Saroj Kumar Amar

Benzophenone 1 induced photogenotoxicity and apoptosis via release of cytochrome c and Smac/DIABLO at environmental UV radiation.

Solar UV radiation is main factor of photocarcinogenesis, photoageing, and phototoxicity; thus, protection from UV radiation is major concern. Sunscreens containing UV filters are suggested as sun safe practices, but safety of UV filters remains in controversies. Benzophenone-1 (BP1) is commonly used in sunscreens as UV blocker. We assessed the photogenotoxicity and apoptotic parameters in human keratinocytes (HaCaT cells) by western blot, immunocytochemistry, flowcytometry, comet assay and TEM imaging. Our results exposed that BP1 photosensitized and generated intracellular ROS (2.02 folds) under sunlight/UVR. Decrease in cell viability was recorded as 80.06%, 60.98% and 56.24% under sunlight, UVA and UVB, respectively. Genotoxic potential of BP1 was confirmed through photomicronuclei and CPDs formation. BP1 enhanced lipid peroxidation and leakage of LDH enzyme (61.7%). Apoptotic cells were detected by AnnexinV/PI staining and sub G1 population of cell cycle. BP1 induced up regulation of apoptotic proteins Bax/Bcl2 ratio, Apaf-1, cytochrome c, Smac/DIABLO and cleaved caspase 3 was noticed. Down regulation of pro caspase 3 was inhibited by Z-VAD-fmk (inhibitor of caspase). Thus, study established the involvement of BP1 in photogenotoxicity and apoptosis via release of cytochrome c and Smac/DIABLO. These findings suggest sunscreen user to avoid BP1 in cosmetics preparation for its topical application.

Role of type I & type II reactions in DNA damage and activation of Caspase 3 via mitochondrial pathway induced by photosensitized benzophenone

Sunscreen users have been increased, since excessive sun exposure increased the risk of skin diseases. Benzophenone (BP) and its derivatives are commonly used in sunscreens as UV blocker. Its photosafety is concern for human health. Our study showed the role of type-I and type-II radicals in activation of caspase 3 and phototoxicity of BP under sunlight/UV radiation. BP photodegraded and formed two photoproducts. BP generates reactive oxygen species (ROS) singlet oxygen ((1)O2), superoxide anion (O2˙(-)) and hydroxyl radical (˙OH) through type-I and type-II photodynamic mechanisms. Photocytotoxicity significantly reduced cell viability under sunlight, UVB and UVA. DCF fluorescence confirmed intracellular ROS generation. BP showed single strand DNA breakage, further proved by cyclobutane pyrimidine dimmers (CPDs) formation. Lipid peroxidation and LDH leakage were enhanced by BP. P21 dependent cell cycle study showed sub G1 population which advocates apoptotic cell death, confirmed through AO/EB and annexin V/PI staining. BP decreased mitochondrial membrane potential, death protein released and activated caspase. We proposed cytochrome c regulated caspase 3 dependent apoptosis in HaCaT cell line through down regulation of Bcl2/Bax ratio. Phototoxicity potential of its photoproducts is essential to understand its total environmental fate. Hence, we conclude that BP may replace from cosmetics preparation of topical application.

Involvement of cathepsin B in mitochondrial apoptosis by p-phenylenediamine under ambient UV radiation

Paraphenylenediamine (PPD), a derivative of paranitroaniline has been most commonly used as an ingredient of oxidative hair dye and permanent tattoos. We have studied the phototoxic potential of PPD under ambient ultraviolet radiation. PPD is photodegraded and form a novel photoproduct under UV A exposure. PPD shows a concentration dependent decrease in cell viability of human Keratinocyte cells (HaCaT) through MTT and NRU test. Significant intracellular ROS generation was measured by DCFDA assay. It caused an oxidative DNA damage via single stranded DNA breaks, micronuclei and CPD formation. Both lysosome and mitochondria is main target for PPD induced apoptosis which was proved through lysosomal destabilization and release of cathepsin B by immunofluorescence, real time PCR and western blot analysis. Cathepsin B process BID to active tBID which induces the release of cytochrome C from mitochondria. Mitochondrial depolarization was reported through transmission electron microscopy. The cathepsin inhibitor reduced the release of cytochrome C in PPD treated cells. Thus study suggests that PPD leads to apoptosis via the involvement of lysosome and mitochondria both under ambient UV radiation. Therefore, photosensitizing nature of hair dye ingredients should be tested before coming to market as a cosmetic product for the safety of human beings.

Cellular and molecular mechanism of ofloxacin induced apoptotic cell death under ambient UV-A and sunlight exposure

Abstract Ofloxacin (OFLX) is a racemic mixture of levofloxacin which revealed phototoxicity in patients exposed with sunlight after medication. Here, we have been addressed the possible cellular and molecular mechanisms of OFLX induced apoptosis under ambient UV-A and sunlight exposure using HaCaT cell line as a model. The results showed that Photodegradation and three photo-products formation of OFLX by LC-MS/MS under ambient intensities of UV-A (1.5 and 2.2 mW/cm2) and sunlight. OFLX produced 1O2, O2.−, and OH radicals via type-II- and type-I-dependent reaction mechanism, which corroborated by its specific quenchers. 2’-dGua degradation in photochemical and % tail DNA formation in cell line using comet test advocated the genotoxic potential of OFLX. Photocytotoxic assays (MTT and NRU) revealed the considerable decline in cell viability by OFLX. OFLX triggered apoptosis, proved by cell cycle, Annexin V/PI double staining along with acridine orange (AO)/ethidium bromide (EB), and Hoechst staining as well as caspase-3 activity by colorimetric assay. OFLX induced lysosomal disruption and mitochondrial membrane destabilization confirmed through fluorescence staining with AO/JC-1. OFLX significantly upregulated the expression of p21 and bax genes. In conclusion, the study revealed that photosensitized OFLX induced apoptosis via ROS-mediated DNA damage, destabilization of lysosomal and mitochondrial membrane, and upregulation of p21, bax, and caspase-3 genes.

Photosensitized 2-amino-3-hydroxypyridine-induced mitochondrial apoptosis via Smac/DIABLO in human skin cells.

The popularity of hair dyes use has been increasing regularly throughout the world as per the demand of hair coloring fashion trends and other cosmetic products. 2-Amino-3-hydroxypyridine (A132) is widely used as a hair dye ingredient around the world. We are reporting first time the phototoxicity mechanism of A132 under ambient environmental UV-B radiation. It showed maximum absorption in UV-B region (317 nm) and forms a photoproduct within an hour exposure of UV-B irradiation. Photocytotoxicity of A132 in human keratinocytes (HaCaT) was measured by mitochondrial (MTT), lysosomal (NRU) and LDH assays which illustrated the significant reduction in cell viability. The role of reactive oxygen species (ROS) generation for A132 phototoxicity was established photo- chemically as well as intracellularly. Noteworthy, formation of tail DNA (comet assay), micronuclei and cyclobutane pyrimidine dimers (CPDs) (immunocytochemistry) formation confirmed the photogenotoxic potential of dye. Cell cycle study (sub-G1peak) and staining with EB/AO revealed the cell cycle arrest and apoptosis. Further, mitochondrial mediated apoptosis was corroborated by reduced MMP, release of cytochrome c and upregulation of caspase-3. Release of mitochondrial Smac/DIABLO in cytoplasm demonstrated the caspase dependent apoptotic cell death by photolabile A132 dye. In-addition increased Bax/Bcl2 ratio again proved the apoptosis. Thus, study suggests that A132 induces photogenotoxicity, phototoxicity and apoptotic cell death through the involvement of Smac/DIABLO in mitochondrial apoptosis via caspase dependent manner. Therefore, the long term use of A132 dye and sunlight exposure jointly increased the oxidative stress in skin which causes premature hair loss, damage to progenitor cells of hair follicles.