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Hemolysis of rabbit erythrocytes induced by cigarette smoke
Life Sciences, Vol. 47, pp. 2207-2215 Printed in the U.S.A.
Pergamon Press
HEMOLYSIS OF RABBIT ERYTHROCYTES INDUCED BY CIGARETTE SMOKE Susumu Minamisawa, Erika Komuro, and Etsuo Niki Department of Reaction Chemistry, Faculty of Engineering, University of Tokyo, Hongo, Tokyo 113, Japan (Received in final form October 5, 1990)
Summarv Cigarette smoke has been found to induce the hemolysis of rabbit erythrocytes. The particulate phase had more profound effect than the gas phase. Neither free radical scavengers such as ascorbic acid, uric acid and water-soluble vitamin E analogue nor antioxidant enzymes such as catalase and superoxide dismutase suppressed the cigarette smoke-induced hemolysis, suggesting that free radicals, hydrogen peroxide, and superoxide were not the active species.
There is now abundant evidence which suggests that smoking is, at least in part, responsible for a variety of diseases such as emphysema and cancer (1,2). The cigarette smoke contains high concentrations of organic free radicals (1,3) and it is now generally accepted that these radicals are involved in smokingrelated diseases. It is in fact known that free radicals attack biological molecules, membranes and tissues and cause a variety of pathological events (4-7). For example, we have previously shown that the free radicals generated chemically from an azo c o m p o u n d attack erythrocyte membranes to oxidize lipids and proteins and eventually cause hemolysis (8-11). The lethal effect of free radicals on cultured HeLa cells has been also observed (12). The toxic effects of cigarette smoke have been studied extensively, most notably by Pryor and his colleagues (1-3), but cigarette smoke is quite a complex system consisting of an e n o r m o u s number of c o m p o u n d s and the mechanism by which it exerts cytotoxicity is not well understood yet. In the course of our study on the effects of free radicals on biological molecules and membranes, we have investigated in this work the hemolysis of rabbit red blood cells induced by cigarette smoke. 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
2208
Hemolysis Induced by Cigarette Smoke
Vol. 47, No. 24, 1990
Caa~n~ge FIImr
Vent
.~ 2
Pump
FIG. 1 Apparatus used in this study for the preparation of smoke-PBS and hemolysis of erythrocytes.
MATERIALS AND METHOD The erythrocytes were obtained from male rabbits: the fresh, heparinized rabbit blood was centrifuged at 1000 x g for 10 min, and erythrocytes were s e p a r a t e d f r o m p l a s m a and b u f f y coat and w a s h e d five t i m e s with a physiological saline as reported previously (8-11). Catalase and superoxide dismutase (SOD) were purchased from Sigma Chemical Co. (St. Louis, MO). Commercial ascorbic acid, 2-carboxy-2,5,7,8-tetramethyl-6-chromanol, uric acid and glutathione were used as received. Smoke-PBS (smoke bubbled phosphate b u f f e r e d saline solution) was prepared as follows. Commercial cigarettes Echo with a filter were smoked using an apparatus shown in Figure 1 and the mainstream smoke was passed through 30 to 40 ml of physiological saline containing 10mM phosphate buffer, pH 7.4, contained in a gas wash bottle. A continuous smoking protocol was used instead of a puff protocol. This difference should have little effect on the results and conclusion. Each cigarette was smoked for 2 min to leave a butt length of 1 cm. Twenty cigarette were used to obtain smoke-PBS. In some experiments, cigarette smoke was passed through a Cambridge filter, a glassfiber filter which retains 99.9 % of all particulate matter with a size greater than 0.11xM (13). The cigarette filter was also removed sometimes to examine its effect. Activated charcoal was used only when specified. The antioxidants were added into smoke-PBS after its preparation.
Vol. 47, No. 24, 1990
Hemolysis Induced by Cigarette Smoke
2209
The hemolysis experiments were performed in two different methods. In one series of experiments, the red blood cells were incubated at 37 °C under air in smoke-PBS. An aliquot (0.5 ml) of the suspension was taken out at specific time interval and the extent of hemolysis was measured as reported previously (8-11). Briefly, the absorption at 540 nm was measured spectrometrically for the supernatant after dilution of the reaction mixture with saline followed by centrifugation, 1000 x g for 10 min. The percent hemolysis is expressed by a ratio of the absorption at 540 nm for the reaction mixture to that for the sample of complete hemolysis obtained by diluting the reaction mixture with water instead of saline. Potassium ion and aspartate transaminase (GOT) were assayed at Teijin Bio-Science Laboratories (Tokyo). In an other series of experiments, cigarette smoke was directly bubbled into a suspension of red blood cells in p h o s p h a t e b u f f e r , pH 7.4, at a flow rate of about 1 L/min and room t e m p e r a t u r e , and an aliquot (0.5 ml) of the s u s p e n s i o n was taken out periodically to measure the extent of hemolysis. The erythrocytes were stable to the bubbled air under the experimental conditions employed (see later text).
RESULTS The rabbit erythrocytes are stable when incubated at 37 °C in air and little oxidation was observed in 5 hours, but when cigarette smoke was bubbled into 50 - -
5 --
100
40
q
80
20
2
qO
10
1
20
0
0
0 60
Time.r~n
120 0
60
120
Time,mln
FIG. 2 Leakages of hemoglobin ( • ) , potassium ion ( • ) and GOT ( • ) from rabbit erythrocytes when cigarette smoke was bubbled through an aqueous suspension of rabbit erythrocytes at 37 °C in air. (A:left) Neither cigarette filter nor Cambridge filter was used. (B:right) With cigarette filter and without Cambridge filter.
180
2210
Hemolysis Induced by Cigarette Smoke
Vol. 47, No. 24, 1990
an aqueous suspension (phosphate buffer, pH 7.4) of 4% rabbit red blood cells, hemolysis took place as shown in Fig. 2 and hemoglobin, potassium ion and GOT leaked out. It may be worth noting that hemoglobin and potassium ion leaked out almost at the same time, although potassium ion leaked out before hemoglobin in the free radical induced hemolysis (11). In Fig. 2A, the cigarette filter was taken off and the cigarette smoke was continuously bubbled into the erythrocyte suspensions. Figure 2 shows that the cigarette filter suppressed the hemolysis. Figure 3 shows the effects of cigarette and Cambridge filters in the preparation of smoke-PBS on the rate of hemolysis observed when the red blood cells were incubated at 37 °C in the smoke-PBS. Hemolysis took place quite rapidly when neither of the two filters was used and both cigarette filter and Cambridge filter retarded the rate of hemolysis. Appreciable hemolysis was not observed in5 hrs when the smoke-PBS was prepared with both filters. This shows that the bubbling of air alone at a flow rate of 1 L/min does not cause hemolysis. In one experiment, air was bubbled for 45 min through a smoke-PBS prepared as usual without using either Cambridge filter or cigarette filter, and the red blood cells were incubated in the above purged smoke-PBS at 37 °C in air. As shown in Fig. 4, little hemolysis was observed in 180 min.
100 8O
K~
6o
O
E 40 -I-
20 0 I
0
60
120
180
240
300
Time, rain
FIG. 3 Effects of cigarette and Cambridge filters in the preparation of smoke-PBS on the rate of hemolysis. ( e ) : No filters; (A) : With only cigarette filter. ( • ) : With both cigarette and Cambridge filter.
Vol. 47, No. 24, 1990
Hemolysis Induced by Cigarette Smoke
2211
100
80
.~
60
20 0I 0
i
I
,
60
I
120
,
180
T i m e , min
FIG. 4 Effect of degassing of smoke-PBS with air before incubation with erythrocytes on the extent of hemolysis. ( • ) : Air was bubbled through smoke-PBS for 45 min ( • ) : Control experiment with normal smoke-PBS.
Substantially the same result was observed when nitrogen was bubbled through a smoke-PBS for 45 min. These results imply that the volatile components which were removed when air or nitrogen was bubbled through a smoke-PBS are responsible for hemolysis, although the possibility that the interaction between volatile and non-volatile components causes hemolysis can not be ruled out. It has been reported (14-16) that superoxide and hydrogen peroxide were formed in a neutral buffer through which cigarette smoke had been bubbled. It was also found in this study that superoxide was indeed formed in a smokePBS. In order to see if the superoxide and hydrogen peroxide formed in the smoke-PBS were responsible for the hemolysis, the effects of catalase and SOD w e r e studied. It was f o u n d that the h e m o l y s i s was not r e t a r d e d appreciably by catalase (0.20, 0.50 and 1.00 mg/ml or 420, 1050, and 2100 U/ml respectively). It was also found that SOD (10-60 U/ml)did not suppress the hemolysis. Furthermore, the combined use of both catalase and SOD also did not significantly influence the extent of hemolysis (data not shown). In the h e m o l y s i s o f e r y t h r o c y t e s i n d u c e d by h y p o x a n t h i n e - x a n t h i n e oxidase, where hydrogen peroxide is responsible and catalase inhibits quite
2212
Hemolysis Induced by Cigarette Smoke
Vol. 47, No. 24, 1990
effectively, the treatment of erythrocytes by carbon monoxide suppressed the h e m o l y s i s (17). However, in the present study, little effect of COtreatment of erythrocytes was observed (data not shown), again suggesting that hydrogen peroxide was not responsible for hemolysis in the present system. It has been observed previously that free radicals generated thermally from a radical initiator induce hemolysis of erythrocytes and that chainbreaking antioxidants suppress the hemolysis (8-11). However, it was found in this study that the water-soluble chain-breaking antioxidants such as ascorbic acid, uric acid, r e d u c e d g l u t a t h i o n e , and 2 - c a r b o x y - 2 , 5 , 7 , 8 tetramethyl -6-chromanol, a water-soluble analogue of vitamin E, did not suppress the cigarette smoke-induced hemolysis appreciably. Figure 5 shows the effect of addition of albumin on the hemolysis of rabbit erythrocytes induced by cigarette smoke. It shows that albumin retarded the hemolysis dose-dependently, possibly by absorbing active species like activated charcoal mentioned later. Furthermore, the blood suppressed the hemolysis markedly and the rate of hemolysis was slow in the whole blood (data not shown).
100
80
.d
60
_>, 0
E
40
20
0
0
60
120
180
240
Time, min
FIG. 5 Effects of addition of albumin on the hemolysis by smoke-PBS. Concentration of albumin : . : 0 ; . : 1 ; • : 3 ; • : 5 mg/ml suspension.
Vol. 47, No. 24, 1990
Hemolysls Induced by Cigarette Smoke
80
t
2213
/
6o ,o
2o 0 0
60
120
180
240
300
Time, min
FIG. 6 Effect of activated charcoal on the extent of hemolysis. Cigarette smoke was passed through activated charcoal packed in the glass tube before bubbling into PBS. In the control experiment ( • ), activated charcoal was not used.
Figure 6 shows the effect of activated charcoal on the hemolysis o f red blood cells. Cigarette smoke was passed through an activated charcoal powder packed in the glass tube (Fig. 1) and b u b b l e d into a p h o s p h a t e buffer. N e i t h e r C a m b r i d g e filter nor cigarette filter was used. The red b l o o d cells were incubated in the above p h o s p h a t e buffer and the extent of h e m o l y s i s was followed. As shown in Fig. 6, little hemolysis was observed in 300 mm when the smoke was passed through an activated charcoal, while a complete hemolysis was observed in about 100 min when activated charcoal was not used.
DISCUSSION The above results show that cigarette smoke induces hemolysis of rabbit erythrocytes. The Cambridge filter had a profound effect, suggesting that cigarette tar has a more toxic effect than the gas phase. The fact that the purged smoke-PBS did not induce hemolysis suggests the important role of volatile components of cigarette smoke. It has been also reported by Nakayama, Kaneko and Kodama (16) that volatile gas components in smoke-PBS had an enhancing effect on inducing single strand break of DNA. Free radicals are contained in both tar and gas phase of cigarette smoke (1)
2214
Hemolysis Induced by Cigarette Smoke
Vol. 47, No. 24, 1990
and these radicals have been assumed to be involved in the pathology induced by smoking. However, the results shown above, especially the inefficiencies of various chain-breaking antioxidants, suggest that the hemolysis of rabbit erythrocyte induced by cigarette smoke is not mediated by free radicals. It was c o n f i r m e d in the p r e s e n t study that, as had been r e p o r t e d in the literatures (14,15), cigarette smoke generated both superoxide and hydrogen peroxide. These species are known to induce hemolysis (17), but the amounts of superoxide and hydrogen peroxide formed were too small to account for the hemolysis observed. Recently, Lee et al. (18) have reported that cigarette smoke increased sister chromatid exchanges in Chinese hamster ovary cells in a dose-dependent manner, but that the oxygen free radicals were not involved in them. Of course, this does not deny the toxic role of free radicals in cigarette smoke. In fact, it was observed that the cigarette smoke induced the free radical chain oxidation of m e t h y l linoleate (Minamisawa, Yamamoto, and Niki, unpublished work). However, the rate of chain initiation was not large enough to account for the hemolysis observed in the present study. In conclusion, although the nature of true active species is not known yet and more work is apparently required to elucidate the mechanism, this work shows that cigarette smoke induced the hemolysis of rabbit erythrocytes. Volatile components, rather than the free radicals, in cigarette smoke were suggested to be responsible for the hemolysis.
REFERENCES 1.
D.F. CHURCH and W. A. PRYOR, Environ. Health Perspect., 64 111-126 (1985), and references cited therein. 2. W.A. PRYOR, Brit J. Cancer, 55 Suppl. VIII, 19-23 (1987). 3. W.A. PRYOR, B. J. HALES, P. I. PREMOVEI, and D. F. CHURCH, Science, 220 425-427 (1983). 4. B. HALLIWELL and J. M. C. GUTTERIDGE, Free Radicals in Biology and Medicince, Clarendon Press, Oxford (1985). 5. H. Sies, (ed.) Oxidative Stress, Academic Press, London (1985). 6. M.G. SIMIC, K. A. TAYLOR, J. F. WARD, and C. von SONNTAG, (eds), Oxygen Radicals in Biology and Medicine, Plenum Press, New York (1989). 7. O. Hayaishi, E. Niki, M. Kondo, and T. Yoshikawa, (eds), Medical, Biochemical and Chemical Aspects of Free Radicals, Elsevier, Amsterdam (1989). 8. Y. YAMAMOTO, E. NIKI, Y. KAMIYA, M. MIKI, H. TAMAI and M. MINO, J. Nutr. Sci. Vitaminol., 32 475-479 (1986). 9. M. MIKI, H. TAMAI, M. MINO, Y. YAMAMOTO and E. NIKI, Arch. Biochem. Biophys. 258 373-380 (1987). 10. E. NIKI, Y. YAMAMOTO, M. TAKAHASHI, K.YAMAMOTO,
Vol. 47, No. 24, 1990
11. 12.
13. 14. 15. 16. 17. 18.
Hemolysis Induced by Cigarette Smoke
2215
Y.YAMAMOTO, E. KOMURO, M. MIKI, H. YASUDA and M. MINO, J. Nutr. Sci. Vitaminol. 34 507-512 (1988). E. NIKI, E. KOMURO, M. TAKAHASHI, S. URANO, E. ITO and K. TERAO, J. Biol. Chem. 263 19809-19814 (1988). M. OIKAWA, E. NIKI and K. TERAO, Medical, Biochemical and Chemical Aspects of Free Radicals, O. Hayaishi, E. Niki, M. Kondo and T. Yoshikawa (eds), 1219-1220, Elsevier, Amsterdam (1989). M . R . GUERIN, Banbury Report. A safe Cigarette? G. B. Gori and F. G. Bock (eds), 191-204, Cold Spring Harbor Laboratory, New York (1980) T. NAKAYAMA, M. KODAMA and C. NAGATA, Gann 75 95-98 (1984). J.P. COSGROVE, E. T. BORISH, D. E CHURCH and W. A. PRYOR, Biochem. Biophys. Res. Commun. 132 390-396 (1985). T. NAKAYAMA, M. KANEKO, and M. KODAMA, Agric. Biol. Chem. 50 3219-3220 (1986). H. TAMAI, M. MIKI and M. MINO, J. Free Rad. Biol. Med. 2 49-56 (1986). C.K. LEE, B. G. BROWN, W. Y. RICE, Jr. and D. J. Doolittle, Environ. Mol. Mutag. 13 54-59 (1989).
Pergamon Press
HEMOLYSIS OF RABBIT ERYTHROCYTES INDUCED BY CIGARETTE SMOKE Susumu Minamisawa, Erika Komuro, and Etsuo Niki Department of Reaction Chemistry, Faculty of Engineering, University of Tokyo, Hongo, Tokyo 113, Japan (Received in final form October 5, 1990)
Summarv Cigarette smoke has been found to induce the hemolysis of rabbit erythrocytes. The particulate phase had more profound effect than the gas phase. Neither free radical scavengers such as ascorbic acid, uric acid and water-soluble vitamin E analogue nor antioxidant enzymes such as catalase and superoxide dismutase suppressed the cigarette smoke-induced hemolysis, suggesting that free radicals, hydrogen peroxide, and superoxide were not the active species.
There is now abundant evidence which suggests that smoking is, at least in part, responsible for a variety of diseases such as emphysema and cancer (1,2). The cigarette smoke contains high concentrations of organic free radicals (1,3) and it is now generally accepted that these radicals are involved in smokingrelated diseases. It is in fact known that free radicals attack biological molecules, membranes and tissues and cause a variety of pathological events (4-7). For example, we have previously shown that the free radicals generated chemically from an azo c o m p o u n d attack erythrocyte membranes to oxidize lipids and proteins and eventually cause hemolysis (8-11). The lethal effect of free radicals on cultured HeLa cells has been also observed (12). The toxic effects of cigarette smoke have been studied extensively, most notably by Pryor and his colleagues (1-3), but cigarette smoke is quite a complex system consisting of an e n o r m o u s number of c o m p o u n d s and the mechanism by which it exerts cytotoxicity is not well understood yet. In the course of our study on the effects of free radicals on biological molecules and membranes, we have investigated in this work the hemolysis of rabbit red blood cells induced by cigarette smoke. 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
2208
Hemolysis Induced by Cigarette Smoke
Vol. 47, No. 24, 1990
Caa~n~ge FIImr
Vent
.~ 2
Pump
FIG. 1 Apparatus used in this study for the preparation of smoke-PBS and hemolysis of erythrocytes.
MATERIALS AND METHOD The erythrocytes were obtained from male rabbits: the fresh, heparinized rabbit blood was centrifuged at 1000 x g for 10 min, and erythrocytes were s e p a r a t e d f r o m p l a s m a and b u f f y coat and w a s h e d five t i m e s with a physiological saline as reported previously (8-11). Catalase and superoxide dismutase (SOD) were purchased from Sigma Chemical Co. (St. Louis, MO). Commercial ascorbic acid, 2-carboxy-2,5,7,8-tetramethyl-6-chromanol, uric acid and glutathione were used as received. Smoke-PBS (smoke bubbled phosphate b u f f e r e d saline solution) was prepared as follows. Commercial cigarettes Echo with a filter were smoked using an apparatus shown in Figure 1 and the mainstream smoke was passed through 30 to 40 ml of physiological saline containing 10mM phosphate buffer, pH 7.4, contained in a gas wash bottle. A continuous smoking protocol was used instead of a puff protocol. This difference should have little effect on the results and conclusion. Each cigarette was smoked for 2 min to leave a butt length of 1 cm. Twenty cigarette were used to obtain smoke-PBS. In some experiments, cigarette smoke was passed through a Cambridge filter, a glassfiber filter which retains 99.9 % of all particulate matter with a size greater than 0.11xM (13). The cigarette filter was also removed sometimes to examine its effect. Activated charcoal was used only when specified. The antioxidants were added into smoke-PBS after its preparation.
Vol. 47, No. 24, 1990
Hemolysis Induced by Cigarette Smoke
2209
The hemolysis experiments were performed in two different methods. In one series of experiments, the red blood cells were incubated at 37 °C under air in smoke-PBS. An aliquot (0.5 ml) of the suspension was taken out at specific time interval and the extent of hemolysis was measured as reported previously (8-11). Briefly, the absorption at 540 nm was measured spectrometrically for the supernatant after dilution of the reaction mixture with saline followed by centrifugation, 1000 x g for 10 min. The percent hemolysis is expressed by a ratio of the absorption at 540 nm for the reaction mixture to that for the sample of complete hemolysis obtained by diluting the reaction mixture with water instead of saline. Potassium ion and aspartate transaminase (GOT) were assayed at Teijin Bio-Science Laboratories (Tokyo). In an other series of experiments, cigarette smoke was directly bubbled into a suspension of red blood cells in p h o s p h a t e b u f f e r , pH 7.4, at a flow rate of about 1 L/min and room t e m p e r a t u r e , and an aliquot (0.5 ml) of the s u s p e n s i o n was taken out periodically to measure the extent of hemolysis. The erythrocytes were stable to the bubbled air under the experimental conditions employed (see later text).
RESULTS The rabbit erythrocytes are stable when incubated at 37 °C in air and little oxidation was observed in 5 hours, but when cigarette smoke was bubbled into 50 - -
5 --
100
40
q
80
20
2
qO
10
1
20
0
0
0 60
Time.r~n
120 0
60
120
Time,mln
FIG. 2 Leakages of hemoglobin ( • ) , potassium ion ( • ) and GOT ( • ) from rabbit erythrocytes when cigarette smoke was bubbled through an aqueous suspension of rabbit erythrocytes at 37 °C in air. (A:left) Neither cigarette filter nor Cambridge filter was used. (B:right) With cigarette filter and without Cambridge filter.
180
2210
Hemolysis Induced by Cigarette Smoke
Vol. 47, No. 24, 1990
an aqueous suspension (phosphate buffer, pH 7.4) of 4% rabbit red blood cells, hemolysis took place as shown in Fig. 2 and hemoglobin, potassium ion and GOT leaked out. It may be worth noting that hemoglobin and potassium ion leaked out almost at the same time, although potassium ion leaked out before hemoglobin in the free radical induced hemolysis (11). In Fig. 2A, the cigarette filter was taken off and the cigarette smoke was continuously bubbled into the erythrocyte suspensions. Figure 2 shows that the cigarette filter suppressed the hemolysis. Figure 3 shows the effects of cigarette and Cambridge filters in the preparation of smoke-PBS on the rate of hemolysis observed when the red blood cells were incubated at 37 °C in the smoke-PBS. Hemolysis took place quite rapidly when neither of the two filters was used and both cigarette filter and Cambridge filter retarded the rate of hemolysis. Appreciable hemolysis was not observed in5 hrs when the smoke-PBS was prepared with both filters. This shows that the bubbling of air alone at a flow rate of 1 L/min does not cause hemolysis. In one experiment, air was bubbled for 45 min through a smoke-PBS prepared as usual without using either Cambridge filter or cigarette filter, and the red blood cells were incubated in the above purged smoke-PBS at 37 °C in air. As shown in Fig. 4, little hemolysis was observed in 180 min.
100 8O
K~
6o
O
E 40 -I-
20 0 I
0
60
120
180
240
300
Time, rain
FIG. 3 Effects of cigarette and Cambridge filters in the preparation of smoke-PBS on the rate of hemolysis. ( e ) : No filters; (A) : With only cigarette filter. ( • ) : With both cigarette and Cambridge filter.
Vol. 47, No. 24, 1990
Hemolysis Induced by Cigarette Smoke
2211
100
80
.~
60
20 0I 0
i
I
,
60
I
120
,
180
T i m e , min
FIG. 4 Effect of degassing of smoke-PBS with air before incubation with erythrocytes on the extent of hemolysis. ( • ) : Air was bubbled through smoke-PBS for 45 min ( • ) : Control experiment with normal smoke-PBS.
Substantially the same result was observed when nitrogen was bubbled through a smoke-PBS for 45 min. These results imply that the volatile components which were removed when air or nitrogen was bubbled through a smoke-PBS are responsible for hemolysis, although the possibility that the interaction between volatile and non-volatile components causes hemolysis can not be ruled out. It has been reported (14-16) that superoxide and hydrogen peroxide were formed in a neutral buffer through which cigarette smoke had been bubbled. It was also found in this study that superoxide was indeed formed in a smokePBS. In order to see if the superoxide and hydrogen peroxide formed in the smoke-PBS were responsible for the hemolysis, the effects of catalase and SOD w e r e studied. It was f o u n d that the h e m o l y s i s was not r e t a r d e d appreciably by catalase (0.20, 0.50 and 1.00 mg/ml or 420, 1050, and 2100 U/ml respectively). It was also found that SOD (10-60 U/ml)did not suppress the hemolysis. Furthermore, the combined use of both catalase and SOD also did not significantly influence the extent of hemolysis (data not shown). In the h e m o l y s i s o f e r y t h r o c y t e s i n d u c e d by h y p o x a n t h i n e - x a n t h i n e oxidase, where hydrogen peroxide is responsible and catalase inhibits quite
2212
Hemolysis Induced by Cigarette Smoke
Vol. 47, No. 24, 1990
effectively, the treatment of erythrocytes by carbon monoxide suppressed the h e m o l y s i s (17). However, in the present study, little effect of COtreatment of erythrocytes was observed (data not shown), again suggesting that hydrogen peroxide was not responsible for hemolysis in the present system. It has been observed previously that free radicals generated thermally from a radical initiator induce hemolysis of erythrocytes and that chainbreaking antioxidants suppress the hemolysis (8-11). However, it was found in this study that the water-soluble chain-breaking antioxidants such as ascorbic acid, uric acid, r e d u c e d g l u t a t h i o n e , and 2 - c a r b o x y - 2 , 5 , 7 , 8 tetramethyl -6-chromanol, a water-soluble analogue of vitamin E, did not suppress the cigarette smoke-induced hemolysis appreciably. Figure 5 shows the effect of addition of albumin on the hemolysis of rabbit erythrocytes induced by cigarette smoke. It shows that albumin retarded the hemolysis dose-dependently, possibly by absorbing active species like activated charcoal mentioned later. Furthermore, the blood suppressed the hemolysis markedly and the rate of hemolysis was slow in the whole blood (data not shown).
100
80
.d
60
_>, 0
E
40
20
0
0
60
120
180
240
Time, min
FIG. 5 Effects of addition of albumin on the hemolysis by smoke-PBS. Concentration of albumin : . : 0 ; . : 1 ; • : 3 ; • : 5 mg/ml suspension.
Vol. 47, No. 24, 1990
Hemolysls Induced by Cigarette Smoke
80
t
2213
/
6o ,o
2o 0 0
60
120
180
240
300
Time, min
FIG. 6 Effect of activated charcoal on the extent of hemolysis. Cigarette smoke was passed through activated charcoal packed in the glass tube before bubbling into PBS. In the control experiment ( • ), activated charcoal was not used.
Figure 6 shows the effect of activated charcoal on the hemolysis o f red blood cells. Cigarette smoke was passed through an activated charcoal powder packed in the glass tube (Fig. 1) and b u b b l e d into a p h o s p h a t e buffer. N e i t h e r C a m b r i d g e filter nor cigarette filter was used. The red b l o o d cells were incubated in the above p h o s p h a t e buffer and the extent of h e m o l y s i s was followed. As shown in Fig. 6, little hemolysis was observed in 300 mm when the smoke was passed through an activated charcoal, while a complete hemolysis was observed in about 100 min when activated charcoal was not used.
DISCUSSION The above results show that cigarette smoke induces hemolysis of rabbit erythrocytes. The Cambridge filter had a profound effect, suggesting that cigarette tar has a more toxic effect than the gas phase. The fact that the purged smoke-PBS did not induce hemolysis suggests the important role of volatile components of cigarette smoke. It has been also reported by Nakayama, Kaneko and Kodama (16) that volatile gas components in smoke-PBS had an enhancing effect on inducing single strand break of DNA. Free radicals are contained in both tar and gas phase of cigarette smoke (1)
2214
Hemolysis Induced by Cigarette Smoke
Vol. 47, No. 24, 1990
and these radicals have been assumed to be involved in the pathology induced by smoking. However, the results shown above, especially the inefficiencies of various chain-breaking antioxidants, suggest that the hemolysis of rabbit erythrocyte induced by cigarette smoke is not mediated by free radicals. It was c o n f i r m e d in the p r e s e n t study that, as had been r e p o r t e d in the literatures (14,15), cigarette smoke generated both superoxide and hydrogen peroxide. These species are known to induce hemolysis (17), but the amounts of superoxide and hydrogen peroxide formed were too small to account for the hemolysis observed. Recently, Lee et al. (18) have reported that cigarette smoke increased sister chromatid exchanges in Chinese hamster ovary cells in a dose-dependent manner, but that the oxygen free radicals were not involved in them. Of course, this does not deny the toxic role of free radicals in cigarette smoke. In fact, it was observed that the cigarette smoke induced the free radical chain oxidation of m e t h y l linoleate (Minamisawa, Yamamoto, and Niki, unpublished work). However, the rate of chain initiation was not large enough to account for the hemolysis observed in the present study. In conclusion, although the nature of true active species is not known yet and more work is apparently required to elucidate the mechanism, this work shows that cigarette smoke induced the hemolysis of rabbit erythrocytes. Volatile components, rather than the free radicals, in cigarette smoke were suggested to be responsible for the hemolysis.
REFERENCES 1.
D.F. CHURCH and W. A. PRYOR, Environ. Health Perspect., 64 111-126 (1985), and references cited therein. 2. W.A. PRYOR, Brit J. Cancer, 55 Suppl. VIII, 19-23 (1987). 3. W.A. PRYOR, B. J. HALES, P. I. PREMOVEI, and D. F. CHURCH, Science, 220 425-427 (1983). 4. B. HALLIWELL and J. M. C. GUTTERIDGE, Free Radicals in Biology and Medicince, Clarendon Press, Oxford (1985). 5. H. Sies, (ed.) Oxidative Stress, Academic Press, London (1985). 6. M.G. SIMIC, K. A. TAYLOR, J. F. WARD, and C. von SONNTAG, (eds), Oxygen Radicals in Biology and Medicine, Plenum Press, New York (1989). 7. O. Hayaishi, E. Niki, M. Kondo, and T. Yoshikawa, (eds), Medical, Biochemical and Chemical Aspects of Free Radicals, Elsevier, Amsterdam (1989). 8. Y. YAMAMOTO, E. NIKI, Y. KAMIYA, M. MIKI, H. TAMAI and M. MINO, J. Nutr. Sci. Vitaminol., 32 475-479 (1986). 9. M. MIKI, H. TAMAI, M. MINO, Y. YAMAMOTO and E. NIKI, Arch. Biochem. Biophys. 258 373-380 (1987). 10. E. NIKI, Y. YAMAMOTO, M. TAKAHASHI, K.YAMAMOTO,
Vol. 47, No. 24, 1990
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