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Effect of several analogs of 2,4,6-triphenyldioxane-1,3 on CYP2B induction in mouse liver
Chemico-Biological Interactions 194 (2011) 134–138
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Effect of several analogs of 2,4,6-triphenyldioxane-1,3 on CYP2B induction in mouse liver Vladimir Pustylnyak a,b,⇑, Yuliya Kazakova a,b, Andrei Yarushkin a, Nikolai Slynko c, Lyudmila Gulyaeva a,b a
Institute of Molecular Biology and Biophysics, SB RAMS, Novosibirsk, Russia Novosibirsk State University, Novosibirsk, Russia c Institute of Cytology and Genetics, SB RAS, Novosibirsk, Russia b
a r t i c l e
i n f o
Article history: Received 2 August 2011 Received in revised form 5 September 2011 Accepted 22 September 2011 Available online 29 September 2011 Keywords: Cyp2b10 TPD Gene regulation CAR
a b s t r a c t 2,4,6-Triphenyldioxane-1,3 (TPD) is a highly effective inducer of CYP2B in rats, but not in mice. Several analogs of TPD were synthesized. All substituents were entered into the same position of TPD (R = H, cisTPD and transTPD; R = N(CH3)2, transpDMA; R = NO2, transpNO2; R = F, transpF; R = OCH3, transpMeO). The purpose of the present study was to investigate the effect of TPD analogs on CYP2B induction in mouse livers. Among the six test compounds, four (R = –N(CH3)2, –NO2, –F, –OCH3) demonstrated a dose-dependent induction of mouse CYP2B. To further characterize the compounds, we determined ED50s using sigmoidal dose–response curves. The dose–response study has shown that all active compounds have similar potencies to induce CYP2B in mouse livers. Western-blot analysis and multiplex RT-PCR have shown that the increase of CYP2B activity in mouse liver is related to the high content of CYP2B proteins and paralleled the increase of cyp2b10 mRNA level. ChIP results have demonstrated that the transcriptional enhancement of cyp2b10 gene in response to compounds is accompanied by the increased recruitment of the constitutive androstane receptor (CAR) to its specific binding site (PBREM) on the target gene. Thus, minor structural changes in TPD cause dramatic changes in its ability to induce mouse CYP2B, and it is likely several TPD analogs act by activation of mouse CAR. Ó 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The CYP2B subfamily is expressed constitutively and inducibly. The induction of CYP2B isoforms by chemical compounds is mediated through effects on the constitutive androstane receptor (CAR, NR1I3), zinc finger-containing transcription factor belonging to the nuclear receptor superfamily [1]. Non-activated CAR is retained in the cytoplasm in a complex with heat shock protein 90 (Hsp90) and CAR cytoplasmic retention protein (CCRP) [2]. In response to chemical compounds, CAR translocates to the nucleus and forms a heterodimer with the retinoid X receptor (RXR). The activation is also associated with recruitment of coactivators, such as SRC-1, Sp1, GRIP1, PGC1a, p160 [3]. The CAR–RXR–coactivator complex binds to the distal specific DNA response elements of target genes leading to an increase in gene expression [4]. It is very important to clarify the mechanism of CYP2B induction because it leads to better models for screening and predicting drug interaction that occur due to the induction of individual CYPs. Generally, such investigations are carried out using experimental animals like rats and mice. A major problem in these studies is ⇑ Corresponding author at: Institute of Molecular Biology and Biophysics, Timakova str. 2, Novosibirsk 630117, Russia. Tel./fax: +7 383 335 98 47. E-mail address: [email protected] (V. Pustylnyak). 0009-2797/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2011.09.003
the extrapolation of data from one animal species to another. Many examples have been documented where various animal species displayed a markedly different response to the same chemical compound. Some species differences in induction of CYP forms have been reported, which are attributable to differences in the ligand binding domain of nuclear receptors between different species [5]. 2,4,6-Triphenyldioxane-1,3 (TPD) is a highly effective activator of CAR and its target genes in rat liver, but it is not able to activate CAR in mice [6]. Our previous study has shown that after a single cisTPD ip administration of 1 mg/kg body weight, a significant increase in CYP2B activity was detected [7]. A maximal CYP2B activity increase was observed after 10 mg/kg body weight ip cisTPD injection [7]. In our recent study, we have synthesized several analogs of TPD (R = H, cisTPD and transTPD; R = N(CH3)2, transpDMA; R = NO2, transpNO2; R = F, transpF; R = OCH3, transpMeO) to determine how minor changes in the inducer structure can cause changes in induction abilities [8]. Substituents were made to minimize changes in the initial structure while changing the hydrophobic properties of the molecule. All substituents were entered into the fourth para position of the second phenyl ring of TPD. Five of the six compounds (cisTPD, transTPD, transpDMA, transpNO2 and transpF) were able to activate CAR and to induce its target genes CYP2B1/2 in rat liver. The dose–response studies have shown that
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cisTPD, transpDMA, transpF and transpNO2 have similar potency, and transTPD is a less potent derivative in rat livers [8]. The purpose of the present study was to investigate the effect of TPD analogs on CYP2B induction in mouse livers.
2.1. Chemicals cisTPD – (4S,6R)-2,4,6-triphenyl-1,3-dioxane, transTPD – (4R,6R) -2,4,6-triphenyl-1,3-dioxane, transpMeO – (4R,6R)-2-(4-methoxyphenyl)-4,6-diphenyl-1,3-dioxane, transpDMA – 4-[(4R,6R)-4,6-diphenyl-1,3-dioxan-2-yl]-N,N-dimethylaniline, transpF – (4R,6R)-2(4-fluorophenyl)-4,6-diphenyl-1,3-dioxane, transpNO2 – (4R,6R)2-(4-nitrophenyl)-4,6-diphenyl-1,3-dioxane (Fig. 1) were synthesized according to Griengl and Geppert [9]. Cis and trans refer to the relative configurations of the aryl fragments near atoms 4 and 6 of the dioxane ring. The compounds were characterized by NMR spectroscopy and shown to be 97–98% pure by LC/MS analysis. All other chemicals and solvents were of analytical grade and were obtained from commercial sources. 2.2. Experimental animals Male C57BL mice (20–25 g) were supplied by the Institute of Clinical Immunology, SB RAMS (Novosibirsk, Russia). Animals were acclimated for 1 week and allowed free access to food and water. All experimental procedures were approved by the Animal Care Committee of the Institute of Molecular Biology and Biophysics, SB RAMS. 2.3. Preparation of microsomal proteins For preparation of microsomal proteins, mice were killed 72 h after a single ip injection of one of the tested compounds (0.5, 1, 5, 10, 30, 50 and 100 mg/kg body weight in corn oil) or an equal amount of corn oil. Five animals per treatment group were used. The microsomal fractions were isolated from freshly excised tissues by standard differential centrifugation. The protein concentrations in microsomes were measured using the Lowry method with a bovine albumin solution as a standard. The microsomal fractions were collected and stored at 80 °C.
transpDMA
2.4. CYP2B catalytic activity The CYP2B specific activity, 7-pentoxyresorufin O-dealkylase (PROD), was measured at 37 °C by fluorimetry [10]. 2.5. SDS–PAGE electrophoresis and Western-blot
2. Materials and methods
cisTPD
135
transTPD
transpF
transpMeO
transpNO2
Fig. 1. Structures of compounds. cisTPD – (4S,6R)-2,4,6-triphenyl-1,3-dioxane, trans TPD – (4R,6R)-2,4,6-triphenyl-1,3-dioxane, transpMeO – (4R,6R)-2-(4-methoxyphe nyl)-4,6-diphenyl-1,3-dioxane, transpDMA – 4-[(4R,6R)-4,6-diphenyl-1,3-dioxan -2-yl]-N,N-dimethylaniline, transpF – (4R,6R)-2-(4-fluorophenyl)-4,6-diphenyl-1, 3-dioxane, transpNO2 – (4R,6R)-2-(4-nitrophenyl)-4,6-diphenyl-1,3-dioxane.
Fifteen micrograms of microsomal proteins per lane were separated in 10% SDS–PAGE and transferred to the nitrocellulose membrane. Membranes were stained with Ponceau S to verify loading and transfer efficiency. Immunodetection was performed with anti-rat CYP2B1 antibodies. Polyclonal antibodies directed against rat CYP2B1 were prepared in rabbits by immunization with the purified CYP2B1 protein. The bands were visualized colorimetrically with 4-chloro-1-naphthol (MP Biomedicals) as substrate. 2.6. cDNA synthesis and multiplex PCR For RNA isolation, mice were killed 24 h after a single ip injection of one of the tested compounds (30 mg/kg body weight in corn oil). Total RNA was isolated from livers frozen in liquid nitrogen with Rneasy Mini Kit (Qiagen) according to the manufacturer’s protocol. The concentration and purity of the RNA were determined by measuring the absorbance at 260 and 280 nm with correction for background at 320 nm in a spectrophotometer, and the integrity was examined by visualizing the 18S and 28S rRNA bands on a denaturating agarose (1%) gel. One microgram of total RNA was used for synthesis of single-stranded cDNA. First strand cDNA synthesis was carried out using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s protocol. Expression level of the cyp2b10 gene was measured by multiplex PCR performed as previously described [11]. The ‘‘house-keeping’’ gene, b-actin, was chosen as an endogenous internal controls to which the cyp2b10 PCR amplification product was normalized. 2.7. ChIP assay For ChIP assay, mice were killed 8 h after a single ip injection of one of the tested compounds (30 mg/kg body weight in corn oil). Livers were rinsed in cold phosphate-buffered saline with 0.4% NP-40 and homogenized in the same solution. Homogenates were treated with 1% formaldehyde at room temperature for 10 min with gentle rotation to cross-link the chromatin and the reaction was stopped by adding glycine to a final concentration of 125 mM. Fixed cells were collected by centrifugation, washed with cold phosphate-buffered saline with 0.4% NP-40, homogenized in cell lysis buffer 50 mM Tris–HCl, pH 8.0, 1% Triton X-100, 1% SDS, 10 mM EDTA supplemented with Complete Mini (Roche Diagnostics) and incubated on ice for 30 min. Lysates were sonicated on ice with three pulses of 20 s and 20% amplitude in Microson™ Ultrasonic Liquid Processor XL-2000 cell disrupter, yielding chromatin fragments of 500–1000 bp in size. Samples were centrifuged at 13000g for 10 min to remove detritus, and the supernatants were collected. To provide a positive control (input), 20 ll of the supernatants was retained. The supernatants were diluted 10-fold with dilution buffer (16.7 mM Tris–HCl, pH 8.0, 1.2 mM EDTA, 150 mM NaCl, 1.1% Triton X-100, 0.01% SDS supplemented with Complete Mini (Roche Diagnostics)). The diluted lysates were pre-cleared by incubating with 20 ll/ml protein G agarose/Salmon sperm DNA (Millipore) for 1 h at 4 °C on rotating plate. Cleared lysates (2 ml) were subjected to overnight incubation with either 5 lg of anti-CAR antibody or normal rabbit IgG at 4 °C under rotation. Immunocomplexes were precipitated by adding 30 ll of protein G agarose/Salmon sperm DNA (Millipore) for 1 h at 4 °C with shaking, washed with low-salt buffer (20 mM Tris–HCl, pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.01% SDS), high-salt
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served in transpDMA (61% of the values measured in transpNO2), transpMeO (22% of the values measured in transpNO2) and transpF (18% of the values measured in transpNO2) treated mouse livers in the dose range of 50–100 mg/kg body weight (Tukey’s post hoc test, p < 0.05). To determine whether the PROD activity level was parallel to the level in cyp2b protein, dose-dependent cyp2b protein induction was investigated by Western-blot analysis (Fig. 3). Westernblot analysis demonstrated a basal level of cyp2b protein in livers of vehicle-treated mice. The protein band corresponded to cyp2b in vehicle-treated mice was not found to be markedly changed in livers from cisTPD- and transTPD-treated mice. On the other hand, the hepatic cyp2b protein level was increased in dose-dependent manner after transpDMA, transpNO2, transpMeO and transpF treatment compared with the level in control animals. Thus, among the six test compounds, four increased CYP2B activity and protein levels. To further characterize the compounds, we determined ED50s using sigmoidal dose–response curves for transpMeO, transpDMA, transpF and transpNO2 (Table 1). All TPD analogs had similar ED50s.
buffer (20 mM Tris–HCl, pH 8.0, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.01% SDS), LiCl buffer (10 mM Tris–HCl, pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP-40) and finally twice with TE buffer (0.25 mM EDTA, 10 mM Tris–HCl, pH 8.0). The immunoselected chromatin was eluted in 500 ll of elution buffer (0.1 M NaHCO3, 1% SDS and 0.3 M NaCl). To reverse cross-linking, the samples were further incubated for 6 h at 65 °C. After protease digestion, DNA was purified by phenol/chloroform extraction. PCR amplification was performed using primers specific for the PBREM region of the mouse cyp2b10 gene (F: 50 -CGAGGACACAATCTTGAAG-30 and R: 50 -GAGCAAGGTCCTGGTGTC-50 ). 2.8. Data analysis The data are presented as mean ± SD. ANOVA followed by Dunnett’s post hoc test was used to compare each group to control. ANOVA followed by Tukey’s post hoc test was used to multiple comparison tests. A p value <0.05 was considered statistically significant. ED50, 95% confidence intervals (CIs) of each chemical were estimated by fitting dose–response data to a sigmoidal function using GraphPAD Prism 5.0.
3.2. Induction of cyp2b10 gene expression 3. Results
Effect of the tested compounds on cyp2b10 gene expression were evaluated in mouse livers by measuring cyp2b10 mRNA levels 24 h after single ip injections of chemical compounds (30 mg/kg body weight in corn oil). As expected, transpDMA (15-fold), transpNO2 (13.5-fold), transpMeO (6.5-fold) and transpF (8-fold) strongly and significantly increased cyp2b10 gene expression compared to the control values (Dunnett’s post hoc test, p < 0.05) (Fig. 4). Moreover, the maximum cyp2b10 mRNA levels produced by transpNO2 and transpDMA were significantly higher than observed in transpMeO and transpF treated mouse livers (Tukey’s post hoc test, p < 0.05). At the same time, cyp2b10 mRNA levels were unchanged in mouse livers after cisTPD and transTPD treatment.
3.1. Dose-dependent CYP2B induction Dose-dependent changes in PROD activity were investigated in mouse livers treated with TPD analogs (0.5, 1, 5, 10, 30, 50 and 100 mg/kg body weight) (Fig. 2). PROD activity was found to be significantly increased in the dose range of 5–100 mg/kg body weight of transpDMA and transpNO2 and in the dose range 30–100 mg/kg body weight of transpMeO and transpF compared to the control values (Dunnett’s post hoc test, p < 0.05). In contrast, the level of CYP2B specific activity was not altered compared to control values after treatment with cisTPD and transTPD in 0.5–100 mg/kg body weight dose range (Dunnett’s post hoc test, p < 0.05). Moreover, transpDMA and transpNO2 evoked dose-dependent increases of PROD activity in the range of 1–50 mg/kg body weight and transpMeO and transpF evoked dose-dependent increases of PROD activity in the range of 10–50 mg/kg body weight (Tukey’s post hoc test, p < 0.05). The maximum of CYP2B induction produced by transpNO2 was significantly higher (was taken as 100%) than ob-
3.3. Recruitment analysis of CAR on PBREM of cyp2b10 gene The expression of CYP2B genes upon inducer treatment is regulated by CAR. The CAR–RXR heterodimer is formed after CAR translocation from cytosol into the nucleus, and the heterodimer is
# #
#
# #
*
16
Fold change
14 12
*
*
10 8
* *
*
6
*
4
*
*
* *
*
2 0,5 1,0 5,0 10,0 30,0 50,0 100,0 0,5 1,0 5,0 10,0 30,0 50,0 100,0 0,5 1,0 5,0 10,0 30,0 50,0 100,0 0,5 1,0 5,0 10,0 30,0 50,0 100,0 0,5 1,0 5,0 10,0 30,0 50,0 100,0 0,5 1,0 5,0 10,0 30,0 50,0 100,0
0
cisTPD
transTPD
pMeO
pDMA
pF
pNO2
Fig. 2. Dose-dependent effect of compounds on PROD activity in mouse livers. Mice were treated with the compounds at 0.5, 1, 5, 10, 30, 50 and 100 mg/kg body weight. For preparation of microsomal proteins, animals were killed 72 h after a single ip injection of one of the tested compounds. Fold change was expressed by taking the corresponding value of the control mice as one. Five animals per treatment group were used. Bars mean ± S.D. ⁄Indicates a significant difference between various doses groups (ANOVA followed by Tukey’s post hoc test, p < 0.05). #Indicates a significant difference between different compounds groups (ANOVA followed by Tukey’s post hoc test, p < 0.05).
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A
CAR
F
RXR
cyp2b10
PBREM R
B
Anti-CAR Normal IgG
Table 1 Potency of TPD analogs. TPD analog
ED50 (mg/kg)
95% CI
transpMeO transpDMA transpF transpNO2
23.5 27.6 24.5 28.4
15.3–36.2 23.4–34.6 12.7–47.7 16.2–49.8
# #
#
* Fold change
pNO2
pF
pDMA
pMeO
transTPD
cisTPD
Cont
Fig. 5. Recruitment of CAR to PBREM of the cyp2b10 gene promoter in mouse livers in response to compound treatments. (A) Schematic representation of cyp2b10 promoter. Location of the PBREM region is shown. (B) Chromatin was isolated and pre-cleared as described under Section 2. Pre-cleared chromatin was immunoprecipitated by antibodies against CAR or normal rabbit IgG. DNA isolated from the input chromatin before precipitation (20% of volume used for precipitation, 20% input) or from the precipitated chromatin was analyzed by PCR using primer set specific for cyp2b10 promoter as indicated in the schematic diagram of the gene. PCR products were resolved on agarose gel.
the binding of CAR to its respective binding site on cyp2b10 promoter, whereas this binding was unchanged by cisTPD and transTPD. These results demonstrate that, in agreement with multiplex PCR results, transpDMA, transpNO2, transpF and transpMeO are able to activate CAR in mouse livers, leading to increased transcription of its target gene cyp2b10.
#
20
*
15
10
100 bp
20% Input Fig. 3. Dose-dependent effects of compounds on CYP2B proteins in mouse livers. Mice were treated with the compounds at 0.5, 1, 5, 10, 30, 50 and 100 mg/kg body weight. For preparation of microsomal proteins, animals were killed 72 h after a single ip injection of one of the tested compounds. Fifteen micrograms of protein were electrophoresed in 10% SDS–PAGE and transferred to nitrocellulose membrane. Immunodetection was performed with anti-rat CYP2B1 polyclonal antibodies.
*
*
5
0
Fig. 4. Effects of compounds on cyp2b10 gene expression in mouse livers. For RNA isolation, mice were killed 24 h after a single ip injection of one of the tested compounds (30 mg/kg body weight). Total RNAs were prepared and subjected to RT-PCR for measurement of cyp2b10 mRNA. Fold change was expressed by taking the corresponding value of the control rats as one. Bars mean ± S.D. Five animals per treatment group were used. ⁄Indicates a significant difference from control animals (ANOVA followed by Dunnett’s post hoc test, p < 0.05). #Indicates a significant difference between animal groups (ANOVA followed by Tukey’s post hoc test, p < 0.05).
required for CAR binding to the 50 -flanking DNA sequence (PBREM) of the target CYP2B genes. ChIP is a powerful approach that allows the analysis of interaction of activated transcriptional factors with DNA in living cells. ChIP assay was performed on the chromatin extracted from mouse livers treated with compounds to demonstrate the recruitment of CAR to its respective binding site on cyp2b10 promoter in vivo. Antibodies directed against CAR were used to immunoprecipitate chromatin fragments. PCR products were generated using a primer pair specific for the PBREM region of the cyp2b10 gene. The results of ChIP showed that in the absence of added compounds, CAR showed weak binding to the promoter (Fig. 5). transpDMA, transpNO2, transpF and transpMeO stimulated
4. Discussion A major problem in the safety evaluation of pharmaceutical agents is the extrapolation of toxicological data from one animal to another. Many examples have been documented where various species displayed a markedly different response to the same chemical, including rat and mouse, two widely used experimental animal models. In the present study, the effect of several analogs of 2,4,6-triphenyldioxane-1,3, a highly effective inducer of CYP2B in rat but not mouse liver [6], was investigated in mouse livers in vivo. Among the six test compounds, four (transpDMA, transpNO2, transpF and transpMeO) demonstrated dose-dependent induction of CYP2B (Fig. 1). CisTPD and transTPD, as expected, failed to induce CYP2B in mouse livers. Thus, minor structural changes in TPD cause dramatic changes in its ability to induce mouse CYP2B. Moreover, it is likely transpDMA, transpNO2, transpF and transpMeO act as partial activators because the maximal TPD analog response is significantly lower than the maximal response to a single ip injection of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) (38-fold compared to the control values) at a dose of 3 mg/ kg body weight (data not shown). Dose–response curves were generated to determine the potency of the TPD analogs in mouse livers (Table 1). The results showed that transpDMA, transpNO2, transpF and transpMeO have similar potencies to induce CYP2B activity in mouse livers. Further studies evaluated the mechanism of the change in PROD activity and protein level with treatment by the compounds. Since most CYPs are regulated transcriptionally [12], this suggests that cyp2b10 gene transcription is stimulated by TPD analogs in a compound-specific manner. To test this hypothesis, relative mRNA levels of cyp2b10 in the animal livers were measured using multiplex RT-PCR (Fig. 4). The results of RT-PCR analyses provide evidence to support the conclusion that compound-specific CYP2B induction in
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mouse livers by TPD analogs occur through differences of transcriptional activation of cyp2b10 gene. It was shown that transcriptional activation of the hepatic cyp2b10 gene in mouse by inducers is carried out by the zinc finger-containing nuclear receptor CAR [13]. CAR activation is a multistep process, where translocation from the cytoplasm to the nucleus has been considered the initial step regulating the induction of CYP2B genes [14]. In normal untreated hepatic cells, CAR is located in the cytoplasm in a complex with CCRP and Hsp90 [2]. In response to inducers, CAR is translocated to the nucleus where it forms a functional heterodimer with the retinoid X receptor (RXR). The CAR–RXR heterodimer recruits p160/SRC-1 and other coactivator proteins [15]. The affinity of CAR–RXR heterodimer to coactivators is enhanced by inducers. Binding of the CAR– RXR heterodimer to NR1-binding sites (DR-4 motifs) in the 50 flanking sequence of the CYP2B genes results in activation of a 51-base pair phenobarbital-responsive enhancer module (PBREM) of the distal promoter [13]. To further characterize TPD analogs as potent CYP2B inducers in mice, we studied the effects of these compounds on mouse CAR in vivo. ChIP was used to examine the recruitment of CAR to the CAR target gene cyp2b10 promoter region. ChIP results demonstrated that transcriptional enhancement of cyp2b10 gene (Fig. 5), protein content and induction of PROD activity in response to transpDMA, transpNO2, transpF and transpMeO is accompanied by the increased recruitment of CAR to its binding site (PBREM) on the target gene (Fig. 5). On the other hand, consistent with our previous study [6,11], we found that cisTPD and transTPD did not have any effects on CYP2B induction and CAR transcriptional activity in mouse livers (Figs. 2–4). Thus, we have identified a series of potent CYP2B inducers in mouse liver. Moreover, our recent and present results have shown species-specific effects of several analogs of TPD on CAR and its target genes CYP2B. Closely related structural variants of chemical compounds have different effects on CAR target gene CYP2B in rat [8] and mouse liver in vivo. Thus, cisTPD and transTPD induced CYP2B activities and CYP2B gene expression only in rat liver, whereas transpMeO – only in mouse liver. The observed speciesspecific features of CYP2B induction by TPD analogs in rats and mice could be caused by differences in the ligand binding domain (LBD) of CAR in these species, although except for a few amino acids differences the mouse LBD is identical to the rat LBD of CAR (89% identity; [3]). However, mouse and rat CAR show sequence variation at amino acid positions corresponding to residues that interact directly with ligands in X-ray crystallographic structures of mouse CAR [16]. Comparison of the ligand binding domains of rat and mouse CAR has revealed that the specificity to TCPOBOP (ligand of mouse CAR) in mice is caused by the presence of Thr350, whereas in rats this position is occupied by Met. Moreover, mouse and rat CAR has shown sequence variation at position 253 (Leu253 in mouse and Ile253 in rat). The amino acid residue in helix 7 at position 253 has a critical role in species difference in response of CAR to 17a-ethynyl-3,17b-estradiol in mouse and human [17]. However, these supposition needs to be investigated in more details using docking study and point-mutated CAR.
Moreover, although our results are consistent with the key role of CAR in the compound-specific CYP2B induction by TPD analogs measured in this work, definitive experiments now should be performed using CAR gene knock out models. Conflict of interest statement There are no conflicts of interest. Acknowledgments This work was supported by RFBR Grant No. 09-04-00801-a and by the Russian Federal Program of the Ministry of Education and Science of the Russian Federation ‘‘Scientific and pedagogical manpower of innovational Russia’’ (GK 14.740.11.1054 and GK 16.740.11.0631). References [1] H. Li, H. Wang, Activation of xenobiotic receptors: driving into the nucleus, Expert Opin. Drug. Metab. Toxicol. 6 (2010) 409–426. [2] K. Kobayashi, T. Sueyoshi, K. Inoue, R. Moore, M. Negishi, Cytoplasmic accumulation of the nuclear receptor CAR by a tetratricopeptide repeat protein in HepG2 cells, Mol. Pharmacol. 64 (2003) 1069–1075. [3] V.O. Pustylnyak, L.F. Gulyaeva, V.V. Lyakhovich, Induction of cytochrome P4502B: role of regulatory elements and nuclear receptors, Biochemistry (Moscow) 72 (2007) 608–617. [4] K. Swales, M. Negishi, CAR, driving into the future, Mol. Endocrinol. 18 (2004) 1589–1598. [5] M. Dickins, Induction of cytochromes P450, Curr. Topics Med. Chem. 4 (2004) 1745–1766. [6] V. Pustylnyak, E. Pivovarova, N. Slynko, L. Gulyaeva, V. Lyakhovich, Speciesspecific induction of CYP2B by 2,4,6-tryphenyldioxane-1,3 (TPD), Life Sci. 85 (2009) 815–821. [7] V.M. Mishin, N.I. Gutkina, V.V. Lyakhovich, L.N. Pospelova, V.V. Chistiakov, Comparison of the inducing effect of triphenyldioxan, bis-(dichloropyridyloxy) benzene and phenobarbital on the liver monooxygenase, Biokhimiya (Russia) 55, 29–36. [8] V. Pustylnyak, A. Yarushkin, E. Kachaylo, N. Slynko, V. Lyakhovich, L. Gulyaeva, Effect of several analogs of 2,4,6-triphenyldioxane-1,3 on constitutive androstane receptor activation, Chem. Biol. Interact. 192 (2011) 177–183. [9] H. Griengl, K.P. Geppert, Prins-reaktionen mit arylaldehyden und arylolefine, Monatshafte fur Chemie 107 (1976) 421–431. [10] M.D. Burke, S. Thompson, C. Elcombe, J. Halpert, T. Haaparanta, R.T. Mayer, Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P450, Biochem. Pharmacol. 34 (1985) 3337–3345. [11] V.O. Pustylnyak, A.N. Lebedev, L.F. Gulyaeva, V.V. Lyakhovich, N.M. Slynko, Comparative study of CYP2B induction in the liver of rats and mice by different compounds, Life Sci. 80 (2007) 324–328. [12] C. Handschin, U. Meyer, Induction of drug metabolism: the role of nuclear receptors, Pharmacol. Rev. 55 (2003) 649–673. [13] K. Swales, M. Negishi, CAR, driving into the future, Mol. Endocrinol. 18 (2004) 1589–1598. [14] T. Kawamoto, T. Sueyoshi, I. Zelko, R. Moore, K. Washburn, M. Negishi, Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B2 gene, Mol. Cell. Biol. 19 (1999) 6318–6322. [15] J. Xia, L. Liao, J. Sarkar, K. Matsumoto, J.K. Reddy, J. Xu, B. Kemper, Redundant enhancement of mouse constitutive androstane receptor transactivation by p160 coactivator family members, Arch. Biochem. Biophys. 468 (2007) 49–57. [16] K. Suino, L. Peng, R. Reynolds, Y. Li, J.-Y. Cha, J.J. Repa, S.A. Kliewer, H.E. Xu, The nuclear xenobiotic receptor CAR: structural determinants of constitutive activation and heterodimerization, Mol. Cell. 16 (2004) 893–905. [17] J. Jyrkkärinne, B. Windshügel, J. Mäkinen, M. Ylisirniö, M. Peräkylä, A. Poso, W. Sippl, P. Honkakoski, Amino acids important for ligand specificity of the human constitutive androstane receptor, J. Biol. Chem. 280 (2005) 5960–5971.
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Effect of several analogs of 2,4,6-triphenyldioxane-1,3 on CYP2B induction in mouse liver Vladimir Pustylnyak a,b,⇑, Yuliya Kazakova a,b, Andrei Yarushkin a, Nikolai Slynko c, Lyudmila Gulyaeva a,b a
Institute of Molecular Biology and Biophysics, SB RAMS, Novosibirsk, Russia Novosibirsk State University, Novosibirsk, Russia c Institute of Cytology and Genetics, SB RAS, Novosibirsk, Russia b
a r t i c l e
i n f o
Article history: Received 2 August 2011 Received in revised form 5 September 2011 Accepted 22 September 2011 Available online 29 September 2011 Keywords: Cyp2b10 TPD Gene regulation CAR
a b s t r a c t 2,4,6-Triphenyldioxane-1,3 (TPD) is a highly effective inducer of CYP2B in rats, but not in mice. Several analogs of TPD were synthesized. All substituents were entered into the same position of TPD (R = H, cisTPD and transTPD; R = N(CH3)2, transpDMA; R = NO2, transpNO2; R = F, transpF; R = OCH3, transpMeO). The purpose of the present study was to investigate the effect of TPD analogs on CYP2B induction in mouse livers. Among the six test compounds, four (R = –N(CH3)2, –NO2, –F, –OCH3) demonstrated a dose-dependent induction of mouse CYP2B. To further characterize the compounds, we determined ED50s using sigmoidal dose–response curves. The dose–response study has shown that all active compounds have similar potencies to induce CYP2B in mouse livers. Western-blot analysis and multiplex RT-PCR have shown that the increase of CYP2B activity in mouse liver is related to the high content of CYP2B proteins and paralleled the increase of cyp2b10 mRNA level. ChIP results have demonstrated that the transcriptional enhancement of cyp2b10 gene in response to compounds is accompanied by the increased recruitment of the constitutive androstane receptor (CAR) to its specific binding site (PBREM) on the target gene. Thus, minor structural changes in TPD cause dramatic changes in its ability to induce mouse CYP2B, and it is likely several TPD analogs act by activation of mouse CAR. Ó 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The CYP2B subfamily is expressed constitutively and inducibly. The induction of CYP2B isoforms by chemical compounds is mediated through effects on the constitutive androstane receptor (CAR, NR1I3), zinc finger-containing transcription factor belonging to the nuclear receptor superfamily [1]. Non-activated CAR is retained in the cytoplasm in a complex with heat shock protein 90 (Hsp90) and CAR cytoplasmic retention protein (CCRP) [2]. In response to chemical compounds, CAR translocates to the nucleus and forms a heterodimer with the retinoid X receptor (RXR). The activation is also associated with recruitment of coactivators, such as SRC-1, Sp1, GRIP1, PGC1a, p160 [3]. The CAR–RXR–coactivator complex binds to the distal specific DNA response elements of target genes leading to an increase in gene expression [4]. It is very important to clarify the mechanism of CYP2B induction because it leads to better models for screening and predicting drug interaction that occur due to the induction of individual CYPs. Generally, such investigations are carried out using experimental animals like rats and mice. A major problem in these studies is ⇑ Corresponding author at: Institute of Molecular Biology and Biophysics, Timakova str. 2, Novosibirsk 630117, Russia. Tel./fax: +7 383 335 98 47. E-mail address: [email protected] (V. Pustylnyak). 0009-2797/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2011.09.003
the extrapolation of data from one animal species to another. Many examples have been documented where various animal species displayed a markedly different response to the same chemical compound. Some species differences in induction of CYP forms have been reported, which are attributable to differences in the ligand binding domain of nuclear receptors between different species [5]. 2,4,6-Triphenyldioxane-1,3 (TPD) is a highly effective activator of CAR and its target genes in rat liver, but it is not able to activate CAR in mice [6]. Our previous study has shown that after a single cisTPD ip administration of 1 mg/kg body weight, a significant increase in CYP2B activity was detected [7]. A maximal CYP2B activity increase was observed after 10 mg/kg body weight ip cisTPD injection [7]. In our recent study, we have synthesized several analogs of TPD (R = H, cisTPD and transTPD; R = N(CH3)2, transpDMA; R = NO2, transpNO2; R = F, transpF; R = OCH3, transpMeO) to determine how minor changes in the inducer structure can cause changes in induction abilities [8]. Substituents were made to minimize changes in the initial structure while changing the hydrophobic properties of the molecule. All substituents were entered into the fourth para position of the second phenyl ring of TPD. Five of the six compounds (cisTPD, transTPD, transpDMA, transpNO2 and transpF) were able to activate CAR and to induce its target genes CYP2B1/2 in rat liver. The dose–response studies have shown that
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cisTPD, transpDMA, transpF and transpNO2 have similar potency, and transTPD is a less potent derivative in rat livers [8]. The purpose of the present study was to investigate the effect of TPD analogs on CYP2B induction in mouse livers.
2.1. Chemicals cisTPD – (4S,6R)-2,4,6-triphenyl-1,3-dioxane, transTPD – (4R,6R) -2,4,6-triphenyl-1,3-dioxane, transpMeO – (4R,6R)-2-(4-methoxyphenyl)-4,6-diphenyl-1,3-dioxane, transpDMA – 4-[(4R,6R)-4,6-diphenyl-1,3-dioxan-2-yl]-N,N-dimethylaniline, transpF – (4R,6R)-2(4-fluorophenyl)-4,6-diphenyl-1,3-dioxane, transpNO2 – (4R,6R)2-(4-nitrophenyl)-4,6-diphenyl-1,3-dioxane (Fig. 1) were synthesized according to Griengl and Geppert [9]. Cis and trans refer to the relative configurations of the aryl fragments near atoms 4 and 6 of the dioxane ring. The compounds were characterized by NMR spectroscopy and shown to be 97–98% pure by LC/MS analysis. All other chemicals and solvents were of analytical grade and were obtained from commercial sources. 2.2. Experimental animals Male C57BL mice (20–25 g) were supplied by the Institute of Clinical Immunology, SB RAMS (Novosibirsk, Russia). Animals were acclimated for 1 week and allowed free access to food and water. All experimental procedures were approved by the Animal Care Committee of the Institute of Molecular Biology and Biophysics, SB RAMS. 2.3. Preparation of microsomal proteins For preparation of microsomal proteins, mice were killed 72 h after a single ip injection of one of the tested compounds (0.5, 1, 5, 10, 30, 50 and 100 mg/kg body weight in corn oil) or an equal amount of corn oil. Five animals per treatment group were used. The microsomal fractions were isolated from freshly excised tissues by standard differential centrifugation. The protein concentrations in microsomes were measured using the Lowry method with a bovine albumin solution as a standard. The microsomal fractions were collected and stored at 80 °C.
transpDMA
2.4. CYP2B catalytic activity The CYP2B specific activity, 7-pentoxyresorufin O-dealkylase (PROD), was measured at 37 °C by fluorimetry [10]. 2.5. SDS–PAGE electrophoresis and Western-blot
2. Materials and methods
cisTPD
135
transTPD
transpF
transpMeO
transpNO2
Fig. 1. Structures of compounds. cisTPD – (4S,6R)-2,4,6-triphenyl-1,3-dioxane, trans TPD – (4R,6R)-2,4,6-triphenyl-1,3-dioxane, transpMeO – (4R,6R)-2-(4-methoxyphe nyl)-4,6-diphenyl-1,3-dioxane, transpDMA – 4-[(4R,6R)-4,6-diphenyl-1,3-dioxan -2-yl]-N,N-dimethylaniline, transpF – (4R,6R)-2-(4-fluorophenyl)-4,6-diphenyl-1, 3-dioxane, transpNO2 – (4R,6R)-2-(4-nitrophenyl)-4,6-diphenyl-1,3-dioxane.
Fifteen micrograms of microsomal proteins per lane were separated in 10% SDS–PAGE and transferred to the nitrocellulose membrane. Membranes were stained with Ponceau S to verify loading and transfer efficiency. Immunodetection was performed with anti-rat CYP2B1 antibodies. Polyclonal antibodies directed against rat CYP2B1 were prepared in rabbits by immunization with the purified CYP2B1 protein. The bands were visualized colorimetrically with 4-chloro-1-naphthol (MP Biomedicals) as substrate. 2.6. cDNA synthesis and multiplex PCR For RNA isolation, mice were killed 24 h after a single ip injection of one of the tested compounds (30 mg/kg body weight in corn oil). Total RNA was isolated from livers frozen in liquid nitrogen with Rneasy Mini Kit (Qiagen) according to the manufacturer’s protocol. The concentration and purity of the RNA were determined by measuring the absorbance at 260 and 280 nm with correction for background at 320 nm in a spectrophotometer, and the integrity was examined by visualizing the 18S and 28S rRNA bands on a denaturating agarose (1%) gel. One microgram of total RNA was used for synthesis of single-stranded cDNA. First strand cDNA synthesis was carried out using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s protocol. Expression level of the cyp2b10 gene was measured by multiplex PCR performed as previously described [11]. The ‘‘house-keeping’’ gene, b-actin, was chosen as an endogenous internal controls to which the cyp2b10 PCR amplification product was normalized. 2.7. ChIP assay For ChIP assay, mice were killed 8 h after a single ip injection of one of the tested compounds (30 mg/kg body weight in corn oil). Livers were rinsed in cold phosphate-buffered saline with 0.4% NP-40 and homogenized in the same solution. Homogenates were treated with 1% formaldehyde at room temperature for 10 min with gentle rotation to cross-link the chromatin and the reaction was stopped by adding glycine to a final concentration of 125 mM. Fixed cells were collected by centrifugation, washed with cold phosphate-buffered saline with 0.4% NP-40, homogenized in cell lysis buffer 50 mM Tris–HCl, pH 8.0, 1% Triton X-100, 1% SDS, 10 mM EDTA supplemented with Complete Mini (Roche Diagnostics) and incubated on ice for 30 min. Lysates were sonicated on ice with three pulses of 20 s and 20% amplitude in Microson™ Ultrasonic Liquid Processor XL-2000 cell disrupter, yielding chromatin fragments of 500–1000 bp in size. Samples were centrifuged at 13000g for 10 min to remove detritus, and the supernatants were collected. To provide a positive control (input), 20 ll of the supernatants was retained. The supernatants were diluted 10-fold with dilution buffer (16.7 mM Tris–HCl, pH 8.0, 1.2 mM EDTA, 150 mM NaCl, 1.1% Triton X-100, 0.01% SDS supplemented with Complete Mini (Roche Diagnostics)). The diluted lysates were pre-cleared by incubating with 20 ll/ml protein G agarose/Salmon sperm DNA (Millipore) for 1 h at 4 °C on rotating plate. Cleared lysates (2 ml) were subjected to overnight incubation with either 5 lg of anti-CAR antibody or normal rabbit IgG at 4 °C under rotation. Immunocomplexes were precipitated by adding 30 ll of protein G agarose/Salmon sperm DNA (Millipore) for 1 h at 4 °C with shaking, washed with low-salt buffer (20 mM Tris–HCl, pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.01% SDS), high-salt
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served in transpDMA (61% of the values measured in transpNO2), transpMeO (22% of the values measured in transpNO2) and transpF (18% of the values measured in transpNO2) treated mouse livers in the dose range of 50–100 mg/kg body weight (Tukey’s post hoc test, p < 0.05). To determine whether the PROD activity level was parallel to the level in cyp2b protein, dose-dependent cyp2b protein induction was investigated by Western-blot analysis (Fig. 3). Westernblot analysis demonstrated a basal level of cyp2b protein in livers of vehicle-treated mice. The protein band corresponded to cyp2b in vehicle-treated mice was not found to be markedly changed in livers from cisTPD- and transTPD-treated mice. On the other hand, the hepatic cyp2b protein level was increased in dose-dependent manner after transpDMA, transpNO2, transpMeO and transpF treatment compared with the level in control animals. Thus, among the six test compounds, four increased CYP2B activity and protein levels. To further characterize the compounds, we determined ED50s using sigmoidal dose–response curves for transpMeO, transpDMA, transpF and transpNO2 (Table 1). All TPD analogs had similar ED50s.
buffer (20 mM Tris–HCl, pH 8.0, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.01% SDS), LiCl buffer (10 mM Tris–HCl, pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP-40) and finally twice with TE buffer (0.25 mM EDTA, 10 mM Tris–HCl, pH 8.0). The immunoselected chromatin was eluted in 500 ll of elution buffer (0.1 M NaHCO3, 1% SDS and 0.3 M NaCl). To reverse cross-linking, the samples were further incubated for 6 h at 65 °C. After protease digestion, DNA was purified by phenol/chloroform extraction. PCR amplification was performed using primers specific for the PBREM region of the mouse cyp2b10 gene (F: 50 -CGAGGACACAATCTTGAAG-30 and R: 50 -GAGCAAGGTCCTGGTGTC-50 ). 2.8. Data analysis The data are presented as mean ± SD. ANOVA followed by Dunnett’s post hoc test was used to compare each group to control. ANOVA followed by Tukey’s post hoc test was used to multiple comparison tests. A p value <0.05 was considered statistically significant. ED50, 95% confidence intervals (CIs) of each chemical were estimated by fitting dose–response data to a sigmoidal function using GraphPAD Prism 5.0.
3.2. Induction of cyp2b10 gene expression 3. Results
Effect of the tested compounds on cyp2b10 gene expression were evaluated in mouse livers by measuring cyp2b10 mRNA levels 24 h after single ip injections of chemical compounds (30 mg/kg body weight in corn oil). As expected, transpDMA (15-fold), transpNO2 (13.5-fold), transpMeO (6.5-fold) and transpF (8-fold) strongly and significantly increased cyp2b10 gene expression compared to the control values (Dunnett’s post hoc test, p < 0.05) (Fig. 4). Moreover, the maximum cyp2b10 mRNA levels produced by transpNO2 and transpDMA were significantly higher than observed in transpMeO and transpF treated mouse livers (Tukey’s post hoc test, p < 0.05). At the same time, cyp2b10 mRNA levels were unchanged in mouse livers after cisTPD and transTPD treatment.
3.1. Dose-dependent CYP2B induction Dose-dependent changes in PROD activity were investigated in mouse livers treated with TPD analogs (0.5, 1, 5, 10, 30, 50 and 100 mg/kg body weight) (Fig. 2). PROD activity was found to be significantly increased in the dose range of 5–100 mg/kg body weight of transpDMA and transpNO2 and in the dose range 30–100 mg/kg body weight of transpMeO and transpF compared to the control values (Dunnett’s post hoc test, p < 0.05). In contrast, the level of CYP2B specific activity was not altered compared to control values after treatment with cisTPD and transTPD in 0.5–100 mg/kg body weight dose range (Dunnett’s post hoc test, p < 0.05). Moreover, transpDMA and transpNO2 evoked dose-dependent increases of PROD activity in the range of 1–50 mg/kg body weight and transpMeO and transpF evoked dose-dependent increases of PROD activity in the range of 10–50 mg/kg body weight (Tukey’s post hoc test, p < 0.05). The maximum of CYP2B induction produced by transpNO2 was significantly higher (was taken as 100%) than ob-
3.3. Recruitment analysis of CAR on PBREM of cyp2b10 gene The expression of CYP2B genes upon inducer treatment is regulated by CAR. The CAR–RXR heterodimer is formed after CAR translocation from cytosol into the nucleus, and the heterodimer is
# #
#
# #
*
16
Fold change
14 12
*
*
10 8
* *
*
6
*
4
*
*
* *
*
2 0,5 1,0 5,0 10,0 30,0 50,0 100,0 0,5 1,0 5,0 10,0 30,0 50,0 100,0 0,5 1,0 5,0 10,0 30,0 50,0 100,0 0,5 1,0 5,0 10,0 30,0 50,0 100,0 0,5 1,0 5,0 10,0 30,0 50,0 100,0 0,5 1,0 5,0 10,0 30,0 50,0 100,0
0
cisTPD
transTPD
pMeO
pDMA
pF
pNO2
Fig. 2. Dose-dependent effect of compounds on PROD activity in mouse livers. Mice were treated with the compounds at 0.5, 1, 5, 10, 30, 50 and 100 mg/kg body weight. For preparation of microsomal proteins, animals were killed 72 h after a single ip injection of one of the tested compounds. Fold change was expressed by taking the corresponding value of the control mice as one. Five animals per treatment group were used. Bars mean ± S.D. ⁄Indicates a significant difference between various doses groups (ANOVA followed by Tukey’s post hoc test, p < 0.05). #Indicates a significant difference between different compounds groups (ANOVA followed by Tukey’s post hoc test, p < 0.05).
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A
CAR
F
RXR
cyp2b10
PBREM R
B
Anti-CAR Normal IgG
Table 1 Potency of TPD analogs. TPD analog
ED50 (mg/kg)
95% CI
transpMeO transpDMA transpF transpNO2
23.5 27.6 24.5 28.4
15.3–36.2 23.4–34.6 12.7–47.7 16.2–49.8
# #
#
* Fold change
pNO2
pF
pDMA
pMeO
transTPD
cisTPD
Cont
Fig. 5. Recruitment of CAR to PBREM of the cyp2b10 gene promoter in mouse livers in response to compound treatments. (A) Schematic representation of cyp2b10 promoter. Location of the PBREM region is shown. (B) Chromatin was isolated and pre-cleared as described under Section 2. Pre-cleared chromatin was immunoprecipitated by antibodies against CAR or normal rabbit IgG. DNA isolated from the input chromatin before precipitation (20% of volume used for precipitation, 20% input) or from the precipitated chromatin was analyzed by PCR using primer set specific for cyp2b10 promoter as indicated in the schematic diagram of the gene. PCR products were resolved on agarose gel.
the binding of CAR to its respective binding site on cyp2b10 promoter, whereas this binding was unchanged by cisTPD and transTPD. These results demonstrate that, in agreement with multiplex PCR results, transpDMA, transpNO2, transpF and transpMeO are able to activate CAR in mouse livers, leading to increased transcription of its target gene cyp2b10.
#
20
*
15
10
100 bp
20% Input Fig. 3. Dose-dependent effects of compounds on CYP2B proteins in mouse livers. Mice were treated with the compounds at 0.5, 1, 5, 10, 30, 50 and 100 mg/kg body weight. For preparation of microsomal proteins, animals were killed 72 h after a single ip injection of one of the tested compounds. Fifteen micrograms of protein were electrophoresed in 10% SDS–PAGE and transferred to nitrocellulose membrane. Immunodetection was performed with anti-rat CYP2B1 polyclonal antibodies.
*
*
5
0
Fig. 4. Effects of compounds on cyp2b10 gene expression in mouse livers. For RNA isolation, mice were killed 24 h after a single ip injection of one of the tested compounds (30 mg/kg body weight). Total RNAs were prepared and subjected to RT-PCR for measurement of cyp2b10 mRNA. Fold change was expressed by taking the corresponding value of the control rats as one. Bars mean ± S.D. Five animals per treatment group were used. ⁄Indicates a significant difference from control animals (ANOVA followed by Dunnett’s post hoc test, p < 0.05). #Indicates a significant difference between animal groups (ANOVA followed by Tukey’s post hoc test, p < 0.05).
required for CAR binding to the 50 -flanking DNA sequence (PBREM) of the target CYP2B genes. ChIP is a powerful approach that allows the analysis of interaction of activated transcriptional factors with DNA in living cells. ChIP assay was performed on the chromatin extracted from mouse livers treated with compounds to demonstrate the recruitment of CAR to its respective binding site on cyp2b10 promoter in vivo. Antibodies directed against CAR were used to immunoprecipitate chromatin fragments. PCR products were generated using a primer pair specific for the PBREM region of the cyp2b10 gene. The results of ChIP showed that in the absence of added compounds, CAR showed weak binding to the promoter (Fig. 5). transpDMA, transpNO2, transpF and transpMeO stimulated
4. Discussion A major problem in the safety evaluation of pharmaceutical agents is the extrapolation of toxicological data from one animal to another. Many examples have been documented where various species displayed a markedly different response to the same chemical, including rat and mouse, two widely used experimental animal models. In the present study, the effect of several analogs of 2,4,6-triphenyldioxane-1,3, a highly effective inducer of CYP2B in rat but not mouse liver [6], was investigated in mouse livers in vivo. Among the six test compounds, four (transpDMA, transpNO2, transpF and transpMeO) demonstrated dose-dependent induction of CYP2B (Fig. 1). CisTPD and transTPD, as expected, failed to induce CYP2B in mouse livers. Thus, minor structural changes in TPD cause dramatic changes in its ability to induce mouse CYP2B. Moreover, it is likely transpDMA, transpNO2, transpF and transpMeO act as partial activators because the maximal TPD analog response is significantly lower than the maximal response to a single ip injection of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) (38-fold compared to the control values) at a dose of 3 mg/ kg body weight (data not shown). Dose–response curves were generated to determine the potency of the TPD analogs in mouse livers (Table 1). The results showed that transpDMA, transpNO2, transpF and transpMeO have similar potencies to induce CYP2B activity in mouse livers. Further studies evaluated the mechanism of the change in PROD activity and protein level with treatment by the compounds. Since most CYPs are regulated transcriptionally [12], this suggests that cyp2b10 gene transcription is stimulated by TPD analogs in a compound-specific manner. To test this hypothesis, relative mRNA levels of cyp2b10 in the animal livers were measured using multiplex RT-PCR (Fig. 4). The results of RT-PCR analyses provide evidence to support the conclusion that compound-specific CYP2B induction in
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mouse livers by TPD analogs occur through differences of transcriptional activation of cyp2b10 gene. It was shown that transcriptional activation of the hepatic cyp2b10 gene in mouse by inducers is carried out by the zinc finger-containing nuclear receptor CAR [13]. CAR activation is a multistep process, where translocation from the cytoplasm to the nucleus has been considered the initial step regulating the induction of CYP2B genes [14]. In normal untreated hepatic cells, CAR is located in the cytoplasm in a complex with CCRP and Hsp90 [2]. In response to inducers, CAR is translocated to the nucleus where it forms a functional heterodimer with the retinoid X receptor (RXR). The CAR–RXR heterodimer recruits p160/SRC-1 and other coactivator proteins [15]. The affinity of CAR–RXR heterodimer to coactivators is enhanced by inducers. Binding of the CAR– RXR heterodimer to NR1-binding sites (DR-4 motifs) in the 50 flanking sequence of the CYP2B genes results in activation of a 51-base pair phenobarbital-responsive enhancer module (PBREM) of the distal promoter [13]. To further characterize TPD analogs as potent CYP2B inducers in mice, we studied the effects of these compounds on mouse CAR in vivo. ChIP was used to examine the recruitment of CAR to the CAR target gene cyp2b10 promoter region. ChIP results demonstrated that transcriptional enhancement of cyp2b10 gene (Fig. 5), protein content and induction of PROD activity in response to transpDMA, transpNO2, transpF and transpMeO is accompanied by the increased recruitment of CAR to its binding site (PBREM) on the target gene (Fig. 5). On the other hand, consistent with our previous study [6,11], we found that cisTPD and transTPD did not have any effects on CYP2B induction and CAR transcriptional activity in mouse livers (Figs. 2–4). Thus, we have identified a series of potent CYP2B inducers in mouse liver. Moreover, our recent and present results have shown species-specific effects of several analogs of TPD on CAR and its target genes CYP2B. Closely related structural variants of chemical compounds have different effects on CAR target gene CYP2B in rat [8] and mouse liver in vivo. Thus, cisTPD and transTPD induced CYP2B activities and CYP2B gene expression only in rat liver, whereas transpMeO – only in mouse liver. The observed speciesspecific features of CYP2B induction by TPD analogs in rats and mice could be caused by differences in the ligand binding domain (LBD) of CAR in these species, although except for a few amino acids differences the mouse LBD is identical to the rat LBD of CAR (89% identity; [3]). However, mouse and rat CAR show sequence variation at amino acid positions corresponding to residues that interact directly with ligands in X-ray crystallographic structures of mouse CAR [16]. Comparison of the ligand binding domains of rat and mouse CAR has revealed that the specificity to TCPOBOP (ligand of mouse CAR) in mice is caused by the presence of Thr350, whereas in rats this position is occupied by Met. Moreover, mouse and rat CAR has shown sequence variation at position 253 (Leu253 in mouse and Ile253 in rat). The amino acid residue in helix 7 at position 253 has a critical role in species difference in response of CAR to 17a-ethynyl-3,17b-estradiol in mouse and human [17]. However, these supposition needs to be investigated in more details using docking study and point-mutated CAR.
Moreover, although our results are consistent with the key role of CAR in the compound-specific CYP2B induction by TPD analogs measured in this work, definitive experiments now should be performed using CAR gene knock out models. Conflict of interest statement There are no conflicts of interest. Acknowledgments This work was supported by RFBR Grant No. 09-04-00801-a and by the Russian Federal Program of the Ministry of Education and Science of the Russian Federation ‘‘Scientific and pedagogical manpower of innovational Russia’’ (GK 14.740.11.1054 and GK 16.740.11.0631). References [1] H. Li, H. Wang, Activation of xenobiotic receptors: driving into the nucleus, Expert Opin. Drug. Metab. Toxicol. 6 (2010) 409–426. [2] K. Kobayashi, T. Sueyoshi, K. Inoue, R. Moore, M. Negishi, Cytoplasmic accumulation of the nuclear receptor CAR by a tetratricopeptide repeat protein in HepG2 cells, Mol. Pharmacol. 64 (2003) 1069–1075. [3] V.O. Pustylnyak, L.F. Gulyaeva, V.V. Lyakhovich, Induction of cytochrome P4502B: role of regulatory elements and nuclear receptors, Biochemistry (Moscow) 72 (2007) 608–617. [4] K. Swales, M. Negishi, CAR, driving into the future, Mol. Endocrinol. 18 (2004) 1589–1598. [5] M. Dickins, Induction of cytochromes P450, Curr. Topics Med. Chem. 4 (2004) 1745–1766. [6] V. Pustylnyak, E. Pivovarova, N. Slynko, L. Gulyaeva, V. Lyakhovich, Speciesspecific induction of CYP2B by 2,4,6-tryphenyldioxane-1,3 (TPD), Life Sci. 85 (2009) 815–821. [7] V.M. Mishin, N.I. Gutkina, V.V. Lyakhovich, L.N. Pospelova, V.V. Chistiakov, Comparison of the inducing effect of triphenyldioxan, bis-(dichloropyridyloxy) benzene and phenobarbital on the liver monooxygenase, Biokhimiya (Russia) 55, 29–36. [8] V. Pustylnyak, A. Yarushkin, E. Kachaylo, N. Slynko, V. Lyakhovich, L. Gulyaeva, Effect of several analogs of 2,4,6-triphenyldioxane-1,3 on constitutive androstane receptor activation, Chem. Biol. Interact. 192 (2011) 177–183. [9] H. Griengl, K.P. Geppert, Prins-reaktionen mit arylaldehyden und arylolefine, Monatshafte fur Chemie 107 (1976) 421–431. [10] M.D. Burke, S. Thompson, C. Elcombe, J. Halpert, T. Haaparanta, R.T. Mayer, Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P450, Biochem. Pharmacol. 34 (1985) 3337–3345. [11] V.O. Pustylnyak, A.N. Lebedev, L.F. Gulyaeva, V.V. Lyakhovich, N.M. Slynko, Comparative study of CYP2B induction in the liver of rats and mice by different compounds, Life Sci. 80 (2007) 324–328. [12] C. Handschin, U. Meyer, Induction of drug metabolism: the role of nuclear receptors, Pharmacol. Rev. 55 (2003) 649–673. [13] K. Swales, M. Negishi, CAR, driving into the future, Mol. Endocrinol. 18 (2004) 1589–1598. [14] T. Kawamoto, T. Sueyoshi, I. Zelko, R. Moore, K. Washburn, M. Negishi, Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B2 gene, Mol. Cell. Biol. 19 (1999) 6318–6322. [15] J. Xia, L. Liao, J. Sarkar, K. Matsumoto, J.K. Reddy, J. Xu, B. Kemper, Redundant enhancement of mouse constitutive androstane receptor transactivation by p160 coactivator family members, Arch. Biochem. Biophys. 468 (2007) 49–57. [16] K. Suino, L. Peng, R. Reynolds, Y. Li, J.-Y. Cha, J.J. Repa, S.A. Kliewer, H.E. Xu, The nuclear xenobiotic receptor CAR: structural determinants of constitutive activation and heterodimerization, Mol. Cell. 16 (2004) 893–905. [17] J. Jyrkkärinne, B. Windshügel, J. Mäkinen, M. Ylisirniö, M. Peräkylä, A. Poso, W. Sippl, P. Honkakoski, Amino acids important for ligand specificity of the human constitutive androstane receptor, J. Biol. Chem. 280 (2005) 5960–5971.