ENVIRONMENTAL
RESEARCH
9, 55-65
Biochemistry
(1975)
of Bisulfite-Sulfur
Dioxide
DAVLLI H. PETERING AND NANCY TING Sm Department
of
Chemisty, Milwaukee, Received
University Wisconsin
of
December
13,
Wisconsin-Milwaukee, 53201 1973
An analysis of the metabolic transformations of sulfur dioxide is made. Then, the biochemical reactivity of two of its forms, bisulfite and S-thiosulfates, is surveyed in order to provide a view of the potential reactivity of sulfur dioxide with biological systems. Finally, these findings are used as a model for the consideration of threshold responses of organisms to agents in the environment.
Sulfur dioxide is a major air pollutant in many large cities. It is, therefore, a major concern to determine the extent of its hazard to health, in particular the with possible long-term effects of exposure. Although numerous experiments animals have been carried out to discover the biological effects of sulfur dioxide, results have been ambivalent concerning its toxicity at low concentrations ( Alarie, 1972; Lewis, 1973; Rylander, 1969; Zarkower, 1972). This may be attributed to at least 3 difficulties in establishing a causal relationship between airborne SO, and detrimental effects in test organisms: (1) At the low concentrations present in urban air, sulfur dioxide does not produce acute, toxic effects in carefully maintained laboratory animals. This does not exclude the presence of subtle, slowly developing effects but does make difficult the observation of a causal relationship between the imposition of the pollutant and organismic responses. (2) Only a selection of an animal’s myriad physiological and biochemical processes may be monitored in any experiment, Important responses may simply be missed because of the limitation of parameters observed or may be lost in the midst of the numerous responses seen. This is particularly true at the biochemical level where the variety of alterations in the concentrations of materials is bewildering (Schmidt, 1969; Sterekhova, 1969). (3) Finally, it is probable that the effects of air pollution are due to the complex of materials in the atmosphere not to chemical species reacting independently of one another (Cassall, 1971). If that is the case, single variable animal experiments may simply not produce readily detectable changes in the test population. In light of these problems, it is suggested that the approach to the question of relating pollutants to disease, which involves exposure of animals to particular pollutants, can be assisted by an understanding of the chemical and biochemical potential for reactivity of these substances so that there might be foci for these studies based on a reasonable chemical model. Therefore, it is the intent here 55 Copyright ICI 1975 hy Academac Printed in the llnited State\.
Pm\\.
Inc. All rIghI\ of reproduction
I” any form rcacrved
56
PETERING
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SHM
to survey the known reactions of the hydrated form of sulfur with biomolecules and to discuss their possible significance.”
dioxide,
bisulfite,’
METABOLISM
Sulfur dioxide ( Eigen, 1961) ,
is hydrated
very rapidly
SO&I) + Hz0 F”
1
according
HSOg-
to the one step reaction
+ H+,
(1)
where k, = 3.4 X 10” M-’ second-l and k-, = 2 X 10s M-I second-’ at 20°C. Therefore, since the pH of physiological systems is generally near 7, and because the acid dissociation constant for bisulfite is 6.24 X lo-* at 25” (Tartar, 1941), the interaction of sulfur dioxide with biological structures in an aqueous medium is equivalent to considering the reactions of the hydrated forms of sulfur dioxide, sulfite and bisulfite. Two routes of entry into mammalian organisms are possible, inhalation of sulfur dioxide and the consumption of foods preserved with bisulfite. In each case, the sulfur oxide encounters a tissue which mediates the transfer of materials from the environment into the blood. The discussion here will focus upon airborne sulfur dioxide. Recently, several studies have examined the biological fate of sulfur dioxide (Gunnison, 1971; Yokoyama, 1971). Confirming earlier work, some 3”S0, inhaled by dogs makes its way into the blood and is only cleared very slowly from it, ultimately to be excreted as sulfate (Yokoyama, 1971). With a concentration of 22-23 ppm SO, in the atmosphere, significant amounts of inspired sulfur dioxide were fixed in the blood of dogs within 1 hour (Yokoyama, 1971). In rabbits subjected to a similar level of sulfur dioxide, plasma levels continued to rise during exposure; at 62 hours, lo-’ M ““SO, had reacted in the blood as follows: R-%-It
+ HSOS-
;t RSSO-
+ RSH
(3
in which RSSR represents disulfide linkages of red cells, plasma proteins, and small dialyzable molecules (Gunnison, 1971). Because of the large concentration of disulfide groups in plasma, no free bisulfite above a control value could be detected after exposure ( Gunnison, 1971). This reaction is well characterized in simple systems. For instance, with cystine a readily established equilibrium develops with an equilibrium constant for all forms of the species present in Reaction (2) of 8.9 X lo-’ at 37°C and pH 7.75 (Stricks, 1951). I n p.10t eins disulfide bond reactivity can vary widely depending upon the contribution of the particular disulfide bond to the proper structure of the protein (Cecil, 1962). Since the cleavage of disulfide bonds in proteins is generally accompanied by significant disruption in structure, such reactions are damaging at the biochemical level at least. In plasma bisulfite becomes bound primarily to the a-globulin fraction (80%) * Bisulfite ’ L. C. background
is used here to designate the sum of SO,I- and HSO,-. Schoepter, “Sulfur Dioxide,” Pergamon Press, New monograph.
York,
1966,
is an
exwllent
BIOCHEMISTRY
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57
some binding in the albumin fraction as well (Yokoyama, 1971). This is a result, for dispite the facts that the weight ratio of albumin to a-globulin in plasma is about 4: 1 and that albumin contains many disulfide groups per mole, bisulfite preferentially attacks components of the a-globulin fraction. Should this reaction be highly specific in terms of moles of bisulfite bound per mole of disulfide in the plasma fraction and also quantitatively significant as presently suggested, then an important site of reaction of bisulfite may be in the blood. Furthermore, the implication of these results is that bisulfite may well be reacting with disulfide groups of proteins in the lung as well. At this level of understanding, an anatomical division of the mammalian organism can be made on the basis of the form of sulfur dioxide available for reaction. At the lung bisulfite is initially present. Once in the blood, however, a bound form predominates, the S-thiosulfate of thiols such as protein cysteinyl residues. The patterns of reactivity of these two species are expected to be markedIy different. Hence, reactions of “bisulfite” beyond the lung need to be considered in this context. What is known about the further metabolism of sulfite by mammalian systems? Ultimately, absorbed sulfur dioxide is excreted primarily as inorganic sulfate (85% in dogs (Yokoyama, 1971) ). This fact has focused attention on the enzyme sulfite oxidase as a detoxification mechanism for converting sulfite directly into sulfate (Cohen, 1972). Its importance has been emphasized by the finding that the hereditary lack of this enzyme was associated with brain deterioration and death in one documented case (Mudd, 1967). However, this now needs reexamination in light of the finding that sulfite is quantitatively converted to Sthiosulfates in the blood. The following metabolic scheme is proposed for the conversion of bound sulfite to sulfate. with
striking
RSH + 2
7
HSO,
--) SOP-
NADPH’
/ SOif HSOt-
RSSOa-
(3)
RH
+ SSO,z-
tissue metabolism
1, occurs in blood; 2-7, occur in liver; 2, Erikson (1968); 3, Sorbo (1958); 4, catalysis by rhodanase or thiosulfate reductase (Villarejo, 1963; Sorbo, 1964); 5, Koj ( 1967) ; 6, Skarzynski ( 1959) ; and 7, catalysis by sulfite oxidase (Cohen, 1972). It has been observed that S-thiosulfates can be metabolized in two ways in the liver, reduction to bisulfite in the presence of NADPH’ or reductive cleavage to
58
PETERING
AND
SHIH
thiosulfate. Thiosulfate may, itself, be reduced to bisulfite and hydrosulfide by the enzyme rhodanase and reduced lipoate or by thiosulfate reductase and reduced glutathione. The liberated sulfide may then be efficiently oxidized to bisulfite. In the absence of sulfite oxidase, a buildup of RSSO,,m, SO,,’ , and SSO:,“- may be expected. In fact, this has been observed in the case of the child with a genetic defect in sulfite oxidasc metabolism (Laster, 1967). It is not clear if the transformations diagrammed above may occur elsewhere besides the liver. BIOCHEMISTRY
With this background of the metabolic transformations of sulfur dioxide, let us consider the interaction of bisulfite and S-thiosulfates with biomolecules:’ (Klayman, 1968). These reactions may be classified in terms of their thermodynamic reversibility. Those which establish simple equilibria by adduct formation will be examined first. The reaction of bisulfite with aldehydes and ketones is applicable to 5 and 6 carbon sugars. 0
OH (4)
The equilibrium constant for the formation of the bisulfite adduct of aliphatic aldehydes is generally less than 10’ (Schroeter, 1966). However, the formation constant for the reaction of the hemiacetal forms of sugars, which must first undergo ring opening, may be expected to be much smaller. Indeed, the constant for D-glucose is 1.6 Mm1 (Schroeter, 1966, p. 198). Nicotinamide adenine dinucleotide rapidly forms a reversible adduct with bisulfite in analogy to its reaction with other nucleophiles such as Hm and CN-. A recent determination of the formation constant is consistent with 1: 1 adduct formation as proposed earlier (Colowick, 1951; Myerhof, 1938; Pfleiderer, 1956; Shih, 1973).
(5)
The adduct has marginal stability at 25°C and pH 7.5, having an equilibrium constant of 36 M-’ (Shih, 1973). It is interesting to note, therefore, that bisulfite has been used to titrate and inactivate the NADr groups at the active sites of lactate dehydrogenase (Holbrook, 1966; Pfleiderer, 1956). Hence, in this enzyme, 3 We are not aware of much work concerning the reactivity of thiosulfite However, as occurs in the mechanism of thiosulfate reductase to bisulfite groups react with thiosulfate to cleave it as follows (Szczepkowski, 1958) RSSOz + HS-.
with biomolecules. and sulfide, thiol : SSO,‘- + RSH +
BIOCHEMISTRY
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59
DIOXIDE
the adduct must be substantially stabilized by the protein environment, for small concentrations of bisulfite react stoichiometrically with protein bound NAD+. A4 second esample of such a stabilization involves the adduct of bisulfite with flnvins. Again a large enhancement in binding constant is found with some enzymes containing flavin coenzymes relative to model isoalloxazines (Massey, 1969; Miiller, 1969). For instance, the formation constants for the sulfite adducts of FAD and FMN bisulfite at 25°C and pH 7 are O.-i and 0.53 hi-l, respectively, whereas those for the sulfite complexes with several flavoproteins are orders of magnitude larger-g.5 X LO’ hi-l for D-amino acid oxidase and 6.7 x lo3 M-I for L-amino acid oxidase. Altiller and Massey identified the site of ndduct formation as the N, atom of the isonlloxazine ring system, the normal site of reduction of the ring by H-. H
A weak complex is formed between bisulfite and pteridyl groups (Albert, 1965). H
H
t HSOI- e ‘\ I II
(6)
OH
$
OH
This observation has been extended to folate and dihydrofolate 1967). H
HzN H=Nv,NJ,
+ HSOa- = R
A
3
;g(Nxso
-
(7)
R
c!
H
(Vonderschmitt,
H + HSOa- s
(8)
The formation constants for these adducts are 3 and 83, respectively, at 31°C. Finally, a water soluble derivative of vitamin K, menadione or vitamin KS, is known to react with bisulfite at a moderate rate under mild conditions (Carmack, 1950; Greenberg, 1971),
60
PETERING
AND
SHIH
0
Vitamin
K,
a;
+ HSOa-ti~$~:
(9) OH
Vitamin
Ka
An equilibrium constant has been measured for this reaction, 1 X 10’; M-I at 25°C and pH 7.5 (Shih, 1973). The direct extrapolation to the fat soluble vitamin K, is not possible, particularly because of the differences in solvent environment expected for the two quinones. The menadione-sulfite adduct is an effective antihemorrhagic agent when given orally, suggesting that it dissociates in viva (Carmack, 1950). Of much current interest is the recent demonstration of the bisulfite catalyzed conversion of cytosine derivatives to uracil compounds. Involved here are a combination of equilibrium and irreversible processes (Hayatsu, 1970b; Hayatsu, 197Oc; Natari, 1967; Shapiro, 1970b). NH2
NH2
+ HSOz-
e
0
0
+
HSOa-
(10)
Bisulfite adds rapidly to the 5,6 double bond of deoxycytidine. At pD = 6.6 and 37°C the formation constant is 0.34 (Shapiro, 1970b). Then, the adduct slowly deaminates, k = 8.6 X lo-” second-i. Finally, the resultant uracil-sulfonate product equilibrates with deoxyuridine (Shapiro, 1970b) at 24°C. The dissociation constant for uracil-sulfonate is 7.7 X lo-’ M-I (Shapiro, 1970b). The scope of these reactions has been extended to polynucleotides. At high concentrations of bisulfite ( > 1 M) and pH ranges from 5 to 7, yeast RNA, and ecoli formylmethionine and glutamine transfer RNA undergo deamination of cytosine residues to uracil (Furuichi, 1970; Goddard, 1972; Shapiro, 1970a; Singhal, 1971). At pH 7 in 1 M NaHSO, polyuridylic acid forms sufficient sulfonate to inhibit helix formation with polyadenylic acid and also to decrease greatly its efficiency as a messenger for polyphenylalanine synthesis (Shapiro, 1972). It appears, however, that little bisulfite binds to bases in double helical structures ( Goddard, 1972). Of direct biological interest are the findings that bisulfite is a moderately strong
BIOCHEMISTRY
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61
DIOXIDE
mutagen in test systems such as Escherichia coli and the bacteriophages X and T, (Hayatsu, 1970a; Mukai, 1970; Summers, 1971). It is specific for G:C pairs as is predicted from chemical studies. Such results have been used to suggest the possibility of bisulfite-induced genetic damage or cancer in higher organisms (Hayatsu, 1970a). Care must be taken to distinguish between reactions in the lung and elsewhere. While it is possible that bisulfite may thus interact with the somatic cells of the lung, it is not clear that such reactions may occur at the remote site of the germ cells. As Eq. ( 1) indicates, once bisulfite passes into the blood it is converted quantitatively to S-thiosulfates. Furthermore, efforts to obtain any reaction between cytidine and cysteine-S-thiosulfate have failed ( Shih, 1973). Therefore, liberation of HSO,- from S-thiosulfates, perhaps by reaction with thiol groups, in the germ cells or other tissues must be envisioned in order to extrapolate the tests of mutagenicity from naked genetic natural or unicellular organisms to a complex higher organism. At lower concentrations ( 10m2M) bisulfite may react with DNA in another way, presumably by an oxygen-dependent free radical reaction to cleave the backbone of the structure (Hayatsu, 1972). Bisulfite undergoes essentially irreversible reactions with thiamine and epinephrine. The cleavage of thiamine by bisulfite has been known for many years (Williams, 1935; Lhoest, 1957).
/
00
+ SOII
--$
I
(11)
and has been of more concern to food chemists in situations in which bisulfite is used as a preservative (Leichter, 1969). The reaction occurs with as little as 10e5 M thiamine and bisulfite (Leichter, 1969). At pH 7 the reaction has a second order rate constant of 6.4 X 1O-3 M-I second-l at 25°C as calculated from data in Leichter ( 1969). Reports have appeared that thiamine supplementation protects against sulfur dioxide toxicity in animals (Hoetzel, 1961; Wilmes, 1966). It may be suggested that a mechanism of protection is simply the above reaction which removes bisulfite from solution. However, unless this reaction is to occur solely in the lungs, this hypothesis ignores the conversion of bisulfite to S-thiosulfates as a key reaction in biological systems, Under basic conditions thiamine reacts with disulfides according to the following reaction (Maier, 1957) :
\ / +OH-tips
mbJHO
with the pK for the pH dependent
7 + RSSR
dN r\SSR \ CHO
+ RS-
(12)
ring opening of 9.3 ( Maier, 1957). The thiolate
62
PETERING
AND
SHIH
anion would be expected to react similarly with cysteine-S-sulfonate. However, under a variety of conditions at pH 7, no reaction is observed (Shih, 1973). Epinephrine also reacts irreversibly with bisulfite (Higuchi, 1960; Riegehnan, 1962; Schroeter, 1958). HO HO
/
CHCH&H&Ha I OH
+ SO?- --) HO HO
(13) /
However, this is an extremely slow reaction; the half-life of 0.082 M epinephrine in the presence of 0.96 M bisulfite is 74 hours at pH 7.5 and 25°C (Riegelman, 1962). DISCUSSION
It is evident that bisulfite is a reactive species with a wide variety of biomolecules, essentially acting as a nucleophile toward electrophilic centers. In this situation how might one examine the relative importance of these reactions? Reactions will be favored which are either thermodynamically or kinetically irreversible. For a set of reactions which equilibrate rapidly, the magnitudes of the equilibrium constants together with the relative concentrations of reactants will determine the distribution of bisulfite among the products. It is clear that the results presented here do not set forth one particular reaction as having a singularly large constant for adduct formation. However, Reaction (1) is conspicuous because of the presence of disulfide groups in many proteins, which, in the case of blood, convert all the measurable bisulfite into S-thiosulfates. Beyond this, the free bisulfite, part of each equilibrium reaction, may react irreversibly with other molecules, notably thiamine. In the lung, therefore, reactions with specific disulfide links and with thiamine may be of importance in the pathology of sulfur dioxide. Furthermore, if cytidine groups react with low concentrations of bisulfite, then surely this may be a damaging reaction to the lung. Other reactions described here also need scrutiny in vioo for the equilibrium and rate constants reported here may vary markedly depending on the environment of the reaction. For instance, the formation constants for NAD .SO:,- and FAD *SO,- increase substantially when these coenzymes are protein bound. Finally, the potential interaction of bisulfite with a host of biomolecules is a particular example of Dinman’s view of the threshold for biological response to a toxic agent (Dinman, 1972). In particular, there are a variety of reactive sites in the cell for bisulfite. and one of two situations may exist: either the site of toxicity is highly specific or toxicity derives from the resultant of the many different reactions. Considering a dose-response curve, at high concentrations, bisulfite may saturate the cellular sites of reaction so that in either case toxicity arises. But as the concentration is decreased, the sites begin to dominate the agent. Then, bisulfite is confronted with a set of possible reactions among which it will distribute itself by equilibrium and irreversible processes.However, in the absence of very large equilibrium constants for some sites, equilibration may not
BIOCHEMISTRY
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lead to much specificity in the products formed. Furthermore, the rates of irreversible reactions will decrease. Finally, when the concentration of bisulfite is lowered sufficiently, little binding will occur with biomolecules. Then the concentration of any product is small compared with the concentration of corresponding, unaltered molecules and there will not be a significant response of the cell as a whole. ACKNOWLEDGMENTS N.T.S. was supported in part by the Graduate School of the University
the National Cancer of Wisconsin-Milwaukee.
Institute,
CA-13099,
and
in part
by
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HAYATSU, H., AND MILLER, R. C. (1972). Th e c 1eavage of DNA by the oxygen-dependent reaction of bisulfite. Biochem. Biophys. Res. Commun. 46, 120. HIGUCHI, T., AND SCHROETER, L. C. (1960). Kinetics and mechanism of formation of sulfonate from epinephrine and bisulfite. J. Amer. Chem. Sot. 82, 1904. HOETZEL, D. (1961). Influence of vitamin B on the toxicity of sulfur dioxide. Verhantl. Deut. Ges. Inn. Med. 67, 868; Chem. Abstr. (1963), 59, 15839f. HOLBROOK, J. J. (1966). The importance of SH-groups for enzymic activity. V. The coenzyme-binding capacity of pig heart lactate dehydrogenase, isozyme 1, after inhibition by various maleinimides. Biochem. Z. 344, 141. KLAYMAN, D. L., AND SHINE, R. J. ( 1968). The chemistry of organic thiosulfates. Quarb.
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Bisulfite-catalyzed transamination of cytosine SHAPIRO, R., AND WEINGRAS, J. hi. (197Oc). and cytidine. Bi~chem. Biophtp. Res. CO~IILU~. 40, 839. acid by bisulfite: SHAPIRO, R., AND BRAVERMAN, B. (1972). M o d’fir ca t’ ion of polyuridylic eifect on double helix formation and coding properties. Bio~hem. Bio$r~s. Res. Commu~ 47, 544. SHIH, N. T., AND PETERING, D. II. ( 1973 ) . hlodel reactions for the study of the interaction of sulfur dioxide with mammalian organisms. Biochcm. Bionkys. Res. Commun. 55, 1319. SINGHAL, R. P. ( 1971). Modification of Escherichia coli glutamate transfer ribonucleic acid with bisulfite. 1. Biol. Chern. 246, 5848. SKARZYNSKI, B., SZCZEPKO~SKI, T. W., AND WEXER, M. ( 1959). Thiosulphatc metabolism in the animal organism. Nature (London) 184, 994. S&no, B. (1958). On the metabolism of thiosulfate esters. Acta Chem. Scud. 12, 1990. SBIIBO, B. (1964). Mechanism of oxidation of inorganic thiosulfate and thiosulfate esters in mammals. Acta Chem. Stand. 18, 821. STEREKHOVA, N. P., AND VORMTSOVA, A. S. (1969). Protein metabolism in copper smelters exposed to the chronic effect of sulfur dioxide. Gig. Prof. Zabol. 13, 52; Chem. Abstr. (1970), 72, 82670s. STNICKS, W., AND KOLTOFF, I. M. (1951). Equilibrium constants of the reactions of sulfite with cystine and with dithiodiglycolic acid. J. Anwr. Clwm. Sot. 73, 4569. SUMMERS, G. A., AND DRAKE, J. W. ( 1971). Bisulfite mutagenesis in bacteriophage T,. Genetics 68, 603. SZCZEPKOWSKI, T. W. (1958). Reaction of thiosulfate with cysteine. Nature (London) 182, 934. TARTAR, H. V., AND GARETSON, H. H. ( 1941). The tl lermodynamic ionization constants of sulfurous acid at 25”. J. Amer. C&n. Sot. 63, 808. VILLAREJO, M., AP\‘D WESTLEY, J. (1963). Mechanism of rhodanase catalysis of thiosulfateiipoate oxidation-reduction. J. Biol. Chem. 238, 4016. (1967). VONDERSCHMIDT, D. J., VITOLS, K. S., HUENNEKENS, F. M., AKD SCRINCEOUR, K. G. Addition of bisulfite to folate and dihydrofolate. Arch. Biochem. Biophys. 122, 488. WILLIAMS, R. R., WATERMAX, R. E., KERESZTESY, J. C., AND BUCHMAN, E. R. (1935). Studies of crystalline vitamin B,. III. Cleavage of vitamin with sulfite. f. Amer. Chem. Sot. 57, 536. WILhlES, G. (1966). Dependence of sulfur dioxide toxicity on thiamine supply. Proc. Int. Congr. Nutr. 7, 540; Chem. Abstr. ( 1969), 70, 18512~. YOKOYAMA, E., YODER, R. E., AND FRANK, N. R. (1971). Distribution of “S in the blood and its excretion in urine of dogs exposed to ‘!“SOZ. Arch. Enuiron. He&h 22, 389. ZARKOWER, A. ( 1972). Alterations in antibody response induced by chronic inhalation of SO, and carbon. Arch. Enuiron. Health 25, 45.
RESEARCH
9, 55-65
Biochemistry
(1975)
of Bisulfite-Sulfur
Dioxide
DAVLLI H. PETERING AND NANCY TING Sm Department
of
Chemisty, Milwaukee, Received
University Wisconsin
of
December
13,
Wisconsin-Milwaukee, 53201 1973
An analysis of the metabolic transformations of sulfur dioxide is made. Then, the biochemical reactivity of two of its forms, bisulfite and S-thiosulfates, is surveyed in order to provide a view of the potential reactivity of sulfur dioxide with biological systems. Finally, these findings are used as a model for the consideration of threshold responses of organisms to agents in the environment.
Sulfur dioxide is a major air pollutant in many large cities. It is, therefore, a major concern to determine the extent of its hazard to health, in particular the with possible long-term effects of exposure. Although numerous experiments animals have been carried out to discover the biological effects of sulfur dioxide, results have been ambivalent concerning its toxicity at low concentrations ( Alarie, 1972; Lewis, 1973; Rylander, 1969; Zarkower, 1972). This may be attributed to at least 3 difficulties in establishing a causal relationship between airborne SO, and detrimental effects in test organisms: (1) At the low concentrations present in urban air, sulfur dioxide does not produce acute, toxic effects in carefully maintained laboratory animals. This does not exclude the presence of subtle, slowly developing effects but does make difficult the observation of a causal relationship between the imposition of the pollutant and organismic responses. (2) Only a selection of an animal’s myriad physiological and biochemical processes may be monitored in any experiment, Important responses may simply be missed because of the limitation of parameters observed or may be lost in the midst of the numerous responses seen. This is particularly true at the biochemical level where the variety of alterations in the concentrations of materials is bewildering (Schmidt, 1969; Sterekhova, 1969). (3) Finally, it is probable that the effects of air pollution are due to the complex of materials in the atmosphere not to chemical species reacting independently of one another (Cassall, 1971). If that is the case, single variable animal experiments may simply not produce readily detectable changes in the test population. In light of these problems, it is suggested that the approach to the question of relating pollutants to disease, which involves exposure of animals to particular pollutants, can be assisted by an understanding of the chemical and biochemical potential for reactivity of these substances so that there might be foci for these studies based on a reasonable chemical model. Therefore, it is the intent here 55 Copyright ICI 1975 hy Academac Printed in the llnited State\.
Pm\\.
Inc. All rIghI\ of reproduction
I” any form rcacrved
56
PETERING
AND
SHM
to survey the known reactions of the hydrated form of sulfur with biomolecules and to discuss their possible significance.”
dioxide,
bisulfite,’
METABOLISM
Sulfur dioxide ( Eigen, 1961) ,
is hydrated
very rapidly
SO&I) + Hz0 F”
1
according
HSOg-
to the one step reaction
+ H+,
(1)
where k, = 3.4 X 10” M-’ second-l and k-, = 2 X 10s M-I second-’ at 20°C. Therefore, since the pH of physiological systems is generally near 7, and because the acid dissociation constant for bisulfite is 6.24 X lo-* at 25” (Tartar, 1941), the interaction of sulfur dioxide with biological structures in an aqueous medium is equivalent to considering the reactions of the hydrated forms of sulfur dioxide, sulfite and bisulfite. Two routes of entry into mammalian organisms are possible, inhalation of sulfur dioxide and the consumption of foods preserved with bisulfite. In each case, the sulfur oxide encounters a tissue which mediates the transfer of materials from the environment into the blood. The discussion here will focus upon airborne sulfur dioxide. Recently, several studies have examined the biological fate of sulfur dioxide (Gunnison, 1971; Yokoyama, 1971). Confirming earlier work, some 3”S0, inhaled by dogs makes its way into the blood and is only cleared very slowly from it, ultimately to be excreted as sulfate (Yokoyama, 1971). With a concentration of 22-23 ppm SO, in the atmosphere, significant amounts of inspired sulfur dioxide were fixed in the blood of dogs within 1 hour (Yokoyama, 1971). In rabbits subjected to a similar level of sulfur dioxide, plasma levels continued to rise during exposure; at 62 hours, lo-’ M ““SO, had reacted in the blood as follows: R-%-It
+ HSOS-
;t RSSO-
+ RSH
(3
in which RSSR represents disulfide linkages of red cells, plasma proteins, and small dialyzable molecules (Gunnison, 1971). Because of the large concentration of disulfide groups in plasma, no free bisulfite above a control value could be detected after exposure ( Gunnison, 1971). This reaction is well characterized in simple systems. For instance, with cystine a readily established equilibrium develops with an equilibrium constant for all forms of the species present in Reaction (2) of 8.9 X lo-’ at 37°C and pH 7.75 (Stricks, 1951). I n p.10t eins disulfide bond reactivity can vary widely depending upon the contribution of the particular disulfide bond to the proper structure of the protein (Cecil, 1962). Since the cleavage of disulfide bonds in proteins is generally accompanied by significant disruption in structure, such reactions are damaging at the biochemical level at least. In plasma bisulfite becomes bound primarily to the a-globulin fraction (80%) * Bisulfite ’ L. C. background
is used here to designate the sum of SO,I- and HSO,-. Schoepter, “Sulfur Dioxide,” Pergamon Press, New monograph.
York,
1966,
is an
exwllent
BIOCHEMISTRY
OF
BISULFITE-SULFUR
DIOXIDE
57
some binding in the albumin fraction as well (Yokoyama, 1971). This is a result, for dispite the facts that the weight ratio of albumin to a-globulin in plasma is about 4: 1 and that albumin contains many disulfide groups per mole, bisulfite preferentially attacks components of the a-globulin fraction. Should this reaction be highly specific in terms of moles of bisulfite bound per mole of disulfide in the plasma fraction and also quantitatively significant as presently suggested, then an important site of reaction of bisulfite may be in the blood. Furthermore, the implication of these results is that bisulfite may well be reacting with disulfide groups of proteins in the lung as well. At this level of understanding, an anatomical division of the mammalian organism can be made on the basis of the form of sulfur dioxide available for reaction. At the lung bisulfite is initially present. Once in the blood, however, a bound form predominates, the S-thiosulfate of thiols such as protein cysteinyl residues. The patterns of reactivity of these two species are expected to be markedIy different. Hence, reactions of “bisulfite” beyond the lung need to be considered in this context. What is known about the further metabolism of sulfite by mammalian systems? Ultimately, absorbed sulfur dioxide is excreted primarily as inorganic sulfate (85% in dogs (Yokoyama, 1971) ). This fact has focused attention on the enzyme sulfite oxidase as a detoxification mechanism for converting sulfite directly into sulfate (Cohen, 1972). Its importance has been emphasized by the finding that the hereditary lack of this enzyme was associated with brain deterioration and death in one documented case (Mudd, 1967). However, this now needs reexamination in light of the finding that sulfite is quantitatively converted to Sthiosulfates in the blood. The following metabolic scheme is proposed for the conversion of bound sulfite to sulfate. with
striking
RSH + 2
7
HSO,
--) SOP-
NADPH’
/ SOif HSOt-
RSSOa-
(3)
RH
+ SSO,z-
tissue metabolism
1, occurs in blood; 2-7, occur in liver; 2, Erikson (1968); 3, Sorbo (1958); 4, catalysis by rhodanase or thiosulfate reductase (Villarejo, 1963; Sorbo, 1964); 5, Koj ( 1967) ; 6, Skarzynski ( 1959) ; and 7, catalysis by sulfite oxidase (Cohen, 1972). It has been observed that S-thiosulfates can be metabolized in two ways in the liver, reduction to bisulfite in the presence of NADPH’ or reductive cleavage to
58
PETERING
AND
SHIH
thiosulfate. Thiosulfate may, itself, be reduced to bisulfite and hydrosulfide by the enzyme rhodanase and reduced lipoate or by thiosulfate reductase and reduced glutathione. The liberated sulfide may then be efficiently oxidized to bisulfite. In the absence of sulfite oxidase, a buildup of RSSO,,m, SO,,’ , and SSO:,“- may be expected. In fact, this has been observed in the case of the child with a genetic defect in sulfite oxidasc metabolism (Laster, 1967). It is not clear if the transformations diagrammed above may occur elsewhere besides the liver. BIOCHEMISTRY
With this background of the metabolic transformations of sulfur dioxide, let us consider the interaction of bisulfite and S-thiosulfates with biomolecules:’ (Klayman, 1968). These reactions may be classified in terms of their thermodynamic reversibility. Those which establish simple equilibria by adduct formation will be examined first. The reaction of bisulfite with aldehydes and ketones is applicable to 5 and 6 carbon sugars. 0
OH (4)
The equilibrium constant for the formation of the bisulfite adduct of aliphatic aldehydes is generally less than 10’ (Schroeter, 1966). However, the formation constant for the reaction of the hemiacetal forms of sugars, which must first undergo ring opening, may be expected to be much smaller. Indeed, the constant for D-glucose is 1.6 Mm1 (Schroeter, 1966, p. 198). Nicotinamide adenine dinucleotide rapidly forms a reversible adduct with bisulfite in analogy to its reaction with other nucleophiles such as Hm and CN-. A recent determination of the formation constant is consistent with 1: 1 adduct formation as proposed earlier (Colowick, 1951; Myerhof, 1938; Pfleiderer, 1956; Shih, 1973).
(5)
The adduct has marginal stability at 25°C and pH 7.5, having an equilibrium constant of 36 M-’ (Shih, 1973). It is interesting to note, therefore, that bisulfite has been used to titrate and inactivate the NADr groups at the active sites of lactate dehydrogenase (Holbrook, 1966; Pfleiderer, 1956). Hence, in this enzyme, 3 We are not aware of much work concerning the reactivity of thiosulfite However, as occurs in the mechanism of thiosulfate reductase to bisulfite groups react with thiosulfate to cleave it as follows (Szczepkowski, 1958) RSSOz + HS-.
with biomolecules. and sulfide, thiol : SSO,‘- + RSH +
BIOCHEMISTRY
OF
BISULFITE-SULFUR
59
DIOXIDE
the adduct must be substantially stabilized by the protein environment, for small concentrations of bisulfite react stoichiometrically with protein bound NAD+. A4 second esample of such a stabilization involves the adduct of bisulfite with flnvins. Again a large enhancement in binding constant is found with some enzymes containing flavin coenzymes relative to model isoalloxazines (Massey, 1969; Miiller, 1969). For instance, the formation constants for the sulfite adducts of FAD and FMN bisulfite at 25°C and pH 7 are O.-i and 0.53 hi-l, respectively, whereas those for the sulfite complexes with several flavoproteins are orders of magnitude larger-g.5 X LO’ hi-l for D-amino acid oxidase and 6.7 x lo3 M-I for L-amino acid oxidase. Altiller and Massey identified the site of ndduct formation as the N, atom of the isonlloxazine ring system, the normal site of reduction of the ring by H-. H
A weak complex is formed between bisulfite and pteridyl groups (Albert, 1965). H
H
t HSOI- e ‘\ I II
(6)
OH
$
OH
This observation has been extended to folate and dihydrofolate 1967). H
HzN H=Nv,NJ,
+ HSOa- = R
A
3
;g(Nxso
-
(7)
R
c!
H
(Vonderschmitt,
H + HSOa- s
(8)
The formation constants for these adducts are 3 and 83, respectively, at 31°C. Finally, a water soluble derivative of vitamin K, menadione or vitamin KS, is known to react with bisulfite at a moderate rate under mild conditions (Carmack, 1950; Greenberg, 1971),
60
PETERING
AND
SHIH
0
Vitamin
K,
a;
+ HSOa-ti~$~:
(9) OH
Vitamin
Ka
An equilibrium constant has been measured for this reaction, 1 X 10’; M-I at 25°C and pH 7.5 (Shih, 1973). The direct extrapolation to the fat soluble vitamin K, is not possible, particularly because of the differences in solvent environment expected for the two quinones. The menadione-sulfite adduct is an effective antihemorrhagic agent when given orally, suggesting that it dissociates in viva (Carmack, 1950). Of much current interest is the recent demonstration of the bisulfite catalyzed conversion of cytosine derivatives to uracil compounds. Involved here are a combination of equilibrium and irreversible processes (Hayatsu, 1970b; Hayatsu, 197Oc; Natari, 1967; Shapiro, 1970b). NH2
NH2
+ HSOz-
e
0
0
+
HSOa-
(10)
Bisulfite adds rapidly to the 5,6 double bond of deoxycytidine. At pD = 6.6 and 37°C the formation constant is 0.34 (Shapiro, 1970b). Then, the adduct slowly deaminates, k = 8.6 X lo-” second-i. Finally, the resultant uracil-sulfonate product equilibrates with deoxyuridine (Shapiro, 1970b) at 24°C. The dissociation constant for uracil-sulfonate is 7.7 X lo-’ M-I (Shapiro, 1970b). The scope of these reactions has been extended to polynucleotides. At high concentrations of bisulfite ( > 1 M) and pH ranges from 5 to 7, yeast RNA, and ecoli formylmethionine and glutamine transfer RNA undergo deamination of cytosine residues to uracil (Furuichi, 1970; Goddard, 1972; Shapiro, 1970a; Singhal, 1971). At pH 7 in 1 M NaHSO, polyuridylic acid forms sufficient sulfonate to inhibit helix formation with polyadenylic acid and also to decrease greatly its efficiency as a messenger for polyphenylalanine synthesis (Shapiro, 1972). It appears, however, that little bisulfite binds to bases in double helical structures ( Goddard, 1972). Of direct biological interest are the findings that bisulfite is a moderately strong
BIOCHEMISTRY
OF
BISULFITE-SULFUR
61
DIOXIDE
mutagen in test systems such as Escherichia coli and the bacteriophages X and T, (Hayatsu, 1970a; Mukai, 1970; Summers, 1971). It is specific for G:C pairs as is predicted from chemical studies. Such results have been used to suggest the possibility of bisulfite-induced genetic damage or cancer in higher organisms (Hayatsu, 1970a). Care must be taken to distinguish between reactions in the lung and elsewhere. While it is possible that bisulfite may thus interact with the somatic cells of the lung, it is not clear that such reactions may occur at the remote site of the germ cells. As Eq. ( 1) indicates, once bisulfite passes into the blood it is converted quantitatively to S-thiosulfates. Furthermore, efforts to obtain any reaction between cytidine and cysteine-S-thiosulfate have failed ( Shih, 1973). Therefore, liberation of HSO,- from S-thiosulfates, perhaps by reaction with thiol groups, in the germ cells or other tissues must be envisioned in order to extrapolate the tests of mutagenicity from naked genetic natural or unicellular organisms to a complex higher organism. At lower concentrations ( 10m2M) bisulfite may react with DNA in another way, presumably by an oxygen-dependent free radical reaction to cleave the backbone of the structure (Hayatsu, 1972). Bisulfite undergoes essentially irreversible reactions with thiamine and epinephrine. The cleavage of thiamine by bisulfite has been known for many years (Williams, 1935; Lhoest, 1957).
/
00
+ SOII
--$
I
(11)
and has been of more concern to food chemists in situations in which bisulfite is used as a preservative (Leichter, 1969). The reaction occurs with as little as 10e5 M thiamine and bisulfite (Leichter, 1969). At pH 7 the reaction has a second order rate constant of 6.4 X 1O-3 M-I second-l at 25°C as calculated from data in Leichter ( 1969). Reports have appeared that thiamine supplementation protects against sulfur dioxide toxicity in animals (Hoetzel, 1961; Wilmes, 1966). It may be suggested that a mechanism of protection is simply the above reaction which removes bisulfite from solution. However, unless this reaction is to occur solely in the lungs, this hypothesis ignores the conversion of bisulfite to S-thiosulfates as a key reaction in biological systems, Under basic conditions thiamine reacts with disulfides according to the following reaction (Maier, 1957) :
\ / +OH-tips
mbJHO
with the pK for the pH dependent
7 + RSSR
dN r\SSR \ CHO
+ RS-
(12)
ring opening of 9.3 ( Maier, 1957). The thiolate
62
PETERING
AND
SHIH
anion would be expected to react similarly with cysteine-S-sulfonate. However, under a variety of conditions at pH 7, no reaction is observed (Shih, 1973). Epinephrine also reacts irreversibly with bisulfite (Higuchi, 1960; Riegehnan, 1962; Schroeter, 1958). HO HO
/
CHCH&H&Ha I OH
+ SO?- --) HO HO
(13) /
However, this is an extremely slow reaction; the half-life of 0.082 M epinephrine in the presence of 0.96 M bisulfite is 74 hours at pH 7.5 and 25°C (Riegelman, 1962). DISCUSSION
It is evident that bisulfite is a reactive species with a wide variety of biomolecules, essentially acting as a nucleophile toward electrophilic centers. In this situation how might one examine the relative importance of these reactions? Reactions will be favored which are either thermodynamically or kinetically irreversible. For a set of reactions which equilibrate rapidly, the magnitudes of the equilibrium constants together with the relative concentrations of reactants will determine the distribution of bisulfite among the products. It is clear that the results presented here do not set forth one particular reaction as having a singularly large constant for adduct formation. However, Reaction (1) is conspicuous because of the presence of disulfide groups in many proteins, which, in the case of blood, convert all the measurable bisulfite into S-thiosulfates. Beyond this, the free bisulfite, part of each equilibrium reaction, may react irreversibly with other molecules, notably thiamine. In the lung, therefore, reactions with specific disulfide links and with thiamine may be of importance in the pathology of sulfur dioxide. Furthermore, if cytidine groups react with low concentrations of bisulfite, then surely this may be a damaging reaction to the lung. Other reactions described here also need scrutiny in vioo for the equilibrium and rate constants reported here may vary markedly depending on the environment of the reaction. For instance, the formation constants for NAD .SO:,- and FAD *SO,- increase substantially when these coenzymes are protein bound. Finally, the potential interaction of bisulfite with a host of biomolecules is a particular example of Dinman’s view of the threshold for biological response to a toxic agent (Dinman, 1972). In particular, there are a variety of reactive sites in the cell for bisulfite. and one of two situations may exist: either the site of toxicity is highly specific or toxicity derives from the resultant of the many different reactions. Considering a dose-response curve, at high concentrations, bisulfite may saturate the cellular sites of reaction so that in either case toxicity arises. But as the concentration is decreased, the sites begin to dominate the agent. Then, bisulfite is confronted with a set of possible reactions among which it will distribute itself by equilibrium and irreversible processes.However, in the absence of very large equilibrium constants for some sites, equilibration may not
BIOCHEMISTRY
OF
BISULFITE-SULFUR
63
DIOXIDE
lead to much specificity in the products formed. Furthermore, the rates of irreversible reactions will decrease. Finally, when the concentration of bisulfite is lowered sufficiently, little binding will occur with biomolecules. Then the concentration of any product is small compared with the concentration of corresponding, unaltered molecules and there will not be a significant response of the cell as a whole. ACKNOWLEDGMENTS N.T.S. was supported in part by the Graduate School of the University
the National Cancer of Wisconsin-Milwaukee.
Institute,
CA-13099,
and
in part
by
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64
PETERING
AND
SHIH
HAYATSU, H., AND MILLER, R. C. (1972). Th e c 1eavage of DNA by the oxygen-dependent reaction of bisulfite. Biochem. Biophys. Res. Commun. 46, 120. HIGUCHI, T., AND SCHROETER, L. C. (1960). Kinetics and mechanism of formation of sulfonate from epinephrine and bisulfite. J. Amer. Chem. Sot. 82, 1904. HOETZEL, D. (1961). Influence of vitamin B on the toxicity of sulfur dioxide. Verhantl. Deut. Ges. Inn. Med. 67, 868; Chem. Abstr. (1963), 59, 15839f. HOLBROOK, J. J. (1966). The importance of SH-groups for enzymic activity. V. The coenzyme-binding capacity of pig heart lactate dehydrogenase, isozyme 1, after inhibition by various maleinimides. Biochem. Z. 344, 141. KLAYMAN, D. L., AND SHINE, R. J. ( 1968). The chemistry of organic thiosulfates. Quarb.
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