Brunhilde Blömeke

Lack of association between the -260 C-->T promoter polymorphism of the endotoxin receptor CD14 gene and the CD14 density of unstimulated human monocytes and soluble CD14 plasma levels.

Abstract.Objective: CD14 is a receptor for endotoxin and binds components of Gram-positive and Gram-negative bacteria. CD14-bearing monocytes respond to stimulation with the increased synthesis and release of cytokines. The recently described –260 C→T promoter polymorphism of the CD14 gene has been found to be related to a risk of myocardial infarction. This study evaluated the role of this polymorphism in the expression of monocyte and soluble CD14. Moreover, the effect of the CD14 –260 genotypes for the ex vivo TNF-α response to endotoxin was analyzed in whole blood. Patients and participants: Ninety-five healthy blood donors were studied. Measurements and results: CD14 –260 genotyping was performed by means of a real-time PCR with fluorescence labeled hybridization probes. CD14 expression on human monocytes (mCD14) was assessed by fluorescence-activated cell sorting analysis with anti-CD14 monoclonal antibodies. Plasma levels of soluble CD14 (sCD14) were measured by ELISA. The TNF-α synthesis was determined by chemiluminescence in whole blood after endotoxin stimulation. There were no differences in mCD14 density, sCD14 levels, or the tumor necrosis factor-α concentrations between individuals with the three different CD14 –260 genotypes CC, CT, and TT. Conclusions: The CD14 –260 polymorphism does not affect the CD14 expression of unstimulated circulating monocytes or soluble CD14 plasma levels.

Potential of coculture in vitro models to study inflammatory and sensitizing effects of particles on the lung.

Exposure to particulate matter (PM) like nanoparticles (NPs) has increased in the last century due to increased combustion processes, road traffic, etc. In addition, the progress in chemical and cosmetic industry led to many new compounds, e.g. fragrances, which humans are exposed to every day. Many chemicals are known to act as contact and some as respiratory sensitizers, causing allergic reactions. Exposure to small particles of less than 100 nm in diameter is linked with an increased risk of respiratory diseases, such as asthma or rhinitis. To date already more than 1000 customer products contain eNPs without knowing much about the health effects. In comparison to chemicals, the mechanisms by which PM and eNPs can cause sensitization are still not fully understood. Validated and regulatory accepted in vitro models to assess this hazard in its full range are still missing. While a huge number of animal studies contributed to our knowledge about sensitization processes, knowledge on involved cellular mechanisms is still limited. In this review relevant in vitro models to study and elucidate these mechanisms in more detail are presented and their potential to serve as part of a tiered testing strategy is discussed.

Formation of genotoxic metabolites from anthraquinone glycosides, present in Rubia tinctorum L.

Rubia tinctorum L., a medicinal plant used for the treatment of kidney and bladder stones, contains a characteristic spectrum of 9,10-anthraquinone derivatives, which are substituted in only one of the aromatic benzo rings. The majority of the anthraquinones present in the plant itself or in plant extracts are glycosides. We investigated the metabolism of two such glycosides, alizarinprimeveroside (AlP) and lucidinprimeveroside (LuP). AlP given orally to rats was metabolized to alizarin (Al) and 1-hydroxyanthraquinone (1-HA). The reductive cleavage of AlP was also observed after treatment of this compound with rat liver enzymes (S9) and NADPH. 1-HA has been reported to induce unscheduled DNA synthesis (UDS) in primary rat hepatocytes (PRH) and intestinal and liver tumors in rats after chronic treatment. The in vitro genotoxicity of 1-HA was confirmed by our present investigations. We also observed that the glycoside AlP was active at inducing UDS in PRH, but the compound was inactive in the Salmonella/microsome assay. Oral administration of LuP to rats resulted in the excretion of lucidin and rubiadin. When LuP was treated with rat liver extract and NADPH, the compound was reduced to rubiadinprimeveroside (RuP), which was hydrolyzed to rubiadin. We have recently shown that lucidin is highly genotoxic in a battery of short-term tests. We now report that rubiadin is also highly genotoxic in Salmonella typhimurium. However, in contrast to lucidin, it requires metabolic activation. In the UDS assay in PRH, rubiadin was even more potent than lucidin and equal to the positive control DMBA. In addition, the glycoside LuP is active in the Salmonella/microsome assay as well as in the UDS assay. The present work demonstrates that the uptake of the anthraquinone glycosides AlP and LuP leads to the rodent carcinogen 1-HA, and to the highly genotoxic compounds lucidin and rubiadin. This extends our previous studies and supports our suggestion that the therapeutic use of Rubia tinctorum may involve a carcinogenic risk.

Cryobanking of viable biomaterials: implementation of new strategies for conservation purposes

Cryobanking, the freezing of biological specimens to maintain their integrity for a variety of anticipated and unanticipated uses, offers unique opportunities to advance the basic knowledge of biological systems and their evolution. Notably, cryobanking provides a crucial opportunity to support conservation efforts for endangered species. Historically, cryobanking has been developed mostly in response to human economic and medical needs — these needs must now be extended to biodiversity conservation. Reproduction technologies utilizing cryobanked gametes, embryos and somatic cells are already vital components of endangered species recovery efforts. Advances in modern biological research (e.g. stem cell research, genomics and proteomics) are already drawing heavily on cryobanked specimens, and future needs are anticipated to be immense. The challenges of developing and applying cryobanking for a broader diversity of species were addressed at an international conference held at Trier University (Germany) in June 2008. However, the magnitude of the potential benefits of cryobanking stood in stark contrast to the lack of substantial resources available for this area of strategic interest for biological science — and society at large. The meeting at Trier established a foundation for a strong global incentive to cryobank threatened species. The establishment of an Amphibian Ark cryobanking programme offers the first opportunity for global cooperation to achieve the cryobanking of the threatened species from an entire vertebrate class.

Multiple Cytochrome P450–Isoenzymes mRNA Are Expressed in Dendritic Cells

Monocytes and dendritic cells (DC) play a critical role in antigen processing and presentation in vivo. Several allergic reactions such as allergic dermatitis as well as drug allergy are mediated most often by small molecular weight compounds. Those haptens must be bound to high molecular weight compounds in order to become antigenic according to the generally accepted hapten concept of Landsteiner. Most haptens however are chemically inert and require metabolic activation. Human cytochrome P450 (CYP) isoenzymes are known to metabolize small molecular weight compounds to highly reactive species which are able to bind to proteins which is considered as a prerequisite for the sensitization by those small molecular weight compounds [1]. In our own studies we were able to show that the response of lymphocytes of sensitized patients to pphenylenediamine is increased after adding murine liver microsomes which contain CYP-dependent isoenzymes in the lymphocyte transformation test [2]. Recently it has been shown that eugenol which is a frequent sensitizer does not sensitize CYP1A1 knockout mice whereas the wild-type mice are sensitized to eugenol suggesting that CYP1A1-dependent metabolites of eugenol are the nominative antigen [3]. Therefore in this study we were interested in which CYP isoenzymes were present in antigen-presenting cells such as DC and compared the results with the presence of these isoenzymes in other antigen-presenting cells such as monocytes.

Evaluation of cytochrome P450 1 (CYP1) and N-acetyltransferase 1 (NAT1) activities in HaCaT cells: Implications for the development of in vitro techniques for predictive testing of contact sensitizers

Xenobiotic metabolizing enzymes like cytochrome P450s and N-acetyltransferase are expressed in keratinocytes and professional antigen-presenting cells. Thus, biotransformation of chemicals applied to the skin can be relevant for their potential to cause skin toxicity and immune responses like allergic contact dermatitis. Considering the keratinocyte cell line HaCaT as a relevant in vitro tool for epidermal biotransformation, we specifically investigated CYP1 (EROD) and N-acetyltransferase 1 (NAT1) activities of three different HaCaT shipments and human primary keratinocytes (NHEK). Solvent treated HaCaT showed EROD levels near the detection limit (0.047 pmol/mg/min), primary keratinocytes (n=4) were in a range between 0 and 0.76 pmol/mg/min. B[a]P (1 microM) induced EROD activities of 19.0+/-0.9 pmol/mg/min (n=11) in HaCaT and 5.8+/-0.5 pmol/mg/min (n=4) in NHEK. N-acetylation activities for para-aminobenzoic acid (PABA) were in average 3.4-fold higher in HaCaT compared to NHEK (8+/-0.5 nmol/mg/min) and varied between the HaCaT shipments (range 12.0-44.5 nmol/mg/min). This was in good agreement with NAT1 promoter P1 dependent mRNA level and N-acetylation of the contact allergen para-phenylenediamine (PPD) under typical cell-based assay conditions. We conclude that HaCaT represent a suitable in vitro model for studying the qualitative contribution of epidermal phase1/phase2 metabolism to toxicological endpoints such as skin sensitization.

Introduction of a methoxymethyl side chain into p-phenylenediamine attenuates its sensitizing potency and reduces the risk of allergy induction

The strong sensitizing potencies of the most important primary intermediates of oxidative hair dyes, p-phenylenediamine (PPD) and p-toluylenediamine (PTD, i.e. 2-methyl-PPD) are well established. They are considered as the key sensitizers in hair dye allergic contact dermatitis. While modification of their molecular structure is expected to alter their sensitizing properties, it may also impair their color performance. With introduction of a methoxymethyl side chain we found the primary intermediate 2-methoxymethyl-p-phenylenediamine (ME-PPD) with excellent hair coloring performance but significantly reduced sensitizing properties compared to PPD and PTD: In vitro, ME-PPD showed an attenuated innate immune response when analyzed for its protein reactivity and dendritic cell activation potential. In vivo, the effective concentration of ME-PPD necessary to induce an immune response 3-fold above vehicle control (EC3 value) in the local lymph node assay (LLNA) was 4.3%, indicating a moderate skin sensitizing potency compared to values of 0.1 and 0.17% for PPD and PTD, respectively. Finally, assessing the skin sensitizing potency of ME-PPD under consumer hair dye usage conditions through a quantitative risk assessment (QRA) indicated an allergy induction risk negligible compared to PPD or PTD.

Cross Talk between Keratinocytes and Dendritic Cells: Impact on the Prediction of Sensitization

Understanding the mechanistic aspects involved in sensitization by chemicals will help to develop relevant preventive strategies. Many potential sensitizers are not directly immunogenic but require activation outside or inside the skin by nonenzymatic oxidation (prehaptens) or metabolic transformation (prohaptens) prior to being able to induce an immune response. This necessary activation step has not yet been actively integrated into a cell line-based prediction approach. We cocultured HaCaT keratinocytes with THP-1 as dendritic cell-like cells allowing intercellular interactions. The sensitizing potential was determined by analyzing differences in the expression of CD86, CD40, and CD54 on cocultured THP-1 cells. This new assay setup allowed (1) to distinguish irritants from allergens without influencing cell viability and (2) to discriminate pre/prohaptens from haptens. Under coculture conditions, the prohaptens eugenol, 2-methoxy-4-methylphenol, and benzo[a]pyrene induced a significantly higher upregulation of CD86 expression on THP-1. In agreement with the hapten concept, responses to 2,4-dinitrochlorobenzene, Bandrowskis base, and the prehapten isoeugenol were not significantly modified. Inhibition of cytochrome P450 or NAD(P)H:quinone oxidoreductase (NQO1) activity reduced the prohapten-mediated upregulation of CD86 on cocultured THP-1 cells. This coculture assay allowing cross talk between HaCaT and THP-1 cells appears to be suitable for the detection of prohaptens, is reproducible, easy to perform, and avoids donor variations. In addition, this assay is a promising approach to understand the impact of cross talk on the prediction of sensitization and once established may be integrated in a future in vitro toolbox to detect potential skin sensitizers and may thus contribute to reduce animal testing.

Extrahepatic metabolism at the body's internal–external interfaces

Abstract In general, xenobiotic metabolizing enzymes (XMEs) are expressed in lower levels in the extrahepatic tissues than in the liver, making the former less relevant for the clearance of xenobiotics. Local metabolism, however, may lead to tissue-specific adverse responses, e.g. organ toxicities, allergies or cancer. This review summarizes the knowledge on the expression of phase I and phase II XMEs and transporters in extrahepatic tissues at the bodys internal–external interfaces. In the lung, CYPs of families 1, 2, 3 and 4 and epoxide hydrolases are important phase I enzymes, while conjugation is less relevant. In skin, phase I-related enzymatic reactions are considered less relevant. Predominant skin XMEs are phase II enzymes, whereby glucuronosyltransferases (UGT) 1, glutathione-S-transferase (GST) and N-acetyltransferase (NAT) 1 are important for detoxification. The intestinal epithelium expresses many transporters and phase I XME with high levels of CYP3A4 and CYP3A5 and phase II metabolism is mainly related to UGT, NAT and Sulfotransferases (SULT). In the kidney, conjugation reactions and transporters play a major role for excretion processes. In the bladder, CYPs are relevant and among the phase II enzymes, NAT1 is involved in the activation of bladder carcinogens. Expression of XMEs is regulated by several mechanisms (nuclear receptors, epigenetic mechanisms, microRNAs). However, the understanding why XMEs are differently expressed in the various tissues is fragmentary. In contrast to the liver – where for most XMEs lower expression is demonstrated in early life – the XME ontogeny in the extrahepatic tissues remains to be investigated.

Impact of aryl hydrocarbon receptor (AhR) knockdown on cell cycle progression in human HaCaT keratinocytes

Abstract While activation of the aryl hydrocarbon receptor (AhR) by exogenous ligands is well investigated, its physiological function is less understood. By extending research in AhR biology, evidence appeared that the receptor generally plays an important role in cell physiology. In keratinocytes, little is known about endogenous functions of the AhR. In order to expand this knowledge, we analyzed the impact of AhR knockdown on cell cycle progression in HaCaT cells and showed that proliferation of siAhR HaCaT cells was significantly decreased. In line with that result, western blot analysis revealed that protein level of the cyclin dependent kinase inhibitor p27KIP1 was increased, whereas protein level of the cyclin dependent kinase (CDK) 2 was reduced. CDK4 and CDK6 protein levels remained unchanged, whereas protein level of the retinoblastoma protein (pRB) was reduced. By measuring ethoxyresorufin-O-deethylase (EROD) activity we showed that endogenous cytochrome P450 1 (CYP1), especially CYP1A1 is required for normal cell cycle in HaCaT cells, as well. To the best of our knowledge, we provide evidence for the first time in human skin cells, that in the absence of exogenous ligands, the AhR promotes cell cycle progression in HaCaT cells and one can speculate that this is the physiological function of this receptor in keratinocytes.