To elucidate the effects of AhR signaling pathways on circadian rhythms, we first investigated the direct effects of TCDD treatment on circadian rhythms in SCN and peripheral tissue explants (). We found that the periods of PER2::LUC expression in liver (), lung (), pituitary (), SCN (), and thymus () were not altered by treatment with TCDD compared to vehicle (DMSO)–treated controls (see Suppl. Table S1
for statistical values). To confirm that the AhR was activated by in vitro TCDD treatment of the explanted tissues, we collected the tissues at the end of the experiment (after 7 days in culture) and analyzed the expression of Cyp1a1
, which is upregulated by TCDD-dependent AhR activation, by semi-quantitative RT-PCR. We found that in vitro treatment with TCDD markedly increased the expression of Cyp1a1
compared to DMSO-treated controls in all tissues we examined, demonstrating that the AhR was activated in our experiment (). Circadian rhythms of Cyp1a1
expression have been observed in SCN and liver, and expression levels vary by 2- to 3-fold each day in untreated mice (Mukai et al. 2008
). Therefore, we sometimes detected Cyp1a1
expression in control samples. In all samples, TCDD treatment increased the expression of Cyp1a1
more than 3-fold compared to controls, demonstrating that we strongly activated AhR signaling above baseline rhythmic levels in all tissues.
We next analyzed the effect of TCDD treatment on the amplitudes of PER2::LUC rhythms in tissues in vitro. We found that the amplitudes of PER2::LUC rhythms in lung (), pituitary (), SCN (), and thymus () were not altered by TCDD treatment compared to vehicle-treated controls (statistical values presented in Suppl. Table S1
). Since TCDD or vehicle was added at the time of culture, the effect of TCDD may be reflected in the damping rate of ex vivo rhythms. We found that the damping rates of PER2::LUC rhythms in lung (), pituitary (), SCN (), and thymus () did not differ between vehicle- and TCDD-treated tissues (see Suppl. Table S1
for statistical values). Amplitude and damping could not be measured in liver because PER2::LUC rhythms in liver explants have spontaneous changes in period and increases in the amplitude of the second cycle relative to the first cycle, which precludes the use of the curve-fitting method for analysis (see Supplemental Online Material
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 (more ...)
Even though we did not detect a direct effect of AhR activation on circadian rhythms in the SCN and peripheral tissues, it was possible that dioxin could indirectly affect the circadian system. To assess the systemic effect of TCDD on the organization of the circadian system, we administered 1 µg/kg TCDD or vehicle (DMSO) to PER2::LUC mice by oral gavage, cultured tissues either 4 days or 14 days later, and measured the phase of PER2::LUC expression in adipose tissue, adrenals, kidney, liver, lung, pituitary, SCN, and thymus (). We found that the phases of these tissues were not altered either 4 or 14 days after administration of TCDD. Furthermore, the expression of Cyp1a1 was strongly activated in the liver 4 and 14 days after the single dose of 1 µg/kg TCDD compared to controls, demonstrating that in vivo TCDD treatment induces long-lasting, potent activation of the AhR ().
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 (more ...)