* Xenogen Corporation, 860 Atlantic Avenue, Alameda, California 94501, and Centre d'Investigacions en Bioquímica i Biologia Molecular (CIBBIM), Hospital Universitari Vall d'Hebron, Barcelona, Spain
Received December 4, 2003; accepted March 1, 2004
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ABSTRACT |
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Key Words: kidney androgen-regulated protein; bioluminescent imaging; in vivo.
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INTRODUCTION |
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The goals of the studies described in this report were first to characterize the expression pattern and hormonal regulation of the Kap-luc transgene and second, to develop a rapid, in vivo model to assess potential endocrine disruption properties of chemicals acting as androgens or anti-androgens.
Biophotonic images were acquired from male and female mice. The bioluminescence detected in vivo was confirmed by euthanizing the animals and removing a variety of tissues for in vitro evaluation. Tissue homogenates were used to perform luminometer assays to assess luciferase expression and Western blot analyses were used to assess endogenous Kap protein levels. The effects of testosterone were evaluated using castrated adult male mice. These mice were imaged before and after castration. Fourteen days following castration, exogenous testosterone was supplied to the mice and the changes in Kap-luc were monitored by biophotonic imaging. The effects of testosterone were also evaluated using intact adult female mice. Female mice were implanted with testosterone time release pellets and monitored by biophotonic imaging for several days, mice were then coadministered either an anti-androgen (cyproterone or vehicle) and biophotonic images were acquired for several more days to assess changes in the Kap-luc transgene expression.
In order to assess the potential to use these transgenic mice and the imaging system to detect hormonal disruption, a 15-day screening assay described by O'Connor et al. (2002) was used as the model system for our anti-androgen evaluation. Two anti-androgens were selected for study, the steroidal anti-androgen cyproterone acetate (Lakshman and Isaac, 1973
; Neri, 1976
) and the nonsteroidal anti-androgen flutamide (Neri, 1976
). Intact male mice were treated with daily doses of the two anti-androgens. Biophotonic images were taken throughout the course of the study. At the conclusion of the study, the mice were euthanized, weighed and various organs were removed, weighed and prepared for in vitro assays. Tissue homogenates were used to perform luminometer assays to assess luciferase expression and Western blot analysis as used to assess endogenous KAP protein levels.
Our results suggest that this model may provide a rapid, noninvasive method to monitor the effect of androgens and anti-androgens on Kap-luc expression and may be useful in testing compounds for endocrine disruption effects.
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MATERIALS AND METHODS |
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In studies reported here, hemizygous mice of greater than eight weeks of age were used. All experimental protocols were approved by the Institutional Animal Care and Use Committee and conform to the ILAR guide for the care and used of laboratory animals (ILAR, 1996).
Chemicals.
Cyproterone, flutamide, testosterone proprionate, and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical Company (St. Louis, MO). D-Luciferin potassium salt was obtained from Biosynth AG (Staad, Switzerland). Testosterone or placebo containing pellet (21-day time release pellets) with 5 mg testosterone/pellet were obtained from Innovative Research of America (Sarasota, FL).
In vivo imaging.
FVB/N-Tg(Kap-luc)Xen animals were injected ip with luciferin (150 mg/kg) 10 min before imaging, anesthetized (13% isoflurane) and placed into a light-tight camera box. Mice were imaged for 2 min from the dorsal and ventral sides at high-resolution settings with a field of view of 20 cm. The light emitted from the transgene was detected by the IVIS Imaging System 100 Series (Xenogen Corporation, Alameda, CA), digitized and displayed on a monitor. The Living Image software (Xenogen Corporation, Alameda, CA) takes the data from the camera and displays the information using a pseudocolor scale with colors representing variations of signal intensity. Signals were quantitated and archived using the Living Image software. Photons of light were quantitated from the kidneys from the dorsal images using oval regions of interest (ROI) 3.38 x 3.53 cm centered over the kidneys. Reproductive organs/lower abdominal luciferase signals were quantitated from oval regions of interest 3.38 x 3.53 cm placed over the lower abdominal area with one edge of the ROI located at the base of the tail.
Absolute intensity calibration of the IVIS Imaging System 100 Series was accomplished utilizing a calibrated 8-inch integrating sphere (OL Series 425 Variable Low-Light-Level Calibration Standard, Optronic Laboratories, Inc.). By imaging and measuring a region of interest on the integrating sphere, counts detected by the CCD camera digitizer can by converted to physical units of radiance in photons/s/cm2/steradian as described by Rice et al. (2001). Background signals are evaluated daily and the Living Image software performs background subtraction calculations.
Assessment of hormonal effects in male and female mice.
All animals used in the experiments represented in these studies were age matched for each study but ranged from 8 to 14 weeks of age. The differences in steady state transgene expression were determined by imaging intact adult male (n = 15) and female (n = 12) mice
In order to determine if steroid hormones regulate the Kap-luc transgene, male (n = 6) mice were castrated and bioluminescent images were acquired before and at intervals following castration. Fourteen days after castration, the mice were divided into two groups (n = 3) and received either testosterone (5 mg/pellet) or placebo 21-day time release pellets. Images were taken at intervals for another 14 days.
Other groups of intact male mice were treated with anti-androgens. Dose response data was generated using adult male mice given daily sc injections of vehicle (DMSO), cyproterone (10, 50, and 100 mg/kg/day), or flutamide (50 and 100mg/kg/day) for seven days (n = 12 for DMSO, n = 5 for 10, 50, and 100 mg/kg/day cyproterone and n = 5 for 50 and 100 mg/kg/day flutamide). Bioluminescent images were acquired on each of the study days.
Each of the compounds, cyproterone acetate and flutamide, were evaluated with concurrent control groups in individual studies. All drugs were dissolved in DMSO as a vehicle and were administered by sc injection into the nape of the neck in a total volume of 50 µl per day.
In the two-week studies, mice were imaged from the dorsal and ventral sides before treatment and at various intervals throughout the treatment period. Cyproterone and flutamide were administered at doses of 50 and 100 mg/kg/day respectively for 16 days (vehicle control [DMSO], n = 8; 50 and 100 mg/kg flutamide, n = 5; 50 mg/kg cyproterone, n = 3; 100 mg/kg cyproterone, n = 5) based on experimental design parameters outlined by O'Connor et al. (2002). Animals were imaged several times each week for the duration of the study. At the conclusion of the study (Day 17) animals were imaged, weighed, and humanely euthanized by cervical dislocation under anesthesia. The liver, kidneys, testes, epididymides, seminal vesicles, and preputial glands were removed and weighed, and relative organ weights (organ weight/body weight) were calculated. Kidneys, testes, epididymides, seminal vesicles, and preputial glands were weighed in pairs. Following weight measurements, tissues were flash frozen in liquid nitrogen and stored at 80°C for further analysis.
Female mice (n = 7) were treated with testosterone containing pellets (5 mg/pellet and one pellet per mouse) implanted into the nape of the neck. Mice were imaged before pellet implantation and at intervals for nine days after pellet implantation. Once the level of luciferase expression was determined to be stable for several days (day +9), the mice were divided into groups for treatment with the anti-androgen cyproterone acetate at 100 mg/kg/day or vehicle control (DMSO) by daily sc injection. At the conclusion of the study animals were imaged, weighed, and humanely euthanized by cervical dislocation under anesthesia. The liver, kidneys, ovaries, and uterus were removed and weighed, and relative organ weights (organ weight/body weight) were calculated. Kidneys and ovaries were weighed in pairs. Following weight measurements, tissues were flash frozen in liquid nitrogen and stored at 80°C for further analysis.
Luciferase assays.
Tissue samples were placed in lysis buffer with inhibitors (Passive Lysis Buffer [Promega] and Complete Mini Protease Inhibitor Cocktail [Roche, Indianapolis, IN]). The tissues were homogenized using a tissue homogenizer (Handishear, Hand-held homogenizer, VirTis, Gardiner, NY). Tissues were further homogenized by brief sonication (60 Sonic Dismembrator, Fisher Scientific, Hampton, NH). Tissue homogenates were centrifuged and clarified lysates were used for luminometer assays and westerns. For the luminometer assays, Luciferase Assay Substrate (Luciferase Assay System, Promega) was prepared as indicated by the manufacturer and placed in disposable cuvettes (Promega). Tissue homgenates (20 µl) and substrate (100 µl) were mixed and measurements were taken in a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA) with the parameters of a 2-s delay, 20-s integration with two repetitions in the standard assay mode. Background luminescence readings were obtained and the background readings were subtracted from the luminescent data. Protein concentrations were determined using the BCA Protein Assay Kit (Pierce, Rockford, IL) following the manufacturer's protocols and analyzed using a VERSAmax tuneable microplate reader and associated Softmax Pro version 3.1.2 software (Molecular Devices, Sunnyvale, CA). The luminescence for each of the protein lysates was calculated as arbitrary units of light per microgram of protein.
Western blot analysis.
Tissue lysates prepared for luminometer assays were analyzed for the presence of the endogenous KAP protein. Samples of 100 µg of protein were separated by electrophoresis on 15% SDS-polyacrylamide gel under reducing conditions. Proteins were transferred to a PVDF (Schleicher & Schuell, Dassel, Germany) membrane, and blots were blocked overnight at 4°C in 55% nonfat dried milk in PBS. Primary rabbit polyclonal antibody (anti-KAP1) was diluted 1:250 in blocking buffer and membranes were probed for 1 h at room temperature. Washes were performed as recommended by the membrane manufacturer. The secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit, DAKO A/S, Glostrup, Denmark) was diluted 1:2000 in blocking buffer and the blot was incubated for 1 h at room temperature. After washing, bands were detected using ECL+chemiluminiscence detection method (Amersham Pharmacia Biotech, Piscataway, NJ) and exposed to X-ray film (Eastman Kodak Company, Rochester, NY). To control for loading, blots were stripped and reprobed for Cyclophilin A, using a polyclonal antibody (Affinity Bioreagents Inc., Golden, CO) following manufacturer's recommendations. Quantification was carried out by a BioRad Model DS800 Calibrated Densitometer (BioRad Laboratories, Hercules, CA).
Statistical analysis.
Data are presented as mean ± standard error about the mean. In order to compare the data from all of the male anti-androgen studies the data are presented as % change from baseline. All significant differences noted from these experiments were also significant when calculated from absolute values. Two to three dorsal and ventral images were obtained for each mouse before treatment with test reagents. Quantitated data from the kidneys or reproductive organs were averaged to obtain an average baseline luciferase reading for each mouse. Percent change from the average baseline was calculated for each mouse and data are presented as mean ± standard error about the mean. Statistical analyses were completed using the Stat View software package (Version 5.0.1; SAS Institute, Cary, NC). Significant changes in the luciferase signal between groups at each time point were determined using unpaired t-tests or ANOVA and Fisher's PLSD.
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RESULTS |
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Hormonal Regulation in Male Kap-luc Transgenic Mice
In order to determine if steroid hormones regulate the Kap-luc transgene, male (n = 6) mice were castrated and bioluminescent images were acquired before and at intervals following castration. Fourteen days after castration, the mice were divided into two groups (n = 3) and received either testosterone (5 mg/pellet) or placebo 21-day time release pellets (one pellet per mouse). Images were taken at intervals for another 14 days. Figure 3a shows that the luciferase expression decreased significantly following castration. Precastration levels of luciferase were restored in the group of mice that was implanted with testosterone pellets but not in the group given placebo pellets. In Figure 3b, the luciferase signal was quantitated using a region of interest placed over the kidneys of the mice. Significant increases in luminescence were shown between the placebo and testosterone treated animals.
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Dose response data was generated using adult male mice given daily sc injections of vehicle (DMSO), cyproterone (10, 50, and 100 mg/kg/day), or flutamide (50 and 100 mg/kg/day) for seven days (n = 12 for DMSO, n = 5 for 10, 50, and 100 mg/kg/day cyproterone and n = 5 for 50 and 100 mg/kg/day flutamide). Bioluminescent images were acquired on each of the study days (Fig. 4).
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DISCUSSION |
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The cellular specificity of our transgenic mice as compared to endogenous Kap message and the transgenic mouse made by Ding et al. can be initially addressed by analysis of the biophotonic images. Biophotonic imaging does not pick up Kap-luc expression from the head (brain), thoracic cavity (lungs and heart), and the upper abdominal region from the ventral side (liver, stomach, spleen, and pancreas). The lack of expression in these areas is consistent with expression data on lungs, heart, brain, liver, and other organs in both wild type and mice transgenic using similar Kap promoter regions by Southern and Northern blotting and RT-PCR (Cebrián et al., 2001; Ding et al., 1997
). Expression in our transgenic mice seems to be limited to the kidneys, androgen regulated and reproductive tissues. Future analysis of the Kap-luc transgenic mouse to assess cellular specificity by Northern blots or RT-PCR will help to increase our understanding of Kap-luc regulation and tissue specificity.
Cebrián et al. (2001), probed several tissues for the presence of the endogenous Kap gene encoded products using RT-PCR/Southern blotting and Western blotting and found that the Kap mRNA was exclusively expressed in the kidney and in the uterus of pregnant female mice from day 13 of gestation. Northern blot analysis by Ding et al. (1997)
also showed high levels of transgene expression in the epididymides. They also showed endogenous Kap message in the epididymides by RT-PCR. In our studies reported here, luciferase assays of organ lysates indicate that expression is detected mainly in the kidney and a variety of reproductive organs. Western blot analysis confirmed the expression of native KAP in the kidney of male mice but not from the other organs or female mice. The Western blot results were consistent with the bioluminescent imaging in demonstrating that the cyproterone treatment decreased both native KAP and Kap-luc expression between 23-fold. Bioluminescent detection of nonrenal Kap-luc signal seems to indicate that bioluminescence may be more sensitive than western blot analysis using the current anti-KAP antibody and western blotting procedures. The anti-KAP antibody is a new polyclonal Ab raised from the same peptides used by Cebrián et al. (2001)
for their monoclonal antibody. The KAP signal only appears in the kidney and mimics the mRNA expression observed under different hormonal conditions. The specificity of this polyclonal anti-KAP antibody was assessed by eliminating the KAP band when the antibody was incubated with the immunizing peptide. Incubating the KAP antibody with other peptides from the KAP protein had no effect on band intensity. Also, the antibody used in this article recognizes a KAP-HA fusion protein with the same specificity as antibodies against the HA epitope. (Messeguer, personal communication) There are several factors that may influence the sensitivity of the antibody that include the extraction buffer, the primary and secondary antibodies and their interaction with the western blotting procedure and membranes, and the ECL reagents used. Studies are underway to optimize Western blotting conditions.
While higher sensitivity of luciferase assays versus Western blot assays constitutes a reasonable explanation for the negative results obtained by Western blotting on female kidneys, the lack of endogenous KAP expression in extra-renal tissues such as the epididymus, which exhibits high bioluminescence values requires other interpretations. We hypothesize that while 1.5 Kb of the Kap proximal promoter contains the necessary elements to drive expression of the reporter gene in the kidney proximal tubule cells in the presence of androgens, it may lack other DNA-binding and regulatory elements further up-stream in the promoter that contribute to negative regulation of the native Kap gene in nonrenal tissues in vivo, or perhaps the regulatory sequences of the Kap promoter may drive other similar genes in a variety of tissues. An alternative hypothesis may be that the insertional site of the transgene is near regulatory regions of a gene expressed in tissues responsive to sex hormonal regulation cannot be overlooked. Analysis of the chromosomal localization will provide insight as to the copy number and localization of transgenic insertions.
Luciferase expression measured in light units/µg protein from luminometer assays showed highest expression in the epididymides and kidneys in male mice. In female mice, expression was highest in the ovaries and lower in the uterus and kidneys. Expression was also observed at lower levels in other organs in the male including testes, seminal vesicles, and preputial glands. The data from the luciferase assays indicate that per µg of protein the epididymides express more light than the kidneys in male mice with 3.4 and 2 Units of light/µg respectively. When light output is considered in the context of whole organs instead of per microgram of protein, the kidneys produce more overall light with 6.5 x 105 Units of light/organ as compared to 2.5 x 105 Units of light/organ from the epididymides. Therefore the kidneys would appear as the brightest feature on the exterior of the mouse by biophotonic imaging.
The native Kap promoter has been shown to regulate the expression of KAP in response to different hormones in specific regions of the kidneys. Testosterone is required for expression in the S1 and S2 segments of the proximal tubules in males and testosterone treated females (Meseguer et al., 1989; Meseguer and Catterall, 1990
). KAP expression in the S3 segment of the proximal tubule is dependent on estrogen and thyroid hormones (Meseguer and Catterall, 1992
; Solé et al., 1994
). Promoter analysis of the Kap promoter sequence used for the generation of these transgenic mice shows that there are putative response elements for the androgen, estrogen, thyroid hormone, glucocorticoid and retinoic acid receptors. The potential for multihormonal regulation exists for this transgenic mouse, but was not addressed by the experiments presented in this article. Future experiments will determine the degree of multihormonal regulation and the tissues that will show differential regulation in the Kap-luc transgenic mice.
Over the past few years, considerable effort has been focused on developing in vivo screening assays for the assessment of chemicals that may have endocrine disrupting activities (EDSTAC, 1998). A 15-day intact male assay has been developed in which the male mice are given daily doses of chemicals (Cook et al., 1997
; O'Connor et al., 2000
, 2002
). Subcutaneous substance administration was used in these studies based on common usage in Kap hormonal regulation studies. For a proof of concept paper this administration route seemed reasonable, however for follow-up studies to assess the capacity of these transgenic mice and biophotonic imaging to respond to a larger group of chemicals, oral gavage will be the administration route of choice. At the conclusion of the study, body weight, serum samples, organ weights, and pathological analyses of selected tissues are used to determine endocrine disruption effects. Biophotonic imaging using Kap-luc mice may accelerate the assessment of potential anti-androgen effects in this intact male assay and provide new and important information regarding the action of anti-androgenic compounds. A reduction in Kap-luc expression was detected as early as 24 h following the administration of cyproterone. Flutamide induced reductions in luciferase expression were demonstrable by eighth day of treatment. The time course of anti-androgenic effects of compounds can be followed in vivo preceding the evaluation of anti-androgenic effects using conventional endpoints of a 15 or 21-day study. Changes in gene expression would generally precede changes in organ architecture and could be used as a new study endpoint.
The preliminary dose response data at 10 mg/kg treatment with cyproterone indicates that even lower doses may be able to inhibit Kap-luc expression. With a limited numbers of mice to use for screening, we chose two doses of flutamide that seemed to fall within the effective dose range of this compound. Studies were found using as low as 33 mg/kg/day (Raghow et al., 2002) to induce anti-androgenic effects in mice, while in rats effective doses started at 20 mg/kg (O'Connor et al., 2002
). Factoring in the increased metabolism of mice and possible strain differences in response to the compound, we chose 50 and 100 mg/kg of flutamide for our studies. Both cyproterone and flutamide will require further dose response data in order to fully assess the sensitivity of Kap-luc transgenic mice to anti-androgens.
We were able to show that significant anti-androgenic effects, detected as reductions in luciferase expression by biophotonic imaging, could be induced by the anti-androgens cyproterone acetate and flutamide. The reduction in luciferase expression was confirmed for cyproterone by luminometer assays of tissue lysates and by Western blot analysis. Flutamide did show reduced in vivo bioluminescence, however there was no change in the units of light produced/µg protein in the kidneys, epididymides, or seminal vesicles. The changes in bioluminescent signal from the surface following flutamide treatment may be attributable to some decreases in luciferase expression and decreases in the weight of organs such as the seminal vesicles. These differences between the action of cyproterone and flutamide in intact mice highlight the differences in how the two compounds induce their affect on the endocrine system. Each anti-androgen represents a different class of anti-androgens. Cyproterone is a synthetic steroidal drug that competes with testosterone and dihydrotestosterone for the androgen receptor. Cyproterone also has progesterone-like activity and reduces pituitary luteinizing hormone and plasma testosterone (McLeod, 1993). Flutamide and other nonsteroidal anti-androgens block the binding of androgens to the androgen receptor. Testosterone levels often increase or remain unchanged following flutamide treatment without co-treatment with luteinizing hormone-releasing hormone (LHRH) agonists (Labrie, 1993
). Also in an article by Shetty et al. (2001)
, the authors look at testosterone, LH, and FSH levels following flutamide, GnRH or both treatments combined in mice. Flutamide treatment alone did not reduce testosterone, LH, or FSH levels. Flutamide did, however, cause a reduction of seminal vesicle weight. For clinical applications, flutamide is generally coadministered with an LHRH agonist or by castration to counter increases in testosterone (Labrie, 1993
). Perhaps a castrated Kap-luc mouse model in which the hypothalamo-hypophysial-testicular axis has been disrupted may be better suited to assess the sensitivity of the system. Nevertheless, significant reductions in luciferase were detected by biophotonic imaging within 24 h after initiation of dosing with cyproterone and eight days following treatment with flutamide.
The changes in biophotonic image data following anti-androgen treatment in our transgenic mice were consistent with changes in animal and organ weights shown previously by O'Connor et al. (2002) in rats. The weights of the epididymides, seminal vesicles, and preputial glands decreased in both mice and rats in a parallel fashion following the course of treatment with cyproterone and flutamide. This concordance of biophotonic imaging with standard endpoints of assessment of endocrine disruption is encouraging that biophotonic imaging may be useful for the screening of androgen disrupting chemicals. Further dose response data to assess the lower limits of sensitivity and screening with a larger group of endocrine disruptive chemical will be essential before performing large-scale screening of compounds. The general toxicity of the compounds was observed as a decrease in body weight and increase in liver size.
In the experiments reported here, we confirmed that Kap-luc expression using biophotonic imaging in our transgenic animals is hormonally regulated and these results are consistent with western blotting data for endogenous KAP. Males experienced rapid decreases in Kap-luc expression following castration suggesting that the presence of androgens is necessary for normal expression. Normal luciferase expression was recovered following exogenous administration of testosterone in castrated males. Renal expression in female transgenic mice using the Kap promoter was near background levels as assayed by bioluminescent imaging of the Kap-luc mice. The low-expressing females were induced to express luciferase at levels comparable to males by the addition of testosterone. Similarly, low Kap promoter/angiotensinogen expression was observed in female mouse kidney that increased significantly in the presence of testosterone (Ding et al., 1997). The data presented in this report are consistent with the observations of (Catterall et al. 1986
) on endogenous Kap promoter function in response to androgen and anti-androgen treatment.
The intact male endocrine disruption assays demonstrated that biophotonic imaging of the Kap-luc mice may be used to assess endocrine disruptive compounds following further characterization this mouse line to endocrine active compounds. These transgenic Kap-luc mice and in vivo biophotonic imaging can provide important information about the timing and localization of anti-androgen action not obtainable from traditional assay endpoints.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (510) 291-6196. E-mail: scott.malstrom{at}xenogen.com.
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