Mutation Research/Genetic Toxicology and Environmental Mutagenesis
Mechanisms of chromosomal aberrations induced by sesamin metabolites in Chinese hamster lung cells
Introduction
Sesame (Sesamum indicum) seeds and oil have been recognized as traditional healthy foods since ancient times, particularly in Asia and Africa. During the refinement of non-roasted sesame seed oil, sesamin, a major lignan found in these foods, is partially epimerized to form episesamin [1]. Mixtures of sesamin and episesamin show multiple physiological effects, including antioxidant [2], [3], [4], [5], hypocholesterolemic and hypolipidemic [6], [7], [8], [9], [10], antihypertensive [11], [12], [13], [14], [15], [16], and hepatoprotective activities [17]. Therefore, sesamin and episesamin have attracted much attention as food constituents with potential health benefits. Nakai et al. [2] have shown that sesamin is metabolized by cytochrome P450-catalyzed oxidation to its mono-chatechol derivative [SC-1; 2-(3,4-methylenedioxyphenyl)-6-(3,4-dihydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane] and then to its di-chatechol derivative [SC-2; 2,6-bis(3,4-dihydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane]; both of these compounds are further metabolized by catechol-O-methyltransferase (COMT) catalyzed methylation to the methoxy metabolites SC-1m and SC-2m, respectively. We confirmed that sesamin is subject to extensive first-pass metabolism and that the plasma concentration of SC-1 is much higher than that of sesamin, after oral administration of sesamin in humans [39]; therefore, SC-1 might be responsible for physiological activities.
We previously reported that sesamin induces chromosomal aberrations (CA) in Chinese hamster lung cells (CHL/IU) in the presence of S9 mix (the supernatant from rat liver homogenate). However, no genotoxicity was detected using an in vitro bacterial reverse mutation assay (Ames test) with or without S9 mix, or in vivo genotoxic tests such as a bone marrow micronucleus (MN) test in mice and a liver cell comet assay in rats [18]. Sesamin only induced CA in the presence of S9 mix, suggesting that a metabolite of sesamin is responsible. No genotoxicity of sesamin has been detected in animals. One reason for this difference may be that the induction of CA in CHL/IU is accompanied by severe cytotoxicity (>50%). This conclusion is consistent with the revised OECD test guideline (TG473), which notes that the effect of cell toxicity should be considered in the interpretation of CA results and that the CA test should be conducted within an appropriate concentration range [19]. In the present study, we have further examined the molecular mechanism underlying CA induction by sesamin in CHL/IU cells.
Previous studies showed that compounds with a catechol structure induce DNA damage and CA in in vitro cell systems [20], [21]. For example, tea catechins gave positive results in an in vitro mouse lymphoma test, CA test, and MN test, but were negative in in vivo tests (bone marrow MN test and transgenic rodent gene mutation test) [20], [22], [23]. Epigallocatechin gallate and dopamine have been reported to generate H2O2, semiquinones, and quinones in vitro, catalyzed by trace metals in the culture medium, inducing DNA damage and cytotoxicity [23], [24], [25], [26], [27], [28], [29], [30], [55], [56]. Sesamin may act through a similar mechanism because its main metabolite, SC-1, has a catechol structure.
Several rodent and human cell lines, including CHL/IU, are commonly used to predict carcinogenicity. However, different results are observed, depending on the cell line used. HepG2 cells, which retain many of the functions of normal liver cells, are believed to be more useful for genotoxicity assessment of chemicals, compared to other mammalian cell lines which require an exogenous metabolizing system [57], [58].
The aim of this study was to clarify the mechanism of CA-induction by sesamin in CHL/IU cells. We identified a major metabolite of sesamin produced by incubation with S9 mix, and evaluated the ability of this metabolite to induce CA in CHL/IU or HepG2 cells. The influence of reducing agents such as reduced glutathione (GSH) and sodium sulfite (Na2SO3) on the ability of sesamin and its metabolite to induce CA in CHL/IU cells was also examined.
Section snippets
Preparation of reagents
Sesamin was purified as described previously [1], [2]. Sesamin metabolites (SC-1 and SC-2) were prepared according to the method of Urata et al. [35]. Benzo[a]pyrene (B[a]P; CAS 50-32-8), dimethyl sulfoxide (DMSO; CAS 67-68-5) and sodium sulfite (Na2SO3; CAS 7757-83-7) were obtained from Wako Pure Chemical Industries (Japan). Mitomycin C (MMC; CAS 50-07-7) was purchased from Kyowa Hakko Kirin Co. Ltd. (Japan). Reduced glutathione (GSH; CAS 70-18-8) and silver oxide (Ag2O; CAS 20667-12-3) were
Determination of the concentrations of sesamin and its metabolites following incubation in cell-free culture medium
Sesamin (100 μg/ml) was incubated with S9 mix in cell-free culture medium at 37 °C for 6 h and the resulting concentrations of sesamin and its metabolites are shown in Fig. 1. Sesamin in the medium decreased rapidly and SC-1 was generated. After 6 h incubation, the concentrations of sesamin and SC-1 were 16.2 and 46.1 μg/ml, respectively. The concentration of SC-1 was about half that of the amount of sesamin added initially and much higher than the concentration of sesamin after 6 h incubation. SC-2
Discussion
The present study evaluated the biological significance of the CA-inducing activity of sesamin, clarifying the mechanisms involved. We confirmed that SC-1 is the main metabolite of sesamin in medium following the incubation of sesamin with S9 mix, and we found that SC-1 induces CA in CHL/IU cells in the absence of S9 mix. This suggests that SC-1 is the major active metabolite of sesamin. However, further studies indicated that SC-1 rapidly decomposed in culture medium, and the addition of GSH
Conflicts of interest
YO, NT, YK, and HS are employees of Suntory Wellness Ltd., which is a manufacturer of foods that contain sesamin. HH is employee of Suntory MONOZUKURI Expert Ltd.
Funding
This work was supported by Suntory Wellness Ltd..
Acknowledgments
We would like to thank Dr. Minako Nagao (Faculty of Pharmacy, Keio University), Dr. Makoto Hayashi (Public Interest Incorporated Foundation, BioSafety Research Center), and Dr. Hiroshi Kawashima (Suntory Wellness Ltd.) for scientific advice and encouragement.
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