Cytotoxicity assessment of beta-lapachone in endothelial cells

The selective activity of an antineoplastic drug is related to its ability to promote cytotoxic action on tumor cells and preserve the integrity of non-neoplastic cells. Beta-lapachone is extracted from the sawdust of Ipe wood, a thick bark tree from the Ipe wood found in the Brazilian Cerrado biome. This study aimed to evaluate the cytotoxic action of beta-lapachone in an endothelial cell line. The EA.hy926 cells were seeded in two groups, G1 and G2, cultured and exposed to beta-lapachone at concentrations of 0.0, 0.01, 0.03, 0.1, 0.3, 1 and 3 μM for 24 hours. G1 remained under normal cultivation conditions and G2 was subjected to oxidative stress through an ischemia and reperfusion assay, in a deoxygenated sealed chamber. The cytotoxicity assay was performed using the tetrazolium reduction method. In G1, the cytotoxicity ranged from 0.0 to 10.0%; and in G2 between 0.0 and 6.3%. No statistically significant difference was observed between the obtained values. Moreover, we found no cytotoxic action of beta-lapachone on endothelial cells, and the results point out that the drug might have preserved the cell’s integrity against oxidative stress under the conditions of this experiment. This promising result suggests the possibility of beta-lapachone as a chemotherapy drug with selective activity.

Assessment of immunohematological, hematological and biochemical responses in cultivable fish Cyprinus carpio exposed to an antibiotic sulfamethoxazole (SMX)

Sulfamethoxazole (SMX) is a member of the sulfonamides group of antibiotics which is used extensively in aquaculture throughout the world. In this study, common carp (Cyprinus carpio) was used as the bioindicator to assess the toxicity potential of SMX. Effects were based on chronic toxicity of environmentally relevant dosages of 25, 50, 100, and 200 μg/L of SMX for 28 days. Cytotoxicity through hematology and biochemistry showed a dose–response relationship. Numerous variations were recorded in blood profile and biochemical parameters in SMX-exposed groups when compared to control. Hemoglobin, platelet, and erythrocyte levels were significantly decreased. Leukocyte level was significantly increased with values ranging from 131 to 303 (×103/μL). Changes in biochemical indices: glucose, total protein, and triglycerides showed biphasic trend, but alanine transaminase secretion was significantly increased from 25.13 to 204 U/L at higher concentration compared to control, suggesting liver damage. Spectrophotometric nitroblue tetrazolium reduction assay showed that respiratory burst activity increased as a function of SMX dose and exposure time (0.48–1.33 absorbance) ultimately leading to reduction in immunity. The present study highlights that prolonged exposure of SMX affects biochemistry, hematology, and immunohematology of fish and these biomarkers act as an effective tool for environmental risk assessment of drugs in the aquatic environment.

A quantitative test for heat-induced cell necrosis in vascular cambium and secondary phloem of Eucalyptus obliqua stems

Aims Exposure of Eucalyptus tree stems to the radiant heat of forest fires can kill cambial cells and their embedded regenerative meristems, thus preventing epicormic resprouting and recovery of the tree. Currently there is no tissue-level method to quantify the viability of cambial cells in Eucalyptus following heat exposure. The first aim of this study was to adapt and validate the tetrazolium reduction method of testing for cell viability in Eucalyptus. The second aim was to apply the method to establish a threshold level of cambium cell viability in Eucalyptus obliqua to enable the identification of a critical temperature. Methods The study used the tetrazolium reduction method to quantitatively determine phloem-cambium cell viability in Eucalyptus. Circular sections of bark with underlying phloem and cambium were cut from mature Eucalyptus obliqua and samples ranging in mass from 1 mg to 30 mg were exposed for 1 minute to temperature treatments ranging from 20°C to 85°C and kept for 20-22 hours at room temperature in 0.8% 2,3,5 triphenyl tetrazolium chloride (TTC) to test for cell viability. The 1,3,5 triphenyl tetrazolium formazan (TPF) formed was cold extracted with ethanol and quantified as absorbance at 485 nm. Important findings The TTC reduction method reliably quantified a decline in cell viability with rising temperature in tissue sections that included vascular cambium, and identified 60°C as the critical temperature for cambium-phloem cells of Eucalyptus species. Cell viability, calculated as [TPF Treatment°C] / [TPF 20°C], declined by 90% between 20°C and 85°C. The cell viability results confirmed that significant tissue necrosis occurred in Eucalyptus at temperatures between 50°C and 70°C, after one minute of in- vitro tissue heating. The decline in cell viability with increasing temperature shown by the TTC method was consistent with an independently derived count of live cells following temperature treatment and neutral red staining.

In vitro methodological standardization of MTT colorimetric assay for evaluation of formazan activity and drug dosage

Background Aiming to develop essential chemotherapeutic drugs to control various types of cancer, one of the primary tests is the tetrazolium reduction method. The present study aims to standardize the electromagnetic spectrum that best discriminates the absorbance and to evaluate the drug dosage of single and daily doses during the cell viability and toxicity analysis by the tetrazolium reduction method, using the ethanol extract of pequi peel in canine osteosarcoma cells. Results The wavelength of 532 nm showed the best results. We found that as the treatment time increased, the formazan conversion reduced. After 72 hours of treatment, we observed clear discrimination of dose-dependent data, with up to five discriminants within 72 hours with a change in absorbance from 0.554 to 0.064 A. The wavelength of 570 nm it was not ideal since we could not discern the difference in the spectral reflectance of the treatments and, therefore, show no statistical difference among treatments. We found no statistical difference for the coefficient of variation at wavelengths of 532 and 570nm, which were 12.77% and 8.80% respectively. Conclusions The wavelength of 532nm best discriminated the absorbance, as it presented better ability to congregate the treatment groups, greater variation between the discriminant and lower coefficient of variation, during the colorimetric test to evaluate the cellular metabolism. Moreover, the ethanol extract of pequi peel in canine osteosarcoma cells showed a statistically equal effect of a single dose administration to the dose reapplied every 24 hours.

The Effect of Administration of Silver Nanoparticles on the Immune Status of Chickens

The purpose of the study was to verify the hypothesis that there are doses of silver nanoparticles (Ag-NPs) that improve immune defence in chickens without compromising their health. To verify this hypothesis, an attempt was made to determine which doses of Ag-NPs (5 nm) consumed by chickens as a result of administration of hydrocolloids during varying time periods at a concentration of 5 or 10 mg Ag/l stimulate immune defence. The experiment was performed on 296 chickens assigned to 3 treatments. Chickens from the control treatment received drinking water without experimental additives. Chickens from the T-5 and T-10 treatments received a hydrocolloid of Ag-NPs at a concentration of 5 mg/l (treatment T-5) or 10 mg/l (treatment T-10) from their second week of life. Blood for analysis was collected at the age of 42 days from 8 birds per treatment. Ceruloplasmin (Cp), leukocyte count (WBC), erythrocyte sedimentation rate (ESR), interleukin IL-6, immunoglobulins IgA, IgY, phagocytic cells (% PC), phagocytic index (PI), nitroblue tetrazolium reduction (NBT), and lysozyme content in the blood was determined. Application of Ag-NPs at a concentration of 5 mg/l or 10 mg/l in the dose range of 2.87-12.25 mg/bird (administration of Ag-NPs in concentration 5 mg/l in weeks: 2; 2 and 3; 2 and 4; 2 and 5; 2 and 6 or concentration 10 mg/l in week 2) resulted in an immunostimulatory effect expressed as an increase in heterophil respiratory burst and an increased concentration of lysozyme. Higher doses of Ag-NPs exerted a pro-inflammatory effect, as indicated by elevated levels of IL-6 and ceruloplasmin, as well as a high ESR. They also stimulated B lymphocytes to produce IgA and IgA immunoglobulins.