Superoxide anion radical induced phototoxicity of 2,4,5,6-Tetraminopyrimidine sulfate via mitochondrial-mediated apoptosis in human skin keratinocytes at ambient UVR exposure.

2,4,5,6-Tetraaminopyrimidine sulfate (TAPS) is worldwide the most commonly used developer in hair dyes. As skin is the major organ, which is directly exposed to these permanent hair dyes, a comprehensive dermal safety assessment is needed. Hereto, we studied the photosensitization potential and mechanism involved in dermal phototoxicity of TAPS exposed to the dark and UVA/UVB/Sunlight by using different in-chemico and mammalian (HaCaT) cells, as test systems. Our experimental outcomes illustrate that TAPS get photodegraded (LC-MS/MS) and specifically generated superoxide anion radical (O2•-) under UVA and UVB via type-I photodynamic reaction. The phototoxic potential of TAPS is measured through MTT, NRU, and LDH assays that depicted a significant cell viability reduction at 25 µg/ml concentration and higher. Different cellular stainings (PI uptake, AO/EB, JC-1, NR uptake) suggested the role of mitochondrial-mediated apoptosis. Further, the transcriptomics study revealed upregulation of Apaf-1, Bax, Cytochrome c, Caspase 3, Caspase 9 and downregulation of Catalase and Bcl-2 by TAPS treated cells that strengthen our findings. Thus, the above findings suggest that chronic application of TAPS may be hazardous for human skin and promote various skin diseases.

Understanding the strain-dependent structure of Cu nanocrystals in Ag-Cu nanoalloys.

The structure of octahedral Ag-Cu nanoalloys is investigated by means of basin hopping Monte Carlo (BHMC) searches involving the optimization of shape and chemical ordering. Due to the significant size mismatch between Ag and Cu, the misfit strain plays a key role in determining the structure of Ag-Cu nanoalloys. At all the compositions, segregated chemical ordering is observed. However, the shape of the Cu nanocrystal and the associated defects are significantly different. At lower amounts of Cu (as little as 2 atom %), defects close to the surface are observed leading to a highly non-compact shape of the Cu nanocrystal which is non-trivial. The number of Cu-Cu bonds is relatively lower in the non-compact shape which is contrary to the preference of bulk Ag-Cu alloys to maximize the homo-atomic bonds. Due to the non-compact shape, {100} Ag-Cu interfaces are observed which are not expected. As the amount of Cu increases, the Cu nanocrystal undergoes a shape transition from non-compact to a compact octahedron. The associated defect structure is also modified. The structural changes due to the strain effects have been explained by calculating the atomic pressure maps and the bond length distributions. The trends relating to the structure have also been verified at larger sizes.

Oxidative stress-mediated photoactivation of carbazole inhibits human skin cell physiology.

Prolonged exposure of the earth's surface to the sun's ultraviolet radiation may result in various skin diseases and cataract. Carbazole (CBZ), as a polycyclic-aromatic hydrocarbon (PAH), is blended with a five-member nitrogen-containing ring. It is found in cigarette smoke, coal, eye kohl, tattoo ink, and wood combustion and affects various types of flora and fauna. Our findings suggest that CBZ generates reactive oxygen species (ROS) like O2•- through type-I photodynamic reaction and causes phototoxicity in the human keratinocyte cell line (HaCaT), which has been proved by mitochondrial dehydrogenase and neutral red uptake assays. CBZ induces single strand DNA damage. We have investigated the involvement of the apoptotic pattern of cell death and confirmed it by cytochrome C release from mitochondria and caspase-9 activation. Similarly, photo-micronuclei formation was associated to CBZ-induced phototoxicity. The results of this study strongly support that the upregulation of bax, cyto-C, apaf-1, casp-9 and down regulation of bcl2, keap-1, nrf-2, and hmox-1 genes cause apoptopic cell death. Downregulation of antioxidant genes showed a significant amount of ROS generation by photosensitized CBZ. Therefore, the current study will be a step forward to safeguard human beings from sunlight-induced photosensitive CBZ prolonged exposure.

Sunscreen-induced expression and identification of photosensitive marker proteins in human keratinocytes under UV radiation.

Solar ultraviolet (UV) radiation is the main factor of photocarcinogenesis, photoaging, and photosensitivity; thus protection from biological damaging UV radiation is a concern. Sunscreens containing UV filters are the most preferred means of photoprotection but the safety and efficacy of UV filters are in question. Benzophenone (BP) and its derivatives, namely, benzophenone 1 (BP1), is commonly used in sunscreens as a UV blocker. The aim of this study was to assess the effects of BP and BP1 on the differential expression of proteins in human keratinocytes (HaCaT cells) under exposure to ultraviolet A radiation. Photosensitive proteins were screened from HaCaT cells by two-dimensional (2-D) gel electrophoresis, and identification of these differentially expressed proteins was performed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)/TOF mass spectrometry. Protein identification was performed using the search program MASCOT and a database made of SUMO and GhJMJ12 amino acid sequences. Our results showed that the proteins involved directly or indirectly in apoptosis are 70 kDa heat shock protein, long-chain specific acyl-CoA dehydrogenase, serine/threonine-protein kinase, and FAM78A protein, which were upregulated in comparison to control HaCaT cells. The expressions of binding immunoglobulin protein, podocalyxin-like protein, actin, cytoplasmic, and calreticulin precursors were downregulated. The altered protein expression indicated that cell growth arrest and apoptosis were potential mechanisms of cytotoxicity and genotoxicity of BPs. The results of 2-D gel electrophoresis followed by mass spectrometry showed expression of novel proteins involved in promoting or initiating apoptotic pathways. Hence, we conclude that BPs should be avoided as a UV blocker from sunscreens because of its potential to promote apoptotic proteins in human skin keratinocytes.

Biochemical regulation and structural analysis of copper-transporting ATPase in a human hepatoma cell line for Wilson disease.

Hepatic copper levels differ among patients with Wilson disease (WD) and normal individuals depending on the dietary intake, copper bioavailability, and genetic factors. Copper chloride (CuCl2 ) caused dose-dependent reduction in cell viability of human teratocarcinoma (HepG2) cell line, measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Cells were exposed to different concentrations of CuCl2 in log doses and maximum cell viability reduction was recorded at 15 µg/mL. Toxic dose of CuCl2 is potent inducer of reactive oxygen species (ROS). Apoptosis as a pattern of cell death was confirmed through sub-G1 fraction and morphological changes such as mitochondrial depolarization, endoplasmic reticulum and lysosomal destabilization, phosphatidylserine translocation, and DNA damage. Our transcriptional and translational results strongly support apoptotic cell death. Using the available data present in dbSNP and bioinformatics tools, three nonsynonymous single nucleotide polymorphisms (nsSNPs) were identified as deleterious, reducing the stability of protein ATP7B. Structural analysis of native and mutant ATP7B proteins was investigated using molecular dynamics simulation (MDS) approach. Mutation in ATP7B gene might disturb the structural conformation and catalytic function of the ATP7B protein may be inducing WD. Hence, excess dietary intake of copper chloride must be avoided for safety of health to prevent from WD.

Photoexcited triclosan induced DNA damage and oxidative stress via p38 MAP kinase signaling involving type I radicals under sunlight/UVB exposure.

Triclosan (TCS) is an antimicrobial preservative used in personal care products. Here, we have studied the phototoxicity, photogenotoxicity of TCS and its molecular mechanism involving p38 mitogen activated protein kinase (MAPK) pathway under UVB/sunlight exposure. We found that TCS showed photodegradation and photoproducts formation under UVB/sunlight. In silico study suggests that photosensitized TCS loses its preservative property due to the formation of its photoproducts. Photosensitized TCS induces significant O2•-, •OH generation and lipid peroxidation via type-I photochemical reaction mechanism under UVB/sunlight exposure. We performed intracellular study of TCS on human skin keratinocytes (HaCaT cell-line) under the ambient intensity of UVB (0.6 mW/cm2) and sunlight exposure. Significant intracellular ROS generation was observed through DCFH2-DA/DHE assays along with a significant reduction in cell viability through MTT and NRU assays in photosensitized TCS. Photosensitized TCS also induces endoplasmic reticulum (ER) stress as shown through ER-tracker/DAPI staining and Ca2+ release. It further induced cell cycle arrest through the sub-G1 phase augmentation and caused lysosomal/mitochondrial destabilization. Photogenotoxicity was shown through significant tail DNA, micronuclei and cyclobutane pyrimidine dimers (CPDs) formations. Cell signaling mechanism implicated upregulated expression of cleaved Caspase-3, Bax, phospho-p38, phospho-JNK and cytochrome C, thereby downregulated Bcl-2 expressions. Results advocate that TCS induces phototoxic effects via type I mediated photodynamic mechanism and activation of MAPK pathway. We conclude that photoexcited TCS may be deleterious to human health at the ambient environmental intensities of sunlight reaching at the earth's surface. Therefore, it may be replaced by alternative safe preservative.

Graphene from discharged dry cell battery electrodes.

Utilization of extracted graphite rods from discharged dry cell batteries for synthesis of graphene oxide / graphene serves two purposes, one is waste management which supports environmental safety and the second is low cost production of graphene oxide / graphene which are highly promising 2D materials in various fields of research. In the present work, a sustainable feasibility for the synthesis of graphene oxide / graphene from graphite rods of waste dry cell batteries is demonstrated. The graphite rods separated from the waste dry cell batteries were subjected to electrochemical exfoliation (ECE) in an acidic media. The graphene oxide (GO) obtained from this method was subjected to reduction heat treatment under argon atmosphere at suitable temperature and time period. Finally, the reduced graphene oxide (rGO) i.e., graphene was characterized using XRD, FTIR, Raman Spectroscopy, TGA, BET, SEM and TEM. The few layer graphene structure is supposed to be less defective in comparison to similar exfoliation techniques due to less oxygen-functional groups associated with the intermediate graphene oxide.

Antioxidant efficacy of chitosan/graphene functionalized superparamagnetic iron oxide nanoparticles.

The antioxidant potential of superparamagnetic iron oxide nanoparticles functionalized with chitosan and graphene were examined in the present work. Coprecipitation technique was followed for the synthesis of iron oxide nanoparticles. Graphene-iron oxide nanocomposites were synthesized by mechanical mixing followed by the heat treatment at moderate temperature. The chitosan coated iron oxide nanoparticles were prepared by dispersing nanoparticles in chitosan solution. The nanoparticles/nanocomposites were characterized using XRD, SEM, TEM and HAADF-STEM for phase structure, morphology and elemental analysis. The superparamagnetic behavior of nanoparticles/nanocomposites were confirmed by magnetic measurements using vibrating sample magnetometry. Antioxidant efficacy of these nanoparticles/nanocomposites were investigated in terms of free radical scavenging and reducing potential using an array of in vitro assay system. Ferric reducing antioxidant power (FRAP) and 2,2'-diphenyl-1-picrylhydrazyl (DPPH) were used for the antioxidant capacity. The investigation suggests that the graphene improves the antiradical response of iron oxide nanoparticles at higher concentration which is almost comparable to the ascorbic acid used as standard.

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

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.

Photosensitized methyl paraben induces apoptosis via caspase dependent pathway under ambient UVB exposure in human skin cells.

Methyl paraben (MP), is a widely used preservative in pharmaceutical, food and cosmetic products. Its molecular mechanism under ambient ultraviolet radiation is not well understood. We investigated photosensitizing mechanism of MP under ambient UVB (0.6 mW/cm2) intensity. MP showed dose dependent decrease in cell viability of human keratinocyte cell line (HaCaT) by MTT and NRU assays. Study showed 40% reduction in antimicrobial activity of UVB irradiated MP through E. coli culture. Photosensitized MP (25 µg/ml) significantly enhanced lipid peroxidation, intracellular ROS generation and disrupted mitochondrial membrane integrity. MP induced loss of lysosomal membrane integrity and endoplasmic reticulum (ER) mediated stress evident from Ca+2 release. Phototoxicity of MP showed nuclear fragmentation, phosphatidylserine translocation, 30% tail DNA and micronuclei formation. Study showed mitochondria mediated apoptosis via upregulation of Bax, Apaf-1, Cytochrome C and Caspase-3. Upregulation of Caspase-12 (2 folds) specifically showed role of ER in apoptosis. Specific caspase inhibitor, Z-VAD-FMK showed involvement of caspase cascade pathway in apoptosis. Results indicate that photosensitive MP leads to oxidative stress mediated DNA damage and apoptosis through mitochondria and ER. MP causes deleterious effects and its long term exposure to human skin may promote skin diseases. Therefore, MP should be replaced by other photosafe preservatives for humans.

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.