Genomics and proteomics analysis of acetaminophen toxicity in mouse liver.
Overdose of acetaminophen (APAP) causes severe centrilobular hepatic necrosis in humans and experimental animals. Here, to explore its mechanism, we administered APAP at subtoxic (150 mg/kg ip) and toxic (500 mg/kg ip) doses to overnight fasted mice. Animals were sacrificed at different time points from 15 min to 4 h postinjection. We assessed liver toxicity by plasma ALT activity and by electron microscopy. Using nylon filter arrays and RTQPCR, we performed genomics analysis in liver. We ran proteomics on liver mitochondrial subfractions using the newly developed quantitative fluorescent 2D-DIGE method (Amersham Pharmacia Biotech UK Limited). As soon as 15 min postinjection, centrilobular hepatocyte mitochondria were already slightly enlarged and GSH total content dropped by a third at top dose. GM-CSF mRNA, which is a granulocyte specific gene likely coming from resident Kupffer cells, was also induced to its maximum of 3-fold at both doses. Chaperone proteins Hsp10 and Hsp60 were readily decreased by half in mitochondria at both doses, most likely by leaking into cytoplasm. Although APAP is known as an apoptotic trigger, no apoptosis was observed at any time point. Most of the protein changes in mitochondria were present at 15 min postinjection, thus preceding most of the gene regulations. The decrease of ATP synthase subunits and beta-oxidation pathway proteins indicated a loss of energy production. As the morphology of mitochondria was also affected very early at top dose, we concluded that APAP toxicity was a direct action of its known reactive metabolite NAPQI, rather than a consequence of gene regulation. However, the latter will either worsen the toxicity or lead toward cell recovery depending on the cellular damage level.
Ruepp
Stefan U
SU
AstraZeneca, Alderley Park, Macclesfield SK10 4TG, Cheshire, United Kingdom.
Tonge
Robert P
RP
Shaw
Joanne
J
Wallis
Nicola
N
Pognan
François
F
D000082:Acetaminophen / Q000097:blood / Q000633:toxicity*; D000818:Animals; D015153:Blotting, Western; D018736:DNA Adducts / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D062787:Drug Overdose; D004591:Electrophoresis, Polyacrylamide Gel; D023281:Genomics; D005978:Glutathione / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D022781:Hepatocytes / Q000473:pathology*; D007728:Kupffer Cells / Q000187:drug effects; D008099:Liver / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D008297:Male; D051379:Mice; D008854:Microscopy, Electron; D008930:Mitochondria, Liver / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D018384:Oxidative Stress; D020543:Proteome; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors
D018736:DNA Adducts; D020543:Proteome; D012333:RNA, Messenger; D000082:Acetaminophen; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D005978:Glutathione