Download fluphenazine and perphenazine impact on melanogenesis and

Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 73 No. 4 pp. 903ñ911, 2016
ISSN 0001-6837
Polish Pharmaceutical Society
FLUPHENAZINE AND PERPHENAZINE IMPACT ON MELANOGENESIS
AND ANTIOXIDANT ENZYMES ACTIVITY IN NORMAL HUMAN
MELANOCYTES
MICHA£ OTR BA, DOROTA WRZEåNIOK, ARTUR BEBEROK, JAKUB ROK
and EWA BUSZMAN*
School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec,
Medical University of Silesia in Katowice, Department of Pharmaceutical Chemistry,
JagielloÒska 4, 41-200 Sosnowiec, Poland
Abstract: Fluphenazine and perphenazine as a phenothiazine-class antipsychotic drugs are widely used to treat
psychoses and schizophrenia, however their use is associated with significant side effects such as extrapyramidal symptoms as well as ocular and skin disorders. The aim of this study was to examine the effect of
fluphenazine and perphenazine on cell viability, melanogenesis and antioxidant defense system in normal
human melanocytes. It has been shown that both phenothiazines induce concentration-dependent loss in cell
viability. The value of EC50 was calculated to be 1.24 and 2.76 µM for fluphenazine and perphenazine, respectively. Fluphenazine in concentration of 1.0 µM and perphenazine in concentrations of 1.0 and 3.0 µM inhibited melanogenesis and decreased microphthalmia-associated transcription factor content. To study the effect of
both analyzed drugs on antioxidant defense system in melanocytes, the level of hydrogen peroxide and the
activities of antioxidant enzymes: superoxide dismutase, catalase and glutathione peroxidase were determined.
Fluphenazine and perphenazine in higher analyzed concentrations caused depletion of melanocytes antioxidant
status, what indicated the induction of oxidative stress. The observed changes in melanization process and
antioxidant defense system in pigmented cells exposed to fluphenazine and perphenazine in vitro suggest a significant role of melanin and melanocytes in the mechanisms of undesirable side effects of these drugs in vivo,
especially directed to pigmented tissues. Moreover, the presented differences in modulation of biochemical
processes in melanocytes may be an explanation for various toxic activity of the analyzed phenothiazine derivatives in vivo.
Keywords: fluphenazine, perphenazine, melanocytes, melanogenesis, oxidative stress
Melanins are synthesized during multistep
complex process called melanogenesis. The process
is localized in specialized cytoplasmic organelles,
melanosomes, in the cytoplasm of melanocytes and
retinal pigment epithelial (RPE) cells. Disturbation
of melanogenesis may determine hypo- and/or
hyperpigmentation defects, which may occur with
or without an altered number of melanocytes (7-10).
Interestingly, fluphenazine therapy may rarely cause
vitiligoid depigmentation and chemical leukoderma,
which are an acquired, hypopigmented dermatosis
that result from repeated, cutaneous application of
an agent that destroys epidermal melanocytes (11,
12).
Specialized
pigment
dendritic
cells,
melanocytes, protect organism from ultraviolet
light, determine pigmentation of skin, hair and eyes,
Fluphenazine and perphenazine belong to the
high-potency antipsychotic drugs with a piperazinyl
structure, which are used for treatment of psychoses
and positive symptoms of schizophrenia, such as
hallucinations and delusions (1, 2). Perphenazine is
also indicated in the manic phases of bipolar disorders (2). The phenothiazine treatment is associated
with high extrapyramidal symptoms (EPS), such as
dystonia and/or akathisia, as well as with other
adverse side effects including movement disorders,
akinesia, dyskinesia, dizziness, drowsiness, sedation, severe toxicity, tachycardia, hypotension, ocular effects (e.g., blurred vision, maculopathy and
cataract) and skin disorders (e.g., rash, exfoliative
dermatitis, drug hypersensitivity syndrome, cutaneous photosensitivity and abnormal skin pigmentation) (1-6).
* Corresponding author: e-mail: [email protected]; phone:+48-32-364-16-11
903
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MICHA£ OTR BA et al.
reversibly bind metal ions, organic amines, cyclic
compounds and drugs, e.g., psychotropic agents or
fluoroquinolones, as well as protect from reactive
oxygen species (ROS) and oxidative stress (9, 1315). The binding of drugs to melanin probably protects organism against undesirable drug side effects,
but also may lead to accumulation of the drug in
melanin-containing tissues (especially in the eye,
ear, skin and brain) causing degeneration of pigmented cells (9, 16).
Cutaneous melanin is synthesized in melanocytes, in specialized organelles called melanosomes
during complex, multistage biochemical pathway
leading to brownish-black eumelanins and/or reddish-yellow pheomelanins. The melanogenesis
process is regulated by very important proteins like
tyrosinase, tyrosinase related proteins TRP1 and
TRP2 as well as transcription factor MITF, which is
also responsible for proliferation, dendrite formation
and induction of antiapoptotic genes expression.
Synthesized melanin is moved from melanocytes to
neighboring epidermal keratinocytes where is
responsible for skin pigmentation, UV light absorption, scavenging free radicals, chelating metal ions
and binding a variety of drugs and other xenobiotics
(7, 9, 17).
To prevent the damage of melanocytes by ROS
induced by endogenous and exogenous sources
(e.g., melanogenesis, mitochondria, lipoxygenases,
peroxisomes, ultraviolet radiation, toxins, chemicals, ionizing radiation and inflammation), cellular
antioxidant enzymes: superoxide dismutase (SOD),
catalase (CAT), glutathione peroxidase (GPx), are
activated to restore normal redox state in cells (18).
Melanin biosynthesis involves oxidation reactions
as well as superoxide anion (O2.-) and hydrogen peroxide (H2O2) generation, which subject melanocytes
to oxidative stress (18). On the other hand, melanin
acts as a free radicals scavenger and possesses
superoxide dismutase activity, what may reduce
ROS content and thus protect pigmented tissues
against cellular damage induced by oxidative stress
(7, 19).
Previously, we have documented that aminoglycoside antibiotics (20, 21) and fluoroquinolones
(22, 23), induce inhibition of cell viability and
melanogenesis in normal human epidermal
melanocytes light pigmented (HEMa-LP). We have
also demonstrated the inhibitory effect of ketoprofen
on melanization process (24), as well as modulation
of melanin biosynthesis and alterations in antioxidant defense system after nicotine treatment (25) in
normal human epidermal melanocytes dark pigmented (HEMn-DP).
The aim of this study was to estimate the effect
of fluphenazine and perphenazine on cell viability,
melanogenesis and antioxidant defense system in
normal human melanocytes dark pigmented
(HEMn-DP).
EXPERIMENTAL
Chemicals
Fluphenazine dihydrochloride, perphenazine,
phosphated-buffered saline (PBS), 3,4-dihydroxyL-phenylalanine (L-DOPA) and amphotericin B
were purchased from Sigma-Aldrich Inc. (USA).
Neomycin sulfate was obtained from Amara
(Poland). Penicillin was acquired from Polfa
Tarchomin (Poland). Growth medium M-254 and
human melanocyte growth supplement-2 (HMGS-2)
were obtained from Cascade Biologics (UK).
Trypsin/EDTA was obtained from Cytogen
(Poland). Cell proliferation reagent WST-1 was purchased from Roche GmbH (Germany). The remaining chemicals were produced by POCH S.A.
(Poland).
Cell treatment
The normal human epidermal melanocytes
(HEMn-DP, Cascade Biologics) were cultured in
M-254 basal medium supplemented with HMGS-2,
penicillin (100 U/mL), neomycin (10 µg/mL) and
amphotericin B (0.25 µg/mL) at 37OC in 5% CO2.
Cells in the passages 6-9 were used in all performed
experiments.
Cell viability assay
The viability of melanocytes was evaluated by
the WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)2H-5-tetrazolio]-1,3-benzene disulfonate) colorimetric assay according to the method described earlier (22, 26).
Measurement of melanin content
Melanin content was measured in normal
human melanocytes cultured in T-25 flasks at a density of 100,000 cells/flask. Cells were incubated for
48 h after seeding and then began 24-h fluphenazine
or perphenazine treatment in a concentration range
from 0.0001 to 1.0 µM and from 0.0001 to 3.0 µM,
respectively. After the three times washings with
PBS, cells were detached with trypsin-EDTA. Cell
pellets were placed into Eppendorf tubes, dissolved
in 100 µL of 1 M NaOH at 80OC for 1 h, and then
centrifuged for 20 min at 16,000 ◊ g. The supernatants were placed into a 96-well microplate, and
absorbance was measured at 405 nm ñ a wavelength
Fluphenazine and perphenazine impact on melanogenesis and...
at which melanin absorbs light (27). A standard synthetic melanin curve (0 to 400 µg/mL) was performed in triplicate for each experiment. Melanin
content in fluphenazine or perphenazine treated cells
was expressed in pg/cell and finally as the percentage of the controls (untreated melanocytes).
Tyrosinase activity assay
Tyrosinase activity in melanocytes was determined by measuring the rate of oxidation of LDOPA to DOPAchrome, according to the method
described previously (22). The cells were cultured at
a density of 100,000 cells/flask for 48 h and then,
incubated with fluphenazine (concentration range
from 0.0001 to 1.0 µM) or perphenazine (concentration range from 0.0001 to 3.0 µM) for 24 h. Before
lysis and clarification (centrifugation at 10,000 ◊ g
for 5 min) cells were washed three times with PBS.
Tyrosinase activity assay was performed in 96-well
plate. A tyrosinase substrate L-DOPA (2 mg/mL)
was prepared in the same lysis phosphate buffer.
Hundred µL of each lysate and 40 µL of L-DOPA
solution were pipetted into each well and incubated
at 37OC for initiation of the enzymatic assay.
Absorbance was measured every 10 min for at least
1.5 h at 475 nm using a microplate reader.
Tyrosinase activity was expressed in µmol/min/mg
protein.
Microphthalmia-associated transcription factor
(MITF) assay
Microphthalmia-associated transcription factor
(MITF) content was measured using ELISA an
assay kit (USCN Life Science Inc, USA) according
905
to the manufacturerís instruction. This kit is a sandwich enzyme immunoassay for in vitro quantitative
measurement of MITF providing a 96-well
microplate pre-coated with a biotin-conjugated antibody specific for MITF. The color change of the
enzyme (horseradish peroxidase) ñ substrate (TMB)
reaction was measured spectrophotometrically at
450 nm using a microplate reader. MITF content in
the samples was expressed in ng/mg protein.
Cellular antioxidant status assay
Superoxide dismutase (SOD), catalase (CAT),
glutathione peroxidase (GPx) activities and hydrogen peroxide (H2O2) content were measured using
an assay kits (Cayman, MI, USA and Cell Biolabs,
Inc., USA) according to the manufacturerís instructions, after 24-h incubation of human melanocytes
HEMn-DP with fluphenazine (in concentration
range from 0.0001 to 1.0 µM) or perphenazine (in
concentration range from 0.0001 to 3.0 µM). The
SOD, CAT, GPx activity and H2O2 content were
measured spectrophotometrically at 441, 540, 340
and 595 nm, respectively, using a microplate reader.
Statistical analysis
In all experiments, mean values of at least three
separate experiments (n = 3) performed in triplicate
± standard deviation (SD) were calculated.
Statistical analysis was performed with one-way
ANOVA followed by Tukey post-hoc test using
GraphPad Prism 6.01 software. The significance
level was estabilished at value of p < 0.05 (*) or p <
0.01 (**), by comparing the data with those for control (cells without drug).
Figure 1. The effect of fluphenazine and perphenazine on viability of melanocytes. Cells were treated with various drug concentrations
(0.0001-10.0 µM) and examined by the WST-1 assay. Data are expressed as percent of the controls. Mean values ± SD from three independent experiments (n = 3) performed in triplicate are presented. * p < 0.05 vs. the control samples, ** p < 0.01 vs. the control samples
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MICHA£ OTR BA et al.
RESULTS
The effect of fluphenazine and perphenazine on
cell viability
The cell viability was determined by the WST1 test after 24-h incubation with fluphenazine or perphenazine in a concentrations range from 0.0001
µM to 10.0 µM. It has been demonstrated that the
analyzed drugs induce concentration-dependent loss
in cell viability (Fig. 1). Melanocytes treated with
fluphenazine in concentrations from 0.001 µM to 10
µM lost 11.3% to 98% in cell viability. After incubation of cells with perphenazine in concentrations
from 0.1 µM to 10 µM, the loss of cell viability was
19.1% to 98.5%. The value of EC50 (the concentration of a drug that produces loss in cell viability by
50%) was calculated to be 1.24 and 2.76 µM, respectively for fluphenazine and perphenazine. At lower
concentrations of fluphenazine (0.0001 µM) and
perphenazine (0.0001, 0.001 and 0.01 µM) the loss
in melanocytes viability was not observed.
The effect of fluphenazine and perphenazine on
melanization process in melanocytes
The effectiveness of melanization process was
estimated by measuring the melanin content, cellular tyrosinase activity and microphthalmia-associated transcription factor (MITF) content in
melanocytes treated with fluphenazine (concentration range from 0.0001 to 1.0 µM) or perphenazine
(concentration range from 0.0001 to 3.0 µM) for 24
h. The melanin content per cell was determined as
Figure 2. The effect of fluphenazine and perphenazine on melanin content (A), tyrosinase activity (B) and microphthalmia-associated transcription factor (MITF) content (C) in melanocytes. Cells were cultured with 0.0001, 0.001, 0.01, 0.1 and 1.0 µM of fluphenazine or
0.0001, 0.001, 0.01, 0.1, 1.0 and 3.0 µM of perphenazine for 24 h. Data are the mean ± SD of at least three independent experiments (n =
3) performed in triplicate. * p < 0.05 vs. the control samples, ** p < 0.01 vs. the control samples
Fluphenazine and perphenazine impact on melanogenesis and...
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Figure 3. The superoxide dismutase (SOD) (A), catalase (CAT) (B), glutathione peroxidase (GPx) (C) activity and hydrogen peroxide
(H2O2) content (D) in HEMn-DP cells after 24-h incubation with 0.0001, 0.001, 0.01, 0.1 and 1.0 µM of fluphenazine or 0.0001, 0.001,
0.01, 0.1, 1.0 and 3.0 µM of perphenazine. Data are the mean ± SD of at least three independent experiments (n = 3) performed in triplicate. * p < 0.05 vs. the control samples, ** p < 0.01 vs. the control samples
57.7 to 65.4 pg/cell and 53.9 to 63.3 pg/cell for
melanocytes treated with fluphenazine and perphenazine, respectively, while the values determined for the control samples of both drugs were
62.5 ± 1.82 pg/cell and 61.3 ± 1.34 pg/cell, respectively. The obtained results, recalculated for culture
(1 ◊ 105 cells), were finally expressed as a percentage of the controls (Fig. 2A). Treatment of HEMnDP cells with 1.0 µM of fluphenazine decreased
melanin content by 7.7%, and perphenazine in concentrations of 1.0 and 3.0 µM also decreased the pig-
ment content by 10.6 and 12.1%, respectively. Both
analyzed drugs in concentrations from 0.0001 to 0.1
µM had no impact on melanin content in
melanocytes.
Tyrosinase activity in melanocytes treated with
fluphenazine or perphenazine decreased in a manner
correlating well with the effect on melanin production (Fig. 2B). The enzyme activity was determined
as 0.93 to 1.06 µmol/min/mg protein and 0.95 to
1.11 µmol/min/mg protein for melanocytes treated
with fluphenazine and perphenazine, respectively,
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MICHA£ OTR BA et al.
while the values determined for the control samples
of both drugs were 1.00 ± 0.03 µmol/min/mg protein
and 1.07 ± 0.03 µmol/min/mg protein, respectively.
The tyrosinase activity decreased by 6.7% for cells
treated with fluphenazine in concentration of 1.0
µM, while perphenazine in concentrations of 1.0 and
3.0 µM decreased the enzyme activity by 10.1% and
11.9%, respectively, as compared with the controls.
Fluphenazine and perphenazine in concentrations
from 0.0001 to 0.1 µM had no impact on cellular
tyrosinase activity.
The MITF content was determined as 0.14 to
0.33 ng/mg protein and 0.20 to 0.29 ng/mg protein
for melanocytes treated with fluphenazine and perphenazine, respectively, while the values determined for the control samples of both drugs were
0.29 ± 0.02 ng/mg protein and 0.26 ± 0.02 ng/mg
protein, respectively (Fig. 2C). Treatment of
HEMn-DP cells with fluphenazine in concentration
of 0.0001 µM increased MITF content by 15.6%,
while the drug in concentrations of 0.01, 0.1 and
1.0 µM decreased this transcription factor content
by 16.9, 29.9 and 52.9%. In contrast to
fluphenazine, perphenazine only decreased the
MITF content by 15.5 and 23.5% in concentrations
of 1.0 and 3.0 µM, respectiviely. Fluphenazine in
concentration of 0.001 µM and perphenazine in
concentrations from 0.0001 to 0.1 µM had no
impact on the cellular MITF content in comparison
to the control cells.
The effect of fluphenazine and perphenazine on
antioxidant defense system in melanocytes
To study the effect of analyzed drugs on reactive oxygen species metabolism in melanocytes, the
activity of antioxidant enzymes and the content of
hydrogen peroxide were determined. Cells were
exposed to fluphenazine (concentration range from
0.0001 to 1.0 µM) or perphenazine (concentration
range from 0.0001 to 3.0 µM) for 24 h.
Both of the analyzed drugs rised SOD activity
(Fig. 3A). The SOD activity was determined as 0.88
to 1.26 U/mg protein and 0.84 to 1.08 U/mg protein
for melanocytes treated with fluphenazine and perphenazine, respectively, while the values determined for the control samples of both drugs were
0.90 ± 0.06 U/mg protein and 0.83 ± 0.03 U/mg protein, respectively. The treatment of cells with 0.1
and 1.0 µM of fluphenazine or 1.0 and 3.0 µM of
perphenazine increased the SOD activity by 21.1
and 40.1% or by 13.3 and 30.4%, respectively, as
compared with the controls. Both analyzed drugs in
concentration range from 0.0001 to 0.01 µM had no
impact on SOD activity.
After 24-h incubation with fluphenazine or
perphenazine the intracellular CAT activity
decreased (Fig. 3B). The CAT activity was determined as 21.60 to 26.83 nmol/min/mg protein and
20.28 to 25.93 nmol/min/mg protein for
melanocytes treated with fluphenazine and perphenazine, respectively, while the values determined for the control samples of both drugs were
26.75 ± 1.50 nmol/min/mg protein and 25.33 ± 1.91
nmol/min/mg protein, respectively. Treatment of
HEMn-DP cells with 0.1 and 1.0 µM of
fluphenazine or 1.0 and 3.0 µM of perphenazine
decreased the CAT activity by 14.1 and 19.2% or by
13.9 and 19.9%, respectively, as compared with the
controls. Both analyzed drugs in concentration
range from 0.0001 to 0.01 µM had no impact on
CAT activity.
Fluphenazine modified GPx activity, while
perphenazine decreased the enzyme activity in
melanocytes (Fig. 3C). The GPx activity was determined as 14.70 to 20.83 nmol/min/mg protein and
13.61 to 15.95 nmol/min/mg protein for cells treated with fluphenazine and perphenazine, respectively, while the values determined for the control
samples of both drugs were 17.84 ± 0.47
nmol/min/mg protein and 15.24 ± 1.10
nmol/min/mg protein, respectively. Treatment of
melanocytes with 0.1 µM of fluphenazine caused
an increase in GPx activity by 16.8%, while the
concentration 1.0 µM decreased the enzyme activity by 17.6%. Perphenazine in concentration of 3.0
µM decreased GPx activity by 10.7%. Both analyzed drugs in lower concentrations had no impact
on GPx activity.
After 24-h incubation of melanocytes with the
analyzed drugs, the hydrogen peroxide (H2O2) content increased in a concentration-dependent manner
(Fig. 3D). The H2O2 content was determined as
158.81 to 276.98 µmol/mg protein and 155.31 to
232.50 µmol/mg protein for melanocytes treated
with fluphenazine and perphenazine, respectively,
while the values determined for the control samples
of both drugs were 153.39 ± 2.09 µmol/mg protein
and 156.41 ± 9.37 µmol/mg protein, respectively.
The treatment of cells with fluphenazine in concentrations from 0.001 to 1.0 µM increased the H2O2
content by 9.2 to 80.6%, while perphenazine in concentrations from 0.01 to 3.0 µM increased the hydrogen peroxide content by 12.7 to 48.7%, as compared
with the controls. Fluphenazine in concentration of
0.0001 µM and perphenazine in concentrations
0.0001 and 0.001 µM had no impact on H2O2 content
in cells.
Fluphenazine and perphenazine impact on melanogenesis and...
DISCUSSION
Like other phenothiazine derivatives,
fluphenazine and perphenazine have been reported
to exhibit a variety of new, important biological
effects such as anticancer, antibacterial, antiprotozoic, antiviral and antimultidrug resistance reversal
activity. The biological activities of phenothiazinebased compounds are strongly dependent on their
chemical structure, intrinsic physical properties and
their ability to aggregate with themselves and solvent molecules (28, 29).
Phenothiazine derivatives treatment may
potentially lead to harmful toxic effects.
Fluphenazine treatment is associated with extrapyramidal symptoms in 97% of patients, incident depression in 55% of patients, blurred vision, excess salivation as well as dose-related changes in prolactin
levels. Similar side effects like acute extrapyramidal
symptoms and hyperprolactinemia are observed
during perphenazine therapy (30).
Interestingly, in the treatment of psychotic disorders, the daily dosage of fluphenazine up to 40
mg/day (31) is relatively lower in comparison to
other phenothiazines: chlorpromazine more than
800 mg/day (32), perphenazine up to 64 mg/day
(33) and thioridazine up to 800 mg/day (34).
Because of the fact that long-term thioridazine therapy with a daily dose of 100 mg is required to trigger chorioretinal changes and the highest dose (800
mg/day) can induce a peripheral retinopathy, as well
as that fluphenazine therapy 2 mg/day may lead to
toxic retinopathy (maculopathy) ñ fluphenazine may
be considered to be more toxic drug than other phenothiazines (5, 35).
Our previous studies showed the ability of phenothiazine derivatives: chlorpromazine, fluphenazine, trifluoperazine and thioridazine to form stable
complexes with model synthetic as well as natural
melanin (14, 36), which may be responsible for
toxic effects of these drugs directed to pigmented
tissues.
In this study, we have used the culture of normal human epidermal melanocytes as an in vitro
experimental model system. It was observed that
fluphenazine in the lowest concentration (0.0001
µM) and perphenazine in concentration range from
0.0001 to 0.01 µM did not have any significant effect
on melanocytes viability. The treatment of cells with
both drugs in higher concentrations resulted in the
loss in cell viability in a concentration-dependent
manner (Fig. 1). The value of EC50 was calculated to
be 1.24 and 2.76 µM for fluphenazine and perphenazine, respectively. For the same cell line the
909
value of EC50 established for chlorpromazine was
2.53 µM (26), what indicates that fluphenazine is
more cytotoxic than chlorpromazine and perphenazine. Taking into account the value of EC50, the
order of drugs cytotoxicity would be as follows:
fluphenazine >> chlorpromazine > perphenazine.
The analysis of melanogenesis process in cells
cultured in the presence of fluphenazine showed that
the drug in concentration of 1.0 µM decreased
tyrosinase activity and melanin content.
Perphenazine in concentrations of 1.0 and 3.0 µM
also decreased the enzyme activity, as well as
melanin content (Fig. 2A and 2B). Our results
demonstrate that both drugs at higher studied concentrations (= 1.0 µM) suppress melanogenesis in
normal human melanocytes, probably due to their
inhibitory effect on tyrosinase activity.
Because of the fact that MITF is a major transcriptional regulator of melanogenesis, cell survival
and differentiation of melanocytes, we have examined the effect of fluphenazine and perphenazine on
the content of this protein. Fluphenazine in concentration of 0.0001 µM increased the MITF content,
while in the concentrations from 0.01 to 1.0 µM significantly decreased the content of this protein.
Perphenazine in concentrations of 1.0 and 3.0 µM
also decreased the content of MITF (Fig. 2C). The
obtained results indicate that fluphenazine possesses
greater inhibitory effect on MITF than perphenazine, what may explain that only during
fluphenazine therapy hypopigmentation disorders,
such as vitiligoid depigmentation and chemical
leukoderma, occur. Moreover, the observed inhibition of melanogenesis process and decrease in cellular viability in the presence of fluphenazine and perphenazine is probably caused by the suppression of
microphthalmia-associated transcription factor.
In contrast to fluphenazine and perphenazine,
chlorpromazine used in lower concentrations
(0.0001, 0.001 and 0.01 µM) increased melanin and
MITF content as well as tyrosinase activity in
HEMn-DP cells, without any impact on melanogenesis in higher drug concentrations (0.1 and 1.0 µM)
(26). This may explain the occurence of photodistributed hyperpigmentation in patients during chlorpromazine therapy.
Reactive oxygen species are produced during
normal cellular metabolic processes, under pathologic conditions as well as by many exogenous factors including UV radiation, xenobiotics and drugs.
The overproduction of pro-oxidant species in cells
and/or reduction of cellular antioxidant capacity
leads to oxidative stress and results in damage of
nucleic acids, lipids, and proteins causing cell muta-
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MICHA£ OTR BA et al.
genesis or death (18, 37, 38). The human body has a
complex antioxidant defense system which contains
small-molecule antioxidants (e.g., glutathione, uric
acid, vitamin C, vitamin E) as well as antioxidant
enzymes such as SOD, GPx and CAT (37).
In the present study, we have examined the
effect of fluphenazine and perphenazine on the
activity of antioxidant enzymes. Fluphenazine in
concentrations of 0.1 and 1.0 µM and perphenazine
in concentrations of 1.0 and 3.0 µM significantly
increased SOD activity, decreased CAT activity and
exerted different effect on GPx activity (Fig. 3A, 3B
and 3C). The observed changes in antioxidant
enzymes activity after exposure of melanocytes to
the analyzed drugs might be the main reason for
overproduction of superoxide anion and elevated
level of H2O2 (Fig. 3D) that cannot be eliminated.
This indicates that antioxidant defense system does
not work properly. The obtained results also indicate
that fluphenazine possesses greater stimulatory
effect on SOD formation, produces higher levels of
hydrogen peroxide and therefore induces oxidative
stress in melanocytes, what confirms that
fluphenazine is more toxic than perphenazine.
The fluphenazine and perphenazine induced
changes in SOD and GPx activities were also stated
by other authors. Abdalla et al. (39) showed the
increased SOD and decreased GPx activity as well
as an accumulation of H2O2 in red blood cells in
schizophrenic patients treated with fluphenazine and
chlorpromazine in combination with 8 different
antipsychotic drugs. Michel et al. (40) demonstrated
the increase of SOD activity in the brain of patients
with schizophrenic psychosis treated with
fluphenazine and Zhang et al. (41) suggested that
increased SOD level in schizophrenic patients, treated with fluphenazine, perphenazine, trifluperazine
and chlorpromazine in combination with 3 different
antipsychotic drugs, may be a response to increased
oxidative stress associated with neuroleptic treatment (40, 41).
One has to take into consideration that
fluphenazine and perphenazine concentrations
found to have a modulatory effect on melanogenesis
and antioxidant defense system in normal human
melanocytes are about 10- to 100-fold higher for
fluphenazine and 20- to 60-fold higher for perphenazine than the concentrations normally
observed in blood (42). However, we have previously demonstrated that phenothiazine derivatives
form complexes with melanin, what may lead to the
accumulation of these drugs in melanin reach tissues
(14, 36). Such accumulation increases the drug concentration and may induce some toxic effects on
melanin containing cells and surrounding tissues.
Thus, it is possible that fluphenazine and perphenazine concentrations in melanocytes may be
significantly higher than that in blood and therefore
the reduction of melanin content, the inhibition of
tyrosinase activity as well as the depletion of cellular antioxidant status in the presence of these drugs
could be observed in vivo.
CONCLUSION
Due to the fact that phenothiazine derivatives
are the subject of new researches, it is important to
explain molecular mechanisms underlying these
drugs toxic effects. The present work provides the
first in vitro study on mechanisms involved in
fluphenazine and perphenazine induced disorders in
HEMn-DP cells. The observed changes in melanization process and antioxidant defense system in pigmented cells exposed to the analyzed drugs in vitro
suggest a significant role of melanin and melanocytes
in the mechanisms of undesirable side effects of these
drugs in vivo. The presented differences in modulation of biochemical processes in melanocytes may be
an explanation for various toxic activity of the analyzed phenothiazine derivatives in vivo.
Declaration of interest
All authors declare that there are no conflicts
of interest.
Acknowledgment
This work was financially supported by the
Medical University of Silesia in Katowice, Poland
(Grants No. KNW-2-002/D/5/K and KNW-1041/N/5/0).
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Received: 20. 07. 2015