Genotoxicity of poorly soluble particles

Poorly soluble particles such as TiO2, carbon black, and diesel exhaust particles have been evaluated for their genotoxicity using both in vitro and in vivo assays, since inhalation of these compounds by rats at high concentrations has been found to lead to tumor formation. Two principle modes of genotoxic action can be considered for particles, referred to as primary and secondary genotoxicity. Primary genotoxicity is defined as genetic damage elicited by particles in the absence of pulmonary inflammation, whereas secondary genotoxicity implies a pathway of genetic damage resulting from the oxidative DNA attack by reactive oxygen/nitrogen species (ROS/RNS), generated during particle-elicited inflammation. Conceptually, primary genotoxicity might operate via various mechanisms, such as the actions of ROS (e.g., as generated from reactive particle surfaces), or DNA-adduct formation by reactive metabolites of particle-associated organic compounds (e.g., polycyclic aromatic hydrocarbons). Currently available literature data, however, merely indicate that the tumorigenesis of poorly soluble particles involves a mechanism of secondary genotoxicity. However, further research is urgently required, since (1) causality between pulmonary inflammation and genotoxicity has not yet been established, and (2) effects of inflammation on fundamental DNA damage responses that orchestrate mutagenesis and carcinogenic outcome,that is, cell cycle arrest, DNA repair, proliferation, and apoptosis, are currently poorly understood. AD - Institut fur umweltmedizinische Forschung an der Heinrich-Heine-Universitat Dusseldorf, Dusseldorf, Germany. [email protected]

Nitrite enhances neutrophil-induced DNA strand breakage in pulmonary epithelial cells by inhibition of myeloperoxidase

Chronic inhalation of environmental particles is associated with pulmonary carcinogenesis. Although the mechanism has not yet been fully elucidated, influx of inflammatory cells, including neutrophils, is suggested to play a major role in this process. Typically, in the particle-exposed lung, influx of neutrophils is accompanied by an accumulation of nitrite. Previous studies indicated that nitrite may affect the toxicity of neutrophils, involving an interaction with neutrophil-derived myeloperoxidase (MPO). To evaluate the possible consequences of this interaction for inflammation-mediated genotoxicity, we investigated the effect of nitrite on neutrophil-induced DNA damage in pulmonary target cells. Therefore, activated neutrophils were co-cultured with alveolar type II epithelial cells (RLE), and DNA strand breakage was evaluated using single-cell gel electrophoresis (comet assay). In this system, addition of nitrite caused an increase in neutrophil-induced DNA strand breakage in RLE cells, which was associated with an inhibition of MPO activity. Similar results were obtained by co-culturing RLE cells with neutrophils in the presence of the specific MPO inhibitor 4-aminobenzoic acid hydrazide (4-ABAH). To further investigate the mechanism underlying these observations, in vitro experiments were performed using mixtures of nitrite, MPO and its substrate H2O2. DNA strand breakage by reagent H2O2 was inhibited when it was allowed to react with MPO before addition to the RLE cells. However, when MPO and H2O2 were pre-mixed in the presence of nitrite or 4-ABAH, the inhibitory effect of MPO on resultant DNA damage was reversed. Further studies using catalase indicated that DNA strand breakage by the pre-mixtures of MPO, H2O2 and nitrite was H2O2-specific, suggesting that nitrite prevents consumption of H2O2 by MPO. Collectively, our results show that nitrite enhances neutrophil-induced DNA strand breakage in pulmonary epithelial cells. This effect is probably due to an inhibition of MPO activity, which increases the availability of its DNA strand breaking substrate H2O2

Neutrophils and respiratory tract DNA damage and mutagenesis: a review.

Inflammation has been recognized as an important factor in cancer development. For the lung, experimental studies with rats, as well as molecular epidemiological studies in humans, have provided evidence that the influx of neutrophils into the airways may be an important process linking inflammation with carcinogenesis. Currently it is believed that the genotoxic capacity of neutrophils is a crucial aetiological factor in this carcinogenic response. In the present review we discuss two major pathways of neutrophil-induced genotoxicity: (i) induction of oxidative DNA damage through release of reactive oxygen species (ROS) and (ii) myeloperoxidase (MPO)-related metabolic activation of chemical carcinogens. So far, direct evidence for a role of neutrophils in pulmonary genotoxicity has largely been derived from in vitro studies using co-cultures of activated neutrophils and target cells. Current evidence from in vivo studies is primarily indirect and additional animal studies are needed to substantiate causality. A further challenge will be to extrapolate results from such studies to humans. Taken together, this will provide a better insight into the role of neutrophils in pulmonary carcinogenicity and may, hence, lead to novel approaches for cancer prevention strategie

Temporal variation of hydroxyl radical generation and 8-hydroxy-2'-deoxyguanosine formation by coarse and fine particulate matter

Aims: To determine the induction of 8-hydroxy-2'-deoxyguanosine (8-OHdG) by fine (<2.5 µm) and coarse (10–2.5 µm) particulate matter (PM) sampled over time at one sampling location, and to relate the observed effects to the hydroxyl radical (•OH) generating activities and transition metal content of these samples, and to meteorological parameters. Methods: Weekly samples of coarse and fine PM were analysed for H(2)O(2) dependent •OH formation using electron spin resonance (ESR) and formation of 8-OHdG in calf thymus DNA using an immuno-dotblot assay. Immunocytochemistry was used to determine 8-OHdG formation in A549 human epithelial lung cells. To determine temporal effects, samples from six weeks in summer and six weeks in autumn/winter were compared using ESR and the dotblot assay. Concentrations of leachable V, Cr, Fe, Ni, and Cu were determined by inductively coupled plasma mass spectrometry. Results: Both PM fractions elicited •OH generation as well as 8-OHdG formation in calf thymus DNA and in A549 cells. 8-OHdG formation in the naked DNA was significantly related to •OH generation, but not to metal concentrations except for copper. A significantly higher •OH generation was observed for coarse PM, but not fine PM collected during the autumn/winter season; this was not due to differences in sampled mass or metal content. Specific weather conditions under which increased •OH formation in the coarse mode was observed suggest that other, as yet unknown, anthropogenic components might affect the radical generating capacity of PM. Conclusions: Both coarse and fine PM are able to generate •OH, and induce formation of 8-OHdG. When considered at equal mass, •OH formation shows considerable variability with regard to the fraction of PM, as well as the sampling season. The toxicological implications of this heterogeneity in •OH formation by PM, as can be easily determined by ESR, need further investigation

Hydroxyl radical generation by electron paramagnetic resonance as a new method to monitor ambient particulate matter composition

Hydroxyl radical generation by electron paramagnetic resonance as a new method to monitor ambient particulate matter composition. Shi T, Schins RP, Knaapen AM, Kuhlbusch T, Pitz M, Heinrich J, Borm PJ. Particle Research, Institut fur Umweltmedizinische Forschung (IUF) at the University of Dusseldorf PO Box 10 30 45, 40021 Dusseldorf, Germany. Epidemiological studies have demonstrated the relationship between exposure to ambient particulate matter (PM) and health effects in those with cardiopulmonary diseases. The free radical generating activity of particles has been suggested as a unifying factor in the biological activity of PM in toxicological studies but so far has not been applied as a method for environmental monitoring of PM. The purpose of this study was to characterize hydroxyl radical (OH*) production by different size fractions of PM, to use as an alternative method for monitoring of PM composition and activity. We have developed a method, using electron paramagnetic resonance (EPR), to measure OH* radical formation in suspensions of particles in the presence of hydrogen peroxide and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a specific spin-trap. Samples of ambient particulate matter (PM) of different size fractions were collected from various sites on various filters. PM deposited on filters as well as suspensions in water retain its ability to generate OH* and this generation is determined by concentration of hydrogen peroxide and soluble metals. However, large variations in OH* radical formation and kinetics were found with different soluble metals and within metals (Fe, V) with different valencies. The method was applied to environmental monitoring in Hettstedt-Zerbst, situated in South-Eastern Germany, where it showed a relation to Cu-content of PM. The method was also applied in Duisburg, where the PMI fraction showed the highest DMPO-OH* generation but was not linked to particle counts. The method integrates metal bioavailability and reactivity and can provide a better understanding of the effect of small variations in mass concentrations on healt

Induction of CYP1A1 in rat lung cells following in vivo and in vitro exposure to quartz

Respirable quartz has been classified as a human lung carcinogen, but the mechanism by which quartz exposure leads to lung cancer has not been clarified. Consistently higher risks of lung cancer are reported in smokers with quartz exposure and we therefore hypothesised that quartz exposure may alter the expression of enzyme systems involved in activation/detoxification of pre-carcinogens in cigarette smoke. More specifically we studied cytochrome P4501A1 (CYP1A1) expression using reverse transcriptase polymerase chain reaction and immunohistochemistry (IHC) upon in vitro and in vivo quartz exposure. In vitro incubation of rat lung epithelial cells with DQ12 quartz for 24 h showed a dose-dependent induction of CYP1A1-mRNA. On the other hand, CYP1A1 message was not increased in lung epithelial cells isolated from rats at 3, 28 or 90 days after intratracheal instillation of 2 mg DQ12. Following IHC for CYP1A1 protein in rat lung sections from later time-points (180 and 360 days), we observed an increase in the number of CYP1A1 positive cells. After in vivo quartz exposure, protein expression of the Aryl hydrocarbon receptor (AhR) was increased and nuclear translocation of AhR was observed at the same time-points. In conclusion, our findings demonstrate an effect of quartz exposure on chronic CYP1A1 expression in vivo, whereas the in vitro models show an immediate upregulation. We suggest that this upregulation of CYP1A1 may act as a co-carcinogenic pathway in quartz exposed workers by activation of pre-carcinogens such as those present in cigarette smoke

Decreased levels of lipid peroxidation-induced DNA damage in the onset of atherogenesis in apolipoprotein E deficient mice

Increased oxidative stress and subsequent lipid peroxidation (LPO) are thought to be critical events in the formation of atherosclerotic lesions in apolipoprotein E deficient mice (ApoE-KO). LPO derived reactive aldehydes react with DNA to form exocyclic etheno-DNA adducts. These pro-mutagenic DNA lesions are known to be involved in the initiation of carcinogenesis, but their role in the development of atherosclerosis is unknown. In the present study we show that levels of the LPO derived 1,N(6)-ethenodeoxyadenosine (varepsilondA) and 3,N(4)-ethenodeoxycytidine (varepsilondC) were both significantly lower in aorta of 12 weeks old ApoE-KO mice as compared to their wild type controls (1.6+/-0.3 versus 3.2+/-0.8 varepsilondA per 10(8) parent nucleotides, P=0.04 and 4.8+/-0.8 versus 9.2+/-2.1 for varepsilondC, P=0.02). Moreover, levels of both DNA adduct types were inversely related with total plasma cholesterol levels. Consequently, lowest etheno-DNA adduct levels were observed in ApoE-KO mice on a high fat diet. Hypercholesterolemia has previously been associated with increased expression of base excision repair (BER) enzymes, which could explain the lower levels of etheno-DNA adducts in ApoE-KO mice as compared to wild type controls. Indeed, increased staining for the BER-specific DNA repair enzyme apurinic/apyrimidinic endonuclease (Ape1/Ref1) was observed by immunohistochemistry in the endothelium and the first layers of arterial smooth muscle cells of ApoE-KO mice as compared to their wild type counterparts. A high fat diet further increased overall Ape1/Ref1 protein expression in ApoE-KO mice. Although these data suggest no role for increased LPO derived DNA damage in the onset of atherogenesis in ApoE-KO mice, the potentially modulating role of Ape1/Ref1 in the arterial wall deserves further attention