ToxSci Advance Access originally published online on November 5, 2007
Toxicological Sciences 2008 101(2):350-363; doi:10.1093/toxsci/kfm275
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o,p'-DDT Elicits PXR/CAR-, Not ER-, Mediated Responses in the Immature Ovariectomized Rat Liver
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* Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824
Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., Shizuoka 437-0065, Japan
Center for Integrative Toxicology, and the National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824
Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan 48824
|| Wellington Laboratories Inc., Guelph, Ontario N1G 3M5, Canada
1 To whom correspondence should be addressed at Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824. Fax: (517) 353-9334. E-mail: tzachare{at}msu.edu.
Received September 27, 2007; accepted November 1, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Technical-grade dichlorodiphenyltrichloroethane (DDT) is an agricultural pesticide and malarial vector control agent that has been designated a potential human hepatocarcinogen. The o,p'-enantiomer exhibits estrogenic activity that has been associated with the carcinogenicity of DDT. The temporal and dose-dependent hepatic estrogenicity of o,p'-DDT was investigated using complementary DNA microarrays in immature ovariectomized Sprague-Dawley rats with complementary histopathology and tissue-level analysis. Animals were gavaged with 300 mg/kg o,p'-DDT either once or once daily for 3 consecutive days. Liver samples were examined 2, 4, 8, 12, 18, or 24 h after a single dose or following three daily doses. For dose-response studies, a single dose of 3, 10, 30, 100, or 300 mg/kg body weight o,p'-DTT was administered for 3 consecutive days. Genes associated with drug metabolism (Cyp2b2 and Cyp3a2), the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR), cell proliferation (Ccnd1, Ccnb1, Ccnb2, and Stmn1), and oxidative stress (Gclm and Hmox1) were significantly induced. Cyp2b2 exhibited dose-dependent regulation and was significantly induced across all time points, while cell proliferation– and oxidative stress–related genes exhibited transient induction. The induction of Cyp2b2 and Cyp3a2 mRNA levels suggest PXR/CAR activation, consistent with expression of genes associated with oxidative stress. Few genes known to be estrogen receptor (ER) regulated were differentially expressed when compared to the hepatic gene expression profile elicited by ethynyl estradiol in immature ovariectomized C57BL/6 mice using the same study design and analysis methods. These data indicate that o,p'-DDT elicits PXR/CAR-, not ER-, mediated gene expression in the rat liver. Based on the species-specific differences in CAR regulation, the extrapolation of rodent DDT hepatocarcinogenicity to humans warrants further investigation.
Key Words: DDT; liver; microarray; CAR; carcinogenesis; estrogen.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Technical-grade dichlorodiphenyltrichloroethane (DDT) is a mixture of enantiomers and related compounds. DDT and its major metabolites, including 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene and 1,1-dichloro-2,2-bis(p,p-chlorophenylethane; DDD), are lipophilic, persistent, and known to bioaccumulate (Bayen et al., 2005
; Mansour, 2004; Minh et al., 2002). The use of DDT was banned in the United States in 1972 due to potential adverse effects in wildlife and humans associated with its estrogenicity and carcinogenicity (Longnecker, 2005; Turusov et al., 2002). Nevertheless, it is still used in many countries, especially for malaria vector control due to its overall cost-effectiveness (Attaran and Maharaj, 2000; Weissmann, 2006).
DDT is reported to be a hepatic tumor promoter (Ito et al., 1983), with inconclusive genotoxicity activity and a nongenotoxic carcinogen in mice and rats. Although considered to be a risk factor (McGlynn et al., 2006), there is no significant correlation between DDT exposure and liver cancer incidence in humans (Cocco et al., 2005). Consequently, the International Agency for Research on Cancer (IARC) classifies it as a "possibly carcinogenic (Group 2B)," based on inadequate evidence of carcinogenicity in humans (IARC, 1991).
In the fruit fly, DDT induced glutathione s-transferase and xenobiotic-metabolizing enzyme genes mediated by the nuclear receptor DHR90, an ortholog of the rodent constitutive androstane receptor (CAR) and pregnane X receptor (PXR) (King-Jones et al., 2006; Pedra et al., 2004; Willoughby et al., 2006). Cyp2b and Cyp3a mRNA levels are induced by p,p'-DDT, mediated by PXR/CAR (Wyde et al., 2003). CAR plays a significant role in tumor carcinogenesis in mice through the induction of drug-metabolizing enzymes and cell proliferation–related genes (Columbano et al., 2005; Huang et al., 2005; Yamamoto et al., 2004). The tumor promotion activity of phenobarbital (PB) is also abolished in CAR null mice (Yamamoto et al., 2004). Consequently, DDT may act as a PB-type hepatic tumor promoter through CAR activation in rats and mice.
In addition to the PB-type enzyme-inducing properties of p,p'-DDT in the rodent liver, the o,p'-enantiomer exhibits estrogenic activities (Hoekstra et al., 2001). Estrogens have also been associated with hepatic tumorigenesis in rodents and humans (Giannitrapani et al., 2006), suggesting that the hepatocarcinogenicity of DDT may involve the estrogenic activity of o,p'--DDT. Several structurally diverse estrogenic compounds including steroids, industrial chemicals, natural products, and environmental pollutants elicit estrogenic responses in the liver (Ciana et al., 2003). Microarray studies have demonstrated that ethynyl estradiol (EE) induces estrogen receptor (ER)–mediated gene expression changes in rodent livers (Boverhof et al., 2004; Kato et al., 2004). Therefore, the temporal and dose-dependent gene expression activity of o,p'-DDT with complementary histopathology and tissue-level analyses were examined to further investigate the PXR-, CAR-, and ER-mediated hepatic activities of o,p'-DDT. Collectively, the data indicate that o,p'-DDT–elicited hepatic gene expression is not mediated by the ER but rather through PXR/CAR-dependent mechanisms.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Husbandry.
Female Sprague-Dawley rats, ovariectomized on postnatal day 20 were obtained from Charles River Laboratories (Raleigh, NC) on day 25. Rats were housed in polycarbonate cages containing cellulose fiber chip bedding (Aspen Chip Laboratory Bedding, Northeastern Products, Warrensberg, NY) and maintained at 40–60% humidity and 23°C in a room with a 12-h dark/light cycle (7:00 A.M.–7:00 P.M.). Animals were allowed free access to deionized water and Harlan Teklad 22/5 Rodent Diet 8640 (Madison, WI) and acclimatized for 4 days prior to dosing.
Treatments and necropsy.
In the dose-response study, rats were orally gavaged once daily for 3 days with 0.1 ml of sesame oil vehicle (Sigma-Aldrich, St Louis, MO) or 3, 10, 30, 100, or 300 mg/kg body weight o,p'-DDT (99.2% purity; Sigma-Aldrich) in 0.1 ml of sesame oil vehicle and were sacrificed 24 h after the last treatment. In the time-course study, rats were orally gavaged once or once daily for 3 days with 300 mg/kg body weight o p'-DDT in 0.1 ml of sesame oil vehicle. An equal number of time-matched vehicle (VEH) control animals were also dosed in the same manner. Rats receiving one dose were sacrificed 2, 4, 8, 12, 18, and 24 h after treatment. Rats receiving three daily doses were sacrificed 24 h after the third treatment. Treated and vehicle groups consisted of five animals each per time point. The o,p'-DDT dose was calculated from average weights of animals prior to treatment. All procedures were performed with the approval of the Michigan State University All-University Committee on Animal Use and Care.
Animals were sacrificed by cervical dislocation, and animal body weights were recorded. Whole liver weights were recorded, and sections of the left lateral lobe (approximately 0.1 g) were snap frozen in liquid nitrogen and stored at – 80°C. The right lateral lobe was placed in 10% neutral-buffered formalin (NBF; VWR International, West Chester, PA) for histopathology and stored at room temperature for at least 24 h prior to further processing.
Histopathology.
Following fixation of the right lateral lobe for at least 24 h in 10% NBF, the samples were embedded in paraffin according to standard techniques. Five-micrometer sections were mounted on glass slides and stained with hematoxylin and eosin. All embedding, mounting, and staining of tissues were performed at the Histology/Immunohistochemistry Laboratory (Michigan State University). The histopathology of each liver section was scored according to the National Toxicology Program Pathology guidelines.
Measurement of hepatic o,p'-DDT and o,p'-DDD levels.
In the time-course study, one sample from a randomly selected control animal and three randomly selected liver samples from o,p'-DDT–treated rats from each time point were processed in parallel with laboratory blanks and a reference or background sample at Wellington Laboratories Inc. (Guelph, Ontario, Canada). Samples were weighed, spiked with 13C12 o,p'-DDT or 13C12 o,p'-DDD surrogate, digested with sulfuric acid, and extracted. Extracts were cleaned, concentrated, and spiked with an injection standard. Analysis was performed on a high-resolution gas chromatograph/high-resolution mass spectrometer (HRMS) using a Hewlett Packard 5890 Series II GC interfaced to a VG 70SE HRMS. The HRMS was operated in the EI/SIR mode at 10,000 resolutions. A 60-m DB5 column (J&W Scientific, Folsom, CA) with an internal diameter of 0.25 mm and film thickness of 0.25 µm was employed. Injection volumes were 2 µl, and a splitless injection was used.
RNA isolation.
Total RNA was isolated from left lateral liver sections using Trizol Reagent (Invitrogen, Carlsbad, CA). Samples were removed from – 80°C storage and immediately homogenized in 1 ml Trizol Reagent using a Mixer Mill 300 tissue homogenizer (Retsch, Germany). Total RNA was isolated according to the manufacturer's protocol and resuspended in The RNA Storage Solution (Ambion, Austin, TX). RNA concentrations were determined by spectrophotometry (A260), and purity was assessed by the A260:A280 ratio and by visual inspection of 3 µg on a denaturing gel.
Microarray platform.
Spotted complementary DNA (cDNA) microarrays were produced in-house from LION Bioscience's Rat cDNA library (LION Bioscience, Heidelberg, Germany). A total of 8565 cDNA features representing 5096 unique genes (confirmed unique Entrez Gene IDs) were selected based on their level of annotation as well as sequence similarity to well-annotated human and mouse genes. Detailed protocols for microarray construction, cDNA probe labeling, sample hybridization, and slide washing can be found at http://dbzach.fst.msu.edu/interfaces/microarray.html. Briefly, PCR-amplified DNA was robotically arrayed onto epoxy-coated glass slides (Schott/Nexterion, Jena, Germany), using an Omnigrid arrayer (GeneMachines, San Carlos, CA) equipped with 32 (8 x 4) Chipmaker 2 pins (Telechem, Sunnyvale, CA) at the Research Technology Support Facility at Michigan State University (http://www.genomics.msu.edu).
Microarray analysis.
Dose-response gene expression changes were analyzed using a spoke design in which samples from o,p'-DDT–treated animals were cohybridized with VEH animals. Temporal changes in gene expression were assessed using an independent reference design in which samples from o,p'-DDT–treated animals were cohybridized with VEH animals. Comparisons were performed between treated and VEH samples using three biological replicates and two independent labelings of each sample (i.e., dye swap) for each time point. Total RNA (25 µg) was reverse transcribed in the presence of Cy3- or Cy5-labeled dUTP (Amersham, Piscataway, NJ) to create fluor-labeled cDNA, which was purified using QIAquick PCR purification kit (Qiagen, Valencia, CA). Cy3- and Cy5-labeled samples were mixed, vacuum dried, and resuspended in 32 µl of hybridization buffer (40% formamide, 4x sodium chloride-sodium citrate, and 1% sodium dodecyl sulfate) with 20 µg polydA and 20 µg of mouse COT-1 DNA (Invitrogen) as a competitor. This probe mixture was heated at 95°C for 2 min and was then hybridized to the array under a 22- x 40-mm Lifterslip (Erie Scientific, Portsmouth, NH) in a light-protected and humidified hybridization chamber (Corning Inc., Lowell, MA). Samples were hybridized for 18–24 h at 42°C in a water bath. Slides were then washed, dried by centrifugation, and scanned at 635 nm (Cy5) and 532 nm (Cy3) on a GenePix 4000B microarray scanner (Molecular Devices, Union City, CA). Images were analyzed for feature and background intensities using GenePix Pro 6.0 (Molecular Devices). All data were managed in the toxicogenomic information management system dbZach relational database (Burgoon et al., 2006; Burgoon and Zacharewski, 2007). Microarray data quality was monitored using the laboratory's quality assurance and control plan (Burgoon et al., 2005).
Microarray data normalization and statistical analysis.
Data were normalized using a semiparametric approach (Eckel et al., 2005). Model-based t-values were calculated from normalized data, comparing treated and vehicle responses per time point. Empirical Bayes analysis was used to calculate posterior probabilities (p1[t] value) of activity on a per gene and time point basis using the model-based t-value (Eckel et al., 2004). Genes were filtered for activity based on the p1(t) value. p1(t) values approaching 1 indicate changes in gene expression that are more robust. In the dose-response study, unique genes with a p1(t) > 0.999 and absolute fold change 
1.5-fold compared to VEH at least at one time point were initially selected for further investigation. In the time-course study, unique genes with a p1(t) > 0.999 in a minimum of two time points and absolute fold change 1.5-fold compared to VEH at least at one time point were selected for further investigation. Functional annotation for differentially expressed genes was obtained using DAVID software (http://david.abcc.ncifcrf.gov/) (Dennis et al., 2003). Gene ontology (GO) molecular function was examined for active genes at each time point. Level 2 GO terms with p < 0.05 were considered significant. Hierarchical clustering analysis was performed by GeneSpring GX 7.3.1 (Agilent Technologies Inc., Santa Clara, CA).
Quantitative real-time PCR.
For each sample, 1.0 µg of total RNA was reverse transcribed by SuperScript II using an anchored oligo-dT primer as described by the manufacturer (Invitrogen). The resultant cDNA (1.0 µl) was used as the template in a 30 µl PCR reaction containing 0.1µM each of forward and reverse gene-specific primers designed using Primer3 (Rozen and Skaletsky, 2000), 3mM MgCl2, 1.0mM deoxynucleoside triphosphates, 0.025 IU AmpliTaq Gold, and 1x SYBR Green PCR buffer (Applied Biosystems, Foster City, CA). Gene names, accession numbers, forward and reverse primer sequences, and amplicon sizes are listed in Table 1. PCR amplification was conducted in MicroAmp Optical 96-well reaction plates (Applied Biosystems) on an Applied Biosystems PRISM 7000 Sequence Detection System using the following conditions: initial denaturation and enzyme activation for 10 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. A dissociation protocol was performed to assess the specificity of the primers and the uniformity of the PCR-generated products. Each plate contained duplicate standards of purified PCR products of known template concentration covering six orders of magnitude to interpolate relative template concentrations of the samples from the standard curves of log copy number versus threshold cycle. The copy number of each unknown sample for each gene was standardized to that of glyceraldehyde-3-phosphate dehydrogenase gene to control for differences in RNA loading, quality, and cDNA synthesis. Microarray data for Ccnd3 gene (Entrez Gene ID: 25193) was not concordant with the quantitative real-time PCR (QRT-PCR) results and therefore excluded from further analysis.
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Correlation analysis between o,p'-DDT–treated rat and EE-treated mouse livers.
Genes with a p1(t) > 0.99 and absolute fold change
1.5-fold at one or more time points in the o,p'-DDT–treated rat liver samples were selected and used for correlation analysis (Burgoon et al., 2006), against our previously published EE-treated mouse liver gene expression data (see http://www.bch.msu.edu/
zacharet/publications/supplementary/index.html for EE data). Orthologous rat and mouse genes were identified using Homologene (http://www.ncbi.nlm.nih.gov/sites/entrez?db=homologene). The filtering criteria used for the correlation analysis were relaxed compared to that used in the GO analysis to include more ortholog comparisons in the correlation analysis and thus be more informative of the overall similarity between the two data sets. The correlation analysis involved a multivariate correlation–based visualization application that was developed in-house (Burgoon et al., 2006) and has been previously used to investigate the estrogenicity of 2,3,7,8-tetrachlrodibenzo-p-dioxin in the mouse uterus when compared to EE (Boverhof et al., 2006). This tool calculates the temporal correlations between gene expression and significance values for orthologous DDT-treated rat (this study) and EE-treated mouse genes (Boverhof et al., 2004) and summarizes the results in a scatterplot.
Statistical analysis.
Body weight, relative liver weight (RLW), and QRT-PCR data are presented as the mean ± SE. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test between VEH and o,p'-DDT–treated groups (p < 0.05). Hepatic o,p'-DDT and o,p'-DDD concentration data were analyzed using a two-way ANOVA to identify significant differences in o,p'-DDT and o,p'-DDD concentrations across time. Pairwise comparisons were performed using Tukey's honestly significant difference post hoc test to control type I error (
= 0.05). For QRT-PCR data, the relative expression levels of target genes were scaled such that the standardized expression level of the time-matched VEH group was equal to 1 for graphing purposes. All statistics were performed using SAS 9.1.3 software (SAS Institute Inc., Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Body Weight, RLW, and Histopathology
There was no significant difference between VEH and o,p'-DDT–treated animal body and RLWs in the dose-response study at 72 h (data not shown). RLWs were significantly higher in 300 µg/kg o,p'-DDT–treated rats at 72 h when compared to VEH in the time-course study in the absence of any effect on body weight (Table 2), consistent with the increase in RLW in rats treated with 106 mg/kg p,p'-DDT (Tomiyama et al., 2003
). Thus, 300 µg/kg o,p'-DDT was used in the time-course study.
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There were also no significant signs of irreversible hepatocellular injury at any time point. Hepatocytes exhibited early centrilobular and midzonal swelling with hypertrophy and eosinophilic staining (Table 3). However, there was no evidence of hepatocyte swelling with hypertrophy and eosinophilia after 18 h, and therefore, the 72 h dose-response sections were not examined.
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Hepatic o,p'-DDT and o,p'-DDD Concentrations in Time-Course Study
The time-dependent accumulation and elimination of hepatic o,p'-DDT has not been previously reported using a comparable study design. Following an initial accumulation, o,p'-DDT and o,p'-DDD levels decreased over time. o,p'-DDT concentrations were only significantly different from o,p'-DDD at 2 h. The levels of o,p'-DDT were highest at 2 h (222 ng/g liver), which dramatically decreased in a time-dependent manner to 0.68 ng/g liver at 72 h (Fig. 1). In contrast, the levels of o,p'-DDD modestly increased to 35.1–52.7 ng/g liver at 12 h and then decreased to 5.8 ng/g liver by 24 h. o,p'-DDT concentrations continued to decrease (2.4 ng/g liver at 72 h), suggesting that treatment enhanced its hepatic clearance. The levels of o,p'-DDD were higher compared to o,p'-DDT after 12 h, and the o,p'-DDD/o,p'-DDT ratio showed a time-dependent increase from 2 h (ratio: 0.16) to 72 h (ratio: 3.5), suggesting that o,p'-DDT was metabolized to o,p'-DDD. o,p'-DDT and o,p'-DDD levels were not examined in the dose-response study samples and were not detected in the liver samples from VEH animals in the time-course study.
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Microarray Analysis
All the microarray data for both the dose response and the time-course studies are provided as Supplementary Tables 1 and 2, respectively. In the time-course study, 327 unique genes were differentially expressed following treatment (Fig. 2, Tables 4
–6). In addition, there was a clear dose-dependent increase in the number of differentially expressed genes (Fig. 3a). Of the 81 genes induced at 72 h in the time-course study, 58 exhibited dose-dependent induction, although none achieved a plateau, thus precluding Effective dose, 50% (ED50) determinations (Fig. 3). GO analysis indicated that genes associated with oxidoreductase activity such as Cyp2b2 or Cyp3a2 were induced as early as 2 h (Table 4), while electron transport genes related to reductive reactions (e.g., Txn1, Txnrd1, Por, Aldh1a1, and Akr7a2) exhibited differential expression as early as 4 h (Table 5), which persisted to 72 h.
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Both the number of active genes and overrepresented GO terms were highest at 18 h. Diverse biological responses such as protein synthesis (GO term: structural constituent of ribosome) and degradation (GO term: proteasome endopeptidase activity) were represented (Table 6). Many eukaryotic translational initiation factors, and proteasome-related and ribosomal protein genes were induced at 18 h. In addition, the stress-responsive heat shock protein (HSP) and Hmox1 genes were induced as well as Gclm and Gpx2 genes, which are involved in glutathione homeostasis, at 18 and 24 h. Genes involved in electron transport or reductive reactions such as Ephx1, Flt1, and Txn1 were also induced at 18 h.
Several sterol metabolism– and cell proliferation–related genes were also differentially expressed (Table 4). The sterol metabolism–related gene, Srebf1, was repressed 4–18 h along with another sterol metabolism–related gene, Cyp17a1, at 12–18 h. Genes associated with cell proliferation, including Ccnb1, Ccnb2, Mdm2_predicted, and Stmn1, exhibited induction by o,p'-DDT.
Quantitative Real-Time PCR
In total, 15 genes identified as being differentially regulated by o,p'-DDT in the microarray time-course study, including other genes known to be regulated by PXR/CAR that were not represented on our cDNA array, were examined by QRT-PCR. QRT-PCR confirmed that the PXR/CAR-regulated drug-metabolizing genes, Cyp2b2, Cyp3a2, and Cyp3a23/3a1, were induced by o,p'-DDT when compared to VEH (Fig. 4). Moreover, the expression of CAR (Nrli3) and PXR (Nrli2) mRNA, which regulate Cyp2b and Cyp3a, respectively, were also induced (Fig. 4). The cell proliferation genes, Ccnb1, Ccnb2, Ccnd1, and Stmn1, also exhibited comparable microarray and QRT-PCR expression profiles (Fig. 5). Overall, there was a good correlation between the 12 genes examined by microarray and QRT-PCR (Figs. 4–6).
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Comparison of o,p'-DDT– and EE-Induced Hepatic Gene Expression Profiles
In order to comprehensively compare hepatic o,p'-DDT–elicited gene expression with a similar study in EE-treated mice, the filtering criteria for the o,p'-DDT rat data were relaxed (p1[t] > 0.99, 1.5-fold change) and the resulting genes that had overlapping orthologs in the mouse EE data set were subsequently compared. In total, 148 orthologous gene pairs were identified between the two data sets (Fig. 7A and Supplementary Table 3). In order to ascertain the similarity of expression profiles between o,p'-DDT in the rat liver and EE in the mouse liver, a Pearson's correlation analysis was performed on the temporal gene expression (fold change) and significance (p1[t] value for rat and transformed t-value for mouse data, respectively) profiles. Note that the o,p'-DDT and EE gene expression studies used the same design, model, microarray platform, and data analysis methods. The paired data, representing 148 orthologous rat and mouse genes, are plotted on a coordinate axis with the x-axis providing an index of the gene expression similarity and the y-axis providing an index of the significance similarity for each orthologous pair present in the o,p'-DDT and EE data sets. Ideally, well-correlated genes (i.e., exhibiting similar gene expression and significance profiles) aggregate in the upper right quadrant. Figure 7B illustrates that the correlations between the orthologous genes appear randomly distributed throughout the plot, indicating a poor correlation between the o,p'-DDT–treated rat liver and EE-treated mouse liver data sets.
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More specifically, Igfbp1, Col4a1, Myh3, Ggt1, Stat5a, and Cyp17a1 exhibited induction following EE treatment, suggesting ER-mediated regulation of the expression of these genes in the mouse liver. However, the rat orthologs of these genes were not induced by o,p'-DDT (Supplementary Table 3). Moreover, the expression of the steroidogenesis-associated gene Cyp17a1 was repressed in the rat liver and verified by QRT-PCR (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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The estrogenicity of o,p-DDT is well established. A comprehensive time-course and dose-response cDNA microarray study with complementary histopathology and o,p'-DDT tissue-level analysis was completed to further investigate its estrogenicity and potential role in hepatocarcinogenicity.
The gene expression profile of o,p'-DDT was characteristic of a PB-type inducer, as opposed to an estrogen. This is consistent with the p,p'-DDT induction of PB-type enzyme activity in rat liver (Wyde et al., 2003). o,p'-DDT induced Cyp2b2, Cyp3a2, and Cyp3a23/3a1 genes, indicative of CAR/PXR regulation (Sparfel et al., 2003). PB is reported to induce Cyp2b2 transcript levels sevenfold in the rat liver (Agrawal and Shapiro, 2003) and Cyp2b1/2 mRNA levels fivefold in primary hepatocytes (Ganem et al., 1999). In the present study, o,p'-DDT induced Cyp2b2 mRNA levels approximately 30-fold in the liver. Dexamethazone, a potent PXR activator, induced Cyp3a2 transcripts fourfold in the male rat liver (Meredith et al., 2003). Cyp3a23/3a1 was also induced 35-fold by dexamethazone and 10-fold by PB in the male rat liver (Martignoni et al., 2004), whereas it is induced sevenfold by 16-carbonitrile, a potent PXR activator, in the adult female rat liver (Guzelian et al., 2006). In the present study, o,p'-DDT induced the Cyp3a2 3.5-fold and Cyp3a23/3a1 12-fold in immature ovariectomized C57BL/6 liver. Collectively, the induction levels of Cyp2b2, Cyp3a2, and Cyp3a23/3a1 mRNA by o,p'-DDT are comparable to other well-established PXR/CAR ligands.
CAR activation is associated with hepatomegaly (Huang et al., 2005) and centrilobular hypertrophy with concomitant induction of Cyp2b1 and Cyp3a2, which is prototypical of PB-type enzyme inducers (Harada et al., 2003). Furthermore, both CAR and PXR transcript levels were induced, which may facilitate receptor-mediated differential gene expression. However, cDNA microarrays and QRT-PCR only assess transcript levels, while increases in mRNA levels may also be due to posttranscriptional events (e.g., mRNA stability).
The activation of PXR/CAR and the induction of cytochrome P450 activity is not only consistent with the increases in RLW and hepatocyte swelling with hypertrophy but also with the hepatic clearance of o,p'-DDT, which bears similarities to the reductive dechlorination of p,p'-DDT to p,p'-DDD by Cyp2b and Cyp3a isozymes in the rat liver (Kitamura et al., 2002). Therefore, the induction of Cyp2b2, Cyp3a2, and Cyp3a23/3a1 mRNA levels likely contributes to the time-dependent clearance of o,p'-DDT and the detection of o,p'-DDD from 2 to 12 h as well as its subsequent decrease, as seen with metabolism of p,p'-DDT (Tebourbi et al., 2006; Tomiyama et al., 2003). Furthermore, p,p'-DDT and its metabolites accumulate in adipocytes with relatively low levels present in the liver (Tebourbi et al., 2006). Collectively, this suggests that o,p'-DDT was metabolized to o,p'-DDD and subsequently stored in peripheral fat stores, as reported for p,p'-DDT metabolism (Tebourbi et al., 2006). Cyp2b and Cyp3a induction may also contribute to sterol metabolism (Tabb and Blumberg, 2006) and alter circulating steroid hormone levels (Gupta et al., 1980), although a poor correlation between hepatic enzyme induction and enhanced hormone clearance has been reported (You, 2004).
The induction of Cyp2b2 and Cyp3a23/3a1 mRNA and the differential expression of electron transport and reductive reaction–related genes such as Ephx1, Gclm, Gpx2, Txn1, and Txnrd1 are also suggestive of an oxidative stress response. Hmox1, HSPs, and poly (ADP-ribose) polymerases are elevated in response to oxidative stress (Bauer and Bauer, 2002; Diller, 2006; Gero and Szabo, 2006) and were induced by o,p'-DDT. Reported increases in hepatic lipid peroxide content (Harada et al., 2003) confirm the significance of these gene expression changes.
CAR activation is associated with cell cycle regulation (Ledda-Columbano et al., 2000) and transient hepatomegaly from the induction of DNA replication and the suppression of apoptosis in response to xenobiotic exposure (Huang et al., 2005). The cell proliferation–related genes, Ccnb1, Ccnb2, Ccnd1, Stmn1, and Mdm2_predicted (Fung and Poon, 2005; Rubin and Atweh, 2004; Tashiro et al., 2007), exhibited increased expression 12–24 h after o,p'-DDT treatment. The induction of Ccnd1 by the potent CAR activator TCPOBOP is abolished in CAR null mice (Columbano et al., 2005). Mdm2, which activates cell cycle progression and blocks apoptosis, is also regulated by CAR (Huang et al., 2005). The induction of cell proliferation–related genes suggests a transient stimulation of hepatocellular proliferation due to CAR activation, consistent with hepatocyte swelling and associated hypertrophy, and the clearance of hepatic o,p'-DDT and o,p'-DDD. Considering that short-term treatments of high-dose p,p'-DDT (500 ppm via diet) or repeated treatments of lower dose p,p'-DDT (50 or 160 ppm via diet) were required to induce cellular proliferation in the rat liver (Harada et al., 2003), our data suggest that o,p'-DDT may also induce hepatocellular proliferation at higher doses or following repeated treatments.
Functional annotation using GO terms suggests preparation for protein synthesis by the expression of ribosomal protein genes at 18 h. The concomitant induction of proteasomal proteolysis genes would facilitate protein turnover and cellular adaptation. Both responses are consistent with the hepatocyte swelling with hypertrophy and the induction of genes associated with oxidative stress. However, the present study primarily focused on differential gene expression. Additional biochemical investigations such as protein-level analysis, enzymatic activity, or metabolite analysis are needed to demonstrate that these gene expression effects elicited by o,p'-DDT correlate with subsequent events.
Binding to the ER has been proposed to contribute to the hepatocarcinogenicity of o,p'-DDT (Giannitrapani et al., 2006; Holsapple et al., 2006). However, a comprehensive comparison of the hepatic differential gene expression profiles of o,p'-DDT–treated rat liver and EE-treated mouse liver (Boverhof et al., 2004) using similar study designs and analysis methods failed to identify a correlation (Fig. 7). This analysis suggests that the hepatic gene expression effects of o,p'-DDT are independent of the ER, despite its estrogenicity in other tissues and models.
The activation of CAR by o,p'-DDT, followed by the induction of transcripts associated with oxidative stress, cell proliferation, and protein turnover, are suggestive of potential roles in the etiology of hepatocarcinogenicity (Yamamoto et al., 2004). However, the carcinogenic modes of action through CAR are significantly different between rodents and humans (Holsapple et al., 2006). Moreover, o,p'-DDT and its metabolites do not elicit agonist effects mediated by human CAR (Kretschmer and Baldwin, 2005). Consequently, the extrapolation of potential nongenotoxic carcinogenicity of DDT mediated by CAR in rodents to humans is questionable.
Furthermore, basal PXR expression is age and sex dependent and is paralleled by Cyp3a mRNA levels in mouse liver (Down et al., 2007). The induction of Cyp3a genes by dexamethazone treatment also shows age and sex dependency (Anakk et al., 2003; Down et al., 2007). Consequently, PXR/CAR activation by o,p'-DDT may be affected by age and sex, which further confounds extrapolations to humans.
In summary, the differential gene expression elicited by o,p'-DDT in the rat liver is consistent with PXR/CAR regulation, and the nongenotoxic carcinogenic properties of DDT appears to be independent of the ER. Given the species differences in CAR-mediated activity, further investigation is warranted in order to more comprehensively assess the acute toxicity and probable carcinogenicity of DDT in humans.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Supplementary Tables 1–3 are available online at http://toxsci.oxfordjournals.org/.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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National Institute of General Medical Sciences (GM075838 [GenBank] ); U.S. Environmental Protection Agency (RD83184701). T.R.Z. is partially supported by the Michigan Agricultural Experiment Station.
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ACKNOWLEDGMENTS |
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The authors would like to acknowledge Edward Dere for critical reading of the manuscript.
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