Real-time PCR analysis of the N-acetyltransferase NAT1 allele *3, *4, *10, *11, *14 and *17 polymorphism in squamous cell cancer of head and neck

Abstract

Although tobacco smoke has been established as a main risk factor in the development of head and neck squamous cell cancer (HNSCC), genetic polymorphisms of xenobiotic metabolizing enzymes are supposed to modulate an individual's susceptibility to smoking-related HNSCC. N-acetyltransferase (NAT) 1 gene is known to be polymorphic and its protein product is implicated in the activation and detoxification of carcinogens, such as aromatic amines, present in tobacco smoke. We developed a rapid and reproducible LightCycler-assisted real-time polymerase chain reaction (PCR) for NAT1 genotyping, which allowed the parallel differentiation of NAT1*3, *4, *10 and *11 alleles and separately of NAT1*14 and *17 alleles within 60 min without the need for further post-PCR processing. In order to investigate the role of the NAT1 gene polymorphism as a risk-modifying factor in HNSCC, we tested for the presence of NAT1*3, *4, *10, *11, *14 and *17 alleles in a case-control study of 291 HNSCC patients and 300 healthy controls of Caucasian origin. Our findings suggest that in Caucasians, the risk of HNSCC is not associated with NAT1 polymorphism. The overall distribution of NAT1 allele frequencies was not significantly different among cases and controls. The presence of the fast acetylator NAT1*10 and NAT1*11 alleles did not significantly increase the risk of HNSCC and no modifying effect of NAT1*10 was observed among smokers. This new approach in NAT1 genotyping substantially increases throughput of sample analysis and, therefore, enhances opportunities to study NAT1 as a risk factor in different cancers in large-scale studies.

Safety, Tolerability, and Pharmacokinetics of Etamicastat, a Novel Dopamine-β-Hydroxylase Inhibitor, in a Rising Multiple-Dose Study in Young Healthy Subjects

“…The apparent doseproportional increase in systemic exposure was confirmed by the statistical analysis using an exponential regression model. Also, the comparison of etamicastat and BIA 5-961 pharmacokinetic parameters after log-transformation and dosenormalization showed that C max , AUC t and AUC 24 following the first dose of etamicastat, and C max , AUC t , AUC 24 , and AUC ¥ following the last dose were not significantly different between dose groups.…”

“…During admission, blood pressure and heart rate measurements using a Dinamap Ò (GE Healthcare, Chalfont St. Giles, UK) blood pressure monitor using the oscillometric method and 12-lead digital ECG recordings were taken in the supine position, after resting for at least 10 minutes, at the following times: (i) day -1 (the day prior to first dosing) -time 0 (24 hours before first dosing) and 1, 2, 3, 4, 6, 8, 10, 12, and 16 hours after; (ii) day 1 (day of first dosing) -pre-dose and 1, 2, 3, 4, 6, 8, 10, 12, and 16 hours post-dose; (iii) from day 2 to day 9 -pre-dose and 2 hours post-dose; and (iv) day 10 (day of last dosing) -pre-dose and 1, 2, 3, 4,6,8,10,12,16,24,48, and 72 hours post-dose. Digital ECGs were performed in triplicate (with an interval of 5 minutes, with a difference of at least 1 minute between each of the three recordings).…”

“…Associated genotypes were defined according to the literature. [17,24] Subjects were classified NAT1 rapid (fast) acetylators if they carried the NAT1 T > C, 590 G > A, and 857 G > A) and the corresponding genotypes determined. Associated genotypes were defined according to the literature.…”

“…A nonlinear power model was used to assess dose proportionality of C max and AUC 24 . The relationship is written as a power function: 24 . Linearization of this relationship gives: log parameter = log a + log dose Á b.…”

“…The relationship is dose proportional when b = 1. The exponent of the power function, with 95% confidence intervals (95% CI), was fitted to the in- dividual C max and AUC 24 . For determination of time to steady-state of plasma concentrations, a pairwise comparison for pre-dose concentrations between day 2 and day 10 was performed by dose group using a paired Student's t-test.…”