and the heart the lowest activity. 2. Pretreatment with phenobarbitone and 3-methylcholanthrene stimulated both the glutathione S-transferase and organic nitrate reductase activities. 3. Glutathione S-transferase activity was competitively inhibited by GTN. 4. A comparison of the plasma and hepatic metabolism of GTN revealed higher drug affinity for the hepatic enzyme.     

Comparison of glutathione S-transferase activity in the rat and birds: tissue distribution and rhythmicity in chicken (Gallus domesticus) liver.

1. Mature, male chickens, Bobwhite quail, and rats differed with respect to glutathione S-transferase (GST) activity in the kidney, duodenum and testis, but species differences were not observed in the liver. 2. GST activity was present in the heart, spleen, liver, duodenum, kidney, testis, cerebral cortex, cerebellum, optic tecta, and medulla oblongata of chickens with differences in tissues and breeds. 3. Renal GST activity was higher in female chickens, whereas enzyme activity in the brain was higher in males. 4. Hepatic GST activity fluctuated about a mean of 784 nmol min-1 mg protein-1 with a 12 hr periodicity which was not a feeding phenomenon. 5. The results demonstrate that GST activity occurs in diverse tissues of the chicken and Bobwhite quail with kidney greater than liver greater than duodenum greater than testis, compared to testis greater than liver greater than duodenum greater than kidney in the rat. Hepatic GST activity exhibits an ultradian periodicity.     

Effects of non-ortho-substituted polychlorinated biphenyls (congeners 77 and 126) on cytochrome p4501a and conjugation activities in rainbow trout (Oncorhynchus mykiss)

Immature rainbow trout (Oncorhynchus mykiss) in two separate experiments received a single intraperitoneal injection of 0.1, 1 and 5 mg/kg of either 3,3′,4,4′-tetra- or 3,3′,4,4′,5-pentachlorobiphenyl (IUPAC congeners 77 and 126, respectively). The experiments were run at water temperatures of 6 °C and 4 °C. Fish were killed 6 days after the injection. Biotransformation enzyme activities and cytochrome P4501A (CYP1A) amount and occurrence in different tissues were assayed. Congeners 77 and 126 strongly induced 7-ethoxyresorufin O-deethylase (EROD) and benzo(α)pyrene hydroxylase (AHH) activities in liver and kidney of rainbow trout. The induction of these cytochrome P4501A dependent monooxygenases was dose-related especially with congener 77 in the kidney. However, in the liver the highest dose of both congeners and in kidney the highest dose of congener 126 did not increase the catalytic monooxygenase activities as much as would have been expected based on the responses obtained with the lower doses. This may be because the monooxygenase activities already had attained their maximal induction capacity at 1 mg/kg dose of each congener. The PCB residues in liver were also determined and found to be highest after 5 mg/kg injections (610 μg/kg wet weight with congener 77 and 220 μg/kg with congener 126). When cytochrome P4501A protein content was measured, the induction of cytochrome P4501A was still on the increase even in those cases where catalytic activity failed to show any further induction. Immunohistochemical samples from liver, kidney and intestine showed cytochrome P4501A staining which strongly correlated with cytochrome P4501A in microsomes. Such observations suggest that the amount and occurrence of P4501A in the tissues can express the induction even when catalytic activities seem to be suppressed. With respect to enzymes mediating conjugation reactions, hepatic and renal UDP-glucuronosyltransferase (UDP-GT) activities showed elevated levels especially with the 1 and 5 mg/kg doses of both congeners. Glutathione S-transferase (GST) activities did not show such a clear trend. Congeners 77 and 126 preferentially affected the P4501A enzymes but to some extent also conjugation activities.     

Stimulation of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase activity by the NS3 protease domain and by HCV RNA-dependent RNA polymerase

Hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses multiple enzyme activities. The N-terminal one-third of NS3 primarily functions as a serine protease, while the remaining two-thirds of NS3 serve as a helicase and nucleoside triphosphatase. Whether the multiple enzyme activities of NS3 are functionally interdependent and/or modulated by other viral NS proteins remains unclear. We performed biochemical studies to examine the functional interdependence of the NS3 protease and helicase domains and the modulation of NS3 helicase by NS5B, an RNA-dependent RNA polymerase (RdRp). We found that the NS3 protease domain of the full-length NS3 (NS3FL) enhances the NS3 helicase activity. Additionally, HCV RdRp stimulates the NS3FL helicase activity by more than sevenfold. However, the helicase activity of the NS3 helicase domain was unaffected by HCV RdRp. Glutathione S-transferase pull-down as well as fluorescence anisotropy results revealed that the NS3 protease domain is required for specific NS3 and NS5B interaction. These findings suggest that HCV RdRp regulates the functions of NS3 during HCV replication. In contrast, NS3FL does not increase NS5B RdRp activity in vitro, which is contrary to a previously published report that the HCV NS3 enhances NS5B RdRp activity.     

Mechanisms of resistance to DDT and pyrethroids in Patagonian populations of Simulium blackflies

Mixed populations of the pest blackflies Simulium bonaerense Coscarón & Wygodzinsky, S. wolffhuegeli (Enderlein) and S. nigristrigatum Wygodzinsky & Coscarón (Diptera: Simuliidae) are highly resistant to DDT and pyrethroids in the Neuquén Valley, a fruit-growing area of northern Patagonia, Argentina. As these insecticides have not been used for blackfly control, resistance is attributed to exposure to agricultural insecticides. Pre-treatment with the synergist piperonyl butoxide (PBO) reduced both DDT and fenvalerate resistance, indicating that resistance was partly due to monooxygenase inhibition. Pre-treatment with the synergist tribufos to inhibit esterases slightly increased fenvalerate toxicity in the resistant population. Even so, biochemical studies indicated almost three-fold higher esterase activity in the resistant population, compared to the susceptible. Starch gel electrophoresis confirmed higher frequency and staining intensity of esterase electromorphs in the resistant population. Incomplete synergism against metabolic resistance indicates additional involvement of a non-metabolic resistance mechanism, such as target site insensitivity, assumed to be kdr-like in this case. Glutathione S-transferase activities were low and inconsistent, indicating no role in Simulium resistance. Knowing these spectra of insecticide activity and resistance mechanisms facilitates the choice of more effective products for Simulium control and permits better coordination with agrochemical operations.     

The influence of hypomagnesemia on erythrocyte antioxidant enzyme defence system in mice

The effect of magnesium deficiency on antioxidant defence system was studied in RBC of mice suffering from hypomagnesemia. The animals were kept for 8, 15 and 22 days on magnesium-deficient diet with consequent reduction of magnesium level in plasma by 38% at the first 8 days and by 64% after 22 days of experiment. The activities of the most important antioxidant enzymes, catalase, glutathione peroxidase, superoxide dismutase, glutathione reductase, glutahione S-transferase were assayed in hemolysates. The level of reduced glutathione in erythrocytes was measured as well. Apart from catalase, the activities of antioxidant enzymes were decreasing. The activity of superoxide dismutase decreased gradually during the experiment and on the 15th and 22nd day of experiment was significantly (P<0,05) lowered by 30 and 32% respectively. The catalase activity was increased on each point of the experiment with the peak value up to 149% on 15th day, and by 32% on 22nd day. Glutathione peroxidase activity was insignificantly reduced. The reduction of Glutatione reductase and Glutathione S-transferase activities by 24 and 21%, respectively, were observed after 8 days of the experiment with a further downward tendency. The reduced glutathione was significantly depleted after 8 days by 33% and was kept on that level in the course of the study. These findings support previous reports on the hypomagnesemia – induced alteration in endogenous enzyme antioxidant defences and glutathione redox cycle of mice.     

The effect of dietary protein and sulfur amino acids on hepatic glutathione concentration and glutathione-dependent enzyme activities in the rat

Hepatic glutathione concentration and glutathione-dependent enzymes, glutathione S-transferase, glutathione peroxidase, and glutathione reductase, are important for protection against toxic compounds. Rats were fed diets containing 4, 7.5, 15, or 45% protein for 2 weeks. Glutathione and cysteine concentrations in rats fed the 4 and 7.5% protein diets were significantly lower (p less than 0.05) than in rats fed the 15 and 45% protein diets. Glutathione S-transferase activity increased with increasing dietary protein. Glutathione peroxidase activity was significantly lower (p less than 0.05) in rats fed 4 and 7.5% protein compared with rats fed 15 and 45% protein, whereas the activity of glutathione reductase was higher in rats fed 4 and 7.5% protein then in rats fed 15 or 45% protein. Dietary sulfur amino acids alone could account for the increase in glutathione concentration resulting from the increase in dietary protein from 7.5 to 15%. The limited availability of glutathione in animals fed the low protein diets could reduce the potential for detoxification of xenobiotics.     

Glutathione S-transferase from the Icelandic scallop (Chlamys islandica): isolation and partial characterization

Glutathione S-transferase from the digestive gland of the cold-adapted marine bivalve Icelandic scallop was purified to apparent homogeneity by single GSTrap chromatography. The enzyme appeared to be a homodimer with subunit M(r) 22,000 having an optimum catalytic activity at pH 6.5-7. Enzymatic analysis of scallop GST using the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione resulted in apparent values for K(m)(GST) and K(m)(CDNB) of 0.3 mM and 0.4 mM, respectively. The scallop GST lost activity faster than porcine GST when exposed to increased temperatures, but both enzymes needed 10 min incubation at 60 degrees C for complete inactivation. A partial coding sequence was identified in cDNA synthesised from digestive gland mRNA. Comparison to known sequences indicates that the gene product is a glutathione S-transferase, and the predicted Icelandic scallop GST protein scores 40% sequence identity and 60% sequence similarity to mu-class proteins.     

Reduction of Brain Antioxidant Defense Upon Treatment with Butylated Hydroxyanisole (BHA) and Sudan III in Syrian Golden Hamster

Treatment with the antioxidant butylated hydroxyanisole (BHA) or the azo dye Sudan III during two weeks led to changes in the brain enzymatic antioxidant defense of Syrian golden hamsters. BHA was able to induce liver superoxide dismutase (SOD) 2-fold but had no effect on the brain SOD activity, whereas SOD activity was reduced to 50% in brain and remained unchanged in liver with Sudan III. These two substances are known inducers of DT-diaphorase and in fact this enzymatic activity was induced 4- and 6-fold in liver with BHA and Sudan III, respectively. However, BHA promoted a significant 40% reduction, whereas no change was observed with Sudan III in brain DT-diaphorase activity. Glutathione(GSH)-related enzymatic activities were also assayed in brain and liver. No induction was observed with BHA or Sudan III for any of the activities tested in hamster brain: GSH S-transferase (GST), GSH peroxidase (GSH-Px) and glutathione disulfide (GSSG) reductase (GR). Only 1.3- and 1. 4-fold increases of GST and GR activities were observed in liver and no change in any of these enzymatic activities in brain with BHA; a partial limitation of permeability to BHA of the blood-brain barrier may explain this results. Furthermore, Sudan III promoted reductions in all these GSH-related enzymatic activities in brain and liver. The possible explanations for these results are discussed.Deceased 4th November 1998     

Nickel-mediated inhibition in the glutathione-dependent protection against lipid peroxidation

Glutathione protects liver microsomes against the rapid onset of lipid peroxidation via a sulfhydryl dependent heat labile factor known as free radical reductase. The administration of nickel to mice resulted in an inhibition in the activity of free radical reductase, and enhanced lipid peroxidation and the activity of glutathione S-transferase in a dose dependent manner. The pretreatment of cyclam, a known specific chelator of nickel restored free radical reductase and glutathione S-transferase activities and alleviated nickel mediated enhancement of lipid peroxidation. Our results indicate that nickel-mediated inhibition in free radical reductase activity and activation of glutathione S-transferase may be due to the interaction of nickel with sensitive-SH groups located on these proteins.