The Influence of Oxidative Stress, Carcinogens and Cloning on DNA ...

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Introduction chromatography with electrochemical detection (HPLC-ECD) (234, 238, 245, 259, 260) , 32 P postlabeled HPLC (261) , 32 P post-labeled-TLC (262) , gas chromatography-mass spectrometry (GC- MS) (263, 264) <strong>and</strong> LC-MS methods (265, 266) . HPLC-ECD <strong>and</strong> GC-MS had high reputation for the quantification <strong>of</strong> oxo-adducts. However, the levels <strong>of</strong> 8-OH-dG estimated by the latter method were found to be 10-50 folds higher than the levels estimated by the former method. <strong>The</strong> sensitivity recorded by GC-MS is about 1 molecule 8-OH-dG per 10 6 molecules deoxyguanosine (245) . However, this technique requires specialized personal <strong>and</strong> expensive instrumentation in addition to the need <strong>of</strong> derivitization step prior to the analyses which can induce an insult <strong>of</strong> artificially oxidative products explaining the high level <strong>of</strong> adducts recorded with GC-MS. <strong>The</strong> silylation step <strong>and</strong> the non-availability <strong>of</strong> isotopically labeled internal st<strong>and</strong>ards are the major disadvantages limiting the application <strong>of</strong> GC-MS technique for the determination <strong>of</strong> DNA base modifications. <strong>The</strong> requirement <strong>of</strong> specific antibody induces a certain difficulty to apply immunologically based methods. Consequently, HPLC-ECD, the pioneer method in this field, is considered so far the only method employed for analysis <strong>of</strong> 8-OH-dG due to its simplicity, sensitivity <strong>and</strong> availability <strong>of</strong> its equipments. However, the only drawback is the difficulty to estimate exact concentration <strong>of</strong> 8-OH-dG. 1.3.3 Exogenous DNA adducts <strong>The</strong>y are formed due to the exposure to genotoxic carcinogens such as polycyclic aromatic hydrocarbons (PAHs), UV photoproducts <strong>and</strong> aflatoxins (267-271) that react with one or more DNA bases producing gene mutation (160) . 1.3.3.1 Environmental chemicals Polycyclic aromatic hydrocarbons are the most extensively studied species as they are widely found, for example in cigarette smoke (272) . <strong>The</strong>y are strongly implicated as causative agents in the development <strong>of</strong> lung cancer (171) . Among the PAHs, benzo[a]pyrene (B[a]P), which occurs in amounts <strong>of</strong> 20 to 40 ng per cigarette, is one <strong>of</strong> the most potent carcinogens. As previously mentioned, the chemicals are not the main causative agents for cancers, but they are metabolically activated by cytochrome P450 enzyme leading to electrophilic species that can bind to DNA (175) . Following this mechanism, B[a]P, a bulky adduct, proceeds in a certain metabolic pathway to produce the ultimate carcinogenic metabolite <strong>of</strong> the isomers <strong>of</strong> 7,8-Diol-9,10-epoxide (BPDE) (273) , which 24
HO O OH N N DNA B[a]P B[a]P-7,8-dihydrodiol-9,10-epoxide O N HO HO DNA NH NH OH cytochrome P450 cytochrome P450 HO 25 O BPDE-10-N 2 -dG OH B[a]P-7,8-epoxide B[a]P-7,8-dihydrodiol Epoxide hydrolase Introduction is an electrophilic metabolite that attacks preferentially the nitrogen sites <strong>of</strong> nucleobase result- ing in the formation <strong>of</strong> N 2 -guanine adducts (171, 226) (Fig. 11). Thus, B[a]P leads to the muta- tions in growth regulatory proto-oncogenes <strong>and</strong> tumour-suppressor genes <strong>and</strong> results in cancer formation (171, 274, 275) . B[a]P was detected along P53 tumour suppressor genes which repre- sents the major mutational hotspots in two third <strong>of</strong> detected lung cancers in humans (171) . Importantly in the pharmaceutical field, B[a]P is present in a high content in coal tars, which are the major incorporated ingredients in the preparation <strong>of</strong> therapeutic ointments used in se- vere cases <strong>of</strong> psoriasis (276, 277) . Recently, coal tars were estimated as being highly involved in the carcinogenicity in humans (278) . Different studies performed in vitro as in vivo to provide an evident conclusion for the contribution <strong>of</strong> PAHs in carcinogenesis (171, 274, 277) . Recently, nitro-PAHs were classified as new mutagens found in some environmental components such as diesel exhaust <strong>and</strong> airborne particles (279) . Figure 11: Proposed metabolic activation pathway <strong>of</strong> B[a]P (273)
Page 1 and 2: The Influe
Page 3 and 4: The work at h<stro
Page 5 and 6: First, I feel always grateful to Al
Page 7: I would like to extend my thanks to
Page 11 and 12: The following acro
Page 13 and 14: FDA Food and drug
Page 15: RIA Radioimmunoassay ROS Reactive o
Page 18 and 19: Index 3.1 Spectrophotometeric deter
Page 20 and 21: Index 5.2.4.2 Dialysis of</
Page 22 and 23: Introduction 1.1.2 DNA methylation
Page 24 and 25: Introduction 1.2 The</stron
Page 26 and 27: Introduction Figure 4: Model for me
Page 28 and 29: Introduction Cancer is classified i
Page 30 and 31: Introduction DNA methylation Gene h
Page 32 and 33: Introduction 1.2.7.1 Clonin
Page 34 and 35: Introduction 1.2.8 Twins an
Page 36 and 37: Introduction chemicals, alkylating
Page 38 and 39: Introduction AP sites are not only
Page 40 and 41: Introduction 1.3.2.1 Oxidat
Page 42 and 43: Introduction Finally, flavo- <stron
Page 46 and 47: Introduction One of</strong
Page 48 and 49: Introduction facturing of</
Page 50 and 51: Introduction 320) ; but due to its
Page 52 and 53: Introduction HO O O Figure 15: Mech
Page 54 and 55: Introduction - Methylation Sensitiv
Page 56 and 57: Introduction 5‘ 5‘ 5 Figure 18:
Page 58 and 59: Introduction - Radioactive-mediated
Page 60 and 61: Aim and objectives
Page 62 and 63: Results and Discus
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Results and Discus
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Summary 4 Summary DNA methylation i
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Summary The same s
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Experimental part Sodium tetrapheny
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Experimental part � Sodium tetrab
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Experimental part framework was fas
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Experimental part propanol
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Experimental part 12.5 mU/µL SPD)
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Experimental part Condition Value D
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Experimental part 5.4 Instrumentati
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Experimental part Graduated cylinde
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Experimental part 3-Nitrobenzanthro
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Appendix In-vitro: Denoting a react
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Appendix Laboratory Document Hydrol
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Appendix BODIPY and</strong
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Appendix Description of</st
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Appendix Transmission [%] 120 100 8
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Appendix Figure 62: Mass spectrum E
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List of figures Fi
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List of tables 8 L
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Literature Literature 1. Egger, G.;
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Literature 41. Bestor, T.H.; "<stro
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Literature 78. Venitt, S.; "Mechani
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Literature 113. Sinclair, K.D.; Mce
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Literature 148. Kadan-Lottick, N.S.
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Literature cyte DNA by gas chromato
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Literature 221. Mirand</str
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Literature 257. Lingaraju, G.M.; Sa
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Literature different tissues <stron
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Literature 324. Vishwanath, B.S.; F
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Literature 359. Wei, L.; Li, R.K.;
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Literature 394. Ramsahoye, B.H.; "M
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Literature 431. Wirtz, M.; Schumann
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Literature 467. Cornelius, M.; Wort
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Literature 502. Issa, J.P.J.; Ottav
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Literature 536. Pinheiro, J. <stron
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