丙泊酚对大鼠肝微粒体细胞色素酶P450的影响

目的:研究丙泊酚对大鼠肝微粒体细胞色素酶P450含量的影响。方法:健康雄性SD大鼠18只,体重180～220g,随机分为苯巴比妥钠组、丙泊酚组、生理盐水组,每组6只,分别给予苯巴比妥钠75mg/kg,丙泊酚3.789mg/kg及等量生理盐水,持续3d。测定肝微粒体蛋白和P450的含量以及氨基比林-N脱甲基酶的活性。结果:与生理盐水组比较,苯巴比妥钠组、丙泊酚组肝微粒体蛋白和P450的含量升高;苯巴比妥钠组氨基比林-N脱甲基酶活性明显增高。结论:丙泊酚对大鼠肝微粒体蛋白、细胞色素P450具有诱导作用,对氨基比林-N脱甲基酶的活性无影响。     

Differential expression and activities of cytochrome P450 3A in the rat brain microsomes and mitochondria

Midazolam (MDZ), a benzodiazepine derivative, is metabolized to 1′- and 4-hydroxylated metabolites (1′-OH-MDZ and 4-OH-MDZ, respectively) by cytochrome P450 3A (CYP3A). The purpose of this study was to investigate the CYP3A-mediated hydroxylation of MDZ in the rat brain mitochondria (MT). Brain microsomes (MC) and MT fractions were prepared from rats (n = 8) using differential and density gradient centrifugations, and the purity of the fractions was evaluated using VDAC1 and calreticulin as markers of MT and MC, respectively. The formation rates of 1′-OH-MDZ and 4-OH-MDZ in the rat brain MC and MT samples were determined using an LC–MS/MS method after validation. Subsequently, Michaelis–Menten kinetics of 1′- and 4-hydroxylation of MDZ were estimated. Western blot (WB) analysis was used to determine the protein expression of CYP3A in the rat brain MC and MT. The MC fractions had 5.93% ± 3.01% mitochondrial impurity, and the MT fractions had 19.3% ± 7.8% microsomal impurity (mean ± SD). The maximum velocity (V_max) values of the formation of the hydroxylated metabolites in the brain MT were 2.4–9-fold higher than those in MC. Further, the V_max values of 4-OH-MDZ in both MC and MT fractions were substantially higher than those of 1′-OH-MDZ. The WB analysis showed that the intensity of the CYP3A immunoreactive band in MT was more than twofold higher than that in MC. It is concluded that compared with MC, rat brain MT contains substantial CYP3A, which may affect the pharmacology or toxicology of centrally acting xenobiotic and endogenous substrates of this enzyme.     

微波辐射对大鼠睾丸P450scc mRNA表达水平的影响

目的探讨微波辐射对成年大鼠睾丸组织中细胞色素P450胆固醇侧链裂解酶(P450scc)表达水平的影响。方法采用放射免疫方法(RIA)分别测定经微波辐射后3,6,24,72h及对照组大鼠的血清睾酮含量,同时运用RTPCR方法测定睾丸组织中P450sccmRNA的表达水平变化。结果辐射组大鼠在微波辐射后3h,其血清睾酮含量及P450sccmRNA水平均显著低于对照组(分别较对照组降低83.1%和53.6%,且均P<0.01),6h后略有回升,但仍明显低于对照组(分别较对照组降低52.6%和29.2%,且均P<0.01),于24h后恢复至正常水平,在72h时再次出现显著降低(分别较对照组降低57.6%和53.5%,且均P<0.01)。结论微波辐射能在转录水平影响睾丸间质细胞P450sccmRNA的表达,从而降低睾酮的含量。     

Cytochrome P450 2E1 activity in diabetic and obese patients as assessed by chlorzoxazone hydroxylation

Summary— Cytochrome P450 2E1 (CYP2E1) is a phase I detoxification enzyme, which is induced by chronic alcohol consumption. It is involved in the activation of numerous carcinogens and in the production of free radicals. As it has previously been shown to be induced in diabetic and obese rats, the aim of this study was to investigate its induction level in poorly-controlled diabetics and in obese patients (Body Mass Index > 30 kg/m²). CYP2E1 activity was determined in 35 diabetic and 17 obese patients by using the in vivo chlorzoxazone hydroxylation test. Even though the glucidic parameters were highly disturbed (mean fasting glycemia > 7.9 mmol/L, post prandial glycemia > 12.2 mmol/L and fructosamine > 326 μmol/L), CYP2E1 activity was not enhanced either in insulin-dependent diabetics (IDDs, n = 7) nor in non-obese non-insulin-dependent diabetics (NTTDDs, n = 15) when compared to controls ( n = 42) (0.21 ± 0.03, 0.33 ± 0.03 and 0.30 ± 0.02, respectively, mean ± SEM). However, this activity was lower in IDDs when compared to NIDDs ( P < 0.05). In obese patients, with ( n = 13) or without ( n = 17) NIDD mellitus, CYP2E1 activity was increased by a mean of 40% when compared to controls. In addition, positive correlations were found in all subjects (controls or patients, n = 74) between CYP2E1 activity and serum cholesterol ( r = 0.42, P < 0.0001), triglycerides ( r = 0.44, P < 0.0001) and BMI ( r = 0.36, P < 0.001). Accordingly, subjects with cholesterol and/or triglyceride serum levels above 6.4 and 1.8 mmol/L, respectively, displayed a mean increase of 40% of their CYP2E1 activity vs subjects within the above values. It is believed that individuals with increased CYP2E1 activity are more susceptible to the adverse effects of CYP2E1-mediated activation of toxins and carcinogens.     

大鼠酒精性肝病细胞凋亡与细胞色素P450 2E1和氧化应激的关系

目的 观察酒精性肝病(ALD)大鼠肝组织病理学改变,探讨细胞凋亡与细胞色素P4502E1(CYP2E1)的表达以及与氧化应激的关系.方法 用乙醇灌胃法制备ALD大鼠模型,模型组(37只)给予体积分数为40%的乙醇8 g·kg-1·d-1分两次灌胃,连续8周;对照组(33只)给予等量生理盐水灌胃.实验第8周末,各组选30只大鼠观察肝组织的病理学改变;用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法(TUNEL)检测肝细胞凋亡,用全自动生化仪检测丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)值,用聚合酶链反应(PCR)法测定肝CYP2E1的表达;分别用硫代巴比妥酸法和黄嘌呤氧化酶法测定血清丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性.结果 模型组凋亡的肝细胞明显增多,主要分布在中央静脉周围、点状和灶状坏死区.对照组CYP2E1的c1基因频率为91.65%、c2基因频率为8.35%;模型组c1基因频率为53.35%、c2基因频率为46.65%,差异均有显著性(P均＜0.05).长期摄入乙醇的大鼠血清MDA含量增加,SOD活性下降,与ALD肝细胞凋亡程度有相关性(rMDA=0.644,rSOD=-0.511,P均＜0.05),且MDA与SOD两指标间呈负相关(r=-0.582,P＜0.05).结论 长期摄入乙醇可引起大鼠ALD及肝功能损伤,肝细胞凋亡明显增加.CYP2E1基因PstⅠ及RsaⅠ限制性片段长度多态性与ALD有关,其中c2基因可能与大鼠ALD的发生有关.MDA含量和SOD活性在ALD的肝细胞凋亡过程及脂质过氧化反应中发挥重要作用.     

雄性大鼠发育期生精小管NOS和P450的表达及意义

目的:探讨一氧化氮合酶(eNOS、iNOS、nNOS)和细胞色素P450(芳香化酶)在雄性SD大鼠生精小管中的表达及意义。方法:选取出生后5周、7周和10周的雄性SD大鼠各6只,左侧睾丸行石蜡切片,运用免疫组化ABC法观察eNOS、iNOS、nNOS和P450在雄性SD大鼠性成熟期睾丸中的表达变化情况。结果:eNOS和P450在出生后5周,生精小管精母细胞免疫表达呈强阳性,在出生后7周和10周阳性程度皆依次减弱;nNOS在出生后5周精母细胞免疫表达为弱阳性表达,出生后7周和10周呈阴性表达;iNOS在三期均为阴性表达。结论:在性成熟期雄性SD大鼠的精子发生过程中,随着性成熟发育的进展过程,eNOS和P450的表达具有关联性,且主要参与了青春期启动时的精母细胞的发育过程。     

Differential expression and induction of two Drosophila cytochrome P450 genes near the Rst(2)DDT locus

Previous studies have shown that the major metabolic resistance locus in the insecticide-resistant Drosophila line Rst(2)DDT(Wisconsin) maps between the markers cn and vg on chromosome 2. Six cytochrome P450 genes exist in this region. We investigated the expression levels of these P450 genes in DDT-resistant and -susceptible fly lines. We report: (i) DDT resistance is significant (> 30-fold) and dominant, (ii) resistance is reduced by the cytochrome P450 inhibitor PBO, (iii) there is constitutive over-expression relative to susceptible flies of two genes encoding cytochrome P450 enzymes within the cn-vg region (CYP6G1 = 4.3-fold; CYP12D1 = 6.0-fold), and (iv) exposure to DDT results in an increased expression of only one of these two P450 genes (CYP12D1 > or = 6-fold above constitutive resistant fly baselines).     

Involvement of cytochrome P450 2C9, 2E1 and 3A4 in trimethadione N-demethylation in human microsomes

BACKGROUND AND OBJECTIVES: Trimethadione (TMO), an antiepileptic drug, may be used as a candidate for estimating hepatic drug-oxidizing activity. While TMO metabolism is mainly catalysed by CYP2C9, CYP2E1 and CYP3A4 the contribution of the different isoforms is unclear. In this study, we determined the percentage contribution of the three CYPs (CYP2C9, CYP2E1 and CYP3A4) to TMO N-demethylation. METHOD: We used human liver microsomes and human recombinant CYPs expressed in human B-lymphoblast cells and baculovirus-infected insect cells. RESULTS: The mean Km, Vmax and Vmax/Km values of TMO N-demethylation in human microsomes were 3.66 (mm), 503 (pmol/min/mg) and 2.61 (mL/h/mg), respectively. In the microsomes from human B-lymphoblast cells or baculovirus-infected insect cells, CYP 2C9, CYP 2E1 and CYP3A4 exhibited similar Km and higher Vmax in baculovirus-infected insect cells than B-lymphoblast cells. In baculovirus-infected insect cells, CYP2C9, CYP2E1 and CYP3A4 exhibited activities of 32, 286 and 77 pmol/min/pmol CYP, respectively. No CYP activity catalysed by CYP1A2 and 2D6 were detected in the two human cDNA expressed CYP isoforms. CONCLUSION: TMO is metabolized not only by CYP2E1 but also CYP3A4 and CYP2C9. The order of this metabolism is as follows: CYP2E1 > CYP3A4 > CYP2C9.     

诱导型一氧化氮合酶和血红素氧合酶-1在百草枯中毒大鼠肾脏中的表达

目的 观察百草枯中毒大鼠肾组织中诱导型一氧化氮合酶(iNOS)和血红素氧合酶-1(HO-1)的表达,探讨其可能的病理生理机制.方法 SD大鼠84只随机分为空白对照组(A组)42只和染毒组(B组)42只.B组百草枯(25 mg/kg)、A组等量盐水腹腔注射.观察2组肾组织病理学变化及iNOS、HO-1表达,并检测HO-1 mRNA.结果 ①A组组织结构清晰.未见充血、水肿及空泡变性等病理变化;B组组织结构清晰度下降,染毒3 h即可见充血、水肿及空泡变性等病理变化,1 d达顶峰,其后缓解趋势不明显.②iNOS:A组多不表达;B组染毒后3 h在皮质部肾小管上皮细胞及肾小球内皮细胞的胞质中出现表达,随时间延长明显增强,1 d达高峰,其后维持较强表达.③HO-1与HO-1 mRNA:A组多不表达;B组染毒3 h在皮质部肾d、管上皮细胞的胞膜及胞质HO-1呈阳性表达,免疫组织化学评分(IHS)升高,HO-1 mRNA的表达增强,与A组相比差异有统计学意义(P<0.05),1 d达顶峰,之后减弱,5 d仍有阳性表达,但与A组相比,IHS差异无统计学意义(P>0.05).结论 iNOS和HO-1参与百草枯中毒肾损伤的发病过程,但其作用方式、调节途径等尚需进一步研究.     

Genetic polymorphisms of drug-metabolizing phase I enzymes CYP2E1, CYP2A6 and CYP3A5 in South Indian population

CYP2E1, CYP2A6 and CYP3A5 enzymes belong to phase I group of drug-metabolizing enzymes, which are involved in the metabolism of various compounds and xenobiotics. Presence of polymorphisms in the genes coding for these enzymes results in interindividual variations in drug metabolism, therapeutic response and susceptibility towards various diseases. The frequencies of these variants in genes differ considerably between ethnic groups. This study was carried out to estimate the allele and genotype frequencies of common variants in CYP2E1, CYP2A6 and CYP3A5 in South Indian population. Six hundred and fifty-two unrelated healthy volunteers of South Indian origin (Andhra Pradesh, Karnataka, Kerala and Tamil Nadu) were included in this study. Polymerase chain reaction-restriction fragment length polymorphism, allele-specific PCR, real-time PCR, SNaPshot and gene sequencing methods were used for the identification of gene polymorphisms. The frequencies of CYP2E1*1B, CYP2E1*5B and CYP2E1*6 alleles in South Indian population were 14.3, 1.3 and 22.4%, respectively. The frequencies of CYP2A6*2, CYP2A6*4A and CYP2A6*5 alleles were found to be 1, 8.9 and 0.7%, respectively. The distribution of CYP3A5*3 allele was 63.5%. There were no variant alleles of CYP3A5*2, CYP3A5*4 and CYP3A5*6 in South Indian population. The frequencies of CYP2E1, CYP2A6 and CYP3A5 in the South Indian population are distinct from Caucasians, Chinese, Japanese, African Americans and other compared populations. This is the first study conducted in the South Indian population with a larger sample size. The findings of our study provide the basic genetic information for further pharmacogenomic investigations in the population.     

沈阳汉族人群细胞色素P450 酶1A2基因多态性与慢性阻塞性肺疾病易感性的关联

目的探讨沈阳市汉族人群细胞色素P450 酶1A2 (CYP1A2)基因多态性与慢性阻塞性肺疾病(COPD)遗传易感性的关系。方法采用聚合酶链反应-限制性片段长度多态性(PCR-RELP)方法检测100 例COPD患者和年龄、性别匹配的100 名健康人CYP1A2 基因不同位点多态性等位基因及基因型频率分布。结果两组不同位点多态性基因型频率分布均符合遗传性Hardy-Weinberg平衡(均P>0.05)。COPD患者和健康人的1D和1F基因型和等位基因频率分布有显著性差异(P<0.05),1C和1E基因型和等位基因频率分布无显著性差异(P>0.05)。结论CYP1A2 基因1D和1F多态性可能与COPD遗传易感性增加有关。     

神经节苷脂对大鼠脑缺血/再灌注后一氧化氮合酶的影响及机制

目的观察单唾液酸四已糖神经节苷脂(GM-1)对脑缺血/再灌注后一氧化氮合酶(NOS,包括cNOS和iNOS)的影响并探讨其机制。方法采用线拴法制作大鼠大脑中动脉闭塞再灌注模型,分别在缺血后即刻和缺血后12 h腹腔注射GM-1,观察GM-1不同时点给药对脑缺血后24 h脑梗死体积、NOS以及NO的作用,并选取诱导型一氧化氮合酶抑制剂氨基胍(AG)作为阳性对照,同样分两个时间点腹腔注射给予AG,比较AG和GM-1两药对脑缺血后24 h NOS和NO的影响。结果①与生理盐水对照组相比,脑缺血后即刻予GM-1处理可有效地缩小脑梗死体积,抑制iNOS和NO的升高,而缺血后12 h GM-1处理则均无变化;AG组在两个时间点给药都可抑制iNOS和NO的升高。②两种药对cNOS都无明显作用。结论GM-1对缺血侧脑中iNOS的抑制作用受起始给药时间影响,这可能是因为GM-1通过减少脑梗死体积、减轻脑损伤而间接影响了iNOS的升高,当延迟给药时,GM-1抗损伤能力下降,进而对iNOS的间接抑制作用降低。     

Polymorphisms of cytochrome P450 1A1, glutathione S-transferase class mu, and tumour protein p53 genes and the risk of developing gallbladder cancer in Japanese

OBJECTIVES: To examine the relationship between genetic polymorphisms of cytochrome P450 1A1 (CYP1A1), glutathione S-transferase class mu (GSTM1), and tumour protein p53 (TP53) genes, and gallbladder cancer (GBC) risk, a case-control study was conducted. DESIGN AND METHODS: Genotypes of CYP1A1 T3801C, CYP1A1 Ile462Val, GSTM1, and TP53 Arg72Pro were determined in 54 cases of GBC and 178 controls. RESULTS: The age-adjusted odds ratios (ORs) for the Ile/Val genotype of CYP1A1 Ile462Val polymorphism in women and the Arg/Pro genotype of TP53 Arg72Pro polymorphism in men were observed to be 2.70 (95% CI: 1.14-6.40) and 4.32 (95% CI: 1.08-17.2), respectively. No significant differences in the genotypic frequencies of CYP1A1 T3801C and GSTM1 polymorphisms were observed between controls and cases in both men and women. CONCLUSION: These results suggest that the Val allele of CYP1A1 Ile462Val polymorphism and the Pro allele of TP53 Arg72Pro polymorphism contribute to an increased risk of GBC among Japanese women and men, respectively.     

Th1/Th2类细胞因子失衡和一氧化氮合酶在内毒素休克性肺损伤中的作用机制

目的　观察内毒素 (LPS)诱导的急性肺损伤 (ALI)大鼠血Th1/Th2类细胞因子浓度以及肺组织一氧化氮合酶 (NOS)活性和NOS基因表达 ,以探讨它们在ALI中的发病机制。方法　采用颈静脉注入LPS复制大鼠急性肺损伤模型 ,用ELISA法检测其外周血单个核细胞产生IFN -γ、IL - 4水平及IFN -γ/IL - 4比值 ;用放射免疫 (RIA)的方法测定LPS注射后大鼠肺组织NOS活性 ;采用逆转录聚合酶链反应 (RT -PCR)检测LPS注射大鼠肺组织内源型一氧化氮合酶 (eNOS)、诱导型一氧化氮合酶 (iNOS)mRNA表达情况。结果　与正常对照组相比 ,急性肺损伤组外周血PBMC产生IFN -γ水平、IFN -γ/IL - 4比值明显升高 (P <0 0 5 ,P <0 0 0 1) ,而IL - 4水平差异无显著性 (P >0 0 5 ) ,同时伴随NOS活性 [( 0 30± 0 0 5 )U/mgpro]明显高于对照组 [( 0 2 4± 0 0 3) ,P <0 0 5 ]。iNOSmRNA表达( 0 70± 0 2 0 )显著高于对照组 (P <0 0 5 ) ,eNOSmRNA表达未见明显变化 (P >0 0 5 )。肺组织病理切片显示大鼠发生ALI。结论　LPS休克肺损伤时 ,体内存在Th1/Th2类细胞因子水平失衡 ,同时伴有NOS活性增高 ,而NOS的主要来源为iNOS基因表达增强     

1-alpha,25-Dihydroxyvitamin D3 regulates inducible nitric oxide synthase messenger RNA expression and nitric oxide release in macrophage-like RAW 264.7 cells

The expression of inducible nitric oxide synthase (iNOS) expression and release of nitric oxide (NO) from macrophages are markedly increased in granulomatous infections. Activation of macrophages 1alpha-hydroxylase results in an increase of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. However, the significance of this increased production is not completely understood. In this study, we analyzed 1,25(OH)(2)D(3) and NO production in patients with tuberculosis infection and hypercalcemia and used lipopolysaccharide (LPS) to stimulate RAW 264.7 cells in an attempt to assess iNOS expression and gaseous NO production regulated by 1,25(OH)(2)D(3). Peroxynitrite (OONO(-)) production and lactate dehydrogenase activity were also examined. Without additional stimulation, peripheral-blood mononuclear cells (PBMCs) from patients with tuberculosis converted more 25-hydroxyvitamin D(3) to 1,25(OH)(2)D(3) than did those from normal controls. These PBMCs released less NO than did those from control subjects, at baseline and in the stimulated state. We found that 1,25(OH)(2)D(3) dose-dependently inhibited iNOS messenger RNA expression of the LPS-stimulated RAW 264.7 cells and also significantly reduced the gaseous NO release and OONO(-) production. Paralleling the 1,25(OH)(2)D(3)-induced inhibition of NO release were reductions in OONO(-) and LDH production. In conclusion, 1,25(OH)(2)D(3) inhibited iNOS expression and reduced NO production by LPS-stimulated macrophages in the range of physiological doses. Inhibition of the NO surge was coupled with a reduction in OONO(-) and LDH production. Increased 1,25(OH)(2)D(3) production and decreased release of NO from the PBMCs of patients with tuberculosis and hypercalcemia were also noted. We propose that 1,25(OH)(2)D(3) production by macrophages may protect themselves against oxidative injuries caused by the NO burst. In the case of tuberculosis infection, increased 1,25(OH)(2)D(3) synthesis may further contribute to the development of an unwanted phenomenon-hypercalcemia.     

CYP1A2*1F基因多态性与肺癌术后化疗疗效及预后的关系

[目的] 探讨细胞色素P4501A2(CYP1A2)*1F基因多态性与肺癌术后化疗疗效及预后的关系.[方法]对100例肺癌患者进行CYP1A2基因多态性检查,分析CYP1A2*1F与肺癌术后化疗效果及预后的相关性.[结果] ①100例患者,部分缓解 24例,稳定 44例,临床获益率为68.00%,患者CYP1A2*1F基因型以A/A型(49.00%)为主,其次为A/C型(38.00%);②临床获益患者中A/A基因型所占比例略高于未获益患者,但差异无显著性(P>0.05);③携带CYP1A2*1F A/A基因型患者总生存时间略高于A/C型、C/C型,但各组之间相比较差异无显著性(P>0.05).[结论] CYP1A2*1F突变基因型与肺癌术后化疗效果及预后无明显相关性.     

Pharmacogenetics of acenocoumarol: CYP2C9, CYP2C19, CYP1A2, CYP3A4, CYP3A5 and ABCB1 gene polymorphisms and dose requirements

BACKGROUND AND OBJECTIVE: Acenocoumarol (AC) is a coumarin derivative, vitamin K antagonist anticoagulant drug. It has a narrow therapeutic index and shows large pharmacokinetic and pharmacodynamic interindividual variability. Our objective was to investigate the association between AC dose requirements to achieve a target level of anticoagulation and genetic polymorphisms of genes possibly associated with its metabolism (CYP1A2, CYP2C9, CYP2C19, CYP3A4, CYP3A5) and transport (ABCB1). METHODS: Ninety-six Bulgarian patients treated orally with AC for at least 3 months were included. They were separated into three groups according to their AC dose requirement, i.e. low, medium and high. RESULTS AND DISCUSSION: CYP2C9*1/*3 (associated with an intermediate CYP2C9 activity), CYP2C9*2/*2, and CYP2C9*2/*3 genotypes (associated with a low CYP2C9 activity) were more prevalent in the group with low dose requirement of AC compared with the other two groups (P = 0.003). The frequency of CYP2C9*1/*1 genotype, which is associated with an extensive CYP2C9 activity, was higher in the group of patients with high dose requirements (79%), compared with the groups of the medium and low dose requirements (67% and 21% respectively). In addition, the ABCB1 2677GG/3435CC haplotype was associated with use of lower AC dose, whereas the 2677TT/3435TT and 2677GT/3435TT haplotypes were associated with use of higher AC dose (P = 0.03). The distribution of polymorphisms of other genes did not show significant differences between the three groups. CONCLUSION: In vivo, cytochromes P450 isoforms other than CYP2C9 [corrected] were not significantly associated with dose requirement of AC. In our Bulgarian patients, the presence of CYP2C9*2 or/and CYP2C9*3 alleles, as well as the ABCB1 2677GG/3435CC haplotype were associated with low dose requirement of AC.