A gamma-tocopherol-rich mixture of tocopherols inhibits colon inflammation and carcinogenesis in azoxymethane and dextran sulfate sodium-treated mice

Abstract

We investigated the effects of a gamma-tocopherol-rich mixture of tocopherols (gamma-TmT, containing 57% gamma-T, 24% delta-T, and 13% alpha-T) on colon carcinogenesis in azoxymethane (AOM)/dextran sulfate sodium (DSS)-treated mice. In experiment 1, 6-week-old male CF-1 mice were given a dose of AOM (10 mg/kg body weight, i.p.), and 1 week later, 1.5% DSS in drinking water for 1 week. The mice were maintained on either a gamma-TmT (0.3%)-enriched or a standard AIN93M diet, starting 1 week before the AOM injection, until the termination of experiment. In the AOM/DSS-treated mice, dietary gamma-TmT treatment resulted in a significantly lower colon inflammation index (52% of the control) on day 7 and number of colon adenomas (9% of the control) on week 7. gamma-TmT treatment also resulted in higher apoptotic index in adenomas, lower prostaglandin E2, leukotriene B4, and nitrotyrosine levels in the colon, and lower prostaglandin E2, leukotriene B4, and 8-isoprostane levels in the plasma on week 7. Some of the decreases were observed even on day 7. In experiment 2 with AOM/DSS- treated mice sacrificed on week 21, dietary 0.17% or 0.3% gamma-TmT treatment, starting 1 week before the AOM injection, significantly inhibited adenocarcinoma and adenoma formation in the colon (to 17-33% of the control). Dietary 0.3% gamma-TmT that was initiated after DSS treatment also exhibited a similar inhibitory activity. The present study showed that gamma-TmT effectively inhibited colon carcinogenesis in AOM/DSS-treated mice, and the inhibition may be due to the apoptosis-inducing, anti-inflammatory, antioxidative, and reactive nitrogen species-trapping activities of tocopherols.

Fig. 1

Structures of tocopherols (A) and effects of γ-TmT treatment (0.3% in the diet) on tocopherol levels in the plasma, colon tissues, and feces of the AOM/DSS–treated mice. Samples from experiment 1 were used. Levels of α-T, γ-T, and δ-T were determined according to Materials and Methods, and typical chromatograms of mouse samples and standard plasma (from human) at the 500 mV channel are shown (B). The detection limit of each tocopherol in our analysis was 0.02 ng. For a clear illustration, the baseline of each sample is set at an arbitrary level. Levels of α-T, γ-T, and δ-T in the plasma (C, n = 10/group) of AOM/DSS–treated mice sacrificed at day 3, day 7, and week 7, as well as in the colon homogenates (D, n = 9/group) and feces (E, n = 3/group) of AOM/DSS–treated mice on week 7, are shown as mean ± SE of each group. All three tocopherols were detected in all the samples, but the value of certain tocopherols were too low to be shown in the figures. Several unknown peaks between 14 and 18 min were observed in the colon samples from the γ-TmT–treated mice (B). Different superscripts (a, b, and c) indicate statistical difference among levels of each specific tocopherol (by one-way ANOVA; P < 0.05). *, statistical difference between the γ-TmT–treated and the control group in a specific tocopherol (P < 0.05 by the two-tailed t test).

Fig. 2

Effects of γ-TmT treatment (0.3% in the diet) on colon adenoma formation in AOM/DSS–treated CF-1 mice. Formalin-fixed visible tumors were dissected, embedded, and sectioned for histopathological analysis. Two H&E-stained sections per tumor were analyzed. All of the individual colon tumors were histologically characterized as tubular adenomas. Scatter plot for the number of colon adenomas per mouse in AOM/DSS–treated mice on week 7 (A, n = 10/group). One mouse in the γ-TmT–treated mice had 15 adenomas in the colon (x), and the number was identified as an extreme outlier (by Q-spread analysis); it was therefore excluded for subsequent statistical analysis. A two-tailed t test yields P = 0.09 and a one-tailed t test yields P = 0.04 when all animals were included. The numbers of colon adenomas with mild, moderate, and severe dysplasia per mouse are also shown as mean ± SE in B. *, the value is different from that in the control group (P ≤ 0.05 by the two-tailed t test).

Fig. 3

Histological characterization of colon adenomas and inflammation in AOM/DSS–treated mice. Tubular adenomas were classified into those with mild (A), moderate (B), and severe (C) dysplasia in the colons of AOM/DSS–treated mice on week 7 (in experiment 1). Well-differentiated adenocarcinomas (D) were found in the colons of AOM/DSS–treated mice on week 21 (in experiment 2). Magnification, ×400. Compared with adenomas from the control group (E), adenomas from the γ-TmT–treated group (F) had an increased number of cleaved caspase-3–positive cells on week 7. Cleaved caspase-3–positive cells displayed in the nucleus of adenoma cells, but rarely in that of normal cells. Magnification, ×400. Effects of γ-TmT on the colonic inflammation in the AOM/DSS–treated mice on day 7 are shown in G and H. G, mild inflammation in the colon of γ-TmT–treated mice showing mononuclear and polymorphonuclear leukocytes infiltration into the basal one third of the mucosa (M). H, severe inflammation in the colon of control AOM/DSS–treated mice showing mononuclear and polymorphonuclear leukocytes infiltration into both mucosa and submucosa (SM), loss of crypts (C, which are seen in panel G), and ulceration (U).

Fig. 4

Effects of γ-TmT treatment on plasma or colonic levels of PGE2, LTB4, 8-isoprostane, and nitrotyrosine. A, PGE2, LTB4, and 8-isoprostane levels were determined in the plasma of AOM/DSS–treated mice (on day 3, day 7, and week 7) and DSS-treated mice (on days 3 and 7). B, PGE2, LTB4, and nitrotyrosine levels were determined in the colon (homogenates) of AOM/DSS–treated mice. Columns, mean of group (n = 9–10 per AOM/DSS–treated group; n = 4–5 per DSS-treated group); bars, SE. Different superscripts (a, b, c, d) indicate statistical difference among levels of AOM/DSS–treated mice (by one-way ANOVA; P < 0.05). Statistical difference between levels of the γ-TmT–treated and the respective control group: **, P < 0.05 by the two-tailed t test; *, P < 0.05 by the one-tailed t test (P = 0.07 by two-tailed t test).