ToxSci Advance Access originally published online on November 27, 2007
Toxicological Sciences 2008 102(1):61-75; doi:10.1093/toxsci/kfm289
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Comparative Toxicogenomic Examination of the Hepatic Effects of PCB126 and TCDD in Immature, Ovariectomized C57BL/6 Mice
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* Department of Biochemistry & Molecular Biology
Center for Integrative Toxicology
Pathobiology and Diagnostic Investigations, Michigan State University, East Lansing, Michigan, 48824-1319
Wellington Laboratories Inc., Guelph, ON N1G 3M5, Canada
1 To whom correspondence should be addressed at Michigan State University, Department of Biochemistry & Molecular Biology, 501 Biochemistry Building, Wilson Road, East Lansing, MI 48824-1319. Fax: (517) 353-9334/(517) 432-2310. E-mail: tzachare{at}msu.edu.
Received August 2, 2007; accepted November 19, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Polychlorinated biphenyls are persistent environmental pollutants that elicit a wide range of effects in humans and wildlife, mediated by the aryl hydrocarbon receptor. 3,3',4,4',5-pentachlorobiphenyl (PCB126) is the most potent congener with relative effect potencies ranging from 0.0026 to 0.857, and a toxic equivalency factor (TEF) of 0.1 set by an expert panel of the World Health Organization. In this study, the hepatic effects elicited by 300 µg/kg PCB126 were compared with 30 µg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in immature, ovariectomized female C57BL/6 mice. Comprehensive hepatic gene expression analyses with complementary histopathology, high-resolution gas chromatograph/high-resolution mass spectrometer tissue analysis, and clinical chemistry were examined. For temporal analysis, mice were orally gavaged with PCB126 or sesame oil vehicle and sacrificed after 2, 4, 8, 12, 18, 24, 72, 120, or 168 h. In the dose–response study, mice were gavaged with 0.3, 1, 3, 10, 30, 100, 300, 1000 µg/kg PCB126, 30 or 100 µg/kg TCDD and sacrificed after 72 h. 251 and 367 genes were differentially expressed by PCB126 at one or more time points or doses, respectively, significantly less than elicited by TCDD. In addition, there was less vacuolization and necrosis, and no immune cell infiltration, despite comparable or higher TEF-adjusted hepatic PCB126 levels. The functional annotation of differentially expressed genes was consistent with the observed histopathology. Collectively, the data indicate that 300 µg/kg PCB126 elicited a subset of weaker effects compared with 30 µg/kg TCDD in immature, ovariectomized C57BL/6 mice.
Key Words: PCB126; TCDD; TEF; liver; mouse; t o x i c o g e n o m i c s.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that are found as mixtures of individual congeners. There are 209 possible PCB congeners with different degrees of chlorination (Mullin et al., 1984
). Many commercial PCB mixtures are known by their industrial name Aroclor, followed by a number designating the number of carbon atoms and the percent chlorine by weight. PCBs were produced between 1930 and 1977 for use as coolants, lubricants, and dielectric insulating fluids for capacitors and transformers, due to their chemical inertness and stability (Mullin et al., 1984). Even though production has ceased, they are still released into the environment through the improper use and disposal of PCB containing products (NTP Technical Report, 2006b). Once released, PCB mixtures are continuously altered through volatilization, partitioning, and biochemical transformations (Ganey and Boyd, 2005; Wu et al., 1998). 3,3',4,4',5-pentachlorobiphenyl (PCB126) is the most potent PCB congener and accounts for 40–60% of the total toxic potency of all dioxin-like PCBs (NTP Technical Report, 2006b).
Dioxin and related compounds elicit a broad spectrum of species- and tissue-specific biochemical and toxic effects including wasting syndrome, dermal toxicity, tumor promotion, teratogenicity, immunotoxicity, and hepatotoxicity (Denison and Heath-Pagliuso, 1998). Many, if not all, of these toxic responses are mediated through the activation of the aryl hydrocarbon receptor (AhR), a basic-helix–loop–helix-PAS (bHLH–PAS) protein (Safe, 2001). Dioxin and related compounds bind to the cytoplasmic AhR, which then translocates to the nucleus to form a heterodimer with the AhR nuclear translocator (ARNT), another member of the bHLH–PAS family (Hankinson, 1995). The activated AhR/ARNT complex interacts with dioxin response elements (DREs) located in the regulatory region of target genes, leading to changes in gene expression (Nebert et al., 2000). The involvement of AhR/ARNT signaling pathway in mediating these responses is supported by several complementary lines of evidence including studies with low affinity AhR allele mice (Okey et al., 1989), structure activity studies (Safe, 1997), and AhR-null mice (Schmidt et al., 1996).
The toxic equivalency factor (TEF)/toxic equivalents (TEQ) approach is used to assess the potential risks associated with exposure to mixtures of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and PCBs. It assumes that PCDDs, PCDFs, and PCBs structurally similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) elicit their toxicity through the AhR. The relative effect potency (REP) for a specific endpoint is determined for individual congeners relative to TCDD, the most potent of the dioxin chemicals (Van den Berg et al., 1998). REPs were used by an expert panel convened by the World Health Organization (WHO) to establish a TEF point estimate with the understanding that it may vary by a half-log unit. The potential toxicity of a mixture could then be represented by the sum of the concentrations of individual congeners multiplied by their corresponding TEFs to obtain an estimated toxicity relative to TCDD. This approach assumes that at submaximal doses, the contributions of individual components are essentially additive (Safe, 1997) and that TEFs are independent of dose, time point, and tissue (Poland and Knutson, 1982; Safe, 1990).
To facilitate the creation of TEFs, the expert panel used the REP2004 Database, a comprehensive listing of REP values for all known dioxins and dioxin-like compounds (Haws et al., 2006; Van den Berg et al., 2006). In vitro data were only considered when there were insufficient in vivo data. For PCB126 there were 318 separate in vivo REPs, from 33 different peer-reviewed publications, a thesis, or government technical report for PCB126. The 2005 WHO expert panel excluded 64 mouse studies due to discrepancies between mouse and rat enzyme activity assay data. Although the official list of studies considered is not available, the Committee stated that they used the REP2004 Database criteria for inclusion and exclusion. The rat studies that were considered included a range of endpoints from short-term enzyme induction to hepatocellular adenomas following chronic exposures (1–2 years). Although the tight range of REPs for PCB126 in rat studies supports a TEF of 0.1, information from mouse and some human studies, especially for enzyme induction, suggests it may have been too high (Van den Berg et al., 2006). The 2005 WHO expert panel concluded that there was insufficient information to change the PCB126 TEF of 0.1, but called for further studies.
In this study, comprehensive time course and dose–response gene expression analyses were conducted with complementary histopathology, clinical chemistry, and high-resolution gas chromatograph/high-resolution mass spectrometer (HRGC/HRMS) tissue level analyses to compare the hepatic effects of 300 µg/kg PCB126 (TEF = 0.1) to 30 µg/kg TCCD (TEF = 1) in immature, ovariectomized female C57BL/6 mice. Comparisons were also made to a previously published TCDD study that used the same animal species, experimental design, cDNA microarray platform, and analysis methods (Boverhof et al., 2005). Collectively, and consistently, 300 µg/kg PCB126 elicited weaker responses and only a subset of effects induced by 30 µg/kg TCDD. However, more comprehensive time optimized dose–response studies are required for each endpoint of interest in order to provide REP data that could be used, in the context of all other available data, when considering the TEF for PCB126.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Animal handling.
Female C57BL/6 mice, ovariectomized by the supplier on postnatal day (PND) 20, with body weights (BW) within 19% of the average, were obtained from Charles Rivers Laboratories (Wilmington, MA) on PND 25. Animals were housed in polycarbonate cages containing cellulose fiber chips (Aspen Chip Laboratory Bedding, Northeastern Products, Warrensberg, NY) with 30–40% humidity and a 12 h light/dark cycle (07:00 A.M.–7:00 P.M.). Mice had free access to deionized water and Harlan Teklad 22/5 Rodent Diet 8640 (Madison, WI). Animals were acclimatized prior to being dosed on PND 28. The immature ovariectomized mouse was used to facilitate comparisons with other data sets obtained in the same model (Boverhof et al., 2005
). The comparisons of the hepatotoxic potency of PCB126 were made to either "internal" TCDD-treated group of mice or to an independent, previously published comprehensive TCDD time course study by Boverhof et al. (2005) that used the same animal model. All procedures were carried out with the approval of the Michigan State University All-University Committee on Animal Use and Care.
Time course and dose–response studies.
A stock solution of PCB126 (99.7% purity, AccuStandard, New Haven, CT) was prepared by first dissolving it in acetone (J.T. Baker), then diluting it with sesame oil (Sigma, St Louis, MO), and evaporating the acetone under a mild stream of nitrogen gas. The PCB126 stock solution was further diluted in sesame oil to achieve the desired dose. For the time course study, mice (n = 5 per group) were orally gavaged with either 300 µg/kg PCB126, 30 µg/kg TCDD (gift from the Dow Chemical Company, Midland, MI), or sesame oil vehicle. PCB126 and vehicle-treated animals were sacrificed at 2, 4, 8, 12, 18, 24, 72, 120, or 168 h postexposure, whereas TCDD animals were sacrificed at 72 h. 30 µg/kg TCDD was initially selected because it elicited maximum induction of Cyp1a1 and 1a2 m RNA levels while not inducing significant changes in BW gain (Boverhof et al., 2005). It was used again in the present study, to facilitate comparisons between studies that employed the same model species, experimental design, cDNA platform, and analysis methods. A concentration of 300 µg/kg PCB126 was used to examine the hypothesis that it would elicit hepatic effects comparable to 30 µg/kg TCDD, based on the PCB126 TEF of 0.1. The vehicle groups were not the same between the current PCB126 study and Boverhof et al. study, but the same vehicle controls were used for the internal TCDD-treated mice in the current PCB126 study.
For the dose–response study, mice were gavaged with a single dose of 0.3, 1, 3, 10, 30, 100, 300, or 1000 µg/kg of PCB126, 30 or 100 µg/kg TCDD, or vehicle and sacrificed 72 h following treatment. All mice were sacrificed by cervical dislocation and tissue samples were removed, weighed, flash frozen in liquid nitrogen, and stored at –80°C. For both the time course and dose–response studies, the right lobe of the liver was fixed in 10% neutral buffered formalin (Sigma) for histological analysis.
Clinical chemistry and histological analysis.
Blood samples were collected by submandibular vein puncture and blood was allowed to clot in the Microtainer Serum Separator Tubes (VWR International, Batavia, IL). Serum was separated by spinning at 10,000 x g for 5 min, after which the samples were stored at –80°C. Serum triglycerides (TG), nonesterified fatty acids (NEFA), cholesterol (CHOL), alanine aminotransferase (ALT), and glucose (GLU) were measured using an Olympus AU640 Automated Chemistry Analyzer (Olympus America Inc., Melville, NY) by the Michigan State University Clinical Pathology Laboratory (http://cvm.msu.edu/clinpath/new.htm).
Fixed liver tissues were sectioned and processed in ethanol, xylene, and paraffin using a Thermo Electron Excelsior tissue processor (Waltham, MA). Tissues were then embedded in paraffin with Miles Tissue Tek II embedding center, after which paraffin blocks were sectioned at 5 µm with a rotary microtome. Sections were placed on glass microscope slides, dried, and stained with the standard hematoxylin and eosin stain. All histological processing was performed at the Michigan State University Histology Laboratory (http://humanpathology.msu.edu/histology/index.html).
Thin layer chromatography.
Liver samples were first homogenized (Polytron PT2100, Kinematica AG, Luzern, CH) in 1% methanol and acidified with concentrated HCl. Lipids were extracted with chloroform: methanol (2:1) containing 1mM 2,6-di-tert-butyl-4-methylphenol (BHT; Sigma). The protein and aqueous phases were re-extracted with chloroform and the organic phases were pooled, dried under nitrogen gas, and resuspended in chloroform with 1mM BHT, and stored at –80°C in glass vials with polytetrafluoroethylene caps (VWR International). Lipid extracts were then fractionated by thin layer chromatography (TLC) on silica gel adsorption plates (LK6D Silica G 60A; Whatman Inc., Florham Park, NJ) with hexane:diethyl ether:acetic acid (90:30:1) and developed with iodine (Sigma). Lipid migrations were compared with triacylglycerol, diacylglycerol, and cholesterol ester standards (Nu-Chek Prep, Elysian, MN).
Quantification of hepatic PCB126 and TCDD levels.
Liver samples were processed in parallel with laboratory blanks and a reference or background sample at Wellington Laboratories, Inc. (Guelph, ON, Canada). Samples were weighed, spiked with 13C12 2,3,7,8-TCDD or 13C12 PCB126 surrogate, digested with sulfuric acid, and extracted. Extracts were cleaned, concentrated, and spiked with 13C12 1,2,3,4-TCDD or 13C12 PCB111 as injection standards. Analysis was performed on a HRGC/HRMS using a Hewlett Packard 5890 Series II GC interfaced to a VG 70SE HRMS. The HRMS was operated in the electron ionization/selective ion recording mode at 10,000 resolution. 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.
Frozen liver samples (on average 
100 mg) were retrieved from –80°C storage and immediately transferred to 1 ml of TRIzol (Invitrogen, Carlsbad, CA) and homogenized using a Mixer Mill 300 tissue homogenizer (Retsch, Germany). Total RNA was isolated according to the manufacturer's protocol with an additional acid phenol:chloroform extraction. Isolated RNA was resuspended in RNA storage solution (Ambion, Inc., Austin, TX), quantified (A260), and quality was assessed by determining the A260/A280 ratio and by visual inspection of 2 µg on a denaturing gel.
cDNA microarray experimental design and protocols.
In the time course study, PCB126-treated samples were cohybridized with time-matched vehicle controls using an independent reference design (Yang and Speed, 2002). Dose-dependent changes in gene expression were analyzed using a common reference design, where PCB126 samples were compared to a common vehicle control. cDNA microarrays were also performed for the "internal" TCDD-treated group of mice, which used the same vehicle controls as in the PCB126 microarray design. In the Boverhof et al. study, independent groups of the TCDD-treated and vehicle control mice were used. All experiments were performed with three biological replicates with two independent labelings of each sample (dye swap) for each time point or dose group, using custom mouse cDNA microarrays containing 13,361 features representing 8516 unique genes (UniGene build 152).
Detailed protocols for microarray preparation, labeling of the cDNA probe, sample hybridization, and washing can be found at http://dbzach.fst.msu.edu/interfaces/microarray.html. Microarrays were printed at the Michigan State University Research Technology Support Facility (http://www.genomics.msu.edu/). Briefly, PCR amplified mouse cDNAs were robotically arrayed onto epoxy-coated glass slides (Schott-Nexterion, Duryea, PA) using an Omnigrid arrayer (GeneMachines, San Carlos, CA) equipped with 48 (4 x 12) Chipmaker 2 pins (TeleChem, Sunnyvale, CA). Total RNA (30 µg) was reverse transcribed in the presence of Cy3- or Cy5-dUTP (Amersham, Piscataway, NJ) to create fluor-labeled cDNA which was purified using a Qiagen PCR purification kit (Qiagen, Valencia, CA). Cy3 and Cy5 samples were mixed, vacuum dried, and resuspended in 48 µl of hybridization buffer (40% formamide, 4x sodium chloride sodium citrate, 1% sodium dodecyl sulfate) with 20 µg polydA and 20 µg of mouse COT-1 DNA (Invitrogen) as competitor. This probe mixture was heated at 95°C for 3 min and hybridized on the array under a 22 x 60 mm lifterslip (Erie Scientific Company, Portsmouth, NH) for 18–24 h in a 42°C water bath. Slides were then washed, dried by centrifugation, and scanned at 635 nm (Cy5) and 532 nm (Cy3) on a GenePix 4100A scanner (Molecular Devices, Union City, CA). Images were analyzed for feature and background intensities using GenePix Pro 6.0 (Molecular Devices).
cDNA microarray data normalization, analysis and feature-to-gene filtering criteria.
All microarray data used within this study passed the laboratory quality assurance protocol (Burgoon et al., 2005). Microarray data were normalized using a semiparametric approach (Eckel et al., 2005), and the posterior probabilities were calculated using an empirical Bayes analysis on a per gene and time point or dose basis (Eckel et al., 2004). Gene expression data were ranked and prioritized using a P1(t) cut-off 
0.9999 and |fold change| 1.5 to identify treatment active genes and to obtain an initial subset of differentially regulated genes for further investigation and data interpretation. Relaxed filtering criteria (from P1(t) 0.9999 and |fold change| 1.5 to P1(t) 0.99 and |fold change| 1.2) were also used to examine overlapping, differentially regulated genes to minimize classifying genes as PCB126 or TCDD specific as a result of using hard cut-offs. Active genes were analyzed by agglomerative hierarchical clustering using a standard correlation distance metric implemented in GeneSpring 6.0 (Agilent Technologies; Santa Clara, CA).
Multiple features spotted on our cDNA microarray may represent the same gene (e.g., Cyp1a1). To obtain the number of unique genes, the features were first screened by their corresponding Entrez Gene IDs. If several features had the same Entrez Gene ID, they were all considered to be representative of the same gene and counted as one gene. Due to this redundancy, and because of the changes to the mouse genome annotation, the 13,361 features spotted on our cDNA microarray correspond to 8516 unique genes based on the annotation provided by UniGene build 152.
Quantitative real-time PCR.
Quantitative real-time PCR (QRTPCR) verification of microarray responses was performed as described (Boverhof et al., 2005). Briefly, 1 µg of total RNA was reverse transcribed by SuperScript II (Invitrogen) using an anchored oligo-dT primer as described by the manufacturer. The cDNA (1.0 µl) was used as a template in a 30 µl PCR reaction containing 0.1µM of forward and reverse gene-specific primers, 3mM MgCl2, 1mM dNTPs, 0.025 IU AmpliTaq Gold, and 1x SYBR Green PCR buffer (Applied Biosystems, Foster City, CA). Supplementary Table 1 provides the gene names, gene abbreviations, accession numbers, forward and reverse primer sequences, and amplicon sizes. PCR amplification was conducted on an Applied Biosystems PRISM 7500 Sequence Detection System. cDNAs were quantified using a standard curve approach and the copy number of each sample was standardized to 3 housekeeping genes (ActB, Gapdh, Hprt) to control for the differences in RNA loading, quality, and cDNA synthesis (Vandesompele et al., 2002). For graphing purposes, the relative expression levels were scaled such that the expression level of the time-matched control group was equal to one.
Dose–response modeling.
A Java application was developed to identify the best-fit dose–response model, by minimizing the Euclidean distance, for differential gene expression responses in the dose–response study. The algorithm uses particle swarm optimization (Shokooh-Saremi and Magnusson, 2007) to identify the best-fit model (i.e., the model with the parameter set that best fits the experimental data) within each of five classes (sigmoidal, exponential, linear, Gaussian, parametric), termed the best in-class model. The algorithm then chooses the best-fit of the five best in-class models. The best-fit model is used to calculate model-specific end points, such as the ED50, ED99, ED01, probabilistic point of departure, and the benchmark dose.
Functional gene annotation and statistical analysis.
Annotation and functional categorization of differentially regulated genes was performed using a Database for Annotation, Visualization and Integrated Discovery (DAVID) (Dennis et al., 2003). All statistical analyses were performed with SAS 8.02 (SAS Institute, Cary, NC). Data were analyzed by analysis of variance followed by Tukey's and Dunnett's post hoc tests. Differences between treatment groups were considered significant when p < 0.05.
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RESULTS |
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Organ and BWs
Increases in liver weight and decreases in BW gain are hallmark, dose-dependent toxic responses following treatment with TCDD and related compounds including PCB126 (Denison and Heath-Pagliuso, 1998
). Significant (p < 0.05) increases in relative liver weights (RLW) were seen with 30 µg/kg TCDD at 72 h and 300 µg/kg PCB126 at 168 h in the time course study (Table 1). In the dose–response study, PCB126 elicited modest increases in RLW that were not significant due to the greater response variance (Table 2). However, TCDD at doses of 30 and 100 µg/kg significantly (p < 0.05) increased RLW at 72 h (Table 2). No other significant treatment-related changes in BW or BW gain were observed at any time point or dose, consistent with a comparable published TCDD study (Boverhof et al., 2005), indicating that neither a single dose of 30 µg/kg TCDD nor 300 µg/kg PCB126 elicits a "wasting syndrome" response within 168 h.
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Hepatic PCB126 and TCDD Tissue Levels
Absolute hepatic PCB126 and TCDD levels per wet weight of three individual liver samples were determined at each time point and dose level. In order to facilitate comparisons between the two compounds, PCB126 TEQs were calculated by multiplying the tissue concentration by the TEF value of 0.1 (Van den Berg et al., 2006
). In the time course study, PCB126 levels continued to increase throughout the study, achieving the highest concentrations at 120 and 168 h (Fig. 1A). In contrast, TCDD levels significantly decreased after 72 h (Boverhof et al., 2005), and approached vehicle control levels by 24 weeks (Boverhof et al., manuscript in preparation). Moreover, PCB126 TEQs increased (p < 0.05) in a dose-dependent manner (Fig. 1B) at 72 h with 30 and 100 µg/kg TCDD achieving similar levels. The absolute concentrations of both PCB126 and TCDD levels from both studies are included in the Supplementary Table 2.
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Hepatic concentrations of PCB126 in this study are comparable with reports in rats using similar exposure regimens. For example, a single bolus dose of 275 µg/kg to male rats resulted in 3,300,000 pg/g of PCB126 in the liver after 7 days (Fisher et al., 2006
), similar to the 3,000,000 pg/g in this study at 168 h (Supplementary Table 2). The National Toxicology Program study on PCB126 in female rats reported that the highest concentrations of PCB126 were discovered in the liver, followed by fat, with the lowest concentrations in blood (NTP Technical Report, 2006b).
Comparison of hepatic PCB126 with published TCDD levels indicates that the TEQ levels are comparable at every time point except for 168 h, where a significant decrease in TCDD hepatic concentration was observed (Boverhof et al., 2005) (Fig. 1C). This single-dose finding is similar to a chronic exposure study (5 days/week over 13 weeks), where PCB126 was found to be sequestered within the liver to a greater extent than TCDD, which was in part mediated by binding to inducible Cyp1a2 (DeVito et al., 1998).
Pathology
Hepatocellular vacuolization was observed in vehicle, PCB126- and TCDD-treated livers, mainly in the periportal and midzonal regions and frequently extended into the centrilobular region in more severely affected animals. Affected hepatocytes were characterized by perinuclear and/or midcellular cytoplasmic loss and replacement by poorly delineated clear vacuoles (Figs. 2A–F). At 72 h TCDD-treated animals also exhibited mixed cell infiltration (neutrophils and mononuclear cells) (Fig. 2F). In the PCB126 time course vacuolization was present in both treated and control animals, however, TCDD elicited increases in vacuole formation were significantly greater than the changes in vehicles and PCB126-treated livers at all time points and doses. PCB126 elicited a pronounced dose-dependent increase in vacuolization (Figs. 2B and 2C), however, microscopic changes (vacuolization and mixed cell infiltration) elicited by TCDD were in any case more severe (Table 3, Figs. 2D and 2E) (Boverhof et al., 2005). Furthermore, PCB126 did not induce necrosis in the time course and dose–response study, whereas TCDD induced necrosis in the time course and dose–response studies (Tables 4 and 5). Together, these data indicate that 300 µg/kg PCB126 is not equivalent to 30 µg/kg TCDD at inducing histopathological changes. For detailed histopathological reports, see Supplementary Tables 3–5.
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Analysis of liver lipid extracts by TLC revealed increases in TG in TCDD and PCB126-treated animals at 72 and 168 h, respectively (Fig. 3), consistent with the increases in RLW, suggesting that fatty accumulation may contribute to increases in liver weight. These findings are consistent with general hallmarks of liver toxicity due to dioxin and PCB exposure (NTP Technical Report (2006a
,b).
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Serum samples from vehicle and PCB126-treated animals were examined for changes in TG, NEFA, CHOL, GLU, and ALT levels at 12, 24, 72, 120, and 168 h. Unlike TCDD, which significantly increased serum TG, NEFA, and ALT, and decreased CHOL levels (Boverhof et al., 2005
), PCB126 only increased (p < 0.05) ALT levels at 168 h, indicative of slight liver injury (Supplementary Fig. 1).
Temporal Gene Expression Changes
Hepatic gene expression was examined using custom mouse cDNA microarrays with 13,361 features representing 8516 unique genes. All PCB126 temporal microarray data are summarized in Supplementary Table 6. In the PCB126 time course, 294 features corresponding to 251 unique genes were differentially regulated (P1(t) 0.9999 and |fold change| 1.5) at one or more time points relative to the time-matched vehicle controls (Fig. 4A). Application of the same filtering criteria for the internal TCDD data set at 72 h identified 221 differentially regulated features, corresponding to 182 unique genes, representing approximately twice as many dysregulated genes than PCB126 (Fig. 4A).
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Fifty-eight genes regulated by 30 µg/kg TCDD and 300 µg/kg PCB126 at 72 h were identified (Supplementary Fig. 2A). TCDD-specific differential expression correlated with the emergence of inflammatory cell aggregates associated with degenerative and necrotic hepatocytes. Examples include lymphocyte antigen 6 complex, locus A (Ly6a) and complement component 1, s subcomponent (C1s) which were downregulated by TCDD, but unaffected following PCB126 treatment. Responses specific to PCB126 typically included marginal differential expression that were selected due to the hard statistical cut-offs. When the selection criteria were relaxed (P1(t)
0.99 and |fold change| 1.2), the number of overlapping genes dramatically increased (Supplementary Fig. 2B). Consequently, the majority of genes differentially expressed following PCB126 treatment simply missed the cut-offs, or were regulated at time points other than 72 h. In contrast, TCDD elicited robust gene expression responses associated with the inflammatory cell infiltration and necrosis, consistent with the histopathology assessment. Clustering of microarray data provides a general view of the similarity of the temporal profiles between the two compounds. Agglomerative hierarchical clustering of temporal gene expression data by time point resulted in three main branches: early (2 and 4 h), mid (8–24 h), and late (72–168 h) time points (Fig. 5A). The clustering of 72 h TCDD with larger clustered group of 8 + 12 h PCB126 and 18 + 24 h PCB126 indicates that the two chemicals are most similar at these time points relative to the other time points, despite the differences in intensity and gene expression patterns (Fig. 5A).
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Dose–Response Gene Expression Changes
Analysis of the dose–response data at 72 h identified 436 microarray features representing 367 unique annotated genes, which were differentially expressed (P1(t)
0.9999 and |fold change| 1.5) relative to vehicle controls, at one or more doses (Fig. 4B, Supplementary Table 7). Among the 244 genes regulated by two TCDD doses and 265 genes regulated by 1000 µg/kg PCB126, 137 genes were commonly regulated by both compounds. 249 of the 436 differentially expressed features, corresponding to 214 unique genes, exhibited a sigmoidal dose–response profile as determined by the particle swarm optimization (Shokooh-Saremi and Magnusson, 2007) Java application. This tool first examines the dose–response data for each gene using sigmoidal, exponential, linear, Gaussian, parametric classes to identify the best-fit dose–response model (i.e., the model with the parameter set that best fits the experimental data). The algorithm then chooses the model that best fits the data and calculates the ED50. ED50 values for differential gene expression ranged from 2.21 to 513 µg/kg dose of PCB126 (Supplementary Table 8). Because the PCB126 and Boverhof et al., TCDD dose–response studies were done at different time points (72 vs. 24 h, respectively), comprehensive comparisons between the ED50s are not possible. In general, PCB126 exhibited higher ED50 values for Cyp1a1 (24.5 vs. 0.3 µg/kg), Nqo1 (301.4 vs. 8.8 µg/kg), and Pck1 (144.1 vs. 0.4 µg/kg) when compared with TCDD.
Hierarchical clustering of the dose–response gene expression data clustered according to lower (1, 3 and 10 µg/kg), intermediate (30 and 100 µg/kg) and high (300 and 1000 µg/kg) PCB126 doses with 30 and 100 µg/kg TCDD clustering with the high PCB126 group (Fig. 5B).
Functional Annotation of Differentially Expressed Genes
Functional annotation of the 251 PCB126 elicited temporal gene expression changes was associated with metabolizing enzymes, lipid metabolism, glucose metabolism/gluconeogenesis, development and differentiation, necrosis, and immune signaling (Table 6). Metabolism functions included the catalytic action of monooxygenases, oxidoreductases, and xenobiotic metabolizing enzymes such as the classical TCDD-inducible "AhR gene battery" members Cyp1a1, Tiparp, and Nqo1. Others included P450 oxidoreductase (Por), epoxide hydrolase 1, microsomal (Ephx1), dehydrogenase/reductase (sdr family) member 3 (Dhrs3), glutaredoxin (Glrx), and xanthine dehydrogenase (Xdh). A majority of the glutathione S-transferase family (Gsta2, Gsta4, Gstm3, Gstt2) were also differentially regulated by PCB126. Genes associated with metabolism exhibited the highest fold change across the time course study. For example, Cyp1a1 was induced 92-fold at the 18-h time point in the PCB126 time course, whereas Tiparp was induced 14-fold at 4 h. PCB126 treatment also induced glutathione S-transferases by 1.7- to 4.4-fold at late time points.
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Lipid metabolism genes like very low density lipoprotein receptor (Vldlr), lipoprotein lipase (Lpl), apolipoprotein (Apoa1), stearoyl-Coenzyme A desaturase 1 (Scd1), arachidonate lipoxygenase 3 (Aloxe3), and sterol regulatory element binding factor 1 (Srebf1) were either induced or repressed by PCB126 treatment. For example, Srebf1 was repressed between 8–24 h, whereas Aloxe3 and Apoa1 were repressed at later time points. In contrast, Vldlr, Lpl, and Scd1 were induced at least 1.6-fold at mid and late time points.
PCB126 and TCDD (Boverhof et al., 2005) elicited gene expression changes associated with development and differentiation, included Notch gene homolog 1 (Drosophila) (Notch1), tumor necrosis factor, alpha-induced protein 2 (Tnfaip2), and Hhypoxia inducible factor 1, alpha subunit (Hif1a). Apart from Notch1, which was continuously upregulated, both Tnfaip2 and Hif1a were induced at early and mid time points with fold changes ranging from 1.7- to 3.6-fold.
Overall, PCB126 differentially regulated the same gene functions as TCDD except for changes associated with immune cell infiltration and hepatocellular necrosis, in agreement with the histopathology observations. A more thorough discussion of the association between differential gene expression and pathology has been previously published (Boverhof et al., 2005, 2006). Moreover, the number of PCB126 elicited gene expression changes was approximately five times lower than that of TCDD (Fig. 6A) when compared with a TCDD study using the same experimental design (Boverhof et al., 2005). After relaxing the filtering criteria (Fig. 6A), the number of TCDD regulated genes was significantly greater, with a majority of the common differentially expressed genes having the same temporal expression pattern (Fig. 6B). Nevertheless, at equipotent doses, based on the TEF of 0.1, 300 µg/kg PCB126 was less effective in eliciting gene expression responses when compared with 30 µg/kg TCDD.
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Verification of Microarray Responses
QRTPCR verified the temporal and dose-dependent changes in transcript levels for a selected subset of differentially regulated genes identified by microarray analysis (Figs. 7A and 7B, respectively). However, data compression was evident for Cyp1a1 due in part to the limited dynamic fluorescence intensity range (0–65,535) of microarrays, which resulted in signal saturation and compression of the true level of induction.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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The present study compared the hepatic effects of 300 µg/kg PCB126 to 30 µg/kg TCDD using a comprehensive time course and dose–response toxicogenomic approach with complementary histopathology, tissue level analysis, and clinical chemistry. In order to use a published report examining the hepatic effects of 30 µg/kg TCDD using the same experimental design (Boverhof et al., 2005
) as a comparator for this study, an equipotent dose of 300 µg/kg PCB126 was used in this study based on the WHO TEF of 0.1. Given the conserved AhR mediated mechanism of action, as well as the use of "equipotent" doses, we examined the hypothesis that 300 µg/kg PCB126 would quantitatively and qualitatively elicit comparable effects when compared with 30 µg/kg TCDD. In general, the similar elicited effects were consistent with the AhR mediated mechanism of action. However, there were also notable qualitative and quantitative differential gene expression, pathology, and clinical chemistry differences suggesting that 30 µg/kg PCB126 does not elicit comparable responses compared with 30 µg/kg TCDD.
For example, PCB126, like TCDD, significantly increased RLW. Complementary microarray, clinical chemistry, and histopathology data suggest the increased RLW was due to fatty accumulation resulting from the disruption of hepatic lipid uptake and metabolism. PCB126-induced lipoprotein lipase (Lpl) expression may involve the hydrolysis of lipids from chylomicrons and very low density lipoproteins, enabling free fatty acid accumulation (Boverhof et al., 2005). PCB126 and TCDD also induced very low density lipoprotein receptor (Vldlr), which is required for Lpl regulation. A decrease of Vldlr may result in hypertriglyceridemia associated with decreased Lpl activity (Yagyu et al., 2002). In addition, apolipoprotein A-1 was inhibited by PCB126 and TCDD, consistent with reported decreases in transcript and activity levels after hepatic fatty acid accumulation (Duplus et al., 2000). PCB126 and TCDD also induced solute carrier family 27 (fatty acid transporter), member 2 (Slc27a2), which supports hepatocellular vacuolization through transport of fatty acids into hepatocytes (Hirsch et al., 1998). Direct comparison of hematoxylin and eosin stained slides indicates that 300 µg/kg PCB126 is less effective in inducing hepatocellular vacuolization than 30 µg/kg TCDD (Boverhof et al., 2005) (Table 3) at later time points (18–168 h). This may also be attributed to the TCDD elicited differential expression of fatty acid synthase (Fasn), lipin 2 (Lpin2), low density lipoprotein receptor-related protein 2 (Lrp2), CD36 antigen (Cd36), and fatty acid binding protein 5, epidermal (Fabp5) (Boverhof et al., 2005), which were not induced by PCB126.
The presence of mixed cell infiltrates at later time points has been associated with the expression of immune signaling genes (Boverhof et al., 2005). Minimal PCB126-mediated inflammation was observed at 168 h and was not observed in the 72 h dose–response study. It was coincident with (C–C motif) ligand 22 (Ccl22) induction, which is produced in response to activated murine B lymphocytes and dendritic cells (Schaniel et al., 1998). Significant downregulation of CD3 antigen, delta polypeptide (Cd3d), and haptoglobin (Hp) at earlier time points preceded histological inflammation. CD antigens are important in select immune signaling functions, including rolling and migration, as well as T-cell activation (Lai et al., 1998), whereas inhibition of haptoglobin is involved in hepatic acute-phase response (Venteclef et al., 2006). Although Ccl22 and Cd3d were comparably regulated by PCB126 and TCDD, haptoglobin showed more significant repression only in response to PCB126. In contrast, TCDD-induced mixed cell infiltration was observed at 72 and 168 h in the time course study and in the dose–response study at 100 µg/kg (Boverhof et al., 2005). Lymphocyte antigen 6 complex, locus A (Ly6a), CD44 antigen (Cd44) involved in T-cell activation (Sumoza-Toledo and Santos-Argumedo, 2004) as well as the major histocompatibility complex (MHC) class II genes, H2-Ab1 and H2-Eb1, involved in mediating antigen presentation and processing (Alfonso et al., 2001) were differentially regulated by TCDD. However, none of these genes were regulated in response to PCB126, suggesting that they are key players in eliciting TCDD-induced hepatic inflammation. The mixed cell infiltrates are likely a response to tissue damage and therefore, the late regulation of immune signaling genes is not directly mediated by the AhR. The attenuated immune signaling gene responses relative to 30 µg/kg TCDD are further evidence of a weaker hepatic response to 300 µg/kg PCB126.
A hallmark of TCDD exposure is feed refusal, BW loss and depletion of energy stores commonly referred to as "wasting syndrome" (Denison and Heath-Pagliuso, 1998). However, because pair-fed animals still experience wasting, feed refusal alone is not sufficient to account for the effect. Like TCDD, PCB126 repressed several gluconeogenesis genes including phosphoenolpyruvate carboxykinase 1 (Pck1), glycerol phosphate dehydrogenase 2, mitochondrial (Gpd2), as well as glutamate oxaloacetate transaminase 1, soluble (Got1), albeit at lower efficacy. For the genes involved in gluconeogenesis, 30 µg/kg TCDD caused more significant repression than 300 µg/kg PCB126. Even though there were no significant changes in BW or BW gain at the doses used, the inhibition of gluconeogenesis may still contribute to hepatotoxicity and an eventual wasting effect (Viluksela et al., 1995).
TCDD also induced minimal to slight necrotic changes at 72 h in this study and in the published TCDD study at 72 and 168 h. Although, there was no evidence of necrosis in the PCB126 time course and dose–response sections, a number of genes involved in necrosis and apoptosis, including BCL2/adenovirus E1B interacting protein 1, NIP3 (Bnip3), hungtingtin interacting protein 1 (Hip1), and myelocytomatosis oncogene (Myc) were regulated by PCB126. Even though upregulation of Hip1 activates apoptosis (Gervais et al., 2002) and Bnip3 mediates apoptosis and oncosis in rodent models (Copple et al., 2004), 300 µg/kg and higher doses of PCB126 were not sufficient to cause necrosis further indicating that 300 µg/kg PCB126 does not elicit comparable effects when compared with 30 µg/kg TCDD.
Other genes of interest also exhibited lower induction by PCB126 when compared with TCDD treatment. For example, members of the "AhR gene battery" were induced by PCB126 and TCDD including Cyp1a1, Nqo1, and Xdh. Their induction serves an important role in detoxification, but may also contribute to reactive oxygen species formation, leading to cellular oxidative stress and DNA fragmentation (Barouki and Morel, 2001; Boverhof et al., 2005; Zimmerman and Granger, 1994). The induction of reactive oxygen species–generating enzymes was accompanied by increases in glutathione transferases (Gsta1, Gsta4, Gstp1, Gstt2, Gstm3). This contributes to the biotransformation of xenobiotics by catalyzing the conjugation of reduced glutathione to electrophiles and products of oxidative stress to facilitate their excretion (Raza et al., 2002). In addition, Notch1 and Tnfaip2, were induced by PCB126 and TCDD. Both exhibit specific patterns of expression in the developing liver, and have been implicated in tissue development (Harper et al., 2003). Their role in hepatotoxicity is unknown, but may be important in normal AhR signaling during hepatic development, because AhR null mice have reduced liver size and distorted hepatic vasculature (Lahvis et al., 2000). Although these genes were differentially expressed by both compounds with similar profiles, in general, PCB126 elicited differential gene expression was lower and for a shorter duration, again suggesting that 300 µg/kg PCB126 does not elicit responses comparable to 30 µg/kg TCDD.
In summary, there were significant qualitative and quantitative differences in the effects elicited by PCB126 when compared with TCDD. This included differences in gene expression, histopathology, and clinical chemistry. Overall, 30 µg/kg TCDD elicited greater fold changes in gene expression compared with 300 µg/kg PCB126. In addition, vacuolization, necrosis, and mixed cell infiltration were less pronounced in mice treated with 300 µg/kg PCB126 compared to 30 µg/kg TCDD. Furthermore, unlike TCDD, PCB126 did not alter circulating NEFA, TG, CHOL, and GLU levels. These differences can not be attributed to metabolism because PCB126 tissue levels were comparable and even exceeded TCDD levels at later time points according to HRGC/HRMS tissue analysis.
The data collectively and consistently indicate that 300 µg/kg PCB126 does not elicit responses comparable to 30 µg/kg TCDD, suggesting that the 0.1 TEF for PCB126 is an overestimate of its hepatotoxicity in immature, ovariectomized C57BL/6 mice. However, other studies have found that the mammalian TEF of 0.1 for PCB126 accurately reflects its toxicity (NTP Technical Report, 2006b). Therefore, outside the context of other studies and given that the TEF is a point estimate that may vary by a half-log unit; additional research is needed to warrant adjusting the PCB126 TEF of 0.1. There are significant species differences in toxicity (Huang and Gibson, 1992), and sensitivity (Silkworth et al., 2005), as well as pharmacokinetic differences between congeners (DeVito et al., 1998; Safe, 1997), that must also be considered. Other factors, including the lack of positionally conserved DREs (Sun et al., 2004), and nonadditive (antagonistic) interactions (DeVito et al., 1998; Safe, 1997), also confound the establishment of appropriate TEFs for human and wildlife risk assessment. Consequently, there continue to be significant gaps in knowledge regarding the validity of the PCB126 TEF and the accuracy of the 0.1 point estimate (Van den Berg et al., 2006). In order to determine a more accurate TEF for the hepatotoxicity of PCB126, comprehensive dose–response studies at multiple times optimal for each specific to endpoints are required. Ideally, these would be comparative and include multiple species account for species specific differences that may not be relevant to human or wildlife toxicity (Boverhof et al., 2006; Sun et al., 2004).
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SUPPLEMENTARY DATA |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Supplementary data 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 Environmental Health Sciences Superfund Basic Research Program (P42ES04911).
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ACKNOWLEDGMENTS |
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We would specially like to thank colleagues Edward Dere and Joshua Kwekel for critical reading of this manuscript.
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