Development of an in vitro reproductive screening assay for novel pharmaceutical compounds
Date
2008
Authors
Edwards, V.
Markovic, E.
Matisons, J.
Young, F.
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Biotechnology and Applied Biochemistry, 2008; 51(2):63-71
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An in vitro reproductive cell-based toxicity assaywas developed using MLTC-1 (murine Leydig tumour cell line) in order to examine the reproductive toxicity of two novel nanopharmaceutical compounds, namely ethylene glycol mono allyl ether and poly(ethylene glycol) octa-functionalized polyhedral oligomeric silsesquioxane. Three commonly used cytotoxicity assays, namely the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide], MTS [3-(4,5-dimethylthiazol-2- yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium] andCrystal Violet assays,were compared, and the MTT assay proved to be the most accurate and reproducible for the MLTC-1 cell line. The doubling rate of the MLTC-1 cells was 30+− 3.5 h and the optimal seeding density for the MTT assay was 20 000 cells per well, and the optimized MTT assay utilized a 4 h cell adherence followed by incubation with 0.5 mg/ml MTT for 1 h. The intra- and inter-assay CV (coefficient of variation) values were 12.3 and 11% respectively. MLTC-1 cells only produce the reproductive hormone progesterone in response to hCG (human chorionic gonadotropin), which stimulated progesterone production dose-dependently from 0 to 100 m.i.u. (milliinternational units)/ml (2706+− 1118 ng/ml). H2O2 as a negative control killed 100% of cells at 1000 μg/ml. The two nanopharmaceutical compounds were cytotoxic at concentrations 0.1 μg/ml, but hCG decreased cytotoxicity to 1000 μg/ml (P<0.001). hCG-stimulated progesterone synthesis afforded some protection against the cytotoxic effects of the two novel nanotechnology compounds; therefore doses 100 μg/ml and an exposure period of 1 h would be recommended for testing in in vivo animal reproductive assays.
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Copyright 2008 Portland Press; International Union of Biochemistry and Molecular Biology