Caffeic acid modulates ultraviolet radiation-B induced oxidative damage in human blood lymphocytes

Journal of Photochemistry and Photobiology B: Biology 95 (2009) 196–203

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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol

Caffeic acid modulates ultraviolet radiation-B induced oxidative damage in human blood lymphocytes Nagarajan Rajendra Prasad *, Kasinathan Jeyanthimala, Samivel Ramachandran Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar 608 002, Tamilnadu, India

a r t i c l e

i n f o

Article history: Received 8 January 2009 Received in revised form 20 March 2009 Accepted 20 March 2009 Available online 29 March 2009 Keywords: Caffeic acid Lymphocytes Antioxidant Oxidative DNA damage UVB-radiation

a b s t r a c t Ultraviolet (UV) radiation causes inflammation, gene mutation and immunosuppressin in the human skin cells. These biological changes are responsible for photocarcinogenesis and photoaging. Normal lymphocytes are highly sensitive to the damaging effect of UV-radiation and undergo cell death. In the present study, the photoprotective effect of caffeic acid (3,4-dihydroxy cinnamic acid), a dietary phenolic compound, has been examined in the UVB (280–320) irradiated human blood lymphocytes. Lymphocytes pretreated with increasing concentration of caffeic acid (l, 5 and 10 lg/mL) for 30 min were irradiated and lipid peroxidation, antioxidant defence status, cell viability (by MTT assay) and DNA damage (by comet assay) were examined. UVB-irradiation causes increased levels of lipid peroxidation, DNA damage and decreased antioxidant status, cell viability in human lymphocytes. Caffeic acid pretreatment significantly reduced the levels of lipid peroxidation markers i.e. thiobarbituric acid reactive substance (TBARS), lipid hydroperoxide (LPH), conjugated diene (CD) and decreased DNA damage (tail length and % tail DNA) in UVB-irradiated lymphocytes. Further, caffeic acid pretreatment significantly maintains antioxidant status and decreased UVB-induced cytotoxicity. The maximum dose of caffeic acid (l0 lg/ mL) normalized the UVB induced cellular changes indicating the photoprotective effect of caffeic acid in irradiated lymphocytes. Ó 2009 Elsevier B.V. All rights reserved.

1. Introduction Ultraviolet radiation (UV), in particular UVB with a wave length range (280–320 nm), represents one of the most important environmental factors affecting human health [1]. The toxic effects of UVB from natural sunlight and therapeutic artificial lamps are a major concern for human health. An additional potential factor is thinning of the ozone layer which results in increased UVB exposure [2]. The effects of UVB irradiation on normal human cellular system comprise sunburn, inflammation (erythema), tanning, immunosuppression, photoaging and photocarcinogenesis [3]. One likely hypothesis for the genesis of pathologies due to exposure to UVB is that such exposure causes the formation of reactive oxygen species (ROS) apart from direct excitation of DNA [4]. If the flux of ROS is high and antioxidant regeneration becomes a limiting factor, then antioxidants will be depleted. The resulting imbalance between oxidants and antioxidants in favor to the former shifts the cellular redox state to the oxidized site and subsequently modulates redox-sensitive cellular signal transduction pathways and gene expression [5]. The use of antioxidants to inhibit photooxida-

* Corresponding author. Tel.: +91 954144 238343; fax: +91 954144 239141. E-mail address: [email protected] (N.R. Prasad). 1011-1344/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2009.03.007

tive toxicity has been suggested to be an important strategy in preventing and treating photodamage [6]. Natural antioxidants, which have a low potential of side effects in physiologic concentrations, appear to be promising in photoprotectors [7]. Caffeic acid (Fig. 1), 3,4-dihydroxy cinnamic acid, is a naturally occurring phytochemical present in a large number of vegetables, medicinal herbs and plants. It has been isolated from Ilex paraguariensis (15 mg/100 g), Melissa officinalis (39.3 mg/100 g), Baccharis genistelloides (8 mg/100 g) and Achyrocline satureioides (4 mg/ 100 g) [8]. Caffeic acid is also present in beverages like wine, tea, coffee and apple juice [8]. Coffee was shown to increase plasma antioxidant capacity in humans [9]. Coffee drinking was shown to increase the incorporation of conjugated forms of caffeic acid into LDL particles, and the oxidation-resistance of LDL was increased [10]. Caffeic acid was reported to have a wide variety of pharmacological activities including antioxidants [11], immunomodulatory, antiviral, anti-HIV [12], anticarcinogenic and antiinflammatory effect [13]. Caffeic acid from Salvia miltiorrhiza is one of the most popular traditional Chinese herbal medicines in some Asian countries, [14] and has been used extensively for treatment of coronary artery disease, myocardial infraction, cerebrovascular disease, various type of hepatitis, chronic renal failure, dysmenorrheal and also improve microcirculation in human body, caffeic acid has been shown to act as a carcinogenic inhibitor [15].

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2.3. Study design

Fig. 1. Structure of caffeic acid (C9H8O4).

Caffeic acid and its derivatives such as chlorogenic acid and caffeic acid phenyl ester are the major phenylpropanoid compounds in plants and act as substrate for polyphenols oxidases or peroxidases [16]. Human peripheral blood lymphocytes are highly sensitive to low doses of ultraviolet exposure [17]. Experiments on human peripheral blood lymphocytes are particularly suitable, being easily obtained, synchronized in G0 because of their circulation, representative of averaged whole body radiation exposure [18]. Lymphocytes are most studied and contain variety of redox and free radical scavenging systems. Earlier report shows that UVB radiation generates ROS in cultured human lymphocytes [19]. Hence, lymphocytes have been used to develop non-invasive bioassays to screen human population for UVB exposure. Studies on cytotoxicity, lipid peroxidation, antioxidants status, DNA damage and apoptotic morphological changes in blood lymphocytes could be of immense significance in identifying intracellular damages in the individuals, who could be at risk of UVB-induced oxidative damage. In this present work, we studied the photoprotective effect of caffeic acid on human blood lymphocytes in vitro.

Isolated lymphocytes were divided into six groups; in each group six samples were processed. Group I: Normal lymphocytes. Group II: Normal + Caffeic acid pretreatment (10 lg/mL). Group III: UVB-irradiated lymphocytes. Group IV: UVB + Caffeic acid pretreatment (1 lg/mL). Group V: UVB + Caffeic acid pretreatment (5 lg/mL). Group VI: UVB + Caffeic acid pretreatment (10 lg/mL). 2.4. Treatment of the cells Thirty min prior to irradiation three test-doses (1, 5 and 10 lg/ mL) of caffeic acid were added to the grouped normal lymphocytes. Preliminary studies were carried out to ensure that whether this concentration had any toxic effect by trypan blue dye exclusion test. Before exposure to UV light, the cells were washed twice with (RPMI-1640) media. Non-irradiated lymphocytes showed no decrease in viability over the 30 min period of incubation. 2.5. Irradiation procedure For UVB irradiation cells were irradiated in 35 mm petridishes containing 2 ml of PBS and covered with a UV permeable membrane to prevent contamination. A battery of TL 20 W/20 fluorescent tubes (Heber Scientific) served as UVB source, which had a wavelength range set 280–320 nm peaked at 312 nm and an intensity of 2.2 mW/cm2 for 9 min. The total UVB-irradiation was 19.8 mJ/cm2, corresponding to an average value of 1.52  103 mJ/cell. After irradiation the lymphocytes were kept at room temperature for 30 min and then subjected to biochemical assays.

2. Materials and methods 2.6. Cell viability assay 2.1. Chemicals Caffeic acid, heat inactivated fetal calf serum (FCS), Ficoll–Histopaque-1017, thiobarbituric acid (TBA), phenozine methosulphate (PMS), nitroblue tetrazolium (NBT), 5,5-dithiobis 2-nitrobenzoic acid (DTNB), 3-(4,5-dimethyl-2-thiaozolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT), nicotinamide adenine dinucleotide (NAD), 2-deoxy-D-ribose, FeCl2, FeCl3, EDTA, H2O2 and ascorbic acid were purchased from Sigma chemicals Co., St. Louis, USA. Other chemicals for blood lymphocyte cultures (RPMI-1640, penicillin, streptomycin, L-glutamine), low melting point agarose (LMPA), normal melting point agarose (NMPA) Ethidium bromide, acridine orange and phosphate buffer saline (PBS) were purchased from Himedia, Mumbai. All other chemicals and solvents of analytical grade were obtained from SD Fine chemicals, Mumbai and Fisher Inorganic and Aromatic Limited, Chennai. 2.2. Isolation of lymphocytes Blood samples were aseptically collected in hepairnized sterile tubes (14–17 U/mL) from median cubital vein of non-smoking healthy individuals (22–25 years). Lymphocytes were isolated using ficoll–histopaque (Sigma, USA) by the method [20]. Blood was diluted 1:1 with phosphate buffered saline (PBS) and layered onto histopaque with the ratio of blood and PBS: Histopaque maintained at 4:3. The blood was centrifuged at 400g for 35 min at room temperature. The lymphocyte layer was removed and washed twice in PBS at 250g for 10 min each, and then washed with (RPMI-1640) media. The number of lymphocytes was counted using a haemocytometer and the viability of the cells was assayed by the trypan blue exclusion test. Approximately 1  106 cells were present in 1.0 mL lymphocyte suspension.

The standard trypan blue viability test was carried out on the isolated lymphocytes [21]. To the UVB-irradiated and caffeic acid pretreated lymphocytes few drops of trypan blue stain (0.4%) were added and the viable cells against dead cells (total 200 cells) were immediately recorded using haemocytometre under a microscope. The percentage of viable cells was calculated as follows:

%Viability ¼

Total number of viable cells  100 Total number of viable and non-viable cells

2.7. MTT assay The MTT test is a colorimetric non-radioactive assay for measuring cell viability through increased metabolisation of tetrazolium salt [22]. Isolated lymphocytes in concentration of 1  106 cells/ mL were taken 200 lL into different eppendroff tubes. Then the cells were pretreated with different concentration (1, 5 and 10 lg) of caffeic acid and added 1 mL of RPMI-1640 medium for 30 min. Then the cells were exposed to UVB-irradiation for 15 min. After irradiation cells were incubated in the presence of 5% CO2 and 95% O2 at 37 °C for 24 h. The incubated cells were added MTT (0.5 mg/mL), further incubated for 4 h. After incubation, all the tubes were centrifuged for 10 min. Under standard conditions the medium with MTT was removed and 200 lL of DMSO were added into each tubes. Absorbance was measured to colorimetry at 570 nm. The OD values are plotted to calculate percentage cell death. 2.8. Biochemical estimation Lymphocytes were suspended in 130 mM KCl plus 50 mM PBS containing 0.1 mL of 0.1 M dithiothreital and centrifuged at

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20,000g for 15 min (4 °C). The supernatant was taken for biochemical estimates. In each group six samples (n = 6) were processed. The level of lipid peroxidation was determined by analyzing TBAreactive substance according to the protocol of Niehaus and Samuelsson [23]. The pink coloured chromogen formed by the reaction of 2-TBA with breakdown products of lipid peroxidation was measured. The lipid hydroperoxides (LHP) levels were determined by analyzing BHT-reactive substance according to the protocol of Jiang and Hunt [24]. Conjugated dienes were assayed by the method of Rao and Recknagel [25]. Superoxide dismutase (SOD) activity was assayed by the method of Kakkar et al. [26], based on the inhibition of the formation of (NADH-PMS-NBT) complex. Catalase (CAT) activity was assayed by the procedure of Sinha [27] quantifying the hydrogen peroxide after reacting with dichromate in acetic acid. The activity of Glutathione peroxidase (GPX) was assayed by method of Rotruck et al. [28] a known amount of enzyme preparation was allowed to react with hydroperoxides (H2O2) and GSH for a specified time period. Then the GSH content remaining after the reaction was measured. The total GSH content was measured by method of Ellman [29]. This method was based on the development of a yellow colour when 5,5-dithiobis 2-nitrobenzoic acid was added to compounds containing sulphydryl groups. The ascorbic acid was estimated by the method of Roe and Kurther [30] the red coloured compound when treated with sulphuric acid and then adding 2,4-dinitrophenyl hydrazine in the presence of thiourea solution. a-Tocopherol was estimated by the method described by Baker et al. [31].

tion, brightly fluorescent, apoptotic nuclei, were easily detected through their high fluorescence and condensed chromatin so called ethidium bromide positive nuclei were scored and the percentage apoptotic cells were calculated.

2.9. Alkaline single-cell gel electrophoresis (Comet assay)

3.2. Effect of caffeic acid on UVB-induced cytotoxicity by MTT assay

DNA damage was estimated by alkaline single-cell gel electrophoresis (Comet assay) according to the method of Singh et al. [32]. A layer of 1% normal melting agarose was prepared on microscope slides. After UVB-irradiation, cells (50 lL) were mixed with 120 lL of 0.5% low melting agarose. The suspension was pipetted onto the precoated slides. Slides were immersed in cold lysis solution at pH 10 (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris pH 10, 1% Triton X-100, 10% DMSO) and kept at 4 °C for 60 min. To allow denaturation of DNA, the slides were placed in alkaline electrophoresis buffer at pH 13 and left for 25 min. Subsequently, slides were transferred to an electrophoresis tank with fresh alkaline electrophoresis buffer and electrophoresis was performed at field strength of 1.33 V/cm for 25 min at 4 °C. Slides were neutralized in 0.4 M Tris pH 7.5 for 5 min and stained with 20 lg/mL ethidium bromide. For visualization of DNA damage, observations were made using a 20 objective on an epifluorescent microscope equipped with an excitation filter of 510–560 nm and a barrier filter of 590 nm. One to two hundred comets on duplicated slides were analyzed. Images were captured with a digital camera with networking capability and analyzed by image analysis software, CASP (http://casp.sourceforge.net). DNA damage was quantified by the tail moment and the tail length.

Yellow MTT (3-(4,5-dimethyl)-2,5-diphenyl-2H tetrazolium bromide) is converted to the blue formazan product only by metabolically active mitochondria, and the absorbance is directly proportional to the number of viable cells. Our result shows that cytotoxicity was greatly increased in UVB-irradiated cells (Fig. 3). Pretreatment with 1, 5 and 10 lg/mL of caffeic acid significantly decreased UVB-induced cytotoxicity. Ten microgram per microlitre of caffeic acid exhibits better cytoprotection in UVB-exposed cells.

Ethidium bromide/acridine orange (EB/AO) staining was carried out to detect morphological evidence of apoptosis on the caffeic acid and UVB-irradiated cells. To the UVB-irradiated and caffeic acid pretreated lymphocytes, cells were fixed in 1:1 ratio of methanol and glacial acetic acid for 1 h at room temperature. After washing with PBS and incubated for 1 min with a solution of ethidium bromide/acridine orange 1:1 ratio of (100 lg/mL) in PBS to take 10 lL (10 lg/mL) and wash immediately. Stained cells were visualized under UV illumination using the 40 objective (Nikon fluorescence microscopes) and their digitized images were captured. The apoptotic cells, with their shrunken, nuclear fragmenta-

Data represent the mean ± standard error (SEM) of the indicated number of experiments. Statistical analysis of the data were carried out by one-way ANOVA on SPSS (Statistical package for social sciences) and the group mean was compared by Duncan’s Multiple Range Test (DMRT). A value of P < 0.05 was considered to be significant.

3. Results 3.1. Effect of caffeic acid on UVB-induced cell death by trypan blue dye exclusion test Cell viability was determined before and immediately after UVB-irradiation by trypan blue dye exclusion test. Fig. 2 shows the effect of UVB and/or caffeic acid pretreatment on cell viability. UVB-irradiated groups show significantly decreased cell viability than the normal cells. Pretreatment with caffeic acid significantly increases the cell viability in a concentration dependent manner.

120 100

a

b

a c d

80 % Cell viability

2.10. Detection of apoptotic nuclei by EB/AO staining

2.11. Statistical Analysis

60

e

40 20 0 Treatments Normal UVB irradiation

Normal + Caffeic acid (10 µg/mL) UVB + Caffeic acid (1 µg/mL)

UVB + Caffiec acid (5 µg/mL)

UVB + Caffeic acid (10 µg/mL)

Fig. 2. Effect of caffeic acid and/or UVB on cell viability in trypan blue dye exclusion assay. Values are given as means ± SD of six experiments in each group. Values not sharing a common superscript differ significantly at P < 0.05 (DMRT).

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Fig. 3. Effects of caffeic acid on UVB-induced cell cytotoxicity, values are given as means ± SD of six experiments in each group. Values not sharing a common superscript differ significantly at P < 0.05 (DMRT).

3.3. Effect of caffeic acid on UVB-induced lipid peroxidation

increased activities of SOD, CAT and GPx in a concentration dependent manner.

The levels of TBARS, LPH and CD were increased significantly in UVB-irradiated human lymphocytes (Fig. 4). Caffeic acid pretreated lymphocytes showed progressively decreased levels of TBARS, LPH and CD when compared with UVB-irradiated cells. Even 1 lg/mL of caffeic acid significantly reduced the levels of lipid peroxidation in UVB-irradiated lymphocytes.

3.4.2. Non-enzymatic antioxidant activity The levels of non-enzymatic antioxidants vitamin-C, vitamin-E and GSH were found to be decreased in UVB-irradiated lymphocytes (Figs. 5b and 5c). Caffeic acid (1, 5 and 10 lg/mL) pretreatment significantly restored the vitamin-C, vitamin-E and GSH levels to normal levels in a concentration dependent manner.

3.4. Effect of caffeic acid on UVB-induced oxidative stress 3.5. Effect of caffeic acid on UVB-induced DNA damage 3.4.1. Enzymatic antioxidant activity Fig. 5a shows the activities of SOD, CAT and GPx in normal, UVBirradiated and caffeic acid pretreatment lymphocytes. The activities of these antioxidant enzymes were significantly decreased in UVB-irradiated groups. Pretreatment with caffeic acid significantly

TBARS

16

LHP

CD

cd

12 nmol/ mg protein

3.6. Effect of caffeic acid on UVB-induced apoptotic morphological changes

ed

14 d

10

b c

8

a a

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b ab

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a a

6 c b

4 2

a ab

Figs. 6a and 6b represents the DNA damage in UVB-irradiated and/or caffeic acid pretreated human lymphocytes. The extent of DNA damage was calculated by means of % tail DNA and tail length in normal, UVB-exposed and caffeic acid pretreated human lymphocytes. UVB-irradiation significantly increased % tail DNA and tail length in lymphocytes. Caffeic acid (1, 5 and 10 lg/mL) pretreatment significantly decreased the levels DNA damage in a concentration dependent manner.

This method provides valuable information on apoptotic morphology in human lymphocytes. Figs. 7a and 7b shows the nuclear morphological changes in caffeic acid pretreated and/or UVB-irradiated human lymphocytes. We observed increased apoptotic cells in UVB-irradiated cells. Caffeic acid pretreatment significantly prevents the formation of condensed nuclei in UVB-irradiated cells.

a

4. Discussion

0 Treatments Normal UVB Irradiation UVB + Caffeic acid (5 µg/mL)

Normal + Caffeic acid (10 µg/mL) UVB + Caffeic acid (1 µg/mL) UVB + Caffeic acid (10 µg/mL)

Fig. 4. Effect of caffeic acid on the levels of TBARS, LPH and CD in normal UVBirradiated and caffeic acid pretreated lymphocytes. Values are given as mean ± SD of six experiments in each group. Values not sharing a common superscript differ significantly at P < 0.05 (DMRT).

Although solar radiation is essential for human life, UV exposure paradoxically constitutes a many hazard to living forms [33]. The UVB exposure generates several reactive oxygen species (ROS) via type I (superoxide radicals) and predominantly type II (singlet oxygen, 1O2) processes, leading to damage of various sub-cellular structures and molecules. Further, UVB-radiation increased the activity of xanthine oxidase and generates superoxide anion in human skin cell [34]. UV light can stimulate donation of

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CAT**

SOD*

GPX***

20 a a

18 16

Units/mg protein

ab

a a

ab

ab

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c

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ab

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b

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b

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d

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8 6 4 2 0

Treatments Normal UVB Irradiation UVB + Caffeic acid (5 µg/mL)

Normal + Caffeic acid (10 µg/mL) UVB + Caffeic acid (1 µg/mL) UVB + Caffeic acid (10 µg/mL)

*Enzyme concentration required for 50% inhibition of nitroblue tetrazolium reduction in one minute. **µmol of hydrogen peroxide consumed per minute. ***µg of glutathione consumed per minute. Fig. 5a. Effect of caffeic acid on the levels of enzymatic antioxidants CAT, SOD and GPX in normal UVB-irradiated and caffeic acid pretreated lymphocytes. Values are given as mean ± SD of six experiments in each group. Values not sharing a common superscript differ significantly at P < 0.05 (DMRT).

an electron to molecular oxygen, present under normal conditions in cells, and produce the free radical, superoxide anion, which can gain another electron from UV light to generate H2O2 [35]. In this study, caffeic acid significantly protects lymphocytes against the cytotoxic effects induced by UV exposure. Caffeic acid is known to exhibit a strong inhibitor effect on xanthine oxidase [36]. In view of these facts, the improvement in cell viability observed in caffeic acid treated cells may be due to the antioxidant action of caffeic acid via the reduction of cellular damages caused by ROS [37]. Similar report shows that caffeic acid protects cell populations against the cytotoxic effect caused by UVC-irradiation [38].

Similarly, recent report shows that dihydrocaffeic acid protects human keratinocyte HaCaT cells from UV radiation-induced cytotoxicity [39]. Loss of mitochondrial membrane potential is a one of the event in UVB-induced cell death and the component of the mitochondrial respiratory chain i.e. cytochrome oxidase have been suggested as possible photoreceptors [40]. Our result shows % cell death was greatly increased in UVB-irradiated cells, and caffeic acid treatment significantly decreased cell death. Nardini et al. showed that caffeic acid significantly increases human skin cell proliferation activity after UVC-irradiation in vitro and the cytoprotective effect of caffeic acid against UVC was more efficient than atocopherol in human skin cells [41].

GSH

25 a

a

VITAMIN-C

4 bc

20

ab

3.5

a

VITAMIN-E

a

c

3

15

b bc

2.5 (mg/dl)

(mg/dl)

d

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Treatments

Treatments Normal

Normal+ Caffeic acid (10 µg/mL)

Normal

UVB radiation

UVB + Caffeic acid (1 µg/mL)

UVB radiation

UVB + Caffeic acid (1 µg/mL)

UVB + Caffeic acid (5 µg/mL)

UVB + caffeic acid (10 µg/mL)

UVB + Caffeic acid (5 µg/mL)

UVB + Caffeic acid (10 µg/mL)

Fig. 5b. Effect of caffeic acid on the GSH levels in normal, UVB-irradiated and caffeic acid pretreated lymphocytes. Values are given as means ± SD of six experiments in each group. Values not sharing a common superscript differ significantly at P < 0.05 (DMRT).

Normal+ Caffeic acid (10 µg/mL)

Fig. 5c. Effect of caffeic acid on the levels of non-enzymatic antioxidants vitamin-C and vitamin-E in normal, UVB-irradiated and caffeic acid pretreated lymphocytes. Values are given as mean ± SD of six experiments in each group. Values not sharing a common superscript differ significantly at P < 0.05 (DMRT).

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TAIL DNA

TAIL LENGTH

90

d

60 d e

40 c

30

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b a

20 10

c

a

b a

a

0

Treatments Normal + Caffeic acid (10 µg/mL)

UVB Irradiation

UVB + Caffeic acid (1 µg/mL)

UVB + Caffeic acid (5 µg/mL)

UVB + Caffeic acid (10 µg/mL)

Fig. 6a. Effect of caffeic acid on DNA damage (% tail DNA, tail length,) in normal, UVB-irradiated and caffeic acid pretreated lymphocytes. Values are given as means ± SD of six experiments in each group. Values not sharing a common superscript differ significantly at P < 0.05 (DMRT).

(C) UVB irradiation

70 c

60 50

b

40 30

ab a

20 10

Normal

(A) Normal

% of apoptotic cells

% Tail DNA, Tail Length

e

50

c

80

(B) Normal + Caffeic acid (10 µg/mL)

(D) UVB + Caffeic acid (1 µg/mL)

0 Treatments Normal Normal+ Caffeic acid (10 µg/mL) UVB radiation UVB Radiation + Caffeic acid (1 µg/mL) UVB Radiation + Caffeic acid (5 µg/mL) UVB Radiation + Caffeic acid (10 µg/mL) Fig. 7a. Effect of caffeic acid on UVB-induced apoptotic cell death. Lymphocytes were incubated with (1, 5 and 10 lg/mL) of caffeic acid for 24 h. Values are given as means ± SD of six experiments in each group. Values not sharing a common superscript differ significantly at P < 0.05 (DMRT).

(A) Normal

(B) Normal + Caffeic acid (10 µg/mL)

(C) UVB irradiation

(D) UVB + Caffeic acid (1 µg/mL)

(E) UVB + Caffeic acid (5 µg/mL)

(E) UVB + Caffeic acid (5 µg/mL)

(F) UVB + Caffeic acid (10 µg/mL)

Fig. 6b. Effect of caffeic acid on UVB-induced DNA damage in human lymphocytes.

UVB radiation induced lipid peroxidation in the epidermis of mice and human beings has been previously demonstrated [42].

(F) UVB + Caffeic acid (10 µg/mL)

Fig. 7b. Effect of caffeic acid on UVB-induced morphological changes in human lymphocytes.

The occurrence of TBARS, LHP and CD in the biological membrane is a free radical-mediated event. Lipid peroxidation induced by UVB radiation is known to be due to the attack of free radicals on the fatty acid component of membrane lipids [43]. Lipid perox-

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idation is a complex multistep process wherein the initially formed lipid radicals get converted to TBARS via the intermediacy of CD and LPH. We assessed the anti-lipid peroxidation potential of the caffeic acid by measuring the TBARS, as well as LPH and CD, two relatively unstable products of lipid peroxidation. As expected, UVB-exposure lymphocytes led to the increased formation of all these parameters, which were effectively reduced by the caffeic acid. This indicated that the caffeic acid could efficiently prevent the UVB-induced lipid peroxidation at its various stages. Previously photoprotective activity of caffeic acid against UVB radiation was reported both in vitro and in vivo [44]. Caffeic acid has been demonstrated to protect phospholipidic membrane from UV induced peroxidation reaction [45]. Ferulic acid, a similar cinnamic acid shown to be strong UV absorber and is employed as a photoprotective agent in a number of skin lotions and sunscreens [46]. The strong inhibition of lipid peroxidation by caffeic acid was shown with prevention of human LDL oxidation [41]. UV protective effect of caffeic acid was also described on both cellular and cell-free systems [47]. This protective effect is probably based on the antioxidant activity of caffeic acid, which can demonstrate to protect phospholipidic biomembranes from UV light induced peroxidation [48]. Antioxidant enzymes protect cells from radiation exposure. Our results show that UVB exposure significantly affects antioxidant defence system. Since UVB induced cellular changes involves ROS, antioxidants might be helpful to minimize UVB-induced photopathologies. In this study, we have demonstrated pretreatment with caffeic acid increases the activities of antioxidant enzymes such as SOD, CAT and GPx in UVB-irradiated lymphocytes. Caffeic acid was also found to show potent hepetoprotective activity by protecting antioxidant enzymes against liver injuries induced by CCl4 and alcohol [49]. Facino et al. [38] demonstrated that caffeic acid effectively inhibits collagen (type III) fragmentation induced by superoxide anion and hydroxyl radicals. Hence, caffeic acid may prevent the synthesis and utilization of antioxidant enzymes. This may be the reason for increased SOD, CAT and GPx activities observed in UVB-irradiated plus caffeic acid pretreated lymphocytes. The levels of non-enzymatic antioxidants such as vitamin-C, vitamin-E and GSH were found to be decreased in UVB-irradiated lymphocytes. The observed decrease in the levels of vitamin-C, vitamin-E and GSH may be due to their increased utilization for scavenging UVB-induced ROS. Vitamin-C and vitamin-E may play a role in preventing oxidative stress under experimental and clinical conditions [50]. Caffeic acid (1, 5 and 10 lg/mL) treatment prior to irradiation protected vitamin-C, vitamin-E depletion resulting from the radiation effects. Glutathione metabolism is also important in quenching the reactive intermediates and radical species generated during oxidative toxicity [51]. Our results show that caffeic acid renders protection against UVB-induced oxidative stress. Caffeic acid as the scavenger of ROS acts in the protection of intracellular glutathione levels. Caffeic acid can double the antioxidant capacity of plasma even in micromolar concentration [52] in this context, caffeic acid minimizes the utilization of endogenous antioxidants and improves the levels of those antioxidants in the irradiated lymphocytes. In recent years, growing evidence suggests that UVB generates ROS and it has been associated with oxidative DNA damage [2]. It has been previously proposed that OH radical could be implicated in the oxidation of the guanine base [53]. In our study, caffeic acid modulates UVB induced DNA damage and decreased % tail DNA and tail length in human lymphocytes. Recent study shows that caffeic acid pretreatment significantly reduced gamma-radiation induced DNA damage and subsequent chromosomal aberrations in cultured human lymphocytes [54]. Protective effect of caffeic acid phenyl ester on tert-butyl hydroperoxides-induced oxi-

dative hepatotoxicity and DNA damage has been recently demonstrated [55]. Gulcin shows that caffeic acid was an effective antioxidant in different antioxidant assays including total antioxidant activity by ferric thiocyanate method, reducing power, ABTS+ scavenging, DPPH scavenging superoxide anion scavenging, and metal chelating activity when compared to standard antioxidant compounds such as BHA, BHT, a-tocopherol and trolox [52]. This antioxidant effect of caffeic acid could be responsible for photoprotective effect against UVB induced DNA damage. Apoptotic cell death is thought to be responsible for numerous physiologic and pathologic events. Ultraviolet radiation is the primary environmental agent that leads to induce apoptosis in human cells. We determined the morphological changes of apoptosis of human peripheral lymphocytes using acridine orange/ethidium bromide staining. UV-irradiation of cells elicits a complex cellular response via cell surface receptor aggregation [56] and, upon prolonged exposure; it induces apoptosis in mammalian cells including keratinocytes and lymphocytes [57]. UV irradiation triggers apoptosis via several independent mechanisms, which include DNA damage followed by the activation of p53 pathway and Fas receptor aggregation-mediated initiation of caspase cell death cascade [58]. In agreement with this, we observed apoptotic morphological changes in UVB-irradiated lymphocytes. On the other hand caffeic acid treated cells showed marked suppression of those apoptotic changes. Previous report shows caffeic acid phenyl ester has antioxidative, anti-inflammatory effects and it may protect myocardial ischemia-reperfusion induced apoptosis [38]. 5. Conclusions Thus, our observations suggest that caffeic acid can prevent UVB-induced oxidative damage in human lymphocytes in vitro. The influence of caffeic acid on the UVB-induced changes might be due to its antioxidant property. Hence caffeic acid might play a beneficial role against toxic effects of ultraviolet radiation. References [1] Z. Assefa, A.V. Laethem, M. Garmyn, P. Agostinis, Ultraviolet radiation-induced apoptosis in keratinocytes: on the role of cytosolic factors, Biochim. Biophy. Acta. 1755 (2005) 90–106. [2] B.M. Coldiron, Thinning of the ozone layer: facts and consequences, J. Am. Acad. Dermatol. 27 (1992) 653–663. [3] L. Marrot, J.R. Meunier, A. Bois, France, skin DNA photodamage and its biological consequences, J. Am. Acad. Dermatol. 58 (2008) S139–S149. [4] J.L. Ravanat, T. Douki, J. Cadet, Direct and indirect effects of UV radiation on DNA and its components, J. Photochem. Photobiol. B: Biol. 63 (2001) 88–102. [5] J. Fuchs, Potentials and limitations of the natural antioxidants rrralphaTocopherol, l-ascorbic acid and b-carotene in cutaneous photoprotection, Free Radical Biol. Med. 25 (7) (1998) 848–873. [6] P.K. Vayalil, C.A. Elmets, S.K. Katiyar, Treatment of green tea polyphenols in hydrophilic cream prevents UVB-induced oxidation of lipids and proteins, depletion of antioxidant enzymes and phosphorylation of MAPK proteins in SKH-1 hairless mouse skin, Carcinogenesis 24 (2003) 927–936. [7] N.R. Prasad, S. Ramachandran, K.V. Pugalendi, V.P. Menon, Ferulic acid inhibits UV-B-induced oxidative stress in human lymphocytes, Nutr. Res. 27 (2007) 559–564. [8] V. Marques, A. Farah, Chlorogenic acid and related compounds in medicinal plants and infusions, Food Chem. 113 (2009) 1370–1376. [9] F. Natella, M. Nardini, I. Giannetti, C. Dattilo, C. Scaccini, Coffee drinking influences plasma antioxidant capacity in humans, J. Agric. Food Chem. 50 (2002) 6211–6216. [10] F. Natella, M. Nardini, F. Belelli, C. Scaccini, Coffee drinking induces incorporation of phenolic acids into LDL and increases the resistance of LDL to ex vivo oxidation in humans, Am. J. Clin. Nutr. 86 (2007) 604–609. [11] M. Nardini, F. Natella, V. Gentili, M. Di Felice, C. Scaccini, Effect of caffeic acid dietary supplementation on the antioxidant defense system in rat: an in vivo study, Arch. Biochem. Biophys. 342 (1999) 157–160. [12] A.A. Johnson, C. Marchand, Y. Pommery, HIV-1 integrase inhibitors a decade of research and two drugs in clinical trial, Curr. Top. Med. Chem. 4 (10) (2004) 1059–1077. [13] M.R. Fesen, Y. Pommier, E. Leteurtre, S. Hiroguchi, J. Yung, K.W. Kohn, Inhibition of HIV-1 integrase by flavones, caffeic acid phenyl ester (CAPE), related compound, Biochem. Pharmacol. 3 (1994) 595–608.

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