Gender Difference in DNA Damage Induced by the Environmental Carcinogen Dibenzo[def,p]chrysene Individually and in Combination with Mouse Papillomavirus Infection in the Mouse Oral Cavity.

Tobacco smoking and human papillomavirus infection are established etiological agents in the development of head and neck squamous cell carcinoma (HNSCC). The incidence and mortality of HNSCC are higher in men than women. To provide biochemical basis for sex differences, we tested the hypothesis that carcinogen treatment using dibenzo[def,p]chrysene, which is an environmental pollutant and tobacco smoke constituent, in the absence or presence of the mouse papillomavirus infection results in significantly higher levels of DNA damage in the oral cavity in male than in female mice. However, the results of the present investigation do not support our hypothesis since we found that females were more susceptible to carcinogen-induced covalent DNA damage than males independent of the viral infection. Since DNA damage represents only a single-step in the carcinogenesis process, additional factors may contribute to sex differences in humans.

The Effects of Black Raspberry as a Whole Food-Based Approach on Biomarkers of Oxidative Stress in Buccal Cells and Urine of Smokers.

Cigarette smoke is a rich source of free radicals that can promote oxidative stress and carcinogenesis, including head and neck squamous cell carcinoma (HNSCC) development; importantly, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-iso-prostaglandin F2α (8-isoprostane) are biomarkers of oxidative stress. Several mechanisms, including the antioxidant properties of black raspberry (BRB), account for their chemopreventive effects. In the present clinical trial, we tested the hypothesis that BRB administration reduces biomarkers levels of oxidative stress in buccal cells and urine of smokers. One week after enrolling 21 smokers, baseline buccal cells and urine samples were collected before the administration of BRB lozenges for 8 weeks (5/day, 1 gm BRB/lozenge). Buccal cells and urine samples were collected at the middle and the end of BRB administration. The last samples were collected after the BRB cessation (washout period). We analyzed levels of 8-oxodG and 8-isoprostane (LC/MS-MS), urinary cotinine (ELISA), and creatinine (spectrophotometry). BRB significantly reduced the levels of 8-oxodG by 17.08% (P = 0.00079) in buccal cells and 12.44% (P = 0.034) in urine at the middle of BRB administration as compared with baseline; the corresponding values at the end of BRB administration were 16.46% (P = 0.026) in buccal cells and 25.72% (P = 0.202) in urine. BRB had no significant effect on the levels of urinary 8-isoprostane. BRB's capacity to inhibit 8-oxodG formation of smokers' buccal cells and urine is clearly evident and the reduction in 8-oxodG suggests that antioxidant abilities are central to BRB's HNSCC chemopreventive properties. PREVENTION RELEVANCE: Cigarette smoke contains highly active components namely free radicals that can promote oxidative stress and oral cancer. We found that black raspberry (BRB) inhibited the formation of oxidative stress markers in the oral cavity and urine of smokers suggesting the antioxidant abilities of BRB in preventing oral cancer.

Immune Responses in Oral Papillomavirus Clearance in the MmuPV1 Mouse Model.

Human papillomavirus (HPV)-induced oropharyngeal cancer now exceeds HPV-induced cervical cancer, with a noticeable sex bias. Although it is well established that women have a more proficient immune system, it remains unclear whether immune control of oral papillomavirus infections differs between sexes. In the current study, we use genetically modified mice to target CCR2 and Stat1 pathways, with the aim of investigating the role of both innate and adaptive immune responses in clearing oral papillomavirus, using our established papillomavirus (MmuPV1) infection model. Persistent oral MmuPV1 infection was detected in Rag1ko mice with T and B cell deficiencies. Meanwhile, other tested mice were susceptible to MmuPV1 infections but were able to clear the virus. We found sex differences in key myeloid cells, including macrophages, neutrophils, and dendritic cells in the infected tongues of wild type and Stat1ko mice but these differences were not observed in CCR2ko mice. Intriguingly, we also observed a sex difference in anti-MmuPV1 E4 antibody levels, especially for two IgG isotypes: IgG2b and IgG3. However, we found comparable numbers of interferon-gamma-producing CD8 T cells stimulated by E6 and E7 in both sexes. These findings suggest that males and females may use different components of innate and adaptive immune responses to control papillomavirus infections in the MmuPV1 mouse model. The observed sex difference in immune responses, especially in myeloid cells including dendritic cell (DC) subsets, may have potential diagnostic and prognostic values for HPV-associated oropharyngeal cancer.

Complementarity between Microbiome and Immunity May Account for the Potentiating Effect of Quercetin on the Antitumor Action of Cyclophosphamide in a Triple-Negative Breast Cancer Model.

Immunotherapy targeting program cell death protein 1 (PD-1) in addition to chemotherapy has improved the survival of triple-negative breast cancer (TNBC) patients. However, the development of resistance and toxicity remain significant problems. Using the translationally relevant 4T1 mouse model of TNBC, we report here that dietary administration of the phytochemical quercetin enhanced the antitumor action of Cyclophosphamide, a cytotoxic drug with significant immunogenic effects that is part of the combination chemotherapy used in TNBC. We observed that quercetin favorably modified the host fecal microbiome by enriching species such as Akkermansia muciniphilia, which has been shown to improve response to anti-PD-1 therapy. We also show that quercetin and, to a greater extent, Cyclophosphamide increased the systemic frequency of T cells and NK cells. In addition, Cyclophosphamide alone and in combination with quercetin reduced the frequency of Treg, which is consistent with an antitumor immune response. On the other hand, Cyclophosphamide did not significantly alter the host microbiome, suggesting complementarity between microbiome- and immune-mediated mechanisms in potentiating the antitumor action of Cyclophosphamide by quercetin. Overall, these results support the potential for microbiota-centered dietary intervention to overcome resistance to chemoimmunotherapy in TNBC.

Lifestyle Modifications and Breast Cancer Risk.

Lifestyle modifications have been shown to be effective in reducing breast cancer [...].

Role of Human Aldo-Keto Reductases in the Nitroreduction of 1-Nitropyrene and 1,8-Dinitropyrene.

1-Nitropyrene (1-NP) and 1,8-dinitropyrene (1,8-DNP) are diesel exhaust constituents and are classified by the International Agency for Research on Cancer as probable (Group 2A) or possible (Group 2B) human carcinogens. These nitroarenes undergo metabolic activation by nitroreduction to result in the formation of DNA adducts. Human aldo-keto reductases (AKRs) 1C1-1C3 catalyze the nitroreduction of 3-nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one, 3-NBA), but the extent of AKR contribution toward the nitroreduction of additional nitroarenes, including 1-NP and 1,8-DNP, is currently unknown. In the present study, we investigated the ability of human recombinant AKRs to catalyze 1-NP and 1,8-DNP nitroreduction by measuring the formation of the respective six-electron reduced amine products in discontinuous ultraviolet-reverse phase high-performance liquid chromatography enzymatic assays. We found that AKR1C1-1C3 were able to catalyze the formation of 1-aminopyrene (1-AP) and 1-amino-8-nitropyrene (1,8-ANP) in our reactions with 1-NP and 1,8-DNP, respectively. We determined kinetic parameters (Km, kcat, and kcat/Km) and found that out of the three isoforms, AKR1C1 had the highest catalytic efficiency (kcat/Km) for 1-AP formation, whereas AKR1C3 had the highest catalytic efficiency for 1,8-ANP formation. Use of ultra-performance liquid chromatography high-resolution mass spectrometry verified amine product identity and provided evidence for the formation of nitroso- and hydroxylamino-intermediates in our reactions. Our study expands the role of AKR1C1-1C3, which are expressed in human lung cells, in the metabolic activation of nitroarenes that can lead to DNA adduct formation, mutation, and carcinogenesis.

Treatment of Human HeLa Cells with Black Raspberry Extracts Enhances the Removal of DNA Lesions by the Nucleotide Excision Repair Mechanism.

As demonstrated by us earlier and by other researchers, a diet containing freeze-dried black raspberries (BRB) inhibits DNA damage and carcinogenesis in animal models. We tested the hypothesis that the inhibition of DNA damage by BRB is due, in part, to the enhancement of DNA repair capacity evaluated in the human HeLa cell extract system, an established in vitro system for the assessment of cellular DNA repair activity. The pre-treatment of intact HeLa cells with BRB extracts (BRBE) enhances the nucleotide excision repair (NER) of a bulky deoxyguanosine adduct derived from the polycyclic aromatic carcinogen benzo[a]pyrene (BP-dG) by ~24%. The NER activity of an oxidatively-derived non-bulky DNA lesion, guanidinohydantoin (Gh), is also enhanced by ~24%, while its base excision repair activity is enhanced by only ~6%. Western Blot experiments indicate that the expression of selected, NER factors is also increased by BRBE treatment by ~73% (XPA), ~55% (XPB), while its effects on XPD was modest (<14%). These results demonstrate that BRBE significantly enhances the NER yields of a bulky and a non-bulky DNA lesion, and that this effect is correlated with an enhancement of expression of the critically important NER factor XPA and the helicase XPB, but not the helicase XPD.

Black Raspberry Extract Enhances Glutathione Conjugation of the Fjord-Region Diol Epoxide Derived from the Tobacco Carcinogen Dibenzo[def,p]chrysene in Human Oral Cells.

In a series of previous studies we reported that black raspberry (BRB) powder inhibits dibenzo[a,l]pyrene (DBP)-induced DNA damage, mutagenesis, and oral squamous cell carcinoma (OSCC) development in mice. In the present study, using human oral leukoplakia (MSK-Leuk1) and squamous cell carcinoma (SCC1483) cells, we tested the hypothesis that BRB extract (BRBE) will enhance the synthesis of glutathione (GSH) and in turn increase GSH conjugation of the fjord-region DBP diol epoxide (DBPDE) derived from DBP leading to inhibition of DBP-induced DNA damage. The syntheses of DBPDE-GSH conjugate, DBPDE-dA adduct, and the corresponding isotope-labeled internal standards were performed; LC-MS/MS methods were used for their quantification. BRBE significantly (p < 0.05) increased cellular GSH by 31% and 13% at 6 and 24 h, respectively, in OSCC cells; in MSK-LeuK1 cells, the levels of GSH significantly (p < 0.05) increased by 55% and 22%, at 1 and 6 h. Since BRBE significantly enhanced the synthesis of GSH in both cell types, subsequent experiments were performed in MSK-Leuk1 cells. Western blot analysis was performed to determine the types of proteins involved in the synthesis of GSH. BRBE significantly (p < 0.05) increased the protein expression (2.5-fold) of the glutamate-cysteine ligase catalytic subunit (GCLC) but had no effect on the glutamate-cysteine ligase modifier subunit (GCLM) and glutathione synthetase (GSS). LC-MS/MS analysis showed that pretreatment of cells with BRBE followed by DBPDE significantly (p < 0.05) increased the levels of DBPDE-GSH conjugate (2.5-fold) and decreased DNA damage by 74% measured by assessing levels of DBPDE-dA adduct formation. Collectively, the results of this in vitro study clearly support our hypothesis, and the LC-MS/MS methods developed in the present study will be highly useful in testing the same hypothesis initially in our mouse model and ultimately in smokers.

Nitroreduction: A Critical Metabolic Pathway for Drugs, Environmental Pollutants, and Explosives.

Nitro group containing xenobiotics include drugs, cancer chemotherapeutic agents, carcinogens (e.g., nitroarenes and aristolochic acid) and explosives. The nitro group undergoes a six-electron reduction to form sequentially the nitroso-, N-hydroxylamino- and amino-functional groups. These reactions are catalyzed by nitroreductases which, rather than being enzymes with this sole function, are enzymes hijacked for their propensity to donate electrons to the nitro group either one at a time via a radical mechanism or two at time via the equivalent of a hydride transfer. These enzymes include: NADPH-dependent flavoenzymes (NADPH: P450 oxidoreductase, NAD(P)H-quinone oxidoreductase), P450 enzymes, oxidases (aldehyde oxidase, xanthine oxidase) and aldo-keto reductases. The hydroxylamino group once formed can undergo conjugation reactions with acetate or sulfate catalyzed by N-acetyltransferases or sulfotransferases, respectively, leading to the formation of intermediates containing a good leaving group which in turn can generate a nitrenium or carbenium ion for covalent DNA adduct formation. The intermediates in the reduction sequence are also prone to oxidation and produce reactive oxygen species. As a consequence, many nitro-containing xenobiotics can be genotoxic either by forming stable covalent adducts or by oxidatively damaging DNA. This review will focus on the general chemistry of nitroreduction, the enzymes responsible, the reduction of xenobiotic substrates, the regulation of nitroreductases, the ability of nitrocompounds to form DNA adducts and act as mutagens as well as some future directions.

Detection of DNA adducts derived from the tobacco carcinogens, benzo[a]pyrene and dibenzo[def,p]chrysene in human oral buccal cells.

Polycyclic aromatic hydrocarbons (PAHs) are recognized as potential etiological agents in the development of oral cancer in smokers. In particular, benzo[a]pyrene (B[a]P) and dibenzo[def,p]chrysene (DB[a,l]P) are detected in cigarette smoke and the environment and can induce DNA damage, mutagenesis and carcinogenesis in the oral cavity of rodents. Consequently, DNA adducts are regarded as the most direct markers of genotoxicity and can be used as biomarkers of cancer risk. Thus, this study used LC-MS/MS analysis with isotope labeled internal standard to detect and quantify DNA adducts derived from B[a]P and DB[a,l]P in buccal cells of cigarette smokers and non-smokers. Participants in this study include 21 smokers and 16 non-smokers. Our data are the first to report that levels (mean ± SD) of BPDE-N2-dG were significantly (P < 0.001) higher in smokers (20.18 ± 8.40 adducts/108 dG) than in non-smokers (0.84 ± 1.02 adducts/108 dG). Likewise, levels of DBPDE-N6-dA in smokers (5.49 ± 3.41 adducts/108 dA) were significantly higher (P = 0.019) than non-smokers (2.76 ± 2.29 adducts/108 dA). Collectively, the results of this clinical study support that PAHs in tobacco smoke can contribute to the development of oral cancer in humans.

Use of Freeze-dried Watercress for Detoxification of Carcinogens and Toxicants in Smokers: Implications of the Findings and Potential Opportunities.

Bonorden and colleagues designed a clinical trial to test the hypothesis that daily consumption of freeze-dried watercress, a rich source of the chemopreventive agent phenethyl isothiocyanate, can enhance the detoxification of well-known tobacco and environmental carcinogens and toxicants. Initial results have validated subject compliance and a positive outcome of this study would further support the use of watercress as a whole food-based approach to cancer chemoprevention. On the basis of the design of the clinical trial and the various biological samples to be collected, we discuss potential opportunities to test future hypotheses. See related article, p. 143.

Black raspberry restores the expression of the tumor suppressor p120ctn in the oral cavity of mice treated with the carcinogen dibenzo[a,l]pyrene diol epoxide.

One of the major risk factors for head and neck squamous cell carcinoma (HNSCC) is tobacco smoke exposure, but the mechanisms that can account for disease development remain to be fully defined. Utilizing our HNSCC mouse model, we analyzed oral squamous cell carcinomas (OSCC) induced by the active metabolite of a common smoke constituent, dibenzo[a,l]pyrene diol-epoxide (DBPDE). Analyzing protein expression by either immunofluorescence or immunohistochemistry, we identified biologic processes that are dysregulated in premalignant and invasive cancer lesions induced by DBPDE. Interestingly, p120ctn expression is downregulated in both stages of the disease. In addition to decreased p120ctn expression, there was also increased proliferation (as measured by Ki67), inflammation (as measured by NFkB (p65) expression), neovascularization (as measured by CD31) and recruitment of Ly6G-positive immune cells as well as strong EGFR expression. We also examined the effect of the chemopreventive agent black raspberry (BRB) on p120ctn and EGFR protein expression in DBPDE treated mice. p120ctn, but not EGFR, protein expression increased in mice treated with BRB. Our results suggest that modulation of p120ctn may, in part, account for the mechanism by which BRB inhibits DBPDE induced OSCC in mice.

Effects of E-Cigarette Aerosols with Varying Levels of Nicotine on Biomarkers of Oxidative Stress and Inflammation in Mice.

To provide insights into the cause of e-cigarette (e-cig) associated lung injury, we examined the effects of propylene glycol (PG) and glycerol (G), two common solvent carriers used to deliver nicotine/flavor, on markers of oxidative stress and inflammation in female B6C3F1 mice which had been used successfully in tobacco smoke (TS)-induced lung carcinogenesis. Mice exposed to air and TS were used as negative and positive controls, respectively. Using LC-MS/MS, we showed that PG/G alone, in the absence of nicotine, significantly increased the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG or its tautomer 8-oxodG), a biomarker of DNA oxidative damage, in lung and plasma of mice; moreover, addition of nicotine (12 and 24 mg/mL) in e-cig liquid appears to suppress the levels of 8-oxodG. Exposure to e-cig aerosols or TS induced nonsignificant increases of plasma C-reactive protein (CRP), a biomarker of inflammation; nonetheless, the levels of fibronectin (FN), a biomarker of tissue injury, were significantly increased by e-cig aerosols or TS. Although preliminary, our data showed that exposure to e-cig aerosols induced a higher score of lung injury than did control air or TS exposure. Our results indicate that the B6C3F1 mouse model may be suitable for an in-depth examination of the impact of e-cig on lung injury associated with oxidative stress and inflammation and this study adds to the growing evidence that the use of e-cig can lead to lung damage.

Lipoxygenase catalyzed metabolites derived from docosahexaenoic acid are promising antitumor agents against breast cancer.

Docosahexaenoic acid (DHA) is known to inhibit breast cancer in the rat. Here we investigated whether DHA itself or select metabolites can account for its antitumor action. We focused on metabolites derived from the lipoxygenase (LOX) pathway since we previously showed that they were superior anti-proliferating agents compared to DHA; 4-OXO-DHA was the most potent. A lipidomics approach detected several LOX-metabolites in plasma and the mammary gland in rats fed DHA; we also identified for the first time, 4-OXO-DHA in rat plasma. In a reporter assay, 4-OXO-DHA and 4-HDHA were more effective activators of PPARÉ£ than DHA. In breast cancer cell lines, 4-OXO-DHA induced PPARÉ£ and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) but inhibited the activity of NF-κB and suppressed PI3K and mTOR signaling. Because of the structural characteristics of 4-OXO-DHA (Michael acceptor), not shared by any of the other hydroxylated-DHA, we used MS and showed that it can covalently modify the cysteine residue of NF-κB. We have also shown that the chemopreventive effect of DHA is associated with significant reduction of PGE2 levels, in both rat mammary tumors induced by MNU and non-involved mammary tissues. Collectively, our results indicate that 4-OXO-DHA is the metabolite of choice in future chemoprevention studies.

Building a Foundation for Precision Onco-Nutrition: Docosahexaenoic Acid and Breast Cancer.

In vivo evidence of heterogeneous effects of n-3 fatty acids (N3FA) on cell signaling pathways associated with the reduced growth of breast cancer has been reported and is consistent with the expectation that N3FA will not exert uniform effects on all molecular subtypes of the disease. Similarly, available evidence indicates that many metabolites of N3FA are synthesized by mammalian cells and that they exert metabolite-specific biological activities. To begin to unravel the complex relationships among molecular subtypes and effects exerted by specific N3FA metabolites on those pathways, proof-of-concept experiments were conducted using cell lines representative of common molecular subtypes of human breast cancer. N3FA differed in anticancer activity with docosahexaenoic acid (DHA) having greater anticancer activity than eicosapentaenoic acid. 4-oxo-docosahexaenoic (4-oxo-DHA), a penultimate metabolite of 5-lipoxygenase mediated DHA metabolism, induced dose-dependent inhibition of cell number accumulation with apoptosis as a primary effector mechanism. Interrogation of protein expression data using the Ingenuity Pathway Analysis (IPA) bioinformatics platform indicated that 4-oxo-DHA differentially impacted six canonical pathways and the cellular functions they regulate across common molecular subtypes of breast cancer. This included the endocannabinoid pathway for cancer inhibition that has not been previously reported. These findings provide a rationale for juxtaposing molecular subtype targeted treatment strategies with the adjuvant use of specific N3FA metabolites as an example of precision onco-nutrition (PON) for the management and control of breast cancer.

The environmental pollutant and tobacco smoke constituent dibenzo[def,p]chrysene is a co-factor for malignant progression of mouse oral papillomavirus infections.

HPV infections in the oral cavity that progress to cancer are on the increase in the USA. Model systems to study co-factors for progression of these infections are lacking as HPVs are species-restricted and cannot grow in preclinical animal models. We have recently developed a mouse papillomavirus (MmuPV1) oral mucosal infection model that provides opportunities to test, for the first time, the hypothesis that tobacco carcinogens are co-factors that can impact the progression of oral papillomas to squamous cell carcinoma (SCC). Four cohorts of mice per sex were included: (1) infected with MmuPV1 and treated orally with DMSO-saline; (2) infected with MmuPV1 and treated orally with the tobacco carcinogen, dibenzo[def,p]chrysene (DBP); (3) uninfected and treated orally with DMSO-saline, and (4) uninfected and treated orally with DBP. Oral swabs were collected monthly for subsequent assessment of viral load. Oral tissues were collected for in situ viral DNA/RNA detection, viral protein staining, and pathological assessment for hyperplasia, papillomas and SCC at study termination. We observed increased rates of SCC in oral tissue infected with MmuPV1 and treated with DBP when compared to mice treated with DBP or virus individually, each of which showed minimal disease. Virally-infected epithelium showed strong levels of viral DNA/RNA and viral protein E4/L1 staining. In contrast, areas of SCC showed reduced viral DNA staining indicative of lower viral copy per nucleus but strong RNA signals. Several host markers (p120 ctn, p53, S100A9) were also examined in the mouse oral tissues; of particular significance, p120 ctn discriminated normal un-infected epithelium from SCC or papilloma epithelium. In summary, we have confirmed that our infection model is an excellent platform to assess the impact of co-factors including tobacco carcinogens for oral PV cancerous progression. Our findings can assist in the design of novel prevention/treatment strategies for HPV positive vs. HPV negative disease.

An Integrated Approach for Preventing Oral Cavity and Oropharyngeal Cancers: Two Etiologies with Distinct and Shared Mechanisms of Carcinogenesis.

Head and neck squamous cell carcinoma (HNSCC) was the 7th most common malignancy worldwide in 2018 and despite therapeutic advances, the overall survival rate for oral squamous cell carcinoma (OSCC; ∼50%) has remained unchanged for decades. The most common types are OSCC and oropharyngeal squamous cell carcinoma (OPSCC, survival rate ∼85%). Tobacco smoking is a major risk factor of HNSCC. In the developed world, the incidence of OSCC is declining as a result of tobacco cessation programs. However, OPSCC, which is also linked to human papillomavirus (HPV) infection, is on the rise and now ranks as the most common HPV-related cancer. The current state of knowledge indicates that HPV-associated disease differs substantially from other types of HNSCC and distinct biological differences between HPV-positive and HPV-negative HNSCC have been identified. Although risk factors have been extensively discussed in the literature, there are multiple clinically relevant questions that remain unanswered and even unexplored. Moreover, existing approaches (e.g., tobacco cessation, vaccination, and chemoprevention) to manage and control this disease remain a challenge. Thus, in this review, we discuss potential future basic research that can assist in a better understanding of disease pathogenesis which may lead to novel and more effective preventive strategies for OSCC and OPSCC.

Black Raspberry Inhibits Oral Tumors in Mice Treated with the Tobacco Smoke Constituent Dibenzo(def,p)chrysene Via Genetic and Epigenetic Alterations.

We previously reported that the environmental pollutant and tobacco smoke constituent dibenzo[def,p]chrysene (DBP) induced DNA damage, altered DNA methylation and induced oral squamous cell carcinoma (OSCC) in mice. In the present study, we showed that 5% dietary black raspberry (BRB) significantly reduced (P < 0.05) the levels of DBP-DNA adducts in the mouse oral cavity with comparable effect to those of its constitutes. Thus, only BRB was selected to examine if aberrant DNA methylation induced by DBP can be altered by BRB. Using comparative genome-wide DNA methylation analysis, we identified 479 hypermethylated and 481 hypomethylated sites (q < 0.01, methylation difference >25%) between the oral tissues of mice treated with DBP and fed control diet or diet containing BRB. Among the 30 differential methylated sites (DMS) induced by DBP, we found DMS mapped to Fgf3, Qrich2, Rmdn2, and Cbarp were hypermethylated by BRB whereas hypomethylated by DBP at either the exact position or proximal sites; DMS mapped to Vamp3, Ppp1rB1, Pkm, and Zfp316 were hypomethylated by BRB but hypermethylated by DBP at proximal sites. In addition to Fgf3, 2 DMS mapped to Fgf4 and Fgf13 were hypermethylated by BRB; these fibroblast growth factors are involved in regulation of the epithelial-mesenchymal transition (EMT) pathway as identified by IPA. Moreover, BRB significantly reduced (P < 0.05) the tumor incidence from 70% to 46.7%. Taken together, the inhibitory effects of BRB on DNA damage combined with its effects on epigenetic alterations may account for BRB inhibition of oral tumorigenesis induced by DBP. SIGNIFICANCE: We provided mechanistic insights that can account for the inhibition of oral tumors by BRB, which could serve as the framework for future chemopreventive trials for addicted smokers as well as non- or former smokers who are exposed to environmental carcinogens.

Customized Prevention Trials Could Resolve the Controversy of the Effects of Omega-3 Fatty Acids on Cancer.

Literature data revealed that the benefits of consuming omega-3 fatty acid (n-3FA) supplements such as fish oil with the goal of reducing the incidence of chronic diseases such as cardiovascular disease and cancer remain controversial. The purpose of this commentary is to discuss factors that may account for the inconsistency of results across different studies. Critical review of the published data, including our own preclinical and clinical studies, strongly suggests that customized clinical prevention trials are needed to resolve the above-mentioned controversy. Specifically, in order to develop a personalized cancer prevention strategy, more attention should be given to multiple factors including the dose of the n-3FA, the specific placebo used as a comparator, duration of administration, type of intervention (primary vs secondary prevention trial), specific compound (DHA vs EPA vs their metabolites), the ratio of n-3FA:n-6FA and the target population tested (high vs average risk).

Effects of the Tobacco Carcinogens N'-Nitrosonornicotine and Dibenzo[a,l]pyrene Individually and in Combination on DNA Damage in Human Oral Leukoplakia and on Mutagenicity and Mutation Profiles in lacI Mouse Tongue.

In previous studies, we showed that the topical application of dibenzo[a,l]pyrene (DB[a,l]P), also known as dibenzo[def,p]chrysene, to the oral cavity of mice induced oral squamous cell carcinoma. We also showed that dA and dG adducts likely account for most of the mutagenic activity of DB[a,l]P in the oral tissues in vivo. Here we report for the first time that the oral treatment of lacI mice with a combination of tobacco smoke carcinogens, DB[a,l]P and N'-nitrosonornicotine (NNN), induces a higher fraction of mutations than expected from a simple sum of their induced individual mutation fractions, and a change in the mutational profile compared with that expected from the sum of the individual agents. The mutational profile of the combination of agents resembled that of the P53 gene in human head and neck cancers more than that of either of the individual agents, in that the percentage of the major class of mutations (GC > AT transitions) is similar to that seen in the P53 gene. A preliminary study was performed to understand the origin of the unexpected mutagenesis observations by measuring specific DNA adducts produced by both NNN and DB[a,l]P in human oral leukoplakia cells. No significant differences in the expected and observed major adduct levels from either agent were observed between individual or combined treatments, suggesting that additional adducts are important in mutagenesis induced by the mixture. Taken together, the above observations support the use of this animal model not only to investigate tobacco smoke-induced oral cancer but also to study chemoprevention.