Nucleotide depletion reveals the impaired ribosome biogenesis checkpoint as a barrier against DNA damage.

Many oncogenes enhance nucleotide usage to increase ribosome content, DNA replication, and cell proliferation, but in parallel trigger p53 activation. Both the impaired ribosome biogenesis checkpoint (IRBC) and the DNA damage response (DDR) have been implicated in p53 activation following nucleotide depletion. However, it is difficult to reconcile the two checkpoints operating together, as the IRBC induces p21-mediated G1 arrest, whereas the DDR requires that cells enter S phase. Gradual inhibition of inosine monophosphate dehydrogenase (IMPDH), an enzyme required for de novo GMP synthesis, reveals a hierarchical organization of these two checkpoints. We find that the IRBC is the primary nucleotide sensor, but increased IMPDH inhibition leads to p21 degradation, compromising IRBC-mediated G1 arrest and allowing S phase entry and DDR activation. Disruption of the IRBC alone is sufficient to elicit the DDR, which is strongly enhanced by IMPDH inhibition, suggesting that the IRBC acts as a barrier against genomic instability.

In Vitro Approaches to Determine the Potential Carcinogenic Risk of Environmental Pollutants.

One important environmental/health challenge is to determine, in a feasible way, the potential carcinogenic risk associated with environmental agents/exposures. Since a significant proportion of tumors have an environmental origin, detecting the potential carcinogenic risk of environmental agents is mandatory, as regulated by national and international agencies. The challenge mainly implies finding a way of how to overcome the inefficiencies of long-term trials with rodents when thousands of agents/exposures need to be tested. To such an end, the use of in vitro cell transformation assays (CTAs) was proposed, but the existing prevalidated CTAs do not cover the complexity associated with carcinogenesis processes and present serious limitations. To overcome such limitations, we propose to use a battery of assays covering most of the hallmarks of the carcinogenesis process. For the first time, we grouped such assays as early, intermediate, or advanced biomarkers which allow for the identification of the cells in the initiation, promotion or aggressive stages of tumorigenesis. Our proposal, as a novelty, points out that using a battery containing assays from all three groups can identify if a certain agent/exposure can pose a carcinogenic risk; furthermore, it can gather mechanistic insights into the mode of the action of a specific carcinogen. This structured battery could be very useful for any type of in vitro study, containing human cell lines aiming to detect the potential carcinogenic risks of environmental agents/exposures. In fact, here, we include examples in which these approaches were successfully applied. Finally, we provide a series of advantages that, we believe, contribute to the suitability of our proposed approach for the evaluation of exposure-induced carcinogenic effects and for the development of an alternative strategy for conducting an exposure risk assessment.

Toxicological Profiling and Long-Term Effects of Bare, PEGylated- and Galacto-Oligosaccharide-Functionalized Mesoporous Silica Nanoparticles.

Mesoporous silica nanoparticles (MSNs) are amongst the most used nanoparticles in biomedicine. However, the potentially toxic effects of MSNs have not yet been fully evaluated, being a controversial matter in research. In this study, bare MSNs, PEGylated MSNs (MSNs-PEG), and galacto-oligosaccharide-functionalized MSNs (MSNs-GAL) are synthesized and characterized to assess their genotoxicity and transforming ability on human lung epithelial BEAS-2B cells in short- (48 h) and long-term (8 weeks) exposure scenarios. Initial short-term treatments show a dose-dependent increase in genotoxicity for MSNs-PEG-treated cells but not oxidative DNA damage for MSNs, MSNs-PEG, or for MSNs-GAL. In addition, after 8 weeks of continuous exposure, neither induced genotoxic nor oxidative DNA is observed. Nevertheless, long-term treatment with MSNs-PEG and MSNs-GAL, but not bare MSNs, induces cell transformation features, as evidenced by the cell's enhanced ability to grow independently of anchorage, to migrate, and to invade. Further, the secretome from cells treated with MSNs and MSNs-GAL, but not MSNs-PEG, shows certain tumor-promoting abilities, increasing the number and size of HeLa cell colonies formed in the indirect soft-agar assay. These results show that MSNs, specifically the functionalized ones, provoke some measurable adverse effects linked to tumorigenesis. These effects are in the order of other nanomaterials, such as carbon nanotubes or cerium dioxide nanoparticles, but they are lower than those provoked by some approved drugs, such as doxorubicin or dexamethasone.

Drosophila as a Suitable In Vivo Model in the Safety Assessment of Nanomaterials.

Nanotechnology is often praised as the future technology that will revolutionize the world as we know it, because nanomaterials (NMs) offer numerous practical applications for a wide range of fields such as medicine, cosmetics, food preservation, paintings, and industry. Produced by nanotechnology, NMs are in the front line of this innovative applied science, while nanoparticles (NPs) refer to materials existing in the natural world and measuring 1-100 nanometers in at least one dimension. The recent surge in the number of endeavors to utilize NMs makes it imperative to identify hazards and risk factors involved as we have yet to know harmful effects of this uncharted territory on the environment and public health. While researchers generally choose to carry out in vitro experiments in an effort to assess toxicity of NMs, in vivo approaches seem to yield better evidence that is more relevant to risk assessment. In that context, Drosophila melanogaster stands out as the most dynamic model organism for biological experiments, since 75% of the genes responsible for human diseases are known to have homologs in D. melanogaster, which facilitates research into various pathologies. This book chapter aims to present the full picture of studies on separate NMs that employed in vivo approaches (toxicity, genotoxicity, internalization, cell uptake, tissue distribution, etc.) using D. melanogaster, attempting to offer an in-depth analysis of risks involved in exposure to NMs, as well as many advantages of other animal models used by nanogenotoxicology studies.

Nanoplastics and Arsenic Co-Exposures Exacerbate Oncogenic Biomarkers under an In Vitro Long-Term Exposure Scenario.

The increasing accumulation of plastic waste and the widespread presence of its derivatives, micro- and nanoplastics (MNPLs), call for an urgent evaluation of their potential health risks. In the environment, MNPLs coexist with other known hazardous contaminants and, thus, an interesting question arises as to whether MNPLs can act as carriers of such pollutants, modulating their uptake and their harmful effects. In this context, we have examined the interaction and joint effects of two relevant water contaminants: arsenic and polystyrene nanoplastics (PSNPLs), the latter being a model of nanoplastics. Since both agents are persistent pollutants, their potential effects have been evaluated under a chronic exposure scenario and measuring different effect biomarkers involved in the cell transformation process. Mouse embryonic fibroblasts deficient for oxidative DNA damage repair mechanisms, and showing a cell transformation status, were used as a sensitive cell model. Such cells were exposed to PSNPLs, arsenic, and a combination PSNPLs/arsenic for 12 weeks. Interestingly, a physical interaction between both pollutants was demonstrated by using TEM/EDX methodologies. Results also indicate that the continuous co-exposure enhances the DNA damage and the aggressive features of the initially transformed phenotype. Remarkably, co-exposed cells present a higher proportion of spindle-like cells within the population, an increased capacity to grow independently of anchorage, as well as enhanced migrating and invading potential when compared to cells exposed to arsenic or PSNPLs alone. This study highlights the need for further studies exploring the long-term effects of contaminants of emerging concern, such as MNPLs, and the importance of considering the behavior of mixtures as part of the hazard and human risk assessment approaches.

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.

Role of As3mt and Mth1 in the genotoxic and carcinogenic effects induced by long-term exposures to arsenic in MEF cells.

DNA damage plays a crucial role in the transforming potential of the human carcinogen arsenic. The arsenic biotransformation enzyme AS3MT is known to participate in the generation of ROS after arsenic exposure, whereas MTH1 sanitizes oxidized dNTP pools to prevent the incorporation of damaged bases into DNA. In this work, we sought to assess the role of these two enzymes in the genotoxic and carcinogenic effects of arsenic exposure. Thus, mouse embryonic fibroblasts (MEF), transformed by chronic arsenite exposure, were monitored for DNA damage by the comet and the micronucleus assays at different time-of-exposure intervals lasting for 50 weeks. Results indicate that the oxidative and DNA damage of chronically exposed MEF cells increased time-dependently up to the point of transformation. As3mt expression followed a pattern like that of DNA damage, and its forced inhibition by shRNA technology before transformation resulted in a DNA damage decrease. On the other hand, Mth1 mRNA levels increased after the transformation point, and its forced knock-down increased significantly the levels of DNA damage and decreased the aggressiveness of the oncogenic phenotype. Thus, As3mt and Mth1 have important differential roles in the accumulation of DNA damage linked to the transformation process: while As3mt contributes to the genotoxic effects before the transformation, Mth1 prevents the DNA damage fixation after the acquisition of the oncogenic phenotype. This study demonstrates the influence of As3mt and Mth1 in arsenic DNA damage induction and it is the first to present Mth1 as a candidate modulator biomarker of the tumoral phenotype.

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.

Interactions of polystyrene nanoplastics with in vitro models of the human intestinal barrier.

The universal presence of micro-nanoplastics (MNPLs) and its relative unknown effects on human health is a concern demanding reliable data to evaluate their safety. As ingestion is one of the main exposure routes for humans, we have assessed their hazard using two in vitro models that simulate the human intestinal barrier and its associated lymphoid system. Two different coculture models (differentiated Caco-2/HT29 intestinal cells and Caco-2/HT29 + Raji-B cells) were exposed to polystyrene nanoparticles (PSNPs) for 24 h. Endpoints such as viability, membrane integrity, NPS localization and translocation, ROS induction, and genotoxic damage were evaluated to have a comprehensive view of their potentially harmful effects. No significant cytotoxic effects were observed in any of the analyzed systems. In addition, no adverse effects were detected in the integrity or in the permeability of the barrier model. Nevertheless, confocal microscopy analysis showed that MNPLs were highly uptaken by both of the barrier model systems, and that translocation across the membrane occurred. Thus, MNPLs were detected into Raji-B cells, placed in the basolateral compartment of the insert. The internalization followed a dose-dependent pattern, as assessed by flow cytometry. Nonetheless, no genotoxic or oxidative DNA damage induction was detected in either case. Finally, no variations in the transcription of oxidative and stress genes could be detected in any of the in vitro barrier models. Our results show that MNPLs can enter and cross the epithelial barrier of the digestive system, as demonstrated when Raji-B cells were included in the model, but without exerting apparent hazardous effects.

MTH1 is involved in the toxic and carcinogenic long-term effects induced by zinc oxide and cobalt nanoparticles.

The nanoparticles (NPs) exposure-related oxidative stress is considered among the main causes of the toxic effects induced by these materials. However, the importance of this mechanism has been mostly explored at short term. Previous experience with cells chronically exposed to ZnO and Co NPs hinted to the existence of an adaptative mechanism contributing to the development of oncogenic features. MTH1 is a well-described enzyme expressed exclusively in cancer cells and required to avoid the detrimental consequences of its high prooxidant microenvironment. In the present work, a significantly marked overexpression was found when MTH1 levels were monitored in long-term ZnO and Co NP-exposed cells, a fact that correlates with acquired 2.5-fold and 3.75-fold resistance to the ZnO and Co NPs treatment, respectively. The forced stable inhibition of Mth1 expression by shRNA, followed by 6 additional weeks of exposure, significantly reduced this acquired resistance and sensitized cells to the oxidizing agents H2O2 and KBrO3. When the oncogenic phenotype of Mth1 knock-down cells was evaluated, we found a decrease in several oncogenic markers, including proliferation, anchorage-independent cell growth, and migration and invasion potential. Thus, MTH1 elicits here as a relevant player in the NPs-induced toxicity and carcinogenicity. This study is the first to give a mechanistic explanation for long-term NPs exposure-derived effects. We propose MTH1 as a candidate biomarker to unravel NPs potential genotoxic and carcinogenic effects, as its expression is expected to be elevated only under exposure conditions able to induce DNA damage and the acquisition of an oncogenic phenotype.

Hazard assessment of three haloacetic acids, as byproducts of water disinfection, in human urothelial cells.

Disinfection by-products (DBPs) are compounds produced in the raw water disinfection processes. Although increased cancer incidence has been associated with exposure to this complex mixture, the carcinogenic potential of individual DBPs remains not well known; thus, further studies are required. Haloacetic acids (HAAs) constitute an important group among DBPs. In this study, we have assessed the in vitro carcinogenic potential of three HAAs namely chloro-, bromo-, and iodoacetic acids. Using a long-term (8 weeks) and sub-toxic doses exposure scenario, different in vitro transformation markers were evaluated using a human urothelial cell line (T24). Our results indicate that long-term exposure to low doses of HAAs did not reproduce the genotoxic effects observed in acute treatments, where oxidative DNA damage was induced. No changes in the transformation endpoints analyzed were observed, as implied by the absence of significant morphological, cell growth rate and anchorage-independent cell growth pattern modifications. Interestingly, HAA-long-term exposed cells developed resistance to oxidative stress damage, what would explain the observed differences between acute and long-term exposure conditions. Accordingly, data obtained under long-term exposure to sub-toxic doses of HAAs could be more accurate, in terms of risk assessment, than under acute exposure scenarios.

Effects of differently shaped TiO2NPs (nanospheres, nanorods and nanowires) on the in vitro model (Caco-2/HT29) of the intestinal barrier.

BACKGROUND: The biological effects of nanoparticles depend on several characteristics such as size and shape that must be taken into account in any type of assessment. The increased use of titanium dioxide nanoparticles (TiO2NPs) for industrial applications, and specifically as a food additive, demands a deep assessment of their potential risk for humans, including their abilities to cross biological barriers. METHODS: We have investigated the interaction of three differently shaped TiO2NPs (nanospheres, nanorods and nanowires) in an in vitro model of the intestinal barrier, where the coculture of Caco-2/HT29 cells confers inherent intestinal epithelium characteristics to the model (i.e. mucus secretion, brush border, tight junctions, etc.). RESULTS: Adverse effects in the intestinal epithelium were detected by studying the barrier's integrity (TEER), permeability (LY) and changes in the gene expression of selected specific markers. Using Laser Scanning Confocal Microscopy, we detected a different behaviour in the bio-adhesion and biodistribution of each of the TiO2NPs. Moreover, we were able to specifically localize each type of TiO2NPs inside the cells. Interestingly, general DNA damage, but not oxidative DNA damage effects, were detected by using the FPG version of the comet assay. CONCLUSIONS: Results indicate different interactions and cellular responses related to differently shaped TiO2NPs, nanowires showing the most harmful effects.

Toxic and genotoxic effects of graphene and multi-walled carbon nanotubes.

Graphene and multi-walled carbon nanotubes (MWCNT) are widely used in nanomedicine, and other fields, due to their unique physicochemical properties including high tensile strength, ultra-light weight, thermal and chemical stability, and reliable semi-conductive electronic properties. Although extensive amount of data exist describing their adverse effects including potential genotoxicity, few studies using gene mutation detection approaches in mammalian cells are available, which represents an important gap for risk estimations. The aim of the present study was to determine the effects of graphene or MWCNT [as pure, carboxyl (COOH) functionalized, and amide (NH2) functionalized] on cytotoxicity, intracellular levels of reactive oxygen species, apoptosis, gene expression changes, and gene mutation induction in L5178Y/Tk+/-3.7.2C mouse lymphoma cell line. Although some adverse effects were observed at concentrations of 350 and 450 µg/ml, which are excessive and not environmentally relevant levels, no marked effects were detected at concentrations of 250 µg/ml and lower. This is the first study reporting cytotoxicity, mutagenicity, and gene expression findings in the mouse lymphoma cell line for graphene and different MWCNT forms at high concentrations; however, the biological relevance of these observations needs to be assessed following chronic in vivo exposure.

Titanium dioxide nanoparticles translocate through differentiated Caco-2 cell monolayers, without disrupting the barrier functionality or inducing genotoxic damage.

The widespread use of titanium dioxide nanoparticles (TiO2 NPs) in commercial food products makes intestinal cells a suitable target. Accordingly, we have used the human colon adenocarcinoma Caco-2 cells to detect their potential harmful effects. Caco-2 cells can differentiate in to enterocytic-like cells, forming consistent cell monolayers and are used as a model of the intestinal barrier. Using both undifferentiated and differentiated Caco-2 cells, we have explored a set of biomarkers, aiming to evaluate undesirable effects associated to TiO2 NP exposure. Results indicate non-toxic effects in exposures ranging 1-200 µg ml-1 . Significant differences were observed in cell uptake, with a higher amount of incorporated TiO2 NPs in undifferentiated cells, as visualized using confocal microscopy. In well-established monolayers, translocation was detected using both confocal microscopy and transmission electron microscopy with energy-dispersive X-ray spectroscopy. In spite of the observed uptake and translocation, TiO2 NP exposures did not modify the integrity of the monolayer, as measured using the transepithelial electrical resistance and Lucifer yellow methods. The potential genotoxic effects in differentiated cells were evaluated in the comet assay, with and without formamidopyrimidine DNA glycosylase enzyme to detect oxidatively the damaged DNA bases. Although some changes were detected at the lower dose (10 µg ml-1 ), no effects were observed at higher doses.

Drosophila melanogaster as a suitable in vivo model to determine potential side effects of nanomaterials: A review.

Despite being a relatively new field, nanoscience has been in the forefront among many scientific areas. Nanoparticle materials (NM) present interesting physicochemical characteristics not necessarily found in their bulky forms, and alterations in their size or coating markedly modify their physical, chemical, and biological properties. Due to these novel properties there is a general trend to exploit these NM in several fields of science, particularly in medicine and industry. The increased presence of NM in the environment warrants evaluation of potential harmful effects in order to protect both environment and human exposed populations. Although in vitro approaches are commonly used to determine potential adverse effects of NM, in vivo studies generate data expected to be more relevant for risk assessment. As an in vivo model Drosophila melanogaster was previously found to possess reliable utility in determining the biological effects of NM, and thus its usage increased markedly over the last few years. The aims of this review are to present a comprehensive overview of all apparent studies carried out with NM and Drosophila, to attain a clear and comprehensive picture of the potential risk of NM exposure to health, and to demonstrate the advantages of using Drosophila in nanotoxicological investigations.

Multi-walled carbon nanotubes (NM401) induce ROS-mediated HPRT mutations in Chinese hamster lung fibroblasts.

Although there is an important set of data showing potential genotoxic effects of nanomaterials (NMs) at the DNA (comet assay) and chromosome (micronucleus test) levels, few studies have been conducted to analyze their potential mutagenic effects at gene level. We have determined the ability of multi-walled carbon nanotubes (MWCNT, NM401), to induce mutations in the HPRT gene in Chinese hamster lung (V79) fibroblasts. NM401, characterized in the EU NanoGenotox project, were further studied within the EU Framework Programme Seven (FP7) project NANoREG. From the proliferation assay data we selected a dose-range of 0.12 to 12µg/cm(2) At these range we have been able to observe significant cellular uptake of MWCNT by using transmission electron microscopy (TEM), as well as a concentration-dependent induction of intracellular reactive oxygen species. In addition, a clear concentration-dependent increase in the induction of HPRT mutations was also observed. Data support a potential genotoxic/ carcinogenic risk associated with MWCNT exposure.

Oxidative DNA damage enhances the carcinogenic potential of in vitro chronic arsenic exposures.

Chronic exposure to arsenic is known to increase the incidence of cancer in humans. Our previous work demonstrated that environmentally relevant arsenic exposures generate an accelerated accumulation of pre-carcinogen 8-OH-dG DNA lesions under Ogg1-deficient backgrounds, but it remains unproved whether this observed arsenic-induced oxidative DNA damage (ODD) is certainly important in terms of cancer. Here, isogenic MEF Ogg1 (+/+) cells and MEF Ogg1 (-/-) cells-unable to properly eliminate 8-OH-dG from DNA-were exposed to 0.5, 1 and 2 µM of sodium arsenite for 40 weeks. The acquisition of an in vitro cancer-like phenotype was assessed throughout the exposure; matrix metalloproteinase (MMP) activities were measured by zymography, colony formation and promotion were evaluated by soft agar assay, and cellular invasiveness was measured by the transwell assay. Alterations in cellular morphology, growth and differentiation status were also included as complementary measures of transformation. MEF Ogg1 (-/-) cells showed a cancer-associated phenotype after 30 weeks of exposure, as indicated by morphological changes, increased proliferation, deregulated differentiation status, increased MMPs secretion, anchorage-independent cell growth and enhancement of tumor growth and invasiveness. Conversely, MEF Ogg1 (+/+) cells did not present changes in morphology or proliferation, exhibited a milder degree of gene deregulation and needed 10 weeks of additional exposure to the highest arsenite doses to show tumor enhancing effects. Thus, Ogg1 genetic background and arsenic-induced 8-OH-dG proved relevant for arsenic-mediated carcinogenic effects. To our knowledge, this is the first study directly linking ODD with arsenic carcinogenesis.

Antioxidant and anti-genotoxic properties of cerium oxide nanoparticles in a pulmonary-like cell system.

Cerium oxide nanoparticles (CeO2-NP) present two different oxidation states what can suppose an auto-regenerative redox cycle. Potential applications of CeO2-NP to quench reactive oxygen species (ROS) in biological systems are currently being investigated. In this context, CeO2-NP may represent a novel agent to protect cells and tissues against oxidative damage by its regenerative free radical-scavenging properties. In this study, we have used a human epithelial lung cell line, BEAS-2B, as a model to study the possible antioxidant and anti-genotoxic effect of CeO2-NP in a pulmonary-like system. We have assessed the protective effect of CeO2-NP pre-treatment in front of a well-defined oxidative stress-inducing agent (KBrO3). Different endpoints like toxicity, intracellular ROS induction, genotoxicity and DNA oxidative damage (comet assay), and gene expression alterations have been evaluated. The obtained results confirmed the antioxidant properties of CeO2-NP. Thus, its pre-treatment significantly reduced the intracellular production of ROS induced by KBrO3. Similarly, a reduction in the levels of DNA oxidative damage, as measured with the comet assay complemented with formamidopyrimidine DNA glycosylase enzyme, was also observed. Pre-treatment of BEAS-2B cells with CeO2-NP (at 2.5 µg/mL) slightly increased the viability of cells treated with KBrO3 as well as down-regulated the expression of the Ho1 and Sod2 genes involved in the oxidative Nrf2 pathway. Our finding would support the potential usefulness of CeO2-NP as a pharmacological agent to be used against diseases caused by oxidative stress.

Acute and long-term in vitro effects of zinc oxide nanoparticles.

Since most of the toxic studies of zinc oxide nanoparticles (ZnO NPs) focused on acute and high-dose exposure conditions, the aim of the present study was to fill the existing knowledge gap of long-term effects of ZnO NPs at sub-toxic doses. To overcome this point, we have evaluated the toxic, genotoxic, and carcinogenic effects of ZnO NPs under long-term treatments (12 weeks), using a sub-toxic dose (1 µg/mL) according to acute 48-h exposure. Preliminarily, oxidative stress and genotoxic/oxidative DNA damage were determined under acute exposure and high-dose conditions. To determine the role of oxidative DNA damage, a wild-type mouse embryonic fibroblast (MEF Ogg1 (+/+)) and its isogenic 8-oxo-guanine DNA glycosylase 1 (Ogg1) knockout partner (MEF Ogg1 (-/-)) cell lines were used. Although short-term exposure (24-h) experiments demonstrated that ZnO NPs were able to induce ROS, genotoxicity, and oxidative DNA damage in both cell lines, no effects were obtained under long-term exposure scenario. Thus, 1 µg/mL exposure over 12 weeks was unable to induce genotoxicity as well as cellular transformation in both cell types, as indicated by the lack of observed morphological cell changes, variations in the secretion of matrix metalloproteinases, and anchorage-independent cell growth ability, regarded as cancer-like phenotypic hallmarks. Our results indicate that short-term effects of ZnO NP exposure are not replicated under long-term and sub-toxic dose conditions. All together, the lack of genotoxic/carcinogenic effects after chronic treatments seem to indicate a reduced risk associated with ZnO NP exposure.

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.