石斛碱在体外肝微粒体代谢的种属差异研究

为了在临床前药效和毒理研究中选择与人有相似代谢性质的实验动物,本实验比较了石斛碱在不同种属肝微粒体间的代谢差异。将石斛碱在SD大鼠、C57BL/6J小鼠、家兔、犬、豚鼠和人的肝微粒体中,分别进行I相和II相代谢。通过底物消除法分析代谢稳定性,并计算体外消除半衰期(t1/2)。通过保留时间、精确质量数、二级碎片离子信息以及色谱峰面积,对石斛碱在各种属肝微粒体中生成代谢产物的情况进行分析。结果表明,在I相反应中,石斛碱体外消除半衰期依次为家兔豚鼠大鼠小鼠犬人;在II相反应中,石斛碱在人、豚鼠肝微粒体中代谢非常慢,而小鼠最快。石斛碱在人肝微粒体体外I相反应中,生成的代谢产物与小鼠、犬的相似。因此,可以选择小鼠和犬作为临床前药代和毒理研究的模型动物。     

喜树碱衍生物NCP4在不同种属肝微粒体中代谢稳定性研究

探讨喜树碱衍生物NCP4在人、SD大鼠、beagle犬肝微粒体中的体外代谢稳定性及代谢产物。应用体外肝微粒体孵育体系,采用HPLC-UV-MS(Q-TOF)法,考察NCP4的代谢稳定性,并推断其代谢产物结构。结果显示,NCP4在Beagle犬和人肝微粒体温孵代谢动力学参数T_(l/2)(min)相当,而在SD大鼠肝微粒体温孵的T_(l/2)(min)相差较大;此外,NCP4在SD大鼠肝微粒体中生成m/z[M+Na]~+为502、460、344的代谢产物,在Beagle犬、人肝微粒体中生成m/z[M+Na]~+为388、390、448的代谢产物。结果表明Beagle犬与人肝微粒体温孵体系中NCP4底物的代谢稳定性基本一致,且NCP4在beagle犬与人的肝微粒体代谢产物相同。可以为NCP4临床前安全性评价的实验动物选择提供依据。     

绒毛崖豆藤中一种查尔酮提取物的体内外代谢及药代动力学研究CSCD

采用UPLC-QE-Orbitrap-MS检测方法,通过比较2,5-二甲氧基呋喃[4″,5″:3,4]查耳酮(1)在体外大鼠、小鼠、恒河猴、Beagle犬和人五个种属肝微粒体体系中孵育0 min和60 min的样品,以及比较静脉注射该化合物及空白的C57小鼠的血浆、粪便、尿液样品,研究其在体内外代谢产物。初步推断孵育后体外代谢产物有M1、M2共2种,体内代谢产物M1、M2、M3、M4、M5共5种,并且M5为M1进一步代谢而来,采用化学合成方法制得代谢产物M1。将大鼠随机分组并给药,于设定的系列时间点取血,采用超高速液相色谱质谱联用(UFLC-MS/MS)检测方法,测定1及M1血药浓度,定量研究该化合物及M1在大鼠体内的变化趋势并计算药代参数。用DAS 2.0软件计算药代动力学参数,1的C_(max)=405.96μg/L,Tmax=0.083 h,AUC_(0-t)=190.64μg/(L·h),T_(1/2)=3.74 h,M1的C_(max)=281.291μg/L,T_(max)=0.083 h,AUC_(0-t)=561.30μg/(L·h),T_(1/2)=3.01 h。研究结果表明,2,5-二甲氧基呋喃[4″,5″:3,4]查耳酮在体内代谢过程中主要发生了还原、去甲基、开环、加羟基等反应,其中主要代谢产物为M1,并且M1进一步代谢为M5,本研究为揭示其药理药效作用奠定了物质基础。     

紫杉醇对食蟹猴和人CYP1A2、CYP3A4及CYP2A6酶代谢的影响

目的探讨紫杉醇对食蟹猴和人肝微粒体CYP1A2、CYP2A6和CYP3A4酶活性的影响。方法采用食蟹猴和人肝脏微粒体,分别以非那西汀、睾丸酮和香豆素分别作为CYP1A2、CYP2A6、CYP3A4的底物,建立CYP1A2、CYP2A6和CYP3A4体外代谢体系。采用不同浓度的紫杉醇分别与上述3种底物共同孵育于肝微粒体代谢体系中。用HPLC法分别测定各底物的代谢产物扑热息痛、6β-羟基睾丸酮、7-羟基香豆素的产生量,计算IC50值,以评估紫杉醇对CYP1A2、CYP2A6和CYP3A4代谢的影响。结果紫杉醇对食蟹猴肝微粒体3种酶的IC50值分别为570±5.9μmol/L、140±2.9μmol/L和无影响;紫杉醇对人肝微粒体3种酶的IC50值分别为193±6.6μmol/L、253±3.6μmol/L和24±1.6μmol/L。结论紫杉醇对食蟹猴肝微粒体CYP1A2和CYP3A4活性具有一定的抑制作用,但对CYP2A6酶的活性几乎没有影响。紫杉醇对人肝微粒体CYP1A2和CYP3A4活性的抑制作用较弱,但对CYP2A6酶的活性抑制作用较强,提示临床上紫杉醇与作为上述酶底物的药物联合用药时应慎重,以避免因中西药物相互作用所导致的不良反应发生。     

实验动物呼吸系统主要器官比较组织学研究

目的比较实验动物呼吸系统主要器官的组织学特征,为制定实验动物病理检测标准、以及毒理学、新药安全性评价提供依据。方法选取实验动物质量国家检测标准检测合格的恒河猴30只、昆明小鼠20只、SD大鼠20只、日本大耳白兔18只、比格犬16只、树鼩20只。除昆明小鼠采用颈椎脱臼致死外,其余动物麻醉后放血处死和病理解剖,对气管、肺脏进行病理大体检查和取材,常规病理制片,进行HE染色、特殊染色和免疫组化染色,显微镜下观察气管、肺脏的组织结构和细胞结构异同。结果 （1）实验动物气管上皮杯状细胞有差异：恒河猴、比格犬、日本大耳白兔杯状细胞较多,大鼠、小鼠、树鼩则较少或无。上皮分泌的黏液类型以中性黏液为主,比格犬杯状细胞分泌的黏液类型有中性黏液和酸性黏液。（2）实验动物黏膜下腺泡分布有差异：比格犬黏膜下层的腺泡最多,恒河猴、大鼠、小鼠、树鼩腺泡数量偏少,日本大耳白兔黏膜下层的混合腺泡最少。（3）实验动物的肺内支气管分支有差异：比格犬、恒河猴、日本大耳白兔由叶支气管、段支气管、小支气管、细支气管、终末细支气管和呼吸性细支气管组成,树鼩、大鼠、小鼠只由细支气管、终末细支气管和呼吸性细支气管组成。（4）实验动物细支气管组织结构有差异：恒河猴、比格犬的细支气管平滑肌为完整环形平滑肌层,没有缺失,而大鼠、小鼠、树鼩及日本大耳白兔的细支气管平滑肌薄或缺失。恒河猴、树鼩、大鼠细支气管有少量杯状细胞,其余实验动物均无杯状细胞。（5）实验动物Clara细胞形态有差异：比格犬Clara细胞呈立方形,其余动物呈柱状。结论实验动物呼吸系统组织结构的质是相同的,差异在于量的不同。研究人员在制定病理学检测标准、实验研究、药物安全性评价时应予充分考虑。     

四个不同种属动物肢体Ⅱ导联心电图比较

目的对比格犬、恒河猴、日本大耳白兔、树鼩四个不同种属实验动物的ECG-II进行分析比较,归纳其ECG-II的特点,旨在为上述动物在安评中的运用提供参考。方法选择成年比格犬、恒河猴、日本大耳白和树鼩,分别对其在清醒状态下的ECG-II进行描记和分析,并对ECG-II各项指标进行测量分析。结果与结论不同种属动物均为窦性心律,在波型方面基本一致,未出现特异性改变,但不同动物在QRS波群和T波形态上具有一定差异;各指标测定结果显示,日本大耳白兔P波、T波振幅和树鼩心率分别小于和快于其他种属动物。     

白皮杉醇苷在大鼠体内外的葡萄糖醛酸结合代谢研究北大核心CSCD

白皮杉醇苷(PG)是藏边大黄中一种天然抗氧化剂,前期研究发现其极易发生代谢。本文主要研究PG在大鼠体内外的葡萄糖醛酸结合代谢特征。SD大鼠经尾静脉注射给予PG(20 mg/kg),采集给药后胆汁样品,采用LC-MS对主要代谢产物进行结构推测。在此基础上,研究大鼠肝微粒体体外温孵体系中PG的葡萄糖醛酸结合代谢,并测定酶促反应动力学参数。实验结果显示SD大鼠经尾静脉注射给予PG,可在胆汁中快速检测到多种PG及其衍生物的葡萄糖醛酸结合代谢产物。在大鼠肝微粒体体外温孵体系中,PG代谢生成两个与体内一致的单葡萄糖醛酸结合代谢物,其葡萄糖醛酸结合代谢的最大反应速率(Vmax)、米氏常数(Km)和肝内清除率CLint(Vmax/Km)分别为10.11 nmol/(min·mg)、0.36 mmol/L和0.028 m L/(min·mg)。PG经静脉途径进入大鼠体内可经肝脏被快速地广泛代谢,葡萄糖醛酸结合代谢是其体内消除的主要途径之一。大鼠肝脏的葡萄糖醛酸转移酶对PG有较强的亲和力,可催化PG发生快速的葡萄糖醛酸结合代谢。     

MSCT-3D技术在动物与人骨骼形态比较研究中的应用

目的应用MSCT-3D显示技术比较正常贵州香猪、Marshall比格犬、恒河猴与人上肢带骨及躯干骨的形态学差异。方法采用MSCT分别对贵州香猪、比格犬和恒河猴进行CT全身扫描并进行图像重建,观察其上肢带骨、躯干骨形态结构与人的异同。结果比格犬、恒河猴、贵州香猪脊椎骨和肋的基本组成与人相同,脊椎骨由椎体和附件组成,肋骨包括真肋、假肋和浮肋。而脊柱曲度、各段椎骨数目、胸骨结构、肋的数目、胸肋连接、上肢带骨的组成与人不同,恒河猴的脊柱曲度和上肢带骨连接与人相同,有颈、胸、腰、骶四个生理性弯曲并由锁骨和肩胛骨共同连接自由上肢骨,比格犬和贵州香猪只有颈、胸腰、骶三个生理性弯曲,仅由肩胛骨连接自由上肢骨。结论恒河猴躯干骨和上肢带骨与人有良好的相似性,而比格犬和贵州香猪与人差别较大。MSCT-3D技术为实验动物形态学比较研究提供了一种相对无创、快速、可以在体研究并动态连续观察的科学有效方法。     

白皮杉醇苷在大鼠体内外的葡萄糖醛酸结合代谢研究

白皮杉醇苷(PG)是藏边大黄中一种天然抗氧化剂,前期研究发现其极易发生代谢。本文主要研究PG在大鼠体内外的葡萄糖醛酸结合代谢特征。SD大鼠经尾静脉注射给予PG(20 mg/kg),采集给药后胆汁样品,采用LC-MS对主要代谢产物进行结构推测。在此基础上,研究大鼠肝微粒体体外温孵体系中PG的葡萄糖醛酸结合代谢,并测定酶促反应动力学参数。实验结果显示SD大鼠经尾静脉注射给予PG,可在胆汁中快速检测到多种PG及其衍生物的葡萄糖醛酸结合代谢产物。在大鼠肝微粒体体外温孵体系中,PG代谢生成两个与体内一致的单葡萄糖醛酸结合代谢物,其葡萄糖醛酸结合代谢的最大反应速率(Vmax)、米氏常数(Km)和肝内清除率CLint(Vmax/Km)分别为10.11 nmol/(min·mg)、0.36 mmol/L和0.028 m L/(min·mg)。PG经静脉途径进入大鼠体内可经肝脏被快速地广泛代谢,葡萄糖醛酸结合代谢是其体内消除的主要途径之一。大鼠肝脏的葡萄糖醛酸转移酶对PG有较强的亲和力,可催化PG发生快速的葡萄糖醛酸结合代谢。     

基于活血化瘀药物有效成分对生物转化酶的影响研究

目的:分析活血化瘀药物有效成分对生物转化酶的影响。方法:选取16只雄性SD大鼠,随机分为4组,每组4只,分别给予DEX色拉油混悬液、PB生理盐水溶液、β-NF色拉油混悬液、色拉油。分析大鼠血浆及肝微粒体温孵液中川芎嗪及其代谢产物的相关性。结果:在大鼠肝微粒体中诱导后的川芎嗪代谢较快,且川芎嗪在DEX、PB、DEX及对照组中的平均代谢消失率分别为(76.25±10.23)%、(19.26±7.13)%、(14.96±1.26)%、(13.49±1.34)%。强效的CYP3A抑制剂Ket能够抑制川芎嗪的代谢,其代谢率与CYP3A的特征性有着相关性。结论:川芎嗪生物转化的CYP450亚酶是CYP3A,且两者抑制或者诱导在药物中可能存在相互作用。     

Pharmacokinetic,metabolic stability,plasma protein binding and CYP450s inhibition/induction assessment studies of N-(2-pyridylmethyl)-2-hydroxiymethyl-1-pyrrolidinyl-4-(3-chloro-4-methoxy-benzylamino)-5-pyrimidine-carboxamide as potential type 5 phosphodiesterase inhibitors

N-(2-pyridylmethyl)-2-hydroxiymethyl-1-pyrrolidinyl-4-(3-chloro-4-methoxy-benzylamino)-5-pyrimidine-carboxamide (NHPPC) is a new potential of type 5 phosphodiesterase (PDE5) inhibitors, synthesized from the avanafil analogue for the treatment of erectile dysfunction. The targets of this article were to assess plasma protein binding, liver microsomal metabolic stability, inhibition and induction on cytochrome P450 isozymes and the pharmacokinetics of NHPPC. Equilibrium dialysis technique was applied to determine Plasma protein binding (PPB) and NHPPC was evaluated in male Sprague–Dawley rats and Beagle dogs in vivo pharmacokinetic. The NHPPC was highly bound to plasma proteins in rats, dogs and human tested and the mean values for PPB rate were 96.2%, 99.6% and 99.4%, respectively. After in vitro liver microsomes incubated for 60?min, the percent remaining of NHPPC was 42.8%, 0.8% and 42.0% in rats, dogs and human, respectively. In vitro intrinsic clearance was found to be 0.0233, 0.1204 and 0.0214 mL/min/mg protein in rat, dog and human liver microsomes of NHPPC, respectively. NHPPC showed no significant inhibitory effects on major CYP450 enzymes, and had no significant induction potential on CYP1A2 and CYP3A4. Following oral administration in rats and dogs, t_max was 6 and 0.5?h, respectively. The clearance for NHPPC was 1.19 and 1.46?L/h/kg in rats and dogs, respectively. And absolute bioavailability in rat and dog were approximately 34.5% and 53.1%, respectively. These results showed that NHPPC has a good development prospect.     

Metabolism of medroxyprogesterone acetate (MPA) via CYP enzymes in vitro and effect of MPA on bleeding time in female rats in dependence on CYP activity in vivo

Medroxyprogesterone acetate (MPA) is a drug commonly used in endocrine therapy for advanced breast cancer, although it is known to cause thrombosis as a serious side effect. Recently, we found that cytochrome P450 3A4 (CYP3A4) mainly catalyzed the metabolism of MPA via CYP in human liver microsomes. However, the metabolic products of MPA in humans and rats have not been elucidated. In addition, it is not clear whether thrombosis could be induced by MPA itself or by its metabolites. In this study, we determined the overall metabolism of MPA as the disappearance of the parent drug from an incubation mixture, and identified the enzymes catalyzing the metabolism of MPA via CYP in rats. Moreover, the effects of CYP-modulators on MPA-induced hypercoagulation in vivo were examined. Intrinsic clearance of MPA in rat liver microsomes was increased by treatment with CYP3A-inducers. The intrinsic clearance of MPA in liver microsomes of rats treated with various CYP-inducers showed a significant correlation with CYP3A activity, but not CYP1A activity, CYP2B activity or CYP2C contents. Among the eight recombinant rat CYPs studied, CYP3A1, CYP3A2 and CYP2A2 catalyzed the metabolism of MPA. However, since CYP3A2 and CYP2A2 are male-specific isoforms, CYP3A1 appears to be mainly involved in the metabolism of MPA in liver microsomes of female rats. In an in vivo study, pretreatment of female rats with SKF525A, an inhibitor of CYPs including CYP3A1, significantly (p < 0.05) enhanced MPA-induced hypercoagulation, whereas pretreatment with phenobarbital, an inducer of CYPs including CYP3A1, reduced it. These findings suggest that CYP-catalyzed metabolism of MPA is mainly catalyzed by CYP3A1 and that MPA-induced hypercoagulation is predominantly caused by MPA itself in female rats.     

A comparison of aroclor 1254-induced and uninduced rat liver microsomes to human liver microsomes in phenytoin O-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, S-mephenytoin 4'-hydroxylation, chloroxazone 6-hydroxylation and testosterone 6beta-hydroxylation

Aroclor 1254-induced rat liver homogenate supernatant (liver S-9) is routinely used as an exogenous metabolic activation system for the evaluation of mutagenicity of xenobiotics. The purpose of this study is to evaluate whether results obtained with Aroclor 1254-induced liver microsomes would be relevant to human. Aroclor 1254-induced and uninduced rat liver microsomes were compared to human liver microsomes in the metabolism of substrates which are known to be selectively metabolized by the major human cytochrome P450 (CYP) isoforms. The activities studied and the major CYP isoforms involved were as follows: phenacetin O-deethylation (CYP1A2); coumarin 7-hydroxylation, (CYP2A6); tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19); dextromethorphan O-demethylation (CYP2D6); chloroxazone 6-hydroxylation (CYP2E1); and testosterone 6beta-hydroxylation (CYP3A4). We found that both induced and uninduced rat liver microsomes were active in all the pathways studied with the exception of coumarin 7-hydroxylation. Coumarin 7-hydroxylation was observed with human liver microsomes but not the rat liver microsomes. Aroclor-1254 was found to induce all activities measured, with the exception of coumarin 7-hydroxylation. Dextromethorphan O-deethylation activity was higher in the rat liver microsomes than the human liver microsomes. Testosterone 6beta-hydroxylation activity was found to be similar between the human liver microsomes and the induced rat liver microsomes. Our results suggest that experimental data obtained with Aroclor 1254-induced rat liver microsomes may not always be relevant to human.     

Preclinical metabolism of LB42908, a novel farnesyl transferase inhibitor, and its effects on the cytochrome P450 isozyme activities

Metabolism of LB42908, a novel farnesyl transferase inhibitor, was investigated for preclinical development. In vitro hepatic metabolism of LB42908 gave rise to at least 9 metabolites via phase I biotransformation pathways, which were characterized by HPLC-UV, LC-MS, and LC-MS/MS analyses. N-Dealkylation was shown to be a major phase I metabolic pathway. Species-specific in vitro metabolism of LB42908 was studied in liver fractions of rat, dog, monkey, and human. Order of metabolic stability is human≈dog>rat≈monkey in both S9 and microsomal fractions. Tissue-specific metabolism of LB42908 in various tissue homogenates of rats demonstrated that the liver was the major organ responsible for phase I metabolism of LB42908. The results from both qualitative and quantitative metabolism studies such as metabolic profiling and metabolic clearance indicated that dog would be the animal model of choice for preclinical toxicology studies. In addition, LB42908 was a potent CYP3A4 inhibitor in human liver microsomes and induced the activities of several CYP isozymes, implying that it has the potential for drug-drug interactions. Repeated dosing of LB42908 in rats did not significantly affect its own metabolism, indicating that long-term administration of LB42908 would not alter its pharmacokinetic profiles.     

Carboxylesterase in the liver and small intestine of experimental animals and human

Native polyacrylamide gel electrophoresis showed carboxylesterase (CES) to be the most abundant hydrolase in the liver and small intestine of humans, monkeys, dogs, rabbits and rats. The liver contains both CES1 and CES2 enzymes in all these species. The small intestine contains only enzymes from the CES2 family in humans and rats, while in rabbits and monkeys, enzymes from both CES1 and CES2 families are present. Interestingly, no hydrolase activity at all was found in dog small intestine. Flurbiprofen derivatives were R-preferentially hydrolyzed in the liver microsomes of all species, but hardly hydrolyzed in the small intestine microsomes of any species except rabbit. Propranolol derivatives were hydrolyzed in the small intestine and liver microsomes of all species except dog small intestine. Monkeys and rabbits showed R-preferential and non-enantio-selective hydrolysis, respectively, for propranolol derivatives in both organs. Human and rat liver showed R- and S-preferential hydrolysis, respectively, in spite of non-enantio-selective hydrolysis in their small intestines. The proximal-to-distal gradient of CES activity in human small intestine (1.1-1.5) was less steep than that of CYP 3A4 and 2C9 activity (three-fold difference). These findings indicate that human small intestine and liver show extensive hydrolase activity attributed to CES, which is different from that in species commonly used as experimental animals.     

Species differences and interindividual variation in liver microsomal cytochrome P450 2A enzymes: effects on coumarin, dicumarol, and testosterone oxidation.

Antibody against purified CYP2A1 recognizes two rat liver microsomal P450 enzymes, CYP2A1 and CYP2A2, that catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone, respectively. In human liver microsomes, this antibody recognizes a single protein, namely CYP2A6, which catalyzes the 7-hydroxylation of coumarin. To examine species differences in CYP2A function, liver microsomes from nine mammalian species (rat, mouse, hamster, rabbit, guinea pig, cat, dog, cynomolgus monkey, and human) were tested for their ability to catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone and the 7-hydroxylation of coumarin. Antibody against rat CYP2A1 recognized one or more proteins in liver microsomes from all mammalian species examined. However, liver microsomes from cat, dog, cynomolgus monkey, and human catalyzed negligible rates of testosterone 7 alpha- and/or 15 alpha-hydroxylation, whereas rat and cat liver microsomes catalyzed negligible rates of coumarin 7-hydroxylation. Formation of 7-hydroxycoumarin accounted for a different proportion of the coumarin metabolites formed by liver microsomes from each of the various species examined. 7-Hydroxycoumarin was the major metabolite (greater than 70%) in human and monkey, but only a minor metabolite (less than 1%) in rat. The 7-hydroxylation of coumarin by human liver microsomes was catalyzed by a single, high-affinity enzyme (Km 0.2-0.6 microM), which was markedly inhibited (greater than 95%) by antibody against rat CYP2A1. The rate of coumarin 7-hydroxylation varied approximately 17-fold among liver microsomes from 22 human subjects. This variation was highly correlated (r2 = 0.956) with interindividual differences in the levels of CYP2A6, as determined by immunoblotting. These results indicate that CYP2A6 is largely or entirely responsible for catalyzing the 7-hydroxylation of coumarin in human liver microsomes. Treatment of monkeys with phenobarbital or dexamethasone increased coumarin 7-hydroxylase activity, whereas treatment with beta-naphthoflavone caused a slight decrease. These results suggest that environmental factors can increase or decrease CYP2A expression in cynomolgus monkeys, which implies that environmental factors may be responsible for the large variation in CYP2A6 levels in humans, although genetic factors may also be important. In contrast to rats and mice, the expression of CYP2A enzymes in cynomolgus monkeys and humans was not sexually differentiated. Despite their structural similarity to coumarin, the anticoagulants dicumarol and warfarin do not appear to be substrates for CYP2A6. The overall rate of dicumarol metabolism varied approximately 5-fold among the human liver microsomal samples, but this variation correlated poorly (r2 = 0.126) with the variation observed in CYP2A6 levels and coumarin 7-hydroxylase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro metabolism of alachlor by human liver microsomes and human cytochrome P450 isoforms.

Alachlor (2-chloro-N-methoxymethyl-N-(2,6-diethylphenyl)acetamide) is a widely used pre-emergent chloroacetanilide herbicide which has been classified by the USEPA as a probable human carcinogen. The putative carcinogenic metabolite, 2,6-diethylbenzoquinone imine (DEBQI), is formed through a complex series of oxidative and non-oxidative steps which have been characterized in rats, mice, and monkeys but not in humans. A key metabolite leading to the formation of DEBQI is 2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA). This study demonstrates that male human liver microsomes are able to metabolize alachlor to CDEPA. The rate of CDEPA formation for human liver microsomes (0.0031 +/- 0.0007 nmol/min per mg) is significantly less than the rates of CDEPA formation for rat liver microsomes (0.0353+/-0.0036 nmol/min per mg) or mouse liver microsomes (0.0106 +/- 0.0007). Further, we have screened human cytochrome P450 isoforms 1A1, 1A2, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4 and determined that human CYP 3A4 is responsible for metabolism of alachlor to CDEPA. Further work is necessary to determine the extent to which humans are able to metabolize CDEPA through subsequent metabolic steps leading to the formation of DEBQI.     

Human cytochrome-P450 enzymes metabolize N-(2-methoxyphenyl)hydroxylamine, a metabolite of the carcinogens o-anisidine and o-nitroanisole, thereby dictating its genotoxicity

N-(2-Methoxyphenyl)hydroxylamine is a component in the human metabolism of two industrial and environmental pollutants and bladder carcinogens, viz. 2-methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-nitroanisole), and it is responsible for their genotoxicity. Besides its capability to form three deoxyguanosine adducts in DNA, N-(2-methoxyphenyl)-hydroxylamine is also further metabolized by hepatic microsomal enzymes. To investigate its metabolism by human hepatic microsomes and to identify the major microsomal enzymes involved in this process are the aims of this study. N-(2-Methoxyphenyl)hydroxylamine is metabolized by human hepatic microsomes predominantly to o-anisidine, one of the parent carcinogens from which N-(2-methoxyphenyl)hydroxylamine is formed, while o-aminophenol and two N-(2-methoxyphenyl)hydroxylamine metabolites, whose exact structures have not been identified as yet, are minor products. Selective inhibitors of microsomal CYPs, NADPH:CYP reductase and NADH:cytochrome-b(5) reductase were used to characterize human liver microsomal enzymes reducing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. Based on these studies, we attribute the main activity for this metabolic step in human liver to CYP3A4, 2E1 and 2C (more than 90%). The enzymes CYP2D6 and 2A6 also partake in this N-(2-methoxyphenyl)hydroxylamine metabolism in human liver, but only to ～6%. Among the human recombinant CYP enzymes tested in this study, human CYP2E1, followed by CYP3A4, 1A2, 2B6 and 2D6, were the most efficient enzymes metabolizing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. The results found in this study indicate that genotoxicity of N-(2-methoxyphenyl)hydroxylamine is dictated by its spontaneous decomposition to nitrenium/carbenium ions generating DNA adducts, and by its susceptibility to metabolism by CYP enzymes.     

Characterization of a phenobarbital-inducible dog liver cytochrome P450 structurally related to rat and human enzymes of the P450IIIA (steroid-inducible) gene subfamily

A cytochrome P450 called PBD-1 isolated from liver microsomes of an adult male Beagle dog treated with phenobarbital (PB) is structurally and functionally similar to members of the P450IIIA gene subfamily in rat and human liver microsomes. The sequence of the first 28 amino-terminal residues of PBD-1 is identical in 15 and 20 positions, respectively, to the P450IIIA forms P450p from rat and P450NF (and HLp) from human. Upon immunoblot analysis, anti-PBD-1 IgG recognizes PCNa (P450p) and PCNb (PB/PCN-E) from rat, P450NF from human, and two proteins in liver microsomes from both untreated and PB-treated dogs. Similarly, anti-PCNb IgG cross-reacts with PBD-1 and with at least one protein in microsomes from untreated dogs and two proteins in microsomes from PB-treated dogs. P450IIIA-form marker steroid 6 beta-hydroxylase activities increase 2.5-fold upon PB-treatment of dogs and are selectively inhibited by anti-PBD-1 IgG. NADPH-dependent triacetyloleandomycin (TAO) complex formation and erythromycin demethylase, also marker activities for P450IIIA forms from rats and humans, increase 4- and 5-fold in dog liver microsomes upon PB treatment, whereas immunochemically reactive PBD-1 is induced 3-fold. In microsomes from PB-treated dogs, 5 mg anti-PBD-1 IgG/nmol P450 inhibits greater than 75 and 50% of TAO complex formation and erythromycin demethylase activity, respectively. TAO complex formation is not inhibited by chloramphenicol, a selective inhibitor of the major PB-inducible dog liver cytochrome P450, PBD-2. These data suggest that PBD-1 or another immunochemically related form is responsible for a major portion of macrolide antibiotic metabolism by microsomes from PB-treated dogs and for steroid 6 beta-hydroxylation by microsomes from both untreated and PB-treated dogs. Major species differences were noted, however, in the apparent Km for 6 beta-hydroxylation of androstenedione by liver microsomes from untreated rats (24 microM), humans (380 microM), and untreated dogs (4700 microM).