uncorrected proof

Mutation Research xxx (2004) xxx–xxx

4 5

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Modulation of genotoxic effects in asbestos-exposed primary human mesothelial cells by radical scavengers, metal chelators and a glutathione precursor Ina Poser a , Qamar Rahman b , Mohtashim Lohani b , Santosh Yadav b , Hans-Henner Becker c , Dieter G. Weiss d , Dietmar Schiffmann d , Elke Dopp e,∗

8 9 10 11 12

a Institute of Pathology, University Hospital Regensburg, Germany Industrial Toxicology Research Centre, Division of Fibre Toxicology, 226 001 Lucknow, India c Department of Ophthalmology, Faculty of Medicine, University of Rostock, 18057 Rostock, Germany Department of Biology, Institute of Cell Physiology and Biosystems Technology, University of Rostock, Germany e Institute of Hygiene and Occupational Health, University of Essen, 45147 Essen, Germany b

F

d

Received 16 January 2003; received in revised form 2 December 2003; accepted 12 December 2003

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Abstract

16

The genotoxicity of asbestos fibers is generally mediated by reactive oxygen species (ROS) and by insufficient antioxidant protection. To further elucidate which radicals are involved in asbestos-mediated genotoxicity and to which extent, we have carried out experiments with the metal chelators deferoxamine (DEF) and phytic acid (PA), and with the radical scavengers superoxide dismutase (SOD), dimethylthiourea (DMTU) and the glutathione precursor NacystelynTM (NAL). We investigated the influence of these compounds on the potency of crocidolite, an amphibole asbestos fiber with a high iron content (27%), and chrysotile, a serpentine asbestos fiber with a low iron content (2%), to induce micronuclei (MN) in human mesothelial cells (HMC) after an exposure time of 24–72 h. Our results show that the number of crocidolite-induced MN is significantly reduced after pretreatment of fibers with PA and DEF. This effect was not observed with chrysotile. In contrast, simultaneous treatment of cells with asbestos and the OH• -scavenging DMTU or the O2 − -scavenging SOD significantly decreased the number of MN induced by chrysotile and crocidolite. In particular, DMTU almost completely suppressed micronucleus induction by both fiber types. A similar effect was observed in the presence of the H2 O2 -scavenging NAL after chrysotile treatment of HMC. By means of kinetochore analysis, it could be shown that the number of clastogenic events is decreased after PA and DEF pretreatment of fibers as well as after application of the above-mentioned scavengers. Our results show that chrysotile asbestos induces an increased release of H2 O2 in contrast to crocidolite. Also, the iron content of the fiber plays

18 19 20 21 22 23 24 25 26 27 28

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1383-5718/$ – see front matter © 2004 Published by Elsevier B.V. doi:10.1016/j.mrgentox.2003.12.006

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Abbreviations: AFC, amniotic fluid cells; DEF, deferoxamine; DMTU, dimethylthiourea; HMC, human mesothelial cells; H2 O2 , hydrogen peroxide; OH• , hydroxyl radical; K, kinetochores; MN, micronucleus; NAL, NacystelynTM ; NO• , nitric oxide; • ONOO, peroxynitrite; PA, phytic acid; RNS, reactive nitrogen species; ROS, reactive oxygen species; O2 − , superoxide anion; SOD, superoxide dismutase; SHE, Syrian hamster embryo fibroblasts ∗ Corresponding author. Tel.: +49-201-723-4574; fax: +49-201-723-4546. E-mail address: [email protected] (E. Dopp).

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I. Poser et al. / Mutation Research xxx (2004) xxx–xxx

32

an important role in radical formation, but nevertheless, chrysotile produces oxy radicals to a similar extent as crocidolite, probably by phagocytosis-mediated oxidative bursting. © 2004 Published by Elsevier B.V.

33

Keywords: Asbestos; Mesothelial cells; Metal chelator; Radical scavenger; Micronuclei

30 31

34

41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74

75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94

2. Materials and methods

95

Crocidolite and Rhodesian chrysotile (UICC standard) were obtained from Dr. Linnainmaa (University of Helsinki, Finland). Average diameter and length of the fibers were 0.10 and 2.24 ␮m, respectively, and the percentage of fibers >5 ␮m was approximately 5%.

96 97 98 99 100 101

2.1. Cell culture and treatment conditions

102

Human mesothelial cells were obtained by effusion of ascites fluid from a non-cancerous patient at the University Hospital, Rostock, Germany. Fluid samples were centrifuged and the pelleted cells were transferred into 25 cm2 tissue culture flasks and grown in a 1:1 mixture (v/v) of M199 and MCDB 105 media (Sigma, Germany) supplemented with 5–10 ng/ml EGF, 0.4 ␮g/ml hydrocortisone and 7% FCS. The

103

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The link between the production of reactive oxygen species (ROS) and the pathogenesis of asbestosmediated diseases has been highlighted in several studies [1–9]. Furthermore, investigations have indicated that ROS such as the superoxide anion (O2 − ) and the hydroxyl radical (OH• ), as well as reactive nitrogen species (RNS) like nitric oxide (NO• ) and peroxynitrite (• ONOO) are involved in asbestosinduced genotoxicity. Asbestos produces ROS in two different ways. The first mechanism is due to the participation of redox-active iron (Fe2+ , Fe3+ ) present in asbestos catalyzing the formation of OH• radicals. The second mechanism is the production of ROS during phagocytosis of fibers. Kamp and Weitzman [2] showed that the chemical nature of asbestos influences the formation of free radicals. All types of asbestos contain iron as an integral component of the crystalline structure or as a substitute cation, or as surface impurity [5]. Amphibole fibers such as crocidolite [Na2 (Fe3+ )2 (Fe2+ )3 Si8 O22 (OH)2 ] contain approximately 27% iron, whereas chrysotile [Mg6 Si4 O10 (OH8 )] contains approximately 1–6% iron, which is primarily present as surface contaminant and substitutes Mg2+ . This iron (especially Fe2+ ) promotes the formation of OH• from hydrogen peroxide (H2 O2 ) (Fenton reaction) as well as the formation of OH• from O2 − and H2 O2 , via the Haber–Weiss reaction [10]. Iron chelators, such as deferoxamine or phytic acid, and antioxidant enzymes (AOE) attenuate asbestosinduced ROS-release, DNA damage, and injury to pulmonary parenchymal cells in vitro [11–15]. Studies using scavengers of reactive oxygen species have shown that crocidolite and chrysotile asbestos induce substantially reduced cytotoxicity in hamster tracheal epithelial cells in the presence of dimethylthiourea (DMTU) or mannitol (both scavengers of hydroxyl radicals) or superoxide dismutase (SOD) in the culture media [16]. Evidence suggests that

OO

36

ROS are also responsible for asbestos toxicity in vivo. Polyethylene glycol-conjugated catalase reduces crocidolite-induced pulmonary inflammation and fibrosis in rats [17]. Recently, Rahman et al. have discussed the role of oxy radicals in the genotoxicity of asbestos [18] and have concluded that iron-catalyzed formation of hydroxyl radicals has the highest significance for asbestos-induced genotoxicity in both acellular and cellular systems. In the present study, primary human mesothelial cells (HMC), the target cells of asbestos-induced mesothelioma, were used to investigate the effect of crocidolite and chrysotile on the formation of ROS. The different radicals were scavenged by NacystelynTM (NAL), a glutathione precursor and quencher of hydrogen peroxide (H2 O2 ), superoxide dismutase, a scavenger of O2 − , and dimethylthiourea, a scavenger of OH• , and the effects on chrysotilemediated as well as crocidolite-mediated genotoxicity were observed.

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1. Introduction

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104 105 106 107 108 109 110


For all treatments human mesothelial cells were grown on coverslips in 8-well plates (Nunc). At the end of the exposure, cells were fixed and stored in cold methanol (−20 ◦ C) for at least 30 min before staining. For the micronucleus assay, the cells were washed with PBS/CMF and their nuclei were stained during 4 min with bisbenzimide (Hoechst 33258, concentration: 5 ␮g/ml). The slides were then mounted for fluorescence microscopy and examined for the

119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145

149 150 151 152 153 154 155 156

2.3. Statistical Analysis

173

Student’s t-test (2-tailed) was used for comparison of the results of the micronucleus assay and the kinetochore analysis with the untreated control in each set of experiments.

174

3. Results

178

Both types of asbestos fibers (crocidolite and chrysotile) induced a concentration- and time-dependent increase of micronucleus formation in HMC. Crocidolite asbestos (concentration: 0.5–1.0 ␮g/cm2 ; 72 h) induced a maximum of 33 MN/1000 cells against control (14 MN/1000 cells) (P < 0.001, Fig. 1A), whereas chrysotile asbestos showed stronger effects and induced up to 41 MN/1000 cells in HMC after exposure at 1 ␮g/cm2 for 72 h (P < 0.001, Fig. 1B). At higher fiber concentrations (>5 ␮g/cm2 ), the number of MN decreased as a result of increased cytotoxicity. No increase in micronucleus formation was observed after treatment of HMC with CaSO4 (negative control, Table 1).

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148

114

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2.2. Micronucleus assay and kinetochore analysis

113

presence of micronuclei. Each data point represents the mean of three treated cultures from three independent experiments with 2000 nuclei evaluated in each case. For further analysis of the induced micronuclei (after treatment of cells with crocidolite or chrysotile), kinetochores were stained by incubating the fixed cell preparations with CREST antibodies (Chemicon, Temecula, CA, USA) for 1 h in a humidified chamber at 37 ◦ C. After rinsing with phosphate-buffered saline (PBS) containing 0.5% Tween 20 (Sigma, Germany), the cells were incubated with FITC-conjugated fluorescein isothiocyanate anti-human IgG (Antibodies Incorporated, Davis, USA) for 30 min before applying bisbenzimide. At least 200 micronuclei were examined for the presence of kinetochores in each case.

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HMC of the first two passages were used for the experiments. Fibers were sterilized at 120 ◦ C for 2 h and suspended in PBS (1 ␮g/␮l). Cells were treated with fibers (0.5, 1.0, 5.0 and 10.0 ␮g/cm2 ) in dependence of the size of culture flasks for different time periods (24, 48, 66, and 72 h). Deferoxamine (DEF) (Sigma, Germany) is an iron chelator as well as a radical scavenger. In the present study, DEF was used to modify the fibers in a previous treatment. DEF was dissolved in double-distilled H2 O at concentrations of 1, 5 and 10 mM under sterile conditions. Subsequently, crocidolite and chrysotile fibers were incubated in these solutions for 24 h at room temperature, then centrifuged and re-suspended in PBS/CMF (phosphate buffered saline/calcium- and magnesium-free). Phytic acid (PA, Sigma Germany) was also dissolved in double-distilled H2 O and the asbestos fibers were incubated with the PA-solution (5 mM) for 24 h at room temperature in the dark and re-suspended in PBS/CMF. NacystelynTM was kindly provided by Dr. M. Coffiner, SMB Laboratories, Brussels, Belgium. NAL was freshly dissolved in double-distilled H2 O prior to every application, and added to the culture media at a final concentration of 0.2 mM. Likewise, superoxide dismutase (PEG-SOD, Sigma, Germany) and dimethylthiourea (DMTU, Sigma, Germany) were dissolved in double-distilled H2 O and added to the culture media at final concentrations of 140 Units/ml and 20 mM, respectively. The treatment with these agents was done simultaneously with the fiber treatment. Commercial calcium sulfate (CaSO4 ) was used as negative control. HMC were exposed to 1 and 5 ␮g/ cm2 CaSO4 , respectively, for 24, 48 and 72 h.

111

3

158 159 160 161 162 163 164 165 166 167 168 169 170 171 172

175 176 177

179 180 181 182 183 184 185 186 187 188 189 190 191 192

3.1. Metal chelators deferoxamine and phytic acid (PA)

193

Pretreatment of crocidolite fibers with different concentrations of DEF (1, 5 and 10 mM) significantly decreased the MN induction (Fig. 2), whereas deferox-

195

MUTGEN 400754 1–9

194

196 197

***

10

Fiber concentration [µg/cm2 ]

0

CaSO4 (␮g/cm2 ) Control 1.0 5.0

Treatment duration (h) 24

48

72

14.25 ± 1.48 15.80 ± 2.48 17.80 ± 1.72

14.25 ± 1.09 15.20 ± 2.04 14.40 ± 2.65

13.00 ± 2.16 16.00 ± 3.03 13.75 ± 2.38

(B)

- PA + PA (5mM)

**

*

30 20 10 0

5.0

Table 1 Occurrence of micronuclei (given in MN/1000 cells; mean ± S.D.) in HMC after treatment with calcium sulfate (CaSO4 , negative control)

2

Fiber concentration [µg/cm ]

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Number of micronuclei/1000 cells

204

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202

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201

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200

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199

amine had no significant influence on micronucleus induction by chrysotile fibers (Fig. 2). The micronucleus frequency was reduced to control levels in all cases in HMC exposed to PA-pretreated (5 mM) crocidolite (1–5 ␮g/cm2 , except 0.5 ␮g/cm2 ) for 48 h (Fig. 3A). This reduction was considerably smaller after an exposure time of 72 h, and no longer observed at a concentration of 5 ␮g/cm2 at

**

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Fig. 1. Induction of micronuclei in human mesothelial cells after exposure to different concentrations of (A) crocidolite asbestos and (B) chrysotile asbestos (exposure time: 24–72 h).

+ PA (5mM) 40

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- PA

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2.0

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72h

Fig. 2. Induction of micronuclei by crocidolite and chrysotile fibers (1 ␮g/cm2 for 48 h) (pretreated for 24 h with different concentrations of deferoxamine before exposure) in human mesothelial cells. ∗∗ P < 0.01, ∗∗∗ P < 0.001 (compared with non-pretreated fibers).

0.5

48h 30

Concentration of deferoxamine [mM ]

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40

***

20

10

5.0

2.0

1.0


0.25

0

0.5

10

Chrysotile

no tre preatm en t

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30

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72h

Crocidolite

**

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30

40


24h 48h


(B)

40

0.25


(A)




4

2


Fig. 3. Induction of micronuclei by crocidolite fibers (0.5– 5.0 ␮g/cm2 ) (pretreated for 24 h with 5 mM phytic acid before exposure) in human mesothelial cells: (A) after 48 h exposure and (B) after 72 h exposure. ∗ P < 0.05, ∗∗ P < 0.01; ∗∗∗ P < 0.001 (comparison of −PA and +PA).

MUTGEN 400754 1–9


- PA

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30

******

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as on bes ly tos

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Fig. 5. Effect of the O2 − -scavenging enzyme superoxide dismutase (SOD, 140 U/ml) on micronucleus induction caused by crocidolite and chrysotile asbestos fibers (1 ␮g/cm2 ) after 48 h exposure. ∗∗∗ P < 0.001 (compared with asbestos-treated cells).

20

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50

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the MN formation induced by both crocidolite and chrysotile asbestos (1 ␮g/cm2 each, 24 and 48 h) almost to control levels (Fig. 5). After 72 h no significant reduction in the MN induction was observed (data not shown). Since O2 − can be converted to OH• in the presence of iron by a modified Haber–Weiss reaction, we also monitored the effect of dimethylthiourea, a scavenger of OH• , on MN induction by asbestos. Simultaneous application of DMTU (20 mM) with both crocidolite and chrysotile asbestos fibers (1 ␮g/cm2 each), markedly decreased the MN induction to the level of the spontaneous rate (untreated control cells). DMTU treatment alone did not cause an increase in MN formation (Fig. 6). We also used dimethyl sulfoxide (DMSO) as OH• scavenger in final concentrations of 1 and 0.05% in the culture medium, to confirm the results with DMTU. With both DMSO concentrations the genotoxic effects of chrysotile and crocidolite, respectively, were significantly reduced to the level of the untreated control (data not shown). The simultaneous treatment of primary HMC with NAL (0.2 mM) and crocidolite fibers (1 ␮g/cm2 , exposure times 48 and 72 h) resulted in a significant reduction (P ≤ 0.001) of MN frequency almost down to the control level after 48 h (Fig. 7). In contrast to the results with the metal chelators DEF and PA, NAL treatment resulted in a significant reduction of MN formation in cells exposed to chrysotile (1 ␮g/cm2 ) also

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(B)

crocidolite chrysotile

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50

+ PA (5mM)

5.0

Fig. 4. Induction of micronuclei by chrysotile fibers (0.5–5.0 ␮g/cm2 ) (pretreated for 24 h with 5 mM phytic acid before exposure) in human mesothelial cells: (A) after 48 h exposure and (B) after 72 h exposure. ∗ P < 0.05, ∗∗ P < 0.01 (comparison of −PA and +PA).

RR

219

SOD (140 U/ml), an enzyme that scavenges O2 − , was effective in significantly (P < 0.001) reducing

CO

218

2.0

217

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216

3.2. Radical scavengers superoxide dismutase, dimethylthiourea and the glutathione precursor NacystelynTM

UN

215

1.0

213 214

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210


209


208

72 h (Fig. 3B). In comparison, DEF-pretreatment of crocidolite was not as effective. The results after exposure of HMC to PA-pretreated chrysotile fibers were different again. After 48 h exposure to PA-pretreated chrysotile fibers (0.5–5 ␮g/cm2 ), just a weak decrease in micronucleus induction was detected (Fig. 4A), which became more prominent after 72 h exposure particularly at 1 ␮g/cm2 concentration of fiber (P < 0.01, Fig. 4B).


207


206

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220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248


50

Table 2 Kinetochore analysis with CREST-staining after exposure of the cells to asbestos fibers (1 ␮g/cm2 ) and the radical scavengers DMTU (20 mM), SOD (140 U/ml) and NAL (0.2 mM) for 24 h


40

No. of MN scored

*** ***

30

400

61.5 ± 6.1

10

Control + DMTU Crocidolite + DMTU Chrysotile + DMTU

400 400 400

63.5 ± 6.4 63.5 ± 6.4 67.0 ± 6.7

Control + SOD Crocidolite + SOD Chrysotile + SOD

400 400 400

69.5 ± 7.3 69.0 ± 4.5 78.0 ± 7.1

Control + NAL Crocidolite + NAL Chrysotile + NAL

400 400 400

64.0 ± 5.4 72.5 ± 6.5 77.0 ± 6.1

Crocidolite Chrysotile

400 400

71.0 ± 7.0 77.5 ± 7.3

50


30

**

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The present study investigated the effects of the metal chelators deferoxamine and phytic acid, the free-radical scavengers superoxide dismutase and dimethylthiourea and the glutathione precursor NacystelynTM on the genotoxicity of asbestos fibers in primary human mesothelial cells, the target cells of asbestos-induced mesothelioma. Both types of asbestos fibers induced a timeand concentration-dependent increase in micronucleus (MN) formation in HMC, with increased cytotoxic effects at fiber concentrations >2 ␮g/cm2 . Similar results were obtained with HMC by Lohani et al. [19]. Phagocytosis plays an important role in asbestos-induced damage of mesothelial cells. HMC are able to phagocytose 94% of fibers within 6 h in contrast to rat liver epithelial cells with 12% [20]. During phagocytosis of fibers, an increased production of free radicals (“oxidative bursts”) can be observed [2]. Also, chrysotile fibers are phagocytosed faster that crocidolite fibers by rat epithelial cells in vitro [21]. The protective effect of the metal chelators PA and DEF observed in the present study may be not only due to their ability to occupy all the coordination sites of iron, thereby preventing redox activation necessary to catalyze OH• formation by the Fenton reaction [22], but also due to depleting the fibers of iron. Kamp

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4. Discussion

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20 10 0

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Fig. 7. The effect of NAL (0.2 mM) on micronucleus induction caused by crocidolite and chrysotile asbestos fibers (1 ␮g/cm2 ) after 48 h exposure. ∗∗ P < 0.01, ∗∗∗ P < 0.001 (compared with asbestos-treated cells).

RR

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co & ntrol NA L

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251 252


250

at 48 h (Fig. 7). No protective effect was observed after 72 h (data not shown). Kinetochore analysis in all groups mentioned above revealed that treatment of human mesothelial cells with asbestos causes formation of micronuclei that mainly contain chromosome fragments (70–74% kinetochore-negative MN [K− ], Table 2). Simultaneous treatment with DMTU, deferoxamine, phytic acid and the other radical scavengers reduced the mean K− rate to values near the control level (data not shown).

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Control

Fig. 6. Effect of the OH• -scavenging dimethylthiourea (DMTU, 20 mM) on micronucleus induction caused by crocidolite and chrysotile asbestos fibers (1 ␮g/cm2 ) after 48 h exposure. ∗∗∗ P < 0.001 (compared with asbestos-treated cells).

249

Mean K− (%)

20

un co trea ntr ted ol


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261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286


Control

500

53.9 ± 4.3

Crocidolite + DEF Chrysotile + DEF

200 200

59.5 ± 6.4 62.5 ± 6.6

Crocidolite + PA Chrysotile + PA

200 200

52.5 ± 4.4 54.0 ± 5.8

Crocidolite Chrysotile

400 400

69.0 ± 6.0 73.5 ± 6.8

a

The fibers were pretreated with the metal chelators DEF (10 mM) and PA (5 mM) for 24 h. The cells were exposed to the pretreated fibers for 48 h.

292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318

TE D

291

EC

290

RR

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288

et al. [12] have shown that phytic acid attenuates the inflammatory and fibrotic effects of asbestos in vivo. We noticed that PA and DEF were able to reduce cytogenetic damage in human mesothelial cells. This reducing effect was dependent on the type of fiber and probably on its iron content. Certain fibers, such as crocidolite, are also capable of binding iron from intracellular sources, which could be as reactive as the intrinsic iron and may be responsible for the increased reactive lifetime of the fiber [14] (Table 3). Recent experiments have shown that PA reduces the iron uptake into cells in vitro [23] and thus protects against oxidative DNA damage [24]. Midorikawa et al. [24] also reported that PA inhibited oxidative DNA damage induced by H2 O2 and copper [Cu(II)], through chelation of copper. Therefore, PA is regarded as metal chelator (iron and copper). Unlike other chelators, PA itself is not genotoxic [25]. The observed effects in HMC after exposure to PA-pretreated crocidolite were time-dependent (exposure time: 48, 72 h) with reduced effects after 72 h exposure. It might be that PA-concentrations higher than 5 mM are necessary for a significant reduction (P < 0.001) of the genotoxic effects of crocidolite in HMC after 72 h exposure. In contrast, a prolonged PA-pretreatment of chrysotile fibers (72 h) reduced the cytogenetic damage significantly. Oxidative DNA damage induced by asbestos and its reduction with metal chelators have been reported previously [11–15]. Faux et al. [26] showed that treatment of isolated DNA with crocidolite significantly increases the concentration of 8-hydroxy-

UN

287

F

Mean K− (%)

OO

No. of MN scored

deoxyguanosine (8-OHdG). It has been demonstrated that the OH• radicals formed during this treatment are able to produce 8-OHdG in DNA, both in cell-free systems and after incubation of mammalian cells with asbestos fibers. In contrast to these findings, Burmeister et al. [27] showed that oxidative DNA damage recognized by the Fpg protein is not detectable up to 24 h after exposure to asbestos in human mesothelial cells. The authors conclude that other radicals than those forming 8-oxo-guanine or 8-oxo-adenine are responsible for the observed early DNA damage. Kamp et al. [11] have reported the role of iron in chrysotile- and amosite-induced DNA strand breaks in A549 and WI-26 cells, showing that iron-catalyzed free radicals mediate asbestos-induced pulmonary toxicity. Fung et al. [28] did not show any protective effect of DEF on the formation of 8-OHdG induced by crocidolite asbestos in rat and human mesothelial cells after 24–72 h exposure. They suggested that the formation of 8-OHdG may not have been caused by iron. The free-radical scavengers SOD and DMTU both caused a significant decrease in MN induction by both asbestos fiber types, further consolidating the role of free radicals in asbestos-induced genotoxicity. SOD was not as effective as DMTU; the reason could be the larger molecular size of SOD, which may have hindered its entrance into the cells. During phagocytosis of the fibers the SOD molecules could have entered the cells. In contrast, DMTU was very effective as it scavenges OH• radicals formed as a result of the conversion of O2 − in the presence of iron by a modified Haber–Weiss reaction [10]. In the present experiments, a partial inhibition of MN formation under the influence of NacystelynTM was noticed. The effect was more pronounced with crocidolite than with chrysotile. NAL, a recently synthesized salt of N-acetylcysteine (a precursor of glutathione), has been reported to enhance cellular glutathione levels and inhibit the formation of H2 O2 in vitro [29]. Glutathione (GSH) is an intracellular thiol present in all tissues, which protects the cells against oxidative stress induced by toxicants [30]. Howden and Faux [31] have reported a protective effect of GSH against the mutagenic effects of mineral fibers in Salmonella typhimurium, whereas Kamp et al. [11] could not show protection against asbestos-induced DNA-single strand breaks applying lyposomal GSH and N-acetyl-l-cysteine (NAC).

PR

Table 3 Kinetochore analysis with CREST-staining after exposure of HMC to pretreated asbestos fibers (1 ␮g/cm2 )a

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MUTGEN 400754 1–9

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374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414

Acknowledgements

419

We thank Prof. J. Emmerich, Prof. M. Barten, Dr. Zendeh and Dr. Heller (University Hospital, Rostock) for providing ascites fluid. We also thank Dr. M. Coffiner, SMB Laboratories, Brussels, Belgium, for providing NAL. This work was supported by the Indo-German CSIR-DLR cooperation program and the Landesforschungsföderungsprogramm of Mecklenburg/Vorpommern (HSP III).

420

References

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[1] T.R. Quinlan, J.P. Marsh, Y.M. Janssen, P.A. Borm, B.T. Mossman, Oxygen radicals and asbestos-mediated disease, Environ. Health Perspect. 102 (1994) 107–110. [2] D.W. Kamp, S.A. Weitzman, The molecular basis of asbestos induced lung injury, Thorax 57 (1999) 638–652. [3] C.B. Manning, V. Vallyathan, B.T. Mossman, Diseases caused by asbestos: mechanisms of injury and disease development, Int. Immunopharmacol. 2 (2002) 191–200. [4] A. Hirvonen, J. Tuimala, K. Ollikainmaa, V. Kinnula, Manganese superoxide dismutase genotypes and asbestosassociated pulmonary disorders, Cancer Lett. 178 (2002) 71– 74. [5] D.W. Kamp, P. Graceffa, W.A. Pryor, S.A. Weitzman, The role of free radicals in asbestos-induced diseases, Free Radic. Biol. Med. 12 (1992) 293–315. [6] N. Mahmood, S.G. Khan, M. Athar, Q. Rahman, Differential role of hydrogen peroxide and organic peroxides in augmenting asbestos-mediated DNA damage: implications for asbestos induced carcinogenesis, Biochem. Biophys. Res. Commun. 200 (1994) 687–694. [7] N. Mahmood, S.G. Khan, S. Ali, M. Athar, Q. Rahman, Asbestos induced oxidative injury to DNA, Ann. Occup. Hyg. 37 (1993) 315–319. [8] M.C. Jaurand, Mechanisms of fiber-induced genotoxicity, Environ. Health Perspect. 105 (1997) 1073–1084. [9] S. Zhu, M. Manuel, S. Tanaka, N. Choe, E. Kagan, S. Matalon, Contribution of reactive oxygen and nitrogen species to particulate-induced lung injury, Environ. Health Perspect. 106 (1998) 1157–1163. [10] J.M. McCord, K. Wong, Phagocyte-produced free radicals: roles in cytotoxicity and inflammation, in: I. Fridovich (Ed.), Oxygen Free Radicals and Tissue Damage, Excerpta Medica, Amsterdam, 1979, pp. 343–367.

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fiber. The iron content of the fiber certainly plays an important role in this context, but does not appear to be the only factor contributing to asbestos-mediated ROS formation.

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The present findings clearly indicate a protective effect by both metal chelators and free-radical scavengers against asbestos-induced genetic damage. McBride et al. [32] have shown that iron-treated DNA shows 20–80-fold more mutations than untreated DNA in the M13mp2 phage-DNA mutation assay. They proposed that Fe2+ /oxygen-induced DNA damage is non-random, having observed a clustering of mutations at specific gene loci. Further, similar to our findings these authors also noticed ameliorating effects of catalase and SOD. Hei et al. [33,34] reported that in the AL human–hamster hybrid cell system, the asbestos-induced mutation spectrum indicates large deletions. They have also demonstrated protective effects of antioxidants. Our results of kinetochore analyses are in agreement with the results of Olofsson and Mark [35], indicating chromosome breakage in HMC treated with asbestos fibers. Pelin et al. [20] also found structural damage to HMC chromosomes treated with amosite. In the SHE- and AFC-model systems, the percentage of K+ -micronuclei increased with fiber exposure [36,37], whereas the percentage decreased in HMC, as shown in the present study (increase of K− -MN). It may be hypothesized that in SHE and AFC cultures, more aneugenic events are going on, whereas mainly clastogenic events are responsible for asbestos-induced MN formation in HMC. Treatment with metal chelators and antioxidants resulted in a decrease in the percentage of K− -MN to the normal level, reflecting the relation between clastogenic action of asbestos fibers in HMC and the role of ROS in this phenomenon. The occurrence of K+ -micronuclei in about 30% of the cases suggests that chromosome malsegregation could also account. This mechanism has been suggested by Jaurand and others [8,18,38]. Altogether, we have shown in our present study which radicals are involved in asbestos-mediated genotoxicity and to which extent. Mainly clastogenic events are responsible for asbestos-induced micronucleus formation. The metal chelators, deferoxamine and phytic acid are able to reduce these effects probably in dependence upon the iron content of the different types of fiber. Free-radical scavengers almost completely suppressed the asbestos-induced genotoxic effects, independent of the chemical composition of the fibers. It is likely that different mechanisms for production of radicals exist, depending on the type of

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[25] P. Whittaker, H.E. Seifried, R.H. San, J.J. Clarke, V.C. Dunkel, Genotoxicity of iron chelators in L5178Y mouse lymphoma cells, Environ. Mol. Mutagen. 38 (2001) 347– 356. [26] S.P. Faux, P.J. Howden, L.S. Levy, Iron-dependent formation of 8-hydroxydeoxy-guanosine in isolated DNA and mutagenicity in Salmonella typhimurium TA102 induced by crocidolite, Carcinogenesis 15 (1994) 1749–1751. [27] B. Burmeister, T. Schwerdtle, I. Poser, A. Hartwig, W.U. Müller, A.W. Rettenmeier, N.H. Seemayer, E. Dopp, Effects of asbestos on initiation of DNA damage, induction of DNA-strand breaks, P53-expression and apoptosis in primary, SV40-transformed and malignant human mesothelial cells, Mutat. Res., in press. [28] H. Fung, Y.W. Kow, B. VanHouten, B.T. Mossman, Patterns of 8-hydroxydeoxy-guanosine formation in DNA and indications of oxidative stress in rat and human pleural mesothelial cells after exposure to crocidolite asbestos, Carcinogenesis 18 (1997) 825–832. [29] A. Gillissen, M. Jaworska, M. Orth, M. Coffiner, P. Maes, E.M. App, A.M. Cantin, G. Schultze-Werninghaus, Nacystelyn, a novel lysine salt of N-acetylcysteine, to augment cellular antioxidant defence in vitro, Respir. Med. 91 (1997) 159–168. [30] A.M. Cantin, R. Begin, Glutathione and inflammatory disorders of the lung, Lung 169 (1975) 123–138. [31] P.J. Howden, S.P. Faux, Glutathione modulates the formation of 8-hydroxydeoxy-guanosine in isolated DNA and mutagenicity in Salmonella typhimurium TA100 induced by mineral fibers, Carcinogenesis 17 (1996) 2275–2277. [32] T.J. McBride, B.D. Preston, L.A. Loeb, Mutagenic spectrum resulting from DNA damage by oxygen radicals, Biochemistry 30 (1991) 207–213. [33] T.K. Hei, C.Q. Piao, Z.Y. He, D. Vannais, C.A. Waldren, Chrysotile fiber is a strong mutagen in mammalian cells, Cancer Res. 52 (1992) 6305–6309. [34] T.K. Hei, Z.Y. He, K. Suzuki, Effects of antioxidants on fiber mutagenesis, Carcinogenesis 16 (1995) 1573–1578. [35] K. Olofsson, J. Mark, Specificity of asbestos-induced chromosomal aberrations in short-term cultures human mesothelial cells, Cancer Genet. Cytogenet. 41 (1989) 33–39. [36] E. Dopp, J. Saedler, H. Stopper, D.G. Weiss, D. Schiffmann, Mitotic disturbances and micronucleus induction in Syrian hamster embryo fibroblast cells caused by asbestos fibers, Environ. Health Perspect. 103 (1995) 268–271. [37] E. Dopp, D. Schiffmann, Analysis of chromosomal alterations induced by asbestos and ceramic fibers, Toxicol. Lett. 96 (97) (1998) 155–162. [38] M. Yegles, L. Saint-Etienne, A. Renier, X. Janson, M.C. Jaurand, Induction of metaphase and anaphase/telophase abnormalities by asbestos fibers in rat pleural mesothelial cells in vitro, Am. J. Respir. Cell Mol. Biol. 2 (1993) 186– 191.

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[11] D.W. Kamp, V.A. Israbian, S.E. Preusen, C.X. Zhang, S.A. Weitzman, Asbestos causes DNA breaks in cultured pulmonary epithelial cells: role of iron-catalyzed free radicals, Am. J. Physiol. 268 (1995) 471–480. [12] D.W. Kamp, V.A. Israbian, A.V. Yeldandi, R.J. Panos, P. Graceffa, S.A. Weitzman, Phytic acid, an iron chelator, attenuates pulmonary inflammation and fibrosis in rats after intratracheal instillation of asbestos, Toxicol. Pathol. 23 (1995) 689–695. [13] B. Fubini, L. Mollo, E. Giamello, Free radical generation at the solid/liquid interface in iron containing minerals, Free Radic. Res. 23 (1995) 593–614. [14] J.A. Hardy, A.E. Aust, The effect of iron binding on the ability of crocidolite asbestos to catalyze DNA single-strand breaks, Carcinogenesis 16 (1995) 319–325. [15] Q. Rahman, N. Mahmood, S.G. Khan, M. Athar, Mechanism of asbestos-mediated DNA damage; role of heme and heme proteins, Environ. Health Perspect. 105 (1997) 1109–1112. [16] B.T. Mossman, J.P. Marsh, A. Sesko, S. Hill, M.A. Shatos, J. Doherty, J. Petruska, K.B. Adler, D. Hemenway, P. Mickey, Inhibition of lung injury, inflammation, and interstitial pulmonary fibrosis by polyethylene glycol-conjugated catalase in a rapid inhalation model of asbestosis, Am. Rev. Respir. Dis. 141 (1990) 1266–1271. [17] B.T. Mossman, Y.M. Janssen, J.P. Marsh, A. Sesko, M.A. Shatos, J. Doherty, K.B. Adler, D. Hemenway, R. Mickey, P. Vacek, Development and characterization of a rapid-onset rodent inhalation model of asbestosis for disease prevention, Toxicol. Pathol. 19 (1991) 412–418. [18] Q. Rahman, E. Dopp, D. Schiffmann, Genotoxic effects of asbestos fibers, in: G.A. Peters, B.J. Peters (Eds.), Sourcebook on Asbestos Diseases, vol. 21, LEXIS Publishing, Charlottesville, USA, 2000, pp. 223–242. [19] M. Lohani, S. Yadav, D. Schiffmann, Q. Rahman, Diallylsulfide attenuates asbestos-induced genotoxicity, Toxicol. Lett. 143 (2003) 45–50. [20] K. Pelin, P. Kivipensas, K. Linnainmaa, Effects of asbestos and man-made vitreous fibers on cell division in cultured human mesothelial cells in comparison to rodent cells, Environ. Mol. Mutagen. 25 (1995) 118–125. [21] T.W. Hesterberg, D.G. Ririe, J.C. Barrett, P. Nettesheim, Mechanisms of cytotoxicity of asbestos fibres in rat tracheal epithelial cells in cultures, Toxicol. In Vitro 1 (1987) 59–65. [22] E. Graf, J.W. Eaton, Antioxidant functions of phytic acid, Free Radic. Biol. Med. 8 (1990) 61–69. [23] R.P. Glahn, G.M. Wortly, P.K. South, D.D. Miller, Inhibition of iron uptake by phytic acid, tannic acid, and ZnCl (2): studies using an in vitro digestion/Caco-2 cell model, J. Agric. Food Chem. 50 (2002) 390–395. [24] K. Midorikawa, M. Murata, S. Oikawa, Y. Hiraku, S. Kawanishi, Protective effect of phytic acid on DNA damage with reference to cancer chemoprevention, Biochem. Biophys. Res. Commun. 288 (2001) 552–557.

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