Mutation Research, 265 (1992) 245-253 © 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00
245
MUT 05048
Oxygen radical-mediated mutagenic effect of asbestos on human lymphocytes: suppression by oxygen radical scavengers Ludmila G. Korkina 1, Andrei D. Durnev 2, Tatjana B. Suslova 1, Zinaida P. Cheremisina 1, Natalia O. Daugel-Dauge i and Igor B. Afanas'ev 3 J 2nd Moscow Medical Institute, Ostrovitjanoua 1, 117869, Moscow, 2 Institute of Pharmacology, Baltiyskaja 8, Moscow and 3All-Union Vitamin Research Institute, Nauchny proezd 14A, GSP-7, 117820, Moscow (U.S.S.R.)
(Received 18 December 1990) (Revision received 25 June 1991) (Accepted 27 August 1991)
Keywords: Oxygen radical-mediated, human lymphocytes; Phagocytes; Asbestos; Zeolite; Antioxidants
Summary The mutagenic effect of chrysotile asbestos fibers and zeolite and latex particles on human lymphocytes in whole blood has been studied. It was concluded that their mutagenic activities were mediated by oxygen radicals because they were inhibited by antioxidant enzymes (SOD and catalase) and oxygen radical scavengers (rutin, ascorbic acid, and bemitil). It was proposed that oxygen radicals were released by phagocytes activated upon exposure to mineral dusts and fibers. The study of lucigenin- and luminol-amplified chemiluminescence of peritoneal macrophages stimulated by chrysotile fibers and zeolite and latex particles has shown that their mutagenic action is probably mediated by different oxygen species, namely, by the iron-oxygen complexes (perferryl ions) plus hydrogen peroxide, hydrogen peroxide, and superoxide ion, respectively. From the oxygen radical scavengers studied, rutin was the most effective inhibitor of the mutagenic effect of mineral fibers and dusts.
Mutagenic effects of oxygen radicals have been shown in many studies, which are now summarized in recent reviews (Meneghini, 1988; Imlay and Linn, 1988). One of the most important in vivo generators of oxygen radicals is the phagocyte which, upon activation with various stimuli, releases active oxygen species. Because of this, activated phagocytes acquire mutagenic activity.
Correspondence: I.B. Afanas'ev, All-Union Vitamin Research Institute, Nauchny proezd 14A, GSP-7, 117820, Moscow
(U.S.S.R.).
For example, it has been shown (Weitzman and Stossel, 1981) that normal human leukocytes were mutagenic to Salmonella typhimurium bacteria, while the leukocytes from a patient with chronic granulomatous disease (which are unable to release oxygen radicals) exhibited no mutagenic activity. An important role of oxygen radicals in mutagenic action of phagocytes was confirmed by the inhibitory effects of antioxidant enzymes (SOD and catalase) and free radical scavengers (a-tocopherol, cysteine, etc.) (Weitzman and Stossel, 1982). Similar results were obtained in studies of mutagenic action of phagocytes on
246
mammalian cells (Weitberg and Weitzman, 1985). It has been shown (Velichkovskiy et al., 1982; Evans and Yano, 1985; Case et al., 1986; Urano et al., 1989; Mossman et al., 1990) that mineral dusts and fibers are effective stimuli of the oxidative burst in macrophages and leukocytes. On the other hand, it is known that they exhibit mutagenic effects on mammalian cells (Huang, 1979; Livingston et al., 1980; Palekar et al., 1987), including human cells (Sincock et al., 1982; Casey, 1983; Lechner et al., 1985; Durnev et al., 1990). Therefore, one may propose that the mutagenic action of mineral dusts and fibers on cells is mediated by oxygen radicals released by the phagocytes activated with these stimuli. In this work we studied the mutagenic effects of chrysotile fibers and zeolite and latex particles on human lymphocytes in whole blood. Since lymphocytes are known to be unable to produce oxygen radicals, one can propose that the mutagenic effect is mediated by oxygen radicals produced by blood phagocytes stimulated with mineral fibers and dusts. For identification of the oxygen species released, we studied the lucigeninand luminol-dependent chemiluminescence (CL) in phagocytes of peritoneal exudate activated with these stimuli. (The content of neutrophils in peritoneal exudate is about 80% and close to that in human peripheral blood.) The results of CL experiments and the inhibition of the mutagenic action of mineral fibers and dusts by oxygen radical scavengers and antioxidant enzymes indicate an important role of oxygen radicals in the mutagenic activity of pathogenic mineral dusts. Materials and methods
Chemicals Rutin (3[6-O(6-dezoxy-a-L-mannopyranosil)-/3D-glycopyranosil)oxy]-2-(3,4-dihydroxyphenyl)-5,7dihydroxy-4-H-l-benzopyran) was manufactured by Khimiko-Farm. Zavod (Tashkent, U.S.S.R.), ascorbic acid was from Vitamin Zavod (Belgorod, U.S.S.R.), bemitil (2-ethylthiobenzimidazole) was a product of PO Darniza (Kiev, U.S.S.R.). Enzymes Superoxide dismutase (SOD) and catalase were from Serva (Germany) and horse radish peroxidase was from Reanal (Hungary). Latex was from
Sigma (U.S.A.), phytohemagglutinin P was from Difco (U.S.A.). Mineral dusts were natural zeolite clinoptilolite (Shivirtuy, U.S.S.R.) with 1-3 /zm average diameter of particles and chrysotile asbestos (Tuva, U.S.S.R.) with 5-10 ~zm fiber length. Before experiments, mineral dusts were rinsed with twice distilled water and suspended in 10 mM Tris-HC1 buffer (pH 7.4). For removal of metal ions, chrysotile fibers and zeolite particles were treated with hot 1 N HC1 for 1 h, rinsed with distilled water, and suspended in physiological solution. Removal of iron ions was checked by titration.
Cell culture Venous blood of healthy donors was drawn into heparinized vacutainer tubes and the cultures were established the same day. Whole blood (0.5 ml) was added to the mixture containing 7.5 ml Medium 199, 1.2 ml fetal calf serum, and 0.02 ml phytohemagglutinin P. Blood was cultured at 37 ° C in the 25 ml culture flasks for 56 h. Mineral dusts or fibers (0.01 or 0.05 mg/ml) were added with or without oxygen radical scavengers to cultures after 52 h. 2.5 h before cell fixation, colchicine was added to a final concentration of 0.25 mg/ml. Fixation was performed by standard cytological procedures. The cells were exposed to 0.075 M KC1 solution for 10-12 min and fixed in ethanol-acetic acid solution (3: 1, v/v). Slide preparation and staining procedure followed the routine protocol. Cell isolation Ceils of peritoneal exudate were isolated from Wistar male rats as described earlier (Korkina et al., 1988). Rats were injected with 2 ml 0.1% sterilized pepton solution intraperitoneally. After 12 h, rats were killed and cells were prepared by peritoneal lavage with 10 ml Hanks' balanced salt solution (HBSS). The lavage fluid was then filtered through a nylon cloth. The filtrate was centrifuged at 300 x g for 10 min and washed twice with cold HBSS. Then, the cells were resuspended and stored at 4 ° C in Medium 199. The cells were counted with a Coulter counter, and their viability was assessed by exclusion of 0.2% trypan blue dye. They were Wright-Giemsastained to obtain a differential cell count. Usu-
247 Luminol-dependent
ally, the cell suspension contained 8 5 - 8 8 % viable polymorphonuclear leukocytes, 8 - 1 0 % macrophages, and less than 2% lymphocytes.
Results
Comparison of the CL responses of phagocytes stimulated by latex, zeofite, and chrysotile As seen from Fig. 1 and Table 1, latex, zeolite, and chrysotile induced different CL responses in peritoneal phagocytes; these differences depended on the nature of both the stimuli and the amplifiers. Latex was nearly equally effective in the stimulation of lucigenin- and luminol-dependent CL, while zeolite and chrysotile induced luminol-dependent CL almost exclusively. In all cases, lucigenin- and luminol-dependent CL was inhibited by SOD. Removing iron ions from zeolite particles and chrysotile fibers by treatment with HC1 made them the efficient stimuli of lucigenin-dependent CL (Fig. 1). The addition of H R P sharply enhanced luminol-dependent CL in the case of zeolite stimulation but an increase was not so evident when chrysotile or latex was applied (Table 1). In all cases, rutin and ascorbic
Lucigenin-dependent
CL
5
Chemiluminescence detection of oxygen radicals and hydrogen peroxide released by mineral duststimulated peritoneal phagocytes Superoxide production was measured by the lucigenin-dependent CL (Gyllenhammar, 1987). Lucigenin (0.5 mM) was incubated with a suspension of phagocytes (1.5 x 105 cells/ml) in HBSS (pH 7.4) at 37 ° C upon continuous mixing in the LKB luminometer cell (Model 1251). After 5 min, the suspension of chrysotile fibers (0.5 m g / m l ) , zeolite particles (0.5 m g / m l ) , or latex particles (0.01%) was added and CL was registered continuously. Similarly, the production of oxygen radicals was determined in the presence of luminol (0.5 mM). For detection of hydrogen peroxide, the suspension containing luminol (0.5 mM), phagocytes (1.5 x 105 cells/ml), and mineral dust particles or fibers (0.5 m g / m l ) was incubated in the presence or absence of horse radish peroxidase (HRP, 0.2 /xg/ml). The amount of H 2 0 2 formed was estimated as the difference in the luminol-dependent CL in the presence and absence of H R P (Dahlgren and Lock, 1988).
CL
2
4
to
~3
/i it\.\ \\~/~ //t \.~1 I/ 3
3
~2
~
#
-
i 0
3
i/~ 0.05
< 0.01
4.4
1.2
5.6_+ 1.5
< 0.01
> 0.05
Zeolite, 0.05 mg/ml Zeolite, 0.05 mg/ml + SOD, 50 ~ g / m l Zeolite, 0.05 mg/ml + catalase, 20 ~zg/ml
8.0
0
8.0 _+1.8
< 0.001
6.5
0.5
7.0_+ 1.8
< 0.001
> 0.05
3.0
0.3
2.0 -+0.8
> 0.05
< 0.01
7.8
0.25
6.8 _+1.3
< 0.001
2.2
0
1.9 -+0.7
> 0.05
< 0.05
3.3
0
3.3 _+1.0
> 0.05
< 0.05
Chrysotile, 0.05 mg/ml Chrysotile, 0.05 mg/ml + SOD, 50/.Lg/ml Chrysotile, 0.05 mg/ml + catalase, 20 tzg/ml
250-400 metaphases counted per data point.
Control
Experiment
250
show that, in the case of zeolite stimulation, a large amount of H 2 0 2 was formed. Significantly smaller amounts of hydrogen peroxide were formed with chrysotile fibers and latex particles. In accordance with the CL experiments, the mutagenic effect of latex was mediated only by the superoxide ion because SOD completely inhibited it and catalase had no effect, and the mutagenic effect of zeolite was suppressed by catalase and not SOD, i.e., it was mediated by hydrogen peroxide (Table 4). The mutagenic effect of chrysotile fibers was inhibited by both SOD (nearly completely) and catalase (about 50%). The CL experiments indicate that chrysotile fibers stimulate the release by phagocytes of active oxygen species different but originating from the superoxide ion (since after removing iron ions from chrysotile fibers, they induced the superoxide release measured by lucigenin-dependent CL); hence, in the presence of absorbed iron ions, the superoxide ion was not released extracellularly by
phagocytes but reacts with iron ions to form new active species, supposedly the perferryl ions FeO 2÷. As catalase only partially inhibited the mutagenic effect of chrysotile fibers, both perferryl ions and hydrogen peroxide apparently participate in the chrysotile mutagenic activity. The mechanisms of the formation of active oxygen species by the phagocytes stimulated with latex, zeolites, and chrysotile particles and fibers are presented in Fig. 2. As it was already pointed out, from the compounds studied, rutin is the most effective inhibitor of mutagenic activities of chrysotile and zeolite (Tables 5 and 6). Similarly, rutin efficiently inhibited the luminol-dependent CL of peritoneal macrophages stimulated by chrysotile fibers or zeolite particles (Table 2). Its high inhibitory activity can be explained by the fact that rutin is simultaneously a free radical scavenger and a chelator (Afanas'ev et al., 1989a). It was also shown (Afanas'ev et al., 1989b) that rutin
TABLE 5 EFFECTS O F F R E E - R A D I C A L SCAVENGERS ON T H E C Y T O G E N E T I C A C T I O N OF C H R Y S O T I L E FIBERS ON T H E C U L T U R E D LYMPHOCYTES Additions to cell cultures, concentrations
Structural chromosome aberrations per 100 cells
% aberrant cells
Statistically significant ( P )
Chromatid breaks
Chromosome breaks
Control
1.0
0
1.0 _+0.7
Chrysotile, 0.05 m g / m l
6.5
1.5
6.5 + 1.7
< 0.01
Chrysotile + ascorbic acid: 0.05 m g / m l 10 -6 M 10 -5 M 10 -4 M 10 -3 M
4.0 1.3 3.3 5.9
0 0.3 0 0.4
4.0 1.3 3.3 5.5
+ 1.1 +0.65 + 1.0 _+ 1.4
< > >
0.05 0.05 > 0.05
Chrysotile, 0.05 m g / m l + rutin: 10 -6 M 3.0 10 -5 M 2.0 10 -4 M 2.0 10 -3 M 0.55
0 0 0 0
2.5 2.0 2.0 0.55
+ 1.1 + 0.9 + 1.0 _+0.55
> > > >
0.05 0.05 0.05 0.05
< < <
> >
0.05 0.05 0.05 0.05
200-300 metaphases counted per data point.
Control
Experiment
251 TABLE 6 EFFECTS O F F R E E - R A D I C A L SCAVENGERS ON T H E C Y T O G E N E T I C A C T I O N OF Z E O L I T E ON T H E C U L T U R E D LYMPHOCYTES
Additions to cell cultures, concentration
Chromatid breaks Control
1.3
Zeolite, 0.05 m g / m l
% aberrant cells
Structural chromosome aberrations per 100 cells
10
Chromosome breaks
Statistically significant ( P ) Control
0
1.3 + 0.7
0
9.0_+ 2.0
< 0.001
Experiment
Zeolite, 0.05 m g / m l + ascorbic acid: 10 -6 M 10 - s M 10 -4 M 10 -3 M
3.3 4.0 3.0 6.0
0.7 0 0 0.5
4.0+ 1.4 4.0+ 1.4 2.5 -+ 1.1 6.5 + 1.7
< > >
0.05 0.05 0.01 0.05
Zeolite, 0.05 m g / m l + rutin: 10 -7 M 10 -6 M 10 5 M 10 4 M
4.5 4.0 3.5 3.0
0 0 0 0
4.5_+ 1.5 4.0_+ 1.4 3.5 _+1.3 3.0+ 1.2
< > > >
0.05 0.05 0.05 0.05
> < <
0.05 < 0.05 < 0.05
200-300 metaphases counted per data point.
efficiently inhibits the cytochrome c reduction and the oxidation of p-nitrophenol by phagocytes stimulated with asbestos fibers. It is possible that the enhanced inhibitory effect of rutin on the mutagenic activity of chrysotile fibers is due to its ability to bind iron ions absorbed on the fibers into inactive iron-rutin complexes.
Bemitil is a compound possessing antioxidative properties (Durnev et al., 1986). It is possible that its antimutagenic activity is explained by its ability to scavenge active oxygen species such as hydroxyl radicals. Indeed, bemitil inhibited luminol-dependent CL produced by cell-free systems such as xanthine plus xanthine oxidase, H~O 2
05
(Lucigenindependent CL)
zeolite Phagocyte
•
•
H202 (Luminol-dependent CL sharply enhanced by HRP)
Fe0~ + + H202
Activation
(Luminol-dependent CL slightly enhanced by HRP)
Release of oxygen species
Fig. 2. Scheme of the release of different oxygen species by latex-, zeolite- and chrysotile-stimulated peritoneal phagocytes.
252
plus HRP, and H202 plus FeSO 4 (Durnev et al., 1986). In contrast to rutin and bemitil, ascorbic acid diminished or enhanced the mutagenic effects of asbestos and zeolite, depending on its concentration. Similarly, ascorbic acid exhibited a double (inhibitory and stimulatory) effect on the luminol-dependent CL of chrysotile-stimulated phagocytes (Table 2). It has already been shown (Weitberg and Weitzman, 1985) that ascorbic acid is able to inhibit the induction of sister-chromatid exchange (SCE) in Chinese hamster ovary cells by the xanthine oxidase-produced oxygen radicals at low concentrations (100/~M) and to enhance the SCE number at higher concentrations. It is possible that the inhibition of the mutagenic effect of oxygen radicals by ascorbic acid is a consequence of its direct interaction with oxygen radicals, while the mutagenic activity of ascorbic acid is explained by the fact that the reduction of ferric ions catalyzed the formation of hydroxyl radicals by the Fenton reaction. In conclusion, it should be stressed that our findings support the idea that the mutagenic activity of mineral particles and dusts is mediated by oxygen species of different types (superoxide ion, hydrogen peroxide, or perferryl ions) (Fig. 2). It is interesting to note that, despite its low reactivity, the mutagenic activity of the superoxide ion can be compared with that of much more reactive oxygen species (hydroxyl radicals or oxygenated iron complexes). It is possible that the mutagenic effect of the superoxide ion is a consequence of its ability to activate a special metabolic route resulting in the subsequent destruction of D N A and not its direct interaction with D N A (Birnboim, 1988). The inhibitory effect of free radical scavengers and chelators on mutagenic activities of pathogenic mineral dusts and particles indicates the possibility to use them for prophylaxis and in the treatment of occupational and environmental diseases. References Afanas'ev, I.B., A.I. Dorozhko, A.V. Brodskii, V.A. Kostyuk and A.I. Potapovich (1989a) Chelating and free radical scavenging mechanisms of inhibitory action of rutin and
quercetin in lipid peroxidation, Biochem. Pharmacol., 38, 1763-1769. Afanas'ev, I.B., L.G. Korkina, K.K. Briviba and B.T. Velichkovskiy (1989b) Rutin is an efficient scavenger of superoxide ion and "crypto-hydroxyl" radicals, in: O. Hayaishi, E. Niki, M. Kondo, and T. Yoshikawa (Eds.), Proc, 4th Biennial General Meeting of the Society for Free Radical Research (Kyoto, Japan, 9-13 April 1988), Elsevier, Amsterdam. Allen. R.C., and G.L. Strong (1980) Lucigenin chemiluminescence: a new approach to the study of polymorphonuclear leukocyte redox activity, Am. Soc. Photobiol., 8, 43-49. Birnboim, H.C. (1988) A superoxide anion-induced DNA strand-break metabolic pathway in human leukocytes: effects of vanadate, Biochem. Cell. Biol., 66, 374-381. Case, B.W., M.P.C. Ip, M. Padilla and J. Kleinerman (1986) Asbestos effect on superoxide production. An in vitro study of hamster alveolar macrophages, Environ. Res., 39, 299-303. Casey, G. (1983) Sister chromatid exchange and cell kinetics in CHO-K1 cells, human fibroblasts and lymphoblastoid cells exposed in vitro to asbestos and glass fibre, Mutation Res., 116, 369-377. Dahlgren, C., and R. Lock (1988) The limitation of the human neutrophil chemiluminescence response by extracellular peroxidase is stimulus dependent: effect of added horseradish peroxidase on the response induced by both soluble and particulate stimuli, J. Clin. Lab. Immunol., 26, 49-53. Durnev, A.D., L.G. Korkina, O.Yu. Dubovskaya, S.B. Seredenin and B.T. Velichkovskyi (1986) Study of the membrane mechanisms of chemical mutagenesis and analysis of antimutagenic properties of some psychotropic drugs, Khim.-Farm. Zh. (in Russian), 12, 1425-1428. Durnev, A.D., T.B. Suslova, Z.P. Cheremisina, O.Yu. Dubovskaya, E.A. Nigarova, L.G. Korkina, S.B. Seredenin and B.T. Velichkovskyi (1990) Investigation of mutagenic action of natural zeolite and chrysotile asbestos dusts, Exper. Oncol. (in Russian), 12, 21-24. Evans, P.H., and E. Yano (1985) Phagocyte-derived reactive oxygen metabolites: stimulation by pathogenic mineral dusts, Biochem. Soc. Trans., 13, 1205-1206. Gyllenhammar, H. (1987) Lucigenin chemiluminescence in the assessment of neutrophil superoxide production, J. lmmunol. Methods, 97, 209-214. Huang, S.L. (1979) Amosite, chrysotile and crocidolite asbestos are mutagenic in Chinese hamster lung cells, Mutation Res., 68, 265-275. Imlay, I.A. and S. Linn (1988) DNA damage and oxygen radical toxicity, Science, 240, 1302-1310. Korkina, L.G., Z.P. Cheremisina, T.B. Suslova, A.D. Durnev, S.B. Seredenin and B.T. Velichkovskyi (1988) Bleomycin and oxygen free radicals, Stud. Biophys., 126, 105-115. Lechner, J.F., T. Tokiwa, M. LaVeck, W.F. Benedict, S. Banks-Schlegel, H. Yeager, A. Beneijee and C.C. Harris (1985) Asbestos-associated chromosomal changes in human mesothelial cells, Proc. Natl. Acad. Sci. (U.S.A.), 82, 3884-3888.
253 Livingston, G.K., W.N. Rom and M.V. Morris (1980) Asbestos-induced sister chromatid exchanges in cultured Chinese hamster ovarian fibroblast cells, J. Environ. Pathol. Toxicol., 4, 473-482. Meneghini, R. (1988) Genotoxicity of active oxygen species in mammalian cells, Mutation Res., 195, 215-230. Mossman, B.T., J. Bignon, M. Corn, A. Seaton and J.B.L. Gee (1990) Asbestos: scientific developments and implications for public policy, Science, 247, 294-301. Palekar, L.D., J.F. Eyre, B.M. Most and D.L. Coffin (1987) Metaphase and anaphase analysis of V79 cells exposed to erionite, UICC chrysotile and UICC crocidolite, Carcinogenesis, 8, 553-560. Sincock, A.M., J.D.A. Delhanty and G. Casey (1982) A comparison of the cytogenetic response to asbestos and glass fibre in Chinese hamster and human cell lines, Mutation Res., 101,257-268. Urano, N., E. Yano, P.H. Evans and G. Ohi (1989) Reactive
oxygen metabolites produced by erionite, a carcinogenic mineral fiber, in: O. Hayaishi, E. Niki, M. Kondo and T. Yoshikawa (Eds.), Proc. 4th Biennial General Meeting of the Society for Free Radical Research (Kyoto, Japan, 9-13 April 1988), Elsevier, Amsterdam, pp. 93-96. Velichkovskyi, B.T., Yu.A. Vladimirov, L.G. Korkina and T.B. Suslova (1982) Physico-chemical mechanism of the interaction of phagocytozing cells with fibrogenic dusts, Vestn. Akad. Nauk SSSR (in Russian), 10, 45-48. Weitberg, A.B., and S.A. Weitzman (1985) The effect of vitamin C on oxygen radical-induced sister-chromatid exchanges, Mutation Res., 144, 23-27. Weitzman, S.A., and T.P. Stossel (1981) Mutation caused by human phagocytes, Science, 212, 546-547. Weitzman, S.A., and T.P. Stossel (1982) Effect of oxygen radical scavengers and antioxidants on phagocyte-induced mutagenesis, J. Immunol., 128, 2770-2772.
245
MUT 05048
Oxygen radical-mediated mutagenic effect of asbestos on human lymphocytes: suppression by oxygen radical scavengers Ludmila G. Korkina 1, Andrei D. Durnev 2, Tatjana B. Suslova 1, Zinaida P. Cheremisina 1, Natalia O. Daugel-Dauge i and Igor B. Afanas'ev 3 J 2nd Moscow Medical Institute, Ostrovitjanoua 1, 117869, Moscow, 2 Institute of Pharmacology, Baltiyskaja 8, Moscow and 3All-Union Vitamin Research Institute, Nauchny proezd 14A, GSP-7, 117820, Moscow (U.S.S.R.)
(Received 18 December 1990) (Revision received 25 June 1991) (Accepted 27 August 1991)
Keywords: Oxygen radical-mediated, human lymphocytes; Phagocytes; Asbestos; Zeolite; Antioxidants
Summary The mutagenic effect of chrysotile asbestos fibers and zeolite and latex particles on human lymphocytes in whole blood has been studied. It was concluded that their mutagenic activities were mediated by oxygen radicals because they were inhibited by antioxidant enzymes (SOD and catalase) and oxygen radical scavengers (rutin, ascorbic acid, and bemitil). It was proposed that oxygen radicals were released by phagocytes activated upon exposure to mineral dusts and fibers. The study of lucigenin- and luminol-amplified chemiluminescence of peritoneal macrophages stimulated by chrysotile fibers and zeolite and latex particles has shown that their mutagenic action is probably mediated by different oxygen species, namely, by the iron-oxygen complexes (perferryl ions) plus hydrogen peroxide, hydrogen peroxide, and superoxide ion, respectively. From the oxygen radical scavengers studied, rutin was the most effective inhibitor of the mutagenic effect of mineral fibers and dusts.
Mutagenic effects of oxygen radicals have been shown in many studies, which are now summarized in recent reviews (Meneghini, 1988; Imlay and Linn, 1988). One of the most important in vivo generators of oxygen radicals is the phagocyte which, upon activation with various stimuli, releases active oxygen species. Because of this, activated phagocytes acquire mutagenic activity.
Correspondence: I.B. Afanas'ev, All-Union Vitamin Research Institute, Nauchny proezd 14A, GSP-7, 117820, Moscow
(U.S.S.R.).
For example, it has been shown (Weitzman and Stossel, 1981) that normal human leukocytes were mutagenic to Salmonella typhimurium bacteria, while the leukocytes from a patient with chronic granulomatous disease (which are unable to release oxygen radicals) exhibited no mutagenic activity. An important role of oxygen radicals in mutagenic action of phagocytes was confirmed by the inhibitory effects of antioxidant enzymes (SOD and catalase) and free radical scavengers (a-tocopherol, cysteine, etc.) (Weitzman and Stossel, 1982). Similar results were obtained in studies of mutagenic action of phagocytes on
246
mammalian cells (Weitberg and Weitzman, 1985). It has been shown (Velichkovskiy et al., 1982; Evans and Yano, 1985; Case et al., 1986; Urano et al., 1989; Mossman et al., 1990) that mineral dusts and fibers are effective stimuli of the oxidative burst in macrophages and leukocytes. On the other hand, it is known that they exhibit mutagenic effects on mammalian cells (Huang, 1979; Livingston et al., 1980; Palekar et al., 1987), including human cells (Sincock et al., 1982; Casey, 1983; Lechner et al., 1985; Durnev et al., 1990). Therefore, one may propose that the mutagenic action of mineral dusts and fibers on cells is mediated by oxygen radicals released by the phagocytes activated with these stimuli. In this work we studied the mutagenic effects of chrysotile fibers and zeolite and latex particles on human lymphocytes in whole blood. Since lymphocytes are known to be unable to produce oxygen radicals, one can propose that the mutagenic effect is mediated by oxygen radicals produced by blood phagocytes stimulated with mineral fibers and dusts. For identification of the oxygen species released, we studied the lucigeninand luminol-dependent chemiluminescence (CL) in phagocytes of peritoneal exudate activated with these stimuli. (The content of neutrophils in peritoneal exudate is about 80% and close to that in human peripheral blood.) The results of CL experiments and the inhibition of the mutagenic action of mineral fibers and dusts by oxygen radical scavengers and antioxidant enzymes indicate an important role of oxygen radicals in the mutagenic activity of pathogenic mineral dusts. Materials and methods
Chemicals Rutin (3[6-O(6-dezoxy-a-L-mannopyranosil)-/3D-glycopyranosil)oxy]-2-(3,4-dihydroxyphenyl)-5,7dihydroxy-4-H-l-benzopyran) was manufactured by Khimiko-Farm. Zavod (Tashkent, U.S.S.R.), ascorbic acid was from Vitamin Zavod (Belgorod, U.S.S.R.), bemitil (2-ethylthiobenzimidazole) was a product of PO Darniza (Kiev, U.S.S.R.). Enzymes Superoxide dismutase (SOD) and catalase were from Serva (Germany) and horse radish peroxidase was from Reanal (Hungary). Latex was from
Sigma (U.S.A.), phytohemagglutinin P was from Difco (U.S.A.). Mineral dusts were natural zeolite clinoptilolite (Shivirtuy, U.S.S.R.) with 1-3 /zm average diameter of particles and chrysotile asbestos (Tuva, U.S.S.R.) with 5-10 ~zm fiber length. Before experiments, mineral dusts were rinsed with twice distilled water and suspended in 10 mM Tris-HC1 buffer (pH 7.4). For removal of metal ions, chrysotile fibers and zeolite particles were treated with hot 1 N HC1 for 1 h, rinsed with distilled water, and suspended in physiological solution. Removal of iron ions was checked by titration.
Cell culture Venous blood of healthy donors was drawn into heparinized vacutainer tubes and the cultures were established the same day. Whole blood (0.5 ml) was added to the mixture containing 7.5 ml Medium 199, 1.2 ml fetal calf serum, and 0.02 ml phytohemagglutinin P. Blood was cultured at 37 ° C in the 25 ml culture flasks for 56 h. Mineral dusts or fibers (0.01 or 0.05 mg/ml) were added with or without oxygen radical scavengers to cultures after 52 h. 2.5 h before cell fixation, colchicine was added to a final concentration of 0.25 mg/ml. Fixation was performed by standard cytological procedures. The cells were exposed to 0.075 M KC1 solution for 10-12 min and fixed in ethanol-acetic acid solution (3: 1, v/v). Slide preparation and staining procedure followed the routine protocol. Cell isolation Ceils of peritoneal exudate were isolated from Wistar male rats as described earlier (Korkina et al., 1988). Rats were injected with 2 ml 0.1% sterilized pepton solution intraperitoneally. After 12 h, rats were killed and cells were prepared by peritoneal lavage with 10 ml Hanks' balanced salt solution (HBSS). The lavage fluid was then filtered through a nylon cloth. The filtrate was centrifuged at 300 x g for 10 min and washed twice with cold HBSS. Then, the cells were resuspended and stored at 4 ° C in Medium 199. The cells were counted with a Coulter counter, and their viability was assessed by exclusion of 0.2% trypan blue dye. They were Wright-Giemsastained to obtain a differential cell count. Usu-
247 Luminol-dependent
ally, the cell suspension contained 8 5 - 8 8 % viable polymorphonuclear leukocytes, 8 - 1 0 % macrophages, and less than 2% lymphocytes.
Results
Comparison of the CL responses of phagocytes stimulated by latex, zeofite, and chrysotile As seen from Fig. 1 and Table 1, latex, zeolite, and chrysotile induced different CL responses in peritoneal phagocytes; these differences depended on the nature of both the stimuli and the amplifiers. Latex was nearly equally effective in the stimulation of lucigenin- and luminol-dependent CL, while zeolite and chrysotile induced luminol-dependent CL almost exclusively. In all cases, lucigenin- and luminol-dependent CL was inhibited by SOD. Removing iron ions from zeolite particles and chrysotile fibers by treatment with HC1 made them the efficient stimuli of lucigenin-dependent CL (Fig. 1). The addition of H R P sharply enhanced luminol-dependent CL in the case of zeolite stimulation but an increase was not so evident when chrysotile or latex was applied (Table 1). In all cases, rutin and ascorbic
Lucigenin-dependent
CL
5
Chemiluminescence detection of oxygen radicals and hydrogen peroxide released by mineral duststimulated peritoneal phagocytes Superoxide production was measured by the lucigenin-dependent CL (Gyllenhammar, 1987). Lucigenin (0.5 mM) was incubated with a suspension of phagocytes (1.5 x 105 cells/ml) in HBSS (pH 7.4) at 37 ° C upon continuous mixing in the LKB luminometer cell (Model 1251). After 5 min, the suspension of chrysotile fibers (0.5 m g / m l ) , zeolite particles (0.5 m g / m l ) , or latex particles (0.01%) was added and CL was registered continuously. Similarly, the production of oxygen radicals was determined in the presence of luminol (0.5 mM). For detection of hydrogen peroxide, the suspension containing luminol (0.5 mM), phagocytes (1.5 x 105 cells/ml), and mineral dust particles or fibers (0.5 m g / m l ) was incubated in the presence or absence of horse radish peroxidase (HRP, 0.2 /xg/ml). The amount of H 2 0 2 formed was estimated as the difference in the luminol-dependent CL in the presence and absence of H R P (Dahlgren and Lock, 1988).
CL
2
4
to
~3
/i it\.\ \\~/~ //t \.~1 I/ 3
3
~2
~
#
-
i 0
3
i/~ 0.05
< 0.01
4.4
1.2
5.6_+ 1.5
< 0.01
> 0.05
Zeolite, 0.05 mg/ml Zeolite, 0.05 mg/ml + SOD, 50 ~ g / m l Zeolite, 0.05 mg/ml + catalase, 20 ~zg/ml
8.0
0
8.0 _+1.8
< 0.001
6.5
0.5
7.0_+ 1.8
< 0.001
> 0.05
3.0
0.3
2.0 -+0.8
> 0.05
< 0.01
7.8
0.25
6.8 _+1.3
< 0.001
2.2
0
1.9 -+0.7
> 0.05
< 0.05
3.3
0
3.3 _+1.0
> 0.05
< 0.05
Chrysotile, 0.05 mg/ml Chrysotile, 0.05 mg/ml + SOD, 50/.Lg/ml Chrysotile, 0.05 mg/ml + catalase, 20 tzg/ml
250-400 metaphases counted per data point.
Control
Experiment
250
show that, in the case of zeolite stimulation, a large amount of H 2 0 2 was formed. Significantly smaller amounts of hydrogen peroxide were formed with chrysotile fibers and latex particles. In accordance with the CL experiments, the mutagenic effect of latex was mediated only by the superoxide ion because SOD completely inhibited it and catalase had no effect, and the mutagenic effect of zeolite was suppressed by catalase and not SOD, i.e., it was mediated by hydrogen peroxide (Table 4). The mutagenic effect of chrysotile fibers was inhibited by both SOD (nearly completely) and catalase (about 50%). The CL experiments indicate that chrysotile fibers stimulate the release by phagocytes of active oxygen species different but originating from the superoxide ion (since after removing iron ions from chrysotile fibers, they induced the superoxide release measured by lucigenin-dependent CL); hence, in the presence of absorbed iron ions, the superoxide ion was not released extracellularly by
phagocytes but reacts with iron ions to form new active species, supposedly the perferryl ions FeO 2÷. As catalase only partially inhibited the mutagenic effect of chrysotile fibers, both perferryl ions and hydrogen peroxide apparently participate in the chrysotile mutagenic activity. The mechanisms of the formation of active oxygen species by the phagocytes stimulated with latex, zeolites, and chrysotile particles and fibers are presented in Fig. 2. As it was already pointed out, from the compounds studied, rutin is the most effective inhibitor of mutagenic activities of chrysotile and zeolite (Tables 5 and 6). Similarly, rutin efficiently inhibited the luminol-dependent CL of peritoneal macrophages stimulated by chrysotile fibers or zeolite particles (Table 2). Its high inhibitory activity can be explained by the fact that rutin is simultaneously a free radical scavenger and a chelator (Afanas'ev et al., 1989a). It was also shown (Afanas'ev et al., 1989b) that rutin
TABLE 5 EFFECTS O F F R E E - R A D I C A L SCAVENGERS ON T H E C Y T O G E N E T I C A C T I O N OF C H R Y S O T I L E FIBERS ON T H E C U L T U R E D LYMPHOCYTES Additions to cell cultures, concentrations
Structural chromosome aberrations per 100 cells
% aberrant cells
Statistically significant ( P )
Chromatid breaks
Chromosome breaks
Control
1.0
0
1.0 _+0.7
Chrysotile, 0.05 m g / m l
6.5
1.5
6.5 + 1.7
< 0.01
Chrysotile + ascorbic acid: 0.05 m g / m l 10 -6 M 10 -5 M 10 -4 M 10 -3 M
4.0 1.3 3.3 5.9
0 0.3 0 0.4
4.0 1.3 3.3 5.5
+ 1.1 +0.65 + 1.0 _+ 1.4
< > >
0.05 0.05 > 0.05
Chrysotile, 0.05 m g / m l + rutin: 10 -6 M 3.0 10 -5 M 2.0 10 -4 M 2.0 10 -3 M 0.55
0 0 0 0
2.5 2.0 2.0 0.55
+ 1.1 + 0.9 + 1.0 _+0.55
> > > >
0.05 0.05 0.05 0.05
< < <
> >
0.05 0.05 0.05 0.05
200-300 metaphases counted per data point.
Control
Experiment
251 TABLE 6 EFFECTS O F F R E E - R A D I C A L SCAVENGERS ON T H E C Y T O G E N E T I C A C T I O N OF Z E O L I T E ON T H E C U L T U R E D LYMPHOCYTES
Additions to cell cultures, concentration
Chromatid breaks Control
1.3
Zeolite, 0.05 m g / m l
% aberrant cells
Structural chromosome aberrations per 100 cells
10
Chromosome breaks
Statistically significant ( P ) Control
0
1.3 + 0.7
0
9.0_+ 2.0
< 0.001
Experiment
Zeolite, 0.05 m g / m l + ascorbic acid: 10 -6 M 10 - s M 10 -4 M 10 -3 M
3.3 4.0 3.0 6.0
0.7 0 0 0.5
4.0+ 1.4 4.0+ 1.4 2.5 -+ 1.1 6.5 + 1.7
< > >
0.05 0.05 0.01 0.05
Zeolite, 0.05 m g / m l + rutin: 10 -7 M 10 -6 M 10 5 M 10 4 M
4.5 4.0 3.5 3.0
0 0 0 0
4.5_+ 1.5 4.0_+ 1.4 3.5 _+1.3 3.0+ 1.2
< > > >
0.05 0.05 0.05 0.05
> < <
0.05 < 0.05 < 0.05
200-300 metaphases counted per data point.
efficiently inhibits the cytochrome c reduction and the oxidation of p-nitrophenol by phagocytes stimulated with asbestos fibers. It is possible that the enhanced inhibitory effect of rutin on the mutagenic activity of chrysotile fibers is due to its ability to bind iron ions absorbed on the fibers into inactive iron-rutin complexes.
Bemitil is a compound possessing antioxidative properties (Durnev et al., 1986). It is possible that its antimutagenic activity is explained by its ability to scavenge active oxygen species such as hydroxyl radicals. Indeed, bemitil inhibited luminol-dependent CL produced by cell-free systems such as xanthine plus xanthine oxidase, H~O 2
05
(Lucigenindependent CL)
zeolite Phagocyte
•
•
H202 (Luminol-dependent CL sharply enhanced by HRP)
Fe0~ + + H202
Activation
(Luminol-dependent CL slightly enhanced by HRP)
Release of oxygen species
Fig. 2. Scheme of the release of different oxygen species by latex-, zeolite- and chrysotile-stimulated peritoneal phagocytes.
252
plus HRP, and H202 plus FeSO 4 (Durnev et al., 1986). In contrast to rutin and bemitil, ascorbic acid diminished or enhanced the mutagenic effects of asbestos and zeolite, depending on its concentration. Similarly, ascorbic acid exhibited a double (inhibitory and stimulatory) effect on the luminol-dependent CL of chrysotile-stimulated phagocytes (Table 2). It has already been shown (Weitberg and Weitzman, 1985) that ascorbic acid is able to inhibit the induction of sister-chromatid exchange (SCE) in Chinese hamster ovary cells by the xanthine oxidase-produced oxygen radicals at low concentrations (100/~M) and to enhance the SCE number at higher concentrations. It is possible that the inhibition of the mutagenic effect of oxygen radicals by ascorbic acid is a consequence of its direct interaction with oxygen radicals, while the mutagenic activity of ascorbic acid is explained by the fact that the reduction of ferric ions catalyzed the formation of hydroxyl radicals by the Fenton reaction. In conclusion, it should be stressed that our findings support the idea that the mutagenic activity of mineral particles and dusts is mediated by oxygen species of different types (superoxide ion, hydrogen peroxide, or perferryl ions) (Fig. 2). It is interesting to note that, despite its low reactivity, the mutagenic activity of the superoxide ion can be compared with that of much more reactive oxygen species (hydroxyl radicals or oxygenated iron complexes). It is possible that the mutagenic effect of the superoxide ion is a consequence of its ability to activate a special metabolic route resulting in the subsequent destruction of D N A and not its direct interaction with D N A (Birnboim, 1988). The inhibitory effect of free radical scavengers and chelators on mutagenic activities of pathogenic mineral dusts and particles indicates the possibility to use them for prophylaxis and in the treatment of occupational and environmental diseases. References Afanas'ev, I.B., A.I. Dorozhko, A.V. Brodskii, V.A. Kostyuk and A.I. Potapovich (1989a) Chelating and free radical scavenging mechanisms of inhibitory action of rutin and
quercetin in lipid peroxidation, Biochem. Pharmacol., 38, 1763-1769. Afanas'ev, I.B., L.G. Korkina, K.K. Briviba and B.T. Velichkovskiy (1989b) Rutin is an efficient scavenger of superoxide ion and "crypto-hydroxyl" radicals, in: O. Hayaishi, E. Niki, M. Kondo, and T. Yoshikawa (Eds.), Proc, 4th Biennial General Meeting of the Society for Free Radical Research (Kyoto, Japan, 9-13 April 1988), Elsevier, Amsterdam. Allen. R.C., and G.L. Strong (1980) Lucigenin chemiluminescence: a new approach to the study of polymorphonuclear leukocyte redox activity, Am. Soc. Photobiol., 8, 43-49. Birnboim, H.C. (1988) A superoxide anion-induced DNA strand-break metabolic pathway in human leukocytes: effects of vanadate, Biochem. Cell. Biol., 66, 374-381. Case, B.W., M.P.C. Ip, M. Padilla and J. Kleinerman (1986) Asbestos effect on superoxide production. An in vitro study of hamster alveolar macrophages, Environ. Res., 39, 299-303. Casey, G. (1983) Sister chromatid exchange and cell kinetics in CHO-K1 cells, human fibroblasts and lymphoblastoid cells exposed in vitro to asbestos and glass fibre, Mutation Res., 116, 369-377. Dahlgren, C., and R. Lock (1988) The limitation of the human neutrophil chemiluminescence response by extracellular peroxidase is stimulus dependent: effect of added horseradish peroxidase on the response induced by both soluble and particulate stimuli, J. Clin. Lab. Immunol., 26, 49-53. Durnev, A.D., L.G. Korkina, O.Yu. Dubovskaya, S.B. Seredenin and B.T. Velichkovskyi (1986) Study of the membrane mechanisms of chemical mutagenesis and analysis of antimutagenic properties of some psychotropic drugs, Khim.-Farm. Zh. (in Russian), 12, 1425-1428. Durnev, A.D., T.B. Suslova, Z.P. Cheremisina, O.Yu. Dubovskaya, E.A. Nigarova, L.G. Korkina, S.B. Seredenin and B.T. Velichkovskyi (1990) Investigation of mutagenic action of natural zeolite and chrysotile asbestos dusts, Exper. Oncol. (in Russian), 12, 21-24. Evans, P.H., and E. Yano (1985) Phagocyte-derived reactive oxygen metabolites: stimulation by pathogenic mineral dusts, Biochem. Soc. Trans., 13, 1205-1206. Gyllenhammar, H. (1987) Lucigenin chemiluminescence in the assessment of neutrophil superoxide production, J. lmmunol. Methods, 97, 209-214. Huang, S.L. (1979) Amosite, chrysotile and crocidolite asbestos are mutagenic in Chinese hamster lung cells, Mutation Res., 68, 265-275. Imlay, I.A. and S. Linn (1988) DNA damage and oxygen radical toxicity, Science, 240, 1302-1310. Korkina, L.G., Z.P. Cheremisina, T.B. Suslova, A.D. Durnev, S.B. Seredenin and B.T. Velichkovskyi (1988) Bleomycin and oxygen free radicals, Stud. Biophys., 126, 105-115. Lechner, J.F., T. Tokiwa, M. LaVeck, W.F. Benedict, S. Banks-Schlegel, H. Yeager, A. Beneijee and C.C. Harris (1985) Asbestos-associated chromosomal changes in human mesothelial cells, Proc. Natl. Acad. Sci. (U.S.A.), 82, 3884-3888.
253 Livingston, G.K., W.N. Rom and M.V. Morris (1980) Asbestos-induced sister chromatid exchanges in cultured Chinese hamster ovarian fibroblast cells, J. Environ. Pathol. Toxicol., 4, 473-482. Meneghini, R. (1988) Genotoxicity of active oxygen species in mammalian cells, Mutation Res., 195, 215-230. Mossman, B.T., J. Bignon, M. Corn, A. Seaton and J.B.L. Gee (1990) Asbestos: scientific developments and implications for public policy, Science, 247, 294-301. Palekar, L.D., J.F. Eyre, B.M. Most and D.L. Coffin (1987) Metaphase and anaphase analysis of V79 cells exposed to erionite, UICC chrysotile and UICC crocidolite, Carcinogenesis, 8, 553-560. Sincock, A.M., J.D.A. Delhanty and G. Casey (1982) A comparison of the cytogenetic response to asbestos and glass fibre in Chinese hamster and human cell lines, Mutation Res., 101,257-268. Urano, N., E. Yano, P.H. Evans and G. Ohi (1989) Reactive
oxygen metabolites produced by erionite, a carcinogenic mineral fiber, in: O. Hayaishi, E. Niki, M. Kondo and T. Yoshikawa (Eds.), Proc. 4th Biennial General Meeting of the Society for Free Radical Research (Kyoto, Japan, 9-13 April 1988), Elsevier, Amsterdam, pp. 93-96. Velichkovskyi, B.T., Yu.A. Vladimirov, L.G. Korkina and T.B. Suslova (1982) Physico-chemical mechanism of the interaction of phagocytozing cells with fibrogenic dusts, Vestn. Akad. Nauk SSSR (in Russian), 10, 45-48. Weitberg, A.B., and S.A. Weitzman (1985) The effect of vitamin C on oxygen radical-induced sister-chromatid exchanges, Mutation Res., 144, 23-27. Weitzman, S.A., and T.P. Stossel (1981) Mutation caused by human phagocytes, Science, 212, 546-547. Weitzman, S.A., and T.P. Stossel (1982) Effect of oxygen radical scavengers and antioxidants on phagocyte-induced mutagenesis, J. Immunol., 128, 2770-2772.
