Ivana Kmetič

Aujeszky's disease virus production in disposable bioreactor

A novel, disposable-bag bioreactor system that uses wave action for mixing and transferring oxygen was evaluated for BHK 21 C13 cell line growth and Aujeszky’s disease virus (ADV) production. Growth kinetics of BHK 21 C13 cells in the wave bioreactor during 3-day period were determined. At the end of the 3-day culture period and cell density of 1.82 × 106 cells ml-1, the reactor was inoculated with 9 ml of gE- Bartha K-61 strain ADV suspension (105.9 TCID50) with multiplicity of infection (MOI) of 0.01. After a 144 h incubation period, 400 ml of ADV harvest was obtained with titre of 107.0 TCID50 ml−1, which corresponds to 40,000 doses of vaccine against AD. In conclusion, the results obtained with the wave bioreactor using BHK 21 C13 cells showed that this system can be considered as suitable for ADV or BHK 21 C13 cell biomass production.

Lindane-induced cytotoxicity and the role of vitamin E in Chinese hamster ovary (CHO-K1) cells

Lindane, a toxic insecticide from the persistent organic pollutants (POP’s) group, may act as an endocrine disrupter affecting crucial tissues of reproductive system. In this study a Chinese Hamster Ovary cell line (CHO-K1) was applied to assess the potential of lindane cytotoxicity at the cellular level. The methods of Trypan blue exclusion, MTT and Kenacid blue assays were used to assess cytotoxicity and confirmed a decrease in the number of viable CHO-K1 cells at 34.4–344 μM lindane during 24, 48 and 72 hours of exposure. The cell proliferation tests showed significant inhibition (p < 0.025–0.001 vs control) and a progressive increase in toxicity with increasing lindane concentrations. Corresponding IC50 values were determined with each applied method. After 72 h of lindane exposure, IC50 values were 184 μM according to the Trypan blue method and 272 and 256 μM with the Kenacid blue and MTT assays, respectively. Morphological changes induced by the cytotoxicity of lindane were followed by the fluorescence microscopy and only necrotic cells were detected. Vitamin E (25 and 50 μg/mL) was used for protection of ovarian cells against lidane-induced oxidative stress damage, and lipid peroxidation was postulated as a possible mechanism of lindane toxicity. The viability of cells pre-incubated with vitamin E was significantly enhanced (up to p < 0.025) compared to the results observed in cells exposed to lindane only, but vitamin E treatment could not prevent complete lindane-induced cytotoxicity. Results suggest that vitamin E may exert a slightly protective role in cell defense against lipophilic pro-oxidant xenobiotics such as lindane.

Natural incidence of zearalenone in Croatian pig feed, urine and meat in 2014

The aim of this study was to determine the levels of zearalenone (ZEN) in different feed materials and feedstuffs for pigs, as well as in pig urine and pig meat following contaminated feed consumption. In total, 253 feed material and feedstuff samples were collected from Croatian pig farms. The results revealed the presence of ZEN in significant concentrations, the maximal being found in maize (5522 µg/kg), wheat (3366 µg/kg) and pig fattening feed (1949 µg/kg). In farms in which high feed contamination and pig hyperestrogenism were observed, samples of pig urine (n = 30) and meat (n = 30) were retrieved as well. The mean ZEN concentrations in pig urine and pig meat were 206 ± 20.6 µg/L and 0.62 ± 0.14 µg/kg, respectively. Despite high contamination of feedstuffs responsible for farmed pigs’ intoxication, ZEN levels determined in pig meat were shown to be of little significance for human safety.

PCB 77 action in ovary cells--toxic effects, apoptosis induction and cell cycle analysis.

Abstract Context: PCB 77 (3,3′,4,4′-tetrachlorobiphenyl), a non-ortho congener with planar configuration, has been identified as potential endocrine disrupter capable to increase the risk of reproductive and developmental failure. Objective: In the present study, in vitro PCB 77 toxic potential, apoptosis induction and cell cycle alterations were investigated to reveal direct toxic effects on ovarian cells. Methods: Chinese Hamster Ovary (CHO-K1) cell line was selected as a model system and decreased cell viability was confirmed by application of four bioassays. Cellular morphology and quantitative analysis of apoptotic, necrotic and viable cells were determined with fluorescent microscopy and cell cycle phase distributions by measuring DNA content using flow cytometry. Results: We have indicated Trypan blue exclusion assay as the most sensitive for quantifying cytotoxicity of PCB 77 in terms of IC50 values, while the results obtained by other methods pointed to a possible localized effect on the lysosomes/endosomes (Neutral red), compromised intracellular metabolic processes (MTT) and possible interferation with the rate of protein synthesis (Kenacid blue). The loss of cell viability, as a consequence of treatment with 10–100 μM PCB 77, fundamentally was due to induction of apoptosis with observed common series of specific morphological changes characteristic to apoptotic phenomenon. The level of alterations of normal cell cycle progression was low without significant changes at analyzed time intervals. Conclusion: These results indicate toxic outcomes of PCB 77 at ovarian cellular level with regard to potential direct adverse effects to female reproductive system.

Determination of ochratoxin A in serum and urine of pigs

The objective of the study was to determine ochratoxin A (OTA) concentrations in serum and urine of pigs during 30-day OTA treatment. OTA was administered orally to the experimental group (n=5) at a dose of 0.78 mg per animal per day, whereas control animals (n=5) were left untreated. OTA concentrations were determined using a validated enzyme-linked immunosorbent assay (ELISA). Method validation resulted in mean recoveries of 93-101% for serum and 98-106% for urine, with acceptable mean inter- and intraday relative standard deviations (<8% for urine and <7% for serum). The ELISA method can be effectively used as a simple screening method to determine OTA exposure in pigs during fattening. The maximum mean OTA concentration in serum was recorded on day 22 (8.75±2.93 ng/ml) and in urine on day 20 (43.56±35.76 ng/ml), indicating significant differences in OTA concentrations between these two matrices.

Effect of porcine brain growth factor on primary cell cultures and BHK-21 [C-13] cell line.

Growth factors from neural tissues have been described as potent mitogens for a wide variety of mesoderm- and ectoderm-derived cells in vitro. We used porcine brain extract for in vitro testing of proliferation properties on primary ovarian cells, uterine cells, and cardiomyocytes in culture as well as for BHK-21 [C-13] cell line. The addition of this extract accelerates proliferation in all examined cultures. It also lowers serum requirement and shortens the cultivation period for BHK-21 [C-13] cells. Fibroblast growth factors from brain of different species, but not porcine, are already characterized and their proliferative effect proved. Therefore, we purified, determined, and confirmed the presence of basic fibroblast growth factor in porcine brain extract by Western blot analysis and showed its biological activity on BHK-21 [C-13] cells.