Damien van Berlo

Effect of prolonged exposure to diesel engine exhaust on proinflammatory markers in different regions of the rat brain.

BackgroundThe etiology and progression of neurodegenerative disorders depends on the interactions between a variety of factors including: aging, environmental exposures, and genetic susceptibility factors. Enhancement of proinflammatory events appears to be a common link in different neurological impairments, including Alzheimers disease, Parkinsons disease, amyotrophic lateral sclerosis, and multiple sclerosis. Studies have shown a link between exposure to particulate matter (PM), present in air pollution, and enhancement of central nervous system proinflammatory markers. In the present study, the association between exposure to air pollution (AP), derived from a specific source (diesel engine), and neuroinflammation was investigated. To elucidate whether specific regions of the brain are more susceptible to exposure to diesel-derived AP, various loci of the brain were separately analyzed. Rats were exposed for 6 hrs a day, 5 days a week, for 4 weeks to diesel engine exhaust (DEE) using a nose-only exposure chamber. The day after the final exposure, the brain was dissected into the following regions: cerebellum, frontal cortex, hippocampus, olfactory bulb and tubercles, and the striatum.ResultsBaseline levels of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1 alpha (IL-1α) were dependent on the region analyzed and increased in the striatum after exposure to DEE. In addition, baseline level of activation of the transcription factors (NF-κB) and (AP-1) was also region dependent but the levels were not significantly altered after exposure to DEE. A similar, though not significant, trend was seen with the mRNA expression levels of TNF-α and TNF Receptor-subtype I (TNF-RI).ConclusionsOur results indicate that different brain regions may be uniquely responsive to changes induced by exposure to DEE. This study once more underscores the role of neuroinflammation in response to ambient air pollution, however, it is valuable to assess if and to what extent the observed changes may impact the normal function and cellular integrity of unique brain regions.

Evaluation of apoptosis induced by nanoparticles and fine particles in RAW 264.7 macrophages: Facts and artefacts

Current hazard characterisation of nanoparticles (NP) is predominantly based on in vitro test systems, being established for small molecules of drugs and chemicals. However, specific physicochemical properties of NP may result in interference with assay components, biomarkers, or detection systems. In the present study, six types of (nano)particles were screened in RAW 264.7 macrophages by common cytotoxicity methods (WST-1, LDH). Our specific focus was on the investigation of apoptosis (analysis of hypodiploid DNA, phosphatidylserine exposure, caspase 3/7 activation, and Cell Death Detection ELISA). Assays were validated by the well-known apoptosis inducer staurosporine. Our results show that ZnO, DQ12 quartz and amorphous silica are cytotoxic with strong indications for apoptotic effects in RAW 264.7 macrophages, whereas toxicity was absent for MgO. For fine as well as ultrafine TiO(2), no apoptotic effects could be detected except for induction of DNA fragmentation. The results of our study demonstrate the necessity to control on a case-by-case basis for assay interference to avoid misinterpretation of specific in vitro test findings. To obtain valid statements on the potential induction of apoptosis by specific NP the measurement of multiple endpoints is a prerequisite.

Neutrophil-derived ROS contribute to oxidative DNA damage induction by quartz particles

The carcinogenicity of respirable quartz is considered to be driven by reactive oxygen species (ROS) generation in association with chronic inflammation. The contribution of phagocyte-derived ROS to inflammation, oxidative stress, and DNA damage responses was investigated in the lungs of C57BL/6J wild-type and p47(phox-/-) mice, 24h after pharyngeal aspiration of DQ12 quartz (100 mg/kg bw). Bone-marrow-derived neutrophils from wild-type and p47(phox-/-) mice were used for parallel in vitro investigations in coculture with A549 human alveolar epithelial cells. Quartz induced a marked neutrophil influx in both wild-type and p47(phox-/-) mouse lungs. Significant increases in mRNA expression of the oxidative stress markers HO-1 and γ-GCS were observed only in quartz-treated wild-type animals. Oxidative DNA damage in lung tissue was not affected by quartz exposure and did not differ between p47(phox-/-) and WT mice. Differences in mRNA expression of the DNA repair genes OGG1, APE-1, DNA Polβ, and XRCC1 were also absent. Quartz treatment of cocultures containing wild-type neutrophils, but not p47(phox-/-) neutrophils, caused increased oxidative DNA damage in epithelial cells. Our study demonstrates that neutrophil-derived ROS significantly contribute to pulmonary oxidative stress responses after acute quartz exposure, yet their role in the associated induction of oxidative DNA damage could be shown only in vitro.

Carbon nanotubes: an insight into the mechanisms of their potential genotoxicity

After the health catastrophe resulting from the widespread use of asbestos which was once hailed as a new miracle material, the increasing use of carbon nanotubes (CNTs) has spawned major concern due to their similarities in terms of size, shape and poor solubility. Assessment of genotoxicity has shown that CNTs can damage DNA in vitro and in vivo. The genotoxic potential of different CNT samples varies considerably, however, with negative findings reported in a number of studies, probably due to the enormous heterogeneity of CNTs. The observed spectrum of genotoxic effects shows similarities with those reported for asbestos fibres. Mutagenicity has been found in vivo but in bacterial assays both asbestos and CNTs have mostly tested negative. An overview of key experimental observations on CNT-induced genotoxicity is presented in the first half of this review. In the second part, the potential mechanisms of CNT-elicited genotoxicity are discussed. Whereas CNTs possess intrinsic ROS-scavenging properties they are capable of generating intracellular ROS upon interaction with cellular components, and can cause antioxidant depletion. These effects have been attributed to their Fenton-reactive metals content. In addition, CNTs can impair the functionality of the mitotic apparatus. A noteworthy feature is that frustrated phagocytosis, which is involved in asbestos-induced pathology, has been observed for specific CNTs as well. The involvement of other mechanisms generally implicated in particle toxicity, such as phagocyte activation and impairment of DNA repair, is largely unknown at present and needs further investigation.

Toxicology of Ambient Particulate Matter

It is becoming increasingly clear that inhalation exposure to particulate matter (PM) can lead to or exacerbate various diseases, which are not limited to the lung but extend to the cardiovascular system and possibly other organs and tissues. Epidemiological studies have provided strong evidence for associations with chronic obstructive pulmonary disease (COPD), asthma, bronchitis and cardiovascular disease, while the evidence for a link with lung cancer is less strong. Novel research has provided first hints that exposure to PM might lead to diabetes and central nervous system (CNS) pathology. In the current review, an overview is presented of the toxicological basis for adverse health effects that have been linked to PM inhalation. Oxidative stress and inflammation are discussed as central processes driving adverse effects; in addition, profibrotic and allergic processes are implicated in PM-related diseases. Effects of PM on key cell types considered as regulators of inflammatory, fibrotic and allergic mechanisms are described.

NF-κB dependent and independent mechanisms of quartz-induced proinflammatory activation of lung epithelial cells

In the initiation and progression of pulmonary inflammation, macrophages have classically been considered as a crucial cell type. However, evidence for the role of epithelial type II cells in pulmonary inflammation has been accumulating. In the current study, a combined in vivo and in vitro approach has been employed to investigate the mechanisms of quartz-induced proinflammatory activation of lung epithelial cells. In vivo, enhanced expression of the inflammation- and oxidative stress-related genes HO-1 and iNOS was found on the mRNA level in rat lungs after instillation with DQ12 respirable quartz. Activation of the classical NF-κB pathway in macrophages and type II pneumocytes was indicated by enhanced immunostaining of phospho-IκBα in these specific lung cell types. In vitro, the direct, particle-mediated effect on proinflammatory signalling in a rat lung epithelial (RLE) cell line was compared to the indirect, macrophage product-mediated effect. Treatment with quartz particles induced HO-1 and COX-2 mRNA expression in RLE cells in an NF-κB independent manner. Supernatant from quartz-treated macrophages rapidly activated the NF-κB signalling pathway in RLE cells and markedly induced iNOS mRNA expression up to 2000-fold compared to non-treated control cells. Neutralisation of TNFα and IL-1β in macrophage supernatant did not reduce its ability to elicit NF-κB activation of RLE cells. In addition the effect was not modified by depletion or supplementation of intracellular glutathione.The results from the current work suggest that although both oxidative stress and NF-κB are likely involved in the inflammatory effects of toxic respirable particles, these phenomena can operate independently on the cellular level. This might have consequences for in vitro particle hazard testing, since by focusing on NF-κB signalling one might neglect alternative inflammatory pathways.

Neutrophils augment LPS-mediated pro-inflammatory signaling in human lung epithelial cells.

BACKGROUND The role of polymorphonuclear neutrophils in pulmonary host defense is well recognized. The influence of a pre-existing inflammation driven by neutrophils (neutrophilic inflammation) on the airway epithelial response toward pro-inflammatory exogenous triggers, however, is still poorly addressed. Therefore, the aim of the present study is to investigate the effect of neutrophils on lipopolysaccharide (LPS)-induced pro-inflammatory signaling in lung epithelial cells. Additionally, underlying signaling pathways are examined. METHODS Human bronchial epithelial cells (BEAS-2B) were co-incubated with human peripheral blood neutrophils or bone-marrow derived neutrophils from either C57BL/6J wild type or nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase deficient (p47(phox-/-)) mice. Upon stimulation with LPS, interleukin (IL)-8 production and reactive oxygen species (ROS) generation were measured. Additionally, activation of the extracellular signal-regulated kinases (ERK) 1/2 and nuclear factor (NF)-κB signaling pathways was analyzed. RESULTS Our studies show that the presence of neutrophils synergistically increases LPS-induced IL-8 and ROS production by BEAS-2B cells without inducing cytotoxicity. The observed IL-8 response to endotoxin increases in proportion to time, LPS-concentration and the number of neutrophils present. Moreover, this synergistic IL-8 production strongly correlated with the chemotactic properties of the co-incubations and significantly depended on a functional neutrophilic NADPH oxidase. The presence of neutrophils also augments LPS-induced phosphorylation of ERK1/2 and IκBα as well as NF-κB RelA DNA binding activity in BEAS-2B cells. CONCLUSIONS Our results indicate that the pro-inflammatory effects of LPS toward lung epithelial cells are amplified during a pre-existing neutrophilic inflammation. These findings support the concept that patients suffering from pulmonary neutrophilic inflammation are more susceptible toward exogenous pro-inflammatory triggers.

Oxidative stress and DNA damage responses in rat and mouse lung to inhaled carbon nanoparticles

Abstract We have investigated whether short-term nose-only inhalation exposure to electric spark discharge-generated carbon nanoparticles (∼60 nm) causes oxidative stress and DNA damage responses in the lungs of rats (152 μg/m3; 4 h) and mice (142 μg/m3; 4 h, or three times 4 h). In both species, no pulmonary inflammation and toxicity were detected by bronchoalveolar lavage or mRNA expression analyses. Oxidative DNA damage (measured by fpg-comet assay), was also not increased in mouse whole lung tissue or isolated lung epithelial cells from rat. In addition, the mRNA expressions of the DNA base excision repair genes OGG1, DNA Polβ and XRCC1 were not altered. However, in the lung epithelial cells isolated from the nanoparticle-exposed rats a small but significant increase in APE-1 mRNA expression was measured. Thus, short-term inhalation of carbon nanoparticles under the applied exposure regimen, does not cause oxidative stress and DNA damage in the lungs of healthy mice and rats.

Investigation of the cytotoxic and proinflammatory effects of cement dusts in rat alveolar macrophages.

Exposure to cement dust, a specifically alkaline and irritant dust, is one of the most common occupational dust exposures worldwide. Although several adverse respiratory health effects have been associated with cement dust exposure, the evidence is not conclusive. In the current study, cytotoxic and pro-inflammatory effects as well as oxidative stress elicited by a number of cement dusts, including a limestone and cement clinker sample, were tested using the NR8383 rat alveolar macrophage cell line and primary rat alveolar macrophages. DQ12 quartz and TiO(2) were included as positive and negative controls, respectively. Cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay and the lactate dehydrogenase assay, oxidative stress was determined by measurement of the depletion of total cellular glutathione, and electron spin resonance was applied to determine reactive oxygen species (ROS) generation. The release of the cytokines tumor necrosis factor-alpha (TNFalpha), interleukin-1 beta (IL-1 beta), and macrophage inflammatory protein-2 (MIP-2) was determined by enzyme-linked immunosorbent assay. None of the dust samples were found to cause toxicity to the macrophages or notable glutathione depletion when compared to DQ12. The cement samples also failed to activate macrophages for the generation of ROS and the production of inflammatory cytokines IL-1 beta and MIP-2. In contrast, however, most of the cement dusts were found to activate macrophage TNFalpha production, and this was significantly associated with their content of CaO. Further research is needed to determine the relevance of these in vitro observations for occupational cement dust exposure settings.

Oxidant generation, DNA damage and cytotoxicity by a panel of engineered nanomaterials in three different human epithelial cell lines

Due to the steeply increased use of nanomaterials (NMs) for commercial and industrial applications, toxicological assessment of their potential harmful effects is urgently needed. In this study, we compared the DNA-damaging properties and concurrent cytotoxicity of a panel of 10 engineered NMs in three different cell lines in relation to their intrinsic oxidant generating properties. The human epithelial cell lines A549, HK-2 and HepG2 were chosen to represent relevant target organs for NMs in the lung, kidney and liver. Cytotoxicity, evaluated by WST-1 assay in the treatment concentration range of 0.3–80 µg/cm2, was shown for Ag and ZnO NM in all three cell lines. Cytotoxicity was absent for all other NMs, i.e. five types of TiO2 and two types of multiwalled carbon nanotubes. DNA damage, evaluated by the alkaline comet assay, was observed with Ag and ZnO, albeit only at cytotoxic concentrations. DNA damage varied considerably with the cell line. The oxidant generating properties of the NMs, evaluated by electron spin resonance spectroscopy in cell free conditions, did not correlate with their cytotoxic or DNA-damaging properties. DNA damage by the nanosilver could be partly attributed to its surfactant-containing dispersant. The coating of a TiO2 sample with the commercial surfactant Curosurf augmented its DNA-damaging properties in A549 cells, while surface modification with serum tended to reduce damage. Our findings indicate that measurement of the intrinsic oxidant-generating capacity of NMs is a poor predictor of DNA damage and that the cytotoxic and DNA-damaging properties of NMs can vary substantially with experimental conditions. Our study also underlines the critical importance of selecting appropriate cell systems and aligned testing protocols. Selection of a cell line on the mere basis of its origin may provide only poor insight on organ-specific hazards of NMs.