Structural and functional characterization of microcystin detoxification-related liver genes in a phytoplanktivorous fish, Nile tilapia ( Oreochromis niloticus)

Abstract

Liver genes related to phase I and phase II detoxification, as well as inhibition of reactive oxygen species (ROS) production, were cloned, and their response to microcystin-LR (MC-LR) and lipopolysaccharide (LPS) exposure via intraperitoneal injection, was determined in a phytoplanktivorous fish, Nile tilapia ( Oreochromis niloticus). The cloned full-length cDNA of tilapia soluble glutathione S-transferase (sGST) was classified as alpha-class GST based on their amino acid sequence identity with other species. The tilapia sGST clone was 861 bp in length, and contained a 25 bp 5′-UTR, a 167 bp 3′-UTR and an open reading frame of 669 bp, encoding a polypeptide of 222 amino acids. Using genome walker method, a 366 bp 5′-flanking sequence of tilapia sGST gene was further obtained, and the possible regulatory elements were identified. Partial cDNA sequences of glutathione peroxidase (GPX) and uncoupling protein 2 (UCP2) were also obtained by PCR using degenerate primers from tilapia liver. To study the transcriptional response of liver genes to microcystin treatment, tilapia were respectively exposed to a single 50 μg kg − 1 body weight (bwt) dose of pure MC-LR, a single 2 mg kg − 1 bwt dose of LPS and a co-exposure MC-LR and LPS (50 μg kg − 1 bwt + 2 mg kg − 1 bwt), and were then sacrificed at 24 h post-exposure. Using beta-actin as external control, a significant increase (about 80%) in sGST mRNA expression was found in response to the MC-LR exposure after 24 h ( P < 0.05), indicating the importance of sGST in microcystin detoxification. A slight decrease of sGST mRNA expression was observed in the liver of tilapia, exposed to LPS and MC-LR + LPS. It seems that the LPS response element (LPSRE), identified in the promoter region of tilapia sGST gene, may be functional at a rather low level. In contrast, the levels of cytochrome P450 1A (CYP1A) mRNA expression were found to keep unchanged to either MC-LR, or LPS, or MC-LR + LPS treatment, indicating that unlike the phase II enzyme (sGST), the phase I enzyme (CYP1A) might not play an important role in the detoxification process of microcystins. Although not significant, the mRNA expression level of GPX tended to increase in the liver of tilapia exposed to both MC-LR and LPS ( P > 0.05). In addition, a significant increase in UCP2 mRNA expression was observed in the liver of tilapia exposed to LPS ( P < 0.05), as well as an obvious but not significant increase in MC-LR exposure group. We suggest that phase II detoxification enzyme, instead of phase I detoxification enzyme, might be responsible for the strong tolerance of the phytoplanktivorous fish to microcystins, and hepatocyte proteins coping with oxidative stress (GPX and UCP2), might also have some auxiliary effect. In addition, the rather low and insignificant response of tilapia sGST gene to the inhibitory effect of LPS exposure, might possibly be critical to the phytoplanktivorous fish to utilize toxic blue-green algae.