Richard Gminski

Cytotoxicity and genotoxicity in human lung epithelial A549 cells caused by airborne volatile organic compounds emitted from pine wood and oriented strand boards.

Due to the massive reduction of air-change rates in modern, energy-saving houses and dwellings, the contribution of volatile organic compound (VOCs) emissions from wood-based materials to indoor air quality has become increasingly important. To evaluate toxicity of VOC mixtures typically emitted from pine wood and oriented strand boards (OSB) and their main constituents (selected terpenes and aldehydes), cytotoxicity and genotoxicity were investigated in human A549 lung cells. To facilitate exposure directly via gas phase, a 250 L emission chamber was combined with a Vitrocell exposure system. VOC exposure concentrations were measured by GC/MSD. Biological effects were determined after an exposure time of 1h by measuring cytotoxicity (erythrosine B staining) and genotoxicity (comet assay). Neither cytotoxic nor genotoxic effects were observed for VOC mixtures emitted from pine wood or OSB at loading factors of approximately 13 m(2)/m(3) (worst case conditions) of the panels (with maximum VOC levels of about 80 mg/m(3)) in comparison to clean air. While alpha-pinene and Delta(3)-carene did not induce toxic effects even at exposure concentrations of up to 1800 mg/m(3) and 600 mg/m(3), respectively, hexanal showed a cytotoxic effect at 2000 mg/m(3). The alpha,beta-unsaturated aldehydes 2-heptenal and 2-octenal caused genotoxic effects in concentrations exceeding 100mg/m(3) and 40 mg/m(3), respectively. In conclusion, high concentrations of VOCs and VOC mixtures emitted from pine wood and OSB did not lead to adverse effects in A549 human lung cells even at concentrations 10(2) to 10(5)-fold higher than those found in normal indoor air. Attention must be paid to mutagenic and possibly carcinogenic alpha,beta-unsaturated aldehydes.

Genotoxic effects of three selected black toner powders and their dimethyl sulfoxide extracts in cultured human epithelial A549 lung cells in vitro.

Until now, the adverse effects of toner powders on humans have been considered to be minimal. However, several recent reports have suggested possible significant adverse health effects from toner dust inhalation. The aim of this study was to evaluate the genotoxic potential of black toner powders in vitro. For the study of DNA damage, A549 cells were exposed to toner‐powder suspensions and to their DMSO extracts, and then subjected to the comet assay and to the in‐vitro cytokinesis block micronucleus test (CB‐MNvit). Cytotoxic effects of the toner samples were assessed by the erythrosin B assay. Furthermore, size, shape, and composition of the toner powders were investigated. None of the three toner powders or their DMSO extracts reduced cell viability; however, they did induce DNA damage and formed micronuclei at concentrations from 80 to 400 μg cm−2, although to a varying extent. All toner powders contain considerable amounts of the pigments carbon black and magnetite (Fe3O4) as well as small amounts of polycyclic aromatic hydrocarbons (PAHs). The overall results of our in‐vitro study suggest that the investigated toner‐powder samples are not cytotoxic but genotoxic. From the results of the physical and chemical characterization, we conclude that metals and metalloids as components of magnetite, or PAHs as components of the carbon‐bearing material, are responsible for the genotoxic effects. Further research is necessary to determine the relevance of these in‐vitro observations for private and occupational toner powder exposure. Environ. Mol. Mutagen., 2011.

Investigations on cytotoxic and genotoxic effects of laser printer emissions in human epithelial A549 lung cells using an air/liquid exposure system

Exposure to emissions from laser printers during the printing process is commonplace worldwide, both in the home and workplace environment. In the present study, cytotoxic and genotoxic effects of the emission from five low to medium‐throughput laser printers were investigated with respect to the release of ozone (O3), volatile organic compounds (VOC), particulate matter (PM), and submicrometer particles (SMP) during standby and operation. Experiments were conducted in a 1 m3 emission chamber connected to a Vitrocell® exposure system. Cytotoxicity was determined by the WST‐1 assay and genotoxicity by the micronucleus test in human A549 lung cells. The five laser printers emitted varying but generally small amounts of O3, VOC, and PM. VOC emissions included 13 compounds with total VOC concentrations ranging from 95 to 280 μg/m3 (e.g., 2‐butanone, hexanal, m,p‐xylene, and o‐xylene). Mean PM concentrations were below 2.4 μg/m3. SMP number concentration levels during standby ranged from 9 to 26 particles/cm3. However, three of the printers generated a 90 to 16 × 103‐fold increase of SMP during the printing process (maximum 294,460 particles/cm3). Whereas none of the printer emissions were found to cause cytotoxicity, emissions from two printers induced formation of micronuclei (P < 0.001), thus providing evidence for genotoxicity. As yet, differences in biological activity cannot be explained on the basis of the specific emission characteristics of the different printers. Because laser printing technology is widely used, studies with additional cytogenetic endpoints are necessary to confirm the DNA‐damaging potency and to identify emission components responsible for genotoxicity. Environ. Mol. Mutagen., 2012.

Oxidative stress and inflammatory response to printer toner particles in human epithelial A549 lung cells.

Reports on adverse health effects related to occupational exposure to toner powder are still inconclusive. Therefore, we have previously conducted an in vitro-study to characterize the genotoxic potential of three commercially available black printer toner powders in A549 lung cells. In these cell-based assays it was clearly demonstrated that the tested toner powders damage DNA and induce micronucleus (MN) formation. Here, we have studied the cytotoxic and proinflammatory potential of these three types of printer toner particles and the influence of ROS and NF-κB induction in order to unravel the underlying mechanisms. A549 cells were exposed to various concentrations of printer toner particle suspensions for 24 h. The toner particles were observed to exert significant cytotoxic effects in the WST-1 and neutral red (NR)-assays, although to a varying extent. Caspase 3/7 activity increased, while the mitochondrial membrane potential (MMP) was not affected. Particles of all three printer toner powders induced concentration-dependent formation of reactive oxygen species (ROS), as measured in the DCFH-DA assay. Furthermore, toner particle exposure enhanced interleukin-6 and interleukin-8 production, which is in agreement with activation of the transcription factor NF-κB in A549 cells shown by the electrophoretic mobility shift assay (EMSA). Therefore, it can be concluded that exposure of A549 lung cells to three selected printer toner powders caused oxidative stress through induction of ROS. Increased ROS formation may trigger genotoxic effects and activate proinflammatory pathways.

Cell-Cycle Changes and Oxidative Stress Response to Magnetite in A549 Human Lung Cells

In a recent study, magnetite was investigated for its potential to induce toxic effects and influence signaling pathways. It was clearly demonstrated that ROS formation leads to mitochondrial damage and genotoxic effects in A549 cells. On the basis of these findings, we wanted to elucidate the origin of magnetite-mediated ROS formation and its influence on the cell cycle of A549 and H1299 human lung epithelial cells. Concentration- and size-dependent superoxide formation, measured by electron paramagnetic resonance (EPR), was observed. Furthermore, we could show that the GSH level decreased significantly after exposure to magnetite particles, while catalase (CAT) activity was increased. These effects were also dependent on particle size, albeit less pronounced than those observed with EPR. We were able to show that incubation of A549 cells prior to particle treatment with diphenyleneiodonium (DPI), a NADPH-oxidase (NOX) inhibitor, leads to decreased ROS formation, but this effect was not observed for the NOX inhibitor apocynin. Soluble iron does not contribute considerably to ROS production. Analysis of cell-cycle distribution revealed a pronounced sub-G1 peak, which cannot be linked to increased cell death. Western blot analysis did not show activation of p53 but upregulation of p21 in A549. Here, we were unexpectedly able to demonstrate that exposure to magnetite leads to p21-mediated G1-like arrest. This has been reported previously only for low concentrations of microtubule stabilization drugs. Importantly, the arrested sub-G1 cells were viable and showed no caspase 3/7 activation.

Recalcitrant pharmaceuticals in the aquatic environment: a comparative screening study of their occurrence, formation of phototransformation products and their in vitro toxicity

Data allowing for a complete environmental risk assessment of pharmaceuticals and their photoderatives in the environment are still scarce. In the present study, in vitro toxicity and both bio- and photopersistence of various pharmaceuticals (aciclovir, allopurinol, cetirizine, cimetidine, fluconazole, hydrochlorothiazide, lisinopril, phenytoin, primidone, ranitidine, sotalol, sulpiride, tramadol and valsartane) as well as their phototransformation products were evaluated in order to fill data gaps and to help prioritise them for further testing. Twelve out of the fourteen compounds investigated were found to be neither readily nor inherently biodegradable in the Organisation of Economic Cooperation and Development-biodegradability tests. The study further demonstrates that the photo-induced transformation of the pharmaceuticals was faster upon irradiation with a Hg lamp (UV light) than with a Xe lamp emitting a spectrum that mimicssunlight.Comparingthenon-irradiatedwiththerespectiveirradiatedsolutions,ahigheracuteandchronictoxicity against bacteria was found for the irradiated solutions of seven compounds (cetirizine, cimetidine, hydrochlorothiazide, ranitidine, sulpiride, tramadol and valsartane). No cyto- and genotoxic effects were found in human cervical (HeLa) and liver(Hep-G2) cellsforanyof theinvestigated compoundsor theirphototransformationproducts. This comparativestudy documents that phototransformation products can arise as a result of UV treatment of wastewater containing these pharmaceuticals. It further demonstrates that some phototransformation products may have a higher environmental risk potential than the respective parent compounds because some phototransformation products exhibited a higher bacterial toxicity.

Chemosensory irritations and pulmonary effects of acute exposure to emissions from oriented strand board

Due to the reduction of air change rates in low-energy houses, the contribution to indoor air quality of volatile organic compounds (VOCs) emitting from oriented strand boards (OSB) has become increasingly important. The aim of this study was to evaluate sensory irritations, pulmonary effects and odor annoyance of emissions from OSB in healthy human volunteers compared to clean air. Twenty-four healthy non-smokers were exposed to clean air and OSB emissions for 2 h under controlled conditions in a 48 m3 test chamber at three different time points: to fresh OSB panels and to the same panels after open storage for 2 and 8 weeks. Chemosensory irritation, exhaled nitric oxide (NO) concentration, eye blink frequency, lung function and subjective perception of irritation of eyes, nose and throat were examined before, during and after exposure. Additionally, olfactory perception was investigated. Total VOC exposure concentrations reached 8.9 ± 0.8 mg/m3 for the fresh OSB panels. Emissions consisted predominantly of α-pinene, ▵3-carene and hexanal. Two-hour exposure to high VOC concentrations revealed no irritating or pulmonary effects. All the subjective ratings of discomfort were at a low level and the medians did not exceed the expression ‘hardly at all.’ Only the ratings for smell of emissions increased significantly during exposure in comparison to clean air. In conclusion, exposure of healthy volunteers to OSB emissions did not elicit sensory irritations or pulmonary effects up to a VOC concentration of about 9 mg/m3. Sensory intensity of OSB emissions in the chamber air was rated as ‘neutral to pleasant.’

Sensory irritations and pulmonary effects in human volunteers following short-term exposure to pinewood emissions

Pinewood (Pinus ssp.) is widely used for furniture and building purposes. However, despite its widespread use, information on possible human sensory irritations and pulmonary effects caused by exposure to volatile organic compounds (VOC) emitted from pinewood is sparse. For this purpose, (1) sensory irritation of eyes, nose and throat, (2) lung function parameters (FVC, FEV1), (3) exhaled nitrogen oxide (NO) concentration, (4) eye blink frequency, and (5) sensory evaluation (using the SD method) were investigated before, after, and partly during exposure of human volunteers to emissions from pinewood panels. Fifteen healthy nonsmokers were exposed for 2 h under controlled conditions to VOCs emitted from pinewood panels in a 48 m3 test chamber. VOC concentrations were about 5 mg/ m3 (loading rate, 1 m2/m3), 8 mg/m3 (loading rate, 2 m2/m3), and 13 mg/m3 (loading rate, 3 m2/m3), respectively. Terpene and aldehyde exposure concentrations ranged from about 3.50 ± 0.51 mg/m3 and 0.07 ± 0.008 mg/m3, 5.00 ± 0.95 mg/ m3, and 0.20 ± 0.02 mg/m3 or 9.51 ± 1.10 mg/m3 and 0.21 ± 0.04 mg/m3 for loading rates of 1, 2, and 3 m2/m3, respectively. The emissions consisted predominantly of α-pinene and δ3-carene. No concentration-dependent effects before or after exposure to the emissions were measured with respect to sensory irritation, pulmonary function, exhaled NO, and eye blink frequency. Only the odor of the emissions was perceived by the study subjects, rated as being closer to “pleasant” than to “unpleasant.” In conclusion, the results of our study suggest that short-term exposure to high VOC concentrations, even up to 13 mg/m3, released from pinewood does not elicit sensory irritation or pulmonary effects in healthy humans under controlled conditions.

Antibiotics and sweeteners in the aquatic environment : biodegradability, formation of phototransformation products, and in vitro toxicity

In the present study, in vitro toxicity as well as biopersistence and photopersistence of four artificial sweeteners (acesulfame, cyclamate, saccharine, and sucralose) and five antibiotics (levofloxacin, lincomycin, linezolid, marbofloxacin, and sarafloxacin) and of their phototransformation products (PTPs) were investigated. Furthermore, antibiotic activity was evaluated after UV irradiation and after exposure to inocula of a sewage treatment plant. The study reveals that most of the tested compounds and their PTPs were neither readily nor inherently biodegradable in the Organisation for Economic Co-operation and Development (OECD)-biodegradability tests. The study further demonstrates that PTPs are formed upon irradiation with an Hg lamp (UV light) and, to a lesser extent, upon irradiation with a Xe lamp (mimics sunlight). Comparing the nonirradiated with the corresponding irradiated solutions, a higher chronic toxicity against bacteria was found for the irradiated solutions of linezolid. Neither cytotoxicity nor genotoxicity was found in human cervical (HeLa) and liver (Hep-G2) cells for any of the investigated compounds or their PTPs. Antimicrobial activity of the tested fluoroquinolones was reduced after UV treatment, but it was not reduced after a 28-day exposure to inocula of a sewage treatment plant. This comparative study shows that PTPs can be formed as a result of UV treatment. The study further demonstrated that UV irradiation can be effective in reducing the antimicrobial activity of antibiotics, and consequently may help to reduce antimicrobial resistance in wastewaters. Nevertheless, the study also highlights that some PTPs may exhibit a higher ecotoxicity than the respective parent compounds. Consequently, UV treatment does not transform all micropollutants into harmless compounds and may not be a large-scale effluent treatment option.

Cement-related particles interact with proinflammatory IL-8 chemokine from human primary oropharyngeal mucosa cells and human epithelial lung cancer cell line A549

Epidemiological studies have shown that respirable exposure to emitted cement particulate matter is associated with adverse health risk for human. The underlying mechanisms, however, are poorly understood. To examine the effect of cement, nine blinded cement‐related particulates (<10 μm) were assessed with regard to their induction of the proinflammatory cytokines IL‐6 and IL‐8 in human primary epithelial cells (pEC) from oropharyngeal mucosa as well as from nonsmall‐cell lung carcinoma (non‐SCLC) cells A549. It was demonstrated that the cement specimens did not act cytotoxic as assessed by the lactate dehydrogenase (LDH) assay. The basal and IL‐1β‐induced IL‐8 expression was suppressed, in contrast to an unchanged IL‐6. At the transcript level the basal and induced IL‐6 and IL‐8 gene expression was not influenced by cement dust. To discover the mechanism by which cement influenced the IL‐8 expression the following experiments were performed. Submerse exposure experiments have shown that the release of IL‐8 was suppressed by cement dust. Furthermore, the incubation of IL‐8 with cement‐related specimens under cell‐free condition led to a loss of immunoreactive IL‐8. An immunological masking of IL‐8 by free soluble components of respiratory epithelial cells was excluded. Thus, the decrease of IL‐8 protein content after cement exposure seems to be a result of the adsorption of IL‐8 protein to cement particles and the inhibition of IL‐8 release. In conclusion, due to absent cytotoxic and inflammatory effects of cement‐related specimens in both human pEC and A549 cell models it remains open how cement exposure may lead to the respiratory adverse effects in humans.