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The efficient entry of nanotechnology-based pharmaceuticals into target cells is highly desired to reach high therapeutic efficiencywhile minimizing the side effects. Despite intensive research, the impact of the surface coating on the mechanism of nanoparticleuptake is not sufficiently understood yet. Herein, we present a mechanistic study of cellular internalization pathways of two magneticiron oxide nanoparticles (MNPs) differing in surface chemistry into A549 cells. The MNP uptake was investigated in the presenceof different inhibitors of endocytosis and monitored by spectroscopic and imaging techniques. The results revealed that theroute of MNP entry into cells strongly depends on the surface chemistry of the MNPs. While serum bovine albumin-coated MNPsentered the cells via clathrin-mediated endocytosis (CME), caveolin-mediated endocytosis (CavME) or lipid rafts were preferentiallyinvolved in the internalization of polyethylene glycol-coated MNPs. Our data indicate that surface engineering can contribute toan enhanced delivery efficiency of nanoparticles.

The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assay (MIRCA) providing recommendations for describing comet assay conditions and results. These recommendations differentiate between ‘desirable’ and ‘essential’ information: ‘essential’ information refers to the precise details that are necessary to assess the quality of the experimental work, whereas ‘desirable’ information relates to technical issues that might be encountered when repeating the experiments. Adherence to MIRCA recommendations should ensure that comet assay results can be easily interpreted and independently verified by other researchers., Here, members of the hCOMET COST Action program provide a consensus statement on the Minimum Information for Reporting Comet Assays (MIRCA).

5-Fluorouracil (5-FU) is an essential chemotherapeutic drug for colorectal cancer (CRC) treatment. However, thefrequent development of drug resistance has dramatically affected its clinical use. Therefore, novel treatmentstrategies are critical to improving patient outcomes. Herein, we investigated the ability of the epigenetic drugSAHA to increase the sensitivity of chemoresistant CRC cells to 5-FU. In addition, we evaluated the potentialgenotoxic risk of SAHA+5-FU combination treatment. As a model system, we used three CRC cell lines, HT-29,SW480, and HT-29/EGFP/FUR, differing in their resistance to 5-FU. CRC cell lines were exposed to sub-toxicSAHA concentrations for 24 h, followed by a 48 h treatment with 5-FU. The cytotoxicity of SAHA, 5-FU, andSAHA+5-FU was measured by the MTT test, the genotoxicity by the comet assay, and the micronucleus test.Theapoptotic/necrotic activity was assessed using morphological criteria. We found a synergic decrease in the viability of HT-29 and SW480 cells, but not the most resistant HT-29/EGFP/FUR cells after combined SAHA+5-FU exposure compared to 5-FU. Remarkably, SAHA most efficientlyinduced apoptosis in HT-29/EGFP/FUR cells compared to HT-29 and SW480 cells. Combined SAHA+5-FUtreatment resulted in a synergistic increase in apoptotic/necrotic cells in HT-29 cell line, while rather additive/sub-additive effect was determined in the SW480 and HT-29/EGFP/FUR cells. At the same time, however, asynergistic rise in micronuclei was found in CRC cell lines (at least at some concentrations). We have shown thatSAHA can sensitize CRC cells to 5-FU; therefore, epigenetic and convential drug combinations could be beneficialfor the patients. However, the increase in micronucleus formation after combined SAHA+5-FU treatment indicatesa potential health hazard. The clastogenic activity could contribute to cancer heterogeneity, favoringprogeny of such aberrant cells to clonal expansion. Therefore, developing new specific epigenetic drugs ornanocarriers for targeted drug delivery might reduce the potential genotoxic risk.

Despite the obvious advantages of gold nanoparticles for biomedical applications, controversial and incomplete toxicological data hamper their widespread use. Here, we present the results from an in vivo toxicity study using gold nanoparticles coated with polyethylene glycol (PEG-AuNPs). The pharmacokinetics and biodistribution of PEG-AuNPs were examined in the rat’s liver, lung, spleen, and kidney after a single i.v. injection (0.7 mg/kg) at different time intervals. PEG-AuNPs had a relatively long blood circulation time and accumulated primarily in the liver and spleen, where they remained for up to 28 days after administration. Increased cytoplasmic vacuolation in hepatocytes 24 h and 7 days after PEG-AuNPs exposure and apoptotic-like cells in white splenic pulp 24 h after administration has been detected, however, 28 days post-exposure were no longer observed. In contrast, at this time point, we identified significant changes in lipid metabolism, altered levels of liver injury markers, and elevated monocyte count, but without marked biological relevance. In blood cells, no DNA damage was present in any of the studied time intervals, with the exception of DNA breakage transiently detected in primary kidney cells 4 h post-injection. Our results indicate that the tissue accumulation of PEG-AuNPs might result in late toxic effects., This paper is supported by European Union’s Horizon 2020 research and innovation program under grant agreement No. 857381, project VISION (Strategies to strengthen scientific excellence and innovation capacity for early diagnosis of gastrointestinal cancers), project HISENTS No. 685817, VEGA grants 2/0055/20, 2/0121/21.

Exposure to pesticides leads to complex, long-lasting adverse effects on human health, and poses a substantial risk to those living in areas devoted to agriculture. Children are particularly vulnerable to the pesticide exposure, due to the developmental, dietary and physiological factors. Small body mass and typical exploratory behavior result in increased risk of intoxication. Thus, even exposure to low concentrations of pesticides, if of sufficient duration, may lead to permanent health disorders and limit their harmonious development. In this study 108 children, living in areas of an intense pesticide use and a control group (n = 92) of children from an agrotouristic area were investigated, whether DNA damage increased due to prolonged pesticide exposure. A presence of DNA breaks and oxidative damage to DNA bases, characterized as Fpg-sensitive sites, were detected by comet assay. Micronuclei (MN) formation was evaluated by cytokinesis-block MN assay. The exposure of children to pesticides resulted in increased number of MN in peripheral blood lymphocytes (P = 0.016), increased DNA strand breaks level (P = 0.002) and oxidative damage to DNA (P 0.001). Negative correlation was demonstrated between the level of DNA strand breaks and acetylcholinesterase (AChE) activity in exposed group. In conclusion, despite just environmental pesticide exposure in the test group of children, significant biological effects were detected.

Microfluidic technology is a valuable tool for realizing more in vitro models capturing cellular and organ level responses for rapid and animal-free risk assessment of new chemicals and drugs. Microfluidic cell-based devices allow high-throughput screening and flexible automation while lowering costs and reagent consumption due to their miniaturization. There is a growing need for faster and animal-free approaches for drug development and safety assessment of chemicals (Registration, Evaluation, Authorisation and Restriction of Chemical Substances, REACH). The work presented describes a microfluidic platform for in vivo-like in vitro cell cultivation. It is equipped with a wafer-based silicon chip including integrated electrodes and a microcavity. A proof-of-concept using different relevant cell models shows its suitability for label-free assessment of cytotoxic effects. A miniaturized microscope within each module monitors cell morphology and proliferation. Electrodes integrated in the microfluidic channels allow the noninvasive monitoring of barrier integrity followed by a label-free assessment of cytotoxic effects. Each microfluidic cell cultivation module can be operated individually or be interconnected in a flexible way. The interconnection of the different modules aims at simulation of the whole-body exposure and response and can contribute to the replacement of animal testing in risk assessment studies in compliance with the 3Rs to replace, reduce, and refine animal experiments., T.K. and Y.K. contributed equally to this work. The authors thank Frank Bauerfeld for system assembly and electrical testing, Werner Haberer for assembly of the microfluidic cartridges, Axel Brenner for the fabrication of the silicon micro cavity chips, Hoffman La Roche for providing the Ro 19-8022, NorGenoTech for providing the Fpg enzyme, and Karen Steenson for proofreading. This research was funded by the European Commission under the Horizon2020 programme (HISENTS, Grant Agreement No. 685817 and VISION, Grant Agreement No. 857381); and by the Norwegian Research Council Norway (RCN) via the European Research Area Network (ERA-NET) EuroNanoMed II project INNOCENT (RCN 271075) and the ERA-NET EuroNanoMed III project Graphene-encapsulated magnetic nanoparticles (RCN 246672/O70). K.K. received Short Term Scientific Mission Grant (ID 42926) under COST Action CA 17140 “Cancer Nanomedicine from the Bench to the Bedside” supported by COST (European Cooperation in Science and Technology). E.E. received funding from the Norwegian Research Council (272412/F40). O.H.M. received funding from the Spanish Ministry of Economy and Competitiveness (SEV-2013-0295-17-3).

Drug-induced nephrotoxicity is a frequent adverse event and a dose-limiting factor in patient treatment and is a leading cause of prospective drug attrition during pharmaceutical development. Despite the obvious benefits of nanotherapeutics in healthcare strategies, the clearance of imaging agents and nanocarriers from the body following their therapeutic or diagnostic application generates concerns about their safety for human health. Considering the potency of nanoparticles and their massive utilization in biomedicine the impact of magnetic nanoparticles (MNPs) on cells forming the filtration apparatus of the kidney was studied. Using primary mouse renal glomerular podocytes and mesangial cells, we investigated their response to exposure to magnetic nanoparticles coated with polyethylene glycol and bovine serum albumin. Cultured podocytes were more sensitive to MNPs than mesangial cells displaying signs of cell damage and stronger inflammatory response. Both types of MNPs induced the remodeling of actin fibers, affected the cell shape and triggered expression of inflammatory cytokines TNFα and IL-6 in podocytes. On the other hand, iNOS was induced in both renal cell types but only by MNPs with a polyethylene glycol coating. Our results have revealed that the type of cell and the type of nanoparticle coating might be the strongest determinants of cellular response toward nanoparticle exposure. Differences in susceptibility of cells to MNPs might be evident also between neighboring renal cell subpopulations integrally forming functional sub-units of this organ.

7H-Dibenzo[c,g]carbazole (DBC), a local and systemic carcinogen in animal studies, is a common environmental pollutant. It generally co-occurs in a variety of organic complex mixtures derived from incomplete combustion of organic matter. Despite high lipophilicity, DBC is more water-soluble and faster metabolized than the homocyclic aromatics. Moreover, greater polarity, high bioaccumulation potential, and persistence in the environment may imply DBC's higher biological significance and impact on human health, even at lower concentrations. The biotransformation pathways of DBC are incompletely known and the ultimate carcinogenic metabolite(s) are not clearly identified as yet. Structure-biological studies suggest two ways of activation: at the ring carbon atoms and at the pyrrole nitrogen. It is supposed that the particular pathway of biotransformation might be connected with the tissue/organ specificity of DBC. Cytochrome P450 (CYP) family of enzymes plays a pivotal role in the metabolism of DBC; though, the one-electron activation and the aldo-keto reductase-catalyzed oxidation are also involved in metabolic activation. Additionally, DBC can be photoactivated even at physiologically relevant doses of UVA light due to the extended aromatic ring system resulting in strong genotoxicity and oxidative stress. The goal of this review is to summarize current knowledge on mechanisms of DBC activation and possible implications for toxicity, genotoxicity, and carcinogenicity.

Nanomedicine is an emerging field that combines knowledge of nanotechnology and material science with pharmaceutical and biomedical sciences, aiming to develop nanodrugs with increased efficacy and safety. Compared to conventional therapeutics, nanodrugs manifest higher stability and circulation time, reduced toxicity and improved targeted delivery. Despite the obvious benefit, the accumulation of imaging agents and nanocarriers in the body following their therapeutic or diagnostic application generates concerns about their safety for human health. Numerous toxicology studies have demonstrated that exposure to nanomaterials (NMs) might pose serious risks to humans. Epigenetic modifications, representing a non-genotoxic mechanism of toxicant-induced health effects, are becoming recognized as playing a potential causative role in the aetiology of many diseases including cancer. This review i) provides an overview of recent advances in medical applications of NMs and ii) summarizes current evidence on their possible epigenetic toxicity. To discern potential health risks of NMs, since current data are mostly based upon in vitro and animal models, a better understanding of functional relationships between NM exposure, epigenetic deregulation and phenotype is required.

Biomonitoring of the effects of environmental and occupational exposure relevant chemical or physical factors on central nervous system is difficult due to the problems with sampling of biological material. Thus, surrogate systems allowing for the estimation of effect intensity are necessary to evaluate a potential risk of exposure. Cancerous neural cells in culture seem to be a reliable trustworthy alternative to ex vivo primary cells culture, where brain tissue is hardly available. In this review we summarized attempts to test genotoxicity of environmentally related xenobiotics or physical factors. Different neural cells of human and non-human origin are described in respect to their use in genotoxicity testing using the comet assay. Surprisingly, despite the large number of commercially available neural cells of different type and origin, only twelve were used for genotoxicity testing by the comet assay. We also recapitulate the environmentally relevant chemical and physical factors tested on neural cell lines in vitro by the comet assay. The most prevalent were fire retardants, plant protection agents, nanoparticles and magnetic field.

Progressive expansion of nanomaterials in our everyday life raises concerns about their safety for human health. Although kidneys are the primary organs of xenobiotic elimination, little attention has been paid to the kidneys in terms of nanotoxicological studies up to now. Here we investigate the cytotoxic and genotoxic potential of four solid-core uncoated inorganic nanoparticles (TiO2NPs, SiO2NPs, Fe3O4NPs and AuNPs) using the human renal proximal tubule epithelial TH1 cells. To mimic the in vivo conditions more realistic, TH1 cells were exposed in vitro to inorganic NPs under static as well as dynamic conditions for 3 h and 24 h. The medium throughput alkaline comet assay (12 minigels per slide) was employed to evaluate the impact of these NPs on genome integrity and their capacity to produce oxidative lesions to DNA. The accumulation and localization of studied inorganic NPs inside the cells was monitored by transmission electron microscopy (TEM) and the efficacy of internalization of particular NPs was determined by atomic absorption spectroscopy (AAS) and inductively coupled plasma mass spectrometry (ICP-MS). From all the tested NPs, only Fe3O4NPs induced a slight cytotoxicity in TH1 cells exposed to high concentrations (>700 μg/ml) for 24 h. On the other hand, the inorganic NPs did not increase significantly the level of DNA strand breaks or oxidative DNA damage regardless of the treatment mode (static vs. dynamic conditions). Interestingly, substantial differences were observed in the internalized amount of inorganic NPs in TH1 cells exposed to equivalent (2.2 μg/ml) concentration. Fe3O4NPs were most efficiently taken up while the lowest quantity of particles was determined in TiO2NPs-treated cells. As the particle size and shape of individual inorganic NPs in culture medium was nearly identical, it is reasonable to suppose that the chemical composition may contribute to the differences in the efficacy of NPs uptake., This article was created by the realization of the H2020 project HISENTS No. 685817, COST Action CA15132, VEGA grant 2/0056/17, project "Center of excellence of environmental health", ITMS No. 26240120033, based on the support of operational Research and development program financed from the European Regional Development Fund; and the EEA projectSK0020. Filip Razga received support within the SASPRO Programme (Project No. 0057/01/02) co-funded by the European Union and the Slovak Academy of Sciences.

7H-Dibenzo[c,g]carbazole (DBC) is a heterocyclic aromatic hydrocarbon that is carcinogenic in many species and tissues. DBC is a common environmental pollutant, and is therefore constantly exposed to sunlight. However, there are limited data exploring the toxicity of DBC photoexcitation products. Here, we investigated the impact of ultraviolet (UV) A radiation on the biological activity of DBC and its methyl derivatives, 5,9-dibenzo[c,g]carbazole and N-methyl dibenzo[c,g]carbazole, on human skin HaCaT keratinocytes. Co-exposure of HaCaT cells to UVA and DBC derivatives resulted in a sharp dose-dependent decrease in cell survival and apparent changes in cell morphology. Under the same treatment conditions, significant increases in DNA strand breaks, intracellular reactive oxygen species, and oxidative damage to DNA were observed in HaCaT cells. Consistent with these results, an apparent inhibition in superoxide dismutase, but not glutathione peroxidase activity, was detected in cells treated with DBC and its derivatives under UVA irradiation. The photoactivation-induced toxicity of individual DBC derivatives correlated with the electron excitation energies approximately expressed as the energy difference between the highest occupied and the lowest vacant molecular orbital. Our data provide the first evidence that UVA can enhance the toxicity of DBC and its derivatives. Photoactivation-induced conversion of harmless chemical compounds to toxic photoproducts associated with reactive oxygen species generation may substantially amplify the adverse health effects of UVA radiation and contribute to increased incidence of skin cancer. Environ. Mol. Mutagen. 56:388–403, 2015. © 2014 Wiley Periodicals, Inc.

Nowadays engineered nanomaterials (ENMs) are increasingly used in a wide range of commercial products and biomedical applications. Despite this, the knowledge of human potential health risk as well as comprehensive biological and toxicological information is still limited. We have investigated the capacity of two frequently used metallic ENMs, nanosilver and magnetite nanoparticles (MNPs), to induce thymidine kinase (Tk+/−) mutations in L5178Y mouse lymphoma cells and transformed foci in Bhas 42 cells. Two types of nanosilver, spherical nanoparticles (AgNM300) and fibrous (AgNM302) nanorods/wires, and MNPs differing in surface modifications [MNPs coated with sodium oleate (SO-MNPs), MNPs coated with SO + polyethylene glycol (SO-PEG-MNPs) and MNPs coated with SO + PEG + poly(lactide-co-glycolic acid) SO-PEG-PLGA-MNPs] were included in this study. Spherical AgNM300 showed neither mutagenic nor carcinogenic potential. In contrast, silver nanorods/wires (AgNM302) increased significantly the number of both gene mutations and transformed foci compared with the control (untreated) cells. Under the same treatment conditions, neither SO-MNPs nor SO-PEG-PLGA-MNPs increased the mutant frequency compared with control cells though an equivocal mutagenic effect was estimated for SOPEG- MNPs. Although SO-MNPs and SO-PEG-MNPs did not show any carcinogenic potential, SO-PEG-PLGA-MNPs increased concentration dependently the number of transformed foci in Bhas 42 cells compared with the control cells. Our results revealed that fibrous shape underlies the mutagenic and carcinogenic potential of nanosilver while surface chemistry affects the biosafety of MNPs. Considering that both nanosilver and MNPs are prospective ENMs for biomedical applications, further toxicological evaluations are warranted to assess comprehensively the biosafety of these nanomaterials. FP7 NANoREG (Grant Agreement No. NMP4-LA-2013–310584); FP7 QualityNano (Grant Agreement No: INFRA-2010–262163) info:eu-repo/semantics/publishedVersion

Comprehensive characterization of nanoparticles associated with investigation of their cellular uptake creates the basis on which fundamental in vitro and in vivo studies can be built. In this work, a complex analysis of various surface-modified magnetite nanoparticles in biologically relevant environment is reported and the promotion of incorrect characterization into the results obtained from model biological experiments leading to false conclusions is demonstrated. Via a bottom-up approach from particle characterization by DLS towards interpretation of biological data based on cellular uptake, this work draws attention to the systematic propagation of errors stemming from inaccurate determination of input parameters for DLS, improper selection of particle size distribution, inadequate sampling, unknown colloidal behavior and the omission of fraction of particles complying with the internalization threshold. In addition, cellular uptake depending on the number of treated cells is shown. The definition of cellular uptake efficacy reflecting the size distribution of particles beside their absolute internalization is postulated.

Iron oxide nanoparticles are one of the most promising types of nanoparticles for biomedical applications, primarily in the context of nanomedicine-based diagnostics and therapy; hence, great attention should be paid to their bio-safety. Here, we investigate the ability of surface-modified magnetite nanoparticles (MNPs) to produce chromosome damage in human alveolar A549 cells. Compared to control cells, all the applied MNPs increased the level of micronuclei moderately but did not cause structural chromosomal aberrations in exposed cells. A rise in endoreplication, polyploid and multinuclear cells along with disruption of tubulin filaments, downregulation of Aurora protein kinases and p53 protein activation indicated the capacity of these MNPs to impair the chromosomal passenger complex and/or centrosome maturation. We suppose that surface-modified MNPs may act as aneugen-like spindle poisons via interference with tubulin polymerization. Further studies on experimental animals revealing mechanisms of therapeutic-aimed MNPs are required to confirm their suitability as potential anti-cancer drugs.

Differences between tissues in the expression of drug-metabolizing enzymes may substantially contribute to tissue-specificity of chemical carcinogens. To verify this hypothesis, the spontaneously immortalized human keratinocytes HaCaT were used, in order to evaluate the genotoxic potential of 7H-dibenzo[c,g]carbazole (DBC), a known hepatocarcinogen and sarcomagen, and its synthetic tissue-specific derivatives, 5,9-dimethyl-DBC (DiMeDBC) and N-methyl-DBC (N-MeDBC), which manifest specific tropism to the liver and skin, respectively. HaCaT cells mainly express cytochrome P4501A1 (CYP1A1), which is involved in metabolism of DBC and N-MeDBC, but not DiMeDBC [10]. Both DBC and the sarcomagen N-MeDBC induced significant levels of DNA strand-breaks, micronuclei, and DNA adducts followed by the phosphorylation of the p53 protein and histone H2AX in HaCaT cells. In contrast, the specific hepatocarcinogen DiMeDBC was devoid of any significant genotoxic activity in this cell line. Our study demonstrates that the absence of drug-metabolizing enzyme(s) involved in DiMeDBC metabolism may contribute substantially to the tissue-specific genotoxicity of this hepatocarcinogen.

The goal of this study was to investigate the genotoxicity of 7H-dibenzo[c,g]carbazole (DBC), a ubiquitous environmental pollutant, and its methyl derivatives, 5,9-dimethylDBC (DiMeDBC), a strict hepatocarcinogen, and N-methylDBC (N-MeDBC), a specific sarcomagen in human hepatoma HepG2 cells, and to infer potential mechanisms underlying the biological activity of particular carcinogen. All dibenzocarbazoles, regardless the tissue specificity, induced significant DNA strand break levels and micronuclei in HepG2 cells; though a mitotic spindle dysfunction rather than a chromosome breakage was implicated in N-MeDBC-mediated micronucleus formation. While DBC and N-MeDBC produced stable DNA adducts followed with p53 protein phosphorylation at Ser-15, DiMeDBC failed. A significant increase in DNA strand breaks following incubation of exposed cells with a repair-specific endonuclease (Fpg protein) suggested that either oxidative DNA damage or unstable DNA-adducts might underlie DiMeDBC genotoxicity in human hepatoma cells. DiMeDBC and N-MeDBC increased substantially also the amount of CYP1A1/2 expression in HepG2 cells. Pretreatment of cells with substances affecting AhR-mediated CYP1A family of enzymes expression; however, diminished DiMeDBC and N-MeDBC genotoxicity. Our data clearly demonstrated differences in the mechanisms involved in the biological activity of DiMeDBC and N-MeDBC in human hepatoma cells; the genotoxicity of these DBC derivatives is closely related to CYP1A1/2 expression. Environ. Mol. Mutagen., 2011. © 2011 Wiley-Liss, Inc.

The free radical generating activity of airborne particulate matter (PM(10)) has been proposed as a primary mechanism in biological activity of ambient air pollution. In an effort to determine the impact of the complex mixtures of extractable organic matter (EOM) from airborne particles on oxidative damage to DNA, the level of 8-oxo-2'-deoxyguanosine (8-oxodG), the most prevalent and stable oxidative lesion, was measured in the human metabolically competent cell line Hep G2. Cultured cells were exposed to equivalent EOM concentrations (5-150microg/ml) and oxidative DNA damage was analyzed using a modified single cell gel electrophoresis (SCGE), which involves the incubation of whole cell DNA with repair specific DNA endonuclease, which cleaves oxidized DNA at the sites of 8-oxodG. EOMs were extracted from PM(10) collected daily (24h intervals) in three European cities: Prague (Czech Republic, two monitoring sites, Libus and Smíchov), Kosice (Slovak Republic) and Sofia (Bulgaria) during 3-month sampling periods in the winter and summer seasons. No substantial time- and dose-dependent increase of oxidative DNA lesions was detected in EOM-treated cells with the exception of the EOM collected at the monitoring site Kosice, summer sampling. In this case, 2h cell exposure to EOM resulted in a slight but significant increase of oxidative DNA damage at three from total of six concentrations. The mean 8-oxodG values at these concentrations ranged from 15.3 to 26.1 per 10(6) nucleotides with a value 3.5 per 10(6) nucleotides in untreated cells. B[a]P, the positive control, induced a variable but insignificant increase of oxidative DNA damage in Hep G2 cell (approximately 1.6-fold increase over control value). Based on these data we believe that EOM samples extracted from airborne particle PM(10) play probably only a marginal role in oxidative stress generation and oxidative lesion formation to DNA. However, adsorbed organic compounds can undergo various interactions (additive or synergistic) with other PM components or physical factors (UV-A radiation) and in this way they might enhance/multiply the adverse health effects of air pollution.

Sensitivity and correlations among three endpoints were evaluated to assess the genotoxic potential of organic complex mixtures in vitro. This study was focused on DNA adduct formation, DNA single strand break induction and tumour suppressor p53 protein up-regulation produced by extractable organic matter (EOM) absorbed on respirable particulate matter PM10 (particulate matter

The genotoxic potential of extractable organic matter (EOM) associated with the respirable particulate matter (PM10 microm) of atmospheric pollution has been determined in three European cities--Prague (Czech Republic, two monitoring sites, Libus and Smíchov), Kosice (Slovak Republic) and Sofia (Bulgaria) using the alkaline single-cell gel electrophoresis (the comet assay). The EOM samples were extracted by dichloromethane from ambient airborne particles collected daily (24 h intervals) during 3-month sampling periods in winter and summer seasons. The human metabolically competent cell line Hep G2 was used as a test system and benzo[a]pyrene (BaP), a known carcinogen, was applied as a positive control (internal standard) in each electrophoretic run. Two-hour exposure of Hep G2 cells to equivalent EOM concentrations ranging from 5 to 150 microg EOM/ml resulted in a linear dose-dependent increase of DNA migration (r0.9, P0.01). A less significant dose-response (r = 0.61) was only induced by the EOM sample from the locality Prague-Libus (PRG-LB) in the winter. Generally, a 1.5 to four-fold increase of DNA strand breaks over the background control level was determined in EOM-exposed cells. In order to compare the genotoxic potential of individual EOMs, a mathematical model was used to correct the 'real' data. No substantial location- or season-related differences were found in EOM genotoxicity (EOM microg/ml), except for the EOM sample from Sofia, collected in the summer. This EOM sample induced a nearly two-fold lower level of DNA damage in comparison with other EOMs. On the other hand, clear statistically significant location- and season-related differences (P0.001) in ambient air genotoxicity were determined when the EOM quantity per cubic meter of air (microg/m3) was taken into account. In that case, the genotoxicity of winter air pollution was six- to 10-fold higher in comparison with summer air. The air pollution genotoxicity in individual localities rose during the winter season in the order: PRG-LBKosicePrague-Smíchov (PRG-SM)Sofia, while during the summer season the highest ambient air genotoxicity was revealed in the locality Prague-Smíchov and approximately equal air pollution genotoxicity was determined among localities Prague-Libus, Kosice and Sofia (PRG-LB approximately Kosice approximately SofiaPRG-SM). The greatest overall air pollution genotoxicity was determined in the locality Sofia during the winter season. In a time course study to evaluate the kinetics of DNA strand break rejoining it was shown that the level of DNA strand breaks in EOM-exposed cells has returned to near the background level within 24 h after the treatment.

N-nitrosomorpholine (NMOR) is a well-known hepatocarcinogen. Since this compound is representative of the group of indirect-acting N-nitrosamines, its metabolic activation should be essential. However, the mechanism of NMOR-induced carcinogenesis is still not completely clear. In this paper we tried to further our understanding of the genotoxic effects of NMOR. The central aim of this study was to elucidate to what extent NMOR requires metabolic activation. For evaluation of the mutagenicity of NMOR, V79 cells were used either in the presence or absence of the microsomal S9 fraction in the mutation assay and formation of reactive oxygen/nitrogen species (ROS/RNS) in Caco-2 cells treated with NMOR was measured by a fluorescent assay. A very weak rise of 6-thioguanine resistant mutations was observed in both NMOR-treated model cells, V79/-S9 and V79/+S9. A significant difference between the level of mutations in V79/-S9 and V79/+S9 cells was recorded on the 7th day of expression only. Data obtained by the fluorescent assay confirmed that NMOR caused generation of ROS/RNS. In summary, the presented results showed that NMOR might induce DNA damage not only indirectly by its activation by drug-metabolizing enzymes but also via direct formation of ROS/RNS.

acetyltransferase. Treatment of the parental cell linesV79MZ and V79NH, which are devoid of any CYPactivity, with DBC and its derivatives did not result indetectable adducts. The highest DNA adduct levelswerefoundinCYP1A1-expressingV79MZh1A1cellsafter DBC and MeDBC treatment (24.5 7.2 and16.2 83.6 adducts/10 nucleotides, respectively).ExposureofthiscelllinetoDBCresultedinfivedistinctspots, while six spots with different chromatographicmobilities were detected in MeDBC-treated cells.DiMeDBC produced only very low levels of DNAadducts in V79MZh1A1 cells. DBC and MeDBCformed relatively low levels of DNA adducts inCYP1A2-expressing V79MZh1A2 cells (0.7 0.2and 2.1 1.2 adducts/10

Exposure to high levels of environmental air pollution is known to be associated with an increased carcinogenic risk. The individual contribution to this risk derived from specific carcinogenic chemicals within the complex mixture of air pollution is less certain, but may be explored by the use of molecular epidemiological techniques. Measurements of biomarkers of exposure, of effect and of susceptibility provide information of potential benefit for epidemiological and cancer risk assessment. The application of such techniques has been mostly concerned in the past with the carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) that are associated with particulate matter in air pollution, and has showed clear evidence of genotoxic effects, such as DNA adducts, chromosome aberrations (CA) and ras oncogene overexpression, in environmentally exposed Czech and Polish populations. We are currently extending these studies by an investigation of populations exposed to environmental pollution in three European countries, Czech Republic, Slovak Republic and Bulgaria. This pays particular attention to PAHs, but also investigates the extent of radically induced (oxidative) DNA damage in the exposed populations. Policemen, bus drivers and controls, who carried personal monitors to determine their exposures to PAHs have been studied, and blood and urine were collected. Antioxidant and dietary status were assessed in these populations. Stationary monitors were also used for ambient air monitoring. Amongst the parameters studied in the biological samples were: (a) exposure biomarkers, such

The antioxidant butylated hydroxyanisole (BHA) is a rodent carcinogen that also reduces the mutagenicity and carcinogenicity of other agents. In this study, we have evaluated possible mechanisms for the antimutagenicity of BHA by investigating its effects on N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-treated Chinese hamster V79 cells. Mutant frequency was determined using the hprt/V79 assay, while plating efficiency was used to measure cytotoxicity, and apoptosis was measured by flow immunofluorocytometry. In addition, DNA strand breaks and the kinetics of strand-break rejoining were investigated by the alkaline elution of DNA and by single-cell gel electrophoresis (SCGE). Although the higher concentration of BHA (0.5 mM) increased the cytotoxicity of MNNG and the lower concentration of BHA (0.25 mM) did not change it, both concentrations were antimutagenic in MNNG-treated cells, with the greater effect occurring at the lower BHA concentration. Neither BHA nor MNNG nor BHA + MNNG increased the level of apoptotic nuclei, and BHA did not change the level of MNNG-induced DNA strand breaks, though it did inhibit their rejoining. Determination of O6-methylguanine-DNA-methyltransferase (MGMT) activity confirmed that V79 cells do not synthesize active MGMT protein; MGMT activity was also undetectable after MNNG and BHA + MNNG treatment. The ability of BHA to reduce the level of MNNG-induced mutations did not correlate with cytotoxicity, induction of apoptosis, the level of DNA strand break induction, or MGMT activity. A modified SCGE assay showed that BHA significantly reduced the level of formamidopyrimidine-DNA-glycosylase + endonucleaseIII-sensitive sites, which at least partially are caused by oxidative DNA lesions. The results suggest that the protective effect of BHA on MNNG-induced mutagenicity is best explained by the antioxidative activity of BHA, which may scavenge free radicals that participate in MNNG-induced mutagenicity. Environ. Mol. Mutagen. 41:28–36, 2003. © 2003 Wiley-Liss, Inc.