High-throughput Screening Of DNA Damages Induced By Smoke And Chemopreventive Mechanism Of Rosemary

There are various chemical carcinogens in polluted air and water bodies,such as benzopyrene,aflatoxins,organochlorophosphorus pesticides,and nitrosamines.These environmental pollutants induce DNA damage,repair of DNA,survival and growth of cells.These are one of the main factors inducing various types of cancer.Smoke from fuel combustion is one of the main causes of air pollution,such as automobile exhaust gas,waste incineration fumes,fire smoke,cooking smoke,etc.The smoke composition is complex and it's quantity is due to the type of fuel and the combustion method so that the harmfulness is related to particle composition and size.Short-term high-dose exposure to harmful smoke can cause acute poisoning,and long-term low-dose exposure has a wide range of effects on human health associated with DNA damage and repair,cellular oxidative stress,inflammatory response,tumorigenesis,as well as affecting the nervous system.However,the hazard is not easy to prevent and often overlooked by people.Therefore,it is of great practical significance to identify hazards in environmental smoke,develop effective methods to reduce the harm of smoke,and enhance people's awareness of protection.Cigarette smoke is a kind of typical environmental smoke,which there are approximately identified 4,800 chemical substances with 69 kinds identified as carcinogens and some of the compounds are tumor-promoting factors or auxiliary carcinogens.The major pollutants in cigarette smoke includes Alkali,CO,HCN,polycyclic aromatic hydrocarbons,tobacco-specific nitrosamines,and volatile aldehydes.Compared with other sources of smoke,such as cooking smoke,mixed aerosols produced by waste incineration,etc.,cigarette smoke has stable release,definite harmful chemical substances,scientific methods of collection methods,and samples are readily available and easy to control,repeatable test,long-term low-dose exposure and other significant advantages.Therefore,this dissertation selected cigarette smoke as a representative and model sample of environmental smoke and researched on how to use a high-throughput screen for detecting smoke-induced genetic damage,and how to use chemical protection agents to effectively counteract smoke hazards.Finally,the chemopreventative mechanism was explored from the transcriptome and protein levels.The dissertation is divided into three parts:The first part is to study the establishment of a high-throughput screening method for DNA damage induced by smoke,while providing a simple method for hazard identification of environmental smoke and water pollution as well as for screening chemopreventative agents.The second part is the screening study of rosemary,which is used as a natural chemopreventative agent with resistance to smoke according to the literature reports.Through the cytotoxicity test and the high-throughput screening method established in the first part,the chemopreventative efficacy of rosemary components'resistance to smoke-induced DNA damage was verified,and rosemarinic acid was screened as a component with low cytotoxicity and strong effect.The third part is the research on the chemopreventative mechanism of rosmarinic acid.The third part is to study the chemical protection mechanism of rosmarinic acid from the level of transcriptome and determine its key signal pathway and verify it,then draw signal network involved in RA preventing from TPM.In the first part of this study,the quantitative identification method of cell scanning,analysis technology,and micro-nuclei were studied;then,we established theflow cytometry,computer image analysis and detection of high content screening systems.TPM samples from 14 cigarettes brands were conducted by utilizing the high-throughput screening method and the results were found to have a good correlation with the artificial microscopy of micronucleus tests.The results are objective and reliable and can be used for the high-throughput detection of micro-nuclei induced by cigarette smoke.Secondly,the high-content cytometer was used to identify the background of cells and the fluorescence of?H2AX by double dyes Hoechst and FITC labeled with?H2AX antibody,to improve the detection accuracy,and to establish a method for high-throughput detection of DNA damage biomarker?H2AX.The results showed that?H2AX induced by TPM exhibited different?H2AX concentrations at different exposure concentrations and exposure times.There was no significant difference in the mean nuclear fluorescence intensity of TPM low-dose group?20?g/ml?exposed for 3h and 24h exposure compared with the control.However,with the increase of TPM exposure concentration,the average fluorescence intensity of single nuclei of?H2AX showed an upward trend.When the concentration of TPM exposure was 80?g/ml,the average fluorescence intensity of single nuclei of?H2AX reached the highest;when the exposure time was 12 hours,the intracellular?H2AX content peaks.In addition,the bacterial reverse mutation?Ames?microtitre fluctuation test was also improved and optimized in the detection of TPM mutagenesis.The established method can rapidly detect TPM-induced gene mutations and has a good correlation with classical plate method.We also applied in vitro micronuclei flow cytometry and Ames microfluidic method to the hazards identification in polluted water bodies,and successfully identified the mutagenic hazards of the polluted water samples.The practicability of this method had been verified.Natural chemopreventative agents need to have non-toxic or low toxicity and clear targets.In the second part of the study,immortalized tumor cells A549 and HepG2,TP53 mutant cells?p53-/-+S+Ras?and wild-type cells?P53-/-+V+Ras?,immortalized normal cells CHO and HPF were used to examine the known components and extract of rosemary including carnosic acid?CA?,carnothine?CS?,ursolic acid?UA?,rosmarinic acid?RA?,rosemary essential oil?RO?and rosemary water-soluble extract?RE?,using CHO and HPF cell line.The aim is to look for a dose range that not have toxic effects on normal cells.The results showed that CA had the lowest IC₅₀0 in CHO cells,followed by HPF cells,possibly CA pairs.Normal cytotoxicity is relatively large;CS has the lowest IC50 values?26.35 and 26.45?g/ml,respectively?in tumor cells A549 and HepG2,whereas RA,RO,and RE have relatively low cytotoxic effects on normal cells?CHO and HPF?.The Annexin V/PI double staining method was used to investigate the apoptosis.The results showed that tthe ratio of necrosis of CHO cells caused by CA,CS,UA,and RA was significantly higher than that of apoptosis.The change in rosmarinic acid was relatively insignificant,resulting in a cell necrosis rate that was almost half that of CA,CS,and UAindicating that the effect on apoptosis is relatively small.From this it can be concluded that carnosic acid,carnosol,ursolic acid are more cytotoxic,and the cytotoxicity of rosmarinic acid,rosemary essential oil and water soluble extract of rosemary is relatively small.In the second part,MTT cytotoxicity method,Ames microtitre fluctuation test and in vitro micro-nucleus high content method were used to verify and screen the protective efficacy and active ingredients of rosemary.The chemopreventative effect was studied by first treating the cells with rosemary for 24 hours and then exposing the cells to TPM for 24 hours.MTT results indicated that CA,CS,UA in the dose range of 70?g/ml,the toxic effects were significantly greater than the protective effect.However,in the dose range of 90?g/ml,RA had obvious protective effect and RO and RE still had certain protective effect in the dose range of 150?g/ml.The protective effect on HPF cells was more obvious.In Ames microtitre fluctuation test,TPM was set strong mutagenicity dose at 250?g/50 well,The results showed that TPM-induced colony counts of TA98 were 47 wells/50 wells.CA,CS,and RA at the maximum dose of 0.8 mg/50 wells reduced TA98 induced reverted colonies to the spontaneous reversion range and have a strong dose-response.whereas UA,RO,and RE have relatively weak anti-mutagenicity.In vitro micronucleus test high content method results showed that within the dose range of 20-80?g/ml RA,TPM significantly reduced the number of micronucleus induced by RA pre-protected HPF cells compared to the unprotected group,indicating that RA has a significant protective effect on TPM induced chromosomal damage.In the third part,we explored the mechanism of chemical protection that has been determined for rosmarinic acid.We firstly used RNA-Seq analysis methods to perform transcriptome sequencing,GO analysis,and GSEA gene enrichment analysis on six groups of samples.These six groups of samples were pure TPM exposure 6h groups?TS?,TPM exposure 24h group?TL?,single cell control group?CC?,RA alone pretreatment group?RC?,RA pretreatment and TPM exposure 6h group?RTS?,RA pretreatment and TPM exposure 24h group?RTL?.The results showed that the number of functional genes of BP,CC,and MF significantly activated by TPM 6h during short-term exposure?TS?was greater than TPM 24h long-term exposure?TL?;the biological events caused by RA protecting TPM for 6 hours were mainly related to early cell stress,and the biological events caused by prevention of TPM for 24 hours by RA are mainly related to DNA repair,chromosomes,and enriched to the G2M checkpoint,angiogenesis,and IL6-JAK-STAT3 signaling pathway.GSEA analysis results showed that differential genes were enriched in multiple gene sets.Based on these gene sets,we speculate that the protective effect of RA on TPM-induced cell damage may play a role through the following aspects:1.the regulation of intracellular oxidation reactions,such as peroxisome,oxidative stress and ROS pathway;2.the regulation of cell metabolism,such as cholesterol and estrogen responsive homomorphism,bile acid metabolism,glycogen,fatty acid metabolism;3.regulation of cell connection and exercise:such as the top connected cells;4.regulation of cell growth and apoptosis,such as P53 pathway,mitotic spindle checkpoint,G2M,DNA repair,KRAS signal pathway,inflammatory reaction.5regulate cell fate,such as myogenic cell formation,KRAS and P53 signal pathway.Because the differentially expressed genes have been enriched into the P53 pathway for many times,the P53 pathway has regulatory effects on DNA damage repair,cell cycle,apoptosis and tumor formation.We also speculate that RA may function through regulating P53 pathway.In the next study,we tested the preventative effect of RA on TPM induced cell damage at the cellular level.By detecting the reactive oxygen species?ROS?level,mitochondrial membrane potential,cell proliferation,apoptosis,cell cycle and DNA damage level,RA was involved in regulating these processes to prevent TPM induced cell damage.The resultsshowed that TPM could induce cell proliferation inhibition in a dose dependent manner.TPM could cause apoptosis and necrosis of HPF cells in a dose-dependent manner.Under oxidative stress,TPM treatment induced the production of ROS in mitochondria,the decrease of mitochondrial membrane potential and the oxidative damage of cells.In addition,TPM treatment could quickly induce the aggregation of gamma H2A.X into the nucleus,indicating that the DNA damage occurred.In addition,TPM induction also induces cell cycle arrest in G0/G1phase.RA pretreatment can significantly reduce the proliferation inhibition,apoptosis,ROS,DNA damage and cell apoptosis induced by TPM.Furthermore,Western Blot results of P53 pathway related proteins showed that TPM can enhance the activation of P53 signaling pathway and down-regulation of SIRTl,increase the expression of p21,Bax,Bcl-2 expression increased;while RA pretreatment can inhibit the production of ROS,SIRTl expression,MDM2 expression decreased,P53 protein phosphorylation and acetylation.From the results of transcriptome and Western Blot results of P53 signaling pathway related proteins,it is speculated that RA can inhibit the production of ROS and activation of SIRTl signaling pathway,and may regulate the MDM2 pathway and SIRTl pathway through phosphorylation and acetylation ofp53 protein.inhibiting the expression of downstream apoptotic proteins Bax and cleaving caspase expression to prevent against the oxidative damage induced by TPM.Otherwise,RA also can increase the cell cycle arrest of p21 protein expression to allow cells to obtain enough self-repair time to resist the damage of TPM.