N-Acetylation of p-Aminobenzoic Acid and p-Phenylenediamine in Primary Porcine Urinary Bladder Epithelial Cells and in the Human Urothelial Cell Line 5637

Abstract

N-Acetyltransferases (NAT) are important enzymes in the metabolism of certain carcinogenic arylamines, as N-acetylation decreases or prevents their bioactivation via N-hydroxylation. To study such processes in the bladder, cell culture models may be used, but metabolic competence needs to be characterized. This study focused on the N-acetylation capacity of two urothelial cell systems, using p-aminobenzoic acid (PABA) and the hair dye precursor p-phenylenediamine (PPD), two well-known substrates of the enzyme NAT1. The constitutive NAT1 activity was investigated using primary cultures of porcine urinary bladder epithelial cells (PUBEC) and in the human urothelial cell line 5637 to assess their suitability for further in vitro studies on PABA and PPD-induced toxicity. N-Acetylation of PABA and PPD was determined by high-performance liquid chromatography (HPLC) analysis in cytosols of the two cell systems upon incubation with various substrate levels for up to 60 min. The primary PUBEC revealed higher N-acetylation rates (2.5-fold for PABA, 5-fold for PPD) compared to the 5637 cell line, based on both PABA conversion to its acetylated metabolite and formation of mono- and diacetylated PPD. The urothelial cell systems may thus be useful as a tool for further studies on the N-acetylation of aromatic amines via NAT1.