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Find video protocols related to scientific articles indexed in Pubmed.
Pathway and Time-Resolved Benzo[a]pyrene Toxicity on Hepa1c1c7 Cells at Toxic and Subtoxic Exposure.
J. Proteome Res.
PUBLISHED: 11-04-2014
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Benzo[a]pyrene (B[a]P) is an environmental contaminant mainly studied for its toxic/carcinogenic effects. For a comprehensive and pathway orientated mechanistic understanding of the effects directly triggered by a toxic (5 ?M) or a subtoxic (50 nM) concentration of B[a]P or indirectly by its metabolites, we conducted time series experiments for up to 24 h to study the effects in murine hepatocytes. These cells rapidly take up and actively metabolize B[a]P, which was followed by quantitative analysis of the concentration of intracellular B[a]P and seven representative degradation products. Exposure with 5 ?M B[a]P led to a maximal intracellular concentration of 1604 pmol/5 × 10(4) cells, leveling at 55 pmol/5 × 10(4) cells by the end of the time course. Changes in the global proteome (>1000 protein profiles) and metabolome (163 metabolites) were assessed in combination with B[a]P degradation. Abundance profiles of 236 (both concentrations), 190 (only 5 ?M), and 150 (only 50 nM) proteins were found to be regulated in response to B[a]P in a time-dependent manner. At the endogenous metabolite level amino acids, acylcarnitines and glycerophospholipids were particularly affected by B[a]P. The comprehensive chemical, proteome and metabolomic data enabled the identification of effects on the pathway level in a time-resolved manner. So in addition to known alterations, also protein synthesis, lipid metabolism, and membrane dysfunction were identified as B[a]P specific effects.
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Identification of lipidomic biomarkers for coexposure to subtoxic doses of benzo[a]pyrene and cadmium: the toxicological cascade biomarker approach.
Environ. Sci. Technol.
PUBLISHED: 08-18-2014
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The search for model bioassay systems indicating activation of different toxicological signaling pathways is one of the paramount goals of modern toxicology. Especially coexposure scenarios need to be investigated with respect to synergistic and interdependent effects for the activation of toxicological signaling pathways. The present study introduces an experimental in vitro model system for nontoxic and low-dose coexposures of human mammary carcinoma MCF-7 cells against polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BP) and heavy metals such as cadmium. For the first time, a multivariate model that identifies 18 metabolic biomarkers has been shown to be sufficient to separate BP-treated cells from coexposed or control cells. A "toxicological pathway color code model" is introduced to visualize the results. Different biomarker subsets can be associated with specific HER2 signaling steps. A tiered cascade biomarker approach is proposed that could be used to identify profiles associated with tumorigenic potency of environmental toxicants in coexposure scenarios, including possible synergistic or additive effects.
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Chemical hazards present in liquids and vapors of electronic cigarettes.
Arch. Toxicol.
PUBLISHED: 06-03-2014
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Electronic (e-)cigarettes have emerged in recent years as putative alternative to conventional tobacco cigarettes. These products do not contain typical carcinogens that are present in tobacco smoke, due to the lack of combustion. However, besides nicotine, hazards can also arise from other constituents of liquids, such as solvents, flavors, additives and contaminants. In this study, we have analyzed 28 liquids of seven manufacturers purchased in Germany. We confirm the presence of a wide range of flavors to enhance palatability. Although glycerol and propylene glycol were detected in all samples, these solvents had been replaced by ethylene glycol as dominant compound in five products. Ethylene glycol is associated with markedly enhanced toxicological hazards when compared to conventionally used glycerol and propylene glycol. Additional additives, such as coumarin and acetamide, that raise concerns for human health were detected in certain samples. Ten out of 28 products had been declared "free-of-nicotine" by the manufacturer. Among these ten, seven liquids were identified containing nicotine in the range of 0.1-15 µg/ml. This suggests that "carry over" of ingredients may occur during the production of cartridges. We have further analyzed the formation of carbonylic compounds in one widely distributed nicotine-free brand. Significant amounts of formaldehyde, acetaldehyde and propionaldehyde were only found at 150 °C by headspace GC-MS analysis. In addition, an enhanced formation of aldehydes was found in defined puff fractions, using an adopted machine smoking protocol. However, this effect was delayed and only observed during the last third of the smoking procedure. In the emissions of these fractions, which represent up to 40 % of total vapor volume, similar levels of formaldehyde were detected when compared to conventional tobacco cigarettes. By contrast, carbonylic compounds were hardly detectable in earlier collected fractions. Our data demonstrate the necessity of standardized machine smoking protocols to reliably address putative risks of e-cigarettes for consumers.
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Metabolomic biomarkers correlating with hepatic lipidosis in dairy cows.
BMC Vet. Res.
PUBLISHED: 05-20-2014
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Hepatic lipidosis or fatty liver disease is a major metabolic disorder of high-producing dairy cows that compromises animal performance and, hence, causes heavy economic losses worldwide. This syndrome, occurring during the critical transition from gestation to early lactation, leads to an impaired health status, decreased milk yield, reduced fertility and shortened lifetime. Because the prevailing clinical chemistry parameters indicate advanced liver damage independently of the underlying disease, currently, hepatic lipidosis can only be ascertained by liver biopsy. We hypothesized that the condition of fatty liver disease may be accompanied by an altered profile of endogenous metabolites in the blood of affected animals.
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Waterpipe smoke: source of toxic and carcinogenic VOCs, phenols and heavy metals?
Arch. Toxicol.
PUBLISHED: 02-20-2014
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The use of the waterpipe, a traditional aid for the consumption of tobacco, has spread worldwide and is steadily increasing especially among the youth. On the other hand, there is a lack of knowledge regarding the composition of mainstream waterpipe smoke and the toxicological risks associated with this kind of smoking habit. Using a standardized machine smoking protocol, mainstream waterpipe smoke was generated and further analyzed for twelve volatile organic compounds (VOCs) and eight phenolic compounds by applying gas chromatography-mass spectrometry and reverse-phase high-performance liquid chromatography-fluorescence detection, respectively. Additionally, seventeen elements were analyzed in waterpipe tobacco and charcoal prior to and after smoking, applying inductively coupled plasma-mass spectrometry to assess the maximum exposure of these elements. For the first time ever, we have been able to show that waterpipe mainstream smoke contains high levels of the human carcinogen benzene. Compared with cigarette smoke yields, the levels were 6.2-fold higher, thus representing a significant health hazard for the waterpipe smoker. Furthermore, we found that waterpipe mainstream smoke contains considerable amounts of catechol, hydroquinone and phenol, each of which causing some health concern at least. The analysis of waterpipe tobacco and charcoal revealed that both matrices contained considerable amounts of the toxic elements nickel, cadmium, lead and chromium. Altogether, the data on VOCs, phenols and elements presented in this study clearly point to the health hazards associated with the consumption of tobacco using waterpipes.
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On the role of co-inhibitory molecules in dendritic cell: T helper cell coculture assays aimed to detect chemical-induced contact allergy.
EXS
PUBLISHED: 01-31-2014
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T cells play a pivotal role in sensitization and elicitation of type IV allergic reactions. While T helper cells sustain and maintain the differentiation of further effector cells, regulatory T cells are involved in control of cytokine release and proliferation, and T killer cells execute cellular lysis, thereby leading to certain levels of tissue damage. According to their central role, the widely applied and OECD-supported test method for the assessment of the sensitization potential of a chemical, i.e., the local lymph node assay (LLNA), relies on the detection of the immune-responsive proliferation of lymphocytes. However, most sensitization assays recently developed take advantage of the initiators of sensitization, dendritic cells (DCs) or DC-like cell lines. Here, we focus on inhibitory molecules expressed on the surface of DCs and their corresponding receptors on T cells. We summarize insight into the function of CTLA-4, the ligands of inducible co-stimulators (ICOSs), and on the inhibitory receptor programmed death (PD). The targeting of immune cell surface receptors by inhibitory molecules holds some promise with regard to the development of T cell-based sensitization assays. Firstly, a broader and more sensitive dynamic range of detection could be achieved by blocking inhibitors or by removing inhibiting regulatory T cells from the assays. Secondly, the actual expression levels of inhibitory molecules could be also a valuable indicator for the process of sensitization. Finally, inhibitory molecules in coculture test systems are supposed to have a major influence on DCs by reverse signaling, thereby affecting their differentiation and maturation status in a feedback loop. In conclusion, inhibitory ligands of DC surface receptors and/or their cognate receptors on T cells could serve as useful tools in cell-based assays, directly influencing toxicological endpoints such as sensitization.
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Method development and inter-laboratory comparison about the determination of titanium from titanium dioxide nanoparticles in tissues by inductively coupled plasma mass spectrometry.
Anal Bioanal Chem
PUBLISHED: 01-05-2014
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Nanosized titanium dioxide (TiO2) is one of the most interesting and valuable nanomaterials for the construction industry but also in health care applications, food, and consumer goods, e.g., cosmetics. Therefore, the properties associated with this material are described in detail. Despite its widespread use, the analytical determination and characterization of nanosized metal oxides is not as straightforward as the comparatively easy-to-detect metallic nanoparticles (e.g., silver or gold). This study presents the method development and the results of the determination of tissue titanium (Ti) levels after treatment of rats with the nanosized TiO2. Total Ti levels were chosen to evaluate the presence and distribution of TiO2 nanoparticles. A procedure consisting of incubation with a mixture of nitric acid (HNO3) and hydrofluoric acid (HF), and heating was developed to digest tissues and TiO2 nanomaterials in order to determine the total Ti content by inductively coupled plasma mass spectrometry (ICPMS). For the inter-laboratory comparison, altogether four laboratories analyzed the same samples upon digestion using the available ICPMS equipment. A major premise for any toxicokinetic study is the possibility to detect the chemical under investigation in biological samples (tissues). So, the study has to be performed with a dose high enough to allow for subsequent tissue level measurement of the chemical under investigation. On the other hand, dose of the chemical applied should not induce over toxicity in the animal as this may affect its absorption, distribution, metabolism, and excretion. To determine a non-toxic TiO2 dosage, an acute toxicity study in rats was performed, and the organs obtained were evaluated for the presence of Ti by ICPMS. Despite the differences in methodology and independent of the sample preparation and the ICPMS equipment used, the results obtained for samples with Ti concentrations >4 ?g Ti/g tissue agreed well.
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MicroRNA profiling as tool for in vitro developmental neurotoxicity testing: the case of sodium valproate.
PLoS ONE
PUBLISHED: 01-01-2014
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Studying chemical disturbances during neural differentiation of murine embryonic stem cells (mESCs) has been established as an alternative in vitro testing approach for the identification of developmental neurotoxicants. miRNAs represent a class of small non-coding RNA molecules involved in the regulation of neural development and ESC differentiation and specification. Thus, neural differentiation of mESCs in vitro allows investigating the role of miRNAs in chemical-mediated developmental toxicity. We analyzed changes in miRNome and transcriptome during neural differentiation of mESCs exposed to the developmental neurotoxicant sodium valproate (VPA). A total of 110 miRNAs and 377 mRNAs were identified differently expressed in neurally differentiating mESCs upon VPA treatment. Based on miRNA profiling we observed that VPA shifts the lineage specification from neural to myogenic differentiation (upregulation of muscle-abundant miRNAs, mir-206, mir-133a and mir-10a, and downregulation of neural-specific mir-124a, mir-128 and mir-137). These findings were confirmed on the mRNA level and via immunochemistry. Particularly, the expression of myogenic regulatory factors (MRFs) as well as muscle-specific genes (Actc1, calponin, myosin light chain, asporin, decorin) were found elevated, while genes involved in neurogenesis (e.g. Otx1, 2, and Zic3, 4, 5) were repressed. These results were specific for valproate treatment and--based on the following two observations--most likely due to the inhibition of histone deacetylase (HDAC) activity: (i) we did not observe any induction of muscle-specific miRNAs in neurally differentiating mESCs exposed to the unrelated developmental neurotoxicant sodium arsenite; and (ii) the expression of muscle-abundant mir-206 and mir-10a was similarly increased in cells exposed to the structurally different HDAC inhibitor trichostatin A (TSA). Based on our results we conclude that miRNA expression profiling is a suitable molecular endpoint for developmental neurotoxicity. The observed lineage shift into myogenesis, where miRNAs may play an important role, could be one of the developmental neurotoxic mechanisms of VPA.
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Low-dose formaldehyde delays DNA damage recognition and DNA excision repair in human cells.
PLoS ONE
PUBLISHED: 01-01-2014
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Formaldehyde is still widely employed as a universal crosslinking agent, preservative and disinfectant, despite its proven carcinogenicity in occupationally exposed workers. Therefore, it is of paramount importance to understand the possible impact of low-dose formaldehyde exposures in the general population. Due to the concomitant occurrence of multiple indoor and outdoor toxicants, we tested how formaldehyde, at micromolar concentrations, interferes with general DNA damage recognition and excision processes that remove some of the most frequently inflicted DNA lesions.
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The evolution of our understanding of endo-xenobiotic crosstalk and cytochrome P450 regulation and the therapeutic implications.
Expert Opin Drug Metab Toxicol
PUBLISHED: 08-13-2013
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Over the past years, there has been increasing evidence that, at least in vertebrates, cytochrome P450-dependent monooxygenases (CYPs) are governed by a most complex regulation. The respective mechanisms comprise structural features such as domain movements, allostery, enzyme-oligomerization as well as numerous transcription factors, non-coding RNAs and extensive regulatory crosstalk.
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The DNT-EST: a predictive embryonic stem cell-based assay for developmental neurotoxicity testing in vitro.
Toxicology
PUBLISHED: 08-06-2013
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As the developing brain is exquisitely vulnerable to chemical disturbances, testing for developmental neurotoxicity of a substance is an important aspect of characterizing its tissue specific toxicity. Mouse embryonic stem cells (mESCs) can be differentiated toward a neural phenotype, and this can be used as a model for early brain development. We developed a new in vitro assay using mESCs to predict adverse effects of chemicals and other compounds on neural development - the so-called DNT-EST. After treatment of differentiating stem cells for 48h or 72h, at two key developmental stages endpoint for neural differentiation, viability, and proliferation were assessed. As a reference, we similarly treated undifferentiated stem cells 2 days after plating for 48h or 72h in parallel to the differentiating stem cells. Here, we show that chemical testing of a training set comprising nine substances (six substances of known developmental toxicity and three without specific developmental neurotoxicity) enabled a mathematical prediction model to be formulated that provided 100% predictivity and accuracy for the given substances, including in leave-one-out cross-validation. The described test method can be performed within two weeks, including data analysis, and provides a prediction of the developmental neurotoxicity potency of a substance.
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Interlaboratory comparison of size measurements on nanoparticles using nanoparticle tracking analysis (NTA).
J Nanopart Res
PUBLISHED: 06-07-2013
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One of the key challenges in the field of nanoparticle (NP) analysis is in producing reliable and reproducible characterisation data for nanomaterials. This study looks at the reproducibility using a relatively new, but rapidly adopted, technique, Nanoparticle Tracking Analysis (NTA) on a range of particle sizes and materials in several different media. It describes the protocol development and presents both the data and analysis of results obtained from 12 laboratories, mostly based in Europe, who are primarily QualityNano members. QualityNano is an EU FP7 funded Research Infrastructure that integrates 28 European analytical and experimental facilities in nanotechnology, medicine and natural sciences with the goal of developing and implementing best practice and quality in all aspects of nanosafety assessment. This study looks at both the development of the protocol and how this leads to highly reproducible results amongst participants. In this study, the parameter being measured is the modal particle size.
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Metabolomics in toxicology and preclinical research.
ALTEX
PUBLISHED: 05-14-2013
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Metabolomics, the comprehensive analysis of metabolites in a biological system, provides detailed information about the biochemical/physiological status of a biological system, and about the changes caused by chemicals. Metabolomics analysis is used in many fields, ranging from the analysis of the physiological status of genetically modified organisms in safety science to the evaluation of human health conditions. In toxicology, metabolomics is the -omics discipline that is most closely related to classical knowledge of disturbed biochemical pathways. It allows rapid identification of the potential targets of a hazardous compound. It can give information on target organs and often can help to improve our understanding regarding the mode-of-action of a given compound. Such insights aid the discovery of biomarkers that either indicate pathophysiological conditions or help the monitoring of the efficacy of drug therapies. The first toxicological applications of metabolomics were for mechanistic research, but different ways to use the technology in a regulatory context are being explored. Ideally, further progress in that direction will position the metabolomics approach to address the challenges of toxicology of the 21st century. To address these issues, scientists from academia, industry, and regulatory bodies came together in a workshop to discuss the current status of applied metabolomics and its potential in the safety assessment of compounds. We report here on the conclusions of three working groups addressing questions regarding 1) metabolomics for in vitro studies 2) the appropriate use of metabolomics in systems toxicology, and 3) use of metabolomics in a regulatory context.
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State of the art in human risk assessment of silver compounds in consumer products: a conference report on silver and nanosilver held at the BfR in 2012.
Arch. Toxicol.
PUBLISHED: 04-04-2013
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In light of the broad spectrum of products containing nanosilver, the harmfulness of nanosilver to human health and the environment was intensively discussed at a conference held in February 2012 at the BfR. The conference agenda covered the aspects of analytics of nanosilver materials, human exposure and toxicology as well as effects on microorganisms and the environment. The discussion recovered major gaps related to commonly agreed guidelines for sample preparation and central analytical techniques. In particular, the characterization of the nanoparticles in complex matrices was regarded as a challenge which might become a pitfall for further innovation and application. Historical and anecdotal records of colloidal silver have been sometimes taken as empirical proof for the general low toxicity of nanosilver. Yet as reported herein, a growing number of animal studies following modern performance standards of toxicity testing have been carried out recently revealing well-characterized adverse effects on different routes of exposure in addition to argyria. Furthermore, recent approaches in exposure assessment were reported. However, consumer exposure scenarios are only starting to be developed and reliable exposure data are still rare. It was further widely agreed on the workshop that the use of silver may lead to the selection of silver resistant bacteria. With respect to its environmental behavior, it was suggested that nanosilver released to wastewater may have negligible ecotoxicological effects. Finally, the presentations and discussion on risk assessment and regulation of nanosilver applications gave insights into different approaches of risk assessment of nanomaterials to be performed under the various regulatory frameworks.
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Waterpipe smoking: analysis of the aroma profile of flavored waterpipe tobaccos.
Talanta
PUBLISHED: 03-12-2013
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In the last years the habit of smoking waterpipes has spread worldwide, especially among young people and emerged as global health issue. Although research is now under way for no less than 40 years in the field of waterpipe smoking, in comparison to cigarette smoking there is still insufficient knowledge on the real composition and the toxicity of the smoke inhaled and the resulting levels of exposure against particular hazardous ingredients. In most cases for waterpipe smoking a highly flavored tobacco called "moassel" is used. However, the number, quantity and toxicity of the added flavorings are widely unknown. In this study the static headspace gas chromatography-mass spectrometry (SHS-GC-MS) was used to identify 79 volatile flavor compounds present in waterpipe tobacco. Among these eleven compounds were analyzed quantitatively. The results show that waterpipe tobacco contains high amounts of the fragrance benzyl alcohol as well as considerable levels of limonene, linalool and eugenol, all of which are known as being allergenic in human skin. The proposed SHS-GC-MS method has been validated and found to be accurate, simple and characterized by low limits of detection (LOD) in the range of 0.016 to 4.3 µg/g tobacco for benzaldehyde and benzyl alcohol, respectively. The identification and characterization of waterpipe tobacco ingredients indeed reveals crucial for the assessment of potential health risks that may be posed by these additives in smokers.
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Effects of triclocarban on the transcription of estrogen, androgen and aryl hydrocarbon receptor responsive genes in human breast cancer cells.
Toxicol In Vitro
PUBLISHED: 03-04-2013
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Triclocarban (TCC) is an antimicrobial agent that is used in detergents, soaps and other personal hygiene products. Similarly to triclosan the widespread use of TCC has raised concerns about its endocrine potential. In luciferase-based reporter assays TCC has been shown to enhance estrogenic and androgenic activities following cellular coexposure with estrogen or dihydrotestosterone, respectively. The present study demonstrates that although coexposure with TCC enhances the estrogenic and androgenic readout of luciferase-based reporter cell lines such as HeLa9908 and MDA-kb2, it fails to act as a xenoandrogen on transcriptional level, nor does it induce cell proliferation in the estrogen sensitive E-screen. In addition TCC did not alter the expression of estrogen responsive genes in human mammary carcinoma MCF-7 cells exposed to 17?-estradiol, bisphenol A, butylparaben or genistein. However, TCC was shown to interfere with the regulon of the aryl hydrocarbon receptor (AhR) as TCC showed a costimulatory effect on transcription of CYP1A1 and CYP1B1, effectively lowering the transcriptional threshold for both genes in the presence of estrogens. It thus seems, that while the induction of the respective luciferase reporter assays by TCC is an unspecific false positive signal caused by luciferase stabilisation, TCC has the potential to interfere with the regulatory crosstalk of the estrogen receptor (ER) and the AhR regulon.
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Evaluation of selected biomarkers for the detection of chemical sensitization in human skin: a comparative study applying THP-1, MUTZ-3 and primary dendritic cells in culture.
Toxicol In Vitro
PUBLISHED: 02-15-2013
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Dendritic cells (DCs) exhibit the unique capacity to induce T cell differentiation and proliferation, two processes that are crucially involved in allergic reactions. By combining the exclusive potential of DCs as the only professional antigen-presenting cells of the human body with the well known handling advantages of cell lines, cell-based alternative methods aimed at detecting chemical sensitization in vitro commonly apply DC-like cells derived from myeloid cell lines. Here, we present the new biomarkers programmed death-ligand 1 (PD-L1), DC immunoreceptor (DCIR), IL-16, and neutrophil-activating protein-2 (NAP-2), all of which have been detectable in primary human DCs upon exposure to chemical contact allergens. To evaluate the applicability of DC-like cells in the prediction of a chemicals sensitization potential, the expression of cell surface PD-L1 and DCIR was analyzed. In contrast to primary DCs, only minor subpopulations of MUTZ-3 and THP-1 cells presented PD-L1 or DCIR at their surface. After exposure to increasing concentrations of nickel and cinnamic aldehyde, the expression level of PD-L1 and DCIR revealed much stronger affected on monocyte-derived DCs (MoDCs) or Langerhans cells (MoLCs) when compared to THP-1 and MUTZ-3 cells. Applying protein profiler arrays we further identified the soluble factors NAP-2, IL-16, IL-8 and MIP-1? as sensitive biomarkers showing the capacity to discriminate sensitizing from non-sensitizing chemicals or irritants. An allergen-specific release of IL-8 and MIP-1? could be detected in the supernatants of MoDCs and MoLCs and also in MUTZ-3 and THP-1 cells, though at much lower levels. On the protein and transcriptional level, NAP-2 and IL-16 indicated sensitizers most sensitively and specifically in MoDCs. Altogether, we have proven the reciprocal regulated surface molecules PD-L1 and DCIR and the soluble factors MIP-1?, NAP-2 and IL-16 as reliable biomarkers for chemical sensitization. We further show that primary DCs are significantly different in their phenotype and function compared to DC-like cell lines. Since they demonstrated higher absolute values and a broader range in biomarker expression, we propose that MoDCs represent an optimal and robust sensor test system well suited to identify and classify chemicals with an allergic potential.
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DNA quality control by a lesion sensor pocket of the xeroderma pigmentosum group D helicase subunit of TFIIH.
Curr. Biol.
PUBLISHED: 01-24-2013
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Nucleotide excision repair is a versatile DNA repair reaction that removes bulky adducts generated by environmental mutagens such as the UV spectrum of sunlight or chemical carcinogens. Current multistep models of this excision repair pathway accommodate its broad substrate repertoire but fail to explain the stringent selectivity toward damaged nucleotides among excess native DNA. To understand the mechanism of bulky lesion recognition, we postulated that it is necessary to analyze the function of xeroderma pigmentosum group D (XPD) protein beyond its well-known role in the unwinding of double-stranded DNA.
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Biomimetic synthesis of chiral erbium-doped silver/peptide/silica core-shell nanoparticles (ESPN).
Nanoscale
PUBLISHED: 10-27-2011
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Peptide-modified silver nanoparticles have been coated with an erbium-doped silica layer using a method inspired by silica biomineralization. Electron microscopy and small-angle X-ray scattering confirm the presence of an Ag/peptide core and silica shell. The erbium is present as small Er(2)O(3) particles in and on the silica shell. Raman, IR, UV-Vis, and circular dichroism spectroscopies show that the peptide is still present after shell formation and the nanoparticles conserve a chiral plasmon resonance. Magnetic measurements find a paramagnetic behavior. In vitro tests using a macrophage cell line model show that the resulting multicomponent nanoparticles have a low toxicity for macrophages, even on partial dissolution of the silica shell.
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Developmental toxicity testing in the 21st century: the sword of Damocles shattered by embryonic stem cell assays?
Arch. Toxicol.
PUBLISHED: 09-29-2011
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Modern society faces an inherent dilemma. In our globalized society, we are spoilt for choice by an ever-increasing number of products, many of which are made of new materials and compound mixtures. At the same time, as consumers we got accustomed to the idea of a life minimized for risk, including our own exposure to chemicals from the environment or to compounds present in and released from everyday products. Chemical safety testing bridges these obviously diverging interests, and the corresponding legislation has hence been tremendously extended (e.g., introduction of the European legislation REACH in 2007). However, the underlying regulatory toxicology still relies mainly on animal testing, which is relatively slow, expensive, and ethically arguable. Meanwhile, recent years have seen a surge in efforts to develop alternative testing systems and strategies. Expectations are particularly high for the applicability of stem cells as test systems especially for developmental toxicity testing in vitro. For the first time in history, test systems can be based on differentiating cells and tissue progenitors in culture, thus bringing the vision of toxicity testing in the 21st century a step closer.
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Waterpipe smoke: a considerable source of human exposure against furanic compounds.
Anal. Chim. Acta
PUBLISHED: 08-16-2011
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Smoking of waterpipes became increasingly popular in the Western hemisphere in recent years. Yet, up to now only little is known about the health hazards and on the composition of waterpipe smoke. To obtain more information on the ingredients present in waterpipe smoke we utilized two different approaches. Based on headspace-solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) instrumentation we identified new compounds present in the waterpipe smoke. Additional reversed-phase-high performance liquid chromatography-diode array detection (RP-HPLC-DAD) then led us to perform reliable quantification of the newly detected chemical species. Upon identification of a range of different furanic compounds such as 5-(hydroxymethyl)-2-furaldehyde (HMF), 2-furaldehyde, and others, we developed an easy-to-perform and fast RP-HPLC-DAD method to quantify these compounds in the complex matrix of waterpipe smoke. The detection limits range from 0.04 ?g for HMF to 7.1 ?g for 3-furan methanol per smoking session. Linearity, intra- and inter-day precision and recovery were determined and proved excellent. We analyzed 5 waterpipe tobacco brands and found up to 62.3±11 mg of HMF generated during one waterpipe smoking session. The applied smoking protocol comprised 171 puffs of 530 mL each and 2.6s duration every 20s. Our results reveal that waterpipe smoking constitutes a major source of HMF exposure. Furthermore, we found a distinct filter effect of the bowl water for all furanic compounds investigated except HMF.
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Nanosilver in consumer products and human health: more information required!
Environ. Sci. Technol.
PUBLISHED: 08-05-2011
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Commenting on "120 Years of Nanosilver History: Implications for Policy Makers" (Environ. Sci. Technol.2011, 45, 1177-1183). The title of the article seduces readers to the impression that we can look back at more than a century of safe use of nanosilver. In this context, colloidal silver and nanosilver have been sometimes used as synonyms. Historically, the term "colloidal silver" refers to dispersed silver particles encompassing a size range of 10-1000 nm. Following scientific definitions, "colloid" stands for freely dispersed particles in a fluid (heterogenic) phase irrespective of its size distribution, while the term "nanosilver" is used for categorization by size. Of course, just the labeling as such neither necessarily implies new hazard properties nor any specific risks; however, uncertainties and data gaps at many levels call for careful consideration and usually should take effect as alert signal for regulatory toxicologists all over the world. Within the frame of this short commentary, we would like to focus on some unclarified issues related to consumer products.
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Regulation of nucleotide excision repair by UV-DDB: prioritization of damage recognition to internucleosomal DNA.
PLoS Biol.
PUBLISHED: 06-03-2011
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How tightly packed chromatin is thoroughly inspected for DNA damage is one of the fundamental unanswered questions in biology. In particular, the effective excision of carcinogenic lesions caused by the ultraviolet (UV) radiation of sunlight depends on UV-damaged DNA-binding protein (UV-DDB), but the mechanism by which this DDB1-DDB2 heterodimer stimulates DNA repair remained enigmatic. We hypothesized that a distinctive function of this unique sensor is to coordinate damage recognition in the nucleosome repeat landscape of chromatin. Therefore, the nucleosomes of human cells have been dissected by micrococcal nuclease, thus revealing, to our knowledge for the first time, that UV-DDB associates preferentially with lesions in hypersensitive, hence, highly accessible internucleosomal sites joining the core particles. Surprisingly, the accompanying CUL4A ubiquitin ligase activity is necessary to retain the xeroderma pigmentosum group C (XPC) partner at such internucleosomal repair hotspots that undergo very fast excision kinetics. This CUL4A complex thereby counteracts an unexpected affinity of XPC for core particles that are less permissive than hypersensitive sites to downstream repair subunits. That UV-DDB also adopts a ubiquitin-independent function is evidenced by domain mapping and in situ protein dynamics studies, revealing direct but transient interactions that promote a thermodynamically unfavorable ?-hairpin insertion of XPC into substrate DNA. We conclude that the evolutionary advent of UV-DDB correlates with the need for a spatiotemporal organizer of XPC positioning in higher eukaryotic chromatin.
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Alternatives to animal testing: current status and future perspectives.
Arch. Toxicol.
PUBLISHED: 05-25-2011
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On the occasion of the 20th anniversary of the Center for Alternative Methods to Animal Experiments (ZEBET), an international symposium was held at the German Federal Institute for Risk Assessment (BfR) in Berlin. At the same time, this symposium was meant to celebrate the 50th anniversary of the publication of the book "The Principles of Humane Experimental Technique" by Russell and Burch in 1959 in which the 3Rs principle (that is, Replacement, Reduction, and Refinement) has been coined and introduced to foster the development of alternative methods to animal testing. Another topic addressed by the symposium was the new vision on "Toxicology in the twenty-first Century", as proposed by the US-National Research Council, which aims at using human cells and tissues for toxicity testing in vitro rather than live animals. An overview of the achievements and current tasks, as well as a vision of the future to be addressed by ZEBET@BfR in the years to come is outlined in the present paper.
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Chemical toxicity testing in vitro using cytochrome P450-expressing cell lines, such as human CYP1B1.
Nat Protoc
PUBLISHED: 04-28-2011
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This protocol describes how to use cytochrome P450-dependent monooxygenase (CYP)-expressing cell lines in toxicity testing of chemicals in vitro. Selected cells amenable to permanently grow in culture are genetically manipulated to stably express single CYP enzymes originating from any species of interest. This expression can be characterized by, for example, determining CYP mRNA content, CYP protein level (western blotting or in situ immunofluorescence) and CYP-mediated enzyme activity (substrate conversion assays). These cells can be used to determine substrate specificities and species differences, e.g., in the bioactivation of drugs. Once constructed, CYP-expressing cells can serve as a straightforward and reliable tool in toxicity testing and the corresponding assays could be adapted for high-throughput analysis. Using these cells, enzyme assays can be performed in a matter of hours. This protocol is exemplified with V79 fibroblasts from Chinese hamster (Cricetulus griseus), modified to express human cytochrome P450 1B1 (CYP1B1). These cells are characterized for their CYP1B1-linked properties by in situ immunofluorescence and their activity in the 7-ethoxyresorufin-O-deethylase enzyme assay. This is followed by an assay showing metabolic activation of the polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene by CYP1B1, along with the toxicological endpoints of cytotoxicity and micronucleus formation.
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Estimation of dermal and oral exposure of children to scented toys: analysis of the migration of fragrance allergens by dynamic headspace GC-MS.
J Sep Sci
PUBLISHED: 04-20-2011
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Fragrances capable of inducing contact allergy in skin potentially can migrate from the toy to the child via oral or dermal contacts. The goal of this work was the developing of an analytical method based on dynamic headspace GC-MS to determine the concentration of 24 fragrances in saliva or sweat simulant. Under optimized conditions, 5 mL of the migration simulant with 2 g sodium chloride were incubated for 10 min at 30°C. The headspace was purged at a flow rate of 50 mL/min. The compounds were quantified by internal calibration resulting in good linearity (>0.991). The recovery was greater than 66.3% for most of the compounds. The limits of detection ranged between 0.5 ng/mL for hydrophobic and 196.0 ng/mL for hydrophilic fragrances. The method was subsequently applied to seven real toys purchased from the market. The highest migration rate could be observed for benzyl benzoate with 268.0 ng/cm(2)/min. Based on the migration data measured, the ranges of dermal and oral exposure of children to fragrances in scented toys were calculated. The maximum oral and dermal exposure levels were estimated at 22.2 ?g per kg body weight (BW) and day (d) for benzyl benzoate and 605.0 ?g/kg BW/d for benzyl alcohol, respectively.
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Mainstream smoke of the waterpipe: does this environmental matrix reveal as significant source of toxic compounds?
Toxicol. Lett.
PUBLISHED: 04-20-2011
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In recent years the number of waterpipe smokers has increased substantially worldwide. Here we report on the concentrations of tobacco-specific nitrosamines (TSNAs) and polycyclic aromatic hydrocarbons (PAHs) in waterpipe smoke and the analysis of selected biomarkers indicative for the body burden in waterpipe users. We further identify high amounts of unburned humectants (glycerol and propylene glycol) in the waterpipe smoke as main part of the so-called "tar" fraction. These results give cause for serious concern. For standardization we applied a machine smoking protocol. Smoke was collected on glass fiber filters and analyzed for nicotine, water, humectants, TSNAs, and PAHs. In addition, we determined carbon monoxide and found high amounts in the smoke being causative for high levels of carboxyhemoglobin (COHb) in the blood of smokers. In comparison to the reference cigarette 3R4F, the nicotine contents were 10-times higher, but TSNA levels were found lower in waterpipe smoke. This finding explained the low levels of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol detected in the urine of waterpipe smokers. Finally, the levels of benzo[a]pyrene were three times higher in waterpipe smoke compared to the reference cigarette. Altogether, the data presented in this study point to the health hazards associated with the consumption of waterpipes.
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Application of laser postionization secondary neutral mass spectrometry/time-of-flight secondary ion mass spectrometry in nanotoxicology: visualization of nanosilver in human macrophages and cellular responses.
ACS Nano
PUBLISHED: 04-02-2011
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Silver nanoparticles (SNP) are the subject of worldwide commercialization because of their antimicrobial effects. Yet only little data on their mode of action exist. Further, only few techniques allow for visualization and quantification of unlabeled nanoparticles inside cells. To study SNP of different sizes and coatings within human macrophages, we introduce a novel laser postionization secondary neutral mass spectrometry (Laser-SNMS) approach and prove this method superior to the widely applied confocal Raman and transmission electron microscopy. With time-of-flight secondary ion mass spectrometry (TOF-SIMS) we further demonstrate characteristic fingerprints in the lipid pattern of the cellular membrane indicative of oxidative stress and membrane fluidity changes. Increases of protein carbonyl and heme oxygenase-1 levels in treated cells confirm the presence of oxidative stress biochemically. Intriguingly, affected phagocytosis reveals as highly sensitive end point of SNP-mediated adversity in macrophages. The cellular responses monitored are hierarchically linked, but follow individual kinetics and are partially reversible.
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Analysis of carcinogenic polycyclic aromatic hydrocarbons in complex environmental mixtures by LC-APPI-MS/MS.
Anal. Chim. Acta
PUBLISHED: 03-30-2011
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Here we developed a highly sensitive, fast and reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the detection and analysis of 16 different polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs that have been identified as carcinogens and classified according to their biological potency. Comparison to standard analysis procedures based on gas chromatography-mass spectrometry (GC-MS) instrumentation demonstrated an improved easiness of sample preparation and sensitivity of detection achieved with the new LC-MS/MS method employing an atmospheric pressure photoionization (APPI) source attached to an API 4000 mass spectrometer (LC-APPI-MS/MS). The favorable outcome could be confirmed by analyzing complex mixtures such as certain Standard Reference Materials (SRMs) obtained from the National Institute of Standards & Technology (NIST), i.e., SRM 1975 and SRM 2975, and several diesel exhaust soots provided by the German automobile industry. Certified concentrations of individual analytes provided by NIST not only could be confirmed, but additional extremely potent carcinogens such as several isomeric hexacyclic dibenzopyrenes (DBPs), 5-methylchrysene (5-MC), and others have been detected in these crude samples in a concentration range down to below 1 ng g(-1) raw material.
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Analysis of primary aromatic amines in the mainstream waterpipe smoke using liquid chromatography-electrospray ionization tandem mass spectrometry.
J Chromatogr A
PUBLISHED: 03-24-2011
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In recent years waterpipe smoking has spread worldwide and emerged as global health issue. Yet only little is known on the composition of waterpipe smoke. Here, we present a study on the identification and quantification of primary aromatic amines (PAAs) in this complex environmental matrix. Smoking of the waterpipe was conducted with a smoking machine and particulate matter was collected on glass fiber pads. We developed a fast, simple and specific liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) approach to simultaneously detect 31 different PAAs in this matrix. The detection limits comprised a range of 0.45-4.50 ng per smoking session, represented by 2-aminobiphenyl and 3,4,5-trichloroaniline, respectively. Intra- and inter-day precision were determined and proved excellent. We detected 31.3 ± 2.2 ng aniline and 28.0 ± 1.6 ng 4,4-oxydianiline in the smoke of one waterpipe session. The water in the bowl exerted a small but considerable filter effect on PAAs. The method worked-out showed excellent sensitivity and specificity and is thus highly suited for the determination of PAAs in mainstream waterpipe smoke.
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Defined culture medium for stem cell differentiation: applicability of serum-free conditions in the mouse embryonic stem cell test.
Toxicol In Vitro
PUBLISHED: 02-23-2011
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The embryonic stem cell test (EST) is a validated method to assess the developmental toxicity potency of chemicals. It was developed to reduce animal use and allow faster testing for hazard assessment. The cells used in this method are maintained and differentiated in media containing foetal calf serum. This animal product is of considerable variation in quality, and individual batches require extensive testing for their applicability in the EST. Moreover, its production involves a large number of foetuses and possible animal suffering. We demonstrate the serum-free medium and feeder cell-free maintenance of the mouse embryonic stem cell line D3 and investigate the use of specific growth factors for induction of cardiac differentiation. Using a combination of bone morphogenetic protein-2, bone morphogenetic protein-4, activin A and ascorbic acid, embryoid bodies efficiently differentiated into contracting myocardium. Additionally, examining levels of intracellular marker proteins by flow cytometry not only confirmed differentiation into cardiomyocytes, but demonstrated significant differentiation into neuronal cells in the same time frame. Thus, this approach might allow for simultaneous detection of developmental effects on both early mesodermal and neuroectodermal differentiation. The serum-free conditions for maintenance and differentiation of D3 cells described here enhance the transferability and standardisation and hence the performance of the EST.
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Silicification of peptide-coated silver nanoparticles--A Biomimetic soft chemistry approach toward chiral hybrid core-shell materials.
ACS Nano
PUBLISHED: 01-31-2011
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Silica and silver nanoparticles are relevant materials for new applications in optics, medicine, and analytical chemistry. We have previously reported the synthesis of pH responsive, peptide-templated, chiral silver nanoparticles. The current report shows that peptide-stabilized nanoparticles can easily be coated with a silica shell by exploiting the ability of the peptide coating to hydrolyze silica precursors such as TEOS or TMOS. The resulting silica layer protects the nanoparticles from chemical etching, allows their inclusion in other materials, and renders them biocompatible. Using electron and atomic force microscopy, we show that the silica shell thickness and the particle aggregation can be controlled simply by the reaction time. Small-angle X ray scattering confirms the Ag/peptide@silica core-shell structure. UV-vis and circular dichroism spectroscopy prove the conservation of the silver nanoparticle chirality upon silicification. Biological tests show that the biocompatibility in simple bacterial systems is significantly improved once a silica layer is deposited on the silver particles.
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Physiologically based toxicokinetic modelling as a tool to assess target organ toxicity in route-to-route extrapolation--the case of coumarin.
Toxicol. Lett.
PUBLISHED: 01-21-2011
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Coumarin (1,2-benzopyrone) is occurring in food, and is also used in cosmetics. In order to perform a risk assessment for both oral and dermal exposure, we applied a physiologically based approach to model kinetics in humans by simulating both routes of exposure. The concentration-time profile in liver revealed a higher peak concentration (C(max-hep)) for the oral when compared to the dermal route. The area under the concentration-time curve in the liver (AUC(hep)) was found the same for both routes if the same extent of absorption is assumed. Dose response information from published rat studies were used to identify the metric relevant for liver toxicity. Liver exposure levels resulting from doses and durations as outlined in the studies were simulated in a rat model. We obtained 31 data pairs of C(max-hep) and AUC(hep). Liver toxicity was observed at doses which resulted in simulated C(max-hep) values exceeding a certain liver concentration whereas we could not identify a clear cut off value of AUC(hep). Our findings support the notion that liver toxicity of coumarin in rats is related to C(max-hep) rather than to AUC(hep). If these findings can be transferred to the situation in humans, the result demonstrates that route specific differences in organ peak concentrations have to be considered when performing route-to-route extrapolation.
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The embryonic stem cell test as tool to assess structure-dependent teratogenicity: the case of valproic acid.
Toxicol. Sci.
PUBLISHED: 01-11-2011
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Teratogenicity can be predicted in vitro using the embryonic stem cell test (EST). The EST, which is based on the morphometric measurement of cardiomyocyte differentiation and cytotoxicity parameters, represents a scientifically validated method for the detection and classification of chemicals according to their teratogenic potency. Furthermore, an abbreviated protocol applying flow cytometry of intracellular marker proteins to determine differentiation into the cardiomyocyte lineage is available. Although valproic acid (VPA) is in worldwide clinical use as antiepileptic drug, it exhibits two severe side effects, i.e., teratogenicity and hepatotoxicity. These limitations have led to extensive research into derivatives of VPA. Here we chose VPA as model compound to test the applicability domain and to further evaluate the reliability of the EST. To this end, we study six closely related congeners of VPA and demonstrate that both the standard and the molecular flow cytometry-based EST are well suited to indicate differences in the teratogenic potency among VPA analogs that differ only in chirality or side chain length. Our data show that identical results can be obtained by using the standard EST or a shortened protocol based on flow cytometry of intracellular marker proteins. Both in vitro protocols enable to reliably determine differentiation of murine stem cells toward the cardiomyocyte lineage and to assess its chemical-mediated inhibition.
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A knowledge-based search engine to navigate the information thicket of nanotoxicology.
Regul. Toxicol. Pharmacol.
PUBLISHED: 06-16-2010
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The risk assessment of nano-sized materials (NM) currently suffers from great uncertainties regarding their putative toxicity for humans and the environment. An extensive amount of the respective original research literature has to be evaluated before a targeted and hypothesis-driven Environmental and Health Safety research can be stipulated. Furthermore, to comply with the European animal protection legislation in vitro testing has to be preferred whenever possible. Against this background, there is the need for tools that enable producers of NM and risk assessors for a fast and comprehensive data retrieval, thereby linking the 3Rs principle to the hazard identification of NM. Here we report on the development of a knowledge-based search engine that is tailored to the particular needs of risk assessors in the area of NM. Comprehensive retrieval of data from studies utilising in vitro as well as in vivo methods relying on the PubMed database is presented exemplarily with a titanium dioxide case study. A fast, relevant and reliable information retrieval is of paramount importance for the scientific community dedicated to develop safe NM in various product areas, and for risk assessors obliged to identify data gaps, to define additional data requirements for approval of NM and to create strategies for integrated testing using alternative methods.
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T-cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays.
Cell. Mol. Life Sci.
PUBLISHED: 04-22-2010
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Chemicals can elicit T-cell-mediated diseases such as allergic contact dermatitis and adverse drug reactions. Therefore, testing of chemicals, drugs and protein allergens for hazard identification and risk assessment is essential in regulatory toxicology. The seventh amendment of the EU Cosmetics Directive now prohibits the testing of cosmetic ingredients in mice, guinea pigs and other animal species to assess their sensitizing potential. In addition, the EU Chemicals Directive REACh requires the retesting of more than 30,000 chemicals for different toxicological endpoints, including sensitization, requiring vast numbers of animals. Therefore, alternative methods are urgently needed to eventually replace animal testing. Here, we summarize the outcome of an expert meeting in Rome on 7 November 2009 on the development of T-cell-based in vitro assays as tools in immunotoxicology to identify hazardous chemicals and drugs. In addition, we provide an overview of the development of the field over the last two decades.
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Mechanistic insights on spider neurotoxins.
EXS
PUBLISHED: 04-03-2010
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In physiology research, animal neurotoxins historically have served as valuable tools for identification, purification, and functional characterization of voltage-dependent ion channels. In particular, toxins from scorpions, sea anemones and cone snails were at the forefront of work aimed at illuminating the three-dimensional architecture of sodium channels. To date, at least six different receptor binding sites have been identified and--most of them--structurally assigned in terms of protein sequence and spatial disposition. Recent work on Australian funnel-web spiders identified certain peptidic ingredients as being responsible for the neurotoxicity of the crude venom. These peptides, termed delta-atracotoxins (delta-ACTX), consist of 42 amino acids and bind to voltage-gated sodium channels in the same way as classical scorpion alpha-toxins. According to the voltage-sensor trapping model proposed in the literature, delta-ACTX isoforms interact with the voltage sensor S4 transmembrane segment of alpha-subunit domain IV, thereby preventing its normal outward movement and concurrent conformational changes required for inactivation of the channel. As consequence prolonged action potentials at autonomic or somatic synapses induce massive transmitter release, resulting in clinical correlates of neuroexcitation (e.g., muscle fasciculation, spasms, paresthesia, tachycardia, diaphoresis, etc.). On the other hand, the major neurotoxin isolated from black widow spiders, alpha-latrotoxin (alpha-LTX), represents a 132 kDa protein consisting of a unique N-terminal sequence and a C-terminal part harboring multiple ankyrin-like repeats. Upon binding to one of its specific presynaptic receptors, alpha-LTX has been shown to tetramerize under physiological conditions to form Ca2+-permeable pores in presynaptic membranes. The molecular model worked out during recent years separates two distinguishable receptor-mediated effects. According to current knowledge, binding of the N terminus of alpha-LTX at one of its specific receptors either triggers intracellular signaling cascades, resulting in phospholipase C-mediated mobilization of presynaptic Ca2+ stores, or leads to the formation of tetrameric pore complexes, allowing extracellular Ca2+ to enter the presynaptic terminal. Alpha-LTX-triggered exocytosis and fulminant transmitter release at autonomic synapses may then provoke a clinical syndrome referred to as latrodectism, characterized by local and incapacitating pain, diaphoresis, muscle fasciculation, tremor, anxiety, and so forth. The present review aims at providing a short introduction into some of the exciting molecular effects induced by neurotoxins isolated from black widow and funnel-web spiders.
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High-molecular weight protein toxins of marine invertebrates and their elaborate modes of action.
EXS
PUBLISHED: 04-03-2010
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High-molecular weight protein toxins significantly contribute to envenomations by certain marine invertebrates, e.g., jellyfish and fire corals. Toxic proteins frequently evolved from enzymes meant to be employed primarily for digestive purposes. The cellular intermediates produced by such enzymatic activity, e.g., reactive oxygen species or lysophospholipids, rapidly and effectively mediate cell death by disrupting cellular integrity. Membrane integrity may also be disrupted by pore-forming toxins that do not exert inherent enzymatic activity. When targeted to specific pharmacologically relevant sites in tissues or cells of the natural enemy or prey, toxic enzymes or pore-forming toxins even may provoke fast and severe systemic reactions. Since toxin-encoding genes constitute "hot spots" of molecular evolution, continuous variation and acquirement of new pharmacological properties are guaranteed. This also makes individual properties and specificities of complex proteinaceous venoms highly diverse and inconstant. In the present chapter we portray high-molecular weight constituents of venoms present in box jellyfish, sea anemones, sea hares, fire corals and the crown-of-thorns starfish. The focus lies on the latest achievements in the attempt to elucidate their molecular modes of action.
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Investigations on the emission of fragrance allergens from scented toys by means of headspace solid-phase microextraction gas chromatography-mass spectrometry.
J Chromatogr A
PUBLISHED: 01-12-2010
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In the revised European toy safety directive 2009/48/EC the application of fragrance allergens in childrens toys is restricted. The focus of the present work lies on the instrumental analytics of 13 banned fragrance allergens, as well as on 11 fragrance allergens that require declaration when concentrations surpass 100 microg per gram material. Applying a mixture of ethyl acetate and toluene solid/liquid extraction was performed prior to quantitative analysis of mass contents of fragrances in scented toys. In addition, an easy-to-perform method for the determination of emitted fragrances at 23 degrees C (handling conditions) or at 40 degrees C (worst case scenario) has been worked out to allow for the evaluation of potential risks originating from inhalation of these compounds during handling of or playing with toys. For this purpose a headspace solid-phase microextraction (HS-SPME) technique was developed and coupled to subsequent gas chromatography-mass spectrometry (GC-MS) analysis. Fragrance allergens were adsorbed (extracted) from the gas phase onto an 85 microm polyacrylate fiber while incubating pieces of the scented toys in sealed headspace vials at 23 degrees C and 40 degrees C. Quantification of compounds was performed via external calibration. The newly developed headspace method was subsequently applied to five perfumed toys. As expected, the emission of fragrance allergens from scented toys depends on the temperature and on the content of fragrance allergens present in those samples. In particular at conditions mimicking worst case (40 degrees C), fragrance allergens in toys may pose a risk to children since considerable amounts of compound might be absorbed by lung tissue via breathing of contaminated air.
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Embryonic stem cell test remastered: comparison between the validated EST and the new molecular FACS-EST for assessing developmental toxicity in vitro.
Toxicol. Sci.
PUBLISHED: 01-23-2009
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The embryonic stem cell test (EST) represents a reliable, scientifically validated in vitro system for the detection and classification of compounds according to their teratogenic potency. However, some serious issues were frequently raised against the widespread implementation and practicability of the EST in its original version. Most importantly, the evaluation of the morphological endpoint of beating cell agglomerates requires extensive experimental experience and is prone to misjudgment. Also, the testing period of 10 days is too long and costly to be attractive for industries interested in high-throughput screening of potential drug candidates. These drawbacks prompted us to work out a new molecular approach based on analysis of the expression of certain marker proteins specific for developing heart tissue. We have previously reported that quantitative flow cytometry of marker proteins (i.e., sarcomeric myosin heavy chain and alpha-actinin) can be performed at day 7 in embryonic stem cells from mice and combined with concurrent cell viability analysis. In the present study, extensive investigations were performed in order to explore the predictive power and validity of the newly established EST, subsequently referred to as molecular fluorescence activated cell sorting (FACS)-EST, by applying and comparing a set of 10 well-known embryotoxicants that encompasses the full range of chemical inherent embryotoxic potencies possible. While the molecular FACS-EST offered the same sensitivity compared to the validated EST protocol, the test duration could be significantly reduced. Due to significant improvements, this new molecular method holds promise as a sensitive, more rapid and reproducible screen highly suited to predict developmental toxicity in vivo from in vitro data.
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On the impact of the molecule structure in chemical carcinogenesis.
EXS
PUBLISHED: 01-23-2009
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Cancer is as a highly complex and multifactorial disease responsible for the death of hundreds of thousands of people in the western countries every year. Since cancer is clonal and due to changes at the level of the genetic material, viruses, chemical mutagens and other exogenous factors such as short-waved electromagnetic radiation that alter the structure of DNA are among the principal causes. The focus of this present review lies on the influence of the molecular structure of two well-investigated chemical carcinogens from the group of polycyclic aromatic hydrocarbons (PAHs), benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP). Although there is only one additional benzo ring present in the latter compound, DBP exerts much stronger genotoxic and carcinogenic effects in certain tumor models as compared to BP. Actually, DBP has been identified as the most potent tumorigen among all carcinogenic PAHs tested to date. The genotoxic effects of both compounds investigated in mammalian cells in culture or in animal models are described. Comparison of enzymatic activation, DNA binding levels of reactive diol-epoxide metabolites, efficiency of DNA adduct repair and mutagenicity provides some clues on why this compound is about 100-fold more potent in inducing tumors than BP. The data published during the past 20 years support and strengthen the idea that compound-inherent physicochemical parameters, along with inefficient repair of certain kinds of DNA lesions formed upon metabolic activation, can be considered as strong determinants for high carcinogenic potency of a chemical.
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Development of a manual method for the determination of mineral oil in foods and paperboard.
J Chromatogr A
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So far the majority of the measurements of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) were obtained from on-line high performance liquid chromatography-gas chromatography-flame ionization detection (on-line HPLC-GC-FID). Since this technique is not available in many laboratories, an alternative method with more easily available tools has been developed. Preseparation on a small conventional liquid chromatographic column was optimized to achieve robust separation between the MOSH and the MOAH, but also to keep out the wax esters from the MOAH fraction. This was achieved by mixing a small portion of silica gel with silver nitrate into highly activated silica gel and by adding toluene into the eluent for the MOAH. Toluene was also added to the MOSH fraction to facilitate reconcentration and to serve as a keeper preventing loss of volatiles during solvent evaporation. A 50 ?l volume was injected on-column into GC-FID to achieve a detection limit for MOSH and MOAH below 1 mg/kg in most foods.
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Esterase activity in excised and reconstructed human skin--biotransformation of prednicarbate and the model dye fluorescein diacetate.
Eur J Pharm Biopharm
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Reconstructed human epidermis (RHE) is used in non-animal testing for hazard analysis and reconstructed human skin (RHS) gains growing interest in preclinical drug development. RHE and RHS have been characterised regarding their barrier function, but knowledge about biotransformation capacity in these constructs and in human skin remains rather poor. However, metabolising enzymes can be highly relevant for the efficacy of topical dermatics as well as genotoxicity and sensitisation. We have compared the esteratic cleavage of the prednisolone diester prednicarbate and the enzyme kinetic parameters (Vmax and S0.5) of the model substrate fluorescein diacetate (FDA) in commercially available RHS and RHE with excised human skin and monolayer cultures of normal and immortalised human keratinocytes and of fibroblasts. Formation of the main metabolite prednisolone and of fluorescein ranked as: RHS~RHE>excised human skin and keratinocytes>fibroblasts, respectively. Because of the aromatic probe, however, Vmax of FDA cleavage did not show a linear relationship with prednicarbate metabolism. In conclusion, RHE and RHS may be useful to quantitatively address esterase activity of human skin in drug development and hazard analysis, although an increased activity compared to native human skin has to be taken into account.
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Metabolically competent human skin models: activation and genotoxicity of benzo[a]pyrene.
Toxicol. Sci.
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The polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BP) is metabolized into a complex pattern of BP derivatives, among which the ultimate carcinogen (+)-anti-BP-7,8-diol-9,10-epoxide (BPDE) is formed to certain extents. Skin is frequently in contact with PAHs and data on the metabolic capacity of skin tissue toward these compounds are inconclusive. We compared BP metabolism in excised human skin, commercially available in vitro 3D skin models and primary 2D skin cell cultures, and analyzed the metabolically catalyzed occurrence of seven different BP follow-up products by means of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). All models investigated were competent to metabolize BP, and the metabolic profiles generated by ex vivo human skin and skin models were remarkably similar. Furthermore, the genotoxicity of BP and its derivatives was monitored in these models via comet assays. In a full-thickness skin, equivalent BP-mediated genotoxic stress was generated via keratinocytes. Cultured primary keratinocytes revealed a level of genotoxicity comparable with that of direct exposure to 50-100 nM of BPDE. Our data demonstrate that the metabolic capacity of human skin ex vivo, as well as organotypic human 3D skin models toward BP, is sufficient to cause significant genotoxic stress and thus cutaneous bioactivation may potentially contribute to mutations that ultimately lead to skin cancer.
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Human Langerhans cells control Th cells via programmed death-ligand 1 in response to bacterial stimuli and nickel-induced contact allergy.
PLoS ONE
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Langerhans cells (LCs) are suspected to initiate inflammatory immune responses to contact allergens and pathogenic bacteria. In chronic infectious diseases, programmed death ligand (PD-L) 1 exhibits both inhibitory and costimulatory functions on T cell-mediated activation and tolerance. Here, we investigated the effects of contact allergens and bacterial stimuli on PD-L1 expression in LCs and the effects of altered PD-L1 expression on cytokine release of subsequently cocultured T cells. Monocyte-derived LCs (MoLCs), LCs, and skin sections of patients suffering from allergic contact dermatitis were challenged with nickel and then analyzed for PD-L1 expression by confocal laser scanning microscopy and flow cytometry. In blocking experiments, we found that the release of Th cell specific cytokines was dependent on both stimulation of LCs and inhibition of PD-L1-PD-1 interactions. Stimulation with peptidoglycan (PGN) or lipopolysaccharide (LPS) and blockage of PD-L1 with a specific antibody triggered the release of high levels of IL-17, IL-22, TNF-?, and IFN-? in CD4(+)T cells. If nickel was used as a stimulus, blockage of PD-L1 led to high amounts of TNF-? and IL-22. A closer look revealed PD-L1-dependent upregulation of IL-17 secretion in FACS-sorted CCR6(+)/CCR4(+) T memory cells. In the presence of anti-PD-L1, PGN induced secretion of IFN-? and IL-17 in total CCR6(+) cells, while nickel triggered secretion of IFN-? and IL-17 exclusively in CCR6(+)/CCR4(+) cells. Our findings suggest that PD-L1 on LCs plays a crucial role in type IV allergic reactions and in response to bacterial stimuli by controlling the nature of inflammatory Th cell responses.
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Risk assessment of nanomaterials in cosmetics: a European union perspective.
Arch. Toxicol.
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In Europe, the data requirements for the hazard and exposure characterisation of chemicals are defined according to the REACH regulation and its guidance on information requirements and chemical safety assessment (Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), and its guidance documents; available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2006:396:0001:0849:EN:PDF ; and at: http://guidance.echa.europa.eu/docs/guidance_document/information_requirements_en.htm ). This is the basis for any related risk assessment. The standard reference for the testing of cosmetic ingredients is the SCCPs Notes of Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation (The SCCPs Notes of Guidance for the testing of cosmetic ingredients and their safety evaluation (2006); available at: http://ec.europa.eu/health/ph_risk/committees/04_sccp/docs/sccp_o_03j.pdf ), which refers to the OECD guidelines for the testing of chemicals (The OECD Guidelines for the Testing of Chemicals as a collection of the most relevant internationally agreed testing methods used by government, industry and independent laboratories to assess the safety of chemical products; available at: http://www.oecd.org/topic/0,2686,en_2649_34377_1_1_1_1_37407,00.html ). According to the cosmetics directive [76/768/EEC], compounds that are classified as mutagenic, carcinogenic or toxic to reproduction are banned for the use in cosmetic products. Since December 2010, the respective labelling is based on the rules of regulation (EC) No. 1272/2008 (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006, Official Journal L 353, 31/12/2008, pages 1-1355; available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:353:0001:1355:en:PDF ) on classification, labelling and packaging of substances and mixtures (CLP). There is no further impact from the CLP regulation on cosmetic products, because regulation (EC) No. 1223/2009 on cosmetic products defines its own labelling rules (Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products; available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:342:0059:0209:en:PDF ). Special notification procedures are mandatory for preservatives, colourants and UV-filters where a safety approval from the European Scientific Committee on Consumer Safety (SCCS) is needed prior to marketing. The risk assessment of nanomaterials in consumer products still poses a significant challenge as highlighted by the example of UV-filters in sunscreens since nanomaterials cannot be classified as a homogenous group of chemicals but still need to be addressed in risk characterisation on a case by case basis.
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"Drugs on oxygen": an update and perspective on the role of cytochrome P450 testing in pharmacology.
Expert Opin Drug Metab Toxicol
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Low hit rates for lead compounds and high attrition remain a major problem for drug development. The reasons for compound failure range from poor pharmacokinetics to toxic metabolites and adverse drug interactions; all of which are frequently mediated by cytochrome P450-dependent monooxygenases (CYPs). However, despite some 30 years of assay development and refinement, CYP metabolism remains a critical issue during drug development. While current testing strategies succeed in characterizing single substance toxicity, they are challenged by practical issues such as assay standardization or complex scenarios such as multidrug usage. This editorial summarizes where we stand and highlights the major challenges we face with CYPs in drug development today. The article also tries to spell out the future direction of CYP testing. The latter will depend on the extended inclusion of polypharmacy into testing strategies, as well as on our capability to make use of upcoming complex in vitro test systems and their inclusion into tiered testing strategies.
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On the role of low-dose effects and epigenetics in toxicology.
EXS
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For a long time, scientists considered genotoxic effects as the major issue concerning the influence of environmental chemicals on human health. Over the last decades, a new layer superimposed the genome, i.e., the epigenome, tremendously changing this point of view. The term "epigenetics" comprises stable alterations in gene expression potential arising from variations in DNA methylation and a variety of histone modifications, without changing the underlying DNA sequence. Recently, also gene silencing by small noncoding RNAs (ncRNAs), in particular by microRNAs, was included in the list of epigenetic mechanisms. Multiple studies in vivo as well as in vitro have shown that a multitude of different environmental factors are capable of changing the epigenetic pattern as well as miRNA expression in certain cell types, leading to aberrant gene expression profiles in cells and tissues. These changes may have extensive effects concerning the proper gene expression necessary in a specified cell type and can even lead into a state of disease. Especially the roles of epigenetic modifications and miRNA alterations in tumorigenesis have been a major focus in research over the last years. This chapter will give an overview on epigenetic features and on the spectrum of epigenetic changes observed after exposure against environmental chemicals and pollutants.
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A personalized life: biomarker monitoring from cradle to grave.
EXS
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Considering the holy grail of future medical treatment being personalized medicines, biomarker research will become more and more the focus for attention not only to develop new medical treatment regimes, based on changes in biomarker patterns, but also for nutritional advice to guarantee a lifelong optimized health condition. The current review gives an outline of how personalized medicine can become established for actual medical treatment using new biomarker concepts. Starting from the development of biomarker research using mainly immunological techniques, the review gives an overview about biomarkers of prediction evolved and focuses on new methodology for the identification of biomarkers using hyphenated analytical techniques like metabolomics and lipidomics. The actual use of multivariate statistical methods in combination with metabolomics and lipidomics is discussed not only for medical treatment but also for precautionary risk identification in human biomonitoring studies.
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Nanomaterials: a challenge for toxicological risk assessment?
EXS
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Nanotechnology has emerged as one of the central technologies in the twenty-first century. This judgment becomes apparent by considering the increasing numbers of people employed in this area; the numbers of patents, of scientific publications, of products on the market; and the amounts of money invested in R&D. Prospects originating from different fields of nanoapplication seem unlimited. However, nanotechnology certainly will not be able to meet all of the ambitious expectations communicated, yet has high potential to heavily affect our daily life in the years to come. This might occur in particular in the field of consumer products, for example, by introducing nanomaterials in cosmetics, textiles, or food contact materials. Another promising area is the application of nanotechnology in medicine fueling hopes to significantly improve diagnosis and treatment of all kinds of diseases. In addition, novel technologies applying nanomaterials are expected to be instrumental in waste remediation and in the production of efficient energy storage devices and thus may help to overcome worlds energy problems or to revolutionize computer and data storage technologies. In this chapter, we will focus on nanomaterials. After a brief historic and general overview, current proposals of how to define nanomaterials will be summarized. Due to general limitations, there is still no single, internationally accepted definition of the term "nanomaterial." After elaborating on the status quo and the scope of nanoanalytics and its shortcomings, the current thinking about possible hazards resulting from nanoparticulate exposures, there will be an emphasis on the requirements to be fulfilled for appropriate health risk assessment and regulation of nanomaterials. With regard to reliable risk assessments, until now there is still the remaining issue to be resolved of whether or not specific challenges and unique features exist on the nanoscale that have to be tackled and distinctively addressed, given that they substantially differ from those encountered with microsized materials or regular chemicals. Based on the current knowledge, we finally provide a proposal on how risk assessment in the nanofield could be achieved and how it might look like in the near future.
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Exposure to polycyclic aromatic hydrocarbons: bulky DNA adducts and cellular responses.
EXS
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Environmental and dietary carcinogens such as polycyclic aromatic hydrocarbons (PAHs) have been intensively studied for decades. Although the genotoxicity of these compounds is well characterized (i.e., formation of bulky PAH-DNA adducts), molecular details on the DNA damage response triggered by PAHs in cells and tissues remain to be clarified. The conversion of hazardous PAHs into carcinogenic intermediates depends on enzyme-catalyzed biotransformation. Certain cytochrome P450-dependent monooxygenases (CYPs) play a pivotal role in PAH metabolism. In particular, CYP1A1 and 1B1 catalyze oxidation of PAHs toward primary epoxide species that can further be converted into multiple follow-up products, both nonenzymatically and enzymatically. Distinct functions between these major CYP enzymes have only been appreciated since transgenic animal models had been derived. Electrophilic PAH metabolites are capable of forming stable DNA adducts or to promote depurination at damaged nucleotide sites. During the following DNA replication cycle, bulky PAH-DNA adducts may be converted into mutations, thereby affecting hot spot sites in regulatory important genes such as Ras, p53, and others. Depending on the degree of DNA distortion and cell cycle progression, PAH-DNA adducts trigger nucleotide excision repair (NER) and various DNA damage responses that might include TP53-dependent apoptosis in certain cell types. In fact, cellular responses to bulky PAH-DNA damage are complex because distinct signaling branches such as ATM/ATR, NER, TP53, but also MAP kinases, interact and cooperate to determine the overall outcome to cellular injuries initiated by PAH-DNA adducts. Further, PAHs and other xenobiotics can also confer DNA damage via an alternative route of metabolic activation, which leads to the generation of PAH semiquinone radicals and reactive oxygen species (ROS). One-electron oxidations mediated by peroxidases or other enzymes can result in PAH radical cations that mainly form unstable DNA adducts subjected to depurination. In addition, generation of ROS can also trigger multiple cellular signaling pathways not directly related to mutagenic or cytotoxic effects, including those mediated by NF?B, SAPK/JNK, and p38. In recent years, it became clear that PAHs may also be involved in inflammatory diseases, autoimmune disorders, or atherosclerosis. Further research is under way to better characterize the significance of such newly recognized systemic effects of PAHs and to reconsider risk assessment for human health.
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Toxicologically relevant phthalates in food.
EXS
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Various phthalates have been detected in a wide range of food products such as milk, dietary products, fat-enriched food, meat, fish, sea food, beverages, grains, and vegetables as well as in breast milk. Here we present an overview on toxicologically considerable phthalate levels in food reported in the literature. The most common phthalates detected are di-(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DnBP), and di-isobutyl phthalate (DiBP). Milk analyses demonstrate that background levels in unprocessed milk are usually low. However, during processing the phthalate contents may significantly increase due to migration from plastic materials in contact with food. Among dietary products fat-enriched food such as cheese and cream were identified with highest levels of DEHP. Plasticized PVC from tubes, conveyor belts, or disposable gloves used in food processing is an important source for contamination of food, especially of fatty food. Paper and cardboard packaging made from recycled fibers are another important source of contamination. In addition, gaskets used in metal lids for glass jars have been identified as possible source for the contamination of foodstuffs with phthalates. The highest concentrations of DEHP reported (>900 mg kg(-1)) were detected in food of high fat content stored in such glass jars. Beyond classical food, DEHP and DnBP were identified in human breast milk samples as the main phthalate contaminants. Phthalate monoesters and some oxidative metabolites were also quantified in breast milk.
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Wind of change challenges toxicological regulators.
Environ. Health Perspect.
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In biomedical research, the past two decades have seen the advent of in vitro model systems based on stem cells, humanized cell lines, and engineered organotypic tissues, as well as numerous cellular assays based on primarily established tumor-derived cell lines and their genetically modified derivatives.
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Neural differentiation of mouse embryonic stem cells as a tool to assess developmental neurotoxicity in vitro.
Neurotoxicology
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Mouse embryonic stem cells (mESCs) represent an attractive cellular system for in vitro studies in developmental biology as well as toxicology because of their potential to differentiate into all fetal cell lineages. The present study aims to establish an in vitro system for developmental neurotoxicity testing employing mESCs. We developed a robust and reproducible protocol for fast and efficient differentiation of the mESC line D3 into neural cells, optimized with regard to chemical testing. Morphological examination and immunocytochemical staining confirmed the presence of different neural cell types, including neural progenitors, neurons, astrocytes, oligodendrocytes, and radial glial cells. Neurons derived from D3 cells expressed the synaptic proteins PSD95 and synaptophysin, and the neurotransmitters serotonin and ?-aminobutyric acid. Calcium ion imaging revealed the presence of functionally active glutamate and dopamine receptors. In addition, flow cytometry analysis of the neuron-specific marker protein MAP2 on day 12 after induction of differentiation demonstrated a concentration dependent effect of the neurodevelopmental toxicants methylmercury chloride, chlorpyrifos, and lead acetate on neuronal differentiation. The current study shows that D3 mESCs differentiate efficiently into neural cells involving a neurosphere-like state and that this system is suitable to detect adverse effects of neurodevelopmental toxicants. Therefore, we propose that the protocol for differentiation of mESCs into neural cells described here could constitute one component of an in vitro testing strategy for developmental neurotoxicity.
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Waterpipe smoking: the role of humectants in the release of toxic carbonyls.
Arch. Toxicol.
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In recent years, the number of waterpipe smokers has increased substantially worldwide. Here, we present a study on the identification and quantification of seven carbonylic compounds including formaldehyde, acetaldehyde and acrolein in the mainstream smoke of the waterpipe. Smoking was conducted with a smoking machine, and carbonyls were scavenged from the smoke with two impingers containing an acidic solution of 2,4-dinitrophenylhydrazine. The derivatives were then analyzed by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). For instance, during one waterpipe smoking session, up to 111 ± 12 ?g formaldehyde could be detected. This value is about 5 times higher when compared to one 2R4F reference cigarette. We also found a distinct filter effect of the bowl water for all carbonyls investigated. Our data further demonstrate that increasing amounts of humectants in the unburned tobacco lowers the temperature in the waterpipe head during smoking, thereby resulting in decreasing levels of carbonyls in the smoke produced. Altogether, considerable amounts of toxic carbonyls are present in the waterpipe smoke, thus conferring a health risk to waterpipe smokers.
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Drug-mediated toxicity: illuminating the bad in the test tube by means of cellular assays?
Trends Pharmacol. Sci.
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Health problems are rising worldwide, be it as a consequence of lifestyle and longevity in increasingly affluent societies or due to a sharp rise in bacterial antibiotic resistance. The pharmaceutical industry is caught between high rates of attrition and the rather slow pace of a historically large regulatory system for pharmacological safety. Meanwhile, the past decade has seen a tremendous evolution of the biological toolbox, most notably of cellular assays, stem-cell differentiation and organ-mimicking systems. These systems were readily adapted for lead-compound identification. However, their use as toxicological test systems is lagging behind, not least because of a lack of regulatory acceptance. This review tries to elucidate the scale of the problem and discusses the applicability of the assays currently available, with particular regard to the use of stem cells.
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Assaying embryotoxicity in the test tube: current limitations of the embryonic stem cell test (EST) challenging its applicability domain.
Crit. Rev. Toxicol.
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Testing for embryotoxicity in vitro is an attractive alternative to animal experimentation. The embryonic stem cell test (EST) is such a method, and it has been formally validated by the European Centre for the Validation of Alternative Methods. A number of recent studies have underscored the potential of this method. However, the EST performed well below the 78% accuracy expected from the validation study using a new set of chemicals and pharmaceutical compounds, and also of toxicity criteria, tested to enlarge the database of the validated EST as part of the Work Package III of the ReProTect Project funded within the 6th Framework Programme of the European Union. To assess the performance and applicability domain of the EST we present a detailed review of the substances and their effects in the EST being nitrofen, ochratoxin A, D-penicillamine, methylazoxymethanol, lovastatin, papaverine, warfarin, ?-aminopropionitrile, dinoseb, furosemide, doxylamine, pravastatin, and metoclopramide. By delineation of the molecular mechanisms of the substances we identify six categories of reasons for misclassifications. Some of these limitations might also affect other in vitro methods assessing embryotoxicity. Substances that fall into these categories need to be included in future validation sets and in validation guidelines for embryotoxicity testing. Most importantly, we suggest conceivable improvements and additions to the EST which will resolve most of the limitations.
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CYP1B1 detection.
Curr Protoc Toxicol
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This unit describes procedures for measuring CYP1B1 gene expression by reverse transcription real-time PCR (qRT-PCR), CYP1B1 protein levels by western blotting, and CYP1B1 enzyme activity through conversion of 7-ethoxyresorufin substrate. To achieve specific measurement of CYP1B1 activity in the presence of CYP1A1 and CYP1A2, CYP1B1 inhibition and a subtractive approach have been adopted. 2,4,3,5-Tetramethoxystilbene (TMS) is a potent and selective competitive inhibitor of CYP1B1 with an IC?? of 3 nM for EROD and ~90 nM for E2 4-hydroxylation. Binding studies with purified CYP1B1 suggests that TMS interferes in the proximity of the heme region of CYP1B1 with high affinity. Compared to other potent inhibitors such as ?-naphthoflavone, which is a known CYP1 family inhibitor with no selectivity between CYP1B1 and CYP1A2, TMS is ~50- and 520-fold selective for inhibition of CYP1B1 when compared to CYP1A1 and CYP1A2, respectively. Thus, TMS can serve as a helpful chemical scalpel for dissecting CYP1B1 activity from the overall activity of CYP1 family members against ethoxyresorufin.
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Effects of silver nanoparticles on primary mixed neural cell cultures: uptake, oxidative stress and acute calcium responses.
Toxicol. Sci.
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In the body, nanoparticles can be systemically distributed and then may affect secondary target organs, such as the central nervous system (CNS). Putative adverse effects on the CNS are rarely investigated to date. Here, we used a mixed primary cell model consisting mainly of neurons and astrocytes and a minor proportion of oligodendrocytes to analyze the effects of well-characterized 20 and 40 nm silver nanoparticles (SNP). Similar gold nanoparticles served as control and proved inert for all endpoints tested. SNP induced a strong size-dependent cytotoxicity. Additionally, in the low concentration range (up to 10 ?g/ml of SNP), the further differentiated cultures were more sensitive to SNP treatment. For detailed studies, we used low/medium dose concentrations (up to 20 ?g/ml) and found strong oxidative stress responses. Reactive oxygen species (ROS) were detected along with the formation of protein carbonyls and the induction of heme oxygenase-1. We observed an acute calcium response, which clearly preceded oxidative stress responses. ROS formation was reduced by antioxidants, whereas the calcium response could not be alleviated by antioxidants. Finally, we looked into the responses of neurons and astrocytes separately. Astrocytes were much more vulnerable to SNP treatment compared with neurons. Consistently, SNP were mainly taken up by astrocytes and not by neurons. Immunofluorescence studies of mixed cell cultures indicated stronger effects on astrocyte morphology. Altogether, we can demonstrate strong effects of SNP associated with calcium dysregulation and ROS formation in primary neural cells, which were detectable already at moderate dosages.
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Degradation of benzo[a]pyrene by bacterial isolates from human skin.
FEMS Microbiol. Ecol.
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Polycyclic aromatic hydrocarbons (PAHs) are some of the most widespread xenobiotic pollutants, with the potentially carcinogenic high molecular weight representatives being of particular interest. However, while in eukaryotes the cytochrome P450-mediated activation of benzo[a]pyrene (B[a]P) has become a model for metabolism-mediated carcinogenesis, the oxidative degradation of B[a]P by microbes is less well studied. This should be reason for concern as the human organ most exposed to environmental PAHs is the skin, which at the same time is habitat to a most diverse population of microbial commensals. Yet, nothing is known about the skins microbiome potential to metabolise B[a]P. This study now reports on the isolation of 21 B[a]P-degrading microbes from human skin, 10 of which were characterised further. All isolates were able to degrade B[a]P as sole source of carbon and energy and degradation was found to be complete in at least 4 isolates. Substrate metabolism involved two transcripts that encode a putative DszA/NtaA-like monooxygenase and a NifH-like reductase, respectively. Analysis of the 16S-rRNA genes showed that the B[a]P-degrading isolates comprise Gram(+) as well as Gram(-) skin commensals, with Micrococci being predominant. Moreover, microbial B[a]P degradation was detected on all volunteers probed, indicating it to be a universal feature of the skins microbiome. This article is protected by copyright. All rights reserved.
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