Effects of tea and chlorophyllin on the mutagenicity of N-hydroxy-IQ: studies of enzyme inhibition, molecular complex formation, and degradation/scavenging of the active metabolites

Abstract

Green tea and black tea inhibit the formation of carcinogen-DNA adducts and colonic aberrant crypts in rats given 2-amino-3-methylimidazo[4, 5-f]quinoline (IQ), a mutagen from cooked meat. The Salmonella mutagenicity assay was used in the present study to test individual constituents of tea as inhibitors of 2-hydroxyamino-3-methylimidazo[4, 5-f]quinoline (N-hydroxy-IQ), a direct-acting metabolite of IQ. Testing of pure compounds at doses relevant to their levels in tea identified epigallocatechin (EGC) and epigalocatechin-3-gallate (EGCG) as the primary antimutagens. Studies of the inhibitory mechanisms established that the rate of degradation of N-hydroxy-IQ under aqueous conditions was not increased significantly in the presence of tea, in contrast to the results obtained with the complexing agent chlorophyllin (CHL), which rapidly degraded the mutagen. Interaction between N-hydroxy-IQ and several tea constituents was detected in spectrophotometric studies, but the binding constants were only on the order of 1 x 10(3) M-1, suggesting that mechanisms other than complex formation might prevail under the conditions of the Salmonella assay. Comparison of the results in two different strains of Salmonella typhimurium, TA98 and TA98/1,8-DNP6, indicated that the antimutagenic activity of EGCG was dependent, at least in part, on a functional O-acetyltransferase activity in the bacteria. These studies suggest that tea constituents inhibit the enzyme(s) which generate the aryl nitrenium ion and directly scavenge the reactive electrophile, whereas CHL complexes with heterocyclic amines and facilitates the degradation of active metabolites.

Fig. 1

Pathway for the conversion of IQ to an aryl nitrenium ion, via N-Hydroxy-IQ (box), and the chemical structures of various compounds in tea. Note: formation of the aryl nitrenium ion from N-Hydroxy-IQ occurs spontaneously, but is facilitated by various enzymes, including bacterial O-acetyltransferase (OAT). Sites labeled i–iii are potential targets for inhibition by antimutagens (see text).

Fig. 2

Antimutagenic activity of individual pure tea constituents toward N-Hydroxy-IQ. Salmonella typhimurium strain TA98 was incubated with mutagen (0.015 nmole) plus inhibitor for 15 min, in the absence of a metabolic activation system, prior to the addition of soft agar. Data are given as means ± SD from the testing of triplicate plates, and are representative of results from two or more such assays.

Fig. 3

Antimutagenic activity of (a) EGCG and (b) CHL in two different strains of Salmonella typhimurium. Inhibitors were preincubated with strain TA98 (filled circles) or the O-acetyltransferase-deficient strain TA98/1,8-DNP₆ (open circles), as described in the legend to Figure 2. Results are given as means ± SD for ≥3 plates, except *duplicates. The shaded area in (a) shows that part of the antimutagenic activity in TA98 not accounted for by inhibition of OAT (see text). Percent inhibition was calculated as follows: $$1-\frac{\text{mean\hspace{0.17em}number\hspace{0.17em}of\hspace{0.17em}induced\hspace{0.17em}revertants\hspace{0.17em}plus\hspace{0.17em}inhibitor}}{\text{mean\hspace{0.17em}number\hspace{0.17em}of\hspace{0.17em}induced\hspace{0.17em}revertants\hspace{0.17em}}\text{minus\hspace{0.17em}inhibitor}}\times 100.$$

Fig. 4

Degradation of N-Hydroxy-IQ under aqueous conditions. (a) Time-dependent changes in the absorption spectrum of mutagen alone in 30 mM Tris-HCl buffer, pH 7.4; 25°C, b = 1 cm. The scan was taken at 3-min intervals. (b) Repeat of the experiment in (a) but with the addition of 10 μl of 2.5% (w/v) green tea to both cuvettes; for clarity, only the spectra at 0 min (solid line) and 15 min (dotted line) are shown. (c) Repeat of (a) in the presence of 10 μM CHL.

Fig. 5

Spectrophotometric titration of N-Hydroxy-IQ with (a) green tea or (b) EGCG. Experiments were conducted in 30 mM Tris-HCl buffer, pH 7.4; b = 25°C. (a) Quenching due to the sequential additions of 1 μL 2.5% (w/v) green tea; (b) titration of mutagen with 25 μM EGCG, each addition; inset: expanded view of the data in the region 260–285 nm, showing an apparent isosbestic point at 272 nm.

Fig. 6

Benesi-Hildebrand plot of the data obtained from Figure 5b for the interaction between EGCG/N-hydroxy-IQ. The binding constant (K_b) was obtained from the x-axis intercept, as described previously [Dashwood et al., 1996]. Similar experiments were conducted with other compounds from tea, and the K_b are presented in Table I.