Cytochrome P450 (CYP) 1A2, sulfotransferase (SULT) 1A1, and N-acetyltransferase (NAT) 2 polymorphisms and susceptibility to urothelial cancer

Purpose Arylamines are suspected to be the primary causative agent of urothelial cancer in tobacco smoke. In the human liver, arylamines are N-hydroxylated by a cytochrome P450 (CYP)1A2-catalyzed reaction, which produces a substrate for O-esterification that can be catalyzed by N-acetyltransferases (NAT) or sulfotransferases (SULT). Recently, several polymorphisms of CYP1A2, SULT1A1, and NAT2 that affect their activities have been reported.Methods In this study, 306 Japanese patients with urothelial transitional cell carcinoma and 306 healthy controls were compared for frequencies of CYP1A2, SULT1A1, and NAT2 genotypes.Results The frequencies of NAT2 intermediate or slow acetylator genotype were significantly higher in the urothelial cancer patients than in the healthy control subjects [odds ratio (OR)=1.49, 95% confidence interval (95% CI) 1.06–2.09, OR=3.23, 95% CI 1.72–6.08, respectively]. Stratifying by amount of smoking, among subjects who consumed >33.5 pack-years and carried the SULT1A1 *1/*1 or NAT2 slow acetylator genotype, the OR was 1.73 (95% CI 1.01–2.97) whereas it was 7.31 (95% CI 1.90–28.05) in non-smokers who carried the homozygous wild genotype, respectively. The relationships between CYP1A2, SULT1A1, and NAT2 polymorphisms and clinical findings including tumor differentiation, stage, and recurrence rate were analyzed. Only associations between NAT2 genotype and pathological findings were admitted, and the higher OR of NAT2 intermediate and slow acetylator genotype was more likely to present to a low-grade tumor (G1) among heavy-smokers.Conclusions Our results suggest that SULT1A1 *1/*1 and NAT2 slow acetylator genotypes might modulate the effect of carcinogenic arylamines contained in tobacco smoke, and that the modulation of NAT2 intermediate and slow acetylator genotype has a tendency to present a higher risk for highly differentiated tumors among heavy-smokers.     

The Highly Expressed and Inducible Endogenous NAD(P)H:quinone Oxidoreductase 1 in Cardiovascular Cells Acts as a Potential Superoxide Scavenger

It has recently been demonstrated that purified NAD(P)H:quinone oxidoreductase 1 (NQO1) is able to scavenge superoxide (O₂^•−) though the rate of reaction of O₂^•− with NQO1 is much lower than the rate of enzymatic dismutation catalyzed by superoxide dismutase (SOD). This study was undertaken to determine if the endogenously expressed NQO1 in cardiovascular cells could scavenge O₂^•−. We observed that NQO1 was highly expressed in cardiovascular cells, including rat aortic smooth muscle A10 and cardiac H9c2 cells, as well as normal human aortic smooth muscle and endothelial cells. NQO1, but not SOD in the cardiovascular cells was highly inducible by 3H-1,2-dithiole-3-thione (D3T). Cytosols from H9c2 and human aortic smooth muscle cells (HASMCs) were isolated to determine the O₂^•− scavenging ability of the endogenously expressed NQO1 by using pyrogallol autooxidation assay. We showed that cytosols from the above cells inhibited pyrogallol autooxidation in an NADPH or NADH-dependent manner. The NADH/NADPH-dependent inhibition of pyrogallol autooxidation by the cytosols was completely abolished by the NQO1-specific inhibitor, ES936, suggesting that the endogenously expressed NQO1 could scavenge O₂^•−. In the presence of NADH/NADPH, cytosols from D3T-treated cells showed increased ability to scavenge O₂^•− as compared to cytosols from untreated cells. This increased ability to scavenge O₂^•− was also completely reversed by ES936. 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin-trapping experiments using potassium superoxide as a O₂^•− generator further confirmed the ability of NQO1 from HASMCs to scavenge O₂^•−. The spin-trapping experiments also showed that induction of NQO1 by D3T in HASMCs augmented the O₂^•− scavenging ability. Taken together, these results demonstrate that the highly expressed and inducible endogenous NQO1 in cardiovascular cells may act as a potential O₂^•− scavenger.     

Up-regulation of NAD（P）H quinone oxidoreductase 1 during human liver injury

AIM:To investigate the expression and activity ofNAD(P)H quinone oxidoreductase 1(NQO1)in humanliver specimens obtained from patients with liver damagedue to acetaminophen(APAP)overdose or primarybiliary cirrhosis(PBC).METHODS:NQO1 activity was determined in cytosolfrom normal,APAP and PBC liver specimens.Westernblot and immunohistochemical staining were used todetermine patterns of NQO1 expression using a specificantibody against NQO1.RESULTS:NQO1 protein was very low in normal humanlivers.In both APAP and PBC livers,there was stronginduction of NQO1 protein levels on Western blot.Correspondingly,significant up-regulation of enzymeactivity(16-and 22-fold,P<0.05)was also observed inAPAP and PBC livers,respectively.Immunohistochemicalanalysis highlighted injury-specific patterns of NQO1staining in both APAP and PBC livers.CONCLUSION:These data demonstrate that NQO1protein and activity are markedly induced in humanlivers during both APAP overdose and PBC.Up-regulationof this cytoprotective enzyme may represent an adaptivestress response tO limit further disease progression bydetoxifying reactive species.     

Dicoumarol enhances gemcitabine-induced cytotoxicity in high NQO1-expressing cholangiocarcinoma cells

AIM: To investigate whether dicoumarol, a potent inhibitor of NAD(P)H quinone oxidoreductase-1 (NQO1), potentiates gemcitabine to induce cytotoxicity in chol-angiocarcinoma cells (CCA) and the role of reactive oxygen generation in sensitizing the cells. METHODS: Four human cell lines with different NQO1 activity were used; the human CCA cell lines, KKU-100, KKU-OCA17, KKU-M214, and Chang liver cells. NQO1 activity and mRNA expression were determined. The cells were pretreated with dicoumarol at relevant con...     

高血压病患者吞噬细胞NAD（P）H氧化酶P22^Phox mRNA的表达水平

目的研究高血压病人及家系中血吞噬细胞NAD(P)H氧化酶P22phoxmRNA表达水平的变化。方法研究对象分为有高血压家族史的高血压组(FH)和正常组(FN),无高血压家族史的高血压组(NFH)和正常组(N)。通过密度梯度离心法获取血中吞噬细胞,采用RT-PCR技术检测吞噬细胞NAD(P)H氧化酶P22phoxmRNA的表达量;比色法检测血清中抗活性氧单位水平。结果FH组(6.60±0.59)、NFH组(6.07±0.56)分别较FN组(4.99±0.29)、N组(4.13±0.51)P22phoxmRNA高表达(P<0.01);FH组较NFH组、FN组较N组P22phoxmRNA高表达(P<0.01)。高血压组血清抗活性氧单位水平较正常组降低。结论高血压组血吞噬细胞NAD(P)H氧化酶P22phoxmRNA较正常组有显著的高表达,且其表达可能受遗传因素的影响。     

Alcohol‐Induced Exacerbation of Ischemic Brain Injury: Role of NAD(P)H Oxidase

Background: Chronic alcohol consumption increases ischemic stroke and exacerbates ischemic brain injury. We determined the role of NAD(P)H oxidase in exacerbated ischemic brain injury during chronic alcohol consumption. Methods: Sprague Dawley rats were fed a liquid diet with or without alcohol (6.4% v/v) for 8 weeks. We measured the effect of apocynin on 2‐hour middle cerebral artery occlusion (MCAO)/24‐hour reperfusion‐induced brain injury. In addition, superoxide production and expression of NAD(P)H oxidase subunit, gp91phox, in the peri‐infarct area were assessed. Results: Chronic alcohol consumption produced a larger infarct volume, worse neurological score, and higher superoxide production. Acute (5 mg/kg, ip, 30 minutes before MCAO) and chronic treatment with apocynin (7.5 mg/kg/d in the diet, 4 weeks prior to MCAO) reduced infarct volume, improved neurological outcome, and attenuated superoxide production in alcohol‐fed rats. Expression of gp91phox at basal conditions and following ischemia/reperfusion was greater in alcohol‐fed rats compared to non‐alcohol‐fed rats. In addition, neurons are partially responsible for upregulated gp91phox during alcohol consumption. Conclusions: Our findings suggest that NAD(P)H oxidase may play an important role in exacerbated ischemic brain injury during chronic alcohol consumption.     

NAD(P)H氧化酶p22phox亚基C242T基因多态性与缺血性脑血管病的关系

目的:探讨在中国人群中NAD(P)H氧化酶p22phox亚基C242T多态性与缺血性脑血管病(ICVD)的相关性。方法:共收集了112例ICVD患者(TIA36例,脑梗死76例)和105例对照者,用聚合酶链反应和限制性片段长度多态性分析(PCR-RFLP)的方法确定其基因型。结果:ICVD组和对照组的TC基因型频率分别为0.152和0.057,未发现有TT基因型。ICVD组的TC基因型频率显著高于对照组(P=0.024,OR2.95,95％CI 1.12-7.81)。ICVD亚组间的分析显示,TIA与对照组之间CT基因型频率无明显差异,而脑梗死与对照组则有显著差异。结论:NAD(P)H氧化酶p22phox亚基C242T多态性可能是脑梗死的一个危险因素。     

Alcohol-induced impairment of neuronal nitric oxide synthase (nNOS)-dependent dilation of cerebral arterioles: role of NAD(P)H oxidase

The goal of the present study was to determine the role of NAD(P)H oxidase in alcohol consumption-induced impairment of nNOS-dependent reactivity in cerebral arterioles. Sprague-Dawley rats were fed an alcohol diet for 2-3 months. We measured the effects of acute (1 hour) and chronic (1 month) treatment with a NAD(P)H oxidase inhibitor, apocynin, on responses of parietal pial arterioles to nNOS-dependent agonists (NMDA and kainate) and an nitric oxide synthase (NOS)-independent agonist (nitroglycerin). In addition, we measured the expression of NAD(P)H oxidase subunits and superoxide production in parietal cortex. Topical application of NMDA and kainate produced dose-related dilation of pial arterioles. However, the magnitude of vasodilation to these agonists was significantly less in alcohol-fed rats. Treatment with apocynin (acute and chronic) did not alter vasodilation in nonalcohol-fed rats, but significantly improved vasodilation in alcohol-fed rats. Response of pial arterioles to nitroglycerin was similar in nonalcohol-fed and alcohol-fed rats, and was not affected by apocynin. In addition, we found an up-regulation of gp91phox and p47phox in parietal cortex of alcohol-fed rats. Finally, alcohol consumption produced an increase in superoxide production under basal conditions and in the presence of NADPH. Acute treatment with apocynin suppressed alcohol consumption-induced superoxide generation. Our findings suggest that NAD(P)H oxidase plays an important role in chronic alcohol consumption-induced impairment of nNOS-dependent dilation of cerebral arterioles.     

A potential mechanism underlying the increased susceptibility of individuals with a polymorphism in NAD(P)H:quinone oxidoreductase 1 (NQO1) to benzene toxicity

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a two-electron reductase that detoxifies quinones derived from the oxidation of phenolic metabolites of benzene. A polymorphism in NQO1, a C609T substitution, has been identified, and individuals homozygous for this change (T/T) have no detectable NQO1. Exposed workers with a T/T genotype have an increased risk of benzene hematotoxicity. This finding suggests NQO1 is protective against benzene toxicity, which is difficult to reconcile with the lack of detectable NQO1 in human bone marrow. The human promyeloblastic cell line, KG-1a, was used to investigate the ability of the benzene metabolite hydroquinone (HQ) to induce NQO1. A concentration-dependent induction of NQO1 protein and activity was observed in KG-1a cells cultured with HQ. Multiple detoxification systems, including NQO1 and glutathione protect against benzene metabolite-induced toxicity. Indeed, exposure to a noncytotoxic concentration of HQ induced both NQO1 and soluble thiols and protected against HQ-induced apoptosis. NQO1 protein and activity increased in wild-type human bone marrow cells (C/C) exposed to HQ, whereas no NQO1 was induced by HQ in bone marrow cells with the T/T genotype. Intermediate induction of NQO1 by HQ was observed in heterozygous bone marrow cells (C/T). NQO1 also was induced by HQ in wild-type (C/C) human bone marrow CD34(+) progenitor cells. Our data suggest that failure to induce functional NQO1 may contribute to the increased risk of benzene poisoning in individuals homozygous for the NQO1 C609T substitution (T/T).     

Inhibition of NAD(P)H:quinone oxidoreductase 1 activity and induction of p53 degradation by the natural phenolic compound curcumin

NAD(P)H:quinone oxidoreductase 1 (NQO1) regulates the stability of the tumor suppressor WT p53. NQO1 binds and stabilizes WT p53, whereas NQO1 inhibitors including dicoumarol and various other coumarins and flavones induce ubiquitin-independent proteasomal p53 degradation and thus inhibit p53-induced apoptosis. Here, we show that curcumin, a natural phenolic compound found in the spice turmeric, induced ubiquitin-independent degradation of WT p53 and inhibited p53-induced apoptosis in normal thymocytes and myeloid leukemic cells. Like dicoumarol, curcumin inhibited the activity of recombinant NQO1 in vitro, inhibited the activity of endogenous cellular NQO1 in vivo, and dissociated NQO1-WT p53 complexes. Neither dicoumarol nor curcumin dissociated the complexes of NQO1 and the human cancer hot-spot p53 R273H mutant and therefore did not induce degradation of this mutant. NQO1 knockdown by small-interfering RNA induced degradation of both WT p53 and the p53 R273H mutant. The results indicate that curcumin induces p53 degradation and inhibits p53-induced apoptosis by an NQO1-dependent pathway.     

Low NAD(P)H:quinone oxidoreductase 1 activity is associated with increased risk of acute leukemia in adults

NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme that detoxifies quinones and reduces oxidative stress. A cysteine-to-threonine (C --> T) substitution polymorphism at nucleotide 609 of the NQO1 complementary DNA (NQO1 C609T) results in a lowering of NQO1 activity. Individuals homozygous for this mutation have no NQO1 activity, and heterozygotes have low to intermediate activity compared with people with wild type. DNA samples from 493 adult de novo acute leukemia patients and 838 matched controls were genotyped for NQO1 C609T. The majority of cases were diagnosed as acute myeloid leukemia (AML) (n = 420); 67 as acute lymphoblastic leukemia (ALL); and 6 as other forms of acute leukemia. The frequency of cases with low or null NQO1 activity (heterozygote + homozygous mutant) was significantly higher among total acute leukemia case subjects compared with their matched controls (odds ratio [OR] = 1.49; 95% confidence interval [CI], 1.17-1.89). Both ALL (OR = 1.93; 95% CI, 0.96-3.87) and AML case subjects (OR = 1.47; 95% CI, 1.13-1.90) exhibited a higher frequency of low or null NQO1 genotypes than controls. For de novo AML, the most significant effect of low or null NQO1 activity was observed among the 88 cases harboring translocations and inversions (OR = 2.39; 95% CI, 1.34-4.27) and was especially high for those harboring inv(16) (OR = 8.13; 95% CI, 1.43-46.42). These findings were confirmed in a second group of 217 de novo AML cases with known cytogenetics. Thus, inheritance of NQO1 C609T confers an increased risk of de novo acute leukemia in adults, implicating quinones and related compounds that generate oxidative stress in producing acute leukemia.     

Polymorphisms in sulfotransferase 1A1 and glutathione S-transferase P1 genes in relation to colorectal cancer risk and patients＇ survival

AIM: To examine whether polymorphisms in SULT1A1 and GSTP1 genes contribute to colorectal cancer development and whether they are associated with clinicopathological variables are not well identified.METHODS: We examined the genotypes of 125 colorectal cancer patients and 666 healthy controls in a Swedish population by using PCR-restriction fragment length polymorphism (RFLP).RESULTS: SULT1A1 *2/*2 genotype (OR=2.49, 95%CI=1.48-4.19, P=0.0002) and *2 allele (OR=1.56, 95%CI=1.16-2.10, P=0.002) had an effect on colorectal cancer susceptibility, while GSTP1 genotype was without effect. However, GSTP1 G-type predicted a worse prognosis in the patients independently of gender, age, Dukes' stage, growth pattern, and differentiation (P=0.03). CONCLUSION:Polymorphism in SULT1A1 may predispose to colorectal cancer and GSTP1 may be a biological indicator of prognosis in the patients.     

The NAD(P)H Oxidase Inhibitor Apocynin Improves Endothelial NO/Superoxide Balance and Lowers Effectively Blood Pressure in Spontaneously Hypertensive Rats: Comparison to Calcium Channel Blockade

The vascular NAD(P)H oxidase contributes to endothelial dysfunction and high blood pressure in the spontaneously hypertensive rat by enhancing superoxide production. We investigated the effects of apocynin, a NAD(P)H oxidase inhibitor, on blood pressure and vascular radical and nitric oxide formation in SHR and compared its effects to the calcium channel blocker nifedipine. Apocynin (over four weeks) lowered systolic blood pressure significantly and as effectively as nifedipine. Both apocynin and nifedipine significantly reduced superoxide production. In parallel, vascular nitric oxide production and ecNOS activity was significantly increased by apocynin treatment. Therefore, apocynin may be an effective antihypertensive drug in essential hypertension.