Distributions for time, interspecies and intraspecies extrapolation for deriving occupational exposure limits.

This work aimed at improving the empirical database of time (i.e., exposure duration), interspecies and intraspecies extrapolation when deriving occupational exposure limits (OELs). For each extrapolation step, a distribution was derived, which can be used to model the associated uncertainties. For time and interspecies extrapolation, distributions of ratios of dose descriptors were derived from studies of different length or species. National Toxicology Program (NTP) study data were manually assessed, and data from REACH (Registration, Evaluation and Authorisation of Chemicals) registration dossiers were evaluated semi-automatically. Intraspecies extrapolation was investigated by compiling published studies on human toxicokinetic and toxicodynamic variability. A new database was established for toxicokinetic differences in interindividual susceptibility, including many inhalation studies. Using NTP data produced more reliable results than using REACH data. The geometric mean (GM) for time extrapolation subacute/chronic agreed with previous evaluations (GM = 4.11), whereas the GM for subchronic/chronic extrapolation was slightly higher (GM = 2.93) than the GMs found by others. No significant differences were observed between systemically and locally acting substances. Observed interspecies differences confirmed the suitability of allometric scaling, with the derived distribution describing remaining uncertainty. Distributions of intraspecies variability at the 1% and 5% incidence level had medians of 7.25 and 3.56, respectively. When compared with assessment factors (AFs) currently used in the EU, probabilities that these AFs are protective enough span a wide range from 10% to 95%, depending on the extrapolation step. These results help to select AFs in a transparent and informed way and, by allowing to compare protection levels achieved, to harmonise methods for deriving OELs.

Derivation of occupational exposure limits: Differences in methods and protection levels.

Frameworks for deriving occupational exposure limits (OELs) and OEL-analogue values (such as derived-no-effect levels [DNELs]) in various regulatory areas in the EU and at national level in Germany were analysed. Reasons for differences between frameworks and possible means of improving transparency and harmonisation were identified. Differences between assessment factors used for deriving exposure limits proved to be one important reason for diverging numerical values. Distributions for exposure time, interspecies and intraspecies extrapolation were combined by probabilistic methods and compared with default values of assessment factors used in the various OEL frameworks in order to investigate protection levels. In a subchronic inhalation study showing local effects in the respiratory tract, the probability that assessment factors were sufficiently high to protect 99% and 95% of the target population (workers) from adverse effects varied considerably from 9% to 71% and 17% to 87%, respectively, between the frameworks. All steps of the derivation process, including the uncertainty associated with the point of departure (POD), were further analysed with two examples of full probabilistic assessments. It is proposed that benchmark modelling should be the method of choice for deriving PODs and that all OEL frameworks should provide detailed guidance documents and clearly define their protection goals by stating the proportion of the exposed population the OEL aims to cover and the probability with which they intend to provide protection from adverse effects. Harmonisation can be achieved by agreeing on the way to perform the methodological steps for deriving OELs and on common protection goals.

Curing of UV prints - Assessment of possible toxicological hazard for consumers.

In an experimental setting a laboratory analysis of substances migrating from UV prints under mechanical stress into sweat and saliva simulant was performed. The influence of paper type and curing degree on UV prints was investigated. Five substances were identified at concentrations above the limit of detection in the simulants PPG-3 glyceryl triacrylate, ethoxylated trimethylolpropane triacrylate, trimethylolpropane triacrylate, 2/4-isopropylthioxanthone (ITX), and 2,4-diethylthioxanthone (DETX). Migration of the acrylates and photoinitiators into saliva and sweat simulants were increased when the UV inks were printed on uncoated paper in comparison to coated paper. With an exposure scenario considering a person to leaf through 80 pages of UV-printed paper per day while touching each page with a licked fingertip, Risk Characterisation Ratios (RCR) for oral exposure well below 1 were obtained for all five substances indicating no risk for the general population. The three acrylates are classified for skin sensitisation. The migrated amounts per skin surface area of these three were compared with the EC3 value for a hypothetical substance that could be categorised as strong sensitiser (EC3 = 0.1%). The results show that the risk of skin sensitisation even under worst case conditions can be considered as negligible.

ERASSTRI - European risk assessment study on synthetic turf rubber infill - Part 3: Exposure and risk characterisation.

As the final part of a Europe-wide study on the risk from synthetic turf infill consisting of rubber granules derived from end-of-life tyres (ELT), exposure of sportspeople was assessed and compared with health-based reference values for various chemical substances. Based on information from previous project phases, exposure scenarios were established and exposure was calculated for oral, dermal and inhalation routes. Calculated cancer risks for exposure to polycyclic aromatic hydrocarbons were below 1:1 million. Risk characterisation ratios (RCRs) for non-carcinogenic substances were below 1, indicating no health concerns. For 2-hydroxybenzothiazole no toxicological data were found from which to derive a substance-specific reference value. A threshold-of-toxicological concern approach revealed maximum RCRs slightly above 1, which are acceptable, given the conservativism of the approach. ERASSTRI substantially improved the data available for assessing human health risks from using ELT-derived infill material. Overall, no health concerns could be identified for the use of synthetic turfs with ELT-derived infill material.

ERASSTRI - European Risk Assessment Study on Synthetic Turf Rubber Infill - Part 2: Migration and monitoring studies.

End-of-life tyre (ELT)-derived rubber granules are used as synthetic turf infill on sports fields. They contain various chemical substances and there are concerns that exposure to these substances might be harmful for human health. In this second part of a Europe-wide study to address these concerns migration of substances from rubber granules to artificial body fluids (sweat, saliva, gastric juice) was tested and exposure measurements at sports fields were performed to improve the database for exposure assessment. Some PAHs, aluminium, cobalt, benzothiazole, tert-butylamine, MIBK, 4-tert-octylphenol, bisphenol A, and the phthalates DINP and DEHP were found in at least some samples of sweat simulant. The migration rates calculated with these data were used to inform the dermal exposure assessment. In artificial saliva and gastric juice, only aluminium, cobalt, 4-tert-octylphenol and MIBK were detected and migrated fractions were calculated. Bioaccessibility from rubber granules in the gastrointestinal tract was estimated conservatively, assuming complete availability for most substances. In addition, air samples from 17 sports fields in six European countries were analysed. There were no increased concentrations of metals (aluminium, cobalt), PAHs, or other semivolatile substances in air samples, but some volatiles (MIBK: 95th percentile: 18 µg/m3, benzothiazole: 95th perc.: 7 µg/m3, tert-butylamine: 95th perc.: 31 µg/m3, 2-heptanone: 95th perc.: 0.4 µg/m3, cyclohexanone: 95th perc.: 1.5 µg/m3, and saturated aliphatic hydrocarbons >C9: 95th perc.: 26 µg/m3) were slightly increased in a few samples. In addition, skin wipe samples were obtained from 43 sportspeople after playing on synthetic turfs. Only aluminium was detected above the limit of quantification in these samples (95th perc.: 0.84 mg/sample). These data are important input for risk characterisation as performed in the final study phase. Bioaccessibility data are used for estimating oral and dermal exposure of sportspeople, and air measurements are essential for inhalation exposure assessment.

ERASSTRI - European Risk Assessment Study on Synthetic Turf Rubber Infill - Part 1: Analysis of infill samples.

End-of-life tyre (ELT)-derived rubber granules are used as synthetic turf infill on sports fields. They contain various chemical substances and there are concerns that exposure to these substances might be harmful for human health. This Europe-wide risk assessment study addresses these concerns. As the first part, chemical substances in samples from recycling companies and from sports fields were analysed. 86 coated and non-coated ELT granule samples from sites in 14 European countries were investigated, together with ten non-ELT materials. An extensive list of potentially relevant substances was compiled, and the infill materials were analysed for these substances, using GC and HPLC methods. Volatilisation of substances was studied in emission chambers. Polycyclic aromatic hydrocarbons (sum of 8 REACH PAHs) were identified at average concentrations below 10 mg/kg. Substances found at higher concentrations in rubber granules were aluminium (arithmetic mean in uncoated samples from sports fields 5383 mg/kg) and cobalt (168 mg/kg), benzothiazole (48 mg/kg) and 2-hydroxybenzothiazole (34 mg/kg), 6PPD (571 mg/kg) and DPG (51 mg/kg), and 4-tert-octylphenol (14 mg/kg). In addition, the following volatiles were found to evaporate from crumb rubber in emission chambers: benzothiazole, tert-butylamine, cyclohexanone, methyl isobutyl ketone, 2-heptanone and saturated aliphatic hydrocarbons higher than C9. With this comprehensive survey we created a profound database on concentrations of chemical substances in ELT-derived infill material, which is essential for a reliable risk assessment. The results were used to inform subsequent investigations (migration studies, exposure monitoring survey).

Consumer behaviour survey for assessing exposure from consumer products: a feasibility study.

Evaluating chemical exposures from consumer products is an essential part of chemical safety assessments under REACH and may also be important to demonstrate compliance with consumer product legislation. Modelling of consumer exposure needs input information on the substance (e.g. vapour pressure), the product(s) containing the substance (e.g. concentration) and on consumer behaviour (e.g. use frequency and amount of product used). This feasibility study in Germany investigated methods for conducting a consumer survey in order to identify and retrieve information on frequency, duration, use amounts and use conditions for six example product types (four mixtures, two articles): hand dishwashing liquid, cockpit spray, fillers, paints and lacquers, shoes made of rubber or plastic, and ball-pens/pencils. Retrospective questionnaire methods (Consumer Product Questionnaire (CPQ), and Recall-Foresight Questionnaire (RFQ)) as well as protocol methods (written reporting by participants and video documentation) were used. A combination of retrospective questionnaire and written protocol methods was identified to provide valid information in a resource-efficient way. Relevant information, which can readily be used in exposure modelling, was obtained for all parameters and product types investigated. Based on the observations in this feasibility study, recommendations are given for designing a large consumer survey.

Fabrication and characterization of chitosan nanoparticles and collagen-loaded polyurethane nanocomposite membrane coated with heparin for atrial septal defect (ASD) closure.

Atrial septal defect (ASD) constitutes 30-40% of all congenital heart diseases in adults. The most common complications in the treatment of ASD are embolization of the device and thrombosis formation. In this research, an occluding patch was developed for ASD treatment using a well-known textile technology called electrospinning. For the first time, a cardiovascular occluding patch was fabricated using medical grade polyurethane (PU) loaded with bioactive agents namely chitosan nanoparticles (Cn) and collagen (Co) which is then coated with heparin (Hp). Fourier transform infrared spectrum showed characteristic vibrations of several active constituents and changes in the absorbance due to the inclusion of active ingredients in the patch. The contact angle analysis demonstrated no significant decrease in contact angle compared to the control and the composite patches. The structure of the electrospun nanocomposite (PUCnCoHp) was examined through scanning electron microscopy. A decrease in nanofiber diameter between control PU and PUCnCoHp nanocomposite was observed. Water uptake was found to be decreased for the PUCnCoHp nanocomposite against the control. The hemocompatibility properties of the PUCnCoHp ASD occluding patch was inferred through in vitro hemocompatibility tests like activated partial thromboplastin time (APTT), prothrombin time (PT) and hemolysis assay. It was found that the PT and APTT time was significantly prolonged for the fabricated PUCnCoHp ASD occluding patch compared to the control. Likewise, the hemolysis percentage was also decreased for the PUCnCoHp ASD patch against the control. In conclusion, the developed PUCnCoHp patch demonstrates potential properties to be used for ASD occlusion.

Cyclophilin-facilitated membrane translocation as pharmacological target to prevent intoxication of mammalian cells by binary clostridial actin ADP-ribosylated toxins.

Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin and Clostridium difficile CDT belong to the family of binary actin ADP-ribosylating toxins and are composed of a binding/translocation component and a separate enzyme component. The enzyme components ADP-ribosylate G-actin in the cytosol of target cells resulting in depolymerization of F-actin, cell rounding and cell death. The binding/translocation components bind to their cell receptors and form complexes with the respective enzyme components. After receptor-mediated endocytosis, the binding/translocation components form pores in membranes of acidified endosomes and the enzyme components translocate through these pores into the cytosol. This step is facilitated by the host cell chaperone heat shock protein 90 and peptidyl-prolyl cis/trans isomerases including cyclophilin A. Here, we demonstrate that a large isoform of cyclophilin A, the multi-domain enzyme cyclophilin 40 (Cyp40), binds to the enzyme components C2I, Ia and CDTa in vitro. Isothermal titration calorimetry revealed a direct binding to C2I with a calculated affinity of 101 nM and to Ia with an affinity of 1.01 µM. Closer investigation for the prototypic C2I revealed that binding to Cyp40 did not depend on its ADP-ribosyltransferase activity but was stronger for unfolded C2I. The interaction of C2I with Cyp40 was also demonstrated in lysates from C2-treated cells by pull-down. Treatment of cells with a non-immunosuppressive cyclosporine A derivative, which still binds to and inhibits the peptidyl-prolyl cis/trans isomerase activity of cyclophilins, protected cells from intoxication with C2, iota and CDT toxins, offering an attractive approach for development of novel therapeutic strategies against binary actin ADP-ribosylating toxins.

Brush head composition, wear profile, and cleaning efficacy: an assessment of three electric brush heads using in vitro methods.

OBJECTIVE: The objective of this research was to evaluate a current store brand (SB) brush head for composition/physical characteristics, Wear Index (WI), and cleaning efficacy versus the previous SB brush head refill design (SB control) and the Oral-B Precision Clean brush head (positive control, PC). METHODS: This research consisted of three parts: 1) Analytical analysis using Fourier Transform Infrared (FT-IR) spectrometry to evaluate the chemical composition of the current SB brush head bristles relative to the SB control. In addition, physical parameters such as bristle count and diameter were determined. 2) Wear Index (WI) investigation to determine the Wear Index scores of in vitro-aged brush heads at four weeks (one month) and 13 weeks (three months) by a trained investigator. To "age" the brush heads, a robot system was used as a new alternative in vitro method to simulate aging by consumer use. 3) Robot testing to determine the cleaning performance of in vitro-aged brush heads, comparing one month-aged current SB brush heads with the SB control (one and three months-aged) and the PC brush heads (three months-aged) in a standardized fashion. RESULTS: 1) FT-IR analysis revealed that the chemical composition of the current and control SB refill brush heads is identical. In terms of physical parameters, the current SB brush head has 12% more bristles and a slightly oval brush head compared to the round brush head of the SB control. 2) Wear Index analysis showed there was no difference in the one month-aged current SB brush head versus the one month-aged SB control (1.67 vs. 1.50, p = 0.65) or versus the three months-aged PC brush head (1.67 vs. 1.50, p = 0.65). The one month-aged current SB brush head demonstrated statistically significantly less wear than the three months-aged SB control (1.67 vs. 2.67, p = 0.01). 3) Analysis of cleaning efficacy shows that the one month-aged current SB brush head had improved cleaning performance over the one month-aged SB control brush head (p < 0.05), despite no statistically significant difference in wear. Both the one month-aged current and control SB brush heads showed statistically significantly lower cleaning performance compared to the three months-aged PC brush heads (p < 0.01). CONCLUSION: While the current SB brush head showed improved cleaning over the SB control, it demonstrated significantly lower durability and cleaning in comparison to the PC brush head. Dental professionals should be aware of these differences, both in durability and in cleaning performance, when recommending brush heads to their patients.

A novel power toothbrush with multi-directional, triple zone cleaning technology.

PURPOSE: Numerous laboratory and clinical studies have proven that certain power toothbrush technologies are more effective in removal of dental plaque and reduction of gingivitis than regular manual toothbrushes. Regardless of this evidence, there is still a large group of individuals who prefer the experience of a manual-like toothbrush. Recently a novel multi-directional power brush has been developed as an alternative for those people who favor the traditional size and shape of a manual toothbrush and prefer the manual brushing technique, but would benefit from the greater cleaning efficiency of the power brush. METHODS: This unique multi-directional power toothbrush with triple-zone cleaning technology has been tested in multiple clinical trials. This special issue introduces the technical features of the brush and presents four clinical investigations conducted with this power toothbrush versus manual and sonic controls. RESULTS: The studies described in this issue demonstrate the superior efficacy of the multi-directional brush in plaque and gingivitis reduction relative to control brushes, even in the hard-to-reach interdental spaces and marginal areas.

Brush head wear, subject-perceived and laboratory cleaning performance of two oscillating-rotating electric toothbrush heads over 3 months.

PURPOSE: To evaluate the progression of wear and the effect of wear on subject-perceived and laboratory cleaning efficacy of two oscillating-rotating electric brush heads, Oral-B Precision Clean (PC) and a store brand Easyflex (SB) brush head, after 4, 6, 8 and 13 weeks of use. METHODS: This research consisted of three phases: (1) Subject questionnaires--A crossover, single-blinded study was conducted among healthy adults who were regular users of Oral-B oscillating-rotating electric toothbrushes. Subjects were recruited from a general population and randomized based on age and gender into one of four cohorts reflecting the time period of use for each product: 4, 6, 8 or 13 weeks. After brushing with their first product (either PC or SB) for the designated period of time, subjects completed a questionnaire evaluating the brush head on 17 attributes related to perceived cleaning performance, brush head condition (i.e., durability) and brush head feel (i.e., gentleness). Subjects then used the second test product for the same period of time and completed the same questionnaire. (2) Wear index investigation--At the end of each time period, subjects' worn brush head pairs were evaluated by an independent, blinded investigator to determine the wear index score. (3) Robot testing--To analyze the laboratory cleaning efficacy of worn refills in the laboratory, a representative sample of 12 subject brush head pairs for each of the four cohorts were evaluated (96 brush heads in total). To analyze the laboratory cleaning efficacy for PC at Week 13 with SB at Week 4, a separate set of 20 subjects (40 brush heads in total) were evaluated. A robot was used to brush standard typodonts (Frasaco A3) covered with plaque substitute with the worn brush head for 2 minutes under standardized, controlled conditions simulating human brushing behavior. A 3D laser scan system was used to measure the area still covered with plaque substitute at different dental sites. RESULTS: Subject questionnaire--267 subjects completed study questionnaires. Statistically significant superior ratings (P < 0.05) were obtained with the PC brush head compared to the SB brush head for virtually all attributes at all four time periods (16/17 attributes for Weeks 4, 6 and 8 and 17/17 at Week 13). Highly significant advantages (P < 0.0001) were seen for 'overall rating', 'overall cleaning' and 'ready to replace brush head' attributes. Wear Index-- A total of 486 brush head samples (243 pairs) were analyzed for wear. At all four time periods, PC brush heads had a statistically significantly lower (P < 0.0005) mean wear index than SB brush heads. Robot Test--136 brush heads were analyzed using a laboratory (robot) test to investigate cleaning efficacy. Directionally higher laboratory cleaning for PC versus SB was observed for all dental sites (35/35) for all time periods. Comparing PC at Week 13 with SB at Week 4 showed statistically significant differences (P < 0.05) in favor of PC for the majority of dental sites and time periods (23/35).

FK506-binding protein 51 interacts with Clostridium botulinum C2 toxin and FK506 inhibits membrane translocation of the toxin in mammalian cells.

The binary Clostridium botulinum C2 toxin consists of the binding/translocation component C2IIa and the separate enzyme component C2I. C2IIa delivers C2I into the cytosol of eukaryotic target cells where C2I ADP-ribosylates actin. After receptor-mediated endocytosis of the C2IIa/C2I complex, C2IIa forms pores in membranes of acidified early endosomes and unfolded C2I translocates through the pores into the cytosol. Membrane translocation of C2I is facilitated by the activities of host cell chaperone Hsp90 and the peptidyl-prolyl cis/trans isomerase (PPIase) cyclophilin A. Here, we demonstrated that Hsp90 co-precipitates with C2I from lysates of C2 toxin-treated cells and identified the FK506-binding protein (FKBP) 51 as a novel interaction partner of C2I in vitro and in intact mammalian cells. Prompted by this finding, we used the specific pharmacological inhibitor FK506 to investigate whether the PPIase activity of FKBPs plays a role during membrane translocation of C2 toxin. Treatment of cells with FK506 protected cultured cells from intoxication with C2 toxin. Moreover, FK506 inhibited the pH-dependent translocation of C2I across membranes into the cytosol but did not interfere with the enzyme activity of C2I or binding of C2 toxin to cells. Furthermore, FK506 treatment delayed intoxication with the related binary actin ADP-ribosylating toxins from Clostridium perfringens (iota toxin) and Clostridium difficile (CDT) but not with the Rho-glucosylating Clostridium difficile toxin A (TcdA). In conclusion, our results support the hypothesis that clostridial binary actin-ADP-ribosylating toxins share a specific FKBP-dependent translocation mechanism during their uptake into mammalian cells.

Membrane translocation of binary actin-ADP-ribosylating toxins from Clostridium difficile and Clostridium perfringens is facilitated by cyclophilin A and Hsp90.

Some hypervirulent strains of Clostridium difficile produce the binary actin-ADP-ribosylating toxin C. difficile transferase (CDT) in addition to Rho-glucosylating toxins A and B. It has been suggested that the presence of CDT increases the severity of C. difficile-associated diseases, including pseudomembranous colitis. CDT contains a binding and translocation component, CDTb, that mediates the transport of the separate enzyme component CDTa into the cytosol of target cells, where CDTa modifies actin. Here we investigated the mechanism of cellular CDT uptake and found that bafilomycin A1 protects cultured epithelial cells from intoxication with CDT, implying that CDTa is translocated from acidified endosomal vesicles into the cytosol. Consistently, CDTa is translocated across the cytoplasmic membranes into the cytosol when cell-bound CDT is exposed to acidic medium. Radicicol and cyclosporine A, inhibitors of the heat shock protein Hsp90 and cyclophilins, respectively, protected cells from intoxication with CDT but not from intoxication with toxins A and B. Moreover, both inhibitors blocked the pH-dependent membrane translocation of CDTa, strongly suggesting that Hsp90 and cyclophilin are crucial for this process. In contrast, the inhibitors did not interfere with the ADP-ribosyltransferase activity, receptor binding, or endocytosis of the toxin. We obtained comparable results with the closely related iota-toxin from Clostridium perfringens. Moreover, CDTa and Ia, the enzyme component of iota-toxin, specifically bound to immobilized Hsp90 and cyclophilin A in vitro. In combination with our recently obtained data on the C2 toxin from C. botulinum, these results imply a common Hsp90/cyclophilin A-dependent translocation mechanism for the family of binary actin-ADP-ribosylating toxins.

Role of CypA and Hsp90 in membrane translocation mediated by anthrax protective antigen.

Bacillus anthracis lethal toxin consists of the protective antigen (PA) and the metalloprotease lethal factor (LF). During cellular uptake PA forms pores in membranes of endosomes, and unfolded LF translocates through the pores into the cytosol. We have investigated whether host cell chaperones facilitate translocation of LF and the fusion protein LF(N)DTA. LF(N) mediates uptake of LF(N)DTA into the cytosol, where DTA, the catalytic domain of diphtheria toxin, ADP-ribosylates elongation factor-2, allowing for detection of small amounts of translocated LF(N)DTA. Cyclosporin A, which inhibits peptidyl-prolyl cis/trans isomerase activity of cyclophilins, and radicicol, which inhibits Hsp90 activity, prevented uptake of LF(N)DTA into the cytosol of CHO-K1 cells and protected cells from intoxication by LF(N)DTA/PA. Both inhibitors, as well as an antibody against cyclophilin A blocked the release of active LF(N)DTA from endosomal vesicles into the cytosol in vitro. In contrast, the inhibitors did not inhibit cellular uptake of LF. In vitro, cyclophilin A and Hsp90 bound to LF(N)DTA and DTA but not to LF, implying that DTA determines this interaction. In conclusion, cyclophilin A and Hsp90 facilitate translocation of LF(N)DTA, but not of LF, across endosomal membranes, and thus they function selectively in promoting translocation of certain proteins, but not of others.

Selective and specific internalization of clostridial C3 ADP-ribosyltransferases into macrophages and monocytes.

The C3 transferases from Clostridium botulinum (C3bot) and Clostridium limosum (C3lim) mono-ADP-ribosylate and thereby inactivate RhoA, -B and -C of eukaryotic cells. Due to their extremely poor cellular uptake, C3 transferases were supposed to be exoenzymes rather than exotoxins, challenging their role in pathogenesis. Here, we report for the first time that low concentrations of both C3lim and C3bot are selectively internalized into macrophages/monocytes in less than 3 h, inducing the reorganization of the actin cytoskeleton by ADP-ribosylation of Rho. We demonstrate that C3 transferases are internalized into the cytosol of macrophages/monocytes via acidified early endosomes. Bafilomycin A1, an inhibitor of endosomal acidification, protected J774A.1 macrophages and human promyelotic leukaemia cells (HL-60) from intoxication by C3. Moreover, confocal laser scanning microscopy revealed colocalization of C3 with early endosomes. An extracellular acidic pulse enabled direct translocation of cell surface-bound C3 across the cytoplasmic membrane to the cytosol. In line with this finding, both C3 proteins exhibited membrane activity in lipid bilayer membranes only under acidic conditions (pH < 5.5). In conclusion, we identified macrophages/monocytes as target cells for clostridial C3 transferases and shed light on their selective uptake mechanism, which might contribute to understand the role of C3 transferases in pathogenesis.

The long-lived nature of clostridium perfringens iota toxin in mammalian cells induces delayed apoptosis.

Mono-ADP ribosylation of actin by bacterial toxins, such as Clostridium perfringens iota or Clostridium botulinum C2 toxins, results in rapid depolymerization of actin filaments and cell rounding. Here we report that treatment of African green monkey kidney (Vero) cells with iota toxin resulted in delayed caspase-dependent death. Unmodified actin did not reappear in toxin-treated cells, and enzyme-active toxin was detectable in the cytosol for at least 24 h. C2 toxin showed comparable, long-lived effects in cells, while a C2 toxin control lacking ADP-ribosyltransferase activity did not induce cell death. To address whether the remarkable stability of the iota and C2 toxins in cytosol was crucial for inducing cell death, we treated cells with C/SpvB, the catalytic domain of Salmonella enterica SpvB. Although C/SpvB also mono-ADP ribosylates actin as do the iota and C2 toxins, cells treated with a cell-permeating C/SpvB fusion toxin became rounded but recovered and remained viable. Moreover, unmodified actin reappeared in these cells, and ADP-ribosyltransferase activity due to C/SpvB was not detectable in the cytosol after 24 h, a result most likely due to degradation of C/SpvB. Repeated application of C/SpvB prevented recovery of cells and reappearance of unmodified actin. In conclusion, a complete but transient ADP ribosylation of actin was not sufficient to trigger apoptosis, implying that long-term stability of actin-ADP-ribosylating toxins, such as iota and C2, in the cytosol is crucial for inducing delayed, caspase-dependent cell death.

Cyclophilin A facilitates translocation of the Clostridium botulinum C2 toxin across membranes of acidified endosomes into the cytosol of mammalian cells.

The binary Clostridium botulinum C2 toxin consists of the binding/translocation component C2IIa and the separate enzyme component C2I, which mono-ADP-ribosylates actin in eukaryotic cells. Pore formation of C2IIa in early endosomal membranes facilitates translocation of unfolded C2I into the cytosol. We discovered earlier that translocation of C2I depends on the activity of the host cell chaperone heat shock protein Hsp90. Here, we demonstrate that cyclosporin A, which inhibits the peptidyl-prolyl cis/trans isomerase activity of cyclophilins, inhibited intoxication of cells with C2 toxin and prevented uptake of C2I into the cytosol. Cyclosporin A blocked the pH-dependent translocation of C2I activity across membranes of intact cells and of partially purified early endosomes. In vitro, the addition of cytosol to C2 toxin-loaded endosomes induced translocation of C2I activity into the cytosol, which was prevented by pretreatment of the cytosol with an antibody against cyclophilin A. Pull-down experiments with lysates from C2 toxin-treated cells revealed specific binding of cyclophilin A to the N-terminal domain of C2I. In conclusion, our results suggest an essential role of cyclophilin A for translocation of C2I across endosomal membranes during the uptake of C2 toxin into mammalian cells.

A cell-permeable fusion toxin as a tool to study the consequences of actin-ADP-ribosylation caused by the salmonella enterica virulence factor SpvB in intact cells.

The virulence factor SpvB is a crucial component for the intracellular growth and infection process of Salmonella enterica. The SpvB protein mediates the ADP-ribosylation of actin in infected cells and is assumed to be delivered directly from the engulfed bacteria into the host cell cytosol. Here we used the binary Clostridium botulinum C2 toxin as a transport system for the catalytic domain of SpvB (C/SpvB) into the host cell cytosol. A recombinant fusion toxin composed of the enzymatically inactive N-terminal domain of C. botulinum C2 toxin (C2IN) and C/SpvB was cloned, expressed, and characterized in vitro and in intact cells. When added together with C2II, the C2IN-C/SpvB fusion toxin was efficiently delivered into the host cell cytosol and ADP-ribosylated actin in various cell lines. The cellular uptake of the fusion toxin requires translocation from acidic endosomes into the cytosol and is facilitated by Hsp90. The N- and C-terminal domains of SpvB are linked by 7 proline residues. To elucidate the function of this proline region, fusion toxins containing none, 5, 7, and 9 proline residues were constructed and analyzed. The existence of the proline residues was essential for the translocation of the fusion toxins into host cell cytosol and thereby determined their cytopathic efficiency. No differences concerning the mode of action of the C2IN-C/SpvB fusion toxin and the C2 toxin were obvious as both toxins induced depolymerization of actin filaments, resulting in cell rounding. The acute cellular responses following ADP-ribosylation of actin did not immediately induce cell death of J774.A1 macrophage-like cells.