ARTICLE
Acne vulgaris is one of the most common disorders
in dermatology [1, 2]. The pathogenesis of acne is multifactorial [3,
4], but inflammation and immunological reactions are mainly the most important
factors studied recently [3, 5].
The proliferating microorganism, Propionibacterium acnes (P. acnes),
in sebum is a potent activator of complement pathways and produces chemotactic
factors resulting in neutrophil chemotaxis [1, 4]. During chemotaxis,
degradative lysosomal enzymes were produced ending with degenerative reactions
and excessive generation of reactive oxygen species (ROS) by neutrophils
may cause further destruction of surrounding tissues [5]. In the most
recent study by Akamatsu et al. [6], neutrophil-derived ROS were
attributed to inflammatory reaction in papulopustular type acne vulgaris.
Thus, much effort should be directed to treatment approaches effective
on ROS and antioxidative enzymes.
Topical benzoyl peroxide (BP) and BP combination with erythromycin (BP/E)
are commonly used medications for the treatment of acne vulgaris. To our
knowledge, studies about the effects of BP and erythromycin (E) on oxidative/antioxidative
system have been performed either under in vitro conditions or
in animals up to date [7-9]. Therefore, in this in vivo, double-blind,
preliminary human study, we investigated whether these formulations affect
the activity of antioxidative defence enzymes.
Materials and methods
Subjects
Forty patients with acne vulgaris of papulopustular type were enrolled
in the study. Patients were ordered not to take any topical or systemic
drugs for acne during the preceding 4 weeks. Those having systemic or
metabolic diseases, smoking or drinking were excluded. Severity of the
disease was determined by using global acne grading system described previously
[10]. Patients were randomized to two groups equally and heparinized fasting
venous blood was drawn at the beginning of the study. For those who agreed
to be biopsied, a papular lesion was excised using a 4 mm dermatologic
punch biopsy device. Another papular lesion was marked for a second biopsy
procedure which would be performed in the posttreatment period. Patients
were instructed to use either 5% BP gel, or a combination of 5% BP/3%
E gel twice daily for up to 4 weeks (Table
I). At the end of 4 weeks, peripheral heparinized blood and tissue
samples from the marked biopsy area were repeated. Superoxide dismutase
(SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) activities and
thiobarbituric acid reactive substance (TBARS) levels in tissues and serum
leukocytes obtained from the samples at the beginning and at the end of
4 weeks of treatment were detected in each group. All patients gave informed
consent for the study.
Sample preparation
Tissue samples were kept in the deep freeze at - 70° C until measuring.
After thawing, they were homogenized for 3 min in a homogenizator (Ultra-Turrax
T25, Germany) under the cold phosphate buffer in order to collect 10%
homogenate. The homogenates, then, were sonicated with ultrasonic cell
disrupter and centrifuged at 6,000 g for 10 min to obtain supernatants
which were used for all tissue measurements.
The separation of mononuclear leukocytes (MNL) from whole blood was
done by using the product of Histopaque-1077 (Sigma Diagnostics, MO, USA,
Product no: 1077). The principle of Histopaque-1077 was based on the method
described by Boyum [11]. Anticoagulated venous blood was layered onto
Histopaque-1077. During centrifugation, erythrocytes and granulocytes
were aggregated by polysucrose whereas lymhocytes and other mononuclear
cells remained at the plasma-Histopaque-1077 interface. Centrifugated
mononuclear leukocytes (MNL) were separated from plasma and washed. Platelets
were removed by low speed centrifugation washing with Hangs' Balanced
Salts (HBSS) (Sigma Diagnostics, MO, USA, Product no: H4891). The MNL
obtained were suspended in 1 ml HBSS and stored at - 70° C. After
the collection of samples, MNL suspensions were thawed, sonicated with
ultrasonic cell disruptor and centrifuged. The levels of TBARS and protein
as well as the activities of SOD, GSH-Px and CAT were determined in supernatant.
Measurements
TBARS was determined by using the double heating method of Draper and
Hadley [12]. The principle of the method was based on the spectrophotometric
measurement of the color which occurred during the reaction to thiobarbituric
acid (TBA) with malondialdehyde (MDA). Determination of GSH-Px activity
was based on the method of Paglia and Valentine [13]. SOD activity was
measured by the degree of inhibition of the reaction in which xanthine
reacts with xanthine oxidase to generate superoxide radicals [14]. CAT
activity was measured according to the method described previously by
Aebi [15]. The autoanalyser, Abbott Aeroset (USA), was used to determine
the concentration of protein, SOD and GSH-Px activities, and the spectrophotometer
by Shimadzu UV-1601 (Japan) was used for measuring the rest of the parameters.
Statistical evaluation
For statistical analysis, non-parametric Wilcoxon Signed Ranks test
and Mann-Whitney U test were used to compare groups, as stated by Dawson-Saunders
and Trapp [16] considering the small number of cases. Since the data obtained
in this research were non-parametric, descriptive statistics - median
(the value that divides the distribution into halves) and the first and
third quartiles (the value that divides the lowest 25% of the observations
from the highest 75%, and the value that divides the highest 25% of the
observations from the lowest 75%, respectively) - instead of mean ±
SD were used. Pearson correlation test was performed to correlate the
data. The significance level was set at p < 0.05.
Results
Twenty patients in the BP-treatment group consisted of 11 females (55%)
and 9 males (45%) whereas this ratio was equal in BP/E-treated group.
The average age was 19.3 ± 3.4 and 19 ± 3 years in BP and BP/E-treated
patients, respectively. The mean duration and severity of acne at the
beginning and at the end of 4 weeks of treatment were similar in both
groups (Table II). Leukocyte
SOD, GSH-Px and CAT activities and TBARS levels before and after (4th
week) treatment in the BP-treated group were shown in Table
III. No difference was detected in leukocyte antioxidant enzyme activities
and TBARS levels due to BP treatment. However, in the BP/E-treated group,
increasing levels of TBARS were associated with decreased SOD, CAT and
GSH-Px activities although not statistically significant in the latter
(Table III). When the
two treatment groups were compared, TBARS levels were found to be significantly
elevated in BP/E-treated group at the end of 4 weeks (p = 0.015).
No correlation was determined in the BP-treated group between enzyme
activities and duration or severity of the disease. In contrast, longer
duration of acne was correlated with higher TBARS levels (r = 0.49, p
= 0.025) and lower CAT activities (r = - 0.44, p = 0.048) in BP/E-treated
patients. Moreover, in the same group, while the severity of acne was
increasing, decreased GSH-Px activity (r = - 0.55, p = 0.025) and enhanced
TBARS levels were detected (r = 0.54, p = 0.013).
In tissue samples, there was no statistically significant difference
between pretreatment and posttreatment enzyme activities in either of
the groups. In addition, no difference was observed comparing BP and BP/E-treated
patients regarding tissue antioxidant enzyme activities (Table
IV).
Discussion
The damage of the follicular epithelium and repeated ROS generation
by neutrophils resulting in tissue injury are proposed to be responsible
for one of the pathogenetic steps in acne [5, 6]. Therefore, inhibition
of ROS production may have been of therapeutic benefit. Treatment success
in acne with antibiotics has been relevant to the reduction in P. acnes
numbers and the inhibition of P. acnes-associated inflammatory
mediators [1, 17]. In vitro studies revealed that tetracyclines
[18-20] and metronidazole used in the presence of palmitoleic acid [21]
were reported to inhibit ROS generated by neutrophils.
Topical BP and BP/E are popular and effective treatment modalities for
mild and moderate papulopustular acne. BP is a lipophilic formulation
and inhibits P. acnes colonization more effectively than topical
E. The formation of new lesions stops after up to 4-6 weeks of treatment
[1, 3, 4, 22]. E has also been reported to have antibacterial activity
against P. acnes [23, 24]. Combination of BP and E has proven to
enhance efficacy and inhibit the development of resistant P. acnes
and Staphylococcus aureus strains [1, 3, 4, 25].
Recently, the therapeutic effect of these topical anti-acne formulations
has also been attributed to their antiinflammatory effects [26]. BP treatment
was reported to increase microsomal lipid peroxidation [9] and the activity
of cutaneous antioxidative enzymes was decreased as mentioned in an animal
study [8]. Topical application of BP resulted in depletion of SOD, GSH-Px
and CAT activities in mouse epidermis which were reversed by a flavonoid
antioxidant [27]. However, lipid peroxidation was observed only in the
presence of Fe2+ with BP [28]. The bactericidal action of BP
as an oxidizing agent on P. acnes population has been proposed
to be due to the production of ROS in the sebaceous follicle during its
breakdown process [29, 30]. On the other hand, BP was reported to inhibit
protein kinase C in mouse [31] and possess direct cytotoxic effects on
leukocytes, resulting in the inhibition of ROS generation by neutrophils
in vitro, in a dose-dependent manner [7]. These effects of BP are
based on its slight antiinflammatory action [23, 32]. In the present study,
no difference was detected in antioxidant enzyme activities, neither in
tissues nor in peripheral blood leukocytes during BP treatment in patients
with papulopustular acne. This might be due to neutralization of the dual
effects of BP on ROS described above. While the antioxidant effects of
BP have been found at higher dose levels than the dose required to kill
P. acnes [7], it can also be suggested that twice daily application
of BP may be inadequate to expose variation in antioxidant enzyme activities
in vivo.
E was found to decrease the activity of lipase and chemotactic factors
by P. acnes [33-35]. The antioxidant effect of E was not attributed
to its capacity for scavenging ROS, but directly on neutrophil cell function
in vitro [18, 36]. With the concomitant use of BP and E in a unique
formulation, inhibition of P. acnes, neutrophil chemotaxis and
production of fatty acids should have been increasingly detected [1, 3,
4, 37].
In the present study, SOD, CAT and GSH-Px activities in leukocytes were
found to be decreased although no difference was demonstrated in tissue
antioxidant enzyme activities due to the treatment with the BP/E. In addition,
TBARS levels were significantly increased at the end of 4 weeks in BP/E-treated
patients. One of the possibilities for the disturbed antioxidant activity
in the BP/E group compared to BP-treated patients could be related to
the commercial preparation of BP/E (Benzamycin®), used
in this study. For this procedure, patients were instructed to prepare
a solution with E powder dissolved in ethyl alcohol 70% and add this solution
into BP gel. This mixture is stirred until homogenous in appearence and
stored in the refrigerator until used. Vermeulen et al. [38] reported
that an overdose of E was used in Benzamycin® gel which
causes E precipitation and BP agglomeration. One of the steps during the
preparation might have been mistaken which could result in damage to the
stability of the formulation.
In this in vivo human study, influence of the vehicle components
or additives in the applied formulation can not be excluded. To our knowledge,
the chemical additives in BP and BP/E (Table
I) other than propylene glycol, ethyl alcohol and sodium hydroxide,
do not induce oxidative stress. Both of the formulations applied in the
present study included sodium hydroxide which is known to increase superoxide
anion production in cultured human gastric mucosal cells [39]. Thus, either
propylene glycol or ethyl alcohol in BP and BP/E formulations respectively,
could have created a difference in the activity of antioxidant defence
enzymes. This suggestion might be supported by the observation of Morisaki
et al. [40] that the inhibition of the superoxide-generating system
in leukocytes was caused mainly by propylene glycol compared to ethyl
alcohol. Additionally, it may be proposed that ethyl alcohol-induced cytotoxicity
by increasing production of ROS as observed in hepatic [41] or gastric
cells [39], could also contribute to the oxidative stress attained during
BP/E treatment. Nevertheless, further pharmacological investigation using
additives or vehicles and pure drugs should provide a better comparison
of these formulations which will clarify the mechanisms of oxidation in
the treatment of papulopustular acne.
Article accepted on 8/10/01
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