Discrimination of Multidrug Resistance in Cancer Cells Achieved Using Single-Cell Analysis.

The biophysical signatures of single cells, such as multidrug resistance (MDR), may easily change during their various disease states. Therefore, there is an ever-growing need for advanced methods to study and analyze the response of cancer cells to therapeutic intervention. To determine the cancer cells and responses to various cancer therapies, from a cell mortality perspective, we report a label-free and real-time method to monitor the in situ responses of ovarian cancer cells using a single-cell bioanalyzer (SCB). The SCB instrument was used to detect different ovarian cancer cells, such as NCI/ADR-RES cells, which are multidrug resistant (MDR), and non-MDR OVCAR-8 cells. The discrimination of ovarian cells has been achieved at the single-cell level by measuring drug accumulation quantitatively in real time, in which the accumulation is high in non-MDR single cells without drug efflux but is low in MDR single cells which are not efflux-free. The SCB was constructed as an inverted microscope for optical imaging and fluorescent measurement of a single cell that was retained in a microfluidic chip. The single ovarian cancer cell retained in the chip offered sufficient fluorescent signals for the SCB to measure the accumulation of daunorubicin (DNR) in the single cell in the absence of cyclosporine A (CsA). The same cell allows us to detect the enhanced drug accumulation due to MDR modulation in the presence of CsA, which is the MDR inhibitor. The measurement of drug accumulation in a cell was achieved after it was captured in the chip for one hour, with the correction of background interference. The detection of accumulation enhancement due to MDR modulation by CsA was determined in terms of either the accumulation rate or enhanced concentration of DNR in the single cell (same cell, p < 0.01). It showed that with the effectiveness of efflux blocking by CsA, the intracellular DNR concentration in a single cell increased by threefold against its same cell control. This single-cell bioanalyzer instrument has the ability to discriminate MDR in different ovarian cells due to drug efflux in them by eliminating the interference of background fluorescence and by using the same cell control.

Targeting Cell Cycle Facilitates E1A-Independent Adenoviral Replication.

DNA replication of E1-deleted first-generation adenoviruses (AdV) in cultured cancer cells has been reported repeatedly and it was suggested that certain cellular proteins could functionally compensate for E1A, leading to the expression of the early region 2 (E2)-encoded proteins and subsequently virus replication. Referring to this, the observation was named E1A-like activity. In this study, we investigated different cell cycle inhibitors with respect to their ability to increase viral DNA replication of dl70-3, an E1-deleted adenovirus. Our analyses of this issue revealed that in particular inhibition of cyclin-dependent kinases 4/6 (CDK4/6i) increased E1-independent adenovirus E2-expression and viral DNA replication. Detailed analysis of the E2-expression in dl70-3 infected cells by RT-qPCR showed that the increase in E2-expression originated from the E2-early promoter. Mutations of the two E2F-binding sites in the E2-early promoter (pE2early-LucM) caused a significant reduction in E2-early promoter activity in trans-activation assays. Accordingly, mutations of the E2F-binding sites in the E2-early promoter in a virus named dl70-3/E2Fm completely abolished CDK4/6i induced viral DNA replication. Thus, our data show that E2F-binding sites in the E2-early promoter are crucial for E1A independent adenoviral DNA replication of E1-deleted vectors in cancer cells. IMPORTANCE E1-deleted AdV vectors are considered replication deficient and are important tools for the study of virus biology, gene therapy, and large-scale vaccine development. However, deletion of the E1 genes does not completely abolish viral DNA replication in cancer cells. Here, we report, that the two E2F-binding sites in the adenoviral E2-early promoter contribute substantially to the so-called E1A-like activity in tumor cells. With this finding, on the one hand, the safety profile of viral vaccine vectors can be increased and, on the other hand, the oncolytic property for cancer therapy might be improved through targeted manipulation of the host cell.

Detection of Oxidative Stress Induced by Nanomaterials in Cells-The Roles of Reactive Oxygen Species and Glutathione.

The potential of nanomaterials use is huge, especially in fields such as medicine or industry. Due to widespread use of nanomaterials, their cytotoxicity and involvement in cellular pathways ought to be evaluated in detail. Nanomaterials can induce the production of a number of substances in cells, including reactive oxygen species (ROS), participating in physiological and pathological cellular processes. These highly reactive substances include: superoxide, singlet oxygen, hydroxyl radical, and hydrogen peroxide. For overall assessment, there are a number of fluorescent probes in particular that are very specific and selective for given ROS. In addition, due to the involvement of ROS in a number of cellular signaling pathways, understanding the principle of ROS production induced by nanomaterials is very important. For defense, the cells have a number of reparative and especially antioxidant mechanisms. One of the most potent antioxidants is a tripeptide glutathione. Thus, the glutathione depletion can be a characteristic manifestation of harmful effects caused by the prooxidative-acting of nanomaterials in cells. For these reasons, here we would like to provide a review on the current knowledge of ROS-mediated cellular nanotoxicity manifesting as glutathione depletion, including an overview of approaches for the detection of ROS levels in cells.

Chemical and Colloidal Dynamics of MnO2 Nanosheets in Biological Media Relevant for Nanosafety Assessment.

Many layered crystal phases can be exfoliated or assembled into ultrathin 2D nanosheets with novel properties not achievable by particulate or fibrous nanoforms. Among these 2D materials are manganese dioxide (MnO2 ) nanosheets, which have applications in batteries, catalysts, and biomedical probes. A novel feature of MnO2 is its sensitivity to chemical reduction leading to dissolution and Mn2+ release. Biodissolution is critical for nanosafety assessment of 2D materials, but the timing and location of MnO2 biodissolution in environmental or occupational exposure scenarios are poorly understood. This work investigates the chemical and colloidal dynamics of MnO2 nanosheets in biological media for environmental and human health risk assessment. MnO2 nanosheets are insoluble in most aqueous phases, but react with strong and weak reducing agents in biological fluid environments. In vitro, reductive dissolution can be slow enough in cell culture media for MnO2 internalization by cells in the form of intact nanosheets, which localize in vacuoles, react to deplete intracellular glutathione, and induce cytotoxicity that is likely mediated by intracellular Mn2+ release. The results are used to classify MnO2 nanosheets within a new hazard screening framework for 2D materials, and the implications of MnO2 transformations for nanotoxicity testing and nanosafety assessment are discussed.

Peroxidase activities of gold nanowires synthesized by TMV as template and their application in detection of cancer cells.

A sensing methodology that combines Au, tobacco mosaic virus (TMV), and folic acid for selective, sensitive, and colorimetric detection of tumor cells based on the peroxidase-like activity was reported in this study. Gold nanowires with a high aspect ratio were synthesized using TMV as a template. Au@TMV nanowire (AT) complex was obtained with diameter of 4 nm and length between 200 and 300 nm. In addition, since TMV was biocompatible and had many amino and carboxyl groups on its surface, AT was conjugated by folate to form a folic acid (FA)-conjugated AT composite (ATF) and tested by FTIR measurements. Furthermore, the peroxidase-like properties were studied and the optimal conditions for mimic enzyme activity were optimized. Finally, HeLa and other tumor cells expressed excessive receptors of folate on the surface, which can specifically bind to folic acid. As the specific binding of ATF with HeLa cells, the peroxidase properties of ATF were used for detection of cancer cells (Scheme 1). The cancer cells were detected not only qualitatively but also quantitatively. In this study, as low as 2000 cancer cells/mL could be detected using the current method.

Comprehensive chemical profiling of Yindan Xinnaotong soft capsule and its neuroprotective activity evaluation in vitro.

Discovering effective combinational components (ECCs) and quality control markers of TCMs is still facing challenges because the holistic healing system comprises hundreds of compounds. Here, taking Yindan Xinnaotong soft capsule (YDXNT), a TCMs preparation composed by 8 herbs, as a case, a strategy that integrated multiple chromatographic analysis and bioactivity assay was proposed for potential ECCs of neuroprotection discovery. Firstly, ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF MS) and gas chromatography-mass spectrometry (GC-MS) were applied for comprehensive profiling of the chemical constituents in YDXNT. Given the fact that the complex matrix interference makes it more difficult to identify potentially active compounds, we proposed a structure-diagnostic ions-oriented strategy to remove interference ions from the raw UHPLC-MS data. The proposed strategy consisted of different filtering methods, including diagnostic fragment ions filtering (DFIF), mass defect filtering (MDF) and neutral loss (NL). Using this strategy, a total of 124 compounds were rapidly identified. Among them, 62 non-volatile and 5 volatile constituents in 30 batches of YDXNT were quantified by UHPLC tandem triple quadrupole mass spectrometry (QQQ-MS) and GC-MS methods, respectively. In order to facilitate the quality control of YDXNT, candidate ECCs were selected based on the threshold setting of absolute -contents, and their neuroprotective effects were examined. Finally, a combination of 16 compounds, accounts for 2.80% (w/w) of original YDXNT, was identified as its potential ECCs, which could be considered for the improvement of quality standardization of YDXNT.

Topography-Induced Cell Self-Organization from Simple to Complex Aggregates.

Self-organization is a fundamental and indispensable process in a living system. To understand cell behavior in vivo such as tumorigenesis, 3D cellular aggregates, instead of 2D cellular sheets, have been employed as a vivid in vitro model for self-organization. However, most focus on the macroscale wetting and fusion of cellular aggregates. In this study, it is reported that self-organization of cells from simple to complex aggregates can be induced by multiscale topography through confined templates at the macroscale and cell interactions at the nanoscale. On the one hand, macroscale templates are beneficial for the organization of individual cells into simple and complex cellular aggregates with various shapes. On the other hand, the realization of these macro-organizations also depends on cell interactions at the nanoscale, as demonstrated by the intimate contact between nanoscale pseudopodia stretched by adjacent frontier cells, much like holding hands and by the variation in the intermolecular interactions based on E-cadherin. Therefore, these findings may be very meaningful for clarifying the organizational mechanism of tumor development, tissue engineering and regenerative medicine.

Cytotoxic and morphological effects of microcystin-LR, cylindrospermopsin, and their combinations on the human hepatic cell line HepG2.

Cylindrospermopsin (CYN) and Microcystin-LR (MC-LR) are toxins produced by different cyanobacterial species, which are found mainly in freshwater reservoirs. Both of them can induce, separately, toxic effects in humans and wildlife. However, little is known about the toxic effects of the combined exposure, which could likely happen, taking into account the concomitant occurrence of the producers. As both cyanotoxins are well known to induce hepatic damage, the human hepatocellular HepG2 cell line was selected for the present study. Thus, the cytotoxicity of both pure cyanotoxins alone (0-5 µg/mL CYN and 0-120 µg/mL MC-LR) and in combination for 24 and 48 h was assayed, as long as the cytotoxicity of extracts from CYN-producing and nonproducing cyanobacterial species. The potential interaction of the combination was evaluated by the isobologram or Chou-Talalay's method, which provides a combination index as a quantitative measure of the two cyanotoxins interaction's degree. Moreover, a morphological study of the individual pure toxins and their combinations was also performed. Results showed that CYN was the most toxic pure cyanotoxin, being the mean effective concentrations obtained ≈4 and 90 µg/mL for CYN and MC-LR, respectively after 24 h. However, the simultaneous exposure showed an antagonistic effect. Morphologically, autophagy, at low concentrations, and apoptosis, at high concentrations were observed, with affectation of the rough endoplasmic reticulum and mitochondria. These effects were more pronounced with the combination. Therefore, it is important to assess the toxicological profile of cyanotoxins combinations in order to perform more realistic risk evaluations.

Nanostructured biomedical selenium at the biological interface (Review).

This paper critically reviews the current evidence of research in biomedical applications of selenium nanoparticles (SeNPs) and their effects at cellular and tissue levels. In recent years, interest in SeNPs as a natural trace element nanomaterial for nanomedicine has resulted in a number of studies evaluating their bioactivities, such as anticancer, antimicrobial, and antioxidant properties. Significant data have been generated to demonstrate the effectiveness of SeNPs alone or in combination with other reagents. Their activities are demonstrated through in vitro and in vivo experimentation; yet, the levels of efficacy need to be improved, particularly when compared with those of pharmaceutical drugs (such as antibiotics and cytotoxic chemotherapeutic drugs). However, promising evidence suggests decreased toxicity when using SeNPs, and more importantly their ability to perform as an interfacing biomaterial with cells and tissues. SeNPs have demonstrated unique antibacterial properties: they inhibit bacterial adhesion, growth, and/or quorum sensing and as a result prevent biofilm formation on medical devices, to name a few. Therefore, as with other nanomaterials, SeNPs warrant further study as part of the biomaterial-based therapeutic toolkit as an alternative to traditional pharmaceutical agents. This paper will provide a succinct review of recent studies on SeNPs to critically assess the findings in the light of effectiveness, particularly highlighting the roles of the cellular interface. Finally, an outlook of the potential of SeNPs will be presented to highlight the need for more intensive studies of material stability, mechanistic understanding at subcellular levels, and investigations into their combinational and/or synergistic effects with other bioactive reagents including pharmaceutical drugs.

Titania nanopores with dual micro-/nano-topography for selective cellular bioactivity.

This letter describes a simple surface modification strategy based on a single-step electrochemical anodization towards generating dual micro- and nano-rough horizontally-aligned TiO2 nanopores on the surface of clinically utilized micro-grooved titanium implants. Primary macrophages, osteoblasts and fibroblasts were cultured on the nano-engineered implants, and it was demonstrated that the modified surfaces selectively reduced the proliferation of macrophages (immunomodulation), while augmenting the activity of osteoblasts (osseo-integration) and fibroblasts (soft-tissue integration). Additionally, the mechanically robust nanopores also stimulated osteoblast and fibroblast adhesion, attachment and alignment along the direction of the pores/grooves, while macrophages remained oval-shaped and sparsely distributed. This study for the first time reports the use of cost-effectively prepared nano-engineered titanium surface via anodization, with aligned multi-scale micro/nano features for selective cellular bioactivity, without the use of any therapeutics.

In Vitro Determination of Protein Conjugates in Human Cells by LC-ESI-MS/MS after Benzyl Isothiocyanate Exposure.

Glucosinolates and their breakdown products, especially isothiocyanates (ITCs), are hypothesized to exert a broad range of bioactivities. However, physiological mechanisms are not yet completely understood. In this study, formation of protein conjugates after incubation with benzyl isothiocyanate (BITC) was investigated in vitro. A survey of protein conjugates was done by determining BITC cysteine and lysine amino acid conjugates after protein digestion. Therefore, a liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated. Stability studies showed that cysteine conjugates are not stable under alkaline conditions, and lysine conjugates did not show any correlation to pH values, although stability increased at low temperatures. Lysine conjugates were the preferred form of protein conjugates, and longer BITC exposure times led to higher amounts. Knowledge about the reaction sites of ITCs in eukaryotic cells may help to understand the mode of action of ITCs leading to health promoting as well as toxicological effects in humans.

Epoxy Stearic Acid, an Oxidative Product Derived from Oleic Acid, Induces Cytotoxicity, Oxidative Stress, and Apoptosis in HepG2 Cells.

In the present study, effects of cis-9,10-epoxy stearic acid (ESA) generated by the thermal oxidation of oleic acid on HepG2 cells, including cytotoxicity, apoptosis, and oxidative stress, were investigated. Our results revealed that ESA decreased the cell viability and induced cell death. Cell cycle analysis with propidium iodide staining showed that ESA induced cell cycle arrest at the G0/G1 phase in HepG2 cells. Cell apoptosis analysis with annexin V and propidium iodide staining demonstrated that ESA induced HepG2 cell apoptotic events in a dose- and time-dependent manner; the apoptosis of cells after treated with 500 µM ESA for 12, 24, and 48 h was 32.16, 38.70, and 65.80%, respectively. Furthermore, ESA treatment to HepG2 cells resulted in an increase in reactive oxygen species and malondialdehyde (from 0.84 ± 0.02 to 8.90 ± 0.50 nmol/mg of protein) levels and a reduction in antioxidant enzyme activity, including superoxide dismutase (from 1.34 ± 0.27 to 0.10 ± 0.007 units/mg of protein), catalase (from 100.04 ± 5.05 to 20.09 ± 3.00 units/mg of protein), and glutathione peroxidase (from 120.44 ± 7.62 to 35.84 ± 5.99 milliunits/mg of protein). These findings provide critical information on the effects of ESA on HepG2 cells, particularly cytotoxicity and oxidative stress, which is important for the evaluation of the biosafety of the oxidative product of oleic acid.

Water extract of Semecarpus parvifolia Thw. leaves inhibits cell proliferation and induces apoptosis on HEp-2 cells.

BACKGROUND: Semecarpus parvifolia Thw is used as an ingredient of poly herbal decoctions to treat cancer in traditional medicine. The present study aims to investigate the antiproliferative activity on HEp 2 cells by the water extract of S. parvifolia leaves and to evaluate potential mechanisms. METHODS: The plant extract was exposed to S. parvifolia for 24 hours and antiproliferative activity was quantified by Sulforhodamine B (SRB), 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and Lactate dehydrogenase (LDH) assays. Morphological changes were observed after staining cells with ethidium bromide/acridine orange (EB/AO) and Giemsa dye. Comet assay was performed to evaluate the DNA damage. The toxicity of the plant extract was determined by brine shrimp lethality assay. RESULTS: S. parvifolia leaves reduced the cell proliferation in a dose and time dependent manner. A two fold increase in NO level was observed at higher concentrations. Morphological changes characteristic to apoptosis were observed in light microscopy, Giemsa and EB/AO stained cells. Fragmented DNA further confirmed its capacity to induce apoptosis. No lethality was observed with brine shrimps. CONCLUSION: The results suggest that Semecarpus parvifolia Thw induces apoptosis in HEp-2 cells through a NO dependent pathway.

Inhibitory effects of selenium on cadmium-induced cytotoxicity in PC12 cells via regulating oxidative stress and apoptosis.

Purpose of this study is to investigate mechanism/s of cyto-protection by selenium (Na2SeO3; Se4+) against cadmium (CdCl2; Cd2+)-induced cytotoxicity using PC12 cells. In addition, Se (5, 10, 20 and 40 µM) and Cd (2.5, 5 and 10 µM)-induced cytotoxicity is determined. Cytotoxicity assays and western blot analyses confirmed that Se (≥10 µM) promotes autophagic cell death via inhibition of mTOR activation and p62 accumulation due to increase of cellular oxidative stress. On the other hand, co-presence of non-toxic Se (5 µM) and toxic Cd (5 µM) showed to increase cell viability, glutathione and glutathione peroxidase 1 (GPx1) levels, and to decrease DNA fragmentation and lactate dehydrogenase (LDH) activity compared to Cd-treated (5 µM) cells alone. Furthermore, western blot analyses of cytochrome c and ERK1 indicated that Cd-induced apoptotic cell death in PC12 cells. However, the co-exposure of Se with Cd significantly decreases the release of cytochrome c into cytosol from mitochondria, and up-regulates ERK1 protein to inhibit Cd-induced apoptosis. In conclusion, Se (≥10 µM) possess cytotoxicity in PC12 cells; however, co-presence of Se (5 µM) with Cd (5 µM) protects against Cd-induced apoptosis in PC12 cells due to inhibition of Cd-induced oxidative stress and subsequently suppression of mitochondrial apoptosis pathway.

A metabolomics strategy to assess the combined toxicity of polycyclic aromatic hydrocarbons (PAHs) and short-chain chlorinated paraffins (SCCPs).

The combined toxicity of mixed chemicals is usually evaluated according to several specific endpoints, and other potentially toxic effects are disregarded. In this study, we provided a metabolomics strategy to achieve a comprehensive understanding of toxicological interactions between mixed chemicals on metabolism. The metabolic changes were quantified by a pseudotargeted analysis, and the types of combined effects were quantitatively discriminated according to the calculation of metabolic effect level index (MELI). The metabolomics strategy was used to assess the combined effects of polycyclic aromatic hydrocarbons (PAHs) and short-chain chlorinated paraffins (SCCPs) on the metabolism of human hepatoma HepG2 cells. Our data suggested that exposure to a combination of PAHs and SCCPs at human internal exposure levels could result in an additive effect on the overall metabolism, whereas diverse joint effects were observed on various metabolic pathways. The combined exposure could induce a synergistic up-regulation of phospholipid metabolism, an additive up-regulation of fatty acid metabolism, an additive down-regulation of tricarboxylic acid cycle and glycolysis, and an antagonistic effect on purine metabolism. SCCPs in the mixture acted as the primary driver for the acceleration of phospholipid and fatty acid metabolism. Lipid metabolism disorder caused by exposure to a combination of PAHs and SCCPs should be an important concern for human health.

Synthesis and antioxidant evaluation of lipophilic oleuropein aglycone derivatives.

Oleuropein is the most important phenolic compound present in olive cultivars, but it is scarcely present in extra virgin olive oil (EVOO) due to its high hydrophilicity and degradability. Thus, a set of oleuropein aglycone derivatives were synthesized by transacetylation under mild conditions with the aim of circumventing these drawbacks and making the active moiety in oleuropein suitable to be added to food fats. The oleuropein aglycone (closed ring form) is obtained by hydrolyzing oleuropein using Lewis acid catalysis. Then, the permeation profiles as well as the antioxidant capacity of the oleuropein aglycone derivatives were evaluated by ORAC, DPPH assays and by ROS formation using the SH-SY5Y cell line. The biological activities of the obtained compounds exhibited a dependence on their level of lipophilicity.

[Preparation, characterization and cytology study of Pluronic-PEI micelles].

Objective: To prepare and characterize Pluronic-PEI micelles as a drug/gene delivery system. Methods: We used the low-molecular-weight PEI as a cross-linking agent to prepare the Pluronic-PEI micelles. The particle size, zeta potential and critical micelle concentration (CMC) were measured by dynamic light scattering (DLS) and pyrene fluorescence probe. The cytotoxicity, transfection efficiency and the impact on the intracellular ATP and P-gp levels of Pluronic-PEI micelles were investigated at the cellular level. Results: Pluronic-PEI micelles were successfully prepared with a suitable particle size (120-180 nm), zeta potential (+6-+9 mv), and a good ability to carry the drug/gene. An in-vitro study showed that Pluronic-PEI had low cytotoxicity, and the P123-PEI600 possessed high gene transfection efficiency and could downregulate the intracellular ATP and P-gp levels. Conclusion: Pluronic-PEI is a good drug/gene delivery system, and P123-PEI600 is an ideal vector, which may be used in the combination therapy for reversing multidrug resistance.

Size-related cytotoxicological aspects of polyvinylpyrrolidone-capped platinum nanoparticles.

The nanotechnological concept is based on size-dependent properties of particles in the 1-100 nm range. Nevertheless, the connection between their size and effect is still not clear. Thus, we focused on reductive colloidal synthesis, characterization and biological testing of Pt nanoparticles (PtNPs) capped with biocompatible polymer polyvinylpyrrolidone (PVP). Synthesized PtNPs were of 3 different primary sizes (approx. ∼10; ∼14 and > 20 nm) and demonstrated exceptional haemocompatibility. In vitro treatment of three different types of malignant cells (prostate - LNCaP, breast - MDA-MB-231 and neuroblastoma - GI-ME-N) revealed that even marginal differences in PtNPs diameter resulted in changes in their cytotoxicity. The highest cytotoxicity was observed using the smallest PtNPs-10, where 24IC50 was lower (3.1-6.2 µg/mL) than for cisplatin (8.1-19.8 µg/mL). In contrast to MDA-MB-231 and LNCaP cells, in GI-ME-N cells PtNPs caused noticeable changes in their cellular structure without influencing their viability. Post-exposure analyses revealed that PtNPs-29 and PtNPs-40 were capable of forming considerably higher amount of reactive oxygen species with consequent stimulation of expression of metallothionein (MT1/2 and MT3), at both mRNA and protein level. Overall, our pilot study demonstrates that in the nanoscaled world even the smallest differences can have crucial biological effect.

[Over-expression of uracil DNA glycosylase 2 (UNG2) enhances the resistance to oxidative damage in HepG2 cells].

Objective To investigate the uracil glycosidic enzyme activity of uracil DNA glycosylase 2 (UNG2) and study the role of UNG2 in the resistance of antioxidant stress of HepG2 cells. Methods The UNG2-expressing vector was built. Western blotting was used to detect the expression of UNG2. Immunofluorescence staining was performed to observe the cellular location of UNG2. Oligonucleotide was used as substrate for the determination of the UNG2 glycosidic enzyme activity. H2O2 toxicity assay was done to study the function of UNG2 in the antioxidant resistance of hepatocellular carcinoma HepG2 cells. Results UNG2 was successfully over-expressed in HEK293FT cells, and UNG2 was found to be mainly located in nucleus. Enzyme activity assay showed that UNG2 had significant oligonucleotide dU glycosidic enzyme activity. H2O2 toxicity assay showed that over-expressed UNG2 could remarkably increase the survival of HepG2 cells after exposed to H2O2. Conclusion UNG2 possesses specific DNA glycosidic enzyme activity, and it can protect HepG2 cells against oxidative stress damage.

Effects of individual and combined toxicity of bisphenol A, dibutyl phthalate and cadmium on oxidative stress and genotoxicity in HepG 2 cells.

Bisphenol A, dibutyl phthalate and cadmium can be found in environment simultaneously. Several studies suggested that they had genotoxic effect. In this study, mono-exposure and co-exposure treatments, designed by 3 × 3 full factorial, were established to determine the individual toxicity and binary mixtures' combined effects on the oxidative stress and genotoxicity in HepG 2 cells. The highest oxidative damage was observed in the Cd treatments groups. Compared with control groups, the maximum level of reactive oxygen species and malondialdehyde were ∼1.4 fold and ∼2.22 fold respectively. And a minimum level of superoxide dismutase activity was found with the decrease of 43%. The mechanism that excessive oxidative stress led to the DNA damage was inferred. However, cells treated with BPA showed the worst DNA damage rather than Cd, which may because Cd mainly damages DNA repairing mechanism. For the joint effect, different interactions can be found in different biological endpoints for different combinations since different mechanisms have been clarified in mixture toxicity studies. It is sure that the co-exposure groups enhanced cytotoxicity, oxidative stress and genotoxicity compared to the mono-exposures. Synergistic and additive interactions were considered, which means greater threat to organisms when exposed to multiple estrogenic endocrine disruptors.