Meta-Analysis of Large-Scale Toxicogenomic Data Finds Neuronal Regeneration Related Protein and Cathepsin D to Be Novel Biomarkers of Drug-Induced Toxicity

<div><p>Undesirable toxicity is one of the main reasons for withdrawing drugs from the market or eliminating them as candidates in clinical trials. Although numerous studies have attempted to identify biomarkers capable of predicting pharmacotoxicity, few have attempted to discover robust biomarkers that are coherent across various species and experimental settings. To identify such biomarkers, we conducted meta-analyses of massive gene expression profiles for 6,567 <i>in vivo</i> rat samples and 453 compounds. After applying rigorous feature reduction procedures, our analyses identified 18 genes to be related with toxicity upon comparisons of untreated versus treated and innocuous versus toxic specimens of kidney, liver and heart tissue. We then independently validated these genes in human cell lines. In doing so, we found several of these genes to be coherently regulated in both <i>in vivo</i> rat specimens and in human cell lines. Specifically, mRNA expression of neuronal regeneration-related protein was robustly down-regulated in both liver and kidney cells, while mRNA expression of cathepsin D was commonly up-regulated in liver cells after exposure to toxic concentrations of chemical compounds. Use of these novel toxicity biomarkers may enhance the efficiency of screening for safe lead compounds in early-phase drug development prior to animal testing.</p></div

The frequency of the <i>VEGF</i> polymorphisms according to age.

*<p>Adjusted by age and gender. NA; not applicable. AOR, adjusted odds ratio. CI, confidence interval.</p

The frequency of the <i>KDR</i> polymorphisms according to age.

*<p>Adjusted by age and gender. NA; not applicable. AOR, adjusted odds ratio. CI, confidence interval.</p

The frequencies of the <i>KDR</i> −604T>C, 1192G>A, and 1719T>A polymorphisms between control subjects and patients with moyamoya disease.

*<p>Adjusted by age and gender. AOR, adjusted odds ratio. CI, confidence interval.</p

Comparison of the <i>VEGF</i> and <i>KDR</i> genotype frequencies according to collateral vessel formation score.

*<p>Adjusted by age and gender. NA; not applicable. AOR, adjusted odds ratio. CI, confidence interval.</p

Haplotype frequencies of the <i>VEGF a</i>nd <i>KDR</i> polymorphisms according to age.

*<p><i>P-values</i> after 10,000 permutation test. NA; not applicable. OR, odds ratio. CI, confidence interval.</p

The frequencies of the <i>VEGF</i> −2578C>A, −1154G>A, −634G>C, and 936C>T polymorphisms between control subjects and patients with moyamoya disease.

*<p>Adjusted by age and gender. AOR, adjusted odds ratio. CI, confidence interval.</p

Overview of toxicity biomarker discovery.

<p>First, we collected toxicogenomic meta-data from public resources, preprocessed gene expression array data, and assigned toxicity classes. Second, we attempted to identify differentially expressed genes (DEGs) through meta-analysis and subsequent multistage feature reductions. DEGs were subjected to systems analysis of biological pathways and networks, and an optimized set of biomarkers was used to generate and validate a prediction model. The final step involved computationally and experimentally testing the applicability of the discovered biomarkers in human cells. GEO, Gene Expression Omnibus at the National Center for Biotechnology Information; ArrayExpress, ArrayExpress at the European Bioinformatics Institute; TG-GATEs, Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System of the National Institute of Health Sciences of Japan; CEBS, Chemical Effects in Biological Systems at the National Institute of Environmental Health Sciences; sPLS-DA, sparse partial least squares discriminant analysis.</p

Linkage disequilibrium(LD) of VEGF and KDR polymorphisms.

<p>There was strong LD between −1154G>A (rs 1570460) and −634G>C (rs2010963) (D′ = 0.92), −2578C>A (rs 699947)and 1154G>A (rs1570360) (D′ = 0.89). There was strong LD between 1719T>A(rs1870377) and 1192G>A (rs2305948) (D′ = 0.79).</p

Functional analysis of DEGs.

<p>(A) Enriched GO terms associated with DEGs from three meta-analysis comparisons. DEGs from MA5 were excluded from the analysis owing to insufficient dataset size. <i>p</i>-value: modified Fisher’s exact test implemented in the Database for Annotation, Visualization and Integrated Discovery (DAVID). (B, C) Highly interconnected subnetworks present within the individual sets of DEGs from MA3 and MA4. A circular node indicates proteins, a diamond node indicates proteins/genes, and solid lines and dashed arrows respectively indicate physical and genetic interactions reported in our input databases (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136698#sec002" target="_blank">Methods</a> for details). Node color indicates the median expression fold-change of the training dataset (level-1/level-0).</p