Cascading Detection of Hydrogen Sulfide and N-Acetyltransferase 2 in Hepatocellular Carcinoma Cells Using a Two-Photon Fluorescent Probe.

Hydrogen sulfide (H2S), a critical gas signaling molecule, and N-acetyltransferase 2 (NAT2), a key enzyme in drug metabolism, are both known active biomarkers for liver function. However, the interactions and effects of H2S and NAT2 in living cells or lesion sites remain unknown due to the lack of imaging tools to achieve simultaneous detection of these two substances, making it challenging to implement real-time imaging and precise tracking. Herein, we report an activity-based two-photon fluorescent probe, TPSP-1, for the cascade detection of H2S and NAT2 in living liver cells. Continuous conversion from TPSP-1 to TPSP-3 was achieved in liver cells and tissues. Significantly, leveraging the outstanding optical properties of this two-photon fluorescent probe, TPSP-1, has been effectively used to identify pathological tissue samples directly from clinical liver cancer patients. This work provides us with this novel sensing and two-photon imaging probe, which can be used as a powerful tool to study the physiological functions of H2S and NAT2 and will help facilitate rapid and accurate diagnosis and therapeutic evaluation of hepatocellular carcinoma.

The Arylamine N-Acetyltransferases as Therapeutic Targets in Metabolic Diseases Associated with Mitochondrial Dysfunction.

In humans, there are two arylamine N-acetyltransferase genes that encode functional enzymes (NAT1 and NAT2) as well as one pseudogene, all of which are located together on chromosome 8. Although they were first identified by their role in the acetylation of drugs and other xenobiotics, recent studies have shown strong associations for both enzymes in a variety of diseases, including cancer, cardiovascular disease, and diabetes. There is growing evidence that this association may be causal. Consistently, NAT1 and NAT2 are shown to be required for healthy mitochondria. This review discusses the current literature on the role of both NAT1 and NAT2 in mitochondrial bioenergetics. It will attempt to relate our understanding of the evolution of the two genes with biologic function and then present evidence that several major metabolic diseases are influenced by NAT1 and NAT2. Finally, it will discuss current and future approaches to inhibit or enhance NAT1 and NAT2 activity/expression using small-molecule drugs. SIGNIFICANCE STATEMENT: The arylamine N-acetyltransferases (NATs) NAT1 and NAT2 share common features in their associations with mitochondrial bioenergetics. This review discusses mitochondrial function as it relates to health and disease, and the importance of NAT in mitochondrial function and dysfunction. It also compares NAT1 and NAT2 to highlight their functional similarities and differences. Both NAT1 and NAT2 are potential drug targets for diseases where mitochondrial dysfunction is a hallmark of onset and progression.

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.

Association of the cytochrome P450 and arylamine N-acetyltransferase gene polymorphisms with the incidence of head and neck cancer in Polish population.

OBJECTIVES: Head and neck cancer (HNC) is one of the most common cancers. Most exogenous HNC is head and neck squamous cell carcinomas. Scientists are striving to develop diagnostic tests that will allow the prognosis of HNC. The aim of the study was to determine the risk of HNC. The research concerned changes caused by polymorphisms in genes encoding proteins responsible for the metabolism of xenobiotics. MATERIAL AND METHODS: In group of 280 patients with HNC, the occurrence of polymorphic variants in NAT1(rs72554606), NAT2(rs1799930), CYP1A(rs1799814), CYP2D(rs3892097) were studied with TaqMan technique. The control group consisted of 260 cancer free people. The TNM scale was analyzed. Gene interactions of genotyped polymorphisms were investigated. The effects of smoking and alcohol consumption on HNC were assessed. RESULTS: The results indicated an increased risk of HNC in NAT1 polymorphisms in the GC genotype (OR = 1.772, 95% CI: 1.184-2.651, p = 0.005) and NAT2 polymorphism in the GA genotype (OR = 1.506, 95% CI: 1.023-2.216, p = 0.037). The protective phenomenon in the CYP1A polymorphism the GT genotype (OR = 0.587, 95% CI: 0.381-0.903, p = 0.015) and the TT genotype (OR = 0.268, 95% CI: 0.159-0.452, p = 0.001). The coexistence of GA-GC polymorphisms (OR = 2.687, 95% CI: 1.387-5.205, p = 0.003) in NAT2-NAT1 genes increases the risk of HNC. Risk-reducing effect in the polymorphism GG-GT (OR = 0.340, 95% CI: 0.149-0.800, p = 0.011), GG-TT (OR = 0.077, 95% CI: 0.028-0.215, p < 0.0001), GA-TT (OR = 0.250, 95% CI: 0.100-0.622, p = 0.002), AA-GT (OR = 0.276, 95% CI: 0.112-0.676, p = 0.002) in NAT2-CYP1A genes. In the CYP2D-CYP1A genes in the polymorphisms CT-CC (OR = 0.338, 95% CI: 0.132-0.870, p = 0.020), TT-GG (OR = 0.100, 95% CI: 0.027-0.359, p = 0.001), TT-GC (OR = 0.190, 95% CI: 0.072-0.502, p = 0.0004), TT-CC (OR = 0.305, 95% CI: 0.107-0.868, p = 0.024). Correlation was noted between cigarette smoking and HNC (OR = 7.297, 95% CI: 4.989-10.674, p < 0.0001) and consuming alcohol (OR = 1.572, 95% CI: 1.003-2.464, p = 0.047). CONCLUSIONS: The CYP1A polymorphism shows a protective association with HNC. On the other hand, NAT2, NAT1 polymorphism influence the susceptibility to developing HNC. The coexistence of the NAT2-NAT1 genotypes increases the risk of HNC. In contrast, NAT1-CYP1A and CYP1A-CYP2D reduce this risk. Smoking and alcohol consumption increase the incidence of HNC. Int J Occup Med Environ Health. 2023;36(6):812-24.

N -acetyltransferase 2 haplotype modifies risks for both dyslipidemia and urinary bladder cancer.

A novel haplotype in N -acetyltransferase 2 ( NAT2 ) composed of seven non-coding variants (rs1495741, rs4921913, rs4921914, rs4921915, rs146812806, rs35246381, and rs35570672) has been linked to dyslipidemia by multiple, independent genome-wide association studies. The haplotype is located approximately 14 kb downstream of NAT2-coding region (ch8:18,272,377-18,272,881; GRCh38/hg38) and represents a non-coding, intergenic haplotype. Interestingly, the same dyslipidemia NAT2 haplotype is also linked to urinary bladder cancer risk. Dyslipidemia risk alleles are associated with rapid acetylator phenotype, whereas bladder cancer risk alleles are associated with slow acetylator, suggesting that the level of systemic NAT2 activity modifies the risk of these pathologies. We speculate that rs1495741 (and its associated haplotype) belongs to a distal regulatory element of human NAT2 gene (e.g., enhancer or silencer), and the genetic variation at the newly discovered haplotype results in a differential level of NAT2 gene expression. Understanding how this NAT2 haplotype contributes to not only urinary bladder cancer but also to dyslipidemia will ultimately help devise strategies to identify and protect susceptible individuals.

Effect of N-acetyltransferase 2 genetic polymorphism on 4,4'-methylenebis(2-chloroaniline)-induced genotoxicity and oxidative stress.

4,4'-Methylenebis(2-chloroaniline) or MOCA is an aromatic amine used primarily in polyurethane and rubber industry. MOCA has been linked to hepatomas in animal studies while limited epidemiologic studies reported the association of exposure to MOCA and urinary bladder and breast cancer. We investigated MOCA-induced genotoxicity and oxidative stress in DNA repair-deficient Chinese hamster ovary (CHO) cells stably transfected with human metabolizing enzymes CYP1A2 and N-acetyltransferase 2 (NAT2) variants as well as in rapid, intermediate, and slow NAT2 acetylator cryopreserved human hepatocytes. N-acetylation of MOCA was highest in UV5/1A2/NAT2*4 followed by UV5/1A2/NAT2*7B and UV5/1A2/NAT2*5B CHO cells. Human hepatocytes showed a NAT2 genotype-dependent response with highest N-acetylation in rapid acetylators followed by intermediate and slow acetylators. MOCA induced higher levels of mutagenesis and DNA damage in UV5/1A2/NAT2*7B compared to UV5/1A2/NAT2*4 and UV5/1A2/NAT2*5B cells (p < 0.0001). MOCA also induced higher levels of oxidative stress in UV5/1A2/NAT2*7B cells. MOCA caused concentration-dependent increase in DNA damage in cryopreserved human hepatocytes (linear trend p < 0.001) which was NAT2 genotype dependent i.e., highest in rapid acetylators, lower in intermediate acetylators, and lowest in slow acetylators (p < 0.0001). Our findings show that N-acetylation and genotoxicity of MOCA is NAT2 genotype dependent and suggest that individuals possessing NAT2*7B are at higher risk to MOCA-induced mutagenicity. DNA damage, and oxidative stress. They confirm significant differences in genotoxicity between the NAT2*5B and NAT2*7B alleles, both of which are associated with slow acetylator phenotype.

Effects of dose and human N-acetyltransferase 1 genetic polymorphism in benzidine metabolism and genotoxicity.

Benzidine undergoes N-acetylation and following CYP1A2-catalyzed N-hydroxylation undergoes O-acetylation catalyzed by N-acetyltransferase 1 (NAT1). Benzidine exposure is associated with urinary bladder cancer but the effect of NAT1 genetic polymorphism on individual risk remains unclear. We used Chinese hamster ovary (CHO) cells transfected with human CYP1A2 and NAT1*4 allele (reference) or NAT1*14B (variant) to investigate the effects of dose and NAT1 polymorphism on benzidine metabolism and genotoxicity. Rates of benzidine N-acetylation in vitro were higher in CHO cells transfected with NAT1*4 compared to NAT1*14B. CHO cells transfected with NAT1*14B exhibited greater N-acetylation rates in situ than cells transfected with NAT1*4 at low doses of benzidine expected with environmental exposures but not at higher doses. NAT1*14B exhibited over tenfold lower apparent KM which resulted in higher intrinsic clearance for benzidine N-acetylation compared to CHO cells transfected with NAT1*4. Benzidine-induced hypoxanthine phosphoribosyl transferase (HPRT) mutations were higher in CHO cells transfected with NAT1*14B than with NAT1*4 (p < 0.001). Benzidine caused concentration-dependent increase in γ-H2AX signal (indicative of DNA double-strand breaks) in CHO cells transfected with NAT1*4 or NAT1*14B. CHO cells transfected with NAT1*14B exhibited significantly higher level of DNA damage than with NAT1*4 (p < 0.0001). Benzidine-induced ROS did not differ significantly (p > 0.05) between CHO cells transfected with NAT1*4 or NAT1*14B except at 50 µM. Levels of benzidine-induced DNA damage and reactive oxygen species (ROS) showed strong dose-dependent correlation. Our findings support human studies associating NAT1*14B with increased incidence or severity of urinary bladder cancer in workers exposed to benzidine.

Altered Arylamine N-acetyltransferase 1 and miR-1290 Levels in Childhood Acute Lymphoblastic Leukemia: A Pilot Study.

BACKGROUND/AIM: Arylamine N-acetyltransferase 1 and 2 (NAT1 and NAT2) are drug-metabolizing enzymes that play a key role in the development of acute lymphoblastic leukemia (ALL). MATERIALS AND METHODS: This study evaluated NAT1 and NAT2 mRNA and protein expression and their enzymatic activity in peripheral blood mononuclear cells (PBMC) from patients with ALL (n=20) and healthy children (n=19) and explored the mechanisms that regulate these enzymes in ALL such as microRNAs (miR-1290, miR-26b) and SNPs. RESULTS: PBMC from patients with ALL showed a decrease in NAT1 mRNA and protein expression. In addition, NAT1 enzymatic activity was decreased in patients with ALL. There was no influence of SNP 559 C>T or 560 G>A on low NAT1 activity. The lower expression of NAT1 might be related to the loss of acetylated histone H3K14 in the NAT1 gene promoter in patients with ALL and the higher relative expression of miR-1290 in the plasma of patients with relapsed ALL compared with healthy controls. There were significantly fewer CD3+/NAT1+ double-positive cells in patients who relapsed compared with control subjects. Based on a t-distributed stochastic neighbor embedding algorithm, CD19+ cells that reappeared in patients with relapse showed low NAT1 expression. In contrast, for NAT2, there were no significant results. CONCLUSION: The expression and function of NAT1 and miR-1290 levels could be involved in modulating immune cells altered in ALL.

NAT2 global landscape: Genetic diversity and acetylation statuses from a systematic review.

Arylamine N-acetyltransferase 2 has been related to drug side effects and cancer susceptibility; its protein structure and acetylation capacity results from the polymorphism's arrays on the NAT2 gene. Absorption, distribution, metabolism, and excretion, cornerstones of the pharmacological effects, have shown diversity patterns across populations, ethnic groups, and even interethnic variation. Although the 1000 Genomes Project database has portrayed the global diversity of the NAT2 polymorphisms, several populations and ethnicities remain underrepresented, limiting the comprehensive picture of its variation. The NAT2 clinical entails require a detailed landscape of its striking diversity. This systematic review spans the genetic and acetylation patterns from 164 articles from October 1992 to October 2020. Descriptive studies and controls from observational studies expanded the NAT2 diversity landscape. Our study included 243 different populations and 101 ethnic minorities, and, for the first time, we presented the global patterns in the Middle Eastern populations. Europeans, including its derived populations, and East Asians have been the most studied genetic backgrounds. Contrary to the popular perception, Africans, Latinos and Native Americans have been significantly represented in recent years. NAT2*4, *5B, and *6A were the most frequent haplotypes globally. Nonetheless, the distribution of *5B and *7B were less and more frequent in Asians, respectively. Regarding the acetylator status, East Asians and Native Americans harboured the highest frequencies of the fast phenotype, followed by South Europeans. Central Asia, the Middle East, and West European populations were the major carriers of the slow acetylator status. The detailed panorama presented herein, expands the knowledge about the diversity patterns to genetic and acetylation levels. These data could help clarify the controversial findings between acetylator states and the susceptibility to diseases and reinforce the utility of NAT2 in precision medicine.

Upregulation of cytidine deaminase in NAT1 knockout breast cancer cells.

PURPOSE: Arylamine N-acetyltransferase 1 (NAT1), a phase II metabolic enzyme, is frequently upregulated in breast cancer. Inhibition or depletion of NAT1 leads to growth retardation in breast cancer cells in vitro and in vivo. A previous metabolomics study of MDA-MB-231 breast cancer cells suggests that NAT1 deletion leads to a defect in de novo pyrimidine biosynthesis. In the present study, we observed that NAT1 deletion results in upregulation of cytidine deaminase (CDA), which is involved in the pyrimidine salvage pathway, in multiple breast cancer cell lines (MDA-MB-231, MCF-7 and ZR-75-1). We hypothesized that NAT1 KO MDA-MB-231 cells show differential sensitivity to drugs that either inhibit cellular pyrimidine homeostasis or are metabolized by CDA. METHODS: The cells were treated with (1) inhibitors of dihydroorotate dehydrogenase or CDA (e.g., teriflunomide and tetrahydrouridine); (2) pyrimidine/nucleoside analogs (e.g., gemcitabine and 5-azacytidine); and (3) naturally occurring, modified cytidines (e.g., 5-formyl-2'-deoxycytidine; 5fdC). RESULTS: Although NAT1 KO cells failed to show differential sensitivity to nucleoside analogs that are metabolized by CDA, they were markedly more sensitive to 5fdC which induces DNA damage in the presence of high CDA activity. Co-treatment with 5fdC and a CDA inhibitor, tetrahydrouridine, abrogated the increase in 5fdC cytotoxicity in NAT1 KO cells, suggesting that the increased sensitivity of NAT1 KO cells to 5fdC is dependent on their increased CDA activity. CONCLUSIONS: The present findings suggest a novel therapeutic strategy to treat breast cancer with elevated NAT1 expression. For instance, NAT1 inhibition may be combined with cytotoxic nucleosides (e.g., 5fdC) for breast cancer treatment.

The rs1801280 SNP is associated with non-small cell lung carcinoma by exhibiting a highly deleterious effect on N-acetyltransferase 2.

PURPOSE: N-acetyltransferase 2 is an enzyme that is involved in the detoxification of carcinogens in the human body, so any damage to this protein may lead to the emergence of several metabolic dysfunctions. This work was conducted to determine the association between NAT2 polymorphism and non-small cell lung carcinoma (NSCLC) that is increasingly reported in the Iraqi population. METHODS: PCR sequencing was conducted to assess the possible association between genetic variants and NSCLC. Several in silico tools were implemented to investigate the effect of the observed SNPs on the structure, function, and stability of the altered NAT2. RESULTS: Five SNPS of NAT2 (rs1208, rs1041983, rs1799929, rs1799930, and rs1801280) were identified in high frequencies in the amplified fragment. These SNPs showed variable distributions of haplotypes between cases and controls. No significant association of rs1208, rs1041983, rs1799929, and rs1799930 with NSCLC was shown in the investigated population. In contrast, rs1801280: CC genotype showed a highly significant (P = 0.009) association with the NSCLC, and individuals with this genotype had 2.19 more chances for developing NSCLC (OR 2.19; Cl95% 1.21-3.94). Association analysis of rs1801280 SNP distribution among the investigated patients showed that patients with CC genotype showed a significant (P = 0.02, OR 2.65) association with family history, which entailed a high hereditary possibility of this genotype among Iraqi patients. It was predicted that this SNP showed high damaging effects on the activity of NAT2 enzyme, with various deleterious outcomes on enzyme structure, function, and stability. CONCLUSION: Data indicated that rs1801280 SNP exerted a tight association with NSCLC since individuals with CC genotype exhibited the most damaging effects on the NAT2 that may be behind the low acetylation rates of this enzyme in patients with NSCLC.

N-acetyltransferase 2 genetic polymorphism modifies genotoxic and oxidative damage from new psychoactive substances.

The use of new psychoactive substances (NPS) as drugs of abuse is common and increasingly popular, particularly among youth and neglected communities. Recent studies have reported acute toxic effects from these chemicals; however, their long-term toxicity is unknown. Genetic differences between individuals likely affect the toxicity risk. Arylamine N-acetyltransferase 2 (NAT2) capacity differs among individuals due to genetic inheritance. The goal of the present study is to investigate the gene-environment interaction between NAT2 polymorphism and toxicity after exposure to these chemicals. We measured N-acetylation by human NAT1 and NAT2 and found that N-acetylation of NPS is carried out exclusively by NAT2. Differences in N-acetylation between NAT2*4 (reference allele) and NAT2*5B (common variant allele) were highly significant (p < 0.0001). Using DNA repair-deficient genetically engineered Chinese hamster ovary (CHO cells), expressing human CYP1A2 and either NAT2*4 or NAT2*5B, we measured the induction of DNA double-strand breaks ([Formula: see text]H2Ax) following treatment of the CHO cells with increasing concentrations of NPS. The induction of [Formula: see text]H2Ax showed a NAT2 allele-dependent response, higher in the NAT2*4 vs NAT2*5B alleles (p < 0.05). Induction of oxidative stress (ROS/RNS) was evaluated; we observed NAT2 allele-dependent response for all compounds in concentrations as low as 10 [Formula: see text]M, where NAT2*4 showed increased ROS/RNS vs NAT2*5B (p < 0.05). In summary, NPS are N-acetylated by NAT2 at rates higher in cells expressing NAT2*4 than NAT2*5B. Exposure to psychoactive chemicals results in genotoxic and oxidative damage that is modified by the NAT2 genetic polymorphism.

Transcriptional Regulation of Human Arylamine N-Acetyltransferase 2 Gene by Glucose and Insulin in Liver Cancer Cell Lines.

Arylamine N-acetyltransferase 2 (NAT2) is well-known for its role in phase II metabolism of xenobiotics and drugs. More recently, genome wide association studies and murine models implicated NAT2 in regulation of insulin sensitivity and plasma lipid levels. However, the mechanism remains unknown. Transcript levels of human NAT2 varied dynamically in HepG2 (hepatocellular) cells, depending on the nutrient status of the culture media. Culturing the cells in the presence of glucose induced NAT2 mRNA expression as well as its N-acetyltransferase activity significantly. In addition, insulin or acetate treatment also significantly induced NAT2 mRNA. We examined and compared the glucose- and acetate-dependent changes in NAT2 expression to those of genes involved in glucose and lipid metabolism, including FABP1, CPT1A, ACACA, SCD, CD36, FASN, ACLY, G6PC, and PCK1. Genes that are involved in fatty acid transport and lipogenesis, such as FABP1 and CD36, shared a similar pattern of expression with NAT2. In silico analysis of genes co-expressed with NAT2 revealed an enrichment of biological processes involved in lipid and cholesterol biosynthesis and transport. Among these, A1CF (APOBEC1 complementation factor) showed the highest correlation with NAT2 in terms of its expression in normal human tissues. The current study shows, for the first time, that human NAT2 is transcriptionally regulated by glucose and insulin in liver cancer cell lines and that the gene expression pattern of NAT2 is similar to that of genes involved in lipid metabolism and transport.

N-acetyltransferase 2 acetylator genotype-dependent N-acetylation and toxicity of the arylamine carcinogen ß-naphthylamine in cryopreserved human hepatocytes.

We used cryopreserved human hepatocytes that express rapid, intermediate, and slow acetylator N-acetyltransferase 2 (NAT2) genotypes to measure the N-acetylation of ß-naphthylamine (BNA) which is one of the aromatic amines found in cigarette smoke including E-cigarettes. We investigated the role of NAT2 genetic polymorphism in genotoxicity and oxidative stress induced by BNA. In vitro BNA NAT2 activities in rapid acetylators was 1.6 and 3.5-fold higher than intermediate (p < 0.01) and slow acetylators (p < 0.0001). BNA N-acetylation in situ was 3 to 4- fold higher in rapid acetylators than slow acetylators, following incubation with 10 and 100 µM BNA (p < 0.01). DNA damage was two to threefold higher in the rapid versus slow acetylators (p < 0.0001) and 2.5-fold higher in intermediate versus slow acetylators following BNA treatment at 100 and 1000 µM, ROS/RNS level was the highest in rapid acetylators followed by intermediate and then slow acetylators (p < 0.0001). Our findings show that the N-acetylation of BNA is NAT2 genotype dependent in cryopreserved human hepatocytes and our data further document an important role for NAT2 genetic polymorphism in modifying BNA-induced genotoxicity and oxidative damage.

Differences in ß-naphthylamine metabolism and toxicity in Chinese hamster ovary cell lines transfected with human CYP1A2 and NAT2*4, NAT2*5B or NAT2*7B N-acetyltransferase 2 haplotypes.

ß-naphthylamine (BNA) is an important aromatic amine carcinogen. Current exposures derive primarily from cigarette smoking including e-cigarettes. Occupational and environmental exposure to BNA is associated with urinary bladder cancer which is the fourth most frequent cancer in the United States. N-acetyltransferase 2 (NAT2) is an important metabolizing enzyme for aromatic amines. Previous studies investigated mutagenicity and genotoxicity of BNA in bacteria and in rabbit or rat hepatocytes. However, the effects of human NAT2 genetic polymorphism on N-acetylation and genotoxicity induced by BNA still need to be clarified. We used nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells that were stably transfected with human CYP1A2 and NAT2 alleles: NAT2*4 (reference allele), NAT2*5B (variant slow acetylator allele common in Europe) or NAT2*7B (variant slow acetylator allele common in Asia). BNA N-acetylation was measured both in vitro and in situ via high-performance liquid chromatography (HPLC). Hypoxanthine phosphoribosyl transferase (HPRT) mutations, double-strand DNA breaks, and reactive oxygen species (ROS) were measured as indices of toxicity. NAT2*4 cells showed significantly higher BNA N-acetylation rates followed by NAT2*7B and NAT2*5B. BNA caused concentration-dependent increases in DNA damage and ROS levels. NAT2*7B showed significantly higher levels of HPRT mutants, DNA damage and ROS than NAT2*5B (p < 0.001, p < 0.0001, p < 0.0001 respectively) although both are slow alleles. Our findings suggest that BNA N-acetylation and toxicity are modified by NAT2 polymorphism. Furthermore, they confirm heterogeneity among slow acetylator alleles for BNA metabolism and toxicity supporting differential risk for individuals carrying NAT2*7B allele.

Enhanced cytotoxicity of a novel family of ATPase inhibitors in colorectal cancer cells with high NAT2 activity.

Loss of heterozygosity (LOH) is a hallmark feature of cancer genomes that reduces allelic variation, thereby creating tumor specific vulnerabilities which could be exploited for therapeutic purposes. We previously reported that loss of drug metabolic arylamine N-acetyltransferase 2 (NAT2) activity following LOH at 8p22 could be targeted for collateral lethality anticancer therapy in colorectal cancer (CRC). Here, we report a novel compound CBK034026C that exhibits specific toxicity towards CRC cells with high NAT2 activity. Connectivity Map analysis revealed that CBK034026C elicited a response pattern related to ATPase inhibitors. Similar to ouabain, a potent inhibitor of the Na+/K+-ATPase, CBK034026C activated the Nf-kB pathway. Further metabolomic profiling revealed downregulation of pathways associated with antioxidant defense and mitochondrial metabolism in CRC cells with high NAT2 activity, thereby weakening the protective response to oxidative stress induced by CBK034026C. The identification of a small molecule targeting metabolic vulnerabilities caused by NAT2 activity provides novel avenues for development of anticancer agents.

Hexavalent chromium increases the metabolism and genotoxicity of aromatic amine carcinogens 4-aminobiphenyl and ß-naphthylamine in immortalized human lung epithelial cells.

Humans are exposed to carcinogenic chemicals via occupational and environmental exposures. Common chemicals of concern that can occur in exposures together are aromatic amines (e.g., 4-aminobiphenyl [4-ABP] and ß-naphthylamine [BNA]) and hexavalent chromium (Cr[VI]). Arylamine N-acetyltransferases 1 and 2 (NAT1 and NAT2) are key to the metabolism of aromatic amines and their genotoxicity. The effects of Cr(VI) on the metabolism of aromatic amines remains unknown as well as how it may affect their ensuing toxicity. The objective of the research presented here is to investigate the effects of Cr(VI) on the metabolism and genotoxicity of 4-ABP and BNA in immortalized human lung epithelial cells (BEP2D) expressing NAT1 and NAT2. Exposure to Cr(VI) for 48 h increased NAT1 activity (linear regression analysis: P < 0.0001) as measured by N-acetylation of para-aminobenzoic acid (PABA) in BEP2D cells but not NAT2 N-acetylation of sulfamethazine, which are prototypic NAT1 and NAT2 substrates respectively. Cr(VI) also increased the N-acetylation of 4-ABP and BNA. In BEP2D cells the N-acetylation of 4-ABP (1-3 µM) exhibited a dose-dependent increase (linear regression analysis: P < 0.05) following co-incubation with 0-3 µM Cr(VI). In BEP2D cells, incubation with Cr(VI) caused dose-dependent increases (linear regression analysis: P < 0.01) in expression of CYP1A1 protein and catalytic activity. For genotoxicity, BEP2D cells were exposed to 4-ABP or BNA with/without Cr(VI) for 48 h. We observed dose-dependent increases (linear regression analysis: P < 0.01) in phospho-γH2AX protein expression for combined treatment of 4-ABP or BNA with Cr(VI). Further using a CYP1A1 inhibitor (α-naphthoflavone) and NAT1 siRNA, we found that CYP1A1 inhibition did not reduce the increased N-acetylation or genotoxicity of BNA by Cr(VI), while NAT1 inhibition did reduce increases in BNA N-acetylation and genotoxicity by Cr(VI). We conclude that during co-exposure of aromatic amines and Cr(VI) in human lung cells, Cr(VI) increased NAT1 activity contributing to increased 4-ABP and BNA genotoxicity.

The role of arylamine N-acetyltransferases in chronic degenerative diseases: Their possible function in the immune system.

Since their discovery, arylamine N-acetyltransferases 1 and 2 (NAT1 and NAT2, respectively) have been associated with the metabolism of xenobiotics. NAT2 is the main factor in the therapeutic success of tuberculosis treatment due to its role in the biotransformation of isoniazid. However, researchers have started to investigate the possible participation of NAT1 and NAT2 (NATs) in carcinogenesis, although the mechanisms have not been elucidated fully. NATs enzymatic activity is essential in some types of cancer, such as breast cancer and acute lymphoblastic leukemia. Whether NAT1 and/or NAT2 participate in insulin resistance level in diabetes mellitus or in the immune system remains to be explored. Therefore, it is clear that its role in cell physiology has more implications than just metabolizing compounds.

The interaction between smoking and bladder cancer genetic variants on urothelial cancer risk by disease aggressiveness.

BACKGROUND: Smoking has shown interactions with bladder cancer (BC) genetic variants, especially N-acetyltransferase-2 (NAT2), a tobacco smoke metabolism gene, on BC risk. The interactions by disease aggressiveness are unknown. METHODS: We investigated the interaction between smoking and 18 single nucleotide polymorphisms (SNPs) for BC, individually and in a genetic risk score (GRS), on urothelial cancer (UC) risk including BC. We analysed data from 25,453 individuals with 520 incident UCs during follow-up, 339 non-aggressive (non-fatal, non-muscle invasive) and 163 aggressive (all other) UCs. Hazard ratios (HRs), absolute risks and additive and multiplicative interactions for two-by-two combinations of never/ever smoking with low/high genetic risk were calculated. RESULTS: Smoking and NAT2 rs1495741 interacted strongly, positively on aggressive UC on both the multiplicative (p = 0.004) and additive (p = 0.0002) scale, which was not observed for non-aggressive UC (pinteractions  ≥ 0.6). This manifested in a higher HR of aggressive UC by ever smoking for the slow acetylation NAT2 genotype (HR, 5.00 [95% confidence interval, 2.67-9.38]) than for intermediate/fast acetylation NAT2 (HR, 1.50 [0.83-2.71]), and in differences in absolute risks by smoking and NAT2 genotype. Smoking also interacted additively and positively with the GRS on any UC (p = 0.01) and non-aggressive UC (p = 0.02), but not on aggressive UC (p = 0.1). Gene-smoking interactions of lesser magnitude than for NAT2 were found for SNPs in APOBEC3A, SLC14A1 and MYNN. CONCLUSIONS: This study suggests that smoking increases UC risk more than expected when combined with certain genetic risks. Individuals with the slow acetylation NAT2 variant might particularly benefit from smoking intervention to prevent lethal UC; however, replication in larger studies is needed.

Expression of arylamine N-acetyltransferase 2 activity in immortalized human bronchial epithelial cells.

Lung cancer is the leading cause of cancer deaths in the United States with high incidence in tobacco smokers. Arylamine N-acetyltransferase 2 (NAT2) is a xenobiotic enzyme that catalyzes both N- and O-acetylation of carcinogens present in tobacco smoke and contributes towards the genotoxicity of these carcinogens. NAT2 allelic variants result in slow, intermediate, and rapid acetylation phenotypes. A recent meta-analysis reported NAT2 non-rapid (slow and intermediate) phenotypes had a significantly increased risk of lung cancer. NAT2 activity in humans is thought to be restricted to liver and gastrointestinal tract, and no studies to our knowledge have reported the expression of NAT2 activity in immortalized human lung epithelial cells. Given the importance of NAT2 in cancer and inhalation of various carcinogens directly into the lungs, we investigated NAT2 activity in human lung epithelial cells. Both NAT1 and NAT2 protein were detected by "in-cell" Western. Arylamine N-acetyltransferase activity was determined with selective substrates for NAT1 (p-aminobenzoic acid; PABA) and NAT2 (sulfamethazine; SMZ) in the presence and absence of a selective NAT1 inhibitor. PABA N-acetylation (NAT1 activity) in cell protein lysates was abolished in the presence of 25 µM of NAT1 inhibitor whereas SMZ N-acetylation (NAT2) was unaffected. Incubation with the NAT1 inhibitor partially reduced the N-acetylation of ß-naphthylamine and the O-acetylation of N-hydroxy-4-aminobiphenyl consistent with catalysis by both NAT1 and NAT2. Immortalized human lung epithelial cells exhibited dose-dependent N-acetylation of 4-ABP with an apparent KM of 24.4 ± 5.1 µM. These data establish that NAT2 is expressed and functional in immortalized human lung epithelial cells and will help us further our understanding of NAT2 in lung cancer.