Food Chemistry 138 (2013) 2146–2151 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Analytical Methods Screening test for rapid food safety evaluation by menadione-catalysed chemiluminescent assay ⇑ Shiro Yamashoji , Naoko Yoshikawa, Masayuki Kirihara, Toshihiro Tsuneyoshi Shizuoka Institute of Science and Technology, 2200-2 Toyosawa, Fukuroi, Shizuoka 437-8555, Japan article info abstract Article history: The chemiluminescent assay of menadione-catalysed H2O2 production by living mammalian cells was Received 2 November 2012 proposed to be useful for rapid food safety evaluation. The tested foods were extracted with water, eth- Received in revised form 10 December 2012 anol and dimethylsulfoxide, and each extract was incubated with NIH3T3, Neuro-2a and HepG2 cells for Accepted 13 December 2012 4 h. Menadione-catalysed H O production by living mammalian cells exposed to each extract was deter- Available online 29 December 2012 2 2 mined by the chemiluminescent assay requiring only 10 min, and the viability of the cells was estimated as percentage based on H2O2 production by intact cells. In this study the cytotoxicity of food was rated in Keywords: order of inhibitory effect on H O production by intact cells. The well known natural toxins such as Fusar- Menadione 2 2 ium mycotoxin, tomato toxin tomatine, potato toxin solanine and marine toxins terodotoxin and breve- Chemiluminescent assay Viable cell number toxin could be detected by the above chemiluminescent assay. Food safety evaluation Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction nescent assay (Yamashoji, Yoshikawa, Kirihara, & Tsuneyoshi, 2012) was applicable to the determination of the cytotoxicity of Food safety evaluation has traditionally relied on toxicological (1) grain foods, (2) nuts and seeds, (3) tubers, (4) sugar, (5) confec- data that have been obtained through animal experiments (Hug- tionery, (6) fat and oil, (7) bean, (8) fruits, (9) green and yellow veg- get, Schilter, Roberfroid, Antignac, & Koeman, 1996). Single sub- etables, (10) other vegetables, (11) mushroom, (12) seaweed, (13) stances, such as food additives, show clear toxicological data, but drink and seasoning, (14) fish and shellfish, (15) meat, egg and the toxicity of whole foods is difficult to measure because the var- milk, and (16) others (spice). We discuss the application of the ious components may induce additive, synergistic or antagonistic chemiluminescent assay to food safety evaluation and the detec- effects (Konemann & Pieters, 1996). Chemical analysis is a conven- tion of well-known natural toxins. This paper is a first study to tional method for quality control of foods but is not applicable to demonstrate the cytotoxicity of the above different foods by using the evaluation of food safety. Even if chemical analysis is con- a screening test with mammalian cells. ducted for food safety, chemical analysis may require a long time to identify the toxic compounds and may miss causative agents, 2. Materials and methods which are not the target of analysis. Cytotoxicity testing cannot identify toxic compounds, but is ex- 2.1. Organisms and growth conditions pected to detect the presence of harmful compounds in foods. Con- ventional cytotoxicity testing has been conducted by colorimetric Contact-inhibited NIH/3T3 Swiss mouse embryo cells, ATCC CRL WST-1 (Ishiyama et al., 1995) or MTT (Mosmann, 1983) assay, 1658 (NIH/3T3 cells) and HepG2 cells were cultured in DMEM sup- and these assays take a few hours to show enough absorbance. plemented with 10% foetal bovine serum. Neuro-2a cells were cul- However, these traditional assays have been little used as screen- tured in RPMI1640 supplemented with 10% foetal bovine serum. ing test for safety evaluation of different foods other than essential The cells were grown in a Falcon tissue flask in a humidified atmo- oils (Huang & Luo, 2009). sphere of 95% air and 5% CO2 at 37 °C. Exponentially growing stock We demonstrated the usefulness of the traditional chemilumi- cells were trypsinised and diluted with the above culture medium nescent assay (Yamashoji & Isshiki, 1998) for the detection of to adjust 400,000 cells/ml. One hundred microlitres of the diluted various food additives and natural toxins in culture medium, but cells were added to each well on a 96-well plate, and cultivated we had no result for food safety evaluation of different foods. In for 18 h, and each well was used for cytotoxicity testing. this study, we demonstrated that the latest developed chemilumi- 2.2. Determination of living cell number by chemiluminescent assay ⇑ Corresponding author. Fax: +81 538 45 0110. Menadione-catalysed H2O2 production by living mammalian E-mail address: [email protected] (S. Yamashoji). cells was determined by chemiluminescent assay (Yamashoji 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.12.037 S. Yamashoji et al. / Food Chemistry 138 (2013) 2146–2151 2147 et al., 2012) and the viability of cells exposed to the extract was Hungerford, & Wekell, 1993). The viability of the treated cells estimated on the basis of the production of H2O2 by cells exposed was determined by chemiluminescent assay. to extract solvent. Mammalian cells were incubated with food extract in a humid- 2.7. Foods and chemicals ified atmosphere of 95% air and 5% CO2 at 37 °C for 4 h, and the cells in each well were washed two times with 100 ll of MEM Foods were purchased from a local supermarket. Wakame, hijiki without phenol red (Medium A), and 100 ll of Medium A were and kelp were not crude, and dried after a wash with water. Chem- added to each well. After that 100 ll of Medium A containing icals were purchased from Sigma and Wako Pure Chemical 0.2 mM menadione were added to each well, and the cells were Industries. incubated for 10 min in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. After the incubation 150 ll of chemiluminescent 3. Results reagent containing 50 mg of TCPO [bis(2,4,6-trichlorophenyl)oxa- late] and 0.14 mg of rhodamine B in 50 ml of acetonitrile were 3.1. Principle of chemiluminescent assay added to each well, and the chemiluminescence intensity was counted for 5 s. The principle of chemiluminescent assay is shown in Fig. 1 (Yamashoji, Nishimoto, Usuda, Kubota, & Isshiki, 1992). Living 2.3. Cytotoxicity test of foods mammalian cells are expected to reduce extracellular menadione by the combination of NAD(P)H and NAD(P)H:quinone reductase, Foods were homogenised with distilled water, ethanol or DMSO and the resulting menadiol may reduce dissolved oxygen to pro- at the ratio of 1–4 (weight/g to volume/ml), and liquid foods were duce H2O2.H2O2 can be specifically determined by chemilumines- mixed with culture medium at the ratio of 1–4 (v/v). After these cent assay with TCPO and fluorescent material, and the mixtures were shaken at 50 °C for 5 h, a supernatant was obtained. concentration of H2O2 is proportional to living cell number. The as- Two microlitres of the supernatant were mixed with 98 ll of cell say with 96-well micro plate was conducted within 15 min. culture medium, and incubated for 4 h under the culture condi- tions. As negative control water was mixed with extracting solvent 3.2. Extracting solvent at the ratio of 1–4 (v/v), and 2 ll of the mixture were mixed with 98 ll of cell culture medium, and incubated for 4 h under the cul- Hydrophobic solvent such as hexane and benzene was not used ture conditions. After incubation the cells in each well were as extracting solvent because hydrophobic solvent is separated washed three times with Medium A in order to remove interfering from culture medium and cells can be little exposed to the ex- substances or H2O2 produced by autoxidation of foods, and the via- tracted compounds. In this test water, ethanol and DMSO were ble cell number was estimated by chemiluminescent assay. The used as extracting solvent to make provide complete mixing of ex- viability was estimated by the following equation: tract and culture medium. In the case of the extraction with DMSO, the mixture was completely dispersed during 4-h incubation even Viability ð%Þ¼ðViable cell number of the cells exposed to if turbidity was observed. The final volume percent of the homog- food extractÞ=ðViable cell number of the cells exposed to enate was 2% in culture medium, in order to minimise the cytotox- extracting solventÞ100: icity of solvent. The viability of cells exposed to the extract from When it was difficult to get 2 ll from the high-viscosity mix- food with extracting solvent was calculated on the basis of the pro- ture, 200 ll of the mixture were mixed with 9800 ll of culture duction of H2O2 by the cells exposed to extracting solvent alone as medium, and 100 ll were added to each well. The final volume described in Section 2. percent of the tested foods was 0.4% in the culture medium, and the toxic compounds in foods were estimated to be diluted 250 3.3. Cytotoxic effects of salt, acetate and ethanol included in foods times in the culture medium. Incubation time of food extract with cells was adjusted to 4 h Salt, acetate and ethanol are contained in various processed because cytotoxic effect of brevetoxin, tomatine, isobutyl p- foods, and we should know the cytotoxicity of these compounds hydroxybenzoate and dibutylhydroxytoluene has been quantita- because these cytotoxic effects increase in a dose-dependent man- tively determined for 4 h-incubation (Yamashoji & Isshiki, 1998). ner (Yamashoji & Isshiki, 1997). As sodium chloride and acetate showed cytotoxic effects on cells at above 0.1%, we have to pay 2.4.