Serum derived from zeranol-implanted ACI rats promotes the growth of human breast cancer cells in vitro.

BACKGROUND: Zeranol (Z) is a non-steroidal anabolic growth promoter with potent estrogenic activity that is widely used as a growth promoter in the US beef industry. Consumption of beef derived from zeranol-implanted cattle may be a risk factor for breast cancer. MATERIALS AND METHODS: The effect of serum on the proliferation of human breast cancer MCF-7 cell line and primary cultured human breast epithelial cells (PCHBECs) was investigated. ACI rats were implanted with 12 mg zeranol pellet and the serum was harvested at day 110 after implantation. The effect of zeranol-serum on mRNA expression of cell cycle regulating gene (cyclin D1) and tumor suppressor genes (p53, and p21) was evaluated using real-time RT-PCR. RESULTS: The serum derived from ACI rats 110 days post-zeranol implantation significantly promoted the proliferation of MCF-7 cells and primary cultured human breast epithelial cells compared to control serum. Zeranol-serum up-regulated cyclin D1 and down-regulated p53 and p21 expression in PCHBECs compared with control serum. CONCLUSION: Bio-active zeranol metabolites contained in meat produced from cattle after zeranol implantation may be a risk factor for breast cancer.

Tissue-specific expression pattern of estrogen receptors (ER): quantification of ER alpha and ER beta mRNA with real-time RT-PCR.

We have examined the tissue-specific mRNA expression of ER alpha and ER beta in various bovine tissues using real-time RT-PCR. The goal of this study was to evaluate the deviating tissue sensitivities and the influence of the estrogenic active preparation RALGRO on the tissue-specific expression and regulation of both ER subtypes. RALGRO contains Zeranol (alpha-Zearalanol), a derivative of the mycotoxin Zearalenon, shows strong estrogenic and anabolic effects, and exhibits all symptoms of hyperestrogenism, in particular reproductive and developmental disorders. Eight heifers were treated over 8 weeks with multiple-dose implantations (0x, 1x, 3x, 10x) of Zeranol. Plasma Zeranol concentration, measured by enzyme immunoassay, of multiple treated heifers was elevated. To quantify ER alpha and ER beta transcripts also in low-abundant tissues, sensitive and reliable real-time RT-PCR quantification methods were developed and validated on the LightCycler. Expression results indicate the existence of both ER subtypes in all 15 investigated tissues. All tissues exhibited a specific ER alpha and ER beta expression pattern and regulation. With increasing Zeranol concentrations, a significant downregulation of ER alpha mRNA expression could be observed in jejunum (p<0.001) and kidney medulla (p<0.05). These data support the hypothesis that ER beta may have different biological functions than ER alpha, especially in kidney and jejunum.

Oestrogenic potencies of Zeranol, oestradiol, diethylstilboestrol, Bisphenol-A and genistein: implications for exposure assessment of potential endocrine disrupters.

We have compared the oestrogenic potency of the synthetic oestrogen Zeranol, used as a growth promoter in meat production, and five related compounds, with the potency of 17beta-oestradiol, diethylstilboestrol (DES), genistein, and Bisphenol-A. The potency was assayed by analysing differences in expression levels of endogenous oestrogen-regulated genes in human MCF7 cells, treated with different concentrations of the compounds. Zeranol, 17beta-oestradiol and DES were about equally potent, genistein was four to six orders of magnitude less potent than 17beta-oestradiol but an order of magnitude more potent than Bisphenol-A. There were gene specific differences, the PS2 and TGFbeta3 genes were about equally sensitive to Zeranol, 17beta-oestradiol and DES whereas a down-regulation of MRG1/p35srj could be detected at fmol/l concentrations of Zeranol whereas 17beta-oestradiol was several orders of magnitude less potent. GST mu3 was sensitive to fmol/l concentrations of 17beta-oestradiol but much less sensitive to Zeranol and DES. The very high potency of Zeranol compared with other potential endocrine disrupters suggests that Zeranol intake from beef products could have greater impact on consumers than the amounts of the known or suspected endocrine disrupters that have been found in food. Since little data is available in man, there is an urgent need for reliable measurements of the concentration of Zeranol in human serum after ingestion of meat products from treated animals.

Thin-layer chromatographic detection of zeranol and estradiol in fortified plasma and tissue extracts with Fast Corinth V.

In an attempt to improve sensitivity of thin-layer chromatographic (TLC) analysis and selectivity of visualizing agents for detection of estrogenic anabolic hormones, several dyes were screened for their chromogenic interactions with estrone, estradiol, diethylstilbestrol (DES), zeranol (zearalanol), zearalanone, and mycotoxins, zearalenone and zearalenol. Fast Corinth V salt was selected for its relatively high sensitivity. These anabolic compounds were separated by TLC and visualized with Corinth V and the results compared to iodine and starch visualization. Fortified bovine plasma and tissues (kidney, liver and muscle) and chicken muscles were analyzed after a clean-up procedure using solid-phase dual columns of alumina and anion-exchange resin. Iodine-starch clearly detected 4 ng of estradiol and DES while zeranol and zearalenone were detected at higher levels (10 ng). Fast Corinth V showed distinct spots with 2 ng of zeranol and 4 ng of zearalenone while faint spots were observed with estradiol and estrone standards. DES was not detectable at these levels. Less background interference was observed with Corinth V than with iodine-starch. The former confirmed spots detected by iodine-starch. This study suggests its selectivity for detection of zeranol and its metabolite, zearalanone, in the presence of steroidal compounds.

Plasma and urinary levels of zearalenone and alpha-zearalenol in a prepubertal gilt fed zearalenone.

One prepubertal gilt, fed 192 micrograms zearalenone/kg body weight/day for 4 days, showed plasma concentrations of alpha-zearalenol 3-4 times higher than of the parent compound during the treatment. Zearalenone and alpha-zearalenol could be traced in plasma until the 5th day and in urine until the 4th day of the posttreatment period. A maximum circulating amount of zearalenone plus alpha-zearalenol, 10.4 ng/ml plasma, was found on the 4th day of treatment followed by an urinary excretion of 305 ng/ml urine. All zearalenone and alpha-zearalenol in plasma and urine were bound to glucuronic acid. On the second day of treatment the animal showed oedema and reddening of the vulva which became more pronounced during the treatment. Hormone analysis, however, showed that the animal had no oestrus cycle during the 3 week experimental period.