Équipe d'Endocrinologie Moléculaire de la Reproduction, UMR CNRS 6026 Université de Rennes I, Campus de Beaulieu, France
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Abstract |
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Key words: growth promoters/in-vitro assays/meat hormones/oestrogen/oestrogenic potency
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Introduction |
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Risk assessment of endocrine disruption by xeno-oestrogens and their metabolites requires accurate evaluation of their oestrogenic potency. To date, several in-vivo and in-vitro bioassays have been used to detect oestrogenic activity of environmental pollutant, e.g. the measurement of uterine weight (uterotrophic assay) in immature or ovariectomized female rats (Odum et al., 1997), production of vitellogenin (VTG) in the liver of oviparous vertebrates (Flouriot et al., 1995
; Knudsen et al., 1997
), cell proliferation response (Soto et al., 1995
) or modulation of endogenous oestrogen-regulated genes (Jorgensen et al., 1998
, 2000
) of the human breast tumour MCF-7 cell line and stimulation of oestrogen-dependent reporter gene expression in genetically modified yeast (Petit et al., 1997
).
The present study was conducted to measure oestrogenic activities of chemicals and also to compare three in-vitro bioassays, displaying different metabolism capabilities.
The first bioassay was a recombinant yeast expressing human or rainbow trout oestrogen receptor (hER and rtER respectively). This bioassay, exhibiting high specificity to oestrogens, could detect low level of oestrogens ranging between 0.1 and 1 nmol/l (Petit et al., 1995). The second bioassay was based on oestradiol-induction of the VTG gene expression in rainbow trout hepatocyte aggregate culture. In this bioassay, the minimal 17ß-oestradiol concentration required for VTG stimulation is ~10 nmol/l 17ß-oestradiol (Flouriot et al., 1993
). The third bioassay was a stable human endometrial carcinoma Ishikawa cell line, which displays a natural oestrogen-inducible alkaline phosphatase (AP) (Littlefield et al., 1990
). Compared with the two other assays, this system showed the highest sensitivity to oestrogens, since oestradiol was capable of inducing AP at concentrations as low as 1 pmol/l (Littlefield et al., 1990
).
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Materials and methods |
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O-Nitrophenyl ß-D-galactopyranoside (ONPG), p-nitrophenyl phosphate (PNPP), lyticase were from Sigma (St Quentin Fallavier, France). Yeast nitrogen base and amino acids (drop-out) were from Difco. Z buffer solution: 60 mmol/l Na2HPO4, 40 mmol/l NaH2PO4, 10 mmol/l KCl, 1 mmol/l MgSO4, 50 mmol/l ß-mercaptoethanol, pH 7.0. Lyticase solution: 0.1 mg/ml lyticase in Z buffer. ONPG substrate: 4 mg/ml ONPG in 0.1 mol/l potassium phosphate buffer, pH 7.0. PNPP substrate: 2 mmol/l PNPP in 0.1 mol/l Tris/HCl buffer pH 9.6
Cell culture
Recombinant yeast and ß-galactosidase assay
The recombinant BJ-ECZ yeast strain previously described (Wrenn and Katzenellenbogen, 1993; Petit et al., 1995
), contains a reporter gene with two oestrogen-responsive elements upstream from the yeast proximal Cytochrome C1 promoter fused to the Lac Z gene. These yeast cells were transformed with rtER or hER expression vectors (Petit et al., 1997
). The activation of the reporter gene is strictly dependent on the presence of ER and oestrogens, and results in the production of the ß-galactosidase (ß-Gal).
Four independent colonies were incubated at 30°C in a shaking incubator at 300 r.p.m. for 36 h. 100 µl of yeast suspension (0.6 OD600) was distributed in each well of conical-bottomed 96-well plates (PS Microplatte 651101; Greiner, Kremsmünster, Austria). After 4 h of incubation at 30°C in the presence of test compounds, plates were centrifuged and the media were discarded. 50 µl of lyticase solution were added to each well, and the plates were incubated at room temperature for 30 min. 100 µl of 0.1% Triton X-100 (Sigma) were added to each well. Plates were then centrifuged at 430 g for 10 min and 100 µl of supernatants were transferred in flat-bottomed 96-well microtitration plates, and 20 µl of ONPG substrate were added to each well. The enzymatic reaction was performed at 30°C for 1 h. The reaction was stopped by adding 50 µl of 1 mol/l Na2CO3 and the OD405 was read after 5 min equilibration. The ß-galactosidase units are defined as OD405/mg protein/min of enzymatic reaction.
Hepatocyte aggregate cultures and dot blot analysis
The hepatocyte aggregates were formed as described (Flouriot et al., 1993) in Dulbecco's modified Eagle's medium/Ham's F-12 nutrient mixture (DMEM/F-12, 1:1) supplemented by 2% Ultroser SF (Biosepra, Villeneuve la Garenne, France). Five-day-old aggregates (12x107cells per 5 cm diameter dish) were treated 48 h with test substances. Total RNA was extracted from hepatocyte cultures using a commercially available kit (Trizol; Gibco BRL, Cergy Pontoise, France). RNA samples (5 µg) were spotted onto nylon Biodyne A (Pall, St Germain en laye, France) membrane, using a Bio-Rad dot blot apparatus. The membranes were hybridized with radiolabelled rainbow trout VTG or actin cDNA as described previously (Pakdel et al., 1989
). The VTG and actin mRNA levels were quantified by counting the radioactivity from dot blots using an Instantimager (Packard, Rungis, France).
Ishikawa cultures and phosphatase alkaline assay
The human Ishikawa cells were routinely maintained in DMEM medium (Sigma) containing 10% fetal bovine serum (FBS) and supplemented with penicillin, streptomycin and fungizone (Littlefield et al., 1990). On the day of experiment, cells were harvested and plated in 96-well plates (2x104 cells/200 µl) in Phenol Red-free DMEM/F-12 medium supplemented with 5% FBS stripped of endogenous oestrogen with dextran-coated charcoal. On day 2 of the experiment, 2 µl of test compounds (dissolved in ethanol) were added. Dose-response was performed by 1/5 serial dilutions. After 48 h treatment, the plates were rinsed with 200 µl Tris/HCl 0.1 mol/l, pH 7.4 and cells were lysed by adding 20 µl Tris/HCl 0.1 mol/l pH 9.6, 0.01% Triton X-100 and by freezing at 80°C for 15 min followed by thawing at room temperature. The alkaline phosphatase activity was revealed at 37°C in the presence of 50 µl of PNPP substrate. The reaction was stopped by adding 50 µl of 1 mol/l Na2CO3 and the optical density (OD) was read at 405 nm.
Mathematical processing of data
Dose-response experiments were analysed mathematically by non-linear regression method. The sigmoidal doseresponse curve (variable slope) was used as model. Stricter criteria was chosen for convergence. The algorithm minimized the sum of squares and convergence was reached when two consecutive iterations changed the sum of squares by <0.01%. The EC50 were calculated from logEC50.
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Results |
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Dose-response of oestrogenic chemicals
Once these chemicals had been classified (oestrogenic or not), doseresponse experiments were performed to determine the EC50 and the relative stimulatory activity (RSA) of oestrogens in recombinant yeast expressing rtER or hER (Figures 3 and 4). The RSA was defined as the ratio of EC50 of ethinyl oestradiol and EC50 of test compound (Table I
). Ethinyl oestradiol is an unmetabolizable 17ß-oestradiol derivative and was chosen in place of 17ß-oestradiol for the calculation of the RSA to avoid misinterpretation of oestrogenic potency when 17ß-oestradiol is metabolized as in the Ishikawa cells (see below). Data showed that all oestrogenic compounds tested displayed a lower activity than ethinyl oestradiol or 17ß-oestradiol (Table I
). 17ß-Oestradiol had the same oestrogenic potency as ethinyl oestradiol (RSA = 100%) with both rtER and hER (RSA = 68 and 83% respectively). Nevertheless, while DES was as potent as ethinyl oestradiol with rtER (RSA = 82%), this chemical was 5-fold less potent than ethinyl oestradiol with hER (RSA = 21%). Oestrone and 17
-oestradiol were revealed as more potent oestrogens with hER (RSA = 29 and 12% respectively) than with rtER (RSA = 16 and 3% respectively). For zearalenone mycotoxin and its derivatives, a large range of activity was measured. With rtER, the ranking was as follows:
-zearalenol >
-zearalanol = zearalanone > zearalenone > ß-zearalanol > ß-zearalenol; and with hER:
-zearalenol >
-zearalanol > zearalanone = zearalenone = ß-zearalanol = ß-zearalenol. Although the ranking was identical for both receptors, in comparison with ethinyl oestradiol these compounds appeared to be 10-fold more potent, with rtER than with hER.
-Zearalenol was the most potent oestrogenic compounds in this group of chemicals with both rtER and hER, and displayed an oestrogenic activity ~29% of that of ethinyl oestradiol activity with rtER and only 2% with hER. Conversely, ß-zearalenol was the less oestrogenic compound of the zearalenone derivative for both receptors.
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Discussion |
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The recombinant yeast system represents a simple assay with low biotransformation activity in which the oestrogenic activity of a given chemical mostly results from its direct interaction with ER. In this system, all compounds tested displayed much lower oestrogenic activity than 17ß-oestradiol, DES or ethinyl oestradiol. We previously demonstrated that rtER binds oestrogens with a lower affinity than hER (Petit et al., 1995). This may explain the lower 17ß-oestradiol sensitivity of rtER (7-fold less) compared with hER in the recombinant yeast system. However, in comparison to 17ß-oestradiol or ethinyl oestradiol, the oestrogenic mycotoxins were more potent with rtER. Indeed, the oestrogenic activity of
-zearalenol represents 30% of the activity obtained with ethinyl oestradiol with rtER versus 2% with hER. Despite these interspecies differences in myco-oestrogen sensitivity, the ranking of oestrogenic potency was identical for both rtER and hER. Our results are in agreement with previous data demonstrating that the binding affinity of
-zearalenol was greater in pig than in rat and chicken and also that
-zearalenol exhibited greater affinity than zearalenone and ß-zearalenol in the three species (Fitzpartrick et al., 1989). Similar results were also reported in trout by in-vitro ER competitive binding assays and in vivo on the induction of VTG and eggshell zona radiata proteins in Salmo salar (Arukwe et al., 1999
).
Hepatocyte cells, particularly in aggregate culture, represent a complex biological system maintaining in vitro most of the biotransformation capacity of the liver (Flouriot et al., 1993). In this bioassay, zearalenone,
-zearalanol and ß-zearalanol display some differences in terms of oestrogenic potency compared with the rtER recombinant yeast system. Although these steroids displayed a strong oestrogenic activity in yeast at 0.1 µmol/l, these compounds showed lower oestrogenic potency in hepatocytes. Indeed, a concentration of 10 µmol/l was required to achieve 60-80% of the maximal VTG gene induction obtained with 1 µmol/l 17ß-oestradiol. This observation suggested that these compounds were metabolized in hepatocyte cells (Pompa et al., 1988
; Bories et al., 1991
) and the oestrogenic activity observed in these cells at 10 µmol/l could result from metabolite(s) or untransformed excess of parent compounds. The metabolism of
-zearalanol has been studied in the rat, rabbit, dog monkey and human following oral administration (Migdalof et al., 1983
), and in the pig after implantation (Bories and Fernandez-Suarez, 1989
). In all species studied,
-zearalanol is oxidized to zearalanone, a less potent metabolite (Bories et al., 1991
). In addition to the formation of zearalanone, a small quantity of ß-zearalanol is always produced (Pompa et al., 1988
; Bories et al., 1991
). These metabolites are subsequently glucuronided or sulphated (Pompa et al., 1988
; Bories et al., 1991
).
Conversely to the yeast system, which does not mimic human metabolism, the Ishikawa cells, as the normal uterine cells, possess important specific metabolism pathways for steroid conversion that modulate the sensitivity of the uterus to oestrogens (Falany and Falany, 1996). The main mechanism for inactivation of oestrogens in uterus is via sulphate conjugation. When physiological concentrations of 17ß-oestradiol or oestrone are incubated with Ishikawa cells, up to 95% of the oestrogens are converted to the sulphated form in a few hours (Chetrite and Pasqualini, 1997
). Another mechanism implicated in the inactivation of 17ß-oestradiol is the transformation to oestrone by the 17ß-hydroxysteroid dehydrogenase. At low nanomolar oestrogen concentration, sulphatation is the major pathway for oestrogen metabolism in Ishikawa cells, whereas the oxidation of 17ß-oestradiol to oestrone by 17ß-hydroxysteroid dehydrogenase becomes important at micromolar concentration (Hata et al., 1987
, 1989
; Chetrite and Pasqualini, 1997
). Four forms of cytosolic sulphotransferase (ST) have been identified in human tissues and three of them have been reported to conjugate oestrogens and hydroxysteroids: the dehydroepiandrosterone-ST (hDHEA-ST), the phenol-ST (hP-ST) and the oestrogen-ST (hE-ST). The hDHEA-ST is involved in the sulphatation of DHEA, pregnenolone, oestrogen and testosterone and many bile acids. The hP-ST conjugate only the 3-phenolic hydroxyl of oestrogens; the hE-ST differs from the other forms in that hE-ST sulphates 17ß-oestradiol and oestrone with higher affinity (Falany and Falany, 1996
). The hE-ST is the major form of human cytosolic ST involved in the inactivation of 17ß-oestradiol during the luteal phase of the menstrual cycle, and hE-ST has a higher affinity for the sulphatation of 17ß-oestradiol than for other oestrogens such as DES (Kotov et al., 1999
). The metabolism of 17ß-oestradiol in the uterus could explain the medium-level oestrogenic activity observed for 17ß-oestradiol, 17
-oestradiol and oestrone in these cells compared with that of DES or ethinyl oestradiol.
Except ß-zearalanol and ß-zearalenol, the mycotoxins displayed a strong oestrogenic activity compared with 17ß-oestradiol in Ishikawa cells. In fact, -zearalenol, with an oestrogenic potency 22-fold that of 17ß-oestradiol, was as potent as the strong oestrogens ethinyl oestradiol and DES (75% of ethinyl oestradiol activity). This result is in accordance with a previous study demonstrating that
-zearalenol possesses a strong oestrogenic potency in vivo on the uterotrophic response in immature female rats (Everett et al., 1987
). One hypothesis to explain this high oestrogenic activity could be that these compounds are poorly sulphated in Ishikawa cells. Alternatively in these cells the strong activity of
-zearalenol could be cell- and promoter-dependent, as observed for some ER ligands such as tamoxifen, which is a strong oestrogen antagonist in the breast, but exhibits oestrogenic activity in the uterus (Jones et al., 1999
).
Zearalenone and its metabolites occur as natural contaminants of cereal grains and derived food products. This chemical is synthesized by moulds and is difficult to avoid in food products. Zearalenone or its derivatives have been associated with reproductive disorders in farm animal feed with mould-infected grain (Korach et al., 1994; Skrinjar et al., 1995
; Meyer et al., 1997
). Zearalenone was found in cattle feed at concentrations between 140 and 960 µg/kg (Skrinjar et al., 1995
) and, in pigs with reproductive problems, zearalenone and
-zearalenol glucuronide conjugates were found in bile at concentrations of up to 40 and 66 µg/l. By comparison, in animals treated with
-zearalanol as growth promoter, the maximum accepted residue limit for
-zearalanol in edible tissue is 2 µg/kg in muscle and 10 µg/kg in liver. The human endometrial carcinoma Ishikawa cell line was found to be highly sensitive to oestrogenic mycotoxins. The EC50 in these cells were 5.8x10-11 mol/l for zearalenone, 6.6x10-12 mol/l for
-zearalenol and 3x10-11 mol/l for
-zearalanol. These concentrations are equivalent to 20, 2 and 10 ng/l respectively. These concentrations are below the concentrations found in mycotoxin-contaminated animal feeds and below the maximum residue limit in edible tissue of animals treated with
-zearalanol. In view of these results, there is some concern about the threshold level for these chemicals and a possible risk for reproductive function in humans.
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Acknowledgements |
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Notes |
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References |
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Submitted on September 6, 2000; accepted on February 22, 2001.