Neurobehavioral Toxicity And Related Mechanisms Of Diazepam To Zebrafish(Danio Rerio)

Benzodiazepines(BZDs),psychoactive drugs,are widely reported to possess anti-anxiety and sedation ability and reduce nervousness,fear,sleep disorders,epilepsy,alcohol dependence,and muscle relaxation.Diazepam(DZP,alias Valium),a typical BZDs drug,is clinically used for sedation,hypnosis,and anti-epileptic treatment.In addition,it is also widely used as anti-stress medicine on animals in the livestock industry.It has been reported that the concentrations of DZP in various aqueous matrix range from 0.47 to 4000 ng/L.Furthermore,DZP can enrich in plasma,liver,muscle,gonads,and brain of fish.Thus,it has great significance to accurately assess the ecological risks of BZDs to formulate relevant prevention and control countermeasures and protect the water ecosystem.In this study,the zebrafish embryos,juveniles,and adults were used as model subjects for revealing the toxicity of DZP on the development,neurology,and behavior of fishes.Furthermore,transcriptome analysis was used to reveal the mechanism involved in the neurobehavioral toxic effects(and their gender differences)induced by diazepam.Here are the main contents:(1).Investigate the developmental and behavioral toxicity of diazepam exposure(0,0.4,4.0,40,400,4000?g/L)on zebrafish embryos(0?120 hpf).An accelerated failure time(AFT)model was used to simultaneously analyze the time-dependent and concentration-dependent effects of the mentioned toxic effects.The survival of embryos had a negative correlation with the concentration of diazepam.Analyzing the death and incubation data of embryos during the 120-hour exposure period with the AFT model,the results showed that 96h-LC₅₀ of DZP was 3302.39(3068.18?3555.56)?g/L.Exposure to DZP can cause bradycardia in zebrafish embryos.4,40,and 400?g/L would significantly delay the hatching time of embryos.The behavioral analysis of zebrafish larvae(120 hpf)demonstrated that DZP would not change the kinematic characteristics of zebrafish larvae under the light-dark alternating condition.The larvae were more active in the dark period comparing to that of the light period.However,larvae exposed to DZP were more sensitive to the conversion of light and dark.Simultaneously,exposure to diazepam could reduce the anxiety response of zebrafish larvae and evoke an anti-anxiety effect.(2).Investigate the neurological and behavioral toxicity of diazepam exposure(0,12,120,1200?g/L)on juvenile zebrafish(2 months old).The experiment consisted of4-day exposure and 21-day recovery,aiming to assess the recovery characteristics of behavioral responses in juvenile fish from DZP exposure and to investigate the links between variations in brain GABA levels and behavioral responses.The results showed that exposure to diazepam(120,1200?g/L)could significantly affect the locomotor ability of juveniles.1200?g/L-DZP exposure disrupted the locomotor ability and activity of zebrafish juveniles.Such persistent hypoactive effects were sustained until the 7th day or even the 21st day of recovery.Exposure to 120 and 12?g/L-DZP enhanced the locomotor activity of juvenile fish.Exposed to 120?g/L-DZP induced the delayed hyperactivity effect on juvenile locomotor activity.DZP exposure exhibited concentration-specific effects on brain GABA levels in zebrafish.For example,the GABA levels decreased in the 1200?g/L-DZP and increased in 120 and 12?g/L-DZP.Correlation analysis suggested that the changes in the brain GABA levels might cause persistent damage to the locomotor activity of juvenile zebrafish.(3).Investigate the mechanisms of neurological and behavioral toxicity and sex difference of 21-day diazepam waterborne exposure(0,12,120?g/L)on adult zebrafish(4 months old).The males and females were separately exposed during the experiment.The results showed that DZP induced a sedative effect on both male and female zebrafish.In addition,DZP exhibited sex-dependent effects on the behaviors of adult fish.The locomotor activity of females was significantly inhibited on the 14-day,and social behavior was severely disrupted.However,the decrease of locomotor ability of males appeared until the 21st day.Exposure to DZP for 21 days significantly disturbed almost all tested behavioral traits associated with courtship when both genders were put together.Sex-dependent responses in brain GABA and ACh E levels due to DZP exposure were also identified.Significant relationships between the brain GABA/ACh E levels and some behavioral parameters related to locomotor activity were detected in females but not in males.The transcriptome analysis showed that the number of down-regulated differentially expressed genes(DEGs)in females was far more than that in the males after exposed to 12?g/L-DZP for 21 days.In addition,the enriched KEGG pathways were different in the fish of different sexes.Those findings at molecular levels provided some new insights into the mechanisms involved in the gender difference in neurobehavioral toxicity of diazepam to zebrafish.