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Chem. Res. Toxicol. 2004, 17, 32-44
Peroxynitrite Delivery Methods for Toxicity Studies Chen Wang† and William M. Deen*,†,‡ Department of Chemical Engineering and Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received July 17, 2003
The endogenous synthesis of peroxynitrite (ONOO-) has been implicated in a number of diseases, but assessments of its cytotoxicity and genotoxicity have been hampered by its extremely short half-life under physiological conditions (100 U/mL.) Because the superoxide assay interferes with the measurement of HX, X, and UA, two separate experiments were performed for each set of conditions. In one, 70 µM cytochrome C was used to measure the O2- concentration at 550 nm (∆550 ) 16.8 mM-1 cm-1 at 23 °C). In the other, concentrations of the substrate and products were monitored in the UV region. To avoid inactivation of XO by O2-, >100 U/mL of SOD was used in this case. Three isosbestic wavelengths (λ1 ) 261.2 nm, λ2 ) 269.6 nm, and λ3 ) 278.4 nm at 23 °C) were chosen to simultaneously measure the concentrations of HX, X, and UA. The concentrations at time t were evaluated using
At - A0 [HX]t ) [HX]0 + | HX - 3 λ3 [X]t ) [X]0 +
At - A0 | X - 2 λ2
[UA]t ) [UA]0 +
At - A 0 | UA - 1 λ1
where A is the absorbance and the subscripts 0 and t indicate initial values and values at time t, respectively. The apparent molar absorptivities were (HX - 3)λ3 ) -6.45, (X - 2)λ2 ) 5.30, and (UA- 1)λ1 ) -5.10 mM-1 cm-1 at 23 °C. Corrections were made for the contribution of XO to the absorbance and for the dilution effect caused by its addition. The kinetics of UA oxidation by uricase were also examined. Uricase activity was routinely measured by monitoring the disappearance of UA at 291 nm with a UV spectrophotometer UA ) 12.2 mM-1 cm-1 at 23 °C). The standard assay condi(291 tions were 3 mL of 120 µM UA in air-saturated 50 mM phosphate buffer, pH 7.4, plus 20 µL of 0.1 g/L uricase stock solution. In the kinetic studies, reactions were initiated by adding various volumes of enzyme stock solution, and the initial rate of oxidation was determined by monitoring the disappearance of UA. The slope of the concentration-time plot was determined over a period of 5 min.
Wang and Deen Enzyme Inhibition. To test whether ONOO- inactivates the key enzymes, 0.1 mL of different concentrations of authentic ONOO- solution was added to 0.9 mL of 211 mU/mL XO or 181 mU/mL uricase solution with rapid vortexing, followed by brief incubation at room temperature. The enzyme activities were then assayed as described above. The activities of the enzymes without any treatment, or treatment with the same concentration of predecomposed ONOO-, were measured as controls. Synthesis of ONOO- in Situ. For experiments involving the complete synthesis system, fresh enzyme stock solutions were first prepared and the activity of XO and uricase was assayed as described above. The NO delivery chamber described previously (40), which consists of a 120 mL Teflon container equipped with a magnetic stirrer and separate loops of Silastic tubing for NO and O2 delivery, was used. NO gas (99%) (scrubbed of higher nitrogen oxides) flowed through a 4 cm loop of tubing (i.d., 1.47 mm; o.d., 1.96 mm), and a 95% O2/5% CO2 gas mixture flowed through a 9 cm loop. The reaction mixture consisted of 1 mM TyrOH, 100 or 200 µM HX, 300 U/mL catalase, and 60 mU/mL uricase in the standard buffer. At t ) 0, 3 mU/mL of XO (final concentration) was added to the reactor via a buffer exchange port, and NO delivery was initiated. The reaction was continued for 50 (100 µM HX) or 100 min (200 µM HX) at 23 °C. At the end of the reactions, a 9 mL sample was withdrawn and mixed with 1 mL of 2 mM allopurinol to terminate the XO reaction. Another sample of 10 mL was taken and stored at 4 °C for later measurements. In one control experiment, NO gas was delivered into the reaction mixture but XO was not added. In this case, the O2 tubing length was reduced to 4 cm. In another control experiment, XO was present but NO was not introduced. The O2 tubing length in that case was 4.5 cm. Both control experiments lasted for 50 or 100 min. Nitrate and nitrite concentrations were measured using an assay kit consisting of nitrate reductase and Griess reagent; the assay procedure was described previously (40). Scavenging of ONOO-. To examine the possible scavenging of peroxynitrite by the purine catabolites involved in the enzyme reactions, experiments were performed using bolus addition of preformed ONOO-. In these, 5 µL of ONOO- stock solution was added as a small droplet to the wall of a test tube containing 495 µL of TyrOH solution supplemented with various concentrations of UA, X, or HX and then mixed rapidly by vortexing. The TyrOH derivatives were assayed as described above.
Kinetic Models Reaction Chemistry. A reaction mechanism proposed by Pfeiffer et al. was used to interpret our data obtained at a physiological pH and CO2 level (25). As shown in eqs 1-3, CO2 converts ONOO- to nitrosoperoxycarbonate (ONOOCO2-), which decomposes either to NO2 and CO3radicals or to NO3- and CO2 (45): k1
ONOO- + CO2 98 ONOOCO2k2
ONOOCO2- 98 NO2 + CO3k3
ONOOCO2- 98 NO3- + CO2
(1) (2) (3)
Reaction 1 is the rate limiting step in this sequence, and the rate for reaction 3 is nearly twice that of reaction 2 (46). The resulting NO2 and CO3- radicals attack TyrOH molecules to generate TyrO•, according to k4
TyrOH + NO2 98 TyrO• + H + + NO2k5
TyrOH + CO3- 98 TyrO• + HCO3-
(4) (5)
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Chem. Res. Toxicol., Vol. 17, No. 1, 2004 35
Figure 2. Reaction pathways leading to TyrOH nitration and dimerization by peroxynitrite. The solid arrows denote reactions that were present in both the infusion and the in situ synthesis experiments, and the dashed arrows represent additional reactions occurring in the synthesis system, due to the presence there of NO, O2-, and UA.
Tyrosyl radicals then either react with NO2 to yield 3-NT or dimerize to give dityr: k6
TyrO• + NO2 98
k7
{ {
0.45k6
98 3-NT 0.55k6
98 other products
TyrO• + TyrO• 98
(6)
0.35k7
98 dityr 0.65k7
98 other products
(7)
The yield of 3-NT in reaction 6 is ∼45% based on the disappearance of TyrO•, as determined from radiation chemical experiments (46); that of dityr in reaction 7 is ∼35%, according to a recent study by Hodges et al. (36). Reactions 1-7 are the core set of reactions directly associated with TyrOH dimerization and nitration. Besides these, several side reactions may occur, including homolysis and dismutation of ONOOH to NO2 and NO3-, dimerization of NO2 to form N2O4, and hydrolysis of N2O4. These side reactions are k8
ONOOH 98 •OH + NO2 k9
ONOOH 98 NO3- + H + k10,k-10
2NO2 798 N2O4 k11
N2O4 + H2O 98 NO2- + NO3- + 2H +
(8) (9) (10) (11)
Thus, eqs 1-11 represent the complete sets of reactions that take place during the infusion experiments. Those reactions are denoted by the solid arrows in Figure 2, and their rate constants are summarized in Table 1. For ONOO- delivery by in situ synthesis, the key additional reactions are the enzymatic generation of O2and the reaction of O2- with NO. The latter is written as k12
NO + O2- 98 ONOO-
(12)
Table 1. Reaction Rate Constants constants
units
values
ref
k1 k2 k3 k4 k5 k6 k7 k8 k9 k10 k-10 k11 k12 kXO kUC
M-1 s-1 s-1 s-1 M-1 s-1 M-1 s-1 M-1 s-1 M-1 s-1 s-1 s-1 M-1 s-1 s-1 s-1 M-1 s-1 M-1 s-1 M-1 s-1
3 × 104 1 × 103 2 × 103 3.2 × 105 4.5 × 107 3 × 109 2.25 × 108 0.24 0.56 4.5 × 108 6.9 × 103 1 × 103 1.6 × 1010 6.7 × 106 3.0 × 107
75 46 46 69 49 69 55 76 76 77 77 77 1 this work this work
Numerous additional reactions were also present in this system, as denoted by the dashed arrows in Figure 2. They include the spontaneous dismutation of O2- (47); NO autoxidation (48); the reactions of ONOO- with NO, N2O3, and CO3- (49-53); the reactions of TyrO• with excess NO or O2- (46, 54, 55); peroxynitrate (O2NOO-) formation and decomposition (56, 57); and the enzymatic oxidation of UA as well as its reactions with ONOO-, NO2, and CO3- (58-60). A set of rate constants for the complete reaction network is given elsewhere (61). Infusion Model. On the basis of the reaction pathways presented above, a kinetic model was formulated to describe the various time-dependent species concentrations during the continuous infusion of ONOO-. The liquid volume was an additional variable. In this model, the instantaneous concentration of each species was assumed to be spatially uniform, due to rapid mixing. As already mentioned, the infusate entered the reaction mixture in a dropwise manner. Because the time interval (ti) for sequential addition of two droplets (∼2 s) was much longer than the ONOO- half-life time (∼20 ms), reactions 1-11 went to completion within ti. As a consequence, concentration-time plots for ONOO- and related radicals had a sawtooth character. Focusing on one time interval between drops, the conservation equations for each species were written as follows. The concentration of total peroxynitrite (CPN, including both ONOO- and ONOOH) was influenced by reactions 1, 8,
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Chem. Res. Toxicol., Vol. 17, No. 1, 2004
Wang and Deen
dityr), the concentration after the droplet was added was
and 9:
dCPN ) -k1CCO2p1CPN - (k8 + k9)(1 - p1)CPN dt
Here, p1 represents the fraction of total peroxynitrite present as the anion (ONOO-), as determined by the acid dissociation equilibrium (pKa1 ) 6.8; ref 10) and given by
10pH-pKa1 p1 ) 1 + 10pH-pKa1
dt
(14)
) k1CCO2p1CPN - (k2 + k3)CONOOCO2- (15)
dCCO3dt dCNO2 dt
) k2CONOOCO2- - k5CTyrOHCCO3-
(16)
) k2CONOOCO2- - (k4CTyrOH + k6CTyrO• +
2k10CNO2)CNO2 + k8(1 - p1)CPN + 2k-10CN2O4 (17)
dCTyrO• ) (k4CTyrOH - k6CTyrO•)CNO2 + dt 2 k5CTyrOHCCO3- - 2k7CTyrO• (18) dCN2O4 dt
2 ) k10CNO - (k-10 + k11)CN2O4 2
dCTyrOH ) -(k4CNO2 + k5CCO3-)CTyrOH dt dCdityr 2 ) 0.35k7CTyrO• dt dC3-NT ) 0.45k6CTyrO•CNO2• dt
(19)
CsVd V0 + 2(V1 + Vd)
For the reactive intermediates (j ) ONOOCO2-, CO3-, NO2, TyrO•, and N2O4), the initial concentrations after adding any droplet were zero:
(21)
(25)
The set of first-order differential equations was solved with the initial conditions shown in eqs 23-25 for each time interval between droplets, using a stiff solver (ode15s) in MATLAB. This procedure was repeated for successive time intervals until the infusion process was completed (i.e., when V1 ) 2 mL). Synthesis Model. Although more complicated chemically than the infusion system, the in situ synthesis system was simpler dynamically, in that all species concentrations varied gradually with time. An important prerequisite for modeling TyrOH modification in the synthesis system is an accurate kinetic model for O2formation. During its oxidation by O2, HX is converted first to X, which is oxidized further to UA; O2- and H2O2 are formed in both steps (Figure 1). As will be shown, a kinetic model based on a ping pong mechanism (62) and competitive inhibition of XO by UA described our experimental results very well. The kinetic equations describing the substrate and product concentrations during HX oxidation are
d[HX] )dt
kHX cat [E]0[HX] KHX m
+ [HX] +
KHX m [X] KXm
(20) d[X] ) dt
+
KHX m [UA]
(23)
where Cs is the stock ONOO- concentration, Vd is the droplet volume, and V0 is the bulk liquid volume before starting the infusion experiment. The factor of 2 in the denominator takes into account the dilution effect of adding equal volumes of HCl and ONOO- solutions. Denoting the concentration of a stable species just before adding the next droplet as Ci′ (i ) TyrOH, 3-NT, and
(26)
KU i
X X HX kHX cat [E]0[HX]Km - kcat[E]0[X]Km X X HX KHX m Km + Km[HX] + Km [X] +
X KHX m Km[UA]
KU i
(22)
The type of initial condition used with each conservation equation depended on whether the species was a reactive intermediate or a relatively stable compound. During every time interval ti, peroxynitrite and all other reactive intermediates were fully depleted prior to addition of the next droplet. In contrast, the stable species (e.g., TyrOH, 3-NT, and dityr) were simply diluted as more ONOO- and HCl were infused. Thus, denoting the volume of ONOO- solution that had already been infused as V1, the concentration of ONOO- immediately after adding a droplet at t ) t′ was
CPN(t′) )
(24)
V0 + 2(V1 + Vd)
Cj(t′) ) 0
The concentrations of the key intermediates and products were determined from:
dCONOOCO2-
Ci′(V0 + 2V1)
Ci(t′) )
(13)
d[UA] ) dt
kXcat[E]0[X] KXm
+
KXm[HX] KHX m
+ [X] +
KXm[UA]
(27) (28)
KU i
X where kHX cat and kcat are the apparent maximum rate constants for HX and X (µmol min-1 U-1), respectively; X KHX m and Km are the apparent Michaelis constants (µM);
KU i is the inhibition constant for UA (µM); and [E]0 is the total active enzyme concentration (mU/mL). The rate of O2- production is
(
)
d[O2-] d[HX] d[UA] ) 2f1 + 2f2 dt dt dt
(29)
where f1 and f2 are the respective fractions of HX and X consumed in forming O2-. These “univalent fractions” must be distinguished from the fractions of HX and X participating in divalent reductions of O2 to H2O2. The other enzyme kinetic information that is needed concerns the rate of UA oxidation to allantoin by uricase. Unlike XO, uricase follows an ordered reaction mecha-
Peroxynitrite Delivery Methods
Chem. Res. Toxicol., Vol. 17, No. 1, 2004 37
Figure 4. Computed concentrations of total peroxynitrite, CO3-, and NO2 during dropwise infusion of ONOO- into a TyrOH solution. The results are based on the addition of a drop at t ) 0. Although not shown, the pattern is repeated every 2 s. The calculations are based on an infusate ONOO- concentration of 246 µM and other conditions as given in Figure 3.
Figure 3. Measured and predicted yields of TyrOH products after infusion of peroxynitrite: (a) dityr and (b) 3-NT. Results are shown for various concentrations of ONOO- in the infusate. Conditions: 2 mL of ONOO- infused into 46 mL of stirred, 1 mM TyrOH solution at a rate of 0.53 mL/min at room temperature and 2 mL of 0.1 N HCl infused at the same rate. The predictions are from eqs 13-25.
nism, and X is a competitive inhibitor (63). In an airsaturated buffer solution, the rate of UA oxidation is given by
d[UA] )dt
(
kcat[E]0[UA]
Km 1 +
)
[X] + [UA] Ki
(30)
where [E]0 is the uricase concentration in mU/mL, kcat is in µmol U-1 min-1, and Km and Ki are in µM. The parameter values in eqs 26-30 were determined by fitting multiple sets of experimental data to eqs 26-29 or 30. Those experiments are described more fully elsewhere (61). To model the complete synthesis system, mass balance equations were written for each reacting species shown in Figure 2, accounting for the various rates of production, physical addition, and consumption. The resulting equations were similar in form to those for the infusion model (eqs 13-22). One set of initial conditions was sufficient in this case (corresponding to t ) 0). The full model for the synthesis system is not presented here, in the interest of space, but is detailed elsewhere (61).
Results TyrOH Nitration and Dimerization during ONOOInfusion. Figure 3 shows the measured and predicted dityr and 3-NT yields when 2 mL of ONOO- solution of varying concentration was infused into 46 mL of 1 mM
TyrOH solution at 0.53 mL/min. The yield of dityr decreased from about 14 to 6% of the added ONOO- as the infusate concentration was increased from 246 to 2500 µM. The opposite trend was observed for 3-NT, where the yield increased from 3 to 6%. Overall, the predicted yields for both products were in remarkably good agreement with the experimental data. The greatest discrepancies were for dityr, where the model tended to underestimate the dityr yield at the lower ONOOconcentrations, but the maximum error was
