Cytotoxicity In Epithelial Cells Research Articles

A novel ion-exchange carrier based upon liposome-encapsulated montmorillonite for ophthalmic delivery of betaxolol hydrochloride.

As a novel ion-exchange carrier with high surface area and excellent exchangeability, montmorillonite (Mt) was intercalated with betaxolol hydrochloride (BH) to form a nanocomposite and then encapsulated by liposomes (Mt-BH-LPs) for an ophthalmic drug-delivery system. The Mt-BH and Mt-BH-LPs were prepared by an acidification process and ethanol injection combined with ammonium sulfate gradient methods. The successful formation of Mt-BH and Mt-BH-LPs was verified by thermogravimetric analysis, X-ray diffraction, Fourier-transform infrared spectra, and transmission electron microscopy. Mt-BH-LPs possessed the favorable physical characteristics of encapsulation efficiency, drug loading, mean particle size, and ζ-potential. In vitro release studies indicated Mt-BH-LPs effectively maintained a relatively sustained slow release. Immortalized human corneal epithelial cell cytotoxicity, in vivo rabbit eye-irritation tests, and chorioallantoic membrane–trypan blue staining all revealed that Mt-BH-LPs had no obvious irritation on ocular tissues. A new in vitro tear-turnover model, including inserts containing human corneal epithelial cells, was designed to evaluate the precorneal retention time of Mt-BH-LPs. The results showed that Mt-BH-LPs maintained a certain BH concentration in tear fluid for a longer period than the BH solution. In vivo precorneal retention studies also indicated Mt-BH-LPs prolonged drug retention on the ocular surface more than the BH solution. Furthermore, pharmacodynamic studies showed that Mt-BH-LPs had a prolonged effect on decreasing intraocular optical pressure in rabbits. Our results demonstrated that Mt-BH-LPs have potential as an ophthalmic delivery system.

Anti-apoptotic effects of Curcuma longa L. extract and its curcuminoids against blue light-induced cytotoxicity in A2E-laden human retinal pigment epithelial cells.

The purpose of the study was to investigate the protective effect of the Curcuma longa L. extract (CLE) and its curcuminoids against blue light-induced cytotoxicity in human retinal pigment epithelial (RPE) cells laded with A2E. A2E has been concerned in age-related macular degeneration (AMD). To perform this study, A2E-accumulated ARPE-19 cells were exposed to blue light to induce cytotoxicity. The cytotoxicity and apoptotic gene expression levels were evaluated using a lactate dehydrogenase (LDH) assay and real-time PCR analysis, respectively. Curcuma longa L. extract was found to exert a protective effect in a dose-dependent manner. At a concentration of 15 μm, curcumin, demethoxycurcumin and bisdemethoxycurcumin exerted significant protective effects against blue light-induced cytotoxicity. Treatment with CLE and curcuminoids meaningfully reduced the mRNA levels of c-Abl and p53, which was known to be augmented in apoptotic RPE cells. Demethoxycurcumin and bisdemethoxycurcumin were found to inhibit p38 expression, which is increased in blue light-irradiated A2E-accumulated RPE cells. Curcuma longa L. extract and its curcuminoids provided significant protection against photooxidative damage and apoptosis in the RPE cells. Our results suggest that curcuminoids may show potential in the treatment of AMD.

Baicalin and chrysin mixture imparts cyto-protection against methylglyoxal induced cytotoxicity and diabetic tubular injury by modulating RAGE, oxidative stress and inflammation.

Protective effect of mixture of flavonoids baicalin and chrysin (BCH) was studied against methylglyoxal (MG, a precursor of AGEs) induced cytotoxicity in NRK 52E kidney epithelial cells. Flow cytometry and microscopic analysis showed increased ROS generation, compromised antioxidant status, depolarization of mitochondria and apoptosis in MG stressed cells which were significantly transformed (p≤0.01) during BCH co-treatment. In vivo studies in streptozotocin induced diabetic rats increased protein levels of iNOS, protein kinase C (PKC) and decreased IκB which was modulated by oral BCH treatment (75mg baicalin and 10mg chrysin/kg b.wt.). Increased levels of AGEs and their receptor proteins (RAGE) in diabetic rats were reduced significantly (p≤0.01) in BCH treated group. Renal tubular injuries and deranged kidney function were significantly improved in BCH treated animals. The results indicate that the protection accorded by BCH through its antioxidant and anti-inflammatory effects can be explored for management of diabetic nephropathy.

The Effect of Sodium Fluoride on Cell Apoptosis and the Mechanism of Human Lung BEAS-2B Cells In Vitro.

Sodium fluoride (NaF) is a source of fluoride ions used in many applications. Previous studies found that NaF suppressed the proliferation of osteoblast MC3T3 E1 cells and induced the apoptosis of chondrocytes. However, little is known about the effects of NaF on human lung BEAS-2B cells. Therefore, we investigated the mode of cell death induced by NaF and its underlying molecular mechanisms. BEAS-2B cells were treated with NaF at concentrations of 0, 0.25, 0.5, 1.0, 2.0, and 4.0mmol/L. Cell viability decreased and apoptotic cells significantly increased as concentrations of NaF increased over specific periods of time. The IC50 of NaF was 1.9 and 0.9mM after 24 and 48h, respectively. The rates of apoptosis increased from 4.8 to 37.7% after NaF exposure. HE staining, electron microscopy, and single cell gel electrophoresis revealed that morphological changes of apoptosis increased with exposure concentrations. RT-PCR and Western blotting were used to detect the apoptotic pathways. The expressions of bax, caspase-3, caspase-9, p53, and the cytoplasmic CytC of the NaF groups increased, while bcl-2 and mitochondrial CytC decreased compared with that of the control group (P<0.05). Further, the fluorescence intensities of ROS in the NaF groups were higher than those in the control group, and the membrane potential of mitochondria in the NaF group was significantly lower than that of the control group (P<0.05). These findings suggested that NaF induced apoptosis in the BEAS-2B cells through mitochondria-mediated signal pathways. Our study provides the theoretical foundation and experimental basis for exploring the mechanisms of human lung epithelial cell damage and cytotoxicity induced by fluorine.

Mitigated NSAID-induced apoptotic and autophagic cell death with Smad7 overexpression.

Non-steroidal anti-inflammatory drugs damaged gastrointestinal mucosa in cyclooxygenase-dependent and -independent pathway, among which apopototic or autophagic cell death in gastrointestinal cells might be one of key cytotoxic mechanisms responsible for NSAID-induced damages. Therefore, alleviating this cell death after NSAIDs can be a rescuing strategy. In this study, we explored the role of Smad7 on NSAID-induced cytotoxicity in gastric epithelial cells. Using RGM1 cells, we have compared biological changes between mock-transfected and Smad7-overexpressed cells. As results, significantly decreased cytotoxicity accompanied with decreased levels of cleaved caspase-3 and poly (ADP-ribose) polymerase, Bax, and autophagic vesicles concurrent with decreased expressions of autophagy protein 5 and microtubule-associated protein light chain 3B-II were noted in Smad7-overexpressed cells with indomethacin administration compared to mock-transfected cells. Contrast to mitigated apoptotic execution, anti-apoptotic Bcl-2 and Beclin-1 were significantly increased in Smad7-overexpressed cells compared to mock-transfected cells. Smad7 siRNA significantly reversed these protective actions of Smad7 against indomethacin, in which p38 mitogen-activated protein kinase was significantly intervened. Furthermore, indomethacin-induced Smad7 degradation through ubiquitin-proteasome pathway was relevant to increased cytotoxicity, while chloroquine as autophagy inhibitor significantly attenuated indomethacin-induced cytotoxicity through Smad7 preservation via repressed ubiquitination. Conclusively, either genetic overexpression or pharmacological induction of Smad7 significantly attenuated indomethacin-induced gastric cell damages.

Inhibition of EGF Uptake by Nephrotoxic Antisense Drugs InVitro and Implications for Preclinical Safety Profiling.

Antisense oligonucleotide (AON) therapeutics offer new avenues to pursue clinically relevant targets inaccessible with other technologies. Advances in improving AON affinity and stability by incorporation of high affinity nucleotides, such as locked nucleic acids (LNA), have sometimes been stifled by safety liabilities related to their accumulation in the kidney tubule. In an attempt to predict and understand the mechanisms of LNA-AON-induced renal tubular toxicity, we established human cell models that recapitulate in vivo behavior of pre-clinically and clinically unfavorable LNA-AON drug candidates. We identified elevation of extracellular epidermal growth factor (EGF) as a robust and sensitive in vitro biomarker of LNA-AON-induced cytotoxicity in human kidney tubule epithelial cells. We report the time-dependent negative regulation of EGF uptake and EGF receptor (EGFR) signaling by toxic but not innocuous LNA-AONs and revealed the importance of EGFR signaling in LNA-AON-mediated decrease in cellular activity. The robust EGF-based in vitro safety profiling of LNA-AON drug candidates presented here, together with a better understanding of the underlying molecular mechanisms, constitutes a significant step toward developing safer antisense therapeutics.

The Protective Effect of Brown-, Gray-, and Blue-Tinted Lenses against Blue LED Light-Induced Cell Death in A2E-Laden Human Retinal Pigment Epithelial Cells

A2E-laden ARPE-19 cells were exposed to a blue light to induce cytotoxicity, in order to investigate the protective effects of various tinted ophthalmic lenses against photo-induced cytotoxicity in human retinal pigment epithelial (RPE) cells laden with A2E, known to be among the etiologies of age-related macular degeneration (AMD). Different-colored tinted lenses with varying levels of tint and different filtering characteristics, such as polarized, blue-cut, and photochromatic lenses, were placed over the cells, and the protective efficacies thereof were evaluated by lactate dehydrogenase assay. When tinted lenses were placed over ARPE-19 cells, there were different reductions in cytotoxicity according to the colors and tint levels. The level of protection afforded by brown-tinted lenses was 6.9, 36.1, and 49% with a tint level of 15, 50, and 80%, respectively. For gray-tinted lenses, the protective effect was 16.3, 35, and 43.4% for the corresponding degree of tint, respectively. In the case of blue-tinted lenses, a protective effect of 20% was observed with 80% tinted lenses, but 15 and 50% tinted lenses provided no significant protection. In addition, photochromic lenses showed a protective effect but blue-cut lenses and polarized lenses provided no significant protection. Tinted lenses significantly reduced cytotoxicity in RPE cells irradiated with blue light. The protection was more efficient in lenses with a brown or gray tint than in blue-tinted lenses. Tinted glasses may provide significant protection against potential blue-light-induced photochemical and photo-oxidative damage in RPE cells.

Glutathione peroxidase 4 plays an important role in oxidative homeostasis and wound repair in corneal epithelial cells.

Oxidative stress is involved in the pathologies of corneal epithelial cells. However, the importance of specific antioxidant enzymes in corneal epithelial cells is not fully understood. The purpose of this study is to elucidate the role of glutathione peroxidase 4 (GPx4) in corneal epithelial cells. For in vitro experiments, an immortalized human corneal epithelial cell line was used. Cytotoxicity measured through LDH activity, lipid peroxidation immunostained for 4‐hydroxynonenal, cell viability, and cell death were compared between cells transfected with either GPx4 siRNA or scrambled control siRNA. In addition, the rescue effects of α‐tocopherol and ferrostatin‐1, a ferroptosis inhibitor, were examined in the cells with deficient GPx4 expression. For in vivo experiments, we applied n‐heptanol on the cornea of GPx4+/+ and GPx4+/− mice to create corneal epithelial wound. The epithelial defect area size was measured up to 48 h after epithelial wound creation. Knockdown of GPx4 strongly induced cytotoxicity and cell death in human corneal epithelial cells. Cell death induced by GPx4 knockdown was characterized by positive staining for both annexin V and propidium iodide, nuclear translocation of AIF, and without activation of caspase 3, and was rescued by α‐tocopherol and ferrostatin‐1. The delayed wound healing of GPx4 siRNA‐transfected cells were ameliorated by α‐tocopherol in vitro. In addition, loss of one GPx4 allele was sufficient to significantly delay the healing of experimental corneal epithelial wounds in vivo. Our results suggest that the antioxidant enzyme GPx4 plays an important role in oxidative homeostasis, cell survival, and wound healing in corneal epithelial cells.

Chemotherapeutic effects of luteolin on radio-sensitivity enhancement and interleukin-6/signal transducer and activator of transcription 3 signaling repression of oral cancer stem cells.

Previously, we successfully identified oral cancer stem cells (OCSC) displaying enhanced stemness and tumorigenic potentials. In the study, we investigated the chemotherapeutic effect of the flavonoid luteolin, commonly found in fruits and vegetables, on targeting OCSC. Oralspheres was applied to isolate OCSC. aldehyde dehydrogenase 1 activity and CD44 positivity of OCSC with luteolin treatment were assessed by flow cytometry analysis. Radio-sensitivity of OCSC treated with luteolin was examined. Invasion and colony-forming assays were performed to assess oncogenicity in OCSC. The expression of interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) was examined by enzyme-linked immunosorbent assay and western blot analysis. We showed that luteolin effectively inhibited the proliferation rate, self-renewal, aldehyde dehydrogenase 1 activity, and CD44 positivity of OCSC but did not cause significant cytotoxicity of normal epithelial cells. Moreover, luteolin restored radio-sensitivity in OCSC. Combined treatment with luteolin and radiation displayed synergistic effect on invasiveness and clonogenicity of OCSC. Mechanistically, treatment of luteolin resulted in inactivation of IL-6/STAT3 signaling. These results suggest that combined treatment of luteolin and radiation therapy can attenuate tumorigenicity of OCSC through IL-6/STAT3 signaling inactivation.

Osmoprotective activity of alpha‐lipoic acid and taurine on hyperosmolar stress in cultured human corneal and conjunctival epithelial cells

PurposeTo characterize the osmoprotective properties of α‐lipoic acid (ALA) alone and in combination with taurine on both human corneal and conjunctival epithelial cells for cell viability and inflammatory biomarkers regulation after hyperosmotic stress.MethodsHuman corneal epithelial (HCE) cells and conjunctival epithelial (WKD) cells were incubated in isotonic media and hyperosmolar media (by addition of 100 mM NaCl), in the presence and absence of ALA, Taurine and ALA + Taurine. The osmoprotective activity on cell viability after 4, 8 and 24 h of hyperosmolar stress was evaluated by XTT assay. The expression of inflammatory biomarkers in cell‐free supernatants was measured using a Human Cytokine 15‐plex ELISA Kit after 4 and 8 h of stress.ResultsThe hyperosmolar conditions significantly reduced the cell viability in a time‐dependent manner and induced an overexpression of a panel of cytokines in corneal and conjunctival cells. Preincubation of cells with ALA alone or in combination with taurine significantly lowered the cell toxicity induced by the stress. The highest cell viability protection was observed within the range of ALA/Taurine ratios from 0.005 to 0.05 in conjunctival cells. In corneal cells the combination of ALA + Taurine, exhibited a higher osmoprotective effect than ALA or Taurine alone. The expression of cytokines was significantly but differentially reduced by ALA, and ALA + Taurine in corneal and conjunctival cells. The combination of ALA + Taurine exhibited a higher anti‐inflammatory effect than ALA or Taurine alone in corneal cells exposed to stress.ConclusionsALA alone or in combination with taurine, was found to protect against cytotoxicity and inflammation of corneal and conjunctival epithelial cells cultured in hyperosmolar media. ALA may have potential effects in protecting ocular surface epithelia from hyperosmotic stress.

Dual-Modal Imaging-Guided Theranostic Nanocarriers Based on Indocyanine Green and mTOR Inhibitor Rapamycin

The development of treatment protocols that resulted in a complete response to photothermal therapy (PTT) was usually hampered by uneven heat distribution and low effectiveness. Here, we reported an NIR fluorescence and photoacoustic dual-modal imaging-guided active targeted thermal sensitive liposomes (TSLs) based on the photothermal therapy agent Indocyanine green (ICG) and antiangiogenesis agent Rapamycin (RAPA) to realize enhanced therapeutic and diagnostic functions. As expected, the in vitro drug release studies exhibited the satisfactory result of drug released from the TSLs under hyperthermia conditions induced by NIR stimulation. The in vitro cellular studies confirmed that the FA-ICG/RAPA-TSLs plus NIR laser exhibited efficient drug accumulation and cytotoxicity in tumor cells and epithelial cells. After 24 h intravenous injection of FA-ICG/RAPA-TSLs, the margins of tumor and normal tissue were accurately identified via the in vivo NIR fluorescence and photoacoustic dual-modal imaging. In addition, FA-ICG/RAPA-TSLs combined with NIR irradiation treated tumor-bearing nude mice inhibited tumor growth to a great extent and possessed much lower side effects to normal organs. All detailed evidence suggested that the theranostic TSLs which were capable of enhancing the therapeutic index might be a suitable drug delivery system for dual-modal imaging-guided therapeutic tools for diagnostics as well as the treatment of tumors.

Extracellular ATP protects pancreatic duct epithelial cells from alcohol-induced damage through P2Y1 receptor-cAMP signal pathway.

Extracellular adenosine-5'-triphosphate (ATP) regulates cell death and survival of neighboring cells. The detailed effects are diverse depending on cell types and extracellular ATP concentration. We addressed the effect of ATP on ethanol-induced cytotoxicity in epithelial cells, the cell type that experiences the highest concentrations of alcohol. Using pancreatic duct epithelial cells (PDEC), we found that a micromolar range of ATP reverses all intracellular toxicity mechanisms triggered by exceptionally high doses of ethanol and, thus, improves cell viability dramatically. Out of the many purinergic receptors expressed in PDEC, the P2Y1 receptor was identified to mediate the protective effect, based on pharmacological and siRNA assays. Activation of P2Y1 receptors increased intracellular cyclic adenosine monophosphate (cAMP). The protective effect of ATP was mimicked by forskolin and 8-Br-cAMP but inhibited by a protein kinase A (PKA) inhibitor, H-89. Finally, ATP reverted leakiness of PDEC monolayers induced by ethanol and helped to maintain epithelial integrity. We suggest that purinergic receptors reduce extreme alcohol-induced cell damage via the cAMP signal pathway in PDEC and some other types of cells.

Chitosan nanoparticles for lipophilic anticancer drug delivery: Development, characterization and in vitro studies on HT29 cancer cells

The aim of this study was to develop chitosan-based nanoparticles that could encapsulate lipophilic molecules and deliver them to cancer cells. Nanoparticles were prepared with different molar ratios of chitosan, hyaluronic acid and sulphobutyl-ether-β-cyclodextrin and with or without curcumin. The nanosystems were characterized in terms of their size, zeta potential, morphology, encapsulation efficiency and stability in different media. Intestinal epithelial and colorectal cancer cells were treated with unloaded nanoparticles in order to study their effect on cellular membrane organization and ROS production. Finally, in vitro assays on both cellular lines were performed in order to evaluate the ability of nanoparticles to promote curcumin internalization and to study their effect on cell proliferation and cell cycle. Results show that nanoparticles were positively charged and their size increased with the increasing amounts of the anionic excipient. Nanoparticles showed good encapsulation efficiency and stability in water. Unloaded nanoparticles led to a change in lipid organization in the cellular membrane of both cell lines, without inducing ROS generation. Confocal microscopy, cell proliferation and cell cycle studies allowed the selection of the best formulation to limit curcumin cytotoxicity in normal intestinal epithelial cells and to reduce cancer cell proliferation. The latter was the result of the increase of expression for genes involved in apoptosis.

Cytotoxicity, oxidative stress, and inflammation in human Hep G2 liver epithelial cells following exposure to gold nanorods

The gold nanorods (GNRs) are great potentials in imaging, therapy, biosensing, and many other commercial applications. However, GNRs interactions with human cells and potential health risks remain not well known. The present investigation aimed to evaluate the in vitro toxicity of 10 and 25 nm GNRs (10–50 μg/mL) following exposure for 48 h in human Hep G2 liver epithelial cells using 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), lactate dehydrogenase (LDH) leakage, glutathione (GSH) estimation, lipid peroxidation (TBARS), caspase-3 levels, and interleukin-8 (IL-8) release assays. Exposure of GNRs to cells results in decrease in cell viability and causes cell membrane damage through LDH leakage results in cytotoxicity. The IC50 (concentration required to inhibit 50% of cells) values of 10 nm GNRs, 25 nm GNRs, and quartz (toxic control)-treated cells were found to be 19.9, 26.8, and 36.35 μg/mL, suggesting the higher cytotoxicity of GNRs. The GNRs exposure to liver cells found in depleted GSH levels, increased lipid peroxidation, and increased caspase-3 levels leads to induction of oxidative stress. In addition, enhanced levels of IL-8 were found, a sign of inflammation. The 10 nm GNRs have shown significant toxicity against all biochemical assays when compare to 25 nm GNRs and quartz-treated cells. Finally, the data indicate that the concentration size-dependent in vitro toxicity of GNRs toward liver Hep G2 cells. The toxicity of GNRs may be due to cell membrane damage, induction of oxidative stress, and inflammatory mediator release. Further investigations are necessitated to elucidate the in vivo toxicity of GNRs.

Translocation of Gold Nanoparticles in Model Epithelial Cells (Caco‐2 Monolayers)

At present, there is limited knowledge about the gastrointestinal fate and potential cytotoxicity of ingested inorganic nanoparticles. In this study, we determined the influence of particle size and concentration of gold nanoparticles (AuNPs) on their intestinal absorption, accumulation and cytotoxicity. AuNPs with different sizes (15, 50, and 100 nm) were subjected to an intestinal epithelium monolayer model to establish their patterns of absorption, accumulation and cytotoxicity. Results from inductively coupled plasma mass spectrometry (ICP‐MS) analysis revealed that as the mean particle diameter of the AuNPs decreased, their rate of absorption by the intestinal epithelium cells increased, but their cellular accumulation in the epithelial cells decreased. Moreover, fluoresce microscopy analysis indicated that accumulation of AuNPs caused cytotoxicity in the intestinal epithelial cells, which was evidenced by depolarization of mitochondria membranes. Our results demonstrated that particle size play an important role in dictating intestinal absorption, accumulation and cytotoxicity of AuNPs.Support or Funding InformationUSDA (2011‐67021, 2014‐67021)

Allicin attenuates H₂O₂-induced cytotoxicity in retinal pigmented epithelial cells by regulating the levels of reactive oxygen species.

Retinal pigmented epithelial cell (RPE) oxidative stress is known to have a vital role in the etiology of age‑related macular degeneration (AMD). The present study aimed to investigate whether allicin, a natural product with antioxidant activity, was able to protect RPEs (ARPE‑19) from hydrogen peroxide (H2O2)‑induced damage, and to determine the underlying mechanisms. The 3-(4,5-dimethylthiazol-2-yl)-2,5‑diphenyl tetrazolium bromide assay was used to determine cellular viability, and reactive oxygen species (ROS) were detected using a ROS Assay kit. The results demonstrated that allicin was able to protect ARPE‑19 cells from H2O2‑induced damage in a dose‑dependent manner. In addition, allicin attenuated oxidative stress by reducing the levels of intracellular ROS and malondialdehyde (MDA), and enhancing the glutathione/glutathione disulfide (GSSG) ratio. With regards to the underlying mechanism, allicin was able to markedly modulate the expression levels of ROS‑associated enzymes, including superoxide dismutase, NADPH oxidase 4 and NAD(P)H dehydrogenase quinone 1, and elevate the activity of nuclear factor erythroid 2‑related factor 2 in the H2O2‑stimulated ARPE‑19 cells. These results suggested that allicin may exert protective effects against H2O2‑induced cytotoxicity in RPEs via ROS regulation.

Abstract B02: Oxidized hemoglobin promotes survival of ROS-stressed and DNA damaged fallopian tube epithelial cells with p53 loss, resulting in a copy number variation phenotype of HGSC.

Abstract Purpose: Ovulation is a strong risk factor for high grade serous carcinoma (HGSC) of ovary known to largely originate from the fallopian tube fimbriae. Previously, we have showed ROS in the mature follicular fluid (FF) is mutagenic and cytotoxic to fimbria epithelial cells, and induces DNA double strand breaks (DSB) in the context of p53 loss. We hypothesized that hemoglobin (Hb) in FF accumulated in cul-de-sac (CDS) after ovulatory bleeding may protect the bathed fimbria from cell death and enhance cancer initiation. Results: Peritoneal fluids collected from the CDS after ovulation contained high level of oxidized Hb and ROS, so did ovarian FFs retrieved from IVF patients. When treating E6/E7-immortalized fimbria secretory cells (FE25) with FFs with various ROS levels ranging from 100-1200 μM of H2O2 equivalence, a positive correlation of Hb levels (ranging from 100-1100 μg/ml) in FF and cell viability was observed. This was further confirmed in treating FE25 cells with H2O2 where Hb dose-dependently rescued cell death. As a consequence, FE25 cells treated with either Hb-high FF or with H2O2 plus Hb accumulated high level of DSB and showed an extensive DNA copy number variation compatible with the copy number variation (CNV) phenotype of HGSC. The resulted DNA aneuploidy rates were 50±5% and 65±7% in FE25 treated with 500 μM H2O2 supplemented with 100 μg/ml Hb or 3% Hb-hgih FF, respectively; as compared to 15±2% at baseline and 31±8% with H2O2 only. In the case of primary fimbria epithelial cells the figures were 2±0.3% and 1.3±0.2%, respectively. Finally, the p53 deficiency dependent rescuing effect of Hb on ROS-stressed tubal epithelial cells, and the consequent CNV phenotype were confirmed ex vivo with oviducts of wild type and Trp53-null mice. Conclusion: While ROS in FF induces DNA damage and cytotoxicity of p53-deficient fimbria epithelial cells, oxidized Hb in peritoneal fluid rescues cells from death and results in a CNV phenotype, a major characteristic of HGSC. Citation Format: Hsuan-Shun Huang, Che-Fang Hsu, Tang-Yuan Chu. Oxidized hemoglobin promotes survival of ROS-stressed and DNA damaged fallopian tube epithelial cells with p53 loss, resulting in a copy number variation phenotype of HGSC. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B02.

Could formaldehyde induce mutagenic and cytotoxic effects in buccal epithelial cells during anatomy classes?

BackgroundDue to increased formaldehyde exposure, carcinogenic to humans, several researches have been studying the potential toxicity and the safe levels for human beings. The aim of this study was to investigate mutagenicity and cytotoxicity in buccal epithelial exfoliated cells (BEC) of students subjected to formaldehyde (FA) during anatomy classes.Material and MethodsBEC were collected periodically from 17 volunteers of undergraduate programs, who had participated in practical anatomy classes, before and after FA exposure. Cells were stained according to Feulgen method and then micronucleus test was applied. A total of 1,500 cells were assessed per individual in this study for the micronucleus frequency and other parameters of cytotoxicity.ResultsThere was statistically significant increase in number of micronucleated BEC after FA exposure (after 1 month p=.034 and after 3.5 months p=.017). However, FA exposure caused no significant increase in other nuclear alterations closely related to cytotoxicity (p≥.05).ConclusionsFA induced mutagenicity during anatomy classes. Cell death increased, but it was not statistically significant. Efforts have to be made to improve air quality and reduce exposures during anatomy classes. Key words:Carcinogens, formaldehyde, micronucleus tests, mutagenicity tests.

Cytotoxicity and autophagy dysfunction induced by different sizes of silica particles in human bronchial epithelial BEAS-2B cells.

The adverse effects of silica nanoparticles are gaining attention due to their wide application in biomedicine. However, information about size-dependent toxicity induced by silica nanoparticles is insufficient. In this study, two size of nano-scale (40 nm, 60 nm) and one size of micro-scale (200 nm) silica particles were studied to investigate the possible mechanism of cytotoxicity and autophagy dysfunction in human bronchial epithelial BEAS-2B cells. The cell viability was decreased in a size- and dose-dependent manner, while the LDH activity, oxidative stress and mitochondrial damage significantly increased, induced by both nano- and micro-scale silica particles. Ultrastructural analysis showed that nano-scale silica particles could induce mitochondrial damage and autophagy, but not micro-scale particles. Verified by the autophagy inhibitor 3-MA, the expression of LC3 and SQSTM1/p62 was upregulated in nano-scale silica particles in a size- and dose-dependent manner, while the micro-scale particles had an inhibitory effect. In addition, autophagy activation and autophagy blockage were triggered by nano-scale silica particles via the PI3K/Akt/mTOR pathway. Our findings first demonstrated that exposure to nano-scale silica particles rather than micro-scale particles could lead to autophagy dysfunction and impair cellular homeostasis.

Induction of Activating Transcription Factor 6 via Activation of ERK and ROS-P38 MAPK is Related to Methylglyoxal-Induced Cytotoxicity in Human Retinal Pigment Epithelial Cells

Methylglyoxal (MGO), a reactive α-oxoaldehyde produced by glucose metabolism, is elevated in several diabetic complications, including diabetic retinopathy. The breakdown of retinal pigment epithelial cells is implicated in the progression of diabetic retinopathy. Increased concentrations of MGO lead to retinal pigment epithelial cell death. In this study, we investigated the involvement of activating transcription factor 6 (ATF6) in MGO-mediated cytotoxicity in ARPE-19 cells. In response to high concentrations of MGO, unfolded protein response-related ATF6 was induced. Interestingly, the MGO also induced the generation of reactive oxygen species (ROS) and the phosphorylation of ERK and p38 MAPK. The induction of ATF6 was inhibited by ERK-specific and p38 MAPK-specific inhibitors (U0126 and SB202190) and by NAC, a well-known ROS scavenger. NAC also attenuated the phosphorylation of p38 MAPK. MGO induced cytotoxicity in the ARPE-19 cells, which was ameliorated by the inhibition of the ERK, p38 MAPK, and ROS pathways. Furthermore, the MGO-mediated cytotoxicity was inhibited by ATF6 siRNA. Taken together, these results clearly show that the induction of ATF6 via the ERK and ROS-p38 MAPK pathways is implicated in MGO-induced cytotoxicity in ARPE-19 cells.