Biological clues to potent DNA-damaging activities in food and flavoring

Abstract

Population differences in age-related diseases and cancer could stem from differences in diet. To characterize DNA strand-breaking activities in selected foods/beverages, flavorings, and some of their constituent chemicals, we used p53R cells, a cellular assay sensitive to such breaks. Substances testing positive included reference chemicals: quinacrine (peak response, 51×) and etoposide (33×); flavonoids: EGCG (19×), curcumin (12×), apigenin (9×), and quercetin (7×); beverages: chamomile (11×), green (21×), and black tea (26×) and coffee (3-29×); and liquid smoke (4-28×). Damage occurred at dietary concentrations: etoposide near 5μg/ml produced responses similar to a 1:1000 dilution of liquid smoke, a 1:20 dilution of coffee, and a 1:5 dilution of tea. Pyrogallol-related chemicals and tannins are present in dietary sources and individually produced strong activity: pyrogallol (30×), 3-methoxycatechol (25×), gallic acid (21×), and 1,2,4-benzenetriol (21×). From structure-activity relationships, high activities depended on specific orientations of hydroxyls on the benzene ring. Responses accompanied cellular signals characteristic of DNA breaks such as H2AX phosphorylation. Breaks were also directly detected by comet assay. Cellular toxicological effects of foods and flavorings could guide epidemiologic and experimental studies of potential disease risks from DNA strand-breaking chemicals in diets.

Fig 1

p53 and γ-H2AX protein levels after treatment with liquid smoke, black tea, and pyrogallol. (A) p53R cells were treated with Wright’s Hickory liquid smoke (Ls W) or Cedar House Hickory liquid smoke (Ls C) at 0.0005x and 0.001x for 18 h. Untreated cells (U) and 2 μg/ml quinacrine-treated cells (Q) served as a negative and a positive control for p53 induction respectively. Cellular p53 and γ-H2AX protein levels were evaluated by immunoblotting. (B) AAV-293 cells were treated with black tea (Tea B) at 0.05x and 0.2x or liquid smoke solutions (Ls W or Ls C) at 0.0005x and 0.001x for 18 h. Cellular γ-H2AX protein levels were assayed by immunoblotting. (C) p53R cells were treated with 0.001x Ls W, various concentrations of pyrogallol, or 2 μg/ml quinacrine for 6, 12, or 20 h. Cellular p53, p21, and γ-H2AX levels were assessed by immunoblotting.

Fig 2

Neutral comet assay after treatment with liquid smoke and pyrogallol. The neutral comet assay was performed on p53R cells treated with 0.0005x Wright’s Hickory liquid smoke (Ls W) or 15 μg/ml pyrogallol. At least 45 cells were analyzed per sample and the mean tail moment of the cell population was calculated using CometScore. (A) Representative pictures of cells. (B) Mean tail moment for each treatment with error bars representing the standard error of the mean (SEM) for the cell population.

Fig 3

p53 reporter activation after treatment with (A) pyrogallol, (B) 3-methoxycatechol, (C) gallic acid, and (D) 1,2,4-benzenetriol. p53R cells were plated in triplicate and treated with various doses of each compound for 18 h. p53 reporter activity was assessed in a luciferase assay. Luminescence was calculated relative to the untreated sample. The mean for each triplet was plotted for each concentration tested.

Fig 3

p53 reporter activation after treatment with (A) pyrogallol, (B) 3-methoxycatechol, (C) gallic acid, and (D) 1,2,4-benzenetriol. p53R cells were plated in triplicate and treated with various doses of each compound for 18 h. p53 reporter activity was assessed in a luciferase assay. Luminescence was calculated relative to the untreated sample. The mean for each triplet was plotted for each concentration tested.