Casuarinin Protects Cultured MDCK Cells from Hydrogen Peroxide-Induced Oxidative Stress and DNA Oxidative Damage

• Abstract
• Introduction
• Materials and Methods
• Plant materials
• Cell culture and treatment
• Measurement of intracellular ROS by flow cytometry
• Measurement of intracellular GSH content by flow cytometry
• Measurement of intracellular 8-oxoguanine by flow cytometry
• Statistical analysis
• Results and Discussion
• Acknowledgements
• References

Abstract

Casuarinin has been shown to be an antioxidant in acellular experiments. This study was designed to assess the ability of casuarinin, extracted from Terminalia arjuna, to protect cultured Madin-Darby canine kidney (MDCK) cells against H₂O₂-mediated oxidative stress. A comparison with trolox, a hydrosoluble vitamin E analogue was performed. MDCK cells were pretreated with casuarinin or trolox for 1 h, then exposed to H₂O₂. After incubation with 0.8 mM H₂O₂ for 1 h, casuarinin caused a decrease in intracellular peroxide production as shown by dichlorofluorescein (DCF) fluorescence in a concentration-dependent manner. After 3 h exposure to 8 mM H₂O₂, the percentage of intracellular glutathione (GSH)-negative cells was reduced in the casuarinin-treated group. Addition of 32mM H₂O₂ to MDCK cells for 3 h induced an increase in the percentage of cells containing 8-oxoguanine but the level of such cells declined in casuarinin-treated cells. These results show that casuarinin is more effective against H₂O₂-induced oxidative damage than trolox. The data suggest that casuarinin attenuates H₂O₂-induced oxidative stress, decreases DNA oxidative damage and prevents the depletion of intracellular GSH in MDCK cells.

Introduction

Terminalia arjuna (Combretaceae) is a bulky woody plant. Recently, some biological effects of the T. arjuna have been demonstrated, such as antioxidant [1], hypocholesterolemic [1], hypolipidemic [2], antimutagenic [3], and antibacterial [4] activities. Casuarinin (Fig. [1]), a compound extracted from T. arjuna has been reported to induce apoptosis in HL-60 cells [5], to be cytotoxic towards PRMI-7951 melanoma cells [6], and to inhibit carbonic anhydrase activity [7].

Aerobic metabolism results in the generation of reactive oxygen species (ROS), such as singlet oxygen, superoxide radical, hydrogen peroxide (H₂O₂), nitric oxide, and hydroxyl radical. At moderate concentrations, ROS function as signal transduction messengers, but their extreme generation induces oxidative damage. Such damage may disturb all types of biological molecules, i. e., DNA, proteins, lipids, and carbohydrates. Cells have thus developed an antioxidant defense system, including antioxidant vitamins, glutathione, sulfhydryls, and antioxidant enzymes such as peroxidases, catalase, and superoxide dismutases [8]. In addition, oxidative stress has been shown to perform a role as a common mediator of apoptosis.

Although casuarinin was shown to exhibit an inhibitory effect on nitric oxide production in the murine macrophage-like cell line, RAW264.7 [9], its antioxidant effects on other ROS-induced cell damage have not yet been reported in detail. In addition, no studies have investigated other possible pathways concerning cytoprotection by casuarinin. The role of ROS has been proposed in many human degenerative diseases of aging, antioxidants have been found to exert some preventive and therapeutic effects on these diseases [10]. H₂O₂, one of the main ROS, was shown to cause lipid peroxidation and DNA oxidative damage in cells [11]. According to these reports we used H₂O₂ in order to evaluate the antioxidant effect of casuarinin in intact cells. The present study was designed to investigate whether casuarinin is capable of reducing the hydrogen peroxide-induced intracellular ROS increase, the intracellular GSH level depletion and the oxidative DNA damage in a renal-derived cell system, namely the tubular epithelial Madin-Darby canine kidney (MDCK) cell line. In parallel, the protective effect of trolox, a cell-permeable and water-soluble derivative of vitamin E with antioxidant properties, was also studied.

Materials and Methods

Plant materials

Casuarinin was isolated from the bark of T. arjuna as described previously [12]. The purity of casuarinin is greater than 98 %. The [α]_D ²⁸ value of casuarinin is + 40.2°.

Cell culture and treatment

The basal medium for MDCK cell culture was DMEM supplemented with 10 % FBS, 100 units/mL penicillin G, 100 μg/mL streptomycin and 250 μg/mL amphotericin B. The stock solution of casuarinin (200 mM) was dissolved in DMSO and different concentrations were prepared in the aforementioned basal medium with a final DMSO concentration of 0.1 %. The solution of trolox (5 mM) was prepared in phosphate buffer saline (PBS). The periods of treatment with H₂O₂ were 1 h for ROS generation, 3 h for intracellular GSH depletion and 8-oxoguanine production. MDCK cells were pretreated with casuarinin or trolox for 1 h prior to the addition of H₂O₂.

Measurement of intracellular ROS by flow cytometry

Production of intracellular ROS was detected by flow cytometry using DCFH-DA [13]. The MDCK cells were cultured in 60-mm tissue culture dishes. The culture medium was replaced with new medium when the cells were 80 % confluent. Cells were treated with 10 μM DCFH-DA for 30 min in the dark, washed once with PBS, detached by trypsinization, collected by centrifugation, and suspended in PBS containing 5 μg/mL of PI for 10 min prior to flow cytometry. Propidium iodide treatment differentiates between integrated and non-integrated cell membranes, since the latter permit the entrance of this dye into the cells, and the former do not. The fluorescence of dichlorofluorescein (DCF), reflecting the level of intracellular ROS, was measured in a Becton-Dickinson FACS-Calibur flow cytometer.

Measurement of intracellular GSH content by flow cytometry

The level of intracellular GSH per cell was determined by flow cytometry after staining with chloromethylfluorescein diacetate (CMF-DA) [14]. CMF-DA, containing a mildly thiol reactive chloromethyl reactive group, is colorless and non-fluorescent. This probe is primarily conjugated to the abundant tripeptide glutathione by glutathione S-transferase. Once inside the cell, cytosolic esterases cleave off their acetates and then the chloromethyl group reacts with intracellular thiols, transforming the probe into a cell-impermeant fluorescent dye-thioether adduct. In our experiments, CMF-DA was prepared as a 25 mM solution in DMSO and stored at -20 °C. It was added at 25 μM to cell suspensions adjusted at 1 - 2 × 10⁶ cells per mL. After 30 min of incubation at 37 °C, cells were washed twice in PBS, resuspended at a concentration of 10⁶ cells/mL in PBS, and analyzed in a Becton-Dickinson FACS-Calibur flow cytometer. The fluorescent dye-thioether adduct was excited at 488 nm and the fluorescence was collected with a 525 nm band-pass filter. Analyses were performed on 10 000 cells and fluorescence was measured on a logarithmic scale of fluorescence of four decades of log. The data were collected, stored, and analyzed with the CellQuest software.

Measurement of intracellular 8-oxoguanine by flow cytometry

Following drug incubation, intracellular 8-oxoguanine was measured using Biotrin OxyDNA Assay kits. Both floating and adherent cells were harvested and fixed using 1 % paraformaldehyde for 15 min on ice. After fixation, cells were permeated with 70 % ethanol for 30 min at -20 °C, followed by one wash with phosphate buffer saline (PBS) and one wash with wash solution (Tris buffered saline/Tween-20). 1 - 2 × 10⁶ cells were added blocking solution and incubated for 1 h at 37 °C. Cells were washed twice, incubated with binding protein-FITC conjugate for 1 h in the dark at room temperature. Cells were washed twice with wash solution, once with PBS, suspended in PBS and read in a Becton-Dickinson FACS-Calibur flow cytometer.

Statistical analysis

Data are presented as means ± standard deviation (SD) and analyzed using one-way ANOVA with Scheffe's test. A P value of less than 0.05 was considered as statistically significant.

Results and Discussion

To investigate the ROS scavenging effect of casuarinin, the level of intracellular ROS was estimated by the changes in DCF fluorescence intensity. The concentration of 0.8 mM H₂O₂ was used to evaluate the ROS scavenging effect of casuarinin. The pretreatment with casuarinin decreased the intracellular ROS level in H₂O₂-treated cells in a dose-dependent manner (Fig. [2]). DCF fluorescence intensity dropped markedly from 245 in cells treated with 0.8 mM H₂O₂ only to values between 239 and 50 in cells which were pretreated with 1 - 50 μM casuarinin. In cells pretreated with 100 μM trolox the generation of ROS was also inhibited. However, the decrease in the level of intracellular ROS caused by casuarinin was greater than that caused by trolox (Fig. [2]). The results suggest that the ROS scavenging effect of casuarinin in MDCK cells increased markedly when the concentration was at least 50 μM. We thus decided to use 50 μM casuarinin for all subsequent experiments.

Glutathione, a low-molecular weight thiol reductant, is present in most cells. It plays an essential role in numerous cell functions, such as protection of cells from toxic oxygen species and detoxification of various xenobiotics [15]. Since casuarinin protected cells from H₂O₂-induced intracellular ROS, we tried to explain whether H₂O₂ could decrease the intracellular GSH content in MDCK cells and whether casuarinin could counteract this event. To evaluate this hypothesis, the MDCK cells were treated with various concentrations of H₂O₂ for 3 h and the intracellular GSH content was measured by flow cytometry with CMF-DA. The effect of various concentrations of H₂O₂ on the amount of intracellular GSH-negative cells was evaluated (Fig. [3]). Under the described conditions 8 mM H₂O₂ induced more than 40 % of intracellular GSH-negative cells thus, we chose this concentration for the next experiment with casuarinin. As shown in Fig. [4], the percentages of intracellular GSH-negative cells in untreated and 8 mM H₂O₂-treated MDCK cells were 4.5 % and 43.0 %, respectively. However, the percentage of GSH-negative cells was markedly less in casuarinin-pretreated cells than in H₂O₂-treated cells. Casuarinin kept the percentage of intracellular GSH-negative cells at 19.3 % during H₂O₂ treatment. The preventive capacity of trolox (50 μM) for intracellular GSH decrease was less than that of casuarinin during the period of H₂O₂ treatment and accounted for 36.6 % of intracellular GSH-negative cells in 50 μM trolox pretreated MDCK cells (Fig. [4]).

It has been demonstrated that oxidative injury to macromolecules is involved in an expansive range of pathological states [16]. ROS may react with DNA inducing reversible and irreversible damage and generate mutations, carcinogenesis, teratogenesis or cell death [16]. In this experiment, we first evaluated the concentration of H₂O₂, which could induce 8-oxoguanine as a product of DNA oxidative damage in MDCK cells using the Biotrin OxyDNA Assay kit and flow cytometry. H₂O₂ showed a concentration-dependent DNA oxidative damage on MDCK cells after treatment for 3 h (Fig. [5]). Thirty-two mM H₂O₂ induced more than 20 % of the cell population expressing 8-oxoguanine products. In order to investigate the effect of casuarinin on the H₂O₂-induced oxidative damage in MDCK cells, we treated cells with 32 mM H₂O₂ for 3 h with or without pretreatment with casuarinin and trolox and preformed an 8-oxoguanine assay using flow cytometry. An increase of the 8-oxoguanine level in MDCK cells treated with 32 mM H₂O₂ (26.4 %) for 3 h compared to the untreated cells (1.4 %) was observed (Fig. [6]). Pretreatment with 50 μM casuarinin for 1 h substantially reduced the H₂O₂-induced 8-oxoguanine level (on content) in MDCK cells to 11.6 %. Trolox (50 μM) pretreatment reduced the production of H₂O₂-induced 8-oxoguanine by 19.7 %, hence, the protective effects of casuarinin remain greater than that of trolox.

In the present study, we have demonstrated that the H₂O₂-induced oxidative damage of MDCK cells occurred in a relatively short time and that the damage was suppressed partially by the addition of casuarinin. Hydrogen peroxide was used as a trigger of oxidative stress since it is known to act as a major component of ROS produced intracellularly during many physiological and pathological processes, and caused oxidative damage [11]. A fluorescent probe, DCFH-DA, was used to measure the intracellular ROS level. DCFH-DA is metabolized intracellularly by esterase to form DCFH. Once ROS, predominantly H₂O₂, are present in the cell, the non-fluorescent DCFH is quickly oxidized to an extremely fluorescent DCF [13]. Thus, DCF fluorescence intensity quickly reflects the intracellular H₂O₂ concentration. It can be observed that casuarinin is very effective in decreasing DCF fluorescence intensity in H₂O₂-treated cells (Fig. [2]). These results imply that casuarinin suppressed (on reduced) intracellular H₂O₂ concentration. It is supposed that H₂O₂ itself is not extremely reactive towards cellular molecules. The principal mechanism of H₂O₂ toxicity in oxidative stress is the generation of highly reactive species, such as hydroxyl radicals, by reaction with transition metal ions or via other mechanisms [17]. The generation of hydroxyl radicals and other ROS initiates lipid peroxidation and induces damage of other macromolecules such as DNA. The decrease of intracellular H₂O₂ concentration leads to lower production of ·OH and attenuation of oxidative damage.

GSH is the most efficient weapon against ROS. In particular, GSH provides a primary protection against oxidative stress by its capability for scavenging free radicals [18], [19], or for the breakdown of H₂O₂ catalyzed by GSH peroxidase, a selenium-dependent enzyme [20]. It was shown that the concentration of H₂O₂, 0.8mM, which induced intracellular ROS formation, produced very small amounts of intracellular GSH-negative cells (Fig. [3]). Hence, we increased the concentration of H₂O₂ to 8 mM to obtain a more marked lowering of the intracellular GSH level. Our data demonstrate that the content of GSH in H₂O₂-treated MDCK cells is protected to some degree by casuarinin (Fig. [4]).

Oxidative DNA damage caused by ROS can result in mutagenesis and carcinogenesis [11]. H₂O₂ is a well-known genotoxic agent able to induce oxidative DNA damage, involving DNA strand breakage and base modification [11]. In this study, H₂O₂-induced DNA damage was analyzed by the measurement of the production of intracellular 8-oxoguanine using an in vitro fluorescent protein binding method. Consistent with the protective effect on intracellular GSH, casuarinin showed to some extent a protective capability against H₂O₂-induced DNA oxidative damage (Fig. [6]). This is the first evidence of such an ability of casuarinin in a cell-cultured system.

In summary, the present findings suggest that the protective effect of casuarinin against H₂O₂-induced oxidative stress, intracellular GSH depletion and DNA damage in MDCK cells observed in the present study is due to a reduction of the intracellular H₂O₂ concentration. The present results also demonstrate that casuarinin reduced these effects more strongly than trolox. It may be used as a potent antioxidant to protect tissues against oxidative damage associated with elevated H₂O₂ production. The antioxidant activity against other ROS and exact mechanisms, however, are yet to be determined.

Acknowledgements

This work was supported by grant NSC 92-2314-B-242-009 from the National Science Council, Taiwan, ROC.

References

• 1 Gupta R, Singhal S, Goyle A, Sharma V N. Antioxidant and hypocholesterolaemic effects of Terminalia arjuna tree-bark powder: a randomised placebo-controlled trial.  J Assoc Physicians India. 2001;  49 231-5
  PubMedSearch in Google Scholar
• 2 Shaila H P, Udupa S L, Udupa A L. Hypolipidemic activity of three indigenous drugs in experimentally induced atherosclerosis.  Int J Cardiol. 1998;  67 119-24
  CrossrefPubMedSearch in Google Scholar
• 3 Kaur S, Grover I S, Kumar S. Antimutagenic potential of extracts isolated from Terminalia arjuna .  J Environ Pathol Toxicol Oncol. 2001;  20 9-14
  PubMedSearch in Google Scholar
• 4 Perumal Samy R, Ignacimuthu S, Sen A. Screening of 34 Indian medicinal plants for antibacterial properties.  J Ethnopharmacol. 1998;  62 173-82
  CrossrefPubMedSearch in Google Scholar
• 5 Yang L L, Lee C Y, Yen K Y. Induction of apoptosis by hydrolysable tannins from Eugenia jambos L. on human leukemia cells.  Cancer Lett. 2000;  157 65-75
  CrossrefPubMedSearch in Google Scholar
• 6 Kashiwada Y, Nonaka G, Nishioka I, Chang J J, Lee K H. Antitumor agents, 129. Tannins and related compounds as selective cytotoxic agents.  J Nat Prod. 1992;  55 1033-43
  CrossrefPubMedSearch in Google Scholar
• 7 Satomi H, Umemura K, Ueno A, Hatano T, Okuda T, Noro T. Carbonic anhydrase inhibitors from the pericarps of Punica granatum L.  Biol Pharm Bull. 1993;  16 787-90
  PubMedSearch in Google Scholar
• 8 Maclin L J, Bendich A. Free radical tissue damage: protective role of antioxidant nutrients.  FASEB J. 1987;  1 441-5
  PubMedSearch in Google Scholar
• 9 Ishii R, Saito K, Horie M, Shibano T, Kitanaka S, Amano F. Inhibitory effects of hydrolysable tannins from Melastoma dodecandrum Lour. on nitric oxide production by murine macrophage-like cell line, RAW264.7, activated with lipopolysaccharide and interferon-gamma.  Biol Pharm Bull. 1999;  22 647-53
  PubMedSearch in Google Scholar
• 10 Ames B N, Shigenaga M K, Hagen T M. Oxidants, antioxidants, and the degenerative diseases of aging.  Proc Natl Acad Sci USA. 1993;  90 7915-22
  CrossrefPubMedSearch in Google Scholar
• 11 Halliwell B, Aruoma O I. DNA damage by oxygen-derived species.  FEBS Lett. 1991;  281 9-19
  CrossrefPubMedSearch in Google Scholar
• 12 Lin T C, Ma Y T, Hsu F L. Tannins from the bark of Terminalia arjuna.  Chin Pharm J. 1996;  48 25-35
  PubMedSearch in Google Scholar
• 13 LeBel C P, Ischiopoulos H, Bondy S C. Evaluation of the probe 2′,7′-dichlorofluorescin as indicator of reactive oxygen species formation and oxidative stress.  Chem Res Toxicol. 1992;  5 227-31
  CrossrefPubMedSearch in Google Scholar
• 14 Chang W H, Chen C H, Lu F J. Different effects of baicalein, baicalin and wogonin on mitochondrial function, glutathione content and cell cycle progression in human hepatoma cell lines.  Planta Medica. 2002;  68 128-32
  Thieme ConnectPubMedSearch in Google Scholar
• 15 Fernandez-Checa J C, Kaplowitz N, Garcia-Ruiz C, Colell A. Mitochondrial glutathione: importance and transport.  Semin Liver Dis. 1998;  18 389-401
  Thieme ConnectPubMedSearch in Google Scholar
• 16 De Zwart L L, Meerman J H, Commandeur J N, Vermeulen N P. Biomarkers of free radical damage applications in experimental animals and in humans.  Free Radical Biol Med. 1999;  26 202-26
  CrossrefPubMedSearch in Google Scholar
• 17 Halliwell B, Gutteridge J MC, Cross C E. Free radicals, antioxidants, and human disease: where are we now?.  J Lab Clin Med. 1992;  119 598-620
  PubMedSearch in Google Scholar
• 18 Winterbourn C C, Metodiewa D. The reaction of superoxide with reduced glutathione.  Arch Biochem Biophys. 1994;  314 284-90
  CrossrefPubMedSearch in Google Scholar
• 19 Tsai C H, Chern C L, Liu T Z. Antioxidant action of glutathione: Its interaction with superoxide anion and hydroxyl radical.  J Biomed Lab Sci (Taiwan). 2000;  12 107-11
  PubMedSearch in Google Scholar
• 20 Chandre J, Samali A, Orrenius S. Triggering and modulation of apoptosis by oxidative stress.  Free Radical Biol Med. 2000;  29 323-33
  CrossrefPubMedSearch in Google Scholar

Professor Chun-Ching Lin

Graduate Institute of Pharmaceutical Science

College of Pharmacy

Kaohsiung Medical University

100 Shih Chuan 1st Road

Kaohsiung 807

Taiwan

ROC

Phone: +886-7-312-1101-2122

Fax: +886-7-3135215

Email: aalin@ms24.hinet.net

References

• 1 Gupta R, Singhal S, Goyle A, Sharma V N. Antioxidant and hypocholesterolaemic effects of Terminalia arjuna tree-bark powder: a randomised placebo-controlled trial.  J Assoc Physicians India. 2001;  49 231-5
  PubMedSearch in Google Scholar
• 2 Shaila H P, Udupa S L, Udupa A L. Hypolipidemic activity of three indigenous drugs in experimentally induced atherosclerosis.  Int J Cardiol. 1998;  67 119-24
  CrossrefPubMedSearch in Google Scholar
• 3 Kaur S, Grover I S, Kumar S. Antimutagenic potential of extracts isolated from Terminalia arjuna .  J Environ Pathol Toxicol Oncol. 2001;  20 9-14
  PubMedSearch in Google Scholar
• 4 Perumal Samy R, Ignacimuthu S, Sen A. Screening of 34 Indian medicinal plants for antibacterial properties.  J Ethnopharmacol. 1998;  62 173-82
  CrossrefPubMedSearch in Google Scholar
• 5 Yang L L, Lee C Y, Yen K Y. Induction of apoptosis by hydrolysable tannins from Eugenia jambos L. on human leukemia cells.  Cancer Lett. 2000;  157 65-75
  CrossrefPubMedSearch in Google Scholar
• 6 Kashiwada Y, Nonaka G, Nishioka I, Chang J J, Lee K H. Antitumor agents, 129. Tannins and related compounds as selective cytotoxic agents.  J Nat Prod. 1992;  55 1033-43
  CrossrefPubMedSearch in Google Scholar
• 7 Satomi H, Umemura K, Ueno A, Hatano T, Okuda T, Noro T. Carbonic anhydrase inhibitors from the pericarps of Punica granatum L.  Biol Pharm Bull. 1993;  16 787-90
  PubMedSearch in Google Scholar
• 8 Maclin L J, Bendich A. Free radical tissue damage: protective role of antioxidant nutrients.  FASEB J. 1987;  1 441-5
  PubMedSearch in Google Scholar
• 9 Ishii R, Saito K, Horie M, Shibano T, Kitanaka S, Amano F. Inhibitory effects of hydrolysable tannins from Melastoma dodecandrum Lour. on nitric oxide production by murine macrophage-like cell line, RAW264.7, activated with lipopolysaccharide and interferon-gamma.  Biol Pharm Bull. 1999;  22 647-53
  PubMedSearch in Google Scholar
• 10 Ames B N, Shigenaga M K, Hagen T M. Oxidants, antioxidants, and the degenerative diseases of aging.  Proc Natl Acad Sci USA. 1993;  90 7915-22
  CrossrefPubMedSearch in Google Scholar
• 11 Halliwell B, Aruoma O I. DNA damage by oxygen-derived species.  FEBS Lett. 1991;  281 9-19
  CrossrefPubMedSearch in Google Scholar
• 12 Lin T C, Ma Y T, Hsu F L. Tannins from the bark of Terminalia arjuna.  Chin Pharm J. 1996;  48 25-35
  PubMedSearch in Google Scholar
• 13 LeBel C P, Ischiopoulos H, Bondy S C. Evaluation of the probe 2′,7′-dichlorofluorescin as indicator of reactive oxygen species formation and oxidative stress.  Chem Res Toxicol. 1992;  5 227-31
  CrossrefPubMedSearch in Google Scholar
• 14 Chang W H, Chen C H, Lu F J. Different effects of baicalein, baicalin and wogonin on mitochondrial function, glutathione content and cell cycle progression in human hepatoma cell lines.  Planta Medica. 2002;  68 128-32
  Thieme ConnectPubMedSearch in Google Scholar
• 15 Fernandez-Checa J C, Kaplowitz N, Garcia-Ruiz C, Colell A. Mitochondrial glutathione: importance and transport.  Semin Liver Dis. 1998;  18 389-401
  Thieme ConnectPubMedSearch in Google Scholar
• 16 De Zwart L L, Meerman J H, Commandeur J N, Vermeulen N P. Biomarkers of free radical damage applications in experimental animals and in humans.  Free Radical Biol Med. 1999;  26 202-26
  CrossrefPubMedSearch in Google Scholar
• 17 Halliwell B, Gutteridge J MC, Cross C E. Free radicals, antioxidants, and human disease: where are we now?.  J Lab Clin Med. 1992;  119 598-620
  PubMedSearch in Google Scholar
• 18 Winterbourn C C, Metodiewa D. The reaction of superoxide with reduced glutathione.  Arch Biochem Biophys. 1994;  314 284-90
  CrossrefPubMedSearch in Google Scholar
• 19 Tsai C H, Chern C L, Liu T Z. Antioxidant action of glutathione: Its interaction with superoxide anion and hydroxyl radical.  J Biomed Lab Sci (Taiwan). 2000;  12 107-11
  PubMedSearch in Google Scholar
• 20 Chandre J, Samali A, Orrenius S. Triggering and modulation of apoptosis by oxidative stress.  Free Radical Biol Med. 2000;  29 323-33
  CrossrefPubMedSearch in Google Scholar

Professor Chun-Ching Lin

Graduate Institute of Pharmaceutical Science

College of Pharmacy

Kaohsiung Medical University

100 Shih Chuan 1st Road

Kaohsiung 807

Taiwan

ROC

Phone: +886-7-312-1101-2122

Fax: +886-7-3135215

Email: aalin@ms24.hinet.net