Ibrahim O. Farah

Accumulation and Toxicity of CuO and ZnO Nanoparticles Through Waterborne and Dietary Exposure of Goldfish (Carassius auratus)

Dietary and waterborne exposure to copper oxide (CuO) and zinc oxide (ZnO) nanoparticles (NPs) was conducted using a simplified model of an aquatic food chain consisting of zooplankton (Artemia salina) and goldfish (Carassius auratus) to determine bioaccumulation, toxic effects, and particle transport through trophic levels. Artemia contaminated with NPs were used as food in dietary exposure. Fish were exposed to suspensions of the NPs in waterborne exposure. ICP‐MS analysis showed that accumulation primarily occurred in the intestine, followed by the gills and liver. Dietary uptake was lower, but was found to be a potential pathway for transport of NPs to higher organisms. Waterborne exposure resulted in about a 10‐fold higher accumulation in the intestine. The heart, brain, and muscle tissue had no significant Cu or Zn. However, concentrations in muscle increased with NP concentration, which was ascribed to bioaccumulation of Cu and Zn released from NPs. Free Cu concentration in the medium was always higher than that of Zn, indicating CuO NPs dissolved more readily. ZnO NPs were relatively benign, even in waterborne exposure (p ≥ 0.05). In contrast, CuO NPs were toxic. Malondialdehyde levels in the liver and gills increased substantially (p < 0.05). Despite lower Cu accumulation, the liver exhibited significant oxidative stress, which could be from chronic exposure to Cu ions.

Probing metabolic stability of CdSe nanoparticles: Alkaline extraction of free cadmium from liver and kidney samples of rats exposed to CdSe nanoparticles

Cadmium selenide nanoparticles (CdSe NPs) exhibit novel optoelectronic properties for potential biomedical applications. However, their metabolic stability is not fully understood because of the difficulties in measurement of free Cd from biological tissues of exposed individuals. In this study, alkaline dissolution with tetramethylammonium hydroxide (TMAH) is demonstrated for selective determination of free Cd and intact NPs from liver and kidney samples of animals that were exposed to thiol-capped CdSe NPs. Aqueous suspensions of CdSe NPs (3.2 nm) were used to optimize the conditions for extracting free Cd without affecting NPs. Nanoparticles were found to aggregate when heated in TMAH without releasing any significant Cd to solution. Performance of the method in discriminating free Cd and intact NPs were verified by Dogfish Liver (DOLT-4) certified reference material. The samples from the animals were digested in 4 mL TMAH at 70°C to extract free Cd followed by analysis of aqueous phase by ICP-MS. Both liver and kidney contained significant levels of free Cd. Total Cd was higher in the liver, while kidney accumulated mostly free Cd such that up to 47.9% of total Cd in the kidney was free Cd when NPs were exposed to UV-light before injection.

Evaluation of alpha and gamma aluminum oxide nanoparticle accumulation, toxicity, and depuration in Artemia salina larvae.

In this study, Artemia salina (crustacean filter feeders) larvae were used as a test model to investigate the toxicity of aluminum oxide nanoparticles (Al2O3 NPs) on marine microorganisms. The uptake, toxicity, and elimination of α‐Al2O3 (50 nm and 3.5 μm) and γ‐Al2O3 (5 nm and 0.4 μm) NPs were studied. Twenty‐four and ninety‐six hour exposures of different concentrations of Al2O3 NPs to Artemia larvae were conducted in a seawater medium. When suspended in water, Al2O3 NPs aggregated substantially with the sizes ranging from 6.3 nm to >0.3 µm for spherical NPs and from 250 to 756 nm for rod‐shaped NPs. The phase contrast microscope images showed that NPs deposited inside the guts as aggregates. Inductively coupled plasma mass spectrometry analysis showed that large particles (3.5 μm α‐Al2O3) were not taken up by Artemia, whereas fine NPs (0.4 μm γ‐Al2O3) and ultra‐fine NPs (5 nm γ‐Al2O3 and 50 nm α‐Al2O3) accumulated substantially. Differences in toxicity were detected as changing with NP size and morphology. The malondialdehyde levels indicated that smaller γ‐Al2O3 (5 nm) NPs were more toxic than larger γ‐Al2O3 (0.4 µm) particulates in 96 h. The highest mortality was measured as 34% in 96 h for γ‐Al2O3 NPs (5 nm) at 100 mg/L (LC50 > 100 mg/L). γ‐Al2O3 NPs were more toxic than α‐Al2O3 NPs at all conditions.

D-Glucose-Induced Cytotoxic, Genotoxic, and Apoptotic Effects on Human Breast Adenocarcinoma (MCF-7) Cells

Introduction Glucose is a simple sugar that plays an important role in energy production in biological systems. However, it has been linked to many long-term health problems including the risk of heart disease and stroke, erectile dysfunction in men and pregnancy complications in women, and damage to the kidneys, nerves, eye and vision. Also, the underlying mechanisms of diabetic complications are poorly understood. Methods In the present study, D-glucose-induced cytotoxic, genotoxic, and apoptotic effects were studied using MCF-7 cells as an in vitro test model. Cell viability was determined by MTT assay. Genotoxic damage was tested by the means of alkaline single cell gel electrophoresis (Comet) assay. Cell apoptosis was measured by flow cytometry assessment (Annexin-V/PI assay). Results The results of MTT assay indicated that D-glucose significantly reduces the viability of MCF-7 cells in a dose and time-dependent manner. Similar trend was obtained with the trypan blue exclusion test. Data obtained from the Comet assay indicated that D-glucose causes DNA damage in MCF-7 cells in a dose-dependent manner. The flow cytometry assessment (Annexin V FITC/PI) showed a strong dose-response relationship between D-glucose exposure and annexin V positive MCF-7 cells undergoing early apoptosis. Conclusion Taking together, these data provide clear evidence that D-glucose induces cytotoxic, genotoxic, and apoptotic effects on MCF-7 cells. This finding represents the basis for further studies addressing the pathophysiological mechanisms of action of glucose overdose.

Abstract 81: Differential metabolic and viability responses of the A549 and MRC-5 cell lines upon exposure to selective organic inhibitors of glycolysis

Lung cancer is a one of the most prevalent and deadly cancers in United States. Research has shown that cancer cells exhibit higher glycolytic rates than normal cells. Studies on the differential role of the interference with glycolysis/energy generation by cancer cells through the use natural glycolytic inhibitors were limited in the literature. Therefore, we hypothesize that exposure of lung cancer cells to natural inhibitors of glycolysis will negatively influence their metabolic activities as well as their cell viabilities due to the inhibition of substrate-level ATP generation, while differentially not affecting the normal lung phenotype. The human lung fibroblast cell line (MRC-5) was selected to represent the normal human lung and the human alveolar epithelial cell line A549 was selected to represent lung cancer in vitro. These cells were maintained and exposed to eleven different organic reagents including fructose diphosphate (FDP), sodium citrate, ascorbic acid, crude honey, sodium bicarbonate, D-glucose, oxalic acid, glycerol, zinc acetate, pyruvic acid, and sodium ascorbate at concentration levels ranging from 31.3-2,000 ug/ml for 24-72 hours in 5% CO 2 and 96 well plates using MTT, Alamar blue and the T4 cellometer assays as well as phase-contrast photo-imaging. Our results indicate that exposure of A549 cells to these organics resulted in concentration dependent differential cell destruction of the A549 cell line. Nine of the eleven organics used showed statistically significant (p 50 using MTT and Alamar blue assays ranged between 161-1041 ug/ml. None of the eleven chemicals used allowed for cell proliferation of the A549 cell line while showing no or minor effects on the MRC-5 cell line. We conclude that nine of the tested organics impaired glycolysis, which is crucial to the generation of cellular energy and survival of the A549 cell line. Supported by NIH-RCMI grant# G12RR13459 and NIH-RISE grant # 63228. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 81.

Recreational water quality control in Mississippi, USA: bacteriological assessment in the Pearl River and Ross Barnett reservoir.

We assessed the bacteriological water quality in the Pearl River and Ross Barnett reservoir, a major source of public raw water for the city of Jackson, Mississippi, USA and an important site for recreational activities for local residents and visitors. Infectious diseases caused by pathogenic bacteria are the most common and widespread health risks associated with such water contact activities as bathing, canoeing, and swimming in recreational waters. Water samples collected twice monthly from April 2004 to April 2005 from five different sites of the Pearl river/Ross Barnett reservoir were tested for heterotrophic bacteria, total coliforms, fecal coliforms, and enterococci using membrane filtration technique. Physicochemical parameters (temperature, pH, turbidity, dissolved oxygen, conductivity) were also analyzed using standard methods. The respective mean concentrations of bacteria in water samples were 8.9 x 10(4) +/- 7.4 x 10(4) colony forming units (CFU) 100 mL(-1), 3.0 x 10(3) +/- 4.1 x 10(3) CFU 100 mL(-1), 2.3 x 10(2) +/- 5.4 x 10(2) CFU 100 mL(-1), and 2.3 x 10(2) +/- 4.8 x 10(2) CFU 100 mL(-1) for heterotrophic bacteria, total coliforms, enterococci, and fecal coliforms. The mean values of the physical and chemical parameters were at acceptable levels. Bacterial densities, however, significantly exceeded federal/state guidelines, raising public health concerns. Hence, control strategies should be developed and implemented to prevent further bacterial contamination of Pearl River-Ross Barnett reservoir water resource system.

Abstract 1168: Impact of some common organics on cellular glycolysis and the differential survival of lung fibroblast and lung carcinoma cell lines

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Tumor growth and abnormal cell survival were shown to be associated with a number of cellular metabolic abnormalities revealed by impaired oral glucose tolerance, depressed lipoprotein lipase activity leading to hypertriglyceridemia, and changes in amino acid profile as evidenced by increased plasma free tryptophan levels in patients with breast, lung, colon, stomach, and other cancers from various origins. The above findings seem to relate to or indicate a shift to non-oxidative metabolic pathways in cancer. In contrast to normal cells, cancer cells may lose the ability to utilize aerobic respiration due to either defective mitochondria or hypoxia within the tumor microenvironments. Glucose was shown to be the major energy source in cancer cells where it utilizes aerobic /anaerobic glycolysis with the resultant lactic acid formation. The role of energetic modulations and use of glycolytic inhibitors on cancer/normal cell survival is not clearly established in the literature. Therefore, the purpose of this study was to evaluate six potential glycolytic inhibitors namely, sodium ascorbate, oxalic acid, oxaloacetic acid, sodium citrate, fructose diphosphate (FDP) and sodium bicarbonate at mM concentrations on growing A549 (lung cancer) and MRC-5 (normal; human lung fibroblast) cell lines with the objective of determining their influence on cell survival. Exposed and non-exposed cells were tested with phase-contrast micro-scanning, survival/death and metabolic activity trends through MTT-assays, as well as death end-point determinations by testing re-growth on complete media and T4 cellometer counts. Results showed that oxalic acid and oxaloacetic acid both influenced the pH of the medium and resulted in differential massive cell debris within the exposure period. Sodium ascorbate, sodium citrate, sodium bicarbonate and FDP did not cause pH changes; however, they caused detectable cell disfigurement and loss of metabolic activity and survival/ death end points with the resultant death of the A549 cell line. MRC-5 cells were differentially unaffected by exposure to sodium ascorbate, sodium citrate, sodium bicarbonate, FDP and oxaloacxetic acid, underwent complete recovery and remained both attached and healthy for 6 weeks upon subculture when transferred to a new complete medium. Oxalic acid did not show differential modulation with the consequent loss of survival and death of the MRC-5 cell line. Phase contrast findings as well as the cell counts confirmed the findings of other tests. These studies show the potential for exploiting cellular metabolic differences in cancer control. Citation Format: Ibrahim O. Farah, Veshell L. Lewis, Zikri Arslan. Impact of some common organics on cellular glycolysis and the differential survival of lung fibroblast and lung carcinoma cell lines. [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 1168. doi:10.1158/1538-7445.AM2015-1168

Trisenox induces cytotoxicity through phosphorylation of mitogen-activated protein kinase molecules in acute leukemia cells: KUMAR et al.

Trisenox (TX) has been used successfully for the treatment of acute promyelocytic leukemia (APL) patients. TX‐induced cytotoxicity in APL cells remains poorly understood. In this study, we investigated the molecular mechanism of TX cytotoxicity using APL cell lines. We assessed TX toxicity by quantitatively measuring lactate dehydrogenase levels. Inhibition of cell cycle progression was assessed by confocal microscopy of Ki‐67 expression. Apoptosis was evaluated by Western blot analysis of apoptotic proteins expression, immunocytochemistry, and confocal imaging of annexin V and propidium iodide. Mitogen‐activated protein kinase (MAPK) signaling cascade was analyzed by Western blot analysis and inhibitor‐based experiments with APL cells. We found that TX‐induced cytotoxicity inhibited APL cell cycle progression. TX also induced significant (P < 0.05) changes in the expression levels of apoptotic molecules and activated the phosphorylation of MAPK signaling pathways in APL cells. Understanding the mechanism of TX cytotoxicity would be helpful in the design of new APL drugs.

Abstract 3524: D-glucose exposure induced DNA damage and apoptosis in MCF-7 cells

D-glucose is the simple carbohydrate sugar that our bodies rely on to produce ATP energy. It has been shown that sustained high glucose burden is related to the promotion of many long-term health problems to various vital target organs including the kidneys, nervous system, eyes, heart (failure and stroke), erectile dysfunction in men and pregnancy complications in women. Epidemiological data have suggested an increased cancer rates in diabetic patients, for which the underlying mechanism is poorly understood. Furthermore, the D-glucose paradox as cancer-promoting energy source as well as being toxic to cancer cells is puzzling. Therefore in the present study, we will investigate the mechanisms of glucose-induced toxicity in MCF-7 cells as an in vitro cellular model to simulate diabetic complications in tumor cells using trypan blue exclusion and MTT assays. Mechanisms of DNA damage and apoptosis were tested by alkaline single cell gel electrophoresis (Comet) assay, and flow cytometry analysis using Annexin V FITC/PI and caspase-3 analysis. The MTT assay indicated that low dose (5 mg/mL) of D-glucose slightly increase cell viability upon 2 h of exposure. On the other hand, high doses (10-80 mg/mL) of D-glucose significantly reduced the viability of MCF-7 cells in a dose and time-dependent manner. Similar trends were seen with the trypan blue exclusion test. Data obtained from the comet assay indicated that D-glucose caused DNA damage in MCF-7 cells in a dose-dependent manner. The flow cytometry assessment (Annexin V FITC/PI) showed a strong dose-response relationship between high glucose exposure and Annexin V positive MCF-7 cells undergoing early stage apoptosis. Similarly, a statistically significant and concentration-dependent increase (p This research is supported by a grant from the National Institutes of Health-NIH (Grant No. NIMHD-G12MD007581). Citation Format: Ibrahim O. Farah, Christine K. Tchounwou, Clement G. Yedjou, Paul B. Tchounwou. D-glucose exposure induced DNA damage and apoptosis in MCF-7 cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3524.

The Effect of Glucocorticoids and LPS on the Functional Activity of RAW Cell Line

It is well documented that glucocorticoids are potent anti-inflammatory and immunosuppressive agents that are known to affect cell mediated inflammation by the inhibition of cellular proliferation and cytokine production. The literature is lacking knowledge in elucidating the mode of action of such agents on the transformed like cells in culture. Therefore, RAW (macrophage like) cells was selected as a model to determine the effects of cortisol administration or cortisol in the presence of LPS on the cells metabolic functions. Cells were treated with physiological concentrations of cortisol or cortisol + LPS for periods of 24, 48 and 72 hours. After each phase, cell numbers, cellular damage and cellular morphology were determined. The results indicated cortisol and cortisol + LPS treated cells inhibited cellular proliferation as well as increased cellular MDA levels as early as 24 hours. Overall, the results indicate that cortisol has a remarkable effect on RAW cellular proliferation similar to the reduction seen in our previous findings using HEP-2 cells. In addition to reduction in cellular number the cells ability to adjust to a bacterial challenge may be directly altered. These results provided important information for patients who are immunosuppressed or chronically exposed to stressful conditions.