Klaus Unfried

Cellular responses to nanoparticles: Target structures and mechanisms

Nanotechnology makes use of the special surface properties of extremely small particles. In this rapidly growing field, many different materials are produced for a multitude of diverse applications. Possible adverse health effects of these materials however are so far scarcely investigated and are therefore a special task of toxicology. Although strategies for risk assessment have been suggested, the authors of the current review emphasize the fact that on the cellular, subcellular and molecular levels, interactions between nanoparticles (NP) and target cells relevant for the induction of possible adverse health effects are poorly understood. On the basis of existing literature, the potentially most relevant cellular target sites of NP as well as the so far known major molecular events specifically induced by these xenobiotics are reviewed. Starting with NP uptake across the cell membrane, mechanisms of generation of reactive oxygen species and the activation of redox-sensitive signalling cascades are described. Besides the cell membrane, mitochondria and cell nucleus are considered as major cell compartments relevant for possible NP-induced toxicity. Finally, an integrated research protocol is proposed to identify fundamental cellular responses to NP in order to complement current toxicological screening strategies with a mechanism-based approach.

The Compatible Solute Ectoine Protects against Nanoparticle-induced Neutrophilic Lung Inflammation

RATIONALE Inflammatory reactions of the airways induced by nanoparticles of occupational and environmental origin contribute to organ-specific and systemic human diseases. Because this kind of exposure in modern societies is often unavoidable, a strategy of molecular prevention on an individual level could help to prevent inflammation-derived secondary diseases. OBJECTIVES To test whether the compatible solute ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid], which is known to reduce cell stress effects on a molecular level, prevents nanoparticle-induced lung inflammation. METHODS Inflammatory parameters were studied in Fischer 344 rats treated with model carbon nanoparticles. The molecular effects of ectoin on proinflammatory signal transduction were demonstrated in the rat and in the human system using cultured lung epithelial cells. MEASUREMENTS AND MAIN RESULTS Ectoine, given with or before the nanoparticles, dose-dependently reduced neutrophil inflammation in the lung. This preventive effect was not observed when lung inflammation was induced by bacterial lipopolysaccharide. Analyses of the underlying mode of action revealed that ectoine acted on lung epithelial cells. Ectoine administration inhibited nanoparticle-induced signaling, which is known to be responsible for proinflammatory reactions in rat lung epithelial cells in vitro as well as in vivo. These findings were corroborated and extended in experiments with cultured human bronchial epithelial cells in which ectoine inhibited nanoparticle-triggered cell signaling and IL-8 induction. CONCLUSIONS Because compatible solutes are compliant natural products without known toxic potential, we propose that this group of substances may be used for the prevention of particle-induced airway inflammation in humans.

Reactive oxygen species as mediators of membrane-dependent signaling induced by ultrafine particles

Cell-membrane-dependent proliferative signal transduction activated by ultrafine carbon particles in lung epithelial cells involves the specific induction of Akt and ERK1/2 phosphorylation. Particle-induced generation of reactive oxygen species (ROS) and oxidative stress are regarded as initial molecular mechanisms leading to the induction of diverse cellular responses. Therefore, we aimed to analyze the ROS dependence of the induced activation of the Akt/ERK1/2 signaling pathway upon exposure to ultrafine particulate matter (UPM). For this, ultrafine carbon black (ufCB) and ferric sulfate (FS) were used as a model representing the carbonaceous core and a nonparticulate Fenton-reactive transition metal salt often found in combustion-derived UPM. Cell-free as well as intracellular particle-induced ROS generation was assessed and related to the induced Akt and ERK1/2 phosphorylation by inhibiting oxidative stress with catalase, superoxide dismutase, and N-acetylcysteine. We show here that the activation of this signal transduction pathway was mainly due to intracellular, rather than extracellular, ROS production induced by both ufCB and FS. Further inhibitor studies on the role of cell membrane receptors pointed to the epidermal growth factor receptor as a common mediator for particle- as well as transition metal-induced signaling, whereas integrin-dependent Akt and ERK1/2 activation seems to be particle-specific.

Carbon nanoparticles induce ceramide- and lipid raft-dependent signalling in lung epithelial cells: a target for a preventive strategy against environmentally-induced lung inflammation.

BackgroundParticulate air pollution in lung epithelial cells induces pathogenic endpoints like proliferation, apoptosis, and pro-inflammatory reactions. The activation of the epidermal growth factor receptor (EGFR) is a key event responsible for signalling events involving mitogen activated protein kinases specific for these endpoints. The molecular events leading to receptor activation however are not well understood. These events are relevant for the toxicological evaluation of inhalable particles as well as for potential preventive strategies in situations when particulate air pollution cannot be avoided. The current study therefore had the objective to elucidate membrane-coupled events leading to EGFR activation and the subsequent signalling cascade in lung epithelial cells. Furthermore, we aimed to identify the molecular target of ectoine, a biophysical active substance which we described to prevent carbon nanoparticle-induced lung inflammation.MethodsMembrane signalling events were investigated in isolated lipid rafts from lung epithelial cells with regard to lipid and protein content of the signalling platforms. Using positive and negative intervention approaches, lipid raft changes, subsequent signalling events, and lung inflammation were investigated in vitro in lung epithelial cells (RLE-6TN) and in vivo in exposed animals.ResultsCarbon nanoparticle treatment specifically led to an accumulation of ceramides in lipid rafts. Detailed analyses demonstrated a causal link of ceramides and subsequent EGFR activation coupled with a loss of the receptor in the lipid raft fractions. In vitro and in vivo investigations demonstrate the relevance of these events for carbon nanoparticle-induced lung inflammation. Moreover, the compatible solute ectoine was able to prevent ceramide-mediated EGFR phosphorylation and subsequent signalling as well as lung inflammation in vivo.ConclusionThe data identify a so far unknown event in pro-inflammatory signalling and contribute to the understanding of particle cell interaction and therefore to risk identification and risk assessment of inhalable xenobiotics. Moreover, as this cellular reaction can be prevented by the well tolerated substance ectoine, a molecular preventive strategy for susceptible persons against airway inflammation is proposed.

The forkhead transcription factor FOXO4 sensitizes cancer cells to doxorubicin-mediated cytotoxicity

The forkhead superfamily of transcription factors, which play major roles in control of cellular proliferation, oxidative stress and apoptosis, are becoming more and more considered as crucial therapeutic targets in cancer. In this study, we addressed the contribution of class O of forkhead box transcription factor (FOXO) 4 transcription factor, a forkhead superfamily member, to cytotoxicity mediated by the anthracyclic drug doxorubicin. FOXO4 can be phosphorylated by phosphatidylinositol-3-kinase/AKT signaling resulting in its inactivation and nuclear exclusion. Under stress conditions, FOXO4 can be phosphorylated via jun N-terminal kinase (JNK) leading to increased transcriptional activation of the transcription factor. Our results show that doxorubicin incubation led to phosphorylation of AKT and concomitantly to AKT-dependent inactivation and nuclear exclusion of the tumor suppressor FOXO4 in Hct-116 cells. We found that inhibition of FOXO4 nuclear exclusion by blockage of AKT phosphorylation following overexpression of dominant-negative AKT enhanced doxorubicin-mediated cytotoxicity. Overexpression of wild-type FOXO4 led to an increase in doxorubicin-mediated cytotoxicity, which was further exacerbated by overexpression of a solely nuclear-localized FOXO4 mutant. In contrast, though doxorubicin resulted in JNK activation, modulation of JNK-dependent regulation of FOXO4 was of no effect to doxorubicin cytotoxicity. These results show for the first time that in Hct-116 cells sustained nuclear localization of FOXO4 seems to be one crucial point enhancing doxorubicin-induced cytotoxicity and apoptosis. Targeting FOXO4 or AKT may lead to new chances in sensitizing cancer cells to cytostatic drugs thereby allowing use of lower drug concentrations and minimizing drug-induced adverse effects in patients.

c-Src-mediated activation of Erk1/2 is a reaction of epithelial cells to carbon nanoparticle treatment and may be a target for a molecular preventive strategy.

Abstract Owing to their specific physico/chemical properties, engineered as well as environmental nanoparticles can induce pathogenic endpoints in humans. Earlier studies demonstrated that pure carbon nanoparticles induce cell signaling events at the level of membrane receptor activation in lung epithelial cells. As a possible link between receptor activation and subsequent MAP-kinase signaling, the involvement of Src family kinases was investigated in cell lines of organs potentially exposed to environmental nanoparticles. Human cells from bronchus, intestine, and skin (keratinocytes) as well as rat lung epithelial cells showed similar time patterns for the activation of mitogen-activated protein kinases Erk1/2 as well as Src family kinases (SFK) when treated with carbon nanoparticles. Moreover, c-Src was identified as an integral part of the signaling mediating the transfer of information from membrane receptors to members of the proliferative signaling cascade in lung epithelial cells. Pretreatment of cells with the compatible solute ectoine, which is known to stabilize macromolecules, reduced the nanoparticle specific phosphorylation of SFK. Together with earlier in vivo and in vitro data, this demonstrates that compatible solutes prevent nanoparticle-induced signaling steps at the level of membrane-coupled signaling.

Recovery of neutrophil apoptosis by ectoine: a new strategy against lung inflammation

The life span of neutrophilic granulocytes has a determining impact on the intensity and duration of neutrophil driven lung inflammation. Based on the compatible solute ectoine, we aimed to prevent anti-apoptotic reactions in neutrophils triggered by the inflammatory microenvironment in the lung. Neutrophils from chronic obstructive pulmonary disease patients and control individuals were exposed to inflammatory mediators and xenobiotics in the presence or absence of ectoine. The in vivo relevance of this approach was tested in xenobiotic-induced lung inflammation in rats. The reduction of apoptosis rates of ex vivo-exposed neutrophils from all study groups was significantly restored in the presence of ectoine. However, natural apoptosis rates not altered by inflammatory stimuli were not changed by ectoine. Mechanistic analyses demonstrated the preventive effect of ectoine on the induction of anti-apoptotic signalling. Neutrophilic lung inflammation induced by single or multiple expositions of animals to environmental particles was reduced after the therapeutic intervention with ectoine. Analyses of neutrophils from bronchoalveolar lavage indicate that the in vivo effect is due to the restoration of neutrophil apoptosis. Ectoine, a compound of the highly compliant group of compatible solutes, demonstrates a reproducible and robust effect on the resolution of lung inflammation.

Upregulation of the RAS-GTPase activating protein (GAP)-binding protein (G3BP) in proliferating RPE cells.

Cultured human retinal pigment epithelial (RPE) cells of different passages (P0 and P3) were used as a model system to examine changes in gene expression in proliferating RPE cells by polymerase chain reaction (PCR)‐based differential expressed mRNA analysis (DEmRNA‐PCR). DEmRNA‐PCR showed enhanced expression of a specific RNA in P3 compared with P0. Sequence alignment displayed its identity with the 3′‐end of the coding sequence of the human RAS‐GTPase activating protein (GAP)‐binding protein (G3BP). Confirmation of the induced expression of G3BP was performed by gene‐specific reverse transcription‐polymerase chain reaction (RT‐PCR) of freshly prepared human RPE cells and of cultured cells of P0, P3 and P8 and by immunohistochemistry of cultivated retinal pigment epithelial cells in an artificial lesion assay. The expression of G3BP mRNA increased with the number of passages. G3BP protein expression increased in cells repopulating the artificial lesion. DEmRNA‐PCR in RPE cells with subsequent sequence analysis led to the characterization of dedifferentiation‐ and proliferation‐dependent expression of a previously undetected gene product in cultured RPE cells with a possible role in modifying signal transduction responses that may have implications on the treatment of proliferative vitreoretinopathy. J. Cell. Biochem. 74:194–201, 1999.

Variation of the N-Acetyltransferase 2 Gene in a Romanian and a Kyrgyz Population

As part of a project on environmental disasters in minority populations, this study aimed to evaluate differences in the sequence of N-acetyltransferase 2 (NAT2) as a metabolic susceptibility gene in yet unexplored ethnicities. Eight single nucleotide polymorphisms (SNP) in the NAT2 coding region and a variant in the 3′ flanking region were analyzed in 290 unrelated Kyrgyz and 140 unrelated Romanians by SNP-specific PCR analysis. The variants 341C, 481T, and 803G were less and 857A more prevalent in Kyrgyz (P < 0.0001). The variant at site 857 indicates Asian descent. 282C>T and 590G>A showed no significant variation by ethnicity. 364G>A and 411A>T turned out to be monomorphic. Database comparisons of the NAT2 minor allele frequencies support that Romanians belong to Caucasians and Kyrgyz are in between Caucasians and East Asians. The distributions of predicted haplotypes differed significantly between the two ethnicities where the Kyrgyz showed a higher genetic diversity. The haplotype without mutations was more common in Kyrgyz (40.1% in Kyrgyz, 29.3% in Romanians). Accordingly, the imputed slow acetylator phenotype was less prevalent in Kyrgyz (35.2% versus 51.4% in Romanians). We found pronounced ethnic differences in NAT2 genotypes with yet unknown effect on the health risks for environmental or occupational exposures in minority populations. (Cancer Epidemiol Biomarkers Prev 2006;(15)1:138–41)

The Compatible Solute Ectoine Reduces the Exacerbating Effect of Environmental Model Particles on the Immune Response of the Airways

Exposure of humans to particulate air pollution has been correlated with the incidence and aggravation of allergic airway diseases. In predisposed individuals, inhalation of environmental particles can lead to an exacerbation of immune responses. Previous studies demonstrated a beneficial effect of the compatible solute ectoine on lung inflammation in rats exposed to carbon nanoparticles (CNP) as a model of environmental particle exposure. In the current study we investigated the effect of such a treatment on airway inflammation in a mouse allergy model. Ectoine in nonsensitized animals significantly reduced the neutrophilic lung inflammation after CNP exposure. This effect was accompanied by a reduction of inflammatory factors in the bronchoalveolar lavage. Reduced IL-6 levels in the serum also indicate the effects of ectoine on systemic inflammation. In sensitized animals, an aggravation of the immune response was observed when animals were exposed to CNP prior to antigen provocation. The coadministration of ectoine together with the particles significantly reduced this exacerbation. The data indicate the role of neutrophilic lung inflammation in the exacerbation of allergic airway responses. Moreover, the data suggest to use ectoine as a preventive treatment to avoid the exacerbation of allergic airway responses induced by environmental air pollution.