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.

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.

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.

Use of L-929 cell line for phototoxicity assessment.

L-929 is an adherent type of mouse fibroblast cell line was known as an alternate test system for toxicity assessment. Its photosensitivity towards ultraviolet radiation (UVR) was studied under the exposure to various intensities of UVA, UVB and sunlight. MTT assay was used for cell viability under UVR alone or in combination with chlorpromazine. UVB intensity below 0.6mW/cm2 did not show phototoxicity till 150min (min) exposure. UVA intensity up to 1.5mW/cm2 for 180min exposure did not alter the cell viability, but at 2.0 and 3.0mW/cm2 showed reduced cell viability beyond 90 and 60min, respectively. Sunlight exposure showed a loss in cell viability beyond 60min. Chlorpromazine showed a dose dependent phototoxic response under UVA, UVB and sunlight exposure. The study suggests the suitability of L-929, as an in vitro test system for the phototoxicity, which was compared with NIH-3T3 cell line. Therefore, L-929 cell line may also be used for phototoxicity assessment. The system provides information regarding the lethality of higher intensities of UVR and its importance in view of increasing UV intensities on the earth's surface due to ozone depletion. Our results suggest that a small change in UVR intensity (especially UVB) in sunlight may increase the risk of phototoxicity.

Phototoxicity assessment of drugs and cosmetic products using E. coli.

A gram negative bacteria Escherichia coli (Dh5alpha strain) was developed as an alternate test system of phototoxicity. Eight drugs (antibiotics) and cosmetic products (eight face creams) were examined for their phototoxicity using this test system. Five known phototoxic compounds were used to validate the test system. UVA-radiation induced phototoxicity of these compounds was tested by agar gel diffusion assay. Decrease in colony forming units (CFU) was taken as an end point of phototoxicity. The phototoxic compounds and antibiotics produced significant reduction in CFU (p<0.001) at 80 microg/ml concentrations under exposure to UVA-radiation (5.4-10.8 J/cm(2)). One face cream was found phototoxic and produced significant decrease in CFU of E. coli at 1.0mg/ml concentration under UVA exposure (10.8 J/cm(2)). The minimum effective concentration of tetracycline and dose of UVA-radiation were also determined by observing growth inhibition of E. coli through disc diffusion assay. The observations suggested that E. coli can be used as an alternative test system for phototoxicity evaluation of chemicals. A battery of test systems is required to conclude the toxic/phototoxic potential of a chemical agent. In view of the speed, easiness, sensitivity and low cost, E. coli is introduced as one of the alternate test system for phototoxicity studies in safety evaluation of various chemical ingredients or formulations used in cosmetics and drugs.

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 microg/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.

Endosulfan degradation by a Rhodococcus strain isolated from earthworm gut.

A Rhodococcus MTCC 6716 bacterial strain was isolated apparently for the first time from the gut microflora of an Indian earthworm (Metaphire posthuma). Endosulfan was used as a carbon source by the strain and degraded it up to 92.58% within 15 days. Furthermore, the isolated strain of the bacterium did not produce the persistent form of the toxic metabolite endosulfan sulfate. This strain exhibits luxury growth in minimal medium with high concentrations of endosulfan (80 microg mL(-1)). Degradation of the endosulfan occurred simultaneously with bacterial growth and an increase in chloride ion (87.1%) in the growth medium, suggesting nearly complete degradation of the insecticide. This strain is able to tolerate 45 degrees C and retain its degradation potential even under sunlight exposure. Since endosulfan is used worldwide for pest control and its residues have been retained for long periods in soil, water, and agricultural products, the strain isolated by us is valuable for bioremediation of endosulfan-contaminated soil and water.

Effect of solar UV radiation on earthworm (Metaphire posthuma).

Human health risks like damage to the eyes, immune system, and skin are known to be associated with increasing ultraviolet radiation (UVR) in the environment. In this study, we evaluated the phototoxic effects of UVR in sunlight and its possible mechanism of action by using earthworm as an alternative model because earthworm skin contains several biomolecules (tetraene and triene sterol) similar to human beings. We studied the generation of reactive oxygen species (ROS), photooxidation of lipids, and histopathological changes in earthworm integument. It was observed that UVR-exposed earthworm skin homogenate produced a significant amount of singlet oxygen ((1)O(2)), superoxide anion (O(2)(*)(-)), hydroxyl radicals ((*)OH), and photooxidation of lipids. The production of ROS and lipid peroxidation product was found dependent on the dose of solar UVR in earthworm integument. Histological anomalies such as thickening, vacuolation, and hypertrophy of epidermal cells were observed when the animals were exposed for 1 to 2h, while a higher exposure period (3h) caused degeneration of circular and longitudinal muscles. Continuous sunlight exposure for more than 3h was found lethal to worms. These observations suggested that the current level of UVR in sunlight may produce significant phototoxic effects in the earthworms probably via the generation of ROS (photodynamic action). Possible increases in UVR in view of ozone depletion may be more detrimental to the biomolecules in the worm's skin. The earthworm thus turned out as a simple, sensitive, and cost-effective test organism for the assessment of the hazardous potential of solar radiation and also for planning safety measures for human beings.

Morphological and metabolic alterations in duckweed (Spirodela polyrhiza) on long-term low-level chronic UV-B exposure.

Laboratory grown duckweed (Spirodela polyrhiza) plants were exposed to 0.72 and 1.44J of UV-B radiation daily for 7 days at 0.4mW/cm(2) intensity. Chlorosis and necrosis were observed along with depletion in protein, pigments (chlorophyll, pheophytin, carotenoids, phycoerythrin, phycocyanin, and flavoxanthin), biomass, root length, and frond size in UV-B-exposed plants. The study confirms morphological and metabolic alterations leading to reduction in the productivity of duckweed following long-term exposure to UV-B radiation.

Effect of UVB radiation on human erythrocytes in vitro.

In order to evaluate the UVB radiation induced phototoxic effect, the human erythrocytes (RBCs) were used an alternate biological model and rate of photohaemolysis was assessed in vitro at various intensities of UVB radiation (0-2.0 mW/cm2) for an exposure period of 0-240 min. The alterations of biochemical activities in RBC membrane (ghosts), caused by its exposure under an average incident intensity of UVB radiation (0.5 mW/cm2) in sunlight at earth surface, were also determined to understand the possible mechanism of photohaemolysis. We observed UVB dose dependent lysis of erythrocytes by recording haemoglobin and methemoglobin (oxidized form of haemoglobin) in photohaemolysate. We also observed significant inhibition in ATPase, acetylcholinesterase, glucose-6-phosphate dehydrogenase activites and an increased amount of thiobarbituric acid-reactive substance (TBA-RS) in RBC ghosts exposed to UVB radiation (0.5 mW/cm2) for a period of 0-100 min (doses: 0, 0.33, 0.66, 1.0, 1.5 and 3.0 J). The changes were found UVB dose dependent. A decrease of glutathione content in RBC ghosts at low dose level of UVB exposure (0.33 J) was found to be recovered at higher dose levels (0.66-1.5 J). These observations suggested, UVB dose dependent toxicity to human erythrocytes in vitro. Thus the erythrocytes can be used for an assessment of UVB induced biological effects and to understand possible mechanism of the phototoxicity.

Tubifex: a sensitive model for UV-B-induced phototoxicity.

The natural increase of UV-B radiation levels due to depletion of the ozone layer in the atmosphere may impose additional stress for the survival of zooplanktons which serve as a major constituent of the aquatic food chain. To study the adverse effects of UV-B radiation on the aquatic biomass, studies were conducted using the aquatic organism Tubifex as a model, as UV-B radiation is known to penetrate into the natural waters. UV-B radiation induced mortality in tubifex and the production of activated oxygen species by these organisms. Alterations in DNA, RNA, protein, glutathione (GSH), hydrogen peroxide H(2)O(2), thiobarbituric acid-reactive substance (TBA-RS), ATPase, AChE, GST, and LDH activities in Tubifex at various doses (0-2.0 J) of UV-B radiation were found. LC(50) value for UV-B-induced mortality of Tubifex was 0.80+/-0.15 J and the threshold dose was 0.35+/-0.05 J; mortality began within 3h postirradiation. UV-B dose-dependent production of singlet oxygen, superoxide anion, and hydroxyl radicals by Tubifex was observed. DNA, RNA, protein, and GSH contents were found to decrease significantly (P<0.001) while H(2)O(2) and TBA-RS increased (P<0.01) under the influence of UV-B radiation. The activities of ATpase, AChE, and GST enzymes were inhibited (P<0.01) and LDH activity was significantly increased (P<0.001) in Tubifex exposed to UV-B radiation. The results suggest that an increase in UV-B radiation alters several biochemical processes, leading to the mortality of the organism. Tubifex could be useful as a sensitive alternate model for studying UV-B-induced phototoxicity and possible mechanisms of action.

Evaluation of UV-radiation induced singlet oxygen generation potential of selected drugs.

Photosensitization reaction of drugs leading to the formation of reactive oxygen species under ultraviolet radiation (UVR) can cause tissue injury, resulting in damage to various cellular macromolecules. The aim of this study was to determine the singlet oxygen generation potential of some commonly used antibiotics so that due precautions can be exercised to minimize their photosensitizing action and oxidative stress potential. The selected antibiotics were examined for their ability to produce singlet oxygen (1O2) under artificial UVA (320-400 nm). Singlet oxygen generation of various screened antibiotics under UVA is of the following order: Nalidixic acid > Amphotericin-B > Cephradine > Cefazolin > Nafcillin > Cephalothin > Ampicillin > Cephalexin > Puromycin > Kanamycin > Lincomycin > Tetracycline > Nystatin > Gentamicin sulphate. Nalidixic acid, the most potent generator of 1O2 among the screened antibiotics, was selected to carry out further studies. Certain specific quenchers of 1O2 such as beta-carotene, 1,4-diazabicyclo[2.2.2] octane (DABCO), and sodium azide (NaN3) accorded significant inhibition in the production of 1O2. The results suggest that precautions are necessary to avoid ultraviolet radiation after the intake of photoreactive drugs, especially in tropical countries such as India. These findings are significant because UVB radiation is reportedly increasing on earth surface in part due to depletion of stratospheric ozone layer. The selected drugs are commonly used for the treatment of various diseases. Thus, the synergistic action of both can lead to undesirable phototoxic responses.

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 earth's 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.

Evaluation of UV-induced superoxide radical generation potential of some common antibiotics.

Photosensitization reaction of drugs leading to the formation of reactive oxygen species (ROS) cause tissue injury causing damage to various cellular macromolecules. The aim of this study was to determine the superoxide anion (O2-) generation potential of commonly used antibiotics so that due precaution could be exercised to minimize their photosensitizing action and oxidative stress potential. The selected antibiotics were examined for their ability to produce (O2-) under sunlight and artificial UVA (320-400 nm) and UVB (290-320 nm). Lincomycin, cephalothin and erythromycin generated significant amount of O2- under various irradiation conditions. Superoxide dismutase (SOD), an enzymatic quencher of O2- inhibited O2- production in all drugs tested. The results suggest that due precaution are necessary to avoid ultraviolet radiation (UVR) after the intake of photoreactive drugs, especially in tropical and sub tropical countries.

An unusual photohaemolytic property of riboflavin.

Riboflavin (RF) is a known photoreactive and phototoxic molecule. However, unlike other photosensitizers, it does not induce photohaemolysis of erythrocytes by itself. On the other hand, illuminated RF caused haemolysis but in the presence of serum or plasma. The kinetics of photohaemolysis in the presence of serum/plasma has been studied by monitoring the rate of haemolysis spectrophotometrically and morphological changes at erythrocytes membrane by scanning electron microscopy. We found that the extent of mammalian RBC membrane damage was dependent on the concentration of RF or hematoporphyrin (HP) (0-20 microgram/ml) and dose of sunlight (0-20 min). The RBC membrane-damaging potential of illuminated HP was not affected by the presence of plasma in the reaction system. Furthermore, RF showed a protective role against photohaemolysis caused by photoexcited HP if erythrocytes were preincubated with RF in the absence of serum/plasma. For mechanistic studies, biochemical parameters such as acetylcholinesterase activity (AChE) and formation of TBA-reactive substance (TBA-RS) were analysed in RBC and RBC+plasma under a similar set of experimental conditions. We observed about a 25% decrease in AchE activity as a synergistic action of RF or HP (20 microgram/ml) and sunlight (30 min) in both cases (RBC or RBC+plasma). Interestingly, illuminated RF caused about a 125% increase of TBA-RS in a reaction system consisting of RBC+plasma. On the other hand, an increase in the production of TBA-RS by illuminated RF was not observed in the absence of plasma/serum, in the reaction system. These results suggested that photooxidation of RBC membrane lipids by illuminated RF, under the influence of plasma/serum, may be one of the causes of membrane modification leading to RBC lysis.

Chromate tolerant bacteria isolated from tannery effluent.

The occurrence of metal tolerant and antibiotic resistant organisms was investigated in tannery effluent. Seventy-seven isolates comprising heterotrophs (41) and coliforms (36) which were tolerant to chromate level of > 50 microg/ml were selected for detailed study. The majority of the coliforms were resistant to higher levels of chromate (200 microg/ml) whereas around 3% of the heterotrophs were resistant to Cr6+ at a level of > 150 microg/ml. All chromate tolerant heterotrophs were also tolerant to Cu2+ (100%) whereas only 58.53% coliforms were tolerant to Cu2+. Except in the case of Cd2+ a higher number of heterotrophs were found tolerant to other heavy metals tested. Both groups of isolates were found sensitive to mercury. Resistance to cephaloridine was more abundant (P < 0.001) in coliforms as compared to heterotrophs. On the other hand a significantly higher number (P < 0.01) of heterotrophs showed resistance to streptomycin and carbencillin. All coliforms were sensitive to chloramphenicol. Around 80%) and 31.70% of coliforms and heterotrophs exhibited a relationship to the combination of metals and antibiotics. Both heterotrophs and coliforms tolerant to Hg2+ were also resistant to polymixin-B.

Effect of lindane on the growth and metabolic activities of cyanobacteria.

The effect of lindane was studied in Cyanobacteria (Anabaena) by estimating its growth pattern, biomass yield, chlorophyll content, and total starch and protein content. The results reveal that exposure of Anabaena with 0.5, 1.0, 1.5, and 2.0 ppm levels of lindane will have drastic effects on biomass production and photosynthetic rate. Since Cyanobacteria are a primary source of aquatic food web and important biofertilizer for rice cultivation, their protection from residual effects of lindane is essential for enriched soil fertility.

Sensitivity of duckweed (Lemna major) to ultraviolet-B radiation.

The sensitivity of an important aquatic macrophyte, duckweed (Lemna major), to UV-B radiation was studied under experimental conditions at three different doses designated as no, mild, and severe injury dose by observing visible injury symptoms and estimating levels of chlorophyll, pheophytin, carotenoids, protein, starch, free sugar, and peroxidase activity. Laboratory-grown duckweed plants were exposed to UV-B radiation at 0.4 mW/cm(2) intensity for different time periods. Mild and severe injury were developed at 6.48 and 8.64 J, respectively. Peroxidase activity increased at all the exposure levels. Dose-dependent decrease in chlorophyll and starch with drastic depletion in protein and free sugar content were observed. Pheophytin and carotenoids content increased at no injury level, but decreased at higher exposure level. The results indicate that ambient UV-B radiation at the indicated level acts as a physiological stress in Lemna major.