A luciferase reporter gene assay was used to investigate for functional implications of sequence variation between zebrafish Pxr variants. For this, two putative zebrafish Pxr in vitro agonists were used, a local anesthetic (4BAB) and an antifungal drug (CLO). Immunoblotting using anti-GAL4 antibodies demonstrated that the levels of the zfPxr variants were similar in the COS7 cells (Supplementary Figure 6). CLO increased luciferase activities by approximately 23–34 million RLUs in cells expressing PxrAB/Tü, PxrTL, and PxrSWT, whereas Rmax in cells expressing PxrUNK was approximately 1.8 million RLUs (Figure 2A, Tables 3 and 4). Butyl-4-aminobenzoate induced a moderate increase in the luciferase activities in cells expressing PxrAB/Tü, PxrTL, or PxrSWT (ΔRLUs in the range of 2.8–6.3 million), whereas luciferase activity in cells expressing PxrUNK did not increase (Figure 2B, Tables 3 and 4).
Dose-Response Dynamics for Activation of Allelic Variants of Zebrafish Pxr by CLO and 4BAB and Ligand-Receptor Interaction
| Zebrafish Pxr Variant . | Clotrimazole (CLO) . | Butyl 4-Aminobenzoate (4BAB) . | |||
|---|---|---|---|---|---|
| Rmax (e6 ΔRLU±SD) . | EC50 (e−7 M) . | Dissociation Constant (Kd) (μM) . | Rmax (e6 ΔRLU±SD) . | EC50 (e−5 M) . | |
| PxrAB/Tü | 22.6 ± 0.28 (b, c, d, *) | 1.42 (b, c, d, *) | 0.04 | 2.75 ± 1.65 (c, *) | 0.96 (b, c, *) |
| PxrTL | 34.0 ± 5.75 (a, d, *) | 1.08 (d, *) | 4.1 | 4.57 ± 2.41 (*) | 1.16 (a, *) |
| PxrSWT | 28.5 ± 6.80 (a, d, *) | 1.28 (a, *) | 260 | 6.31 ± 2.86 (a, *) | 1.18 (a, *) |
| PxrUNK | 1.81 ± 0.67 (a, b, c) | 3.48 (a, b) | 180 | N/D | N/D |
| Zebrafish Pxr Variant . | Clotrimazole (CLO) . | Butyl 4-Aminobenzoate (4BAB) . | |||
|---|---|---|---|---|---|
| Rmax (e6 ΔRLU±SD) . | EC50 (e−7 M) . | Dissociation Constant (Kd) (μM) . | Rmax (e6 ΔRLU±SD) . | EC50 (e−5 M) . | |
| PxrAB/Tü | 22.6 ± 0.28 (b, c, d, *) | 1.42 (b, c, d, *) | 0.04 | 2.75 ± 1.65 (c, *) | 0.96 (b, c, *) |
| PxrTL | 34.0 ± 5.75 (a, d, *) | 1.08 (d, *) | 4.1 | 4.57 ± 2.41 (*) | 1.16 (a, *) |
| PxrSWT | 28.5 ± 6.80 (a, d, *) | 1.28 (a, *) | 260 | 6.31 ± 2.86 (a, *) | 1.18 (a, *) |
| PxrUNK | 1.81 ± 0.67 (a, b, c) | 3.48 (a, b) | 180 | N/D | N/D |
Maximum response (Rmax) and effective concentration 50 (EC50) of Pxr-mediated induction of luciferase activities in COS7 cells calculated from dose-response curves fitted by nonlinear regression (GraphPad Prism 7.0). Dissociation constants were determined from experimental data from surface plasmon resonance. Statistical significance differences of Rmax or EC50 were tested with Students t test (p < .05). Letter indicators signify more than 95% probability for difference of Rmax or EC50 for response via PxrAB/Tü (a), PxrTL (b), PxrSWT (c), or PxrUNK (d), whereas (*) indicates statistical significant difference between the Rmax or EC50 for CLO and 4BAB responses via the same zfPxr variant.
Dose-Response Dynamics for Activation of Allelic Variants of Zebrafish Pxr by CLO and 4BAB and Ligand-Receptor Interaction
| Zebrafish Pxr Variant . | Clotrimazole (CLO) . | Butyl 4-Aminobenzoate (4BAB) . | |||
|---|---|---|---|---|---|
| Rmax (e6 ΔRLU±SD) . | EC50 (e−7 M) . | Dissociation Constant (Kd) (μM) . | Rmax (e6 ΔRLU±SD) . | EC50 (e−5 M) . | |
| PxrAB/Tü | 22.6 ± 0.28 (b, c, d, *) | 1.42 (b, c, d, *) | 0.04 | 2.75 ± 1.65 (c, *) | 0.96 (b, c, *) |
| PxrTL | 34.0 ± 5.75 (a, d, *) | 1.08 (d, *) | 4.1 | 4.57 ± 2.41 (*) | 1.16 (a, *) |
| PxrSWT | 28.5 ± 6.80 (a, d, *) | 1.28 (a, *) | 260 | 6.31 ± 2.86 (a, *) | 1.18 (a, *) |
| PxrUNK | 1.81 ± 0.67 (a, b, c) | 3.48 (a, b) | 180 | N/D | N/D |
| Zebrafish Pxr Variant . | Clotrimazole (CLO) . | Butyl 4-Aminobenzoate (4BAB) . | |||
|---|---|---|---|---|---|
| Rmax (e6 ΔRLU±SD) . | EC50 (e−7 M) . | Dissociation Constant (Kd) (μM) . | Rmax (e6 ΔRLU±SD) . | EC50 (e−5 M) . | |
| PxrAB/Tü | 22.6 ± 0.28 (b, c, d, *) | 1.42 (b, c, d, *) | 0.04 | 2.75 ± 1.65 (c, *) | 0.96 (b, c, *) |
| PxrTL | 34.0 ± 5.75 (a, d, *) | 1.08 (d, *) | 4.1 | 4.57 ± 2.41 (*) | 1.16 (a, *) |
| PxrSWT | 28.5 ± 6.80 (a, d, *) | 1.28 (a, *) | 260 | 6.31 ± 2.86 (a, *) | 1.18 (a, *) |
| PxrUNK | 1.81 ± 0.67 (a, b, c) | 3.48 (a, b) | 180 | N/D | N/D |
Maximum response (Rmax) and effective concentration 50 (EC50) of Pxr-mediated induction of luciferase activities in COS7 cells calculated from dose-response curves fitted by nonlinear regression (GraphPad Prism 7.0). Dissociation constants were determined from experimental data from surface plasmon resonance. Statistical significance differences of Rmax or EC50 were tested with Students t test (p < .05). Letter indicators signify more than 95% probability for difference of Rmax or EC50 for response via PxrAB/Tü (a), PxrTL (b), PxrSWT (c), or PxrUNK (d), whereas (*) indicates statistical significant difference between the Rmax or EC50 for CLO and 4BAB responses via the same zfPxr variant.
| A . | Compared Responses . | PxrAB/Tü . | PxrTL . | PxrSWT . | PxrUNK . |
|---|---|---|---|---|---|
| CLO | DMSO vs 4.5 μM | 0.0001 | 0.0001 | 0.0001 | 0.0001 |
| DMSO vs 3.0 μM | 0.0001 | 0.0001 | 0.0001 | 0.0001 | |
| DMSO vs 0.5 μM | 0.0001 | 0.0001 | 0.0001 | 0.0001 | |
| DMSO vs 83 nM | 0.0001 | 0.0001 | 0.0001 | 0.5961 | |
| DMSO vs 14 nM | 0.9231 | 0.8682 | 0.9316 | 0.9995 | |
| DMSO vs 0.2 nM | 0.9999 | 0.9998 | 0.9996 | 0.9971 | |
| DMSO vs 0.03 nM | 0.9999 | 0.9997 | 0.9998 | 0.9996 | |
| 4BAB | DMSO vs 50 μM | 0.0001 | 0.0001 | 0.0001 | 0.9996 |
| DMSO vs 33 μM | 0.0001 | 0.0001 | 0.0001 | 0.5488 | |
| DMSO vs 11 μM | 0.0001 | 0.0073 | 0.0001 | 0.9998 | |
| DMSO vs 3.7 μM | 0.7763 | 0.9997 | 0.953 | 0.6689 | |
| DMSO vs 1.2 μM | 0.9958 | 0.9995 | 0.9995 | 0.8995 | |
| DMSO vs 0.4 μM | 0.9999 | 0.8796 | 0.9929 | 0.9999 | |
| DMSO vs 0.1 μM | 0.9994 | 0.9979 | 0.9996 | 0.993 |
| A . | Compared Responses . | PxrAB/Tü . | PxrTL . | PxrSWT . | PxrUNK . |
|---|---|---|---|---|---|
| CLO | DMSO vs 4.5 μM | 0.0001 | 0.0001 | 0.0001 | 0.0001 |
| DMSO vs 3.0 μM | 0.0001 | 0.0001 | 0.0001 | 0.0001 | |
| DMSO vs 0.5 μM | 0.0001 | 0.0001 | 0.0001 | 0.0001 | |
| DMSO vs 83 nM | 0.0001 | 0.0001 | 0.0001 | 0.5961 | |
| DMSO vs 14 nM | 0.9231 | 0.8682 | 0.9316 | 0.9995 | |
| DMSO vs 0.2 nM | 0.9999 | 0.9998 | 0.9996 | 0.9971 | |
| DMSO vs 0.03 nM | 0.9999 | 0.9997 | 0.9998 | 0.9996 | |
| 4BAB | DMSO vs 50 μM | 0.0001 | 0.0001 | 0.0001 | 0.9996 |
| DMSO vs 33 μM | 0.0001 | 0.0001 | 0.0001 | 0.5488 | |
| DMSO vs 11 μM | 0.0001 | 0.0073 | 0.0001 | 0.9998 | |
| DMSO vs 3.7 μM | 0.7763 | 0.9997 | 0.953 | 0.6689 | |
| DMSO vs 1.2 μM | 0.9958 | 0.9995 | 0.9995 | 0.8995 | |
| DMSO vs 0.4 μM | 0.9999 | 0.8796 | 0.9929 | 0.9999 | |
| DMSO vs 0.1 μM | 0.9994 | 0.9979 | 0.9996 | 0.993 |
| B . | Compared Responses . | 0.4 nM . | 2.3 nM . | 14 nM . | 83 nM . | 0.5 μM . | 3.0 μM . | 4.5 μM . |
|---|---|---|---|---|---|---|---|---|
| CLO | PxrAB/Tü vs PxrTL | 0.0167 | 0.2374 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | 0.0018 |
| PxrAB/Tü vs PxrSWT | 0.7707 | 0.0511 | 0.6888 | 0.0496 | 0.0294 | 0.8725 | 0.0393 | |
| PxrAB/Tü vs PxrUNK | 0.8965 | 0.9998 | 0.0002 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | |
| PxrTL vs PxrSWT | 0.1551 | 0.8774 | 0.0017 | 0.0281 | 0.0002 | 0.0007 | 0.6801 | |
| PxrTL vs PxrUNK | 0.0023 | 0.2755 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | |
| PxrSWT vs PxrUNK | 0.3548 | 0.0627 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
| B . | Compared Responses . | 0.4 nM . | 2.3 nM . | 14 nM . | 83 nM . | 0.5 μM . | 3.0 μM . | 4.5 μM . |
|---|---|---|---|---|---|---|---|---|
| CLO | PxrAB/Tü vs PxrTL | 0.0167 | 0.2374 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | 0.0018 |
| PxrAB/Tü vs PxrSWT | 0.7707 | 0.0511 | 0.6888 | 0.0496 | 0.0294 | 0.8725 | 0.0393 | |
| PxrAB/Tü vs PxrUNK | 0.8965 | 0.9998 | 0.0002 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | |
| PxrTL vs PxrSWT | 0.1551 | 0.8774 | 0.0017 | 0.0281 | 0.0002 | 0.0007 | 0.6801 | |
| PxrTL vs PxrUNK | 0.0023 | 0.2755 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | |
| PxrSWT vs PxrUNK | 0.3548 | 0.0627 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
| . | Compared Responses . | 0.1 μM . | 0.4 μM . | 1.2 μM . | 3.7 μM . | 11 μM . | 33 μM . | 50 μM . |
|---|---|---|---|---|---|---|---|---|
| 4BAB | PxrAB/Tü vs PxrTL | 0.1840 | 0.0013 | 0.9996 | 0.4299 | 0.9637 | 0.0751 | 0.4159 |
| PxrAB/Tü vs PxrSWT | 0.3229 | 0.0542 | 0.9938 | 0.9556 | 0.0175 | 0.0040 | 0.0083 | |
| PxrAB/Tü vs PxrUNK | 0.9071 | 0.9983 | 0.7314 | 0.1045 | 0.0011 | 0.0038 | 0.0123 | |
| PxrTL vs PxrSWT | 0.9875 | 0.5249 | 0.9985 | 0.1888 | 0.0570 | 0.6704 | 0.2806 | |
| PxrTL vs PxrUNK | 0.5165 | 0.0022 | 0.6710 | 0.8485 | 0.0002 | <0.0001 | 0.0001 | |
| PxrSWT vs PxrUNK | 0.7190 | 0.0793 | 0.5724 | 0.0322 | <0.0001 | <0.0001 | <0.0001 | |
| . | Compared Responses . | 0.1 μM . | 0.4 μM . | 1.2 μM . | 3.7 μM . | 11 μM . | 33 μM . | 50 μM . |
|---|---|---|---|---|---|---|---|---|
| 4BAB | PxrAB/Tü vs PxrTL | 0.1840 | 0.0013 | 0.9996 | 0.4299 | 0.9637 | 0.0751 | 0.4159 |
| PxrAB/Tü vs PxrSWT | 0.3229 | 0.0542 | 0.9938 | 0.9556 | 0.0175 | 0.0040 | 0.0083 | |
| PxrAB/Tü vs PxrUNK | 0.9071 | 0.9983 | 0.7314 | 0.1045 | 0.0011 | 0.0038 | 0.0123 | |
| PxrTL vs PxrSWT | 0.9875 | 0.5249 | 0.9985 | 0.1888 | 0.0570 | 0.6704 | 0.2806 | |
| PxrTL vs PxrUNK | 0.5165 | 0.0022 | 0.6710 | 0.8485 | 0.0002 | <0.0001 | 0.0001 | |
| PxrSWT vs PxrUNK | 0.7190 | 0.0793 | 0.5724 | 0.0322 | <0.0001 | <0.0001 | <0.0001 | |
One-way ANOVA and Dunnett’s test were used to determine the probability that the means of responses in exposed cells were different from exposures in nonexposed/DMSO treated cells (A). One-way ANOVA and Tukey-Kramer test were used to test for statistical differences between means of responses produced by the same exposure via different zfPxr variants (B). GraphPad Prism7.0 was used for statistical analyses. Tests that returned a greater than 5% probability for equal means (p value > .05) were shaded in gray in the tables.
| A . | Compared Responses . | PxrAB/Tü . | PxrTL . | PxrSWT . | PxrUNK . |
|---|---|---|---|---|---|
| CLO | DMSO vs 4.5 μM | 0.0001 | 0.0001 | 0.0001 | 0.0001 |
| DMSO vs 3.0 μM | 0.0001 | 0.0001 | 0.0001 | 0.0001 | |
| DMSO vs 0.5 μM | 0.0001 | 0.0001 | 0.0001 | 0.0001 | |
| DMSO vs 83 nM | 0.0001 | 0.0001 | 0.0001 | 0.5961 | |
| DMSO vs 14 nM | 0.9231 | 0.8682 | 0.9316 | 0.9995 | |
| DMSO vs 0.2 nM | 0.9999 | 0.9998 | 0.9996 | 0.9971 | |
| DMSO vs 0.03 nM | 0.9999 | 0.9997 | 0.9998 | 0.9996 | |
| 4BAB | DMSO vs 50 μM | 0.0001 | 0.0001 | 0.0001 | 0.9996 |
| DMSO vs 33 μM | 0.0001 | 0.0001 | 0.0001 | 0.5488 | |
| DMSO vs 11 μM | 0.0001 | 0.0073 | 0.0001 | 0.9998 | |
| DMSO vs 3.7 μM | 0.7763 | 0.9997 | 0.953 | 0.6689 | |
| DMSO vs 1.2 μM | 0.9958 | 0.9995 | 0.9995 | 0.8995 | |
| DMSO vs 0.4 μM | 0.9999 | 0.8796 | 0.9929 | 0.9999 | |
| DMSO vs 0.1 μM | 0.9994 | 0.9979 | 0.9996 | 0.993 |
| A . | Compared Responses . | PxrAB/Tü . | PxrTL . | PxrSWT . | PxrUNK . |
|---|---|---|---|---|---|
| CLO | DMSO vs 4.5 μM | 0.0001 | 0.0001 | 0.0001 | 0.0001 |
| DMSO vs 3.0 μM | 0.0001 | 0.0001 | 0.0001 | 0.0001 | |
| DMSO vs 0.5 μM | 0.0001 | 0.0001 | 0.0001 | 0.0001 | |
| DMSO vs 83 nM | 0.0001 | 0.0001 | 0.0001 | 0.5961 | |
| DMSO vs 14 nM | 0.9231 | 0.8682 | 0.9316 | 0.9995 | |
| DMSO vs 0.2 nM | 0.9999 | 0.9998 | 0.9996 | 0.9971 | |
| DMSO vs 0.03 nM | 0.9999 | 0.9997 | 0.9998 | 0.9996 | |
| 4BAB | DMSO vs 50 μM | 0.0001 | 0.0001 | 0.0001 | 0.9996 |
| DMSO vs 33 μM | 0.0001 | 0.0001 | 0.0001 | 0.5488 | |
| DMSO vs 11 μM | 0.0001 | 0.0073 | 0.0001 | 0.9998 | |
| DMSO vs 3.7 μM | 0.7763 | 0.9997 | 0.953 | 0.6689 | |
| DMSO vs 1.2 μM | 0.9958 | 0.9995 | 0.9995 | 0.8995 | |
| DMSO vs 0.4 μM | 0.9999 | 0.8796 | 0.9929 | 0.9999 | |
| DMSO vs 0.1 μM | 0.9994 | 0.9979 | 0.9996 | 0.993 |
| B . | Compared Responses . | 0.4 nM . | 2.3 nM . | 14 nM . | 83 nM . | 0.5 μM . | 3.0 μM . | 4.5 μM . |
|---|---|---|---|---|---|---|---|---|
| CLO | PxrAB/Tü vs PxrTL | 0.0167 | 0.2374 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | 0.0018 |
| PxrAB/Tü vs PxrSWT | 0.7707 | 0.0511 | 0.6888 | 0.0496 | 0.0294 | 0.8725 | 0.0393 | |
| PxrAB/Tü vs PxrUNK | 0.8965 | 0.9998 | 0.0002 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | |
| PxrTL vs PxrSWT | 0.1551 | 0.8774 | 0.0017 | 0.0281 | 0.0002 | 0.0007 | 0.6801 | |
| PxrTL vs PxrUNK | 0.0023 | 0.2755 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | |
| PxrSWT vs PxrUNK | 0.3548 | 0.0627 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
| B . | Compared Responses . | 0.4 nM . | 2.3 nM . | 14 nM . | 83 nM . | 0.5 μM . | 3.0 μM . | 4.5 μM . |
|---|---|---|---|---|---|---|---|---|
| CLO | PxrAB/Tü vs PxrTL | 0.0167 | 0.2374 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | 0.0018 |
| PxrAB/Tü vs PxrSWT | 0.7707 | 0.0511 | 0.6888 | 0.0496 | 0.0294 | 0.8725 | 0.0393 | |
| PxrAB/Tü vs PxrUNK | 0.8965 | 0.9998 | 0.0002 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | |
| PxrTL vs PxrSWT | 0.1551 | 0.8774 | 0.0017 | 0.0281 | 0.0002 | 0.0007 | 0.6801 | |
| PxrTL vs PxrUNK | 0.0023 | 0.2755 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | |
| PxrSWT vs PxrUNK | 0.3548 | 0.0627 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
| . | Compared Responses . | 0.1 μM . | 0.4 μM . | 1.2 μM . | 3.7 μM . | 11 μM . | 33 μM . | 50 μM . |
|---|---|---|---|---|---|---|---|---|
| 4BAB | PxrAB/Tü vs PxrTL | 0.1840 | 0.0013 | 0.9996 | 0.4299 | 0.9637 | 0.0751 | 0.4159 |
| PxrAB/Tü vs PxrSWT | 0.3229 | 0.0542 | 0.9938 | 0.9556 | 0.0175 | 0.0040 | 0.0083 | |
| PxrAB/Tü vs PxrUNK | 0.9071 | 0.9983 | 0.7314 | 0.1045 | 0.0011 | 0.0038 | 0.0123 | |
| PxrTL vs PxrSWT | 0.9875 | 0.5249 | 0.9985 | 0.1888 | 0.0570 | 0.6704 | 0.2806 | |
| PxrTL vs PxrUNK | 0.5165 | 0.0022 | 0.6710 | 0.8485 | 0.0002 | <0.0001 | 0.0001 | |
| PxrSWT vs PxrUNK | 0.7190 | 0.0793 | 0.5724 | 0.0322 | <0.0001 | <0.0001 | <0.0001 | |
| . | Compared Responses . | 0.1 μM . | 0.4 μM . | 1.2 μM . | 3.7 μM . | 11 μM . | 33 μM . | 50 μM . |
|---|---|---|---|---|---|---|---|---|
| 4BAB | PxrAB/Tü vs PxrTL | 0.1840 | 0.0013 | 0.9996 | 0.4299 | 0.9637 | 0.0751 | 0.4159 |
| PxrAB/Tü vs PxrSWT | 0.3229 | 0.0542 | 0.9938 | 0.9556 | 0.0175 | 0.0040 | 0.0083 | |
| PxrAB/Tü vs PxrUNK | 0.9071 | 0.9983 | 0.7314 | 0.1045 | 0.0011 | 0.0038 | 0.0123 | |
| PxrTL vs PxrSWT | 0.9875 | 0.5249 | 0.9985 | 0.1888 | 0.0570 | 0.6704 | 0.2806 | |
| PxrTL vs PxrUNK | 0.5165 | 0.0022 | 0.6710 | 0.8485 | 0.0002 | <0.0001 | 0.0001 | |
| PxrSWT vs PxrUNK | 0.7190 | 0.0793 | 0.5724 | 0.0322 | <0.0001 | <0.0001 | <0.0001 | |
One-way ANOVA and Dunnett’s test were used to determine the probability that the means of responses in exposed cells were different from exposures in nonexposed/DMSO treated cells (A). One-way ANOVA and Tukey-Kramer test were used to test for statistical differences between means of responses produced by the same exposure via different zfPxr variants (B). GraphPad Prism7.0 was used for statistical analyses. Tests that returned a greater than 5% probability for equal means (p value > .05) were shaded in gray in the tables.
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