Oxidative stress and apoptosis are induced in human endothelial cells exposed to urban particulate matter

Correlations between exposure to particle matter (PM) with an aerodynamic diameter ⩽ 2.5 or 10 μm (PM_2.5 and PM₁₀, respectively) with cardiovascular effects have been demonstrated recently. Endothelial cells seem to play a relevant role in the responses to PM due to their participation in pro-inflammatory events. In this study we determined the effect of PM_2.5 and PM₁₀ from Mexico City on human endothelial cells by means of evaluating reactive oxygen species (ROS), nitric oxide (NO), NF-κB translocation and cell death. For this purpose we used human umbilical vein endothelial cells (HUVEC) as a model.The production of ROS was determined by the reduction of H₂DCFDA and NO by Griess reagent. The translocation of NF-κB was evaluated by Electrophoretic Mobility Shift Assay (EMSA) and the cellular death by the translocation of phosphatidylserine. TNF-α was used as a positive control for endothelial cell activation.PM_2.5 and PM₁₀ induced the production of ROS (77% and 126% increase, respectively, vs. control) and NO (up to 132% and 233% increase, respectively, vs. control). PM_2.5 and PM₁₀ also induced the nuclear translocation of NF-κB. All these events were associated with apoptosis. In conclusion, the activation of HUVEC induced by PM_2.5 and PM₁₀ is related with an oxidative stress, suggesting that these particles may participate in the development of cardiovascular and inflammatory diseases.     

Impact of probable interaction of low temperature and ambient fine particulate matter on the function of rats alveolar macrophages

The present study aimed to explore the probable interaction of low temperature and ambient fine particulate matter (PM_2.5) on rat alveolar macrophages (AMs). AMs were separated from rat BALF and exposed to PM_2.5 (0, 25, 50, 100 μg/ml) under different temperature (18, 24, 30, 37 °C) for 8 h. Results indicated that viability and phagocytosis function of AMs decreased with the decline of temperature and the rise of PM_2.5 dose, and the strongest toxicity was shown in the highest PM_2.5 (100 μg/ml) exposure group at 18 °C. Both PM_2.5 and lower temperature increased the releasing of tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein 1α (MIP-1α) and interleukin-6 (IL-6), while significant interaction was only found in MIP-1α production. No obvious change was found in granulocyte-macrophage colony-stimulating factor (GM-CSF) detection. These results indicated that both the two factors are harmful to rat AMs and lower temperature could increase the toxicity of PM_2.5 on the AMs.     

PM2.5-induced oxidative stress triggers autophagy in human lung epithelial A549 cells

Exposure to higher levels of air pollution particulate matter (PM) with an aerodynamic diameter of less than 2.5 μm (PM_2.5) links with an increased risk of cardiovascular and respiratory deaths and hospital admission as well as lung cancer. Although the mechanism underlying the correlation between PM_2.5 exposure and adverse effects has not fully elucidated, PM_2.5-induced oxidative stress has been considered as an important molecular mechanism of PM_2.5-mediated toxicity. In this work, human lung epithelial A549 cells were used to further investigate the biological effects of PM_2.5 on autophagy. The cell viability showed both time- and concentration-dependent decrease when exposure to PM_2.5, which can be attributed to increase of the levels of extracellular lactate dehydrogenase (LDH) release and intracellular reactive oxygen species (ROS) generation in A549 cells. Moreover, PM_2.5-induced oxidative damage in A549 cells was observed through the alteration of superoxide dismutase (SOD) and catalase (CAT) activities compared to the unexposed control cells. PM_2.5-induced autophagy was indicated by an increase in microtubule-associated protein light chain-3 (LC3) puncta, and accumulation of LC3 in both time- and concentration-dependent manner. PM_2.5-induced mRNA expression of autophagy-related protein Atg5 and Beclin1 was also observed compared with those of the unexposed control cells. These results suggest the possibility that PM_2.5-induced oxidative stress probably plays a key role in autophagy in A549 cells, which may contribute to PM_2.5-induced impairment of pulmonary function.     

Reactive oxygen species induced by beauvericin,patulin and zearalenone in CHO-K1 cells

The cytotoxic effects of mycotoxins, induction of reactive oxygen species (ROS) and generation of lipid peroxidation products in CHO-K1 cells were determined as function of increasing time of exposure and concentrations of beauvericin (BEA), patulin (PAT) and zearalenone (ZEA). The end points were evaluated after 24 h of exposure, by the tetrazolium salt (MTT) and neutral red (NR) assays. The IC₅₀ values obtained on the MTT and NR assays ranged from 0.69 to 79.40 μM and 4.40 to 108.76 μM, respectively. To determine the intracellular production of ROS, the intensity of fluorescence emitted from the probe H₂-DCFDA was measured. The relative intensity of fluorescence from cells incubated with BEA, PAT and ZEA was approximately 4-, 7- and 4-fold higher in comparison with control cells at 0 min, respectively. Lipid peroxidation induced by ROS generation was assessed using the thiobarbituric acid reactive substances (TBARS) method for 2, 24 and 48 h. The malondialdehyde (MDA) production was increased with BEA and PAT exposure in a concentration- and time-dependent manner. MDA was not increased after 1 and 5 μM ZEA exposures for 2 h, but was slightly increased at 50 μM.In conclusion, PAT was the most cytotoxic mycotoxin to CHO-K1 cells, followed by BEA and ZEA. Mycotoxins reduce cell viability correlated with the increases of ROS generation and MDA formation in concentration- and time-dependent manner. These data suggested that cytotoxicity and ROS generation are mechanisms of mycotoxins mediated toxicity.     

Chitosan oligosaccharides attenuate hydrogen peroxide-induced stress injury in human umbilical vein endothelial cells

Chitosan oligosaccharides (COS) have been reported to have anticancer activity, immuno-enhancing effect and antimicrobial activity. However, other biological activities are unknown. Herein, we investigated the protective effects of COS against hydrogen peroxide (H₂O₂)-induced oxidative damage on human umbilical vein endothelial cells (HUVEC, ECV304 cells). After 24 h pre-incubation with COS (25–200 μg/ml), the viability loss in ECV304 cells induced by H₂O₂ (300 μM) for 12 h was markedly restored in a concentration-dependent manner as measured by MTT assay. This effect was accompanied by a marked decrease in intracellular reactive oxygen species (ROS) by measuring intensity of DCFH fluorescence. COS also exerted preventive effects on suppressing the production of lipid peroxidation such as malondialdehyde (MDA), restoring activities of endogenous antioxidants including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), along with the capacity of increasing levels of nitric oxide (NO) and nitric oxide synthase (NOS), as were determined by commercial regent kits. In addition, pre-incubation of COS with ECV304 cells for 24 h resulted in the reduction of apoptosis and the induction of cell cycle arrest in G₁/S + M phase as assayed quantitatively by Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit using flow cytometry. Taken together, our findings suggest that COS can effectively protect HUVECs against oxidative stress by H₂O₂, which might be of importance in the treatment of cardiovascular diseases.     

In vitro and in vivo assessment of pulmonary risk associated with exposure to combustion generated fine particles

Strong correlations exist between exposure to PM_2.5 and adverse pulmonary effects. PM_2.5 consists of fine (≤2.5 μm) and ultrafine (≤0.1 μm) particles with ultrafine particles accounting for >70% of the total particles. Environmentally persistent free radicals (EPFRs) have recently been identified in airborne PM_2.5. To determine the adverse pulmonary effects of EPFRs associated with exposure to elevated levels of PM_2.5, we engineered 2.5 μm surrogate EPFR-particle systems. We demonstrated that EPFRs generated greater oxidative stress in vitro, which was partly responsible for the enhanced cytotoxicity following exposure. In vivo studies using rats exposed to EPFRs containing particles demonstrated minimal adverse pulmonary effects. Additional studies revealed that fine particles failed to reach the alveolar region. Overall, our study implies qualitative differences between the health effects of PM size fractions.     

Ginsenoside Rg3 enhances the chemosensitivity of tumors to cisplatin by reducing the basal level of nuclear factor erythroid 2-related factor 2-mediated heme oxygenase-1/NAD(P)H quinone oxidoreductase-1 and prevents normal tissue damage by scavenging cisplatin-induced intracellular reactive oxygen species

The clinical use of cisplatin (cis-diamminedichloroplatinum II) has been limited by the frequent emergence of cisplatin-resistant cell populations and numerous other adverse effects. Therefore, new agents are required to improve the therapy and health of cancer patients. Oral administration of ginsenoside Rg3 significantly inhibited tumor growth and promoted the anti-neoplastic efficacy of cisplatin in mice inoculated with CT-26 colon cancer cells. In addition, Rg3 administration remarkably inhibited cisplatin-induced nephrotoxicity, hepatotoxicity and oxidative stress. In cell-based experiments, Rg3 inhibited cisplatin-induced cytotoxicity in LLC-RK1 kidney and NCTC1469 liver cells but not in CT-26 cancer cells and significantly decreased cisplatin-induced intracellular ROS levels in these cells. In normal cells with cytoplasmically localized Nrf2 and negligible levels of HO-1 and NQO-1, Rg3 substantially decreased cisplatin-induced elevation in HO-1/NQO-1 levels and inhibited cisplatin-induced translocation of Nrf2 into the nucleus. In chemoresistant cancer cells with high levels of HO-1/NQO-1 and nuclear Nrf2, both basal and cisplatin-induced levels of HO-1/NQO-1 and nuclear Nrf2 were decreased by Rg3 treatment, thereby enhancing the susceptibility of cancer cells to cisplatin. Collectively, Rg3 promotes the efficacy of cisplatin by inhibiting HO-1 and NQO-1 expression in cancer cells and protects the kidney and liver against tissue damage by preventing cisplatin-induced intracellular ROS generation.     

Toxic effects induced by mycotoxin fumonisin B1 on equine spermatozoa: Assessment of viability,sperm chromatin structure stability,ROS production and motility

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium species that exerts its toxic effect through interference with the sphingolipid pathway by inhibiting ceramide synthase. A FB1-dependent sperm toxicity was reported in boars. No information on FB1-related reproductive toxicity in stallions, the most sensitive animal species, has been reported. The aim of the present study was to assess the in vitro toxicity of FB1 on fresh and frozen-thawed equine spermatozoa by analyzing sperm viability, chromatin stability (SCSA) and reactive oxygen species (ROS) production by flow cytometry and sperm motility by CASA system. Fumonisin B1 did not affect viability of fresh spermatozoa after 2 h exposure up to 25 μM. Damage on sperm chromatin structure was observed only in one frozen sample after exposure up to 2.5 × 10⁻⁵ μM FB1 without associated increase of ROS. Increase of ROS, at FB1 levels up to 2.5 × 10⁻⁴ μM, was found on another frozen-thawed sperm sample, may be as a consequence of seminal plasma removal. At 7.5 and 15 μM, FB1 induced reduction of total and progressive motility.     

Oxidized casein impairs antioxidant defense system and induces hepatic and renal injury in mice

ScopeOxidized protein products (OPPs) can be easily found in meat and milk during processing and storage. Evidence supports that accumulation of endogenous OPPs plays a negative role in physiological metabolism. However, the impacts of dietary OPPs and the mechanisms have not been elucidated yet. The present study evaluated whether oral oxidized casein would destruct the antioxidant defense system and cause potential oxidized injury in mice liver and kidney.Methods and resultsWe performed oxidized casein (modified respectively by H₂O₂–Cu and HClO) feeding experiments using KM mice (20–22 g). A 10-weeks feeding of oxidized casein as basal protein caused oxidative stress by increasing protein carbonylation (PC), advanced oxidation protein products (AOPPs), dityrosine (Dityr), lipid peroxidation and ROS levels in mice liver, kidney and blood (P < 0.05). In mice liver and kidney, the mRNA expression of Nrf2, γ-GCS, HO-1, GPX-3, and GPX-4 up-regulated, the protein level of Nrf2 in nucleus increased. However, activities of anti-oxidant enzymes (CAT, SOD, and GPX) decreased (P < 0.05). Moreover, histopathological examination displayed the formation of fibrous septa in mice liver and kidney after oxidized casein feeding.ConclusionOxidized casein impairs antioxidant defense system and induces hepatic and renal fibrosis.     

Reduced in vitro toxicity of fine particulate matter collected during the 2008 summer Olympic Games in Beijing: The roles of chemical and biological components

Beijing has implemented systematic air pollution control legislation to reduce particulate emissions and improve air quality during the 2008 Summer Olympics, but whether the toxicity of fine fraction of particles (PM_2.5) would be changed remains unclear. In present study we compared in vitro biological responses of PM_2.5 collected before and during the Olympics and tried to reveal possible correlations between its chemical components and toxicological mechanism(s). We measured cytotoxicity, cytokines/chemokines, and related gene expressions in murine alveolar macrophages, MH-S, after treated with 20 PM_2.5 samples. Significant, dose-dependent effects on cell viability, cytokine/chemokine release and mRNA expressions were observed. The cytotoxicity caused at equal mass concentration of PM_2.5 was notably reduced (p < 0.05) by control measures, and significant association was found for viability and elemental zinc in PM_2.5. Endotoxin content in PM_2.5 correlated with all of the eight detected cytokines/chemokines; elemental and organic carbon correlated with four; arsenic and chromium correlated with six and three, respectively; iron and barium showed associations with two; nickel, magnesium, potassium, and calcium showed associations with one. PM_2.5 toxicity in Beijing was substantially dependent on its chemical components, and lowering the levels of specific components in PM_2.5 during the 2008 Olympics resulted in reduced biological responses.     

Epigallocatechin gallate potentially abrogates fluoride induced lung oxidative stress,inflammation via Nrf2/Keap1 signaling pathway in rats: An in-vivo and in-silico study

BackgroundSince this Nrf2-dependent cellular defense response is able to protect multi-organs, including cancer, neurodegenerative diseases, cardiovascular diseases, inflammation and chronic lung injury. The antioxidant and anti-inflammatory potential of Epigallocatechin gallate (EGCG) and Nrf2/Keap1 signaling mechanisms in pulmonary toxicity have not been clarified. In the present study, we demonstrated that protective efficacy of EGCG against fluoride (Fl) induced oxidative stress mediated lung injury in rats.MethodsThe animals were divided in to four groups. Group 1: Control rats received normal saline; Group 2 rats received EGCG (40 mg/kg/bw) alone for four weeks; Group 3 rats received Fl (25 mg/kg/bw) alone for four weeks, Group 4 rats received EGCG (90 min before administration) along with Fl for four weeks.ResultsOral administration of Fl (25 mg/kg/bw) significantly (p < 0.05) increased the ROS, inflammatory cytokines, lung edema, melonaldehyde (MDA) and myeloperoxidase (MPO) in rats. In addition, upon administration of Fl significantly (p < 0.05) decreased the antioxidant status, Nrf2, and HO-1 with increased Keap1 protein. Histological and immunohistochemical (iNOS) study also revealed the Fl induced significant (p < 0.05) changes in the lung tissue of rats. Pre-administration of EGCG significantly (p < 0.05) improved the antioxidant status, and inhibited the oxidative stress, inflammatory cytokines, and Keap1 protein via the activation of Nrf2 translocation in to the nucleus. Moreover, the molecular docking studies also support the antioxidant potential of EGCG and Nrf2 activation.ConclusionTaken together, our data indicate that EGCG potentially abrogates Fl induced oxidative lung injury by activation of the Nrf2/Keap1 pathway in rats.     

Ascorbic acid reduces HMGB1 secretion in lipopolysaccharide-activated RAW 264.7 cells and improves survival rate in septic mice by activation of Nrf2/HO-1 signals

High mobility group box 1 (HMGB1) is now recognized as a late mediator of sepsis. We tested hypothesis that ascorbic acid (AscA) induces heme oxygenase (HO)-1 which inhibits HMGB1 release in lipopolysaccharide (LPS)-stimulated cells and increases survival of septic mice. AscA increased HO-1 protein expression in a concentration- and time-dependent manner via Nrf2 activation in RAW 264.7 cells. HO-1 induction by AscA was significantly reduced by Nrf2 siRNA-transfected cells. Mutation of cysteine to serine of keap-1 proteins (C151S, C273S, and C288S) lost the ability of HO-1 induction by AscA, due to failure of translocation of Nrf-2 to nucleus. The PI3 kinase inhibitor, LY294002, inhibited HO-1 induction by AscA. Oxyhemoglobin (HbO₂), LY294002, and ZnPPIX (HO-1 enzyme inhibitor) reversed effect of AscA on HMGB1 release. Most importantly, administration of AscA (200 mg/kg, i.p.) significantly increased survival in LPS-induced endotoxemic mice. In cecal ligation and puncture (CLP)-induced septic mice, AscA reduced hepatic injury and serum HMGB1 and plasminogen activator inhibitor (PAI)-1 in a ZnPPIX-sensitive manner. In addition, AscA failed to increase survival in Nrf2 knockout mice by LPS. Thus, we concluded that high dose of AscA may be useful in the treatment of sepsis, at least, by activation of Nrf2/HO-1 signals.     

Cadmium (Cd2+) exposure differentially elicits both cell proliferation and cell death related responses in SK-RC-45

Cadmium (Cd²⁺) is a major nephrotoxic environmental pollutant, affecting mostly proximal convoluted tubule (PCT) cells of the mammalian kidney, while conditionally Cd²⁺ could also elicit protective responses with great variety and variability in different systems. The present study was designed to evaluate the molecular mechanism of Cd²⁺ toxicity on human PCT derived Renal Cell Carcinoma (RCC), SK-RC-45 and compare its responses with normal human PCT derived cell line, NKE. Exposure of SK-RC-45 cells with different concentrations of CdCl₂ (e.g. 0, 10 and 20 μM) in serum free medium for 24 h generate considerable amount of ROS, accompanied with decreased cell viability and alternations in the cellular and nuclear morphologies, heat shock responses and GCLC mediated protective responses. Also phosphatidylserine externalization, augmentation in the level of caspase-3, PARP, BAD, Apaf1 and cleaved caspase-9 along with decreased expression of Bcl2 and release of cytochrome c confirmed that, Cd²⁺ dose dependently induces solely intrinsic pathway of apoptosis in SK-RC-45, independent of JNK. Furthermore, the non-toxic concentration (10 μM) of Cd²⁺ induced nuclear translocation of Nrf2 and increased expression in the level of HO-1 enzyme suggesting that at the milder concentration, Cd²⁺ induces protective signaling pathways. On the other hand, exposure of NKE to different concentrations of CdCl₂ (e.g. 0, 10, 20, 30 and 50 μM) under the same conditions elevate stronger heat shock and SOD2 mediated protective responses. In contrary to the RCC PCT, the normal PCT derived cell follows JNK dependent and extrinsic pathways of apoptosis. Cumulatively, these results suggest that Cd²⁺ exposure dose dependently elicit both cell proliferative and cell death related responses in SK-RC-45 cells and is differentially regulated with respect to normal kidney epithelia derived NKE cells.     

H3K9 acetylation change patterns in rats after exposure to traffic-related air pollution

Traffic-related air pollution (TRAP) has been acknowledged as a potential risk factor for numerous respiratory disorders including lung cancer; however, the exact mechanisms involved are still unclear. Here we investigated the effects of TRAP exposure on the H3K9 acetylation in rats. The exposure was performed in both spring and autumn with identical study procedures. In each season, 48 healthy Wistar rats were exposed to different levels of TRAP for 4 h, 7 d, 14 d, and 28 d, respectively. H3K9 acetylation levels in both the peripheral blood mononuclear cells (PBMCs) and lung tissues were quantified. Multiple linear regression was applied to assess the influence of air pollutants on H3K9 acetylation levels. The levels of PM_2.5, PM₁₀, and NO₂ in the tunnel and crossroad groups were significantly higher than in the control group. The H3K9 acetylation levels were not significantly different between spring and autumn. When spring and autumn data were analyzed together, no significant association between the TRAP and H3K9 acetylation was found in 4 h exposure window. However, in the 7 d exposure window, PM_2.5 and PM₁₀ exposures were associated with changes in H3K9 acetylation ranging from 0.276 (0.053, 0.498) to 0.475 (0.103, 0.848) per 1 μg/m³ increase in the pollutant concentration. In addition, prolonged exposure of the rats in the tunnel showed that both PM_2.5 and PM₁₀ concentrations were positively associated with H3k9 acetylation in both PBMCs and lung tissues. The findings showed that 7-d and prolonged TRAP exposure could effectively increase the H3K9 acetylation level in both PBMCs and lung tissues of rats.     

PM2.5-induced alterations of cell cycle associated gene expression in lung cancer cells and rat lung tissues

The aim of the current study was to investigate the expression of cell cycle-associated genes induced by fine particulate matter (PM_2.5) in lung cancer cell line and tissues. The pulmonary lymph node metastasis cells (H292) were treated with PM_2.5 in vitro. Wistar rats were used to perform an in vivo study. Rats were randomly assigned to experiment and control groups and those in the experiment group were exposed to PM_2.5 once every 15 d, while those in the control group were exposed to normal saline. The cell cycle-associated genes expression was analyzed by real-time PCR. Trachea and lung tissues of rats were processed for scanning electron microscopic (SEM) examinations. Exposure of H292 cells to PM_2.5 dramatically increased the expressions of p53 and cyclin-dependent kinase 2 (CDK2) after 24 h of exposure (p < 0.01) and markedly increased the expressions of the cell division cycle 2 (Cdc2) and cyclin B after 48 h of exposure (p < 0.01), while those genes expressions were significantly reduced after 72 h of exposure, at which time the expression of p21 was predominant (p < 0.01). In vivo studies further demonstrated these results. The results of SEM suggested that both of the trachea and lung tissues were damaged and the degree of damage was time-dependent. In conclusion, PM_2.5 can induce significantly alterations of p53 and CDK2 in the early phase, Cdc2 and cyclin B in mid-term and p21 in long-term exposure. The degree of PM_2.5-induced damage to the trachea and lung tissue was time-dependent.     

Cell damage induced by copper: An explant model to study anemone cells

Sea anemones are benthic organisms, of low mobility and can be directly affected by water pollution. This work studied the defense mechanisms and DNA damage caused by copper toxicity in cells from the anemone Bunodosoma cangicum. For this, exposure of anemones cells were held, kept in primary culture through explant of podal disk to copper (7.8 and 15.6 μg/L), and the control group, for 6 and 24 h. Cytotoxicity was seen through the viability and cell number, MXR phenotype through the accumulation of rhodamine-B, ROS generation by H₂DCF-DA and DNA damage by comet assay. The results obtained show that there is a drop in viability and number of cells, especially after exposure of 24 h in 15.6 μg/L. There is an induction of the MXR activity only at 7.8 μg/L for 24 h. As for ROS, there is an increase in the generation of reactive species in greatest concentration of copper for 6 h, and in both for 24 h, which leads to oxidative stress, which culminates with a DNA damage. What was evidenced by the increase of the tail size, % DNA presented and moment of tail. Therefore, the copper represents an adversity to the anemones cells, being cytotoxic and genotoxic.     

Troubleshooting the dichlorofluorescein assay to avoid artifacts in measurement of toxicant-stimulated cellular production of reactive oxidant species

IntroductionThe dichlorofluorescein (DCF) assay is a popular method for measuring cellular reactive oxidant species (ROS). Although caveats have been reported with the DCF assay and other compounds, the potential for artifactual results due to cell-free interactions between the DCF compound and toxicants has hardly been explored. We evaluated the utility of the DCF assay for measuring ROS generation by the toxicants mono-(2-ethylhexyl) phthalate (MEHP), and tetrabromobisphenol A (TBBPA).MethodsDCF fluorescence was measured spectrofluorometrically after a 1-h incubation of toxicants with 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H₂DCFDA). MEHP was incubated with carboxy-H₂DCFDA in cell-free solutions of Hank's buffered salt solution (HBSS), or in Royal Park Memorial Institute (RPMI) medium with or without fetal bovine serum. TBBPA was incubated with carboxy-H₂DCFDA in cell-free HBSS and with human trophoblast cells (HTR8/SVneo cells).ResultsMEHP did not increase fluorescence in solutions of carboxy-H₂DCFDA in HBSS or RPMI medium without serum. However, MEHP (90 and 180 μM) increased DCF fluorescence in cell-free RPMI medium containing serum. Furthermore, serum-free and cell-free HBSS containing 25 μM TBBPA exhibited concentration-dependent increased fluorescence with 5–100 μM carboxy-H₂DCFDA (p < 0.05), but not 1 μM carboxy-H₂DCFDA. In addition, we observed increased fluorescence in HTR8/SVneo cell cultures exposed to TBBPA (0.5–25 μM) (p < 0.05), as we had observed in cell-free buffer.DiscussionMEHP demonstrated an interaction with serum in cell-free generation of DCF fluorescence, whereas TBBPA facilitated conversion of carboxy-H₂DCFDA to the fluorescent DCF moiety in the absence of serum. Because TBBPA increased fluorescence in the absence of cells, the increased DCF fluorescence observed with TBBPA in the presence of cells cannot be attributed to cellular ROS and may, instead, be the result of chemical activation of carboxy-H₂DCFDA to the fluorescent DCF moiety. These data illustrate the importance of including cell-free controls when using the DCF assay to study toxicant-stimulated cellular production of ROS.