DCFH Assay Research Articles

Perfluoroalkyl substances and immunotoxicity: An in vitro structure-activity relationship study in THP-1-derived monocytes and macrophages

Recently, PFASs toxicity for the human immune system has become a growing concern. However, there is currently limited information on PFASs immunotoxicity beyond PFHxS, PFOA, PFOS, and PFNA. Therefore, it is urgent to close the present knowledge gap by testing a wider range of compounds. In the present study, twelve compounds were tested for a relationship between the chain-length and headgroup of a PFAS and its cytotoxic for THP-1. As such, THP-1, either as monocytes or differentiated macrophages, were exposed to PFASs in a concentration range of 0–800 μM for either 3 or 24 h. After that, cell viability and reactive oxygen species (ROS) generation were assessed using MTT and DCFH assay, respectively. PFASs' cytotoxicity is dependent on both their chain-length and headgroups. Cell viability decreased with increasing chain-length, and FTOHs displayed markedly higher toxicity than PFCAs and PFSAs. PFASs were ranked based on their calculated Relative Potency Factor. The ranking for the cytotoxicity data on monocytes appears to be 6:2 FTOH ≫ PFNA > PFDA > PFOS > PFOA >4: 2 FTOH > PFHxS = PFHxA > PFBA. For macrophages, this ranking was as follows: 6:2 FTOH >4:2 FTOH > PFOS > PFDA > PFNA > PFOA > PFHxS. The results observed for the ROS generating potential differed as FTOHs generated no ROS. Here, the ranking in monocytes was PFOA > PFNA > PFOS > PFHxS > PFDA > PFHxA = PFBS = PFBA. The ranking for macrophages was PFNA > PFDA ≥ PFOA > PFOS > PFHxA > PFHxS > PFBA = PFBS. In conclusion, the carbon chain-length and functional headgroup of a PFAS are major determinants for their toxicity to THP-1 cells. Furthermore, our study demonstrates the most potent cytotoxic effect for FTOHs in vitro, which has not been observed before to the authors’ knowledge.

Improving the dichloro-dihydro-fluorescein (DCFH) assay for the assessment of intracellular reactive oxygen species formation by nanomaterials

To facilitate Safe and Sustainable by Design (SSbD) strategies during the development of nanomaterials (NMs), quick and easy in vitro assays to test for hazard potential at an early stage of NM development are essential. The formation of reactive oxygen species (ROS) and the induction of oxidative stress are considered important mechanisms that can lead to NM toxicity. In vitro assays measuring oxidative stress are therefore commonly included in NM hazard assessment strategies. The fluorescence-based dichloro-dihydro-fluorescein (DCFH) assay for cellular oxidative stress is a simple and cost-effective assay, making it a good candidate assay for SSbD hazard testing strategies. It is however subject to several pitfalls and caveats. Here, we provide further optimizations to the assay using 5-(6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA-AE, referred to as DCFH probe), known for its improved cell retention.We measured the release of metabolic products of the DCFH probe from cells to supernatant, direct reactions of CM-H2DCFDA-AE with positive controls, and compared the commonly used plate reader-based DCFH assay protocol with fluorescence microscopy and flow cytometry-based protocols. After loading cells with DCFH probe, translocation of several metabolic products of the DCFH probe to the supernatant was observed in multiple cell types. Translocated DCFH products are then able to react with test substances including positive controls. Our results also indicate that intracellularly oxidized fluorescent DCF is able to translocate from cells to the supernatant. In either way, this will lead to a fluorescent supernatant, making it difficult to discriminate between intra- and extra-cellular ROS production, risking misinterpretation of possible oxidative stress when measuring fluorescence on a plate reader.The use of flow cytometry instead of plate reader-based measurements resolved these issues, and also improved assay sensitivity. Several optimizations of the flow cytometry-based DCFH ISO standard (ISO/TS 19006:2016) were suggested, including loading cells with DCFH probe before incubation with the test materials, and applying an appropriate gating strategy including live-death staining, which was not included in the ISO standard.In conclusion, flow cytometry- and fluorescence microscopy-based read-outs are preferred over the classical plate reader-based read-out to assess the level of intracellular oxidative stress using the cellular DCFH assay.

PH-Responsive self-assembled polymer-photosensitizer conjugate for activable photodynamic therapy.

This paper reports synthesis, aqueous self-assembly and relevance of the pH-triggered activable photodynamic therapy of amphiphilic polyurethane (P1S) functionalized with a heavy-atom free organic photosensitizer. Condensation polymerization between 1,4-diisocyanatobutane and two different dihydroxy monomers (one having a pendant hydrophilic wedge and the other having 1,3-dihydroxypropan-2-one with a reactive carbonyl group) in the presence of a measured amount of (S)-2-methylbutan-1-ol (chain-stopper) and DABCO catalyst produces a reactive pre-polymer P1. Hydrazide-functionalized thionated-naphthalenemonoimide (NMIS), which acts as a photosensitizer, reacted with the carbonyl-functionality of P1 to obtain the desired polymer-photosensitizer conjugate P1S in which the dye was attached to the polymer backbone via an acid-labile hydrazone linker. In water, P1S adopted an intra-chain H-bonding stabilized folded structure, which further assembled to produce a polymersome structure (Dh ≈ 200 nm), in which the hydrophobic membrane consists of aggregated NMIS and trialkoxy-benzene chromophores, as evident from UV/vis, CD and small-angle X-ray diffraction studies. In the aggregated state, NMIS loses its reactive oxygen species (ROS) generation ability and remains in a dormant state. However, under acidic conditions (pH 5.5), the photosensitizer is detached (presumably because of the cleavage of the hydrazone linker) and regains its full ROS-generation activity under photoirradiation, as evidenced from the standard DCFH assay. To test the possibility of such pH-activable intra-cellular ROS generation, P1S was treated with HeLa cells, as it is known that cancer cells are more acidic than normal cells. Indeed, photoirradiation-induced intra-cellular ROS generation was evident by the DCFH assay, resulting in efficient cell killing.

Investigating the cytotoxicity of per- and polyfluoroalkyl substances in HepG2 cells: A structure-activity relationship approach

Per- and polyfluoroalkyl substances (PFAS) are a family of man-made chemicals with currently over 4'700 compounds identified. While toxicological data are available for some of the legacy PFAS, such as PFOA and PFOS, a knowledge gap remains concerning both emerging and legacy PFAS' toxicity due to the diversity of the PFAS. Therefore, a better understanding of the PFAS structure-activity relationship may prove helpful. The present study investigated a potential structure-activity relationship between PFAS and hepatotoxicity. As such, the effects of thirteen PFAS with varying carbon chain-length and functional head-groups (in a concentration range of 0–800 µM) on the cell viability of HepG2 cells and intracellular reactive oxygen species formation have been tested using the MTT and DCFH assay, respectively. The exposure times were either 3 or 24 h. In addition, intracellular PFAS levels were determined in HepG2 after 24 h exposure. The present study demonstrated that the cytotoxicity of PFAS is dependent on their chain-length as cell viability decreased with increasing chain-length at both exposure times. Calculated Relative Potency Factors (RPF), based on the TC50 values, were used for a tentative ranking of PFAS regarding their hepatotoxicity: PFNA ˃ PFDA ˃ PFOS ≥ PFOA ˃ PFHxS ˃ PFBS ˃˃ PFHpA = PFHxA = PFBA = PFPrA = 6:2 FTOH = 4:2 = FTOH = 3:1 FTOH. Similar results were observed regarding intracellular reactive oxygen species generation at both exposure times, with a tentative ranking of: PFNA ˃ PFOS ˃ PFOA ≥ PFDA ˃ PFHxS ˃ PFBS ˃ PFBA ˃ PFHpA ≥ PFHxA ˃ PFPrA ˃ 6:2 FTOH = 4:2 FTOH = 3:1 FTOH. Moreover, a concentration-dependent reactive oxygen species generation has been observed for all PFSAs and PFCAs, but not for the FTOHs. In conclusion, the carbon chain-length and functional head-group of a PFAS determine their in vitro toxicity for the two toxicological endpoints assessed in the present study. Moreover, no effects were observed for the tested FTOHs. As such, the present study established a potential structure-activity relationship that opens the possibility of developing a predictive model to help with the risk assessment of PFAS in the future.

P12-35 Optimized DCFH assay for measuring intracellular ROS formation in response to nanomaterials in a safe-by-design approach

P12-35 Optimized DCFH assay for measuring intracellular ROS formation in response to nanomaterials in a safe-by-design approach

Apoptotic and antioxidant effects in HCT-116 colorectal carcinoma cells by a spiro-acridine compound, AMTAC-06.

Acridine compounds have been described as promising anticancer agents. Previous studies showed that (E)-1'-((4-chlorobenzylidene)amino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-06), a spiro-acridine compound, has antitumor activity on Ehrlich tumor and low toxicity. Herein, we investigated its antitumor effect against human cells in vitro. MTT assay was used to assess cytotoxicity of AMTAC-06 (3.125-200µM) against tumor and non-tumor cells, and the half-maximal inhibitory concentration (IC50) and the selectivity index (SI) were calculated. The effects on the cell cycle (propidium iodide-PI-staining), apoptosis (Annexin V-FITC/PI double staining by flow cytometry), and production of reactive oxygen species, ROS (DCFH assay) were also evaluated. Statistical analysis was achieved using ANOVA followed by Tukey's post-test. AMTAC-06 showed higher cytotoxicity against colorectal carcinoma HCT-116 cells (IC50: 12.62µM). The SI showed that AMTAC-06 was more selective for HCT-116 cells (HaCaT SI: 1.41; PBMC SI: 0.62) than doxorubicin (HaCaT SI: 0.10; PBMC SI: 0.01). AMTAC-06 (15 and 30µM) induced an increase in the sub-G1 peak (p < 0.000001) and cell cycle arrest in S phase (p = 0.003547). Moreover, treatment with this compound (15 and 30µM) resulted in increased early (p < 0.000001) and late apoptotic cells (p < 0.000001). In addition, there was a reduction on ROS production (p < 0.000001). AMTAC-06 presents anticancer activity against HCT-116 cells by regulating the cell cycle, inducing apoptosis and an antioxidant action.

Seasonal and diurnal variation of PM2.5 HULIS over Xi'an in Northwest China: Optical properties, chemical functional group, and relationship with reactive oxygen species (ROS)

Humic-like substances (HULIS) in particulate matter (PM) play critical roles in the atmospheric changes in our environment. In this study, high time resolution PM2.5 samples were collected to insight the abundances, spectroscopic characters, chemical groups, and oxidative potential of HULIS in Xi'an, China. The average mass concentrations of HULIS in term of carbon (HULIS-C) was 11.55 ± 5.85 and 8.28 ± 2.23 μg C m−3 in winter and summer, respectively. The diurnal variations of HULIS displayed three peaks (03:00–07:00, 10:00–14:00, and 18:00–21:00 LT) in winter, but a single peak (08:00–12:00 LT) in summer. The optical parameters show obvious difference between winter and summer. Fourier Transform infrared (FT-IR) spectra revealed that the HULIS mainly consisted of aliphatic chains, aromatic rings, and carboxylic groups. The FT-IR spectra proved that the aromatic compounds and carboxylic acids dominated the diurnal variation in winter, while carboxylic acids were responsible for that in summer. The normalized oxidative potential of HULIS exhibited an inverse trend with the HULIS-C concentration measured by the DCFH assay. The diurnal reactive oxygen species (ROS) productions from HULIS presents three valleys (03:00–07:00, 10:00–14:00, and 18:00–21:00 LT) in winter but only a single valley (08:00–12:00 LT) in summer. In addition, a positive correlation (R2 ≈ 0.6, p＜0.01) between the oxidative potential and specific ultraviolet absorbance at a wavelength of 254 nm (SUVA254) in winter suggested that the oxidative power of HULIS could be more related to the compounds with high light absorbance, high aromaticity, and molecular weight. The results of this study offer more solid knowledge on the spectral and chemical characteristics and oxidative potential of the HULIS in PM2.5 of the typical city in northwest China.

138 Environmental stress protection and inflammaging prevention: A novel synergistic antioxidant blend

We present an efficient approach to slowing the progression of skin damage using a multi-mechanistic antioxidant blend suitable for cosmetic formulations and validate its performance in vitro. Three different mechanisms were targeted simultaneously: scavenging reactive oxygen species (ROS), mitigating intracellular ROS and reducing ROS-induced inflammaging markers. Using a Design of Experiment approach, a novel, non-phototoxic synergistic blend of antioxidants was identified and characterized in vitro for its ability to quench free radicals (DPPH assay) and intracellular ROS generated by UVA (DCFH assay). Further, we demonstrated that use of the blend results in a mitigation of markers correlated with inflammaging (photoaging and hyperpigmentation) caused by environmental oxidative stress (PGE2, IL-8, MMP-1).3 These results support the notion that a multi-mechanistic antioxidant blend may effectively alleviate environmentally induced skin damage and be easily incorporated into skin care formulations providing an anti-inflammaging benefit. Brewer, M.S. “Natural Antioxidants: Sources, Compounds, Mechanisms of Action and Potential Applications.” Comprehensive Reviews in Food Science and Food Safety, vol. 10, 2011; McMullen, Roger L. Antioxidants and the Skin. Allured Books, 2013; Oswald, T., Crane, C.M., Dueva-Koganov, O., Bianchini, R. Design of Experiments to Optimize a Novel Antioxidant Blend [conference presentation]. Innovations in Dermatological Sciences, Rutgers, NJ 2018.

Adsorption of Horseradish Peroxidase on Metallic Nanoparticles: Effects on Reactive Oxygen Species Detection Using 2',7'-Dichlorofluorescin Diacetate.

The fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) together with the enzyme horseradish peroxidase (HRP) is widely used in nanotoxicology to study acellular reactive oxygen species (ROS) production from nanoparticles (NPs). This study examined whether HRP adsorbs onto NPs of Mn, Ni, and Cu and if this surface process influences the extent of metal release and hence the ROS production measurements using the DCFH assay in phosphate buffered saline (PBS), saline, or Dulbecco’s modified Eagle’s medium (DMEM). Adsorption of HRP was evident onto all NPs and conditions, except for Mn NPs in PBS. The presence of HRP resulted in an increased release of copper from the Cu NPs in PBS and reduced levels of nickel from the Ni NPs in saline. Both metal ions in solution and the adsorption of HRP onto the NPs can change the activity of HRP and thus influence the ROS results. The effect of HRP on the NP reactivity was shown to be solution chemistry dependent. Most notable was the evident affinity/adsorption of phosphate toward the metal NPs, followed by a reduced adsorption of HRP, the concomitant reduction in released manganese from the Mn NPs, and increased levels of released metals from the Cu NPs in PBS. Minor effects were observed for the Ni NPs. The solution pH should be monitored since the release of metals can change the solution pH and the activity of HRP is known to be pH-dependent. It is furthermore essential that solution pH adjustments are made following the addition of NaOH during diacetyl removal of DCFH-DA. Even though not observed for the given exposure conditions of this study, released metal ions could possibly induce agglomeration or partial denaturation of HRP, which in turn could result in steric hindrance for H2O2 to reach the active site of HRP. This study further emphasizes the influence of HRP on the background kinetics, its solution dependence, and effects on measured ROS signals. Different ways of correcting for the background are highlighted, as this can result in different interpretations of generated results. The results show that adsorption of HRP onto the metal NPs influenced the extent of metal release and may, depending on the investigated system, result in either under- or overestimated ROS signals if used together with the DCFH assay. HRP should hence be used with caution when measuring ROS in the presence of reactive metallic NPs.

New indole-7-aldehyde derivatives as melatonin analogues; synthesis and screening their antioxidant and anticancer potential

Over the last decade, there has been substantial interest in the use of melatonin (MLT) and MLT-like compounds in the treatment of several diseases. MLT can scavenge different reactive oxygen species and can also stimulate the synthesis of antioxidant enzymes.Our ongoing study relies on changing the groups in the different modifiable sites of the indole ring to increase the antioxidant activity. In this study a new approach for substitution of indole ring as indole based MLT analogue was proposed. We report the synthesis and characterization of a series of new indole-7-aldehyde hydrazide/hydrazone derivatives as indole-based MLT analogues. Anticancer potential of the compounds were evaluated both by their antioxidant and CYP1 inhibitory activities. In vitro antioxidant capacity of the compounds was investigated both in a cell-based (DCFH assay) and a cell-free (DPPH assay) assay. Potential inhibitory effects of the compounds on CYP1 catalytic activity were investigated via EROD assay. Cytotoxic activity of the compounds was further evaluated by the MTT assay in CHO-K1 cells. MLT analogues having an o-halogenated aromatic moiety exhibited effective antioxidant properties without having any cytotoxic effect. In conclusion, MLT derivatives represent promising scaffolds for discovery of effective antioxidant agents.

MP01-08 GPX2 PROMOTES THE DEVELOPMENT OF BLADDER CANCER WITH SQUAMOUS DIFFERENTIATION THROUGH APOPTOSIS CONTROL

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (MP01)1 Apr 2020MP01-08 GPX2 PROMOTES THE DEVELOPMENT OF BLADDER CANCER WITH SQUAMOUS DIFFERENTIATION THROUGH APOPTOSIS CONTROL Taku Naiki*, Aya Naiki-Ito, Toshiki Etani, Keitaro Iida, Ryosuke Ando, Takashi Nagai, Noriyasu Kawai, Satoru Takahashi, and Takahiro Yasui Taku Naiki*Taku Naiki* More articles by this author , Aya Naiki-ItoAya Naiki-Ito More articles by this author , Toshiki EtaniToshiki Etani More articles by this author , Keitaro IidaKeitaro Iida More articles by this author , Ryosuke AndoRyosuke Ando More articles by this author , Takashi NagaiTakashi Nagai More articles by this author , Noriyasu KawaiNoriyasu Kawai More articles by this author , Satoru Takahashi Satoru Takahashi More articles by this author , and Takahiro YasuiTakahiro Yasui More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000815.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Reactive oxygen species (ROS) have been identified as important chemical mediators in cell growth and differentiation. The glutathione redox system is the main mechanism protecting against the damage caused by ROS. We investigated the role and therapeutic potential of the glutathione redox system in bladder cancer. METHODS: The expression levels of glutathione peroxidase 2 (GPX2) and Ki-67 proteins were analyzed in human TUR specimens obtained from urethral carcinoma (UC) patients using immunohistochemistry; correlations between the GPX2 expression and prognosis were also analyzed. Further, male F344 rats were given 0.05% BBN in drinking water and 0.1% phenyl isothiocyanate in their diet for 36 weeks. Bladder tissue samples were collected from each animal for analyses. Furthermore, the rat cell line BC31 and human cell lines, were transfected with GPX2 siRNA and negative control siRNA (NC). Subsequently, the cell proliferation rates and ROS levels were investigated by cell counting, DCFH assay, western blotting, and flow cytometry. siRNA- or NC- transfected BC31 cells were subcutaneously implanted into nude mice. RESULTS: GPX2 was strongly expressed in low-grade and low-MIB-1 index cancers. PFS rates were significantly better in patients with higher GPX2 than in those with lower GPX2. Furthermore, GPX2 expression was significantly lower in the normal epithelium of the control group animals with bladder cancer than in the treated group. GPX2 expression was significantly higher in UC than in the normal epithelium with the strongest expression observed in cells with squamous differentiation. BC31 and RT4 cells strongly expressed GPX2 compared to the other cell lines. Silencing the GPX2 caused significant growth inhibition of BC31 and RT4 cells; the DCFH assay revealed significant reductions in the ROS levels in the siRNA-treated cells. Caspase-dependent apoptosis was found to be the cause of the decreased proliferation rates in the siRNA group. Notably, tumor growth was significantly inhibited in the BC31-implanted nude mice using the siRNA strategy for Gpx2. CONCLUSIONS: Our findings demonstrated that GPX2 plays several important roles in carcinogenesis through apoptosis regulation against intracellular ROS and may be a novel marker or therapeutic target in bladder cancer. Source of Funding: Grant-in-Aid for Scientific Research C (17K11153) The Ministry of Education, Culture, Sports, Science and Technology © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e4-e4 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Taku Naiki* More articles by this author Aya Naiki-Ito More articles by this author Toshiki Etani More articles by this author Keitaro Iida More articles by this author Ryosuke Ando More articles by this author Takashi Nagai More articles by this author Noriyasu Kawai More articles by this author Satoru Takahashi More articles by this author Takahiro Yasui More articles by this author Expand All Advertisement PDF downloadLoading ...

RETRACTED: Oxidative potential of particulate matter components generated by specific emission sources

Abstract Different acellular assays are currently used for the determination of the oxidative potential (OP) of particular matter (PM). In order to better understand the existing correlations between OP and the PM generated by specific emission sources, dusts generated from seven different sources (urban dust, soil, road dust, brake dust, pellet ash, coke, and desert dust) were chemically characterized and analysed using three OP assays: dithiothreitol (DTT), acid ascorbic (AA) and 2′,7′-dichlorofluorescin (DCFH). In agreement with some previous literature studies, these assays provided very different results for each selected dust: their reliability as a proxy of ROS generation in biological system need then to be further investigated. When applied to the soluble fraction of these dusts, the DTT and AA assays were particularly sensitive toward pellet ash, while the highest OP DCFH values were measured for road dust. Multivariate analysis was used to confirm the correlations between the OP assay results and the chemical characteristics of the dust soluble fractions. DTT and AA assays appeared mostly influenced by organic and inorganic components, respectively. In the case of the DCFH assay, no clear correlations were highlighted, but, differently from the other assays, relevant OP values were measured also for dust of crustal origin. The insoluble fraction of the dusts, which is often not considered in this kind of study, generated, for all the considered assays, OP values even higher than those related to the soluble fraction.

Particle-bound reactive oxygen species (PB-ROS) emissions and formation pathways in residential wood smoke under different combustion and aging conditions

Abstract. Wood combustion emissions can induce oxidative stress in the human respiratory tract by reactive oxygen species (ROS) in the aerosol particles, which are emitted either directly or formed through oxidation in the atmosphere. To improve our understanding of the particle-bound ROS (PB-ROS) generation potential of wood combustion emissions, a suite of smog chamber (SC) and potential aerosol mass (PAM) chamber experiments were conducted under well-determined conditions for different combustion devices and technologies, different fuel types, operation methods, combustion regimes, combustion phases, and aging conditions. The PB-ROS content and the chemical properties of the aerosols were quantified by a novel ROS analyzer using the DCFH (2′,7′-dichlorofluorescin) assay and a high-resolution time-of-flight aerosol mass spectrometer (HR-ToF-AMS). For all eight combustion devices tested, primary PB-ROS concentrations substantially increased upon aging. The level of primary and aged PB-ROS emission factors (EFROS) were dominated by the combustion device (within different combustion technologies) and to a greater extent by the combustion regimes: the variability within one device was much higher than the variability of EFROS from different devices. Aged EFROS under bad combustion conditions were ∼ 2–80 times higher than under optimum combustion conditions. EFROS from automatically operated combustion devices were on average 1 order of magnitude lower than those from manually operated devices, which indicates that automatic combustion devices operated at optimum conditions to achieve near-complete combustion should be employed to minimize PB-ROS emissions. The use of an electrostatic precipitator decreased the primary and aged ROS emissions by a factor of ∼ 1.5 which is however still within the burn-to-burn variability. The parameters controlling the PB-ROS formation in secondary organic aerosol were investigated by employing a regression model, including the fractions of the mass-to-charge ratios m∕z 44 and 43 in secondary organic aerosol (SOA; f44−SOA and f43−SOA), the OH exposure, and the total organic aerosol mass. The regression model results of the SC and PAM chamber aging experiments indicate that the PB-ROS content in SOA seems to increase with the SOA oxidation state, which initially increases with OH exposure and decreases with the additional partitioning of semi-volatile components with lower PB-ROS content at higher OA concentrations, while further aging seems to result in a decay of PB-ROS. The results and the special data analysis methods deployed in this study could provide a model for PB-ROS analysis of further wood or other combustion studies investigating different combustion conditions and aging methods.

Abstract 1049: Combinatorial effects of thymoquinone on the anticancer activity of doxorubicin in adult T-cell leukemia

Abstract Doxorubicin (Dox) is a clinically approved drug which suffers from drug resistance and cardiotoxicity. Recent studies have shown that thymoquinone (TQ) in combination with Dox can reduce Dox toxic side effects in vitro as well as in vivo. Both TQ and Dox have shown promising anticancer effects against aggressive adult T-cell leukemia (ATL), however, the anticancer potential of TQ and Dox combination treatment has never been tested against ATL. We hypothesized that co-treatment with TQ could enable the use of lower doses of Dox to achieve similar or enhanced anticancer activity. The effects of TQ and Dox combination on cell death and cell cycle were evaluated by trypan blue and propidium iodide. TUNEL assay was used to investigate the mode of cell death. The levels of reactive species (ROS) were determined using DCFH assay, and mitochondrial membrane potential was measured by rhodamine assay. The regulation of key proteins involved in cell cycle regulation and cell death was determined by Western blot. The results reveal that the human T-lymphotropic virus (HTLV-1) positive HuT-102 cells are more resistant to treatment with Dox alone, than the HTLV-1 negative Jurkat cells. However, treatment with high doses of TQ and low doses of Dox simultaneously, enhances cell death in both cancer cell lines as compared to treatment with Dox alone. TUNEL assay on Jurkat and HuT-102 cells further indicated that the combination of TQ and Dox caused cell death by apoptosis. An increase in ROS production was noted in response to treatment with TQ alone and with TQ and Dox combination in both Jurkat and HuT-102 cells. The oxidative stress; however, was only shown to play a role in the disruption of the mitochondria of Jurkat cells. Similarly, caspase activation was involved in the disruption of the mitochondria of Jurkat cells. The expression levels of key regulatory proteins were modulated in response to treatment with the combination. In conclusion, the combination of TQ and Dox effectively inhibits ATL leukemic cancer cell growth at lower doses of Dox which can potentially lower the side effects of the drug. In vivo studies are still warranted to assess the adjuvant chemotherapeutic potential of TQ in combination with Dox. Citation Format: Hala Gali-Muhtasib, Isabelle Fakhoury, Maamoun Fatfat, Rasha Mismar, Regine Schneider-Stock. Combinatorial effects of thymoquinone on the anticancer activity of doxorubicin in adult T-cell leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1049. doi:10.1158/1538-7445.AM2017-1049

Optimization of the Measurement of Particle-Bound Reactive Oxygen Species with 2′,7′-dichlorofluorescin (DCFH)

The 2′,7′-dichlorofluorescin (DCFH) assay is widely used to measure particle-bound reactive oxygen species (ROS), which are considered as a major contributor leading to the adverse health effects upon exposure to atmospheric particulate matter. DCFH, a non-fluorescent substance that can be oxidized to highly fluorescent dichlorofluorescein (DCF) in the presence of horseradish peroxidase (HRP), is usually used as a probe for ROS determination due to its response to diverse and relevant oxidant species. However, there is limited literature that reports the effects of different experimental conditions in the performance of this assay. In our work, various experimental conditions, such as pH value, incubation temperature, reagent concentration and stability, reaction time, linearity range, and extraction method, were examined and optimized as a pilot study for developing an online system for atmospheric ROS measurement. The results showed that pH value, reagent concentration, and extraction method significantly affect the performance of DCFH assay, while incubation at a specified temperature (37 °C) did not increase the oxidization extent of DCFH. After optimization, some practical samples were measured using different experimental parameters to check the performance of the optimized assay. The comparisons of these measurements showed that optimization can greatly improve the detection limit and reproducibility of the DCFH assay, which can then be employed to better the accuracy of offline and online ROS measurement.

MP84-17 LUTEOLIN HAS THERAPEUTIC POTENTIAL THROUGH THE REGULATION OF OXIDATIVE STRESS IN CASTRATION-RESISTANT PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Reactive oxygen species (ROS) have been identified as important chemical mediators of cell growth and differentiation, and the glutathione redox system is the main mechanism protecting against the damage caused by ROS in human body. In previous study, we established a new castration resistant prostate cancer (CRPC) animal model. Results of cDNA microarray analysis using this model revealed that glutathione peroxidase 2 (GPX2) had higher expression in CRPC. In this study, we investigated the role of the glutathione redox system in CRPC and the therapeutic potential of the antioxidant properties of luteolin. METHODS: (i) GPX2 siRNA and negative control siRNA (NC) were used to transfect castration resistant PCai1 and PC3 cells, respectively. After transfection, we investigated the proliferation rate and ROS levels through cell counts, DCFH assay, western blotting, and flow cytometry. (ii) siRNA or NC transfected cells were subcutaneously implanted into normal and castrated nude mice. Three weeks after implantation, the mice were sacrificed, and the subcutaneous tumors were isolated. (iii) The effects of luteolin on cell proliferation and apoptosis in PCai cells and on tumor growth of the subcutaneously implanted PCai1 cells in nude mice were analyzed. RESULTS: (i) Silencing of Gpx2 caused significant growth inhibition of PCai1 and PC3 cells, and the DCFH assay revealed that intracellular ROS were significantly elevated in the siRNA treated groups. The decrease in proliferation rate in the siRNA group was attributed to cyclin B1 dependent cell cycle arrest as assessed with flowcytometry. (ii) On PCai1 implantation in nude mice, the tumor growth of PCai1 was significantly inhibited by silencing Gpx2 in the castrated nude mice. (iii) Luteolin suppressed the cell proliferation in a dose dependent manner in vitro, and inhibited tumor growth in a xenograft model via induction of apoptosis and downregulation of Gpx2. CONCLUSIONS: These results suggest that GPX2 plays an important role in cell growth in the presence of ROS in prostate cancer, and the glutathione redox system can regulate cancer cell growth in castration environment. GPX2 expression may be a therapeutic target in CRPC. Luteolin may be a potentially effective chemotherapeutic agent for CRPC via regulation of expression of GPX2 and induction of apoptosis.

MP84-18 CORRELATION BETWEEN NUCLEAR PPARG EXPRESSION AND BIOCHEMICAL RECURRENCE IN PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Reactive oxygen species (ROS) have been identified as important chemical mediators of cell growth and differentiation, and the glutathione redox system is the main mechanism protecting against the damage caused by ROS in human body. In previous study, we established a new castration resistant prostate cancer (CRPC) animal model. Results of cDNA microarray analysis using this model revealed that glutathione peroxidase 2 (GPX2) had higher expression in CRPC. In this study, we investigated the role of the glutathione redox system in CRPC and the therapeutic potential of the antioxidant properties of luteolin. METHODS: (i) GPX2 siRNA and negative control siRNA (NC) were used to transfect castration resistant PCai1 and PC3 cells, respectively. After transfection, we investigated the proliferation rate and ROS levels through cell counts, DCFH assay, western blotting, and flow cytometry. (ii) siRNA or NC transfected cells were subcutaneously implanted into normal and castrated nude mice. Three weeks after implantation, the mice were sacrificed, and the subcutaneous tumors were isolated. (iii) The effects of luteolin on cell proliferation and apoptosis in PCai cells and on tumor growth of the subcutaneously implanted PCai1 cells in nude mice were analyzed. RESULTS: (i) Silencing of Gpx2 caused significant growth inhibition of PCai1 and PC3 cells, and the DCFH assay revealed that intracellular ROS were significantly elevated in the siRNA treated groups. The decrease in proliferation rate in the siRNA group was attributed to cyclin B1 dependent cell cycle arrest as assessed with flowcytometry. (ii) On PCai1 implantation in nude mice, the tumor growth of PCai1 was significantly inhibited by silencing Gpx2 in the castrated nude mice. (iii) Luteolin suppressed the cell proliferation in a dose dependent manner in vitro, and inhibited tumor growth in a xenograft model via induction of apoptosis and downregulation of Gpx2. CONCLUSIONS: These results suggest that GPX2 plays an important role in cell growth in the presence of ROS in prostate cancer, and the glutathione redox system can regulate cancer cell growth in castration environment. GPX2 expression may be a therapeutic target in CRPC. Luteolin may be a potentially effective chemotherapeutic agent for CRPC via regulation of expression of GPX2 and induction of apoptosis.

In vitro antiproliferative and apoptosis-inducing properties of a mononuclear copper(II) complex with dppz ligand, in two genotypically different breast cancer cell lines.

In the background that there is concerted effort to discover newer metal-based cancer chemotherapeutic agents that could overcome the limitations in cisplatin and that copper, a biocompatible and redox-active metal, offers potential as alternative to cisplatin, the present study was undertaken to investigate the in vitro anti-proliferative properties of the mononuclear copper(II)complex [Cu(L)(diimine)]+where LH=2-[(2-dimethylaminoethylimino)methyl]phenol and diimine=dipyrido[3,2-a:2',3'-c]phenazine (dppz) using breast cancer cell lines MCF-7 (ER(+ve) and p53(WT)) and MDA-MB-231(ER(-ve) and p53(mutant)) when cisplatin was used as positive control. The complex affected the viability of both the cell lines in dose-as well as duration-dependent manner as revealed in the MTT assay. The 24 and 48h IC50 of the complex were several times lesser than those of cisplatin, and within this huge difference the efficacy of the complex was much superior with MCF-7 cell compared to MDA-MB-231 cell. The cell death was preferentially apoptosis, though necrosis also occurred to a certain extent. These inferences were substantiated by AO/EB fluorescent staining, Hoechst staining, assessment of mitochondrial transmembrane potential, comet assay for DNA damage, DCFH assay for reactive oxygen species (ROS) generation and Western blot of apoptosis-related proteins. Thus, the copper(II) dppz complex under investigation is much more efficient than cisplatin in affecting viability of the breast cancer cells. The underlying mechanism appears to be DNA damage-primed (in view of the known intercalation mode of binding of the complex with DNA) and ROS-associated mitochondria-mediated intrinsic apoptosis to a great extent but necrosis also has a role to a certain extent, which may also be a PARP-mediated cell death independent of apoptosis. Within the purview of this conclusion, the results indicate that the ER and/or p53 genotypes have a bearing on the efficacy of the complex as a cytotoxic agent since the response in the ER(-ve) and p53(mutant) MDA-MB-231 cell was not so prominent as in ER(+ve) and p53(WT) MCF-7 cell. Taken together, the complex has been shown to be a potential DNA damaging agent and, in the light of the superiority of the complex over cisplatin, we are further investigating the possibility of targeted nano-delivery of the complex to the tumor cells. When tested on a normal cell, 3T3, Cu(II)dppz was found to affect its viability but at concentrations very high compared to those for the breast cancer cells. Yet, this is a cause of concern and, therefore, we are working out a strategy for targeted delivery of this complex to the cancer cells only.

Abstract 2564: The anticancer molecule TPEN induces DNA damage in human colon cancer cells

Abstract The maintenance of optimal metal levels is an essential aspect of cell homoeostasis. However, in many types of cancer these metal levels especially iron, zinc and copper diverge from normal levels. We have recently shown that the zinc chelator TPEN increases the generation of reactive oxygen species (ROS) which selectively kills colon cancer cells. We have also provided evidence that the redox cycling of copper is responsible for TPEN anticancer effects. In this study, we aimed to further decipher the mechanism of TPEN-induced cell death in colon cancer cells through studying its effect on DNA damage. HCT116 p53+/+ human colon cancer cells were seeded were treated with 5μM TPEN at 50% confluence The inhibition of cell growth was measured by MTT, while ROS production was measured by the DCFH Assay using flow cytometry. siRNAs against DNApk and Chk2 was used to investigate the involvement of these DNA damage sensors in TPEN activity. DNA damage was assessed by the comet assay. Phosphorylation of ATM, ATR, Chk1, Chk2 and H2AX by TPEN were detected immunocytochemically by multiparameter cytometry. Expression levels of Chk1/2, ATR and DNApK were determined by western blotting. We show that cell death by TPEN is associated with significant DNA damage, an effect that was dependent on ROS generation and on the redox cycling of copper, as evidenced by reversal of DNA damage in the presence of antioxidants (NAC, CAT) or the copper chelator neocuproine (Neo). DNA damage was associated with increased expression of p-H2AX and a significant activation of ATM/ATR signaling molecules, specifically p-ATM, p-ATR and p-Chk1. Interestingly, silencing DNApk and Chk2 reversed DNA damage caused by TPEN, suggesting the involvement of DNApk and ATM/ATR pathways in TPEN-mediated effects. This study shows for the first time the involvement of DNApk and Chk2 in TPEN-induced DNA damage and confirms our previous findings that the redox cycling of copper is the main mechanism by which TPEN induces cell death in human colon cancer cells. Citation Format: Hala Gali-Muhtasib, Omar Rahal, Maamoun Fatfat, Carla Hankache, Bassam Osman, Hala Khalife, Khaled Machaca. The anticancer molecule TPEN induces DNA damage in human colon cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2564. doi:10.1158/1538-7445.AM2015-2564

Physicochemical properties and ability to generate free radicals of ambient coarse, fine, and ultrafine particles in the atmosphere of Xuanwei, China, an area of high lung cancer incidence

The link between the high incidence of lung cancer and harmful pollutants emitted by local coal combustion in Xuanwei, Yunnan province, China, has been a focus of study since the 1980s. However, the mechanisms responsible for the high lung cancer rate remain unclear, necessitating further study. Since a close relationship between ambient air particle pollution and respiratory diseases exists, we sampled size-resolved ambient particles from the atmosphere of Xuanwei. In our indoor experiment, cutting-edge methods, including scanning electron microscopy coupled with energy dispersive X-ray detection (SEM/EDX), particle-induced X-ray emission (PIXE), electronic paramagnetic resonance (EPR) and the cell-free DCFH-DA assay, were employed to investigate the physicochemical properties, the potential to generate free radicals and the oxidative potential of ambient coarse (diameter, 1.8–10 μm), fine (diameter, 0.1–1.8 μm), and ultrafine (diameter, <0.1 μm) particles. We found the total mass concentrations of the size-resolved particles collected in spring were higher than that in early winter. Mass percentage of fine particles accounted for 68% and 61% of the total particulate mass in spring and in early winter samples, respectively, indicating that fine particles were the major component of the Xuanwei ambient particulate matters. On the other hand, the results of SEM/EDX analysis showed that the coarse particles were dominated by minerals, the fine particles by soot aggregates and fly ashes, and the ultrafine particles by soot particles and unidentified particles. Our PIXE results revealed that crustal elements (Ca, Ti Si, Fe) were mainly distributed in coarse particles, while trace metals (Cr, Mn, Ni, Cu, Zn, Pb) dominated in the fine particle fraction, and S, a typical element emitted by coal combustion, mainly resided in fine particles collected from the winter atmosphere. EPR results indicated that the magnitude of free radical intensity caused by size-resolved particles followed these patterns: fine particles > coarse particles > ultrafine particles for spring samples and ultrafine particles > fine particles > coarse particles for winter samples. Cell-free DCFH assay results conclusively showed that all of the measured particle suspensions displayed a higher oxidative potential than the negative control. The correlation coefficient (R2) between free radical intensity and fluorescent intensity generated by the size-resolved particles was 0.535 and 0.507 for the spring and winter seasons, respectively, implying that ambient air particles in the Xuanwei atmosphere have the ability to generate free radicals, and fine and ultrafine particles could be hazardous to local residents.