Differential Effect of 7,12-Dimethylbenz[a]anthracene on Human and Mouse CYP1B1 from Livers of Castrated Transgenic Mice

Abstract

Humanized transgenic mice coexpressing tetracycline-controlled transactivator (tTA) and human cytochrome P450 1B1 (CYP1B1) (hCYP1B1) have been created by this group. The aims of this study was to determine if 7,12-dimethylbenz[a]anthracene (DMBA) functions as testosterone or doxycycline in its ability to induce or reduce expression of hCYP1B1 or endogenous mouse CYP1B1 (mCYP1B1). This was tested in the livers by treating castrated transgenic males and hCYP1B1/luciferase-transfected cells with DMBA. Herein, DMBA-treated group exhibited (i) gradual reduction of hCYP1B1 expression at the transcript, protein, and activity levels but gradually induced its transcript level during DMBA release; (ii) gradual reduction of hCYP1B1 at the transcript and protein levels, as in the case of doxycycline or testosterone; (iii) gradual induction of mCYP1B1 expression at the transcript and protein levels but gradually reduced its transcript level during DMBA release. In parallel, DMBA-treated transfected cells exhibited gradual increase in luciferase activity in a time- and dose-dependent manner. Thus, castrated transgenic males or in vitro system could be useful as models for the detection of polycyclic aromatic hydrocarbons (PAHs) or environmental toxicants by measuring either hCYP1B1 or mCYP1B1 expressions.

Of the members of the cytochrome P450 (CYP) superfamily, 1A1, 1A2, and 1B1 are induced through the aryl hydrocarbon receptor (AhR) activation by environmental toxins such as poly-chlorinated biphenyls (PCBs), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and polycyclic aromatic hydrocarbons (PAHs) (Rowlands and Gustafsson 1997; Savas et al. 1994; Shen et al. 1994; Sutter et al. 1994). In this family, CYP1B1 and CYP1A1 appear to be principally extrahepatic and can easily be detected after administering PAHs, adrenocorticotrophins, and peptide hormones (Otto et al. 1991; Otto, Bhattacharyya, and Jefcoate 1992; Bhattacharyya et al. 1995), whereas CYP1A2 is primarily a hepatic enzyme (Sutter et al. 1994; Sesardic et al. 1990). There are two prototypic PAHs, 7,12-dimethylbenz[a]anthracene (DMBA) and benzo(a)pyrene (BP), that are known carcinogens and immunosuppressants, and the metabolism of DMBA and BP by CYP1B1 produces a distinctly different ratio of hydroxylated products compared with the CYP1A1-dependent metabolism of these toxins (Otto et al. 1991; Otto, Bhattacharyya, and Jefcoate 1992).

CYP1B1 is the predominant enzyme in the metabolic activation of DMBA to carcinogenic metabolites in vivo, instead of CYP1A1 (Butters et al. 1999). Exposure to DMBA has toxic effects on spermatogenesis in the male reproductive processes, and exposure to TCDD decreases the level of testosterone production (Chahoud et al. 1989; Ford and Huggins 1963; Georgellis et al. 1990). In particular, reduced steroid production was observed as a result of exposure to DMBA (Mandal et al. 2001). The xenobiotic responsive element (XRE) or dioxin responsive element (DRE) are cis-acting enhancer elements located in the promoter region of CYP1B1. The expression of these xenobiotic responsive genes can be regulated via a pathway involving aryl hydrocarbon receptor (AhR). In this pathway, the activated AhR translocates to the nucleus, dimerizes with AhR, and interacts with a second basic helix-loop protein aryl hydrocarbon receptor nuclear translocator (ARNT), which activates gene transcription by its binding to DRE or XRE (Spink et al. 1994; Gonzalez 1989).

Humanized transgenic mice coexpressing tetracycline-controlled transactivator (tTA) and human CYP1B1 (hCYP1B1) have been created by this group (Hwang et al. 2001). The tTA-driven hCYP1B1 level in transgenic males is enhanced by release of doxycycline or testosterone, and an enhanced level is similar with that of castrated transgenic males in the absence of doxycycline or testosterone (Hwang et al. 2003). In this study, DMBA was chosen as an inducer because it has been shown to be important in the bioactivation of CYP1B1. The aim of this study was to examine whether the levels of hCYP1B1 induced by DMBA were similar with those in the livers from castrated transgenic male treated with doxycycline or testosterone. In parallel, the endogenous promoter-linked mCYP1B1 and heterologous tet promoter–linked hCYP1B1 were studied to determine if these are regulated in a different way or same way in response to DMBA. Finally, the DMBA-mediated activation of the hCYP1B1 promoter–driven Luc reporter gene was examined in HepG2 cells.

MATERIALS AND METHODS

Materials

Doxycycline and testosterone propionate were obtained from Sigma (St Louis, MO). DMBA was purchased from Amersham Pharmacia (NJ, USA).

Humanized Transgenic Mice

Humanized doubly transgenic mice were produced by mating the first lineage of the Tet-tTA transgenic lines with the second lineage of the Tet-hCYP1B1 transgenic lines (Hwang et al. 2001). The double-transgenic mice were then crossed with the age-matched control mice for subsequent quantitative production. All the humanized transgenic mice expressing tTA and hCYP1B1 were handled in an accredited Korea Food and Drug Administration (FDA) animal facility, in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International Animal Care Policies (Accredited Unit-Korea Food and Drug Administration: Unit Number 000936), and were maintained in a specific pathogen–free environment. All the mice were housed in cages under a strict light cycle (lights on at 08:00 h and off at 20:00 h), and were given a standard irradiated chow diet (Purina Mills) ad libitum.

Treatments

In order to determine the effects of DMBA (0.4 mg/kg/day), doxycycline (0.4 mg/kg/day), and testosterone propionate (0.4 mg/kg/day), the transgenic males were castrated after being anesthetized with pentobarbital (0.02 mg/kg, intraperitoneally [i.p.]) (Sigma, St Louis, MO), and castrated transgenic males were consecutively injected intraperitoneally into the mice in a 0.2-ml volume diluted in corn oil. These doses for DMBA, doxycycline, and testosterone were based on the reports as described (Hwang et al. 2001, 2003). The control mice were injected with the corn oil vehicle.

RT-PCR

The total RNA was isolated from the livers using RNAzol (Tel-Test; CS104) according to the manufacturer’s instructions. Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed as described (Hwang et al. 2001). hCYP1B1 was synthesized using the sense primer 5′-GGCCA CTGAT CGGAA ACG-3′ and the antisense primer 5′-AGGCG GATCT GGAAA ACGT-3′, with a sequence complementary to hCYP1B1, ranging from 1310 to 1900 nucleotides, used as the DNA template. The sense primer 5′-CTTAG TGCAG ACAGT CCACA G-3′ and the antisense primer 5′-GAAAG CACGC ATCGT GCTAT AG-3′ were used in the synthesis of mCYP1B1.

Real-Time PCR

Real-time PCR analysis was performed using an ABI PRISM Sequence Detection System (ABI-SDS) (Applied Biosystems). After reverse transcription, PCR of each mixture with the synthesized cDNA added to the TaqMan probes and primers was carried out in triplicate. With the exception of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene–specific primers and TaqMan probes, PCR was carried out simultaneously for the GAPDH. The TaqMan probes contain two fluorescent dyes at the 5′-terminal (Reporter, R) and the 3′-terminal (Quencher, Q) for displacing the strand and blocking the extension, respectively. The reaction mixture was then subjected to amplification involving 1 cycle at 50°C for 2 min (annealing stage), 1 cycle at 95°C for 10 min (denaturation stage), 40 cycles at 95°C for 15 s, and 1 cycle at 60°C for 2 min (extension stage). In all cases, an initial denaturation step was carried out at 95°C for 5 min to inhibit the dimerization of the primer under these PCR conditions. The ABI-SDS was programmed to measure the fluorescence of the R and Q fluorescent dye after each cycle at a temperature several degrees (60°C) lower than the melting temperature of the target amplicon. This step at a 60°C was carried out in order to avoid or minimize any potential contributions of primer-dimer formation to the overall fluorescent dye signal. Calibration was constructed by plotting the R/Q ratio against the amounts of hCYP1B1 cDNA synthesized based on the RNA isolated from the livers of the transgenic mice treated with the androgens and antiandrogens and from the livers of the nontransgenic mice used as the control.

Western Blot Analysis

The microsomal proteins were prepared as previously described elsewhere (Hwang et al. 2001, 2003). Briefly, the liver tissue was homogenized in an ice-cold 0.25 M sucrose solution, and the homogenate centrifuged at 10,000 × g for 15 min, followed by 15,000 × g for 15 min. The final supernatant was centrifuged at 27,000 × g for 10 min, and the resulting microsomal pellet was suspended in 1 ml of a 0.25 M sucrose solution. The protein concentration was determined using the bicinchroninic acid (BCA) method using Protein Assay Kit (Pierce, IL, USA). The microsomal proteins (50 μg) were separated in a 10% poly-acrylamide gel for 3 h, and transferred to a nitrocellulose membrane using an electroblot at 40 V, for 4 h. A solution containing 5% powdered nonfat milk, 25 mM Tris (pH 7.5), and 150 mM NaCl was used to block the nonspecific binding sites for 1 h at room temperature. The membrane was incubated with a 1:1000 dilution of the primary anti-human CYP1B1 (GeneTest, MA) in a blocking buffer for 3 h at room temperature. The membranes were then washed in a washing buffer containing 10 mM Tris (pH 8.0), 150 mM NaCl, and 0.05% Tween-20. This was followed by incubation with a secondary antibody, horseradish peroxidase–conjugated goat anti-rabbit immunoglobulin G (IgG) (GenTest, MA), at a 1:1000 dilution for 1 h at room temperature. The anti-human CYP1B1 could also detect the mCYP1B1 from Western blot analysis, when used as recommended by the manufacturer.

Gene Constructions

The phCYP1B1/Luc combining hCYP1B1 promoter and Luc reporter gene were reconstructed by modifying pGEM/hCYP1B1, which was used to construct phCYP1B1/LacZ (Kim et al. 2002). pGEM/hCYP1B1 was then digested with SalI/XbaI, and the resulting hCYP1B1 promoter was then cloned into the SalI/XbaI site in a 5′ to 3′ orientation with respect to the Luc transcription unit in the pGL3-Basic (Clontech).

DNA Transfection

The human HepG2 cells were maintained in a minimum essential medium (MEM) (Eagle; Sigma, St. Louis, USA), and DNA transfection was performed as described (Lee et al. 2003). Each dish was washed with Opti-MEM and exposed to a mixture containing 50 μg lipofectamine (Life Technologies) with 10 μg of DNA (Petri dish), according to the manufacturer’s instructions. After the cells were incubated for 5 h, the mixture of lipofectamine and DNA was removed, and the cultures were incubated with 1 ml of the complete growth medium containing the DMBA for 12 h. The medium was then aspirated, and the cells were washed in phosphate-buffered saline. The harvested cells were collected by centrifugation at 15,000 rpm for 10 min at 4°C. The resulting protein concentration was determined using the bicinchroninic method with a bovine serum albumin (BSA) standard.

Benzyloxyresorufin O-Dealkylase Activity

The benzyloxyresorufin O-debenzylase (BROD) (Sigma, MO) activity was determined by measuring the dealkylation of benzyloxyresorufin (Nerurka et al. 1993), using 0.5 mM benzyloxyresorufin as the substrate. In the metabolism of the CYP enzyme substrate, BROD is relatively specific to hCYP1B1 (Hwang et al. 2001, 2003), although it is also a substrate for CYP2B (Nerurkar et al. 1993). The resorufin fluorescence was recorded with excitation and emission wavelengths of 532 and 586 nm, respectively. The fluorescence was corrected using a blank (incubation mixture) prior to interpretation, and then compared with a resorufin calibration curve. The rate of resorufin formation was defined as the CYP1B1 activity, and expressed in pmol/min/mg protein.

Reporter Gene Assays

Extracts from the transfected cells were centrifuged for 5 min, and the protein concentration was determined using the bicinchroninic acid (BCA) Protein Assay Kit (Pierce). The luciferase enzyme assay (Luciferase Assay System; Promega) was performed by mixing 20 μl of each lysate with 100 μl of a Luciferase Assay Reagent per well. Light was produced as a result of the conversion of chemical energy from luciferin oxidation through an electron transition to form the product molecule, oxyluciferin. Its intensity was measured for 10 s using a Monolight 2010B Lumonometer (Micro Lumat, LB96V; Bertold Technologies). Here, the firefly luciferase catalyzes luciferin oxidation using ATPMg²⁺ as a cosubstrate. The RL/Luc activity was measured in order to normalize the transcription efficiency in each experiment. The luciferase activity of each plate was measured in triplicate for luciferase activity.

Statistical Analysis

In all experiments, tests for significance among the groups were performed using one-way analysis of variance (ANOVA) (SPSS for Windows, release10.01, standard version; Chicago, IL). All the values are reported as a mean ± standard deviation (SD). A p value of <.05 was considered significant. The dose-response results were fitted by a logistic equation using the Table Curve version 1.0 for Windows.

RESULTS

Doxycycline Regulation in the Transgenic Mice

The humanized double-transgenic mice expressing tTA and hCYP1B1 were previously produced by this group (Hwang et al. 2001). In the absence of doxycycline, the minimum base activity of the human cytomegalovirus (CMV) (hCMV) promoter in the livers of this transgenic line resulted in very low levels of the tTA protein. However, in the presence of doxycycline, the production of the tTA protein was blocked by doxycycline from binding to the tet promoter, and the expressions of the hCYP1B1 gene were maintained at low levels. When the doxycycline was removed, small amounts of tTA proteins were bound to the tetracycline-regulated promoter, stimulating the expression of the tTA gene. Higher levels of the tTA protein then stimulated higher hCYP1B1 expression levels (Figure 1A ). Endogenous levels of mCYP1B1 transcript and protein were not present in the nontransgenic mouse liver, but high levels of hCYP1B1 expression were observed in the livers of the transgenic mice upon the removal of doxycycline removal (Figure 1B, C, D ).

DMBA Leads to a Decrease in hCYP1B1 Expression in the Livers of the Castrated Male Mice

Noticeably, the difference in the levels of the mCYP1B1 between noncastrated and castrated nontransgenic males was not observed, but the expression of tTA and hCYP1B1 appears to be most readily enhanced after castrating transgenic males, which is similar level with those in noncastrated doxyclycline-treated group (Hwang et al. 2003). It suggests that testosterone removal induces tTA-driven hCYP1B1 expression in the noncastrated transgenic livers, as in case of doxycycline (Hwang et al. 2003). Because treatment of castrated transgenic males with testosterone causes an inhibition in tTA-driven hCYP1B1, the transgenic males were castrated, and the castrated mice were then used to determine if DMBA (Figure 2A ) is responsible for inhibiting the hCYP1B1 transgene as in case of testosterone or doxycycline removal. To test this, DMBA was injected into the castrated males for 1, 3, and 6 days, and the transcript, protein, and activity levels of the hCYP1B1 in the livers were measured. The hCYP1B1 expression levels began to decrease with increasing amounts of DMBA (Figure 2B, C, D, E ). In the activity assay, the metabolism of the CYP450 enzyme substrate is relatively specific for CYP1B1 (Hwang et al. 2001, 2003). This suggests that the DMBA treatment caused a decrease in hCYP1B1 expression in the castrated transgenic males. Next, a novel strategy was designed to detect an inducible level of DMBA-mediated hCYP1B1 expression after treatment (Figure 2F ). In typical strategy, CYP reporter gene system was used to detect compounds using human cell lines (Hamers et al. 2000: Jones, Anderson, and Tukey 2000). Here, castrated transgenic males were injected for 3 and 9 days after which the injections were stopped for 3 and 9 days. The RNA was then prepared from the liver tissues for real-time PCR and RT-PCR analysis. The hCYP1B1 expression level was inhibited by DMBA, and its transcript level gradually increased during the DMBA release (Figure 2G ). This suggests that the castrated transgenic males were able to use for detecting induced level of tTA-driven hCYP1B1 in response to DMBA.

Testosterone and Doxycycline Also Lead to a Decrease in hCYP1B1 Expression in the Livers of the Castrated Transgenic Males

Treatment of the castrated adult males with DMBA resulted in the decreased expression of hCYP1B1. Therefore, testosterone and doxycycline were administered to the castrated transgenic males in order to determine if they could also inhibit the hCYP1B1 expression as in case of DMBA. The castrated transgenic males were given an intraperitoneal injection of either testosterone or doxycyline for 1, 3, and 6 days, and the transcript and protein levels of the hCYP1B1 were measured using real-time PCR, RT-PCR and Western blot analysis. The hCYP1B1 expression level gradually decreased in a dose-dependent manner (Figure 3). This indicates that the DMBA has a similar inhibitory effect compared to testosterone or doxycycline.

DMBA Leads to an Increase in mCYP1B1 Expression in the Livers of the Castrated Transgenic Males

The hCYP1B1 gene was linked to the tet regulatable promoter, and the mCYP1B1 gene was linked to its own promoter containing the xenobiotic response element (XRE) or the dioxin response element (DRE) (Figure 4A ) (Lee et al. 2003). This observation suggests that mCYP1B1 expression in castrated transgenic livers may be regulated by DMBA. To test this, the castrated transgenic males were treated with DMBA for 1, 2, and 6 days with 0.2 mg/kg DMBA, and the level of the mCYP1B1 transcript was measured by real-time PCR and RT-PCR using the mCYP1B1-specific primers. As shown in Figure 4B, C , the mCYP1B1 expressions level increased gradually after the DMBA treatments. This suggests that DMBA-AhR/ARNT heterodimer activated expression of mCYP1B1 by binding to DRE on promoter. A further study was examined according to identical design (Figure 2F ) to determine if the increased level was gradually decreased after stopping treatment. The transcriptional level of mCYP1B1 gradually decreased during the DMBA release (Figure 4D, E ).

DMBA Leads to Induction of the hCYP1B1/Luc Reporter Gene

XRE or DRE sequence is also contained within the promoter of the hCYP1B1 gene (Figure 5A ) (Lee et al. 2003). The DMBA-mediated promoter activity was examined by fusing the hCYP1B1 promoter to the Luc reporter gene (hCYP1B1/Luc) (Figure 5B ). The HepG2 cells derived from an adult human hepatocarcinoma were transfected with hCYP1B1/Luc and the transfected cells were then treated with between 25 and 100 nM DMBA for 2 to 8 h (100 nM). The levels of the Luc transcript gradually increased in a dose- and time-dependent manner (Figure 5D ). Luc activity level was also increased in a dose-dependent manner (Figure 5E ). This suggests that the hCYP1B1 promoter directs the transcription and activity of the Luc reporter gene by binding of DMBA-AhR/ARNT heterodimer to DRE sequences within the hCYP1B1 promoter.

DISCUSSION

In this study, castrated doubly transgenic males expressing the tetracycline-controlled tTA and hCYP1B1 gene were used to test the hypothesis that DMBA would reduce hCYP1B1 as in case of doxycycline and testosterone. This approach offers a critical insight which may have an impact on developing a strategy for testing PAHs and other environmental toxicants using both mCYP1B1 and hCYP1B1 levels.

We found that DMBA, as well as testosterone and doxycycline, inhibited hCYP1B1 in the castrated transgenic males. It appears that DMBA binds to the doxycyline-binding site on the tTA protein, which is a fusion protein between the Tet repressor DNA-binding domain (207 amino acids) and the VP16 transcriptional activation domain (130 amino acids) of the herpes simplex virus. The DMBA-tTA or testosterone-tTA complex, like the doxycycline-tTA complex, then binds the tet sequences, which brings the VP16 activation domain into close proximity with the minimal CMV promoter, thereby activating the hCYP1B1 gene in the absence of either DMBA or testosterone/doxycycline. This possibility was based on the result from a doxycyline experiment that suppressed the expression of the hCYP1B1 gene (Figure 3 B ), but its mechanism is unknown.

The treatment of castrated transgenic males with DMBA leads to an increase in endogenous mCYP1B1 expression. This shows that DMBA induces mCYP1B1 directly by binding of DMBA-AhR/ARNT complex on the endogenous mCYP1B1 promoter in transgenic mice. This result was supported by a report that PAHs have been shown to induce CYP1B1 expression (Savas et al. 1994; Shen et al. 1994; Sutter et al. 1994) even though it is a relatively weak ligand (Bigelow and Nebert 1982). Although DMBA is not directly linked to the induction of CYP1B1 (Mandal et al. 2001), it is possible that DMBA is involved in the activation of the endogenous mCYP1B1 gene.

This study found that DMBA leads to an induction of expression of the hCYP1B1-driven Luc reporter gene. This finding suggests that DMBA binds activated AhR. In its DMBA-AhR complex, the AhR interacts with a ARNT, and the resultant AhR/ARNT heterodimer binds to core recognition motifs contained within the DRE or XRE (GCGTG) domain on the hCYP1B1 promoter, and stimulates the linked hCYP1B1 or Luc reporter gene. In addition, the hCYP1B1 promoter–driven Luc reporter gene assay system is useful for both identifying and assessing the PAHs-mediated promoter activity in vitro.

Indeed, mammalian, yeast, insect, and bacterial systems have been used to assess the role of CYP in chemical toxicants as in vitro model. As an in vivo animal, the high dosage application of various chemicals has used in homogenous animals under the controlled-conditions for short periods. However, these typical strategies may not meet the requirement of a novel in vivo transgenic model. Regulatable tet promoter linked to a human CYP expressed in transgenic mice provides a means to assess the effects of a single gene product at the basal and inducible levels. It was reported that adult transgenic males are useful for testing antiandrogens including flutamide, di(2-ethylhexyl)phthalate (DEHP), di-n-butyl phthalate (DBP), diethyl phthalate (DEP), Linuron (3-(4-dichlorophenyl)-methoxy-1-methylurea), and 2,4′-DDE (1,1-dichloro-2(p-chlorophenyl)-2-2(o-chlorophenyl)ethylene) (Hwang et al. 2005). An additional use of hCYP1B1 and mCYP1B1 expressions in castrated transgenic males was found in response to DMBA in this study.

In conclusion, DMBA functions as testosterone or doxycycline and when given to castrated transgenic males leads to the distinguishable detections of both basal and inducible levels of endogenous mCYP1B1 and exogenous hCYP1B1 expression after release of DMBA treatment.