Pathway and Time-Resolved Benzo[<i>a</i>]pyrene Toxicity on Hepa1c1c7 Cells at Toxic and Subtoxic Exposure

Benzo­[a]­pyrene (B­[a]­P) is an environmental contaminant mainly studied for its toxic/carcinogenic effects. For a comprehensive and pathway orientated mechanistic understanding of the effects directly triggered by a toxic (5 μM) or a subtoxic (50 nM) concentration of B­[a]P or indirectly by its metabolites, we conducted time series experiments for up to 24 h to study the effects in murine hepatocytes. These cells rapidly take up and actively metabolize B­[a]­P, which was followed by quantitative analysis of the concentration of intracellular B­[a]P and seven representative degradation products. Exposure with 5 μM B­[a]P led to a maximal intracellular concentration of 1604 pmol/5 × 104 cells, leveling at 55 pmol/5 × 104 cells by the end of the time course. Changes in the global proteome (>1000 protein profiles) and metabolome (163 metabolites) were assessed in combination with B­[a]P degradation. Abundance profiles of 236 (both concentrations), 190 (only 5 μM), and 150 (only 50 nM) proteins were found to be regulated in response to B­[a]P in a time-dependent manner. At the endogenous metabolite level amino acids, acylcarnitines and glycerophospholipids were particularly affected by B­[a]­P. The comprehensive chemical, proteome and metabolomic data enabled the identification of effects on the pathway level in a time-resolved manner. So in addition to known alterations, also protein synthesis, lipid metabolism, and membrane dysfunction were identified as B­[a]P specific effects.