Internalization and toxicity of polystyrene nanoplastics on inmortalized human neural stem cells.

Global plastic production has increased exponentially in recent decades, and a significant part of it persists in the environment, where it degrades into microplastics and nanoplastics (MPs and NPs). These can enter in humans by ingestion, inhalation, and dermal routes, and there is scientific evidence that they are able to reach the systemic circulation and penetrate and accumulate in various tissues and organs. Neurodevelopmental toxicity of NPs is one of the most worrying effects, as they can cross the blood-brain barrier. In the following study, we analyzed, by transmission electron microscopy, the in vitro uptake of 30-nm polystyrene nanoplastics (PS-NPs) into human neural stem cells (NSCs), their accumulation and subcellular localization within the cell. Furthermore, we studied the effects of different concentrations of PS-NPs on cell death, proliferation, and cell differentiation using immunocytochemistry and quantitative real time PCR for specific markers. This study demonstrated that PS-NPs were able to enter the cell, probably by endocytosis, accumulate, and aggregated in human NSCs, without being detected in the nucleus, causing cell death by apoptosis and decreased cell proliferation. This study provides new insights into the interaction and effects of PS-NPs in human NSC and supports the scientific evidence for the involvement of nanoplastic in neurodevelopmental disorders.

Molecular effects of polystyrene nanoplastics on human neural stem cells.

Nanoplastics (NPs) have been found in many ecological environments (aquatic, terrestrial, air). Currently, there is great concern about the exposition and impact on animal health, including humans, because of the effects of ingestion and accumulation of these nanomaterials (NMs) in aquatic organisms and their incorporation into the food chain. NPs´ mechanisms of action on humans are currently unknown. In this study, we evaluated the altered molecular mechanisms on human neural stem cell line (hNS1) after 4 days of exposure to 30 nm polystyrene (PS) NPs (0.5, 2.5 and 10 µg/mL). Our results showed that NPs can induce oxidative stress, cellular stress, DNA damage, alterations in inflammatory response, and apoptosis, which could lead to tissue damage and neurodevelopmental diseases.

DNA damage and molecular level effects induced by polystyrene (PS) nanoplastics (NPs) after Chironomus riparius (Diptera) larvae.

In this work, we analyzed the early molecular effects of polystyrene (PS) nanoplastics (NPs) on an aquatic primary consumer (larvae of Chironomus riparius, Diptera) to evaluate their potential DNA damage and the transcriptional response of different genes related to cellular and oxidative stress, endocrine response, developmental, oxygen transport, and immune response. After 24-h exposures of larvae to doses of PS NPs close to those currently found in the environment, the results revealed a large genotoxic effect. This end was evidenced after significant increases in DNA strand breaks of C. riparius larvae quantified by the comet assay, together with results obtained when analyzing the expression of four genes involved in DNA repair (xrrc1, ATM, DECAY and NLK) and which were reduced in the presence of these nanomaterials. Consequently, this reduction trend is likely to prevent the repair of DNA damage caused by PS NPs. In addition, the same tendency to reduce the expression of genes involved in cellular stress, oxidative stress, ecdysone pathway, development, and oxygen transport was observed. Taken together, these results suggest that PS NPs reduce the expression of hormonal target genes and a developmental gene. We show, for the first time, effects of PS NPs on the endocrine system of C. riparius and suggest a possible mechanism of blocking ecdysteroid hormones in insects. Moreover, the NPs were able to inhibit the expression of hemoglobin (Hb C), a protein involved in oxygen transport, and activate a gene of the humoral immune system. These data reveal for the first time the genomic effects of PS NPs in the aquatic invertebrate C. riparius, at the base of the food chain.

Graphene Oxides (GOs) with Different Lateral Dimensions and Thicknesses Affect the Molecular Response in Chironomus riparius.

Graphene oxide (GO) materials possess physicochemical properties that facilitate their application in the industrial and medical sectors. The use of graphene may pose a threat to biota, especially aquatic life. In addition, the properties of nanomaterials can differentially affect cell and molecular responses. Therefore, it is essential to study and define the possible genotoxicity of GO materials to aquatic organisms and their ecosystems. In this study, we investigated the changes in the expression of 11 genes in the aquatic organism Chironomus riparius after 96 h of exposure to small GOs (sGO), large GOs (lGO) and monolayer GOs (mlGO) at 50, 500 and 3000 µg/L. Results showed that the different genes encoding heat shock proteins (hsp90, hsp70 and hsp27) were overexpressed after exposure to these nanomaterials. In addition, ATM and NLK-the genes involved in DNA repair mechanisms-were altered at the transcriptional level. DECAY, an apoptotic caspase, was only activated by larger size GO materials, mlGO and lGO. Finally, the gene encoding manganese superoxide dismutase (MnSOD) showed higher expression in the mlG O-treated larvae. The lGO and mlGO treatments indicated high mRNA levels of a developmental gene (FKBP39) and an endocrine pathway-related gene (DRONC). These two genes were only activated by the larger GO materials. The results indicate that larger and thicker GO nanomaterials alter the transcription of genes involved in cellular stress, oxidative stress, DNA damage, apoptosis, endocrine and development in C. riparius. This shows that various cellular processes are modified and affected, providing some of the first evidence for the action mechanisms of GOs in invertebrates. In short, the alterations produced by graphene materials should be further studied to evaluate their effect on the biota to show a more realistic scenario of what is happening at the molecular level.

Neurotoxicity and endocrine disruption caused by polystyrene nanoparticles in zebrafish embryo.

Nanoplastics (NP) are present in aquatic and terrestrial ecosystems. Humans can be exposed to them through contaminated water, food, air, or personal care products. Mechanisms of NP toxicity are largely unknown and the Zebrafish embryo poses an ideal model to investigate them due to its high homology with humans. Our objective in the present study was to combine a battery of behavioral assays with the study of endocrine related gene expression, to further explore potential NP neurotoxic effects on animal behavior. Polystyrene nanoplastics (PSNP) were used to evaluate NP toxicity. Our neurobehavioral profiles include a tail coiling assay, a light/dark activity assay, two thigmotaxis anxiety assays (auditory and visual stimuli), and a startle response - habituation assay in response to auditory stimuli. Results show PSNP accumulated in eyes, neuromasts, brain, and digestive system organs. PSNP inhibited acetylcholinesterase and altered endocrine-related gene expression profiles both in the thyroid and glucocorticoid axes. At the whole organism level, we observed altered behaviors such as increased activity and anxiety at lower doses and lethargy at a higher dose, which could be due to a variety of complex mechanisms ranging from sensory organ and central nervous system effects to others such as hormonal imbalances. In addition, we present a hypothetical adverse outcome pathway related to these effects. In conclusion, this study provides new understanding into NP toxic effects on zebrafish embryo, emphasizing a critical role of endocrine disruption in observed neurotoxic behavioral effects, and improving our understanding of their potential health risks to human populations.

Molecular effects of polystyrene nanoplastics toxicity in zebrafish embryos (Daniorerio).

Plastics pose a health hazard to living beings and the environment. Plastic degradation produces nano-sized plastic particles (NPs) that end up in terrestrial and aquatic ecosystems, including oceans, rivers, and lakes. Their presence in air, drinking water, sediments, food, and personal care products leads to a variety of exposure routes for living beings, including humans. The toxicity mechanisms of these nanomaterials (NMs) in living organisms and ecosystems are currently unknown, making it a priority to understand their effects at the molecular and cellular levels. The zebrafish (Zf) (Danio rerio) is a model organism which has a high homology with humans and has been widely used to assess the hazard of different xenobiotics. In this study, the expression changes of different genes in 120 hpf Zf embryos (Zfe) after exposure to polystyrene (PS) NPs (30 nm) at concentrations of 0.1, 0.5 and 3 ppm were investigated. The results showed that the gene encoding heat shock protein (hsp70) was down-regulated in a dose-dependent manner. The genes encoding superoxide dismutase (SOD 1 and SOD 2), apoptotic genes (cas 1 and cas 8) and interleukin 1-ß (il1ß) were activated at the concentration of 3 ppm PS NP, while the anti-apoptotic gene Bcl2α was inhibited at 0.5 and 3 ppm. In addition, the neurotransmitter-related gene Acetyl-Cholinesterase (ache) was significantly inhibited and the DNA repair genes (gadd45α and rad51) were also down-regulated. In contrast, the mitochondrial metabolism-related gene cox1 did not alter its expression in any of the treatments. Most of the changes in gene expression occurred at the highest concentration of NPs. Overall, the results indicated that NPs generated cellular stress that caused certain alterations in normal gene expression (oxidative stress, apoptotic and inflammatory processes, neurotoxicity and anti-apoptotic proteins), but did not cause any mortality after 120 hpf exposure at the three concentrations assayed. These results highlight the need for further studies investigating the effects, at the molecular level, of these materials in humans and other living organisms.

Toxicological effects of three different types of highly pure graphene oxide in the midge Chironomus riparius.

Graphene oxide (GO) is a carbon nanomaterial used in electronics, biomedicine, environmental remediation and biotechnology. The production of graphene will increase in the upcoming years. The carbon nanoparticles (NPs) are released into the environment and accumulated in aquatic ecosystems. Information on the effects of GO in aquatic environments and its impact on organisms is still lacking. The aim of this study was to synthesise and characterise label-free GO with controlled lateral dimensions and thickness - small GO (sGO), large GO (lGO) and monolayer GO (mlGO) - and determine their impact on Chironomus riparius, a sentinel species in the freshwater ecosystem. Superoxide dismutase (SOD) and lipid peroxidation (LPO) was evaluated after exposures for 24 h and 96 h to 50, 500, and 3000 µg/L. GOs accumulated in the gut of C. riparius and disturbed its antioxidant metabolism. We suggest that all types of GO exposure can upregulate of SOD. Moreover, both lGO and mlGO treatments caused LPO damage in C. riparius in comparison to sGO, proving its favourable lateral size impact in this organism. Our results indicate that GOs could accumulate and induce significant oxidative stress on C. riparius. This work shows new information about the potential oxidative stress of these NMs in aquatic organisms.

BPA and its analogues (BPS and BPF) modify the expression of genes involved in the endocrine pathway and apoptosis and a multi drug resistance gene of the aquatic midge Chironomus riparius (Diptera).

Many countries are limiting the use of bisphenol A (BPA) because evidence shows it is dangerous to human health and wildlife. For the manufacturing of polycarbonate plastics, bisphenol S (BPS) and bisphenol F (BPF) are proposed as safer alternatives. They have already been released into the aquatic environment without previously available information about their potential adverse effects. In this study, we compared the effects of BPA, BPS and BPF exposure to the expression profile of genes involved in the endocrine pathway (EcR and E74), ecdysone metabolism (Cyp18a1 and Shadow), apoptosis (DRONC) and the multidrug resistance-associated protein 1 gene (MRP1) in the midge, Chironomus riparius (Diptera). The three toxicants increased Shadow expression, which is involved in ecdysone synthesis, but only BPF significantly altered Cyp18a1, which is implicated in ecdysone degradation. BPS and BPF modified EcR and E74 expression; BPF upregulated the effector caspase DRONC. Furthermore, BPA significantly increased MRP1 expression. This study provides insights into the action of bisphenols at the molecular level and highlights the potential risks of BPS and BPF as BPA alternatives.

Effects of single and mixture exposure of cadmium and copper in apoptosis and immune related genes at transcriptional level on the midge Chironomus riparius Meigen (Diptera, Chironomidae).

Metals and heavy metals are natural contaminants with an increasing presence in aquatic ecosystems as a result of human activities. Although they are mixed in the water, research is usually focused on analyzing them in isolation, so there is a lack of knowledge about their combined effects. The aim of this work was to assess the damage produced by mixtures of cadmium and copper, two frequent metals used in industry, in the harlequin midge Chironomus riparius (Diptera). The effects of acute doses of cadmium and copper were evaluated in fourth instar larvae by analyzing the mRNA levels of six genes related to apoptosis (DRONC, IAP1), immune system (PO1, Defensin), stress (Gp93), and copper homeostasis (Ctr1). DRONC, Ctr1, and IAP1 transcripts are described here for first time in this species. Individual fourth instar larvae were submitted to 10 µM, 1 µM and 0.1 µM of CdCl2 or CuCl2, and mixture. The employed individuals came from different egg masses. Real-time PCR analysis showed a complex pattern of alterations in transcriptional activity for two genes, DRONC and Gp93, while the rest of them did not show any statistically significant differences. The effector caspase DRONC showed upregulation with the highest concentration tested of the mixture. In case of gp93, chaperone involved in regulation of immune response, differences in expression levels were found with 1 and 10 µM Cu and 0.1 and 10 µM of mixtures, compared to control samples. These results suggest that mixtures affect the transcriptional activity differently and produce changes in apoptosis and stress processes, although it is also possible that Gp93 alteration could be related to the immune system since it is homologous to human protein Gp96, which has been related with Toll-like receptors. In conclusion, cadmium and copper mixtures can affect the population by affecting the ability of larvae to respond to the infection and the apoptosis, an important process in the metamorphosis of insects.

Ultraviolet filters and heat shock proteins: effects in Chironomus riparius by benzophenone-3 and 4-methylbenzylidene camphor.

Benzophenone-3 (BP3) and 4-methylbenzylidene camphor (4MBC) are common ultraviolet filters (UV filters), compounds considered as emergent contaminants, used in different products like plastics and personal care products. The levels of these compounds are rising in the wild, but the effects they have on invertebrates are poorly understood. Chironomus riparius is a benthic insect widely used in toxicology, and several studies have been previously performed in our laboratory to determine the effects these compounds have on this organism at the molecular level. We have shown that UV filters can alter the mRNA levels of heat shock protein 70 (Hsp70), one of the most studied heat shock proteins. Although these proteins are crucial for the survival of organisms, little data is available on the effects these emergent contaminants have on them, especially in invertebrates. Here, we analyzed the transcriptional activity of 12 genes covering the different groups of heat shock protein [Hsp10, Hsp17, Hsp21, Hsp22, Hsp23, Hsp24, Hsp27, Hsp34, Hsp40, Hsp60, Hsc70 (3), and Hsc70 (4)] in response to 0.1 and 1 mg/L concentrations of BP3 and 4MBC at 8 and 24 h. The results showed that some small Hsp (sHsp) genes were altered by these compounds, while the genes of proteins present in mitochondria, Hsp10 and Hsp60, did not change. sHsps are also involved in developmental processes, so the observed variations could be due to the endocrine disruption activity described for these compounds rather than to a stress response.

Cadmium alters the expression of small heat shock protein genes in the aquatic midge Chironomus riparius.

Cadmium (Cd) is a widespread and highly toxic heavy metal of particular ecotoxicological relevance for aquatic ecosystems. It occurs naturally in the environment but is also an industrial pollutant with extensively researched carcinogenic potentials. Heat shock proteins (HSPs) are chaperones that play an important role in maintaining protein homeostasis under stress conditions. Small heat shock proteins (sHSPs) comprise the most diverse group of the HSPs family. They are expressed both constitutively and by stress-induction. The midge Chironomus riparius is widely used as a test species in aquatic toxicology. In the present study, Reverse Transcription Polymerase Chain Reaction (RT-PCR) was used to evaluate the effects of acute Cd exposure to the expression profile of seven shsp genes (hsp17, hsp21, hsp22, hsp23, hsp24, hsp27, and hsp34) in C. riparius larvae. Results show a specific pattern of response with a rapid response by hsp27, which was downregulated at 2-6 h, while the rest of the shsp genes remained unaltered except for hsp17 at 2 h, which was upregulated. However, at 24 h of exposure are observed high levels of hsp23, hsp24, hsp27, and hsp34 transcription while hsp22 mRNA levels were downregulated and hsp17 and hsp21 remained unaltered. These changes in gene expression suggest a functional diversity between the sHSPs in the cellular response to heavy metal stress. The differential pattern in comparison with heat shock supports a specific profile depending on the stress supporting the use of shsp genes as suitable biomarkers for ecotoxicological studies on aquatic systems.

Characterization of six small HSP genes from Chironomus riparius (Diptera, Chironomidae): Differential expression under conditions of normal growth and heat-induced stress.

Small heat shock proteins (sHSPs) comprise the most numerous, structurally diverse, and functionally uncharacterized family of heat shock proteins. Several Hsp genes (Hsp 90, 70, 40, and 27) from the insect Chironomus riparius are widely used in aquatic toxicology as biomarkers for environmental toxins. Here, we conducted a comparative study and characterized secondary structure of the six newly identified sHsp genes Hsp17, Hsp21, Hsp22, Hsp23, Hsp24, and Hsp34. A characteristic α-crystallin domain is predicted in all the new proteins. Phylogenetic analysis suggests a strong relation to other sHSPs from insects and interesting evidence regarding evolutionary origin and duplication events. Comparative analysis of transcription profiles for Hsp27, Hsp70, and the six newly identified genes revealed that Hsp17, Hsp21, and Hsp22 are constitutively expressed under normal conditions, while under two different heat shock conditions these genes are either not activated or are even repressed (Hsp22). In contrast, Hsp23, Hsp24, and Hsp34 are significantly activated along with Hsp27 and Hsp70 during heat stress. These results strongly suggest functional differentiation within the small HSP subfamily and provide new data to help understand the coping mechanisms induced by stressful environmental stimuli.

Experimental infection of young adult European breed sheep with Rift Valley fever virus field isolates.

The increasing interest in Rift Valley fever virus (RVFV) and its potential impact on naive animal populations deserve revisiting experimental reproduction of RVFV infection, particularly in those animal breeds for which no data about their susceptibility to RVFV infection have ever been recorded. In this study we show the susceptibility of 9-10 weeks old European sheep (Ripollesa breed) to RVFV infection, showing a mild, subacute form of disease. Four different viral isolates efficiently replicated in vivo after subcutaneous experimental inoculation, and consistent viral loads in blood and virus shedding (variable in length depending on the RVFV isolate used) were detected, showing horizontal transmission to a noninfected, sentinel lamb. RVFV infection caused transient pyrexia in adult lambs and no other clinical symptoms were observed, with the exception of corneal opacity ("blue eye") found in 3 out of 16 subcutaneously inoculated sheep. In conclusion, adult sheep from this European breed are readily infected with RVFV without apparent clinical manifestations.

Development and characterization of monoclonal antibodies against Rift Valley fever virus nucleocapsid protein generated by DNA immunization.

This paper describes the generation of monoclonal antibodies directed to immunogenic nucleoprotein N epitopes of Rift Valley fever virus (RVFV), and their application in diagnostics, both for antibody detection in competitive ELISA and for antigen capture in a sandwich ELISA. Monoclonal antibodies (mAbs) were generated after DNA immunization of Balb/c mice and characterized by western blot, ELISA and cell immunostaining assays. At least three different immunorelevant epitopes were defined by mAb competition assays. Interestingly, two of the mAbs generated were able to distinguish between RVFV strains from Egyptian or South African lineages. These monoclonal antibodies constitute useful tools for diagnosis, especially for the detection of serum anti-RVFV antibodies from a broad range of species by means of competitive ELISA.

Protection against lethal Rift Valley fever virus (RVFV) infection in transgenic IFNAR(-/-) mice induced by different DNA vaccination regimens.

In this work, plasmid constructs encoding two different M segment ORFs, as well as the nucleoprotein N, have been used in different vaccination regimes to test protection against a RVFV-MP12 virus challenge in a transgenic mouse model with impaired interferon type I response (IFNAR(-/-)). We obtained dose dependent protection in animals immunized with a construct encoding both mature glycoproteins (pCMV-M4), whereas only partial protection in animals vaccinated with either N construct (pCMV-N) or a combination of both plasmids (pCMV-M4+pCMV-N). The protection elicited by the expression of the mature glycoproteins could be directly related to the induction of neutralizing antibodies against them. Interestingly, the combination of both vaccine constructs induced specific lymphoblast proliferation upon stimulation with a recombinant nucleoprotein.

Priming with DNA plasmids encoding the nucleocapsid protein and glycoprotein precursors from Rift Valley fever virus accelerates the immune responses induced by an attenuated vaccine in sheep.

In this work we tested the ability of plasmid DNA constructs encoding structural Rift Valley fever virus (RVFV) antigens to induce specific immune responses in sheep. The sole immunization of DNA constructs encoding the glycoprotein precursor NSm/G2/G1 did not suffice to induce a detectable antibody response. In contrast, immunization of sheep with a plasmid vector encoding the viral nucleocapsid protein N elicited a potent and long lasting induction of antibodies but with low neutralizing titers. After DNA immunization, no antigen-specific proliferating cells were detected in sheep PBLs. Boosting with the attenuated vaccine strain MP12 was able to increase the levels of proliferating memory cell pools and induction of IFN-gamma in response to purified virus or recombinant proteins, particularly in sheep vaccinated with a combination of both plasmid constructs. These results open the possibility to exploit this strategy to improve the induction of immune responses against RVFV in sheep.