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.

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.

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.

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.

Phototoxicity evaluation--Tetrahymena thermophila as an alternative model.

Interest in utilizing an alternative to animal method for toxicological evaluation has received considerable attention due to cost effectiveness and the ethical issues involving animal experimentation. Alternative methods for phototoxicity evaluation are significant because of growing concern over increasing health effects due to stratospheric ozone depletion resulting in an increasing penetration of ultraviolet light-B radiation (UVB, 290-320 nm) which contributes to activation of chemical and biological molecules to potential phototoxic agents. The classic rabbit eye-irritancy test referred to as Draize test has been the subject of severe criticism by animal welfare groups. Dermal toxicity test using guinea pigs and mouse tail phototoxicity test is time consuming and requires a large number of laboratory animals. In photohaemolysis assay some of the phototoxic agents (such as riboflavin) react with the membrane proteins of the erythrocyte. However, in vitro test system using protozoa offers a promising alternative means of phototoxicity evaluation. Our previous studies have demonstrated that synergistic action of photochemically reactive agents and sunlight produces lethal effects to Paramecium but the protozoan has not received serious consideration for use as an alternative model for phototoxicity evaluation. In the present communication we have described the potential application of Tetrahymena as an alternative model to study the radiation-induced changes both in the presence or absence of photoreactive chemical agents. This model is likely to provide scope for studying the biological effects of environmental UVB radiation, DNA damage and defence against oxidative stress.

Ultraviolet-B effects on ocular tissues.

The enhancement of UVB on the surface of the earth due to stratospheric ozone depletion may increase the risk of photochemically induced cataract. In this study, changes in the content of certain biological parameters were used as a marker to study ocular toxicity. A significant enhancement in the level of H2O2, LP and Pi, along with the depletion of GSH (antioxidant-defence system) in serum, aqueous humor and lens of albino mice exposed to UVB was observed. The level of Ca++ increased in serum and aqueous humor but decreased considerably in the lens due to increase in UVB exposure. These observations provide further clues to support the involvement of oxidative stress and Ca++ in the events leading to the formation of cataract.

Membrane damaging potential of photosensitized riboflavin.

Riboflavin upon exposure to UV and visible radiations has been shown to produce active oxygen species. The present work deals with erythrocyte membrane as model system to study the damaging potential of photosensitized riboflavin. Membrane preparations (2.5 mg protein/ml) following exposure to sunlight in presence of riboflavin for different time intervals revealed significant inhibition of ATPases, p-nitrophenyl phosphatase and acetylcholinesterase. Considerable increase in lipid peroxidation was caused by the photosensitized riboflavin. Quenching studies using specific scavengers indicated remarkable inhibition. The production and identification of reactive oxygen species by photosensitized riboflavin and their possible involvement in membrane damaging effect has been discussed.

Photosensitizing potential of benzanthrone.

In skin-photosensitization studies on guinea-pigs benzanthrone induced significant erythema and oedema, depending upon the doses both of benzanthrone and of sunlight or artificially simulated UVA radiation. Maximum sensitization and delayed tanning response on the guinea-pig skin were observed 24-36 hr after irradiation. Photosensitized benzanthrone was found to produce significant amounts of singlet oxygen in vitro, as assessed by the bleaching of N,N-dimethyl-p-nitrosoaniline. As with haematoporphyrin and rose bengal, both of which are potent generators of singlet oxygen, the production of singlet oxygen by benzanthrone was dependent on both the concentration of the test chemical and the dose of solar radiation. Benzanthrone also produced a significant yield of superoxide anion radicals on exposure to simulated solar radiation or sunlight. Photosensitized benzanthrone induced photohaemolysis of rat erythrocytes and lipid peroxidation of erythrocyte ghosts, in vitro, probably largely through involvement of singlet oxygen (1O2). The rate of lipid peroxidation by photosensitized benzanthrone was inhibited by 64-80% with 1,4-diazabicyclo(2,2,2)octane and sodium azide, 15% with superoxide dismutase but was not affected by mannitol and sodium benzoate. Equimolar concentrations of haematoporphyrin and rose bengal also produced considerable photohaemolysis of erythrocytes and lipid peroxidation in erythrocyte ghosts; in both cases rose bengal was the most active and benzanthrone the least active of the three compounds.

Evaluation of chemically-induced phototoxicity to aquatic organism using Paramecium as a model.

Phototoxicity evaluation using Paramecium aurelia as a model revealed that 4 out of 21 pesticides produced lethal toxicity to cells. Four commonly used synthetic dyes (bromophenol blue, rose bengal, benzanthrone and methylene blue) also exhibited toxicity. Well known phototoxic agents like hematoporphyrin, riboflavin, and anthracene produced positive phototoxic response. Psoralen, a DNA cross-linking agent, also produced phototoxicity to the cells. The results clearly demonstrate that the synergistic action of chemical agents and sunlight produce lethal effects to aquatic organism.