TOXICOLOGY
AND
Participation
APPLIED
PHARMACOLOGY
of Superoxide
46,29-38
(1978)
Free Radical Oxidation’
and Mn*+ in Sulfite
BUNJI INOUYE, MIKIKO IKEDA, TATSUO ISHIDA, MASANA SITSUO AKIYAMA,~ AND Kozo UTSUMI~
OGATA,~
Okayama Prefectural Research Centerfor Environment and Public Health I -1-l 7 Furugyoucho, Okayama-shi, Okayama 703, Japan, and Department of Biochemistry, Cancer Institute, and Department of Public Health, Okayama University Medical School, 2-S-I Shikata-rho, Okayama-shi, Okayama 700, Japan Received
September
29.1977;
accepted
January
23,1978
Participation of Superoxide Free Radical and Mn *+ in Sulfite Oxidation. INOUYE. B., IKEDA, M., ISHIDA, T., OGATA, M., AKIYAMA, J., AND UTSUMI, K. (1978). Toxicol. Appl. Pharmacol. 46, 29-38. Sulfurous acid gas is a well-known air pollutant. The participation of superoxide (Oi), a species of activated oxygen, in sulfite oxidation was investigated in relationship to this health hazard. The reduction of nitroblue tetrazolium (NBT) was markedly accelerated in the presence of the xanthine-xanthine oxidase system (X-X0), Mn2+ and SO:-, but not by X-X0 and Mn2+ or X-X0 and SO:- alone. This accelerated NBT reduction was partially suppressed by superoxide dismutase and was completely suppressed by allopurinol. Oxygen consumption was also markedly accelerated under the condition which caused the increase in NBT reduction. Lipid peroxidation of rat liver homogenate increased in the presence of X-X0, SO:-, or both. This increased lipid peroxidation was definitely suppressed by Mnl+. From these observations, it is suggested that chain reactions involving sulfite oxidation are imtiated by 0~ generated from X-X0, and Mn*+ acts as a catalyst in the process.
With the increased activity of the chemical industry, various noxious materials are exhausted into the air in large quantities giving rise to environmental destruction and creating a health hazard. Sulfurous acid gas (SOJ has been regarded as one of the primary causes of air pollution as well as nitrogen oxide, hydrogen carbon, and oxidants which are considered constituents of photochemical smog. The problems of air pollution by SO, have been frequently pointed out and a close epidemiological correlation between SO, concentration in the air and certain respiratory diseases has been made by several systematic investigations (Yoshida et al., 1966; Imai et al., 1967). However, there is a dearth of fundamental studies on the mechanism by which harmful actions of SO, develop in vim because of the complexity of its reactivity. It is notable that several radicals are formed in the process of the oxidation of SO, and that their formation is stimulated in the presence of transition metal ions such as Mn2+ (Hayatsu and Miller, 1972). On the other hand, the formation of superoxide i This work was presented in part at the Third Meeting for the Study of Toxic Effects, November 24, 1976, Tokyo. * Department of Public Health, Okayama University Medical School. 3 Niihama Medical Research Center. 4 Department of Biochemistry, Cancer Institute, Okayama University Medical School. 0041-008X/78/0461-002YKl2,0U~0 29 Copyright Q 1978 by Academic Press. inc. All rights of reproduction in any form reserved. Printed in Great Britain
30
INOUYE
ET AL.
radical (0,) in the course of redox reactions in uivo has been found (McCord and Fridovich, 1969; Massey et al., 1969; Hirata and Hayaishi, 1971; Rotilio et al., 1973; Fridovich, 1974). Studies on its harmful action (Lavelle et al., 1973) and protective mechanisms against it in living organisms (Fridovich, 1975) have been reported. The 0, participates also in the autooxidation of sulfite (Asada and &so, 1973; Yang, 1970), and sulfite ion or the resulting sulfite radical modifies DNA (Hayatsu and Miller, 1972) or RNA (Shapiro et al., 1970; Furuichi et al., 1970) in vitro and induces the mutation of phages (Hayatsu and Miura, 1970; Summers and Drake, 1971) or bacteria (Mukai et al., 1970). Moreover, these radicals react with important constituents of living organisms such as proteins (Cecil, 1963; Yang, 1970) and lipids (Pryor, 1971; Utsumi et al., 1973), suggesting that these radicals damage the maintenance of physiolgical functions of cells. This communication presents the results of experimental studies on the interaction between the oxidation of SO, and the Oi-producing system in living organisms. METHODS Measurement
of the 0, resulting from the xunthine-xunthine
oxiduse system. 0,
production by the X-X0 system was measured by the reduction of nitroblue tetrazolium (NBT) according to a modification of the method of Beauchamp and Fridovich (1971). The reaction was carried out in medium containing 150 mrvr choline chloride, 10 mM Tris-HCl (pH 7.4), 25 ,BM NBT, 0.1 mM xanthine, and 4 x 10mg M xanthine oxidase at 25 o C. Measurement of oxygen consumption. The change of oxygen concentration in the reaction medium was measured using a galvanic electrode in connection with an oxygen monitor. Measurement of lipid peroxidution. Rat liver homogenates were used as a lipid source. Liver was homogenized in 9 vol of 10 mM phosphate buffer (pH 7.4) with a Potter homogenizer. The amount of malondialdehyde produced by lipid peroxidation was measured according to the method of Hunter et al. (1963). Determination of protein concentration. Protein concentration was determined by the biuret reaction (Gornal et al., 1948) using bovine serum albumin as a standard. Reagents. Xanthine (grade I), xanthine oxidase (grade I), and NBT (grade III) were purchased from Sigma Chemical Co. Crude superoxide dismutase (SOD) was prepared from human blood cells according to the method of McCord and Fridovich (1969). Other chemicals used were commercial products of reagent grade. RESULTS 0, Production by X-X0
System and the E#ect of SOi- and Mn2+
Figure 1 shows the NBT reduction due to 0, production by the X-X0 system and the effect of SO:- and Mn2+ on NBT reduction. The NBT reduction induced by the X-X0 system was remarkably accelerated by successive addition of Mn2+ and SO:-. As shown in Figs. 2 and 3, the acceleration of NBT reduction depends on the concentrations of SO:- and Mn*+. On the other hand, this accelerated NBT reduction decreased to approximately 70%
SUPEROXIDE IN SULFITE OXIDATION
10unitimi SOD
Mn*+
1 0
TlilE
so;-
1 IN
;/
:: ; /
- Xanthine c/ L ..- . . . . ..___...r! ..___. M:N”TES 12 “‘0
16
FIG. 1. NBT reduction by the xanthine-xanthine oxidase system and the effect of SO:- and Mn?‘. Incubation was carried out in reaction medium containing 150 mM choline chloride. 10 mM Tris-HC1 buffer (pH 7.4), 0.1 mM xanthine, and 25 pM NBT at 25’C. Total incubation mixture was 3.0 ml. Additives were introduced at the points indicated and the absorbance at 560 nm was recorded.
FIG. 2. SOi- concentration dependence of NBT reduction by the xanthine-xanthine Basic experimental conditions were the same as in Fig. 1.
oxidasc system.
of the maximum level upon addition of SOD which decomposesthe 0; radical enzymatically (Fig. 1). The effect of allopurinol, an inhibitor of xanthine oxidase, on the NBT reduction is shown in Fig. 4. By the addition of allopurinol, the NBT reduction
32
INOUYEET
AL.
50
Imy
0
8 TIME
IN
025mM
12 MINUTES
16
20
FIG. 3. MI-?+ concentration dependence of NBT reduction by the xanthine-xanthine Basic experimental conditions were the same as in Fig. 1.
10 unit/ml SOD
-0.02
0
oxidase systkm.
1, T:ME
IN
12 MINUTES
16
20
FIG. 4. Acceleration of NBT reduction by SO:- and Mn2+ in the xanthine-xanthine the effect of allopurinol. Basic experimental conditions were the same as in Fig. 1.
oxidase system and
was suppressed to 27% of the maximum accelerated level in the presence of Mn*+ and SO:- (Fig. 4a). Addition of allopurinol prior to SO:- inhibited the NBT reduction almost completely. Relation between 0; Production by X-X0
System and Oxygen Consumption
Increase in NBT reduction which depends on 0, production by the X-X0 system suggests considerable oxygen consumption in the absence of NBT. As shown in Fig. 5a no notable change of oxygen concentration in the medium containing 0.1 mM xanthine could be induced by addition of xanthine oxidase and Mn*+. However, a remarkable acceleration of oxygen consumption was caused by successive addition of SO:- to the medium. A similar phenomenon was observed by changing the order of addition of Mn*+ and SOi- (Fig. 5b). A considerable, but slower oxygen consumption than in the two cases above was observed when only Mn*+ and SO:- were present in the
SUPEROXIDE
IN SULFITE
33
OXIDATION
I
200 natoms Oxygen
/ml
!
0
2
6 TIME
I 0
IN fiIN”TES
5. Oxygen consumption with the SOi- oxidation in the xanthine-xanthine oxidase system. Basic experimental conditions were the same as in Fig. 1 except there was no NBT in the medium. FIG.
FIG. 6. Effect of the xanthine-xanthine oxidase system on the lipid peroxidation of rat liver homogenate. Reaction medium contained 150 mM KCl, 10 mM Tris-HCI buffer (pH 7.4), liver homogenate (20 mg of protein), 1 mM xanthine and xanthine oxidase. The incubation mixture was 20 ml in volume and was incubated at 37°C with gentle shaking. After incubation, a 2-ml aliquot was withdrawn from the incubation mixture at 15, 30, 60, and 120 min, respectively, and delivered into a lo-ml Pyrex tube. The thiobarbituric acid reaction was carried out according to the method of Hunter et al. (1963). The color was read at 535 and 600 nm. The absorbance reading A,,,_,,, was graphed directly as lipid peroxide formation.
medium without X-X0 system (Fig. 5~). However, it was markedly accelerated to almost the same level as that shown in Fig. 5b by addition of the X-X0 system. From these results, it seems clear that the acceleration of 0; production by Mn*+ and SO:- in the presence of the X-X0 system is accompanied by oxygen consumption.
34 Effect of X-X0
INOUYE
ET
AL.
System, Mnzi, and SOi- on Lipid Peroxidation
It is assumed that the 0; resulting from several enzymatic reactions in vivo produces hydroxy radical (HO.), which induces peroxidation of unsaturated lipids of the biological membranes (Kellogg and Fridovich, 1975). Thus the effect of the 0, resulting from the X-X0 system on lipid peroxidation of rat liver homogenate was examined. As shown in Fig. 6, the amount of malondialdehyde (TBA reactant) formed in rat liver homogenate was small even after incubation for 2 hr in 150 mrvr KCl/lO mM TrisHCl buffer (pH 7.4) at 37V. The TBA value (A,,,-,,) in the presence of the X-X0 system increased remarkably depending on the xanthine oxidase concentration. The TBA reaction was accelerated also by adding SO:- (2 mM) with or without the X-X0
0 n 3 0
0
30
60 TIME
120 IN
MINUTES
FIG. 7. Effect of SOi- and Mn*+ on the lipid peroxidation of rat liver homogenate. Basic experimental conditions were the same as in Fig. 6.
system (Fig. 7). The increase in TBA reaction by SO:-, the X-X0 definitely suppressed by Mn*+ (2 mM) to under the control level.
system, or both was
DISCUSSION
In aerobic organisms, oxygen molecules are used as an electron acceptor for energy production under the control of enzymatic systems. However, without protective mechanisms against activated oxygen released from intermediate steps of various redox reactions in vivo, no living organisms could escape from damage by the toxicity of activated oxygen. 0, is formed by adding one electron to an oxygen molecule and is released in the intermediate steps of enzymatic reactions in vivo such as that catalyzed by xanthine oxidase and aldehyde oxidase (Massey et al., 1969; Hirata and Hayaishi, 1971; Rotilio et al., 1973). As to the toxicity of activated oxygen, 0, is most important because it is transformed nonenzymatically into other forms of activated oxygen as shown in the following equations (Haber and Weiss, 1934): Of+H,O,+H++HO~+H,O+‘O,, O;+H,O,+Oz+OH-+HO..
(1)
(2)
35
SUPEROXIDE IN SULFITE OXIDATION
SOD is found in every aerobic cell. It catalyzes reaction (3) as shown, and therefore protects cells against the toxic actions of 0; and other radicals derived from it (McCord and Fridovich, 1969; Fridovich, 1974, 1975): 20; + 2H+ -
The O,-producing
reactions of the X-X0
SOD
0, + H,O,
system are shown as follows:
Xanthine G-+
uric acid.
20,
2H+
(4)
20,
It is proposed that the remarkable acceleration of NBT reduction by adding SO:- in the presence of MnZ+ is due to the chain reactions as shown in Fig. 8 (Asada and Kiso,
2H’ -‘j Formazan
sot-
FIG. 8. Schematic reactions of SO:- oxidation in the presence of the xanthine-xanthine MIPf.
oxidase
and
1973; Yang, 1970). Mn*+ probably acts as a catalyst in these reactions though further investigations are necessary for a sufficient explanation of the definite mechanism. An exceedingly rapid oxygen consumption by Mn*+ and SO:- in the presence of the X-X0 system (Fig. 5a) is comprehensible from the chain reactions as shown in Fig. 8. However, in spite of a considerable oxygen consumption induced by Mn*+ and SO:without the X-X0 system (Fig. SC), no NBT reduction was observed (Fig. lc). This is probably due to a low 0; production in this condition. Several species of radicals resulting from the following reactions (Fig. 8) are likely to be associated with the acceleration of NBT reduction by Mn*+ and SO:- in the presence of the X-X0 system. For example, uric acid formation by the X-X0 system was inhibited in the presence of both Mn*+ and SO:-, and the NBT reduction was suppressed by hydroquinone, a . HSO, scavenger (unpublished observation). These suggest that the accelerated NBT reduction by Mn*+ and SO:- in the presence of the X-X0 system depends not only on 0, but also on other radicals such as . HSO, formed in the process of SO:- oxidation.
36
INOUYE
ET
AL
Suppression by allopurinol of the NBT reduction by the X-X0 system (Fig. 4c) suggests that 0, is required for the initiation of NBT reduction in the presence of Mn2+ and SO:-. Based on the fact that uric acid formation was suppressed in this case, it is assumed that a trace amount of 0, acts as an initiator. Therefore, it is probable that, once 0, is formed, successive reactions proceed automatically. Incomplete inhibition of the accelerated NBT reduction by SOD or allopurinol is probably due to the reason mentioned above. This consideration is supported by the fact that addition of allopurinol prior to SO:- suppressed NBT reduction.almost completely. On the other hand, it can be expected that 0; and other radicals derived from 0, in uivo accelerate the peroxidation of unsaturated lipids contained in biological membranes (Kellogg and Fridovich, 1975; Pederson and Aust, 1973; Zimmermann et al., 1973; Tyler, 1975). Acceleration of lipid peroxidation was demonstrated in the peroxidative reactions of linoleic acid with the acetaldehyde-X0 system (Kellogg and Fridovich, 1975) or of mitochondrial lipids with SO:- (Tyler, 1975). Our result that the TBA reaction in liver homogenate was accelerated by the X-X0 system or SO:- agrees with their data. However, the TBA reactions was decreased remarkably by Mn*+ in spite of the considerable increase in NBT reduction produced by Mn2+ and SO:-. Kaplan et al. (1975) obtained a similar result in their experiments. They hypothesized that this phenomenon was due to an initial oxidation via catalysis by a transition metal contained as a trace contaminant in their buffer and by free radical-induced peroxidation, which produced products that were not TBA-reactive materials. Further oxidation of these products to TBA-reactive materials may then be inhibited by added Mn2+. Furthermore the stimulated radical production by Mn2+ and SO:- may be utilized for other reactions, for example, the reduction of cytochrome c (Yang, 1970) or Fe3+ (Williams et al., 1974) or the oxidation of epinephrine (McCord and Fridovich, 1969) or catecholamine (Miller and Rapp, 1973). In examining the relationship between the acceleration of NBT reduction and lipid peroxidation, the catabolic reactions of lipid peroxides as well as the SOD-like action of Mn2+ must be considered. This communication describes the results of a model experiment undertaken to elucidate the toxic effect of SO, on living organisms. It is notable that NBT reduction and lipid peroxidation can be altered at the SO:- concentration which is within the range experienced during acute episodes of SO, exposure.5 In addition, it should be emphasized that the effect of various kinds of pollutants on living organisms must be investigated from the point of view that their harmful actions depend on their interaction with other substances rather than on each one’s individual action. REFERENCES ASADA, K., AND KISO, K. (1973). Initiation of aerobic oxidation of sulfite by illuminated spinach
chloroplasts. Eur. J. Biochem.33, 253-257. BEAUCHAMP, C., AND FRIDOVICH, I. (1971). Superoxide dismutase: Improved assays and an
assay applicable to acrylamide gels. Anal. Biochem.44, 276-287. bonds in proteins. I. The role of sulfur in proteins. In The Proteins, Composition,Structure, Function (H. Neurath, ed.), 2nd ed., Vol. 1, pp. 379-476. Academic Press, New York.
CECIL, R. (1963). Intramolecular
5 Criteria for a Recommended Standard. Occupational Department of Health, Education.
Exposure to Sulfur Dioxide (1974). U.S.
SUPEROXIDE
IN SULFITE
37
OXIDATION
I. (1974). Superoxide dismutases. Advan. Enzymol. 41,35-97. I. (1975). Superoxidedismutases. Annu. Rev. Biochem.44, 147-159. Y., WATAYA, Y., HAYATSU, H., AND UKITA, T. (1970). Chemicalmodificationof tRNAF$ with bisulfite. A new method to modify isopentenyladenosine residue.Biochem. Biophys.Res.Commun.41, 1185-l 191. GORNAL, A. G., BARDAWILL, C. J., AND DAVID, M. M. (1948).Determinationof serumproteins by meansof the biuret reaction.J. Biol. Chem.177, 75 l-766. HABER, F., AND WEISS, J. (1934). The catalytic decompositionof hydrogenperoxideby iron salts.Proc. Roy. Sot. LondonA 147, 332-35 1. HAYATSU, H., AND MILLER, R. C. JR. (1972). The cleavageof DNA by the oxygen-dependent reactionof bisulfite.Biochem.Biophys.Res.Commun.46, 120- 124. HAYATSU, H., AND MIURA, A. (1970). The mutagenicaction of sodiumbisulfite.Biochem. Biophys.Res.Commun.39, 156-160. HIRATA, F., AND HAYAISHI, 0. (1971). Possibleparticipation of superoxideanion in the intestinaltryptophan 2,3-dioxygenasereaction.J. Biol. Chem.246, 7825-7826. HUNTER, F. F., JR., GEBICKI, J. M., HOFFSTEN, P. E., WEINSTEIN, J., AND SCOTT, A. (1963). Swellingandlysis of rat liver mitochondriainducedby ferrousions.J. Biol. Chem.238, 828FRIDOVICH, FRIDOVICH, FURUICHI,
835.
IMAI, M., OSHIMA,H., TAKATSUKA,Y., ANDYOSHIDA,K. (1967). On the Yokkaichi-asthma (Review).Japan.J. Hyg. 22, 323-335. KAPLAN, D., MCJILTON, C., AND LUCHTEL. D. (1975).Bisulfite inducedlipid oxidation. Arch. Environ. Health 30, 507-509. KELLOGG, E. W., AND FRIDOVICH, I. (1975).Superoxide,hydrogenperoxideand singletoxygen in lipid peroxidationby xanthineoxidasesystem.J. Biol. Chem.250, 8812-8817. LAVELLE,F., MICHELSON, A. M., AND DIMITRIJEVIC, L. (1973). Biologicalprotectionby superoxide dismutase. Biochem.Biophys.Res.Commun.55, 350-357. MASSEY, V., STRICKLAND, S., MAYHEW, S. G., HOWELL, L. G., ENGEL, P. C., MATTHEWS, R. G., SCHUMAN, M., AND SULLIVAN, P. A. (1969). The production of superoxideanion radicalsin the reactionof reducedflavins and flavoproteinswith molecularoxygen.Biochem. Biophys.Res.Commun.36,89 l-897. MCCORD, J. M., AND FRIDOVICH, I. (1969). Superoxidedismutase.An enzymic function for erythrocuprein(hemocuprein).J. Biol. Chem.244, 6049-6055. MILLER, R. W., AND RAPP, U. (1973). The oxidation of catecholsby reducedflavins and dehydrogenases. An electron spin resonancestudy of the kinetics and initial products of oxidation.J. Biol. Chem.248, 60846090. MUKAI, F., HAWRYLUK, I., AND SCHAPIRO, R. (1970). The mutagenicspecificity of sodium bisulfite.Biochem.Biophys.Res.Commun.39,983-988. PEDERSON. T. C., AND AUST, S. D. (1973). The role of superoxideand singletoxygen in lipid peroxidation promoted by xanthine oxidase.Biochem.Biophys. Res. Commun.52, 10711078. PRYOR, W. A. (1971). Free radical pathology. Chem.Eng. News49,34-36. ROTILIO, G., CALABRESE, L., FINAZZI-AGR~, A., ARGENTO-CERU, M. P., AUTUORI, F., AND MONDOVi, B. (1973). Intracellular localizationof superoxidedismutaseand its relation to the
distribution and mechanismof hydrogen peroxide-producingenzymes.Biochim. Biophys. Acta 321, 98-102. SHAPIRO, R., COHEN, B. I., AND SERVIS, R. E. (1970). Specificdeamination of RNA by sodium bisulfite.Nature (London) 227, 1047-1048. SUMMERS, G. A., AND DRAKE, J. W. (1971).Bisulfite mutagenesis in bacteriophage T4. Genetics 68,603-607. TYLER, D. D. (1975). Role of superoxide radicals membranes. FEBS Lett. 51, 180-183. UTSUMI, K., HASEGAWA, T., AND OGATA, M. (1973).
in the Bisulfite
lipid
peroxidation
induced
lipid
of intracellular peroxidation
of rat
liver mitochondriaand the inhibition of the reaction by radical scavenger.Photosensitising Dyes83, 3l-36 lin Japanese]. WILLIAMS, D. M., LEE, G. R., AND CARTWRIGHT, G. E. (1974). The role of superoxideanion radicalin the reductionof ferritin iron by xanthineoxidase.J. CZin.Invest. 53, 665-667.
38
INOUYE
ET AL.
S. F. (1970). Sulfoxide formation from methionine or its sulfide analogs during aerobic oxidation of sulfite. Biochemistry 9, 5008-5014. YOSHIDA, K., OSHIMA, H., AND IMAI, M. (1966). Air pollution and asthma in Yokkaichi. Arch. Environ. Health 13, 763-768. ZIMMERMANN, R., FLOHB, L., WESER, U., AND HARTMANN, H.-J. (1973). Inhibition of lipid peroxidationin isolatedinner membraneof rat liver mitochondriaby superoxidedismutase. FEBS Lett. 29, 117- 120. YANG,
AND
Participation
APPLIED
PHARMACOLOGY
of Superoxide
46,29-38
(1978)
Free Radical Oxidation’
and Mn*+ in Sulfite
BUNJI INOUYE, MIKIKO IKEDA, TATSUO ISHIDA, MASANA SITSUO AKIYAMA,~ AND Kozo UTSUMI~
OGATA,~
Okayama Prefectural Research Centerfor Environment and Public Health I -1-l 7 Furugyoucho, Okayama-shi, Okayama 703, Japan, and Department of Biochemistry, Cancer Institute, and Department of Public Health, Okayama University Medical School, 2-S-I Shikata-rho, Okayama-shi, Okayama 700, Japan Received
September
29.1977;
accepted
January
23,1978
Participation of Superoxide Free Radical and Mn *+ in Sulfite Oxidation. INOUYE. B., IKEDA, M., ISHIDA, T., OGATA, M., AKIYAMA, J., AND UTSUMI, K. (1978). Toxicol. Appl. Pharmacol. 46, 29-38. Sulfurous acid gas is a well-known air pollutant. The participation of superoxide (Oi), a species of activated oxygen, in sulfite oxidation was investigated in relationship to this health hazard. The reduction of nitroblue tetrazolium (NBT) was markedly accelerated in the presence of the xanthine-xanthine oxidase system (X-X0), Mn2+ and SO:-, but not by X-X0 and Mn2+ or X-X0 and SO:- alone. This accelerated NBT reduction was partially suppressed by superoxide dismutase and was completely suppressed by allopurinol. Oxygen consumption was also markedly accelerated under the condition which caused the increase in NBT reduction. Lipid peroxidation of rat liver homogenate increased in the presence of X-X0, SO:-, or both. This increased lipid peroxidation was definitely suppressed by Mnl+. From these observations, it is suggested that chain reactions involving sulfite oxidation are imtiated by 0~ generated from X-X0, and Mn*+ acts as a catalyst in the process.
With the increased activity of the chemical industry, various noxious materials are exhausted into the air in large quantities giving rise to environmental destruction and creating a health hazard. Sulfurous acid gas (SOJ has been regarded as one of the primary causes of air pollution as well as nitrogen oxide, hydrogen carbon, and oxidants which are considered constituents of photochemical smog. The problems of air pollution by SO, have been frequently pointed out and a close epidemiological correlation between SO, concentration in the air and certain respiratory diseases has been made by several systematic investigations (Yoshida et al., 1966; Imai et al., 1967). However, there is a dearth of fundamental studies on the mechanism by which harmful actions of SO, develop in vim because of the complexity of its reactivity. It is notable that several radicals are formed in the process of the oxidation of SO, and that their formation is stimulated in the presence of transition metal ions such as Mn2+ (Hayatsu and Miller, 1972). On the other hand, the formation of superoxide i This work was presented in part at the Third Meeting for the Study of Toxic Effects, November 24, 1976, Tokyo. * Department of Public Health, Okayama University Medical School. 3 Niihama Medical Research Center. 4 Department of Biochemistry, Cancer Institute, Okayama University Medical School. 0041-008X/78/0461-002YKl2,0U~0 29 Copyright Q 1978 by Academic Press. inc. All rights of reproduction in any form reserved. Printed in Great Britain
30
INOUYE
ET AL.
radical (0,) in the course of redox reactions in uivo has been found (McCord and Fridovich, 1969; Massey et al., 1969; Hirata and Hayaishi, 1971; Rotilio et al., 1973; Fridovich, 1974). Studies on its harmful action (Lavelle et al., 1973) and protective mechanisms against it in living organisms (Fridovich, 1975) have been reported. The 0, participates also in the autooxidation of sulfite (Asada and &so, 1973; Yang, 1970), and sulfite ion or the resulting sulfite radical modifies DNA (Hayatsu and Miller, 1972) or RNA (Shapiro et al., 1970; Furuichi et al., 1970) in vitro and induces the mutation of phages (Hayatsu and Miura, 1970; Summers and Drake, 1971) or bacteria (Mukai et al., 1970). Moreover, these radicals react with important constituents of living organisms such as proteins (Cecil, 1963; Yang, 1970) and lipids (Pryor, 1971; Utsumi et al., 1973), suggesting that these radicals damage the maintenance of physiolgical functions of cells. This communication presents the results of experimental studies on the interaction between the oxidation of SO, and the Oi-producing system in living organisms. METHODS Measurement
of the 0, resulting from the xunthine-xunthine
oxiduse system. 0,
production by the X-X0 system was measured by the reduction of nitroblue tetrazolium (NBT) according to a modification of the method of Beauchamp and Fridovich (1971). The reaction was carried out in medium containing 150 mrvr choline chloride, 10 mM Tris-HCl (pH 7.4), 25 ,BM NBT, 0.1 mM xanthine, and 4 x 10mg M xanthine oxidase at 25 o C. Measurement of oxygen consumption. The change of oxygen concentration in the reaction medium was measured using a galvanic electrode in connection with an oxygen monitor. Measurement of lipid peroxidution. Rat liver homogenates were used as a lipid source. Liver was homogenized in 9 vol of 10 mM phosphate buffer (pH 7.4) with a Potter homogenizer. The amount of malondialdehyde produced by lipid peroxidation was measured according to the method of Hunter et al. (1963). Determination of protein concentration. Protein concentration was determined by the biuret reaction (Gornal et al., 1948) using bovine serum albumin as a standard. Reagents. Xanthine (grade I), xanthine oxidase (grade I), and NBT (grade III) were purchased from Sigma Chemical Co. Crude superoxide dismutase (SOD) was prepared from human blood cells according to the method of McCord and Fridovich (1969). Other chemicals used were commercial products of reagent grade. RESULTS 0, Production by X-X0
System and the E#ect of SOi- and Mn2+
Figure 1 shows the NBT reduction due to 0, production by the X-X0 system and the effect of SO:- and Mn2+ on NBT reduction. The NBT reduction induced by the X-X0 system was remarkably accelerated by successive addition of Mn2+ and SO:-. As shown in Figs. 2 and 3, the acceleration of NBT reduction depends on the concentrations of SO:- and Mn*+. On the other hand, this accelerated NBT reduction decreased to approximately 70%
SUPEROXIDE IN SULFITE OXIDATION
10unitimi SOD
Mn*+
1 0
TlilE
so;-
1 IN
;/
:: ; /
- Xanthine c/ L ..- . . . . ..___...r! ..___. M:N”TES 12 “‘0
16
FIG. 1. NBT reduction by the xanthine-xanthine oxidase system and the effect of SO:- and Mn?‘. Incubation was carried out in reaction medium containing 150 mM choline chloride. 10 mM Tris-HC1 buffer (pH 7.4), 0.1 mM xanthine, and 25 pM NBT at 25’C. Total incubation mixture was 3.0 ml. Additives were introduced at the points indicated and the absorbance at 560 nm was recorded.
FIG. 2. SOi- concentration dependence of NBT reduction by the xanthine-xanthine Basic experimental conditions were the same as in Fig. 1.
oxidasc system.
of the maximum level upon addition of SOD which decomposesthe 0; radical enzymatically (Fig. 1). The effect of allopurinol, an inhibitor of xanthine oxidase, on the NBT reduction is shown in Fig. 4. By the addition of allopurinol, the NBT reduction
32
INOUYEET
AL.
50
Imy
0
8 TIME
IN
025mM
12 MINUTES
16
20
FIG. 3. MI-?+ concentration dependence of NBT reduction by the xanthine-xanthine Basic experimental conditions were the same as in Fig. 1.
10 unit/ml SOD
-0.02
0
oxidase systkm.
1, T:ME
IN
12 MINUTES
16
20
FIG. 4. Acceleration of NBT reduction by SO:- and Mn2+ in the xanthine-xanthine the effect of allopurinol. Basic experimental conditions were the same as in Fig. 1.
oxidase system and
was suppressed to 27% of the maximum accelerated level in the presence of Mn*+ and SO:- (Fig. 4a). Addition of allopurinol prior to SO:- inhibited the NBT reduction almost completely. Relation between 0; Production by X-X0
System and Oxygen Consumption
Increase in NBT reduction which depends on 0, production by the X-X0 system suggests considerable oxygen consumption in the absence of NBT. As shown in Fig. 5a no notable change of oxygen concentration in the medium containing 0.1 mM xanthine could be induced by addition of xanthine oxidase and Mn*+. However, a remarkable acceleration of oxygen consumption was caused by successive addition of SO:- to the medium. A similar phenomenon was observed by changing the order of addition of Mn*+ and SOi- (Fig. 5b). A considerable, but slower oxygen consumption than in the two cases above was observed when only Mn*+ and SO:- were present in the
SUPEROXIDE
IN SULFITE
33
OXIDATION
I
200 natoms Oxygen
/ml
!
0
2
6 TIME
I 0
IN fiIN”TES
5. Oxygen consumption with the SOi- oxidation in the xanthine-xanthine oxidase system. Basic experimental conditions were the same as in Fig. 1 except there was no NBT in the medium. FIG.
FIG. 6. Effect of the xanthine-xanthine oxidase system on the lipid peroxidation of rat liver homogenate. Reaction medium contained 150 mM KCl, 10 mM Tris-HCI buffer (pH 7.4), liver homogenate (20 mg of protein), 1 mM xanthine and xanthine oxidase. The incubation mixture was 20 ml in volume and was incubated at 37°C with gentle shaking. After incubation, a 2-ml aliquot was withdrawn from the incubation mixture at 15, 30, 60, and 120 min, respectively, and delivered into a lo-ml Pyrex tube. The thiobarbituric acid reaction was carried out according to the method of Hunter et al. (1963). The color was read at 535 and 600 nm. The absorbance reading A,,,_,,, was graphed directly as lipid peroxide formation.
medium without X-X0 system (Fig. 5~). However, it was markedly accelerated to almost the same level as that shown in Fig. 5b by addition of the X-X0 system. From these results, it seems clear that the acceleration of 0; production by Mn*+ and SO:- in the presence of the X-X0 system is accompanied by oxygen consumption.
34 Effect of X-X0
INOUYE
ET
AL.
System, Mnzi, and SOi- on Lipid Peroxidation
It is assumed that the 0; resulting from several enzymatic reactions in vivo produces hydroxy radical (HO.), which induces peroxidation of unsaturated lipids of the biological membranes (Kellogg and Fridovich, 1975). Thus the effect of the 0, resulting from the X-X0 system on lipid peroxidation of rat liver homogenate was examined. As shown in Fig. 6, the amount of malondialdehyde (TBA reactant) formed in rat liver homogenate was small even after incubation for 2 hr in 150 mrvr KCl/lO mM TrisHCl buffer (pH 7.4) at 37V. The TBA value (A,,,-,,) in the presence of the X-X0 system increased remarkably depending on the xanthine oxidase concentration. The TBA reaction was accelerated also by adding SO:- (2 mM) with or without the X-X0
0 n 3 0
0
30
60 TIME
120 IN
MINUTES
FIG. 7. Effect of SOi- and Mn*+ on the lipid peroxidation of rat liver homogenate. Basic experimental conditions were the same as in Fig. 6.
system (Fig. 7). The increase in TBA reaction by SO:-, the X-X0 definitely suppressed by Mn*+ (2 mM) to under the control level.
system, or both was
DISCUSSION
In aerobic organisms, oxygen molecules are used as an electron acceptor for energy production under the control of enzymatic systems. However, without protective mechanisms against activated oxygen released from intermediate steps of various redox reactions in vivo, no living organisms could escape from damage by the toxicity of activated oxygen. 0, is formed by adding one electron to an oxygen molecule and is released in the intermediate steps of enzymatic reactions in vivo such as that catalyzed by xanthine oxidase and aldehyde oxidase (Massey et al., 1969; Hirata and Hayaishi, 1971; Rotilio et al., 1973). As to the toxicity of activated oxygen, 0, is most important because it is transformed nonenzymatically into other forms of activated oxygen as shown in the following equations (Haber and Weiss, 1934): Of+H,O,+H++HO~+H,O+‘O,, O;+H,O,+Oz+OH-+HO..
(1)
(2)
35
SUPEROXIDE IN SULFITE OXIDATION
SOD is found in every aerobic cell. It catalyzes reaction (3) as shown, and therefore protects cells against the toxic actions of 0; and other radicals derived from it (McCord and Fridovich, 1969; Fridovich, 1974, 1975): 20; + 2H+ -
The O,-producing
reactions of the X-X0
SOD
0, + H,O,
system are shown as follows:
Xanthine G-+
uric acid.
20,
2H+
(4)
20,
It is proposed that the remarkable acceleration of NBT reduction by adding SO:- in the presence of MnZ+ is due to the chain reactions as shown in Fig. 8 (Asada and Kiso,
2H’ -‘j Formazan
sot-
FIG. 8. Schematic reactions of SO:- oxidation in the presence of the xanthine-xanthine MIPf.
oxidase
and
1973; Yang, 1970). Mn*+ probably acts as a catalyst in these reactions though further investigations are necessary for a sufficient explanation of the definite mechanism. An exceedingly rapid oxygen consumption by Mn*+ and SO:- in the presence of the X-X0 system (Fig. 5a) is comprehensible from the chain reactions as shown in Fig. 8. However, in spite of a considerable oxygen consumption induced by Mn*+ and SO:without the X-X0 system (Fig. SC), no NBT reduction was observed (Fig. lc). This is probably due to a low 0; production in this condition. Several species of radicals resulting from the following reactions (Fig. 8) are likely to be associated with the acceleration of NBT reduction by Mn*+ and SO:- in the presence of the X-X0 system. For example, uric acid formation by the X-X0 system was inhibited in the presence of both Mn*+ and SO:-, and the NBT reduction was suppressed by hydroquinone, a . HSO, scavenger (unpublished observation). These suggest that the accelerated NBT reduction by Mn*+ and SO:- in the presence of the X-X0 system depends not only on 0, but also on other radicals such as . HSO, formed in the process of SO:- oxidation.
36
INOUYE
ET
AL
Suppression by allopurinol of the NBT reduction by the X-X0 system (Fig. 4c) suggests that 0, is required for the initiation of NBT reduction in the presence of Mn2+ and SO:-. Based on the fact that uric acid formation was suppressed in this case, it is assumed that a trace amount of 0, acts as an initiator. Therefore, it is probable that, once 0, is formed, successive reactions proceed automatically. Incomplete inhibition of the accelerated NBT reduction by SOD or allopurinol is probably due to the reason mentioned above. This consideration is supported by the fact that addition of allopurinol prior to SO:- suppressed NBT reduction.almost completely. On the other hand, it can be expected that 0; and other radicals derived from 0, in uivo accelerate the peroxidation of unsaturated lipids contained in biological membranes (Kellogg and Fridovich, 1975; Pederson and Aust, 1973; Zimmermann et al., 1973; Tyler, 1975). Acceleration of lipid peroxidation was demonstrated in the peroxidative reactions of linoleic acid with the acetaldehyde-X0 system (Kellogg and Fridovich, 1975) or of mitochondrial lipids with SO:- (Tyler, 1975). Our result that the TBA reaction in liver homogenate was accelerated by the X-X0 system or SO:- agrees with their data. However, the TBA reactions was decreased remarkably by Mn*+ in spite of the considerable increase in NBT reduction produced by Mn2+ and SO:-. Kaplan et al. (1975) obtained a similar result in their experiments. They hypothesized that this phenomenon was due to an initial oxidation via catalysis by a transition metal contained as a trace contaminant in their buffer and by free radical-induced peroxidation, which produced products that were not TBA-reactive materials. Further oxidation of these products to TBA-reactive materials may then be inhibited by added Mn2+. Furthermore the stimulated radical production by Mn2+ and SO:- may be utilized for other reactions, for example, the reduction of cytochrome c (Yang, 1970) or Fe3+ (Williams et al., 1974) or the oxidation of epinephrine (McCord and Fridovich, 1969) or catecholamine (Miller and Rapp, 1973). In examining the relationship between the acceleration of NBT reduction and lipid peroxidation, the catabolic reactions of lipid peroxides as well as the SOD-like action of Mn2+ must be considered. This communication describes the results of a model experiment undertaken to elucidate the toxic effect of SO, on living organisms. It is notable that NBT reduction and lipid peroxidation can be altered at the SO:- concentration which is within the range experienced during acute episodes of SO, exposure.5 In addition, it should be emphasized that the effect of various kinds of pollutants on living organisms must be investigated from the point of view that their harmful actions depend on their interaction with other substances rather than on each one’s individual action. REFERENCES ASADA, K., AND KISO, K. (1973). Initiation of aerobic oxidation of sulfite by illuminated spinach
chloroplasts. Eur. J. Biochem.33, 253-257. BEAUCHAMP, C., AND FRIDOVICH, I. (1971). Superoxide dismutase: Improved assays and an
assay applicable to acrylamide gels. Anal. Biochem.44, 276-287. bonds in proteins. I. The role of sulfur in proteins. In The Proteins, Composition,Structure, Function (H. Neurath, ed.), 2nd ed., Vol. 1, pp. 379-476. Academic Press, New York.
CECIL, R. (1963). Intramolecular
5 Criteria for a Recommended Standard. Occupational Department of Health, Education.
Exposure to Sulfur Dioxide (1974). U.S.
SUPEROXIDE
IN SULFITE
37
OXIDATION
I. (1974). Superoxide dismutases. Advan. Enzymol. 41,35-97. I. (1975). Superoxidedismutases. Annu. Rev. Biochem.44, 147-159. Y., WATAYA, Y., HAYATSU, H., AND UKITA, T. (1970). Chemicalmodificationof tRNAF$ with bisulfite. A new method to modify isopentenyladenosine residue.Biochem. Biophys.Res.Commun.41, 1185-l 191. GORNAL, A. G., BARDAWILL, C. J., AND DAVID, M. M. (1948).Determinationof serumproteins by meansof the biuret reaction.J. Biol. Chem.177, 75 l-766. HABER, F., AND WEISS, J. (1934). The catalytic decompositionof hydrogenperoxideby iron salts.Proc. Roy. Sot. LondonA 147, 332-35 1. HAYATSU, H., AND MILLER, R. C. JR. (1972). The cleavageof DNA by the oxygen-dependent reactionof bisulfite.Biochem.Biophys.Res.Commun.46, 120- 124. HAYATSU, H., AND MIURA, A. (1970). The mutagenicaction of sodiumbisulfite.Biochem. Biophys.Res.Commun.39, 156-160. HIRATA, F., AND HAYAISHI, 0. (1971). Possibleparticipation of superoxideanion in the intestinaltryptophan 2,3-dioxygenasereaction.J. Biol. Chem.246, 7825-7826. HUNTER, F. F., JR., GEBICKI, J. M., HOFFSTEN, P. E., WEINSTEIN, J., AND SCOTT, A. (1963). Swellingandlysis of rat liver mitochondriainducedby ferrousions.J. Biol. Chem.238, 828FRIDOVICH, FRIDOVICH, FURUICHI,
835.
IMAI, M., OSHIMA,H., TAKATSUKA,Y., ANDYOSHIDA,K. (1967). On the Yokkaichi-asthma (Review).Japan.J. Hyg. 22, 323-335. KAPLAN, D., MCJILTON, C., AND LUCHTEL. D. (1975).Bisulfite inducedlipid oxidation. Arch. Environ. Health 30, 507-509. KELLOGG, E. W., AND FRIDOVICH, I. (1975).Superoxide,hydrogenperoxideand singletoxygen in lipid peroxidationby xanthineoxidasesystem.J. Biol. Chem.250, 8812-8817. LAVELLE,F., MICHELSON, A. M., AND DIMITRIJEVIC, L. (1973). Biologicalprotectionby superoxide dismutase. Biochem.Biophys.Res.Commun.55, 350-357. MASSEY, V., STRICKLAND, S., MAYHEW, S. G., HOWELL, L. G., ENGEL, P. C., MATTHEWS, R. G., SCHUMAN, M., AND SULLIVAN, P. A. (1969). The production of superoxideanion radicalsin the reactionof reducedflavins and flavoproteinswith molecularoxygen.Biochem. Biophys.Res.Commun.36,89 l-897. MCCORD, J. M., AND FRIDOVICH, I. (1969). Superoxidedismutase.An enzymic function for erythrocuprein(hemocuprein).J. Biol. Chem.244, 6049-6055. MILLER, R. W., AND RAPP, U. (1973). The oxidation of catecholsby reducedflavins and dehydrogenases. An electron spin resonancestudy of the kinetics and initial products of oxidation.J. Biol. Chem.248, 60846090. MUKAI, F., HAWRYLUK, I., AND SCHAPIRO, R. (1970). The mutagenicspecificity of sodium bisulfite.Biochem.Biophys.Res.Commun.39,983-988. PEDERSON. T. C., AND AUST, S. D. (1973). The role of superoxideand singletoxygen in lipid peroxidation promoted by xanthine oxidase.Biochem.Biophys. Res. Commun.52, 10711078. PRYOR, W. A. (1971). Free radical pathology. Chem.Eng. News49,34-36. ROTILIO, G., CALABRESE, L., FINAZZI-AGR~, A., ARGENTO-CERU, M. P., AUTUORI, F., AND MONDOVi, B. (1973). Intracellular localizationof superoxidedismutaseand its relation to the
distribution and mechanismof hydrogen peroxide-producingenzymes.Biochim. Biophys. Acta 321, 98-102. SHAPIRO, R., COHEN, B. I., AND SERVIS, R. E. (1970). Specificdeamination of RNA by sodium bisulfite.Nature (London) 227, 1047-1048. SUMMERS, G. A., AND DRAKE, J. W. (1971).Bisulfite mutagenesis in bacteriophage T4. Genetics 68,603-607. TYLER, D. D. (1975). Role of superoxide radicals membranes. FEBS Lett. 51, 180-183. UTSUMI, K., HASEGAWA, T., AND OGATA, M. (1973).
in the Bisulfite
lipid
peroxidation
induced
lipid
of intracellular peroxidation
of rat
liver mitochondriaand the inhibition of the reaction by radical scavenger.Photosensitising Dyes83, 3l-36 lin Japanese]. WILLIAMS, D. M., LEE, G. R., AND CARTWRIGHT, G. E. (1974). The role of superoxideanion radicalin the reductionof ferritin iron by xanthineoxidase.J. CZin.Invest. 53, 665-667.
38
INOUYE
ET AL.
S. F. (1970). Sulfoxide formation from methionine or its sulfide analogs during aerobic oxidation of sulfite. Biochemistry 9, 5008-5014. YOSHIDA, K., OSHIMA, H., AND IMAI, M. (1966). Air pollution and asthma in Yokkaichi. Arch. Environ. Health 13, 763-768. ZIMMERMANN, R., FLOHB, L., WESER, U., AND HARTMANN, H.-J. (1973). Inhibition of lipid peroxidationin isolatedinner membraneof rat liver mitochondriaby superoxidedismutase. FEBS Lett. 29, 117- 120. YANG,
