Asbestos-induced decomposition of hydrogen peroxide

ENVIRONMENTAL

RESEARCH

Asbestos-Induced MANFRED

37, 187-292 (198.0

Decomposition

of Hydrogen

Peroxide

K. EBERHARDT,

ANGEL A. ROMAN-FRANCO, AND MARGARITA R. QUILES

Puerto Rico Cuncev Center. Universify of Puerto Rico-Medicctl Sun Juan. Puerto Rico 00936

Sciences Cutnptts.

Received August 31, 1982 Decomposition of H,Oz by chrysotile asbestos was demonstrated employing titration with KMnO,. The participation of OH radicals in this process was delineated employing the OH radical scavenger dimethyl sulfoxide (DMSO). A mechanism involving the Fenton and Haber-Weiss reactions as the pathway for the H?Oz decomposition and OH radical produc& 1985 Academic Pre?\. Inc. tion is postulated.

INTRODUCTION

Asbestos fibers of selected sizes are known to be cytotoxic and cocarcinogenic (1). Such fibers, when they reach the deep respiratory spaces, excite phagocytic activity. Because of the size of the fibers, as well as because of their cytotoxicity, failed phagocytosis is known to occur, causing the release of intracellular enzymes of affected phagocytes (2). It has been also demonstrated that during the phagocytosis of asbestos there is enhanced chemiluminescence and reactive oxygen products are generated (3). Several mechanisms have been postulated by which asbestos acts as a cocarcinogen. Some authors have postulated that asbestos is directly mutagenic to cells (4). Other authors have suggested that asbestos concentrates on its surface precarcinogens, and in this manner expedites their entry into cells and their bioactivation into ultimate carcinogens both via the action of microsomal monooxygenases (5). We have recently presented evidence supporting a mechanism of biotransformation via the nonenzymatic interaction between precarcinogenic polynuclear aromatic hydrocarbons and reactive oxygen released from macrophages during the failed phagocytosis of asbestos (6). Asbestos fibers, particularly chrysotile, contain varying concentrations of contaminating metalic ions (7). The phagocytic event is accompanied by the production and eventual release of H,Oz into the cell’s microenvironment. Metals, such as Fe?+ cause the decomposition of H202 by a Fenton-type reaction. The result is the production of OH radicals that then become available to attack a variety of biologically important substrates (8). The present experiments were conducted to analyze whether asbestos can lead to the production of OH radicals from HzO, as a putative first step to the nonenzymatic biotransformation of precarcinogenic PAH’ (9). ’ Abbreviations asbestos.

used: PAH, polynuclear aromatic hydrocarbon(s);

DMSO. dimethylsulfoxide;

A.

387 OOl3-9351185 $3.00 Copynght All rights

‘0 1985 by Academic Press. Inc. of reproduction in any form reserved

EBERHARDT,

288

ROMAN-FRANCO,

MATERIALS

AND QUILES

AND METHODS

Asbestos-induced decompositiorz of H202 in aqueous solutions. Fibers of chrysotile asbestos (Borinquen Asbestos Corp., San Juan, Puerto Rico) ranging from 5 to 100 pm were prepared according to the method of Badollet and Gantt (IO). Table 1 lists the concentrations of asbestos, H,O,, and the time periods during which the reactants were allowed to interact. Changes in the concentration of TABLE EFFECT

OF CONCENTRATION

AND

HYDROGEN

SHAKING PEROXIDE

IN AQUEOUS

Conditions” Asbestos (mg) 10 10 10 10

1

ON THE ASBESTOS-INDUCED

time (hr)

Still

0.42 0.42 0.42 0.42

2 4 6 24

5 13 17 67

10 10 10 10

4.2 4.2 4.2 4.2

x x x x

10-l lo-? lo-? lo-?

2 4 6 24

13 27 41 96

10 10 10 10

4.2 4.2 4.2 4.2

x x x x

1O-3 1O-3 10m3 10m3

2 4 6 24

20 41 48 85

20 20 20 20

0.42 0.42 0.42 0.42

2 4 6 24

13 25 37 65

25 25 25 25 25

0.42 0.42 0.42 0.42 0.42

2 4 6 24 48

3 25 37 76 99

x x x x

2 4 6 24

42 62 78 98

2 4

76’ 9oc

4.2 4.2 4.2 4.2

lo-? lo-? lo-? lo-?

25 25

4.2 x 1O-3 4.2 x 1O-3

OF

HzOz decomposition (%)b

HzOz LW

25 25 25 25

DECOMPOSITION

SOLUTIONS

With shaking’ 41

75

67

68

71 85

a All reactions were carried out in a 5-ml screw cap reaction vial with a Teflon-coated septum. To the asbestos was added 2 ml water and 100 ~1 H,O, 130, 3, or 0.3%). The reaction vessels were left standing in the dark. All experiments were carried out in duplicate and triplicate. All the asbestos fibers were washed thoroughly with distilled water prior to drying and sieving. b Without asbestos no H,O, decomposition was detectable in any of the above solutions. c These results were obtained if the reaction vials were shaken to achieve better mixing. This leads to a faster rate of decomposition.

ASBESTOS-INDUCED

DECOMPOSITION

OF HZOZ

289

H,O, were determined by titration with 0. I, 0.01, and 0.001 N KMnO, solutions (12). Asbestos-induced decomposition of H,O, in dimethylsulfoxide (DMSO). Asbestos fibers, H202 and dimethylsulfoxide were allowed to react under the conditions listed in Table 3. For assaying for aOH radicals the method of Repine et al. was employed (11). This method is based upon the reaction of .OH with DMSO to yield methane. For the methane analysis, l-ml samples were withdrawn from the reaction vials with a gas-tight syringe. The samples were analyzed using a 6ft Carbosieve B Column (Supelco Inc.) at 12O”C, a He flow of 25 ml/min and a hydrogen flame detector. Standard methane samples (Supelco, Inc.) were analyzed for comparison. RESULTS AND DISCUSSION

The results are summarized in Tables 1 and 2. From Table 1 we can see that asbestos fibers cause a rapid decomposition of H,O,. There seems to be a short induction period (ca. 2 hr), but after 24 hr a high percentage of the H,O, has been decomposed, while in absence of asbestos under otherwise identical conditions no significant decomposition could be observed. We propose the following chain mechanism:

TABLE EFFECT

OF DIFFERENT

PRETREATMENT

OF ASBESTOS

2 FIBERS

ON THE RATE

OF

H,O,

DECOMPOSITION

Conditions” Asbestos’

(mg) Asbestos A 10 10 10 10 Asbestos B IO 10 10 10 Asbestos C IO 10 IO 10

H?O,

CM)

Time (hr)

0.42 0.42 0.42 0.42

2 4 6 24

5 19 22 71

0.42 0.42 0.42 0.42

2 4 6 24

5 26 33 80

0.42 0.42 0.42 0.42

3 4 6 24

22 42 94

HzOz decomposition

U All reactions were carried out in a 5-ml screw cap reaction vial with a Teflon-coated septum. To the asbestos was added 2 ml water and 100 PI H,O, _ _ (30, 3, or 0.3%). The reaction vessels were left standing in the dark. All experiments were carried out in duplicate and triplicate. All the asbestos fibers were washed thoroughly with distilled water prior to drying and sieving. h Asbestos A. unwashed fibers; asbestos B, fibers washed thoroughly with double-distilled water before drying and sieving: asbestos C, fibers washed with ethanol and acetone before drying and sieving.

290

EBERHARDT,

ROMAN-FRANCO,

AND

A + H,O,+A+ + OH- + *OH OH + H202 + H,O, + HO,; HO? @ H+ + 0; 02; + H,O, + 0, + OH- + .OH

QUILES

initiation

(1)

propagation

w

(3)

The initiation consists of an electron transfer from asbestos (A) to H,Oz. This probably involves an active metal-containing site on the asbestos surface. The rate of decomposition is certainly dependent on the surface area. This can be deduced from the results (Table 1) of our experiments using a mechanical shaker. In most experiments the asbestos fibers are clumped together at the bottom of the reaction vial or sometimes they float to the top. In the experiments carried out under shaking a considerably greater percentage of H202 decomposition was observed due to the more intimate contact between the fibers and the bulk of the solution. With increasing fiber concentration an increase in decomposition was observed. The rate of H20, decomposition does not vary significantly with differently pretreated fibers, where asbestos fibers unwashed, washed with water, and washed with ethanol and acetone were used. These results make it unlikely that the decomposition is due to surface impurities. In order to show the involvement of OH radicals we have scavenged the OH radicals with DMSO. DMSO has recently been recommended as an OH radical probe (11). It reacts with OH to produce CH, radicals (13) and subsequently CH,, which can easily be identified by gas chromatography. aOH + CH, - SO-CH, -+ CH,. + CH, - SOOH CH3. + CH3-SOOH + CH, + CH, -SOO. Our results with DMSO are shown in Table 3. In presence of asbestos significant amounts of methane are produced whereas in the absence of asbestos the methane yields are much smaller. From Table 3 we can see that even at H,O, concentrations as low as 4.5 mM production of OH radicals still takes place. In presence of DMSO no chain decomposition takes place because the DMSO scavenges the chain propagating OH radicals, thus producing only one CH, for each *OH radical. In the absence of asbestos some HzOz decomposition and some OH radical formation occur, but no chain decomposition takes place. This is probably due to the fact that the chain carrying Haber-Weiss reaction is very slow (14, 15) in the absence of asbestos, but is catalyzed by asbestos according to the following reaction sequence: A(Me+‘“+“) + 01:* A(Me+“) + O2 A(Me+“) + HzO, -+A (Me+‘“+‘)) + OH0,

+ H,O, --+ O2 + OH-

+ .OH

+ -OH.

The catalytic effect of metal ions on the Haber-Weiss reaction is well known (16, 17). The above reactions are analogous to those discussed by McCord and Day (17) with the iron-EDTA complex. It has been proposed that the formation of OH radicals during the phagocytosis

ASBESTOS-INDUCED

DECOMPOSITION TABLE

ASBESTOS-INDUCED

DECOMPOSITION

OF

291

H,O,

- -

3

OF HYDROGEN

PEROXIIIE

IN DIMETHYLSULFOXIDE

Conditions” Asbestos (mg)

H,O, 04)

25 25 25

4.2 4.2 4.2 4.2

0.42 0.42 x 10-l x lo-’ x IO-” x 10-j

Methane yield” (pmol) 32,000 3,000 7.100 I .600 3.000 750

0 All reactions were carried out in 5-ml screw cap reaction vials with Teflon-lined septums through which the gas could be withdrawn for analysis. To the asbestos was added 2 ml of dimethylsulfoxide and 100 pJ HzO, (30, 3. or 0.3%). The reaction vessels were left standing in the dark at room temperature for 10 days. The results were reproducible within ? 10%. b The methane content of air, which was ca. 100 pmoliml was substracted.

of asbestos may play an important part in the activation of PAH which may be adsorbed on the surface of the asbestos fibers or may find themselves in the cells microenvironment (6, 9). It has been shown that PAH coated on asbestos can enter into the cytoplasm of cells (18). Furthermore, since it is well known that OH radicals are the species responsible for radiation-induced cell killing, the formation of OH radicals by asbestos and H202 could be partly responsible for cell damage in asbestos-induced diseases. Asbestos fibers inside or near cells could then be considered analogous to a low-dose radiation source, causing slow (possibly over periods of years) cell damage and eventually cell death. ACKNOWLEDGMENTS This work was supported in part by Grants CA 16598-07. CA 28894-02, and SO-6RR 08102-10 from the National institutes of Health.

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3. 4. 5. 6. 7.

McCarthy D. J.. and Kozin, F. (1975). An overview of cellular and molecular mechanisms in crystal-induced inflammation. Arthritis Ri~errm. (.Supp/.) 18, 757. Gaumer R. H., Cairo, R. M.. and Salvaggio. J. E. (1979). Chemiluminescent response to asbestos fibers. Clin. Res. 27, 37A. Chamberlain. M., and Tarmy, E. M. (1977). Asbestos and glass fibers in bacterial mutation tests. Mutar. Res. 43, 159. Lankowicz. J. R., Englund, F., and Hidmark, A. (1978). Particle-enhanced membrane uptake of a polynuclear aromatic hydrocarbon: A possible role in cocarcinogenesis. J. Ncrtl. Crrnccr Ins/. 61, 1155. RomBn-Franc0 A. A., Carrasco, J.. Garcia, S.. and Valldejulli. D. (1981). Bioactivation of precarcinogens during phagocytosis. In “ASM: Abstracts of the Annual Meeting of the American Society of Microbiology. 1981,” E-36, page 61. Pooley F. D. (1981). Mineralogy of asbestos: The physical and chemical properties of the dusts they form. Serb. Oncd. 8, 243.

292

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AND QUILES

8. Tauber A. I., and Babior, B. M. (1978). O?- and host defense: The production and fate of O?in neutrophils. Photochern. Photobiol. 28, 701. 9. Roman-Franc0 A. A. (1982). Mechanism of action of carcinogenic fibers. J. Theor. Bio/. 97, 543-555. 10. Badollet M. S., and Gantt, W. A. (1965). Preparation of asbestos fibers for experimental use. Ann. N. Y. Acad. Sci. 132, 451. 11. Repine J. E., Eaton, .I. W., Anders, M. W., Hoidal, J. R., and Fox, R. B. (1979). Generation of hydroxyl radical by enzymes, chemicals, and human phagocytes in vitro. J. Clin. Intvsr. 64, 1642. 12. Bergmayer H. U. (1975). “Methods of Enzymatic Analysis,” p. 2247. Academic Press, New York. 13. Gilbert B. C., Norman, R. 0. C., and Scaly. R. C. (1975). Electron spin resonance studies. Part XLIII. Reaction of dimethyl sulfoxide with hydroxyl radicals. J. Chew. Sot. Perhin Trans. 2, 1975, 303. 14. Ferradini, C., Foos, J., Houee, C., and Pucheault, J. (1978). The reaction between superoxide anion and hydrogen peroxide. Photochern. Photobiol. 28, 697-700. 15. Weinstein J., and Bielski, B. H. J. (1979). Kinetics of the interaction of HO: and 02- radicals with hydrogen peroxide. The Haber-Weiss reaction. J. Amer. Chem. So<,. 101, 58-62, and earlier references cited therein. 16. Haber, E. and Weiss, J. (1934). The catalytic decomposition of hydrogen peroxide by iron salts. Proc. R. Sot. London Ser. A 147, 332-351. 17. McCord, J. M.. and Day, E. D. (1978). Superoxide dependent production of hydroxyl radical catalyzed by iron-EDTA complex. FEES Left. 86, 139-142. 18. Mossman, B. T., and Craighead, J. E. (1981). Mechanisms of asbestos carcinogenesis. Environ. Res. 25, 269.