Toward the stereochemical identification of prohibited characterizing flavors in tobacco products: the case of strawberry flavor.

With the revision of the European Tobacco Products Directive (2014/40/EU), characterizing flavors such as strawberry, candy, vanillin or chocolate will be prohibited in cigarettes and fine-cut tobacco. Product surveillance will therefore require analytical means to define and subsequently detect selected characterizing flavors that are formed by supplemented flavors within the complex matrix tobacco. We have analyzed strawberry-flavored tobacco products as an example for characterizing fruit-like aroma. Using this approach, we looked into aroma components to find indicative patterns or features that can be used to satisfy obligatory product information as requested by the European Directive. Accordingly, a headspace solid-phase microextraction (HS-SPME) technique was developed and coupled to subsequent gas chromatography-mass spectrometry (GC/MS) to characterize different strawberry-flavored tobacco products (cigarettes, fine-cut tobacco, liquids for electronic cigarettes, snus, shisha tobacco) for their volatile additives. The results were compared with non-flavored, blend characteristic flavored and other fruity-flavored cigarettes, as well as fresh and dried strawberries. Besides different esters and aldehydes, the terpenes linalool, α-terpineol, nerolidol and limonene as well as the lactones γ-decalactone, γ-dodecalactone and γ-undecalactone could be verified as compounds sufficient to convey some sort of strawberry flavor to tobacco. Selected flavors, i.e., limonene, linalool, α-terpineol, citronellol, carvone and γ-decalactone, were analyzed further with respect to their stereoisomeric composition by using enantioselective HS-SPME-GC/MS. These experiments confirmed that individual enantiomers that differ in taste or physiological properties can be distinguished within the tobacco matrix. By comparing the enantiomeric composition of these compounds in the tobacco with that of fresh and dried strawberries, it can be concluded that non-natural strawberry aroma is usually used to produce strawberry-flavored tobacco products. Such authenticity control can become of interest particularly when manufacturers claim that natural sources were used for flavoring of products. Although the definition of characterizing flavors by analytical means remains challenging, specific compounds or features are required to be defined for routine screening of reported information. Clarifications by sensory testing might still be necessary, but could be limited to a few preselected samples.

Chemical hazards present in liquids and vapors of electronic cigarettes.

Electronic (e-)cigarettes have emerged in recent years as putative alternative to conventional tobacco cigarettes. These products do not contain typical carcinogens that are present in tobacco smoke, due to the lack of combustion. However, besides nicotine, hazards can also arise from other constituents of liquids, such as solvents, flavors, additives and contaminants. In this study, we have analyzed 28 liquids of seven manufacturers purchased in Germany. We confirm the presence of a wide range of flavors to enhance palatability. Although glycerol and propylene glycol were detected in all samples, these solvents had been replaced by ethylene glycol as dominant compound in five products. Ethylene glycol is associated with markedly enhanced toxicological hazards when compared to conventionally used glycerol and propylene glycol. Additional additives, such as coumarin and acetamide, that raise concerns for human health were detected in certain samples. Ten out of 28 products had been declared "free-of-nicotine" by the manufacturer. Among these ten, seven liquids were identified containing nicotine in the range of 0.1-15 µg/ml. This suggests that "carry over" of ingredients may occur during the production of cartridges. We have further analyzed the formation of carbonylic compounds in one widely distributed nicotine-free brand. Significant amounts of formaldehyde, acetaldehyde and propionaldehyde were only found at 150 °C by headspace GC-MS analysis. In addition, an enhanced formation of aldehydes was found in defined puff fractions, using an adopted machine smoking protocol. However, this effect was delayed and only observed during the last third of the smoking procedure. In the emissions of these fractions, which represent up to 40 % of total vapor volume, similar levels of formaldehyde were detected when compared to conventional tobacco cigarettes. By contrast, carbonylic compounds were hardly detectable in earlier collected fractions. Our data demonstrate the necessity of standardized machine smoking protocols to reliably address putative risks of e-cigarettes for consumers.

Soluble and transmembrane TNF-like weak inducer of apoptosis differentially activate the classical and noncanonical NF-kappa B pathway.

TNF-like weak inducer of apoptosis, TWEAK, is a typical member of the TNF ligand family. Thus, it is initially expressed as a type II transmembrane protein from which a soluble variant can be released by proteolytic processing. In this study, we show that membrane TWEAK is superior to soluble variant of TWEAK (sTWEAK) with respect to the activation of the classical NF-kappaB pathway, whereas both TWEAK variants are potent inducers of TNFR-associated factor-2 depletion, NF-kappaB-inducing kinase accumulation and p100 processing, hallmarks of activation of the noncanonical NF-kappaB pathway. Like other soluble TNF ligands with a poor capability to activate their corresponding receptor, sTWEAK acquires an activity resembling those of the transmembrane ligand by oligomerization or cell surface-immobilization. Blockade of the Fn14 receptor inhibited NF-kappaB signaling irrespective of the TWEAK form used for stimulation, indicating that the differential activities of the two TWEAK variants on classical and noncanonical NF-kappaB signaling is not related to the use of different receptors.

Risk assessment of nanomaterials in cosmetics: a European union perspective.

In Europe, the data requirements for the hazard and exposure characterisation of chemicals are defined according to the REACH regulation and its guidance on information requirements and chemical safety assessment (Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), and its guidance documents; available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2006:396:0001:0849:EN:PDF ; and at: http://guidance.echa.europa.eu/docs/guidance_document/information_requirements_en.htm ). This is the basis for any related risk assessment. The standard reference for the testing of cosmetic ingredients is the SCCP's 'Notes of Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation' (The SCCP's Notes of Guidance for the testing of cosmetic ingredients and their safety evaluation (2006); available at: http://ec.europa.eu/health/ph_risk/committees/04_sccp/docs/sccp_o_03j.pdf ), which refers to the OECD guidelines for the testing of chemicals (The OECD Guidelines for the Testing of Chemicals as a collection of the most relevant internationally agreed testing methods used by government, industry and independent laboratories to assess the safety of chemical products; available at: http://www.oecd.org/topic/0,2686,en_2649_34377_1_1_1_1_37407,00.html ). According to the cosmetics directive [76/768/EEC], compounds that are classified as mutagenic, carcinogenic or toxic to reproduction are banned for the use in cosmetic products. Since December 2010, the respective labelling is based on the rules of regulation (EC) No. 1272/2008 (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006, Official Journal L 353, 31/12/2008, pages 1-1355; available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:353:0001:1355:en:PDF ) on classification, labelling and packaging of substances and mixtures (CLP). There is no further impact from the CLP regulation on cosmetic products, because regulation (EC) No. 1223/2009 on cosmetic products defines its own labelling rules (Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products; available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:342:0059:0209:en:PDF ). Special notification procedures are mandatory for preservatives, colourants and UV-filters where a safety approval from the European 'Scientific Committee on Consumer Safety' (SCCS) is needed prior to marketing. The risk assessment of nanomaterials in consumer products still poses a significant challenge as highlighted by the example of UV-filters in sunscreens since nanomaterials cannot be classified as a homogenous group of chemicals but still need to be addressed in risk characterisation on a case by case basis.

The role of DNA-binding and ARNT dimerization on the nucleo-cytoplasmic translocation of the aryl hydrocarbon receptor.

The human aryl hydrocarbon receptor (AHR) is predominantly located in the cytoplasm, while activation depends on its nuclear translocation. Binding to endogenous or xenobiotic ligands terminates the basal nucleo-cytoplasmic shuttling and stabilizes an exclusive nuclear population. The precise mechanisms that facilitate such stable nuclear accumulation remain to be clarified as essential step in the activation cascade. In this study, we have tested whether the sustained nuclear compartmentalization of ligand-bound or basal AHR might further require heterodimerization with the AHR-nuclear translocator (ARNT) and binding to the cognate XRE-motif. Mutagenesis of the DNA-binding motif or of selected individual residues in the ARNT-binding motif did not lead to any variation in AHR's nucleo-cytoplasmic distribution. In response to ligands, all mutants were retained in the nucleus demonstrating that the stable compartmentalization of activated AHR in the nucleus is neither dependent on interactions with DNA, nor ARNT. Knocking down the ARNT gene using small interfering RNA confirmed that ARNT does not play any role in the intracellular trafficking of AHR.

The extracellular domains of FasL and Fas are sufficient for the formation of supramolecular FasL-Fas clusters of high stability.

Using fluorescent variants of Fas and FasL, we show that membrane FasL and Fas form supramolecular clusters that are of flexible shape, but nevertheless stable and persistent. Membrane FasL-induced Fas clusters were formed in caspase-8- or FADD-deficient cells or when a cytoplasmic deletion mutant of Fas was used suggesting that cluster formation is independent of the assembly of the cytoplasmic Fas signaling complex and downstream activated signaling pathways. In contrast, cross-linked soluble FasL failed to aggregate the cytoplasmic deletion mutant of Fas, but still induced aggregation of signaling competent full-length Fas. Moreover, membrane FasL-induced Fas cluster formation occurred in the presence of the lipid raft destabilizing component methyl-beta-cyclodextrin, whereas Fas aggregation by soluble FasL was blocked. Together, these data suggest that the extracellular domains of Fas and FasL alone are sufficient to drive membrane FasL-induced formation of supramolecular Fas-FasL complexes, whereas soluble FasL-induced Fas aggregation is dependent on lipid rafts and mechanisms associated with the intracellular domain of Fas.

Validation of the 3D Skin Comet assay using full thickness skin models: Transferability and reproducibility.

Recently revised OECD Testing Guidelines highlight the importance of considering the first site-of-contact when investigating the genotoxic hazard. Thus far, only in vivo approaches are available to address the dermal route of exposure. The 3D Skin Comet and Reconstructed Skin Micronucleus (RSMN) assays intend to close this gap in the in vitro genotoxicity toolbox by investigating DNA damage after topical application. This represents the most relevant route of exposure for a variety of compounds found in household products, cosmetics, and industrial chemicals. The comet assay methodology is able to detect both chromosomal damage and DNA lesions that may give rise to gene mutations, thereby complementing the RSMN which detects only chromosomal damage. Here, the comet assay was adapted to two reconstructed full thickness human skin models: the EpiDerm™- and Phenion® Full-Thickness Skin Models. First, tissue-specific protocols for the isolation of single cells and the general comet assay were transferred to European and US-American laboratories. After establishment of the assay, the protocol was then further optimized with appropriate cytotoxicity measurements and the use of aphidicolin, a DNA repair inhibitor, to improve the assay's sensitivity. In the first phase of an ongoing validation study eight chemicals were tested in three laboratories each using the Phenion® Full-Thickness Skin Model, informing several validation modules. Ultimately, the 3D Skin Comet assay demonstrated a high predictive capacity and good intra- and inter-laboratory reproducibility with four laboratories reaching a 100% predictivity and the fifth yielding 70%. The data are intended to demonstrate the use of the 3D Skin Comet assay as a new in vitro tool for following up on positive findings from the standard in vitro genotoxicity test battery for dermally applied chemicals, ultimately helping to drive the regulatory acceptance of the assay. To expand the database, the validation will continue by testing an additional 22 chemicals.

Activation of the cold-receptor TRPM8 by low levels of menthol in tobacco products.

Activation of the cold-receptor TRPM8 by menthol or other tobacco additives can suppress natural defense reactions such as coughing that usually would become effective as involuntary resistance against the inhalation of fumes. In Europe menthol is only regulated as flavor, but can be used as additive as long as no characteristic mint-like aroma will become noticeable in the end-product tobacco. The question needs to be addressed of whether such comparatively minor contents would be sufficient to trigger a measurable activation of TRPM8. In this study, we have analyzed both the contents of menthol and other natural TRPM8 agonists in tobacco products and developed a bioassay to determine the minimum concentrations of selected agonists to activate the TRPM8 receptor in cultured cells. The data confirm menthol as strongest natural agonist investigated. Based on these experiments and previously published data, we have estimated both the minimum menthol concentrations in cigarette smoke and in tobacco that are expected to trigger measurable physiological effects. According to our assessments, TRPM8 activation is likely to occur when cigarettes contain more than 50 micrograms of menthol. Importantly, menthol contents in cigarettes far below the typical levels that require declaration as "mentholated" would be sufficient to activate sensory receptors.

Oxidative and inert pyrolysis on-line coupled to gas chromatography with mass spectrometric detection: On the pyrolysis products of tobacco additives.

According to European legislation, tobacco additives may not increase the toxicity or the addictive potency of the product, but there is an ongoing debate on how to reliably characterize and measure such properties. Further, too little is known on pyrolysis patterns of tobacco additives to assume that no additional toxicological risks need to be suspected. An on-line pyrolysis technique was used and coupled to gas chromatography-mass spectrometry (GC/MS) to identify the pattern of chemical species formed upon thermal decomposition of 19 different tobacco additives like raw cane sugar, licorice or cocoa. To simulate the combustion of a cigarette it was necessary to perform pyrolysis at inert conditions as well as under oxygen supply. All individual additives were pyrolyzed under inert or oxidative conditions at 350, 700 and 1000°C, respectively, and the formation of different toxicants was monitored. We observed the generation of vinyl acrylate, fumaronitrile, methacrylic anhydride, isobutyric anhydride and 3-buten-2-ol exclusively during pyrolysis of tobacco additives. According to the literature, these toxicants so far remained undetectable in tobacco or tobacco smoke. Further, the formation of 20 selected polycyclic aromatic hydrocarbons (PAHs) with molecular weights of up to 278Da was monitored during pyrolysis of cocoa in a semi-quantitative approach. It was shown that the adding of cocoa to tobacco had no influence on the relative amounts of the PAHs formed.

The Q-rich/PST domain of the AHR regulates both ligand-induced nuclear transport and nucleocytoplasmic shuttling.

The aryl hydrocarbon receptor (AHR) shuttles continuously between cytoplasm and nucleus, unless ligand-binding triggers association with the AHR nuclear translocator (ARNT) and subsequent binding to cognate DNA motifs. We have now identified Val 647 as mandatory residue for export from the nucleus and AHR-function. This residue prevents inactivation of the receptor as a consequence of nuclear sequestration via constitutive import. Concomitantly mutants lacking this residue are exclusively localised in the nucleus. Although ligands accelerate nuclear import transiently, stable nuclear transition depends on a motif adjacent to Val 647 that comprises residues 650-661. Together, this defined region within the Q-rich domain regulates intracellular trafficking of the AHR in context of both nucleocytoplasmic shuttling and receptor activation. Nuclear export therefore depends on the previously characterised N-terminal NES and the newly identified motif that includes V647. Nucleocytoplasmic distribution of full-length human AHR is further affected by a section of the PST domain that shows sequence similarities with nuclear export signals. In concert, these motifs maintain a predominant cytoplasmic compartmentalisation, receptive for ligand binding.

Esterase activity in excised and reconstructed human skin--biotransformation of prednicarbate and the model dye fluorescein diacetate.

Reconstructed human epidermis (RHE) is used in non-animal testing for hazard analysis and reconstructed human skin (RHS) gains growing interest in preclinical drug development. RHE and RHS have been characterised regarding their barrier function, but knowledge about biotransformation capacity in these constructs and in human skin remains rather poor. However, metabolising enzymes can be highly relevant for the efficacy of topical dermatics as well as genotoxicity and sensitisation. We have compared the esteratic cleavage of the prednisolone diester prednicarbate and the enzyme kinetic parameters (Vmax and S0.5) of the model substrate fluorescein diacetate (FDA) in commercially available RHS and RHE with excised human skin and monolayer cultures of normal and immortalised human keratinocytes and of fibroblasts. Formation of the main metabolite prednisolone and of fluorescein ranked as: RHS~RHE>excised human skin and keratinocytes>fibroblasts, respectively. Because of the aromatic probe, however, Vmax of FDA cleavage did not show a linear relationship with prednicarbate metabolism. In conclusion, RHE and RHS may be useful to quantitatively address esterase activity of human skin in drug development and hazard analysis, although an increased activity compared to native human skin has to be taken into account.

Metabolically competent human skin models: activation and genotoxicity of benzo[a]pyrene.

The polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BP) is metabolized into a complex pattern of BP derivatives, among which the ultimate carcinogen (+)-anti-BP-7,8-diol-9,10-epoxide (BPDE) is formed to certain extents. Skin is frequently in contact with PAHs and data on the metabolic capacity of skin tissue toward these compounds are inconclusive. We compared BP metabolism in excised human skin, commercially available in vitro 3D skin models and primary 2D skin cell cultures, and analyzed the metabolically catalyzed occurrence of seven different BP follow-up products by means of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). All models investigated were competent to metabolize BP, and the metabolic profiles generated by ex vivo human skin and skin models were remarkably similar. Furthermore, the genotoxicity of BP and its derivatives was monitored in these models via comet assays. In a full-thickness skin, equivalent BP-mediated genotoxic stress was generated via keratinocytes. Cultured primary keratinocytes revealed a level of genotoxicity comparable with that of direct exposure to 50-100 nM of BPDE. Our data demonstrate that the metabolic capacity of human skin ex vivo, as well as organotypic human 3D skin models toward BP, is sufficient to cause significant genotoxic stress and thus cutaneous bioactivation may potentially contribute to mutations that ultimately lead to skin cancer.

Exposure to polycyclic aromatic hydrocarbons: bulky DNA adducts and cellular responses.

Environmental and dietary carcinogens such as polycyclic aromatic hydrocarbons (PAHs) have been intensively studied for decades. Although the genotoxicity of these compounds is well characterized (i.e., formation of bulky PAH-DNA adducts), molecular details on the DNA damage response triggered by PAHs in cells and tissues remain to be clarified. The conversion of hazardous PAHs into carcinogenic intermediates depends on enzyme-catalyzed biotransformation. Certain cytochrome P450-dependent monooxygenases (CYPs) play a pivotal role in PAH metabolism. In particular, CYP1A1 and 1B1 catalyze oxidation of PAHs toward primary epoxide species that can further be converted into multiple follow-up products, both nonenzymatically and enzymatically. Distinct functions between these major CYP enzymes have only been appreciated since transgenic animal models had been derived. Electrophilic PAH metabolites are capable of forming stable DNA adducts or to promote depurination at damaged nucleotide sites. During the following DNA replication cycle, bulky PAH-DNA adducts may be converted into mutations, thereby affecting hot spot sites in regulatory important genes such as Ras, p53, and others. Depending on the degree of DNA distortion and cell cycle progression, PAH-DNA adducts trigger nucleotide excision repair (NER) and various DNA damage responses that might include TP53-dependent apoptosis in certain cell types. In fact, cellular responses to bulky PAH-DNA damage are complex because distinct signaling branches such as ATM/ATR, NER, TP53, but also MAP kinases, interact and cooperate to determine the overall outcome to cellular injuries initiated by PAH-DNA adducts. Further, PAHs and other xenobiotics can also confer DNA damage via an alternative route of metabolic activation, which leads to the generation of PAH semiquinone radicals and reactive oxygen species (ROS). One-electron oxidations mediated by peroxidases or other enzymes can result in PAH radical cations that mainly form unstable DNA adducts subjected to depurination. In addition, generation of ROS can also trigger multiple cellular signaling pathways not directly related to mutagenic or cytotoxic effects, including those mediated by NFκB, SAPK/JNK, and p38. In recent years, it became clear that PAHs may also be involved in inflammatory diseases, autoimmune disorders, or atherosclerosis. Further research is under way to better characterize the significance of such newly recognized systemic effects of PAHs and to reconsider risk assessment for human health.

The role of oxidative stress in carcinogenesis induced by metals and xenobiotics.

In addition to a wide range of adverse effects on human health, toxic metals such as cadmium, arsenic and nickel can also promote carcinogenesis. The toxicological properties of these metals are partly related to generation of reactive oxygen species (ROS) that can induce DNA damage and trigger redox-dependent transcription factors. The precise mechanisms that induce oxidative stress are not fully understood. Further, it is not yet known whether chronic exposures to low doses of arsenic, cadmium or other metals are sufficient to induce mutations in vivo, leading to DNA repair responses and/or tumorigenesis. Oxidative stress can also be induced by environmental xenobiotics, when certain metabolites are generated that lead to the continuous release of superoxide, as long as the capacity to reduce the resulting dions (quinones) into hydroquinones is maintained. However, the specific significance of superoxide-dependent pathways to carcinogenesis is often difficult to address, because formation of DNA adducts by mutagenic metabolites can occur in parallel. Here, we will review both mechanisms and toxicological consequences of oxidative stress triggered by metals and dietary or environmental pollutants in general. Besides causing DNA damage, ROS may further induce multiple intracellular signaling pathways, notably NF-kB, JNK/SAPK/p38, as well as Erk/MAPK. These signaling routes can lead to transcriptional induction of target genes that could promote proliferation or confer apoptosis resistance to exposed cells. The significance of these additional modes depends on tissue, cell-type and is often masked by alternate oncogenic mechanisms being activated in parallel.

Implementation and enforcement of the 3Rs principle in the field of transgenic animals used for scientific purposes. Report and recommendations of the BfR expert workshop, May 18-20, 2009, Berlin, Germany.

In 2007, 2.7 million vertebrates were used for animal experiments and other scientific purposes in Germany alone. Since 1998 there has been an increase in the number of animals used for research purposes, which is partly attributable to the growing use of transgenic animals. These animals are, for instance, used as in vivo models to mimic human diseases like diabetes, cancer or Alzheimer's disease. Here, transgenic model organisms serve as valuable tools, being instrumental in facilitating the analysis of the molecular mechanisms underlying human diseases, and might contribute to the development of novel therapeutic approaches. Due to variable and, sometimes low, efficiency (depending on the species used), however, the generation of such animals often requires a large number of embryo donors and recipients. The experts evaluated methods that could possibly be utilised to reduce, refine or even replace experiments with transgenic vertebrates in the mid-term future. Among the promising alternative model organisms available at the moment are the fruit fly Drosophila melanogaster and the roundworm Caenorhabditis elegans. Specific cell culture experiments or three-dimensional (3D) tissue models also offer valuable opportunities to replace experiments with transgenic animals or reduce the number of laboratory animals required by assisting in decision-making processes. Furthermore, at the workshop an in vitro technique was presented which permits the production of complete human antibodies without using genetically modified ("humanised") animals. Up to now, genetically modified mice are widely used for this purpose. Improved breeding protocols, enhanced efficiency of mutagenesis as well as training of laboratory personnel and animal keepers can also help to reduce the numbers of laboratory animals. Well-trained staff in particular can help to minimise the pain, suffering and discomfort of animals and, at the same time, improve the quality of data obtained from animal experiments. This, in turn, can lead to a reduction in the numbers of animals needed for each experiment. The experts also came to the conclusion that the numbers of laboratory animals can be reduced by open access to a central database that provides detailed documentation of completed experiments involving transgenic animals. This documentation should not be restricted to experiments with substantial scientific results that warrant publication, but should also include those with "negative" outcome, which are usually not published. Capturing all kinds of results within such a database provides added value to the respective scientists and the scientific community as a whole; it could also help to stimulate collaborations and to ensure funding for future research. An important aspect to be considered in the generation of this kind of database is the quality and standardisation of the information provided on existing in vitro models and the respective opportunities for their use. The experts felt that the greatest potential for reducing the numbers of laboratory animals in the near future realistically might not be offered by the complete replacement of transgenic animal models but by opportunities to examine specific questions to a greater degree using in vitro models, such as cell and tissue cultures including organotypic models. The use of these models would considerably reduce the number of in vivo experiments using transgenic animals. However, the overall number of experimental animals may still be increasing or remain unaffected, e.g. when transgenic animals continue to serve as the source of primary cells and organs/tissues for in vitro experiments.

Intracellular localization and transcriptional regulation of tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4).

To gain insight in the subcellular localization of tumor necrosis factor receptor-associated factor (TRAF4) we analyzed GFP chimeras of full-length TRAF4 and various deletion mutants derived thereof. While TRAF4-GFP (T4-GFP) was clearly localized in the cytoplasm, the N-terminal deletion mutant, T4(259-470), comprising the TRAF domain of the molecule, and a C-terminal deletion mutant consisting mainly of the RING and zinc finger domains of TRAF4 were both localized predominantly to the nucleus. Passive nuclear localization of T4(259-470) can be ruled out as the TRAF domain of TRAF4 was sufficient to form high molecular weight complexes. T4(259-470) recruited full-length TRAF4 into the nucleus whereas TRAF4 was unable to change the nuclear localization of T4(259-470). Thus, it seems that individual T4(259-470) mutant molecules are sufficient to direct the respective TRAF4-T4(259-470) heteromeric complexes into the nucleus. In cells forming cell-cell contacts, TRAF4 was recruited to the sites of contact via its C-TRAF domain. The expression of some TRAF proteins is regulated by the NF-kappaB pathway. Thus, we investigated whether this pathway is also involved in the regulation of the TRAF4 gene. Indeed, in primary T-cells and Jurkat cells stimulated with the NF-kappaB inducers TNF or phorbol 12-myristate 13-acetate (PMA), TRAF4-mRNA was rapidly up-regulated. In Jurkat T-cells deficient for I-kappaB kinase gamma (IKKgamma, also known as NEMO), an essential component of the NF-kappaB-inducing-IKK complex, induction of TRAF4 was completely inhibited. In cells deficient for RIP (receptor interactive protein), an essential signaling intermediate of TNF-dependent NF-kappaB activation, TNF-, but not PMA-induced up-regulation of TRAF4 was blocked. These data suggest that activation of the NF-kappaB pathway is involved in up-regulation of TRAF4 in T-cells.