ABSTRACT:Cultured human hepatocytes are a valuable in vitro system for evaluating new molecular entities as inducers of cytochrome P450 (P450) enzymes. The present study summarizes data obtained from 62 preparations of cultured human hepatocytes that were treated with vehicles (saline or dimethylsulfoxide, 0.1%), ␤-naphthoflavone (33 M), phenobarbital (100 or 250 M), isoniazid (100 M) and/or rifampin (20 or 50 M), and examined for the expression of P450 enzymes based on microsomal activity toward marker substrates, or in the case of CYP2C8, the level of immunoreactive protein. The results show that CYP1A2 activity was markedly induced by ␤-naphthoflavone (on average 13-fold, n ‫؍‬ 28 preparations), and weakly induced by phenobarbital (1.9-fold, n ‫؍‬ 25) and rifampin (2.3-fold, n ‫؍‬ 22); CYP2A6 activity tended to be increased with phenobarbital (n ‫؍‬ 7) and rifampin (n ‫؍‬ 3) treatments, but the effects were not statistically significant; CYP2B6 was induced by phenobarbital (6.5-fold, n ‫؍‬ 13) and rifampin (13-fold, n ‫؍‬ 14); CYP2C8 was induced by phenobarbital (4.0-fold, n ‫؍‬ 4) and rifampin (5.2-fold, n ‫؍‬ 4); CYP2C9 was induced by phenobarbital (1.8-fold, n ‫؍‬ 14) and rifampin (3.5-fold, n ‫؍‬ 10); CYP2C19 was markedly induced by rifampin (37-fold, n ‫؍‬ 10), but relatively modestly by phenobarbital (7-fold, n ‫؍‬ 9); CYP2D6 was not significantly induced by phenobarbital (n ‫؍‬ 5) or rifampin (n ‫؍‬ 5); CYP2E1 was induced by phenobarbital (1.7-fold, n ‫؍‬ 5), rifampin (2.2-fold, n ‫؍‬ 5), and isoniazid (2.3-fold, n ‫؍‬ 5); and, CYP3A4 was induced by phenobarbital (3.3-fold, n ‫؍‬ 42) and rifampin (10-fold, n ‫؍‬ 61), but not by ␤-naphthoflavone. Based on these observations, we generalize that ␤-naphthoflavone induces CYP1A2 and isoniazid induces CYP2E1, whereas rifampin and, to a lesser extent phenobarbital, tend to significantly and consistently induce enzymes of the CYP2A, CYP2B, CYP2C, CYP2E, and CYP3A subfamilies but not the 2D subfamily. Drugs and NMEs5 are often screened for their ability to induce P450 and other drug-metabolizing enzymes with the aim of predicting or explaining drug-drug interactions and pharmacokinetic tolerance.

Acetaminophen (APAP) overdose is the most prevalent cause of drug-induced liver injury in western countries. Numerous studies have been conducted to investigate the mechanisms of injury after APAP overdose in various animal models; however, the importance of these mechanisms for humans remains unclear. Here we investigated APAP hepatotoxicity using freshly isolated primary human hepatocytes (PHH) from either donor livers or liver resections. PHH were exposed to 5mM, 10mM or 20mM APAP over a period of 48 hours and multiple parameters were assessed. APAP dose-dependently induced significant hepatocyte necrosis starting from 24h, which correlated with the clinical onset of human liver injury after APAP overdose. Interestingly, cellular glutathione was depleted rapidly during the first 3h. APAP also resulted in early formation of APAP-protein adducts (measured in whole cell lysate and in mitochondria) and mitochondrial dysfunction, indicated by the loss of mitochondrial membrane potential after 12h. Furthermore, APAP time-dependently triggered c-Jun N-terminal kinase (JNK) activation in the cytosol and translocation of phospho-JNK to the mitochondria. Both co-treatment and post-treatment (3h) with the JNK inhibitor SP600125 reduced JNK activation and significantly attenuated cell death at 24h and 48h after APAP. The clinical antidote N-acetylcysteine offered almost complete protection even if administered 6 hours after APAP and a partial protection when given at 15h. Conclusion: These data highlight important mechanistic events in APAP toxicity in PHH and indicate a critical role of JNK in the progression of injury after APAP in humans. The JNK pathway may represent a therapeutic target in the clinic.

Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models.

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