Department of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark
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Abstract |
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Key words: ATB0+/GST mu3/p35srj/PS2/Zeranol
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Introduction |
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Recently there has been much focus on potential endocrine disrupters that, because of their hormone-like properties, may have adverse effects on humans and wildlife (Kavlock et al., 1996; Toppari et al., 1996
). Many of the suspected endocrine disrupters are oestrogenic and could interfere with the endocrine functions of endogenous oestrogens (Sharpe and Skakkebæk, 1993). A number of chemicals with oestrogenic properties have been identified and the oestrogenic potency of the different compounds can thus be estimated. The potencies differ by almost eight orders of magnitude (Soto et al., 1995
; Jobling et al., 1995
; Zava et al., 1997
; Jørgensen et al., 2000
) which has a significant impact on the `endocrine disrupting potential' of the chemicals. For example, the most oestrogenic phthalate, dibutylphthalate (DBP), is seven to eight orders of magnitude less oestrogenic than diethylstilboestrol (DES) or 17ß-oestradiol (Jobling et al., 1995
; Jørgensen et al., 2000
). Thus, the serum concentration of DBP should be in the millimolar range before its oestrogenicity exceeds the oestrogenicity of the endogenous 17ß-oestradiol in adult men; such concentrations of DBP would probably be insoluble in water. Note that DBP and other phthalates have other properties, including anti-androgenic, that may be more adverse than their oestrogenicity (Sohoni and Sumpter, 1998
; Gray et al., 1999
; Ashby and Lefevre, 2000
). The affinity to the hormone-binding proteins in serum, including sex hormone binding protein (SHBG), albumin and
-fetoprotein strongly affect the in-vivo oestrogenicity of a compound. It is estimated that about 98% of the endogenous 17ß-oestradiol is bound to the binding proteins, especially SHBG, resulting in only a small percentage being available to the cells (Ben-Rafael et al., 1986
). Thus, since most exogenous hormone-like chemicals, including Zeranol and the other synthetic growth promoting hormones, exhibit limited or no binding to carrier proteins (Mastri et al., 1985
; Shrimanker et al., 1985
; Nagel et al., 1998
), their potential potency is much larger than their actual concentrations suggest (up to 50 times).
Zeranol constitutes a special case among potential endocrine disrupters, because Zeranol, in contrast to all other oestrogenic `endocrine disrupting' chemicals, is present in human food because it is deliberately used in the production of consumer products. Furthermore, Zeranol is designed to be a potent, fairly persistent, oestrogen whereas the oestrogenic properties of the chemicals that are considered potential endocrine disrupters is accidental. Although Zeranol is a synthetic derivative of the myco-oestrogen zearalanone, it is sometimes called a `nature-identical' oestrogen (Linsay, 1985). There are, however, six major forms of oestrogenic beta resorcylic acid lactones (Figure 1), and each can be metabolized into all the other forms, although with very different efficiencies (Thouvenot et al., 1981
; Migdalof et al., 1983
). To estimate the total oestrogenicity of these compounds it is therefore important to know both the amount that is formed of each isoform and the oestrogenic potency of each of the six compounds. Analyses of American beef products have shown that Zeranol and some of its metabolites can be detected in the finished consumer products (Stephany and André, 2000).
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Note that results of oestrogen-dependent gene-regulation is highly cell type specific; results from one cell type (here MCF7) in most cases cannot be transferred to other cell types or to animals or humans (Kledal et al., 2000).
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Materials and methods |
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Test chemicals
The test compounds were: Zeranol (-zearalanol) (Sigma, Z-0292, St Louis, MO, USA); 17ß-oestradiol (Sigma, E-2758); diethylstilboestrol (DES) (Sigma, D-4628); Bisphenol-A (Sigma, I-0635); and genistein (Sigma, G-6649). Samples treated with 100 nmol/l ICI 182.780 (ICI) (Zeneca Pharmaceuticals, London, UK) were included as an anti-oestrogen control in all experiments.
-zearalanol and its metabolites (
-zearalenol, ß-zearalanol, ß-zearalenol, zearalenone and zearalanone) were obtained both from Sigma (Z0292, Z0166, Z0417, Z2000, Z2125, Z0167 respectively) and from The European Reference Laboratory (Laboratory for Residue Analysis, NL 3720 BA Bilthoven, The Netherlands).
The tested concentrations depended on the potencies of the compounds, which were determined in pilot experiments. For 17ß-oestradiol, DES and Zeranol the range was from 1015 mol/l (1 fmol/l) to 1011 mol/l (10 pmol/l), for neutral and cationic amino acid transporter B(0+) (ATB0+) 1010 mol/l (100 pmol/l) Zeranol was also included. For genistein the range was from 1010 mol/l (0.1 nmol/l) to 105 mol/l (10 µmol/l) and for Bisphenol-A the range was from 109 mol/l (1 nmol/l) to 105 mol/l (10 µmol/l).
Isolation of RNA, cDNA synthesis, competitive polymerase chain reaction and amplification of cDNA fragments from differential display gels
Cells were harvested and the RNA was extracted as described by Jørgensen et al. (2000). Total RNA was dissolved in diethylpyrocarbonate-treated H2O at a concentration of 15 µg/µl and stored at 80°C. cDNA synthesis, competitive polymerase chain reaction (PCR) and amplification of cDNA fragments from differential display gels were made as described previously (Jørgensen et al., 1999, 2000
). Sequencing reactions were performed as cycle sequencing using the ThermoSequenase enzyme (Amersham Pharmacia Biotech, Uppsala, Sweden). All sequencing and DDRTPCR gel electrophoresis were run on ALFexpress sequenators (Amersham Pharmacia Biotech). Detailed step-by-step manuals for all procedures related to DDRT-PCR can be obtained from our web site: www.biobase.dk/~ddbase/DD-Manuals.html
Quantification of band intensities and data analysis
DDRTPCR gels were analysed on a STORM 820 phosphor imager (Amersham Pharmacia Biotech). Exposure time was adjusted to the level of radioactivity on the gels (624 h). Lane-to-lane variation in intensity was normalized according to either the background above or below the quantified band or a constant band. We used the following formula for normalization:
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If the background was used for normalization, BG replaces CB. The choice of normalization parameter (background or constant band) did not affect the results. All values were then calculated as fold induction or reduction compared with the average of the four (or two) samples from cells treated with 100 nmol/l of the anti-oestrogen ICI 182.780. The average and the standard deviation were then calculated and all data points that are derived from four independent samples (due to occasional bad lanes a few of the results are only based on three samples) are represented as average ± standard deviation. For results obtained from analysis of duplicate samples (Zeranol metabolites), the values represent the average of the two samples.
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Results |
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Induction of PS2 and ATB0+
Induction of the PS2 gene has been used extensively as a marker for oestrogen exposure (Jakowlew et al., 1984; Soto et al., 1995
; Zava et al., 1997
; Jørgensen et al., 2000
). The PS2 mRNA was displayed with a primer combination that was designed to display this mRNA (Table I
; Jørgensen et al., 2000
). A significant induction could be detected at a concentration of about 1 pmol/l of 17ß-oestradiol, DES and Zeranol, whereas almost 10 nmol/l genistein and 100 nmol/l Bisphenol-A were required for a similar induction (Figure 3A
; Table II
). A slight induction of PS2 could, in some experiments, be detected at 0.1 pmol/l of 17ß-oestradiol, DES and Zeranol (not shown). The PS2 mRNA can be induced almost 200-fold by high concentrations of many oestrogens and the apparently smaller induction with the high-potency oestrogens, as compared with genistein (Figure 3A
), was caused by the chosen concentrations; higher concentrations of 17ß-oestradiol, DES and Zeranol (and genistein and Bisphenol-A) led to higher induction. The maximum induction of the PS2 mRNA by different oestrogens varied, but high concentrations of the three high-potency oestrogens always led to larger induction of PS2 than high concentrations of medium- and low-potency oestrogens (such as genistein, Bisphenol-A and DBP) (Jørgensen et al., 2000
; results not shown). `Fold induction' is very sensitive to differences in the level in ICI 182.780 treated cells, which always is low, but a little variable, leading to different `fold induction' despite essentially similar expression levels, in treated samples.
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Oestrogenicity determined from monoamine oxidase A and TGFß3
To further elucidate differences in potency on different genes, we analysed the expression of two other oestrogen-regulated genes, monoamine oxidase A and TGFß3. Both showed minor differences compared to PS2 and ATB0+ (Table II). For monoamine oxidase A the potency of 17ß-oestradiol was greater than that of DES, and Zeranol was slightly less oestrogenic than DES. For TGFß3, the potency of 17ß-oestradiol, DES and Zeranol was essentially identical. The expression of TGFß3 was less sensitive to genistein and Bisphenol-A than the other four genes; a down-regulation could be detected with 0.010.1 µmol/l genistein and 0.11 µmol/l Bisphenol-A 1.
GST mu3 and MRG1/p35srj are extremely sensitive to oestrogens
A few genes responded to very low concentrations of the high potency oestrogens. One encoded glutathione S-tranferase (GST) mu3, a member of the phase-two detoxification enzymes that protect the cell, and especially the DNA, against damage from free oxygen radicals (Hayes and McLellan, 1999). GST mu3 was down-regulated by all the oestrogens we have tested and was especially sensitive to the three high potency compounds (Figure 4A
; Table II
). GST mu3 was sometimes significantly down-regulated by ICI 182.780, and the level in ICI 182.780 treated samples was always lower that in the ethanol treated sample (Figure 4A
). Down-regulation could be detected already at 110 fmol/l concentrations of 17ß-oestradiol whereas a concentration of about 1 pmol/l of Zeranol and almost 10 pmol/l DES was required for a similar down-regulation (Figure 4A
). For medium and low potency oestrogens the results were less consistent, with relatively large differences between results obtained with GST mu3 and other genes (not shown). Furthermore, almost all other oestrogen regulated genes we have assayed responded to oestrogens within 28 h (M.Jørgensen et al., unpublished results) whereas a down-regulation of GST mu3 required about 16 h of treatment.
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Oestrogenicity of Zeranol metabolites
To investigate the oestrogenic potencies of Zeranol metabolites, MCF7 cells were exposed to increasing concentrations of six different compounds: -zearalanol,
-zearalenol, ß-zearalanol, ß-zearalenol, zearalenone, and zearalanone (Figure 1
). All samples were prepared in duplicates, using compounds from both Sigma and from The European Reference Laboratory. The potencies of compounds from Sigma and from The European Reference Laboratory were identical. The expression levels of the six genes described above were subsequently analysed (Table III
) and the results from analysis of ATB0+ are shown in Figure 5
.
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Discussion |
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Different response from different genes
The sensitivity of the different genes to the three high-potency oestrogens varied. For most, a response could be detected at a concentration of about 1 pmol/l. Almost all the regulation occurred within the physiological range of 17ß-oestradiol (<1 pmol/l2 nmol/l), i.e. expression was strongly affected by small changes in concentration within this range, whereas expression was essentially unaffected at concentrations below, and reached a plateau at concentrations above, the physiological range. This was especially the case for PS2, monoamine oxidase A and TGFß3, whereas ATB0+ was regulated within a more narrow concentration range. For all the tested oestrogens, there were only 12 orders of magnitude between the lowest concentration that affected the expression of ATB0+ to the concentration where it reached its highest effect (Figure 3B). ATB0+ is involved in regulating proliferation of hepatoma cells (Bode et al., 1998
) and its up-regulation by oestrogens could suggest that it exerts the same function in MCF7 breast cancer cells.
Highly sensitive genes
GST mu3 and MRG1/p35srj were more sensitive to low concentrations of 17ß-oestradiol than any of the other genes we have assayed; a down-regulation could be detected at 10 fmol/l 17ß-oestradiol, which corresponds to only about 500 molecules per cell, provided all the available 17ß-oestradiol is taken up by the cells. There are other reports that show that MCF7 cells respond to a few 17ß-oestradiol molecules since femto molar concentrations can induce maximal proliferation of hypersensitized MCF7 cells (Masamura et al., 1995).
GST mu3 is the only oestrogen-regulated gene we have detected that seems to show an oestrogen-like response to ICI 182.780, leading to a slight down-regulation by ICI 182.780. Together with the late response (after 16 h treatment) and the occasional lack of a consistent dose-response relationship (Figure 4A), this may suggest that the down-regulation of GST mu3 is not a direct result of activation of the oestrogen receptor but rather a down-stream event. This could, for example, be through an initial down-regulation of a transcription factor that then leads to a down-regulation of GST mu3. Alternatively, GST mu3 may be sensitive to the redox status in the cells, and the high sensitivity to 17ß-oestradiol may be caused by changes in redox status rather than through a direct regulation by the oestrogen receptor. GST mu3 was significantly less sensitive to DES and Zeranol, which further suggests that its regulation may not reflect a response, mediated directly by oestrogens. Several other members of the phase-two detoxification enzymes, including another member of the GST mu family, were also down-regulated by oestrogens (unpublished results). The down-regulation of phase-two enzymes may have severe consequences for the cells, since their function is to protect the cells against DNA damage by free oxygen radicals (Prestera and Talalay, 1995
; Hayes and McLellan, 1999
). Furthermore, reactive catecholoestrogen metabolites of 17ß-oestradiol are produced by phase-one detoxification enzymes (Liehr, 2000
), including cytochrome p4501b1 which is rapidly and strongly induced by ethanol, the most used vehicle for oestrogens (unpublished results). The combined effect of down-regulation of phase-two enzymes and up-regulation of p4501b1 would be an increase in the level of reactive 17ß-oestradiol metabolites and less protection against their DNA damaging activity. This should be taken into account when the genotoxic potential, and thus the carcinogenicity, of 17ß-oestradiol (Liehr, 2000
, review) is considered.
MRG1/p35srj is the most Zeranol-sensitive gene we have identified. A significant down-regulation could be detected in cells treated with 10 fmol/l Zeranol whereas MRG1/p35srj was much less sensitive to 17ß-oestradiol (Figure 4B). The regulation of MRG1/p35srj was similar to `standard' oestrogen regulated genes, with a rapid response (within 4 h), reverse response with the anti-oestrogen ICI 182.780 and consistent dose-dependent regulation by all the tested oestrogens (not shown). MRG1/p35srj is a p300/CBP binding protein and it is involved in regulation of the activity of p300/CBP (Bhattacharya et al., 1999
) and a down-regulation of MRG1/p35srj could have significant consequences for the cell, since most nuclear receptors require p300/CBP for transcriptional activation of target genes (McKenna et al., 1999
). Activation/repression of the activity of p300/CBP may thus affect the activity of both the oestrogen receptors and other nuclear receptors.
Potency of Zeranol metabolites
The most potent beta resorcylic acid lactones were Zeranol (-zearalanol) and
-zearalenol, which were about equally potent, although
-zearalenol was slightly more potent than Zeranol for some genes. The myco-oestrogen zearalenone was at least two orders of magnitude less potent than Zeranol and other metabolites were between 1 and 5 orders of magnitude less potent.
The myco-oestrogen zearalenone can be metabolically converted into both Zeranol and -zearalenol although most of the ingested zearalenone is not converted (Thouvenot et al., 1981
; Migdalof et al., 1983
). Thus, calling Zeranol a `nature identical' oestrogen (Linsay, 1985) does not seem to reflect the much higher potency of Zeranol. However, there are four beta resorcylic acid lactones with a fairly high potency (within 23 orders of magnitude of 17ß-oestradiol) (Zeranol,
-zearalenol, zearalanone and zearalenone; Table II
) and the combined level of at least these four compounds should be considered when assaying for residual levels of Zeranol and its metabolites in beef products.
Does Zeranol pose a threat to consumers?
Zeranol is a potent oestrogen and its effects in animals suggests that its tissue specificity is similar to 17ß-oestradiol and DES. This includes stimulation of proliferation of breast tissue (Sheffield and Welsch, 1985), induction of lesions in the testes (Veeramachaneni et al., 1988
; Pérez-Martínez et al., 1996
), and induction of hepatic neoplasia (Coe et al., 1992
). Because of its high potency, the Zeranol present in beef products may constitute the major exposure to exogenous oestrogens for consumers in the USA and other countries where it is used legally or illegally. The amount of Zeranol that is allowed in the USA is 2 µg/kg1 in muscle and 10 µg/kg1 in liver (FAO/WHO, 1995
), however, in the USA, concentrations of 150 µg/kg1 and 600 µg/kg1 in muscle and fat respectively are considered safe (Code of Federal Regulations, 1999
). This corresponds to concentrations of 465 nmol/l and 1.9 µmol/l respectively. However, the oral activity may be relatively low. In rodents it is about 150 times less than 17ß-oestradiol (Everett et al., 1987
), but it has never been investigated in humans or when ingested dissolved in meat and fat tissue. Furthermore, in contrast to the natural hormones, Zeranol does not bind the carrier proteins (Mastri et al., 1985
; Shrimanker et al., 1985
), and all may therefore be available to the cells. Therefore Zeranol could pose a risk to humans and especially to pre-pubertal children where the endogenous 17ß-oestradiol concentration is very low (Klein et al., 1994
). Currently there are no reports in the literature of actual measurements of serum concentrations of Zeranol in humans since the relatively low concentrations, until recently, could not be assayed. Furthermore, although we, in this paper, have not discussed the other synthetic growth-promoting hormones (trenobolone and melengoestrol acetate) or the use of natural hormones for growth promotion, the same concerns apply to the use of all the hormones.
In conclusion, because the synthetic and the natural hormones, used as anabolic growth promoters in meat production, are by far the most potent hormones found in human food, an assessment of their concentrations in serum from humans and especially children, before and after ingestion of treated products, must be given a very high priority. Before such values are available, it is not possible to estimate the potential threat the use of hormones for growth promotion pose to human health.
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Acknowledgements |
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Notes |
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References |
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Submitted on January 15, 2001;