慢性间断性低氧对大鼠肝脏CYP3A_2和CYP2E_1的影响研究

目的研究慢性间断性低氧对大鼠肝脏CYP3A2和CYP2E1的影响。方法将大鼠随机分为对照组、低氧3d组、低氧7d组、低氧14d组、低氧28d组,低氧处理结束24h后,常规腹腔注射麻醉,摘取眼球血液2ml制备血清,并测定丙氨酸氨基转移酶(ALT)、天门冬酸氨基转移酶(AST)、红霉素N-脱甲基酶(ERD)、苯胺羟化酶(ANH)活性;取新鲜肝组织以制备微粒体和提取核糖核酸(RNA),并以RT-PCR进行基因片段扩增以检测大鼠肝脏细胞色素CYP3A2、CYP2E1的mRNA表达水平。结果慢性间断性低氧对血清ALT、AST活性无明显影响;低氧7d后,大鼠肝脏ERD、ANH活性明显升高,28d时诱导率分别为155.5%、42.2%;同时,CYP3A2、CYP2E1mRNA的表达水平也分别增加了220.5%、102.8%。结论慢性间断性低氧能显著增加大鼠肝脏ERD、ANH活性,其机制可能与其在转录水平上提高肝脏CYP3A2和CYP2E1的基因表达水平有关。     

CYP2E1 mediated isoniazid-induced hepatotoxicity in rats

INTRODUCTIONIsoniazid (INH) in the treatment of all types oftuberculosis (TB) is associated with mild to moderateelevation of liver enzyme activity in plasma, and severehepatotoxicity in approximate 1 %-2 % of patients. Acetyl-hydrazine, the metabolite of INH, has been suggested tobe the cause of hepatic damage in patients. Recently,hydrazine, not INH or acetylhydrazine, has been reportedto be most likely involved in the pathogenic mechanismof hepatic necrosis of INH-induced hepatot…     

Bromuconazole-induced hepatotoxicity is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1 gene expression

Despite widespread use of bromuconazole as a pesticide for food crops and fruits, limited studies have been done to evaluate its toxic effects. Here, we evaluated the hepatotoxic effect of bromuconazole using classical toxicological (biochemical analysis and histopathological examination) and gene-based molecular methods. Male rats were treated either orally or topically with bromuconazole at doses equal to no observed adverse effect level (NOAEL) and 1/10 LD50 for 90?d. Bromuconazole increased activities of liver enzymes (ALT, AST, ALP, and ACP), and levels of bilirubin. It also induced hepatic oxidative stress as evidenced by significant decrease in the activities of superoxide dismutase (SOD), and significant increase in levels of malondialdehyde (MDA) in liver. In addition, bromuconazole caused an increase in liver weights and necrobiotic changes (vacuolation and hepatocellular hypertrophy). It also strongly induced the expression of PXR and its downstream target CYP3A1 gene as well as the activity of CYP3A1. However, it inhibited the expression of CAR and its downstream target CYP2B1 gene without significant changing in CYP2B1 activity. Overall, the oral route showed higher hepatotoxic effect and molecular changes than the dermal route and all changes were dose dependent. This is the first investigation to report that bromuconazole-induced liver oxidative damage is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1.     

复方银杏叶胶囊对肝损伤大鼠CYP2E1、CYP3A4的影响

目的研究复方银杏叶胶囊(CGB)对酒精性肝损伤大鼠CYP2E1、CYP3A4活性的影响。方法正常组和酒精性肝损伤模型组均以CGB[(250 mg/(kg.d)]灌胃,分别在灌胃CGB前及灌胃1周后,灌胃探针药氯唑沙宗(50 mg/kg)及氨苯砜(20 mg/kg),于探针药灌后24 h内不同时间点采血,测定各探针药血药浓度。结果灌胃CGB前,模型组氯唑沙宗和氨苯砜的AUC0-24、Cmax均显著低于正常组(P<0.05或P<0.01)。灌胃CGB后,模型组氯唑沙宗和氨苯砜的AUC0-24、Cmax均较灌胃前显著升高(P<0.05或P<0.01);且氯唑沙宗的t1/2灌胃CGB后明显高于灌胃前(P<0.05)。结论 CGB能够明显抑制酒精性肝损伤大鼠CYP2E1、CYP3A4酶活性。     

An interaction between the cytochrome P450 probe substrates chlorzoxazone (CYP2E1) and midazolam (CYP3A)

AIMS: The use of multiple probe substrates to evaluate the activity of drug metabolizing enzymes requires that there are no inter-substrate interactions. As part of a series of studies to develop a clinically useful collection of probe substrates that could be given alone or in any combination, we observed an interaction between midazolam (MDZ) and another component of the six-drug cocktail. Published data indicated that the interacting component was likely to be chlorzoxazone. This was investigated as part of a second study. The data relating to the interaction from both studies are reported here. METHODS: Both studies were performed in 16 healthy subjects. All treatments were given orally after an overnight fast. In study 1, which was performed to a four-period, open, crossover design, subjects received on separate occasions MDZ 5 mg, diclofenac 25 mg, a four drug cocktail (caffeine 100 mg, mephenytoin 100 mg, debrisoquine 10 mg and chlorzoxazone 250 mg) and a six drug cocktail (caffeine 100 mg, mephenytoin 100 mg, debrisoquine 10 mg, chlorzoxazone 250 mg, diclofenac 25 mg and MDZ 5 mg). In study 2, which was performed to a two-period, open, crossover design, subjects received a five drug cocktail (as the six drug cocktail in the first study, but without chlorzoxazone and with diclofenac dose increased to 50 mg) and a six drug cocktail (as five drug cocktail, with chlorzoxazone 250 mg). In both studies, blood samples were taken for measurement of plasma MDZ and 1-hydroxy MDZ (1-OH MDZ) concentrations. In study 1, blood samples were taken up to 12 h post-dose while in study 2 a single sample was taken 2 h after dosing. In study 1, the potential interaction between MDZ and the other components of the six drug cocktail was assessed by comparing AUClast ratios (1-OH MDZ/MDZ) between the two treatments. Additionally, a single sampling timepoint of 2 h post-dose for determination of concentration, rather than AUC, ratios was established. The 2 h plasma concentration ratios from studies 1 and 2 were combined and a pooled analysis performed to compare ratios within each study (to determine the change in ratio when MDZ was dosed with and without chlorzoxazone) and between studies (to determine the consistency of the ratios when MDZ was given either as part of the two six drug cocktails or when given alone and as part of the five drug cocktail). RESULTS: In study 1, both the AUClast ratio and the 2 h post-dose plasma concentration ratio were reduced when MDZ was given as part of the six drug cocktail in comparison with those for MDZ alone. This was the result of an increase in MDZ, rather than decrease in 1-OH MDZ, concentrations and was considered to result from a reduction in first pass metabolism of MDZ. The geometric mean AUClast values (with 95% CI) for MDZ were 95.6 (79.0, 115.7) and 160.4 (133.6, 192.6) microg l(-1) h when given alone and as part of the six drug cocktail, respectively. The corresponding values for 1-OH MDZ were 789.6 (697.6, 893.6) and 791.4 (701.7, 892.6) microg l(-1) h. The ratio of adjusted geometric mean AUClast ratios for the two treatments was 1.82 (90% CI 1.48, 2.23, P < 0.001). The pooled plasma 1-OH MDZ/MDZ ratio data from both studies showed that the differences in MDZ metabolism observed in study 1 were replicated in study 2. The adjusted geometric mean 1-OH MDZ/MDZ ratios when MDZ was given alone and as part of the six drug cocktail were 7.79 and 4.59, respectively, for study 1 (ratio 1.70, 95% CI 1.36, 2.11, P < 0.001) and 7.64 and 4.60 for study 2 (ratio 1.66, 95% CI 1.34, 2.06, P < 0.001). These data indicate that when given orally chlorzoxazone interacts with MDZ, increasing plasma MDZ concentrations. In contrast, there was no difference between the plasma 1-OH MDZ/MDZ ratios when MDZ was given alone and as part of the five drug cocktail indicating that there were no interactions between MDZ and any of the other components of that cocktail. CONCLUSIONS: Chlorzoxazone appears to significantly influence the pharmacokinetics of oral MDZ, probably through inhibition of first pass metabolism by CYP3A in the GI tract. Data from these studies and literature evidence showing a further interaction between chlorzoxazone and CYP1A2 substrates and questions concerning the specificity of chlorzoxazone as a probe substrate for CYP2E1, indicate that the use of chlorzoxazone in multisubstrate probe cocktails should be avoided.     

三氯乙烯对3种细胞色素P450酶基因表达的影响

目的 探讨三氯乙烯（Trichloroethylene,TCE)对人体淋巴细胞瘤细胞株（MCL-5）中3种细胞色素P450酶基因（CYP1A1、CYP2E1、CYP3A4）表达的影响,并研究剂量反应关系和时间反应关系,方法 用常规的细胞培养方法,0.5、1.0、1.5、2.0mmol/L TCE处理细胞12、24、48、72h。利用提纯RNA和cDNA的药盒,合成cDNA,然后逆转录聚合酶反应（RT-PCR）表达3种CYP450基因,以β-Actin作为内对照,分析不同处理剂量和时间时基因表达的强度。结果 在MCL-5细胞株中都有基本的表达,CYP1A1表达在用1.0、1.5、2.0mmol/LTCE处理48h后有被上调的趋势,而且上调趋势随处理时间延长耐加强;CYP2E1、CYP3A4表达不受TCE处理时间长短的影响。3种CYP450酶基因的表达受TCE剂量的影响。3随0.5mol/L,1.0、1.5、2.0mmol/L剂量的增加有上调的趋势,结论 TCE对CYP450酶系统中的CYP2E1、CYP1A1、CYP3A4基因有明显的诱导作用。这些基因被诱导后的结果。可能会导致相对应酶活性的增加,同时加强对TCE的代谢,使TCE的代谢产物增加。     

Molecular mechanism of trichloroethylene-induced hepatotoxicity mediated by CYP2E1

Cytochrome P450 (CYP) 2E1 was suggested to be the major enzyme involved in trichloroethylene (TRI) metabolism and TRI-induced hepatotoxicity, although the latter molecular mechanism is not fully understood. The involvement of CYP2E1 in TRI-induced hepatotoxicity and its underlying molecular mechanism were studied by comparing hepatotoxicity in cyp2e1^+/+ and cyp2e1^−/− mice. The mice were exposed by inhalation to 0 (control), 1000, or 2000 ppm of TRI for 8 h a day, for 7 days, and TRI-hepatotoxicity was assessed by measuring plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and histopathology. Urinary metabolites of trichloroethanol and trichloroacetic acid (TCA) were considerably greater in cyp2e1^+/+ compared to cyp2e1^−/− mice, suggesting that CYP2E1 is the major P450 involved in the formation of these metabolites. Consistent with elevated plasma ALT and AST activities, cyp2e1^+/+ mice in the 2000 ppm group showed histopathological inflammation. TRI significantly upregulated PPARα, which might function to inhibit NFκB p50 and p65 signalling. In addition, TRI-induced NFκB p52 mRNA, and significantly positive correlation between NFκB p52 mRNA expression and plasma ALT activity levels were observed, suggesting the involvement of p52 in liver inflammation. Taken together, the current study directly demonstrates that CYP2E1 was the major P450 involved in the first step of the TRI metabolism, and the metabolites produced may have two opposing roles: one inducing hepatotoxicity and the other protecting against the toxicity. Intermediate metabolite(s) from TRI to chloral hydrate produced by CYP2E1-mediated oxidation may be involved in the former, and TCA in the latter.     

St. John's wort extract induces CYP3A and CYP2E1 in the Swiss Webster mouse.

This investigation was designed to determine the ability of St. John's wort (SJW), a readily available antidepressant, to induce various hepatic drug metabolizing enzymes. SJW (140 or 280 mg/kg/day) was administered to male Swiss Webster mice for 1, 2, or 3 weeks. Enzymatic activity was analyzed in hepatic microsomes for all of the following drug metabolizing enzymes: CYP3A, CYP1A, CYP2E1, and UDP-glucuronosyltransferase (UDPGT). The catalytic activity of CYP1A was unchanged from control following any dose or duration of SJW, while both CYP3A and CYP2E1 catalytic activities were increased 2-fold by both SJW concentrations but only following 3 weeks of administration. Results from Western immunoblotting studies supported the changes in catalytic activity, as protein levels for CYP2E1 and CYP3A were increased (2.5-fold and 6-fold, respectively) following 3 weeks of SJW administration. Additionally, the catalytic activity of the conjugation enzyme UDPGT was unchanged from control following all SJW treatments. These results indicate that in the mouse moderate doses of SJW cause an increase in the catalytic activity and polypeptide levels of CYP2E1 and CYP3A but only following 21 days of administration, while the catalytic activity of CYP1A and UDPGT activity remain unaffected.     

Modification of CYP2E1 and CYP3A4 activities in haemoglobin E-beta thalassemia patients

Objective Thalassemia disease is a genetic haemoglobinopathy usually associated with an iron overload and some degree of organ impairment. The impact of the disease on the drug metabolising enzyme cytochrome P450 (CYP) is not known. CYP2E1 and CYP3A4 are responsible for the metabolism of a large number of drugs and changes in their activities may have clinical consequences. Methods Haemoglobin E-β thalassemia paediatric, blood transfusion-dependent patients apparently without complications (n = 35) and healthy controls (n = 42) were recruited in this study. The ratios of plasma 6-hydroxychlorzoxazone to chlorzoxazone, and urinary 6-beta-hydroxycortisol (6β-OHF) to cortisol were used as indices for CYP2E1 and CYP3A4 activities, respectively. Blood and plasma samples were assayed for parameters of clinical biochemistry, oxidants and antioxidants. Results There were significant increases in serum iron, protein carbonyl and lipid peroxidation in thalassemia patients, whereas there was a decrease in blood glutathione, but unchanged plasma nitric oxide metabolites. CYP2E1 activity in the patients was unchanged; however, when the patients were stratified by splenectomy status, CYP2E1 activity was increased in non-splenectomised patients in comparison with the controls and splenectomised subjects. On the other hand, 6β-OHF/cortisol ratios increased markedly in patients associated with depressed growth hormone levels. There were no correlations between CYP2E1 activity and oxidant stress or antioxidant parameters. Conclusions This report is the first demonstration that thalassemia major is associated with an alteration of CYP2E1 and CYP3A4 activities; this could modify the sensitivity of thalassemia patients to the toxic or therapeutic effects of drugs.     

Selective inhibitory effects of HYIpro‐3‐1 on CYP1A2 in human liver microsomes

CYP1A2 is one of the main Cytochrome P450 enzymes in the human liver associated with the metabolism of several xenobiotics. CYP1A2 is especially involved in the metabolic activation of different procarcinogens. Therefore, the development of cancer may be inhibited by inhibiting CYP1A2 activity. Here, the inhibitory effect of HYIpro‐3‐1 and its derivatives on CYP1A2 activity in human liver microsomes (HLM) was studied through LC‐MS/MS using a cocktail assay. Among the four compounds, HYIpro‐3‐1 showed the most selective and strongest inhibitory effect on CYP1A2 at IC₅₀ values of 0.1 µM in HLMs and inhibition was confirmed using purified human CYP1A2. It was determined that inhibition is reversible because the inhibitory effect of HYIpro‐3‐1 is not dependent on preincubation time. HYIpro‐3‐1 showed a typical pattern of competitive inhibition for CYP1A2‐catalyzed phenacetin O‐deethylation, based on the Lineweaver‐Burk plot, with a Ki value of 0.05 μM in HLMs; the secondary plot also showed a linear pattern. In our study, HYIpro‐3‐1 was proposed as a novel inhibitor with the capacity to selectively inhibit CYP1A activity in HLMs.     

MiR‐30a‐5p ameliorates spinal cord injury‐induced inflammatory responses and oxidative stress by targeting Neurod 1 through MAPK/ERK signalling

Spinal cord injury (SCI) is a major disability requiring more effective treatment than is currently available. MicroRNAs have been shown to effectively regulate gene expression at the translational level. The aim of the present study was to explore the potential role of miR‐30‐5p and possible mechanism in SCI. We found that miR‐30‐5p was notably down‐regulated, while Neurod 1 expression was highly elevated in microglia from the mouse model of SCI. Additionally, overexpression of miR‐30a‐5p significantly suppressed inflammatory responses as reflected by a decrease in the secretion of the cytokines TNF‐α, IL‐1β and IL‐10 triggered by SCI. Furthermore, introduction of miR‐30a‐5p strengthened the scavenging of oxygen free radicals accompanied by an increase in the expression of SEPN1, TXNL1 and GPX1. More importantly, our study explored that Neurod 1 was a direct and functional target of miR‐30a‐5p, which was validated by the dual luciferase reporter assay. qRT‐PCR and western blot analysis further validated that miR‐30a‐5p negatively regulated the expression of Neurod 1. Mechanistically, overexpression of miR‐30a‐5p or silencing of the Neurod 1 gene prevented the MAPK/ERK signalling and inhibited inflammatory responses, meanwhile activated SEPN1, TXNL1 and GPX1. These findings indicate that miR‐30a‐5p ameliorates inflammatory responses and oxidative stress by targeting Neurod 1 through MAPK/ERK signalling.     

Different regulation of miR‐29a‐3p in glomeruli and tubules in an experimental model of angiotensin II‐dependent hypertension: potential role in renal fibrosis

The aim of this study was to evaluate the role of the angiotensin II (Ang II) induced‐differential miRNA expression in renal glomerular and tubulo‐interstitial fibrosis in an experimental model of Ang II‐dependent hypertension. To clarify this issue, Sprague Dawley rats were treated with Ang II (200 ng/kg per minute, n = 15) or physiological saline (n = 14) for 4 weeks. Systolic blood pressure and albuminuria were measured every 2 weeks. At the end of the experimental period, renal glomerular and tubulo‐interstitial fibrosis was evaluated by histomorphometric analysis, after Sirius‐Red and Masson's trichrome staining. Ang II increased systolic blood pressure (P < 0.0001), albuminuria (P < 0.01) and both glomerular and tubulo‐interstitial fibrosis (P < 0.01). Using laser capture microdissection and miRNA microarray analysis this study showed that miR‐29a‐3p was down‐regulated in renal tubules and up‐regulated in glomeruli. Real‐time polymerase chain reaction (PCR) experiments confirmed in Ang II‐treated rats a down‐regulation of miR‐29a‐3p in tubules (P < 0.01), while no significant changes were observed in glomeruli. Matrix metalloproteinase‐2 (MMP‐2) was identified as putative miR‐29a‐3p target (by TargetScan, miRanda, Tarbase software) and functionally confirmed by luciferase activity assay. These data demonstrate that the effects of Ang II on miR‐29a‐3p expression in renal tubules is different from the one exerted in the glomeruli and that miR‐29a‐3p targets MMP‐2. These results suggest that the development of renal fibrosis at glomerular and tubulo‐interstitial level depends on different molecular mechanisms.     

Effect of piperine on CYP2E1 enzyme activity of chlorzoxazone in healthy volunteers

1.?The purpose of the present study was to investigate the effect of piperine (PIP) on CYP2E1 enzyme activity and pharmacokinetics of chlorzoxazone (CHZ) in healthy volunteers.

2.?An open-label, two period, sequential study was conducted in 12 healthy volunteers. A single dose of PIP 20?mg was administered daily for 10 days during treatment phase. A single dose of CHZ 250?mg was administered during control and after treatment phases under fasting conditions. The blood samples were collected at predetermined time intervals after CHZ dosing and analyzed by HPLC.

3.?Treatment with PIP significantly enhanced maximum plasma concentration (C_max) (3.14–4.96?μg/mL)_, area under the curve (AUC) (10.46–17.78?μg h/mL), half life (T_1/2) (1.26–1.82?h) and significantly decreased elimination rate constant (K_el) (0.57–0.41?h?^??¹), apparent oral clearance (CL/F) (24.76–13.65?L/h) of CHZ when compared to control. In addition, treatment with PIP significantly decreased C_max (0.22–0.15?μg/mL), AUC (0.94–0.68?μg h/mL), T_1/2 (2.54–1.68?h) and significantly increased K_el (0.32–0.43?h?^??¹) of 6-hydroxychlorzoxazone (6-OHCHZ) as compared to control. Furthermore, treatment with PIP significantly decreased metabolite to parent (6-OHCHZ/CHZ) ratios of C_max, AUC, T_1/2 and significantly increased K_el ratio of 6-OHCHZ/CHZ, which indicate the decreased formation of CHZ to 6-OHCHZ.

4.?The results suggest that altered pharmacokinetics of CHZ might be attributed to PIP mediated inhibition of CYP2E1 enzyme, which indicate significant pharmacokinetic interaction present between PIP and CHZ. The inhibition of CYP2E1 by PIP may represent a novel therapeutic benefit for minimizing ethanol induced CYP2E1 enzyme activity and results in reduced hepatotoxicity of ethanol.     

抑制CYP2E1增强何首乌水提物肝毒性作用及其毒性成分筛选研究

目的 研究肝细胞色素氧化酶P450(CYP450)亚型CYP2E1与何首乌肝毒性的关系并筛选有关毒性成分.方法 MTT法检测CYP2E1特异性抑制剂二乙基二硫代氨基甲酸钠(DDTC)作用后何首乌水提物(PMR)对人肝细胞L02的毒性作用,及PMR和6种成分2,3,5,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷(2,...     

Long non‐coding RNA ANRIL enhances mitochondrial function of hepatocellular carcinoma by regulating the MiR‐199a‐5p/ARL2 axis

Although the roles of long non‐coding RNA (lncRNA) ANRIL (Antisense non‐coding RNA in the INK4A locus) have been established in various tumors, its roles in mitochondrial metabolic reprogramming of hepatocellular carcinoma (HCC) cells are still unclear. This work aims to explore lncRNA ANRIL roles in regulating the mitochondrial metabolic reprogramming of liver cancer cells. First, we found that lncRAN ANRIL expression was significantly increased in HCC tissues or cells compared with the normal adjacent tissues and normal tissues or cells. Functional experiment showed that overexpression of lncRNA ANRIL promoted mitochondrial function in HCC cells, evident by the increased mitochondrial DNA copy numbers, ATP (Adenosine triphosphate) level, mitochondrial membrane potential, and the expression levels of mitochondrial markers, while ANRIL knockdown exerted the opposite effects. Mechanistically, lncRNA ANRIL acted as a competing endogenous RNA to increase ARL2 (ADP‐ribosylationfactor‐like 2) expression via sponging miR‐199a‐5p. Notably, the miR‐199a‐5p/ARL2 axis is necessary for ANRIL‐mediated promoting effects on HCC cell mitochondrial function. This work reveals a novel ANRIL‐miR‐199a‐5p‐ARL2 axis in HCC cell progression, which might provide potential targets for HCC treatment.     

Pogostone inhibits the activity of CYP3A4, 2C9, and 2E1 in vitro

ContextPogostone possesses various pharmacological activities, which makes it widely used in the clinic. Its effect on the activity of cytochrome P450 enzymes (CYP450s) could guide its clinical combination.ObjectiveTo investigate the effect of pogostone on the activity of human CYP450s.Materials and methodsThe effect of pogostone on the activity of CYP450s was evaluated in human liver microsomes (HLMs) compared with blank HLMs (negative control) and specific inhibitors (positive control). The corresponding parameters were obtained with 0–100 μM pogostone and various concentrations of substrates.ResultsPogostone was found to inhibit the activity of CYP3A4, 2C9, and 2E1 with the IC₅₀ values of 11.41, 12.11, and 14.90 μM, respectively. The inhibition of CYP3A4 by pogostone was revealed to be performed in a non-competitive and time-dependent manner with the K_i value of 5.69 μM and the KI/K_inact value of 5.86/0.056/(μM/min). For the inhibition of CYP2C9 and 2E1, pogostone acted as a competitive inhibitor with the K_i value of 6.46 and 7.67 μM and was not affected by the incubation time.Discussion and conclusionsThe inhibitory effect of pogostone on the activity of CYP3A4, 2C9, and 2E1 has been disclosed in this study, implying the potential risk during the co-administration of pogostone and drugs metabolized by these CYP450s. The study design provides a reference for further in vivo investigations to validate the potential interaction.     

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.     

醋氨酚所致精密肝切片损伤模型的建立及CYP2E1的调节作用

目的：利用精密肝切片（PCLS）技术,建立醋氨酚所致肝切片损伤的体外模型,并探讨CYP2E1活性变化对肝损伤的调节作用,为研究和筛选肝保护药物提供实验依据。方法：利用振荡切片机制备PCLS,建立培养系统。将终浓度为500μmol/L的醋氨酚分别与切片共孵育0、2、4、6h,测定培养基和切片中的谷胱甘肽S-转移酶（GST）和乳酸脱氢酶（LDH）活性。在体给予不同剂量（0.25、0.5、1.0g/kg）的CYP2E1诱导剂乙醇,每天一次,连续3d。末次给药8h后处死动物,取其肝脏制备PCLS,再与500μmol/L醋氨酚共同孵育6h,检测ALT和LDH漏出率。正常PCLS与不同浓度的二乙基二硫代氨基甲酸酯盐（DDTC,CYP2E1抑制剂）（5、10、20μmol/L）、500μmol/L醋氨酚共同孵育6h,检测ALT和LDH漏出率。结果：与常规培养组比较,醋氨酚孵育4、6h组的GST和LDH漏出率显著升高（P〈0.01）。与醋氨酚模型对照组比较,乙醇中、大剂量组LDH漏出率和小、大剂量组ALT漏出率皆升高（P〈0.01,P〈0.05）;DDTC各浓度组ALT漏出率则降低（P〈0.05）。结论：PCLS与500μmol/L醋氨酚共孵育4h即能成功地建立体外肝切片损伤的模型。抑制CYP2E1活性可能对醋氨酚所致的肝损伤有保护作用,而上调CYP2E1活性则可能加重醋氨酚所致的肝损伤。     

酒精性肝损伤大鼠细胞色素P450 CYP2E1和细胞色素P450 CYP3A的代谢活性

目的观察酒精性肝损伤对大鼠细胞色素P450CYP3A(CYP3A)和细胞色素P450CYP2E1(CYP2E1)代谢活性的影响。方法采用ig给予白酒制备大鼠酒精性肝损伤模型,检测血清中谷丙转氨酶(GPT)和谷草转氨酶(GOT)活性,采用HE染色法光镜下观测酒精对肝脏损伤程度。大鼠ip给予CYP3A探针药物咪达唑仑10mg·kg-1或ig给予CYP2E1探针药物氯唑沙宗50mg·kg-1后,采用高效液相色谱法测定不同时间点大鼠血浆中咪达唑仑和氯唑沙宗的血药浓度,并应用3P87软件计算其药代动力学参数,以考察CYP2E1和CYP3A的代谢活性的变化。大鼠ig给予氯唑沙宗80mg·kg-1后,热板方法测定大鼠添足次数和添足反射潜伏期。结果酒精性肝损伤可致大鼠肝小叶结构不清,肝索排列紊乱,肝细胞体积增大,呈弥漫性中度水变性,肝窦受压,大部分肝细胞胞浆内见大小不等的脂肪空泡;与正常对照组相比,酒精性肝损伤组大鼠GPT和GOT活性分别增加了16.0%和20.0%(P<0.05,P<0.01)。酒精性肝损伤致大鼠CYP2E1对探针药物氯唑沙宗的代谢活性增强,AUC,t1/2和cmax分别降低了38.0%,30.5%和35.0%(P<0.05);酒精肝损伤组大鼠氯唑沙宗镇痛效果明显降低;酒精性肝损伤致大鼠CYP3A对探针药物咪达唑仑的代谢活性增强,AUC,t1/2和cmax分别降低了122.6%,54.9%和56.9%(P<0.01,P<0.05)。结论酒精性肝损伤可使大鼠CYP2E1和CYP3A代谢活性增强。     

Protective effects of diallyl disulfide on carbon tetrachloride‐induced hepatotoxicity through activation of Nrf2

This study was conducted to investigate the potential effects of diallyl disulfide (DADS) on carbon tetrachloride (CCl₄)‐induced acute hepatotoxicity and to determine the molecular mechanisms of protection offered by DADS in rats. DADS was administered orally at 50 and 100 mg/kg/day once daily for 5 consecutive days prior to CCl₄ administration. The single oral dose of CCl₄ (2 mL/kg) caused a significant elevation in serum aspartate and alanine aminotransferase activities, which decreased upon pretreatment with DADS. Histopathological examinations showed extensive liver injury, characterized by extensive hepatocellular degeneration/necrosis, fatty changes, inflammatory cell infiltration, and congestion, which were reversed following pretreatment with DADS. The effects of DADS on cytochrome P450 2E1 (CYP2E1), the major isozyme involved in CCl₄ bioactivation, were also investigated. DADS pretreatment resulted in a significant decrease in CYP2E1 protein levels in dose‐dependent manner. In addition, CCl₄ caused a decrease in protein level of cytoplasmic nuclear factor E2‐related factor 2 (Nrf2) and suppression of nuclear translocation of Nrf2 concurrent with downregulation of detoxifying phase II enzymes and a decrease in antioxidant enzyme activities. In contrast, DADS prevented the depletion of cytoplasmic Nrf2 and enhanced nuclear translocation of Nrf2, which, in turn, upregulated antioxidant and/or phase II enzymes. These results indicate that the protective effects of DADS against CCl₄‐induced hepatotoxicity possibly involve mechanisms related to its ability to induce antioxidant or detoxifying enzymes by activating Nrf2 and block metabolic activation of CCl₄ by suppressing CYP2E1. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 538–548, 2015.