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|Title:||Inter-laboratory Comparison of Human Renal Proximal Tubule (HK-2) Transcriptome Alterations Due to Cyclosporine A Exposure and Medium Exhaustion|
|Authors:||JENNINGS Paul; AYDIN Sonia; BENNET Jason; MCBRIDE Rachael; WEILAND Christina; TUITE Niamh; GRUBER Leo; PERCO Paul; O'GAORA Peadar; ELLINGER-ZEIGELBAUER Heidrun; AHR Hans-Juergen; VAN KOOTEN Cees; DAHA Moh; PRIETO PERAITA Maria Del Pilar; RYAN Michael; PFALLER Walter; MCMORROW Tara|
|Citation:||TOXICOLOGY IN VITRO vol. 23 p. 486-499|
|Publisher:||PERGAMON-ELSEVIER SCIENCE LTD|
|Type:||Articles in periodicals and books|
|Abstract:||There is an acknowledged need to promote and further develop in vitro techniques in order to achieve the 36 goal of improved risk assessment of chemicals and pharmaceuticals to humans. The EU 6th framework 37 project ¿¿PREDICTOMICS¿ was established in order to contribute to the further development of in vitro 38 toxicology, with a particular focus on emerging techniques including toxicogenomics. DNA microarray 39 technology is being used more frequently in the in vitro field, however, only very few studies have 40 assessed the reproducibility of this technique with respect to in vitro toxicology. To this end we conducted an interlaboratory comparison to test the reproducibility of transcriptomic 42 changes induced by the immunosuppressive agent, Cyclosporine A (CsA) on the human renal proximal 43 tubular cell line, HK-2 cell. Four European laboratories took part in this study. Under standardised con- 44 ditions, each laboratory treated HK-2 cells with 5 lM CsA for 12 and 48 h. RNA was isolated and hybri- 45 dised to Affymetrix HGU-133 plus two arrays at three different sites. Analysis of the transcription profiles demonstrated that one laboratory clustered away from the other 47 laboratories, potentially due to an inclusion of a trypsinisation step by this laboratory. Once the genes 48 responsible for this separate clustering were removed all laboratories showed similar expression profiles. 49 There was a major impact of time since feed, due to medium exhaustion in the 48 h arrays compared to 50 the 12 h arrays, regardless of CsA treatment. Biological processes including general vesicle transport, 51 amino acid metabolism, amino acid transport and amino acid biosynthesis were over represented due 52 to time since feed, while cell cycle, DNA replication, mitosis and DNA metabolism were under-repre- 53 sented. CsA responsive genes were involved in cell cycle, the p53 pathway and Wnt signaling. Addition- 54 ally there was an overlap of differentially expressed genes due to CsA and medium exhaustion which is 55 most likely due to CsA induced glycolysis. The glucose deprivation dependent genes HspA5 and GP96 and 56 the Hsp70 chaperones DNAJ/Hsp40, DNAJ/HspB9, DNAJ/HspC3 DNAJ/HspC10 were induced by both CsA 57 and medium exhaustion. We conclude that under standardised conditions the application of Affymetrix DNA microarrays to 59 in vitro toxiciological studies are satisfactorily reproducible. However, confounding factors such as med- 60 ium exhaustion must also be considered in such analyses.|
|JRC Directorate:||Institute for Health and Consumer Protection Historical Collection|
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