Lysosomal Membrane Destabilization Research Articles

Vanillic acid protects mortality and toxicity induced by N-ethyl-N-nitrosourea in mice; in vivo model of chronic lymphocytic leukemia

Alkylating agents such as N-Ethyl-N-Nitrosourea (ENU) are ubiquitous within living cells and in the environment. This study designed to evaluate the chemopreventive activity of vanillic acid on ENU-induced toxicity and carcinogenesis in mice as an animal model of chronic lymphocytic leukemia (CLL). The female, Swiss albino mice were divided into three groups each with 7 mice, group I received normal saline, group II, mice received ENU at a dose of 80 mg/kg body weight i.p. to induce CLL on the 31th day of the study, and group III, the mice pretreated with vanillic acid at a dose of 20 mg/kg body weight/day, i.p. up to 30 days and received ENU. The animals were monitored for weight changes and mortality during 120 days, and then were sacrificed for isolation of lymphocytes, as target cells in CLL. Cellular parameters like reactive oxygen species (ROS) formation, malondialdehyde (MDA) production, depletion of glutathione (GSH), mitochondrial membrane potential (MMP) and lysosomal membrane integrity were studied. We found that pretreatment with vanillic acid significantly increased the survival of mice up to 57%, delay in death time (30%) and prevented weight changes after exposure to ENU. In addition, it was found that vanillic acid protected ROS formation, lipid peroxidation mitochondrial dysfunction, and lysosomal membrane destabilization in isolated lymphocytes. These data suggest that vanillic acid exhibited significant protection against ENU-induced toxicity and carcinogenicity, which might be related to the protection of the mitochondria and lysosomes and the reduction of ROS formation and oxidative stress.

Antioxidants and mitochondrial/lysosomal protective agents reverse toxicity induced by titanium dioxide nanoparticles on human lymphocytes.

Most of the literature has focused on titanium dioxide (TiO2) nanoparticles (NPs) toxicity, showing the importance of oxidative stress, mitochondrial dysfunction, and cell death in TiO2-induced toxicity. For this purpose, in the current study, we investigated the protective role of antioxidant and mitochondrial/lysosomal protective agents to minimize TiO2 NPs-induced toxicity in human lymphocytes. Human lymphocytes were obtained from heathy individuals and treated with different concentrations (80, 160, and 320µg/mL) of TiO2 NPs, and then human lymphocytes preincubated with butylated hydroxytoluene (BHT), cyclosporin A (CsA), and chloroquine separately were exposed to TiO2 NPs for 6h. In all the above-mentioned treated groups, adverse parameters such as cytotoxicity, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), lysosomal membrane destabilization, the levels of malondialdehyde (MDA), and glutathione (GSH) were measured. The results showed that TiO2 nanoparticles induced cytotoxicity through ROS formation, MMP collapse, lysosomal damages, depletion of GSH, and lipid peroxidation. However, BHT as an antioxidant, CsA as a mitochondrial permeability transition (MPT) pore sealing agent, and chloroquine as a lysosomotropic agent, significantly inhibited all the TiO2 NPs-induced cellular and organelle toxicities. Thus, it seems that antioxidant and mitochondrial/lysosomal protective agents are promising preventive strategies against TiO2 NPs-induced toxicity.

Toxicopathic effects of waterborne Cd in sole juveniles, a prospective pollution monitoring sentinel for estuarine ecosystems

IntroductionEstuarine ecosystems are under high anthropogenic pressure and receive a wide Q7 variety of contaminants, including metals. They can remain trapped in estuarine sediments at high concentrations for long periods but eventually they can be released to the overlying water and become toxic for the resident benthic biota. As a consequence, biomonitoring programs surveying the health status of estuaries and coastal areas count with benthic species for the assessment of health disturbances in these specific ecosystems. The present investigation aims at recognizing toxicopathic effects elicited in estuarine life stages of the benthic fish Solea senegalensis on exposure to waterborne Cd, a model compound for metal contamination.MethodsBiological responses to metal exposure were assessed based on the application of a “biomarker + histopathology” approach. Two-year old sole juveniles were exposed to various concentrations of waterborne Cd for 3 and 7 days (Control; Low Cd: 1 µg/l; Mid Cd: 10 µg/l and High Cd: 1000 µg/l). Liver samples were collected for chemical analysis at day 7. Biological samples were collected at days 3 and 7 for biochemical (brain and liver), histochemical (liver) and histopathological analysis (liver, gills and gonad).ResultsBrain acetyl cholinesterase was inhibited after 7 days exposure, indicating neurotoxic effects on exposure to 1000 µg Cd/l. Hepatic catalase and glutathione-Stransferase were induced at day 3 and inhibited at day 7, which suggests a bell-shaped response. A dose-dependent lysosomal membrane destabilization in hepatocytes was recorded at day 7. In parallel, histopathological lesions in gills, liver and gonad were more frequent at day 7 in soles exposed to high Cd concentrations.DiscussionOverall, the “biomarker + histopathology” approach revealed that waterborne Cd causes toxicopathic effects in sole juveniles upon exposure to environmentally relevant concentrations (10 µg Cd/l). The effects were clearly time dependent, and thus it is conceivable that more severe biological effects would be elicited by these low concentrations of Cd at longer exposure periods. Therefore, biomarkers and histopathology in combination provide early-warning indications of altered health status after waterborne Cd exposure in sole juveniles, a prospective sentinel for biological effects assessment of metal contamination in pollution monitoring programs in estuaries and coastal areas.

Impact of polyethylene microplastics on the clam Ruditapes decussatus (Mollusca: Bivalvia): examination of filtration rate, growth, and immunomodulation.

The present study was conducted to assess, for the first time, the effects of a 14 days experimental exposure to polyethylene (PE) based MPs (40-48 µm) on the clam Ruditapes decussatus. Clams were exposed to three different concentrations of MPs in controlled laboratory conditions: 10 µg/L (low), 100 µg/L (medium), and 1000 µg/L (high). The effects of MPs were assessed using a multi-marker approach, including the filtration rate, growth, and the integrity of immune cells (such as haemocyte numbers, viability, and lysosomal membrane destabilization). The results revealed that as the concentration of PE-MPs increased, the filtration rate decreased, indicating that PE-MPs hindered the clams' ability to filter water. Furthermore, there was a noticeable decrease in the overall weight of the clams, particularly in the group exposed to 1000 µg/L. This decrease could be attributed to the impairment of their nutrient filtration function. In terms of immune system biomarkers, exposure to PE-MPs led to immune system disruption, characterized by a significant increase in the number of haemocytic cells, especially in the group exposed to the high concentration. Additionally, there was a notable reduction in the viability of haemocytes, resulting in the destabilization of their lysosomal membranes, particularly in the groups exposed to medium and high PE-MPs concentrations. The findings of this study indicate that the sensitivity of hemolymph parameter changes and filtration rate in R. decussatus exposed to PE-MPs (100 and 1000 µg/L), surpasses that of growth performance and can serve as reliable indicators to assess habitat conditions and contaminant levels.

GPX4 overexpressed non-small cell lung cancer cells are sensitive to RSL3-induced ferroptosis

Ferroptosis can be induced by inhibiting antioxidant enzymes GPX4 or system Xc−, increased intracellular iron concentrations, and lipid peroxidation. Recently, it has been suggested that ferroptosis can be an effective way to induce cancer cell death, although the specific relevance and mechanism of ferroptosis have not been fully elucidated. Here, we investigated the anticancer effects of ferroptosis inducers erastin and RSL3 on non-small cell lung cancer (NSCLC) cells. RSL3 induced cell death more effectively in NSCLC cells than erastin, with limited cytotoxicity in BEAS-2B normal bronchial epithelial cells. The sensitivity of NSCLC cells to RSL3 induced death was dependent on GPX4 expression levels; the effect of RSL3 was reversed by ferrostatin-1 (a ferroptosis inhibitor) but not by Z-VAD-FMK, chloroquine, bafilomycin A1, or necrostatin-1. RSL3 induced ferroptosis by promoting lipid peroxidation, elevating intracellular LIP concentration and ROS level, and blocking GSH-to-GSSH conversion through the inhibition of GPX4 and induction of Nrf2/HO1. Furthermore, RSL3 induced autophagosomes but disrupted the formation of autolysosomes with lysosomal membrane destabilization. GPX4 knockdown had a similar effect on ferroptosis phenotypes as RSL3. Taken together, RSL3-induced ferroptosis depends on the regulation of GPX4-Nrf2/HO1 in NSCLC cells. These results may be useful in predicting the ferroptosis response in NSCLC as well as drug resistant cancer cells.

Clostridium perfringens virulence factors are nonredundant activators of the NLRP3 inflammasome.

Inflammasome signaling is a central pillar of innate immunity triggering inflammation and cell death in response to microbes and danger signals. Here, we show that two virulence factors from the human bacterial pathogen Clostridium perfringens are nonredundant activators of the NLRP3 inflammasome in mice and humans. C. perfringens lecithinase (also known as phospolipase C) and C. perfringens perfringolysin O induce distinct mechanisms of activation. Lecithinase enters LAMP1+ vesicular structures and induces lysosomal membrane destabilization. Furthermore, lecithinase induces the release of the inflammasome-dependent cytokines IL-1β and IL-18, and the induction of cell death independently of the pore-forming proteins gasdermin D, MLKL and the cell death effector protein ninjurin-1 or NINJ1. We also show that lecithinase triggers inflammation via the NLRP3 inflammasome in vivo and that pharmacological blockade of NLRP3 using MCC950 partially prevents lecithinase-induced lethality. Together, these findings reveal that lecithinase activates an alternative pathway to induce inflammation during C. perfringens infection and that this mode of action can be similarly exploited for sensing by a single inflammasome.

Morphological and functional alterations in hemocytes of Mytilus galloprovincialis exposed in high-impact anthropogenic sites

The work aimed to study the induction of morphological alterations in M. galloprovincialis in the field and its suitability to be integrated into a sensitive, simple, and cost-effective cell-based multimarker approach for the detection of the stress status induced by pollution in coastal marine environments in view of ecotoxicological biomonitoring and assessment application. Cellular morphometric alterations was paralleled by the analysis of standardized biomarkers such as lysosomal membrane destabilization, and genotoxocity biomarkers such as micronuclei and binuclated cells frequencies were investigated. The study was carried out by means of a transplanting experiment in the field, using caged organisms from an initial population exposed in the field in two multi-impacted coastal sites of the central Mediterranean area, Bagnoli in the eastern Tyrrhenian Sea and Augusta-Melilli-Priolo in the western Ionian Sea. Capo Miseno (NA) for the Tyrrhenian area and Brucoli (ME) for the Ionian area were chosen as control sites.Hemocyte enlargement and filopodial elongation increased frequencies were observed in organisms exposed to the impacted sites. These morphometric alterations showed strong agreement with the lysosomal membrane destabilization and biomarkers of genotoxicity, suggesting their usefulness in detecting the pollutant-induced stress syndrome related to genotoxic damage.

Abstract 678: Investigating the mechanisms involved in HMGB1-dependent DNA uptake and STING activation in dendritic cells

Abstract Background: The cGAS-STING pathway is critical for the development of anti-tumor immunity. Activation of host STING can occur by sensing of extracellular DNA by intratumoral dendritic cells (DCs). After chemotherapy and under conditions of chromosomal instability, tumor cells release double-stranded DNA. We have previously shown DCs are able to internalize DNA in a dynamin-dependent manner, suggesting that a membrane receptor might mediate DNA endocytosis. DNA uptake also requires the presence of the high mobility group box protein 1 (HMGB1). HMGB1 has multiple known receptors such as TLR2, TLR4, RAGE and CD24. However, whether these receptors are involved in DNA engulfment by DCs remains unknown. Here, we sought to elucidate the mechanisms facilitating DNA uptake and subsequent STING activation in DCs. Methods: Bone marrow derived (BM)DCs obtained from wild type (WT), RAGE, TLR2 or TLR4-deficient mice, as well as and WT or CD24-deficient MutuDC1940 cells, were cultured in the presence or absence of Cy5-labeled plasmid DNA, a recombinant Flag-tagged HMGB1 protein (rHMGB1) and/or DMXAA. Cy5-DNA uptake, Flag-HMGB1 binding and IRF3 phosphorylation was analyzed by flow cytometry or immunofluorescence microscopy. MutuDC1940 cells were also treated with plasmid DNA, rHMGB1, tumor debris (HS) and/or L-Leucyl-L-Leucine methyl ester (LLOMe) and galectin-3 clustering was quantified by immunofluorescence. Statistically significant differences were determined by ANOVA test. Results: DCs take-up Cy5-DNA when rHMGB1 was added, and it was HMGB1 dose-dependent, supporting HMGB1 as a key factor for DNA uptake. We found that TLR2, TLR4, RAGE or CD24 deficient DCs were capable of internalizing Cy5-DNA in the presence of rHMGB1 to the same extent as WT DCs, indicating that none of these receptors are necessary for HMGB1-mediated DNA uptake. DNA-HMGB1 stimulation triggered IRF3 phosphorylation in DCs, suggesting extracellular DNA enters the cytosol and is sensed by cGAS, leading to STING activation. In support of this, DNA-HMGB1 treatment induced an increase in lysosomal membrane permeabilization, as measured by the extent of galectin-3 clusters within cells. Conclusion: HMGB1 promotes DNA uptake by DCs independently of TLR2, TLR4, RAGE or CD24, suggesting other receptor(s) mediate the process of HMGB1-dependent DNA internalization. Further, DNA-HMGB1 induced lysosomal membrane destabilization which may facilitate DNA release into the cytosol for sensing and trigger activation of the cGAS-STING pathway. Citation Format: Daiana P. Celias, Kay Hänggi, Brian Ruffell. Investigating the mechanisms involved in HMGB1-dependent DNA uptake and STING activation in dendritic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 678.

Modulation of innate immune responses in the flame scallop Ctenoides scaber (Born, 1778) caused by exposure to used automobile crankcase oils

The used automobile crankcase oils are potential sources of contaminant elements for the coastal-marine ecosystems, affecting mainly the immunological system of organisms that feed by filtration, e. g. scallops. This study examined the effects of a water-soluble fraction of used automobile crankcase oils (WSF-UACO) on innate cellular- and humoral immune responses of the flame scallop Ctenoides scaber. The scallops were exposed to ascending concentrations of 0, 0.001, 0.01, and 0.1 of WSF-UACO under a static system of aquaria during 7 and 13 d. The viability, haemocyte total count (HTC), lysosomal membrane destabilization (LMD), phagocytosis, and protein concentration in hemolymph samples withdrawn taken from the blood sinus as well as lysozyme activity of the digestive gland were measured as immune endpoints. A decrease in cellular immune competence in scallops exposed to WSF-UACO was observed, with significant impairment of viability, HTC, and phagocytosis. LMD index increased about exposure concentrations, and plasma protein concentrations augmented to 0.01 and 0.1% during 13 d. Lysozyme activity increased in scallops exposed to WSF-UVCO during 7 d, to level off in the chronic period. Lysozyme activity and enhanced plasma proteins could act as compensatory responses when cell parameters tend to fall, helping to the regulation of microbial microflora and possible invasion of pathogenic microbes as well as defense against xenobiotics. The results demonstrate that the immunological responses of C. scaber are highly sensitive to the complex chemical mixture of contaminants, and it could be used for evaluating biological risks of hazardous xenobiotics in tropical marine environments. Republic of Ecuador

Ceramide Aminoethylphosphonate as a New Molecular Target for Pore-Forming Aegerolysin-Based Protein Complexes.

Ostreolysin A6 (OlyA6) is a 15 kDa protein produced by the oyster mushroom (Pleurotus ostreatus). It belongs to the aegerolysin family of proteins and binds with high affinity to the insect-specific membrane sphingolipid, ceramide phosphoethanolamine (CPE). In concert with its partnering protein with the membrane-attack-complex/perforin domain, pleurotolysin B (PlyB), OlyA6 can form bicomponent 13-meric transmembrane pores in artificial and biological membranes containing the aegerolysin lipid receptor, CPE. This pore formation is the main underlying molecular mechanism of potent and selective insecticidal activity of OlyA6/PlyB complexes against two economically important coleopteran plant pests: the western corn rootworm and the Colorado potato beetle. In contrast to insects, the main sphingolipid in cell membranes of marine invertebrates (i.e., molluscs and cnidarians) is ceramide aminoethylphosphonate (CAEP), a CPE analogue built on a phosphono rather than the usual phosphate group in its polar head. Our targeted lipidomic analyses of the immune cells (hemocytes) of the marine bivalve, the mussel Mytilus galloprovincialis, confirmed the presence of 29.0 mol% CAEP followed by 36.4 mol% of phosphatidylcholine and 34.6 mol% of phosphatidylethanolamine. Further experiments showed the potent binding of OlyA6 to artificial lipid vesicles supplemented with mussel CAEP, and strong lysis of these vesicles by the OlyA6/PlyB mixture. In Mytilus haemocytes, short term exposure (max. 1 h) to the OlyA6/PlyB mixture induced lysosomal membrane destabilization, decreased phagocytic activity, increased Annexin V binding and oxyradical production, and decreased levels of reduced glutathione, indicating rapid damage of endo-lysosomal and plasma membranes and oxidative stress. Our data suggest CAEP as a novel high-affinity receptor for OlyA6 and a target for cytolytic OlyA6/PlyB complexes.

Investigation of toxicity effect of TiCN coated on 304 SS and 410 SS substrates in rat fibroblasts and B-lymphocytes.

In the present study, TiCN thin films were coated on AISI 304 and AISI 410 stainless steel (SS) substrates by Cathodic Arc Physical Vapor Deposition method. TiCN-coated substrates were confirmed by the XRD analysis results. Dense morphology and fine-grained surface of TiCN film were established by SEM images. Cellular toxicity of the coated 304 SS and 410 SS substrates was investigated in the fibroblasts and B-lymphocyte. In respect to that, we have shown coated substrates cytotoxicity, oxidative stress as well as cell viability, reactive oxygen species (ROS), lipid peroxidation (MDA), protein carbonyl, glutathione oxidase (GSSG), and glutathione reductase (GSH) assessment, releasing cytochrome c (Cytc), lysosomal membrane destabilization (AO) may lead to cell death signaling. Our results showed that the coated 304 SS and 410 SS substrates induced cells dysfunction via a significant increase in ROS production, MDA (P<0.01 and P<0.001), protein carbonyl (P<0.05), and GSSG (P<0.05 and P<0.01) that correlated to cytochrome c release (P<0.01). In addition, increased disturbance in oxidative phosphorylation was also shown by the decrease in cell viability (P<0.001) and GSH (P<0.01 and P<0.001) in the coated 304 SS and 410 SS substrates-treated fibroblast and B-lymphocytes. The coated 304 SS and 410 SS substrates contacted cells and trafficked to the lysosomes and this is followed by lysosomal damage, leading to apoptosis/Necrosis. Our results indicated that these materials cause cellular dysfunction and subsequent oxidative stress leading to cognitive impairment in the rat fibroblasts and B-lymphocytes cells.

Abstract PO-110: Plant isolate dibenzyl trisulfide induces caspase-independent death in triple negative breast cancer cells derived from patients of West African descent

Abstract Patients with triple negative breast cancer (TNBC) possess tumors that lack estrogen receptor, progesterone receptor, and human epidermal growth receptor expression. While these patients traditionally receive chemotherapy to combat this aggressive breast cancer subtype, others use natural remedies. Dibenzyl trisulfide (DTS) is derived from Petiveria alliacea, a perennial shrub that grows in tropical regions of the world. Many TNBC patients residing in the tropics are of West African descent. Therefore, we evaluated the anticancer actions of DTS in TNBC cells, including those derived from patients of West African descent. We found that DTS inhibited TNBC cell viability, migration and proliferation in a dose-dependent manner. Interestingly, DTS blocked the propensity of pro-carcinogen benzo-A-pyrene to induce proliferation of immortalized breast epithelial cells. Moreover, we found that DTS induced early apoptosis in TNBC cells, which was only partially attenuated following pretreatment with pan-caspase inhibitor zVAD-fmk. Though DTS induced pro-apoptotic gene and protein expression along with PARP cleavage, it failed to produce appreciable caspase 3 cleavage and promote significant apoptotic body formation. This suggests that this plant isolate induces caspase-independent and non-apoptotic death of TNBC cells. Furthermore, DTS promoted lysosomal membrane destabilization and cathepsin B release in TNBC cells. Taken together, DTS exhibits promising chemotherapeutic and chemopreventive ability by inducing non-apoptotic TNBC cell death and thwarting TNBC progression, supporting its evaluation in clinical trials as an agent to combat TNBC among patients of West African descent. Citation Format: Jonathan Wooten, Shaniece Wooten, Cristina Araújo, Joyce Aja, Nicole Mavingire, Rupika Delgoda, Eileen Brantley. Plant isolate dibenzyl trisulfide induces caspase-independent death in triple negative breast cancer cells derived from patients of West African descent [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-110.

Biological responses and toxicopathic effects elicited in Solea senegalensis juveniles by waterborne exposure to benzo[a]pyrene

Polycyclic aromatic hydrocarbons (PAHs) are priority contaminants in coastal and estuarine ecosystems under anthropogenic pressure. Although PAHs tend to accumulate in the sediment, toxicity for benthic flat fish such as soles may be caused by PAHs released from the sediment to the water column. Within this context, the present investigation aims at recognizing toxicopathic effects elicited after waterborne exposure to benzo[a]pyrene B[a]P, a model individual PAH compound, in juvenile Solea senegalensis. Sole juveniles were exposed to various concentrations of waterborne B[a]P for 3 and 7 days. Brain, liver, gills and gonad were the target tissues selected to determine biochemical and lysosomal biomarkers, and histopathology. Biological responses and toxicopathic effects were consistent with B[a]P concentration and exposure time. From day 3, hepatic catalase inhibition indicated potential oxidative effects of B[a]P. At day 7, contaminant exposure produced hepatic glutathione-S-transferase induction at low concentrations and inhibition at higher levels, evidencing a bell-shaped response. A clear gradient in lysosomal membrane destabilisation was observed in relation with B[a]P concentrations. Histopathological lesions were more frequent at day 7 and at higher contaminant levels. It seems that environmentally relevant waterborne concentrations of B[a]P (1000 ng/l) would suffice to cause toxicopathic effects on sole juveniles in relatively short exposure times. In agreement, the Integrative Biological Response index (IBR/n) indicated a dose-dependent decline in health condition upon exposure to B[a]P (IBR/nHighB[a]P > IBR/nMidB[a]P > IBR/nLowB[a]P > IBR/nDMSO > IBR/nControl). Overall, changes in antioxidant enzymes activity, lysosomal biomarkers and gill and liver histopathology are responsive early-warning signs of health disturbance in sole juveniles exposed to waterborne PAHs.

ZnO, Ag and ZnO-Ag nanoparticles exhibit differential modes of toxic and oxidative action in hemocytes of mussel Mytilus galloprovincialis

The present study investigates the cytotoxic and oxidative effects of custom-made nanoparticles (NPs) on hemocytes of Mytilus galloprovincialis, utilizing hemolymph serum (HS) as exposure medium. Specifically, hemocyte lysosomal membrane destabilization (in terms of neutral red retention time assay/NRRT), superoxide anion (O2−), nitric oxide (NO, in terms of nitrites) and lipid peroxidation content (in terms of malondialdehyde/MDA equivalents) were determined in cells treated for 1 h with different concentrations (0.1–50 μg mL−1) of ZnO NPs, Ag NPs and ZnO-Ag NPs, as well as AgNO3 and/or ZnCl2 (bulk ions, respectively). According to the results, Ag NPs were more cytotoxic than ZnO-Ag NPs and/or ZnO NPs, while NRRT values observed in AgNO3 treated cells were lower than those of ZnCl2. Furthermore, high levels of both O2− and MDA were detected in cells treated with Ag NPs, ZnO-Ag NPs, and AgNO3 at concentrations lower than 5 μg mL−1, while high NO generation was observed only in cells treated with 5–25 μg mL−1 of ZnO NPs or ZnCl2. Despite the absence of data, regarding the formation of NP-serum protein corona complexes that could mediate NP surface energy and uptake efficiency, the current study firstly revealed that ZnO NPs, probably via their surface charge, particle agglomeration, and NP Zn+ release could promote an immune-related generation of O2− and NO via the respiratory burst stimulation, a process that is questioned in the case of Ag NPs and/or ZnO-Ag NPs. Moreover, ZnO-Ag NP interaction with biological membranes and their oxidative mode of action seemed to be regulated by the release and the antagonistic/synergistic response of its ionic counterparts (ZnO+ and Ag+), but further studies are needed to elucidate the oxidative mode of action of NP metal ions in complex NP mixtures.

The Response of the Planorbid Snail Isidorella newcombi to Chronic Copper Exposure Over a 28-Day Period: Linking Mortality, Cellular Biomarkers, and Reproductive Responses.

The native freshwater gastropod Isidorella newcombi attacks the roots of developing rice plants in southern Australia and is controlled using copper sulphate. The apparent tolerance of this species to moderate levels of copper (Cu) exposure led us to investigate its potential usefulness as a biomonitor species. To assess its response to chronic Cu exposure, adult I. newcombi were exposed to 0-120µgL-1 of Cu for 28days. Lethal and sublethal responses were investigated. The relationships between subcellular biomarkers and life history traits also were explored. At exposure concentrations of 60µgL-1 Cu and above, 100% mortality was observed during the 28-day exposure period. In these treatments, there was an exposure concentration dependent decrease in the time that the snails survived. In the surviving snails, there was an exposure concentration-dependent increase in tissue Cu concentration. In the snails exposed to Cu concentrations above 15µgL-1, no eggs were produced during the final week of copper exposure, indicating that populations would not persist at Cu concentrations above 15µgL-1. The general stress biomarker lysosomal membrane destabilisation (LD) indicated organisms exposed to 10µgL-1 Cu and above were experiencing Cu induced stress. This suggests that LD could act as an early warning system for responses at higher levels of biological organisation in I. newcombi exposed to copper.

Effects of lysosomal iron involvement in the mechanism of mitochondrial apoptosis on postmortem muscle protein degradation

This study investigated the effect of lysosomal iron involvement in the mechanism of mitochondrial apoptosis on bovine muscle protein degradation during postmortem aging. Six crossbred cattle were studied to evaluate intracellular reactive oxygen species (ROS), antioxidant enzyme activity, lysosomal membrane stability, mitochondrial dysfunction-induced apoptosis, desmin and troponin-T degradation in both control and iron chelator desferrioxamine (DFO) groups. Results showed that lysosomal iron induced ROS accumulation and lysosomal membrane destabilization by decreasing the antioxidant enzyme activity (P < 0.05). Subsequently, lysosomal dysfunction mediated by iron increased mitochondrial membrane permeability and decreased mitochondrial membrane potential, thereby enhancing Bid and cytochrome c release and caspase-9/-3 activation (P < 0.05). Ultimately, lysosomal iron mediated lysosomal-mitochondrial apoptosis increased the postmortem bovine muscle desmin and troponin-T degradation (P < 0.05). The results indicated that lysosomal iron contributes to postmortem meat tenderization through the lysosomal-mitochondrial dysfunction-induced apoptosis pathway.

Trifluoperazine an Antipsychotic Drug and Inhibitor of Mitochondrial Permeability Transition Protects Cytarabine and Ifosfamide-Induced Neurotoxicity.

The link between Ca2+ dysregulation, mitochondria damages, oxidative stress and cellular derangement is particularly evident in neurotoxicity induced by chemotherapeutic agents. In the current study, we investigated effects of trifluoperazine (TFP) as an inhibitor of calmodulin against the cytotoxicity induced by cytarabine (Ara-C) and Ifosfamide (IFOS) on isolated rat neurons and also the mechanisms involved in this toxicity. Isolated rat neurons were pretreated with TFP (100 µM) for 5 min at 37°C, then Ara-C (226 µM) and IFOS (290 µM) were added in separate experiments. After 3 h, the cytotoxicity, reactive oxygen species (ROS), lysosomal membrane destabilization, mitochondrial membrane potential (MMP), lipid peroxidation (LP), glutathione (GSH) and glutathione disulfide (GSSG) levels were measured. Ara-C and IFOS treatments caused a significant decrease in cellular viability, which was accompanied by ROS generation, GSSG/GSH ratio, lipid peroxidation and lysosomal and mitochondrial damages. On the other hand, TFP (100 µM) pre-treatment attenuated Ara-C and IFOS -induced decrease in cell viability. In addition, TFP (100 µM) pre-treatment significantly protected against Ara-C and IFOS -induced increase in ROS generation, lysosomal and mitochondrial damages, lipid peroxidation levels and decrease in GSH/GSSG ratio. Our data provided insights into the mechanism of protection by TFP against Ara-C and IFOS neurotoxicity, which is related, to neuronal ROS formation and mitochondrial damages.

Biological responses and toxicopathic effects elicited in Solea senegalensis juveniles on exposure to contaminated sediments under laboratory conditions

Whole-sediment toxicity assays contribute to elucidating the intricate association between the presence of contaminants in sediments and their toxicopathic effects in benthic fish. In the present study, Solea senegalensis juveniles were exposed under laboratory conditions to contaminated whole-sediments for 7 and 28 days. Sediments were obtained from a low to moderately polluted estuary, a highly polluted harbour and from the mixture of both field-collected sediments. Biometry data were recorded. Liver, brain, gills, and gonads were dissected out and processed to determine markers of oxidative stress, neurotoxicity and lysosomal biomarkers, and histopathology. Analyses of sediment granulometry and chemical profiles indicated different degrees of toxicity and suggested a distinct release of pollutants from each sediment in relation with their physicochemical properties. Interestingly, biological responses were in agreement with contaminant levels reported in source sediments. The most distinct toxicopathic effects were detected upon exposure to the harbour's sediment and particularly on day 28. Overall, enhanced hepatic glutathione-S-transferase activity and lysosomal enlargement were detected in all experimental groups, demonstrating a toxic effect from all sediments whilst catalase inhibition, lysosomal membrane destabilisation, changes in lysosomal content and liver histopathology were most pronounced in soles exposed to the harbour's sediment. The Integrative Biomarker Response index (IBR/n) evidenced that exposure to the three sediments caused an impact of diverse magnitude in sole health (IBR/nHarbour > IBR/nMixture > IBR/nEstuary). The magnitude of biological responses essentially depended on the presence of contaminants in source sediments, which seemed to be altered by the conditions imposed by whole-sediment toxicity assays.

Integrated characterization and risk management of marine sediments: The case study of the industrialized Bagnoli area (Naples, Italy)

The aim of the present work is to demonstrate the practical importance of a multidisciplinary approach and weighted criteria to synthesize and integrate different typologies of data (or lines of evidence, LOEs), including chemical levels in marine sediments, their bioavailability to specific indicator species, ecotoxicological effects measured through subcellular biomarkers and batteries of bioassays, and potential impacts of pollution on local benthic communities. The area of Bagnoli (Gulf of Naples, Southern Italy) was selected as a model case-study, as it is a coastal area chronically impacted by massive industrial contamination (trace metals and hydrocarbons), and dismissed decades ago without any subsequent remediation or habitat restoration. The results of each LOE were elaborated to provide specific hazard indices before their overall integration in a weight of evidence (WOE) evaluation. Levels of some trace metals and PAHs revealed a severe contamination in the entire study area. Bioavailability of hydrocarbons was evident particularly for high molecular weight PAHs, which also caused significant variations of cellular biomarkers, such as cytochrome P450 metabolization in fish, lysosomal membrane destabilization in mussels, genotoxic effects both in fish and molluscs. The results of a battery of bioassays indicated less marked responses compared to those obtained from chemical and biomarkers analyses, with acute toxicity still present in sediments close to the source of contamination. The analysis of benthic assemblages showed limited evidence of impact in the whole area, indicating a good functioning of local ecosystems at chronic contamination. Overall, the results of this study confirm the need of combining chemical and biological data, the quantitative characterization of various typologies of hazard and the importance of assessing an integrated environmental WOE risk, to orientate specific and scientifically-supported management options in industrialized areas.

Effects of quercetin loaded nanostructured lipid carriers on the paraquat-induced toxicity in human lymphocytes

Paraquat (PQ) as a herbicide and an environmental pollutant with increasing importance due to its toxicity to humans and animals. This study aimed to evaluate the protective and antioxidant activity of quercetin loaded Nanostructured Lipid Carriers (QNLC) against toxicity induced by PQ. Blood lymphocytes were prepared using Ficoll polysaccharide and subsequently by gradient centrifugation. The QNLC was prepared using an ultra-sonication method, which was characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM).The viability, reactive oxygen species (ROS), lipid peroxidation (LPO), mitochondrial membrane potential (MMP), lysosome membrane integrity, Bax and Bcl2 gene expression were evaluated in human isolated lymphocytes.The results showed spherical QNLCs with nano-size range (52.7 nm) and high drug encapsulation efficiency (98.5% -96%). The results also indicated that PQ induced cell death, as well as ROS production, decreased by QNLC in human lymphocytes. Also, QNLC meaningfully restored MMP reduction, lysosomal membrane destabilization, and lipid peroxidation and were capable of preventing PQ-treated change in Bax and Bcl2 gene expression. We report that QNLC, have a significantly higher capacity to prevent PQ-induced toxicity than Q itself. It is suggested that the QNLC is a promising antioxidant for drug delivery to be used as a therapeutic and prophylactic agent for PQ poisoning.