In vitro cell-transforming potential of secondary polyethylene terephthalate and polylactic acid nanoplastics.

Continuous exposure to plastic pollutants may have serious consequences on human health. However, most toxicity assessments focus on non-environmentally relevant particles and rarely investigate long-term effects such as cancer induction. The present study assessed the carcinogenic potential of two secondary nanoplastics: polyethylene terephthalate (PET) particles generated from plastic bottles, and a biodegradable polylactic acid material, as respective examples of environmentally existing particles and new bioplastics. Pristine polystyrene nanoplastics were also included for comparison. A broad concentration range (6.25-200 µg/mL) of each nanoplastic was tested in both the initiation and promotion conditions of the regulatory assessment-accepted in vitro Bhas 42 cell transformation assay. Parallel cultures allowed confirmation of the efficient cellular internalisation of the three nanoplastics. Cell growth was enhanced by polystyrene in the initiation assay, and by PET in both conditions. Moreover, the number of transformed foci was significantly increased only by the highest PET concentration in the promotion assay, which also showed dose-dependency, indicating that nano PET can act as a non-genotoxic tumour promotor. Together, these findings support the carcinogenic risk assessment of nanoplastics and raise concerns regarding whether real-life co-exposure of PET nanoplastics and other environmental pollutants may result in synergistic transformation capacities.

Impact of physico-chemical properties on the toxicological potential of reduced graphene oxide in human bronchial epithelial cells.

The increasing use of graphene-based materials (GBM) requires their safety evaluation, especially in occupational settings. The same physico-chemical (PC) properties that confer GBM extraordinary functionalities may affect the potential toxic response. Most toxicity assessments mainly focus on graphene oxide and rarely investigate GBMs varying only by one property. As a novelty, the present study assessed the in vitro cytotoxicity and genotoxicity of six reduced graphene oxides (rGOs) with different PC properties in the human bronchial epithelial 16HBE14o - cell line. Of the six materials, rGO1-rGO4 only differed in the carbon-to-oxygen (C/O) content, whereas rGO5 and rGO6 were characterized by different lateral size and number of layers, respectively, but similar C/O content compared with rGO1. The materials were characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, laser diffraction and dynamic light scattering, and Brunauer-Emmett-Teller analysis. Cytotoxicity (Luminescent Cell Viability and WST-8 assays), the induction of reactive oxygen species (ROS; 2',7'-dichlorofluorescin diacetate-based assay), the production of cytokines (enzyme-linked immunosorbent assays) and genotoxicity (comet and micronucleus assays) were evaluated. Furthermore, the internalization of the materials in the cells was confirmed by laser confocal microscopy. No relationships were found between the C/O ratio or the lateral size and any of the rGO-induced biological effects. However, rGO of higher oxygen content showed higher cytotoxic and early ROS-inducing potential, whereas genotoxic effects were observed with the rGO of the lowest density of oxygen groups. On the other hand, a higher number of layers seems to be associated with a decreased potential for inducing cytotoxicity and ROS production.

Insights into the potential carcinogenicity of micro- and nano-plastics.

There is a growing concern regarding the potential health effects that continuous exposure to environmental micro- and nano-plastics (MNPLs) may cause on humans. Due to their persistent nature, MNPLs may accumulate in different organs and tissues and may induce in the long term the development of cancer. The present study aimed to review the existing literature on the carcinogenic potential of MNPLs. As studies directly assessing carcinogenicity were expected to be scarce, studies dealing with indirect outcomes associated with the carcinogenic process were considered in the literature search. Of the 126 studies screened, 19 satisfied the inclusion criteria. Besides, 7 additional cross-referenced articles, identified through a careful reading of the previously selected papers, also met the inclusion criteria and, consequently, were included in the review. Most of the selected studies were performed using in vitro models whereas about 40% of the studies were done in rodents, although none of them included a 2-year carcinogenicity assay. Most of the reviewed studies pointed out the potential of MNPLs to induce inflammation and genotoxicity, the latter being recognized as a strong predictor of carcinogenicity. These, along with other important findings such as the MNPLs' ability to accumulate into cells and tissues, or their capacity to induce fibrosis, may suggest an association between MNPLs exposures and the carcinogenic potential. Nevertheless, the limited number of available studies precludes reaching clear conclusions. Therefore, this review also provides several recommendations to cover the current knowledge gaps and address the future evaluation of the MNPLs' carcinogenic risk.

High aspect ratio nanomaterial-induced macrophage polarization is mediated by changes in miRNA levels.

Introduction: Inhalation of nanomaterials may induce inflammation in the lung which if left unresolved can manifest in pulmonary fibrosis. In these processes, alveolar macrophages have an essential role and timely modulation of the macrophage phenotype is imperative in the onset and resolution of inflammatory responses. This study aimed to investigate, the immunomodulating properties of two industrially relevant high aspect ratio nanomaterials, namely nanocellulose and multiwalled carbon nanotubes (MWCNT), in an alveolar macrophage model. Methods: MH-S alveolar macrophages were exposed at air-liquid interface to cellulose nanocrystals (CNC), cellulose nanofibers (CNF) and two MWCNT (NM-400 and NM-401). Following exposure, changes in macrophage polarization markers and secretion of inflammatory cytokines were analyzed. Furthermore, the potential contribution of epigenetic regulation in nanomaterial-induced macrophage polarization was investigated by assessing changes in epigenetic regulatory enzymes, miRNAs, and rRNA modifications. Results: Our data illustrate that the investigated nanomaterials trigger phenotypic changes in alveolar macrophages, where CNF exposure leads to enhanced M1 phenotype and MWCNT promotes M2 phenotype. Furthermore, MWCNT exposure induced more prominent epigenetic regulatory events with changes in the expression of histone modification and DNA methylation enzymes as well as in miRNA transcript levels. MWCNT-enhanced changes in the macrophage phenotype were correlated with prominent downregulation of the histone methyltransferases Kmt2a and Smyd5 and histone deacetylases Hdac4, Hdac9 and Sirt1 indicating that both histone methylation and acetylation events may be critical in the Th2 responses to MWCNT. Furthermore, MWCNT as well as CNF exposure led to altered miRNA levels, where miR-155-5p, miR-16-1-3p, miR-25-3p, and miR-27a-5p were significantly regulated by both materials. PANTHER pathway analysis of the identified miRNA targets showed that both materials affected growth factor (PDGF, EGF and FGF), Ras/MAPKs, CCKR, GnRH-R, integrin, and endothelin signaling pathways. These pathways are important in inflammation or in the activation, polarization, migration, and regulation of phagocytic capacity of macrophages. In addition, pathways involved in interleukin, WNT and TGFB signaling were highly enriched following MWCNT exposure. Conclusion: Together, these data support the importance of macrophage phenotypic changes in the onset and resolution of inflammation and identify epigenetic patterns in macrophages which may be critical in nanomaterial-induced inflammation and fibrosis.

Occupational exposure and markers of genetic damage, systemic inflammation and lung function: a Danish cross-sectional study among air force personnel.

Air force ground crew personnel are potentially exposed to fuels and lubricants, as raw materials, vapours and combustion exhaust emissions, during operation and maintenance of aircrafts. This study investigated exposure levels and biomarkers of effects for employees at a Danish air force military base. We enrolled self-reported healthy and non-smoking employees (n = 79) and grouped them by exposure based on job function, considered to be potentially exposed (aircraft engineers, crew chiefs, fuel operators and munition specialists) or as reference group with minimal occupational exposure (avionics and office workers). We measured exposure levels to polycyclic aromatic hydrocarbons (PAHs) and organophosphate esters (OPEs) by silicone bands and skin wipes (PAHs only) as well as urinary excretion of PAH metabolites (OH-PAHs). Additionally, we assessed exposure levels of ultrafine particles (UFPs) in the breathing zone for specific job functions. As biomarkers of effect, we assessed lung function, plasma levels of acute phase inflammatory markers, and genetic damage levels in peripheral blood cells. Exposure levels of total PAHs, OPEs and OH-PAHs did not differ between exposure groups or job functions, with low correlations between PAHs in different matrices. Among the measured job functions, the UFP levels were higher for the crew chiefs. The exposure level of the PAH fluorene was significantly higher for the exposed group than the reference group (15.9 ± 23.7 ng/g per 24 h vs 5.28 ± 7.87 ng/g per 24 h, p = 0.007), as was the OPE triphenyl phosphate (305 ± 606 vs 19.7 ± 33.8 ng/g per 24 h, p = 0.011). The OPE tris(1,3-dichlor-2-propyl)phosphate had a higher mean in the exposed group (60.7 ± 135 ng/g per 24 h) compared to the reference group (8.89 ± 15.7 ng/g per 24 h) but did not reach significance. No evidence of effects for biomarkers of systemic inflammation, genetic damage or lung function was found. Overall, our biomonitoring study show limited evidence of occupational exposure of air force ground crew personnel to UFPs, PAHs and OPEs. Furthermore, the OH-PAHs and the assessed biomarkers of early biological effects did not differ between exposed and reference groups.

Genotoxicity and cellular uptake of nanosized and fine copper oxide particles in human bronchial epithelial cells in vitro.

We studied the genotoxicity and cellular uptake of nanosized (<50 nm) and fine (<10 µm) copper oxide (CuO) particles in vitro in human bronchial epithelial (BEAS-2B) cells. In addition, the effect of dispersing the particles using bovine serum albumin (BSA) on DNA damage induction was investigated. DNA damage was assessed by the alkaline comet (single cell gel electrophoresis) assay after 3-h, 6-h and 24-h exposures. The cytokinesis-block micronucleus assay was applied to study chromosome damage. Both fine- and nanosized CuO particles induced a dose-dependent increase in DNA damage at all timepoints tested. However, nanosized CuO damaged DNA at lower doses and higher levels compared with fine CuO. Dispersing the nanoparticles in the presence of BSA (0.6 mg/mL) resulted in a small and inconsistent decrease in DNA damage compared with dispersions in serum-free cell culture medium only. CuO nanoparticles induced a clear dose-dependent increase in micronucleated cells at doses that strongly increased cytostasis and were markedly cytotoxic at 24 and 48 h. Fine CuO showed a slight induction of micronuclei. Hyperspectral microscopy indicated a substantial cellular uptake of both types of particles after a 3-h exposure to a dose of 20 µg/cm2. The number of particles internalized by the cells was higher for nanosized than fine CuO, as quantified by the frequency of spectral matches in the total cell area and by the number of spectrally matched visible particles or agglomerates per cell. The particle uptake was limited by particle size. The stronger genotoxic activity of nanosized than fine CuO particles is likely to derive from the higher cellular uptake and more effective intracellular dissolution of nanoparticles.

Genotoxic and inflammatory effects of nanofibrillated cellulose in murine lungs.

Nanofibrillated cellulose (NFC) is a sustainable and renewable nanomaterial, with diverse potential applications in the paper and medical industries. As NFC consists of long fibres of high aspect ratio, we examined here whether TEMPO-(2,2,6,6-tetramethyl-piperidin-1-oxyl) oxidised NFC (length 300-1000nm, thickness 10-25nm), administrated by a single pharyngeal aspiration, could be genotoxic to mice, locally in the lungs or systemically in the bone marrow. Female C57Bl/6 mice were treated with four different doses of NFC (10, 40, 80 and 200 µg/mouse), and samples were collected 24h later. DNA damage was assessed by the comet assay in bronchoalveolar lavage (BAL) and lung cells, and chromosome damage by the bone marrow erythrocyte micronucleus assay. Inflammation was evaluated by BAL cell counts and analysis of cytokines and histopathological alterations in the lungs. A significant induction of DNA damage was observed at the two lower doses of NFC in lung cells, whereas no increase was seen in BAL cells. No effect was detected in the bone marrow micronucleus assay, either. NFC increased the recruitment of inflammatory cells to the lungs, together with a dose-dependent increase in mRNA expression of tumour necrosis factor α, interleukins 1ß and 6, and chemokine (C-X-C motif) ligand 5, although there was no effect on the levels of the respective proteins. The histological analysis showed a dose-related accumulation of NFC in the bronchi, the alveoli and some in the cytoplasm of macrophages. In addition, neutrophilic accumulation in the alveolar lung space was observed with increasing dose. Our findings showed that NFC administered by pharyngeal aspiration caused an acute inflammatory response and DNA damage in the lungs, but no systemic genotoxic effect in the bone marrow. The present experimental design did not, however, allow us to determine whether the responses were transient or could persist for a longer time.

Effect of particle size and dispersion status on cytotoxicity and genotoxicity of zinc oxide in human bronchial epithelial cells.

Data available on the genotoxicity of zinc oxide (ZnO) nanoparticles (NPs) are controversial. Here, we examined the effects of particle size and dispersion status on the cytotoxicity and genotoxicity of nanosized and fine ZnO, in the presence and absence of bovine serum albumin (BSA; 0.06%) in human bronchial epithelial BEAS-2B cells. Dynamic light scattering analysis showed the most homogenous dispersions in water alone for nanosized ZnO and in water with BSA for fine ZnO. After a 48-h treatment, both types of ZnO were cytotoxic within a similar, narrow dose range (1.5-3.0µg/cm(2)) and induced micronuclei at a near toxic dose range (1.25-1.75µg/cm(2)), both with and without BSA. In the comet assay, nanosized ZnO (1.25-1.5µg/cm(2)), in the absence of BSA, caused a statistically significant increase in DNA damage after 3-h and 6-h treatments, while fine ZnO did not. Our findings may be explained by better uptake or faster intracellular dissolution of nanosized ZnO without BSA during short treatments (3-6h; the comet assay), with less differences between the two ZnO forms after longer treatments (>48h; the in vitro micronucleus test). As ZnO is genotoxic within a narrow dose range partly overlapping with cytotoxic doses, small experimental differences e.g. in the dispersion of ZnO particles may have a substantial effect on the genotoxicity of the nominal doses added to the cell culture.

In vitro and in vivo genotoxic effects of straight versus tangled multi-walled carbon nanotubes.

Some multi-walled carbon nanotubes (MWCNTs) induce mesothelioma in rodents, straight MWCNTs showing a more pronounced effect than tangled MWCNTs. As primary and secondary genotoxicity may play a role in MWCNT carcinogenesis, we used a battery of assays for DNA damage and micronuclei to compare the genotoxicity of straight (MWCNT-S) and tangled MWCNTs (MWCNT-T) in vitro (primary genotoxicity) and in vivo (primary or secondary genotoxicity). C57Bl/6 mice showed a dose-dependent increase in DNA strand breaks, as measured by the comet assay, in lung cells 24 h after a single pharyngeal aspiration of MWCNT-S (1-200 µg/mouse). An increase was also observed for DNA strand breaks in lung and bronchoalveolar lavage (BAL) cells and for micronucleated alveolar type II cells in mice exposed to aerosolized MWCNT-S (8.2-10.8 mg/m(3)) for 4 d, 4 h/d. No systemic genotoxic effects, assessed by the γ-H2AX assay in blood mononuclear leukocytes or by micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow or blood, were observed for MWCNT-S by either exposure technique. MWCNT-T showed a dose-related decrease in DNA damage in BAL and lung cells of mice after a single pharyngeal aspiration (1-200 µg/mouse) and in MNPCEs after inhalation exposure (17.5 mg/m(3)). In vitro in human bronchial epithelial BEAS-2B cells, MWCNT-S induced DNA strand breaks at low doses (5 and 10 µg/cm(2)), while MWCNT-T increased strand breakage only at 200 µg/cm(2). Neither of the MWCNTs was able to induce micronuclei in vitro. Our findings suggest that both primary and secondary mechanisms may be involved in the genotoxicity of straight MWCNTs.

Extensive temporal transcriptome and microRNA analyses identify molecular mechanisms underlying mitochondrial dysfunction induced by multi-walled carbon nanotubes in human lung cells.

Understanding toxicity pathways of engineered nanomaterials (ENM) has recently been brought forward as a key step in twenty-first century ENM risk assessment. Molecular mechanisms linked to phenotypic end points is a step towards the development of toxicity tests based on key events, which may allow for grouping of ENM according to their modes of action. This study identified molecular mechanisms underlying mitochondrial dysfunction in human bronchial epithelial BEAS 2B cells following exposure to one of the most studied multi-walled carbon nanotubes (Mitsui MWCNT-7). Asbestos was used as a positive control and a non-carcinogenic glass wool material was included as a negative fibre control. Decreased mitochondrial membrane potential (MMP↓) was observed for MWCNTs at a biologically relevant dose (0.25 µg/cm(2)) and for asbestos at 2 µg/cm(2), but not for glass wool. Extensive temporal transcriptomic and microRNA expression analyses identified a 330-gene signature (including 26 genes with known mitochondrial function) related to MWCNT- and asbestos-induced MMP↓. Forty-nine of the MMP↓-associated genes showed highly similar expression patterns over time (six time points) and the majority was found to be regulated by two transcription factors strongly involved in mitochondrial homeostasis, APP and NRF1. In addition, four miRNAs were correlated with MMP↓ and one of them, miR-1275, was found to negatively correlate with a large part of the MMP↓-associated genes. Cellular processes such as gluconeogenesis, mitochondrial LC-fatty acid ß-oxidation and spindle microtubule function were enriched among the MMP↓-associated genes and miRNAs. These results are expected to be useful in the identification of key events in ENM-related toxicity pathways for the development of molecular screening techniques.

Genotoxic and immunotoxic effects of cellulose nanocrystals in vitro.

Nanocellulosics are among the most promising innovations for a wide-variety of applications in materials science. Although nanocellulose is presently produced only on a small scale, its possible toxic effects should be investigated at this early stage. The aim of the present study was to examine the potential genotoxicity and immunotoxicity of two celluloses in vitro - cellulose nanocrystals (CNC; mean fibril length 135 nm, mean width 7.3 nm) and a commercially available microcrystalline (non-nanoscale) cellulose (MCC; particle size ∼50 µm). Both celluloses showed 55% cytotoxicity at approximately 100 µg/ml after 4-h, 24-h, and 48-h treatment of human bronchial epithelial BEAS 2B cells, as determined by luminometric detection of ATP and cell count (dead cells identified by propidium iodide). Neither of the materials was able to induce micronuclei (MN) in binucleate or mononucleate BEAS 2B cells after a 48-h treatment (2.5-100 µg/ml). In human monocyte-derived macrophages, MCC induced a release (measured by enzyme-linked immunosorbent assay; ELISA) of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and (after lipopolysaccharide-priming) interleukin 1ß (IL-1ß) after a 6-h exposure to a dose of 300 µg/ml, but CNC (30-300 µg/ml) did not. In conclusion, our results show that nanosized CNC is neither genotoxic nor immunotoxic under the conditions tested, whereas non-nanosized MCC is able to induce an inflammatory response. More studies are needed, especially in vivo, to further assess if CNC and other nanocelluloses induce secondary genotoxic effects mediated by inflammation.

Free radical scavenging and formation by multi-walled carbon nanotubes in cell free conditions and in human bronchial epithelial cells.

BACKGROUND: Certain multi-walled carbon nanotubes (MWCNTs) have been shown to elicit asbestos-like toxicological effects. To reduce needs for risk assessment it has been suggested that the physicochemical characteristics or reactivity of nanomaterials could be used to predict their hazard. Fibre-shape and ability to generate reactive oxygen species (ROS) are important indicators of high hazard materials. Asbestos is a known ROS generator, while MWCNTs may either produce or scavenge ROS. However, certain biomolecules, such as albumin - used as dispersants in nanomaterial preparation for toxicological testing in vivo and in vitro - may reduce the surface reactivity of nanomaterials. METHODS: Here, we investigated the effect of bovine serum albumin (BSA) and cell culture medium with and without BEAS 2B cells on radical formation/scavenging by five MWCNTs, Printex 90 carbon black, crocidolite asbestos, and glass wool, using electron spin resonance (ESR) spectroscopy and linked this to cytotoxic effects measured by trypan blue exclusion assay. In addition, the materials were characterized in the exposure medium (e.g. for hydrodynamic size-distribution and sedimentation rate). RESULTS: The test materials induced highly variable cytotoxic effects which could generally be related to the abundance and characteristics of agglomerates/aggregates and to the rate of sedimentation. All carbon nanomaterials were found to scavenge hydroxyl radicals (•OH) in at least one of the solutions tested. The effect of BSA was different among the materials. Two types of long, needle-like MWCNTs (average diameter >74 and 64.2 nm, average length 5.7 and 4.0 µm, respectively) induced, in addition to a scavenging effect, a dose-dependent formation of a unique, yet unidentified radical in both absence and presence of cells, which also coincided with cytotoxicity. CONCLUSIONS: Culture medium and BSA affects scavenging/production of •OH by MWCNTs, Printex 90 carbon black, asbestos and glass-wool. An unidentified radical is generated by two long, needle-like MWCNTs and these two CNTs were more cytotoxic than the other CNTs tested, suggesting that this radical could be related to the adverse effects of MWCNTs.

Genotoxicity of short single-wall and multi-wall carbon nanotubes in human bronchial epithelial and mesothelial cells in vitro.

Although some types of carbon nanotubes (CNTs) have been described to induce mesothelioma in rodents and genotoxic effects in various cell systems, there are few previous studies on the genotoxicity of CNTs in mesothelial cells. Here, we examined in vitro DNA damage induction by short multi-wall CNTs (MWCNTs; 10-30 nm × 1-2 µm) and single-wall CNTs (SWCNTs; >50% SWCNTs, ~40% other CNTs; <2 nm × 1-5 µm) in human mesothelial (MeT-5A) cells and bronchial epithelial (BEAS 2B) cells, using the single cell gel electrophoresis (comet) assay and the immunoslot blot assay for the detection of malondialdehyde (M1dG) DNA adducts. In BEAS 2B cells, we also studied the induction of micronuclei (MN) by the CNTs using the cytokinesis-block method. The cells were exposed to the CNTs (5-200 µg/cm(2), corresponding to 19-760 µg/ml) for 24 and 48h in the comet assay and for 48 and 72 h in the MN and M1dG assays. Transmission electron microscopy (TEM) showed more MWCNT fibres and SWCNT clusters in BEAS 2B than MeT-5A cells, but no significant differences were seen in intracellular dose expressed as area of SWCNT clusters between TEM sections of the cell lines. In MeT-5A cells, both CNTs caused a dose-dependent induction of DNA damage (% DNA in comet tail) in the 48-h treatment and SWCNTs additionally in the 24-h treatment, with a statistically significant increase at 40 µg/cm(2) of SWCNTs and (after 48 h) 80 µg/cm(2) of both CNTs. SWCNTs also elevated the level of M1dG DNA adducts at 1, 5, 10 and 40 µg/cm(2) after the 48-h treatment, but both CNTs decreased M1dG adduct level at several doses after the 72-h treatment. In BEAS 2B cells, SWCNTs induced a statistically significant increase in DNA damage at 80 and 120 µg/cm(2) after the 24-h treatment and in M1dG adduct level at 5 µg/cm(2) after 48 h and 10 and 40 µg/cm(2) after 72 h; MWCNTs did not affect the level of DNA damage but produced a decrease in M1dG adducts in the 72-h treatment. The CNTs did not affect the level of MN. In conclusion, MWCNTs and SWCNTs induced DNA damage in MeT-5A cells but showed a lower (SWCNTs) or no (MWCNTs) effect in BEAS 2B cells, suggesting that MeT-5A cells were more sensitive to the DNA-damaging effect of CNTs than BEAS 2B cells, despite the fact that more CNT fibres or clusters were seen in BEAS 2B than MeT-5A cells. M1dG DNA adducts were induced by SWCNTs but decreased after a 3-day exposure to MWCNTs and (in MeT-5A cells) SWCNTs, indicating that CNTs may lead to alterations in oxidative effects within the cells. Neither of the CNTs was able to produce chromosomal damage (MN).

Genotoxicity of polyvinylpyrrolidone-coated silver nanoparticles in BEAS 2B cells.

Silver nanoparticles (AgNPs) are widely utilized in various consumer products and medical devices, especially due to their antimicrobial properties. However, several studies have associated these particles with toxic effects, such as inflammation and oxidative stress in vivo and cytotoxic and genotoxic effects in vitro. Here, we assessed the genotoxic effects of AgNPs coated with polyvinylpyrrolidone (PVP) (average diameter 42.5±14.5 nm) on human bronchial epithelial BEAS 2B cells in vitro. AgNPs were dispersed in bronchial epithelial growth medium (BEGM) with 0.6 mg/ml bovine serum albumin (BSA). The AgNP were partially well-dispersed in the medium and only limited amounts (ca. 0.02 µg Ag(+) ion/l) could be dissolved after 24h. The zeta-potential of the AgNPs was found to be highly negative in pure water but was at least partially neutralized in BEGM with 0.6 mg BSA/ml. Cytotoxicity was measured by cell number count utilizing Trypan Blue exclusion and by an ATP-based luminescence cell viability assay. Genotoxicity was assessed by the alkaline single cell gel electrophoresis (comet) assay, the cytokinesis-block micronucleus (MN) assay, and the chromosomal aberration (CA) assay. The cells were exposed to various doses (0.5-48 µg/cm(2) corresponding to 2.5-240 µg/ml) of AgNPs for 4 and 24 h in the comet assay, for 48 h in the MN assay, and for 24 and 48 h in the CA assay. DNA damage measured by the percent of DNA in comet tail was induced in a dose-dependent manner after both the 4-h and the 24-h exposures to AgNPs, with a statistically significant increase starting at 16 µg/cm(2) (corresponding to 60.8 µg/ml) and doubling of the percentage of DNA in tail at 48 µg/cm(2). However, no induction of MN or CAs was observed at any of the doses or time points. The lack of induction of chromosome damage by the PVP-coated AgNPs is possibly due to the coating which may protect the cells from direct interaction with the AgNPs, either by reducing ion leaching from the particles or by causing extensive agglomeration of the nanoparticles, with a possible reduction of the cellular uptake.

Nano-specific genotoxic effects.

It cannot be generally assumed that nanoparticles are genotoxic or that nanoscale size would increase the genotoxicity of the material. The large-scale in vivo genotoxicity testing of the various nanomaterials does not seem realistic. Information on nanoparticle characteristics associated with genotoxicity, carcinogenicity and mechanisms involved can probably be used in risk assessment.

Micronucleus assay for mouse alveolar Type II and Clara cells.

The objective of our study was to develop a micronucleus (MN) assay for detecting genotoxic damage after inhalation exposure in mouse alveolar Type II and Clara cells, potential target cells for lung carcinogens. Ten male C57BL/6J mice were exposed to ethylene oxide (630 mg/m(3)) for 4 hr via inhalation; 10 unexposed mice serving as controls. 72 hr after the exposure, Clara cells and alveolar Type II cells were isolated using two different methods. Method 1 included a 15-min trypsin lavage and a 2-hr incubation of cell suspension. Method 2 involved a 30-min trypsin lavage, Percoll gradient centrifugation, and a 48-hr incubation for cell attachment. Nitro blue tetrazolium (NBT) -staining was applied to distinguish Clara cells. The frequency of micronuclei (MNi) was scored in NBT-negative cells (defined as Type II cells in Method 2) and NBT-positive cells (Clara cells). To detect possible differences between the techniques, MNi in Clara cells were analyzed from samples prepared by both methods. With Method 2, a clear increase in the mean frequency of micronucleated cells was seen in the exposed mice as compared with the controls, for both alveolar Type II and Clara cells. However, no significant increase in MN frequency was seen in Clara cells analyzed from samples prepared by Method 1. Based on our findings, mouse alveolar Type II and Clara cells seem to be suitable for MN analysis in studies aimed at identifying genotoxic lung carcinogens. Both alveolar Type II and Clara cells can be isolated using Method 2.

Chromosomal aberrations in railroad transit workers: effect of genetic polymorphisms.

Complex chemical mixtures are transported by train from Russia to Finland for further shipment. Here, we studied if exposure to genotoxic components among these substances could affect chromosomal aberrations (CAs) in peripheral lymphocytes of workers handling the tank cars. An initial survey among 48 railroad workers and 39 referents (male smokers and nonsmokers) showed an elevation of CAs. A campaign was started to reduce exposures through preventive measures. Five years later, 51 tank car workers and 40 age-matched referents (all nonsmoking men) were studied for CAs and genetic polymorphisms of xenobiotic metabolism (EPHX1, GSTM1, GSTP1, GSTT1, NAT1, NAT2), DNA repair (ERCC2, ERCC5, XPA, XPC, XRCC1, XRCC3), and folate metabolism (MTHFR, MTR). No increase in CAs was seen in the exposed group, suggesting that the preventive measures had been successful. However, a positive association existed between exposure duration and CA level among the exposed subjects. The level of chromosome-type breaks was actually lower in the exposed workers than the referents, particularly among MTHFR wild-type homozygotes or XRCC3 codon 241 variant allele carriers, suggesting modulation of CA frequency by folate metabolism and DNA repair. An interaction was observed between the occupational exposure and MTHFR, EPHX1, and MTR genotypes in determining CA level. The NAT2, ERCC2 exon 10, and XRCC1 codon 194 polymorphisms also affected CA frequency. Our findings suggest that handling of tank cars containing complex chemical mixtures poses a genotoxic risk, which may be reduced by preventive measures. Several genetic polymorphisms seem to modify the genotoxic effect or baseline CA level.

Interaction of MAP17 with NHERF3/4 induces translocation of the renal Na/Pi IIa transporter to the trans-Golgi.

The function of the NaPiIIa renal sodium-phosphate transporter is regulated through a complex network of interacting proteins. Several PDZ domain-containing proteins interact with its COOH terminus while the small membrane protein MAP17 interacts with its NH(2) end. To elucidate the function of MAP17, we identified its interacting proteins using both bacterial and mammalian two-hybrid systems. Several PDZ domain-containing proteins, including the four NHERF proteins, as well as NaPiIIa and NHE3, were found to bind to MAP17. The interactions of MAP17 with the NHERF proteins and with NaPiIIa were further analyzed in opossum kidney (OK) cells. Expression of MAP17 alone had no effect on the NaPiIIa apical membrane distribution, but coexpression of MAP17 and NHERF3 or NHERF4 induced internalization of NaPiIIa, MAP17, and the PDZ protein to the trans-Golgi network (TGN). This effect was not observed when MAP17 was cotransfected with NHERF1/2 proteins. Inhibition of protein kinase C (PKC) prevented expression of the three proteins in the TGN. Activation of PKC in OK cells transfected only with MAP17 induced complete degradation of MAP17 and NaPiIIa. When lysosomal degradation was prevented, both proteins accumulated in the TGN. When the dopamine D1-like receptor was activated with fenoldopam, both NaPiIIa and MAP17 also accumulated in the TGN. Finally, cotransfection of MAP17 and NHERF3 prevented the adaptive upregulation of phosphate transport activity in OK cells in response to low extracellular phosphate. Therefore, the interaction between MAP17, NHERF3/4, and NaPiIIa in the TGN could be an important intermediate or alternate path in the internalization of NaPiIIa.

Rat kidney MAP17 induces cotransport of Na-mannose and Na-glucose in Xenopus laevis oocytes.

Renal reabsorption is the main mechanism that controls mannose homeostasis. This takes place through a specific Na-coupled uphill transport system, the molecular identity of which is unknown. We prepared and screened a size-selected rat kidney cortex cDNA library through the expression of mannose transport in Xenopus laevis oocytes. We have identified a membrane protein that induces high-affinity and specific Na-dependent transport of d-mannose and d-glucose in X. laevis oocytes, most likely through stimulation of the capacity of an endogenous transport system of the oocyte. Sequencing has revealed that the cDNA encodes the counterpart of the human membrane-associated protein MAP17, previously known by its overexpression in renal, colon, lung, and breast carcinomas. We show that MAP17 is a 12.2-kDa nonglycosylated membrane protein that locates to the brush-border plasma membrane and the Golgi apparatus of transfected cells and that it is expressed in the proximal tubules of the kidney cortex and in the spermatids of the seminiferous tubules. It spans twice the cell membrane, with both termini inside the cell, and seems to form homodimers through intracellular Cys55, a residue also involved in transport expression. MAP17 is responsible for mannose transport expression in oocytes by rat kidney cortex mRNA. The induced transport has the functional characteristics of a Na-glucose cotransporter (SGLT), because d-glucose and alpha-methyl-d-glucopyranoside are also accepted substrates that are inhibited by phloridzin. The corresponding transporter from the proximal tubule remains to be identified, but it is different from the known mammalian SGLT-1, -2, and -3.