Abstract
Dietary supplementation with natural chemoprotective agents is receiving considerable attention because of health benefits and lack of toxicity. In recent in vivo and in vitro experimental studies, diets rich in n-3 polyunsaturated fatty acids have been shown to provide significant anti-tumor action. In this investigation, the effects of control fatty acids (oleic acid (OA), linoleic acid (LA)) and n-3 PUFA, e.g., docosahexaenoic acid (DHA) on the uptake and metabolism of the carcinogenic polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP) was investigated in A549 cells, a human adenocarcinoma alveolar basal epithelial cell line. A549 cells activate BaP through the cytochrome P450 enzyme system to form reactive metabolites, a few of which covalently bind to DNA and proteins. Therefore, multiphoton microscopy spectral analysis combined with linear unmixing was used to identify the parent compound and BaP metabolites formed in cells, in the presence and absence of fatty acids. The relative abundance of select metabolites was associated with altered P450 activity as determined using ethoxyresorufin-O-deethylase activity in cells cultured in the presence of BSA-conjugated fatty acids. In addition, the parent compound within cellular membranes increases significantly in the presence of each of the fatty acids, with the greatest accumulation observed following DHA treatment. DHA treated cells exhibit significantly lower pyrene-like metabolites indicative of lower adducts including DNA adducts compared to control BSA, OA or LA treated cells. Further, DHA reduced the abundance of the proximate carcinogen BaP 7,8-dihydrodiol and the 3-hydroxybenzo[a]pyene metabolites compared to other treatments. The significant changes in BaP metabolites in DHA treated cells may be mediated by the effects on the physicochemical properties of the membrane known to affect enzyme activity related to phase I and phase II metabolism. In summary, DHA is a highly bioactive chemo-protective agent capable of modulating BaP-induced DNA adducts.
Highlights
Exposure to polycyclic aromatic hydrocarbons (PAHs) usually occurs by breathing contaminated air or by eating grilled foods
The relative abundance of several major metabolites changed when cells were cultured in the presence of bovine serum albumin (BSA)-conjugated fatty acids (OA, linoleic acid (LA) or docosahexaenoic acid (DHA), each at 50 mM along with BSA as carrier control) for 24 h in advance of 2 mM BaP exposure for 24 h (Figure 2)
Accumulation of the parent compound (BaP) within cellular membranes increased significantly in the presence of each of the three fatty acids (OA, LA, and DHA) when compared to control, with the greatest accumulation observed in DHA treated cells (Figure 3A)
Summary
Exposure to polycyclic aromatic hydrocarbons (PAHs) usually occurs by breathing contaminated air or by eating grilled foods. Several PAHs have been listed by the U.S Environmental Protection Agency as probable human carcinogens and this includes the prototype carcinogenic PAH, benzo[a]pyrene (BaP) [1]. As a ligand for the aryl hydrocarbon receptor (AhR), BaP upregulates the expression of phase I bioactivation genes and phase II conjugation genes [2,3]. Induction of biotransformation enzymes including CYP1A1, CYP1B1 and epoxide hydrolase metabolically activate BaP to different types of metabolites including hydroxylated intermediates, epoxides, quinones, dihydrodiols, dihydrodiol epoxides and various metabolite-conjugates in cells [4,5]. BaP toxicity results from the bioactivation of BaP to the ultimate toxic compound, benzo[a]pyrene-7,8-dihydrodiol9,10-epoxide (BPDE). BPDE can alkylate DNA to form BPDEDNA adducts (BPDE-N(2)-deoxyguanosine [BPDE-dG]), which have been associated with BaP-induced carcinogenesis [6]. Many of the major metabolites can be conjugated to glucuronic acid, sulfate and glutathione to become more water soluble facilitating excretion [7]
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