Establishment of human endometriai stromal cell cultures as in-vitro models to test the effects of metalloestrogens in women with or without endometriosis

Abstract

INTRODUCTION: Effects of metalloestrogens like cadmium (Cd), lead (PbJ and nickel (Ni) are difficult to demonstrate in-vivo due to toxicity and therefore requires suitable in-vitro models. AIMS: To establish endometriai stromal cell (ESC) cultures as in-vitro models to demonstrate the effects of metalloestrogens in women with and without endometriosis. METHODS: Stromal cells were isolated from eutopic endometriai samples from five women with endometriosis (patients) and five women without endometriosis (controlsj. ESC cultures were established and maintained in RPMI medium. Cultures were treated with Cd, Pb and Ni at concentrations of 10-6M, 10-9M and 10-9M respectively. At 24 h and 48 h, cell number was counted using the Neubauer haemocytometer. Sulphorhodamine(SRB) cytotoxicity assay was used to test the effect of different concentrations of metals on ESC cultures. After 24 h of treatment, caspase levels in ESC cultures were evaluated with a commercially available ELISA kit Relative cell proliferation, SRB assay results and caspase levels were analyzed with ANOVA. RESULTS: ESC cultures were maintained up to 30 days. In both patients and controls Cd, but not Pb or Ni increased the relative proliferation in ESC cultures (p<0.05). At 48 h, Cd induced ESC proliferation was higher in patients than in controls (p=0.02). SRB assay results and caspase levels were similar in the two groups. CONCLUSIONS: Established ESC cultures served as stable in-vitro models. Cd induced the proliferation of ESC from women with endometriosis which appears to be independent of reduced apoptosis. Similar effects of Pb and Ni could not be demonstrated in this in vitro model.