ToxSci Advance Access originally published online on October 4, 2006
Toxicological Sciences 2007 95(1):172-181; doi:10.1093/toxsci/kfl126
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Effects of Tryptophan Photoproducts in the Circadian Timing System: Searching for a Physiological Role for Aryl Hydrocarbon Receptor
,1
* Department of Veterinary Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 61802
Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802
1 To whom correspondence should be addressed at Department of Veterinary Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 2001 S. Lincoln Avenue, Veterinary Medicine Basic Sciences Building, Urbana, IL 61802. Fax: +1-217-244-1652. E-mail: tischkau{at}uiuc.edu.
Received September 1, 2006; accepted September 30, 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The aryl hydrocarbon receptor (AhR) mediates adverse effects of dioxins, but its physiological role remains ambiguous. The similarity between AhR and canonical circadian clock genes suggests potential involvement of AhR in regulation of circadian timing. Photoproducts of tryptophan (TRP), including 6-formylindolo[3,2-b]carbazole (FICZ), have high affinity for AhR and are postulated as endogenous ligands. Although TRP photoproducts activate AhR signaling in vitro, their effects in vivo have not been investigated in mammals. Because TRP photoproducts may act as transducers of light, we examined their effects on the circadian clock. Intraperitoneal injection of TRP photoproducts or FICZ to C57BL/6J mice dose dependently induced AhR downstream targets, cytochrome P4501A1 (CYP1A1) and cytochrome P4501B1 mRNA expression, in liver. c-fos mRNA, a commonly used marker for light responses, was also induced with FICZ, and all responses were AhR dependent. A rat-immortalized suprachiasmatic nucleus (SCN) cell line, SCN 2.2, was used to examine the direct effect of TRP photoproducts on the molecular clock. Both TRP photoproducts and FICZ-increased CYP1A1 expression and prolonged FICZ incubation altered the circadian expression of clock genes (Per1, Cry1, and Cry2) in SCN 2.2 cells. Furthermore, FICZ inhibited glutamate-induced phase shifting of the mouse SCN electrical activity rhythm. Circadian light entrainment is critical for adjustment of the endogenous rhythm to environmental light cycle. Our results reveal a potential for TRP photoproducts to modulate light-dependent regulation of circadian rhythm through triggering of AhR signaling. This may lead to further understanding of toxicity of dioxins and the role of AhR in circadian rhythmicity.
Key Words: dioxin; suprachiasmatic nucleus; circadian rhythm; light entrainment; phase shift.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The aryl hydrocarbon receptor (AhR) belongs to a family of transcriptional regulatory proteins named for their common basic helix-loop-helix/Per-ARNT-Sim domain. Extensive research has focused on the role of AhR in mediating toxic effects of polycyclic aromatic hydrocarbons and dioxin-like compounds, including the highly toxic and ubiquitous environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, endogenous ligands and a physiological function of AhR remain a mystery.
Potential endogenous ligands have been discovered throughout the last decade, of which many are tryptophan (TRP) derivatives such as indirubin and indigo (Adachi et al., 2001
), trypthantrin and malassezin (Schrenk et al., 1997, 1999), indolo[3,2-b]carbazole (Bjeldanes et al., 1991), and 6-formylindolo[3,2-b]carbazole (FICZ) (Rannug et al., 1995; Wei et al., 1998, 2000). One of several light-induced photoproducts of TRP, FICZ, has generated considerable interest, due to its high binding affinity towards AhR (Kd = 0.07nM), which exceeds that of the prototypic exogenous ligand, TCDD (Kd = 0.48nM) (Rannug et al., 1987, 1995). Recently, FICZ has been detected in ordinary culture medium exposed to light (Oberg et al., 2005) and in TRP solution exposed to window light (Diani-Moore et al., 2006). FICZ binds to and activates AhR in vitro. It efficiently induced expression of the AhR downstream target gene cytochrome P4501A1 (CYP1A1) in human keratinocytes (HaCaT), fresh human peripheral blood cells (Wei et al., 1998), and mouse Hepa-1 cells (Wei et al., 2000). The induction was transient because FICZ itself is metabolized quickly by CYP1A1 (Wei et al., 2000), which supports the possibility that FICZ may act as an endogenous natural ligand for AhR. Although this compound has yet to be identified in plasma or tissues after light exposure and it is likely that concentrations in vivo will prove difficult to detect, the high affinity toward AhR suggests that even very low concentrations have the potential to be physiologically relevant. Furthermore, FICZ is not the only TRP photoproduct capable of acting as an AhR agonist (Diani-Moore et al., 2006). It is possible that a combination of TRP photoproducts act in concert to activate AhR in response to light under physiological conditions. Thus, we examined whether AhR can be activated in vivo by the constellation of TRP photoproducts acting together and by FICZ acting alone.
Phylogenetic analysis of the molecular evolution of AhR has revealed that this ancient (450–510 million years old) protein is present in all vertebrate and some invertebrate groups (Hahn et al., 1997) and suggests an important physiological role. AhR is speculated to function as a part of the circadian timing system due to the fact that many other Per-ARNT-Sim (PAS) domain family proteins, which are also highly conserved phylogenetically, play critical roles in circadian rhythmicity (Kewley et al., 2004). Intron/exon splice pattern comparisons of PAS domain family genes demonstrated that the splice pattern of the circadian clock gene, Bmal1 (brain and muscle ARNT-like), most closely matches that of the AhR (Yu et al., 1999). Bmal1 is critical for generation of circadian rhythmicity in mammals (Bunger et al., 2000); Bmal1 forms heterodimers with another canonical circadian clock gene, CLOCK (circadian locomotor output cycles kaput) to drive the molecular circadian clock. However, PAS domain family members bind promiscuously to one another, forming various alternative hetero/homodimers (Hogenesch et al., 1998; Probst et al., 1997). BMAL1, also known as MOP3, interacts with AhR in vitro (Hogenesch et al., 1997). Although the functional significance of this interaction has not been investigated, it certainly raises the possibility that AhR may function within the circadian timing system. Together with the fact that TRP photoproducts are potent AhR ligands, it has been proposed that photoproducts of TRP may act as light hormones and that a physiological role of the AhR is to mediate the signaling of light and to regulate biological rhythms (Huang et al., 2002; Rannug et al., 2003; Wei et al., 1999). Examination of the effects of FICZ (and other TRP photoproducts) in vivo will test this hypothesis.
Circadian rhythmicity is controlled by the suprachiasmatic nucleus (SCN), the master clock, located in the basal hypothalamus of the brain in mammals. Neuronal cells in the SCN maintain a near 24-h firing rhythm, and output from the SCN likely synchronizes all peripheral clocks, such as liver, heart, and kidney (for review see Reppert and Weaver [2002]). Surgical lesion of the rodent SCN causes arrhythmicity of behavioral circadian rhythms, which can be restored with SCN transplantation (LeSauter et al., 1996; Matsumoto et al., 1996). Light is the most important regulator of behavioral circadian rhythms (Reppert and Weaver, 2002). In mammals, the light signal is mainly transmitted through the retina reaching the SCN primarily via glutamatergic input from the retinohypothamic tract, although existence of extraocular phototransduction has been the subject of debate (Campbell et al., 2001).
This study tests whether TRP photoproducts can induce AhR signaling targets in vivo in a mammalian species and examines their potential to act directly on the master clock to influence light regulation of the SCN circadian rhythm. Our results suggest that TRP photoproducts induce AhR signaling targets in vivo in mice. With the use of SCN 2.2 cells, a rat-immortalized SCN cell line, and brain slices containing SCN, we conclude that there is a potential for TRP photoproducts to act directly on the master clock to alter the light regulation of circadian rhythms.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Animals.
Wildtype C57BL/6J mice were obtained at 6 weeks of age from Jackson laboratory, and AhR knockout mice (AhRKO) on a C57BL/6J background were generated from our colony at the University of Illinois, Urbana-Champaign. AhR+/– males and females were crossed, and genotypes of pups were determined by polymerase chain reaction (PCR) using genomic DNA obtained from ear punches as previously described (Benedict et al., 2000
). All animals were 2–3 months of age at the time of sacrifice. All animals were entrained for at least 2 weeks under controlled lighting (12:12 light/dark cycle), temperature (22°C), and humidity (39%) in light-tight chambers before initiation of the experiments described below. Under these conditions, zeitgeber time 0 (ZT0) indicates the time of lights on in the colony. Circadian time is used for designation of time under the constant conditions present in the brain slice chamber. For in vitro brain slice experiments, circadian time 0 (CT0) refers to the time of lights on in the donor colony prior to sacrifice. These abbreviations are used throughout the article as indicators of time. All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Illinois and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
TRP photoproducts.
L-Tryptophan (Fluka, Buchs, Switzerland; 25 mg/ml) dissolved in dimethylsulfoxide (DMSO; Fisher Scientific, Fairlawn, NJ) contained in glass scintillation vials (RPI corp., Mount Prospect, IL) was exposed to direct window light facing south for 120 h and stored at 4°C until use (TRP + WL). Specific AhR agonists, including but not limited to FICZ, were found by HPLC fractionation in a light-activated TRP solution that was prepared similarly to our methods (Diani-Moore et al., 2006). The authors report 72 pM of FICZ from light-activated TRP at 69µM. We assume conversion to FICZ was similar in our experiment. TRP solution covered with aluminum foil to prevent the exposure to light was used as a treatment control (TRP) and vehicle exposed to the same light was used as a vehicle control (DMSO). These solutions were used both for in vivo (Fig. 1) and in vitro studies (Fig. 3).
|
|
Animal treatments.
C57BL/6J were injected with TRP + WL (50 mg/kg BW, ip), TRP, or DMSO at ZT16, and liver was collected at ZT17, frozen in liquid nitrogen, and then stored in – 80°C until further analysis. To study the dose and AhR dependency of gene induction by TRP photoproducts in vivo, FICZ (BIOMOL, Plymouth Meeting, PA) was dissolved in DMSO and injected (0, 1, 10 and 100 µg/kg BW, ip) to wild type (WT) and AhRKO mice (0 and 100 µg/kg BW, ip) at ZT16 (4 h after lights off). SCN and liver tissues were collected at ZT17 and stored at – 80°C until further analysis. ZT16 was selected for time of treatment because light causes maximal phase shifting of activity rhythm in rodents at this time (Daan and Pittendrigh, 1976
).
Cell cultures.
SCN 2.2 cells were plated at 0.6 x 106 cells per well in 1 ml of medium on Falcon flat-bottomed 24-well polystyrene cell culture plates (Becton Dickinson, Franklin Lakes, NJ), which were coated with laminin (1 µg/cm2) prior to use. Medium used for cell culture consisted of Eagle's minimum essential medium (Biowhittaker, Waltersville, MD) supplemented with 10% fetal bovine serum, penicillin (1 x 104 U/ml), streptomycin (1 x 104 µg/ml), and fungizone (250 µg/ml). The cells were grown at 37°C in a 5% CO2 environment.
When the cells achieved 90–100% confluency, medium was removed and replaced with new medium containing 0, 0.5, 5, 50nM FICZ dissolved in DMSO. Final DMSO content was kept at 0.25% to avoid cytotoxicity (SCN neurons incubated with the same DMSO content showed robust activity for at least 24 h). The cells were incubated for 3 h, harvested, and stored at – 80°C overnight for further analysis. To study the effect of FICZ on the circadian expression pattern of genes, cells were incubated at same conditions as above with 0 or 50nM FICZ and cells were harvested after 0, 0.5, 1, 3, 6, 12, 18, and 24 h. For the TRP photoproduct study, TRP solutions prepared as previously described were used. SCN 2.2 cells were grown at 1.3 x 106 cells per well on a 6-well plate until confluency and treated with 25nM TRP + WL, TRP, or DMSO (final DMSO concentration 0.25%) for 3 h. Cells were then collected for mRNA analysis.
Real-time quantitative PCR.
Total RNA was isolated using TRIzol reagent (GIBCO BRL, Carlsbad, CA) for liver tissues, RNeasy Mini Kits (QIAGEN Inc, Valencia, CA) for SCN 2.2 cells or RNAqueous (Ambion, Austin, TX) for SCN tissues following the manufacturers' guides. The RNA was diluted in RNase-free H2O, quantified using NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE), and 1 µg of total RNA sample was reverse transcribed for 50 min at 42°C in a 20-µl reaction with 200 U of SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) and 0.5 µg of Oligo (dT)12–18 primer following the manufacturer's protocol. The synthesized cDNA was diluted 1:25, and 4 µl of this diluted cDNA was used for subsequent 20-µl quantitative PCR (qPCR) reactions with 2x SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and 300nM of each forward and reverse primer (Tables 1 & 2). Primers were designed across two exons to inhibit false positives by amplification of genomic DNA using Primer Express 3 software (PE Applied Biosystems, Foster City, CA). Quantitative real-time PCR analysis was done using ABI Prism 7000 Sequence Detector. The Ct (threshold cycle) value was obtained, and relative amount of amplicon was calculated using the relative standard curve method described in Applied Biosystems User Bulletin 2. For detection of AhR in SCN 2.2 cells, the PCR product was run on a 1.5% agarose gel and visualized with ethidium bromide.
|
|
Brain slices.
Animals were euthanized at CT7–9, and 500 µm coronal brain slices containing SCN were made with a mechanical chopper and placed in a Hatton-style brain slice chamber. The slices were continuously perfused with Earle's essential balanced salt solution (Life Technologies, Gaithersburg, MD), supplemented with 24.6mM glucose and 26.2mM NaHCO3 at 34°C, pH7.4, in an environment of 95% O2 and 5% CO2. Slices were treated with FICZ (50nM) or vehicle at CT13.5 for 30 min and subsequently treated with glutamate (2 µl of 10mM glutamate was applied directly to SCN) or vehicle at CT14. The perfusion pump was paused at the time of treatment, and the standard medium was replaced with medium containing FICZ. After 10 min of glutamate incubation, the medium was completely replaced with fresh medium, and perfusion was reinitiated. Single-unit activity of the SCN neuronal ensemble was recorded starting from CT3 on the following day.
Single-unit recordings of SCN neuronal activity.
Neuronal activity of single SCN cells was obtained with the use of extracellular single-unit recording technique as previously described (Prosser and Gillette, 1989; Tischkau et al., 2004). Briefly, a glass microelectrode containing 5M NaCl was inserted into SCN using a hydraulic microdrive until single-cell activity was encountered. The firing activity was recorded for 4 min, and then, the electrode was moved to a different area until another SCN neuron was located. The recording was continued at least until 3 h after maximal firing rate activity was achieved. Sliding window averages were obtained by calculating the mean firing rate of all cells recorded in a 2-h time slot, then shifting ahead 15 min, and calculating the mean firing rate for the subsequent 2-h time slot. Under these conditions, the SCN generates a stable, approximate 24-h rhythm in neuronal activity with a peak around midday (CT6–7) in mice (Buchanan and Gillette, 2005). The difference between peak times of treatment group versus control group was calculated to determine the extent of phase shifts.
Statistics.
Statistical analysis was performed by ANOVA followed by Tukey's test for pairwise comparisons using SYSTAT (SSI, Richmond, CA). For time-course mRNA expression, Student's t-test was used to compare treatment and control group at designated time points. Power regression was used to examine the dose-dependent relationships. When the normality assumption was not met, the data were transformed into natural log. All p values were two sided and considered statistically significant at p < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Effect of TRP Photoproducts In Vivo
TRP solution exposed to light has been shown to induce CYP1A1 expression in human keratinocytes (Wei et al., 1999
), chick hepatocytes, and embryos in vitro (Diani-Moore et al., 2006). Injection of TRP photoproducts (TRP + WL, 50 mg/kg BW, ip) likewise caused induction of CYP1A1 in vivo in the mouse liver (Fig. 1). There was a fourfold increase (p < 0.01) of CYP1A1 mRNA expression in liver of mice that were injected with TRP exposed to window light for 5 days compared to DMSO control. Injection of TRP alone had no effect on CYP1A1 expression levels.
Next, a specific TRP photoproduct, FICZ, with the highest known affinity for AhR (Rannug et al., 1995), was injected into mice in vivo. Although this compound causes rapid and transient CYP1A1 induction in various cell lines (Wei et al., 1998, 1999, 2000) at picomolar concentrations, effects of FICZ on AhR activation had yet to be investigated in vivo. Our results show that FICZ dose dependently increases CYP1A1 mRNA expression in liver in vivo (Fig. 2A). CYP1A1 expression levels were significantly elevated in all WT groups treated with FICZ (p < 0.05) compared to vehicle-treated controls and to those groups treated with lower doses (1 ± 0.1, 2.5 ± 0.1, 7.0 ± 3.3, and 14.7 ± 2.9 for 0, 1, 10, and 100 mg/kg BW, respectively). AhRKO mice had lower basal levels of CYP1A1 (0.29 ± 0.03), and FICZ treatment was unable to increase CYP1A1 expression in AhRKO mice. Increased mRNA levels of another AhR target gene, cytochrome P4501B1 (CYP1B1), was also observed in liver of WTs at the higher two doses (2.0 ± 0.6 and 2.1 ± 0.3 for 10 and 100 µg/kg BW, respectively; Fig. 2B) compared to control (1 ± 0.15; p < 0.05). In contrast to CYP1A1, AhRKO mice had higher basal levels of CYP1B1 (2.2 ± 0.48), and FICZ treatment did not significantly affect this basal expression, although a trend toward a decrease was observed.
|
As both c-fos, a proto-oncogene, and period 1 (Per1) are induced in the SCN immediately after light exposure in vivo, these genes were also examined. In WTs, there was a nonlinear trend of increase of c-fos expression with FICZ dose (p < 0.02; Fig. 2C) using power regression analysis, although there was no statistical significance with ANOVA. c-fos was not changed in AhRKO with FICZ treatment. Per1 expression was not affected with any dose of FICZ in WT liver, and similar basal expression was observed in AhRKO liver (not shown). Finally, CYP1A1 and CYP1B1 expression in WT SCN was not changed after ip injection of FICZ (not shown).
Effect of TRP Photoproducts in SCN 2.2 Cells
To determine the direct effects of TRP photoproducts on the master clock in the SCN, we used the rat-immortalized SCN cell line (SCN 2.2). SCN 2.2 expresses AhR as determined by RT-PCR (Fig. 3A). Similar to the results in vivo, TRP exposed to window light for 5 days increased CYP1A1 mRNA expression by threefold (1 ± 0.2 and 3.0 ± 0.2 in DMSO and TRP + WL, respectively; Fig. 3B) in SCN 2.2 cells (p < 0.001). FICZ also increased CYP1A1 mRNA expression in a dose-dependent manner (Fig. 4, p < 0.01).
|
A 24-h time course of the expression patterns of CYP1A1, c-fos, and several clock genes (Bmal1, Per1, Cry1, and Cry2) was performed in SCN 2.2 cells incubated with FICZ to determine whether AhR activation can alter the circadian rhythms of clock gene expression in these cells. CYP1A1 mRNA expression (Fig. 5A) remained increased after 3 h of FICZ incubation compared to controls at same time points and was highest at 24 h. c-fos showed significantly decreased expression at 12 h (Fig. 5B). There were no significant differences in either Bmal1 or Per1 mRNA expression between treatment groups (Figs. 5C and 5D); however, there was trend toward a decreased expression in both of these genes at 0.5 and 1 h with FICZ treatment. The overall amplitude of Per1 expression was dampened by FICZ incubation (2.43- and 1.92-fold at peak from trough, control, and FICZ, respectively), suggesting a potential for an alteration in the amplitude of the circadian rhythm of Per1. Comparison of Bmal1 and Per1 profiles shows an out-of-phase relationship between these two genes, which is indicative of a functional clock and validates SCN 2.2 as a model system. Furthermore, both Cry1 and Cry2 showed a significant increase at 12 h with FICZ treatment (Figs. 5E and 5F), which may also reflect an alteration in rhythmic expression pattern of these genes.
|
Effect of FICZ on Light-Induced Phase Shifting in the SCN In Vitro
To determine the effects of FICZ on light regulation of the circadian rhythm, we obtained brain slices and tested whether glutamate-induced phase shifting can be altered by preincubation of the slices with FICZ. Our results demonstrate that preincubation with FICZ blocks the effect of glutamate on phase shifting (Fig. 6A). Treatment of the slice with only glutamate caused a –3.0 ± 0.4 h shift (delay shift, Fig. 6B) in the peak of neuronal firing activity of SCN compared to control (0 ± 0.2 h). Preincubation with FICZ by itself did not cause significant phase shifts (–0.2 ± 0.1 h). However, preincubation with FICZ prevented the phase shifts caused by treatment with glutamate (–0.13 ± 0.25 h).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Our data reveal dose-dependent induction of CYP1A1 and CYP1B1 by TRP photoproducts in mice in vivo. Although previously demonstrated in human keratinocytes (Wei et al., 1999
), and in chick hepatocytes and embryos (Diani-Moore et al., 2006), our data provide the first evidence that TRP photoproducts can induce these genes in vivo in a mammalian species. Experiments in AhRKO mice clearly demonstrated that induction of CYP1A1 and CYP1B1 by TRP photoproduct requires AhR. TRP is the strongest near-UV absorbing of all amino acids. Induction of CYP1A1 expression in cultured cells after medium change has often been attributed to photooxidation products of TRP (Kocarek et al., 1993; Oberg et al., 2005; Paine, 1976). UV light–induced activation of AhR signaling in both skin and in internal organs such as liver (Goerz et al., 1996) has also been ascribed to TRP photoproducts (Heath-Pagliuso et al., 1998; Helferich and Denison, 1991; Rannug et al., 1987). Our results corroborate these observations. Thus, it is plausible that TRP photoproducts are synthesized upon light exposure in vivo, transferred through the bloodstream, and bind to AhR in internal organs.
TRP solution, which we used in vivo (122mM or 25 mg/ml), was higher than what is generally contained in food products; however, it is not unreasonably higher. Some dairy products in the United States can contain up to about 25mM TRP (http://www.nal.usda.gov/). Based on the conversion rate reported previously (Diani-Moore et al., 2006), we have estimated that our light-exposed TRP contained about 0.13µM of FICZ. In comparison, the lowest FICZ dose used for our in vivo study was 14x, the FICZ concentration (1.76µM). However, the CYP1A1 induction was higher in mice injected with light-exposed TRP than in mice injected with lowest dose of FICZ (4- and 2.5-fold, respectively). This further verifies the existence of other AhR agonists present in the light-exposed TRP.
The physiological significance of light-induced AhR activation remains an open question. Although relatedness of AhR to canonical circadian clock genes suggests its potential involvement in the circadian timing system, this hypothesis has yet to be fully investigated. However, PAS domain proteins mediate organismic sensitivity to environmental signals, including a central role in regulation of circadian rhythms (Gu et al., 2000). FICZ and other TRP photoproducts may act as chemical messengers of light by binding to AhR (Rannug et al., 1987; Wei et al., 2000), thus, providing a mechanism for mediation of light entrainment of endogenous rhythms to the environmental light/dark cycle. Although AhR is expressed in various regions of the rat brain including SCN (Petersen et al., 2000), ip injection of FICZ did not reveal any evidence of AhR activation in the SCN in vivo. Rapid metabolism of FICZ or inability to cross the blood brain barrier may have prevented a sufficient dose from reaching the SCN, although the lipophilic nature of the compound makes the latter less likely. The lack of response in SCN could have resulted from our inability to isolate specific light-responsive regions for qPCR. The SCN is complex, with multiple of cell types that range in their responsiveness to light signals (Hamada et al., 2004). Limitations of sensitivity in qPCR may have also hampered our ability to detect change in the SCN. Finally, the single collection time at 1 h after injection of FICZ may not have been sufficient to detect changes in the SCN. In fact, no significant differences in gene expression were detected in SCN 2.2 cells at 1 h with FICZ treatment.
Effects of TRP photoproducts on the master clock were revealed by time-course studies in the rhythmic rat-immortalized SCN cell line (Earnest et al., 1999; Hurst et al., 2002b). Bmal1, Per1, Cry1, and Cry2 are expressed in these cells (Hurst et al., 2002a). AhR was also expressed in SCN 2.2 cells, and incubation of these cells with both window light–activated TRP and FICZ induced CYP1A1 mRNA expression. Bmal1, Per1, and Cry1 mRNA expression has been reported to have rhythmicity in SCN 2.2 similarily to the SCN in vivo (Earnest and Cassone, 2005). Robust circadian expression of Per1, Bmal1, and Cry1 mRNA demonstrates the circadian function of SCN 2.2 cells in our study.
FICZ treatment altered the circadian expression patterns of clock genes in these cells. Although expression of Per1 was not significantly different in FICZ-incubated cells at any individual time point, the amplitude of the rhythm seemed to dampen with FICZ incubation. Because rhythmic Per1 transcription occurs downstream of BMAL1 and CLOCK heterodimerization, a dampening of the Per1 rhythm implies a disruption of the molecular clock mechanism. However, Bmal1 mRNA expression was not altered by FICZ treatment.
We demonstrated trends toward decreased expression of CYP1A1, Bmal1, Per1, and Cry1 at earlier time points 0.5 and 1 h in FICZ-incubated cells. Although further experimentation is required to evaluate this effect, overall suppression of transcription is not a likely cause because c-fos increased during the same period. Immediate upregulation of Per1 is common in response to serum shock in vitro and light pulses in vivo. Our data suggest that activation of AhR may not contribute to the mechanism that leads to Per1 upregulation during circadian light responses. Interestingly, other triggers of circadian synchronization, including glucose (Hirota et al., 2002), are accompanied by immediate Per1 downregulation, which is similar to our findings.
Early increases in c-fos in both control and FICZ groups is likely attributable to serum-containing medium change (Bird et al., 1990). The potential presence of TRP photoproducts within the medium used for both control and FICZ groups may have diminished the effectiveness of FICZ treatment. The slight increase of Per1 at 0.5 h in the control group could also be attributed to this as a simple medium change can phase shift Per1 expression in human neuroblastoma cells (Maronde and Motzkus, 2003). Future studies will use serum-free medium to test this hypothesis.
The most significant effect of FICZ on clock gene expression in these studies was on Cry1 and Cry2 genes. Cryptochromes are predicted to be evolutionary descendents of DNA photolyases that act as photoreceptors both in plants and animals (Sancar, 2003). Cry is an essential component of the light response in the Drosophila circadian clock (Ceriani et al., 1999). In mammals, Cry, in conjunction with opsins, is also important in light-dependent regulation of circadian rhythm (Selby et al., 2000). Thus, alteration of Cry expression after FICZ treatment provides a potential mechanism for AhR to influence light-induced circadian phase resetting. Further studies will investigate potential interactions between AhR and the Cry genes during light-induced phase resetting of the SCN.
Light is the most prominent environmental signal used to affect circadian timing. In response to nocturnal light, the SCN triggers a phase resetting to realign the animal's behavior with the perceived change in light and darkness. Light during early subjective night causes phase delay (Gillette and Mitchell, 2002). FICZ blocks the glutamate-induced phase delay in the SCN, an equivalent of light-triggered phase delay in vivo. These data provide the first physiological evidence that an AhR agonist can directly inhibit light responses in terms of phase shifting. Further investigations, including direct intracerebrospinal injection of FICZ, are necessary to determine whether this occurs in vivo. These studies demonstrate that activation of AhR may play a role in the circadian timing system by influencing the animal's responsiveness to light.
Recently, a new PAS domain protein that plays a role in circadian rhythm has been discovered. Neuronal PAS domain protein 2 (NPAS2) is considered as a peripheral replacement for the core clock gene, CLOCK, and seems to be important in the food-entrainable oscillator (Dudley et al., 2003). NPAS2 mouse mutants cannot adapt well to restricted feeding during daylight, leading ultimately to death (Dudley et al., 2003), although robust circadian activity rhythms were observed. This finding raises the question of whether these ancient proteins, including AhR, have evolved to play different roles in circadian timing among different tissues and organisms. AhR is expressed abundantly in various tissues, including the SCN, other parts of the brain, and the rest of the body. Although AhRKO mice seem to have robust circadian rhythm (Mukai and Tischkau, unpublished data), it is possible that, like NPAS2, AhR has evolved to have a specific role in circadian rhythm of certain tissues, such as modulating a light response as suggested by our data. Activation of AhR using the prototypical environmental contaminant, TCDD, alters rhythms in activity, feeding (Kelling et al., 1985; Seefeld et al., 1984), hormones (Jones et al., 1987; Pohjanvirta et al., 1989; Yellon et al., 2000), and clock gene expression (Garrett and Gasiewicz, 2006; Miller et al., 1999). Whether these ubiquitous environmental contaminants cause inhibition of light-dependent regulation of circadian rhythm, remains an important question.
Mechanisms underlying SCN regulation of rhythmicity in target peripheral tissues is not fully characterized, but neural, hormonal, and behavioral signals are involved (Hirota and Fukada, 2004). Our data demonstrate that TRP photoproducts have the potential to affect both peripheral and master clocks. Specifically, FICZ inhibition of light-like phase shifts in the SCN suggests that TRP photoproducts are capable of modulating light-dependent regulation of the circadian rhythm. Although the mechanism for FICZ modulation of light responsiveness remains elusive, it is intriguing considering that TRP is an important precursor for serotonin and melatonin, which are both important in regulation of circadian rhythms.
Although AhR is known to mediate the majority of the toxic effects of dioxins, mechanisms downstream of AhR still remain ambiguous. Our results provide evidence of a potential physiological role for AhR in regulation of circadian rhythmicity through actions on both peripheral and master clocks. Deciphering the role of AhR in circadian rhythms will shed light not only into unknown mechanism of dioxin toxicity but unexplored mechanism of light-dependent regulation of circadian rhythm.
|
ACKNOWLEDGMENTS |
|---|
We thank Drs Richard E. Peterson and Tien-Min Lin (University of Wisconsin, Madison) for providing the original breeders of the AhRKO mice, Dr David J. Earnest for SCN 2.2 cells, Dr Martha U. Gillette for use of recording equipment and Pilar Silva and Ben Haley for technical assistance with cell culture experiments. We are also grateful to Drs Paul S. Cooke and Jodi A. Flaws for critical reading of the manuscript and Dr David J. Schaeffer for assistance with statistics. Certain primer sets for qPCR were designed by Bethany Karman and Jason Hickok. This research was supported by National Institute of Environmental Health Sciences grant ES012948 (to S.A.T.), Eli Lilly Predoctoral Fellowship (to M.M.), and Illinois State Governor's Venture Technology Fund.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Adachi J, Mori Y, Matsui S, Takigami H, Fujino J, Kitagawa H, Miller CA III, Kato T, Saeki K, Matsuda T. (2001) Indirubin and indigo are potent aryl hydrocarbon receptor ligands present in human urine. J. Biol. Chem. 276:31475–31478.
Benedict JC, Lin TM, Loeffler IK, Peterson RE, Flaws JA. (2000) Physiological role of the aryl hydrocarbon receptor in mouse ovary development. Toxicol. Sci. 56:382–388.
Bird RC, Kung TY, Wu G, Young-White RR. (1990) Variations in c-fos mRNA expression during serum induction and the synchronous cell cycle. Biochem. Cell Biol. 68:858–862.[ISI][Medline]
Bjeldanes LF, Kim JY, Grose KR, Bartholomew JC, Bradfield CA. (1991) Aromatic hydrocarbon responsiveness-receptor agonists generated from indole-3-carbinol in vitro and in vivo: Comparisons with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Proc. Natl. Acad. Sci. U.S.A. 88:9543–9547.
Buchanan GF and Gillette MU. (2005) New light on an old paradox: Site-dependent effects of carbachol on circadian rhythms. Exp. Neurol. 193:489–496.[CrossRef][ISI][Medline]
Bunger MK, Wilsbacher LD, Moran SM, Clendenin C, Radcliffe LA, Hogenesch JB, Simon MC, Takahashi JS, Bradfield CA. (2000) Mop3 is an essential component of the master circadian pacemaker in mammals. Cell 103:1009–1017.[CrossRef][ISI][Medline]
Campbell SS, Murphy PJ, Suhner AG. (2001) Extraocular phototransduction and circadian timing systems in vertebrates. Chronobiol. Int. 18:137–172.[CrossRef][ISI][Medline]
Ceriani MF, Darlington TK, Staknis D, Mas P, Petti AA, Weitz CJ, Kay SA. (1999) Light-dependent sequestration of TIMELESS by CRYPTOCHROME. Science 285:553–556.
Daan S and Pittendrigh CS. (1976) A functional analysis of circadian pacemakers in nocturnal rodents. II. The variability of phase response curves. J. Comp. Physiol. 143:527–539.[CrossRef]
Diani-Moore S, Labitzke E, Brown R, Garvin A, Wong L, Rifkind AB. (2006) Sunlight generates multiple tryptophan photoproducts eliciting high efficacy CYP1A induction in chick hepatocytes and in vivo. Toxicol. Sci. 90:96–110.
Dudley CA, Erbel-Sieler C, Estill SJ, Reick M, Franken P, Pitts S, McKnight SL. (2003) Altered patterns of sleep and behavioral adaptability in NPAS2-deficient mice. Science 301:379–383.
Earnest DJ and Cassone VM. (2005) Cell culture models for oscillator and pacemaker function: Recipes for dishes with circadian clocks? Methods Enzymol. 393:558–578.[CrossRef][ISI][Medline]
Earnest DJ, Liang FQ, Ratcliff M, Cassone VM. (1999) Immortal time: Circadian clock properties of rat suprachiasmatic cell lines. Science 283:693–695.
Garrett RW and Gasiewicz TA. (2006) The aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin alters the circadian rhythms, quiescence, and expression of clock genes in murine hematopoietic stem and progenitor cells. Mol. Pharmacol. 69:2076–2083.
Gillette MU and Mitchell JW. (2002) Signaling in the suprachiasmatic nucleus: Selectively responsive and integrative. Cell Tissue Res. 309:99–107.[CrossRef][ISI][Medline]
Goerz G, Barnstorf W, Winnekendonk G, Bolsen K, Fritsch C, Kalka K, Tsambaos D. (1996) Influence of UVA and UVB irradiation on hepatic and cutaneous P450 isoenzymes. Arch. Dermatol. Res. 289:46–51.[CrossRef][ISI][Medline]
Gu YZ, Hogenesch JB, Bradfield CA. (2000) The PAS superfamily: Sensors of environmental and developmental signals. Annu. Rev. Pharmacol. Toxicol. 40:519–561.
Hahn ME, Karchner SI, Shapiro MA, Perera SA. (1997) Molecular evolution of two vertebrate aryl hydrocarbon (dioxin) receptors (AHR1 and AHR2) and the PAS family. Proc. Natl. Acad. Sci. U.S.A. 94:13743–13748.
Hamada T, Antle MC, Silver R. (2004) Temporal and spatial expression patterns of canonical clock genes and clock-controlled genes in the suprachiasmatic nucleus. Eur. J. Neurosci. 19:1741–1748.[CrossRef][ISI][Medline]
Heath-Pagliuso S, Rogers WJ, Tullis K, Seidel SD, Cenijn PH, Brouwer A, Denison MS. (1998) Activation of the Ah receptor by tryptophan and tryptophan metabolites. Biochemistry 37:11508–11515.[CrossRef][Medline]
Helferich WG and Denison MS. (1991) Ultraviolet photoproducts of tryptophan can act as dioxin agonists. Mol. Pharmacol. 40:674–678.[Abstract]
Hirota T and Fukada Y. (2004) Resetting mechanism of central and peripheral circadian clocks in mammals. Zool. Sci. 21:359–368.[CrossRef][ISI][Medline]
Hirota T, Okano T, Kokame K, Shirotani-Ikejima H, Miyata T, Fukada Y. (2002) Glucose down-regulates Per1 and Per2 mRNA levels and induces circadian gene expression in cultured Rat-1 fibroblasts. J. Biol. Chem. 277:44244–44251.
Hogenesch JB, Chan WK, Jackiw VH, Brown RC, Gu YZ, Pray-Grant M, Perdew GH, Bradfield CA. (1997) Characterization of a subset of the basic-helix-loop-helix-PAS superfamily that interacts with components of the dioxin signaling pathway. J. Biol. Chem. 272:8581–8593.
Hogenesch JB, Gu YZ, Jain S, Bradfield CA. (1998) The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors. Proc. Natl. Acad. Sci. U.S.A. 95:5474–5479.
Huang P, Ceccatelli S, Rannug A. (2002) A study on diurnal mRNA expression of CYP1A1, AHR, ARNT, and PER2 in rat pituitary and liver. Environ. Toxicol. Pharmacol. 11:119–126.[CrossRef]
Hurst WJ, Earnest D, Gillette MU. (2002a) Immortalized suprachiasmatic nucleus cells express components of multiple circadian regulatory pathways. Biochem. Biophys. Res. Commun. 292:20–30.[CrossRef][ISI][Medline]
Hurst WJ, Mitchell JW, Gillette MU. (2002b) Synchronization and phase-resetting by glutamate of an immortalized SCN cell line. Biochem. Biophys. Res. Commun. 298:133–143.[CrossRef][ISI][Medline]
Jones MK, Weisenburger WP, Sipes IG, Russell DH. (1987) Circadian alterations in prolactin, corticosterone, and thyroid hormone levels and down-regulation of prolactin receptor activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 87:337–350.[CrossRef][ISI][Medline]
Karman BN and Tischkau SA. (2006) Circadian clock gene expression in the ovary: Effects of luteinizing hormone. Biol Reprod 75:624–632.
Kelling CK, Christian BJ, Inhorn SL, Peterson RE. (1985) Hypophagia-induced weight loss in mice, rats, and guinea pigs treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fundam. Appl. Toxicol. 5:700–712.[CrossRef][ISI][Medline]
Kewley RJ, Whitelaw ML, Chapman-Smith A. (2004) The mammalian basic helix-loop-helix/PAS family of transcriptional regulators. Int. J. Biochem. Cell Biol. 36:189–204.[CrossRef][ISI][Medline]
Kocarek TA, Schuetz EG, Guzelian PS. (1993) Transient induction of cytochrome P450 1A1 mRNA by culture medium component in primary cultures of adult rat hepatocytes. In Vitro Cell Dev. Biol. 29A:62–66.
LeSauter J, Lehman MN, Silver R. (1996) Restoration of circadian rhythmicity by transplants of SCN "micropunches.". J. Biol. Rhythms 11:163–171.
Maronde E and Motzkus D. (2003) Oscillation of human period 1 (hPER1) reporter gene activity in human neuroblastoma cells in vivo. Chronobiol. Int. 20:671–681.[CrossRef][ISI][Medline]
Matsumoto S, Basil J, Jetton AE, Lehman MN, Bittman EL. (1996) Regulation of the phase and period of circadian rhythms restored by suprachiasmatic transplants. J. Biol. Rhythms 11:145–162.
Miller JD, Settachan D, Frame LT, Dickerson R. (1999) 2,3,7,8-Tetracholorodibenzo-p-dioxin phase advances the deer mouse (Peromyscus maniculatus) circadian rhythm by altering expression of clock proteins. Organohalogen Compd. 42:23–28.
Oberg M, Bergander L, Hakansson H, Rannug U, Rannug A. (2005) Identification of the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole, in cell culture medium, as a factor that controls the background aryl hydrocarbon receptor activity. Toxicol. Sci. 85:935–943.
Paine AJ. (1976) Effect of amino acids and inducers on the activity of the microsomal mono-oxygenase system in rat liver cell culture. Chem. Biol. Interact. 13:307–315.[CrossRef][ISI][Medline]
Petersen SL, Curran MA, Marconi SA, Carpenter CD, Lubbers LS, McAbee MD. (2000) Distribution of mRNAs encoding the arylhydrocarbon receptor, arylhydrocarbon receptor nuclear translocator, and arylhydrocarbon receptor nuclear translocator-2 in the rat brain and brainstem. J. Comp. Neurol. 427:428–439.[CrossRef][ISI][Medline]
Pohjanvirta R, Tuomisto J, Linden J, Laitinen J. (1989) TCDD reduces serum melatonin levels in Long-Evans rats. Pharmacol. Toxicol. 65:239–240.[ISI][Medline]
Probst MR, Fan CM, Tessier-Lavigne M, Hankinson O. (1997) Two murine homologs of the Drosophila single-minded protein that interact with the mouse aryl hydrocarbon receptor nuclear translocator protein. J. Biol. Chem. 272:4451–4457.
Prosser RA and Gillette MU. (1989) The mammalian circadian clock in the suprachiasmatic nuclei is reset in vitro by cAMP. J. Neurosci. 9:1073–1081.[Abstract]
Rannug A, Rannug U, Rosenkranz HS, Winqvist L, Westerholm R, Agurell E, Grafstrom AK. (1987) Certain photooxidized derivatives of tryptophan bind with very high affinity to the Ah receptor and are likely to be endogenous signal substances. J. Biol. Chem. 262:15422–15427.
Rannug A, Tuominen R, Warholm M, Rannug U. (2003) A proposed physiological role of the aryl hydrocarbon receptor. Organohalogen Compd. 65:60–65.
Rannug U, Rannug A, Sjoberg U, Li H, Westerholm R, Bergman J. (1995) Structure elucidation of two tryptophan-derived, high affinity Ah receptor ligands. Chem. Biol. 2:841–845.[CrossRef][ISI][Medline]
Reppert SM and Weaver DR. (2002) Coordination of circadian timing in mammals. Nature 418:935–941.[CrossRef][Medline]
Sancar A. (2003) Structure and function of DNA photolyase and cryptochrome blue-light photoreceptors. Chem. Rev. 103:2203–2237.[CrossRef][ISI][Medline]
Schrenk D, Riebniger D, Till M, Vetter S, Fiedler HP. (1997) Tryptanthrins: A novel class of agonists of the aryl hydrocarbon receptor. Biochem. Pharmacol. 54:165–171.[CrossRef][ISI][Medline]
Schrenk D, Riebniger D, Till M, Vetter S, Fiedler HP. (1999) Tryptanthrins and other tryptophan-derived agonists of the dioxin receptor. Adv. Exp. Med. Biol. 467:403–408.[ISI][Medline]
Seefeld MD, Corbett SW, Keesey RE, Peterson RE. (1984) Characterization of the wasting syndrome in rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 73:311–322.[CrossRef][ISI][Medline]
Selby CP, Thompson C, Schmitz TM, Van Gelder RN, Sancar A. (2000) Functional redundancy of cryptochromes and classical photoreceptors for nonvisual ocular photoreception in mice. Proc. Natl. Acad. Sci. U.S.A. 97:14697–14702.
Semo M, Lupi D, Peirson SN, Butler JN, Foster RG. (2003) Light-induced c-fos in melanopsin retinal ganglion cells of young and aged rodless/coneless (rd/rd cl) mice. Eur. J. Neurosci. 18:3007–3017.[CrossRef][ISI][Medline]
Tischkau SA, Mitchell JW, Pace LA, Barnes JW, Barnes JA, Gillette MU. (2004) Protein kinase G type II is required for night-to-day progression of the mammalian circadian clock. Neuron 43:539–549.[CrossRef][ISI][Medline]
Wei YD, Bergander L, Rannug U, Rannug A. (2000) Regulation of CYP1A1 transcription via the metabolism of the tryptophan-derived 6-formylindolo[3,2-b]carbazole. Arch. Biochem. Biophys. 383:99–107.[CrossRef][ISI][Medline]
Wei YD, Helleberg H, Rannug U, Rannug A. (1998) Rapid and transient induction of CYP1A1 gene expression in human cells by the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole. Chem. Biol. Interact. 110:39–55.[CrossRef][ISI][Medline]
Wei YD, Rannug U, Rannug A. (1999) UV-induced CYP1A1 gene expression in human cells is mediated by tryptophan. Chem. Biol. Interact. 118:127–140.[CrossRef][ISI][Medline]
Yellon SM, Singh D, Garrett TM, Fagoaga OR, Nehlsen-Cannarella SL. (2000) Reproductive, neuroendocrine, and immune consequences of acute exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in the Siberian hamster. Biol. Reprod. 63:538–543.
Yu W, Ikeda M, Abe H, Honma S, Ebisawa T, Yamauchi T, Honma K, Nomura M. (1999) Characterization of three splice variants and genomic organization of the mouse BMAL1 gene. Biochem. Biophys. Res. Commun. 260:760–767.[CrossRef][ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Mukai, T.-M. Lin, R. E. Peterson, P. S. Cooke, and S. A. Tischkau Behavioral Rhythmicity of Mice Lacking AhR and Attenuation of Light-Induced Phase Shift by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin J Biol Rhythms, June 1, 2008; 23(3): 200 - 210. [Abstract] [PDF]
|
|
| |||||||||||||||||||||||||||||||