ToxSci Advance Access originally published online on October 25, 2006
Toxicological Sciences 2007 95(1):98-107; doi:10.1093/toxsci/kfl144
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Human CYP1A1GFP Expression in Transgenic Mice Serves as a Biomarker for Environmental Toxicant Exposure
Laboratory of Environmental Toxicology, Departments of Chemistry and Biochemistry and Pharmacology, University of California, San Diego, La Jolla, California 92093-0722
1 To whom correspondence should be addressed at University of California, San Diego, Leichtag Biomedical Research Building, Room 211, La Jolla, CA 92093-0722. Fax: (858) 822-0363. E-mail: rtukey{at}ucsd.edu.
Received September 27, 2006; accepted October 23, 2006
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ABSTRACT |
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The human CYP1A1 gene is regulated by the aryl hydrocarbon receptor (AhR), and induction of CYP1A1 is known to play an important role in xenobiotic metabolism. To examine the regulation of human CYP1A1 in vivo, we created a transgenic mouse strain (Tg-CYP1A1GFP) expressing a chimeric gene consisting of the entire human CYP1A1 gene (15 kb) fused with a GFP reporter gene. The treatment of Tg-CYP1A1GFP mice with a single intraperitoneal dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo[a]pyrene (B[a]P) led to the induction of CYP1A1GFP in both the liver and the lung as determined by fluorescence and Western blot analysis. The localization of induced fluorescence in liver also demonstrated the usefulness of cultured hepatocytes in examining the actions of AhR agonists toward induction of CYP1A1GFP. Other routes of B[a]P administration, such as by oral exposure at 100 mg/kg for 3 days, led to reduced induction of CYP1A1GFP in liver and lung. In liver, expression of CYP1A1GFP was a sensitive marker for oral exposure, while mouse CYP1A1 was not induced at these doses. While first pass metabolism of B[a]P in the gastrointestinal tract reduces the potential of the AhR to induce CYP1A1GFP in the liver, adequate concentrations reach the hepatic circulation as demonstrated by induction of human UGT1A proteins in transgenic mice that express the human UGT1 locus. The capability to identify fluorescently labeled CYP1A1 in vivo provides a sensitive measurement of gene response and links exposure to potential environmental toxicants and activation of the AhR.
Key Words: CYPIAI; UGT; TCDD; gene expression; Ah receptor; flourescent.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Exposure of mice to classical aryl hydrocarbon receptor (AhR) ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or any of the family of polycyclic aromatic hyrdrocarbons like benzo[a]pyrene (B[a]P), results in activation of the AhR (Okino et al., 1992
; Tukey et al., 1982) and induction of selective target genes such as those represented by the family of CYP1 proteins (Nelson et al., 1996). The transcriptional mechanism of CYP1 induction begins with ligand binding to the AhR and culminates in translocation to the nucleus where the AhR dimerizes with the aryl hydrocarbon receptor nuclear translocator (Arnt) (Hoffman et al., 1991; Reyes et al., 1992). The AhR/Arnt complex binds to highly conserved DNA-enhancer sequences known as xenobiotic response elements (Fujisawa-Sehara et al., 1986; Gonzalez and Nebert, 1985), resulting in the propagation of the induction signal to the promoter region, alteration of chromatin structure (Okino and Whitlock, 1995; Wu and Whitlock, 1992) and subsequent enhanced gene expression.
Induction of the CYP1A family of proteins, in particular CYP1A1, is a hallmark of exposure to combustion products and other environmental contaminants that activate the AhR. Cell-based assays expressing portions of the CYP1A1-promoter region (Anderson et al., 1995; Postlind et al., 1993) or dioxin-responsive elements (El-Fouly et al., 1995; Nagy et al., 2002; Yueh et al., 2005) fused to reporter genes have been used to screen for potential AhR ligands. These cell-based assays are sensitive, and for the most part can be used to identify agonists/antagonists capable of interacting with the AhR. To complement this approach, transgenic mice expressing reporter genes driven by AhR-enhancer elements have also proven to be useful in equating gene expression patterns when mice have been exposed to AhR ligands (Campbell et al., 1996; Galijatovic et al., 2004). Based upon the route of administration, induction of reporter gene activity in vivo not only is the end result of AhR activation but also reflects the contribution of circulating and activated humoral factors toward those processes involved in gene expression. The only potential drawback with the use of reporter gene assays in vivo is that they monitor transcriptional activity that is based upon the steady-state levels of the selected reporter that is being analyzed, and not functional protein.
As an additional tool to examine the regulation of the human CYP1A1 gene, we have generated a transgenic mouse line expressing the human CYP1A1 gene fused in frame to a GFP reporter gene inserted at the last codon located in exon 7 (Tg-CYP1A1GFP). This transgenic model serves as a biological tool to explore the mechanisms associated with the regulation and expression of the human CYP1A1 gene in vivo as well as in cultured cells by measuring GFP fluorescence. Since the measurement of induction results from the cellular accumulation of the human CYP1A1 protein, induction is based upon transcriptional response and the steady-state balance of protein accumulation. These mice allow for the visualization of spatial and temporal induction of human CYP1A1GFP, which can be used as a biomarker for environmental toxicant exposure to substances that serve as AhR ligands.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Reagents.
TCDD was purchased from Wellington Laboratories. B[a]P, 3-methylcholanthrene, ß-naphthoflavone (ßNF), and an anti-ß-actin antibody were purchased from Sigma Aldrich, ST Louis, MO. Human HepG2 cells were purchased from American Type Tissue Culture (http://www.atcc.org/) and the hepa1c1c7 cells were provided by Dr Oliver Hankinson University of California Los Angeles. The antihuman CYP1A1 antibody (Soucek et al., 1995
) was kindly provided by Dr Fred Guengerich (Vanderbilt University) and the antihuman UGT1A1, UGT1A4, and UGT1A6 antibodies (Chen et al., 2005; Ritter et al., 1999) from Dr Joseph Ritter (Virginia Commonwealth University). All other products are referenced by the company name and the information can be obtained by an Internet search.
Generation of the CYP1A1GFP transgenic mice.
The human CYP1A1 gene was cloned previously from a human liver genomic library constructed in the replacement vector 
-EMBL-3 (McManus et al., 1990) and the 15-kb insert removed by SalI digestion followed by cloning into pBluescript II (Stratagene, la Jolla, CA). A selective mutation was made at the stop codon located in exon 7 and the gene subcloned into the pEGFPN2 vector (Invitrogen, Carlsbad, CA), generating a chimeric gene that encoded the full-length CYP1A1 gene fused in frame with the GFP reporter gene (Fig. 1). The CYP1A1GFP plasmid DNA was purified from Escherichia coli using Qiagen Plasmid Purification columns (Qiagen, Valencia, CA) followed by linearization with SalI digest. The linearized DNA was microinjected into fertilized CB6F1 mouse eggs and transplanted into the oviduct of pseudopregnant C57BL/6N mice for the production of transgenic mice. All procedures for the generation of the transgenic mice were carried out by the University of California, San Diego (UCSD) Superfund Transgenic Core Facility. For genotype analysis, DNA from tail clippings was screened by PCR using specific GFP sense 5'-cacatgaagcagcacgactt-3' and antisense 5'-tgctcaggtagtggttgtcg-3' primers. For PCR analysis, DNA was amplified in a 50-µl reaction containing 25 µl of Qiagen Hotstart Master Mix, 5 ng of template DNA, and 0.4µM concentration of each primer. A 15-min activation of the polymerase was carried out at 95°C followed by 30 cycles from 95°C for 30 s to 58°C for 30 s and 72°C for 45 s followed by final extension at 72°C for 7 min. PCR reactions were carried out in a PerkinElmer Life Sciences GeneAmp DNA thermocycler PCR system. From 13 litters and 57 offspring, 12 founders were CYP1A1GFP positive and five of the founders were bred to produce F1 offspring. Three out of the five founders had F1 offspring that produced inducible hepatic CYP1A1GFP protein following a single intraperitoneal (ip) dose of 16 µg/kg TCDD.
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Animal studies.
Animal studies were performed in compliance with the rules and regulations for the care and use of laboratory animals from the National Institutes of Health. All animals were 8–12 weeks of age and fed a standard chow diet. TCDD was dissolved in DMSO and given as a single ip dose of 16 µg/kg. B[a]P was dissolved in DMSO:corn oil (50:50 vol/vol) and was given as a single ip dose of 100 mg/kg or 100 mg/kg as an oral dose (po).
Generation of G418-resistant CYP1A1GFP cells.
HepG2 and hepa1c1c7 cells were cultured as previously described (Chen et al., 2003) and transfected with the CYP1A1GFP expression plasmid and after 48 h were trypsinized and plated at 1/10 volume. The cells were then exposed to media containing 0.8 mg/ml G418 (Invitrogen). After approximately 2–3 weeks, individual colonies were selected and recultured with continued exposure to G418. A number of the expanded colonies were treated with 5nM TCDD for 24 h followed by analysis of induced GFP. In addition, cell populations expressing CYP1A1GFP was also calibrated by flow cytometry (Chen et al., 2003). Cells were collected after 24 h following trypsin treatment, washed twice in DPBS (Gibco, Carlsbad, CA) and suspended to a concentration of 1 x 106 cells/ml. The suspended cells were acquired on a FACS Calibur flow cytometer and analyzed using CELLQuest software. Conditions for GFP detection were established by setting the excitation maximum at 488 nm, and the emission maximum at 507 nm.
Microsomal protein isolation.
Tissues isolated from three mice per treatment group were combined and frozen in liquid nitrogen. Samples were pulverized using a porcelain mortar and pestle under liquid nitrogen. Five volumes of ice-cold 1.15% KCl was added to the pulverized tissue and homogenized using a motorized glass-teflon homogenizer. The tissue homogenate was first centrifuged at 3000 x g for 10 min at 4°C. The supernatant was removed and then centrifuged at 10,000 x g for 10 min at 4°C and the resulting supernatant then centrifuged at 100,000 x g for 60 min at 4°C. The pellet was resuspened in 20% glycerol, 100mM potassium phosphate buffer, pH 7.4 containing 10mM EDTA and 1mM PMSF.
Western blot analysis.
All Western blots were performed using NuPAGE Bis-Tris polyacrylamide gels as outlined by the supplier (Invitrogen). Protein was heated at 70°C for 10 min in loading buffer and resolved in 4–12% Bis-Tris gels under denaturing conditions (50mM MOPS, 50mM Tris-base, pH 7.7, 0.1% SDS, 1mM EDTA). The resolved protein was transferred onto nitrocellulose membrane using a semidry transfer system. The membrane was blocked with 5% nonfat dry milk in 10mM Tris-HCl, pH 8, 0.15M NaCl, and 0.05% Tween 20 (Tris-buffered saline) for 1 h at room temperature. The membrane was washed in the Tris-buffered saline solution and incubated with primary antibodies in Tris-buffered saline, shaking at 4°C overnight. Membranes were then washed five times with Tris-buffered saline solution and incubated with horseradish peroxidase–conjugated secondary antibodies in Tris-buffered saline solution with 2% nonfat milk for 1 h at room temperature. Membranes were then washed five times with Tris-buffered saline solution and visualized using chemiluminscent reagents according to the manufacturer's instructions (PerkinElmer, Wellesley, MA) followed by exposure to X-ray film.
Isolation and treatment of transgenic mouse primary hepatocytes.
Primary hepatocytes were isolated from 8- to 12-week-old mice as previously described (Chen et al., 2005). Mice were anesthetized by isoflurane inhalation. The portal vein was cannulated and perfused with Hank's Balanced Salt Solution (Ca2+ free and Mg2+ free) containing 0.1mM EGTA and 10mM HEPES at pH 7.4 for 5 min at a flow rate of 7 ml/min. The anterior vena cava was cut immediately after the perfusion began to allow for continuous flow of the solution out of the liver. After the 5-min initial perfusion, the perfusate was changed to 20 µg/ml Liberase Blendzyme (Roche Applied Science, Indianapolis, IN) in Hank's Balanced Salt Solution (with Ca2+ and Mg2+) for 5 min at a flow rate of 7 ml/min. The liver was removed and placed in low-glucose DMEM media, and the hepatocytes were isolated by mechanical dissection followed by filtration through a sterile 70-µm filter. The cells were collected by centrifugation at 50 x g for 30 s and washed with low-glucose DMEM media. The hepatocytes were cultured in six-well collagen-treated plates (Discovery Labware) in 3 ml of low-glucose DMEM media containing penicillin/streptomycin and supplemented with 10% fetal bovine serum. Three hours after plating the media was removed and replaced with fresh media and the hepatocytes used for subsequent experiments after a 24-h recovery period. The hepatocytes were treated with various chemicals for 48 h, and the media containing the chemical compounds refreshed after 24 h. For Western blot analysis, hepatocytes were collected and lysed in a buffer containing 0.05M Tris-HCl, pH 7.4, 0.15M NaCl, 0.25% deoxycholic acid, and 1% Nonidet P-40 with a compliment of protease and phosphatase cocktail inhibitors (Sigma Aldrich). After incubation in lysis buffer for 1 h on ice, the supernatant was collected after centrifugation for 20 min in a refrigerated Eppendorf centrifuge at 16,000 x g, and used directly for Western blot analysis.
Tissue section fluorescence microscopy.
Transgenic mice were anesthetized with the general anesthetic, nembutol. The mice were then intracardially perfused with Ringer's solution at a rate of 4 ml/min until the liver was clear of blood, followed by perfusion with 4% paraformaldehyde in PBS. The organs were then removed and placed in 4% paraformaldehyde on ice for 2 h to allow for complete fixation. Each organ was sectioned using a vibratome at 100 µm thickness. Tissue sections were washed 3x with PBS and mounted on glass slides with gelvatol (antifade medium). The slides were stored at 4°C until imaged. Samples were visualized by confocal microscopy using an MRC-1024 system (Bio Rad Laboratories, Hercules, CA) attached to an Axiovert 35M (Zeiss AG) and a 40x NA objective. Excitation illumination was with 488 nm light from a krypton/argon laser. Individual images (1024 x 1024 pixels) were converted to PICT format and merged as pseudo-color RGB images using Adobe Photoshop.
Fluorescence microscopy.
Primary hepatocytes were isolated and cultured on collagen-treated plates as previously described. Hepatocytes were treated with the following CYP1A1 inducers: TCDD at 0.1, 1, and 10nM, B[a]P, 3-methylcholanthrene, and ßNF at 0.1, 1, and 10µM. Hepatocytes were treated for 48 h and media changed every 24 h with fresh compound. Cells were then imaged using the Axiovert 200 microscope (Zeiss, Ismaning, Germany) with Endow GFP Bandpass filter set.
Measuring tissue fluorescence in microsomal preparations.
Fluorescence of 50 µg of microsomal protein from various organs was measured using a Tecan Safire fluorescence plate reader (Tecan, San Jose, CA) in a 96-well UV plate (Costar, Corning, NY). CYP1A1GFP was excited at 488 nm and emission intensity was measured at 525 nm using an excitation and emission slit widths of 5 nm in a top read mode at room temperature.
Measurement of 7-ethoxyresorfin O-deethylase activity.
A marker of CYP1A1/CYP1A2 activity, 7-ethoxyresorufin O-deethylase (EROD) activity, was measured using 20 µg of liver microsomes containing 100mM potassium phosphate buffer (pH 7.4), 5mM MgCl2, 8µM 7-ethoxyresorufin and 1mM NADPH. Reactions were incubated for 15 min at 37°C and then terminated by the addition of 100 µl cold methanol. The formation of resorufin was measured fluorometrically with an excitation at 530 nm and emission at 590 nm and normalized to a resorufin standard.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Characterization of CYP1A1GFP Tissue Expression in Tg-CYP1A1GFP Mice
We have previously demonstrated that the human CYP1A1 gene could be regulated in CYP1A1N transgenic mice by the identification of TCDD-induced human CYP1A1 in tissues such as liver and lung (Galijatovic et al., 2004
), but not small intestine. In attempts to design a CYP1A1 marker protein that could be identified by fluorescence, a CYP1A1-GFP chimeric gene (CYP1A1GFP) was first constructed with the stop codon in exon 7 interrupted and replaced in frame with the GFP gene (Fig. 1). This new construct contains 9 kb of regulatory sequence, and when stably integrated into human HepG2 or mouse hepa1c1c7 cells produced a TCDD-inducible CYP1A1GFP fusion protein that could be detected with either an antihuman CYP1A1 antibody or an anti-GFP antibody (Fig. 2). The anti-CYP1A1 antibody recognizes mouse and human CYP1A1, which can be seen to have slightly different mobilities, as previously shown (Galijatovic et al., 2004). The increase in CYP1A1GFP was also represented in the HepG2 cells following a 24-h exposure to TCDD as demonstrated by separation of those cells eliciting fluorescence by flow cytometry (Fig. 2).
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With expression of CYP1A1GFP demonstrated in tissue culture, the CYP1A1GFP gene was used to generate transgenic mice (Tg-CYP1A1GFP). Following the selection of a transgenic founder line that transmitted the gene, the Tg-CYP1A1GFP mice were treated with a single ip dose of TCDD and expression of CYP1A1GFP monitored in microsomes from different tissues. The inducibility of CYP1A1GFP was first demonstrated in whole liver when a transgenic mouse was treated with TCDD and the liver exposed and imaged under blue light optics (Fig. 3A). The entire liver was fluorescent green in contrast to the liver from the nontreated transgenic mouse. Isolation of microsomes from TCDD-treated mice followed by fluorometric analysis confirmed a four- to six-fold increase in liver fluorescence over DMSO-treated Tg-CYP1A1GFP mice (Fig. 3B). A two-fold increase in CYP1A1GFP fluorescence was observed in lung microsomes, while no detectable induction of fluorescence was seen in kidney, small intestine, large intestine, brain, or spleen.
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When Western blots were conducted, TCDD injection led to the prominent induction of mouse CYP1A1 (mCYP1A1) (Fig. 4). In addition, the anti-CYP1A1 antibody detected induction of an 85-kDa protein that was also identified with the anti-GFP antibody, confirming that this induced protein represented the CYP1A1GFP fusion protein. TCDD-induced mCYP1A1 and CYP1A1GFP were also detected in microsomes from lung tissue.
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Fluorescence Analysis of Tissue Sections of Liver and Lung
The Western blot and fluorometry data indicated that CYP1A1GFP was inducible in both the liver and the lung. Tissue sections were then performed on these tissues to determine the cellular localization of the GFP fluorescence. Following injection of a single ip dose of TCDD (16 µg/kg) for 24 h, the mice were whole body perfused and fixed with 4% formaldehyde. Tissues were sectioned at 100 µm thickness and imaged using confocal microscopy with excitation at 488 nm with a krypton/argon laser. Very little background fluorescence was observed in liver and lung tissue from Tg-CYP1A1GFP mice that were treated with DMSO (Figs. 5A and 5C). In mice that were treated with TCDD, abundant GFP fluorescence was observed in liver hepatocytes, signifying that activation of the AhR led to adequate expression of CYP1A1GFP (Fig. 5B). When lung tissue was examined, primary fluorescence was noted in a population of cuboidal-shaped cells, signifying that induction in the lung is restricted to a selective cell type that morphologically resembles Clara cells (Fig. 5D).
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CYP1A1GFP Expression in Primary Hepatocytes
Since TCDD treatment demonstrated induction of CYP1A1GFP in liver tissue, experiments were conducted to examine the potential for induction in primary hepatocytes. To initiate these experiments, primary hepatocytes were isolated from Tg-CYP1A1GFP mice and cultured on collagen-coated tissue culture plates. The cells were treated with 10nM TCDD for 8, 24, and 48 h and fluorescence images were captured following each treatment with a Zeiss inverted microscope using a GFP filter set. As shown in Figure 6, DMSO-treated hepatocytes exhibited very little background fluorescence. At 8 h of TCDD exposure, slight GFP fluorescence was observed compared to the DMSO-treated hepatocytes. At 24 h, the GFP signal increased and maximum GFP fluorescence was observed following 48 h of exposure to TCDD.
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To determine the sensitivity of CYP1A1GFP induction with other AhR agonists, primary hepatocytes were isolated from Tg-CYP1A1GFP mice and the cells treated with agents known to activate the AhR. After 48 h of exposure, the cells were collected, resuspended in PBS, and fluorescence was measured using a 96-well fluorometer (Fig. 7A). TCDD induced maximal fluorescence at 0.1nM (lower concentrations were not tested), while B[a]P and 3-methylcholanthrene (3MC) induced fluorescence at 1µM and ß-Naphthoflavone (ßNF) facilitated induction at 10µM. The induced fluorescence profiles matched induction of CYP1A1GFP as determined by Western blot analysis (Fig. 7B). In addition, the induction of CYP1A1GFP with each of the AhR agonists was paralleled by increases in mCYP1A1. These results confirm that Tg-CYP1A1GFP hepatocytes can be used to examine the potential of chemicals and xenobiotics to induce human CYP1A1.
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Induction and Function of CYP1A1GFP following Oral Administration of B[a]P
Following the ip injection of B[a]P into Tg-CYP1A1GFP mice, induction of CYP1A1GFP was apparent by detection of the fusion protein by Western blot analysis as well as fluorescence in microsomal preparations. However, the ip administration of drugs and other xenobiotics is not representative of normal routes of exposure. To examine the effects of oral B[a]P administration on CYP1A1GFP induction, Tg-CYP1A1GFP mice received B[a]P (100 mg/kg) every day for 3 days and tissues were collected 24 h after the last dose. Western blots using liver and lung microsomal preparations demonstrated induction of CYP1A1GFP in these tissues as indicated with the anti-GFP and anti-CYP1A1 antibody (Fig. 8). It was interesting to note that minimal induction of mCYP1A1 was observed in liver microsomes after oral administration of B[a]P although induction was observed in lung. Since mCYP1A1 was not detected in liver at an oral dose of 100 mg/kg, the appearance of CYP1A1GFP at this dose leads us to speculate that the induction of fluorescence in these mice may be a more sensitive indicator of AhR activation than induction of mCYP1A1.
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The preferential induction of liver CYP1A1GFP by oral B[a]P administration led us to examine the contribution of induced CYP1A1GFP toward EROD activity, a relatively specific microsomal monooxygenase activity that has been attributed to expressed CYP1A1/1A2 (Fisher et al., 1992
; Guo et al., 1994; Sandhu et al., 1994). When we measured liver microsomal EROD activity from wild type (WT) litter mates treated orally with B[a]P, there was a two- to three-fold induction of activity in microsomes (Table 1). Since we were unable to detect by immunoblot-induced mCYP1A1 (Fig. 8), this activity may result from mild induction of a faster mobility band observed by Western blot analysis (Fig. 8, lane 4), which may correspond to mCYP1A2. However, when EROD activity was measured in liver microsomes from Tg-CYP1A1GFP mice given B[a]P by oral administration, EROD activity was induced 8- to 10-fold over control (Table 1), and was comparable to those values measured following ip treatment with B[a]P. The contrasting differences in induced EROD activity between WT and Tg-CYP1A1GFP mice suggest that induction of CYP1A1GFP by oral B[a]P administration is responsible for the majority of the induced EROD activity in transgenic mice, confirming that the fusion protein is functional.
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As another means to examine the potential of oral B[a]P treatment to induce other xenobiotic metabolizing proteins, we examined the expression of several human UGT1A proteins in transgenic mice (Tg-UGT1) that express the human UGT1 locus. In Tg-UGT1 mice, functional expression of all nine human UGTs have been demonstrated, with expression of the genes targeted by activation of xenobiotic receptors such as the pregnane X-receptor, the liver X-receptor alpha, and the AhR (Chen et al., 2005
; Verreault et al., 2006). In previous studies (Chen et al., 2005), the UGT1A proteins have been shown to be expressed and induced in the GI tract following treatment with TCDD. When Tg-UGT1 mice were treated orally with B[a]P for 3 days, Western blot analysis using microsomes from the small intestine confirmed the induction of human UGT1A1, UGT1A4, and UGT1A6 (Fig. 9). Interestingly, UGT1A1, UGT1A4, and UGT1A6 were also induced in liver, indicating that adequate concentrations of B[a]P were being absorbed through the portal circulation to the liver resulting in activation of the AhR. These results indicate that oral B[a]P treatment is sufficient to induce human CYP1A1 and UGT1A gene expression in liver, but has limited potential to induce mCYP1A1 in liver.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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The development of transgenic mouse strains that express human genes provides a novel opportunity to examine the regulatory properties of the genes but also the functional role of the regulated gene product. The current study demonstrates that the entire human CYP1A1 gene spliced to a reporter GFP gene produces a functional and inducible CYP1A1GFP fusion protein in transgenic mice. Isolation of primary hepatocytes from Tg-CYP1A1GFP mice provides a sensitive biological tool to quantitate CYP1A1 gene induction through the simple and rapid measurement of induced GFP fluorescence. Following the ip treatment of Tg-CYP1A1GFP mice with TCDD, induced fluorescence was observed primarily in liver and lung tissue. This corresponded with the induction of human CYP1A1 as previously demonstrated in transgenic mice that express only the human CYP1A1 gene (Galijatovic et al., 2004
). Similar induction patterns were seen when mice were treated by oral administration with B[a]P, confirming that absorption of AhR ligands into circulation through the oral cavity serves as an efficient mechanism to examine induction of the CYP1A1 gene in these tissues. In addition, CYP1A1GFP fluorescence was not observed in the gastrointestinal tract or kidney, similar to the expression patterns of the human CYP1A1 gene and CYP1A1-luciferase gene in transgenic mice expressing these DNA constructs (Galijatovic et al., 2004). Thus, consistency in the induction pattern of the human CYP1A1 genes in different transgenic strains is corroborated.
One of the unique aspects of undertaking regulatory gene experiments in transgenic mice is the ability to examine expression patterns in different tissues following routes of administration that contrast those to classical treatment regimes such as the administration of xenobiotics by the oral route. When expression experiments were conducted following oral administration of B[a]P, liver CYP1A1GFP levels were slightly less abundant than CYP1A1GFP levels following ip administration. Interestingly, the induced human CYP1A1GFP by oral administration in liver was in contrast to undetectable levels of mCYP1A1 induction. The lack of expression that we have observed at oral doses of 100 mg/kg is consistent with the minimal induction of liver mCYP1A1 following consecutive oral doses of 125 mg/kg (Uno et al., 2006). Thus, in Tg-CYP1A1GFP mice, these findings indicate that the induction of mouse and human CYP1A1 genes in liver may result from differential sensitivity of these genes to activated AhR. In support of these findings, induced EROD activity in WT mice were minimal following oral treatment with B[a]P. Under similar treatment conditions, EROD activity was induced 8- to 10-fold in liver microsomes from Tg-CYP1A1GFP mice. This compared favorably to the four- to six-fold induction of CYP1A1GFP-induced fluorescence. This finding suggests that the human CYP1A1GFP gene was more sensitive to AhR activation than the rodent Cyp1a1/Cyp1a2 genes. However the increased sensitivity of the CYP1A1GFP gene toward response may reflect a gene dosage phenomenon, indicative of the transgene being integrated into the mouse genome in multiple copies. Regardless, sufficient concentrations of B[a]P reach the liver through the portal circulation, a finding which was also confirmed by induction of the AhR-dependent family of human UGT1A genes in Tg-UGT1 mice. The reduced induction of mCYP1A1 and CYP1A1GFP following oral administration of B[a]P may in part be a reflection of extensive metabolism in the gastrointestinal tract.
As a tool to monitor CYP1A1 induction, primary hepatocytes cultured from Tg-CYP1A1GPF mice can also be cultured and the induction of fluorescence was used as a tool to identify agents that lead to the activation of the AhR. Measurement of fluorescence following exposure to several AhR agonists correlated with the induction of mCYP1A1, confirming that the increase in CYP1A1GFP was commensurate with the regulation of the Cyp1a1 gene. Primary hepatocytes from Tg-CYP1A1GFP mice also allow for the real-time monitoring of CYP1A1GFP protein induction and turnover after toxicant exposure, a property that may be useful to assess persistent biological induction of CYP1A1. The treatment of hepatocytes isolated from transgenic mice with TCDD demonstrated that concentrations as low as 10–11M elicited maximal fluorescence. These values are equal in sensitivity to those reported for induction of reporter gene activities constructed with XRE- or CYP1A1-promoter elements (Anderson et al., 1995; Nagy et al., 2002; Postlind et al., 1993), and provide an additional tool for the detection and measurement of potent AhR ligands and environmental toxicants. The fold induction of CYP1A1GFP fluorescence observed in liver and primary hepatocytes was comparable to those values measured by GFP fluorescence using XRE promoter–driven reporter gene activities in mouse hepatoma cells (Nagy et al., 2002). However, a significant difference is noted in the fold induction between CYP1A1GFP fluorescence in primary hepatocytes and liver and those values noted in these same tissues by expression of luciferase activity in a transgenic CYP1A1 promoter–driven animal model (Galijatovic et al., 2004). The lower CYP1A1GFP response (100-fold) may be attributed to differences in the steady-state accumulation of the CYP1A1GFP protein when compared to those of the promoter-driven luciferase accumulation. Alternatively, these differences may be a reflection of the inherent sensitivity between the measurement of luciferase-generated light output and GFP-generated fluorescence. Yet, measurement of CYP1A1GFP induction (four- to sixfold) as measured by fluorescence closely resembles the increases in mCYP1A1-induced EROD activity (eightfold) in Tg-CYP1A1GFP mice (Table 1). Thus, coupled with the ease of measuring fluorescence in liver tissue, CYP1A1GFP expression following induction would appear to be a good indicator in mice of those events that lead to environmental toxicant exposure and activation of the AhR.
Although other strains of transgenic mice that express CYP1A1 reporter gene constructs have been developed (Campbell et al., 1996; Galijatovic et al., 2004), expression of CYP1A1GFP in liver and lung provides the opportunity to link gene expression with CYP1A1 protein production, as monitored by fluorescence. Expression of CYP1A1GFP in tissues such as the liver and the lung can be used as a biomarker of exposure to agents that regulate the AhR in vivo. Unlike tissue culture, the physiological impact of the in vivo environment may dramatically influence expression patterns. An example of this stems from observations that were made in mice following treatment with phorbol esters and TCDD. While TCDD induced Cyp1a1/Cyp1a2, the co-administration of phorbol esters and TCDD completely blocked the actions of TCDD on Cyp1 gene response (Okino et al., 1992). However, when TCDD and phorbol esters were added to cultures of human HepG2 cells, the actions of TCDD on human CYP1A1 gene expression were enhanced (Chen and Tukey, 1996). The differences in CYP1 gene expression between the tissue culture experiments and those from in vivo experiments may be attributed to unknown humoral factors that play an important role in gene control. Thus, examining expression of CYP1A1GFP in vivo takes advantage of the regulatory and signaling processes engaged in the control of the AhR, in addition to potential humoral processes that may be associated with the activation of the human CYP1A1 gene.
In efforts to examine the impact of environmental toxicant exposure on activation of the human AhR in vivo, a humanized animal model had recently been developed by homologous recombination of a human AhR cDNA downstream of the mouse AhR promoter (Moriguchi et al., 2003). Integration of the human cDNA allowed for expression of only the human AhR. Results indicated that the humanized AhR mice when compared to WT mice were less responsive to the induction of AhR-directed target genes, a finding that implicates the human AhR as a weaker sensor of known AhR ligands. Since our findings indicate that the human CYP1A1 gene may be a more sensitive predictor of activated AhR than the mCyp1a1 gene, future experiments may allow us to measure human CYP1A1 gene expression in the context of the humanized AhR animal model providing a more accurate prediction of the impact of human toxicant exposure to humans.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Supplementary data showing figures in color are available online at http://toxsci.oxfordjournals.org/.
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ACKNOWLEDGMENTS |
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This work was supported in part by grant GM49135 and a Superfund Basic Research Program Grant ES10337 from the NIH. The authors appreciate the technical assistant provided by Dr Mason Mackey at the UCSD Superfund Imaging Core for help with confocal microscopy, as well as the staff at the Superfund Mouse Transgenic Core for generation of the Tg-CYP1A1GFP mice.
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REFERENCES |
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