Alencar Machado

Superoxide-hydrogen peroxide imbalance interferes with colorectal cancer cells viability, proliferation and oxaliplatin response

The role of superoxide dismutase manganese dependent enzyme (SOD2) in colorectal cancer is presently insufficiently understood. Some studies suggest that high SOD2 levels found in cancer tissues are associated with cancer progression. However, thus far, the role of colorectal cancer superoxide-hydrogen peroxide imbalance has not yet been studied. Thus, in order to address this gap in extant literature, we performed an in vitro analysis using HT-29 colorectal cell line exposed to paraquat, which generates high superoxide levels, and porphyrin, a SOD2 mimic molecule. The effect of these drugs on colorectal cancer cell response to oxaliplatin was evaluated. At 0.1 μM concentration, both drugs exhibited cytotoxic and antiproliferative effect on colorectal cancer cells. However, this effect was more pronounced in cells exposed to paraquat. Paraquat also augmented the oxaliplatin cytotoxic and antiproliferative effects by increasing the number of apoptosis events, thus causing the cell cycle arrest in the S and M/G2 phases. The treatments were also able to differentially modulate genes related to apoptosis, cell proliferation and antioxidant enzyme system. However, the effects were highly variable and the results obtained were inconclusive. Nonetheless, our findings support the hypothesis that imbalance caused by increased hydrogen peroxide levels could be beneficial to cancer cell biology. Therefore, the use of therapeutic strategies to decrease hydrogen peroxide levels mainly during oxaliplatin chemotherapy could be clinically important to the outcomes of colorectal cancer treatment.

The in vitro genotoxic effect of Tucuma (Astrocaryum aculeatum), an Amazonian fruit rich in carotenoids.

Tucuma (Astrocaryum aculeatum) is an Amazonian fruit that presents high levels of carotenoids and other bioactive compounds such as quercetin. The extracts of tucuma peel and pulp present strong antioxidant activity which illustrate an elevated concentration that causes cytotoxic effects in human peripheral blood mononuclear cells (PBMCs). This study performed additional investigations to analyze the potential genotoxic effects of the tucuma extracts on PBMCs. The genotoxicity was evaluated by DNA fragmentation, Comet assay, and chromosomal instability G-band assays. The acute tucuma extract treatment showed genoprotective effects against DNA denaturation when compared with untreated PBMC cells. However, in the experiments with 24 and 72 h treatments to tucuma treatments, we observed low genotoxicity through a concentration of 100 μg/mL, some genotoxic effects related to intermediary concentrations (100-500 μg/mL), and more pronounced genotoxic effects on higher tucuma extract concentrations. After 24 h of treatment, the reactive oxygen species were similar among treatments and PBMC control groups. However, the caspase-1 activity related to the apoptosis and pyroptosis process increased significantly in higher tucuma concentrations. In summary, tucuma extracts, despite their higher antioxidant content and antioxidant activity, would present PBMCs genotoxic effects that are dependent on concentration and time exposition. These results need to be considered in future in vitro and in vivo studies of tucuma effects.

Haloperidol and Risperidone at high concentrations activate an in vitro inflammatory response of RAW 264.7 macrophage cells by induction of apoptosis and modification of cytokine levels

Antipsychotic drugs, such as haloperidol and risperidone, are used in long-term treatment of psychiatric patients and thus increase the risk of obesity and other metabolic dysfunctions. Available evidence suggests that these drugs have pro-inflammatory effect, which contributes to the establishment of endocrine disturbances. However, results yielded by extant studies are inconsistent. Therefore, in this work, we tested the in vitro effects of different high concentrations of haloperidol and risperidone on the activation of isolated macrophages (RAW 264.7 cell line). The results indicated that macrophages were activated by both drugs. In addition, the activation involved an increase in nitric oxide levels and apoptosis events by modulation of caspases 8 and 3 levels and a decrease of the Bcl-2/BAX gene expression ratio. Cells treated with haloperidol and risperidone also presented higher concentrations of inflammatory cytokines (IL-1β, IL-6, TNFα) and low levels of IL-6 anti-inflammatory cytokine in a dose-dependent manner. Despite the limitation of cell line studies based solely on macrophages cells, we suggest that antipsychotic drugs could potentially exacerbate inflammatory processes in peripheral tissues (blood and fat). The continued activation of macrophages could contribute to the development of obesity and other endocrine disturbances caused by the use of antipsychotic drugs.

Cytoprotective and genoprotective effects of β-glucans against aflatoxin B1-induced DNA damage in broiler chicken lymphocytes

The polysaccharide β-glucan presents beneficial effects on the immune system, although the mechanisms of the immunomodulatory effect remain poorly understood. The potential cytoprotective and genoprotective effects of β-glucans were evaluated in broiler chicken lymphocytes exposed to increasing concentrations of aflatoxin B₁ (AFB₁) and/or β-glucans. AFB₁ significantly decreased cell viability at the concentrations of 10 and 20 μg/ml at 72 h of incubation (p<0.01 and p<0.001, respectively). Moreover, the AFB₁ concentrations of 1, 10 and 20 μg/ml increased DNA fragmentation levels at 24 h (p<0.001). Conversely, lymphocyte death was prevented by β-glucans at the concentrations of 1% and 10%, indicating a cytoprotective effect. Reactive oxygen species levels were increased in the cells treated with 20 μg/ml AFB₁ at 24 h (p<0.05) and 10% β-glucans with or without AFB₁ at 24, 48 and 72 h of incubation (p<0.001). DNA damage increased by more than 100% in AFB₁-treated lymphocytes when compared to control group. β-glucans at 1% was able to fully revert the AFB₁-induced lymphocyte DNA damage, indicating a genoprotective effect and maintaining DNA integrity. In conclusion, β-glucans showed in vitro dose-dependent cytoprotective and genoprotective effects in broiler chicken lymphocytes exposed to AFB₁.

Neuroprotective Effects of Açaí (Euterpe oleracea Mart.) against Rotenone In Vitro Exposure

Neuropsychiatric diseases, such as bipolar disorder (BD) and schizophrenia (SCZ), have a very complex pathophysiology. Several current studies describe an association between psychiatric illness and mitochondrial dysfunction and consequent cellular modifications, including lipid, protein, and DNA damage, caused by cellular oxidative stress. Euterpe oleracea (açaí) is a powerful antioxidant fruit. Açaí is an Amazonian palm fruit primarily found in the lowlands of the Amazonian rainforest, particularly in the floodplains of the Amazon River. Given this proposed association, this study analyzed the potential in vitro neuropharmacological effect of Euterpe oleracea (açaí) extract in the modulation of mitochondrial function and oxidative metabolism. SH-SY5Y cells were treated with rotenone to induce mitochondrial complex I dysfunction and before and after we exposed the cells to açaí extract at 5 μg/mL. Treated and untreated cells were then analyzed by spectrophotometric, fluorescent, immunological, and molecular assays. The results showed that açaí extract can potentially increase protein amount and enzyme activity of mitochondrial complex I, mainly through NDUFS7 and NDUFS8 overexpression. Açaí extract was also able to decrease cell reactive oxygen species levels and lipid peroxidation. We thus suggest açaí as a potential candidate for drug development and a possible alternative BD therapy.

Genomodifier capacity assay: a non-cell test using dsDNA molecules to evaluate the genotoxic/genoprotective properties of chemical compounds

We describe here an ultrasensitive and fast protocol called a GEMO assay (genomodifier capacity assay). This non-cell method was developed to identify chemicals with genomodifier (genotoxic and/or genoprotective) capacity. The assay is performed in a black 96-well plate using calf thymus dsDNA exposed to different concentrations of chemicals tested (CT) for 30 minutes with and without the addition of a prooxidant substance that causes dsDNA damage (H2O2, 3 M). Furthermore, PicoGreen®, a highly sensitive dsDNA dye is added and so the fluorescence is emitted according to the concentration of intact dsDNA. Chemicals that cause a break in dsDNA are identified by a decrease in fluorescence in comparison with the fluorescence observed in an untreated dsDNA (control group) indicating genotoxic capacity. In contrast, attenuation of dsDNA degradation caused by H2O2 exposition indicates CT genoprotective capacity. The GEMO assay was validated by comparing peripheral blood mononuclear cells (PBMCs) and an HT29 colorectal cell line exposed to similar conditions where the effect on dsDNA was also evaluated by a DNA alkaline comet assay. Vitamin C was used as CT and other variables were also evaluated to confirm the cytotoxic action of H2O2. The results showed a strong negative correlation between the GEMO assay and the comet assay performed in PBMCs (r2 = −0.828; p < 0.0001) since higher dsDNA fluorescence measured by the GEMO assay was associated with lower index damage measured by the DNA alkaline comet assay. Therefore, the GEMO assay could be useful for early screening of genoprotective and genotoxic effects of chemicals and plant extracts without interfering cell biological variables.

Hypercholesterolemia and Ecto-enzymes of Purinergic System: Effects of Paullinia cupana.

Hypercholesterolemia is a metabolic disorder characterized by high levels of low‐density lipoprotein and blood cholesterol, causing inflammatory lesion. Purinergic signaling modulates the inflammatory and immune responses through adenine nucleotides and nucleoside. Guaraná has hypocholesterolemic and antiinflammatory properties. Considering that there are few studies demonstrating the effects of guaraná powder on the metabolism of adenine nucleotides, we investigated its effects on the activity of ecto‐nucleoside triphosphate diphosphohydrolase (E‐NTPDase) and ecto‐adenosine deaminase activity in lymphocytes of rats with diet‐induced hypercholesterolemia. The rats were divided into hypercholesterolemic and normal diet groups. Each group was subdivided by treatment: saline, guaraná powder 12.5, 25, or 50 mg/kg/day and caffeine concentration equivalent to highest dose of guaraná, fed orally for 30 days. An increase in adenosine triphosphate hydrolysis was observed in the lymphocytes of rats with hypercholesterolemia and treated with 25 or 50 mg/kg/day when compared with the other groups. The hypercholesterolemic group treated with the highest concentration of guaraná powder showed decreased ecto‐adenosine deaminase activity compared with the normal diet groups. Guaraná was able to reduce the total cholesterol and low‐density lipoprotein cholesterol to basal levels in hypercholesterolemic rats. High concentrations of guaraná associated with a hypercholesterolemic diet are likely to have contributed to the reduction of the inflammatory process. Copyright

Guaraná a Caffeine-Rich Food Increases Oxaliplatin Sensitivity of Colorectal HT-29 Cells by Apoptosis Pathway Modulation

We investigated the in vitro effects of guaraná and its main metabolites (caffeine, theobromine and catechin) on cytotoxicity and cell proliferation on colorectal cancer (CRC) line HT-29 cells and on oxaliplatin sensitivity. The cells were exposed to different concentrations of guaraná extract with and without oxaliplatin. The concentrations of bioactive molecules were also estimated considering their potential proportion on guaraná hydro-alcoholic extract. Apoptosis effect was analyzed by annexin V quantification using flow cytometry, while apoptosis pathway gene modulation (p53, Bax/Bcl-2 genes ratio, caspases 8 and 3) was determined by qRT-PCR analysis. Cells exposed to guaraná at a concentration of 100 μg/mL presented a similar cytotoxic effect as HT-29 cells treated with oxaliplatin and did not affect the sensitivity of the drug. Guaraná presented cell anti-proliferative effect and increased anti-proliferative oxaliplatin sensitivity at all concentrations tested here. Guaraná was able to induce apoptosis and up-regulate the p53 and Bax/Bcl-2 genes.

Reactive, proactive, and extensible situation-awareness in ambient assisted living

The identification and prediction of situations in smart houses.An approach developed with the principles of reactive and proactive behavior.A case study presented with sensors and appliances.The proactivity and reasoning about uncertainty supported by probabilistic ontology. Considering that the world population is aging, health-support issues are in evidence, and many dangerous situations concerning users in their living environment may arise. This paper presents an approach to allow Ambient Assisted Living (AAL) systems to identify and predict situations that may endanger users in their living environment. Elderly people generally have difficulties to perform daily activities as using some electronic device, cooking, dressing among others. This paper offers an alternative to acquire comprehension of a former persons behavioral providing this knowledge when cognitive impairments will occur. This is a significant improvement to traditional expert systems applied to AAL as the handling of the knowledge base is adapted to the failing users behavior. We consider the user profile dynamics where new and different situations not initially planned in the development of the systems may occur. This approach was developed considering the principles of reactive and proactive behavior and the extensibility capacity of the system. Systems developed following the above-described principles can react to the current situations and anticipate proactively a dangerous situation eliminating it or controlling its impact. The approach is evaluated and verified by a case study where unwanted, dangerous situations were created and subsequently detected. The case study is based on a simulated smart house fulfilled with sensors and appliances. An application was build based on the developed approach, and it was capable of choosing the most appropriate actions to solve the unwanted situations. The main contributions of the present work are the identification and prediction of situations in smart houses, an approach developed with the principles of reactive and proactive behavior validated with a case study and supported by a probabilistic ontology.

Proactive Domain Data Querying based on Context Information in Ambient Assisted Living Environments

Ubiquitous computing defines a set of technologies to make computing omnipresent in real life environments. In the area of ambient assisted living, ubiquitous technologies have been used to improve the life quality and expectancy for elderly people. Recently, researches have shown that the use of context-awareness combined with proactive actions can cause systems to act more appropriately in assistance to the user. In this paper, we present a new persistent and proactive data retrieval model for ambient assisted living systems. This model provides an architecture that is able to integrate information that is gathered from the user environment and considers the current user context to act in a proactive manner. The model was implemented on a service inte-grated in a Situation as a Service middleware and it was applied in a case study for evaluation and validation.