Investigation of sex-based differences in the immunotoxicity of silver nanoparticles.

The growing application of silver nanoparticles (AgNPs) in consumer, healthcare, and industrial products has raised concern over potential health implications due to increasing exposure. The evaluation of the immune response to nanomaterials is one of the key criteria to assess their biocompatibility. There are well-recognized sex-based differences in innate and adaptive immune responses. However, there is limited information available using human models. The aim was to investigate the potential sex-based differences in immune functions after exposure to AgNPs using human peripheral blood mononuclear cells (PBMCs) and plasma from healthy donors. These functions include inflammasome activation, cytokine expression, leukocyte proliferation, chemotaxis, plasma coagulation, and complement activation. AgNPs were characterized by dynamic light scattering and transmission electron microscopy. Inflammasome activation by AgNPs was measured after 6- and 24-hours incubations. AgNPs-induced inflammasome activation was significantly higher in the females, especially for the 6-hour exposure. No sex-based differences were observed for Ag ions controls. Younger donors exhibited significantly more inflammasome activation than older donors after 24-hours exposure. IL-10 was significantly suppressed in males and females after exposure. AgNPs suppressed leukocyte proliferation similarly in males and females. No chemoattractant effects, no alterations in plasma coagulation, or activation of the complement were observed after AgNPs exposure. In conclusion, the results highlight that there are distinct sex-based differences in inflammasome activation after exposure to AgNPs in human PBMCs. The results highlight the importance of considering sex-based differences in inflammasome activation induced by exposure to AgNPs in any future biocompatibility assessment for products containing AgNPs.

Radiation impacts on toxicity of cobalt-chromium (CoCr) implant debris.

Particulate and soluble debris are generated by mechanical and non-mechanical degradation of implanted medical devices. Debris containing cobalt and chromium (CoCr) is known to cause adverse biological reactions. Implant-related complications are often diagnosed using radiography, which results in more frequent patient exposure to ionizing radiation. The aim of this study was to evaluate the potential for increased toxicity due to combined radiation and CoCr exposure. This was investigated using a controlled in vitro model consisting of commercially available CoCr debris that was generated from components of hip replacements and human cell lines relevant to the joint environment: endothelial HMEC-1 and synovial SW982. Particle sizes and shapes were heterogenous. Cells tended to internalize smaller particles, as observed by electron microscopy. Indicators of toxicity were measured after short (24 h after radiation) or extended (12-14 d after radiation) exposure timelines. In the short-term, CoCr reduced cell viability, increased apoptosis, and increased oxidative stress. The effects of radiation were not apparent until the timeline was extended. CoCr and radiation reduced cell survival, with both additive and synergistic effects. Mechanisms for reduced survival included rapid cell death caused by CoCr and senescence caused by radiation. In conclusion, results showed combined toxicological effects of CoCr and radiation at the doses and timelines used for this in vitro model. These observations warrant further investigation using other experimental models to determine translational impact.

Development of benchmark datasets for text mining and sentiment analysis to accelerate regulatory literature review.

In the field of regulatory science, reviewing literature is an essential and important step, which most of the time is conducted by manually reading hundreds of articles. Although this process is highly time-consuming and labor-intensive, most output of this process is not well transformed into machine-readable format. The limited availability of data has largely constrained the artificial intelligence (AI) system development to facilitate this literature reviewing in the regulatory process. In the past decade, AI has revolutionized the area of text mining as many deep learning approaches have been developed to search, annotate, and classify relevant documents. After the great advancement of AI algorithms, a lack of high-quality data instead of the algorithms has recently become the bottleneck of AI system development. Herein, we constructed two large benchmark datasets, Chlorine Efficacy dataset (CHE) and Chlorine Safety dataset (CHS), under a regulatory scenario that sought to assess the antiseptic efficacy and toxicity of chlorine. For each dataset, ∼10,000 scientific articles were initially collected, manually reviewed, and their relevance to the review task were labeled. To ensure high data quality, each paper was labeled by a consensus among multiple experienced reviewers. The overall relevance rate was 27.21% (2,663 of 9,788) for CHE and 7.50% (761 of 10,153) for CHS, respectively. Furthermore, the relevant articles were categorized into five subgroups based on the focus of their content. Next, we developed an attention-based classification language model using these two datasets. The proposed classification model yielded 0.857 and 0.908 of Area Under the Curve (AUC) for CHE and CHS dataset, respectively. This performance was significantly better than permutation test (p < 10E-9), demonstrating that the labeling processes were valid. To conclude, our datasets can be used as benchmark to develop AI systems, which can further facilitate the literature review process in regulatory science.

DNase I Induces Other Endonucleases in Kidney Tubular Epithelial Cells by Its DNA-Degrading Activity.

Endonuclease-mediated DNA fragmentation is both an immediate cause and a result of apoptosis and of all other types of irreversible cell death after injury. It is produced by nine enzymes including DNase I, DNase 2, their homologs, caspase-activated DNase (CAD) and endonuclease G (EndoG). The endonucleases act simultaneously during cell death; however, regulatory links between these enzymes have not been established. We hypothesized that DNase I, the most abundant of endonucleases, may regulate other endonucleases. To test this hypothesis, rat kidney tubular epithelial NRK-52E cells were transfected with the DNase I gene or its inactive mutant in a pECFP expression vector, while control cells were transfected with the empty vector. mRNA expression of all nine endonucleases was studied using real-time RT-PCR; DNA strand breaks in endonuclease genes were determined by PCR and protein expression of the enzymes was measured by Western blotting and quantitative immunocytochemistry. Our data showed that DNase I, but not its inactive mutant, induces all other endonucleases at varying time periods after transfection, causes DNA breaks in endonuclease genes, and elevates protein expression of several endonucleases. This is the first evidence that endonucleases seem to be induced by the DNA-degrading activity of DNase I.

Mechanism of graphene-induced cytotoxicity: Role of endonucleases.

Graphene, a crystalline allotrope or carbon, presents numerous useful properties; however, its toxicity is yet to be determined. One of the most dramatic and irreversible toxic abilities of carbon nanomaterials is the induction of DNA fragmentation produced by endogenous cellular endonucleases. This study demonstrated that pristine graphene exposed to cultured kidney tubular epithelial cells is capable of inducing DNA fragmentation measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which is usually associated with cell death. TUNEL (cell death) and endonuclease activity measured using a near infrared fluorescence probe was significantly higher in cells containing graphene aggregates detected by Raman spectroscopy. The elevation of TUNEL coincided with the increased abundance of heme oxygenase 1 (HO-1), heat shock protein 90 (HSP90), active caspase-3 and endonucleases (deoxyribonuclease I [DNase I] and endonuclease G [EndoG]), as measured by quantitative immunocytochemistry. Specific inhibitors for HO-1, HSP90, caspase-3, DNase I and EndoG almost completely blocked the DNA fragmentation induced by graphene exposure. Therefore, graphene induces cell death through oxidative injury, caspase-mediated and caspase-independent pathways; and endonucleases DNase I and EndoG are important for graphene toxicity. Inhibition of these pathways may ameliorate cell injury produced by graphene. Copyright © 2017 John Wiley & Sons, Ltd.

Photoacoustic in vitro flow cytometry for nanomaterial research.

Conventional flow cytometry is a versatile tool for drug research and cell characterization. However, it is poorly suited for quantification of non-fluorescent proteins and artificial nanomaterials without the use of additional labeling. The rapid growth of biomedical applications for small non-fluorescent nanoparticles (NPs) for drug delivery and contrast and therapy enhancement, as well as research focused on natural cell pigments and chromophores, demands high-throughput quantification methods for the non-fluorescent components. In this work, we present a novel photoacoustic (PA) fluorescence flow cytometry (PAFFC) platform that combines NP quantification though PA detection with conventional in vitro flow cytometry sample characterization using fluorescence labeling. PAFFC simplifies high-throughput analysis of cell-NP interactions, optimization of targeted nanodrugs, and NP toxicity assessment, providing a direct correlation between NP uptake and characterization of toxicity markers for every cell.

Mutant Profilin1 transgenic mice recapitulate cardinal features of motor neuron disease.

The recent identification of profilin1 mutations in 25 familial ALS cases has linked altered function of this cytoskeleton-regulating protein to the pathogenesis of motor neuron disease. To investigate the pathological role of mutant profilin1 in motor neuron disease, we generated transgenic lines of mice expressing human profilin1 with a mutation at position 118 (hPFN1G118V). One of the mouse lines expressing high levels of mutant human PFN1 protein in the brain and spinal cord exhibited many key clinical and pathological features consistent with human ALS disease. These include loss of lower (ventral horn) and upper motor neurons (corticospinal motor neurons in layer V), mutant profilin1 aggregation, abnormally ubiquitinated proteins, reduced choline acetyltransferase (ChAT) enzyme expression, fragmented mitochondria, glial cell activation, muscle atrophy, weight loss, and reduced survival. Our investigations of actin dynamics and axonal integrity suggest that mutant PFN1 protein is associated with an abnormally low filamentous/globular (F/G)-actin ratio that may be the underlying cause of severe damage to ventral root axons resulting in a Wallerian-like degeneration. These observations indicate that our novel profilin1 mutant mouse line may provide a new ALS model with the opportunity to gain unique perspectives into mechanisms of neurodegeneration that contribute to ALS pathogenesis.

Serotonin transporter protects the placental cells against apoptosis in caspase 3-independent pathway.

Serotonin (5-HT) and its specific transporter, SERT play important roles in pregnancy. Using placentas dissected from 18d gestational SERT-knock out (KO), peripheral 5-HT (TPH1)-KO, and wild-type (WT) mice, we explored the role of 5-HT and SERT in placental functions in detail. An abnormal thick band of fibrosis and necrosis under the giant cell layer in SERT-KO placentas appeared only moderately in TPH1-KO and minimally present in WT placentas. The majority of the changes were located at the junctional zone of the placentas in SERT. The etiology of these findings was tested with TUNEL assays. The placentas from SERT-KO and TPH1-KO showed 49- and 8-fold increase in TUNEL-positive cells without a concurrent change in the DNA repair or cell proliferation compared to WT placentas. While the proliferation rate in the embryos of TPH1-KO mice was 16-fold lower than the rate in gestational age matched embryos of WT or SERT-KO mice. These findings highlight an important role of continuous 5-HT signaling on trophoblast cell viability. SERT may contribute to protecting trophoblast cells against cell death via terminating the 5-HT signaling which changes cell death ratio in trophoblast as well as proliferation rate in embryos. However, the cell death in SERT-KO placentas is in caspase 3-independent pathway.

Impact of Hydroxychloroquine on Atherosclerosis and Vascular Stiffness in the Presence of Chronic Kidney Disease.

Cardiovascular disease is the largest cause of morbidity and mortality among patients with chronic kidney disease (CKD) and end-stage kidney disease, with nearly half of all deaths attributed to cardiovascular disease. Hydroxychloroquine (HCQ), an anti-inflammatory drug, has been shown to have multiple pleiotropic actions relevant to atherosclerosis. We conducted a proof-of-efficacy study to evaluate the effects of hydroxychloroquine in an animal model of atherosclerosis in ApoE knockout mice with and without chronic kidney disease. Forty male, 6-week-old mice were divided into four groups in a 2 x 2 design: sham placebo group; sham treatment group; CKD placebo group; and CKD treatment group. CKD was induced by a two-step surgical procedure. All mice received a high-fat diet through the study duration and were sacrificed after 16 weeks of therapy. Mice were monitored with ante-mortem ultrasonic echography (AUE) for atherosclerosis and vascular stiffness and with post-mortem histology studies for atherosclerosis. Therapy with HCQ significantly reduced the severity of atherosclerosis in CKD mice and sham treated mice. HCQ reduced the area of aortic atherosclerosis on en face examination by approximately 60% in HCQ treated groups compared to the non-treated groups. Additionally, therapy with HCQ resulted in significant reduction in vascular endothelial dysfunction with improvement in vascular elasticity and flow patterns and better-preserved vascular wall thickness across multiple vascular beds. More importantly, we found that presence of CKD had no mitigating effect on HCQ's anti-atherosclerotic and vasculoprotective effects. These beneficial effects were not due to any significant effect of HCQ on inflammation, renal function, or lipid profile at the end of 16 weeks of therapy. This study, which demonstrates structural and functional protection against atherosclerosis by HCQ, provides a rationale to evaluate its use in CKD patients. Further studies are needed to define the exact mechanisms through which HCQ confers these benefits.

Human UDP-Glucuronosyltransferases: Effects of altered expression in breast and pancreatic cancer cell lines.

Increased aerobic glycolysis and de novo lipid biosynthesis are common characteristics of invasive cancers. UDP-glucuronosyltransferases (UGTs) are phase II drug metabolizing enzymes that in normal cells possess the ability to glucuronidate these lipids and speed their excretion; however, de-regulation of these enzymes in cancer cells can lead to an accumulation of bioactive lipids, which further fuels cancer progression. We hypothesize that UGT2B isoform expression is down-regulated in cancer cells and that exogenous re-introduction of these enzymes will reduce lipid content, change the cellular phenotype, and inhibit cancer cell proliferation. In this study, steady-state mRNA levels of UGT isoforms from the 2B family were measured using qPCR in 4 breast cancer and 5 pancreatic cancer cell lines. Expression plasmids for UGT2B isoforms known to glucuronidate cellular lipids, UGT2B4, 2B7, and 2B15 were transfected into MCF-7 and Panc-1 cells, and the cytotoxic effects of these enzymes were analyzed using trypan blue exclusion, annexin V/PI staining, TUNEL assays, and caspase-3 immunohistochemistry. There was a significant decrease in cell proliferation and a significant increase in the number of dead cells after transfection with each of the 3 UGT isoforms in both cell lines. Cellular lipids were also found to be significantly decreased after transfection. The results presented here support our hypothesis and emphasize the important role UGTs can play in cellular proliferation and lipid homeostasis. Evaluating the effect of UGT expression on the lipid levels in cancer cell lines can be relevant to understanding the complex regulation of cancer cells, identifying the roles of UGTs as "lipid-controllers" in cellular homeostasis, and illustrating their suitability as targets for future clinical therapy development.

Regulation of Apoptotic Endonucleases by EndoG.

Cells contain several apoptotic endonucleases, which appear to act simultaneously before and after cell death by destroying the host cell DNA. It is largely unknown how the endonucleases are being induced and whether they can regulate each other. This study was performed to determine whether apoptotic mitochondrial endonuclease G (EndoG) can regulate expression of other apoptotic endonucleases. The study showed that overexpression of mature EndoG in kidney tubular epithelial NRK-52E cells can increase expression of caspase-activated DNase (CAD) and four endonucleases that belong to DNase I group including DNase I, DNase X, DNase IL2, and DNase γ, but not endonucleases of the DNase 2 group. The induction of DNase I-type endonucleases was associated with DNA degradation in promoter/exon 1 regions of the endonuclease genes. These results together with findings on colocalization of immunostained endonucleases and TUNEL suggest that DNA fragmentation after EndoG overexpression was caused by DNase I endonucleases and CAD in addition to EndoG itself. Overall, these data provide first evidence for the existence of the integral network of apoptotic endonucleases regulated by EndoG.

Novel high-throughput deoxyribonuclease 1 assay.

Deoxyribonuclease I (DNase I), the most active and abundant apoptotic endonuclease in mammals, is known to mediate toxic, hypoxic, and radiation injuries to the cell. Neither inhibitors of DNase I nor high-throughput methods for screening of high-volume chemical libraries in search of DNase I inhibitors are, however, available. To overcome this problem, we developed a high-throughput DNase I assay. The assay is optimized for a 96-well plate format and based on the increase of fluorescence intensity when fluorophore-labeled oligonucleotide is degraded by the DNase. The assay is highly sensitive to DNase I compared to other endonucleases, reliable (Z' ≥ 0.5), and operationally simple, and it has low operator, intraassay, and interassay variability. The assay was used to screen a chemical library, and several potential DNase I inhibitors were identified. After comparison, 2 hit compounds were selected and shown to protect against cisplatin-induced kidney cell death in vitro. This assay will be suitable for identifying inhibitors of DNase I and, potentially, other endonucleases.

Single-walled carbon nanotube and graphene nanodelivery of gambogic acid increases its cytotoxicity in breast and pancreatic cancer cells.

Graphene and single-walled carbon nanotubes were used to deliver the natural low-toxicity drug gambogic acid (GA) to breast and pancreatic cancer cells in vitro, and the effectiveness of this complex in suppressing cellular integrity was assessed. Cytotoxicity was assessed by measuring lactate dehydrogenase release, mitochondria dehydrogenase activity, mitochondrial membrane depolarization, DNA fragmentation, intracellular lipid content, and membrane permeability/caspase activity. The nanomaterials showed no toxicity at the concentrations used, and the antiproliferative effects of GA were significantly enhanced by nanodelivery. The results suggest that these complexes inhibit human breast and pancreatic cancer cells grown in vitro. This analysis represents a first step toward assessing their effectiveness in more complex, targeted, nanodelivery systems.

Circulating tumor cell identification by functionalized silver-gold nanorods with multicolor, super-enhanced SERS and photothermal resonances.

Nanotechnology has been extensively explored for cancer diagnostics. However, the specificity of current methods to identify simultaneously several cancer biomarkers is limited due to color overlapping of bio-conjugated nanoparticles. Here, we present a technique to increase both the molecular and spectral specificity of cancer diagnosis by using tunable silver-gold nanorods with narrow surface-enhanced Raman scattering (SERS) and high photothermal contrast. The silver-gold nanorods were functionalized with four Raman-active molecules and four antibodies specific to breast cancer markers and with leukocyte-specific CD45 marker. More than two orders of magnitude of SERS signal enhancement was observed from these hybrid nanosystems compared to conventional gold nanorods. Using an antibody rainbow cocktail, we demonstrated highly specific detection of single breast cancer cells in unprocessed human blood. By integrating multiplex targeting, multicolor coding, and multimodal detection, our approach has the potential to improve multispectral imaging of individual tumor cells in complex biological environments.