Applied

Appl Magn Reson (2015) 46:239249

DOI 10.1007/s00723-014-0631-6

Magnetic Resonance

Probing Radical Chemistry in Salmonella typhimurium

Cells Under Oxidative Stress Using Spin Traps
and Nitroxyl Radicals
Marco Bellinazzi Stephen N. Batchelor
Georg Gescheidt

Received: 18 August 2014 / Revised: 16 October 2014 / Published online: 13 January 2015
Springer-Verlag Wien 2015

Abstract The interaction between tert-butyl hydroperoxide and Salmonella typhimurium cell strain TA1547 as monitored by electron spin resonance (ESR) is
reported. Spin traps (DMPO and POBN) show the formation of methyl and alkoxyl
radicals in the bacterial culture. The kinetic behavior of the radical formation was
followed by the dose-dependent decay of the ESR spectra of the stable nitroxide
radicals TEMPOL and 3-aminomethyl-PROXYL, which are quenched by shortlived radicals. Whereas the spin traps provide qualitative characterization of some
of the radicals produced by the interaction between the bacteria and the hydroperoxide, our investigation indicates that the use of nitroxides provides further
quantitative kinetic data.

1 Introduction
Oxidative stress may be generally defined as the excess formation and/or
insufficient removal of highly reactive species from living cells [1]. Reactive
oxygen species (ROS) are believed to be the key culprits in oxidative stress and
include peroxides, hydroperoxides and the radicals that may be formed from them
[24]. If not properly controlled, ROS react with components of the cell leading to
cell damage, mutations and death [5]. Methods to monitor the chemistry occurring
in this process are required, and considerable attention has naturally been given to
electron spin resonance (ESR). The very low concentration of radicals in these
M. Bellinazzi  G. Gescheidt (&)
Institute of Physical and Theoretical Chemistry, NAWI Graz, Graz University of Technology,
Technikerstrasse 4/I, 8010 Graz, Austria
e-mail: g.gescheidt-demner@tugraz.at
S. N. Batchelor (&)
Unilever Research, Port Sunlight, Quarry Road East, Bebington, Wirral CH63 3JW, UK
e-mail: stephen.batchelor@unilever.com

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chronic processes coupled with the wide variability in many biological processes
makes this a difficult task. Spin trapping has been a technique of choice; however,
further methods are required to be developed to give more quantitative insights.
Here, a model system is investigated using classical spin probing, and a simple new
approach, monitoring of nitroxide quenching.
To probe the mechanism of oxidative stress, the oxidant tert-butyl hydroperoxide
has been a popular choice as a model hydroperoxide [6, 7]. Hydroperoxides are
generated as intermediates in lipid oxidation [8], can originate in amino acids [9],
and may also be formed by lipoxygenase [10]. The behavior of tert-butyl
hydroperoxide is also of interest in its own right as a high production volume
chemical, widely used in organic synthesis (HPV chemicals are classified as those
chemicals produced or imported in the United States in quantities of 1 million
pounds or more per year. US Environmental Protection Agency). In intact human
endothelial cells, it has been shown by spin trapping ESR experiments that, at high
concentrations, tert-butyl hydroperoxide rapidly decomposes to form peroxyl,
alkoxyl and methyl radicals, in a cleavage process driven by mitochondrial electron
transport chain in the cell [11, 12]. Work in bacterial strains of Salmonella
typhimurium has shown that bacterial cells protect themselves from hydroperoxides
using an alkyl peroxide reductase (peroxidase), which reduces alkyl hydroperoxides
to the corresponding alcohol using redox-active disulfides within each protein
[1315]. S. typhimurium strains are widely used in the bacterial reverse mutation
test (Ames test, OECD 471), which is one of the battery of tests used to assess the
mutagenic potential of chemicals. It has been shown that agents, which promote
the formation of reactive oxygen species, also induce mutagenicity in S. typhimurium [16, 17]. Tert-butyl hydroperoxide is mutagenic in bacterial cell-based Ames
test [18, 19] and the extent of this may be reduced by the use of anti-oxidants [14,
20, 21].
Despite all this work, to our knowledge, no ESR investigation has been reported
on the tert-butyl hydroperoxide/S. typhimurium system. Such a study is presented
here to probe the direct radical formation in the bacteria. The TA1537 strain was
chosen, which detects frameshift mutations, because it gives the largest mutagenic
response to peroxide and hydroperoxides of the standard set of strains used in the
Ames test [22].
In the cells, the reactive radicals are produced at a steady-state concentration too
low to be observed directly, so two indirect approaches were adopted. Firstly, they
are converted into more stable, long-lasting radicals using a spin trap. Subsequently,
the persistent radicals may be observed by ESR and the corresponding hyperfine
coupling constants, a, are characteristic for the nature of the trapped radical, see,
e.g., [23].
However, it is not straightforward to use the intensities of the spin trap ESR
spectra to gain kinetic insights into the chemical processes occurring. Therefore, we
also studied the impact on the decay of the ESR signal size of stable nitroxide
radicals in the cell culture upon addition of different doses of tert-butyl
hydroperoxide. These approaches provide a quantitative monitor of the radical
chemistry, on the minute time scale.

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2 Materials and Methods

The bacterial strain, S. typhimurium TA1537, was provided by Unilever.
Nutrient broth was purchased from Oxoid (Oxoid nutrient broth No. 2). A culture
of bacterial strain S. typhimurium TA1537 0.25 ml, which had been frozen and
preserved, was inoculated into 30 ml of nutrient broth, in a flask and the culture was
incubated at 37 C for 10 h. The concentration of cells was measured by means of a
Thoma counting chamber and adjusted through centrifugation at 1,500 rpm for
10 min or by addition of fresh nutrient broth.
In the Ames test, 0.1 ml of bacteria solution (2 9 109 bacteria/ml) is added to
0.5 ml of phosphate buffer and 0.1 ml of mutagenic chemical. In the current
experiments, the volume ratio of the three components was maintained but the
concentration of bacteria was increased tenfold (2 9 1010 bacteria/ml instead of
2 9 109), to increase the concentration of any radical species that are formed.
Salmonella typhimurium is a pathogenic organism and proper microbiological
technique was followed. Cell work was done in a secure microbiological facility and
for ESR measurements were placed in small volume in sealed contains for transfer
and to the ESR laboratory for immediate measurement. Scrupulous cleaning and
disinfection of all ESR glassware were carried out after each measurement.
tert-Butyl hydroperoxide (70 % in H2O) was purchased from Sigma-Aldrich.
All the ESR spectra were recorded on a X-band Bruker EMX Spectrometer
equipped with a TE102 cavity (ER 4102 ST). The measurements were carried out at
room temperature with a flat quartz cell positioned in the middle of the ESR cavity.
Chemicals were obtained from Aldrich and used as received (in the case of the spin
traps it was verified that no ESR signal was present before the trapping experiment).

3 Results and Discussion

3.1 Spin Trapping Experiments with DMPO
Many reactive radicals, R, undergo an addition reaction with DMPO to form
a persistent nitroxide radical (Scheme 1), which is observed in the ESR spectrum
[2428].
Figure 1 shows the ESR spectrum recorded from an aqueous solution of S.
typhimurium cells after the addition of tert-butyl hydroperoxide and the spin trap
DMPO. The signal was observed immediately after the mixing of the ingredients

Scheme 1 Spin traping with

DMPO

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M. Bellinazzi et al.

Gauss
3440

3460

3480

3500

3520

3540

Fig. 1 ESR spectrum obtained from a sample of S. typhimurium after addition of 15 mM tert-butyl
hydroperoxide and 0.02 M DMPO as spin trap. The superimposed simulation uses two radical species
(see Table 1)

Table 1 Experimental data

obtained for species 1 and
species 2 in Fig. 1 (DMPO) by
spectral simulation and their
comparison with literature data

Radical

14

Species 1

14.7

16.4

Species 2

16.6

23.9

(CH3)3CO-DMPO [29, 30, 41]

14.85

16.40

H3C-DMPO [29, 42]

16.45

23.4

ROO-DMPO [29]

14.25

10.55

N hfc/Gauss

H hfc/Gauss

and remained for several hours. The spectra were reproducible when generated
using different batches of cells; however, the intensity of the signal varied widely.
Control experiments performed in the absence of tert-butyl hydroperoxide or the
absence of cells did not lead to the detection of an ESR signal. Consequently, tertbutyl hydroperoxide must decompose in the presence of the bacteria to form
radicals.
To identify the radicals, the spectrum was simulated and an excellent match
obtained, assuming the presence of two radical species with hyperfine couplings
listed in Table 1. Comparison to literature values [29, 30] indicates that an alkoxyradical and a methyl radical derivative of DMPO were formed. The production of
these radical species is thought to occur as follows:
CH3 3 COOH ! CH3 3 CO   OH

CH3 3 CO ! CH3 2 CO H3 C

Decomposition of tert-butyl hydroperoxide via a 1-electron reduction driven by a

cell component [29] forms tert-butyloxyl radicals ((CH3)3CO), reaction (1). These
radicals are then trapped by DMPO to yield species 1 (Table 1). It has been well
documented that tert-butyloxyl radicals undergo rapid b-cleavage [31] to give

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Probing Radical Chemistry in Salmonella typhimurium Cells

243

acetone and a methyl radical (H3C), a reaction that takes place in the microsecond
time regime in water, vide supra. Subsequent addition of the methyl radical to
DMPO produces the second species observed in the spectrum. Methyl and alkoxyl
radicals were also found in related spin trapping studies of intact human endothelial
cells [7, 12, 29, 32].
The component of bacteria, which drives the 1-electron reduction of the
hydroperoxide is unknown. Alkylhydroperoxide reductase enzymes are present in
Salmonella to reduce hydroperoxides to alcohols and protect the cell from damage
[14]. These enzymes show strong similarity with those in mammals [33]. The
reductase enzymes are ubiquitous peroxiredoxins [21, 34], non-haem oxidases,
which reduce hydroperoxides via a nucleophilic attack by cysteine (Cys-SH), in
which the SH may be first deprotonated. No radicals are formed in this process.
CysS-H CH3 3 COOH ! CyS-OH CH3 3 COH

In the current experiments, these enzymes must be overwhelmed, presumably

then allowing a 1-electron reduction of the hydroperoxide to occur, perhaps driven
by the electron transport chain and an electron donor such as NADH. Other
mechanism, such as metal ion-driven decomposition of the peroxide may also occur.
This simple explanation of the observed signals deserves a little more discussion
as both radicals observed have a complex set of reactions they can undergo, which
compete with reaction (12) [3537]. An important question is whereabouts in the
cells the radicals are formed. If the radicals are formed in a lipophilic part, the
(CH3)3CO and H3C radical would be expected to rapidly abstract hydrogen from
anti-oxidants such as vitamin E and from fatty acids giving rise to very different
ESR signals [38]. There is no evidence for this in the spectra, pointing to the
observed radicals being formed in an aqueous environment.
Oxygen effects must be considered as carbon-centered radicals such as H3C
react rapidly with oxygen to form hydroperoxyl radicals:
H3 C  O2 ! H3 C-OO

However, no hydroperoxyl radicals were observed in the spectra [32]. At the

normal concentration of oxygen in water, 0.3 mM, the methyl radical would react
with oxygen in under a microsecond, because the reaction is diffusion controlled.
Clearly, oxygen effects will disappear when all the O2 is removed by reaction (4)
and the current results indicate that rapid radical formation removing oxygen has
occurred allowing the (CH3)3CO and H3C radical to be observed in the ESR
spectrum.
To provide further confirmation of radical formation, the spin trap a-(4-pyridyl
N-oxide)-N-tert-butylnitrone (POBN) was used. Again, an ESR signal was
obtained (Fig. 2); however, only one radical was observed, which by comparison
to other reports in the literature [39] and the spin trap database (http://EPR.niehs.
nih.gov) was a methyl radical, which can be typically detected upon the administration of t-butyl hydroperoxide [cf. reaction (1)] [40]. Further confirmation by
Mass Spectroscopic identification of the end products of the spin traps was not
performed.

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M. Bellinazzi et al.

Gauss
3460

3480

3500

3520

Fig. 2 ESR spectrum obtained from a sample of S. typhimurium after addition of 15 mM tert-butyl
hydroperoxide and 0.01 M POBN as spin trap. The superimposed simulation considers the presence of
one species (see Table 2)

3.2 Experiments with TEMPOL and 3-Aminomethyl-PROXYL Nitroxide

Radicals
To provide a further probe of the chemistry, persistent nitroxide radicals were added
to the cells with and without the presence of the tert-butyl hydroperoxide. Two
different radicals, TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) and
AMP (3-aminomethyl-PROXYL, 3-aminomethyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl) were chosen to see if molecular topology would influences the behavior.

TEMPOL is a neutral nitroxide based on a six-membered ring, whereas AMP is

based on a five-membered ring and will be positively charged at acidic and neutral pH,
since the exocyclic amino group is protonated [44]. There is some evidence that fivemembered ring positive charged nitroxides provide more protection then neutral
nitroxides to cells under oxidative stress [45], and additionally are reduced more
slowly [45]. For both nitroxides, a typical isotropic three-line ESR spectrum was
observed [46] and the measured nitrogen hyperfine constant, aN, was 17.0 and 16.1 G
for TEMPOL and AMP, respectively. These aN values (which have a well defined
solvent dependence) indicate that both radicals are in an aqueous environment [47];
this is corroborated by the spectral linewidth, which corresponds to that of water [47].

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Probing Radical Chemistry in Salmonella typhimurium Cells

245

0.24

0mM
0.15mM
1.5mM
15mM

[TEMPOL] / mM

0.22

0.20

0.18

0.16

0.14

0.12
0

10

20

30

40

50

60

time / minutes

Fig. 3 ESR signal intensities (proportional to the concentration) of TEMPOL depending on the
concentration of tert-butylhydroperoxide vs. time. Error bars are 95 % confidence limits based on the
standard deviation of three independent repeats for 0.15, 1.5 and 15 mM and eight repeated for 0 mM.
TEMPOL concentrations were calculated from the ESR signal intensities by extrapolating to t = 0 and
defining this as the initial concentration of 0.25 mM of TEMPOL). A linear fit is shown for 0 mM and an
exponential fit for 1.5 and 15 mM

The kinetic plot for TEMPOL is shown in Fig. 3. In the absence of bacterial cells,
the ESR signal intensity was constant; however, in the bacterial culture, the ESR
signal reduced linearly with time in an apparently zero-order reaction in the absence
of tert-butyl hydroperoxide (Fig. 3, black square). On the time scale of the
experiment (*5 min mixing time), no lag phase could be observed indicating that
the reactions started immediately. There appears to be no data on nitroxide
reduction rates in Salmonella, but the rates observed here are very similar to those
seen with Bacillus subtilis [48].
Essentially, this is in line with an enzymatic driven reduction of the radicals
following MichaelisMenten kinetics, as has been found for water-soluble
nitroxides in Staphylococcus aureus bacterial cells [49]. Other non-enzymatic
components of the cell may also contribute or drive the reduction, such as ascorbic
acid, metal ions or free thiols; however, concentration of these are generally low and
enzymatic reduction is generally believed to dominate [50]. Assuming the
enzymatic route dominates, the nitroxide radicals (NO) rapidly form a complex
with the enzyme (E) which may fall apart to give the starting materials or react to
give the non-paramagnetic product:
k1

k2

NO E
NOE ! E product
k1

In the limit that [NO] is much greater than KM (=(k-1 ? k2)/k1), the rate of loss
of nitroxide is k2[E]0, i.e., zero order provided the concentration of enzyme does not
change. When hydroperoxide is added significant dose-dependent changes are seen

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M. Bellinazzi et al.

Table 2 Experimental data

obtained for species 1 (Fig. 2,
POBN) by spectral simulation
and comparison with literature
data

Table 3 Intensity loss of the

ESR signal of TEMPOL and
AMP (lower concentration of
AMP was used then TEMPOL,
due to solubility issues)

Radical

14

Species 1

15.5

2.6

H3C-POBN [43]

15.9

2.7

N hfc/Gauss

H hfc/Gauss

[(CH3)3COOH] (mM) % loss in nitroxide ESR signal after 20 min

TEMPOL (0.25 mM)

AMP (0.1 mM)

14

0.15

14

1.5

26

15

29

10

in the nitroxide decay profile (Fig. 2) while the shape and the ESR parameters of the
spectrum remain unchanged. At high peroxide concentration (1.5 and 15 mM), the
decay profile is no longer zero order, with the initial nitroxide loss rate faster, but by
50 min is clearly slower than the cell alone. At low peroxide concentration
(0.15 mM), the reaction appears to remain essentially first order and within error
matches that of the cell alone.
The nitroxide decay can be rationalized in the following way (Table 3): at
0.15 mM, the defense mechanism of the cell, the alkyl peroxide reductases, are
sufficient to prevent the hydroperoxide from interfering with the enzymatic
reduction of the nitroxide. At higher concentrations of the hydroperoxide, the initial
nitroxide loss rate increases. The higher rate is not due to direct reaction of the
hydroperoxide with the nitroxide, as the control experiments without the cells
present show that the parent peroxide does not react with TEMPOL. Therefore, the
radicals (R), which finally react with the nitroxide, must be first formed by
decomposition of the hydroperoxide by the bacteria (reaction 6).

R
N

The coupling of the nitroxide with the radical is very rapid, close to the diffusion
controlled rate (k * 5 9 108 M-1 s-1 [51, 52]) and gives the methyl radical a
maximum lifetime of *1 ls (1/k[NO]), at the current nitroxide concentrations.
This would provide a reasonable explanation for the increased decay rate and
directly shows that the nitroxide can effectively scavenge radicals in the cells.
Alternatively, the presence of hydroperoxide is known to upregulate a number of
enzymes, which may alter the rate; however, the timescale of only a few minutes
appears too slow for this explanation [53]. It is very interesting that there is little
initial difference between the two highest hydroperoxide concentrations, indicating
that once the defense enzymes are deactivated, radical production and damage are

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Probing Radical Chemistry in Salmonella typhimurium Cells

247

not strongly dependent on the hydroperoxide level. Probably, this is because the
reduction process leading to radical production is working at maximum chemical
capacity.
The AMP probe shows similar effects to TEMPOL (Table 3) with a doubling of
the initial loss rate on addition of 15 mM hydroperoxide. Intriguingly, the absolute
loss of AMP radicals is smaller, indicating that this radical is less capable of
intercepting the alkyl and alkoxy radicals, i.e., is a less effective anti-oxidant for the
cell. The lower concentration of AMP does not account for the difference as on a
simple chemical basis, the initial % loss of nitroxide is proportional to k[R], where
R is the radicals reacting with the nitroxide and k is the rate constant. Chemically,
the radical coupling reaction (6) would be expected to occur at the same rate for
both nitroxides and tentatively points to the difference lying in different locations of
the nitroxides in the cells. However, it should be noted that the oxidation potential,
?
Eox
1/2 (RN=O /RN-O) of TEMPOL is 810 mV, lower than 853 mV for AMP [54],
which means that TEMPOL is more readily reduced.
After 2030 mins of hydroperoxide exposure, the decrease of TEMPOL
concentrations slows and starts to level off (Fig. 3). Some of the radicals produced
will react and damage components of the cell including the enzyme reducing the
nitroxide, as the enzyme is deactivated, the rate of loss will slow. Additionally, the
rate of radical production from the hydroperoxide may also decrease with time. A
stronger but still limited dose response is found than at earlier time. Hydroperoxides
can damage cells by non-radical routes, for example, it is used as an oxygen atom
transfer agent in epoxidations, and these reactions would become apparent at higher
concentrations and longer times, because they are chemically slower. Thus the later
time data also supports the notion that there is a limited total amount of radicals that
can be formed the hydroperoxide which saturates at above *12 mM.
It has to be borne in mind that the ESR signal arises from nitroxides residing in
an aqueous environment, both in and outside of the cell. That nitroxide radicals can
cross the cell wall is evidenced by the fact they are mutagenic in S. typhimurium,
i.e., can directly act on the bacterias DNA [55]. S. typhimurium cells are rod shaped
with a diameter of *1 lm and length of *5 lm giving a volume of 4 9 10-18 m3/
cell, combining this with the number of cells per ml of 2 9 1010 vide supra, means
*8 % of the sample volume are cells and the remainder water. Thus, at any one
time at a maximum 8 % of the nitroxides are inside the cell. With our experiments,
we are not able to directly distinguish the sites at which reactive radicals are
generated and quench the nitroxides. However, it is reasonable to assume that the
nitroxide must be inside, or near, the cell to react with the radicals formed from the
hydroperoxide, as the control experiments without the cell show no effect. The
decay curves show that the nitroxides in the sample are being quenched, and thus
nitroxide diffusion through the cell wall is not a rate-limiting step. The EPR signal
has to originate from nitroxide radicals in and outside the cell, whilst the radical
recombination very likely occurs at the cell membranes or inside the cell.
Accordingly, care must be taken in the absolute values, i.e., the hydroperoxide
effects inside the cells may well be larger than the ESR decay displayed in the
curves of Fig. 3 suggest.

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4 Conclusions
The well-established pro-oxidant tert-butylhydroperoxide induces the production of
radicals when administered to S. typhimurium. It is noteworthy, that related alcohols
do not lead to such an effect [56]. The applications of spin traps (DMPO, POBN)
indicate the presence of methyl and alkoxyl radicals; however, little information on
the reaction kinetics could be obtained [57]. On the other hand, the use of persistent
nitroxides (TEMPOL, AMP) provides a clear cut response showing the decay of the
ESR signal depending on the concentration of the hydroperoxide. Accordingly, the
use of nitroxides provides a very promising approach, when quantitative information is desired [58]. The decay curves certainly comprise several components
(diffusion of the hydroperoxide and the nitroxide through the cell membranes,
interfacial phenomena at the cell surface, etc.) which could not be resolved in this
work. Nevertheless, our results indicate that this procedure could provide valuable
information for determining kinetic parameters in these complex systems. The, thus,
determined time profiles afford further analysis to obtain a comprehensive picture
combining the occurrence of radicals, cell mutation and death.
Acknowledgments We are indebted to Prof. Peter Macheroux, Institute of Biochemistry, Graz
University of Technology for his help in preparing the cultures of S. typhimurium. Unilever (Safety and
Environmental Assurance Centre) is thanked for funding this study.

References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.

J.L. Martindale, N.J. Holbrook, J. Cell. Physiol. 192, 1 (2002)

T. Finkel, N.J. Holbrook, Nature 408, 239 (2000)
E. Cadenas, K.J.A. Davies, Free Radic. Biol. Med. 29, 222 (2000)
P.L. Oliveira, M.F. Oliveira, FEBS Lett. 525, 3 (2002)
R.E. Shackelford, W.K. Kaufmann, R.S. Paules, Free Radic. Biol. Med. 28, 1387 (2000)
J.P. Kehrer, L.G. Lund, Free Radic. Biol. Med. 17, 65 (1994)
C. Hazlewood, M.J. Davies, J. Chem. Soc. Perkin Trans. 2, 895 (1995)
R. Stocker, V.W. Bowry, B. Frei, Proc. Natl. Acad. Sci. USA 88, 1646 (1991)
A.S. Rahmanto, P.E. Morgan, C.L. Hawkins, M.J. Davies, Free Radic. Biol. Med. 48, 1071 (2010)
S. Yamamoto, Biochim. Biophys. Acta 1128, 117 (1992)
C.H. Kennedy, J.M. Dyer, D.F. Church, G.W. Winston, W.A. Pryor, Biochem. Biophys. Res.
Commun. 160, 1067 (1989)
V. Odonnell, M.J. Burkitt, Biochem. J. 304, 707 (1994)
S.B. Farr, T. Kogoma, Microbiol. Rev. 55, 561 (1991)
F.S. Jacobson, R.W. Morgan, M.F. Christman, B.N. Ames, J. Biol. Chem. 264, 1488 (1989)
L.B. Poole, H.R. Ellis, Biochemistry 35, 56 (1996)
T.M. de Kok, J.M. van Maanen, J. Lankelma, F. ten Hoor, J.C. Kleinjans, Carcinogenesis 13, 1249
(1992)
T.M.C.M. de Kok, J.M.S. van Maanen, J. Lankelma, F. ten Hoor, J.C.S. Kleinjans, Cardnogenesis 13,
1249 (1992)
B.N. Ames, M.K. Shigenaga, T.M. Hagen, Proc. Natl. Acad. Sci. USA 90, 7915 (1993)
K. Mortelmans, E. Zeiger, Mutat. Res. 455, 29 (2000)
M. Minnunni, U. Wolleb, O. Mueller, A. Pfeifer, H.U. Aeschbacher, Mutat. Res. 269, 193 (1992)
E.R. Assessment Tertiary butyl hydroperoxide CAS [75-91-2] (2008)
A.C. Bulmer, K. Ried, J.S. Coombes, J.T. Blanchfield, I. Toth, K.H. Wagner, Mutat. Res. 629, 122
(2007)
M.J. Davies, C.L. Hawkins, Free Radic. Biol. Med. 36, 1072 (2004)

123

Probing Radical Chemistry in Salmonella typhimurium Cells

249

24. A. Alberti, D. Macciantelli, Spin Trapping (2009), p. 287

25. G.M. Rosen, B.E. Britigan, H.J. Halpern, S. Pou, Free Radicals: Biology and Detection by Spin
Trapping (Oxford University Press, New York, 1999)
26. J.A. Degray, R.P. Mason, Biological Spin Trapping (1994), p. 246
27. K. Ranguelova, R.P. Mason, Magn. Reson. Chem. 49, 152 (2011)
28. M. Kopani, P. Celec, L. Danisovic, P. Michalka, C. Biro, Clin. Chim. Acta 364, 61 (2006)
29. W. Chamulitrat, N. Takahashi, R.P. Mason, J. Biol. Chem. 264, 7889 (1989)
30. NIEHS, N.I.E.H.S. Spin-Trap DataBase. NIEHS, Research Triangle Park (1998)
31. D.V. Avila, C.E. Brown, K.U. Ingold, J. Lusztyk, J. Am. Chem. Soc. 115, 466 (1993)
32. J. Van der Zee, D.P. Barr, R.P. Mason, Free Radic. Biol. Med. 20, 199 (1996)
33. H.Z. Chae, K. Robison, L.B. Poole, G. Church, G. Storz, S.G. Rhee, Proc. Natl. Acad. Sci. USA 91,
7017 (1994)
34. L. Flohe, J.R. Harris, in Subcellular Biochemistry, ed. by J.R. Harris (Springer, New York, 2007)
35. F. Leinisch, K. Ranguelova, E. DeRose, J. Jiang, R.P. Mason, Free Radic. Biol. Med. 51, S146 (2011)
36. F. Leinisch, K. Ranguelova, E.F. DeRose, J. Jiang, R.P. Mason, Chem. Res. Toxicol. 24, 2217 (2011)
37. M. Pazos, M.L. Andersen, L.H. Skibsted, J. Agric. Food Chem. 54, 10215 (2006)
38. N. Yanai, S. Shiotani, S. Hagiwara, H. Nabetani, M. Nakajima, Biosci. Biotechnol. Biochem. 72,
3100 (2008)
39. G.R. Buettner, Free Radic. Biol. Med. 3, 259 (1987)
40. K.R. Maples, C.H. Kennedy, S.J. Jordan, R.P. Mason, Arch. Biochem. Biophys. 277, 402 (1990)
41. A.S.W. Li, K.B. Cummings, H.P. Roethling, G.R. Buettner, C.F. Chignell, J. Magn. Reson. 79, 140
(1988)
42. P.J. Thornalley, R.J. Trotta, A. Stern, Biochim. Biophys. Acta 759, 16 (1983)
43. A. Zeghdaoui, B. Tuccio, J.P. Finet, V. Cerri, P. Tordo, J. Chem. Soc. Perkin Trans. 2, 2087 (1995)
44. J. Fuchs, W.H. Nitschmann, L. Packer, O.H. Hankovszky, K. Hideg, Free Radic. Res. Commun. 10,
315 (1990)
45. S.M. Hahn, L. Wilson, C.M. Krishna, J. Liebmann, W. Degraff, J. Gamson, A. Samuni, D. Venzon,
J.B. Mitchell, Radiat. Res. 132, 87 (1992)
46. C.S. Wilcox, A. Pearlman, Pharmacol. Rev. 60, 418 (2008)
47. B.R. Knauer, J.J. Napier, J. Am. Chem. Soc. 98, 4395 (1976)
48. K. Jung, S. Ristori, E. Gallori, G. Martini, Biochim. Biophys. Acta 1425, 387 (1998)
49. J.S. Goldberg, E.J. Rauckmann, G.M. Rosen, Biochem. Biophys. Res. Commun. 79, 1998 (1977)
50. N. Kocherginsky, H.M. Swartz, Nitroxide Spin Labels Reaction in Biology and Chemistry (CRC
Press, Boca Raton, 1995)
51. A.L.J. Beckwith, V.W. Bowry, K.U. Ingold, J. Am. Chem. Soc. 114, 4983 (1992)
52. V.W. Bowry, K.U. Ingold, J. Am. Chem. Soc. 114, 49924996 (1992)
53. S.B. Farr, T. Kogoma, Microbiol. Rev. 55, 561 (1991)
54. M.C. Krishna, D.A. Grahame, A. Samuni, J.B. Mitchell, A. Russo, Proc. Natl. Acad. Sci. USA 89,
5537 (1992)
55. H. Sies, R.J. Mehlhorn, Arch. Biochem. Biophys. 252, 393 (1986)
56. D.B. McGregor, G. Cruzan, R.D. Callander, K. May, M. Banton, Mutat. Res. Genet. Toxicol.
Environ. Mutagen. 565, 181 (2005)
57. A. Samouilov, V. Roubaud, P. Kuppusamy, J.L. Zweier, Anal. Biochem. 334, 145 (2004)
58. L. Valgimigli, G.F. Pedulli, M. Paolini, Free Radic. Biol. Med. 31, 708 (2001)

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