Abstract 915: Effects of black raspberry extract (BRB) and related compounds on mutagenesis induced by metabolites of the tobacco carcinogen, dibenzo(a,l)pyrene in cultured rat oral fibroblasts; and on oxidative stress in human oral cancer cells

Abstract

Abstract BRB powder, extracts and metabolites have been shown to inhibit tumorigenesis in oral and other sites in animal models. Here we examined the effects of an anthocyanin-enriched BRB extract, and protocatechuic acid (PA), a major BRB anthocyanin metabolite, on mutagenesis and DNA adduct formation by DBP-dihydrodiol, and DBP diolepoxide (DBPDE) (the proximate and ultimate carcinogenic metabolites, resp. of DBP) in lacI rat oral fibroblasts. DBP was poorly metabolized to genotoxic metabolites by the oral fibroblasts, but DBP-dihydrodiol was mutagenic and produced DNA adducts. DBP-dihydrodiol (0.5 uM) induced a mutant fraction of 14.1 +/- 3.9 mutants/10E5 pfu in the target cII gene. This was reduced in a concentration-dependent fashion to 9.1 +/- 2.4 by 0.1-1uM PA– a 35% reduction. Mutagenesis by DBP-dihydrodiol was also reduced in a concentration-dependent fashion by BRB extract and at the highest concentration (20ug/ml) mutagenesis was reduced by 68%. The major DNA adduct produced by 0.5 uM DBP-dihydrodiol was anti-DBPDE-dA, as assessed by L.C.-M.S.-M.S. Its levels were reduced from 13.7 +/- 1.2 to 7.8 +/- 1.9 adducts/10E6 dA by 3uM protocatechuic acid, and from 6.8 +/- 0.7 to 3.2 +/- 0.4 by 66 ug/ml BRB extract. The inhibitions were statistically significant. In contrast, no significant effect PA on mutagenesis by 140 nM DBPDE was observed, with mutant fractions similar to that of DBPDE (3.2 +/- 0.45 mutants/10E5 pfu) being obtained over a range of 0.1 - 1 uM PA. Similarly 10-50 ug/ml BRB extract had no effect on DBPDE-induced mutagenesis. Consistent with these results, levels of total DBPDE adducts were unchanged over a range of PA concentrations, with the high dose of 1uM PA yielding, 4.6 +/- 0.7 adducts/10E5 nucleotides and 4.5 +/- 0.9 without PA. Presumably the short halflife of DBPDE precludes detoxification, or scavenging of DBPDE. The effects of PA and BRB extract on oxidative stress in a human oral cancer cell line (SCC 1483, retromolar trigone) were also examined. The redox sensitive fluorescent probe, dihydrodichlorofluorescein diacetate (DHDCF) was added to the cells at 5uM and BRB (0.5-5 ug/ml) or PA (0.02-0.2 uM) was added shortly afterwards. 2 hr later all doses of BRB extract and PA inhibited the oxidation of DHDCF to its fluorescent product, in a dose-dependent manner. At 5ug/ml BRB extract the increase in fluorescence was reduced from 304 +/- 3.3 to 177 +/- 7.1 relative fluorescence units (RFU) and at 0.2 uM PA the increase was reduced to 224 +/- 25 RFU. In summary, BRB extract and PA inhibited mutagenesis and DNA adduct formation by DBP-dihydrodiol, but not DBPDE. BRB extract and PA were also effective inhibitors of oxidative stress. These mechanisms may a play a role in the protective effects of BRB on oral and other cancers in experimental animals. Supported by NIH grant #CA173465. Citation Format: Joseph B. Guttenplan, Seungjin Kim, Ying Zhou, Wieslawa Kosinska, Ana-Vera Golgotiu, Shantu Amin, Gary D. Stoner, Yuan-Wan Sun, Kun-Ming Chen, Karam El-Bayoumy. Effects of black raspberry extract (BRB) and related compounds on mutagenesis induced by metabolites of the tobacco carcinogen, dibenzo(a,l)pyrene in cultured rat oral fibroblasts; and on oxidative stress in human oral cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 915. doi:10.1158/1538-7445.AM2015-915