Christoph Hutzler

Metabolically Competent Human Skin Models: Activation and Genotoxicity of Benzo[a]pyrene

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 50–100nM 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.

Investigations on the emission of fragrance allergens from scented toys by means of headspace solid-phase microextraction gas chromatography-mass spectrometry.

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.

Development of a manual method for the determination of mineral oil in foods and paperboard.

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 μl volume was injected on-column into GC-FID to achieve a detection limit for MOSH and MOAH below 1 mg/kg in most foods.

Inhalation exposure of children to fragrances present in scented toys.

UNLABELLED When utilized in the perfuming of childrens 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.

Estrogenic Activity of Mineral Oil Aromatic Hydrocarbons Used in Printing Inks

The majority of printing inks are based on mineral oils (MOs) which contain complex mixtures of saturated and aromatic hydrocarbons. Consumer exposure to these oils occurs either through direct skin contacts or, more frequently, as a result of MO migration into the contents of food packaging that was made from recycled newspaper. Despite this ubiquitous and frequent exposure little is known about the potential toxicological effects, particularly with regard to the aromatic MO fractions. From a toxicological point of view the huge amount of alkylated and unsubstituted compounds therein is reason for concern as they can harbor genotoxicants as well as potential endocrine disruptors. The aim of this study was to assess both the genotoxic and estrogenic potential of MOs used in printing inks. Mineral oils with various aromatic hydrocarbon contents were tested using a battery of in vitro assays selected to address various endpoints such as estrogen-dependent cell proliferation, activation of estrogen receptor α or transcriptional induction of estrogenic target genes. In addition, the comet assay has been applied to test for genotoxicity. Out of 15 MOs tested, 10 were found to potentially act as xenoestrogens. For most of the oils the effects were clearly triggered by constituents of the aromatic hydrocarbon fraction. From 5 oils tested in the comet assay, 2 showed slight genotoxicity. Altogether it appears that MOs used in printing inks are potential endocrine disruptors and should thus be assessed carefully to what extent they might contribute to the total estrogenic burden in humans.

Identification and hazard prediction of tattoo pigments by means of pyrolysis-gas chromatography/mass spectrometry.

The implementation of regulation for tattoo ink ingredients across Europe has generated the need for analytical methods suitable to identify prohibited compounds. Common challenges of this subject are the poor solubility and the lack of volatility for most pigments and polymers applied in tattoo inks. Here, we present pyrolysis coupled to online gas chromatography and electron impact ionization mass spectrometry (py-GC/MS) as quick and reliable tool for pigment identification using both purified pigments and tattoo ink formulations. Some 36 organic pigments frequently used in tattoo inks were subjected to py-GC/MS with the aim to establish a pyrogram library. To cross-validate pigment identification, 28 commercially available tattoo inks as well as 18 self-made pigment mixtures were analyzed. Pyrograms of inks and mixtures were evaluated by two different means to work out the most reliable and fastest strategy for an otherwise rather time-consuming data review. Using this approach, the declaration of tattoo pigments currently used on the market could be verified. The pyrolysis library presented here is also assumed suitable to predict decomposition patterns of pigments when affected by other degradation scenarios, such as sunlight exposure or laser irradiation. Thus, the consumers’ risk associated with the exposure to toxicologically relevant substances that originate from pigment decomposition in the dermal layers of the skin can be assessed. Differentiation between more or less harmful pigments for this field of application now will become feasible.

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

ABSTRACT 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.

Toward the stereochemical identification of prohibited characterizing flavors in tobacco products: the case of strawberry flavor

With the revision of the European Tobacco Products Directive (2014/40/EU), characterizing flavors such as strawberry, candy, vanillin or chocolate will be prohibited in cigarettes and fine-cut tobacco. Product surveillance will therefore require analytical means to define and subsequently detect selected characterizing flavors that are formed by supplemented flavors within the complex matrix tobacco. We have analyzed strawberry-flavored tobacco products as an example for characterizing fruit-like aroma. Using this approach, we looked into aroma components to find indicative patterns or features that can be used to satisfy obligatory product information as requested by the European Directive. Accordingly, a headspace solid-phase microextraction (HS-SPME) technique was developed and coupled to subsequent gas chromatography–mass spectrometry (GC/MS) to characterize different strawberry-flavored tobacco products (cigarettes, fine-cut tobacco, liquids for electronic cigarettes, snus, shisha tobacco) for their volatile additives. The results were compared with non-flavored, blend characteristic flavored and other fruity-flavored cigarettes, as well as fresh and dried strawberries. Besides different esters and aldehydes, the terpenes linalool, α-terpineol, nerolidol and limonene as well as the lactones γ-decalactone, γ-dodecalactone and γ-undecalactone could be verified as compounds sufficient to convey some sort of strawberry flavor to tobacco. Selected flavors, i.e., limonene, linalool, α-terpineol, citronellol, carvone and γ-decalactone, were analyzed further with respect to their stereoisomeric composition by using enantioselective HS-SPME–GC/MS. These experiments confirmed that individual enantiomers that differ in taste or physiological properties can be distinguished within the tobacco matrix. By comparing the enantiomeric composition of these compounds in the tobacco with that of fresh and dried strawberries, it can be concluded that non-natural strawberry aroma is usually used to produce strawberry-flavored tobacco products. Such authenticity control can become of interest particularly when manufacturers claim that natural sources were used for flavoring of products. Although the definition of characterizing flavors by analytical means remains challenging, specific compounds or features are required to be defined for routine screening of reported information. Clarifications by sensory testing might still be necessary, but could be limited to a few preselected samples.

Toxification of polycyclic aromatic hydrocarbons by commensal bacteria from human skin

The ubiquitous occurrence of polycyclic aromatic hydrocarbons (PAHs) leads to constant human exposure at low levels. Toxicologically relevant are especially the high-molecular weight substances due to their (pro-)carcinogenic potential. Following ingestion or uptake, the eukaryotic phase I metabolism often activates these substances to become potent DNA binders, and unsurprisingly metabolism and DNA-adduct formation of model substances such as benzo[a]pyrene (B[a]P) are well studied. However, apart from being subjected to eukaryotic transformations PAHs are also carbon and energy sources for the myriads of commensal microbes inhabiting man’s every surface. Yet, we know little about the microbiome’s PAH-metabolism capacity and its potentially adverse impact on the human host. This study now shows that readily isolable skin commensals transform B[a]P into a range of highly cyto- and genotoxic metabolites that are excreted in toxicologically relevant concentrations during growth. The respective bacterial supernatants contain a mixture of established eukaryotic as well as hitherto unknown prokaryotic metabolites, the combination of which leads to an increased toxicity. Altogether we show that PAH metabolism of the microbiome has to be considered a potential hazard.

Polycyclic Aromatic Hydrocarbons in Newspaper Inks: Migration, Metabolism, and Genotoxicity in Human Skin

Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BP), are detectable in certain consumer products, including newspapers. We investigated the release of PAHs from newspapers and the fate and effects of BP in human skin models. The migration and penetration of BP into the dermal layers was shown using pig skin. Upon treatment the stratum corneum was gradually removed by tape strips and all samples were subsequently analyzed. BP could be detected in newspapers at levels of up to 52 μg/kg via LC-APPI-MS/MS. We also could show that PAHs such as BP are able to penetrate from the surface into deeper layers of the skin. In addition, the migration of different PAHs including BP from newspapers into a sheet of polyethylene as skin simulant was demonstrated. We further report on the biotransformation of BP in various three-dimensional human skin models and cultures of human keratinocytes or fibroblasts (5). The metabolic profiles observed by means of LC-APCI-MS/MS contained seven major BP follow-up products (i.e., 3- and 7-phenol, 1,6- and 7,8-dione, 7,8-, and 9,10-diol, 7,8,9,10-tetraol) and thus resembled the profiles detected in human skin ex vivo. In the frame of these studies, we also confirmed the genotoxicity of BP and its metabolites in the skin models via comet assay.