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

Abstract

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.

Figure 1.

Structures of DBP, DBP-diol, and two isomers of anti-DBPDE.

Figure 2.

Dose response for DNA adducts in MSK cells. (A) Dependence of DNA adduct level on concentration of DBP. (B) Dependence of DNA adduct level on concentration of DBPDE. (C) A representative HPLC-MS/MS chromatogram of DBP-induced dA adducts. (D) A representative HPLC-MS/MS chromatogram of DBPDE-induced dA adducts. (E) A representative HPLC-MS/MS chromatogram of DNA from control cells. Values in (A) represent the mean and SEM from DNA extracted from cells on triplicate plates and in (B) represent DNA adducts from single plates, which were used to establish a dose range.

Figure 3.

Effect of (A) BE, (B) delphinidin Cl (DLP), pelargonidin Cl (PGA) and BE on removal of DBP-dA adducts at two concentrations of DBP in MSK cells. Cells were treated with DBP. One day later BE, delphinidin chloride, or pelargonidin chloride was added. One day later (48 h after addition of DBP) adducts were analyzed. *, P ≤ 0.05 in a one-tailed t test; **, P ≤ 0.01 in a one-tailed t test vs corresponding group with no additive Structures of DLP and PGA.

Figure 4.

Effect of time of addition of 160 μg/mL BE on mutagenesis induced by DBPDE in lacI rat oral fibroblasts. BE was added either 1 h before or 1 h after DBPDE. Experiments were conducted at two different concentrations of DBPDE on different days and are expressed as relative levels of mutagenesis. The mutant factions for the DBPDE-alone treated cells were 50.1 and 53.8 mutants/10⁵ pfu at 50 and 100 nM DBPDE, respectively.

Figure 5.

Effect of 160 μg/mL BE on UV-induced cyclobutanepyrimidine dimer levels after 4 and 8 min of exposure in 96 well plates. The absorbance (ordinate) represents the signal for cyclobutanepyrimidine dimers in the ELISA assay.

Figure 6.

Effect of 160 μg/mL BE on mutagenesis induced in lacI oral fibroblasts at different irradiation times. *, P ≤ 0.05 in a one-tailed t test vs the group with no BE.