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High-density real-time PCR-based in vivo toxicogenomic screen to predict organ-specific toxicity.

ABSTRACT: Toxicogenomics, based on the temporal effects of drugs on gene expression, is able to predict toxic effects earlier than traditional technologies by analyzing changes in genomic biomarkers that could precede subsequent protein translation and initiation of histological organ damage. In the present study our objective was to extend in vivo toxicogenomic screening from analyzing one or a few tissues to multiple organs, including heart, kidney, brain, liver and spleen. Nanocapillary quantitative real-time PCR (QRT-PCR) was used in the study, due to its higher throughput, sensitivity and reproducibility, and larger dynamic range compared to DNA microarray technologies. Based on previous data, 56 gene markers were selected coding for proteins with different functions, such as proteins for acute phase response, inflammation, oxidative stress, metabolic processes, heat-shock response, cell cycle/apoptosis regulation and enzymes which are involved in detoxification. Some of the marker genes are specific to certain organs, and some of them are general indicators of toxicity in multiple organs. Utility of the nanocapillary QRT-PCR platform was demonstrated by screening different references, as well as discovery of drug-like compounds for their gene expression profiles in different organs of treated mice in an acute experiment. For each compound, 896 QRT-PCR were done: four organs were used from each of the treated four animals to monitor the relative expression of 56 genes. Based on expression data of the discovery gene set of toxicology biomarkers the cardio- and nephrotoxicity of doxorubicin and sulfasalazin, the hepato- and nephrotoxicity of rotenone, dihydrocoumarin and aniline, and the liver toxicity of 2,4-diaminotoluene could be confirmed. The acute heart and kidney toxicity of the active metabolite SN-38 from its less toxic prodrug, irinotecan could be differentiated, and two novel gene markers for hormone replacement therapy were identified, namely fabp4 and pparg, which were down-regulated by estradiol treatment.

SUBMITTER: Fabian G

PROVIDER: S-EPMC3189772 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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High-density real-time PCR-based in vivo toxicogenomic screen to predict organ-specific toxicity.

Fabian Gabriella G Farago Nora N Feher Liliana Z LZ Nagy Lajos I LI Kulin Sandor S Kitajka Klara K Bito Tamas T Tubak Vilmos V Katona Robert L RL Tiszlavicz Laszlo L Puskas Laszlo G LG

International journal of molecular sciences 20110919 9

Toxicogenomics, based on the temporal effects of drugs on gene expression, is able to predict toxic effects earlier than traditional technologies by analyzing changes in genomic biomarkers that could precede subsequent protein translation and initiation of histological organ damage. In the present study our objective was to extend in vivo toxicogenomic screening from analyzing one or a few tissues to multiple organs, including heart, kidney, brain, liver and spleen. Nanocapillary quantitative re ...[more]

PMID: 22016648

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Project description:Purpose: Transvaginal meshes for the treatment of Pelvic Organ Prolapse (POP) have been associated with severe adverse events and have been banned for clinical use in many countries. We recently reported the design of degradable poly (L-lactic acid)-co-poly (e-caprolactone) nanofibrous mesh (P nanomesh) bioengineered with endometrial mesenchymal stem/stromal cells (eMSC) for POP repair. We showed that such bioengineered meshes had high tissue integration as well as immunomodulatory effects in vivo. This study aimed to determine the key molecular players enabling eMSC- based foreign body response modulation. Methods: SUSD2+ eMSC were purified from single cell suspensions obtained from endometrial biopsies from cycling women by magnetic bead sorting. Electrospun P nanomeshes with and without eMSC were implanted in a NSG mouse skin wound repair model for 1 and 6 weeks. Quantitative PCR was used to assess the expression of extracellular matrix (ECM), cell adhesion, angiogenesis and inflammation genes as log2 fold changes compared to sham controls. Histology and immunostaining was used to visualize the ECM, blood vessels and multinucleated foreign body giant cells around implants. Results: Bioengineered P nanomesh/ eMSC constructs explanted after 6 weeks showed significant increase in 35 genes associated with ECM, ECM regulation, cell adhesion angiogenesis and immune response in comparison to P nanomesh alone. In the absence of eMSC, acute inflammatory genes were significantly elevated at 1 week. However, in presence of eMSC, there was an increased expression of anti-inflammatory genes including Mrc1 and Arg1 by 6 weeks. There was formation of multinucleated foreign body giant cells around both implants at 6 weeks that expressed CD206, a M2 macrophage marker. Conclusion: This study reveals that eMSC modulate the foreign body response to degradable P nanomeshes in vivo by altering the expression profile of mouse genes. eMSC reduce acute inflammatory and increase ECM synthesis, angiogenesis and anti-inflammatory gene expression at 6 weeks while forming newly synthesized collagen within the nanomeshes and neo-vasculature in close proximity. From a tissue engineering perspective, this is a hallmark of a highly successful implant, suggesting significant potential as alternative surgical constructs for the treatment of POP.

Dataset Information

High-density real-time PCR-based in vivo toxicogenomic screen to predict organ-specific toxicity.

Publications

High-density real-time PCR-based in vivo toxicogenomic screen to predict organ-specific toxicity.

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