Study Of Embryotoxicity Induced By Deoxynivalenol And The Role Of Placental Oxidative Stress During The Process

Deoxynivalenol (DON) is one of the important pollutants in grains, and barley, wheat, corn and other cereal crops can be detected high levels of DON. Besides, some agricultural and sideline products (such as beer, bread, eggs, milk, meat, etc.) also contain detectable DON. DON is a member of the family of type B trichothecene, the toxins in this family are produced by fusarium, stachybotrys and myrothecium in the process of growth, and the food poisoning incidents induced by them has been a key point of concern all the world. DON is difficult to be degraded by common cooking and heating, that is to say, its toxicity is extremely difficult to be destroyed. Therefore, its residual concentration in food and environment is rather high. Once formed, the toxin transfers through the food chain and becomes a great threat to humans and animals.In the context of the current studies, DON can cause multiple organ injuries in numerous species, mainly manifests as relatively mild reactions such as nausea, vomiting, food refusal, diarrhea, digestive disorder and weight loss, etc. Besides, it also can induce serious adverse reactions such as skin damage, immunosuppression and even bone marrow damage. On the other hand, DON has no target organs in the body, but it has strong cytotoxicity. At the same time, DON has various biological toxicities, mainly including cytotoxicity, immunotoxicity, genotoxicity, teratogenicity, carcinogenicity, etc. In recent years, in vivo and in vitro models, DON was found to influence different animals and cells on reproduction and embryonic development, swine and murine are widely used as objects in vivo study, while Be Wo cells and oocytes are both important research objects in vitro experiments. Although the embryotoxicity of DON has been confirmed by numerous research, the mechanism of its embryotoxicity still needs further discussion, and it is one of the key points of this study.Former experiments have confirmed that oxidative stress which is caused by intracellular ROS excessive accumulation is one of the important early mechanisms of DON-induced toxic effects. This study will focus on whether this toxic mechanism exists in the model of DON-induced placenta oxidative damage. The placenta is the organ that exchanges substance between maternal and fetal, and fetus in the uterus can only rely on the maternal nutrition that crosses the placenta. HO-1 is the key enzyme of HO metabolism, its increased expression level is one of the adaptive mechanisms to protect themselves. To a certain extent, it protects cells from oxidative damages caused by different kinds of factors. For this experiment, on the one hand, HO-1 widely distributes in the microsomes in tissues and cells throughout the body (including the uterus and placenta) and can be induced by oxidative stress which can be caused by a variety of stimulating factors (such as Hypoxia state, endotoxin and ultraviolet radiation, etc.). This is one of the important mechanisms that the body protects itself from oxidative damage. On the other hand, HO-1 defects will lead to a variety of adverse obstetric outcomes, such as recurrent abortion, intrauterine growth retardation, stillbirth, preeclampsia, etc. Therefore, we believe that HO-1 can be used as the effect biomarker of the present study, and discussing the expression of related signal pathway, namely Nrf2/HO-1, is also one of the focuses of this study.In conclusion, in the present study we aimed to evaluate the embryotoxicity of DON in late pregnancy and analyze its molecular mechanism. This study will cast a new light on the molecular mechanism of DON-induced developmental toxicities, and it may provide fundamental evidence to early warning and risk assessment of DON-induced developmental damage in various foods over the world.Part1:Evaluation of the embryotoxicity of Deoxynivalenol through exposure in pregnant C57BL/6 mice.Objectives:The aim of this study was to evaluate the embryotoxicity of DON in late pregnancy through exposure in pregnant C57BL/6 mice.Methods:After one-week acclimation, the female mice were mated with males, and gestation day 0.5 d (GD 0.5 d) was designated when a vaginal plug was detected. The pregnant mice were randomly divided into 4 groups:control group (0 mg/kg/day, Control); DON low dose group (1.0 mg/kg/day, DON-L); DON medium dose group (2.5 mg/kg/day, DON-M); DON high dose group (5.0 mg/kg/day, DON-H). At GD9.5-11.5 d, pregnant mice in each group were given by gavage DON following the above doses respectively. All the pregnant mice were killed by cervical dislocation at GDI8.5 d, and the placenta and embryos were rapidly separated. Furthermore, skeletons and viscera were dyed to observe the malformations, and related indexes of embryonic development were calculated and statistically analyzed.Results:(1) Effects of oral administration of DON for GD9.5-11.5 d on maternal mice: DON-induced food refusal and weight loss during pregnancy were not significant. No significant differences were observed when compared total litter weight, total placenta weight and average placenta weight in DON-treated groups with the Control (P> 0.05). (2) Effects of oral administration of DON for GD9.5-11.5 d on embryos:Percentage of live embryos and resorbed embryos in DON-H were significantly different with the Control (P < 0.01). Embryotoxicity of DON may show up as deformities of skeletal instead of appearance and organs, and the obvious deformities can be detected in bones through the whole body.Conclusion:(1) The embryotoxicity of DON has been preliminary confirmed, and it is mainly presented as resorbed embryos (mainly in 5.0 mg/kg DON-treated group) and skeletal deformities (mainly in 2.5 mg/kg DON-treated group). No significant difference has been observed in 1.0 mg/kg DON-treated group. (2) DON-induced skeletal deformities can be detected in bones (such as cranial and cervical skeletons, axial skeletons, clavicle and limbs) all over the body, and the incidence of prehensile tail is the highest.Part2:Evaluation of placenta oxidative stress during Deoxynivalenol exposure in pregnant C57BL/6 mice.Objectives:The aim of this study was to observe the oxidative stress of placenta and the expression of Nrf2/HO-1 signal transduction pathway. To investigate the role of oxidative stress and Nrf2/HO-1 pathway in DON-induced embryotoxicity.Methods:After one-week acclimation, the female mice were mated with males, and GD0.5 d was designated when a vaginal plug was detected. The pregnant mice were randomly divided into 4 groups:control group (0 mg/kg/day, Control); DON low dose group (1.0 mg/kg/day, DON-L); DON medium dose group (2.5 mg/kg/day, DON-M); DON high dose group (5.0 mg/kg/day, DON-H). At GD9.5-11.5 d, pregnant mice in each group were given by gavage DON following the above doses respectively. All the pregnant mice were killed by cervical dislocation at GD18.5 d, and the placenta and embryos were rapidly separated. The placenta tissue was fixed and then stained with H&E, dihydroethidium was used to reflect the level of ROS in the placenta and the levels of MDA, GSH and activities of SOD were measured using commercial assay kits. Besides, the expressions of Nrf2/HO-1 pathway related genes were measured by PCR and WB, HO-1 levels were measured by ELISA. Immunohistochemistry was used to observe the translocation of Nrf2 in the placenta tissue. Finally, the expressions of inflammation and apoptosis related genes were measured by PCR.Results:(1) Structural and functional damages were discovered in placenta during DON exposure, especially in DON-M and DON-H groups.(2) DON exposure induced placenta ROS generation, especially in DON-H group. MDA in DON-H group was significantly higher than the Control (P< 0.01). The activities of SOD in DON-M and DON-L groups significantly increased (P< 0.05). However, GSH in DON-treated groups were not significantly different with the Control.(3) The level of PIGF mRNA in DON-treated groups was lower than the Control(P<0.05), but the significance was only found in the DON-H group(P<0.01).(4) Under the condition that DON-treated groups were lower than the Control, HO-1 level showed an increased trend from low to high dose (P< 0.05).(5) The mRNA level of HO-1 was significantly higher (P< 0.05) in the DON-L group and was significantly lower (P< 0.01) in the DON-H group than the Control. The mRNA level of Nrf2 was significantly higher both in the DON-L and DON-M groups than the Control (P< 0.05). The protein level of HO-1 was significantly higher both in the DON-L (P< 0.01) and DON-M (P< 0.05) groups than the Control. The protein level of HO-1 was significantly higher both in the DON-L (P< 0.01)、DON-M (P<0.01) and DON-H (P< 0.05) groups than the Control. However, no significant difference was found when examined the protein expression of Keap-1. Furthermore, we discovered the translocation of Nrf2 from cytoplasm to nucleus in the placenta.(6) The mRNA expressions of inflammation related genes IL-6, IL-8, TNF-a and Cox-2 were rather complicated, for example TNF-a significantly increased in the DON-L group (P < 0.05), Cox-2 significantly decreased in the DON-M group (P< 0.05), while IL-6 and IL-8 respectively decreased (P< 0.05) and increased (P< 0.05) in the DON-H group.Conclusion:(1) DON exposure during pregnancy leads to structural and functional damages of placenta in C57BL/6 mice.(2) The placental damages may relate to oxidative stress induced by DON, inflammation and apoptosis also appears, but the role which they play in the process still needs further investigations.(3) The expression of Nrf2/HO-1 pathway changes during the process of DON exposure and this change may protect the placental and embryonic development from DON-induced embryotoxicity.Part3:Molecular mechanism of the toxicity of Deoxynivalenol to the Be Wo cellsObjectives:Be Wo cells were treated with different doses of DON to investigate the effects of DON-induced oxidative stress on cells. On the other hand, HO-1 activator (Hemin) and inhibitor (Znpp) were used as well to discuss the effect of HO-1 expression change on Be Wo cells.Methods:The cells was cultured in the culture medium after recovery and observed the growth and adherence conditions everyday. When the confluence of cell reached 80%, passage culture can be achieved. Then cells were plated on 12-well dishes or 96-well transwell inserts according to the experiment, and the culture medium was changed every other day. The cell viability was measured with CCK-8 kit, the groups were divided according to the result of cell viability:(1) negative control:0 nM DON; (2)DON treatment group:50 nM DON; (3)HO-1 activator group:50 nM DON+10μM Hemin; (4)HO-1 inhibitor group:50 nM DON+10 μM Znpp. After 1 h (only PCR),3 h,12 h and 24 h incubation, the cells could be used for future experiments. ELISA kits were used to measure the levels of P-HCG and HO-1 in the placenta. Cellular ROS level was measured with automatic microplate reader, while MDA, SOD and GSH levels with commercial kits. PCR result reflected the mRNA expression of Nrf2/HO-1 pathway. Besides, immunohistochemistry was used to observe the translocation of Nrf2 in the placenta tissue. At last, the expressions of inflammation and apoptosis related genes were measured with PCR.Results:(1) Significant decreases of P-HCG were found in all DON-treated groups when compared with control (P< 0.01), but no significant differences were discovered among these groups in 3 h and 12 h. However, P-HCG in HO-1 activator and inhibitor groups were significantly lower than the DON treatment group in 24 h (P< 0.05).(2) ROS accumulation appeared in cells when treated with DON (P< 0.01), but Hemin could improve it (P< 0.01). The level of MDA in HO-1 activator and inhibitor groups were significantly higher than the DON treatment group in 3 h (P< 0.01), but the level reversed in 12 h in the activator group (P< 0.05). Hemin increased the level of SOD in 3 h (P< 0.05) while decrease occurred after that (P< 0.01), and Znpp increased SOD only in 12 h (P< 0.01). Hemin significantly decreased GSH level in 3 h (P< 0.01) while Znpp increased it in 12h(P<0.01).(3) In 3 h, Hemin and Znpp increased and decreased HO-1 concentration respectively (P< 0.01), but no significant difference was found among 3 DON-treated groups in 12 h, then Hemin and Znpp both significantly increased HO-1 level in 24 h (P< 0.01).(4) Hemin and Znpp both increased mRNA expressions of HO-1 at all time points (P< 0.01), but mRNA expression of Nrf2 was up-regulated by Hemin in 3 h (P< 0.05) while by Znpp in 12 h and 24 h (P< 0.01). Besides, when exposed to DON for a long time (12 h and 24 h), Nrf2 in BeWo cells that pretreated with Hemin and Znpp would translocated from cytoplasm to nucleus.(5) With the increase of DON intervention time and dose, cells’ inflammatory response enhanced, and the expression trend of all the inflammatory cytokines is complicated.Conclusion:(1) During early intervention of DON, Hemin improves DON-induced cell dysfunction, but this effect will disappear over time, instead Znpp plays a protective role.(2) The level of ROS has time-and dose-effect relationship with HO-1 expression in DON-treated embryonic trophoblastic cells.(3) Placental oxidative stress is one of the mechanisms of DON-induced embryotoxicity, up-regulation activity of HO-1 will increase the antioxidant capacity of cells in early time, but excessive increase of HO-1 even aggravates oxidative damage. In other words, the protective effect of HO-1 for oxidative stress has a "threshold effect".