Comparison of transcriptional responses in liver tissue and primary hepatocyte cell cultures after exposure to hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine

Abstract

Background: Cell culture systems are useful in studying toxicological effects of chemicals such as Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), however little is known as to how accurately isolated cells reflect responses of intact organs. In this work, we compare transcriptional responses in livers of Sprague-Dawley rats and primary hepatocyte cells after exposure to RDX to determine how faithfully the in vitro model system reflects in vivo responses.

Results: Expression patterns were found to be markedly different between liver tissue and primary cell cultures before exposure to RDX. Liver gene expression was enriched in processes important in toxicology such as metabolism of amino acids, lipids, aromatic compounds, and drugs when compared to cells. Transcriptional responses in cells exposed to 7.5, 15, or 30 mg/L RDX for 24 and 48 hours were different from those of livers isolated from rats 24 hours after exposure to 12, 24, or 48 mg/Kg RDX. Most of the differentially expressed genes identified across conditions and treatments could be attributed to differences between cells and tissue. Some similarity was observed in RDX effects on gene expression between tissue and cells, but also significant differences that appear to reflect the state of the cell or tissue examined.

Conclusion: Liver tissue and primary cells express different suites of genes that suggest they have fundamental differences in their cell physiology. Expression effects related to RDX exposure in cells reflected a fraction of liver responses indicating that care must be taken in extrapolating from primary cells to whole animal organ toxicity effects.

Figure 1

Hierarchical clustering analysis of the correlation matrix of normalized data. Blue squares represent low correlations and red represents high correlations.

Figure 2

Principal components analysis of relatedness between microarray data. Each colored box represents an individual hybridization. Blue = liver tissue, Red = 24 hr exposed cells, and Green = 48 hr exposed cells. Prin1 = first principal component, Prin2 = second principal component, and Prin3 = third principal component.

Figure 3

Volcano plot illustrating predominant sources of differentially expressed genes. Differentially expressed genes are highlighted according to a Bonferroni cutoff (red dashed line) between the high dose treatments and controls within the condition e.g. 24 hr cell (A), 48 hr cell (B) and rat (C), and between the controls of the three conditions ((24 hr cell – 48 hr cell (D), 24 hr cell -rat (E), and 48 hr cell -rat (F)).

Figure 4

Hierarchical clustering of significant differentially expressed genes according to Bonferroni correction, total 2929 genes. Red represents up and green represents down-regulated genes relative to the mean of all samples. Cluster enriched GO terms are listed adjacent to each cluster.

Figure 5

Common differentially expressed genes among all doses of exposed primary hepatic cells and liver tissue determined by ANOVA. Numbers in overlapping circles represent genes common to respective cells or tissue. 24 cell = 24 hr exposed cells, 48 cell = 48 hr exposed cells, and Liver = liver tissue. A. ANOVA analysis (p < 0.01). B. ANOVA analysis (p < 0.001).

Figure 6

Common KEGG pathway terms among all doses of exposed primary hepatic cells and liver tissue. Numbers represent pathways common to respective cells or tissue. 24 cell = 24 hr exposed cells, 48 cell = 48 hr exposed cells, and Liver = liver tissue. A. ANOVA analysis (p < 0.01). B. ANOVA analysis (p < 0.001).

Figure 7

Looped hybridization scheme for analysis of liver and hepatic cell transcriptional responses. Circles represent treatment samples. For liver tissue samples, 0.x = solvent control biological replicate x, 1.x = 12 mg/kg dose biological replicate x, 2.x = 24 mg/kg dose biological replicate x, 3.x = 48 mg/kg dose biological replicate x. For 24 and 48 hr exposures of primary hepatic cells to RDX, 0.x = solvent control biological replicate x, 1.x = 7.5 mg/L dose biological replicate x, 2.x = 15 mg/L dose biological replicate x, 3.x = 30 mg/L dose biological replicate x. Arrows represent array hybridizations between respective samples where the arrowhead indicates dye labelling with Alexa 647 and the base of arrows indicate dye labelling with Cy3.