ABCB1 gene in Aporrectodea caliginosa - a potential ecotoxicological tool

Abstract

Earthworms are exposed to a variety of agrochemicals due to strict export requirements and maintenance of high productivity. Aporrectodea caliginosa (grey earthworm) is the most abundant species in New Zealand pastures. In biological systems, the first-line defence mechanism against accumulation of toxicants within cells is mediated by drug efflux transporters of the ATP Binding Cassette (ABC) transporter family. This study describes the discovery of a partial sequence homologous to ABCB1 gene that encodes the efflux transporter P-glycoprotein (P-gp). The regulation of ABCB1 gene expression has been associated with exposure to polluted environments in aquatic organisms. The expression of the ABCB1 gene in annelids is a potential candidate as a molecular biomarker for soil pollution monitoring. The lack of A. caliginosa ABCB1 genetic information or any other Lumbricidae family member has been a significant drawback. The novel sequence was identified by using a combined approach of degenerate PCR and sequence retrieval from the draft Lumbricus rubellus genome LUMBRIBASE. The novel translated sequence of 134 amino acids (encoding 10.7% of exogenic sequence) has high amino acid sequence identity to P-gp (>78%) in vertebrate and invertebrate species and possesses some of the classical domains of P-gp. A real-time quantitative PCR (RT-qPCR) was developed to measure ABCB1 gene transcription. In the process of PCR optimisation, another partial gene sequence, homologous to β-actin gene was identified in A. caliginosa and used as the reference gene for the normalisation of samples. Preliminary analyses in RT-qPCR were performed with earthworms exposed to modulators (rifampicin, cyclosporin A, Ivermectin™) on ABCB1 gene and P-gp expression used in mammals. Fold-changes in the ABCB1 transcriptional levels were measured by the comparison between exposed and non-exposed earthworms. The P-gp activity was measured by a biochemical assay using a specific P-gp substrate (fluorescent dye Rhodamine B) and two P-gp inhibitors (verapamil and cyclosporin A). Rifampicin induced the ABCB1 expression in A. caliginosa. However, it did not increase P-gp activity. This observation suggests the involvement of post-transcriptional mechanisms to produce the functional P-gp. Ivermectin™ also increased ABCB1 expression in A. caliginosa, showing that this drug may have the same effect on ABCB1 expression observed in studies with nematodes and mammals. In contrast, cyclosporin A did not increase ABCB1 gene expression, suggesting that P-gp inhibition alone does not lead to overexpression of ABCB1 to compensate for the inactive P-gps. The presence of an ABCB1 gene homologue in A. caliginosa and its expression in response to known modulators supported the hypothesis that P-gp is present in A. caliginosa. Moreover, the P-gp activity in A. caliginosa was demonstrated in the results of the biochemical assay. The results of this research provide baseline data in A. caliginosa ABCB1 and P-gp expression, and may assist in the advancement of ABC transporter research in earthworms and development of a monitoring system in toxicological risk assessments.