Estimating Hepatotoxic Doses Using High-Content Imaging in Primary Hepatocytes

Abstract

Using in vitro data to estimate point of departure (POD) values is an essential component of new approach methodologies (NAM)-based chemical risk assessments. In this case study, we evaluated a NAM for hepatotoxicity based on rat primary hepatocytes, high-content imaging (HCI), and toxicokinetic modeling. First, we treated rat primary hepatocytes with 10 concentrations (0.2 to 100 µM) of 51 chemicals that produced hepatotoxicity in repeat-dose subchronic and chronic exposures. Second, we used HCI to measure endoplasmic reticulum stress, mitochondrial function, lysosomal mass, steatosis, apoptosis, DNA texture, nuclear size, and cell number at 24, 48, and 72 h and calculated concentrations at 50% maximal activity (AC50). Third, we estimated administered equivalent doses (AEDs) from AC50 values using toxicokinetic modeling. AEDs using physiologically-based toxicokinetic models were 4.1-fold (SD 6.3) and 8.1-fold (SD 15.5) lower than subchronic and chronic lowest observed adverse effect levels (LOAELs), respectively. In contrast, AEDs from ToxCast and Tox21 assays were 89.8-fold (SD 149.5) and 168-fold (SD 323.7) lower than subchronic and chronic LOAELs. Individual HCI end-points also estimated AEDs for specific hepatic lesions that were lower than in vivo PODs. Lastly, AEDs were similar for different in vitro exposure durations, but steady-state toxicokinetic models produced 7.6-fold lower estimates than dynamic physiologically-based ones. Our findings suggest that NAMs from diverse cell types provide conservative estimates of PODs. In contrast, NAMs based on the same species and cell type as the adverse outcome may produce estimates closer to the traditional in vivo PODs.

Recent progress in cytometric technologies and their applications in ecotoxicology and environmental risk assessment

“…Rapid assessment of cytotoxic effects of chemicals, in line with paradigms outlined by the TT21C, can be performed at large scale on a number of established human, murine and even insect and fish cell lines using both flow and imaging cytometry [19,[86][87][88][89]. When using a panel of cell lines from diverse linages, one can also perform rapid prioritization screening of organ-specific cytotoxicity.…”

In vitro to in vivo extrapolation and high-content imaging for simultaneous characterization of chemically induced liver steatosis and markers of hepatotoxicity

“…There are several recent studies concerning chemically induced steatosis in vitro (Anthérieu et al 2011 , 2012 ; Rogue et al 2014 ; Klein et al 2016 ; Tolosa et al 2016 ; Angrish et al 2017 ; Cuykx et al 2018 ; Luckert et al 2018 ; Bucher et al 2018 ; Allard et al 2020 ; Lichtenstein et al 2020 ; Lasch et al 2021 ). These include at least four studies in which key events along the liver steatosis AOP were quantified in vitro (Supplementary table 1) (Donato et al 2012 ; Tolosa et al 2016 ; Luckert et al 2018 ; Shah et al 2021 ). Most recently, Shah et al quantified mitochondrial function, lipid accumulation, endoplasmic reticulum stress, lysosomal mass, DNA texture, nuclear size, apoptosis, and cell number using a high-content imaging approach with rat hepatocytes and 51 hepatotoxicants.…”

“…Most recently, Shah et al quantified mitochondrial function, lipid accumulation, endoplasmic reticulum stress, lysosomal mass, DNA texture, nuclear size, apoptosis, and cell number using a high-content imaging approach with rat hepatocytes and 51 hepatotoxicants. Moreover, these in vitro data were extrapolated to in vivo administered equivalent doses and compared to in vivo (rat) data (Shah et al 2021 ). Relating in vitro bioactivity data to exposure values by means of physiologically based pharmacokinetic modeling (PBPK) and reverse dosimetry is critical in order that in vitro bioassays can be accepted for chemical hazard and eventually risk assessment by regulatory agencies (Wetmore 2015 ; Hartung 2018 ).…”

Chemical screening of food-related chemicals for human fatty liver risk: Combining high content imaging of cellular responses with in vitro to in vivo extrapolation

“…There are at least four examples of quantitative in vitro assays for steatosis hazard assessment available ( Table 1 ) (Luckert et al ., 2018; Tolosa et al ., 2016; Shah et al ., 2021; Donato et al ., 2012). In a previous study (Luckert et al ., 2018), we characterized the molecular signature of cyproconazole-induced steatosis in a metabolically competent model of human liver cells (HepaRG) by investigating key events along the liver steatosis AOP (Mellor et al ., 2015).…”

“…Finally, in a recent study Shah et al quantified mitochondrial function, lipid accumulation, endoplasmic reticulum stress, lysosomal mass, DNA texture, nuclear size, apoptosis, and cell number using a high content imaging approach in rat hepatocytes exposed to 51 hepatotoxicants. Moreover, the in vitro data was extrapolated to in vivo administered equivalent doses and compared to in vivo rat data (Shah et al ., 2021). Thus, a major benefit was simultaneous quantification of several cellular responses as well as extrapolation to in vivo doses.…”