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Acute Gene Induction by Tienilic Acid in the Male Sprague Dawley Rat: Possible Role for Danger

ABSTRACT: Tienilic acid (TA) was withdrawn from the US market due to numerous cases of liver necrosis. Two major hypotheses currently used to understand the mechanisms of idiosyncratic reactions such as TA-induced hepatotoxicity are the hapten and danger hypotheses. Both human cytochrome (CYP) P450 2C9 and the rat ortholog CYP 2C11 metabolize TA, and it was reported that a reactive metabolite of TA binds almost exclusively to these enzymes, thus acting as a mechanism-based inhibitor. TA-induced liver toxicity is associated with antibodies against CYP 2C9, thus TA appears to act as a hapten. However, if the binding were limited to CYP 2C, it is unlikely that this would lead to significant cell stress. Thus, if TA does not cause cell stress it would suggest that a drug does not have to generate a “danger signal” in order to cause an idiosyncratic drug reaction and acting as a hapten is sufficient. In order to test whether TA can cause cell stress, male Sprague Dawley rats were orally dosed with TA, and hepatic gene expression was profiled at 6 and 24 h after drug administration. Results showed that TA induced robust changes in genes involved in oxidative stress (e.g. aldo-keto reductase, glutathione-S-transferase, thioredoxin reductase, epoxide hydrolase), inflammation and cytotoxicity (e.g. IL-1b, interferon regulatory factor 1, macrophage stimulating protein 1, caspase-12), as well as those promoting liver regeneration (e.g. p27Kip1, DUSP6, serine dehydratase, spectrin bII, inhibin bA). These data support the hypothesis that danger signals in the form of cell-stress proteins and the generation of oxidative stress may be involved in initiating the immune response observed in TA-induced toxicity. Other recent data also indicate that the reactive metabolite of TA binds to other proteins in addition to CYP 2C Keywords: time course, stress response

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE8252 | GEO | 2007/06/23

SECONDARY ACCESSION(S): PRJNA101199

REPOSITORIES: GEO

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Project description:Tienilic acid (TA) was withdrawn from the US market due to numerous cases of liver necrosis. Two major hypotheses currently used to understand the mechanisms of idiosyncratic reactions such as TA-induced hepatotoxicity are the hapten and danger hypotheses. Both human cytochrome (CYP) P450 2C9 and the rat ortholog CYP 2C11 metabolize TA, and it was reported that a reactive metabolite of TA binds almost exclusively to these enzymes, thus acting as a mechanism-based inhibitor. TA-induced liver toxicity is associated with antibodies against CYP 2C9, thus TA appears to act as a hapten. However, if the binding were limited to CYP 2C, it is unlikely that this would lead to significant cell stress. Thus, if TA does not cause cell stress it would suggest that a drug does not have to generate a âdanger signalâ in order to cause an idiosyncratic drug reaction and acting as a hapten is sufficient. In order to test whether TA can cause cell stress, male Sprague Dawley rats were orally dosed with TA, and hepatic gene expression was profiled at 6 and 24 h after drug administration. Results showed that TA induced robust changes in genes involved in oxidative stress (e.g. aldo-keto reductase, glutathione-S-transferase, thioredoxin reductase, epoxide hydrolase), inflammation and cytotoxicity (e.g. IL-1b, interferon regulatory factor 1, macrophage stimulating protein 1, caspase-12), as well as those promoting liver regeneration (e.g. p27Kip1, DUSP6, serine dehydratase, spectrin bII, inhibin bA). These data support the hypothesis that danger signals in the form of cell-stress proteins and the generation of oxidative stress may be involved in initiating the immune response observed in TA-induced toxicity. Other recent data also indicate that the reactive metabolite of TA binds to other proteins in addition to CYP 2C Experiment Overall Design: Rats were orally dosed with TA or control sodium bicarbonate. Eight animals were divided into two groups: Group 1 consisted of two animals that received a single dose of TA, and the two control animals received only sodium bicarbonate. Group 1 was sacrificed 6 h after drug/vehicle administration. Group 2 also comprised of two animals, and they were given two doses of TA (given at t=0 and t=16 h) and sacrificed at 24 h. Control rats in group 2 received two oral doses of sodium bicarbonate. For microarray analysis, six Affymetrix chips were used: Group 1 samples (3 chips) was comprised of pooled RNA from control rats C1 and C2, and an individual chip for each treated animal T1 and T2; Group 2 (3 chips) pooled RNA from control rats C3 and C4, and an individual chip for each treated animal T3 and T4.

Project description:Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics and drug-drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically-labelled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labelled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II and 8 control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11 and 3a13, carboxylesterase (Ces) 2a and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of import to xenobiotic metabolism, with improved utility for the study of drug pharmacokinetics, pharmacodynamics, and of chemically-treated and genetically-modified mouse models.

Project description:Direct comparison of the genome-level expression patterns of THP-1 cells exposed to either LPS or heat shock; Peripheral blood mononuclear cells (PBMC) serve a sentinel role allowing the host to efficiently sense and adapt to the presence of danger signals. Herein we have directly compared the genome-level expression patterns (microarray) of human PBMC (THP-1 cells) subjected to one of two canonical danger signals, heat shock and lipopolysaccharide (LPS). Based on sequential expression and statistical filters, and in comparison to control cells, we found that 3,988 genes were differentially regulated in THP-1 cells subjected to LPS stress, and 2,921 genes were differentially regulated in THP-1 cells subjected to heat shock stress. Venn analyses demonstrated that the majority of differentially regulated genes (greather than or equal to 70%) were uniquely expressed in response to one of the two danger signals. Functional analyses demonstrated that the two danger signals induced expression or repression of genes corresponding to unique pathways, molecular functions, biological processes, and gene networks. In contrast, there were 184 genes that were commonly upregulated by both stress signals, and 430 genes that were commonly downregulated by both stress signals. Interestingly, the 184 commonly upregulated genes corresponded to a gene network broadly related to inflammation, and more specifically to chemokine signaling. These data demonstrate that the mononuclear cell responses to the canonical stress signals, heat shock and LPS, are highly divergent. However, there is a heretofore unrecognized common pattern of gene network expression corresponding to chemokine-related biology. The data also serve as a reference database for investigators in the field of stress signaling. Experiment Overall Design: In vitro exposure of THP-1 cells to control conditions, LPS (1 micogram/ml for 4 hrs), or heat shock (43 deg C for 1 hour, followed by a 4 hour recovery at 37 deg C)

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Acute Gene Induction by Tienilic Acid in the Male Sprague Dawley Rat: Possible Role for Danger

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