David W. Hein

NAT2 slow acetylation, GSTM1 null genotype, and risk of bladder cancer: results from the Spanish Bladder Cancer Study and meta-analyses

BACKGROUND Many reported associations between common genetic polymorphisms and complex diseases have not been confirmed in subsequent studies. An exception could be the association between NAT2 slow acetylation, GSTM1 null genotype, and bladder-cancer risk. However, current evidence is based on meta-analyses of relatively small studies (range 23-374 cases) with some evidence of publication bias and study heterogeneity. Associations between polymorphisms in other NAT and GST genes and bladder-cancer risk have been inconsistent. METHODS We investigated polymorphisms in NAT2, GSTM1, NAT1, GSTT1, GSTM3, and GSTP1 in 1150 patients with transitional-cell carcinoma of the urinary bladder and 1149 controls in Spain; all the participants were white. We also carried out meta-analyses of NAT2, GSTM1, and bladder cancer that included more than twice as many cases as in previous reports. FINDINGS In our study, the odds ratios for bladder cancer for individuals with deletion of one or two copies of the GSTM1 gene were 1.2 (95% CI 0.8-1.7) and 1.9 (1.4-2.7) respectively (p for trend <0.0001). Compared with NAT2 rapid or intermediate acetylators, NAT2 slow acetylators had an increased overall risk of bladder cancer (1.4 [1.2-1.7]) that was stronger for cigarette smokers than for never smokers (p for interaction 0.008). No significant associations were found with the other polymorphisms. Meta-analyses showed that the overall association for NAT2 was robust (p<0.0001), and case-only meta-analyses provided support for an interaction between NAT2 and smoking (p for interaction 0.009). The overall association for GSTM1 was also robust (p<0.0001) and was not modified by smoking status (p=0.86). INTERPRETATION The GSTM1 null genotype increases the overall risk of bladder cancer, and the NAT2 slow-acetylator genotype increases risk particularly among cigarette smokers. These findings provide compelling evidence for the role of common polymorphisms in the aetiology of cancer. RELEVANCE TO PRACTICE Although the relative risks are modest, these polymorphisms could account for up to 31% of bladder cancers because of their high prevalence.

Molecular genetics and function of NAT1 and NAT2: role in aromatic amine metabolism and carcinogenesis

Aromatic and heterocyclic amines require metabolic activation to electrophilic intermediates that initiate carcinogenesis. N-Acetyltransferase 1 (NAT1) and 2 (NAT2) are important enzymes in the biotransformation of these carcinogens and exhibit genetic polymorphism. Human NAT1 and NAT2 alleles are listed at: http://www.louisville.edu/medschool/pharmacology/NAT.html by an international gene nomenclature committee. The high frequency of the NAT1 and NAT2 acetylation polymorphisms in human populations together with ubiquitous exposure to aromatic and heterocyclic amines suggest that NAT1 and NAT2 acetylator genotypes are important modifiers of human cancer susceptibility. For cancers in which N-acetylation is a detoxification step such as aromatic amine-related urinary bladder cancer, NAT2 slow acetylator phenotype is at higher risk. Multiple studies have shown that the urinary bladder cancer risk is particularly high in the slowest NAT2 acetylator phenotype or genotype (NAT2(*)5). In contrast, for cancers in which N-acetylation is negligible and O-acetylation is an activation step such as for heterocyclic amine-related colon cancer, NAT2 rapid acetylator phenotype is at higher risk. Although studies have found associations between NAT1 genotype and various cancers, the findings are less consistent and are not well understood. Since cancer risk requires exposure to aromatic and/or heterocyclic amine carcinogens modified by NAT1 and/or NAT2 acetylator genotype, the results from human epidemiology studies are dependent upon the quality and accuracy of the exposure assessment and genotype determination. Conclusions require understanding the relationship between genotype and phenotype, as well as the role of genetic variation in carcinogen metabolism, DNA repair, and host susceptibility. Investigations have been carried out in rapid and slow acetylator rodent models in which both exposure and genetic variability are tightly controlled. Human NAT1 and NAT2 alleles have been characterized by recombinant expression to further understand the effects of nucleotide polymorphisms on function and phenotype.

Nomenclature for N-acetyltransferases.

A consolidated classification system is described for prokaryotic and eukaryotic N-acetyltransferases in accordance with the international rules for gene nomenclature. The root symbol (NAT) specifically identifies the genes that code for the N-acetyltransferases, and NAT* loci encoding proteins with similar function are distinguished by Arabic numerals. Allele characters, denoted by Arabic numbers or by a combination of Arabic numbers and uppercase Latin letters, are separated from gene loci by an asterisk, and the entire gene-allele symbols are italicized. Alleles at the different NAT* loci have been numbered chronologically irrespective of the species of origin. For designation of genotypes at a single NAT* locus, a slash serves to separate the alleles; in phenotype designations, which are not italicized, alleles are separated by a comma.

Acetylator genotype and arylamine-induced carcinogenesis

A diverse array of arylamine chemicals derived from industry, diet, cigarette smoke and other environmental sources are carcinogenic. These chemicals require metabolic activation by host enzymes to chemically reactive electrophiles to initiate the carcinogenic response. Genetic regulation of activation and/or deactivation pathways are thought to account in large measure for corresponding differences in tumor incidence from these chemicals between tissues, between species, or between individuals within a species. Various acetyltransfer reactions are involved in arylamine metabolism and much has been learned regarding their enzymology, genetic regulation, and toxicological significance. The small amount of human data are supported by systematic investigations carried out in animal models characterized with respect to the acetylation polymorphism. Enzymological and genetic investigations suggest that common enzymes encoded by the acetyltransferase gene carry out a diverse set of acetyltransferase reactions. Thus, the acetylation polymorphism can influence both activation and deactivation pathways in arylamine metabolism. Of particular significance recently have been reports documenting the O-acetylation of N-hydroxyarylamine carcinogens and its genetic coregulation with the well-characterized arylamine N-acetylation polymorphism. The toxicological consequences of this polymorphic pathway have yet to be fully explored. Epidemiological investigations show associations between acetylator phenotype and the incidence and/or severity of tumors in the urinary bladder, colon and larynx. Associations between acetylator phenotype and breast cancer are more equivocal and require further study. The divergent influence of acetylator phenotype on the incidence of tumors in different organ sites suggests an important role for extrahepatic acetyltransferases, and further characterization of them in human and animal tissues is needed. The advent of newer methodologies to monitor chemical exposures and to measure acetylator phenotype (rapid, intermediate and slow) using less invasive and more standardized protocols should soon result in a much more definitive understanding regarding the role of acetylator status in arylamine-induced carcinogenesis.

Clinical Pharmacokinetics of Isoniazid

SummaryThe pharmacokinetics of isoniazid in man are described. Pronounced interindividual variation in circulating isoniazid concentration and clearance which occur after dosing with the drug are associated with hereditary differences in the acetylalor status. The variations in rate of isoniazid inactivation and elimination in different (rapid and slow) acetylator phenotypes are primarily due to differences in the rate of acetylation of isoniazid by a genetically controlled polymorphic N-acetyltransferase in liver and small intestine. An appreciable ‘first-pass’ effect is observed following oral isoniazid administration, particularly in the rapid acetylator phenotype.Liver disease can cause a significant prolongation in the clearance of isoniazid; in the acutely ill patient, the prolongation correlates most closely with serum bilirubin elevation, although the degree of prolongation is less important than the intrinsic genetic difference between acetylator phenotypes. The effect of renal impairment on isoniazid excretion is relatively unimportant, even in slow acetylators.Methods for monitoring blood and urine concentrations of isoniazid and for acetylator phenotype determination which are convenient for the patient and clinician are available. Implications of phenotypic differences in acetylator status for the optimal management of tuberculosis with isoniazid are considered. Attempts to devise new isoniazid formulations for this purpose are being evaluated.

Etiologic heterogeneity among non-Hodgkin lymphoma subtypes.

Understanding patterns of etiologic commonality and heterogeneity for non-Hodgkin lymphomas may illuminate lymphomagenesis. We present the first systematic comparison of risks by lymphoma subtype for a broad range of putative risk factors in a population-based case-control study, including diffuse large B-cell (DLBCL; N = 416), follicular (N = 318), and marginal zone lymphomas (N = 106), and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; N = 133). We required at least 2 of 3 analyses to support differences in risk: (1) polytomous logistic regression, (2) homogeneity tests, or (3) dichotomous logistic regression, analyzing all 7 possible pairwise comparisons among the subtypes, corresponding to various groupings by clinical behavior, genetic features, and differentiation. Late birth order and high body mass index (>/= 35) kg/m(2)) increased risk for DLBCL alone. Autoimmune conditions increased risk for marginal zone lymphoma alone. The tumor necrosis factor G-308A polymorphism (rs1800629) increased risks for both DLBCL and marginal zone lymphoma. Exposure to certain dietary heterocyclic amines from meat consumption increased risk for CLL/SLL alone. We observed no significant risk factors for follicular lymphoma alone. These data clearly support both etiologic commonality and heterogeneity for lymphoma subtypes, suggesting that immune dysfunction is of greater etiologic importance for DLBCL and marginal zone lymphoma than for CLL/SLL and follicular lymphoma.

Functional characterization of human N-acetyltransferase 2 (NAT2) single nucleotide polymorphisms.

N-Acetyltransferase 2 (NAT2) catalyses the activation and/or deactivation of a variety of aromatic amine drugs and carcinogens. Polymorphisms in the N-acetyltransferase 2 (NAT2) gene have been associated with a variety of drug-induced toxicities, as well as cancer in various tissues. Eleven single nucleotide polymorphisms (SNPs) have been identified in the NAT2 coding region, but the specific effects of each of these SNPs on expression of NAT2 protein and N-acetyltransferase enzymatic activity are poorly understood. To investigate the functional consequences of SNPs in the NAT2 coding region, reference NAT2*4 and NAT2 variant alleles possessing one of the 11 SNPs in the NAT2 coding region were cloned and expressed in yeast (Schizosaccharomyces pombe). Reductions in catalytic activity for the N-acetylation of a sulfonamide drug (sulfamethazine) and an aromatic amine carcinogen (2-aminofluorene) were observed for NAT2 variants possessing G191A (R64Q), T341C (I114T), A434C (E145P), G590A (R197Q), A845C (K282T) or G857A (G286T). Reductions in expression of NAT2 immunoreactive protein were observed for NAT2 variants possessing T341C, A434C or G590A. Reductions in protein stability were noted for NAT2 variants possessing G191A, A845C, G857A or, to some extent, G590A. No significant differences in mRNA expression or transformation efficiency were observed among any of the NAT2 alleles. These results suggest two mechanisms for slow acetylator phenotype(s) and more clearly define the effects of individual SNPs on human NAT2 expression, stability and catalytic activity.

Metabolic activation of aromatic and heterocyclicN-hydroxyarylamines by wild-type and mutant recombinant human NAT1 and NAT2 acetyltransferases

Recombinant human NAT1 and polymorphic NAT2 wild-type and mutantN-acetyltransferases (encoded byNAT2 alleles with mutations at 282/857, 191, 282/590, 341/803, 341/481/803, and 341/481) were expressed inEscherichia coli strains XA90 and/or JM105, and tested for their capacity to catalyze the metabolic activation (viaO-acetylation) of theN-hydroxy (N-OH) derivatives of 2-aminofluorene (AF), and the heterocyclic arylamine mutagens 2-amino-3-methylimidazo [4,5-f]quinoline (IQ), 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Both NAT1 and NAT2 (including all mutant human NAT2s tested) catalyzed the metabolic activation of each of theN-hydroxyarylamines to products that bound to DNA. Metabolic activation of N-OH-AF was greater than that of the heterocyclicN-hydroxyarylamines. The relative capacity of NAT1 versus NAT2 to catalyze activation varied withN-hydroxyarylamine substrate. N-OH-MeIQx and N-OH-PhIP exhibited a relative specificity for NAT2. These results provide mechanistic support for a role of the genetic acetylation polymorphism in the metabolic activation of heterocyclic amine mutagens and carcinogens.

Inactivation of GSK-3β by Metallothionein Prevents Diabetes-Related Changes in Cardiac Energy Metabolism, Inflammation, Nitrosative Damage, and Remodeling

OBJECTIVE Glycogen synthase kinase (GSK)-3β plays an important role in cardiomyopathies. Cardiac-specific metallothionein-overexpressing transgenic (MT-TG) mice were highly resistant to diabetes-induced cardiomyopathy. Therefore, we investigated whether metallothionein cardiac protection against diabetes is mediated by inactivation of GSK-3β. RESEARCH DESIGN AND METHODS Diabetes was induced with streptozotocin in both MT-TG and wild-type mice. Changes of energy metabolism–related molecules, lipid accumulation, inflammation, nitrosative damage, and fibrotic remodeling were examined in the hearts of diabetic mice 2 weeks, 2 months, and 5 months after the onset of diabetes with Western blotting, RT-PCR, and immunohistochemical assays. RESULTS Activation (dephosphorylation) of GSK-3β was evidenced in the hearts of wild-type diabetic mice but not MT-TG diabetic mice. Correspondingly, cardiac glycogen synthase phosphorylation, hexokinase II, PPARα, and PGC-1α expression, which mediate glucose and lipid metabolisms, were significantly changed along with cardiac lipid accumulation, inflammation (TNF-α, plasminogen activator inhibitor 1 [PAI-1], and intracellular adhesion molecule 1 [ICAM-1]), nitrosative damage (3-nitrotyrosin accumulation), and fibrosis in the wild-type diabetic mice. The above pathological changes were completely prevented either by cardiac metallothionein in the MT-TG diabetic mice or by inhibition of GSK-3β activity in the wild-type diabetic mice with a GSK-3β–specific inhibitor. CONCLUSIONS These results suggest that activation of GSK-3β plays a critical role in diabetes-related changes in cardiac energy metabolism, inflammation, nitrosative damage, and remodeling. Metallothionein inactivation of GSK-3β plays a critical role in preventing diabetic cardiomyopathy.

Metallothionein Suppresses Angiotensin II-Induced Nicotinamide Adenine Dinucleotide Phosphate Oxidase Activation, Nitrosative Stress, Apoptosis, and Pathological Remodeling in the Diabetic Heart

OBJECTIVES We evaluated metallothionein (MT)-mediated cardioprotection from angiotensin II (Ang II)-induced pathologic remodeling with and without underlying diabetes. BACKGROUND Cardiac-specific metallothionein-overexpressing transgenic (MT-TG) mice are resistant to diabetic cardiomyopathy largely because of the antiapoptotic and antioxidant effects of MT. METHODS The acute and chronic cardiac effects of Ang II were examined in MT-TG and wild-type (WT) mice, and the signaling pathways of Ang II-induced cardiac cell death were examined in neonatal mouse cardiomyocytes. RESULTS Acute Ang II administration to WT mice or neonatal cardiomyocytes increased cardiac apoptosis, nitrosative damage, and membrane translocation of the nicotinamide adenine dinucleotide phosphate oxidase (NOX) isoform p47(phox). These effects were abrogated in MT-TG mice, MT-TG cardiomyocytes, and WT cardiomyocytes pre-incubated with peroxynitrite or superoxide scavengers and NOX inhibitors, suggesting a critical role for NOX activation in Ang II-mediated apoptosis. Prolonged administration of subpressor doses of Ang II (0.5 mg/kg every other day for 2 weeks) also induced apoptosis and nitrosative damage in both diabetic and nondiabetic WT hearts, but not in diabetic and nondiabetic MT-TG hearts. Long-term follow-up (1 to 6 months) of both WT and MT-TG mice after discontinuing Ang II administration revealed progressive myocardial fibrosis, hypertrophy, and dysfunction in WT mice but not in MT-TG mice. CONCLUSIONS Metallothionein suppresses Ang II-induced NOX-dependent nitrosative damage and cell death in both nondiabetic and diabetic hearts early in the time course of injury and prevents the late development of Ang II-induced cardiomyopathy.