Use of LUCS (Light-Up Cell System) as an alternative live cell method to predict human acute oral toxicity.

LUCS (Light-Up Cell System) is a new live cell test that allows assessment of a cell's homeostasis and its alteration by a toxic agent. To evaluate the effectiveness of LUCS as an alternative test method for acute oral toxicity, we compared EC50s determined in HepG2 cells treated with 53 chemicals selected from the ACuteTox EU database with corresponding human blood LC50s derived from human acute poisoning cases. Linear regression analysis showed that LUCS results predict human data to 69 %. Rodent oral LD50s and LUCS EC50s were then correlated to human LC50s using shared data sets. Linear regression analyses comparing LUCS and animal data clearly showed that LUCS always predicts human toxicity better than animal data do. These successful prediction values prompted us to simplify the LUCS test, adapting it to regulatory and high throughput applications, resulting in a new protocol with consistent dose-response profiles and EC50s. This study demonstrates that the LUCS test method could be relevant for assessing human acute oral toxicity with a simplified protocol adapted to commercially available fluorescence readers. We suggest that this new alternative method can be used for acute systemic toxicity testing in combination with other tests under European REACH and other regulations, wherever pertinent alternative methods are still lacking.

Granulosa cell tumors express erbB4 and are sensitive to the cytotoxic action of heregulin-beta2/PE40.

The molecular genetic events involved in the etiology of human granulosa cell (GC) tumors, which represent approximately 7% of all malignant ovarian neoplasms, are unknown. Amplification and/or overexpression of the ERBB genes are a feature of many cancer types, and overexpression of erbB2 correlates with poor prognosis in epithelial ovarian cancer. In the present study, we used immunohistochemistry to determine the level and frequency of expression of different erbB receptors in GC tumors. Ten of 12 tumors expressed erbB4 at moderate to high levels in >50% of cancer cells, whereas erbB2 (6 of 12) and erbB3 (2 of 12) were expressed less frequently. Western blot experiments showed that the only available GC tumor cell line, COV434, also expressed erbB receptors. Heregulin (HRG)-beta2, a ligand for erbB3 and erbB4 receptors, stimulated tyrosine phosphorylation of the erbB receptors, which was accompanied by activation of Erk1 and Erk2, two mitogen-activated protein kinases with a functional role in mitogenesis. Importantly, HRG increased cell proliferation in COV434 cells, and treatment with HRG/PE40, a ligand toxin shown previously to be cytotoxic against human breast cancer cells overexpressing erbB receptors, led to a dramatic and irreversible decrease in cell number. These results indicate that erbB receptor signaling pathways may be critical in the control of GC tumor cell proliferation and that HRG/PE40 is a potential therapeutic agent for the treatment of GC tumors.

Human granulosa cells in culture exhibit functional cyclic AMP-regulated gap junctions.

Numerous gap junctions exist between granulosa cells, between cumulus cells and between cumulus cells and the oocyte. They may play a role in the regulation of both follicular development and oocyte status. We used primary cultures of human granulosa cells to study the molecular nature and functionality of these gap junctions. As shown by a cinemicrographic technique, during the first 3 days of culture, cells flattened and extended in several directions by means of cytoplasmic extensions. An ultrastructural study showed the presence of both intercellular and annular gap junctions after 48 h of culture. As revealed by immunodetection analyses, connexin 43 was present. An analysis using a functional procedure, the gap fluorescence recovery after photobleaching (FRAP) method, indicated that: (i) diffusional communication existed among granulosa cells; (ii) the communication was delayed by treatment with 1-heptanol, a well-documented inhibitor of gap junction permeability; and (iii) permeability was up-regulated by incubation with 8-Br-cAMP, an analogue of cyclic AMP. The detection of connexin 43 and functional gap junctions in networks of cytoplasmic extensions indicated junction formation among cells during culture. In conclusion, our results show that human granulosa cells in culture exhibited functional gap junctions. Connexin 43 was present and the permeability of the gap junctions was up-regulated by cyclic AMP, an important modulator of human granulosa cell function.

Cell shape change reveals the cyclic AMP-mediated action of follicle stimulating hormone, human chorionic gonadotrophin and vasoactive intestinal peptide in primary cultured human granulosa-lutein cells.

Granulosa cells are known to be the site of action of various hormones and agents that regulate ovarian function. This study was conducted to evaluate the effects of gonadotrophins, vasoactive intestinal peptide (VIP), prostaglandin (PG) F2 alpha and angiotensin II on the cyclic AMP (c-AMP) signalling transduction pathway in human granulosa-lutein cells. Exposure to agents that elevate c-AMP or mimic c-AMP action caused the cells to become rounded in a process that was rapid and reversible. We were able to demonstrate this cell rounding process in the presence of gonadotrophins and VIP, but not in the presence of PGF 2 alpha or angiotensin II. In addition, incubation of the cells with various selective phosphodiesterase (PDE) inhibitors revealed that the PDE type IV isoform, but not type III, catalyses c-AMP degradation in human granulosa-lutein cells. Alteration in c-AMP-dependent cytomorphology appears to be a convenient method to analyse the regulation of c-AMP-mediated events in the human granulosa-lutein cells.

Gonadotrophin-releasing hormone and triptorelin inhibit the follicle stimulating hormone-induced response in human primary cultured granulosa-lutein cells.

Cyclic AMP (c-AMP)-dependent cell shape changes can be used to study the gonadotrophin response in cultured human granulosa-lutein cells. The same approach has been developed here to determine the direct potential antigonadotrophic effect of gonadotrophin-releasing hormone (GnRH) and a GnRH agonist (triptorelin) on human granulosa-lutein cells. Treatment with triptorelin or GnRH alone for 1 h did not affect granulosa-lutein cell morphology. However, in the presence of stimulatory doses of follicle stimulating hormone (FSH), triptorelin (5 x 10(-7)-5 x 10(-6) M) and GnRH (10(-11)-10(-9) M) inhibited the FSH-induced c-AMP-dependent response. The antigonadotrophic effect of triptorelin was prevented by two GnRH antagonists, indicating that triptorelin acts via specific GnRH binding sites. On the other hand, triptorelin failed to inhibit human chorionic gonadotrophin- and forskolin-mediated morphological changes. Our results suggest that the GnRH agonist interacts specifically with the FSH-induced c-AMP-dependent cascade of events, at a site located ahead of that of c-AMP generation. In conclusion, GnRH and triptorelin strongly inhibit FSH-mediated function in human granulosa-lutein cells in culture. This inhibition might play a role in the low follicular development rates observed in some patients treated with GnRH agonist + gonadotrophins for ovarian stimulation.

Endothelins inhibit FSH-mediated function via ETA receptors in cultured human granulosa-lutein cells.

Endothelins (ETs) are a family of vasoactive peptides involved in granulosa and luteal cell function in some animal species. However, the potential relevance of ETs in ovarian physiology remains unclear, and the direct action of the peptides in the human ovary has not been studied to date. Experiments were conducted to determine whether ET-1 and ET-3 could regulate the follicle stimulating hormone (FSH)-induced cell response in human granulosa-lutein cells in culture. The FSH-mediated cell rounding process was used as an indicator of cell response, as previously described (Lawrence et al., 1979). Forskolin, cholera toxin, 8-Br-cyclic AMP, isobutylmethylxanthine (IBMX), rolipram and FSH all stimulated similar cell morphological changes, indicating that the cell rounding process was mediated by cyclic AMP. Although ET-1 and ET-3 alone failed to alter cell shape, the FSH-induced cell response was totally inhibited by treatment with ET-1 (10(-10) mol/l) and ET-3 (10(-7) mol/l). In addition, treatment of the cells with BQ123, an antagonist of ET binding on ETA receptor subtype, totally prevented the inhibitory effects of ET-1 and ET-3 on the FSH-induced response. The data presented here show that human granulosalutein cells are a site of ET reception and action. Endothelins inhibit cyclic AMP-dependent FSH-mediated function and the ET(A) receptor participates in this effect.