The aim of this study was to analyze the photostability and phototoxicity mechanism of anthracene (ANT) in a human skin epidermal cell line (HaCaT) at ambient environmental intensities of sunlight/UV-R (UV-A and UV-B). Photomodification of ANT under sunlight/UV-R exposure produced two photoproducts, anthrone and 9,10 anthracenedione. Generation of (1)O(2), O(2)(•-) and (•)OH was measured under UV-R/sunlight exposure. Involvement of reactive oxygen species (ROS) was further substantiated by their quenching with free radical quenchers. Photodegradation of 2-deoxyguanosine and linoleic acid peroxidation showed that ROS were mainly responsible for ANT phototoxicity. ANT generates significant amount of intracellular ROS in cell line. Maximum cell viability (85%) was reduced under sunlight exposure (30 min). Results of MTT assay accord NRU assay. ANT (0.01 μg mL(-1)) induced cell-cycle arrest at G1 phase. RT-PCR demonstrated constitutive inducible mRNA expression of CYP 1A1 and 1B1 genes. Photosensitive ANT upregulates CYP 1A1 (2.2-folds) and 1B1 (4.1-folds) genes. Thus, the study suggests that ROS and DNA damage were mainly responsible for ANT phototoxicity. ANT exposure may be deleterious to human health at ambient environmental intensities reaching the earth's surface through sunlight.
The present study illustrates the photosensitizing behavior of mefloquine (MQ) in human skin keratinocytes under ambient doses of UVB and sunlight exposure. Photochemically, MQ generated reactive oxygen species superoxide radical, hydroxyl radical, and singlet oxygen through type I and type II photodynamic reactions, respectively, which caused photooxidative damage to DNA and formed localized DNA lesions cyclobutane pyrimidine dimers. Photosensitized MQ reduced the viability of keratinocytes to 25 %. Significant level of intracellular reactive oxygen species (ROS) generation was estimated through fluorescence probe DCF-H2. Increased apoptotic cells were evident through AO/EB staining and phosphatidyl serine translocation in cell membrane. Single-stranded DNA damage was marked through single-cell gel electrophoresis. Mitochondrial membrane depolarization and lysosomal destabilization were evident. Upregulation of Bax and p21 and downregulation of Bcl-2 genes and corresponding protein levels supported apoptotic cell death of keratinocyte cells. Cyclobutane pyrimidine dimers (CPDs) were confirmed through immunofluorescence. In addition, hallmarks of apoptosis and G2/M phase cell cycle arrest were confirmed through flow cytometry analysis. Our findings suggest that MQ may damage DNA and produce DNA lesions which may induce differential biological responses in the skin on brief exposure to UVB and sunlight.
Levofloxacin (LVFX) is a broad spectrum third generation fluoroquinolone antibiotic, used in the treatment of severe or life-threatening bacterial infections. Photosensitizing mechanism of LVFX was investigated under the ambient environmental intensities of UV-A, UV-B and sunlight exposure. Phototoxic effects of LVFX were assessed on NIH-3T3 and HaCaT cell lines. Results identified first time three photoproducts of LVFX at ambient levels of UV-R by LC-MS/MS. The generation of reactive oxygen species (ROS) was investigated photochemically as well as intracellularly in HaCaT cell line. ROS were significantly quenched by specific quenchers like DABCO, NaN(3), D-mannitol and NAC. Photosensitized LVFX caused lipid peroxidation at different concentrations. Quenching study with superoxide dismutase confirms the LVFX-induced lipid photoperoxidation. Further, photocytotoxicity of LVFX showed significant reduction in cell viability by MTT and neutral red uptake assays. LVFX caused cell arrest in G2/M phases as well as induced apoptosis through ROS-dependent pathway. In addition, photosensitized LVFX also induced upregulation of p21 and Bax/Bcl-2 genes ratio. India is a tropical country and most of the human activities such as agriculture, commerce, sports, etc. take place in bright sunlight; therefore, photosensitive LVFX may lead to skin/ocular disorders and immune suppression. Information is needed regarding the phototoxicity of LVFX for human safety.
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