In vitro and in vivo cytochrome P450 3A enzyme inhibition by Aframomum melengueta and Dennettia tripetala extracts

Objective: To evaluate the in vitro and in vivo inhibitory effects of two commonly used herbs, Aframomum melengueta(A. melengueta) and Dennettia tripetala(D. tripetala) on CYP 3A enzymes. Methods: In vitro inhibition of the enzymes were assessed with microsomes extracted from female albino rats using erythromycin-N-demethylation assay(EMND) method while their in vivo effects were measured by estimating simvastatin plasma concentrations in rats. Pharmacokinetic parameters were determined using non-compartmental anaysis as implemented in Win Nonlin pharmacokinetic program. Results: EMND assay with intestinal microsomes indicated that aqueous extracts of D. tripetala and A. melengueta significantly(P < 0.05) inhibited intestinal CYP 3A activity at both 50 μg and 100 μg concentrations. Petroleum ether extract of D. tripetala and ethanol extracts of A. melengueta inhibited intestinal CYP3 A activity at 100 μg but not at 50 μg concentrations. All the extracts showed an in vitrodose dependent CYP 3A inhibition with liver microsomes. In vivo analysis showed that pretreatment with the extracts enhanced systemic absorption of simvastatin with reductions in metabolizing enzymes activity as indicated in significant increases in maximal concentration, area under curve, area under moment curve and mean resident time of simvastatin(P < 0.05). Conclusions: Herbal preparations containing these plants' extracts should be used with caution especially in patients on CYP450 3A substrate medications.

Influences of V5-epitope tag on the metabolic activation of AFB1 by human cytochrome P450 2A13

Objective： To explore the impact of V5-epitope tag inserted in the commercial pcDNA5/FRT/V5-His TOPO expression vector on the metabolic activation of AFB1 by human CYP2A13. Methods ： A C-terminal 6 × Histag was first introduced into CYP2A13 cDNA by PCR and subsequently transferred into the expressing vector pcDNA5/FRT. Another commercial pcDNA5/FRT/V5-His TOPO expression vector was used to develop the construct directly via PCR. Both of the constructs were then transfected into Flp-In CHO and allowed for the stable expression of CYP2A13. The mouse CYP2A5 and the vector alone were used as positive and negative control, respectively. The presence of CYP2A5 and CYP2A13 cDNA and their protein expression in the stable transfectant cells were deterrrfined by immunoblotting assay using a monoclonal antibody against 6 × Histag. The AFBl-induced cytotoxicity in these tranfected CHO cells were conducted by MTS assay and the IC50 of cell viability was used to compare the CYP enzyme metabolic activity in AFB1 metabolism among these cells. Results： In accordance with the Flp-In system working mechanism, all the transfectant cells presented same protein expression level. The CHO cells expressing CYP2A5 was more sensitive to AFB1 treatment than those cells expressing CYP2A13, there was about 30-fold ICs0 difference between the two cells （2.1 nmol/L vs 58 nmol/L）. Interestingly, CYP2A13 fused with V5-Histag had the lost of metabolic activity to AFB1 than that fused with Histag alone, the ICa, of the viability in CHO-2A13-His-V5 cells was about 20-fold less than CHO-2A13- His （〉 1 000 nmol/L vs 58 nmol/L）. However, there was no change between CYP2A5 fused with V5-Histag and Histag alone （2.4 nmol/L vs 2.1 nmol/L）. Conclusion： The results demonstrate that CYP2A13 fused with V5-epitope has a significant impact on its metabolic activation to AFB1, which indicated that it should be careful to select a new expressing vector for evaluating the enzyme activity in carcinogen metabolism.

Hepatoprotective effects of S-adenosyl-L-methionine against alcohol-and cytochrome P450 2E1-induced liver injury

S-adenosyl-L-methionine (SAM) acts as a methyl donor for methylation reactions and participates in the synthesis of glutathione. SAM is also a key metabolite that regulates hepatocyte growth, differentiation and death. Hepatic SAM levels are decreased in animal models of alcohol liver injury and in patients with alcohol liver disease or viral cirrhosis. This review describes the protection by SAM against alcohol and cytochrome P450 2E1-dependent cytotoxicity both in vitro and in vivo and evaluates mechanisms for this protection.

Cardioprotection of Shenfu preparata on cardiac myocytes through cytochrome P450 2J3

To evaluate whether Shenfu injection （SFI） protects against cardiac myocyte injury induced by Fupian injection （FPI） in vitro. METHODS： H9c2 cells were separately treated with FPI, Renshen injection （RSI） and SFI. Cell viability, lactate dehydrogenase （LDH） release, spontaneous beating rate of primative cardical cells, caspase-3/7 activity, cell apoptosis, and cytochrome P450 2J3 （CYP2J3） mRNA expression were analyzed. RESULTS： The viability of H9c2 cells treated with SFI （37 and 75 mg/mL） was significantly higher than that of H9c2 cells treated with FPI （25 and 50 mg/mL） （P〈0.05, P〈0.01, respectively）. LDH activity of H9c2 cells treated with SFI （75 mg/mL） was significantly decreased （P〈0.01） compared with that of H9c2 cells treated with FPI （50 mg/mL）. SFI （150 mg/mL） significantly attenuated FPI （100 mg/mL）-induced spontaneous beating rate decrease in primary myocardial cells after 4-hour treatment. Compared with FPI （12 and 25 mg/mL）, SFI （18 and 37 mg/mL） treatment could effectively reverse the change of caspase-3/7 activity （P〈0.01 and P〈0.01, respectively）. Compared with FPI （6 and 25 mg/mL）, apoptotic cells decreased significantly （P〈0.05, P〈0.01, respectively） when H9c2 cells were incubated with SFI （9 and 37 mg/mL）. The expression of CYP2J3 mRNA was down-regulated by FPI, while RSI and SFI could up-regulate the expression of CYP2J3 （P〈0.01）, which suggested the potential mechanism of protection of RSI against cardiac myocyte damage induced by FPI treatment. CONCLUSION： These observations indicate that SFI has the potential to exert cardioprotective effects against FPI toxicity. The effect was possibly correlated with the activation of CYP2J3.

Cytochrome P450 2E1 RsaI/PstI and DraI Polymorphisms Are Risk Factors for Lung Cancer in Mongolian and Han Population in Inner Mongolia

Objective： To explore the relationship between cytochrome P450 2E1 （CYP2E1） RsaI/PstI and DraI polymorphism and lung cancer susceptibility in Mongolian and Han population in Inner Mongolia of China. Methods： CYP2E1 RsaI/PstI and DraI polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism in 64 lung cancer patients, 150 healthy Mongolian and 150 healthy Han individuals. The distribution of genotype and allele frequencies of CYP2E1 RsaI/PstI and DraI polymorphisms were studied. Results： The risk of lung cancer was increased in individuals with CYP2E1 （cl/cl） and CYP2E1 （DD） with OR values of 2.431 （95%CI=1.082-5.460） and 2.778 （95%CI=1.358-5.683） respectively （P0.05）. When CYP2E1 RsaI/PstI and DraI polymorphisms were combined, the risk of lung cancer was reduced in individuals with CYP2E1 （cl/c2＋c2/c2 and DD＋CC） with OR values of 0.233 （95%CI=0.088-0.615, P0.05）. In smokers, the susceptibility to lung cancer was higher in the individuals with CYP2E1 （c1/c1） and CYP2E1 （DD） than in the individuals with c2 and C allele （P0.05, OR=2.643 and 4.308 respectively）. There was no significant difference in distribution of CYP2E1 genotype frequency between healthy Mongolian, Han population and lung cancer patients, healthy controls in Inner Mongolia. Conclusion： CYP2E1 （c1/c1） and CYP2E1 （DD） are predisposing factors of lung cancer in population in Inner Mongolia. CYP2E1 （c2﹢C） co-mutation may decrease the risk of lung cancer. Smoking exerts synergetic effect with CYP2E1 （c1/c1） and CYP2E1 （DD） on the occurrence of lung cancer.

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。

酮康唑对健康成人肝细胞微粒体细胞色素P450同工酶3A4、1A2活性的作用

目的观察酮康唑对健康成人肝细胞微粒体细胞色素P450同工酶3A4、1A2活性的作用,为临床上安全有效地联合用药提供实验依据。方法采用健康成人肝细胞微粒体,分为对照组和处理组。酮康唑处理组分别加入不同浓度的酮康唑1 ml,对照组仅加入培养液,孵育15 min后再加入CYP450同工酶3A4和1A2的相应底物(分别为睾酮和非那西丁)再孵育20 min。反应终止后用高效液相色谱仪测量代谢产物(分别为6β-羟基睾酮与对乙酰氨基酚)的生成量,分别代表3A4和1A2的活性。结果细胞色素P450同工酶3A4的相对活性百分比减小到对照组50%时(IC50)酮康唑的质量浓度为0.16 mg/L。而同工酶3A4的相对活性百分比随质量浓度酮康唑增加逐渐减小,各处理组与对照组比较差异均有显著性意义(P<0.05)。低剂量细胞色素P450同工酶1A2的相对活性百分比处理组与对照组比较明显降低(P<0.05),高剂量细胞色素P450同工酶1A2的相对活性百分比处理组与对照组比较则明显升高(P<0.05)。结论酮康唑对健康成人肝细胞微粒体细胞色素P450同工酶3A4的活性有抑制作用,然而对细胞色素P450同工酶1A2的活性则是低剂量有抑制作用,而高剂量有诱导作用。

注射用丹参总酚酸(冻干)对人CYP450酶和P-糖蛋白体外抑制作用及对大鼠CYP1A2和CYP3A体内诱导作用(英文)

目的探讨注射用丹参总酚酸(冻干)(SLI)对人CYP450酶和P-糖蛋白体外抑制作用以及对大鼠CYP1A2和CYP3A体内诱导作用。方法①应用P450-GloTMCYP450检测试剂盒,通过化学发光法测定SLI和经典抑制剂对细胞色素P4501A2(CYP1A2),CYP2D6,CYP3A4,CYP2C19和CYP2C9的IC50值,通过比较SLI和经典抑制剂对相应细胞色素P450亚型的IC50值来判断SLI对人CYP450酶的体外抑制作用。②Wistar大鼠分别iv给予SLI 3,10和30 mg·kg-1和诱导剂苯巴比妥钠20 mg·kg-1,采用探针底物法,通过比较代谢产物的生成速率来评价SLI对大鼠CYP1A2和CYP3A的诱导作用。③应用ATP酶检测试剂盒,通过化学发光法测定ATP酶活性来评价SLI是否为P-gp的底物或抑制剂。结果①CYP1A2,CYP2C9,CYP2C19,CYP2D6和CYP3A4抑制剂的IC50与SLI对其的IC50进行比较(CYP1A2:0.12μmol·L-1vs 840μmol·L-1;CYP2C9:3.362μmol·L-1vs 704μmol·L-1;CYP2C19:3.236μmol·L-1vs 306μmol·L-1;CYP2D6:0.117μmol·L-1vs 2660μmol·L-1;CYP3A4:0.078μmol·L-1vs 1780μmol·L-1)。②与空白对照组(86.4±6.3)nmol·g-1.min-1相比,SLI 3,10和30 mg·kg-1组CYP1A2活性分别为83.4±6.6,82.5±4.0和(83.4±6.6)nmol·g-1.min-1。与空白对照组(16.1±0.9)nmol·g-1.min-1比较,SLI 3,10和30 mg·kg-1组CYP3A活性分别为15.7±0.6,15.9±0.7和(15.9±1.0)nmol·g-1.min-1,无显著性差异。③以临床血药浓度为依据设计的一系列浓度的SLI 0.0002,0.0006,0.002,0.006,0.017,0.052,0.156和0.468 g.L-1的ATP酶活性分别与空白对照组进行比较(5.8,5.3,5.8,5.5,5.8,5.2,,5.8,5.3,vs 5.75μmol·g-1.min-1),无显著性差异。结论SLI临床给药剂量既不能体外抑制人CYP1A2,CYP2D6,CYP3A4,CYP2C19和CYP2C9酶活性,也不能诱导大鼠CYP1A2和CYP3A,同时也不是P-gp的体外抑制剂或底物。

伊曲康唑对健康成人肝细胞微粒体细胞色素P450同工酶1A2、3A4活性的作用

目的观察伊曲康唑对健康成人肝细胞微粒体细胞色素P450同工酶1A23、A4活性的作用,为临床上安全有效地联合用药提供实验依据。方法采用健康成人肝细胞微粒体,分为对照组和伊曲康唑处理组。处理组分别加入0.1、0.2、1.0、2.0、10.0、20.0 mg.L-1的伊曲康唑1mL,对照组仅加入培养液,孵育15 min后再加入CYP450同工酶1A2和3A4的相应底物(分别为非那西丁和睾酮)再孵育20min。反应终止后用高效液相色谱仪测量代谢产物(分别为对乙酰氨基酚与6β-羟基睾酮)的生成量代表1A2和3A4的活性。结果细胞色素P450同工酶1A2的相对活性百分比各处理组与对照组比较差异无显著性意义(P>0.05),而同工酶3A4的相对活性百分比随伊曲康唑质量浓度增加逐渐减小,各处理组与对照组比较差异均有显著性意义(P<0.05)。细胞色素P450同工酶3A4的相对活性百分比减小到对照组50%时(IC50)伊曲康唑的质量浓度为0.16 mg.L-1。结论伊曲康唑在临床应用血药质量浓度范围内,对健康成人肝细胞微粒体细胞色素P450同工酶1A2的活性无显著影响,然而对细胞色素P450同工酶3A4的活性有抑制作用。

3—甲基胆蒽对原代培养大鼠肝细胞细胞色素P4501A1／2的诱导作用

目的：研究3—甲基胆蒽(3—methylcholanthrene，3—MC)对原代培养大鼠肝细胞细胞色素P4501A1／2的诱导作用，并探讨其时间—效应和剂量—效应关系。方法：原位两步胶原酶灌流法分离大鼠肝细胞，无血清条件下培养细胞，并用不同剂量的3—MC诱导肝细胞24h或48h。乙氧基试卤灵(ethoxyresomn，EOR)为CYP1A酶活性探针药，高效液相色谱法(HPLC)测定试卤灵(resomfin，RSF)浓度。Taqman RT—PCR法测定CYP1A1基因表达，Westem bolt法分析CYP1A酶蛋白表达。结果：实验条件下对照组和3—MC诱导组的CYP1A1基因和酶蛋白，CYP1A1／2酶活性均可被检测。3—MC对原代培养大鼠肝细胞CYP1A1／2基因及蛋白表达、酶活性的诱导作用呈明显的剂量依赖性。结论：3—MC对大鼠原代培养肝细胞的CYP1A1／2有明显的诱导作用。

柴胡水提液对大鼠肝CYP450酶含量及其亚型CYP3A、CYP2E1和CYP1A2活性的影响

目的:研究柴胡水提物对大鼠细胞色素CYP450酶含量及对亚型CYP3A、CYP2E1和CYP1A2活性的影响。方法:大鼠灌胃给予柴胡水提液7d后取肝脏称重并制备肝微粒体,紫外分光光度法测定肝微粒体细胞色素b5(Cytb5)、P450的含量及CYP3A的活性,高效液相法测定CYP2E1和CYP1A2的活性。结果:与空白对照组比较,柴胡水提液组大鼠肝指数无显著变化,CYP450含量减少但统计学意义(P>0.05),CYP1A2活性极显著降低(P<0.01),CYP2E1活性明显降低(P<0.05)。结论:柴胡水提液降低大鼠肝脏CYP450酶含量并抑制CYP2E1和CYP1A2的活性,由于抑制CYP450酶活性,减少对其他药代谢,提示若柴胡水提液与经CYP2E1和CYP1A2代谢的药物同用,有可能会影响这些药物的临床疗效,临床用药时应慎重考虑。

注射用盐酸头孢他美对大鼠肝微粒体CYP1A2、CYP3A4和CYP2E1活性的影响

目的研究注射用盐酸头孢他美对大鼠肝微粒体细胞色素P450（CYP450）酶系CYPlA2、CYP3A4和CYP2E1活性的影响。方法将SD大鼠分成盐酸头孢他美给药组和生理盐水空白组，每组6只，雌雄各半，给药组尾静脉注射盐酸头孢他美50mg／（kg·d），连续给药7d，每天2次。HPLC法同时测定CYP450的3种同工酶特异性探针底物在SD大鼠肝微粒体内代谢产物生成量和原型探针底物降解量，判断酶亚型活性变化。分析柱DiamonsilC，s（150mm×4．6mm，5／tm），流速1．0mL／min。CYPlA2代谢样品测定条件为甲醇（O．1％甲酸）（A）一水（0．1％甲酸）（B），0～5min：18VooA，5～10min：18％～60％A，10～15min：60oAA，检测波长为247nm；CYP3A4代谢样品测定条件为甲醇（A）-水（0．02％甲酸）（B），0～11min：40％～60％A，检测波长为223nm；CYP2E1代谢样品测定条件为甲醇（A）-水（B），0～10min：37oA～75％A，检测波长为287nm。结果探针底物及其代谢产物在测定浓度范围内线性关系良好（r≥0．9997），精密度RSD〈6％（n=5），提取回收率83．2％～97．5％。SD大鼠连续注射给予盐酸头孢他美后，CYP3A4的活性与对照组比较差异有统计学意义（P〈0．05），可能存在诱导作用；CYPlA2和CYP2E1的活性与对照组比较差异无统计学意义（P〉0．05）。结论静注给予盐酸头孢他美能诱导SD大鼠肝微粒体CYP3A4，对CYPlA2和CYP2E1没有诱导或抑制作用。提示经CYP3A4代谢的药物在临床上与注射用盐酸头孢他美合用时，可能存在潜在药物一药物相互作用。

Functionalized selenium nanoparticles ameliorated acetaminophen-induced hepatotoxicity through synergistically triggering PKCδ/Nrf2 signaling pathway and inhibiting CYP 2E1

Selenium nanoparticles(SeNPs)have been demonstrated potential for use in diseases associated with oxidative stress.Functionalized SeNPs with lower toxicity and higher biocompatibility could bring better therapeutic activity and clinical application value.Herein,this work was conducted to investigate the protective effect of Pleurotus tuber-regium polysaccharide-protein complex funtionnalized SeNPs(PTR-SeNPs)against acetaminophen(APAP)-induced oxidative injure in HepG2 cells and C57BL/6J mouse liver.Further elucidation of the underlying molecular mechanism,in particular their modulation of Nrf2 signaling pathway was also performed.The results showed that PTR-SeNPs could significantly ameliorate APAP-induced oxidative injury as evidenced by a range of biochemical analysis,histopathological examination and immunoblotting study.PTR-SeNPs could hosphorylate and activate PKCδ,depress Keap1,and increase nuclear accumulation of Nrf2,resulting in upregulation of GCLC,GCLM,HO-1 and NQO-1 expression.Besides,PTR-SeNPs suppressed the biotransformation of APAP to generate intracellular ROS through CYP 2E1 inhibition,restoring the mitochondrial morphology.Furthermore,the protective effect of PTR-SeNPs against APAP induced hepatotoxicity was weakened as Nrf2 was depleted in vivo,indicating the pivotal role of Nrf2 signaling pathway in PTR-SeNPs mediated hepatoprotective efficacy.Being a potential hepatic protectant,PTR-SeNPs could serve as a new source of selenium supplement for health-promoting and biomedical applications.

检测VKORC1、CYP2C9^(*)3基因多态性指导急性肺血栓栓塞症患者华法林抗凝治疗的价值

目的探讨维生素K环氧化物还原酶复合物1(VKORC1)、细胞色素P450酶2C9^(*)3(CYP2C9^(*)3)基因多态性检测在指导急性肺血栓栓塞症(PTE)患者接受华法林抗凝治疗中的临床价值。方法回顾性收集2016年1月~2018年12月四川大学华西医院呼吸与危重症医学科住院接受华法林抗凝治疗的411例成人PTE患者为研究对象。根据是否行VKORC1、CYP2C9^(*)3基因多态性检测并据此制定华法林初始剂量及调整方案分为指导组(基因检测指导组,n=119)和对照组(常规处理组,n=292)。以开始使用华法林到国际标准化比值(INR)第一次达标(2-3)的时间(d)为主要观察指标;以稳定时(INR 2-3)的华法林维持剂量为次要观察指标,比较两组差异。随访两组自接受华法林抗凝开始到第6个月末时的不良反应。结果基因多态性,VKORC1以AA野生型多见(n=89,74.8%);其次为GA杂合突变型(n=29,24.4%);纯合突变GG型仅1例(0.8%)。CYP2C9^(*)3野生型(AA型)为106例(89.1%);杂合突变(AC型)13例(10.9%)。从两个基因多态性组合看,VKORC1 AA型合并CYP2C9^(*)3 AA型81例,占68.1%,即大部分患者两个基因均为野生型;其次为VKORC1 GA型合并CYP2C9^(*)3 AA型24例(20.2%);VKORC1、CYP2C9^(*)3均突变的5例(4.20%)。指导组INR达到目标范围的时间为(7.96±4.02)d,对照组INR达标的时间为(7.68±3.58)d,二者无统计学差异(P>0.05);指导组达到目标INR的华法林维持剂量为(5.30±0.99)mg,对照组的华法林维持剂量为(5.22±0.64)mg,二者无显著性差异(P>0.05)。两组患者出现死亡、大出血、新发血栓的不良事件发生率均无统计学意义(均P>0.05)。结论VKORC1、CYP2C9^(*)3基因多态性检测在指导急性PTE患者接受华法林抗凝治疗中的意义有限。

甘草与海藻提取液合用对CYP3A1/2酶活性及mRNA表达的影响

目的:研究甘草与海藻提取液合用对CYP3A1/2酶活性的影响及在mRNA水平的调控作用。方法:采用紫外-可见分光光度法测定CYP3A1/2活性;采用RT-PCR评价药物对CYP3A1、CYP3A2 mRNA水平的影响。结果:甘草、海藻提取液合用后诱导了CYP3A1/2的酶活性;合用后诱导CYP3A1、CYP3A2 mRNA表达。结论:甘草、海藻提取液合用后诱导CYP3A1/2酶活性,酶活性增强可能主要由诱导其mRNA水平来实现。

中药藜芦及其与玄参合用对大鼠肝脏CYP3A1/2酶活性的影响

目的研究藜芦及其与玄参合用对P450同工酶CYP3A1/2酶活性的影响。方法采用紫外分光光度法测定大鼠肝微粒体细胞色素P450与细胞色素b5含量及CYP3A1/2酶活性。结果玄参与藜芦合用可明显降低P450蛋白含量,藜芦对CYP3A1/2酶活性有一定的诱导作用,配伍应用时恢复到正常水平。结论玄参与藜芦配伍前后对CYP3A1/2亚型调控作用发生明显变化,可能存在基于药物代谢酶机理的相互作用,需进一步结合代谢研究加以综合分析,阐明相互作用的机理。

Self-sufficient Cytochrome P450s and their potential applications in biotechnology

Cytochrome P450s(CYPs)are ubiquitously found in all kingdoms of life,playing important role in various biosynthetic pathways as well as degradative pathways;accordingly find applications in a vast variety of areas from organic synthesis and drug metabolite production to modification of biomaterials and bioremediation.Significantly,CYPs catalyze chemically challenging CAH and CAC activation reactions using a reactive high-valent iron-oxo intermediate generated upon dioxygen activation at their heme center,while the other oxygen atom is reduced to the level of water by electrons provided through a reductase partner protein.Self-sufficient CYPs,encoding their heme domain and reductase protein in a single polypeptide,facilitate increased catalytic efficiency and render a less complicated system to work with.The self-sufficient CYP enzyme from CYP102A family(CYP102A1,BM3)is among the earliest and most-investigated model enzymes for mechanistic and structural studies as well as for biotechnological applications.An increasing number of self-sufficient CYPs from the same CYP102 family and from other families have also been reported in last decade.In this review,we introduce chemistry and biology of CYPs,followed by an overview of the characteristics of self-sufficient CYPs and representative reactions.Enzyme engineering efforts leading to novel self-sufficient CYP variants that can catalyze synthetically useful natural and non-natural(nature-mimicking)reactions are highlighted.Lastly,the strategy and efforts that aim to circumvent the challenges for improved thermostability,regio-and enantioselectivity,and total turnover number;associated with practical use of self-sufficient CYPs are reviewed.

人参皂苷Re对H9c2心肌细胞CYP450酶的影响

目的观察人参皂苷Re对H9c2心肌细胞色素P450酶的影响,旨在发现人参皂苷Re对心肌细胞作用的分子机制。方法实验分对照组、人参皂苷Re各剂量组(1、5、10、50、100μmol·L^(-1))处理组,人参皂苷Re作用6、24、36、48及60 h后提取RNA或蛋白进行测定。利用实时定量PCR法检测H9c2心肌细胞CYP2C11、CYP2J3、CYP4A1、CYP4A3、CYP4F4及ANP mRNA的表达;采用Western blot法检测心肌细胞CYP4A1和CYP2J3蛋白的表达。结果人参皂苷Re明显上调心肌细胞CYP2C11、CYP2J3 mRNA的表达至正常对照的1.6、1.8倍,明显下调CYP4A1、CYP4A3及CYP4F4 mRNA的表达至正常对照的0.4、0.15、0.3倍。人参皂苷Re明显上调ANP基因表达水平至正常对照的3.2倍。随着药物浓度的增加,人参皂苷Re对CYP4A1蛋白表达产生明显的下调作用,而对CYP2J3蛋白产生明显的上调作用。结论人参皂苷Re可影响心肌细胞的CYP450酶和ANP基因的表达。

CYP2C9及VKORC1基因多态性对心脏瓣膜置换术后华法林个体剂量差异的研究

目的研究湖北地区汉族人细胞色素P450酶2C9(CYP2C9)基因和维生素K环氧化物还原酶复合体1(VKORC1)基因多态性分布特点及其对华法林稳态剂量的影响。方法收集2012年3—8月湖北地区汉族临床使用华法林的患者108例作为华法林组,选择同期健康受试者85名为对照组。采用聚合酶链反应-限制性内切酶片段长度多态性(PCR-RFLP)技术检测CYP2C9和VKORC1基因型,并比较不同基因型华法林稳态剂量。结果两组基因型频率和等位基因频率分布比较差异无统计学意义(P>0.05)。华法林组中CYP2C9基因*1/*3型较*1/*1型华法林稳态剂量小,VKORC1基因AA型较GA型华法林稳态剂量小(P<0.05,P<0.01)。除了检测的3个多态位点,还发现了50多个变异位点。结论在湖北地区汉族人群中,存在CYP2C9和VKORC1基因多态性,且不同基因型患者间华法林稳态剂量存在差异,华法林基因相关变异位点需要进一步证实。

表没食子儿茶素-3-没食子酸酯对细胞色素P450酶活性的影响

目的研究表没食子儿茶素-3-没食子酸酯(EGCG)对人肝微粒体中细胞色素P450(CYP450)酶活性的影响。方法以未处理的肝微粒体作为阴性对照组,CYP450酶各亚型的特异性抑制剂作为阳性对照组,通过EGCG或特异性抑制剂与探针底物共同孵育,高效液相色谱法定量分析特异性底物的代谢产物,分析EGCG对CYP450酶各亚型活性的影响,并通过统计学分析拟合获得相应的动力学参数。结果与阴性对照组相比,EGCG显著抑制了CYP1A2酶和CYP3A4酶的活性,但抑制作用远小于阳性抑制剂的抑制作用。EGCG对CYP1A2酶和CYP3A4酶的抑制作用受EGCG浓度的影响,IC50值分别为8.69和14.07μmol/L。EGCG能够竞争性抑制CYP1A2酶的活性,而对CYP3A4酶为非竞争性抑制作用。此外,EGCG对CYP3A4酶的抑制作用具有时间依赖性,随着培养时间的延长,抑制作用逐渐增强。结论EGCG可显著抑制CYP1A2酶及CYP3A4酶的活性。因此,在EGCG的应用中应充分考虑可能出现的药物相互作用及潜在风险。