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.

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.

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.

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.

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.

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).

Omega-3 Fatty Acids Responsive Proteins and Reduction in Breast Density in Obese Postmenopausal Women.

We reported that breast density (BD) was inversely correlated with the plasma level of DHA in postmenopausal obese, but not in nonobese, women given Lovaza (n-3FA). To identify protein biomarkers for the possible differential effect of n-3FA on BD between obese and nonobese women, an iTRAQ method was performed to analyze plasma from obese and lean women at each time point (baseline, 12 and 24-months, n = 10 per group); 173 proteins with >95% confidence (Unuses Score >1.3 and local false discovery rate estimation <5%) were identified. Comparative analysis between various groups identified several differentially expressed proteins (hemopexin precursor, vitamin D binding protein isoform 1 precursor [VDBP], fibronectin isoform 10 precursor [FN], and α-2 macroglobulin precursor [A2M]). Western blot analysis was performed to verify the differential expression of proteins in the iTRAQ study, and those found to be altered in a tumor protective fashion by an n-3FA rich diet in our previous preclinical study; gelsolin, VDBP, and FN were altered by n-3FA in a manner consistent with reduction in inflammation in obese women. To test the impact of our findings on breast cancer risk reduction by n-3FA, a posthoc analysis revealed that n-3FA administration reduced BD selectively in obese postmenopausal women.

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.

Comparative Tumorigenicity and DNA Damage Induced by Dibenzo[ def,p]chrysene and Its Metabolites in the Mouse Ovary.

Ovarian cancer ranked second in incidence among gynecologic cancers, but it causes more deaths than any other gynecologic cancer; at present there is no curative treatment beyond surgery. Animal models that employ carcinogens found in the human environment can provide a realistic platform to understand the mechanistic basis for disease development and to design rational chemopreventive/therapeutic strategies. We and others have shown that the administration of the environmental pollutant and tobacco smoke constituent dibenzo[ def,p]chrysene (DBP) to mice by several routes of exposure can induce tumors in multiple sites including the ovary. In the present study we compared, for the first time, the tumorigenicity and DNA damage induced by DBP and its metabolites DBP-dihydrodiol (DBPDHD) and DBP-dihydrodiol epoxide (DBPDE) in the mouse ovary. Compounds were dissolved in dimethyl sulfoxide (DMSO) as the vehicle and administered by topical application into the mouse oral cavity three times per week for 38 weeks. No tumors were observed in mice treated with DMSO. At equal dose (24 nmol/30 µL DMSO), the incidence of ovarian tumors induced by DBPDHD was higher (60.7%), although not significantly, than that induced by DBP (44.8%). Similarly the levels of DNA damage induced by DBPDHD in the ovary were higher than those observed with DBP. We did not observe any histological abnormality in the ovary of mice treated with DBPDE, which is consistent with lack of DNA damage. Our results suggested that both DBP and DBPDHD can be metabolized in the mouse ovary leading to the formation of DBPDE that can damage DNA, which is a prerequisite step in the initiation stage of carcinogenesis.

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

Worldwide, cancers of the oral cavity and pharynx comprise the sixth 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 7,12-dimethylbenz[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 humans could provide a realistic platform. To achieve this goal, we introduced a novel nonsurgical 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-11,12,13,14-tetrahydrodibenzo[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, 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 the 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.

Effects of Black Raspberry Extract and Berry Compounds on Repair of DNA Damage and Mutagenesis Induced by Chemical and Physical Agents in Human Oral Leukoplakia and Rat Oral Fibroblasts.

Black raspberries (BRB) have been shown to inhibit carcinogenesis in a number of systems, with most studies focusing on progression. Previously we reported that an anthocyanin-enriched black raspberry extract (BE) enhanced repair of dibenzo-[a,l]-pyrene dihydrodiol (DBP-diol)-induced DNA adducts and inhibited DBP-diol and DBP-diolepoxide (DBPDE)-induced mutagenesis in a lacI rat oral fibroblast cell line, suggesting a role for BRB in the inhibition of initiation of carcinogenesis. Here we extend this work to protection by BE against DNA adduct formation induced by dibenzo-[a,l]-pyrene (DBP) in a human oral leukoplakia cell line (MSK) and to a second carcinogen, UV light. Treatment of MSK cells with DBP and DBPDE led to a dose-dependent increase in DBP-DNA adducts. Treatment of MSK cells with BE after addition of DBP reduced levels of adducts relative to cells treated with DBP alone, and treatment of rat oral fibroblasts with BE after addition of DBPDE inhibited mutagenesis. These observations showed that BE affected repair of DNA adducts and not metabolism of DBP. As a proof of principle we also tested aglycones of two anthocyanins commonly found in berries, delphinidin chloride and pelargonidin chloride. Delphinidin chloride reduced DBP-DNA adduct levels in MSK cells, while PGA did not. These results suggested that certain anthocyanins can enhance repair of bulky DNA adducts. As DBP and its metabolites induced formation of bulky DNA adducts, we investigated the effects of BE on genotoxic effects of a second carcinogen that induces bulky DNA damage, UV light. UV irradiation produced a dose-dependent increase in cyclobutanepyrimidine dimer levels in MSK cells, and post-UV treatment with BE resulted in lower cyclobutanepyrimidine dimer levels. Post-UV treatment of the rat lacI cells with BE reduced UV-induced mutagenesis. Taken together, the results demonstrate that BE extract reduces bulky DNA damage and mutagenesis and support a role for BRB in the inhibition of initiation of carcinogenesis.

Effects of chronic alcohol consumption on DNA damage and immune regulation induced by the environmental pollutant dibenzo[a,l]pyrene in oral tissues of mice.

Previously, we showed that oral application of the environmental pollutant dibenzo[a,l]pyrene (DB[a,l]P) induces oral tumors in mice. Thus, in the present investigation we examined the effect of alcohol on DB[a,l]P-induced DNA damage and immune regulation; we showed that alcohol (6.4% v/v in the diet, 35% of Calories) significantly enhanced the levels of (-)-anti-trans-DB[a,l]P-dA while decreased the levels of GSH in the mouse oral tissues. Analysis of RNA expression revealed that DB[a,l]P alone upregulates inflammatory genes while alcohol suppresses several markers of immune surveillance. Collectively, these results suggest that alcohol may enhance oral carcinogenesis induced by DB[a,l]P.

Docosahexaenoic Acid in Combination with Dietary Energy Restriction for Reducing the Risk of Obesity Related Breast Cancer.

There is strong evidence that obesity poses a significant risk factor for postmenopausal breast cancer. There are multiple mechanisms by which obesity can predispose to breast cancer, prominent among which is the creation of a pro-inflammatory milieu systemically in the visceral and subcutaneous tissue, as well as locally in the breast. Although dietary intervention studies have shown in general a favorable effect on biomarkers of breast cancer risk, it is still unclear whether losing excess weight will lower the risk. In this manuscript, we will review the evidence that omega-3 fatty acids, and among them docosahexaenoic acid (DHA) in particular, may reduce the risk of obesity related breast cancer primarily because of their pleotropic effects which target many of the systemic and local oncogenic pathways activated by excess weight. We will also review the evidence indicating that intentional weight loss (IWL) induced by dietary energy restriction (DER) will augment the tumor protective effect of DHA because of its complementary mechanisms of action and its ability to reverse the obesity-induced alterations in fatty acid metabolism predisposing to carcinogenesis. We believe that the combination of DER and DHA is a promising safe and effective intervention for reducing obesity-related breast cancer risk which needs to be validated in appropriately designed prospective, randomized clinical trials.

Proteomic profiling of human plasma identifies apolipoprotein E as being associated with smoking and a marker for squamous metaplasia of the lung.

Biomarkers to identify subjects at high-risk for developing lung cancer will revolutionize the disease outlook. Most biomarker studies have focused on patients already diagnosed with lung cancer and in most cases the disease is often advanced and incurable. The objective of this study was to use proteomics to identify a plasma biomarker for early detection of lung lesions that may subsequently be the harbinger for cancer. Plasma samples were obtained from subjects without lung cancer grouped as never, current, or ex-smokers. An iTRAQ-based proteomic analysis was performed on these pooled plasma samples. We identified 31 proteins differentially abundant in current smokers or ex-smokers relative to never smokers. Western blot and ELISA analyses confirmed the iTRAQ results that demonstrated an increase of apolipoprotein E (APOE) in current smokers as compared to both never and ex-smokers. There was a strong and significant correlation of the plasma APOE levels with development of premalignant squamous metaplasia. Additionally, we also showed that higher tissue levels of APOE are seen with squamous metaplasia, supporting a direct relationship. Our analysis reveals that elevated plasma APOE is associated with smoking, and APOE is a novel predictive protein biomarker for early morphological changes of squamous metaplasia in the lung.

Tissue Distribution, Excretion and Pharmacokinetics of the Environmental Pollutant Dibenzo[def,p]chrysene in Mice.

Dibenzo[def,p]chrysene (DBP), a representative example of the class of polycyclic aromatic hydrocarbon (PAH), is known to induce tumors in multiple organ sites including the ovary, lung, mammary glands, and oral cavity in rodents. The goal of this study was to test the hypothesis that the levels of DBP and its metabolites that reach and retain the levels for an extended time in the target organs as well as the capacity of these organs to metabolize this carcinogen to active metabolites that can damage DNA may account for its tissue selective tumorigenicity. Therefore, we used the radiolabeled [(3)H] DBP to accurately assess the tissue distribution, excretion, and pharmacokinetics of this carcinogen. We also compared the levels of DBPDE-DNA adducts in a select target organ (ovary) and nontarget organs (kidney and liver) in mice treated orally with DBP. Our results showed that after 1 week, 91.40 ± 7.23% of the radioactivity was recovered in the feces; the corresponding value excreted in the urine was less than 2% after 1 week. After 24 h, the stomach had the highest radioactivity followed by the intestine and the liver; however, after 1 week, levels of the radioactivity in these organs were the lowest among tissues examined including the ovary and liver; the pharmacokinetic analysis of DBP was conducted using a one compartment open model. The level of (-)-anti-trans-DBPDE-dA in the ovaries (8.91 ± 0.08 adducts/10(7) dA) was significantly higher (p < 0.01) than the levels of adducts in kidneys (0.69 ± 0.09 adducts/10(7) dA) and livers (0.63 ± 0.11 adducts/10(7) dA). Collectively, the results of the tissue distribution and pharmacokinetic analysis may not fully support our hypothesis, but the capacity of the target organs vs nontarget organs to metabolize DBP to active intermediates that can damage DNA may account for its tissue selective tumorigenicity.

Fish Oil Enhances T Cell Function and Tumor Infiltration and Is Correlated With a Cancer Prevention Effect in HER-2/neu But Not PyMT Transgenic Mice.

Few studies have explored the effects of omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation on immune modulation in murine models of mammary carcinogenesis. HER-2/neu and PyMT mice were randomized to 2 dietary interventions: AIN-93G-based diet with 1) 11% of diet (per gram weight) as corn oil (CO) or 2) 10% of diet as menhaden fish oil plus 1% of diet as corn oil (FO). FO significantly reduced the incidence and multiplicity of tumors (P < 0.001) in HER-2/neu, but not PyMT mice. FO-fed mice had significantly larger splenocyte counts than CO-fed mice in both the HER-2/neu and PyMT models; and in both models this was comprised of an increase in most cell types, including Gr-1(+)/CD11b(+) cells. T cells from FO-fed HER-2/neu mice produced significantly more interleukin-2 (P = 0.004) and interferon-γ (P = 0.012) in response to in vitro stimulation with anti-CD3 (0.5 µg/ml). Lastly, FO-fed HER-2/neu mice had significantly more tumor immune infiltrates than CO-fed mice, including NK1.1(+), F4/80(+), and Gr-1(+)/CD11b(+) cells (P ≤ 0.05). Greater Th1 cytokine production and significantly more tumor immune infiltrates in FO-fed Her2/neu mice may account for the cancer prevention effect of fish oil in this model.

Influence of omega-3 fatty acids on Tamoxifen-induced suppression of rat mammary carcinogenesis.

We report here a detailed time course study of the individual and combined chemopreventive effects of Tamoxifen (Tam) and a high fish oil (FO) diet on multiple histologic parameters of mammary carcinogenesis. Groups of female Sprague-Dawley rats were injected ip with 1-methyl-1-nitrosourea at 50 days of age and assigned to either a control diet (20% corn oil [CO]) or a FO-rich diet (10% FO + 10% CO) in the presence and absence of Tam in the diet (0.6 ppm). Rats were sacrificed at weeks 4 (before palpable tumors), 8 and 12 (when ∼90% of control rats had palpable tumors). Our results demonstrate a major effect of Tam in inhibiting the development of early preneoplastic lesions. FO, while having a marginal protective effect of it own, enhanced the antitumor action of Tam on all histologic parameters of carcinogenesis, although the effects of the combination were not statistically different from those of Tam alone. The combination of FO and Tam was the only intervention that induced regression of established preneoplastic lesions. We also found that in contrast to plasma, only target tissue n-3 fatty acids (FAs) levels correlated with select tissue biomarkers of carcinogenesis whose expression was altered in a manner predictive of a protective effect. Our results demonstrating the potentially superior chemopreventive efficacy of Tam and n-3FA have important translational implications. Our data also emphasize the importance of local factors in affecting target tissue levels and biologic effects of n-3FA.

Differential impact of body mass index on absolute and percent breast density: implications regarding their use as breast cancer risk biomarkers.

Percent breast density (PBD), a commonly used biomarker of breast cancer risk (BCR), is confounded by the influence of non-dense breast tissue on its measurement and factors, such as BMI, which have an impact on non-dense tissue. Consequently, BMI, a potent BCR factor, is, paradoxically, negatively correlated with PBD. We propose that absolute breast density (ABD) is a more accurate biomarker of BCR. We used a volumetric method to compare the correlation between PBD and ABD with baseline demographics and dietary and physical activity variables in a group of 169 postmenopausal women enrolled in a clinical trial prior to any intervention. As expected, a strong negative correlation between PBD and BMI was observed (Rho = -0.5, p < 5e(-12)). In contrast, we observed a strong, previously not well established, positive correlation of BMI with ABD (Rho = 0.41, p < 2.5e(-8)), which supports the use of ABD as a more accurate indicator of BCR. Correction of PBD by BMI did not frequently provide the same information as ABD. In addition, because of the strong influence of BMI on ABD, many correlations between dietary variables and ABD did not emerge, until adjustment was made for BMI. ABD corrected by BMI should be the gold standard BD measurement. These findings identify the optimal measurement of BD when testing the influence of an intervention on BD as a biomarker of BCR.

Simultaneous detection of deoxyadenosine and deoxyguanosine adducts in the tongue and other oral tissues of mice treated with Dibenzo[a,l]pyrene.

We were the first to demonstrate that direct application of the environmental pollutant and tobacco smoke constituent dibenzo[a,l]pyrene (DB[a,l]P) into the oral cavity of mice induced squamous cell carcinoma (SCC) in oral tissues but not in the tongue; however, the mechanisms that can account for the varied carcinogenicity remain to be determined. Furthermore, we also showed that not only dA adducts, but also dG adducts can account for the mutagenic activity of DB[a,l]P in the oral tissues in vivo. In this study, we initially focused on DB[a,l]P-induced genotoxic effects in both oral and tongue tissues. Therefore, to fully assess the contribution of these DNA adducts in the initiation stage of carcinogenesis induced by DB[a,l]P, an LC-MS/MS method to simultaneously detect and quantify DB[a,l]PDE-dG and -dA adducts was developed. Mice were orally administered with DB[a,l]P (24 nmole, 3 times per week for 5 weeks) or its fjord region diol epoxide, (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE, 12 nmole, single application); animals were sacrificed at 2, 7, 14, and 28 days after the last dose of carcinogen administration. Oral and tongue tissues were obtained and DNA were isolated followed by enzymatic hydrolysis. Following the development of an isotope dilution LC-MS/MS method, we successfully detected (-)-anti-cis- and (-)-anti-trans-DB[a,l]PDE-N(2)-dG, as well as (-)-anti-cis- and (-)-anti-trans-DB[a,l]PDE-N(6)-dA in oral and tongue tissues of mice treated with DB[a,l]P. Levels of (-)-anti-trans-DB[a,l]PDE-N(6)-dA were ≥2 folds higher than (-)-anti-cis-DB[a,l]PDE-N(6)-dA adduct and those of dG adducts in the oral tissues and tongue at all time points selected after the cessation of DB[a,l]P treatment. Levels of dG adducts were comparable in both tissues. Collectively, our results support that DB[a,l]P is predominantly metabolized to (-)-anti-DB[a,l]PDE, and the levels and persistence of (-)-anti-trans-DB[a,l]PDE-N(6)-dA may, in part, explain the carcinogenicity of DB[a,l]P in the oral tissues but not in the tongue.

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.