JOURNAL OFENDODONTICS
Copyright 0 2000 by The American Association of Endodontists
Characterization of Reactive Oxygen Species
Generated from the Mixture of NaClO and H,O,
Used as Root Canal lrrigants
Atsushi Shiozawa, DDS
Characterization of a mixed solution of sodium hy-
pochlorite (NaCIO) and hydrogen peroxide (H202),
which have been used as root canal irrigants, was
investigated using electron spin resonance spec-
troscopy combined with a spin-trapping technique.
In the solution, two kinds of reactive oxygen spe-
cies, the superoxide anion radical (02--) and the
hydroxyl radical (.OH), were detected. The pH
changes and dissolved oxygen values in the Na-
CIO-H202 reaction mixture were investigated. The
pH influenced the generation of 02-.and .OH, and
O2 was generated from H202. It is suggested that
root canal irrigation with NaClO and H202induces
biologicaleffects, such as tissue irritation (from the
chemical reactions of 02--and .OH) and physical
effects, such as O2 bubbling.
Sodium hypochlorite (NaClO) and hydrogen peroxide (H202) have
been widely used for root canal irrigation in endodontic therapy.
Tissue emphysema sometimes results from careless use of NaClO
and H,O, for root canal irrigation. Before using medicaments,
dentists need to have enough information and understanding about
them. It is well known that NaClO and hypochlorous acid (HClO)
are at equilibrium in water (1). Change in the equilibrium depends
on the pH. In an acid condition, NaClO easily changes to HC10.
HClO is strong oxidant and exhibits marked bactericidal activity.
H,O, is one of the active oxygens. The reaction of both chemicals
is postulated as follows:
NaClO + H202+NaCl + H 2 0 + 2.0..
-0-is the oxygen atom, which is reactive and changes to oxygen
(0,). However, the detailed reaction mechanism has not been
clarified yet.
Shiozawa et al. ( 2 ) detected the superoxide anion radical (O,-.)
and the hydroxyl radical (-OH) in the NaClO-H,O, reaction mix-
ture by using the electron spin resonance (ESR) spin-trapping
technique (3). This method is a powerful tool, which is used for
research into free radicals and some kinds of active oxygens.
Active oxygens, such as O,-., .OH, and H,O,, have been
11
Printed in U.S.A.
VOL. 26, No. 1. JANUARY 2000
studied by many investigators because they have a bactericidal
effect (4) and induce various abnormal biological phenomena, such
as inflammation (3,radiation damage (6), cell aging, and mutation
(7).
The detailed mechanisms of reactive oxygen species (ROS)
from the NaCIO-H,O, reaction mixture have not been reported
yet.
The aim of this study is to clarify more details in the interaction
mechanisms between NaClO and H,O,.
MATERIALS AND METHODS
Chemicals
The reagents-6% (8.4 X lo-' M) NaClO, 30% (10 M) H,O,,
and CuS0,-were obtained from Wako Pure Chemicals (Osaka,
Japan). 5,SDirnethyl- 1-pyrroline-N-oxide (DMPO; Dojin Chemi-
cals, Kumamoto, Japan) was used as a spin-trapping agent, and
4-hydroxyl-2,2,6,6,-tetramethylpiperidine-N-oxyl (TEMPO), used
as a standard for spin concentration, was obtained from Labotec
(Tokyo, Japan).
Measurements of ROS
ROS generated from the reaction between NaClO and H,O,,
Cuz+ and H,O, or NaClO were measured by the ESR spin-
trapping technique (3). The ESR spectrum was recorded with a
Radical Biosensor FR-80 (JEOL, Tokyo, Japan). Conditions for
ESR were: microwave power, 5 mW; magnetic field, 335.4 2 5
mT; sweep time, 2 min; field modulation, 0.1 kHz; and time
constant, 0.03 s.
Detection of ROS from NaClO-H,O, Reaction Mixture
ROS, identified as free radicals, are detected as spin adducts of
DMPO by the ESR spin-trapping technique. The reaction mixture
(200 pl) contained 1% (1.4 X lo-' M) NaClO, 3% (1.OM) H,O,,
and 89 X lop3 M DMPO as a spin-trapping agent.
After rapid stirring, the reaction mixture was placed into an ESR
flat cell. Recording of the ESR spectrum was started 40 s after the
addition of DMPO. The spin concentrations of ROS were deter-
12 Shiozawa Journal of Endodontics

mined by the method described by Kohno et al. (8) using the ESR
signal relative intensity of the DMPO adduct and TEMPO as the
known concentration to obtain the area of the spin adduct as the
spin concentration by double integration.

Relationship Between Generated ROS and pH

For examination of pH alteration, the pH of the NaCIO-H,O,

H
I.0mT

mc
kc A.
OH

reaction mixture was measured. The reaction mixture (200 pl)

contained 89 X l o p 3 M DMPO, 1.0 M H,O,, and various con-
centrations of NaClO in the presence or absence of 13 X M
phosphate buffer (p-buffer; pH 7.4). After these solutions were
1.0 mT
mixed, pH was measured with a pH meter 240 (Corning, New
York, NY). The generated free radicals and active oxygen species
m
H
JlH3C
H
c3
were also measured by the ESR spin-trapping technique, and spin
concentrations were calculated.

Quantitative Analyses of Dissolved Oxygen (DO) in the

NaClO-H,O, Mixture
H
1.0 mT
Bubbling over was observed in alternate irrigation with NaClO
and H,O,. For examination of 0, generation from various con-
centrations of H,O, and 1.0 X 10-3-l.0 X M NaClO
reaction mixtures, DO values were measured with a DO meter
"i(&
H3C
A-
(DO-25A; Toa Electrics, Tokyo, Japan).
FIG 1. ESR spectra and detected adducts of DMPO from the NaCIO-
H,O, reaction mixture and the structure of these DMPO adducts. (A)
.OH Generation Process from H,O, or NaClO DMPO-OHCOH), (€3) DMPO-O,-(O,-.), and (C) DMPO-X.

Usually, H,O, and metal ions have been used for the study of 9.0 1
*OH generation (9). In this experiment, the amounts of -OH gen-
erated from NaCIO-H,O, and NaClO-CuSO, reaction mixtures
were measured by the ESR spin-trapping technique. The reaction
E
a
. 0
mixtures (200 pl) contained various concentrations of NaClO and W

1.0 M H,O,, or 5.0 X M or 1.0 X lop3 M CuSO,. 8 6.0 - 0

Ti
RESULTS b 0

Detection of ROS Generated from the NaCIO-H,O, 0

Reaction Mixture g3.0 - 0
0 0
0
Typical ESR spectra of DMPO-0,- (O,-.), DMPO-OH (.OH) c
.d
0

and from the NaCIO-H,O, reaction mixture were detected with the a
r/3
. 0

ESR spin-trapping technique (Fig. 1). Hyperfine coupling con-

stants and the g-values of these signals were analyzed and con-
firmed as: 10-6 10-5 10-4 10-3 10-2 10-1

02-*:
aN= 14.3(mT), apH= 11.7(mT), g-value = 2.0064
*OH:aN= 14.9(mT), apH= 14.9(mT), g-value = 2.0050
DMPO-X: a, = 7.l(mT), apH= 4.2(mT), g-value = 2.0048.
These results agreed well with previously reported values (lo),
and the signals were confirmed as O,--, -OH, and DMPO-X. The
intensities of these spectra changed with the concentrations of
added NaCIO. When NaClO in concentrations from 1.4 X to
1.4 X lo-' M was added to 1.O M H,O,, both -OH and O,-* were
detected, and the amounts of both increased in a concentration-
dependent manner (Fig. 2). The amounts of generated -OH detected
were higher than those of O,-. when concentrations of up to 7.0 X
lop3 M NaClO were used. On the other hand, mostly DMPO-X
NaClO (M)
FIG 2. Generation of O,-. and .OH from the NaCIO-H,O, reaction
mixture. Amounts are dependent on the concentration of NaCIO. (0)
O,-*, (0)*OH.
was detected when various concentrations (1.0 X lo-" M to 1.0 X
10-'M) of H,O, were added to 1.4 X lo-' M (1.0%)NaClO (data
not shown).
Relationship Between Generated ROS and pH
In the absence of the p-buffer in the reaction mixture, pH was
stable at 6.4 when the concentration of NaClO was <1.4 X lop3
Vol. 26, No. 1, January 2000 Reactive Oxygen during Canal Irrigation 13

PH 9

8 -
0
0
1 z
w 0.6

0
( 00 00 00 00 oo

-- I
-
------------____ -
I I
w

DO value of pure water

0.2 ,
10-6 10-5 104 10-3 10-2 10-1 10“ 10-3 10-2 10-1 1.o
NaClO (M)
H20, (a)
FIG3. Alteration of pH on the NaCIO-H,O, reaction mixture with or
FIG5. Effect of DO on the H,O,-NaCIO reaction. (0)1 X M
without pH 7.4 p-buffer. (0)Presence of p-buffer, (0)absence of
NaCIO, (m) 1.O x 1 O-, M NaCIO, (0)1.O x 1 0-5 M NaCIO.
p-buffer.

E
9.0

- 0.7

0.6
0.5

W
a x
0.4
c
0
6.0
2 0.3
+J
.r(

ce
+- 0.2
“0
& (/3
0 0.25 0.5 0.75 1.0 1.25 1.5
c (/3

5 NaClO (mM)
8 FIG6. Effect of DO in NaClO and 1.0 x M H,O,.
3.0
0
c
.3
a M NaClO, but was not detected in the mixture without p-buffer.
m More O,-. was generated from the mixture without p-buffer than
with p-buffer in 1.4 X lo-’ M and 1.4 X lo-’ M NaClO.

1.4X10-1 1.4X10-I 1.4X10-1 1.4X10-1 1.4X10-1 1.4X10-’

NaClO (M)
FIG4. Effect of pH 7.4 p-buffer on O,-. and .OH generated by the
NaCIO-H,O, reaction mixture. (B).OH values,
presence of p-buffer, (0) 0,-- values, 0 O,-. values in the pres-
ence of p-buffer.
M. However, a remarkable increase in pH was observed when
NaClO in concentrations up to 1.4 X M were added to 1.O M
H,O, (Fig. 3). On the other hand, pH was not changed with the
concentration of NaClO in the presence of p-buffer (pH 7.4) in
these reaction mixtures.
The quality and quantity of generated ROS were examined in
various concentrations of NaClO and 1.0 M H,O, reaction mix-
tures in the presence and absence of p-buffer (Fig. 4).When 1.4 X
M NaClO was added to 1.O M H,O,, more .OH was generated
than from the mixture with p-buffer; however, inverse amounts of
.OH were generated in 1.4 X lo-’ M and 1.4 X lo-’ M NaC10.
O,-- was generated from the mixture with p-buffer in 1.4 X
Quantitative Analyses of DO in the NaCIO-H,O, Reaction
Mixture
The amounts of DO in the NaClO-H,O, reaction mixtures are
shown in Fig. 5 . The DO value increased with the addition of
various concentrations of H,O, to 1.O X M NaClO. However,
DO values almost plateaued when 1.0 X lo-, M and 1.0 X
M NaClO were used. On the other hand, when various concentra-
tions of NaClO were added to 1.0 X lop3M H,O,, the DO value
increased in NaClO concentration (Fig. 6).
.OH Generation Process from H,O, or NaClO
As shown in Fig. 7, *OHwas detected in both NaClO-H,O, and
NaClO-CuSO, reaction mixtures. However, more *OH was gen-
erated from the NaClO-CuSO, than the NaCIO-H,O, reaction
mixture. In this case, the CuSO, concentration used in the reaction
was lower than the H,O, concentration.
14 Shiozawa
30 1 I
- I
E
0
104 10-5 104 10-3 10-2 10-1
NaClO (M)
FIG 7. Amounts of .OH generated in the NaCIO-H,O, and the
NaCIO-CuSO, reaction mixtures. Amounts are dependent on the
concentration of NaCIO. (0)1.O x M CuSO,, (B)5.0 x 1O-, M
CuSO,, (A) 1.O M HO, .,
DISCUSSION
In endodontic therapy, a combination of NaClO and H,O,
solutions has been used for root canal irrigation. Their interaction
in the canal produces a transient but energetic effervescence that
mechanically forces out debris and microorganisms (1 1). The
interaction mechanism of these solutions has been described as:
NaClO + H202+NaCl + H 2 0 + 02.
The first observation of the ROS and DMPO-X generated from the
NaClO-H,O, reaction mixtures was reported by Shiozawa et al.
(2). In the current study, more detailed mechanisms of the NaClO-
H,O, reaction system were investigated. First, generation of O,-*,
*OH, and DMPO-X from the NaCIO-H,O, reaction system were
confirmed, as previously reported (2). The amounts of generated
O,-- and -OH were changed by the addition of various concentra-
tions of NaClO to 1.0 M H,O,. Oxidation and reduction reactions
occurred in the NaClO-H,O, reaction system. When various con-
centrations of H,O, were added to 1.4 X lo-' M NaClO, mostly
DMPO-X was detected and the mixture could not be analyzed for
O,-* and -OH. Bernofsky et al. reported that DMPO-X was gen-
erated from a secondary oxidation-reduction process in the inter-
action between strong oxide substances and DMPO (12, 13).
Therefore, this result suggested that strong oxide substances, such
as C 1 and ClO., existed on DMPO spin adducts in the NaClO-
H,O, reaction mixture.
Usually, NaClO is used as the final irrigant in root canal im-
gation. Because of pressure, H,O, must always be neutralized by
NaClO and not sealed in the canal. Measurement of pH has an
important role in the study of generation of ROS. In this experi-
ment, pH in various concentrations of NaClO and 1.0 M H,O,
mixtures was measured for examination of the relationship be-
tween the amount of ROS and pH in the NaClO-H,O, reaction
mixture (Figs. 3 and 4).
The range of pH was changed by the addition of various con-
centrations of NaClO to H,O, (Fig. 3). When NaClO at low
concentrations was added to H,O,, pH plateaued at 6.4 (Fig. 3),
Journal of Endodontics
and O,-* and *OHwere detected in almost the same amounts (Fig.
2). When high concentrations of NaClO were added to H,O,, pH
increased to the alkaline range (Fig. 3). When pH was adjusted by
the addition of p-buffer, pH was not changed with the concentra-
tion of NaClO in the reaction mixture (Fig. 3). Higher 0,-* values
were detected in the reaction mixture with high concentrations of
NaClO without pH adjustment than in the mixture with pH ad-
justed; however, the reverse was observed in mixtures with low
concentrations of NaClO. .OH was detected in mixtures with high
concentrations of NaClO with pH adjusted than in mixtures with-
out pH adjusted; however, the reverse was observed with low
concentrations of NaClO (Fig. 4).
It is well known that 0,-- changes to H,O, by spontaneous
dismutation, and second-order rate constants are 7.6 X 105/M/sin
the acidic range, 8.5 X 107/M/s in the neutral range and 1.0 X
102/M/s in the alkaline range (14, 15). Therefore, as more O,-.
changed to H,O, by spontaneous dismutation in the neutral and
acidic ranges, less O,-- was detected than in the alkaline range.
Conversely, much .OH was detected in neutral and alkaline mix-
tures. The reason for these phenomena is thought to be that the
Haber-Weiss reaction (O,-. + H,O, +-OH + OH- +0,) might
occur (16) in these conditions with surplus H,O, and finally the
total amount of .OH would be increased. Usually, NaClO is used
for final irrigation to neutralize H,O,. Using equal amounts of
NaClO and H,O, inhibits the antibacterial action of the irrigants
(17). The results of this study (Fig. 4) showed that more O,-. and
-OH were detected in the neutral and alkaline ranges in the NaClO-
H,O, reaction mixture than in the acidic range. It is well known
that .OH has a strong antibacterial effect, as well as being a tissue
irritant. It has been reported that .OH has a stronger antibacterial
effect on Streptococcus species and Staphylococcus aureus than
H,O, alone (4, 18). Therefore, these results suggest that the Na-
ClO-H,O, reaction mixture exerts antibacterial effects by gener-
ating .OH. The detailed mechanisms are yet to be studied.
It is well known that -OH is also generated from the C U ~ ~ - H , O ,
reaction system (19). In this experiment, -OH generation from
NaClO-CuSO, reaction mixtures was investigated. The amounts
of generated .OH were more than those from the NaClO-H,O,
reaction mixtures (Fig. 7). It is considered that the -OH generated
from NaClO-CuSO, was caused by HClO originating from Na-
C10. The process is the decomposition of HClO to .OH + C 1 by
metal ions. It is also considered that some *OH was derived from
the decomposition of NaClO by H,O, in the NaClO-H,O, reaction
mixture. However, it is not possible to confirm this reaction pro-
cess, because a reliable measurement method has not been estab-
lished at this point.
A possible mechanism of O,-* and .OH generation from the
NaClO-H,O, reaction mixture can be postulated as follows (Fig.
8):
(I) HClO (NaClO) was decomposed to *OH + *C1 (or C1-) by
HZO,.
(11) H,O, was decomposed to .OH + .OH by HClO.
(111) generated O,-. acted on H,O,, and -OH was generated by the
Haber-Weiss reaction.
(IV) HClO extracted hydrogen ions from H,O, and this generated
*OOH (O,--).
These reactions were thought to change, depending on the
concentrations of the reaction mixtures and pH.
Vol. 26, No. 1, January 2000
f. OH)
\/
FIG 8. Speculated scheme for generation of ROS from the NaCIO-
, , reaction mixture. (I) HClO (NaCIO) is decomposed to .OH + -CI
HO
, , was decomposed to .OH + .OH by HCIO.
(or CI-) by H,O,. (11) HO
(Ill) Generated O,-. acts on HO ,, and .OH is generated by the
Haber-Weiss reaction. (Iv) HClO extracts hydrogen ion from HO,,
and then .OOH (O,-.) is generated. (V) HO ,, is decomposed by
NaCIO, and then 0, is generated.
To investigate the amounts of 0, generated from the interaction
of NaClO and H20,, it is helpful to measure DO, because gener-
ated 0, will be dissolved in the solution rapidly. DO values were
increased in various concentrations of H,O, added to 1.0 X
M NaClO. As shown in Fig. 6, DO values were not increased in
low concentrations of NaCIO, which could not decompose H,O,.
To investigate the concentration of NaClO required to decompose
H,O,, various concentrations of NaClO were added to 1.0 X lop3
M H,O, (Fig. 7). Up to 2.5 X lop4 M NaClO was required to
decompose 1.O X lop3M H,O,. These results also suggest that if
H,O, is decomposed by NaCIO, 0, generation will occur [Fig. 8
(V)]. In endodontic therapy, NaClO and H,O, solution are used
alternately for root canal irrigation. When H,O, is added to NaClO
in the root canal, bubbling over is observed. At this time, the
concentration of NaClO will be low. When NaClO is continuously
added to the first mixture, bubbling over is observed again. This
phenomenon can be explained thus: a high concentration of NaClO
can decompose H,O,, with 0, bubbles occurring, and a low
concentration of NaClO cannot decompose H,O,.
Grossman et al. (1 1) showed that the advantages of the NaCIO-
H,O, reaction mixture were: (a) the effervescent reaction, in which
the mixture mechanically bubbles and pushes debris out of the root
canal through the least-resistant orifice into the chamber; (b) the
solvent action of the NaClO on the organic debris of the pulp
tissue; and (c) the disinfecting and bleaching action of both solu-
tions (1 1). When NaClO or H,O, are used for imgation, irritation
is a problem. The fear of toxicity of NaClO as an irritant of
periapical tissue has tended to discourage its use, also H,O, gen-
erates 0, with a catalase reaction that might cause periapical
discomfort. The study suggests that distilled water or pure water
should be used as an imgant to eliminate O,-., -OH, NaCIO, and
H,O, in the canal.
In this study, O,-. and .OH generation and 0, generation by the
NaClO-H,O, reaction mixture were confirmed, and possible
causes were speculated upon. However, further details of the
Reactive Oxygen during Canal Irrigation 15
interaction mechanisms of these solutions remain unclear, and
further studies in this area are currently under way in our labora-
tory.
I gratefully acknowledge the help of Dr. M. Kohno (JEOL, Japan) in ESR
work, and Dr. Y. Tsujimoto and Professor M. Yamazaki for critical reading of
the paper and helpful comments.
Dr. Shiozawa is affiliated with the Department of Endodontics, Nihon
University School of Dentistry at Matsudo, Matsudo, Japan. Address requests
for reprints to Dr. Atsushi Shiozawa, Department of Endodontics. Nihon
University School of Dentistry at Matsudo, 870-1, Sakaecho, Nishi-2, Mat-
sudo, Chiba 271-8587, Japan.
References
1. Hostynek JJ, Wilhelm KP, Cua AB, Maibach HI. Irritation factors of
sodium hypochlorite solutions in human skin. Contact Dermatitis 1990;23:
316-24.
2. Shiozawa A, Kimura K, Hashizume H, Tsujimoto Y, Yamazaki M. Gen-
eration of active oxygen species by NaOCI-H,O, reaction system. Jpn J
Conserv Dent 1996;39:1171-7.
3. Ueta H, Sugiyama M, Kagiyama A, Shin G, Shoji T, Mimori S, Ogura R,
Kohno M. Studies on the quantitative and qualitative characterization of
superoxide anion using spin trapping methods by electron spin resonance
spectrometry. Kurume Med Assoc 1988;51:1032-8.
4. Hashizume H, Kimura K, Tomita T, Matsushima K, Tsujimoto Y,
Yamazaki M. Effect of sterilization of Streptococcus species by the hydroxyl
radicals. Jpn J Consetv Dent 1994;37:1254-60.
5. McCord JM. Free radicals and inflammation: protection of synovial fluid
by superoxide dismutase. Science 1974;185:529-31.
6. Hodgkins PS, Fairman MP, O'Neill P. Rejoining of gamma-radiation-
induced single-strand breaks in plasmid DNA by human cell extracts: depen-
dence on the concentrationof the hydroxyl radical scavenger, Tris. Radiat Res
1996;145:1:24-30.
7. Harman D. Role of free radicals in mutation, cancer, aging and the
maintenance of life. Radiat Res 1962;16:753-63.
8. Kohno M, Mizuta Y, Kusai M, Masumizu T, Makino K. Measurements of
superoxide anion radical and superoxide anion scavenging activity by elec-
tron spin resonance spectroscopy coupled with DMPO spin trapping. Bull
Chem SOCJpn 1994;67:1085-90.
9. Graf E, Mahoney JR. Bryant RG, Eaton JW. Iron-catalyzed hydroxyl
radical formation. Stringent requirement for free iron coordination site. J Biol
Chem 1984;259:6:3620-4.
10. Buettner GR. Spin trapping: ESR parameters of spin adducts. Free
Radic Biol Med 1987;5:259-303.
11. Grossman LI, Oliet S, DelRio CE. Endodontic practice. 1l t h ed. Phil-
adelphia: Lea & Febiger, 1988:188-189.
12. Bernofsky C, Bandara BMR, Hinojosa 0. Electron spin resonance
studies of the reaction of hypochlorite with 5,5,-dimethyl-l -pyrroline-N-oxide.
Free Radic Biol Med 1990;8:231-9.
13. Bernofsky C , Olavesen EY, Felix CC, Kalyanaraman B. Evidence for a
hydroxyl radical from hypochlorite-modified adenosine 5'-monophosphate
(AMP). Biochem Arch 1988;4:103-7.
14. Ohnishi T, Hirata F, Hayashi 0. lndoleamine 2,3-dioxygenase. Potas-
sium superoxide as substrate. J Biol Chem 1977;252:4643-7.
15. Taniguchi T, Hirata F, Hayashi 0. lntracellular utilization of superoxide
anion by indoleamine 2,3-dioxygenase of rabbit enterocytes. J Biol Chem
1977;252:2774-6.
16. Haber F, Weiss J. The catalytic decomposition of hydrogen peroxide
by iron salts. Proc R SOCLond Ser A Math Phys Sci 1934;147:332-51.
17. Harrison JW, Hand RE. The effect of dilution and organic matter on the
antibacterial property of 5.25% sodium hypochlorite. J Endodon 1981;7:128-
32.
18. Yamamoto H, Kimura K, Tsujimoto Y, Yamazaki M. The bactericidal
effect of hydroxyl radical generated from CUCI,-H,O, mixed solution on
Staphylococcus aureus. Jpn J Conserv Dent 1997;40:1247-54.
19. Seacat AM, Kuppusamy P, Zweier JL, Yager JD. ESR identificationof
free radicals formed from the oxidation of catechol estrogens by Cu". Arch
Biochem Biophys 1997;347:45-52.