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.

Household plastic waste habits and attitudes: A pilot study in the city of Valencia.

Bearing in mind that only 42% of plastic packaging post-consumer waste is recycled in Europe, the European Directive 2018/852 established the key target of a 55% plastic packaging waste recycling rate by 2030. For this reason, PlastiCircle, funded by the European Union's Horizon 2020 research and innovation program project, aims to foster the recycling of packaging, improve all stages of the waste collection, and promote responsible consumption. Three European cities have been selected as locations for pilot implementation: Valencia (Spain), Utrecht (The Netherlands) and Alba Iulia (Romania). The main objective of the present study has been to evaluate the participants' opinion and attitudes on plastic recycling. This paper presents the results from the district of San Marcelino in the city of Valencia, the first PlastiCircle pilot to face the challenges of encouraging households to participate more in plastic waste sorting and recycling.

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.

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.

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.

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.

Mechanisms of oral carcinogenesis induced by dibenzo[a,l]pyrene: an environmental pollutant and a tobacco smoke constituent.

We previously reported that dibenzo[a,l]pyrene (DB[a,l]P), the most potent known environmental carcinogen among polycyclic aromatic hydrocarbons (PAH) congeners, is carcinogenic in the oral tissues of mice. We have now developed a new mouse model which employs the oral application of the fjord region diol epoxide, (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE), a metabolite of the tobacco smoke constituent DB[a,l]P, and we show its specific induction of oral squamous cell carcinoma (OSCC) in both tongue and other oral tissues. Groups of B6C3F1 mice (20/group) received 6 or 3 nmol of (±)-anti-DB[a,l]PDE administered into the oral cavity; 3 times per week for 38 weeks. Additional groups received the vehicle alone or were left untreated. Mice were sacrificed 42 weeks after the first carcinogen administration. The high dose induced 74 and 100% OSCC in the tongue and other oral tissues, respectively; the corresponding values at the lower dose were 45 and 89%. Using immunohistochemistry, we showed that DB[a,l]PDE resulted in overexpression of p53 and COX-2 proteins in malignant tissues when compared to normal oral tissues and tongues. Consistent with the carcinogenicity, we demonstrated powerful mutagenicity in cII gene in B6C3F1 (Big Blue) mouse tongue. The mutational profile in lacI reporter gene is similar to those detected in human head and neck cancer, and p53 mutations were observed in mouse oral tumor tissues. Taken together, we conclude that the formation of diol epoxides plays a major role among the mechanisms by which DB[a,l]P exerts its oral mutagenicity and tumorigenicity.

Changes in proteomic profiles in different prostate lobes of male rats throughout growth and development and aging stages of the life span.

BACKGROUND: Aging-related changes in important cellular pathways in the prostate may promote a permissive environment for an increased risk for prostatic disease development such as prostate cancer. Our objectives were to examine for such changes, by systematically determining the effects of growth and development and aging on proteomic profiles in different lobes of the rat prostate. METHODS: Prostate lobes (dorsolateral lobe, DL and ventral lobe, VL) were obtained from male Fisher rats of various ages representing young (4 months), mature (12 months), old (18 months), and very old (24 months). Differentially expressed proteins between age groups in each lobe were identified using a proteomic approach, isobaric Tags for Relative and Absolute Quantitation (iTRAQ). Select changes in the DL and VL were verified by immunoblot analysis. RESULTS: iTRAQ identified 317 proteins with high confidence. iTRAQ discovered 12 and 6 proteins significantly modulated in response to growth and development in the DL and VL, respectively, and 42 and 29 proteins significantly modulated in response to aging in the DL and VL, respectively. Proteins modulated during growth and development in the DL and VL are involved in a variety of biological processes including cell communication and development, whereas proteins modulated during aging were predominantly related to antioxidant activity and immunity. Immunoblot analysis verified age-related changes for α-1 antitrypsin, annexin A1, hypoxia up-regulated protein 1, and 78 kDa glucose-regulated protein. CONCLUSIONS: Aging results in changes in numerous prostatic proteins and pathways which are mainly linked to inflammation and may lead to prostatic disease development.

Therapeutic efficacy of FTY720 in a rat model of NK-cell leukemia.

NK-cell leukemia is a clonal expansion of NK cells. The illness can occur in an aggressive or chronic form. We studied cell lines from human and rat NK-cell leukemias (aggressive NK-cell leukemia) as well as samples from patients with chronic NK-cell leukemia to investigate pathogenic mechanisms. Here we report that Mcl-1 was overexpressed in leukemic NK cells and that knockdown of Mcl-1 induced apoptosis in these leukemic cells. In vitro treatment of human and rat NK leukemia cells with FTY720 led to caspase-dependent apoptosis and decreased Mcl-1 expression in a time- and-dose-dependent manner. These biologic effects could be inhibited by blockade of reactive oxygen species generation and the lysosomal degradation pathway. Lipidomic analyses after FTY720 treatment demonstrated elevated levels of sphingosine, which mediated apoptosis of leukemic NK cells in vitro. Importantly, systemic administration of FTY720 induced complete remission in the syngeneic Fischer rat model of NK-cell leukemia. Therapeutic efficacy was associated with decreased expression of Mcl-1 in vivo. These data demonstrate that therapeutic benefit of FTY720 may result from both altered sphingolipid metabolism as well as enhanced degradation of a key component of survival signaling.

First Generation of Antioxidant Precursors for Bioisosteric Se-NSAIDs: Design, Synthesis, and In Vitro and In Vivo Anticancer Evaluation.

The introduction of selenium (Se) into organic scaffolds has been demonstrated to be a promising framework in the field of medicinal chemistry. A novel design of nonsteroidal anti-inflammatory drug (NSAID) derivatives based on a bioisosteric replacement via the incorporation of Se as diacyl diselenide is reported. The antioxidant activity was assessed using the DPPH radical scavenging assay. The new Se-NSAID derivatives bearing this unique combination showed antioxidant activity in a time- and dose-dependent manner, and also displayed different antiproliferative profiles in a panel of eight cancer cell lines as determined by the MTT assay. Ibuprofen derivative 5 was not only the most antioxidant agent, but also selectively induced toxicity in all the cancer cell lines tested (IC50 < 10 µM) while sparing nonmalignant cells, and induced apoptosis partially without enhancing the caspase 3/7 activity. Furthermore, NSAID derivative 5 significantly suppressed tumor growth in a subcutaneous colon cancer xenograft mouse model (10 mg/kg, TGI = 72%, and T/C = 38%) without exhibiting any apparent toxicity. To our knowledge, this work constitutes the first report on in vitro and in vivo anticancer activity of an unprecedented Se-NSAID hybrid derivative and its rational use for developing precursors for bioisosteric selenocompounds with appealing therapeutic applications.

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.

Mutagenesis and carcinogenesis induced by dibenzo[a,l]pyrene in the mouse oral cavity: a potential new model for oral cancer.

Cancer of the oral cavity is a serious disease, affecting about 30,000 individuals in US annually. There are several animal models of oral cancer, but each has certain disadvantages. As a new model, we investigated whether topical application of the tobacco smoke carcinogen, dibenzo[a,l]pyrene (DB[a,l]P) is mutagenic and carcinogenic in the oral cavity of the B6C3F1 lacI and B6C3F1 mouse, respectively. B6C3F1 lacI mice received DB[a,l]P (0, 3, 6, 12 nmol) 3× per week. B6C3F1 mice received the same doses and also 24 nmol. At 38 weeks mutagenesis was measured in oral tissues in lacI mice. For the high dose group, the mutant fraction (MF) in upper mucosa and tongue increased about twofold relative to that in vehicle-alone. The increases were statistically significant. The mutational profile in the DB[a,l]P-induced mutants was compared with that induced by benzo[a]pyrene (BaP) in oral tissue. BaP is mutagenic in many tissues when administered by gavage. The mutational profile for DB[a,l]P was more similar to that reported for p53 mutations in head and neck cancers than was that of BaP. At 47 weeks, oral squamous cell carcinomas (OSCC) were found in 31% of the high-dose B6C3F1 group. Elevations of p53 and COX-2 protein were observed in tumor and dysplastic tissue. As DB[a,l]P induces mutations and tumors in the oral cavity, and has a mutational profile in oral tissue similar to that found in p53 in human OSCC, the treatment protocol described here may represent a new and relevant model for cancer of the oral cavity.

Targeting of survivin by nanoliposomal ceramide induces complete remission in a rat model of NK-LGL leukemia.

The natural killer (NK) type of aggressive large granular lymphocytic (LGL) leukemia is a fatal illness that pursues a rapid clinical course. There are no effective therapies for this illness, and pathogenetic mechanisms remain undefined. Here we report that the survivin was highly expressed in both aggressive and chronic leukemic NK cells but not in normal NK cells. In vitro treatment of human and rat NK-LGL leukemia cells with cell-permeable, short-chain C6-ceramide (C6) in nanoliposomal formulation led to caspase-dependent apoptosis and diminished survivin protein expression, in a time- and dose-dependent manner. Importantly, systemic intravenous delivery of nanoliposomal ceramide induced complete remission in the syngeneic Fischer F344 rat model of aggressive NK-LGL leukemia. Therapeutic efficacy was associated with decreased expression of survivin in vivo. These data suggest that in vivo targeting of survivin through delivery of nanoliposomal C6-ceramide may be a promising therapeutic approach for a fatal leukemia.

Induction of ovarian cancer and DNA adducts by Dibenzo[a,l]pyrene in the mouse.

Tobacco smoking is an etiological factor of ovarian cacner; however, the mechanisms remain largely undefined. Therefore, as an initial investigation, we examined the carcinogenicity and DNA adducts formation in the ovary of mice treated with DB[a,l]P, a tobacco smoke constituent and environmental pollutant. Ovarian tumors in B6C3F1 mice were induced by direct application of DB[a,l]P (24, 12, 6, and 3 nmol/mouse, three times a week for 38 weeks) into the oral cavity of mice. At 6 nmol, DB[a,l]P induced the highest total ovarian tumor incidence (79%), but the incidence of malignancy was only 15%. However, at the dose of 12 nmol, the total ovarian tumor incidence was 75%, and the incidence of malignancy was 65%. In addition to ovarian tumors, at the dose of 24 nmol, DB[a,l]P induced lesions in sites distal from the ovaries including the skin, mammary, lung, and oral tissues, which were rare at doses lower than 24 nmol. Another bioassay was conducted to detect and quantify DNA adducts induced by DB[a,l]P (24 nmol, three times a week for 5 weeks) in the ovary at 48 h and 1, 2, and 4 weeks after the last administration of DB[a,l]P. DNA was isolated, and the dibenzo[a,l]pyrene-11,12-dihydrodiol-13,14-epoxide (DB[a,l]PDE)-DNA adducts were analyzed by a LC-MS/MS method. DB[a,l]P resulted in the formation of (-)-anti-cis-DB[a,l]PDE-dA and (-)-anti-trans-DB[a,l]PDE-dA adducts, which were 0.8 and 1.6 fmol/10(6) dA, respectively, in ovaries of mice within 48 h, and the level of adducts decreased over a week. Our results indicated that DB[a,l]P can be metabolized to form (-)-anti-DB[a,l]PDE; the latter may, in part, account for DB[a,l]P-induced ovarian cancer. This animal model should assist to better understand the mechanisms, account for the induction of ovarian cancer by tobacco carcinogens, and facilitate the development of chemopreventive agents against ovarian cancer.

Fish oil alters tamoxifen-modulated expression of mRNAs that encode genes related to differentiation, proliferation, metastasis, and immune response in rat mammary tumors.

We have previously shown that a fish oil (FO)-rich diet increased the chemopreventive efficacy of tamoxifen (Tam) against N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinogenesis. Herein, we provide evidence that Tam treatment modifies gene expression of mammary tumors depending upon the type of dietary fat fed to the animals. Rats initiated with MNU and treated with Tam were fed a diet rich in corn oil or FO. After 8 wk, cribriform tumors were collected and gene expression analysis was performed. Increased RNA expression of genes such as SerpinB10, Wisp2, and Apod in tumors from FO-treated rats is indicative of highly differentiated tumors. Decreased expression of H19 and Igf2 mRNA in Tam-treated groups, and Gamma Synuclein mRNA in the FO + Tam group may be related to tumor growth impairment and lower metastatic capacity. Change in the expression of genes associated with immunity in animals in the FO + Tam group may suggest a shift in the immune response. These data show that, although Tam modulates the expression of genes leading to tumor growth impairment, further modulations of genes are influenced by FO. FO modulation of Tam changes in gene expression accounts for its enhancing chemopreventive effect against MNU-induced mammary carcinogenesis. Supplemental materials are available for this article. Go to the publisher's online edition of Nutrition and Cancer to view the supplemental file.

Identification and quantification of DNA adducts in the oral tissues of mice treated with the environmental carcinogen dibenzo[a,l]pyrene by HPLC-MS/MS.

Tobacco smoking is one of the leading causes for oral cancer. Dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke constituent, is the most carcinogenic polycyclic aromatic hydrocarbon (PAH) tested to date in several animal models (target organs: skin, lung, ovary, and mammary tissues). We have recently demonstrated that DB[a,l]P is also capable of inducing oral cancer in mice; however, its metabolic activation to the ultimate genotoxic metabolite dibenzo[a,l]pyrene-11,12-dihydrodiol-13,14-epoxide (DB[a,l]PDE) in mouse oral cavity has not been examined. Here we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect and quantify (±)-anti-DB[a,l]PDE-dA adducts in oral tissues of mice treated with DB[a,l]P. [(15)N(5)]-(±)-anti-DB[a,l]PDE-N(6)-dA adducts were synthesized as internal standards. The stereoisomeric adducts were characterized by MS, NMR, and CD analysis. The detection limit of the method is 8 fmol with 100 µg of digested DNA as the matrix. Two adducts were detected and identified as (-)-anti-cis and (-)-anti-trans-DB[a,l]PDE-dA in the oral tissues of mice following the direct application of DB[a,l]P (240 nmol per day, for 2 days) into the oral cavity, indicating that DB[a,l]P is predominantly metabolized into (-)-anti-DB[a,l]PDE in this target organ. We also compared the formation and removal of adducts as a function of time, following the direct application of DB[a,l]P (24 nmol, 3 times per week for 5 weeks) into the oral cavity of mice. Adducts were quantified at 48 h, 1, 2, and 4 weeks after the last dose. Maximal levels of adducts occurred at 48 h, followed by a gradual decrease. The levels (fmol/µg DNA) of (-)-anti-trans adducts (4.03 ± 0.27 to 1.77 ± 0.25) are significantly higher than (-)-anti-cis-DB[a,l]PDE-dA adduct (1.63 ± 0.42 to 0.72 ± 0.04) at each time point (p < 0.005). The results presented here indicate that the formation and persistence of (-)-anti-DB[a,l]PDE-dA adducts may, in part, contribute to the initiation of DB[a,l]P-induced oral carcinogenesis.

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.