Development of an in vitro reproductive screening assay for novel pharmaceutical compounds

Abstract

An in vitro reproductive cell-based toxicity assay was 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-2H-tetrazolium bromide], MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and Crystal 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 20000 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). H(2)O(2) as a negative control killed 100% of cells at 1000 microg/ml. The two nanopharmaceutical compounds were cytotoxic at concentrations > or =0.1 microg/ml, but hCG decreased cytotoxicity to > or =1000 microg/ml (P<0.001). hCG-stimulated progesterone synthesis afforded some protection against the cytotoxic effects of the two novel nanotechnology compounds; therefore doses < or =100 microg/ml and an exposure period of 1 h would be recommended for testing in in vivo animal reproductive assays.

Effect of Chemotherapeutics and Tocopherols on MCF-7 Breast Adenocarcinoma and KGN Ovarian Carcinoma Cell LinesIn Vitro

“…An extract from a marine snail was significantly cytotoxic to a human Jar choriocarcinoma placental cell line. As the number of viable cells decreased, secreted progesterone increased [77]. Gross et al [78] also described dying primary-derived granulosa cells increasing progesterone production.…”

An in vitro high‐throughput assay for screening reproductive and toxic effects of anticancer compounds

“…Previous studies have shown that the MTT assay requires extensive incubation periods of anything from 4 to 17 H for the production of the formazan product . MTT incubation periods vary greatly depending on the cell line . The optimized MTT assay for the JAr cell line in the present study adopted a 2 H attachment using 20,000 cells per well, followed by 1 H MTT incubation at a concentration of 0.5 mg/mL.…”

Recent Developments in the Chemistry of Cubic Polyhedral Oligosilsesquioxanes

“…760 An in vitro study of the reproductive toxicity of poly(ethylene glycol)-substituted POSS cage has been carried out, and the cages were found to be cytotoxic at concentrations of g0.1 µg mL -1 , but human chorionic gonadotropin reduced cytotoxicity to g1000 µg mL -1 . 256 See section 4.4.1 for a further description of the uses of POSS-containing materials with biological applications.…”