P 003 - Ambient UV-B exposure attenuate the binding affinity of ofloxacin with bacterial DNA gyrase and induced apoptosis in human keratinocytes via Reactive Oxygen Species mediated pathway

Abstract

Ofloxacin (OFLX) is a known synthetic broad spectrum antibiotics which inhibit bacterial DNA topoisomerase activity. Previous studies showed reduced antimicrobial activity of photosensitized OFLX and its photo-products. Here, we have addressed the binding affinity of OFLX and its photo-products against DNA gyrase to measure the antimicrobial activity. Further, the study was extended to explore the molecular mechanism of photogenotoxicity on human keratinocyte cells (HaCaT) under environmental UV-B irradiation. Photochemical experiments showed the generation of Reactive Oxygen Species (ROS) such as 1O2, O2•- and •OH by photosensitized OFLX. OFLX shows a concentration dependent decrease in cell viability of HaCaT through MTT and NRU tests. Significant intracellular ROS generation was measured by DCFDA assay. ROS caused an oxidative DNA damage via single stranded DNA breaks, micronuclei and CPD formation. OFLX induced cell cycle arrest in G1 phase with appearance of sub-G1 peak. OFLX triggered apoptosis via permeabilization of mitochondrial membrane with the downregulation of anti-apoptotic Bcl-2 and caspase-3 whereas, upregulation of pro-apoptotic Bax and Cyto-C proteins. Our study illustrated that binding affinity of photo-products of OFLX with DNA gyrase is mainly responsible for attenuated antimicrobial activity. Thus, study suggests that sunlight exposure should avoid by drug users during peak hours.