Synergistic activity of acetohydroxamic acid on prokaryotes under oxidative stress: The role of reactive nitrogen species

Article abstract of DOI:10.1016/j.freeradbiomed.2014.09.020

One-electron oxidation of acetohydroxamic acid (aceto-HX) initially gives rise to nitroxyl (HNO), which can be further oxidized to nitric oxide (NO) or react with potential biological targets such as thiols and metallo-proteins. The distinction between th

Full text of DOI:10.1016/j.freeradbiomed.2014.09.020

Free Radical Biology and Medicine 77 (2014) 291–297
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Free Radical Biology and Medicine
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Original Contribution
Synergistic activity of acetohydroxamic acid on prokaryotes under
oxidative stress: The role of reactive nitrogen species
Reeta Yadav a, Sara Goldstein b, Mohamed O. Nasef a, Wendy Lee a, Uri Samuni a,
n
a Department of Chemistry and Biochemistry, Queens College, City University of New York, Flushing, NY 11367, USA
b Chemistry Institute, the Accelerator Laboratory, the Hebrew University of Jerusalem, Jerusalem 91904, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
One-electron oxidation of acetohydroxamic acid (aceto-HX) initially gives rise to nitroxyl (HNO), which
can be further oxidized to nitric oxide (NO) or react with potential biological targets such as thiols and
metallo-proteins. The distinction between the effects of NO and HNO in vivo is masked by the reversible
redox exchange between the two congeners and by the Janus-faced behavior of NO and HNO.
The present study examines the ability of aceto-HX to serve as an HNO donor or an NO donor when
added to Escherichia coli and Bacillus subtilis subjected to oxidative stress by comparing its effects to
those of NO and commonly used NO and HNO donors. The results demonstrate that: (i) the effects of NO
and HNO on the viability of prokaryotes exposed to H2O2 depend on the type of the bacterial cell; (ii) NO
synergistically enhances H2O2-induced killing of E. coli, but protects B. subtilis depending on the extent of
cell killing by H2O2; (iii) the HNO donor Angeli's salt alone has no effect on the viability of the cells;
(iv) Angeli's salt synergistically enhances H2O2-induced killing of B. subtilis, but not of E. coli; (v) aceto-
HX alone (1–4 mM) has no effect on the viability of the cells; (vi) aceto-HX enhances the killing of both
cells induced by H2O2 and metmyoglobin, which may be attributed in the case of B. subtilis to the
formation of HNO and to further oxidation of HNO to NO in the case of E. coli; (vii) the synergistic activity
of aceto-HX on the killing of both cells induced by H2O2 alone does not involve reactive nitrogen species.
The effect of aceto-HX on prokaryotes under oxidative stress is opposite to that of other hydroxamic
acids on mammalian cells.
Received 18 June 2014
Received in revised form
16 September 2014
Accepted 16 September 2014
Available online 28 September 2014
Keywords:
HNO
NO
B. subtilis
E. coli
H2O2
Metmyoglobin
Angeli's salt
Nitroxide radical
& 2014 Elsevier Inc. All rights reserved.
Introduction
due to the redox exchange between the two congeners and by the
Janus-faced behavior of NO and HNO [17–24]. Opposing effects of
Hydroxamic acids (RC(O)NHOH, HXs) are important com-
pounds used in the clinic [1,2]. Their therapeutic activities may
be explained by their ability to bind metal ions, thus treating metal
poisoning [3,4], and inhibiting metallo-enzymes as in the treat-
ment of cutaneous T cell lymphoma by suberoylanilide hydro-
xamic acid (SAHA, Vorinostat) [5,6]. In addition, their physiological
effects are attributed to their capacity to generate nitric oxide (NO)
and/or its reduced form HNO (nitroxyl, azanone) [7–12]. NO and
HNO play diverse roles in physiological and pathophysiological
processes [13–16]. The distinction between their effects is difficult
NO have been observed in nearly every area of its research, which
were mostly ascribed to differences in its tissue level, or rates and
duration of its formation [21,23,25].
Recently, we have demonstrated that oxidation of acetohy-
droxamic acid (aceto-HX) by radiolytically borne radicals and by
the metmyoglobin (MbFeIII) and H2O2 reactions system initially
gives rise to HNO, which in the latter system is partially oxidized
to NO by compound II (MbFeIV) [11,12]. Hence, aceto-HX might be
considered as a NO donor if HNO oxidation to NO is more efficient
than its reaction with other biological targets such as thiols and
metallo-proteins [16].
The present work examined the ability of aceto-HX to serve as
an HNO donor or an NO donor on prokaryotes under oxidative
stress by comparing its effects to those of authentic NO and
commonly used NO and HNO donors. The effect of HNO on
prokaryotes subjected to oxidative stress has never been studied.
The only reported work is on mammalian cells (MCF-7) where the
HNO donor Angeli's salt and H2O2 displayed synergistic cytotoxic
effects [26]. NO itself demonstrates opposite effects on cells sub-
jected to oxidative stress [19,22,27–39]. NO predominately protects
Abbreviations: aceto-HX, acetohydroxamic acid; AS, 3-amino-1,2,4-triazole,
Angeli’s salt; ATZ, 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonate, ABTS2–
;
DTNB, 5-50-dithio-bis-(2-nitrobenzoic acid); BSA, bovine serum albumin; GSH,
glutathione; MbFeIV¼O, ferryl myoglobin; HX, hydroxamic acid; Tempol,
4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl; MbFeIII, metmyoglobin; PB, phos-
phate buffer; PBS, phosphate buffer saline; SNAP, S-nitroso-N- acetylpenicillamine;
GSNO, S-nitroso-GSH; SNP, sodium nitroprusside; SAHA, suberoylanilide hydro-
xamic acid; t-BuOOH, tert-butyl-hydroperoxide.
n
Corresponding author. Fax: þ1 718 997 5531.
E-mail address: uri.samuni@qc.cuny.edu (U. Samuni).
http://dx.doi.org/10.1016/j.freeradbiomed.2014.09.020
0891-5849/& 2014 Elsevier Inc. All rights reserved.

292
R. Yadav et al. / Free Radical Biology and Medicine 77 (2014) 291–297
eukaryotes from H2O2 and alkyl peroxide [22,27,30,33–35], protects
Bacillus subtilis and Neisseria meningitides against H2O2 cytotoxicity
[36,37], but enhances the killing of Escherichia coli [28,29,32,37,
40–42]. Moreover, NO protected Staphylococcus aureus exposed to
370 mM H2O2 [36], but enhanced the killing when the cells were
exposed to 10 mM H2O2 [43]. Here, we studied the effects of NO,
S-nitrosothiols and Angeli’s salt on H2O2-induced killing of B. subtilis
and E. coli and compared their effects to those of aceto-HX on cells
exposed to H2O2 and MbFeIII.
H2O2, plated in triplicates on LB agar, and incubated overnight
at 37 1C (E. coli) or 30 1C (B. subtilis) for clonogenic assay. All
experiments were repeated at least 3 times and each survival
curve represents a typical experiment.
Analysis of thiols in LB medium
HNO readily reacts with thiols [16], and therefore it is essential
to determine the potential contamination of LB medium with
thiols. No traces of thiols were detected using Ellman’s reagent
(DTNB) [52,53] in LB medium whereas thiols were readily detect-
able when the LB medium was deliberately contaminated with
10 mM cysteine or 0.5 mM BSA. We also examined any accumula-
tion of nitrite in the LB medium containing 4 mM SNP in the dark,
which is extremely sensitive to the presence of thiols [54,55].
Nitrite was not accumulated unless we deliberately contaminated
the LB medium with GSH or cysteine.
Materials and methods
Chemicals
Aceto-HX, glutathione (GSH), N-acetylpenicillamine, cysteine,
bovine serum albumin (BSA), myoglobin from horse heart, 3-amino-
1,2,4-triazole (ATZ), 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonate)
(ABTS2–), bovine serum albumin (BSA), 5-50-dithio-bis-(2-nitrobenzoic
acid) (DTNB), sodium nitroprusside (SNP), 4-hydroxy-2,2,6,6-tetra-
methylpiperidin-1-oxyl (Tempol), tert-butyl-hydroperoxide (t-BuOOH),
and Griess reagent were purchased from Sigma-Aldrich (St. Louis, MO,
USA). Catalase was purchased from Boehringer Biochemicals. Sepha-
dex G-25 for gel-filtration chromatography was purchased from
Pharmacia (Uppsala, Sweden). MbFeIII was prepared by adding an
excess of ferricyanide to myoglobin in 5-50 mM phosphate buffer (PB)
at pH 7 followed by chromatographic separation through a Sephadex
G-25 column. The concentrations of MbFeIII were determined spectro-
photometrically using ε408¼188 mM–1 cm–1 [44]. Angeli’s salt (AS)
was purchased from Cayman Chemicals Co. Stock solutions of AS were
prepared in 10 mM NaOH and the concentration was determined
Results
Effects of NO and HNO on bacterial cells subjected to oxidative stress
NO demonstrates opposing effects on E. coli and B. subtilis
exposed to peroxides [28,29,32,37,40–42] whereas the effect of
HNO on prokaryotes has not been studied. These bacterial cells
were selected as model systems for studying the ability of aceto-
HX to serve as an HNO donor or an NO donor by performing
comparative studies utilizing authentic NO, GSNO, SNAP, and
Angeli’s salt.
by the absorbance at 248 nm (
ε
¼8300 Mꢀ1 cmꢀ1) [45]. NO was
B. subtilis
purchased from Matheson Gas Products and was purified by
passing the gas through a series of traps containing deaerated
50% w/v NaOH and purified water in this order. Stock solutions of
NO solutions were prepared in gas tight syringes containing
10 mM PB, pH 6.8, and the concentration of NO was determined
immediately before use employing a spectroscopic assay with
ABTS2– as a reductant (ε660¼12000 M–1 cm–1 and 60% yield [46]).
S-Nitrosothiols were prepared daily by mixing equimolar concen-
trations of the thiol with HNO2 in 0.1 N H2SO4 stored in an ice
bath. The concentration of S-nitroso-GSH (GSNO) was determined
spectrophotometrically at 336 nm (ε336¼770 M–1 cm–1) and that
A previous study of the effect of NO on B. subtilis subjected to
oxidative stress involved high cell concentrations (OD660¼0.5)
treated with 10 mM H2O2 [36]. Under such conditions the cells
were protected from oxidative stress only when exposed to NO
shortly before the addition of H2O2 [36]. We show that at such high
cell concentrations the oxidant is diminished to subtoxic levels
within less than 4 min, thus terminating the oxidative stress (Fig. 1).
When the cells were grown in the presence of 10 mM ATZ,
which irreversibly inhibits catalase [56], the depletion of H2O2 was
slowed down prolonging the time window of the oxidative
damage, thereby increasing the duration and rate of cell killing
of S-nitroso-N-acetylpenicillamine (SNAP) at 340 nm
(
ε340¼
815 M–1 cm–1) [47]. Visible light was used to release NO from
GSNO or SNAP [48–50], and the rate of its release was evaluated by
determining the accumulation rate of nitrite. Nitrite concentration
was assayed with the Griess reagent. The absorption at 540 nm
was read 15 min after mixing the sample with the reagent.
Calibration curves were prepared using known concentrations of
nitrite. The concentrations of H2O2 and t-BuOOH were assayed
10
10
10
8
10
6
iodometrically at 352 nm (
the relatively slow oxidation of iodide by t-BuOOH, the buildup of
I–3 was followed at 352 nm until a plateau value was reached.
ε
¼ 25,800 M–1 cm–1) [51]. In view of
10
0
10
20
Time (min)
30
4
2
0
Cell cultures
B. subtilis PY79 and E. coli 25922 were cultured aerobically in
Luria-Bertani (LB) medium adjusted to pH 7 by 40 mM PB in a
vigorously shaking incubator at 37 1C. Cells were diluted 1:100 in
fresh LB and grown aerated at 37 1C until OD660 ꢁ 0.5. In some
experiments the cells were diluted in LB or 1:100 in saline (0.9%
NaCl) or phosphate buffered saline (PBS, 40 mM PB, 0.65% NaCl) to
the desired cell concentration and challenged with various sub-
strates. Cells cultures were sampled at various time points, diluted
in sterile water containing 60 U/mL catalase to remove residual
0
2
4
6
8
Time (min)
Fig. 1. Effect of high cell concentrations on the depletion of H2O2. Cultures of
exponentially growing B. subtilis (OD660 ꢁ 0.5, 7 ꢂ 107 cells/mL) were challenged
with 10 mM H2O2 in LB at 37 1C, and samples were taken for assaying residual H2O2
and for clonogenic assay (inset).

R. Yadav et al. / Free Radical Biology and Medicine 77 (2014) 291–297
293
as compared to cells exposed to the same initial concentration of
H2O2 but in the absence of ATZ (Fig. 2).
induce cell killing whether given alone or in combination with
H2O2 in LB medium, in saline, or in PBS. Conversely, NO either
added as a bolus or released by GSNO or SNAP enhanced the cell
The implications are that in cell survival experiments involving
high cell concentrations there is an exceedingly short time
window in which the cells are actually exposed to oxidative stress.
Therefore, we carried out our experiments using sufficiently low
cell concentrations, avoiding significant oxidant depletion.
Exposure of the cells to NO alone caused a growth delay, but did
not induce cell killing in agreement with previous reports [36,37].
The protective effect of NO was independent of the order of the
exposure of the cells to NO and H2O2 as demonstrated in Fig. 3.
The lack of effect of the order of the addition of NO and the
oxidant is also demonstrated when H2O2 was replaced by
t-BuOOH, which was only slightly depleted even in the presence
of high cell concentrations (Fig. 4).
1
0.1
When bolus addition of NO was replaced by GSNO or SNAP,
which continuously and slowly release NO under visible illumina-
tion [48–50], both NO donors protected the cells (Fig. 5) indepen-
dent of the order of their addition (Fig. 6). Photolysis of the cells
exposed to GSNO or SNAP alone did not induce cell killing,
indicating that any intermediates or end-products formed during
the photolysis of these nitrosothiols are not toxic.
NO-H2O2
H2O2-NO
H2O2
0.01
0
5
10
15
20
25
Time (min)
The HNO donor AS alone (0.05–1 mM) caused cell growth
delay, but did not induce any cell killing in LB medium, in saline
or in PBS. In the presence of H2O2 it synergistically potentiated the
cell killing in LB medium (Fig. 7) as well as in saline or in PBS (data
not shown).
It should be noted that despite systematic attempts to rigor-
ously apply similar oxidative stress by a careful control of experi-
mental conditions, the extent of the cell killing greatly varied
between apparently similar experiments. In most experiments NO
protected B. subtilis against extensive killing predominantly
induced by high concentrations of H2O2. In some cases where
the cells suffered only minor viability loss, NO demonstrated no
protection or even potentiated cell killing. The poor reproducibility
of H2O2-induced cell killing precluded drawing any clear
correlation.
1.0
0.5
0
5
10
15
20
25
E. coli
Time (min)
E. coli cells deplete H2O2 at lower rates compared to those
induced by B. subtilis. For instance, more than 75% oxidant is left
when H2O2 at 4.4 mM is incubated with E. coli culture at ca.
107 cells/mL over 60 min. The HNO donor AS (0.05–1 mM) did not
Fig. 3. Effect of NO on survival of B. subtilis at low concentrations exposed to H2O2
(A) and on H2O2 depletion (B). Cultures of exponentially growing B. subtilis (OD660
ꢁ
0.03, 6 ꢂ 106 cells/mL) in LB at 37 1C were challenged with 4 mM H2O2 alone (■) or
with 90 μM NO added 10 s before (●) or after (O) the challenge with the oxidant.
100
100
10-1
10-2
10
8
6
10-1
4
2
10-2
0
0
5
10
15
Time (min)
10-3
10-4
10-5
t-BuOOH
NO-t-BuOOH
t-BuOOH-NO
0
20
40
Time (min)
60
ATZ & H2O2
control
H2O2
Fig. 2. Effect of ATZ on survival of B. subtilis exposed to H2O2 and on oxidant
depletion. Cultures of exponentially growing B. subtilis (OD660 ꢁ 0.5, 7 ꢂ 107 cells/mL)
in LB at 37 1C were challenged for 15 min with 10 mM H2O2 alone and in the
presence of 10 mM ATZ. The inset of the figure shows the kinetics of H2O2 depletion
in the absence (circle) and presence of ATZ (square).
Fig. 4. Effect of NO on survival of B. subtilis exposed to t-BuOOH. Cultures of
exponentially growing B. subtilis (OD660 ꢁ 0.5, 5 ꢂ 107 cells/mL) in LB at 37 1C were
challenged with 1.5 mM t-BuOOH alone (■) or with 60 μM NO added 10 s before (●)
or after (O) the challenge with the oxidant. The consumption of the peroxide within
60 min was less than 30% in the absence and presence of NO.

294
R. Yadav et al. / Free Radical Biology and Medicine 77 (2014) 291–297
100
100
10-1
10-2
10-3
10-3
10-6
0
5
10
15
20
SNAP
H2O2
NO
H2O2
SNAP
H2O2
GSNO
H2O2
Time (min)
Fig. 7. Synergic pro-oxidative effect of AS on B. subtilis subjected to oxidative stress.
Cultures of exponentially growing B. subtilis (OD660 ꢁ 0.012, 6 ꢂ 106 cells/mL) in LB
at 37 1C were challenged with 10 mM H2O2 (□), 15 mM H2O2 (Δ), 0.2 mM AS (●),
10 mM H2O2 and 0.2 mM AS (■), and 15 mM H2O2 and 0.2 mM AS (▲). AS was
added 10 s before H2O2 addition. The rate of the decomposition 0.2 mM AS in
40 mM phosphate buffer at pH 7.4 and 37 1C was determined spectrophotome-
trically at 240 nm to be (1.970.1) ꢂ 10–3 s–1 (i.e., half-life ca. 6 min).
Fig. 5. Effect of NO and NO donors on survival of B. subtilis exposed to H2O2.
Cultures of exponentially growing B. subtilis (OD660 ꢁ 0.013, 3.5 ꢂ 106 cells/mL) in
LB at 37 1C were challenged for 20 min with 10 mM H2O2 alone or in combination
with NO, either adding 40 μM NO 10 s before H2O2 or illuminating with visible light
0.79 mM SNAP and 0.78 mM GSNO starting 10 s before H2O2 addition. The rates of
nitrite accumulation during the experiment were 0.3870.05 and 0.3470.03 μM/min
for GSNO and SNAP, respectively.
100
10-1
10-2
10-3
100
10-1
H2O2
GSNO/H2O2
AS/H2O2
10-2
H2O2
SNAP-H2O2
H2O2-SNAP
10-4
0
10
20
Time (min)
30
0
5
10
15
20
25
30
Time (min)
Fig. 8. Effects of GSNO and AS on E. coli subjected to oxidative stress. Cultures of
exponentially growing E. coli (OD660 ꢁ 0.01, 3 ꢂ 106 cells/mL) in LB at 37 1C were
challenged with 4 mM H2O2 alone (□) and in combination with either 0.1 mM AS
added 10 s before H2O2 addition (▲) or 1 mM GSNO illuminated with visible light
starting 10 s before H2O2 addition (●). The rate of nitrite accumulation induced by
GSNO was 0.6370.05 μM/min. The rate of AS decomposition was determined to be
(1.970.1) ꢂ 10–3 s–1 (i.e., half-life ca. 6 min) by following the decay of 0.2 mM AS at
240 nm in 40 mM phosphate buffer at pH 7.4 and 37 1C.
Fig. 6. SNAP protects B. subtilis from H2O2 independent of the order of their
delivery. Cultures of exponentially growing B. subtilis (OD660 ꢁ 0.01, 1.5ꢂ 106 cells/mL)
in LB at 37 1C were challenged with 4 mM H2O2 alone (■) or in combination with
1 mM SNAP illuminated with visible light 20 s before (O) and after (●) H2O2
addition.
killing induced by H2O2 (Fig. 8) as previously reported for NON-
Oates [28,29,32,37,40–42]. Photolysis of either GSNO or SNAP
alone caused a growth delay, but did not induce cell killing,
indicating that no intermediate or product formed during the
photolysis of these S-nitrosothiols is toxic to the cells.
demonstrated to protect from metal-mediated oxidative stress
[57,58], inhibited the H2O2-induced cell killing (Fig. 11).
Discussion
Effect of aceto-HX on bacterial cells subjected to oxidative stress
Aceto-HX alone (0.5–4 mM) did not induce cell killing in both
cells, but potentiated cell killing induced by H2O2 and MbFeIII in a
dose-dependent manner (Figs. 9 and 10).
Oxidation of aceto-HX by radiolytically borne radicals and by the
MbFeIII/H2O2 reactions system initially gives rise to HNO, which in
the latter system is partially oxidized to NO [11,12]. Aceto-HX,
which is a HNO donor, might be considered as a NO donor if HNO
conversion into NO is more efficient than its reactions with other
biological targets such as thiols and metallo-proteins [16]. While NO
effects have been primarily studied using E. coli and B. subtilis, those
of HNO have not been studied on bacterial cells. A previous
publication reported a dramatic protective effect of NO added
shortly before the exposure of high concentrations of B. subtilis to
10 mM H2O2, and concluded that this phenomenon is general for
Nitrite accumulates in these cell cultures, and its initial rate of
accumulation increases on increasing [MbFeIII] or [aceto-HX], and
is hardly affected by [H2O2]¼2–10 mM, e.g., 0.8970.08
μ
M/min in
cell culture containing 4 mM H2O2, 5
μ
M MbFeIII, and 1 mM aceto-
HX. Aceto-HX also potentiates the cell killing induced by H2O2
alone (Figs. 9 and 10), but in this case nitrite is not accumulated.
This synergic effect might involve the metal-chelating properties
of hydroxamic acids since Tempol, which has been previously

R. Yadav et al. / Free Radical Biology and Medicine 77 (2014) 291–297
295
100
10-1
10-2
10-3
subjected to oxidative stress to determine whether aceto-HX acts as
an HNO donor or an NO donor.
Our results indicate that in the absence of oxidative stress
neither NO nor HNO induce bactericidal effects. These observa-
tions imply that nitrite, which is the product of NO reaction with
O2 and of AS decomposition, is not involved in killing of prokar-
yotes. Also under oxidative stress AS does not potentiate killing of
E. coli, indicating that nitrite is not toxic also in the presence of
H2O2. This is also the case with B. subtilis where it has been shown
that nitrite has no effect on the cells exposed to H2O2 [36,37].
There is a clear distinction between the effects of NO and HNO on
H2O2-induced killing of E. coli where only NO, but not HNO,
demonstrates a synergistic pro-oxidative effect. NO greatly inhibits
extensive killing of B. subtilis by H2O2 while HNO demonstrates a
synergistic pro-oxidative effect. The dependence of NO effect on
the extent of cell injury, which was observed for S. aureus cells
exposed to 370 mM H2O2 [36] vs 10 mM H2O2 [43], is intriguing
and deserves further elucidation.
Aceto-HX (pKa¼9 [11,59]) is cell permeable and an efficient
metal chelator [60]. Since it enhances H2O2-induced cell killing,
but does not modify cell viability in the absence of oxidative stress,
its synergic pro-oxidative activity should not be attributed to
inhibition of essential metallo-enzymes. Instead, the synergic
effect of aceto-HX on the killing of cells exposed to H2O2/MbFeIII
might be ascribed to the formation of reactive nitrogen species.
In this case aceto-HX undergoes a one-electron oxidation to yield
initially the respective transient nitroxide radical [11,12].
10-4
4
10
-
4
4
10
4
-
-
4
-
4
5
4
-
H2O2, mM
4
-
4
5
MbFeIII,
M
-
30
4
1
1
4
-
-
HX, mM
time, min
30
30
30
15
15
30
15
15
Fig. 9. Synergic pro-oxidative effect of aceto-HX on E. coli subjected to oxidative
stress. Cultures of exponentially growing E. coli (OD660¼0.013, 2 ꢂ 107 cells/mL) in
LB at 37 1C were challenged with mixtures of H2O2, MbFeIII, and aceto-HX for 15 or
30 min as indicated below the columns. The exposure time was measured once
H2O2 was added to the reaction mixture.
100
10-1
10-2
10-3
10-4
ꢀe–
–Hþ
CH3CðOÞNHOH ⟹ H2OþCH3CðOÞNHOꢃ
ð1Þ
Our recent work has demonstrated that the transient nitroxide
radical generates HNO through the hydrolysis of CH3C(O)N¼O
formed via the dismutation of the nitroxide radical or its oxidation
by compound II (MbFeIV) [12].
-
-
4
H2O2, mM
2
5
3
4
9
4
9
4
2
-
-
2
5
-
2
-
3
4
9
-
4
9
2
10 10
11 11
MbFeIII,
HX, mM
time, min
M
1
-
4
30
30
30 30
16
16
30
16
16
10 10
The results of the current study show that AS exhibits a
synergistic pro-oxidative effect when added to B. subtilis cells
exposed to H2O2 similar to the effect of aceto-HX on the cell killing
induced by H2O2/MbFeIII, indicating that in this case aceto-HX acts
as a HNO donor. Testing this conclusion using thiols as HNO
scavengers [16] is impossible because thiols are readily oxidized by
compounds I and II [61]; i.e., elimination of the synergistic activity
of aceto-HX would result from the competition of thiols with
aceto-HX for the oxidizing species rather than from HNO scaven-
ging. The use of cyclic nitroxides or other one-electron oxidants as
HNO scavengers is further complicated by oxidation of HNO to NO
[62,63]. In the case of E. coli, aceto-HX demonstrates a synergistic
pro-oxidative effect, thus resembling the effect of NO donors.
A substantiation for this conclusion using specific NO scavengers,
e.g., nitronyl nitroxides, is complicated since these nitroxides also
oxidize HNO to NO [64]. Whether aceto-HX acts as an HNO donor
or an NO donor is determined by the competition between HNO
oxidation to NO and its reaction with thiols and heme proteins
[16], which could depend on the type of the bacteria.
Fig. 10. Synergic pro-oxidative effect of aceto-HX on B. subtilis subjected to
oxidative stress. Cultures of exponentially growing B. subtilis (OD660
ꢁ 0.012,
5 ꢂ 106 cells/mL) in LB at 37 1C were challenged with mixtures of H2O2, MbFeIII, and
aceto-HX for different periods of time as indicated below the columns. The
exposure time was measured once H2O2 was added to the reaction mixture.
B.subtilis
E. coli
100
10-1
10-2
10-3
The synergic pro-oxidative effect of aceto-HX on cell killing
induced by H2O2 alone cannot be ascribed to the formation of reactive
nitrogen species; i.e., H2O2 does not oxidize directly the hydroxamate
moiety and in this system nitrite is not accumulated. The mechanism
underlying the pro-oxidative activity of aceto-HX is not fully under-
stood. Aceto-HX is an efficient metal chelator, which does not
necessarily imply cytoprotection from metal-catalyzed oxidative
stress. Metal chelators, which do not render transition metal redox
inactive, but rather increase their solubility and availability for binding
to critical cellular targets, actually potentiate biological injury. Since
aceto-HX enhanced H2O2-induced cell killing, we assume that it acts
by releasing redox-inactive metals from cellular stores, rending them
redox active, thus enhancing bactericidal effects via the Fenton-type
H2O2, mM
HX, mM
Tempol, mM
time, min
2
-
2
2
2
10 10 10
3
-
3
-
3
2
-
3
2
2
-
4
4
-
-
4
-
6
4
-
4
-
4
4
6
-
2
10 10 10 10
6
36 36 36 36
Fig. 11. Effect of Tempol on the synergic pro-oxidative effect of aceto-HX on cells
exposed to H2O2. Cultures of exponentially growing cells (ꢁ 5 ꢂ 106 cells/mL) in LB
at 37 1C were challenged with mixtures of H2O2, aceto-HX, and Tempol for different
periods of time as indicated below the columns.
prokaryotes [35]. However, this report has overlooked previous
studies demonstrating the synergic effect of NO on E. coli
[28,29,32,37,40–42]. The present work examined the effect of NO,
NO donors, HNO donor, and aceto-HX on E. coli and B. subtilis

296
R. Yadav et al. / Free Radical Biology and Medicine 77 (2014) 291–297
reaction. Tempol has several biological effects including oxidation of
reduced metals thus preemptying the Fenton-like reaction [57,58].
Our results demonstrate that Tempol inhibit the potentiation by
aceto-HX of H2O2-induced cell killing (Fig. 11), and this protective
effect supports our assumption. The full elucidation of this pro-
oxidative synergic activity is beyond the scope of this study and is
currently under investigation.
Aceto-HX synergistically enhances the bactericidal effect of H2O2
with and without MbFeIII through more than a single mechanism.
Such a synergy is intriguing since SAHA and its structural analog
Trichostatin A provided protection against H2O2-induced killing of
mammalian cells [10].
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potential: not just the Janus face of NO. Pharmacol. Ther. 113:442–458; 2007.
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damage. Gastroenterology 113:1405–1407; 1997.
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The results obtained from the present study can be summarized
as follows: (i) the effects of NO and HNO on prokaryotes under
oxidative stress depend on the type of the bacterial cell; (ii) NO
synergistically enhances H2O2-induced killing of E. coli, and protect
B. subtilis depending on the extent of cell killing by the oxidant; (iii)
HNO has no effect on the viability of the cells; (iv) HNO does not
modify the killing of E. coli subjected to oxidative stress, but
synergistically enhances H2O2-induced killing of B. subtilis; (v)
aceto-HX alone has no effect on the viability of both cells; (vi)
aceto-HX demonstrates synergic pro-oxidative effects on both cells
exposed to H2O2 and MbFeIII, which may be attributed in the case of
B. subtilis to the formation of HNO and to further oxidation of HNO
to NO in the case of E. coli; (vii) aceto-HX-induced synergy in the
presence of H2O2 alone does not involve reactive nitrogen species.
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Acknowledgments
This work has been supported by the Israel Science Foundation,
the Queens College Research Enhancement Funds, and PSC-CUNY
Research Awards.
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