Ashish Dwivedi

UVB-induced apoptosis and DNA damaging potential of chrysene via reactive oxygen species in human keratinocytes.

Chrysene is one of the basic polycyclic aromatic hydrocarbon (PAH) which is toxic environmental pollutant and consistently exposed to sunlight. However, little information is available on its photogenotoxicity. The objective of the present study was to analyze the effects of chrysene, under environmental intensity of UVB (0.6mW/cm(2)) in human skin epidermal cell line (HaCaT). Kinetic of chrysene showed that the highest intracellular uptake of chrysene occurred after 24h of incubation. The intracellular reactive oxygen species (ROS) was increased in a concentration dependent manner in chrysene treated cells under UVB irradiation. It was observed that UVB-irradiated chrysene induced apoptosis through activation of caspases-3 and phosphatidylserine translocation. Glutathione reduced (GSH) and catalase activity were decreased while apoptosis and DNA damage were induced significantly (P>0.01) as concentration of chrysene increased. Thus our results suggest that chrysene may be phototoxic as well as photogenotoxic under UVB irradiation.

Photosensitizing mechanism and identification of levofloxacin photoproducts at ambient UV radiation.

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, NaN3, 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.

Singlet oxygen mediated apoptosis by anthrone involving lysosomes and mitochondria at ambient UV exposure.

Anthrone a tricyclic aromatic hydrocarbon which is toxic environmental pollutant comes in the environment through photooxidation of anthracene. We have studied the photomodification of anthrone under environmental conditions. Anthrone generates reactive oxygen species (ROS) like (1)O2 through Type-II photodynamic reaction. Significant intracellular ROS generation was measured through dichlorohydrofluorescein fluorescence intensity. The generation of (1)O2 was further substantiated by using specific quencher like sodium azide. UV induced photodegradation of 2-deoxyguanosine and photoperoxidation of linoleic acid accorded the involvement of (1)O2 in the manifestation of anthrone phototoxicity. Phototoxicity of anthrone was done on human keratinocytes (HaCaT) through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and neutral red uptake assays. Anthrone induced cell cycle arrest (G2/M-phase) and DNA damage in a concentration dependent manner. We found apoptosis as a pattern of cell death which was confirmed through sub-G1 fraction, morphological changes, caspase-3 activation, acridine orange/ethidium bromide staining and phosphatidylserine translocation. Mitochondrial depolarization and lysosomal destabilization was parallel to apoptotic process. Our RT-PCR results strongly supports our view point of apoptotic cell death through up-regulation of pro-apoptotic genes p21 and Bax, and down regulation of anti-apoptotic gene Bcl2. Therefore, much attention should be paid to concomitant exposure of anthrone and UV-R for its total environmental impact.

MicroRNA: a new and promising potential biomarker for diagnosis and prognosis of ovarian cancer.

Epithelial ovarian cancer (EOC) is the leading cause of death among all gynecological malignancies. Despite the technological and medical advances over the past four decades, such as the development of several biological markers (mRNA and proteins biomarkers), the mortality rate of ovarian cancer remains a challenge because of its late diagnosis, which is specifically attributed to low specificities and sensitivities. Under this compulsive scenario, recent advances in expression biology have shifted in identifying and developing specific and sensitive biomarkers, such as microRNAs (miRNAs) for cancer diagnosis and prognosis. MiRNAs are a novel class of small non-coding RNAs that deregulate gene expression at the posttranscriptional level, either by translational repression or by mRNA degradation. These mechanisms may be involved in a complex cascade of cellular events associated with the pathophysiology of many types of cancer. MiRNAs are easily detectable in tissue and blood samples of cancer patients. Therefore, miRNAs hold good promise as potential biomarkers in ovarian cancer. In this review, we attempted to provide a comprehensive profile of key miRNAs involved in ovarian carcinoma to establish miRNAs as more reliable non-invasive clinical biomarkers for early detection of ovarian cancer compared with protein and DNA biomarkers.

Production of ROS by Photosensitized Anthracene Under Sunlight and UV‐R at Ambient Environmental Intensities

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 1O2, O2•− 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.

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.

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.

Attenuated neuroprotective effect of riboflavin under UV-B irradiation via miR-203/c-Jun signaling pathway in vivo and in vitro.

BackgroundRiboflavin (RF) or vitamin B2 is known to have neuroprotective effects. In the present study, we report the attenuation of the neuroprotective effects of RF under UV-B irradiation. Preconditioning of UV-B irradiated riboflavin (UV-B-RF) showed attenuated neuroprotective effects compared to that of RF in SH-SY5Y neuroblostoma cell line and primary cortical neurons in vitro and a rat model of cerebral ischemia in vivo.ResultsResults indicated that RF pretreatment significantly inhibited cell death and reduced LDH secretion compared to that of the UV-B-RF pretreatment in primary cortical neuron cultures subjected to oxygen glucose deprivation in vitro and cortical brain tissue subjected to ischemic injury in vivo. Further mechanistic studies using cortical neuron cultures revealed that RF treatment induced increased miR-203 expression which in turn inhibited c-Jun expression and increased neuronal cell survival. Functional assays clearly demonstrated that the UV-B-RF preconditioning failed to sustain the increased expression of miR-203 and the decreased levels of c-Jun, mediating the neuroprotective effects of RF. UV-B irradiation attenuated the neuroprotective effects of RF through modulation of the miR-203/c-Jun signaling pathway.ConclusionThus, the ability of UV-B to serve as a modulator of this neuroprotective signaling pathway warrants further studies into its role as a regulator of other cytoprotective/neuroprotective signaling pathways.

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.

Ambient UVA-Induced Expression of p53 and Apoptosis in Human Skin Melanoma A375 Cell Line by Quinine

This study aimed to analyze the phototoxic mechanism and photostability of quinine in human skin cell line A375 under ambient intensities of UVA (320–400 nm). Photosensitized quinine produced a photoproduct 6‐methoxy‐quinoline‐4‐ylmethyl‐oxonium identified through LC‐MS/MS. Generation of 1O2, O2•−, and •OH was measured and further substantiated through their respective quenchers. Photosensitized Quinine (Q) caused degradation of 2‐deoxyguanosine, the most sensitive nucleotide to UV radiation. The intracellular ROS was increased in a concentration‐dependent manner. Significant reduction in metabolic status measured in terms of cell viability (54%) at 25 μg mL−1 was observed through MTT assay. Results of MTT assay accord NRU assay. Single strand DNA breaks and apoptosis were increased significantly (P < 0.01) as observed through comet assay and EB/AO double staining. Photosensitized quinine caused cells to arrest in G2 phase of cell cycle and induced apoptosis (5.08%) as revealed through FACS. Real‐Time PCR showed upregulation of p21 (4.56 folds) and p53 (2.811 folds) genes expression. Thus, our study suggests that generation of reactive oxygen species by quinine under ambient intensity of UVA may result into deleterious phototoxic effects among human population.