Acute toxicity test provides useful information on a wide range of concentrations that can be adopted in the following toxicity testing and the assessment of the therapeutic impacts of toxins [24]. Former reports have displayed that the 4-day LC50 value of AFB1 to zebrafish larvae is 0.51 mg a.i.L− 1, which is not consistent with our findings [25]. Such a prominent difference might be likely attributed to the different life periods. Moreover, our data showed that AFB1 possessed stronger toxicity to zebrafish in comparison to DON. The determined mycotoxins had prominently different toxicities, which might be attributed to deviations in toxicokinetics and dynamics, such as uptakes, supersession, excretion, or binding to the target sites in D. rerio [26]. Therefore, the exposure of AFB1 and DON had latent risks to aquatic animals. Nevertheless, previous investigations on AFB1 and DON have mainly focused on their single toxic effect, while the toxicity of their combinations remains largely unknown.
Because of the natural co-occurrence in the food of mycotoxin, a great deal of attention has been paid to the effects of the mixture exposure [27]. Zebrafish embryos are fertilized ex utero and evolve independently of the mother, making zebrafish embryos an adequate animal model for compound exposure study [28]. Besides, due to its morphological transparency, the zebrafish embryo favors real-time histological observation in vivo [29]. However, more importantly, previous reports have shown that the genetic similarity between humans and zebrafish is more than 80%, and zebrafish has similar signal transduction pathways with suckler [30]. Because of these, zebrafish toxicity studies can serve as an important reference to humans [31]. Consequently, embryonic zebrafish were adopted to evaluate the mixture toxic effect of AFB1 and DON in this study. An additive effect was observed by exposing zebrafish embryos to AFB1 and DON mixtures, indicating that the mixture of AFB1 and DON had stronger mixture toxicity in comparison to their single compounds [19]. Our findings manifested that it was imminently essential to assess the combined toxicity of mycotoxins because the toxicity evaluation of mycotoxins is generally performed barely on single compounds, which might result in an underrated toxic effect in realistic situations.
With the wide existence of mycotoxins in food, it is very necessary to clarify the detoxification mechanism of mycotoxins in humans and how mycotoxins impair and influence human’s detoxification mechanism [32]. Our findings indicated that changed CYP 450 and GST activities might contribute to the toxic action of AFB1 in D. rerio, and the elevated GST activity might be the toxic mechanism of DON in the animals. Furthermore, the decreased CarE and GST activities in the high-concentration group of JOT in comparison to the corresponding single exposure group of DON, and the enhanced CYP450 activity in the high-concentration group of JOT might result in the overall additive toxicity of AFB1 and DON [33]. Therefore, all the determined three detoxification enzymes were involved in the detoxification mechanism of mixture toxicity.
As a cell suicide mechanism, apoptosis can get rid of superfluous or undesired cells [34]. The caspase family has a critical function in cell apoptosis, and caspase-3 has been shown as an important executor that is activated downstream in apoptosis pathways [35]. As a transcription factor, the P53 protein can prevent tumorigenesis via inducing cell cycle arrest and apoptosis [36]. Apaf-1 is the molecular core of the apoptosome, which is the executioner of mitochondria-dependent apoptosis [37]. Caspase-9 can bind to the apoptosome where it can induce executioner caspases, such as caspase-3 [38]. As a cardinal proapoptotic member of the BCL-2 family, the BCL-2-associated X protein (bax) can mediate the critical equilibrium in a cell cycle [39]. We showed that the caspase-3 activity was markedly induced in the AFB1 exposure, while no prominent change in caspase-9 was observed, which was not in line with the gene expression pattern. AFB1 apparently stimulated the expressions of cas3, cas9, bax, and P53 at the mRNA level, showing that this mycotoxin caused apoptosis via activating P53, which directly triggered the expression of gene encoding the proapoptotic protein (bax), then induced the critical executor of apoptosis (cas3) activation and subsequently resulted in cell apoptosis [35]. Moreover, the expressions of cas3, cas9, and apaf-1 were markedly altered in the JOT group, implying that regular apoptosis was influenced by the co-exposure of AFB1 and DON.
Environmental stressors can immediately stimulate oxidative stress via a complex physiological process, and oxidative stress occurs when the level of reactive oxygen species ( e.g., O2·−, H2O2, ·OH, etc.) is higher than the scavenging activities of antioxidants [40]. The antioxidants, including SOD, CAT, and POD, can protect the cells from oxidative stress through their important role in the defense mechanism [41]. SOD can scavenge O2·−and catalyze it to O2 and H2O2 [34]. The H2O2 generated is further detoxified via CAT and other enzymes, such as POD. The changes in their contents may indicate the extent of damage to the organism [42]. Both SOD and CAT activities in the single mycotoxin exposure groups were not markedly altered, indicating that the determined concentration of single mycotoxin was insufficient to induce prominent oxidative stress. However, a prominent reduction was found in the high-concentration group of JOT in comparison to the corresponding single exposure group of DON, which could be attributed to an overwhelmed antioxidant capacity [43]. LPO is a reliable oxidative stress indicator since it plays a critical role in denoting cell damage. MDA is the primary by-product of LPO, and an elevation in its content incarnates the degree of the cell damage caused by free radicals [44]. However, the MDA level was reduced in the AFB1 and JOT exposure groups, potentially suggesting that LPO was reduced by antioxidants. The expressions of anti-oxidative genes are necessary for evaluating antioxidant capacity [35]. In our current study, AFB1 and DON exposures up-regulated the expressions of Mn-sod and Cu/Zn-sod at the mRNA level, which was not consistent with the SOD activity, suggesting that SOD was primarily modulated at the protein level. The increased expressions of Mn-sod and Cu/Zn-sod may induce the SOD activity and eliminate the superoxide anion radical caused by the exposure of AFB1 and DON. Besides, the up-regulation of gpx at the mRNA level could also activate the gpx activity. Such discrepancy between antioxidant activity and gene expression might also be attributed to the presence of multiple gene copies in the species, a time-lag, and post-transcriptional modifications [45].
THs play an important role in the growth and development of fish and the thyroid endocrine system, while the HPT axis can regulate the thyroid system [46]. In the current study, the levels of T3 and T4 were markedly changed after exposure to AFB1 or DON, and their contents were also distinctly altered in the JOT exposure groups. Besides, the T4 level was markedly increased in the JOT exposure. The expression of the TRα gene was dramatically enhanced in the low-concentration group of DON, indicating that a mechanism negatively responded to the reduced T4 level [47]. On the contrary, a marked up-regulation of the TRα gene was found in the low-concentration group of JOT, implying that there was a positive feedback mechanism because of the induced T4 level [48]. Noticeably increased expression of tsh was found in the high-concentration groups of AFB1 and DON in comparison to the control group. Iodothyroinine deiodinases (Dio) possess an important function in modulating the circulation of peripheral TH levels and maintaining the ratios of T4 and T3 in vertebrates [49]. Dio1 affects iodine recovery and TH degradation. The expression of dio1 was negatively associated with T3 and T4 levels in fish upon exposure to AFB1, DON, and their mixture. Consequently, the changes in T3 and T4 contents in zebrafish indicate the maladjustment of THs [50]. The expressions of thyroid-associated genes were also considerably changed, indicating that AFB1, DON, and their mixture were thyroid disruptors in embryonic zebrafish.
VTG is considered a biomarker of estrogen-associated endocrine disorders, and the generation of VTG is regulated via 17β-estradiol activation of estrogen receptors (ERs) [51, 52]. The VTG content was obviously reduced in the AFB1 and DON exposure groups, indicating that the two mycotoxins had the anti-estrogenic effect. Conversely, the VTG content was increased in the JOT exposure group, implying that the mixture of AFB1 and DON possessed the estrogenic effect [53]. HPG axis modulates sex hormones, which are tightly associated with procreation in fish [54]. Surprisingly up-regulated expressions of cyp17 and cyp19a were found in the AFB1, DON, or JOT group. Besides, the cyp19a expression was remarkably up-regulated in the low-concentration group of JOT implies that AFB1, DON, and their mixture could impair the reproduction of zebrafish.
The main function of the HPA axis is to modulate the adaptive stress response of organisms [55]. The expressions of crh and gr were noticeably varied in most of AFB1 and JOT exposure groups, implying that AFB1 and its combination with DON could impact the development of zebrafish. Environmental pollutants can elevate or reduce the expressions of immune-related genes [56]. Our present study exhibited that the single and JOT exposures increased the expressions of IL-8, cc-chem, and cxcl-cic at the mRNA level. This finding might imply that latent tissue damage triggered by AFB1, DON, and their blend provoked the up-regulation of the investigated cytokines in this study [57]. Moreover, AFB1, DON, and their mixture might impair the immune function and elevate the susceptivity to toxicants for fish.
A test of multiple toxicities in zebrafish is helpful in comprehensively understanding the toxic mechanisms of environmental pollutants [36, 52]. To comprehensively assess the mixture mechanisms of AFB1 and DON in fish, we attempted to assess the toxic effects of mixture mycotoxins on oxidative stress, immunotoxicity, cell apoptosis, and endocrine system in the early developmental stage of zebrafish at various endpoints. Our data provided solid evidence on the mixture toxic mechanism of AFB1 and DON in aquatic organisms. In the present study, the altered enzyme activity reflected the extent of cellular damage. Nevertheless, different biochemical enzymes have different sensitivity to mycotoxins. To validate this hypothesis, further study is still required to evaluate the changes of these enzymes at the protein level. The zebrafish can adapt to various stimuli induced by exogenous mycotoxins via a potential mechanism by up/down-regulating genes. Therefore, it is indispensable to evaluate the mechanism underlying the different alternations in immune-associated genes.