Interplay of metabolizing enzymes and transporter of xenobiotics

Abstract

1. Xenobiotics are metabolized and eliminated through the coordinated interplay of their metabolizing enzymes and transporters. However, these two activities in vitro are measured separately, with the addition of ATP as a pre-requisite.

2. We propose a human renal cell-line model which integrates the sulfate and glutathione conjugation of xenobiotics with the efflux of their respective conjugates. Sulfation and glutathionylation represent two major Phase II detoxification of xenobiotics in man. The reactions are catalyzed, respectively, by phenolsulfotransferase and glutathione-S-transferase followed by extrusion of their respective conjugates.

3. Using Ko-143, a specific inhibitor of breast cancer resistance protein (BCRP), an ATP-binding cassette (ABC) transporter, we identified this transporter to be responsible for the efflux of p-cresol sulfate, harmol sulfate and the glutathione conjugate of 1-chloro-2,4-dinitrobenzene.

4. The conjugation-cum-efflux was inhibited by oligomycin and uncouplers, which highlights the role of cellular mitochondria in providing ATP for the biosynthesis of their conjugating agents, 3′-phosphoadenosine-5′-phosphosulfate (PAPS) and reduced glutathione as well as for the transport function of BCRP.

5. The human 786-O renal cell-line provides a “3-in-1” system linking ATP biosynthesis to metabolism of xenobiotics and their ultimate transport and elimination by BCRP; this integrated system was not apparent in other human cell-lines examined.

• 2,4-dinitrophenyl-S-glutathione
• breast cancer resistance protein (BCRP or ABCG2)
• glutathione-S-transferase
• mitochondrial ATP
• p-cresyl sulfate
• phenolsulfotransferase

Acknowledgements

We are grateful to Assoc. Prof. Deng Lih Wen for the 786-O cells, Xue Fei for his help in the chemical synthesis of PCS, Dr. Patrick Goo Chong Kiat for his technical assistance and Assoc. Prof Chan Yiong Huak, Head of Biostatistics Unit in the Yong Loo Lin School of Medicine, for his guidance in the analysis of data. We thank Dr. Rebecca Anne Jackson for her editing of this manuscript.

Declaration of interest

We thank the National University of Singapore for the research grant (R-183-000-327-112) and the National Kidney Foundation (NKFRC/2008/09) for their financial support.

Supplementary material available online

Supplementary figures.