Estrogenic in vitro evaluation of zearalenone and its phase I and II metabolites in combination with soy isoflavones.

Humans and animals are exposed to multiple substances in their food and feed that might have a negative health impact. Among these substances, the Fusarium mycoestrogen zearalenone (ZEN) and its metabolites α-zearalenol (α-ZEL) and α-zearalanol (α-ZAL) are known to possess endocrine disruptive properties. In a mixed diet or especially animal feed, these potential contaminants might be ingested together with naturally occurring phytoestrogens such as soy isoflavones. So far, risk assessment of potential endocrine disruptors is usually based on adverse effects of single compounds whereas studies investigating combinatorial effects are scarce. In the present study, we investigated the estrogenic potential of mycoestrogens and the isoflavones genistein (GEN), daidzein (DAI) and glycitein (GLY) as well as equol (EQ), the gut microbial metabolite of DAI, in vitro alone or in combination, using the alkaline phosphatase (ALP) assay in Ishikawa cells. In the case of mycoestrogens, the tested concentration range included 0.001 to 10 nM with multiplication steps of 10 in between, while for the isoflavones 1000 times higher concentrations were investigated. For the individual substances the following order of estrogenicity was obtained: α-ZEL > α-ZAL > ZEN > GEN > EQ > DAI > GLY. Most combinations of isoflavones with mycoestrogens enhanced the estrogenic response in the investigated concentrations. Especially lower concentrations of ZEN, α-ZEL and α-ZAL (0.001-0.01 nM) in combination with low concentrations of GEN, DAI and EQ (0.001-0.1 µM) strongly increased the estrogenic response compared to the single substances.

The mycoestrogen zeranol at high dosage antagonizes transient receptor potential channel activities in 3T3 L1 cells.

Recent findings have revealed that exposure to environmental contaminants may result in obesity and pose a health threat to the general public. As the activity of transient receptor potential channels (TRPs) plays a permissive role in adipogenesis, the interactions between TRPs and some food pollutants, i.e. bisphenol A, di (2-ethylhexyl) phthalate, zearalenone, and zeranol at 10 µM were investigated in the present study. TRP-V1,-V3, -C4 and -C6 are reported to be differentially expressed in the adipocyte differentiation, and immunoblotting was performed to quantify changes in these TRPs affected by the pollutants. Our result indicated that the mycoestrogen zeranol or α-zearalanol suppressed the expression of the V1 and C6 isoforms. Subsequently, confocal microscopy was used to measure the calcium inflow repressed by zeranol from 0.1 µM to 10 µM. Oil Red O staining was used to determine the differentiation of 3T3 L1 preadipocytes. Zeranol could suppress the expression of TRP-V1 and -C6 protein and inhibit the associated flow of calcium into the cytosol of 3T3 L1 cells. Its IC50 value for inhibiting calcium inflow stimulated by 40 µM capsaicin or 10 µM GSK1702934A was estimated to be around 6 µM. Reduced TRP-V1 or -C6 activity might result in promoting adipogenesis. In conclusion, this study demonstrated that zeranol could potentiate fat cell differentiation through antagonizing TRP-V1 and -C6 activities.

Coffee Silverskin and Spent Coffee Suitable as Neuroprotectors against Cell Death by Beauvericin and α-Zearalenol: Evaluating Strategies of Treatment.

Coffee silverskin and spent coffee have been evaluated in a neuroblastoma cell line (SH-SY5Y cells) against beauvericin (BEA) and α-zearalenol (α-ZEL)-induced cytotoxicity with different strategies of treatment. First, the direct treatment of mycotoxins and coffee by-products extracts in SH-SY5Y cells was assayed. IC50 values for α-ZEL were 20.8 and 14.0 µM for 48 h and 72 h, respectively and, for BEA only at 72 h, it was 2.5 µM. Afterwards, the pre-treatment with spent coffee obtained by boiling water increased cell viability for α-ZEL at 24 h and 48 h from 10% to 16% and from 25% to 30%, respectively; while with silverskin coffee, a decrease was observed. Opposite effects were observed for BEA where an increase for silverskin coffee was observed at 24 h and 48 h, from 14% to 23% and from 25% to 44%, respectively; however, a decrease below 50% was observed for spent coffee. Finally, the simultaneous treatment strategy for the highest concentration assayed in SH-SY5Y cells provided higher cytoprotection for α-ZEL (from 44% to 56% for 24 h and 48 h, respectively) than BEA (30% for 24 h and 48 h). Considering the high viability of coffee silverskin extracts for SH-SY5Y cells, there is a forthcoming promising use of these unexploited residues in the near future against mycotoxins effects.

Metabolism of Zearalenone in the Rumen of Dairy Cows with and without Application of a Zearalenone-Degrading Enzyme.

The mycotoxin zearalenone (ZEN) is a frequent contaminant of animal feed and is well known for its estrogenic effects in animals. Cattle are considered less sensitive to ZEN than pigs. However, ZEN has previously been shown to be converted to the highly estrogenic metabolite α-zearalenol (α-ZEL) in rumen fluid in vitro. Here, we investigate the metabolism of ZEN in the reticulorumen of dairy cows. To this end, rumen-fistulated non-lactating Holstein Friesian cows (n = 4) received a one-time oral dose of ZEN (5 mg ZEN in 500 g concentrate feed) and the concentrations of ZEN and ZEN metabolites were measured in free rumen liquid from three reticulorumen locations (reticulum, ventral sac and dorsal mat layer) during a 34-h period. In all three locations, α-ZEL was the predominant ZEN metabolite and ß-zearalenol (ß-ZEL) was detected in lower concentrations. ZEN, α-ZEL and ß-ZEL were eliminated from the ventral sac and reticulum within 34 h, yet low concentrations of ZEN and α-ZEL were still detected in the dorsal mat 34 h after ZEN administration. In a second step, we investigated the efficacy of the enzyme zearalenone hydrolase ZenA (EC 3.1.1.-, commercial name ZENzyme®, BIOMIN Holding GmbH, Getzersdorf, Austria) to degrade ZEN to the non-estrogenic metabolite hydrolyzed zearalenone (HZEN) in the reticulorumen in vitro and in vivo. ZenA showed a high ZEN-degrading activity in rumen fluid in vitro. When ZenA was added to ZEN-contaminated concentrate fed to rumen-fistulated cows (n = 4), concentrations of ZEN, α-ZEL and ß-ZEL were significantly reduced in all three reticulorumen compartments compared to administration of ZEN-contaminated concentrate without ZenA. Upon ZenA administration, degradation products HZEN and decarboxylated HZEN were detected in the reticulorumen. In conclusion, endogenous metabolization of ZEN in the reticulorumen increases its estrogenic potency due to the formation of α-ZEL. Our results suggest that application of zearalenone hydrolase ZenA as a feed additive may be a promising strategy to counteract estrogenic effects of ZEN in cattle.

Zearalenone and Metabolites in Livers of Turkey Poults and Broiler Chickens Fed with Diets Containing Fusariotoxins.

Zearalenone (ZEN) and metabolites were measured in livers of turkeys and broilers fed a control diet free of mycotoxins, a diet that contained 0.5 mg/kg ZEN (ZEN diet), and a diet that contained 0.5, 5, and 20 mg/kg of ZEN, fumonisins, and deoxynivalenol, respectively (ZENDONFB diet). The feed was individually distributed to male Grade Maker turkeys from the 55th to the 70th day of age and to male Ross chickens from the 1st to the 35th day of age, without any signs of toxicity. Together, the free and conjugated forms of ZEN, α- and ß-zearalenols (ZOLs), zearalanone (ZAN), and α- and ß-zearalanols (ZALs) were measured by UHPLC-MS/MS with [13C18]-ZEN as an internal standard and immunoaffinity clean-up of samples. ZAN and ZALs were not detected. ZEN and ZOLs were mainly found in their conjugated forms. α-ZOL was the most abundant and was found at a mean concentration of 2.23 and 1.56 ng/g in turkeys and chickens, respectively. Consuming the ZENDONFB diet significantly increased the level of total metabolites in the livers of chickens. Furthermore, this increase was more pronounced for the free forms of α-ZOL than for the conjugated forms. An investigation of the presence of ZEN and metabolites in muscle with the methods validated for the liver failed to reveal any traces of these contaminants in this tissue. These results suggest that concomitant dietary exposure to deoxynivalenol (DON) and fumonisins (FB) may alter the metabolism and persistence of ZEN and its metabolites in the liver.

A rapid method for the determination of mycotoxins in edible vegetable oils by ultra-high performance liquid chromatography-tandem mass spectrometry.

An analytical method based on a QuEChERS procedure (quick, easy, cheap, effective, rugged and safe) has been developed for the determination of mycotoxins (α-zearalenol and zearalenone, and aflatoxins B1, B2, G1 and G2) in edible oils. The analysis was performed by ultra-high performance liquid chromatography coupled to triple quadrupole analyser (UHPLC-QqQ-MS/MS). The method was fully validated and the quantification limit is 0.5 µg kg-1 for aflatoxins and 1 µg kg-1 for α-zearalenol and zearalenone. Suitable recoveries were obtained at low concentration levels (0.5-25 µg kg-1 for aflatoxins and 1-25 µg kg-1 for α-zearalenol and zearalenone), ranging from 80 to 120%. Intra and inter-day precision values were also evaluated and relative standard deviation was lower than 20%. The expanded uncertainty, U, was also evaluated ant it was below 32% at 25 µg kg-1. The validated method has been applied to monitor the presence of mycotoxins in 194 samples belonging to different types of edible oils (olive oil, sunflower oil, soy oil and corn oil). Zearalenone was detected in 25% of the analysed samples at concentrations up to 25.6 µg kg-1, and aflatoxin G1 and G2 in 3% and 14% of the samples at a maximum concentration of 1.9 and 6.8 µg kg-1 respectively.

The protective effect of zeranol in cerebral ischemia reperfusion via p-CREB overexpression.

AIMS: Cerebral ischemia reperfusion (I/R) is a neurovascular disease leading to cerebral damage. It was found that postmenopausal women are liable to more dangerous effects than men at same age in stroke. The objective of this study is to investigate the neuroprotective effect of zeranol against cerebral ischemia reperfusion in ovariectomized rats. MAIN METHODS: 36 female wistar rats divided in to 3 groups: sham group, I/R group (where I/R was induced 7 weeks after ovariectomy), zeranol group (0.5 mg/kg every 3 days for 5 weeks before I/R). Cerebral ischemia reperfusion (I/R) was performed by bilateral common carotid artery occlusion then de-ligated to restore blood flow. After 24 h of reperfusion, rats performed cylinder test to evaluate behavioral dysfunction followed by decapitation. Brain tissues were collected for biochemical measures such as oxidative stress marker malondialdehyde, antioxidant markers reduced glutathione, inflammatory markers (interleukin-1 beta, tumor necrosis factor alpha, and inducible nitric oxide synthase), matrix metalloproteinase-9, adenosine triphosphate, brain derived neurotrophic factor, glucose transporter-3, phosphorylated c-AMP response element binding protein and finally nissl staining for histopathological examination. KEY FINDINGS: The zeranol administered group showed a reversal of neuronal damage caused by ischemia evidenced by the decrease in MDA, IL-1ß, TNF-α, and MMP-9 levels, increase GSH, and ATP levels, decrease expression of iNOS in both regions cortex and hippocampus, increase protein level of p-CREB, GLUT-3 and BDNF, increase number of intact neuron cells in both regions and attenuated histological changes in both cortex and hippocampus regions. SIGNIFICANCE: Zeranol has neuroprotective potential against cerebral ischemia reperfusion in ovariectomized rats.

[Determination of zearalenone and α-zearalenol in vegetable oil and grain products by C_(18)-Al_2O_3 solid phase extraction column purification coupled with ultra-performance liquid chromatography tandem mass spectrometry].

OBJECTIVE: To develop a method for simultaneous determination of zearalenone( ZEN) and α-zearalenol( α-ZEL) in vegetable oil and grain products by solid phase extraction column purification coupled with ultra-performance liquid chromatography tandem mass spectrometry. METHODS: Firstly, ZEN and α-ZEL in grain products were extracted by hexane/ethyl acetate( 50 : 50, V/V), and then extracted as vegetable oil by acetonitrile-water solution( 90: 10, V/V), and purified by C_(18)-Al_2O_3 solid phase extraction column. ZEN and α-ZEL was separated by UPLC with acetonitrile-water gradient elution on C_(18) column( 2. 1 mm × 100 mm, 1. 6 µm), and qualified/quantified by mass spectrometry with ESI negative MRM mode with ~(13)C_(18)-zearalenone as internal standard. RESULTS: The linearity of ZEN and α-ZEL ranged from 1. 0-500 ng/mL. The limit of detection for ZEN and α-ZEL in vegetable oil and grain products was 0. 3 and 0. 2 µg/kg, respectively. The limit of quantification for ZEN and α-ZEL in vegetable oil and grain products was 1. 0 and 0. 5 µg/kg. The average recoveries of ZEN and α-ZEL for spiked samples of 1. 0-100 µg/kg were 93. 5%-108. 0% and 92. 0%-105. 0%. The relative standard deviations of ZEN and α-ZEL were 3. 2%-8. 5% and 4. 6%-7. 8%( n = 6). 55 samples sold in Hangzhou supermarkets were analyzed. ZEN was detected in all corn germ oil with median and maximum contents of 126. 2 and 453. 1 µg/kg. α-ZEL was detected in 50% corn germ oil with median and maximum contents of 2. 0 and 5. 0µg/kg. CONCLUSION: The method possesses several advantages including sensitivity, precision, good efficiency of purification, simplicity and economy, and it is applicable to the batch analysis of zearalenone and α-zearalenol in vegetable oil and grain products.

Phytoestrogen alpha-zearalanol attenuate endoplasmic reticulum stress to against cultured rat hippocampal neurons apoptotic death induced by amyloid beta25-35.

OBJECTIVE: Our previous studies demonstrated both phytoestrogen α-zearalanol (α-ZAL) and estrogen is effective decrease Alzheimer's disease (AD)-like apoptotic neuron death, but α-ZAL showed significantly less side-effect on breast and endometrial tissue compared to estrogen, it suggested that α-ZAL can be used as a potential substitute for estrogen. However, the molecular mechanism by which α-ZAL prevents neuron damage remains unclear. Growing evidence suggests that endoplasmic reticulum (ER) stress plays an important role in the process of cell apoptosis in AD; in addition, our published data indicated that α-ZAL possessed the potential ability to stabilize ER function. We therefore hypothesized that ER-stress mechanism maybe involved in the antiapoptotic effect of α-ZAL in this study. METHODS: Primary rat hippocampal neurons have been cultured and subsequently followed exposed to ß-peptide fragment 25-35(Aß25-35) with or without α-ZAL pre-treatment, and then western blot and flow cytometry techniques has been used to evaluate the intracellular calcium balance, ER stress and apoptotic cell death. RESULTS: The results showed that Aß25-35 treatment for 24h induced dramatic neuronal apoptosis, accompanied by an increase in calpain2 expression, a marker of intracellular calcium overload. On the other hand, ER stress sensitive hallmarks, glucose-regulated protein 78 (GRP78), double-stranded RNA-dependent protein kinase (PKR)-like ER-resident kinase (PERK) and C/EBP homologous protein-10 (CHOP10) expressions were up-regulated after Aß25-35 administration. Importantly, α-ZAL pre-treatment effectively attenuated above changes. CONCLUSION: These results demonstrated that α-ZAL protects cells against AD-like apoptosis and the effects at least partially by attenuating severely ER stress.

Phytoestrogen α-zearalanol exerts antiapoptotic effects in differentiated pc12 cells via oestrogen receptor α.

Our previous studies have demonstrated that phytoestrogen α-zearalanol (α-ZAL) possesses potential benefits in alleviating cell apoptotic death just like oestrogen. However, the underlying mechanism is not fully understood. This study was designed to test the hypothesis that the neuroprotective effect of α-ZAL is mediated by oestrogen receptor (ER) as α-ZAL owns the benzene ring structure may interact with ER. The present results showed a significant increase in apoptosis in differentiated PC12 cells after a 24-hr exposure to amyloid ß-peptide fragment 25-35 (Aß25-35 ), accompanied by decreasing of bcl-2 expression and increasing bax expression, whereas a pre-treatment with α-ZAL ameliorated these changes induced by Aß25-35 . In addition, the α-ZAL-mediated cytoprotection was abrogated by ERα antagonist but not by ERß antagonist. In summary, these data suggest that α-ZAL intervenes against Aß-induced apoptosis via intersecting bcl-2-bax apoptotic pathway in an ERα-sensitive manner.

Phytoestrogen α-zearalanol improves memory impairment and hippocampal neurogenesis in ovariectomized mice.

Estrogen is known to provide robust protection of memory in postmenopausal women, but the fact that estrogen may increase the incidence of uterine and breast tumors has undoubtedly limited the clinical use of estrogen. In the present study, the effect of α-zearalanol (α-ZAL), a plant-derived phytoestrogen with low side-effect on uterine and breast, on memory has been evaluated in ovariectomized (OVX) mice when using 17ß-estradiol (17ß-E2) as an estrogen positive control. Our findings demonstrated that OVX resulted in impaired spatial learning and memory and reduced numbers of newborn neurons in the dentate gyrus of the hippocampus, while 17ß-E2 or α-ZAL treatment significantly improved memory performance and restored hippocampal neurogenesis. We also found the reduction of brain derived neurotrophic factor (BDNF) and TrkB expression in OVX mice, which were ameliorated by 17ß-E2 or α-ZAL supplementation. These results indicated that α-ZAL may improve memory impairments induced by OVX and modulate the expression of BDNF-TrkB benefit to neurogenesis which may be involved in the memory protection from α-ZAL, in a manner similar to that of 17ß-E2. The present findings suggested that α-ZAL may be a plausible substitute of 17ß-E2 in improving memory in postmenopausal women.

Alleviation of plasma homocysteine level by phytoestrogen α-zearalanol might be related to the reduction of cystathionine ß-synthase nitration.

Hyperhomocysteinemia is strongly associated with cardiovascular diseases. Previous studies have shown that phytoestrogen α-zearalanol can protect cardiovascular system from hyperhomocysteinemia and ameliorate the level of plasma total homocysteine; however, the underlying mechanisms remain to be clarified. The aim of this research is to investigate the possible molecular mechanisms involved in ameliorating the level of plasma homocysteine by α-zearalanol. By the successfully established diet-induced hyperhomocysteinemia rat models, we found that, after α-zearalanol treatment, the activity of cystathionine ß-synthase, the key enzyme in homocysteine metabolism, was significantly elevated and level of nitrative stress in liver was significantly reduced. In correlation with this, results also showed a decreased nitration level of cystathionine ß-synthase in liver. Together data implied that alleviation of plasma homocysteine level by phytoestrogen α-zearalanol might be related to the reduction of cystathionine ß-synthase nitration.

Phytoestrogen α-zearalanol ameliorates memory impairment and neuronal DNA oxidation in ovariectomized mice.

OBJECTIVE: The aim of this study was to evaluate the effect of a novel phytoestrogen, α-Zearalanol, on Alzheimer's disease-related memory impairment and neuronal oxidation in ovariectomized mice. METHODS: Female C57/BL6 mice were ovariectomized or received sham operations and treatment with equivalent doses of 17ß-estradiol or α-Zearalanol for 8 weeks. Their spatial learning and memory were analyzed using the Morris water maze test. The antioxidant enzyme activities and reactive oxygen species generation, neuronal DNA oxidation, and MutT homolog 1 expression in the hippocampus were measured. RESULTS: Treatment with 17ß-estradiol or α-Zearalanol significantly improved spatial learning and memory performance in ovariectomized mice. In addition, 17ß-estradiol and α-Zearalanol attenuated the decrease in antioxidant enzyme activities and increased reactive oxygen species production in ovariectomized mice. The findings indicated a significant elevation in hippocampi neuronal DNA oxidation and reduction in MutT homolog 1 expression in estrogen-deficient mice, but supplementation with 17ß-estradiol or α-Zearalanol efficaciously ameliorated this situation. CONCLUSION: These results demonstrate that α-Zearalanol is potentially beneficial for improving memory impairments and neuronal oxidation damage in a manner similar to that of 17ß-estradiol. Therefore, the compound may be a potential therapeutic agent that can ameliorate neurodegenerative disorders related to estrogen deficiency.

Phytoestrogen -zearalanol ameliorates memory impairment and neuronal DNA oxidation in ovariectomized mice

OBJECTIVE: The aim of this study was to evaluate the effect of a novel phytoestrogen, α-Zearalanol, on Alzheimer's disease-related memory impairment and neuronal oxidation in ovariectomized mice. METHODS: Female C57/BL6 mice were ovariectomized or received sham operations and treatment with equivalent doses of 17β-estradiol or α-Zearalanol for 8 weeks. Their spatial learning and memory were analyzed using the Morris water maze test. The antioxidant enzyme activities and reactive oxygen species generation, neuronal DNA oxidation, and MutT homolog 1 expression in the hippocampus were measured. RESULTS: Treatment with 17β-estradiol or α-Zearalanol significantly improved spatial learning and memory performance in ovariectomized mice. In addition, 17β-estradiol and α-Zearalanol attenuated the decrease in antioxidant enzyme activities and increased reactive oxygen species production in ovariectomized mice. The findings indicated a significant elevation in hippocampi neuronal DNA oxidation and reduction in MutT homolog 1 expression in estrogen-deficient mice, but supplementation with 17β-estradiol or α-Zearalanol efficaciously ameliorated this situation. CONCLUSION: These results demonstrate that α-Zearalanol is potentially beneficial for improving memory impairments and neuronal oxidation damage in a manner similar to that of 17β-estradiol. Therefore, the compound may be a potential therapeutic agent that can ameliorate neurodegenerative disorders related to estrogen deficiency. .

Analysis of zearalenone and α-zearalenol in 100 foods and medicinal plants determined by HPLC-FLD and positive confirmation by LC-MS-MS.

BACKGROUND: Mycotoxins, which may contaminate many foods and medicinal plants, are poisonous to humans. A high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method was successfully developed for analysing the contamination levels of zearalenone (ZON) and its metabolite α-zearalenol (α-ZOL) in 100 widely consumed foods and medicinal plants in China. Samples were extracted with methanol-water (80:20, v/v), and cleaned up by using an immunoaffinity column. RESULTS: The limits of detection of this developed method for ZON and α-ZOL were 4 µg kg(-1) and 2.5 µg kg(-1) , respectively. Recoveries for the samples spiked with three levels (30, 60 and 300 µg kg(-1) for ZON and α-ZOL) ranged from 85.8% to 96.1% with relative standard deviation (RSD) of 2.6-7.1% for ZON, and from 89.9% to 98.7% with RSD of 1.9-9.2% for α-ZOL. Twelve (12%) of these tested samples were contaminated with ZON at levels ranging from 5.3 to 295.8 µg kg(-1). The most contaminated samples were Semen coicis, four of them in a concentration level exceeding 60 µg kg(-1) 'maximum level' (range 68.9-119.6 µg kg(-1)). Positive samples were further confirmed by liquid chromatography-tandem mass spectrometry. CONCLUSION: The results suggest that it is necessary to control ZON contamination in medicinal plants, especially Semen coicis. This is a successful study on the analysis of ZON and α-ZOL in medicinal plants in China by HPLC-FLD. Immunoaffinity clean-up and HPLC-FLD proved to have broad applicability in the field of simultaneously detecting ZON and α-ZOL in foods and medicinal plants and other complicated matrices.

Effect of growth promotants on the occurrence of endogenous and synthetic steroid hormones on feedlot soils and in runoff from beef cattle feeding operations.

Supplements and growth promotants containing steroid hormones are routinely administered to beef cattle to improve feeding efficiency, reduce behavioral problems, and enhance production. As a result, beef cattle manure will contain both synthetic steroids as well as a range of endogenous steroids including androgens, estrogens, and progestogens. A two-year controlled study was conducted in which beef cattle were administered steroid hormones via subcutaneous implants and feed additives and the occurrence of 16 endogenous and synthetic steroid hormones and metabolites was evaluated in runoff from beef cattle feedlots and in manure and soil collected from feedlot surfaces. Samples were extracted and analyzed using liquid chromatography tandem mass spectrometryfor metabolites of the synthetic androgen trenbolone acetate, 17α-trenbolone, 17ß-trenbolone, for the nonsteroidal semisynthetic estrogen agonist, α-zearalanol, and the synthetic progesterone melengesterol acetate, as well as a wide range of endogeneous estrogens, androgens, and fusarium metabolites. Synthetic steroids including trenbolone metabolites and melengestrol acetate were detected in fresh manure and in feedlot surface soils from cattle administered synthetic steroids at concentrations up to 55 ± 22 ng/g dry weight (dw) (17α-trenbolone) and 6.5 ± 0.4 ng/g dw (melengesterol acetate). Melengesterol acetate was detected in 6% of runoff samples from feedlots holding cattle administered synthetic steroids at concentrations ranging up to 115 ng/L. The presence of melengesterol acetate in runoff from beef cattle feeding operations has not been previously reported. Synthetic steroids were not detected in manure or runoff from control cattle. A wide range of endogenous hormones were detected in runoff and feedlot surface soils and manure from cattle given synthetic steroids and from control cattle, with no statistically significant differences in concentration. These results indicate that runoff from confined animal production facilities is of environmental and public health concern regardless of the use of growth promotants.

The role of α-zearalanol in reversing bone loss induced by ovarian hormone deficiency in rats.

To assess the ability of α-zearalanol (α-ZAL) to prevent bone loss in an ovariectomized (OVX) rat model of osteoporosis, α-ZAL was administered intragastrically to rats. After 35 days, the total body bone mineral density (BMD) was assessed in all rats. All sections were processed for immunohistochemistry and hematoxylin and eosin staining. One-way ANOVA and an LSD multiple-range test were used to determine the significant differences between groups. BMD was lower in the OVX and OVX + α-ZAL high-dose (OVX + High) groups compared to the sham-operated (Sham), OVX + 17ß-ethinylestradiol (OVX + E(2)), OVX + α-ZAL medium-dose (OVX + Medium) and OVX + α-ZAL low-dose (OVX + Low) groups (P < 0.05). Clear bone trabeculae arrangements were observed in the OVX + E(2,) OVX + Medium and OVX + Low groups. The expressions of bone morphogenetic proteins and basic fibroblast growth factor were up-regulated in the OVX + E(2), OVX + Medium and OVX + Low groups compared to the OVX and OVX + High groups (P < 0.05). The OVX + E(2), OVX + Medium and OVX + Low groups showed lower levels of bone Gla protein, bone alkaline phosphatase, tartrate-resistant acid phosphatase and tumor necrosis factor α expressions than the OVX and OVX + High groups (P < 0.05). The administration of α-ZAL to ovariectomized rats reverses bone loss and prevents osteoporosis.

ZEN and the art of breast health maintenance.

Zearalenone (ZEN) is a non-steroidal mycoestrogen that widely contaminates agricultural products. ZEN and its derivatives share similar molecular mechanisms and activity with estrogens and interact with ERα and ERß leading to changes in the reproductive system in both animals and humans. The reduced form of ZEN, α-ZEA ralenol, has been used as an anabolic agent for animals and also proposed as hormonal replacement therapy in postmenopausal women. Furthermore, both zearelanol ZEN and derivatives have been patented as oral contraceptives. ZEN has been widely used in the United States since 1969 to improve fattening rates in cattle by increasing growth rate and feed conversion efficiency. Evidence of human harm from this practice is provided by observations of central precocious puberty. As a result, this practice has been banned by the European Union. As ZEN has been associated with breast enlargement in humans, it has been included in many bust-enhancing dietary supplements but epidemiological evidence is lacking with regard to breast cancer risk. Extensive work with human breast cancer cell lines has shown estrogenic stimulation in those possessing ER but a reduction in DMBA-induced breast cancers in rodents given ZEN. Protein disulfide isomerase provides a molecular biomarker of dietary exposure to ZEN and its derivatives allowing the detection and control of harmful food intake. The interaction of ZEN with anti-estrogens, anticancer agents and antioxidants requires further investigation.

Changes in intramuscular fat, fatty acid profile and cholesterol content induced by zeranol implantation strategy in hair lambs.

BACKGROUND: The effect of zeranol implantation strategy on intramuscular fat, fatty acid profile and cholesterol content of the longissimus dorsi muscle of hair lambs was studied. Four treatments were tested: C, control group; Z12, 12 mg zeranol; Z24, 24 mg zeranol in a single application; and RZ12, 12 mg zeranol given twice. One-way analysis of variance was employed to estimate the effect of treatments (P < 0.05). To separate the effect of the mean, orthogonal contrasts were tested: C1, C versus Z12 + Z24 + RZ12; C2, Z12 versus Z24 + RZ12; and C3, Z24 versus RZ12. RESULTS: A decrease (P < 0.05) in intramuscular fat content was observed from implanting (C1 effect) and zeranol reimplantation (C3 effect). Implanted lambs exhibited an increase (P < 0.05) in monounsaturated fatty acids compared with control group (40.60% versus 35.35%). All contrasts were significant for the sum of n-6 and n-3, with values lower (P < 0.05) in the control (n-6: 0.84% and n-3: 1.38%) and higher in the RZ12 treatment (n-6: 7.55% and n-3: 14.9%). Cholesterol decreased by 78% with implantation and increasing the dose. CONCLUSION: The results indicate that it is possible to induce favorable changes in the fatty acid profile and cholesterol content using a zeranol implantation strategy on hair lambs.

The neuroprotective effects of phytoestrogen α-zearalanol on ß-amyloid-induced toxicity in differentiated PC-12 cells.

Although favorable effects of estrogen replacement therapy on Alzheimer's disease on postmenopausal women have been recognized, an associated increased incidence of uterine and breast tumors has jeopardized the clinical use of estrogen. Phytoestrogen α-zearalanol (α-ZAL) is a reductive product of the Gibberella zeae metabolite and abundant in plants and vegetables, which has been shown to protect cell injury with low side-effects on uterine and breast. This study was designed to evaluate the neuroprotective effects of α-ZAL, on the cultured differentiated PC-12 cells, while 17ß-estradiol (17ß-E2) has been used as an estrogen positive control. Following a 24 h exposure of the cells to amyloid ß-peptide fragment 25-35 (Aß25₋35), a significant reduction in cell survival and activities of total superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), as well as increased of malondialdehyde (MDA) were observed. However, preincubation of the cells with α-ZAL or 17ß-E2 prior to Aß25₋35 exposure elevated the cell survival and SOD and GSH-Px activities, and decreased the level of MDA. In addition, Aß25₋35 caused a significant cell apoptosis and increased apoptotic rate, accompanied by decreasing of bcl-2 expression and increasing bax, caspase-3 expression, pretreatment of the cells with α-ZAL or 17ß-E2 ameliorated these changes induced by Aß25₋35. Taken together, these data indicated that the phytoestrogen α-ZAL may effectively antagonize Aß25₋35-induced cell toxicity by attenuating oxidative stress and apoptotic cell death, in a manner similar to 17ß-E2. Our results suggested that α-ZAL can be used as a potential substitute of 17ß-E2 in postmenopausal women for Alzheimer's disease prevention.