Lysosomal trapping of 4-dimethylamino-1-{3-(1-methyl-1H-imidazole-2-yl)propanoyl}piperidine, a hydrophilic and weakly basic amine, in human aortic vascular smooth muscle cells.

Some weakly basic compounds lead to cell death accompanied by cellular vacuolation. The novel analgesic agent, 4-dimethylamino-1-{3-(1-methyl-1H-imidazole-2-yl)propanoyl}piperidine (DMIP), is a hydrophilic and weakly basic compound that induces vacuolation in the vascular smooth muscle cells in dogs. Here, we investigated the vacuolation mechanism and the potential cytotoxicity of DMIP using human aortic vascular smooth muscle cells. When cells were treated with DMIP (0.1, 0.3, and 1 mM) for 6, 24, and 48 h, clear cytoplasmic vacuolation was observed at 1 mM after 24 and 48 h, along with an increase in the intracellular DMIP concentration. The vacuolation and intracellular DMIP were markedly reduced by bafilomycin A1, a vacuolar H+-ATPase inhibitor. The late endosome marker Rab7 and lysosome marker LAMP-2 were highly expressed but the early endosome marker Rab5 and autophagosome marker LC3 were not expressed specifically on the vacuolar membranes. These results suggested that the most vacuoles were enlarged late endosomes/lysosomes, resulting from the accumulation of DMIP by ion trapping. Moreover, DMIP did not affect lysosomal membrane integrity and was less cytotoxic than chloroquine, an inducer of phospholipidosis. The current study provides further insight into the mechanisms of vacuolation and lysosomal trapping induced by the hydrophilic and weakly basic amine DMIP.

Balance dysfunction the most significant cause of in-hospital falls in patients taking hypnotic drugs: A retrospective study.

PURPOSE: Preventing falls in patients is one of the most important concerns in acute hospitals. Balance disorder and hypnotic drugs lead to falls. The Standing Test for Imbalance and Disequilibrium (SIDE) is developed for the evaluation of static standing balance ability. There have been no reports of a comprehensive assessment of falls risk including hypnotic drugs and SIDE. The purpose of this study was to investigate the fall rate of each patient who took the hypnotic drug and the factor associated with falls. METHODS: Fall rates for each hypnotic drug were calculated as follows (number of patients who fell/number of patients prescribed hypnotic drug x 100). We investigated the hypnotic drugs as follows; benzodiazepine drugs, Z-drugs, melatonin receptor agonists, and orexin receptor antagonists. Hypnotic drug fall rate was analyzed using Pearson's chi-square test. Decision tree analysis is the method we used to discover the most influential factors associated with falls. RESULTS: This study included 2840 patients taking hypnotic drugs. Accidents involving falls were reported for 211 of inpatients taking hypnotic drugs. Z-drug recipients had the lowest fall rate among the hypnotic drugs. We analyzed to identify independent factors for falls, a decision tree algorithm was created using two divergence variables. The SIDE levels indicating balance disorder were the initial divergence variable. The rate of falls in patients at SIDE level ≦ 2a was 14.7%. On the other hand, the rate of falls in patients at SIDE level ≧ 2b was 2.9%. Gender was the variable for the second classification. In this analysis, drugs weren't identified as divergence variables for falls. CONCLUSION: The SIDE balance assessment was the initial divergence variable by decision tree analysis. In order to prevent falls, it seems important not only to select appropriate hypnotic drugs but also to assess patients for balance and implement preventive measures.

The impact of sarcopenia on low back pain and quality of life in patients with osteoporosis.

PURPOSE: Osteoporosis combined with sarcopenia contributes to a high risk of falling, fracture, and even mortality. However, sarcopenia's impact on low back pain and quality of life (QOL) in patients with osteoporosis is still unknown. The purpose of this study is to investigate low back pain and QOL in osteoporosis patients with sarcopenia. METHODS: We assessed 100 ambulatory patients who came to our hospital for osteoporosis treatment. Low back pain was evaluated using the Visual Analogue Scale (VAS) with 100 being an extreme amount of pain and 0 no pain. The Japanese Orthopaedic Association Back Pain Evaluation Questionnaire (JOABPEQ) score was used to assess QOL after adjustment for age, history of vertebral fracture, and adult spinal deformity. Differences in low back pain intensity assessed by VAS between groups were evaluated by the Willcoxon rank-sum test. Covariance analysis was used to assess QOL. All data are expressed as either median, interquartile range, or average, standard error. RESULTS: Patients were classified into the sarcopenia group (n = 32) and the non-sarcopenia group (n = 68). Low back pain intensity assessed by VAS was significantly higher in the sarcopenia group than in the non-sarcopenia group (33.0 [0-46.6] vs. 8.5 [0-40.0]; p < 0.05). The subscales of the JOABPEQ for low back pain were significantly lower in the sarcopenia group than in the non-sarcopenia group (65.0 ± 4.63 vs. 84.0 ± 3.1; p < 0.05). CONCLUSION: In this cross-sectional study, sarcopenia affected low back pain and QOL in ambulatory patients with osteoporosis. Sarcopenia may exacerbate low back pain and QOL.

Electrically stimulated eccentric contraction during non-weight bearing knee bending exercise in the supine position increases oxygen uptake: A randomized, controlled, exploratory crossover trial.

It is well known that prolonged bed rest induces muscle weakness, muscle atrophy, cardiovascular deconditioning, bone loss, a loss of functional capacity, and the development of insulin resistance. Neuromuscular electrical stimulation is anticipated to be an interventional strategy for disuse due to bed rest. A hybrid training system (HTS), synchronized neuromuscular electrical stimulation for voluntary exercise using an articular motion sensor, may increase the exercise load though bed rest. We assessed oxygen uptake or heart rate during knee bending exercise in the supine position on a bed both simultaneously combined with HTS and without HTS to evaluate exercise intensity on different days in ten healthy subjects (8 men and 2 women) by a randomized controlled crossover trial. The values of relative oxygen uptake during knee bending exercise with HTS were significantly greater than those during knee bending exercise without HTS (7.29 ± 0.91 ml/kg/min vs. 8.29 ± 1.06 ml/kg/min; p = 0.0115). That increment with HTS was a mean of 14.42 ± 13.99%. Metabolic equivalents during knee bending exercise with HTS and without HTS were 2.08 ± 0.26 and 2.39 ± 0.30, respectively. The values of heart rate during knee bending exercise with HTS were significantly greater than those during knee bending exercise without HTS (80.82 ± 9.19 bpm vs. 86.36 ± 5.50 bpm; p = 0.0153). HTS could increase exercise load during knee bending exercise which is easy to implement on a bed. HTS might be a useful technique as a countermeasure against the disuse due to bed rest, for example during acute care or the quarantine for infection prophylaxis.

Collagen vitrigel promotes hepatocytic differentiation of induced pluripotent stem cells into functional hepatocyte-like cells.

Differentiation of stem cells to hepatocytes provides an unlimited supply of human hepatocytes and therefore has been vigorously studied. However, to date, the stem cell-derived hepatocytes were suggested to be of immature features. To obtain matured hepatocytes from stem cells, we tested the effect of culturing human-induced pluripotent stem (hiPS) cell-derived endoderm cells on collagen vitrigel membrane and compared with our previous reported nanofiber matrix. We cultured hiPS cell-derived endoderm cells on a collagen vitrigel membrane and examined the expression profiles, and tested the activity of metabolic enzymes. Gene expression profile analysis of hepatocytic differentiation markers revealed that upon culture on collagen vitrigel membrane, immature markers of AFP decreased, with a concomitant increase in the expression of mature hepatocyte transcription factors and mature hepatocyte markers such as ALB, ASGR1 Mature markers involved in liver functions, such as transporters, cytochrome P450 enzymes and phase II metabolic enzymes were also upregulated. We observed the upregulation of the liver markers for at least 2 weeks. Gene array profiling analysis revealed that hiPS cell-derived hepatocyte-like cells (hiPS-hep) resemble those of the primary hepatocytes. Functions of the CYP enzyme activities were tested in multi-institution and all revealed high CYP1A, CYP2C19, CYP2D6, CYP3A activity, which could be maintained for at least 2 weeks in culture. Taken together, the present approach identified that collagen vitrigel membrane provides a suitable environment for the generation of hepatocytes from hiPS cells that resemble many characteristics of primary human hepatocytes.

Discovery of Peripheral κ-Opioid Receptor Agonists as Novel Analgesics.

κ-Opioid receptor agonists with high selectivity over the µ-opioid receptor and peripheral selectivity are attractive targets in the development of drugs for pain. We have previously attempted to create novel analgesics with peripheral selective κ-opioid receptor agonist on the basis of TRK-820. In this study, we elucidated the biological properties of 17-hydroxy-cyclopropylmethyl and 10α-hydroxy derivatives. These compounds were found to have better κ-opioid receptor selectivity and peripheral selectivity than TRK-820.

Hepatic microsomal UDP-glucuronosyltransferase (UGT) activities in the microminipig.

The microminipig, a small minipig, was bred as a novel experimental animal for nonclinical pharmacology/toxicology studies by Fuji Micra Inc. (Shizuoka, Japan). Species differences in drug metabolism between humans and the microminipig need to be elucidated in more detail in order to discuss the results of nonclinical studies. Glucuronidation catalysed by UDP-glucuronosyltransferase (UGT) is an important pathway in the metabolism of a wide variety of compounds. The aim of the present study was to identify the characteristics of hepatic UGT activity in the microminipig and compare them with those in humans and other experimental animals. This study examined in vitro UGT activities using liver microsomes from microminipigs (8 months old and 1 day old), humans, mice, rats, dogs, monkeys and minipigs. The glucuronides of estradiol, imipramine, serotonin, propofol, 3'-azido-3'-deoxythymidine (AZT) and morphine, selective substrates of human UGT1A1, 1A4, 1A6, 1A9 and 2B7 (AZT and morphine), respectively, were measured using LC-MS/MS. Estradiol-3-glucuronidation activity was higher in the microminipig than in humans and the other animals. High levels of estradiol-3-glucuronidation were observed in the microsomes of 1-day-old microminipigs. Imipramine-N-glucuronidation, a distinctive conjugation by human UGT1A4, was catalysed by microminipig liver microsomes, but not by dog liver microsomes. Although AZT-glucuronidation activity was low in the microminipig compared with humans, morphine-3-glucuronidation activity in the microminipig was higher than that in humans. The UGT activities in the microminipig were similar to those in the minipig. The results of the present study have provided useful information for selecting an appropriate animal model for nonclinical studies.

Discovery of 1-oxa-4,9-diazaspiro[5.5]undecane-based trisubstituted urea derivatives as highly potent soluble epoxide hydrolase inhibitors and orally active drug candidates for treating of chronic kidney diseases.

We identified 1-oxa-4,9-diazaspiro[5.5]undecane-based trisubstituted ureas as highly potent soluble epoxide hydrolase (sEH) inhibitors and orally active agents for treating chronic kidney diseases. Compound 19 exhibited excellent sEH inhibitory activity and bioavailability. When administered orally at 30 mg/kg, 19 lowered serum creatinine in a rat model of anti-glomerular basement membrane glomerulonephritis but 2,8-diazaspiro[4.5]decane-based trisubstituted ureas did not. These results suggest that 19 is an orally active drug candidate for treating chronic kidney diseases.

Discovery of 2,8-diazaspiro[4.5]decane-based trisubstituted urea derivatives as highly potent soluble epoxide hydrolase inhibitors and orally active drug candidates for treating hypertension.

We identified 2,8-diazaspiro[4.5]decane-based trisubstituted urea derivatives as highly potent soluble epoxide hydrolase (sEH) inhibitors and orally active agents for treating hypertension. Docking studies using human and murine sEH X-ray crystal structures revealed steric hindrance around the side chain of Phe406 of murine sEH. The trifluoromethyl moiety (11) was replaced with a trifluoromethoxy moiety (12) to prevent steric clash, and improved murine sEH inhibitory activity was observed. The oral administration of 12, 20, and 37 at a dose of 30mg/kg reduced blood pressure in spontaneously hypertensive rat, but had little effect on blood pressure in normotensive rat.

Focused DNA microarray analysis for sex-dependent gene expression of drug metabolizing enzymes, transporters and nuclear receptors in rat livers and kidneys.

Cytochrome P450(CYP)s are known to show a sexual dimorphic expression in rat livers. However, the comprehensive analysis for the sex-dependent gene expressions of drug metabolizing enzymes except for CYPs, transporters and nuclear receptors in rat livers and kidneys has not been investigated yet. The purpose of the present study was to identify the novel drug metabolizing and pharmacokinetics (DMPK)-related gene(s) which show the sex difference in the mRNA expressions in rat livers and kidneys. Total RNAs were prepared from livers and kidneys in both male and female rats (Crl:CD(SD) and Crlj:WI). A DNA microarray analysis using a "GeneSQUARE Multiple Assay DNA Microarray Drug Metabolism Gene Expression for Rat" was performed. DMPK-related genes which showed sex differences in the mRNA expression were identified in rat livers or kidneys. Especially, the female dominant expressions of UDP glucuronosyltransferase (UGT) s were seen in rat livers and kidneys. The sex difference of UGT expressions in rats might be one of the causal factors of the sex difference of the biological response to UGT substrates.

Human CYP2A6 is regulated by nuclear factor-erythroid 2 related factor 2.

Human CYP2A6 is responsible for the metabolism of nicotine and coumarin as well as the metabolic activation of tobacco-related nitrosamines. Earlier studies revealed that CYP2A6 activity was increased by dietary cadmium or cruciferous vegetables, but the underlying mechanisms remain to be clarified. In the present study, we investigated the possibility that Nrf2 might be involved in the regulation of CYP2A6. Real-time RT-PCR analysis revealed that the CYP2A6 mRNA level in human hepatocytes was significantly (P<0.01, 1.4-fold) induced by 10µM sulforaphane (SFN), a typical activator of Nrf2. A computer-based search identified three putative antioxidant response elements (AREs) in the 5'-flanking region of the CYP2A6 gene at positions -1212, -2444, and -3441, termed ARE1, ARE2, and ARE3, respectively. Electrophoretic mobility shift assays demonstrated that Nrf2 bound only to ARE1. Luciferase assays using HepG2 cells revealed that the overexpression of Nrf2 significantly increased the reporter activities of the constructs containing a 30-bp fragment that included ARE1. However, the activity of the construct containing the intact 5'-flanking region (-1 to -1395) including ARE1 was not increased by the overexpression of Nrf2. In contrast, when the reporter construct was injected into mice via the tail vein, the reporter activity in the liver was significantly (P<0.05, 1.9-fold) increased by SFN (1mg/head) administration. In conclusion, we found that human CYP2A6 is regulated via Nrf2, suggesting that CYP2A6 is induced under oxidative stress.

Genetic polymorphisms in the 5'-flanking region of human UDP-glucuronosyltransferase 2B7 affect the Nrf2-dependent transcriptional regulation.

OBJECTIVES: Human uridine diphosphate-glucuronosyltransferase 2B7 (UGT2B7) plays important roles in the metabolism of some clinical drugs, carcinogens, and steroid hormones. The molecular mechanisms of the inducible expression of UGT2B7 in response to xenobiotics have not been fully clarified. We sought to investigate whether the UGT2B7 is under the control of NF-E2 p45-related factor 2 (Nrf2), a key transcriptional factor regulating the expression of cytoprotective enzymes. METHODS: HepG2, HuH7, HLE, and Caco-2 cells were treated with sulforaphane (SFN), and the UGT2B7 mRNA levels were determined by real-time reverse transcriptase PCR. These cells were genotyped for the UGT2B7*2 (H268Y) allele using the PCR-restriction fragment length polymorphism method. Luciferase analyses and gel shift analyses were performed to identify the responsive regions for Nrf2 signaling. RESULTS: The UGT2B7 mRNA was induced by SFN in HepG2 and HuH7 genotyped as UGT2B7*1/*1, but not in HLE and Caco-2 cells genotyped as UGT2B7*2/*2. In HepG2 cells, the UGT2B7 protein level and morphine glucuronosyltransferase activity were also significantly induced by SFN. The induction was prominently decreased with small interfering RNA for Nrf2. In the 5'-flanking region (-2.5 kb) of the UGT2B7*2 allele, a 324-base pair insertion at -2067 and 12 single nucleotide polymorphisms simultaneously existed. Luciferase analyses and gel shift analyses revealed that an antioxidant responsive element at -1170 was responsible for the transactivation by Nrf2. In addition, a region from -990 to -858 on the UGT2B7*1 allele was also responsible for the transactivation by Nrf2. Abrogation of the Nrf2-dependent transactivation of the UGT2B7*2 allele was owing to the single nucleotide polymorphism -900A>G. CONCLUSION: UGT2B7 is transcriptionally regulated by Nrf2, but the mechanism is hindered by polymorphisms in the promoter region of UGT2B7*2. The allele-specific mechanism may cause variability of the glucuronidation in response to oxidative stress.

Human CYP2A6 is induced by estrogen via estrogen receptor.

Human CYP2A6, which is predominantly expressed in liver, is a key enzyme responsible for the metabolism of nicotine, coumarin, and some pharmaceutical drugs. CYP2A6 is also expressed in sex steroid-responsive tissues such as breast, ovary, uterus, testis, and adrenal grand. In this study, we examined the regulation of CYP2A6 gene by estrogen. Reverse transcription-polymerase chain reaction (RT-PCR) assays revealed that CYP2A6 mRNA was induced by estradiol in estrogen receptor (ER)-positive MCF-7 (2.9-fold) and HepG2 (1.3-fold) cells, but not in ER-negative MDA-MB-435 cells. Real-time RT-PCR assays revealed the CYP2A6 induction by estradiol in human hepatocytes (1.2- to 1.5-fold). Computer-assisted homology search identified a putative estrogen response element (ERE) at -2436 on the CYP2A6 gene. Electrophoretic mobility shift assays demonstrated specific binding of ERalpha to this element. Luciferase assays using MCF-7 cells revealed that the transcriptional activity of the CYP2A6 promoter was significantly activated by estradiol in an ERalpha-dependent manner, in which ERE was responsible for the activation. Chromatin immunoprecipitation assays verified the in vivo association of ERalpha with the ERE on the CYP2A6 gene. Immunohistochemical analyses using human endometrial tissues indicated that the CYP2A6 protein level in glandular cells was significantly higher in the proliferative phase than in the secretory phase, concomitant with local estrogen secretion during the menstrual cycle. These findings clearly demonstrated that CYP2A6 is directly induced by estrogen in an ERalpha-dependent manner, implying a biological role of CYP2A6 in estrogen-responsive tissues. Furthermore, this mechanism can also explain clinical aspects of increased nicotine metabolism under estrogen-rich environments.

Inhibitory effects of neurotransmitters and steroids on human CYP2A6.

Human CYP2A6 catalyzes the metabolism of nicotine, cotinine, and coumarin as well as some pharmaceutical drugs. CYP2A6 is highly expressed in liver and, also, in brain and steroid-related tissues. In this study, we investigated the inhibitory effects of neurotransmitters and steroid hormones on CYP2A6 activity. We found that coumarin 7-hydroxylation and cotinine 3'-hydroxylation by recombinant CYP2A6 expressed in baculovirus-infected insect cells were competitively inhibited by tryptamine (both K(i) = 0.2 microM), serotonin (K(i) = 252 microM and 167 microM), dopamine (K(i) = 49 microM and 22 microM), and histamine (K(i) = 428 microM and 359 microM). Cotinine formation from nicotine was inhibited by tryptamine (K(i) = 0.7 microM, competitive), serotonin (K(i) = 272 microM, noncompetitive), dopamine, noradrenaline, and adrenaline (K(i) = 11 microM, 54 microM, and 81 microM, uncompetitive). Estrogens (K(i) = 0.6-3.8 microM), androgens (K(i) = 60-149 microM), and corticosterone (K(i) = 36 microM) also inhibited cotinine formation, but coumarin 7-hydroxylation and cotinine 3'-hydroxylation did not. Nicotine-Delta(5'(1'))-iminium ion formation from nicotine was not affected by these steroid hormones, indicating that the inhibition of cotinine formation was due to the inhibitory effects on aldehyde oxidase. The nicotine-Delta(5'(1'))-iminium ion formation was competitively inhibited by tryptamine (K(i) = 0.3 microM), serotonin (K(i) = 316 microM), dopamine (K(i) = 66 microM), and histamine (K(i) = 209 microM). Thus, we found that some neurotransmitters inhibit CYP2A6 activity, being related with inter- and intraindividual differences in CYP2A6-dependent metabolism. The inhibitory effects of steroid hormones on aldehyde oxidase may also contribute to interindividual differences in nicotine metabolism.

Comprehensive evaluation of variability in nicotine metabolism and CYP2A6 polymorphic alleles in four ethnic populations.

Human cytochrome P450 (CYP) 2A6 metabolizes nicotine to cotinine and is a possible modulator of nicotine addiction. Quantitative and qualitative differences in nicotine addiction have been observed between ethnic groups. However, there are few data on the ethnic influences of the CYP2A6-nicotine metabolism relationship, particularly with regard to black subjects. We determined the nicotine metabolism and CYP2A6 genotype in 176 white subjects and 160 black subjects, comparing them with our previous data from 209 Korean subjects and 92 Japanese subjects. Large interindividual differences were observed in the cotinine/nicotine ratios in plasma calculated as an index of nicotine metabolism in white subjects (range, 0.6-36.5) and in black subjects (range, 0.9-30.4). No ethnic difference in the metabolic ratio was observed among white subjects (mean, 7.2 +/- 5.0), black subjects (mean, 7.1 +/- 4.7), and Korean subjects (mean, 8.7 +/- 11.9), whereas Japanese subjects showed a significantly (P < .005) lower metabolic ratio (mean, 3.8 +/- 3.1) compared with the other populations. Women showed significantly (P < .05) higher metabolic ratios than men in the black population (8.0 +/- 5.3 versus 6.0 +/- 3.7). Obvious ethnic differences in the CYP2A6 alleles were observed among these 4 populations. The combined frequencies of the alleles lacking or showing reduced enzymatic activity (CYP2A6*2, CYP2A6*4, CYP2A6*5, CYP2A6*7, CYP2A6*9, CYP2A6*10, CYP2A6*11, CYP2A6*17, CYP2A6*19, and CYP2A6*20) were 9.1%, 21.9%, 42.9%, and 50.5% in white, black, Korean, and Japanese subjects, respectively. These CYP2A6 alleles were associated with reduced nicotine metabolism. Among the homozygotes of CYP2A6*1, interindividual and ethnic differences in the metabolic ratio were still observed. Thus some factors other than genetic ones might also contribute to the interindividual and ethnic differences. This comprehensive study of 4 populations extends our understanding of nicotine metabolism and the impact of genetic polymorphisms of the CYP2A6 gene.

Induction of human CYP2A6 is mediated by the pregnane X receptor with peroxisome proliferator-activated receptor-gamma coactivator 1alpha.

CYP2A6 plays important roles in the metabolism of nicotine and some clinically used drugs. Interindividual variability in the CYP2A6 expression level in human liver might be caused by an inducible property, but the molecular mechanism of induction is unclear. Rifampicin, phenobarbital, and 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime, which are activators of pregnane X receptor (PXR) and constitutive androstane receptor (CAR), induced CYP2A6 mRNA in human hepatocytes. We identified three direct repeat separated by four nucleotides (DR4)-like elements at -6698, -5476, and -4618 in the CYP2A6 gene, to which PXR and CAR could bind after dimerization with retinoid X receptor (RXR)-alpha. In luciferase assays, overexpression of PXR or CAR could not activate the transcriptional activity of CYP2A6 promoter constructs (-6754 to -1) in HepG2 cells. Cotransfection of hepatocyte nuclear factor-4alpha did not affect the transcriptional activities in the absence or presence of PXR or CAR. Interestingly, cotransfection of peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha) as well as PXR significantly enhanced the transcriptional activity (3.9-fold of control). By the deletion of a possible suppresser region (-4533 to -185), the effects of PXR/PGC-1alpha on the transcriptional activity were increased (6.9-fold of control). Deletion or mutation analyses revealed that two DR4-like elements at -5476 and -4618 are essential for transactivation by PXR/PGC-1alpha. Chromatin immunoprecipitation assay revealed that PXR and PGC-1alpha bind to CYP2A6 chromatin. In conclusion, we found that CYP2A6 is induced via PXR and PGC-1alpha through the DR4-like element at the distal response region. This is the first study to report the molecular mechanism of the induction of CYP2A6.

A novel CYP2A6*20 allele found in African-American population produces a truncated protein lacking enzymatic activity.

Human CYP2A6 is a cytochrome P450 (CYP) isoform responsible for the metabolism of nicotine, coumarin, tegafur, and valproic acid, and metabolic activation of nitrosamines. Genetic polymorphisms of the CYP2A6 gene are a major causal factor of the large interindividual differences in nicotine metabolism. In the present study, we identified a novel allele, termed CYP2A6*20, in an African-American population. The allele possesses the deletion of two nucleotides in exon 4 resulting in a frame-shift from codon 196 and an early stop codon at 220 (exon 5) as well as three synonymous SNPs of G51A (G51A in cDNA), T5684C (T1191C), and C6692G (C1546G, 3'-untranslated region). The allele frequency in the African-American population (n=96) was 1.6% (95% confidence interval, 0.6-4.5%). In contrast, the CYP2A6*20 allele was not found in Caucasians (European-American) (n=185), Japanese (n=184) and Korean (n=209) populations. To investigate the effects of the polymorphism on the enzymatic activities, we expressed a wild type or variant (deletion of two nucleotides) CYP2A6 together with NADPH-CYP reductase in Escherichia coli. SDS-PAGE and immunoblot analyses demonstrated that truncated CYP2A6 protein was produced from the variant allele, although detected mRNA was the predicted size by reverse transcriptional-polymerase chain reaction. Coumarin 7-hydroxylation and nicotine C-oxidation, which are typical CYP2A6 activities, were completely abolished in the E. coli membrane expressing the variant allele. In vivo nicotine metabolism was evaluated using the cotinine/nicotine ratio 2 h after the chewing of one piece of nicotine gum. Two CYP2A6*1/CYP2A6*20 heterozygotes and a single CYP2A6*17/CYP2A6*20 heterozygote revealed lower cotinine/nicotine ratios compared with CYP2A6*1/CYP2A6*1 subjects (1.6 and 4.5, and 1.8 versus 9.5+/-5.4, n=52, respectively). We found a novel CYP2A6*20 allele in African-American subjects which codes a truncated protein lacking enzymatic activity.

Characterization of novel CYP2A6 polymorphic alleles (CYP2A6*18 and CYP2A6*19) that affect enzymatic activity.

Genetic polymorphisms of CYP2A6 gene are known as a causal factor of the interindividual differences in nicotine metabolism. We found three novel CYP2A6 alleles. The CYP2A6(*)18A allele has a single nucleotide polymorphism (SNP) of A5668T (A1175T, Y392F) in exon 8. The CYP2A6(*)18B allele has synonymous SNPs of G51A (G51A), T5684C (T1191C), and T5702C (T1209C) in addition to A5668T (A1175T, Y392F). The CYP2A6(*)19 allele has the SNPs of A5668T (A1175T, Y392F), T6354C (intron 8), and T6558C (T1412C, I471T) as well as the conversion with the CYP2A7 sequence in the 3'-untranslated region, in which the latter two changes correspond to CYP2A6(*)7. Ethnic differences in the frequencies of these alleles were observed between whites, African-Americans, Japanese, and Koreans. Wild or variant CYP2A6 (CYP2A6(*)18, CYP2A6(*)19, and CYP2A6(*)7) were expressed in Escherichia coli. For coumarin 7-hydroxylation and 5-fluorouracil formation from tegafur, the K(m) values were increased, and V(max) values were decreased in CYP2A6.18 compared with those in CYP2A6.1, resulting in decreased clearance to 50 and 35% of that of the wild type, respectively. The K(m) and V(max) values for nicotine C-oxidation were both increased, resulting in no change of clearance. In CYP2A6.19, the effects on the coumarin 7-hydroxylation and 5-fluorouracil formation (increased K(m) and decreased V(max)) were prominent, resulting in decreased clearance to 8% of those of the wild type. For nicotine C-oxidation, the K(m) and V(max) values were both decreased, resulting in decreased clearance to 30% of that of the wild type. The changes of the kinetics in CYP2A6.19 were similar to those in CYP2A6.7. In vivo nicotine metabolism was evaluated in whites (n = 56) and Koreans (n = 40). Although the CYP2A6(*)18 and CYP2A6(*)19 alleles were found only heterozygously, a subject with CYP2A6(*)7/CYP2A6(*)19 showed a lower cotinine/nicotine ratio of the plasma concentration compared with homozygotes of the CYP2A6(*)1A, supporting the in vitro results that the CYP2A6(*)19 allele leads to decreased enzymatic activity.