Fluorescent labeling of micro/nanoplastics for biological applications with a focus on "true-to-life" tracking.

The increased environmental presence of micro-/nanoplastics (MNPLs) and the potential health risks associated with their exposure classify them as environmental pollutants with special environmental and health concerns. Consequently, there is an urgent need to investigate the potential risks associated with secondary MNPLs. In this context, using "true-to-life" MNPLs, resulting from the laboratory degradation of plastic goods, may be a sound approach. These non-commercial secondary MNPLs must be labeled to track their presence/journeys inside cells or organisms. Because the cell internalization of MNPLs is commonly analyzed using fluorescence techniques, the use of fluorescent dyes may be a sound method to label them. Five different compounds comprising two chemical dyes (Nile Red and Rhodamine-B), one optical brightener (Opticol), and two industrial dyes (Amarillo Luminoso and iDye PolyPink) were tested to determine their potential for such applications. Using commercial standards of polystyrene nanoplastics (PSNPLs) with an average size of 170 nm, different characteristics of the selected dyes such as the absence of impact on cell viability, specificity for plastic staining, no leaching, and lack of interference with other fluorochromes were analyzed. Based on the overall data obtained in the wide battery of assays performed, iDye PolyPink exhibited the most advantages, with respect to the other compounds, and was selected to effectively label "true-to-life" MNPLs. These advantages were confirmed using a proposed protocol, and labeling titanium-doped PETNPLs (obtained from the degradation of milk PET plastic bottles), as an example of "true-to-life" secondary NPLs. These results confirmed the usefulness of iDye PolyPink for labeling MNPLs and detecting cell internalization.

The potential effects of in vitro digestion on the physicochemical and biological characteristics of polystyrene nanoplastics.

The presence of plastic waste in our environment has continued growing and become an important environmental concern. Because of its degradation into micro- and nanoplastics (MNPLs), MNPLs are becoming environmental pollutants of special environmental/health concern. Since ingestion is one of the main exposure routes to MNPLs, the potential effects of digestion on the physicochemical/biological characteristics of polystyrene nanoplastics (PSNPLs) were determined. The results indicated a high tendency of digested PSNPLs to agglomerate and a differential presence of proteins on their surface. Interestingly, digested PSNPLs showed greater cell uptake than undigested PSNPLs in all three tested cell lines (TK6, Raji-B, and THP-1). Despite these differences in cell uptake, no differences in toxicity were observed except for high and assumed unrealistic exposures. When oxidative stress and genotoxicity induction were determined, the low effects observed after exposure to undigested PDNPLs were not observed in the digested ones. This indicated that the greater ability of digested PSNPLs to internalize was not accompanied by a greater hazard. This type of analysis should be performed with other MNPLs of varying sizes and chemical compositions.

Titanium-doped PET nanoplastics of environmental origin as a true-to-life model of nanoplastic.

The increased presence of secondary micro/nanoplastics (MNPLs) in the environment requires urgent studies on their potentially hazardous effects on exposed organisms, including humans. In this context, it is essential to obtain representative MNPL samples for such purposes. In our study, we have obtained true-to-life NPLs resulting from the degradation, via sanding, of opaque PET bottles. Since these bottles contain titanium (TiO2NPs), the resulting MNPLs also contain embedded metal. The obtained PET(Ti)NPLs were extensively characterized from a physicochemical point of view, confirming their nanosized range and their hybrid composition. This is the first time these types of NPLs are obtained and characterized. The preliminary hazard studies show their easy internalization in different cell lines, without apparent general toxicity. The demonstration by confocal microscopy that the obtained NPLs contain Ti samples offers this material multiple advantages. Thus, they can be used in in vivo approaches to determine the fate of NPLs after exposure, escaping from the existing difficulties to follow up MNPLs in biological samples.

A new source of representative secondary PET nanoplastics. Obtention, characterization, and hazard evaluation.

Micro and nanoplastics (MNPLs) are emergent environmental pollutants requiring urgent information on their potential risks to human health. One of the problems associated with the evaluation of their undesirable effects is the lack of representative samples, matching those resulting from the environmental degradation of plastic wastes. To such end, we propose an easy method to obtain polyethylene terephthalate nanoplastics from water plastic bottles (PET-NPLs) but, in principle, applicable to any other plastic goods sources. An extensive characterization indicates that the proposed process produces uniform samples of PET-NPLs of around 100 nm, as determined by using AF4 and multi-angle and dynamic light scattering methodologies. An important point to be highlighted is that to avoid the metal contamination resulting from methods using metal blades/burrs for milling, trituration, or sanding, we propose to use diamond burrs to produce metal-free samples. To visualize the toxicological profile of the produced PET-NPLs we have evaluated their ability to be internalized by cells, their cytotoxicity, their ability to induce oxidative stress, and induce DNA damage. In this preliminary approach, we have detected their cellular uptake, but without the induction of significant biological effects. Thus, no relevant increases in toxicity, reactive oxygen species (ROS) induction, or DNA damage -as detected with the comet assay- have been observed. The use of representative samples, as produced in this study, will generate relevant data in the discussion about the potential health risks associated with MNPLs exposures.

Selenite Downregulates STAT3 Expression and Provokes Lymphocytosis in the Liver of Chronically Exposed Syrian Golden Hamsters.

Arsenic is considered a worldwide pollutant that can be present in drinking water. Arsenic exposure is associated with various diseases, including cancer. Antioxidants as selenite and α-tocopherol-succinate have been shown to modulate arsenic toxic effects. Since changes in STAT3 and PSMD10 gene expression have been associated with carcinogenesis, the aim of this study was to evaluate the effect of arsenic exposure and co-treatments with selenite or α-tocopherol-succinate on the expression of these genes, in the livers of chronically exposed Syrian golden hamsters. Animals were divided into six groups: (i) control, (ii) chronically treated with 100 ppm arsenic, (iii) treated with 6 ppm α-tocopherol-succinate (α-TOS), (iv) treated with 8.5 ppm selenite, (v) treated with arsenic + α-TOS, and (vi) treated with arsenic + selenite. Urine samples and livers were collected after 20 weeks of continuous exposure. The urine samples were analyzed for arsenic species by atomic absorption spectrophotometry, and real-time RT-qPCR analysis was performed for gene expression evaluation. A reduction in STAT3 expression was observed in the selenite-treated group. No differences in PSMD10 expression were found among groups. Histopathological analysis revealed hepatic lymphocytosis in selenite-treated animals. As a conclusion, long-term exposure to arsenic does not significantly alter the expression of STAT3 and PSMD10 oncogenes in the livers of hamsters; however, selenite down-regulates STAT3 expression and provokes lymphocytosis.

Biological effects, including oxidative stress and genotoxic damage, of polystyrene nanoparticles in different human hematopoietic cell lines.

In recent years the terms "micro-/nanoplastics" (MNPLs) have caught special attention due to the increasing levels by which humans are exposed. Among MNPLs, polystyrene nanoparticles (PSNPs) are one of the most represented MNPLs in the environment. These tiny particles may enter into the human body, translocate through human barriers, interacting with blood and lymphatic immune cells, and reaching secondary organs. By using three different human leukocytic cell lines: Raji-B (B-lymphocytes), TK6 (lymphoblasts) and THP-1 (monocytes), we pursued to determine the effects of these PSNPs on the immune cell population. With this aim, the three cell lines were exposed to spherical PSNPs of about 50 nm of diameter and cytotoxicity, cellular uptake, reactive oxygen species (ROS) production, and genotoxicity were assessed at different time-points. Results show differences in all the measured endpoints, among the selected cell lines. Thus, whilst the monocytic THP-1 cells showed the highest particle internalization, no adverse effects were observed in such cells. On the other side, although Raji-B and TK6 showed lesser PSNPs uptake, mild toxicity, ROS production and genotoxicity were detected. These results highlight the importance of the cell line selection when the biological effects of PSNPs are evaluated.

Potential adverse health effects of ingested micro- and nanoplastics on humans. Lessons learned from in vivo and in vitro mammalian models.

In recent years, increasing global attention has focused on "microplastics" (MPs) and "nanoplastics" (NPs) resulting in many studies on the effects of these compounds on ecological and environmental aspects. These tiny particles (<5000 µm), predominantly derived from the degradation of plastics, pollute the marine and terrestrial ecosystems with the ability to enter into the food chain. In this manner, human consumption of food contaminated with MPs or NPs is unavoidable, but the related consequences remain to be determined. The aim of this review is to complement previous reviews on this topic by providing new studies related to exposure, absorption, and toxicity in mammalian in vivo and in vitro systems. With respect to novel information, gaps and limitations hindering attainment of firm conclusions as well as preparation of a reliable risk assessment are identified. Subsequently, recommendations for in vivo and in vitro testing methods are presented in order to perform further relevant and targeted research studies.

Assessing the effectiveness of green synthetized silver nanoparticles with Cryptocarya alba extracts for remotion of the organic pollutant methylene blue dye.

In the present work, silver nanoparticles (AgNPs) synthetized with Cryptocarya alba (Peumo) leaf extract were studied. The fabrication method was fast, low cost, and eco-friendly, and the final properties of AgNPs were determined by experimental parameters, such as AgNO3 and Peumo extract concentrations used. Setting suitable experimental conditions, crystalline AgNPs with apparent spherical forms and average diameter around 3.5 nm were obtained. In addition, the capability of synthesized Peumo-AgNPs to remove methylene blue dye (MB) in aqueous solution as well as their catalytic effectiveness was also investigated. The results showed that green synthesized AgNPs can remove fast and effectively the MB dye from aqueous medium by itself, but better results were found acting like catalyst by using sodium borohydride (NaBH4) in the reaction. In addition, this green nanomaterial can be recycling several times maintaining initial properties for removal of MB. Thus, AgNPs synthetized with Peumo leaf extracts could be an excellent catalyst candidate for degradation of blue methylene dye in chemical industries.

Genotoxicity of Copper and Nickel Nanoparticles in Somatic Cells of Drosophila melanogaster.

Copper and nickel nanoparticles (Cu-NPs and Ni-NPs, respectively) are used in a variety of industrial applications, such as semiconductors, catalysts, sensors, and antimicrobial agents. Although studies on its potential genotoxicity already exist, few of them report in vivo data. In the present study we have used the wing-spot assay in Drosophila melanogaster to determine the genotoxic activity of Cu-NPs and Ni-NPs, and these data have been compared with those obtained with their microparticle forms (MPs). Additionally, a complete physical characterization of NPs using transmission electronic microscopy (TEM), dynamic light scattering (DLS), and laser Doppler velocimetry (LDV) techniques was also performed. Results obtained with Cu-NPs and Cu-MPs indicate that both failed to induce an increase in the frequency of mutant spots formation in the wings of the adults, suggesting a lack of genotoxicity in somatic cells of D. melanogaster. However, when Ni-NPs and Ni-MPs were evaluated, a significant increase of small single spots and total mutant spots was observed only for Ni-NPs (P<0.05) at the highest dose assessed. Thus, the genotoxicity of Ni-NPs seem to be related to their nanoscale size, because no genotoxic effects have been reported with their microparticles and ions. This study is the first assessing the in vivo genotoxic potential of Cu-NPs and Ni-NPs in the Drosophila model.

Selenite restores Pax6 expression in neuronal cells of chronically arsenic-exposed Golden Syrian hamsters.

Arsenic is a worldwide environmental pollutant that generates public health concerns. Various types of cancers and other diseases, including neurological disorders, have been associated with human consumption of arsenic in drinking water. At the molecular level, arsenic and its metabolites have the capacity to provoke genome instability, causing altered expression of genes. One such target of arsenic is the Pax6 gene that encodes a transcription factor in neuronal cells. The aim of this study was to evaluate the effect of two antioxidants, α-tocopheryl succinate (α-TOS) and sodium selenite, on Pax6 gene expression levels in the forebrain and cerebellum of Golden Syrian hamsters chronically exposed to arsenic in drinking water. Animals were divided into six groups. Using quantitative real-time reverse transcriptase (RT)-PCR analysis, we confirmed that arsenic downregulates Pax6 expression in nervous tissues by 53 ± 21% and 32 ± 7% in the forebrain and cerebellum, respectively. In the presence of arsenic, treatment with α-TOS did not modify Pax6 expression in nervous tissues; however, sodium selenite completely restored Pax6 expression in the arsenic-exposed hamster forebrain, but not the cerebellum. Although our results suggest the use of selenite to restore the expression of a neuronal gene in arsenic-exposed animals, its use and efficacy in the human population require further studies.

Tocopherol and selenite modulate the transplacental effects induced by sodium arsenite in hamsters.

Human studies suggest that in utero exposure to arsenic results in adverse pregnancy outcomes. The use of dietary supplements, such as sodium selenite (SS) or α-tocopherol succinate (α-TOS), is a reasonable approach to ameliorate such health effects. Sodium arsenite at 100ppm was administered via drinking water to female hamsters from gestational days 1 or 8 to the time of delivery. Viable fetuses, fetal resorptions and non-viable fetuses were recorded during and after pregnancy and total arsenic and its metabolites were characterized in pregnant animals, placentas and fetuses. Arsenic was found to accumulate in the placenta and fetus, increasing fetal mortality, non-viable fetuses and resorptions. Co-administration of SS and α-TOS significantly reduced the observed teratogenic effects. SS influenced arsenic biotransformation by reducing the MMA/InAs index and increasing the DMA/MMA, whereas α-TOS more likely exerts its protective effect through its potent antioxidant activity.

Genotoxic and oxidative stress potential of nanosized and bulk zinc oxide particles in Drosophila melanogaster.

Zinc oxide nanoparticles (ZnONP) are manufactured on a large scale and can be found in a variety of consumer products, such as sunscreens, lotions, paints and food additives. Few studies have been carried out on its genotoxic potential and related mechanisms in whole organisms. In the present study, the in vivo genotoxic activity of ZnONP and its bulk form was assayed using the wing-spot test and comet assay in Drosophila melanogaster Additionally, a lipid peroxidation analysis using the thiobarbituric acid assay was also performed. Results obtained with the wing-spot test showed a lack of genotoxic activity of both ZnO forms. However, when both particle sizes were tested in the comet assay using larvae haemocytes, a significant increase in DNA damage was observed for ZnONP treatments but only at the higher dose applied. In addition, the lipid peroxidation assay showed significant malondialdehyde (MDA) induction for both ZnO forms, but the induction of MDA for ZnONP was higher for the ZnO bulk, suggesting that the observed DNA strand breaks could be induced by mediated oxidative stress. The overall data suggest that the potential genotoxicity of ZnONP in Drosophila can be considered weak according to the lack of mutagenic and recombinogenic effects and the induction of primary DNA damage only at high toxic doses of ZnONP. This study is the first assessing the genotoxic and oxidative stress potential of nano and bulk ZnO particles in Drosophila.

Analyses of the genotoxic and mutagenic potential of the products formed after the biotransformation of the azo dye Disperse Red 1.

Azo dyes constitute the largest class of synthetic dyes. Following oral exposure, these dyes can be reduced to aromatic amines by the intestinal microflora or liver enzymes. This work identified the products formed after oxidation and reduction of the dye Disperse Red 1, simulating hepatic biotransformation and evaluated the mutagenic potential of the resultant solution. Controlled potential electrolysis was carried out on dye solution using a Potentiostat/Galvanostat. HPLC-DAD and GC/MS were used to determine the products generated after the oxidation/reduction process. The Salmonella/microsome assay with the strains TA98 and YG1041 without S9, and the mouse lymphoma assay (MLA) using the thymidine kinase (Tk) gene, were used to evaluate the mutagenicity of the products formed. Sulfate 2-[(4-aminophenyl)ethylamino]-ethanol monohydrate, nitrobenzene, 4-nitro-benzamine and 2-(ethylphenylamino)-ethanol were detected. This dye has already being assigned as mutagenic in different cell system. In addition, after the oxidation/reduction process the dye still had mutagenic activity for the Salmonella/microsome assay. Nevertheless, both the original dye Disperse Red 1 and its treated solutions showed negative results in the MLA. The present results suggest that the ingestion of water and food contaminated with this dye may represent human and environmental health problem, due to the generation of harmful compounds after biotransformation.

Genotoxicity of heptachlor and heptachlor epoxide in human TK6 lymphoblastoid cells.

The genotoxic potential of the organochlorine insecticides heptachlor (HC) and its metabolite heptachlor epoxide (HCE) has been evaluated in TK6 cells, a well-established human lymphoblastoid cell line. Genotoxicity has been determined by scoring the induction of DNA breaks in the comet assay and by measuring the frequency of micronuclei (MN) in binucleated cells. The results indicate that both compounds are able to induce significant increases in the percentage of DNA in the tail, the parameter used in the comet assay, with a direct dose-response relationship. Nevertheless, both compounds were unable to induce an increase in the frequency of MN. The comet assay measures primary DNA damage, while the induction of MN measures fixed damage. Thus, our results would suggest that the DNA damage induced by the two insecticides is not fixed as chromosome damage, which would be detectable by means of the MN assay (chromosome breaks and aneuploidy).