Shiro Yamashoji

Chemiluminescent assay for determination of viable cell density of yeast, mammalian, and plant cells

The production of H2O2 by intact cells is promoted in the presence of menadione and is proportional to the density of viable cells. The concentration of H2O2 produced is determined by the measurement of chemiluminescence which is generated in the mixture of H2O2, pyrene, and bis(2,4,6-trichlorophenyl)oxalate. This method is applied to the measurement of viable yeast, mammalian, and plant cells. For example, viable yeast cell density above 10(4) cells/ml is determined for 2 min, and mammalian cell density and the activity of plant tissues are determined for 10 and 5 min, respectively.

Application of the chemiluminescent assay to cytotoxicity test: Detection of menadione-catalyzed H2O2 production by viable cells

Menadione-catalyzed H2O2 production by viable cells is proportional to viable cell number. The correlations between the viable cell number and the concentration of H2O2 produced are determined with the rapid chemiluminescent assay (S. Yamashoji, T. Ikeda, and K. Yamashoji, 1989, Anal. Biochem. 181, 149-152). This chemiluminescent assay of viable cells requires only 10 min and is much faster than NR (neutral red) inclusion and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assays, which require 3-5 h. When viable cells are incubated with antitumor drugs, detergents, mycotoxins, and glycoalkaloids for 24-48 h, a decrease in menadione-catalyzed H2O2 production in a dose- or incubation time-dependent manner is observed. In general, the 50% inhibition concentration determined by the chemiluminescent assay is lower than that determined by NR inclusion and MTT reduction assays, and the order of relative cytotoxic effects of agents is the same among these assays. Furthermore, clear cytotoxic effects are observed by the chemiluminescent assay after 1 h exposure of trypsinized cells to toxic compounds. Therefore, the chemiluminescent assay is expected to be more useful for the rapid detection of cytotoxic compounds than NR inclusion and MTT reduction assays.

Extracellular reduction of menadione and ferricyanide in yeast cell suspension

Abstract Extracellular reduction of menadione and ferricyanide were promoted by glucose rather than ethanol in a yeast cell (Saccharomyces cerevisiae IFO 2044) suspension. NAD(P)H: menadione oxidoreductase from yeast plasma membrane possessing ferricyanide reductase activity had higher substrate specificity for NADH than for NADPH. Glucose produced a transient increase in NADH content in the absence of menadione, but neither glucose nor ethanol restored the NADH content upon menadione-catalyzed ferricyanide reduction. These findings suggest that the NADH content is controlled by the metabolism of glucose and ethanol, and that the excess of NADH generated by glucose and ethanol is effectively consumed by NADH: menadione oxidoreductase in the yeast transplasma membrane redox system.

Rapid assay of cell activity of yeast cells

Abstract Yeast cells produce H2O2 in the presence of exogenous menadione, and the extracellular concentration of H2O2 produced is proportional to the viable cell number or cell activity. Chemiluminescent assay of menadione-catalyzed H2O2 production requires only a few minutes, and is useful for rapid determination of the viability of yeast cells.

Cytotoxicity Testing for Food Safety Evaluation

We have been interested in studying the factors affecting the quality of food. It is important to detect toxic substances in foods as well as to control food-born disease microorganisms. We have been applied in vitro bioassay to evaluate the quality of food. We tried the following assay procedure: Alamar Blue reduction assay, lactose dehydrogenase assay, neutral red inclusion assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl2H-tetrazolium bromide(MIT) reduction assay, WST-1 reduction assay, and others. We have developed a chemiluninescent cytotoxicity assay and applied it to food component and toxins. This assay was sensible and rapid. WST-1 reduction assay was easy to operate.

Cytotoxicity Testing of Tomatine

Glycoalkaloids, e.g. solanine and tomatine are found in potato, tomato and other plants. They are toxic to animals. It is difficult to analyze them in food. We developed a bioassay of tomatine. A combination method of HepG2 cell with a chemiluminescent was more easy to operate and rapid than others. Recently, it has been reported that tomato contains dehydrotomatine, which has C-5-C-6 double bond in the aglycon. We purified crude tomatine and obtained pure tomatine and dehydrotomatine. We measured the cytotoxicity of these compounds with chemiluminescent assay. As the animal cells for bioassay, HepG2, NIH3T3 and U937 cells were used. Cell viability in the presence of pure tomatine was just a little lower than that in the presence of pure dehydrotomatine.

A Rapid Cytotoxicity Assay of Metabolic Inhibitors

H202 was produced upon the addition of menadione to mammalian cell culture. Chemiluminescent assay (CL assay) of menadione-catalyzed H202 production by viable cells was useful for the rapid detection of the cytotoxic inhibitors because the assay require only 10 min and detected the immidiate cytotoxic effect occurring a few minutes after the addition of inhibitors to cell culture. This study demonstrated that the chemiluminescent assay was superior to NR (neutral red) inclusion assay and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay on the basis of the sensitivity and rapidicity for the detection of cytotoxic effects of antibiotics and metabolic inhibitors such as DNA synthesis inhibitors, protein synthesis inhibitors, ATPase inhibitors and ionophorous antibiotics. Furthermore, the trypsinized cells were found to be more sensitive to the above inhibitors than the adherent cells with the chemiluminescent assay.

Bioassay Method for Glycoalkaloids in Food with Animal Cell Cultures

Glycoalkaloids, e.g. solanine and tomatine are found in potato, tomato or other plants. They are toxic to animals. It is difficult to analyze them in food. We tried to detect cytotoxicity of tomatine in tomato. Cell lines of NIH3T3, HepG2, HuH-6KK and U937 were tested for detecting cytotoxicity of tomatine. The following detection methods were compered; Alamar Blue, MIT, WST-1 and chemiluminescent method. These method were possible to detect cytotoxicity of tomatine. Particularly, a combination of HepG2 cell with the chemiluminescent method was more easy to operate and rapid than others.