Cigarette Smoking Induced DNA Damage Evaluation In Vitro And Screening Of Natural Protective Compounds

Cigarette smoking is harmful to health and can induce several diseases such as cancer, carotid and autoimmune diseases, etc. Most of the diseases are associated with genetic mutation arising from DNA damage. It is difficult to forbid cigarette smoking in a short time because of the great profit from tobacco industry. Therefore, research for the cigarette smoke-induced DNA damage make great sense for the environment monitoring, new-drug development and early-prevention of the hereditary diseases. Recently, many researchers are seeking for stable biomarker to establish sensitive estimation technology of DNA damage. Meanwhile, as the natural product investigation developed with great pace, there are numerous natural compounds found every year and some of them have been proved good pharmacological activities in many fields. In order to find the good pharmacological activity drug in the numerous natural compounds, the high-throughput screening assay is very important. Traditional screening assays such as animals, cells are time-costing and require many types of equipment. So they are not quite suitable for high-throughput screening.Herein we focus on developing a sensitive technology to monitor cigarette smoke-induced DNA damage, and aim to investigate mechanism of DNA damage as well as to screen natural compounds for protecting DNA with high-throughput.The evaluation of DNA damage here is mainly based on the immobilized DNA on magnetic beads (MB) and the chemiluminescence (CL) detection technology. MB is an efficient carrier with feature of fast-separation and easy-operation, and CL detection offers unique advantages including simple instrumentation, high sensitivity and feasibility for apparatus miniaturization in analytical tests. The innovative idea in this paper combined the two technologies and an auto-smoker, developed the detection method for cigarette smoke-induced DNA damage, screened some natural compounds, approached DNA damage mechanism and validated the DNA protective natural compounds using plasmid DNA and cells. Description of research in my thesis is presented as follows:Chapter 1:This thesis first introduces the development of investigation for DNA damage including types of DNA damage, traditional evaluation methods of DNA damage and several specific DNA damage detection reported recent years. The second phase of this chapter reviews current research for cigarette smoke induced DNA damage, reactive oxygen species (ROS) in the smoke which can attack the DNA is the culprit to DNA damage. Following that, objectives and significance of this research are summarized.Chapter 2:Initial establishment of 8-hydroxy-2’-deoxyguanosine (8-OHdG,DNA damage biomarker) detection method in-vitroLarge amount of ROS from smoke can attack the C-8 position of 2’-deoxyguanosine and form 8-OHdG, which has often been used as a biomarker of oxidative DNA damage. In the first phase of this chapter, verified the specificity of the 8-OHdG antibody via the competitive ELISA, offered a rationale for the further study, which use ELISA to detect 8-OHdG. In the second phase, taking the hydroxyl radical arising from Fenton reagent as simple oxidation source, which can attack DNA and produce 8-OHdG, we establish a new method to evaluate 8-OHdG production and investigate the effects of 9 natural compounds. The whole method is composed of following four steps:(1)DNA oxidative damage:DNA is immobilized on the surface of MB by covalent binding, DNA oxidation is carried out with Fenton reagent and natural compounds are added for the protection of DNA against oxidative damage; (2) impurity elimination and 8-OHdG purification by a magnetic force; (3) 8-OHdG-antibody immunoreaction:anti-8-OHdG antibody is added, which reacts with 8-OHdG induced by oxidative DNA damage. An HRP-labeled secondary antibody is then added; (4) CL detection:the CL signal on the surface of magnetic beads is directly determined with CL HRP substrate. The results showed that the CL intensity from the groups with Fenton reagents added was obviously higher than the no-oxidation groups and had a dependence with the amount of Fenton reagent, and most of the 9 natural compounds had a effectively inhibition to the 8-OHdG production, correlated to their scavenging ability for the hydroxyl radicals from the Fenton reagents. In the third phase, based on the above 8-OHdG evaluation system, we changed the Fenton reagent to the’off-line’smoking products, i.e. the cigarette smoke was first collected in some carrier (gas bag or fiberglass membrane) as the auto-smoker ’smoked’,then dissolve in buffers. The results showed the’off-line’smoke can induce DNA to produce 8-OHdG, and quercetin, emodin, ferulaic acid, ginkgolic acid, panaxsaponin, astragaloside, naringenin, etc. had obvious inhibition to 8-OHdG production in our reaction systems, probably because of their scavenging ability to the superoxide anion and hydroxyl radical.Chapter 3:8-OHdG formation induced by on-line smoke and screening of natural protective compoundsThe most harm to the smoker comes from the mainstream cigarette smoke which includes the tar phase and gas phase, besides part of the ROS produced by’off-line’ smoke which includes much short-life radicals and have more attacking ability. In this chapter, we combined the previous method with a gas-liquid reactor to investigate the on-line smoking induced 8-OHdG. The main improvement is that, immobilized DNA dispersed in the gas-liquid reactor which connected to the airintake channel of the auto-smoker, the mainstream produced by smoking was instantly trapped in the gas-liquid reactor and reacted with the dispersing DNA. In this system, DNA suffered more intensive oxidation,after short-time contact between DNA and smoke,we detected obvious signal from 8-OHdG. The production efficiency of 8-OHdG and the easy operation of the on-line smoking made this technology more feasible to high-throughput screening of natural compounds.After optimizing the reaction condition, a library of 32 natural compounds including flavonoids, anthraquinones, organic acids,and organic alkalis,etc. was then screened and quercetin, baicalein and sodium danshensu were found to protect DNA from intensive oxidative damage and thus may be promising compounds for the development of new drugs.Moreover, the protection effect of these three natural compounds against DNA oxidation damage was successfully classified by directly spiking them in the reference cigarettes.In addition, the potential to screen a mixture in a complex sample matrix, such as crude extracts, was also demonstrated, and hence the proposed technique can screen compound within a complex matrix and enhance the screening throughput.Chapter 4:DNA single strand breakage induced by on-line smoke and screening of natural protective compoundsAccording to the above results, as the amount of smoke increased, the 8-OHdG increased and then almost level off or decreased gradually for some DNA. Therefore, mainstream smoke did not only induce DNA to form 8-OHdG, it also induced other DNA damage, such as DNA strand breakage which can cause parts of 8-OHdG divorced from the immobilized DNA and result in lower of 8-OHdG signal.In this chapter, we investigated the DNA single strand breakage induced by on-line smoke using immobilized FAM-DNA on MB combined with CL ELISA. The process is presented as follows:fluorescein (FAM) labeled target DNA was immobilized on the MB via carboxyl-amino reaction; With on-line cigarette smoking, the mainstream smoke attacked the immobilized FAM-DNA, DNA strand breakage make the broken part strand including FAM divorce from the MB;After extensive washing, HRP-labeled anti-FAM was added and reacted with the left immobilized FAM-DNA; CL HRP substrate was added and the CL intensity from the unbroken DNA was recorded by CL instrument, thus the broken DNA were indirectly calculated and the sensitivity can up to pmol-grade. The extensive washing after the DNA oxidation removed the interference of impurity to the detection. The results showed that mainstream smoke indeed induced DNA strand breakage, and to some extent the breakage amount have a positive correlation with the amount of cigarette smoke and the contact time between DNA and smoke. This technology could be not only used for evaluating the DNA damage induced by cigarette smoke but also the screening of the natural protective compounds against DNA breakage. The library of 30 natural compounds above mentioned was screened here and found that quercetin and baicalein had obvious inhibition to the cigarette smoke-induced DNA breakage, which had a correlation with the results in the last chapter.Chapter 5:Mechanism of DNA damage induced by on-line cigarette smokeThe complicated ROS from the cigarette smoke can not only induce DNA modified bases but also cause DNA breakage. In this chapter, we investigated the mechanism of DNA damage in our DNA model, including the relationship between 8-OHdG and DNA single strand breakage. The main work is as follows:Firstly, FITC, which has the same chromophore to the FAM, was linked to the carboxyl MB via the ethanediamine. The results of oxidation by on-line smoke showed that the mainstream smoke had no obvious influence on the structure of FAM and the combination of carboxyl and amino, which further supported that, as described in the last chapter, CL decrease from FAM-DNA oxidation represented the break of DNA main strand. Moreover, the LC-MS detected the similar DNA probe indeed damaged by mainstream cigarette smoke.In an effort to more clearly define the mechanism of action of DNA stand break induced by ROS from cigarette smoke, different DNA labeling FAM are immobilized on the MB,and react with smoke.The results indicated that the same FAM-DNA produced both 8-OHdG and single strand breakage, different DNA got different degree of breakage:10-mer single strand probe with different number of G (0,1,4) got an similar breakage,30-mer oligonucleotide probe with random sequence and the 10bp double strand DNA had relatively more intensive stand breakage. From these results we can presume that H-abstraction probability from deoxyribose residue in the backbone of a DNA strand was the main mechanism of DNA breakage for single strand DNA:longer DNA has more deoxyribose residues and more H-abstraction probability which resulted in more intensive DNA breakage; in the case of double DNA, due to the more complex biomolecule polymer matrix, the lifetimes of ROS between the two strands had been extended,enabling different chemical pathways to became available resulting more DNA strand break.Chapter 6:The protection effects of natural protective compounds on plasmid DNA & Cell DamageAim to develop the protection of natural protective compounds screened in above chapters to real DNA or cell DNA, in this chapter, we investigated their effects on plasmid DNA and cell DNA using traditional evaluation methods. Agarose gel electrophoresis (AGE) detection for plasmid DNA and SAGE detection for two kinds of cells showed that mainstream cigarette smoke indeed induced plasmid DNA and cell DNA strand breakage, and the breakage degree positively depend on the amount of smoke and the contact time between smoke and plasmid DNA or cells, quercetin, baicalein and sodium dansensu could also obviously protect the real DNA against oxidative damage from mainstream cigarette smoke. In addition, MTT detection for cells showed that mainstream smoke could severely damage the cells and cut down the cell vital force, and the previous protective compounds can also obviously protect the cells and maintain their vital force obviously. All these have convinced that the high-throughput screening method for natural protective compounds we established is reliable. And it also proved that DNA damage evaluation technology we’ve described in is paper is an ideal method compared to the cell or real DNA evaluation model. Besides, this method can overcome the problems of traditional methods, which are more complex to collect samples and often less sensitivity. It will lead to a great potential to screening natural protective compounds for DNA with high-throughput.