CYP1A2 activity, gender and smoking, as variables influencing the toxicity of caffeine

We have investigated several factors that might be related to the occurrence of toxic effects during the performance of a urinary test with caffeine (300  mg p.o), in 120 healthy volunteers. A total of 218 toxic effects were self-reported by eighty-two (68%) subjects. Females and nonsmokers were at the highest risk (chi-square test, P =0.01). Furthermore, two nonsmoking females experienced a symptomatology with delirium, restlessness, muscle tremor, vomiting and wakefulness. Among females and nonsmokers, those subjects who experienced toxic effects had lower caffeine N3-demethylation index (CYP1A2 activity) compared with unaffected females (1.87±0.51 vs 1.47±0.27, P <0.0005) and nonsmokers (1.69±0.23 vs 1.49±0.31, P <0.02). Caffeine N1- and N7-demethylations indices were also lower among females ( P <0.0005) and nonsmokers ( P <0.02) who reported toxic symptoms. We conclude that CYP1A2 activity, gender and smoking are variables to be considered as influencing the toxicity of caffeine.     

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.     

Involvement of human cytochrome P450 3A4 in reduced haloperidol oxidation

Objective: The present study was conducted to identify in vitro the cytochrome P450(CYP) isoform involved in the metabolic conversion of reduced haloperidol to haloperidol using microsomes derived from human AHH-1 TK +/− cells expressing human cytochrome P450s. The inhibitory and/or stimulatory effects of reduced haloperidol or haloperidol on CYP2D6-catalyzed carteolol 8-hydroxylase activity were also investigated. Results: The CYP isoform involved in the oxidation of reduced haloperidol to haloperidol was CYP3A4. CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 2E1 were not involved in the oxidation. The k_M value for the CYP3A4 expressed in the cells was 69.7 μmol · l⁻¹, and the V_max was 4.87 pmol · min⁻¹ · pmol⁻¹ P450. Troleandomycin, a relatively selective probe for CYP3A enzymes, inhibited the CYP3A4-mediated oxidation of reduced haloperidol in a dose-dependent manner. Quinidine and sparteine competitively inhibited the oxidative reaction with a k_i value of 24.9 and 1390 μmol · l⁻¹, respectively. Carteolol 8-hydroxylase activity, which is a selective reaction probe for CYP2D6 activity, was inhibited by reduced haloperidol with a k_i value of 4.3 μmol · l⁻¹. Haloperidol stimulated the CYP2D6-mediated carteolol 8-hydroxylase activity with an optimum concentration of 1 μmol · l⁻¹, whereas higher concentrations of the compound (>10 μmol · l⁻¹) inhibited the hydroxylase activity. Conclusion: It was concluded that CYP3A4, not CYP2D6, is the principal isoform of cytochrome P450 involved in the metabolic conversion of reduced haloperidol to haloperidol. It was further found that reduced haloperidol is a substrate of CYP3A4 and an inhibitor of CYP2D6, and that haloperidol has both stimulatory and inhibitory effects on CYP2D6 activity. Received: 10 April 1997 / Accepted in revised form: 16 December 1997     

Restriction fragment length polymorphisms of the CYP11B1 gene in the Japanese population

Summary Restriction fragment length polymorphisms (RFLPs) of the CYP11B1 gene were studied in Japanese using cDNA clone P450c11 as a probe. Genomic DNAs from 60 unrelated Japanese individuals were digested with 8 different restriction enzymes and analyzed by Southern blot hybridization. Two RFLPs were detected inMspI digests of the DNA. One(A) was characterized by polymorphic bands at 3.4 and 2.5 kilobasepairs (kb) and the other (B) by polymorphic bands at 1.7 and 1.2 kb. The third RFLP was observed inPvuII-digested samples and was polymorphic at 5.8 and 4.0 kb bands. Two of the three RFLPs found, RFLP (A) and (C), have not been described in the only previous report which was based on Caucasian samples. We also examined the RFLPs of a 3 generation family of 11-hydroxylase deficiency caused by an abnormality of the CYP11B1 gene. All the family members were homozygous in all three RFLPs and was thus not informative.     

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CYP3A4第9外显子基因突变频率的检测

目的建立一种简便、准确、实用的人CYP3A4第9外显子基因突变频率的检测方法，并了解汉族人CYP3A4第9外显子的分布特点．方法应用聚合酶链反应（PCR）特异性扩增人CYP3A4第9外显子基因序列，扩增产物用限制性内切酶Hinf Ⅰ酶切，琼脂糖凝胶电泳后，观察酶切位点的限制性片段长度多态性（RFLP）图谱．结果运用PCR—RFLP法检测了92名汉族人CYP3A4第9外显子基因点突变，其中野生型纯合子频率为85．9％，杂合子频率为14．1％，突变型纯合子频率为O．突变等位基因频率为0．0706．结论该方法简便、快速、准确，适合于一般实验室检测及大规模的人群调查，汉族人CYP3A4第9外显子也存在相同的突变位点．