Effects of tebuconazole on cytochrome P450 enzymes,oxidative stress,and endocrine disruption in male rats

The major objective of the present study was to determine the ability of a triazole fungicide tebuconazole to induce cytochrome P450‐dependent monooxygenases, oxidative stress, and endocrine‐disrupting activity using male rats treated with tebuconazole at 10, 25, and 50 mg/kg p.o. once daily for 28 days. In liver, tebuconazole dose‐dependently increased microsomal contents of cytochrome P450 and cytochrome b₅ and the activities of NADPH‐cytochrome P450 reductase, 7‐ethoxyresorufin O‐deethylase, methoxyresorufin O‐demethylase, pentoxyresorufin O‐dealkylase, 7‐ethoxycoumarin O‐deethylase, aniline hydroxylase, and erythromycin N‐demethylase. In kidney, tebuconazole increased 7‐ethoxycoumarin O‐deethylase activity without affecting other monooxygenase activities. In marked contrast to liver and kidney, tebuconazole decreased testicular 7‐ethoxyresorufin O‐deethylase, methoxyresorufin O‐demethylase, 7‐ethoxycoumarin O‐deethylase, aniline hydroxylase, and erythromycin N‐demethylase activities. The results of immunoblot analysis of liver microsomes of controls and tebuconazole‐treated rats revealed that tebuconazole induced CYP1A1/2, CYP2B1/2, CYP2E1, and CYP3A proteins in liver. Additions of tebuconazole to liver microsomes inhibited microsomal 7‐ethoxycoumarin O‐deethylase activity in vitro (IC₅₀ = 1.50–1.69 µM). Treatment of rats with tebuconazole decreased glutathione content and increased glutathione S‐transferase, superoxide dismutase, catalase, and glutathione peroxidase activities in liver; increased superoxide dismutase activities in kidney and testis; but decreased glutathione S‐transferase activity in testis. Treatments with tebuconazole decreased serum testosterone concentration and cauda epididymal sperm count. The present study demonstrates that tebuconazole induces a multiplicity of CYPs and oxidative stress in liver; inhibits testicular P450 and glutathione S‐transferase activities; and produces anti‐androgenic effects in male rats.     

Tumour necrosis factor‐α inhibition with lenalidomide alleviates tissue oxidative injury and apoptosis in ob/ob obese mice

Lenalidomide (Revlimid; Selleck Chemicals, Houston, TX, USA), an analogue of thalidomide, possesses potent cytokine modulatory capacity through inhibition of cytokines such as tumour necrosis factor (TNF)‐α, a cytokine pivotal for the onset and development of complications in obesity and diabetes mellitus. The present study was designed to evaluate the effect of lenalidomide on oxidative stress, protein and DNA damage in multiple organs in an ob/ob murine model of obesity. To this end, C57BL/6 lean and ob/ob obese mice were administered lenalidomide (50 mg/kg per day, p.o.) for 5 days. Oxidative stress, protein and DNA damage were assessed using the conversion of reduced glutathione (GSH) to oxidized glutathione (GSSG), carbonyl formation and Comet assay, respectively. Apoptosis was evaluated using caspase 3 activity, and levels of Bax, Bcl‐2, Bip, caspase 8, caspase 9 and TNF‐α were assessed using western blot analysis. Lenalidomide treatment did not affect glucose clearance in lean or ob/ob mice. Obese mice exhibited a reduced GSH/GSSG ratio in the liver, gastrocnemius skeletal muscle and small intestine, as well as enhanced protein carbonyl formation, DNA damage and caspase 3 activity in the liver, kidney, skeletal muscle and intestine; these effects were alleviated by lenalidomide, with the exception of obesity‐associated DNA damage in the liver and kidney. Western blot analysis revealed elevated TNF‐α, Bax, Bcl‐2, Bip, caspase 8 and caspase 9 in ob/ob mice with various degrees of reversal by lenalidomide treatment. Together, these data indicate that lenalidomide protects against obesity‐induced tissue injury and protein damage, possibly in association with antagonism of cytokine production and cytokine‐induced apoptosis and oxidative stress.     

PCB-associated alteration of hepatic steroid metabolism in harbor seals (Phoca vitulina)

Hepatic cytochrome P-450 (CYP450) isozymes are involved in xenobiotic detoxification and steroid metabolism. Seals are highly exposed to polychlorinated biphenyls (PCBs) in the environment, resulting in CYP450 induction, which may have concomitant effects on CYP450 steroid metabolism. Experiments were conducted to determine rates of steroid metabolism in harbor seal (Phoca vitulina) liver. CYP450 was induced with increasing liver PCB concentrations between 0.2 and 5.73 microg/g (wet weight). Progesterone metabolism was significantly decreased with increasing liver PCB concentration. Testosterone metabolism was also increased with increasing liver PCB concentration. The results indicate a change in steroid metabolism associated with increasing PCB burden, suggesting that PCBs may have a modulating effect on hepatic steroid biotransformation in environmentally exposed seals. The possible implications of this for endocrine homeostasis in seals are discussed.     

Rossicaside B Protects against Carbon Tetrachloride‐induced Hepatotoxicity in Mice

Abstract: The protective effect of rossicaside B, the major phenylpropanoid glycoside from Boschniakia rossica, on CCl₄‐induced hepatotoxicity and the mechanisms underlying its protective effect were investigated. The mice were administered orally with rossicaside B (100 or 200 mg/kg of body weight) 48, 24 and 1 hr before CCl₄ (0.5 ml/kg of body weight) administration. The CCl₄ challenge caused a marked increase in the levels of serum aspartate aminotransferase, alanine aminotransferase and of tumour necrosis factor‐α, and propagated lipid peroxidation with a concomitant reduction in reduced glutathione (GSH) and antioxidative enzyme activities in the liver. The administration of rossicaside B to CCl₄‐treated mice not only decreased the serum toxicity marker enzymes and TNF‐α but also reduced hepatic oxidative stress, as demonstrated by decreased lipid hydroperoxide and thiobarbituric acid‐reactive substance concentrations, combined with elevated GSH content and antioxidative enzyme activities in the liver tissues. Furthermore, the contents of hepatic nitrite, inducible nitric oxide synthase (iNOS), cyclooxygenase‐2 (COX‐2) and haem oxygenase‐1 (HO‐1) were elevated after CCl₄ treatment while the cytochrome P450 2E1 (CYP2E1)‐specific monooxygenase activity was suppressed. Rossicaside B treatment inhibited the formation of liver nitrite, reduced the over‐expression of iNOS and COX‐2 proteins, but increased the CYP2E1 function compared with the CCl₄‐treated mice. However, the protein expression of HO‐1 was further elevated by rossicaside B treatment. The results demonstrate that rossicaside B provides a protective action on CCl₄‐induced acute hepatic injury, which may be related to its antioxidative activity, suppressed inflammatory responses, induced HO‐1 expression and improved CYP2E1 function in the liver.     

Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species

Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and β-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from β-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.     

Cytotoxicity of luteolin in primary rat hepatocytes: the role of CYP3A‐mediated ortho‐benzoquinone metabolite formation and glutathione depletion

Luteolin (LUT), an active ingredient in traditional Chinese medicines and an integral part of the human diet, has shown promising pharmacological activities with a great potential for clinical use. The purpose of this study was to evaluate the role of cytochrome P450 (CYP450)‐mediated reactive ortho‐benzoquinone metabolites formation and glutathione (GSH) depletion in LUT‐induced cytotoxicity in primary rat hepatocytes. A reactive ortho‐benzoquinone metabolite was identified by liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) in rat liver microsomes (RLMs) and rat hepatocytes. Using a specific chemical inhibitor method, the CYP3A subfamily was found to be responsible for the reactive metabolite formation in RLMs. Induction of CYP3A by dexamethasone enhanced LUT‐induced cytotoxicity, whereas inhibition of CYP3A by ketoconazole (Keto) decreased the cytotoxicity. The cytotoxicity and cell apoptosis induced by LUT were related to the amount of reactive metabolite formation. Furthermore, Keto inhibited the LUT‐induced GSH exhaustion. The cytotoxicity was significantly enhanced by pretreatment with L‐buthionine sulfoximine to deplete the intracellular GSH. A time course experiment showed that GSH depletion by LUT was not via oxidation of GSH and occurred prior to the increase in 2', 7'‐dichlorofluorescein in hepatocytes. Collectively, these data suggest that CYP3A‐mediated reactive metabolite formation plays a critical role in LUT‐induced hepatotoxicity, and the direct GSH depletion is an initiating event in LUT‐mediated cytotoxicity in primary rat hepatocytes. Copyright © 2015 John Wiley & Sons, Ltd.     

Differential toxicity of heterocyclic aromatic amines and their mixture in metabolically competent HepaRG cells

Human exposure to heterocyclic aromatic amines (HAA) usually occurs through mixtures rather than individual compounds. However, the toxic effects and related mechanisms of co-exposure to HAA in humans remain unknown. We compared the effects of two of the most common HAA, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), individually or in combination, in the metabolically competent human hepatoma HepaRG cells. Various endpoints were measured including cytotoxicity, apoptosis, oxidative stress and DNA damage by the comet assay. Moreover, the effects of PhIP and/or MeIQx on mRNA expression and activities of enzymes involved in their activation and detoxification pathways were evaluated. After a 24 h treatment, PhIP and MeIQx, individually and in combination, exerted differential effects on apoptosis, oxidative stress, DNA damage and cytochrome P450 (CYP) activities. Only PhIP induced DNA damage. It was also a stronger inducer of CYP1A1 and CYP1B1 expression and activity than MeIQx. In contrast, only MeIQx exposure resulted in a significant induction of CYP1A2 activity. The combination of PhIP with MeIQx induced an oxidative stress and showed synergistic effects on apoptosis. However, PhIP-induced genotoxicity was abolished by a co-exposure with MeIQx. Such an inhibitory effect could be explained by a significant decrease in CYP1A2 activity which is responsible for PhIP genotoxicity. Our findings highlight the need to investigate interactions between HAA when assessing risks for human health and provide new insights in the mechanisms of interaction between PhIP and MeIQx.     

Repeated administration of oxycodone modifies the gene expression of several drug metabolising enzymes in the hepatic tissue of male Sprague‐Dawley rats,including glutathione S‐transferase A‐5 (rGSTA5) and CYP3A2

Objectives Clinical use and illicit abuse of the potent opioid agonist oxycodone has dramatically increased over the past decade. Yet oxycodone remains one of the least studied opioids, particularly its interactions on the genomic level. The aim of this study was to examine potential alterations in gene expression of drug metabolising enzymes in the liver tissue of male Sprague‐Dawley rats chronically treated with oxycodone. Methods Rats were administered saline or oxycodone 15 mg/kg i.p. twice a day for 8 days. Changes in RNA levels were detected using microarray analysis validated by quantitative real‐time PCR; consequent changes in protein expression and functionality were further assessed by Western blotting and activity assays. Key findings The expression of several drug metabolising enzymes was modulated by oxycodone treatment: cytochrome P450 (CYP) 2B2, CYP2C13, CYP17A1, epoxide hydrolase 2, carboxylesterase 2, flavin‐containing monooxygenase 1, glutathione S‐transferase alpha 5 (rGSTA5) and CYP3A2. In particular, the mRNA level of rGSTA5 (formerly GSTYc₂) was up‐regulated by approximately 6.5 fold and CYP3A2 was down‐regulated by approximately 7.0 fold. Immunoblotting assays demonstrated a corresponding significant elevation of rGSTA5 protein and repression of CYP3A2 protein. The apparent cytosolic GST activity towards 1‐chloro‐2,4‐dinitrobenzene conjugation and reduction of cumene hydroperoxide were significantly higher in liver from oxycodone‐treated rats than that of saline‐treated rats. In addition, the microsomal activity of CYP3A2, measured via 6β‐hydroxylation of testosterone, was significantly decreased in oxycodone‐treated rats. Conclusions Repeated oxycodone administration is associated with a significant up‐regulation of rGSTA5 and concomitant down‐regulation of CYP3A2 mRNA, protein expression and functionality. These results support further in‐vivo studies into the clinical impact of our findings.     

Metabolic pathways leading to detoxification of triptolide,a major active component of the herbal medicine Tripterygium wilfordii

Triptolide (TP) shows promising anti‐inflammatory and antitumor activity but with severe toxicity. TP is a natural reactive electrophile containing three epoxide groups, which are usually linked to hepatotoxicity via their ability to covalently bind to cellular macromolecules. In this study, metabolic pathways leading to detoxification of TP were evaluated in glutathione (GSH)‐depleted (treated with L‐buthionine‐S,R‐sulfoxinine, BSO) and aminobenzotriazole (ABT; a non‐specific inhibitor for P450s)‐treated mice. The toxicity of TP in mice was evaluated in terms of mortality and levels of serum alanine transaminase (ALT). In incubates with NADPH‐ and GSH‐supplemented liver microsomes, seven GSH conjugates derived from TP were detected. In mice, these hydrolytically unstable GSH conjugates underwent γ‐glutamyltranspeptidase/dipeptidases‐mediated hydrolysis leading to two major cysteinylglycine conjugates, which underwent further hydrolysis by dipeptidases to form two cysteine conjugates of TP. In ABT‐treated mice, the hydroxylated metabolites of TP were found at a lower level than normal mice, and their subsequent conjugated metabolites were not found. The level of cysteinylglycine and cysteine conjugates derived from NADPH‐independent metabolism increased in mice treated with both TP and BSO (or ABT), which could be the stress response to toxicity of TP. Compared with normal mice, mortality and ALT levels were significantly higher in TP‐treated mice, indicating the toxicity of TP. Pretreatment of ABT increased the toxicity caused by TP, whereas the mortality decreased in GSH‐depleted mice. Metabolism by cytochrome P450 enzymes to less reactive metabolites implied a high potential for detoxification of TP. The GSH conjugation pathway also contributed to TP's detoxification in mice. Copyright © 2013 John Wiley & Sons, Ltd.     

Beetroot juice protects against N-nitrosodiethylamine-induced liver injury in rats

Red beetroot, a common ingredient of diet, is a rich source of a specific class of antioxidants, betalains. Our previous studies have shown the protective role of beetroot juice against carcinogen induced oxidative stress in rats. The aim of this study was to examine the effect of long term feeding (28 days) with beetroot juice on phase I and phase II enzymes, DNA damage and liver injury induced by hepatocarcinogenic N-nitrosodiethylamine (NDEA). Long term feeding with beetroot juice decreased the activities of enzymatic markers of cytochrome P450, CYP1A1/1A2 and CYP2E1. NDEA treatment also reduced the activities of these enzymes, but increased the activity of CYP2B. Moreover, combined treatment with beetroot juice and NDEA enhanced significantly CYP2B only. Modulation of P450 enzyme activities was accompanied by changes in the relevant proteins levels. Increased level and activity of NQO1 was the most significant change among phase II enzymes. Beetroot juice reduced the DNA damage increased as the result of NDEA treatment, as well as the biomarkers of liver injury.     

The discovery of the microsomal ethanol oxidizing system and its physiologic and pathologic role

Oxidation of ethanol via alcohol dehydrogenase (ADH) explains various metabolic effects of ethanol but does not account for the tolerance. This fact, as well as the discovery of the proliferation of the smooth endoplasmic reticulum (SER) after chronic alcohol consumption, suggested the existence of an additional pathway which was then described by Lieber and DeCarli, namely the microsomal ethanol oxidizing system (MEOS), involving cytochrome P450. The existence of this system was initially challenged but the effect of ethanol on liver microsomes was confirmed by Remmer and his group. After chronic ethanol consumption, the activity of the MEOS increases, with an associated rise in cytochrome P450, especially CYP2E1, most conclusively shown in alcohol dehydrogenase negative deer mice. There is also cross-induction of the metabolism of other drugs, resulting in drug tolerance. Furthermore, the conversion of hepatotoxic agents to toxic metabolites increases, which explains the enhanced susceptibility of alcoholics to the adverse effects of various xenobiotics, including industrial solvents. CYP2E1 also activates some commonly used drugs (such as acetaminophen) to their toxic metabolites, and promotes carcinogenesis. In addition, catabolism of retinol is accelerated resulting in its depletion. Contrasting with the stimulating effects of chronic consumption, acute ethanol intake inhibits the metabolism of other drugs. Moreover, metabolism by CYP2E1 results in a significant release of free radicals which, in turn, diminishes reduced glutathione (GSH) and other defense systems against oxidative stress which plays a major pathogenic role in alcoholic liver disease. CYP1A2 and CYP3A4, two other perivenular P450s, also sustain the metabolism of ethanol, thereby contributing to MEOS activity and possibly liver injury. CYP2E1 has also a physiologic role which comprises gluconeogenesis from ketones, oxidation of fatty acids, and detoxification of xenobiotics other than ethanol. Excess of these physiological substrates (such as seen in obesity and diabetes) also leads to CYP2E1 induction and nonalcoholic fatty liver disease (NAFLD), which includes nonalcoholic fatty liver and nonalcoholic steatohepatitis (NASH), with pathological lesions similar to those observed in alcoholic steatohepatitis. Increases of CYP2E1 and its mRNA prevail in the perivenular zone, the area of maximal liver damage. CYP2E1 up-regulation was also demonstrated in obese patients as well as in rat models of obesity and NASH. Furthermore, NASH is increasingly recognized as a precursor to more severe liver disease, sometimes evolving into "cryptogenic" cirrhosis. The prevalence of NAFLD averages 20% and that of NASH 2% to 3% in the general population, making these conditions the most common liver diseases in the United States. Considering the pathogenic role that up-regulation of CYP2E1 also plays in alcoholic liver disease (vide supra), it is apparent that a major therapeutic challenge is now to find a way to control this toxic process. CYP2E1 inhibitors oppose alcohol-induced liver damage, but heretofore available compounds are too toxic for clinical use. Recently, however, polyenylphosphatidylcholine (PPC), an innocuous mixture of polyunsaturated phosphatidylcholines extracted from soybeans (and its active component dilinoleoylphosphatidylcholine), were discovered to decrease CYP2E1 activity. PPC also opposes hepatic oxidative stress and fibrosis. It is now being tested clinically.     

Impact of zinc oxide nanoparticles on thioredoxin‐interacting protein and asymmetric dimethylarginine as biochemical indicators of cardiovascular disorders in gamma‐irradiated rats

Nanoparticle is a microscopic particle that has been existed in a wide range of biotechnological purposes. Zinc oxide nanoparticles (ZnO‐NPs) have fewer environmental hazards and have shown positive impacts in the medical field. This work aimed to observe the effects of low and high doses of ZnO‐NPs on heart injury induced by ionizing radiation (IR). Animals were irradiated by 8 Gy of gamma rays and ZnO‐NPs (10 and 300 mg/Kg/day) were orally delivered to rats 1 hour after irradiation. Animals were dissected on 15th day postirradiation. Data showed that the oxidative damage resulted from radiation exposure, appeared by marked increments in the malondialdehyde (MDA) content and the level and protein expression of thioredoxin‐interacting protein (TXNIP) with a noticeable decline in the level and expression of thioredoxin 1 (Trx‐1) and thioredoxin reductase (TrxR), as well as glutathione (GSH) level and the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Moreover, radiation‐induced inflammation, manifested by a noticeable elevation in the level of tumor necrotic factor‐alpha (TNF‐α), interleukin‐18 (IL‐18), and C‐reactive protein (CRP). Additionally, endothelial dysfunction marked with a high level of asymmetric dimethylarginine (ADMA), total nitrite/nitrate (NOx), intercellular adhesion molecule 1 (ICAM‐1), homocysteine (Hcy), creatine kinase (CK‐MB), cardiac troponin‐I (cTn‐I), and lactate dehydrogenase (LDH). In addition, a decrease of zinc (Zn) level in the cardiac tissue was recorded. ZnO‐NPs treatment (10 mg/kg) mitigated the oxidative stress and inflammation effects on the cardiovascular tissue through the positive modulations in the studied parameters. In contrast, ZnO‐NPs treatment (300 mg/kg) induced cardiovascular toxicity of normal rats and elevated the deleterious effects of radiation. In conclusion, ZnO‐NPs at a low dose could mitigate the adverse effects on cardiovascular tissue induced by radiation during its applications, while the high dose showed morbidity and mortality in normal and irradiated rats.     

Induction of CYP1A1 and CYP2E1 in rat liver by histamine: binding and kinetic studies

Histamine (HA) may bind to cytochrome P450 (CYP450) in rat liver microsomes. The CYP450-HA complex seems to regulate some cellular processes such as proliferation. In the present work, it is shown that HA increases the activity and protein level of CYP1A1 and CYP2E1, in vivo. CYP1A1 is associated with polycyclic aromatic hydrocarbon-mediated carcinogenesis and CYP2E1 with liver damage by oxidative stress. Studies of enzyme kinetics and binding with rat liver microsomes and supersomes were carried out to determine whether HA is a substrate of CYP1A1 and/or CYP2E1. The lack of NADPH oxidation in the presence of HA showed that it is not a substrate for CYP1A1. Activity measurements using the O-dealkylation of ethoxyresorufin indicated that HA is a mixed-type inhibitor of CYP1A1 in both microsomes and supersomes. On the other hand, HA induced a significant NADPH oxidation catalyzed by CYP2E1 supersomes, strongly suggesting that HA is a substrate for this isoform. Furthermore, HA is consumed in the presence of CYP2E1-induced microsomes and supersomes, as determined by o-phtalaldehyde complexes with HA by HPLC. The present findings may contribute to understand better the physiological function of CYP450 in relation with inflammation and other physiological processes in which HA may have a relevant role.     

Differential detoxifying responses to crude oil water-accommodated fraction in Hyallela curvispina individuals from unpolluted and contaminated sites

Sublethal effects of water-accommodated fraction (WAF) from crude oil of Neuquén basin, Northern Patagonia-Argentina, were examined on both antioxidant and detoxification system of Hyalella curvispina adults collected in Los Barreales (LB) lake and in an oil-polluted stream (DS). The effects of WAF exposure during 6, 24 and 48 h were evaluated in the glutathione content (GSH) and glutathione S-transferase (GST), catalase (CAT) and cytochrome P450 (CYP450) activities. Populations from DS and LB showed not only different basal GSH content and enzyme activities but also different behavior to WAF exposure. LB population exposed to WAF showed a significant increase in GSH content, CAT and CYP450 activities, compared to control group. DS population presented high basal levels in CAT and CYP activity compared with LB population, but their response to WAF exposure was minor. Amphipods from DS, chronically exposed to hydrocarbons, were adapted to their environment.     

Effect of cytochrome P450 inducers on the metabolism and toxicity of thiram in rats

Thiram is a dithiocarbamate compound widely used as an agricultural fungicide. This study examined the effect of cytochrome P450 (CYP) inducers on the metabolism and toxicity of thiram in rats. Rats were pretreated with 3-methyl cholathrene (3-MC), phenobarbital (PB), isoniazid (INH), or pregnenolone-16a-carbonitrile (PCN) as selective inducers of CYP 1A1, 2B1, 2E1 and 3A2, respectively. Thiram was administered ip to induced rats at 0.1 or 0.5 mmol/kg, and the animals were sacrificed 3 or 24 h later to assess P450 interaction and liver damage, respectively. No significant inhibition of 3-me-induced CYP1A1 was observed with either thiram dose at 3 or 24 h after treatment; similar results were noted for rats induced with PB or PCN. By contrast, when INH was the selective inducer of CYP2E1, there was significant inhibition by thiram 3 h and 24 h after treatment, suggesting that thiram was metabolized by the induced CYP2E1; there was a significant increase in ALT activity reflective of liver damage in the rats treated with thiram. The results suggest that CYP2EI induced by INH may be significantly involved in the metabolism of thiram, and the associated liver damage.     

细胞色素P450 2E1在依达拉奉减轻免疫性肝损伤氧化应激中的作用

目的观察细胞色素P450 2E1(CYP 2E1)在依达拉奉作用于刀豆蛋白A(Con A)致小鼠免疫性肝损伤模型中的变化。方法先期给予依达拉奉,之后用Con A腹腔注射致敏小鼠产生急性免疫性肝损伤,观察肝脏病理变化,分光光度法测血清AST和ALT浓度,肝匀浆中SOD、MDA、GSH含量,RT-PCR技术检测肝脏组织CYP 2E1 mRNA水平,Westernblot技术检测肝脏组织CYP 2E1蛋白表达水平。结果与模型组比较,依达拉奉能降低免疫性肝损伤小鼠血清中升高的ALT、AST含量;降低肝匀浆中的MDA水平,升高其降低的SOD、GSH水平;CYP 2E1 mRNA水平明显降低,CYP 2E1蛋白表达水平明显下调。结论依达拉奉对小鼠急性免疫性肝损伤具有一定的保护作用,这可能与它降低CYP 2E1的表达,清除自由基,增强机体抗脂质过氧化能力有关。     

Biochemical basis for the age-related sensitivity of broilers to aflatoxin B1

In this study, we investigated the mechanism underlying age-related susceptibility in broilers to aflatoxin B1 (AFB1). The results showed that AFB1 induced significant changes in serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT) activity &; liver superoxide dismutase (SOD), malonaldehyde (MDA), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST) activity at day 7, 21 and 42 relative to control group. However, AFB1-induced changes in serum biochemical parameters and liver antioxidant activities become less severe with increasing age of broilers. Particularly, liver cytosolic GST activity increases with the age of broilers, crucial for the detoxification of AFB1. The mRNA expression level of Cytochrome P450 (CYP) enzymes was significantly higher at day 7, and decreases at day 21 and 42. While, the mRNA expression level of liver GSTA3, GSTA4 and EPHX1 increases with age of broilers. Maximum AFB1 residues level was detected at day 42 relative to day 7 and 21. While, AFM1 residues level increases (p?p?>?0.05) at day 42. Most importantly, our data confirmed the efficient AFB1-bioactivation by CYP enzymes and deficient detoxification of GST enzymes at younger age (～7-day old) compared to older age. In summary, the age-related changes particularly in phase-I and phase-II enzymes mainly responsible for AFB1 bioactivation and detoxification may be partially accountable for the increased susceptibility of younger broilers (～7-day old) compared to older broilers.     

Retinoid X receptor alpha regulates glutathione homeostasis and xenobiotic detoxification processes in mouse liver

Retinoid X receptor alpha (RXRalpha) plays a pivotal role in regulating liver metabolism. RXRalpha-mediated gene expression involved in amino acid metabolism was examined using the NIA Mouse 15K cDNA microarray containing 15,000 different expressed sequence tags. Seven amino acid metabolic genes, three of which encode enzymes involved in phase II detoxification process, were identified as RXRalpha target genes in mouse liver. Glutamate-cysteine ligase catalytic subunit (GCLC), glutathione S-transferasemu, and glutathione peroxidase 1 were down-regulated in the liver of hepatocyte RXRalpha-deficient mice. The down-regulation of GCLC in RXRalpha-deficient mice led to 40% and 45% reductions in the rate of glutathione (GSH) synthesis and level of hepatic GSH, respectively. Primary hepatocytes from RXRalpha-deficient mice were more sensitive to t-butylhydroperoxide-induced oxidative stress. However, GSH diminished RXRalpha-deficient mice were resistant to acetaminophen (APAP)-induced hepatotoxicity. Analysis of phase I detoxification genes revealed that CYP1A2 and CYP3A11 were up-regulated in wild-type mice but down-regulated in RXRalpha-deficient mice after APAP administration. Taken together, the data indicate that RXRalpha centrally regulates both phase I and phase II drug metabolism and detoxification. Regulation of hepatic GSH levels by RXRalpha is essential to protect hepatocytes from oxidative stress, whereas up-regulation of phase I drug metabolism genes by RXRalpha may render the liver more sensitive to APAP-induced toxicity.     

Effect of thymoquinone on hepatorenal dysfunction and alteration of CYP3A1 and spermidine/spermine N-1-acetyl-transferase gene expression induced by renal ischaemia-reperfusion in rats

Objectives Renal ischaemia–reperfusion (I/R) is a well‐characterised model of acute renal failure that causes both local and remote organ injury. The aim of this work was to investigate the effect of thymoquinone, the main constituent of the volatile oil extracted from Nigella sativa seeds, on renal and hepatic changes after renal ischaemia–reperfusion. Methods Male Sprague‐Dawley rats were divided into sham I/R vehicle‐treated groups, and I/R thymoquinone‐treated groups. Thymoquinone (10 mg/kg, p.o.) was administered for ten consecutive days to the I/R thymoquinone group before injury. I/R and I/R thymoquinone groups were subjected to 30‐min ischaemia followed by 4‐h reperfusion. Key findings I/R resulted in a significant increase in malondialdehyde (MDA) level and decreases in glutathione‐S‐transferase (GST) and superoxide dismutase (SOD) activity in liver and kidney tissues. Thymoquinone treatment caused the reversal of I/R‐induced changes in MDA as well as GST and SOD activity. Moreover, I/R caused a significant rise in creatinine and alanine aminotransferase serum levels. CYP3A1 mRNA expression was induced significantly by I/R in both liver and kidney tissues compared with sham group. Thymoquinone reduced significantly this increase. I/R caused induction of mRNA expression of spermidine/spermine N‐1‐acetyl‐transferase (SSAT), a catabolic enzyme that participates in polyamine metabolism, in liver and kidney tissues. Thymoquinone reduced SSAT mRNA expression significantly in liver and markedly in kidney. Conclusions These findings suggested that thymoquinone protected against renal I/R‐induced damage through an antioxidant mechanism as well as the decrease of CYP3A1 and SSAT gene expression.