Transcriptomic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in liver: comparison of rat and mouse

Abstract

Background: Mouse and rat models are mainstays in pharmacology, toxicology and drug development -- but differences between strains and between species complicate data interpretation and application to human health. Dioxin-like polyhalogenated aromatic hydrocarbons represent a major class of environmentally and economically relevant toxicants. In mammals dioxin exposure leads to a broad spectrum of adverse affects, including hepatotoxicity of varying severity. Several studies have shown that dioxins extensively alter hepatic mRNA levels. Surprisingly, though, analysis of a limited portion of the transcriptome revealed that rat and mouse responses diverge greatly (Boverhof et al. Toxicol Sci 94:398-416, 2006).

Results: We employed oligonucleotide arrays to compare the response of 8,125 rat and mouse orthologs. We confirmed that there is limited inter-species overlap in dioxin-responsive genes. Rat-specific and mouse-specific genes are enriched for specific functional groups which differ between species, conceivably accounting for species-specificities in liver histopathology. While no evidence for the involvement of copy-number variation was found, extensive inter-species variation in the transcriptional-regulatory network was identified; Nr2f1 and Fos emerged as candidates to explain species-specific and species-independent responses, respectively.

Conclusion: Our results suggest that a small core of genes is responsible for mediating the similar features of dioxin hepatotoxicity in rats and mice but non-overlapping pathways are simultaneously at play to result in distinctive histopathological outcomes. The extreme divergence between mouse and rat transcriptomic responses appears to reflect divergent transcriptional-regulatory networks. Taken together, these data suggest that both rat and mouse models should be used to screen the acute hepatotoxic effects of drugs and toxic compounds.

Figure 1

TCDD-Induced Changes in mRNA in Rat and Mouse. The hepatic mRNA abundance profiles of C57BL/6 mice and L-E rats were determined using microarray methods. Following GCRMA pre-processing, ProbeSet-wise linear-models were fit to identify differentially expressed genes. A) A plot of the number of distinct genes (Entrez Gene IDs) called differentially-expressed in each species (y-axis) as a function of threshold (x-axis) suggests more mouse genes than rat genes are TCDD-responsive. B) In a threshold-independent manner (x-axis) a larger fraction of mouse genes than rat genes are induced by TCDD. C) The variance of each rat ProbeSet was calculated and those having a variance above 1.0 were mean-centered and root-mean-square-scaled and subjected to divisive hierarchical clustering using the DIANA algorithm. TCDD-treated animals (rows with red annotation bars) cluster independently from vehicle controls (rows with blue annotation bars) in this unsupervised analysis. D) The variance of each mouse ProbeSet was calculated and those having a variance above 1.0 were mean-centered and root-mean-square-scaled and subjected to divisive hierarchical clustering using the DIANA algorithm. TCDD-treated animals (rows with red annotation bars) cluster independently from vehicle controls (rows with blue annotation bars) in this unsupervised analysis.

Figure 2

Correlation of Rat and Mouse Responses to TCDD. To assess the variability of the response to TCDD between mice and rats we took the pre-processed and linearly-modeled data and selected all ProbeSets with evidence for differential mRNA abundances (p_adjusted < 0.01) in at least one species. We mapped homologs between the two species using the Homologene database. A) To determine if genes showed similar trends in their profiles we plotted the fold-change in log₂ space (M-values) for all homologs. The two profiles are well-correlated (Spearman's rho = 0.32, p < 2.2 × 10^-16), showing similar trends. B) To determine if these similarities were the strongest pattern within the dataset we selected all genes with evidence for TCDD-induced changes in mRNA abundance (p_adjusted < 0.01 in one or both species) and selected the pre-processed values for all animals in both species. These values were then median-centered and root-mean-square-scaled separately within species before being subjected to divisive hierarchical clustering using the DIANA algorithm. The rat (pink) and mouse (red) TCDD animals cluster together, separately from the rat (light blue) and mouse (dark blue) controls. This unsupervised clustering suggests that species differences are less prominent than conserved TCDD-induced changes in mRNA abundances.

Figure 3

Gene-Wise Differences in Rat and Mouse Responses to TCDD. A) The specific identity of genes showing TCDD-induced changes in mRNA abundances diverges significantly between rats and mice. Only 33 genes are found altered in both species and, of these, 3 are altered in different directions. Thus only 15% of rat responses (30/200) and 10.8% of mouse responses (30/278) to TCDD are conserved in homologs from the other species. B) To test if genes with strong evidence (low p-values) for differential expression in one species would have similarly strong evidence (low p-values) in the other we plotted the adjusted p-values for the two species in log₁₀ space. Surprisingly these values are strongly anti-correlated (Spearman's rho = -0.60, p < 2.2 × 10^-16), suggesting that genes that have strong evidence for differential expression in one species are generally likely to have weak or no evidence in the other.

Figure 4

Genome-Wide Mapping of Differential Expression. To determine if differentially-expressed ProbeSets were localized to specific portions of the rat or mouse genome we plotted the entire genome, with one chromosome per line. Each gene was plotted with a white bar representing its location on the chromosome and its position on the plus (up) or minus (down) strand. Genes showing differential abundances (p_adjusted < 0.01) in the rat are colour-coded in red, those in the mouse in blue, and those in both species in black. No prominent clusters of expression are observed in either the rat (A) or mouse (B).

Figure 5

Analysis of Transcription-Factor Binding-Sites. To rationalize the observed patterns of species-dependent and species-independent expression we performed two separate transcription-factor binding-site analyses. A) We employed a library-based position-weight matrix enrichment analysis using the JASPAR library and the clover algorithm. We separately tested rat (rows with yellow boxes) and mouse (rows with blue boxes) promoter sequences from rat-specific, mouse-specific, and common genes. Thus the first row indicates the analysis of the promoter sequences of the rat orthologs of genes displaying mouse-specific TCDD expression. Each column represents a separate position-weight matrix, and only matrices enriched or depleted in at least two datasets are included. The colour bar indicates the p-value based on 10,000 randomizations. B) and C) We also performed de novo motif discovery using the MotifSampler algorithm. Two matrices enriched in genes showing species-independent responses to TCDD were converted into sequence logos and are displayed here. Neither sequence appears to match a known transcription-factor binding-site.

Figure 6

Comparison With Previously Published Data. We compared our findings with those of Boverhof and co-workers by performing a gene-by-gene comparison. Responsive genes were identified in our dataset as those having p_adjusted < 0.05. A) First, we looked at the 32 genes that responded to TCDD in both species in the Boverhof et al. study and assessed their response in our dataset. Four genes could not be mapped to homologs on arrays, while 57% (16/28) genes responded to TCDD in one or two species in our study. We then repeated this analysis looking at the rat-specific (B) and mouse-specific (C) genes identified in the Boverhof study. In total 24 of the 185 rat-specific genes and 31 of the 225 mouse-specific genes could not be mapped to the 8,125 ortholog pairs present on our oligonucleotide arrays. Next we mapped the specific fold-changes observed in the two studies for both species (D). Highly similar patterns were observed for both mouse (Spearman's rho = 0.83, p = 4.4 × 10^-8) and rat (Spearman's rho = 0.88, p = 1.06 × 10^-9).