A Novel Statistical Algorithm for Gene Expression Analysis Helps Differentiate Pregnane X Receptor-Dependent and Independent Mechanisms of Toxicity

Genome-wide gene expression profiling has become standard for assessing potential liabilities as well as for elucidating mechanisms of toxicity of drug candidates under development. Analysis of microarray data is often challenging due to the lack of a statistical model that is amenable to biological variation in a small number of samples. Here we present a novel non-parametric algorithm that requires minimal assumptions about the data distribution. Our method for determining differential expression consists of two steps: 1) We apply a nominal threshold on fold change and platform p-value to designate whether a gene is differentially expressed in each treated and control sample relative to the averaged control pool, and 2) We compared the number of samples satisfying criteria in step 1 between the treated and control groups to estimate the statistical significance based on a null distribution established by sample permutations. The method captures group effect without being too sensitive to anomalies as it allows tolerance for potential non-responders in the treatment group and outliers in the control group. Performance and results of this method were compared with the Significant Analysis of Microarrays (SAM) method. These two methods were applied to investigate hepatic transcriptional responses of wild-type (PXR+/+) and pregnane X receptor-knockout (PXR−/−) mice after 96 h exposure to CMP013, an inhibitor of β-secretase (β-site of amyloid precursor protein cleaving enzyme 1 or BACE1). Our results showed that CMP013 led to transcriptional changes in hallmark PXR-regulated genes and induced a cascade of gene expression changes that explained the hepatomegaly observed only in PXR+/+ animals. Comparison of concordant expression changes between PXR+/+ and PXR−/− mice also suggested a PXR-independent association between CMP013 and perturbations to cellular stress, lipid metabolism, and biliary transport

Serum perfluorooctanoic acid (PFOA) concentrations in normal and hyperlipidemic female hamsters dosed orally with ammonium perfluorooctanoate (APFO) for up to 30 days

In epidemiology studies, the presence of perfluorooctanoate (PFOA) in human blood has been associated with higher serum cholesterol concentrations. A possible explanation for these results is that elevated serum cholesterol might reduce clearance of PFOA. In this study, female hamsters, which transport and regulate cholesterol in a manner similar to humans, were fed normal diet or diet supplemented with 0.05% cholesterol and 10% coconut oil (high-fat diet) resulting in hyperlipidemia throughout the study in supplemented animals. Hamsters on either a normal and high-fat diet were given oral doses of 0.1, 1.0, or 10 mg APFO/kg for 30 days. Serum PFOA concentrations evaluated 24 h after 1, 10, 20, and 30 doses of APFO were not altered in hyperlipidemic hamsters compared to those fed normal diet. For a given dose group, serum concentrations of PFOA were highest following the 10 doses (except for the 10 mg/kg group where concentrations were the highest after the first dose) and were lowest after 20 and 30 doses. Under the condition of this study, higher serum lipids did not affect the absorption and clearance of serum PFOA. Serum PFOA concentrations declined over the course of the study despite continued daily dosing with APFO. This does not support the hypothesis that higher serum lipids might increase the retention of PFOA in the body

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Organic iodides have been shown to induce thyroid hypertrophy and increase alterations in colloid in rats, although the mechanism involved in this toxicity is unclear. To evaluate the effect that free iodide has on thyroid toxicity, we exposed rats for 2 weeks by daily gavage to sodium iodide (NaI). To compare the effects of compounds with alternative mechanisms (increased thyroid hormone metabolism and decreased thyroid hormone synthesis, respectively), we also examined phenobarbital (PB) and propylthiouracil (PTU) as model thyroid toxicants. Follicular cell hypertrophy and pale-staining colloid were present in thyroid glands from PB-treated rats, and more severe hypertrophy/colloid changes along with diffuse hyperplasia were present in thyroid glands from PTU-treated rats. In PBand PTU-treated rats, thyroid-stimulating hormone (TSH) levels were significantly elevated, and both thyroxine and triiodothyronine hormone levels were significantly decreased. PB induced hepatic uridine diphosphate-glucuronyltransferase (UDPGT) activity almost 2-fold, whereas PTU reduced hepatic 5´-deiodinase I (5´-DI) activity to < 10% of control in support of previous reports regarding the mechanism of action of each chemical. NaI also significantly altered liver weights and UDPGT activity but did not affect thyroid hormone levels or thyroid pathology. Thyroid gene expression analyses using Affymetrix U34A GeneChips, a regularized t-test, and Gene Map Annotator and Pathway Profiler demonstrated significant changes in rhodopsin-like G-protein-coupled receptor transcripts from all chemicals tested. NaI demonstrated dose-dependent changes in multiple oxidative stress-related genes, as also determined by principal component and linear regression analyses. Differential transcript profiles, possibly relevant to rodent follicular cell tumor outcomes, were observed in rats exposed to PB and PTU, including genes involved in Wnt signaling and ribosomal protein expression. could be obtained that correlate with clinical and pathological end points in rats, and determine whether profiles are predictive of the carcinogenic potential of each chemical in rats. Materials and Methods In Vivo Studies Adult male Crl:CD (SD)IGS BR rats, approximately 8 weeks of age, were treated with NaI, PB, and PTU for 14 consecutive days. NaI, PB, and PTU were purchased from Sigma Chemical Company (St. Louis, MO). Rats (n = 20/group) were dosed by oral gavage with vehicle (water or 0.25% methylcellulose), NaI (0.1, 1, 10, or 100 mg/kg/day), PB (100 mg/kg/day), or PTU (10 mg/kg/day) at a dose volume of 5 mL/kg. NaI was dissolved in water, whereas PB and PTU were dissolved in methylcellulose. On day 15, all rats were euthanized by carbon dioxide anesthesia and exsanguination. Blood samples were collected from the inferior vena cava of each animal at necropsy to measure serum levels of TSH, T 4 , T 3 , and reverse T 3 (rT 3 ). Terminal body, thyroid gland, and liver weights were recorded for the first 10 animals of each dose group. The thyroid gland and surrounding tissue from the first 10 animals of each dose group were processed for histopathological evaluation. A liver sample from the first five animals of each dose group was processed to measure 5´-deiodinase I (5´-DI) and uridine diphosphateglucuronyltransferase (UDPGT) activity. Thyroid glands from the last 10 animals (five from methylcellulose group) from each dose group were removed and placed in RNALater (Ambion, Austin, TX) overnight at 4°C. The next day, thyroids were removed from the RNALater and stored at -80°C until processed for total RNA. The research described in this publication was conducted in a laboratory accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International, and the investigators complied with the regulations and standards of the Animal Welfare Act and adhered to the principles of the Guide for the Care and Use of Laboratory Animals (National Research Council 1996). Pathological Evaluations After euthanization the thyroid glands and surrounding tissue from the first 10 animals from each group were removed and placed into formalin fixative for at least 48 hr before trimming and weighing. After fixation, one individual performed a final dissection under a dissecting microscope. This was done in order to reduce the variability of the dissection procedure, thereby reducing the variability of the thyroid gland weights. Organ weights were calculated relative to body weight. The formalin-fixed thyroid glands were examined microscopically. Hormonal Measurements Blood was collected at the time of euthanization from all animals. Serum was prepared and stored between -65°C and -85°C until analyzed for serum hormone concentrations. Serum TSH (Amersham Biosciences Corp., Piscataway, NJ), T 3 and T 4 (Diagnostic Products Corp., Los Angeles, CA), and rT 3 (Polymedco Corp., Cortlandt Manor, NY) concentrations were measured using commercially available RIA kits. Microsomal Preparations At necropsy, a section of the liver from the first five animals from each group was removed, and hepatic microsomes were prepared for biochemical evaluation. A portion of the liver was homogenized (1 g tissue/8 mL buffer) in buffer containing 50 mM Tris-HCl, 0.25 M sucrose, and 5.4 mM EDTA, pH 7.4. The homogenates were centrifuged at 15,000 × g for 15 min at 4°C. The resulting supernatants were removed and centrifuged at 100,000 × g for 70 min at 4°C; these pellets contained the microsomal fractions. The microsomal pellets were resuspended in the homogenization buffer at a protein concentration of 10-20 mg/mL, aliquoted, and stored between -65°C and -85°C until analyzed for UDPGT and 5´-DI. The protein content of the microsomes was measured before and after analyses by the BioRad method 5´-Deiodenase I Measurements Microsomal 5´-DI activity was determined using modifications of the methods of PazosMoura et al. (1991) and UDPGT Measurements Microsomal UDPGT activity was determined spectrophotometrically using a modification of the method of Microarray Analysis RNA preparation and analysis was done according to the Affymetrix-recommended protocol (Affymetrix 2002). Briefly, total RNA from four animals from each dose group was prepared individually using the TRIzol procedure (Invitrogen, Carlsbad, CA) and cleaned using the Qiagen RNeasy mini RNA cleanup protocol (Qiagen, Valencia, CA). The integrity of each RNA sample was determined using an Agilent 2100 Bioanalyzer (Agilent, Foster City, CA). After this, double-stranded cDNA from three of the four samples was prepared from 16 µg of total RNA using Superscript II reverse transcriptase (Invitrogen) and a T7 primer (Genset, Boulder, CO) for first-strand synthesis, and DNA polymerase and ligase (Invitrogen) for second-strand synthesis. Subsequently, labeled cRNA was synthesized from the cDNA using the Enzo RNA transcript labeling kit (Affymetrix, Santa Clara, CA) according to the manufacturer's instructions. Approximately 20 µg of biotin-labeled cRNA was then fragmented in a solution of 40 mM Tris-acetate, pH 8.1, 100 mM KOAc, and 30 mM MgOAc at 94°C for 35 min. Labeled cRNA was hybridized to the Affymetrix GeneChip Test2 Array (Affymetrix) to verify the quality of labeled cRNA. After this, cRNA was hybridized to the Affymetrix Rat Genome U34A GeneChip Probe Array (RG-U34A; Affymetrix). The cRNA in hybridization Gene expression profiles of rat thyroid toxicity Environmental Health Perspectives • VOLUME 113 | NUMBER 10 | October 2005 1355 cocktail was incubated overnight at 45°C while rotating in a hybridization oven. After approximately 16 hr of hybridization, the cocktail was removed and the arrays were washed and stained in a Fluidics Station 400 (Affymetrix) according to the Affymetrixrecommended protocol (Affymetrix 2002). Briefly, several cycles of washes were done initially with a nonstringent buffer (1 M NaCl, 67 mM NaH 2 PO 4 , 6.7 mM EDTA, 0.01% Tween 20) at 25°C and then with stringent buffer [100 mM MES, 0.1 M Na + , 0.01% Tween 20] at 50°C. The arrays were then stained in streptavidin phycoerythrin (SAPE) solution (10 µg/mL SAPE, 2 mg/mL acetylated BSA, 100 mM MES, 1 M Na + , 0.05% Tween 20) at 25°C, washed in nonstringent buffer, stained in antibody solution (2 mg/mL acetylated BSA, 100 mM MES, 1 M [Na + ], 0.05% Tween 20, 0.1 mg/mL normal goat IgG, 3 µg/mL anti-streptavidin biotinylated antibody) at 25°C, stained again in SAPE solution at 25°C, and then washed again in nonstringent buffer at 30°C. Arrays were then scanned on a GeneArray scanner (Agilent). Image analysis, quantification of raw gene expression values, mismatched probe background subtraction, and present/absent calls were performed using the Microarray Suite software (version 5.0; Affymetrix). Data Analysis Differential gene expression was determined by the regularized t-test, which uses a Bayesian procedure (Baldi and Long 2001). Briefly, the expression level of each gene is assumed to be from a normal distribution with µ and σ 2 . Using a conjugate prior, the mean of the posterior (MP) estimate of µ is the sample mean. The MP estimate of σ 2 is where n is the sample size, s 2 is the sample variance, v 0 is the degrees of freedom of the prior (a value of 10 is used in the analysis), and σ 0 2 is the mean of sample variances of genes in the neighborhood of the gene under consideration. The neighborhood is the 50 genes with sample means immediately above and below the sample mean of the gene under consideration; that is, the neighborhood consists of the 101 genes centered on the gene. After the MP estimates of µ and σ 2 are obtained, the t-test of unequal variances is used to calculate a p-value of differential expression. Multiple linear regressions are used to determine dose-dependent expression after NaI treatments of 0.1, 1, 10, or 100 mg/kg/day. Some genes respond to NaI linearly, but for other genes, the induction or repression of expression may become saturated after some dose levels. Therefore, two types of multiple linear regressions were performed. The first type was the linear regression of the gene expression levels and the dose levels, and the other type was the linear regression of the gene expression levels and the logarithms of the dose levels. A principal component analysis was also performed on the data. Three animals were measured within each treatment for each gene. The treatment means were then subjected to principal component analysis. The components were thus determined on a per-treatment basis rather than a per-gene basis, as in Results Liver Weights and Hormone Metabolism After the 2-week exposure period, liver weights were increased in a dose-dependent manner and were significantly higher in rats administered 10 and 100 mg/kg/day NaI (8-13% increase) and 100 mg/kg/day PB (44% increase) compared with control rats that received water alone UDPGT activity was significantly higher (99% increase) in rats administered 100 mg/ kg/day PB compared with controls ( Thyroid Hormone Levels and Histopathology Treatment-related effects on thyroid hormone levels were observed in the 100 mg/kg/day PB and 10 mg/kg/day PTU groups. Compared with controls, T 3 , T 4 , and rT 3 levels were reduced 23, 40, and 28%, respectively, in PBtreated rats and 80, 99, and 56%, respectively, in PTU-treated rats Treatment-related changes in thyroid gland histopathology were observed in the PB and PTU treatment groups gland weights (percent of body weight) were also significantly increased (~3-fold) in the PTU treatment group compared with controls Thyroid Gland Gene Expression Principal component analysis. Thyroid gene expression data were analyzed using principal component analysis, a regularized t-test and multiple linear regressions. Principal component analysis of gene expression data from all 24 samples demonstrated grouping according to treatment. Six principal components were identified To understand these principal components, it is helpful to express each as a linear combination of the means of the six treatments Large negative values (i.e., negative numbers large in absolute value) of this component tend to be associated with down-regulation in one of more NaI treatments. The second component is an indicator for an effect due to PB and PTU. A large value of Pcomp2 (principal component 2) indicates an up-regulation, whereas a large negative value indicates a downregulation. Component 3 is primarily a contrast between the PB and PTU treatments. The fourth principal component is primarily an indicator of effect at low doses of NaI. Based on these principal component analysis findings, further gene ontology work was directed to the first two principal components, namely, genomic profiles associated with NaI exposure or PB and PTU exposure. Multiple linear regressions. Dose-dependent expression, as determined by multiple linear regressions, was observed after NaI treatment. Transcript levels most influenced by dose (p ≤ 0.001), included the NIS [Slc5a5; GenBank accession no. U60282; (http:// www.ncbi.nlm.gov)] and antioxidant enzymes such as glutathione peroxidase 2 (Gpx2), thioredoxin reductase (Txnrd1), and glutathione S-transferase pi (GST-pi; Gstp2) ( Regularized t-test (Bayesian procedure). In a separate analysis using the regularized t-test, 872, 948, and 1552 gene transcripts (of 8,740 transcripts present in all samples) were significantly (p < 0.01) changed by 100 mg/kg/day NaI, 100 mg/kg/day PB, and 10 mg/kg/day PTU administration compared with controls, respectively. To further characterize these genomic changes according to biological function and identify molecular pathways involved in the mode of action of each chemical, these gene lists were uploaded into GenMAPP (Gene Map Annotator and Pathway Profiler, version 1.0

Molecular Characterization of Thyroid Toxicity: Anchoring Gene Expression Profiles to Biochemical and Pathologic End Points-1

<p><b>Copyright information:</b></p><p>Taken from "Molecular Characterization of Thyroid Toxicity: Anchoring Gene Expression Profiles to Biochemical and Pathologic End Points"</p><p>Environmental Health Perspectives 2005;113(10):1354-1361.</p><p>Published online 12 May 2005</p><p>PMCID:PMC1281279.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.</p

Molecular Characterization of Thyroid Toxicity: Anchoring Gene Expression Profiles to Biochemical and Pathologic End Points-0

<p><b>Copyright information:</b></p><p>Taken from "Molecular Characterization of Thyroid Toxicity: Anchoring Gene Expression Profiles to Biochemical and Pathologic End Points"</p><p>Environmental Health Perspectives 2005;113(10):1354-1361.</p><p>Published online 12 May 2005</p><p>PMCID:PMC1281279.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.</p