Systems Toxicology Approach to Understand the Kinetics of Benzo(a)pyrene Uptake, Biotransformation, and DNA Adduct Formation in a Liver Cell Model

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.

A 3D fish liver model for aquatic toxicology: Morphological changes and Cyp1a induction in PLHC-1 microtissues after repeated benzo(a)pyrene exposures

“…Using both gene and protein expression of Cyp1a, we were able to demonstrate the sensitivity of our model to the exposure to aromatic hydrocarbons. Aromatic hydrocarbons like benzo(a)pyrene enter cells via passive diffusion and can be observed by visualization of their autofluorescence, allowing us to observe uptake into the 3D microtissue (Barhoumi et al, 2000; Madureira et al, 2014). Unlike cells in monolayer, which are exposed through direct contact with the medium, the multilayered structure of a 3D microtissue is a barrier to toxicant diffusion or transport similar to in vivo liver (Godoy et al, 2013; Olinga et al, 2001).…”

“…The persistent upregulation of cyp1a gene expression observed after high exposure (>100 nM benzo(a)pyrene) may be the result of benzo(a)pyrene or benzo(a)pyrene metabolites remaining in the microtissue, with continued activation of the aryl hydrocarbon receptor and induced gene expression. Compared to a lower, more readily metabolized dose, the high concentrations of benzo(a)pyrene are likely beyond the cell’s ability to be completely metabolized in 24 h, leading to both higher and prolonged intra-cellular exposure (Madureira et al, 2014; Thibaut et al, 2009). The continued upregulation of cyp1a during the recovery period may be important for the survival of cells after subsequent or chronic exposures.…”

“…While the differences in monolayer cell number are likely due to cell death or limited proliferation (Madureira et al, 2014), microtissues show a greater complexity in their morphological changes. These studies revealed that repeated benzo(a)pyrene exposure results in altered microtissue architecture and signs of liver pathology including altered cytoskeletal organization, compensatory proliferation, and both necrotic and apoptotic cell death.…”

“…To limit adsorptive loss of benzo(a)pyrene to plastic surfaces, all benzo(a)pyrene stock solutions and exposure media were prepared in glass, and all spheroid exposures were conducted in glass containers (Chlebowski et al, 2016; Madureira et al, 2014). …”

The role of phytochemicals in modulating intrinsic human cellular defence processes

“…The group of induced enzymes includes NAD(P)H:NQO1 (quinone reductase) and the family of GSTs, both of which are required for the detoxification of steroids and the ubiquitous environmental toxin, benzo(a)pyrene. [491][492][493] Zhang et al…”