Photoprotective effect of flax seed oil (Linum usitatissimum L.) against ultraviolet C-induced apoptosis and oxidative stress in rats

Toxicology and Industrial Health 28(2) 99–107 ª The Author(s) 2012 Reprints and permission: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233711407239 tih.sagepub.com

¨ zkol1 and Ismail _ Yasin Tu¨lu¨ce1, Halil O Koyuncu2

Abstract The aim of this study is to determine antioxidant and antiapoptotic effects of flax seed oil (FSO) on rats exposed to ultraviolet C (UVC). Malondialdehyde (MDA), protein carbonyl (PC) and reduced glutathione (GSH) levels as well as glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were measured in lens, skin and serum. In addition, b-carotene, vitamin A, C and E contents were measured in serum, while apoptosis was determined in retina. Rats were divided into three groups as control, UVC and UVC þ FSO. UVC and UVC þ FSO groups were exposed to UVC light for 1 h twice a day for 4 weeks. FSO (4 ml/kg bw) was given by gavage before each irradiation period to the UV þ FSO group. While MDA and PC levels of the UVC group increased compared to the control group, their levels decreased in the UVC þ FSO group compared with the UVC group in skin, lens and serum. Skin GSH level decreased significantly in the UVC and UVC þ FSO groups. As GPx and SOD activities of the UVC group were lower, their activities were higher in the UVC þ FSO group in skin, lens and serum. There was only marked elevation of vitamin A level in the UVC group compared to the control group. Apoptosis increased in the UVC group and the UVC þ FSO groups in retina. However, retinal apoptosis were lower in the UVC þ FSO group compared with the UVC group. This investigation demonstrated that UVC exposure led to oxidative stress and apoptosis in rats as reflected by increased MDA, PC contents and decreased enzymatic and nonenzymatic antioxidant levels, FSO may be useful for preventing photoreactive damage. Keywords Ultraviolet, flax seed oil, apoptosis, oxidative stress, photoprotective

Introduction Ultraviolet (UV) leads to different health problems, including erythema/edema and subsequent pigmentation (sunburn), inflammation, premature skin aging (photoaging) and melanogenesis (Biesalski and Obermueller-Jevic, 2001; Ichihashi et al., 2003; Matsumura and Ananthaswamy, 2002). The UV spectrum range consists of ultraviolet A (UVA; 320– 400 nm), ultraviolet B (UVB; 280–320 nm) and ultraviolet C (UVC; 200–280 nm; Holden, 1983). Among them the most powerful and dangerous one is UVC which also has sterilization, non-ionising and biocidal properties. Although UVA and UVB cellular damage is well documented, UVC damage is rarely reported owing to its almost complete absorption via molecular oxygen and ozone layer (Basti et al., 2009). Nevertheless, during the past few decades, depletion of the ozone

layer gave rise to an elevation in the level of UVC received by the people on earth. Skin is the largest organ of the human body that is in contact with UV. Moreover, eyes also suffer from UV directly so onset of lens opacification and cataract processes occur (Kohli et al., 1996). UV causes an increase of free radicals and initiates photooxidation in lens (Bardak et al., 2000). Reactive oxygen species (ROS) can damage the cellular 1

Department of Medical Biology, Faculty of Medicine, Yuzuncu Yil University, Van, Turkey 2 Department of Biology, Faculty of Science and Art, Harran University, Sanliurfa, Turkey Corresponding author: Yasin Tu¨lu¨ce, Department of Medical Biology, Faculty of Medicine, Yuzuncu Yil University, Van 65080, Turkey Email: [email protected]

100

components and inhibit DNA repair (Fuchs et al., 1989a; Kripke, 1981; Nishi et al., 1991; Strickland, 1986; Wang et al., 1992). Lipid peroxides causes changes in nucleic material such as DNA–protein crosslinking, pyrimidine dimers and single-strand breaks. In another study, it is reported that UV radiation also induces apoptosis (Aragane et al., 1998; Kulms et al., 1999). Apoptosis is an important process for eliminating UV-damaged cells that potentially could become cancerous. Antioxidants have been reported to provide protection against many different types of apoptosis (Buttke and Sandstrom, 1994). In case ROS could not be eliminated by the antioxidant systems in the skin, they would affect the other body parts and tissues by the circulation and peroxidate the membrane lipids and oxidate the cell proteins. The endogenous antioxidant defense system consists of catalase, superoxide dismutases and glutathione redox cycle enzymes as enzymatic antioxidants in skin as well as nonenzymatic antioxidants including glutathione, ubiquinol, b-carotene, ascorbate, a-tocopherol, uric acid and a-lipoic acid (Fuchs, 1998; Ichihashi et al., 2000; Jurkovic et al., 2003; Shindo et al., 1994). Although the skin possesses an elaborate antioxidant system to deal with UV-induced oxidative damage, the use of active photoprotectives is recommended. In recent years, natural compounds which possess antioxidant and anti-inflammatory properties have created considerable interest as protective agents for reducing radiation-induced skin damage (Cemek et al., 2006; Enginar et al., 2007; F’guyer et al., 2003; Pinnell, 2003; Svobodova et al., 2003). Many studies have to be conducted on preserving human health against the deleterious impacts of UV radiation. An approach to protect the occurrence of damage is to augment endogenous photoprotection through topical or oral administration of botanical antioxidants that have photoprotective properties. For this purpose the protective impact of flax seed oil (FSO) with oral administration was investigated in the current study. FSO is obtained from Linum usitatissimum L. belonging to family Linaceae. It consists of palmitic acid 6.0%, stearic acid 2.5%, arachidic acid 0.5%, oleic acid 19.0%, linoleic acid 24.1%, a-linolenic acid 47.4%, other 0.5% (Morris, 2003). We postulated that FSO might be a useful antioxidant nutrient because of its high lignan content. The oxygen radical scavenging properties of lignans were shown in vitro by either direct hydroxyl radical scavenging activity (Prasad, 1997, 2000) or inhibiting lipid peroxidation (LPO; Kitts et al., 1999).

Toxicology and Industrial Health 28(2)

In the current study, the activity of antioxidant enzymes including glutathione peroxidase (GPx) and superoxide dismutase (SOD) and the alterations in the levels of reduced glutathione (GSH), malondialdehyde (MDA) and protein carbonyl (PC) in rat serum, skin and lens as well as serum antioxidant vitamin levels with retinal apoptosis were investigated to find out whether FSO alters UVC effect by scavenging free radicals.

Materials and methods Determination of FSO dose FSO was obtained from a plant store in Sanliurfa, Turkey. Optimal dose of FSO against UVC radiation was determined as 4 ml/kg body weight as reported in Bhatia et al. (2007).

Animals and experimental groups Experiments were performed on 21 Sprague-Dawley male albino rats (260 + 25 g) that were housed in plastic cages (7 rats per cage) in a temperature-controlled room (21 C) and given free access to laboratory chow with water. Rats were shaved with a safety razor and this operation was repeated before the UV periods. The light–dark cycle was from 7 a.m. to 7 p.m. The animals were divided into three groups. (1) Control group: received 1 ml physiological water by intragastric administration during the 28 days before they were killed. (2) UVC group: exposed to UVC irradiation (1.25 mW/cm2) for 1 h twice a day and received 1 ml physiological water by intragastric administration for 4 weeks. (3) UVC þ FSO group: received 1 ml FSO (4 ml/kg bw) by intragastric administration and exposed to UVC light for 1 h twice a day for this time. Animal experiments were carried out following the guidelines of the Animal Ethics Committee of Harran University.

Exposure to ultraviolet radiation In this experiment UVC was produced by a lamp (Mazda TG model) with 30 Watt power and 90 cm length. Rats were exposed to UVC in a 182  68  50 box which was painted flat dark as described by Brainard et al. (1986) and Turker (2004). The lamp was mounted on the lid of the box at a distance of 44 cm from the animals. The animals were subjected to UVC light radiated from the lamp for 28 days. The intensity of the UVC coming from the lamp was measured by a spectrophotometer to have a wavelength with a peak value of 254 nm. In this experiment,

O¨zkol et al.

energy of the UVC light emitted from the lamp per cm2 for 1 second was found as 0.0014 joule/cm2. This amount was adjusted according to Turker (2004).

Preparation of lens, retina, skin and serum samples At the end of the treatments, rats were anesthetized with ketamine (100 mg/kg ip). Blood samples were taken from the animals into tubes with K3EDTA by injectors from the heart under light anesthesia then the rats were euthanized with inhalation of CO2. The serum samples were obtained by centrifuging the blood samples at 3000 rpm for 15 min at 4 C. After the eye enucleation, lens and retina were carefully removed using a posterior approach and rinsed with 6.3-mM EDTA phosphate buffer. Skin was dissected and put in petri dishes for washing with physiological saline (0.9% NaCl). All tissue samples were kept at 80 C until analysis. Tissues were homogenized in ice-cold phosphate buffer solution for 5 min using both an ultrasonic (Bandelin, UW 2070) and a mechanic (Heidolf, silent crusher M) homogenizers and then centrifuged at 7000-g force for 15 min. All processes were carried out at 4 C. While apoptosis was assessed in homogenate of retina samples, lens and skin supernatants as well as serum were used to determine protein concentration and oxidant/ antioxidant parameters. In addition, b-carotene, vitamin A (retinol), C, E (a-tocopherol) levels were measured in serum.

Determination of GPx and SOD activities GPx activity was determined by using the Cayman Chemical Glutathione Peroxidase Assay Kit (Cat. no 703102) that measures the GPx activity indirectly by a coupled reaction with glutathione reductase. SOD activity was assessed by the Cayman Chemical SOD Assay kit (Cat. no 706002) measuring the dismutation of superoxide radicals generated by xanthine oxidase and hypoxanthine.

Reduced glutathione determination GSH concentration was assayed by reacting with O-phthaldialdehyde (OPT, 10 mg/10 ml methanol) according to the modified method of Lee and Chung (1999). Pure reduced GSH was used as standard for calibration. GSH samples were measured by using spectrofluorimetry (Jasco 6000, USA), with excitation at 345 nm and emission at 425 nm.

101

Antioxidant vitamin determination Vitamin A. The levels of b-carotene and vitamin A (retinol) were detected at 425 and 325 nm according to method of Suzuki and Katoh (1990). Serum (100 ml) was placed in a dark brown test tube. Butylated hydroxyl toluene solution (100 ml) and 300 ml of n-hexane were added to the serum, respectively. All tubes were shaken mechanically for 10 min and then centrifuged at 2000 rpm for 10 min. After centrifugation, 250 ml of hexane was removed from tubes and absorbance was measured. Vitamin C. For vitamin C analysis, serum sample (50 ml) and 6% perchloric acid (450 ml) were placed in a polypropylene tube and incubated for 15 min and then centrifuged at 2500 rpm for 10 min. Supernatant (100 ml) was removed into a separate tube and reaction solution (30 ml) including 2,4-dinitrophenylhydrazine was added. Later tube was incubated at 90 C for 20 min. Consequently 65% H2SO4 (150 ml) was supplemented after incubation in ice. Optical density was measured at 520 nm. Six per cent of perchloric acid was used as a blank according to the method of Omaye et al. (1979). Vitamin E. It was analyzed colorimetrically (Martinek, 1964). Serum, absolute ethanol and xylene (1:1:1) were placed in a glass tube and mixed well. Then centrifuged at 2500 rpm for 5 min. Supernatant (50 ml) was removed into a separate tube and 2,4,6-tripridyl-striazin (50 ml) was added. First absorbance was read at 460 nm. Second absorbance was read at 600 nm after FeCl3 (10 ml) supplementation. Finally the concentration of vitamin E was calculated using these optic densities with standard and blank values. All antioxidant vitamins measured in a microplate reader spectrophotometer.

Malondialdehyde determination The LPO product MDA was estimated using the modified thiobarbituric acid-reactive substance method by Hegde et al. (2003). MDA was measured by spectrofluorimetry, with excitation at 520 nm and emission at 555 nm. Calculations were performed using a linear regression from tetraethoxypropane for the MDA standard curve.

Protein carbonyl determination Protein oxidation was measured by Cayman’s Protein Carbonyl Assay Kit (Cat. no 10005020) as carbonyl content in the samples. This kit bases on the principle which utilizes the 2,4-dinitrophenylhydrazine reaction

102

Toxicology and Industrial Health 28(2)

Table 1. The photoprotective effect of FSO on UVC-induced oxidative stress in ratsa Tissue Skin

Lens

Serum

Time (h) MDA (nmol/g) PC (nmol/mg prot.) GSH (nmol/g) GPx (nmol/min/g) SOD (U/g) MDA (nmol/g) PC (nmol/mg prot.) GSH (nmol/g) GPx (nmol/min/g) SOD (U/g) MDA (nmol/g) PC (nmol/mg prot.) GSH (nmol/ml) GPx (nmol/min/g) SOD (U/g)

Control 8.31 4.11 1.03 16.39 2.32 3.88 2.77 14.63 6.22 2.84 7.15 6.66 4.38 15.51 2.68

+ 2.02 + 0.31 + 0.12 + 3.55 + 0.41 + 0.78 + 0.32 + 3.17 + 0.82 + 0.27 + 0.56 + 1.33 + 0.35 + 2.21 + 0.38

UVC þ FSO

UVC 16.04 + 9.57 + 0.69 + 6.60 + 1.70 + 5.73 + 6.30 + 12.82 + 2.63 + 1.35 + 13.52 + 12.1 + 3.83 + 9.31 + 1.17 +

b

4.48 1.46b 0.15b 1.81b 0.52 1.01b 1.33b 2.79 0.53b 0.35b 1.01b 0.93b 1.09 2.30b 0.14b

11.48 6.18 0.75 12.67 1.95 4.57 3.56 12.50 4.15 1.86 10.26 8.63 4.71 13.11 1.79

+ + + + + + + + + + + + + + +

1.66b 1.49b,c 0.07b 2.23c 0.63 1.24 0.87c 1.19 0.92b,c 0.57b 2.05b,c 0.58b,c 1.20 2.97c 0.15b,c

FSO: flax seed oil, GSH: reduced glutathione, GPx: glutathione peroxidase, MDA: malondialdehyde, PC: protein carbonyl, SOD: superoxide dismutase, UVC: ultraviolet C. a Values are given as the means + SD. b Control versus treated p < 0.05 level. c UVC versus treated p < 0.05 level.

to measure the PC content in homogenate (Levine et al., 1990). The amount of protein-hydrozone produced is quantified spectrophotometrically at an absorbance 360 nm by 96-well plate reader (Spectra Max M5). The carbonyl content was standardized to protein concentration.

Protein determination The protein content in the samples was measured by the method of Lowry et al. (1951) with bovine serum albumin as the standard.

Assessment of retinal apoptosis Roche Apoptotic DNA Ladder Kit (Cat. no 11 835 246 001) was used which enabled the isolation of apoptotic DNA fragments for DNA ladder analysis. Apoptosis was identified by separating DNA fragments on agarose gel. At the end of electrophoresis, the gel was visualized by UV fluorescence and then photographed.

Statistical analysis The statistical analyses were realized using the Minitab 13 for windows package program. All data were presented as means + standard deviation (SD). One-way analysis of variance (ANOVA) statistical test and Tukey’s posttest were used to determine the

differences between means of the experimental groups accepting the significance level at p < 0.05.

Results Effect on MDA, PC and GSH levels Results for MDA, PC and GSH are shown in Table 1. MDA and PC levels of the UVC group increased compared to the control group in all tissues. MDA content was markedly higher in the UVC þ FSO group than in the control group in skin and serum. However, its level was lower in the UVC þ FSO group than in the UVC group in serum. PC level decreased significantly in the UVC þ FSO group compared to the UVC group in all tissues. GSH content decreased markedly in the UVC and UVC þ FSO groups compared with the control in skin (Table 1).

Effect on antioxidant enzymes GPx activity of the UVC group was significantly lower compared with the control group in all tissues. Its activity in the UVC þ FSO group was markedly lower than the control group in serum, and higher than the UVC group in all tissues. SOD activity of the UVC and UVC þ FSO groups significantly diminished in comparison with the control group in lens and serum. But its activity was markedly higher in the UVC þ FSO group compared with the UVC group in serum (Table 1).

O¨zkol et al.

103

Table 2. The photoprotective effect of FSO on antioxidant vitamin levels of UVC irradiated ratsa Parameters b-Carotene (mg/dl) Vitamin A (mg/dl) Vitamin C (mg/dl) Vitamin E (mg/dl)

Control 18.76 114.18 0.043 2.44

+ 1.05 + 3.52 + 0.01 + 0.65

UVC þ FSO

UVC 20.83 + 121.54 + 0.039 + 2.42 +

2.28 5.52b 0.01 0.46

18.29 + 116.64 + 0.035 + 2.30 +

1.27 1.74 0.01 0.77

FSO: flax seed oil, UVC: ultraviolet C. a Values are given as the means + SD. b Control versus treated p < 0.05 level.

Figure 1. Retinal apoptosis/agarose gel electrophoresis of DNA ladder formation in retina of rats from the control (C), UVC (U) and UVC þ FSO (F) groups. (M) indicates the lane of the molecular size marker (0.072–1.35 kbp). (þC) represents positive control.

As shown in Table 2, there was statistically significant elevation of vitamin A level in the UVC group compared with the control group (Table 2).

Agarose gel electrophoresis of DNA ladder formation in retina of rats from the control (C), UVC (U) and UVC þ FSO (F) groups. (M) indicates the lane of the molecular size marker (0.072–1.35 kbp). (þC) represents positive control.

Effect on retinal apoptosis

Discussion

Apoptosis increased in the UVC group compared to the control and the UVC þ FSO groups in retina. However, retinal apoptosis were lower in the UVC þ FSO group compared with the UVC group (Figure 1).

The skin and eyes are tissues that come in contact with UVC due to the thinning of the ozone layer and other artificial sources such as sterilization lamps. In this study, we decided to investigate the alterations

Effect on antioxidant vitamin levels

104

in oxidant/antioxidant status of these organs after UVC exposure. This status was also investigated in serum owing to its close relation with organs via circulation. Moreover the protective effect of FSO against UVCinduced oxidative damage and retinal apoptosis were determined. Although many in vivo studies have been performed on mammals concerning the influences of UVA and UVB, this is the first study investigating the effects of UVC. Therefore, we compared the data of this study with the results of UVC applied in vitro studies and UVA and UVB administrated in vivo investigations regarding oxidative stress and apoptosis. ROS generation plays an important role in UV radiation-induced skin damage and can cause injury by reacting with various molecules such as lipids, proteins and nucleic acids, and also by depleting the skin of endogenous enzymatic and nonenzymatic antioxidants (Darr and Fridovich, 1994; Shindo et al., 1993). Some of the nonenzymatic scavengers of free radicals include vitamins A, C and E, and enzymatic scavengers include superoxide dismutase, catalase and glutathione peroxidase. Besides, ROS may also induce apoptosis (Ramakrishnan et al., 1993) It has been demonstrated that UV light induces LPO in mitochondria and sustains free radical reactions. MDA (end product of LPO) and/or other aldehydes are formed in oxidative stress. These can react with amino acids and DNA (Ames et al., 1982) and introduce cross-linkages between proteins (Nielsen, 1981) and nucleic acids, resulting in alterations in replication, transcription (Perchellet and Perchellet, 1989). ROS are believed to be critical mediators of the photoaging and photocarcinogenesis processes (Bowden, 2004; Droge, 2002; F’guyer et al., 2003). It can modify proteins, for example, collagen cross-linking in tissue to form carbonyl derivatives, which accumulate in the papillary dermis of photo damaged skin (Sander et al., 2002). In our study, while MDA and PC levels of the UVC group increased compared to the control group, their levels decreased in the UVC þ FSO group compared with the UVC group in all tissues. Elevated MDA and PC levels show consistency with previous studies (Anwar and Moustafa, 2001; Cerutti, 1985; Inal and Kahraman, 2000; Oberley and Oberley, 1986; Sander et al., 2002). MDA and PC were accumulated remarkably by UVC irradiation, which indirectly suggested that the large amount of oxygen radicals was generated from UVC irradiation. In the presence of FSO, the decreased levels of MDA and PC suggest that FSO could scavenge oxygen radicals.

Toxicology and Industrial Health 28(2)

There is an antioxidant system known as GSH redox cycle. Decrease in the function of this system appears to be closely associated with lipid peroxidation and protein oxidation (Yu et al., 2007). GSH is the most important cellular nonenzymatic antioxidant of this cycle (Rennenberg, 1980). In the current study its level diminished significantly in the UVC and UVC þ FSO groups compared to the control in skin. This result is in accordance with other studies (Katiyar et al., 2001; Psotova et al., 2006; Schneide et al., 2006). GSH level decreases owing to its ROS scavenging activity by hydrogen transfer (Casagrande et al., 2006). While GPx and SOD activities of the UVC group were lower compared with the control group, their activities were higher in the UVC þ FSO group than the UVC group in all tissues. In this study diminished GPx and SOD activities by UV show consistency with previous investigations (Anwar and Moustafa, 2001; Fuchs et al., 1989b; Inal and Kahraman, 2000; Shindo et al., 1993). Our data clearly shows that UV exposure markedly suppressed GPx and SOD activities. Due to trapping O2 and OH radicals by FSO, formation of peroxy radicals was prevented. Further FSO broke peroxy radical chains. Thus, GPx and SOD activities elevated. As for vitamins, there was only marked elevation of vitamin A level in the UVC group compared to the control group. As it has been reported that vitamins A, E and C exhibit photoprotective effects on skin due to their antioxidant activities (Eberlein-Konig et al., 1998; Mittal et al., 2003; Stahl et al., 2000), endogenous production of vitamin A might be enhanced in the liver. Therefore the usual transition of retinol from the liver to plasma might have increased. Another presumption is that the absorption of dietary b-carotene and retinol in the bowel might have been augmented which may also clarify why their levels are higher in serum for the UVC group. Apoptosis is an active process of cell destruction characterized by cell shrinkage, cytoplasmic blebbing, cell rounding, chromatin condensation with extensive nuclear fragmentation and nuclear pyknosis. Apoptosis plays an important role in both physiological and pathological conditions. ROS exacerbates formation of the highly reactive hydroxyl radical, which in turn can cause DNA strand breaks, damage protein residues, initiate lipid peroxidation and triggers apoptotic cellular death process. Antioxidants may prevent apoptosis by depletion of ROS (Buttke and Sandstrom, 1994). The apoptosis in retinal cells as a consequence of photic injury proceeds by both

O¨zkol et al.

caspase-dependent and caspase-independent pathways (Donovan et al., 2001; Grimm et al., 2000). UVCirradiated cultured cells exhibited an elevated number of apoptotic cells (Chathoth et al., 2009; Lonskaya et al., 1997; Nagira et al., 2002; Radziszewska et al., 2000; Wang et al., 1997). In this study, retinal apoptosis increased in the UVC group compared to the control and the UVC þ FSO groups. However, it was lower in the UVC þ FSO group compared with the UVC group. FSO treatment might reduce UVC-induced apoptosis either by activation of the DNA repair systems or scavenging ROS. In conclusion, the current study demonstrates that UVC light damages the antioxidant defense system and induces apoptosis in different tissues of rats. In addition, oral consumption of FSO is of benefit by preventing cells from the detrimental effects of UVC light or at least it reduces the injury. Acknowledgements The authors would like to thank Prof Dr Nihat Dilsiz for laboratory equipment and biologists Elif Aktas¸ , Fatma Tarkan, M Emin Alpay, Canan Durak, Sinan Soral and Fuat Karakus for their help in the course of laboratory studies.

Funding This research was supported by a grant from the TUBITAKBIDEB, Turkey.

References Ames BN, Hollstein MC, and Cathcart R (1982) Lipid peroxidation and oxidative damage in DNA. In: Yagi K (ed.) Lipid Peroxides in Biology and Medicine. New York: Academic Press, 339–351. Anwar MM, Moustafa MA (2001) The effect of melatonin on eye lens of rats exposed to ultraviolet radiation. Comparative Biochemistry and Physiology 129(1): 57–63. Aragane Y, Kulms D, Metze D, et al. (1998) Ultraviolet light induces apoptosis via direct activation of CD95 (Fas/APO-1) independently of its ligand CD95L. Cell Biology 140(1): 171–182. Bardak Y, Ozerturk Y, Ozguner F, Durmus M, and Delibas N (2000) Effect of melatonin against oxidative stress in ultraviolet-B exposed rat lens. Current Eye Research 20(3): 225–230. Basti D, Bricknell I, and Bouchard D (2009) Recovery from a near-lethal exposure to ultraviolet-C radiation in a scleractinian coral. Journal of Invertebrate Pathology 101(1): 43–48. Bhatia AL, Sharma A, Patni S, and Sharma AL (2007) Prophylactic effect of flaxseed oil against

105 radiation-induced hepatotoxicity in mice. Phytotherapy Research 21(9): 852–859. Biesalski HK, Obermueller-Jevic UC (2001) UV light, hcarotene and human skin-Beneficial and potentially harmful effects. Archives of Biochemistry and Biophysics 389(1): 1–6. Bowden GT (2004) Prevention of non-melanoma skin cancer by targeting ultraviolet-B-light signaling. Nature Reviews Cancer 4(1): 23–35. Brainard GC, Podolin PL, Leivey SW, Rollag MD, Cole C, and Barker FMF (1986) Near ultraviolet radiation suppresses pineal melatonin content. Endocrinology 119(5): 2201–2205. Buttke TM, Sandstrom PA (1994) Oxidative stress as a mediator of apoptosis. Immunology Today 15(1): 7–10. Casagrande R, Georgetti SR, Verri WA, Dorta DJ, dos Santos AC, and Fonseca MJV (2006) Protective effect of topical formulations containing quercetin against UVB-induced oxidative stress in hairless mice. Journal of Photochemistry and Photobiology B: Biology 84(1): 21–27. Cemek M, Enginar H, Karaca T, and Unak P (2006) In vivo radioprotective effects of Nigella sativa L oil and reduced glutathione against irradiation-induced oxidative injury and number of peripheral blood lymphocytes in rats. Photochemistry and Photobiology 82(6): 1691– 1696. Cerutti PA (1985) Prooxidant states and cancer. Science 227(4685): 375–381. Chathoth S, Thayyullathil F, Hago A, Shahin A, Patel M, and Galadari S (2009) UVC-induced apoptosis in Dubca cells is independent of JNK activation and p53 (Ser-15) phosphorylation. Biochemical and Biophysical Research Communications 383(4): 426–432. Darr D, Fridovich I (1994) Free radicals in cutaneous biology. Journal of Investigative Dermatology 102(5): 671– 675. Donovan M, Carmody RJ, and Cotter TG (2001) Lightinduced photoreceptor apoptosis in vivo requires neuronal nitricoxide synthase and guanylate cyclase activity and is caspase-3-independent. Journal of Biological Chemistry 276(25): 23000–23008. Droge W (2002) Free radicals in the physiological control of cell function. Physiological Reviews 82(1): 47–95. Eberlein-Konig B, Placzek M, and Przybilla B (1998) Protective effect against sunburn of combined systemic ascorbic acid (vitamin C) and d-alpha-tocopherol (vitamin E). Journal of the American Academy of Dermatology 38(1): 45–48. Enginar H, Cemek M, Karaca T, and Unak P (2007) Effect of grape seed extract on lipid peroxidation, antioxidant

106 activity and peripheral blood lymphocytes in rats exposed to x-radiation. Phytotherapy Research 21(11): 1029–1035. F’guyer S, Afaq F, and Mukhtar H (2003) Photochemoprevention of skin cancer by botanical agents. Photodermatology, Photoimmunology and Photomedicine 19(2): 56–72. Fuchs J (1998) Potentials and limitations of the natural antioxidants RRR-alpha tocopherol, L-ascorbic acid and betacarotene in cutaneous photoprotection. Free Radical Biology and Medicine 25(7): 848–873. Fuchs J, Huflejt ME, Rothfuss LM, Wilson DS, Carcamo R, and Packer L (1989a) Impairment of enzymic and nonenzymic antioxidants in skin by UVB irradiation. Journal of Investigative Dermatology 93(6): 769–773. Fuchs J, Huflejt ME, Rothfuss LM, Wilson DS, Carcamo R, and Packer L (1989b) Acute effects of near ultraviolet and visible light on the cutaneous antioxidant defense system. Photochemistry and Photobiology 50(6): 739–744. Grimm C, Wenzel A, Hafezi F, and Reme CE (2000) Gene expression in the mouse retina: the effect of damaging light. Molecular Vision 6: 252–260. Hegde KR, Henein MG, and Varma SD (2003) Establishment of mouse as an animal model for study of diabetic cataracts: biochemical studies. Diabetes, Obesity and Metabolism 5(2): 113–119. Holden WO (1983) UV: Black light measurement for NDT. The American Society for Nondestructive Testing, Inc. Reprinted from Materials Evaluation 41(3): 244–449. Ichihashi M, Ahmed N, Budiyanto A, et al. (2000) Preventive effect of antioxidant on ultraviolet-induced skin cancer in mice. Journal of Dermatological Science 23(suppl 1): 45–50. Ichihashi M, Ueda M, Budiyanto A, et al. (2003) UVinduced skin damage. Toxicology 189(1–2): 21–39. Inal ME and Kahraman A (2000) The protective effect of flavonol quercetin against ultraviolet a induced oxidative stress in rats. Toxicology 154(1–3): 21–29. Jurkovic P, Sentjurc M, Gasperlin M, Kristl J, and Pecar S (2003) Skin protection against ultraviolet induced free radicals with ascorbyl palmitate in microemulsions. European Journal of Pharmaceutics and Biopharmaceutics 56(1): 59–66. Katiyar SK, Afaq F, Perez A, and Mukhtar H (2001) Green tea polyphenol (-)-epigallocatechin-3-gallate treatment of human skin inhibits ultraviolet radiation-induced oxidative stress. Carcinogenesis 22(2): 287–294. Kitts DD, Yuan YV, Wijewickreme AN, and Thompson LU (1999) Antioxidant activity of the flaxseed lignan secoisolariciresinol diglycoside and its mammalian

Toxicology and Industrial Health 28(2) lignan metabolites enterodiol and enterolactone. Molecular and Cellular Biochemistry 202(1–2): 91–100. Kohli KS, Rai DV, and Sanyal SN (1996) Near ultraviolet radiation-induced changes in goat lens. Indian Journal of Biochemistry and Biophysics 33(5): 403–408. Kripke ML (1981) Immunologic mechanisms in UV radiation carcinogenesis. Advanced Cancer Research 34: 70–107. Kulms D, Poppelmann B, Yarosh D, Luger TA, Krutmann J, and Schwarz T (1999) Nuclear and cell membrane effects contribute independently to the induction of apoptosis in human cells exposed to UVB radiation. Proceedings of the National Academy of Sciences United States of America 96(14): 7974–7979. Lee AYW, Chung SSM (1999) Contributions of polyol pathway to oxidative stress in diabetic cataract. Federation of American Societies for Experimental Biology 13(1): 23– 30. Levine RL, Garland D, Oliver CN, et al. (1990) Determination of carbonyl content in oxidatively modified proteins. Methods in Enzymology 186: 464–478. Lonskaya IA, Afanasev VN, and Pechatnikov VA (1997) Induction and inhibition of rat thymocyte rat apoptosis by ultraviolet irradiation. Biofizika 42(3): 680–686. Lowry OH, Rosebrough NJ, and Farr AL (1951) Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193(1): 265–175. Martinek R (1964) Method for determination of vitamin E (total tocopherol) in serum. Clinical Chemistry 10: 1078–1086. Matsumura Y, Ananthaswamy HN (2002) Molecular mechanisms of photocarcinogenesis. Frontiers in Bioscience 7: 765–783. Mittal A, Elmets CA, and Katiyar SK (2003) Dietary feeding of proanthocyanidins from grape seeds prevents photocarcinogenesis in SKH-1 hairless mice: Relationship to decreased fat and lipid peroxidation. Carcinogenesis 24(8): 1379–1388. Morris D (2003) Flax: A Health and Nutrition Primer. Winnipeg: Flax Council of Canada, 11. Nagira T, Narisawa J, Teruya K, et al. (2002) Suppression of UVC-induced cell damage and enhancement of DNA repair by the fermented milk, Kefir. Cytotechnology 40(1–3): 125–137. Nielsen H (1981) Covalent binding of peroxidized lipid to protein: III. Reaction of individual phospholipids with different proteins. Lipids 16: 215. Nishi J, Ogura R, Sugiyama M, Hidaka T, and Kohno M (1991) Involvement of active oxygen in lipid peroxide radical reaction of epidermal homogenate following ultraviolet light exposure. Journal of Investigative Dermatology 97(1): 115–119.

O¨zkol et al. Oberley LW, Oberley TD (1981) Free radicals, cancer and aging. In: Liss AR (ed.) Free Radicals, Aging and Degenerative Diseases. 325–371. Oberley L, Oberley T (1986) Free radicals cancer and aging. In: Johnson JE, Walford Jr R, Harman D, and Miguel J (eds.) Free Radicals, Aging and Degenerative Diseases. New York: Alan R Liss, 325–371. Omaye ST, Turnbul JD, and Savberlich HE (1979) Ascorbic acid analysis. II. Determination after derivatisation with 2.2. dinitrophenylhydrazine. Selected methods for determination of ascorbic acid in animal cells, tissues and fluids. Methods in Enzymology 62: 3–11. Perchellet JP, Perchellet M (1989) Antioxidant and multistage carcinogenesis in mouse skin. Free Radical Biology and Medicine 7(4): 377–408. Pinnell S (2003) Cutaneous photodamage., oxidative stress., and topical antioxidant protection. Journal of the American Academy of Dermatology 48(1): 1–19. Prasad K (1997) Hydroxyl radical-scavenging property of secoisolariciresinol diglucoside (SDG) isolated from flax-seed. Molecular and Cellular Biochemistry 168(1–2): 117–123. Prasad K (2000) Antioxidant activity of secoisolariciresinol diglucoside-derived metabolites, secoisolariciresinol, enterodiol, and enterolactone. International Journal of Angiology 9(4): 220–225. Psotova J, Svobodova A, Kolarova H, and Walterova D (2006) Photoprotective properties of Prunella vulgaris and rosmarinic acid on human keratinocytes. Journal of Photochemistry and Photobiology B: Biology 84(3): 167–174. _ Radziszewska E, Piwocka K, Bielak-Zmijewska A, Skierski J, and Sikora E (2000) Effect of aging on UVC-induced apoptosis of rat splenocytes. Acta Biochimica Polonica 47(2): 339–347. Ramakrishnan N, McClain DE, and Catravas GN (1993) Membranes as sensitive targets in thymocyte apoptosis. International Journal of Radiation Oncology 63(6): 693–701. Rennenberg H (1980) Glutathione metabolism and possible biological roles in higher plants. Phytochemistry 21: 2771–2781. Sander CS, Chang H, Salzmann S, et al. (2002) Photoaging is associated with protein oxidation in human skin in vivo. Journal of Investigative Dermatology 118(4): 618–625.

107 Schneide LA, Dissemond J, Brenneisen P, et al. (2006) Adaptive cellular protection against UVA-1-induced lipid peroxidation in human dermal fibroblasts shows donor-to-donor variability and is glutathione dependent. Archives of Dermatological Research 297(7): 324–328. Shindo Y, Witt E, Han D, Epstein W, and Packer L (1994) Enzymic and non-enzymic antioxidants in epidermis and dermis of human skin. Journal of Investigative Dermatology 102(1): 122–124. Shindo Y, Witt E, and Packer L (1993) Antioxidant defance mechanisms in murine epidermis and dermis and their responses to ultraviolet light. Journal of Investigative Dermatology 100(3): 260–265. Stahl W, Heinrich U, Jungmann H, Sies H, and Tronnier H (2000) Carotenoids and carotenoids plus vitamin E protect against ultraviolet light-induced erythema in humans. The American Journal of Clinical Nutrition 71(3): 795–798. Strickland PT (1986) Photocarcinogenesis by nearultraviolet (UVA) radiation in Sencar Mice. Journal of Investigative Dermatology 87(2): 272–275. Suzuki I, Katoh NA (1990) Simple and cheap methods for measuring serum vitamin A in cattle using spectrophotometer. Japanese Journal of Veterinary Science 52(6): 1281–1283. Svobodova A, Psotova J, and Walterova D (2003) Natural phenolics in the prevention of UV-induced skin damage. Biomedical papers of the Medical Faculty of the University Palacky´, Olomouc, Czech republic 147(2): 137–145. Turker H (2004) Effect of ultraviolet radiation on total plasma T3, total plasma T4 and TSH hormones in molerat (Spalax leucodon). Gazi University Journal of Science 17(2): 1–8. Wang B, Fujita K, Watanabe K, Mitani H, Yamada T, and Shima A (1997) Induction of apoptosis in cultured midbrain cells from embryonic mice. Radiation Research 147(3): 304–308. Wang YZ, Huang MT, Ferraro T, et al. (1992) Inhibitory effect of green tea in the drinking water on tumorigenesis by ultraviolet light and 12-O-tetradecanoylphorbol-13acetate in the skin of SKH-1 mice. Cancer Research 52(5): 1162–1170. Yu X, Wang W, and Yang M (2007) Antioxidant activities of compounds isolated from Dalbergia odorifera T. Chen and their inhibition effects on the decrease of glutathione level of rat lens induced by UV irradiation. Food Chemistry 104: 715–720.