Toxicity in Liver Microtissues
3D InSight™ human liver microtissues (InSphero) were exposed to acetaminophen concentrations between 9.77 µM and 2,500 µM. The IC50 was 2,300 µM determined by a cell viability assay. A specific assay library for human liver microtissues was generated using LC-MS/MS and contained over 2,400 proteins. Many proteins included in the library were characteristic liver proteins.
HRM-MS analysis resulted in 2,361 full protein profiles over all samples in the tested concentration range. As sample materials are commonly precious, all analyses were carried out using as little as 12,000 cells as starting material.
Pairwise comparison of acetaminophen-exposed liver microtissues at the different concentrations showed that with increasing acetaminophen concentration more proteins were significantly regulated while most of the proteins in the library remained, as expected, unchanged (Figure 2A). Functional annotation revealed cellular processes that were activated or deactivated, such as oxidation reduction for upregulated proteins and fatty acid metabolism for downregulated candidates. These findings corresponded well with the known effects of oxidative stress and liver damage occurring in liver after acetaminophen exposure.
The most striking change in protein expression level was observed for cytochrome P450 1A2 (gene name CYP1A2), a protein that is known to be involved in phase I metabolism of acetaminophen. A significant upregulation was found even at the lowest concentration (Figure 2). This is especially noteworthy as the traditional cell viability assay did not show any effect at that concentration, which is known to be well below the therapeutic acetaminophen concentration in vivo.
The HRM-MS method revealed a promising candidate for very early marker of liver toxicity upon acetaminophen treatment. Raw data from HRM-MS generated with Spectronaut™ are displayed in Figure 2B.
In addition to the global analysis, HRM-MS allows for a targeted analysis of any protein of interest that is covered in the assay library. Enzymes involved in phase II metabolism such as UDP-glucuronosyltransferases (UGTs) or sulfotransferases (SULTs) play an important role for the conjugation and elimination of acetaminophen. These enzymes can be monitored over the entire concentration range and show significant increases at higher doses of acetaminophen (Figure 2C).