Targeted impact of cyproterone acetate on the sexual reproduction of female rotifers.

Monogonont rotifers constitute, depending on the moment of the year, most of the zooplankton in many freshwater ecosystems. Sexual reproduction is essential in the development cycle of these organisms as it enables them to constitute stocks of cysts which can withstand adverse environmental conditions and hatch when favorable conditions return. However, endocrine disrupting compounds (EDCs) can interfere with the reproduction of organisms. The present work aimed to investigate the effects of cyproterone acetate (CPA, anti-androgen and progestogen synthetic steroid) at 0.5 mg L-1, on the sexual reproduction of Brachionus calyciflorus in a cross-mating experiment. Results show no impact on mixis whereas the fertilization rate and resting egg production were higher in females exposed to CPA (from embryogenesis to adult stage), regardless of the treatment applied to the males with which they were mating (i.e. males hatched from CPA-treated females or from control females). Moreover, neonate females which mothers has been exposed to 0.5 mg L-1 CPA had more oocytes in their germarium than control neonates. Our results suggest that the effects of CPA observed are not related to toxicity but rather are consistent with an endocrine disruption-related impact, probably through disturbance of the mate recognition protein (MRP) production and through interference with a steroid receptor. Moreover, the absence of effect on mixis rate indicates that mixis induction on the one hand and mating process and resting production on the other hand are not controlled by the same hormonal pathways.

Impact of three phthalate esters on the sexual reproduction of the Monogonont rotifer, Brachionus calyciflorus.

Phthalate esters are widespread contaminants that can cause endocrine disruption in vertebrates. Studies showed that molecules with hormonal activities in vertebrates and invertebrates can affect asexual and sexual reproduction in rotifers. We investigated the impact of di-hexylethyl phthalate (DEHP), di-butyl phthalate (DBP) and butylbenzyl phthalate (BBP), on the asexual and sexual reproduction of the freshwater monogonont rotifer Brachionus calyciflorus in order to determine a potential environmental risk for sexual reproduction. We observed that DEHP has no significant impact on both asexual and sexual reproduction up to 2 mg/L. DBP has a positive effect on asexual reproduction at concentrations from 0.05 to 1 mg/L, but depresses it at 2 mg/L. Sexual reproduction is only affected at 2 mg/L and the impact observed is negative. BBP displayed a negative impact on both asexual and sexual reproduction at 1 and 2 mg/L. However we showed that the impacts of BBP on mixis and fertilization rates observed are due to the decrease in population growth rates at these concentrations and not to a direct impact of BBP on the mixis and the fertilization processes. Our results show that sexual reproduction in B. calyciflorus is not more sensitive than asexual reproduction to any of the substances tested which indicates the mode of action of these molecules is related to general toxicity and not to an interference with potential endocrine regulation of sexual reproduction. Comparison of effect concentrations and surface water contamination by phthalate esters suggests these compounds do not constitute a risk for primary consumers in these environments.

The vertebrate connexin family.

Connexins are chordate-specific transmembrane proteins that can form gap junctional channels between adjacent cells. With the progress in vertebrate genome sequencing, it is now possible to reconstruct the main lines in the evolution of the connexin family from fishes to mammals. Four connexin groups are only found in fishes. Otherwise, the differences between fishes and mammals can be explained by two gene losses (Cx39.9 and Cx43.4) after the divergence of the Reptilia, and three gene duplications (the generation of Cx26 and 30 from a preCx26/30 sequence, Cx30.3 and 31.1 from a preCx30.3/ 31.1 sequence, and Cx31.3 from an uncertain origin). Orthologs of most connexins can be found throughout the vertebrates from fishes to mammals. As judged from the recently defined connexins in tunicates, the original connexin might be related to the ortholog groups of Cx36, 39.2, 43.4, 45 or 47.

Pharmacological evidence for system-dependent involvement of protein kinase C isoenzymes in phorbol ester-suppressed gap junctional communication.

Several phorbol esters are potent activators of protein kinase C. They down-regulate gap junctional intercellular communication and induce phosphorylation of connexin43, but the sensitivity and extent of responses vary much between systems. We asked whether the total protein kinase C enzyme activity or the protein kinase C isoenzyme constitution was of importance for such variations. Some fibroblastic culture systems were compared. It was concluded that the total protein kinase C enzyme activity did not determine the sensitivity to phorbol esters. Furthermore, the use of isotype-specific inhibitors of protein kinase C indicated that protein kinase C alpha, delta, and epsilon may be involved to different extents in different fibroblastic systems in the response to phorbol esters.

Phosphorylation of connexin43 and inhibition of gap junctional communication in 12-O-tetradecanoylphorbol-13-acetate-exposed R6 fibroblasts: minor role of protein kinase C beta I and mu.

12-O:-tetradecanoylphorbol-13-acetate (TPA) inhibits gap junctional communication in many cell culture systems, but TPA-induced phosphorylation of the gap junction protein connexin43 (Cx43) varies much between systems. We have here studied whether these responses and their sensitivities can be correlated with total protein kinase C (PKC) enzyme activity and if specific PKC isoenzymes are involved. Rat R6 fibroblasts transfected with the cDNA sequence encoding PKC beta I (R6-PKC3) had a total PKC activity 7- to 16-fold higher than the corresponding control cells (R6-C1), depending on the selection pressure (G418 concentration). Still, R6-PKC3 cells were no more sensitive than R6-C1 cells to TPA-induced down-regulation of communication, except at the highest selection pressure (500 micrograms/ml G418). Thus, total PKC activity does not indicate absolute sensitivity of a cell system to TPA-induced suppression of communication, but within a certain cell system increasing PKC activity may enhance the sensitivity to TPA in this respect. The results also suggest that PKC beta I is of minor importance for TPA-induced regulation of communication. Experiments with the Lilly compound 379196, a PKC beta-specific inhibitor, further supported this conclusion. Except for PKC beta I in R6-PKC3 cells, both cell lines contained the TPA-responsive PKC isoenzymes alpha, delta, epsilon and mu. Long-term treatment with TPA caused strong down-regulation of PKC alpha, delta and epsilon, but little down-regulation of PKC mu. Concurrently, the cells became refractory to repeated exposure to TPA, indicating that PKC mu is of minor importance. Experiments with the general PKC inhibitor GF109203X and the PKC alpha (and beta/gamma) inhibitor Gö6976 suggested that both classical (alpha) and novel PKCs (delta and epsilon) might be involved in TPA-induced suppression of intercellular communication, while phosphorylation of Cx43 may mainly be mediated by PKC alpha in the present systems.

Phosphatases involved in modulation of gap junctional intercellular communication and dephosphorylation of connexin43 in hamster fibroblasts: 2B or not 2B?

12-O-Tetradecanoylphorbol-13-acetate (TPA) caused strong suppression of gap junctional intercellular communication, altered phosphorylation status of the gap junction protein, connexin43, and disappearance of immunorecognizible connexin43-containing gap junction plaques in V79 fibroblasts. When TPA was removed, all parameters normalized during a 3- to 4-h period. The normalizations were independent of protein synthesis, suggesting the possible involvement of phosphatases. None of the phosphatase inhibitors okadaic acid, calyculin A, cyclosporin A, or FK506 affected intercellular communication or connexin43 phosphorylation status on their own. In sequential exposures to TPA and phosphatase inhibitors, only the protein-phosphatase 2B (PP2B) inhibitors cyclosporin A and FK506 delayed the recovery of the studied parameters. Rapamycin binds to the same set of proteins as does FK506, but without inhibiting PP2B. Rapamycin did not affect the recovery of intercellular communication, but it delayed the normalization of connexin43 band pattern and immunorecognition of gap junction plaques. Dephosphorylation of immunoprecipitated connexin43 was studied using PP1, 2A, 2B, and 2C. PP2A was the most efficient (by 100-fold on a molar basis). Connexin43 immunoprecipitated from TPA-exposed cells was a poor substrate for PP1, 2B, and 2C. Thus, PP2B appeared to play a role in normalization of intercellular communication, but not necessarily in direct dephosphorylation of connexin43. Peptidyl-prolyl isomerase activity of cyclosporin/FK506/rapamycin-binding proteins may promote the dephosphorylation of connexin43 in cells.

Stimulated phosphorylation of intracellular connexin43.

A monoclonal antibody, Zymed 13-8300, was previously reported to only detect nonphosphorylated connexin43 (Nagy et al., Exp. Cell Res. 236, 127-136, 1997). We show that 13-8300 can detect several phosphorylated species of connexin43 in Western blots after stimulation of two fibroblast cell systems with fresh growth medium, 12-O-tetradecanoyl phorbol-13-acetate, pervanadate, or permolybdate. In one of the cell systems, at least three forms of phosphorylated connexin43 could migrate at the same position during electrophoresis. The comigration of differentially phosphorylated species may complicate the molecular and functional analysis of phosphorylation sites in Cx43. Immunofluorescence experiments indicated that the newly generated phosphorylated Cx43 forms mainly had a perinuclear location. Also, in cells treated with brefeldin A for 8 h, in which the majority of connexin43 was intracellular, phosphorylation was induced by the agents. Phosphorylation of intracellular connexin43 can therefore be induced by several stimuli.

Peroxisome Proliferator-induced Transformation of Syrian Hamster Embryo Cells: Influence of Experimental Procedures.

We compared the ability of four hepatic peroxisome proliferators (HPPs) to induce morphological transformation (MT) in Syrian hamster embryo (SHE) cells under different experimental conditions including the composition of the test medium (DMEM at pH7.35 and 7.0, and in LeBoeuf's modified DMEM at pH6.7) and the modalities of exposure. The HPPs studied were two structurally-related hypolipidaemic agents, clofibrate and methyl clofenapate (MCP), an industrial plasticizer, di(2-ethylhexyl)phthalate (DEHP) and one of its primary active metabolite in vivo, mono(2-ethylhexyl)phthalate (MEHP). SHE cells were exposed to the HPP tested either alone, or in sequential treatments with other carcinogens such as benzo[a]pyrene (BaP) or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in order to study possible interactions. A two-stage exposure assay was applied with DMEM at pH7.35 and 7.0. Structural analogues did not give similar results using the same experimental conditions. Indeed, while MCP was more potent at acidic pH, the transforming potency of clofibrate was higher at pH7.0 and 7.35. DEHP and MEHP also behaved differently: in contrast to DEHP, MEHP was more active at pH6.7 than at pH7.0. The MT induction, resulting from the interaction between MCP and BaP or TPA, appeared pH-dependent and higher at pH7.0 than at pH7.35. This study showed that: (i) pH actually influences SHE cell response to HPPs, (ii) the use of acidic medium (pH6.7) does not guarantee a better detection of HPPs' transforming effects and (iii) repeated applications of the test-medium within the 7 days of the assay are more efficient in detecting a transforming potency.

Two-stage exposure of Syrian-hamster-embryo cells to environmental carcinogens: superinduction of ornithine decarboxylase correlates with increase of morphological-transformation frequency.

As part of environmental toxicology, it is important to assess both the carcinogenic potential of xenobiotics and their mode of action on target cells. Since dysregulation of ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine biosynthesis, is considered as an early and essential component in the process of multistage carcinogenesis, we have studied the mode of ODC induction in Syrian-hamster-embryo(SHE) cells stage-exposed to carcinogens and to non-carcinogens. One-stage (5 hr) treatment of SHE cells with 50 microM clofibrate (CLF), a non-genotoxic carcinogen, or with 0.4 microM benzo(a)pyrene (BaP), a genotoxic carcinogen, slightly decreased basal ODC activity. Using the 2-stage exposure, 1 hr to carcinogen, then replacement by TPA for 5 hr, the ODC activity was higher than that obtained with TPA alone. This ODC superinduction was not observed when SHE cells were similarly pre-treated with non-carcinogenic compounds. Several environmental chemicals, pesticides, solvents, oxidizers and drugs were investigated with this SHE cell model. With one-stage exposure, some xenobiotics decreased basal ODC activity, while for others ODC changes were not noticeable. With 2-stage exposure (chemical followed by TPA), all carcinogens amplified the TPA-inducing effect, resulting in ODC superinduction. Comparative studies of the action of carcinogens and of non-carcinogens, using 2-stage exposure protocols, clearly show a close relationship between ODC induction rate and morphological transformation frequency.

Effects of peroxisome proliferators and 12-O-tetradecanoyl phorbol-13-acetate on intercellular communication and connexin43 in two hamster fibroblast systems.

Effects of 12-O-tetradecanoyl phorbol 13-acetate (TPA) and the hepatic peroxisome proliferators (HPPs) clofibrate, methyl clofenapate (MCP), di(2-ethylhexyl)phthalate (DEHP) and mono(2-ethylhexyl)phthalate (MEHP) were studied in 2 gap junctional intercellular communication (GJIC) systems, metabolic cooperation in V79 cells and microinjection/dye transfer in Syrian hamster embryo (SHE) cells and V79 cells. TPA inhibited GJIC in both systems but was considerably more potent in V79 cells. SHE cells showed a rapid and transient inhibition of GJIC after exposure to HPPs, with maximal inhibition occurring at 5-15 min. The transient inhibition could be caused by metabolization of the compounds. Clofibrate and MEHP produced strong inhibition of metabolic cooperation in V79 cells at high concentrations, while the effect of MCP and DEHP was lower. However, DEHP, MEHP and clofibrate strongly inhibited dye transfer in V79 cells after a 30 min exposure. Clofibrate also showed a dose- and time-dependent effect on dye transfer in V79 cells. The phosphorylation status of the gap junction protein connexin43 (Cx43) changed minimally in SHE cells after exposure to TPA or HPPs. Cx43 from V79 cells was strongly affected by TPA, but not by HPPs. Immunofluorescence of Cx43 disappeared in both cell types when they were exposed to TPA and MEHP, but not to the other HPPs. Thus, there is no direct correlation between the inhibition of GJIC and changes in the phosphorylation status of Cx43 or the appearance of Cx43 in immunofluorescence experiments. The discrepancies may partly be explained by binding of accessory proteins to Cx43. We point out sequences that may be involved in such binding.

Comparative effects of clofibrate and methyl clofenapate on morphological transformation and intercellular communication of Syrian hamster embryo cells.

The Syrian hamster embryo (SHE) cell system was used to evaluate the ability of two hepatocarcinogenic structurally related peroxisome proliferators (PPs) to induce morphological transformation (MT) of SHE colonies and to inhibit gap junctional intercellular communication (GJIC). Clofibrate and methyl clofenapate (MCP), which was shown to be a more active PP and a more potent carcinogen in vivo than clofibrate, were compared. MCP appeared slightly more active in vitro than clofibrate in affecting MT and GJIC of SHE cells. The morphological transformation of SHE colonies was induced by 50 microM MCP, against 100 microM clofibrate. Moreover, 50 microM MCP potentiated the transforming effects of both benzo[a]pyrene and 12-O-tetradecanoylphorbol-13-acetate. The inhibition of GJIC, measured by transfer of lucifer yellow, was transient and occurred at concentrations inducing morphological transformation. MCP inhibited dye transfer at 50 microM and the inhibition lasted up to 24 h at 100 microM. Inhibition of communication lasted only 4 h with clofibrate and occurred at a higher concentration (175 microM). This study showed that both the SHE cell transformation and dye transfer assays were able to display the different activities of the two PPs, even though the difference in potency observed was smaller than in vivo. It also revealed interactions between non-genotoxic carcinogens and the ability of the SHE cell transformation assay to detect these combined effects.