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Chem. Res. Toxicol. 2006, 19, 668-673
Effect of Heparin on Viologen-Stimulated Enzymatic NADH Depletion Jacek Zielonka, Małgorzata Rybak, Joanna Celin´ska, Jan Adamus, Andrzej Marcinek, and Jerzy Gebicki* Institute of Applied Radiation Chemistry, Technical UniVersity, 90-924 Lodz, Poland ReceiVed December 2, 2005
Paraquat and diquat undergo redox cycling mediated by xanthine oxidase in the NADH-dependent manner. In these processes, the rates of NADH oxidation and superoxide formation are increased almost 10-fold. The addition of heparin can substantially inhibit these processes. A protective role of heparin against oxygen radicals formation can be rationalized in terms of its ability to bind paraquat or diquat. The binding process has been investigated by means of the pulse radiolysis technique. Biological consequences of the binding processes are discussed. Introduction (V2+),1
(PQ2+)
The viologens like paraquat and diquat (DQ2+), are widely used in agriculture as effective herbicides. They exert herbicidal activity by interfering with the intracellular electron transfer systems in plants through the inhibition of the reduction of nicotinamide coenzymes, NADP+ to NADPH, during photosynthesis and the formation of reactive oxygen species (ROS) that cause the destruction of plant organelles (1).
One of the most important drawbacks of the use of viologen herbicides is their toxicity to humans and animals. There have been numerous reports of human death following the accidental or intentional ingestion of viologens (2, 3). The main organs of PQ2+ intoxication in the human body are the lungs and to a lesser extent the liver, kidneys, and heart. On the other hand, DQ2+ does not exhibit accumulation in the lungs. Moreover, DQ2+ can be metabolized in part to more easily excreted pyridone derivatives; hence, it exhibits a lower toxicity than PQ2+ (4-6). Because both dications can be easily reduced to the corresponding radical cations, most of the proposed mechanisms of their toxicity involve the enzymatic reduction of viologen dication V2+ to its one-electron reduced form V•+ (reaction 1) (7).
V2+ + e- f V•+
(1)
The enzymatic systems include NAD(P)H:cytochrome P450 reductase, NADH:ubiquinone oxidoreductase (complex I), nitric oxide synthase, and xanthine oxidase (XO) (8-13). The viologen radical cation V•+ formed in these enzymatic reactions can exert its deleterious effects in several ways. The most * To whom correspondence should be addressed. Tel: +48-42-6313171. Fax: +48-42-6365008. E-mail:
[email protected]. 1 Abbreviations: ROS, reactive oxygen species; XO, xanthine oxidase; GAGs, glycosoaminoglycans; SOD, superoxide dismutase; DPI, diphenyliodonium; Hep, heparin; V2+, viologen; PQ2+, paraquat; DQ2+, diquat.
important is the reaction with oxygen to produce the superoxide radical anion O2•- (reaction 2), and subsequently hydrogen peroxide H2O2, as well as hydroxyl and peroxyl radicals. It has been postulated that V•+ can also release iron from ferritin enabling the Fenton reaction and •OH formation. In these reactions, V2+ act as electron carriers contributing to redox cycling processes (7, 14).
V•+ + O2 f V2+ + O2•-
(2)
PQ2+ redox cycling leads also to the potential depletion of intracellular NAD(P)H. Because this could perturb important cell processes, it has been suggested that PQ2+-stimulated NAD(P)H depletion may be a primary toxic event (7). It has been postulated that NADPH-cytochrome c3+ reductase as well as XO may play important roles in PQ2+-induced dysfunction of endothelial cells and lung injury (13, 15-19). Because there are no pharmacological antagonists for viologens, different strategies in the management of the intoxication were applied. Those include administration of the inhibitors of PQ2+-mediated enzymatic reactions or use of enzymatic and low-molecular antioxidants. However, most of the antioxidants were ineffective in preventing the toxicity of these herbicides (20). Many strategies against intoxication involve modification of the pharmacokinetics of viologens by either decrease of their absorption or enhancement of their excretion. Such procedures involve gastric lavage, hemodialysis, hemoperfusion, and administration of oral adsorbents (20). Different attempts to reduce toxicity of viologens are based on an application of polymeric sulfates, including glycosaminoglycans (GAGs). The positive effect of their administration has been attributed to the trapping capabilities of these polyanions toward viologens (21-23). As glycosaminoglycans also exert a protective effect against free radicals injury of endothelium (24, 25), the major aim of this study was to determine the possible mechanisms of GAG action against PQ2+ and DQ2+ toxicity, using heparin (Hep) as a model for GAG and XO as a model for the viologens-stimulated enzymatic system.
Materials and Methods Compounds. NADH, XO from buttermilk, cytochrome c3+ from equine heart, superoxide dismutase (SOD) from bovine erythrocytes,
10.1021/tx050336s CCC: $33.50 © 2006 American Chemical Society Published on Web 03/22/2006
Effect of Heparin on Viologen-Stimulated XO Hep sodium salt from porcine intestinal mucosa (catalog no. H9399), diphenyliodonium (DPI) chloride, Sepharose CL-6B, Na2EDTA, KH2PO4, Na2HPO4, AgCl, and NaCl were from Sigma (St. Louis, MO). NaOH, HClO4, PQ2+ (1,1′-dimethyl-4,4′-bipyridinium dichloride), and KSCN were obtained from Aldrich (Steinheim, Germany), and DQ2+ (1,1′-ethylene-2,2′-bipyridinium dibromide) was from Chem Service, Inc. (West Chester, PA). Hep immobilized on sepharose CL-6B was from Amersham Pharmacia Biotech (Uppsala, Sweden). All solutions were prepared using water purified by a Millipore Milli-Q system. Hep Characteristics. The Hep from porcine intestinal mucosa is a mixture of polymer chains of different molecular masses, most of them in the range of 17-19 kDa. The average molecular mass was assumed to be 18 kDa. The equivalent molecular mass of Hep 178 g per unit of charge was used (26) giving the average number of 101 equivalents per Hep macromolecule. The average charge per disaccharide unit was calculated as 3.5 in accordance with other reports (26, 27). The calculated average number of disaccharide units per polymer chain was 29. The concentration of Hep was expressed as the number of moles of polymer chains per dm3 ([Hep]0). Spectrophotometric and pH Measurements. The spectrophotometric measurements were carried out using Perkin-Elmer Lambda 40P double-beam spectrophotometer equipped with a thermostated cell holder. The pH of the solutions was adjusted with perchloric acid, sodium hydroxide, or phosphate buffer and measured with the ORION 420A pH meter (Orion Research, Inc., Beverly, MA). Spectrophotometric Assay of the Binding of Viologens to Immobilized Hep. The solutions of viologens (20 µM) were mixed with the aqueous suspension of sepharose-immobilized Hep (25 mg/mL). The mixture was shaken for about 1 min and then centrifuged at 13000 rpm for 4 min using a FORCE 1418 microcentrifuge from Labnet Int. (Edison, NJ). The changes of viologens concentrations in solution were determined spectrophotometrically. Enzymatic Assay. XO activity was measured using NADH as a reducing substrate. The NADH decay was followed spectrophotometrically at the wavelength of 340 nm [absorption coefficient 6.22 × 103 M-1 cm-1 (28)]. Superoxide radical anion formation was measured by the reduction of cytochrome c3+ (30 µM), following the absorbance build-up at 550 nm [the difference in absorption coefficients of reduced and oxidized forms of cytochrome was 2.1 × 104 M-1 cm-1 (29)]. For every measurement, the blank solution without XO was placed in the reference beam and the rates were determined in the first 3 min of the reactions. The XO-mediated reactions were run at T ) 25°C, in aqueous solutions of pH 7.8 containing 2 U/mL XO, 200 µM NADH, 1 mM EDTA, and 50 mM phosphate buffer. NaCl was used for the correction of the ionic strength of the solution. To minimize the effect of ionic strength on the binding process in experiments with Hep, the phosphate buffer concentration was 1 mM with 0.4 mM EDTA and no NaCl was added. The solutions were thermostated at 25 °C for 10 min before measurement, and the reaction was started by the addition of NADH. To remove oxygen, the solutions were bubbled with nitrogen in the septum-sealed cells for at least 15 min before they were thermostated. Pulse Radiolysis. Pulse radiolysis experiments were carried out with high-energy (6 MeV) 3 ns electron pulse generated from an ELU-6 linear electron accelerator. The dose absorbed per pulse was determined with a N2O-saturated aqueous solution of KSCN (0.01 M), assuming G[(SCN)2•-] ) 6.0 and [(SCN)2•-] ) 7600 M-1 cm-1 (G represents yield of radicals per 100 eV of energy absorbed and is the absorption coefficient at 475 nm). The dose delivered per pulse was 4 Gy. Details of pulse radiolysis system are given elsewhere (30, 31). The pulse radiolysis of neutral water produced three highly reactive species (32), the hydrated electron (eaq) (2.6), •OH (2.7), and H• (0.6), in addition to the formation of less reactive products, H2O2 (0.7), H2 (0.45), and H3O+ (2.6) (the numbers in parentheses are the G values 100 ns after electron pulse).
Chem. Res. Toxicol., Vol. 19, No. 5, 2006 669
Figure 1. Dependence of the initial rates of NADH oxidation under aerobic conditions on the PQ2+ concentration in the system containing XO (0.2 U/mL), NADH (200 µM), EDTA (1 mM), and phosphate buffer (50 mM), pH 7.8.
To study the reactivity of eaq toward viologen dications, the solutions were deoxygenated by bubbling with Ar for at least 10 min. The hydroxyl radical scavengers were not used since in the investigated system the eaq lifetime was not affected by their presence or absence. The reaction was monitored at 720 nm, at which the eaq absorption band exhibited the maximum (32). The reactivity of viologen radical cations V•+ was studied in the aqueous solutions containing 50 mM HCOONa, 50 mM phosphate buffer (pH 7.8), 0.2 mM EDTA, and 2 mM viologen saturated with an appropriate gas (Ar, air, or O2) to secure the oxygen concentration at the levels ∼0, 0.25, and 1.25 mM, respectively. In such solutions, pulse radiolysis lead to the formation of V•+ via reactions 3-6. V2+ + eaq- f V•+
(3)
HCOO- + •OH f CO2•- + H2O
(4)
HCOO- + H• f CO2•- + H2
(5)
V2+ + CO2•- f V•+ + CO2
(6)
The reactivity of V•+ toward cytochrome c3+ was studied in the Ar- and air-saturated solutions containing cytochrome c3+ at the concentration range of 10-50 µM. A desired ionic strength of the solution was maintained by NaCl addition. The concentration of Na+ was calculated as the sum of NaCl added and the concentration of sodium cations of the Hep sodium salt, assuming 1 mol of Na+ per 1 mol of Hep equivalent. Kinetic analysis was done with the Levenberg-Marquardt algorithm. The first-order rate constant values (kobs) were evaluated from the plot of ∆A vs time. The bimolecular rate constants were determined from the slope of the linear plot of kobs vs solute concentration.
Results and Discussion Viologen-Stimulated Enzymatic NADH Depletion. The XO is an enzyme involved in the conversion of hypoxanthine to xanthine and xanthine to uric acid, as the final catabolism of purines. In the absence of reducing substrates, the enzyme is capable of catalyzing the oxidation of NADH coenzyme by oxygen. Some xenobiothics can additionally enhance this activity of the enzyme (33-36). In Figure 1, the influence of the PQ2+ concentration on the XO-mediated NADH oxidation is presented. As can be seen in this figure, PQ2+ is able to increase the rate of enzymatic oxidation of NADH almost 10-fold. The enzymatic oxidation of NADH enhanced by PQ2+ is also observed in the absence of oxygen.
670 Chem. Res. Toxicol., Vol. 19, No. 5, 2006
Figure 2. Absorption spectra of the radical cation PQ•+ formed after 15 and 40 min of enzymatic XO (0.02 U/mL) oxidation of NADH (200 µM) in the presence of PQ2+ (0.7 mM). Degassed aqueous solution, EDTA (1 mM), and phosphate buffer (50 mM), pH 7.8, were used.
Figure 3. Dependence of the initial rates of cytochrome c3+ reduction under aerobic conditions on the PQ2+ concentration in the system containing XO (0.2 U/mL), cyt c3+ (30 µM), NADH (200 µM), EDTA (1 mM), and phosphate buffer (50 mM), pH 7.8.
The UV-vis absorption spectrum of the reaction mixture (deoxygenated solution containing NADH, XO, and PQ2+) and the changes observed in the course of the enzymatic reaction are presented in Figure 2. No reaction is observed in the solutions without the enzyme added. It is evident from this figure that decay of the absorbance associated with NADH (band at 340 nm) is accompanied by the build-up of the absorption bands with the maxima at 396 and 604 nm, which are characteristic for PQ•+ radical cation. A similar behavior has been observed for DQ2+ (data not shown) whose radical cation absorbs at 380 and 765 nm (37-40). Because the NADH-oxidase activity of XO is enhanced by PQ2+ in the presence and absence of oxygen, it is evident that PQ2+ is an effective competitor of oxygen in the XO enzymatic reaction. Despite this fact, the yield of superoxide radical anion O2•- increases through the one-electron reduction of oxygen by viologen radical cations (reaction 2). In Figure 3, the initial rates of cytochrome c3+ reduction as a function of PQ2+ concentration are shown. It is evident from this figure that the higher rate of cyt c3+ reduction correlates with the higher PQ2+ concentration. A similar behavior has been observed for DQ2+, for which the relative increase in the rate of cyt c3+ reduction approached the value 11 at a 2 mM concentration of DQ2+. Two possible pathways of cyt c3+ reduction should be considered as follows: (i) reduction of O2 by V•+ (reaction 2) and subsequent reaction of O2•- with cytochrome c3+ (reaction 7) and (ii) direct reduction of cyt c3+ by V•+ (reaction 8).
Zielonka et al.
O2•- + cytochrome c3+ f O2 + cytochrome c2+
(7)
V•+ + cytochrome c3+ f V2+ + cytochrome c2+
(8)
The pulse radiolysis experiments were conducted to determine directly the rate constants of these reactions, and values were found as follows: k7 ) 2.1 × 105 M-1 s-1 and k8 ) 6.1 × 108 or 3.5 × 108 M-1 s-1 for PQ2+ or DQ2+, respectively. As the rate constant for the reaction of V•+ radical cations with O2 is high [k2 ) 8.0 × 108 M-1 s-1 for PQ•+ and 4.7 × 108 M-1 s-1 for DQ•+ (37-40)], it can be expected that in the investigated systems, V•+ reacts with O2 (0.25 mM) much faster than with cyt c3+ (0.05 mM). In the experiments in which V•+ species were generated in the presence of both cytochrome c3+ and oxygen, the radical cations disappeared within tens of microseconds [20-40 µs; as measured by the decay of the absorbance at 396 nm (PQ•+) and at 380 nm (DQ•+)] while cytochrome c3+ reduction was observed with some delay on the miliseconds time scale (up to 500 ms). Moreover, the radical cation V•+ decays were independent of cytochrome concentration in contrast to the reduction of the cytochrome itself. The rate constant of the latter process remained in agreement with the literature value of k7 for the reaction of cytochrome with oxygen (41). This observation corresponds to the mechanism of the formation of O2•- as a secondary transient species, which reduces cyt c3+ in the subsequent reaction. Because the concentrations of oxygen and cytochrome c3+ were the same as in enzymatic assay, these observations provide a direct support for the notion that V•+ formed in XO-mediated processes undergoes reoxidation in the presence of oxygen and that the O2•- formed is a reducing species for cyt c3+. The participation of O2•- in the reduction of cytochrome c3+ finds support in experiments with SOD. SOD at a concentration of 15 U/mL inhibited cyt c3+ reduction while it had no effect on the rate of NADH oxidation by PQ2+-activated XO (0.07 U/mL). This is in agreement with the expectation that SOD should eliminate O2•- produced in the viologen-stimulated reactions while the rate of XO enzymatic oxidation of NADH should remain unaffected. The XO-mediated oxidation of NADH cannot be inhibited by a specific XO inhibitor, as allopurinol, since the flavin center of the enzyme is involved in the NADH oxidation (33). An enzymatic process can, however, be inhibited by nonselective flavoenzymes inhibitors as, for example, DPI cation and Ag+. Addition of DPI or AgCl almost completely inhibited the PQ2+activated XO enzymatic reaction at a concentration of 250 µM or 30 nM, respectively, as measured by the decay of the NADH absorption and cyt c3+ reduction. On the other hand, activation of XO activity was canceled by the inactivation of viologen dications through their complexation with polyanionic species, like Hep. In Figure 4C, the effect of Hep on the relative rate of NADH oxidation in the presence of PQ2+ is shown. Similarly, in a dose-dependent manner, cyt c3+ reduction is also observed (the relative rate decreased to 40% upon addition of 300 µM of Hep; IC50 ) 100 µM). Apparently, the addition of Hep results in cancellation of the PQ2+ effect on XO activity. At high Hep concentrations, the rate of enzymatic reaction becomes close to the value characteristic for the system without PQ2+. The same effects have been observed on cyt c3+ reduction for DQ2+ (oxidation of NADH cannot be monitored directly since its absorption at 340 nm overlaps with the absorption of DQ2+ itself). As Hep itself had
Effect of Heparin on Viologen-Stimulated XO
Figure 4. Relative rates of PQ2+ (1 mM)-activated XO enzymatic oxidation of NADH (200 µM) in the presence of (A) DPI, (B) Ag+ (AgCl), and (C) Hep.
Figure 5. UV-vis spectrum of aqueous solution of PQ2+ (20 µM) before and after (dashed line) mixing with sepharose-immobilized Hep (25 mg/mL).
no effect on the rate of enzymatic reaction, these observations indicate that Hep can decrease the “effective” concentration of viologens in solution. This can be straightforwardly presented by the use of water-insoluble sepharose-immobilized Hep (see Materials and Methods). The assay based on the spectrophotometric measurement of the viologen concentration in the solution before and after mixing with sepharose-immobilized Hep shows tremendous change presented in Figure 5. Because Hep has no (or little) effect on the UV-vis absorption spectra of the viologens in aqueous solution and sepharose alone does not bind those dications, it is evident from the results presented in Figure 5 that in the systems investigated all PQ2+ molecules have been bound to Hep and removed from the solution. Pulse Radiolysis Studies of the Binding Process. The complexation of polyanions with viologens changes the reactivity and redox properties of the latter species. Therefore, the reactivity of viologens toward hydrated electron eaq was used as a measure of the binding properties of viologens to Hep. There are numerous examples of the successful application of such methodology in the studies of the interaction of biologically important cations with polyanions (42-44). The reactivity of PQ2+ and DQ2+ toward hydrated electron (reaction 1) was determined in the presence and absence of Hep. The second-order rate constants for this reaction have been found from the dependence of the pseudo-first-order rate constant of eaq decay on the viologen concentrations. The very good scavenging properties of viologens toward eaq [k1 ) 6.6 × 1010 M-1 s-1 and 6.2 × 1010 M-1 s-1 for PQ2+ and DQ2+,
Chem. Res. Toxicol., Vol. 19, No. 5, 2006 671
Figure 6. Dependence of the observed pseudo-first-order rate constant for the reaction of eaq with PQ2+ (5 × 10-5 M) on Hep concentration [Hep]0. Degassed aqueous solution and 1 mM phosphate buffer, pH 8.1, were used.
Figure 7. Dependence of the observed pseudo-first-order rate constant of eaq scavenging on PQ2+ concentration in the absence (b) and presence (9) of Hep (1 × 10-5 M). Aqueous solution and 1 mM phosphate buffer, pH 7.8, were used.
respectively (38, 40)] deteriorated with Hep added (see Figure 6). In the investigated systems, the major contribution to decrease of these rate constants comes from the binding of PQ2+ and DQ2+ to Hep. From the results of experiments conducted at fixed polyanion concentration and various concentrations of the viologens studied, presented for PQ2+ in Figure 7, it can be seen that the rate of reaction is strongly inhibited at a low concentration of V2+. However, at certain values of [V2+], the rate of reaction sharply increases to the value characteristic for the reaction in the absence of polyanions. It can be concluded that after binding of a certain number of cation molecules to the polymer chain, no more molecules can be bound due to a saturation effect and the reaction rate is restored. Therefore, from this plot, the number of V2+ molecules bound to Hep chain can be evaluated (45). From the intersection of the lines representing the slopes at low and large concentrations of PQ2+, the value of 30 dications per polyanion chain was estimated. The experimental kinetic results were also analyzed in terms of the concentrations of the “bound” ([V2+]b) and “free” ([V2+]f) dications. It can be assumed that observed pseudo-first-order rate constant of electron scavenging in the presence of Hep (kHep) increases by the addition of viologens (kobs). This change in rate of the reaction is directly related to the concentration of “free” dications and the second-order rate constant of their reaction with eaq (2k3) as presented in the eq 9 (44-46).
672 Chem. Res. Toxicol., Vol. 19, No. 5, 2006
[V++]f )
kobs - kHep 2
k3
Zielonka et al.
(9)
In this equation, kobs represents the observed pseudo-firstorder rate constant of eaq decay in the presence of viologens and Hep, kHep represents the pseudo-first-order rate constant of electron decay in the system with Hep only, and 2k3 represents the second-order rate constant for the reaction of PQ2+ with electrons corrected for the ionic strength of the solution. In the case of experiments with different Hep concentrations used, the kHep was calculated using eq 10.
kHep ) k0 + k11[Hep]0
(10)
The k0 represents the rate constant of hydrated electron decay in pure water, while k11 is the rate constant for the reaction of hydrated electrons with Hep found at a constant ionic strength of the solution.
eaq- + Hep salt f product(s)
(11)
The value of k11 was determined using Hep concentrations in the range of 0.01-1.6 mM and equals 8.4 × 108 M-1 s-1 when the Hep concentration is expressed as [Hep]0. The concentration of dications bound to Hep [V2+]b was calculated as the difference between total concentration of the viologen ([V2+]0) and the value of the concentration of the “free” dications [V2+]f (eq 12).
[V2+]b ) [V2+]0 - [V2+]f
Figure 8. Scatchard plot [PQ2+]b/[PQ2+]f vs [PQ2+]b. Degassed aqueous solution, Hep (1 × 10-5 M), and 1 mM phosphate buffer, pH 8.1, were used.
in the blood stream. PQ-mediated endothelial cell dysfunction has been demonstrated in rat aorta or lung endothelium (18, 19, 48). Taking into account that XO may also be bound to the endothelial surface (49, 50), one could expect “site-specific” generation of ROS directly on the endothelial surface and, therefore, their low susceptibility to antioxidant defense system (14). Because Hep is known to release XO from the endothelial surface and because it should compete with endothelium for V2+ dications, it might be an additional beneficial mode of Hep action against the viologens poisoning and oxidative damage to the endothelial cells.
(12)
Analysis of the binding process in terms of Scatchard plot (the dependence of [V2+]b/[V2+]f on [V2+]b) is presented for PQ2+ in Figure 8. From this plot, the association constant KN ) 6.9 × 104 M-1 and the total number of binding sites equal to 37 were evaluated (26, 46). Only qualitative results were obtained for DQ2+, but it may be concluded that its binding properties remain alike. It is evident that Hep could be an efficient antidote against PQ2+ intoxication since a single average polyanion chain is capable of binding tens of viologens molecules.
Conclusions The results presented in this paper indicate that enzymatic oxidation of NADH by the XO is effectively activated by the viologens. This process cannot be inhibited by a classical molybdenum site XO inhibitor, allopurinol, but it is inhibited by the flavoenzymes inhibitors, DPI and Ag+. Because similar effects have been observed for many flavoenzymes, including NAD(P)H oxidase and NO synthase, it is difficult to estimate a contributing role of XO to PQ2+ toxic action (33). The beneficial effect attributed to the interactions between polyanions and viologen dications may arise not only from the limitation of the absorption of viologens from the digestive tract but also from the minimization of the redox cycling activity of viologens, as has been shown for the system involving NADH and XO. The effects above could be of some physiological significance expressed by potentially a lower toxicity of viologens in the presence of Hep. It can be expected that nonspecific electrostatic interactions as with Hep may exist also between V2+ and negatively charged glycosoaminoglycans (GAGs) present on the vascular endothelium surface (47). Therefore, the local concentration of the viologens on the endothelial surface might be much higher than
Acknowledgment. This work was supported by the grant (PBZ-KBN-101/T09/2003) from the Ministry of Science and Informatization.
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