We determined the protective activity of melanoidins against DNA damage using the comet assay. We used nitric oxide (NO) as a DNA damaging agent and HL60 (human promyelocytic leukemic cell line). Melanoidins were prepared from soy sauce and soy paste (miso). When the cells were incubated with NO (1 mM) and melanoidin (0-0.25%) for 30 min, soy sauce melanoidin strongly inhibited NO-induced DNA damage in a dose dependent manner. Miso melanoidin and melanoidin prepared from a glycine/glucose model system also inhibited the DNA damage. Melanoidins showed the protective effect even when cells were subsequently incubated with NO after the treatment and removal of melanoidins. As melanoidins did not trap NO under the condition used, they seemed to inhibit the DNA damage indirectly through a modification of some cellular function.Keywords: melanoidin, DNA damage, comet assay, nitric oxide (NO), soy sauce, miso, HL60Maillard reaction is one of the most important reactions in chemical changes of food components during storage and processing of foods. Melanoidins, advanced glycation end products of Maillard reaction, are brown polymerized pigments that are produced in food systems containing proteins or amino acids and reducing sugars (Ikan et al., 1996). They are contained in many foods such as soy sauce, miso, roasted coffee etc., and play an important role in adding desirable flavor and color to food. In addition to making food more desirable, there have been many reports about their physiological functions , such as promotion of faecal bacterial growth (Ames et al., 1999), suppression of tumor cell growth (Kamei et al., 1997), inhibition of digestive enzymes (Hirano et al., 1994(Hirano et al., , 1996, antioxidative activity (Chuyen et al., 1998), and antimutagenic activity (Lee et al., 1994). We examined DNA damage-protecting activities of melanoidins using the comet assay, a single cell gel electrophoresis assay which has been used to detect the genotoxic effect of irradiation of various foods (Cerda et al., 1997;Singh, 1988). Subsequent improvements of the sensitivity and methodology have made it possible to detect single-and doublestrand breaks, alkali labile sites, incomplete excision repair sites and genomic structural discontinuities. Recently we have shown that the comet assay is an easy, quick and highly sensitive method to detect the protective effect of food components against DNA damage induced by several chemical mutagens (Miwa & Hongo, 2000;Miwa et al., 1999Sugai & Miwa, 1999). In this paper, we evaluated the DNA damage-protecting activity of melanoidins derived from soy sauce, miso paste and glycine-glucose model melanoidin against DNA damage of HL60 cells induced by nitric oxide using the comet assay.
Materials and MethodsCell preparation The cell line HL60 (human promyelocytic leukemia) was used in this experiment. Cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a CO 2 incubator (air: 95%, CO 2 : 5%). Before the assay, cells were centrifuged ...