Selective Inhibition of Methylbenzylnitrosamine-induced Formation of Esophageal O⁶-Methylguanine by Dietary Ellagic Acid in Rats¹

Ellagic acid is a naturally occurring plant phenol which has been shown to reduce the incidence of a number of carcinogen-induced tumors including methylbenzylnitrosamine (MBN)-induced esophageal carcinoma in the rat. The postulated mechanism of MBN-induced esophageal carcinogenesis is through oxidation of MBN to form benzaldehyde and an activated metabolite which methylates DNA forming a variety of methylated DNA adducts including O⁶-methylguanine (O⁶-mGua) and 7-methylguanine (m⁷Gua). O⁶-mGua adducts have been shown to induce DNA mutations which can lead to cancer, while m⁷Gua adducts do not appear to be related to tumor induction. In this study, we examined whether the decreased incidence of MBN-induced esophageal carcinoma observed with dietary ellagic acid was associated with a decrease in the in vivo and in vitro formation of MBN-induced DNA adducts and whether this reduction was specific to O⁶-mGua or due to a reduction in total methylation. Weanling male Sprague-Dawley rats were fed a nutritionally complete diet with and without the addition of 0.4 g of ellagic acid per kg of diet. This dose of dietary ellagic acid has previously been shown to reduce the incidence of MBN-induced esophageal carcinoma by 30 to 50%. After 3 wk on the diets, rats were given injections of a single dose of MBN (2.0 mg/kg of body weight i.p.) and sacrificed 1 h after injection. Dietary ellagic acid significantly reduced the MBN-induced in vivo formation of esophageal O⁶-mGua, without significantly reducing the formation of esophageal m⁷Gua. Examination of this effect in an in vitro methylation assay demonstrated that dietary ellagic acid did not reduce the ability of esophageal microsomes to methylate purified calf thymus DNA; however, pretreatment of the calf thymus DNA with ellagic acid selectively reduced the MBN-induced formation of O⁶-mGua by microsomes from both ellagic acid-fed and control animals without altering the in vitro formation of m⁷Gua. These results suggest that ellagic acid bound to DNA selectively blocks methylation of the O⁶-position of guanine without inhibiting the activation of MBN or the ability of MBN to methylate DNA.