Abstract 503: Enhanced repair of bulky DNA adducts induced by a tobacco carcinogen and UV light in human oral epithelial cells by black raspberry extract
Authors: Guttenplan Joseph B. , Chen Kun-Ming , Sun Yuan-Wan , Teicher Ross , Kosinska Wieslawa , Gowda Krishne , Amin Shantu , Stoner Gary D. , El-Bayoumy Karam 
Published: July 2017

Abstract
We were the first to report that an anthocyanin-enriched black raspberry extract (BE) inhibited mutagenesis, and reduced levels of DNA adducts induced by metabolites of the tobacco carcinogen dibenzo(a,l)pyrene (DBP), in a rat oral fibroblast cell line, by enhancing removal of DNA adducts (Guttenplan, et al., Cancer Prev Res; 9(8) August 2016). Here we extend these findings to the repair of DBP-induced DNA damage in a human oral leukoplakia cell line (MSK leuk1), and a reduction in the toxicity (assayed by dead cell numbers and cell survival) of DBP diolepoxide (DBPDE) in these cells. In addition, we examined the effect of BE on DNA damage and toxicity induced by UV light. Treatment of the MSK cells with DBP, and 2 of its metabolites, DBP-dihydrodiol and DBPDE led to a major adenine adduct, (-)-anti-trans-DB[a,l]PDE-dA (DBPDE-dA). The order of potency was DBPDE&gt;DBP&gt;DBP-dihydrodiol with one µM DBP, producing adduct levels of about 20/10E6 dA. BE in the range of 75-150 µg/ml significantly inhibited adduct formation when cells were pretreated with BE before addition of DBP. However, when cells were first treated with DBP and one day later BE was added, adduct levels were reduced by about 50% two days after treatment with DBP, indicating that the BE was enhancing DNA repair. This conclusion results from the fact that BE was not present during the metabolic activation of DBP to DBPDE, and hence couldn’t modulate the activation steps. We also tested whether BE, added 4 hr after treatment with the short-lived DBPDE, inhibited toxicity to MSK cells. Initial toxicity was measured by counting the numbers of dead cells in the medium 24 hr after addition of DBPDE; and the relative levels of surviving cells were determined using an MTT cell viability assay. It was found that 100ug/ml BE reduced toxicity induced by 25 - 200 nM DBPDE by about 50%, with a concomitant increase in survival in the BE-treated cells. As DBP produces bulky DNA adducts we also investigated the effects of BE on toxicity and levels of DNA adducts produced by UV light. Cells were irradiated for 30 - 120 seconds with 254 nm light and 15 minutes later, treated with 50 - 150 µg/ml BE. Toxicity was measured, as above, and a logarithmic decrease in cell death was observed with increasing concentration of BE. Similar to results with DBPDE, BE increased cell survival. The effects of BE on relative levels of the UV-light-induced cyclopyrimidine adducts were also determined, using an ELISA assay. BE at levels of 150 - 300 µg/ml reduced adduct levels by 30 - 60%. These results indicate that BE may provide chemopreventive effects on initiation of carcinogenesis by environmental agents that produce bulky DNA adducts - by enhancing DNA repair, likely via the nucleotide-excision repair pathway. Supported by NIH grant #CA173465.
Note: This abstract was not presented at the meeting.
Citation Format: Joseph B. Guttenplan, Kun-Ming Chen, Yuan-Wan Sun, Ross Teicher, Wieslawa Kosinska, Krishne Gowda, Shantu Amin, Gary D. Stoner, Karam El-Bayoumy. Enhanced repair of bulky DNA adducts induced by a tobacco carcinogen and UV light in human oral epithelial cells by black raspberry extract [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 503. doi:10.1158/1538-7445.AM2017-503