The Inhibitory Effect of Phytochemicals on the Oxidative DNA Damage in Lymphocytes by Chrysotile
A-Reum Ryu · Jum-Ji Kim · Mi-Young Lee
Received: 18 May 2012 / Accepted: 6 August 2012 / Published Online: 30 September 2012
© The Korean Society for Applied Biological Chemistry 2012
Abstract We investigated the cytotoxicity and oxidative DNA damage by chrysotile, one of the asbestos, in this investigation.
Chrysotile enhanced malondialdehyde (MDA) levels and intracellular reactive oxygen speices generation in human airway epithelial cells. Furthermore, asbestos-induced oxidative DNA damage in lymphocytes was evaluated by single cell gel electrophoresis and quantified as DNA tail moment. Notably, phytochemicals such as curcumin, berberine, and sulforaphane presented inhibitory effect on the asbestos-induced oxidative DNA damage in lymphocytes.
Keywords asbestos · lipid peroxidation · phytochemicals · reactive oxygen speices
Introduction
Asbestos, including chrysotile, amosite, crocidolite, tremolite, anthophyllite and actinolite, is a family of naturally occurring silicate fibers used commercially in such applications as electrical insulation and in building insulation (Frank, 1993). Asbestos exposure in humans is associated with inflammatory and malignant disease in the lung, including malignant lung cancer, mesothelioma and asbestosis (Park et al., 2008; Loomis et al., 2010; Seo et al., 2012). Cytokines and growth factors, such as tumor necrosis factor (TNF-α), interleukin (IL)-1α, IL-8, and platelet-derived growth factor, are strongly implicated as mediators of asbestos-related pathophysiologic responses (Bonner et al., 1993; Driscoll et al., 1993; Li et al., 1993; Perkins et al., 1993).
The exact molecular mechanisms responsible for these processes are not clear (Rom et al., 1991), but largely involved in the reactive oxygen species (ROS) generation via the interaction of iron, present as a silicate-iron complex on the fiber surface (Simeonova and Luster, 1995; 1996). Generally, high level of
ROS induces cytotoxicity and plays a direct role in tissue damage, whereas moderate oxidative stress can activate events in the cell signal transduction pathways leading to the production of inflammatory mediators (Simeonova et al., 1997). Previous studies have yielded conflicting data on the cytotoxic effects of asbestos on mesothelial cells. For example, increase in intracellular oxidation have not been found in studies of asbestos-exposed mesothelial cells (Gabrielson et al., 1986; Ollikainen et al., 1999), although upregulation of antioxidant enzymes and oxidative changes in DNA suggest a role for oxidative damage to the cell and to cellular DNA (Janssen et al., 1994). In addition, DNA strand breakage, an expected toxic effect of oxidative damage to DNA, has not been found in previous studies of asbestos-exposed mesothelial cells (Gabrielson et al., 1986; Kinnula et al., 1994), although other DNA lesions an indirect measures of DNA damage have been reported (Renier et al., 1990; Liu et al., 2000).
A variety of antioxidants and medicinal herbs have been known to reduce the generation of reactive oxygen species. Curcumin is the principal curcuminoid of the popular Indian spice tumeric (Choi, 2009). Its antioxidant, anti-inflammatory, antiviral, and antifungal actions, as well as an anticancer effect were reported (Biswas and Rahman, 2008; Seo and Lee, 2011). Berberine, the major constituent of Coptis Chinensis, is an isoquinolin alkaloid which has wide range of pharmacological and biochemical effects. Berberine has demonstrated the significant antimicrobial activity and antitumor activity (Letasiová et al., 2005). Sulforaphane, isolated from broccoli, is not a direct-acting antioxidant or prooxidant, since it is unlikely that the isothiocyanate group might participate in oxidation or reduction reactions under physiological conditions (Barton and Ollis, 1979). There is, however, substantial and growing evidence that sulforaphane administration acts indirectly to increase the antioxidant capacity of animal cells, and their abilities to cope with oxidative stress (Fahey and Talalay, 1999). In this study, chrysotile-induced cytotoxicity in NCI-H292 cells and oxidative DNA damage in lymphocytes were measured.
The suppressive effect of phytochemicals such as curcumin, berbrine, and sulforaphane on the DNA damage by chrysotile was also investigated.
A-R. Ryu · J.-J. Kim · M.-Y. Lee ()
Department of Medical Biotechnology, SoonChunHyang University, Asan, Chungnam, 336-745, Republic of Korea
E-mail: miyoung@sch.ac.kr ORIGINAL ARTICLE
chrysotile on the NCI-H292 cells, a trypan blue exclusion assay was performed. NCI-H292 cells were seeded and treated with 0, 0.1, 0.5, 1, 2.5 and 5µg/cm2 of asbestos for 24 h. We evaluated cell viability using trypan blue exclusion assay.
Malondialdehyde (MDA) determination. MDA level in NCI- H292 cell was measured by the thiobarbituric acid method (Heath and Packer, 1968).
Confocal analysis of intracellular ROS. ROS detection is additional tools in the assessment of cytotoxicity caused by asbestos fibers (Park et al., 2007). NCI-H292 cells seeded into poly-L-lysine coated cover slip and treated with 2.5 and 5µg/cm2 asbestos for 24 h. Asbestos treated NCI-H292 cells were washed twice with PBS and then incubated with 5µM 2',7'-dichloro- fluorescein diacetate (DCFH-DA) for 30 min at 37oC in 5% CO2 incubator, and were then washed with PBS. DCFH-DA, aliquoted in DMSO at a stock solution of 5 M, was stored in the dark at
−20oC, and then diluted in PBS immediately before the experiment.
The intracellular ROS was detected by Confocal microscope (460–495 nm/510 nm excitation/emission).
Determination of DNA damage by comet assay. The alkaline comet assay was performed according to Singh et al. (1988) with slight modifications, as reported previously (Park et al., 2005).
The Comet assay has been considered a sensitive method in the assessment of DNA single-strand breaks in the oxidant-exposed cells (Seo et al., 2010). The isolated lymphocytes from rat were mixed with 75µL of 0.7% low-melting-point agarose and added to slides precoated with 1.0% normal melting-point agarose. After the agarose solidified, the slides were covered with 100µL of 0.7% low-melting-point agarose and immersed in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% sodium lauryl sarcosine, 1% Triton X-100, and 10% DMSO) for 1 h at 4oC. The slides were placed in an electrophoresis tank containing 300 mM NaOH and 10 mM Na2EDTA (pH 13.0) for 20 min to allow the DNA to unwind. Electrophoresis was performed at 25 V/300 mA for 20 min at 4oC. The slides were washed with neutralizing buffer (0.4 M Tris·HCl pH 7.5) three times for 5 min at 4oC, and then treated with ethanol for 5 min.
Image analysis and statistical analysis. The slides were stained with ethidium bromide (20µg/mL) and cover slipped. Measurements were made by image analysis using Komet 5.5 software (Kinetic
Results
Cytotoxicity of chrysotile in human airway epithelial cells. The cytotoxicity of chrysotile in human airway epithelial cells (NCI- H292) was determined using trypan blue dye exclusion assay. The assay is based on the principle that live cells possess intact cell membranes that exclude trypan blue dye, following exposure to
Fig. 1 Cytotoxicity of chrysotile in NCI-H292 cells. Values not sharing the same letter are significantly different from one another. *p <0.05, significantly different from the chrysotile-induced cytotoxic damage in NCI-H292 cells.
Fig. 2 Intracellular MDA levels of NCI-H292 cells with exposure to chrysotile. Values not sharing the same letter are significantly different from one another (p <0.05).
various concentrations of chrysotiles for 24 h. Chrysotile exposure reduced cell viability in a concentration-dependent manner as shown in Fig. 1. The exposure of NCI-H292 cell to 1, 2.5 and 5 mg/cm2 asbestos resulted in an approximately 15.5, 28.1 and
50.2% decrease in cell viability, respectively.
MDA level and ROS generation in NCI-H292 cells exposed to chrysotile. In order to determine intracellular MDA levels of NCI-H292 cells following chrysotile exposure, we performed modified thiobarbituric acid method (Heath and Packer, 1968). As shown in Fig. 2, chrysotile enhanced MDA levels of NCI-H292 cells, showing lipid peroxidation increase by chrysotile in dose- dependent manners. To analyze whether the generation of reactive oxygen species (ROS) may be related with asbestos-induced lipid peroxidation in NCI-H292 cells, the cells were incubated with an Fig. 3 The confocal images of intracellular ROS levels of NCI-H292
cells exposed with chrysotile.
Fig. 4 (A) Chrysotile-induced oxidative DNA damages of lymphocytes by Comet assay. (B) Inhibitory effect of catalase on the chrysotile- induced oxidative DNA damage. Values not sharing the same letter are significantly different from one another (p <0.05) according to Duncan's multiple range test
Fig. 5 Inhibitory effect of (A) curcumin, (B) berberine, and (C) sulforaphane on the chrysotile-induced oxidative DNA damage. Values not sharing the same letter are significantly different from one another (p <0.05).
oxidative-sensitive fluorescent probe DCFH-DA using Confocal microscope. Figure 3 shows a marked and dose dependent increase in the ROS levels in NCI-H292 cells treated with 2.5 and 5µg/
cm2 chrysotile, suggesting that enhanced lipid peroxidation might be attributable to the generation of intracellular ROS. Chrysotile- treated cells were strongly positive for DCFDA staining compared to the medium control.
Chrysotile-induced oxidative DNA damage in lymphocytes.
Chrysotile-induced oxidative DNA damage of lymphocytes was evaluated by Comet assay. Lymphocytes treated with 5, 10, and 20µg/mL chrysotile exhibited DNA damage, as determined by the olive tail moment (Fig. 4A). Chrysotile at low concentrations did not cause DNA damage, whereas high concentrations of chrysotile induced severe DNA damage. The olive tail moment at 20µg/mL chrysotile was about 28.04±2.03, compared with 3.03±1.23 in the PBS-treated control, indicating an approximately 9-fold increase in DNA damage at 20µg/mL chrysotile. The in vitro oxidative DNA damage caused by chrysotile increased in a dose-dependent manner. When catalase was treated (Fig. 4B), however, the tail lengths of lymphocyte DNA was reduced to the control, These results served as positive control for the current experiment to assess the antioxidant activities of phytochemicals.
Suppressive effects of phytochemicals on the chrysotile- induced DNA damage. Curcumin, berberine, and sulforaphane were used for their suppressive effects against chrysotile-induced oxidative DNA damage in lymphocytes (Fig. 5). The characteristics of the phytochemicals used in this investigation were described in Table 1. The addition of phytochemicals significantly inhibited the oxidative DNA damage caused by 20µg/cm2 chrysotile, as demonstrated by a reduction of the olive tail moment in comet assay. Sulforaphane effectively suppressed the oxidative DNA damage in concentration-dependent manners. 3 and 5µg/mL curcumin could suppress the DNA damage, on the contrary, 8 µg/
mL curcumin could not inhibit the damage in this experimental condition. Berberine was the most effective in reducing the olive tail moment in this system. Vitamin C, a general antioxidant, also inhibited the DNA damage. This result raises the possibility that
curcumin, berberine, and sulforaphane could be developede as therapeutic chemicals to suppress the chrysotile-induced oxidative DNA damage and asbestos-related diseases.
Discussion
Asbestos fiber has been heavily used in industry since World war II because of their durability, heat-resistance, and low cost.
However, in 1987, the International Agency for Research on Cancer (IARC) designated of asbestos fibers as a Group 1 (definite) carcinogen for human, and asbestos fibers were banned in many western countries in the 1990s (Roggli et al., 2004;
Dadson and Hammnar, 2006).
Previous studies have suggested the importance of hydroxyl radicals and irons in the fenton reaction in the pathogenesis of asbestos-related cell and DNA damage (Goodglick and Kane, 1990; Kamp et al., 1992). Another potential mechanism leading to asbestos-related cell and DNA damage occurred via nitric oxide (NO). NO itself has been shown to cause oxidant-related DNA damage, inhibition of repair processes, and/or increase of various genotoxic species in cells (Wink et al., 1998). However, the role of NO-mediated mechanisms in the development of DNA damage due to asbestos fibers has remained unclear (Puhakka et al., 2002).
The measurement of oxidant generation is difficult, since most previous methods are non-specific. They detect mainly the net release of various oxidants, such as H2O2, form the cells or measure the products of oxidant-mediated reaction, such as lipid peroxidation. Most recent studies have used an oxidation-sensitive fluorescent probe, 2',7'-dichlorodihydtrofluorescin (DCDHF)- diacetate (DA), which diffuses into cells and is oxidized to a fluorescent compound by ROS and reactive nitrogen species (RNS) (Liu et al., 2000).
The present study confirms the recent findings that asbestos fibers cause an increase in the mean tail extent moment in the Comet assay, a similar result shown in DNA single-strand breaks in cultured human mesothelioma cells (Ollikainen et al., 1999).
Chrysotile-induced cytotoxicity and intracellular ROS and MDA
generation of cells were enhanced in a dose dependent manner.
Next, we performed a comet assay to investigate the suppressive effects of phytochemicals on the chrysotile-induced oxidative DNA damage. Notably, however, various phytochemicals inhibited the chrysotile-induced oxidative DNA damage. Similar inhibitory effects were reported previously with red-ginseng and Acantopanax in oxidative DNA damage induced by herbicide (Ollikainen et al., 1999).
There have been considerable evidences that asbestos may initiate ROS production by Fenton-like reaction in exposed cells, stimulating phagocytic cells to release extracellular ROS through membrane-associated-NADPH oxidase, and by mitochondrial oxidative phosphorylation (Huang et al., 2012). In this study, treatment with chrysotile caused DNA damage, indicated by increased tail lengths, however, catalase repaired the injury, evidenced by decreased tail lengths to the control level. The repair of the H2O2-induced DNA damage by catalase was reported in showing antioxidative effect of mush room (Park et al., 2009). Our results indicate that phytochemicals have antioxidant properties in oxidative stress by radical scavenging. Therefore, those data presented in this study may provide the possible therapeutic values of the curcumin, berberine, and sulforaphane for the prevention of oxidative DNA damage progression by chrysotile.
Acknowledgments This work was supported in part by the SoonChunHyang University Research Fund.
References
Barton D and Ollis WD (1979) Comprehensive Organic Chemistry: The Synthesis and Reactions of Organic Compounds. pp. 461–77, Pergamon, USA.
Biswas S and Rahman I (2008) Modulation of steroid activity in chronic inflammation: a novel anti-inflammatory role for curcumin. Mol Nutr Food Res 52, 987–94.
Bonner JC, Goodell AL, Coin PG, and Brody AR (1993) Chrysotile asbestos upregulates gene expression and production of alpha-receptors for platelet-derived growth factor (PDGF-AA) on rat lung fibroblasts. J Clin Invest 92, 425–30.
Choi H, Chun YS, Kim SW, Kim MS, and Park JW (2006) Curcumin inhibits hypoxia-inducible factor-1 by degrading aryl hydrocarbon receptor nuclear translocator: a mechanism of tumor growth inhibition. Mol Pharmacol 70, 1664–71.
Choi HY (2009) Antioxidant activity of curcuma longa L., novel foodstuff.
Mol Cell Toxicol 5, 237–42.
Dadson RF and Hammnar SP (2006) In Asbestos: Risk Assessment, Epidemiology, and Health Effects. CRC Press, Taylor & Francis Group, USA.
Driscoll KE, Hassenbein DG, Carter J, Poynter J, Asquith TN, Grant RA et al.
(1993) Macrophage inflammatory proteins 1 and 2: expression by rat alveolar macrophages, fibroblasts, and epithelial cells and in rat lung after mineral dust exposure. Am J Respir Cell Mol Biol 8, 311–8.
Fahey JW and Talalay P (1999) Antioxidant functions of sulforaphane: a potent inducer of Phase II detoxication enzymes. Food Chem Toxicol 37, 973–9.
Fahey JW, Haristoy X, Dolan PM, Kensler TW, Scholtus I, Stephenson KK, et al. (2002) Sulforaphane inhibits extracellular, intracellular, and antibiotic-resistant strains of Helicobacter pylori and prevents benzo[a]pyrene-induced stomach tumors. Proc Natl Acad Sci USA 99, 7610–5.
Frank AL (1993) Global problems from exposure to asbestos. Environ Health Perspect 101, 165–67.
Gabrielson EW, Rosen GM, Grafstrom RC, Strauss KE, and Harris CC (1986) Studies on the role ofoxygen radicals in asbestos-induced cytopathology of cultured human lung mesothelial cells. Carcinogenesis 7, 1161–4.
Goodglick LA and Kane AB (1990) Cytotoxicity of long and short crocidolite asbestos fibers in vitro and in vivo. Cancer Res 50, 5153–63.
Hayes JD, Kelleher MO, and Eggleston IM (2008) The cancer chemopreventive actions of phytochemicals derived from glucosinolates.
Eur J Nutr 2, 73–88.
Heath RL and Packer L (1968) Photoperoxidation in isolated chloroplasts. I.
Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125, 189–98.
Huang SX, Partridge MA, Ghandhi SA, Davidson MM, Amundson SA, and Hei TK (2012) Mitochondria-derived reactive intermediate species mediate asbestos-induced genotoxicity and oxidative stress-responsive signaling pathways. Environ Health Perspect 120, 840–7.
Janssen YM, Marsh JP, Absher MP, Gabrielson E, Borm PJ, Driscoll K et al.
(1994) Oxidant stress responses in human pleural mesothelial cells exposed to asbestos. Am J Respir Crit Care Med 149, 795–802.
Kamp DW, Graceffa P, Pryor WA, and Weitzman SA (1992) The role of free radicals in asbestos-induced diseases. Free Radic Biol Med 12, 293–315.
Kinnula VL, Aalto K, Raivio KO, Walles S, and Linnainmaa K (1994) Cytotoxicity of oxidants and asbestos fibers in cultured human mesothelial cells. Free Radic Biol Med 16, 169–76.
Kuo CL, Chi CW, and Liu TY (2004) The anti-inflammatory potential of berberine in vitro and in vivo. Cancer Lett 203, 127–37.
Letasiová S, Jantová S, Múcková M, and Theiszová M (2005) Antiproliferative activity of berberine in vitro and in vivo. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 149, 461–3.
Li XY, Lamb D, and Donaldson K (1993) The production of TNF-alpha and IL-1-like activity by bronchoalveolar leucocytes after intratracheal instillation of crocidolite asbestos. Int J Exp Pathol 74, 403–10.
Liu W, Ernst JD, and Broaddus VC (2000) Phagocytosis of crocidolite asbestos induces oxidative stress, DNA damage, and apoptosis in mesothelial cells. Am J Respir Cell Mol Biol 23, 371–8.
Loomis D, Dement J, Richardson D, and Wolf S (2010) Asbestos fibre dimensions and lung cancer mortality among workers exposed to chrysotile. Occup Environ Med 67, 580–4.
Marin-Neto JA, Maciel BC, Secches AL, and Gallo Júnior L (1988) Cardiovascular effects of berberine in patients with severe congestive heart failure. Clin Cardiol 11, 253–60.
Ollikainen T, Linnainmaa K, and Kinnula VL (1999) DNA single strand breaks induced by asbestos fibers in human pleural mesothelial cells in vitro. Environ Mol Mutagen 33, 153–60.
Park BJ, Lim YS, Lee HJ, Eum WS, Park J, Han KH et al. (2009) Anti- oxidative effects of Phellinus linteus and red ginseng extracts on oxidative stress-induced DNA damage. BMB Rep 42, 500–5.
Park EJ, Ryoo KK, Lee YB, Lee JK, and Lee MY (2005) Protective effect of electrolyzed reduced water on the paraquat-induced oxidative damage of human lymphocyte DNA. J Korean Soc Appl Biol Chem 48, 155–60.
Park EK, Hannaford-Turner KM, Hyland RA, Johnson AR, and Yates DH (2008) Asbestos-related occupational lung diseases in NSW, Australia and potential exposure of the general population. Ind Health 46, 535–40.
Park SK, Kim JJ, Jeon YM, and Lee MY (2007) Suppressive effects of Korean indigenous Acanthopanax divaritacus on the allergic inflammation. J Appl Biol Chem 50, 155–9.
Perkins RC, Scheule RK, Hamilton R, Gomes G, Freidman G, and Holian A (1993) Human alveolar macrophage cytokine release in response to in vitro and in vivo asbestos exposure. Exp Lung Res 19, 55–65.
Puhakka A, Ollikainen T, Soini Y, Kahlos K, Säily M, Koistinen P et al.
(2002) Modulation of DNA single-strand breaks by intracellular glutathione in human lung cells exposed to asbestos fibers. Mutat Res 514, 7–17.
Renier A, Lévy F, Pillière F, and Jaurand MC (1990) Unscheduled DNA synthesis in rat pleural mesothelial cells treated with mineral fibres.
Mutat Res 241, 361–7.
ischemic effect of curcumin in rat brain. Neurochem Res 33, 1036–43.
Simeonova PP and Luster MI (1995) Iron and reactive oxygen species in the asbestos-induced tumor necrosis factor-alpha response from alveolar macrophages. Am J Respir Cell Mol Biol 12, 676–83.
Simeonova PP and Luster MI (1996) Asbestos induction of nuclear transcription factors and interleukin 8 gene regulation. Am J Respir Cell Mol Biol 15, 787–95.
Simeonova PP, Toriumi W, Kommineni C, Erkan M, Munson AE, Rom WN et al. (1997) Molecular regulation of IL-6 activation by asbestos in lung
congestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy. Am J Cardiol 92, 173–6.
Zhang Q, Cai L, Zhong G, and Luo W (2010) Simultaneous determination of jatrorrhizine, palmatine, berberine, and obacunone in Phellodendri Amurensis Cortex by RP-HPLC. Zhongguo Zhong Yao Za Zhi 35, 2061–
4.
Zhang Y, Talalay P, Cho CG, and Posner GH (1992) A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure. Proc Natl Acad Sci USA 89, 2399–403.
