Metabolomic and transcriptomic analysis reveals endogenous substrates and metabolic adaptation in rats lacking Abcg2 and Abcb1a transporters.

Samit Ganguly,Timothy I Shaw,Ryan D Michalek,John D Schuetz,Kamalika Mukherjee,Yu Fukuda,Kimberly M Zorn,Kazuto Yasuda,Sean Ekins,Erin G Schuetz,David Finkelstein,Catherine Mounier

doi:10.1371/journal.pone.0253852

Abstract

Abcg2/Bcrp and Abcb1a/Pgp are xenobiotic efflux transporters limiting substrate permeability in the gastrointestinal system and brain, and increasing renal and hepatic drug clearance. The systemic impact of Bcrp and Pgp ablation on metabolic homeostasis of endogenous substrates is incompletely understood. We performed untargeted metabolomics of cerebrospinal fluid (CSF) and plasma, transcriptomics of brain, liver and kidney from male Sprague Dawley rats (WT) and Bcrp/Pgp double knock-out (dKO) rats, and integrated metabolomic/transcriptomic analysis to identify putative substrates and perturbations in canonical metabolic pathways. A predictive Bayesian machine learning model was used to predict in silico those metabolites with greater substrate-like features for either transporters. The CSF and plasma levels of 169 metabolites, nutrients, signaling molecules, antioxidants and lipids were significantly altered in dKO rats, compared to WT rats. These metabolite changes suggested alterations in histidine, branched chain amino acid, purine and pyrimidine metabolism in the dKO rats. Levels of methylated and sulfated metabolites and some primary bile acids were increased in dKO CSF or plasma. Elevated uric acid levels appeared to be a primary driver of changes in purine and pyrimidine biosynthesis. Alterations in Bcrp/Pgp dKO CSF levels of antioxidants, precursors of neurotransmitters, and uric acid suggests the transporters may contribute to the regulation of a healthy central nervous system in rats. Microbiome-generated metabolites were found to be elevated in dKO rat plasma and CSF. The altered dKO metabolome appeared to cause compensatory transcriptional change in urate biosynthesis and response to lipopolysaccharide in brain, oxidation-reduction processes and response to oxidative stress and porphyrin biosynthesis in kidney, and circadian rhythm genes in liver. These findings present insight into endogenous functions of Bcrp and Pgp, the impact that transporter substrates, inhibitors or polymorphisms may have on metabolism, how transporter inhibition could rewire drug sensitivity indirectly through metabolic changes, and identify functional Bcrp biomarkers.

Highlights

Breast cancer resistant protein (Bcrp; Abcg2) and P-glycoprotein (Pgp; Abcb1) are multidrug efflux transporters with a substantial overlap in their substrates [1, 2] and a similar tissue distribution [3]
In the absence of Bcrp/Pgp, alterations in some metabolites in plasma and cerebrospinal fluid (CSF) might have occurred for reasons such as they were transporter substrates, or represented compensatory transcriptional response to the altered metabolome, and some may result from reprogramming of the gut microbiome by transporter absence
Altered CSF metabolites in double knock-out (dKO) rats suggest Bcrp and Pgp may contribute to the regulation of a healthy central nervous system in rats

Summary

Introduction

Breast cancer resistant protein (Bcrp; Abcg2) and P-glycoprotein (Pgp; Abcb1) are multidrug efflux transporters with a substantial overlap in their substrates [1, 2] and a similar tissue distribution [3]. Bcrp and Pgp transport controls substrate (endogenous substances, dietary constituents, and drugs) absorption from the intestine into blood and from the blood into brain and cerebrospinal fluid (CSF). These transporters enhance substrate clearance from the liver, kidney and other tissues, thereby regulating tissue exposure of their substrates. The absence or inhibition of these transporters may lead to accumulation of their substrates, leading to toxicity from endogenous compounds or dietary components or altered efficacy of their substrate drugs These properties make Bcrp and Pgp among the most important FDA-regulated efflux transporters for drug-approval processes [4]. Understanding of the transporter’s effect on the metabolome may help interpret whether metabolomic signatures attributed to drug response, toxicities or side effects (pharmacometabolomics) [17, 18], are revealing a drug’s inhibition/competition with these transporters

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