Estrogenic activity of commercial milk as revealed in immature hamster uterotrophic assay ­ pilot study

Background: The risk for public health posed by endocrine disruptors present in food is relatively new issue. Our current understanding of human exposure is mainly based on the residue analysis of selected compounds. With such approach potential, effects of mixtures, including so-far unidentified compounds are not taken into consideration. Therefore, the knowledge of overall hormonal activity in food samples is of big importance. Objective: Milk and dairy products are a rich source of estrogens but very rarely undergo testing for estrogenic activity. For this reason the rodent uterotrophic bioassay is one of the most useful tool. This preliminary study was conducted in immature hamsters to assess commercially available milk. The endpoint measured was uterine weight increase. Material and methods: Fifteen-day old females received ad libitum throughout 7 days commercially available milk i.e. raw goat's, raw cow's, processed 3.2% UHT, and for comparison soy milk. The animals of negative control group received water but positive control group got 17ß - estradiol (E2) at the concentration of 100 ng/ml. Results: All samples of milk showed estrogenic activity as follow: goat's >cow's >soy >processed milk. Significant increase of uteri weights were recorded in goat's (p<0.001) and cow's milk (p<0.01). However, the activity was approximately 5-fold lower than induced by 17ß-estradiol. The ratio uterine weight/body weight (%) in negative control was 0.096%, in milk experimental groups ranged from 0.112% to 0.153% and in positive control this value was 0.493%. Conclusion: The results suggest that commercially available milk has a weak uterotrophic activity. Further in vivo and in vitro studies are warranted to gain more insight into the estrogenic risk from milk and other dairy products.

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.

Comparison of Albendazole Cytotoxicity in Terms of Metabolite Formation in Four Model Systems.

INTRODUCTION: Albendazole is used to treat endoparasitic diseases in animals and humans. After oral administration, it is quickly oxidised into its pharmacologically active metabolite albendazole sulfoxide and then to sulfone. However, it is not clear which compound is responsible for toxic effects towards mammalian cells. MATERIAL AND METHODS: The model systems comprised cultures of isolated rat hepatocytes, two hepatoma cell lines (FaO, HepG2), and non-hepatic Balb/c 3T3 line. Cells were exposed for 24, 48, and 72 h to eight concentrations of albendazole ranging from 0.05 to 100 µg/mL. At all three time points cytotoxic effects were assessed by MTT assay and metabolites in the culture media were determined by LC-MS/MS analysis. RESULTS: The effective concentrations EC50-72h showed that Balb/c 3T3 cells were the most sensitive to albendazole (0.2 ±0.1 µg/mL) followed by FaO (1.0 ±0.4 µg/mL), and HepG2 (6.4 ±0.1 µg/mL). In the case of isolated hepatocytes this value could not be attained up to the highest concentration used. Chemical analysis revealed that the concentrations of albendazole in hepatocytes and HepG2 and FaO culture media gradually decreased with incubation time, while the concentrations of its metabolites increased. The metabolism in isolated hepatocytes was dozens of times greater than in HepG2 and FaO cells. Two metabolites (albendazole sulfoxide, albendazole sulfone) were detected in isolated hepatocytes and HepG2 culture medium, one (albendazole sulfoxide) in FaO culture medium and none in Balb/c 3T3. CONCLUSION: The obtained data indicate that metabolism of albendazole leads to its detoxification. The lower cytotoxic potential of metabolites was confirmed in the independent experiments in this study.

Application of the yeast-based reporter gene bioassay for the assessment of estrogenic activity in cow's milk from Poland.

Milk contain compounds acting through the estrogen receptor signaling. The still open question whether such estrogens pose a risk for human health, encouraged us to measure the overall estrogenic activity of cow's milk in the in vitro yeast reporter bioassay. First, we assessed the ability of the bioassay to detect estrogens frequently detected in milk. The relative potencies of 16 compounds descended in the order: 17ß-estradiol (17ß-E2), 17α-ethinylestradiol, diethylstilbestrol, dienestrol, 17α-E2, estrone, zearalenone, estriol, equol, genistein, 17ß-E2 glucuronide, bisphenol A, apigenin, daidzein. Flavone, 4-n-nonylphenol and 4-t-octylphenol shown no activity in the bioassay.The estrogenic activities of milk samples without hydrolysis were below the detection limit, whereas in 50% of the deconjugated samples they varied between 0.29 and 0.49 ng EEQ mL(-1). We also compared the estrogenic activity in raw cow's milk collected from rural and industrial locations in Poland. In our pilot study we did not observe statistically significant difference in estrogenic activities in milk collected from the two locations. We found that the daily intake of estrogens with milk may be higher than estrogen levels in human serum. Further studies are warranted to evaluate the significance of milk and dairy as a source of estrogens for humans.

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 17ß-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 17ß-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 (CCß). In assessing the performance of the estrogenic activity in these diets evaluated by comparison with the 17ß-estradiol calibration curve, 17ß-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 CCß values established and all other samples showed no estrogenic activity with responses below the CCα value, which corresponds to levels below 2µg EEQkg(-1).

Developmental toxicity of Ochratoxin A in rat embryo midbrain micromass cultures.

Embryonic midbrain micromass cultures were exposed for five days to ochratoxin A (OTA) at seven concentrations (ranging from 0.16 to 10 microg/mL). Cell viability was assessed in neutral red uptake test (NRU), and differentiation - by immunoenzymatic determination of structural proteins (beta(III)-tubulin, MAP2, GFAP) expression level as well as by computer image analysis. Dose dependent decrease in cell number and differentiation was observed. Concentration-response curves were analysed and the mean inhibition concentrations (microg/mL) for cytotoxicity (IC(50)) and differentiation (ID(50)) were calculated. There were no significant differences in the sensitivity of neurons in early and late stage of differentiation and astrocytes to the toxic activity of this compound. For all endpoints ID(50) value was very low (< 10 microg/mL) so OTA was classified as a strong teratogen. IC(50)/ ID(50) ratios <2 pointed out that with harmful action of OTA the basic cytotoxicity should be connected.

Inhibition of cell differentiation by quinolones in micromass cultures of rat embryonic limb bud and midbrain cells.

Micromass cultures (MMC) of rat embryonic limb bud (LB) and midbrain (CNS) cells were applied to compare the developmental toxicity of three quinolone antimicrobials: norfloxacin (Nor), enrofloxacin (Enr) and ciprofloxacin (Cip). Cultures were exposed for 5 days to seven concentrations of drugs. Cytotoxicity was assessed by quantifying neutral red uptake; differentiation-by quantifying alcian blue uptake (LB) or by image analysis of Gill's haematoxylin stained foci (CNS). Both, LB and CNS cultures showed dose-dependent reduction in total cell number and differentiation. To distinguish specific effect on differentiation, IC(50) for proliferation (P) and differentiation (D) were calculated and P/D ratios were compared. In LB cultures all three drugs were cytotoxic (P/D ratios were 1). In CNS cultures P/D ratios were 1 (up to 2.7 for Nor, up to 4.4 for Enr and up to 16 for Cip) what can suggest specific action on differentiation. Ciprofloxacin was the most toxic and CNS cells were more sensitive than LB. The ranges of IC(50)-D values (microg/ml) were as follows: Nor (79-14), Enr (127-179), Cip (91-101) in LB cultures; Nor (22-52), Enr (38-91), Cip (3-17) in CNS cultures. With one exception (Cip in CNS culture) all drugs were classified as weak embryotoxic.