Ex vivo and in vitro testis and ovary explants: utility for identifying steroid biosynthesis inhibitors and comparison to a Tier I screening battery.

Testis and ovary explants have been proposed as in vitro screens for identifying potential inhibitors of steroid biosynthesis. The goals of the current study were to optimize the conditions of the two assays, to characterize these assays using several compounds with well-defined endocrine activity, and to compare the responses from the explant assays with an in vivo male battery currently undergoing validation using the Crl:CD BR rat in order to evaluate their utility as test systems for screening unknown compounds for possible steroid biosynthesis inhibition activity. There were two components to the testis/ovary assays: ex vivo and in vitro. The ex vivo component used testes/ovaries from animals dosed with the test compounds in vivo, and the in vitro component used testes/ovaries from control animals. For the testis assays, decapsulated testis explants (50 mg) were placed into glass scintillation vials, +/-1.0 IU/ml hCG for 3 h in a shaking water bath (34 degrees C). Following the incubation period, medium was removed, centrifuged, and frozen until assayed for hormone concentrations. A similar procedure was used for the ovary explant assay except that each ovary was incubated separately. The testis explants were evaluated using the following compounds: ketoconazole (KETO), a testosterone biosynthesis inhibitor; aminoglutethimide (AG) (only in vitro) and anastrozole (ANA), aromatase inhibitors; finasteride (FIN), a 5alpha-reductase inhibitor; 17beta-estradiol (17beta-E2), an estrogen receptor agonist; flutamide (FLUT), an androgen receptor antagonist; ICI-182,780 (ICI), an estrogen receptor antagonist; haloperidol (HALO), a D2 receptor antagonist; and reserpine (RES), a dopamine depletor. In the ovary assay, AG (only in vitro), ANA, ICI, and HALO (only in vitro) were evaluated. Addition of fetal calf serum to the medium allowed measurement of estradiol (E2) in the testis assay, but production was not inhibited by ANA or AG. In the ovary explant assay, only AG was identified as inhibiting E2 production in vitro. Hence, both the testis and ovary explant assays appear to have limited utility for detecting aromatase inhibitors. Screening of these nine diverse endocrine-active compounds resulted in all of them being identified as altering the endocrine system when assessed by ex vivo and in vitro testis explants. Using only the in vitro assessment with the criteria of steroid biosynthesis inhibition, four of nine compounds were correctly identified in the testis explant assay (17beta-E2, KETO, FLUT, and HALO). The predictability of both the in vitro and ex vivo ovary assay was 50%, suggesting a 50% false positive or negative rate with unknown compounds. However, of the seven compounds assessed to date (17beta-E2, ICI, ANA, KETO, FLUT, HALO, and RES), all were correctly identified using an in vivo male battery, which also has the capability to detect other endocrine activities. Therefore, the testis and ovary explant assay would not be necessary if one were using an in vivo male battery, since this screen would identify steroid biosynthesis inhibitors and would also identify several other endocrine activities. Because of the difficulties in assessing cytotoxicity and the high false positive/negative rates, the ovary and testis explant assays are not useful as routine screening procedures for detecting steroid biosynthesis inhibitors; however, they may have utility in confirming in vivo findings.

Toxicity of cadmium in human trophoblast cells (JAr choriocarcinoma): role of calmodulin and the calmodulin inhibitor, zaldaride maleate.

Cadmium (Cd), the heavy metal, is toxic to the placenta. The objectives of this study were to determine if Cd toxicity is due to inhibition of placental or trophoblast cell proliferation through interactions with the intracellular calcium binding protein, calmodulin (CaM). Cd can replace calcium and thus interfere with CaM's function. Also, CaM inhibitors reverse selected toxic effects of Cd. The CaM inhibitor, zaldaride maleate, was used to determine if Cd inhibits trophoblast cell proliferation through interactions with CaM. JAr choriocarcinoma cells, a neoplastic trophoblast cell line which is similar to early human trophoblast cells, were selected to study this question. Cd (20 and 40 microM) inhibits JAr cell proliferation, as measured by cell number and BrdU incorporation. Zaldaride (10 and 20 microM) inhibits proliferation to a lesser extent; 100 microM is lethal. To determine if zaldaride alters actions of Cd, zaldaride and Cd are added simultaneously. Zaldaride (20 microM) and Cd (20 microM) together inhibit proliferation less than Cd alone, thus partially protecting cells. Metallothionein is induced in cells exposed to Cd, while zaldaride does not cause induction of this cellular defense mechanism protein. To determine if Cd inhibits proliferation through alterations of cell cycle, JAr cells enriched for G0/G1 phase were exposed to 20 microM Cd, 20 microM zaldaride, or 20 microM Cd plus 20 microM zaldaride for 24 hr. Cells remain in G0/G1 following Cd exposure; cells treated with 20 microM zaldaride progress through S phase and into G2. Zaldaride and Cd together allow JAr cells to leave G1 and enter S phase, partially relieving the cycle block produced by Cd. This study demonstrates a role for calmodulin in mediating the toxicity of Cd in trophoblast cell proliferation.

In vitro differentiation and ultrastructure of human extravillous trophoblast (EVT) cells.

Tissue explants of anchoring villi from the first trimester placentae cultured on extracellular matrix (Matrigel) give rise to EVT cells in vitro. This study was designed to address two issues important for further application of the described in vitro model: first, were the observed EVT cells derived by cell proliferation in vitro and second, what is the degree of homology between the in vivo and the in vitro differentiated EVT cells. The cultures (tissue and matrix) were prepared for light and electron microscopic (EM) examinations. Semi-thin sections from Spurr epoxy resin-embedded tissue were used to 'pop-off' the selected area for EM examination. Cell proliferation in vitro was assessed immunohistochemically using proliferative cell nuclear antigen (PCNA) antibodies. Since positive hPL immunostaining has been consistently demonstrated in the invasive subpopulation of EVT cells from placental bed in situ, hPL staining was used as a marker of EVT cell differentiation in vitro. It has been demonstrated that PCNA antibodies immunostained nuclei of cytotrophoblast cells from cell column at the base of the anchoring villi, indicating that these cells expressed proliferative activity in vitro. Cytotrophoblast proliferation resulted in the formation of the flattened zone of cell outgrowths at the tip of anchoring villi. Cells from the distal layer of the cell column detached gradually and migrated into the surrounding matrix. These cells appeared as individual, round-shaped EVT cells with smooth surface cell membrane. Their cytoplasm was rich in glycogen and contained large lipid droplets and flattened cisternae of the RER. Positive PCNA immunostaining, along with the presence of mitotic figures, indicated that EVT cells in vitro retained the ability for cell proliferation. As a result of cell proliferation and migration, the number of EVT cells increased during the culture period of 4 days. EVT cell glycogen content and lipid stores decreased progressively as they migrated into the matrix. Individual EVT cells, as well as EVT cell clusters, became surrounded by the clear zone of digested matrix. Some cells started to express strong positive staining with hPL antibodies as soon as they had migrated outside the villous explant. By day 4 of culture, a small percentage of EVT cells (about 5-10%) ceased to migrate, firmly attached to the substratum and appeared as irregular shaped cells with filopodia-like projections. Their cytoplasm contained dilated cisternae of RER, a small number of glycogen granules and bundles of actin-like filaments located in the cytoplasm inside the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)