Hepatotoxicity of Cadmium Telluride Quantum Dots Induced by Mitochondrial Dysfunction.

The aim of this study was to investigate the detailed mechanisms of hepatotoxicity induced by cadmium telluride quantum dots (CdTe-QDs) in BALB/c mice after intravenous injection. The study investigated oxidative stress, apoptosis, and effects on mitochondria as potential mechanistic events to elucidate the observed hepatotoxicity. Oxidative stress in the liver, induced by CdTe-QD exposure, was demonstrated by depletion of total glutathione, an increase in superoxide dismutase activity, and changes in the gene expression of several oxidative stress-related biomarkers. Furthermore, CdTe-QD treatment led to apoptosis in the liver via both intrinsic and extrinsic apoptotic pathways. Effects on mitochondria were evidenced by the enlargement and increase in the number of mitochondria in hepatocytes of treated mice. CdTe-QDs also caused changes in the levels and gene expression of electron transport chain enzymes, depletion of ATP, and an increase in the level of the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a regulator of mitochondrial biogenesis. The findings from this study suggest that CdTe-QDs-induced hepatotoxicity might have originated from mitochondrial effects which resulted in oxidative stress and apoptosis in the liver cells. This study provides insight into the biological effects of CdT-QDs at the tissue level and the detailed mechanisms of their toxicity in animals. The study also provides important data for bridging the gap between in vitro and in vivo testing and risk assessment of these NPs.

Biodistribution and Systemic Effects in Mice Following Intravenous Administration of Cadmium Telluride Quantum Dot Nanoparticles.

Quantum dots (QDs) are engineered nanoparticles (NPs) of semiconductor structure that possess unique optical and electronic properties and are widely used in biomedical applications; however, their risks are not entirely understood. This study investigated the tissue distribution and toxic effects of cadmium telluride quantum dots (CdTe-QDs) in male BALB/c mice for up to 1 week after single-dose intravenous injections. CdTe-QDs were detected in the blood, lung, heart, liver, spleen, kidney, testis and brain. Most CdTe-QDs accumulated in the liver, followed by the spleen and kidney. At high doses, exposure to CdTe-QDs resulted in mild dehydration, lethargy, ruffled fur, hunched posture, and body weight loss. Histological analysis of the tissues, upon highest dose exposures, revealed hepatic hemorrhage and necrotic areas in the spleen. The sera of mice treated with high doses of CdTe-QDs showed significant increases in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin levels, as well as a reduction in albumin. CdTe-QD exposure also led to a reduced number of platelets and elevated total white blood cell counts, including monocytes and neutrophils, serum amyloid A, and several pro-inflammatory cytokines. These results demonstrated that the liver is the main target of CdTe-QDs and that exposure to CdTe-QDs leads to hepatic and splenic injury, as well as systemic effects, in mice. By contrast, cadmium chloride (CdCl2), at an equivalent concentration of cadmium, appeared to have a different pharmacokinetic pattern from that of CdTe-QDs, having minimal effects on the aforementioned parameters, suggesting that cadmium alone cannot fully explain the toxicity of CdTe-QDs.

Incorporating Aptamers in the Multiple Analyte Profiling Assays (xMAP): Detection of C-Reactive Protein.

Aptamers are short oligonucleotide sequences used in detection systems because of their high affinity binding to a variety of macromolecules. With the introduction of aptamers over 25 years ago came the exploration of their use in many different applications as a substitute for antibodies. Aptamers have several advantages; they are easy to synthesize, can bind to analytes for which it is difficult to obtain antibodies, and in some cases bind better than antibodies. As such, aptamer applications have significantly expanded as an adjunct to a variety of different immunoassay designs. The Multiple-Analyte Profiling (xMAP) technology developed by Luminex Corporation commonly uses antibodies for the detection of analytes in small sample volumes through the use of fluorescently coded microbeads. This technology permits the simultaneous detection of multiple analytes in each sample tested and hence could be applied in many research fields. Although little work has been performed adapting this technology for use with apatmers, optimizing aptamer-based xMAP assays would dramatically increase the versatility of analyte detection. We report herein on the development of an xMAP bead-based aptamer/antibody sandwich assay for a biomarker of inflammation (C-reactive protein or CRP). Protocols for the coupling of aptamers to xMAP beads, validation of coupling, and for an aptamer/antibody sandwich-type assay for CRP are detailed. The optimized conditions, protocols and findings described in this research could serve as a starting point for the development of new aptamer-based xMAP assays.

Composition and pathogenic potential of a microbial bioremediation product used for crude oil degradation.

A microbial bioremediation product (MBP) used for large-scale oil degradation was investigated for microbial constituents and possible pathogenicity. Aerobic growth on various media yielded >108 colonies mL-1. Full-length 16S rDNA sequencing and fatty acid profiling from morphologically distinct colonies revealed ≥13 distinct genera. Full-length 16S rDNA library sequencing, by either Sanger or long-read PacBio technology, suggested that up to 21% of the MBP was composed of Arcobacter. Other high abundance microbial constituents (>6%) included the genera Proteus, Enterococcus, Dysgonomonas and several genera in the order Bacteroidales. The MBP was most susceptible to ciprofloxacin, doxycycline, gentamicin, and meropenam. MBP exposure of human HT29 and A549 cells caused significant cytotoxicity, and bacterial growth and adherence. An acellular MBP filtrate was also cytotoxic to HT29, but not A549. Both MBP and filtrate exposures elevated the neutrophil chemoattractant IL-8. In endotracheal murine exposures, bacterial pulmonary clearance was complete after one-week. Elevation of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α, and chemokines KC and MCP-1 occurred between 2h and 48h post-exposure, followed by restoration to baseline levels at 96h. Cytokine/chemokine signalling was accompanied by elevated blood neutrophils and monocytes at 4h and 48h, respectively. Peripheral acute phase response markers were maximal at 24h. All indicators examined returned to baseline values by 168h. In contrast to HT29, but similar to A549 observations, MBP filtrate did not induce significant murine effects with the indicators examined. The results demonstrated the potentially complex nature of MBPs and transient immunological effects during exposure. Products containing microbes should be scrutinized for pathogenic components and subjected to characterisation and quality validation prior to commercial release.

Toxicological evaluation of representative silver nanoparticles in macrophages and epithelial cells.

Silver nanoparticles (Ag-NPs) are highly relevant for human and environmental exposure due to their widespread use in consumer and medical products and various applications. Thus, there is a need for evaluating potential toxicity of these NPs. The objective of this study was to investigate the toxic effects of the OECD (Organization for Economic Co-operation and Development) representative Ag-NPs, NM300K, in mouse macrophage J774A.1 and human colonic epithelial HT29 cells, using multiple endpoint assays. Exposure of test cells to different concentrations (1-250 µg/mL; total silver content) of NM300K for 24h showed a dose-dependent decrease in cell viability. At high doses, NM300K altered cell shape and induced the formation of vacuolar structures, as examined by confocal and electron microscopy. Moreover, NM300K induced inflammation as evidenced by the elevated levels of pro-inflammatory cytokines. Finally, high doses of NM300K led to increased production of reactive oxygen species and induction of oxidative stress, leading to oxidative DNA damage and apoptosis in test cells. At equivalent silver concentrations, NM300K were less cytotoxic than AgNO3. However, the similar patterns in the effects of NM300K and AgNO3 throughout the assessed toxicological endpoints suggest that Ag(+) released from these NPs by dissolution could be a primary contributor to toxicity. This study is among the first to characterize the potential toxicity of OECD representative AgNPs in vitro, and provides additional insight into the biological mechanisms associated with Ag-NP toxicity.

Virulence Attributes and Host Response Assays for Determining Pathogenic Potential of Pseudomonas Strains Used in Biotechnology.

Pseudomonas species are opportunistically pathogenic to humans, yet closely related species are used in biotechnology applications. In order to screen for the pathogenic potential of strains considered for biotechnology applications, several Pseudomonas strains (P.aeruginosa (Pa), P.fluorescens (Pf), P.putida (Pp), P.stutzeri (Ps)) were compared using functional virulence and toxicity assays. Most Pa strains and Ps grew at temperatures between 28°C and 42°C. However, Pf and Pp strains were the most antibiotic resistant, with ciprofloxacin and colistin being the most effective of those tested. No strain was haemolytic on sheep blood agar. Almost all Pa, but not other test strains, produced a pyocyanin-like chromophore, and caused cytotoxicity towards cultured human HT29 cells. Murine endotracheal exposures indicated that the laboratory reference strain, PAO1, was most persistent in the lungs. Only Pa strains induced pro-inflammatory and inflammatory responses, as measured by elevated cytokines and pulmonary Gr-1 -positive cells. Serum amyloid A was elevated at ≥ 48 h post-exposure by only some Pa strains. No relationship was observed between strains and levels of peripheral leukocytes. The species designation or isolation source may not accurately reflect pathogenic potential, since the clinical strain Pa10752 was relatively nonvirulent, but the industrial strain Pa31480 showed comparable virulence to PAO1. Functional assays involving microbial growth, cytotoxicity and murine immunological responses may be most useful for identifying problematic Pseudomonas strains being considered for biotechnology applications.

Phagocytosis-coupled flow cytometry for detection and size discrimination of anionic polystyrene particles.

Flow cytometry was evaluated for its capacity to detect and distinguish a wide size range (20-2000 nm) of fluorescent polystyrene particles (PSPs). Side scatter and fluorescence parameters could predict dispersed PSP sizes down to 200 nm, but the forward scatter parameter was not discriminatory. Confocal microscopy of flow-sorted fractions confirmed that dispersed PSPs appeared as a single sharp peak on fluorescence histograms, whereas agglomerated PSPs were detected as smaller adjacent peaks. Particles as small as 200 nm could also be detected by flow cytometry after they were first phagocytized by J774A.1 murine macrophages. Confocal microscopy demonstrated that these PSPs were internalized within the cytoplasm. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and calcein-AM (acetoxymethyl ester) assays showed that they were not cytotoxic. Internalized PSP size correlated to both cellular side scatter (R(2)=0.9821) and fluorescence intensity (R(2)=0.9993). Furthermore, PSPs of various sizes could be distinguished when J774A.1 cells were loaded with a single size of PSP and mixed with cells containing other sizes. However, spectra of cells loaded with a mixture of PSP sizes resembled those containing only the largest PSP. These data demonstrate the capacity and limitations of phagocytosis-coupled flow cytometry to distinguish between dispersed and agglomerated states and detect a wide size range of particles.

Mitochondrial Toxicity of Cadmium Telluride Quantum Dot Nanoparticles in Mammalian Hepatocytes.

There are an increasing number of studies indicating that mitochondria are relevant targets in nanomaterial-induced toxicity. However, the underlying mechanisms by which nanoparticles (NPs) interact with these organelles and affect their functions are unknown. The aim of this study was to investigate the effects of cadmium telluride quantum dot (CdTe-QD) NPs on mitochondria in human hepatocellular carcinoma HepG2 cells. CdTe-QD treatment resulted in the enlargement of mitochondria as examined with transmission electron microscopy and confocal microscopy. CdTe-QDs appeared to associate with the isolated mitochondria as detected by their inherent fluorescence. Further analyses revealed that CdTe-QD caused disruption of mitochondrial membrane potential, increased intracellular calcium levels, impaired cellular respiration, and decreased adenosine triphosphate synthesis. The effects of CdTe-QDs on mitochondrial oxidative phosphorylation were evidenced by changes in levels and activities of the enzymes of the electron transport chain. Elevation of peroxisome proliferator-activated receptor-γ coactivator levels after CdTe-QD treatment suggested the effects of CdTe-QDs on mitochondrial biogenesis. Our results also showed that the effects of CdTe-QDs were similar or greater to those of cadmium chloride at equivalent concentrations of cadmium, suggesting that the toxic effects of CdTe-QDs were not solely due to cadmium released from the NPs. Overall, the study demonstrated that CdTe-QDs induced multifarious toxicity by causing changes in mitochondrial morphology and structure, as well as impairing their function and stimulating their biogenesis.

Development of a bead-based aptamer/antibody detection system for C-reactive protein.

A multiplexing bead-based platform provides an approach for the development of assays targeting specific analytes for biomonitoring and biosensing applications. Multi-Analyte Profiling (xMAP) assays typically employ a sandwich-type format using antibodies for the capture and detection of analytes of interest, and the system permits the simultaneous quantitation of multiple targets. In this study, an aptamer/antibody assay for the detection of C-reactive protein (CRP) was developed. CRP is an acute phase marker of inflammation whose elevated basal levels are correlated with an increased risk for a number of pathologies. For this assay, an RNA aptamer that binds CRP was conjugated to beads to act as the capture agent. Biotinylated anti-CRP antibody coupled to fluorescently labeled streptavidin was used for quantification of CRP. The detection limit of the CRP assay was 0.4 mg/L in diluted serum. The assay was then used to detect spiked CRP samples in the range of 0.4 to 10mg/L in diluted serum with acceptable recoveries (extrapolated values of 70-130%), including that of a certified reference material (129% recovery). The successful incorporation of the CRP aptamer into this platform demonstrates that the exploration of other aptamer-target systems could increase the number of analytes measurable using xMAP-type assays.

Critical experimental parameters related to the cytotoxicity of zinc oxide nanoparticles.

The increasing use of zinc oxide nanoparticles (ZnO-NPs) has raised concerns about their potential hazards to human and environmental health. In this study, the characterization and cytotoxicity of two ZnO-NPs products (Z-COTE and Z-COTE HP1) were investigated. The zinc content of Z-COTE and Z-COTE HP1 was 82.5 ± 7.3 and 80.1 ± 3.5 %, respectively. Both ZnO-NP samples contained sub-cytotoxic levels of iron and copper, and silicon was detected from the surface coating of Z-COTE HP1. All samples were highly agglomerated, and the primary particles appeared as variable polyhedral structures. There was no significant difference in size distribution or average diameter of Z-COTE (53 ± 23 nm) and Z-COTE HP1 (54 ± 26 nm). A dose-dependent cytotoxicity was observed 24 h after exposure to ZnO-NPs, and monocytes were more sensitive than lung epithelial cells or lymphoblasts in both human and mouse cells. There was a significant difference in cytotoxicity between nano- and fine-forms, but only at the threshold cytotoxic dose with cellular metabolism assays. Compared to uncoated ZnO-NPs, the surface coating with triethoxycaprylylsilane marginally attenuated cellular oxidative stress and protected cellular metabolic activity. These results demonstrate the importance of model cell type, dose selection, and cytotoxicity assessment methodology to accurately evaluate the potential toxicity of various nanoparticles in vitro.

Nanosilver cytotoxicity in rainbow trout (Oncorhynchus mykiss) erythrocytes and hepatocytes.

Silver nanoparticles (AgNPs) are present in a multitude of consumer and medical products; however, the toxicity of AgNPs is not fully understood. This research aimed to elucidate the relationship between AgNP cytotoxicity and oxidative stress and damage in rainbow trout (Oncorhynchus mykiss) hepatocytes and erythrocytes in comparison to silver ions (Ag(+)). Generally the cytotoxicity of AgNPs and Ag(+) was similar, such that both silver types generated reactive oxygen species, decreased glutathione levels, and decreased activities of glutathione reductase and glutathione-S-transferase. Nonetheless, the two silver types had different cellular targets; AgNPs increased lipid peroxidation without apparent uptake into the cells whereas Ag(+) increased DNA damage. Furthermore, the toxicity of both silver types was generally decreased in cells treated with cysteine while treatment with buthionine sulfoximine increased the toxicity of both silver types.

Cadmium telluride quantum dots cause oxidative stress leading to extrinsic and intrinsic apoptosis in hepatocellular carcinoma HepG2 cells.

The mechanisms of toxicity related to human hepatocellular carcinoma HepG2 cell exposures to cadmium telluride quantum dots (CdTe-QDs) were investigated. CdTe-QDs caused cytotoxicity in HepG2 cells in a dose- and time-dependent manner. Treated cells showed an increase in reactive oxygen species (ROS). Altered antioxidant levels were demonstrated by depletion of reduced glutathione (GSH), a decreased ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) and an increased NF-E2-related Factor 2 (Nrf2) activation. Enzyme assays showed that superoxide dismutase (SOD) activity was elevated whereas catalase (CAT) and glutathione-S-transferase (GST) activities were depressed. Further analyses revealed that CdTe-QD exposure resulted in apoptosis, indicated by changes in levels of caspase-3 activity, poly ADP-ribose polymerase (PARP) cleavage and phosphatidylserine externalization. Extrinsic apoptotic pathway markers such as Fas levels and caspase-8 activity increased as a result of CdTe-QD exposure. Involvement of the intrinsic/mitochondrial apoptotic pathway was indicated by decreased levels of B-cell lymphoma 2 (Bcl2) protein and mitochondrial cytochrome c, and by increased levels of mitochondrial Bcl-2-associated X protein (Bax) and cytosolic cytochrome c. Further, mitogen-activated protein kinases (MAPKs) such as c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinases (Erk1/2), and p38 were all activated. Our findings reveal that CdTe-QDs cause oxidative stress, interfere with antioxidant defenses and activate protein kinases, leading to apoptosis via both extrinsic and intrinsic pathways. Since the effects of CdTe-QDs on selected biomarkers were similar or greater compared to those of CdCl2 at equivalent concentrations of cadmium, the study suggests that the toxicity of CdTe-QDs arises from a combination of the effects of cadmium and ROS generated from the NPs.

Cadmium telluride quantum dot nanoparticle cytotoxicity and effects on model immune responses to Pseudomonas aeruginosa.

This study examines dose effects of cadmium telluride quantum dots (CdTe-QDs) from two commercial sources on model macrophages (J774A.1) and colonic epithelial cells (HT29). Effects on cellular immune signalling responses were measured following sequential exposure to QDs and Pseudomonas aeruginosa strain PA01. At CdTe-QD concentrations between 10(-2) and 10 µg/ml, cells exhibited changes in metabolism and morphology. Confocal imaging revealed QD internalisation and changes in cell-cell contacts, shapes and internal organisations. QD doses below 10(-2) µg/ml caused no observed effects. When QD exposures at 10(-7) to 10(-3) µg/ml preceded PA01 (10(7) bacteria/ml) challenges, there were elevated cytotoxicity (5-22%, p < 0.05) and reduced levels (two- to fivefold, p < 0.001) of nitric oxide (NO), TNF-α, KC/CXC-1 and IL-8, compared with PA01 exposures alone. These results demonstrate that exposures to sub-toxic levels of CdTe-QDs can depress cell immune-defence functions, which if occurred in vivo would likely interfere with normal neutrophil recruitment for defence against bacteria.

Comparison of the virulence potential of Acinetobacter strains from clinical and environmental sources.

Several Acinetobacter strains have utility for biotechnology applications, yet some are opportunistic pathogens. We compared strains of seven Acinetobacter species (baumannii, Ab; calcoaceticus, Ac; guillouiae, Ag; haemolyticus, Ah; lwoffii, Al; junii, Aj; and venetianus, Av-RAG-1) for their potential virulence attributes, including proliferation in mammalian cell conditions, haemolytic/cytolytic activity, ability to elicit inflammatory signals, and antibiotic susceptibility. Only Ah grew at 10(2) and 10(4) bacteria/well in mammalian cell culture medium at 37°C. However, co-culture with colonic epithelial cells (HT29) improved growth of all bacterial strains, except Av-RAG-1. Cytotoxicity of Ab and Ah toward HT29 was at least double that of other test bacteria. These effects included bacterial adherence, loss of metabolism, substrate detachment, and cytolysis. Only Ab and Ah exhibited resistance to killing by macrophage-like J774A.1 cells. Haemolytic activity of Ah and Av-RAG-1 was strong, but undetectable for other strains. When killed with an antibiotic, Ab, Ah, Aj and Av-RAG-1 induced 3 to 9-fold elevated HT29 interleukin (IL)-8 levels. However, none of the strains altered levels of J774A.1 pro-inflammatory cytokines (IL-1ß, IL-6 and tumor necrosis factor-α). Antibiotic susceptibility profiling showed that Ab, Ag and Aj were viable at low concentrations of some antibiotics. All strains were positive for virulence factor genes ompA and epsA, and negative for mutations in gyrA and parC genes that convey fluoroquinolone resistance. The data demonstrate that Av-RAG-1, Ag and Al lack some potentially harmful characteristics compared to other Acinetobacter strains tested, but the biotechnology candidate Av-RAG-1 should be scrutinized further prior to widespread use.

Early murine immune responses from endotracheal exposures to biotechnology-related Bacillus strains.

An immunology-based in vivo screening regime was used to assess the potential pathogenicity of biotechnology-related microbes. Strains of Bacillus cereus (Bc), Bacillus subtilis (Bs), Bacillus thuringiensis (Bt), and Bt commercial products (CPs) were tested. Balb/c mice were endotracheally instilled with purified spores, diluted CP, or vegetative cells (VC) (live or dead). Exposed mice were evaluated for changes in behavioral and physical symptoms, bacterial clearance, pulmonary granulocytes, and pulmonary and circulatory pyrogenic cytokines (interleukins (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α), as well as acute phase biomarkers (fibrinogen and serum amyloid A). Except for some differences in clearance rates, no marked effects were observed in mice exposed to any spore at 10(6) or 10(7) colony forming units (cfu). In contrast, live Bc or Bt VCs (10(5) or 10(6) cfu) produced shock-like symptoms (lethargy, hunched appearance, ruffled fur, and respiratory distress), and 11-200-fold elevations in pyrogenic cytokines at 2-h post-exposure. In the study, 4-h effects included increased lethargy, ocular discharge, and 1.5-4-fold rise in circulatory acute phase markers, but no indications of recovery. Bs VC did not produce any changes in symptoms or biomarkers. After 2 or 4 h of exposure to dead VC, increases of only plasma IL-1? and TNF-α (4.6- and 12.4-fold, respectively) were observed. These findings demonstrate that purified spores produced no marked effects in mice compared to that of metabolically active bacteria. This early screening regime was successful in distinguishing the pathogenicity of the different Bacillus species, and might be useful for assessing the relative hazard potential of other biotechnology-related candidate strains.