Carcinogenesis of the Oral Cavity - American Chemical Society

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Carcinogenesis of the Oral Cavity: Environmental Causes and Potential Prevention by Black Raspberry Karam El-Bayoumy, Kun-Ming Chen, Shang-Min Zhang, YuanWan Sun, Shantu Amin, Gary David Stoner, and Joseph B Guttenplan Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00306 • Publication Date (Web): 14 Nov 2016 Downloaded from http://pubs.acs.org on November 18, 2016

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Chemical Research in Toxicology

Carcinogenesis of the Oral Cavity: Environmental Causes and Potential Prevention by Black Raspberry

Karam El-Bayoumy*† Kun-Ming Chen†, Shang-Min Zhang§, Yuan-Wan Sun†, Shantu Amin‡, Gary Stoner┴, Joseph B. Guttenplan║ †

Department of Biochemistry and Molecular Biology, and ‡Department of Pharmacology, College of Medicine, Pennsylvania State University, Hershey, PA 17033 §

Department of Pathology Yale University, Yale School of Medicine, New Haven, CT 06510

┴

Department of Medicine, Medical College of Wisconsin, Milwaukee, WI 53226

║

Department of Basic Science, New York University College of Dentistry, New York, NY 10010; Department of Environmental Medicine, New York University School of Medicine, New York, NY 10019

CORESPONDING AUTHOR* Tel: 717-531-1005. E-mail: [email protected].

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Abstract Worldwide, cancers of the oral cavity and pharynx comprise the 6th most common malignancies. Histologically, more than 90% of oral cancers are squamous cell carcinoma (SCC). Epidemiologic data strongly support the role of exogenous factors such as tobacco, alcohol and human papilloma virus infection as major causative agents. Avoidance of risk factors has only been partially successful and survival rates have not improved despite advances in therapeutic approaches. Therefore new or improved approaches to prevention and/or early detection are critical. Better understanding of the mechanisms of oral carcinogenesis can assist in the development of novel biomarkers for early detection and strategies for disease prevention. Toward this goal several animal models for carcinogenesis in the oral cavity have been developed. Among these are xenograft, and transgenic animal models, and others employing the synthetic carcinogens such as 7,12dimethylbenz[a]anthracene in hamster cheek pouch, and 4-nitroquinoline-N-oxide in rats and mice. Additional animal models employing environmental carcinogens such as benzo[a]pyrene and N`-nitrosonornicotine have been reported. Each model has certain advantages and disadvantages. Models that 1) utilize environmental carcinogens 2) reflect tumor heterogeneity and 3) accurately represent the cellular and molecular changes involved in the initiation and progression of oral cancer in human could provide a realistic platform. To achieve this goal we introduced a novel non-surgical mouse model to study oral carcinogenesis induced by dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and tobacco smoke constituent, and its diol epoxide metabolite (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14tetrahydrodibenzo[a,l]pyrene [(±)-anti-DB[a,l]PDE]. On the basis of a detailed comparison of oral cancer induced by DB[a,l]P with that induced by the other above-mentioned oral carcinogens with respect to dose, duration, species and strain, cellular and molecular targets, 3



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and relative carcinogenic potency, our animal model may offer a more realistic platform to study oral carcinogenesis. In this perspective we also discuss our preclinical studies to demonstrate the potential of black raspberry extracts on the prevention of OSCC. Specifically, we were the first to demonstrate that black raspberry inhibited DB[a,l]P-DNA binding and of particular importance its capacity to enhance repair of DB[a,l]P-induced bulky lesions in DNA. We believe that the information presented in this perspective will stimulate further research on the impact of environmental carcinogens in the development of oral cancer and may lead to novel strategies toward the control and prevention of this disease.

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Contents 1. Introduction 2. Animal Models for Oral Cancer 2.1. Xenograft and Transgenic Animal Models 2.2. Chemically-Induced Oral Cancer in Animal Models 3. Metabolic Activation of Oral Carcinogens 3.1. Benzo[a]pyrene 3.2. N`-Nitrosonornicotine 3.3. 4-Nitroquinoline-N-Oxide 3.4. 7,12-Dimethybenz[a]anthracene 3.5. Dibenzo[a,l]pyrene 4. Repair of DB[a,l]P-DNA adducts 5. Mutagenesis in the Oral Cavity in Rodents: Effects of DB[a,l]P and DB[a,l]PDE 6. The Effect of DB[a,l]P on p53 Mutations and Gene Expression 7. The Effects of Black Raspberry on Oral Cancer 8. Summary and Future Direction 9. Funding Sources 10. Abbreviation Section 11. Authors Biographies 12. References

1. Introduction Worldwide, head and neck cancer is the 6th most common human cancer, and oral cancer is the most common type of this disease.1-2 For statistical purposes, oral cancer is often 5



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grouped together with cancers of the pharynx (the back of the throat). In Asian populations, 40-50% of oral cancers are located in the buccal mucosa. However, in the US and European populations tongue is the most common site for intraoral carcinoma, and it accounts for 4050% of all cases. The posterior lateral border and ventral surfaces are the most frequently affected sites of the tongue. The next most common site is the floor of the mouth. Surgical removal of oral tumors frequently leads to serious facial disfiguration.3 Cancer of the oral cavity is a deadly disease and can strip away the patient’s voice and certain basic needs in life such as eating and drinking; there are currently over 300,000 cases, and more than 145,500 deaths occurred in 2012 worldwide; and in the USA over 30,000 cases and over 6,000 deaths from the disease occur annually2, 4-5. In 2016, the number of new cases of oropharyngeal carcinomas in the USA is estimated to be 48,330 and the estimated deaths from the disease 9,570.5 Incidence and mortality rates are higher in men than women; higher in blacks than in whites.6 The incidence varies among geographical regions. It is less common in developed than developing countries. Oral cancer ranks among the three most common types of cancer in south-central Asia. Sharp increases in incidence rates have been reported for regions such as Denmark, France, Germany, central and Eastern Europe, which largely reflects the ongoing tobacco epidemic and the heavy consumption of alcohol and tobacco in these countries.2, 7 The most common histological type of oral cancers is oral squamous cell carcinoma (OSCC); it accounts for more than 90% of oral cancers.1-2 Early diagnosis for oral cancer has not improved over time; up to 77% of oral cancer cases were diagnosed at advanced stages.8 The conventional treatments include surgery, radiotherapy, and chemotherapy.9 However, approximately one-third of treated patients will experience local or regional recurrence and/or

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distant metastasis.10 The survival rate is stagnant at about 50%; it varies greatly, with the stage of disease detection.8 The survival rate of oral cancer patients who have metastatic disease at diagnosis is only 28%, because of their high risk of developing a second primary cancer. Collectively, late presentation, lack of biomarkers for early diagnosis and frequent lack of post therapeutic monitoring all contribute to the poor prognosis for oral cancer patients.11 The main risk factors for oral cancer are exposure to exogenous carcinogens, such as tobacco smoke, smokeless tobacco, excess alcohol and human papilloma virus (HPV). These factors are estimated to account for 90% of oral cancers.1-2 Cases of HPV-associated oropharyngeal cancer (cancer on the back and sides of the throat, tonsils, and the base of the tongue) have been rising in young men at an alarming rate.12 Worldwide, 30% of oropharygeal cancers are now thought to be related to HPV-infection, which is linked to sexual practices, such as oral sex. A recent study in the US show that over the past 20 years, the rate of HPV detection in oropharyngeal tumor specimens increased from 16% to 70%.13 HPV-negative HNSCC is generally diagnosed in older patients and has significantly worse outcomes compared with HPV-positive cancer.12, 14 HPV-16 is the type most often linked to throat cancer.15 The two HPV vaccines available (Gardasil, Cervarix) prevent infections with both HPV-16 and HPV-18 and other preliminary, but promising results16 suggesting that HPV vaccines might prevent most oral HPV infections and may also have a significant impact on the prevalence of throat cancer. While HPV infection is increasingly common as an etiologic factor in a subset of HNSCC17, cancers of the oral cavity are infrequently associated with the expression of viral agents.18 In general, avoidance of risk factors has only been partially successful – largely because of the addictive power of tobacco smoking and alcohol consumption. The combination of heavy smoking and heavy drinking results in a 35-fold 7



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increase in risk of developing oral cancer.19 Smokeless tobacco has been shown to cause oral cancer and esophageal cancer.20 More than 60 carcinogens (animal or human) have been identified in tobacco smoke, and 28 chemicals in smokeless tobacco have been found to cause cancer.20 Smokeless tobacco use, such as tobacco chewing and snuff dipping, is quite common in India and other Asian countries, as well as parts of Africa and northern Europe.21 Marijuana smoking and betel quid chewing have also been shown to increase the risk of developing oral cancer.21 The risk to develop oral cancer increases with age and most often occurs in people over the age of 40. Cancer of the lip can be caused by sun exposure.22 It has been shown that diets lacking in fruit and fresh vegetables are associated with increased risk of head and neck squamous cell carcinoma (HNSCC), including oral cancer. A diet high in animal fat may increase the risk of oral cancer.23

2. Animal Models for Oral Cancer The lack of appropriate animal models that reflect human OSCC hampers progress in the prevention and control of oral cancer. Biologically and clinically relevant laboratory animal models that mimic human exposure to environmental carcinogens can provide important information at the cellular and molecular levels on the role of environmental agents in disease progression, and assist in the design of effective preventive and treatment strategies. Several animal models of oral cancer have been reported, including xenograft, transgenic and chemically-induced animal models; the advantages and disadvantages have been reported24 and will be briefly discussed in this perspective.

2.1. Xenograft and Transgenic Animal Models

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Orthotopic xenograft models of OSCC are superior to ectopic subcutaneous xenograft models for their ability to restore the specific pattern of interactions between the tumor and host organ that are absent when the tumors are established in ectopic sites.25 Orthotopic xenograft models of OSCC involve injecting OSCC cells into the tongues of nude mice subcutaneously or into the floor of mouth transcutaneously. Therefore, orthotopic models can be more technically challenging to establish and may cause animal death.26 Orthotopic implantation of tumor cells can result in high rates of spontaneous tumor metastasis whereas ectopic subcutaneous implantation of tumor cells rarely results in metastasis.26 The disadvantage of xenograft models is that the use of immunodeficient mice precludes the study of tumor-host immune interactions. Evading immune destruction is considered as a hallmark of cancer.27 A limitation of xenograft models is that the initiation and early-tumor formation stages are not represented.28 Cell transformation from premalignant to malignant stage will also not be observed during the progressive growth of the xenografts. Recently, patientderived xenografts (PDX) are emerging as a model platform that may better reflect human cancer compared with xenografts derived from immortalized cells that have been propagated indefinitely in vivo.29-31 Transgenic animals can be used to study the premaligant process.28 Several transgenic mouse models of oral cancer have been reported which utilized the keratin 5 (K5) or keratin 14 (k14) promoter to overexpress the oncogene K-rasG12D or Akt in oral epithelium of mice.28 K5 or K14 promoter were selected for targeting the expression of transgenes to the oral cavity because K5 is expressed within the basal epithelium of the tongue and the forestomach and K14 is mainly expressed in the basal layer of the oral mucosa and tongue.28 In these models, the expression of K-rasG12D or Akt driven by K5 or K14 promoter was placed under the control of tet-responsive elements24 or Cre recombinase.32-33 There are several drawbacks in OSCC 9



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transgenic models. For example, the transgenic mice usually have a heterologous promoter driving transgenic expression, leading to non-physiological levels of transgene product. Also the intended tissue specificity is not absolute despite the use of oral-mucosa specific promoters such as K5 or K14.

Lastly, no single gene predominates in the process of oral carcinogenesis.

Therefore, the use of one or two specific genes such as K-ras or Akt, to derive the tumor formation in these transgenic mice may not necessarily reflect the carcinogenic process in human.18

2.2. Chemically-Induced Oral Cancer in Animal Models Several agents including coal tar, cigarette smoke, benzo[a]pyrene (B[a]P), 7, 12dimethylbenz(a)anthracene (DMBA) and 3-methylcholanthrene have been used to induce oral cancer in immunocompetent animals.24 The animal models, which used tobacco carcinogens, can mimic human exposure to environmental carcinogens and also can recapitulate the tumor heterogeneity.34 The DMBA-hamster cheek pouch model was introduced in 1954 by Salley35 and further optimized by Morris in 1961.36 Although DMBA (Figure 1) is a commonly used chemical carcinogen, when applied locally in the hamster cheek pouch, it induces an inflammatory response, necrosis and the appearance of granulation tissue. This makes it difficult to study early lesions. In the early 1970s, the synthetic water-soluble 4-nitroquinoline1-oxide (4-NQO) was used to induce oral carcinogenesis in rats and later in mice. It doesn’t cause an extensive inflammatory response.24, 34, 37-38 As in the hamster model, cancer initiation and progression were correlated with abnormal expression of H-ras, Bcl-2, Bax, p53, E- and Pcadherin.17, 27 Both 4-NQO and DMBA have been commonly used as model carcinogens in the study of factors involved in the development of OSCC; however, neither DMBA nor 4-NQO 10


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are found in the human environment. In contrast to the DMBA hamster model, the human oral cavity doesn’t have a cheek pouch.17, 27, 39 Furthermore, DMBA induces H-ras mutations40, which are found in fewer than 5% of oral cancers in the western world.41 Although it is not clear which compounds in tobacco smoke contribute in the development of human oral cancer, certain classes of chemical carcinogens such as tobaccospecific nitrosamines (TSNA) and polycyclic aromatic hydrocarbons (PAH) are recognized as potential etiological agents for oral cancer.42-44 Table 1 presents the levels and sources of human exposure to environmental oral carcinogens. The prototypical PAH, B[a]P (Figure 2), when fed in the diet by Beland and his team, utilized a 2 year bioassay in order to induce tongue papillomas and carcinomas, but it also induces tumors in distal sites (forestomach is the main target) in mice and B[a]P is toxic at the high doses necessary to induce tongue tumors.45 In an earlier study B[a]P was tested in the hamster cheek pouch model; it induced both papilloma and carcinoma.46 Studies using snuff and certain TSNA, such as 4(methylnitrosamino)-1-(-3-pyridyl)-1-butanone (NNK) and N’-nitrosonornicotine (NNN) to induce oral cancer in rats were pioneered by a team of investigators, including Hecht and the late Hoffmann at the American Health Foundation in Valhalla, New York.47-48 Further studies by Hecht’s group have demonstrated that (S)-NNN and racemic NNN, present in smokeless tobacco products, induce oral cancer in rats.49 Oral cancer models using both classes of tobacco carcinogens (TSNA and PAH) require long duration protocols (about 2 years), certain protocols require surgical procedure and more importantly detailed molecular characterizations of tumors have not been fully defined. Therefore, our efforts were focused on developing an animal model that mimics human exposure to environmental carcinogens and reflects tumor heterogeneity; and in 2012 we introduced a new mouse model to study oral carcinogenesis.50 We chose to focus on the 11



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tobacco smoke constituent and the environmental pollutant, the fjord-region PAH, dibenzo[a,l]pyrene (DB[a,l]P).50 This PAH has been identified in mainstream cigarette smoke, diesel exhaust particulate matter and soil.51-53 Briefly, DB[a,l]P (3, 6, 12 and 24 nmol) and its ultimate diol epoxide metabolites (±)-anti- DB[a,l]PDE (3, 6 nmol) were topically administered (3 times per week, for 38 weeks) into the oral cavity of B6C3F1 mice.50, 54 At 42 weeks after the first dose, mice were sacrificed and at the highest dose, DB[a,l]P-induced hyperplasia (94%), dysplasia (69%) and OSCC (31%) in the mouse oral cavity. The high dose of (±)-anti- DB[a,l]PDE induced 74% and 100% OSCC in tongue and oral tissues, respectively. In tumor and dysplastic tissues induced by both DB[a,l]P and (±)-antiDB[a,l]PDE, we observed similar elevations of p53 and COX-2 protein expression.50, 54 For comparison, studies of oral carcinogenesis induced by synthetic (4-NQO, DMBA) and environmental (B[a]P, DB[a,l]P and NNN) agents in rodents are summarized in Table 2. Assuming that DB[a,l]P, B[a]P , [S]-NNN, 4-NQO and DMBA induce oral tumors in a linear dose-response manner, the relative carcinogenic potency was calculated as the percent incidence per µmol of the compound per gram body weight of the animals used in these studies. The results (Figure 3) appear to suggest that other carcinogens tested are less potent than DB[a,l]P in the rodent oral cavity; this is consistent with the higher potency of DB[a,l]P than B[a]P in the induction of tumors in other organs.55 However, the routes of administration were not identical, so comparisons are approximate. Balbo et al 49 estimated that human exposure to NNN would be about 31 µg/day based on the consumption of a half-tin per day (17g) of a popular smokeless tobacco product which contains about 3 µg NNN per gram tobacco and on extraction efficiency of 60%. Therefore, after 30 years of using smokeless tobacco, the total dose of NNN was estimated to be 340 mg (5mg/kg body weight); [S]-NNN-induced oral center in F344 rats at a total dose of 123 mg 12


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(336 mg/kg body weight).49. All cigarettes also deliver NNN in their smoke (5-270 ng/cigarette); cigarette smoking is a known risk factor for oral cancer and [S]-NNN is the predominant form in smoke.56 For a smoker of 20 cigarettes per day for 40 years and assuming an average level of NNN as 100 ng/cigarette, we estimate a total dose of 28.8 mg (~ 0.5 mg/kg). DB[a,l]P and B[a]P have been identified in numerous environmental sources including combustion systems, urban air pollution, soil sediments, cooked foods, and cigarette smoke. In smokeless tobacco, levels of B[a]P but not DB[a,l]P have been reported57. It was reported that levels of B[a]P (1.0-15.2 ng/cigarette) in cigarette smoke are at least 10-fold higher than DB[a,l]P; however in soil sediments levels of B[a]P are two orders of magnitude higher than those of DB[a,l]P.58 For a smoker of 20 cigarettes per day for 40 years the total inhaled B[a]P would be about 28.8 mg (0.48 mg/kg body weight). Using the approach described above by Balbo et al49 and based on the levels of B[a]P in smokeless tobacco (24-56 ng/g) a consumer of smokeless tobacco for 30 years would be exposed to B[a]P at levels of 0.25 mg/kg body weight. In non-smokers, food is the major source of B[a]P and DB[a,l]P. If a person consumes once per week broiled steak for about 60 years (B[a]P = 50 mg/g broiled steak), the total amount of B[a]P is estimated to be 28.8 mg (0.48 mg/kg). Table 3 summarizes these results; this table also includes the total dose (mg/kg body weight) of these carcinogens that induced oral cancer in rodents. The rodent studies suggest that DB[a,l]P is a more potent oral carcinogen than B[a]P, and NNN. However, future studies should compare these carcinogens under identical conditions to further support this suggestion.

3. Metabolic Activation of Oral Carcinogens 3.1. Benzo[a]pyrene 13



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B[a]P, the most widely studied PAH, is now classified as a human carcinogen (Group 1) by the IARC.59-60 As described above, B[a]P induces tumors in the mouse tongue and in hamster cheek pouch.45-46 To our knowledge, detailed metabolic activation of B[a]P in the oral cavity in rodents has not been explored. Nevertheless, based on literature data, we envision three routes of metabolic activation of B[a]P that may account for its tumorigenic effects in the oral cavity. In the 1st pathway, B[a]P is sequentially monooxygenated by cytochrome P4501A1/1B1 (CYP1A1/1B1 - both proteins are expressed in the oral cavity) via the (-)7R,8RB[a]P-dihydrodiol (B[a]P-7,8-DHD) intermediate to yield (+)-anti-B[a]P-diol epoxide (antiB[a]PDE) , which reacts with dG to form (+)-anti-B[a]PDE-N2-dG adducts (Figure 4).61-68 In the 2nd pathway, B[a]P-7,8-DHD undergoes an NAD(P)+ dependent oxidation catalyzed by human AKR1A1, AKR1C1-AKR1C3 to yield a ketol which spontaneously rearranges to B[a]P-7,8-catechol.69-71 Because the catechol is unstable, it undergoes a 1 electron-oxidation to form a ortho-semiquinone anion radical and a 1electron-oxidation to form B[a]P-7,8-dione with the concomitant production of ROS72; the dione can be enzymatically reduced to its catechol or form covalent adducts with DNA in in vitro systems.73-76 ROS may be involved in the initiation and more so in the post-initiation phase of carcinogenesis.77 In the 3rd pathway, cytochrome P-450 peroxidases catalyze the formation of B[a]P radical cations78 which can react with guanine bases leading to the formation of depurinating adducts; all of these adducts derived from B[a]P and ROS can lead to G to T transversions observed in p53 in HNSCC.79

3.2. N`-Nitrosonornicotine Because of the chiral center at the 2`-position, N`-Nitrosonornicotine (NNN) exists as two enantiomers, namely [R]-NNN and [S]-NNN; the latter is the major form in currently marketed smokeless tobacco products.80-81 Metabolism and DNA binding studies of [R]-NNN 14


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and [S]-NNN were compared in the rat.82-84 These previous studies in the rat demonstrate that [S]-NNN is metabolized by 2`-hydroxylation to a significantly greater extent than [R]-NNN in the esophagus, oral cavity and liver; this pathway is known to lead to pyridyloxobutyl-DNA adduct formation82-84 (Figure 4). Building on these studies Balbo et al compared [S]-NNN and racemic-NNN and demonstrated they are powerful oral cavity carcinogens in the male F344 rats.49 Characterization of the cytochrome P-450(s) responsible for the metabolic activation of NNN and assessment of DNA adduct distribution in the oral cavity of rats administered NNN remains to be examined.

3.3. 4-Nitroquinoline-N-Oxide Nitroreduction of 4-nitroquinoline-N-oxide (4-NQO) by NADH:4-NQO nitroreductase and NAD(P)H:quinone reductase leads to the formation of 4-hydroxyaminoquinoline-1-oxide (4-HAQO).85 4-HAQO can be further metabolized and acetylated by seryl-tRNA-synthetase to form seryl-AMP enzyme complex. This complex and 4-HAQO can form DNA adducts at various positions. In vitro two guanine and one adenine adducts have been identified. However, in vivo, 4-NQO preferentially reacts with guanine residues in DNA to form C8- and N2-dG adducts (Figure 4). These covalent adducts, as well as those derived from oxidative stress induced by 4-NQO, have not been examined in the rat and in the mouse oral cavity.

3.4. 7,12-Dimethybenz[a]anthracene Extensive previous studies demonstrate that the liver and mammary glands are capable of metabolizing DMBA to reactive diol epoxides which are prerequisite for the formation of DNA adducts and initiation of carcinogenesis86-87 (Figure 4). Using the 32P-postlabelling

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technique, DMBA-DNA adducts were found in the hamster cheek pouch. However, structural confirmation and accurate quantification of individual adducts has not been reported.88

3.5. Dibenzo[a,l]pyrene Similar to B[a]P, there are three different pathways proposed to be involved in the activation of DB[a,l]P: (1) the diol epoxides pathway to form stable bulky diol-epoxide adducts, which is the most widely accepted pathway of PAH activation leading to DNA damage (Figure 5); (2) the PAH ortho-quinone pathway (mediated by aldo-keto reductases) which can give rise to reactive oxygen species leading to the formation of 8-hydroxy- 2’deoxyguanosine (8-oxo-dG); and (3) the radical cation pathway to yield depurinating adducts. The carcinogenic metabolites of different PAHs are formed through cytochrome P-450mediated initial monooxygenation, hydrolysis to trans-diols by epoxide hydrolase and subsequent epoxidation of trans-diols to vicinal bay or fjord diol-epoxides.89 To study the metabolism of DB[a,l]P and its DNA binding activities, different cell lines have been used, including MCF-7 cancer cells or liver preparations from rats pretreated with inducers of CYP1A1 and 1B1.90-92 It has been shown that DB[a,l]P is predominantly metabolized to antiand syn- 11,12-diol -13, 14-epoxides. Both CYP and EPHX1 proteins operate with high enantio- and diastereoselectivity. Thus in mammalian systems, only (+)-syn and (-)-antiDB[a,l]PDE enantiomers with (S, R, S, R) and (R, S, S,R) configuration are detected 55, 91-94 (Figure 6). It is known that in mouse skin, CYP1B1 plays a major role in the activation of DB[a,l]P, inducing DNA damage and carcinogenesis, while murine CYP1A1 is critical to the activation of other planar carcinogens such as B[a]P.94-95 Metabolic activation of DB[a,l]P in human cells, mouse embryo cells, and recombinant cell lines expressing human CYP1A1 or 16


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CYP1B1, predominantly leads to the formation of (-)-anti-DB[a,l]PDE-DNA adducts, which is in line with the results obtained in mouse skin treated with DB[a,l]P.55, 92-95 Lesser amounts of (+)-syn-DB[a,l]PDE-DNA adducts were detected upon treatment with high doses, but not at low doses of DB[a,l]P.96-97 In mice, DB[a,l]P has been shown to induce CYP1A1 and CYP1B1 in several tissues.98-99 Previous studies have demonstrated that CYP1A1 and CYP1B1 differ in their regio- and stereochemical selectivity of activation of DB[a,l]P, with higher levels of (-)-antiDB[a,l]PDE formed by CYP1B1.96 Furthermore, Buters et al have shown that CYP1B1dependent formation of the DNA-reactive (-)-anti-DB[a,l]PDE represents the critical step in DB[a,l]P-induced tumor formation.94 Whether CYP1B1 is much more prevalent than CYP1A1 in mouse oral tissues remains to be explored. The balance between the metabolic activation and detoxification processes influences the levels of the intracellular DNA adducts (Figure 6). Among the important cellular defenses against PAH diol epoxides are glutathione S-transferase (GST)-catalyzed conjugation and uridine-5’-diphosphate glucuronosyltransferase (UGT)-catalyzed glucuronidation. The latter process transfers a glucuronic acid group from uridine-5’-diphosphate glucuronic acid to a target PAH-derived substrate. These detoxification processes increase the water solubility of the PAH metabolites and allow the elimination of the compounds from the body through the bile and/or urine.100-101 The higher carcinogenicity of DB[a,l]P than B[a]P is a result of its fjord region chemical structure (composed of five carbon atoms), which allows its diol epoxide to bind to purine nucleotides without distorting the DNA structure. This leads to the formation of DNA adducts which are not easily recognized by DNA repair enzymes and are more persistent than the adducts derived from bay-region PAHs like B[a]P (see below, Section 4). 101-102 DB[a,l]P 17



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is a potent mutagen in bacteria, mammalian cells and in rodent models, and has been shown to initiate tumors to a greater degree than other PAHs tested.101 In addition, because of its intrinsic chemical structure and metabolism, DB[a,l]P induces both dG and dA adducts, which is different from B[a]P, which predominantly form dG adducts. For instance in the human lung epithelial carcinoma A549 cells, DB[a,l]P induced dA adducts over dG adducts in a 4:1 ratio.103 To further characterize our animal model and evaluate the potential contribution of DB[a,l]P in human oral carcinogenesis, we focused on studying the DNA damage induced by DB[a,l]P in the oral cavity of mice. Prior to our study, nothing was known about the metabolism of DB[a,l]P and its ability to cause DNA damage in the oral cavity of mice. We hypothesize that DNA adducts formed by DB[a,l]P may contribute to the mutagenic and carcinogenic properties of DB[a,l]P. In fact, as will be discussed later (Section 5), studies in lacI mice have shown that DB[a,l]P is mutagenic in the oral cavity of mice.50 The types of mutations induced by different DNA adducts (mutational spectra) reflect a number of factors such as mispairing by adducts, error-prone DNA repair, and depurination resulting from unstable adducts. The ability of a mutagen to lead to premutagenic adducts in addition to some of the above factors contributes to the potency of the mutagen. It is possible that DB[a,l]PDNA adducts induce mutations in critical cancer genes, which are involved in the development of oral cancer. To detect and quantify DNA adducts in the oral cavity of mice treated with DB[a,l]P, we chose to use the HPLC-MS/MS method because it can offer structural and stereochemical information about the DNA adducts induced by this carcinogen. We initially focused on developing an LC-MS/MS method to detect and quantify antiDB[a,l]PDE-N6-dA in vivo (Figure 4).104 The rationale was based on the intrinsic structural properties of sterically hindered fjord region dihydrodiol epoxides, which primarily bind to dA 18


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residues in DNA.105 Animal studies have generally shown that fjord region PAHs are more tumorigenic than bay region PAHs, but there are exceptions, such as benzo[c]phenanthrene. Fjord region PAHs, such as DB[a,l]P, predominantly bind covalently to dA residues in DNA, while binding to dG residues occurs at a lower levels.55, 106 In contrast, bay region PAHs, such as B[a]P, are known to form N2-dG adducts at a much higher frequency than N6- dA adducts (Figure 4).55 Two adducts derived from (-)-anti- DB[a,l]PDE in oral tissues of mice treated with DB[a,l]P were detected (Figure 4). This result appears to support a mechanism by which DB[a,l]P can be metabolized to (-)-anti-DB[a,l]PDE, under our experimental conditions. Our finding is consistent with others reported in the literature; it has been shown that in human cells, mouse skin, mouse embryo cells, and cells expressing recombinant CYP enzymes, metabolic activation of DB[a,l]P leads predominantly to the formation of (-)-anti-DB[a,l]PDEDNA adducts.55,91, 107-109 Similarly, using 32P-postlabeling method, Mahadevan et al showed that the majority of DNA adducts formed in the lungs of mice exposed to DB[a,l]P were derived from (-)- anti-DB[a,l]PDE.110 4. Repair of DB[a,l]P-DNA adducts In general, nucleotide excision repair (NER) is known to remove bulky PAH-DNA adducts.111-112 It has been reported that dA adducts derived from several fjord PAH diol epoxides, including DB[a,l]PDE-N6-dA adducts are resistant to NER.113-114 Studies using human cell extracts also showed that DB[a,l]PDE-N6-dA adducts are more resistant to NER than DB[a,l]PDE-N2-dG adducts, which suggest that DB[a,l]PDE-N6-dA adducts may persist longer and have higher mutagenic potential than DB[a,l]PDE-N2-dG adducts.115-117 The predominant formation of dA adducts, combined with their higher resistance to DNA repair suggests the importance of these adducts in mutagenesis and carcinogenesis

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induced by DB[a,l]P. These observations indicate that certain DB[a,l]PDE-N6-dA adducts may be critical during the initiation of DB[a,l]P-induced oral carcinogenesis. Regarding removal efficiency, we reported that after treatment of mice with DB[a,l]P (3x a week for 5 weeks) the levels of (-)-anti-trans-DB[a,l]PDE-dA adduct decreased significantly throughout the time course examined, while the levels of the (-)-anti-cisDB[a,l]PDE-dA adduct in oral tissue were not altered significantly until 4 weeks after the last carcinogen treatment.106 Our results is consistent with a report by Kropacheb et al that high genotoxic activity of DB[a,l]P is related to the formation of NER-resistant and persistent DB[a,l]P-derived adenine adducts.115 In our previous study, we found a faster rate of removal of (-)-anti-trans-DB[a,l]PDE-dA adduct within the first 2 weeks after treatment, followed by a slower rate of removal from 2 to 4 weeks after administration. The efficiencies of DNA repair depend on the stereochemical properties of adducts. Dreij et al reported that when A549 human epithelial lung carcinoma cells were incubated with 0.1 µM of (-)-anti-DB[a,l]PDE for up to 6 h, the levels of trans- adducts do not change, while there are significant increases for ()-anti-cis-DB[a,l]PDE-dA.103 Furthermore, levels of (-)-anti-cis-DB[a,l]PDE-N2- dG also remained constant for 6h. Apparently the structural features of the (-)-anti-cis-DB[a,l]PDE-dA adduct render it relatively resistant to repair, but these structural features have not yet been defined. It seems reasonable that the adduct can adopt a stable structure in the DNA duplex which is less recognizable by cellular sensors and DNA repair systems than other DB[a,l]PDE adducts. Our results also demonstrated that following the administration of DB[a,l]P, we were able to detect and quantify (-)-anti-cis-DB[a,l]PDE-N2-dG and (-)-anti-trans-DB[a,l]PDE-N2dG in DNA from the oral tissue of mice.106 Spencer et al118 detected two major guanine adducts with unknown stereochemistry which were resistant to removal by NER system for up 20


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to 34 h, following the incubation of racemic (±)-anti-DB[a,l]PDE with several human NER proficient (NER+) cell lines. Prahalad et al have shown that the levels of anti-DB[a,l]PDE-dG adducts have also been correlated with the levels of mutations and tumor initiation in the A/J mouse lung119; they have shown that at the time point of maximum binding, in A/J mice treated with DB[a,l]P, the ratio of dA and dG adducts in the lung was close to 1:1. Spencer et al118 have shown in human cell lines, the majority of DNA adducts following treatment of (±)-antiDB[a,l]PDE, were formed with guanine. These results are consistent with the prevalence of guanine adducts in calf thymus DNA and mouse skin when treated with (±)-antiDB[a,l]PDE.120 However, other reports have shown that (-)-anti-DB[a,l]PDE reacts predominantly with adenine in human epithelial cells, resulting primarily in mutations at A:T base pairs.103,121-122 Based on these studies, it is likely that (+)-anti-DB[a,l]PDE is responsible for the large numbers of dG adducts formed in studies reported by Spencer et al when those human cell lines were treated with 1nM (±)- anti-DB[a,l]PDE.118 Collectively it is apparent that the variations in adduct distributions among different studies, may be due to different doses, differences in metabolic capacities of different cell types and treatment duration, time points at which these adducts were measured, as well as the capacity of DNA repair machinery in various organs.

5. Mutagenesis in the Oral Cavity in Rodents: Effects of DB[a,l]P and DB[a,l]PDE Most of the agents that induce oral cancer in rodents do not appear to have an unusual specificity for the oral cavity. Rather, it appears that deposition is a major factor. In studies where cancer of the oral cavity was induced, the carcinogen was applied directly by topical application in the oral cavity or administered in the drinking water or in the diet, or by gavage.24, 28, 35-38, 45, 49-50 When administered in diet, or in the drinking water, other organs such 21



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as esophagus or forestomach are usually also targeted.45, 49-50 4-NQO administered in drinking water is unusual in that it appears to target the oral cavity in rodents, likely because it is very efficiently metabolized to its ultimate carcinogen in the oral cavity.123 Similar target specificities between mutagenesis and carcinogenesis have been observed when mutagenesis in the oral cavity of lac rodents is monitored.124-126 In general, it is not possible to measure mutagenesis in non-cancer tissue because mutational frequencies are so low (10-4 – 10-5) that current sequencing techniques do not have the extremely high fidelity necessary to distinguish mutations from noise. However several transgenic animal models have been developed with mutagenesis reporter genes that allow the detection of mutations in normal or precancerous tissue127 and the most frequently employed are the lacI and lacZ rodents.127 Using two of these models we have reported that all of the known oral carcinogens are also mutagens in the mouse and/or rat oral cavity.124 With the exception of 4-NQO the specificity of the agents for the oral cavity is not absolute. For instance NNN and NNK when given in drinking water are similarly mutagenic in tongue, other oral tissue and esophagus (likely reflecting deposition), but mutagenesis in liver and lung is even greater.125 When B[a]P is given by gavage there is a good, but not unequivocal correlation between targets of mutagenesis and carcinogenesis.128 4-NQO exhibits a very strong mutational preference for the oral cavity in lac rodents, similar to that observed in its carcinogenic effects.124 Sites of increased mutagenesis presumably represent initiated sites; the likelihood of tumors developing at a particular site will depend on additional factors, including promotion, progression, immunological response, apoptosis and the ability of the animal to survive long enough for tumors to develop.

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As mutagenesis is considered an important step in carcinogenesis, it can represent an early biomarker for cancer and precancer. We assayed mutagenesis induced by DB[a,l]P in the oral cavity of the B6C3F1 lacI mice (0, 3, 6, 12 nmol/treatment).50 There was a dosedependent increase in mutagenesis in tongue and upper oral mucosa. We have previously shown that B[a]P is mutagenic in the mouse oral cavity.129 Our data have shown that compared to B[a]P, DB[a,l]P is a more powerful mutagen and carcinogen in the oral cavity of mice although the routes of administration were different.45, 50 B[a]P has also been tested for carcinogenesis in lacZ mice, and did not induce tumors of the oral cavity, but was carcinogenic in other organs.128 However, the oral cavity appears not to have been examined, and the mice may have been euthanized before tumors in the oral cavity were apparent. In Table 4 we have compiled a summary of mutational spectra of the oral carcinogens in Table 3 in lac rodents and in one case from a lacI cell line. Where possible the mutational spectra were taken from the oral cavity. However, mutational spectra in non-tumor tissues tend to be similar in different organs, as they are largely dependent on the chemical reaction of the ultimate carcinogen with DNA, and not with any specific organ. For comparison we have also included a column listing the mutational spectra in the p53 gene of human HNSCC and control oral tissue from lacI mice. There appear to be no common patterns in the spectra of mutations induced by oral carcinogens. This is not surprising given the different chemistry of the carcinogens and in some cases, different animals and target organs. However, all of the mutagens induce much lower percentages of GC to AT transitions than are found in control oral tissue, indicating they lead to mutagenesis via DNA damage, rather than simply enhancing clonal expansion of preexisting mutations. It may be significant that of all the agents in Table 4, the ones whose ratio of point mutations at GC base pairs to those at AT base pairs is most similar to that in p53, are DB[a,l]P and DB[a,l]PDE. More specifically about 60% of the 23



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mutations DB[a,l]P or DB[a,l]PDE-induced mutations are G:C→T:A and G:C→A:T substitutions and around 30% of the mutations at AT base pairs and these percentages are similar to those found in the p53 gene in HNSCC in humans.130-131 When compared with the other environmental carcinogen in Table 4, B[a]P the major difference was that a significantly higher fraction of mutations at AT base pairs were induced by DB[a,l]P.50,132 Mutations in tumors are filtered by selection, as are those in the cII gene, so comparisons of mutational spectra are only approximate representations of the originally induced mutational spectra. However, thus far these results appear to support a contribution of the environmental pollutant and tobacco smoke constituent, DB[a,l]P to the development of oral cancer in humans, and that our model is appropriate for tobacco smoke-induced cancer of the oral cavity.

6. Effect of DB[a,l]P on p53 Mutations and Gene Expression We have shown overexpression of p53 by immunohistochemistry (IHC) in oral tissues of mice treated with DB[a,l]P50 and its diol epoxide.54 Over-expression of p53 protein assessed by IHC may result from p53 gene mutations or exposure to genotoxic stress. To determine whether overexpression of p53 protein is associated with mutations, we isolated DNA from frozen tumor tissues induced by DB[a,l]PDE, and exons 5-8 of the p53 gene were purified by PCR and sequenced to screen for mutations.54 In mice treated with (±)-anti-DB[a,l]PDE , we identified two p53 mutations in DNA samples isolated from five OSCC tissues, using direct sequencing of exons 5-8. This represents the sequence-specific DNA binding domain, where approximately 90% of the missense mutations occur in p53. This result was confirmed by three independent sequencings of the forward and reverse strand.54 The p53 mutations identified include a G→T transversion at codon 155 from exon5, leading to an arginine to leucine change and an A→T transversion at 24


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codon 232 from exon 7, resulting in change from lysine to stop codon. The mutations identified by direct sequencing were further confirmed by SSCP.54 To further elucidate the mechanisms by which DB[a,l]P and presumably its diol epoxides may be involved in oral carcinogenesis, the effects of DB[a,l]P on gene expression in mouse oral cavity was explored using a focused real-time cancer pathway PCR array.54 The duration and dose of treatment with DB[a,l]P in this experiment were identical to those previously shown to form maximum DB[a,l]P-DNA adduct formation in the oral tissues of mouse.106 Among the 84 representative genes in the array, 22 of them were up-regulated 2 or more fold and only one gene was downregulated.54 As mutations in p53 were observed, genes affecting or modulated by p53 were of interest. We observed that 12 of the genes modulated by DB[a,l]P have been reported to affect expression of, or be modulated by p53 (S100A4, CHEK2, CDKN2A, E2F1, CDK4, TNFRSF10B, BAX, SERPINE1, TERT, MTA2, MUC1 and CCNE1), and all of them were upregulated. Although future studies are required to explore in detail, the relevance of selected gene expression to oral carcinogenesis, we have demonstrated that DB[a,l]P influences the expression of several genes related to cell cycle, DNA damage and repair and apoptosis among other cellular events that are critical in the carcinogenic process. It has been reported that the expression of S100A4, CHEK2, CDKN2A, and E2F1 result in the stabilization, activation or overexpression of p53 protein.133-136 Overexpression of CDKN2A (p21) is a p53- mediated response to DNA damage which slows progression through the cell cycle and allows more time for DNA repair.137 Tnfrsf10b, Bax and SERPINE1 were found to be activated or up-regulated by p53.138140

It has been reported that high TERT immunostaining is associated with p53 mutations in

human breast tumors.141 Furthermore, it has been shown that the amplification of CCNE1 is 25



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associated with p53 mutations and smoking in a series of non-small-cell lung carcinomas.142 Therefore, the overexpression of TERT and CCNE1 could result from mutated p53. The MUC1 oncogene is overexpressed in human carcinomas and suppresses p53-dependent apoptotic response to DNA damage.143 The expression of MTA2 strongly represses p53dependent transcriptional activation, and modulates p53- mediated cell growth arrest and apoptosis.144 We propose that the induction of MUC1 and MTA2 might inhibit p53-dependent processes. This model will be especially valuable to test the efficacy of chemopreventive and therapeutic agents which can modulate p53, COX-2 and its derived PGE2 and PGE-M or other molecular targets that are critical in oral carcinogenesis.145

7. Effects of Black Raspberry on Oral Cancer It remains challenging to treat cancer including OSCC at late stages, even with recent advances in targeted therapies. The ultimate goal of cancer prevention is to reverse or prevent the development and progression of early stage disease before it becomes aggressive. Consumption of diets rich in fruits and vegetables may contribute to a lower risk of developing oral cancer. It has been demonstrated that synthetic or naturally occurring agents that can inhibit the initiation stage of carcinogenesis and/or the progression of premalignant lesions are attractive and plausible approaches for cancer prevention.146-147 To develop novel chemopreventive agents with high anti-carcinogenic properties and low toxicity remains a high priority in chemoprevention research. For oral cancer chemoprevention, initial studies employed retinoids. When 13-cisretinoic acid was administered for 3 months, it resulted in a 67% regression of oral leukoplakia vs. 10% for placebo after 3 months. Unfortunately, the toxicity of this agent is considerable, and there was a very high rate of relapse within 3 months of stopping treatment.1 Other agents, 26


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including vitamin E, Bowman-Birk inhibitor concentrate derived from soybeans, curcumin, and green tea polyphenol epigallocatechin-3-gallate, have been tested in clinical trials to evaluate their chemoprevention activity in oral leukoplakia patients.1 Therefore, the search for effective, safe and easy to administer chemopreventive agents continues to be a high research priority in several laboratories. For example several studies explored the tolerability and feasibility of black raspberry (BRB); in an early phase pharmacokinetic study in healthy subjects who received 45g of BRB powder once daily for 7 days, Stoner et al148 demonstrated the presence, in urine and plasma samples of ellagic acid and several anthocyanin metabolites. A report by Kresty et al showed that administration of BRB powder daily (32-45g) for 26 weeks to 10 Barrett esophagus patients reduced systemic oxidative damage measured in the urine as 8-epi-prostaglandin F2α and 8-oxo-dG.149 Mallery and her team and Ugalde et al demonstrated the feasibility of delivering a “mucoadhesive gel” into the oral cavity of healthy volunteers.150-152 In the human oral cavity, when dysplastic lesions were treated topically with a 10% BRB gel, four times per day for six weeks; there was a histologic regression of about 60% of the lesions.153-154 In a recent clinical trial Knobloch et al demonstrated that BRB phytochemical-rich troche suppressed proinflammatory and prosurvival markers in oral cancer patients.155 Using human SCC-9 cancer cells, Wen et al compared several flavonoids including a flavonoid, 5,7-dimethoxyflavone ; the latter was an unusually effective inhibitor of CYP1B1, and inhibited B[a]P- induced DNA adduct formation, while resveratrol had no effect on CYP1B1 in SCC-9 cells.156 Later, based on their abilities to affect CYP1A1/1A2/1B1 activities, polyphenols, such as quercetin, resveratrol and ellagic acid were tested in a bioengineered human oral-tissue model which was treated with B[a]P; and these compounds resulted in reduced B[a]P-DNA adducts.157 27



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Resveratrol is present in grapes, berries and peanuts. Polyphenols are divided into several groups, one of which is represented by flavonoids.158 Flavonols, such as kaempferol, are present in raspberries and can account, in part, for BRB’s chemopreventive activities.158 Kaempferol has been detected in tea, broccoli, grapefruit, berries and other plant sources. Its anti-carcinogenic potential comes from its anti-oxidant, anti-inflammatory and pro-apoptotic properties.159-160 Both kaempferol and resveratrol are known to inhibit the enzymatic activities of phase I enzymes including CYP1A1, CYP1B1, CYP1A2, CYP3A4. The presence of numerous potential chemopreventive agents in BRB, such as vitamins A, C and E, folic acid, calcium, selenium, β-sitosterol, ellagic and ferulic acids and quercetin, makes BRB an attractive chemopreventive agent.88,146-147 In DMBA-induced OSCC in hamster cheek pouch model, a 5% BRB diet resulted in a 45% reduction in tumor incidence, which was associated with reduced formation of DNA adducts in epithelial cells of the oral mucosa88; however, accurate adducts quantification and adducts distribution remain to be assessed. Our LC- MS/MS method was used to test the hypothesis that naturally-occurring agents that can alter the levels of phase I/ phase II enzymes and/or enhance DNA repair, will reduce the levels of DNA damage caused by DB[a,l]P in the oral cavity. Therefore, in a recent study we examined the effect of black raspberry extract (BRBE) and protocatechuic acid (PCA), a major metabolite of the anthocyanin component of BRBE, on carcinogen-DNA adducts and mutagenesis, and oxidative stress in both rat oral fibroblasts and human oral leukoplakia cells.161 The DB[a,l]P metabolites, (+)-anti-11,12-dihydroxy -11,12,dihydrodibenzo[a,l]pyrene (DBP-diol) and 11,12-dihydroxy-13,14-epoxy-11,12,13,14tetrahydrodibenzo[a,l]pyrene (DBPDE) induced dose-dependent DNA adducts and mutations. DBPDE was considerably more potent than DBP-diol, while the parent compound had no significant effect. Treatment with BRB extract (BRBE) and PCA resulted in reduced DBP28


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derived DNA adduct levels and reduced mutagenesis induced by DBP-diol, but only BRBE was similarly effective against DBPDE. BRBE did not directly inactivate DBPDE, but rather induced a cellular response – enhanced DNA repair. When BRBE was added to cells one day after the DBP-diol treatment, it greatly enhanced removal of DBP-derived DNA adducts. As oxidative stress can contribute to several stages of carcinogenesis, BRBE and PCA were investigated for their abilities to reduce oxidative stress in a human leukoplakia cell line by monitoring the redox indicator, 2',7'- dichlorodihydrofluorescein diacetate (H2DCF) in cellular and acellular systems. BRBE effectively inhibited the oxidation, but PCA was only minimally effective at best against oxidation of H2DCF. Our in vitro results demonstrate that BRBE and PCA can inhibit carcinogen-induced DNA damage; an important step in the initiation of carcinogenesis. BRBE also enhanced DNA repair and reduced oxidative stress. Taken together, we hypothesize that BRBE is a potential chemopreventive agent against the development of oral carcinogenesis, and ongoing studies in our laboratory are testing this hypothesis. Furthermore, the impact of BRBE on DB[a,l]Pinduced genetic and epigenetic alterations in mice is being explored. While the chemopreventive effect of BRB powder and BRB extract are promising, there are some concerns regarding the formulations of whole berry powder for cancer prevention.146-147 To identify the bioactive constituents, bio-fractionation studies of BRB have been carried out and it appears that the chemopreventive activity of BRB is due primarily to anthocyanins (cyanidin 3-glucoside, cyanidin 3-xylosylrutinoside, cyanidin 3-glucoside, and cyanidin 3-sambubioside).147,160 The anthocyanins are the predominant (3%-5% BRB weight) polyphenolic compounds in BRB, which are responsible for the color of the berries as well as for much of their antioxidant activity.146-147,153, 160 It has been shown that anthocyaninenriched fraction is the BRB component responsible for the inhibition of B[a]PDE induced 29



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activation of pleiotropic transcription activating factors NF-κB and AP-1.162 However, the anthocyanins are poorly absorbed in rodents and in humans, and their systemic levels are very low.163 This is likely why they exhibit the strongest chemopreventive effects in organs such as the oral cavity, esophagus and colon, where they can be absorbed locally from the diet. The major concern in using anthocyanins as chemopreventive agents is that they are difficult to synthesize and therefore can be expensive for routine chemoprevention. In addition, they are unstable both at alkaline pH and at high temperatures, so that chemical processing to enhance their biological effects may be somewhat constrained.164

8. Summary and Future Direction Development of OSCC is the consequence of a complex interaction between environmental exposure (tobacco, alcohol), HPV, genetic and epigenetic factors. A recent study by Wu et al reported a substantial contribution of extrinsic risk factors (tobacco, alcohol) to cancer development including head and neck cancer.165 However, Noble et al166 argue that Wu et al overestimated the role of environment in cancers. Clearly, mechanistic studies in preclinical animal models which mimic human exposure to environmental agents and human tumor heterogeneity will continue to be essential to understand the multistep carcinogenesis in the oral cavity, from initiation, promotion, progression and metastasis. Nevertheless, it would not be realistic to believe that a single animal model would provide the ultimate mechanistic understanding of oral cancer. Better understanding of the interaction between the environment and the host at the cellular and molecular levels will assist in the discovery of novel biomarkers for early detection and in the design of effective strategies for cancer prevention and treatment. A recent study further demonstrates the link between environmental exposure to agents such as

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those found in tobacco smoke (e. g. PAH) and aryl hydrocarbon receptor in the development of OSCC167 using a novel orthotopic xenograft model.168 The animal model developed in our laboratory can provide a realistic platform to study the impact of preventive and therapeutic agents on signaling pathways involved in this heterogeneous disease such as EGFR and the hepatocyte growth factor (HGF) receptor, and the mesenchymal epithelial transition (Met) factor receptor.169 Met activation is known to drive proliferation, migration, invasion and angiogenesis in HNSCC170; furthermore, HGF/Met activation is a known mechanism of resistance to anti-EGFR therapy.171 We have shown that oral application of DB[a,l]PDE induces tumors in the oral cavity of mice and was more potent than the parent DB[a,l]P. We suggest that in preclinical animal models, DB[a,l]P appears to be more potent than other oral carcinogens tested so far. However, a comparison of these agents under identical experimental condition is required to support our suggestion. Tumors induced by DB[a,l]PDE closely resemble those induced by DB[a,l]P. Both of them similarly affect protein expression of COX-2 and p53 in the mouse oral cavity, and have mutation profiles in p53 not very different than those found in human OSCC.79, 131 We have also demonstrated that both DB[a,l]PDE and DB[a,l]P can lead to the formation of stable bulky DB[a,l]PDE-dA and -dG adducts in mouse oral tissues. The higher levels of DB[a,l]PDE-dA adducts in oral tissues compared to that of tongue may, in part, contribute to the higher level of tumor incidence in oral tissues compared to that in tongue of mice treated with DB[a,l]P. In terms of the other two possible pathways involved in the activation of DB[a,l]P (Figure 5), the one-electron oxidation pathway can yield radical cations, forming apurinic (AP) sites in DNA via depurinating adducts. Melendez-Colon et al have used an aldehyde reactive probe and a slot blot method to analyze AP sites induced by DB[a,l]P in human cell cultures.107 They found that after exposure to DB[a,l]P for 24h, levels of AP sites 31



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remained low, and the levels of stable adducts derived from the diol epoxide are more than 100-fold higher than levels of AP sites. They conclude that metabolism of DB[a,l]P is catalyzed by P450 enzymes leading to diol epoxides that form predominantly stable DNA adducts but only low levels of AP sites.107 In addition, DeMarini et al have compared the mutation spectra with stable and unstable DNA adducts induced by B[a]P and DB[a,l]P in Salmonella and concluded that B[a]P induced abasic sites while DB[a,l]P did not induce abasic sites, and the proportion of mutations induced by DB[a,l]P at adenine and guanine paralleled the proportion of stable DNA adducts induced by DB[a,l]P at adenine and guanine.172 We did not study the adducts induced through aldo-keto reductase pathway in our mouse model. However, it is possible that this pathway may lead to the generation of ROS-mediated genotoxicity induced by DB[a,l]P. To test this hypothesis, future studies will measure the levels of 8-oxo-dG lesions in the oral tissues of mice treated with DB[a,l]P. It may be optimistic to believe that the induction of OSCC following animal exposure to a single environmental carcinogen is the ideal model to fully understand disease progression. Therefore, future studies need to refine the existing animal models by considering the combined effects of more than one factor in the development of OSCC in rodents. In fact, the results of a previous study indicate that NNN, when combined with subcarcinogenic doses of 4-NQO and/or DMBA, is a promoter in the development of OSCC.173 Studies on the effect of BRB and some of its components on DNA repair are scarce.174175

Ferulic acid (a phenolic acid in berries) dose-dependently decreased DNA strand breaks in

peripheral blood leukocytes and bone marrow cells of mice exposed to whole-body γ-radiation. When administered after irradiation of mice, ferulic acid resulted in disappearance of DNA strand breaks at a faster rate than was observed in irradiated controls, suggesting enhanced DNA repair in ferulic acid-treated animals.156 Further studies demonstrate that ellagic acid 32


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may have weak or no effect on stimulation of the DNA repair protein, O6 methylguanineDNA-methyltransferase in human lymphocytes and in glioblastoma and colon cancer cells.157 However, to our knowledge, detailed studies on the effects of BRB and components on the repair of bulky DNA adducts such as those derived from DB[a,l]P and B[a]P in vitro and in vivo have not been explored. Our recent observation demonstrates for the first time the inhibition of DB[a,l]P-DNA binding by BRB and of particular importance the capacity of the BRB to enhance DNA repair.161 However, our studies were limited to DB[a,l]P and to the whole BRB extract using rat and human oral leukoplakia cells in vitro. Clearly, there is an urgent need to extend these studies beyond those reported in our laboratory.161 Specifically, studies should be extended to examine the effects of BRB and some of its relevant constituents in animal models using carcinogens such as B[a]P and DB[a,l]P. Furthermore, examining the effects of BRB and components on DNA repair at the gene and protein levels would provide important mechanistic insights at the molecular level. The inhibitory effects of BRB on formation and/or accumulation of DB[a,l]P-DNA adducts has been demonstrated in vitro.161 Studies aimed at determining the effects of BRB on DB[a,l]P-induced covalent adducts and oxidative lesions such as 8-OHdG in our animal model in vivo remain to be explored. Furthermore, the effect of BRB on DB[a,l]P-induced epigenetic alterations is unknown. DNA methylation, one of the best known epigenetic modifications, shares critical roles with DNA mutations in carcinogenesis.176 Tobacco smoking induces alterations in DNA methylation across multiple tissues and organ systems.177 Epigenetic changes could predispose cells to further genetic instability and can be targeted early by chemopreventive agents.178-179

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On the basis of our in vitro studies, we showed that BRB can inhibit DNA damage, suggesting its potential effect as a chemopreventive agent in our oral cancer model during the initiation phase of carcinogenesis. Successful achievement of future chemoprevention of BRB against DB[a,l]P-induced OSCC (ongoing study) can provide the basis to examine the effect of BRB on the initiation phase of carcinogenesis – assessed by the extent of DNA damage induced by B[a]P and DB[a,l]P in tobacco smoke – in smokers who are considered at high risk of developing this disease. 9. Funding Sources Studies conducted by the authors and discussed in this perspective were supported by NIH grant # R01-CA173465. 10. Abbreviation Section 7,12-dimethylbenz[a]anthracene (DMBA), 4-nitroquinoline-N-oxide (4-NQO), benzo[a]pyrene (B[a]P), dibenzo[a,l]pyrene (DB[a,l]P), N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone (NNK), polycyclic aromatic hydrocarbon (PAH), 10-(deoxyguanosin-N2-yl)-7,8,9trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-B[a]PDE-N2-dG), 10-(deoxyadenosin-N6-yl)-7,8,9trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-B[a]PDE-N6-dA), (11R,12S,13R,14S)-14(deoxyadenosin-N6-yl)-11,12,13-trihydroxy-11,12,13,14-tetrahydrodiben[def,p]chrysene ((-)-anti-transDB[a,l]PDE-N6-dA), (-)-anti-cis- (11R,12S,13R,14R)-14-(deoxyadenosin-N6-yl)-11,12,13-trihydroxy11,12,13,14-tetrahydrodiben[def,p]chrysene ((-)-anti-cis-DB[a,l]PDE-N6-dA), (-)-anti-trans(11R,12S,13R,14S)-14-(deoxyguanosin-N2-yl)-11,12,13-trihydroxy-11,12,13,14tetrahydrodiben[def,p]chrysene ((-)-anti-trans-DB[a,l]PDE-N2-dG), (-)-anti-cis- (11R,12S,13R,14R)14-(deoxyguanosin-N2-yl)-11,12,13-trihydroxy-11,12,13,14-tetrahydrodiben[def,p]chrysene ((-)-anticis-DB[a,l]PDE-N2-dG), O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-PoB-dThd), 7-[4-(3-pyridyl-4oxobut-1-yl]guanine (7-PoB-Gua), N-(deoxyguanosin-C8-yl)-4-aminoquinoline-1-oxide, N-dG-C8-4AQO; 3-(deoxyguanosin-N2-yl)-4-aminoquinoline-1-oxide (3-dG-N2-4-AQO), (1R,2S,3S,4S)-1-

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(deoxyguanosin-N2-yl)- 2,3,4-trihydroxy-1,2,3,4-tetrahydrotetraphen (anti-dG-N2-DMBADE), (1R,2R,3R,4S)-1-(deoxyadenosin-N6-yl)-2,3,4-trihydroxy-1,2,3,4-tetrahydrotetraphene (syn-dA-N6DMBADE), and (1R,2S,3S,4S)-1-(deoxyadenosin-N6-yl)-2,3,4-trihydroxy-1,2,3,4-tetrahydrotetraphene (anti-dA-N6-DMBADE).

11. Authors Biographies Karam El-Bayoumy, Ph.D. Dr. El-Bayoumy is a distinguished professor (Penn State College of Medicine); and the Associate Director of Basic Research in the Cancer Institute. His research focuses on understanding the causes of select cancers and developing the means of their prevention in preclinical and clinical settings. Specifically, his research is focused on the prevention of tobacco- and environmentally-related cancers by various synthetic and naturally-occurring chemopreventive agents, combined with dietary manipulation. More recent studies focus on the chemopreventive action of black raspberry on oral cancer in a novel mouse model developed in his laboratory.

Kun-Ming Chen, Ph.D. Dr. Kun-Ming Chen is an Associate Professor at the Penn State University College of Medicine. He received his Ph.D. degree from St. John's University majored in Medicinal Chemistry. He has involved in the research of oral and ovarian carcinogenesis induced by chemical carcinogens such as dibenzo[a,l]pyrene (DB[a,l]P). He is also interested in detection, structure identification and quantification of macro- or small molecules using HPLC or GC mass spectrometry. He also has the expertise in developing analytical methods to detect DNA and 35



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protein modifications, or secondary metabolites resulted from ROS/RNS derived from environmental pollutants. Shang-Min Zhang, Ph.D. Dr. Shang-Min Zhang is currently a postdoctoral associate at Yale. My Ph.D. training was in the laboratory of Dr. Karam El-Bayoumy, a world-renowned scientist in chemical carcinogenesis and chemoprevention. I published 5 papers during my graduate training at Penn State College of Medicine. I developed the first LC-MS/MS method to identify and quantify DNA adducts induced by carcinogen DB[a,l]P in a mouse oral cancer model. An R01 grant was awarded based on my thesis work on the carcinogenesis and chemoprevention for oral cancer. As the result of my outstanding work, I won Graduate Alumni Endowed Scholarship Award from Penn State University in 2012. Yuan-Wan Sun, Ph.D. Yuan-Wan Sun, Ph.D. is an Assistant Professor at the Pennsylvania State University, College of Medicine since 2014. She holds a Ph.D. in Medicinal Chemistry from St. John’s University, New York and then worked as a Postdoctoral Fellow/Research Associate in the field of Chemical Carcinogenesis and Prevention at American Health Foundation (2002-2004) and Penn State University (2004-2013). Her research involves chemical analysis and elucidation of molecular mechanisms in tumor development. Dr. Sun is the author of numerous original research papers on oral cancer and other topics. Shantu Amin, Ph.D. Dr. Shantu Amin received his Ph.D. from Stevens Institute of Technology (NJ) in 1975. At present, he is a Professor of Pharmacology at Penn State College of Medicine. The major focus of his research is on developing novel anti-cancer agents, elucidating their efficacy, and 36


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investigating the drug metabolism. Dr. Amin has also been involved in structure-activity studies with mutagenic and carcinogenic compounds and using this knowledge to develop appropriate chemopreventive agents. His laboratory is nationally recognized for developing synthetic strategies for novel small drug-like molecules for cancer prevention and therapy. Gary Stoner, Ph.D. Dr. Gary Stoner is a Professor Emeritus from the Ohio State University and the Medical College of Wisconsin. He pioneered the development of a food-based approach to cancer prevention using freeze-dried black raspberries and, along with his colleagues, demonstrated the ability of the berries to prevent cancer in the rodent oral cavity, esophagus, colon and skin. Biofractionation studies indicated that the anthocyanins and fiber in black raspberries are responsible for much of their chemopreventive activity. Human trials provide evidence of the ability of black raspberries to inhibit the progression of premalignant lesions in the oral cavity, esophagus and colon. Joseph B. Guttenplan, Ph.D. Dr. Guttenplan is a Professor of Basic Science and a pt. Res. Assoc. Professor at NYU Dental and Medical Schools, resp. His research focusses on several areas: 1) Inhibition of carcinogenesis and mutagenesis, and enhancement of DNA repair, most recently using black raspberries, 2) Effects of tobacco products on carcinogenesis and mutagenesis in oral cancer, 3) Effects of different tobacco products on metabolism of tobacco carcinogens, 4) Effects of potential carcinogens on mutagenesis in oral cavity and other organs of lac mice, 5) Research on the causes and prevention of spontaneous carcinogenesis.

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138. Chipuk, J. E.; Kuwana, T.; Bouchier-Hayes, L.; Droin, N. M.; Newmeyer, D. D.; Schuler, M.; Green, D. R., Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science (New York, N.Y 2004, 303 (5660), 1010-4. 139. Takimoto, R.; Wang, W.; Dicker, D. T.; Rastinejad, F.; Lyssikatos, J.; el-Deiry, W. S., The mutant p53-conformation modifying drug, CP-31398, can induce apoptosis of human cancer cells and can stabilize wild-type p53 protein. Cancer biology & therapy 2002, 1 (1), 47-55. 140. Shetty, S.; Shetty, P.; Idell, S.; Velusamy, T.; Bhandary, Y. P.; Shetty, R. S., Regulation of plasminogen activator inhibitor-1 expression by tumor suppressor protein p53. The Journal of biological chemistry 2008, 283 (28), 19570-80. 141. Bodvarsdottir, S. K.; Steinarsdottir, M.; Hilmarsdottir, H.; Jonasson, J. G.; Eyfjord, J. E., MYC amplification and TERT expression in breast tumor progression. Cancer genetics and cytogenetics 2007, 176 (2), 93-9. 142. Blons, H.; Pallier, K.; Le Corre, D.; Danel, C.; Tremblay-Gravel, M.; Houdayer, C.; FabreGuillevin, E.; Riquet, M.; Dessen, P.; Laurent-Puig, P., Genome wide SNP comparative analysis between EGFR and KRAS mutated NSCLC and characterization of two models of oncogenic cooperation in nonsmall cell lung carcinoma. BMC medical genomics 2008, 1, 25. 143. Wei, X.; Xu, H.; Kufe, D., Human MUC1 oncoprotein regulates p53-responsive gene transcription in the genotoxic stress response. Cancer cell 2005, 7 (2), 167-78. 144. Luo, J.; Su, F.; Chen, D.; Shiloh, A.; Gu, W., Deacetylation of p53 modulates its effect on cell growth and apoptosis. Nature 2000, 408 (6810), 377-81. 145. Kekatpure, V. D.; Bs, N.; Wang, H.; Zhou, X. K.; Kandasamy, C.; Sunny, S. P.; Suresh, A.; Milne, G. L.; Kuriakose, M. A.; Dannenberg, A. J., Elevated Levels of Urinary PGE-M Are Found in Tobacco Users and Indicate a Poor Prognosis for Oral Squamous Cell Carcinoma Patients. Cancer prevention research (Philadelphia, Pa 2016, 9 (6), 428-36. 146. Stoner, G. D.; Wang, L. S.; Zikri, N.; Chen, T.; Hecht, S. S.; Huang, C.; Sardo, C.; Lechner, J. F., Cancer prevention with freeze-dried berries and berry components. Seminars in cancer biology 2007, 17 (5), 403-10. 147. Stoner, G. D., Foodstuffs for preventing cancer: the preclinical and clinical development of berries. Cancer prevention research (Philadelphia, Pa 2009, 2 (3), 187-94. 148. Stoner, G. D.; Sardo, C.; Apseloff, G.; Mullet, D.; Wargo, W.; Pound, V.; Singh, A.; Sanders, J.; Aziz, R.; Casto, B.; Sun, X., Pharmacokinetics of anthocyanins and ellagic acid in healthy volunteers fed freeze-dried black raspberries daily for 7 days. Journal of clinical pharmacology 2005, 45 (10), 1153-64. 149. Kresty, L. A.; Frankel, W. L.; Hammond, C. D.; Baird, M. E.; Mele, J. M.; Stoner, G. D.; Fromkes, J. J., Transitioning from preclinical to clinical chemopreventive assessments of lyophilized black raspberries: interim results show berries modulate markers of oxidative stress in Barrett's esophagus patients. Nutrition and cancer 2006, 54 (1), 148-56. 150. Mallery, S. R.; Stoner, G. D.; Larsen, P. E.; Fields, H. W.; Rodrigo, K. A.; Schwartz, S. J.; Tian, Q.; Dai, J.; Mumper, R. J., Formulation and in-vitro and in-vivo evaluation of a mucoadhesive gel containing freeze dried black raspberries: implications for oral cancer chemoprevention. Pharmaceutical research 2007, 24 (4), 728-37. 151. Mallery, S. R.; Tong, M.; Shumway, B. S.; Curran, A. E.; Larsen, P. E.; Ness, G. M.; Kennedy, K. S.; Blakey, G. H.; Kushner, G. M.; Vickers, A. M.; Han, B.; Pei, P.; Stoner, G. D., Topical application of a mucoadhesive freeze-dried black raspberry gel induces clinical and histologic regression and reduces loss of heterozygosity events in premalignant oral intraepithelial lesions: results from a multicentered, placebo-controlled clinical trial. Clin Cancer Res 2014, 20 (7), 1910-24. 152. Ugalde, C. M.; Liu, Z.; Ren, C.; Chan, K. K.; Rodrigo, K. A.; Ling, Y.; Larsen, P. E.; Chacon, G. E.; Stoner, G. D.; Mumper, R. J.; Fields, H. W.; Mallery, S. R., Distribution of anthocyanins delivered from a bioadhesive black raspberry gel following topical intraoral application in normal healthy volunteers. Pharmaceutical research 2009, 26 (4), 977-86.

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153. Shumway, B. S.; Kresty, L. A.; Larsen, P. E.; Zwick, J. C.; Lu, B.; Fields, H. W.; Mumper, R. J.; Stoner, G. D.; Mallery, S. R., Effects of a topically applied bioadhesive berry gel on loss of heterozygosity indices in premalignant oral lesions. Clin Cancer Res 2008, 14 (8), 2421-30. 154. Mallery, S. R.; Zwick, J. C.; Pei, P.; Tong, M.; Larsen, P. E.; Shumway, B. S.; Lu, B.; Fields, H. W.; Mumper, R. J.; Stoner, G. D., Topical application of a bioadhesive black raspberry gel modulates gene expression and reduces cyclooxygenase 2 protein in human premalignant oral lesions. Cancer research 2008, 68 (12), 4945-57. 155. Knobloch, T. J.; Uhrig, L. K.; Pearl, D. K.; Casto, B. C.; Warner, B. M.; Clinton, S. K.; SardoMolmenti, C. L.; Ferguson, J. M.; Daly, B. T.; Riedl, K.; Schwartz, S. J.; Vodovotz, Y.; Buchta, A. J., Sr.; Schuller, D. E.; Ozer, E.; Agrawal, A.; Weghorst, C. M., Suppression of Proinflammatory and Prosurvival Biomarkers in Oral Cancer Patients Consuming a Black Raspberry Phytochemical-Rich Troche. Cancer prevention research (Philadelphia, Pa 2015, 9 (2), 159-71. 156. Wen, X.; Walle, T., Preferential induction of CYP1B1 by benzo[a]pyrene in human oral epithelial cells: impact on DNA adduct formation and prevention by polyphenols. Carcinogenesis 2005, 26 (10), 1774-81. 157. Walle, T.; Walle, U. K.; Sedmera, D.; Klausner, M., Benzo[A]pyrene-induced oral carcinogenesis and chemoprevention: studies in bioengineered human tissue. Drug metabolism and disposition: the biological fate of chemicals 2006, 34 (3), 346-50. 158. Calderon-Montano, J. M.; Burgos-Moron, E.; Perez-Guerrero, C.; Lopez-Lazaro, M., A review on the dietary flavonoid kaempferol. Mini reviews in medicinal chemistry 2011, 11 (4), 298-344. 159. Chow, H. H.; Garland, L. L.; Hsu, C. H.; Vining, D. R.; Chew, W. M.; Miller, J. A.; Perloff, M.; Crowell, J. A.; Alberts, D. S., Resveratrol modulates drug- and carcinogen-metabolizing enzymes in a healthy volunteer study. Cancer prevention research (Philadelphia, Pa 2010, 3 (9), 1168-75. 160. Bruno, R. D.; Njar, V. C., Targeting cytochrome P450 enzymes: a new approach in anti-cancer drug development. Bioorganic & medicinal chemistry 2007, 15 (15), 5047-60. 161. Guttenplan, J.; Chen, K. M.; Sun, Y. W.; Kosinska, W.; Zhou, Y.; Kim, S. A.; Sung, Y.; Gowda, K.; Amin, S.; Stoner, G.; El-Bayoumy, K., Effects of black raspberry extract and protocatechuic acid on carcinogen-DNA adducts and mutagenesis, and oxidative stress in rat and human oral cells. Cancer prevention research (Philadelphia, Pa 2016, 9 (8), 704-12. 162. !!! INVALID CITATION !!! 160. 163. Manach, C.; Williamson, G.; Morand, C.; Scalbert, A.; Remesy, C., Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. The American journal of clinical nutrition 2005, 81 (1 Suppl), 230S-242S. 164. Ichiyanagi, T.; Oikawa, K.; Tateyama, C.; Konishi, T., Acid mediated hydrolysis of blueberry anthocyanins. Chemical & pharmaceutical bulletin 2001, 49 (1), 114-7. 165. Wu, S.; Powers, S.; Zhu, W.; Hannun, Y. A., Substantial contribution of extrinsic risk factors to cancer development. Nature 2016, 529 (7584), 43-7. 166. Noble, R. J.; Kaltz, O.; Nunney, L.; Hochberg, M. E., Overestimating the role of environment in cancers. Cancer prevention research (Philadelphia, Pa 2016. 167. Stanford, E. A.; Ramirez-Cardenas, A.; Wang, Z.; Novikov, O.; Alamoud, K.; Koutrakis, P.; Mizgerd, J. P.; Genco, C. A.; Kukuruzinska, M.; Monti, S.; Bais, M. V.; Sherr, D. H., Role for the Aryl Hydrocarbon Receptor and Diverse Ligands In Oral Squamous Cell Carcinoma Migration and Tumorigenesis. Mol Cancer Res 2016, 14 (8), 696-706. 168. Bais, M. V.; Kukuruzinska, M.; Trackman, P. C., Orthotopic non-metastatic and metastatic oral cancer mouse models. Oral oncology 2015, 51 (5), 476-82. 169. Hartmann, S.; Bhola, N. E.; Grandis, J. R., HGF/Met Signaling in Head and Neck Cancer: Impact on the Tumor Microenvironment. Clin Cancer Res 2016, 22 (16), 4005-4013.

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170. Lin, D. T.; Subbaramaiah, K.; Shah, J. P.; Dannenberg, A. J.; Boyle, J. O., Cyclooxygenase-2: a novel molecular target for the prevention and treatment of head and neck cancer. Head & neck 2002, 24 (8), 792-9. 171. Morton, R. S.; Dongari-Bagtzoglou, A. I., Cyclooxygenase-2 is upregulated in inflamed gingival tissues. Journal of periodontology 2001, 72 (4), 461-9. 172. DeMarini, D. M.; Hanley, N. M.; Warren, S. H.; Adams, L. D.; King, L. C., Association between mutation spectra and stable and unstable DNA adduct profiles in Salmonella for benzo[a]pyrene and dibenzo[a,l]pyrene. Mutation research 2011, 714 (1-2), 17-25. 173. Altuwairgi, O. S.; Papageorge, M. B.; Doku, H. C., The cancer-promoting effect of Nnitrosonornicotine used in combination with a subcarcinogenic dose of 4-nitroquinoline-N-oxide and 7,12-dimethylbenz (A) anthracene. J Oral Maxillofac Surg 1995, 53 (8), 910-3; discussion 914. 174. Maurya, D. K.; Salvi, V. P.; Nair, C. K., Radiation protection of DNA by ferulic acid under in vitro and in vivo conditions. Molecular and cellular biochemistry 2005, 280 (1-2), 209-17. 175. Niture, S. K.; Velu, C. S.; Smith, Q. R.; Bhat, G. J.; Srivenugopal, K. S., Increased expression of the MGMT repair protein mediated by cysteine prodrugs and chemopreventative natural products in human lymphocytes and tumor cell lines. Carcinogenesis 2007, 28 (2), 378-89. 176. Pavanello, S.; Bollati, V.; Pesatori, A. C.; Kapka, L.; Bolognesi, C.; Bertazzi, P. A.; Baccarelli, A., Global and gene-specific promoter methylation changes are related to anti-B[a]PDE-DNA adduct levels and influence micronuclei levels in polycyclic aromatic hydrocarbon-exposed individuals. International journal of cancer 2009, 125 (7), 1692-7. 177. Barros, S. P.; Offenbacher, S., Epigenetics: connecting environment and genotype to phenotype and disease. Journal of dental research 2009, 88 (5), 400-8. 178. Sharma, S.; Kelly, T. K.; Jones, P. A., Epigenetics in cancer. Carcinogenesis 2010, 31 (1), 27-36. 179. Tommasi, S.; Zheng, A.; Yoon, J. I.; Besaratinia, A., Epigenetic targeting of the Nanog pathway and signaling networks during chemical carcinogenesis. Carcinogenesis 2014, 35 (8), 1726-36. 180. Hoffmann, D.; Hoffmann, I.; El-Bayoumy, K., The less harmful cigarette: a controversial issue. a tribute to Ernst L. Wynder. Chemical research in toxicology 2001, 14 (7), 767-90. 181. El-Bayoumy, K., Environmental carcinogens that may be involved in human breast cancer etiology. Chemical research in toxicology 1992, 5 (5), 585-90. 182. Schwartz, J.; Baker, V.; Larios, E.; Desai, D.; Amin, S., Inhibition of experimental tobacco carcinogen induced head and neck carcinogenesis. Oral oncology 2004, 40 (6), 611-23. 183. Tanaka, T.; Makita, H.; Kawabata, K.; Mori, H.; El-Bayoumy, K., 1,4phenylenebis(methylene)selenocyanate exerts exceptional chemopreventive activity in rat tongue carcinogenesis. Cancer research 1997, 57 (17), 3644-8. 184. Bauman, J. E.; Zang, Y.; Sen, M.; Li, C.; Wang, L.; Egner, P. A.; Fahey, J. W.; Normolle, D. P.; Grandis, J. R.; Kensler, T. W.; Johnson, D. E., Prevention of Carcinogen-Induced Oral Cancer by Sulforaphane. Cancer prevention research (Philadelphia, Pa 2016, 9 (7), 547-57. 185. Shklar, G.; Schwartz, J. L., Vitamin E inhibits experimental carcinogenesis and tumour angiogenesis. European journal of cancer 1996, 32B (2), 114-9. 186. Gorelick, N. J.; Andrews, J. L.; Gu, M.; Glickman, B. W., Mutational spectra in the lacl gene in skin from 7,12-dimethylbenz[a]anthracene-treated and untreated transgenic mice. Molecular Carcinogenesis 1995, 14 (1), 53-62. 187. Ryu, J.; Youn, J.; Kim, Y.; Kwon, O.; Song, Y.; Kim, H.; Cho, K.; Chang, I., Mutation spectrum of 4-nitroquinoline N-oxide in the lacI transgenic Big Blue Rat2 cell line. Mutation Research 1999, 445 (1), 127-35.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Levels and Sources of Representative Oral Carcinogens Source B[a]P DB[a,l]P NNN Cigarette Smoke 1.0-16.2 nga 0.1 nga 5-270 nga Smokeless Tobacco: NA Moist Snuff 56 ± 22 ngb 1-10 µgc 23 ± 26.2 ngb Spit-free Cooked Foods 1-200 ngd l Urban Air, soil 400 pge 10-100f -sediments Combustion Systems 208-558 µgg 136-254 µgg --

a. Concentrations are expressed as ng/cigarette180. b. Concentrations per g dry weight57. c. Concentrations per g dry weight 49. d. Concentrations per gm weight181 and references therein. e. Concentrations per cubic meter 181 and references therein. f. ng/g soil sample58 g. Concentrations per g Diesel Particulate Extract52. l. Levels are 100-1000 times less in food and beer than those found in smokeless tobacco.

49



1 2 3 Table 2. Oral Tumorigenesis Induced by Environmental and Synthetic Agents 4 5 6 Agent Source Species & Route of Total Dose 7 Strain Administration/Dose/Dura 8 tion 9 B[a]P Cigarette smoke, Female Topical application/100 µg (3 X 8.1 mg 10 smokeless Syrian week)/6 months (32µmol) 11 tobacco, cooked Hamster 12 foods, combustion cheek pouch 13 systems, urban air 14 B[a]P Cigarette smoke, Female Diet/100 ppm (430 µg/day)/24 309.6 mg 15 smokeless B63F1 mice months (1313 µmol) 16 tobacco, cooked 17 foods, combustion 18 systems, urban air 19 DB[a,l]P Urban air, soil Female Topical application into oral 0.83 mg 20 sediments, B63F1 mice cavity/24 nmol, 3 X week/38 (2.9 µmol) 21 cigarette smoke weeks 22 Urban air, soil Female Topical application 0.01 µM 0.453 mg 23DB[a,l]P sediments, Syrian (5 X week)/30 weeks (1.5 µmol) 24 cigarette smoke Hamster 25 cheek pouch 26 DB[a,l]PDE Metabolite of Female Topical application into oral 0.24 mg 27 DB[a,l]P B63F1 mice cavity/6 nmol, 3 X week/38 (0.68 µmol) 28 weeks 29 Tobacco smoke, Male F344 Drinking water/14 ppm (14 123 mg 30[S]-NNN 31 smokeless rats µg/ml)/20 months (694 µmol) 32 tobacco, food, 33 beer 34 Male F344 Drinking water/20 ppm/8 22.4 mg 35 rats weeks (117.8 µmol) 36 4-NQO Synthetic 37 C57BL/6 14 mg 38 mice Drinking water/100 ppm/8 (73.7 µmol) 39 weeks 40 DMBA Synthetic Male or Painting/0.6 mg in mineral oil 28.8 mg 41 Female (3 X week)/16 weeks (112.5 µmol) 42 Syrian 43 Hamster 44 cheek pouch 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

50


Page 50 of 60

in Laboratory Animals Incidence (Histology)

Reference

61% (squamous cell papilloma/carcino ma)

46

47.9% (Tongue papillomas and/or carcinomas)

45

31% (OSCC)

50

85.7% (SCC)

182

100% (OSCC), 70% (Tongue SCC) 30% (SCC in tongue, oral, soft palate, epiglottis, pharynx) 47% (tongue SCC)

54

49

183

34, 184

35.3% (tongue SCC) 100% (SCC)

24, 185

Page 51 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 3. Comparison of Estimated Levels of Human Exposure to NNN, B[a]P, and DB[a,l]P from Several Sources to those Inducing Oral Cancer in Rodentsa

a

Carcinogen

Cigarette Smoke

Smokeless Tobacco

Foods and Beverages

Dose That Induced Oral Cancer in Rodents

NNN

0.025

5

0.05 – 0.005

B[a]P

0.048

≤ 0.25

0.48

12360

DB[a,l]P

≤0.04

NA

0.05

33.2

b

Levels presented in mg/kg body weight Levels in foods and beer are 100-1000 times less than those found in smokeless tobacco

b

51


336


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 52 of 60

Table 4. Mutational spectra of carcinogens that induce cancer in the oral cavity in

rodents Percentage of mutations induced by carcinogens DB[a,l]P 1 DBPDE2 BaP1 Control1 NNN3 DMBA4 4-NQO5 P536 class GC:AT

17

12

31

63

14

11

21

40

GC:TA

33

38

40

14

5

28

44

17

GC:CG

7

16

10

3

0

2

19

8

AT:GC

7

8

0

3

14

2

4

12

AT:CG

10

4

0

3

24

2

2

4

AT:TA

14

20

4

6

38

42

4

7

in/del5

12

4

15

9

5

13

6

13

Mutations in combined tongue and other oral tissue of female B6C3F1 lacI mice50 Mutations in combined tongue and other oral tissue of female B6C3F1 lacI mice54 Mutations in liver of male CD-2 lacZ mice (Guttenplan, J.B., unpublished data) Mutations in skin of C57/BL6 lacI male mice186 Mutations in Big Blue Rat2 embryonic fibroblast cell line 187 6. From the P53 Handbook131

1. 2. 3. 4. 5.

52


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure Legends Fig. 1. Synthetic agents (DMBA and 4-NQO) known to induce oral cancer in rodents. Fig. 2 Environmental agents (B[a]P, DB[a,l]P, NNN, and NNK) known to induce oral cancer in rodents. Fig. 3. Relative potency of synthetic and environmental agents known to induce oral cancer in rodents. Fig. 4. Structures of carcinogen-DNA adducts detected in mammalian systems. Fig. 5. Schematic representation of pathways leading to DB[a,l]P-induced DNA damage. In the first pathway, P450 peroxidases can catalyze the formation of DB[a,l]P radical cation which can react with DNA bases leading to the formation of depurinating adducts. In the second pathway, DB[a,l]P is sequentially monooxygenated by P450 1A1/1B1 (both enzymes are expressed in the oral cavity) leading to the formation of (+)-syn-DB[a,l]PDE and (-)-antiDB[a,l]PDE which can react with DNA to form stable bulky DNA adducts. In the third pathway (±)-DB[a,l]P-11,12-dihydrodiol undergoes an NAD(P)+ dependant oxidation catalyzed by human AKR1A1, AKR1C1-AKR1C3 to yield a ketol which spontaneously rearranges to DB[a,l]P-11,12-catechol, followed by a 1 electron-oxidation to form an orthosemiquinone anion radical, and a 1 electron oxidation to form DB[a,l]P-11,12-quinone with concomitant production of ROS; both quinone and ROS can lead to DNA damage. Fig. 6. Metabolic activation and detoxification of DB[a,l]P. DB[a,l]P is metabolized sequentially by CYP1A1, CYP1B1 and epoxide hydrolase leading to the formation of (+)(11S,12S)-dihydrodiol and (-)-(11R,12R)-dihydrodiol; further metabolism of these dihydrodiols, catalyzed by CYP1A1 and CYP1B1, leads to the formation of the diol-epoxides 53 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 54 of 60

(+)-syn-(11S,12R,13S,14R)-DB[a,l]PDE and (-)-anti-(11R,12S,13S,14R)-DB[a,l]PDE. The detoxification processes of the dihydrodiols and diol epoxides catalyzed by several phase II enzymes (GST, glucuronidase, sulfatase) increase the water solubility of these metabolites and allows their elimination from the body through the bile and/or urine. The balance between the metabolic activation and detoxification processes influences the intracellular DNA adduct formation.

54


Page 55 of1 60 Figure 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


NO2

CH3

N CH3

O

DMBA

4-NQO


Figure 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


Bay

Page 56 of 60

Fjord

B[a]P

DB[a,l]P

O

N O

N N

CH3

N O

N

NNN

NNK


Page 57 of3 60 Figure

Relative Potency

1000

Mouse

100

10

1

Relative Potency

B[a]P DB[a,l]P 4-NQO 100

Rat

10

1

0.1

[S]-NNN

Relative Potency

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58


1000

4-NQO

Hamster

100

10

1


B[a]P DB[a,l]P DMBA

Figure 4


Carcinogens 1 2 3 B[a]P 4 5 6 7 8 9 10 11 12 13 14 DB[a,l]P 15 16 17 18 19 20 21 22 23 24 25 NNN 26 27 28 29 30 31 32 4-NQO 33 34 35 36 37 38 39 40 41 42 43 44 DMBA 45 46 47 48 49 50 51 52 53 54 55 56 57 58

Page 58 of 60

DNA Adducts O N

NH

N

N

N dR

NH

N

dR

N

N

NH

HO

HO

HO

HO OH

OH

Anti-B[a]PDE-N2-dG

Anti-B[a]PDE-N6-dA O

O

N

N

N

N

N

N

N

NH

N

N

N

dR

dR

NH

NH

HO

HO

HO

HO

N

N

dR

NH

N

NH

N

dR

HO

HO

HO

OH

OH

OH

OH

NH

HO

(-)-anti-cis(-)-anti-trans(-)-anti-cis(-)-anti-trans6 6 2 DB[a,l]PDE-N -dA DB[a,l]PDE-N -dA DB[a,l]PDE-N -dG DB[a,l]PDE-N2-dG O

O CH3

N

O

N N

NH

N

O

N dR

N

O

O2-PoB-dThd

NH2

N

7-PoB-Gua

O N

O

N

N

HN

NH

HN

H 2N

N

NH2

N

NH

dR

dR

N

N

O

O

3-(deoxyguanosin-N2-yl)-4aminoquinoline-1-oxide

N-(deoxyguanosin-C8-yl)-4aminoquinoline-1-oxide O N

N NH

N N

N

dR

N

NH

NH

N OH

CH3

dR

N

N

OH OH

N

N

OH OH

CH3

NH

N dR

OH

OH

OH

CH3

anti-dG

CH3

syn-dA ACS Paragon Plus Environment

OH

CH3

CH3

anti-dA

Page 59 of 60

Chemical Research in Toxicology 3

Figure 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

2

4 5

1

6

14

P-450 Peroxidase Depurinated DNA adducts

7

13 8

12 11

10

9

P4501A1/1B1, Epoxide Hydrolase

Radical Cation

P4501A1/1B1 OH

O

O

+

OH

OH

OH

OH

(±)-DB[a,l]P-11,12-dihydrodiol

Stable Bulky Diol Epoxide-DNA Adducts

Oxidative DNA Damage

H2O2

e-

HO OH

Catechol

(-)-anti-DB[a,l]PDE

(+)-syn-DB[a,l]PDE

aldo-keto reductases

O2

OH

Oxidative DNA Damage Covalent DNA Adducts

O2

O2

e-

O-

O O Paragon Plus Environment ACS

Semiquinone anion radical

O

o-quinone

Figure 6


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OH 21 22 OH 23 24 (+)-(11S,12S)-dihydrodiol 25 26 (+)-anti27 DB[a,l]PDE 28 29 30 31 32 O 33 34 35 36 HO 37 38 OH 39 40 (+)-syn-(11S,12R,13S,14R)41 DB[a,l]PDE 42

CYP 1A1, 1B1

CYP 1A1 CYP 1B1

Page 60 of 60

Epoxide hydrolase Glucuronidase Sunphatase Glucuronide, and sulphate conjugates OH OH

(-)-(11R,12R)-dihydrodiol (-)-synDB[a,l]PDE

O

HO OH

CYP 1A1 CYP 1B1 GST Glucuronidase Glutathione, Sunphatase Glucuronide, and suphate conjugates

ACS Paragon Plus Environment (-)-anti-(11R,12S,13S,14R)DB[a,l]PDE

Carcinogenesis of the Oral Cavity - American Chemical Society

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