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.

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.

Evaluation of R- (-) and S- (+) Clenbuterol enantiomers during a doping cycle or continuous ingestion of contaminated meat using chiral liquid chromatography by LC-TQ-MS.

Clenbuterol is known to improve competition resistance and muscular growth in athletes. Although it is an illegal drug, its use by farmers is widely spread to induce growth of their cattle. Thus, when clenbuterol is found in the urine of an athlete, there is doubt whether it was consumed with doping purposes or if it is due to the consumption of meat from a clenbuterol-fed animal. Previous studies suggest that enantiomeric relationship of clenbuterol may be different according to the intake source. However, the enantiomeric relationship throughout a doping cycle or a continuous intake of contaminated meat has not yet been explored. In this first approximation, our aim was the development and validation of a sensitive and rapid method for the determination of S- (+) and R- (─) clenbuterol enantiomers to be used in a controlled study in rats fed for one week with contaminated meat or simulating a doping cycle. Enantiomers were measured using liquid chromatography coupled to mass spectrometry with a triple quadrupole analyzer (LC-TQ-MS) and were separated on an AGP Chiralpak column. The method was fully validated following the VICH (Veterinary International Conference on Harmonization guidelines) and was linear in the range of 12.5-800 pg/mL with a correlation coefficient of ≥0.98 for each enantiomer, and with a limit of quantitation and detection (LOQ and LOD) of 12.5 pg/mL and 6.5 pg/mL, respectively, for both enantiomers. The application of this method pointed out the shift of the enantiomeric relationship in urine from rats during the first five days of the doping cycle compared to those fed with contaminated meat. This finding can be of substantial importance in further doping studies.