Ratan Singh Ray

Photosensitizing Potential of Ciprofloxacin at Ambient Level of UV Radiation

Ciprofloxacin is a widely used fluoroquinolone drug with broad spectrum antibacterial activities. Clinical experience has shown incidences of adverse effects related to skin, hepatic, central nervous system, gastrointestinal and phototoxicity. India is a tropical country and sunlight is abundant throughout the day. In this scenario exposure to ambient levels of ultraviolet radiation (UV‐R) in sunlight may lead to harmful effects in ciprofloxacin users. Phototoxicity assessment of ciprofloxacin was studied by two mouse fibroblast cell lines L‐929 and NIH‐3T3. Generation of reactive oxygen species (ROS) like singlet oxygen (1O2), superoxide anion radical (O2ḃ−) and hydroxyl radical (ḃOH) was studied under the exposure of ambient intensities of UV‐A (1.14, 1.6 and 2.2 mW cm−2), UV‐B (0.6, 0.9 and 1.2 mW cm−2) and sunlight (60 min). The drug was generating 1O2, O2ḃ− and ḃOH in a concentration and dose‐dependent manner. Sodium azide (NaN3) and 1,4‐diazabicyclo 2‐2‐2‐octane (DABCO) inhibited the generation of 1O2. Superoxide dismutase (SOD) inhibited 90–95% O2ḃ− generation. The drug (5–40 μg mL−1) was responsible for linoleic acid peroxidation. Quenching study of linoleic acid peroxidation with SOD (25 and 50 U mL−1) confirms the involvement of ROS in drug‐induced lipid peroxidation. The generation of ḃOH radical was further confirmed by using specific quenchers of ḃOH such as mannitol (0.5 m) and sodium benzoate (0.5 m). 2′‐deoxyguanosine (2′‐dGuO) assay and linoleic acid peroxidation showed that ROS were mainly responsible for ciprofloxacin‐sensitized photo‐degradation of guanine base. L‐929 cell line showed 29%, 34% and 54% reduced cell viability at higher drug concentration (300 μg mL−1) under UV‐A, UV‐B and sunlight, respectively. 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay in NIH‐3T3 cell line at higher drug concentration (300 μg mL−1) showed a decrease in cell viability by 54%, 56% and 59% under UV‐A, UV‐B and sunlight, respectively. Results of neutral red uptake assay (NRU) in L‐929 cell line were in accordance with MTT assay. The NIH‐3T3 cell line showed a higher photosensitizing potential than L‐929. The phototoxicity end point shows a time‐ and concentration‐dependent statistically significant (P < 0.001) damage. Ciprofloxacin produced ROS by Type I and Type II photodynamic reactions, interacted with nucleic acid moiety and inhibited cell viability. Further, UV‐induced photo‐peroxidation of linoleic acid accorded the involvement of ROS in the manifestation of drug phototoxicity. Appearance of ciprofloxacin‐induced phototoxicity at the ambient level of sunlight is a real risk for the people of India and for those of other tropical countries. We suggest that sunlight exposure should be avoided (especially peak hours) during ciprofloxacin treatment.

DDT and HCH Residues in Basmati Rice (Oryza sativa) Cultivated in Dehradun (India)

Organochlorine pesticides were used earlier for agricultureproduction. Their residues may still be present in soil and mayaccumulate in food crops, posing potential health problems to consumers. DDT, HCH, their isomers and metabolites were analyzedin samples of soil and rice plants collected from ten differentvillages of a well-known Basmati rice growing area in Dehradun.Residues of both pesticides were found in all samples ofsoil and different parts of rice plants except for a few grainsamples. Maximum residue was observed in husk and minimum ingrains. The average concentration of DDT in soil ranged from0.013 to 0.238 ppm. p,p′-DDE was the major metabolite (>63%). Theaverage concentration of DDT in rice grain varied from 0.002 to 0.040 ppm. o,p′-DDT was the main isomer (>93%). Theaverage concentration of HCH in soil ranged from 0.122 to 0.638 ppm. β-HCH was the predominant (43%) isomerfollowed by α-HCH (21%). The average HCH concentrationin rice grain ranged between 0.013 and 0.113 ppm. All four isomers were present in grains. The levels of DDT and CHCin grains were similar in magnitude as those from differentIndian states, but well below the maximum residue limit of 0.1 ppm for DDT and 0.05 ppm for HCH prescribed by the Government ofIndia and WHO/FAO. As such, the pesticide residue levels in thisexport commodity are not of hazardous nature.

Activation of Autophagic Flux against Xenoestrogen Bisphenol-A-induced Hippocampal Neurodegeneration via AMP kinase (AMPK)/Mammalian Target of Rapamycin (mTOR) Pathways

Background: The effects of xenoestrogen bisphenol-A on autophagy, and association with oxidative stress and apoptosis are still elusive. Results: Transient activation of autophagy protects against bisphenol-A-induced neurodegeneration via AMPK activation and mTOR down-regulation. Conclusion: Autophagy induction against bisphenol-A is an early cells tolerance response. Significance: Autophagy provides an imperative biological marker for evaluation of neurotoxicity by xenoestrogen. The human health hazards related to persisting use of bisphenol-A (BPA) are well documented. BPA-induced neurotoxicity occurs with the generation of oxidative stress, neurodegeneration, and cognitive dysfunctions. However, the cellular and molecular mechanism(s) of the effects of BPA on autophagy and association with oxidative stress and apoptosis are still elusive. We observed that BPA exposure during the early postnatal period enhanced the expression and the levels of autophagy genes/proteins. BPA treatment in the presence of bafilomycin A1 increased the levels of LC3-II and SQSTM1 and also potentiated GFP-LC3 puncta index in GFP-LC3-transfected hippocampal neural stem cell-derived neurons. BPA-induced generation of reactive oxygen species and apoptosis were mitigated by a pharmacological activator of autophagy (rapamycin). Pharmacological (wortmannin and bafilomycin A1) and genetic (beclin siRNA) inhibition of autophagy aggravated BPA neurotoxicity. Activation of autophagy against BPA resulted in intracellular energy sensor AMP kinase (AMPK) activation, increased phosphorylation of raptor and acetyl-CoA carboxylase, and decreased phosphorylation of ULK1 (Ser-757), and silencing of AMPK exacerbated BPA neurotoxicity. Conversely, BPA exposure down-regulated the mammalian target of rapamycin (mTOR) pathway by phosphorylation of raptor as a transient cells compensatory mechanism to preserve cellular energy pool. Moreover, silencing of mTOR enhanced autophagy, which further alleviated BPA-induced reactive oxygen species generation and apoptosis. BPA-mediated neurotoxicity also resulted in mitochondrial loss, bioenergetic deficits, and increased PARKIN mitochondrial translocation, suggesting enhanced mitophagy. These results suggest implication of autophagy against BPA-mediated neurodegeneration through involvement of AMPK and mTOR pathways. Hence, autophagy, which arbitrates cell survival and demise during stress conditions, requires further assessment to be established as a biomarker of xenoestrogen exposure.

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.

Resveratrol enhances ultraviolet B-induced cell death through nuclear factor-κB pathway in human epidermoid carcinoma A431 cells

Resveratrol has been reported to suppress cancer progression in several in vivo and in vitro models, whereas ultraviolet B (UVB), a major risk for skin cancer, is known to induce cell death in cancerous cells. Here, we investigated whether resveratrol can sensitize A431 human epidermoid carcinoma cells to UVB-induced cell death. We examined the combined effect of UVB (30 mJ/cm(2)) and resveratrol (60 microM) on A431 cells. Exposure of A431 carcinoma cells to UVB radiation or resveratrol can inhibit cell proliferation and induce apoptosis. However, the combination of resveratrol and UVB exposure was associated with increased proliferation inhibition of A431 cells compared with either agent alone. Furthermore, results showed that resveratrol and UVB treatment of A431 cells disrupted the nuclear factor-kappaB (NF-kappaB) pathway by blocking phosphorylation of serine 536 and inactivating NF-kappaB and subsequent degradation of IkappaBalpha, which regulates the expression of survivin. Resveratrol and UVB treatment also decreased the phosphorylation of tyrosine 701 of the important transcription factor signal transducer activator of transcription (STAT1), which in turn inhibited translocation of phospho-STAT1 to the nucleus. Moreover, resveratrol/UVB also inhibited the metastatic protein LIMK1, which reduced the motility of A431 cells. In conclusion, our study demonstrates that the combination of resveratrol and UVB act synergistically against skin cancer cells. Thus, resveratrol is a potential chemotherapeutic agent against skin carcinogenesis.

UVA-induced cyototoxicity and DNA damaging potential of benz (e) acephenanthrylene

The toxicity of benz (e) acephenanthrylene (BeA) has been studied earlier with regard to the carcinogenicity of its metabolites, but its phototogenotoxicity is not well understood. Present study aimed to analyze the photodynamic response of BeA in human skin cell line (A375) under ambient environmental intensity of UVA (1.40 mW/cm(2)). Kinetic of BeA showed that the highest intracellular uptake of BeA occurred after 24h of incubation. Cell viability, generation of reactive oxygen species (ROS), oxidative stress and DNA damage induced by BeA under UVA irradiation were assessed. BeA generates singlet oxygen ((1)O(2)), superoxide anion radical (O(2)(ⁱ-)) and hydroxyl radical (ⁱOH) in a concentration-dependent manner. It was observed that glutathione reduced (GSH) and catalase activity were decreased while DNA damage and cell death were induced significantly (P>0.01) as concentration of BeA increased. Thus our results suggest that BeA may be phototoxic as well as photogenotoxic under UVA irradiation.

Expression and inducibility of endosulfan metabolizing gene in Rhodococcus strain isolated from earthworm gut microflora for its application in bioremediation.

The metabolizing potential of a bacterial strain Rhodococcus MTCC 6716, isolated from the gut of an Indian earthworm (Metaphire posthuma) was studied for endosulfan bioremediation. In the present work, the optimum conditions for the maximum growth, kinetic of endosulfan degradation, regression equation, half life and correlation coefficient were studied. Endosulfan induced alterations in the expression of mRNA and protein of specific endosulfan metabolizing marker gene (Esd) was studied. Maximum growth of bacteria was observed at pH 7.0, 30°C and 0.085 M sodium chloride concentration in a liquid culture medium. Endosulfan was degraded by Rhodococcus strain up to 97.23% within 15 days without producing toxic metabolite and with strong correlation coefficient (-0.728) and half life 5.99 days. Endosulfan degradation was mediated through gene(s) present in genomic DNA. Expression of marker gene was found endosulfan concentration dependent. The results suggest that this novel strain (Rhodococcus) may be utilized for bioremediation of endosulfan.

Radiation-Induced in Vitro Phototoxic Potential of Some Fluoroquinolones

Photosensitizing drugs that can damage cellular biomolecules is a matter of concern. Lomefloxacin, norfloxacin, ofloxacin, and enoxacin (broad-spectrum antibiotics of fluoroquinolone group) are used for the treatment of Gram-positive and Gram-negative bacterial infections. Phototoxicity and possible mechanism of their action was assessed under the exposure of ambient levels of UV-A, UV-B, and sunlight at a concentration generally used in the treatment of various diseases. Singlet oxygen (1O2), superoxide anion radical (O2·−) generation, DNA damage, and lipid peroxidation in human blood were studied. All the fluoroquinolones tested in this study produced 1O2 and O2·− under exposure to UV-A, UV-B, and sunlight depending on the concentrations (0 to 60 μg/mL) of the drugs. Enoxacin showed a higher yield of 1O2 and O2·− than other drugs. These materials also degraded deoxyguanosine and induced lipid peroxidation in vitro under exposure to UV-A, UV-B, and sunlight (depending on the dose of radiation). The formation of the reactive oxygen species (ROS) by the photoexcited drugs may be considered as a possible mechanism of their action.

Effect of UV-B radiation on some common antibiotics.

Some of the commonly used antibiotics such as cephaloridine, cephalexin, cephradine, nystatin and nafcillin were tested for generation of singlet oxygen (1O(2)) under UV-B (290-320 nm) exposure and the order for 1O(2) generation was obtained: cephaloridine>cephalexin>nystatin>cephradine>nafcillin. In vitro study with deoxyguanosine (dGuo) showed that 1O(2) was responsible for drug-sensitized photodegradation of the guanine base of DNA and RNA. Sodium azide (NaN(3)) and 1,4-diazabicyclo [2.2.2] octane (DABCO) accorded significant inhibition (76-98%) in the production of (1)O(2) and photo-oxidation of dGuo. The combined effect of drug and UV-B irradiation is of paramount importance in view of cell-damaging reactions by 1O(2). Our findings are important because of increasing UV-B radiation on the earths surface due to depletion of the stratospheric ozone layer. The selected drugs are used routinely for the treatment of various diseases and their combined action may cause undesirable phototoxic responses. Our study suggests that exposure to sunlight should be avoided after the intake of the photosensitive drugs.

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.