Minor structural modifications of bisphenol A strongly affect physiological responses of HepG2 cells.

Bisphenols represent a large group of structurally similar compounds. In contrast to bisphenol A (BPA) and bisphenol S (BPS), however, toxicological data are usually scarce, thus making bisphenols an ideal candidate for read-across assessments. BPA, bisphenol C (BPC) and a newly synthesized bisphenol A/C (BPA/C) differ only by one methyl group attached to the phenolic ring. Their EC50 values for cytotoxicity and logPOW values are comparable. However, the estrogenic activities of these bisphenols are not comparable and among this group only BPC leads to a decrease of the mitochondrial membrane potential and ATP concentration in HepG2 cells. Conversely, the cell division rate was decreased by BPS, BPA, BPC and BPA/C at 10% toxicity (EC10). At lower concentrations, only BPC significantly affected proliferation. The pro-inflammatory cytokines TGFB1 and TNF were significantly upregulated by BPC only, while SPP1 was upregulated by BPA, BPA/C and BPS. BPC led to the release of cytochrome c from mitochondria, indicating that this compound is capable of inducing apoptosis. In conclusion, the read-across approach revealed non-applicable in the case of the various structurally and physicochemically comparable bisphenols tested in this study, as the presence of one or two additional methyl group(s) attached at the phenol ring profoundly affected cellular physiology.

Nuclear transport of the human aryl hydrocarbon receptor and subsequent gene induction relies on its residue histidine 291.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor involved in the metabolism of physiological substances and xenobiotics, representing an interesting target in both toxicology and pharmacology. In this study, we investigated the ligand-dependent conjunction of nuclear import of the human AHR in living cells and target gene induction. Our findings strengthen the theory that the AHR triggers a precisely defined and rapid reaction upon binding to endogenous ligands, while the xenobiotic ß-naphthoflavone only induces rather unspecific and slow effects. To better illuminate the ligand-mediated responses of the human AHR, we applied site-directed mutagenesis and identified histidine 291 as key residue for AHR functionality, essential for both nuclear import and target gene induction. Contrary, replacing histidine at position 291 by alanine did not affect nucleo-cytoplasmic shuttling, showing that permanent endogenous import and ligand-induced import of the AHR into the nucleus are two independent and differently regulated processes. Combining these observations with our structural investigations using a homology model of the AHR-PAS B domain, we suggest a dual role of histidine 291: (1) a major role for shaping the ligand binding site including direct interactions with ligands and, (2) an essential role for the conformational dynamics of a PAS B loop, which most likely influences the association of the AHR with the AHR nuclear translocator through interference with their protein-protein interface.

Chemical stabilization of polymers: Implications for dermal exposure to additives.

Technical benefits of additives in polymers stand in marked contrast to their associated health risks. Here, a multi-analyte method based on gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) was developed to quantify polymer additives in complex matrices such as low-density polyethylene (LDPE) and isolated human skin layers after dermal exposure ex vivo. That way both technical aspects and dermal exposure were investigated. The effects of polymer additivation on the material were studied using the example of LDPE. To this end, a tailor-made polymer was applied in aging studies that had been furnished with two different mixtures of phenol- and diarylamine-based antioxidants, plasticizers and processing aids. Upon accelerated thermo-oxidative aging of the material, the formation of LDPE degradation products was monitored with attenuated total reflectance-Fourier transformed infrared (ATR-FTIR) spectroscopy. Compared to pure LDPE, a protective effect of added antioxidants could be observed on the integrity of the polymer. Further, thermo-oxidative degradation of the additives and its kinetics were investigated using LDPE or squalane as matrix. The half-lives of additives in both matrices revealed significant differences between the tested additives as well as between LDPE and squalane. For instance, 2-tert-butyl-6-[(3-tert-butyl-2-hydroxy-5-methylphenyl)methyl]-4-methylphenol (Antioxidant 2246) showed a half-life 12 times lower when incorporated in LDPE as compared to squalane. As a model for dermal exposure of consumers, human skin was brought into contact with the tailor-made LDPE containing additives ex vivo in static Franz diffusion cells. The skin was then analyzed for additives and decomposition products. This study proved 10 polymer additives of diverse pysicochemical properties and functionalities to migrate out of the polymer and eventually overcome the intact human skin barrier during contact. Moreover, their individual distribution within distinct skin layers was demonstrated. This is exemplified by the penetration of the procarcinogenic antioxidant N-phenylnaphthalen-2-amine (Neozon D) into the viable epidermis and the permeation through the skin of the neurotoxic plasticizer N-butylbenzenesulfonamide (NBBS). In addition, the analyses of additive degradation products in the isolated skin layers revealed the presence of 2-tert-butyl-4-methylphenol in all layers after contact to a polymer with substances of origin like Antioxidant 2246. Thus, attention needs to be paid to absorption of polymer additives together with their degradation products when it comes to dermal exposure assessment.

A simple metal staining procedure for identification and visualization of single cells by LA-ICP-MS.

High lateral resolution of metal detection in single cells by use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) demands powerful staining methods. In this work different staining procedures for the single cell analysis with LA-ICP-MS were optimized. An iridium intercalator was utilized to stain the cell nuclei whereas the whole cell was stained by the use of maleimido-mono-amide-DOTA (mDOTA) complexing lanthanide(iii) ions. The content of the artificially introduced metals per cell was quantified using a matrix matched calibration approach based on cellulose membranes onto which standards were spotted by a microarray spotter. Absolute metal stain amounts in the range of 2.34 to 9.81 femtomole per cell were determined. The metal staining procedures allow direct identification and visualization of single cells and their cell compartments by element microscopy without the use of bright field images of the sample.

Skin permeation of polycyclic aromatic hydrocarbons: A solvent-based in vitro approach to assess dermal exposures against benzo[a]pyrene and dibenzopyrenes.

Consumer products with high contents of polycyclic aromatic hydrocarbons (PAHs) were repeatedly identified by market surveillance authorities. Since several of the individual compounds have been identified as genotoxic carcinogens, there might be health risks associated with the usage of these items. It therefore becomes reasonable to argue to reduce PAH contents in consumer products to a level as low as possible. This study presents data on the migration of PAHs from consumer products into aqueous sweat simulant or aqueous ethanol and on its combined migration and penetration into human skin. Product specimens were either submerged in simulant, or placed directly on test skins in Franz cell chambers to simulate dermal contacts. Migration of hexacyclic dibenzopyrenes became detectable by using ethanolic simulant, but not in aqueous sweat simulant. Similarly, migration of the pentacyclic model carcinogen benzo[a]pyrene (B[a]P) into aqueous sweat simulant was significantly lower when compared with human skin or skin models. The results point to a gross underestimation (about two orders of magnitude) when using aqueous sweat simulant instead of human skin for assessing PAH migration. On the other side, the usage of 20% ethanol as simulant revealed good agreement to the actual exposure of human skin against B[a]P migrating out of contaminated products. Our results underline that aqueous sweat simulant is not suitable to study dermal migration of highly lipophilic compounds.

Allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro methods and regulatory aspects. Current knowledge assembled at an international workshop at BfR, Germany.

Contact allergies are complex diseases, and one of the important challenges for public health and immunology. The German 'Federal Institute for Risk Assessment' hosted an 'International Workshop on Contact Dermatitis'. The scope of the workshop was to discuss new discoveries and developments in the field of contact dermatitis. This included the epidemiology and molecular biology of contact allergy, as well as the development of new in vitro methods. Furthermore, it considered regulatory aspects aiming to reduce exposure to contact sensitisers. An estimated 15-20% of the general population suffers from contact allergy. Workplace exposure, age, sex, use of consumer products and genetic predispositions were identified as the most important risk factors. Research highlights included: advances in understanding of immune responses to contact sensitisers, the importance of autoxidation or enzyme-mediated oxidation for the activation of chemicals, the mechanisms through which hapten-protein conjugates are formed and the development of novel in vitro strategies for the identification of skin-sensitising chemicals. Dendritic cell cultures and structure-activity relationships are being developed to identify potential contact allergens. However, the local lymph node assay (LLNA) presently remains the validated method of choice for hazard identification and characterisation. At the workshop the use of the LLNA for regulatory purposes and for quantitative risk assessment was also discussed.

A novel type of silver nanoparticles and their advantages in toxicity testing in cell culture systems.

Silver nanoparticles (SNPs) are among the most commercialized nanoparticles worldwide. Often SNP are used because of their antibacterial properties. Besides that they possess unique optic and catalytic features, making them highly interesting for the creation of novel and advanced functional materials. Despite its widespread use only little data exist in terms of possible adverse effects of SNP on human health. Conventional synthesis routes usually yield products of varying quality and property. It thus may become puzzling to compare biological data from different studies due to the great variety in sizes, coatings or shapes of the particles applied. Here, we applied a novel synthesis approach to obtain SNP of well-defined colloidal and structural properties. Being stabilized by a covalently linked small peptide, these particles are nicely homogenous, with narrow size distribution, and form monodisperse suspensions in aqueous solutions. We applied these peptide-coated SNP in two different sizes of 20 or 40 nm (Ag20Pep and Ag40Pep) and analyzed responses of THP-1-derived human macrophages while being exposed against these particles. Gold nanoparticles of similar size and coating (Au20Pep) were used for comparison. The cytotoxicity of particles was assessed by WST-1 and LDH assays, and the uptake into the cells was confirmed via transmission electron microscopy. In summary, our data demonstrate that this novel type of SNP is well suited to serve as model system for nanoparticles to be tested in toxicological studies in vitro.

[Assessment of the sensitizing potency of cosmetic ingredients and commodities. How will the ingredients of cosmetics and commodities be tested in Europe today and tomorrow?]. / Bewertung des sensibilisierenden Potenzials von Stoffen in Kosmetika und Bedarfsgegenständen. Wie werden Inhaltsstoffe von Kosmetika und Bedarfsgegenständen heute und morgen in Europa getestet?

Cosmetics and certain commodities are applied or used by consumers directly on the skin. Creams may remain on the skin for longer periods, hair is dyed multiple times per year, nickel ions can be released from studs and piercings in areas of skin damage or migrate from toy materials into the skin of children. Accordingly, using or handling such products always entails a risk for developing a contact allergy. Moreover, daily usage and repeated contacts to certain cosmetics and commodities might lead to repeated elicitation of contact eczema in people already sensitized against allergenic ingredients. Unfortunately, contact allergy is not curable. For the assessment of the allergenic potential of chemicals, only testing based on animal experiments was available in the past. In 2003, the 7(th) amendment of the Cosmetics Directive 76/768/EWG laid down a ban on animal testing of cosmetic ingredients and from 2013 a general marketing ban of such products as well. Therefore, the development and validation of non-animal methods for assessing the toxicological endpoint sensitization/allergenic potency of chemicals is a major task for the years ahead and remains equally a challenge for industry and regulatory agencies.

Inhalation exposure of children to fragrances present in scented toys.

UNLABELLED: When utilized in the perfuming of children's toys, fragrances capable of inducing contact allergy in human skin may also become bioavailable to children via the inhalation route. The aim of this study was to determine the area-specific emission rates of 24 fragrances from a plasticized PVC reference material that was meant to mimic a real plastic toy. This material was introduced into an emission chamber for 28 days at handling conditions or at worst-case conditions. As a result, fragrances can be separated into three categories according to their emission rates ranging from 0.0041 to 16.2 mg/m² × h, i.e., highly volatile, semivolatile, and low-volatile compounds. Compounds of the first and second categories were monitored with decreasing emission rates. Substances of the third category were detected with increasing emission rates over time. Further, higher temperatures led to higher emission rates. The emission concentration of fragrances from four real scented toys varied between 1.10 and 107 µg/m³ at day 1 in the test chamber. Therefore, short-term inhalation exposure to fragrances originating from toys was in the range of 0.53-2700 ng/kg BW/d for the children of age 1 and older. Long-term exposure to these fragrances was calculated in the range of 2.2-220 ng/kg BW/d. PRACTICAL IMPLICATIONS: Besides household products and cosmetics, fragrances can be found in toys for children. Some fragrances are known contact allergens in the skin, but there is a lack of information on their effects in the human respiratory tract. Here, we analyzed and categorized fragrances present in a plasticized PVC reference material according to their emission profiles and volatility. We also demonstrate that volatile fragrances are being emitted from real toys and thus may get inhaled under consumer conditions to different extents.

A prospective whole-mixture approach to assess risk of the food and chemical exposome.

Many widely used chemicals result in ubiquitous human exposure from multiple sources, including diet. Legislation mainly deals with the toxicological evaluation of single substances owing to a methodological and conceptual lack of alternatives, and does so within defined silos subject to over 40 distinct regulations in the EU alone. Furthermore, much of the research and many of the initiatives concerned with the assessment and evaluation of chemical mixtures and their potential effects on human health rely on retrospective analysis. Here we propose an approach for the prospective identification, assessment and regulation of mixtures relevant to human health. We address two distinct aspects of toxicology-which chemicals actually do occur together, and how potential mixture-related health hazards can be predicted-with an adapted concept of the exposome and large-scale hazard screens. The proactive use of the likelihood of co-exposure, together with the new approach of methods-based testing, may be a timely and feasible way of identifying those substances and mixtures where hazards may have been overlooked and regulatory action is needed. Ideally, we would generate co-exposure patterns for specific consumer groups, depending on lifestyle and dietary habits, to assess the specific risk of identified mixtures.

Phosphine fumigation - Time dependent changes in the volatile profile of table grapes.

Industrial and agricultural goods are fumigated in transport containers in order to control pest infestations and to avoid the transmission of alien species. Phosphine is increasingly used prior to the export as fumigant for table grapes, fruit cultures and dried fruits to control active table grapevine insect pests. Less knowledge exists for fumigants about the desorption time of toxic gases and factors that affect the composition of the fumigated good. Therefore, red and white table grapes (´Thompson seedless´, ´Scarlotta´ and ´Flame seedless´) were chosen to represent the allowed group of phosphine fumigated foods and were treated with a concentration of 2000 vpm phosphine (PH3) at different temperatures. In the present study, sorption and desorption behavior of PH3 by table grapes and possible changes in their VOC (volatile organic compounds) profiles were investigated. The PH3 concentration was monitored before and after the fumigation process and was determined under the maximum residue level 0.005 ppm after 35 days. The adsorbed amount of PH3 was not influenced by fumigation parameters. For analysis of the influences on the volatile profile after fumigation, a headspace solid-phase micro-extraction coupled to gas chromatography mass spectrometry (HS-SPME-GC/MS) was used. Small differences in volatile profiles of fumigated and subsequently outgassed table grapes compared to non-fumigated table grapes could be observed. A slight influence on the aldehyde group directly after fumigation could be perceived by a decrease of hex-2-en-1-ol and 1- hexanol in PH3-treated table grapes. The concentrations of both compounds increase again after completion of the desorption process. On the other hand terpenes are not significantly influenced by the fumigation process. Overall these changes are likely to affect table grape aroma characteristics directly after a treatment with PH3 and it could be demonstrated that phosphine alters the volatile profile of fumigated table grapes qualitatively and quantitatively.

Characterization of aluminum, aluminum oxide and titanium dioxide nanomaterials using a combination of methods for particle surface and size analysis.

The application of appropriate analytical techniques is essential for nanomaterial (NM) characterization. In this study, we compared different analytical techniques for NM analysis. Regarding possible adverse health effects, ionic and particulate NM effects have to be taken into account. As NMs behave quite differently in physiological media, special attention was paid to techniques which are able to determine the biosolubility and complexation behavior of NMs. Representative NMs of similar size were selected: aluminum (Al0) and aluminum oxide (Al2O3), to compare the behavior of metal and metal oxides. In addition, titanium dioxide (TiO2) was investigated. Characterization techniques such as dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) were evaluated with respect to their suitability for fast characterization of nanoparticle dispersions regarding a particle's hydrodynamic diameter and size distribution. By application of inductively coupled plasma mass spectrometry in the single particle mode (SP-ICP-MS), individual nanoparticles were quantified and characterized regarding their size. SP-ICP-MS measurements were correlated with the information gained using other characterization techniques, i.e. transmission electron microscopy (TEM) and small angle X-ray scattering (SAXS). The particle surface as an important descriptor of NMs was analyzed by X-ray diffraction (XRD). NM impurities and their co-localization with biomolecules were determined by ion beam microscopy (IBM) and confocal Raman microscopy (CRM). We conclude advantages and disadvantages of the different techniques applied and suggest options for their complementation. Thus, this paper may serve as a practical guide to particle characterization techniques.

Comparison of cytochrome P450- and peroxidase-dependent metabolic activation of the potent carcinogen dibenzo[a,l]pyrene in human cell lines: formation of stable DNA adducts and absence of a detectable increase in apurinic sites.

The potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) has been reported to form both stable and depurinating DNA adducts upon activation by cytochrome P450 enzymes and/or cellular peroxidases. Only stable DB[a,l]P-DNA adducts were detected in DNA after reaction of DB[a,I]P-11,12-diol-13,14-epoxides in solution or cells in culture. To determine whether DB[a,l]P can be activated to metabolites that form depurinating adducts in cells with either high peroxidase (human leukemia HL-60 cell line) or cytochrome P450 activity (human mammary carcinoma MCF-7 cell line), cultures were treated with DB[a,l]P for 4 h, and the levels of stable adducts and apurinic (AP) sites in the DNA were determined. DNA samples from DB[a,l]P-treated HL-60 cells contained no detectable levels of either stable adducts or AP sites. MCF-7 cells exposed to 2 microM DB[a,l]P for 4 h contained 4 stable adducts per 10(6) nucleotides, but no detectable increase in AP sites. The results indicate that metabolic activation of DB[a,l]P by cytochrome P450 enzymes to diol epoxides that form stable DNA adducts, rather than one-electron oxidation catalyzed either by cytochrome P450 enzymes or peroxidases to form AP sites, is responsible for the high carcinogenic activity of DB[a,l]P.

The K-region trans-8,9-diol does not significantly contribute as an intermediate in the metabolic activation of dibenzo[a,l]pyrene to DNA-binding metabolites by human cytochrome P450 1A1 or 1B1.

Metabolic activation of the K-region trans-8,9-diol of the highly carcinogenic hexacyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P) by human cytochrome P-450 (P450) 1A1 and 1B1 was investigated in Chinese hamster V79 cell lines expressing human P450 1A1 or 1B1. P450 1A1 and 1B1 are the major P450s involved in metabolic activation of polycyclic aromatic hydrocarbons in human cells. The major DNA adducts formed by metabolism of DB[a,l]P in cultures expressing P450 1A1 or 1B1 resulted mainly from the fjord region (-)-anti-DB[a,l]P-11,12-diol 13,14-epoxide [(-)-anti-DB[a,l]PDE] and, to a lesser extent, (+)-syn-DB[a,l]PDE. In V79 cells expressing human P450 1A1, high amounts of as yet unidentified highly polar DNA adducts are formed in addition to the DNA adducts derived from DB[a,l]PDEs. Human P450 1A1 has been found to metabolize DB[a,l]P on its K-region to the trans-8,9-diol, and it has been proposed that the DNA binding of the parent compound in P450 1A1-expressing tissues may be partially mediated by activation of the K-region trans-8,9-diol to form bis-diol epoxides. V79 cells expressing human P450 1A1 or 1B1 formed only low amounts of DNA adducts after treatment with high doses of the K-region trans-8,9-diol. None of the adducts formed were identical to the main adducts formed in the same cell lines by metabolic activation of DB[a,l]P or (-)-DB[a,l]P-trans-11,12-diol. These results demonstrate that the K-region trans-8,9-diol does not significantly contribute to the genotoxicity of the very potent carcinogen DB[a,l]P in human cells or tissues expressing P450 1A1 or 1B1.

The potent carcinogen dibenzo[a,l]pyrene is metabolically activated to fjord-region 11,12-diol 13,14-epoxides in human mammary carcinoma MCF-7 cell cultures.

Dibenzo[a,l]pyrene (DB[a,l]P), an environmental hydrocarbon and very potent carcinogen in rodent bioassays, could be activated to DNA-binding intermediates in cells through formation of three different regioisomeric bay- or fjord-region diol-epoxides or other more highly oxidized metabolites. The mechanism of metabolic activation of DB[a,l]P in the human mammary carcinoma cell line MCF-7 was elucidated by analyzing the DB[a,l]P-DNA adducts formed by [35S]phosphorothioate postlabeling, immobilized boronate chromatography, and high-performance liquid chromatography. Six DB[a,l]P-DNA adducts were detected. Comparison with those formed in cells by DB[a,l]P-11,12-diol and by reaction of DNA with syn- and anti-(benzylic hydroxyl and epoxide oxygen cis and trans, respectively) DB[a,l]P-11,12-diol-13,14-epoxide (DB[a,l]PDE) demonstrated that all DB[a,l]P-DNA adducts in MCF-7 cells were formed by these diol-epoxide isomers. Cellular DNA contained large amounts of two syn- and one anti-DB[a,l]PDE-DNA adducts and small amounts of one syn- and two anti-DB[a,l]PDE-DNA adducts. The ability of human cells to activate DB-[a,l]P to its fjord-region 11,12-diol 13,14-epoxides suggests that environmental exposure to DB[a,l]P could pose a risk for humans.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS)-based analysis and imaging of polyethylene microplastics formation during sea surf simulation.

Plastic particles smaller than 5mm, so called microplastics have the capability to accumulate in rivers, lakes and the marine environment and therefore have begun to be considered in eco-toxicology and human health risk assessment. Environmental microplastic contaminants may originate from consumer products like body wash, tooth pastes and cosmetic products, but also from degradation of plastic waste; they represent a potential but unpredictable threat to aquatic organisms and possibly also to humans. We investigated exemplarily for polyethylene (PE), the most abundant constituent of microplastic particles in the environment, whether such fragments could be produced from larger pellets (2mm×6mm). So far only few analytical methods exist to identify microplastic particles smaller than 10µm, especially no imaging mass spectrometry technique. We used at first time-of-flight secondary ion mass spectrometry (ToF-SIMS) for analysis and imaging of small PE-microplastic particles directly in the model system Ottawa sand during exposure to sea surf simulation. As a prerequisite, a method for identification of PE was established by identification of characteristic ions for PE out of an analysis of grinded polymer samples. The method was applied onto Ottawa sand in order to investigate the influence of simulated environmental conditions on particle transformation. A severe degradation of the primary PE pellet surface, associated with the transformation of larger particles into smaller ones already after 14days of sea surf simulation, was observed. Within the subsequent period of 14days to 1month of exposure the number of detected smallest-sized particles increased significantly (50%) while the second smallest fraction increased even further to 350%. Results were verified using artificially degraded PE pellets and Ottawa sand.

Tumor-initiating activity of the (+)-(S,S)- and (-)-(R,R)- enantiomers of trans-11,12-dihydroxy-11,12-dihydrodibenzo[a,l]pyrene in mouse skin.

A single administration of enantiomerically pure 11,12-dihydrodiols of dibenzo[a,l]pyrene (DB[a,l]P) on the back of NMRI mice and subsequent chronic treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) (initiation/promotion assay) revealed strikingly different carcinogenic activities of both enantiomers. Tumor-initiating activity of (-)-(11R,12R)-DB[a,l]P-dihydrodiol, which is the metabolic precursor of the (-)-anti-(11R,12S)-dihydrodiol (13S,14R)-epoxide, was exceptionally higher than the corresponding effect of (+)-(11S,12S)-DB[a,l]P-dihydrodiol, the metabolic precursor of (+)-syn-(11S,12R)-dihydrodiol (13S,14R)-epoxide. After topical application of 10 nmol (-)-11,12-dihydrodiol and promotion with TPA twice weekly for a further 18 weeks 93% of treated animals exhibited four to five tumors. In contrast, no neoplasms were observed after treatment with 10 nmol (+)-11,12-dihydrodiol, whereas in the group exposed to 20 nmol of this enantiomer only 13% of mice developed neoplasms (0.1 tumors/survivor). For DB[a,l]P, considered as the most potent carcinogenic polycyclic aromatic hydrocarbon to date, stereoselective formation of (+)-syn- and (-)-anti-11,12-dihydrodiol 13,14-epoxides via the corresponding enantiomeric 11,12-dihydrodiols has been found to be the principal metabolic activation pathway leading to DNA adducts and mutagenicity. Our study demonstrates that the striking difference in carcinogenic activity in mouse skin of (+)-(11S,12S)- and (-)-(11R,12R)-DB[a,l]P-dihydrodiol convincingly reflects the different genotoxicity, i.e. DNA binding and mutagenicity, of both enantiomers observed earlier.

Dual beam organic depth profiling using large argon cluster ion beams.

Argon cluster sputtering of an organic multilayer reference material consisting of two organic components, 4,4'-bis[N-(1-naphthyl-1-)-N-phenyl- amino]-biphenyl (NPB) and aluminium tris-(8-hydroxyquinolate) (Alq3), materials commonly used in organic light-emitting diodes industry, was carried out using time-of-flight SIMS in dual beam mode. The sample used in this study consists of a ∽400-nm-thick NPB matrix with 3-nm marker layers of Alq3 at depth of ∽50, 100, 200 and 300 nm. Argon cluster sputtering provides a constant sputter yield throughout the depth profiles, and the sputter yield volumes and depth resolution are presented for Ar-cluster sizes of 630, 820, 1000, 1250 and 1660 atoms at a kinetic energy of 2.5 keV. The effect of cluster size in this material and over this range is shown to be negligible. © 2014 The Authors. Surface and Interface Analysis published by John Wiley & Sons Ltd.

Monitoring oxidative stress biomarkers in the lipidome: is there a roadmap for "human inspection"?

Oxidative stress is more and more recognized as the underlying motif for a broad variety of diseases including cancer. Medicine faces the paramount task to develop better diagnostic tools and drug treatment prediction models in the future to significantly enhance the quality of life. Special interest will focus on earlystage disease biomarkers and biomarkers that could predict healing success at the earliest time point after the treatment started. The accelerated formation of so-called reactive oxygen species (ROS) is becoming widely regarded as the underlying process associated with many diseases like myocardial infarction, Alzheimer's, Parkinson's and kidney disease, etc. Once generated within cells and tissues, ROS can react with a variety of cellular metabolites like fatty acids, proteins or DNA. This review investigates the possibilities for various oxidized metabolites as well as proteomics, genomics and bioimaging biomarkers to serve as early-stage disease biomarkers or biomarkers for drug treatment success. We also assess the value of a step-by-step or cascade biomarker approach as a new paradigm in medical diagnostics. Examples are given for possible analytical methodology and tools as well as statistical methods that could be applied. Such an approach may straighten the road toward new medical diagnostics and treatment regimes, which ultimately could lead to a significantly enhanced medical service for patients suffering from chronic and debilitating or deadly diseases including cancer. Examples from recent research are given to show the progress and possibilities for the proposed model.

Activation of the aryl hydrocarbon receptor sensitises human keratinocytes for CD95L- and TRAIL-induced apoptosis.

In this study, we have analysed the apoptotic effects of the ubiquitous environmental toxin benzo[a]pyrene (BP) in HaCaT cells and human keratinocytes. Although prolonged exposure to BP was not cytotoxic on its own, a strong enhancement of CD95 (Fas)-mediated apoptosis was observed with BP at concentrations activating the aryl hydrocarbon receptor (AhR). Importantly, the ultimately mutagenic BP-metabolite, that is, (+)-anti-BP-7,8-diol-9,10-epoxide (BPDE), failed to enhance CD95-mediated cell death, suggesting that the observed pro-apoptotic effect of BP is neither associated with DNA adducts nor DNA-damage related signalling. CD95-induced apoptosis was also enhanced by ß-naphtoflavone, a well-known agonist of the AhR that does not induce DNA damage, thus suggesting a crucial role for AhR activation. Consistently, BP failed to sensitise for CD95L-induced apoptosis in AhR knockdown HaCaT cells. Furthermore, inhibition of CYP1A1 and/or 1B1 expression did not affect the pro-apoptotic crosstalk. Exposure to BP did not increase expression of CD95, but led to augmented activation of caspase-8. Enhancement of apoptosis was also observed with the TRAIL death receptors that activate caspase-8 and apoptosis by similar mechanisms as CD95. Together, these observations indicate an interference of AhR signalling with the activity of receptor-associated signalling intermediates that are shared by CD95 and TRAIL receptors. Our data thus suggest that AhR agonists can enhance cytokine-mediated adversity upon dermal exposure.