ToxSci Advance Access originally published online on July 14, 2007
Toxicological Sciences 2007 99(2):612-627; doi:10.1093/toxsci/kfm181
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Subchronic Toxicity and Toxicogenomic Evaluation of Tamoxifen Citrate + Bexarotene in Female Rats
* Life Sciences Group, IIT Research Institute, Chicago, Illinois 60616
Charles River Laboratories, Inc., Pathology Associates, Chicago, Illinois 60612
Chemopreventive Agent Development Research Group, Division of Cancer Prevention, National Cancer Institute, Bethesda, Maryland 20892
1 To whom correspondence should be addressed at Life Sciences Group, IIT Research Institute, 10 West 35th St, Chicago, IL 60616. Fax: (312) 567-4021. E-mail: dmccormick{at}iitri.org.
Received May 30, 2007; accepted July 9, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Tamoxifen (TAM) is a nonsteroidal antiestrogen that prevents estrogen receptor–positive breast cancer in rodents and humans. Bexarotene (BEX), a selective agonist for retinoid X receptors, inhibits mammary carcinogenesis in rodents. The present study was conducted to support the preclinical development of TAM (tamoxifen citrate) + BEX for use in breast cancer chemoprevention, and to investigate the influence of these agents on hepatic gene expression. Female CD rats (20 per group) received daily oral (gavage) exposure to TAM (0 or 60 µg/kg/day) and/or BEX (0, 5, 15, or 45 mg/kg/day) for a minimum of 90 days. BEX induced mild, dose-related anemia and dose-related increases in serum alkaline phosphatase, cholesterol, triglycerides, and calcium levels, and increased platelet counts. TAM had no biologically significant effect on any clinical pathology parameter and did not alter the effects of BEX on these endpoints. Microscopic alterations induced by BEX included epidermal hyperplasia, hyperkeratosis (stomach), and cytoplasmic clearing (liver). Microscopic changes in TAM-treated rats were limited to mucous cell hypertrophy in the cervix and vagina. The toxicity of administration of the combination of TAM + BEX can generally be predicted on the basis of the toxicity of each drug as a single agent. BEX induced dose-related alterations in the expression of several genes involved in steroid, drug, and/or fatty acid metabolism; TAM did not alter these effects of BEX. Differential expression of genes involved in drug and lipid metabolism may underlie the observed effects of BEX on cholesterol and triglyceride levels and its effects on liver histology.
Key Words: tamoxifen; bexarotene; preclinical toxicity; toxicogenomics; rat; chemoprevention.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Tamoxifen (TAM) is a nonsteroidal antiestrogen with known activity in the prevention of human breast cancer. Through its binding to the estrogen receptor (ER), TAM inhibits mammary carcinogenesis and induces the regression of existing mammary cancers. TAM has been used clinically in breast cancer therapy for more than 20 years, and has been shown to prevent the appearance of additional primary cancers in breast cancer patients (Fisher et al., 2005
). However, TAM increases the risk for endometrial cancer (Fisher et al., 2005), and may support the outgrowth of hormone-independent breast cancers (Nayfield, 1995; Osborne et al., 1994; Wakeling, 1995). To preclude such toxicity, efforts continue to identify combination approaches to breast cancer prevention and therapy in which activity can be maintained using lower dose levels of TAM (Nayfield, 1995).
The results of preclinical studies in several model systems have demonstrated significantly enhanced chemopreventive activity when TAM or related SERMs (selective ER modulators) are combined with agonists for retinoid X receptors (RXR; Anzano et al., 1994, 1996; Suh et al., 2002). Bexarotene (BEX; Targretin) is a selective RXR agonist with demonstrated activity in the prevention of mammary carcinogenesis in both mice and rats (Bischoff et al., 1998, 1999; Gottardis et al., 1996; Lubet et al., 2005; Wu et al., 2002a,b; Yen et al., 2004). Recent in vitro experiments using human breast cancer cells demonstrated that when BEX was given in combination with chemotherapeutic agents (including TAM), the cells remained chemosensitive, whereas cells that were not cotreated with BEX became resistant to chemotherapy. Further, drug-resistant cells showed increased sensitivity to chemotherapeutic agents that were coadministered with BEX (Yen and Lamph, 2005). Of particular interest is a report using a rat model in which the addition of BEX increased therapeutic activity in mammary tumors that had developed TAM resistance (Bischoff et al., 1999). These data suggest that administration of TAM + BEX may provide a useful new approach to breast cancer prevention, and to the treatment of breast cancers that no longer respond to TAM alone.
The present study was conducted to support preclinical development of TAM (as tamoxifen citrate) + BEX for use as a breast cancer chemoprevention regimen in women. The subchronic toxicity of TAM alone, BEX alone, and TAM + BEX was evaluated in female rats using a 2 x 4 matrix study design. A complete battery of toxicologic evaluations was performed, and toxicogenomic analyses were performed using microarray and polymerase chain reaction (PCR) techniques to determine the effects of these agents on hepatic gene expression. Toxicogenomics studies were conducted using the livers of treated animals to identify differences in gene expression that (1) may provide insight into the mechanisms of chemopreventive action of these agents and (2) may underlie observed toxicities and thereby support the interpretation of toxicity data.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Chemicals and reagents.
TAM and BEX were supplied by the Chemopreventive Agent Repository maintained by the Division of Cancer Prevention, National Cancer Institute. Corn oil was purchased from MP Biomedicals (Aurora, OH) and Sigma-Aldrich (St Louis, MO). Absolute ethanol was purchased from Pharmco Products (Brookfield, CT). RNeasy Mini Kits were purchased from Qiagen (Valencia, CA), and Quant-iT RiboGreen RNA Assay Kits were purchased from Invitrogen (Carlsbad, CA). The BioArray High Yield RNA Transcript Labeling Kit was purchased from Enzo Diagnostics (Farmingdale, NY). All remaining reagents were purchased from Sigma-Aldrich or Fisher Scientific (Pittsburgh, PA).
Animals and animal husbandry.
Prior to the initiation of in vivo work, the study protocol was reviewed and approved by the IIT Research Institute Animal Care and Use Committee. All aspects of the study involving animal care, use, and welfare were performed in compliance with U.S. Department of Agriculture regulations and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Female CD IGS rats [Crl:CD(SD)], approximately 4 weeks of age upon receipt, were purchased from virus-free colonies maintained at Charles River Laboratories (Portage, MI). During a 14-day quarantine period, rats were assigned to experimental groups using a randomization process designed to ensure a comparable initial mean body weight in each group. Rats were individually housed in stainless steel cages suspended over absorbent cage boards, and were held in a temperature-controlled room maintained on a 12-h light/12-h dark cycle. Rats were permitted free access to City of Chicago drinking water (supplied by automatic watering system) at all times during the study. Rats were also allowed free access to Certified Rodent Diet 5002 (PMI Nutrition International, Brentwood, MO) throughout the study, except during an overnight fast prior to scheduled necropsies. Animals were observed a minimum of twice daily to monitor their general health status. Detailed clinical examinations and measurements of body weight and food consumption were performed once weekly.
Experimental design.
Female rats (20/group) received daily oral (gavage) exposure to TAM (in 0.5% ethanol in corn oil) at doses of 0 or 60 µg/kg/day and/or BEX (in corn oil) at doses of 0, 5, 15, or 45 mg/kg/day for a minimum of 90 days using a dosing volume of 5 ml/kg body weight. Dose levels of TAM and BEX were selected on the basis of the results of a preliminary 28-day range-finding study. After the completion of the dosing period, surviving rats were euthanized and necropsied approximately 24 h after the final dose of TAM and/or BEX.
Quantitation of plasma drug levels.
Blood samples for plasma drug level analyses were collected from 10 rats per group at approximately 3- to 4-h postdosing during the final week of exposure.
For analysis of plasma levels of BEX, plasma samples and calibrators were extracted with methanol, chilled for 1 h at – 20°C to precipitate proteins, and centrifuged at 5000 x g at 4°C for 10 min. The resulting supernatant was evaporated under nitrogen. Dried extracts were reconstituted in a mixture of 80% acetonitrile and 20% 10mM ammonium acetate buffer/glacial acetic acid (100/1 vol/vol). Samples were analyzed via high-performance liquid chromatography (HPLC) using a Brownlee New Guard, RP-18, 5-µm precolumn and a Varian Microsorb MV, C18, 4.6 x 250 mm, 5-µm column. BEX was quantitated at an ultraviolet (UV) absorbance of 262 nm; retention time was approximately 14 min. The limit of quantitation for the assay was 0.13 µg/ml. The concentration of BEX in plasma was determined by comparing the peak area of BEX to a standard curve prepared using bulk drug as the standard.
For analysis of plasma levels of TAM, N-desmethyltamoxifen, and 4-hydroxytamoxifen, plasma samples and calibrators were extracted following addition of internal standard (1.25 µg/ml propranolol in ethanol) with ethyl ether, vortexed for 1 min and centrifuged at 5000 x g at 4°C for 10 min. The ether layer was then reextracted, and ether layers were combined and evaporated under nitrogen. The dried extracts were reconstituted in a mixture of 100mM phosphate buffer (pH 2) and acetonitrile (65/35, vol/vol). Samples were analyzed via HPLC using a Zorbax CN, 4.6 x 250 mm, 5-µm column (Agilent Technologies, Inc., Palo Alto, CA), a photochemical reactor for enhanced detection (PHRED) and fluorescent detection. Column eluants were exposed to UV light at 254 nm using the PHRED device and TAM was detected using fluorescence with excitation at 258 nm and emission at 378 nm. Limits of quantitation for the assay were 0.66 ng/ml for TAM, 0.91 ng/ml for N-desmethyltamoxifen, and 2 ng/ml for 4-hydroxytamoxifen.
Postmortem evaluations.
After a minimum of 90 days of exposure to TAM ± BEX, rats were fasted overnight, bled for hematology, coagulation, and clinical chemistry evaluations, euthanized, and subjected to a complete necropsy with tissue collection. Immediately after euthanasia, samples were harvested from a standardized site in the liver of five animals per group, weighed, snap-frozen in liquid nitrogen, and stored at – 70°C for use in toxicogenomics assays. The liver was selected for toxicogenomics studies (1) in consideration of the substantial existing database for genomics studies in this organ and (2) because transcriptional changes in hepatic enzymes could underlie alterations in carbohydrate and lipid metabolism that have been reported for several retinoids.
After collection of livers for toxicogenomic analyses, remaining liver and approximately 45 other tissues were collected from each animal and fixed in 10% neutral buffered formalin for histopathologic evaluation. Tissues collected from all rats in the vehicle control group, TAM only group, high-dose BEX group, and TAM + high-dose BEX group were processed by routine histologic methods, cut at 5 µm, stained with hematoxylin and eosin, and evaluated histopathologically. Histopathologic evaluations in remaining groups were limited to gross lesions and identified target tissues only.
Hematologic parameters (erythrocyte count and morphology, hemoglobin, mean cell volume, platelet count, absolute and relative reticulocyte count, total white blood cell count, and absolute and relative differential white blood cell counts) were quantitated using the ADVIA System 120 Hematology Analyzer (Bayer, Tarrytown, NY). Hematocrit, mean cellular hemoglobin, and mean cellular hemoglobin concentration were calculated on the basis of the automated hematology data. Coagulation parameters (prothrombin time, fibrinogen, and activated partial thromboplastin time) were quantitated using an MLA Electra 900 Automatic Coagulation Timer (Hemoliance, Raritan, NJ). Serum chemistry parameters (sodium, potassium, chloride, calcium, inorganic phosphorus, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, lactate dehydrogenase, total bilirubin, blood urea nitrogen, creatinine, glucose, total protein, albumin, cholesterol, and triglycerides) were evaluated using a Beckman Synchron CX5 analyzer (Beckman Instruments, Brea, CA). Serum globulin, albumin:globulin ratio, and blood urea nitrogen:creatinine ratio were calculated from the automated clinical chemistry data.
RNA isolation and quality determination.
Total RNA was isolated from individual rat livers using the RNeasy Mini Kit. RNA quality was evaluated by gel electrophoresis and with an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA concentration was quantitated using the Quant-iT RiboGreen RNA Assay Kit.
Microarray assays.
Microarray assays were performed using Affymetrix Rat 230 2.0 oligonucleotide array platform (Affymetrix, Santa Clara, CA). This platform contains 31,099 probe sets representing transcripts and variants from > 28,000 rat genes. Single-stranded and double-stranded complementary DNA (cDNA) were synthesized from total RNA and then purified. Purified cDNA was used to synthesize biotin-labeled antisense cRNA using the BioArray High Yield RNA Transcript Labeling Kit. cRNA was fragmented, added to temperature equilibrated, prewetted probe arrays, and hybridized. Arrays were washed and stained using a GeneChip Fluidics Station (Affymetrix) and then scanned using a GeneChip Scanner 3000 (Affymetrix).
Microarray quality control.
Reproducibility of hybridization was evaluated using Affymetrix Gene Chip Operating System (GCOS) software, dChip software, and R language package (Bioconductor). GCOS cutoffs for the scale factor, background, noise, and 3'/5' ratio were < 5, 300, 100, and 3, respectively. Comparisons of expression values on each array versus the median expression value for each probe set in all arrays were performed using the Probe Level Model package for R (Bioconductor), which preprocesses intensity values using the Robust Multichip Average (RMA) method.
Microarray data normalization and evaluation of reproducibility.
After outlier arrays were removed, RMA preprocessing was performed using GeneSpring software (Agilent). Intensity values for individual genes were normalized to adjust for differences in array signal intensities. GeneSpring was used to evaluate sample reproducibility (weighted Spearman correlation), hierarchical clustering (Pearson correlation), and Principal Component Analysis. Only samples that passed this level of quality control were used for statistical analysis to identify differentially expressed genes.
Confirmation by quantitative reverse transcription–PCR.
Twelve genes demonstrating differential expression in BEX-treated rats and four control genes (albumin; calcium/calmodulin-dependent protein kinase IV; ferritin heavy polypeptide 1; and tryptophan 5-monooxygenase) were selected for subsequent validation with quantitative real-time reverse transcription–PCR (qRT-PCR). Total RNA samples were converted to cDNA by RT using the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). PCR was performed using a 7900HT Fast Real-Time PCR System (Applied Biosystems), and data were analyzed using Sequence Detection System (SDS) software (Applied Biosystems).
Data analyses.
Body weight, body weight gain, food consumption, clinical pathology, and organ weights were compared by analysis of variance (ANOVA) followed by Dunnett's test for comparing multiple treatment groups to a single control group. Statistical comparisons for plasma drug level data were performed (where appropriate) by ANOVA, followed by pairwise comparisons using Tukey's test or a two-sample t-test assuming unequal variances. A minimum significance level of p < 0.05 was used in all comparisons.
Prior to statistical analysis for differential expression, microarray data were filtered using GeneSpring to eliminate (1) unexpressed genes; (2) genes with raw signals < 100 in all samples; (3) genes whose expression profile was unchanged across all samples (standard correlation 
0.997); and (4) genes demonstrating a < 1.5-fold change in treated versus vehicle control groups. Statistical comparisons of gene expression in livers from control and BEX-treated rats were performed using a one-sample t-test and the Benjamini and Hochberg false discovery correction set at a false discovery rate of 0.01. Dose-related effects of BEX on gene expression were evaluated using K-means clustering and a standard correlation (GeneSpring, Affymetrix). Significantly enriched canonical pathways and biological functions were identified through the use of Ingenuity Pathways Analysis v2.0 (Ingenuity Systems, Redwood City, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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In-life Toxicologic Evaluations
No treatment-related deaths were noted during the study. In comparison to vehicle controls, statistically significant decreases in mean terminal body weight were observed in groups exposed to TAM only, the high dose of BEX only, and TAM + the high dose of BEX (Fig. 1). By contrast, mean terminal body weights in groups exposed to the low or mid doses of BEX only were slightly, but not significantly, increased from control. Group mean body weights in rats exposed to TAM + the low or mid doses of BEX were comparable to control at all times during the study. Since the mean terminal body weights in groups receiving TAM only, the high dose of BEX only, or TAM + the high dose of BEX were not significantly different from one another, no additive or synergistic effects of TAM and BEX on body weights or body weight gains were present. No treatment-related effects on food consumption were observed in any experimental group at any point in the study.
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Several nonspecific but dose-dependent clinical signs (e.g., discolored inguinal fur, unkempt appearance) were observed in animals treated with BEX (± TAM). During the final week of the study, posterior incipient cataracts were observed in both eyes in one animal each in mid- and high-dose BEX groups, and in five animals treated with TAM + the high dose of BEX.
Hematology evaluations performed on samples collected during study weeks 4 and 13 demonstrated that treatment with BEX (± TAM) induced a mild but dose-related anemia (Table 1). At both sampling times, groups exposed to the mid and/or high doses of BEX demonstrated decreases in RBC count (6–8%; high-dose BEX only); hemoglobin (9–16%; mid- and high-dose BEX groups); hematocrit (6–11%; mid- and high-dose BEX groups); and mean cellular hemoglobin (3–5%, mid- and high-dose BEX groups). Concomitant increases in the incidence of hypochromia (10–20%) were also seen in these groups. In addition, rats exposed to all doses of BEX + TAM demonstrated small but statistically significant reductions in mean cell volume (3–5%), mean cellular hemoglobin (5–9%), and mean cellular hemoglobin concentration (3–6%). Although these effects were clearly related to BEX exposure, they were of minimal to mild in severity and do not suggest limiting hematologic toxicity of the retinoid.
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Exposure to BEX also impacted coagulation parameters (Table 1). Activated partial thromboplastin time was increased by 55% in rats receiving the high dose of BEX and 78% in rats receiving TAM + the high dose of BEX. Prothrombin time was increased by 32% in the group receiving the high dose of BEX + TAM, and fibrinogen levels were increased in dose-related fashion (29–47%) in all groups exposed to BEX ± TAM.
Subchronic administration of BEX increased both serum cholesterol and triglycerides in dose-related fashion (Table 1). At 4 weeks, dose-related increases in serum cholesterol were observed in groups exposed to BEX alone (45, 71, and 153% in low-, mid-, and high-dose BEX groups, respectively) and BEX + TAM (37, 61, and 111% in groups treated with TAM + low, mid, and high doses of BEX, respectively). At 4 weeks, alkaline phosphatase was also increased by 51% in rats exposed to TAM + the high dose of BEX.
Similar changes were seen in samples collected at 13 weeks. Serum cholesterol levels were increased by 68, 141, and 241% in rats exposed to BEX alone, and by 74, 115, and 191% in groups exposed to BEX + TAM. Triglyceride levels were increased by more than 10-fold in rats exposed to the high dose of BEX alone, and by approximately fivefold and sevenfold in rats exposed to TAM + BEX. Alkaline phosphatase was increased by 180% by the high dose of BEX alone, and by 164 and 212% in groups exposed to TAM + the mid or high doses of BEX, respectively.
Plasma Drug Levels
Plasma levels of BEX increased linearly with increasing administered dose; coadministration of TAM had no statistically significant effect on plasma BEX levels (Table 2). Mean plasma BEX concentrations in groups exposed to the low, mid, and high doses of BEX only were 0.252, 0.417, and 0.854 µg/ml, respectively, yielding a linear regression line with the formula y = 0.0747x + 0.184 (r2 = 0.999). These data were comparable to the mean plasma BEX concentrations in groups exposed to TAM + BEX; plasma concentrations in groups exposed to TAM + the low, mid, and high doses of BEX were 0.272, 0.636, and 0.993 µg/ml, respectively [y = 0.0831x + 0.274 (r2 = 0.920)].
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Levels of TAM and major metabolites (N-desmethyltamoxifen and 4-hydroxytamoxifen) in many plasma samples were below their limits of quantitation, thus precluding the calculation of mean plasma levels of these agents.
Postmortem Evaluations
At necropsy, gross pathology was limited to enlarged livers observed in 5/20 rats in the group exposed to the high dose of BEX only. This gross observation correlated with modest, but statistically significant and dose-related increases in liver weights in all groups exposed to BEX (± TAM; Table 2). A general pattern was seen in which concomitant exposure to TAM resulted in modest reductions in the effects of BEX on liver weight (Table 2). However, although this pattern was seen in all TAM + BEX dose groups, the reductions in mean liver weights were not significant at the 5% level of confidence when compared to mean liver weights in groups treated with the same dose of BEX only.
As expected on the basis of its pharmacologic activity, administration of TAM resulted in a significant reduction in mean uterine weight in all exposed groups (Table 2). This finding is consistent with the results of previous studies in which rats received subchronic exposure to TAM either in the diet (Carthew et al., 1996) or by gavage (Kennel et al., 2003). Administration of BEX had no effect on the influence of TAM on uterine weights.
Microscopic pathology in groups exposed to TAM (± BEX) was limited to mucous cell hypertrophy and individual cell necrosis (with or without hyperplasia) in the vagina and cervix (Table 3). Coadministration of the high dose of BEX appeared to decrease the severity of TAM-induced necrosis in the vagina (reduction in mean severity score from 1.50 in the TAM only group to 0.75 in the group exposed to TAM + high-dose BEX). Lesion severity scores were comparable in groups exposed to TAM alone versus TAM + either the low or mid doses of BEX.
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In the liver, groups exposed to BEX (± TAM) demonstrated increased incidences and severity of periportal cytoplasmic clearing (Table 3); cytoplasmic clearing commonly results from glycogen accumulation, and could underlie increases in liver weight observed in these groups. Interestingly, the high dose of BEX decreased the incidence of chronic inflammation of the hepatic parenchyma. Other microscopic changes induced by BEX included increased hyperkeratosis in the squamous mucosa of the stomach (high-dose BEX); increased pseudocysts in the pituitary pars intermedia (low-, mid-, and high-dose BEX); and increased epidermal hyperplasia (mid and high-dose BEX). Coadministration of TAM had no effect on histopathologic changes induced by BEX.
Microarray Analysis of Gene Expression
Microarray analyses were performed on individual liver samples to identify patterns of differential hepatic gene expression in animals exposed to TAM and/or BEX. Of the 31,099 probe sets included within the microarray, 16,487 probe sets were filtered out (removed from the analysis) due to (1) absence of expression; (2) raw signals < 100; or (3) high correlation to a nonchanging expression profile in the samples evaluated. On this basis, 14,812 probe sets remained as the starting gene list for further analysis.
A one-sample t-test was performed on genes that passed the gene filtering criteria in order to identify those that were up- or downregulated by 1.5-fold (p < 0.01) between each treatment group and the vehicle control group. This analysis identified a total of 2972 probe sets that were significantly deregulated in at least one treatment group (369 sets in rats exposed to TAM only; 868, 1295, and 1851 sets in rats exposed to the low, mid, and high doses of BEX only, respectively; and 699, 1157, and 2011 sets in rats exposed to TAM + the low, mid, and high doses of BEX, respectively).
Reproducibility within treatment groups was good. Spearman correlation analysis demonstrated that patterns of gene expression in groups exposed to TAM only clustered close to the vehicle control group (Fig. 2), while gene expression in groups receiving BEX only or TAM + BEX clustered farther from controls as a function of increasing BEX dose (Fig. 2).
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= vehicle control; 
= TAM only; 
= 5 mg BEX/kg/day (± TAM); 
= 15 mg BEX/kg/day (± TAM); 
= 45 mg BEX/kg/day (± TAM).To obtain a more quantitative evaluation of dose-related effects of BEX, and to assess the influence of concomitant exposure to TAM on BEX effects on gene expression, a Standard Correlation (Pearson Correlation around Zero) was calculated for all 2972 differentially expressed genes. Of these 2972 genes (1) expression of 23 annotated genes was upregulated (Table 4) and expression of 13 annotated genes was downregulated (Table 5) in dose-related fashion by BEX, regardless of the presence of TAM; (2) expression of 22 annotated genes was altered in dose-related fashion by BEX only, and not in groups exposed to BEX + TAM (Table 6) suggesting TAM may inhibit the effects of BEX on the expression of these 22 genes; and (3) expression of 30 annotated genes was altered in dose-related fashion by BEX + TAM, but not by administration of BEX alone (Table 7) suggesting that TAM may enhance the effects of BEX on the expression of these genes.
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K-means clustering was used to identify dose-related effects of BEX on hepatic gene expression. Analysis was limited to 494 genes (180 upregulated and 314 downregulated) that demonstrated highly significant (> twofold; p < 0.00001) differential expression in at least one group exposed to BEX only. A total of 141 upregulated genes and 248 downregulated genes exhibited dose-related alterations in expression in response to BEX. Although many differentially expressed genes are not annotated, genes demonstrating dose-related alterations in expression in BEX-treated rats (Tables 4–7) included numerous genes that regulate drug or lipid metabolism.
Canonical pathway analysis using Ingenuity software demonstrated correlations between increasing BEX dose (± TAM) and expression of genes involved in the regulation of cell growth and proliferation, cell death, cancer, and regulation of cell morphology (Fig. 3). Differential expression of genes associated with regulation of these cellular functions is consistent with the demonstrated chemopreventive and therapeutic activity of BEX (± TAM) in the rat mammary gland (Bischoff et al., 1998, 1999; Gottardis et al., 1996; Lubet et al., 2005). Decreasing enrichment with BEX dose (± TAM) was noted for genes involved in fatty acid synthesis and metabolism, sterol biosynthesis, bile acid biosynthesis, and tryptophan metabolism (Fig. 4). Alterations in the expression of genes involved in fatty acid and sterol metabolism may underlie the hypertriglyceridemia, hypercholesterolemia, and other toxicities of BEX observed in the present study in rats as well as in the clinic (Duvic et al., 2001; Esteva et al., 2003; Lowe and Plosker, 2000; Querfeld et al., 2004).
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= TAM + BEX (5 mg/kg/day); 
= TAM + BEX (15 mg/kg/day); 
= TAM + BEX (45 mg/kg/day).
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qRT-PCR Confirmation of Differentially Expressed Hepatic Genes
In order to confirm the specificity of responses identified through the microarray analysis, four control genes and 12 genes identified by microarray as being differentially expressed in livers from BEX-treated rats were selected for confirmation by qRT-PCR. Genes were selected for PCR analysis primarily on the basis of their possible role in BEX-associated alterations in plasma lipid profiles, as identified in the present toxicology bioassay. The results of microarray assays and qRT-PCR assays demonstrated comparable qualitative (directional) changes in the expression of all 12 genes (up- or downregulation vs. vehicle controls), as well as similar quantitative (dose–response) patterns of gene expression in animals exposed to TAM alone or BEX alone (Table 8), or to the two agents in combination (Table 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Recent results from several laboratories, including ours, suggest that differences in the chemopreventive activity and toxicity of natural and synthetic analogs of vitamin A (retinoids) may result from their relative affinities for specific intracellular receptors. Retinoid action may be mediated via binding to retinoic acid receptors (RAR) and/or RXR. Some retinoids are pure RAR or RXR agonists, while others (such as 9-cis-retinoic acid) bind to both receptors and are considered to be pan-agonists.
RXR agonists such as BEX have biological and toxicologic effects that differ from those of pure RAR agonists (e.g., all-trans- and 13-cis-retinoic acid) or RAR/RXR pan-agonists such as 9-cis-retinoic acid. Our data demonstrate that subchronic administration of BEX does not induce many of the toxic effects (e.g., bone fractures, alterations in mucous membranes) that are well-established toxicities of high-dose administration of RAR agonists (Penniston and Tanumihardio, 2006; Silverman et al., 1987). However, the results of the present study, as well as clinical trials with BEX (Duvic et al., 2001; Lowe and Plosker, 2000) demonstrate that toxic effects such as hypercholesterolemia and hypertriglyceridemia are induced by both RXR and RAR agonists.
The chemopreventive activities of these classes of agents also differ. Whereas 13-cis-retinoic acid (an RAR agonist) and 9-cis-retinoic acid (an RAR/RXR pan-agonist) are both effective chemopreventive agents in the rat prostate, BEX (a pure RXR agonist) has no chemopreventive activity in this site (McCormick et al., 1999, 2007). By contrast, BEX confers significant protection against carcinogenesis in both ER-positive and ER-negative models of mammary tumorigenesis (Wu et al., 2002a,b), while RAR agonists appear to have less activity in these models.
The enhanced chemopreventive and chemotherapeutic activities of BEX administered in combination with TAM or other SERMs in the rat mammary tumor model (Bischoff et al., 1998, 1999) suggest a possible role for this agent combination in the management of human breast cancer. The results of the present study demonstrate that when BEX and TAM are administered in combination, the toxic effects of each agent are largely independent of the presence of the other agent. Restated, no evidence of additive or synergistic toxicities was identified in the present study, suggesting that the toxicity of the agent combination can be predicted with reasonable accuracy on the basis of the toxicity of each agent alone. In fact, the results of the present study suggest that toxicologic interactions between BEX and TAM are limited to partial attenuation by each agent of the toxicity of the other agent. This possible interaction is evidenced by: (1) the decreased severity of hepatomegaly in BEX + TAM groups versus groups exposed to BEX alone; (2) the reduction in BEX-induced hypercholesterolemia in rats coexposed to TAM (p < 0.05 at week 4 in rats treated with high-dose BEX + TAM vs. high-dose BEX only); and (3) the decreased incidence of cellular necrosis in the vagina of rats exposed to TAM + high-dose BEX when compared to rats treated with TAM alone.
Because alterations in toxicologic parameters were observed at all BEX doses used in the present study a No Observed Adverse Effect Level could not be determined for BEX or for BEX + TAM. Effects seen at all doses of BEX (± TAM) included hepatomegaly (associated with increased cytoplasmic clearing in periportal regions of the liver), increased numbers of pseudocysts in the pars intermedia of the pituitary, increased serum cholesterol, and increased fibrinogen levels. Statistically significant, dose-related alterations in each of these parameters were seen in groups exposed to all doses of BEX or BEX + TAM.
Subchronic administration of BEX (± TAM) to rats resulted in dose-related increases in serum cholesterol and triglyceride levels in the present study. These results are similar to data reported in preclinical studies with a number of retinoids, and to the results of several clinical trials with BEX. In clinical studies, hyperlipidemia was a common side effect in a phase II clinical trial for cutaneous T-cell lymphoma following daily oral BEX administration (Querfeld et al., 2004). Similarly, increases in triglyceride levels were reported in 4% of patients receiving topical BEX administration (Heald et al., 2003) and in more than 80% of patients receiving oral BEX (Esteva et al., 2003; Querfeld et al., 2004). In a recent clinical review, elevated cholesterol and triglyceride levels were cited as common side effects following BEX administration (Farol and Hymes, 2004).
By contrast to these experimental and clinical data, Lalloyer et al. (2006) recently reported decreases in plasma total cholesterol levels in female apolipoprotein E2 knockin mice (a mixed dyslipidemic murine model) fed a BEX-supplemented diet (0.018% wt/wt, which corresponds to a dose of approximately 35 mg/kg body weight/day) for 11 weeks. The authors attribute the observed decrease in plasma cholesterol levels to decreased circulating cholesterol-containing lipoproteins (non-high-density lipoproteins) in BEX-treated mice, and to decreased intestinal absorption of cholesterol. These investigators did, however, report increased serum triglycerides in BEX-treated mice.
Consistent with its pharmacologic actions and previous reports of its toxicity (Carthew et al., 1996; Dragan et al., 1996; Kennel et al., 2003; Wade and Heller, 1993; Wallen et al., 2001, 2002), administration of TAM (60 µg/kg/day) reduced body weight gains and induced mucous cell hypertrophy and individual cell necrosis in the vagina and cervix. The generalized suppression of body weight gain in female rodents exposed to TAM has been reported in numerous studies (Dragan et al., 1996; Kennel et al., 2003; Wade and Heller, 1993; Wallen et al., 2001, 2002), including those in which TAM has been studied as a breast cancer chemopreventive agent (McCormick and Moon, 1986). Suppression of body weight gain by TAM is seen over a wide range of doses, and is commonly observed in chemoprevention studies in which no other evidence of toxicity is identified (McCormick and Moon, 1986).
Interrogation of rat liver transcriptomes using microarray technology (> 30,000 probe sets representing > 28,000 rat genes) identified 2972 annotated genes and transcribed loci whose expression was apparently modulated by BEX and/or TAM. The very large number of genes demonstrating altered expression in response to BEX is perhaps not surprising in consideration of the well-known promiscuity of RXR in the formation of heterodimers with other nuclear receptors (reviewed in Germain et al., 2006). Through such interactions, an RXR agonist may induce biological effects that are associated with activation of a relatively large number of other nuclear receptors, including peroxisome proliferator–activated receptors (PPARs), Vitamin D receptor, thyroid hormone receptors, nuclear orphan receptors such as LXR and OR-1, RAR, and many others. Through mechanisms initiated by heterodimerization with nuclear receptors such as PPAR
, RXR may serve as a regulator of a broad range of hepatic physiologic processes (Ouamrane et al., 2003; Wan et al., 2000).
Statistical analysis of the nearly 3000 genes that were differentially expressed in the livers of BEX-exposed rats identified 109 genes whose expression was reproducibility up- or downregulated in a dose-related fashion. Of this total, differential expression of 26 genes was seen in response to BEX only, 36 genes were differentially expressed in animals exposed to BEX + TAM but not to BEX alone, and 47 genes were differentially expressed in response to BEX, regardless of the presence or absence of TAM. Functional pathway analysis demonstrated significant, dose-related alterations by BEX in the expression of genes involved in the regulation of cell growth and proliferation, apoptosis, maintenance of cellular morphology, and cancer; modulation of these pathways could clearly underlie the cancer preventive and therapeutic activities of this agent. Pathway analysis also revealed significant, dose-related alterations in the expression of genes involved in fatty acid synthesis and metabolism, sterol biosynthesis, bile acid synthesis, and tryptophan metabolism; changes in the expression of genes underlying regulation of these processes could be responsible for the hypercholesterolemia, hypertriglyceridemia, and other toxic effects of BEX that were observed in this study and which have been observed in clinical trials with this agent.
Consistent with the hypercholesterolemia, hypertriglyceridemia, and hepatic cytoplasmic clearing observed in the in vivo toxicity bioassay, a number of genes involved in lipid metabolism were overexpressed in dose-related fashion in the livers of BEX-treated rats. Overexpressed genes included carboxylesterase-2 (lipid metabolism; 5- to 10-fold overexpression); acetyl-coenzyme A carboxylase (lipid synthesis; 2.4- to 3.5-fold overexpression); cytosolic acyl-CoA thioesterase 1 (lipid metabolism; 16- to 27-fold overexpression); fatty acid synthase (lipid synthesis; 4- to 10-fold overexpression); stearoyl-coenzyme A desaturase 1 (lipid metabolism; 26- to 35-fold overexpression); and stearoyl-coenzyme A desaturase 2 (lipid metabolism; 26- to 63-fold overexpression). In addition, several genes involved in steroid metabolism were also overexpressed in BEX-treated rats. These genes included cyp2a1 (testosterone 15 hydroxylase; steroid metabolism; 17- to 43-fold overexpression); cyp2b2 (phenobarbital-inducible P450; drug metabolism; 8- to 11-fold overexpression); cyp17a1 (aromatase; steroid metabolism; 1.5- to 4-fold overexpression); and cyp26a1 (retinoic acid hydroxylase; retinoid metabolism; 3- to 17-fold overexpression).
PPAR is expressed at high levels in the liver and other tissues that are sites of fatty acid catabolism (Michalik et al., 2006). Formation of RXR–PPAR heterodimers is a key step in the transcriptional activation of hepatic PPAR and its subsequent regulation of hepatic physiology. The observed upregulation by BEX on the expression of hepatic genes that regulate lipid metabolism suggests a possible role for the formation of RXR–PPAR heterodimers in BEX toxicity; the pattern of upregulation of genes involved in fatty acid metabolism in the present study is generally similar to that reported in primates exposed to known PPAR agonists (Cariello et al., 2005). Although the experimental approaches used were quite different, our results are also consistent with the recent report of Kang et al. (2007) who found that PPAR-associated genes involved in fatty acid metabolism were downregulated in vitamin A–deficient animals. It should be noted, however, that while RXR–PPAR heterodimerization provides an attractive mechanism for the effects of BEX on lipid homeostatis, other mechanisms could also underlie the observed effects. For example, similar effects on lipid metabolism could be induced by RXR homodimers alone (rather than by RXR–PPAR heterodimers), since activation of PPAR target genes has been demonstrated by RXR homodimers in nullizygous PPAR knockout mice (IJpenberg et al., 2004).
Twelve genes demonstrating dose-related differential expression of at least twofold at the high dose of BEX (as determined by microarray) were selected for confirmation by qRT-PCR. For all 12 genes, microarray and qRT-PCR analyses demonstrated similar qualitative and quantitative patterns of gene expression as a result of treatment. Based on this relatively high concordance, it is concluded that genes that are identified as being differentially expressed by microarray analyses have a high likelihood of being confirmed by qRT-PCR. This allows one to screen for mechanistically relevant genes or potential pharmacodynamic biomarkers based on microarray data, and supports the hypothesis that changes identified by microarray will be confirmed by RT-PCR.
The results of the present study demonstrate that coadministration of BEX + TAM for 90 days does not induce additive or synergistic toxicity in rats, and that the toxic effects of the combined regimen are predictable on the basis of the toxicity of each agent alone. On this basis, the enhanced antitumor activity of the drug combination appears unlikely to be limited by any toxicologic interactions between the two drugs. Through toxicogenomic evaluations, we have identified a number of possible mechanisms of BEX toxicity. Although these toxic effects may be mediated simply by the binding of BEX to RXR, heterodimer formation between RXR and PPAR and/or other members of the steroid receptor superfamily cannot be excluded as a possibly critical step in the biological activity and toxicity of BEX.
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Supplementary data are available online at www.toxsci.oxfordjournals.org.
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Chemopreventive Agent Development Research Group, Division of Cancer Prevention, National Cancer Institute contracts (N01-CN-35121 and N01-CN-43304).
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