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Phenols are regarded as highly toxic chemicals. Their effects are difficult to study in in vitro systems because of their ambiguous fate (degradation, auto-oxidation and volatility). In the course of in vitro studies of a series of redox-cycling phenols, we found evidences of cross-contamination in several in vitro high-throughput test systems, in particular by trimethylbenzene-1, 4-diol/trimethylhydroquinone (TMHQ) and 2,6-di-tertbutyl-4-ethylphenol (DTBEP), and investigated in detail the physicochemical basis for such phenomenon and how to prevent it. TMHQ has fast degradation kinetics followed by significant diffusion rates of the resulting quinone to adjacent wells, other degradation products being able to air-diffuse as well. DTBEP showed lower degradation kinetics, but a higher diffusion rate. In both cases the in vitro toxicity was underestimated because of a decrease in concentration, in addition to cross-contamination to neighbouring wells. We identified four degradation products for TMHQ and five for DTBEP indicating that the current effects measured on cells are not only attributable to the parent phenolic compound. To overcome these drawbacks, we investigated in detail the physicochemical changes occurring in the course of the incubation and made use of gas-permeable and non-permeable plastic seals to prevent it. Diffusion was greatly prevented by the use of both plastic seals, as revealed by GC–MS analysis. Gas non-permeable plastic seals, reduced to a minimum compounds diffusion as well oxidation and did not affect the biological performance of cultured cells. Hence, no toxicological cross-contamination was observed in neighbouring wells, thus allowing a more reliable in vitro assessment of phenol-induced toxicity., The authors wish to acknowledge the support of the European Union’s Horizon 2020 research and innovation 493 program under Grant Agreement No 681002 (EU-ToxRisk). We are indebted to CIBEREHD (ISCIII), for supporting our research program on hepatotoxicity of xenobiotics. L.T. was supported by the Miguel 494 Servet I Program financed by the Institute of Health Carlos III (Plan Estatal de I+D+i 2013-2016) 495 and co-financed by the European Regional Development Fund "A way to achieve Europe" (FEDER) 496 through grant CP16/00097. TMS acknowledges her personal predoctoral grant (FI18/00260, 497 ISCIII, Spain).

Microplastics (MPs) represent a worldwide emerging relevant concern toward human and environmental health due to their intentional or unintentional release. Human exposure to MPs by inhalation is predicted to be among the most hazardous. MPs include both engineered, or primary MPs, and secondary MPs, materials obtained by fragmentation from any plastic good. The major part of the environmental MPs is constituted by the second ones that are irregular in size, shape and composition. These features make the study of the biological impact of heterogenous MPs of extremely high relevance to better estimate the real toxicological hazards of these materials on human and environmental organisms. The smallest fractions of plastic granules, relying on the micron-sized scale, can be considered as the most abundant component of the environmental MPs, and for this reason, they are typically used to perform toxicity tests using in vitro systems representative of an inhalation exposure scenario. In the present work, MPs obtained from industrial treatment of waste plastics (wMPs < 50 μm) were investigated, and after the physico-chemical characterization, the cytotoxic, inflammatory and genotoxic responses, as well as the modality of wMPs interactions with alveolar lung cells, were determined. Obtained results indicated that, at high concentrations (100 μg/ml) and prolonged exposure time (48 h), wMPs affect biological responses by inducing inflammation and genotoxicity, as a result of the cell–wMP interactions, also including the uptake of the smaller particles.

Road traffic is one of the main sources of particulate emissions into the environment and has an increasing, negative impact on the release of potentially dangerous materials. Vehicle brakes release a significant amount of wear particles, and knowledge regarding their possible adverse effects is limited. One of the most dangerous elements contained in brake pads is copper (Cu), known to be toxic for human health. Therefore, our aim was to study the cell toxicity of particulate matter (PM) produced by different combinations of braking discs and pads containing different amounts of Cu. We investigated whether brake-derived microparticles have toxic effects on lung cells proportionally to their Cu content. Analyte content was measured in friction materials by XRFS and in PM2.5 captured during braking tests using SEM/EDX. The biological impact of brake-derived PM2.5 was investigated on a human epithelial alveolar cell line (A549). Cell viability, oxidative stress, mitochondrial membrane potential, apoptosis, and the pro-inflammatory response of the cells, as well as gene expression, were assessed following exposure to increasing PM2.5 concentrations (1, 10, 100, 200, and 500 µg/ml). The brake debris with the lowest Cu content did not induce significant changes in biological effects on A549 cells compared to normal controls, except for ROS production and IL6 gene expression. PM2.5 containing higher Cu quantities induced cell toxicity that correlated with Cu concentration. Our data suggest that the toxicity of PM2.5 from the brake system is mainly related to Cu content, thus confirming that eliminating Cu from brake pads will be beneficial for human health in urbanized environments.

The use of tobacco products is a major global public health issue, as it is the leading cause of preventable death worldwide. In addition, nicotine (NIC) is a key component of electronic and conventional cigarettes. Although nicotine’s addictive potential is well known, its health effects are not entirely understood. Thus, the main objective of the present study was to evaluate its toxicological profile both in vitro, at the level of three healthy cell lines, and in ovo, at the level of the chorioallantoic membrane. Five different concentrations of nicotine were used in keratinocytes, cardiomyocytes, and hepatocytes for the purpose of evaluating cell viability, cell morphology, and its impact on nuclei. Additionally, the hen’s egg test on the chorioallantoic membrane (HET-CAM) method was used to assess the biocompatibility and irritant potential of the chorioallantoic membrane. Across all cell lines studied, nicotine was proven to be significantly damaging to cell viability, with the highest concentration tested resulting in less than 2% viable cells. Moreover, the morphology of cells changed dramatically, with alterations in their shape and confluence. Nicotine-induced cell death appears to be apoptotic, based on its impact on the nucleus. In addition, nicotine was also found to have a very strong irritating effect on the chorioallantoic membrane. In conclusion, nicotine has an extremely strong toxicological profile, as demonstrated by the drastic reduction of cell viability and the induction of morphological changes and nuclear alterations associated with cellular apoptosis. Additionally, the HET-CAM method led to the observation of a strong irritating effect associated with nicotine.

Information about the potential oral health effects of vaping from electronic cigarettes (e-cigs) is still sparse and inconsistent. The purpose of this study was to compare the safety and cytotoxicity of e-cig liquid aerosols versus traditional cigarette (t-cig) smoke on human epithelial oral cells. T-cig smoke and e-cig aerosols were generated by a newly developed automated smoking instrument in order to simulate realistic user puffing behaviors. Air–liquid interface transwell cell cultures were exposed to standardized puff topography (puff duration: 2 s, puff volume: 35 mL, puff frequency: 1 puff every 60 s) of reference t-cigs or commercially available e-cigs at different air dilutions. Cell viability, morphology, and death rate were evaluated with MTT and TUNEL assays. The inflammatory cytokine gene expression of inflammatory genes was assessed by quantitative RT-PCR. E-cigs and t-cigs indicated similar adverse effects by enhancing cytotoxicity and cell death in a dose-dependent manner. E-cig aerosol and t-cig smoke treatment expressed upregulation of inflammatory cytokines up to 3.0-fold (p < 0.05). These results indicate that e-cig smoking may contribute to oral tissue–cell damage and tissue inflammation. Our approach allows the production of e-cig aerosol and t-cig smoke in order to identify harmful effects in oral tissues in vitro.

Propylene glycol (PG) and glycerin (G) are the most widely used humectants in electronic nicotine delivery system (ENDS) devices. Carbonyls are present in aerosols produced when ENDS devices heat PG and G. Whether aerosolized PG and G are innocuous to the lungs has not been established. Here, we determined the chemical profiles of ENDS aerosols containing three humectant ratios (30/70, 50/50 and 70/30, PG/VG), for three flavors (strawberry, vanilla and Catalan cream) containing either 12 or 18 mg/mL of nicotine. Additionally, we examined the in vitro toxicity of the strawberry- and vanilla-flavored ENDS aerosol in human lung epithelial cells (BEAS-2B) exposed at the air-liquid interface for 1 h. For strawberry- and vanilla-flavored aerosols produced by a 3rd-generation ENDS device with the same PG/G ratio, the e-liquid nicotine content of 12 and 18 mg/mL did not transfer to the aerosol with substantial differences in concentrations. Our data also indicate the presence of carbonyls in all three flavored e-cig aerosols analyzed, with levels exceeding 1 µg/puff for acetone, butyraldehyde, and acetaldehyde, in strawberry-, vanilla, and Catalan cream-flavored e-cig aerosols, respectively. Furthermore, closed-system ENDS of the fourth generation emitted trace levels of carbonyls in the aerosols (1 µg/puff), formaldehyde (>5 µg/puff), and m- & p-tolualdehyde (>4 µg/puff). Moreover, under non-cytotoxic conditions, BEAS-2B cells exposed to strawberry-flavored aerosols exhibited significantly increased reactive oxygen and nitric oxide species (ROS/NOS) levels in cell media compared to air controls, while vanilla-flavored ENDS aerosols up-regulated the expression of pro-inflammatory and oxidative stress markers. Our data suggest (a) that ENDS aerosol chemical composition will vary based upon the presence and concentration of the initial e-liquid ingredients, with a pronounced impact of the flavoring components; and (b) short-term exposures to flavored ENDS aerosols may impair lung cells’ redox signaling in a flavor-specific manner.

Among environmental endocrine-active chemicals, phthalates, commonly known as plasticizers, disrupt the development of the male reproductive tract. In this study, the effects of phthalates (DIBP, BBP, DINP, DBP, DEP, DEHP and DMP) were evaluated on cultures of bovine primary male reproductive cells (n = 3) and spermatozoa (n = 4). Epididymal (caput and corpus epididymis), testicular (parenchymal and mediastinal/tubular) and vas deferens cells (VDC) were prepared from samples collected from slaughterhouse. Second part of caput epididymis which have fewer amount of principal cells, were found to be less affected compared to the first part except DEHP; while corpus epididymis was found to be more affected with IC(50) values below 0.976 ng/mL (except for DEP at 4.97 ng/mL). In testicular parenchymal cells, IC(50) ranged from 0.15 to 4.11 ng/mL and for mediastinum from 0.01 to 7.31 ng/mL; where cytotoxic effects were more evident in mediastinal section. Least cytotoxic and even proliferational effects (DEHP, DMP and DEP) were observed in VDC, the muscular tube carrying sperm from epididymis to the ejaculatory duct. Least spermiotoxic phthalate was DBP (3.928 ng/mL); while DINP (0.550 ng/mL) induced highest cytotoxic effect on bovine spermatozoa. Differences in the cellular structure and/or the androgen receptor distribution effect the toxicity of phthalates. Our preliminary findings on bovine spermatozoa indicate possible morphological and motility alterations; which challenges further investigation of the transition of phthalates on semen straws used in cryopreservation. Increase of exposure to environmental contaminants raise the issue of the requirement of a new perspective on reproductive health, species and tissue specific differences should further be emphasized.

Phenols are regarded as highly toxic chemicals. Their effects are difficult to study in in vitro systems because of their ambiguous fate (degradation, auto-oxidation and volatility). In the course of in vitro studies of a series of redox-cycling phenols, we found evidences of cross-contamination in several in vitro high-throughput test systems, in particular by trimethylbenzene-1, 4-diol/trimethylhydroquinone (TMHQ) and 2,6-di-tertbutyl-4-ethylphenol (DTBEP), and investigated in detail the physicochemical basis for such phenomenon and how to prevent it. TMHQ has fast degradation kinetics followed by significant diffusion rates of the resulting quinone to adjacent wells, other degradation products being able to air-diffuse as well. DTBEP showed lower degradation kinetics, but a higher diffusion rate. In both cases the in vitro toxicity was underestimated because of a decrease in concentration, in addition to cross-contamination to neighbouring wells. We identified four degradation products for TMHQ and five for DTBEP indicating that the current effects measured on cells are not only attributable to the parent phenolic compound. To overcome these drawbacks, we investigated in detail the physicochemical changes occurring in the course of the incubation and made use of gas-permeable and non-permeable plastic seals to prevent it. Diffusion was greatly prevented by the use of both plastic seals, as revealed by GC–MS analysis. Gas non-permeable plastic seals, reduced to a minimum compounds diffusion as well oxidation and did not affect the biological performance of cultured cells. Hence, no toxicological cross-contamination was observed in neighbouring wells, thus allowing a more reliable in vitro assessment of phenol-induced toxicity.

Faster, cheaper, sensitive, and mechanisms-based animal alternatives are needed to address the safety assessment needs of the growing number of nanomaterials (NM) and their sophisticated property variants. Specifically, strategies that help identify and prioritize alternative schemes involving individual test models, toxicity endpoints, and assays for the assessment of adverse outcomes, as well as strategies that enable validation and refinement of these schemes for the regulatory acceptance are needed. In this review, two strategies 1) the current nanotoxicology literature review and 2) the adverse outcome pathways (AOPs) framework, a systematic process that allows the assembly of available mechanistic information concerning a toxicological response in a simple modular format, are presented. The review highlights 1) the most frequently assessed and reported ad hoc in vivo and in vitro toxicity measurements in the literature, 2) various AOPs of relevance to inhalation toxicity of NM that are presently under development, and 3) their applicability in identifying key events of toxicity for targeted in vitro assay development. Finally, using an existing AOP for lung fibrosis, the specific combinations of cell types, exposure and test systems, and assays that are experimentally supported and thus, can be used for assessing NM-induced lung fibrosis, are proposed.

The uncertainty of potential risks associated with micro- and nanoplastics (MNPs) are of growing public concern. However, the diversity of MNPs in the environment makes a systematic analysis of potential health effects challenging. New tools and approaches are necessary to investigate biological effects of MNPs. With this quick scoping review, we aim to analyse the suitability of in vitro models for assessing the interaction of MNPs with intestinal cells. Our analysis revealed that currently the majority of in vitro tests are based on the three cell lines Caco-2, HT-29, and HCT-116. They have particularly been used to assess endpoints related to basal cytotoxicity, the internalisation of MNPs and effects on the intestinal barrier. When co-cultured with various cell lines, they also allow to investigate additional effects such as inflammation, metabolic actions and the relevance of the intestinal mucus. However, methodological gaps remain regarding the assessment of a potential accumulation of MNPs, leaching of additives/impurities and in resulting long-term effects as well as cell-type specific toxicities. In addition, only few in vitro studies investigated effects of MNPs on the microbiome. Stem cell-based assays using, for example, the emerging organoid technology are promising for analysing MNP effects on tissue-like structures, while avoiding the particular characteristics of the currently used cancer derived cell lines. The various cell lines and culture techniques can be combined in testing strategies, to better elucidate potential biological interaction of MNPs with biological systems. We suggest to implement a tiered testing strategy, in which monocultures can serve as a tool for high-throughput testing of MNPs. In the next steps co-cultures can be used to assess the potential of a systemic uptake of MNPs and organ-on-a-chip models will provide more reliable insights into relevant doses triggering biological effects. Finally, organoids can help to discover new and more complex reactions initiated by MNPs.

Chemical safety assessment requires information on both chronic and acute effects of toxicants. Traditionally, such information has been provided by a set of animal studies conducted over different durations, ranging from a single dose with observation of effects over a few days, to repeat daily dosing and observations made over many months. With the advent of modern mechanistic approaches to toxicology, the role of in vitro studies within alternative approaches has never been more prominent. Typical in vitro experiments are conducted over short durations with measurements of response at a single time point, with a focus on providing effect and concentration-response information as input to hazard and risk assessment. This limits the usefulness of such data since potential chronic effects that cumulate over time are not usually considered. To address this, an experimental design is presented to characterise the toxicodynamics of a response not only in terms of concentration, but also as a function of time. Generation of concentration-time-effect responses allows both the extrapolation of points of departure from an acute to chronic exposure, and the determination of a chronicity index that provides a quantitative measure of a chemical's potential to cause cumulative effects over time. In addition, the approach provides a means to characterise the dynamics of key event relationships for the development of quantitative adverse outcome pathways., Highlights • Extrapolation of points-of-departure from acute to chronic exposure in vitro through time-concentration-response analysis • Introduction of a chronicity index as a quantitative measure of the cumulative effects a chemical can cause over time • Experimental approach to characterise the dynamics of key event relationships for quantitative Adverse Outcome Pathways

Copper mining generates large quantities of waste, tailings, and acid outflows causing long-term environmental impacts and potential threats to human health. Valea Şesei is the largest tailing impoundment in Romania, created by flooding the valley (known as Valea Şesei) of the Metalliferous Mountains (a division of the Apuseni Mountains) with copper mining waste. The present study (i) estimated the total volume of tailings in this area; (ii) screened the concentration of 65 elements (rare earth and platinum group elements, alkali metals and alkali earth metals, transition and post-transition metals and metalloids) and cyanide concentrations in wastewater samples collected from tailing impoundment; (iii) evaluated the toxicity of these water samples using five in vitro bioassays employing human cells isolated from healthy donors and a short-term (1 h) exposure model. The sampled waters were highly acidic (pH 2.1–4.9) and had high electrical conductivity (280–1561 mS cm−1). No cyanides were detected in any sample. Water samples collected from the stream (AMD) inflowing to the tailing impoundment were characterized by the greatest concentrations of alkali metals, alkaline earth metals, transition and post-transition metals, metalloids, rare earth elements, and noble metal group. At other sites, the elemental concentrations were lower but remained high enough to pose a relevant risk. The greatest magnitude of in vitro toxic effects was induced by AMD. Observed alterations included redox imbalance in human neutrophils followed by lipid peroxidation and decreased cell survival, significant aggregation of red blood cells, and increased prothrombin time. The study highlights that Valea Şesei is a large sink for toxic elements, posing environmental and health risks, and requiring action to prevent further release of chemicals and to initiate restoration of the area. Electronic supplementary material The online version of this article (doi:10.1007/s11356-017-9782-y) contains supplementary material, which is available to authorized users.

Aphanizomenon gracile and Cylindrospermopsis raciborskii are extensively studied Nostocales of wide geographical distribution and have potential to produce toxins. However, a number of knowledge gaps regarding their toxicity and related health risks in certain locations, including Europe, exists. The present study applied a polyphasic approach to screen the toxicity of different strains of C. raciborskii (LBY-Cr, LBO-Cr and LKM-Cr) and A. gracile (LBY-Ag, LBN-Ag and LWI-Ag) isolated from five freshwater lakes of Western Poland. The following investigations were carried out: (i) in vitro toxicological studies employing human cells isolated from healthy donors; (ii) analytical screening for the presence of cylindrospermopsin (CYN), guanidinoacetate (GAA; initial CYN precursor and postulated general cyanobacterial metabolite), three microcystin (MC) analogues, β- N -methylamino- l -alanine (BMAA) and its isomer α-γ,-diaminobutyric acid (DAB), anatoxin-a (ATX) and ten saxitoxin (STX) analogues; and (iii) molecular studies of genes involved in CYN, GAA, MCs and ATX biosynthesis. Extracts of C. raciborskii LBY-Cr and A. gracile LBN-Ag caused a significant increase in the intracellular reactive oxygen content in human neutrophils during short-term (1 h) exposure and also led to lipid peroxidation and cell death. No cytotoxic effects were noted for the other tested strains. None of the toxin genes ( cyrA , cyrJ , anaF and mcyE ) and toxins (CYN, GAA, MCs, BMAA, ATX and STX) were detected. The only exception was DAB found at a concentration below 1.0 μg g − 1 dw in A. gracile LWI-Ag. It is the first time that cyanobacterial DAB producer has been identified in the Central European region. The study points to the production of as yet unknown metabolite(s) that may pose a relevant threat to human health through strains of C. raciborskii and A. gracile isolated from two Polish lakes, and adds to the general understanding of the toxicity of European strains of both species.

Synthetic polymer-based materials are ubiquitous in aquatic environments, where weathering processes lead to their progressive fragmentation and the leaching of additive chemicals. The current study assessed the chemical content of freshwater and marine leachates produced from car tire rubber (CTR), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS) and polyvinyl chloride (PVC) microplastics, and their adverse effects on the microalgae Raphidocelis subcapitata (freshwater) and Skeletonema costatum (marine) and the Mediterranean mussel Mytilus galloprovincialis. A combination of non-target and target chemical analysis revealed a number of organic and metal compounds in the leachates, including representing plasticizers, antioxidants, antimicrobials, lubricants, and vulcanizers. CTR and PVC materials and their corresponding leachates had the highest content of tentatively identified organic additives, while PET had the lowest. The metal content varied both between polymer leachates and between freshwater and seawater. Notable additives identified in high concentrations were benzothiazole (CTR), phthalide (PVC), acetophenone (PP), cobalt (CTR, PET), zinc (CTR, PVC), lead (PP) and antimony (PET). All leachates, except PET, inhibited algal growth with EC50 values ranging from 0.5% (CTR) and 64% (PP) of the total leachate concentration. Leachates also affected mussel endpoints, including the lysosomal membrane stability and early stages endpoints as gamete fertilization, embryonic development and larvae motility and survival. Embryonic development was the most sensitive parameter in mussels, with EC50 values ranging from 0.8% (CTR) to 65% (PET) of the total leachate. The lowest impacts were induced on D-shell larvae survival, reflecting their ability to down-regulate motility and filtration in the presence of chemical stressors. This study provides evidence of the relationship between chemical composition and toxicity of plastic/rubber leachates. Consistent with increasing contamination by organic and inorganic additives, the leachates ranged from slightly to highly toxic to mussels and algae, highlighting the need for a better understanding of the overall impact of plastic-associated chemicals on aquatic ecosystems.

The enormous technological relevance of titanium dioxide (TiO2) nanoparticles (NPs) and the consequent concerns regarding potentially hazardous effects that exposure during production, use, and disposal can generate, encourage material scientists to develop and validate intrinsically safe design solution (safe-by-design). Under this perspective, the encapsulation in a silica dioxide (SiO2) matrix could be an effective strategy to improve TiO2 NPs safety, preserving photocatalytic and antibacterial properties. In this work, A549 cells were used to investigate the toxic effects of silica-encapsulated TiO2 having different ratios of TiO2 and SiO2 (1:1, 1:3, and 3:1). NPs were characterized by electron microscopy and dynamic light scattering, and cell viability, oxidative stress, morphological changes, and cell cycle alteration were evaluated. Resulting data demonstrated that NPs with lower content of SiO2 are able to induce cytotoxic effects, triggered by oxidative stress and resulting in cell necrosis and cell cycle alteration. The physicochemical properties of NPs are responsible for their toxicity. Particles with small size and high stability interact with pulmonary cells more effectively, and the different ratio among silica and titania plays a crucial role in the induced cytotoxicity. These results strengthen the need to take into account a safe(r)-by-design approach in the development of new nanomaterials for research and manufacturing.

A MIL series metal‒organic framework (MOF), MIL-100(Fe), was successfully synthesized at the nanoscale and fully characterized by TEM, TGA, XRD, FTIR, DLS, and BET. A toxicological assessment was performed using two different cell lines: human normal liver cells (HL-7702) and hepatocellular carcinoma (HepG2). In vitro cytotoxicity of MIL-100(Fe) was evaluated by the MTT assay, LDH releasing rate assay, DAPI staining, and annexin V/PI double staining assay. The safe dose of MIL-100(Fe) was 80 μg/mL. It exhibited good biocompatibility, low cytotoxicity, and high cell survival rate (HL-7702 cells’ viability &gt, 85.97%, HepG2 cells’ viability &gt, 91.20%). Therefore, MIL-100(Fe) has a potential application as a drug carrier.

The health hazard represented by the exposure to asbestos may also concern other minerals with asbestos-like crystal habit. One of these potentially hazardous minerals is fibrous glaucophane. Fibrous glaucophane is a major component of blueschist rocks of California (USA) currently mined for construction purposes. Dust generated by the excavation activities might potentially expose workers and the general public. The aim of this study was to determine whether fibrous glaucophane induces in vitro toxicity effects on lung cells by assessing the biological responses of cultured human pleural mesothelial cells (Met-5A) and THP-1 derived macrophages exposed for 24 h and 48 h to glaucophane fibres. Crocidolite asbestos was tested for comparison. The experimental configuration of the in vitro tests included a cell culture without fibres (i.e., control), cell cultures treated with 50 μg/mL (i.e., 15.6 μg/cm2) of crocidolite fibres and 25-50−100 μg/mL (i.e., 7.8−15.6–31.2 μg/cm2) of glaucophane fibres. Results showed that fibrous glaucophane may induce a decrease in cell viability and an increase in extra-cellular lactate dehydrogenase release in the tested cell cultures in a concentration dependent mode. Moreover, it was found that fibrous glaucophane has a potency to cause oxidative stress. The biological reactivity of fibrous glaucophane confirms that it is a toxic agent and, although it apparently induces lower toxic effects compared to crocidolite, exposure to this fibre may be responsible for the development of lung diseases in exposed unprotected workers and population.

Silver selenide (Ag2Se) is a promising nanomaterial due to its outstanding optoelectronic properties and countless bio-applications. To the best of our knowledge, we report, for the first time, a simple and easy method for the ultrasound-assisted synthesis of Ag2Se nanoparticles (NPs) by mixing aqueous solutions of silver nitrate (AgNO3) and selenous acid (H2SeO3) that act as Ag and Se sources, respectively, in the presence of dissolved fructose and starch that act as reducing and stabilizing agents, respectively. The concentrations of mono- and polysaccharides were screened to determine their effect on the size, shape and colloidal stability of the as-synthesized Ag2Se NPs which, in turn, impact the optical properties of these NPs. The morphology of the as-synthesized Ag2Se NPs was characterized by transmission electron microscopy (TEM) and both &alpha, and &beta, phases of Ag2Se were determined by X-ray diffraction (XRD). The optical properties of Ag2Se were studied using UV&ndash, Vis spectroscopy and its elemental composition was determined non-destructively using scanning electron microscopy&ndash, energy-dispersive spectroscopy (SEM&ndash, EDS). The biological activity of the Ag2Se NPs was assessed using cytotoxic and bactericidal approaches. Our findings pave the way to the cost-effective, fast and scalable production of valuable Ag2Se NPs that may be utilized in numerous fields.

Diesel exhaust particles (DEP) are responsible for both respiratory and cardiovascular effects. However many questions are still unravelled and the mechanisms behind the health effects induced by the exposure to ultrafine particles (UFP) need further investigations. Furthermore, different emission sources can lead to diverse biological responses. In this perspective, here we have compared the effects of three DEPs, two standard reference materials (SRM 1650b and 2975) and one DEP directly sampled from a EuroIV vehicle without Diesel Particulate Filter (DPF). For the biological investigations, different in vitro lung models involving both epithelial and vascular endothelial cells, were used. Cell viability, oxidative stress, inflammation, DNA damage and endothelial activation markers were investigated at sub-cytotoxic DEP doses. The data obtained have shown that only DEP EuroIV, which had the major content of polycyclic aromatic hydrocarbons (PAHs) and metals, was able to induce oxidative stress, inflammation and consequent endothelial activation, as demonstrated by the expression of adhesion molecules (ICAM-1 and VCAM-1) and the release of inflammatory markers (IL-8) from endothelial cells. Standard reference materials were not effective under our experimental conditions. These data suggest that oxidative stress, endothelial activation and systemic inflammatory cytokines release are crucial events after DEP exposure and that the source of DEP emission, responsible of the particle chemical fingerprint, may have a key role in the resulting adverse biological outcomes.

Silver nanoparticles (AgNPs) have important antimicrobial activity. These nanoparticles are present in several commercial products, leading to human and environmental exposure. This review presents a critical view of the toxic effects of Ag NPs on in vitro and in vivo biological models. It is noteworthy that the toxicity of AgNPs is dependent on several parameters, such as the preparation mode of the colloidal suspension of the nanoparticles, their state of aggregation, the chemical nature of the nanoparticles, surface size, morphology, dose, cell type, nature of the living organism, among others. In general, low concentrations of AgNPs do not present significant toxicity in in vitro and in vivo assays, however with the increase of the concentration of AgNPs there is a significant increase of nanoparticle toxicity and its accumulation in diverse tissues/organs. It should be emphasized that in living organisms, exposure routes should be taken into account, such as ingestion and inhalation. At high concentrations, inhalation or ingestion of AgNPs can cause adverse effects and even lead to human death. In this context, the importance of further studies of the toxicity of AgNPs in several in vitro and in vivo models is emphasized.

The zero (0-D) and one-dimensional (1-D) carbon nanomaterials have gained attention among researchers because they exhibit a larger surface area to volume ratio, and a smaller size. Furthermore, carbon is ubiquitously present in all living organisms. However, toxicity is a major concern while utilizing carbon nanomaterials for biomedical applications such as drug delivery, biosensing, and tissue regeneration. In the present review, we have summarized some of the recent findings of cellular and animal level toxicity studies of 0-D (carbon quantum dot, graphene quantum dot, nanodiamond, and carbon black) and 1-D (single-walled and multi-walled carbon nanotubes) carbon nanomaterials. The in vitro toxicity of carbon nanomaterials was exemplified in normal and cancer cell lines including fibroblasts, osteoblasts, macrophages, epithelial and endothelial cells of different sources. Similarly, the in vivo studies were illustrated in several animal species such as rats, mice, zebrafish, planktons and, guinea pigs, at various concentrations, route of administrations and exposure of nanoparticles. In addition, we have described the unique properties and commercial usage, as well as the similarities and differences among the nanoparticles. The aim of the current review is not only to signify the importance of studying the toxicity of 0-D and 1-D carbon nanomaterials, but also to emphasize the perspectives, future challenges and possible directions in the field.

Amphotericin B (AmB) is a very efficient drug against serious diseases such as leishmaniasis and systemic fungal infections. However, its oral bioavailability is limited due to its poor solubility in water. Nevertheless, it is marketed as high-cost lipid parenteral formulations that may induce serious infusion-related side effects. In this study, oil-in-water (O/W) microemulsions (MEs) were developed and characterized with a view to their use as solubility enhancers and oral delivery systems for AmB. Therefore, different nonionic surfactants from the Tween® and Span® series were tested for their solubilization capacity in combination with several oils. Based on pseudoternary phase diagrams, AmB-loaded MEs with mean droplet sizes about 120nm were successfully produced. They were able to improve the drug solubility up to 1000-fold. Rheological studies showed the MEs to be low-viscosity formulations with Newtonian behavior. Circular dichroism and absorption spectra revealed that part of the AmB in the MEs was aggregated as an AmB reservoir carrier. Cytotoxicity studies revealed limited toxicity to macrophage-like cells that may allow the formulations to be considered as suitable carriers for AmB.

Nanoparticles use in nano-biotechnology applications have increased significantly with Aminated polystyrene amine (AmPs NP), Zinc oxide (ZnO NP), and Silver (Ag NP) nanoparticles utilized in wide variety of consumer products. This has presented a number of concerns due to their increased exposure risks and associated toxicity on living systems. Changes in the structural and physicochemical properties of nanoparticles can lead to changes in biological activities. This study investigates, compares, and contrasts the potential toxicity of AmPs, ZnO and Ag NPs on an in vitro model (HeLa cells) and assesses the associated mechanism for their corresponding cytotoxicity relative to the surface material. It was noted that NPs exposure attributed to the reduction in cell viability and high-level induction of oxidative stress. All three test particles were noted to induce ROS to varying degrees which is irrespective of the attached surface group. Cell cycle analysis indicated a G2/M phase cell arrest, with the corresponding reduction in G0/G1 and S phase cells resulting in caspase-mediated apoptotic cell death. These findings suggest that all three NPs resulted in the decrease in cell viability, increase intracellular ROS production, induce cell cycle arrest at the G2/M phase and finally result in cell death by caspase-mediated apoptosis, which is irrespective of their differences in physiochemical properties and attached surface groups.

The present study was designed to evaluate and compare the time- and dose-dependent cellular response of human periodontal ligament fibroblasts (hPDLFs), and mouse dermal fibroblasts (mDFs) to three different types of nanoparticles (NPs); fullerenes (C60), single walled carbon nanotubes (SWCNTs) and iron (II,III) oxide (Fe3O4) nanoparticles via in vitro toxicity methods, and impedance based biosensor system. NPs were characterized according to their morphology, structure, surface area, particle size distribution and zeta potential by using transmission electron microscopy, X-ray diffraction, Brunauer–Emmett–Teller, dynamic light scattering and zeta sizer analyses. The Mössbauer spectroscopy was used in order to magnetically characterize the Fe3O4 NPs. The hPDLFs and mDFs were exposed to different concentrations of the NPs (0.1, 1, 10, 50 and 100 μg/mL) for predetermined time intervals (6, 24 and 48 h) under controlled conditions. Subsequently, NP exposed cells were tested for viability, membrane leakage and generation of intracellular reactive oxygen species. Additional to in vitro cytotoxicity assays, the cellular responses to selected NPs were determined in real time using an impedance based biosensor system. Taken together, information obtained from all experiments suggests that toxicity of the selected NPs is cell type, concentration and time dependent.

Magnetic oxide nanoparticles provide a fascinating tool for biological research and medicine, serving as contrast agents, magnetic carriers, and core materials of theranostic systems. Although the applications rely mostly on iron oxides, more complex oxides such as perovskite manganites may provide a much better magnetic performance. To assess the risk of their potential use, in vitro toxicity of manganite nanoparticles was thoroughly analysed and compared with another prospective system of Mn-Zn ferrite nanoparticles. Magnetic nanoparticles of La0.63Sr0.37 MnO3 manganite were prepared by two distinct methods, namely the molten salt synthesis and the traditional sol-gel route, whereas nanoparticles of Mn0.61Zn0.42Fe1.97O4 ferrite, selected as a comparative material, were synthesized by a new procedure under hydrothermal conditions. Magnetic cores were coated with silica and, moreover, several samples of manganite nanoparticles with different thicknesses of silica shell were prepared. The size-fractionated and purified products were analysed using transmission electron microscopy, dynamic light scattering, measurement of the zeta-potential dependence on pH, IR spectroscopy, and SQUID magnetometry. The silica-coated products with accurately determined concentration by atomic absorption spectroscopy were subjected to a robust evaluation of their cytotoxicity by four different methods, including detailed analysis of the concentration dependence of toxicity, analysis of apoptosis, and experiments on three different cell lines. The results, comparing two manganese-containing systems, clearly indicated superior properties of the Mn-Zn ferrite, whose silica-coated nanoparticles show very limited toxic effects and thus constitute a promising material for bioapplications. Magnetické oxidové nanočástice poskytují fascinující nástroj pro biologický výzkum a medicínu, slouží jako kontrastní látky, magnetické nosiče a materiály jádra teranostických systémů. Pro vyhodnocení rizika jejich potenciálního použití, in vitro toxicita manganitových nanočástic byla důkladně analyzována a porovnána s jiným potenciálním systémem Mn-Zn feritových nanočástic. Magnetické nanočástice La0,63Sr0,37MnO3 manganitu byly připraveny dvěma různými metodami, a to syntézou z taveniny a tradiční sol-gel cestou, zatímco nanočástice Mn0,61Zn0,42Fe1,97O4 feritu vybrané jako srovnávací materiál, byly syntetizovány novým postupem za hydrotermálních podmínek. Magnetická jádra byla obalena silikou a navíc několik vzorků manganitových nanočástic s různými tloušťkami silikové slupky bylo připraveno. Tyto silikou obalené produkty byly podrobeny důkladnému vyhodnocení jejich cytotoxicity různými metodami zahrnujícími podrobnou analýzu koncentrační závislost toxicity, analýzu apoptózy a experimentů na třech různých buněčných linií. Výsledky porovnání dvou systémů obsahujících mangan jasně indikovaly vynikající vlastnosti Mn-Zn feritu, jehož silikou obalené nanočástice vykazují velmi omezené toxické účinky, a tudíž představují slibný materiál pro bioaplikace.

There is a widespread awareness that the wealth of preclinical toxicity data that the pharmaceutical industry has generated in recent decades is not exploited as efficiently as it could be. Enhanced data availability for compound comparison ("read-across"), or for data mining to build predictive tools, should lead to a more efficient drug development process and contribute to the reduction of animal use (3Rs principle). In order to achieve these goals, a consortium approach, grouping numbers of relevant partners, is required. The eTOX ("electronic toxicity") consortium represents such a project and is a public-private partnership within the framework of the European Innovative Medicines Initiative (IMI). The project aims at the development of in silico prediction systems for organ and in vivo toxicity. The backbone of the project will be a database consisting of preclinical toxicity data for drug compounds or candidates extracted from previously unpublished, legacy reports from thirteen European and European operation-based pharmaceutical companies. The database will be enhanced by incorporation of publically available, high quality toxicology data. Seven academic institutes and five small-to-medium size enterprises (SMEs) contribute with their expertise in data gathering, database curation, data mining, chemoinformatics and predictive systems development. The outcome of the project will be a predictive system contributing to early potential hazard identification and risk assessment during the drug development process. The concept and strategy of the eTOX project is described here, together with current achievements and future deliverables.

A simple and green chemical method has been developed to synthesize stable bare and capped silver nanoparticles based on the reduction of silver ions by glucose and capping by poly(α,γ,L-glutamic acid) (PGA). The use of ammonia during synthesis was avoided. PGA has had a dual role in the synthesis and was used as a capping agent to make the silver nanoparticle more biocompatible and to protect the nanoparticles from agglomerating in the liquid medium. The synthesized PGA-capped silver nanoparticles in the size range 5–45 nm were stable over long periods of time, without signs of precipitation. Morphological examination has shown that the silver nanoparticles had a nearly spherical, multiply twinned structure. The effects of the reaction temperature and the reaction time during the synthesis were investigated too. The biocompatibility of the PGA-capped silver nano-particles is discussed in terms of in vitro toxicity with human intestinal Caco-2 cells. The samples were characterized by UV–Visible spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, and zeta potential measurements. Published version: [https://hdl.handle.net/21.15107/rcub_dais_487] This is the peer-reviewed version of the article: Stevanović, Magdalena, Savanović, Igor, Uskoković, Vuk, Škapin, Srečo Davor, Bračko, Ines, Jovanović, Uroš, Uskoković, Dragan, "A new, simple, green, and one-pot four-component synthesis of bare and poly(α,γ, L-glutamic acid)-capped silver nanoparticles" in Colloid and Polymer Science, 290, no. 3 (2012):221-231, [https://doi.org/10.1007/s00396-011-2540-7]

The development of carbon nanotubes (CNTs) for biomedical and biotechnological applications has gained great promise recently, especially for their interesting use in the delivery of therapeutically active molecules to targeted cells. The interaction between cells and this nanomaterial is a critical feature that is responsible for the pharmacological effect as well as for any eventual toxicity. With respect to the latter aspect, in this manuscript we have evaluated a few parameters that seem to be involved in the cytotoxic profile of CNTs (both Single-walled (SWCNTs) and Multi-walled (MWCNTs) nanotubes), such as their sidewall functionalization, tubes' length, solubility, concentration and purity. Among them, we identified the last as the most crucial factor: we have shown that our ultrapure, totally dispersible, carbon nanotubes not only display lack of toxicity in the range of concentrations normally used (10-150 ?g/ml), but they also pave the way for an extensive use of this material for several biomedical purposes. � 2010 Bentham Science Publishers Ltd.

Oral administration of yessotoxin (YTX) has been reported to induce ultrastructural alterations in rodent cardiac muscle. To study its effects on various fundamental aspects of cardiac muscle cells activity, that is, cell beating, Ca(2+) and cyclic adenosine 3',5'-monophosphate (cAMP) levels, as well as cell vitality, a primary culture of rat cardiomyocytes was used. Patch-clamp recordings, Ca(2+) imaging, and cAMP assays were performed on cultured cardiomyocytes to characterize YTX effects on the cell beating frequency. 3-(4,5-Dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) and sulforhodamine B (SRB) tests were carried out to determine its effect on cardiomyocytes viability. Videoimaging techniques showed a time- and concentration-dependent reduction in the beating frequency after 1, 5, and 24 h incubation with YTX (0.1-1 microM). This effect was neither associated to the uncoupling between the membrane electrical activity and Ca(2+) release from intracellular stores nor to the impairment of the mechanisms controlling the Ca(2+) homeostasis. In addition, 1 microM YTX did not modify basal cAMP levels in cardiomyocytes. MTT and SRB assays revealed that incubation of cardiomyocytes with YTX (0.01-1 microM; 24, 48, and 72 h) caused a decrease in cell viability in a concentration- and time-dependent way. This effect was still evident in cardiomyocytes exposed to YTX for 1, 5, and 24 h and cultured up to 72 h in YTX-free medium. Our results demonstrate that, at nanomolar concentrations, a short incubation with YTX causes an inhibition of the beating activity and an irreversible reduction of viability of cardiac cells in vitro.

Methylmercury (MeHg) and polychlorinated biphenyls (PCBs) are widespread environmental pollutants and food contaminants, and known developmental neurotoxicants. Aim of this study was to evaluate the effects of MeHg, PCB 126 and PCB 153 in a battery of in vitro cell systems. A total of 17 cell types were utilized, including nervous system (neuronal and astroglial) and non-nervous system cells. End-points measured included MTT reduction, Trypan blue exclusion and (3)H-thymidine incorporation into DNA. Results indicate that this approach would identify these three compounds as neurotoxicants, and would also point out to the thyroid (for PCB 126 and MeHg) and the prostate (for both PCBs) as important additional targets. Tests of binary combinations of MeHg and PCBs indicated no interaction and an additive response, in agreement with other recent reports. Cerebellar granule neurons from mice with genetically determined low glutathione levels were more sensitive than wild-type neurons to the toxicity of all three compounds, supporting a role for oxidative stress in their neurotoxicity. These findings provide initial evidence that a relatively rapid in vitro screening approach can be developed, that would provide initial information useful for assessing neurotoxicity, as well as indication on potential other targets of biological action or toxicity.

Toxic effects of the antineoplastic drug irinotecan on human blood cells at concentrations of 9.0 microg/ml and 4.6 microg/ml were evaluated in vitro. Using the alkaline and neutral comet assay significantly increased levels of primary DNA damage in lymphocytes were detected. The induction of apoptosis/necrosis, as determined by a fluorescent assay, was also notably increased. Cytogenetic outcomes of the treatment were assessed by the analysis of structural chromosome aberrations and fluorescence in situ hybridization. A significantly higher incidence of chromatid breaks and complex quadriradials was observed. Painted chromosomes 1, 2 and 4 were equally involved in translocations, but only the chromosome 1 was involved in the formation of quadriradials. Sister chromatid exchange analysis was performed in parallel with the analysis of lymphocyte proliferation kinetics. The higher concentration of irinotecan caused almost seven-time increase, while the lower one caused a five-time increase of the basal sister chromatid exchange frequency, accompanied with significant lowering of the lymphocyte proliferation index. Using the cytokinesis-block micronucleus assay, a dose-dependent increase in micronucleus frequency along with the formation of nuclear buds and nucleoplasmic bridges was noticed. Inhibitory effects of irinotecan on enzyme acetylcholinesterase (AChE) were studied in erythrocytes. An IC(50) value of 5.0 x 10(-7) was established. Irinotecan was found to be strong inhibitor of the acetylcholine hydrolysis and to cause a continuous decrease of catalytic activity of AChE. The results obtained on a single donor may contribute to the understanding of irinotecan toxicity, but further in vitro and in vivo studies are essential in order to clarify remaining issues, especially on possible inter-individual variability in genotoxic responses to the drug.

The distribution of silver (Ag) into remote organs secondary to the application of Ag nanoparticles (Ag-NP) to the lung is still incompletely understood and was investigated in the rat with imaging methods. Dose-finding experiments were carried out with 50 nm- or 200 nm-sized polyvinyl pyrrolidine (PVP)-coated Ag-NP using alveolar macrophages in vitro and female rats, which received Ag-NP via intratracheal instillation. In the main study, we administered 37.5-300 µg per rat lung of the more toxic Ag50-PVP and assessed the broncho-alveolar lavage fluid (BALF) for inflammatory cells, total protein and fibronectin after three and 21 days. In parallel, lung tissue was analysed for DNA double-strand breaks and altered cell proliferation. While 75-150 µg Ag50-PVP per rat lung caused a reversible inflammation, 300 µg led to DNA damage, accelerated cell proliferation and progressively increasing numbers of neutrophilic granulocytes. Ag accumulation was significant in homogenates of liver and other peripheral organs upon lung dose of ≥75 µg. Quantitative laser-ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS) combined with enhanced dark field microscopy and autometallography revealed focal accumulations of Ag and/or Ag-NP in sections of peripheral organs: mediastinal lymph nodes contained Ag-NP especially in peripheral macrophages and Ag in argyrophilic fibres. In the kidney, Ag had accumulated within proximal tubuli, while renal filter structures contained no Ag. Discrete localizations were also observed in immune cells of liver and spleen. Overall, the study shows that concentrations of Ag-NP, which elicit a transient inflammation in the rat lung, lead to focal accumulations of Ag in peripheral organs, and this might pose a risk to particular cell populations in remote sites.

The high-quality in vivo preclinical safety data produced by the pharmaceutical industry during drug development, which follows numerous strict guidelines, are mostly not available in the public domain. These safety data are sometimes published as a condensed summary for the few compounds that reach the market, but the majority of studies are never made public and are often difficult to access in an automated way, even sometimes within the owning company itself. It is evident from many academic and industrial examples, that useful data mining and model development requires large and representative data sets and careful curation of the collected data. In 2010, under the auspices of the Innovative Medicines Initiative, the eTOX project started with the objective of extracting and sharing preclinical study data from paper or pdf archives of toxicology departments of the 13 participating pharmaceutical companies and using such data for establishing a detailed, well-curated database, which could then serve as source for read-across approaches (early assessment of the potential toxicity of a drug candidate by comparison of similar structure and/or effects) and training of predictive models. The paper describes the efforts undertaken to allow effective data sharing intellectual property (IP) protection and set up of adequate controlled vocabularies) and to establish the database (currently with over 4000 studies contributed by the pharma companies corresponding to more than 1400 compounds). In addition, the status of predictive models building and some specific features of the eTOX predictive system (eTOXsys) are presented as decision support knowledge-based tools for drug development process at an early stage. The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement n° 115002 (eTOX), resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contributions. The authors would like to formally acknowledge the contribution to the eTOX project of all scientists and other staff involved

Deep eutectic solvents (DESs) have been dramatically expanding in popularity as a new generation of environmentally friendly solvents with possible applications in various industrial fields, but their ecological footprint has not yet been thoroughly investigated. In the present study, three choline chloride-based DESs with glucose, glycerol and oxalic acid as hydrogen bond donors were evaluated for in vitro toxicity using fish and human cell line, phytotoxicity using wheat and biodegradability using wastewater microorganisms through closed bottle test. Obtained in vitro toxicity data on cell lines indicate that choline chloride: glucose and choline chloride:glycerol possess low cytotoxicity (EC50>10 mM for both cell lines) while choline chloride:oxalic acid possess moderate cytotoxicity (EC50 value 1.64 mM and 4.19 mM for fish and human cell line, respectively). Results on phytotoxicity imply that tested DESs are non-toxic with seed germination EC50 values higher than 5000 mg l −1. All tested DESs were classified as′readily biodegradable′ based on their high levels of mineralization (68–96%). These findings indicate that DESs have a green profile and a good prospect for a wider use in the field of green technologies.

Studies of in vitro activity of synthetic peptides derived from the A-gliadin structure were carried out using assays based on cultures of foetal chick intestinal mucosa and on incubation with rat liver lysosomes. The peptide corresponding to residues 11–19, displayed very high activity in the chick intestinal assay, but was only weakly active in the lysosomal assay. Peptide 9–19 was highly active in the chick intestinal assay but was only mildly active in the lysosomal assay. Peptide 8–19 was still appreciably active in both assays. The results on this group of peptides suggest the importance of residues 8–12 to activity and possibly also of a N-terminal glutamine residue. The peptide 213–227, found in a sub-fraction of fraction 9, was only weakly active in both assays, indicating that the PSQQ motif was not solely responsible for toxicity. Thus, as the peptide 208–219 was shown previously to be active in the chick intestinal assay, it is likely that the 208–212 region of this peptide is of prime importance in conferring activity. The results show, for the first time, that a nonapeptide from the N-terminal region of A-gliadin is very active in an in vitro model of toxicity in coeliac disease.

Objectives: Ethanol exposure during pregnancy has toxic effects on the neural stem cells of the developing brain. Differential effects of the alcohol on the neural precursor cells from different regions of the brain have not been established. This study was planned to find out the difference in the morphology of the neurosphere (NS) from the embryonic mouse cortex and cerebellum when exposed to ethanol. The dose of ethanol was comparable to blood alcohol concentration in binge drinking. Materials and methods: The germinal tissues around the lateral ventricle and fourth ventricle of the embryo of pregnant albino mice (E14) were dissected out. Primary cultures were plated using adequate density of cells in media hormone mix (MHM) and growth factors. After 48 hours, ethanol was added to the cultures (low dose = 80 mg/dl; high dose = 400 mg/dl). NS from different groups were analyzed using phase-contrast microscopy, Trypan blue exclusion assay and immunostaining with 4'-6-Diamidino-2-phenylindol (DAPI). Results: With low dose ethanol treatment, the mean area of cortical neurospheres was reduced by 6.9% whereas that of the cerebellar neurospheres was reduced by 51.2%. The mean count of pyknotic cells increased significantly (p, Amaç: Gebelikte etanol maruziyeti, gelişen beyindeki nöral kök hücreler üzerine toksik etkilere sahiptir. Beynin değişik bölgelerindeki nöral öncül hücreler üzerine alkolün farklı etkileri henüz belirlenmemiştir. Bu çalışma fare embriyonik korteks ve serebellumundan alınan nörosferin etanole maruz kaldığında morfolojideki farklılığı araştırmak için planlandı. Etanol dozu aşırı alkol alımındaki kan konsantrasyonuna benzer idi. Gereç ve yöntem: Gebe albino fare (E14) embriyosunun lateral ventrikül ve dördüncü ventrikülü çevresindeki germinal doku alındı. Primer kültürler, ortam hormon karışımındaki yeterli yoğunluktaki hücreler kullanılarak ekim yapıldı. Kırksekiz saat sonra kültür ortamına etanol (düşük doz = 80 mg/dl; yüksek doz = 400 mg/dl) eklendi. Farklı grupların nörosferleri faz-kontrast mikroskop, triptan mavısı dışlama testi ve 4'-6-Diamidino-2-fenilindol immunoboyama ile analiz edildi. Bulgular: Düşük doz etanol ile kortikal nörosferde %6,9 azalma gözlenirken, serebellar nörosferde %51,2 azalma oldu. Ortalama piknotik hücre sayıları hem düşük hem de yüksek doz etanol uygulanan gruplarda, hem kortikal hem de serebellar kültürlerde, kontrol gruplarına göre anlamlı artış gösterdi (p

Cidofovir (HPMPC) was recently reported to exert a valuable antineoplastic activity against primary effusion lymphoma (PEL), a B-cell neoplasm associated with Human Herpesvirus-8 (HHV-8) infection. In this study, we developed and characterized liposomes encapsulating HPMPC to increase drug efficacy reducing the administered dose and the related toxicity, which actually hamper its clinical therapeutic use in patients affected with PEL. The liposomes, obtained using different formulations of neutral and cationic lipids, were analyzed by microscopical (AFM) and spectroscopical (PCS and NMR) techniques. Using an in vitro model of PEL (BCBL-1 cell line), the carrier toxicity and the antineoplastic efficacy of liposomes were evaluated by flow cytometry applying apoptosis and cell death analysis. The in vitro study showed the applicability of the liposomes within a restricted range of lipidic concentrations according to the lipids used during the preparation. The moderate increases in the percentage of apoptotic/necrotic cells suggests that liposomal delivery allows the release of HPMPC into BCBL-1 cells enabling an unexpected antineoplastic activity of this drug even at lower doses.

The in vitro toxicity of neem seed oil (Azadirachta indica A. Juss, family: Meliaceae, Dogon yaro in Hausa language in Nigeria) was tested against the larvae of a one-host tick, Boophilus decoloratus (family: Ixodidae or hard tick, commonly known as blue tick) parasitic mainly to cattle generally found in savannas of tropical equatorial Africa. The 20, 40, 60, 80 and 100% concentrations of neem seed oil were found to kill all (100% mortality) the larvae after 27, 27, 27, 27 and 24 h respectively.

Journal of Antimicrobial Chemotherapy, 61 (2), ISSN:0305-7453, ISSN:1460-2091

This study investigated whether mouthwash dilutions, recommended by the manufacturers, may contribute to oral mucosa irritation. Twenty frequently used mouthwash brands were examined by the pollen tube growth test, an alternative in vitro method for assessment of the irritating potential of ingredients of cosmetic formulations. The test is based on the photometric quantification of pollen tube growth inhibition. This parameter is expressed by IC50 values that characterize the cytotoxic potential of a product. The IC50s clearly revealed that none of the tested mouthwashes should cause acute irritation if used in the recommended dilution. However, at least 4 of the mouthwashes evaluated in this study could probably irritate the oral mucosa acutely if used undiluted (original concentration). Este estudo investigou se soluções de bochecho nas diluições recomendadas pelos fabricantes podem contribuir para a irritação das mucosas orais. Vinte soluções de bochecho frequentemente utilizadas foram avaliadas por meio do análise do crescimento do tubo polínico. Este teste constitui um método alternativo in vitro para estimar o potencial irritante dos ingredientes de formulações cosméticas. O teste baseia-se numa medição fotométrica da inibição do crescimento de tubos polínicos. A inibição do crescimento é expressa por valores IC50 que caracterizam o potencial citotóxico de uma dada substância. Os valores de IC50 aqui apresentados demonstraram que nenhuma das soluções de bochecho estudadas deve causar irritação aguda das mucosas orais, desde que utilizadas nas diluições recomendadas pelo fabricante. No entanto, verificou-se que pelo menos quatro dos colutórios podem causar irritação aguda das mucosas orais ao serem utilizados na a forma concentrada.

International audience; 3,4-Dihydroxyphenylacetaldehyde (DOPAL) is formed by the oxidative deamination of dopamine (DA) catalyzed by monoamine oxidases (MAO); then, the aldehyde is oxidized to 3,4-dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenases (ALDH) or reduced to 3,4-dihydroxyphenylethanol (DOPET) by aldose/aldehyde reductases. The present work aimed at evaluating the in vitro toxicity of DOPAL on catecholaminergic neuroblastoma SH-SY5Y cells which accumulate DA. DOPAL synthesis was stimulated by incubating cells with DA and blocking DOPAL oxidation by disulfiram, an irreversible inhibitor of ALDH. As evidenced by MTT reduction assays, DA and disulfiram treatments produced cell losses which increased with time. 10(-2)M DA reduced by 40% cell viability after a 1h treatment, when its TC(50) (concentration reducing viability by 50%) value was 7.3 x 10(-5) M after a 24 h treatment. For the same treatment periods, TC(50) values for disulfiram were 8 x 10(-5) and 8.7 x 10 (-7) M, respectively. MTT reduction assay performed after a 24h treatment followed by a 24h incubation in a drug-free medium evidenced that the toxicity of 10(-4)M DA or 10(-6)M disulfiram was potentiated by the second drug. HPLC measurements showed that DOPAL was produced at the early stages of the treatment by DA and disulfiram. This was evidenced by the significant increase in the ((DOPAL + DOPET)/DOPAC ratio observed after a combined 3h treatment by 10(-4)M DA and 10(-6)M disulfiram. Total contents in DA and DOPAL were greatly reduced at the end of a 15 h treatment, and disulfiram did not significantly enhanced the (DOPAL + DOPET)/DOPAC ratio. For both treatment durations, DOPAL and DOPET were detectable only in the extracellular medium. So, these results suggest that an early production of DOPAL could produce delayed toxic effects on SH-SY5Y cells. Production of DOPET and release of DOPAL could be important means for reducing DOPAL concentrations in dopaminergic neurons.

Protocols for evaluating chemical toxicity at the cellular level using fish cell lines are described in this unit. Routine methodologies for growing salmonid cell lines, and using them in aquatic toxicology studies that support the mandate of the Organization for Economic Co-operation and Development (OECD) to reduce the use of whole animals in toxicity testing, are presented. Rapid, simple, cost-effective tests evaluating viability of cells with three indicator dyes per sample provides a broad overview of the sensitivity of cells to chemical contaminants. This fluorometric assay involves: (1) alamar blue for metabolic activity, (2) CFDA-AM for membrane integrity, and (3) neutral red for lysosomal function. These protocols are conveniently performed in semi-unison within the same multiwell plates and read at three different wavelengths. Detailed step-by-step descriptions of the assays, parameters to consider, troubleshooting, and guidelines for data interpretation are provided as essential tools for investigating environmental aquatic contaminants at the cellular level.