| Chemical pollutants widely existed in the natural environment,affecting the normal function of life body and even inducing tumorigenesis.Identifying chemicals associated with cancer has become an important part of the field of environmental science.Traditional methods for evaluating whether the chemicals could promote tumor progression included in vitro cell or subcellular detection,rodent in vivo testing.Among them,the results of cell and subcellular detection that indirectly predicted the risk of tumor were often inaccurate,and the rodent live test was difficult to perform the test of a large number of chemicals due to the difficulty of operation and high cost.Therefore,it is necessary to establish a in vivo evaluation assay to rapidly and efficiently screen the cancer-promoting chemicals.Zebrafish is a model organism for toxicology research and has been employed to establish a variety of disease models of people in recent years.In this study,a transgenic zebrafish-induced liver cancer model was used to establish a method for rapid and efficient screening of environmental pollutants with liver tumor promotion,and to test the potential of promoting liver tumor progression of four types of environmental pollutants.Then,according to the screening results,with the help of liver histological examination,transcriptome sequencing analysis,RT-q PCR,hormone content determination and related verification experiments,the effects and mechanisms of tris(1,3-dichloro-2-propyl)phosphate(TDCIPP)and prochloraz(PCZ)on liver tumor progression are explored.The main results are as follows:(1)Based on the liver cancer induction model of kras transgenic zebrafish,a 7-day breeding exposure system was established.Lipopolysaccharide,which was reported to promote the liver tumor progression,was chosen for exposure testing.The field fluorescence area and the field average fluorescence intensity were determined,and the test results were verified by combining with histopathological observation.The tested results of the two plates were consistent,which showed exposure to lipopolysaccharide increased the expression of hepatic fluorescence in a dose-dependent manner,and 5μg/L lipopolysaccharide could significantly promote the field area and field average intensity.The results of histopathological examination showed that the addition of 5μg/L lipopolysaccharide could promote the liver tumor progression of kras larvae.These results show that the method has good operability and reproducibility.(2)Based on the established high-throughput screening method for chemicals with the potential to promote liver tumor progression,the test of the potential of 32 typical environmental pollutants to promote liver cancer was carried out.Histopathological examination was performed and it was found that 13 of the 15 chemicals,which were reported to promote cancer development,were also able to promote the liver tumor progression in kras larvae.While in the other 17 chemicals,which were not reported to be associated with cancer growth,five of the pollutants could promote the liver tumor progression in kras larvae.In addition,prochloraz and abamectin have the potential to inhibit the liver tumor progression in kras larvae.(3)Kras larvae were first exposed to 0.1,0.3 and 1 mg/L TDCIPP,and it was found that 0.3 mg/L TDCIPP started to promote the fluorescent protein expression.Then,0.3mg/L TDCIPP was used to expose male and female kras zebrafish,and it was found that both male and female fish were induced to develop liver tumor,and it had obvious gender differences.The addition of TDCIPP could significantly increase liver size and hepatic somatic index of kras males and females.The results of histological examination showed TDCIPP could promote the liver tumor progression both in kras males and females.According to the results of transcriptome sequencing,TDCIPP promoted the development of liver tumor in kras fishes by up-regulating the expression of inflammatory response-related genes and DNA replication pathway-related genes.After that,kras and lyz double transgenic zebrafishes were exposed to TDCIPP and the inflammatory inhibitor ketoprofen to confirm that TDCIPP could lead to massive infiltration of neutrophils into the liver and induce an inflammatory response to promote the liver tumor progression of kras zebrafish.(4)Taking PCZ as the research object,three time points(4,8 and 12 days of exposure)were selected to study its relationship with liver tumor growth and its underlying mechanism.Due to the induction time of 4 days,kras males and females could not induce obvious liver tumor,and the effect of PCZ was not significant.After induction for 8 days,both males and females could induce green fluorescent protein,and PCZ could attenuate the expression of fluorescent protein and hepatic somatic index.The results of histological examination confirmed that PCZ could inhibit liver tumor progression of kras males and females.However,after exposure for 12 days,PCZ could significantly reduce the fluorescence expression of males,but not in females.Histopathological results also found that PCZ could inhibit the development of liver tumor in kras males,but not in females.The determination of 19 steroid hormone levels and the correlation analysis of cortisol with parameters related to liver tumor progression identified that cortisol might be the key hormone that PCZ affected the liver tumor progression in kras zebrafish.The combination of PCZ and cortisol exposure to kras and lyz double transgenic zebrafish confirmed that PCZ could inhibit the promotion of cortisol induced liver tumor development in kras zebrafish. |
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