Role of cytochrome P450 1A2 and N-acetyltransferase 2 in 2, 6-dimethylaniline induced genotoxicity

Abstract The purpose of the current work was to assess a possible role of cytochrome P450 1A2 (CYP1A2) and N-acetyltransferase 2 (NAT2) in the metabolic activation of 2,6-dimethylaniline (2,6-DMA) and also clarify the function of DNA repair in affecting the ultimate mutagenic potency. Two cell lines, nucleotide excision repair (NER)-deficient 5P3NAT2 and proficient 5P3NAT2R9 both expressing CYP1A2 and NAT2, were treated with 2,6-DMA for 48 h or its metabolites for 1 h. Cell survival determined by trypan blue exclusion and MTT assays, and 8-azaadenine-resistant mutants at the adenine phosphoribosyltransferase (aprt) gene locus were evaluated. 5P3NAT2 and 5P3NAT2R9 cells treated with 2,6-DMA and its metabolites showed a dose-dependent increase in cytotoxicity and mutant fraction; N-OH-2,6-DMA and 2,6-DMAP in serum-free α-minimal essential medium (MEM) are more potent than 2,6-DMA in complete MEM. 5P3NAT2 cells was more sensitive to the cytotoxic and mutagenic action than 5P3NAT2R9 cells. H2DCFH-DA assay showed dose-dependent ROS production under 2,6- DMAP treatment. These findings indicate that the genotoxic effects of 2,6-DMA are mediated by CYP1A2 activation via N-hydroxylation and the subsequent esterification by the phase II conjugation enzyme NAT2, and through the generation of ROS by hydroxylamine and/or aminophenol metabolites. NER status is also an important contributor

Homozygotes NAT2*5B slow acetylators are highly associated with hepatotoxicity induced by anti-tuberculosis drugs

BACKGROUND Distinct N-acetyltransferase 2 (NAT2) slow acetylators genotypes have been associated with a higher risk to develop anti-tuberculosis drug-induced hepatotoxicity (DIH). However, studies have not pointed the relevance of different acetylation phenotypes presented by homozygotes and compound heterozygotes slow acetylators on a clinical basis. OBJECTIVES This study aimed to investigate the association between NAT2 genotypes and the risk of developing DIH in Brazilian patients undergoing tuberculosis treatment, focusing on the discrimination of homozygotes and compound heterozygotes slow acetylators. METHODS/FINDINGS The frequency of NAT2 genotypes was analysed by DNA sequencing in 162 patients undergoing tuberculosis therapy. The mutation analyses revealed 15 variants, plus two new NAT2 mutations, that computational simulations predicted to cause structural perturbations in the protein. The multivariate statistical analysis revealed that carriers of NAT2*5/*5 slow acetylator genotype presented a higher risk of developing anti-tuberculosis DIH, on a clinical basis, when compared to the compound heterozygotes presenting NAT2*5 and any other slow acetylator haplotype [aOR 4.97, 95% confidence interval (CI) 1.47-16.82, p = 0.01]. CONCLUSION These findings suggest that patients with TB diagnosis who present the NAT2*5B/*5B genotype should be properly identified and more carefully monitored until treatment outcome in order to prevent the occurrence of anti-tuberculosis DIH.

Arylamine-acetyltransferase 2 genotype-dependent-acetylation of isoniazid in cryopreserved human hepatocytes

Cryopreserved human hepatocytes were used to investigate the role of arylamine-acetyltransferase 2 (NAT2; EC 2.3.1.5) polymorphism on the-acetylation of isoniazid (INH).genotype was determined by Taqman allelic discrimination assay and INH-acetylation was measured by high performance liquid chromatography. INH-acetylation ratesexhibited a robust and highly significant (<0.005) NAT2 phenotype-dependent metabolism.-acetylation rateswere INH concentration- and time-dependent. Following incubation for 24 h with 12.5 or 100 µmol/L INH, acetyl-INH concentrations varied significantly (= 0.0023 and= 0.0002) across cryopreserved human hepatocytes samples from rapid, intermediate, and slow acetylators, respectively. The clear association betweengenotype and phenotype supports use ofgenotype to guide INH dosing strategies in the treatment and prevention of tuberculosis.

Polymorphisms in NAT2 (N-acetyltransferase 2) gene in patients with systemic lupus erythematosus / Polimorfismos no gene NAT2 (N-acetiltransferase 2) em pacientes com lúpus eritematoso sistêmico

ABSTRACT Objective: To investigate potential associations of four substitutions in NAT2 gene and of acetylator phenotype of NAT2 with systemic lupus erythematosus (SLE) and clinical phenotypes. Methods: Molecular analysis of 481C>T, 590G>A, 857G>A, and 191G>A substitutions in the NAT2 gene was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, from DNA extracted from peripheral blood samples obtained from patients with SLE (n = 91) and controls (n = 97). Results and conclusions: The 857GA genotype was more prevalent among nonwhite SLE patients (OR = 4.01, 95% CI = 1.18-13.59). The 481T allele showed a positive association with hematological disorders that involve autoimmune mechanisms, specifically autoimmune hemolytic anemia or autoimmune thrombocytopenia (OR = 1.97; 95% CI = 1.01-3.81).

RESUMO Objetivo: Investigar potenciais associações de quatro substituições do gene NAT2 (N-acetiltransferase 2) e do fenótipo acetilador de NAT2 com o lúpus eritematoso sistêmico (LES) e os fenótipos clínicos. Métodos: A análise molecular das substituições 481C > T, 590G > A, 857G > A e 191G > A do gene NAT2 foi feita com a técnica de PCR-RFLP, usando DNA extraído de amostras de sangue periférico obtidas de pacientes com LES (n = 91) e controles (n = 97). Resultados e conclusões: O genótipo 857GA foi mais prevalente entre pacientes com LES não brancas (OR = 4,01, IC 95% = 1,18-13,59). O alelo 481 T apresentou associação positiva com as alterações hematológicas que envolvem mecanismos autoimunes, especificamente anemia hemolítica autoimune ou trombocitopenia autoimune (OR = 1,97; IC 95% = 1,01-3,81).

The association of genetic polymorphisms of N-acetyltransferase 2 gene with hepatotoxicity and efficacy in Chinese Han patients with tuberculosis / 中华传染病杂志

Objective To investigate the association of the polymorphism of the N-acetyltransferase 2 (NAT2 )gene with isoniazid-induced hepatotoxicity and anti-tuberculous treatment efficacy in Chinese Han patients with tuberculosis(TB).Methods A total of 108 TB patients who received initial anti-TB treatment were followed up prospectively.A polymerase chain reaction direct sequencing approach was used to detect genetic polymorphisms of the NAT2 gene.Associations between NAT2 genotype and isoniazid-induced hepatitis/early treatment were analyzed.Chi-square test was used for statistical analysis. Results Among the 108 TB patients, intermediate-acetylators (IA ) was the most frequent NAT2 genotype with the proportion of 54.63%(59/108).The proportion of rapid-acetylators(RA)was 33.33%(36/108),slow-acetylators (SA)was 10.19%(11/108)and super-rapid acetylators was 1 .85 % (2/108). Among the 20 patients who developed drug-induced hepatitis,2 were RA,5 were SA and 13 were IA. Regarding NAT2 genotype,RA patients had a lower incidence of hepatotoxicity (OR =0.176,95 %CI :0.038-0.809,P =0.014)and SA patients were more likely to developed drug-induced hepatic injury (OR=4.556,95 %CI :1 .231 -16.854,P =0.044 ).Statistical analysis revealed that the frequency of variant diplotypes,NAT2*4/*6A (OR=7.741 ,95 %CI :2.653-22.586,P <0.01 )and NAT2 *6A/*6A (OR=15 .353,95 %CI :1 .506 -156.552,P =0.020)were significantly increased in TB patients with hepatotoxicity.NAT2 *4/*4 was less likely to developed hepatic injury (OR =0.176,95 %CI :0.038-0.809,P =0.014).Among the 58 culture-positive patients,12(31 .03%)were persistent culture positive after 2 months standard therapy.Early treatment failure was observed with significantly higher incidence rate in RA than other genotypes (OR = 7.200, 95 % CI :1 .794-28.900, P = 0.008). Conclusions In Chinese Han TB patients,IA is the most frequent NAT2 genotype.The SA status of NAT2 is a risk factor of isoniazid-induced hepatotoxicity.The diplotype of NAT2 *6A has clearly high risk of isoniazid-induced hepatotoxicity.In contrast,NAT2 * 4/* 4 is protective diplotype.RA is associated with early treatment failure in culture-positive patients.

Effects of Tibetan Medicine Zuotai on the Activities of CYP1A2 and NAT2 in Rats / 中国药房

OBJECTIVE:To study the effects of Tibetan medicine Zuotai on the activities of cytochrome oxidase (CYP1A2) and drug metabolism enzyme N-acetyltransferase 2(NAT2)in rats. METHODS:70 SD rats were equally randomized into a normal control (normal saline) group,the groups of single administration of low,middle and high-dose (1.2,3.8 and 12 mg/kg) Zuotai and the groups of multiple administrations thereof(once daily for 12 consecutive days). The rats were given drugs ig. caffeine(25 mg/kg)was given ig to the rats in the normal control group and the groups of single administration on the 2nd day,and to those in the groups of multiple administrations on the 13th day. 5 h later,their urine was collected and added with vitamin C based on 10 mg/ml. High performance liquid chromatography (HPLC) was adopted to determine the cafeine metabolites contents of 5-acetami-do-6-formamido-3-methyl-uric acid(AFMU),1-methylxanthine(1X),1-methyl-uric acid(1U)and 1,7-dimethyl uric acid(17U) in rats’urine,and the activities of CYP1A2 and NAT2 were reflected through (AFMU+1X+1U)/17U and AFMU/(AFMU+1X+1U). RESULTS:Compared with normal control group,the(AFMU+1X+1U)/17U and AFMU/(AFMU+1X+1U)in rats were de-creased,namely the activities of CYP1A2 and NAT2 were lower in the groups of single administration of middle-dose Zuotai and multiple administrations of middle and high-dose Zuotai than in the normal control group. There was statistical difference (P<0.05). CONCLUSIONS:Zuotai can obviously inhibit the activities of CYP1A2 and NAT2 in rats.

A study on the relationship between NAT2 genetic polymorphisms and biomarkers related to anti -tuberculosis drug -induced liver injury / 预防医学

Objective To know the relationship between NAT2 genetic polymorphisms and biomarkers related to anti -tuberculosis drug -induced liver injury (ATLI).Methods A total of 41 pulmonary tuberculosis patients with ATLI who received treatment within three months and 88 patients without ATLI were enrolled.The NAT2 genotypes were analyzed by pyrosequencing,and the biomarkers of liver function were analyzed.Results After anti -tuberculosis treatment,six biomarkers of liver function of pulmonary tuberculosis patients with different NAT2 genotypes were significantly higher than those before treatment(P <0.01 ).And there were significant differences in ALT level between different genotypes(P <0.05),the ALT levels of the fast acetylation genotype were significantly lower than that of the slow acetylation genotype(P<0.05 ),but no significant differences were observed in the other 5 biomarkers of liver function between different genotypes(P >0.05).Conclusion There is a certain relationship between NAT2 genetic polymorphisms and the elevated ALT levels among pulmonary tuberculosis patients after treatment.

Relationship between polymorphism of arylamine N-acetyltransferase 2 and genetic susceptibility to hair dye dermatitis / 中华医学美学美容杂志

Objective To investigate the relationship between polymorphism of arylamine N-acetyltransferase 2 (NAT2) and hair dye dermatitis in a Chinese population. Methods Polymorphism chain-restriction fragment length polymorphism (PCR-RFLP) was used and the wild-type allele (NAT2 * 4) and three mutant alleles (NAT2 * 5A, 6B and 7A) were determined in 60 patients with hair dye dermatitis and 73 age-matched control subjects in Tianjin region. Results In hair dye dermatitis cases, the frequency of NAT2 * 4, NAT2 * 5A, NAT2 * 6B, NAT2 * 7A was 52. 5 % , 5. 0 % ,26.7 % and 15. 8 %, respectively, and no statistically significant difference of the frequencies was found between the hair dye dermatitis patients and controls (P>0. 05). The frequency of rapid genotype, intermediate genotype and slow genotype was 26. 7 % , 51. 7 % and 21. 7 % in hair dye dermatitis cases, 30. 1 %, 50. 7 % and 19. 2 % in control subjects, respectively, and no statistically significant difference of the frequencies between the two groups (P>0. 05). Conclusions Our study suggests that there might be no relationship between polymorphism of NAT2 and genetic susceptibility to hair dye dermatitis in a Chinese population.

Association of genetic polymorphisms in CYP1A1 and NAT2 with susceptibility to renal cancer: A case control study / 第二军医大学学报

Objective: To study the effect of genetic polymorphisms of cytochrome P4501A1 (CYP1A1), N-acetyltransferase 2 (NAT2) on the susceptibility to renal cancer. Methods: The genetic polymorphisms of CYP1A1 and NAT2 were examined in the renal cancer patients and controls using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) methods. Results: The distribution of CYP1A1 (W/M) and NAT2 (intermediate) genotypes was significantly different between the renal cancer patients and controls (P<0.05). Individuals carrying CYP1A1 (W/M) or NAT2 (intermediate) genotypes had an increased risk for renal cancer(OR=2.487, 95% CI: 1.493-4. 142; OR=1.970, 95% CI: 1.128-3.442,respectively). Multivariate analysis showed increased risk for renal cancer patients carrying CYP1A1 (W/M) or NAT2 (intermediate) genotypes and those who smoke. Conclusion: The genotypes CYP1A1 (W/M) and NAT2 (intermediate) are the risk factors of renal cancer, and the 2 genotypes have a interactive effect and both have a joint effect with smoking.

The research progress of the computer simulation technology of three-dimension structure of human N-acetyltransferase2 protein / 中国药理学通报

N-acetyltransferase 2 is an important metabolic enzyme in the human body,participating mainly in the metabolism of medicine containing nitrogen.Presently a lot of researches have been undertaken in the respects like the determination of NAT2 genotype and drug metabolism and so on.In the recent years,more and more attention is paid to the simulation of three-dimension(solid)structure of protein,but the study is just at the outset on three-dimension structure of the NAT2 as well as its structure-function relationship when combined with drug.The research progress of construction of the three-dimension structure of NAT2 is summarized,emphasizing on its methods and tools.

Three-dimensional simulation of the structures of human N-acetyltransferase 2 protein molecule / 医学研究生学报

Objective:To simulate human N-acetyltransferase 2 protein 3D model by computer technology. Methods: 3D model of human N-acetyltransferase 2 protein was simulated with protein list and computer technology,the physical,chemical,biological characteristics and the functions were analyzed.Results: The possible structure of NAT2 was simulated using ANTHEPORT5.0,a software for determining the sequence of protein,forecasting the pHi and molecular weight of NAT2 were 5.495 and(33 544) respectively.The titration curve and physics-chemistry characteristic curve of NAT2 were also obtained.A Helical Wheel chart was plotted,aiming at the fragment of NAT2 around amino acid 282 nd.Conclusion:The possible structure of NAT2 was simulated using computer,analysis and forecast was also carried out,the method was feasible to genetic analysis of NAT2.

The Effects of the Genetic Polymorphisms of N-acetyltransferase 2 and Glutathione S-transferase mu on the Development of Bladder Tumor / 대한비뇨기과학회지

PURPOSE: Activity of enzymes involved in the metabolism of various carcinogenic xenobiotics is one of the most important host factor for cancer occurrence. N-acetyltransferase (NAT) and glutathione S-transferase(GST) are enzymes which reduce the toxicity of activated carcinogenic metabolites. Slow N-acetylation and lack of glutathione S-transferase mu(GSTM1) were reported as risk factors of bladder tumor. Since the cause of bladder cancer is not fully explained by single risk factor, many kinds of enzymes would be involved in the metabolism of carcinogens excreted in urine. This study was performed to investigate whether the polymorphisms of N-acetyltransferase type 2(NAT2) and GSTM1 are risk factors of bladder tumor and to evaluate the effects of their interaction on bladder tumor development. MATERIALS AND METHODS: 113 bladder tumor and 221 non-cancer patients, hospitalized in the Chungbuk National University Hospital, Samsung Medical Center, and Asan Medical Center from March to December 1996 participated in this case-control study. Questionnaire interview was done and the genotypes of NAT2 and GSTM1 were identified using PCR methods with DNA extracted from the venous blood. The effects of the polymorphism of NAT2 and GSTM1 and their interaction on bladder cancer were statistically tested after controlling the other risk factors. RESULTS: The frequencies of slow, intermediate, and rapid acetylators were 7.1%, 37.5%, and 55.4% for the cases, and 11.0%, 43.4%, and 45.7% for the controls, respectively. The risk of bladder tumor was not associated with the increase of NAT2 activity (chi2trend=3.16, p-value>0.05). GSTM1 was deleted in 69.6% of the cases and 55.9% of the controls (Chi2=5.86, p-value<0.05), and the odds ratio (95% CI) was 1.81 (1.12 - 2.93). Smoking history turned out to be insignificant as a risk factor of bladder tumor (OR=1.34, 95%CI: 0.78 - 2.31), and occupation could not be tested because of the extremely small number of occupational history related to the increase of bladder tumor. Medical history of tuberculosis and bronchial asthma were significant risk factors for bladder tumor, and their ORs (95% CI) were 3.61 (1.57-8.26) and 4.15 (1.61-10.75), respectively. In multiple logistic analysis controlling the effects of other risk factors, GSTM1 deletion (OR: 1.80, 95% CI: 1.07-3.05), and histories of tuberculosis (OR: 2.99, 95% CI: 1.22-7.32) and of bronchial asthma (OR: 3.38, 95% CI: 1.24-9.22) were the significant risk factors for bladder tumor, but slow acetylation was not. CONCLUSIONS: These results suggest that GSTM1 deletion may be a significant risk factor of bladder tumor. The medications for tuberculosis and bronchial asthma could possibly cause bladder tumor, or immune mechanism might be involved in the development of bladder tumor.

Effects of Polymorphism of Genes related to Metabolism of Xenobiotics on Bladder Tumor Occurrence / 대한비뇨기과학회지

PURPOSE: Cancer development depends on activities of enzymes involved in metabolisms of various carcinogenic xenobiotics. N-acetyltransferase(NAT) and glutathione S-transferases(GST) are enzymes, reducing the toxicity of activated carcinogenic metabolites. Carcinogens of bladder cancer can not be fully explained by single risk factor and many kinds of enzymes would be involved in the metabolism of carcinogens excreted in urine. This study was performed to investigate whether the polymorphisms of NAT2, GSTM1, and GSTT1 were risk factors of bladder cancer and to evaluate the effects of their interaction on bladder cancer occurrence. MATERIALS AND METHODS: One hundred and thirteen bladder cancer and 221 non-cancer patients hospitalized in the Chungbuk National University Hospital participated in the present case-control study. Questionnaire interview was performed and the genotypes of NAT2, GSTM1 and GSTT1 were identified using PCR methods with DNA extracted from venous blood. Effects of the polymorphism of NAT2, GSTM1 and GSTT1 and their interaction on bladder cancer were statistically analyzed after controlling other risk factors. RESULTS: The frequencies of slow, intermediate, and rapid acetylators were 7.1%, 37.5%, and 55.4% for the cases, and 11.0%, 43.4%, and 45.7% for the controls, respectively. The risk of bladder cancer was not associated with the increase of NAT2 activity(x2trend=3.16, p>0.05). GSTM1 was deleted in 69.6% of the cases and 55.9% of the controls(x2=5.86, P<0.05), and the mean odds ratio(95% CI) was 1.81 (1.12 - 2.93). The GSTT1 deletion, the rate of which were 42.0% for the bladder cancer patients and 45.9% for the controls, showed the protective effect against bladder cancer, but was not statistically significant. Smoking history turned out to be insignificant as a risk factor of bladder cancer(OR=1.34, 95%CI: 0.78 - 2.31), and occupation was not analyzed due to the extremely small number of occupational history related to the increase of bladder cancer. Medical histories of tuberculosis and bronchial asthma were significant risk factors for bladder cancer, and their average ORs(95% CI) were 3.61(1.57-8.26) and 4.15(1.61-10.75), respectively. In multiple logistic analysis controlling the effects of other risk factors, GSTM1 deletion (OR: 1.80, 95% CI: 1.07-3.05), and histories of tuberculosis(OR: 2.99, 95% CI: 1.22-7.32) and of bronchial asthma(OR: 3.38, 95% CI: 1.24-9.22) were the significant risk factors for bladder cancer, but slow acetylation and GSTT1 deletion were not. CONCLUSIONS: These results suggest that GSTM1 deletion may be a significant risk factor of bladder cancer, and GSTT1 deletion may have the protective effect against bladder tumor development. Since tuberculosis and bronchial asthma, in the present study, appeared to be involved in the development of bladder tumor, possible associated relationships are under experimental investigation in every aspect.

NAT2 Polymorphism and Individual Genetic Susceptibility to Colorectal Cancer Patients in Korea

To determine the frequencies of the genotypes of NAT2 gene in healthy Korean populations and to identify the high-risk genotypes of NAT2 gene in colorectal cancer patients, 115 healthy controls and 109 cancer patients were analyzed using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). The distribution of NAT2 polymorphism in healthy Korean was found to be 7.8% of S/S genotype, 48.7% of S/F genotype, and 43.5% of F/F genotype. And the frequency of phenotypes was 8% of slow acetylator and 92% of rapid acetylator. S/S genotype of colorectal cancer patients was slightly more frequent than that of healthy controls(11.9% vs 7.8%). The relative risk of S/S genotype to colorectal cancer was estimated to be 1.41, taking the risk of F/F genotype as a baseline(1.00). These results suggest that the distribution of frequencies of NAT2 genotypes is very unique in Korean characterized by extremely low frequency of slow acetylator geno type(S/S) in comparison to the other ethnic groups. And the slow acetylator genotype(S/S) in Korean was found to be more susceptible to colon cancer. Therefore, S/S genotype may have a certain role an colonic carcinogenesis in Korean.