Enhanced activity of punicalagin delivered via polymeric implants against benzo[a]pyrene-induced DNA adducts

Abstract

We investigated the effect of punicalagin (PC) on benzo[a]pyrene (BP)-induced DNA adducts in vitro and in vivo. Incubation of BP (1μM) with rat liver microsomes, appropriate co-factors and DNA in the presence of vehicle or punicalagin (1–40μM) showed dose-dependent inhibition of the resultant DNA adducts, with essentially complete (97%) inhibition at 40μM. However, PC failed to inhibit anti-BPDE-induced DNA adducts when tested in an in vitro non-microsomal system, suggesting that the inhibition of the microsomal BP–DNA adducts occurred due to inhibition of P450 1A1 by PC. To determine its efficacy in vivo, female S/D rats were administered punicalagin via the diet (1500ppm; ∼19mg/day/animal) or subcutaneous polymeric implants (two 2-cm, 200mg with 20% drug load; 40mg PC/implant) and then treated with continuous low-dose of BP by a subcutaneous polymeric implant (2cm, 200mg with 10% load; 20mg BP/implant) and euthanized after 10 days. Analysis of the lung DNA by 32P-postlabeling showed significant (60%; p=0.029) inhibition of DNA adducts by PC administered via the implants; the dietary route showed modest (34%) but statistically insignificant inhibition. Furthermore, total PC administered by implants was approximately 38-fold lower compared with the dietary route. Analysis of the lung microsomes showed significant inhibition of cytochrome P450 1A1 activity and induction of glutathione. Release of PC from the implants was found to be biphasic starting with a burst release, followed by a gradual decline. Ultra performance liquid chromatography analysis showed no detectable PC in the plasma but its hydrolyzed product, ellagic acid was readily detected. The plasma concentration of ellagic acid was over two orders of magnitude higher (589±78ng/mL) in the implant group compared with diet (4.36±0.83ng/mL). Together, our data show that delivery of PC by implants can reduce its effective dose substantially, and that the inhibition of DNA adducts in vivo occurred presumably due to the conversion of PC to ellagic acid.