Lidia Radko

Cytotoxicity of monensin, narasin and salinomycin and their interaction with silybin in HepG2, LMH and L6 cell cultures.

The cytotoxic effect of monensin, narasin and salinomycin followed by their co-action with silybin in the cell line cultures of human hepatoma (HepG2), chicken hepatoma (LMH) or rat myoblasts (L6) have been investigated. The effective concentration of the studied ionophoric polyethers has been assessed within two biochemical endpoints: mitochondrial activity (MTT assay) and membrane integrity (LDH assay) after 24h incubation of each compound and farther, the cytotoxicity influenced in course of their interaction with silybin was determined. The most affected endpoints were found for inhibition of mitochondrial activity of the hepatoma cell lines and their viability depended on concentration of the ionophoric polyether, as well as on the cell line tested. The rat myoblasts were more sensitive target for cellular membrane damage when compared to inhibition of mitochondrial activity. An interaction between the ionophoric polyethers and silybin resulted a considerable cytotoxicity decrease within all studied cell lines; the combination index (CI) showed differences of interaction mode and dependence on cell culture, concentration of silybin, as well as the assay used. The obtained results are of interest in respect to recent findings on applicability of salinomycin and monensin for human therapy.

Evaluation of estrogenic activity in animal diets using in vitro assay.

A yeast estrogen bioassay (RIKILT REA) was in-house validated for feed on the 5μg 17β-estradiol-equivalents per kg level according to EC Decision 2002/657/EC. All the performance characteristics met the criteria as defined in the Decision and the REA is able to detect 17β-estradiol in animal feed at a low level of 1.15-2μgkg(-1). Subsequently, the developed and validated procedure was applied to determine the estrogenic activity in 24 feed samples intended for food producing animals, pets and laboratory animals. Two batches of rodent diet Murigran and one dog feed have been presented as a suspect, i.e. gave responses above the determined decision limit (CCα) and detection capability (CCβ). In assessing the performance of the estrogenic activity in these diets evaluated by comparison with the 17β-estradiol calibration curve, 17β-estradiol-equivalence levels of 7.07μg EEQkg(-1) and 9.54μg EEQkg(-1) in two batches of rodent diet and 5.3μg EEQkg(-1) in dog feed have been established. The activities observed in the rodent feed could be explained by chemical analysis, revealing high amounts of genistein, daidzein and trace amounts of zearalenone. In addition, the estrogenic activity in one of rodent feed was above the established CCα, but below the CCβ values established and all other samples showed no estrogenic activity with responses below the CCα value, which corresponds to levels below 2μg EEQkg(-1).

The protective effect of silybin against lasalocid cytotoxic exposure on chicken and rat cell lines.

Lasalocid, an ionophore coccidiostat, extensive use implies a risk of toxicological impacts. Protective effects of silybin, a herbal compound of Silybum marianum, are reported elsewhere. The aim of this study was to compare effects of the combined use of lasalocid and silybin in chicken hepatoma cells (LMH) and rat myoblasts (L6) cell lines cultures. The cytoprotective effect resulting from an interaction of both pharmaceuticals was measured with the help of MTT reduction and, coomassie brilliant blue binding (CBB) and LDH release assays. Isobolography and the combination index (CI) estimated the nature and scale of interaction. In all performed tests, the lowest lasalocid EC50-values were obtained for chicken hepatocytes. In the rat myoblasts cultures, the lowest lasalocid EC50-values were found with LDH test. Simultaneously, a lack of silybin cytotoxic effect was proven for the studied cell lines. An interaction between both substances led to a considerable decrease of lasalocid cytotoxicity. The isobolograms and combination index showed a significant antagonistic nature of silybin effect in the course of lasalocid cytotoxicity. It is concluded that the mechanism of cytoprotection results from complex reaction at biochemical and biophysical endpoints during chicken hepatocytes and rat myoblasts cell lines exposure to silybin and lasalocid co-action.

Influence of fluoroquinolones on viability of Balb/c 3T3 and HepG2 cells

Abstract The cytotoxic potential of fluoroquinolones (enrofloxacin, ciprofloxacin, difloxacin, sarafloxacin, danofloxacin, norfloxacin and marbofloxacin) was investigated using mouse fibroblasts Balb/c 3T3 and human hepatoma HepG2 cell lines. The cells were exposed for 24, 48, and 72 h to drugs at eight concentrations ranged from 0.78 to 100 μg/mL. Four independent cytotoxicity assays were applied, in which various endpoints were assessed: mitochondrial activity - MTT reduction, lysosomal activity - neutral red uptake, total protein content, and cellular membrane integrity - lactate dehydrogenase release. Mean effective cytotoxic concentrations (EC50) calculated at different time points from concentration-response curves ranged from 10 to 100 μg/mL. The most affected endpoint in both cell lines was mitochondrial activity. The EC50-MTT-72 h <10 μg/mL was found for difloxacin, marbofloxacin (fibroblasts), sarafloxacin, and norfloxacin (HepG2). The data shows that cytotoxicity of the fluoroquinolones appears after longer exposure of both cell cultures to these compounds.

Identification of metabolites of anticancer candidate salinomycin using liquid chromatography coupled with quadrupole time-of-flight and hybrid triple quadrupole linear ion trap mass spectrometry

RATIONALE Salinomycin is an ionophore antibiotic with potential anticancer activity. The history of its use in veterinary medicine shows large differences in species susceptibility to its toxicity. At the same time, the results of research to date suggest a correlation between the extent and pathways of ionophore biotransformation and its toxicity. The biotransformation pattern of salinomycin has not been studied so far. METHODS Extracts from culture media of human hepatoma cells (HepG2) exposed to salinomycin were analysed with two mass spectrometry techniques. For the first one, micro-liquid chromatography coupled with a quadrupole time-of-flight (Q-TOF) mass spectrometer was used. In the second approach, high-performance liquid chromatography was coupled with a hybrid triple quadrupole linear ion trap. Both experiments were operated in positive electrospray ionization mode. To identify unknown salinomycin metabolites, information-dependent acquisition was applied. RESULTS Metabolites identified with tandem mass spectrometry included hydroxylated, demethylated and hydroxylated-demethylated derivatives, in total 14 compounds. Using high resolution, only eight isomers of hydroxysalinomycin were detected. The efficiency of biotransformation was low, and so was the abundance of the signals; only for two metabolites did the signal exceed 1% of the salinomycin signal. The analysis of fragmentation patterns narrowed the structure combinations but the actual modification site could not be specified. CONCLUSIONS Tandem mass spectrometry was more sensitive in the identification of salinomycin metabolites in comparison to the Q-TOF approach. Because of low efficiency of biotransformation of the applied model, the obtained fragmentation data are not sufficient to fully characterize the detected compounds. A study with more metabolically active primary hepatocytes is needed.

Nonsteroidal mycotoxin alternariol is a full androgen agonist in the yeast reporter androgen bioassay

Alternariol (AOH) is a toxic metabolite of phytopathogenic fungi of the Alternaria spp. and important contaminant of agricultural commodities. According to the recent studies, AOH has a potential to modulate the endocrine system of humans and animals. In the view of these reports, our study addressed the effects of AOH on human estrogen receptor (hERα) and androgen receptor (hAR) signaling with the use of the yeast estrogen and androgen reporter bioassays. Our results show that, apart from a weak estrogenic response, AOH induces full androgenic response of the bioassay with the EC50 of 269.4μM. The androgenic potency of AOH relative to testosterone (T) is 0.046%. Moreover, in the presence of T, AOH at 5μM acts as a weak antiandrogen, whereas at higher concentrations AOH sum up with the androgenic activity of T in a dose-dependent manner, suggesting additive effect. To our knowledge it is the first report of the androgenic potency of natural, nonsteroidal substance and may have the impact on the direction of the further studies. Further research is warranted to clarify the role of AOH in disruption of AR signaling in humans and animals.

Differential toxicities of albendazole and its two main metabolites to Balb/c 3T3, HepG2, and FaO lines and rat hepatocytes

Abstract Introduction: The cytotoxicity of anthelmintic agent, albendazole (ABZ) and its two major metabolites, sulfoxide (ABZSO) and sulfone (ABZ-SO2), on non-hepatic Balb/c 3T3 line, two hepatoma cell lines (FaO, HepG2), and isolated rat hepatocytes was investigated. Material and Methods: Cell cultures were exposed for 24, 48, and 72 h to eight concentrations of the compounds ranging from 0.05 to 100 μg/mL (ABZ) and from 0.78 to 100 μg/mL (ABZ-SO and ABZ-SO2). Three different assays were applied in which various biochemical endpoints were assessed: lysosomal activity - neutral red uptake (NRU) assay, proliferation - total protein contents (TPC) assay and lactate dehydrogenase (LDH) leakage assay. Results: The most toxic was albendazole whose EC50 values calculated from the concentration effect curves ranged from 0.2 to 0.5 μg/mL (Balb/c 3T3 ) and from 0.4 to 73.3 μg/mL (HepG2). Rat hepatoma line and isolated rat hepatocytes were less sensitive to the impact of ABZ. Toxic action expressed as EC50 was recorded after 72 h exposure only in LDH release assay at 0.8 μg/mL and 9.7 μg/mL respectively. The toxicity of metabolites was much lower. The most sensitive to ABZ-SO were fibroblasts and EC50-72h values were similar in all three assays used, i.e. NRU (14.1 μg/mL), TPC (15.8 μg/mL), and LDH (20.9 μg/mL). In the case of ABZ-SO2 the mean effective concentrations were the highest, and could be reached only in one LDH assay. These values (μg/mL) were as follows: 65.3 (FaO), 65.4 (HepG2), 75.8 (hepatocytes), and 77.4 (Balb/c 3T3). Conclusion: The differences in in vitro toxicity of albendazole depend on metabolic ability of the cellular models. Primary cultured rat hepatocytes represent a valuable tool to study the impact of biotransformation on the cytotoxicity of drugs.

Cytotoxic effects of the synthetic oestrogens and androgens on Balb/c 3T3 and HepG2 cells

Abstract The aim of the study was to test and compare the cytotoxic potential of two synthetic oestrogens: diethylstilboestrol (DES) and ethinyloestradiol (EE2) and two androgens: testosterone propionate (TP) and trenbolone (TREN) on two cell lines. The fibroblast cell line Balb/c 3T3 and the hepatoma cell line HepG2 were selected. To get more insight into the mode of toxic action, four methods were used, which evaluated different biochemical endpoints: mitochondrial activity (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide reduction assay), lysosomal activity (neutral red uptake assay), total protein content, and lactate dehydrogenase release. Cytotoxicity was assessed after 24, 48, and 72 h exposure to eight concentrations ranging from 0.78 to 100 μg/mL. Concentration- and time- dependent effects were observed. Depending on the line and assay used, half maximal effective concentration after 72 h (EC50-72h) values ranged as follows: DES 1-13.7 μg/mL (Balb/c 3T3) and 3.7-5.2 μg/mL (HepG2); EE2 2.1-14.3 μg/mL (Balb/c 3T3) and 1.8-7.8 μg/mL (HepG2); TP-14.9-17.5 μg/mL (Balb/c 3T3), and 63.9- 100 μg/mL (HepG2); and TREN 11.3-31.4 μg/mL (Balb/c 3T3) and 12.5-59.4 μg/mL (HepG2). The results revealed that oestrogens were more toxic than androgens and the most affected endpoint was mitochondrial activity. In contrast to oestrogens, for which EC50-72h values were similar in both lines and by all assays used, Balb/c 3T3 cells were more sensitive than HepG2 cells to TP.

Influence of dietary soy isoflavones on immature hamster uterotrophic and Hershberger assays

Abstract To select appropriate diet for hamsters used in the uterotrophic and Hershberger assays two rodent diets were compared: Murigran (Agropol, Poland) and Altromin 7010 (Altromin Spezialfutter GmbH&Co., Germany). The contents of bioactive compounds in feeds were evaluated by liquid chromatography, and their oestrogenic activity by yeast enhanced green fluorescent protein assay. In opposition to Altromin, Murigran contained high amounts (μg/kg) of genistein (765 600) and daidzein (132 000), and the oestrogenic activity of these compounds, expressed as 17β-oestradiol equivalent concentration (EEQ), was found to be 9.54 μg EEQ/kg. In in vivo study, Murigran induced a high degree of oestrogenisation in immature hamsters, and females failed to exhibit a normal uterine response to recommended dose of a model oestrogen agonist 17α-ethinyloestradiol. There was no influence of the diet on the weight of five accessory sex organs (ASO): ventral prostate, seminal vesicles with coagulating glands, levator ani bulbocavernosus muscles, Cowper`s glands, and glans penis of control males. However, the impact on ASO response to model androgen agonist, testosterone propionate was observed. The obtained results provide the evidence that phytooestrogen-rich feed modulates the oestrogenic and androgenic response to chemicals.

Cytotoxicity of Some Nitroimidazole Derivatives - Comparative Studies on Human and Rat Hepatoma Cell Lines

Abstract The cytototoxic potential of metronidazole, tinidazole, ronidazole, and ornidazole, using human and rat hepatoma cell lines (HepG2 and FaO) in culture was assessed. The cells were treated with drugs for 24, 48 and 72 h at 37 °C in 5% CO2 at concentrations of 0.1 to 200 μg/mL. Following the treatment period, the cells were assayed by four independent assays: MTT reduction, neutral red uptake (NRU), total protein content (TPC), and LDH leakage. The results suggest that nitroimidazoles are of low cytotoxic potential (EC50 >200μg/mL). The exception was ronidazole, which demonstrated a distinct endpoint sensitivity related to the species. EC50 (μg/mL) in human cells were: in MTT assay - 196±5.5 and 122±9.3 at 24 and 48 h, respectively, and in NRU assay - 150±1.25 at 72 h. Based on minimal toxic concentrations (EC20) for ronidazole, determined by all methods used in HepG2 cells, it could be concluded that their sensitivity was as follows: MTT>NRU>LDH>TPC.