Carbon tetrachloride affects inflammation-related biochemical networks in the mouse liver as identified by a customized cDNA microarray system

Abstract

Objectives: We have attempted to upgrade and validate an in-house cDNA microarray system developed by our group for the evaluation of chemical toxicity.

Methods: To establish an in-house microarray, we selected genes that play pivotal roles in detoxifying exogenous substances and maintaining homeostasis in the liver. To validate the system, we examined gene expression profiles in mouse liver following treatment with different doses of carbon tetrachloride (CCl(4)). The data were also analyzed by pathway analysis tools.

Results: We upgraded our array system by collecting genes that are responsive to xenobiotic receptors, apoptosis-related genes, and stress-responsive genes. The acute toxicity of CCl(4) was confirmed by elevated levels of serum transaminase and histopathological findings. The microarray data showed the CCl(4) treatment induced significant changes in gene expression in the mouse liver, and the ingenuity pathways analysis revealed alterations in gene expression in inflammation-related networks.

Conclusions: We have established a focused microarray system that may be useful for use in toxicogenomics studies. Using this array system, we gained insight into the mechanisms by which CCl(4) exerts its toxic effects. The results of our study also indicate that the combination of focused arrays and bioinformatics tools is helpful in the mechanistic analysis of chemical toxicity.

Fig. 1

a Effect of carbon tetrachloride (CCl₄) treatment on serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. Serum AST (white bars) and ALT (black bars) were determined as described in the Materials and methods. Values represent the mean ± standard error of the mean (SEM). *p < 0.05 versus vehicle control. b Paraffin-embedded liver sections were stained with hematoxylin–eosin. Original magnification 100×

Fig. 2

Cluster analysis of gene expression profiles after treatment with CCl₄. Gene expression profiles represent up-regulated genes (red) and down-regulated genes (green) after treatment with CCl₄. Increasing redintensity denotes genes whose expression increased with respect to vehicle, and green intensity denotes genes that decreased in expression with respect to vehicle. Each row denotes a single gene and each column represents an individual dose. The color scale shown indicates the ratios of the signal for CCl₄ (Cy5 signal, red) to that for the vehicle (Cy3 signal, green) (color figure online)

Fig. 3

Dose–response changes of the certain genes that showed at least a two-fold up-regulation at any given dose. OSP oxidative stress-induced protein, Ddit3 DNA-damage inducible transcript 3, HSP60 heat shock 60-kDa protein, Fth ferritin heavy chain, Gadd45a growth arrest and DNA-damage-inducible 45 alpha, Scyb14 small inducible cytokine subfamily B member 14

Fig. 4

Ingenuity pathway analysis of gene expression. Biological findings are assigned to each gene and network based on the information in the Ingenuity Pathways Knowledge Base, which was extracted from the scientific literature. The pathways least likely to have occurred by chance following treatment with 100 µl/kg CCl₄ are presented. a Tumor necrosis factor alpha (TNF-α)-related pathway, b transforming growth factor beta (TGF-β)-related pathway. Red Up-regulation, green down-regulation (color figure online)