A Nanopore Sequencing-based Pharmacogenomic Panel to Personalize Tuberculosis Drug Dosing.

Rationale: Standardized dosing of antitubercular drugs leads to variable plasma drug levels, which are associated with adverse drug reactions, delayed treatment response, and relapse. Mutations in genes affecting drug metabolism explain considerable interindividual pharmacokinetic variability; however, pharmacogenomic assays that predict metabolism of antitubercular drugs have been lacking. Objectives: We sought to develop a Nanopore sequencing panel and validate its performance in patients with active tuberculosis (TB) to personalize treatment dosing. Methods: We developed a Nanopore sequencing panel targeting 15 SNPs in five genes affecting the metabolism of antitubercular drugs. For validation, we sequenced DNA samples (n = 48) from the 1,000 Genomes Project and compared the variant calling accuracy with that of Illumina genome sequencing. We then sequenced DNA samples from patients with active TB (n = 100) from South Africa on a MinION Mk1C and evaluated the relationship between genotypes and pharmacokinetic parameters for isoniazid (INH) and rifampin (RIF). Measurements and Main Results: The pharmacogenomic panel achieved 100% concordance with Illumina sequencing in variant identification for the samples from the 1,000 Genomes Project. In the clinical cohort, coverage was more than 100× for 1,498 of 1,500 (99.8%) amplicons across the 100 samples. Thirty-three percent, 47%, and 20% of participants were identified as slow, intermediate, and rapid INH acetylators, respectively. INH clearance was 2.2 times higher among intermediate acetylators and 3.8 times higher among rapid acetylators, compared with slow acetylators (P < 0.0001). RIF clearance was 17.3% (2.50-29.9) lower in individuals with homozygous AADAC rs1803155 G→A substitutions (P = 0.0015). Conclusions: Targeted sequencing can enable the detection of polymorphisms that influence TB drug metabolism on a low-cost, portable instrument to personalize dosing for TB treatment or prevention.

Evaluating interactions of polygenic risk scores and NAT2 genotypes with tobacco smoking in bladder cancer risk.

Tobacco smoking is the most important risk factor for bladder cancer. Previous studies have identified the N-acetyltransferase (NAT2) gene in association with bladder cancer risk. The NAT2 gene encodes an enzyme that metabolizes aromatic amines, carcinogens commonly found in tobacco smoke. In our study, we evaluated potential interactions of tobacco smoking with NAT2 genotypes and polygenic risk score (PRS) for bladder cancer, using data from the UK Biobank, a large prospective cohort study. We used Cox proportional hazards models to measure the strength of the association. The PRS was derived using genetic risk variants identified by genome-wide association studies for bladder cancer. With an average of 10.1 years of follow-up of 390 678 eligible participants of European descent, 769 incident bladder cancer cases were identified. Current smokers with a PRS in the highest tertile had a higher risk of developing bladder cancer (HR: 6.45, 95% CI: 4.51-9.24) than current smokers with a PRS in the lowest tertile (HR: 2.41, 95% CI: 1.52-3.84; P for additive interaction = <.001). A similar interaction was found for genetically predicted metabolizing NAT2 phenotype and tobacco smoking where current smokers with the slow NAT2 phenotype had an increased risk of developing bladder cancer (HR: 5.70, 95% CI: 2.64-12.30) than current smokers with the fast NAT2 phenotype (HR: 3.61, 95% CI: 1.14-11.37; P for additive interaction = .100). Our study provides support for considering both genetic and lifestyle risk factors in developing prevention measures for bladder cancer.

Application of the Hollow-Fiber Infection Model to Personalized Precision Dosing of Isoniazid in a Clinical Setting.

BACKGROUND: The hollow-fiber infection model (HFIM) is a valuable tool for evaluating pharmacokinetics/pharmacodynamics relationships and determining the optimal antibiotic dose in monotherapy or combination therapy, but the application for personalized precision medicine in tuberculosis treatment remains limited. This study aimed to evaluate the efficacy of adjusted antibiotic doses for a tuberculosis patient using HFIM. METHODS: Model-based Bayesian forecasting was utilized to assess the proposed reduction of the isoniazid dose from 300 mg daily to 150 mg daily in a patient with an ultra-slow-acetylation phenotype. The efficacy of the adjusted 150-mg dose was evaluated in a time-to-kill assay performed using the bacterial isolate Mycobacterium tuberculosis (Mtb) H37Ra in a HFIM that mimicked the individual pharmacokinetic profile of the patient. RESULTS: The isoniazid concentration observed in the HFIM adequately reflected the target drug exposures simulated by the model. After 7 days of repeated dose administration, isoniazid killed 4 log10 Mtb CFU/mL in the treatment arm, while the control arm without isoniazid increased 1.6 log10 CFU/mL. CONCLUSION: Our results provide an example of the utility of the HFIM for predicting the efficacy of specific recommended doses of anti-tuberculosis drugs in real clinical setting.

Procainamide pharmacokinetics during extracorporeal membrane oxygenation.

Procainamide is a useful agent for management of ventricular arrhythmia, however its disposition and appropriate dosing during extracorporeal membrane oxygenation (ECMO) is unknown. We report experience with continuous procainamide infusion in a critically ill adult requiring venoarterial ECMO for incessant ventricular tachycardia. Pharmacokinetic analysis of procainamide and its metabolite, N-acetylprocainamide (NAPA), was performed using serum and urine specimens. Kidney function was preserved, and sequencing of the N-acetyltransferase 2 gene revealed the patient was a phenotypic slow acetylator. Procainamide volume of distribution and half-life were calculated and found to be similar to healthy individuals. However, despite elevated serum procainamide concentrations, NAPA concentrations remained far lower in the serum and urine. The magnitude of procainamide and NAPA discordance suggested alternative contributors to the deranged pharmacokinetic profile, and we hypothesized NAPA sequestration by the ECMO circuit. Ultimately, the patient received orthotopic cardiac transplantation and was discharged home in stable condition. Procainamide should be used cautiously during ECMO, with close therapeutic drug monitoring of serum procainamide and NAPA concentrations. The achievement of therapeutic NAPA concentrations while maintaining safe serum procainamide concentrations during ECMO support may be challenging.

Influence of N-acetyltransferase 2 (NAT2) genotype/single nucleotide polymorphisms on clearance of isoniazid in tuberculosis patients: a systematic review of population pharmacokinetic models.

PURPOSE: Significant pharmacokinetic variabilities have been reported for isoniazid across various populations. We aimed to summarize population pharmacokinetic studies of isoniazid in tuberculosis (TB) patients with a specific focus on the influence of N-acetyltransferase 2 (NAT2) genotype/single-nucleotide polymorphism (SNP) on clearance of isoniazid. METHODS: A systematic search was conducted in PubMed and Embase for articles published in the English language from inception till February 2022 to identify population pharmacokinetic (PopPK) studies of isoniazid. Studies were included if patient population had TB and received isoniazid therapy, non-linear mixed effects modelling, and parametric approach was used for building isoniazid PopPK model and NAT2 genotype/SNP was tested as a covariate for model development. RESULTS: A total of 12 articles were identified from PubMed, Embase, and hand searching of articles. Isoniazid disposition was described using a two-compartment model with first-order absorption and linear elimination in most of the studies. Significant covariates influencing the pharmacokinetics of isoniazid were NAT2 genotype, body weight, lean body weight, body mass index, fat-free mass, efavirenz, formulation, CD4 cell count, and gender. Majority of studies conducted in adult TB population have reported a twofold or threefold increase in isoniazid clearance for NAT2 rapid acetylators compared to slow acetylators. CONCLUSION: The variability in disposition of isoniazid can be majorly attributed to NAT2 genotype. This results in a trimodal clearance pattern with a multi-fold increase in clearance of NAT2 rapid acetylators compared to slow acetylators. Further studies exploring the generalizability/adaptability of developed PopPK models in different clinical settings are required.

N-Acetyltransferase 2, glutathione S-transferase gene polymorphisms and susceptibility to hepatocellular carcinoma in an Algerian population.

This study was conducted to investigate the potential association of genetic polymorphisms of glutathione S-transferase M1/T1 (GSTM1, GSTT1), and N-acetyltransferase 2 (NAT2) genes and epidemiological parameters with the risk of HCC in the Algerian population.A case-control study including 132 confirmed HCC patients and 141 cancer-free controls was performed. Genotyping analysis was performed using conventional multiplex PCR and PCR-RFLP. Statistical analysis was performed using the Chi-square test. Logistic regression analysis was used to estimate odds ratios and 95% confidence intervals (95% CI).GSTM1 null and NAT2 slow acetylator genotypes confer an increased risk to HCC (OR = 1.88, 95% CI 1.16-3.05; OR = 2.30, 95% CI 1.26-4.18, respectively). This association was prevalent in smokers (OR = 2.00, 95% CI 1.05-3.8 and OR = 2.55, 95% CI 1.22-5.34, respectively). No significant association was observed for GSTT1 null genotype in the contribution to HCC risk (OR = 0.76, 95% CI 0.46-1.27).In conclusion, the GSTM1 and NAT2 gene polymorphisms are positively associated with the risk of HCC in older men and especially in smokers.

Relevance of gene polymorphisms of NAT2 and NR1I2 to anti-tuberculosis drug-induced hepatotoxicity.

The recommended treatment regimen for tuberculosis is a combination of agents with antitubercular activity, during which hepatotoxicity is one of the most common side effects. In addition to the N-acetyltransferase 2 (NAT2) genotype, rs3814055 in nuclear receptor subfamily 1, group I, member 2 (NR1I2) has been demonstrated to be associated with anti-tuberculosis drug-induced hepatotoxicity (ATDH), but previous results have been inconsistent.A retrospective nested hospital-based case-control study was performed to investigate the association between genetic polymorphisms and the risk of ATDH. Fifteen genetic variants (13 SNPs and two null genotypes) in cytochrome P450 2E1, NR1I2, UDP-glucuronosyltransferase 1A1, NAT2, superoxide dismutase 1, superoxide dismutase 2, and glutathione S-transferases (GSTT1, GSTM1, GSTP1) were genotyped. Odds ratios with 95% confidence intervals were calculated with drug doses, body mass index comorbidity of diabetes mellitus, and baseline alanine transaminase value as covariates.Conditional logistic regression demonstrated that the NAT2 slow acetylation genotype and the T allele of rs3814055 in NR1I2 may contribute to susceptibility to ATDH.Stratified association analysis demonstrated that in NAT2 non-slow acetylators, the T allele of rs3814055 was a risk factor for ATDH, whereas the T allele did not increase the susceptibility to ATDH in slow acetylators.

A Rapid Pharmacogenomic Assay to Detect NAT2 Polymorphisms and Guide Isoniazid Dosing for Tuberculosis Treatment.

Rationale: Standardized dosing of antitubercular drugs contributes to a substantial incidence of toxicities, inadequate treatment response, and relapse, in part due to variable drug concentrations achieved. SNPs in the NAT2 (N-acetyltransferase-2) gene explain the majority of interindividual pharmacokinetic variability of isoniazid (INH). However, an obstacle to implementing pharmacogenomic-guided dosing is the lack of a point-of-care assay. Objectives: To develop and test a NAT2 classification algorithm, validate its performance in predicting isoniazid clearance, and develop a prototype pharmacogenomic assay. Methods: We trained random forest models to predict NAT2 acetylation genotype from unphased SNP data using a global collection of 8,561 phased genomes. We enrolled 48 patients with pulmonary tuberculosis, performed sparse pharmacokinetic sampling, and tested the acetylator prediction algorithm accuracy against estimated INH clearance. We then developed a cartridge-based multiplex quantitative PCR assay on the GeneXpert platform and assessed its analytical sensitivity on whole blood samples from healthy individuals. Measurements and Main Results: With a 5-SNP model trained on two-thirds of the data (n = 5,738), out-of-sample acetylation genotype prediction accuracy on the remaining third (n = 2,823) was 100%. Among the 48 patients with tuberculosis, predicted acetylator types were 27 (56.2%) slow, 16 (33.3%) intermediate, and 5 (10.4%) rapid. INH clearance rates were lowest in predicted slow acetylators (median 14.5 L/h), moderate in intermediate acetylators (median 40.3 L/h), and highest in fast acetylators (median 53.0 L/h). The cartridge-based assay accurately detected all allele patterns directly from 25 µl of whole blood. Conclusions: An automated pharmacogenomic assay on a platform widely used globally for tuberculosis diagnosis could enable personalized dosing of INH.

Quantitative Analysis of Isoniazid and Its Four Primary Metabolites in Plasma of Tuberculosis Patients Using LC-MS/MS.

Isoniazid and its metabolites are potentially associated with hepatotoxicity and treatment outcomes in patients who receive antituberculosis (TB) therapy. To further understand the pharmacokinetic profiles of these molecules, a method based on LC-MS/MS was developed to determine the concentration of these compounds in human plasma. Isoniazid, acetylisoniazid, and isonicotinic acid were directly analyzed, whereas hydrazine and acetylhydrazine were determined after derivatization using p-tolualdehyde. Chromatographic separation was conducted on reversed-phase C18 columns with gradient elution, and detection was carried out in multiple reaction monitoring mode. The calibration curves were linear with correlation coefficients (r) greater than 0.9947 for all analytes. The intra- and inter-day precision was less than 13.43%, and the accuracy ranged between 91.63 and 114.00%. The recovery and matrix effect of the analytes were also consistent (coefficient of variation was less than 9.36%). The developed method successfully quantified isoniazid and its metabolites in TB patients. The method has broad applications in clinical research, including isoniazid one-point-based therapeutic drug monitoring, genotype-phenotype association studies of isoniazid metabolic profile and isoniazid-induced hepatotoxicity, and the initial dose prediction of isoniazid using population pharmacokinetic modeling.

A Method to Determine Xenobiotic Acetylation Rate by Taq SNP rs1495741.

Drug acetylation plays an important role in the medical practice. Modern methods of acetylation phenotype prediction are based on genotyping of polymorphisms in the second exon of the gene NAT2. Some disadvantages of these methods limit their application in the clinical practice. We developed a method of human genotyping based on identification of NAT2 gene polymorphism rs1495741 by real-time PCR. This method of genotype determination has a number of advantages: high sensitivity, simplicity, possibility of automated interpretation of the results, and feasibility in clinical laboratories.

Population Pharmacokinetic Analysis of Isoniazid among Pulmonary Tuberculosis Patients from China.

The blood concentration of isoniazid (INH) is evidently affected by polymorphisms in N-acetyltransferase 2 (NAT2), an enzyme that is primarily responsible for the trimodal (i.e., fast, intermediate, and slow) INH elimination. The pharmacokinetic (PK) variability, driven largely by NAT2 activity, creates a challenge for the deployment of a uniform INH dosage in tuberculosis (TB) patients. Although acetylator-specific INH dosing has long been suggested, well-recognized dosages according to acetylator status remain elusive. In this study, 175 blood samples were collected from 89 pulmonary TB patients within 0.5 to 6 h after morning INH administration. According to their NAT2 genotypes, 32 (36.0%), 38 (42.7%), and 19 (21.3%) were fast, intermediate, and slow acetylators, respectively. The plasma INH concentration was detected by liquid chromatography-tandem mass spectrometry. Population pharmacokinetic (PPK) analysis was conducted using NONMEM and R software. A two-compartment model with first-order absorption and elimination well described the PK parameters of isoniazid. Body weight and acetylator status significantly affected the INH clearance rate. The dosage simulation targeting three indicators, including the well-recognized efficacy-safety indicator maximum concentration in serum (Cmax; 3 to 6 µg/ml), the reported area under the concentration-time curve from 0 h to infinity (AUC0-∞; ≥10.52 µg·h/ml), and the 2-h INH serum concentrations (≥2.19 µg/ml), was associated with the strongest early bactericidal activity. The optimal dosages targeting the different indicators varied from 700 to 900 mg/day, 500 to 600 mg/day, and 300 mg/day for the rapid, intermediate, and slow acetylators, respectively. Furthermore, a PPK model for isoniazid among Chinese tuberculosis patients was established for the first time and suggested doses of approximately 800 mg/day, 500 mg/day, and 300 mg/day for fast, intermediate, and slow acetylators, respectively, after a trade-off between efficacy and the occurrence of side effects.

Population Pharmacokinetics of Isoniazid, Pyrazinamide, and Ethambutol in Pregnant South African Women with Tuberculosis and HIV.

Tuberculosis is an important cause of maternal morbidity, but little is known about the effects of pregnancy on antituberculosis drug concentrations. We developed population pharmacokinetic models to describe drug dispositions of isoniazid, pyrazinamide, and ethambutol in pregnant women with tuberculosis and HIV. HIV-positive pregnant women with tuberculosis receiving standard first-line tuberculosis treatment and participating in Tshepiso, a prospective cohort study in Soweto, South Africa, underwent sparse pharmacokinetic sampling at >36 weeks of gestation and 7 weeks postpartum. The effects of pregnancy on the pharmacokinetics of isoniazid, pyrazinamide, and ethambutol were investigated via population pharmacokinetic modeling. Isoniazid, pyrazinamide, and ethambutol concentrations were available for 29, 18, and 18 women, respectively. Their median weight was 66 kg while pregnant and 64 kg postpartum. No significant differences were observed in drug clearance, volume of distribution, or bioavailability during and after pregnancy. The model-estimated isoniazid, pyrazinamide, and ethambutol area under the concentration-time curve from 0 to 24 h (AUC0-24) medians were, respectively, 6.88, 419, and 16.5 mg · h/liter during pregnancy versus 5.01, 407, and 19.0 mg · h/liter postpartum. The model-estimated maximum concentration (Cmax) medians for isoniazid, pyrazinamide, and ethambutol were, respectively, 1.39, 35.9, and 1.82 mg/liter during pregnancy versus 1.43, 34.5, and 2.11 mg/liter postpartum. A posteriori power calculations determined that our analysis was powered 91.8%, 59.2%, and 90.1% at a P of <0.01 to detect a 40% decrease in the AUCs of isoniazid, pyrazinamide, and ethambutol, respectively. Pregnancy does not appear to cause relevant changes in the exposure to isoniazid, pyrazinamide, and ethambutol. Additional studies of antituberculosis drugs in pregnancy are needed.

Studying polymorphic variants of the NAT2 gene (NAT2*5 and NAT2*7) in Nenets populations of Northern Siberia.

BACKGROUND: N-acetyltransferase 2 plays a crucial role in the metabolism of a wide range of xenobiotics, including many drugs, carcinogens, and other chemicals in the human environment. The article presents for the first time data on the frequency of two important "slow" variants of NAT2 gene (NAT2*5, rs1801280 and NAT2*7, rs1799931), which significantly affect the rate of xenobiotics acetylation, among representatives of indigenous populations of Forest and Tundra Nenets in Northern Siberia. The aim of this study was to identify the frequencies of these variants and compare them with frequencies in other ethnic populations. RESULTS: NAT2*5 (T341C) genotyping revealed frequencies of 28,0% and 38,6% for Tundra and Forest Nenets, respectively. The frequencies of NAT2*7 (G857A) variant were 9,8% and 8,2% for Tundra and Forest Nenets, respectively. Polymorphic variants frequencies for Nenets are intermediate between those in populations of Europeans and Asians. These results can probably be explained by the presence of both European and Asian components in Nenets gene pools. CONCLUSIONS: The results of this study expand the knowledge of NAT2 polymorphism in world populations. These data may also help assess the genetic predisposition of Nenets to multifactorial diseases associated with polymorphism in the NAT2 gene and, in general, contribute to the development of personalized medicine in reference to native people of Siberia.

Altered activity of xenobiotic detoxifying enzymes at menopause - A cross-sectional study.

Xenobiotic metabolism at menopause is an under-investigated topic, albeit women spend one-third of their life in the postmenopausal period. The present study examined the effect of menopause on the in vivo activities of CYP1A2, CYP2A6, xanthine oxidase (XO) and N-acetyltransferase-2 (NAT2) xenobiotic metabolizing enzymes. Enzyme activity was determined in 152 non-smoking volunteers following oral intake of a single dose of 200 mg caffeine and subsequent determination of caffeine metabolite ratios (CMRs) in a 6-h urine sample as follows: CYP1A2: (AFMU+1U+1X)/17U, CYP2A6: 17U/(17U + 17X), XO: 1U/(1U+1X) and NAT2: AFMU/(AFMU+1U+1X). CMRs among groups were analyzed using one-way ANOVA. Significantly lower CYP1A2 and higher CYP2A6 CMRs were observed in postmenopausal compared to premenopausal women and age-matched men. These changes could be attributed to menopause rather than chronological aging since an age-related effect was not observed in premenopausal women or men of any age group. XO CMRs were higher in postmenopausal women and men>50 compared to premenopausal women and men<50, respectively, suggesting an age-related increase in XO activity. No significant alterations were discerned in NAT2 CMRs, in either slow- or rapid-acetylators, indicating that menopause exerts minimal modulation of xenobiotics metabolized by this enzyme. This study provides evidence that the transition to menopause induces significant alterations in xenobiotic-metabolizing enzymes independent of chronological aging suggesting altered metabolism of pharmaceutical and environmental agents.

The role of NAT2 polymorphism and methylation in anti-tuberculosis drug-induced liver injury in Mongolian tuberculosis patients.

WHAT IS KNOWN AND OBJECTIVE: Anti-tuberculosis drug-induced liver injury (ATLI) is one of the most significant adverse reactions for this line of therapy. N-acetyltransferase 2 (NAT2) is an important metabolic enzyme involved in drug metabolism and detoxification. Genetic polymorphism and DNA methylation have been proven to be key factors that affect the expression of NAT2. Therefore, the objective of the study was to investigate the relationship between NAT2 gene polymorphism and DNA methylation in the promoter region with ATLI risk in Mongolian tuberculosis patients. METHODS: Our study is a case-control design. Chi-square test, Mann-Whitney U non-parametric test and Pearson test were all used to analyse existing relationships. The association between NAT2 gene acetylation phenotype and the total methylation of the NAT2 promoter region was analysed by means of binary logistic regression analysis. The general situation of the patients was evaluated by questionnaire, and the NAT2 genotyping of the three major polymorphism loci of gene coding was carried out by a gene sequencing technique. The methylation status of the NAT2 gene promoter region was detected by bisulphite sequencing and mass spectrometry. RESULT AND DISCUSSION: Our study found that the detection rate of ATLI in Mongolian tuberculosis patients was 27.6%. There were no significant differences in demographic characteristics and living habits amongst the two groups, while significant differences were observed in the polymorphism of the NAT2 genes 481 (rs1799929) and 590 (rs1799930) and the acetylation phenotype. Moreover, the composition and distribution of the NAT2*4/4 and NAT2*4/5 genotypes were found in the two groups. The risk of ATLI in the slow acetylation type was 3.56 times higher than that of the fast acetylation type. Compared with the control group, the CpG5, CpG10, CpG11.12 and total methylation of the NAT2 promoter region in the ATLI group showed a hypermethylated pattern (P < .05). However, on performing binary logistic regression, neither the slow acetylation, intermediate acetylation nor rapid acetylation were found to be associated with ATLI (P > .05). It was found that the total methylation of NAT2 gene promoter region was an independent influencing factor of ATLI in Mongolian tuberculosis patients. With the increase of the total methylation level of NAT2 gene promoter region, the risk of ATLI increased gradually. (OR = 8.371, 95% CI: 2.391 ~ 29.315). CpG1, CpG4, CpG9, CpG10 and CpG11.12 were positively correlated with a total methylation level in the ATLI group. WHAT IS NEW AND CONCLUSION: The detection rate of ATLI in Mongolian tuberculosis patients was 27.6%, and there were differences in the NAT2 genotypes and acetylated phenotypes. The slow acetylated type was the risk factor for ATLI. Methylation in the promoter region of the NAT2 gene has an effect on the risk of ATLI. After adjusting for the interference of three acetylation types, it was found that the total methylation of the promoter region of NAT2 gene in Mongolian tuberculosis patients is an independent influencing factor of ATLI. Furthermore, there is a moderate to high correlation between some sites and the overall level of methylation.

Effects of microRNA-217 on proliferation, apoptosis, and autophagy of hepatocytes in rat models of CCL4-induced liver injury by targeting NAT2.

Liver injury is an important cause of serious liver disease. This study aims to explore the effects of miR-217 targeting NAT2 on hepatocyte proliferation, apoptosis, and autophagy following carbon tetrachloride (CCL4)-induced liver injury. Rat models of CCL4-induced liver injury were established. Healthy Wistar rats were randomized into the normal, blank, negative control (NC), microRNA-217 (miR-217) mimic, miR-217 inhibitor, small interfering RNA (siRNA)-N-acetyltransferase 2 (NAT2), and miR-217 inhibitor + siRNA-NAT2 groups. NAT2 activity was evaluated with reversed-phase high-performance liquid chromatographic method. Immunohistochemistry was used to detect NAT2 protein positive rate. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to examine expressions of miR-217, NAT2, Bcl-2, Bax, p35, LC3-II, Becline-1, and the ratio of caspase-3/cleaved caspase-3. Autophagy, proliferation, and cell cycle distribution were determined by electron microscope, CCK-8, and flow cytometry. NAT2 protein positive rate and miR-217, NAT2, Bcl-2, and p35 expressions were higher and Bax, LC3-II, and Becline-1 expressions and the ratio of caspase-3/cleaved caspase-3 lower in the normal group than the other six groups. Compared with the blank and NC groups, in the miR-217 mimic and siRNA-NAT2 groups, Bax, LC3-II, and Becline-1 expressions and the ratio of caspase-3/cleaved caspase-3, and hepatocyte apoptosis and autophagy increased, while NAT2, Bcl-2, and p35 expressions and hepatocyte proliferation decreased; opposite results were observed in the miR-217 inhibitor group. Collectively, miR-217 targeting NAT2 inhibits proliferation and promotes apoptosis and autophagy of hepatocytes in CCL4-induced liver injury.

N-acetyltransferase 2 polymorphism and acetylation profiles in Buginese ethnics of Indonesia.

BACKGROUND: N-acetyltransferase 2 (NAT2) is a key enzyme involved in the phase II metabolism of aromatic amines and heterocyclic aromatic amines present in a wide range of xenobiotics. The aim of this study was to investigate the NAT2 polymorphism in the Buginese ethnic group of Indonesia to determine the frequency of NAT2 alleles in this population. RESULTS: We found six haplotypes consisting of six single-nucleotide polymorphisms and 12 NAT2 genotype variations. NAT2*6A haplotype (42%) showed the highest frequency, followed by NAT2*4 (33%), NAT2*7B (15%), NAT2*5B (5%), NAT2*12A (3%), and NAT2*13 (2%). In terms of phenotypes, the Buginese population comprised 18% rapid acetylators, 40% intermediate acetylators, and 42% slow acetylators. CONCLUSION: We confirmed the high-frequency slow acetylator phenotype in the Buginese population. The NAT2*6A/*6A genotype was the most frequent slow acetylator genotype, followed by NAT2*6A/*7B. The pattern of NAT2 alleles of Buginese is similar to Southeast Asian populations but not Northeast Asian populations. However, the slow acetylator frequencies in the Buginese population were higher than those in Northeast Asian populations and lower than those in Caucasians and some American populations.

Genomewide Association Study Confirming the Association of NAT2 with Susceptibility to Antituberculosis Drug-Induced Liver Injury in Thai Patients.

Antituberculosis drug-induced liver injury (ATDILI) is a common side effect leading to tuberculosis (TB) treatment disruption. The mechanism of the disease remains poorly understood. We conducted a genomewide association study (GWAS) to investigate all possible genetic factors of ATDILI in Thai patients. This study was carried out in Thai TB patients, including 79 ATDILI cases and 239 tolerant controls from our network hospitals in Thailand. Nearly 1 million single-nucleotide polymorphisms (SNPs) were genotyped across the whole genome using an Illumina OmniExpress Exome BeadChip array. In the discovery stage, we identified strong association signals on chromosome 8 originating from the N-acetyltransferase (NAT2) region. The A allele of rs1495741, the top SNP in the intergenic region of NAT2 and PSD3 (14 kb from NAT2), was significantly associated with ATDILI (recessive model, odds ratio of 6.01 [95% confidence interval, 3.42 to 10.57]; P = 6.86E-11). This particular SNP was reported as a tag SNP for NAT2 inferred phenotypes. The AA, AG, and GG genotypes represented NAT2 slow acetylators, intermediate acetylators, and fast acetylators, respectively. The tag SNP genotypes demonstrated a concordance rate of 94.98% with NAT2 acetylator phenotypes. This GWAS shows that NAT2 is the most important risk factor for ATDILI in the Thai population.

Relevance of NAT2 genotype to anti-tuberculosis drug-induced hepatotoxicity in a Chinese Han population.

BACKGROUND: Anti-tuberculosis drug-induced hepatotoxicity (ATDH) is a serious adverse drug reaction. The slow acetylator status of N-acetyl transferase 2 (NAT2) is a well-established risk factor for ATDH. One novel tagging single nucleotide polymorphism (tagging SNP), rs1495741, in NAT2 has been found to be highly predictive of the NAT2 phenotype. The present study aimed to validate the relationships between tagging SNP rs1495741 and ATDH in a Chinese Han population. METHODS: A 1:2 matched case-control study was conducted using 235 ATDH cases and 470 controls. Conditional or unconditional logistic regression analysis was used to estimate the association between genotypes and the risk of ATDH according to the odds ratio (OR) with a 95% confidence interval (CI). RESULTS: Patients carrying the AA genotype of tagging SNP rs1495741 were at higher risk of ATDH than those carrying the GG genotype (OR = 1.653, 95% CI = 1.050-2.601; p = 0.030). Subgroup analysis suggested that the AA genotype was a risk factor for ATDH in patients aged older than 50 years (OR = 2.486, 95% CI = 1.313-4.706; p = 0.005), weighing over 50 kg (OR = 1.757, 95% CI = 1.016-3.038; p = 0.044) or using a hepatoprotectant (OR = 1.611, 95% CI = 1.009-2.572; p = 0.046). Tagging SNP rs1495741 was not a significant risk factor for moderate and severe hepatotoxicity but appears to be relevant to risk of mild hepatotoxicity specifically. CONCLUSIONS: The present study is the first to validate the relationships between the tagging SNP rs1495741 and ATDH in a Chinese population. Based on this case-control study, the NAT2 rs1495741 polymorphism is a risk factor for mild but not more severe ATDH in Chinese Han patients.

Role of the human N-acetyltransferase 2 genetic polymorphism in metabolism and genotoxicity of 4, 4'-methylenedianiline.

4, 4'-Methylenedianiline (MDA) is used extensively as a curing agent in the production of elastomers and is classified as reasonably anticipated to be a human carcinogen based on sufficient evidence in animal experiments. Human N-acetyltransferase 1 (NAT1) and 2 (NAT2) catalyze the N-acetylation of aromatic amines and NAT2 is subjected to a common genetic polymorphism in human populations separating individuals into rapid, intermediate, and slow acetylator phenotypes. Although MDA is known to undergo N-acetylation to mono- and di-acetyl metabolites, very little is known regarding whether this metabolism is subject to the NAT2 genetic polymorphism. We investigated the N-acetylation of MDA by recombinant human NAT1, NAT2, genetic variants of NAT2, and cryoplateable human hepatocytes obtained from rapid, intermediate and slow acetylators. MDA N-acetylation was catalyzed by both recombinant human NAT1 and NAT2 exhibiting a fivefold higher affinity for human NAT2. N-acetylation of MDA was acetylator genotype dependent as evidenced via its N-acetylation by recombinant human NAT2 genetic variants or by cryoplateable human hepatocytes. MDA N-acetylation to the mono-acetyl or di-acetyl-MDA was highest in rapid, lower in intermediate, and lowest in slow acetylator human hepatocytes. MDA-induced DNA damage in the human hepatocytes was dose-dependent and also acetylator genotype dependent with highest levels of DNA damage in rapid, lower in intermediate, and lowest in slow acetylator human hepatocytes under the same MDA exposure level. In summary, the N-acetylation of MDA by recombinant human NAT2 and cryopreserved human hepatocytes support an important role for the NAT2 genetic polymorphism in modifying MDA metabolism and genotoxicity and potentially carcinogenic risk.