 |
INTRODUCTION |
Human phagocytes utilize respiratory burst-derived
H2O2 in combination with a peroxidase and
halides to generate reactive oxidants that can kill bacterial, fungal,
metazoan, and viral pathogens but also damage host tissue. The overall
reaction is: H2O2 + X
+ H+
HOX + H2O, where X
= Cl
, Br
, SCN
, or
I
, and HOX is the corresponding hypohalous
acid. Two distinct human phagocytic peroxidases have been identified:
neutrophil myeloperoxidase (MPO)1 and a related (70%
amino acid homology (1, 2)) eosinophil peroxidase (EPO (donor:hydrogen
peroxide oxidoreductase, EC 1.11.1.7)). Neutrophil MPO predominantly
catalyzes the two-electron oxidation of the most abundant halide,
Cl
, to generate the potent bleaching oxidant hypochlorous
acid (HOCl), a highly but indiscriminantly reactive substance with
potent cytocidal capacity. This powerful "bleaching" oxidant reacts
rapidly with a wide variety of cell membrane components, disrupts
membrane integrity, and lyses cells (3-5). In view of these
characteristics, HOCl seems well suited for fulfilling the primary
function of the neutrophil: to kill relatively small internalized
microbes within the confines of a phagocytic vacuole.
In contrast to neutrophils, eosinophils mediate the extracellular
destruction of large metazoan pathogens such as helminthic parasites
(6). Eosinophils contain large quantities (15 µg/106
eosinophils, 40% by weight of total specific granule protein (7, 8))
of EPO, a 77,000-dalton, two-subunit enzyme that, although related to
MPO, is both structurally (1, 2, 8) and functionally (9-12) distinct
from MPO. EPO plays a critical role in eosinophil killing of parasites
under physiologic conditions, because EPO inhibitors nearly completely
block the killing of antibody- and complement-opsonized schistosomules
by intact eosinophils in serum conditions (13).
The physiologic substrate for eosinophil EPO, although clearly
different from that of MPO, is less certain. EPO oxidizes
Cl
poorly, if at all (9-12). In physiologic fluids such
as serum and extracellular fluid, the concentrations of halides are 100 mM Cl
, 20-100 µM
Br
, 20-120 µM SCN
, and <1
µM I
(14-16). Considering other potential
substrates, it has been shown that the
EPO/I
/H2O2 system has pronounced
toxicity for bacteria (9), parasites (17, 18), and mammalian cells (10,
19), but only at I
concentrations far above physiologic.
Alternatively, in the presence of physiologically relevant
concentrations of Cl
and Br
(i.e. 100 mM and 20-100 µM,
respectively) EPO preferentially oxidizes Br
to form
another potent bleaching oxidant, HOBr, suggesting that Br
might be the predominant natural substrate (11, 12,
62, 63). However, other work has shown (9, 10, 22, 23) that the
pseudohalide thiocyanate (SCN
) can also be a substrate
for the EPO system. We found (23) that in fluids of physiologic halide
composition (i.e. 100 mM Cl
,
20-100 µM Br
, 10-100 µM
SCN
, and 1 µM I
) and in
serum, SCN
is the virtually exclusive substrate for EPO
oxidation, both by purified EPO and by activated eosinophils. Nitrite
(NO2
) can also serve as a substrate
for EPO, producing a bactericidal product (21) that nitrates protein
tyrosine residues (20), but SCN
is preferentially
oxidized over NO2
as well (20). Based
upon these data, we propose that, unexpectedly, SCN
is
the primary substrate oxidized by EPO and H2O2
under physiologic circumstances.
The product(s) of this potentially important reaction have, however,
not yet been directly identified. Previous work (22, 25, 26) on the
closely related
lactoperoxidase/SCN
/H2O2 system
has shown that it generates a relatively weak, predominantly sulfhydryl
(SH)-reactive oxidant that is both bacteriostatic and bactericidal. By
analogy with the other halide/hypohalous systems, it was proposed that
this oxidant might be HOSCN or hypothiocyanous acid. The product(s) of
the EPO/SCN
/H2O2 system react(s)
with the sulfhydryl dye 5-thio-2-nitrobenzoic acid (TNB) (22-26) and
is profoundly less toxic (on a molar basis) for mammalian cells than is
HOBr or HOCl (23, 27), a profile similar to the product(s) of the
lactoperoxidase/SCN
/H2O2 system.
However, even in the better-characterized
lactoperoxidase/SCN
/H2O2 system,
based upon nuclear magnetic and electron spin resonance studies, at
least four different compounds have been proposed to be the major
reaction products: HOSCN (28, 29), CN
(29),
NCS-O-SCN
(28), and
OSC·N
(30). We now
use nuclear magnetic resonance (NMR) and electrospray ionization mass
spectometry (ESI-MS) to identify the major stable reaction products of
the EPO/SCN
/H2O2 system and
demonstrate its capacity to inflict sulfhydryl reactivity-based
toxicity. These findings suggest that the eosinophil EPO system employs
a radically different biologic strategy to kill extracellular pathogens
than the neutrophil MPO system does to kill intracellular pathogens.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Human EPO was kindly provided by Dr. Gerald J. Gleich (Mayo Clinic and Research Foundation, Rochester, MN). EPO was
isolated from granule extracts of purified eosinophil suspensions
obtained from patients with hypereosinophilic syndrome as described
previously (31). Granule extracts were then chromatographed on a
Sephadex G-50 column equilibrated with 0.25 M acetate
buffer (pH 4.3, 0.15 M NaCl). The fractions eluting with
the void volume were then collected and purified to an
A415/280 ratio of >0.9 by chromatography on carboxymethyl-Sepharose as described by Carlson et al.
(8). SDS-polyacryamide gel electrophoresis was used to confirm
homogeneity of the EPO preparation. Two discrete bands (molecular mass
of ~78 and ~14 kDa) that correspond to the heavy and light chains of EPO, with no contaminating bands, were present. EPO activity was
assayed with guaiacol oxidation and converted to international units
(the amount of the enzyme that oxidizes 1 µmol of electron donor/min
at 25 °C (32). The specific activity of the EPO utilized in the
course of these experiments was 133-250 units/mg of protein. Preparations were stored at
70 °C until needed for use and were then maintained at 4 °C wrapped in foil. 13C-Labeled
(99-atom %) potassium thiocyanate was obtained from Cambridge Isotope
Laboratories (Andover, MA). Unlabeled potassium thiocyanate was from
Fisher. [14C]SCN
as the potassium salt,
specific activity 55.1 mCi/mmol, was from Amersham Pharmacia Biotech.
Potassium cyanate was from J. T. Baker Inc.. Percoll was from
Amersham Pharmacia Biotech. CD-16 magnetic microbeads were obtained
from Miltenyi Biotech Inc (Sunnyvale, CA). PMA was from Consolidated
Midland Corporation (Brewster, NY). Hanks' buffered salt solution was
obtained from Life Technologies, Inc. All other reagents were obtained
from Sigma.
Temperature- and pH Dependence of
EPO/SCN
/H2O2 System
Oxidants--
Oxidants were quantified by titration of TNB as
described previously (22). TNB was generated from
5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) by the addition of 2 µl
of
-mercaptoethanol to 50 ml of a 1 mM DTNB solution.
Freshly prepared TNB is calibrated at A412 and
combined with the EPO reaction mixture to a total volume of 1 ml to
determine HOSCN/OSCN
concentration using a molar
extinction coefficient of 26,000 M
1 cm
1.
Independent studies with reagent OCN
confirmed that this
assay does not detect OCN
(not shown). EPO-catalyzed
oxidation of SCN
was performed using conditions similar
to those described by Modi et al. (29) using the
lactoperoxidase system. KSCN (2 mM) and EPO
(0.4 µM) were added to 0.1 M sodium phosphate
buffer at pH 6.0 or 7.4 (total volume 1 ml), and 5 consecutive 250 µM increments of H2O2 were added
at 1-min intervals at room temperature (25 °C), vortexing between
additions. One minute after the final bolus addition, 20 µl of
catalase (2 mg/ml) was added to consume excess unreacted
H2O2. Specimens were incubated then at 25 or
4 °C, and after 0, 30, 90, 120, and 240 min, 100-µl aliquots were
removed for assay of oxidants by TNB titration.
NMR Analysis of
EPO/[13C]SCN
/H2O2
System Reaction Products and [13C]Urea--
The
[13C]SCN
spectra were collected either on a
Bruker AMX-600 MHz apparatus using an 8-mm broad-band probe or on
Varian Inova-600 using a 5-mm broad-band probe. Spectra were obtained
of
EPO/[13C]SCN
/H2O2
reaction mixture in 10% D2O 90% 0.1 M sodium
phosphate buffer at either pH 6.0 or 7.4. Reactions were mixed as
described above to 2 ml total volume at appropriate pH at room
temperature. One minute after the final H2O2
bolus addition, 50 µl of catalase (2 mg/ml) was added to the reaction
tube, and reaction mixtures were transferred to ice. Samples were
placed in an 8-inch, 8-mm thin-walled NMR sample tube (Wilmad Glass,
Buena NJ). One-dimensional spectra were collected using the
following parameters: sweep width = 15,009, number of points = 32,000, acquisition time = 1.066 s, recycle delay of 2 s,
number of scans was either 1500 or 2000. The data were processed using
gaussian function 0.099. Chemical shifts were referenced relative to
external (CH3)4Si. For determination of cyanate
spectrum, a 1 M solution of 13C-labeled urea
was prepared in phosphate-buffered saline (PBS) buffer (pH 7.4)
supplemented with 10% D2O and incubated for 40 min in a
water bath at 85 °C to promote equilibration between urea and
cyanate. Assay of OCN
by chemical analytic assay (see
below) in this preparation confirmed a cyanate concentration of 5 mM (not shown). NMR spectra were then obtained as described above.
ESI-MS and Collision-induced Dissociation Analysis of the
EPO/SCN
/H2O2 System Reaction
Products--
Negative ion ESI-MS was carried out on a triple-stage
quadrupole mass spectrometer (PerkinElmer Life Sciences). The
instrument was tuned and calibrated for negative ion operation using
polypropylene glycol with the Sciex IonSpray® source. EPO (0.4 µM) and either [13C]SCN
or
[12C] SCN
potassium thiocyanate (2 mM) were combined in 10 mM ammonium acetate
buffer (pH 7.4. Five 200 µM boluses of
H2O2 were added at room temperature at 1-min
intervals with mixing, and 40 µg/ml catalase was added to destroy
excess H2O2. The samples were then placed in
Microcon concentrators (Amicon, Beverly MA) with a molecular mass
cutoff of 10 kDa, centrifuged for 30 min (13,000 × g
at 4 °C) to remove protein, and kept cold until ESI-MS analysis in the negative ion mode. Alternatively, for some experiments, samples were filtered through a 0.22 µM exclusion Acrodisc to
remove particles and immediately analyzed. Samples were infused into
the IonSpray® source at a flow rate of 5-10 µl/min using a syringe
pump (Harvard Apparatus model 22, South Natick, MA, USA). The various
instrumental parameters involved in efficient nebulization and charging
of droplets were optimized to the following values: spray needle voltage (
3500 V); interface plate (
650 V); orifice skimmer (
50 to
l00V); Q0 rod offset voltage (
30V); gas curtain interface (1.2 liters/min of N2 at 60 °C); nebulizer gas (air at 1.5 liters/min). Data were acquired in the range of 10 to 100 m/z at a step size of 0.2 atomic mass units and
5-10-mc dwell time; 10-30 scans were summed over time.
Collision-induced dissociation tandem mass spectrometry (MS/MS) studies
were performed on a Micromass Quatro II triple quadruple mass
spectrometer (Altrincham, UK). Collision-induced mass spectra
were determined in the negative ion mode by direct infusion of reaction
products (formed by the
EPO/SCN
/H2O2 system as described
for the ESI/MS studies above) at a flow rate of 10 µl/min (Harvard
Apparatus pump) and analyzed with a cone potential of 20 eV, collision
energy of 30 eV, collision gas (Ar) cell at 1.7 × 10
3 millibar, source temperature 70 °C,
capillary 3500 V, high voltage lens 690 V, skimmer offset of 5 V.
Analytic Quantitation of Cyanate--
OCN
was
assayed using the method of Guilloton and Karst (33). Briefly, 250 ml
of a freshly prepared 10 mM solution of 2-aminobenzoic acid
(anthranilic acid) prepared in 50 mM sodium phosphate
buffer at pH 4.4 was incubated with 250 µl of the EPO system reaction mixture for 10 min at 40 °C and then added to 500 µl of 10 N HCl. The mixture was then boiled for 1 min, allowed to
cool to room temperature, and assayed spectrophotometrically at 310 nm
with a molar extinction coefficient of 3.56 mM
1
cm
1. A standard curve using reagent
KOCN
was used to determine specific concentration. This
assay does not detect HOSCN/OSCN
because EPO system
reaction mixtures contained the same amount of cyanate before and
immediately after selective titration of HOSCN/OSCN
by
the addition of dithiothreitol (not shown).
Production of Oxidants and Cyanate by the
EPO/SCN
/H2O2 System: Time Course
and Peroxidase Dependence--
To determine the relative time courses
of HOSCN and cyanate generation (Fig. 5), H2O2
was added to a reaction mixture containing PBS with 1 mM
KSCN (pH 7.4) and 0.4 µM EPO by continuous infusion at 50 µM/min for 20 min to simulate the continuous generation of H2O2 by the respiratory burst of an
activated eosinophil. An aliquot was removed from the reaction mixture
before and at various time intervals after initiating the addition of
H2O2, supplemented with 40 µg/ml catalase to
consume unreacted H2O2, and placed on ice until
subsequent assay of oxidants and cyanate as described above. To
establish the peroxidase dependence of oxidant and cyanate generation
by the EPO/SCN
/H2O2 system (Fig.
7), the effect of either omitting EPO or adding the EPO inhibitor azide
(1 mM) was assayed in a system composed of PBS (pH 7.4),
0.4 µM EPO, and 1 mM KSCN to
which five consecutive increments of 100 µM
H2O2 were added at 1-min intervals, and the reaction was terminated by adding 100 µg/ml catalase and placing the
specimen on ice before assaying oxidants and cyanate.
Purification of Peripheral Blood Eosinophils--
The anti-CD-16
immunomagnetic bead cell sorting system (MACS, Miltenyi Biotec Inc.)
was used as described by Ide et al. (34). Minor
modifications are delineated here. 60 ml of citrated blood from
eosinophilic donors (n = 3) undergoing interleukin-2
immunotherapy were mixed with 30 ml of 6% Hetastarch in a 0.9%
sodium chloride solution (Abbott Laboratories). Erythrocytes
were allowed to sediment for 45 min at room temperature, and the
leukocyte-rich fraction was collected. This fraction was diluted with
an equal volume of PBS (137 mM NaCl, 3 mM KCl,
4.2 mM sodium phosphate, and 1.5 mM potassium
phosphate (pH 7.4)) supplemented with 2% fetal bovine serum, layered
atop a half-volume of isotonic Percoll (density 1.082 g/ml) in 50-ml
conical tubes, and centrifuged for 30 min and 1000 × g
at 4 °C. The supernatant and mononuclear cells at the interface were
carefully aspirated, and the inside wall of the tube was wiped with
sterile cotton tip applicators to remove residual mononuclear cells.
The pellet of granulocytes and remaining erythrocytes were subjected to
a hypotonic lysis by exposure to 20 ml of ice-cold sterile water for
30 s. The granulocytes were rescued with 20 ml of a 2× isotonic
buffer (40 mM HEPES, 10 mM KCl, 10 mM D-glucose, 280 mM NaCl (pH 7.4))
and pelleted. The lysis was repeated if erythrocytes remained. The
specimen was then incubated with anti-CD-16-coupled immunomagnetic
beads and processed as described previously (35). The resulting
preparations all contained
98% eosinophils.
Eosinophil Production of Oxidants and Cyanate--
Purified
eosinophils were washed, suspended at 4 × 106/ml in a
modified Hanks' balanced salt solution composed of 5.3 mM
KCl, 138 mM NaCl, 0.4 mM potassium phosphate,
5.3 mM sodium phosphate, 5.5 mM
D-glucose, 1.3 mM CaCl2, and 0.5 mM MgCl2 (pH 7.4). supplemented with 1 mM KSCN. Triplicate groups were then supplemented with nothing (control), 1 µg/ml phorbol 12-myristate 13-acetate (PMA), or
1 µg/ml PMA and 5 mM azide, then incubated at 37 °C
for 60 min in capless 5-ml round-bottom tubes. The tubes were vortexed every 15 min to aid oxygenation. After the incubation, the samples were
centrifuged for 10 min, 1000 × g at 4 °C. The
supernatants were placed in Microcon concentrators (Amicon) with
membranes of 3000-Dalton molecular mass cut off and centrifuged for 30 min, 13,000 × g at 4 °C to remove degranulated
proteins. The filtrates were assayed for HOSCN and cyanate quantities
as described above.
OSCN
/OCN
Effects upon RBC Glutathione
and Enzymes--
Human RBCs were suspended at a hematocrit of 2% in
PBS (pH 7.4)) supplemented with increasing concentrations of
OSCN
/OCN
generated as above by the
EPO/SCN
/H2O2 system. After 30 min, cells were pelleted by centrifugation at 2000 × g
for 3 min and washed 3 times in ice-cold H/H buffer. Under these
conditions, only at
200 µM
OSCN
/OCN
was there detectable hemolysis, as
assayed by spectrophometric assay of supernatant hemoglobin or
methhemoglobin. RBC lysates and membranes were then prepared by
hypotonic lysis and assayed for glutathione and glyceraldehyde
3-phosphate dehydrogenase (GAPDH), glutathione S-transferase
(GST), and lactate dehydrogenase activity as described by Beutler (47)
and for ATPase activity as described below. Hemolysates were incubated
15 min further either with or without 10 mM dithiotheitol
and reassayed for GAPDH, GST, and ATPases.
Comparative Oxidant Inactivation of Red Blood Cell ATPase
Activity--
Human RBC membranes were prepared by hypotonic
lysis. RBC membranes were suspended at 0.2 mg of protein/ml in membrane
buffer (20 mM Hepes, 5.5 mM NaCl, with 1 g/liter D-glucose, 0.10 g/liter MgCl2·6H2O, 0.10 g/liter
MgSO4·7H2O, and 0.4 g/liter KCl (pH 7.4)) supplemented with increasing concentrations of
H2O2, HOCl (diluted from concentrated
hypochlorite (Sigma)), HOBr (prepared adding excess bromide to stock
HOCl, as described previously (56)) or
OSCN
/OCN
(prepared by EPO as described
above). RBC membranes were incubated with the oxidants for 15 min at
37 °C then pelleted and washed three times in ATPase buffer without
oxidant. Total (i.e. Ca2+-, Mg2+-,
calmodulin-, and Na+/K+-dependent)
ATPase activity was assayed in 0.2 mg of protein RBC membrane aliquots
suspended in 500 µl of ATPase buffer (90 mM histidine, 90 mM imidazole, 15 mM MgCl2, 400 mM NaCl2, 0.5 mM EGTA, 75 mM KCl, 1 mM CaCl2, 300 nM calmodulin (pH 7.4)) by adding 3 mM ATP,
incubating 10 min at 37 °C, then quenching phosphate release by
adding 1000 µl of 1% ascorbate in 10% trichloroacetic acid.
Phosphate release was quantitated as described (57) by the addition of
250 µl of 1% ammonium molybdate tetrahydrate, incubating 5 min, then
adding 500 µl of 2% sodium citrate dihydrate and 2% sodium arsenite
in 2% acetic acid. The resulting solution was assayed with a
spectrophotometer at 700 nm, and the concentration of phosphate
was calculated from a standard curve generated using H2KPO4. Values were corrected for both
spontaneous membrane phosphate release (i.e. membranes
without ATP added) and spontaneous ATP hydrolysis (i.e. ATP
in the absence of membranes).
PAGE Gels of GST Exposed to
[14C]OSCN
/OCN
--
10 µg
of human GST-
was exposed to 200 µM
[14C]OSCN
/OCN
(generated
using [14C] SCN
in the
EPO/SCN
/H2O2 system described
above) or H/H buffer. Samples were suspended in Laemmli buffer with or
without
-mercaptoethanol (BME) and separated on a 3-15% SDS/PAGE
gel (58). Gels were stained with Coomassie Blue, and autoradiograms
were developed by fluorographically enhanced exposure of x-ray film
(Kodak X-AR).
 |
RESULTS |
pH and Temperature Dependence of
EPO/SCN
/H2O2 System
Oxidants--
To characterize the oxidant reaction product(s)
generated by the EPO/SCN
/H2O2
system, we assayed TNB-titratable oxidant stability over time as a
function of pH and temperature (Fig. 1).
Under the conditions employed, more total oxidant is initially
generated at pH 6.0 than at pH 7.4 (350 µM
versus 275 µM). When subsequently
incubated either at 25 or 4 °C at pH 7.4, the oxidant half-life was
more than 6 h at 4 °C and 60 min at 25 °C. At pH 6.0 the
half-life was approximately 4 h at 4 °C but less than 30 min at
25 °C. Thus, oxidant generation by the
EPO/SCN
/H2O2 system is favored at
acidic versus physiologic pH; in contrast, oxidant stability
is favored at 4 °C and physiologic pH. These stability
characteristics are similar to those of the oxidant product of the
lactoperoxidase/SCN
/H2O2 system
(22, 25, 26).

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Fig. 1.
pH and temperature dependence of oxidants
generated by the EPO/SCN /H2O2
system. Five consecutive 250 µM increments of
H2O2 were added at 1-min intervals to 1 ml of a
solution containing 2 mM KSCN and 0.4 µM EPO
in 0.1 M sodium phosphate buffer at either pH 6.0 (squares) or 7.4 (triangles) at 25 °C. One
minute after the addition of the final bolus, 20 µl of catalase (2 mg/ml) was added to consume excess unreacted
H2O2. After the addition of catalase, reaction
mixtures were incubated either at 25 or 4 °C, and aliquots were
sampled at the indicated time intervals for total TNB titratable
oxidants. , (pH 6.0) 25 °C; , (pH 6.0) 4 °C; , (pH 7.4)
25 °C; , (pH 7.4) 4 °C.
|
|
NMR analysis of EPO/SCN
/H2O2
System Reaction Products--
To enumerate and characterize the major
products, whether oxidant or non-oxidant, of the
EPO/SCN
/H2O2 system, we employed
13C NMR analysis of solutions containing
[13C]SCN
substrate (Fig.
2). Analysis was performed both at pH 7.4 (panels A, B, and C) and pH 6.0 (panels D and E). At pH 7.4, spectra accumulated over the first 90 min show the expected large parent
[13C]SCN
resonance (a) at 133.4 ppm. In addition, two new peaks are discernible: b at 128.6 ppm, along with a more intense resonance, c, at 127.3 ppm.
These two latter peaks were absent from the NMR spectrum of
[13C] SCN
alone or of
[13C]SCN
in the presence of added
H2O2 without EPO (panel A) and are
therefore EPO-dependent. Both products are still detectable
after incubation overnight at 4 °C at pH 7.4 (panel C).
In contrast, at pH 6.0 (panels D and E), although
the first spectrum shows peaks b and c
(panel D), after overnight incubation, peak b
remains detectable, but peak c has disappeared and a new
peak, d, is seen at 124.6 ppm. Thus, peak c has a
pH and temperature stability profile compatible with that of the
TNB-titratable oxidant depicted in Fig. 1. By contrast, peak
b is relatively stable, at least at 4 °C. Peak d, which appears coincident with the disappearance of peak
c, may represent a decomposition product of peak
c. There are therefore two major initial stable reaction
products (i.e. peaks b and c) of the
EPO/SCN
/H2O2 system.

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Fig. 2.
[13C]SCN NMR
analysis of EPO/SCN /H2O2 system
reaction products. Using the protocol described for Fig. 1,
reaction products of the
EPO/SCN /H2O2 system were
generated either at pH 7.4 (panels A, B, and
C) or pH 6.0 (panels E and F) at room
temperature, then maintained thereafter at 4 °C for subsequent
evaluation of products and their stabilities. Reaction mixtures were
generated and NMR spectra obtained in buffers supplemented with 10%
D2O. Spectra (1500-2000) were obtained with a Bruker
AMX-600 apparatus using an 8-mm broad-band probe for 90-120 min.
Chemical shifts were referenced relative to an external
(CH3)4Si standard. Panel A, pH 7.4, SCN /H2O2 as described in the
legend for Fig. 1 without EPO. 10,000 scans were obtained to maximize
sensitivity. Panel B, pH 7.4, reaction products of the
EPO/SCN /H2O2 system analyzed over
the first 90 min after generation. Panel C, pH 7.4, reaction
products from panel B after incubation overnight (16 h) at
4 °C. Panel D, pH 6.0, reaction products of the
EPO/SCN /H2O2 system analyzed over
the first 90 min after generation. Panel E, pH 6.0, reaction
products from panel D after incubation overnight (16 h) at
4 °C. Peak assignments, based on our subsequently described studies
and previous NMR studies (24, 28), are: a = SCN ; b = OCN ;
c = OSCN ; d = CO2.
|
|
Mass Spectometry Analysis of
EPO/SCN
/H2O2 System Reaction
Products--
Although these NMR studies suggest there are two major
stable products of the
EPO/SCN
/H2O2 system, they do not
identify their structures. Moreover, because of the relative
insensitivity of this technique, they do not rule out the possibility
that other products exist. We therefore used ESI-MS to analyze the
reaction mixture (Fig. 3). To confirm
that putative reaction products resulted from oxidation of
SCN
, we employed both
[12C]SCN
and
[13C]SCN
as substrates and sought the
predicted isotope shift in the resultant carbon-containing products.
Before the addition of H2O2 to EPO and
SCN
, a large parent SCN
ion peak with
m/z 58 was seen (not shown). Panel A
shows the ESI-MS spectrum of an acetate buffer,
H2O2, and SCN
control solution in
the absence of EPO. A large contaminant ion is seen at
m/z 77 as are several smaller ions, including two
with m/z 74 and 75. In the presence of the
complete EPO/SCN
/H2O2 system
using [12C]SCN
(panel B), an ion
with m/z 74 peak is formed, consistent with generation of OSCN
. When
[13C]SCN
was substituted for
[12C]SCN
in the complete
EPO/SCN
/H2O2 system (panel
C), no significant ion with at m/z 74 was generated (i.e. ion intensity is the same as in the absence
of EPO (panel A)), but there was a large new ion at
m/z 75, consistent with formation of
[13C]OSCN
. These results are compatible
with OSCN
(m/z 74) being one major
product of the EPO/SCN
/H2O2
system.

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Fig. 3.
Negative ion ESI-MS analysis of the
EPO/SCN /H2O2 system reaction
products. EPO/SCN /H2O2
reaction products were generated at pH 7.4 in 10 mM
ammonium acetate buffer supplemented with 0.4 µM EPO and
2 mM potassium thiocyanate, either
[12C]SCN or
[13C]SCN . Five consecutive 200 µM increments of H2O2 were added
at 1-min intervals to 1 ml of a solution containing 2 mM
KSCN and 0.4 µM EPO in 0.1 M sodium phosphate
buffer at either pH 6.0 or 7.4 at 25 °C. One minute after the
addition of the last bolus of H2O2, catalase
(40 µg/ml) was added to consume unreacted excess
H2O2. A syringe pump was then used to spray
solutions at a flow rate of 5-10 µl/min into the SCIEX API III
triple quadrupole mass spectrometer. Results are expressed as relative
intensity as percent of the largest peak in the
m/z 10-180 spectrum, which in all cases was the
[12C]- or [13C]thiocyanate ion,
m/z 58 or m/z 59, respectively. Upper panels (A, B, and
C), m/z range 70-80. Panel
A, control composed of complete system as described above except
lacking EPO; panel B, complete system in the presence of
[12C]SCN ; panel C, complete
system in the presence of [13C] SCN .
Lower panels (D, E, and F),
m/z range 40-50. Panel D, complete
system lacking EPO; Panel E, complete system in presence of
[12C]SCN ; panel F, complete
system in presence of [13C]SCN . *,
contaminant peaks in areas of interest not dependent upon presence of
EPO.
|
|
In scanning the range m/z 10-180, the only other
new peak developing in the presence of the
EPO/SCN
/H2O2 system was at
m/z = 42 (Fig. 3, panels
D and E). In the acetate buffer,
H2O2 and SCN
control in the
absence of EPO (panel D, only a low intensity (i.e. background) ion at m/z 42 is
seen. An ion with m/z 42 was generated by the
EPO/SCN
/H2O2 system using
[12C] SCN
, consistent with the formation of
[12C] OCN
. Confirming that this ion arises
from SCN
, substitution of
[13C]SCN
for
[12C]SCN
yields a prominent new ion with
m/z 43 instead of 42 (panel F). Under
these conditions, the m/z 42 ion intensity is the
same as in the control lacking EPO (panel D), indicating
that this background m/z 42 ion is likely a
contaminant in our buffer system. Based on these results we propose
that OCN
(m/z 42) is the second
major product of the EPO/SCN
/H2O2 system.
However, ESI-MS analyses at low m/z range are
difficult, because it is extremely difficult to get rid of all
background ions in this low mass range. Moreover, a number of potential
compounds have an m/z of 74 and 42. To
characterize these compounds further, we therefore employed
collision-induced dissociation MS/MS. As shown in Fig.
4, panel A, when
[12C]SCN
was the substrate, the parent ion
with m/z 74 fragmented to yield a predominant
daughter ion of m/z 26 (compatible with
CN
) as well as less abundant ions at
m/z 58 (compatible with SCN
) and
m/z 42 (compatible with OCN
).
Similarly, the m/z 42 ion also fragmented,
although less extensively, to yield a daughter ion of
m/z 26 (panel B). In parallel MS/MS studies of ions at m/z 75 and at
m/z 43 arising from substitution of
[13C]SCN
for
[12C]SCN
, both yielded daughter ions
consistent with these assignments (not shown). Collectively, these data
strongly suggest that the ions representing the major catalysis
products of the EPO/SCN
/H2O2
system are attributable to OSCN
and
OCN
.

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Fig. 4.
Collision-induced dissociation MS/MS analysis
of the major EPO/SCN /H2O2 system
reaction products. The complete
EPO/SCN /H2O2 system was run in
the presence of [12C]SCN as described in
the legend for Fig. 3. Panel A, MS/MS spectrum of
m/z 74 parent ion. Panel B, MS/MS
spectrum of m/z 42 parent ion. Ion assignments
are indicated in brackets.
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|
Quantitative Detection of OSCN
and Cyanate as Major
Products of Both the EPO/SCN
/H2O2
System and Activated Human Eosinophils: Kinetic Relationship between
OSCN
and OCN
Generation--
Detection of
an ion with m/z compatible with OCN
by mass spectrometry could represent an artifact of OSCN
fragmentation during the ionization process. Therefore, to ascertain whether OCN
is present in the
EPO/SCN
/H2O2 system reaction
product before mass spectrometry analysis, we used a well characterized
analytical assay based upon the reaction of OCN
with
2-aminobenzoic acid and subsequent cyclization in acid conditions to
form 2,4 (1H,3H)-quinazolinedione, which is detected at 310 nm (33). In
experiments not shown, we first showed that this assay for
OCN
did not also detect HOSCN by selectively titrating
the reaction mixture with dithiothreitol, confirming the absence of
HOSCN by ESI-MS and showing that the OCN
assay detected
similar amounts of OCN
before and after the selective
depletion of HOSCN.
Using this assay, we find that the
EPO/SCN
/H2O2 system generates
large amounts of cyanate with a time course that lags behind that of
OSCN
. Fig. 5 shows
simultaneous determinations of oxidant (as TNB-titratable material) and
OCN
(as determined in the aminobenzoic acid reaction) at
various time points during the progression of the
EPO/SCN
/H2O2 reaction.
H2O2 was continuously infused at 50 µM/min for 20 min, simulating the continuous generation
of H2O2 by the respiratory burst of an
activated eosinophil. Aliquots were removed from the reaction mixture
at 1-min intervals, and catalase was added to destroy excess unreacted
H2O2 before assay of TNB-reactive substances and cyanate. OCN
generation initially lags behind that of
HOSCN by 5 min. HOSCN production plateaus at
175 µM,
whereas cyanate accumulation continues only so long as
H2O2 infusion continues (i.e. 20 min), then abruptly ceases thereafter. This plateauing of HOSCN levels
during H2O2 infusion reflects a balance between
rapid generation by the EPO and consumption by some secondary reaction,
because the addition of the EPO inhibitor azide at this stage of the
progression curve causes an abrupt collapse of HOSCN levels (not
shown). Taken together, these data suggest that HOSCN is the initial
product of the EPO/SCN
/H2O2
system and OCN
results from a subsequent reaction of
HOSCN, e.g. reaction with excess
H2O2, reaction with a second molecule of HOSCN,
or spontaneous decomposition.

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Fig. 5.
Time course of OSCN and cyanate
production by the
EPO/SCN /H2O2. To solutions
containing 0.4 µM EPO, 1 mM KSCN in potassium
phosphate buffer 15 mM (pH 7.4)
H2O2 was added by continuous infusion of 50 µM/min for 20 min then discontinued. At the indicated
time points aliquots were withdrawn, supplemented with 40 µg/ml
catalase to consume excess unreacted H2O2,
placed on ice, and subsequently assayed for oxidant ( ) by TNB
titration and cyanate ( ) by derivativization of 2-aminobenzoic acid
(see "Experimental Procedures"). Reactions took place at room
temperature.
|
|
To determine which of the NMR resonance peaks (i.e.
b or c) detected in Fig. 2 represents
OCN
, we analyzed the NMR spectrum of
[13C]urea. Although [13C]OCN
is not commercially available, urea exists in equilibrium with small
(approximately 0.5% at physiologic pH) amounts of cyanate (43). The
NMR spectrum of [13C]urea at pH 7.4 (Fig.
6) shows the expected large main urea
peak at 162.5 ppm (36) as well as a smaller OCN
peak at
128.6 PPM. The shift of this peak is indistinguishable from that of
peak b in Fig. 2, supporting its identification as OCN
. Since sulfur is less electron-withdrawing than
oxygen, the upfield resonance (peak c in Fig. 2) is likely
[13C]OSCN
.

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Fig. 6.
[13C]Urea NMR analysis to
establish shift of OCN . A 1 M solution
of 13C-labeled urea was prepared in a PBS buffer (pH 7.4)
supplemented with 10% D2O and incubated for 40 min in a
water bath at 85 °C to promote equilibration with OCN .
Concomitant assay of OCN by chemical analytic assay in
this preparation showed a cyanate concentration of 5 mM
(not shown). After cooling on ice, this preparation was then analyzed
by NMR as described "Experimental Procedures." All peaks were
referenced relative to a (CH3)4Si standard. The
large resonance at 162.5 ppm represents the parent urea peak. The only
other major peak is observed at 128.6 ppm. nMR analysis of freshly
prepared [13C]urea before heating did not show any
significant feature at 128.6 ppm (not shown).
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|
To rule out non-enzymatic oxidation of SCN
by
H2O2 as the basis for OCN
generation by the EPO/SCN
/H2O2
system, we either omitted EPO or, alternatively, blocked EPO catalytic
activity with the potent inhibitor azide (Fig.
7.) Under both conditions, generation of
both HOSCN and OCN
was nearly completely blocked,
suggesting that generation of OCN
as well as HOSCN
depends upon the catalytic action of EPO.

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Fig. 7.
Peroxidase activity dependence of
OSCN and OCN production by the
EPO/SCN /H2O2 system.
Solutions containing 0.4 µM EPO, 1 mM KSCN,
50 mM potassium phosphate buffer (pH 7.4) were supplemented
with H2O2 (added as 100 µM
increments/min ×5), subsequent to which catalase (40 µg/ml) was
added to consume excess unreacted H2O2.
OSCN and OCN were subsequently determined
in the presence of the complete system (left pair of
bars), the complete system in the presence of the peroxidase
inhibitor azide, 5 mM (middle pair of
bars), or in a system lacking added EPO (right
pair of bars). Data represent mean ± S.E.
n = 3 for each group.
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|
As a gauge of the potential physiologic relevance of the above
findings, all made with a purified reagent system, we determined whether intact, activated human eosinophils also generate HOSCN and
OCN
(Fig. 8). Peripheral
blood eosinophils were isolated from cancer patients undergoing
experimental therapy with subcutaneous injections of interleukin-2, a
circumstance known to be associated with the secondary elevation of
blood interleukin-5 with consequent eosinophilia and activation of
circulating eosinophils (37). Eosinophils isolated from such patients
spontaneously generated low but detectable amounts of HOSCN and
OCN
(left pair of bars). When
maximally stimulated by the addition of phorbol myristate acetate,
eosinophils generated greatly increased and roughly equimolar
amounts of both HOSCN and OCN
(middle pair of
bars). The addition of 5 mM azide severely
attenuated phorbol myristate acetate-stimulated generation of both
products, although HOSCN more so than OCN
. Thus,
activated human eosinophils generate both HOSCN and OCN
by an EPO-dependent mechanism.

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Fig. 8.
OSCN and OCN
production by PMA-activated eosinophils. Purified human
eosinophils (4 × 106/ml) isolated from three
different donors were suspended in modified H/H buffer (pH 7.4)
supplemented with 1 mM KSCN. Cells were then incubated for
60 min at 37 °C in the absence of further additions (left
pair of bars), after addition of 100 ng/ml of PMA
(center pair of bars), or in the concomitant
presence of both 5 mM sodium azide and 100 ng/ml of PMA
(right pair of bars). Supernatant buffer obtained
by spinning 5000 × g for 5 min was deproteinated by
Microcon concentrators (Amicon) with membranes of 3000-kDa molecular
mass cut off and centrifugation for 30 min at 13,000 × g at 4 °C. Filtrates were assayed for oxidants and
cyanate as described previously.
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Because the products of the
lactoperoxidase/H2O2/SCN
system
have been shown to react almost exclusively with SH groups (see "Discussion"), we asked whether the
EPO/SCN
/H2O2 system products
react similarly and inflict SH-based toxicity. As a model we employed
intact human RBCs exposed to increasing concentrations of
OSCN
/OCN
generated by the
EPO/SCN
/H2O2 system and assayed
intracellular levels of the SH-containing oxidant scavenger
glutathione. In addition, we assayed the activities of three enzymes
(GST, GAPDH, and membrane ATPases) that are known (59-61) to be
vulnerable to SH oxidation-based inactivation as well as one, lactate
dehydrogenase, not prone to such inactivation (Fig.
9). Exposure of RBCs to of
OSCN
/OCN
under these conditions produced no
detectable hemolysis. With increasing concentrations of
OSCN
/OCN
, glutathione was first depleted,
followed by inactivation of GST and GAPDH, then membrane ATPases.
Lactate dehydrogenase activity was completely unaffected. This
inactivation of GST, GAPDH, and ATPase activity is based on reversible
SH oxidation, because subsequent treatment of lysates and membranes
prepared from RBCs exposed to 100 µM
OSCN
/OCN
with 10 mM
dithiothreitol, an SH-reducing agent, completely restored the
activities of all three enzymes (not shown). In aggregate, these
findings suggest that
EPO/SCN
/H2O2 products diffuse
through intact RBC membranes to oxidize intracellular SH-containing
compounds.

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Fig. 9.
Inactivation of RBC enzymes by products of
the EPO/SCN /H2O2 system.
Human RBCs were suspended at a hematocrit of 2% in PBS buffer (pH 7.4)
and the indicated concentration (titrated by TNB oxidation) of
OSCN , generated by the
EPO/SCN /H2O2 system using the
protocol described in the legend for Fig. 6 for 30 min at 37 °C.
After pelleting and washing three times rapidly in ice-cold PBS buffer,
lysates were prepared and assayed for GSH, GAPDH, GST, and lactate
dehydrogenase (LDH); membrane preparations were assayed for
ATPase activity. Under these conditions there was no detectable
hemolysis.
|
|
Given the potential specificity of OSCN
/OCN
for SH reactivity, we hypothesized that these substances, despite being
far less reactive for other biologic materials than the potent
bleaching oxidants HOCl and HOBr, might more effectively mediate
SH-based toxicity. We therefore compared the capacity of these oxidants as well as H2O2 to inactivate ATPases in
isolated RBC membranes (Fig. 10).
Membrane preparations rather than intact RBC were used to allow all
oxidants equal access to intra- and extracellular membrane components.
In this model, OSCN
/OCN
inhibits RBC
ATPases much more effectively than in intact RBCs (cf. Fig.
9) with an ID50 of 2 µM, one-tenth that of
HOCl and HOBr; H2O2 fails to inactivate ATPases
even at 1 mM. Thus, the failure of
OSCN
/OCN
to react indiscriminantly with a
wide array of other membrane components allows it to function more
effectively than do more potent oxidants as a SH-based metabolic
inhibitor.

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Fig. 10.
Comparative oxidant inactivation of red
blood cell membrane ATPases. 100-µg aliquots of RBC membranes
were exposed to the indicated concentrations of OSCN
(titrated by TNB oxidation and generated by the
EPO/SCN /H2O2 system) or reagent
HOBr, HOCl, or H2O2 in H/H buffer 15 min at
37 °C, washed three times in ice-cold H/H buffer, then assayed for
total ATPase activity by quantitation of phosphate release as described
under "Experimental Procedures."
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|
To examine the structural basis of SH-dependent
inactivation of GST by OSCN
/OCN
, we exposed
human GST-
, the isoform found in RBCs, to
[14C]OSCN
/OCN
. Proteins were
then analyzed by SDS/PAGE for electrophoretic mobility and
14C incorporation under reducing and non-reducing
conditions (Fig. 11). As shown in the
left panel (lanes 1-4), GST not exposed to [14C]OSCN
/OCN
ran as a single
band (lane 1), with a mobility of 23 kDa under reducing
conditions; under non-reducing conditions (lane 2), an additional faint band of higher mobility, presumably representing a
species containing intramolecular disulfide bonds, is evident. After
exposure to [14C]OSCN
/OCN
,
under nonreducing conditions (lane 4), the 23 kDa band is
reduced in intensity, that of the higher mobility band increased, and an additional faint band of ~48 kDa mobility is also seen. That this
last band represents disulfide-bonded intermolecular dimers is shown by
the fact that it, along with the high mobility band, disappeared upon
reduction (lane 3). Autoradiograms of lanes 3 and
4 (right panel, lanes 3A and
4A) show heavy, nearly completely SH reductant-reversible of
14C into all three of these bands and a faint band of even
higher mobility. Thus, exposure of GST to
[14C]OSCN
/OCN
causes intra-
and intermolecular disulfide bond formation, as well as SH
reductant-reversible bonding of a 14C-containing
moiety.

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Fig. 11.
SDS/PAGE analysis of human
GST- exposed to
[14C]OSCN /OCN . 10 µg
of human GST- was exposed to 200 µM
[14C]OSCN /OCN (generated
using [14C]SCN in the
EPO/SCN /H2O2 system described
above) or H/H buffer. Samples were suspended in Laemmli buffer with or
without BME and separated on a 3-15% SDS/PAGE gel. Gels were stained
with Coomassie Blue (left four lanes 1-4), and
autoradiograms were developed by fluorographically enhanced exposure of
x-ray film (right two lanes 3A and 4A). Left
panel: lane 1, buffer + BME; lane 2, buffer
without BME; lane 3, 200 µM
[14C]OSCN /OCN + BME;
lane 4, 200 µM
[14C]OSCN /OCN , no BME.
Right panel: autoradiograms of lanes 3 (3A) and 4 (4A).
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|
 |
DISCUSSION |
Products of the EPO/SCN
/H2O2
System--
Peroxidase-catalyzed oxidation of SCN
has
hitherto been best characterized in the case of lactoperoxidase, an
enzyme present in large quantities in milk, saliva, and tears.
Lactoperoxidase has extensive structural and therefore presumably
functional similarities to EPO based upon primary amino acid sequence
(1, 2, 8) and prosthetic group resonance characteristics (48-51).
SCN
, termed a pseudohalide because it reacts as a halide
in several peroxidase-catalyzed reactions (25), is present in human
serum at concentrations of 10-100 µM (25). Serum
SCN
derives from a detoxification reaction between
cyanide and thiosulfates and, more importantly, from ingestion of
SCN
and related compounds from dietary sources (25). In
saliva, milk, and tears, SCN
concentrations reach
millimolar levels, and SCN
is the principal substrate for
lactoperoxidase. The weak oxidant products of the
lactoperoxidase/SCN
/H2O2 system
are bacteriostatic and bactericidal. By this mechanism the
lactoperoxidase/SCN
/H2O2 system
appears to play a critical role in preventing bacterial overgrowth in
the oral cavity, gut, and eyes (25, 38). In addition, the product(s) of
this system are cytocidal for candida fungus (52) and certain viruses
(53, 54) but relatively innocuous for mammalian cells (27).
The predominant oxidative product(s) of this reaction has been
suggested, by analogy with the hypohalous acids HOCl and HOBr, to be
hypothiocyanous acid (HOSCN) (22, 25, 26). However, efforts to identify
definitively the species produced by the
lactoperoxidase/SCN
/H2O2 system
system have been hampered by the inability to produce authentic HOSCN
by chemical synthesis (25). Modi et al. (29) characterized the products of this system using
[15N]SCN
NMR. These studies demonstrated
the formation of one major reaction product with a resonance of 179 ppm. However, in the presence of excess H2O2,
this peak disappeared, and a new one at 128 ppm appeared, which they
attributed to CN
. In contrast, Pollock and Goff (28),
using 13C-labeled SCN
, found that the initial
stable reaction product of this reaction appeared at 126.5 ppm, which
they attributed to dicyanate ether (NCS-O-SCN
). Twenty to
30 min later this initial product peak declined, coincident with the
appearance of a new species at 128.6 ppm, which they attributed to
OSCN
. Lövaas (30), based on electron spin resonance
studies of the
lactoperoxidase/SCN
/H2O2 system,
suggested that the major stable product of the reaction was
OSC·N
. Thus,
spectral analysis of the products of the
lactoperoxidase/SCN
/H2O2 system
have resulted in three different species other than HOSCN being
proposed as the initial reaction product.
We now present evidence that the major products of the
EPO/SCN
/H2O2 system are
OSCN
and OCN
. Preliminary experiments using
ESI-MS suggest that OSCN
and OCN
are also
the predominant products of the
lactoperoxidase/SCN
/H2O2 system
as well (not shown). We therefore predict that the two major product
resonance peaks previously detected by
[15N]SCN
NMR (29) and
[13C]OCN
NMR (28) studies of this system
will also prove to be attributable to OSCN
and
OCN
. If so, both EPO- and lactoperoxidase-mediated
oxidation of their principal physiologic substrate, SCN
,
by H2O2 yields two compounds with limited but
specific reactivities (see below).
Mechanism of HOSCN
and OCN
Generation
by the EPO/SCN
/H2O2
System--
Our data suggest, but do not prove, that the
EPO/SCN
/H2O2 system first
generates HOSCN, which then subsequently reacts with excess
H2O2, a second molecule of HOSCN, or decomposes
to generate OCN
secondarily. The first possibility is
supported by Fig. 5, in which generation of OCN
clearly
lags behind generation of HOSCN, which plateaus at ~150 µM, whereas OCN
accumulation continues, but
only so long as H2O2 is being added. A reaction
sequence that follows this scenario has been proposed by Aune et
al. (26) and Thomas (25).
|
(Eq. 1)
|
|
(Eq. 2)
|
|
(Eq. 3)
|
A number of other mechanisms are conceivable. Because our
experiments were designed to detect relatively stable products of the
EPO/SCN
/H2O2 system, they do not
rule in or out the transient existence of what are likely highly
reactive and unstable intermediates such as HOOSCN (cyanosulfurous acid).
EPO/SCN
/H2O2 as a Sulfhydryl
Reactivity-based Cytotoxic System--
In a model cytotoxic system
employing human RBCs, we find that graded exposure to the products of
the EPO/SCN
/H2O2 system first
depletes glutathione, then causes SH reductant-reversible inactivation
of GAPDH and GST, then membrane ATPases without detectable hemolysis
(Figs. 9 and 10). This sequence suggests a mechanism of toxicity based
largely, if not exclusively, upon SH reactivity of a membrane-permeant,
diffusible agent. This pattern, also seen with the products of the
lactoperoxidase/SCN
/H2O2 system
(39), contrasts strikingly with that of HOCl, which reacts rapidly with
a variety of cell surface biologic targets, rapidly disrupting membrane
integrity and lysing cells before oxidizing intracellular proteins to a
significant degree (3-5). A weak, but relatively SH-specific reactive
oxidant such as HOSCN can more effectively function as an
SH-dependent metabolic poison than a potent, but
nonspecifically reactive oxidant such as HOCl (Fig. 10).
Potential mechanisms underlying such toxicity are suggested using
GST-
as a model protein target (Fig. 11), where it is shown that
[14C]OSCN
/OCN
causes intra-
and intermolecular disulfide bonds and, in addition, covalent
incorporation of 14C that is nearly completely reversible
by SH reductants. 14C incorporation may reflect sulfenyl
thiocyanate bond formation (R-S-SCN), which can either persist, be
reversed by a SH reductant or react further with another SH to yield a
disulfide: R-S-SCN+ R'SH = RSSR' + SCN
+ H+ (25). 14C incorporation could theoretically
also reflect carbamylation reactions of OCN
with SH
groups (see below).
What, if any, contribution OCN
makes to the cytotoxicity
of the EPO/SCN
/H2O2 system
remains to be determined. It may simply be an inert decomposition
product of HOSCN. However, the large amounts of OCN
produced by the EPO/SCN
/H2O2
system and the known chemical reactivity and toxicity of OCN
raise the possibility (not addressed by our
experiments) that it could play a role by modifying target proteins.
Unlike the electrophile oxidants HOCl and HOSCN, OCN
is a
nucleophile that reacts irreversibly with amino groups and slowly but
reversibly with thiol groups of proteins through carbamylation reactions (40). Thus, OCN
has been shown to react with
-amino,
-amino, and thiol moieties of amino acids, peptides, and
proteins (41). These carbamylation reactions can irreversibly
inactivate enzymes such as glucose-6-phosphate dehydrogenase
(40, 41). Moreover, OCN
can directly react with
glutathione (40), and RBCs exposed to OCN
have decreased
levels of intracellular glutathione (42). An alternative potential role
for OCN
is suggested by the finding that 100-1000
µM OCN
strongly inhibits MPO generation of
HOCl by an H2O2-dependent mechanism, suggesting autoinactivation (44). We find that
OCN
also inhibits HOSCN production by EPO but only at
concentrations
1 mM (not shown). Despite these
possibilities, however, our data showing SH reactivity based toxicity
can be accounted for solely by the proposed oxidant reactivity of
OSCN
without invoking a role for OCN
,
because OCN
reaction with SH groups is slow in comparison
with that of OSCN
(40). On the other hand,
OCN
, unlike OSCN
, reacts rapidly with amino
groups (40). Experiments with selective scavenging agents may permit
dissection of the contribution of each of these species to cytotoxicity.
Potential Physiologic Significance of Findings--
Both
OSCN
and OCN
have relatively specific
reactivities that may bespeak a novel biologic strategy for the EPO
system in carrying out the putative role of eosinophils,
i.e. extracellular destruction of metazoan parasites (6). In
preliminary studies we find (55) that the ED50 for killing
of cultured human and mammalian cells, including cardiac myocytes and
several types of endothelial cells, is approximately 10-fold higher
(200-300 µM oxidant) for
EPO/H2O2/SCN
system-derived
products than for HOCl or HOBr; however, the ED50 for
killing of freshly transformed schistosomules, a model of parasitic
disease, is nearly identical (20-30 µM oxidant). It is
therefore conceivable that OSCN
and OCN
comprise a cytotoxic system that exploits subtle, sulfhydryl-based metabolic differences between host and parasites to effect selective killing of the latter. These weakly, but specifically, reactive compounds may be better suited for selective extracellular killing of
pathogens than is a potent but nonspecific oxidant such as HOCl.
Supporting the plausibility of a sulfhydryl-based cytotoxic system, the
efficacy of several anti-parasite drugs, such as oltipraz (45) and
certain arsenicals (46), derives from selective oxidative depletion and
impaired regeneration of parasite glutathione, a compound also readily
oxidized by both OSCN
and OCN
. Our findings
also have implications for the potential role of EPO in the
pathogenesis of human diseases, such as asthma and other allergic
states, in which eosinophils are known to play an important role.
Covalent modification of crucial cellular or extracellular
SH-containing biological targets by the
EPO/SCN
/H2O2 system, as we have
shown to occur in RBCs, could contribute to the initiation and
propagation of the host tissue damage. Carbamylation of host cell
amine-containing protein and lipid targets in the setting of chronic
eosinophilic inflammatory states could play a similar role.
Identification and quantitation of stable OSCN
- or
OCN
- modified biologic targets containing SH or amino
groups might serve as a sensitive and specific marker of the
EPO/SCN
/H2O2 system participation
in these diseases. Given these possibilities, the potential roles of
OSCN
/OCN
as mediators of
eosinophil-mediated toxicity, both for pathogens and host tissue,
warrants further scrutiny.