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Combined toxic effects of T-2 toxin and propiconazole on the early life stages of zebrafish (Danio rerio)
Environmental Sciences Europe volume 35, Article number: 8 (2023)
Agricultural products are frequently co-contaminated by mycotoxins and pesticides. Most studies have only focused on the single toxicities of these chemicals, while their combined effects are neglected. Therefore, we investigated the combined toxic effects of T-2 toxin (T-2) and propiconazole (PRO) on zebrafish (Danio rerio).
Our data exhibited that T-2 had higher toxicity to embryonic fish (96-h LC50 value of 0.39 mg a.i. L−1) than PRO (96-h LC50 value of 17.16 mg a.i. L−1). The mixture of T-2 and PRO showed an acute synergistic effect on zebrafish. Meanwhile, indicators associated with oxidative stress (SOD, Mn-sod, and cat) displayed significant variations in most exposures to T-2 and PRO mixtures (MTP) compared with the single exposures. The expressions of apoptosis-related genes cas3 and cas9 were also substantially elevated in the high-dose MTP exposure compared with the corresponding T-2 exposure. Besides, the expressions of endocrine system-related genes (TRβ, tsh, crh, cyp19a, and vtg1) were markedly varied in most MTP exposures compared with the corresponding single exposures. Our present results suggested that the mixture of T-2 and PRO could cause enormous effects on oxidative stress, cellular apoptosis, and the hypothalamic–pituitary–thyroid/hypothalamic–pituitary–gonadal (HPG/HPT) axis of zebrafish.
Our results provided new insights into the development of combined pollution standards for agricultural products. Taken together, the impact of the combined effects could be considered and regulated as priorities.
Mycotoxins and pesticides are common contaminants in agricultural products, which are widely present in almost all aspects of the production, processing, and storage processes [1]. These chemicals can cause food pollution and exert negative effects on animal and human health [2]. Previous surveys have indicated that about 25% of global agricultural products are polluted by mycotoxin each year, and long-term exposure to them presents a substantial risk to human health [3, 4]. Pesticides are used to achieve high yields and high quality in agricultural products, and the toxic effects caused by their residues have been reported frequently [5]. Only very few studies have reported the combined effects of pesticides and mycotoxins on animals. Aflatoxin and cypermethrin have been shown to increase mortality in rats after combined exposure compared with their individual exposures [6]. Similarly, patulin, in combination with chlorpyrifos has been reported to synergistically induce hepatotoxicity in AML12 mouse liver cells [7]. Nonetheless, most studies merely focus on the toxicities of pesticides or mycotoxins individually [8,9,10]. Considering that mycotoxins and pesticides are often present as complex mixtures in agricultural products, it is crucial to investigate their combined toxicities to human health [11].
As a secondary metabolite generated by the fungus Fusarium oxysporum, T-2 toxin (T-2) is one of the most toxic food-derived toxins [12, 13]. It has a high heat resistance and non-volatile properties and will not be inactivated during food processing [14, 15]. A previous investigation has demonstrated that T-2 can cause reproductive organ atrophy and a decline in testicular function in mice [16]. In addition, exposure to T-2 increases reactive oxygen species (ROS) production and induces mitochondrial dysfunction in the neuronal cells, suggesting that T-2 has neurotoxicity effects [17]. The increase in leukocyte number and the decrease in bactericidal activity indicate that the T-2 can affect both non-specific cellular and humoral immunity in rainbow trout (Oncorhynchus mykiss) [18]. Ingestion of T-2-contaminated food not only can cause damage to tissues and organs, but also is closely related to degenerative joint diseases, such as Kashin–Beck disease (KBD) [19,20,21]. Propiconazole (PRO) is a triazole fungicide commonly used on corn, wheat, and vegetables worldwide [22]. It is characterized by a long half-life and easy diffusion in the environment [23, 24]. It has been confirmed that PRO can cause neurobehavioral toxicity in mammals [25, 26]. PRO can also disturb the endocrine system and reproductive function of zebrafish [27, 28]. Previous examinations on T-2 and PRO have mainly focused on their individual effects, while their combined toxicities are poorly documented [29, 30].
Recent evidence has exhibited that T-2 and PRO often co-exist in the same agricultural products [31]. Therefore, increasing attention has been paid to their joint toxic effect on human health [32]. Zebrafish (Danio rerio) have the advantages of a short reproductive cycle and a high spawning capacity, making them an ideal model in the field of toxicology [33]. Moreover, its genome is up to 87% homologous relative to the human genome, and the biosynthetic pathway is similar to that of humans [34]. When zebrafish are exposed to contaminants, they can respond physiologically, with gene expression, and behavior [35]. Therefore, the results obtained from zebrafish model research have high compatibility with human detection data and have great reference value [36, 37]. In this context, we determined the changes in enzymatic activity and gene expression when D. rerio were exposed to the mixture of T-2 and PRO. The data obtained would be helpful in understanding the overall toxicity of T-2 and PRO mixtures for zebrafish and promote the further exploration of the underlying hazards in other co-existing mycotoxins and pesticides for agricultural products. In addition, EU (EC) 396/2005 stipulates that the synergistic effects of multiple pesticides should be considered when developing pesticide MRL (maximum residue limit) standards [38]. Therefore, combined toxicity assessment of mycotoxins and pesticides played a vital role in developing more realistic agricultural product quality standards and monitoring guidelines.
Materials and methods
T-2 (purity ≥ 98%, CAS number: 21259-20-1) was obtained from Sangon Biotech Co., Ltd. (Shanghai, China). PRO (purity ≥ 99%, CAS number: 60207-90-1) was purchased from Hubei Marvel Co., Ltd. (Wuhan, China). The stock solutions of chemical were formulated in dimethyl sulfoxide (DMSO, purity > 99.9%; Amresco, Solon, OH, USA) and 10% Tween-80 (Wt: Vol). All stock solutions were diluted with reconstituted water. The composition of reconstituted water was 2 mmol/L Ca2+,0.5 mmol/L Mg2+, 0.75 mmol/L Na+, and 0.074 mmol/L K+ [39].
Collection of the zebrafish embryos
Adult zebrafish of the AB strain were purchased from the Zebrafish Research Center of China (Wuhan, China) and were maintained in a recirculation system at 27 ± 1 ℃ with a 14/10-h light/dark photoperiod. The fish were fed a commercial brine shrimp flake twice daily. Mature female and male zebrafish (ratio of 1:2) were placed in the egg-laying boxes to obtain embryos overnight. Light stimulation was initiated the next morning, and the eggs were collected 30 min later. Normal embryos were selected for subsequent experiments by microscopic observation. All procedures were authorized by the Independent Animal Ethics Committee of the Zhejiang Academy of Agricultural Science.References: Citation details for Reference [38, 39, 40] are incomplete. Please supply the complete details of this reference. Otherwise, kindly advise us on how to proceedThe three references are the standards of EC, ISO and OECD, respectively. They are okay.
Acute toxicity test
Individual toxicity assay
Acute toxicity assay of individual chemicals was performed according to the previous experimental protocol and OECD test guideline 236 [40]. In order to maintain the appropriate concentration of chemical and water quality, the semi-static method was adopted. All exposure solutions were refreshed every 24 h. Healthy embryos at 3 h post-fertilization (hpf) were placed into 96-well plates, and each well contained one embryo and 0.2 mL of exposure solution. Reconstituted water was set to the blank control. Six different concentrations with a geometrical ratio that caused 10%-90% mortality according to the results from pre-trials were set for T-2 and PRO exposures, respectively. Each concentration consisted of three replicates. Each replicate contained 24 embryos. Mortality was recorded after 96 h of exposure.
Mixture toxicity assay
The interaction mode of T-2 and PRO was assessed. Significant level of mean separation (P < 0.05) was set based on non-overlap between the 95% confidence interval of two LC50 values. Briefly, zebrafish embryos at 3 hpf were exposed to T-2 and PRO mixtures at a fixed equitoxic constant mixture ratio with serial dilutions based on the determined individual LC50 values [41]. The other procedures of the equitoxic assay were similar to those of the individual assay.
The combined toxicity was evaluated according to the additive index method of Markings [42]. The formula is as follows:
S is the sum of the additive effect of biotoxicity; Ai and Bi are the LC50 values of toxicity of the individual A and B toxicants, respectively; and Am and Bm are the LC50 values of A or B in the mixture, respectively. When S ≤ 1: AI = 1/S − 1.0; when S > 1: AI = 1.0-S. The summation index method (AI) was used to assess the combined effects of pesticides: AI > 0.25 for synergistic effects, AI ≤ − 0.2 for antagonistic effects, and − 0.2 < AI ≤ 0.25 for additive effects [43].
Cell- and gene-level determinations
Exposure protocols
Low, middle, and high concentrations were set according to 1/320, 1/80, and 1/20 of 96-h LC50 of T-2 and PRO single concentrations, respectively. The low, middle, and high concentrations of the mixture were combinations of T-2 and PRO (MTP) at the low, middle, and high concentrations, respectively. That is, the nominal concentration of T-2 was 1.2 μg a.i. L−1, 4.8 μg a.i. L−1 and 19.5 μg a.i. L−1; the nominal concentration of PRO was 53.6 μg a.i. L−1, 214.5 μg a.i. L−1 and 858 μg a.i. L−1; the nominal concentration of MTP was (1.2 + 53.6) μg a.i. L−1, (4.8 + 214.5) μg a.i. L−1, (19.5 + 858) μg a.i. L−1.
Final DMSO concentrations were 0.05% in the exposure solution. The use of 0.05% DMSO as a solvent control in the pre-experimental stage did not significantly differ in enzyme activity and gene expression compared to the blank control. Therefore, reconstituted water was used as blank control in this study.
Sample collection
Normally developed embryos (3 hpf) were chosen and placed into crystal dishes containing 500 mL of solution, with about more than 300 embryos in each dish as a replicate. Three replicates were given for each concentration. Dead zebrafish have been removed immediately from the exposure solution. The exposed embryos were placed in an incubator at 27 ± 1 °C for 7 days with a light–dark cycle of 14 h:10 h. In order to maintain the concentration of chemical and water quality, the chemical solutions were refreshed every 24 h in these experiments. After the exposure, zebrafish from each treatment group were collected, 250 fish were designated for determining enzyme activity, and 50 fish were used for RNA extraction. The captured zebrafish were kept in a freezer at − 80 °C.
Cell-level determinations
Briefly, 250 fish were placed into a 2.0-mL centrifuge tube at 7 dpf. Each tube was homogenized (1:10, w/v) using 1 mL PBS (pH 7.4) and centrifuged at 12,000 rpm for 15 min at 5 ℃. After centrifugation, the supernatant was transferred into a new tube, and the biomarker assay was performed immediately.
The activities of SOD, CAT, CYP450, GST, caspase 3, and CarE were measured by respective commercial kits (Shanghai Sangon Biotech Co., Ltd., Shanghai, China) based on the manufacturer's instructions. The content of MDA was also detected using reagent kits from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). In addition, the content of VTG was measured by adopting enzyme-linked immunosorbent assay (ELISA) kits (Enzyme-linked Biotechnology Co., Ltd., Shanghai, China). Protein concentration was determined by a BCA method (Beyotime Biotech Co., Ltd., Shanghai, China). The data of all the biomarkers were normalized to total protein content.
Gene expression analysis
The tested genes, including detoxification-related genes, endocrine disruption-related genes, and immune-related genes, were detected by quantitative real-time PCR as previously described [44]. Briefly, 50 larvae at 7 dpf were used for RNA extraction. Total mRNA was extracted from each sample using the TransZol reagent (Transgen Biotech Co., Ltd., Beijing, China). The first-strand cDNA was synthesized with a vazyme kit (Vazyme Biotech Co., Ltd., Nanjing, China). β-Actin was adopted as the housekeeping gene. Sequences of gene-specific primers were selected according to previous studies [45,46,47,48]. The primer sequences of genes are shown in Additional file 1: Table S1. The relative expression levels of the target genes were calculated by adopting the 2−ΔΔCt method [49].
Chemical analysis
To verify the actual, T-2, PRO and their mixture exposure solution were determined at the start (0 h) and before water renewal (24 h) during the experimental period. Every concentration has three replicates. The concentration of exposure solution was detected by liquid chromatography–tandem mass spectrometry (SHIMADZU, LC–MS-8050) with a mobile phase composed of an aqueous solution (5 mmol/L ammonium formate (A) and methanol (B). The separation was conducted using a gradient elution program as follows: 0–0.5 min, 95% A; 0.5–2.0 min, 5% A; 2.0–5.0 min, 5% A; 5.0–5.1 min, 95% A; 5.1–7.0 min, 95% A. Mass spectrometric detection was conducted in positive electrospray ionization (ESI) with multiple reaction monitoring (MRM) modes. For T-2, and the product ions were m/z 484.4/245.3 for quantization and m/z 484.4/215.5 for confirmation with collision energies of 27 and 25 eV, respectively. For PRO, the product ions were m/z 172.9/145.0 for quantization and m/z 172.9/74 for confirmation with collision energies of 16 and 11 eV, respectively. Analysis results revealed that the deviations between the nominal and actual concentrations of T-2, PRO, and their mixtures were less than 20%. Therefore, the nominal concentration was used as the actual concentration in the present study.
Statistical analysis
The acute toxicities of mycotoxin and pesticides to zebrafish were analyzed according to the procedure developed by Chi [50]. Data were expressed as means ± standard error of the mean (SEM). All data were statistically analyzed using the SPSS 18.0 software. One-way analysis of variance (ANOVA) for multiple groups and Dunnett's post hoc comparison for two treatments were conducted to compare the data. In all cases, P < 0.05 was considered statistically significant. The ΔCt method of the reference gene was used to calculate the expression of interest genes, and performed logarithmic conversion. The housekeeping gene (β-actin) was applied relative to Ct Q. Statistical analysis of the qPCR with 2−ΔΔCt method [49]. Gene data of the heatmap were transformed for Log10 and homogenized.
Determinations of single and joint toxicities
Table 1 presents the acute toxicity of T-2 and PRO in zebrafish embryos. Results exhibited that the 96-h LC50 value of T-2 and PRO was 0.39 mg a.i. L−1 and 17.16 mg a.i. L−1, respectively, indicating that T-2 had higher toxicity to zebrafish embryos compared with PRO. To explore the combined effect, we also tested the LC50 values of T-2 and PRO in their mixture after 96 h of exposure. Based on the LC50 values of T-2 and PRO individually and in the mixture, the mixture of T-2 and PRO exerted acute synergetic toxicity to zebrafish with an AI value of 0.63. The mortalities of every chemical to zebrafish larvae, individually and combined, are presented in Additional file 1: Table S2.
Determinations of biochemical level
Determinations of oxidative stress
The SOD activity was markedly increased in all the individual exposures (except for the high-dose PRO exposure) compared with the control group. By contrast, a remarkable decrease was found in the low-dose MTP exposure compared with the corresponding T-2 and PRO exposures. Its activity was also prominently weakened in the middle- and high-dose MTP exposures compared with the corresponding T-2 exposures (Fig. 1A). The MDA content was dramatically reduced in all the individual exposures (except for the low-dose PRO exposure) and the high-dose MTP exposure compared with the control group. In contrast, a significant enhancement was noticed in the middle-dose MTP exposure compared with the control group. Its content was also dramatically elevated in the middle-dose MTP exposure compared with the individual exposures (Fig. 1B). The CAT activity was steeply increased in the low- and middle-dose T-2 exposures, as well as the high-dose PRO exposure, compared with the control group. However, a marked weakening was detected in the low-dose MTP exposure compared with the corresponding T-2 exposure (Fig. 1C).
Apoptotic and detoxification enzyme activities
The caspase 3 activity was raised in all MTP exposures compared with the control group. A rise was also registered in the low- and middle-dose MTP exposures compared with the corresponding T-2 exposures (Fig. 2A). The activity of CYP450 was inhibited in all the T-2 and PRO exposures (except for the middle-dose PRO exposure) compared with the control group. Contrarily, its activity was noticeably increased in the middle-dose MTP exposure compared with the corresponding PRO exposure (Fig. 2B). The activity of CarE was considerably stimulated in the middle-dose T-2 exposure compared with the control group. Nevertheless, a considerable decrement was monitored in the middle-dose MTP exposure compared with the corresponding T-2 exposure (Fig. 2C). The GST activity was enormously elevated in all the T-2 exposure compared with the control group. Its activity was also more increaser in all the PRO exposure than the control group. Nonetheless, an enormous decline was detected in all the MTP exposures compared with the corresponding T-2 exposures. Moreover, its activity also declined in the low-dose MTP exposure compared with the corresponding PRO exposure (Fig. 2D).
VTG content
The content of VTG was apparently reduced in all individual exposures (except for the middle-dose T-2 and PRO exposures) compared with the control group. An apparent reduction was also monitored in the high-dose MTP exposure compared with the control group. Nonetheless, its content was apparently induced in the high-dose MTP exposure compared with the corresponding T-2 and PRO exposures (Fig. 3).
Gene expression analysis
Expressions of genes related to oxidative stress
The expression of Mn-sod was remarkably diminished in the high-dose T-2 exposure and the middle-dose MTP exposure compared with the control group. A diminution was also seen in the low- and middle-dose MTP exposures compared with the corresponding individual exposures. However, its expression was augmented in the high-dose MTP exposure compared with the corresponding T-2 exposure. Significant increase was also noticed in the low-dose PRO exposure compared to the control (Fig. 4A). The expression of cat was stimulated in the low-dose T-2 and PRO exposures compared with the control group. However, its expression was dramatically decreased in the high-dose PRO exposure and the middle-dose MTP exposure compared with the control group. A dramatic inhibition was also observed in the middle-dose MTP exposure compared with the corresponding individual exposures (Fig. 4B).
Expressions of genes associated with cellular apoptosis and immune system
Figure 5 summarizes the changes in the expressions of genes involved in cellular apoptosis and the immune system. The expression of cas9 was observably diminished in the middle-dose PRO and MTP exposures compared with the control group. Concurrently, a noticeable decline was also seen in the low-dose MTP exposure compared with the corresponding PRO exposure. Conversely, its expression was elevated in the high-dose MTP exposure compared with the corresponding T-2 exposure. The expression of cas9 was also significantly elevated in the low-dose PRO exposure compared with the control group (Fig. 5A). The expression of p53 was notably elevated in the middle- and high-dose T-2 exposures compared with the control group. However, its expression was notably reduced in the middle- and high-dose MTP exposures compared with the corresponding T-2 exposures (Fig. 5B). The expression of bax was notably raised in the low-dose PRO exposure compared with the control group. In contrast, its expression was notably reduced in the low-dose MTP exposure compared with the corresponding PRO exposure (Fig. 5C).
The expression of cas3 was diminished in the high-dose of T-2 exposure and the middle-dose of PRO and MTP exposure compared with the control group. Its expression was diminished in the middle-dose PRO and MTP exposures compared with the corresponding T-2 exposure. A diminution was also noted in all the MTP exposures (except for the high-dose MTP exposure) compared with the corresponding PRO and T-2 exposures. By contrast, its expression was markedly increased in the low-dose PRO exposure compared with the control group (Fig. 5D). The expression of bcl-2 was substantially reduced in the middle-dose PRO and MTP exposures compared with the control group. A substantial reduction was monitored in the high-dose T-2 exposure compared with the control group. However, its expression was substantially elevated in the low- and high-dose MTP exposures compared with the corresponding T-2 exposures (Fig. 5E). The expression of IL-8 was pronouncedly induced in the middle- and high-dose PRO exposures compared with the control group. In contrast, a pronounced down-regulation was monitored in the middle- and high-dose MTP exposures compared with the corresponding PRO exposures (Fig. 5F).
Expressions of genes involved in the endocrine system
The expression of TRα was promoted in the low-dose PRO and MTP exposures compared with the control group. A significant increase was also monitored in the low-dose MTP exposure compared with the corresponding T-2 exposure. Nonetheless, its expression was decreased in the high-dose MTP exposure compared with the corresponding PRO exposure (Fig. 6A). The expression of TRβ was pronouncedly up-regulated in all exposures (except for the high-dose MTP exposure) compared with the control group. On the contrary, a pronounceable inhibition was discovered in the middle- and high-dose MTP exposures compared with the corresponding PRO exposures (Fig. 6B). The expression of tsh was prominently improved in all exposures (except for the high-dose MTP exposure) compared with the control group. However, a steep inhibition was observed in the high-dose MTP exposure compared with the corresponding T-2 and PRO exposures. Its expression was also steeply inhibited in the middle-dose MTP exposure compared with the corresponding PRO exposure (Fig. 6C).
The expression of crh was appreciably enhanced in all exposures compared with the control group. An appreciable enhancement was also noted in the high-dose MTP exposure compared with the corresponding T-2 exposure. In contrast, its expression was appreciably weakened in the high-dose MTP exposure compared with the corresponding T-2 exposure. It was worth noting that the expression of crh increased with the increase of concentration in the T-2 treatment, but its expression was negatively correlated with the dose of PRO and MTP treatment (Fig. 6D). The expression of cyp19a was surprisingly elevated in the low-dose T-2 exposure compared with the control group. In contrast, its expression was surprisingly diminished in the low- and high-dose MTP exposures compared with the control group, as well as the corresponding T-2 and PRO exposures (Fig. 6E). The expression of vtg1 was significantly induced in the individual and MTP exposures (except for the middle-dose T-2 exposure and the high-dose MTP exposure) compared with the control group. An induction was also noticed in the low- and middle-dose MTP exposures compared with the corresponding T-2 exposures. On the contrary, its expression was markedly inhibited in the high-dose MTP exposure compared with the corresponding PRO exposure (Fig. 6F).
Throughout mRNA expression variations
A heatmap analysis consisting of 16 genes was established upon the exposures to T-2, PRO, and their mixtures for exploring the overall changes in gene expressions. The results showed that the distance between the experimental groups was far apart. T-2 and PRO individual exposure groups were closed together, followed by single exposure group and combined exposure group, which can be clustered together. The control group was far away from most treatment groups. It was shown that gene expression in each treatment group was specific compared to the control group. At the gene clustering level, the gene expression of bax and TRα was closed together, meaning that gene expression was similar. In addition, the expression of tsh and vtg1 genes was an up-regulated trend, with consistent expression changes (Fig. 7).
Acute toxicity test on zebrafish provides a fundamental understanding of the primary effects of chemicals [51]. Our results demonstrated that T-2 had stronger acute toxicity to zebrafish compared with PRO. A previous study has demonstrated that the 96-h LC50 value of PRO to zebrafish juveniles is 8.25 mg a.i. L−1 [52], which is not consistent with our data (the 96-h LC50 value of PRO to zebrafish larvae was 17.16 mg a.i. L−1). This slight difference might likely be attributed to the different life stages of zebrafish determined [53]. Another investigation has demonstrated that T-2 has developmental toxicity in zebrafish [54]. In the present study, the synergistic effects of T-2 and PRO suggested that there might be a potential risk in the co-existence of these chemicals in agricultural products. Consequently, it is urgently necessary to elucidate the combined toxic mechanism of T-2 and PRO to avoid underestimating the combined toxicity.
There are oxidation and antioxidant regulation mechanisms in organisms [55]. When exogenous pollutants stimulate the organism, the levels of antioxidant defense factors change, and the organism enters a state of oxidative stress. CAT and SOD are critical antioxidant enzymes [56]. The present study found that the SOD activity was enhanced when exposed to low doses of T-2 and PRO. This transient increase suggests that zebrafish embryos responded to the concentration of T-2 and PRO in the form of antioxidant, which may be a mechanism of self-protection [57]. However, the SOD activity in zebrafish was dramatically inhibited by MTP exposure compared with individual chemicals, suggesting that MTP exposure increased oxidative stress in zebrafish. MDA is the main by-product of lipid peroxidation, and its level may reflect the status of lipid peroxidation in organisms [58]. The content of MDA was significantly induced in the middle-dose MTP exposure, suggesting that combined exposure resulted in lipid peroxidation. Exposure to low-dose T-2 significantly increased the CAT content compared with the control group, which might be attributed to the fact the induced CAT activity abolished oxidative damage. Based on the results above, we deduced that T-2, PRO, and MTP exposures enhanced the ROS production in zebrafish embryos and activated antioxidant defense afterward. As suggested by Li et al. T-2 induces apoptosis through ROS-mediated mitochondrial pathway in poultry [59]. Exposure to PRO induced mouse liver oxidative stress [60]. In contrast, antioxidant responses could not completely eliminate the excess ROS in MTP exposures, rendering oxidative impairment.
In response to exogenous toxic substances, ROS production and apoptosis are closely related in zebrafish [61]. Apoptosis affects the growth and development of organisms, as well as different systemic mechanisms of toxicity. Caspase 3 is an important executioner of apoptosis and is activated to cleaved-caspase 3 in an apoptotic state [62]. In this study, we demonstrated that the caspase 3 activity was notably induced after exposure to MTP compared with the control group, implying that T-2 and PRO exerted a toxic effect on apoptosis in zebrafish embryos. The expression of p53 was considerably enhanced in the medium- and high-dose T-2 exposures compared with the control group, revealing that the apoptotic effect caused by T-2 was enhanced. Our data supported the previous finding of Xian et al. [63] that exposure to T-2 induces apoptosis in the human renal tubular epithelial cells HK-2. An increased amount of ROS can cause apoptosis during embryogenesis [64]. The continuation of apoptosis signaling leads to lipid peroxidation and cellular death [65]. Oxidative stress mediates apoptosis through mitochondria, death receptors (TN-FR1, Fas, TRAILR2, DR3, DR4, DR5), and endoplasmic reticulum stress and may also induce apoptosis by activating the mitogen-activated protein kinase pathway, nuclear transcription factor κB, and caspase [66, 67]. CYP450, CarE, and GST are the primary detoxification enzymes in organisms [68]. CarE catalyzes carboxylate hydrolysis in phase-I biotransformation to produce alcohols and acids. The CarE activity was appreciably increased in the middle-dose T-2 exposure, implying that exposure to the middle-dose T-2 enhanced the detoxification in embryos. GST is a superfamily of enzymes that catalyze detoxification reactions, commonly used as a marker of detoxification in vertebrate and invertebrate studies [69, 70]. The CarE and GST activities were significantly inhibited by simultaneous exposure to T-2 and PRO, suggesting that the detoxification system of zebrafish embryos was compromised when exposed to MTP. We found that exposure to the middle-dose MTP increased the activity of CYP450, which might boost the active metabolism of T-2 and PRO in zebrafish.
VTG is a biomarker of environmental estrogen exposure and provides a source of nutrition for fish embryos at the early stages of larval development [71]. Our results found that the level of VTG was appreciably lower in the low- and high-dose individual chemical exposures compared with the control group. However, it was significantly increased in the high-dose MTP exposure, indicating that T-2 and PRO could greatly affect estrogen levels in zebrafish. T-2 has exhibited a concentration-dependent inhibition effect on the cell reproductive system [72]. Besides, the sex hormone is regulated by the hypothalamic–pituitary–gonadal (HPG) axis. Consequently, our results showed that the zebrafish endocrine system was affected by T-2 and PRO, may leading to disrupted fish reproduction. Apoptosis is continuously induced when cells are subjected to oxidative stress or DNA damage [73]. The expressions of apoptosis-related genes such as p53, bax, cas3, and cas9, were inhibited. In addition, the expressions of cas3 and cas9 were more strongly inhibited in the middle-dose MTP exposure. Additionally, oxidative stress can also affect immune function. The expression of IL-8 was conspicuously decreased in the MTP exposure, indicating that the immune system was compromised when zebrafish were exposed to MTP. T-2 has been shown to have immunomodulatory functions, and in this study, the expression of IL-8 tended to increase in the medium and high concentration T-2 treatments [74]. Its expression was significantly increased in the middle -dose of PRO, indicating that the PRO could lead to immunity boosting. However, with the further increase of PRO concentration, the immune promoting effect tended to decrease, which means that the immune effect in the high concentration of PRO on zebrafish was hindered. Endocrine disruptors interact with endocrine properties in animals by either mimicking or blocking, which can affect organismal immunity, growth, metabolism, and reproduction [75]. Accumulating evidence has summarized that food intake is the primary source of exposure in humans, which can lead to female infertility, precocious puberty, and other related diseases, while such exposure can reduce sperm and sexual functions for men [76, 77]. The six genes associated with endocrine disruption we analyzed were TRα, TRβ, tsh, crh, cyp19a, and vtg1. The HPG is a crucial regulator of reproduction in mammals [78]. Under normal conditions without exposure to external pollutants, the level of vtg in fish larvae is low. When fish larvae are exposed to exogenous environmental pollutants, vtg is synthesized in the liver of fish larvae. Therefore, the gene of vtg has been used as a sensitive molecular biological biomarker for detecting the endocrine-disrupting effects of estrogen. The expression of vtg1 was appreciably increased in all the T-2 and PRO exposures (except for the medium-dose T-2 exposure) compared with the control group, implying that both T-2 and PRO exerted estrogenic disrupting capacity on zebrafish embryos [79, 80]. The expression of vtg1 was also greatly up-regulated in the low- and medium-dose MTP exposures compared with the corresponding single exposures, confirming that T-2 and PRO had an enhancing effect on the estrogen of zebrafish. The gene expression in response to chemical exposure represents the interaction sites of drugs and zebrafish [81]. cyp19a is mainly expressed in the sex gland. It encodes aromatase that alters the ratio of estrogens to androgens in fish, affecting the sex of the fish [82]. Its expression was tremendously down-regulated in the MTP exposure compared with the corresponding T-2 and PRO exposures, indicating that MTP exposure probably affected the reproduction of D. rerio.
The hypothalamic–pituitary–thyroid (HPT) axis regulates fish growth and development [83]. In the HPT axis, corticotropin-releasing crh secreted by the hypothalamus induces the pituitary gland to secrete tsh [84]. The tsh is an essential gene for the secretion of thyroid hormones. Genes of TRα and TRβ are receptors for thyroid hormones [85]. In the present study, the expressions of crh and tsh were conspicuously induced in the T-2 and PRO exposures compared with the control group, indicating that both T-2 and PRO had an inductive effect on the hypothalamus and pituitary gland of zebrafish. However, the expressions of tsh and crh were significantly suppressed when exposed to high-dose MTP compared with the T-2 exposure, implying that a high concentration of T-2 and PRO had negative feedback on the HPT axis. In addition, the expressions of TRα and TRβ were also remarkably altered by the MTP exposures. The varying degrees of inhibition in the high-dose MTP exposure implied that T-2 and PRO had potential disruptive effects on the thyroid gland of zebrafish. Similar to the obtained results, PRO and T-2 have been reported that induced thyroid and metabolism disruption [86, 87]. Inhibition of thyroid axis-related genes might weaken the synthesis and secretion of thyroid hormones, thereby inhibiting the development of fish larvae.
Investigation of mixed toxicity of T-2 and PRO at multiple endpoints contributes to a comprehensive understanding of the effects of mycotoxin and pesticide mixtures on zebrafish [88, 89]. Enzymatic activities of SOD, GST, and caspases 3 were obviously impacted when exposed to T-2 PRO or their mixture. Moreover, genes associated with the endocrine system such as tsh, crh, and vtg1, were significantly changed after exposure to MTP. When an organism is exposed to chemical contaminants, changes in enzyme activity can reflect the degree of cell damage, and gene expression can provide changes at the mRNA level [90]. Therefore, we deduced that the mixture of T-2 and PRO might disturb the growth, development, and reproduction of human beings. However, the mixture mechanism of T-2 and PRO at the transcription and metabolomics levels needs to be further explored.
T-2 exhibited higher acute toxicity to zebrafish embryos than PRO. The combination of T-2 and PRO had an acute synergistic effect on zebrafish. Most individual and combined exposures significantly changed the SOD and GST activities compared with the control group. Furthermore, the expressions of HPT and HPG axis-related genes (tsh, crh, TRβ, cyp19a, and vtg1) immune system-related genes (bcl-2 and IL-8), and cellular apoptosis-related genes (cas3 and cas9) were tremendously disturbed in the combined exposure group compared with individual exposures. These changes of indexes determined might provide early warning on the mixture effects of mycotoxin and pesticides. Collectively, the impacts of chemical synergy on the toxicity of combined contaminants should be considered prior to assessing the risk in agriculture products.
Availability of data and materials
Not applicable.
T-2 toxin
The mixture of T-2 toxin and propiconazole
Median lethal concentration
Additive index
Low concentration
Middle concentration
High concentration
Superoxide dismutase
Cytochrome P450
- CarE:
Elfadil D, Lamaoui A, Pelle FD, Amine A, Compagnone D (2021) Molecularly imprinted polymers combined with electrochemical sensors for food contaminants analysis. Molecules 26:4607
Kresse M, Drinda H, Romanotto A, Speer K (2019) Simultaneous determination of pesticides mycotoxins and metabolites as well as other contaminants in cereals by LC-LC-MS/MS. J Chromatogr B 1117:86–102
Streit E, Naehrer K, Rodrigues I, Schatzmayr G (2013) Mycotoxin occurrence in feed and feed raw materials worldwide: Long-term analysis with special focus on Europe and Asia. J Sci Food Agric 93(12):2892–2899
Chen Z, Zhang F, Jiang L, Chen Z, Sun H (2021) Toxic effects of mycotoxin fumonisin B1 at six different doses on female balb/c mice. Toxins 14(1):21
Zhu T, Liu X, Song J, Li D, Pang X, Wang S, Li Q, Fu D, Zhang S, Xie H (2021) Ras/Raf/MEK/ERK pathway axis mediated neurotoxicity induced by high-risk pesticide residue-Avermectin. Environ Toxicol 36(5):984–993
Hussain S, Khan M, Khan A, Javed I, Asi M (2009) Toxico-pathological effects in rats induced by concurrent exposure to aflatoxin and cypermethrin. Toxicon 53:33–41
Lu S, Liu S, Cui J, Liu X, Zhao C, Fan L, Yin S, Hu H (2019) Combination of patulin and chlorpyrifos synergistically induces hepatotoxicity via inhibition of catalase activity and generation of reactive oxygen species. J Agric Food Chem 67(41):11474–11480
Jia K, Cheng B, Huang L, Xiao J, Bai Z, Liao X, Cao Z, Shen T, Zhang C, Hu C, Lu H (2020) Thiophanate-methyl induces severe hepatotoxicity in zebrafish. Chemosphere 248:125941
Jia M, Wang Y, Wang D, Teng M, Yan J, Yan S, Meng Z, Li R, Zhou Z, Zhu W (2019) The effects of hexaconazole and epoxiconazole enantiomers on metabolic profile following exposure to zebrafish (Danio rerio) as well as the histopathological changes. Chemosphere 226:520–533
Juan-García A, Bind MA, Engert F (2020) Larval zebrafish as an in vitro model for evaluating toxicological effects of mycotoxins. Ecotoxicol Environ Saf 202:110909
Feng P, Ye Z, Kakade A, Virk AK, Li X, Liu P (2018) A review on gut remediation of selected environmental contaminants: possible roles of probiotics and gut microbiota. Nutrients 11(1):22
Edyta J, Marcin N, Marcin P, Michal C, Maksymilian S, Michal B (2021) T-2 toxin—the most toxic trichothecene mycotoxin: Metabolism toxicity and decontamination strategies. Molecules 26:6868
Huang L, Zhu L, Ou Z, Ma C, Kong L, Huang Y, Chen Y, Zhao H, Wen L, Wu J, Yuan Z, Yi J (2021) Betulinic acid protects against renal damage by attenuation of oxidative stress and inflammation via Nrf2 signaling pathway in T-2 toxin-induced mice. Int Immunopharmacol 101:108210
Chilaka CA, De Boevre B, Atanda O, De Saeger S (2018) Stability of fumonisin B1 deoxynivalenol zearalenone and T-2 toxin during processing of traditional Nigerian beer and spices. Mycotoxin Res 34(4):229–239
Sun L, Cui S, Deng Q, Liu H, Cao Y, Wang S, Yu J (2019) Selenium content and/or T-2 toxin contamination of cereals soil and children's hair in some areas of Heilongjiang and Gansu provinces China. Biol Trace Elem Res 191:294–299
Yang X, Zhang X, Yao Q, Song M, Han F, Shao B, Li Y (2019) T-2 toxin impairs male fertility by disrupting hypothalamic-pituitary-testis axis and declining testicular function in mice. Chemosphere 234:909–916
Dai C, Xiao X, Sun F, Zhang Y, Hoyer D, Shen J, Tang S, Velkov T (2019) T-2 toxin neurotoxicity: role of oxidative stress and mitochondrial dysfunction. Arch Toxicol 93(11):3041–3056
Modra H, Palikova M, Hyrsl P, Bartonkova J, Papezikova I, Svobodova Z, Blahova J, Mares J (2020) Effects of trichothecene mycotoxin T-2 toxin on haematological and immunological parameters of rainbow trout (Oncorhynchus mykiss). Mycotoxin Res 36(3):319–326
Wang L, Fu Y, Shi Y, Wang W (2011) T-2 toxin induces degenerative articular changes in rodents: link to Kaschin-Beck disease. Toxicol Pathol 39:502–507
Chandratre G, Telang A, Badgujar P, Raut S, Sharma A (2014) Toxicopathological alterations induced by high dose dietary T-2 mycotoxin and its residue detection in Wistar rats. Arch Environ Contam Toxicol 67:124–138
Shi M, He Y, Zhang Y, Guo X, Lin J, Wang W, Chen J (2021) LncRNA MIAT regulated by selenium and T-2 toxin increases NF-κB-p65 activation promoting the progress of Kashin-Beck Disease. Hum Exp Toxicol 40:869–881
Wang Y, Ning X, Li G, Sang N (2022) New insights into potential estrogen agonistic activity of triazole fungicides and coupled metabolic disturbance. J Hazard Mater 424:127479
Cheng Y, Zheng Y, Dong F, Li J, Zhang Y, Sun S, Li N, Cui X, Wang Y, Pan X, Zhang W (2017) Stereoselective analysis and dissipation of propiconazole in wheat grapes and soil by supercritical fluid chromatography-tandem mass spectrometry. J Agric Food Chem 65:234–243
Ju C, Li X, He S, Shi L, Yu S, Wang F, Xu S, Cao D, Fang H, Yu Y (2020) Root uptake of imidacloprid and propiconazole is affected by root composition and soil characteristics. J Agric Food Chem 68(52):15381–15389
Elhady MA, Khalaf AAA, Kamel MM, Noshy PA (2019) Carvacrol ameliorates behavioral disturbances and DNA damage in the brain of rats exposed to propiconazole. Neurotoxicology 70:19–25
Teng M, Chen X, Wang C, Song M, Zhang J, Bi S, Wang C (2020) Life cycle exposure to propiconazole reduces fecundity by disrupting the steroidogenic pathway and altering DNA methylation in zebrafish (Danio rerio). Environ Int 135:105384
Svanholmm S, Säfholmm M, Brande-Lavridsenm N, Larssonmm E, Bergm C (2021) Developmental reproductive toxicity and endocrine activity of propiconazole in the Xenopus tropicalis model. Sci Total Environ 753:141940
Yang R, Wang X, Wang J, Chen P, Liu Q, Zhong W (2022) Insights into the sex-dependent reproductive toxicity of 2-ethylhexyl diphenyl phosphate on zebrafish (Danio rerio). Environ Int 158:106928
Nesnow S, Padgett WT, Moore T (2011) Propiconazole induces alterations in the hepatic metabolome of mice: Relevance to propiconazole-induced hepatocarcinogenesis. Toxicol Sci 120(2):297–309
Yang J, Guo W, Wang J, Yang X, Zhang Z, Zhao Z (2020) T-2 toxin-induced oxidative stress leads to imbalance of mitochondrial fission and fusion to activate cellular apoptosis in the human liver 7702 cell line. Toxins 12(1):43
Cladière M, Delaporte G, Le Roux E, Camel V (2018) Multi-class analysis for simultaneous determination of pesticides mycotoxins process-induced toxicants and packaging contaminants in tea. Food Chem 242:113–121
Eze UA, Huntriss J, Routledge MN, Gong YY, Connolly L (2019) The effect of individual and mixtures of mycotoxins and persistent organochloride pesticides on estrogen receptor transcriptional activation using in vitro reporter gene assays. Food Chem Toxicol 130:68–78
Tao Y, Li Z, Yang Y, Jiao Y, Qu J, Wang Y, Zhang Y (2022) Effects of common environmental endocrine-disrupting chemicals on zebrafish behavior. Water Res 208:117826
Zhang J, Wang K, Guo J, Huang Y, Wei Y, Jia K, Peng Y, Lu H (2023) Study on the mechanism of liver toxicity induced by acenaphthene in zebrafish. Ecotoxicol Environ Saf 249:114441
Xu Q, Wu D, Dang Y, Yu L, Liu C, Wang J (2017) Reproduction impairment and endocrine disruption in adult zebrafish (Danio rerio) after waterborne exposure to TBOEP. Aquat Toxicol 182:163–171
Chen H, Chen K, Qiu X, Xu H, Mao G, Zhao T, Feng W, Okeke E, Wu X, Yang L (2020) The reproductive toxicity and potential mechanisms of combined exposure to dibutyl phthalate and diisobutyl phthalate in male zebrafish (Danio rerio). Chemosphere 258:127238
Yang K, Feng T, Han Y, Li G, Li J, Ma H (2022) Bioaccumulation, metabolism and endocrine-reproductive effects of metolachlor and its S-enantiomer in adult zebrafish (Danio rerio). Sci Total Environ 802:149826
Commission E (2005) Regulation (EC) No 396/2005 of the European parliament and of the council of 23 February 2005 concerning on maximum levels of pesticides in or on food and feed of plant and animal origin and amending directive 91/414/EEC concerning the placing of plant protection products on the market and repealing council directives 79/117/EEC and 91/414/EEC. Official Journal of European Community 1–4
ISO (1996) Water quality-determination of the acute lethal toxicity of substances to a freshwater fish [Brachy Danio rerio Hamilton-Buchanan (Teleostei Cyprinidae)]-Part3: flow-through method. ISO 7346–3
OECD (2013) OECD guidelines for the testing of chemicals fish embryo acute toxicity (FET) test. OECD, Paris, France. No. 236
Wang Y, Dai D, Yu Y, Yang G, Shen W, Wang Q, Weng H, Zhao X (2018) Evaluation of joint effects of cyprodinil and kresoxim-methyl on zebrafish Danio rerio. J Hazard Mater 352:80–91
Marking L (1985) Toxicity of chemical mixtures. In: Rand G, Petroceli S (eds) Fundamentals of Aquatic Toxicology. Hemisphere Publishing Corporation, Washington DC, pp 164–176
Wang Y, Xu C, Wang D, Weng H, Yang G, Guo D, Yu R, Wang X, Wang Q (2020) Combined toxic effects of fludioxonil and triadimefon on embryonic development of zebrafish (Danio rerio). Environ Pollut 260:114105
Yang G, Wang Y, Wang T, Wang D, Weng H, Wang Q, Chen C (2021) Variations of enzymatic activity and gene expression in zebrafish (Danio rerio) embryos co-exposed to zearalenone and fumonisin B1. Ecotoxicol Environ Saf 222:112533
Yang Y, Qi S, Wang D, Wang K, Zhu L, Chai T, Wang C (2016) Toxic effects of thifluzamide on zebrafish (Danio rerio). J Hazard Mater 307:127–136
Jin Y, Zhang X, Shu L, Chen L, Sun L, Qian H, Liu W, Fu Z (2010) Oxidative stress response and gene expression with atrazine exposure in adult female zebrafish (Danio rerio). Chemosphere 78:846–852
Lv L, Gao Z, Mao L, Liu X, Wang Q, Shen W, Wang Y (2022) Insights into the combined toxic impacts of phoxim and deltamethrin on the embryo-larval stage of zebrafish (Danio rerio). Environ Sci Eur 34:90
Zheng S, Liu C, Huang Y, Bao M, Huang Y, Wu K (2017) Effects of 2,2ʹ,4,4′-tetrabromodiphenyl ether on neurobehavior and memory change and bcl-2, c-fos, grin1b and lingo1b gene expression in male zebrafish (Danio rerio). Toxicol Appl Pharmacol 333:10–16
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−△△ CT method. Method 25:402–408
Chi H (1997) Computer program for the probit analysis. National Chung Hsing University Taichung, Taichung
Li D, Wu J, Liu J, Li A, Meng F (2021) Natural attenuation characteristics and comprehensive toxicity changes of C9 aromatics under simulated marine conditions. J Environ Sci 109:26–35
Ge J, Jiang J, Cai L (2018) Study on toxicity of three kinds of triazole fungicides on zebrafish (Danio rerio). Acta Agriculaturae Zhejiangensis 30:744–755
Liu S, Lai H, Wang Q, Martínez R, Zhang M, Liu Y, Huang J, Deng M, Tu W (2021) Immunotoxicity of F-53B an alternative to PFOS on zebrafish (Danio rerio) at different early life stages. Sci Total Environ 790:148165
Yuan G, Wang Y, Yuan X, Zhang T, Zhao J, Huang L, Peng S (2014) T-2 toxin induces developmental toxicity and apoptosis in zebrafish embryos. J Environ Sci 26(4):917–925
Ismail M, Srivastava V, Marimani M, Ahmad A (2022) Carvacrol modulates the expression and activity of antioxidant enzymes in Candida Auris. Res Microbiol 173:103916
Ren R, Zeng H, Mei Q, Xu Z, Mazhar M, Qin L (2022) Effects of Monascus purpureus-fermented tartary buckwheat extract on the blood lipid profile glucose tolerance and antioxidant enzyme activities in KM mice. J Cereal Sci 105:103465
Pei X, Jiang H, Liu X, Li L, Li C, Xiao X, Li D, Tang S (2021) Targeting HMGB1 inhibits T-2 toxin-induced neurotoxicity via regulation of oxidative stress, neuroinflammation and neuronal apoptosis. Food Chem Toxicol 151:112134
Shubin AD, Felong TJ, Graunke D, Ovitt CE, Benoit DS (2015) Development of poly (ethylene glycol) hydrogels for salivary gland tissue engineering applications. Tissue Eng Part A 21(11–12):1733–1751
Li S, Zhang G, Xue B, Ding Q, Han L, Huang J, Wu F, Li C, Yang C (2022) T-2 induces apoptosis through ROS-mediated mitochondrial pathway in poultry. Food Chem Toxicol 169:113392
Ortiz P, Bruno M, Moore T, Nesnow S, Winnik W, Ge Y (2010) Proteomic analysis of propiconazole responses in mouse liver: comparison of genomic and proteomic profiles. J Proteome Res 9:1268–1278
Hernandez M, Recalde S, González-Zamora J, Bilbao-Malavé V, Sáenz de Viteri M, Bezunartea J, Moreno-Orduña M, Belza I, Barrio-Barrio J, Fernandez-Robredo P, García-Layana A (2021) Anti-inflammatory and anti-oxidative synergistic effect of vitamin D and nutritional complex on retinal pigment epithelial and endothelial cell lines against age-related macular degeneration. Nutrients 13(5):1423
Cheng Q, Cao X, Yuan F, Li G, Tong T (2014) Knockdown of WWP1 inhibits growth and induces apoptosis in hepatoma carcinoma cells through the activation of caspase 3 and p53. Biochem Biophys Res Commun 448(3):248–254
Xian T, Han X, Han C, Li F, Xu W, Zhang H, Tu K (2019) Effects of T-2 toxin on in vitro proliferation and apoptosis of human renal tubular epithelial cell HK-2. Food Ferment Ind 45:86–92
Wang M, Wang H, Chen G, Liu J, Hu T (2022) Spiromesifen conferred abnormal development in zebrafish embryos by inducing embryonic cytotoxicity via causing oxidative stress. Aquat Toxicol 252:106324
Parlak V (2018) Evaluation of apoptosis oxidative stress responses AChE activity and body malformations in zebrafish (Danio rerio) embryos exposed to deltamethrin. Chemosphere 207:397–403
Li X, Lin Y, Yao J, Pan B, Zhan X, Chen Z, Bai Y, Zhang H, Wang B, Chen S, Li J, Liu C (2022) Protegrin-1 inhibits porcine ovarian granulosa cell apoptosis from H2O2-induced oxidative stress via the PERK/eIF2a/CHOP signaling pathway in vitro. Theriogenology 179:117–127
Chen H, Tang X, Zhou B, Xu N, Wang Y (2016) Mechanism of Deca-BDE-induced apoptosis in Neuro-2a cells: Role of death-receptor pathway and reactive oxygen species-mediated mitochondrial pathway. J Environ Sci 46:241–251
Jia W, Mao L, Zhang L, Zhang Y, Jiang H (2018) Effects of two strobilurins (azoxystrobin and picoxystrobin) on embryonic development and enzyme activities in juveniles and adult fish livers of zebrafish (Danio rerio). Chemosphere 207:573–580
Yan S, Wang J, Zhu L, Chen A, Wang J (2015) Toxic effects of nitenpyram on antioxidant enzyme system and DNA in zebrafish (Danio rerio) livers. Ecotoxicol Environ Saf 122:54–60
Liu C, Song Y, Ren H, Sun G, Liu R, Jiang P, Zhang LS, X, Wang Z, Cui J, (2017) Cloning and expression of a Trichinella spiralis putative glutathione S-transferase and its elicited protective immunity against challenge infections. Parasit Vectors 10(1):448
Guo D, Liu W, Yao T, Ma M, Wang Q, Qiu J, Qian Y (2021) Combined endocrine disruptive toxicity of malathion and cypermethrin to gene transcription and hormones of the HPG axis of male zebrafish (Danio rerio). Chemosphere 267:128864
Yuan Z, Matias FB, Yi JE, Wu J (2016) T-2 toxin-induced cytotoxicity and damage on TM3 Leydig cells. Comp Biochem Physiol C Toxicol Pharmacol 181:47–54
Wang Y, Wang Q, Ji C, Guo X, Yang G, Wang D, Weng H, Qian Z, Chen C (2021) Mixture toxic impacts and the related mechanism of aflatoxin B1 and deoxynivalenol on embryonic zebrafish (Danio rerio). Environ Sci Eur 33:86
Wang X, Liu Q, Ihsan A, Huang L, Dai M, Hao H, Cheng G, Liu Z, Wang Y, Yuan Z (2012) JAK/STAT pathway plays a critical role in the proinflammatory gene expression and apoptosis of RAW264.7 cells induced by trichothecenes as DON and T-2 toxin. Toxicol Sci 127:412–424
Sax L (2010) Polyethylene terephthalate may yield endocrine disruptors. Environ Health Persp 118(4):445–448
Gálvez-Ontiveros Y, Páez S, Monteagudo C, Rivas A (2020) Endocrine disruptors in food: Impact on gut microbiota and metabolic diseases. Nutrients 12(4):1158
Stukenborg JB, Mitchell R, Söder O (2021) Endocrine disruptors and the male reproductive system. Best Pract Res Clin Endocrinol Metab 35(5):101567
Mohapatra S, Mukherjee J, Banerjee D, Das PK, Ghosh PR, Das K (2021) RFamide peptides the novel regulators of mammalian HPG axis: a review. Vet World 14(7):1867–1873
Costa N, Vieira M, Sgarioni V, Pereira M, Montagnini B, Mesquita S, Gerardin D (2015) Evaluation of the reproductive toxicity of fungicide propiconazole in male rats. Toxicology 335:55–61
Wu Q, Wang X, Yang W (2014) Oxidative stress-mediated cytotoxicity and metabolism of T-2 toxin and deoxynivalenol in animals and humans: an update. Arch Toxicol 88:1309–1326
Jiang J, Wu S, Liu X, Wang Y, An X, Cai L, Zhao X (2015) Effect of acetochlor on transcription of genes associated with oxidative stress apoptosis immunotoxicity and endocrine disruption in the early life stage of zebrafish. Environ Toxicol Pharmacol 40:516–523
Risalde M, Molina AM, Lora A, Ayala N, Gómez-Villamandos J, Moyano M (2021) Immunohistochemical expression of aromatase cyp19a1a and cyp19a1b in the ovary and brain of zebrafish (Danio rerio) exposed to different concentrations of bisphenol A. Aquat Toxicol 237:105876
Kang H, Lee Y, Kim B, Kim C, Jeong C, Lee J (2017) Adverse effects of BDE-47 on in vivo developmental parameters thyroid hormones and expression of hypothalamus-pituitary-thyroid (HPT) axis genes in larvae of the self-fertilizing fish Kryptolebias marmoratus. Chemosphere 176:39–46
Guo D, Liu W, Qiu J, Li Y, Chen L, Wu S, Wang Q, Qian Y (2020) Changes in thyroid hormone levels and related gene expressions in embryo-larval zebrafish exposed to binary combinations of bifenthrin and acetochlor. Ecotoxicology 29:584–593
Lema S, Dickey J, Schultz I, Swanson P (2009) Thyroid hormone regulation of mRNAs encoding thyrotropin β-subunit glycoprotein α-subunit and thyroid hormone receptors α and β in brain pituitary gland liver and gonads of an adult teleost Pimephales promelas. J Endocrinol 202:43–54
Teng M, Zhao W, Chen X, Wang C, Zhou L, Wang C, Xu Y (2022) Parental exposure to propiconazole at environmentally relevant concentrations induces thyroid and metabolism disruption in zebrafish (Danio rerio) offspring: an in vivo, in silico and in vitro study. Ecotoxicol Environ Saf 242:113865–113865
Liu X, Huang D, Guo P, Wu Q, Dai M, Cheng G, Hao H, Xie S, Yuan Z, Wang X (2017) PKA/CREB and NF-κB pathway regulates AKNA transcription: a novel insight into T-2 toxin-induced inflammation and GH deficiency in GH3 cells. Toxicology 392:81–95
Yuan Z, Matias F, Yi J, Wu J (2016) T-2 toxin-induced cytotoxicity and damage on TM3 Leydig cells. Comp Biochem Physiol C Toxicol Pharmacol 181–182:47–54
Perveen A, Shen J, Ali Kaka N, Li C (2020) Maternal exposure to T-2 toxin affects puberty genes and delays estrus cycle in mice offspring. Animals (Basel) 10(3):471
Shen W, Yang G, Guo Q, Lv L, Liu L, Wang X, Lou B, Wang Q, Wang Y (2021) Combined toxicity assessment of myclobutanil and thiamethoxam to zebrafish embryos employing multi-endpoints. Environ Pollut 269:116116
The authors acknowledge the technical assistance of Yancen Lou and Liying Pan (Zhejiang Academy of Agricultural Sciences).
The research was supported by Scientific and Technological Innovation Project of Yantai City, China (Grant No. 2020MSGY053) and Shanghai Agriculture Applied Technology Development Program, China (Grant No. 2021, NO. 3-2).
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SZ: conceptualization, data curation, formal analysis, writing—original draft, writing–review and editing. XL: data curation, formal analysis, writing–original draft, project administration. LL: conceptualization, data curation, formal analysis, writing–original draft, project administration. CL: conceptualization, investigation, methodology, resources. TL: conceptualization, data curation, formal analysis. HZ: investigation, methodology, resources. JZ: supervision. YW: conceptualization, writing—original draft, supervision, funding acquisition, project administration, writing—review and editing. All authors read and approved the final manuscript.Authors' contributions: Journal standard instruction requires the statement "All authors read and approved the final manuscript." in the Authors' contributions section. In order to insert this text at the end of the paragraph of the said section, the contribution paragraph has been modified. Please check if appropriate.They are okay.
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Additional file 1
: Table S1. Gene primer sequences in real-time quantitative PCR reaction. Table S2. Lethal toxicity of T-2 toxin and propiconazole to zebrafish, separately and combined.
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Zhang, S., Liu, X., Lv, L. et al. Combined toxic effects of T-2 toxin and propiconazole on the early life stages of zebrafish (Danio rerio). Environ Sci Eur 35, 8 (2023). https://doi.org/10.1186/s12302-023-00714-7
DOI: https://doi.org/10.1186/s12302-023-00714-7


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Best, matt On 10/02/2013 10:49 PM, Keith Brown wrote:
What version does the startd have to run? And more important what version of collector needs to run? On Wed, Oct 2, 2013 at 10:04 AM, Matthew Farrellee <matt@xxxxxxxxxx <mailto:matt@xxxxxxxxxx>> wrote: On 10/02/2013 08:07 AM, Keith Brown wrote: I would like to each machine broadcast nic=1000 on each machine. Since I have a 1G backbone. Then as a user, I would like to specify nic=500 (this job takes 50% of network traffic). Is that possible to do? Some machine have 10000 btw You sure can, see http://research.cs.wisc.edu/__htcondor/manual/v8.0/3___5Policy_Configuration.html#__sec:Configuring-SMP <http://research.cs.wisc.edu/htcondor/manual/v8.0/3_5Policy_Configuration.html#sec:Configuring-SMP> (search for MACHINE_RESOURCE_NAMES) and https://htcondor-wiki.cs.wisc.__edu/index.cgi/tktview?tn=2905 <https://htcondor-wiki.cs.wisc.edu/index.cgi/tktview?tn=2905> Best, matt

subset: commoncrawl


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HackSpace Magazine — Issue 47: 50 best 3D prints
If your 3D printer is looking a little dusty and unloved, now's the time to put it to work: we've 50 of the best 3D prints to improve your home, office, workshop and more. From functional to frivolous, we've got ideas for you. It's time to unleash the awesome power of your printer!
- Oskitone: where 3D printing meets analogue synth goodness
- Pure Data: make music with this awesome graphical programming language
- Surface mount soldering: solder the way the professionals do it
- Cardboard tubes: do something useful with toilet roll tubes
Read more, download PDF, buy now, subscribe.
Get a free Raspberry Pi Pico board with a new 12 month subscription.


text: Alpha Princess GTR Battles TX2K20 – She Fights for Gold! (1.6s 0-60MPH) Drag & Track by turboandstance - April 16, 2020April 16, 2020 Here we have the Alpha Princess and her freshly built R35 GT-R! She participated in roll races and drag races to reset the stock turbo record, 8.9 sec @151mph! She tried to dive a little deeper into 8's during the weekend. Watch the video below to see this beast in action and leave a comment. Follow us also on Facebook, Pinterest or Instagram! Leave a comment


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What we do
Sustainability instead of crises
Climate crisis, climate change, global warming – many names, the etymology of which is not worth delving into here, but the impact of which on food security and livelihoods in East Africa is undeniable.
In the spring of 2023, six consecutive rainy seasons had failed. Famine had been declared in most parts of Somalia. In Kenya, the cost of food had made three meals a day a utopia for the majority of families – for example, the price of essential maize flour had more than doubled in five years.
There are numerous challenges: East African countries often rely on food imports, especially wheat from Ukraine and Russia. Erratic rainfall and extended dry periods have made cultivation difficult. The cost of fertilizers has become unaffordable for most small-scale farmers.
The climate crisis is also linked to women's rights. Women are often the first to compromise their nutrition. Girls are more likely to be taken out of school to help with food production and procurement. Girls might be married off at younger ages, shifting the responsibility of their food security to the husband's family.
Hence, turning away from the climate crisis is not an option: humanity must find ways to adapt to its consequences and ensure that the escalation of the climate crisis is halted. In our projects, we primarily focus on building sustainable food production and climate resilience.
In its projects, International Solidarity Foundation seeks to ensure that the prerequisites for local sustainable food production are enhanced. This entails several aspects, such as:
The significance of this kind of work is enormous at the local level. It increases food security, economic opportunities in the food market, and simultaneously fosters hope amidst a changing climate. For instance, the most important achievement of the water project in Somaliland was that those involved felt they could now remain in their hometowns.
ISF also engages in advocacy and stakeholder engagement, where we aim to highlight disparities in global South regarding food markets and climate financing. On the other hand, we establish connections between universities, businesses, and our projects, consistently striving to find more impactful ways to adapt to the consequences of the climate crisis.
Women's status in the labor market improves
Sustainable farming practices and food production enhance women's standing in the labor market. Simultaneously, the well-being of families and the community improves.
Bringing crises under control
Instead of moving from crisis to crisis, communities can better prepare for challenging weather conditions and exceptional years.
Enhancing local production value-addition
By developing local food production and improving value-addition, women gain significant opportunities for additional income.
Location: Kenya & Somaliland
Implementation period: 2021-
Partners: North Nyamira Women Sacco, ADO
Livelihood – Field Coordinator, Kenya
Location: Kisii
Livelihood Field Coordinator, Somaliland
Location: Burao


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News Story
Bill to empower Education Department to investigate discrimination against students gets killed
Gov. Kate Brown told the bill's sponsor, State Sen. Sara Gelser Blouin, that there was not enough time in the current session to pass it
Sen. Sara Gelser Blouin, D-Corvallis, speaks on the Senate floor with Sens. Jeff Golden, D-Ashland, and Deb Patterson, D-Salem, watching on Wednesday, Feb. 16, 2022. (Ron Cooper/Oregon Capital Chronicle)
A plan to empower the Oregon Department of Education to investigate allegations of discrimination against students with disabilities was killed Tuesday by legislators because of time.
State Sen. Sara Gelser Blouin, D-Corvallis, said she was on the Senate floor when Gov. Kate Brown called to share with her that Senate Bill 1578, which Gelser Blouin authored and sponsored, was dead due to the time constraints of the current session.
The bipartisan bill had already been approved by the Senate Education Committee on Feb. 11 and was in the Joint Committee on Ways and Means.
"We lose bills all the time. This is different," Gelser Blouin said in an interview. "We spent so much time talking about our commitment to diversity, equity and inclusion during this session."
Charles Boyle, deputy communications director for Brown, wrote in an email that based on the governor's conversations with legislators, it was clear that the bill wasn't moving forward this session and she passed the message to Gelser Blouin as a courtesy.
Gelser Blouin does not know who made the decision to kill her proposal or why it was chosen among many.
The bill was a response to more than three dozen parents across the state who had reached out to Gelser Blouin saying their kids with disabilities weren't getting equal class time as their peers, a violation of state and federal laws. The pandemic exacerbated teacher shortages, especially among special education teachers and paraprofessionals, which led some districts to cancel entire days of instruction each week for students with disabilities, adding up to weeks and months worth of lost class time.
Gelser Blouin launched an online campaign called #AllMeansAll to help parents file complaints and to bring attention to the issue.
Currently, parents who believe their children aren't getting equal class time can either file a complaint with the federal Department of Education, which can take months or years to be investigated, or with the local school district where an alleged violation took place. The district then has a responsibility to investigate and decide whether to refer the case to the state Education Department.
In proposing the bill, Gelser Blouin wanted to make it easier for parents to report allegations of discrimination and lost class time, and to take investigations to an agency outside the school district. She also hoped to speed up the investigation process and get students back into school quickly.
Gelser Blouin said she was heartbroken to learn the bill would no longer be considered.
Shortly after getting the news from Brown, in a brief and impassioned speech on the Senate floor, Gelser Blouin told her peers that going forward, she would be voting on bills based solely on their perceived urgency.
"I have recently learned that we may be leaving some very important kinds of legislation on the table under the argument that we just don't have time," she said. "So I am now thinking about the legislation on which we are voting, relative to how urgent and emergent it is compared to, say, ensuring the right of children with disabilities to not continue to be denied access to school like their non-disabled peers."
Gelser Blouin's bill had the support of the Education Department's director, Colt Gill, as well as leaders of the Coalition of Oregon School Administrators and the Oregon School Boards Association.
Gelser Blouin said she is confused why her seven-page bill was killed to save time, and why the issue isn't more urgent. She pointed to the uproar from parents over the Portland Public Schools' proposal to go to a four-day school week.
"Imagine what they would say if their student got an hour a day with an unlicensed teacher," she said.
Gelser Blouin held back tears talking about what would happen next.
"I don't know what to say to these people," she said of the families who have reached out to her for help getting their kids equal class time.
"Your kids have a right to attend school, we know it's being abridged and we are actively choosing to not move forward."
Our stories may be republished online or in print under Creative Commons license CC BY-NC-ND 4.0. We ask that you edit only for style or to shorten, provide proper attribution and link to our web site.


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When: 15 June 2011 19.30 – 10.00 pm.
Where: Sally's Rift Cave, Bathford.
Who: Darren T, Rich T. Dave G, Tim T, Ivan E, Mad, Digdug.
After going on a few Mine exploring trips Rich and myself thought we would have a go at this caving game. Dave G posted a trip on Darkplaces to go to sally's Rift Cave in Bathford. This is the second longest Gull cave in Britain. We met at the layby at the foot of the hill and followed Dave to the entrance. Dave decided to rig a ladder and lifeline to get us all down the entrance hole. This was our first time on a wire ladder but we managed it well and were soon all at the bottom and set fourth on our mission to explore this rather sporting cave.
The survey is deceptive as movement in the cave is rarely straightforward – squeezes and climbs bar progress continually. Digdub pointed out the scaffolding bar that marked the route into the boulder chamber. Ivan, Digdub and Dave climbed up with the ladder and lifeline bag into the boulder chamber. Mad had a few problems climbing up but managed it in the end.
They rigged the vertical squeeze by looping the rope around a boulder and hanging the ladder down the vertical squeeze. The rift and squeeze are quite tight but it soon opens out into a nice wide rift. The way on is not very clear and as Dave had left the survey in the car we decided to head back and come back another day. we exited the cave problem free and were soon walking back down the hill to the cars.
So what did Rich and myself think of caving, well you probably guessed it we got hooked and will be doing many more trips.


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Fitness and health is definitely regarded an important part of keeping yourself healthful. It signifies the chance to do various types of athletics and workouts with minimal probability of harm. The meaning of fitness might be stretched to add exercises that are carried out at sleep, including browsing, or maybe slumbering. Health and fitness is defined as a condition of whole well-being and health and, furthermore, the capability to undertake tasks, including actual kinds, with minimal risk of damage.
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Washington, Dec 11 (IANS): Former Republican Liz Cheney (from Wyoming) warned the US that all of former President Donald Trump's remarks at an event last week that he would be a "dictator" only on "day one" of a second term should be taken "literally and seriously".
"We have to take everything that Donald Trump says literally and seriously," Cheney said in an interview that aired Sunday on ABC's "This Week".
Trump mocked questions about authoritarianism at an event billed as a "town hall" with Fox News host Sean Hannity last week. The former President said he would be a dictator only on "day one".
"After that I'm not a dictator OK?" Trump remarked.
Trump's comments come as his rhetoric invites closer scrutiny since his reference to political opponents as "vermin", a term many politicians across the aisle feel echoes authoritarian and fascist leaders such as Adolf Hitler and Benito Mussolini, media reports said.
Cheney, ousted by House Republicans for her criticism of Trump, pointed to the former President's efforts to overturn his loss in the 2020 election while arguing that Trump's dictator comments should not be taken as a joke.
"People need to remember that when Donald Trump woke up on the morning of January 6, he thought he was going to remain as President. And we saw the extent to which he was willing to attempt to seize powers when he lost an election," Cheney said.
Cheney added that a second Trump presidency would be "worse" than his first "because he has had practice and because those people who were around him who actually did stop the worst he was trying to do would not be around him again". USA TODAY said quoting Liz as having said to some agencies.
"Our institutions don't protect themselves. It's the people who do," she added.
Cheney told USA TODAY in an interview last week that she has not ruled out running for President in 2024 and joining a bipartisan ticket, such as one proposed by the group 'No Labels'. However, she said she wouldn't run if it would help Trump's re-election chances and siphon votes away from President Joe Biden.
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Street Fighter 6 A batch of new information about the upcoming fighting video game is coming today as part of the Tokyo Game Show 2022 presentation. There was more information about the video game's World Tour and Battle Hub among other details, but perhaps the most important reveal for fans of the franchise was the confirmation of four fighters Two different returns will appear in Street Fighter 6.
More specifically, today Capcom revealed the return of popular Street Fighter characters Ken, Blanka, Dhalsim, and E. Honda. Street Fighter 6. Given how regularly all four appear in major Street Fighter titles, and an increasingly accurate leak earlier this year, their appearances in Street Fighter 6 Not entirely surprising, but it's still remarkable to see them confirm what's supposed to be the title's launch roster. You can check out the four in the new Street Fighter 6 Trailer included below:
Get a detailed look at the world tour, battle center, and extreme battle with the game mode trailer.
The return of Kane, Blanca, Dalcim and e Honda # Streetfighter 6!
🌐 https://t.co/3TmEHBJFPN pic.twitter.com/3AeWPK1H7Y
Street Fighter September 15, 2022
Here's how Capcom officially describes the four return letters of Street Fighter 6:
Ken: "Accusations of orchestrating a criminal conspiracy have forced Kane, a former U.S. National Fighting Champion and former Vice President of the Masters Foundation, to give up his family and his business to go into hiding. As a legend in the Street Fighter series, Kane presents a classic action movie with new additions that keep things fresh."
Blanca: An adventurous, kind-hearted tour guide and defender of nature, Blanka features his signature electric and moving thunder attacks, as well as new Blanka-chan bombs and the return of the Lightning Beast action.
Delseem: "A monk and expert in yoga, Dhalsim channels his familiar shooting techniques from previous games while also mastering moves like Yoga Fire, Yoga Arch, Yoga Comet, and Yoga Flame."
H.Honda: "Touring sumo wrestler, E. Honda combines old and new, bringing back his trademark moves like Hundred Hand Slap and Sumo Headbutt, as well as his brand new Sumo Dash as he moves forward with traditional sumo mixing."
Generally, Street Fighter 6 It is set for release for PlayStation 5, PlayStation 4, Xbox Series X, Xbox Series S, and PC in 2023. A final release date has not yet been announced. The game includes several different game modes such as Fighting Ground, World Tour, and Battle Hub. You can check out all of our previous coverage of the upcoming fighting video game here.
What do you think of all the newly confirmed returning fighters? Do you hope to Street Fighter 6 To confirm the return of any particular fighters? Tell us in the comments, or feel free to reach out to me and hit me up right on Twitter at Tweet embed In order to talk about all things gaming!


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Carlos Gardel. Su vida, su música, su época
Biography of one of the leading figures of Argentine tango, Carlos Gardel.
Biography of one of the leading figures of Argentine tango, Carlos Gardel.
How do keyboards make music playable? Drawing on theories of media, systems, and cultural techniques, Keys to Play spans Greek myth and contemporary Japanese digital games to chart a genealogy of musical play and its animation via improvisation, performance, and recreation. As a paradigmatic digital interface, the keyboard forms a field of play on which the book's diverse objects of inquiry-from clavichords to PCs and eighteenth-century musical dice games to the latest rhythm-action titles-enter into analogical relations. Remapping the keyboard's topography by way of Mozart and Super Mario, who head an expansive cast of historical and virtual actors, Keys to Play invites readers to unlock ludic dimensions of music that are at once old and new.
In Playful Identities, eighteen scholars examine the increasing role of digital media technologies in identity construction through play. Going beyond computer games, this interdisciplinary collection argues that present-day play and games are not only appropriate metaphors for capturing postmodern human identities, but are in fact the means by which people create their identity. From discussions of World of Warcraft and Foursquare to digital cartographies, the combined essays form a groundbreaking volume that features the most recent insights in play and game studies, media research, and identity studies.


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8135 Severn Dr # D, Boca Raton FL 33433
Boca RatonMap
Active With Contract
Price | $389,000.00 |
Listing ID | A11523309 |
Status | Active With Contract |
Address Map |
8135 Severn Dr # D, Boca Raton FL 33433 |
Area | Boca Raton |
Beds | 2 |
Baths | 2 |
Square Ft | 1,302 |
Lot Size | 0 |
Year Built | 1986 |
Taxes | $2,879 |
Beautifully updated 2 bed 2 and 1 half bath Fee Simple Townhome in the heart of Boca Raton with A-Rated schools. 2 EnSuite bedrooms with balcony, granite countertops, updated baths, stainless steel appliances, laminate and tile flooring, custom blinds, banyan shutters and private, fenced-in patio. Smart Home features, LED lighting, crown molding, convenient assigned parking right out front and washer and dryer. Community pool, tennis courts, hot tub, sauna, fitness center, playground and playroom. And the peace of mind that comes with Roaming Security 7 days a week. Info here-in deemed accurate and is subject to errors/omissions. Listing info can be updated/revised at any time without notice.
Map Location
Copyright 2020 Miami Association of Realtors MLS. IDX information is provided exclusively for consumers' personal, non-commercial use, that it may not be used for any purpose other than to identify prospective properties consumers may be interested in purchasing, and that data is deemed reliable but is not guaranteed accurate by the MLS.
Miami Association of Realtors MLS data last updated on 3/1/2024 1:00:00 AM