Algorithmic assessment of cellular senescence in experimental and clinical specimens.

The development of genetic tools allowed for the validation of the pro-aging and pro-disease functions of senescent cells in vivo. These discoveries prompted the development of senotherapies-pharmaceutical interventions aimed at interfering with the detrimental effect of senescent cells-that are now entering the clinical stage. However, unequivocal identification and examination of cellular senescence remains highly difficult because of the lack of universal and specific markers. Here, to overcome the limitation of measuring individual markers, we describe a detailed two-phase algorithmic assessment to quantify various senescence-associated parameters in the same specimen. In the first phase, we combine the measurement of lysosomal and proliferative features with the expression of general senescence-associated genes to validate the presence of senescent cells. In the second phase we measure the levels of pro-inflammatory markers for specification of the type of senescence. The protocol can help graduate-level basic scientists to improve the characterization of senescence-associated phenotypes and the identification of specific senescent subtypes. Moreover, it can serve as an important tool for the clinical validation of the role of senescent cells and the effectiveness of anti-senescence therapies.

Water dispersible ZnSe/ZnS quantum dots: Assessment of cellular integration, toxicity and bio-distribution.

Quantum dots (QDs) comprise an emerging group of materials with innumerable number of possibilities in biological research including cellular labelling. Among the leading members in this category, ZnSe/ZnS quantum dots (QDs) hold greater attractive possibilities in imaging primarily due to their higher biocompatibility and dispersibility. Nevertheless, the inherent toxicity of ZnSe/ZnS QDs is not yet completely explored which largely compromise most of their biomedical application potential. Strong blue emitting water soluble QDs effectively synthesized by aqueous phase route. Synthesized QDs further subjected to various optical and physicochemical characterization. Approximately 5-6 nm sized ZnSe/ZnS QDs illuminated bluish green fluorescence under UV lamp. Present study addresses possible adverse effects of ZnSe/ZnS QDs in hepatic system using HepG2 cells; which is the routinely employed in vitroliver cell model. A bundle of assays wasperformed out to reveal the cytotoxic nature of ZnSe/ZnS QDs and the mechanism behind it. Herein, absorption, distribution, metabolism, excretion and toxicity (ADME and T) of ZnSe/ZnS in mice were profiled in detail followed by intravenous (i.v.) and intraperitoneal (i.p.) administration at a dose of 10 mg/kg body weight. In a short review, it could be state that ZnSe/ZnS QDs did not exhibit any significant in vivo toxicity outcome in mice.

Assessment of the cytotoxic impact of cyanotoxin beta-N-methylamino-L-alanine on a fish immune cell line.

Beta-N-methylamino-L-alanine (BMAA) is a non-proteinogenic amino acid produced by several cyanobacteria species. It is considered to be a potent neurotoxin. Although its neurotoxic effects are well studied, other negative effects of BMAA have not yet been completely elucidated. In the present study, we studied the cytotoxic effects of a wide range of concentrations of BMAA (0.25-2.0 mM) on a stable fish immune cell line (CLC) obtained from carp monocytes. The cells exposed to higher concentrations of BMAA exhibited an altered morphology, changed ATP levels, and reduced proliferation. On the basis of toxic effects of BMAA on lysosomes, mitochondrial dehydrogenases activity, and cell membrane integrity, we determined its cytotoxic concentrations. We also investigated effects of the toxin at non-cytotoxic concentrations on the basic functions of CLC cells. BMAA did not affect the production and release of IL-1ß or phagocytic activity of the cells. However, higher non-toxic BMAA concentrations altered the levels of extracellular and intracellular total proteins compared to those in control cells.

Assessment of metal contamination in estuarine surface sediments from Dongying City, China: Use of a modified ecological risk index.

Surface sediments and clam Meretrix meretrix were collected from a northern estuarine region in Dongying City, China. Sediments were analysed for heavy metals (Hg, As, Cd, Cr, Cu, Pb, and Zn) and the clams were tested for metallothioneins (MTs) and lysosomal membrane stability (LMS). The heavy metal total concentrations decreased in the order of Cr>Zn>Cu>Pb>As>Cd>Hg. The results of Bureau Communautaire de Référence (BCR) sequential extraction of heavy metals showed that the geochemical speciation of all heavy metals was dominated by residual fraction. According to the responses of biomarkers in M. meretrix, the modified potential ecological risk index (PERI-B) can more accurately reflect heavy metals pollution. PERI-B showed all sediment samples have low or moderate risk, except at site S10 (considerable risk), and the main contribution of ecological risk heavy metals were Cd and Hg.

Quantitative Assessment of Drug Delivery to Tissues and Association with Phospholipidosis: A Case Study with Two Structurally Related Diamines in Development.

Drug induced phospholipidosis (PLD) may be observed in the preclinical phase of drug development and pose strategic questions. As lysosomes have a central role in pathogenesis of PLD, assessment of lysosomal concentrations is important for understanding the pharmacokinetic basis of PLD manifestation and forecast of potential clinical appearance. Herein we present a systematic approach to provide insight into tissue-specific PLD by evaluation of unbound intracellular and lysosomal (reflecting acidic organelles) concentrations of two structurally related diprotic amines, GRT1 and GRT2. Their intratissue distribution was assessed using brain and lung slice assays. GRT1 induced PLD both in vitro and in vivo. GRT1 showed a high intracellular accumulation that was more pronounced in the lung, but did not cause cerebral PLD due to its effective efflux at the blood-brain barrier. Compared to GRT1, GRT2 revealed higher interstitial fluid concentrations in lung and brain, but more than 30-fold lower lysosomal trapping capacity. No signs of PLD were seen with GRT2. The different profile of GRT2 relative to GRT1 is due to a structural change resulting in a reduced basicity of one amino group. Hence, by distinct chemical modifications, undesired lysosomal trapping can be separated from desired drug delivery into different organs. In summary, assessment of intracellular unbound concentrations was instrumental in delineating the intercompound and intertissue differences in PLD induction in vivo and could be applied for identification of potential lysosomotropic compounds in drug development.

Fish cell lines as a tool for the ecotoxicity assessment and ranking of engineered nanomaterials.

Risk assessment of engineered nanomaterials (ENMs) is being hindered by the sheer production volume of these materials. In this regard, the grouping and ranking of ENMs appears as a promising strategy. Here we sought to evaluate the usefulness of in vitro systems based on fish cell lines for ranking a set of ENMs on the basis of their cytotoxicity. We used the topminnow (Poeciliopsis lucida) liver cell line (PLHC-1) and the rainbow trout (Oncorhynchus mykiss) fibroblast-like gonadal cell line (RTG-2). ENMs were obtained from the EU Joint Research Centre repository. The size frequency distribution of ENM suspensions in cell culture media was characterized. Cytotoxicity was evaluated after 24 h of exposure. PLHC-1 cells exhibited higher sensitivity to the ENMs than RTG-2 cells. ZnO-NM was found to exert toxicity mainly by altering lysosome function and metabolic activity, while multi-walled carbon nanotubes (MWCNTs) caused plasma membrane disruption at high concentrations. The hazard ranking for toxicity (ZnO-NM > MWCNT ≥ CeO2-NM = SiO2-NM) was inversely related to the ranking in size detected in culture medium. Our findings reveal the suitability of fish cell lines for establishing hazard rankings of ENMs in the framework of integrated approaches to testing and assessment.

Assaying Cellular Viability Using the Neutral Red Uptake Assay.

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

Assessment of immune status of yellowfin seabream (Acanthopagrus latus) during short term exposure to phenanthrene.

The aim of the present investigation was to assess the immune status in yellowfin seabream (Acanthopagrus latus) exposed to different concentrations of phenanthrene (Phe) for 14days. In addition, the Phe accumulation in the fish muscle was measured during the experiment. Fish were injected with different concentrations (0, 2, 20 and 40mg/kg) of Phe and samples were taken from tissue and blood of fish 1, 4, 7 and 14days after injection. Exposure of fish to Phe caused a significant decrease in white blood cells, C3 and C4 levels, lysosomal membrane stability, lysozyme activity after 4days and antibacterial activity after 7days of the experiment. In contrast, cortisol level significantly increased after 4days. The concentration of Phe in fish muscle increased rapidly after 4days. The main tissue changes observed in the head kidney including increase in melanomacrophage centers (MMCs), empty spaces between cells and hemorrhage. The degree of tissue changes ranged from normal to moderate in Phe-treated fish. The size and number of MMCs in treated fish were significantly higher than control. In conclusion, Phe toxicity in yellowfin seabream can induce increased cortisol level, tissue changes and immune suppression.

Ecotoxicological assessment of cobalt using Hydra model: ROS, oxidative stress, DNA damage, cell cycle arrest, and apoptosis as mechanisms of toxicity.

The mechanisms underlying cobalt toxicity in aquatic species in general and cnidarians in particular remain poorly understood. Herein we investigated cobalt toxicity in a Hydra model from morphological, histological, developmental, and molecular biological perspectives. Hydra, exposed to cobalt (0-60 mg/L), were altered in morphology, histology, and regeneration. Exposure to standardized sublethal doses of cobalt impaired feeding by affecting nematocytes, which in turn affected reproduction. At the cellular level, excessive ROS generation, as the principal mechanism of action, primarily occurred in the lysosomes, which was accompanied by the upregulation of expression of the antioxidant genes SOD, GST, GPx, and G6PD. The number of Hsp70 and FoxO transcripts also increased. Interestingly, the upregulations were higher in the 24-h than in the 48-h time-point group, indicating that ROS overwhelmed the cellular defense mechanisms at the latter time-point. Comet assay revealed DNA damage. Cell cycle analysis indicated the induction of apoptosis accompanied or not by cell cycle arrest. Immunoblot analyses revealed that cobalt treatment triggered mitochondria-mediated apoptosis as inferred from the modulation of the key proteins Bax, Bcl-2, and caspase-3. From this data, we suggest the use of Hydra as a model organism for the risk assessment of heavy metal pollution in aquatic ecosystems.

Toxicity assessment of diesel- and metal-contaminated soils through elutriate and solid phase assays with the slime mold Dictyostelium discoideum.

A suite of organisms from different taxonomical and ecological positions is needed to assess environmentally relevant soil toxicity. A new bioassay based on Dictyostelium is presented that is aimed at integrating slime molds into such a testing framework. Toxicity tests on elutriates and the solid phase developmental cycle assay were successfully applied to a soil spiked with a mixture of Zn, Cd, and diesel fuel freshly prepared (recently contaminated) and after 2 yr of aging. The elutriates of both soils provoked toxic effects, but toxicity was markedly lower in the aged soil. In the D. discoideum developmental cycle assay, both soils affected amoeba viability and aggregation, with fewer multicellular units, smaller fruiting bodies and, overall, inhibition of fruiting body formation. This assay is quick and requires small amounts of test soil, which might facilitate its incorporation into a multispecies multiple-endpoint toxicity bioassay battery suitable for environmental risk assessment in soils. Environ Toxicol Chem 2016;35:1413-1421. © 2015 SETAC.

Use of a pro-fibrogenic mechanism-based predictive toxicological approach for tiered testing and decision analysis of carbonaceous nanomaterials.

Engineered carbonaceous nanomaterials (ECNs), including single-wall carbon nanotubes (SWCNTs), multiwall carbon nanotubes (MWCNTs), graphene, and graphene oxide (GO), are potentially hazardous to the lung. With incremental experience in the use of predictive toxicological approaches, seeking to relate ECN physicochemical properties to adverse outcome pathways (AOPs), it is logical to explore the existence of a common AOP that allows comparative analysis of broad ECN categories. We established an ECN library comprising three different types of SWCNTs, graphene, and graphene oxide (two sizes) for comparative analysis according to a cell-based AOP that also plays a role in the pathogenesis of pulmonary fibrosis. SWCNTs synthesized by Hipco, arc discharge and Co-Mo catalyst (CoMoCAT) methods were obtained in their as-prepared (AP) state, following which they were further purified (PD) or coated with Pluronic F108 (PF108) or bovine serum albumin (BSA) to improve dispersal and colloidal stability. GO was prepared as two sizes, GO-small (S) and GO-large (L), while the graphene samples were coated with BSA and PF108 to enable dispersion in aqueous solution. In vitro screening showed that AP- and PD-SWCNTs, irrespective of the method of synthesis, as well as graphene (BSA) and GO (S and L) could trigger interleukin-1ß (IL-1ß) and transforming growth factor-ß1 (TGF-ß1) production in myeloid (THP-1) and epithelial (BEAS-2B) cell lines, respectively. Oropharyngeal aspiration in mice confirmed that AP-Hipco tubes, graphene (BSA-dispersed), GO-S and GO-L could induce IL-1ß and TGF-ß1 production in the lung in parallel with lung fibrosis. Notably, GO-L was the most pro-fibrogenic material based on rapid kinetics of pulmonary injury. In contrast, PF108-dispersed SWCNTs and -graphene failed to exert fibrogenic effects. Collectively, these data indicate that the dispersal state and surface reactivity of ECNs play key roles in triggering a pro-fibrogenic AOP, which could prove helpful for hazard ranking and a proposed tiered testing approach for large ECN categories.

Assessment and optimization of autophagy monitoring methods in Arabidopsis roots indicate direct fusion of autophagosomes with vacuoles.

Autophagy is a degradation pathway that recycles cell materials upon encountering stress conditions or during specific developmental processes. To better understand the physiological roles of autophagy, proper monitoring methods are very important. In mammals and yeast, monitoring of autophagy is often performed with a green fluorescent protein (GFP)-ATG8 fusion protein or with acidotropic dyes such as monodansylcadaverine (MDC) and LysoTracker Red (LTR). To evaluate these monitoring methods, here we examined these systems by inducing autophagy in Arabidopsis thaliana roots as a model for monitoring autophagy in planta. Under carbon- and nitrogen-starved conditions, the number and size of vesicles labeled by GFP-ATG8 was increased for several hours and then gradually decreased to a level higher than that observed before the start of the experiment. We also observed the disappearance of GFP-ATG8-labeled vesicles after treatment with wortmannin, a phosphatidylinositol 3-kinase inhibitor known as an autophagy inhibitor, showing that the GFP-ATG8 transgenic line constitutes an excellent method for monitoring autophagy. These data were compared with plants stained with MDC and LTR. There was no appreciable MDC/LTR staining of small organelles in the root under the induction of autophagy. Some vesicles were eventually observed in the root tip only, but co-localization experiments, as well as experiments with autophagy-deficient atg mutants, provided the evidence that these structures were located in the vacuole and were not manifestly autophagosomes and/or autolysosomes. Extreme caution should therefore be used when monitoring autophagy with the aid of MDC/LTR. Additionally, our observations strongly suggest that autophagosomes fuse directly to vacuoles in Arabidopsis roots.

A biomarker of contaminant exposure is effective in large scale assessment of ten estuaries.

Cost-effective and sensitive measures of anthropogenic stress are necessary tools in any environmental monitoring program. When implementing new monitoring tools in a region, rigorous laboratory and field studies are essential for characterizing the sensitivity and efficacy of the approach. We exposed the oyster Saccostrea glomerata to various individual contaminants through multiple exposure pathways (water- and food-borne) in the laboratory and measured two biomarker responses, lysosomal membrane stability (LMS) and lipid peroxidation (LPO). LMS was sensitive to both contaminant exposure pathways. We subsequently measured this biomarker in oysters which had been experimentally deployed at multiple sites in each of ten estuaries with varying levels of contamination associated with re-suspended sediments. There was a strong association between LMS and metal exposure, despite substantial natural variation in water quality parameters. Our results illustrate the potential use of LMS as a pragmatic indicator of biotic injury in environmental monitoring programs for re-suspended contaminated sediments.

Assessment of cation trapping by cellular acidic compartments.

All nucleated cells, from yeast to animal cells, concentrate cationic chemicals (weak bases with a pKa~8-10) into acidic cell compartments (low retro-diffusion under a protonated form at low pH=ion trapping). The proton pump vacuolar (V)-ATPase is the driving force of this pseudotransport that concerns acidic organelles (mainly late endosomes and lysosomes). The latter rapidly become swollen (osmotic vacuolization) and macroautophagic. Cation concentration in cells is not proved to involve membrane transporters, but is prevented or reversed by inhibitors of V-ATPase, such as bafilomycin A1. Lipophilicity is a major determinant of the apparent affinity of this pseudotransport because simple diffusion of the uncharged form supports it. Quinacrine is a formerly used antiparasitic drug that is intensely fluorescent, lipophilic, and a tertiary amine. The drug, at micromolar concentrations, is proposed as a superior probe for assessing cation trapping by cellular acidic compartments, being readily quantified using fluorometry in cell extracts and analyzed using microscopy and cytofluorometry (fluorescence settings for fluorescein being applicable). Further, cells respond to micromolar levels of quinacrine by autophagic accumulation (e.g., accumulation of the activated macroautophagic effector LC3 II, immunoblots), an objective and universal response to sequestered amines.

Beyond amyloid: getting real about nonamyloid targets in Alzheimer's disease.

For decades, researchers have focused primarily on a pathway initiated by amyloid beta aggregation, amyloid deposition, and accumulation in the brain as the key mechanism underlying the disease and the most important treatment target. However, evidence increasingly suggests that amyloid is deposited early during the course of disease, even prior to the onset of clinical symptoms. Thus, targeting amyloid in patients with mild to moderate Alzheimer's disease (AD), as past failed clinical trials have done, may be insufficient to halt further disease progression. Scientists are investigating other molecular and cellular pathways and processes that contribute to AD pathogenesis. Thus, the Alzheimer's Association's Research Roundtable convened a meeting in April 2012 to move beyond amyloid and explore AD as a complex multifactorial disease, with the goal of using a more inclusive perspective to identify novel treatment strategies.

Proteases inhibition assessment on PC12 and NGF treated cells after oxygen and glucose deprivation reveals a distinct role for aspartyl proteases.

Hypoxia is a severe stressful condition and induces cell death leading to neuronal loss both to the developing and adult nervous system. Central theme to cellular death is the activation of different classes of proteases such as caspases calpains and cathepsins. In the present study we investigated the involvement of these proteases, in the hypoxia-induced PC12 cell death. Rat PC12 is a model cell line for experimentation relevant to the nervous system and several protocols have been developed for either lethal hypoxia (oxygen and glucose deprivation OGD) or ischemic preconditioning (IPS). Nerve Growth Factor (NGF) treated PC12 differentiate to a sympathetic phenotype, expressing neurites and excitability. Lethal hypoxia was established by exposing undifferentiated and NGF-treated PC12 cells to a mixture of N(2)/CO(2) (93:5%) in DMEM depleted of glucose and sodium pyruvate for 16 h. The involvement of caspases, calpains and lysosomal cathepsins D and E to the cell death induced by lethal OGD was investigated employing protease specific inhibitors such as z-VAD-fmk for the caspases, MDL28170 for the calpains and pepstatin A for the cathepsins D and E. Our findings show that pepstatin A provides statistically significant protection from cell death of both naive and NGF treated PC12 cells exposed to lethal OGD. We propose that apart from the established processes of apoptosis and necrosis that are integral components of lethal OGD, the activation of cathepsins D and E launches additional cell death pathways in which these proteases are key partners.

Assessment of lysosomal membrane stability and peroxisome proliferation in the head kidney of Atlantic cod (Gadus morhua) following long-term exposure to produced water components.

There is a need for sensitive biological effect methods by which to detect impacts of chronic exposure to low concentrations of contaminants. Two methods shown to be potentially useful for monitoring purposes in fish include lysosomal membrane stability and peroxisome proliferation. These biological endpoints were assessed in Atlantic cod (Gadus morhua) head kidney following exposure to a mixture of produced water components including polycyclic aromatic hydrocarbons, phenol, and alkylphenols. Lysosomal damage of head kidney cells occurred within the first two weeks and did not recover during the entire exposure period (32 weeks). Lysosomal membrane stability was not affected by gender and was responsive at low concentrations of contamination, indicating that lysosomal membrane stability measured in the head kidney could be a useful biomarker for effects of offshore pollution. Peroxisome proliferation, measured as acyl-CoA oxidase activity in the head kidney, appeared to be a potential biomarker in male cod exposed less than 16 weeks.

Simultaneous assessment of autophagy and apoptosis using multispectral imaging cytometry.

Multiple stress pathways result in the induction of autophagy and apoptosis. Current methods (e.g., protein gel blot, microscopy) do not offer quantitative single-cell resolution, thus making it difficult to discern if these pathways are mutually exclusive or, in some situations, cooperative in executing cell death. We report a novel method that enables high-throughput, high-content assessment of LC3 puncta and caspase-3 cleavage at the single cell level.

Integrated biomarker assessment of the effects exerted by treated produced water from an onshore natural gas processing plant in the North Sea on the mussel Mytilus edulis.

The biological impact of a treated produced water (PW) was investigated under controlled laboratory conditions in the blue mussel, Mytilus edulis. Mussel health status was assessed using an integrated biomarker approach in combination with chemical analysis of both water (with SPMDs), and mussel tissues. Acyl-CoA oxidase activity, neutral lipid accumulation, catalase activity, micronuclei formation, lysosomal membrane stability in digestive cells and haemocytes, cell-type composition in digestive gland epithelium, and the integrity of the digestive gland tissue were measured after 5 week exposure to 0%, 0.01%, 0.1%, 0.5% and 1% PW. The suite of biomarkers employed were sensitive to treated PW exposure with significant sublethal responses found at 0.01-0.5% PW, even though individual chemical compounds of PW were at extremely low concentrations in both water and mussel tissues. The study highlights the benefits of an integrated biomarker approach for determining the potential effects of exposure to complex mixtures at low concentrations. Biomarkers were integrated in the Integrative Biological Response (IBR/n) index.

Assessment of nanomaterials cytotoxicity and internalization.

The impact that nanotechnology may have on life and medical sciences is immense and includes novel therapies as much as novel diagnostic and imaging tools, often offering the possibility to combine the two. It is, therefore, of the essence to understand and control the interactions that nanomaterials can have with cells, first at an individual level, focusing on, e.g., binding and internalization events, and then at a tissue level, where diffusion and long-range transport add further complications. Here, we present experimental methods based on selective labeling techniques and the use of effectors for a qualitative and quantitative evaluation of endocytic phenomena involving nanoparticles. The understanding of the cell-material interactions arising from these tests can then form the basis for a model-based evaluation of nanoparticles behavior in 3D tissues.