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.

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.

Effects of Black Raspberry on Dibenzo[a,l]Pyrene Diol Epoxide Induced DNA Adducts, Mutagenesis, and Tumorigenesis in the Mouse Oral Cavity.

We previously showed that metabolic activation of the environmental and tobacco smoke constituent dibenzo[a,l]pyrene (DB[a,l]P) to its active fjord region diol epoxide (DB[a,l]PDE) is required to induce DNA damage, mutagenesis, and squamous cell carcinoma (SCC) in the mouse oral cavity. In contrast to procarcinogens, which were employed previously to induce SCC, DB[a,l]PDE does not require metabolic activation to exert its biological effects, and thus, this study was initiated to examine, for the first time, whether black raspberry powder (BRB) inhibits postmetabolic processes, such as DNA damage, mutagenesis, and tumorigenesis. Prior to long-term chemoprevention studies, we initially examined the effect of BRB (5% added to AIN-93M diet) on DNA damage in B6C3F1 mice using LC/MS-MS and on mutagenesis in the lacI gene in the mouse oral cavity. We showed that BRB inhibited DB[a,l]PDE-induced DNA damage (P < 0.05) and mutagenesis (P = 0.053) in the oral cavity. Tumor incidence in the oral cavity (oral mucosa and tongue) of mice fed diet containing 5% BRB was significantly (P < 0.05) reduced from 93% to 66%. Specifically, the incidence of benign tumor was significantly (P < 0.001) reduced from 90% to 31% (62% to 28% in the oral cavity and 28% to 2% in the tongue), a nonsignificant reduction of malignant tumors from 52% to 45%. Our preclinical findings demonstrate for the first time that the chemopreventive efficacy of BRB can be extended to direct-acting carcinogens that do not require phase I enzymes and is not just limited to procarcinogens. Cancer Prev Res; 11(3); 157-64. ©2017 AACR.

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 Black Raspberry Extract and Protocatechuic Acid on Carcinogen-DNA Adducts and Mutagenesis, and Oxidative Stress in Rat and Human Oral Cells.

Effects of black raspberry (BRB) extract and protocatechuic acid (PCA) on DNA adduct formation and mutagenesis induced by metabolites of dibenzo[a,l]pyrene (DBP) were investigated in rat oral fibroblasts. The DBP metabolites, (±)-anti-11,12-dihydroxy-11,12,-dihydrodibenzo[a,l]pyrene (DBP-diol) and 11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DBPDE) induced dose-dependent DNA adducts and mutations. DBPDE was considerably more potent, whereas the parent compound had no significant effect. Treatment with BRB extract (BRBE) and PCA resulted in reduced DBP-derived DNA adduct levels and reduced mutagenesis induced by DBP-diol, but only BRBE was similarly effective against (DBPDE). BRBE did not directly inactivate DBPDE, but rather induced a cellular response-enhanced DNA repair. When BRBE was added to cells 1 day after the DBP-diol, the BRBE greatly enhanced removal of DBP-derived DNA adducts. As oxidative stress can contribute to several stages of carcinogenesis, BRBE and PCA were investigated for their abilities to reduce oxidative stress in a human leukoplakia cell line by monitoring the redox indicator, 2',7'-dichlorodihydrofluorescein diacetate (H2DCF) in cellular and acellular systems. BRBE effectively inhibited the oxidation, but PCA was only minimally effective against H2DCF. These results taken together provide evidence that BRBE and PCA can inhibit initiation of carcinogenesis by polycyclic aromatic hydrocarbons; and in addition, BRBE reduces oxidative stress. Cancer Prev Res; 9(8); 704-12. ©2016 AACR.