Ethanol-Induced Enhancement of Cocaine Bioactivation and Irreversible Protein Binding: Evidence Against a Role of Cytochrome P-450IIE1

Chronic ethanol consumption potentiates cocaine-induced liver injury in rodents. Since cocaine has to be bioactivated by a cytochrome P-450-dependent N-oxidative pathway to exert its hepatotoxic effects, we studied the role of the ethanol-inducible P-450IIE1 for cocaine metabolism. Male Sprague-Dawley rats were pretreated with either a liquid diet containing ethanol (30% of calories) for 4 weeks or injected with pyrazole (200 mg/kg/day, ip, for 3 days). Both agents induced microsomal p-nitrophenol hydroxylation which is a probe for the catalytic activity of P-450IIE1. However, only ethanol, but not pyrazole, increased both microsomal cocaine N-demethylase activity (by 47%) and the extent of irreversible binding of [3H]-cocaine to microsomal proteins (by 100%), which was taken as a quantitative endpoint for the formation of a reactive metabolite. Cocaine N-demethylation and irreversible protein binding of cocaine were not inhibited by P-450IIE1 isozyme-selective substrates, nor was the rate of cocaine metabolism and binding decreased by functionally active polyclonal anti-rat P-450IIE1 antibodies. Furthermore, pyrazole pretreatment sensitized cultured hepatocytes to the glutathione-dependent cytotoxic effects of nontoxic concentrations of cocaine. These results indicate that (a) cocaine is not a major substrate for the ethanol-inducible P-450IIE1, (b) the enhancing effects of ethanol on cocaine bioactivation may be due to induction of other P-450 isoforms, and (c) induction of P-450IIE1 may potentiate cocaine-induced hepatocellular toxicity in vitro independently of cocaine metabolism, e.g., by P-450IIE1-dependent oxidative stress.     

Effect of ketoconazole and terbinafine on the pharmacokinetics of caffeine in healthy volunteers

The effects of single oral doses of ketoconazole 400 mg and terbinafine 500 mg on the hepatic microsomal system have been investigated in 8 healthy male volunteers. Microsomal activity caffeine was assessed by following the metabolism of 3 mg/kg bodyweight i.v. administered 1 h after the drug. The inhibitory effect of terbinafine was more pronounced than that of ketoconazole: clearance was decreased from 1.34 ml.kg-1.min-1 in controls to 1.06 and 1.21 ml.kg-1.min-1, respectively, and the corresponding half-life was increased from 5.8 h in controls to 7.6 and 6.7 h, respectively. The apparent volume of distribution remained unchanged. The serum levels of the antimycotics were within the therapeutic range in each subject. Although all three substances are metabolised by microsomes, the kinetic parameters (Cmax, half-life, elimination constant) of the antimycotics were poorly if at all correlated with the elimination of caffeine.     

Identification of N2 as a metabolite of acetylhydrazine in the rat

 In the pathogenesis of isoniazid-induced hepatic injury, cytochrome P450-dependent metabolic activation of the metabolite, acetylhydrazine (AcHz), is the crucial step. Exhalation of [¹⁴C]-carbon dioxide has previously been used to quantify indirectly this pathway. In contrast, according to the current concept of AcHz bioactivation, molecular nitrogen is produced directly, but has not yet been identified. Here, we measured [¹⁵N]-nitrogen and ¹⁴CO₂ exhalation, after the administration of [¹⁵N₂]-[¹⁴C]-AcHz, in rats. Laser magnetic resonance (LMR) spectroscopy, a new sensitive and specific technique for the measurement of ¹⁵N and ¹⁴N in gas samples, was used. To demonstrate the involvement of cytochrome P450, rats were treated with phenobarbital (PB) or PB + cobalt(II) chloride (CoCl₂) (n=3 in each group). Time-dependent ¹⁵N₂ exhalation differed significantly between treatment groups (p<0.001). At 240 min, cumulative exhalation of ¹⁵N was 1.92±0.43% (mean±SE) of the dose in the control group, 2.53±0.23% in the PB group, and 1.00±0.15% in the PB+CoCl₂ group (p<0.05 compared to controls, p<0.01 compared to PB). Cumulative exhalation of ¹⁴CO₂ in 24 h ranged from 15.1 to 21.9%, with no significant difference between treatment groups. In conclusion, N₂ is a metabolite of AcHz. N₂ formation reflects the cytochrome P450-mediated activation of AcHz and can be used as an index of this pathway. Generally, LMR spectroscopy is valuable for monitoring any N₂-liberating process in vivo. Received: 14 March 1995/Accepted: 15 August 1995     

Formation of pyridinium species of haloperidol in human liver and brain

Recent interest in the neurotoxicity of haloperidol is based on its oxidation in rodents to the pyridinium derivative, HPP⁺, a structural analog of the neurotoxin, 1-methyl-4-phenylpyridinium (MPP⁺). Recently, we reported that HPP⁺ and a newly identified reduced pyridinium, RHPP⁺, were present in blood and urine of haloperidol-treated schizophrenics and that the concentrations of RHPP⁺ exceeded those of HPP⁺. In this study, we examined pathways for formation of RHPP⁺ in subcellular fractions of human liver (n=5) and brain (basal ganglia;n=5). The major pathway was reduction of HPP⁺ (20 µM) to RHPP⁺ in cytosol (0.17–0.39 and 0.03–0.07 µM RHPP⁺/g cytosolic protein per h in liver and brain, respectively). The reactions were inhibited significantly by menadione and in brain also by daunorubicin. The inhibition profile, cytosolic location and strict NADPH dependence suggest that the enzymes involved are ketone reductases. A second pathway was oxidation of reduced haloperidol (50 µM), a major metabolite of haloperidol in blood and brain, to RHPP⁺. In liver microsomes, 0.17–0.63 µmol RHPP⁺ was formed /g microsomal protein per h. A potent inhibitor of the pathway was ketoconazole (IC₅₀, 0.8 µM), which suggests that P-450 3A isozymes could be involved. In brain mitochondria but not microsomes, reduced haloperidol (120 µM) was oxidised to RHPP⁺ at a small but significant rate (0.005–0.020 µmol RHPP⁺/g mitochondrial protein per h) which was not attenuated by SKF 525A, quinidine, ketoconazole, or monoamine oxidase inhibitors. Further studies are warranted to establish the biological importance of these metabolites in vivo.     

中国汉族雄激素过多症女性21-羟化酶缺陷症携带者基因检测

目的　初步了解中国汉族女性雄激素过多症患者中2 1-羟化酶缺陷症(2 1- hydroxylasedeficiency,2 1- OHD)携带者发生率,探讨促肾上腺皮质激素(adrenocorticotropic hormone,ACTH)兴奋试验结果与基因突变检测结果的相关性。方法　82例汉族女性雄激素过多症患者及14名健康女性进行ACTH兴奋试验,并应用PCR扩增产生限制性酶切位点方法检测已知的9个2 1- OHD常见突变位点。结果　雄激素过多组(n=82 ) F0 显著高于正常对照组(P<0 .0 1) ;17- OHP0 及17- OHP6 0 也显著高于对照组(P<0 .0 1) ,而F6 0 差异没有统计学意义(P>0 .0 5 )。比较17- OHP净增值及17- OHP净增值/ F净增值,雄激素过多组也均显著高于正常对照组(P<0 .0 1)。正常对照组未检测出细胞色素P4 5 0 (cytochrome P4 5 02 1,CYP2 1)基因突变。发现雄激素过多组4例CYP2 1基因突变携带者(4/ 82 ,4 .9% ) ,分别携带V2 81L(2例) ,i2 g及Q318X(各1例) ,携带者的ACTH兴奋试验结果与正常对照及未检出突变的雄激素过多症患者的结果存在一定的交叉。结论　82例汉族雄激素过多症女性中2 1- OHD携带者为4例,占4 .9%。ACTH兴奋试验不能用以发现携带者,应进行基因检测确定。     

The effect of ethanol-induced CYP2E1 on proteasome activity: the role of 4-hydroxynonenal

Previous studies have shown that the induction of P450 cytochrome 2E1 (CYP2E1) is associated with the loss of proteasomal activities. To correlate the loss of proteasomal activity with CYP2E1 induction, ethanol was fed intragastrically for 1, 3, 7, and 15 days. The maximum induction of CYP2E1 (3.5-fold) occurred after 15 days of ethanol feeding. However, there was no significant decrease in the 26 S chymotrypsin-like and trypsin-like activity over this period of time. When ethanol was given to rats for 1 month, CYP2E1 was significantly induced, and the proteasomal activity was significantly decreased. These results indicate that proteasomal activity was not directly affected by ethanol or CYP2E1 induction. Since 4-hydroxynonenal (4-HNE) concentration was significantly increased at 1 month of ethanol feeding, it was suspected that 4-HNE adduct formation with proteasome subunits could be the mechanism of proteasome inhibition. Using an antibody to 4-HNE adducted proteins in Western blot analysis of the 26 S proteasome fraction isolated from the liver of alcohol fed rats, one extra band appeared around 44 kDa. When the antibody to an ATPase Rpt4 was used to stain the stripped membrane, the same band that was detected with the 4-HNE antibody was detected with the Rpt4 antibody. An adduct of 4-HNE formed with the Rpt4 subunit of 26 S could impede the association of 19 S and 20 S and thus account for the observed decrease of proteasomal activity.     

中国广东人CYP2F1基因遗传多态性研究

目的检测广东地区正常人群和鼻咽癌患者中细胞色素P450酶系CYP2F1基因的多态性，并分析该基因遗传多态性与鼻咽癌易感性的关联。方法采用直接测序法检测40例鼻咽癌患者全血标本中CYP2F1基因全部10个外显子的多态性变化。对于等位基因频率较高的多态性位点，进一步采用错配聚合酶链反应．限制性片段长度多态性检测368例鼻咽癌患者和344名正常对照人群中该位点的等位基因频率。结果在40例鼻咽癌样本中，共检测到CYP2F1基因的35个单核苷酸多态性。其中，10个单核甘酸引起编码的氨基酸改变，1个移码突变，15～16bp之间插入C引起移码突变（15-16ins C），该等位基因频率为25％。但病例-对照分析却未能显示该位点突变与鼻咽癌易感的相关性（P〉0．05）。结论中国广东人的CYP2F1基因遗传多态性位点较多，但暂未发现与鼻咽癌的易感性关联的单一多态位点，多个多态性位点或不同基因多态性位点的协同互补作用可能才是鼻咽癌发生发展的关键影响因素。     

Study of CYP2D6 gene in children with autoimmune hepatitis and P450 IID6 autoantibodies.

Cytochrome P450 IID6 is an autoantigen recognized by the sera of children affected with a subtype of autoimmune hepatitis. It was hypothesized that a mutation in the CYP2D6 gene could explain the autoimmune response in these patients. To examine this question, genomic DNA from peripheral lymphocytes (n = 9) and liver (n = 1) of 10 patients with anti-LKM-1 antibody was analysed by Southern blot for genetic association studies between a particular CYP2D6 haplotype and autoimmune hepatitis. In addition, a region of CYP2D6, from the same genomic DNA, was amplified by polymerase chain reaction (PCR) and digested by BstNI, in a search for the most prevalent 29B mutation, described in subjects who do not express the P450 IID6. Total RNA and proteins, prepared from the liver of an anti-LKM-1+ patient, were analysed by Northern and Western (immunoblot) blots respectively. Our results do not reveal any major structural change in the DNA of this patient at the CYP2D6 locus that could explain their autoimmune response. Corroborating this observation, no changes were noted either in P450 IID6 mRNA size or in the corresponding protein. However, these data do not exclude the possibility of subtle changes in the protein due to point mutations in critical regions that might trigger an autoimmune response.     

人细胞色素P450 前mRNA的可变剪接研究进展

Human genes typically contain multiple introns, and in many cases the exons can be joined more than one way to generate multiple rnRNAs, encoding distinct protein isoforms. This process is called alternative splicing. The article summarized the human cytochrome P450 pre-mRNA alternative splicing and their regulatory mechanism and impacts on biological functions.