Extrapolation of cytotoxic masked effects in planar in vitro assays.

The masking of specific effects in in vitro assays by cytotoxicity is a commonly known phenomenon. This may result in a partial or complete loss of effect signals. For common in vitro assays, approaches for identifying and quantifying cytotoxic masking are partly available. However, a quantification of cytotoxicity-affected signals is not possible. As an alternative, planar bioassays that combine high-performance thin layer chromatography with in vitro assays, such as the planar yeast estrogen screen (p-YES), might allow for a quantification of cytotoxically affected signals. Affected signals form a typical ring structure with a supressed or completely lacking centre that results in a double peak chromatogram. This study investigates whether these double peaks can be used for fitting a peak function to extrapolate the theoretical, unaffected signals. The precision of the modelling was evaluated for four individual peak functions, using 42 ideal, undistorted peaks from estrogenic model compounds in the p-YES. Modelled ED50-values from bisphenol A (BPA) experiments with cytotoxically disturbed signals were 13 times higher than for the apparent data without compensation for cytotoxicity (320 ± 63 ng versus 24 ± 17 ng). This finding has a high relevance for the modelling of mixture effects according to concentration addition that requires unaffected, complete dose-response relationships. Finally, we applied the approach to results of a p-YES assay on leachate samples of an elastomer material used in water engineering. In summary, the fitting approach enables the quantitative evaluation of cytotoxically affected signals in planar in vitro assays and also has applications for other fields of chemical analysis like distorted chromatography signals.

Planar chromatography-bioassays for the parallel and sensitive detection of androgenicity, anti-androgenicity and cytotoxicity.

Anti-androgens entering the aquatic environment, e.g., by effluents from wastewater treatment plants or agricultural settings are contributing to endocrine disruption in wildlife and humans. Due to the simultaneous presence of agonistic compounds, common in vitro bioassays can underestimate the risk posed by androgen antagonists. On the other hand, cytotoxic effects might lead to false positive assessments of anti-androgenic effects in conventional bioassays. In the present study, a combination of normal phase high-performance thin-layer chromatography (NP-HPTLC) with a yeast-based reporter gene assay is established for the detection of anti-androgenicity as a promising tool to reduce interferences of androgenic and anti-androgenic compounds present in the same sample. To avoid a misinterpretation of anti-androgenicity with cytotoxic effects, cell viability was assessed in parallel on the same plate using a resazurin viability assay adapted to HPTLC plates. The method was characterized by establishing dose-response curves for the model compounds flutamide and bisphenol A. Calculated effective doses at 10% (ED10) were 27.9 ± 1.3 ng zone-1 for flutamide and 20.1 ± 5.1 ng zone-1 for bisphenol A. Successful distinction between anti-androgenicity and cytotoxicity was exemplarily demonstrated with 4-nitroquinoline 1-oxide. As a proof of concept, the detection and quantification of anti-androgenicity in an extract of a landfill leachate is demonstrated. This study shows that the hyphenation of HPTLC with the yeast anti-androgen screen is a matrix-robust, cost-efficient and fast screening tool for the sensitive and simultaneous detection of anti-androgenic and cytotoxic effects in environmental samples. The method offers a wide range of possible applications in environmental monitoring and contributes to the identification of anti-androgenicity drivers in the course of an effect-directed analysis.

Development and validation of a parent-proxy health-related quality of life survey for Australian First Nations children.

OBJECTIVE: Within Australia, Aboriginal and Torres Strait Islander (First Nations) populations perceive health and well-being differently to non-Indigenous Australians. Existing health-related quality of life (HR-QoL) measurement tools do not account for these differences. The objective of this study was to develop and validate a culturally specific parent-proxy HR-QoL measurement tool for First Nations children. DESIGN: Scale development was informed by parents/carers of children with a chronic illness and an expert panel. The preliminary 39-item survey was reviewed (n=12) and tested (n=163) with parents/carers of First Nations children aged 0-12 years at baseline with comparative scales: the Kessler Psychological Distress Scale, generic HR-QoL (Paediatric QoL Inventory 4.0, PedsQL4.0) and Spence Children's Anxiety Scale, and repeated (n=46) 4 weeks later. Exploratory Factor Analysis was used for scale reduction. Reliability and validity were assessed by internal consistency, test-retest, and correlations with comparison scales. RESULTS: Items within our First Nations-Child Quality of Life (FirstNations-CQoL) were internally consistent with Cronbach's alpha coefficients of ≥0.7 (quality of life, 0.808; patient experience, 0.880; patient support, 0.768) and overall test-retest reliability was good (r=0.75; 95% CI 0.593 to 0.856). Convergent validity was observed with the PedsQL4.0 with Pearson's coefficients of r=0.681 (ages 2-4 years); r=0.651 (ages 5-12 years) and with the Kessler Psychological Distress scale (r=-0.513). Divergent validity against the Spence Anxiety Scale was not demonstrated. CONCLUSIONS: The FirstNations-CQoL scale was accepted by the participants, reliable and demonstrated convergent validity with comparison measures. This tool requires further evaluation to determine responsiveness, its minimal important difference and clinical utility.

UV aged epoxy coatings - Ecotoxicological effects and released compounds.

Organic coatings can guarantee long-term protection of steel structures due to causing a physical barrier against water and oxygen. Because of their mechanical properties and resistances to heat and chemicals, epoxy resin-based coatings are widely used for corrosion protection. Despite of the aromatic backbone and the resulting susceptibility to UV degradation, epoxy resins are frequently used as binding agent in top layers of anti-corrosion coating systems. Consequently, these organic polymers are directly exposed to sunlight and thus UV radiation. The present study was designed to investigate if toxic effects of epoxy resin-based-coatings are changed by UV-A irradiation. For this purpose, two epoxide-based top coatings were examined with and without UV aging for their bacterial toxicity and estrogenicity. In addition, chemical analyses were performed to identify released compounds as well as photolytic degradation products and to assign toxic effects to individual substances. UV-A irradiation of epoxy resin based top coatings resulted in an overall decrease of acute and specific ecotoxicological effects but as well to the formation of toxic transformation products. Both, in leachates of untreated and UV-A irradiated coatings, 4tBP was identified as the main driver of estrogenicity and toxicity to luminescent bacteria. BPA and structural analogs contributing to estrogenic effects in leachates were formed by UV-A irradiation. The combination of HPTLC coupled bioassays and LC-MS analyses supported the identification of bioactive compounds in terms of an effect-directed analysis. The present findings indicate that epoxide-based coatings are less suitable for the application as top coatings and more UV stable coatings like aliphatic polyurethanes should be preferred.

Determination of metal uptake in single organisms, Corophium volutator, via complementary electrothermal vaporization/inductively coupled plasma mass spectrometry and laser ablation/inductively coupled plasma mass spectrometry.

RATIONALE: (Eco-)toxicological effects are mostly derived empirically and are not correlated with metal uptake. Furthermore, if the metal content is determined, mostly bulk analysis of the whole organism population is conducted; thus, biological variability is completely disregarded, and this may lead to misleading results. To overcome this issue, we compared two different solid sampling techniques for the analysis of single organisms. METHODS: In this study, complementary electrothermal vaporization/inductively coupled plasma mass spectrometry (ETV/ICP-MS) ⇔ laser ablation/inductively coupled plasma mass spectrometry (LA/ICP-MS)-based methods for the analysis of individual organisms were developed and the results obtained were compared with the concentrations obtained after digestion and measured using ICP-MS. For this purpose, a common (eco-)toxicological test organism, the mud shrimp Corophium volutator, was selected. As proof-of-concept application, these organisms were incubated with environmentally relevant metals from galvanic anodes, which are often used for protection against metal corrosion in, for example, offshore wind farms. RESULTS: The bulk analysis revealed that large quantities of the incubated elements were detectable. Using the ETV/ICP-MS method, we could identify a high biovariability within the population of organisms tested. Using the LA/ICP-MS method, it could be determined that the large quantities of the elements detected were due to adsorption of the metals and not due to uptake, which correlates well with the absence of (eco-)toxicological effects. CONCLUSIONS: The results obtained imply the efficiency of complementary methods to explain the absence or presence of (eco-)toxicological effects. In particular, methods that allow for single-organism analysis or provide even a spatial resolution support the interpretation of ecotoxicological findings.

Ecotoxicological characterization of emissions from steel coatings in contact with water.

In order to prevent corrosion damage, steel structures need to be protected. Coating systems achieve this by the isolation of the steel from its environment. Common binding agents are epoxide and polyurethane resins which harden by polyaddition reactions. In contact with water, various organic substances might be leached out and released into the aquatic environment potentially causing adverse effects. So far, no legal requirements are mandatory for the environmental sustainability of coating systems. To characterize emissions from steel coatings, recommendations for the ecotoxicological assessment of construction products were utilized. Seven different coating systems based on epoxide or polyurethane resins were leached in 8 steps (6 h-64 d), followed by the testing of acute toxic effects on bacteria and algae as well as estrogen-like and mutagenic effects. In addition, chemical analysis by GC-MS was performed to identify potentially toxic compounds released from the coating systems. Two systems tested did not show any significant effects in the bioassays. One coating system caused significant algal toxicity, none was found to cause mutagenic effects. The other coating systems mainly showed estrogenic effects and bacterial toxicity. The effects increased with increasing leaching time. 4-tert-butylphenol, which is used in epoxy resins as a hardener, was identified as the main contributor to acute and estrogenic effects in two coatings. The release mechanism of 4-tert-butylphenol was characterized by two different modelling approaches. It was found that the release from the most toxic coating is not explainable by an elevated content of 4-tert-butylphenol but more likely by the release mechanism that - in contrast to the less toxic coating - is controlled not only by diffusion. This finding might indicate a sub-optimal formulation of this coating system resulting in a less stable layer and thus an increased release of toxic compounds.

Bonding of fibre-reinforced composite post to root canal dentin.

OBJECTIVE: The aim of this study was to determine bonding properties of two types of fibre-reinforced composite (FRC) posts cemented into root canals of molars. Serrated titanium posts served as reference. METHODS: Prefabricated carbon/graphite FRC posts with cross-linked polymer matrix and individually formed glass FRC posts with interpenetrating polymer network (IPN) polymer matrix were compared. The crowns of extracted third molars were removed and post space (diameter: 1.5mm) was drilled, etched and bonded. The posts were treated with dimethacrylate adhesive resin, light-polymerized and cemented with a dual-polymerizing composite resin luting cement. After thermocycling (6000x) the samples were cut into discs of thicknesses: 1, 2 and 4mm (n=12/group). Push-out force was measured by pushing the post from one end. Assessment of failure mode was made under a stereomicroscope (1, adhesive failure between post and cement; 2, cohesive failure of post-system; 3, adhesive failure between cement and dentin). RESULTS: The push-out force increased with increased height of dentin disc in all groups (ANOVA, p<0.001). In the 4mm thick dentin discs the individually formed glass FRC posts showed highest push-out force and the difference to that of the titanium posts was significant (ANOVA, p<0.001). The other differences were not statistically significant. None of the individually formed glass FRC posts showed adhesive failures between the post and the cement. CONCLUSIONS: Contrary to the other posts, there were no adhesive (post-cement) failures with the individually formed glass FRC posts, suggesting better interfacial adhesion of cement to these posts.

Flexural properties of fiber reinforced root canal posts.

OBJECTIVES: Fiber-reinforced composite (FRC) root canal posts have been introduced to be used instead of metal alloys and ceramics. The aim of this study was to investigate the flexural properties of different types of FRC posts and compare those values with a novel FRC material for dental applications. METHODS: Seventeen different FRC posts of various brands (Snowpost, Carbopost, Parapost, C-post, Glassix, Carbonite) and diameters, (1.0-2.1 mm) and a continuous unidirectional E-glass FRC polymerized by light activation to a cylindrical form (everStick, diameter 1.5 mm) as a control material were tested. The posts (n=5) were stored at room's humidity or thermocycled (12.000 x, 5 degrees C/55 degrees C) and stored in water for 2 weeks before testing. A three-point bending test (span=10 mm) was used to measure the flexural strength and modulus of FRC post specimens. RESULTS: Analysis of ANOVA revealed that thermocycling, brand of material and diameter of specimen had a significant effect (p<0.001) on the fracture load and flexural strength. The highest flexural strength was obtained with the control material (everStick, 1144.9+/-99.9 MPa). There was a linear relationship between fracture load and diameter of posts for both glass fiber and carbon fiber posts. Thermocycling decreased the flexural modulus of the tested specimens by approximately 10%. Strength and fracture load decreased approximately 18% as a result of thermocycling. SIGNIFICANCE: Considerable variation can be found in the calculated strength values of the studied post brands. Commercial prefabricated FRC posts showed lower flexural properties than an individually polymerised FRC material.

Bonding of composite resin luting cement to fiber-reinforced composite root canal posts.

PURPOSE: The aim of this study was to compare bonding of composite resin luting cement to a fiber-reinforced composite (FRC) root canal post with either a cross-linked or a semi-interpenetrating polymer network (IPN) polymer matrix. MATERIALS AND METHODS: Four different types of prefabricated FRC posts with a cross-linked polymer matrix and two types of FRC posts with a semi-IPN polymer matrix which were individually formed were tested. Serrated titanium posts served as the reference. An auto-polymerizing resin luting cement was used for cementing the posts into the holes of composite resin disks. The pull-out force was measured using a universal testing machine after the post/cement/disk system had been stored dry or thermocycled (5 degrees C/55 degrees C, 6000x) in water. The bonding sites of the posts were examined with SEM. RESULTS: The FRC posts with a semi-IPN polymer matrix gave significantly higher pull-out force values than the prefabricated FRC posts with a smooth surface and a cross-linked polymer matrix (p < 0.004). The highest pull-out force was obtained with serrated titanium posts. Both the type of FRC post and thermocycling had a significant effect on the pull-out force (n = 8, ANOVA p < 0.001 and p < 0.007, respectively). SEM photomicrographs confirmed the results. CONCLUSION: This study showed that FRC posts with a semi-IPN polymer matrix bonded better to composite resin luting cement than did prefabricated FRC posts with a cross-linked polymer matrix, although their pull-out force was not as high as that of the mechanically interlocked serrated titanium posts.

Depth of light-initiated polymerization of glass fiber-reinforced composite in a simulated root canal.

PURPOSE: The possibility of polymerizing glass fiber-reinforced composite (FRC) material into the root canal was preliminarily evaluated by determining the depth of light-initiated polymerization of FRC. MATERIALS AND METHODS: The material used was polymer-preimpregnated E-glass fiber reinforcement, which was further impregnated with light-polymerizable dimethacrylate monomer resin. The same resin without fiber reinforcement was used as a control. Six different lengths (range 4 to 24 mm) of light-protected cylinders filled with the test materials were light polymerized from one end. The degree of monomer conversion was determined from the other end by FT-IR spectrometry. Infrared spectra were recorded at six time points from the beginning of polymerization. The microhardness of the test materials was measured from the light-exposure surface toward the other end of the cylinder. RESULTS: Both groups showed a reduction in the degree of conversion with increased lengths of the cylinder. The FRC group showed a higher degree of conversion in the longest sample group compared to the resin group. Microhardness measurement confirmed the constant reduction of the degree of conversion by the reduced Vickers hardness values with increased cylinder length of the FRC. CONCLUSION: Generally, the glass FRC showed an almost equal degree of conversion after light curing as monomer resin without fibers. However, in the longest cylinders, FRC showed a slightly higher degree of conversion compared to resin only; this might be due to the fibers' ability to conduct light.