Ajeet K. Srivastav

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.

Synergistic effect of piperine and paclitaxel on cell fate via cyt-c, Bax/Bcl-2-caspase-3 pathway in ovarian adenocarcinomas SKOV-3 cells

BACKGROUND AND AIMS Ovarian cancer is fourth most common and lethal among all gynecologic malignancies. The chemotherapy usually requires in all stages of ovarian cancer but drugs have several side effects. We hypothesized that use of combination therapy of paclitaxel (PTX) and phytochemical piperine (PIP) may reduce the PTX dose as well as toxicity. The human ovarian adenocarcinomas SKOV3 cell treated with PTX-5nM and PIP-10µm after determination of IC50 by MTT assay. Reactive oxygen species generation, mitochondrial membrane potential (MMP), DNA damage, cell death pathway markers as release of cyt-c, Bax/Bcl2-caspase-3 and cell cycle arrest were analyzed. The dose dependent treatment of SKOV-3 cells showed IC50 and synergism at combination of 5nM-PTX and 10µm-PIP in cell viability assay. PTX and PIP increases the accumulation of reactive oxygen species which subsequently leading to increase in JC-1 and fragmented nuclei in mitotracker/DAPI staining. Comet assay showed 4.4-fold increase of tail formation in combined treated cells as compared to control. PTX-PIP arrests the cell cycle in sub-G1 phase. Immunocytochemistry of Bax showed increase in red fluorescence intensity whereas decrease in green fluorescence i.e Bax/Bcl-2 ratio increased. Moreover morphological EB/AO and Hoechst staining confirmed the enhanced apoptosis in combined treatment. Significant upregulation of apoptotic genes, cyt-c (3.4 fold) Bax (2.8 fold), caspase-3 (3.6 fold) whereas no change occurred in Bcl2 mRNA expression and protein expressions. The combination of PTX with PIP produces synergistic effects in SKOV-3 cells via the modulation of pro and anti-apoptotic gene and may compensate the toxicity and side effects of PTX.

ROS mediated crosstalk between endoplasmic reticulum and mitochondria by Phloxine B under environmental UV irradiation.

Phloxine B (PhB) is a most commonly used dye in cosmetic products throughout the world. It shows an absorption in visible and ultraviolet radiations. PhB was photodegraded within 4h of UV exposure. It generates reactive oxygen species (ROS) photochemically and intracellularly. Photosensitized PhB caused dose dependent cell viability reduction of human keratinocyte cell line (HaCaT) which was measured through MTT (75.4%) and NRU (77.3%) assays. It also induces cell cycle arrest and DNA damage. Photosensitized PhB induces Ca(2+) release from endoplasmic reticulum (ER). It causes the upregulation of ER stress marker genes ATF6 (1.79 fold) and CHOP (1.93 fold) at transcription levels. The similar response of ATF6 (3.6 fold) and CHOP (2.38 fold) proteins was recorded at translation levels. CHOP targeted the mitochondria and reduced the mitochondrial membrane potential analyzed through JC-1 staining. It further increases Bax/Bcl2 ratio (3.58 fold) and promotes the release of cytochrome c, finally leads to caspase-dependent apoptosis. Upregulation of APAF1 (1.79 fold) in PhB treated cells under UV B exposure supports the mitochondrial-mediated apoptotic cell death. The results support the involvement of ER and mitochondria in ROS mediated PhB phototoxicity. Therefore, the use of PhB in cosmetic products may be deleterious to users during sunlight exposure.

Combined effect of Benzophenone-2 and ultraviolet radiation promote photogenotoxicity and photocytotoxicity in human keratinocytes

ABSTRACT Benzophenone‐2 (BP2), a common ingredient of sunscreens formulation is widely used as UV filter. We have assessed the photogenotoxic and photocytotoxic potential of BP2. Photostability test showed that BP2 is unstable under UV exposure. Cell proliferation assay revealed that viability of HaCaT cells significantly reduced under UVA, UVB and sunlight exposure. DCF fluorescence intensity proved intracellular ROS generation capacity of BP2 under sunlight, UVA and UVB irradiation. Photodynamic degradation of guanine base of DNA is promoted by BP2 under UV treatment. Genotoxicity assessed by comet assay, showed that photosensitized BP2 enhanced DNA damage, which is measured in term of % tail DNA and olive tail moment. Genotoxic potential of BP2 was further validated with photomicronuclei assay. Photogenotoxicity of BP2 was lastly confirmed by formation of CPDs (Cyclo butane pyrimidine dimmers). DNA damage induced by BP2 was irreversible and extended incubation periods (6–12 h) not favored the recovery from damaged DNA. JC 1 staining showed significant reduction in mitochondrial membrane potential. Membrane integrity compromisation of HaCaT cells was established by AO (Acridine orange), EtBr (Ethidium bromide) staining and confirmed with sub G1 population of cell cycle. Thus, results suggest that BP2 should be avoided in topical application for safe sunscreen practices. Graphical abstract Figure. No Caption available. HighlightsUV filter Benzophenone 2 (BP2) is not stable in UV radiation.Photosensitized BP2 induced photogenotoxicity.BP2 induced cytotoxicity in HaCaT cells at environmental UV radiation.Sunscreen induced DNA damage at environmental UVR.Formation of CPDs and photomicronuclei by photosensitized BP2.