- Email: [email protected]
Oxidative stress-induced cellular damage caused by UV and methyl viologen in Euglena gracilis and its suppression with rutin
Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116–129 www.elsevier.com / locate / jphotobiol
Oxidative stress-induced cellular damage caused by UV and methyl viologen in Euglena gracilis and its suppression with rutin Helen Palmer, Mari Ohta, Masumi Watanabe, Tetsuya Suzuki* Laboratory of Food Wholesomeness, Department of Life Sciences, Graduate School of Fisheries Science, Hokkaido University, 3 -1 -1 Minato, Hakodate 041 -8611, Japan Received 13 June 2001; accepted 30 January 2002
Abstract The effects of ultraviolet radiation (UV-A: 320–400 nm and UV-B: 280–320 nm) and methyl viologen (MV) single or combined exposure, on the cell growth, viability and morphology of two strains of the unicellular flagellate Euglena gracilis, using the Z strain as a plant model and the achlorophyllous mutant SMZ strain as an animal model were investigated. Cell growth was not affected by MV only, whereas UV-A or UV-B single and combined exposure with MV inhibited the cell growth or decreased the viability. The SMZ strain had a higher number of abnormal cells than the Z strain after the third dose of UV-B was delivered simultaneously with MV. The abnormal cell number decreased when E. gracilis SMZ cells were preincubated with 100 mM rutin prior to the UV-B and MV exposure. There were higher abnormal cell numbers with groups exposed to UV rather than MV single exposure. Combined exposure to UV-B and 200 mM MV induced the highest levels of TBARS in both strains, and with the supplementation of rutin these high levels were suppressed. These results suggest that UV-A or UV-B irradiation alone or combined with MV cause considerable oxidative damage in E. gracilis cells, and rutin supplementation may suppress their adverse effects. 2002 Elsevier Science B.V. All rights reserved. Keywords: Euglena; Methyl viologen; Oxidative stress; Rutin; TBARS; Ultraviolet ray irradiation
1. Introduction Depletion of the ozone layer causes an increase in UV radiation that can cause serious damage to all living organisms on the earth [1]. Until recently, UV-B radiation (280–320 nm) was difficult to measure [1–3] because of its natural variability in UV radiation levels across the Earth’s surface. But UV-B radiation can cause damage to both aquatic and terrestrial habitants [4,5]. It has been demonstrated that UV-B reduces phytoplankton productivity [6], and in the Gulf sea UV-A (320–400 nm) radiation penetrates to depths of 23 m and UV-B penetrating from 7 to 12 m, resulting in mortality of developing embryos of larvae of Atlantic cod through oxidative stress [7]. On the other hand, organic pollutants that are discharged into wastewater have complex interactions with UV irradiation, which in turn affect algae [8]. In agriculture, numerous pesticides and herbicides are used to sterilize seeds for the prevention of seed-borne diseases. However, pesticides and herbicides are major environmental pollu-
*Corresponding author. Tel. / fax: 181-138-405-564. E-mail address: [email protected] (T. Suzuki).
tants, and the distribution of toxic substances in the environment is a growing concern. Methyl viologen (1,19-dimethyl-4,49-bipyridinium-dichloride), which is commercially known as paraquat, is a water-soluble herbicide that is widely used in many areas of the environment. It is one of the most potent pulmonary toxins [9]. The herbicidal effect of methyl viologen (MV) depends on light and oxygen; MV interferes with the photosystem by disrupting electron transport and catalyzing the production of active oxygen within the chloroplast [10–12]. Furthermore, according to Babbs et al. [13], MV readily produces hydroxyl radicals through either a superoxide-driven Fenton reaction or a Fenton-like reaction, which explains its potent toxicity. Numerous studies have been carried out on the adverse effects of environmental pollutants, and excess exposure to UV rays, which are a threat to almost all living organisms including humans. As a primary producer, phytoplankton constitutes the first level of the intricate food web in the ocean and is the main basis of the diet of fishes, crustaceans and mollusks [5]. The generation of cyclobutane pyrimidine dimers is a major type of DNA damage induced by UV-B irradiation [14]. Although UV-B wavelength range is more photo-
1011-1344 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S1011-1344( 02 )00271-3
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
chemically active than UV-A [15], it is now evident that chronic UV-A exposure at low doses also induces damage in human and animal skin [16]. Furthermore, UV-A also produces oxidative DNA damage mainly to guanine [14], therefore, adverse effects of UV-A are also causing concern. Here, we have investigated the hypothesis that cellular damage is induced through interactions of reactive oxygen species generated by UV irradiation and MV that has been discharged into the environment. We examined the adverse effects of exposure to UV irradiation at different wavelengths, 320–400 nm (UV-A) and 280–320 nm (UV-B) and MV when exposed alone or simultaneously. The eucaryote unicellular flagellate Euglena gracilis, a type of fresh water plankton, was used to assess the effects of UV and MV single or combined exposure. E. gracilis Z strain can photosynthesize, however, antibiotic streptomycin is able to eliminate chloroplasts from E. gracilis [17], to form a mutant achlorophyllous SMZ strain without interfering with the cell division. Notably, both strains have highly sophisticated subcellular organella equivalent to those of higher mammals; the E. gracilis Z strain can be regarded as a plant model, and its mutant strain SMZ as an animal model. Considering their properties, E. gracilis Z and SMZ strains were used as the model organisms in this study comparatively, to assess the effects of UV-A or UV-B, and MV when each were exposed either alone (UV-A, UV-B, MV), or combined (UV-A or UV-B1MV) on the cell morphology, viability and growth. The effect as mentioned above, of UV radiation and MV inducing oxidative stress or generating free radicals [10,18], are considered highly likely to affect biomembrane lipids [19]. Thus, to assess the deterioration of membrane lipids, we examined levels of thiobarbituric acid reactive substance (TBARS) after UV irradiation or MV single or combined exposure in this study. It has been reported previously that flavonoids are effective reactive oxygen species scavengers that protect plants from potentially harmful UV-B radiation [20]. Investigations also proved that flavonoids including rutin, can capture O 2 2 [21]. Rutin has also been reported to be effective in scavenging reactive oxygen species and suppressing lipid peroxidation [22]. Therefore, using rutin as a potential natural antioxidant scavenger, we also examined its suppressant effect by measuring the levels of TBARS in rutin-supplemented cells exposed to MV and UV-irradiation. In order to identify whether rutin could also be effective in protecting against UV- and MV-induced cellular damage, cell growth, viability and morphology were assessed with rutin preincubated E. gracilis cells. 2. Materials and methods
2.1. Reagents and chemicals Methyl viologen (1,19-dimethyl-4,49-bipyridinium-di-
117
chloride), purchased from Nacalai Tesque (Kyoto, Japan), was dissolved in Koren Hutner (KH) medium and used in the assay at concentrations of 100 and 200 mM. Control solutions without MV were also prepared. Rutin, purchased from Nacalai Tesque, was used to determine whether or not UV- and MV-induced cellular damage could be suppressed by its supplementation or preincubation. Rutin was prepared in phosphate buffer solution, pH 7.4. All other chemicals used were guaranteed reagent grade.
2.2. Organism used and incubation conditions E. gracilis Z and SMZ strains (kindly provided by Professor Y. Nakano, Laboratory of Nutritional Chemistry, Osaka Prefectural University) were grown at 29 8C (61 8C) in 5 ml of KH medium [17], in test-tubes under illuminated (3200 lx) fluorescent light for plant growth on a light / dark cycle of 12 / 12 h until early logarithmic growth phase. Illumination intensity was measured regularly using a Digital Lux Meter Model: LX-1330 / LX-1332 (Custom Corporation Tokyo, Japan).
2.3. UV irradiation UV-A (365 nm, 4 W Black Light Matsushita Electric Co., Osaka) or UV-B (model UVM-57, 302 nm, 6 W, equipped with 2 UVG filter to cut UV-C; UVP Inc., Upland, CA, USA), were used for UV irradiation in the present study. Cells in the petri-dishes were exposed to UV-A or UV-B as follows. The distance between the petri-dishes and the UV source was adjusted, and then the cells were exposed to UV-A or UV-B rays at 3 W m 22 ; UV-A and UV-B dose delivered was 0.36 J cm 22 for each exposure. The UV-A and UV-B lamps were fixed in dark black boxes and all other fluorescent light or daylight was excluded during the irradiation of the petri-dish samples. The UV exposures were delivered once a day for 3 consecutive days, and UV output was monitored simultaneously using a radiometer (VLX-3W Vilber lourmat, Torcy, France), with a 365 nm and 305 nm detector placed at the same distance from the UV source as the petri-dish samples. Approximately 1 h after UV irradiation, the cells were observed under a microscope before being placed in the light cycle of the incubator. The UV exposure of all samples was carried out daily from 14:00 to 16:00 h for 3 days. Cellular damage in E. gracilis was compared between cells undergoing UV-A or UV-B irradiation only, cells exposed singularly to 200 mM of MV, and those undergoing combined UV and MV treatment. The morphological change of cells preincubated with 100 mM rutin prior to UV-A or UV-B single exposure, or combined with MV were also assessed. The experimental procedures are described in Section 2.10. After the UV irradiation cells were observed, hypertrophy, V-shaped, starfish-shaped and those that were not completely divided were considered as abnormal. The morphology of both strains were observed
118
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
under a video microscope using an Olympus IMT-2 inverted microscope (Olympus Optic Co., Inc., Tokyo) equipped with an image processor, ARGUS-100 (Hamamatsu Photonics Co., Hamamatsu, Japan).
2.4. Exposure to MV To examine the effect of MV exposure on E. gracilis, cells from a liquid culture (10 5 –10 6 cells ml 21 ), were adjusted to a density of 1.6310 4 cells ml 21 , and 50 ml of the cell suspensions were inoculated into 3 ml of KH medium containing either 100 mM or 200 mM MV in small glass petri-dishes. These petri-dishes were then exposed to UV-A or UV-B as described in Section 2.3. For samples exposed to UV-A or UV-B single exposure, cells were inoculated into 3 ml of KH medium without MV.
2.5. MV single exposure The effect of MV single exposure on the cell morphology and growth of E. gracilis Z and SMZ strains was
investigated in order to compare single and combined stress-induced cellular damage. For the morphological assessment, cells were inoculated into microtiter wells containing either 100 mM of MV in KH medium or KH medium alone (as control cells), and incubated under illuminated conditions as described in Section 2.2. Observations for morphological changes were carried out daily under the video microscope, as explained in Section 2.3.
2.6. Cell growth and viability of E. gracilis exposed to single or simultaneous UV and MV exposure The effect of MV single exposure on cell growth was assessed by inoculating 30 ml of 10 6 cells ml 21 of E. gracilis into 5 ml of KH medium containing 100 mM MV in test-tubes. Cells were incubated under either light / dark conditions (described in Section 2.2) or a 24-h dark cycle, as the herbicidal effect of MV is oxygen- and lightdependent. The cell density was measured using a Bosch and Lomb spectrometer (Shimadzu Instruments, Kyoto,
Fig. 1. Effect of methyl viologen exposure on the cell growth of Euglena gracilis. Control, the cell growth of E. gracilis cells not exposed to MV under 12 / 12 h light dark cycle; 100 mM MV, 12 / 12 h light dark cycle exposed to 100 mM MV; control / dark, not exposed to MV under dark cycle; 100 mM MV, exposed to 100 mM under dark cycle at 28 8C for 3 days. Cell density at 610 nm was measured daily.
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
Japan) over 3 days (results shown in Fig. 2). The growth of E. gracilis Z and SMZ strains exposed to either UV-A or UV-B single exposure, or combined exposure with MV was determined by measuring the cell abundance in 10 ml of cell suspension taken from the petri-dish samples. The estimation of cell numbers was performed under the microscope (n518).
2.7. Cell viability To estimate cell viability, 100 ml of E. gracilis cell suspension were taken from each petri-dish, centrifuged at 10003g (23 8C for 3 min), washed twice with 100 ml of phosphate saline buffer (PBS, pH 7.2), and then stained with 20 ml of 0.4% Trypan Blue solution at 28 8C for 1 h. After staining, cells were washed again with 100 ml of
119
PBS solution. The non-viable blue-stained cells were detected by an Olympus IMT-2. The numbers of nonviable cells in six randomly selected frames were counted in triplicate (cell values were expressed as the average viable cell percentage of the total cell number6S.D., n536 measurements). Viable cells were calculated as the difference between the total number of cells and the number of non-viable cells. Cell viability was expressed as the percentage of viable cells over total cells.
2.8. Estimation of abnormal cell occurrence V-shaped, hypertrophied and starfish-shaped cells were classified as abnormal cells (Fig. 4). The frequency of abnormal cells was estimated by counting the number of abnormal cells in six frames for each sample. Values of
Fig. 2. Effect of UV-A or UV-B irradiation and MV exposure on the cell growth of E. gracilis. Cell growth was measured after each exposure to UV irradiation. (A and C) Z strain; (B and D) SMZ strain. Cell growth of cells exposed to: no UV irradiation or MV (control), UV-A irradiation (UV-A), UV-A irradiation and 100 mM MV (UV-A1100 mM MV), UV-A irradiation and 200 mM MV (UV-A1200 mM MV), UV-B irradiation (UV-B), UV-B irradiation and 100 mM MV (UV-B1100 mM MV), UV-B irradiation and 200 mM MV (UV-B1200 mM MV), MV represents methyl viologen. Each daily exposure of UV-A or UV-B irradiation was 0.36 J cm 22 . Each bar represents the mean6S.D. (n518). Symbols a, b, c, d indicate a significant difference between each group (P,0.005) after the third day of exposure.
120
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
abnormal cells were expressed as the mean percentage of the total observed frames obtained from 36 measurements.
2.9. Estimation of TBARS levels It was considered that assessing the TBARS level of UV-A or UV-B with or without MV exposed E. gracilis cells may be an indication of induced damage to lipid components. Cells were delivered UV-A or UV-B doses of 0.78 J cm 22 at 3 W m 22 exposed with or without 200 mM MV. TBARS values of cells exposed to 200 mM MV only were also assessed.
2.10. Rutin supplementation To assess whether the UV-B- and MV-induced lipid peroxidation could be suppressed by rutin, either 50 mM or 100 mM rutin was added to E. gracilis cells immediately before exposure to UV irradiation and MV. After an acute exposure of UV-B irradiation at a dosage of 0.78 J cm 22 (3 W m 22 ), the cell suspension samples were taken and immediately centrifuged at 10003g (23 8C for 3 min), placed in a cryogenic tube, and kept at 280 8C until TBARS analysis. The levels of TBARS were estimated following the method of Kikugawa et al. [23].
Fig. 3. Effect of UV irradiation and MV exposure on the cell viability of E. gracilis. Cell viability was tested after each exposure to UV irradiation. (A and C) Z strain; (B and D) SMZ strain. The viable cell percentages on the third day are also shown separately in E to H. (E and G) Z strain; (F and H) SMZ strain. Viable cells exposed to no UV or MV (control), UV-A irradiation (UV-A), UV-A irradiation and 100 mM MV (UV-A1100 mM MV), UV-A irradiation and 200 mM MV (UV-A1200 mM MV), UV-B irradiation (UV-B), UV-B irradiation and 100 mM MV (UV-B1100 mM MV), UV-B irradiation and 200 mM MV (UV-B1200 mM MV), MV represents methyl viologen. Each daily exposure of UV-A or UV-B irradiation was 0.36 J cm 22 . Each bar represents the mean6S.D. (n536). Symbols, a, b, c, d indicate a significant difference between each group (P,0.005) after the third day of exposure of UV irradiation.
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
2.11. E. gracilis cells preincubated with rutin The cell growth, viability and morphology of E. gracilis cells incubated with 100 mM of rutin was also assessed to determine whether rutin would suppress cellular damage induced by UV single exposure or when combined with MV. E. gracilis cells were preincubated with 100 mM of rutin (adjusted pH 7.4 with PBS buffer) for 24 h, washed three times with KH medium, after which they were resuspended in petri-dishes with or without MV, as described in Section 2.4. UV irradiation was carried out
121
according to the protocol of Section 2.3 and the determination of cell growth, viability and the estimation of abnormal cells following the procedure of Sections 2.6, 2.7 and 2.8.
2.12. Statistical analysis The data were evaluated by analysis of variance (ANOVA). A value of P,0.005 was considered to be significant.
Fig. 3. (continued)
122
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
3. Results
3.1. Cell growth and viability The cell growth of cells exposed to MV alone at 100 mM is shown in Fig. 1A and B. For both Z and SMZ strains there was no growth inhibition observed for the cells incubated either in light / dark or dark conditions over the 3-day period when the cell density was monitored. This reflects that under light / dark or under dark incubation conditions, oxidative stress derived from MV exposure alone was probably not strong enough to inhibit the cell growth. UV single irradiation and UV exposed with MV inhibited cell growth, and there was a greater inhibition after UV-B irradiation than UV-A. As there was little significant difference (P,0.005) between the cell growths of UV-B only, and UV-B combined with MV exposed cells in both strains, UV-B may have been the dominant cause of the inhibition (Fig. 2C and D). The UV-B dosage in the present study inhibited growth to almost the same extent in both strains (Fig. 2C and D). The cell viability of E. gracilis Z and SMZ strains was also affected by both UV-A and UV-B irradiation (Fig. 3). Cell viability in both strains decreased gradually after exposure to UV-A irradiation, however the viable cell numbers decreased significantly (P,0.005) in the higher concentration of MV in comparison with lower concentration of MV (Fig. 3A and B). In contrast, UV-B exposure had different effects on both strains, causing a more marked decrease in the viability of the SMZ strain than in the Z strain, after the third UV-B dosage (Fig. 3C and D). For both strains, UV-B irradiation caused a considerably higher reduction of viable cells after 3 days than UV-A (Fig. 3E to H).
3.2. Cell morphology In both Z and SMZ strains, the most frequently observed abnormal cell was V-shaped, followed by hypertrophied and starfish-shaped cells under the experimental conditions. The normal shape of E. gracilis cell (a), V-shaped (b), starfish-shaped (c), hypertrophy (d) cells that were observed under different exposure conditions are shown (Fig. 4). UV-A or MV alone induced between 6 and 8% abnormal cell outbreak (Fig. 5A and B), whereas combined exposure of UV-A and MV significantly (P,0.005) increased abnormality by twofold after the third exposure (Fig. 5A and B). (The abnormal cell percentages on the third day are also shown separately in Fig. 5E–H.) There was no significant difference in abnormality between cells exposed to 100 mM and those exposed to 200 mM MV even when combined with UV-A exposure. Single or combined exposure to UV-B and MV caused a similar cell abnormality to the UV-A and MV in the Z strain (Fig. 5A and C). However, there was a marked
Fig. 4. Cell morphology of E. gracilis cells exposed to UV irradiation and MV. Abnormal cell occurrence of E. gracilis cells exposed to UV-A or UV-B single or combined with MV exposure was observed. The normal shape of E. gracilis cell (a), V-shaped (b), starfish-shaped (c), hypertrophy (d) cells that occurred. Bar in each photograph represents 20 mm.
difference between UV-A and UV-B exposed groups in the SMZ strain (Fig. 5B and D). Distinctly more abnormal cells (27%) were observed after UV-B irradiation alone;
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
123
Fig. 5. Effect of UV irradiation and MV exposure on the cell morphology of E. gracilis. (A and C) Z strain; (B and D) SMZ strain. Abnormal cell percentages on the third day are also shown separately in E to H. (E and G) Z strain; (F and H) SMZ strain. The abnormal cell percentage of cells exposed to no UV or MV (control), UV-A irradiation (UV-A), methyl viologen only (MV), UV-A irradiation and 100 mM MV (UV-A1100 mM MV), UV-A irradiation and 200 mM MV (UV-A1200 mM MV), UV-B irradiation (UV-B), UV-B irradiation and 100 mM MV (UV-B1100 mM MV), UV-B irradiation and 200 mM MV (UV-B1200 mM MV). Each daily exposure of UV-A or UV-B irradiation was 0.36 J cm 22 . Each bar represents mean percentage of abnormal cells6S.D. (n536). Symbols a, b, c, d indicate a significant difference between each group with P,0.005 after the second and the third day of exposure.
furthermore, the combined exposure of UV-B and MV, especially 200 mM MV, greatly increased the number of abnormal cells (76%) after the third exposure (Fig. 5D and H). On the third day, significant difference (P,0.005) was recognized between sole UV-B exposure and combined exposure of UV-B and MV; i.e. more abnormal cells with UV-B and 200 mM MV than with UV-B and 100 mM MV exposed cell groups.
3.3.2. The effect of rutin supplementation Cells supplemented with 100 mM rutin immediately before the UV-B exposure showed an extremely marked decrease in TBARS levels. This was apparent in both Z and SMZ strains. The 50 mM supplementation also suppressed the TBARS levels to an extent (Fig. 6A and B). 3.4. Effects of preincubation with rutin on cell growth, viability, and morphology
3.3. TBARS 3.3.1. Exposure to MV and /or UV-A or UV-B Simultaneous exposure to UV-B and MV (UV-B1MV) resulted in the highest levels of TBARS in both the Z and SMZ strains of E. gracilis (Fig. 6A and B). A considerably higher TBARS level was obtained with the Z strain. Furthermore, with the exception of the UV-A exposed cell group, all exposed groups displayed a significant difference (P,0.005) (Fig. 6A). The SMZ strain, however, had far lower TBARS levels than the Z strain in all the exposed groups.
E. gracilis cells preincubated with rutin prior to the UV-A or UV-B exposure did not display any growth recovery compared to the non-treated cells (Fig. 2A–D; Fig. 7A and B). Both UV-A and UV-B irradiation alone and when combined with MV significantly (P,0.005) inhibited the cell growth compared to the control group in the Z strain, however, UV-A was not significantly different from the control group in the SMZ strain (Fig. 7B). The cell viability of the cells preincubated with rutin appeared slightly higher than the non-preincubated cell groups, after the third exposure to sole UV-B irradiation or combined
124
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
Fig. 5. (continued)
with MV exposure (Fig. 3E and F; Fig. 8A and B) in both strains. Abnormal cells appeared to be lower in cells preincubated with rutin when cells were simultaneously exposed to UV-A or UV-B and MV in the SMZ strain (Fig. 9B), than the non-treated cells (Fig. 5D).
4. Discussion The adverse effects of UV irradiation on the growth and motility [24] and the photosynthetic efficiency [8] of E.
gracilis have been reported previously. Results showed that UV irradiation inhibits photosynthesis and cell growth. However, there are few studies reporting the combined effects of UV irradiation and toxic substances on E. gracilis. The lack of growth inhibition by 100 mM of MV, coupled with the severe growth inhibition by UV-A or UV-B with or without MV in either strain (Fig. 2), indicates that UV-A or UV-B are the dominant contributors to the inhibition of cell growth. UV-B irradiation alone or combined with exposure to MV, had an adverse effect on
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
Fig. 6. Levels of TBARS in E. gracilis cells exposed to UV and MV and the effect of rutin supplementation. (A) Z strain; (B) SMZ strain. Cells exposed to no UV or MV (control), UV-A irradiation (UV-A), UV-B irradiation (UV-B), 200 mM methyl viologen (200 mM MV), UV-A irradiation and 200 mM methyl viologen exposure (UV-A1MV), UV-B irradiation and 200 mM methyl viologen exposure (UV-B1MV), UV-B irradiation and 200 mM methyl viologen exposure with 50 mM rutin (UV-B1MV1R 50 mM), UV-B irradiation and 200 mM methyl viologen with 100 mM rutin (UV-B1MV1R 100 mM). The total exposure energy of UV-A and UV-B was 0.72 J cm 22 . Each bar represents the mean value6S.D. (n59). Cells were analyzed immediately after UV irradiation.
125
cell viability in both the Z and SMZ strains of E. gracilis (Fig. 3C and D). Furthermore, the SMZ strain had a lower survival ratio than the Z strain after UV-B irradiation with or without MV on the third day (Fig. 3C, D, G, H). Shigeoka et al. reported a peroxidase in E. gracilis Z that requires ascorbic acid as the electron donor in the cytosol that scavenges H 2 O 2 , as E. gracilis does not contain catalase [25]. Ascorbate peroxidase (APX) is found only in higher plants and algae, but no report in animals. It was found that the APX content in E. gracilis Z strain, a plant model, was 5.157 mmol / mg protein / min and in SMZ strain, an animal model, 0.826 mmol / mg protein / min, in cells incubated under almost the same conditions as that of the present study (unpublished data). Therefore, the lower APX content may have resulted in less viable cells in the SMZ strain after the third dosage of UV-B with or without MV, indicating different reactive oxygen scavenging abilities between the two strains. On the other hand, the viable cell percentages were higher in cells preincubated with 100 mM rutin prior to UV-B single or combined MV exposure (Fig. 8A and B) than non-preincubated cells in both strains (Fig. 3E and F). The percentage of abnormal cells increased when cells were exposed simultaneously to MV and either UV-A or UV-B, suggesting that two consecutive daily exposures of the combined exposure generated sufficient reactive oxygen species to induce teratogenicity in E. gracilis cells. The combined exposure of UV-B and 200 mM MV enhanced the occurrence of abnormal cells in SMZ strain after the third exposure (Fig. 5H). This high incidence of abnormal cell proliferation in the SMZ might result from the lower potential of its reactive oxygen scavenging system as mentioned previously. Abnormal cell proliferation must be linked to dysfunction of the cell-cycle regulation system, which frequently involves impairment of DNA duplication, protein synthesis, membrane lipid synthesis and organization of subcellular organella and their function. Scheuerlein et al. reported the evidence for UV-B-induced DNA degeneration in E. gracilis mediated by activation of metal-dependent nucleases [26]. On the other hand, cells preincubated with 100 mM of rutin before the combined exposure of UV and MV, displayed a considerably lower abnormal cell number after the third UV-A or UV-B dosage (Fig. 9B) than non-treated cells (Fig. 5H). Whether the decrease was a result of rutin, acting as intracellular or extracellular reactive oxygen scavenger or not is not yet clear, and this decrease was only observed in the SMZ strain. UV-A alone, or combined with exposure to MV, did not increase the levels of TBARS as much as combined with UV-B and MV. This might be due to the shorter wavelength of UV-B which may have a greater threat to unprotected molecules [15]. Cell growth (Fig. 1) and abnormal cell proliferation (Fig. 5) were either unaffected or only slightly affected by simple exposure to 200 mM MV in both Z and SMZ strains. Furthermore, simple exposure to MV did not signifi-
126
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
Fig. 7. Effect of UV and methyl viologen exposure on the cell growth of Euglena gracilis preincubated with rutin. E. gracilis cells preincubated with rutin were exposed to UV and MV and the cell growth measured after the third exposure to UV irradiation. (A) Z strain; (B) SMZ strain. After the third exposure of UV, the cell growth of cells exposed to no UV or MV (control), UV-A irradiation (UV-A), UV-A irradiation and 200 mM MV (UV-A1200 mM MV), UV-B irradiation (UV-B), UV-B irradiation and 200 mM MV (UV-B1200 mM MV), MV represents methyl viologen. Each daily exposure of UV-A or UV-B irradiation was 0.36 J cm 22 . Each bar represents the mean6S.D. (n518). Symbols a, b, c, d indicate a significant difference between each group (P,0.005) after the third exposure.
cantly increase TBARS levels in the SMZ strain, however, a significant (P,0.005) increase was observed in the Z strain (Fig. 6). The highest TBARS level (136 nmol g 21 ) was obtained from combined exposure to UV-B and MV in the Z strain; by contrast, in the SMZ strain this level was below 100 nmol g 21 . Comparing the results from the TBARS (single exposure at a higher UV energy level) (Fig. 6) with those of the cell growth (Figs. 1 and 2), viability (Fig. 3) and morphology assessment (chronic exposure at lower UV energy level) (Fig. 5), UV- and MV-induced oxidative damage may have differed between the two strains. The TBARS levels were
higher in the Z strain (Fig. 6), however, abnormal cell occurrence was generally higher in the SMZ strain (Figs. 5 and 9). Pretreatment with rutin definitely decreased the frequency of the abnormal cells in both strains, especially in combined exposure of UV-B and 200 mM MV in SMZ. The abnormal cells in SMZ under the combined exposure of UV-B and 200 mM MV without rutin treatment, remarkably decreased by almost 60% with pretreatment with rutin. However, the effect of rutin was not so remarkable in Z strain. This may be due to different reactive oxygen scavenging systems and membrane lipid components between the E. gracilis Z and SMZ strains.
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
Fig. 8. Effect of UV irradiation and MV exposure on the viability of E. gracilis preincubated with rutin. E. gracilis cells preincubated with rutin were exposed to UV and MV and the cell viability tested after the third exposure to UV irradiation. (A) Z strain; (B) SMZ strain. After the third exposure of UV cell viability of cells exposed to no UV or MV (control), UV-A irradiation (UV-A), UV-A irradiation and 200 mM MV (UV-A1200 mM MV), UV-B irradiation (UV-B), UV-B and 200 mM MV (UV-B1200 mM MV), MV represents methyl viologen. Each daily exposure of UV-A or UV-B irradiation was 0.36 J cm 22 . Each bar represents the mean6S.D. (n536). Symbols, a, b, c, d indicate a significant difference between each group (P,0.005) after the third day.
127
Fig. 9. Effect of UV irradiation and MV in the cell morphology of E. gracilis preincubated with rutin. (A) Z strain; (B) SMZ strain. After the third exposure of UV cell morphology of cells exposed to no UV or MV (control), UV-A irradiation (UV-A), UV-A irradiation and 200 mM MV (UV-A1200 mM MV), UV-B irradiation (UV-B), UV-B irradiation and 200 mM MV (UV-B1200 mM MV), MV represents methyl viologen. Each daily exposure of UV-A or UV-B irradiation was 0.36 J cm 22 . Each bar represents mean percentage of abnormal cells6S.D. (n536) after the third exposure to UV irradiation. Symbols a, b, c, d indicate a significant difference between each group with P,0.005.
128
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
Acute or chronic exposure of UV irradiation also could cause oxidative stress in different respects. Regarding the protection effects of rutin, flavonoids are known to have a protective effect, suppressing UV damage by absorbing light in the UV region [27–29]. However, in the present study this was not investigated. Halliwell and Gutteridge reported previously that secondary products such as flavonols and flavonoids have powerful antioxidant actions, such as scavenging O 2 and inhibiting lipid 2 peroxidation [30]. Rutin is known to function as an O 22 scavenger [21,28], and here we found that the levels of TBARS in cells exposed to MV and UV-B were suppressed by the addition of rutin compared with non rutin supplementation. The Z and SMZ strains exposed to UV-B and MV in the presence of 100 mM of rutin showed remarkably low levels of TBARS (Fig. 6A and B). From the experiments carried out in the present study, full speciation of the reactive oxygen species that were entangled in the cellular damage is difficult. Regarding the fact that rutin was able to scavenge O 2 [21,29], and suppress lipid 2 peroxidation [22], there are possibilities, however, that it may have scavenged generated O 2 2 . In the experiment the result of which is shown in Fig. 6, rutin was added immediately before exposure to UV-B and MV, however, it is not fully clear whether the scavenging effect took place inside or outside the cell. In order to clarify the mechanism of the protecting role of rutin against UV- and MV-induced oxidative stress, further investigations on the incorporation and intracellular / extracellular behavior of rutin are necessary. The preventive effect of antioxidants on UV-induced skin cancer in mice has been reported recently by Ichihashi [31]. It is worthwhile investigating whether E. gracilis Z and SMZ strains are protected from UV-B- and MVinduced cellular damage by the enrichment of other natural antioxidants under various oxidative stress conditions. Investigations concerning the effects of these UV and chemical reagents on the DNA, lipid membranes and proteins of E. gracilis cells are now under way. In the present study, the authors have learnt that the combined exposure to UV and MV can cause considerable cellular damage to smaller organisms, such as phytoplankton in the hydrosphere, by an increasing adverse effect of either one, through interaction with environmental pollutants. In summary, we have shown that for the unicellular flagellate E. gracilis, combined exposure of UV radiation and the herbicide MV affected cell morphology significantly more than exposure to UV radiation or MV exposure alone. Furthermore, it was demonstrated that TBARS values were increased by combined exposure of UV and MV presumably owing to interactions between the radiation and MV that generate reactive oxygen species. UV-B clearly affected the cell growth and viability more than UV-A, however, simple exposure of UV-A or UV-B and combined exposure with MV had a similar adverse effect
for both rutin preincubated and non-preincubated cells, which could support the fact that UV irradiation alone can cause considerable oxidative damage to smaller living organisms. In addition, the antioxidant rutin could suppress the adverse effects caused by exposure to UV-B and MV. The alterations in the membrane lipid caused by UV-B and / or MV are currently under investigation by means of chromatographic analyses.
References [1] C. Nolan, G. Amantidis, European Commission research on the fluxes and effects of environmental UV-B radiation, J. Photochem. Photobiol. B: Biol. 31 (1995) 3–7. [2] J.B. Kerr, C.T. McElroy, Evidence for large upward trends of UV-B radiation linked to ozone depletion, Science 26 (1993) 1032–1034. [3] C.S. Zerefos, C. Meleti, A. Bais, The recent UV-B variability over southern Eastern Europe, J. Photochem. Photobiol. B: Biol. 31 (1995) 15–19. ¨ [4] L.O. Bjorn, Effects of ozone depletion and increased UV-B on terrestrial ecosystems, Int. J. Environ. Stud. 51 (1996) 217–243. ¨ [5] D.P. Hader, Penetration and effects of solar UV-B on the phytoplankton and microalgae, Plant Ecol. 128 (1997) 4–13. [6] O. Holm-Hansen, E.W. Helbling, D. Lubin, Ultraviolet radiation in Antarctica: inhibition of primary production, Photochem. Photobiol. 58 (1993) 567–570. [7] M.P. Lessser, J.H. Farrrell, C.W. Walker, Oxidative stress DNA damage, and p53 expression in the larvae of Alantic cod exposed to ultraviolet (290–400 nm) radiation, J. Exp. Biol. 204 (2001) 157– 164. [8] R. Danilov, N.G.A. Ekelund, Influences of waste water from the paper industry and UV-B radiation on the photosynthetic efficiency of Euglena gracilis, J. Appl. Phycol. 11 (1999) 157–163. [9] J.K. Howard, Recent experience with paraquat poisoning in Great Britain: a review of 68 cases, Vet. Hum. Toxicol. 21 (1979) 213– 216. [10] A.D. Dodge, The role of light and oxygen in the action of photosynthetic inhibitor herbicides, in: Biochemical Responses Induced by Herbicides, ACS Symposium Series, Vol. 181, 1982, pp. 57–77. [11] B. Halliwell, in: 2nd Edition, Chloroplast Metabolism, Clarendon Press, Oxford, 1984. [12] B. Halliwell, J. Gutteridge, Reactions of the superoxide radical, in: Free Radicals in Biology and Medicine, Clarendon Press, Oxford, 1989, pp. 301–307. [13] C.F. Babbs, J.A. Pham, R.C. Coolbaugh, Lethal hydroxyl radical production in paraqaut-treated plants, Plant Physiol. 90 (1989) 1267–1270. ´ W. Woollons, J.E. Lowe, [14] P.H. Clingen, M. Berneburg, C. Petit-Frere, C.F. Arlett, M.H.L. Green, Contrasting effects of an ultraviolet B and ultraviolet A tanning lamp on interleukin-6, necrosis factor alpha and intracellular adhesion molecule-1 expression, Br. J. Dermatol. 145 (2001) 54–62. [15] F. de Gruijl, Photocarcinogenesis: UVA vs. UVB methods in enzymology, Photocarcinogenesis 319 (2000) 359–367. ´ ´ ˆ [16] S. Seite, D. Moyal, S. Richard, J. de Rigal, J.L. Leveque, C. Hourseau, A. Fourtanier, Mexoryl, SX: abroad absorption UV-A filter protects human skin from the effects of repeated suberythemal doses of UVA, J. Photochem. Photobiol. B: Biol. 44 (1998) 69–76. [17] Y. Oda, Y. Nakano, S. Kitaoka, Utilization of toxicity of exogenous amino acids in Euglena gracilis, J. Gen. Microbiol. 12 (1982) 853–858. [18] O.I. Aruoma, B. Halliwell, Molecular Biology of Free Radicals In Human Diseases, OICA, London, 1998.
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129 [19] T. Schwartz, UV light effects cell membrane and cytoplasmic targets, J. Photochem. Photobiol. B: Biol. 44 (2) (1998) 191–195. [20] A. Koostra, Protection from UV-B induced DNA damage by flavonoids, PMBIDB 26 (1994) 771–774. [21] Y.T. Chen, R.L. Zheng, Z.J. Jia, Y. Ju, Flavonoids as superoxide scavengers antioxidants, Free Rad. Biol. Med. 9 (1) (1990) 19–21. [22] I.B. Afanas’eva, A.I. Dorozhko, A.V. Brodskii, V.A. Kostyuk, A.I. Potapovitch, Chelating and free radical scavenging mechanisms of inhibitory action of rutin, quercetin in lipid peroxidation, Biochem. Pharmacol. 38 (1989) 1763–1769. [23] K. Kikugawa, T. Kojima, S. Yamaki, H. Kosugi, Interpretation of the thiobarbituric acid reactivity of rat liver and brain homogenates in the presence of ferric ion and ethylenediaminetetraacetic acid, Anal. Biochem. 202 (2) (1992) 249–255. [24] N.G.A. Ekelund, The effect of UV-B radiation and humic substances on the growth and motility of the flagellate, Euglena gracilis, J. Plankton Res. 15 (1993) 715–722. [25] S. Shigeoka, Y. Nakano, S. Kitaoka, Metabolism of hydrogen peroxide in Euglena gracilis Z by L-ascorbic acid peroxidase, J. Biochem. 186 (1) (1980) 377–380.
129
¨ [26] R. Scheuerlein, S. Treml, B. Thar, U.K. Tirlapur, D.-P. Hader, Evidence for UV-B-induced DNA degradation in Euglena gracilis mediated by activation of metal-dependent nucleases, J. Photochem. Photobiol. B: Biol. 31 (1995) 113–123. [27] E.M. Middleton, A.H. Teramura, The role of flavonol glycosides and carotenoids in protecting soy bean from Ultraviolet B damage, Plant Physiol. 103 (1993) 741–752. [28] A.E. Stapleton, Ultraviolet radiation and plants: burning questions, Plant Cell 4 (1992) 1353–1358. [29] Y. Hanasaki, S. Ogawa, S. Fukui, The correlations between active oxygens scavenging and antioxidative effects of flavonoids, Free Rad. Biol. Med. 16 (6) (1994) 845–850. [30] B. Halliwell, J. Gutteridge, Reactions of the superoxide radical, in: Free Radicals in Biology and Medicine, Clarendon Press, Oxford, 1989, pp. 275–288. [31] M. Ichihashi, N.U. Ahmed, A. Budiyanto, A. Wu, T. Bito, M. Ueda, T. Osawa, Preventive effect of antioxidant on ultraviolet-induced skin cancer in mice, J. Dermatol. Sci. 23 (Suppl. 1) (2000) 45–50.
Oxidative stress-induced cellular damage caused by UV and methyl viologen in Euglena gracilis and its suppression with rutin Helen Palmer, Mari Ohta, Masumi Watanabe, Tetsuya Suzuki* Laboratory of Food Wholesomeness, Department of Life Sciences, Graduate School of Fisheries Science, Hokkaido University, 3 -1 -1 Minato, Hakodate 041 -8611, Japan Received 13 June 2001; accepted 30 January 2002
Abstract The effects of ultraviolet radiation (UV-A: 320–400 nm and UV-B: 280–320 nm) and methyl viologen (MV) single or combined exposure, on the cell growth, viability and morphology of two strains of the unicellular flagellate Euglena gracilis, using the Z strain as a plant model and the achlorophyllous mutant SMZ strain as an animal model were investigated. Cell growth was not affected by MV only, whereas UV-A or UV-B single and combined exposure with MV inhibited the cell growth or decreased the viability. The SMZ strain had a higher number of abnormal cells than the Z strain after the third dose of UV-B was delivered simultaneously with MV. The abnormal cell number decreased when E. gracilis SMZ cells were preincubated with 100 mM rutin prior to the UV-B and MV exposure. There were higher abnormal cell numbers with groups exposed to UV rather than MV single exposure. Combined exposure to UV-B and 200 mM MV induced the highest levels of TBARS in both strains, and with the supplementation of rutin these high levels were suppressed. These results suggest that UV-A or UV-B irradiation alone or combined with MV cause considerable oxidative damage in E. gracilis cells, and rutin supplementation may suppress their adverse effects. 2002 Elsevier Science B.V. All rights reserved. Keywords: Euglena; Methyl viologen; Oxidative stress; Rutin; TBARS; Ultraviolet ray irradiation
1. Introduction Depletion of the ozone layer causes an increase in UV radiation that can cause serious damage to all living organisms on the earth [1]. Until recently, UV-B radiation (280–320 nm) was difficult to measure [1–3] because of its natural variability in UV radiation levels across the Earth’s surface. But UV-B radiation can cause damage to both aquatic and terrestrial habitants [4,5]. It has been demonstrated that UV-B reduces phytoplankton productivity [6], and in the Gulf sea UV-A (320–400 nm) radiation penetrates to depths of 23 m and UV-B penetrating from 7 to 12 m, resulting in mortality of developing embryos of larvae of Atlantic cod through oxidative stress [7]. On the other hand, organic pollutants that are discharged into wastewater have complex interactions with UV irradiation, which in turn affect algae [8]. In agriculture, numerous pesticides and herbicides are used to sterilize seeds for the prevention of seed-borne diseases. However, pesticides and herbicides are major environmental pollu-
*Corresponding author. Tel. / fax: 181-138-405-564. E-mail address: [email protected] (T. Suzuki).
tants, and the distribution of toxic substances in the environment is a growing concern. Methyl viologen (1,19-dimethyl-4,49-bipyridinium-dichloride), which is commercially known as paraquat, is a water-soluble herbicide that is widely used in many areas of the environment. It is one of the most potent pulmonary toxins [9]. The herbicidal effect of methyl viologen (MV) depends on light and oxygen; MV interferes with the photosystem by disrupting electron transport and catalyzing the production of active oxygen within the chloroplast [10–12]. Furthermore, according to Babbs et al. [13], MV readily produces hydroxyl radicals through either a superoxide-driven Fenton reaction or a Fenton-like reaction, which explains its potent toxicity. Numerous studies have been carried out on the adverse effects of environmental pollutants, and excess exposure to UV rays, which are a threat to almost all living organisms including humans. As a primary producer, phytoplankton constitutes the first level of the intricate food web in the ocean and is the main basis of the diet of fishes, crustaceans and mollusks [5]. The generation of cyclobutane pyrimidine dimers is a major type of DNA damage induced by UV-B irradiation [14]. Although UV-B wavelength range is more photo-
1011-1344 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S1011-1344( 02 )00271-3
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
chemically active than UV-A [15], it is now evident that chronic UV-A exposure at low doses also induces damage in human and animal skin [16]. Furthermore, UV-A also produces oxidative DNA damage mainly to guanine [14], therefore, adverse effects of UV-A are also causing concern. Here, we have investigated the hypothesis that cellular damage is induced through interactions of reactive oxygen species generated by UV irradiation and MV that has been discharged into the environment. We examined the adverse effects of exposure to UV irradiation at different wavelengths, 320–400 nm (UV-A) and 280–320 nm (UV-B) and MV when exposed alone or simultaneously. The eucaryote unicellular flagellate Euglena gracilis, a type of fresh water plankton, was used to assess the effects of UV and MV single or combined exposure. E. gracilis Z strain can photosynthesize, however, antibiotic streptomycin is able to eliminate chloroplasts from E. gracilis [17], to form a mutant achlorophyllous SMZ strain without interfering with the cell division. Notably, both strains have highly sophisticated subcellular organella equivalent to those of higher mammals; the E. gracilis Z strain can be regarded as a plant model, and its mutant strain SMZ as an animal model. Considering their properties, E. gracilis Z and SMZ strains were used as the model organisms in this study comparatively, to assess the effects of UV-A or UV-B, and MV when each were exposed either alone (UV-A, UV-B, MV), or combined (UV-A or UV-B1MV) on the cell morphology, viability and growth. The effect as mentioned above, of UV radiation and MV inducing oxidative stress or generating free radicals [10,18], are considered highly likely to affect biomembrane lipids [19]. Thus, to assess the deterioration of membrane lipids, we examined levels of thiobarbituric acid reactive substance (TBARS) after UV irradiation or MV single or combined exposure in this study. It has been reported previously that flavonoids are effective reactive oxygen species scavengers that protect plants from potentially harmful UV-B radiation [20]. Investigations also proved that flavonoids including rutin, can capture O 2 2 [21]. Rutin has also been reported to be effective in scavenging reactive oxygen species and suppressing lipid peroxidation [22]. Therefore, using rutin as a potential natural antioxidant scavenger, we also examined its suppressant effect by measuring the levels of TBARS in rutin-supplemented cells exposed to MV and UV-irradiation. In order to identify whether rutin could also be effective in protecting against UV- and MV-induced cellular damage, cell growth, viability and morphology were assessed with rutin preincubated E. gracilis cells. 2. Materials and methods
2.1. Reagents and chemicals Methyl viologen (1,19-dimethyl-4,49-bipyridinium-di-
117
chloride), purchased from Nacalai Tesque (Kyoto, Japan), was dissolved in Koren Hutner (KH) medium and used in the assay at concentrations of 100 and 200 mM. Control solutions without MV were also prepared. Rutin, purchased from Nacalai Tesque, was used to determine whether or not UV- and MV-induced cellular damage could be suppressed by its supplementation or preincubation. Rutin was prepared in phosphate buffer solution, pH 7.4. All other chemicals used were guaranteed reagent grade.
2.2. Organism used and incubation conditions E. gracilis Z and SMZ strains (kindly provided by Professor Y. Nakano, Laboratory of Nutritional Chemistry, Osaka Prefectural University) were grown at 29 8C (61 8C) in 5 ml of KH medium [17], in test-tubes under illuminated (3200 lx) fluorescent light for plant growth on a light / dark cycle of 12 / 12 h until early logarithmic growth phase. Illumination intensity was measured regularly using a Digital Lux Meter Model: LX-1330 / LX-1332 (Custom Corporation Tokyo, Japan).
2.3. UV irradiation UV-A (365 nm, 4 W Black Light Matsushita Electric Co., Osaka) or UV-B (model UVM-57, 302 nm, 6 W, equipped with 2 UVG filter to cut UV-C; UVP Inc., Upland, CA, USA), were used for UV irradiation in the present study. Cells in the petri-dishes were exposed to UV-A or UV-B as follows. The distance between the petri-dishes and the UV source was adjusted, and then the cells were exposed to UV-A or UV-B rays at 3 W m 22 ; UV-A and UV-B dose delivered was 0.36 J cm 22 for each exposure. The UV-A and UV-B lamps were fixed in dark black boxes and all other fluorescent light or daylight was excluded during the irradiation of the petri-dish samples. The UV exposures were delivered once a day for 3 consecutive days, and UV output was monitored simultaneously using a radiometer (VLX-3W Vilber lourmat, Torcy, France), with a 365 nm and 305 nm detector placed at the same distance from the UV source as the petri-dish samples. Approximately 1 h after UV irradiation, the cells were observed under a microscope before being placed in the light cycle of the incubator. The UV exposure of all samples was carried out daily from 14:00 to 16:00 h for 3 days. Cellular damage in E. gracilis was compared between cells undergoing UV-A or UV-B irradiation only, cells exposed singularly to 200 mM of MV, and those undergoing combined UV and MV treatment. The morphological change of cells preincubated with 100 mM rutin prior to UV-A or UV-B single exposure, or combined with MV were also assessed. The experimental procedures are described in Section 2.10. After the UV irradiation cells were observed, hypertrophy, V-shaped, starfish-shaped and those that were not completely divided were considered as abnormal. The morphology of both strains were observed
118
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
under a video microscope using an Olympus IMT-2 inverted microscope (Olympus Optic Co., Inc., Tokyo) equipped with an image processor, ARGUS-100 (Hamamatsu Photonics Co., Hamamatsu, Japan).
2.4. Exposure to MV To examine the effect of MV exposure on E. gracilis, cells from a liquid culture (10 5 –10 6 cells ml 21 ), were adjusted to a density of 1.6310 4 cells ml 21 , and 50 ml of the cell suspensions were inoculated into 3 ml of KH medium containing either 100 mM or 200 mM MV in small glass petri-dishes. These petri-dishes were then exposed to UV-A or UV-B as described in Section 2.3. For samples exposed to UV-A or UV-B single exposure, cells were inoculated into 3 ml of KH medium without MV.
2.5. MV single exposure The effect of MV single exposure on the cell morphology and growth of E. gracilis Z and SMZ strains was
investigated in order to compare single and combined stress-induced cellular damage. For the morphological assessment, cells were inoculated into microtiter wells containing either 100 mM of MV in KH medium or KH medium alone (as control cells), and incubated under illuminated conditions as described in Section 2.2. Observations for morphological changes were carried out daily under the video microscope, as explained in Section 2.3.
2.6. Cell growth and viability of E. gracilis exposed to single or simultaneous UV and MV exposure The effect of MV single exposure on cell growth was assessed by inoculating 30 ml of 10 6 cells ml 21 of E. gracilis into 5 ml of KH medium containing 100 mM MV in test-tubes. Cells were incubated under either light / dark conditions (described in Section 2.2) or a 24-h dark cycle, as the herbicidal effect of MV is oxygen- and lightdependent. The cell density was measured using a Bosch and Lomb spectrometer (Shimadzu Instruments, Kyoto,
Fig. 1. Effect of methyl viologen exposure on the cell growth of Euglena gracilis. Control, the cell growth of E. gracilis cells not exposed to MV under 12 / 12 h light dark cycle; 100 mM MV, 12 / 12 h light dark cycle exposed to 100 mM MV; control / dark, not exposed to MV under dark cycle; 100 mM MV, exposed to 100 mM under dark cycle at 28 8C for 3 days. Cell density at 610 nm was measured daily.
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
Japan) over 3 days (results shown in Fig. 2). The growth of E. gracilis Z and SMZ strains exposed to either UV-A or UV-B single exposure, or combined exposure with MV was determined by measuring the cell abundance in 10 ml of cell suspension taken from the petri-dish samples. The estimation of cell numbers was performed under the microscope (n518).
2.7. Cell viability To estimate cell viability, 100 ml of E. gracilis cell suspension were taken from each petri-dish, centrifuged at 10003g (23 8C for 3 min), washed twice with 100 ml of phosphate saline buffer (PBS, pH 7.2), and then stained with 20 ml of 0.4% Trypan Blue solution at 28 8C for 1 h. After staining, cells were washed again with 100 ml of
119
PBS solution. The non-viable blue-stained cells were detected by an Olympus IMT-2. The numbers of nonviable cells in six randomly selected frames were counted in triplicate (cell values were expressed as the average viable cell percentage of the total cell number6S.D., n536 measurements). Viable cells were calculated as the difference between the total number of cells and the number of non-viable cells. Cell viability was expressed as the percentage of viable cells over total cells.
2.8. Estimation of abnormal cell occurrence V-shaped, hypertrophied and starfish-shaped cells were classified as abnormal cells (Fig. 4). The frequency of abnormal cells was estimated by counting the number of abnormal cells in six frames for each sample. Values of
Fig. 2. Effect of UV-A or UV-B irradiation and MV exposure on the cell growth of E. gracilis. Cell growth was measured after each exposure to UV irradiation. (A and C) Z strain; (B and D) SMZ strain. Cell growth of cells exposed to: no UV irradiation or MV (control), UV-A irradiation (UV-A), UV-A irradiation and 100 mM MV (UV-A1100 mM MV), UV-A irradiation and 200 mM MV (UV-A1200 mM MV), UV-B irradiation (UV-B), UV-B irradiation and 100 mM MV (UV-B1100 mM MV), UV-B irradiation and 200 mM MV (UV-B1200 mM MV), MV represents methyl viologen. Each daily exposure of UV-A or UV-B irradiation was 0.36 J cm 22 . Each bar represents the mean6S.D. (n518). Symbols a, b, c, d indicate a significant difference between each group (P,0.005) after the third day of exposure.
120
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
abnormal cells were expressed as the mean percentage of the total observed frames obtained from 36 measurements.
2.9. Estimation of TBARS levels It was considered that assessing the TBARS level of UV-A or UV-B with or without MV exposed E. gracilis cells may be an indication of induced damage to lipid components. Cells were delivered UV-A or UV-B doses of 0.78 J cm 22 at 3 W m 22 exposed with or without 200 mM MV. TBARS values of cells exposed to 200 mM MV only were also assessed.
2.10. Rutin supplementation To assess whether the UV-B- and MV-induced lipid peroxidation could be suppressed by rutin, either 50 mM or 100 mM rutin was added to E. gracilis cells immediately before exposure to UV irradiation and MV. After an acute exposure of UV-B irradiation at a dosage of 0.78 J cm 22 (3 W m 22 ), the cell suspension samples were taken and immediately centrifuged at 10003g (23 8C for 3 min), placed in a cryogenic tube, and kept at 280 8C until TBARS analysis. The levels of TBARS were estimated following the method of Kikugawa et al. [23].
Fig. 3. Effect of UV irradiation and MV exposure on the cell viability of E. gracilis. Cell viability was tested after each exposure to UV irradiation. (A and C) Z strain; (B and D) SMZ strain. The viable cell percentages on the third day are also shown separately in E to H. (E and G) Z strain; (F and H) SMZ strain. Viable cells exposed to no UV or MV (control), UV-A irradiation (UV-A), UV-A irradiation and 100 mM MV (UV-A1100 mM MV), UV-A irradiation and 200 mM MV (UV-A1200 mM MV), UV-B irradiation (UV-B), UV-B irradiation and 100 mM MV (UV-B1100 mM MV), UV-B irradiation and 200 mM MV (UV-B1200 mM MV), MV represents methyl viologen. Each daily exposure of UV-A or UV-B irradiation was 0.36 J cm 22 . Each bar represents the mean6S.D. (n536). Symbols, a, b, c, d indicate a significant difference between each group (P,0.005) after the third day of exposure of UV irradiation.
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
2.11. E. gracilis cells preincubated with rutin The cell growth, viability and morphology of E. gracilis cells incubated with 100 mM of rutin was also assessed to determine whether rutin would suppress cellular damage induced by UV single exposure or when combined with MV. E. gracilis cells were preincubated with 100 mM of rutin (adjusted pH 7.4 with PBS buffer) for 24 h, washed three times with KH medium, after which they were resuspended in petri-dishes with or without MV, as described in Section 2.4. UV irradiation was carried out
121
according to the protocol of Section 2.3 and the determination of cell growth, viability and the estimation of abnormal cells following the procedure of Sections 2.6, 2.7 and 2.8.
2.12. Statistical analysis The data were evaluated by analysis of variance (ANOVA). A value of P,0.005 was considered to be significant.
Fig. 3. (continued)
122
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
3. Results
3.1. Cell growth and viability The cell growth of cells exposed to MV alone at 100 mM is shown in Fig. 1A and B. For both Z and SMZ strains there was no growth inhibition observed for the cells incubated either in light / dark or dark conditions over the 3-day period when the cell density was monitored. This reflects that under light / dark or under dark incubation conditions, oxidative stress derived from MV exposure alone was probably not strong enough to inhibit the cell growth. UV single irradiation and UV exposed with MV inhibited cell growth, and there was a greater inhibition after UV-B irradiation than UV-A. As there was little significant difference (P,0.005) between the cell growths of UV-B only, and UV-B combined with MV exposed cells in both strains, UV-B may have been the dominant cause of the inhibition (Fig. 2C and D). The UV-B dosage in the present study inhibited growth to almost the same extent in both strains (Fig. 2C and D). The cell viability of E. gracilis Z and SMZ strains was also affected by both UV-A and UV-B irradiation (Fig. 3). Cell viability in both strains decreased gradually after exposure to UV-A irradiation, however the viable cell numbers decreased significantly (P,0.005) in the higher concentration of MV in comparison with lower concentration of MV (Fig. 3A and B). In contrast, UV-B exposure had different effects on both strains, causing a more marked decrease in the viability of the SMZ strain than in the Z strain, after the third UV-B dosage (Fig. 3C and D). For both strains, UV-B irradiation caused a considerably higher reduction of viable cells after 3 days than UV-A (Fig. 3E to H).
3.2. Cell morphology In both Z and SMZ strains, the most frequently observed abnormal cell was V-shaped, followed by hypertrophied and starfish-shaped cells under the experimental conditions. The normal shape of E. gracilis cell (a), V-shaped (b), starfish-shaped (c), hypertrophy (d) cells that were observed under different exposure conditions are shown (Fig. 4). UV-A or MV alone induced between 6 and 8% abnormal cell outbreak (Fig. 5A and B), whereas combined exposure of UV-A and MV significantly (P,0.005) increased abnormality by twofold after the third exposure (Fig. 5A and B). (The abnormal cell percentages on the third day are also shown separately in Fig. 5E–H.) There was no significant difference in abnormality between cells exposed to 100 mM and those exposed to 200 mM MV even when combined with UV-A exposure. Single or combined exposure to UV-B and MV caused a similar cell abnormality to the UV-A and MV in the Z strain (Fig. 5A and C). However, there was a marked
Fig. 4. Cell morphology of E. gracilis cells exposed to UV irradiation and MV. Abnormal cell occurrence of E. gracilis cells exposed to UV-A or UV-B single or combined with MV exposure was observed. The normal shape of E. gracilis cell (a), V-shaped (b), starfish-shaped (c), hypertrophy (d) cells that occurred. Bar in each photograph represents 20 mm.
difference between UV-A and UV-B exposed groups in the SMZ strain (Fig. 5B and D). Distinctly more abnormal cells (27%) were observed after UV-B irradiation alone;
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
123
Fig. 5. Effect of UV irradiation and MV exposure on the cell morphology of E. gracilis. (A and C) Z strain; (B and D) SMZ strain. Abnormal cell percentages on the third day are also shown separately in E to H. (E and G) Z strain; (F and H) SMZ strain. The abnormal cell percentage of cells exposed to no UV or MV (control), UV-A irradiation (UV-A), methyl viologen only (MV), UV-A irradiation and 100 mM MV (UV-A1100 mM MV), UV-A irradiation and 200 mM MV (UV-A1200 mM MV), UV-B irradiation (UV-B), UV-B irradiation and 100 mM MV (UV-B1100 mM MV), UV-B irradiation and 200 mM MV (UV-B1200 mM MV). Each daily exposure of UV-A or UV-B irradiation was 0.36 J cm 22 . Each bar represents mean percentage of abnormal cells6S.D. (n536). Symbols a, b, c, d indicate a significant difference between each group with P,0.005 after the second and the third day of exposure.
furthermore, the combined exposure of UV-B and MV, especially 200 mM MV, greatly increased the number of abnormal cells (76%) after the third exposure (Fig. 5D and H). On the third day, significant difference (P,0.005) was recognized between sole UV-B exposure and combined exposure of UV-B and MV; i.e. more abnormal cells with UV-B and 200 mM MV than with UV-B and 100 mM MV exposed cell groups.
3.3.2. The effect of rutin supplementation Cells supplemented with 100 mM rutin immediately before the UV-B exposure showed an extremely marked decrease in TBARS levels. This was apparent in both Z and SMZ strains. The 50 mM supplementation also suppressed the TBARS levels to an extent (Fig. 6A and B). 3.4. Effects of preincubation with rutin on cell growth, viability, and morphology
3.3. TBARS 3.3.1. Exposure to MV and /or UV-A or UV-B Simultaneous exposure to UV-B and MV (UV-B1MV) resulted in the highest levels of TBARS in both the Z and SMZ strains of E. gracilis (Fig. 6A and B). A considerably higher TBARS level was obtained with the Z strain. Furthermore, with the exception of the UV-A exposed cell group, all exposed groups displayed a significant difference (P,0.005) (Fig. 6A). The SMZ strain, however, had far lower TBARS levels than the Z strain in all the exposed groups.
E. gracilis cells preincubated with rutin prior to the UV-A or UV-B exposure did not display any growth recovery compared to the non-treated cells (Fig. 2A–D; Fig. 7A and B). Both UV-A and UV-B irradiation alone and when combined with MV significantly (P,0.005) inhibited the cell growth compared to the control group in the Z strain, however, UV-A was not significantly different from the control group in the SMZ strain (Fig. 7B). The cell viability of the cells preincubated with rutin appeared slightly higher than the non-preincubated cell groups, after the third exposure to sole UV-B irradiation or combined
124
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
Fig. 5. (continued)
with MV exposure (Fig. 3E and F; Fig. 8A and B) in both strains. Abnormal cells appeared to be lower in cells preincubated with rutin when cells were simultaneously exposed to UV-A or UV-B and MV in the SMZ strain (Fig. 9B), than the non-treated cells (Fig. 5D).
4. Discussion The adverse effects of UV irradiation on the growth and motility [24] and the photosynthetic efficiency [8] of E.
gracilis have been reported previously. Results showed that UV irradiation inhibits photosynthesis and cell growth. However, there are few studies reporting the combined effects of UV irradiation and toxic substances on E. gracilis. The lack of growth inhibition by 100 mM of MV, coupled with the severe growth inhibition by UV-A or UV-B with or without MV in either strain (Fig. 2), indicates that UV-A or UV-B are the dominant contributors to the inhibition of cell growth. UV-B irradiation alone or combined with exposure to MV, had an adverse effect on
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
Fig. 6. Levels of TBARS in E. gracilis cells exposed to UV and MV and the effect of rutin supplementation. (A) Z strain; (B) SMZ strain. Cells exposed to no UV or MV (control), UV-A irradiation (UV-A), UV-B irradiation (UV-B), 200 mM methyl viologen (200 mM MV), UV-A irradiation and 200 mM methyl viologen exposure (UV-A1MV), UV-B irradiation and 200 mM methyl viologen exposure (UV-B1MV), UV-B irradiation and 200 mM methyl viologen exposure with 50 mM rutin (UV-B1MV1R 50 mM), UV-B irradiation and 200 mM methyl viologen with 100 mM rutin (UV-B1MV1R 100 mM). The total exposure energy of UV-A and UV-B was 0.72 J cm 22 . Each bar represents the mean value6S.D. (n59). Cells were analyzed immediately after UV irradiation.
125
cell viability in both the Z and SMZ strains of E. gracilis (Fig. 3C and D). Furthermore, the SMZ strain had a lower survival ratio than the Z strain after UV-B irradiation with or without MV on the third day (Fig. 3C, D, G, H). Shigeoka et al. reported a peroxidase in E. gracilis Z that requires ascorbic acid as the electron donor in the cytosol that scavenges H 2 O 2 , as E. gracilis does not contain catalase [25]. Ascorbate peroxidase (APX) is found only in higher plants and algae, but no report in animals. It was found that the APX content in E. gracilis Z strain, a plant model, was 5.157 mmol / mg protein / min and in SMZ strain, an animal model, 0.826 mmol / mg protein / min, in cells incubated under almost the same conditions as that of the present study (unpublished data). Therefore, the lower APX content may have resulted in less viable cells in the SMZ strain after the third dosage of UV-B with or without MV, indicating different reactive oxygen scavenging abilities between the two strains. On the other hand, the viable cell percentages were higher in cells preincubated with 100 mM rutin prior to UV-B single or combined MV exposure (Fig. 8A and B) than non-preincubated cells in both strains (Fig. 3E and F). The percentage of abnormal cells increased when cells were exposed simultaneously to MV and either UV-A or UV-B, suggesting that two consecutive daily exposures of the combined exposure generated sufficient reactive oxygen species to induce teratogenicity in E. gracilis cells. The combined exposure of UV-B and 200 mM MV enhanced the occurrence of abnormal cells in SMZ strain after the third exposure (Fig. 5H). This high incidence of abnormal cell proliferation in the SMZ might result from the lower potential of its reactive oxygen scavenging system as mentioned previously. Abnormal cell proliferation must be linked to dysfunction of the cell-cycle regulation system, which frequently involves impairment of DNA duplication, protein synthesis, membrane lipid synthesis and organization of subcellular organella and their function. Scheuerlein et al. reported the evidence for UV-B-induced DNA degeneration in E. gracilis mediated by activation of metal-dependent nucleases [26]. On the other hand, cells preincubated with 100 mM of rutin before the combined exposure of UV and MV, displayed a considerably lower abnormal cell number after the third UV-A or UV-B dosage (Fig. 9B) than non-treated cells (Fig. 5H). Whether the decrease was a result of rutin, acting as intracellular or extracellular reactive oxygen scavenger or not is not yet clear, and this decrease was only observed in the SMZ strain. UV-A alone, or combined with exposure to MV, did not increase the levels of TBARS as much as combined with UV-B and MV. This might be due to the shorter wavelength of UV-B which may have a greater threat to unprotected molecules [15]. Cell growth (Fig. 1) and abnormal cell proliferation (Fig. 5) were either unaffected or only slightly affected by simple exposure to 200 mM MV in both Z and SMZ strains. Furthermore, simple exposure to MV did not signifi-
126
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
Fig. 7. Effect of UV and methyl viologen exposure on the cell growth of Euglena gracilis preincubated with rutin. E. gracilis cells preincubated with rutin were exposed to UV and MV and the cell growth measured after the third exposure to UV irradiation. (A) Z strain; (B) SMZ strain. After the third exposure of UV, the cell growth of cells exposed to no UV or MV (control), UV-A irradiation (UV-A), UV-A irradiation and 200 mM MV (UV-A1200 mM MV), UV-B irradiation (UV-B), UV-B irradiation and 200 mM MV (UV-B1200 mM MV), MV represents methyl viologen. Each daily exposure of UV-A or UV-B irradiation was 0.36 J cm 22 . Each bar represents the mean6S.D. (n518). Symbols a, b, c, d indicate a significant difference between each group (P,0.005) after the third exposure.
cantly increase TBARS levels in the SMZ strain, however, a significant (P,0.005) increase was observed in the Z strain (Fig. 6). The highest TBARS level (136 nmol g 21 ) was obtained from combined exposure to UV-B and MV in the Z strain; by contrast, in the SMZ strain this level was below 100 nmol g 21 . Comparing the results from the TBARS (single exposure at a higher UV energy level) (Fig. 6) with those of the cell growth (Figs. 1 and 2), viability (Fig. 3) and morphology assessment (chronic exposure at lower UV energy level) (Fig. 5), UV- and MV-induced oxidative damage may have differed between the two strains. The TBARS levels were
higher in the Z strain (Fig. 6), however, abnormal cell occurrence was generally higher in the SMZ strain (Figs. 5 and 9). Pretreatment with rutin definitely decreased the frequency of the abnormal cells in both strains, especially in combined exposure of UV-B and 200 mM MV in SMZ. The abnormal cells in SMZ under the combined exposure of UV-B and 200 mM MV without rutin treatment, remarkably decreased by almost 60% with pretreatment with rutin. However, the effect of rutin was not so remarkable in Z strain. This may be due to different reactive oxygen scavenging systems and membrane lipid components between the E. gracilis Z and SMZ strains.
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
Fig. 8. Effect of UV irradiation and MV exposure on the viability of E. gracilis preincubated with rutin. E. gracilis cells preincubated with rutin were exposed to UV and MV and the cell viability tested after the third exposure to UV irradiation. (A) Z strain; (B) SMZ strain. After the third exposure of UV cell viability of cells exposed to no UV or MV (control), UV-A irradiation (UV-A), UV-A irradiation and 200 mM MV (UV-A1200 mM MV), UV-B irradiation (UV-B), UV-B and 200 mM MV (UV-B1200 mM MV), MV represents methyl viologen. Each daily exposure of UV-A or UV-B irradiation was 0.36 J cm 22 . Each bar represents the mean6S.D. (n536). Symbols, a, b, c, d indicate a significant difference between each group (P,0.005) after the third day.
127
Fig. 9. Effect of UV irradiation and MV in the cell morphology of E. gracilis preincubated with rutin. (A) Z strain; (B) SMZ strain. After the third exposure of UV cell morphology of cells exposed to no UV or MV (control), UV-A irradiation (UV-A), UV-A irradiation and 200 mM MV (UV-A1200 mM MV), UV-B irradiation (UV-B), UV-B irradiation and 200 mM MV (UV-B1200 mM MV), MV represents methyl viologen. Each daily exposure of UV-A or UV-B irradiation was 0.36 J cm 22 . Each bar represents mean percentage of abnormal cells6S.D. (n536) after the third exposure to UV irradiation. Symbols a, b, c, d indicate a significant difference between each group with P,0.005.
128
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129
Acute or chronic exposure of UV irradiation also could cause oxidative stress in different respects. Regarding the protection effects of rutin, flavonoids are known to have a protective effect, suppressing UV damage by absorbing light in the UV region [27–29]. However, in the present study this was not investigated. Halliwell and Gutteridge reported previously that secondary products such as flavonols and flavonoids have powerful antioxidant actions, such as scavenging O 2 and inhibiting lipid 2 peroxidation [30]. Rutin is known to function as an O 22 scavenger [21,28], and here we found that the levels of TBARS in cells exposed to MV and UV-B were suppressed by the addition of rutin compared with non rutin supplementation. The Z and SMZ strains exposed to UV-B and MV in the presence of 100 mM of rutin showed remarkably low levels of TBARS (Fig. 6A and B). From the experiments carried out in the present study, full speciation of the reactive oxygen species that were entangled in the cellular damage is difficult. Regarding the fact that rutin was able to scavenge O 2 [21,29], and suppress lipid 2 peroxidation [22], there are possibilities, however, that it may have scavenged generated O 2 2 . In the experiment the result of which is shown in Fig. 6, rutin was added immediately before exposure to UV-B and MV, however, it is not fully clear whether the scavenging effect took place inside or outside the cell. In order to clarify the mechanism of the protecting role of rutin against UV- and MV-induced oxidative stress, further investigations on the incorporation and intracellular / extracellular behavior of rutin are necessary. The preventive effect of antioxidants on UV-induced skin cancer in mice has been reported recently by Ichihashi [31]. It is worthwhile investigating whether E. gracilis Z and SMZ strains are protected from UV-B- and MVinduced cellular damage by the enrichment of other natural antioxidants under various oxidative stress conditions. Investigations concerning the effects of these UV and chemical reagents on the DNA, lipid membranes and proteins of E. gracilis cells are now under way. In the present study, the authors have learnt that the combined exposure to UV and MV can cause considerable cellular damage to smaller organisms, such as phytoplankton in the hydrosphere, by an increasing adverse effect of either one, through interaction with environmental pollutants. In summary, we have shown that for the unicellular flagellate E. gracilis, combined exposure of UV radiation and the herbicide MV affected cell morphology significantly more than exposure to UV radiation or MV exposure alone. Furthermore, it was demonstrated that TBARS values were increased by combined exposure of UV and MV presumably owing to interactions between the radiation and MV that generate reactive oxygen species. UV-B clearly affected the cell growth and viability more than UV-A, however, simple exposure of UV-A or UV-B and combined exposure with MV had a similar adverse effect
for both rutin preincubated and non-preincubated cells, which could support the fact that UV irradiation alone can cause considerable oxidative damage to smaller living organisms. In addition, the antioxidant rutin could suppress the adverse effects caused by exposure to UV-B and MV. The alterations in the membrane lipid caused by UV-B and / or MV are currently under investigation by means of chromatographic analyses.
References [1] C. Nolan, G. Amantidis, European Commission research on the fluxes and effects of environmental UV-B radiation, J. Photochem. Photobiol. B: Biol. 31 (1995) 3–7. [2] J.B. Kerr, C.T. McElroy, Evidence for large upward trends of UV-B radiation linked to ozone depletion, Science 26 (1993) 1032–1034. [3] C.S. Zerefos, C. Meleti, A. Bais, The recent UV-B variability over southern Eastern Europe, J. Photochem. Photobiol. B: Biol. 31 (1995) 15–19. ¨ [4] L.O. Bjorn, Effects of ozone depletion and increased UV-B on terrestrial ecosystems, Int. J. Environ. Stud. 51 (1996) 217–243. ¨ [5] D.P. Hader, Penetration and effects of solar UV-B on the phytoplankton and microalgae, Plant Ecol. 128 (1997) 4–13. [6] O. Holm-Hansen, E.W. Helbling, D. Lubin, Ultraviolet radiation in Antarctica: inhibition of primary production, Photochem. Photobiol. 58 (1993) 567–570. [7] M.P. Lessser, J.H. Farrrell, C.W. Walker, Oxidative stress DNA damage, and p53 expression in the larvae of Alantic cod exposed to ultraviolet (290–400 nm) radiation, J. Exp. Biol. 204 (2001) 157– 164. [8] R. Danilov, N.G.A. Ekelund, Influences of waste water from the paper industry and UV-B radiation on the photosynthetic efficiency of Euglena gracilis, J. Appl. Phycol. 11 (1999) 157–163. [9] J.K. Howard, Recent experience with paraquat poisoning in Great Britain: a review of 68 cases, Vet. Hum. Toxicol. 21 (1979) 213– 216. [10] A.D. Dodge, The role of light and oxygen in the action of photosynthetic inhibitor herbicides, in: Biochemical Responses Induced by Herbicides, ACS Symposium Series, Vol. 181, 1982, pp. 57–77. [11] B. Halliwell, in: 2nd Edition, Chloroplast Metabolism, Clarendon Press, Oxford, 1984. [12] B. Halliwell, J. Gutteridge, Reactions of the superoxide radical, in: Free Radicals in Biology and Medicine, Clarendon Press, Oxford, 1989, pp. 301–307. [13] C.F. Babbs, J.A. Pham, R.C. Coolbaugh, Lethal hydroxyl radical production in paraqaut-treated plants, Plant Physiol. 90 (1989) 1267–1270. ´ W. Woollons, J.E. Lowe, [14] P.H. Clingen, M. Berneburg, C. Petit-Frere, C.F. Arlett, M.H.L. Green, Contrasting effects of an ultraviolet B and ultraviolet A tanning lamp on interleukin-6, necrosis factor alpha and intracellular adhesion molecule-1 expression, Br. J. Dermatol. 145 (2001) 54–62. [15] F. de Gruijl, Photocarcinogenesis: UVA vs. UVB methods in enzymology, Photocarcinogenesis 319 (2000) 359–367. ´ ´ ˆ [16] S. Seite, D. Moyal, S. Richard, J. de Rigal, J.L. Leveque, C. Hourseau, A. Fourtanier, Mexoryl, SX: abroad absorption UV-A filter protects human skin from the effects of repeated suberythemal doses of UVA, J. Photochem. Photobiol. B: Biol. 44 (1998) 69–76. [17] Y. Oda, Y. Nakano, S. Kitaoka, Utilization of toxicity of exogenous amino acids in Euglena gracilis, J. Gen. Microbiol. 12 (1982) 853–858. [18] O.I. Aruoma, B. Halliwell, Molecular Biology of Free Radicals In Human Diseases, OICA, London, 1998.
H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 – 129 [19] T. Schwartz, UV light effects cell membrane and cytoplasmic targets, J. Photochem. Photobiol. B: Biol. 44 (2) (1998) 191–195. [20] A. Koostra, Protection from UV-B induced DNA damage by flavonoids, PMBIDB 26 (1994) 771–774. [21] Y.T. Chen, R.L. Zheng, Z.J. Jia, Y. Ju, Flavonoids as superoxide scavengers antioxidants, Free Rad. Biol. Med. 9 (1) (1990) 19–21. [22] I.B. Afanas’eva, A.I. Dorozhko, A.V. Brodskii, V.A. Kostyuk, A.I. Potapovitch, Chelating and free radical scavenging mechanisms of inhibitory action of rutin, quercetin in lipid peroxidation, Biochem. Pharmacol. 38 (1989) 1763–1769. [23] K. Kikugawa, T. Kojima, S. Yamaki, H. Kosugi, Interpretation of the thiobarbituric acid reactivity of rat liver and brain homogenates in the presence of ferric ion and ethylenediaminetetraacetic acid, Anal. Biochem. 202 (2) (1992) 249–255. [24] N.G.A. Ekelund, The effect of UV-B radiation and humic substances on the growth and motility of the flagellate, Euglena gracilis, J. Plankton Res. 15 (1993) 715–722. [25] S. Shigeoka, Y. Nakano, S. Kitaoka, Metabolism of hydrogen peroxide in Euglena gracilis Z by L-ascorbic acid peroxidase, J. Biochem. 186 (1) (1980) 377–380.
129
¨ [26] R. Scheuerlein, S. Treml, B. Thar, U.K. Tirlapur, D.-P. Hader, Evidence for UV-B-induced DNA degradation in Euglena gracilis mediated by activation of metal-dependent nucleases, J. Photochem. Photobiol. B: Biol. 31 (1995) 113–123. [27] E.M. Middleton, A.H. Teramura, The role of flavonol glycosides and carotenoids in protecting soy bean from Ultraviolet B damage, Plant Physiol. 103 (1993) 741–752. [28] A.E. Stapleton, Ultraviolet radiation and plants: burning questions, Plant Cell 4 (1992) 1353–1358. [29] Y. Hanasaki, S. Ogawa, S. Fukui, The correlations between active oxygens scavenging and antioxidative effects of flavonoids, Free Rad. Biol. Med. 16 (6) (1994) 845–850. [30] B. Halliwell, J. Gutteridge, Reactions of the superoxide radical, in: Free Radicals in Biology and Medicine, Clarendon Press, Oxford, 1989, pp. 275–288. [31] M. Ichihashi, N.U. Ahmed, A. Budiyanto, A. Wu, T. Bito, M. Ueda, T. Osawa, Preventive effect of antioxidant on ultraviolet-induced skin cancer in mice, J. Dermatol. Sci. 23 (Suppl. 1) (2000) 45–50.