CELL-BASED BIOASSAYS FOR TESTING BIOACTIVE COMPOUNDS IN FARM ANIMALS

Cell-based assays can be adopted as in vitro method to evaluate the bioavailability and functionality of different nutraceutical and bioactive compounds, particularly in view of the need to use alternatives to animal studies. The interest in these bioactive compounds in animal sciences is not only related to medical research. It also represents an enormous benefit for health food companies and the animal produce sector in general. The general aim of my PhD study was to study nutraceutical effects at a cellular level in response to different stress challenges. In the first section of my thesis, the protective role of α-tocopherol in counteracting the cytotoxicity and DNA damage induced by Ochratoxin A (OTA) in primary porcine fibroblast cell cultures (ear and embryo), was determined by using the MTT assay, LDH release, DNA fragmentation, and TUNEL stain. The aim of the second section was to evaluate the protective role of bovine Lactoferrin (bLf), added to the culture medium, against lipopolysaccharide (LPS) cytotoxicity using the established bovine mammary epithelial cell line BME-UV1 as an in vitro model of the bovine mammary epithelium. In addition, we assessed whether BME-UV1 cells were able to express endogenous bLf after in vitro exposure to LPS. A further objective of my thesis work was to use cell-based bioassays to investigate the plasmin-plasminogen system. This system plays a key role in cellular responses, and is involved both in physiological and in pathological conditions in the mammary gland. The aim of the third section was to determine the effect of growth factors (IGF-1 and EGF) and three hormones (insulin, dexamethasone, and prolactin) on the expression of plasminogen activator (PA)-related genes (u-PA, u-PAR, PAI-1, PAI-2) and BME-UV1 cell proliferation. In addition we investigated the effects of E. coli LPS on cell viability, the modulation of cell-associated u-PA activity and the regulation of u-PA and u-PAR RNA expression in BME-UV1 cells. Below are more details on what each section covers: The first section reports how the role of α-tocopherol in counteracting OTA toxicity was evaluated in various experimental conditions using primary porcine fibroblasts. Cells showed a dose-, time- and origin-dependent (ear vs. embryo) sensitivity to ochratoxin A. Pre-incubation for 3 h with 1 nM α-tocopherol significantly (P < 0.01) reduced OTA cytotoxicity, lactate dehydrogenase release and DNA damage in both fibroblast cultures. These findings indicate that α-tocopherol administration may counteract short-term OTA toxicity, thus supporting its defensive role at a cell membrane level. The second section describes how BME-UV1 was used as an in vitro model to evaluate the protective role of exogenous bovine Lf (bLf) against the cytotoxic damage induced by bacterial lipopolysaccharides (LPS). Exogenous bLf showed a protective effect against endotoxin cytotoxicity, which could be mediated by the LPS-neutralizing capability of bLf. In addition, in BME-UV1 cells the response to LPS exposure did not involve endogenous bLf mRNA expression, suggesting that this cell line lacks functional LPS-responsive elements. The third section details how cell proliferation was measured using the MTT assay and direct cell enumeration on BME-UV1 treated with physiological stimuli. Results showed that both IGF-1 and EGF increased cell proliferation. Neither of the growth factors had any effect on the expression of PAI-1 and PAI-2. In line with changes in gene expression, EGF and IGF-1 up regulated total cell-associated, membrane-bound and secreted u-PA activity. Dexamethasone alone and when combined with insulin or prolactin up regulated the gene expression of both PAI- and PAI-2, but not that of u-PA and u-PAR without affecting cell proliferation. Total decreased cell-associated, membrane-bound and secreted u-PA activity was detected in cells cultured in the presence of dexamethasone when combined with insulin or prolactin. However no such effect was observed in the presence of dexamethasone alone. This thus suggests that when dexamethasone acts synergistically with prolactin or insulin it inhibits the activation of the plasmin-plasminogen system, but this inhibition is not correlated with any changes in cell proliferation. In addition, the plasmin-plasminogen system was examined, using the BME-UV1 cell model, in order to evaluate the effects of Escherichia coli LPS on cell viability, the modulation of cell-associated u-PA activity, and the regulation of u-PA and u-PA receptor (u-PAR) RNA expression. LPS did not affect cell viability, but led to an increase in u-PA activity, with the maximum response after 6 h of incubation. In addition, u-PA and u-PAR mRNA expression were both up-regulated in BME-UV1 cells after 3 h of incubation with LPS. These data indicated that E. coli LPS increased u-PA activity and RNA expression of u-PA and u-PAR in BME-UV1 cells, thus strengthening the role of the PA system during pathological processes. In conclusion, the application of appropriate in vitro models represents a fundamental requirement for the study of cellular responses to different stimuli as in the case covered in my thesis regarding nutraceutical compounds. Cell-based assays are a valuable tool for assessing fundamental regulatory mechanisms at a cellular level, such as the plasmin-plasminogen system. Cell-based assays allow both the functionality of nutraceutical and cellular response mechanisms to be evaluated reducing use animal models in the preliminary study phase. Obviously, data obtained in cell culture models must be interpreted carefully, since this system represents a simplification of the intricacies of the numerous reactions and interactions that occur in vivo.