Characterization of human sulfotransferases catalyzing the formation of p-cresol sulfate and identification of mefenamic acid as a potent metabolism inhibitor and potential therapeutic agent for detoxification

Abstract

p-Cresol sulfate, the primary metabolite of p-cresol, is a uremic toxin that has been associated with toxicities and mortalities. The study objectives were to i) characterize the contributions of human sulfotransferases (SULT) catalyzing p-cresol sulfate formation using multiple recombinant SULT enzymes (including the polymorphic variant SULT1A1*2), pooled human liver cytosols, and pooled human kidney cytosols; and ii) determine the potencies and mechanisms of therapeutic inhibitors capable of attenuating the production of p-cresol sulfate. Human recombinant SULT1A1 was the primary enzyme responsible for the formation of p-cresol sulfate (Km = 0.19 ± 0.02 μM [with atypical kinetic behavior at lower substrate concentrations; see text discussion], Vmax = 789.5 ± 101.7 nmol/mg/min, Ksi = 2458.0 ± 332.8 μM, mean ± standard deviation, n = 3), while SULT1A3, SULT1B1, SULT1E1, and SULT2A1 contributed negligible or minor roles at toxic p-cresol concentrations. Moreover, human recombinant SULT1A1*2 exhibited reduced enzyme activities (Km = 81.5 ± 31.4 μM, Vmax = 230.6 ± 17.7 nmol/mg/min, Ksi = 986.0 ± 434.4 μM) compared to the wild type. The sulfonation of p-cresol was characterized by Michaelis-Menten kinetics in liver cytosols (Km = 14.8 ± 3.4 μM, Vmax = 1.5 ± 0.2 nmol/mg/min) and substrate inhibition in kidney cytosols (Km = 0.29 ± 0.02 μM, Vmax = 0.19 ± 0.05 nmol/mg/min, Ksi = 911.7 ± 278.4 μM). Of the 14 investigated therapeutic inhibitors, mefenamic acid (Ki = 2.4 ± 0.1 nM [liver], Ki = 1.2 ± 0.3 nM [kidney]) was the most potent in reducing the formation of p-cresol sulfate, exhibiting noncompetitive inhibition in human liver cytosols and recombinant SULT1A1, and mixed inhibition in human kidney cytosols. Our novel findings indicated that SULT1A1 contributed an important role in p-cresol sulfonation (hence it can be considered a probe reaction) in liver and kidneys, and mefenamic acid may be utilized as a potential therapeutic agent to attenuate the generation of p-cresol sulfate as an approach to detoxification.