亚硝基谷胱甘肽对大鼠肝微粒体谷胱甘肽转移酶的激活及机制

目的　探索一氧化氮供体亚硝基谷胱甘肽(GSNO)能否在体外通过S 亚硝酰化机制激活大鼠肝微粒体谷胱甘肽转移酶 (mGST)。方法　微粒体粗提物与GSNO体外共孵育 ,测定mGST催化动力学改变 ,结合N 乙基马来酰亚胺 (NEM )再激活实验和二巯基苏醇 (DTT)逆转实验 ,以及酶蛋白游离巯基和酶S 亚硝酰化蛋白的改变 ,研究酶的激活机制。结果　GSNO在 0 .12 5～ 2mmol·L- 1浓度范围内呈浓度和时间 (3～ 15min)依赖性地激活mGST ,NEM对酶的再激活效应消失 ,DTT可以逆转上述激活作用 ,同时酶蛋白游离巯基浓度依赖性减少 ,而S 亚硝酰化蛋白浓度依赖性增多。结论　GSNO体外可激活大鼠肝mGST ,激活机制可能与mGST第 4 9位半胱氨酸 (Cys4 9)的巯基被亚硝酰化形成S 亚硝基硫醇结构有关。     

The NO donor DETA-NONOate reversibly activates an inward current in neurones and is not mediated by the released nitric oxide

Background and purpose:

It has been previously shown that high levels of nitric oxide (NO), from NO donors, kill neurones, but the mechanisms are unclear.

Experimental approach:

The effects of NO donors on the electrical properties of rat cultured cerebellar granule cells (CGC neurones) were investigated using the whole-cell patch-clamp technique.

Key results:

The NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate or NOC-18) caused a rapid, persistent, but fully reversible inward current that was associated with an increase in baseline noise and was concentration dependent (100 µM–10 mM). The response to 3 mM DETA-NONOate was completely inhibited by 1 mM gadolinium, but not by NO scavengers (1 mM haemoglobin or 1 mM PTIO) or glutamate receptor antagonists (10 µM MK-801 or 60 µM CNQX). Application of decomposed 3 mM DETA-NONOate or 3 mM nitrite had no effect. In contrast, the NO donor S-nitrosoglutathione (GSNO) caused a rapid, persistent, but fully reversible outward current that was also concentration dependent (1–10 mM). The 3 mM GSNO response was unaltered by NO scavengers, glutamate antagonists or gadolinium, but was mimicked by decomposed 3 mM GSNO and 3 mM oxidized glutathione.

Conclusions and implications:

These results suggest that DETA-NONOate directly activates cation-selective channels, causing an inward current in CGCs. In contrast, GSNO causes an outward current in these cells. Some of the effects of these NO donors are independent of NO, and thus caution is required in interpreting results when using high concentrations of these compounds.     

Effect of 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one on oxidative stress in cerebral cortex of rats

The objective of this study was to verify the effect of the organochalcogen 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one on some parameters of oxidative stress in the brain of 10-day-old rats. Cerebral cortex was incubated for 1 h in the presence or absence of 1, 10 or 30 μM of 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one and thiobarbituric acid reactive substances (TBARS), carbonyl, sulfhydryl, catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST), nitric oxide (NO) production and the release of the cytosolic enzyme lactate dehydrogenase (LDH) were measured. The organotellurium was not capable to alter TBARS and carbonyl assays. In contrast, the compound at 10 and 30 μM provoked a reduced of protein thiol groups measured by the sulfhydryl assay. Furthermore, the activity of the antioxidant enzyme CAT (10 and 30 μM) and GPx (1, 10 and 30 μM) was reduced by the organochalcogen. On the other hand, the activity of SOD and GST were enhanced respectively by 1, 10 and 30 μM of the compound. Furthermore, NO production was also increased by 30 μM of this organochalcogen. Finally, we verified that the organotellurium was capable of enhance the LDH release at 30 μM concentration. Our findings indicate that this organotellurium compound induces in vitro oxidative stress in the cerebral cortex of rats being potentially toxic for the brain of rats.     

Activation of microsomal glutathione s-transferase by peroxynitrite

Peroxynitrite (ONOO-) toxicity is associated with protein oxidation and/or tyrosine nitration, usually resulting in inhibition of enzyme activity. We examined the effect of ONOO- on the activity of purified rat liver microsomal glutathione S-transferase (GST) and found that the activity of reduced glutathione (GSH)-free enzyme was increased 4- to 5-fold by 2 mM ONOO-; only 15% of this increased activity was reversed by dithiothreitol. Exposure of the microsomal GST to ONOO- resulted in concentration-dependent oxidation of protein sulfhydryl groups, dimer and trimer formation, protein fragmentation, and tyrosine nitration. With the exception of sulfhydryl oxidation, these modifications of the enzyme correlated well with the increase in enzyme activity. Nitration or acetylation of tyrosine residues of the enzyme using tetranitromethane and N-acetylimidazole, respectively, also resulted in increased enzyme activity, providing additional evidence that modification of tyrosine residues can alter catalytic activity. Addition of ONOO--treated microsomal GST to microsomal membrane preparations caused a marked reduction in iron-induced lipid peroxidation, which raises the possibility that this enzyme may act to lessen the degree of membrane damage that would otherwise occur under pathophysiological conditions of increased ONOO- formation.     

Collagen gel sandwich and immobilization cultures of rat hepatocytes: Problems encountered in expressing glutathione S-transferase activities

Collagen gel sandwich and immobilization cultures of rat hepatocytes are two recently developed organotypical culture models. Basic information with respect to the maintenance of xenobiotic biotransformation pathways and the expression of key enzyme activities, however, is lacking, making their use in pharmaco-toxicological studies rather speculative. The expression of the glutathione S-tranferase (GST; EC 2.5.1.18) activity, a key phase II enzyme, has been chosen to study the various problems that may arise in expressing the results of cytosolic enzyme activities when rat hepatocytes are cultured using both new culture models. Collagen gel matrix easily entraps culture medium proteins. These interfere with the cytosolic protein content, a parameter versus which cytosolic enzyme activities, including GSTs, are usually expressed. The following solutions are proposed: expression of the cytosolic enzyme activity results versus either (i) microsomal proteins, these are not contaminated by medium proteins, or versus (ii) cytosolic proteins after a complete collagenase digestion (0.05% collagenase type I of Sigma, 45 min, 37 °C) of the collagen matrix. Expression of enzyme activities versus cellular DNA appears to be unacceptable since unreliable results were obtained due to entrapped DNA in the collagen matrix. Once it was known how to express cytosolic enzyme activity, the maintenance of GST activities was investigated in both culture models using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) as substrates for total and Mu class GSTs, respectively. Two culture media were compared, control medium (DMEM) with and without supplementation of -proline (final concentration 60 μg/ml). In both culture models, after an initial decrease, total GST activities increased significantly up to values higher than those observed for freshly isolated cells. The Mu class GST activities were maintained constant for 7 days and increased thereafter. -Proline supplementation of the culture medium prevented the initial decline in total and Mu class GST activities in both culture configurations but did not seem to be of crucial importance in the maintenance of GST activities in both culture models.     

Expression of glutathione S-transferases in fetal lung and liver tissue from parental strains and F1 crosses between C57BL/6 and BALB/c F1 mice following in utero exposure to 3-methylcholanthrene

GST isoforms have been extensively studied in adult tissues but little is known about the composition and levels of these enzymes in fetal tissues. As part of our ongoing studies to determine the potential role of metabolic enzymes in mediating the differential susceptibility of different strains of mice to lung tumorigenesis following in utero exposure to 3-methylcholanthrene (MC), we screened for GST enzyme activity and for expression of the individual GSTalpha, pi, mu, and theta isoforms in murine fetal lung and liver tissues isolated from the parental strains and F1 crosses between C57BL/6 (B6) and BALB/c (C) mice. Using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate, we found that treatment with MC had no effect on the levels of GST enzyme activity in either the fetal lung or liver in either of the two parental strains or their F1 crosses. Low levels of expression of each of the four enzymes were detected by Western blotting in both fetal lung and liver tissues in all four strains. A statistically significant 3.5-fold induction was observed only for GSTmu in the fetal lung of the parental strain of BALB/c mice 48 h after exposure to MC. None of the other enzymes showed any significant differences in the levels of expression following exposure to MC. Although strain-specific differences in the expression of the GSTs that were independent of MC treatment were observed, they could not account for the differences previously observed in either the Ki-ras mutational spectrum or lung tumor incidence in the different strains of mice. Similar results were obtained when the maternal metabolism of MC was assayed in liver microsomal preparations. The results are consistent with previous studies showing low levels and poor inducibility of phase II enzymes during gestation, and demonstrate for the first time that all four of the major GST enzymes are expressed in fetal tissues. While the high inducibility of activating enzymes, such as Cyp1a1, and low, uninducible levels of phase II conjugating enzymes probably account for the high susceptibility of the fetus to transplacentally induced tumor formation, the results also suggest that factors other than metabolism may account for the strain-specific differences in susceptibility to carcinogen-mediated lung tumor induction following in utero exposure to chemical carcinogens.     

Nitric oxide inhibits insulin-degrading enzyme activity and function through S-nitrosylation

Insulin-degrading enzyme (IDE) is responsible for the degradation of a number of hormones and peptides, including insulin and amyloid β (Aβ). Genetic studies have linked IDE to both type 2 diabetes and Alzheimer's disease. Despite its potential importance in these diseases, relatively little is known about the factors that regulate the activity and function of IDE. Protein S-nitrosylation is now recognized as a redox-dependent, cGMP-independent signaling component that mediates a variety of actions of nitric oxide (NO). Here we describe a mechanism of inactivation of IDE by NO. NO donors decreased both insulin and Aβ degrading activities of IDE. Insulin-degrading activity appeared more sensitive to NO inhibition than Aβ degrading activity. IDE-mediated regulation of proteasome activity was affected similarly to insulin-degrading activity. We found IDE to be nitrosylated in the presence of NO donors compared to that of untreated enzyme and the control compound. S-nitrosylation of IDE enzyme did not affect the insulin degradation products produced by the enzyme, nor did NO affect insulin binding to IDE as determined by cross-linking studies. Kinetic analysis of NO inhibition of IDE confirmed that the inhibition was noncompetitive. These data suggest a possible reversible mechanism by which inhibition of IDE under conditions of nitrosative stress could contribute to pathological disease conditions such as Alzheimer's disease and type 2 diabetes.     

Effects of chitosan oligosaccharides on drug-metabolizing enzymes in rat liver and kidneys

To investigate the effect of chitosan oligosaccharides (COS) on drug-metabolizing enzymes in rat liver and kidneys, male Spraque–Dawley rats were fed a diet containing 1% or 3% COS for 5 weeks. The activities of cytochrome P450 (CYP) enzymes, UDP-glucurosyltransferase (UGT) and glutathione S-transferase (GST) in the liver and kidneys were determined. Significant decreases in microsomal CYP3A-catalyzed testosterone 6β-hydroxylation, CYP2C-catalyzed diclofenac 4-hydroxylation, and CYP4A-catalyzed lauric acid 12-hydroxylation in the liver of rats fed the COS diets were observed compared with those rats fed the control diet. Immunoblot analyses of CYP proteins showed the same trend as with enzyme activities. Increased glutathione content in liver was found in rats fed the 1% COS diet. Increased hepatic NADPH: quinone oxidoreductase 1 (NQO1) activity was found in rats fed the COS diets. In kidneys, COS had little or no effect on CYP enzyme activities. However, increased GST activity was observed in rats fed the COS diets. Moreover, a higher UGT activity was found in rats fed the 1% COS diet. Our results indicate that COS may suppress hepatic CYP enzymes and induce phase II detoxifying reactions in the liver and kidneys of rats.     

Glutathione S-transferase expression and isoenzyme composition during cell differentiation of Caco-2 cells

The human colon adenocarcinoma cell line Caco-2 is frequently used to study human intestinal metabolism and transport of xenobiotica. Previous studies have shown that both Caco-2 cells and human colon cells constitutively express the multigene family of detoxifying enzymes glutathione S-transferases (GSTs), particularly GST alpha and GST pi. GSTs may play a fundamental role in the molecular interplay between phase I, II enzymes and ABC-transporters. The gut fermentation product, butyrate, can modulate the potential for detoxification. The aim of this study was to investigate the basal expression of further cytosolic GSTs in Caco-2 cells during cell differentiation. In addition, a comparison was made with expression levels in MCF-7 and HepG2, two other cell types with barrier functions. Finally, the butyrate-mediated modulation of gene and protein expression was determined by real time PCR and western blot analysis.     

Role of microsomal glutathione transferase 1 in the mechanism-based biotransformation of glyceryl trinitrate in LLC-PK1 cells

Although glyceryl trinitrate (GTN) has been used in the treatment of angina for many years, details of its conversion to the proximal activator (presumed to be NO or an NO congener) of soluble guanylyl cyclase (sGC) are still unclear. We reported previously that purified microsomal glutathione transferase 1 (MGST1) mediates the denitration of GTN. In the current study, we investigated in intact cells whether this enzyme also converts GTN to species that activate sGC (mechanism-based biotransformation). We utilized LLC-PK1 cells, a cell line with an intact NO/sGC/cGMP system, and generated a stable cell line that overexpressed MGST1. MGST1 in the stably transfected cells was localized to the endoplasmic reticulum, and microsomes from these cells exhibited markedly increased GST activity. Although incubation of these cells with GTN resulted in a 3-4-fold increase in GTN biotransformation, attributed primarily to an increase in formation of the 1,3-glyceryl dinitrate metabolite, GTN-induced cGMP accumulation in cells overexpressing MGST1 was not different than that observed in wild type cells or in cells stably transfected with empty vector. To determine whether overexpression of NADPH cytochrome P450 reductase might act in concert with MGST1 to generate activators of sGC, we assessed GTN-induced cGMP accumulation in MGST1-overexpressing cells that had been transiently transfected with CPR. In this case, GTN-induced cGMP accumulation was also not different than that observed in wild type cells. We conclude that although MGST1 mediates the biotransformation of GTN in intact cells, this biotransformation does not contribute to the formation of activators of sGC.     

Hydrolysis of pyrethroids by human and rat tissues: examination of intestinal, liver and serum carboxylesterases

Hydrolytic metabolism of pyrethroid insecticides in humans is one of the major catabolic pathways that clear these compounds from the body. Rodent models are often used to determine the disposition and clearance rates of these esterified compounds. In this study the distribution and activities of esterases that catalyze pyrethroid metabolism have been investigated in vitro using several human and rat tissues, including small intestine, liver and serum. The major esterase in human intestine is carboxylesterase 2 (hCE2). We found that the pyrethroid trans-permethrin is effectively hydrolyzed by a sample of pooled human intestinal microsomes (5 individuals), while deltamethrin and bioresmethrin are not. This result correlates well with the substrate specificity of recombinant hCE2 enzyme. In contrast, a sample of pooled rat intestinal microsomes (5 animals) hydrolyze trans-permethrin 4.5-fold slower than the sample of human intestinal microsomes. Furthermore, it is demonstrated that pooled samples of cytosol from human or rat liver are approximately 2-fold less hydrolytically active (normalized per mg protein) than the corresponding microsomal fraction toward pyrethroid substrates; however, the cytosolic fractions do have significant amounts (approximately 40%) of the total esteratic activity. Moreover, a 6-fold interindividual variation in carboxylesterase 1 protein expression in human hepatic cytosols was observed. Human serum was shown to lack pyrethroid hydrolytic activity, but rat serum has hydrolytic activity that is attributed to a single CE isozyme. We purified the serum CE enzyme to homogeneity to determine its contribution to pyrethroid metabolism in the rat. Both trans-permethrin and bioresmethrin were effectively cleaved by this serum CE, but deltamethrin, esfenvalerate, alpha-cypermethrin and cis-permethrin were slowly hydrolyzed. Lastly, two model lipase enzymes were examined for their ability to hydrolyze pyrethroids. However, no hydrolysis products could be detected. Together, these results demonstrate that extrahepatic esterolytic metabolism of specific pyrethroids may be significant. Moreover, hepatic cytosolic and microsomal hydrolytic metabolism should each be considered during the development of pharmacokinetic models that predict the disposition of pyrethroids and other esterified compounds.     

Differential mechanisms of urethral smooth muscle relaxation by several NO donors and nitric oxide

We have examined the mechanisms of action of a broad spectrum of nitric oxide (NO) donors, including several S-nitrosothiols, sodium nitroprusside (SNP) and nitroglycerine (GTN), in relation to their relaxant activity of urethral smooth muscle. For all the compounds examined, NO release (in solution and in the presence of urethral tissue), relaxation responses, elevations in cGMP levels and the effect of thiol modulators were evaluated and compared with the effect of NO itself. Whilst all NO donors, except GTN, released NO in solution due to photolysis or chemical catalysis, this release was not correlated with their relaxant activity in sheep urethral preparations, which were furthermore not affected by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (cPTIO; 0.3 mM). A substantial NO-generating activity was found for S-nitroso-L-cysteine (CysNO) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) in the presence of urethral cytosolic fractions, suggesting metabolic activation to NO in the cytosol of the target tissue. In contrast, NO generation from S-nitroso-N-acetyl-L-cysteine (N-ac-CysNO), S-nitrosoglutathione (GSNO) and SNP were reduced by the presence of urethral homogenate and/or subcellular fractions, suggesting direct NO transfer to tissue constituents. NO donors and NO gas induced dissimilar degrees of cGMP accumulation in urethral tissue, while they were essentially equipotent as urethral relaxants. Furthermore, 1H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ; 10 μM) inhibited both relaxation and cGMP accumulations, but with different potency for the different compounds. Oxidation of sarcolemmal thiol groups with 5-5′-dithio-bis[2-nitrobenzoic acid] (DTNB; 0.5 mM) enhanced relaxations to GSNO, an effect that was reversed by dithiotreitol (DTT; 1 mM), suggesting a direct effect through nitrosylation/oxidation reactions at the cell membrane, while relaxations to NO and to all the other compounds were not affected by these treatments. Finally, photodegradation of SNP induced the formation of a stable intermediate that still evoked NO-cGMP-mediated relaxations. This indicates that the assumption that SNP is fully depleted of NO by exposure to light should be revised. It can be concluded that important differences exist in the mechanisms by which distinct NO donors relax urethral smooth muscle and they cannot be regarded simply as NO-releasing prodrugs. Received: 28 December 1998 / Accepted: 14 April 1999 / Published online: 22 June 1999     

苯丁酸氮芥及环磷酰胺对大鼠肝微粒体谷胱甘肽S-转移酶的激活

目的探索苯丁酸氮芥(CHB)和环磷酰胺(CP)在体外是否通过烷化激活大鼠肝微粒体谷胱甘肽S-转移酶(mGST)。方法微粒体粗提物与CHB或CP体外共孵育,测定mGST催化动力学改变,结合N-乙基马来酰亚胺(NEM)再激活实验和结合二硫苏糖醇(DTT)逆转实验,研究酶激活机制。结果CHB或CP浓度(0～5mmol.L-1)与时间(0～5min)依赖性地激活mGTS。增强的mGST活性能被NEM进一步增强,不被二硫键断裂剂DTT逆转,NEM对CHB或CP活化后的mGST活性的增强效应与NEM单独的增强效应无差异。结论CHB或CP体外可激活大鼠肝mGST,激活机制可能与mGST的Cys49的巯基被CHB或CP修饰激活有关。     

Dynamics of glutathione-S-transferases in Mytilus galloprovincialis exposed to toxic Microcystis aeruginosa cells, extracts and pure toxins

Molluscs and especially bivalves are able to accumulate dinoflagelates, diatoms and cyanobacteria toxins, and, being vectors for these toxins, transfer them along food chains. The data obtained from laboratory experiments showed that bivalve molluscs are resistant to cyanobacteria toxins. In this work, we wanted to test if Mytilus galloprovincialis organs react to microcystins and other cyanobacteria compounds by inducing or decreasing its GST activity. Acclimated mussels M. galloprovincialis were exposed to the toxic Microcystis aeruginosa M13 strain. Exposure of mussels to toxins was done in three ways: living Microcystis cells, crude Microcystis extracts and pure toxins. The measurement of soluble and microsomal GST activity in the different mussel organs was done by using the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 2,4-dichloro-1-nitrobenzene (DCNB). Analysis of the GST activity of the control mussels using CDNB as a substrate showed that cytosolic activity is much more significant than microsomal. Intact M. aeruginosa cells did not induce any significant response from the mussels, showing that these animals are quite resistant to the cyanobacteria if they are intact. On the other hand, cell extracts caused an important effect in the gut, in the gills and in the labial palps, although in different ways. There was an increase in GST activity in the gut and gills of mussels exposed to Microcystis extracts, showing a response of this detoxication pathway, but in the labial palps a severe reduction in GST activity occurred. Pure MC LR+YR induced an increase in GST activity in all organs but the labial palps. The results showed that other substances apart from microcystins may cause stress to mussels and affect detoxication enzymes such as GST.     

Bilirubin and S-nitrosothiols interaction: evidence for a possible role of bilirubin as a scavenger of nitric oxide

Bilirubin (BR), the final product of heme catabolism, plays a crucial role in the defense against reactive oxygen species in various cell types. In this study, we addressed the hypothesis that BR can act as a physiological scavenger of nitric oxide (NO), a gaseous mediator involved in many cellular functions and able to trigger the formation of reactive nitrogen species with pro-oxidant activity. We found that S-nitrosocysteine (SNOC) and S-nitrosoglutathione (GSNO), which have a half-life of 0.52+/-0.07 hr and 38+/-5 hr and release NO at a constant rate of 1.42+/-0.2 hr(-1) and 0.018+/-0.002 hr(-1), respectively, were able to decrease BR half-life in a concentration-dependent manner under physiological conditions. This effect appears to be dependent on NO formation as L-cysteine and GSH did not affect BR consumption and nitrite was four to five times less efficient than SNOC in reducing BR half-life. Oxyhemoglobin, a well-known scavenger of NO, protected BR from SNOC-mediated degradation. In addition, the reaction between SNOC/GSNO and BR modified the absorption spectrum of the bile pigment showing a gradual increase in the absorbance at 316 nm. This change in the BR spectrum indicates that the bile pigment could be a target for N-nitrosation reactions, since it resembles the modifications occurred when other molecules such as di-peptides and uric acid are nitrosated. Taken together, these data suggest that BR should not be considered only as an endogenous antioxidant but also as a molecule with the potential ability to counteract intracellular nitrosative stress reactions.     

Nitric oxide donors prevent while the nitric oxide synthase inhibitor L-NAME increases arachidonic acid plus CYP2E1-dependent toxicity

Polyunsaturated fatty acids such as arachidonic acid (AA) play an important role in alcohol-induced liver injury. AA promotes toxicity in rat hepatocytes with high levels of cytochrome P4502E1 and in HepG2 E47 cells which express CYP2E1. Nitric oxide (NO) participates in the regulation of various cell activities as well as in cytotoxic events. NO may act as a protectant against cytotoxic stress or may enhance cytotoxicity when produced at elevated concentrations. The goal of the current study was to evaluate the effect of endogenously or exogenously produced NO on AA toxicity in liver cells with high expression of CYP2E1 and assess possible mechanisms for its actions. Pyrazole-induced rat hepatocytes or HepG2 cells expressing CYP2E1 were treated with AA in the presence or absence of an inhibitor of nitric oxide synthase L-N(G)-Nitroarginine Methylester (L-NAME) or the NO donors S-nitroso-N-acetylpenicillamine (SNAP), and (Z)-1-[-(2-aminoethyl)-N-(2-aminoethyl)]diazen-1-ium-1,2-diolate (DETA-NONO). AA decreased cell viability from 100% to 48+/-6% after treatment for 48 h. In the presence of L-NAME, viability was further lowered to 23+/-5%, while, SNAP or DETA-NONO increased viability to 66+/-8 or 71+/-6%. The L-NAME potentiated toxicity was primarily necrotic in nature. L-NAME did not affect CYP2E1 activity or CYP2E1 content. SNAP significantly lowered CYP2E1 activity but not protein. AA treatment increased lipid peroxidation and lowered GSH levels. L-NAME potentiated while SNAP prevented these changes. Thus, L-NAME increased, while NO donors decreased AA-induced oxidative stress. Antioxidants prevented the L-NAME potentiation of AA toxicity. Damage to mitochondria by AA was shown by a decline in the mitochondrial membrane potential (MMP). L-NAME potentiated this decline in MMP in association with its increase in AA-induced oxidative stress and toxicity. NO donors decreased this decline in MMP in association with their decrease in AA-induced oxidative stress and toxicity. These results indicate that NO can be hepatoprotective against CYP2E1-dependent toxicity, preventing AA-induced oxidative stress.     

Effects of simultaneous repeated exposure at high levels of arsenic and malathion on hepatic drug-biotransforming enzymes in broiler chickens

Groundwater contamination with arsenic is a major global health concern. The organophosphorus insecticide malathion has gained significance as an environmental pollutant due to its widespread use in agriculture, grain storage, ectoparasite control and public health management. The deleterious effects produced by arsenic or malathion alone are documented, but very little is known about the consequences of their coexposure. The aim of the current study was to examine the effects of repeated simultaneous exposure to arsenic and malathion on drug-biotransforming enzymes in the liver of broiler chickens. One-month-old broiler chickens were exposed daily to arsenic (50 ppm)-supplemented drinking water, malathion (500 ppm)-mixed diet or in a similar fashion coexposed to these agents for 28 days. At the term, changes in body weight, organ weights, and levels of hepatic cytochrome P450 (CYP), cytochrome b₅, microsomal and cytosolic proteins; aminopyrine N-demethylase (ANDM), aniline P-hydroxylase (APH), glutathione S-transferase (GST) and uridine diphosphate glucuronosyltransferase (UGT) were assessed. Arsenic, malathion or their coexposure decreased the body weight gain and liver weight. Brain weight (relative) was increased with arsenic or malathion, but not with the coexposure. Treatment with arsenic decreased the CYP and cytochrome b₅ contents by 39 and 36%, than with malathion by 54 and 22% and the coexposure by 45 and 28%, respectively. The ANDM activity was decreased with arsenic (44%), malathion (23%) and the coexposure (32%). Arsenic (23%) and the coexposure (37%), but not malathion (14%), reduced the APH activity. The activities of hepatic microsomal and cytosolic GST were increased with all the three treatments [Arsenic (microsomal: 88% cytosolic: 113%), malathion (microsomal: 137%, cytosolic: 94%) and coexposure (microsomal: 140%, cytosolic: 148%)]. These treatments did not significantly affect the hepatic UGT activity, but reduced the hepatic microsomal (arsenic: 28%, malathion: 34% and coexposure: 43%) and cytosolic (17–19%) protein contents. The effects of coexposure on the activities of various phase I and phase II drug-biotransforming enzymes were almost similar to that of arsenic or malathion. This study provides evidence that repeated coexposure to arsenic and malathion may influence the extent of drug metabolism in chickens.     

The mechanism of the nitric oxide-mediated enhancement of tert-butylhydroperoxide-induced DNA single strand breakage

1. Caffeine (Cf) enhances the DNA cleavage induced by tert-butylhydroperoxide (tB-OOH) in U937 cells via a mechanism involving Ca²⁺-dependent mitochondrial formation of DNA-damaging species (Guidarelli et al., 1997b). Nitric oxide (NO) is not involved in this process since U937 cells do not express the constitutive nitric oxide synthase (cNOS).
2. Treatment with the NO donors S-nitroso-N-acetyl-penicillamine (SNAP, 10 μM), or S-nitrosoglutathione (GSNO, 300 μM), however, potentiated the DNA strand scission induced by 200 μM tB-OOH. The DNA lesions generated by tB-OOH alone, or combined with SNAP, were repaired with superimposable kinetics and were insensitive to anti-oxidants and peroxynitrite scavengers but suppressed by iron chelators.
3. SNAP or GSNO did not cause mitochondrial Ca²⁺ accumulation but their enhancing effects on the tB-OOH-induced DNA strand scission were prevented by ruthenium red, an inhibitor of the calcium uniporter of mitochondria. Furthermore, the enhancing effects of both SNAP and GSNO were identical to and not additive with those promoted by the Ca²⁺-mobilizing agents Cf or ATP.
4. The SNAP- or GSNO-mediated enhancement of the tB-OOH-induced DNA cleavage was abolished by the respiratory chain inhibitors rotenone and myxothiazol and was not apparent in respiration-deficient cells.
5. It is concluded that, in cells which do not express the enzyme cNOS, exogenous NO enhances the accumulation of DNA single strand breaks induced by tB-OOH via a mechanism involving inhibition of complex III.

     

Inhibition of nitric oxide/cyclic GMP-mediated relaxation by purified flavonoids, baicalin and baicalein, in rat aortic rings

The dried roots of Scutellaria baicalensis Georgi (Huangqin) are widely used in traditional Chinese medicine. We purified two flavonoids, baicalin and baicalein from S. baicalensis Georgi and examined their effects on isolated rat aortic rings. Baicalin (3-50 microM) inhibited endothelium/nitric oxide (NO)-dependent relaxation induced by acetylcholine (Ach) or cyclopiazonic acid (CPA). Baicalein at 50 microM abolished Ach-induced relaxation and markedly reduced CPA-induced relaxation. Treatment with 1mM L-arginine partially but significantly reversed the effects of baicalin (50 microM) or baicalein (50 microM) on Ach-induced relaxation. In endothelium-denuded rings, treatment with baicalin, baicalein or methylene blue partially inhibited relaxations induced by the NO donors, sodium nitroprusside (SNP) and hydroxylamine. Both flavonoids markedly reduced the increase in cyclic GMP levels stimulated by Ach in endothelium-intact rings and by SNP in endothelium-denuded rings. In contrast, exposure of endothelium-denuded rings to baicalin or baicalein did not affect relaxations induced by pinacidil or NS 1619, putative K+ channel activators. Neither flavonoids affected agonist-induced increase in the endothelial [Ca2+]i. Our results indicate that baicalin and baicalein attenuated NO-mediated aortic relaxation and cyclic GMP increases, likely through inhibition of NO-dependent guanylate cyclase activity.