Development of an in vitro reproductive screening assay for novel pharmaceutical compounds.
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.
Edwards
Vicki
V
Department of Medical Biotechnology, School of Medicine, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia. edwa0223@flinders.edu.au
Markovic
Elda
E
Matisons
Janis
J
Young
Fiona
F
D000818:Animals; D002452:Cell Count; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D019855:Ethylene Glycol / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D005840:Gentian Violet / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D015394:Molecular Structure; D017646:Organosilicon Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D011374:Progesterone / Q000032:analysis; D011863:Radioimmunoassay; D015203:Reproducibility of Results; D013778:Tetrazolium Salts / Q000494:pharmacology*; D013844:Thiazoles / Q000494:pharmacology*; D018675:Toxicity Tests / Q000379:methods*
D006063:Chorionic Gonadotropin; D017646:Organosilicon Compounds; D013778:Tetrazolium Salts; D013844:Thiazoles; C070380:3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; D011374:Progesterone; C022616:thiazolyl blue; D019855:Ethylene Glycol; D005840:Gentian Violet