Identification of platform-independent gene expression markers of cisplatin nephrotoxicity - Genomics and Risk Assessment: Mini-Monograph

Environmental Health Perspectives, March 15, 2004 by Karol L. Thompson, Cynthia A. Afshari, Rupesh P. Amin, Timothy A. Bertram, Bruce Car, Michael Cunningham, Clive Kind, Jeffrey A. Kramer, Michael Lawton, Michael Mirsky, Jorge M. Naciff, Victor Oreffo, P. Scott Pine, Frank D. Sistare

Within the International Life Sciences Institute Committee on Genomics, a working group was formed to focus on the application of microarray technology to preclinical assessments of drug-induced nephrotoxicity. As part of this effort, Sprague-Dawley rats were treated with the nephrotoxicant cisplatin at doses of 0.3-5 mg/kg over a 4- m 144-hr time course. RNA prepared from these animals was run on a variety of microarray formats at multiple sites. A set of 93 differentially expressed genes associated with cisplatin-induced renal injury was identified on the National Institute of Environmental Health Sciences (NIEHS) custom cDNA microarray platform using quadruplicate measurements of pooled animal RNA. The reproducibility of this profile of statistically significant gene changes on other platforms, in pooled and individual animal replicate samples, and in an independent study was investigated. A good correlation in response between platforms was found among the 48 genes in the NIEHS data set that could be matched to probes on the Affymetrix RGU34A array by UniGene identifier or sequence alignment. Similar results were obtained with genes that could be linked between the NIEHS and Incyte or PHASE-1 arrays. The degree of renal damage induced by cisplatin in individual animals was commensurate with the number of differentially expressed genes in this data set. These results suggest that gene profiles linked to specific types of tissue injury or mechanisms of toxicity and identified in well-performed replicated microarray experiments may be extrapolatable across platform technologies, laboratories, and in-life studies. Key words: cisplatin, cross-platform, kidney, microarrays, nephrotoxicity. Environ Health Perspect 112:488-494 (2004), doi: 10.1289/txg.6676 available via http://dx.doi.org/[Online 15 January 2004]

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The International Life Science Institute (ILSI) Health and Environmental Sciences Institute (HESI) Committee on Application of Genomics and Proteomics to Mechanism-Based Risk Assessment was established to advance the scientific basis for the development and application of genomic and proteomic methodologies to mechanism-based risk assessment (Robinson et al. 2003). One of the long-term objectives of the subcommittee is to relate changes in gene and protein expression to other measures of toxicity. Three toxicity working groups were formed to assess the application of microarray technology to nephrotoxicity, hepatotoxicity, and genotoxicity. The HESI Nephrotoxicity Working Group conducted studies using three nephrotoxicants--cisplatin, gentamicin, and puromycin--with different mechanisms or target regions within the kidney (Kramer et al. 2004). RNA was prepared at one site and sent to other laboratories for analysis on multiple microarray formats, using individual and pooled animal samples.

One of the primary objectives of toxicogenomics is the development of gene profiles characteristic of discrete toxicities that are potentially measurable on any well-designed and well-annotated microarray platform (Hamadeh et al. 2002; Thomas et al. 2001; Waring et al. 2001). The development of toxicity profiles and specific gene expression signatures requires the use of standardized conditions and the testing of a number of drugs of distinct classes and mechanisms, and therefore they are usually developed on one platform technology. Analytical validation of the veracity of gene changes within a signature is usually performed with an independent gene expression detection technology such as quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), An additional approach to analytical gene profile validation involves the detection of the identified gene signature on microarray platforms that use different types of probes to capture and detect specific RNA transcripts within samples. Deposition in a public repository of genomic data annotated with toxicity data that was generated by the ILSI working groups is one of the goals of this project (Robinson et al. 2003). The potential to mine gene expression data from diverse sources that will eventually populate public databases will be unrealized if gene profiles of toxicity cannot be extrapolated from one microarray platform to another. It is encouraging that meta-analysis of microarray data collected from different sources and on different microarray formats has shown that cohorts of genes dysregulated in prostate cancer can be consistently identified across data sets (Rhodes et at. 2002). However, more effort must be devoted to evaluating and improving data comparisons across platforms, including verifying probe annotations, gene family and splice variant analysis, and comparability of statistical methods.

In this article, we examine the cross-platform and cross-experiment reproducibility of gene changes induced in rat kidney by cisplatin treatment and identified as statistically significant on a custom cDNA array. Of the studies performed by the HESI Nephrotoxicity Working Group, the most complete data set with the largest number of microarray formats was available for the study involving the antineoplastic agent and regionally specific nephrotoxicant cisplatin. RNA prepared from the kidneys of rats on day 7 after a single injection of 5 mg/kg cisplatin, a dose and time point coincident with proximal tubule necrosis and regeneration, was tested on three different cDNA-spotted arrays (the NIEHS custom rat cDNA array, the Incyte Rat Toxicology GEM, and the PHASE-1 Rat 700 array) and one high-density oligonucleotide array (the Affymetrix RGU34A array).