BackgroundPolybrominated 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.ObjectiveBecause 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.MethodsWe 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.ResultsPBDEs 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.ConclusionPBDEs disturb development of hNPCs in vitro via endocrine disruption of cellular TH signaling at concentrations that might be of relevance for human exposure.

RAW 264.7 macrophages, when challenged with a combination of lipopolysaccharide (10 g/ml) and interferon-␥ (100 units/ml), respond with endogenous NO ⅐ formation, which ultimately results in apoptotic cell death. Apoptosis is detected morphologically by chromatin condensation. Concomitantly we noticed the accumulation of the tumor suppressor protein p53. NO ⅐ -derived apoptosis was blocked by the NO ⅐ -synthase inhibitor N Gmonomethyl-L-arginine. Repetitive treatment of RAW 264.7 macrophages with lipopolysaccharide/interferon-␥, followed by subculturing viable cells, allowed us to select resistant macrophages which we called RES. RES cells still produced comparable amounts of nitrite/nitrate in response to agonist treatment but showed no apoptotic markers, i.e. chromatin condensation or p53 accumulation. However, RES macrophages undergo apoptosis in the presence of exogenously supplied NO ⅐ , released from the NO-donors S-nitrosoglutathione or spermine-NO. Assessment of cytochrome c reduction established that RES cells released twice the amount of superoxide compared to RAW 264.7 macrophages under both resting and stimulated conditions. We linked increased superoxide production to cellular macrophage resistance by demonstrating decreased apoptosis after simultaneous application of S-nitrosoglutathione or spermine-NO and the redox cycler 2,3-dimethoxy-1,4-naphthoquinone. Our results suggest that macrophage resistance toward NO ⅐ -mediated apoptosis is, at least in part, due to increased superoxide formation. Therefore, the balance between reactive nitrogen and reactive oxygen species regulates RAW 264.7 macrophage apoptosis.Nitric oxide (NO) 1 is recognized for its participation in diverse biological processes in nearly all aspects of life (1-3). The formation of NO ⅐ occurs under both physiological and pathophysiological settings. The molecule is synthesized by a family of enzymes termed NO ⅐ synthases (NOS), which utilize arginine as their substrate in the generation of NO ⅐ and stoichiometric amounts of citrulline (4). For convenience, two types of NOS are recognized; constitutive isoforms, which are active for a relatively short time in response to intracellular Ca 2ϩ fluctuations, and a cytokine-inducible isoform. For the latter to be active, mRNA translation and protein synthesis are required. The inducible NOS generates large amounts of NO ⅐ for an extended period. However, once NO ⅐ is produced by the action of NOS, it is extremely susceptible to both oxidation and reduction. This results in the concomitant formation of species with NO ϩ -like activity (nitrosonium ion) or NO Ϫ (nitroxyl anion), respectively (5). In addition to reacting with oxygen, superoxide, and transition metals, NO ⅐ causes biological signaling via interactions with sulfhydryl groups. Because multiple NO surrogates are formed, transduction pathways are classified as either cyclic GMP-dependent or -independent. Cyclic GMP formation is initiated by NO ⅐ binding to the heme group of soluble guanylyl cyclase, thus causing enzyme activati...

Skin is important for the absorption and metabolism of exposed chemicals such as cosmetics or pharmaceuticals. The Seventh Amendment to the EU Cosmetics Directive prohibits the use of animals for cosmetic testing for certain endpoints, such as genotoxicity; therefore, there is an urgent need to understand the xenobiotic metabolizing capacities of human skin and to compare these activities with reconstructed 3D skin models developed to replace animal testing. We have measured Phase I enzyme activities of cytochrome P450 (CYP) and cyclooxygenase (COX) in ex vivo human skin, the 3D skin model EpiDermÔ (EPI-200), immortalized keratinocyte-based cell lines and primary normal human epidermal keratinocytes. Our data demonstrate that basal CYP enzyme activities are very low in whole human skin and EPI-200 as well as keratinocytes. In addition, activities in monolayer cells differed from organotypic tissues after induction. COX activity was similar in skin, EPI-200 and NHEK cells, but was significantly lower in immortalized keratinocytes. Hence, the 3D model EPI-200 might represent a more suitable model for dermatotoxicological studies. Altogether, these data help to better understand skin metabolism and expand the knowledge of in vitro alternatives used for dermatotoxicity testing.Abbreviations: CYP, cytochrome P450-monooxygenase; COX, cyclooxygenase; PGE 2, prostaglandin E2; 3-MC, 3-methylcholanthrene; EPI-200, reconstituted epidermis model EpiDermÔ (MatTek); NHEK, normal human keratinocytes; HLM, human liver microsomes.

BackgroundHuman 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 FindingsLabel-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/SignificanceThe 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.