Phototoxic interactions of tattoo pigments with laser and natural light in vitro

Tattooing, a process in which colorants are permanently embedded in human skin to generate long-lasting images, enjoys a high popularity nowadays. Today, every fifth to fourth person is tattooed. This popularity seems not to be affected by the high number of tattoo related side effects, which especially occur when tattooed skin encounters light. Light induced side effects make up to about 60 % of tattoo associated side effects. Consequently, the interactions of ultraviolet light and laser light on tattoo pigments and the resulting effects on human skin cells were investigated in this thesis to increase the understanding of photo-induced side effects found in tattoos. Firstly, we treated postmortem tattooed pig skin with laser used in tattoo removal. We consequently analyzed cleavage products using gas chromatographic separation coupled to mass spectrometric detection. 3,3’-dichlorobenzidine, a carcinogen produced through cleavage of Pigment Orange (P.O.)13, was found to be cytotoxic and genotoxic in terms of DNA double strand breaks in human fibroblast and keratinocyte cell lines even beneath identified concentrations. While photo-induced cleavage is a key event that leads to tattoo clearing during laser removal, it can also occur on a smaller scale when tattooed skin is exposed to natural light, particularly in the ultraviolet (UV) range. Since light-induced effects are the most common side effects associated with tattoos, our goal was to investigate the underlying pathomechanisms accordingly. However, no in vitro model that accurately resembles the architecture of tattooed human skin has been described in the literature so far. Therefore, we established a 3D in vitro ‘tattooed’ full thickness skin model (TatSFT) as an animal replacement model for tattoo research. The uptake of the tattoo pigments used in this study, titanium dioxide (TiO2) anatase, TiO2 rutile, Pigment Orange (P.O.)13 and carbon black by dermal fibroblasts was proven by electron microscopy in TatSFT. Despite this uptake, the pigments showed no effect on the viability of TatSFT nor its dermal compartment (TatSDE). Concordantly, cytokine secretion, general histology, and the expression of important skin homeostasis markers was unaffected by tattoo pigments in TatSFT. Contrary to the absence of toxicity in 3D, TiO2 anatase significantly decreased cell viability and increased interleukin-8 release in 2D monolayer cultured fibroblasts. Due to the inherent differences in toxicity sensitivity of 2D monolayered cultured fibroblasts and 3D cultured models, we investigated phototoxicity of tattoo pigments in both, 2D and 3D. Concordantly with particle toxicity, phototoxic effects were bigger in monolayer cultured fibroblasts than in 3D. While TiO2 showed strong phototoxic effects in 2D, these effects were absent in TatSFT. However, UVB induced DNA damage marker levels in the dermis of TatSFT were reduced by pigments. Combined with photoprotective effects found in TatSDE concerning viability, these data suggest photoprotective properties of tattoo pigments for the tattooed dermis and its underlying tissue. Contrary to these results, we found P.O.13 to alter cytokine secretion upon UV irradiation in both, 2D and TatSDE. While minor amounts of cleavage products of P.O.13 were identified after UV irradiation, we were unable to proof that these cleavage products might have resulted in adverse effects. The data in this thesis not only highlight the need for 3D test systems for tattoo phototoxicity research, but also present a highly modifiable 3D in vitro test system for this purpose. This work also strengthens concerns regarding TiO2 anatase and azo pigments like P.O.13 and their use in tattoo inks.Mit dem Tätowieren erfreut sich heutzutage ein Verfahren größter Beliebtheit, bei dem Farbstoffe dauerhaft in die menschliche Haut eingebettet werden, um permanente Bilder in dieser zu erzeugen. Momentan ist jeder fünfte bis vierte Mensch tätowiert. Dieser Beliebtheit scheint die hohe Zahl an tätowierungsbedingten Nebenwirkungen keinen Abbruch zu tun. Viele Nebenwirkungen treten insbesondere dann auf, wenn die tätowierte Haut mit Licht in Berührung kommt. Insgesamt machen lichtinduzierte Nebenwirkungen etwa 60 % der tätowierungsassoziierten Nebenwirkungen aus. In dieser Arbeit wurden daher die Wechselwirkungen von ultraviolettem Licht und Laserlicht auf Tätowierpigmente und die daraus resultierenden Effekte auf menschliche Hautzellen untersucht, um das Verständnis für lichtinduzierte Nebenwirkungen in tätowierter Haut zu erhöhen. Zunächst behandelten wir postmortem tätowierte Schweinehaut mit Lasern, die zur Tattooentfernung verwendet werden. Anschließend analysierten wir die Spaltprodukte mittels gaschromatographischer Trennung gekoppelt mit massenspektrometrischer Detektion. 3,3'-Dichlorbenzidin, ein Karzinogen, welches durch die Spaltung von Pigment Orange (P.O.)13 entsteht, erwies sich in menschlichen Fibroblasten- und Keratinozyten-Zelllinien bereits unterhalb identifizierter Konzentrationen als zytotoxisch und genotoxisch in Form von DNA-Doppelstrangbrüchen. Während die photoinduzierte Spaltung ein Schlüsselereignis ist, das bei der Laserentfernung zum Verblassen der Tätowierungen führt, kann sie auch in kleinerem Umfang auftreten, wenn tätowierte Haut natürlichem Licht, insbesondere im ultravioletten (UV) Bereich, ausgesetzt wird. Da lichtinduzierte Effekte die häufigsten Nebenwirkungen im Zusammenhang mit Tätowierungen sind, war es unser Ziel, die zugrunde liegenden Pathomechanismen entsprechend zu untersuchen. Allerdings wurde in der Literatur bisher kein in vitro Modell beschrieben, das die Architektur tätowierter menschlicher Haut genau nachbildet. Daher etablierten wir ein 3D in vitro "tätowiertes" Vollhautmodell (TatSFT) als Tierersatzmodell für die Tätowiermittelforschung. Die Aufnahme der in dieser Studie verwendeten Tattoo-Pigmente Titandioxid (TiO2) Anatas, TiO2 Rutil, Pigment Orange (P.O.)13 und Carbon Black durch dermale Fibroblasten wurde elektronenmikroskopisch in TatSFT nachgewiesen. Trotz dieser Aufnahme zeigten die Pigmente keinen Einfluss auf die Lebensfähigkeit von TatSFT oder dessen dermales Kompartiment (TatSDE). Auch die Zytokinsekretion, die allgemeine Histologie und die Expression wichtiger Marker der Hauthomöostase wurden durch die Tattoo-Pigmente in TatSFT nicht beeinflusst. Im Gegensatz zur fehlenden Toxizität in 3D verringerte TiO2-Anatas signifikant die Zellviabilität und erhöhte die Interleukin-8-Ausschüttung in 2D-monolayer kultivierten Fibroblasten. Aufgrund der inhärenten Unterschiede in der Toxizitätsempfindlichkeit von 2D-monolayer kultivierten Fibroblasten und 3D kultivierten Modellen, untersuchten wir die Phototoxizität von Tattoo-Pigmenten sowohl in 2D als auch in 3D. Übereinstimmend mit der Partikeltoxizität waren die phototoxischen Effekte in Einzelschicht-kultivierten Fibroblasten größer als in 3D. Während TiO2 starke phototoxische Effekte in 2D zeigte, waren diese Effekte in TatSFT nicht vorhanden. Allerdings wurden die UVB-induzierten DNA-Schadensmarker in der Dermis von TatSFT durch die Pigmente reduziert. In Kombination mit den photoprotektiven Effekten, die in TatSFT hinsichtlich der Lebensfähigkeit gefunden wurden, deuten diese Daten auf photoprotektive Eigenschaften von Tätowierpigmenten für die tätowierte Dermis und das darunter liegende Gewebe hin. Im Gegensatz zu diesen Ergebnissen fanden wir, dass P.O.13 die Zytokinsekretion bei UV-Bestrahlung sowohl in 2D als auch in TatSDE verändert. Obwohl geringe Mengen an Spaltprodukten von P.O.13 nach UV-Bestrahlung identifiziert wurden, konnten wir nicht nachweisen, dass diese Spaltprodukte nachteilige Effekte hervorgerufen haben könnten. Die Daten in dieser Arbeit unterstreichen nicht nur die Notwendigkeit von 3D-Testsystemen für die Forschung zur Phototoxizität von Tätowierungen, sondern stellen auch ein hochgradig modifizierbares 3D in vitro Testsystem für diesen Zweck dar. Zusätzlich stärkt diese Arbeit auch die Bedenken bezüglich der Verwendung von TiO2 Anatas und Azopigmenten, wie P.O.13, in Tätowiertinten

Indirect co-cultivation of HepG2 with differentiated THP-1 cells induces AHR signalling and release of pro-inflammatory cytokines

HepG2 and THP-1 cells, the latter differentiated by phorbol 12-myristate 13-acetate (PMA), were co-cultured and characterized for typical liver-specific functions, such as xenobiotic detoxification, lipid and cholesterol metabolism. Furthermore, liver injury-associated pathways, such as inflammation, were studied. In general, the co-cultivation of these cells produced a pro-inflammatory system, as indicated by increased levels of cytokines (IL-8, TGF-alpha, IL-6, GM-CSF, G-CSF, TGF-beta, and hFGF) in the respective supernatant. Increased expression levels of target genes of the aryl hydrocarbon receptor (AHR), e.g., CYP1A1, CYP1A2 and CYP1B1, were detected, accompanied by the increased enzyme activity of CYP1A1. Moreover, transcriptome analyses indicated a significant upregulation of cholesterol biosynthesis, which could be reduced to baseline levels by lovastatin. In contrast, total de novo lipid synthesis was reduced in co-cultured HepG2 cells. Key events of the adverse outcome pathway (AOP) for fibrosis were activated by the co-cultivation, however, no increase in the concentration of extracellular collagen was detected. This indicates, that AOP should be used with care. In summary, the indirect co-culture of HepG2/THP-1 cells results in an increased release of pro-inflammatory cytokines, an activation of the AHR pathway and an increased enzymatic CYP1A activity

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

Oral and Fecal Campylobacter concisus Strains Perturb Barrier Function by Apoptosis Induction in HT-29/B6 Intestinal Epithelial Cells

Campylobacter concisus infections of the gastrointestinal tract can be accompanied by diarrhea and inflammation, whereas colonization of the human oral cavity might have a commensal nature. We focus on the pathophysiology of C. concisus and the effects of different clinical oral and fecal C. concisus strains on human HT-29/B6 colon cells. Six oral and eight fecal strains of C. concisus were isolated. Mucus-producing HT-29/B6 epithelial monolayers were infected with the C. concisus strains. Transepithelial electrical resistance (Rt) and tracer fluxes of different molecule size were measured in Ussing chambers. Tight junction (TJ) protein expression was determined by Western blotting, and subcellular TJ distribution was analyzed by confocal laser-scanning microscopy. Apoptosis induction was examined by TUNEL-staining and Western blot of caspase-3 activation. All strains invaded confluent HT-29/B6 cells and impaired epithelial barrier function, characterized by a time- and dose-dependent decrease in Rt either after infection from the apical side but even more from the basolateral compartment. TJ protein expression changes were sparse, only in apoptotic areas of infected monolayers TJ proteins were redistributed. Solely the barrier-forming TJ protein claudin-5 showed a reduced expression level to 66±8% (P<0.05), by expression regulation from the gene. Concomitantly, Lactate dehydrogenase release was elevated to 3.1±0.3% versus 0.7±0.1% in control (P<0.001), suggesting cytotoxic effects. Furthermore, oral and fecal C. concisus strains elevated apoptotic events to 5-fold. C. concisus-infected monolayers revealed an increased permeability for 332 Da fluorescein (1.74±0.13 vs. 0.56±0.17 10−6 cm/s in control, P<0.05) but showed no difference in permeability for 4 kDa FITC-dextran (FD-4). The same was true in camptothecin-exposed monolayers, where camptothecin was used for apoptosis induction

Evaluating expert-based habitat suitability information of terrestrial mammals with GPS-tracking data

Aim Macroecological studies that require habitat suitability data for many species often derive this information from expert opinion. However, expert-based information is inherently subjective and thus prone to errors. The increasing availability of GPS tracking data offers opportunities to evaluate and supplement expert-based information with detailed empirical evidence. Here, we compared expert-based habitat suitability information from the International Union for Conservation of Nature (IUCN) with habitat suitability information derived from GPS-tracking data of 1,498 individuals from 49 mammal species. Location Worldwide. Time period 1998-2021. Major taxa studied Forty-nine terrestrial mammal species. Methods Using GPS data, we estimated two measures of habitat suitability for each individual animal: proportional habitat use (proportion of GPS locations within a habitat type), and selection ratio (habitat use relative to its availability). For each individual we then evaluated whether the GPS-based habitat suitability measures were in agreement with the IUCN data. To that end, we calculated the probability that the ranking of empirical habitat suitability measures was in agreement with IUCN's classification into suitable, marginal and unsuitable habitat types. Results IUCN habitat suitability data were in accordance with the GPS data (> 95% probability of agreement) for 33 out of 49 species based on proportional habitat use estimates and for 25 out of 49 species based on selection ratios. In addition, 37 and 34 species had a > 50% probability of agreement based on proportional habitat use and selection ratios, respectively. Main conclusions We show how GPS-tracking data can be used to evaluate IUCN habitat suitability data. Our findings indicate that for the majority of species included in this study, it is appropriate to use IUCN habitat suitability data in macroecological studies. Furthermore, we show that GPS-tracking data can be used to identify and prioritize species and habitat types for re-evaluation of IUCN habitat suitability data

Femtosecond Operation of the LCLS for User Experiments

In addition to its normal operation at 250pC, the LCLS has operated with 20pC bunches delivering X-ray beams to users with energies between 800eV and 2 keV and with bunch lengths below 10 fs FWHM. A bunch arrival time monitor and timing transmission system provide users with sub 50 fs synchronization between a laser and the X-rays for pump/probe experiments. We describe the performance and operational experience of the LCLS for short bunch experiments

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 (Ti

Operational Performance of LCLS Beam Instrumentation

The Linac Coherent Light Source (LCLS) X-ray FEL utilizing the last km of the SLAC linac has been operational since April 2009 and finished its first successful user run last December. The various diagnostics for electron beam properties including beam position monitors, wire scanners, beam profile monitors, and bunch length diagnostics are presented as well as diagnostics for the X-ray beam. The low emittance and ultra-short electron beam required for X-ray FEL operation has implications on the transverse and longitudinal diagnostics. The coherence effects of the beam profile monitors and the challenges of measuring fs long bunches are discussed

Surface Characterization of the LCLS RF Gun Cathode

The first copper cathode installed in the LCLS RF gun was used during LCLS commissioning for more than a year. However, after high charge operation (&gt; 500 pC), the cathode showed a decline of quantum efficiency within the area of drive laser illumination. They report results of SEM, XPS and XAS studies that were carried out on this cathode after it was removed from the gun. X-ray absorption and X-ray photoelectron spectroscopy reveal surface contamination by various hydrocarbon compounds. In addition they report on the performance of the second installed cathode with emphasis on the spatial distribution of electron emission