Species-specific differences in coumarin-induced hepatotoxicity as an example toxicogenomics-based approach to assessing risk of toxicity to humans

Abstract

One expected result from toxicogenomics technology is to overcome the barrier because of species-specific differences in prediction of clinical toxicity using animals. The present study serves as a model case to test if the well-known species-specific difference in the toxicity of coumarin could be elucidated using comprehensive gene expression data from rat in-vivo, rat in-vitro, and human in-vitro systems. Coumarin 150 mg/kg produced obvious pathological changes in the liver of rats after repeated administration for 7 days or more. Moreover, 24 h after a single dose, we observed minor and transient morphological changes, suggesting that some early events leading to hepatic injury occur soon after coumarin is administered to rats. Comprehensive gene expression changes were analyzed using an Affymetrix GeneChip^® approach, and differentially expressed probe sets were statistically extracted. The changes in expression of the selected probe sets were further examined in primary cultured rat hepatocytes exposed to coumarin, and differentially expressed probe sets common to the in-vivo and in-vitro datasets were selected for further study. These contained many genes related to glutathione metabolism and the oxidative stress response. To incorporate human data, human hepatocyte cultured cells were exposed to coumarin and changes in expression of the bridging gene set were examined. In total, we identified 14 up-regulated and 11 down-regulated probe sets representing rat–human bridging genes. The overall responsiveness of these genes to coumarin was much higher in rats than humans, consistent with the reported species difference in coumarin toxicity. Next, we examined changes in expression of the rat–human bridging genes in cultured rat and human hepatocytes treated with another hepatotoxicant, diclofenac sodium, for which hepatotoxicity does not differ between the species. Both rat and human hepatocytes responded to the marker genes to the same extent when the same concentrations of diclofenac sodium were exposed. We conclude that toxicogenomics-based approaches show promise for overcoming species-specific differences that create a bottleneck in analysis of the toxicity of potential therapeutic treatments.

Keywords

coumarin hepatocyte hepatotoxicity human liver rat toxicogenomics

References

1 Urushidani T Nagao T . Toxicogenomics: the Japanese initiative. In: J Borlak , (Ed.), Handbook of toxicogenomics – strategies and applications. Wiley: VCH; 2005. p. 623–31.

2 Takashima K Mizukawa Y Morishita K Okuyama M Kasahara T Toritsuka N . Effect of the difference in vehicles on gene expression in the rat liver-analysis of the control data in the Toxicogenomics Project Database . Life Sci 2006; 78: 2787 – 2796 .

3 Waters MD Fostel JM . Toxicogenomics and systems toxicology: aims and prospects . Nat Rev Genet 2004; 5: 936 – 948 .

4 Mattingly CJ Colby GT Forrest JN Boyer JL . The Comparative Toxicogenomics Database (CTD) . Environ Health Perspect 2003; 111: 793 – 795 .

5 Liu G Loraine AE Shigeta R Cline M Cheng J Valmeekam V . NetAffx: Affymetrix probesets and annotations . Nucleic Acids Res 2003; 31: 82 – 86 .

6 Kiyosawa N Shiwaku K Hirode M Omura K Uehara T Shimizu T . Utilization of a one-dimensional score for surveying chemical-induced changes in expression levels of multiple biomarker gene sets using a large-scale toxicogenomics database . J Toxicol Sci 2006; 31: 433 – 448 .

7 Hedges LV . Distribution theory for Glass’s estimator of effect size and related estimators . J Edu Statis 1981; 6: 107 – 128 .

8 Gomez-Lechon MJ Ponsoda X O’Connor E Donato T Castell JV Jover R . Diclofenac induces apoptosis in hepatocytes by alteration of mitochondrial function and generation of ROS . Biochem Pharmacol 2003; 66: 2155 – 2167 .

9 Amin A Hamza AA . Oxidative stress mediates drug-induced hepatotoxicity in rats: a possible role of DNA fragmentation . Toxicology 2005; 208: 367 – 375 .

10.

10 National Toxicology Program . NTP toxicology and carcinogenesis studies of coumarin (CAS No. 91-64-5) in F344/N rats and B6C3F1 mice (Gavage Studies) . Natl Toxicol Program Tech Rep Ser 1993; 422: 1 – 340 .

11.

11 Lake BG . Investigations into the mechanism of coumarin-induced hepatotoxicity in the rat . Arch Toxicol Suppl 1984; 7: 16 – 29 .

12.

12 Lake BG Gray TJ Evans JG Lewis DF Beamand JA Hue KL . Studies on the mechanism of coumarin-induced toxicity in rat hepatocytes: comparison with dihydrocoumarin and other coumarin metabolites . Toxicol Appl Pharmacol 1989; 97: 311 – 323 .

13.

13 Kiyosawa N Uehara T Gao W Omura K Hirode M Shimizu T . Identification of glutathione depletion-responsive genes using phorone-treated rat liver . J Toxicol Sci 2007; 32: 469 – 486 .

14.

14 Vassallo JD Hicks SM Daston GP Lehman-McKeeman LD . Metabolic detoxification determines species differences in coumarin-induced hepatotoxicity . Toxicol Sci 2004; 80: 249 – 257 .

15.

15 Felter SP Vassallo JD Carlton BD Daston GP . A safety assessment of coumarin taking into account species-specificity of toxicokinetics . Food Chem Toxicol 2006; 44: 462 – 475 .

16.

16 Born SL Hu JK Lehman-McKeeman LD . o-hydroxyphenylacetaldehyde is a hepatotoxic metabolite of coumarin . Drug Metab Dispos 2000a; 28: 218 – 223 .

17.

17 Born SL Caudill D Smith BJ Lehman-McKeeman LD . In vitro kinetics of coumarin 3,4-epoxidation: application to species differences in toxicity and carcinogenicity . Toxicol Sci 2000b; 58: 23 – 31 .