| Although disinfection of drinking water eliminates most of the pathogenic microorganisms,the disinfectants respond to natural organic matter or environmental contaminants,generating a large number of disinfection byproducts(DBPs).Halobenzoquinones(HBQs)are an emerging class of DBPs detected in both disinfected drinking water and swimming pool water.Among the HBQ species,2,6-dichloro-1,4-benzoquinone(DCBQ)is the most frequently detected and has the highest concentration in water.Toxicological experiments have proved that HBQs are extremely cytotoxic,and the toxicity of HBQs is between 10-1000 times higher than that of the regulated DBPs,so the health risks of HBQs cannot be ignored.However,the toxicological evaluation of HBQs still needs to be further improved,and there are relatively few reports on their potential neurodevelopmental toxicity,especially the lack of in vivo studies as the most direct evidence of neurotoxicity.This study adopts an integrated method of in vivo and in vitro experiments.A rat model was established for the entire gestation and lactation period to study the neurodevelopmental toxicity of DCBQ in offspring rats.SH-SY5Y cells were exposed to DCBQ during differentiation to investigate the effects of DCBQ on cell proliferation,neural differentiation,oxidative damage and apoptosis.And deeply explore the role of oxidative stress-induced mitochondrial apoptosis pathway and PI3K/AKT/mTOR pathway in the neurodevelopmental toxicity of DCBQ to further reveal the action targets and molecular mechanisms of the neurodevelopmental toxicity of HBQs.It also provides new ideas for further studies of the neurodevelopmental toxic effects and mechanisms of other DBPs.Part Ⅰ Effect of DCBQ on neurodevelopmental toxicity in offspring rats and neural differentiating SH-SY5Y cellsObjective:A rat model of DCBQ exposure throughout pregnancy and lactation was established to clarify the effect of DCBQ on the neurodevelopmental toxicity of offspring rats.Retinoic acid(RA)induced SH-SY5Y cells investigated the effects of DCBQ on neural cell proliferation,neural differentiation,oxidative damage and apoptosis,and further clarified the neurotoxic effects of DCBQ.Methods:1.In vivo experimentsForty female specific pathogen-free(SPF)adult SD rats,with weights of 240-260 g were selected.After a week of SPF barrier feeding,the SD rats were paired 1:1 by gender,and the pregnant rats were identified by the presence of a vaginal plug the next morning.The pregnant rats were randomly divided into four groups exposed to different doses of DCBQ:the control group with medical castor oil solvent,0.5 mg/kg DCBQ group,5 mg/kg DCBQ group,and 50 mg/kg DCBQ group,with 10 rats in each group.Starting from successful conception,the rats were exposed to DCBQ until the end of lactation,lasting 6 weeks,and were administered drugs through gastric irrigation once a day at a relatively fixed time in the morning.The status of the maternal rats was observed daily,and the weight was recorded every two days,then the maternal rats were killed.After weaning,the offspring rats were housed separately by gender.At 4 weeks and 8 weeks of age,the Morris water maze test was used to test their spatial learning and memory functions;The open field test was used to detect the autonomous motility of offspring rats.After the behavioral tests were completed,the offspring rats were sacrificed,and their brains were collected and weighed.HE staining and Nissl staining were used to observe the pathological changes in the cerebral cortex of4-week-old and 8-week-old offspring rats.2.In vitro experimentsThe CCK-8 method was used to determine the effects of different concentrations of DCBQ on the viability of neural differentiating SH-SY5Y cells after 24 h,48 h,and 72 h exposure.Based on the experimental results,the subsequent exposure dose was determined.The low,medium,and high dose groups of DCBQ exposure were set at 6.75,13.5,and 27.0μM respectively.During the process of RA-induced SH-SY5Y cell neurodifferentiation,DCBQ was given to cells at different doses.Flow cytometry assay and Western-Blot methods were used to detect changes in the content of β-Tubulin in differentiating SH-SY5Y cells exposed to DCBQ.The DCFH-DA method was applied to evaluate the generation level of ROS in cells.The effect of DCBQ exposure on the mitochondrial membrane potential of differentiating SH-SY5Y cells was measured using Rhodamine 123.The apoptosis of cells was detected by flow cytometry using the Annexin V-FITC and PI double staining method.Data analysis was performed using SPSS 25.0 and Graph Pad Prism 8.0 software.Data were expressed as mean ± standard deviation(mean ± SD).The difference between multiple groups was analyzed by one-way ANOVA,and pairwise comparisons were made using the LSD test.P < 0.05 was referred to statistically significant.Results:1.In vivo experiments(1)Since the 14 th day of gestation,the maternal rat weight in the 50 mg/kg DCBQ group showed a decreasing trend compared to other groups,and throughout the lactation period,especially the first 14 days of lactation.(2)Compared with the control group,the brain organ coefficient of the offspring rats in the 5 mg/kg and 50 mg/kg DCBQ exposure groups were significantly decreased at both 4weeks and 8 weeks old(P<0.05).(3)The Morris water maze test found that with the increase of maternal rat DCBQ exposure dose,the movement trajectory of offspring rats deviated significantly from the platform and quadrant,and the activity around the pool edge increased significantly.Compared with the control group,the number of times that offspring rats in the 5 mg/kg and50 mg/kg DCBQ groups entered the target quadrant and crossed the platform was significantly reduced(P<0.05).The open field test found that with the increase of DCBQ exposure dose,the activity of offspring rats around the edge area increased significantly.Compared with the control group,the total distance and average velocity of offspring rats in the 5 mg/kg and 50 mg/kg DCBQ groups were significantly reduced(P<0.05).(4)After DCBQ exposure,the offspring rats showed disordered and loose neural fiber structure,nuclear contraction,deep staining,vacuolation,dissolution and disappearance of Nissl bodies,especially in the 5 mg/kg and 50 mg/kg DCBQ groups.2.In vitro experiments(1)DCBQ had a significant proliferation inhibitory effect on the proliferation of differentiating SH-SY5Y cells,and this effect showed a dose-and time-dependent manner,as the concentration of poisoning increased and the duration of action extended,the viability of the cells showed a significant downward trend.(2)Flow cytometry assay revealed that with the increase in DCBQ exposure dose,the content of β-Tubulin in differentiating SH-SY5Y cells decreased,with statistically significant differences between high-dose group with the control group and low-dose group(P<0.05);Western blotting detected β-Tubulin protein expression levels,which showed a more significant trend,with the increase in DCBQ exposure dose,the β-Tubulin protein levels in the cells decreased significantly in the medium and high-dose groups,with statistically significant differences between the control group and the medium-dose group,high-dose group with other groups(P<0.05).(3)DCBQ significantly increased the production of ROS in differentiating SH-SY5Y cells and the levels of ROS generation in all dose groups were significantly higher than those in the control group(P<0.05).(4)With the increase in DCBQ exposure dose,the mitochondrial membrane potential in differentiating SH-SY5Y cells decreased,and the levels in the medium and high-dose groups were significantly lower than those in the control group(P<0.05),and the levels in the high-dose group were also significantly lower than those in the low and medium-dose groups(P<0.05).(5)The rates of early,late and total apoptosis in the high-dose DCBQ group were significantly higher compared with the control group.With the increase of DCBQ exposure dose,the total apoptosis rate showed an increasing trend,and the difference between the total apoptosis rate in the low,medium and high dose groups was statistically significant(P<0.05).Conclusions:1.DCBQ exposure caused a decrease in the organ coefficient of the offspring rat brain.2.Maternal DCBQ exposure altered the pathological structure of the offspring rat cerebral cortex.3.Maternal DCBQ exposure impaired the spatial learning and memory function of offspring rats.4.DCBQ inhibited the proliferation of differentiating SH-SY5Y cells.5.DCBQ inhibited the expression of β-Tubulin in differentiating SH-SY5Y cells,suggesting that DCBQ could inhibit the neural differentiation level of SH-SY5Y cells.6.DCBQ can increase intracellular ROS,decrease mitochondrial membrane potential and increase the rate of apoptosis.DCBQ exposure had toxic effects on SH-SY5Y cells during differentiation.Part Ⅱ The role and mechanism of the oxidative stress-mediated mitochondrial apoptosis pathway in DCBQ neurodevelopmental toxicity Objective:The study examined the changes of oxidative damage index and mitochondrial apoptosis pathway in the cerebral cortex at 4 weeks and 8 weeks after DCBQ exposure.Using the SH-SY5Y cells undergoing differentiation as an in vitro model,introduce the antioxidant N-acetyl-L-cysteine(NAC)and clarify the toxic effects of DCBQ with or without NAC on the differentiating SH-SY5Y cells.Explore the role of oxidative stress in the effects of DCBQ on neural differentiation and potential mechanisms.Methods:1.In vivo experimentsThe group and DCBQ exposure were the same as in Part I.The TBA method was used to determine the content of MDA in the cerebral cortex of offspring rats.Ultraviolet spectrophotometry was used to determine the content of protein carbonyl groups in the cerebral cortex.Immunohistochemistry was used to detect the expression of i NOS in the cerebral cortex.Real-time quantitative PCR and Western blot were used to detect the mRNA and protein expression levels of key genes in the mitochondrial apoptosis pathway(Cyt-C,Caspase 9,and Caspase 3)in the cerebral cortex of offspring rats.2.In vitro experimentsIn this part of the study,the concentration of 13.5 μM was selected as the dose of DCBQ exposure.The experimental groups included: the negative control group(Control),the NAC group,the DCBQ group,and the NAC + DCBQ group.The methods for detecting cell proliferation,protein expression of β-Tubulin,mitochondrial membrane potential,and apoptosis rate were the same as in Part I.Real-time quantitative PCR was used to detect the mRNA expression levels of key genes in the mitochondrial apoptosis pathway(Cyt-C,Caspase 9,and Caspase 3)and the PI3K/AKT/mTOR pathway(PI3K,AKT,and mTOR)in differentiating SH-SY5Y cells.Western-Blot was used to detect the protein expression levels of Cyt-C,Caspase 9,Caspase 3,PI3K,AKT,and mTOR in the cells.Results:1.In vivo experiments(1)After maternal rats were exposed to DCBQ,the level of MDA in the offspring rat cerebral cortex increased gradually with the increase of DCBQ exposure dose,and the content of MDA in the cerebral cortex of offspring rats in the 0.5 mg/kg,5 mg/kg,and 50mg/kg DCBQ exposure groups was significantly higher than that of the control group.The MDA content in the 50 mg/kg DCBQ group was also significantly higher than that in the 0.5mg/kg and 5 mg/kg DCBQ groups(P<0.05).(2)Compared with the control group,the protein carbonyl content in the cerebral cortex of offspring rats in the 0.5 mg/kg,5 mg/kg,and 50 mg/kg DCBQ exposure groups showed a significant increase(P<0.05).(3)Compared with the control group,the immunopositive reaction of i NOS in the cerebral cortex of offspring rats was significantly enhanced after maternal exposure to DCBQ.With increasing exposure doses,the expression levels of i NOS-positive cells showed an increasing trend,with the highest expression level in the 50 mg/kg DCBQ group.The expression levels between groups were statistically different(P<0.05).(4)After maternal rats were exposed to DCBQ,the mRNA and protein expression levels of Cyt-C,Caspase 9,and Caspase 3 in the cerebral cortex of offspring rats were significant up-regulated.2.In vitro experiments(1)The cell viability of the NAC+DCBQ group was significantly improved compared to the DCBQ group(P<0.05),returning to the level of the control group.(2)The expression level of β-Tubulin protein in the NAC+DCBQ group was significantly increased compared to the DCBQ group(P<0.05).(3)The mitochondrial membrane potential level in the NAC+DCBQ group cells was significantly improved compared to the DCBQ group,with a statistically significant difference(P<0.05).(4)Compared to the DCBQ exposure group,the percentage of late and total apoptotic cells in the NAC+DCBQ group was significantly reduced(P<0.05).(5)After exposure to DCBQ,the mRNA and protein expression levels of Cyt-C,Caspase 9,and Caspase 3 in differentiating SH-SY5Y cells were significantly increased compared to the control group(P<0.05).In the NAC+DCBQ group,the cells were pretreated with NAC before being exposed to DCBQ,and the mRNA and protein expression levels of Cyt-C,Caspase 9,and Caspase 3 were significantly lower than in the DCBQ group(P<0.05).(6)After exposure to DCBQ,the mRNA and protein expression levels of PI3K and AKT in differentiating SH-SY5Y cells were significantly decreased compared to the control group(P<0.05),and the mTOR expression level also showed a downward trend.In the NAC+DCBQ group,the cells were pretreated with NAC before being exposed to DCBQ,and the mRNA and protein expression levels of PI3K,AKT,and mTOR were significantly increased than in the DCBQ group(P<0.05).Conclusions:1.Maternal DCBQ exposure aggravated oxidative stress injury in the cerebral cortex of offspring rats.2.Maternal DCBQ exposure affected the expression of key genes in the mitochondrial apoptosis pathway in the cerebral cortex of offspring rats.3.Inhibition of oxidative stress increased cell viability,reduced ROS production,promoted neural differentiation,restored mitochondrial membrane potential,and decreased apoptotic rate in SH-SY5Y cells induced by DCBQ,indicating that inhibition of oxidative stress has a protective effect on differentiating SH-SY5Y cells exposed to DCBQ.4.DCBQ induced activation of the Cyt-C/Caspase3 mitochondrial apoptotic pathway in neural differentiating SH-SY5Y cells,and inhibition of oxidative stress downregulated the expression of key genes in this pathway,suggesting that oxidative stress-mediated activation of mitochondrial apoptotic pathway is one of the important molecular mechanisms underlying the neurotoxic effects of DCBQ.5.DCBQ inhibited the PI3K/AKT/mTOR signaling pathway in neural differentiating SH-SY5Y cells,and inhibition of oxidative stress upregulated the expression of key genes in this pathway,indicating that oxidative stress-mediated downregulation of PI3K/AKT/mTOR signaling plays an important role in the process of DCBQ affecting neural differentiation.Part Ⅲ The role and mechanism of PI3K/AKT/mTOR signaling pathway in the influence of DCBQ on neural differentiation Objective:The study examined the changes of PI3K/AKT/mTOR pathway in the cerebral cortex of4 weeks and 8 weeks after DCBQ exposure.And in this section,the PI3K pathway specific inhibitor LY294002 was introduced to observe the effects of DCBQ on the proliferation,differentiation,oxidative stress and apoptosis of differentiating SH-SY5Y cells after inhibition of PI3K signaling pathway,and to gain insight into the role and possible mechanisms of PI3K/AKT/mTOR signaling pathway in DCBQ neurotoxicity.Methods:1.In vivo experimentsThe group and DCBQ exposure were the same as in Part I.Real-time quantitative PCR and Western blot were used to detect the mRNA and protein expression levels of key genes in the PI3K/AKT/mTOR pathway in the cerebral cortex of offspring rats.2.In vitro experimentsThe concentration of 13.5 μM was still chosen in this part as the toxic dose of DCBQ.The experimental groups were: negative control group(Control),LY294002 group(LY group),DCBQ group,and LY294002+DCBQ group(LY+DCBQ group).The methods for detecting cell proliferation,β-Tubulin protein expression,ROS generation,mitochondrial membrane potential,and cell apoptosis rate were the same as in Part I.The methods for measuring the mRNA and protein expression levels of key genes in the mitochondrial apoptosis pathway,including Cyt-C,Caspase 9,and Caspase 3,were the same as in Part II.Results:1.After maternal rats were exposed to DCBQ,the mRNA and protein expression levels of PI3K,AKT,and mTOR in the cerebral cortex of offspring rats were significantly downregulated.2.The cell viability of the LY+DCBQ group was significantly lower than that of the LY group and the DCBQ group(P<0.05).3.The expression level of β-Tubulin protein in the LY+DCBQ group was lower than that of the DCBQ group(P<0.05).4.Compared with the DCBQ-exposed group,the intracellular ROS content in the LY+DCBQ group was significantly increased(P<0.05).5.The mitochondrial membrane potential of the cells in the LY+DCBQ group was further decreased compared with the DCBQ group(P <0.05).6.Compared with the DCBQ-exposed group,the percentages of early,late,and total apoptosis in the LY+DCBQ group were significantly increased(P<0.05).7.After pretreatment with LY294002,the mRNA and protein expression levels of Cyt-C,Caspase 9,and Caspase 3 in the LY+DCBQ group were significantly higher than those in the DCBQ group,and much higher than those in the LY group(P<0.05).Conclusions:1.Maternal DCBQ exposure affected the expression of key genes in PI3K/AKT/mTOR pathway in the cerebral cortex of offspring rats.2.Inhibition of the PI3K/AKT/mTOR signaling pathway enhanced the inhibitory effect of DCBQ on the proliferation of differentiating SH-SY5Y cells.3.Inhibition of the PI3K/AKT/mTOR signaling pathway can further enhance the effect of DCBQ in reducing the level of SH-SY5Y cell differentiation.4.Inhibition of the PI3K/AKT/mTOR signaling pathway further promoted the generation of ROS,decreased the mitochondrial membrane potential and increased the apoptosis rate induced by DCBQ in SH-SY5Y cells.5.After inhibition of the PI3K signaling pathway,the expression levels of key genes in the mitochondrial apoptosis pathway induced by DCBQ were further increased,and the PI3K/AKT/mTOR pathway plays an important role in DCBQ affecting the processes of neural differentiation. |
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