236 BMB reports http://bmbreports.org *Corresponding Author. Tel: 91-3222-283764 Fax: 91-3222-278433 E-mail: kundu@hijli.iitkgp.ernet.in # These authors contributed equally to this work. Received 28 February 2007, Accepted 14 September 2007 Keywords: Antioxidant, Fibroblasts, Hydrogen peroxide, Oxidative stress, Silk protein sericin Antioxidant potential of silk protein sericin against hydrogen peroxide-induced oxidative stress in skin fibroblasts Rupesh Dash#, Chitrangada Acharya#, PC Bindu# & SC Kundu* Department of Biotechnology, Indian Institute of Technology, Kharagpur, India-721 302 The antioxidant potential of silk protein sericin from the non-mulberry tropical tasar silkworm Antheraea mylitta co- coon has been assessed and compared with that of the mul- berry silkworm, Bombyx mori. Skin fibroblast cell line (AH927) challenged with hydrogen peroxide served as the positive con- trol for the experiment. Our results showed that the sericin ob- tained from tasar cocoons offers protection against oxidative stress and cell viability is restored to that of control on pre-in- cubation with the sericin. Fibroblasts pre-incubated with non-mulberry sericin had significantly lower levels of catalase lactate dehydrogenase and malondialdehyde activity when compared to untreated ones. This report indicates that the silk protein sericin from the non-mulberry tropical tasar silkworm, A. mylitta can serve as a valuable antioxidant. [BMB reports 2008 41(3): 236-241] INTRODUCTION Free radical theory of aging has given much impetus to the role of reactive oxygen species (ROS) such as superoxide, hy- drogen peroxide, and hydroxyl radicals in the initiation and progression of the aging process (1). Increased levels of ROS can damage various cellular processes. The hydroxyl radical generated from hydrogen peroxide reacting with different tran- sition metals is particularly damaging to DNA, leading to mu- tagenesis and carcinogenesis (2). Voluminous research has been carried out to discover new antioxidant compounds from plant and animal origin to prevent free radical damage (3-6). Polypeptide with antioxidant property has also been reported in Chlamys farreri (7, 8). Lepidopteran insects of family Bombycidae and Saturniidae produce commercially important silks. The domesticated mul- berry silkworm Bombyx mori belongs to family Bombycidae whereas the wild non-mulberry silkworms including Antheraea mylitta belong to family Saturniidae. Cocoons of silkworms belonging to both families consist of two major pro- teins, fibroin and sericin. Fibroin and sericin, obtained from mulberry silkworm Bombyx mori are now recognized as ex- cellent biomaterials in the field of tissue engineering and therapy. Fibroin, the water-insoluble protein, from mulberry silkworm, has been recognized as a substrate for growth and adherence of cells in culture (9-13). Sericin, the water-soluble component of silk, from the mulberry silkworm, is used as a biomaterial due to its antibacterial and UV resistant properties (14). Sericin is also reported to suppress in vitro lipid perox- idation (15) and possesses antitumor properties (16) with no immunogenicity (17). All the biomaterial related applications of silk proteins in- volve in vitro studies on cells prior to their implantation in vivo. The present study reports the antioxidant effects of silk protein sericin obtained from the cocoons of the non-mul- berry, tropical tasar silkworm Antheraea mylitta, in skin fibro- blast cell line AH927 exposed to hydrogen peroxide for 24 hrs. RESULTS Sensitivity to H2O2 Cell viability strikingly decreased in a concentration-depend- ant manner on treatment with various concentrations of H2O2. The LC50 for 24 hr exposure was 0.2 mM, indicating that AH927 cells were sensitive to H2O2-induced cell damage. Treatment with 0.5 mM H2O2 reduced cell viability to 27.3 % and with 1.0 mM decreased the viability to 9.07% (Fig. 1a). Morphological changes on hydrogen peroxide exposure Preliminary MTT assay (Fig. 1a) revealed that viability was sig- nificantly less at hydrogen peroxide concentrations of 0.5 mM. Propidium iodide staining was used to study the morpho- logical changes in cells at concentration of 0.5 mM H2O2 to assess the damage caused by oxidative stress. Under phase contrast microscope, the morphology of the cells exposed to 0.5 mM H2O2 were shrunk and rounded as compared to nor- mal cells. Fluorescence staining of cells revealed that cells ex- posed to 0.5 mM H2O2 exhibited nuclear condensation (Fig. 1b).

Protection by silk sericin against oxidative stress Rupesh Dash, et al. 237 http://bmbreports.org BMB reports a b Fig. 1. Hydrogen peroxide stress upon feline fibroblast cells. a) Addition of hydrogen peroxide decreased the cell viability of fe- line fibroblast cells (AH927) as assessed by MTT assay. Results are expressed as a percentage of the corresponding untreated con- trol as the mean �� SEM. (n = 4), *P ��� 0.05 (ANOVA followed by Tukeys test). b) Effect of H2O2 on morphology of feline fibroblast cells (AH927) (A) Phase contrast micrograph of normal untreated cell, (B) Fluorescence micrograph of normal cell, (C) Phase con- trast micrograph of H2O2 treated cell, (D) Fluorescence micro- graph of H2O2 treated cell. Cells were micro photographed at a magnification of 100 ��. Fig. 2. Effect of silk protein sericin (from mulberry silkworm, B. mori and non-mulberry silkworm, A. mylitta) treatment on fibro- blast (AH927) cell viability. Incubation of cells with sericin was carried out for 24 hrs, prior to the exposure to 0.5 mM hydrogen peroxide for 24 hrs, and cell viability was assayed by MTT. Results are expressed as the mean �� SEM. (n = 4), *P ��� 0.05 compared to hydrogen peroxide treated cells (one-way ANOVA followed by Tukeys test). Effect of pre-treatment with sericin on oxidative damage The effect of pre-treatment of cells with sericin for 24 hrs is given in Fig. 2. One-way analysis of variance revealed that there was an overall significant difference in cell viability be- tween controls, H2O2-treated and sericin treated fibroblasts (F = 44.47, P ��� 0.001). Subsequent multiple comparisons by Tukeys test indicated that cell viability was significantly lower (P ��� 0.01) in hydrogen peroxide-treated when compared with control and sericin treated fibroblasts. Pre-incubation with A. mylitta sericin from 5 to 30 ng/ml had no protective effect (data not shown). It was observed that concentrations of 35, 50 and 100 ng/ml significantly (P ��� 0.05) increased the cell viability. Cells treated with sericin at 150 ng/ml showed cell viability comparable to that of control group (P ��� 0.05), in- dicating that pre-incubation with 150 ng/ml restored the cell viability to normal. On the other hand sericin from B. mori could not restore cell viability to normal at the same concen- tration (Fig. 2). Cell viability study also showed that pretreat- ment of gelatin with the cells for 24 hrs did not show sig- nificant protection against H2O2 induced oxidative stress. LDH activity Fig. 3 depicts the percentage of LDH activity released into the medium in normal cells, cells treated with 0.5 mM hydrogen peroxide for 24 hrs, and cells pre-incubated with 35 ng/ml and 100 ng/ml of sericin from A. mylitta. There was a significant increase (P ��� 0.01) in the release of enzyme in hydrogen per- oxide treated cells as compared to untreated control and cells pre-incubated with 100 ng/ml of sericin before H2O2 treat- ment. Catalase activity The rate of release of catalase activity in various treated cells and control are presented in Fig. 3. Catalase activity was sig- nificantly high (P ��� 0.01) in medium of cells treated with hy- drogen peroxide (81%) compared to control. The fibroblasts pre-incubated with sericin at both 35 and 100 ng/ml had sig- nificantly lower amount (P ��� 0.01) of enzyme activity, indicat- ing the protective effect of silk protein against oxidative stress.