Oral Cancer Induced by The Tobacco Carcinogen Dibenzo[a,l]pyrene in Mice

Open Access
Zhang, Shang-min
Graduate Program:
Biochemistry and Molecular Biology
Doctor of Philosophy
Document Type:
Date of Defense:
Committee Members:
  • Karam E El Bayoumy, Dissertation Advisor
  • Kristin Ann Eckert, Committee Member
  • John Peter Richie Jr., Committee Member
  • Thomas E Spratt, Committee Member
  • Oral Cancer
  • Tobacco Carcinogen
  • DB[a
  • l]P
  • DNA adducts
  • LC-MS/MS
  • p53 mutations
  • epigenetics
  • alcohol
  • chemoprevention
Oral cancer is the most common type of head and neck cancer, which is the sixth most common cancer worldwide. More than 90% of oral cancers are a type called oral squamous cell carcinoma (OSCC). Tobacco use is the most important risk factor. Chronic and/or heavy use of alcohol is another major risk factor. Early detection and prevention of oral cancer is very pivotal. Progress in the prevention and control of oral cancer has been hampered by the lack of appropriate animal models that could reflect human exposure. Therefore, experimental animal models that can accurately represent the cellular and molecular changes associated with the initiation and progression of human oral cancer are of crucial importance to better understand the mechanisms of oral carcinogenesis and to identify novel chemopreventive, as well as chemotherapeutic agents. We recently developed a novel mouse model to study oral carcinogenesis. We showed for the first time that direct application of DB[a,l]P into the oral cavity, induced SCC in oral tissues; (±)-anti-DB[a,l]PDE-diol epoxide, the ultimate metabolite of DB[a,l]P, is a remarkably potent carcinogen in the oral cavity (both oral tissues and tongue). The mechanisms that can account for oral cancer-induced by this tobacco carcinogen are the focus of this dissertation. Our working hypothesis is that both genetic and epigenetic alterations induced by DB[a,l]P can contribute to the development of oral cancer. To test our hypothesis, we focused initially on assessing the effect of this carcinogen on genetic alterations. We have developed a new stable isotope dilution HPLC-MS/MS method to identify and quantify DB[a,l]PDE-dA and -dG adducts in oral tissue of mice. Our method is sensitive enough to detect DNA adducts in vivo. We detected (-)-anti-cis- and (-)-anti-trans-DB[a,l]PDE-N6-dA and (-)-anti-cis- and (-)-anti-trans-DB[a,l]PDE-N2-dG adducts in oral and tongue tissues of mice treated with DB[a,l]P; the results indicate that the levels of dA adducts are significantly higher than dG adducts and that DB[a,l]P is predominantly metabolized to (-)-anti-DB[a,l]PDE. Using immunohistochemistry (IHC), we have shown over-expression of p53 and COX-2 protein in OSCC and dysplastic tissues induced by DB[a,l]P and DB[a,l]PDE in mice. P53 protein overexpression detected by IHC may result from p53 gene mutation or exposure to genotoxic stress. To determine whether p53 over-expression is, in part, due to p53 mutations, Exons 5 to 8 of p53 from representative tumor tissues, were analyzed by polymerase chain reaction single-strand conformation polymorphisms (PCR-SSCP) and direct sequencing. G:C→T:A transversion was detected in Exon 5, leading to mutation of codon 155 Arg to Leu; A:T → T:A transversion was detected in Exon 7, resulting in mutation of codon 232 Lys to stop codon. To further test our hypothesis, we examined the epigenetic effect of DB[a,l]P exposure. Methylation specific PCR and bisulfite sequencing were used to examine methylation alteration of p16 and RAR-β promoters. Promoter hypermethylation of both p16 and RAR-β was detected in OSCC induced by DB[a,l]PDE; also in oral tissues of mice treated with DB[a,l]P at early stage, prior to the appearance of detectable tumors. Employing our newly developed LC-MS/MS method, we tested the effect of alcohol on genetic damage-induced by DB[a,l]P in the oral cavity of mice. Our working hypothesis is that alcohol may deplete GSH in the oral cavity and in turn may increase the levels of the DB[a,l]PDE, that can enhance levels of genetic damage. The results support our hypothesis and indicate that alcohol can enhance DNA adduct formation induced by DB[a,l]P, through depletion of GSH. Even with recent advances in targeted therapies, it is still challenging to treat cancer including OSCC at late stage. The ultimate goal of cancer prevention is to reverse or prevent the development and progression of early stage disease before it becomes aggressive. Using our LC-MS/MS method, we tested the hypothesis that naturally-occuring agents that can alter the levels of phase I/ phase II enzymes in a manner that can reduce the formation of the active electrophilic metabolites derived from DB[a,l]P, will reduce the levels of DNA damage. Diets rich in fruits and vegetables can contribute to a lower risk of developing oral cancer. Resveratrol is a polyphenol present in grapes and berries. Kaempferol is a flavonoid found in tea, broccoli, grapefruit, berries and other plant sources. Both Resveratrol and kaemferol have been shown to inhibit the enzymatic activities of some phases I drug metabolizing enzymes, such as CYP1A1, CYP1B1 and CYP1A2, which are known to activate DB[a,l]P to its ultimate carcinogenic forms that can damage DNA. We have tested the chemopreventive potential of 5% Black Resberry, 0.025% of Kaempferol and 0.002% Resveratrol in our mouse oral cancer model. Our results indicated that these agents can inhibit DB[a,l]P-DNA adduct formation, and thus further studies in our laboratory will examine the chemopreventive efficacy of these agents against the development of oral cancer in our newly developed animal model.