Case-Control Study of Nodding Syndrome in Acholiland: Urinary Multi-Mycotoxin Screening.

This case-control study adds to the growing body of knowledge on the medical, nutritional, and environmental factors associated with Nodding Syndrome (NS), a seizure disorder of children and adolescents in northern Uganda. Past research described a significant association between NS and prior history of measles infection, dependence on emergency food and, at head nodding onset, subsistence on moldy maize, which has the potential to harbor mycotoxins. We used LC-MS/MS to screen for current mycotoxin loads by evaluating nine analytes in urine samples from age-and-gender matched NS cases (n = 50) and Community Controls (CC, n = 50). The presence of the three mycotoxins identified in the screening was not significantly different between the two groups, so samples were combined to generate an overall view of exposure in this community during the study. Compared against subsequently run standards, α-zearalenol (43 ± 103 µg/L in 15 samples > limit of quantitation (LOQ); 0 (0/359) µg/L), T-2 toxin (39 ± 81 µg/L in 72 samples > LOQ; 0 (0/425) µg/L) and aflatoxin M1 (4 ± 10 µg/L in 15 samples > LOQ; 0 (0/45) µg/L) were detected and calculated as the average concentration ± SD; median (min/max). Ninety-five percent of the samples had at least one urinary mycotoxin; 87% were positive for two of the three compounds detected. While mycotoxin loads at NS onset years ago are and will remain unknown, this study showed that children with and without NS currently harbor foodborne mycotoxins, including those associated with maize.

Changes in Th1 and Th2 cytokine concentrations in ileal Peyer's patches in gilts exposed to zearalenone.

GALT induces tolerance to foreign food antigens and plays an important role in the development of food allergies and the inflammatory bowel disease. The immune function of GALT is significantly influenced by an equilibrium between Th1 and Th2 subpopulations and the cytokines they produce. Th1 cytokines participate in the induction of a cell-mediated immune response, whereas Th2 cytokines induce powerful antibody-mediated responses. Changes in Th1/Th2 cell polarization of an immune response are associated with susceptibility to autoimmune and infectious diseases. This experiment investigated changes in cytokine levels produced by Th1 and Th2 cells in ileal Payer's patches in gilts exposed to ZEN doses below the NOEL (approximately 8 microg kg(-1) BW) for 14, 28 and 42 days. A significant linear increase in IL-4 (40.32 +/- 1.55 ng mg(-1)--137.60 +/- 29.96 ng mg(-1)), and IL-10 (5.99 +/- 0.15 ng mg(-1)--16.39 +/- 1.11 ng mg(-1)) concentrations was observed. An increase in Th1 (IL-2 and IFN-gamma) cytokine levels was also noted in the experimental group, but it was not statistically significant. An HPLC analysis of Peyer's patches in group E animals revealed a linear increase in ZEN concentrations (3.65 +/- 0.91 ng g(-1)--4.72 +/- 1.85 ng g(-1)) and an absence of alpha-ZEL. IL-4 stimulates monocytes and macrophages, it induces the production of proinflammatory cytokines and it may directly and indirectly contribute to the development of inflammatory foci. Higher IL-4 levels could shift polarization toward Th2 cells, stimulate B cells to undergo class switching to produce IgE and contribute to the development of allergies.

Zeranol induces deleterious effects on the testes and the prostate gland of mature rats.

BACKGROUND: Endocrine disrupters have been shown to affect the male and female reproductive systems and to alter potential fertility. OBJECTIVES: This study was conducted to evaluate the effect of a continuous-release pellet containing 12 mg of zeranol for 30 days on the testes and the prostate gland of mature male rats. RESULTS: Zeranol treatment induced significant decrease of the testes and the prostate gland weights which were associated with a remarkable atrophy of the testicular seminiferous tubules and prominent regression of the glandular compartment of the prostate gland. However, zeranol treatment increased the thickness of the periductal layer of stromal cells of the prostate gland from a thin layer that express intense immunostaining of SM-actin and mild vimentin to a thicker layer of cells that exhibited intense immunostaining for both SM-actin and vimentin. CONCLUSION: These findings suggest that zeranol-induced changes to the prostate gland could result from either a direct effect of zeranol on the prostate gland or an indirect effect by interfering with testosterone production through disruption of testicular function.

Effects of long-term in vitro exposure of ejaculated boar sperm to zearalenone and α-zearalenol in sperm liquid storage medium.

The effects of in vitro exposure of porcine spermatozoa to zearalenone (ZEN) and α-zearalenol (α-ZOL) were studied by evaluating several parameters of an in vitro fertilization (IVF) system. For this purpose, boar spermatozoa cultured with semen storage medium containing 0 (control), 10 and 1000 µg/L of ZEN and α-ZOL for 1 week at 5°C were used for IVF of in vitro matured oocytes. Overall, there were no significant differences in the rates of total penetration, monospermic fertilization, and polyspermic fertilization of oocytes inseminated with spermatozoa from the different groups. Similarly, ZEN and α-ZOL at 10 and 1000 µg/L did not have detrimental effects on the cleavage and development to blastocysts of oocytes after in vitro fertilization. Although the motility, viability, and plasma membrane integrity of spermatozoa significantly decreased after 3 weeks of storage compared to non-stored spermatozoa (P < 0.05), ZEN and α-ZOL at the evaluated concentrations did not exert detrimental effects on the above parameters, even after 3 weeks of storage. These results indicate that prolonged exposure of boar spermatozoa to ZEN and α-ZOL up to 1000 µg/L under reduced metabolic conditions does not affect their in vitro function.

Catechol metabolites of zeranol and 17ß-estradiol: a comparative in vitro study on the induction of oxidative DNA damage and methylation by catechol-O-methyltransferase.

α-Zearalanol (α-ZAL, zeranol) is a highly estrogenic macrocyclic ß-resorcylic acid lactone, which is used as a growth promotor for cattle in various countries. We have recently reported that α-ZAL and its major metabolite zearalanone (ZAN) are hydroxylated at the aromatic ring by microsomes from human liver in vitro, thereby forming two catechol metabolites each. Thus, the oxidative metabolism of α-ZAL and ZAN resembles that of the endogenous steroidal estrogens 17ß-estradiol (E2) and estrone (E1), which also give rise to two catechols each. As these catechol metabolites are believed to mediate the carcinogenicity of E2 and E1 by causing oxidative DNA damage and DNA adducts, their methylation by catechol-O-methyltransferase (COMT) is an important inactivation pathway. Here we report that hepatic microsomes from five species generate catechol metabolites of α-ZAL and ZAN, the highest amounts being formed by human liver microsomes, followed by rat, mouse, steer and swine. The microsomal extracts and the individual catechols of α-ZAL, ZAN, E2 and E1 were found to induce oxidative DNA damage, as measured by the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in a cell-free system. The ranking of pro-oxidant activity was 15-HO-ZAN>15-HO-α-ZAL≈4-HO-E2/E1≈2-HO-E2/E1>13-HO-ZAN>13-HO-α-ZAL. With respect to the rate of methylation by human hepatic COMT, the ranking was 2-HO-E2/E1>>4-HO-E2/E1>15-HO-α-ZAL/ZAN>>13-HO-α-ZAL/ZAN. Thus, some catechol metabolites of α-ZAL and ZAN are better pro-oxidants and poorer substrates of COMT than the catechols of E2 and E1. These findings warrant further investigations into the genotoxic potential of α-ZAL, which may constitute another biological activity in addition to its well-known estrogenicity.

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.

Urinary mycoestrogens, body size and breast development in New Jersey girls.

BACKGROUND: Despite extensive research and interest in endocrine disruptors, there are essentially no epidemiologic studies of estrogenic mycotoxins, such as zeranol and zearalenone (ZEA). ZEA mycoestrogens are present in grains and other plant foods through fungal contamination, and in animal products (e.g., meat, eggs, dairy products) through deliberate introduction of zeranol into livestock to enhance meat production, or by indirect contamination of animals through consumption of contaminated feedstuff. Zeranol is banned for use in animal husbandry in the European Union and other countries, but is still widely used in the US. Surprisingly, little is known about the health effects of these mycoestrogens, including their impact on puberty in girls, a period highly sensitive to estrogenic stimulation. OBJECTIVES AND METHODS: We conducted a cross-sectional analysis among 163 girls, aged 9 and 10 years, participating in the Jersey Girl Study to measure urinary mycoestrogens and their possible relationship to body size and development. RESULTS: We found that mycoestrogens were detectable in urine in 78.5% of the girls, and that urinary levels were predominantly associated with beef and popcorn intake. Furthermore, girls with detectable urinary ZEA mycoestrogen levels tended to be shorter and less likely to have reached the onset of breast development. CONCLUSIONS: Our findings suggest that ZEA mycoestrogens may exert anti-estrogenic effects similar to those reported for isoflavones. To our knowledge, this was the first evaluation of urinary mycoestrogens and their potential health effects in healthy girls. However, our findings need replication in larger studies with more heterogeneous populations, using a longitudinal approach.

Oestrogenic mycotoxin exposures and precocious pubertal development.

Since the 1970s, there has been a worldwide scientific discussion on the potential health consequences of human exposure to endocrine disrupters: many environmentally persistent compounds are oestrogen agonists and/or androgen antagonists. Thus, they can dysregulate the hypothalamic-pituitary-gonadal axis potentially affecting human puberty timing. Zearalenone (ZEA) is a non-steroidal mycotoxin produced by Fusarium species on several grains. Despite its low acute toxicity and carcinogenicity, ZEA exhibits oestrogenic and anabolic properties in several animal species. ZEA food contamination is caused either by direct contamination of grains, fruits and their based-products or by 'carry-over' of mycotoxins in animal tissues, milk and eggs after intake of contaminated feedstuff. In addition, zeranol (alpha-ZAL), a resorcyl lactone derived from ZEA, has been widely used in the USA as a growth promoter to improve fattening rates in cattle. From 1978 to 1984, a great epidemic of premature thelarche and precocious puberty occurred in Puerto Rico. To explain this condition, it was suggested that dairy and meat products could be contaminated with anabolic oestrogens such as ZEA or alpha-ZAL. Subsequently, worldwide other groups have also reported causative associations between oestrogenic mycotoxins and development of early thelarche and/or precocious puberty in exposed children. In addition to animal data, epidemiological studies strongly support the hypothesis that human pubertal development may be induced by foetal/early or prepubertal exposure to oestrogenic compounds. Indeed, ZEA and its metabolites are able to adopt molecular conformation, which sufficiently resembles 17beta-oestradiol to allow it to bind to oestrogen receptors (ERs) in target cells exerting oestrogenic (agonist) actions. In this view, oestrogenic mycotoxins are suspected as triggering factor for precocious pubertal development at least in prepubertal exposed girls.

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.

The environmental impact of growth-promoting compounds employed by the United States beef cattle industry: history, current knowledge, and future directions.

The current state of knowledge regarding the environmental impact of growth-promoting compounds associated with the U.S. beef cattle industry is extensive in some areas but virtually nonexistent in others. The compounds administered to the cattle are quite well understood, as are bovine metabolism and excretion. If the sex and age of the cattle on the feedlot are known, the metabolites excreted by the cattle should be predictable with a great deal of accuracy. The fate, transport, and biological effects of growth-promoting compounds are just beginning to be studied. Most of the research conducted on the fate and transport of growth-promoting compounds has focused on 17beta-E2; however, much of this research was not conducted using feedlot runoff or manure. Studies are needed that focus specifically on manures and runoff from experimental or commercial feedlots. To date, the degree to which growth-promoting compounds are released from feedlots in a bioavailable form remains a point of speculation. The environmental fate and transport of TBA, P, and MGA have not been well studied. Comparisons between the fate and transport of T and 17beta-E2, however, make it clear that compounds with similar structure may behave very differently once released into the environment. Considering that 17beta-E2 is a naturally occurring estrogen and that TBA is a nonaromatizable androgen, it is not surprising that these compounds directly impact the reproductive physiology of fishes. The effects of these two compounds have been well documented, as has been described here; however, the effects of P and MGA exposures have gone largely uninvestigated. This is a serious critical gap in our knowledge base because progestogins play an important role in sex steroid synthesis and reproduction. Clearly, additional research on the consequences of exposures to P and MGA is warranted. The majority of research investigating the effects of 17beta-E2 and TBA metabolites on fish has been conducted in the laboratory and has typically focused on continuous, pharmacological exposures to single compounds. These exposures may not bear much similarity to environmentally relevant exposures, and as such may offer little information regarding biological effects seen in nature. Cattle feedlot runoff is likely to contain a suite of growth-promoting compounds rather than any single compound. Clearly, deciphering the biological effects of exposure to complex mixtures containing androgenic, estrogenic, and progestogenic compounds will remain an important area of study for the next few years. A second complexity associated with the biological runoff from cattle feedlots is the discontinuous nature of the release. It is likely that inadvertent entry of growth-promoting compounds will follow spring snowmelt or rainstorm events. These events will result in intermittent, pulsed exposures to high concentrations of these compounds interspersed by long-term exposures to lower concentrations. The effects of exposure timing and duration should be considered to generate a clearer understanding of the biological consequences of exposures to growth-promoting compounds. To date, a very limited number of studies (only one!) have sought to determine whether fish living in waterways receiving runoff from cattle feedlots are adversely affected by growth-promoting compounds associated with the runoff. Clearly, more field studies need to be conducted before a relationship between cattle feedlot effluent and biological consequences can be elucidated.

The influence of the mycotoxins deoxynivalenol and zearalenol on in vitro maturation of pig oocytes and in vitro culture of pig zygotes.

The aim of the present study was to investigate the influence of specific toxins on in vitro maturation and embryo culture. alpha- and beta-zearalenol were tested at increasing levels from 3.75 to 90 microM and deoxynivalenol from 0.94 to 7.5 microM in order to evaluate the effect on in vitro maturation rate of porcine cumulus-oocyte complexes. Furthermore, the influence of alpha-zearalenol (3.75-30 microM) was appraised on the developmental competence of in vivo-derived zygotes during 5 days of in vitro culture. All three substances affected maturation and degeneration rates in a dose-dependent manner, but to different extents. Significant differences were obtained at a concentration of 7.5 microM alpha-zearalenol and higher. beta-zearalenol negatively affected the process of oocyte development beginning at a concentration of 30.0 microM (P<0.05). Deoxynivalenol had significant influence on oocyte maturation at a concentration of 1.88 microM (31.4 vs 79.3% for control). Differences in embryonic development in vitro were observed at a concentration of 15 microM alpha-zearalenol (P<0.05). These data demonstrate a negative effect of alpha-zearalenol on embryonic development of zygotes, and a compound-specific, dose-dependent negative effect of the three substances on meiotic progression of porcine oocytes.

Quantitative assessment of foetal exposure to trenbolone acetate, zeranol and melengestrol acetate, following maternal dosing in rabbits.

1. Residues of commonly used growth-promoting agents found in animal meat can be hormonally active and they have been implicated as possible endocrine disruptors in man. Although these compounds could be potentially detrimental to the developing foetus, it is not clear whether and to what extent they pass through placental barrier. 2. This issue was addressed using the rabbit as an animal model. Pregnant rabbits were treated with trenbolone acetate, zeranol or melengestrol acetate beginning at gestation day 14. Levels of active substances in plasma were screened by means of specific ELISA systems. The residues of parent compounds and their metabolites were quantified in maternal and foetal tissues on gestation day 27 using validated, sensitive HPLC/ELISA methods. 3. All three compounds crossed the placental barrier and were detectable in foetal tissues. The extent of tissue concentration varied depending on the compound and tissue analysed. Gender differences were observed in some instances.

Effects of in utero exposure to nonsteroidal estrogens on mouse testis.

Male mice exposed in utero to alpha-zearalanol (zeranol) or diethylstilbestrol (DES) were analyzed postnatally to evaluate the possible changes on their testicular morphology as part of an examination of the effects of transplacental exposure to non-steroidal estrogens on sensitive tissues. Pregnant NMRI mice were injected subcutaneously with ethyl oleate (0.1 mL) alone (negative control) or with 150 micrograms/kg of body weight of zeranol or DES (positive control) on days 9 and 10 of gestation. Experimental and control male offspring were euthanized at days 45 (n = 47), 90 (n = 44), 180 (n = 40) and 365 (n = 26) after birth and their gonads were examined by light and electron microscopy. The results suggested that prenatal zeranol or DES exposure induced more severe and earlier (at 45 d) testicular abnormalities than in negative control (at 6 mo). These age-related alterations were characterized by regressive changes in the germinal epithelium and Sertoli's cells as well as foci of Leydig's cells around atrophied seminiferous tubules and dysplasia of the rete testis epithelium. On the contrary, the presence of Leydig's cells with immature morphology and their arrangement in sheet could be attributable exclusively to estrogen treatment. The presence of no neoplasm was confirmed.

Occupational exposure to zeranol, an animal growth promoter.

Zeranol (3,4,5,6,7,8,9,10,11,12-Decahydro-7,14,16-trihydroxy-3-methyl-1H-2 - benzoxacyclotetradecin-1-one) is a synthetic oestrogenic agent used as an animal growth promoter. The effects of occupational exposure to zeranol in 11 exposed workers from a pelletising plant and 14 nonexposed subjects were assessed. A questionnaire showed that more breast symptoms were reported by male and female plant workers compared with non-exposed subjects, although the difference was not statistically significant. Clinical assessment showed no cases of gynaecomastia in all the male participants. Blood samples analysed by high performance liquid chromatography for zeranol, its precursor zearalenone, and its main metabolites did not show any of these compounds above the laboratory limit of detection. Serum levels of follicle stimulating hormone (FSH), luteinising hormone (LH), prolactin, and oestradiol showed no striking differences between the exposed and the non-exposed subjects. Total and low density lipoprotein cholesterol (LDL cholesterol) levels did not significantly differ between the two groups but mean high density lipoprotein cholesterol (HDL cholesterol) levels were higher in the exposed group; this could be due to relatively high HDL cholesterol in two women exposed to zeranol or relatively low HDL cholesterol in three non-exposed men.

Should we worry about animal growth promoters?

Estrogens and compounds with estrogenic activity have been used for many years as animal growth promoters and to synchronize estrus in cows. The resorcylic acid lactones (RALS), which include zearalenone, are being used increasingly for these purposes. These estrogenic mycotoxins, now produced commercially, have teratogenic, mutagenic, and carcinogenic properties. There is some evidence that their residues could remain in certain organs at the time the animal products come to market, and this may be of medical significance.