Cytotoxicity against fish and mammalian cell lines and endocrine activity of the mycotoxins beauvericin, deoxynivalenol and ochratoxin-A.

The toxicity of mycotoxins is well recognized in mammals, but their effects on fish have received less attention. Moreover, in the last years several studies have reported that some mycotoxins may act as endocrine disruptors. The aim of this study was to determine the cytotoxic effects and endocrine activities of three mycotoxins: beauvericin, deoxynivalenol and ochratoxin-A. Cytotoxicity in two fish hepatoma and one mammalian hepatoma cell lines was determined by the AlamarBlue, Neutral Red Uptake and CFDA-AM assays. For the assessment of androgenic, estrogenic and thyroidal agonistic/antagonistic effects three cell lines stably expressing luciferase as reporter gene under the control of hormone receptors were used. Results showed that both fish and mammalian cell lines were very sensitive to the mycotoxins tested. OTA was the least toxic mycotoxin and DON and BEA showed similar acute toxicity. None of the three mycotoxins tested presented agonistic effects at the receptors studied, but all of them showed strong antagonistic effect at the thyroid receptor. BEA showed a weak antagonistic effect at the androgen receptor and OTA produced a biphasic dose-response curve at the estrogen receptor. The data obtained in this work are of high interest for aquaculture industries and for regulators.

Development of a new tool for the long term in vitro ecotoxicity testing of nanomaterials using a rainbow-trout cell line (RTL-W1).

The current wide use of manufactured nanomaterials (MNs) is leading to the release of nanoparticles (NPs) to water bodies. Aquatic organisms, including fish, are exposed to low concentrations of NPs for long periods of time being necessary to develop laboratory toxicity tests reflecting realistic conditions. Additionally, today there is a demand of in vitro assays respecting the 3Rs principle. Thus, the main aim of this work was to stablish an in vitro tool for the assessment of long-term NPs ecotoxicity. Considering the key role of liver in detoxification, a rainbow trout liver cell line, RTL-W1, was used. CuO NPs were chosen to validate this tool taking into account their important production level. Cells were exposed for 21 days to 25 or 100 µg CuO NPs/ml. Every seven days cells were split and one fourth of them transferred to a new plate with appropriate concentrations of NPs in culture medium. Lower concentrations of CuO NPs did not cause any deleterious effect, whereas higher concentrations led to significant mortality after 14 days and to the intracellular accumulation of Cu particles. Identical results were observed in cells exposed to CuSO4 at the same Cu concentrations. Therefore, the observed toxic effects might be mainly due to Cu2+ ions.

Determining the presence of chemicals with suspected endocrine activity in drinking water from the Madrid region (Spain) and assessment of their estrogenic, androgenic and thyroidal activities.

Endocrine disruptors (EDs) are natural or man-made chemicals that can affect the health of organisms by interfering with their normal hormonal functions. Many of these substances can cause their effects at very low doses and, considering the key role played by the endocrine system on development, organisms in early phases of growth (foetal, childhood, puberty) are especially sensitive to the action of EDs. In addition, when combined, they can show additive, antagonistic and synergistic activities. Taking all this into account it is essential to determine the presence of this kind of compounds in drinking water. Thus the main aim of the present study was to monitor the presence of substances with suspected or known endocrine activity in drinking water of the Madrid Region (MR) (Central Spain) and determine possible estrogenic, androgenic, or thyroidal activities. Water samples were collected at different times from a number of supply points that received water from reservoirs or rivers. The sampling point with the highest concentration of the analysed substances (up to 30 compounds) was DW1 (1203 ng L-1). This sampling point receives water from a drinking water treatment plant (DWTP) that serves the population from the south of the MR with treated water from the Tajuña River. DW2 was the second point with the highest concentration of the analysed substances (1021 ng L-1). DW2 receives water from one of the reservoirs in the north of the MR. The highest daily concentrations detected corresponded to the flame retardant Tris (2-chloroethyl)phosphate (TCEP) (266.55 ng L-1) and to the nonylphenol diethoxylate (188.57 ng L-1) at points DW1 and DW4, respectively, both of which are supplied with treated river water. None of the water samples exhibited androgenic, oestrogenic, or thyroidal activities in in vitro assays based on cells stably transfected with the receptors of interest and luciferase as reporter gene. These results demonstrate that water quality in the MR is high and does not present a health risk for the population, although the concentrations of some substances justify the need for local authorities to continually monitor the presence of these contaminants in order to implement any corrective measures if necessary.

Thyroid signaling in immune organs and cells of the teleost fish rainbow trout (Oncorhynchus mykiss).

Thyroid hormones are involved in modulating the immune system in mammals. In contrast, there is no information on the role played by these hormones in the immune system of teleost fish. Here we provide initial evidence for the presence of active thyroid signaling in immune organs and cells of teleosts. We demonstrate that immune organs (head kidney and spleen) and isolated leukocytes (from head kidney and peripheral blood) of the rainbow trout (Oncorhynchus mykiss) express both thyroid receptor α (THRA) and ß (THRB). Absolute mRNA levels of THRA were significantly higher than those of THRB. THRA showed higher expression in immune organs and isolated immune cells compared to the reference organ, liver, while THRB showed the opposite. In vivo exposure of trout to triiodothryronine (T3) or the anti-thyroid agent propylthiouracil (PTU) altered THR expression in immune organs and cells. Effect of T3 and PTU over the relative expression of selected marker genes of immune cell subpopulations was also studied. Treatments changed the relative expression of markers of cytotoxic, helper and total T cells (cd4, cd8a, trb), B lymphocytes (mIgM) and macrophages (csf1r). These findings suggest that the immune system of rainbow trout is responsive to thyroid hormones.

In vitro assessment of thyroidal and estrogenic activities in poultry and broiler manure.

Among the many chemicals found in avian manure, endocrine disruptors (EDs), of natural or anthropogenic origin, are of special environmental concern. Nowadays, an increasing amount of estrogens is being released into the environment via the use of manure to fertilize agricultural land. While most research in this field has focused on estrogenic phenomena, little is known about alterations related to other endocrine systems, such as the thyroidal one. Here we simultaneously assessed the potential estrogenic and thyroidal activity of poultry and broiler litter manure using in vitro approaches based on estrogen receptor (Er) and thyroid receptor (Tr) transactivation assays. In addition, leaching experiments were performed to assess whether the EDs present in the manure pass through a soil column and potentially reach the groundwater. Manure from four broiler and four poultry farms was collected in two sampling campaigns carried out in two seasons (fall and spring). Extracts from broiler and poultry manure exhibited strong thyroidal activity. Only poultry manure showed estrogenic activity, which is consistent with the low levels of estrogens expected in hatchlings. Leakage experiments were performed in columns with two kinds of arable soils: sandy and loamy. No estrogenicity or thyroidal activity was detectable in soils treated with the manure or in the corresponding leachates. These results indicate that substances with estrogenic or thyroidal activity were degraded in the soil under our experimental conditions. However, the long-term effects associated with the constant and intensive application of manure to agricultural land in some regions require further research.

Differences in the induction of cyp1A and related genes in cultured rainbow trout Oncorhynchus mykiss. Additional considerations for the use of EROD activity as a biomarker.

Two rainbow trout Oncorhynchus mykiss fish farms were repeatedly sampled in order to observe the variability of ethoxyresorufin-O-deethylase (EROD) activity and of related genes in the liver. Fish coming from fish farm A exhibited EROD levels that could be considered as basal according to the scientific literature, however, EROD activity in fish coming from fish farm B was significantly increased. This was accompanied by augmented aryl hydrocarbon receptor (ahr) and cytochrome P4501A (cyp1A) messenger RNA expression and reduced oestrogen receptor (er) and vitellogenin (vtg) transcription. Only sediment extracts from the entry channel of fish farm B induced EROD activity in O. mykiss cultured cells, however, this induction could not be explained by the levels of polyaromatic hydrocarbons (PAH) and polychlorinated biphenyls (PCB) measured in the sediments. The results of this study point out that O. mykiss cultured in fish farms could be used as sentinels for indication of pollution. In this particular work, however, no conclusive evidence has been found for a relationship between the presence of PAHs and PCBs and the observed EROD induction.