Table 1.
Clinical characteristics of the liver donors.
Table 2.
Primer sequences.
Fig 1.
(A) Luciferase activity in HepG2-C3 cells transiently transfected with the pTXINV-XRE reporter plasmid and incubated with 100 μM skatole for 2, 4, 8 or 24 h (n = 3). Luciferase activity measured in HepAhLH (B) or HAhLH (C) cells, in which the luciferase reporter gene is controlled by the XRE 5(TnGCGTG)3, after incubation with skatole (from 1.10−7 M to 1.10−4 M) or TCDD (from 1.10−11 M to 1.10−7 M) for 8 h (n = 3). Luciferase activity was expressed as the percentage of the activity obtained by incubation with 1.10−7 M TCDD. (D) Relative luciferase activity measured in HepG2-C3 cells transfected with pTXINV-XRE and incubated with 1, 10 or 100μM skatole for 2, 4, 8 or 24 h, following 30 min pre-incubation with DMSO (control) or 3 μM CH223191 (a specific AhR antagonist) (n = 3). In Fig 1D, are all data points significantly different (p < 0.05) form its time-matched CH 223191 treated counterpart, except for control and 100 μM skatole at time point 24 hours.
Fig 2.
Skatole increases CYP1A1 expression in HepG2-C3 cells and PHHs.
RT-qPCR analysis of CYP1A1 (A) and AhR (B) mRNA expression following incubation of HepG2-C3 cells with 1, 10 or 100 μM skatole for 2, 4, 8 or 24 h (n = 3). (C) CYP1A and actin protein expression in PHHs (Donor #400) after incubation with 10, 50 or 100 μM for 24 h. * Significantly different from time-matched control cells (no treatment) (Student’s t–test; p < 0.001).
Table 3.
CYP1A1, CYP1A2, CYP1B1 and AhR mRNA expression in PHHs (n = 5) incubated with 1, 10, 100 μM skatole or 10 nM TCDD for 8 or 24 hours.
Fig 3.
AhR is required for skatole-mediated CYP1A1 and CYP1A2 up-regulation.
(A) RT-qPCR analysis of AhR, CYP1A1 and CYP1A2 mRNA expression in PHHs (Donor #391) after siRNA-mediated AhR down-regulation. (B) RT-qPCR analysis of CYP1A1 (B) and CYP1A2(C) mRNA expression in PHHs after siRNA-mediated AhR down-regulation and incubation with 50 or 100 μM skatole, or 10 nM TCDD. * significantly different from cells transfected with non-target siRNA.
Fig 4.
Skatole reduces TCDD-induced AhR activity and CYP1A1 expression.
RT-qPCR analysis of (A) CYP1A1 and (B) AhR mRNA expression following incubation of HepG2-C3A cells with 10 nM TCDD in the presence or not of 100 μM skatole for 2, 4, 8 or 24 h (n = 3). RT-qPCR analysis of CYP1A1, CYP1A2, CYP1B1 and AhR mRNA expression in PHHs treated for 8 (C) or 24 h (D) with TCDD in the presence or not of 100 μM skatole (n = 5). Results are expressed as the percentage of the induction observed with 10 nM TCDD. (E) CYP1A and actin protein expression in PHHs after incubation with 10 nM TCDD in the presence or not of 10, 50 or 100 μM skatole for 24 h (Donor #401). (F) Luciferase activity in HepG2-C3 cells transfected with pTXINV-XRE and incubated with TCDD alone or with skatole, in the presence or not of 3 μM CH223191 for 2, 4, 8 or 24 h (n = 3). * Significantly different from time-matched control cells (DMSO); # significantly different from time-matched TCDD treated cells (Student’s t–test; p < 0.01).
Fig 5.
Effect of skatole metabolism on CYP1A induction in HepG2-C3 cells.
(A) RT-qPCR analysis of CYP1A1 mRNA expression in HepG2-C3 cells following incubation with 4 μM actinomycin D for 1 h and incubation with 10 nM TCDD or 10, 50 or 100 μM skatole for 8 h (n = 3). (B) Relative CYP1A1 activity following incubation with 1 mM ABT for 1 h (n = 3). (C) RT-qPCR analysis of CYP1A1 mRNA expression in HepG2-C3 cells that were pre-incubated or not with 1 mM ABT for 1 h before incubation with 10, 50 or 100μM skatole for 8 h (n = 3). Insert shows CYP1A1 mRNA expression in HepG2-C3 cells after incubation or not with ABT. (D) RT-qPCR analysis of CYP1A1 mRNA expression in HepG2-C3 cells that were pre-incubated or not with 1 mM ABT for 1 h before incubation with 10 nM TCDD alone or together with 10, 50 or 100μM skatole for 8 h (n = 3). * significantly different from control cells; § significantly different from its time-matched CH 223191 treated counterpart.
Fig 6.
Indole-3-carbinol (I3C) induces CYP1A1 expression and activates AhR.
(A) RT-qPCR analysis of CYP1A1 mRNA expression in HepG2-C3 cells incubated with 0.1, 1 or 10 μM I3C for 24 h (n = 3). (B) Relative luciferase activity measured in HAhLH or HepAhLH cells incubated with I3C (from 1.10−7 M to 1.10−4 M) for 8 h (n = 3). (C) RT-qPCR analysis of CYP1A1 mRNA expression in HepG2-C3 cells incubated with 10 nM TCDD alone or in the presence of 0.1, 1 or 10 μM I3C (n = 3). Bars not sharing subscription are significantly different (p < 0.05).
Fig 7.
Schematic model of the impact of skatole on AhR activity, as suggested from the findings of this work.