Unknown,Transcriptomics,Genomics,Proteomics

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Minor compensatory changes in SAGE Mdr1a (P-gp), Bcrp, and Mrp2 knockout rats do not detract from their utility in the study of transporter-mediated pharmacokinetics.

ABSTRACT: Mdr1a-, Bcrp-, and Mrp2-knockout rats are a more practical species for ADME studies than murine models and previously demonstrated expected alterations in pharmacokinetics of various probe substrates. At present, gene expression and pathology changes were systematically studied in small intestine, liver, kidney, and brain tissue from male SAGE Mdr1a-, Bcrp-, and Mrp2-knockout rats versus wild-type Sprague Dawley controls. Gene expression data supported the relevant knockout genotype. As expected, Mrp2-knockout rats were hyperbilirubinemic and exhibited upregulation of hepatic Mrp3. Overall, few alterations were observed within 137 ADME-relevant genes. The two most consequential changes were upregulation of intestinal carboxylesterase in Mdr1a-knockouts and catechol-O-methyltransferase in all tissues of Bcrp-knockout rats. Previously reported upregulation of hepatic Mdr1b P-glycoprotein in proprietary Wistar Mdr1a-knockout rats was not observed in the SAGE counterpart investigated herein. Relative liver and kidney weights were 22-53% higher in all three knockouts, with microscopic increases in hepatocyte size in Mdr1a- and Mrp2-knockout rats, and glomerular size in Bcrp- and Mrp2-knockouts. Increased relative weight of clearing organs is quantitatively consistent with reported increases in clearance of drugs that are not substrates of the knocked-out transporter. Overall, SAGE knockout rats demonstrated modest compensatory changes, which do not preclude their general application to study transporter-mediated pharmacokinetics. However until future studies elucidate the magnitude of functional change, caution is warranted in rare instances of extensive metabolism by catechol-O-methyltransferase in Bcrp-knockouts and intestinal carboxylesterase in Mdr1a-knockout rats, specifically for molecules with free catechol groups and esters subject to gut wall hydrolysis. 3 groups of knockout animals compared to wild-type male Sprague Dawley control rats. 4 animals per group. Assayed the following tissues: brain, duodenum, ileum, jejunum, kidney (cortex-anterior pole), kidney (medulla), liver. Hybridized to Affymetrix Rat Genome 230 v2 [Rat230_2] arrays.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Keith Goldstein

PROVIDER: E-GEOD-44962 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:This study assessed gene expression changes associated with inflammation in the colon tissue of multi-drug resistance targeted mutation (Mdr1a-/-) mice, a model of human IBD. It also tested the anti-inflammatory potential of curcumin in the colon of these mice with a particular focus on the proteome. A colonic histological injury score was determined for each mouse to assess the level of colon inflammation in Mdr1a-/- mice, and to establish the effect of curcumin on inflammation. Insights into mechanisms of colonic inflammation in the Mdr1a-/- mice were gained using transcriptome (microarray) and proteome (2-D gel electrophoresis and LCMS protein identification) analyses. These data were compared to changes in the colon proteome in response to curcumin, and to previously described transcriptomic analyses [Nones et al, PMID: 18761777; GSE10684] in response to curcumin, in this model. A total of 24 male Mdr1a-/- mice, and 24 male FVB/N mice, purchased from Taconic (Hudson, NY, USA) at 4-5 weeks of age, were used for this study. 12 mice were randomly assigned to each of two different dietary groups: control (AIN-76A powdered diet), or curcumin (AIN-76A + 0.2% curcumin). At 21 and 24 weeks of age, mice were euthanized and colon samples taken for histological, microarray, and proteomics analyses. The total histology score (HIS) in the colon was determined according to previously described criteria. Total RNA was isolated using TRIzolM-BM-. reagent. Colon RNA from four Mdr1a-/- mice (with high HIS) on the control diet (i.e. AIN-76A powdered diet) was compared with colon RNA from four FVB/N mice with low HIS also on the control diet (i.e. AIN-76A powdered diet). All individual RNA samples were hybridized against a common reference RNA on separate slides. The reference RNA was prepared using equimolar RNA extracts from small intestine, colon, kidney and liver of normal healthy growing Swiss mice plus RNA extracts from Swiss mouse fetuses. These data were compared with previously reported transcriptomics analysis of colon RNA from the same four Mdr1a-/- mice on the AIN-76A diet (with high HIS) compared with colon RNA from four Mdr1a-/- mice on the curcumin diet (with low HIS; Nones et al 2009, PMID: 18761777)

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Minor compensatory changes in SAGE Mdr1a (P-gp), Bcrp, and Mrp2 knockout rats do not detract from their utility in the study of transporter-mediated pharmacokinetics.

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