- Email: [email protected]
Ecotoxicology of ozone: Bioactivation of extracellular ascorbate
Available online at www.sciencedirect.com
Biochemical and Biophysical Research Communications 366 (2008) 271–274 www.elsevier.com/locate/ybbrc
Mini Review
Ecotoxicology of ozone: Bioactivation of extracellular ascorbate Heinrich Sandermann Ecotox.freiburg, Schubertstr. 1, D-79104 Freiburg, Germany GSF-National Research Center, Institute of Biochemical Plant Pathology, D-85764 Neuherberg, Germany Received 21 November 2007 Available online 17 December 2007
Abstract The ascorbate pools of the extracellular respiratory lining fluids and the plant apoplast are currently considered as part of the first line of defence against ambient ozone. Ozone is in fact rapidly decomposed by ascorbate, but the only product identified so far (singlet oxygen) is toxic. Peroxy-L-threonic and peroxy-oxalic acids are now derived as further decomposition products on the basis of Criegee-type ozone chemistry. These secondary toxicants may resemble known ozone induced transmitter molecules by participating in signalling events affecting plant and animal innate immunity. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Ascorbate; Respiratory lining fluid; Plant apoplast; Singlet oxygen; Secondary toxicants
Extracellular ascorbate as first line of defence against ozone Ambient tropospheric ozone is known to be toxic for man, animals and plants, the recommended safety levels being frequently exceeded. In animals and man, lung functions are affected, pre-inflammatory mediators are increased and airway inflammation occurs. Epidemiological studies have detected increased premature mortality [1,2]. In plants, acute and chronic exposure to ozone can lead to plant stress responses, visible symptoms, premature senescence, crop loss and alterations of plant community structure [3,4]. After stomatal uptake, ozone dissolves in the apoplastic fluid, and the reaction with apoplastic ascorbate has been considered as a first line of plant defence [5] on the basis of three main types of evidence: the intercellular concentration of ozone was found to be near zero [6], ozone is scavenged by ascorbate with a high rate constant [7–9], and the ozone sensitivity of mutant vtc1 of Arabidopsis appeared to be caused by its total ascorbate level of only 30% of normal [10]. The extracellular ascorbate in respiratory lining fluids of animals and humans has also been considered as part of the first line of defence against ozone E-mail address: [email protected] 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.12.018
[11,12], again due to its function as one of the primary ozone absorption targets [8,9,13]. Two reaction pathways for toxicants derived from ozone The extracellular compartments and plasma membranes of plants and animals are essentially the only cellular components directly exposed to ozone. Due to the rapid extracellular quenching of ozone, the intracellular compartments are mainly exposed to secondary toxicants derived from one of two pathways, namely ozone decomposition in aqueous solution, or ozonolysis of double bonds [1]. It is well known that ozone will in aqueous solutions decompose to hydrogen peroxide, singlet oxygen and hydroxyl radicals, biomolecules such as ascorbate [14] or phenolics [15] enhancing these reactions. The toxic effects and the detoxification of these secondary reactive oxygen species have been well studied, ascorbate being involved in both extra- and intracellular detoxification [1,16]. This beneficial role involves electron transfer reactions leading to monodehydroascorbate and dehydroascorbate, followed by regeneration of ascorbate. The protective systems are most abundant in intracellular organelles where reactive oxygen species are formed as metabolic side-products.
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H. Sandermann / Biochemical and Biophysical Research Communications 366 (2008) 271–274
The ozonolysis pathway is best studied for unsaturated fatty acids [17–19] and cholesterol [20–22]. Older studies have pointed out that ozone is also likely to attack the double bond of ascorbate [7,23]. However, it appears that the Criegee mechanism of ozonolysis [24,25] has never been applied to ascorbate.
mechanism is, like ozonolysis, in agreement with the 1:1 stoichiometry determined for the overall reaction between ozone and ascorbate [9]. The formation of singlet oxygen from ozone and olefins is thought to proceed by transfer of an oxygen atom to yield an unstable epoxide [14]. This mechanism is consistent with the 5,6-epoxide of cholesterol being a major reaction product detected after ozone exposure of lung surfactant [22]. The epoxide of ascorbic acid could add water to yield dehydroascorbic acid. However, the reactivity of the hypothetical ascorbyl-epoxide may be so high that C–C bond cleavage and formation of aldehydes occur. Singlet oxygen in turn is able to react with ascorbate, one of the elucidated main reaction products being depicted in Fig. 1 [29]. The isomeric peroxy-ketones are highly electrophilic, their reaction with water leading to dehydroascorbate and hydrogen peroxide [30]. Reactions with sulfhydryl and other oxidizable groups have apparently not been characterized.
Chemistry of the ascorbate/ozone reaction Ozone attack on double bonds is known to proceed initially by 1,3-dipolar cycloaddition. The resulting primary ozonide opens to a zwitter ion which is unstable and rearranges or adds to other molecules. Most ozonolysis studies have been carried out in non-polar solvents and at low temperatures [24,25]. The same general principles also apply to aqueous systems although water may participate as a reactant. Ozonolysis of an olefinic sulphone [26] and of cholesterol [20] may be cited as early case studies. The seco-derivative derived from the initial zwitter ion of cholesterol was used as a specific chemical marker to demonstrate a role of endogenous ozone in the formation of atherosclerotic plaques [21]. One of two expected zwitter ions from the ozonolysis of ascorbate is shown in Fig. 1. Rearrangement and cleavage by an apoplastic esterase or a non-enzymatic mechanism [27] lead to peroxy-L-threonic acid and oxalic acid. The second expected zwitter ion (not shown) leads to peroxy-oxalic acid and L-threonic acid. Oxalic acid and L-threonic acid are normal plant and animal degradation products of ascorbate that are formed via dehydroascorbate [27,28]. On the other hand, the reaction of ozone with ascorbate is known to lead to high yields of singlet oxygen [14]. This
Evidence against first line defence by ascorbate The decomposition of ozone by extracellular ascorbate has in plants, animals and humans been considered as part of the first line of defence and detoxification [5,11,12]. In contrast, the above chemical considerations point to a pro-oxidant role of extracellular ascorbate. It seems difficult to gather evidence for this new function because ascorbate always has its basic protective functions against reactive oxygen species. However, some initial evidence appears to exist. In plants, extracellular antioxidants other than ascorbate appeared to be protective [31,32]. The Arabidopsis mutant vtc2 was ozone tolerant in spite of resem-
CH2OH
CH2OH
CHOH
CHOH O
HO CH O
O C OOH Peroxy - L - threonic Acid
H
+ H-O O O
CH2OH O3
COOH
OH O
+
COOH Oxalic Acid
Zwitter Ion
CHOH O CH2OH
O
CHOH
H
OH
OH
1
O2
O O
H
L-Ascorbic Acid O
O O H OH Peroxy - Ketone
Fig. 1. Reaction between L-ascorbic acid and ozone according to the Criegee mechanism [24,25] (upper part) and reaction between L-ascorbic acid and singlet oxygen [29] (lower part).
H. Sandermann / Biochemical and Biophysical Research Communications 366 (2008) 271–274
bling the sensitive mutant vtc1 by containing only 30% of normal total ascorbate [33]. In humans, ozone exposure decreased extracellular ascorbate [34], but dietary ascorbate failed to protect against ozone symptoms [35]. Under different experimental conditions, dietary ascorbate had a protective effect for pulmonary functions [36]. The best evidence for a new pro-oxidant role of ascorbate was obtained when ozone damage of erythrocyte membranes covered by simulated lung lining fluid was found to depend on the addition of ascorbate [12]. This pro-oxidant effect of ascorbate was ‘‘surprisingly’’ [12] not prevented by catalase, superoxide dismutase or mannitol so that the known secondary reactive oxygen species, namely hydrogen peroxide, superoxide anion and singlet oxygen did not appear to be responsible. The peroxy-products of Fig. 1 may offer a explanation for these findings.
273
PLANTS
ANIMALS / MAN
Stomatal uptake
Inhalation
Decomposition in apoplast/ plasma membrane (Ascorbate, phenolics, lipids)
Decomposition in lining fluids/ plasma membrane (Ascorbate, glutathione, lipids)
Secondary toxicants, signalling
Ethylene, salicylic acid Oxidative burst Inhibition of photosynthesis
Tumor necrosis factor Pre-inflammatory mediators Disruption of epithelial barrier
Functions of secondary toxicants The various peroxy-compounds formed by ozone from ascorbate are expected to have toxic potential [37]. These often short-lived toxicants need to be chemically characterized. The methods employed to characterize the in-vitro and in-vivo ozonolysis products derived from unsaturated fatty acids [17–19] and cholesterol [20–22] have set excellent examples. The use of 18O-labeled ozone may provide an additional experimental strategy. The reaction between ascorbate and ozone has been followed by HPLC [9] but no reaction products were identified. The secondary products originating from the reaction between lipids and ozone have previously been postulated to mediate ozone toxicity in what has been termed a cascade mechanism [17] Animal studies have indeed revealed signalling effects of lipid peroxidation and ozonolysis products [18,19,38] and of the seco-derivative of cholesterol. The latter affected cellular apoptosis [39] and b-amyloid aggregation [40]. It remains to be studied whether the peroxy-derivatives of Fig. 1 also possess signalling activity. A hypothetical view of the mode of action of ozone is schematically summarized in Fig. 2 for plants, animals and man. In plant studies, ozone-induced transmitter molecules such as ethylene, salicylic acid and lipid peroxides led to altered gene expression and biochemical defence status [3,4,41]. Moreover, plant innate immunity was changed as determined by pathogen resistance induced by ozone [42] as well as by singlet oxygen and hydrogen peroxide [43]. When considering animals and man, ozone induced transmitter molecules such as tumor necrosis factor, cytokines, prostaglandins and the transcription factor, nuclear factor jB [44–46]. In addition, inflammatory pathways, parameters of innate immunity and epithelial barrier functions were affected [44–46]. Recent research indicates that Toll-like receptors [47] and the type I interleukin 1 receptor [48] are involved in pulmonary ozone responses. In conclusion, ozone-derived secondary toxicants may feed into signalling networks that affect the innate immunity systems of plants, animals and man.
Changed innate immunity Fig. 2. Possible role of secondary toxicants in ozone action in plants, animals and man.
Acknowledgments This work has received financial support from the Helmholtz Society, and academic support from Professor Heinz Rennenberg, Chair of Tree Physiology, University of Freiburg, Germany. References [1] M.G. Mustafa, Biochemical basis of ozone toxicity, Free Radic. Biol. Med. 9 (1990) 245–265. [2] K.E. Pinkerton, Ozone, a malady for all ages, Am. J. Respir. Crit. Care Med. 176 (2007) 07–108. [3] H. Sandermann, Ozone and plant health, Annu. Rev. Phytopathol. 34 (1996) 347–366. [4] S. Krupa, M.T. McGrath, C.P. Andersen, F.L. Booker, K.O. Burkey, A.H. Chappelka, B.I. Chevone, E.J. Pell, B.A. Zilinskas, Ambient ozone and plant health, Plant Dis. 85 (2001) 4–12. [5] P.L. Conklin, C. Barth, Ascorbic acid, a familiar small molecule intertwined in the response of plants to ozone, pathogens, and the onset of senescence, Plant Cell Environ. 27 (2004) 959–970. [6] A. Laisk, O. Kull, H. Moldau, Ozone concentration in leaf intercellular air spaces is close to zero, Plant Physiol. 90 (1989) 1163–1167. [7] W.L. Chameides, The chemistry of ozone deposition to plant leaves: role of ascorbic acid, Environ. Sci. Technol. 23 (1989) 595–600. [8] J.R. Kanofsky, P.D. Sima, Reactive absorption of ozone: an assay for reaction rates of ozone with sulfhydryl groups and with other biological molecules, Methods Enzymol. 319 (2000) 505–512. [9] S. Kermani, A. Ben-Jebria, J.S. Ultman, Kinetics of ozone reaction with uric acid, ascorbic acid, and glutathione at physiologically relevant conditions, Arch. Biochem. Biophys. 451 (2006) 8–16. [10] P.L. Conklin, E.H. Williams, R.L. Last, Environmental stress sensitivity of an ascorbic acid-deficient Arabidopsis mutant, Proc. Natl. Acad. Sci. USA 93 (1996) 9970–9974. [11] C.E. Cross, A. van der Vliet, C.A. O’Neill, S. Louie, B. Halliwell, Oxidants, antioxidants and respiratory tract lining fluids, Environ. Health Perspect. 102 (Suppl. 10) (1994) 185–191.
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[12] C.A. Ballinger, R. Cueto, G. Squadrito, J.F. Coffin, L.W. Velsor, W.A. Pryor, E.M. Postlethwait, Antioxidant-mediated augmentation of ozone-induced membrane oxidation, Free Radic. Biol. Med. 38 (2005) 515–526. [13] S.D. Langford, A. Bidani, E.M. Postlethwait, Ozone-reactive absorption by pulmonary epithelial lining fluid constituents, Toxicol. Appl. Pharmacol. 132 (1995) 122–130. [14] J.R. Kanofsky, P. Sima, Singlet oxygen production from the reactions of ozone with biological molecules, J. Biol. Chem. 266 (1991) 9039– 9042. [15] H.D. Grimes, K.K. Perkins, W.F. Boss, Ozone degrades into hydroxyl radical under physiological conditions. A spin trapping study, Plant Physiol. 72 (1983) 1016–1020. [16] C.H. Foyer, G. Noctor, Redox homeostasis and antioxidant signalling: a metabolic interface between stress perception and physiological responses, Plant Cell 17 (2005) 1866–1875. [17] W.A. Pryor, G.L. Squadrito, M. Friedman, The cascade mechanism to explain ozone toxicity: the role of lipid ozonation products, Free Radic. Biol. Med. 19 (1995) 935–941. [18] G.L. Squadrito, M.G. Salgo, F.R. Fronczeck, W.A. Pryor, Synthesis of inflammatory signal transduction species formed during ozonation and/or peroxidation of tissue lipids, Methods Enzymol. 319 (2000) 570–582. [19] L.I. Szweda, P.A. Szweda, A. Holian, Detection of 4-hydroxy-2nonenol adducts following lipid peroxidation from ozone exposure, Methods Enzymol. 319 (2000) 562–570. [20] J. Gomulka, L.L. Smith, Ozonization of cholesterol, A. Amer. Chem. Soc. 105 (1983) 1972–1979. [21] P. Wentworth, J. Nieva, C. Takeuchi, R. Galve, A.D. Wentworth, R.B. Dilley, G.A. DeLaria, A. Saven, B.M. Babior, K.D. Janda, A. Eschenmoser, R.A. Lerner, Evidence for ozone formation in human atherosclerotic arteries, Science 302 (2003) 1053–1056. [22] M.K. Pulfer, R.C. Murphy, Formation of biologically active oxysterols during ozonolysis of cholesterol present in lung surfactant, J. Biol. Chem. 279 (2004) 26331–26338. [23] V.C. Runeckles, B.I. Chevone, Crop responses to ozone, in: A.S. Lefohn (Ed.), Surface Level Ozone Exposures and their Effects on Vegetation, Lewis Publishers, Chelsea, 1992, pp. 189–270. [24] P.S. Bailey, The reactions of ozone with organic compounds, Chem. Rev. 58 (1958) 925–1010. [25] R. Criegee, Mechanism of ozonolysis, Angew. Chem. Int. Ed. 14 (1975) 745–752. [26] G. Lohaus, Die Ozonide ungesa¨ttigter cyclischer Sulfone, J. Liebigs Annalen der Chemie 583 (1953) 12–19. [27] M.A. Green, S.C. Fry, Vitamin C degradation in plant cells via enzymatic hydrolysis of 4-O-oxalyl-L-threonate, Nature 433 (2005) 83–87. [28] M.B. Davies, J. Austin, D.A. Partridge, Vitamin C: Its Chemistry and Biochemistry, Royal Society of Chemistry, Cambridge, 1991 (pp. 94– 96). [29] B.M. Kwon, C.S. Foote, Chemistry of singlet oxygen. 50. Hydroperoxide intermediates in the photooxygenation of ascorbic acid, J. Am. Chem. Soc. 110 (1988) 6582–6583. [30] G.G. Kramarenko, S.G. Hummel, S.M. Martin, G.R. Buettner, Ascorbate reacts with singlet oxygen to produce hydrogen peroxide, Photochem. Photobiol. 82 (2006) 1634–1637. [31] D. d’Haese, K. Vandermeiren, H. Asard, N. Horemans, Other factors than apoplastic ascorbate contribute to the differential ozone tolerance of two clones of Trifolium repens L., Plant Cell Environ. 28 (2005) 623–632.
[32] K.O. Burkey, H.S. Neufeld, L. Souza, A.H. Chappelka, A.W. Davison, Seasonal profiles of leaf ascorbic acid content and redox state in ozone-sensitive wildflowers, Environ. Pollut. 143 (2006) 427– 434. [33] P.L. Conklin, S.A. Saraco, S.R. Norris, R.L. Last, Identification of ascorbic acid-deficient Arabidopsis thaliana mutants, Genetics 154 (2000) 847–856. [34] I.S. Mudway, M.T. Krishna, A.J. Frew, D. MacLeod, T. Sandstrom, S.T. Holgate, F.J. Kelly, Compromised concentrations of ascorbate in fluid lining the respiratory tract in human subjects after exposure to ozone, Occup. Environ. Med. 56 (1999) 473–481. [35] I.S. Mudway, A.F. Behndig, R. Helleday, J. Pourazar, A.J. Frew, F.J. Kelly, A. Blomberg, Vitamin supplementation does not protect against symptoms in ozone-responsive subjects, Free Radic. Biol. Med. 40 (2006) 1702–1712. [36] J.M. Samet, G.E. Hatch, D. Hortman, S. Steck Scott, L. Arab, P.A. Bromberg, M. Levine, W.F. McDonnell, R.B. Devlin, Effect of antioxidant supplementation on ozone-induced lung injury in human subjects, Am. J. Respir. Crit. Care Med. 164 (2001) 819–825. [37] R.B. Reid JB, Organic peroxides, in: E. Bingham, B. Cohrssen, C.H. Powell (Eds.), Patty’s Toxicology, vol. 6, John Wiley Sons, New York, 2001, pp. 1147–1246. [38] C. Uhlson, K. Harrison, C.B. Allen, S. Ahmad, C.W. White, R.C. Murphy, Oxidized phospholipids derived from ozone-treated lung surfactant extract reduce macrophage and epithelial cell viability, Chem. Res. Toxicol. 15 (2002) 896–906. [39] K. Sathishkumar, M. Haque, T.E. Perumal, J. Francis, R.M. Uppa, A major ozonation product of cholesterol, 3b-hydroxy-5-oxo-5,6secocholestan-6-al, induces apoptosis in H9c2 cardiomyoblasts, FEBS Lett. 579 (2005) 6444–6450. [40] K. Sathishkumar, R. Martin, R.M. Uppu, Cholesterol secoaldehyde, an ozonation product of cholesterol, induces amyloid aggregation and apoptosis in murine GT1-7 hypothalamic neurons, Alzheimers Dis. 11 (2007) 261–274. [41] J. Kangasja¨rvi, P. Jaspers, H. Kollist, Signalling and cell death in ozone-exposed plants, Plant Cell Environ. 28 (2005) 1021– 1036. [42] C. Langebartels, D. Ernst, J. Kangasja¨rvi, H. Sandermann, Ozone effects on plant defence, Methods Enzymol. 319 (2000) 520–535. [43] H. Sandermann, Molecular ecotoxicology of plants, Trends Plant Sci. 9 (2004) 406–413. [44] K.F. Chung, I.M. Adcock, Induction of nuclear factor-jB by exposure to ozone and inhibition by glucocorticoids, Methods Enzymol. 319 (2000) 551–562. [45] J.W. Hollingworth, S.R. Kleeberger, W.M. Foster, Ozone and pulmonary innate immunity, Proc. Am. Thorac. Soc. 4 (2007) 240– 246. [46] J.W. Hollingworth, S. Maruoka, Z. Li, E.N. Potts, D.M. Brass, S. Garantziotis, A. Fong, W.M. Foster, D.A. Schwartz, Ambient ozone primes pulmonary innate immunity in mice, J. Immunol. 179 (2007) 4367–4375. [47] A.S. Williams, S.Y. Leung, P. Nath, N.M. Khorasani, P. Bhavsar, R. Issa, J.A. Mitchell, I.M. Adcock, K.F. Chung, Role of TLR2, TLR4 and MyD88 in murine ozone-induced airway hyperresponsiveness and neutrophilia, J. Appl. Physiol. 103 (2007) 1189–1195. [48] R.A. Johnston, J.P. Mizgerd, L. Flynt, L.J. Quinton, E.S. Williams, S.A. Shore, Type I interleukin-1 receptor is required for pulmonary responses to subacute ozone exposure in mice, Am. J. Respir. Crit. Care Med. 37 (2007) 477–484.
Biochemical and Biophysical Research Communications 366 (2008) 271–274 www.elsevier.com/locate/ybbrc
Mini Review
Ecotoxicology of ozone: Bioactivation of extracellular ascorbate Heinrich Sandermann Ecotox.freiburg, Schubertstr. 1, D-79104 Freiburg, Germany GSF-National Research Center, Institute of Biochemical Plant Pathology, D-85764 Neuherberg, Germany Received 21 November 2007 Available online 17 December 2007
Abstract The ascorbate pools of the extracellular respiratory lining fluids and the plant apoplast are currently considered as part of the first line of defence against ambient ozone. Ozone is in fact rapidly decomposed by ascorbate, but the only product identified so far (singlet oxygen) is toxic. Peroxy-L-threonic and peroxy-oxalic acids are now derived as further decomposition products on the basis of Criegee-type ozone chemistry. These secondary toxicants may resemble known ozone induced transmitter molecules by participating in signalling events affecting plant and animal innate immunity. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Ascorbate; Respiratory lining fluid; Plant apoplast; Singlet oxygen; Secondary toxicants
Extracellular ascorbate as first line of defence against ozone Ambient tropospheric ozone is known to be toxic for man, animals and plants, the recommended safety levels being frequently exceeded. In animals and man, lung functions are affected, pre-inflammatory mediators are increased and airway inflammation occurs. Epidemiological studies have detected increased premature mortality [1,2]. In plants, acute and chronic exposure to ozone can lead to plant stress responses, visible symptoms, premature senescence, crop loss and alterations of plant community structure [3,4]. After stomatal uptake, ozone dissolves in the apoplastic fluid, and the reaction with apoplastic ascorbate has been considered as a first line of plant defence [5] on the basis of three main types of evidence: the intercellular concentration of ozone was found to be near zero [6], ozone is scavenged by ascorbate with a high rate constant [7–9], and the ozone sensitivity of mutant vtc1 of Arabidopsis appeared to be caused by its total ascorbate level of only 30% of normal [10]. The extracellular ascorbate in respiratory lining fluids of animals and humans has also been considered as part of the first line of defence against ozone E-mail address: [email protected] 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.12.018
[11,12], again due to its function as one of the primary ozone absorption targets [8,9,13]. Two reaction pathways for toxicants derived from ozone The extracellular compartments and plasma membranes of plants and animals are essentially the only cellular components directly exposed to ozone. Due to the rapid extracellular quenching of ozone, the intracellular compartments are mainly exposed to secondary toxicants derived from one of two pathways, namely ozone decomposition in aqueous solution, or ozonolysis of double bonds [1]. It is well known that ozone will in aqueous solutions decompose to hydrogen peroxide, singlet oxygen and hydroxyl radicals, biomolecules such as ascorbate [14] or phenolics [15] enhancing these reactions. The toxic effects and the detoxification of these secondary reactive oxygen species have been well studied, ascorbate being involved in both extra- and intracellular detoxification [1,16]. This beneficial role involves electron transfer reactions leading to monodehydroascorbate and dehydroascorbate, followed by regeneration of ascorbate. The protective systems are most abundant in intracellular organelles where reactive oxygen species are formed as metabolic side-products.
272
H. Sandermann / Biochemical and Biophysical Research Communications 366 (2008) 271–274
The ozonolysis pathway is best studied for unsaturated fatty acids [17–19] and cholesterol [20–22]. Older studies have pointed out that ozone is also likely to attack the double bond of ascorbate [7,23]. However, it appears that the Criegee mechanism of ozonolysis [24,25] has never been applied to ascorbate.
mechanism is, like ozonolysis, in agreement with the 1:1 stoichiometry determined for the overall reaction between ozone and ascorbate [9]. The formation of singlet oxygen from ozone and olefins is thought to proceed by transfer of an oxygen atom to yield an unstable epoxide [14]. This mechanism is consistent with the 5,6-epoxide of cholesterol being a major reaction product detected after ozone exposure of lung surfactant [22]. The epoxide of ascorbic acid could add water to yield dehydroascorbic acid. However, the reactivity of the hypothetical ascorbyl-epoxide may be so high that C–C bond cleavage and formation of aldehydes occur. Singlet oxygen in turn is able to react with ascorbate, one of the elucidated main reaction products being depicted in Fig. 1 [29]. The isomeric peroxy-ketones are highly electrophilic, their reaction with water leading to dehydroascorbate and hydrogen peroxide [30]. Reactions with sulfhydryl and other oxidizable groups have apparently not been characterized.
Chemistry of the ascorbate/ozone reaction Ozone attack on double bonds is known to proceed initially by 1,3-dipolar cycloaddition. The resulting primary ozonide opens to a zwitter ion which is unstable and rearranges or adds to other molecules. Most ozonolysis studies have been carried out in non-polar solvents and at low temperatures [24,25]. The same general principles also apply to aqueous systems although water may participate as a reactant. Ozonolysis of an olefinic sulphone [26] and of cholesterol [20] may be cited as early case studies. The seco-derivative derived from the initial zwitter ion of cholesterol was used as a specific chemical marker to demonstrate a role of endogenous ozone in the formation of atherosclerotic plaques [21]. One of two expected zwitter ions from the ozonolysis of ascorbate is shown in Fig. 1. Rearrangement and cleavage by an apoplastic esterase or a non-enzymatic mechanism [27] lead to peroxy-L-threonic acid and oxalic acid. The second expected zwitter ion (not shown) leads to peroxy-oxalic acid and L-threonic acid. Oxalic acid and L-threonic acid are normal plant and animal degradation products of ascorbate that are formed via dehydroascorbate [27,28]. On the other hand, the reaction of ozone with ascorbate is known to lead to high yields of singlet oxygen [14]. This
Evidence against first line defence by ascorbate The decomposition of ozone by extracellular ascorbate has in plants, animals and humans been considered as part of the first line of defence and detoxification [5,11,12]. In contrast, the above chemical considerations point to a pro-oxidant role of extracellular ascorbate. It seems difficult to gather evidence for this new function because ascorbate always has its basic protective functions against reactive oxygen species. However, some initial evidence appears to exist. In plants, extracellular antioxidants other than ascorbate appeared to be protective [31,32]. The Arabidopsis mutant vtc2 was ozone tolerant in spite of resem-
CH2OH
CH2OH
CHOH
CHOH O
HO CH O
O C OOH Peroxy - L - threonic Acid
H
+ H-O O O
CH2OH O3
COOH
OH O
+
COOH Oxalic Acid
Zwitter Ion
CHOH O CH2OH
O
CHOH
H
OH
OH
1
O2
O O
H
L-Ascorbic Acid O
O O H OH Peroxy - Ketone
Fig. 1. Reaction between L-ascorbic acid and ozone according to the Criegee mechanism [24,25] (upper part) and reaction between L-ascorbic acid and singlet oxygen [29] (lower part).
H. Sandermann / Biochemical and Biophysical Research Communications 366 (2008) 271–274
bling the sensitive mutant vtc1 by containing only 30% of normal total ascorbate [33]. In humans, ozone exposure decreased extracellular ascorbate [34], but dietary ascorbate failed to protect against ozone symptoms [35]. Under different experimental conditions, dietary ascorbate had a protective effect for pulmonary functions [36]. The best evidence for a new pro-oxidant role of ascorbate was obtained when ozone damage of erythrocyte membranes covered by simulated lung lining fluid was found to depend on the addition of ascorbate [12]. This pro-oxidant effect of ascorbate was ‘‘surprisingly’’ [12] not prevented by catalase, superoxide dismutase or mannitol so that the known secondary reactive oxygen species, namely hydrogen peroxide, superoxide anion and singlet oxygen did not appear to be responsible. The peroxy-products of Fig. 1 may offer a explanation for these findings.
273
PLANTS
ANIMALS / MAN
Stomatal uptake
Inhalation
Decomposition in apoplast/ plasma membrane (Ascorbate, phenolics, lipids)
Decomposition in lining fluids/ plasma membrane (Ascorbate, glutathione, lipids)
Secondary toxicants, signalling
Ethylene, salicylic acid Oxidative burst Inhibition of photosynthesis
Tumor necrosis factor Pre-inflammatory mediators Disruption of epithelial barrier
Functions of secondary toxicants The various peroxy-compounds formed by ozone from ascorbate are expected to have toxic potential [37]. These often short-lived toxicants need to be chemically characterized. The methods employed to characterize the in-vitro and in-vivo ozonolysis products derived from unsaturated fatty acids [17–19] and cholesterol [20–22] have set excellent examples. The use of 18O-labeled ozone may provide an additional experimental strategy. The reaction between ascorbate and ozone has been followed by HPLC [9] but no reaction products were identified. The secondary products originating from the reaction between lipids and ozone have previously been postulated to mediate ozone toxicity in what has been termed a cascade mechanism [17] Animal studies have indeed revealed signalling effects of lipid peroxidation and ozonolysis products [18,19,38] and of the seco-derivative of cholesterol. The latter affected cellular apoptosis [39] and b-amyloid aggregation [40]. It remains to be studied whether the peroxy-derivatives of Fig. 1 also possess signalling activity. A hypothetical view of the mode of action of ozone is schematically summarized in Fig. 2 for plants, animals and man. In plant studies, ozone-induced transmitter molecules such as ethylene, salicylic acid and lipid peroxides led to altered gene expression and biochemical defence status [3,4,41]. Moreover, plant innate immunity was changed as determined by pathogen resistance induced by ozone [42] as well as by singlet oxygen and hydrogen peroxide [43]. When considering animals and man, ozone induced transmitter molecules such as tumor necrosis factor, cytokines, prostaglandins and the transcription factor, nuclear factor jB [44–46]. In addition, inflammatory pathways, parameters of innate immunity and epithelial barrier functions were affected [44–46]. Recent research indicates that Toll-like receptors [47] and the type I interleukin 1 receptor [48] are involved in pulmonary ozone responses. In conclusion, ozone-derived secondary toxicants may feed into signalling networks that affect the innate immunity systems of plants, animals and man.
Changed innate immunity Fig. 2. Possible role of secondary toxicants in ozone action in plants, animals and man.
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