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Genotoxicity and DNA damage signaling in response to complex mixtures of PAHs in biomass burning particulate matter from cashew nut roasting
2020
Approximately 3 billion people world-wide are exposed to air pollution from biomass burning. Herein, particulate matter (PM) emitted from artisanal cashew nut roasting, an important economic activity worldwide, was investigated. This study focused on: i) chemical characterization of polycyclic aromatic hydrocarbons (PAHs) and oxygenated (oxy-) PAHs; ii) intracellular levels of reactive oxygen species (ROS); iii) genotoxic effects and time- and dose-dependent activation of DNA damage signaling, and iv) differential expression of genes involved in xenobiotic metabolism, inflammation, cell cycle arrest and DNA repair, using A549 lung cells. Among the PAHs, chrysene, benzo[a]pyrene (B[a]P), benzo[b]fluoranthene, and benz[a]anthracene showed the highest concentrations (7.8–10 ng/m³), while benzanthrone and 9,10-anthraquinone were the most abundant oxy-PAHs. Testing of PM extracts was based on B[a]P equivalent doses (B[a]Pₑq). IC₅₀ values for viability were 5.7 and 3.0 nM B[a]Pₑq at 24 h and 48 h, respectively. At these low doses, we observed a time- and dose-dependent increase in intracellular levels of ROS, genotoxicity (DNA strand breaks) and DNA damage signaling (phosphorylation of the protein checkpoint kinase 1 – Chk1). In comparison, effects of B[a]P alone was observed at micromolar range. To our knowledge, no previous study has demonstrated an activation of pChk1, a biomarker used to estimate the carcinogenic potency of PAHs in vitro, in lung cells exposed to cashew nut roasting extracts. Sustained induction of expression of several important stress response mediators of xenobiotic metabolism (CYP1A1, CYP1B1), ROS and pro-inflammatory response (IL-8, TNF-α, IL-2, COX2), and DNA damage response (CDKN1A and DDB2) was also identified. In conclusion, our data show high potency of cashew nut roasting PM to induce cellular stress including genotoxicity, and more potently when compared to B[a]P alone. Our study provides new data that will help elucidate the toxic effects of low-levels of PAH mixtures from air PM generated by cashew nut roasting.
Show more [+] Less [-]Identification of inhalable rutile and polycyclic aromatic hydrocarbons (PAHs) nanoparticles in the atmospheric dust
2020
Gallego-Hernández, Ana L. | Meza-Figueroa, Diana | Tanori, Judith | Acosta-Elías, Mónica | González-Grijalva, Belem | Maldonado-Escalante, Juan F. | Rochín-Wong, Sarai | Soto-Puebla, Diego | Navarro-Espinoza, Sofia | Ochoa-Contreras, Roberto | Pedroza-Montero, Martín
Addressing the presence of rutile nanoparticles (NPs) in the air is a work in progress, and the development of methodologies for the identification of NPs in atmospheric dust is essential for the assessment of its toxicological effects. To address this issue, we selected the fast growing desertic city of Hermosillo in northern Mexico. Road dust (n = 266) and soils (n = 10) were sampled and bulk Ti-contents were tested by portable X-ray fluorescence. NPs were extracted from atmospheric dust by PM₁.₀-PTFE filters and further characterized by Confocal Raman Microscopy, Energy-dispersive X-ray spectroscopy (EDS) coupled to Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). Results showed (i) the average concentration of Ti in road dust (3447 mg kg⁻¹) was similar to natural values and worldwide urban dusts; (ii) the bulk geochemistry was not satisfactory for Ti-NPs identification; (iii) 76% of the total extracted PM₁.₀ sample corresponded to NPs; (iv) mono-microaggregates of rutile NPs were identified; (v) ubiquitous polycyclic aromatic hydrocarbons (PAHs) were linked to NPs. The genotoxicity of rutile and PAHs, in connection with NPs content, make us aware of a crucial emerging environmental issue of significant health concern, justifying further research in this field.
Show more [+] Less [-]Application of advanced HepG2 3D cell model for studying genotoxic activity of cyanobacterial toxin cylindrospermopsin
2020
Hercog, Klara | Štampar, Martina | Štern, Alja | Filipič, Metka | Žegura, Bojana
Cylindrospermopsin (CYN) is an emerging cyanotoxin increasingly being found in freshwater cyanobacterial blooms worldwide. Humans and animals are exposed to CYN through the consumption of contaminated water and food as well as occupational and recreational water activities; therefore, it represents a potential health threat. It exhibits genotoxic effects in metabolically active test systems, thus it is considered as pro-genotoxic. In the present study, the advanced 3D cell model developed from human hepatocellular carcinoma (HepG2) cells was used for the evaluation of CYN cyto-/genotoxic activity. Spheroids were formed by forced floating method and were cultured for three days under static conditions prior to exposure to CYN (0.125, 0.25 and 0.5 μg/mL) for 72 h. CYN influence on spheroid growth was measured daily and cell survival was determined by MTS assay and live/dead staining. The influence on cell proliferation, cell cycle alterations and induction of DNA damage (γH2AX) was determined using flow cytometry. Further, the expression of selected genes (qPCR) involved in the metabolism of xenobiotics, proliferation, DNA damage response, apoptosis and oxidative stress was studied. Results revealed that CYN dose-dependently reduced the size of spheroids and affected cell division by arresting HepG2 cells in G1 phase of the cell cycle. No induction of DNA double strand breaks compared to control was determined at applied conditions. The analysis of gene expression revealed that CYN significantly deregulated genes encoding phase I (CYP1A1, CYP1A2, CYP3A4, ALDH3A) and II (NAT1, NAT2, SULT1B1, SULT1C2, UGT1A1, UGT2B7) enzymes as well as genes involved in cell proliferation (PCNA, TOP2α), apoptosis (BBC3) and DNA damage response (GADD45a, CDKN1A, ERCC4). The advanced 3D HepG2 cell model due to its more complex structure and improved cellular interactions provides more physiologically relevant information and more predictive data for human exposure, and can thus contribute to more reliable genotoxicity assessment of chemicals including cyanotoxins.
Show more [+] Less [-]Effects of different mobile phone UMTS signals on DNA, apoptosis and oxidative stress in human lymphocytes
2020
Gulati, Sachin | Kosik, Pavol | Durdik, Matus | Skorvaga, Milan | Jakl, Lukas | Markova, Eva | Belyaev, Igor
Different scientific reports suggested link between exposure to radiofrequency radiation (RF) from mobile communications and induction of reactive oxygen species (ROS) and DNA damage while other studies have not found such a link. However, the available studies are not directly comparable because they were performed at different parameters of exposure, including carrier frequency of RF signal, which was shown to be a critical for appearance of the RF effects. For the first time, we comparatively analyzed genotoxic effects of UMTS signals at different frequency channels used by 3G mobile phones (1923, 1947.47, and 1977 MHz). Genotoxicity was examined in human lymphocytes exposed to RF for 1 h and 3 h using complimentary endpoints such as induction of ROS by imaging flow cytometry, DNA damage by alkaline comet assay, mutations in TP53 gene by RSM assay, preleukemic fusion genes (PFG) by RT-qPCR, and apoptosis by flow cytometry. No effects of RF exposure on ROS, apoptosis, PFG, and mutations in TP53 gene were revealed regardless the UMTS frequency while inhibition of a bulk RNA expression was found. On the other hand, we found relatively small but statistically significant induction of DNA damage in dependence on UMTS frequency channel with maximal effect at 1977.0 MHz. Our data support a notion that each specific signal used in mobile communication should be tested in specially designed experiments to rule out that prolonged exposure to RF from mobile communication would induce genotoxic effects and affect the health of human population.
Show more [+] Less [-]Ecotoxicity of trace elements to chicken GALLUS gallus domesticus exposed to a gradient of polymetallic-polluted sites
2020
Kribi-Boukhris, Sameh EL. | Boughattas, Iteb | Zitouni, Nesrine | Helaoui, Sondes | Sappin-Didier, Valérie | Coriou, Cécile | Bussiere, Sylvie | Banni, Mohamed
Mining activity may cause heavy metal accumulation, which threatens human and animal health by their long-term persistence in the environment. This study aims to assess the impact of polymetallic pollution on chicken (Gallus domesticus) from old lead mining sites in northeast of Tunisia: Jebel Ressas (JR). Samples of soil and chickens were collected from five sites being ranked along a gradient of heavy metal contamination. Heavy metal loads were evaluated in soil samples and in chicken liver and kidney. Biochemical evaluation of oxidative stress parameters termed as Catalase (CAT), Glutathione-S-Transferase (GST), and Malondialdehydes (MDA) accumulation was monitored. Metallothionein protein level was assessed as a specific response to heavy metals. DNA alteration was achieved using MNi frequency in the investigated tissues. Finally, the evaluation of gene expression levels of CAT, GST, mt1, mt4, P53, bcl2, caspase3 and DNA-ligase was performed. Our data showed the highest loads of Cd, Cu, Zn and Pb in tissues of animals from site 3, being more pronounced in kidney. Biochemical data suggested a significant increase in antioxidant enzymes activities in all sites respect to control except in site 3 were CAT and GST were inhibited. DNA alteration was observed in all tissues being very pronounced in animals from site 3. Overall, transcriptomic data showed that genes involved in apoptosis were up-regulated in animals exposed to the most contaminated soils. Our data suggest that chicken and selected biomarkers offer a suitable model for biomonitoring assessment of heavy metals transfer through the food web in mining sites. Finally, the obtained results of heavy metals accumulation and related alterations should be carefully considered in view of the controversial relationship between distribution and toxicology of contaminants in exposed organisms.
Show more [+] Less [-]Adverse effects of in vitro GenX exposure on rat thyroid cell viability, DNA integrity and thyroid-related genes expression
2020
Coperchini, Francesca | Croce, Laura | Denegri, Marco | Pignatti, Patrizia | Agozzino, Manuela | Netti, Giuseppe Stefano | Imbriani, Marcello | Rotondi, Mario | Chiovato, Luca
The hexafluoropropylene-oxide-dimer-acid (GenX) is a short-chain perfluoroalkyl substance that was recently introduced following the phase out of PFOA, as an alternative for the process of polymerization. GenX was detected at high concentrations in rivers, drinking water and in sera of exposed workers and recent findings suggested its potential dangerousness for human health.Aim of the study was to assess the consequences of GenX exposure on in vitro thyroid cells with particular attention to the effects on cell-viability, proliferation, DNA-damage and in the thyroid-related genes expression.FRTL-5 rat-thyroid cell line were incubated with increasing concentrations of GenX for 24 h, 48 h and 72 h to assess cell viability by WST-1. DNA-damage was assessed by comet assay and further confirmed by micronucleus assay. The proliferation of survived cells was measured by staining with crystal violet and evaluation of its optical density after incubation with SDS. Changes in TTF-1, Pax8, Tg, TSH-R, NIS and TPO genes expression were evaluated by RT-PCR.GenX exposure reduced FRTL-5 viability in a time and dose-dependent manner (24 h: ANOVA F = 22.286; p < 0.001; 48 h: F = 43.253, p < 0.001; 72 h: F = 49.708, p < 0.001). Moreover, GenX exerted a genotoxic effect, as assessed by comet assay (significant increase in tail-length, olive-tail-moment and percentage of tail-DNA) and micronucleus assay, both at cytotoxic and non-cytotoxic concentrations. Exposure to GenX at concentrations non-cytotoxic exerted a significant lowering of the expression of the regulatory gene TTF-1 (p < 0.05 versus untreated) and higher expression of Pax-8 (p < 0.05 versus untreated) and a down-regulation of NIS (p < 0.05 versus untreated). In addition, cells survived to GenX exposure showed a reduced re-proliferation ability (24 h: ANOVA F = 11,941; p < 0,001; 48 h: F = 93.11; p < 0.001; 72 h F = 21.65; p < 0.001).The exposure to GenX produces several toxic effects on thyroid cells in vitro. GenX is able to promote DNA-damage and to affect the expression of thyroid transcription-factor genes.
Show more [+] Less [-]A full evaluation of chiral phenylpyrazole pesticide flufiprole and the metabolites to non-target organism in paddy field
2020
Gao, Jing | Wang, Fang | Jiang, Wenqi | Miao, Jingwen | Wang, Peng | Zhou, Zhiqiang | Liu, Donghui
Pesticides applied to paddy fields may pose considerable danger to non-target aquatic organisms and further threaten human health. Flufiprole is a pesticide used in rice fields; considering the widespread existence of rice-fish-farming ecosystems, the acute toxicities of flufiprole enantiomers and its six metabolites (fipronil, flufiprole sulfide, flufiprole sulfone, detrifluoromethylsulfinyl flufiprole, desulfinyl flufiprole, and flufiprole amide) to four common aquatic organisms in rice fields including Misgurnus anguillicaudatus (pond loach), Carassius gibelio (Prussian carp), Pelophylax nigromaculatus (black-spotted frog), and Daphnia magna (water flea) were investigated. Genotoxicity, pathological changes and the effects on the antioxidant system of M. anguillicaudatus were also evaluated after exposure. The LC₅₀ (EC₅₀) values showed that fipronil and desulfinyl flufiprole were the most toxic compounds and were approximately about six times as toxic as flufiprole. No enantioselective toxicity was observed between the two enantiomers. The activity of antioxidant defense enzymes and the content of malondialdehyde (MDA) in the liver and gills of M. anguillicaudatus were significantly increased by the chemicals in most cases. In addition, fipronil and desulfinyl flufiprole were found to induce an increase in the micronucleus rate in M. anguillicaudatus. Histopathological analysis showed that the liver of M. anguillicaudatus was not significantly affected by flufiprole. Our study demonstrated a potential negative effect on flufiprole-treated aquatic organisms. As an alternative to fipronil, the environmental risk of flufiprole and its metabolites to non-target organisms in rice fields cannot be ignored.
Show more [+] Less [-]Uptake and detoxification of diesel oil by a tropical soil Actinomycete Gordonia amicalis HS-11: Cellular responses and degradation perspectives
2020
Sowani, Harshada | Kulkarni, Mohan | Zinjarde, Smita
A tropical soil Actinomycete, Gordonia amicalis HS-11, has been previously demonstrated to degrade unsaturated and saturated hydrocarbons (squalene and n-hexadecane, respectively) in an effective manner. In present study, G. amicalis HS-11 degraded 92.85 ± 3.42% of the provided diesel oil [1% (v/v)] after 16 days of aerobic incubation. The effect of different culture conditions such as carbon source, nitrogen source, pH, temperature, and aeration on degradation was studied. During degradation, this Actinomycete synthesized surface active compounds (SACs) in an extracellular manner that brought about a reduction in surface tension from 69 ± 2.1 to 30 ± 1.1 mN m⁻¹ after 16 days. The morphology of cells grown on diesel was monitored by using a Field Emission Scanning Electron Microscope. Diesel-grown cells were longer and clumped with smooth surfaces, possibly due to the secretion of SACs. The interaction between the cells and diesel oil was studied by Confocal Laser Scanning Microscope. Some cells were adherent on small diesel droplets and others were present in the non-attached form thus confirming the emulsification ability of this organism. The fatty acid profiles of the organism grown on diesel oil for 48 h were different from those on Luria Bertani Broth. The genotoxicity and cytotoxicity of diesel oil before and after degradation were determined. Cytogenetic parameters such as mitotic index (MI); mitosis distribution and chromosomal aberration (type and frequency) were assessed. Oxidative stress was evaluated by measuring levels of catalase, superoxide dismutase and concentration of malondialdehyde. On the basis of these studies it was deduced that the degradation metabolites were relatively non-toxic.
Show more [+] Less [-]Visible-light reduced silver nanoparticles’ toxicity in Allium cepa test system
2020
Souza, Irisdoris R. | Silva, Lucas R. | Fernandes, Letícia S.P. | Salgado, Lilian D. | Silva de Assis, Helena C. | Firak, Daniele S. | Bach, Larissa | Santos-Filho, Ronaldo | Voigt, Carmen L. | Barros, Ariana C. | Peralta-Zamora, Patricio | Mattoso, Ney | Franco, Celia Regina C. | Soares Medeiros, Lia C. | Marcon, Bruna H. | Cestari, Marta M. | Sant’Anna-Santos, Bruno F. | Leme, Daniela M.
Silver nanoparticles (AgNPs) are widely used in consumer products due to their antibacterial property; however, their potential toxicity and release into the environment raises concern. Based on the limited understanding of AgNPs aggregation behavior, this study aimed to investigate the toxicity of uncoated (uc-AgNP) and coated with polyvinylpyrrolidone (PVP-AgNP), at low concentrations (0.5–100 ng/mL), under dark and visible-light exposure, using a plant test system. We exposed Allium cepa seeds to both types of AgNPs for 4–5 days to evaluate several toxicity endpoints. AgNPs did not cause acute toxicity (i.e., inhibition of seed germination and root development), but caused genotoxicity and biochemical alterations in oxidative stress parameters (lipid peroxidation) and activities of antioxidant enzymes (superoxide dismutase and catalase) in light and dark conditions. However, the light exposure decreased the rate of chromosomal aberration and micronuclei up to 5.60x in uc-AgNP and 2.01x in PVP-AgNP, and 2.69x in uc-AgNP and 3.70x in PVP-AgNP, respectively. Thus, light exposure reduced the overall genotoxicity of these AgNPs. In addition, mitotic index alterations and morphoanatomical changes in meristematic cells were observed only in the dark condition at the highest concentrations, demonstrating that light also reduces AgNPs cytotoxicity. The light-dependent aggregation of AgNPs may have reduced toxicity by reducing the uptake of these NPs by the cells. Our findings demonstrate that AgNPs can be genotoxic, cytotoxic and induce morphoanatomical and biochemical changes in A. cepa roots even at low concentrations, and that visible-light alters their aggregation state, and decreases their toxicity. We suggest that visible light can be an alternative treatment to remediate AgNP residues, minimizing their toxicity and environmental risks.
Show more [+] Less [-]In vitro evaluation of the cytotoxicity, mutagenicity and DNA damage induced by particle matter and gaseous emissions from a medium-duty diesel vehicle under real driving conditions using palm oil biodiesel blends
2020
Botero, Maria L. | Mendoza, Carolina | Arias, Silvana | Hincapié, Oscar D. | Agudelo, John R. | Ortiz, Isabel C.
The influence of palm oil biodiesel content on the cytotoxicity, mutagenicity and genotoxicity of particle- and gas-phase diesel vehicle emissions was investigated. The emissions were collected on-board of a EURO IV diesel truck, fuelled with mixtures of 10% (B10), 20% (B20) and 100% (B100) of palm oil biodiesel, under real driving conditions. Organic extracts of the particulate matter (PM) and gases were characterised for 17 PAH (including EPA priority) and used for the biological assay. Increasing biodiesel content in the fuel mixture results in a decrease in the PM and PAH emission factors, both in the particulate and gas-phase. The majority of the PAH are present in the gas-phase. The mutagenic potencies, in TA98 bacteria, are higher for B20 in both phases, whereas the mutagenicity emission factor, that takes into account the lower emission of PM and PAH, is not significantly different between the fuels. Higher direct mutagenicity (TA98 + S9) is observed in all the tested fuels, indicating the action of carcinogenic compounds other than non-substituted PAH. The gas-phase extracts present higher cytotoxicity and genotoxicity in lung epithelial cell A549, which may be related to the higher PAH content in the gas-phase. The increase in biodiesel content have a different impact on cytotoxicity, being larger in the gas-phase and lower in the particle-phase. This indicates that pulmonary toxicity may be higher for the gaseous emissions, due to the role of different toxic compounds compared to the PM. The adverse biological effects when biodiesel content increases are not consequent with the reduction of the PAH characterised, indicating that other toxic compounds are more relevant. Further investigations to identify these compounds are required in order to update and focus the efforts regarding emission targets and controls.
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