Direct oxygenation of hepatocytes through an oxygen-permeable membrane enhances in vitro detection of repeated-dose acetaminophen-induced hepatotoxicity

抄録

Hepatotoxicity remains a major challenge in drug development. Conventional in vitro hepatocyte cultures often lack physiologically relevant oxygenation, which may impair metabolic function and decrease the detectability of drug-induced liver injury (DILI). This study employed oxygen-permeable plates to supply oxygen directly to hepatocyte monolayers and evaluated the impact of this approach on acetaminophen (APAP) metabolism and resulting toxicity during two weeks of repeated administration, comparing results with those from conventional tissue-culture-treated polystyrene (TCPS) cultures. Direct oxygenation increased basal albumin secretion by approximately 2- to 3-fold and markedly increased urea production across the observation period. Simultaneously, direct oxygenation enhanced APAP-induced functional decline over the exposure period, thereby enabling more sensitive detection of APAP hepatotoxicity. Mechanistically, direct oxygenation increased the major APAP metabolites and approximately doubled the formation of APAP-protein adducts at 1 mM and 3 mM APAP. This increased intracellular formation of such APAP-protein adducts is the most direct evidence of APAP bioactivation-linked toxicity through increased NAPQI formation. Furthermore, the overall APAP metabolite profile under direct oxygenation better recapitulated the in vivo profile than that under the TCPS culture, although the improvement was marginal. These findings demonstrate that direct oxygenation of hepatocytes enhances the detection of metabolism-dependent DILI by increasing hepatocyte function and drug metabolism, showing that improved oxygenation mimicking in vivo conditions is beneficial for in vitro DILI evaluation.