Fluoride Vol.30 No.1 5-15 1997. Research Report 5
SPIN TRAPPING TECHNIQUE STUDIES ON ACTIVE OXYGEN
RADICALS FROM HUMAN POLYMORPHONUCLEAR
LEUKOCYTES DURING FLUORIDE-STIMULATED
RESPIRATORY BURST
Y Y Wang, B L Zhao, X J Li, Z Su and W J Xi
Beijing, China
SUMMARY: By means of the ESR spectrum technique it was demonstrated
that fluoride (F-) stimulated human polymorphonuclear leukocytes (PMN) in a
respiratory burst to generate active oxygen radicals (• 02 and -OH) and to
consume oxygen (02). The experiments' using the spin trapping technique
with the nitrone DMPO as the trapping agent indicated that the PMN produce
the adduct DMPO-OOH under high concentrations of F- and the adduct
DMPO-OH under low concentrations of F-. Under medium concentrations of F-,
DMPO-OOH was produced which then decreased in amount as a decrease
occurred in F- concentration. The results of tests of scavenging by superoxide dismutase (SOD) and catalase suggested that the active oxygen radicals
generated in the respiratory burst originated mainly from superoxide (-02). By
the spin probe oximetry method (CTPO and CrOX) it was shown that the 02
consumed is from the intercellular medium.
Key words: ESR (electron spin resonance); Fluoride stimulation; Oxygen radicals;
PMN (polymorphonuclear leukocyte); Respiratory burst; Spin trapping.
INTRODUCTION
Earlier papers from our laboratory reported the use of the spin label ESR
(electron spin resonance) spectrum technique to discover various effects of fluoride
(F-) exposure on the membrane of human red blood cells (erythrocytes). 1 -3 In
this new study we report on the use of the same technique to examine the
production of active oxygen radicals by certain human white blood cells, namely
polymorphonuclear leukocytes (PMN) during a fluoride-stimulated respiratory
burst. The term "respiratory burst" refers to the simultaneous increases in oxygen
uptake, superoxide ( . 02-) and hydrogen peroxide (H2 02 ) production, and oxidation
of glucose (via the "hexose monophosphate shunt") which are observed when
certain leukocytes are exposed to appropriate stimuli.4
In 1975 Curnutte and Babior found, unexpectedly, that F-, in the high concentration of 20 mM, was a potent stimulus for • 02 production from PMN. 5 The
response was rapid with all the ferricytochrome c used in the experiment to
measure the rate of • Of production being reduced within 10 minutes after the start
of the reaction. The rate of • 02 production was 5 times faster than the rate obtained
when the PMN were incubated with bacteria alone and 20 times greater than the
rate for resting PMN. These results were foreshadowed by the finding by Sbarra
and Karnovsky in 1959 that 20 mM NaF caused a 2.5 fold increase in 0 2 consumption in resting guinea pig PMN.6
Little information is available about the effect of F- on 0 2 consumption by
human PMN, the mechanism of • Of production from human PMN, or the influence of fluorosis or other fluoride-related diseases on these effects.
Beijing Municipal Institute of Environmental Protection, Fu Wei Avenue, Beijing,
100037 China.
6 Y Y Wang, B L Zhao, X J Li, Z Su and W J Xi
The spin trapping Electron Spin Resonance (ESR) technique allows short-lived
free radicals to be measured and can be used to study the process of superoxide,
• 02, and hydroxyl radical, • OH, production from F--stimulated PMN, and the
relationship of this to the F- concentration. The spin probe method also allows
measurement of the consumption of 02 and the difference in 02 between the
extracellular and intracellular compartments. This allows calculation of the
generation of the active oxygen radicals 02 and • OH, the correlation between
them, their relationship to 02 consumption, and the relationships between F- and
the levels of • O2 and • OH produced. Information about the behaviour of active
oxygen radicals formed during the respiratory burst in human PMN stimulated by
F- may be relevant to understanding the effects of fluoride consumed with
fluoridation, and fluoride containing drugs and toothpastes, and preventing
fluoride toxicity and fluoride-related diseases.
MATERIALS AND METHODS
Reagents
Spin trapping reagent DMPO, (5,5-dimethyl-l-pyrroline-N-oxide), purchased
from Sigma Co., USA, was purified with active charcoal before use and found to
have no endogenous ESR signal. Spin probe C1'P0 (3-carbamoy1-2,2,5,5tetramethy1-3-pyrrolidin-l-yloxyl) and TEMPOL (2,2,6,6-tetramethyl-piperidine-Noxyl-4-ol), products of Sigma Co., were dissolved in a little alcohol and then diluted
by 0.05 M phosphate buffer (PBS, pH 7.4). Stimulus PMA (phorbol 12-myristate
13-acetate), from Sigma Co., was dissolved in a little acetone, stored at -20°C and
diluted with PBS before use. Widening reagent CrOX (potassium trioxalochrome),
from Alfa Co., was dissolved by PBS. SOD (superoxide dismutase) and catalase
were purchased from Donfony Medical Factory in China. Each solution was prepared with PBS. Other reagents were purchased in China and were of AR grade.
Isolation of human PMN
Whole fresh blood from healthy people (ACD-B) was obtained from the
Municipal Blood Centre of the Red Cross in Beijing. The red cells were allowed to
sediment to the bottom of the container for 1 hour after the addition of one third
of the blood volume of 6% dextran 70 in 154 mM NaCl. The PMN were purified to
the 99% level by isolating the white cells by centrifuging the suspension containing
the PMN and then using Ficoll solution and gradient centrifugation to separate the
PMN from other mononuclear cells. The PMN were washed with PBS and then
suspended in PBS in a dilution of 5 x 10 7 cells/mL and kept at 4°C until used.
FIGURE 1. (opposite page) ESR spectra of DMPO spin trapping radicals from PMN
stimulated by F-.
A-1,2,3,4,5: addition of 0.5, 0.2, 0.1, 0.05 and 0.0 pL 0.5 mM F- solution after adding
PMN, 0.1 mM DMPO and 1.0 mM DETAPAC.
B-1,2,3,4: addition of 15, 10, 5, and 0 pL 0.5 F- solution after adding PMN, 0.1 M
DMPO and 1.0 mM DETAPAC.
Fluoride 30 (1) 1997
Spin trapping studies on F--stimulated human PMN 7
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Fluoride 30
(1) 1997
8 Y Y Wang, B L Zhao, X J Li, Z Su and W J Xi
Procedure
The samples for the measurement of active oxygen radicals were prepared by
treating a mixture of 10 piL of PMN and 0.1 mM of CTPO with various doses of F-.
A period of incubation at 37°C followed. The duration of the incubation was three
minutes, so as to give the maximal height for the first peak measured, in accordance
with the data in Table 1. To the sample was then added 0.1 mM DMPO.
TABLE 1. Effect of incubation time at 37°C on the height of the first peak
Time in minutes
H* (G)
1
2
3
4
1.6
2.1
2.9
2.1
* Height of first peak measured in gauss
The samples for measuring 0 2 consumption were prepared by incubating
10 !AL of PMN and 0.1 mM of TEMPOL at 37°C for 30 minutes so that equilibrium
was reached between the intracellular and extracellular TEMPOL concentrations.
Various doses of F- were then added, with and without CrOX, and the mixture
incubated for a further 20 minutes at 37°C.
In the control experiments F- was replaced by PBS.
ESR measurement
The samples were transferred to a quartz capillary and measured with a Varian
E-109 spectrometer. The conditions for measuring active oxygen radicals were:
microwave power 15 mW, 100 kHz field modulation, modulation amplitude 1 gauss
(G), central magnetic field 3400 G, scan width 200 G, scan time 4 minutes, and
temperature 25°C. The conditions for the measurement of 0 2 consumption were
the same apart from: microwave power 1 mW, modulation amplitude 0.056 G, and
scan width 10 G.7
RESULTS AND DISCUSSION
The ESR spectra of active oxygen radicals trapped by DMPO are shown in
Figure 1. There are two distinct components shown in the spectra. One is the spin
adduct DMPO-OOH of superoxide, • 02, and DMPO. The other is the spin adduct
DMPO-OH of hydroxyl radical, -OH and DMPO. The ESR parameters of DMPOOOH and DMPO-OH are shown in Table 2.
TABLE 2. ESR parameters
Parameter
of DMPO-OOH and DMPO-OH
DMPO-OOH
DMPO-OH
g
2.0055
2.0053
aN
14.3 G
14.9 G
aR H
11.3 G
aTH
1.26 G
aH
14.9 G
These results are consistent with those obtained by computer simulation
(Figure 2) and with data reported in another study.8
Fluoride 30 (1) 1997
Spin trapping studies on F--stimulated human PMN 9
The interaction of the unpaired spin electron in the DMPO-OOH adduct with
the N (I= 1) nucleon on the nitroxide causes a split of the ESR spectrum into two
parts, each with 2 lines, with the splitting constant having a value of 14.3 G. The
unpaired electron further interacts with the nucleon of a-H (I =1/2) to split each of
the above lines in two, producing a total of 8 lines, the value of the splitting
constant being 11.7 G. Finally, in turn, 4 of these 8 lines, two pairs of two lines, are
split, to produce 8 lines, by the interaction of the unpaired electron with the
nucleon of 0-H (I = 1/2), the splitting constant being 1.26 G. The total number of
lines in the spectra, comprising 4 plus 8, is 12 (Figure 2-B).
With DMPO-OH the interaction of the unpaired electron with the N (I = 1)
nucleon results in the spectrum being split into three lines with a superfine splitting
constant of 14.9 G. When the unpaired electron progressively interacts with H
(I = 1/2), however, the constant of this second splitting is still 14.9 G resulting in the
overlap of two lines in the middle interval. The split spectrum thus has a strength
ratio of 1:2:2:1 (Figure 2-A).
Whether a spectrum was obtained corresponding to DMPO-OOH or to DMPOOH depended on the F- concentration or the volume of the F- solution when its
concentration was kept constant at 0.5 mM. With high concentrations of F- the
spectrum type of DMPO-OOH was present. This represents the spin adduct of
superoxide, • 02, and DMPO. With medium concentrations of F- the spectrum type
of DMPO-OH was present. This represents the spin adduct of hydroxyl radical,
• OH, and DMPO.
FIGURE 2. ESR spectra of computer simulation for hydroxyl radical adduct and
superoxide anion adduct.
A: DMPO-OH, aN = a H = 14.9 G.
B: DMPO-OOH, aN = 14.3 G, al3H = 11.7 G, aYH = 1.25 G.
C: Composite of DMPO-OH and DMPO-OOH with an intensity ratio of 1:2.
B
A ,____J
Fluoride
30 (1) 1997
10 Y Y Wang, B L Zhao, X J Li, Z Su and W J Xi
To clarify the origin of the active oxygen radicals, scavenger tests were performed. As shown in Figure 3 catalase decreases the DMPO-OOH signals from
PMN stimulated by 0.05 mM F-. Treatment with SOD results in only weak signals
being emitted from DMPO-OH. 7 With combined treatment by both catalase and
SOD the signals of active oxygen radicals are almost eliminated. The dominant
signals with trapping of the PMN-F- reaction system, when PMN are stimulated
with a high concentration of F-, originate from DMPO-OOH with only a minor
contribution resulting from the DMPO-OH formed in the dismutation of superoxide, • 0 2- . Thus superoxide, -0 2-, is the main species of active oxygen produced
from human PMN under the strong stimulation of F-.
The volume of 0.05 mM F- which stimulated the maximal production of
DMPO-OOH from the PMN was 10 uL as shown in Table 3.
TABLE 3. Height of the first peak measured and volume of 0.05 mM FVolume of 0.5 mM F- in uL
0
Height*
0
5
2.5 ± 0.18
10
15
4.4 ± 0.25
3.9 ± 0.37
*Mean height of first peak measured ± S.E. in gauss
FIGURE 3. Active oxygen radicals from human PMN stimulated with F- and scavenging
ESR spectra of oxygen radicals.
A: stimulated with 15 pL 0.5 mM F-.
B: treated further with 600 unit/mL catalase.
C: with 20 pg/mL SOD.
D: with 600 unit/mL catalase and 20 pg/mL SOD.
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Fluoride 30 (1) 1997
Spin trapping studies on F--stimulated human PMN 11
Oxygen consumption was measured by incubating PMN with CTPO, with and
without F-, at 37°C for 15 minutes (Figure 4). The ESR spectra in Figure 4-B,
without the addition of F-, are indicative of the hyperfine splitting of the nitrogen
nucleon caused from the widened interaction of the spin moment of paramagnetic
oxygen in the media with the spin moment of the CIPO nitroxide. With the
addition of F-, causing a respiratory burst in the PMN, the spectra altered with
splitting into more than 10 superhyperfine peaks with four methylhydrogen
photons contributing to the superhyperfine structure. The mechanism by which the
sirperhyperfine peaks developed involved F- stimulating a respiratory burst with
the production of active oxygen radicals and the consumption of oxygen leading to
a decrease in the oxygen concentration in the media and a weakening of the spinspin interaction of oxygen with the spin probe CTPO which can not penetrate the
PMN membrane. Thus the appearance of a superhyperfine structure in Figure 4-A
is seen to reflect the oxygen consumption in the intercellular medium during the
production of active oxygen radicals by PMN stimulated by F-.
FIGURE 4. ESR spectra of CTPO in 0 2 consumption during human PMN respiratory
burst (PMN, 10- 4M CTPO 14pL)
A: 0.05 mM F- 10pL
B: PBS 10pL
A
B
0.5 G
--->
Fluoride 30 (1) 1997
12 Y Y Wang, B L Zhao, X J Li, Z Su and W J Xi
Although these results indicate that intercellular oxygen is consumed when
PMN are stimulated with F-, the consumption of intracellular oxygen in this
process can not be excluded. This was examined by an experiment in which a
mixture of PMN, TEMPOL, F- and CrOX were incubated at 37°C, after prior
incubation of the PMN and TEMPOL for 30 minutes at 37 C to allow equilibrium
to be reached between the intracellular and extracellular TEMPOL concentrations. When the PMN were incubated with F-, CrOX and TEMPOL, the ESR
spectra were a straight line of TEMPOL with the transition element Cr 3+ producing widening with spin moment. TEMPOL can dissolve in both the intercellular
and intracellular media while CrOX can not penetrate into the cell and can only
widen the ESR spectra of TEMPOL in the intercellular media. In Figure 5-A are
shown overlap spectra contributed by TEMPOL outside the cell with the addition
of F- and showing a superhyperfine splitting structure from the hydrogen nucleon
of a methyl group. When the addition of F- was followed by CrOX the spectrum in
Figure 5-A widened due to the the action of the spin moment of the paramagnetic
CrOX with a change from superhyperfine structure to the hyperfine splitting
structure shown in Figure 5-B. These findings with TEMPOL and CrOX suggest
that the 02 consumed, when PMN release active oxygen radicals with F- stimulation, originates from the 0 2 in the intercellular medium.
FIGURE 5. ESR spectra of TEMPOL during human PMN respiratory burst
(PMN, 10-3M TEMPOL, 0.5 mM F-)
C: TEMPOL and CrOX
B: 50 mM CrOX
A: no CrOX
B
0.5 G
Fluoride 30 (1) 1997
Spin trapping studies on F--stimulated human PMN 13
CONCLUSIONS
Four results emerged from the experiments.
1. For human polymorphonuclear leukocytes, PMN, fluoride (F-) is a stimulus
which has the property of being able to induce a respiratory burst with the
formation of superoxide, • 02-, and the consumption of oxygen, 02.
2. Of the active oxygen radicals produced by fluoride acting on PMN, the
proportions of superoxide, • 02, and hydroxyl radical, • OH, are affected by the Fdose. With stimulation by a high F- concentration , the radical trapped by the
adducts is DMPO-OOH, while with stimulation by a low F- concentration the main
radical trapped by the adducts is DMPO-OH. As a transition in the F- concentration occurs from high to low, the spectrum gradually changes from the DMPOOOH adduct to that of DMPO-OH.
3. The volume of the F- solution also affects the strength of the signals of DMPOOOH trapped by the adducts when the PMN are being stimulated by a high
concentration of F-. Because of this volume-effect relationship, 10 !IL of 0.5 mM
fluoride solution was found to be the optimal volume for giving a maximal H value.
4. From experiments using the spin probe CI BO and TEMPOL together with the
widening agent CrOX, it was found that the 0 2 being consumed, when PMN were
stimulated by F- to produce active oxygen radicals, originated in the intercellular
medium, outside of the PMN, rather than in the intracellular medium.
Thus by using the spin trapping Electron Spin Resonance (ESR) technique it
has been possible to show the existence in human PMN of two experimental
findings previously reported. The first involved oxygen, 0 2, consumption in the
release of superoxide, • 02, and hydroxyl radical, • OH, from guinea pig PMN
stimulated by F". 6 The second involved the production of superoxide, • 02, when
human PMN were stimulated by F-. The oxygen consumed during the respiratory
burst stimulated by F- is seen to come from oxygen outside the cell. The
intracellular enzymes involved in the generation or scavenging of active oxygen
radicals are seen to be able to be activated by fluoride so that oxygen, 0 2, is
reduced to superoxide, • 02-, and then altered further to hydroxyl radical, • OH, by
dismutation and/or the Haber-Weiss reaction. The basic biochemical process
involves the activation of an enzyme by fluoride, F-, during the respiratory burst to
catalyse the one-electron reduction of oxygen, 0 2, to superoxide, 02, at the
expense of a pyridine nucleotide. Most of the superoxide, • 02-, is seen to react with
itself either spontaneously or catalysed by superoxide dismutase to generate
hydrogen peroxide, H202. In turn hydrogen peroxide, H202, and superoxide, • 02-,
are catalysed by ferrous ions, Fe2+ , in the Haber-Weiss reaction to generate
hydroxyl radical, • OH.9
Mainly DMPO-OOH is trapped from PMN when they are stimulated by a high
F- concentration and mainly DMPO-OH is trapped with stimulation by a low Fconcentration. The efficiency of trapping the hydroxyl radical, • OH, with DMPO is
10 6 times greater than that of superoxide, • 02. The production rate of superoxide,
• 02, from PMN with stimulation by a high concentration of F- is at least 10 6 greater
than that with a low F- concentration. Superoxide, • Of, is the primary free radical
Fluoride 30 (1) 1997
14 Y Y Wang, B L Zhao, X J Li, Z Su and W J Xi
with the hydroxy radical, OH, appearing secondarily. When active oxygen radicals
are generated during a respiratory burst, superoxide, • 02, appears first followed by
hydroxyl radical, • OH. When a high concentration of F- is placed initially in the
reaction system the dominant spectrum type in the ESR spectrum is DMPO-OOH.
With time, as the F- concentration decreases to a sufficiently low level, the dominant spectrum in the ESR becomes that of DMPO-OH.
By using catalase and superoxide dismutase, SOD, it was shown that various
active oxygen radicals released from PMN by F- were derived from superoxide,
•02. If the oxygen, 0 2 , used in the production of superoxide, • 02, came from the
medium inside the cell it should result in the consumption of the oxygen dissolved
in the intracellular medium. The present study showed however that the active
oxygen was generated outside the cell.
These results together with other data from the literature suggest three aspects
deserving of further study.
1. Fluoride (F-) in high concentration is able to stimulate PMN to produce superoxide, • 02, despite the presence of scavenger activity by catalase or superoxide
dismutase, SOD, which would should stop the buildup of significant levels of superoxide. The question thus arises as to whether fluoride in high dose has the property
of inhibiting intercellular SOD and thus obstructing the dismutation pathway.
2. Questions exist as to whether fluoride has a physiological role and at what level
it has toxic effects. The fluoride dose affects the level of production of superoxide,
•02, and hydroxyl radical, • OH. Debate may exist as to whether fluoride may be a
nutrient in very small amounts but it is accepted that it is an accumulative toxin in
larger quantities. Similarly , superoxide, • 02, and hydroxyl radical, • OH, may have
a useful protective function in limited quantities but produce toxicity when present
in larger quantities. Hydroxyl radical, • OH, has strong oxidation properties and
greater toxicity than the superoxide, • 02, from which it is derived. It is produced
immediately when PMN are exposed to fluoride (F-). Thus whether fluoride (F-)
acts as a nutrient or a toxin may relate, to some extent, to the levels of superoxide,
• Of, and hydroxyl radical, • OH, present.
3. A question arises as to whether, in addition to acting on PMN, fluoride is also
able to act on collagen. 1 ° Collagens are very sensitive to the action of the
superoxide radical." Fluoride (F-) itself is an effective binding agent. 1 -3 In
addition, as shown in the present study, fluoride, F-, can stimulate the cell acted on
to produce active oxygen. radicals. Thus fluoride could affect the structure and
function of collagen by acting directly on collagen and also on cells to produce
active oxygen radicals. The detailed mechanisms whereby fluoride might affect
collagen are not clear. Superoxide, • 02, and its active derivatives such as hydroxyl
radical, • OH, might damage the collagenoblast by affecting the synthesis of
collagen protein by reacting with 02, SO2, NON, or Si. 12 For example, hydroxyl
radical, .0H, acts as an initiating factor in the process in which proline, a main
component of collagen, is converted into N-glutamate.13
The study of these aspects may help to .clarify the behaviour of active oxygen
radicals produced by fluoridation, and in fluorosis and other fluoride-related
Fluoride 30 (1) 1997
Spin trapping studies on F--stimulated human PMN 15
diseases. As better understanding emerges of the effects of fluoride it may be
possible for new preventive measures to be taken to prevent fluoride-related illness
and for more effective treatments to be developed such as obstructing the reactions
at the stage of the generation of superoxide, • Of, by the the use of antioxidants.
These new perspectives may encourage more research on the biological effects of
fluoride on plant, animal and human life.
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10 Waldbott GL. Editorial: Fluoride's effect on collagen. Fluoride 12 111-113 1979.
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12 Shi XL, Dalai NS, Vallyathan V. ESR evidence for the hydroxyl radical formation in
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Fluoride Vol.30 No.1 1997. Published by the International Society for Fluoride Research
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