First evidence of the P-glycoprotein gene expression and multixenobiotic resistance modulation in earthworm

Ivana Bošnjak,Sanja Babić,Natalija Topić Popović,Ivančica Strunjak-Perović,Ana Bielen,Roberta Sauerborn Klobučar,Lidija Šver,Rozelinda Čož-Rakovac

doi:10.2478/10004-1254-65-2014-2421

Abstract

Multixenobiotic resistance (MXR) is an important mechanism of cellular efflux mediated by ATP binding cassette (ABC) transporters that bind and actively remove toxic substrates from the cell. This study was the first to identify ABC transporter P-glycoprotein (P-gp/ABCB1) as a representative of the MXR phenotype in earthworm (Eisenia fetida). The identified partial cDNA sequence of ABCB1 overlapped with ABCB1 homologues of other organisms from 58.5 % to 72.5 %. We also studied the effect of five modulators (verapamil, cyclosporine A, MK571, probenecid, and orthovanadate) on the earthworm's MXR activity by measuring the accumulation of model substrates rhodamine B and rhodamine 123 in whole body tissue of the adult earthworm. MK571, orthovanadate, and verapamil significantly inhibited MXR activity, and rhodamine 123 turned out to better reflect MXR activity in that species than rhodamine B. Our results show that E. fetida can serve well as a test organism for environmental pollutants that inhibit MXR activity.

Highlights

Multixenobiotic resistance (MXR) is an important mechanism of cellular efflux mediated by ATP binding cassette (ABC) transporters that bind and actively remove toxic substrates from the cell
The amplified fragment corresponds to the Walker A module and the translated 51 amino acids were: IVQLLERFYDIPEGQVMVDGR DVKSLNVAWLRAQLGIVSQEPTLFDCSIRE. This partial Polymerase chain reaction (PCR) product showed a high degree of identity with P-gp from various other organisms (Figure 1), and this homology was confirmed with National Center for Biotechnology Information (NCBI) Blast2 Protein Database Query
We used phylogenetic analysis to establish the relationship between the identified P-gp/ABCB1 transport protein sequence and other available ABC transporters

Summary

Introduction

Multixenobiotic resistance (MXR) is an important mechanism of cellular efflux mediated by ATP binding cassette (ABC) transporters that bind and actively remove toxic substrates from the cell. The most studied efflux transporter proteins involved in the so-called multixenobiotic resistance (MXR) mechanism are ATP-binding cassette (ABC) transporters These transmembrane proteins, present in all living organisms, interact with a wide number of chemicals, including xenobiotics, and pump them across the cell membrane, preventing their accumulation in the cell [8,9,10]. The aim of our study was 1) to identify the sequence of P-gp/ ABCB1 gene mRNA transcript in the earthworm and 2) to determine the modulation effects of MXR efflux activity in E. fetida by five different types of inhibitors

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Discussion

Conclusion