Measurement of the relative binding affinity of zearalenone, α-zearalenol and β-zearalenol for uterine and oviduct estrogen receptors in swine,rats and chickens: An indicator of estrogenic potencies

1. The relative binding affinity of zearalenone, α-zearalenol and β-zearalenol for estrogen receptors was determined in the pig, rat and chicken.2. Similar relative binding patterns were observed, with a-zearalenol exhibiting greater affinity than zearalenone and β-zearalenol the least binding affinity in all species.3. The relative binding affinity of α-zearalenol was greater in pig, than in rat and significantly greater than in chicken.4. Interspecies differences in zearalenone sensitivity may be due to the binding affinity of α-zearalenol for estrogen receptors and differences in zearalenone metabolites formed.     

Production and analytical properties of antibodies with high specificity to zearalenone

The time course of production, specificity, and analytical potential of anti-zearalenone polyclonal rabbit antibody synthesized by formaldehyde condensation and conjugated to bovine serum albumin were investigated. The relative cross-reactivities with natural analogues were: zearalenone, 100%; alpha-zearalenol, 0.15%; and beta-zearalenol, < 0.02%. With synthetic analogues: zearalanone, 31.7% and alpha-zearalanol, 0.12%. An enzyme immunoassay for zearalenone with a sensitivity of 0.1 ng/ml was developed on the basis of these antibodies and solid-phase conjugates homologous to the immunogen in the method of synthesis.     

Indirect enzyme-linked immunosorbent assay for the mycotoxin zearalenone.

A competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of zearalenone, an estrogenic mycotoxin. Zearalenone was converted to zearalenone-6'-carboxymethyloxime and conjugated to bovine serum albumin and poly-L-lysine for use as immunogen and solid-phase marker, respectively. Immunization of rabbits with the bovine serum albumin conjugate resulted in zearalenone antibody titers of 20,480 in 11 weeks. A competitive indirect ELISA was conducted by simultaneously incubating zearalenone with zearalenone antiserum over zearalenone-6'-carboxymethyloxime poly-L-lysine solid phase and then determining the bound rabbit immunoglobulin with goat anti-rabbit peroxidase conjugate. Response range for zearalenone in the resulting competition curve was between 1 and 50 ng/ml. Reactivities of this antiserum for alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were, respectively, 50, 12, 6, and 3% of that found for zearalenone. By using the competitive indirect ELISA, zearalenone was detectable in methanol-water extracts of corn, wheat, and pig feed samples.     

Indirect enzyme-linked immunosorbent assay for the mycotoxin zearalenone

A competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of zearalenone, an estrogenic mycotoxin. Zearalenone was converted to zearalenone-6'-carboxymethyloxime and conjugated to bovine serum albumin and poly-L-lysine for use as immunogen and solid-phase marker, respectively. Immunization of rabbits with the bovine serum albumin conjugate resulted in zearalenone antibody titers of 20,480 in 11 weeks. A competitive indirect ELISA was conducted by simultaneously incubating zearalenone with zearalenone antiserum over zearalenone-6'-carboxymethyloxime poly-L-lysine solid phase and then determining the bound rabbit immunoglobulin with goat anti-rabbit peroxidase conjugate. Response range for zearalenone in the resulting competition curve was between 1 and 50 ng/ml. Reactivities of this antiserum for alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were, respectively, 50, 12, 6, and 3% of that found for zearalenone. By using the competitive indirect ELISA, zearalenone was detectable in methanol-water extracts of corn, wheat, and pig feed samples.     

Brain CCK receptors: species differences in regional distribution and selectivity

The binding of 125I-CCK-33 to its receptors prepared from cerebral cortex and cerebellum was studied in four species: mouse, rat, hamster, and guinea pig. Only the guinea pig showed significant binding to membranes from cerebellum and this binding was comparable to that observed for cerebral cortex. In all four species, the order of potency of unlabeled analogs to compete for the binding site was CCK-8 greater than CCK-33 greater than desulfated CCK-8 greater than CCK-4. While the affinity for CCK-8 and CCK-33 was similar in the various species, the relative affinity for desulfated CCK-8 and CCK-4 was less for hamster and guinea pig, indicating species differences in receptor specificity, as well as in regional localization.     

Differential estrogen receptor binding of estrogenic substances: a species comparison

The study investigated the ability of 34 natural and synthetic chemicals to compete with [3H]17beta-estradiol (E2) for binding to bacterially expressed glutathione-S-transferase (GST)-estrogen receptors (ER) fusion proteins from five different species. Fusion proteins consisted of the ER D, E and F domains of human alpha (GST-hERalphadef), mouse alpha (GST-mERalphadef), chicken (GST-cERdef), green anole (GST-aERdef) and rainbow trout ERs (GST-rtERdef). All five fusion proteins displayed high affinity for E2 with dissociation constants (K(d)) ranging from 0.3 to 0.9 nM. Although, the fusion proteins exhibited similar binding preferences and binding affinities for many of the chemicals, several differences were observed. For example, alpha-zearalenol bound with greater affinity to GST-rtERdef than E2, which was in contrast to other GST-ERdef fusion proteins examined. Coumestrol, genistein and naringenin bound with higher affinity to the GST-aERdef, than to the other GST-ERdef fusion proteins. Many of the industrial chemicals examined preferentially bound to GST-rtERdef. Bisphenol A, 4-t-octylphenol and o,p' DDT bound with approximately a ten-fold greater affinity to GST-rtERdef than to other GST-ERdefs. Methoxychlor, p,p'-DDT, o,p'-DDE, p,p'-DDE, alpha-endosulfan and dieldrin weakly bound to the ERs from the human, mouse, chicken and green anole. In contrast, these compounds completely displaced [3H]E2 from GST-rtERdef. These results demonstrate that ERs from different species exhibit differential ligand preferences and relative binding affinities for estrogenic compounds and that these differences may be due to the variability in the amino acid sequence within their respective ER ligand binding domains.     

Relation of 8-ketotrichothecene and zearalenone analog structure to inhibition of mitogen-induced human lymphocyte blastogenesis.

The 50% effective doses of fusarenon X, nivalenol, deoxynivalenol, and 15-acetyldeoxynivalenol required to reduce [3H]thymidine uptake in mitogen-stimulated human lymphocytes by 50% were 18, 72, 140, and 240 ng/ml, respectively. These results indicated that lymphotoxicity of 8-ketotrichothecenes decreased according to the C-4 substituent order acetyl greater than hydroxyl greater than hydrogen, whereas acetylation of position C-15 of deoxynivalenol caused a slight decrease in in vitro toxicity. The 50% effective doses for zearalenone, alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were 3,500, 6,300, 36,000, 3,750, and 33,000 ng/ml, respectively, suggesting that a keto group or alpha-hydroxyl at the position C-6' contributed to the lymphotoxicity of the parent molecule. The inhibitory effects of zearalenone analogs observed in the blastogenesis assay did not correlate with the estrogenic potencies of these compounds. All 8-ketotrichothecenes and zearalenone analogs tested were capable of inhibiting B- and T-cell subsets stimulated by a mitogen panel of leukoagglutinin, concanavalin A, and pokeweed mitogen.     

Species comparison of adenosine A1 receptors in isolated mammalian atrial and ventricular myocardium

Various species have been used as models to study the effects of adenosine (ADO) on atrial and ventricular myocardium, but few direct tissue comparisons between species have been made. This study further characterizes adenosine A(1) receptor binding, adenylate cyclase activity and direct and indirect A(1) receptor-mediated functional activity in atrial and ventricular tissue from Sprague-Dawley rats and Hartley guinea pigs. Rat right atria (RA) were found to be significantly more sensitive to cyclopentyladenosine (CPA), while guinea pig left atria (LA) were more sensitive to CPA. After the addition of isoproterenol (ISO), the reduction of CPA response in rat RA was significantly greater than in guinea pig; however, after ISO treatment, the guinea pig LA was more sensitive to CPA than the rat. Adenylate cyclase inhibition by CPA was significantly greater in atria and ventricles obtained from guinea pig than rat. In competition binding experiments, guinea pig RA had significantly more high affinity sites than rat, but the K(i)s were not significantly different. There were no significant differences between guinea pig LA and rat LA. Guinea pig ventricular tissue had fewer high affinity sites than rat without any differences in their K(i) values. In antagonist saturation experiments, the density and affinity of A(1) receptors in guinea pig cardiac membranes were significantly greater than in rat. Our results indicate definite species differences as well as tissue differences between rat and guinea pig. These differences must be considered when interpreting studies using rat and guinea pig tissue as models for cardiac function.     

Interaction of ring B unsaturated estrogens with estrogen receptors of human endometrium and rat uterus

The present investigation was undertaken to compare the binding affinities (Ka) of the ring B unsaturated equine estrogens (equilin [Eq], equilenin [Eqn], 17 beta-dihydroequilin [17 beta-Eq], 17 beta-dihydroequilenin [17 beta-Eqn], 17 alpha-dihydroequilin [17 alpha-Eq], and17 alpha-dihydroequilenin [17 alpha-Eqn]) and the classic estrogens (estrone [E1], 17 beta-estradiol [17 beta-E2], and 17 alpha-estradiol [17 alpha-E2]) for estrogen receptors in human endometrium and rat uterus. In both species, the ring B unsaturated estrogens bind with cytosol and nuclear receptors with high affinity (Ka x 10(9) M-1). The relative binding affinities of these estrogens were measured by determining the amount of unlabeled estrogen required to reduce by 50% the specific binding of [3H]17 beta-Eq to endometrial cytosol receptors. The order of activity found was 17 beta-Eq greater than 17 beta-E2 greater than 17 beta-Eqn greater than E1 greater than Eq greater than 17 alpha-Eq greater than 17 alpha-E2 greater than 17 alpha-Eqn greater than Eqn. Essentially the same order of activity was observed when the apparent affinity constants of these estrogens for human and rat cytosol and nuclear receptors were determined by a competitive (inhibition) binding assay. Sucrose density gradient analysis indicated that these estrogens form protein complexes with cytosol and nuclear preparation that sediment at approximately 8S and 4S, respectively. The affinity constants for 17 beta-Eq were approximately two- to six-fold higher than E2 in both species. In a rat uterotropic assay, all nine estrogens were uterotropic. These data indicate that all ring B unsaturated estrogens present in conjugated equine estrogen preparations are biologically active and they express their biologic effects in the human endometrium by mechanisms similar to those described for the classic estrogens.     

alpha-Zearalenol, a major hepatic metabolite in rats of zearalenone, an estrogenic mycotoxin of Fusarium species

The chemical and biological properties of the hepatic metabolite of zearalenone, an estrogenic and non-steroidal fungal toxin produced by Fusarium species, were investigated by employing TLC, GC/MS, high pressure liquid chromatography and fluorospectral analyses, as well as uterine weight bioassay in immature mice. All the chemical and physical data supported the view that the major metabolite, obtained by incubating zearalenone with S-9 and microsomes of rat liver in the presence of NADPH, is C-6'-alpha-hydroxylated zearalenone (alpha-zearalenol). The estrogenic activity of this metabolite was several times higher than that of the parent zearalenone, and the results of biological and toxicological evaluations of alpha-zearalenol are discussed.     

Effect of estrogens on follicular development and ovarian and uterine estrogen receptors in the immature rabbit, guinea pig and mouse

Immature rabbits, guinea pigs and mice were injected with estradiol cyclopentylpropionate (ECP) or diethylstilbestrol (DES) for 3 days to evaluate whether estrogen enhances follicular maturation. Also, estrogen receptors in the ovary and uterus from these animals were measured. Uterine weight increased in all animals treated with ECP or DES, whereas actual ovarian weight increased only in the guinea pig. This correlated with the ability of estrogens to significantly increase the number of antral follicles in the guinea pig ovary. In the rabbit and mouse, estrogen increased only the number of small or large preantral follicles. However, the number of estrogen binding sites in the ovarian cytosol and nucleus was greater in the rabbit and the mouse than in the guinea pig. The affinity of ovarian cytosol receptors was the lowest for the guinea pig among the 3 species. Thus it is seen that estrogen does not enhance follicular maturation in all animal species. The ovarian response to estrogen is not only dependent upon estrogen receptors but also unknown mechanism(s) that may be related to paracrine or autocrine functions.     

Electrophoretic characterization of purified bovine, porcine, murine, rat, and human uterine estrogen receptors

The calf uterine estrogen receptor (E2R) in the presence of sodium molybdate has been purified, 7,000-fold by a single passage over an estradiol affinity column. A dominant 70,000-dalton band and two minor bands at 50,000 and 30,000 daltons were observed by electrophoretic analysis. These bands had been eluted using estradiol, sodium sulfocyanate, CHAPS, and HEPES (pH 7.4) with insulin as a carrier protein. The identities of the protein bands were initially confirmed by their failure to bind the affinity column when saturated with estradiol. This single step purification procedure was reproducible and rapid, with yields of 10-20%, providing 25% purity. Diffusion blot analysis, with specific 35S- and 125I-labeled monoclonal antibodies to E2R, confirmed that the 70,000-dalton band represented the estrogen receptor. Specificity was demonstrated by inhibition of binding of purified E2R by both estradiol and diethylstilbestrol but not testosterone, progesterone, corticosterone, aldosterone, or hydrocortisone. The relative binding affinity of the purified receptor was: ethynyl estradiol greater than 17 beta estradiol greater than estriol greater than or equal to estrone greater than or equal to 17 alpha-estradiol greater than mestranol. Pig, human, mouse, and rat uterine estrogen receptors were similarly purified with the affinity column. As with the calf uterine preparations, a dominant 70,000-dalton band with minor bands at 50,000 and 30,000 daltons was identified by diffusion blot analysis in all the species examined.     

Studies of the human, rat, and guinea pig Y4 receptors using neuropeptide Y analogues and two distinct radioligands

The neuropeptide Y-family receptor Y4 differs extensively between human and rat in sequence, receptor binding, and anatomical distribution. We have investigated the differences in binding profile between the cloned human, rat, and guinea pig Y4 receptors using NPY analogues with single amino acid replacements or deletion of the central portion. The most striking result was the increase in affinity for the rat receptor, but not for human or guinea pig, when amino acid 34 was replaced with proline; [Ahx(8-20),Pro(34)]NPY bound to the rat Y4 receptor with 20-fold higher affinity than [Ahx(8-20)]NPY. Also, the rat Y4 tolerates alanine in position 34 since p[Ala(34)]NPY bound with similar affinity as pNPY while the affinity for hY4 and gpY4 decreased about 50-fold. Alanine substitutions in position 33, 35, and 36 as well as the large loop-deletion, [Ahx(5-24)]NPY, reduced the binding affinity to all three receptors more than 100-fold. NPY and PYY competed with (125)I-hPP at Y4 receptors expressed in CHO cells according to a two-site model. This was investigated for gpY4 by saturation with either radiolabeled hPP or pPYY. The number of high-affinity binding-sites for (125)I-pPYY was about 60% of the receptors recognized by (125)I-hPP. Porcine [Ala(34)]NPY and [Ahx(8-20)]NPY bound to rY4 (but not to hY4 or gpY4) according to a two-site model. These results suggest that different full agonists can distinguish between different active conformations of the gpY4 receptor and that Y4 may display functional differences in vivo between human, guinea pig, and rat.     

Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria

The effect of rumen microbes on six mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol ) considered to be health risks for domestic animals was investigated. The mycotoxins were incubated with intact rumen fluid or fractions of rumen protozoa and bacteria from sheep and cattle in the presence or absence of milled feed. Rumen fluid had no effect on aflatoxin B1 and deoxynivalenol . The remaining four mycotoxins were all metabolized, and protozoa were more active than bacteria. Metabolism of ochratoxin A, zearalenone, and diacetoxyscirpenol was moderately or slightly inhibited by addition of milled feed in vitro. The capacity of rumen fluid to degrade ochratoxin A decreased after feeding, but this activity was gradually restored by the next feeding time. Ochratoxin A was cleaved to ochratoxin alpha and phenylalanine; zearalenone was reduced to alpha-zearalenol and to a lesser degree to beta-zearalenol; diacetoxyscirpenol and T-2 toxin were deacetylated to monoacetoxyscirpenol and HT-2 toxin, respectively. Feeding of 5 ppm (5 mg/kg) of ochratoxin A to sheep revealed 14 ppb (14 ng/ml) of ochratoxin A and ochratoxin alpha in rumen fluid after 1 h, but neither was detected in the blood. Whether such conversions in the rumen fluid may be considered as a first line of defense against toxic compounds present in the diet is briefly discussed.     

Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria.

The effect of rumen microbes on six mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol ) considered to be health risks for domestic animals was investigated. The mycotoxins were incubated with intact rumen fluid or fractions of rumen protozoa and bacteria from sheep and cattle in the presence or absence of milled feed. Rumen fluid had no effect on aflatoxin B1 and deoxynivalenol . The remaining four mycotoxins were all metabolized, and protozoa were more active than bacteria. Metabolism of ochratoxin A, zearalenone, and diacetoxyscirpenol was moderately or slightly inhibited by addition of milled feed in vitro. The capacity of rumen fluid to degrade ochratoxin A decreased after feeding, but this activity was gradually restored by the next feeding time. Ochratoxin A was cleaved to ochratoxin alpha and phenylalanine; zearalenone was reduced to alpha-zearalenol and to a lesser degree to beta-zearalenol; diacetoxyscirpenol and T-2 toxin were deacetylated to monoacetoxyscirpenol and HT-2 toxin, respectively. Feeding of 5 ppm (5 mg/kg) of ochratoxin A to sheep revealed 14 ppb (14 ng/ml) of ochratoxin A and ochratoxin alpha in rumen fluid after 1 h, but neither was detected in the blood. Whether such conversions in the rumen fluid may be considered as a first line of defense against toxic compounds present in the diet is briefly discussed.     

Comparison of insulin and insulin-like growth factor I receptors from rat skeletal muscle and L-6 myocytes

Insulin and IGF-I receptors were solubilized from fused L-6 myocytes, a rat skeletal muscle derived cell line, and compared to rat skeletal muscle receptors. In skeletal muscle, 125I-insulin binding was competed by insulin greater than IGF-I greater than MSA, whereas in L-6 cells IGF-I greater than insulin greater than MSA. 125I-IGF-I binding was competed by IGF-I greater than insulin = MSA in both tissues. On electrophoresis, differences in Mr were observed between skeletal muscle and L-6 derived receptors both in the alpha- and beta-subunits. Six antibodies directed against the human insulin receptor beta-subunit recognized the rat skeletal muscle insulin receptor, while only two reacted strongly with L-6 derived receptors. Skeletal muscle has receptors with relative specificity for insulin and IGF-I respectively; L-6 cells also have two classes of receptors, one is kinetically similar to the IGF-I receptor from skeletal muscle; the other, which binds insulin with relatively high affinity has even greater affinity for IGF-I. This unusual receptor may represent a developmental stage in muscle or the transformed nature of L-6 cells.     

Effects of PHI on vasoactive intestinal peptide receptors and adenylate cyclase activity in lung membranes. A comparison in man, rat, mouse and guinea pig

The presence of receptors, recognized by vasoactive intestinal peptide (VIP) as well as by PHI (a peptide with N-terminal histidine and C-terminal isoleucine amide), was documented in lung membranes from rat, mouse, guinea pig and man by the ability of these receptors, once occupied, to stimulate adenylate cyclase. In lung membranes from rat, mouse and guinea pig, the capacity of VIP, PHI and secretin to stimulate the enzyme and the potency of the same peptides to compete with 125I-VIP for binding to VIP receptors were similar, the affinity decreasing in the order: VIP greater than PHI greater than secretin. In addition, dose-effect curves were compatible with the coexistence of high-affinity and low-affinity VIP receptors, in the four animal species considered. If PHI was able to recognize all VIP receptors it could not, however, discriminate the subclasses of VIP receptors.     

The interaction of nivazol with the glucocorticoid receptor from rat and rhesus monkey target tissues

Steroid hormone receptor competition techniques were used to evaluate the glucocorticoid receptor binding properties of nivazol and its 11 beta-hydroxy derivative, Win 44577 in rat and monkey target tissues. These agents competitively inhibited the binding of 3H-dexamethasone to the glucocorticoid receptor from the liver and anterior pituitary from both rat and monkey with relative binding affinities of Win 44577 greater than dexamethasone greater than nivazol greater than cortisol in all cases. However, nivazol and Win 44577 had approximately twice the affinity for the anterior pituitary glucocorticoid receptor from both species. Neither compound demonstrated any significant binding to rat estrogen, progestin or androgen receptors. These results are consistent with a glucocorticoid receptor mediated mechanism of action for nivazol and Win 44577; however, the difference in the endocrine profile of nivazol in the rhesus monkey versus the rat does not appear to be due to a species selectivity in the affinity of nivazol for the glucocorticoid receptor from central or peripheral target tissue.     

Estrogenic effects of environmental chemicals: an interspecies comparison

The development of various in vitro screening methods has led to identification of novel estrogenic chemicals of natural and anthropogenic origin. In this study, the (anti)estrogenic potential of several environmental chemicals were compared in an array of in vitro test systems comprising: (i) competitive binding to estrogen receptors derived from the human breast cancer cell line MCF-7 (hER) and rainbow trout (Oncorhynchus mykiss) (rtER), (ii) a proliferation assay with MCF-7 cells (E-SCREEN), and iii) induction of vitellogenin (rtVtg) in isolated rainbow trout hepatocytes. The results showed substantial differences in assay sensitivity for potent estrogens like 17beta-estradiol, diethylstilbestrol and zearalenone (ranking order of sensitivity: E-SCREEN > hER approximately rtER approximately rtVtg). Chemicals like 4-n-nonylphenol and bisphenol A had higher relative binding affinity to the hER, whereas 4-t-butylphenol and 4-n-butylphenol showed highest affinity to the rtER. Zearalenone and the novel estrogen 4-t-butylhexanol displayed a considerable higher relative potency in the E-SCREEN than the rtVtg assay, whereas alkylphenols and the novel estrogen mimic 4-t-butyl-nitrobenzene were most potent in fish cells. Correlation analysis of data from the test systems suggest that interspecies differences is largely due to inter-assay variation of the ER-dependent cellular responses, whereas binding to the ER are fairly similar in the two species tested.     

Comparative in vitro effects of guinea pig VIP and common VIP on liver and lung membranes from guinea pig and rat and on human lymphoblastic SUP-T1 membranes

Guinea pig VIP differs from VIP of several mammals by its amino acids in positions 5, 9, 19 and 26. We tested a) its ability to occupy VIP receptors in liver and lung membranes of rat and guinea pig and in the human lymphoblastic SUP-T1 cell line and b) the ensuing adenylate cyclase stimulation. In liver and lung membranes from rat, guinea pig VIP was less potent than common VIP to occupy high and low affinity VIP receptors. In rat liver both VIP activated adenylate cyclase mostly through high affinity receptors. In rat lung, guinea pig VIP activated the enzyme mostly through high affinity receptors and was less efficient than common VIP acting through both classes of receptors. In guinea pig liver and lung membranes, binding inhibition curves were steeper than with rat preparations and adenylate cyclase appeared to be mostly activated through high affinity VIP receptors in liver and through both classes of receptors in lung. On human lymphoblastic SUP-T1 membranes both VIP were equally potent and efficient to inhibit tracer binding and activate adenylate cyclase.