Evaluation of acute-to-chronic ratios of fish and Daphnia to predict acceptable no-effect levels.

BACKGROUND: Acute-to-chronic extrapolation is an important approach to predict acceptable no-effect levels from acute data which has some uncertainties, but is valuable for risk assessment of chemical substances. With regard to the still limited and heterogenic data of chronic fish tests, conclusions on aquatic hazard estimation need to be checked and the question arises whether the chronic toxicity to fish can be adequately derived from acute data. A comprehensive dataset including ecotoxicological studies of 203 substances was used to investigate acute-to-chronic ratios (ACR) for both fish and Daphnia. To address potential uncertainty parameters of the approach, the influence of the octanol-water partition coefficient Kow, the mode of action, and the acute toxicity levels on the ACR was evaluated. RESULTS: For industrial chemicals, median ACRs of 12.0 for fish and 8.8 for Daphnia and 90th percentiles of 68.0 and 50.2, respectively, were determined. The ACR for the most sensitive aquatic trophic level (ACRaqu) is derived by comparing the lowest acute and chronic effect value of Daphnia and fish. The median ACRaqu was 9.9, and the 90th percentile was determined to 58.5. The influence of the Kow on the ACR value was analysed and a correlation could not be confirmed. Non-polar narcosis was associated with a lower ACR, whereas polar narcosis was associated with an increased ACR. CONCLUSIONS: The result suggests that an acute-to-chronic extrapolation factor of 100 is protective for more than 90 % of the chemicals. Polar narcosis may represent a predictor for an increased ACR and an increased uncertainty of the approach. The result further suggests that a high Kow is probably not associated with increased ACRs and does not necessarily represent a determinant for chronic toxicity testing within this context.

Evaluation of the integrated testing strategy for PNEC derivation under REACH.

Species sensitivity evaluation represents an approach to avoid chronic toxicity testing of aquatic vertebrates in accordance with the animal welfare concept of the EU chemicals regulation. In this study a data set of chemicals is analysed for relative species sensitivity between Daphnia and fish in chronic testing to evaluate under what condition chronic fish tests can be waived without underestimating the environmental hazard. Chronic fish toxicity is covered in 84% of the evaluated substances by the chronic invertebrate test and an assessment factor of 50. Thus, animal testing can be avoided in environmental hazard assessment for many chemicals. Moreover, it is shown that species sensitivity in chronic testing is associated with species sensitivity in acute testing. The more sensitive species in chronic testing is predicted with a high probability if a species is >5x more sensitive in acute testing. If substances are comparable or more toxic to Daphnia in acute testing than to fish chronic fish toxicity is covered by the chronic Daphnia test and an assessment factor of 50 in about 95% of the evaluated cases. To provide decision support for the regulation of chemicals a categorization scheme on relative sensitivity comparison is presented.

Rac1-dependent recruitment of PAK2 to G2 phase centrosomes and their roles in the regulation of mitotic entry.

During mitotic entry, the centrosomes provide a scaffold for initial activation of the CyclinB/Cdk1 complex, the mitotic kinase Aurora A, and the Aurora A-activating kinase p21-activated kinase (PAK). The activation of PAK at the centrosomes is yet regarded to happen independently of the Rho-GTPases Rac/Cdc42. In this study, Rac1 (but not RhoA or Cdc42) is presented to associate with the centrosomes from early G2 phase until prometaphase in a cell cycle-dependent fashion, as evidenced by western blot analysis of prepared centrosomes and by immunolabeling. PAK associates with the G2/M-phase centrosomes in a Rac1-dependent fashion. Furthermore, specific inhibition of Rac1 by C. difficile toxinB-catalyzed glucosylation or by knockout results in inhibited activation of PAK1/2, Aurora A, and the CyclinB/Cdk1 complex in late G2 phase/prophase and delayed mitotic entry. Inhibition of PAK activation at late G2-phase centrosomes caused by Rac1 inactivation coincides with impeded activation of Aurora A and the CyclinB/Cdk1 complex and delayed mitotic entry.

Expression and cytoprotective activity of the small GTPase RhoB induced by the Escherichia coli cytotoxic necrotizing factor 1.

RhoB is the only member of the Rho subfamily of small GTPases, which is classified as an immediate early gene product. RhoB is up-regulated in response to growth factors as well as cytotoxic and genotoxic agents. Clostridial glucosylating toxins have been reported to evoke pronounced RhoB expression, based on the inactivation of Rho/Ras proteins. In this study, we report on a long lasting expression of RhoB in cultured cells upon activation of Rho proteins by the cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli. The observations of this study highlight a new pathway involving Rac1, which positively regulates the activity of the rhoB promoter and RhoB expression. Conversely, the isomeric cytotoxic necrotizing factor from Yersinia pseudotuberculosis (CNFy) drives GTP-loading of basal RhoB but fails to cause activation of the rhoB promoter and thus its expression. CNF1 inhibits cytokinesis and induces the formation of bi-nucleated (tetraploid) cells. Upon long term treatment with CNF1, RhoB(-/-) mouse embryonic fibroblasts (MEFs) exhibit DNA fragmentation, phosphatidylserine exposure, and loss of membrane integrity, while RhoB(+/-) MEFs persist as bi-nucleated (tetraploid) cells without any signs of cell death. In conclusion, the cytoprotective RhoB response is not only evoked by bacterial protein toxins inactivating Rho/Ras proteins but also by the Rac1-activating toxin CNF1.

Difference in F-actin depolymerization induced by toxin B from the Clostridium difficile strain VPI 10463 and toxin B from the variant Clostridium difficile serotype F strain 1470.

Clostridium difficile toxin A (TcdA) and toxin B (TcdB) are the causative agent of the C. difficile-associated diarrhea (CDAD) and its severe form, the pseudomembranous colitis (PMC). TcdB from the C. difficile strain VPI10463 mono-glucosylates (thereby inactivates) the small GTPases Rho, Rac, and Cdc42, while Toxin B from the variant C. difficile strain serotype F 1470 (TcdBF) specifically mono-glucosylates Rac but not Rho(A/B/C). TcdBF is related to lethal toxin from C. sordellii (TcsL) that glucosylates Rac1 but not Rho(A/B/C). In this study, the effects of Rho-inactivating toxins on the concentrations of cellular F-actin were investigated using the rhodamine-phalloidin-based F-actin ELISA. TcdB induces F-actin depolymerization comparable to the RhoA-inactivating exoenzyme C3 from C. limosum (C3-lim). In contrast, the Rac-glucosylating toxins TcdBF and TcsL did not cause F-actin depolymerization. These observations led to the conclusion that F-actin depolymerization depends on the toxin's capability of glucosylating RhoA. Furthermore, the integrity of focal adhesions (FAs) was analyzed using paxillin and p21-activated kinase (PAK) as FA marker proteins. Paxillin dephosphorylation was observed upon treatment of cells with TcdB, TcdBF, or C3-lim. In conclusion, the Rho-inactivating toxins induce loss of cell shape by either F-actin depolymerization (upon RhoA inactivation) or the disassembly of FAs (upon Rac1 inactivation).

Cytoprotective effect of the small GTPase RhoB expressed upon treatment of fibroblasts with the Ras-glucosylating Clostridium sordellii lethal toxin.

Mono-glucosylation of (H/K/N)Ras by Clostridium sordellii lethal toxin (TcsL) blocks critical survival signaling pathways, resulting in apoptosis. In this study, TcsL and K-Ras knock-down by siRNA are presented to result in expression of the cell death-regulating small GTPase RhoB. TcsL-induced RhoB expression is based on transcriptional activation involving p38(alpha) MAP kinase. Newly synthesized RhoB protein is rapidly degraded in a proteasome- and a caspase-dependent manner, providing first evidence for caspase-dependent degradation of a Rho family protein. Although often characterised as a pro-apoptotic protein, RhoB suppresses caspase-3 activation in TcsL-treated fibroblasts. The finding on the cytoprotective activity of RhoB in TcsL-treated cells re-enforces the concept that RhoB exhibits cytoprotective rather than pro-apoptotic activity in a cellular background of inactive Ras.

Increased Cell-Matrix Adhesion upon Constitutive Activation of Rho Proteins by Cytotoxic Necrotizing Factors from E. Coli and Y. Pseudotuberculosis.

Cytotoxic necrotizing factors (CNFs) encompass a class of autotransporter toxins produced by uropathogenic E. coli (CNF1) or Y. pseudotuberculosis (CNFy). CNF toxins deamidate and thereby constitutively activate RhoA, Rac1, and Cdc42. In this study, the effects of CNF1 on cell-matrix adhesion are analysed using functional cell-adhesion assays. CNF1 strongly increased cell-matrix binding of suspended Hela cells and decreased the susceptibly of cells to trypsin-induced cell detachment. Increased cell-matrix binding was also observed upon treatment of Hela cells with isomeric CNFy, that specifically deamidates RhoA. Increased cell-matrix binding thus appears to depend on RhoA deamidation. In contrast, increased cell spreading was specifically observed upon CNF1 treatment, suggesting that it rather depended on Rac1/Cdc42 deamidation. Increased cell-matrix adhesion is further presented to result in reduced cell migration of adherent cells. In contrast, migration of suspended cells was not affected upon treatment with CNF1 or CNFy. CNF1 and CNFy thus reduced cell migration specifically under the condition of pre-established cell-matrix adhesion.

Inhibition of macrophage migration by C. botulinum exoenzyme C3.

C3-like exoenzymes are produced by various microorganism including Clostridium botulinum (C3bot), Bacillus cereus and Staphylococcus aureus. C3bot is the prototype of C3-like exoenzymes that specifically ADP-ribosylates and thereby inactivates Rho(A/B/C). C3-like exoenzymes are not yet regarded as virulence factors, as the lack of cell entry domains results in a poor accessibility of the C3-like exoenzymes to cells. In this study, the sensitivity of various cell lines to C3bot has been reinvestigated. Primary monocytes as well as cultured macrophage-like cells including J774A.1 cells and RAW macrophages exhibit a tenfold higher sensitivity to C3bot than fibroblasts and epithelial cells. RhoA ADP-ribosylation by C3bot resulted in the formation of pronounced bipolar protrusions based on defective tail retraction. The formation of bipolar protrusion resulted in inhibited macrophage migration. These findings suggested that macrophages appear to be target cells of C3bot. Migration of macrophage is a prerequiste for their recruitment to the site of pathogen invasion or tissue damage. Inhibition of macrophage migration likely preserves the survival of C3-producing microorganisms. The observations of this study reinforce the paradigm of a role of C3-like exoenzymes as virulence factors.

The SCF-FBXW5 E3-ubiquitin ligase is regulated by PLK4 and targets HsSAS-6 to control centrosome duplication.

Deregulated centrosome duplication can result in genetic instability and contribute to tumorigenesis. Here, we show that centrosome duplication is regulated by the activity of an E3-ubiquitin ligase that employs the F-box protein FBXW5 (ref. 3) as its targeting subunit. Depletion of endogenous FBXW5 or overexpression of an F-box-deleted mutant version results in centrosome overduplication and formation of multipolar spindles. We identify the centriolar protein HsSAS-6 (refs 4,5) as a critical substrate of the SCF-FBXW5 complex. FBXW5 binds HsSAS-6 and promotes its ubiquitylation in vivo. The activity of SCF-FBXW5 is in turn negatively regulated by Polo-like kinase 4 (PLK4), which phosphorylates FBXW5 at Ser 151 to suppress its ability to ubiquitylate HsSAS-6. FBXW5 is a cell-cycle-regulated protein with expression levels peaking at the G1/S transition. We show that FBXW5 levels are controlled by the anaphase-promoting (APC/C) complex, which targets FBXW5 for degradation during mitosis and G1, thereby helping to reset the centrosome duplication machinery. In summary, we show that a cell-cycle-regulated SCF complex is regulated by the kinase PLK4, and that this in turn restricts centrosome re-duplication through degradation of the centriolar protein HsSAS-6.

Difference in the biological effects of Clostridium difficile toxin B in proliferating and non-proliferating cells.

Toxin A (TcdA) and toxin B (TcdB) from Clostridium difficile are the causative agents of the C. difficile-associated diarrhea (CDAD) and its severe form, the pseudomembranous colitis. TcdA and TcdB both glucosylate and thereby inactivate low molecular weight GTP-binding proteins of the Rho, Rac, and Cdc42 subfamilies. In cultured cell lines, TcdB induces actin re-organization and bi-nucleation ("cytopathic effects") and cell death ("cytotoxic effects"). In this study, the role of cell cycle progression in the cytopathic and the cytotoxic effects of TcdB is evaluated by a differential analysis of these effects in proliferating and non-proliferating cells. Density-synchronized murine fibroblasts and confluent HT29 colonocytes are exploited as cell culture models for non-proliferating cells. Cell death is analyzed in terms of a loss of cell viability, phosphatidylserine exposure, and DNA fragmentation. In proliferating cells, TcdB blocks cell proliferation and induces apoptotic cell death. In contrast, TcdB induces non-apoptotic cell death in non-proliferating cells. TcdB-induced cell rounding turns out to be independent of cell cycle progression. Cell cycle progression is an important determinant in the biological effects of TcdB. With respect to the pathology of CDAD, this study leads to the new hypothesis that necrotic cell death of terminally differentiated colonocytes and inhibition of epithelial renewal of the colon contribute to the pathogenesis of CDAD.

Actin re-organization induced by Chlamydia trachomatis serovar D--evidence for a critical role of the effector protein CT166 targeting Rac.

The intracellular bacterium Chlamydia trachomatis causes infections of urogenital tract, eyes or lungs. Alignment reveals homology of CT166, a putative effector protein of urogenital C. trachomatis serovars, with the N-terminal glucosyltransferase domain of clostridial glucosylating toxins (CGTs). CGTs contain an essential DXD-motif and mono-glucosylate GTP-binding proteins of the Rho/Ras families, the master regulators of the actin cytoskeleton. CT166 is preformed in elementary bodies of C. trachomatis D and is detected in the host-cell shortly after infection. Infection with high MOI of C. trachomatis serovar D containing the CT166 ORF induces actin re-organization resulting in cell rounding and a decreased cell diameter. A comparable phenotype was observed in HeLa cells treated with the Rho-GTPase-glucosylating Toxin B from Clostridium difficile (TcdB) or HeLa cells ectopically expressing CT166. CT166 with a mutated DXD-motif (CT166-mut) exhibited almost unchanged actin dynamics, suggesting that CT166-induced actin re-organization depends on the glucosyltransferase motif of CT166. The cytotoxic necrotizing factor 1 (CNF1) from E. coli deamidates and thereby activates Rho-GTPases and transiently protects them against TcdB-induced glucosylation. CNF1-treated cells were found to be protected from TcdB- and CT166-induced actin re-organization. CNF1 treatment as well as ectopic expression of non-glucosylable Rac1-G12V, but not RhoA-G14A, reverted CT166-induced actin re-organization, suggesting that CT166-induced actin re-organization depends on the glucosylation of Rac1. In accordance, over-expression of CT166-mut diminished TcdB induced cell rounding, suggesting shared substrates. Cell rounding induced by high MOI infection with C. trachomatis D was reduced in cells expressing CT166-mut or Rac1-G12V, and in CNF1 treated cells. These observations indicate that the cytopathic effect of C. trachomatis D is mediated by CT166 induced Rac1 glucosylation. Finally, chlamydial uptake was impaired in CT166 over-expressing cells. Our data strongly suggest CT166's participation as an effector protein during host-cell entry, ensuring a balanced uptake into host-cells by interfering with Rac-dependent cytoskeletal changes.

Inhibition of cytokinesis by Clostridium difficile toxin B and cytotoxic necrotizing factors--reinforcing the critical role of RhoA in cytokinesis.

Low molecular weight GTP-binding proteins of the Rho family control the organization of the actin cytoskeleton in eukaryotic cells. RhoA governs the formation of actin stress fibers and is responsible for the formation of the contractile ring in cytokinesis. Cytokinesis completion requires RhoA inactivation resulting in disassembly of the contractile ring. Cytokinesis thus requires switching of RhoA activity. This switch of RhoA activity is blocked by Rho-modifying bacterial protein toxins that either activate or inactivate RhoA by covalent modifications. Exoenzyme C3 from Clostridium limosum (C3-lim) and Clostridium difficile toxin B (TcdB) inactivate RhoA by mono-ADP-ribosylation and mono-glucosylation, respectively. Cytotoxic necrotizing factors (CNF), produced by either Yersinia pseudotuberculosis (CNFY) or uropathogenic strains of E. coli (CNF1), deamidate and thereby activate RhoA. This study provides evidence that RhoA-activating as well as RhoA-inactivating toxins cause inhibition of cytokinesis and cell division. The toxins' effects on cytokinesis were analyzed in Hela cells synchronized using the thymidine double block technique. Treatment of G2-phase cells with either the RhoA-activating CNFY or CNF1 or the RhoA-inactivating C3-lim or TcdB resulted in cytokinesis inhibition, as evidenced by the formation of a 4N population on flow cytometry, the inhibition of contractile ring formation, and the formation of bi-nucleated cells. While TcdB and CNF1 modify a broad-spectrum of Rho proteins, C3-lim and CNFY specifically target RhoA. Since C3-lim and CNFY both caused cytokinesis inhibition, our study re-inforces the critical role of RhoA (not Rac1 or Cdc42) in cytokinesis and cell division.

Transition from longitudinal to transversal patterns in an anisotropic system.

Periodic stripe patterns which form when an electric field is applied to a thin nematic liquid crystal layer with a very low conductivity are discussed. In this case the dielectric electroconvection mode persists down to very low frequencies of the driving voltage. A Lifschitz point, i.e., a transition from normal to oblique rolls is detected in the dielectric regime. A crossover from electroconvection to flexoelectric domains occurs for extremely low frequencies of about 0.1 Hz . The crossover scenario yields pattern morphologies characteristic for both mechanisms, i.e., electroconvection and flexoelectric domains which appear consecutively within one period of the driving voltage. A theoretical description of the onset characteristics of dielectric convection, which is based on an extended model including flexoelectricity, is also presented.

Prevention of the cytopathic effect induced by Clostridium difficile Toxin B by active Rac1.

Clostridium difficile Toxin B (TcdB) glucosylates low molecular weight GTP-binding proteins of the Rho subfamily and thereby causes actin re-organization (cell rounding). This "cytopathic effect" has been generally attributed to RhoA inactivation. Here we show that cells expressing non-glucosylatable Rac1-Q61L are protected from the cytopathic effect of TcdB. In contrast, cells expressing RhoA-Q63L or mock-transfected cells are fully susceptible for the cytopathic effect of TcdB. These findings are extended to the Rac1/RhoG mimic IpgB1 and the RhoA mimic IpgB2 from Shigella. Ectopic expression of IpgB1, but not IpgB2, counteracts the cytopathic effect of TcdB. These data strongly suggest that Rac1 rather than RhoA glucosylation is critical for the cytopathic effect of TcdB.