RESUMO
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.
Assuntos
Arilamina N-Acetiltransferase , Metilenobis (cloroanilina) , Cricetinae , Animais , Humanos , Cricetulus , Células CHO , Arilamina N-Acetiltransferase/genética , Arilamina N-Acetiltransferase/metabolismo , Dano ao DNA , Acetiltransferases/genética , Genótipo , Estresse Oxidativo , Polimorfismo Genético , AcetilaçãoRESUMO
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.
Assuntos
Arilamina N-Acetiltransferase , Neoplasias da Bexiga Urinária , Cricetinae , Animais , Humanos , Citocromo P-450 CYP1A2/metabolismo , Cricetulus , Células CHO , Espécies Reativas de Oxigênio , Arilamina N-Acetiltransferase/genética , Arilamina N-Acetiltransferase/metabolismo , Dano ao DNA , Polimorfismo Genético , Benzidinas/toxicidade , AcetilaçãoRESUMO
N-acetyltransferase 1 (NAT1) is a xenobiotic metabolizing enzyme that uses acetyl coenzyme A (AcCoA) as a cofactor for N-acetylation of many carcinogens including aromatic amines and alkylanilines. NAT1 is characterized by single nucleotide polymorphisms (SNPs) that may modulate affinity towards AcCoA. In the current study, we used Chinese hamster ovary (CHO) cells stably transfected with human NAT1*4 (reference allele) or NAT1*14B (variant allele) to measure AcCoA kinetic parameters for N-acetyltransferase activity measurements towards p-aminobenzoic acid (PABA), 4-aminobiphenyl (4-ABP), ß-naphthylamine (BNA), benzidine and 3,4-dimethylaniline (3,4-DMA). Our results showed higher N-acetylation rates for each substrate catalyzed by NAT1*4 compared to NAT1*14B. NAT1*4 exhibited higher affinity to AcCoA when catalyzing the N-acetylation of BNA and benzidine compared to NAT1*14B. The results of the current study provide further insights into differences in carcinogen metabolism among individuals possessing the NAT1*14B haplotype.
RESUMO
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.
Assuntos
Arilamina N-Acetiltransferase , Sistemas Eletrônicos de Liberação de Nicotina , Humanos , Carcinógenos/toxicidade , Arilamina N-Acetiltransferase/genética , Arilamina N-Acetiltransferase/metabolismo , 2-Naftilamina , Acetilação , Espécies Reativas de Oxigênio , Genótipo , Hepatócitos/metabolismo , Acetiltransferases/genética , AminasRESUMO
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.
Assuntos
Arilamina N-Acetiltransferase , Sistemas Eletrônicos de Liberação de Nicotina , 2-Naftilamina , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Arilamina N-Acetiltransferase/genética , Arilamina N-Acetiltransferase/metabolismo , Células CHO , Carcinógenos/toxicidade , Cricetinae , Cricetulus , Citocromo P-450 CYP1A2/metabolismo , Genótipo , Haplótipos , Humanos , Hipoxantina Fosforribosiltransferase/genética , Hipoxantinas , Coelhos , Ratos , Espécies Reativas de OxigênioRESUMO
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.
Assuntos
Arilamina N-Acetiltransferase , Carcinógenos , 2-Naftilamina , Acetilação , Acetiltransferases/metabolismo , Aminas/toxicidade , Compostos de Aminobifenil , Arilamina N-Acetiltransferase/genética , Arilamina N-Acetiltransferase/metabolismo , Carcinógenos/metabolismo , Carcinógenos/toxicidade , Cromo , Citocromo P-450 CYP1A1/metabolismo , Células Epiteliais/metabolismo , Humanos , Isoenzimas/genética , Pulmão/metabolismoRESUMO
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.
Assuntos
Arilamina N-Acetiltransferase , Neoplasias Pulmonares , Ácido 4-Aminobenzoico/metabolismo , Acetilação , Arilamina N-Acetiltransferase/genética , Arilamina N-Acetiltransferase/metabolismo , Carcinógenos/metabolismo , Células Epiteliais/metabolismo , Humanos , Isoenzimas/genéticaRESUMO
Arylamine N-acetyltransferases catalyze the transfer of acetyl groups from the endogenous cofactor acetyl coenzyme A (AcCoA) to arylamine (N-acetylation) and N-hydroxy-arylamine (O-acetylation) acceptors. Humans express two arylamine N-acetyltransferase isozymes (NAT1 and NAT2) which catalyze both N- and O-acetylation but differ in genetic regulation, substrate selectivity, and expression in human tissues. We investigated recombinant human NAT1 and NAT2 expressed in an Escherichia coli JM105 and Schizosaccharomyces pombe expression systems as well as in Chinese hamster ovary (CHO) cells to assess the relative affinity of AcCoA for human NAT1 and NAT2. NAT1 and NAT2 affinity for AcCoA was higher for recombinant human NAT1 than NAT2 when catalyzing N-acetylation of aromatic amine carcinogens 2-aminofluroene (AF), 4-aminobiphenyl (ABP), and ß-naphthylamine (BNA) and the metabolic activation of N-hydroxy-2-aminofluorene (N-OH-AF) and N-hydroxy-4-aminobiphenyl (N-OH-ABP) via O-acetylation. These results suggest that AcCoA level may influence differential rates of arylamine carcinogen metabolism catalyzed by NAT1 and NAT2 in human tissues. Affinity was higher for NAT2 than for NAT1 using N-OH-AF and N-OH-ABP as substrate consistent with a larger active site for NAT2. In conclusion, following recombinant expression in bacteria, yeast, and CHO cells, we report significant differences in affinity between human NAT1 and NAT2 for its required co-factor AcCoA, as well as for N-hydroxy-arylamines activated via O-acetylation. The findings provide important information to understand the relative contribution of human NAT1 vs NAT2 towards N-acetylation and O-acetylation reactions in human hepatic and extrahepatic tissues.
RESUMO
Arylamine N-acetyltransferase 1 (NAT1) plays a pivotal role in the metabolism of carcinogens and is a drug target for cancer prevention and/or treatment. A protein-ligand virtual screening of 2 million chemicals was ranked for predicted binding affinity towards the inhibition of human NAT1. Sixty of the five hundred top-ranked compounds were tested experimentally for inhibition of recombinant human NAT1 and N-acetyltransferase 2 (NAT2). The most promising compound 9,10-dihydro-9,10-dioxo-1,2-anthracenediyl diethyl ester (compound 10) was found to be a potent and selective NAT1 inhibitor with an in vitro IC50 of 0.75 µM. Two structural analogs of this compound were selective but less potent for inhibition of NAT1 whereas a third structural analog 1,2-dihydroxyanthraquinone (a compound 10 hydrolysis product also known as Alizarin) showed comparable potency and efficacy for human NAT1 inhibition. Compound 10 inhibited N-acetylation of the arylamine carcinogen 4-aminobiphenyl (ABP) both in vitro and in DNA repair-deficient Chinese hamster ovary (CHO) cells in situ stably expressing human NAT1 and CYP1A1. Compound 10 and Alizarin effectively inhibited NAT1 in cryopreserved human hepatocytes whereas inhibition of NAT2 was not observed. Compound 10 caused concentration-dependent reductions in DNA adduct formation and DNA double-strand breaks following metabolism of aromatic amine carcinogens beta-naphthylamine and/or ABP in CHO cells. Compound 10 inhibited proliferation and invasion in human breast cancer cells and showed selectivity towards tumorigenic versus non-tumorigenic cells. In conclusion, our study identifies potent, selective, and efficacious inhibitors of human NAT1. Alizarin's ability to inhibit NAT1 could reduce breast cancer metastasis particularly to bone.
Assuntos
Arilamina N-Acetiltransferase/antagonistas & inibidores , Neoplasias da Mama/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Isoenzimas/antagonistas & inibidores , Animais , Antraquinonas/farmacologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Células CHO , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Simulação por Computador , Cricetinae , Cricetulus , Adutos de DNA/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Hepatócitos/enzimologia , Humanos , Concentração Inibidora 50RESUMO
Recent reports suggest that arylamine N-acetyltransferases (NAT1 and/or NAT2) serve important roles in regulation of energy utility and insulin sensitivity. We investigated the interaction between diet (control vs. high-fat diet) and acetylator phenotype (rapid vs. slow) using previously established congenic rat lines (in F344 background) that exhibit rapid or slow Nat2 (orthologous to human NAT1) acetylator genotypes. Male and female rats of each genotype were fed control or high-fat (Western-style) diet for 26 weeks. We then examined diet- and acetylator genotype-dependent changes in body and liver weights, systemic glucose tolerance, insulin sensitivity, and plasma lipid profile. Male and female rats on the high fat diet weighed approximately 10% more than rats on the control diet and the percentage liver to body weight was consistently higher in rapid than slow acetylator rats. Rapid acetylator rats were more prone to develop dyslipidemia overall (i.e., higher triglyceride; higher LDL; and lower HDL), compared to slow acetylator rats. Total cholesterol (TC)-to-HDL ratios were significantly higher and HDL-to-LDL ratios were significantly lower in rapid acetylator rats. Our data suggest that rats with rapid systemic Nat2 (NAT1 in humans) genotype exhibited higher dyslipidemia conferring risk for metabolic syndrome and cardiovascular dysfunction.
RESUMO
Arylamine N-acetyltransferases are xenobiotic-metabolizing enzymes responsible for detoxification of many drugs and carcinogens. Two N-acetyltransferase proteins (NAT1 and NAT2) are expressed in humans and they both N-acetylate aromatic amine carcinogens such as 4-aminobiphenyl. Arylamines such as 4-aminobiphenyl represent a large class of chemical carcinogens. Exposure to 4-aminobiphenyl occurs in the chemical, dye and rubber industries as well as in hair dyes, paints, and cigarette smoke. NAT2 is subject to a genetic polymorphism resulting in rapid, intermediate and slow acetylator phenotypes. We investigated the role of the NAT2 genetic polymorphisms on the N-acetylation of 4-aminobiphenyl in cryopreserved human hepatocytes in which NAT2 genotype and deduced phenotype were determined. Differences in sulfamethazine (selectively N-acetylated via NAT2) and 4-aminobiphenyl (N-acetylated by both NAT1 and NAT2) N-acetylation rates among rapid, intermediate, and slow NAT2 acetylator genotypes were tested for significance by one-way analysis of variance. In vitro 4-aminobiphenyl N-acetyltransferase activities differed significantly between rapid, intermediate and slow acetylators at 10 µM (P = 0.0102) or 100 µM (P = 0.0028). N-acetylation of 4-aminobiphenyl in situ also differed significantly between human hepatocytes from rapid, intermediate, and slow acetylators at 10 µM (P = 0.0015) and 100 µM (P = 0.0216). A gene dose-response relationship was exhibited as intermediate acetylators catalyzed 4-aminobiphenyl N-acetylation both in vitro and in situ at rates arithmetically between rapid and slow acetylators. In conclusion, N-acetylation of 4-aminobiphenyl is NAT2 genotype-dependent in human hepatocytes. These results suggest refinement of the exposure limit and safety for arylamine carcinogens according to NAT2 genotype.