Development of a common carp (Cyprinus carpio) pregnane X receptor (cPXR) transactivation reporter assay and its activation by azole fungicides and pharmaceutical chemicals

Abstract

In mammals, the pregnane X receptor (PXR) is a transcription factor with a key role in regulating expression of several genes involved in drug biotransformation. PXR is present in fish and some genes known to be under its control can be up-regulated by mammalian PXR ligands. Despite this, direct involvement of PXR in drug biotransformation in fish has yet to be established. Here, the full length PXR sequence was cloned from carp (Cyprinus carpio) and used in a luciferase reporter assay to elucidate its role in xenobiotic metabolism in fish. A reporter assay for human PXR (hPXR) was also established to compare transactivation between human and carp (cPXR) isoforms. Rifampicin activated hPXR as expected, but not cPXR. Conversely, clotrimazole (CTZ) activated both isoforms and was more potent on cPXR, with an EC50 within the range of concentrations of CTZ measured in the aquatic environment. Responses to other azoles tested were similar between both isoforms. A range of pharmaceuticals tested either failed to activate, or were very weakly active, on the cPXR or hPXR. Overall, these results indicate that the cPXR may differ from the hPXR in its responses and/or sensitivity to induction by different environmental chemicals, with implications for risk assessment because of species differences.

Keywords: Azole fungicides; Common carp; Human; Pharmaceuticals; Pregnane X receptor; Transient transactivation assay.

Fig. 1

Sequence of the 6xPXRE reporter construct. Nucleotides shown in green are PXRE sequences. Capital letters mark the conserved motifs. The boxed section is a DR4 based on that published by Xie et al. (2000). This sequence was inserted into the pGL4.24 vector using KpnI and HindIII restriction sites (underlined) to create the PXRE reporter vector. Arrows indicate the direction of the nuclear response element repeat motif.

Fig. 2

cPXR amino acid sequence aligned with PXR sequences of other animal species. The highly conserved DBD (blue outline) consists of two C4-type zinc fingers and includes a P-box motif (red outline). The conserved LBD (green outline) includes the AF-2 motif (purple outline). Accession numbers of sequences used for alignment: AAH17304 (H. sapiens); NP_443212 (R. norvegicus); NP_035066 (M. musculus); NP_001092087 (D. rerio).

Fig. 3

Evolutionary relationships of PXR. The neighbour-joining phylogenetic tree was constructed based on full length amino acid sequences. The scale bar represents 0.05 substitutions per site.

Fig. 4

Activation of cPXR and hPXR by rifampicin (RIF) and dexamethasone (DEX) mediated by two different luciferase reporter vectors. An asterisk denotes the treatment is significantly different from the corresponding DMSO control group (p < 0.05).

Fig. 5

Concentration-response curves for carp (solid line) and human (dashed line) PXR activation on exposure to (A) rifampicin and (B) clotrimazole for 44 h at concentration between 10 μM and 10 pM (10^− 5 and 10^− 11 M). Data are presented as x-fold activation relative to DMSO control.

Fig. 6

Concentration–response profiles of cPXR and hPXR exposure to: the NSAIDs diclofenac (A), ibuprofen (B) and ketoprofen (C); the fibrates clofibric acid (D) and gemfibrozil (E); the β-blockers atenolol (F) and propranolol (G); the (anti-)oestrogens 17α-ethinyloestradiol (H) and tamoxifen (I); and the antifungals ketoconazole (J), miconazole (H) and propiconazole (L). Results are expressed as mean change normalised against their relevant respective controls ± SEM. Dose-response-curves were fitted by non-linear regression.