A study on diurnal mRNA expression of CYP1A1, AHR, ARNT, and PER2 in rat pituitary and liver

Environmental Toxicology and Pharmacology 11 (2002) 119– 126 www.elsevier.com/locate/etap

A study on diurnal mRNA expression of CYP1A1, AHR, ARNT, and PER2 in rat pituitary and liver Ping Huang a,b, Sandra Ceccatelli a, Agneta Rannug b,* a

Di6ision of Toxicology and Neurotoxicology, National Institute of En6ironmental Medicine, Karolinska Institutet, S-171 77 Stockholm, Sweden b Di6ision of Genetic Toxicology, National Institute of En6ironmental Medicine, Karolinska Institutet, S-171 77 Stockholm, Sweden Received 11 July 2001; received in revised form 26 October 2001; accepted 2 November 2001

Abstract The ligand activated basic-helix-loop-helix (bHLH)-PAS transcription factor, the aryl hydrocarbon receptor (AHR) protein, heterodimerizes with its partner protein the aryl hydrocarbon receptor nuclear translocator (ARNT). The heterodimer activates transcription via xenobiotic responsive elements to regulate the transcription of a battery of biotransformation genes as well as genes involved in growth, differentiation, and cellular homeostasis. In this study we have investigated the diurnal expression of cytochrome P450 1A1, one of the genes in the AHR target gene battery, in rat pituitary and liver. The mRNA expression patterns of AHR, ARNT, and the periodic gene (PER2) were also analyzed. PER2 belongs to another group of bHLH-PAS transcription factor complexes, which are involved in the control of circadian rhythms. Diurnal variation of cytochrome P450 1A1 (CYP1A1) mRNA expression was observed in the anterior and posterior pituitary and in the liver. The accumulation of CYP1A1 mRNA occurred during different times of the day and exhibited an opposite expression in anterior and posterior pituitary, respectively. A daily upregulation of CYP1A1 and PER2 mRNAs that was in antiphase to the AHR and ARNT mRNAs was seen in the liver. The AHR/ARNT system is considered a defense system against toxic chemicals. The high inducibility of CYP1A1 in the pituitary, shown in an earlier study, as well as the tissue specific expression patterns shown here, suggest that AHR and CYP1A1 may play a physiological role in controlling neuroendocrine functions. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Diurnal rhythms; Cytochrome P450 1A1; Aryl hydrocarbon receptor; Aryl hydrocarbon receptor nuclear translocator; Period; Gene expression; RT-PCR

1. Introduction The aryl hydrocarbon receptor (AHR), the aryl hydrocarbon receptor nuclear translocator (ARNT), and the periodic protein (PER) belong to a subclass of transcription factors named basic-helix-loop-helix (bHLH)-PAS proteins. The functions of bHLH-PAS proteins seem to have their origin in early photoreceptor proteins (Pellequer et al., 1998). bHLH-PAS proteins regulate fundamental biological processes involving the maintenance of homeostasis, adaptation to environmental stimuli, circadian rhythmicity, and development (Wenger and Gassmann, 1997; Schmidt and Bradfield, 1996; Dunlap, 1999; Crews, 1998). * Corresponding author. Tel.: +46-8-728-7629; fax: + 46-8-314124. E-mail address: [email protected] (A. Rannug).

The AHR and ARNT proteins mediate the aryl hydrocarbon-dependent induction of cytochrome P450 1A1 (CYP1A1) (for reviews see Nebert et al., 2000; Gu et al., 2000). The most potent inducer of CYP1A1, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), elicits a wide range of biological effects through the activation of AHR. The AHR is located in the cytoplasm as a complex with other proteins. After the binding of a ligand, the AHR translocates to the nucleus where it heterodimerizes with ARNT. This heterodimer binds to the xenobiotic responsive element (XRE) and activates the transcription of a large number of genes, the AHR battery, leading to the synthesis of the CYP1A1 protein and several other proteins involved in xenobiotic metabolism, growth, differentiation, and cellular homeostasis (Nebert, 1994; Nebert et al., 2000). A feedback process regulating the expression of the CYP1A1 gene and other genes in the AHR battery by

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a CYP1A1-dependent oxidation of an endogenous substrate/AHR ligand has been suggested by the studies of CYP1A1 deficient mouse hepatoma cells (Hankinson et al., 1985; RayChaudhuri, et al., 1990; Chang and Puga, 1998). The normal cellular functions of the AHR receptor and the AHR/ARNT heterodimer are not yet known. A photoproduct derived from tryptophan (6-formylindolo[3,2-b]carbazole, FICZ), which binds to the AHR with very high affinity, however, has been described and suggested to be an endogenous ligand for the AHR (Rannug et al., 1987, 1995). The photoproduct, FICZ, is metabolized by CYP1A1, and in contrast to TCDD, and other slowly metabolized AHR ligands, causes a transient rise in CYP1A1 gene transcripts (Wei et al., 1998, 1999, 2000). Thus, the formation of the CYP1A1 protein seems to autoregulate CYP1A1 gene expression after exposure to the photoproduct FICZ. Based on the very high affinity of the photoproduct to the AHR and the rapid, transient expression of a battery of response genes, we have suggested that oxidation products of tryptophan are mediators of light via binding to the AHR. In this regard, they may have a role in light-regulated biological rhythms (Rannug et al., 1998 Wei et al., 1999). The bHLH-PAS protein PER2 is involved in the control of circadian rhythms. Cellular oscillators or clock genes are expressed after binding of the bHLHPAS heterodimer of the two proteins CLOCK and BMAL to the recognition sequence (E-box) in DNA, giving rise to transiently elevated levels of several mRNAs and proteins. The proteins feed back, after a lag phase, to depress the level of their own transcripts and thereby sustain a circadian rhythmicity (Dunlap, 1999). PER2, together with another PER protein (PER1) and cryptochrome proteins (CRY1, CRY2), is an essential component of negative regulation of the two clock proteins CLOCK and BMAL1 (Chang and Reppert, 2001). We have previously observed a strong expression of CYP1A1 mRNA and protein in rat pituitary after treatment with the AHR ligand TCDD (Huang et al., 2000). The aim of this study was to characterize in more detail the expression of CYP1A1 in the anterior and posterior pituitary in relation to the expression of AHR and ARNT in the same tissues. An additional objective was to investigate the diurnal rhythms in the expression of the CYP1A1 gene and of AHR and ARNT. In parallel, we characterized the expression pattern of the PER2 gene. The mRNA levels in anterior pituitary, posterior pituitary, and liver of male Sprague–Dawley rats were analyzed at six different time points 08:00, 12:00, 16:00, 20:00, 00:00, and 04:00 hours by a semi-quantitative RT-PCR method.

2. Materials and methods

2.1. Materials We used the RNeasy™ Mini Kit and QIAshredder from QIAGEN GmbH (Germany). The RNase-free DNase I was purchased from Boehringer Mannheim. SuperScript™ II RNase H− Reverse Transcriptase (RT), RNaseOUT™ Recombinant Ribonuclease Inhibitor, Taq DNA Polymerase (recombinant), and dNTP Set were purchased from GibcoBRL (Life Technology). Random hexamers were acquired from Pharmacia Biotech.

2.2. Experimental animals and sample preparation Male Sprague–Dawley rats (B and K Stockholm, Sweden) with body weights of 200 g were housed in polypropylene cages and checked twice daily. Animals were kept in air-conditioned quarters at a temperature of 25 °C, under a controlled photoperiod (14 h light– 10 h darkness) and with free access to food and tap water. Procedures used in animal experimentation comply with the Karolinska Institute’s regulations for the care and use of laboratory animals. In the biological rhythm experiment, rats were sacrificed at six different time points (08:00 12:00, 16:00, 20:00, 00:00, and 04:00 hours). The pituitary was removed from the skull, and the anterior and posterior lobes were dissected. The tissue collection and dissection were completed within a time period of 1–2 min. The tissue samples were frozen in liquid nitrogen and stored at − 70 °C until further use. For each of the six time points, five independent RNA preparations from five different rats were made from anterior pituitary, posterior pituitary, and liver.

2.3. RNA preparation Total RNA was isolated from less than 20 mg of tissue with QIAGEN’s RNeasy Mini Kit. For complete homogenization of the sample, tissue lysates were passed 15 times through a 20-G (¥ 0.9 mm) needle fitted to a syringe, then pipetted directly onto a QIAshredder column and spun down. RNA titration curves were done in order to establish protocols that allowed quantification of PCR products. To minimize the contamination of genomic DNA, 1.2 U of RNase-free DNase I was used to treat approximately 1.2 mg RNA of the sample prior to cDNA synthesis in a total volume of 12 ml at room temperature for 15 min. The reaction was terminated at 90 °C for 5 min. Approximately 1.2 mg treated total RNA was directly used for cDNA synthesis.

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2.4. Re6erse transcriptase-linked polymerase chain reaction (RT-PCR) 2.4.1. cDNA synthesis First-strand cDNA was produced following a protocol of SuperScript™ II RT with slight modification. In the reverse transcription reaction system, 1.2 mg total RNA, 1X First strand buffer, 75 pmol of random hexamers, 1 mM of all four deoxynucleotide triphosphates (dNTPs), 10 mM dithiothreitol (DTT), 0.72 ml (28.8 U) RNaseOUT™ Recombinant Ribonuclease Inhibitor and 0.72 ml (144 U) SuperScript™ II RNase H− Reverse Transcriptase were contained in a total 30 ml reaction volume. The mixture was incubated at room temperature for 10 min, then at 42 °C for 60 min, and the reverse transcriptase was inactivated by heating to 99 °C for 5 min. The cDNA product was stored at −20 or − 70 °C until used. 2.4.2. PCR amplification The sequences of the primers were as follows: b-actin FP, 5%-TGC AGA AGG AGA TTA CTG CC-3%; b-actin RP, 5%-GCA GCT CAG TAA CAG TCC G-3%; CYP1A1 FP, 5%-CCA TGA CCA GGA ACT ATG GG-3%; CYP1A1 RP, 5%-TCT GGT GAG CAT CCA GGA CA-3%; AHR FP, 5%-CCC CAA TTC CCT TAT GAG TGC3%; AHR RP, 5%-GGA GGA GTC GGT TCG GAA GA-3%; ARNT1 FP, 5%-GTC TCC CTC CCA GAT GAT GA-3%; ARNT1 RP, 5%-AAG AGC TCC TGT GGC TGG TA-3%; PER2 FP 5%-CTC CCC AAG TCC CAC CAG TC-3%; PER2 RP 5%-CGT CCC GTG GAG CAG TTC TC-3%; Two microliters first-strand cDNA (corresponding to 80 ng of initial total RNA that had been reverse transcribed) were amplified in each 20 ml PCR reaction volume. The final concentration of reagents in the PCR reaction system were as follows: 1X PCR buffer, 1.5 mM MgCl2; 200 mM of each dNTP; 200 nM each primer and 0.5 U/20 ml Taq DNA polymerase. PCR cycling conditions were as follows: 94 °C 45 s, 60 °C 30 s, 72 °C 35 s, 20 cycles for b-actin; 95 °C 45 s, 60 °C 35 s, 72 °C 45 s, 35 cycles for CYP1A1; 94 °C 35 s, 57 °C 30 s, 72 °C 45 s, 29 cycles for AHR; 94 °C 30 s, 58 °C 60 s, 72 °C 60 s, 30 cycles for ARNT1; 95 °C 30 s, 61 °C 30 s, 72 °C 30 s, 31 cycles for rPER2. All reactions had an initial denaturation step at 94 °C for 3 min and a final extension at 72 °C for 7 min.

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No product was obtained when RT was carried out in the absence of the reverse transcriptase enzyme, indicating that there was no amplification from any genomic DNA that may have been present in the RNA preparation.

2.4.3. mRNA semi-quantitation The PCR products (20 ml) were electrophoresed in 1.6% agarose gel and visualized with ethidium bromide staining. Specificity was confirmed by the size of the amplified products, corresponding to 212 bp for bactin, 341 bp for CYP1A1, 340 bp for AHR, 218 bp for ARNT1, and 123 bp for PER2. Densitometry was used for quantitation of the intensity of the RT-PCR products. After the electrophoresis, the gels were directly analyzed using a Gel-Doc instrument (BIO-RAD) connected to the MOLECULAR ANALYST software. The levels of CYP1A1, AHR, ARNT1, and PER2 mRNA were plotted as the relative ratio between the target-specific band and the b-actin band individually. The intensity of each band was measured by a volume analysis technique (MOLECULAR ANALYST program). All PCR reactions were linear at the PCR cycling conditions given above. Within each run, linearity was ensured by varying the amount of cDNA. PCR reactions were linear in all present experimental PCR cycling conditions. 2.5. Statistical analysis Values are given as mean9 SD in table and mean9 SEM in figures. As a result of non-normally distributed mRNA values, the nonparametric test Kruskall– Wallis one-way ANOVA was used to compare mRNA levels from the different time points. The pair-wise Mann– Whiney U-tests were then used for comparison between the groups. Reported P-values are two-tailed and PB 0.05 was considered statistically significant.

3. Results

3.1. CYP1A1 expression When the level of CYP1A1 expression was normalized to that of b-actin, a diurnal variation in CYP1A1 mRNA expression was observed in the anterior and posterior pituitary, as well as in the liver (Table 1, Figs. 1A and 2). The diurnal accumulation of CYP1A1 occurred during different times of the day, and exhibited an opposite pattern of expression in anterior and posterior pituitary, respectively (Fig. 1A). At midnight (00:00 hours time point) CYP1A1 mRNA exhibited the highest level in anterior pituitary, while the lowest level was recorded in posterior pituitary. In contrast, at the early night (20:00 hours time point) the highest level

F

8:00 12:00 16:00 20:00 0:00

4:00 ANOVAc

ARNT

F

4:00

ANOVAc

A B C D E

8:00 12:00 16:00 20:00 0:00

A B C D E F

4

3 5 4 5 3

5

5 5 5 5 5

5 5 5 5 5 5

5 5 5 5 5 5

n

H(5, N= 24) =22.81; P =0.0004

0.549 0.13

0.379 0.08 0.949 0.15 0.959 0.09 0.8190.19 0.5090.11 A–B** A–C** A–D** B–E**; C–E**; D–E** A–F*; B–F**; C–F**; D–F* 3

4 5 4 5 3

3

0.87 9 0.14 H(5, N= 30)= 5.82; P =0.32

4 5 5 5 3 3

4 5 5 5 2 3

n

H(5, N= 24) = 7.31; P =0.20

0.93 9 0.41

5

5 5 5 5 5

0.61 9 0.35 0.68 9 0.18 1.16 9 1.24 1.17 9 0.37 1.59 90.71

5 4 5 5 5 5

B–D*

B–E*

5 5 5 5 5 5

0.52 90.10 H(5, N= 24) = 3.45; P =0.63

0.74 9 0.49 0.65 9 0.22 0.75 9 0.21 0.88 90.38 0.89 9 0.58

0.70 9 0.28 0.74 9 0.11 0.73 90.41 0.54 90.26 0.48 90.06 0.55 90.12 H(5, N =25) =7.36; P =0.20

A–C*; B–C* A–D*

5 5 5 5 5 5

0.25 90.04 0.35 90.17 0.55 90.12 0.69 9 0.41 0.23 90.22 0.32 9 0.29 H(5, N =24) = 10.83; P= 0.055

H(5, N =30) =12.69; P= 0.027

0.469 0.21

0.43 9 0.15 0.20 9 0.10 0.38 9 0.21 0.73 9 0.31 0.42 9 0.08

0.52 9 0.19 H(5, N= 29)= 16.38; P = 0.006

0.81 9 0.18 0.93 9 0.17 0.87 9 0.31 0.89 9 0.35 0.41 9 0.13

0.71 9 0.27 1.019 0.15 0.78 9 0.26 0.55 9 0.17 0.46 9 0.09 0.42 9 0.09 H(5, N= 30)= 18.46; P = 0.002

0.55 9 0.31 0.20 9 0.10 0.53 9 0.27 0.65 9 0.15 0.51 9 0.08 0.44 9 0.10 H(5, N= 30)= 15.15; P =0.0098

Mean9 SDb

Liver n

Mann–Whitney test P valuea

Mean 9SDb

Posterior pituitary

4 5 4 5 3

A–F*

B–E*; C–E*; D–E*

A–B*

Mann–Whitney test P valuea

0.739 0.10 0.8390.18 0.7590.20 0.829 0.16 0.989 0.20

0.679 0.13 0.629 0.06 0.659 0.20 0.62 90.11 0.699 0.19 0.52 9 0.09 H(5, N= 30)= 4.66; P =0.46

0.489 0.11 0.2990.09 0.34 90.12 0.319 0.14 0.639 0.21 0.44 9 0.14 H(5, N= 30) =12.36; P= 0.030

Mean 9SDb

Anterior pituitary

b

Mann–Whitney nonparametric test, two-sided P value. Results from pair-wise tests are given as indicated by the group label (A–F), *PB0.05; **PB0.01. Relative mRNA levels present the ratio of target mRNA/b-actin mRNA. c Kruskal–Wallis ANOVA test.

a

PER2

A B C D E

8:00 12:00 16:00 20:00 0:00 4:00 ANOVAc

AHR

A B C D E F

8:00 12:00 16:00 20:00 0:00 4:00 ANOVAc

CYP1A1

Group

Time

Gene

Table 1 Diurnal variations in gene expression of CYP1A1, AHR, ARNT, and PER2 mRNA relative levels in rat pituitary and liver

B–F*

B–D** B–E*; D–E*

A–B*

A–E**; B–E*; C–E**; D–E* A–F*; B–F*; C–F*

B–E**; C–E** B–F**; C–F**

A–B* B–C* B–D** B–E** B–F**; D–F*

Mann–Whitney test P valuea

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P. Huang et al. / En6ironmental Toxicology and Pharmacology 11 (2002) 119–126

was observed in the posterior pituitary, whereas a low level of expression was observed in the anterior pituitary. Moreover, we observed a pronounced daily timedependent change in CYP1A1 expression in the liver (Fig. 2). The peak level of CYP1A1 mRNA in the liver was seen at 20:00 hours. The CYP1A1 was found to oscillate with a 12 h period in the liver and a 24 h period in the pituitary. ANOVA analysis (Table 1) showed that the apparent daily cycle of CYP1A1 mRNA concentration was statistically significant in the liver (P B 0.01) and in the anterior pituitary (P = 0.03). In the posterior pituitary a borderline significance was observed (P= 0.055). Several of the mean values of the time points were also found to differ significantly as indicated in the table.

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3.2. AHR expression AHR shared an oscillation trend in the pituitary similar to CYP1A1, but with a lower magnitude of statistically non-significant (P\ 0.05) oscillation (Table 1 and Fig. 1B). AHR also exhibited an antiphase expression between the anterior and the posterior pituitary. There was a maximal expression in anterior pituitary and the lowest expression in the posterior pituitary at 00:00 hours. The AHR expression was found to precede the maximal expression of CYP1A1 by 4–8 h in the posterior pituitary and in the liver. AHR mRNA fluctuated significantly in liver as indicated by the ANOVA test (PB 0.01), with a pattern opposite that of CYP1A1. The highest AHR mRNA

Fig. 1. Diurnal mRNA expression of several PAS genes, as well as the CYP1A1 gene in the anterior and posterior pituitary of male Sprague– Dawley rats. Total RNA was isolated at six different time points, indicated on the abscissa. The amount of target gene expression was quantified by semi-quantitative RT-PCR, as described in Section 2 and calibrated in reference to b-actin mRNA levels using a Gel-Doc instrument (BIO-RAD) connected to the MOLECULAR ANALYST software. Diurnal cycles of gene expression were graphed using Microsoft Excel 6.0 with the curve smoothing function of a scatter plot. All values are presented as the means 9SEM (n = 3 – 5). The graph illustrates the relative ratio of transcript level of target genes normalized to b-actin. (A) CYP1A1 mRNA/b-actin mRNA; (B) AHR mRNA/b-actin mRNA; (C) ARNT mRNA/b-actin mRNA; (D) PER2 mRNA/b-actin mRNA.

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Fig. 2. Diurnal oscillation in mRNA levels of CYP1A1, AHR, ARNT, and PER2 in rat liver. RNA preparations from five different rats were analyzed in each group. The relative mRNAs levels of target genes were normalized to b-actin. The figure illustrates that the expression of PER2 and CYP1A1 is coordinated in time and antiphase to the expression of AHR and ARNT in the rat liver.

expression in the liver was seen at the 12:00 hours time point and the lowest level in early morning at the 04:00 hours time point (Table 1 and Fig. 2).

3.3. ARNT expression No significant difference in the expression of ARNT mRNA was found in the anterior or posterior pituitary (Table 1 and Fig. 1C). ARNT mRNA showed a significant oscillating pattern only in the liver, with the higher levels observed during day time (08:00, 12:00, 16:00, and 20:00 hours) and a drop in expression at midnight (00:00 hours) (Table 1 and Fig. 2).

3.4. PER2 expression The PER2 mRNA abundance, comparing the highest and lowest levels in the daily cycle, was found to change 2.6-fold in both anterior and posterior pituitary (Table 1 and Fig. 1D) and 3.7-fold in liver (Table 1 and Fig. 2). There was a tissue-dependent difference observed in the expression rhythm of PER2, with peak mRNA levels observed at 12:00 and 16:00 hours in the anterior pituitary, and at 00:00 hours in the posterior pituitary (Table 1 and Fig. 1D). The peak mRNA levels in the liver was observed at 20:00 hours (Table 1 and Fig. 2). In the liver, PER2 and CYP1A1 expression were coordinated in time. The lowest levels of both transcripts were observed at 12:00 hours, while the

highest levels were seen at 20:00 hours time point (PB 0.05) (Fig. 2).

4. Discussion The results of this study show temporal patterns in the mRNA expression of CYP1A1, AHR, and ARNT in pituitary and liver from Sprague–Dawley rats. In these samples, the diurnal expression of the PER2 gene, coding for a PAS-protein, involved in the regulation of the mammalian clock genes CLOCK/BMAL1, was demonstrated. Furthermore, the data provide a detailed analysis and comparison of the expression of PER2, AHR, ARNT, and CYP1A1 in the anterior and posterior pituitary, which were earlier indicated as particularity sensitive to the induction of CYP1A1 caused by TCDD (Huang et al., 2000). We observed a pronounced antiphase expression of some of the genes between the anterior and the posterior pituitary. Our present data are in agreement with the previous findings of temporal peaks in CYP1A1 protein or enzyme activity (Tredger and Chhabra, 1977; Plewka et al., 1992), as well as temporal changes of AHR and ARNT protein levels in the liver (Richardson et al., 1998). CYP1A1, AHR, and ARNT mRNAs are constitutively expressed in rat brain and pituitary, suggesting that both brain and pituitary may be direct targets for TCDD induced toxicity (Huang et al., 2000). TCDD

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increases the turnover of brain serotonin, along with increased levels of the serotonin precursor, tryptophan, in brain and plasma (Pohjanvirta and Tuomisto 1994; Rozman et al., 1991; Tuomisto et al., 1990; Unkila et al., 1994, 1999). Although very few measurements have been performed on the effect of TCDD on diurnal rhythms in endocrine functions, there have been some observations of a shift in circadian feeding rhythms of rats (Pohjanvirta et al., 1988; Christian et al., 1986; Pohjanvirta and Tuomisto, 1990), on prolactin secretion and response to prolactin (Jones et al., 1987), and corticosterone levels (DiBartolomeis et al., 1987). These reports suggest that TCDD could interfere with several biological rhythms. Recently, it was reported that TCDD also has an effect on circadian rhythms in deer mice (Miller et al., 1999). Molecular circadian pacemakers consist of genes specifying proteins that can activate or repress gene transcription. The AHR/ARNT heterodimeric protein shares a great similarity with the known component of the mammalian circadian response pathway, the CLOCK/BMAL1 complex. They are bHLH-PAS proteins that utilize promoter sequences, the XRE-sequence and the E-box sequence, which share high sequence homology. Additionally, their transcriptional products, the CYP1A1 protein and the PER proteins (by interaction with CRY proteins), act as repressors that turn off transcription of their own genes. The repressor proteins, CYP1A1 and PER, have a limited lifespan so they eventually decay, and when activated again a new wave of repressor gene transcription commences. The present study found that the expression of CYP1A1 and PER2 was highly coordinated in the posterior pituitary and liver in the rats. This observation may suggest a possible interaction between the AHR/ARNT and the CLOCK/BMAL systems, or a similar response to the daily environmental changes. The PER2 oscillation that we observed in rat liver, with a peak expression seen in early night, is consistent with earlier published data (Oishi et al., 1998; Zylka et al., 1998). These authors described similar PER2 mRNA expression rhythms in all tissues investigated (skeletal muscle, brain, eye, heart, kidney, and lung) in mice and rats. Our results, however, indicate that the mRNA expression rhythm in the anterior pituitary, with the maximal expression in the middle of the day (12:00 and 16:00 hours), differs considerably from the other tissues analyzed. In conclusion, this study illustrates that the CYP1A1 gene, as well as the bHLH-PAS genes, AHR, ARNT, and PER2, display daily variations in mRNA levels. The results increase our knowledge on the biological role of the AHR/ARNT signaling pathway and improve our understanding of the mechanisms underlying toxic effects of exogenous chemicals such as TCDD and other polycyclic aromatic compounds. Moreover, the

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data support the hypothesis that AHR and CYP1A1 may play a role in controlling neuroendocrine functions, as was also suggested by our previous study showing the presence of AHR and CYP1A1 in the hypothalamus and pituitary (Huang et al., 2000).

Acknowledgements This work was supported by the grant 12X-10815 from the Swedish Medical Research Council, and by Swedish Match. We thank Dr Eva Ahlbom for her assistance in tissue sample collection and Ms Margareta Sandstro¨ m for technical assistance in total RNA extraction.

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