Uridine uptake inhibition as a cytotoxicity test for a human hepatoma cell line (HepG2 cells): comparison with the neutral red assay.

This study describes a sensitive microassay for measuring cytotoxicity based on the degree of inhibition of RNA synthesis in HepG2 cells. RNA synthesis is measured by the kinetic uptake of radiolabeled uridine. A large number of compounds were tested in a wide range of concentrations. The concentration required to induce 50% inhibition of HepG2 uridine uptake rates (IC(50)) was determined for each compound and used to rank its potency. These IC(50)s were compared with IC(50)s measured with the neutral red assay. 2-acetylaminofluorene, benzo[a]pyrene and methylnitrosourea were not cytotoxic in the neutral red assay. Uridine uptake was always inhibited at lower concentrations than those required in the neutral red assay, suggesting that the uridine uptake assay is a more sensitive indicator of toxic action than the neutral red inclusion. Uridine uptake assay provides a rapid and quantitative method for assessing toxicity in a human cell line. Application of this method to bottled spring waters are described. Due to its high sensitivity and reproducibility, this method provides a suitable tool for screening a great number of samples and will be a helpful test for evaluating food safety and controlling the recycling process of wrapping materials.

[1]  T. Mosmann Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.

[2]  M. Argus,et al.  Use of high concentrations of dimethylnitrosamine in bacterial lethality, mutagenesis, and enzymological studies. , 1978, Cancer research.

[3]  A. Collins,et al.  Preculturing of Chinese hamster V79 cells with sublethal concentration of theophylline sensitizes cells to cytotoxic effects of MNNG. , 1998, Mutation research.

[4]  T. Norseth The carcinogenicity of chromium and its salts. , 1986, British journal of industrial medicine.

[5]  K. Ziegler-Skylakakis,et al.  Mutagenicity of hydrogen peroxide in V79 Chinese hamster cells. , 1987, Mutation research.

[6]  P. Giangrande,et al.  Reduction of chromium(VI) by ascorbate leads to chromium-DNA binding and DNA strand breaks in vitro. , 1995, Biochemistry.

[7]  J A Miller,et al.  Carcinogenesis by chemicals: an overview--G. H. A. Clowes memorial lecture. , 1970, Cancer research.

[8]  G. Michalopoulos,et al.  Genotoxic effects of 2-acetylaminofluorene on rat and human hepatocytes. , 1983, Environmental health perspectives.

[9]  H Babich,et al.  Comparisons of two in vitro cytotoxicity assays-The neutral red (NR) and tetrazolium MTT tests. , 1988, Toxicology in vitro : an international journal published in association with BIBRA.

[10]  E Borenfreund,et al.  Toxicity determined in vitro by morphological alterations and neutral red absorption. , 1985, Toxicology letters.

[11]  G Williamson,et al.  Use of metabolically competent human hepatoma cells for the detection of mutagens and antimutagens. , 1998, Mutation research.

[12]  A. Lichtenstein,et al.  Apoptotic vs. nonapoptotic cytotoxicity induced by hydrogen peroxide. , 1997, Free radical biology & medicine.

[13]  C. Kurland,et al.  The distribution of soluble and ribosomal RNA as a function of growth rate , 1963 .

[14]  P J Dierickx,et al.  Cytotoxicity testing of 114 compounds by the determination of the protein content in Hep G2 cell cultures. , 1989, Toxicology in vitro : an international journal published in association with BIBRA.

[15]  C. Cross,et al.  Free radicals, antioxidants, and human disease: where are we now? , 1992, The Journal of laboratory and clinical medicine.

[16]  J. Goodman,et al.  Quantitative studies on the in vitro metabolic activation of dimethylnitrosamine by rat liver postmitochondrial supernatant. , 1984, Environmental health perspectives.

[17]  E. Rozengurt,et al.  Uptake of nucleosides in density-inhibited cultures of 3T3 cells , 1977, Nature.

[18]  P. Fournier [Biotransformation of dimethylnitrosamine]. , 1990, Journal de toxicologie clinique et experimentale.

[19]  A Wolfreys,et al.  The ability of the Comet assay to discriminate between genotoxins and cytotoxins. , 1998, Mutagenesis.

[20]  C C Howe,et al.  Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. , 1980, Science.

[21]  A. Natarajan,et al.  Metabolic activation of chemicals to mutagenic carcinogens by human hepatoma microsomal extracts in Chinese hamster ovary cells (in vitro). , 1993, Mutagenesis.

[22]  G. Cohen,et al.  Studies on the metabolism and excretion of benzo(a)pyrene in isolated adult rat hepatocytes. , 1978, Biochemical pharmacology.

[23]  P. O'Brien,et al.  Cytotoxicity induced by N-methyl-N'-nitro-N-nitrosoguanidine may involve S-nitrosyl glutathione and nitric oxide. , 1995, Xenobiotica; the fate of foreign compounds in biological systems.

[24]  R. Fautz,et al.  Application of the neutral red assay (NR assay) to monolayer cultures of primary hepatocytes: rapid colorimetric viability determination for the unscheduled DNA synthesis test (UDS). , 1991, Mutation research.

[25]  J. Pounds,et al.  Potentiation of dimethylnitrosamine genotoxicity in rat hepatocytes isolated following ethanol treatment in vivo. , 1984, Chemico-biological interactions.

[26]  H. Rosenkranz,et al.  Mutagenicity, unscheduled DNA synthesis, and metabolism of 1-nitropyrene in the human hepatoma cell line HepG2. , 1987, Cancer research.

[27]  C Shopsis,et al.  Uridine uptake inhibition as a cytotoxicity test: correlations with the Draize test. , 1984, Toxicology.

[28]  A. Natarajan,et al.  Use of human hepatoma cells for in vitro metabolic activation of chemical mutagens/carcinogens. , 1991, Mutagenesis.

[29]  R. Koren The relevance of the state of growth and transformation of cells to their patterns of metabolite uptake. , 1980, International review of cytology.

[30]  T. Decker,et al.  A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. , 1988, Journal of immunological methods.

[31]  D. E. Pritchard,et al.  Chromium-induced genotoxicity and apoptosis: relationship to chromium carcinogenesis (review). , 1998, Oncology reports.

[32]  C. Danglot,et al.  Rapidity of RNA synthesis in human cells. A highly sensitive parameter for water cytotoxicity evaluation , 1985 .

[33]  R. Barale,et al.  The genetic toxicology of styrene and styrene oxide. , 1991, Mutation research.

[34]  E. Borenfreund,et al.  A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90) , 1985 .

[35]  H. Birnboim,et al.  Mutagenicity and cytotoxicity of reactive oxygen and nitrogen species in the MN-11 murine tumor cell line. , 1997, Mutation research.