An Xu

Rapid Synthesis of Stable and Functional Conjugates of DNA/Gold Nanoparticles Mediated by Tween 80

Gold nanoparticles conjugated with DNA represent an attractive and alternative platform for broad applications in biosensors, medical diagnostic, and biological analysis. However, current methods to conjugate DNA to gold nanoparticles are time-consuming. In this study, we report a novel approach to rapidly conjugate DNA to gold nanoparticles (AuNPs) to form functional DNA/AuNPs in 2-3 h using Tween 80 as protective agent. With a fluorescence-based technique, we determine that the DNA density on the surface of AuNPs achieves about ∼60 strands per particles, which is comparable to the loading density in the current methods. Moreover, the DNA/AuNPs synthesized by our approach exhibit an excellent stability as a function of temperature, pH, and freeze-thaw cycle, and the functionality of DNA/AuNPs conjugates is also verified. The work presented here has important implications to develop the fast and reproducible synthesis of stable DNA-functionalized gold nanoparticles.

Selection of DNA aptamers against polychlorinated biphenyls as potential biorecognition elements for environmental analysis.

Polychlorinated biphenyls (PCBs) have been of major concerns for decades due to their potential toxicity to human health. To trace the PCBs efficiently and sensitively, many detection methods have been developed. Aptamers, a new class of diagnostic tools, are considered to be such additional candidates for detection of pollutants. In the current study, we report the DNA aptamers, isolated by FluMag-SELEX (a modified SELEX [systematic evolution of ligands by exponential enrichment] technology), that recognize PCBs with the dissociation constants (Kd values) down to the micromolar range. Using the selected aptamers, a highly sensitive aptamer-based fluorescent assay for detection of PCBs was established using gold nanoparticles, with a widely linear range from 0.1 to 100 ng/ml. Moreover, our aptamer-based gold nanoprobe displays specificity toward 3,3,4,4-tetrachlorobiphenyl (PCB77) compared with a few common PCB77 structural analogs. These results open the possibility of using aptamers as biorecognition elements for easy and fast environmental monitoring.

Mitochondrial dysfunction resulting from loss of cytochrome c impairs radiation-induced bystander effect

Cytochrome c is a pivotal protein that resides in mitochondria as component of mitochondria respiration and apoptosis initiator. Using murine cells lacking cytochrome c, we showed here that cytochrome c-deficient cells had attenuated reactive oxygen species/nitric oxide and micronuclei induction to radiation-induced bystander signals, indicating cytochrome c is essential for the bystander effect.

PFOS-induced apoptosis through mitochondrion-dependent pathway in human–hamster hybrid cells

Perfluorooctane sulfonate (PFOS) was listed as one of the persistent organic pollutants (POPs) in Stockholm Convention in 2009. Recent evidence showed that PFOS could induce apoptosis both in vivo and in vitro. However, the apoptotic mechanisms induced by PFOS as well as the possible relationship between apoptosis and other PFOS-induced endpoints, remain unclear. In the present study, normal human-hamster hybrid (AL) cells and mtDNA-depleted (ρ(0) AL) cells were exposed to PFOS, and assayed for cytotoxicity, mutagenicity, and apoptosis (caspase-3/7, caspase-9 activities). Our results showed that PFOS decreased cell viability in a time- and concentration-dependent manner in AL cells, but not in ρ(0) AL cells. However, long-term exposure to PFOS failed to induce the mutagenic effects at the CD59 locus in AL cells. Exposure to 200 μM PFOS significantly increased the activities of caspase-3/7 and caspase-9 in AL cells, but the activities of these caspases were not affected in ρ(0) AL cells. In addition, PFOS increased the levels of reactive oxygen species (ROS), superoxide anion (O2(-)), as well as nitric oxide (NO), and decreased mitochondrial membrane potential (MMP) at the concentrations of 100 and 200μM in AL cells. On the other hand, exposure to PFOS had no effect on intracellular ROS, O2(-), and NO production in ρ(0) AL cells. Caspase-3/7 activity, which was increased by 200 μM PFOS, could be suppressed by ROS/O2(-) scavengers and nitric oxide synthases (NOSs) inhibitors in AL cells. These results implicate that PFOS-induced apoptosis and oxidative stress is mediated by a mitochondrion-dependent pathway and that the induction of apoptosis might be a protective function against mutagenesis in AL cells exposed to PFOS.

Mutagenicity of PFOA in Mammalian Cells: Role of Mitochondria-Dependent Reactive Oxygen Species

Mutagenicity is often a prerequisite to the development of malignancy. Evidences have shown that exposure to perfluorooctanoic acid (PFOA) results in various cancer inductions. However, whether any mutagenic base exists is still puzzling. In the present study, we exposed exponentially growing AL cells to PFOA and assayed the cells for survival, mutation induction, and caspase-3/7, -9 activities. Mitochondrial-DNA deficient human-hamster hybrid (ρ(0) AL) cells and reactive oxygen species (ROS) inhibitor were used to elucidate the possible mechanism. Our results showed that treatment of AL cells with PFOA for 16 days induced significant mutagenic effects together with the increment of ROS, superoxide anions (O2(.-)), and nitrogen oxide (NO) levels, while treatment of ρ(0) AL cells did not have much change. Concurrent treatment of AL cells with ROS inhibitor significantly decreased the mutagenic potential of PFOA. In addition, caspase activities in AL cells were increased by PFOA exposure and suppressed by ROS/RNS (reactive oxygen/nitrogen species) inhibitors. Our results suggest that exposure to PFOA lead to mutagenicity induction in AL cells, and mitochondria-dependent ROS plays an important role in this process. This provides a direct base for PFOA mediated cancer induction.

Mechanism of genotoxicity induced by targeted cytoplasmic irradiation

Background:Direct damage to DNA is generally accepted as the main initiator of mutation and cancer induced by environmental carcinogens or ionising radiation. However, there is accumulating evidence suggesting that extracellular/extranuclear targets may also have a key role in mediating the genotoxic effects of ionising radiation. As the possibility of a particle traversal through the cytoplasm is much higher than through the nuclei in environmental radiation exposure, the contribution to genotoxic damage from cytoplasmic irradiation should not be ignored in radiation risk estimation. Although targeted cytoplasmic irradiation has been shown to induce mutations in mammalian cells, the precise mechanism(s) underlying the mutagenic process is largely unknown.Methods:A microbeam that can target the cytoplasm of cells with high precision was used to study mechanisms involved in mediating the genotoxic effects in irradiated human–hamster hybrid (AL) cells.Results:Targeted cytoplasmic irradiation induces oxidative DNA damages and reactive nitrogen species (RNS) in AL cells. Lipid peroxidation, as determined by the induction of 4-hydroxynonenal was enhanced in irradiated cells, which could be suppressed by butylated hydroxyl toluene treatment. Moreover, cytoplasmic irradiation of AL cells increased expression of cyclooxygenase-2 (COX-2) and activation of extracellular signal-related kinase (ERK) pathway.Conclusion:We herein proposed a possible signalling pathway involving reactive oxygen/nitrogen species and COX-2 in the cytoplasmic irradiation-induced genotoxicity effect.

Graphene Oxide Attenuates the Cytotoxicity and Mutagenicity of PCB 52 via Activation of Genuine Autophagy

Graphene oxide (GO), owing to its large surface area and abundance of oxygen-containing functional groups, is emerging as a potential adsorbent for polychlorinated biphenyls (PCBs), which accumulate over time and are harmful to both natural ecosystems and human health. However, the effect of GO against PCB-induced toxicity remains largely unexplored. The present study aimed to investigate the protective effect of GO against PCB 52 induced cytotoxic and genotoxic response in mammalian cells at various exposure conditions and clarify the protective role of autophagy. Pretreatment with GO dramatically decreased PCB 52 induced cytotoxicity and CD59 gene mutation in human-hamster hybrid (AL) cells. The toxic response in cells either pretreated with PCB 52 and then treated with GO or concurrently treated with GO and PCB 52 did not differ significantly from the toxic response in the cells treated with PCB 52 alone. Using autophagy inhibitors (3-methyladenine and wortmannin) and inducers (trehalose and rapamycin), we found that genuine autophagy induced by GO was involved in decreasing PCB 52 induced toxicity. These findings suggested that GO has an antagonistic effect against the toxicity of PCB 52 mainly by triggering a genuine autophagic process, which might provide new insights into the potential application of GO in PCB disposal and environmental and health risk assessment.

Reproductive Toxicity of Endosulfan: Implication From Germ Cell Apoptosis Modulated by Mitochondrial Dysfunction and Genotoxic Response Genes in Caenorhabditis elegans.

Endosulfan as a new member of persistent organic pollutants has been shown to induce reproductive dysfunction in various animal models. However, the action mechanism of endosulfan-produced reproductive toxicity remains largely unknown. This study was focused on investigating the reproductive toxicity induced by α-endosulfan and clarifying the role of mitochondria and genotoxic response genes in germ cell apoptosis of Caenorhabditis elegans. Our data showed that endosulfan induced a dose-dependent decrease of life span, fecundity, and hatchability, whereas the germ cell apoptosis was dose-dependently increased. The mitochondria membrane potential was disrupted by endosulfan, leading to a significant increase of germ cell apoptosis in mev-1(kn-1) mutant. However, the apoptotic effects of endosulfan were blocked in mutants of cep-1(w40), egl-1(n487), and hus-1(op241), indicating conserved genotoxic response genes played an essential role in endosulfan-induced germ cell apoptosis. Furthermore, exposure to endosulfan induced the accumulation of HUS-1::GFP foci and the germ cell cycle arrest. These findings provided clear evidence that endosulfan caused significant adverse effects on the reproduction system of C. elegans and increased germ cell apoptosis, which was regulated by mitochondrial dysfunction and DNA damage response genes. This study may help to understand the signal transduction pathways involved in endosulfan-induced reproductive toxicity.

Mutagenicity of ZnO nanoparticles in mammalian cells: Role of physicochemical transformations under the aging process.

Abstract Zinc oxide nanoparticles (ZnO NPs) potentially undergo physicochemical transformation in the environment, which may lead to unexpected environmental and health risks. The “aging” process is essential for better understanding the toxicity and fate of NPs in the environment. However, the mutagenic effects of aged ZnO NPs are still unexplored. The present study focused on investigating the physicochemical transformation during aging process and clarifying the mutagenicity of naturally aged ZnO NPs in human–hamster hybrid (AL) cells. It was found that ZnO NPs underwent sophisticated physicochemical transformations with aging regardless of original morphology or size, such as the microstructural changes, the formation of hydrozincite (Zn5(CO3)2(OH)6) and the release of free zinc ions. Interestingly, the aged ZnO NPs were investigated to be able to result in much lower cytotoxicity while relatively high degree mutation than fresh ZnO NPs. With characterization of the soluble and insoluble fractions of aged ZnO NPs suspension, together with the control measurements using metal chelator (TPEN) and endocytosis inhibitor (Nystatin), it was revealed that the release of zinc ions and nanoparticle uptake made significantly different contributions to the mutagenicity of fresh and aged ZnO NPs. This study clearly demonstrated that the physicochemical transformation of ZnO NPs with aging plays important and comprehensive roles in the ZnO NPs-induced mutagenicity in mammalian cells.

Endosulfan Isomers and Sulfate Metabolite Induced Reproductive Toxicity in Caenorhabditis elegans Involves Genotoxic Response Genes

Endosulfan is enlisted as one of the persistent organic pollutants (POPs) and exists in the form of its α and β isomers in the environment as well as in the form of endosulfan sulfate, a toxic metabolite. General endosulfan toxicity has been investigated in various organisms, but the effect of the isomers and sulfate metabolites on reproductive function is unclear. This study was aimed at studying the reproductive dysfunction induced by endosulfan isomers and its sulfate metabolite in Caenorhabditis elegans (C. elegans). We also determined a role for the DNA-damage-checkpoint gene hus-1. Compared to β-endosulfan and its sulfate metabolite, α-endosulfan caused a dramatically higher level of germ cell apoptosis, which was regulated by DNA damage signal pathway. Both endosulfan isomers and the sulfate metabolite induced germ cell cycle arrest. Loss-of-function studies using hus-1, egl-1, and cep-1 mutants revealed that hus-1 specifically influenced the fecundity, hatchability, and sexual ratio after endosulfan exposure. Our data provide clear evidence that the DNA-checkpoint gene hus-1 has an essential role in endosulfan-induced reproductive dysfunction and that α-endosulfan exhibited the highest reproductive toxicity among the different forms of endosulfan.