Biochemical parameters, oxidative stress biomarkers, and anatomopathological changes in Wistar rats treated with 3'-demethoxy-6-O-demethylisoguaiacin and norisoguaiacin.

Artemisia cina (Ac) is a plant with anthelmintic compounds such as 3'-demethoxy-6-O-demethylisoguaiacin (D) and norisoguaiacin (N). Three major objectives were proposed: (1) To evaluate biochemical parameters in blood (2) to determine the tissue oxidative stress by biomarkers as TBARS and glutathione peroxidase activity, and (3) to evaluate anatomopathological changes in organs such as the brain, liver, kidney, and lung after oral administration of n-hexane extract of Ac and D and N. D and N were administrated following the OECD guides for acute oral toxicity evaluation (Guide 420). Fifty Wistar rats were distributed into ten groups as follows: Group 1 (G1): 4 mg/Kg; G2: 40 mg/Kg; G3: 240 mg/Kg; G4: 1600 mg/Kg of n-hexane extract of Ac. G5: 2 mg/Kg; G6: 20 mg/Kg; G7: 120 mg/Kg; G8: 800 mg/Kg of D and N, G9: water and G10: polyvinylpyrrolidone at 2000 mg/Kg. At 14 days, the rats were euthanized, and the blood, liver, brain, kidney, and lung were taken for biochemical analysis, anatomopathological changes, and TBARS and GSH evaluation. Glucose, cholesterol, and phosphorus were altered. Histopathological analysis showed multifocal neuronal degeneration in the brain (G2). The kidney and lungs had changes in G7. The GSH and TBARS increased in G6 and G7. The TBARS activity was higher in G1 and G2. In conclusion, extract and D and N of Ac did not have damage at therapeutic doses. D, N, and n-hexane extract of A. cina do not cause histopathological damage at pharmaceutical doses. Still, the brain, kidney, and liver are related to biochemical parameters at higher doses. However, compounds are proposed as antioxidant agents.

Study of the Early Effects of Chitosan Nanoparticles with Glutathione in Rats with Osteoarthrosis.

Due to cartilage's limited capacity for regeneration, numerous studies have been conducted to find new drugs that modify osteoarthrosis's progression. Some evidence showed the capability of chitosan nanoparticles with glutathione (Np-GSH) to regulate the oxide-redox status in vitro in human chondrocytes. This work aimed to evaluate the capacity of Np-GSH in vivo, using Wistar rats with induced surgical osteoarthritis. Radiographic, biochemical (GSH and TBARS quantification), histopathological, and immunohistochemical (Col-2 and MMP-13) analyses were performed to evaluate the progress of the osteoarthritic lesions after the administration of a single dose of Np-GSH. According to the results obtained, the GSH contained in the NPs could be vectored to chondrocytes and used by the cell to modulate the oxidative state reduction, decreasing the production of ROS and free radicals induced by agents oxidizing xenobiotics, increasing GSH levels, as well as the activity of GPx, and decreasing lipid peroxidation. These results are significant since the synthesis of GSH develops exclusively in the cell cytoplasm, and its quantity under an oxidation-reduction imbalance may be defective. Therefore, the results allow us to consider these nanostructures as a helpful study tool to reduce the damage associated with oxidative stress in various diseases such as osteoarthritis.

Excessive Consumption Hibiscus sabdariffa L. Increases Inflammation and Blood Pressure in Male Wistar Rats via High Antioxidant Capacity: The Preliminary Findings.

Hibiscus sabdariffa L. (HSL) has high amounts of antioxidants and many beneficial effects in several pathologies. However, few studies describe the possible harmful effects of high concentrations of HSL. Here we evaluate the effect of excessive and chronic consumption of infusions with different percentages of HSL on some oxidative stress markers in serum, and the possible association with inflammation and increased systolic blood pressure (SBP), in healthy rats. A total of 32 male Wistar rats were used to form 4 groups with 8 animals each. Group 1 control (drinking tap water), group 2, 3 and 4, drinking water supplemented with 15, 30 and 60 g/L of HSL calyxes respectively. SBP was evaluated and determinations in serum of the NO3-/NO2- ratio, glutathione (GSH), total antioxidant capacity (TAC), selenium (Se), TNF-α, IL-1α/IL-1F1, IL-1ß, IL-10, extracellular superoxide dismutase (EcSOD), thioredoxin reductase (TrxR) and glutathione peroxidase (GPx) activities, were evaluated. The SBP (p = 0.01), GPx activity, GSH, TAC, Se, TNF-α and EcSOD activities (p ≤ 0.001) and IL-1α/IL-1F1, IL-1ß, TrxR and NO3-/NO2- (p ≤ 0.05), were increased but IL-10 (p < 0.001) was decreased in rats that consumed the 3 and 6% HSL infusions. The excessive and chronic consumption of HSL may increase the TAC that could lead to a proinflammatory state which is associated with hypertension.

Precision-cut liver slices as a model for assess hepatic cellular response of chitosan-glutathione nanoparticles on cultures treated with zilpaterol and clenbuterol.

Zilpaterol and clenbuterol are two ß-adrenergic agonist drugs used in animal production. Both drugs have anabolic effects with advantages on carcass yield. Meanwhile, zilpaterol is approved for animal feed in authorized countries. Clenbuterol is a banned substance due to the risk of toxicity; however, it is still being used in unknown dose levels in many farm species. Therefore, the use and abuse of these substances should be closely monitored, considering the clenbuterol ability and the not proved yet of zilpaterol to produce reactive oxygen and nitrogen species. Regarding glutathione which is the main intracellular antioxidant plays detoxification functions on liver metabolism; in this work, it is our interest to know the capacity of chitosan-glutathione nanoparticles (CS/GSH-NP) as a complementary source of exogenous GSH to modify the oxide-reduction status on bovine precision-cut liver slice cultures (PCLS) exposed to clenbuterol and zilpaterol. A single drug assay was performed in first instance by adding clenbuterol, zilpaterol, chitosan nanoparticles (CS-NP), and CS/GSH-NP. Then combinate drug assay was carried out by testing clenbuterol and zilpaterol combined with CS-NP or CS/GSH-NP. The results showed that both ß-adrenergic agonists modify in a dose-dependent manner in oxide-reduction response through ROS generation. The activity or content of glutathione peroxidase activity, intracellular GSH, gamma glutamyl-transpeptidase, aspartate aminotrasnferase and alanine aminotrasnferase were modified. The exogenous GSH delivered by nanoparticles could be used to modulate these markers.

Modification of Proliferation and Apoptosis in Breast Cancer Cells by Exposure of Antioxidant Nanoparticles Due to Modulation of the Cellular Redox State Induced by Doxorubicin Exposure.

In this report, we investigated whether the use of chitosan-carrying-glutathione nanoparticles (CH-GSH NPs) can modify proliferation and apoptosis, and reduce cell damage induced by doxorubicin on breast cancer cells. Doxorubicin is a widely used antineoplasic agent for the treatment of various types of cancer. However, it is also a highly toxic drug because it induces oxidative stress. Thus, the use of antioxidant molecules has been considered to reduce the toxicity of doxorubicin. CH-GSH NPs were characterized in size, zeta potential, concentration, and shape. When breast cancer cells were treated with CH-GSH nanoparticles, they were localized in the cellular cytoplasm. Combined doxorubicin exposure with nanoparticles increased intracellular GSH levels. At the same time, decreasing levels of reactive oxygen species and malondialdehyde were observed and modified antioxidant enzyme activity. Levels of the Ki67 protein were evaluated as a marker of cell proliferation and the activity of the Casp-3 protein related to cell apoptosis was measured. Our data suggests that CH-GSH NPs can modify cell proliferation by decreasing Ki67 levels, induce apoptosis by increasing caspase-3 activity, and reduce the oxidative stress induced by doxorubicin in breast cancer cells by modulating molecules associated with the cellular redox state. CH-GSH NPs could be used to reduce the toxic effects of this antineoplastic. Considering these results, CH-GSH NPs represent a novel delivery system offering new opportunities in pharmacy, material science, and biomedicine.

Extracellular and intracellular zilpaterol and clenbuterol quantification in Hep G2 liver cells by UPLC-PDA and UPLC-MS/MS.

Zilpaterol and Clenbuterol are ß-adrenergic agonists that have been widely used to feed cattle. Although the use of Zilpaterol has been approved, Clenbuterol is still used illegally at unknown doses. However, the research of both substances has been based mainly on the evaluation of residues. To our knowledge, this is the first time that a cellular model using Hep G2 cells treated with Zilpaterol and Clenbuterol is presented as an alternative approach to quantify both drugs at the cellular level. Thus, a complete analytical methodology has been developed for the accurate quantitation of these ß-adrenergic agonists in both cellular compartments. We propose the use of ultra-performance liquid chromatography with photodiode array detector (UPLC-PDA) for extracellular determinations while UPLC coupled to a tandem mass spectrometer (UPLC-MS/MS) for intracellular analysis. The methods were fully validated in terms of selectivity, linearity, accuracy, and precision, limits of detection and quantitation (LOD and LOQ, respectively), stability, carryover, and matrix effect. The method for intracellular content was linear ranging from 0.25 to 8 ng/mL while for extracellular content, the concentration of Zilpaterol and Clenbuterol ranged from 0.125 to 4 µg/mL, with correlation coefficients of R > 0.98 and >0.99, respectively. The combination of the two methodologies in the cellular model showed intracellular concentrations of 0.344 ± 0.06 µg/mL and 2.483 ± 0.36 µg/mL for Zilpaterol and Clenbuterol, respectively. Extracellular concentration was 0.728 ± 0.14 µg/mL and 0.822 ± 0.11 µg/mL for Zilpaterol and Clenbuterol, respectively. This work shows the potential applications of cellular modelling in the study of toxicity for the mentioned drugs.

Combinatorial Use of Chitosan Nanoparticles, Reversine, and Ionising Radiation on Breast Cancer Cells Associated with Mitosis Deregulation.

Breast cancer is the most commonly occurring cancer in women worldwide and the second most common cancer overall. The development of new therapies to treat this devastating malignancy is needed urgently. Nanoparticles are one class of nanomaterial with multiple applications in medicine, ranging from their use as drug delivery systems and the promotion of changes in cell morphology to the control of gene transcription. Nanoparticles made of the natural polymer chitosan are easy to produce, have a very low immunogenic profile, and diffuse easily into cells. One hallmark feature of cancer, including breast tumours, is the genome instability caused by defects in the spindle-assembly checkpoint (SAC), the molecular signalling mechanism that ensures the timely and high-fidelity transmission of the genetic material to an offspring. In recent years, the use of nanoparticles to treat cancer cells has gained momentum. This is in part because nanoparticles made of different materials can sensitise cancer cells to chemotherapy and radiotherapy. These advances prompted us to study the potential sensitising effect of chitosan-based nanoparticles on breast cancer cells treated with reversine, which is a small molecule inhibitor of Mps1 and Aurora B that induces premature exit from mitosis, aneuploidy, and cell death, before and after exposure of the cancer cells to X-ray irradiation. Our measurements of metabolic activity as an indicator of cell viability, DNA damage by alkaline comet assay, and immunofluorescence using anti-P-H3 as a mitotic biomarker indicate that chitosan nanoparticles elicit cellular responses that affect mitosis and cell viability and can sensitise breast cancer cells to X-ray radiation (2Gy). We also show that such a sensitisation effect is not caused by direct damage to the DNA by the nanoparticles. Taken together, our data indicates that chitosan nanoparticles have potential application for the treatment of breast cancer as adjunct to radiotherapy.

Detoxification of Aflatoxin-Contaminated Maize by Neutral Electrolyzed Oxidizing Water.

Aflatoxins, a group of extremely toxic mycotoxins produced by Aspergillus flavus, A. parasiticus and A. nomius, can occur as natural contaminants of certain agricultural commodities, particularly maize. These toxins have been shown to be hepatotoxic, carcinogenic, mutagenic and cause severe human and animal diseases. The effectiveness of neutral electrolyzed oxidizing water (NEW) on aflatoxin detoxification was investigated in HepG2 cells using several validation methodologies such as the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay, the induction of lipid peroxidation, the oxidative damage by means of glutathione modulation, the Ames test and the alkaline Comet assay. Our results showed that, after the aflatoxin-contaminated maize containing 360 ng/g was soaked in NEW (60 mg/L available chlorine, pH 7.01) during 15 min at room temperature, the aflatoxin content did not decrease as confirmed by the immunoaffinity column and ultra performance liquid chromatography methods. Aflatoxin fluorescence strength of detoxified samples was similar to untreated samples. However, aflatoxin-associated cytotoxicity and OPEN ACCESS Toxins 2015, 7 4295 genotoxicity effects were markedly reduced upon treatment. According to these results, NEW can be effectively used to detoxify aflatoxin-contaminated maize.

Cytotoxic and genotoxic evaluation of tortillas produced by microwave heating during alkaline-cooking of aflatoxin-contaminated maize.

UNLABELLED: In vitro cytotoxicity and genotoxicity induction by aflatoxin B1 (AFB1) from maize (ME) and tortillas (TE) produced by microwave nixtamalization were investigated in monkey kidney (Vero cells) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the induction of lipid peroxidation, the oxidative damage by means of glutathione (GSH) depletion, and the Salmonella-microsomal screening system (Ames test). Our results showed that, at higher concentrations, both ME and TE extracts that contained varying amounts of aflatoxin caused a considerable decrease in Vero cell viability (up to 37%) after 4 h of exposure. Aflatoxins from ME induced greater oxidative damage by enhancing lipid peroxidation (up to 6.05 ± 0.14 µmol/mg protein) as compared to TE; however, TE also induced significant malondialdehyde formation in particular at the higher aflatoxin concentration tested (up to 2.7 ± 0.19 µmol/mg protein). The decrease in GSH level was also more pronounced in ME as compared to TE. Moreover, the Ames test results indicated that the mutagenic activity of TE was greatly reduced compared with that of ME based on his(-) → his(+) reversions in the Salmonella TA100 strain. According to these results, it is concluded that the microwave nixtamalization procedure reduced aflatoxins and their in vitro toxicity and mutagenic activity. PRACTICAL APPLICATION: In Mexico, aflatoxins are often found in maize destined for the tortilla industry; consequently, tortilla consumption invariably leads to an important intake of intact and/or modified aflatoxin molecules caused by the thermal-alkaline treatment used during production. Therefore, it is of the highest importance to check whether such intake has the potential to lead to higher risk for adverse human health effects. In view of these considerations, in vitro tests may thus be useful for predicting the potential cytotoxicity and genotoxicity of tortillas produced for human consumption using aflatoxin-contaminated maize.