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) 116–129www.elsevier.com/ locate / jphotobiol

Oxidative stress-induced cellular damage caused by UV and methylviologen in Euglena gracilis and its suppression with rutin

*Helen Palmer, Mari Ohta, Masumi Watanabe, Tetsuya SuzukiLaboratory 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 combinedexposure, on the cell growth, viability and morphology of two strains of the unicellular flagellate Euglena gracilis, using the Z strain as aplant 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 hada higher number of abnormal cells than the Z strain after the third dose of UV-B was delivered simultaneously with MV. The abnormalcell number decreased when E. gracilis SMZ cells were preincubated with 100 mM rutin prior to the UV-B and MV exposure. There werehigher abnormal cell numbers with groups exposed to UV rather than MV single exposure. Combined exposure to UV-B and 200 mM MVinduced the highest levels of TBARS in both strains, and with the supplementation of rutin these high levels were suppressed. Theseresults suggest that UV-A or UV-B irradiation alone or combined with MV cause considerable oxidative damage in E. gracilis cells, andrutin 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 tants, and the distribution of toxic substances in theenvironment is a growing concern.

Depletion of the ozone layer causes an increase in UV Methyl viologen (1,19-dimethyl-4,49-bipyridinium-di-radiation that can cause serious damage to all living chloride), which is commercially known as paraquat, is aorganisms on the earth [1]. Until recently, UV-B radiation water-soluble herbicide that is widely used in many areas(280–320 nm) was difficult to measure [1–3] because of of the environment. It is one of the most potent pulmonaryits natural variability in UV radiation levels across the toxins [9]. The herbicidal effect of methyl viologen (MV)Earth’s surface. But UV-B radiation can cause damage to depends on light and oxygen; MV interferes with theboth aquatic and terrestrial habitants [4,5]. It has been photosystem by disrupting electron transport and catalyz-demonstrated that UV-B reduces phytoplankton productivi- ing the production of active oxygen within the chloroplastty [6], and in the Gulf sea UV-A (320–400 nm) radiation [10–12]. Furthermore, according to Babbs et al. [13], MVpenetrates to depths of 23 m and UV-B penetrating from 7 readily produces hydroxyl radicals through either ato 12 m, resulting in mortality of developing embryos of superoxide-driven Fenton reaction or a Fenton-like re-larvae of Atlantic cod through oxidative stress [7]. action, which explains its potent toxicity. Numerous

On the other hand, organic pollutants that are discharged studies have been carried out on the adverse effects ofinto wastewater have complex interactions with UV ir- environmental pollutants, and excess exposure to UV rays,radiation, which in turn affect algae [8]. In agriculture, which are a threat to almost all living organisms includingnumerous pesticides and herbicides are used to sterilize humans. As a primary producer, phytoplankton constitutesseeds for the prevention of seed-borne diseases. However, the first level of the intricate food web in the ocean and ispesticides and herbicides are major environmental pollu- 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*Corresponding author. Tel. / fax: 181-138-405-564.E-mail address: [email protected] (T. Suzuki). [14]. Although UV-B wavelength range is more photo-

1011-1344/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S1011-1344( 02 )00271-3

H. Palmer et al. / Journal of Photochemistry and Photobiology B: Biology 67 (2002) 116 –129 117

chemically active than UV-A [15], it is now evident that chloride), purchased from Nacalai Tesque (Kyoto, Japan),chronic UV-A exposure at low doses also induces damage was dissolved in Koren Hutner (KH) medium and used inin human and animal skin [16]. Furthermore, UV-A also the assay at concentrations of 100 and 200 mM. Controlproduces oxidative DNA damage mainly to guanine [14], solutions without MV were also prepared. Rutin, purchasedtherefore, adverse effects of UV-A are also causing con- from Nacalai Tesque, was used to determine whether orcern. not UV- and MV-induced cellular damage could be sup-

Here, we have investigated the hypothesis that cellular pressed by its supplementation or preincubation. Rutin wasdamage is induced through interactions of reactive oxygen prepared in phosphate buffer solution, pH 7.4. All otherspecies generated by UV irradiation and MV that has been chemicals used were guaranteed reagent grade.discharged into the environment. We examined the adverseeffects of exposure to UV irradiation at different wave- 2.2. Organism used and incubation conditionslengths, 320–400 nm (UV-A) and 280–320 nm (UV-B)and MV when exposed alone or simultaneously. E. gracilis Z and SMZ strains (kindly provided by

The eucaryote unicellular flagellate Euglena gracilis, a Professor Y. Nakano, Laboratory of Nutritional Chemistry,type of fresh water plankton, was used to assess the effects Osaka Prefectural University) were grown at 29 8Cof UV and MV single or combined exposure. E. gracilis Z (61 8C) in 5 ml of KH medium [17], in test-tubes understrain can photosynthesize, however, antibiotic strep- illuminated (3200 lx) fluorescent light for plant growth ontomycin is able to eliminate chloroplasts from E. gracilis a light /dark cycle of 12/12 h until early logarithmic[17], to form a mutant achlorophyllous SMZ strain without growth phase. Illumination intensity was measured regular-interfering with the cell division. Notably, both strains ly using a Digital Lux Meter Model: LX-1330/LX-1332have highly sophisticated subcellular organella equivalent (Custom Corporation Tokyo, Japan).to those of higher mammals; the E. gracilis Z strain can beregarded as a plant model, and its mutant strain SMZ as an 2.3. UV irradiationanimal model. Considering their properties, E. gracilis Zand SMZ strains were used as the model organisms in this UV-A (365 nm, 4 W Black Light Matsushita Electricstudy comparatively, to assess the effects of UV-A or Co., Osaka) or UV-B (model UVM-57, 302 nm, 6 W,UV-B, and MV when each were exposed either alone equipped with 2 UVG filter to cut UV-C; UVP Inc.,(UV-A, UV-B, MV), or combined (UV-A or UV-B1MV) Upland, CA, USA), were used for UV irradiation in theon the cell morphology, viability and growth. present study. Cells in the petri-dishes were exposed to

The effect as mentioned above, of UV radiation and MV UV-A or UV-B as follows. The distance between theinducing oxidative stress or generating free radicals petri-dishes and the UV source was adjusted, and then the

22[10,18], are considered highly likely to affect biomem- cells were exposed to UV-A or UV-B rays at 3 W m ;22brane lipids [19]. Thus, to assess the deterioration of UV-A and UV-B dose delivered was 0.36 J cm for each

membrane lipids, we examined levels of thiobarbituric acid exposure. The UV-A and UV-B lamps were fixed in darkreactive substance (TBARS) after UV irradiation or MV black boxes and all other fluorescent light or daylight wassingle or combined exposure in this study. excluded during the irradiation of the petri-dish samples.

It has been reported previously that flavonoids are The UV exposures were delivered once a day for 3effective reactive oxygen species scavengers that protect consecutive days, and UV output was monitored simul-plants from potentially harmful UV-B radiation [20]. taneously using a radiometer (VLX-3W Vilber lourmat,Investigations also proved that flavonoids including rutin, Torcy, France), with a 365 nm and 305 nm detector placed

2can capture O [21]. Rutin has also been reported to be at the same distance from the UV source as the petri-dish2

effective in scavenging reactive oxygen species and sup- samples. Approximately 1 h after UV irradiation, the cellspressing lipid peroxidation [22]. Therefore, using rutin as a were observed under a microscope before being placed inpotential natural antioxidant scavenger, we also examined the light cycle of the incubator. The UV exposure of allits suppressant effect by measuring the levels of TBARS in samples was carried out daily from 14:00 to 16:00 h for 3rutin-supplemented cells exposed to MV and UV-irradia- days. Cellular damage in E. gracilis was compared be-tion. In order to identify whether rutin could also be tween cells undergoing UV-A or UV-B irradiation only,effective in protecting against UV- and MV-induced cel- cells exposed singularly to 200 mM of MV, and thoselular damage, cell growth, viability and morphology were undergoing combined UV and MV treatment. The mor-assessed with rutin preincubated E. gracilis cells. phological change of cells preincubated with 100 mM rutin

prior to UV-A or UV-B single exposure, or combined withMV were also assessed. The experimental procedures are

2. Materials and methods described in Section 2.10. After the UV irradiation cellswere observed, hypertrophy, V-shaped, starfish-shaped and2.1. Reagents and chemicalsthose that were not completely divided were considered as

Methyl viologen (1,19-dimethyl-4,49-bipyridinium-di- 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 investigated in order to compare single and combinedinverted microscope (Olympus Optic Co., Inc., Tokyo) stress-induced cellular damage. For the morphologicalequipped with an image processor, ARGUS-100 assessment, cells were inoculated into microtiter wells(Hamamatsu Photonics Co., Hamamatsu, Japan). containing either 100 mM of MV in KH medium or KH

medium alone (as control cells), and incubated under2.4. Exposure to MV illuminated conditions as described in Section 2.2. Ob-

servations for morphological changes were carried outTo examine the effect of MV exposure on E. gracilis, daily under the video microscope, as explained in Section

5 6 21cells from a liquid culture (10 –10 cells ml ), were 2.3.4 21adjusted to a density of 1.6310 cells ml , and 50 ml of

the cell suspensions were inoculated into 3 ml of KH 2.6. Cell growth and viability of E. gracilis exposed tomedium containing either 100 mM or 200 mM MV in single or simultaneous UV and MV exposuresmall glass petri-dishes. These petri-dishes were thenexposed to UV-A or UV-B as described in Section 2.3. For The effect of MV single exposure on cell growth was

6 21samples exposed to UV-A or UV-B single exposure, cells assessed by inoculating 30 ml of 10 cells ml of E.were inoculated into 3 ml of KH medium without MV. gracilis into 5 ml of KH medium containing 100 mM MV

in test-tubes. Cells were incubated under either light /dark2.5. MV single exposure conditions (described in Section 2.2) or a 24-h dark cycle,

as the herbicidal effect of MV is oxygen- and light-The effect of MV single exposure on the cell morpholo- dependent. The cell density was measured using a Bosch

gy and growth of E. gracilis Z and SMZ strains was 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 under12/12 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.


Japan) over 3 days (results shown in Fig. 2). The growth of PBS solution. The non-viable blue-stained cells wereE. gracilis Z and SMZ strains exposed to either UV-A or detected by an Olympus IMT-2. The numbers of non-UV-B single exposure, or combined exposure with MV viable cells in six randomly selected frames were countedwas determined by measuring the cell abundance in 10 ml in triplicate (cell values were expressed as the averageof cell suspension taken from the petri-dish samples. The viable cell percentage of the total cell number6S.D., n536estimation of cell numbers was performed under the measurements). Viable cells were calculated as the differ-microscope (n518). ence between the total number of cells and the number of

non-viable cells. Cell viability was expressed as the2.7. Cell viability percentage of viable cells over total cells.

To estimate cell viability, 100 ml of E. gracilis cell 2.8. Estimation of abnormal cell occurrencesuspension were taken from each petri-dish, centrifuged at10003g (23 8C for 3 min), washed twice with 100 ml of V-shaped, hypertrophied and starfish-shaped cells werephosphate saline buffer (PBS, pH 7.2), and then stained classified as abnormal cells (Fig. 4). The frequency ofwith 20 ml of 0.4% Trypan Blue solution at 28 8C for 1 h. abnormal cells was estimated by counting the number ofAfter staining, cells were washed again with 100 ml 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 UVirradiation. (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-Airradiation 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 irradiationand 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

22UV-A or UV-B irradiation was 0.36 J cm . Each bar represents the mean6S.D. (n518). Symbols a, b, c, d indicate a significant difference between eachgroup (P,0.005) after the third day of exposure.


abnormal cells were expressed as the mean percentage of 2.10. Rutin supplementationthe total observed frames obtained from 36 measurements.

To assess whether the UV-B- and MV-induced lipid2.9. Estimation of TBARS levels peroxidation could be suppressed by rutin, either 50 mM or

100 mM rutin was added to E. gracilis cells immediatelyIt was considered that assessing the TBARS level of before exposure to UV irradiation and MV. After an acute

22UV-A or UV-B with or without MV exposed E. gracilis exposure of UV-B irradiation at a dosage of 0.78 J cm (322cells may be an indication of induced damage to lipid W m ), the cell suspension samples were taken and

components. Cells were delivered UV-A or UV-B doses of immediately centrifuged at 10003g (23 8C for 3 min),22 220.78 J cm at 3 W m exposed with or without 200 mM placed in a cryogenic tube, and kept at 280 8C until

MV. TBARS values of cells exposed to 200 mM MV only TBARS analysis. The levels of TBARS were estimatedwere also assessed. 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 andC) 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) SMZstrain. 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-Airradiation 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

22and 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 . Each barrepresents 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 ofUV irradiation.


2.11. E. gracilis cells preincubated with rutin according to the protocol of Section 2.3 and the de-termination of cell growth, viability and the estimation of

The cell growth, viability and morphology of E. gracilis abnormal cells following the procedure of Sections 2.6, 2.7cells incubated with 100 mM of rutin was also assessed to and 2.8.determine whether rutin would suppress cellular damageinduced by UV single exposure or when combined withMV. E. gracilis cells were preincubated with 100 mM of 2.12. Statistical analysisrutin (adjusted pH 7.4 with PBS buffer) for 24 h, washedthree times with KH medium, after which they were The data were evaluated by analysis of varianceresuspended in petri-dishes with or without MV, as de- (ANOVA). A value of P,0.005 was considered to bescribed in Section 2.4. UV irradiation was carried out significant.

Fig. 3. (continued)


3. Results

3.1. Cell growth and viability

The cell growth of cells exposed to MV alone at 100mM is shown in Fig. 1A and B. For both Z and SMZstrains there was no growth inhibition observed for thecells incubated either in light /dark or dark conditions overthe 3-day period when the cell density was monitored. Thisreflects that under light /dark or under dark incubationconditions, oxidative stress derived from MV exposurealone was probably not strong enough to inhibit the cellgrowth. UV single irradiation and UV exposed with MVinhibited cell growth, and there was a greater inhibitionafter UV-B irradiation than UV-A. As there was littlesignificant difference (P,0.005) between the cell growthsof UV-B only, and UV-B combined with MV exposed cellsin both strains, UV-B may have been the dominant causeof the inhibition (Fig. 2C and D). The UV-B dosage in thepresent study inhibited growth to almost the same extent inboth strains (Fig. 2C and D).

The cell viability of E. gracilis Z and SMZ strains wasalso affected by both UV-A and UV-B irradiation (Fig. 3).Cell viability in both strains decreased gradually afterexposure to UV-A irradiation, however the viable cellnumbers decreased significantly (P,0.005) in the higherconcentration of MV in comparison with lower concen-tration of MV (Fig. 3A and B). In contrast, UV-B exposurehad different effects on both strains, causing a moremarked decrease in the viability of the SMZ strain than inthe Z strain, after the third UV-B dosage (Fig. 3C and D).For both strains, UV-B irradiation caused a considerablyhigher 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 observedabnormal cell was V-shaped, followed by hypertrophiedand starfish-shaped cells under the experimental condi-tions. The normal shape of E. gracilis cell (a), V-shaped(b), starfish-shaped (c), hypertrophy (d) cells that wereobserved under different exposure conditions are shown(Fig. 4).

UV-A or MV alone induced between 6 and 8% abnormalcell outbreak (Fig. 5A and B), whereas combined exposure Fig. 4. Cell morphology of E. gracilis cells exposed to UV irradiationof UV-A and MV significantly (P,0.005) increased abnor- and MV. Abnormal cell occurrence of E. gracilis cells exposed to UV-A

or UV-B single or combined with MV exposure was observed. Themality by twofold after the third exposure (Fig. 5A and B).normal shape of E. gracilis cell (a), V-shaped (b), starfish-shaped (c),(The abnormal cell percentages on the third day are alsohypertrophy (d) cells that occurred. Bar in each photograph represents 20shown separately in Fig. 5E–H.) There was no significantmm.

difference in abnormality between cells exposed to 100mM and those exposed to 200 mM MV even whencombined with UV-A exposure.

Single or combined exposure to UV-B and MV caused a difference between UV-A and UV-B exposed groups in thesimilar cell abnormality to the UV-A and MV in the Z SMZ strain (Fig. 5B and D). Distinctly more abnormalstrain (Fig. 5A and C). However, there was a marked cells (27%) were observed after UV-B irradiation alone;


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 cellpercentages 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 exposedto 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-Airradiation 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

22and 200 mM MV (UV-B1200 mM MV). Each daily exposure of UV-A or UV-B irradiation was 0.36 J cm . Each bar represents mean percentage ofabnormal 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 ofexposure.

furthermore, the combined exposure of UV-B and MV, 3.3.2. The effect of rutin supplementationespecially 200 mM MV, greatly increased the number of Cells supplemented with 100 mM rutin immediatelyabnormal cells (76%) after the third exposure (Fig. 5D and before the UV-B exposure showed an extremely markedH). On the third day, significant difference (P,0.005) was decrease in TBARS levels. This was apparent in both Zrecognized between sole UV-B exposure and combined and SMZ strains. The 50 mM supplementation also sup-exposure of UV-B and MV; i.e. more abnormal cells with pressed the TBARS levels to an extent (Fig. 6A and B).UV-B and 200 mM MV than with UV-B and 100 mM MVexposed cell groups. 3.4. Effects of preincubation with rutin on cell growth,

viability, and morphology3.3. TBARS

E. gracilis cells preincubated with rutin prior to the3.3.1. Exposure to MV and /or UV-A or UV-B UV-A or UV-B exposure did not display any growth

Simultaneous exposure to UV-B and MV (UV-B1MV) recovery compared to the non-treated cells (Fig. 2A–D;resulted in the highest levels of TBARS in both the Z and Fig. 7A and B). Both UV-A and UV-B irradiation aloneSMZ strains of E. gracilis (Fig. 6A and B). A considerably and when combined with MV significantly (P,0.005)higher TBARS level was obtained with the Z strain. inhibited the cell growth compared to the control group inFurthermore, with the exception of the UV-A exposed cell the Z strain, however, UV-A was not significantly differentgroup, all exposed groups displayed a significant differ- from the control group in the SMZ strain (Fig. 7B). Theence (P,0.005) (Fig. 6A). The SMZ strain, however, had cell viability of the cells preincubated with rutin appearedfar lower TBARS levels than the Z strain in all the slightly higher than the non-preincubated cell groups, afterexposed groups. the third exposure to sole UV-B irradiation or combined


Fig. 5. (continued)

with MV exposure (Fig. 3E and F; Fig. 8A and B) in both gracilis have been reported previously. Results showedstrains. Abnormal cells appeared to be lower in cells that UV irradiation inhibits photosynthesis and cell growth.preincubated with rutin when cells were simultaneously However, there are few studies reporting the combinedexposed to UV-A or UV-B and MV in the SMZ strain (Fig. effects of UV irradiation and toxic substances on E.9B), than the non-treated cells (Fig. 5D). gracilis.

The lack of growth inhibition by 100 mM of MV,coupled with the severe growth inhibition by UV-A or

4. Discussion UV-B with or without MV in either strain (Fig. 2),indicates that UV-A or UV-B are the dominant contributors

The adverse effects of UV irradiation on the growth and to the inhibition of cell growth. UV-B irradiation alone ormotility [24] and the photosynthetic efficiency [8] of E. combined with exposure to MV, had an adverse effect on


cell viability in both the Z and SMZ strains of E. gracilis(Fig. 3C and D). Furthermore, the SMZ strain had a lowersurvival ratio than the Z strain after UV-B irradiation withor without MV on the third day (Fig. 3C, D, G, H).Shigeoka et al. reported a peroxidase in E. gracilis Z thatrequires ascorbic acid as the electron donor in the cytosolthat scavenges H O , as E. gracilis does not contain2 2

catalase [25]. Ascorbate peroxidase (APX) is found only inhigher plants and algae, but no report in animals. It wasfound that the APX content in E. gracilis Z strain, a plantmodel, was 5.157 mmol /mg protein /min and in SMZstrain, an animal model, 0.826 mmol /mg protein /min, incells incubated under almost the same conditions as that ofthe present study (unpublished data). Therefore, the lowerAPX content may have resulted in less viable cells in theSMZ strain after the third dosage of UV-B with or withoutMV, indicating different reactive oxygen scavengingabilities between the two strains. On the other hand, theviable cell percentages were higher in cells preincubatedwith 100 mM rutin prior to UV-B single or combined MVexposure (Fig. 8A and B) than non-preincubated cells inboth strains (Fig. 3E and F).

The percentage of abnormal cells increased when cellswere exposed simultaneously to MV and either UV-A orUV-B, suggesting that two consecutive daily exposures ofthe combined exposure generated sufficient reactive oxy-gen species to induce teratogenicity in E. gracilis cells.The combined exposure of UV-B and 200 mM MVenhanced the occurrence of abnormal cells in SMZ strainafter the third exposure (Fig. 5H). This high incidence ofabnormal cell proliferation in the SMZ might result fromthe lower potential of its reactive oxygen scavengingsystem as mentioned previously. Abnormal cell prolifer-ation must be linked to dysfunction of the cell-cycleregulation system, which frequently involves impairmentof DNA duplication, protein synthesis, membrane lipidsynthesis and organization of subcellular organella andtheir function. Scheuerlein et al. reported the evidence forUV-B-induced DNA degeneration in E. gracilis mediatedby activation of metal-dependent nucleases [26]. On theother hand, cells preincubated with 100 mM of rutin beforethe combined exposure of UV and MV, displayed aconsiderably lower abnormal cell number after the thirdUV-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 oxygenscavenger or not is not yet clear, and this decrease wasonly observed in the SMZ strain.

Fig. 6. Levels of TBARS in E. gracilis cells exposed to UV and MV and UV-A alone, or combined with exposure to MV, did notthe effect of rutin supplementation. (A) Z strain; (B) SMZ strain. Cells

increase the levels of TBARS as much as combined withexposed to no UV or MV (control), UV-A irradiation (UV-A), UV-BUV-B and MV. This might be due to the shorter wave-irradiation (UV-B), 200 mM methyl viologen (200 mM MV), UV-A

irradiation and 200 mM methyl viologen exposure (UV-A1MV), UV-B length of UV-B which may have a greater threat toirradiation and 200 mM methyl viologen exposure (UV-B1MV), UV-B unprotected molecules [15]. Cell growth (Fig. 1) andirradiation and 200 mM methyl viologen exposure with 50 mM rutin abnormal cell proliferation (Fig. 5) were either unaffected(UV-B1MV1R 50 mM), UV-B irradiation and 200 mM methyl viologen

or only slightly affected by simple exposure to 200 mMwith 100 mM rutin (UV-B1MV1R 100 mM). The total exposure energy22 MV in both Z and SMZ strains.of UV-A and UV-B was 0.72 J cm . Each bar represents the mean

value6S.D. (n59). Cells were analyzed immediately after UV irradiation. Furthermore, simple exposure to MV did not signifi-


Fig. 7. Effect of UV and methyl viologen exposure on the cell growth of Euglena gracilis preincubated with rutin. E. gracilis cells preincubated with rutinwere 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 exposureof 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

22UV-B irradiation was 0.36 J cm . 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, higher in the Z strain (Fig. 6), however, abnormal cella significant (P,0.005) increase was observed in the Z occurrence was generally higher in the SMZ strain (Figs. 5

21strain (Fig. 6). The highest TBARS level (136 nmol g ) and 9). Pretreatment with rutin definitely decreased thewas obtained from combined exposure to UV-B and MV in frequency of the abnormal cells in both strains, especiallythe Z strain; by contrast, in the SMZ strain this level was in combined exposure of UV-B and 200 mM MV in SMZ.

21below 100 nmol g . The abnormal cells in SMZ under the combined exposureComparing the results from the TBARS (single exposure of UV-B and 200 mM MV without rutin treatment,

at a higher UV energy level) (Fig. 6) with those of the cell remarkably decreased by almost 60% with pretreatmentgrowth (Figs. 1 and 2), viability (Fig. 3) and morphology with rutin. However, the effect of rutin was not soassessment (chronic exposure at lower UV energy level) remarkable in Z strain. This may be due to different(Fig. 5), UV- and MV-induced oxidative damage may have reactive oxygen scavenging systems and membrane lipiddiffered between the two strains. The TBARS levels were components between the E. gracilis Z and SMZ strains.


Fig. 8. Effect of UV irradiation and MV exposure on the viability of E.gracilis preincubated with rutin. E. gracilis cells preincubated with rutin Fig. 9. Effect of UV irradiation and MV in the cell morphology of E.were exposed to UV and MV and the cell viability tested after the third gracilis preincubated with rutin. (A) Z strain; (B) SMZ strain. After theexposure to UV irradiation. (A) Z strain; (B) SMZ strain. After the third third exposure of UV cell morphology of cells exposed to no UV or MVexposure of UV cell viability of cells exposed to no UV or MV (control), (control), UV-A irradiation (UV-A), UV-A irradiation and 200 mM MVUV-A irradiation (UV-A), UV-A irradiation and 200 mM MV (UV-A1200 (UV-A1200 mM MV), UV-B irradiation (UV-B), UV-B irradiation andmM MV), UV-B irradiation (UV-B), UV-B and 200 mM MV (UV-B1200 200 mM MV (UV-B1200 mM MV), MV represents methyl viologen.

22mM MV), MV represents methyl viologen. Each daily exposure of UV-A Each daily exposure of UV-A or UV-B irradiation was 0.36 J cm . Each

22or UV-B irradiation was 0.36 J cm . Each bar represents the mean6S.D. bar represents mean percentage of abnormal cells6S.D. (n536) after the(n536). Symbols, a, b, c, d indicate a significant difference between each third exposure to UV irradiation. Symbols a, b, c, d indicate a significantgroup (P,0.005) after the third day. difference between each group with P,0.005.


Acute or chronic exposure of UV irradiation also could for both rutin preincubated and non-preincubated cells,which could support the fact that UV irradiation alone cancause oxidative stress in different respects.cause considerable oxidative damage to smaller livingRegarding the protection effects of rutin, flavonoids areorganisms. In addition, the antioxidant rutin could suppressknown to have a protective effect, suppressing UV damagethe adverse effects caused by exposure to UV-B and MV.by absorbing light in the UV region [27–29]. However, inThe alterations in the membrane lipid caused by UV-Bthe present study this was not investigated. Halliwell andand/or MV are currently under investigation by means ofGutteridge reported previously that secondary productschromatographic analyses.such as flavonols and flavonoids have powerful antioxidant

2actions, such as scavenging O and inhibiting lipid22peroxidation [30]. Rutin is known to function as an O2

Referencesscavenger [21,28], and here we found that the levels ofTBARS in cells exposed to MV and UV-B were suppressed

[1] C. Nolan, G. Amantidis, European Commission research on theby the addition of rutin compared with non rutin supple-fluxes and effects of environmental UV-B radiation, J. Photochem.mentation. The Z and SMZ strains exposed to UV-B andPhotobiol. B: Biol. 31 (1995) 3–7.

MV in the presence of 100 mM of rutin showed re- [2] J.B. Kerr, C.T. McElroy, Evidence for large upward trends of UV-Bmarkably low levels of TBARS (Fig. 6A and B). From the radiation linked to ozone depletion, Science 26 (1993) 1032–1034.

[3] C.S. Zerefos, C. Meleti, A. Bais, The recent UV-B variability overexperiments carried out in the present study, full speciationsouthern Eastern Europe, J. Photochem. Photobiol. B: Biol. 31of the reactive oxygen species that were entangled in the(1995) 15–19.

cellular damage is difficult. Regarding the fact that rutin ¨[4] L.O. Bjorn, Effects of ozone depletion and increased UV-B on2was able to scavenge O [21,29], and suppress lipid terrestrial ecosystems, Int. J. Environ. Stud. 51 (1996) 217–243.2

¨peroxidation [22], there are possibilities, however, that it [5] D.P. Hader, Penetration and effects of solar UV-B on the phyto-2 plankton and microalgae, Plant Ecol. 128 (1997) 4–13.may have scavenged generated O . In the experiment the2

[6] O. Holm-Hansen, E.W. Helbling, D. Lubin, Ultraviolet radiation inresult of which is shown in Fig. 6, rutin was addedAntarctica: inhibition of primary production, Photochem. Photobiol.

immediately before exposure to UV-B and MV, however, it 58 (1993) 567–570.is not fully clear whether the scavenging effect took place [7] M.P. Lessser, J.H. Farrrell, C.W. Walker, Oxidative stress DNAinside or outside the cell. In order to clarify the mechanism damage, and p53 expression in the larvae of Alantic cod exposed to

ultraviolet (290–400 nm) radiation, J. Exp. Biol. 204 (2001) 157–of the protecting role of rutin against UV- and MV-induced164.oxidative stress, further investigations on the incorporation

[8] R. Danilov, N.G.A. Ekelund, Influences of waste water from theand intracellular /extracellular behavior of rutin are neces- paper industry and UV-B radiation on the photosynthetic efficiencysary. of Euglena gracilis, J. Appl. Phycol. 11 (1999) 157–163.

The preventive effect of antioxidants on UV-induced [9] J.K. Howard, Recent experience with paraquat poisoning in GreatBritain: a review of 68 cases, Vet. Hum. Toxicol. 21 (1979) 213–skin cancer in mice has been reported recently by Ichihashi216.[31]. It is worthwhile investigating whether E. gracilis Z

[10] A.D. Dodge, The role of light and oxygen in the action ofand SMZ strains are protected from UV-B- and MV- photosynthetic inhibitor herbicides, in: Biochemical Responsesinduced cellular damage by the enrichment of other natural Induced by Herbicides, ACS Symposium Series, Vol. 181, 1982, pp.antioxidants under various oxidative stress conditions. 57–77.

[11] B. Halliwell, in: 2nd Edition, Chloroplast Metabolism, ClarendonInvestigations concerning the effects of these UV andPress, Oxford, 1984.chemical reagents on the DNA, lipid membranes and

[12] B. Halliwell, J. Gutteridge, Reactions of the superoxide radical, in:proteins of E. gracilis cells are now under way. Free Radicals in Biology and Medicine, Clarendon Press, Oxford,

In the present study, the authors have learnt that the 1989, pp. 301–307.combined exposure to UV and MV can cause considerable [13] C.F. Babbs, J.A. Pham, R.C. Coolbaugh, Lethal hydroxyl radical

production in paraqaut-treated plants, Plant Physiol. 90 (1989)cellular damage to smaller organisms, such as phyto-1267–1270.plankton in the hydrosphere, by an increasing adverse

´[14] P.H. Clingen, M. Berneburg, C. Petit-Frere, W. Woollons, J.E. Lowe,effect of either one, through interaction with environmental C.F. Arlett, M.H.L. Green, Contrasting effects of an ultraviolet Bpollutants. and ultraviolet A tanning lamp on interleukin-6, necrosis factor

In summary, we have shown that for the unicellular alpha and intracellular adhesion molecule-1 expression, Br. J.Dermatol. 145 (2001) 54–62.flagellate E. gracilis, combined exposure of UV radiation

[15] F. de Gruijl, Photocarcinogenesis: UVA vs. UVB methods inand the herbicide MV affected cell morphology signifi-enzymology, Photocarcinogenesis 319 (2000) 359–367.

cantly more than exposure to UV radiation or MV exposure ´ ´ ˆ[16] S. Seite, D. Moyal, S. Richard, J. de Rigal, J.L. Leveque, C.alone. Furthermore, it was demonstrated that TBARS Hourseau, A. Fourtanier, Mexoryl, SX: abroad absorption UV-Avalues were increased by combined exposure of UV and filter protects human skin from the effects of repeated suberythemal

doses of UVA, J. Photochem. Photobiol. B: Biol. 44 (1998) 69–76.MV presumably owing to interactions between the radia-[17] Y. Oda, Y. Nakano, S. Kitaoka, Utilization of toxicity of exogenoustion and MV that generate reactive oxygen species. UV-B

amino acids in Euglena gracilis, J. Gen. Microbiol. 12 (1982)clearly affected the cell growth and viability more than 853–858.UV-A, however, simple exposure of UV-A or UV-B and [18] O.I. Aruoma, B. Halliwell, Molecular Biology of Free Radicals Incombined exposure with MV had a similar adverse effect Human Diseases, OICA, London, 1998.


¨[19] T. Schwartz, UV light effects cell membrane and cytoplasmic [26] R. Scheuerlein, S. Treml, B. Thar, U.K. Tirlapur, D.-P. Hader,targets, J. Photochem. Photobiol. B: Biol. 44 (2) (1998) 191–195. Evidence for UV-B-induced DNA degradation in Euglena gracilis

[20] A. Koostra, Protection from UV-B induced DNA damage by mediated by activation of metal-dependent nucleases, J. Photochem.flavonoids, PMBIDB 26 (1994) 771–774. Photobiol. B: Biol. 31 (1995) 113–123.

[21] Y.T. Chen, R.L. Zheng, Z.J. Jia, Y. Ju, Flavonoids as superoxide [27] E.M. Middleton, A.H. Teramura, The role of flavonol glycosidesscavengers antioxidants, Free Rad. Biol. Med. 9 (1) (1990) 19–21. and carotenoids in protecting soy bean from Ultraviolet B damage,

[22] I.B. Afanas’eva, A.I. Dorozhko, A.V. Brodskii, V.A. Kostyuk, A.I. Plant Physiol. 103 (1993) 741–752.Potapovitch, Chelating and free radical scavenging mechanisms of [28] A.E. Stapleton, Ultraviolet radiation and plants: burning questions,inhibitory action of rutin, quercetin in lipid peroxidation, Biochem. Plant Cell 4 (1992) 1353–1358.Pharmacol. 38 (1989) 1763–1769. [29] Y. Hanasaki, S. Ogawa, S. Fukui, The correlations between active

[23] K. Kikugawa, T. Kojima, S. Yamaki, H. Kosugi, Interpretation of oxygens scavenging and antioxidative effects of flavonoids, Freethe thiobarbituric acid reactivity of rat liver and brain homogenates Rad. Biol. Med. 16 (6) (1994) 845–850.in the presence of ferric ion and ethylenediaminetetraacetic acid, [30] B. Halliwell, J. Gutteridge, Reactions of the superoxide radical, in:Anal. Biochem. 202 (2) (1992) 249–255. Free Radicals in Biology and Medicine, Clarendon Press, Oxford,

[24] N.G.A. Ekelund, The effect of UV-B radiation and humic substances 1989, pp. 275–288.on the growth and motility of the flagellate, Euglena gracilis, J. [31] M. Ichihashi, N.U. Ahmed, A. Budiyanto, A. Wu, T. Bito, M. Ueda,Plankton Res. 15 (1993) 715–722. T. Osawa, Preventive effect of antioxidant on ultraviolet-induced

[25] S. Shigeoka, Y. Nakano, S. Kitaoka, Metabolism of hydrogen skin cancer in mice, J. Dermatol. Sci. 23 (Suppl. 1) (2000) 45–50.peroxide in Euglena gracilis Z by L-ascorbic acid peroxidase, J.Biochem. 186 (1) (1980) 377–380.

Documents

Oxidative stress-induced cellular damage caused by UV and methyl viologen in Euglena gracilis and its suppression with rutin