Christine Götz

Polybrominated diphenyl ethers induce developmental neurotoxicity in a human in vitro model: evidence for endocrine disruption.

Background Polybrominated diphenyl ethers (PBDEs) are persistent and bioaccumulative flame retardants, which are found in rising concentrations in human tissues. They are of concern for human health because animal studies have shown that they possess the potential to be developmentally neurotoxic. Objective Because there is little knowledge of the effects of PBDEs on human brain cells, we investigated their toxic potential for human neural development in vitro. Moreover, we studied the involvement of thyroid hormone (TH) disruption in the effects caused by PBDEs. Methods We used the two PBDE congeners BDE-47 and BDE-99 (0.1–10 μM), which are most prominent in human tissues. As a model of neural development, we employed primary fetal human neural progenitor cells (hNPCs), which are cultured as neurospheres and mimic basic processes of brain development in vitro: proliferation, migration, and differentiation. Results PBDEs do not disturb hNPC proliferation but decrease migration distance of hNPCs. Moreover, they cause a reduction of differentiation into neurons and oligodendrocytes. Simultaneous exposure with the TH receptor (THR) agonist triiodothyronine rescues these effects on migration and differentiation, whereas the THR antagonist NH-3 does not exert an additive effect. Conclusion PBDEs disturb development of hNPCs in vitro via endocrine disruption of cellular TH signaling at concentrations that might be of relevance for human exposure.

Xenobiotic metabolism capacities of human skin in comparison with a 3D epidermis model and keratinocyte-based cell culture as in vitro alternatives for chemical testing: activating enzymes (Phase I)

Abstract:  The 7th Amendment to the EU Cosmetics Directive prohibits the use of animals in cosmetic testing for certain endpoints, such as genotoxicity. Therefore, skin in vitro models have to replace chemical testing in vivo. However, the metabolic competence neither of human skin nor of alternative in vitro models has so far been fully characterized, although skin is the first‐pass organ for accidentally or purposely (cosmetics and pharmaceuticals) applied chemicals. Thus, there is an urgent need to understand the xenobiotic‐metabolizing capacities of human skin and to compare these activities to models developed to replace animal testing. We have measured the activity of the phase II enzymes glutathione S‐transferase, UDP‐glucuronosyltransferase and N‐acetyltransferase in ex vivo human skin, the 3D epidermal model EpiDerm 200 (EPI‐200), immortalized keratinocyte‐based cell lines (HaCaT and NCTC 2544) and primary normal human epidermal keratinocytes. We show that all three phase II enzymes are present and highly active in skin as compared to phase I. Human skin, therefore, represents a more detoxifying than activating organ. This work systematically compares the activities of three important phase II enzymes in four different in vitro models directly to human skin. We conclude from our studies that 3D epidermal models, like the EPI‐200 employed here, are superior over monolayer cultures in mimicking human skin xenobiotic metabolism and thus better suited for dermatotoxicity testing.

Elucidation of Xenobiotic Metabolism Pathways in Human Skin and Human Skin Models by Proteomic Profiling

Background Human skin has the capacity to metabolise foreign chemicals (xenobiotics), but knowledge of the various enzymes involved is incomplete. A broad-based unbiased proteomics approach was used to describe the profile of xenobiotic metabolising enzymes present in human skin and hence indicate principal routes of metabolism of xenobiotic compounds. Several in vitro models of human skin have been developed for the purpose of safety assessment of chemicals. The suitability of these epidermal models for studies involving biotransformation was assessed by comparing their profiles of xenobiotic metabolising enzymes with those of human skin. Methodology/Principal Findings Label-free proteomic analysis of whole human skin (10 donors) was applied and analysed using custom-built PROTSIFT software. The results showed the presence of enzymes with a capacity for the metabolism of alcohols through dehydrogenation, aldehydes through dehydrogenation and oxidation, amines through oxidation, carbonyls through reduction, epoxides and carboxylesters through hydrolysis and, of many compounds, by conjugation to glutathione. Whereas protein levels of these enzymes in skin were mostly just 4–10 fold lower than those in liver and sufficient to support metabolism, the levels of cytochrome P450 enzymes were at least 300-fold lower indicating they play no significant role. Four epidermal models of human skin had profiles very similar to one another and these overlapped substantially with that of whole skin. Conclusions/Significance The proteomics profiling approach was successful in producing a comprehensive analysis of the biotransformation characteristics of whole human skin and various in vitro skin models. The results show that skin contains a range of defined enzymes capable of metabolising different classes of chemicals. The degree of similarity of the profiles of the in vitro models indicates their suitability for epidermal toxicity testing. Overall, these results provide a rational basis for explaining the fate of xenobiotics in skin and will aid chemical safety testing programmes.

Photoprotection against UVAR: effective triterpenoids require a lipid raft stabilizing chemical structure

Abstract:  UVA(Ultraviolet A)‐induced gene expression is supposed to be a hallmark for inflammation, for immunosuppression and in long‐term cancer formation. In previous studies, we have shown for keratinocytes that physiological doses of UVA radiation result in the upregulation of gene expression mediated by ceramide formation from sphingolipids/cholesterol‐rich microdomains (rafts), which can be blocked by preloading keratinocytes with cholesterol or plant sterols. Here, we show that besides stigmasterol and ß‐sitosterol, also sterols like 14‐dehydroergosterol, ergosterol‐peroxide and 29‐norcycloartenol inhibit the UVA response. Moreover, we present evidence that natural material‐derived triterpenoids such as oleanolic acid can abrogate UVA‐induced gene expression by raft stabilization. This effect depends on the structure of the molecule, because its isomer ursolic acid also integrates within the rafts without inhibiting ceramide formation and upregulation of gene expression.

Regulation of dioxin receptor function by different beta-carboline alkaloids

The dioxin receptor, also known as arylhydrocarbon receptor (AhR), is a ligand-activated transcription factor that mediates the toxicity of dioxins and related environmental contaminants. In addition, there is a growing list of natural compounds, mainly plant polyphenols that can modulate AhR function and downstream signaling with quite unknown consequences for cellular function. We investigate the potential of four different β-carboline alkaloids to stimulate AhR signaling in human hepatoma cells and keratinocytes. Three test substances, namely rutaecarpine, annomontine and xestomanzamine A, increase AhR-driven reporter gene activity as well as expression of two AhR target genes in a dose-dependent and time-dependent manner. Additionally, the three test alkaloids stimulate cytochrome P450 (CYP) 1 enzyme activity without showing any antagonistic effects regarding benzo(a)pyrene-stimulated CYP1 activation. The AhR-activating property of the β-carbolines is completely abrogated in AhR-deficient cells providing evidence that rutaecarpine, annomontine and xestomanzamine A are natural stimulators of the human AhR. The toxicological relevance of beta-carboline-mediated AhR activation is discussed.

Epigallocatechin-3-gallate does not affect the activity of enzymes involved in metabolic activation and cellular excretion of benzo[a]pyrene in human colon carcinoma cells.

Benzo[a]pyrene (B[a]P) and related procarcinogens found in cigarette smoke and roasted foodstuff require metabolic activation to build mutagenic DNA adducts that may cause tumor diseases like colorectal cancer. The major B[a]P-activating enzymes belong to the cytochrome-P450 (CYP)-1 family and are regulated by the aryl hydrocarbon receptor (AhR). Previous studies have indicated that an inhibition of AhR is accompanied with a reduced metabolic activation of B[a]P and therefore may act protective against carcinogenesis. We investigated if the green tea flavonoid (-)-epigallocatechin-3-gallate (EGCG), a known AhR inhibitor, is able to influence B[a]P-metabolizing and B[a]P-transporting enzymes in human Caco-2 colon carcinoma cells. Strikingly, treatment with EGCG did neither affect constitutive and B[a]P-inducible expression of CYP1A1 and UDP-glucuronosyltransferase (UGT)-1A1 nor overall CYP1 and UGT enzyme activities, indicating that EGCG does not antagonize the AhR in Caco-2 cells. Since flavonoids were also identified to enhance the activity of B[a]P-carrying transporter, we analyzed if EGCG exposure alters cellular excretion of B[a]P conjugates. In contrast to the positive control fisetin, EGCG did not affect cellular excretion of B[a]P metabolites. Our data provide evidence that EGCG does not alter the metabolism and transport of B[a]P in Caco-2 cells, and thus may not protect against procarcinogenic food contaminants.

Luteolin enhances the bioavailability of benzo(a)pyrene in human colon carcinoma cells.

We investigated the effect of luteolin, a plant-derived flavonoid, on benzo(a)pyrene (B(a)P)-stimulated drug metabolism and transport in human colon carcinoma cells. While luteolin treatment inhibited B(a)P-induced expression and activity of arylhydrocarbon receptor-dependent cytochrome P450 enzymes, the overall activity of UDP-glucuronosyltransferases and sulfotransferases was not affected by luteolin, indicating that luteolin affects phase-I but not phase-II function. Luteolin exposure decreased apical transport of B(a)P metabolites due to its interaction with the transporter breast cancer resistance protein. Inhibitor studies provide a first clue to the mechanism of luteolin-mediated inhibition of this transporter. The inhibition of both phase-I metabolism as well as phase-III transport by luteolin resulted in a 3-fold intracellular accumulation of radioactively labeled B(a)P. Our data reveal that luteolin is able to interfere with crucial steps of drug metabolism and thereby enhances the bioavailability of B(a)P. These findings are of special importance regarding future benefit-risk evaluations of preventive flavonoid usage.

Effects of the genotoxic compounds, benzo[a]pyrene and cyclophosphamide on phase 1 and 2 activities in EpiDerm™ models

The micronucleus assay in the 3D human reconstructed EpiDerm™ skin model (RSMN) is a promising new assay for evaluating genotoxicity of dermally applied chemicals. To complement the testing of metabolically activated chemicals, such as cyclophosphamide (CPA) and benzo[a]pyrene (B[a]P), we measured phase 1 (ethoxyresorufin O-deethylation (EROD) and testosterone metabolism) and 2 activities (UGTs and GSTs) in non-treated and genotoxin treated EpiDerm™ models in a study design which mimics the RSMN assay. The assay involved a three-dose dosing regimen over 72 h to take into account effects e.g. enzyme induction, which requires longer than the standard 2 dose 48-h assay. These studies demonstrated the presence of basal phase 1 and 2 activities of EpiDerm™ models. With the exception of GST, all of the activities measured did not reproducibly change over time. It was possible to measure enzyme induction using this assay design. EROD activity was significantly induced by B[a]P but not by CPA. CPA and B[a]P had little or no reproducible effects on GST and UGT activities. In conclusion, a number of metabolic enzyme activities were present in the EpiDerm™ skin model and at least the CYP1 family was inducible.

Filling the gaps: need for research on cell-specific xenobiotic metabolism in the skin

The removal and metabolism of organic molecules is apivotal body function, usually performed by phase I andphase II enzymes. While detoxification is beneficial andnecessary, at the same time, the metabolic intermediatesand breakdown products generated in the process can haveimmunogenic, cancerogenic or apoptogenic potential,which differs from the respective parental chemical.Whether and which adverse health effects ensue dependson the affected cell types and tissue. For instance, ifdamage caused by a chemical induces apoptosis, easilyregenerating tissues may cope better than slowly prolifer-ating or rare precursor cells. On the other hand, potentialextent of damage is conceivably dependent on the xeno-biotic metabolizing activity of cell types, which in turnmight be governed by exposure, that is, tissues with highexposure could be more competent in xenobiotic metabo-lism. There is, however, a surprising lack of research toidentify cell-specific xenobiotic metabolism. Research onthe molecular activity of xenobiotic metabolizing enzymes(XME) has traditionally a strong focus on the liver as themain metabolizing and detoxifying organ of the body. Theskin is less well studied, albeit it forms a major barrieragainst the environment (including the chemical environ-ment) and is a target of occupational, accidental andintended-use (i.e., cosmetics and pharmaceuticals) chemi-cal exposure. Risks of exposure are irritation, inflamma-tion, allergies and cancer. Conceivably, skin cells maypossess yet underestimated strategies to balance XME-mediated danger.In a recent paper, the team around Michael Girardi(Modi et al. 2012) presented the exciting finding that dif-ferential expression of phase I enzymes CYP4501A1 and1B1 in skin Langerhans cells versus keratinocytes leads togeneration of DNA-modifying metabolites from the car-cinogen 7,12-Dimethylbenz[a]anthracen (DMBA) in theskin and eventually non-melanoma skin cancer. Theyfound that skin Langerhans cells efficiently metabolizeDMBA to DMBA-3,4-diol which leads to oncogenic Hrasmutations. Mice lacking skin Langerhans cells had fewerskin tumours upon DMBA treatment. They conclude‘‘ Thus, tissue-associated dendritic cells can enhancechemical carcinogenesis via PAH metabolism, highlightingthe complex relation between immune cells and carcino-genesis.’’ While the paper of Girardi and colleagues looksat chemical-induced (DMBA-induced) cancer, it has alsoimportant implications for understanding skin physiologyand skin toxicology.Sun light, that is, its UV radiation component, is themost important stressor of skin (Fritsche et al. 2007) andhas long been known to be genotoxic and a major riskfactor in skin cancer (Narayanan et al. 2010). UV and UV-induced DNA damage are the primary cause not only formelanoma with increasing incidence and new casesworldwide every year (Little and Eide 2012), but also ofskin basal cell carcinoma, squamous cell carcinoma,Kaposi