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.

High growth rate of girls with precocious puberty exposed to estrogenic mycotoxins.

OBJECTIVE: To test the hypothesis that human puberty timing can be advanced by environmental estrogen exposure. STUDY DESIGN: We analyzed serum mycoestrogen contamination via high-performance liquid chromatography (HPLC) in 32 girls affected by central precocious puberty (CPP) and in 31 healthy female control subjects. All 32 patients received triptorelin (TR) for more than 12 months after diagnosis. RESULTS: Increased serum levels of zearalenone (ZEA; 933.7 +/- 200.3 pg/mL; 95% CI, 723.5-1143.9) and of its congener alpha-zearalenol (106.5 +/- 1.9 pg/mL; 95% CI, 104.5-108.5) contaminated 6 girls with CPP, who were from a bounded Tuscany area. At diagnosis, ZEA levels correlated with patient height (r = 0.906, P < .05) and weight (r = 0.887, P < .05), but not with bone age. In patients who were mycotoxin-positive, height (F = 4.192; P < .01), weight (F = 3.915; P < .01), and height velocity (F = 2.777, P < .05) were higher than patients who were mycotoxin-negative during 12-months TR treatment. Height correlated with weight both in patients who were mycotoxin-positive (r = 0.986, P < .001) and in patients who were mycotoxin-negative (r = 0.994, P < .001). Body mass index, bone age, and gonadal secretion was not different in patient groups before and during TR treatment (P > .05). CONCLUSIONS: Mycoestrogenic zearalenone is suspected to be a triggering factor for CPP development in girls. Because of its chemical resemblance to some anabolic agents used in animal breeding, ZEA may also represent a growth promoter in exposed patients.

Level of zearalenone in blood serum and lesions in ovarian follicles of sexually immature gilts in the course of zearalenone micotoxicosis.

The aim of the study was to determine how a low dose of zearalenone applied orally for eight days influences the level of zearalenone (ZEN) and alpha-zearalenole in blood plasma and causes the occurrence of histopathological changes in the cells of the ovarian follicles in sexually immature gilts. The animals were divided into 2 groups (control, C; n = 4 and experimental, E; n = 4). The gilts from group E were treated daily with zearalenone at a dose of 200 microg/kg b.w. The level of zearalenone and alpha-zearalenole (ZON as the sum of the levels of both zearalenone and alpha-zearalenole) was measured daily. On day eight of the experiment the animals were sacrificed and their ovaries were taken for histopathological examination. The tissue sections obtained were HE- and PAS-stained according to McManus. The presence of PCNA antigen was also estimated. The highest concentration of ZON was noted on day 5 in group E (8.16 +/- 2.49 ng/ml). External estrus symptoms without standing reflex were observed in group E on day 4. In group C there were no pathological changes in the ovaries. In group E, a few ovarian follicles were found, but they were located in the cortical layer. They were filled with a liquid substance rich in protein and without the granulosa layer. There was disintegration with apoptotic-like changes of the PCNA-negative cells in the granulosa layer of single mature follicles. On day 4 the dose of zearalenone caused disturbances in the process of development and maturation of some of the best developed ovarian follicles. This probably occurred through the activation of on apoptosis-like process of the granulosa cells with simultaneous manifestation of estrus without standing reflex.

Kinetics and metabolism of zearalenone in young female pigs.

The fate of a single bolus of the Fusarium mycotoxin zearalenone (ZON) given intravenously to pigs was followed up. Pigs were equipped with duodenal re-entrant cannulas, post-valvular T-shape cannulas and with a urinary bladder balloon catheter. The animals were divided into three groups. Pigs of the control group were injected with ZON (Co), and pigs of the second group were also injected with ZON but their duodenal digesta was quantitatively exchanged for 12 h with corresponding pigs of the third group, not injected with ZON. Therefore, the second group had a disrupted entero-hepatic cycling of ZON (DEHC) and the third one had an induced entero-hepatic cycling of ZON (IEHC). The kinetic profile of ZON and its metabolites in plasma and their flow with urine, duodenal and ileal digesta and with faeces was examined over the next 72 h after the bolus was given. Eleven days later, pigs were slaughtered for collection of bile, urine and liver to analyse ZON residues. In all specimens examined, alpha-zearalenol (ZOL) was detected as the only metabolite of ZON. Kinetic evaluation of the plasma data revealed that the terminal elimination half-life of ZON was reduced from 2.63 h in pigs of Co-group to 1.1 h when EHC of ZON was disrupted for 12 h (DEHC-group). The maximum ZON concentration in plasma of pigs with the IEHC was found at 2.73 h after the bolus was given to their counterparts. The percentage of the alpha-ZOL- and ZON-area under the curves (AUC) estimated for the IEHC-group amounted to approximately 18% of the corresponding AUC of the Co-group which would suggest that a substantial proportion of both substances are re-cycled via entero-hepatic re-circulation. Cumulative recovery of ZON and alpha-ZOL, expressed as percentage of the ZON-bolus was characterized by a saturation kinetics in urine and duodenal digesta, and after 72 h, the respective values for Co-, DEH-, and IEHC-groups were 70%, 55% and 12%; and 35%, 22% and 11%. Faecal excretion started to increase steeply after 48 h and still continued to increase after 72 h when the cumulative excretion was 6%, 3% and 2% for Co-, DEHC- and IEHC-groups respectively. Fourteen days after the bolus injection, ZON and alpha-ZOL concentrations in bile, liver and urine were lower than the detection limits of the applied method. The results would suggest that within this period of time a massive single bolus of ZON is nearly completely eliminated from the body.

Influence of zearalenone on reproductive system cell proliferation in gilts.

Zearalenone (ZEA) is a macrocyclic lactone, estrogenic, diet-depending and fusaric micotoxin, which is produced on many kinds of cereals and feeds in the favourable conditions of humidity and temperature. The structure of ZEA is similar to the structure of estrogens and it enables binding to the estrogenic receptors. The stimulation of protein synthesis in the cells of the reproductive system, which causes intensification of cell proliferation, is one of the effects of ZEA actions. Oedema and vulva reddening are the clinical, external signs of ZEA intoxication in pigs. The aim of this study was to designate the degree of reproductive cell proliferation after low doses of ZEA were applied per os in sexually immature gilts with simultaneous monitoring of zearalenone and alpha-zearalenol levels in peripheral blood. The following were observed in the gilts examined fluctuations of zearalenone and alpha-zearalenol levels in blood, which were connected with entero-hepatic circulation and also numerous histopathological changes in ovarian follicle structure. These changes were present in the reproductive system of sexually immature gilts with a big contribution of PCNA-positive cells. The studies show that zearalenone application in sexually immature gilts caused ovarian follicle atresia and apoptoso-like changes in granule cells. Intensified cell proliferation, which was expressed with the growth of PCNA index, was observed in uterus and oviduct.

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.

Improved thin-layer chromatographic detection of diethylstilbestrol and zeranol in plasma and tissues isolated with alumina and ion-exchange membrane columns in tandem.

Clean-up procedures for the isolation of zeranol and diethylstilbestrol (DES) were modified to reduce the analysis time and to increase the efficiency of purification. Several dyes (Fast Blue BB, Fast Corinth V, Fast Blue RR, Fast Blue B, Fast Red Violet B and Fast Violet B) were evaluated, and their minimum detectabilities were determined. Conditions for non-instrumental, semi-quantitative thin-layer chromatography were optimized. Zeranol and DES in plasma and tissues were determined using modified procedures. Enzyme digestion brought about significant improvement in detectabilities of zeranol and DES in both fortified and incurred plasma, serum and tissues. Minimum detectabilities for zeranol and DES were 25 ppb in fortified plasma and tissues. The amount of incurred zeranol measured in the serum of an experimental cow was increased four times, i.e. from 50 to 200 ppb, after protease digestion. Glucuronidase digestion showed an eight-fold increase in detection of incurred zeranol levels in bovine liver eight times. These results suggest that digestion releases zeranol and DES from protein and glucuronide complexes, thereby allowing detection of low levels of zeranol and DES which may not be detectable without digestion. Further modification of the purification with an ion-exchange membrane reduced the analysis time by 25%, and the membranes were regenerated up to ten times without loss of activity, allowing an automated process. This method utilizes inexpensive equipment and avoids use of organic solvent, in this case diethyl ether.

Measurement of zeranol in plasma from three blood vessels in steers implanted with zeranol.

Zeranol (Z) is a widely used growth promotant; however, plasma Z profiles in cattle implanted with Z have not been characterized. This study was conducted to determine bovine plasma Z profiles. In Exp. 1, four steers (BW = 284.8 +/- 5.6 kg) were implanted with 108 mg of Z (Ralgro). To determine the effect of sampling site on plasma Z concentrations, blood was sampled by venipuncture from the maxillary vein ipsilateral (IMV) to the ear in which Z was implanted and from the ipsilateral (IJV) and contralateral (CJV) jugular veins of each steer. Samples were collected on d 1, 4, 6, 8, 11, and 13 after implantation and Z was assayed by RIA. There was an effect of sampling site (P < .01). The overall mean plasma Z concentrations and 95% confidence intervals (CI) for each vessel were 282 (CI: 172 to 463), 135 (CI: 85 to 215), and 67 (CI: 42 to 106) pg/mL for the IMV, IJV, and CJV, respectively. Plasma Z concentration was higher (P < .05) in IMV than in IJV and higher (P < .05) in IJV than in CJV. In Exp. 2, nine steers (BW = 316.7 +/- 10.0 kg) were implanted with 108 mg of Z and IMV blood was collected on d 0, 1, 3, 7, 10, 14, 17, 21, 28, 35, 42, 56, 63, 73, and 91 after implantation. Day affected plasma Z concentration (P < .01); plasma Z was elevated above preimplantation levels for 91 d (P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Profiling of free and conjugated [3H]zeranol metabolites in pig plasma.

Extraction and high-performance liquid chromatographic (HPLC) procedures are described that permit the complete analysis of free and conjugated zeranol metabolites in plasma from pigs implanted with [3H]zeranol. Free metabolites (9.0%) are extracted and then analysed by radio-HPLC on a reversed phase C18 column. They are distributed between three compounds that have been identified by gas chromatography-mass spectrometry as taleranol, zeranol and zeralanone. Direct radio-HPLC of the pre-extracted, deproteinated and Sep-Pak C18-purified plasma on a reversed-phase C18 column using tetrabutylammonium as an ion-pairing agent showed four main peaks: one corresponds to a weakly retained unidentified compound(s) (20%) and the other three were identified as the taleranol, zeranol and zeralanone glucuro conjugates. However, the total recovery is only about 25% owing to strong affinity of this polar material for the plasma proteins. Enzymatic deconjugation of the pre-extracted plasma followed by radio-HPLC analysis of the freed metabolites led to a good recovery of the radioactivity (81.8%) and allowed the quantitation of the different metabolites. These preliminary results indicate that zeranol is metabolized in the pig following pathways similar to those in other tested species.

Detection of zeranol in the blood of ram lambs by a radioimmunoassay technique.

Plasma separated from jugular blood samples from six weaned ram lambs was examined for zeranol after the lambs had been implanted, behind the left ear, with a 12 mg pellet. The results showed that the ipsilateral vein contained significantly more zeranol for up to 100 days after implantation; by 145 to 175 days the difference between the ipsi- and contralateral veins was no longer significant. Massage of the implant site did not increase the rate of release of zeranol from the residual mass.

Metabolism of high concentrations of dietary zearalenone by young male turkey poults.

Six male turkey poults (3 weeks of age) were fed a starter ration artificially contaminated with 800 mg zearalenone/kg for a 2-week period to examine zearalenone metabolism and residues in various tissues, excreta, and blood plasma. Zearalenone had no effect on either feed consumption or body weight gain. All the birds fed zearalenone frequently showed strutting behavior, displayed an increased size and coloration of caruncles and dewlaps, and had swollen vent tissue. None of these signs were seen among six control birds fed uncontaminated starter feed. Hormone analysis, however, revealed that the testosterone concentrations in blood plasma were the same in both controls and treated birds. Analysis after 14 days of feeding showed that most of the dietary zearalenone had been metabolized into alpha-zearalenol. Levels of zearalenone and alpha-zearalenol were: blood plasma 66 +/- 27 and 194 +/- 80 ng/ml, excreta 182 +/- 33 and 644 +/- 86 micrograms/g, lung 56 +/- 45 and 202 +/- 161 ng/g, heart 57 +/- 40 and 238 +/- 121 ng/g, kidney 122 +/- 25 and 477 +/- 53 ng/g, and liver 276 +/- 54 and 2715 +/- 590 ng/g, respectively. Only traces of beta-zearalenol could be detected in plasma, excreta, and the various tissues. The percentage alpha-zearalenol of total zearalenone plus alpha-zearalenol rose significantly in both blood plasma and excreta during the experimental period. Almost all zearalenone and alpha-zearalenol was found conjugated in blood plasma, and the conjugates consisted of both glucuronides and sulfate conjugates. Approximately 65% of all zearalenone and alpha-zearalenol in excreta was found to be conjugated.

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.

Transformation of zearalenone and zearalenol by rat erythrocytes.

The interconversion of zearalenone and zearalenol by rat erythrocytes in vitro has been investigated. The major metabolite obtained by incubating zearalenone with erythrocytes or whole blood from Sprague-Dawley rats was alpha-zearalenol. beta-Zearalenol was also formed but at levels several times lower than those of alpha-zearalenol. In the cell-free haemolysate NADPH was much more effective than NADH as a co-factor in the reduction of zearalenone. The maximal transformation of zearalenone to zearalenol by haemolysates occurred at pH 8.0. Both NAD+ and NADP+ were effective as co-factors in the oxidation of alpha-zearalenol to zearalenone. However, only NADP+ was effective as a co-factor in the oxidation of beta-zearalenol. Conversion of alpha-zearalenol and beta-zearalenol to the corresponding epimer was observed in both erythrocyte suspensions and in cell-free haemolysates. The significance of these findings to the metabolism of zearalenone in vivo is discussed.

Biotransformation of zeranol: disposition and metabolism in the female rat, rabbit, dog, monkey and man.

The disposition of [3H]zeranol has been studied in the female Wistar rat, New Zealand rabbit, beagle dog, rhesus monkey and man. The blood elimination half-life of total radioactivity in rabbit was 26 h, monkey 18 h and man 22 h. In all species studied the drug was absorbed, oxidized and/or conjugated, and was extensively excreted via the bile in all species except rabbit and man, in which urinary excretion predominated. Blood total radioactivity in man probably consisted entirely of conjugates of zeranol and/or its metabolites. Urinary metabolites in all species included conjugates (beta-glucuronides and/or sulphates) of zeranol and the major metabolite zearalanone. A more polar minor metabolite was isolated from human urine and was shown to be hydroxy-zeranol. Taleranol (7 beta-zearalanol, the lower-melting diastereoisomer), a probable metabolite of zeranol (7 alpha-zearalanol, the higher-melting diastereoisomer) in animals and in man, was shown to be a urinary metabolite in a female New Zealand white rabbit which had received [3H]zeranol (8 mg/kg per day) for seven days. A reverse isotope dilution method was developed for the quantification of both diastereoisomers of zearalanol, and also zearalanone, in urine.

Radioimmunoassay for zearalenone and zearalanol in human serum: production, properties, and use of porcine antibodies.

To produce antigens susceptible to raise antibodies for resorcylic acid lactones, the 6'-carboxymethyloxime derivatives of zearalenone and zearalanone were bound to bovine serum albumin. Pigs could be immunized by using these antigens, the best titer in antibodies being obtained with the zearalenone antigen. The porcine antibodies were specific for the resorcylic acid lactones of structural resemblance with zearalenone. This specificity made the antibodies usable for a radioimmunoassay of zearalenone and zearalanol, which may be found in human and animal sera. The range of the assay was between 0.25 and 10 ng. The limit of detection was 5 ppb (5 ng/ml) in human serum.

High performance liquid chromatographic method using fluorescence detention for quantitative analysis of zearalenone and alpha-zearalenol in blood plasma.

A sensitive, high performance liquid chromatographic method is described for quantitative determination of zearalenone and alpha-zearalenol in blood plasma. Blood plasma is extracted with 2-propanol in ether, the extract is evaporated to dryness, and the residue is dissolved in 0.18N NaOH. The aqueous phase is washed with chloroform, dichloromethane, and benzene, neutralized with 0.10M H3PO4, and extracted with benzene. The extract is evaporated, dissolved in methanol, and injected onto a reverse phase column containing LiChrosorb RP-8 under the following conditions: methanol-acetonitrile-water mobile phase, fluorescence detector, excitation wavelength 236 nm, and 418 nm cut-off emission filter. The limit of detectability (twice background) is 0.5 ng standard which is equivalent to 0.6 ng standard/mL blood plasma. Linear standard curves are observed over the range of 0-35 ng of injected zearalenone and alpha-zearalenol. The recoveries from blood plasma are 76-101% in the range of 1.5-6.0 ng standard/mL blood.