The mammalian circadian system is resistant to dioxin

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that is bound and activated by many toxic ubiquitous environmental contaminants, including the halogenated aromatic hydrocarbon, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The AhR belongs to a family of proteins that contain basic helix-loop-helix/Per-ARNT-SIM (bHLH/PAS) domains. The circadian clock protein, BMAL1, is also a bHLH-PAS transcription factor and has been shown to interact with the AhR. AhRs are expressed in nearly every mammalian tissue, including the suprachiasmatic nuclei (SCN), and previous studies have suggested that activation of the AhR with dioxins affects rhythmicity in circadian clocks. In this study, the authors tested the hypothesis that activation of the aryl hydrocarbon receptor with the potent dioxin, TCDD, alters the organization of the mammalian circadian system by measuring bioluminescence from tissues explanted from PER2::LUCIFERASE mice. They found that in vitro treatment of explanted tissues with TCDD did not alter the periods, amplitudes, or damping rates of the PER2::LUC rhythms compared with controls. Likewise, in vivo treatment with TCDD had no effect on the phase relationship between central and peripheral oscillators. Together, these data demonstrate that activation of the AhR with TCDD does not directly or systemically alter the mouse circadian system.

Figure 1

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) does not directly affect the periods of circadian rhythms in cultured tissues. Representative detrended bioluminescence rhythms in liver (A, B), lung (E, F), pituitary (I, J), SCN (M, N), and thymus (Q, R) explants treated in vitro with DMSO (control, A, E, I, M, Q) or 100 nM TCDD (B, F, J, N, R). The mean period (± SD) of the PER2::LUC rhythm with DMSO (control) or TCDD treatment (1 nM, 10 nM, or 100 nM as indicated) is shown for each tissue (C, G, K, O, S). The first period measured in liver explants is shown (C; secondary period shown in Suppl. Fig. S2). The number of samples for each condition is indicated in the bar graph. Representative examples of the expression of Cyp1a1 and Gapdh in tissues treated in vitro with DMSO (C) or with 100 nM TCDD (T) were assessed by RT-PCR (D, H, L, P, T). Water (−) was added to the PCR reaction as a negative control.

Figure 2

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) does not directly affect the amplitudes and damping rates of circadian rhythms in cultured tissues. The mean (± SD) amplitudes (A, C, E, G) and damping rates (B, D, F, H) of PER2::LUC rhythms in lung (A, B), pituitary (C, D), SCN (E, F), and thymus (G, H) explants did not differ between tissues treated in vitro with DMSO (control) or TCDD (1 nM, 10 nM, or 100 nM as indicated). The number of samples for each condition is indicated in the bar graph.

Figure 3

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) does not systemically affect the organization of the circadian system. (A) Phase map for circadian oscillators from PER2::LUC mice administered DMSO (white circles) or 1 µg/kg TCDD (filled symbols) by oral gavage. Tissues were cultured either 4 days (white circles and black squares) or 14 days (black triangles) after treatment. The mean phases (± SD) were determined from the first peak of PER2::LUC expression in vitro and are plotted relative to the time of last lights-on, where 0 h is lights-on and 12 h is lights-off (black and white bar at top). The sample size is shown (number of rhythmic tissues/number of tissues tested). (B) The expression of Cyp1a1 and Gapdh in the liver of mice 4 days after treatment with DMSO (control) or 4 or 14 days after treatment with TCDD was assessed by RT-PCR.