Multiphoton spectral analysis of benzo[a]pyrene uptake and metabolism in a rat liver cell line

Abstract

Dynamic analysis of the uptake and metabolism of polycyclic aromatic hydrocarbons (PAHs) and their metabolites within live cells in real time has the potential to provide novel insights into genotoxic and non-genotoxic mechanisms of cellular injury caused by PAHs. The present work, combining the use of metabolite spectra generated from metabolite standards using multiphoton spectral analysis and an “advanced unmixing process”, identifies and quantifies the uptake, partitioning, and metabolite formation of one of the most important PAHs (benzo[a]pyrene, BaP) in viable cultured rat liver cells over a period of 24 hr. The application of the advanced unmixing process resulted in the simultaneous identification of 8 metabolites in live cells at any single time. The accuracy of this unmixing process was verified using specific microsomal epoxide hydrolase inhibitors, glucuronidation and sulfation inhibitors as well as several mixtures of metabolite standards. Our findings prove that the two-photon microscopy imaging surpasses the conventional fluorescence imaging techniques and the unmixing process is a mathematical technique that seems applicable to analysis of BaP metabolites in living cells especially for analysis of changes of the ultimate carcinogen benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide. Therefore, the combination of the two-photon acquisition with the unmixing process should provide important insights into the cellular and molecular mechanisms by which BaP and other PAHs alter cellular homeostasis.

Effects of Fatty Acids on Benzo[a]pyrene Uptake and Metabolism in Human Lung Adenocarcinoma A549 Cells

“…Many of the BaP metabolites and their time-dependent changes in liver cells have been detected in living cells using multiphoton microscopy with the linear unmixing process [13]. Similar time-dependent changes in BaP levels and metabolites in A549 cells are shown in Figure 1 .…”

Revisiting Polyarenes and Related Molecules: An Update of Synthetic Approaches and Structure‐Activity‐Mechanistic Correlation for Carcinogenesis

“…However B[ a ]P and its derivatives are photosensitive and are known for their instability in presence of UV light including confocal microscopy. Barhoumi et al . reported on tackling this problem by merging multiphoton spectral analysis and an “advanced un‐mixing process” to detect metabolites of B[ a ]P. The method enabled them to not only detect metabolite formation, but also the uptake and partitioning of B[ a ]P, which could ultimately provide more insights on PAH homeostasis and its mechanisms of action.…”

Differential gene expression analysis of benzo(a)pyrene toxicity in the clam, Ruditapes philippinarum

“…The metabolic process of BaP in vertebrates was studied more clearly than that in invertebrates (Barhoumi et al, 2011;Cavalieri and Rogan, 1995;Harris et al, 2009;Jacques et al, 2010;Ramesh et al, 2001). It has been reported that once BaP enters the cells of mammals, it can be oxidated to various BaP epoxides and phenols catalyzed by cytochrome P450 (CYP450).…”

“…Studies on the toxicity of PAHs are focused on several fields such as genotoxicity, hepatotoxicity, developmental toxicity, carcinogenesis and others (Barhoumi et al, 2011;Harris et al, 2009;Jacques et al, 2010;Wang and Jin, 2010), the toxicity effects of PAHs have been detected from a wide range of organisms (Song et al, 2011;Yin et al, 2011;Pan et al, 2008;Wang et al, 2011b;Tuvikene, 1995;Zhang et al, 2009;Kannan and Perrotta, 2008). It is reported that PAHs can cause harmful effects on the skin, body fluids, and the human ability to fight disease after both short-and long-term exposure (U.S. Department of health and human services, 1995).…”