Systems Toxicology Approach to Understand the Kinetics of Benzo(a)pyrene Uptake, Biotransformation, and DNA Adduct Formation in a Liver Cell Model Danielle J. Madureira, †,‡ Frederik T. Weiss, † Paul Van Midwoud, § Damian E. Helbling, †,⊥ Shana J. Sturla, § and Kristin Schirmer* ,†,‡,∥ † Eawag, Swiss Federal Institute of Aquatic Science and Technology, Dü bendorf, Switzerland ‡ Institute of Biogeochemistry and Pollutant Dynamics, Department of Environmental Systems Science, ETH Zü rich, Zü rich, Switzerland § Department of Health Sciences and Technology, Laboratory of Food and Nutrition Toxicology, ETH Zü rich, Zü rich, Switzerland ∥ School of Architecture, Civil and Environmental Engineering, EPF Lausanne, Lausanne, Switzerland * S Supporting Information ABSTRACT: Cell-based models are important for deriving mechanistic information about stress response pathways that have evolved to protect cells from toxic insult, such as exposure to environmental pollutants. One determinant of the stress response is the amount of chemical entering the cell and the cell’s ability to detoxify and remove the chemical. If the stress response is overwhelmed, an adverse outcome will ensue. It was the goal of our study to quantify uptake and elimination rates of benzo(a)pyrene (BaP), a ubiquitous environmental pollutant, in a murine liver cell line. We evaluated the kinetic behavior in the context of BaP uptake, biotransformation, DNA adduct formation and repair along with the transcriptional and cell proliferation response. A low (50 nM) and a high (5 μM) BaP concentration were chosen in order to differentiate the role of exposure concentration in the time-resolved interaction of BaP with cells. While rates of uptake and the initial transcriptional response were similar for both BaP concentrations, cells exposed to 50 nM BaP completely recovered from exposure within 24 h, whereas cells exposed to 5 μM BaP did not. Biotransformation proceeded faster on 50 nM BaP, and the few DNA adducts formed were completely repaired after transient cell cycle arrest. In contrast, DNA adducts greatly accumulated in cells exposed to 5 μM BaP, despite significant biotransformation; complete cell cycle arrest and toxicity evolved. On the basis of the kinetic rate constants and cellular response, we conclude that at least short-term, pulsed exposures to 50 nM BaP, which we consider environmentally relevant, can be handled by cells without adverse outcome. Further studies are needed to determine the ability of cells to recover from repeated exposure. Our study emphasizes the importance of quantifying chemical uptake and fate in cell models to differentiate a stress response from an adverse outcome for better risk assessment. ■ INTRODUCTION Benzo(a)pyrene (BaP) is a widely studied and well-known environmental carcinogenic pollutant belonging to the class of polycyclic aromatic hydrocarbons (PAHs). BaP is formed by incomplete combustion of organic matter, being identified as a major tumor producing agent in coal tar in 1932. 1 Cigarette smoke and car exhaust are prominent sources of BaP, but it can also be found in some occupational atmospheres and food. Despite vast general knowledge on BaP toxicity, significant uncertainties for quantitative risk assessment remain. This is due, in part, to a lack of knowledge of the kinetics of the chemical in exposed cells and the subsequent time-resolved induction of a stress or toxic response leading to recovery or irreversible DNA damage and potentially cell death. This study addresses this knowledge gap by using a systems toxicology approach that combines analytical chemistry and molecular and cell biology to decipher stress- and toxicity response profiles of a liver cell model on exposure to low and high concentrations of BaP. It is generally accepted that BaP requires biological activation through oxidative biotransformation to be toxic and carcino- genic. 2 BaP induces its own biotransformation by activating the aryl hydrocarbon receptor (AhR). The AhR is a cytosolic transcription factor that, upon activation, moves to the nucleus where it binds to ARNT (aryl hydrocarbon nuclear trans- locator), the heterodimeric partner of the AhR. This complex Special Issue: Systems Toxicology Received: December 3, 2013 Published: January 21, 2014 Article pubs.acs.org/crt © 2014 American Chemical Society 443 dx.doi.org/10.1021/tx400446q | Chem. Res. Toxicol. 2014, 27, 443−453