Alteration of cytochrome P-450 by prolonged administration of imipramine and/or lithium to rats

Summary The aim of this study was to investigate imipramine-induced alterations of cytochrome P-450 and to determine whether prolonged concomitant administration of imipramine and lithium results in a pharmacokinetic interaction.Male Wistar rats received imipramine (10 mg/kg i. p.) at 12 h intervals or lithium chloride (100 mg/kg in drinking water) or they were treated with the combination of these drugs for 2 weeks. The long term treatment with imipramine produced a very complex alteration of cytochrome P-450: imipramine increased the level of the cytochrome, but it decreased the rate of its own aromatic hydroxylation in position 2. The rate of N-demethylation in the side chain was not changed. Consequently, in the case of both hydroxylation and demethylation, calculated molecular activities were decreased to 48% and 70% respectively. This differential change in activities corresponded well to the observed decrease of absorption in difference spectra (type I) produced in microsomes by imipramine. Carbamazepine-induced type I difference spectra were also decreased by imipramine pretreatment, but to a lesser extent. In contrast, hexobarbital type I binding was increased by imipramine treatment while type II difference spectra produced by metyrapone were not affected. The preliminary SDS-PAGE analysis of cytochrome P-450 isoenzymes of control and imipramine treated rats showed that the investigated antidepressant markedly intensified a protein band at 50.11 kD while bands at 51.28 kD, 56.20 kD and 56.88 kD were less intensive. These results indicate that the alteration of cytochrome P-450 by imipramine treatment is not only of quantitative but also of qualitative character. Lithium alone given to rats affected neither the concentration of cytochrome P-450 in microsomal protein nor the rate of imipramine metabolism in vitro. Lithium given jointly with imipramine reduced imipramine-induced elevation of cytochrome P-450. This, however, did not cause any change in the rate of imipramine metabolism in vitro and accordingly in imipramine pharmacokinetics in vivo. The concentration of lithium in the blood plasma tended to increase by concurrent administration of imipramine.Send offprint requests to K. J. Netter at the above address     

Synthesis and Evaluation of Azole-Substituted Tetrahydronaphthalenes as Inhibitors of P450 arom,P450 17, and P450 TxA2

In search of potential drugs for the treatment of estrogen- and androgen-dependent cancer as well as the prophylaxis of metastases, tetralones, tetralins, and dihydronaphthalenes bearing a OCH₃ substituent at the benzene nucleus and an imidazol-4-yl, imidazol-1-yl, or 1,2,4-triazol-1-yl substituent in 2-position were synthesized with and without C₁-spacer between the rings (compounds 2 – 26 ). The compounds were tested in vitro for inhibition of the three target enzymes P450 arom (human placental microsomes), P450 17 (rat testicular microsomes), and P450 TxA₂ (citrated human whole blood). To examine selectivity, some compounds were further tested in vitro for inhibition of P450 18 (bovine adrenal mitochondria), P450 see (bovine adrenal mitochondria) and corticoid formation (aldosterone, corticosterone; ACTH stimulated rat adrenal tissue). In vivo, selected compounds were examined in Sprague Dawley rats regarding P450 TxA₂ inhibition, reduction of plasma testosterone concentration, antiuterotrophic activity (inhibition of the uterotrophic activity of androstenedione), reduction of plasma estradiol concentration (pregnant mares' serum gonadotropin-primed rats), and mammary tumor inhibiting activity (dimethylbenzanthracene-induced tumor; pre- and postmenopausal model). In the series of imidazol-4-yl compounds, which represent a novelty in the field of azole inhibitors of steroidogenic P450 enzymes, strong inhibitors of P450 arom and/or P450 17 were found: 7-OCH₃-2-(imidazol-4-ylmethylene)-1-tetralone ( 4 ) and 7-OCH₃-2-(imidazol-4-ylmethyl)-tetralin ( 12 ) are among the most potent inhibitors of P450 arom in vitro known so far. Compound 4 is a selective inhibitor, whereas 12 shows in addition strong inhibition of P450 17. In contrast to 12 , the 6-OCH₃ derivative (compound 11 ) is a selective inhibitor of P450 17, being 50 times more potent than ketoconazole. Some imidazol-1-yl compounds show a marked inhibition of P450 TxA₂: 2-(imidazol-1-ylmethyl)-1-tetralone ( 13 ) is a selective inhibitor of P450 TxA₂, whereas 7-OCH₃-2-(imidazol-1-ylmethyl)-tetralin ( 17 ) as well as 2-(imidazol-1-ylmethyl)-tetralin ( 16 ) and 7-OCH₃-2-imidazol-1-yl-3,4-dihydronaphthalene ( 25 ) additionally show strong inhibition of P450 arom and P450 17. Regarding the other steroidogenic P450 enzymes as well as corticosterone formation, the compounds show only little inhibitory activity. Aldosterone formation, however, is inhibited at low concentrations. Nevertheless, 4 and 12 are more selective, i.e. inhibit aldosterone synthesis less than the well known inhibitor of P450 arom fadrozole. The compounds show activity in the aforementioned in vivo tests.     

A rapid and sensitive method for measuring monooxygenase activities in hepatocytes cultured in 96-well plates

Summary Measurement of biotransformation activities in cells is of great importance for drug metabolism and toxicologic studies. It is currently done by measuring the enzymatic activities in partially purified microsomes. In the present work we report on a rapid, easy, sensitive, and reproducible fluorimetric assay for quantifying cytochrome P450-dependent monooxygenase activities (P450IA1, P450IIB1) in hepatocytes cultured in 96-well plates. The procedure involves the direct determination of enzymatic activities in intact hepatocytes while avoiding cell homogenization, thereby permitting use of a the reduced number of cells and allowing cultured cells to be used in later experiments. Substrates (7-ethoxyresorufin, 7-pentoxyresorufin) are added to culture medium and metabolized by hepatocytes. After enzymatic deconjugation, the fluorescent resorufin present in culture medium is quantified by means of a microplate fluorimetric reader. Major advantages of this technique, as compared to other available methods, are: a) no cell disruption is required; b) activity can be measured with a very small number of cells; c) rapid processing time; and d) possibility of performing repeated assays with the same cell monolayer.     

Proline reduces brain cytochrome c oxidase: prevention by antioxidants

In the present study, we initially investigated the in vivo (acute and chronic) and in vitro effects of proline on cytochrome c oxidase (complex IV) activity in rat cerebral cortex to test the hypothesis that proline might alter energy metabolism and that this alteration could be provoked by oxidative stress. The action of alpha-tocopherol and ascorbic acid on the effects produced by proline was also evaluated. For acute administration, 29- and 60-day-old rats received one subcutaneous injection of proline (18.2 micromol/g body weight) or an equivalent volume of 0.9% saline solution (control) and were sacrificed 1h later. For chronic treatment, proline was injected subcutaneously twice a day at 10h intervals from the 6(th) to the 28(th) day of age. Rats were sacrificed 12h (29(th)) or 31 days (60(th)) after the last injection. Results showed that acute administration of proline significantly diminished the activity of cytochrome c oxidase in the cerebral cortex of 29- and 60-day-old rats. On the other hand, chronic hyperprolinemia reduced this complex activity only on day 29, but not on the 60(th) day of life. In another set of experiments, 22-day-old rats or 53-day-old rats were pretreated for 1 week with daily intraperitoneal administration of alpha-tocopherol (40 mg/kg) and ascorbic acid (100mg/kg) or saline. Twelve hours after the last antioxidant injection, rats received a single injection of proline or saline and were killed 1h later. In parallel to chronic treatment, rats received a daily intraperitoneal injection of alpha-tocopherol and ascorbic acid from the 6(th) to the 28(th) day of life and were killed 12h after the last injection. Results showed that the pretreatment with alpha-tocopherol and ascorbic acid before acute proline administration or concomitant to chronic proline administration significantly prevented these effects. We also observed that proline (3.0 microM-1.0 mM) when added to the incubation medium (in vitro studies) did not alter cytochrome c oxidase activity. Data suggest that the inhibitory effect of proline on cytochrome c oxidase activity is possibly associated with oxidative stress and that this parameter may be involved in the brain dysfunction observed in hyperprolinemia.     

Absence of a differential induction of cytochrome P450 2E1 by different alcoholic beverages in rats: implications for the aetiology of human oesophageal cancer

Alcohol is an important risk factor for human oesophageal cancer. There is evidence from epidemiological studies that some specific alcoholic drinks, e.g. Calvados apple brandy, are associated with a greater risk than others. Alcohol induces cytochrome P450 2E1 (CYP2E1) and the hypothesis was tested that different alcoholic beverages, containing a variety of alcoholic compounds, could differentially induce expression of cytochrome P450 enzymes. Twelve groups of five rats each were treated for 3 days with different alcoholic beverages (ethanol alone, whisky, farm-produced or commercial Calvados brandy, beer, cider, wine) adjusted to 4, 10 or 20% of ethanol in drinking water. Immunoblotting using a monoclonal antibody specific for rat CYP2E1 revealed a single protein band in liver microsomes. Densitometric quantitation of microsomal proteins demonstrated a significant two-, three- and sixfold increase in band intensity after treatment with ethanol concentrations of 4, 10 and 20% respectively, compared to control rats drinking water alone. There was a dose-dependent increase in liver microsomal metabolism of CYP2E1 substrates (para-nitrophenol and dimethylnitrosamine) in ethanol-treated rats. However, there were no significant differences in the level of CYP2E1 protein or enzymatic activity between the different alcoholic beverages at the same ethanol concentration. There was a slight increase in hepatic CYP1A-related enzymatic activities in the alcohol-treated rats compared to the controls, but no difference between the treated groups either with dose of ethanol or type of beverage. These data show that induction of CYP2E1 with acute alcohol treatment is predominantly determined by the ethanol content of the beverage. Received: 10 February 1997 / Accepted: 26 May 1997     

Integration of new regulatory strategies into the network of an endocrine control system: limitation of androgen secretion by rat testis is achieved by substrate-dependent modulation of P450XVII enzyme concentration and catalytic efficiency.

In addition to the well-known control circuits involved in the regulation and adaptation of testicular androgen biosynthesis, it is proposed that two new control strategies are involved in the maintenance of steady-state testosterone secretion rates by testicular Leydig cells. Cytochrome P450XVII (steroid-17 alpha-monooxygenase/steroid-17,20-lyase), one key enzyme in steroid hormone biosynthesis, responds to external human choriogonadotropin stimulation with an oxygen-dependent and substrate flux-dependent inactivation and decomposition, and increased substrate availability decreases the efficiency of androgen formation in favour of abortive intermediate leakage. These results are discussed as a paradigm of substrate-dependent modulation of cytochrome P450 activities.     

细胞色素P450调节剂对DNA加合物形成的影响

人羊膜上皮细胞ＦＬ系分别接触ａ－萘黄酮（０．６ｍｍｏｌ·Ｌ￣（－１））β－萘黄酮（２０ｐｍｏｌ·Ｌ￣（－１））２４ｈ后，再用苯并（ａ）芘［Ｂ（ａ）Ｐ，１０ｕｍｏｌ·Ｌ￣（－１）］处理２４ｈ，用３２Ｐ后标记技术测定以Ｂ（ａ）－ＤＮＡ加合物。结果发现，阳性对照组，ａ－萘黄酮预处理组及β－萘黄酮预处理组加合物的量分别为（加合物个数／１０’个核苷酸）：４．７±０．２（１００％），１．８±０．９（３８．３％），１６．０±２．２（３４０．１％）．该实验结果直接显示了纳胞色素Ｐ４５０调节剂对肿瘤发生影响的作用水平。亦为药物对致癌物代谢影响的研究提供了一种方法．     

Altered enzyme activities of xenobiotic biotransformation in kidneys after subchronic administration of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) to rats

Activities of the xenobiotic metabolizing enzymes were measured in the liver, kidney, duodenum and lung microsomes and cytosol fractions of Wistar rats after subchronic administration of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a potent bacterial mutagen in chlorinated drinking water. MX was administered by gavage at the dose level of 30 mg/kg for 18 weeks (low dose), or at the dose level which was raised gradually from 45 mg/kg for 7 weeks via 60 mg/kg for 2 weeks to a clearly toxic dose of 75 mg/kg for 5 weeks (high dose). Microsomal and cytosolic preparations were made and the activities of 7-ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-dealkylase (PROD), NADPH-cytochrome-c-reductase, UDP-glucuronosyltransferase (UDPGT) and glutathione-S-transferase (GST) were measured. Kidneys were affected most. A dose-dependent decrease was observed in EROD (90% in males, 80% in females at the high dose) and in PROD (58% in females, at the high dose) in kidneys. An increase was, however, detected in kidney NADPH-cytochrome-c-reductase (66% in females at high dose), UDPGT (89% in males and 97% in females at high dose) and GST activities (56% in males and 50% in females at high dose). MX caused only a few changes in the enzyme activities of the liver. The EROD activity was decreased 25% to 37%, both in the livers of males and females, but the total content of P450s was not altered. Hepatic GST activity was elevated in females in a dose-dependent manner (31% and 44%). GST activity was elevated in duodenum in females (59%) at the high dose. There were no marked changes in the enzyme activities in the lungs. MX was a weak inhibitor of EROD activity both in the liver and kidney microsomes in vitro, decreasing the EROD activity by 53% and 43%, respectively at the concentration of 0.9 mM. The results indicate that MX decreases the activity of phase I metabolism enzymes, but induces phase II conjugation enzyme activities, particularly in kidneys in vivo. It is possible that these changes contribute to metabolism of MX in kidneys and renders them susceptible to MX in the course of repeated exposure.     

利培酮治疗与细胞色素P4502D6/C188T酶基因多态性的关联分析

目的　研究利培酮临床效应的个体差异与其代谢酶细胞色素P4 5 0 2D6 (cytochromeP4 5 02D6 ,CYP2D6 )酶基因多态性的相关性。方法　对 88例符合CCMD 3精神分裂症诊断标准的患者和 96例健康对照者作病例 -对照分析。精神分裂症患者给予利培酮治疗 8周 ,用阳性和阴性症状量表 (posi tiveandnegativesymptomscale ,PANSS)评分评价利培酮疗效。采用聚合酶链反应扩增及限制性片段长度多态性 (PCR RFLP)技术对CYP2D6exonⅠ的C188T位点突变进行检测 ,分析利培酮临床效应与其主要代谢酶CYP2D6 /C188T酶基因多态性的相关性。结果　中国上海地区人群的CYP2D6 /C188T突变率(弱代谢型 )为 36 .3% ,病例组和正常对照组间基因型频率总体分布比较无显著差异 (χ2 =1.15 ,df=2 ,P >0 .0 5 ) ,两组间的等位基因频率之间比较也无显著性差异 (χ2 =0 .78,df=1,P >0 .0 5 )。进行性别及有否家族史分组后分析 ,亦无差异存在 ,且CYP2D6 /C188T突变与利培酮临床效应之间并无相关性 (χ2 =1.12 ,df=2 ;χ2 =0 .0 3,df=1,P >0 .0 5 )。结论　未发现中国人CYP2D6 /C188T多态性与利培酮临床效应的个体差异有相关性。