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Developmental toxicity and estrogenic potency of zearalenone in zebrafish (Danio rerio)
Abstract
Zearalenone (ZEA, F2) is one of the most common mycotoxins and the only known mycoestrogen. It enters the food and feed chain from contaminated cereals and infiltrates into sewage or natural waters posing potential threat to exposed livestock, wildlife and humans. Therefore evaluation of its biological effects is of international importance. We performed toxicological tests on zebrafish (Danio rerio) larvae and adults. Developmental toxicity was assessed by an extended (5 days) fish embryo toxicity test (FET). Effects of early ZEA exposure were concentration-dependent with LC50 and LC10 values of 893 and 335μg/L. In larvae exposed to 500μg/L and above, ZEA induced similar phenotype to has (heart-and soul) showing defects in heart and eye development and upward curvature of the body axis. From 250μg/L at 72hpf the gap in the melanophore streak at the base of the tail fin was missing and the fin fold was abnormal, suggesting disturbance in the development of the adult tail fin primordium. Estrogenic potency was measured on the basis of Vitellogenin (Vtg) protein (adults) levels and relative abundance of vitellogenin-1 mRNA (vtg-1) (larvae and adults). qRT-PCR in larvae proved to be sufficient substitute to adult tests and sensitive enough to detect ZEA in 0.1μg/L concentrations, that is close to levels observed in wastewaters. Developmental defects reveal that besides direct estrogenic effects, zearalenone might interact with other ontogenic pathways.
... Showing a high affinity for estrogen receptors, ZEA toxicity has been historically associated with hormonal effects and the regulation of genes involved in cell survival and apoptosis in both in vitro and in vivo experiments [11]. The biological consequences of ZEA exposure have been studied using an array of fish species, as well as various in vitro systems [12][13][14]. Commonly reported effects have included altered reproductive function, such as intersex males, disrupted sperm production, and morphological deformities in early life stages [12,14]. Reproductive alterations resulting from ZEA exposure have been largely associated with the xenoestrogen properties of the compound and its high affinity for estrogen receptors (ER) in hormone-sensitive tissues [15,16]. ...
... The biological consequences of ZEA exposure have been studied using an array of fish species, as well as various in vitro systems [12][13][14]. Commonly reported effects have included altered reproductive function, such as intersex males, disrupted sperm production, and morphological deformities in early life stages [12,14]. Reproductive alterations resulting from ZEA exposure have been largely associated with the xenoestrogen properties of the compound and its high affinity for estrogen receptors (ER) in hormone-sensitive tissues [15,16]. ...
... Extending previous studies of ZEA in the zebrafish embryo model [12,19,30,31], the present study investigated the toxicity of ZEA in embryonic stages of not only zebrafish but two additional marine species, namely olive flounder and yellowtail snapper. Additionally, it is, indeed, the first study to assess toxicity in these two ecologically and economically relevant species. ...
Zearalenone (ZEA) is a mycotoxin, commonly found in agricultural products, linked to adverse health impacts in humans and livestock. However, less is known regarding effects on fish as both ecological receptors and economically relevant “receptors” through contamination of aquaculture feeds. In the present study, a metabolomics approach utilizing high-resolution magic angle spinning nuclear magnetic resonance (HRMAS NMR) was applied to intact embryos of zebrafish (Danio rerio), and two marine fish species, olive flounder (Paralichthys olivaceus) and yellowtail snapper (Ocyurus chrysurus), to investigate the biochemical pathways altered by ZEA exposure. Following the assessment of embryotoxicity, metabolic profiling of embryos exposed to sub-lethal concentrations showed significant overlap between the three species and, specifically, identified metabolites linked to hepatocytes, oxidative stress, membrane disruption, mitochondrial dysfunction, and impaired energy metabolism. These findings were further supported by analyses of tissue-specific production of reactive oxygen species (ROS) and lipidomics profiling and enabled an integrated model of ZEA toxicity in the early life stages of marine and freshwater fish species. The metabolic pathways and targets identified may, furthermore, serve as potential biomarkers for monitoring ZEA exposure and effects in fish in relation to ecotoxicology and aquaculture.
... Any disruption in the HPG axis can adversely affect the reproductive endocrine function (Xu et al., 2017). The reproductive toxicity of ZEA has been reported in several fish species such as carp (Cyprinus carpio) (Sandor & Vanyi, 1990) Atlantic salmon (Salmo salar) (Arukwe, Grotmol, Haugen, Knudsen, & Goksøyr, 1999), rainbow trout (Oncorhynchus mykiss) (Olsen, Meussen-Elholm, Hongslo, Stenersen, & Tollefsen, 2005), fathead minnow (Pimephales promelas) (Johns et al., 2011), zebrafish (Danio rerio) (Bakos et al., 2013;Schwartz, Thorpe, Bucheli, Wettstein, & Burkhardt-Holm, 2013). ZEA and its metabolites competitively binds with estrogen receptors (ER) resulting in estrogenicity. ...
... Its presence has been examined in various agricultural products and in the environment (Bennett & Klich, 2003). Several studies shows that ZEA could cause genotoxic (Gao et al., 2013), hepatotoxic (Stadnik & Borzecki, 2009 reproductive toxicity (Bakos et al., 2013). Though studies have shown reproductive toxicity of ZEA, the detailed mechanisms of action on HPG axis and steroidogenesis pathway in female zebrafish have not been reported. ...
... The mycoestrogen, ZEA exposed at 5 and 10 μg l −1 to the female zebrafish revealed that Vtg expression was drastically increased in liver. In accordance to our result, Bakos et al. (2013) found a similar induction of Vtg expression in the liver of male zebrafish exposed to 10 and 1000 μg l −1 ZEA. ...
In the present study, we aimed to assess the adverse effects of zearalenone (ZEA) at environmentally relevant concentrations (0.5, 1, 5 and 10 μg l⁻¹) on hypothalamic–pituitary–gonadal axis associated reproductive function using zebrafish model. ZEA was exposed to female zebrafish for 21 days to assess growth indices such as condition factor, hepatosomatic index, gonadosomatic index and caspase 3 activity. Further, expression of estrogen receptor (ER) α and CYP19a1b genes in the brain, ERα and vitellogenin (Vtg) genes in the liver and follicle‐stimulating hormone receptor, luteinizing hormone receptor, ERα, steroidogenic acute regulatory protein, 3β‐hydroxysteroid dehydrogenase (HSD), 17‐βHSD and CYP19a1 genes in the ovary were also investigated. Our results showed that there were no significant changes in the condition factor and hepatosomatic index, whereas a significant (P < .05) reduction in the gonadosomatic index, increase in caspase 3 activities and Vtg expression was observed at higher concentration. However, no significant changes were observed at lower treatment levels. Further, we also observed significant (P < .05) upregulation in ERα, Vtg, luteinizing hormone receptor, steroidogenic acute regulatory protein, 3β‐HSD, 17β‐HSD, CYP19a1 and CYP19a1b genes in treatment groups with higher levels of ZEA. Moreover, in histopathological examination, we observed oocyte atresia and oocyte membrane detachment in ovaries at the highest concentration. In conclusion, the present study revealed the negative impact of ZEA on zebrafish reproductive system by involvement of the hypothalamic–pituitary–gonadal axis‐associated reproductive function.
... Therefore, the exposure of ZEA in aquatic systems may be linked with adverse effect to aquatic organisms and to affect the subsequent ecosystem (Tatay et al., 2017). The previous reports have shown that ZEA could cause genotoxic, hepatotoxic, immunotoxic, neurotoxic and estrogenic effects (Bakos et al., 2013;Venkataramana et al., 2014;Sellamani et al., 2016). Reproductive toxicity of ZEA was reported in various fish species like studies using carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and fathead minnow (Pimephales promelas) (Bakos et al., 2013). ...
... The previous reports have shown that ZEA could cause genotoxic, hepatotoxic, immunotoxic, neurotoxic and estrogenic effects (Bakos et al., 2013;Venkataramana et al., 2014;Sellamani et al., 2016). Reproductive toxicity of ZEA was reported in various fish species like studies using carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and fathead minnow (Pimephales promelas) (Bakos et al., 2013). To this extent, Bakos et al. (2013) reported that, ZEA induced defects in heart and eye development as well as upward curvature of the body axis in zebrafish embryos at a concentration above 500 mg/L. ...
... Reproductive toxicity of ZEA was reported in various fish species like studies using carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and fathead minnow (Pimephales promelas) (Bakos et al., 2013). To this extent, Bakos et al. (2013) reported that, ZEA induced defects in heart and eye development as well as upward curvature of the body axis in zebrafish embryos at a concentration above 500 mg/L. Further, Schwartz et al. (2010) reported that, short-term exposure of ZEA with 3200 ng/L does not affected fertility, hatch, and embryo survival of zebrafish. ...
In the present study, we evaluated the zearalenone induced adverse effects in zebrafish embryos using various endpoints like embryo toxicity, heart rate, oxidative stress indicators (reactive oxygen species (ROS), lipid peroxidation (LPO), Nitric oxide (NO)), antioxidant responses (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase enzyme (GST) and reduced glutathione (GSH), metabolic biomarkers (lactate dehydrogenase (LDH) and Nitric oxide (NO)), neurotoxicity (acetylcholinesterase (AChE)), genotoxicity (comet assay and acridine orange staining (AO)) and histological analysis. In this study, four concentrations 350, 550, 750 and 950 μg/L of ZEA were chosen based on LC10 and LC50 values of the previous report. The results shows that ZEA induces developmental defects like pericardial edema, hyperemia, yolk sac edema, spine curvature and reduction in heart rate from above 550 μg/L exposure and the severity was increased with concentration and time dependent manner. Significant induction in oxidative stress indices (ROS, LPO and NO), reduction in antioxidant defence system (SOD, CAT, GPx, GST and GSH) and changes in metabolic biomarkers (LDH and AP) were observed at higher ZEA exposed concentration. Neurotoxic effects of ZEA were observed with significant inhibition of AChE activity at higher exposure groups (750 and 950 μg/L). Moreover, we also noticed DNA damage, apoptosis and histological changes in the higher ZEA treatments at 96 h post fertilization (hpf) embryos. Hence, in the present study we concluded that oxidative stress is the main culprit in ZEA induced developmental, genotoxicity and neurotoxicity in zebrafish embryos.
... Another compound that may interact with estrogen receptors in fishes is the known mycoestrogen-Zearalenone (ZEA). In a study by Bakos et al. [87] zebrafish larvae exposed to environmentally relevant concentrations (0.1 μg/L) of ZEA developed anatomical defects of the heart muscle associated with disturbed gene expression. In studies by Orozco-Hernández et al. [59], exposition of Common carps (Cyprinus carpio) to the different concentrations of the E2 (1 ng/L, 1 μg/L and 1 mg/L) in water environment have induced cyto-genotoxicity effect on fish blood through influence on apoptotic signaling pathways. ...
... Fat metabolism is also influenced by the expression level of CD36, a fatty acid transport (FA) protein that also acts as a receptor for low density oxygenated lipoproteins [87,88]. A study on triploid rainbow trout showed no change in FA translocase (FAT/CD36) expression after exposure to an increased concentration of E2, but an increase in the expression of one of the FA binding proteins (FABP)-FABP3, which binds to the aforementioned CD36. ...
Estrogen receptors (ERs) play a key role in many biochemical and physiological processes, that are involved in maintaining organism homeostasis. At the most basic level, they can be divided into nuclear estrogen receptors and membrane estrogen receptors that imply their effect in two ways: slower genomic, and faster non-genomic. In these ways, estrogens and xenoestrogens can negatively affect animal health and welfare. Most of the available literature focuses on human and mammalian physiology, and clearly, we can observe a need for further research focusing on complex mutual interactions between different estrogens and xenoestrogens in aquatic animals, primarily fishes. Understanding the mechanisms of action of estrogenic compounds on the ERs in fishes and their negative consequences, may improve efforts in environmental protection of these animals and their environment and benefit society in return. In this review, we have summarized the ER-mediated effects of xenoestrogens and estrogens on teleost fishes metabolism, their carcinogenic potential, immune, circulatory, and reproductive systems.
... Estrogens in general bind to estrogen receptors (ERs) in steroidogenic tissues and up-regulate expression of cytochrome P450 aromatase (cyp19a1), which converts androgens to estrogens, thus increasing endogenous estrogen levels and promoting ovary formation (Leet et al., 2011;Li et al., 2019). Similarly, ZEN has been shown to bind to fish ERs and induce ER-dependent gene expression (Arukwe et al., 1999;Celius et al., 1999;Woźny et al., 2008;Schwartz et al., 2010;Bakos et al., 2013;Muthulakshmi et al., 2018a;Dellafiora et al., 2020). Thus, for these reasons, we believe that the morphological anomalies in the gonads of the exposed rainbow trout were most likely due to the estrogenic potential of ZEN or its metabolite(s), which critically affected the early stage of the fishes' reproductive development. ...
... Although the content of ZEN in eggs was not measured in the present study, the mycotoxin is transferred from the alimentary tract to the reproductive system of rainbow trout, including the oocytes (Woźny et al., 2017). Moreover, subjecting fertilized zebrafish eggs to waterborne exposure to ZEN induces developmental abnormalities in the embryos (Bakos et al., 2013;Khezri et al., 2018;Muthulakshmi et al., 2018b). Therefore, the transfer of ZEN to eggs, and subsequently, the mycotoxin's direct effect on embryonic development are likely explanations for the increase in offspring mortality after maternal ZEN-exposure observed in our study. ...
To assess the effects on rainbow trout reproduction of feed contamination with zearalenone (ZEN) at the current European guidance value (2 mg∙kg⁻¹ feed), we performed a life-cycle feeding trial under aquaculture conditions. The trial began when the fish larvae commenced external feeding and continued for 96 weeks, up to the point when the fish reached sexual maturity. To compare the reproductive development of the exposed and control fish, histological and molecular observations and analyses were performed at 12, 72, and 96 weeks of the feeding trial. To investigate the effect of ZEN exposure on the fertility of male and female fish, gametes obtained from mature fish were fertilized, after which offspring mortality was tracked at the eyed egg, hatched, and swimming larva stages. The mortality data were also used to estimate the risk of offspring death depending on the parental exposure scenario. We observed that feed-borne exposure to ZEN resulted in abnormal development of the fishes' gonads, and the occurrence of intersex fish and sex-reversed (feminized) males. These observations suggest that ZEN could have interfered with sex differentiation of the exposed fish at an early stage of their reproductive development. Our data indicate that the feed-borne exposure to ZEN probably did not affect the timing of sexual maturation or the relative fecundity of the female fish, but they suggest it might have led to advanced ovarian development. Moreover, ZEN-exposed male fish had a higher mean sperm concentration and markedly higher plasma levels of vitellogenin than males in the control group. These findings suggest that males could be more susceptible to the detrimental effects of ZEN than females. Importantly, offspring of fish that were fed with ZEN-contaminated feed throughout their whole reproductive cycle had a higher mortality risk than offspring of fish that were fed blank feed. Moreover, the offspring of parents that were only exposed to ZEN during the last 17 weeks of feeding before spawning also had a markedly increased mortality risk. Together, these findings not only demonstrate that ZEN harms rainbow trout reproductive outcomes, but also indicate that fish at sexual maturation may be especially vulnerable to ZEN contamination in feed. Our findings raise concerns about the safety of the current recommended guidance values for ZEN in feed and feedstuff used in aquaculture and suggest a need to consider lowering the maximum allowable levels of this mycotoxin.
... The effect of mycotoxins such as zearalenone (ZEA) (Bakos et al. 2013), aflatoxin (Troxel et al. 1997), citrinin and patulin (Wu et al. 2012) have also been conducted. Bakos et al. (2013) referred to the early effects of ZEA exposure being concentration-dependent with LC 50% and LC 10% values of 893 and 335μg/L. ...
... The effect of mycotoxins such as zearalenone (ZEA) (Bakos et al. 2013), aflatoxin (Troxel et al. 1997), citrinin and patulin (Wu et al. 2012) have also been conducted. Bakos et al. (2013) referred to the early effects of ZEA exposure being concentration-dependent with LC 50% and LC 10% values of 893 and 335μg/L. In larvae exposed to 500μg/L and above, ZEA induced phenotype changes showing defects in heart and eye development and upward curvature of the body axis. ...
Toxicity studies in mammals continue to be the most appropriate model for predicting risk in humans, but they tend to be expensive and time-consuming. In the aftermath of the genetic sequencing of zebrafish (Danio rerio), this species is highly genetically homologous to humans. The use of the zebrafish model to assess food toxicity is already a reality as it is capable of biological processes difficult to reproduce in vitro. Studies of complex mechanisms of absorption, distribution, metabolism, and excretion as well as cellular and tissue interactions are of great information value resulting in time, space and cost savings, when compared to studies with rodents. This review addresses the relevance of zebrafish model in food safety research, both in the use of ingredients and approved and generally recognized as safe food additives as well as for establishing levels of safe food contaminant residues present in the environment. Toxicological screening using the zebrafish model integrate the evaluation of teratogenicity, cardiotoxicity, hepatotoxicity, genotoxicity, neurotoxicity, endocrine toxicity, reproductive and behavioral aspects. These are important endpoints for food safety assessment, which take substantially less time than in mammalian tests. Furthermore, it serves well as a screening test follow-up for validating favorable results in murine models, hence accelerating the risk assessment process of products submitted for approval and registration, prioritizing safe compounds and reducing unnecessary costs in subsequent mammalian studies. In conclusion, the zebrafish model can be a useful tool for food safety tests; however, additional studies are needed to further validate this model for registration of new food ingredients and additives.
... Mycotoxins such as ZEN are known to contribute to the estrogenic potential in aquatic systems (Bucheli et al. 2005). Accordingly, estrogenic effects have also been described in several fish species (Johns et al. 2009;Schwartz et al. 2010;Bakos et al. 2013), although physiological effects are lacking in other species (Pietsch et al. 2015a(Pietsch et al. , 2015bPietsch 2017). Estrogenic effects have therefore also been included in the calculations for Fig. 7a, which led to a mean LOEL for this endpoint of 1256 ± 982 μg/kg ZEN (mean ± SEM). ...
... For example, dietary exposure of carp to ZEN resulted in an impaired quality and number of sperm (Sándor and Ványi 1990). Developmental problems in zebrafish (Danio rerio) and early life stages of fathead minnow (Pimephales promelas) due to ZEN exposure have also been described (Johns et al. 2009;Schwartz et al. 2010;Bakos et al. 2013). Developmental effects often included the occurrence of edema and lacking pigmentation in early ontogenic life stages or impaired development of the bony parts of the body. ...
Mycotoxins are difficult to monitor continuously, and a tool to assess the risk would help to judge if there is a particular risk due to the inclusion of certain feed ingredients. For this, the toxin contents of 97 commercial fish feeds have been estimated, and the most prominent toxins in fish feed are calculated to be deoxynivalenol, zearalenone, fumonisins and enniatins. These pose a risk to fish well-being, as can be calculated by the Bayesian models for determining the critical concentrations 5% (CC5) for the different toxins. Besides fishmeal, wheat, soybean products and corn are regularly used as fish feed ingredients. The calculated scenarios show that fish are at high risk of toxin contamination if feed ingredients of low quality are chosen for feed production. Due to this, specific maximum allowable levels for several mycotoxins in fish feeds should be established.
... Vitellogenin genes are expressed in an estrogen-dependent manner therefore only mature females are able to produce vitellogenin in larger quantities. On the other hand estrogenic molecules trigger vitellogenin synthesis in males and larvae as well (48)(49)(50). In this study vitellogenin levels increased in exposure groups. ...
... In this study vitellogenin levels increased in exposure groups. DEHP's estrogenic activity is still unclear (50,51). We have previously shown that DEHP (2,5 μg/L) did not increase vitellogenin levels in zebrafish embryos (52). ...
Objective: Polyethylene terephthalate (PET) is a material that is most commonly used for production of clear plastic bottles. Adenomatous polyposis coli (APC) and β-catenin have been related with cancer. Aim was to investigate the effects of PET bottled water samples that were exposed to sunlight and hot water on zebrafish embryos. Moreover the effects of these water samples on APC knockdown zebrafish embryos were also evaluated.Methods: Phthalate concentrations in water samples were determined using ELISA. Immunohistochemical method and RT-PCR were used to analyse the expressions of proliferative cell nuclear antigen (PCNA), β catenin, Wnt 3a and Gsk3β. Biochemical parameters were measured using spectrophotometric methods. Vitellogenin concentrations were measured using ELISA and apoptotic cells were evaluated by Acridine Orange staining.Results: Increased PCNA, β-catenin, Wnt 3a, Gsk3β expressions, vitellogenin, nitric oxide, apoptosis and impaired oxidant-antioxidant balance were observed in the exposure groups with these increases being more profound in APC knockdown groups.Conclusion: APC knockdown embryos were more prone to the deleterious effects of water samples used in this study.
... Moreover, ZEA induces various genital and cellular abnormalities, such as abnormal mouse sperm morphology, atrophy of seminiferous tubules, abnormal ovaries, hypertrophy of reproductive organs in female pigs, abnormal embryonic development of zebrafish. (Fig. 3) (Bakos et al., 2013;Zatecka et al., 2014;Chen et al., 2015;Adibnia et al., 2016;Gao et al., 2017). In this article, we will review reproductive damage of ZEA and related mechanisms. ...
... suspended development and even decomposition in embryos. The lower concentrations of ZEA treatment caused pericardial edema, yolk sac edema, tail deformity, and curvature of the spine (Bakos et al., 2013;Belli et al., 2010). ...
... Addressing how to minimize the harmful effects of unavoidable mycotoxin exposure is a pressing issue. Previous reports indicated that the LC 50 value for ZEA exposure over 96 h post-fertilization (hpf) was 1099.4 μg/L [19]. Therefore, the single ZEA doses used in this study were 0.25, 0.5, and 1 mg/L. ...
The contamination of food and feed by mycotoxins, particularly zearalenone (ZEA) and deoxynivalenol (DON), is a global issue. Prenatal exposure to ZEA and DON can result in congenital cardiac malformations in fetuses. Addressing the prevention and mitigation of embryonic cardiotoxicity caused by these toxins is crucial. Citrus limonoid nomilin (NOM) is an extract known for its pathological properties in various diseases. This study investigated the potential mechanism of NOM in mitigating cardiotoxicity caused by ZEA and DON co-exposure in a zebrafish model. The findings indicated that NOM pretreatment alleviated cardiac developmental toxicity induced by ZEA and DON and normalized the expression of key genes involved in heart development, including gata4, vmhc, nkx2.5, and sox9b. Co-exposure to NOM, ZEA, and DON enhanced SOD and catalase activity, increased glutathione levels, and reduced ROS and malondialdehyde production. Furthermore, NOM reduced cardiac oxidative damage by activating the Keap1/Nrf2 signaling pathway. In summary, this study offers new insights for preventive interventions against congenital heart disease caused by mycotoxin exposure.
... mechanism of individual mycotoxins. According to previous studies, mycotoxins cause different types of toxicological effects in zebrafish, such as abnormal behavior (Khezri et al., 2018), teratogenicity (Haq et al., 2016), developmental toxicity (Bakos et al., 2013), and hepatotoxicity (Wang, Liu, et al., 2021). In Table 2 and Figure 1, we summarize the toxicological effects of mycotoxins on zebrafish or zebrafish embryos. ...
Food safety has long been an area of concern. The selection of stable and efficient model organisms is particularly important for food toxicology studies. Zebrafish (Danio rerio) are small model vertebrates, and 70% of human genes have at least one zebrafish ortholog. Zebrafish have advantages as model organisms due to their short life cycle, strong reproductive ability, easy rearing, and low cost. Zebrafish embryos have the advantage of being sensitive to the breeding environment and thus have been used as biosensors. Zebrafish and their embryos have been widely used for food toxicology assessments. This review provides a systematic and comprehensive summary of food toxicology studies using zebrafish as model organisms. First, we briefly introduce the multidimensional mechanisms and structure–activity relationship studies of food toxicological assessment. Second, we categorize these studies according to eight types of hazards in foods, including mycotoxins, pesticides, antibiotics, heavy metals, endocrine disruptors, food additives, nanoparticles, and other food‐related ingredients. Finally, we list the applications of zebrafish in food toxicology studies in line with future research prospects, aiming to provide a valuable reference for researchers in the field of food science.
... This finding suggested that F-2, TFS, and their mixture had potent anti-oestrogenic effects on D. rerio (Kawan et al., 2019). A previous study has reported that F-2 may interact with other oncogenic pathways (Bakos et al., 2013). Therefore, we hypothesized that exposure to F-2, TFS, and their combination altered ER expressions, leading to impaired generation of VTG and weakened reproduction of D. rerio. ...
Although humans and animals are usually exposed to combinations of toxic substances, little is known about the interactive toxicity of mycotoxins and farm chemicals. Therefore, we can not precisely evaluate the health risks of combined exposure. In the present work, using different approaches, we examined the toxic impacts of zearalenone and trifloxystrobin on zebrafish (Danio rerio). Our findings showed that the lethal toxicity of zearalenone to embryonic fish with a 10-day LC50 of 0.59 mg L-1 was lower than trifloxystrobin (0.037 mg L-1). Besides, the mixture of zearalenone and trifloxystrobin triggered acute synergetic toxicity to embryonic fish. Moreover, the contents of CAT, CYP450, and VTG were distinctly altered in most single and combined exposures. Transcriptional levels of 23 genes involved in the oxidative response, apoptosis, immune, and endocrine systems were determined. Our results implied that eight genes (cas9, apaf-1, bcl-2, il-8, trb, vtg1, erβ1, and tg) displayed greater changes when exposed to the mixture of zearalenone and trifloxystrobin compared with the corresponding individual chemicals. Our findings indicated that performing the risk assessment based on the combined impact rather than the individual dosage response of these chemicals was more accurate. Nevertheless, further investigations are still necessary to reveal the modes of action of mycotoxin and pesticide combinations and alleviate their effects on human health.
... Так, при включении в корма Danio rerio 0,1-3 мг/кг ДОН выявлено повышение генетических биомаркеров печени и влияние на репродуктивную систему (78). По наблюдениям за содержанием вителлогенина и молекулярного биомаркера vitellogenin-1 mRNA (vtg-1) на взрослых особях D. rerio удалось установить, что, кроме прямых эстрогенных эффектов, ЗЕН способен влиять и на другие пути онтогенеза (79,80). ...
... Так, при включении в корма Danio rerio 0,1-3 мг/кг ДОН выявлено повышение генетических биомаркеров печени и влияние на репродуктивную систему (78). По наблюдениям за содержанием вителлогенина и молекулярного биомаркера vitellogenin-1 mRNA (vtg-1) на взрослых особях D. rerio удалось установить, что, кроме прямых эстрогенных эффектов, ЗЕН способен влиять и на другие пути онтогенеза (79,80). ...
Современная аквакультура рыб относится к масштабным и стремительно развивающимся
отраслям мирового производства (FAO, 2018). С целью повышения качества выращиваемой продукции активно ведется поиск эффективных способов контроля безопасности искусственных кормов (J. Bostock с соавт., 2010). По результатам мониторинговых проектов, выполненных в Аргентине, Бразилии, США, Китае, Корее и странах Центральной Европы (C. Pietsch с соавт., 2013;
B.T.C. Barbosa с соавт., 2013; M. Greco с соавт., 2015; L. Pinotti с соавт., 2016), ситуация по
загрязненности комбикормов для рыб микотоксинами признана чрезвычайно серьезной как по распространенности и содержанию, так и по сочетанной встречаемости (I. Matejova с соавт., 2017;
C. Pietsch, 2019). В российском рыбоводстве, которое в последние годы стало многопрофильным
направлением сельского хозяйства, специалистами академической и вузовской науки, а также отраслевых НИИ разработаны рецептуры комбикормов, учитывающие возрастные и видовые особенности рыб (Ю.А. Желтов, 2006; В.Я. Скляров, 2008; И.Н. Остроумова, 2012; Гамыгин Е.А. с
соавт., 2013), и подробно рассмотрена проблема их микробной контаминации (И.В. Бурлаченко,
2008). В Российской Федерации для комбикормовой продукции введены обязательные требования
на соответствие показателям качества и безопасности (ГОСТ 10385-2014) и создана современная
методическая база для проведения микотоксикологического контроля (ГОСТ 31653-2012, ГОСТ
31691-2012, ГОСТ 32587-2013, ГОСТ 34108-2017, ГОСТ Р 51116-2017). Цель настоящего обзора — актуализация сведений о контаминации микотоксинами сырья для производства аквакормов, обобщение мировых данных по характеру острого действия наиболее вероятных контаминантов, а также анализ клинических признаков, патологоанатомических и биохимических изменений,
сопровождающих острые и хронические микотоксикозы рыб. За последние годы получены убедительные подтверждения того, что в группу наиболее распространенных контаминантов отечественных сырьевых ингредиентов (пшеничной, ячменной и кукурузной муки, отрубей, подсолнечного
жмыха и шрота) входят Т-2 токсин, дезоксиниваленол, фумонизины группы В и зеараленон, относящиеся к фузариотоксинам, а также альтернариол, охратоксин А, цитринин, циклопиазоновая
кислота, микофеноловая кислота и эмодин (Г.П. Кононенко с соавт., 2018, 2019). Анализ массива
мировых данных по экспериментальным микотоксикозам карпа обыкновенного (Cyprinus carpio),
канального сома (Ictalurus punctatus), амура белого (Ctenopharyngodon idella), нильской тиляпии
(Oreochromis niloticus), радужной форели (Oncorhynchus mykiss), атлантического лосося (Salmo
salar) разных возрастных групп показывает, что фузариотоксины следует причислить к ключевым
факторам риска и направить усилия на определение их безопасных пороговых значений. Интоксикации, вызванные охратоксином А, остаются недостаточно изученными, а ситуация в отношении
остальных возможных контаминантов комбикормового сырья — неясной. Обоснованные предложения по регламентации количества микотоксинов в кормах для рыб приведены лишь по Т-2 токсину для карпа обыкновенного (В.Т. Галаш, 1988), дезоксиниваленолу для белого амура (C. Huang
с соавт., 2018, 2019, 2020) и атлантического лосося (A. Bernhoft с соавт., 2018), фумонизину B1
для канального сома (M.N. Li с соавт., 1994, S. Lumlertdacha с соавт., 1995). Данные по степени
сохранения этих микотоксинов в тканях рыб ограничены (C. Pietsch с соавт., 2014, 2015;
A. Ananter с соавт., 2016), поэтому регламенты по их остаточным количествам в продукции до сих
пор не приняты. Однако поиск новых подходов к корректной оценке последствий негативного действия микотоксинов и трансмиссии в рыбную продукцию продолжается.
... Therefore, ZEN has adverse effects on swine ovarian atrophy, reproductive problems, and abortion (Minervini and Aquila 2008;Zhu et al. 2012;Gao et al. 2017;Hennig-Pauka et al. 2018). Furthermore, ZEN causes several cellular and reproductive deformities, such as abnormal ovaries, rats sperms morphological abnormalities, sow reproductive hypertrophy, zebra fish abnormal embryo, and atrophy of seminiferous tubules (Bakos et al. 2013;Zatecka et al. 2014;Chen et al. 2015;Gao et al. 2017). Studies showed that gametogenesis and embryo development was affected by ZEN toxicity (Zhu et al. 2014;Dänicke and Winkler 2015;Ren et al. 2016;Salem et al. 2016). ...
Zearalenone (ZEN) contamination of various foods and feeds is an important global problem. In some animals and humans, ZEN causes significant health issues in addition to massive economic losses, annually. Therefore, removal or degradation of the ZEN in foods and feeds is required to be done. The conventional physical and chemical methods have some serious issues including poor efficiency, decrease in nutritional value, palatability of feed, and use of costly equipment. Research examined microbes from diverse media for their ability to degrade zearalenone and other toxins, and the findings of several investigations revealed that enzymes produced from microbes play a significant role in the degradation of mycotoxins. In established bacterial hosts, genetically engineered technique was used to enhance heterologously produced degrading enzymes. Then, the bio-degradation of ZEN by the use of microorganisms or their enzymes is much more advantageous and is close to nature and ecofriendly. Furthermore, an effort is made to put forward the work done by different scientists on the biodegradation of ZEN by the use of fungi, yeast, bacteria, and/or their enzymes to degrade the ZEN to non-toxic products.
... Therefore, ZEN has adverse effects on swine ovarian atrophy, reproductive problems, and abortion (Minervini and Aquila 2008;Zhu et al. 2012;Gao et al. 2017;Hennig-Pauka et al. 2018). Furthermore, ZEN causes several cellular and reproductive deformities, such as abnormal ovaries, rats sperms morphological abnormalities, sow reproductive hypertrophy, zebra fish abnormal embryo, and atrophy of seminiferous tubules (Bakos et al. 2013;Zatecka et al. 2014;Chen et al. 2015;Gao et al. 2017). Studies showed that gametogenesis and embryo development was affected by ZEN toxicity (Zhu et al. 2014;Dänicke and Winkler 2015;Ren et al. 2016;Salem et al. 2016). ...
Zearalenone (ZEN) contamination of various foods and feeds is an important global problem. In some animals and humans, ZEN causes significant health issues in addition to massive economic losses, annually. Therefore, removal or degradation of the ZEN in foods and feeds is required to be done. The conventional physical and chemical methods have some serious issues including poor efficiency, decrease in nutritional value, palatability of feed, and use of costly equipment. Research examined microbes from diverse media for their ability to degrade zearalenone and other toxins, and the findings of several investigations revealed that enzymes produced from microbes play a significant role in the degradation of mycotoxins. In established bacterial hosts, genetically engineered technique was used to enhance heterologously produced degrading enzymes. Then, the bio-degradation of ZEN by the use of micro-organisms or their enzymes is much more advantageous and is close to nature and ecofriendly. Furthermore, an effort is made to put forward the work done by different scientists on the biodegradation of ZEN by the use of fungi, yeast, bacteria, and/or their enzymes to degrade the ZEN to non-toxic products.
Key points
•Evolved microbial strains degraded ZEA more quickly
•Different degrading properties were studied
... On a study on mice by Boussabbeh et al., patulin's cardiotoxic effects are discussed: oxidative stress and cell apoptosis, which can also lead to alterations of the cardio-vascular system [60]. The fact that mycotoxins exhibit cardiotoxic effects on zebrafish embryos heart are shown in several studies: pericardial edema [61][62][63][64], decrease in heartbeat rate [63,64]. After being exposed to fungi that produce kojic acid, rodent myocardium exhibited hemorrhagic lesions [57]. ...
Machine learning is a branch of artificial intelligence that allows computer systems to learn directly from examples, data, and experience. Statistical modeling is more about finding connections between variables and consequently the impact of these relationships, while also catering for prediction. It should be clear that these two methodologies are different in terms of their purpose, despite the fact that they use similar means to get there. The evaluation of the machine learning algorithm uses a set of tests to validate its accuracy. Although, for a statistical model, the analysis of regression parameters by confidence intervals, significance tests and other tests can be used to assess the legitimacy of the model. To demonstrate the applications and usefulness of this theory, an experimental study was conducted on zebrafish exposed to mycotoxin. Methods: Patulin (70 µg/L) and kojic acid (100 mg/L, 204 mg/L, and 284 mg/L) were administered by immersion to zebrafish once daily for a period of 7 days before the behavior testing. The following behavioral tests were performed: a novel tank test (NTT) (to assess the explorative behavior and anxiety); and a Y-maze test (which measures the spontaneous explorative behavior). Behavioral tests were performed on separate days. For the behavior tests, the statistical analysis was performed using ANOVA variation analysis (two-way ANOVA). All results are expressed as the mean ± standard error of the mean. The values of the general index F for which p < 0.05 were considered statistically significant. Results: Y-maze—patulin exposure led to an intensification of the locomotor activity and an increased traveled distance and number of arm entries. By increasing the spontaneous alternation between the aquarium’s arms, patulin has shown a stimulating effect on spatial memory. In the case of zebrafish exposed to 100 mg/L kojic acid, the traveled distance was shorter by 27% than the distance attained by those in the control group. The higher doses of kojic acid (204 mg/L and 284 mg/L) led to an increased locomotor activity, distance traveled, number of arm entries, and the spontaneous alternation. The increase in spontaneous alternation demonstrates that 204 mg/L and 284 mg/L kojic acid doses had a stimulating effect on spatial memory. Novel tank test—compared to the control group, the traveled distance of the patulin-exposed fish is slightly reduced. Compared to the control group, the traveled distance of the kojic acid-exposed fish is reduced, due to a shorter mobile time (by 25–27% in the case of fish exposed to 204 mg/L and 284 mg/L kojic acid). Patulin and kojic acid exhibit toxic effects on zebrafish liver, kidney, and myocardium and leads to severe alteration. We continued the analysis by trying some machine learning algorithms on the classification problems in the case of the two behavioral tests MAZE and NTT, after which we concluded that the results were better in the case of the NTT test relative to the MAZE test and that the use of decision tree algorithms leads to amazing results, knowing that their hierarchical structure allows them to learn signals from both classes. Conclusions: The groups exposed to patulin and kojic acid show histological changes in the liver, kidneys, and myocardial muscle tissue. The novel tank test, which assesses exploratory behavior, has been shown to be conclusive in the behavioral analysis of fish that have been given toxins, demonstrating that the intoxicated fish had a decreased explorative behavior and increased anxiety. We were able to detect a machine learning algorithm in the category of decision trees, which can be trained to classify the behavior of fish that were given a toxin in the category of those used in the experiment, only by analyzing the characteristic features of the NTT Behavior Test.
... ZEN can greatly impact zebrafish embryos because such a compound can be hardly metabolized by aquatic organisms (Muthulakshmi et al., 2018a). However, most of these reports on ZEN and FB1 have only examined their single toxic effects on fish, while their mixture toxic actions have not been identified (Bakos et al., 2013;Azman et al., 2020). Humans and animals often encounter a mixture of ZEN and FB1 during environmental mycotoxin exposure, rather than individual compounds (Al-Jaal et al., 2019;Carballo et al., 2019). ...
The natural co-occurrence of multiple mycotoxins has been reported in cereals and cereal products worldwide. Even though the dietary exposure to mycotoxins constitutes a serious human health, most reports are limited to the toxic effect of individual mycotoxins. The purpose of the present study was to assess the combined toxic effects of zearalenone (ZEN) and fumonisin B1 (FB1) and the potential interaction of their mixture on zebrafish (Danio rerio) embryos. Our results showed that ZEN possessed the higher toxicity to embryonic zebrafish (7-day LC50 value of 0.78 mg a.i. L⁻¹) compared with FB1 (7-day LC50 value of 227.7 mg a.i. L⁻¹). The combination of ZEN and FB1 exerted an additive effect on zebrafish embryos. Meanwhile, the activities of antioxidant CAT, caspase-3, and detoxification enzyme CYP450, as well as the expressions of six genes (Mn-sod, cas9, bax, cc-chem, ERα, and crh) associated with oxidative stress, cellular apoptosis, immune system, and endocrine system were prominently altered in the mixture exposure compared with the corresponding single treatment group of ZEN or FB1. Taken together, the regulatory standards of mycotoxins in food and feed should be updated based on the mixture effects of mycotoxins, and there is an increased need on effective detoxification methods for controlling and reducing the toxicity of multiple mycotoxins in animal feed and throughout the food supply chain.
... This highlighted concerns about the growth, health, and immunity of farmed fish that might be adversely influenced by the existence of mycotoxins in their diets [7][8][9]. Reports showed that feeding fish on ZEN-contaminated diets induced developmental and reproductive toxicity in zebrafish (Danio rerio) [10,11], reduced immune responses and modulation of physiological responses of common carp (Cyprinus carpio) [12,13], histopathological alterations, apoptotic changes and oxidative stress injury in the intestinal tract of grass carp (Ctenopharyngodon idella) [14], histopathological alterations of hepatopancreatic tissues of rainbow trout (Oncorhynchus mykiss) [15], and haemato-biochemical alterations and immunotoxicity of European seabass (Dicentrarchus labrax) [7]. Hence, there is an urgent necessity to prevent the harmful consequences of ZEN toxicity on fish health and immune status. ...
The present study was performed to evaluate the toxic effects of feed-born zearalenone (ZEN) on antioxidative status, immunity, transcriptomic responses of European seabass, and the modulating roles of dietary garlic and/or chitosan powders. Fish (30.7 ± 0.6 g) were randomly arranged in five experimental groups (in triplicates), whereas the first group was fed on the control diet only without any supplements (control), and the second group was fed on the basal diet contaminated with ZEN (0.725 mg/kg diet). Three other groups were fed on ZEN-contaminated diets and simultaneously supplemented with garlic powder (GP) (30 g/kg diet) (ZEN+GP), chitosan powder (CH) (10 g/kg diet) (ZEN+CH), and a mixture of GP and CH (ZEN+GP+CH). Fish were fed the experimental diets thrice a day for 4 weeks. Two-way ANOVA revealed a gradual decline in serum superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) enzyme activities in the ZEN group and reaching their lowest levels at the 4th week. Contrariwise, malondialdehyde levels were markedly higher in the ZEN group reaching their highest values at the end of the experiment. A significant decline of total immunoglobulins (P < 0.05) was observed in the serum of the ZEN group, especially after the 4th week. Moreover, significant down-regulation of interleukin-4 (IL-4) and interleukin 1 beta (IL-1β) genes (P < 0.05) alongside significant up-regulation of tumor necrosis factor-alpha (TNF-α) and heat shock protein 70 (HSP70) genes (P < 0.05) in the liver and anterior kidney of ZEN-intoxicated group. Interestingly, dietary supplementation with GP and/or CH significantly attenuated ZEN-induced oxidative stress, immunosuppression, and modulated transcriptomic responses of ZEN-exposed fish. Moreover, combined dietary supplementation of both feed additives resulted in better effects than each one alone.
... For example, a study of Shao et al. (2019) demonstrated the influence of GEN on the embryo mortality of zebrafish. ZEN and its metabolites were shown to induce a rise in vitellogenin levels (Schwartz et al., 2010) and potentially cause intersex and decreased sperm production in fish (Bakos et al., 2013). Woźny et al. (2020) also observed abnormal development of rainbow trout gonads after feedborne exposure to ZEN. ...
Estrogenic substances are today among the contaminants of emerging concern. Besides naturally occurring estrogens, other natural and synthetic substances can mimic a hormonal action due to their structural resemblance to hormones, possibly affecting the endocrine system of living organisms. Estrogens have been detected in inland water bodies such as influents and effluents of waste water treatment plants as well as in rivers, but data on their distribution and variability in the marine ecosystem are still limited. Surface water samples obtained during two research cruises on the northern shelf of the South China Sea (SCS) near the Pearl River Estuary, in September 2018 and in August 2019, were investigated for estrogenic substances, namely estrone (E1), 17β-estradiol (E2), 17α-ethinylestradiol (EE2), genistein (GEN), daidzein (DAI) and zearalenone (ZEN). Among the target analytes, the natural hormones E1 and E2, as well as the synthetic EE2, were the most abundant with maximum concentrations of 1.1 ng L⁻¹, 0.7 ng L⁻¹ and 0.6 ng L⁻¹, respectively. Of substances produced by plants and fungi, GEN, DAI and ZEN, only GEN was detected (1.2 ng L⁻¹). High concentrations occurred predominantly close to the coast, which was also reflected in the calculated estradiol equivalent quotients (up to 1.4 ng L⁻¹). In general, the distribution of estrogenic substances observed in both years shows a regional and inter-annual variability consistent with the modeled surface current data for the SCS. Regarding single estrogenic compounds and estradiol equivalents, marine organisms in the northern SCS might be exposed to high potential risk.
... In an earlier study, the lethal effects of ZEN on zebrafish embryos were examined in a concentration-dependent way, which aligned with dose-dependent mortality in the present study. Our results confirmed the sublethal symptoms described earlier including the curvature of body axis (dose-dependent) with abnormal heart and eye development (typical for estrogenic materials), pericardial and yolk edema, and reduced pigmentation (melanophore streak at the base of the caudal fin) [60]. However, hyperemia was not characterized in this study [61]. ...
The multimycotoxin-degrading efficiency of the Rhodococcus erythropolis NI1 strain was investigated with a previously developed three-step method. NI1 bacterial metabolites, single and combined mycotoxins and their NI1 degradation products, were injected into one cell stage zebrafish embryos in the same doses. Toxic and interaction effects were supplemented with UHPLC-MS/MS measurement of toxin concentrations. Results showed that the NI1 strain was able to degrade mycotoxins and their mixtures in different proportions, where a higher ratio of mycotoxins were reduced in combination than single ones. The NI1 strain reduced the toxic effects of mycotoxins and mixtures, except for the AFB1+T-2 mixture. Degradation products of the AFB1+T-2 mixture by the NI1 strain were more toxic than the initial AFB1+T-2 mixture, while the analytical results showed very high degradation, which means that the NI1 strain degraded this mixture to toxic degradation products. The NI1 strain was able to detoxify the AFB1, ZEN, T-2 toxins and mixtures (except for AFB1+T-2 mixture) during the degradation experiments, which means that the NI1 strain degraded these to non-toxic degradation products. The results demonstrate that single exposures of mycotoxins were very toxic. The combined exposure of mycotoxins had synergistic effects, except for ZEN+T-2 and AFB1+ZEN +T-2, whose mixtures had very strong antagonistic effects.
... Meanwhile, another mycotoxin exposure on zebrafish embryo was T-2 toxin where higher concentration of T-2 toxin can significantly increase the mortality rate and also caused the malformations on tails and cardiovascular defects (Yuan et al. 2014). Zeralenone exposure on zebrafish showing defects on eye development and also upward curvature of the body axis (Bakos et al. 2013). ...
Fumonisin B1 (FB1) is a common mycotoxin produced by Fusarium species particularly F. proliferatum and F. verticillioides. The toxin produced can cause adverse effects on humans and animals. The objectives of this study were to detect the production of FB1 based on the amplification of FUM1 gene, to quantify FB1 produced by the isolates using Ultra-fast Liquid Chromatography (UFLC) analysis, to examine the embryotoxicity effect of FB1 and to determine EC50 toward the larvae of zebrafish (Danio rerio). Fifty isolates of Fusarium species were isolated from different hosts throughout Malaysia. Successful amplification of the FUM1 gene showed the presence of this gene (800 bp) in the genome of 48 out of 50 isolates. The highest level of FB1 produced by F. proliferatum isolate B2433 was 6677.32 ppm meanwhile F. verticillioides isolate J1363 was 954.01 ppm. From the assessment of embryotoxicity test of FB1 on larvae of zebrafish, five concentrations of FB1 (0.43 ppm, 0.58 ppm, 0.72 ppm, 0.87 ppm and 1.00 ppm) were tested. Morphological changes of the FB1 exposed-larvae were observed at 24 to 168 hpf. The mortality rate and abnormality of zebrafish larvae were significantly increased at 144 hpf exposure. Meanwhile, the spontaneous tail coiling showed a significant difference. There were no significant differences in the heartbeat rate. As a conclusion, the presence of FUM1 in every isolate can be detected by FUM1 gene analysis and both of the species produced different concentrations of FB1. This is the first report of FB1 produced by Fusarium species gave a significant effect on zebrafish development.
... In zebrafish (Danio rerio), ZEN reduced spawning frequency [126] or changed their relative fecundity from one generation to another [127]. In another study, defects in the heart No effects on growth and may accelerate sexual maturation of female fish Woźny et al. [130] International Journal of Microbiology and eye development and upward curvature of the body axis of were observed when zebrafish larvae were exposed to 500 µg·L − 1 or higher of ZEN [135]. By considering the few studies made in aquaculture species (Table 4), we would presume that ingestion of ZEN may affect growth performance, but it depends on species, dose, and duration of exposure, and it can result in complications in broodstocks of farmed species and monosex-cultured species. ...
The rapid population growth in developing countries has led to strong pressure on capture fisheries. However, capture fisheries have reached their maximal limits of fish production and are supplemented by farmed fish. The growth in aquaculture has led to high demand for fish feeds, which play a very important role in fish nutrition and health. Use of animal protein in fish feeds is expensive; hence, a majority of farmers from developing countries use local feed ingredients from plant origin as a source of dietary protein. However, these ingredients of plant origin provide the best natural substrates for fungi, which can be easily accompanied by mycotoxin development under suitable conditions. The locally made feed comprises ingredients such as soybeans, cottonseed cake, and wheat and maize bran which are mixed together and ground after which the compounded feed is pelleted and stored. Among the ingredients, maize and oilseeds are more susceptible for mycotoxigenic fungi compared to other ingredients. The outcomes of mycotoxin contamination in fish feeds are not different from other animal species intended for human consumption, and they are directly associated with production losses, particularly decreased weight gain and feed conversion, impaired immune system and reproductive performance, and increased fish mortality. Fish may also carry mycotoxin residues along the food chain, thus compromising human health. Hence, it is important to ensure the control of mycotoxin contamination in fish feeds, especially during the production and storage.
... T-2 toxin has a mean toxicity of 3201 ± 1236 μg/kg (±SEM) in fish, based on the currently available LOEL for different fish species (Figure 2). This level is considerably higher than the actual contamination level found in salmonid fish feed [176][177][178][179][180][181][182][183][184] reporting LOEL for 5 different species, 10 studies [185][186][187][188][189][190][191][192][193] reporting effects of different levels of T-2 toxins on 4 different fish species, and 3 studies [162,194,195] for 3 different species exposed to MON. in South America [28], and much lower than the guidance levels of 250 mg/kg for T-2 toxin set by the European Commission for cereal products in compound feeds [61] and individual recommendations in other countries (max. 80-100 mg/kg) for T-2 toxin in complete feed and all grains [49]. ...
... Changes in embryonic formation have been observed in the larvae of zebrafish (Danio rerio) exposed to concentrations of 500 mg/L and above, which is characterized by defects in the heart and eye development [209], developmental disorders such as malformation of the spine and reduced heart rate due to oxidative stress, and a decrease in the antioxidant defense system (SOD, CAT, GPx, GST, and GSH) [210]. ...
There are alarming reports of the presence of mycotoxins and the induction of mycotoxicoses in the vital organs of humans and animals, as well as economic loss due to reduced crop yields. In light of these problems, mycotoxin detoxification has become mandatory for the production of healthy products. As the potential for prevention can be absent or limited in the field or during long storage periods, other methods based on several protocols have shown potential usefulness for efficient mycotoxin control; however, few are accepted for practical use. Detoxification can be performed by lactic acid bacteria, Bacillus, moulds, actinobacteria, edible fungi and medicinal plants. Another lower-cost method based on nanoparticles has recently been discovered, and its profitability and efficiency have been proven.
Keywords: Mycotoxins, detoxification, nanoparticles, nanocontrol method
... Changes in embryonic formation have been observed in the larvae of zebrafish (Danio rerio) exposed to concentrations of 500 mg/L and above, which is characterized by defects in the heart and eye development [209], developmental disorders such as malformation of the spine and reduced heart rate due to oxidative stress, and a decrease in the antioxidant defense system (SOD, CAT, GPx, GST, and GSH) [210]. ...
Mycotoxins and mycotoxinogenic molds are responsible for mycotoxicosis in humans and animals. Some mycotoxicoses are chronic and lead to very serious and worrying diseases, such as cancer. High humidity and the ambient temperature are key factors in the biosynthesis of these toxins in foods, but the toxins can be detected in foodstuffs in dry areas, which makes ingestion the most common mode of exposure. Following recent developments, exposure by inhalation of mycotoxins in the air, especially for people who work in storage silos, or by a dermal route during handling in the laboratory, is no longer negligible. Long-term exposure at high doses causes nephrotoxicity, hepatotoxicity, neurotoxicity, gastrointestinal toxicity, immunotoxicity, genotoxicity and teratogenicity. The majority of these diseases result from alteration of repair systems and activations of molecules responsible for inflammation and apoptosis induced by ROS formed after intoxication of cells. This chapter deals with mycotoxicosis and toxicities induced by four mycotoxins: aflatoxin, ochratoxin, fumonisin and zearalenone, as well as their mode of action in vivo and in vitro.
Keywords: Mycotoxins, mycotoxigenic molds, mycotoxicosis, toxicities, mode of action.
... Our study showed that diets with a ZEA content ≥ 1041 µg/kg caused body malformation, such as body line irregularities, upward curvature of the tail, caudal fin deformity, operculum abnormality, and skeletal anomalies in juvenile grass carp. A similar study reported that ZEA could cause upward curvature of the body axis in zebrafish [31]. The deformities observed in ZEA-treated grass carp in the present study were probably related to genotoxicity. ...
Zearalenone (ZEA) is a prevalent mycotoxin with high toxicity in animals. In order to study its effect on juvenile grass carp (Ctenopharyngodon idella), six diets supplemented with different levels of ZEA (0, 535, 1041, 1548, 2002, and 2507 μg/kg diet) for 10 weeks were studied to assess its toxicity on intestinal structural integrity and potential mechanisms of action. Our report firstly proved that ZEA led to growth retardation and body deformity, and impaired the intestinal structural integrity of juvenile grass carp, as revealed by the following findings: (1) ZEA accumulated in the intestine and caused histopathological lesions; (2) ZEA resulted in oxidative injury, apoptosis, and breached tight junctions in the fish intestine, which were probably associated with Nuclear factor-erythroid 2-related factor 2 (Nrf2), p38 mitogen activated protein kinases (p38MAPK), and myosin light chain kinase (MLCK) signaling pathways, respectively. ZEA had no influence on the antioxidant gene levels of Kelch-like ECH associating protein 1 (Keap1)b (rather than Keap1a), glutathione-S-transferase (GST)P1, GSTP2 (not in the distal intestine (DI)), tight junctions occludin, claudin-c (not in the proximal intestine (PI)), or claudin-3c (not in the mid intestine (MI) or DI).
... It can introduce into natural waters thorough contaminated cereals and can create a serious threat to animals and humans. A negative impact on heat and eye early development as well as upward curvature of the body axis in larvae exposed to ZEA has been reported (Bakos et al., 2013). It is also responsible for reproduction problems in cows and ovine species (Nezami et al., 2002). ...
Mycotoxins are a group of naturally occurring toxins that are produced by different filamentous fungi genera such as Aspergillus, Penicillium, Fusarium, etc. The word mycotoxin literally is derived from Greek word “myke” meaning fungus and “toxicum” meaning toxin. These contaminants can develop on different food and feed commodities during different stages including pre-harvest, harvest, and storage. Mycotoxins are of concern because their outbreak result in animal and human diseases and economic losses. It has been estimated that global post-harvest losses are approximately at 50%. Human exposure to mycotoxins is typically through consumption of contaminated agricultural products or indirectly by consumption of animal products containing mycotoxins or their metabolites. The chapter provides the latest information on mycotoxin issues and challenges related to food and feed safety.
... However, the benefit of using acute toxicity tests for fish is represented by the applicability to a wide range of substances, this methodology is also used to compare the sensitivity to other aquatic species (Schirmer et al., 2008;Bakos et al., 2013). ...
The objective of the study was to devise a 96 h test of sterlet embryos (Acipenser ruthenus) in order to evaluate if this species is suitable for water pollution investigation as an evaluation tool. Replicate concentrations of lead acetate were selected from 400, 200, 100 and 50 µgL-1. Each mirroraquarium was filled with 10 L of lead acetate dilutions, excluding the control, the free sterlet embryos were inserted as follows: 50 / aquarium, i.e. 100 / concentration and control group). The fish were examined at time 0, then at: 12, 24, 48, 72 and 96 hours, every change in behavior or in the body somatic was statistically analyzed, according to ANOVA (significant when p < 0.05, or less). Evaluation revealed significant connection among the embryos length (p < 0.05), weight (p = 0.01; p < 0.01) and the dead embryos proved the deleterious feature of lead at 400 and 200 µgL-1. The main critical points were: the delayed resorption of the yolk sac; deformed tail; undeveloped eyes; massive haemorrhages; atypical / altered pigmentation and exitus observed at 12 h. Although A. ruthenus is not considered as a standard subject in the pollution assays, it was observed that, changes can be applied for early fish stages as a parallel to other accepted tests
... ZEN is a highly stable compound and could be induced hyperestrogenic and other pathological symptoms like reduced fertility; increase number of absorbed or dead embryos in cattle, sheep, and pigs; and induce severe toxicity to children in the early phases of growth (Gajecki 2002;Hennig-Pauka et al. 2018;Metzler et al. 2010). Additionally, ZEN causes hyperestrogenic syndrome in humans and could be considered a source for plant estrogen (phytoestrogens) that could lead to many external and development deformities in the female genital tract, changes in estrous cycle, and sexual behavior (Gromadzka et al. 2008;Bakos et al. 2013;Gao et al. 2018). ZEN also has genotoxic and cytotoxic effects either in vivo or in vitro, including chromosomal aberrations, DNA fragmentation, and micronuclei production in the bone marrow (Lioi et al. 2004;Liu et al. 2017a). ...
The aims of this study were to determine the polyphone content of grape seed extract (GSE) and to assess their protective effects against zearalenone (ZEN)-induced maternal toxicity and in utero development defects in mice. Five groups of pregnant mice were treated orally during days 6–13 of gestation as follows: control group, corn oil as vehicle (0.1 ml/mice)-treated group, ZEN-treated group (25 mg/kg b.w), GSE-treated group (150 mg/kg b.w.), and ZEN plus GSE-treated group. All animals were sacrificed on the 19th day of gestation and samples of bone marrow were collected for the micronucleus assay. The maternal and developmental toxicity were carried out. The HPLC analyses revealed that GES is rich in gallic acid, syringic acid, vanillin, quercetin, and coumaric acid. ZEN administration resulted in severe maternal and developmental toxicity which included an increase of micronuclei formation in bone marrow, decreased maternal weight gain, and litter weight. It also induces fetal growth retardation, increased number of the aborted dams and resorbed fetuses, abnormality of fetal bone ossification, and number of fetuses with a hematoma. GSE showed positive effects on the pregnant mice and the developing fetuses. Moreover, it counteracted the detrimental effects of ZEN in dams and fetuses. It could be concluded that polyphenols in GSE are a promising candidate to protect against ZEN toxicity in highly endemic areas.
... ZEN is a highly stable compound and could be induced hyperestrogenic and other pathological symptoms like reduced fertility; increase number of absorbed or dead embryos in cattle, sheep, and pigs; and induce severe toxicity to children in the early phases of growth (Gajecki 2002;Hennig-Pauka et al. 2018;Metzler et al. 2010). Additionally, ZEN causes hyperestrogenic syndrome in humans and could be considered a source for plant estrogen (phytoestrogens) that could lead to many external and development deformities in the female genital tract, changes in estrous cycle, and sexual behavior (Gromadzka et al. 2008;Bakos et al. 2013;Gao et al. 2018). ZEN also has genotoxic and cytotoxic effects either in vivo or in vitro, including chromosomal aberrations, DNA fragmentation, and micronuclei production in the bone marrow (Lioi et al. 2004;Liu et al. 2017a). ...
The aims of this study were to determine the polyphone content of grape seed extract (GSE) and to assess their protective effects against zearalenone (ZEN)-induced maternal toxicity and in utero development defects in mice. Five groups of pregnant mice were treated orally during days 6–13 of gestation as follows: control group, corn oil as vehicle (0.1 ml/mice)-treated group, ZEN-treated group (25 mg/kg b.w), GSE-treated group (150 mg/kg b.w.), and ZEN plus GSE-treated group. All animals were sacrificed on the 19th day of gestation and samples of bone marrow were collected for the micronucleus assay. The maternal and developmental toxicity were carried out. The HPLC analyses revealed that GES is rich in gallic acid, syringic acid, vanillin, quercetin, and coumaric acid. ZEN administration resulted in severe maternal and developmental toxicity which included an increase of micronuclei formation in bone marrow, decreased maternal weight gain, and litter weight. It also induces fetal growth retardation, increased number of the aborted dams and resorbed fetuses, abnormality of fetal bone ossification, and number of fetuses with a hematoma. GSE showed positive effects on the pregnant mice and the developing fetuses. Moreover, it counteracted the detrimental effects of ZEN in dams and fetuses. It could be concluded that polyphenols in GSE are a promising candidate to protect against ZEN toxicity in highly endemic areas.
Filamentous fungi, mainly those belonging to genera Aspergillus, Fusarium, and Penicillium, the main producers of mycotoxins and responsible for food and feed contamination, exhibit metabolites that are highly toxic to animals and humans. This scoping review compiles data on occurrence of mycotoxins in fish feed, assesses distribution of metabolites across continents by the hierarchical cluster analysis, and establishes risks posed to the main global orders of farmed fish by exposure to mycotoxins. Resulting data showed that fish of different orders in both fingerling and juvenile phases are exposed to high concentrations of mycotoxins.
Exposure to low-levels of some chemical the contaminants or xenobiotics present in the wastewater which is released from the industries, houses, agricultural and non-agricultural lands discharges as a source of endocrine-disrupting chemicals to the freshwater, marine water, and brackish aquatic environment shows potential effects on the endocrine system especially disruption of endocrine functions in animals, such as reproduction. This is done by modulating, mimicking, or interfering with normal hormonal activity. The present review shows that the effect of endocrine-disrupting chemicals (Biotoxins and Pesticides) on fish reproductive endpoints including spermatogenesis, steroidogenesis, activities of antioxidants, fecundity potential, egg production, and estradiol levels of male and female fishes. In the present review study, the exposure to pesticides combined with mycotoxins further deteriorated the reproductive endpoints indicating additive effects of these endocrine-disrupting chemicals significantly decreased almost overall reproductive parameters like tissue indices reproductive organs, sperm quantity and quality, steroidogenic enzyme activities, levels of testosterone, histology of testis, lipid peroxidation levels and antioxidant enzyme activities are inflated by pesticides and biotoxins in male and female fishes. It is concluded that these toxicants deteriorate the reproductive ability of male and female fishes.
In the past decade, mycotoxin zearalenone (ZEN)-induced gastrointestinal adverse effects have been increasingly attracting worldwide attention. This study aimed to determine the gastrointestinal adverse effects of ZEN in Drosophila melanogaster (D. melanogaster) and reveal possible mechanisms of action of ZEN in insects. Here, chronic exposure of D. melanogaster to ZEN killed flies in a dose-dependent manner (2-20 µM). ZEN (20 µM) decreased the survival rates and climbing ability of flies, and activated immune deficiency-mediated intestinal immunity in midgut, leading to the production of antimicrobial peptides. Meanwhile, ZEN exposure induced morphological alteration of adult midgut. Further study suggested that high levels of oxidative stress was observed in ZEN-treated midgut due to the imbalance between the production of reactive oxygen species and the expression and activities of cellular antioxidant enzyme, including superoxide dismutase and catalase. ZEN-induced oxidative stress then caused cell death, impaired gut barrier function and increased gut permeability, leading to oxidative injury in midgut. Subsequently, ZEN-induce midgut injury further disrupted intestinal stem cell (ISC) homeostasis, stimulating ISC proliferation and tissue regeneration, but did not alter cell fate specification of ISC. Additionally, activation of Jun N-terminal kinase pathway was involved in ZEN-induced oxidative injury and tissue regeneration in midgut. Antioxidant vitamin E alleviated ZEN-induced oxidative injury to midgut epithelium. Collectively, this study provided additional evidences for ZEN-induced gastrointestinal adverse effects from an invertebrate model, extended our understanding of the mechanisms mediating mycotoxin toxicity in organisms.
Co-existence of mycotoxins may pose a greater risk. It remains less known about the toxic effect of co-exposure of zearalenone (ZEA) and deoxynivalenol (DON) on aquatic life. In the present study, the toxic effects of the combine treatment of ZEA and DON on zebrafish (Danio rerio) embryos were investigated. The results showed that the combined treatment of ZEA (200, 400, 800 μg/L) and DON (4000 μg/L) did not cause apparent deaths, but induced a developmental toxicity as indicated by decreased movement times and heartbeat. At 96 h post-fertilization (hpf), co-exposure of ZEA and DON (Z400 + D4000 and Z800 + D4000 group) led to significant oxidative stress as evidenced by the increased ROS level and MDA content, as well as the changes of antioxidant enzymes (SOD, CAT and GPX) and their genes. Besides, the combined treatment of ZEA and DON triggered hepatotoxicity as shown by the changes of Fabp10a, Gclc, Gsr, Nqo1 genes, apoptosis through upregulating apoptosis-related genes (p53, Caspase-9, Caspase-3) and downregulating Bcl-2 gene, as well as inflammation by promoting the expression of IL-1β, IL-6, TNF-α, TLR4, MyD88, NF-κBp65 genes. These results indicated the co-exposure of ZEA and DON caused oxidative stress, leading to stronger potential toxic effects to zebrafish embryos than their respective single treatment. Therefore, more attention should be paid to risk management of the co-contamination of mycotoxins.
Waste from livestock farms, including manure, is a significant source of estrogenic pollutants in the environment. These wastes have complex matrices, necessitating the implementation of in vivo and in vitro tests in order to investigate their estrogenic effects. However, most current in vivo methods are limited by the toxic effect of livestock waste due to their high concentrations of organic matter. Here we propose a novel in vivo microinjection method which is able to avoid this limitation. In this study, the estrogen content of slurry-based irrigation water extracts from dairy cattle farms was examined using a classical in vitro and the newly developed in vivo method. The limitations of the in vitro system, with its absence of endogenous steroid hormone receptors and subsequent lack of elucidating complex interactions involving the estrogen receptor (ER), are complemented by the in vivo fish test, which allows for a more complete assessment of estrogenicity and toxicity to vertebrate animals. In vitro screenings were performed with the ISO 19040–1:2018 Yeast Estrogen Screen (YES). The YES test showed estrogenic activity in all 32 tested samples, which ranged from 5 to 50518 ng/L in EEQ (E2-Estradiol equivalents). The in vivo microinjection method was developed using a Tg(vtg1:mCherry) transgenic zebrafish embryo model. This model is able to eliminate secondary symptoms of hypoxia that may occur during normal aqueous exposure to high organic matter extracts. Using the microinjection method, a total of 12 samples, out of the 32 samples examined, presented no observable estrogenic effects in fish embryos based on integrated density values. In samples where the fish test showed no estrogenic effect, the liver of the larvae was significantly damaged due to sample toxicity. Our results clearly show that the combination of these methods provides a highly effective screening tool for samples containing high concentrations of organic matter.
Zearalenone (ZEN) is a common contaminant in animal feed, but the adverse effects of ZEN on the intestinal microbiota of fish have not yet been investigated. To reveal the effects of ZEN on serum biochemistry, hepatic and intestinal histology, and intestinal microbiota of fish, Dabry′s sturgeon (Acipenser dabryanus) received feed containing 1030 μg kg⁻¹ ZEN (ZEN-treated group) for 7 weeks and were compared to a control feed that have not been fortified with ZEN (control group). The results showed that dietary supplementation with ZEN did not significantly affect the serum contents of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, high-density lipoprotein cholesterol (HDLC) and estradiol (E2; p > .05). The serum contents of total protein, albumin, triglycerides, total cholesterol and low-density lipoprotein cholesterol in the ZEN-treated group were significant lower than the control group (p < .05). The serum contents of glucose in the ZEN-treated group was significant higher than the control group (p < .05). Intestinal histology was normal in the ZEN-treated group with comparsion to the control group. Compared to the control group, the appearance of nuclear pyknosis and vacuoles in the hepatic cell in the ZEN-treated group. The α-diversity index (Chao 1, Faith pd and Shannon diversity index) significant increased in the ZEN-treated group compared to the control group (p < .05). Simpson diversity index was not affected by the dietary ZEN-treated (p > .05). Principal coordinates analysis (PCA) showed different clustering of prokaryotic communities in the ZEN-treated group compared to the control group. Further Anosim (Analysis of similarities) found that significnat differences in species community structure composition (R > .44) between the control group and the ZEN-treated group (p < .05). The number of operational taxonomic units (OTUs) was decreased after Dabry's sturgeon were fed diets supplemented with ZEN. At the genus level, the differences in the relative abundances of the first 20 main microbiota genera were considerable. In summary, this study suggests that dietary containing 1030 μg kg⁻¹ ZEN may be interfere with physiological parameters, and also affect the intestinal microbiota α-diversity, the numbers of OTUs and the microbiota compostion at the genus level of the juvenile Dabry′s sturgeon.
The occurrence of immuno-compromised status in animals with zearalenone (ZEA) exposure may be a critical contributor to associated mucosal (gastrointestinal tract) diseases. However, it is difficult to assess the associated risks with limited reference data. This study comprehensively discussed the effects of ZEA on intestinal immune components, cytokines and molecular mechanism of juvenile grass carp infected with Aeromonas hydrophila. Specifically, the fish were fed six graded levels of dietary ZEA (0-2507 μg kg-1 diet) for 70 d. The results pointed out that the average residual amount of ZEA in the intestines increased with dose level after ZEA feeding. We further performed an infection assay using A. hydrophila. After 14 d, ZEA groups increased enteritis morbidity rate compared with controls. The acid phosphatase (ACP), lysozyme (LZ) activities and immunoglobulin M (IgM) content were significantly decreased in three intestinal segments. Furthermore, ZEA could reduce the transcription of β-defensin-1, Hepcidin, liver expressed antimicrobial peptide 2A/2B (LEAP-2A/2B) and Mucin-2. We next confirmed the loss of these immune components accompanied by the invasion of the intestinal barrier by bacteria, as indicated by activation of the nuclear factor κB (NF-κB) and the expression of downstream cytokines. Notably, the phosphorylated target of rapamycin (TOR) plays an important role in regulating these genes, thus indicating a possible target caused by ZEA. In summary, the extensive inhibition of immune components by ZEA promotes the spread of pathogens, which may increase the possibility of intestinal mucosa exposure and the risk of transforming disease.
Endocrine disrupting compounds and in particular estrogenic substances have the ability to interact with the hormone system of organisms. Among them are not only synthetic but also natural substances that potentially stress the aquatic ecosystem. High human population densities such as around the Pearl River Estuary (PRE) are suspected of exerting significant anthropogenic pressure onto coastal areas. In this study, natural and synthetic estrogens as well as estrogen-like substances derived from plants and fungi were investigated in the PRE and at the adjacent northern shelf of the South China Sea. Maximum concentration of 3.6 ng L⁻¹ for estrone (E1), 0.7 ng L⁻¹ for 17α-ethinylestradiol (EE2), 12.9 ng L⁻¹ for genistein (GEN), 11.9 ng L⁻¹ for daidzein (DAI) and 1.9 ng L⁻¹ for zearalenone (ZEN) were observed. While E1 and EE2 were detected in fresh and saltwater samples, GEN, DAI and ZEN were observed only at freshwater sampling sites. During the investigations, the analysis of 17β-estradiol (E2) and EE2 indicated a strong matrix dependence. Additionally, an estrogen screen observation showed estrogenic activity in form of estradiol equivalent quotients up to 0.18 ng L⁻¹.
In this study, laboratory experiments have addressed the acute toxicity of two common mycotoxins, deoxynivalenol (DON) and zearalenone (ZON), in a range of freshwater organisms (including rotifers Brachionus calyciflorus, insects Chironomus riparius (larvae), crustaceans Daphnia pulex and Thamnocephalus platyurus, cnidarians Hydra vulgaris, molluscs Lymnaea stagnalis (embryos) and Protozoa Tetrahymena thermophila). Acute EC50 values highlight crustaceans as the most sensitive organisms to DON, with T. platyurus having a 24 h EC50 of 0.14 and D. magna having a 48 h EC50 of 0.13 mg DON/L. During exposures to ZON, H. vulgaris and L. stagnalis embryos showed the highest sensitivity; mortality EC50 values were 1.1 (96 h) and 0.42 mg ZON/L (7 d), respectively. Combining these novel invertebrate toxicity results, along with recent published data for freshwater plant and fish toxicity for analysis of Species Sensitivity Distributions, provides freshwater HC5 values of 5.2 μg DON/L and 43 μg ZON/L, respectively. Using highest reported environmental concentrations and following REACH guidelines, risk ratios calculated here show the risk of ZON to freshwater organisms is low. In contrast, DON may periodically because for concern in streams subject to high agricultural run-off, likely during certain times of year where cereal crops are susceptible to higher fungal infections rates and may pose increased risks due to climate change.
The presence of mycotoxins in food has created concern. Mycotoxin prevalence in our environment has changed in the last few years maybe due to climatic and other environmental changes. Evidence has emerged from in vitro and in vivo models: some mycotoxins have been found to be potentially carcinogenic, embryogenically harmful, teratogenic, and to generate nephrotoxicity. The risk assessment of exposures to mycotoxins at early life stages became mandatory. In this regard, the effects of toxic compounds on zebrafish have been widely studied, and more recently, mycotoxins have been tested with respect to their effects on developmental and teratogenic effects in this model system, which offers several advantages as it is an inexpensive and an accessible vertebrate model to study developmental toxicity. External post-fertilization and quick maturation make it sensitive to environmental effects and facilitate the detection of endpoints such as morphological deformities, time of hatching, and behavioral responses. Therefore, there is a potential for larval zebrafish to provide new insights into the toxicological effects of mycotoxins. We provide an overview of recent mycotoxin toxicological research in zebrafish embryos and larvae, highlighting its usefulness to toxicology and discuss the strengths and limitations of this model system.
Com a intensificação produtiva da piscicultura, a substituição parcial ou total da farinha de peixe por ingredientes de origem vegetal tornou-se realidade na indústria de ração, com o objetivo de reduzir custos. No entanto, essa prática aumentou o impacto da contaminação por micotoxinas. Estudos demonstraram que as micotoxinas podem induzir vários distúrbios em peixes, como alterações celulares e orgânicas, além de prejudicar o desenvolvimento funcional e morfológico e, em casos mais graves, a mortalidade. Assim, estudos foram realizados para avaliar e desenvolver estratégias para prevenir a formação de micotoxinas, bem como para induzir sua eliminação, inativação ou redução de sua disponibilidade em alimentos para animais.
Zearalenone is a xenoestrogenic mycotoxin produced by Fusarium species. High exposure with zearalenone induces reproductive disorders worldwide. Cyclodextrins are ring-shaped host molecules built up from glucose units. The apolar cavity of cyclodextrins can entrap so-called guest molecules. The formation of highly stable host-guest type complexes with cyclodextrins can decrease the biological effect of the guest molecule. Therefore, cyclodextrins may be suitable to decrease the toxicity of some xenobiotics even after the exposure. In this study, the protective effect of beta-cyclodextrins against zearalenone-induced toxicity was investigated in HeLa cells and zebrafish embryos. Fluorescence spectroscopic studies demonstrated the formation of stable complexes of zearalenone with sulfobutyl-, methyl-, and succinyl-methyl-substituted beta-cyclodextrins at pH 7.4 (K = 1.4-4.7 × 10^4 L/mol). These chemically modified cyclodextrins considerably decreased or even abolished the zearalenone-induced loss of cell viability in HeLa cells and mortality in zebrafish embryos. Furthermore, the sublethal effects of zearalenone were also significantly alleviated by the co-treatment with beta-cyclodextrins. To test the estrogenic effect of the mycotoxin, a transgenic bioindicator zebrafish model (Tg(vtg1:mCherry)) was also applied. Our results suggest that the zearalenone-induced vitellogenin production is partly suppressed by the hepatotoxicity of zearalenone in zebrafish. This study demonstrates that the formation of stable zearalenone-cyclodextrin complexes can strongly decrease or even abolish the zearalenone-induced toxicity, both in vitro and in vivo. Therefore, cyclodextrins appear as promising new mycotoxin binders.
Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin synthesized in Fusarium species, mainly F. graminearum and F. culmorum, and it has strong estrogenic activity and causes genotoxic effects, reproductive disorders and immunosuppressive effects. Neutrophil extracellular trap (NET) have been studied for many years. Initially, NET were considered a form of the innate response that combats invading microorganisms. However, NET are involved in a series of pathophysiological mechanisms, including thrombosis, tissue necrosis, autoinflammation, and even autoimmunity. We recently found that polymorphonuclear neutrophils (PMNs) response to ZEA exposure by undergoing NET formation. However, the molecular mechanisms involved in this process remain poorly characterized. Here, we analyzed whether estrogen receptors (ERs) could affect NET formation after ZEA stimulation. The involvement of ERs was investigated with the selective ER antagonists. Moreover, we investigated the mechanisms of NET formation using immunofluorescence staining, fluorescence microplate and Western blot analysis. Our results showed that ERs (ERα and ERβ) were not involved in ZEA-induced NET, but reactive oxygen species (ROS), ERK and p38 were postulated to be involved. Specifically, we provided data demonstrating that ZEA-induced reactive oxygen species (ROS) might promote activation of ERK and p38 as well as subsequent NET release. We are the first to demonstrate this new mechanism of ZEA-induced NET formation, which might help in understanding the role of ZEA in overexposure diseases and provide a relevant basis for therapeutic applications.
Mycotoxins are an important class of chemicals of emerging concern, recently detected in aquatic environments, potentially reflecting the influence of fungicide resistance and climatic factors on fungal diseases in agricultural crops. Zearalenone (ZON) is a mycotoxin formed by Fusarium spp. and is known for its biological activity in animal tissues; both in vitro and in vivo. ZON has been reported in US and Polish surface waters at 0.7 − 96 ng/L, with agricultural run-off and wastewater treatment plants being the likely sources of mycotoxins. As some mycotoxins can induce phytotoxicity, laboratory studies were conducted to evaluate the toxicity of ZON (as measured concentrations) to freshwater algae (Pseudokirchneriella subcapitata) and macrophytes (Lemna minor) following OECD test guidelines 201 and 221, respectively. Zinc sulphate was used as a positive control. In the OECD 201 algal static study (72 h at 24 ± 1 °C), exposure to ZON gave average specific growth rate (cell density) EC50 and yield (cell density) EC50 values of > 3.1 and 0.92 (0.74 − 1.8) mg/L, respectively. ZON was less toxic in the OECD 221 static study and after 7 d at 24 ± 1 °C. L. minor growth was significantly reduced based on frond number and frond area at 11.4 mg ZON/L, showing a higher tolerance than reported for other mycotoxins with Lemna spp. Chlorophyll fluorescence parameters were used as biomarkers of impacts on photosystem II efficiency, with no effect seen in algae but, with responses being observed in L. minor between 5.2 − 14.4 mg ZON/L. ZON toxicity seen here is not of immediate concern in context with environmental levels, but this study highlights that other freshwater organisms including algae are more sensitive to mycotoxins than Lemna sp., the only current source of toxicity data for freshwater plants.
Mycotoxins are secondary metabolites produced by varieties of fungi that contaminate food and feed resources and are capable of inducing a wide range of toxicity. In the current study, we investigated developmental and behavioural toxicity in zebrafish larvae after exposure to six different mycotoxins; ochratoxin A (OTA), type A trichothecenes mycotoxin (T-2 toxin), type B trichothecenes mycotoxin (deoxynivalenol - DON), and zearalenone (ZEN) and its metabolites alpha-zearalenol (α-ZOL) and beta-zearalenol (β-ZOL). Developmental defects, hatching time, and survival were monitored until 96 h post fertilisation (hpf). The EC50, LC50, and IC50 values were calculated. Subsequently, to assess behavioural toxicity, new sets of embryos were exposed to a series of non-lethal doses within the range of environmental and/or developmental concern. Results indicated that all the tested mycotoxins were toxic, they all induced developmental defects, and with the exception of OTA, all affected hatching time. Behavioural effects were only observed following exposure to OTA and ZEN and its metabolites, α ZOL and β ZOL. These results demonstrate that mycotoxins are teratogenic and can influence behaviour in a vertebrate model.
In the article the method of the analysis of zearalenone in the aqueous environment using GC-MS chromatography with electron ionization (EI) is described. The separation of zearalenone from the aqueous matrix was performed by means of solid phase extraction with the use of Supelclcan™ ENVI-18 tubes. Extraction efficiency was determined by the analysis of deionized and tap water samples of volume ranging from 100 to 500 cm3, and zearalenone concentrations varying from 50 to 200 ng/dm3. Chromatography analysis was preceded by the dcrivatization of samples via silylation reaction using a ternary mixture of BSTFA/TMCS/DTE. The developed method enabled us to determine the content of zearalenone in aqueous environment at a concentration level from 0.3 to 0.5 ng/dm3, with the repeatability of the analysis 4-8%. The efficiency of the extraction exceeded 62% and depended on the investigated matrix and the volume of analyzed sample. In the final stage of the study the analysis of 500 cm3 samples of natural water collected in the Upper Silesia Region was performed. The determined concentration of zearalenone was in the range from 0 to 3.2 ng/dm3, depending on the character of the analyzed sample.
Zebrafish (Danio rerio) embryos treated with a small plant molecule, genistein generated a mimic of zebrafish VEGF-A loss of function model. These embryos develop with an enlarged pericardium and major blood vessel deficiencies and their morphological assessment after 48 h post fertilization indicates a nearly complete absence of both axial and intersegmental vasculature with no or reduced numbers of circulating red blood cells resulting in distinctive phenotype of "heart and soul". Thus, zebrafish provides an excellent vertebrate model system to study developmental process and identify novel genes involved during development.
Fungi cause human illness in different ways. Mycoses are the best-known diseases of fungal etiology, but toxic secondary metabolites produced by saprophytic species are also an important health hazard. The term mycotoxin is an artificial rubric used to describe pharmacologically active mold metabolites characterized by vertebrate toxicity. They fall into several chemically unrelated classes, are produced in a strain-specific way, and elicit some complicated and overlapping toxigenic activities in sensitive species that include carcinogenicity, inhibition of protein synthesis, immunosuppression, dermal irritation, and other metabolic perturbations. Mycotoxins usually enter the body via ingestion of contaminated foods, but inhalation of toxigenic spores and direct dermal contact are also important routes. It is difficult to prove that a disease is a mycotoxicosis. Molds may be present without producing any toxin. Thus, the demonstration of mold contamination is not the same thing as the demonstration of mycotoxin contamination. Moreover, even when mycotoxins are detected, it is not easy to show that they are the etiological agents in a given veterinary or human health problem. Nevertheless, there is sufficient evidence from animal models and human epidemiological data to conclude that mycotoxins pose an important danger to human and animal health, albeit one that is hard to pin down. The incidence of mycotoxicoses may be more common than suspected. It is easy to attribute the symptoms of acute mycotoxin poisoning to other causes; the opposite is true of etiology. It is not easy to prove that cancer and other chronic conditions are caused by mycotoxin exposure. In summary, in the absence of appropriate investigative criteria and reliable laboratory tests, the mycotoxicoses will remain diagnostically daunting diseases.
This study provides the first broad-scale investigation on the spatial and temporal occurrence of phytoestrogens and mycotoxins in streams in the United States. Fifteen stream sites across Iowa were sampled five times throughout the 2008 growing season to capture a range of climatic and crop-growth conditions. Basin size upstream from sampling sites ranged from 7 km2 to > 836,000 km2. Atrazine (herbicide) also was measured in all samples as a frame-of-reference agriculturally derived contaminant. Target compounds were frequently detected in stream samples: atrazine (100%), formononetin (80%), equol (45%), deoxynivalenol (43%), daidzein (32%), biochanin A (23%), zearalenone (13%), and genistein (11%). The nearly ubiquitous detection of formononetin (isoflavone) suggests a widespread agricultural source, as one would expect with the intense row crop and livestock production present across Iowa. Conversely, the less spatially widespread detections of deoxynivalenol (mycotoxin) suggest a more variable source due to the required combination of proper host and proper temperature and moisture conditions necessary to promote Fusarium spp. infections. Although atrazine concentrations commonly exceeded 100 ng L(-1) (42/75 measurements), only deoxynivalenol (6/56 measurements) had concentrations that occasionally exceeded this level. Temporal patterns in concentrations varied substantially between atrazine, formononetin, and deoxynivalenol, as one would expect for contaminants with different source inputs and processes of formation and degradation. The greatest phytoestrogen and mycotoxin concentrations were observed during spring snowmelt conditions. Phytoestrogens and mycotoxins were detected at all sampling sites regardless of basin size. The ecotoxicological effects from long-term, low-level exposures to phytoestrogens and mycotoxins or complex chemicals mixtures including these compounds that commonly rake place in surface water are poorly understood and have yet to be systematically investigated in environmental studies.
In our country, there is present extensive, semi-intensive and intensive growing of cyprinid fish species. The quality of food is an essential prerequisite for obtaining optimal production results in fish production. Fish food is being produced as a complete pellet meal, and raw materials used are of plant, animal, mineral and vitamin origin. Out of plant feed, the most commonly used ones are corn, wheat, barley, oats, soy and others. By applying additional carbohydrate food, energetic needs of an organism are being met. In this paper, we presented the results of hygienic safety of carbohydrate feed (corn, wheat, barley) investigated in the laboratory of Veterinary Research Institute of Serbia in Belgrade within regular control, or with the aim of establishing the causes of disturbance of health status and decreased production results in the pond. During 2004 we performed microbiology and mycotoxicology investigations of the total of 43 samples, namely: 31 corn samples, 8 barley samples and 4 wheat samples. The obtained results point at a high level of mould contamination (Aspergillus, Penicillium, Fusarium, and Rhizopus) and the presence of their secondary mycotoxin metabolites (aflatoxin, ochratoxin, trichothecenes and zearalenone) in feed.
The nuclear receptors encompass a group of regulatory proteins involved in a number of physiological processes. The estrogen receptors (ERs), of which one alpha and one beta form exist in mammals function as transcription factors in response to 17beta-estradiol (E2). In zebrafish there are three gene products of estrogen receptors and they are denoted esr1 (ERalpha), esr2a (ERbeta2) and esr2b (ERbeta1). Total RNA of zebrafish early life stages (<3, 6, 12, 24, 48, 72, 96 and 120 hours post fertilization) and of adult fish (liver, intestine, eye, heart, brain, ovary, testis, gill, swim bladder and kidney) were isolated following in vivo exposures. Using specific primers for each of the three zebrafish ERs the expression levels were quantified using real time PCR methodology. It was shown that in absence of exposure all three estrogen receptors were expressed in adult fish. The levels of expression of two of these three ER genes, the esr1 and esr2a were altered in organs such as liver, intestine, brain and testis in response to ligand (E2, diethylstilbestrol or 4-nonylphenol). During embryogenesis two of the three receptor genes, esr1 and esr2b were expressed, and in presence of ligand the mRNA levels of these two genes increased. The conclusions are i) estrogen receptor genes are expressed during early development ii) altered expression of esr genes in response to ligand is dependent on the cellular context; iii) the estrogenic ligand 4-nonylphenol, a manufactured compound commonly found in sewage of water treatment plants, acts as an agonist of the estrogen receptor during development and has both agonist and antagonist properties in tissues of adult fish. This knowledge of esr gene function in development and in adult life will help to understand mechanisms of interfering mimicking endocrine chemicals in vivo.
The objective of this study was to characterize the mechanisms of action of the model environmental toxicant methyl mercury (MeHg) in the zebrafish embryo. Zebrafish embryos were exposed to MeHg, and the effective concentration and window of exposure were determined in wild-type and fluorescent reporter transgenic zebrafish embryos. Genes were systematically assessed for altered expression in response to MeHg by in situ hybridization. MeHg impairs development of the fin fold and the tail fin primordium. Alterations in transgene expression were noted at 6 μg/l MeHg, making this shh:gfp line the most sensitive biosensor of MeHg exposure. The matrix metalloproteases mmp9 and mmp13 and eight other genes are induced in the embryonic tail in response to MeHg. Our data suggest that MeHg impairs tail development at least partially by activation of the tissue remodeling proteases Mmp9 and Mmp13. © The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected]
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Zearalenone is a mycotoxin produced by some Fusarium species in food and feed. Toxicity of ZEA and its metabolites is related to the chemical structure of the mycotoxins, similar to naturally occurring estrogens. Currently, there is increasing awareness of the significance of fungi and their toxic metabolites in water. Thus it was considered essential to determine the concentration of estrogenic toxin in surface waters, groundwater and wastewater in Poland. The application of ZearalaTest immunoaffinity columns for zearalenone determination by HPLC method in water samples characterized by different natural organic matter (NOM) content gave the recovery rate ranging from 74 to 86%. Obtained results showed that the natural organic micropollutants of water with low molecular masses have an effect on zearalenone recovery. Zearalenone was present in water samples in the range from 0 to 43.7 ngL(-1).
A rapidly increasing number of chemicals, or their degradation products, are being recognized as possessing estrogenic activity, albeit usually weak. We have found that effluent from sewage treatment works contains a chemical, or mixture of chemicals, that induces vitellogenin synthesis in male fish maintained in the effluent, thus indicating that the effluent is estrogenic. The effect was extremely pronounced and occurred at all sewage treatment works tested. The nature of the chemical or chemicals causing the effect is presently not known. However, we have tested a number of chemicals known to be estrogenic to mammals and have shown that they are also estrogenic to fish; that is, no species specificity was apparent. Many of these weakly estrogenic chemicals are known to be present in effluents. Further, a mixture of different estrogenic chemicals was considerably more potent than each of the chemicals when tested individually, suggesting that enhanced effects could occur when fish are exposed simultaneously to various estrogenic chemicals (as is likely to occur in rivers receiving effluent). Subsequent work should determine whether exposure to these chemicals at the concentrations present in the environment leads to any deleterious physiological effects.
Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
Three in-vitro bioassays were used to compare the oestrogenic potency of chemicals used as growth promoter in beef cattle
in certain non-European Union countries (17β-oestradiol, α-zearalanol, testosterone, trenbolone, trenbolone acetate, melengestrol
acetate) or found as food contaminant such as the mycotoxin zearalenone and some of their metabolites (17α-oestradiol, oestrone,
17α-epitestosterone, 19-nortestosterone, androstendione, zearalanone, α-zearalanol, β-zearalanol, α-zearalenol, β-zearalenol).
The strong oestrogens 17α-ethinyl oestradiol and diethylstilboestrol were used as standards. The first bioassay was based
on the activation of a reporter gene by oestrogens in recombinant yeast expressing human or rainbow trout oestrogen receptor.
In the second bioassay, the vitellogenin gene induction of rainbow trout hepatocyte cultures was used as a biomarker for the
exposure to oestrogens. The third bioassay was based on the alkaline phosphatase gene induction by oestrogens in the human
endometrial Ishikawa cell line. The assessment of oestrogenic potency of these chemicals clearly demonstrates the strong oestrogenicity
of the mycotoxin zearalenone and its metabolites and particularly α-zearalenol which was as potent as ethinyl oestradiol and
diethylstilboestrol in the human endometrial Ishikawa cell line.
Real-time reverse transcription followed by polymerase chain reaction (RT–PCR) is the most suitable method for the detection
and quantification of mRNA. It offers high sensitivity, good reproducibility and a wide quantification range. Today, relative
expression is increasingly used, where the expression of a target gene is standardised by a non-regulated reference gene.
Several mathematical algorithms have been developed to compute an expression ratio, based on real-time PCR efficiency and
the crossing point deviation of an unknown sample versus a control. But all published equations and available models for the
calculation of relative expression ratio allow only for the determination of a single transcription difference between one
control and one sample. Therefore a new software tool was established, named REST© (relative expression software tool), which
compares two groups, with up to 16 data points in a sample and 16 in a control group, for reference and up to four target
genes. The mathematical model used is based on the PCR efficiencies and the mean crossing point deviation between the sample
and control group. Subsequently, the expression ratio results of the four investigated transcripts are tested for significance
by a randomisation test. Herein, development and application of REST© is explained and the usefulness of relative expression
in real-time PCR using REST© is discussed. The latest software version of REST© and examples for the correct use can be downloaded
at http://www.wzw.tum.de/gene-quantification/.
Last years researches focused on several natural and synthetic compounds that may interfere with the major functions of the endocrine system and were termed endocrine disrupters. Endocrine disrupters are defined as chemical substances with either agonist or antagonist endocrine effects in human and animals. These effects may be achieved by interferences with the biosynthesis or activity of several endogenous hormones. Recently, it was demonstrated that heavy metals such as cadmium (Cd), arsen (As), mercury (Hg), nickel (Ni), lead (Pb) and zinc (Zn) may exhibit endocrine-disrupting activity in animal experiments. Emerging evidence of the intimate mechanisms of action of these heavy metals is accumulating. It was revealed, for example, that the Zn atom from the Zn fingers of the estrogen receptor can be replaced by several heavy metal molecules such as copper, cobalt, Ni and Cd. By replacing the Zn atom with Ni or copper, binding of the estrogen receptor to the DNA hormone responsive elements in the cell nucleus is prevented. In both males and females, low-level exposure to Cd interferes with the biological effects of steroid hormones in reproductive organs. Arsen has the property to bind to the glucocorticoid receptor thus disturbing glucocorticoids biological effects. With regard to Hg, this may induce alterations in male and female fertility, may affect the function of the hypothalamo-pituitary-thyroid axis or the hypothalamo-pituitary-adrenal axis, and disrupt biosynthesis of steroid hormones.
Zearalenone (ZEN) is an estrogenic secondary metabolite produced by certain fungi that commonly infest important cereal crops. The ability of ZEN to move from contaminated crops to surface waters has been demonstrated previously. This article reports the development and application of a method for the measurement of ZEN in surface waters from the central part of Illinois, USA. The method uses a cleanup procedure based on tandem reverse-phase disks and immunoaffinity columns, separation by liquid chromatography and detection by a combination of absorbance and fluorescence. ZEN was frequently found in samples of waters from lakes, streams and a field ditch. Although the frequency of detection was high (32% above the limit of detection, 0.4 ng L(-1)), the levels found were low, with the highest sample having 5.7 ng L(-1). Therefore, although fungi can contribute to the exposure to environmental estrogens, the contribution from zearalenone in water is likely small.
Zearalenone (ZEA) is a macrocyclic beta-resorcylic acid lactone produced by numerous species of Fusarium It frequently contaminates corn and cereal products in many regions of the world The biological activity of ZEA is dominated by its pronounced oestrogenicity, which is even enhanced in certain reductive metabolites This review updates the metabolism in fungi, plants and mammalian systems, as well as the pharmacokinetics of ZEA The present evidence for the hormonal effects of the parent mycoestrogen and some of its metabolites in vitro and in farm and experimental animals in vivo is reviewed, together with its association with endocrine-disruptive effects in humans Possible mechanisms of the oestrogenic and carcinogenic activity of ZEA are discussed and future areas of research proposed
An enzyme-linked immunosorbent assay (ELISA) based oncarp (Cyprinus carpio) vitellogenin (c-VTG, an estrogen-dependent precursor of egg yolk) was developed to quantify VTG in the fathead minnow (Pimephales promelas) and was employed to establish an in vivo testing system for estrogens in fish early life stages. The working range of the c-VTG ELISA was between 1 and 50 ng/ml (20%–80% binding). The inter- and intraassay variations were 9.7 ± 3% and 8.9 ± 3.4% (n = 15), respectively. Plasma and whole body homogenates from females and from estrogenized male fathead minnow diluted parallel with the c-VTG standard. Measurements of plasma VTG fractionated on sodium dodecyl sulphate gels and quantified using densitometry were compared with measurements with the same plasma samples using the c-VTG ELISA and they were similar for the carp and fathead minnow. The plasma concentration of VTG in the male fathead minnow was 25 ± 6 ng/ml, whereas in females, plasma concentrations of VTG ranged from 5.3 ± 1 μg/ml to 182 ± 7.6 μg/ml, depending on the state of maturity. Exposure of fathead minnows to estradiol-17β in the water (nominal concentrations of 25 ng/L, 50 ng/L, and 100 ng/L) from 24 h postfertilization to 30 d posthatch induced VTG synthesis in a dose-dependent manner. Shorter periods of exposure to 100 ng/L estradiol-17β (24 h postfertilization to 10 or 20 d posthatch) similarly induced a vitellogenic response. This work demonstrates that fathead minnow are sensitive to estrogen and are able to synthesize VTG very early in development; this provides the basis for the adaptation of existing fish early-life-stage tests for the in vivo testing of estrogenic chemicals.
Zearalenone (ZEA) is a mycotoxin of worldwide occurrence, and it has been shown to produce numerous adverse effects in both laboratory and domestic animals. However, regardless of recent achievements, the molecular mechanisms underlying ZEA toxicity remain elusive, and little is known about transcriptome changes of fish cells in response to ZEA occurrence. In the present study, differential display PCR was used to generate a unique cDNA fingerprint of differentially expressed transcripts in the liver and ovary of juvenile rainbow trout after either 24, 72, or 168h of intraperitoneal exposure to ZEA (10mg/kg of body mass). From a total of 59 isolated cDNA bands (ESTs), 5 could be confirmed with Real-Time qPCR and their nucleotide sequences were identified as mRNAs of: acty (β-centractin), the cytoskeleton structural element; bccip, responsible for DNA repair and cell cycle control; enoa (α-enolase), encoding enzyme of the glycolysis process; proc (protein C), that takes part in the blood coagulation process; and frih, encoding the heavy chain of ferritin, the protein complex important for iron storage. Further qPCR analysis of the confirmed ESTs expression profiles revealed significant mRNA level alterations in both tissues of exposed fish during the 168h study. The results revealed a complex network of genes associated with different biological processes that may be engaged in the cellular response to ZEA exposure, i.e. blood coagulation or iron-storage processes.
There is increasing concern about man-made chemicals in the aquatic environment that mimic oestrogens because they may disrupt
reproductive function. Vitellogenin, a precursor of egg-yolk in fish and other oviparous animals, may be used as a biomarker
for “oestrogen” exposure. This study investigated the use of a radioimmunoassay developed to carp (Cyprinus carpio) vitellogenin to measure vitellogenin in other species of fish, especially cyprinids that would be of value for field and
laboratory studies on oestrogenic xenobiotics. Of the nine families of fish studied, only vitellogenin from cyprinids (to
which the carp belongs) showed good cross-reactivity in the carp vitellogenin radioimmunoassay. Vitellogenin from cyprinids
native to Europe that cross reacted in the carp vitellogenin radioimmunoassay included: bream (Abramis brama), roach (Rutilus rutilus), rudd (Scardinius erythropthalmus), gudgeon (Gobio gobio) and minnow (Phoxinus phoxinus). Vitellogenin from cyprinids used widely in ecotoxicology that cross reacted in the carp vitellogenin radioimmunoassay included:
fathead minnow (Pimephales promelas), zebrafish (Brachydanio rerio) and goldfish (Carassius auratus). In the cyprinids studies, the concentrations of vitellogenin in mature females were between a few hundred and a thousand
microgram per millilitre. Concentrations of plasma vitellogenin in immature females were always greater than 200 ng·m-1, whereas in males (with the exception of the fathead minnow) plasma vitellogenin concentrations were less than 20 ng·ml-1 (and generally, much lower). The results suggest that the structure of vitellogenin is highly conserved within the cyprinid
family and that the carp vitellogenin radioimmunoassay may be used to measure the concentrations of vitellogenin in plasma
from a wide variety of cyprinids.
In this study, a method for assessing the occurrence of trace amounts of 12 representative estrogenic compounds in sewage and surface waters was developed. The selected substances were the phytoestrogens daidzein, genistein and biochanin A, the alkylphenols bisphenol A and 4-nonylphenol, the natural hormones 17β-estradiol, estrone, estriol and the synthetic hormone 17α-ethynylestradiol and the mycoestrogens zearalenone and two of its metabolites (α-zearalanol and β-zearalanol). The procedure consists in solid phase extraction (SPE) performed with OASIS cartridges followed by liquid chromatography coupled with tandem mass spectrometry (LC–MS–MS). Recoveries were all above 80% for each analyzed aqueous matrices. The developed method was applied to verify the occurrence of endocrine disrupters in environmental samples of sewage influents and effluents of an Italian STP. Phytoestrogens were present in effluents at concentrations ranging from 3 to 83 ng/l, whereas the levels detected for alkylphenols were in the range 13–36 ng/l for bisphenol A and up to 1 μg/l for nonylphenol. Estrogens and zeranols were determined at levels below 30 ng/l. Analysis of a river (Tiber) receiving effluent waters revealed high quantities of bisphenol A (15–29 ng/l) and nonylphenol (up to 1.2 μg/l), whereas the presence of all the other compounds were at levels of few ng/l.
Adult male zebrafish (Danio rerio) were exposed to 17β-estradiol (E2) or 17α-ethinylestradiol (EE2) in flow-through systems for 8 days. This was done to compare the sensitivity of the estrogen inducible vitellogenin (Vtg) biomarker system of this proposed OECD test guideline species to other relevant test species. Vtg was quantified in whole body homogenate by a species-specific ELISA. Actual water concentrations of E2 and EE2 were quantified by LC-MS, with detection limits of 1.0 and 0.6 ng/l, respectively. Vtg induction (LOEC) occurred in whole body homogenate at actual water concentrations of 21 ng E2/l and 3.0 ng EE2/l, respectively. As an alternative to the ANOVA approach, the relationship between the percentage of responding fish (Vtg) and the external E2 or EE2 concentration was determined by logistic regression analysis. Based on the regression analysis, EC-values could be determined: EC10, EC50 and EC90 were 15.4, 41.2 and 67.1 ng E2/l, respectively and 0.92, 2.51 and 4.09 ng EE2/l, respectively. Comparisons of these response limits to corresponding values for rainbow trout (Oncorhynchus mykiss), fathead minnow (Pimephales promelas) and Japanese medaka (Oryzias latipes) revealed the zebrafish as a sensitive test species.
The yolk protein, lipovitellin (Lv) was purified from ovaries of mature female zebrafish (Danio rerio) by gel filtration and anion exchange chromatography. Polyclonal antibodies against Lv were raised in rabbits. Anti-Lv IgG was purified by affinity chromatography. SDS-PAGE followed by Western blotting was performed to analyse the specificity of the antibody and the immunological similarities between Lv and vitellogenin (Vtg). Anti-Lv IgG was used to develop a direct non-competitive sandwich ELISA to measure Vtg concentrations of whole body homogenate (WBH) in zebrafish. The intra- and interassay variabilities were 5.8% and 10.4%, respectively. The sensitivity was 0.2 ng Vtg ml−1 and the practical detection limit was 40 ng Vtg g−1 fish (wet weight). Adult male zebrafish were exposed to a nominal water concentration of 10 ng l−1 of ethinylestradiol (EE2) in a semi-static exposure system for 7 days. Compared with the control group, exposure to 10 ng EE2 l−1 induced a 200-fold increase in Vtg levels.
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 17β-estradiol, diethylstilbestrol and zearalenone (ranking order of sensitivity: E-SCREEN > hER ≈ rtER ≈ 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.
The objectives of this research were: (1) to assess the effects of waterborne 17β-estradiol [E2; (17β)-estra-1,3,5(10)-triene-3,17-diol; CAS RN 50-28-2] on the reproduction of fathead minnows (Pimephales promelas) as a benchmark to which xeno-estrogens can be compared, and (2) to correlate the effects on reproductive function with plasma vitellogenin expression, measured as alkaline-labile phosphorous. Histopathological changes were also noted but are reported elsewhere. Duplicate groups of six fish (3 male and 3 female) were exposed to waterborne E2 at nominal concentrations of 10, 1, and 0.1 nM (2724, 272.4, and 27.24 ng l−1) administered via a flow-through proportional diluter apparatus for 19 days. An ethanol carrier solvent was used at a final tank concentration of 1 ppm v/v in the treated tanks and in the solvent control tanks; the latter did not receive E2. Duplicate control tanks received neither ethanol nor E2. Dissolved E2 concentrations, measured throughout the exposure period using an ELISA, averaged 79% of nominal concentrations in the treated tanks. ELISA-detectable concentrations of E2 were found in all tanks (ranging from 3.5 to 15 ng E2 l−1), including the control and solvent control tanks, which indicated that fish in the untreated tanks may have been the source of some E2. The EC50 (concentration expected to cause 50% effect), based on measured E2 concentrations, for inhibition of egg production was 120 ng E2 l−1 (log10 EC50=2.08±1.22, ±S.E.). The EC50 for induction of vitellogenin (measured as plasma alkaline-labile phosphate) in males was 251 ng E2 l−1 (log10 EC50=2.40±0.33, ±S.E.). No vitellogenin induction plateau was observed in females, therefore no EC50 could be calculated. Egg production, expressed as eggs laid per female, was significantly correlated with plasma vitellogenin in both males (linear r2=0.46, P<0.03) and females (linear r2=0.81, P<0.0004), though the relationship was stronger with female plasma vitellogenin expression than with males. The primary effect of E2 exposure on female fathead minnows appeared to be alteration of the timing of recrudescence including vitellogenin production. Spawning was inhibited in a way that indicated that exposure to waterborne E2 may have `reset' the cycle of recrudescence toward the beginning of the oogenic cycle. Vitellogenin induction in male fathead minnows was strongly correlated with E2 exposure, but less so with egg production. The results of this experiment link a biochemical indicator of waterborne estrogen exposure, vitellogenin, with a reproductive performance indicator, egg production, an important parameter affecting fish populations in the environment.
The study investigated the ability of 34 natural and synthetic chemicals to compete with [3H]17β-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-hERαdef), mouse alpha (GST-mERαdef), 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 (Kd) 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, α-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, α-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.
Zearalenone (ZON) is one of the worldwide most common mycotoxin and exhibits estrogenic activity in the range of natural steroid estrogens. The occurrence of ZON has been reported in soil, drainage water, wastewater effluents, and rivers, but its ecotoxicological effects on fish have hardly been investigated. The consequences of continuous long-term ZON exposure, including a subsequent depuration period, as well as transgenerational effects of F0 short-term exposure on F1 generation were investigated. Effects on growth, reproduction activity, physiology, and morphology of zebrafish (Danio rerio) were examined in a 182 day live-cycle experiment. Life-long exposure to ZON for 140 days increased wet weight, body length, and condition factor of female fish at 1000 ng/L, and sex ratio was shifted toward female from 320 ng/L ZON. Only females at 1000 ng/L ZON revealed a 1.5-fold induction of plasma vitellogenin (VTG). Relative fecundity at 1000 ng/L recovered significantly during the depuration period. An increased condition factor in adult female F1 fish implies that exposure of F0 generation to 1000 ng/L ZON affected growth of F1 generation. A negative correlation between relative fecundity in the F1 generation (all groups exposed to 320 ng/L ZON) and the nominal ZON concentrations of the F0 exposure might indicate an influence of F0 exposure on reproductive performance of F1 generation. No exposure scenario affected fertility, hatch, embryo survival, and gonad morphology of zebrafish. Evaluating the environmental relevance of this data, the risk for fish to be harmed by exposure to ZON solely seems rather marginal, but ZON might contribute to the overall estrogenicity in the environment. © 2011 Wiley Periodicals, Inc. Environ Toxicol 2011.