FOXO3a/PI3K/Akt pathway participates in the ROS- induced apoptosis triggered by α-ZEL and ß-ZEL.

Zearalenone (ZEN), an estrogenic mycotoxin, is one of the most common food and feed contaminants. Also, its metabolites α-zearalenol (α-ZEL) and ß-zearalenol (ß-ZEL) are considered to induce oxidative stress, however its effect in prostate cells is not known yet. Our previous observations showed that forehead box transcription factor 3a (FOXO3a) expression is modified in hormone- sensitive cells in the response to mycotoxins, similar to the phosphoinositide 3-kinase (PI3K)/ protein kinase B (Akt) pathway. Thus, this study evaluated the direct molecular effect of α-ZEL and ß-ZEL in a dose of 30 µM in hormone-dependent human prostate cancer (PCa) cells with the focus of the involvement of FOXO3a and PI3K/Akt signaling pathway in that effect. We observed that both active metabolites of ZEN reduced cell viability, induced oxidative stress, cell cycle arrest and apoptosis in PCa cells. Furthermore, we observed that FOXO3a as well as PI3K/Akt signaling pathway participate in ZELs induced toxicity in PCa cells, indicating that this signaling pathway might be a regulator of mycotoxin-induced toxicity generally.

A mechanistic toxicology study to grasp the mechanics of zearalenone estrogenicity: Spotlighting aromatase and the effects of its genetic variability.

Zearalenone (ZEN) is a mycoestrogen produced by Fusarium fungi contaminating cereals and in grain-based products threatening human and animal health due to its endocrine disrupting effects. Germane to the mechanisms of action, ZEN may activate the estrogen receptors and inhibit the estrogens-producing enzyme aromatase (CYP19A1). Both show single nucleotide variants (SNVs) among humans associated with a diverse susceptibility of being activated or inhibited. These variations might modify the endocrine disrupting action of ZEN, requiring dedicated studies to improve its toxicological understanding. This work focused on human aromatase investigating via 3D molecular modelling whether some of the SNVs reported so far (n = 434) may affect the inhibitory potential of ZEN. It has been also calculated the inhibition capability of α-zearalenol, the most prominent and estrogenically potent phase I metabolite of ZEN, toward those aromatase variants with an expected diverse sensitivity of being inhibited by ZEN. The study: i) described SNVs likely associated with a different susceptibility to ZEN and α-zearalenol inhibition - like T310S that is likely more susceptible to inhibition, or D309G and S478F that are possibly inactive variants; ii) proofed the possible existence of inter-individual susceptibility to ZEN; iii) prioritized aromatase variants for future investigations toward a better comprehension of ZEN xenoestrogenicity at an individual level.

Precise Modeling of the Protective Effects of Quercetin against Mycotoxin via System Identification with Neural Networks.

Cell cytotoxicity assays, such as cell viability and lactate dehydrogenase (LDH) activity assays, play an important role in toxicological studies of pharmaceutical compounds. However, precise modeling for cytotoxicity studies is essential for successful drug discovery. The aim of our study was to develop a computational modeling that is capable of performing precise prediction, processing, and data representation of cell cytotoxicity. For this, we investigated protective effect of quercetin against various mycotoxins (MTXs), including citrinin (CTN), patulin (PAT), and zearalenol (ZEAR) in four different human cancer cell lines (HeLa, PC-3, Hep G2, and SK-N-MC) in vitro. In addition, the protective effect of quercetin (QCT) against various MTXs was verified via modeling of their nonlinear protective functions using artificial neural networks. The protective model of QCT is built precisely via learning of sparsely measured experimental data by the artificial neural networks (ANNs). The neuromodel revealed that QCT pretreatment at doses of 7.5 to 20 µg/mL significantly attenuated MTX-induced alteration of the cell viability and the LDH activity on HeLa, PC-3, Hep G2, and SK-N-MC cell lines. It has shown that the neuromodel can be used to predict the protective effect of QCT against MTX-induced cytotoxicity for the measurement of percentage (%) of inhibition, cell viability, and LDH activity of MTXs.

Estrogenic activity of zearalenone, α-zearalenol and ß-zearalenol assessed using the E-screen assay in MCF-7 cells.

Mycotoxins, including zearalenone (ZEA), can occur worldwide in cereals. They can enter the food chain and cause several health disorders. ZEA and its derivatives (α-zearalenol, α-ZOL and ß-zearalenol, ß-ZOL) have structural analogy to estrogen, thus they can bind to estrogen receptors (ERs). In order to characterize the estrogenic activity of ZEA, α-ZOL and ß-ZOL, the proliferation of ER-positive human breast cancer cells (MCF-7) exposed to these mycotoxins was measured. After exposure at levels ranging from 6.25 to 25 µM, cell proliferation was evaluated by using the E-Screen bioassay. In accordance with previous studies, our results show the estrogenic activity of ZEA, α-ZOL and ß-ZOL in MCF-7 cells. This effect is related to ZEA and its metabolites being flexible enough to bind to mammalian ERs. The relative proliferative effect (RPE) ranged from 10% to 91%. The α-ZOL induced the highest proliferative effect due to its higher affinity for the ERs compared to the other mycotoxins.

Effects of α-zearalenol on the metabolome of two breast cancer cell lines by 1H-NMR approach.

INTRODUCTION: Zearalenone (ZEN) is one of the most widely distributed toxins that contaminates many crops and foods. Its major metabolites are α-Zearalenol (α-zol) and ß-Zearalenol. Previous studies showed that ZEN and α-zol have estrogenic properties and are able to induce growth promoting effect in breast tissues. OBJECTIVIES: Considering that tumorigenesis is dependent on the reprogramming of cellular metabolism and that the evaluation of the cellular metabolome is useful to understand the metabolic changes that can occur during the cancer development and progression or after treatments, aim of our work is to study, for the first time, the effects of α-zol on the metabolomic profile of an estrogen positive breast cancer cell line, MCF-7, and of an estrogen negative breast cancer cell lines MDA-MB231. METHODS: Firstly, we tested the effects of α-zol on the cell viability after 24, 48 and 72 h of treatments with 10-10, 10-8 and 10-6 M concentrations on breast cancer MCF-7 and MDA-MB231 cell lines in comparison to human non-cancerous breast MCF10A cell line. Then, we evaluated cell cycle progression, levels of reactive oxygen species (ROS) and the metabolomic profiling by 1H-NMR approach on MCF-7 and MDA-MB231 before and after 72 h treatments. Principal component analysis was used to compare the obtained spectra. RESULTS: α-zol is resulted able to induce: (i) an increase of the cell viability on MCF-7 cells mainly after 72 h treatment, (ii) a slight decrease of the cell viability on MDA-MB231 cells, and (iii) an increase of cells in S phase of the cell cycle and of ROS only in MCF-7 cells. Moreover, the evaluation of metabolomics profile evidenced that after treatment with α-zol the levels of some metabolites increased in MCF-7 cells whereas decreased slightly in MDA-MB231 cells. CONCLUSIONS: Our results showed that α-zol was able to increase the protein biosynthesis as well as the lipid metabolism in MCF-7 cells, and, hence, to induce an estrogen positive breast cancer progression.

Zearalenone and alpha-zearalenol inhibit the synthesis and secretion of pig follicle stimulating hormone via the non-classical estrogen membrane receptor GPR30.

Zearalenone (ZEA) is one of the most popular endocrine-disrupting chemicals and is mainly produced by fungi of the genus Fusarium. The excessive intake of ZEA severely disrupts human and animal fertility by affecting the reproductive axis. However, most studies on the effects of ZEA and its metabolite α-zearalenol (α-ZOL) on reproductive systems have focused on gonads. Few studies have investigated the endocrine-disrupting effects of ZEA and α-ZOL on pituitary gonadotropins, including follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The present study was designed to investigate the effects of ZEA and α-ZOL on the synthesis and secretion of FSH and LH and related mechanisms in female pig pituitary. Our in vivo and in vitro results demonstrated that ZEA significantly inhibited the synthesis and secretion of FSH in the pig pituitary gland, but ZEA and α-ZOL had no effects on LH. Our study also showed that ZEA and α-ZOL decreased FSH synthesis and secretion through non-classical estrogen membrane receptor GPR30, which subsequently induced protein kinase cascades and the phosphorylation of PKC, ERK and p38MAPK signaling pathways in pig pituitary cells. Furthermore, our study showed that the LIM homeodomain transcription factor LHX3 was involved in the mechanisms of ZEA and α-ZOL actions on gonadotropes in the female pig pituitary. These findings elucidate the mechanisms behind the physiological alterations resulting from endocrine-disrupting chemicals and further show that the proposed key molecules of the α-ZOL signaling pathway could be potential pharmacological targets.

Cytotoxic effects of zearalenone and its metabolites and antioxidant cell defense in CHO-K1 cells.

Zearalenone (ZEA) and its metabolites (α-zearalenol; α-ZOL, ß-zearalenol; ß-ZOL) are secondary metabolites of Fusarium fungi that produce cell injury. The present study explores mycotoxin-induced cell damage and cellular protection mechanisms in CHO-K1 cells. Cytotoxicity has been determined by reactive oxygen species (ROS) production and DNA damage. ROS production was determined using the fluorescein assay and DNA strand breakage by comet assay. Intracellular protection systems were glutathione (GSH), glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD). The results demonstrated that all mycotoxins increased the ROS levels up to 5.3-fold the control levels in CHO-K1 cells. Zearalenone metabolites, but not ZEA, increased DNA damage 43% (α-ZOL) and 28% (ß-ZOL) compared to control cells. The GSH levels decreased from 18% to 36%. The GPx and SOD activities respectively increased from 26% to 62% and from 23% to 69% in CHO-K1 cells, whereas CAT activity decreased from 14% to 52%. In addition, intracellular ROS production was induced by ZEA and its metabolites. The endogenous antioxidant system components GSH, GPx and SOD were activated against ZEA and its metabolites. These antioxidant system components thus could contribute to decrease cell injury by ZEA and its metabolites.

Zeranol induces COX-2 expression through TRPC-3 activation in the placental cells JEG-3.

Transient Receptor Potential Channels (TRPs) are commonly expressed in the reproductive tissues in human. Many female reproductive processes have been associated with these TRPs. The mycotoxin zeranol or α-zearalanol is derived from fungi in the Fusarium family. Limited exposure to zeranol appears to be safe. In North America, farmers are using synthetic zeranol to promote growth in livestock. As the health risks of exposure to residual zeranol have not been determined, this practice is disallowed in the European Community. In the present study the cellular calcium levels were elevated in JEG-3 cells treated with zeranol at or above 10nM. Subsequent study indicated that expressions of TRP channels were induced. In response to the calcium flow, ERK, P38 and PKCß were activated and COX-2 expression was increased. Specific TRP inhibitors were employed to establish the connection between the ion channel activity and COX-2 expression, and TRPC-3 appeared to be the triggering mechanism. Since the involvement of COX-2 is implicated in placental development and parturition, exposure to this mycotoxin poses a potential threat to pregnant women.

Zeranol stimulates proliferation and aromatase activation in human breast preadipocytes.

Aromatase is a crucial enzyme for the biosynthesis of estrogens and is involved in the process of breast carcinogenesis. Concerns have been raised regarding the effects of environmental estrogens as potential regulators of aromatase expression in human breast cells. Zeranol is a non­steroidal agent with potent estrogenic activity, which is widely used as a growth promoter for cattle in certain countries. The present study hypothesized that aromatase expression and activity may be elevated by low dose zeranol exposure, providing a source of estrogens that may stimulate cell proliferation. In the present study, primary cultured human breast preadipocytes were used as an in vitro model. The effects of zeranol on cell proliferation were measured using the MTS assay, aromatase expression levels were determined by immunocytochemical staining and reverse transcription­polymerase chain reaction, and aromatase enzyme activity and estrogen production were analyzed using corresponding assay kits. The results demonstrated that low dose zeranol (2­50 nM) was able to significantly promote cell proliferation, aromatase mRNA expression, aromatase activity and estrogen production in primary cultured human breast preadipocytes, thus suggesting that zeranol may act as an aromatase activator. The findings of the present study suggest that zeranol promotes breast cancer cell growth by stimulating aromatase activation and increasing estrogen biosynthesis in adipose tissue.

The effects of α-zearalanol on the proliferation of bone-marrow-derived mesenchymal stem cells and their differentiation into osteoblasts.

The aim of this study was to explore the effects of α-zearalanol (α-ZAL) on the proliferation of mouse bone-marrow-derived mesenchymal stem cells (BMSCs) and their differentiation into osteoblasts. Six- to eight-week-old BALB/C mice were used either as recipients or as bone marrow donors. BMSCs were isolated and collected using a differential adhesion method, with use of 10 % fetal bovine serum and Iscove's modified Dulbecco's medium. After the third generation, the BMSCs were randomly placed into the following subgroups: a control group, an osteogenic medium (OM) group, a 17ß-estradiol group, an α-ZAL 10(-7) mol/L group, an α-ZAL 10(-6) mol/L group, and an α-ZAL 10(-5) mol/L group. Flow cytometry was used to identify the BMSCs collected from the bone marrow. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test was performed, and markers of the osteoblasts were measured in the different subgroups. In addition, expression of osteoprotegerin and expression of receptor activator of nuclear factor κB ligand were examined using Western blot. In contrast to the control and OM groups, BMSCs in the α-ZAL groups exhibited long fusiform shapes, and contact inhibition was observed when the cells were closely packed. After induction, the BMSCs grew well and exhibited triangular, star, polygonal, or irregular shapes. Clumps and multiple cells were evident. The trends of the proliferation and differentiation for the control, OM, 17ß-estradiol, and α-ZAL groups were similar. Compared with the control and OM groups, in the α-ZAL groups the expression levels of alkaline phosphatase, procollagen type I N-terminal propeptide, bone morphogenetic protein 2, and osteocalcin were significantly increased (p < 0.05). In addition, α-ZAL inhibited osteoclastogenesis by increasing the expression of osteoprotegerin and decreasing the expression of nuclear factor κB ligand. In conclusion, α-ZAL can increase the proliferation of BMSCs and their differentiation into osteoblasts and can effectively suppress osteoclastogenesis.

An alternative purification method for human serum paraoxonase 1 and its interactions with anabolic compounds.

In this study, an alternative purification method for human paraoxonase 1 (hPON1) enzyme was developed using two-step procedures, namely, ammonium sulfate precipitation and Sepharose-4B-L-tyrosine-3-aminophenantrene hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent M(W) of 43 kDa. The enzyme was purified 219-fold with a final specific activity of 4,408,400 U/mg and a yield of 10%. Furthermore, we examined the in vitro effects of some anabolic compounds, such as zeranol, 17 ß-estradiol, diethylstilbestrol, oxytocin, and trenbolone on the enzyme activity to understand the better inhibitory properties of these molecules. The five anabolic compounds dose dependently decreased the activity of hPON1 with inhibition constants in the millimolar-micromolar range. The results show that these compounds exhibit inhibitory effects on hPON1 at low concentrations with IC50 values ranging from 0.064 to 16.900 µM.

Activation of ER stress and apoptosis by α- and ß-zearalenol in HCT116 cells, protective role of Quercetin.

Zearalenone (ZEN) and its metabolites are found in many food products and are known to induce many toxic effects. The major ZEN metabolites are α-zearalenol (α-ZOL) and ß-zearalenol (ß-ZOL). The mechanisms by which they mediate their cytotoxic effects are not well known and seem to differ depending on the type of cells. We investigated the possible underlying mechanism in α-ZOL and ß-ZOL-induced toxicity in HCT116 cells. We showed that cell treatment with α-ZOL/ß-ZOL generated endoplasmic reticulum (ER) stress and activated the Unfolded Protein Response (UPR) as evidenced by XBP1 mRNA splicing and up-regulation of GADD34, GRP78, ATF4 and CHOP. Apoptosis was triggered by ZEN metabolites-induced ER stress, and executed through a mitochondria-dependent pathway, characterized by a loss of mitochondrial transmembrane potential (ΔΨm), a downstream generation of O2•(-) and caspase 3 activation. Cellular deficiency of the pro-apoptotic proteins Bax and Bak protected cells against α/ß-ZOL-induced toxicity. However, treatment with α-ZOL or ß-ZOL combined with Quercetin (QUER), a common dietary flavonoid with well-known antioxidant activity, significantly reduced damage induced by α and ß-ZOL in all tested markers. We concluded that QUER protects against the cellular toxicity of α and ß-ZOL.×.

The nonsteroidal mycoestrogen zearalenone and its five metabolites suppress LH secretion from the bovine anterior pituitary cells via the estradiol receptor GPR30 in vitro.

Picomolar concentrations of estradiol produce nongenomic suppression of GnRH-induced LH secretion from the anterior pituitary (AP) of cattle via G-protein-coupled receptor 30 (GPR30). Zearalenone (ZEN) is the nonsteroidal mycoestrogen produced by Fusarium fungi and has been detected in cereal grains, animal feed, and ruminant urine worldwide. Zearalenone has a prolonged blood half-life that results from enterohepatic cycling. There are five metabolites of ZEN: α-zearalanol (α-ZAL), ß-zearalanol (ß-ZAL), α-zearalenol (α-ZOL), ß-zearalenol (ß-ZOL), and zearalanone, which may persist for long periods in animals and humans after consumption of ZEN-contaminated feed. We recently reported that GPR30 bound with α-ZAL decreases cytoplasmic cAMP but not gene expression of LHα and LHß subunits, and GPR30 bound with α-ZAL suppresses GnRH-induced LH release in bovine AP cells in vitro. We tested the hypothesis that GPR30 bound with ZEN or one of the four previously untested metabolites suppresses GnRH-induced LH release from the bovine AP cells in vitro. Anterior pituitary cells were cultured for 3 days under steroid-free conditions and were then incubated with various concentrations (0.001-10 nM) of estradiol or one of the ZEN analogs for 5 minutes before GnRH stimulation. Gonadotropin-releasing hormone-stimulated LH secretion from AP cells was inhibited by all of the test concentrations of ZEN, 0.001 to 1 nM of α-ZAL, and 0.001 to 0.1 nM of the remaining four analogs. Pretreatment for 5 minutes with a GPR30-specific antagonist, G36, inhibited estradiol- and the ZEN analog-induced suppression of LH secretion from cultured AP cells. G36 alone had no significant effect on LH secretion. The estimated order of the nongenomic inhibiting effect was ZEN, α-ZAL, zearalanone, α-ZOL, ß-ZOL, and ß-ZAL, which is quite different from the reported order for their genomic effects. Therefore, ZEN and all of its metabolites suppress LH secretion from the bovine AP cells via GPR30 in vitro.

The non-steroidal mycoestrogen zeranol suppresses luteinizing hormone secretion from the anterior pituitary of cattle via the estradiol receptor GPR30 in a rapid, non-genomic manner.

Picomolar concentrations of estradiol produce rapid suppression of GnRH-induced luteinizing hormone (LH) secretion from the anterior pituitary (AP) of cattle via G-protein-coupled receptor 30 (GPR30). Zeranol is a strong estrogenic metabolite derived from zearalenone, a non-steroidal mycoestrogen produced by Fusarium that induces reproductive disorders in domestic animals. The hypothesis was tested that zeranol suppresses GnRH-induced LH release from the AP of cattle via GPR30 in a rapid, non-genomic manner. The AP cells (n=15) were cultured for 3 days in steroid-free conditions and then treated them with estradiol (0.001-10nM) or zeranol (0.001-100nM) for 5min before GnRH stimulation. Pre-treatment with 0.001-0.1nM estradiol suppressed GnRH-stimulated LH secretion. Pre-treatment with zeranol at concentrations of 0.001nM (P<0.01), 0.01nM (P<0.01), 0.1nM (P<0.05), and 1nM (P<0.05), but not at concentrations of 10 and 100nM, also inhibited GnRH-stimulated LH secretion from AP cells. Pre-treatment for 5min with a GPR30-specific antagonist, G36, inhibited estradiol or zeranol suppression of LH secretion from cultured AP cells. Cyclic AMP measurements and quantitative PCR analyses revealed that pre-treatment with small amounts of estradiol (P<0.05) or zeranol (P<0.01) decreased cAMP, but not gene expressions of the LHα, LHß, or FSHß subunits in the AP cells. Hence, zeranol may suppress luteinizing hormone secretion from the AP of cattle via GPR30 in a rapid, non-genomic manner.

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.

Effect of zeranol on expression of apoptotic and cell cycle proteins in murine placentae.

Mycotoxins are chemicals produced by fungus and many of them are toxic to humans. Zeranol is a mycotoxin used to promote growth in cattle in North America; yet such a practice draws concern about the residual compound in meat in European countries. In the present study, the toxicity of zeranol was tested in a mouse model for reproduction. Pregnant ICR mice were given p.o. daily doses of zeranol at 0, 1, 10, 100mg/kg for 4 days (from E13.5 to E16.5). Increased rates of fetal resorption at late gestation (E17.5) and preterm birth (

Three-dimensional dynamic culture of pre-osteoblasts seeded in HA-CS/Col/nHAP composite scaffolds and treated with α-ZAL.

Pre-osteoblast MC3T3-E1 cells were cultured in hyaluronic acid-modified chitosan/collagen/nano-hydroxyapatite (HA-CS/Col/nHAP) composite scaffolds and treated with phytoestrogen α-zearalanol (α-ZAL) to improve bone tissue formation for bone tissue engineering. Perfusion and dynamic strain were applied to three-dimensional (3D) cultured cells, which simulates mechanical microenvironment in bone tissue and solves mass transfer issues. The morphology of cell-scaffold constructs in vitro was then examined and markers of osteogenesis were assessed by immunohistochemistry staining and western blotting. The results showed that cells expanded their pseudopodia in an irregular manner and dispersed along the walls in 3D-dynamic culture. Osteogenic phenotype was increased or maintained by enhanced collagen I (COLI) levels, decreased osteopontin expression and having little effect on osteocalcin expression during the 12 days of in vitro culture. In response to α-ZAL, the cell-scaffold constructs showed inhibited cellular proliferation, enhanced the alkaline phosphatase (ALP) activity and increased ratio of osteoprotegerin to receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL). Application of perfusion and dynamic strain to cells-scaffold constructs treated with α-ZAL represents a promising approach in the studies of osteogenesis stimulation of bone tissue engineering.

Mycotoxin alpha-zearalenol impairs the quality of preimplantation porcine embryos.

Alpha-Zearalenol (α-ZEA) is one of derivatives from Zearalenone (ZEA) which impacts mammalian reproduction and development. Previous studies have shown that pigs are sensitive to the estradiol-like effects of α-ZEA. However, the effect of α-ZEA for the early embryonic development has not been fully studied. The objective of this study was to identify the direct toxicity of α-ZEA on porcine preimplantation embryonic development, embryo quality and expression of developmentally important genes. Presumptive zygotes were cultured in porcine zygote medium 3 (PZM-3) in the presence of α-ZEA (n=2,957) or 17ß-estradiol (E2) (n=1,333) dissolved in 0.1% Dimethyl Sulfoxide (DMSO) from 24 to 84 h post insemination followed by determination of apoptotic cell numbers and transcript levels of BAX, BCL2L1 and POU5F1 in blastocysts. Cleavage rates on day 2 were significantly decreased in 10, 30 and 60 µM α-ZEA groups; whereas blastocyst rates on day 6 were significantly decreased in the 30 and 60 µM of α-ZEA groups. Only the 100 µM E2 group significantly decreased cleavage and blastocyst rates. Total cell numbers (TCN) in blastocysts were significantly lower in the 10 µM α-ZEA group, but no differences in apoptotic cell rates were found. The expression levels of POU5F1 and BCL2L1 transcripts were similar; however, levels of BAX transcripts and the BAX/BCL2L1 ratio were increased in both α-ZEA groups. Since α-ZEA and E2 did not elicit similar effects, results suggest that α-ZEA might impact porcine preimplantation embryonic development through pathways other than estrogen receptor binding.

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.