Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN, USA and
1 Division of Environmental Health Sciences, Ohio State University School of Medicine and Public Health, Columbus, OH, USA
Abstract
Recent studies have examined and demonstrated the potential cancer chemopreventive activity of freeze-dried berries including strawberries and black raspberries. Although ellagic acid, an abundant component in these berries, has been shown to inhibit carcinogenesis both in vivo and in vitro, several studies have reported that other compounds in the berries may also contribute to the observed inhibitory effect. In the present study, freeze-dried strawberries (Fragara ananassa, FA) or black raspberries (Rubus ursinus, RU) were extracted, partitioned and chromatographed into several fractions (FA-F001, FA-F003, FA-F004, FA-F005, FA-DM, FA-ME from strawberries and RU-F001, RU-F003, RU-F004, RU-F005, RU-DM, RU-ME from black raspberries). These extracts, along with ellagic acid, were analyzed for anti-transformation activity in the Syrian hamster embryo (SHE) cell transformation model. None of the extracts nor ellagic acid by themselves produced an increase in morphological transformation. For assessment of chemopreventive activity, SHE cells were treated with each agent and benzo[a]pyrene (B[a]P) for 7 days. Ellagic acid, FA-ME and RU-ME fractions produced a dose-dependent decrease in transformation compared with B[a]P treatment only, while other fractions failed to induce a significant decrease. Ellagic acid, FA-ME and RU-ME were further examined using a 24 h co-treatment with B[a]P or a 6 day treatment following 24 h with B[a]P. Ellagic acid showed inhibitory ability in both protocols. FA-ME and RU-ME significantly reduced B[a]P-induced transformation only when co-treated with B[a]P for 24 h. These results suggest that a methanol extract from strawberries and black raspberries may display chemopreventive activity. The possible mechanism by which these methanol fractions (FA-ME, RU-ME) inhibited cell transformation appear to involve interference of uptake, activation, detoxification of B[a]P and/or intervention of DNA binding and DNA repair.
Abbreviations: B[a]P, benzo[a]pyrene; EA, Ellagic acid; FA, Fragara ananassa; RU, Rubus ursinus; SHE, Syrian hamster embryo.
Introduction
Chemoprevention has been acknowledged as an important and practical strategy for the management of cancer. Many naturally occurring substances present in the human diet have been identified as potential chemopreventive agents (14). Animal investigations supported by epidemiological studies have suggested that consuming relatively large amounts of vegetables and fruit can prevent the development of cancers (5). Block et al. reviewed numerous epidemiological investigations and found an inverse association between the consumption of fruit and vegetables and the incidence of cancers in multiple organs including lung, larynx, oral pharynx, gastrointestinal tract and pancreas (6). Constituents and micronutrients in vegetables and fruit, including phytochemicals, vitamins, vitamin precursors and minerals, have been found to possess both complementary and overlapping mechanisms of chemopreventive activity in multistage carcinogenesis (5).
Recent studies have demonstrated potential cancer chemopreventive activity of berry derivatives including strawberries and black raspberries. Endogenous formation of N-nitrosoamino acids was reported to be significantly inhibited by the administration of strawberry juice in humans and this inhibitory activity was not solely related to the ascorbate content of the juice as was initially expected (7). Strawberries have also been shown to be high in antioxidant activity and thus the consumption of strawberries could increase the antioxidant capacity in humans (8,9).
Studies by Stoner et al. and Kresty et al. have shown that the supplementation of strawberries or black raspberries in the diet reduced the multiplicity and incidence of esophageal tumors in N-nitrosomethylbenzylamine-treated rats (10,11). Further, the decrease in the O6-methylguanine level found in esophageal DNA in rats fed strawberries or black raspberries suggested that a component(s) in these berries influenced the metabolism of N-nitrosomethylbenzylamine (10,11). The chemopreventive effects of these berries have primarily been attributed to ellagic acid, an abundant component in various fruits and nuts including strawberries and black raspberries. Ellagic acid by itself has been shown to inhibit cancers in rodents induced by several carcinogens (12). The inhibition of mutagenesis and carcinogenesis by ellagic acid appears to involve blockage of carcinogen metabolic activation, interference in the binding of reactive metabolites of carcinogens to DNA and the stimulation of detoxification enzymes (1315).
The identification and development of cancer chemopreventive agents should proceed in a stepwise, mechanistic-based fashion. Several short-term in vitro and in vivo test systems have been used to assess the mutagenicity and carcinogenicity of chemicals. These tests can also be applied to identify and rank the potency of chemopreventive agents (3,16,17). In vitro cell transformation occurs by mechanisms similar to those involved in multistage in vivo carcinogenesis. Therefore, Syrian hamster embryo (SHE) cell transformation assay has been widely used both to predict the carcinogenicity of chemicals and to study the mechanisms by which chemicals induce cellular transformation (1820). This assay can also be a potentially useful in vitro model for assessing the chemopreventive ability of chemicals and investigating their mechanisms of action (21,22).
In the N-nitrosomethylbenzylamine-induced esophageal tumorigenesis model, Stoner et al. (23) found that the tumor inhibitory effects of the berries could not be solely attributed to their ellagic acid content. Other components in the berries appeared to contribute to their chemopreventive effects. In the present study, we utilized transformation in SHE cells by benzo[a]pyrene (B[a]P) to examine the potential chemopreventive activity of ellagic acid and selected strawberry and black raspberry extracts.
Ripe strawberries [Fragara ananassa (FA)] and ripe black raspberries [Rubus ursinus (RU)] were washed immediately after picking, frozen at 20°C, then freeze-dried as described by Stoner et al. (23). Freeze-dried strawberries were extracted with methanol. The extract was filtered and then dried under vacuum at 60°C (Fraction F001). The residue from Fraction F001 (F002) was not processed further. A portion of F001 was partitioned with water:dichloromethane (1:1). The aqueous layer was concentrated under vacuum and dried (Fraction F003). The organic (dichloromethane) layer was vacuumed dried at 60°C resulting in a water insoluble fraction (F004). A small amount of insoluble fraction, F005, was obtained from the interface between the aqueous and organic layer. Additional F001 was dissolved in methanol and allowed to evaporate. The resulting precipitate was chromatographed on a silica gel column and eluted by dichloromethane:methanol (1:1). The resulting non-polar eluate (DM) and polar fraction (ME) were obtained. All extracts were stored at 20°C until examined in the transformation assay. Chemical analysis showed there was no ellagic acid in the ME fractions. All other fractions contained minor amounts of ellagic acid. For cell treatment each extract was dissolved in DMSO to give a final concentration of 200 mg of extract/ml, and was further used in cell transformation studies. The SHE cell transformation assay was conducted as described previously (24). Following 7 days incubation, SHE cell colonies were fixed, stained and scored for morphological transformation (MT). DMSO (0.2%) was used as a solvent control. Two protocols were used to access anti-transformation activity of the extracts and ellagic acid. B[a]P (10 µg/ml) treatment for 24 h or 7 days was included as a positive control. In the 7 day dosing protocol, cells were treated with non-toxic concentrations of either ellagic acid or each extract with and without B[a]P co-treatment for 7 days. For the agent(s) showing the anti-transformation activity in the 7 day treatment studies, a 24 h co-treatment with B[a]P and a 6-day treatment following a 24 h treatment of B[a]P were performed.
Initial studies determined that maximum subtoxic concentration of the various berry extracts on SHE cell colony formation was 100 µg/ml and for ellagic acid was 4.5 µg/ml, which was subsequently used as the highest concentration in subsequent assays (data not shown). The effects of ellagic acid and all test berry extracts by themselves on SHE cell transformation following 7-day treatment was examined. Neither berry extracts nor ellagic acid produced an increase in morphological transformation. For chemopreventive activity assessment, SHE cells were treated with B[a]P and either ellagic acid or each extract continuously for 7 days. B[a]P (10 µg/ml) induced SHE cell transformation after 7 days of continuous treatment. Ellagic acid inhibited B[a]P-induced morphological transformation in a dose-dependent manner. Concentrations of ellagic acid from 0.3 to 4.5 µg/ml reduced B[a]P-induced transformation from 24 to 83% (Table I). Of the strawberry extracts, FA-001, FA-003, FA-004, FA-005 and FA-DM did not produce a statistically significant decrease in B[a]P-induced morphological transformation at any of the doses examined (data not shown). However, a concentration-dependent reduction in transformation was seen with these extracts, albeit not one that was statistically significant. In contrast, FA-ME, at 5 µg/ml and higher concentrations, produced a significant decrease (6084%) in B[a]P-induced transformation frequency (Table II
). Similar effects were observed for black raspberry extracts. RU-F001, RU-F003, RU-F004, RU-F005 and RU-DM did not exhibit significant inhibitory effects on the transformation frequency (data not shown). As observed with strawberry extracts, black raspberry extracts produced a concentration-related reduction in transformation. RU-ME (2100 µg/ml) decreased B[a]P-induced morphological transformation 1884% in a dose-dependent manner (Table III
).
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Stoner et al. (11,23) previously found that esophageal tumor formation was inhibited more in rats fed dietary strawberries or black raspberries than with ellagic acid only. Thus, there appear to be compounds other than ellagic acid in the berries that contribute to the observed chemopreventive effects. This was confirmed in the present study. Among the tested extracts, FA-ME from strawberries and RU-ME from black raspberries showed a significant inhibitory effect on B[a]P-induced morphological transformation, suggesting their potential chemopreventive activity. For all other fractions examined (FA-F001, FA-F003, FA-F004, FA-F005, FA-DM, RU-F001, RU-F003, RU-F004, RU-F005 and RU-DM), decreases of 3050% in transformation frequency were also found, but not statistically significant. These fractions contained low but detectable levels of ellagic acid that may be contributory to the anti-transforming activity seen. Further evaluation of this issue needs to be performed. In contrast, both FA-ME and RU-ME fractions were determined to be devoid of ellagic acid and thus, the chemopreventive activity seen is related to other chemical components.
The mechanism by which ellagic acid, FA-ME or RU-ME inhibited the B[a]P-induced transformation in SHE cells were further studied through the two treatment protocols. (Co-cultured with B[a]P for 24 h or B[a]P treatment for 24 h followed by treatment with extracts for 6 days.) When SHE cells were treated with B[a]P and either ellagic acid, FA-ME or RU-ME in different stages, only ellagic acid decreased the cell transformation frequency in both treatment procedures. This is consistent with previous studies which have revealed that ellagic acid appears to function as an anticarcinogen at both the initiation and post-initiation stages (28). FA-ME clearly showed an inhibitory effect in the early stage and to a lesser extent when administered following treatment with B[a]P, suggesting an effect predominantly on the `initiation' stage of B[a]P-induced transformation. FA-ME fraction might therefore be functioning to inhibit uptake or activation of B[a]P, to interfere with B[a]P-DNA binding, to enhance B[a]P detoxification and/or to increase DNA repair. A similar mechanism was also suggested for RU-ME, as this fraction also exhibited its main effect in the early stage of the transformation process. Compared with ellagic acid, which functioned at both `initiation' and `promotion' stages and the inhibition in the latter was higher, FA-ME and RU-ME showed their most inhibitory effects in the `initiation' step. This supports the theory that other active component(s) in berries besides ellagic acid contribute to their chemopreventive effects.
Further studies are needed to identify specific compound(s) in the FA-ME and RU-ME fractions that are responsible for their chemopreventive activity and to further clarify the possible mechanisms involved in this process. The identification of compounds in these fractions is currently in progress.
Notes
2 To whom correspondence should be addressed Email: jklauni{at}iupui.edu
References