TatS: a novel in vitro tattooed human skin model for improved pigment toxicology research

Reports of tattoo-associated risks boosted the interest in tattoo pigment toxicity over the last decades. Nonetheless, the influence of tattoo pigments on skin homeostasis remains largely unknown. In vitro systems are not available to investigate the interactions between pigments and skin. Here, we established TatS, a reconstructed human full-thickness skin model with tattoo pigments incorporated into the dermis. We mixed the most frequently used tattoo pigments carbon black (0.02 mg/ml) and titanium dioxide (TiO2, 0.4 mg/ml) as well as the organic diazo compound Pigment Orange 13 (0.2 mg/ml) into the dermis. Tissue viability, morphology as well as cytokine release were used to characterize TatS. Effects of tattoo pigments were compared to monolayer cultures of human fibroblasts. The tissue architecture of TatS was comparable to native human skin. The epidermal layer was fully differentiated and the keratinocytes expressed occludin, filaggrin and e-cadherin. Staining of collagen IV confirmed the formation of the basement membrane. Tenascin C was expressed in the dermal layer of fibroblasts. Although transmission electron microscopy revealed the uptake of the tattoo pigments into fibroblasts, neither viability nor cytokine secretion was altered in TatS. In contrast, TiO2 significantly decreased cell viability and increased interleukin-8 release in fibroblast monolayers. In conclusion, TatS emulates healed tattooed human skin and underlines the advantages of 3D systems over traditional 2D cell culture in tattoo pigment research. TatS is the first skin model that enables to test the effects of pigments in the dermis upon tattooing

Synchrotron-based nu-XRF mapping and mu-FTIR microscopy enable to look into the fate and effects of tattoo pigments in human skin

The increasing prevalence of tattoos provoked safety concerns with respect to particle distribution and effects inside the human body. We used skin and lymphatic tissues from human corpses to address local biokinetics by means of synchrotron X-ray fluorescence (XRF) techniques at both the micro (mu) and nano (nu) scale. Additional advanced mass spectrometry-based methodology enabled to demonstrate simultaneous transport of organic pigments, heavy metals and titanium dioxide from skin to regional lymph nodes. Among these compounds, organic pigments displayed the broadest size range with smallest species preferentially reaching the lymph nodes. Using synchrotron mu-FTIR analysis we were also able to detect ultrastructural changes of the tissue adjacent to tattoo particles through altered amide I alpha-helix to beta-sheet protein ratios and elevated lipid contents. Altogether we report strong evidence for both migration and long-term deposition of toxic elements and tattoo pigments as well as for conformational alterations of biomolecules that likely contribute to cutaneous inflammation and other adversities upon tattooing

Investigation of Adverse Reactions in Tattooed Skin through Histological and Chemical Analysis

Background: Just as the number of tattooed people has increased in recent years, so has the number of adverse reactions in tattooed skin. Tattoo colourants contain numerous, partly unidentified substances, which have the potential to provoke adverse skin reactions like allergies or granulomatous reactions. Identification of the triggering substances is often difficult or even impossible. Methods: Ten patients with typical adverse reactions in tattooed skin were enrolled in the study. Skin punch biopsies were taken and the paraffin-embedded specimens were analysed by standard haematoxylin and eosin and anti-CD3 stainings. Tattoo colourants provided by patients and punch biopsies of patients were analysed with different chromatography and mass spectrometry methods and X-ray fluorescence. Blood samples of 2 patients were screened for angiotensin-converting enzyme (ACE) and soluble interleukin-2 receptor (sIL-2R). Results: Histology showed variable skin reactions such as eosinophilic infiltrate, granulomatous reactions, or pseudolymphoma. CD3+ T lymphocytes dominated the dermal cellular infiltrate. Most patients had adverse skin reactions in red tattoos (n = 7), followed by white tattoos (n = 2). The red tattooed skin areas predominantly contained Pigment Red (P.R.) 170, but also P.R. 266, Pigment Orange (P.O.) 13, P.O. 16, and Pigment Blue (P.B.) 15. The white colourant of 1 patient contained rutile titanium dioxide but also other metals like nickel and chromium and methyl dehydroabietate – known as the main ingredient of colophonium. None of the 2 patients showed increased levels of ACE and sIL-2R related to sarcoidosis. Seven of the study participants showed partial or complete remission after treatment with topical steroids, intralesional steroids, or topical tacrolimus. Conclusions: The combination of the methods presented might be a rational approach to identify the substances that trigger adverse reactions in tattoos. Such an approach might help make tattoo colourants safer in the future if such trigger substances could be omitted

Tätowiermittelpigmente: Bioverteilung und Toxizität ihrer laserinduzierten Zersetzungsprodukte

Due to the continuous popularity of tattoos in Germany and other countries one fifth to a quarter of the population is already carrying this permanent body decoration. Despite this high incidence, numerous toxicological endpoints, especially in the case of the color giving pigments, are missing for an adequate risk assessment of tattoo inks. In this thesis, photostability and biokinetics are investigated as two of the key elements of tattoo pigment pharmacokinetics. The light-induced decomposition of six organic pigments was investigated using laser irradiation, which is commonly used for tattoo removal. Decomposition products were analyzed using gas chromatographic separation coupled to mass spectrometric detection. Additionally, the photothermal decomposition as occurring with laser irradiation was mimicked by pyrolysis. Data for pigment biokinetics could only be obtained by analysis of human samples since animal testing for tattoo applications was declined in Germany. Here, pigment and element distribution in skin and lymph nodes, as well as other peripheral organs, were assessed using mass spectrometric devices and synchrotron x-ray fluorescence techniques. Upon laser irradiation, all organic pigments were cleaved into benzene and hydrogen cyanide. Also, potentially carcinogenic and sensitizing compounds were found for each pigment specifically. The same decomposition products were also found in pigment pyrolysis. In in vitro cytotoxicity tests, hydrogen cyanide showed an impairment of the skin cell metabolism in the expected concentrations. The analysis of skin and lymph node samples revealed a preferential transport of smaller particles of organic and inorganic pigments. Associated to tattoo pigments, potentially carcinogenic and sensitizing elements like Ni, Cr and Cd are transported to the draining lymph nodes. No increased element concentrations were detected in other peripheral organs investigated so far. The data obtained from laser irradiation and pyrolysis in combination with information from other publications allow an extrapolation of the decomposition of non-investigated pigments of the same chemical classes. This facilitates the exclusion of pigments degrading into toxins out of the several hundred potentially used in tattoo inks. The data on distribution of tattoo inks do not display a full data set for biokinetics under the given circumstances but confirm life-long exposition to potentially harmful material in the lymph nodes. Since the distribution of other insoluble pigments including white titanium dioxide is well described in literature upon subcutaneous and intradermal application, this data might be used to extrapolate the distribution of tattoo pigments.Durch die anhaltende Beliebtheit von Tätowierungen tragen in Deutschland und anderen Ländern zwischen einem Fünftel und einem Viertel aller Menschen diesen permanenten Körperschmuck. Trotz dieser hohen Inzidenz stehen viele toxikologische Daten für eine ausreichende Risikobewertung der Inhaltstoffe von Tätowiermitteln, insbesondere der Pigmente, nicht zur Verfügung. In dieser Arbeit wurden zwei Kernelemente der Pharmakokinetik von Tätowiermittelpigmenten, die Stabilität unter Lichteinfluss und die Biokinetik, untersucht. Die lichtinduzierte Zersetzung von sechs organischen Pigmenten unter Laserbestrahlung, welche zur Entfernung von Tätowierungen eingesetzt wird, wurde mit Hilfe von Gaschromatographie mit massenspektrometrischer Detektion untersucht. Zudem wurde die photothermische Zersetzung unter Laserbestrahlung durch Pyrolyse simuliert. Biokinetische Daten konnten nur durch die Analyse humaner Proben erhoben werden, da Tierversuche für diesen Anwendungsbereich in Deutschland nicht genehmigt wurden. Hier wurde die Verteilung von organischen Pigmenten und Elementen in Haut und Lymphknoten, sowie anderen peripheren Organen mit Hilfe massenspektrometrischer Methoden und Synchrotron-Röntgenfluoreszenz analysiert. In den Laserversuchen konnte gezeigt werden, dass generell aus allen untersuchten organischen Pigmenten Benzol und Blausäure freigesetzt werden können. Zudem entstehen pigmentspezifisch potentiell krebserregende und allergieauslösender Substanzen. Die gleichen Zersetzungsprodukte zeigten sich ebenfalls durch Pyrolyse der Pigmente. In vitro Zytotoxizitätstests konnten zeigen, dass der Hautzellmetabolismus durch Blausäure in den zu erwartenden Konzentrationen eingeschränkt wird. Die Analysen der Haut und Lymphknotenproben zeigten einen bevorzugten Transport von kleinen Partikeln organischer und anorganischer Pigmente. Zusammen mit diesen Pigmenten gelangen auch potentiell krebserregende und allergieauslösende Elementverunreinigung wie Ni, Cr und Cd in die Lymphknoten. In peripheren Organen konnten noch keine Pigmente oder erhöhte Elementgehalte festgestellt werden. Durch die gewonnen Daten der Laserbestrahlung, Pyrolyse und anhand der Literatur lassen sich für einzelne chemische Pigmentklassen auch die Zersetzungsprodukte nicht untersuchter Pigmente extrapolieren. Die Daten können dazu genutzt werden, unter Hunderten verfügbaren Pigmenten diejenigen mit toxischen Zersetzungsprodukten zu identifizieren und für die Anwendung in Tätowiermitteln auszuschließen. Die unter den gegebenen Umständen gewonnenen Daten in Bezug auf die Verteilung von Tätowiermittelpigmenten im Körper stellen keine vollständige Biokinetik dar, belegen jedoch die lebenslange Exposition gegenüber potentiell gesundheitsschädlichen Stoffen in den Lymphknoten. Da die Verteilung anderer unlöslicher Partikel, inklusive dem Weißpigment Titandioxid, auch in subkutaner und intradermaler Applikation in der Literatur gut beschrieben ist, können diese Daten ebenfalls zur Extrapolation der Verteilung von Tätowiermittelpigmenten herangezogen werden

Schreiver et al 2019 Pigment_pyrograms_elu_files

These data files are pyrograms of different pigments at 800°C. Double entries relate to different purity grades, manufacturers or repetitions at different time points. The Amdis .elu file format can be opened with open source software such as OpenChrom (Lablicate GmBH & Scientific community). However, the software does not allow pyrogram similarity searches (see data file "MSChromSearch_Library_Pigments_Polymers")

Schreiver et al 2019 ChemStation_Library_AMS_Pigments_Polymers.L

This library contains average mass spectra (AMS) of specific pigment pyrolysis GC-MS runs. Select this library (file format “.L”) in the GC-MS Software Enhanced ChemStation (Agilent Technologies, Waldbronn, Germany) for fast screening for pigment identities. Create an AMS of the unknown pyrogram and start the search. The files are specific for the corresponding software but may be converted into other file types

Data from: Two-step pyrolysis-gas chromatography method with mass spectrometric detection for identification of tattoo ink ingredients and counterfeit products

Tattoo inks are complex mixtures of ingredients. Each of them possesses different chemical properties which have to be addressed upon chemical analysis. In this method for two-step pyrolysis online coupled to gas chromatography mass spectrometry (py-GC-MS) volatile compounds are analyzed during a first desorption run. In the second run, the same dried sample is pyrolyzed for analysis of non-volatile compounds such as pigments and polymers. These can be identified by their specific decomposition patterns. Additionally, this method can be used to differentiate original from counterfeit inks. Easy screening methods for data evaluation using the average mass spectra and self-made pyrolysis libraries are applied to speed up substance identification. Using specialized evaluation software for pyrolysis GS-MS data, a fast and reliable comparison of the full chromatogram can be achieved. Since GC-MS is used as separation technique, the method is limited to volatile substances upon desorption and after pyrolysis of the sample. The method can be applied for quick substance screening in market control surveys since it requires no sample preparation steps

Data from: Two-step pyrolysis-gas chromatography method with mass spectrometric detection for identification of tattoo ink ingredients and counterfeit products

Tattoo inks are complex mixtures of ingredients. Each of them possesses different chemical properties which have to be addressed upon chemical analysis. In this method for two-step pyrolysis online coupled to gas chromatography mass spectrometry (py-GC-MS) volatile compounds are analyzed during a first desorption run. In the second run, the same dried sample is pyrolyzed for analysis of non-volatile compounds such as pigments and polymers. These can be identified by their specific decomposition patterns. Additionally, this method can be used to differentiate original from counterfeit inks. Easy screening methods for data evaluation using the average mass spectra and self-made pyrolysis libraries are applied to speed up substance identification. Using specialized evaluation software for pyrolysis GS-MS data, a fast and reliable comparison of the full chromatogram can be achieved. Since GC-MS is used as separation technique, the method is limited to volatile substances upon desorption and after pyrolysis of the sample. The method can be applied for quick substance screening in market control surveys since it requires no sample preparation steps

Schreiver et al 2019 ChemStation_Library_Pigments_Polymers

This library contains spectra of specific pigment pyrolysis products that are not yet included in standard GC-MS libraries. Most decomposition products are unknown. The file format “.MSP” can be opened with open source software such as OpenChrom (Lablicate GmBH & Scientific community)

Schreiver et al 2019 ChemStation_Library_AMS_Pigments_Polymers

This library contains average mass spectra (AMS) of specific pigment pyrolysis GC-MS runs. The file format “.MSP” can be opened with open source software such as OpenChrom (Lablicate GmBH & Scientific community)