Alteration of Acetaminophen-induced Cytotoxicity in Mouse Hepatocytes during Primary Culture

Abstract

The present study investigated issues regarding cytotoxicity tests using mouse hepatocytes in primary culture. The results indicated that the cytotoxicity of acetaminophen (APAP) markedly varied depending on culture period for up to 5 days. P450 isoforms involved in the activation of APAP, such as CYP1A2, CYP2E1, and CYP3A, showed a marked decrease in expressions of respective mRNA. Their expression levels were a few percent within 24 hr after the start of the culture, and remained low throughout. Meanwhile, the mRNA expression of enzymes involved in the detoxification of APAP, UGT1A6 and SULT1A1, declined moderately and then recovered to approximately 40% of the in vivo level later in the culture. Treatment with APAP at non-cytotoxic concentrations increased expressions of CYP1A2, CYP2E1, CYP3A11 and CYP3A41 mRNA, while it decreased that of SULT1A1 mRNA. Using the lactate dehydrogenase (LDH) assay, the cells treated with 25-mM APAP for 24-48 or 48-72 hr showed higher cytotoxicity values than those for 72-96 or 96-120 hr, but the values after 50-mM treatment were completely inverted. By employing the methylthiazolyl tetrazolium (MTT) assay, culture period-dependent cytotoxicity was also observed on 25-mM APAP treatment, but this was not clear at 50 mM. Lower LDH or higher MTT activities than the control were observed in cells treated with lower concentrations of APAP, becoming the more prominent the longer the culture period. The observations suggested that, since the expression of metabolizing enzymes alters markedly during the primary culture and the range of cytotoxic concentrations of a test compound varies depending on the culture period, it is necessary to take account of these fluctuating parameters when assessing the cytotoxic character of a compound.

In vitro to in vivo acetaminophen hepatotoxicity extrapolation using classical schemes, pharmacodynamic models and a multiscale spatial-temporal liver twin

“…The here presented in vivo data of APAP are in agreement with previous reports on APAP-induced liver injury in mice (McGill et al, 2012;Ghallab et al, 2022). The in vitro experiments showed slightly higher APAP-induced cytotoxicity compared to previous reports (Jemnitz et al, 2008;Jaruchotikamol et al, 2009). A possible explanation of this discrepancy is that we started incubation with APAP already at 2 hours after hepatocyte isolation, the time when Cyp2e1 expression is still preserved.…”