Intracellular oxidative stress and cadmium ions release induce cytotoxicity of unmodified cadmium sulfide quantum dots

ObjectiveTo fully understand the cytotoxicity of after-degradation QDs, we synthesized CdS QDs and investigated its toxicity mechanism.MethodsBiomimetic method was proposed to synthesize cadmium sulfide (CdS) QDs. Thereafter MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay was conducted to evaluate their cytotoxicity. To investigate the toxicity mechanism, we subsequently conducted intracellular reactive oxygen species (ROS) measurement with DCFH-DA, glutathione (GSH) measurement with DTNB, and cellular cadmium assay using atomic absorption spectrometer. Microsized CdS were simultaneously tested as a comparison.ResultsMTT assay results indicated that CdS QDs are more toxic than microsized CdS especially at concentrations below 40 μg/ml. While microsized CdS did not trigger ROS elevation, CdS QDs increase ROS by 20–30% over control levels. However, they both deplete cellular GSH significantly at the medium concentration of 20 μg/ml. In the presence of NAC, cells are partially protected from CdS QDs, but not from microsized particles. Additionally, nearly 20% of cadmium was released from CdS nanoparticles within 24 h, which also accounts for QDs’ toxicity.ConclusionIntracellular ROS production, GSH depletion, and cadmium ions (Cd²⁺) release are possible mechanisms for CdS QDs’ cytotoxicity. We also suggested that with QD concentration increasing, the principal toxicity mechanism changes from intracellular oxidative stress to Cd²⁺ release.     

Differential effects of silver nanoparticles on DNA damage and DNA repair gene expression in Ogg1-deficient and wild type mice

Due to extensive use in consumer goods, it is important to understand the genotoxicity of silver nanoparticles (AgNPs) and identify susceptible populations. 8-Oxoguanine DNA glycosylase 1 (OGG1) excises 8-oxo-7,8-dihydro-2-deoxyguanine (8-oxoG), a pro-mutagenic lesion induced by oxidative stress. To understand whether defects in OGG1 is a possible genetic factor increasing an individual’s susceptibly to AgNPs, we determined DNA damage, genome rearrangements, and expression of DNA repair genes in Ogg1-deficient and wild type mice exposed orally to 4?mg/kg of citrate-coated AgNPs over a period of 7?d. DNA damage was examined at 3 and 7?d of exposure and 7 and 14?d post-exposure. AgNPs induced 8-oxoG, double strand breaks (DSBs), chromosomal damage, and DNA deletions in both genotypes. However, 8-oxoG was induced earlier in Ogg1-deficient mice and 8-oxoG levels were higher after 7-d treatment and persisted longer after exposure termination. AgNPs downregulated DNA glycosylases Ogg1, Neil1, and Neil2 in wild type mice, but upregulated Myh, Neil1, and Neil2 glycosylases in Ogg1-deficient mice. Neil1 and Neil2 can repair 8-oxoG. Thus, AgNP-mediated downregulation of DNA glycosylases in wild type mice may contribute to genotoxicity, while upregulation thereof in Ogg1-deficient mice could serve as an adaptive response to AgNP-induced DNA damage. However, our data show that Ogg1 is indispensable for the efficient repair of AgNP-induced damage. In summary, citrate-coated AgNPs are genotoxic in both genotypes and Ogg1 deficiency exacerbates the effect. These data suggest that humans with genetic polymorphisms and mutations in OGG1 may have increased susceptibility to AgNP-mediated DNA damage.     

Connexin32 deficiency is associated with liver injury,inflammation and oxidative stress in experimental non‐alcoholic steatohepatitis

Non‐alcoholic steatohepatitis is a highly prevalent liver pathology featured by hepatocellular fat deposition and inflammation. Connexin32, which is the major building block of hepatocellular gap junctions, has a protective role in hepatocarcinogenesis and is downregulated in chronic liver diseases. However, the role of connexin32 in non‐alcoholic steatohepatitis remains unclear. Connexin32^?/? mice and their wild‐type littermates were fed a choline‐deficient high‐fat diet. The manifestation of non‐alcoholic steatohepatitis was evaluated based on a battery of clinically relevant read‐outs, including histopathological examination, diverse indicators of inflammation and liver damage, in‐depth lipid analysis, assessment of oxidative stress, insulin and glucose tolerance, liver regeneration and lipid‐related biomarkers. Overall, more pronounced liver damage, inflammation and oxidative stress were observed in connexin32^?/? mice compared to wild‐type animals. No differences were found in insulin and glucose tolerance measurements and liver regeneration. However, two lipid‐related genes, srebf1 and fabp3, were upregulated in Cx32^?/? mice in comparison with wild‐type animals. These findings suggest that connexin32‐based signalling is not directly involved in steatosis as such, but rather in the sequelae of this process, which underlie progression of non‐alcoholic steatohepatitis.     

Cadmium sulfate and CdTe-quantum dots alter DNA repair in zebrafish (Danio rerio) liver cells

Increasing use of quantum dots (QDs) makes it necessary to evaluate their toxicological impacts on aquatic organisms, since their contamination of surface water is inevitable. This study compares the genotoxic effects of ionic Cd versus CdTe nanocrystals in zebrafish hepatocytes. After 24 h of CdSO₄ or CdTe QD exposure, zebrafish liver (ZFL) cells showed a decreased number of viable cells, an accumulation of Cd, an increased formation of reactive oxygen species (ROS), and an induction of DNA strand breaks. Measured levels of stress defense and DNA repair genes were elevated in both cases. However, removal of bulky DNA adducts by nucleotide excision repair (NER) was inhibited with CdSO₄ but not with CdTe QDs. The adverse effects caused by acute exposure of CdTe QDs might be mediated through differing mechanisms than those resulting from ionic cadmium toxicity, and studying the effects of metallic components may be not enough to explain QD toxicities in aquatic organisms.     

Identification of small molecules inducing apoptosis by cell-based assay using fission yeast deletion mutants

The cell-based assay using yeast deletion mutants has been recognized as an efficient analysis to discover therapeutic compounds and reveal their mode of action. In this study, S. pombe deletion mutants-based HTS screening was carried out to identify potential anti-cancer agents. The NCI chemical library of 5700 compounds was screened using kit strains, which consisted of S. pombe mutants harboring deletions in genes involved in DNA repair and mitotic control. During the screening, we identified 40 compounds conferring growth inhibition of S. pombe. Their anti-tumorigenic properties were examined by phenotypic effect on S. pombe, flow cytometry and apoptosis analysis of human cancer. Here, we report hit compounds inducing apoptosis for development of anti-cancer agents suggesting that S. pombe deletion mutants are useful in identifying potential anti-cancer agents in human cancer therapeutics.     

1,3,5,8‐Tetrahydroxy‐9H‐xanthen‐9‐one exerts its antiageing effect through the regulation of stress‐response genes and the MAPK signaling pathway

The antioxidative effects of 30 xanthone derivatives (XDs) ( XD‐ n , n = 1–30) in HepG2 cells were evaluated by the cellular antioxidant activity assay. Results showed that all XDs were antioxidants and 1,3,5,8‐tetrahydroxy‐9H‐xanthen‐9‐one ( XD‐2 ) was the most active antioxidant. The all‐oxygenated substituted xanthones extended the lifespan of wild‐type N2 nematodes under normal culture conditions and XD‐2 was the best one. XD‐2 eliminated excessive intracellular reactive oxygen species and enhanced the expression levels and activities of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase. XD‐2 inhibited the H₂O₂‐increased phosphorylation levels of c‐JUN N‐terminal kinase, extracellular signal‐regulated kinase, and p38 in HepG2 cells. In vivo, XD‐2 also extended the lifespan of wild‐type N2 nematodes under oxidative stress induced by paraquat, but failed in extending the lifespan of CF1038 (daf‐16 deletion) and AY102 (pmk‐1 deletion) mutant nematodes. It was revealed by real‐time polymerase chain reaction that the genes daf‐16, sir‐2.1, akt‐1, and age‐1 were all inhibited by paraquat stimuli, while XD‐2 reversed these inhibitions; in contrast, paraquat stimuli upregulated both the skn‐1 and pmk‐1 genes. However, treatment by  XD‐2 further increased the levels of both genes. These pieces of evidence implied that XD‐2 promotes longevity through endogenous signaling pathways rather than through the antioxidative activity alone. Taken all together, it may be concluded that XD‐2 is a promising antiageing agent.     

Volatile organic compounds from fungi isolated after hurricane katrina induce developmental defects and apoptosis in a Drosophila melanogaster model

In previous work, our laboratory developed a Drosophila model for studying the adverse effects of fungal volatile organic compounds (VOCs) emitted by growing cultures of molds. In this report, we have extended these studies and compared the toxic effects of fungal VOCs emitted from living cultures of four molds isolated after Hurricane Katrina from a flooded home in New Orleans. Strains of Aspergillus, Mucor, Penicillium, and Trichoderma were grown with wild‐type larvae and the toxic effects of volatile products on the developmental stages of Drosophila larvae were evaluated. Furthermore, heterozygous mutants of Drosophila carrying the apoptotic genes, reaper and dronc, were used to assess the role of apoptosis in fungal VOCs mediated toxicity. Third‐instar larvae of Drosophila carrying these apoptotic genes were exposed to fungal VOCs emitted from growing mold cultures for 10 days. The larval strains carrying apoptopic genes survived longer than the control wild type larvae; moreover, of those that survived, heterozygous reaper and dronc strains progressed to pupae and adult phases more rapidly, suggesting that fungal VOCs may induce apoptotic changes in flies. These data lend support to the use of Drosophila as an inexpensive and genetically versatile toxicological model to investigate the mechanistic basis for some of the human illnesses/symptoms associated with exposure to mold‐contaminated indoor air, especially after hurricanes. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 614–620, 2015.     

Antiageing properties of Damaurone D in Caenorhabditis elegans

Objectives

This study was conducted to evaluate the longevity potential of damaurone D (DaD), a component of the damask rose, in the animal model Caenorhabditis elegans .

Methods

To investigate the effect of DaD on the longevity, lifespan assay was carried out. Fluorescence intensity of transgenic mutants was quantified to test the expression levels of stress proteins. A genetic study using single gene knockout mutants was designed to determine the target genes of DaD.

Key findings

DaD prolonged the mean lifespan of wild‐type nematodes by 16.7% under normal conditions and also improved their stress endurance under thermal, osmotic, and oxidative stress conditions. This longevity‐promoting effect could be attributed to in vivo antioxidant capacity and its up‐regulating effects on the expressions of stress‐response proteins such as SOD‐3 and HSP‐16.2. In addition, DaD treatment attenuated food intake, body length, lipofuscin accumulation and age‐dependent decline of motor ability. Gene‐specific mutant studies showed the involvement of genes such as daf‐2 , age‐1 , and daf‐16 .

Conclusions

These results suggest that DaD has beneficial effects on the longevity, and thus it can be a valuable plant origin lead compound for the development of nutraceutical preparations targeting ageing and ageing‐related diseases.

     

In vitro immunotoxicology of quantum dots and comparison with dissolved cadmium and tellurium

The increasing use of products derived from nanotechnology has raised concerns about their potential toxicity, especially at the immunocompetence level in organisms. This study compared the immunotoxicity of cadmium sulfate/cadmium telluride (CdS/Cd‐Te) mixture quantum dots (QDs) and their dissolved components, cadmium chloride (CdCl₂)/sodium telluride (NaTeO₃) salts, and a CdCl₂/NaTeO₃ mixture on four animal models commonly used in risk assessment studies: one bivalve (Mytilus edulis), one fish (Oncorhynchus mykiss), and two mammals (mice and humans). Our results of viability and phagocytosis biomarkers revealed that QDs were more toxic than dissolved metals for blue mussels. For other species, dissolved metals (Cd, Te, and Cd‐Te mixture) were more toxic than the nanoparticles (NPs). The most sensitive species toward QDs, according to innate immune cells, was humans (inhibitory concentration [IC₅₀] = 217 μg/mL). However, for adaptative immunity, lymphoblastic transformation in mice was decreased for small QD concentrations (EC₅₀ = 4 μg/mL), and was more sensitive than other model species tested. Discriminant function analysis revealed that blue mussel hemocytes were able to discriminate the toxicity of QDs, Cd, Te, and Cd‐Te mixture (Partial Wilk's λ = 0.021 and p < 0.0001). For rainbow trout and human cells, the immunotoxic effects of QDs were similar to those obtained with the dissolved fraction of Cd and Te mixture. For mice, the toxicity of QDs markedly differed from those observed with Cd, Te, and dissolved Cd‐Te mixture. The results also suggest that aquatic species responded more differently than vertebrates to these compounds. The results lead to the recommendation that mussels and mice were most able to discriminate the effects of Cd‐based NPs from the effects of dissolved Cd and Te at the immunocompetence level. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 9–25, 2015.     

Multiple substrate-binding sites are retained in cytochrome P450 3A4 mutants with decreased cooperativity

1. The basis of decreased cooperativity in substrate binding in the cytochrome P450 3A4 mutants F213W, F304W, and L211F/D214E was studied with fluorescence resonance energy transfer and absorbance spectroscopy.

2. Although in the wild type enzyme, the absorbance changes reflecting the interactions with 1-pyrenebutanol exhibit a Hill coefficient (n_H) around 1.7 (S₅₀?=?11.7 µM), the mutants showed no cooperativity (n_H ≤ 1.1) with unchanged S₅₀ values.

3. Contrary to the premise that the mutants lack one of the two binding sites, the mutants exhibited at least two substrate binding events. The high-affinity interaction is characterized by a dissociation constant (K_D) ≤ 1.0 µM, whereas the K_D of the second binding has the same magnitude as the S₅₀.

4. Theoretical analysis of a two-step binding model suggests that n_H values may vary from 1.1 to 2.2 depending on the amplitude of the spin shift caused by the first binding event.

5. Alteration of cooperativity in the mutants is caused by a partial displacement of the “spin-shifting” step. Although in the wild type the spin shift occurs in the ternary complex only, the mutants exhibit some spin shift on binding of the first substrate molecule.

     

Low Toxicity of Deoxynivalenol-3-Glucoside in Microbial Cells

Host plants excrete a glucosylation enzyme onto the plant surface that changes mycotoxins derived from fungal secondary metabolites to glucosylated products. Deoxynivalenol-3-glucoside (DON3G) is synthesized by grain uridine diphosphate-glucosyltransferase, and is found worldwide, although information on its toxicity is lacking. Here, we conducted growth tests and DNA microarray analysis to elucidate the characteristics of DON3G. The Saccharomyces cerevisiae PDR5 mutant strain exposed to DON3G demonstrated similar growth to the dimethyl sulfoxide control, and DNA microarray analysis revealed limited differences. Only 10 genes were extracted, and the expression profile of stress response genes was similar to that of DON, in contrast to metabolism genes like SER3, which encodes 3-phosphoglycerate dehydrogenase. Growth tests with Chlamydomonas reinhardtii also showed a similar growth rate to the control sample. These results suggest that DON3G has extremely low toxicity to these cells, and the glucosylation of mycotoxins is a useful protective mechanism not only for host plants, but also for other species.     

Deletion of the distal COOH-terminus of the A2B adenosine receptor switches internalization to an arrestin- and clathrin-independent pathway and inhibits recycling

Background and purpose:

We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A_2B adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5′-(N-ethylcarboxamido)-adenosine.

Experimental approach:

The trafficking of the wild type A_2B adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy.

Key results:

The wild type A_2B adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln³²⁵-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu³³⁰-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln³²⁵-stop, Ser³²⁶-stop and Phe³²⁸-stop receptors. Following internalization, the wild type A_2B adenosine receptor recycled rapidly to the cell surface, whereas the Gln³²⁵-stop receptor did not recycle.

Conclusions and implications:

Deletion of the COOH-terminus of the A_2B adenosine receptor beyond Leu³³⁰ switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A_2B adenosine receptor following prolonged agonist addition.     

Acute toxicity and tissue distribution of CdSe/CdS‐MPA quantum dots after repeated intraperitoneal injection to mice

Quantum dots (QDs) are novel tools with multiple biological and medical applications because of their superior photoemission and photostability characteristics. However, leaching of toxic metals from QDs is of great concern. Therefore, for the successful application of QDs in bioscience, it is essential to understand their biological fate and toxicity. We investigated toxicological effects and tissue distribution of mercaptopropionic acid‐conjugated cadmium selenide/cadmium sulfide (CdSe/CdS‐MPA) QDs after repeated intraperitoneal injection into BALB/c mice. The mice were injected every 3 days with various doses of QDs (0, 5, 10 and 25 mg kg^?1). The subsequent effects of QDs on plasma levels of various biomarkers were evaluated at different time points (at 0, 1, 4, 7, 10, 13 and 15 days). Various tissue samples (spleen, liver, lung, kidneys, brain, heart and thymus) were collected for toxicity analysis, distribution testing, histopathological examination and inflammation assessment. No abnormal clinical signs or behaviors were recorded but the body weight of mice treated with 25 mg kg^?1 QDs was significantly decreased from day 7 compared with control mice. QDs were observed in the liver, spleen, lung and kidneys, but not in brain or heart. Significantly higher levels of lactate dehydrogenase and nicotinamide adenine dinucleotide phosphate oxidase were found in the plasma, liver and spleen. Histopathological examination did not show any tissue toxicity but the levels of interleukin‐6, a pro‐inflammatory marker, were increased in the plasma, liver and spleen. All of these findings provide insight into the observed toxicological effect levels and tissue‐specific distribution of CdSe/CdS‐MPA QDs. Copyright © 2012 John Wiley & Sons, Ltd.     

ATP‐binding cassette transporter A1 (ABCA1) promotes arsenic tolerance in human cells by reducing cellular arsenic accumulation

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Heme oxygenase expression as a biomarker of exposure to amphiphilic polymer-coated CdSe/ZnS quantum dots

Abstract

Because of their unique optical properties, quantum dots (QDs) have become a preferred system for ultrasensitive detection and imaging. However, since QDs commonly contain Cd and other heavy metals, concerns have been raised regarding their toxicity. QDs are thus commonly synthesised with a ZnS cap structure and/or coated with polymeric stabilisers. We recently synthesised amphiphilic polymer-coated tri-n-octylphosphine oxide - poly(maleic anhydride-alt-1-tetradecene (TOPO-PMAT) QDs, which are highly stable in aqueous environments. The effects of these QDs on viability and stress response in five cell lines of mouse and human origins are reported here. Human and mouse macrophages and human kidney cells readily internalised these QDs, resulting in modest toxicity. TOPO-PMAT QD exposure was highly correlated with the induction of the stress response protein heme oxygenase-1 (HMOX1). Other stress biomarkers (glutamate cysteine ligase modifier subunit, NAD(P)H, necrosis) were only moderately affected. HMOX1 may thus be a useful biomarker of TOPO-QDOT QD exposure across cell types and species.     

Gene expression profile changes induced by acute toxicity of [C16mim]Cl in loach (Paramisgurnus dabryanus)

Ionic liquids (ILs) are widely used as reaction media in various commercial applications. Many reports have indicated that most ILs are poorly decomposed by microorganisms and are toxic to aquatic organisms. In this study, differential gene expression profiling was conducted using a suppression subtraction hybridization cDNA library from hepatic tissue of the loach (Paramisgurnus dabryanus) after exposure to 1‐hexadecyl‐3‐methylimidazolium chloride ([C₁₆mim]Cl), a representative IL. Two hundred and fifty‐nine differentially expressed candidate genes, whose expression was altered by >2.0‐fold by the [C₁₆mim]Cl treatment, were identified, including 127 upregulated genes and 132 downregulated genes. A gene ontology analysis of the known genes isolated in this study showed that [C₁₆mim]Cl‐responsive genes were involved in cell cycle, stimulus response, defense response, DNA damage response, oxidative stress responses, and other biological responses. To identify candidate genes that may be involved in [C₁₆mim]Cl‐induced toxicity, 259 clones were examined by Southern blot macroarray hybridization, and 20 genes were further characterized using quantitative real‐time polymerase chain reaction. Finally, six candidate genes were selected, including three DNA damage response genes, two toxic substance metabolic genes, and one stress protein gene. Our results indicate that these changes in gene expression are associated with [C₁₆mim]Cl‐induced toxicity, and that these six candidate genes can be promising biomarkers for detecting [C₁₆mim]Cl‐induced toxicity. Therefore, this study demonstrates the use of a powerful assay to identify genes potentially involved in [C₁₆mim]Cl toxicity, and it provides a foundation for the further study of related genes and the molecular mechanism of [C₁₆mim]Cl toxicity. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 404–416, 2017.     

Neuroglobin mitigates mitochondrial impairments induced by acute inhalation of combustion smoke in the mouse brain

Abstract

Context: Acute inhalation of combustion smoke adversely affects brain homeostasis and energy metabolism. We previously showed that overexpressed neuroglobin (Ngb), neuron specific globin protein, attenuates the formation of smoke inhalation-induced oxidative DNA damage, in vivo, in the mouse brain, while others reported protection by Ngb in diverse models of brain injury, mainly involving oxidative stress and hypoxic/ischemic insults.

Objective: To determine to what extent elevated Ngb ameliorates post smoke-inhalation brain bioenergetics and homeostasis in Ngb overexpressing transgenic mouse.

Methods: Smoke inhalation induced changes in bioenergetics were measured in the wild type and Ngb transgene mouse brain. Modulations of mitochondrial respiration were analyzed using the Seahorse XF24 flux analyzer and changes in cytoplasmic energy metabolism were assessed by measuring enzymatic activities and lactate in the course of post smoke recovery.

Results: Cortical mitochondria from Ngb transgene, better maintained ATP synthesis-linked oxygen consumption and unlike wild type mitochondria did not increase futile oxygen consumption feeding the proton leak, reflecting lesser smoke-induced mitochondrial compromise. Measurements revealed lesser reduction of mitochondrial ATP content and lesser compensatory increases in cytosolic energy metabolism, involving pyruvate kinase and lactate dehydrogenase activities as well as cytosolic lactate levels. Additionally, induction of c-Fos, the early response gene and key neuronal stress sensor, was attenuated in Ngb transgene compared to wild type brain after smoke.

Conclusion: Considered together, these differences reflect lesser perturbations produced by acute inhalation of combustion smoke in the Ngb overexpressing mouse, suggesting that Ngb mitigates mitochondrial dysfunction and neurotoxicity and raises the threshold of smoke inhalation-induced brain injury.