In vivo hepatotoxicity study of rats in comparison with in vitro hepatotoxicity screening system.
For the establishment of a high throughput screening system using primary cell cultures, investigation of elucidated toxicities to assess the correlation between in vitro and in vivo hepatotoxicity is necessary in the safety evaluation of the compound. In the previous study, we reported the usability of rat primary cultured hepatocytes for establishment of high throughput screening system. To confirm the reliability of rat primary hepatocytes culture screening system, we conducted a single-dose in vivo study with relatively high dose of hepatotoxicant in rats using 4 reference compounds (acetaminophen, amiodarone, tetracycline, carbon tetrachloride), and investigated histopathological changes and expression of oxidative stress-related proteins by immunohistochemistry. We also carried out a proteomics analysis for estimating the reliable and sensitive biomarkers. Histopathologically, compound-specific hepatotoxicity was detected at 24 hr after administration in all compounds except amiodarone, which is known to induce phospholipidosis. Immunohistochemically, oxidative stress-related proteins were increased within 6 hr after administration in all treated groups. Proteomics analysis revealed several protein biomarkers related to oxidative stress and mitochondrial metabolism-regulation, which had been previously detected by proteomics analysis in in vitro screening system. Oxidative stress-related proteins were considered as useful biomarkers of hepatotoxicity; since they were detected by immunohistochemistry and proteomics analysis prior to appearance of compound-specific histopathological changes detected by light microscopy. Considering the relevance of in vitro system to in vivo system from the aspect of new biomarkers related to the toxicogenomics/toxicoproteomics, in vitro primary cell culture system would be sufficient to detect hepatotoxicity in the early stage of drug discovery.
Kikkawa
Rie
R
Worldwide Safety Sciences, Pfizer Global Research and Development, Nagoya Laboratories, Pfizer Inc., 5-2 Taketoyo, Aichi 470-2393, Japan. rie.kikkawa@pfizer.com
Fujikawa
Masaaki
M
Yamamoto
Toshinori
T
Hamada
Yoshimasa
Y
Yamada
Hiroshi
H
Horii
Ikuo
I
D000082:Acetaminophen / Q000493:pharmacokinetics / Q000633:toxicity; D000638:Amiodarone / Q000493:pharmacokinetics / Q000633:toxicity; D000818:Animals; D015415:Biomarkers; D002251:Carbon Tetrachloride / Q000633:toxicity; D002478:Cells, Cultured; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D007728:Kupffer Cells / Q000187:drug effects / Q000378:metabolism; D008099:Liver / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008297:Male; D018384:Oxidative Stress; D011506:Proteins / Q000378:metabolism; D040901:Proteomics; D051381:Rats; D011919:Rats, Inbred Strains; D013752:Tetracycline / Q000493:pharmacokinetics / Q000633:toxicity; D018675:Toxicity Tests / Q000379:methods*
D015415:Biomarkers; D011506:Proteins; D000082:Acetaminophen; D002251:Carbon Tetrachloride; D013752:Tetracycline; D000638:Amiodarone