INFECTION AND IMMUNITY, June 1984, p. 581-586 Vol. 44, No.

3
0019-9567/84/060581-06$02.00/0
Copyright © 1984, American Society for Microbiology

Bactericidal and Cytotoxic Effects of Hypothiocyanite-Hydrogen

Peroxide Mixtures
JAN CARLSSON,'* MAY-BRITT K. EDLUND1, AND LENNART HANSTROM2
Departments of Oral Microbiology1 and Periodontology,2 University of Umea, S-90187 Umea, Sweden
Received 14 December 1983/Accepted 24 February 1984

Lactoperoxidase catalyzes the oxidation of thiocyanate by hydrogen peroxide into hypothiocyanite, a

reaction which can protect bacterial and mammalian cells from killing by hydrogen peroxide. The present
study demonstrates, however, that lactoperoxidase in the presence of thiocyanate can actually potentiate
the bactericidal and cytotoxic effects of hydrogen peroxide under specific conditions, such as when
hydrogen peroxide is present in the reaction mixtures in excess of thiocyanate. The toxic agent was also

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formed in the absence of lactoperoxidase in a reaction between hypothiocyanite and hydrogen peroxide.
Sulfate, sulfite, cyanate, carbonate, and ammonia, which have been postulated to be formed in the chemical
oxidation of hypothiocyanite by hydrogen peroxide, were not bactericidal and did not potentiate the
bactericidal effect of hydrogen peroxide. Cyanosulfurous acid, the only other postulated product of the
chemical oxidation of hypothiocyanite by hydrogen peroxide, may be the killing agent.

Lactoperoxidase catalyses the oxidation of thiocyanate by
hydrogen peroxide into hypothiocyanite (OSCN-) (4, 19).
Lactoperoxidase and thiocyanate are components of the
human salivary secretions (25), and hydrogen peroxide is
excreted by oral bacteria (20). The OSCN- ion inhibits
bacterial glyceraldehyde 3-P dehydrogenases (11, 24) and
thereby stops the bacterial production of acids from sugars.
The inhibition of bacterial acid production by OSCN- has
been implicated as playing an important role in the preven-
tion of dental caries (17).
Hydrogen peroxide can be highly toxic to mammalian cells
(3, 13, 14) and bacteria (2, 10). This effect of hydrogen
peroxide is alleviated, however, in the presence of lactoper-
oxidase and thiocyanate (2, 8, 13). It has therefore been
suggested that lactoperoxidase and thiocyanate of the sali-
vary secretions play an important role in protecting the
epithelial cells of the oral mucous membranes against the
hydrogen peroxide excreted by bacteria colonizing the oral
surfaces (2, 13). In this role, lactoperoxidase in the presence
of thiocyanate detoxifies hydrogen peroxide by converting it
into OSCN-, and OSCN- prevents bacteria from excreting
hydrogen peroxide by inhibiting glyceraldehyde 3-P dehy-
drogenase. Because of this inhibition, no NADH is generat-
ed in the bacteria, and the hydrogen peroxide-producing
NADH oxidases become short of their substrate, NADH
(11). This inhibition of glycolysis usually has a bacteriostatic
effect.
In recent studies, significant levels of OSCN- have been
found in saliva collected directly from the ducts of the
salivary glands (22, 29). This indicates that hydrogen perox-
ide is actually produced within the salivary glands; thus,
lactoperoxidase and thiocyanate may also play an important
role in protecting the salivary glands and ducts against
hydrogen peroxide toxicity.
The products of the lactoperoxidase-thiocyanate-hydro-
gen peroxide reaction have also been reported to be bacteri-
cidal (6, 7, 33, 36, 37). This effect has been ascribed to
OSCN-, but it has also been suggested that higher oxyacids
*
Corresponding author.
581
of the thiocyanate ion, cyanosulfurous and cyanosulfuric
acids, may be formed in the lactoperoxidase reaction, and
these acids may be the effective molecular species in the
killing (6, 15, 31).
We now report that a mixture of OSCN- and hydrogen
peroxide in the absence of lactoperoxidase was more bacte-
ricidal and more toxic to mammalian cells than was hydro-
gen peroxide alone.
MATERIALS AND METHODS
Microorganisms. Peptostreptococcus anaerobius VPI
4330 (ATCC 27337) was kept on blood agar plates, and
Escherichia coli K-12 K37, an Str/r mutant strain of W 3102
(5), was kept on a minimal agar medium at 4°C in an
anaerobic box with an atmosphere of 10% hydrogen in
nitrogen.
Mammalian cells. HeLa cells were kindly provided by E.
Lundgren, University of Umea, Umea, Sweden. The cells
were cultured in an atmosphere of 5% carbon dioxide in air
in Eagle minimal essential medium containing 10% fetal calf
serum, penicillin (60 mg liter-1 streptomycin (100 mg
liter-'), and gentamicin (200 mg liter-').
Media. Peptone-yeast extract-glucose broth was prepared
as described by Holdeman et al. (16), but instead of being
prereduced and saturated with carbon dioxide before being
autoclaved, the broth was autoclaved and then stored in the
anaerobic box for at least 2 days before it was used. The
MOPS (3-N-morpholino propanesulfonic acid) buffer solu-
tion was a modification of a medium devised by Neidhardt et
al. (26) and contained 40 mM MOPS, 4 mM tricine, 0.3 mM
potassium sulfate, 0.4 ,uM calcium chloride, 0.5 mM magne-
sium chloride, and 50 mM sodium chloride. This solution
was adjusted to pH 7.4 by KOH and sterilized by filtration.
The phosphate-buffered saline contained 137 mM sodium
chloride, 2.7 mM potassium chloride, 1.5 mM monopotas-
sium phosphate, and 7.7 mM disodium phosphate (pH 7.3).
A minimal medium contained the MOPS buffer solution, 20
mM glucose, 10 mM ammonium bicarbonate, 1 mM dipotas-
sium phosphate, and 0.02 mM ferric chloride. The minimal
agar medium was supplemented with 16 g of agar (Difco
Laboratories, Detroit, Mich.) per liter. Blood agar medium
was prepared as described by Holdeman et al. (16). The
582 CARLSSON, EDLUND, AND HANSTROM
defibrinated horse blood in the medium was hemolyzed by
freeze-thawing.
Evaluation of cytotoxicity and bactericidal effects. The
mammalian cells were cultured in 25-cm2 plastic culture
flasks until the growth was confluent. They were detached
with 0.1% trypsin-0.2% EDTA in phosphate-buffered saline,
washed in phosphate-buffered saline, and suspended in the
culture medium; portions of the suspension were added to
the culture medium of 25-cm2 culture flasks to obtain a final
volume of 5 ml and a cell density of 100 cells per cm2. The
number of cells attached to the bottom surface of the flask
was counted with an inverted phase-contrast microscope
supplied with a grid with squares (1 by 1 mm). After 4 h, ca.
70% of the cells had attached to the bottom surface. After 20
h, the cells were counted, the medium was decanted, and 5
ml of phosphate-buffered saline (at 22°C) containing various
combinations of hydrogen peroxide, thiocyanate, lactoper-
oxidase, and a lactoperoxidase-free OSCN- preparation was
added. The solution was decanted after 30 min, and the cells
were exposed to fresh test solution for another 30 min. The
test solution was then replaced by culture medium. The
numbers of cells attached to the bottom surface of the flasks
were counted after 2 and 3 days. Triplicate flasks were run in
all experiments.
P. anaerobius was grown at 37°C in peptone-yeast extract-
glucose broth, and E. coli was grown in the minimal medium
under anaerobic conditions. When the cultures were in
exponential growth phase and had a density of 108 cells
ml-,, they were diluted in MOPS buffer solution or in
phosphate-buffered saline to a density of ca. 1 x 104 to 1.5 x
104 cells ml-'. Within 2 min after the start of the dilution
procedure, a 0.2-ml sample of this suspension was added to
0.8 ml of a reaction mixture containing MOPS buffer solution
or phosphate-buffered saline with various additions. In most
experiments hydrogen peroxide was added to the reaction
mixture after the bacteria. Samples (0.1 ml) were taken at
various times from the reaction mixtures and spread over the
surface of duplicate blood agar plates. The plates were
incubated for at least 2 days under anaerobic conditions, and
the numbers of surviving organisms were determined.
Preparation of lactoperoxidase-free OSCN- solution. Each
stirred 10-ml ultrafiltration cell (Amicon Corp., Lexington,
Mass.) fitted with a Diaflo membrane (PM 30) contained 9 ml
of MOPS buffer solution or phosphate-buffered saline, 0.1 or
1 mM KSCN and lactoperoxidase (10 jig ml-'). Hydrogen
peroxide was added to this reaction mixture to give a final
concentration of 0.1 or 1 mM, and 5 min after this addition
the solution was filtered. The concentration of OSCN- in
the filtered solution was ca. 40 or 400 jiM, respectively, as
determined with the 2-nitro-5-thiobenzoic acid reagent. This
reagent was prepared as previously described (11), and the
concentration of 2-nitro-5-thiobenzoic acid was calculated
assuming an extinction coefficient of 14,130 M-1cm-1 at 412
nm (34). Before the solution containing OSCN- was used in
the reaction mixture, it was passed through a 0.22-p.m filter
(Millipore Corp., Bedford, Mass.).
Chemicals. Hydrogen peroxide (30%[wt/wt]; Perhydrol)
was from E. Merck AG, Darmstadt, Federal Republic of
Germany; lactoperoxidase (from milk), tricine, and MOPS
were from Sigma Chemical Co., St. Louis, Mo.; potassium
thiocyanate was from Riedel De Haen AG, Seele-Hannover,
Federal Republic of Germany; streptomycin sulfate was
from Glaxo Laboratories Ltd., England; benzyl penicillin
sodium salt were from Astra, Sodertalje, Sweden; gentami-
cin was from Flow Laboratories, Svenska AB, Stockholm,
Sweden; and 1,4-diaza-bicyclo(2.2.2)octane (DABCO) was
INFECT. IMMUN.
from EGA-Chemie Gesellschaft mbH & Co., Steinheim,
Federal Republic of Germany.
RESULTS
Hydrogen peroxide (80 ,uM) killed 90% of the cells of P.
anaerobius within ca. 40 min (Fig. 1, line 2). Lactoperoxi-
dase in the presence of 100 ,uM thiocyanate protected the
cells from the bactericidal effect of hydrogen peroxide (Fig.
1, line 3), whereas lactoperoxidase potentiated the bacteri-
cidal effect of hydrogen peroxide in the absence of thiocya-
nate (Fig. 1, line 4).
In a mixture of lactoperoxidase, 100 ,uM KSCN, and 80
,uM hydrogen peroxide in MOPS buffer solution, ca. 40 ,uM
OSCN- was formed. P. anaerobius survived in this solution
after lactoperoxidase had been removed from it (Fig. 1, line
5). When P. anaerobius was stored in this lactoperoxidase-
free solution containing 32 ,uM OSCN- for 5 min and then
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exposed to 80 ,uM hydrogen peroxide, the toxicity of hydro-
gen peroxide was highly potentiated (Fig. 1, line 6).
When cells of P. anaerobius were exposed to 80 puM
hydrogen peroxide in the presence of lactoperoxidase and
various concentrations of KSCN, only concentrations higher
than 80 jiM KSCN fully protected the cells from killing by
1.0 5
3
1
z
0
u
- .1
Lc
0
cc
z
CO)
2
.01
0 20 40 60
TIME (min)
FIG. 1. Killing of P. anaerobius after exposure to various combi-
nations of lactoperoxidase-thiocyanate-hydrogen peroxide, showing
the surviving fraction of cells, under the following conditions: after
storage for 1 h in MOPS buffer solution (line 1); in the presence of 80
,uM hydrogen peroxide (line 2); in the presence of lactoperoxidase
(10 ,ug ml-'), 80 F.M hydrogen peroxide, and 0.1 mM KSCN (line 3);
in the presence of lactoperoxidase (10 jig ml-') and 80 ,uM hydrogen
peroxide (line 4); after storage for 1 h in a 32 I.M lactoperoxidase-
free OSCN- preparation (line 5); after storage for 5 min in this
preparation and subsequent exposure at time zero to 80 ,uM hydro-
gen peroxide (line 6). The dotted line indicates the level at which the
surviving fraction was 0.1, i.e., 90% of the cells were killed.
VOL. 44, 1984 TOXICITY OF HYPOTHIOCYANITE-HYDROGEN PEROXIDE 583

hydrogen peroxide (Fig. 2). At lower concentrations of TABLE 1. Killing of P. anaerobius by hydrogen peroxide in the
KSCN, the initial killing rates were higher than in the presence of a lactoperoxidase-free OSCN- preparationa
absence of KSCN. At 20 or 40 ,uM KSCN, however, the
killing stopped after a few minutes, and a significant fraction OSCN- Hydrogen Killing time
(p.M) peroxide (min)
of the cells survived (Fig. 2). (p.M)
When the cells of P. anaerobius were stored in a lactoper- 32 80 2.1 0.5
oxidase-free solution containing various concentrations of 32 40 6.1 ± 3.2
OSCN-, as little as 8 ,uM OSCN- potentiated the bactericid- 32 20 10.1 ± 3.1
al effect of 80 p.M hydrogen peroxide (Table 1). In the 32 10 38.2 ± 9.2
presence of 32 ,uM lactoperoxidase-free OSCN-, 10 ,uM 24 80 3.3 ± 2.5
hydrogen peroxide had a bactericidal effect similar to that of 16 80 3.6 t 3.0
80 ,uM hydrogen peroxide alone (Table 1). When the lacto- 8 80 8.6 ± 0.6
peroxidase-free OSCN- solution was mixed with hydrogen 4 80 39.1 ± 5.5
0 80 36.1 ± 2.0
peroxide and the mixture was stored for various times before
cells of P. anaerobius were added to it, the bactericidal a Cells were stored for 5 min in 0.9 ml of MOPS buffer solution
effect of the mixture remained high, even after a 60-min containing various concentrations of OSCN- before various
storage, although the amount of OSCN- detected in the amounts of hydrogen peroxide (0.1 ml) were added. The killing time

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reaction mixture at that time was very low (Table 2). was defined as that time after the hydrogen peroxide addition at
which the surviving fraction of cells was 0.1. Means ± standard
Sulfate, sulfite, cyanate, cyanide, carbonate, and ammo- deviations are given for three independent experiments. The killing
nia have been postulated as products of the chemical or the time was determined in experiments similar to those described in the
enzymic oxidation of thiocyanate by hydrogen peroxide (12, legend to Fig. 1. The killing time was defined by the intersection of
15, 27, 38). These substances were not toxic to P. anaero- the dotted line in Fig. 1 with the killing curve of the organism.
bius at a concentration of 0.1 mM, and they did not potenti-
ate the bactericidal effect of 80 ,uM hydrogen peroxide (data
not shown). Cyanosulfurous and cyanosulfuric acids have (data not shown) but not by superoxide dismutase, methio-
also been postulated as products of these oxidations (6, 15, nine, tryptophan, histidine, or mannitol (Table 3). Reduced
30). Since there are no reliable assays for these substances, it glutathione and 2-mercaptoethanol did not influence the
was not possible to evaluate their possible role in the killing bactericidal effect of 80 ,uM hydrogen peroxide, but they
of the cells. eliminated the potentiating effect of OSCN- on hydrogen
The bactericidal effect of a mixture of OSCN--hydrogen peroxide toxicity (Table 3). DABCO significantly decreased
peroxide was abolished by catalase and by lactoperoxidase the toxicity of the OSCN--hydrogen peroxide mixture (Ta-
ble 3). This effect of DABCO would suggest that singlet
oxygen mediated the bactericidal effect of the mixture. No
KSCN
oxygen could, however, be detected with an oxygen elec-
1.0 trode when OSCN- in concentrations up to 400 ,uM was
mixed with 1 mM hydrogen peroxide. The concentration of
0*
-------
80VM OSCN- decreased 7 ± 2% during a 1-h storage in MOPS
buffer solution. DABCO did not influence the rate of
-- * OSCN- breakdown or the stability of hydrogen peroxide in
40VM MOPS buffer solution (data not shown). The bactericidal
effects of OSCN--hydrogen peroxide mixtures were similar
in MOPS buffer solution and phosphate-buffered saline (data
2OPM not shown). E. coli was much more resistant to hydrogen
peroxide than was P. anaerobius, but OSCN- also potenti-
z ated the bactericidal effect of hydrogen peroxide to this
0.1 organism (Fig. 3).
When HeLa cells were exposed to 30 ,uM hydrogen
peroxide or to a mixture of 30 ,uM hydrogen peroxide and 20
U-
LL
,uM of lactoperoxidase-free OSCN- in phosphate-buffered
z saline for 1 h, their capacity to proliferate was reduced
5: OPM significantly more by the OSCN--hydrogen peroxide mix-
cc
co
ture than by hydrogen peroxide alone (Fig. 4).
3 DISCUSSION
1OpM
Lactoperoxidase in the presence of thiocyanate could
5pM protect bacteria (2, 8) and cultured mammalian cells (13)
from killing by hydrogen peroxide. There are, however, also
.01

several reports in which lactoperoxidase in the presence of

thiocyanate has potentiated the bactericidal effect of hydro-
gen peroxide (6, 7, 21, 23, 33, 36).
I I I I I
The effect of lactoperoxidase-thiocyanate-hydrogen per-
0 20 40 6( oxide mixtures on bacteria is dependent on the experimental
TIME (min) conditions. If the bacteria are cultured after the exposure to
FIG. 2. Killing of P. anaerobius after exposure to 80 ,M hydro- lactoperoxidase-thiocyanate-hydrogen peroxide on nutrient
gen peroxide in the presence of lactoperoxidase (10 ,ug ml-') and agar under aerobic conditions, they may not grow, whereas
various concentrations of KSCN. they grow readily on blood agar under anaerobic conditions
584 CARLSSON, EDLUND, AND HANSTROM INFECT. IMMUN.

TABLE 2. Killing of P. anaerobius by mixtures of various ages hydrogen peroxide and thiocyanate at neutral pH and at a
of hydrogen peroxide and a lactoperoxidase-free OSCN- concentration of hydrogen peroxide which does not exceed
preparationa that of thiocyanate. A highly toxic agent was, however,
Age of reaction OSCN- (,uM) Killing time formed in the present study, in which the concentration of
mixture (min) OSCin)M hydrogen peroxide exceeded that of thiocyanate in lactoper-
120 <1.0 36.0 ± 5.0 oxidase-thiocyanate-hydrogen peroxide mixtures. This
60 <1.0 20.0 ± 4.1 agent was also formed in a reaction between OSCN- and
30 1.0 ± 1.2 12.2 ± 6.1 hydrogen peroxide in the absence of lactoperoxidase. It was
15 6.7 ± 0.3 7.1 ± 3.1 not possible to determine the nature of this agent. The
10 8.5 ± 1.4 4.8 ± 2.2 postulated products of the chemical oxidation of OSCN- by
5 12.0 ± 0.5 3.8 ± 1.4 hydrogen peroxide, sulfate, sulfite, cyanate, carbonate, and
0.5 16.4 ± 0.4 2.1 ± 0.4 ammonia (38) were not bactericidal, and they did not potenti-
a Hydrogen peroxide (0.4 ml; 200 ,uM) was mixed with 0.4 ml of ate the effect of hydrogen peroxide. The only other postulat-
35 ,uM OSCN- in MOPS buffer solution; at various times thereafter, ed product of the chemical oxidation of OSCN- by hydrogen
0.2 ml of a bacterial suspension was added. The levels of OSCN- in peroxide is HOOSCN, and it stands out as the suspected
the reaction mixtures at the time of the bacterial addition were killing agent. There was, however, no method available for
assayed in parallel reaction tubes. The definition of killing time is demonstrating the presence of this compound in the reaction

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given in footnote a of Table 1. Means ± standard deviations are
given for three independent experiments. The killing time was 39 ±
2.5 min when the bacteria were exposed to 80 F.M hydrogen
peroxide in the absence of OSCN-.
(2). The explanation may be that the OSCN- formed in the
mixture oxidizes bacterial sulfhydral groups (36), and the
bacteria may not be able to rereduce these vital groups when
cultured on nutrient agar under aerobic conditions. The
present study demonstrated another situation, in which
lactoperoxidase-thiocyanate-hydrogen peroxide was more
bactericidal than hydrogen peroxide alone. This happened
when hydrogen peroxide was in excess of thiocyanate in the
mixture.
There are several potentially toxic products that may be
formed. In studies of the chemical oxidation of thiocyanate
by hydrogen peroxide, Wilson and Harris (38) found sulfate,
cyanate, carbonate, and ammonia as final products and
postulated the following mechanisms:
SCN- + H202-+ HOSCN + OH-
HOSCN + H202-* HOOSCN + H20
HOOSCN + H202 -* H2SO3 + HOCN
HOCN + 2H20-* HC03- + NH4+
H2SO3 + H202-- H2S04 + H20
where reaction 1 is the rate-determining step. It is this
reaction which is catalyzed by lactoperoxidase (4, 19). From
the stoichiometry of reactions in lactoperoxidase-thiocya-
nate-hydrogen peroxide mixtures it has been postulated that
reaction 2 could be catalyzed by lactoperoxidase and that
cyanosulfurous acid (HOOSCN) could be further oxidized
by hydrogen peroxide into cyanosulfuric acid (HO3SCN)
(15, 29).
HOOSCN + H202 -*
HO3SCN + H20
These compounds have been suggested to be more toxic
than OSCN- (6).
In the experiments in which the enzymatic formation of
these compounds has been postulated, the possibility was
overlooked, however, that lactoperoxidase might have cata-
latic activity (9). Oxygen and OSCN- may be the only
products of the lactoperoxidase-catalyzed reaction between
mixtures. In a recent polarographic study it has been demon-
strated, however, that in addition to OSCN- another oxida-
tion product of thiocyanate is formed in lactoperoxidase-
thiocyanate-hydrogen peroxide mixtures when the
concentration of hydrogen peroxide exceeds that of thiocya-
nate (31). This product might be HOOSCN.
The use of putative antagonists of various other possible
toxic agents showed that hydroxyl radicals (mannitol), su-
peroxide radicals (superoxide dismutase), or thiocyanogen
anion radicals (tryptophan) (1) were not likely the effective
molecular species. DABCO, a singlet oxygen quencher (32),
decreased the toxicity of the OSCN--hydrogen peroxide
mixture. The effective molecular species of the mixture
could not possibly be singlet oxygen, since no oxygen was
formed in these mixtures and the singlet oxygen traps,
tryptophan, histidine, and methionine, did not have any
protective effects. DABCO is, however, not specific for
singlet oxygen. It also reacts with peroxy radicals (28). The
protective effect of DABCO could not be ascribed to a
reaction of DABCO with OSCN- or hydrogen peroxide,
since OSCN- and hydrogen peroxide were as stable in the
(1) presence of DABCO as in its absence. Catalase completely
annihilated the killing effect of the OSCN--hydrogen perox-
(2) ide mixtures, and this could be ascribed to the decomposi-
(3)
(4) TABLE 3. Killing of P. anaerobius by mixtures (with added
substances) of hydrogen peroxide and a lactoperoxidase-free
(5) OSCN- preparation'
OSCN-H202 Addition Killing time
mixture (min)
+ None 5.0 ± 2.8
+ 0.1 mM mercaptoethanol 32.1 ± 3.8
+ 0.1 mM reduced glutathione 38.5 ± 3.2
+ 10 mM mannitol 7.2 ± 0.8
+ 10 mM histidine 2.7 ± 0.5
+ 1 mM methionine 10.1 ± 3.1
+ 1 mM tryptophan 5.5 ± 3.0
+ 10 mM DABCO 25.3 ± 3.5
_ 80 puM H202 36.1 ± 2.0
(6) - 80 p.M H202 + 0.1 mM 35.5 ± 3.5
mercaptoethanol
a Hydrogen peroxide (0.05 ml; 1.6 mM) was mixed with 0.75 ml of
44 mM OSCN- in MOPS buffer solution. Various substances (10 p.1)
were added 5 min after hydrogen peroxide and OSCN- had been
mixed; after another 5 min, 0.2 ml of a bacterial suspension was
added. The definition of killing time is given in footnote a of Table 1.
Means ± standard deviations are given for three independent
experiments.
VOL. 44, 1984 TOXICITY OF HYPOTHIOCYANITE-HYDROGEN PEROXIDE 585

tion of hydrogen peroxide in the mixture. Reduced glutathi- 700

one and 2-mercaptoethanol decreased the toxicity of the
mixture as expected, since these compounds readily convert
OSCN- into thiocyanate (18, 35). To settle the nature of the
killing agent, it is important that the actual products of the 600
chemical oxidation of OSCN- by hydrogen peroxide and
those of the lactoperoxidase-catalyzed reaction between
thiocyanate and hydrogen peroxide be fully identified. Since i
the lactoperoxidase-catalyzed reaction has an optimum pH CO,
-J 500
of 4.5 and oxygen is only formed at neutral and alkaline pHs -J
(9), the reaction products at various pHs should be studied.
In the absence of thiocyanate, lactoperoxidase potentiated w
LL
the bactericidal effect of hydrogen peroxide. It is possible LU 400
0
that various components of the bacterial cell or of the buffer
solution served as substrates in reactions catalyzed by
lactoperoxidase in the presence of hydrogen peroxide, re- CD 300
sulting in bactericidal products (2).

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The present results show that the biological effect of z
lactoperoxidase-thiocyanate-hydrogen peroxide mixtures
will be determined by the actual concentration of the individ-
ual components. In the oral cavity, where the salivary 200
secretions contain more than 1 mM thiocyanate and the
production of hydrogen peroxide will rarely produce a higher
concentration than 0.1 mM OSCN- (35), lactoperoxidase
100

1.0 3
0
0 1 2 3
TIME (days)
FIG. 4. Number of HeLa cells per cm2 attached to the surface of
culture flasks after 1 h of exposure to phosphate-buffered saline (line
1); 30 pFM hydrogen peroxide (line 2); or 30 ,uM hydrogen peroxide-
20 ,uM lactoperoxidase-free OSCN- (line 3). Exposure of the cells
to 20 ,uM lactoperoxidase-free OSCN- or to a mixture of 30 ,uM
hydrogen peroxide, 100 ,uM KSCN, and lactoperoxidase (10 ,ug
z 2 ml-', produced results almost identical to those shown in line 1.
0
.1

and thiocyanate could be considered as an efficient protec-

LL.
tive system of the mucous membranes against hydrogen
z
peroxide toxicity. Hydrogen peroxide is not only converted
cc
5; into the less toxic OSCN-; this compound also stops the
cc
bacterial production of hydrogen peroxide (11). It is, of
course, possible that this protective system may fail in
individual cases, in which the lactoperoxidase or thiocyanate
level of saliva is low, or the capacity of the bacteria at
specific sites to produce hydrogen peroxide is very high.
Lactoperoxidase-thiocyanate-hydrogen peroxide is usual-
.01
4 ly referred to as an antimicrobial system of saliva. It is
certainly true that OSCN- formed by this system reversibly
stops glycolysis and consequently the acid production in
many oral bacteria, but we feel that this effect is only a part
0 20 40 60 of a more important function of salivary lactoperoxidase and
TIME (min) thiocyanate, viz., to protect the salivary glands and the oral
FIG. 3. Killing of E. coli after exposure to hydrogen peroxide mucous membranes against hydrogen peroxide toxicity.
or OSCN--hydrogen peroxide mixtures under anaerobic condi- ACKNOWLEDGMENTS
tions, showing the surviving fraction of cells under the following
conditions: after storage for 1 h in phosphate-buffered saline (line 1); This study was supported by the Swedish Medical Research
in the presence of 500 p.M hydrogen peroxide (line 2); in the Council (project no. 4977).
presence of 280 p.M OSCN- (line 3); in the presence of a mixture of LITERATURE CITED
500 p.M hydrogen peroxide and 280 p.M lactoperoxidase-free
OSCN- in phosphate-buffered saline (line 4). Hydrogen peroxide 1. Adams, G. E., J. E. Aldrich, R. H. Bisby, R. B. Cundall, J. L.
and lactoperoxidase-free OSCN- were mixed 10 min before the Redpath, and R. L. Willson. 1972. Selective free radical reac-
cells were added. tions with proteins and enzymes: reactions of inorganic radical
586 CARLSSON, EDLUND, AND HANSTROM
anions with amino acids. Radiat. Res. 49:278-289.
2. Adamson, M., and J. Carlsson. 1982. Lactoperoxidase and
thiocyanate protect bacteria from hydrogen peroxide. Infect.
Immun. 35:20-24.
3. Ananthaswamy, H. N., and A. Eisenstark. 1977. Repair of
hydrogen peroxide-induced single-strand breaks in Escherichia
coli deoxyribonucleic acid. J. Bacteriol. 130:187-191.
4. Aune, T. M., and E. L. Thomas. 1977. Accumulation of hypoth-
iocyanite ion during peroxidase-catalysed oxidation of thiocya-
nate ion. Eur. J. Biochem. 80:209-214.
5. Bachmann, B. J. 1972. Pedigrees of some mutant strains of
Escherichia coli K-12. Bacteriol. Rev. 36:525-557.
6. Bjoerck, L., and 0. Claesson. 1980. Correlation between con-
centration of hypothiocyanate and antibacterial effect of the
lactoperoxidase system against Escherichia coli. J. Dairy Sci.
63:919-922.
7. Bjorck, L., C.-G. Rosen, V. Marshall, and B. Reiter. 1975.
Antibacterial activity of the lactoperoxidase system in milk
against pseudomonads and other gram-negative bacteria. Appl.
Microbiol. 30:199-204.
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