Sulfation of ractopamine and salbutamol by the human cytosolic sulfotransferases

Abstract

Feed additives such as ractopamine and salbutamol are pharmacologically active compounds, acting primarily as β-adrenergic agonists. This study was designed to investigate whether the sulfation of ractopamine and salbutamol may occur under the metabolic conditions and to identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating two major feed additive compounds, ractopamine and salbutamol. A metabolic labelling study showed the generation and release of [(35)S]sulfated ractopamine and salbutamol by HepG2 human hepatoma cells labelled with [(35)S]sulfate in the presence of these two compounds. A systematic analysis using 11 purified human SULTs revealed SULT1A3 as the major SULT responsible for the sulfation of ractopamine and salbutamol. The pH dependence and kinetic parameters were analyzed. Moreover, the inhibitory effects of ractopamine and salbutamol on SULT1A3-mediated dopamine sulfation were investigated. Cytosol or S9 fractions of human lung, liver, kidney and small intestine were examined to verify the presence of ractopamine-/salbutamol-sulfating activity in vivo. Of the four human organs, the small intestine displayed the highest activity towards both compounds. Collectively, these results imply that the sulfation mediated by SULT1A3 may play an important role in the metabolism and detoxification of ractopamine and salbutamol.

Fig. 1

Chemical structures of ractopamine and salbutamol.

Fig. 2

Analysis of the [³⁵S]sulfated products generated and released by HepG2 human hepatoma cells labelled with [³⁵S]sulfate in the presence of ractopamine or salbutamol. The autoradiograph taken from the TLC plate used for TLC analysis of the labelling media is shown. Confluent HepG2 cells were labelled with [³⁵S]sulfate for 18 h in the presence of different concentrations (5 or 50 µM) of ractopamine and salbutamol. Lane 1 shows the control labelling medium without added compound. Lanes 2–5 correspond to the labelling media containing 5 or 50 µM ractopamine and 5 or 50 µM salbutamol, respectively. The figure is representative of three independent experiments.

Fig. 3

pH dependency of the sulfating activity of human SULT1A3 with (A) ractopamine and (B) salbutamol as substrates. Enzymatic assays with a final concentration of 50 µM for each substrate were carried out under standard assay conditions as described in Materials and Methods, using different buffer systems as indicated. Data shown represent calculated mean ± standard deviation derived three experiments.

Fig. 4

Kinetic analysis for the sulfation of ractopamine and salbutamol by human SULT1A3. (A) and (B) The hyperbolic curve analyses of the sulfation of ractopamine and salbutamol. The fitting curves were generated using sigmoidal program and the Hill coefficients (h) were determined by Hill equation. Hill plots are inserted under each fitting curve. (C) and (D) The saturation curve analyses of the sulfation of ractopamine and salbutamol. The fitting curves were generated using substrate inhibition (C) and Michaelis–Menten kinetics (D). Eadie–Hofstee plots are inserted under each fitting curve. Data shown represent calculated mean ± standard deviation derived three experiments.

Fig. 5

Inhibitory effects of ractopamine or salbutamol on the sulfation of dopamine by SULT1A3. Enzymatic assays using SULT1A3 with 5 µM dopamine as a substrate in the presence of varying concentrations of ractopamine (0.1–1,000 µM) or salbutamol (0.1–4,000 µM) were carried out. Data from three experiments were calculated based on the activity determined in the absence of inhibitor as 100%.

Fig. 6

Sulfating activities of the human lung, liver, kidney and small intestine cytosol or S9 fractions towards ractopamine and salbutamol. Enzymatic assays using human tissues cytosol or S9 fractions based on the procedure described in Materials and Methods. Data shown represent calculated mean ± standard deviation derived three experiments.