cell biochemistry and function

Cell Biochem Funct 2010; 28: 394–402.

Published online in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/cbf.1669

Testosterone suppresses oxidative stress in human neutrophils

Douglas Popp Marin 1*, Anaysa Paola Bolin 2, Rita de Cassia Macedo dos Santos 2,
Rui Curi 3 and Rosemari Otton 1,2
1
Postgraduate Program—Human Movement Sciences Institute of Physical Activity and Sport Sciences, Cruzeiro do Sul University,
São Paulo, SP 01506-000, Brazil
2
Biological Science and Health Sciences, Cruzeiro do Sul University, São Paulo, SP 03342-000, Brazil
3
Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Av. Prof. Lineu Prestes,
1524, 05508-900, Butantan, São Paulo, SP, Brazil

The in vitro effect of testosterone on human neutrophil function was investigated. Blood neutrophils from healthy male subjects were isolated
and treated with 10 nM, 0.1 and 10 mM testosterone for 24 h. As compared with untreated cells, the testosterone treatment produced a
significant decrease of superoxide production as indicated by the measurement of extra- and intracellular superoxide content. An increment in
the production of nitric oxide was observed at 0.1 and 10 mM testosterone concentrations, whereas no effect was found for 10 nM. Intracellular
calcium mobilization was significantly increased at 10 nM, whereas it was reduced at 10 mM testosterone. There was an increase in phagocytic
capacity at 10 nM and a decrease of microbicidal activity in neutrophils treated with testosterone at 10 mM. Glutathione reductase activity was
increased by testosterone treatment, whereas no effect was observed in other antioxidant enzyme activities. An increase in the content of thiol
groups was observed at all testosterone concentrations. Lipid peroxidation in neutrophils evaluated by levels of TBARS was decreased at
10 nM and 0.1 mM testosterone. These results indicate the antioxidant properties of testosterone in neutrophils as suggested by reduction of
superoxide anion production, and lipid peroxidation, and by the increase in nitric oxide production, glutathione reductase activity and the
content of thiol groups. Therefore, the plasma levels of testosterone are important regulators of neutrophil function and so of the inflammatory
response. Copyright # 2010 John Wiley & Sons, Ltd.

key words — testosterone; leukocytes; antioxidant; oxidative stress; free radicals

INTRODUCTION risk factors such as age, smoking and sedentary life.5 In

Testosterone is a major circulating androgen, which is
synthesized by testicular Leydig’s cells and in small
amounts by the adrenal glands. It is now well accepted
that total and bioavailable serum testosterone decline
progressively with aging in men. Low serum levels of
testosterone have been associated with numerous age related
adverse health conditions including abdominal obesity,
diabetes, and prediabetic states (such as insulin resistance,
impaired glucose tolerance, and metabolic syndrome),
dyslipidemia, low bone and muscle mass, impaired sexual
function, depressed mood, frailty, and decreased quality of
life.1–4 Also, reduced androgen levels associated with the
aging or androgen deficiencies increase cardiovascular risk
factors and produce marked adverse effects on cardiovas-
cular function. Recently, low levels of testosterone are
associated with carotid atherosclerosis in men regardless the
* Correspondence to: D. P. Marin, Postgraduate Program—Human Move-
ment Sciences Institute of Physical Activity and Sport Sciences, Cruzeiro do
Sul University, São Paulo, SP 01506-000, Brazil.
E-mail: douglas.marin@metodista.br
addition, low testosterone levels may predict increased
cardiovascular risk in men.6 Patients with cardiovascular
disease treated with exogenous testosterone showed
improvement in arterial vasodilatation7 and lipid profile
(decrease of serum triglyceride, LDL, and increase of HDL
cholesterol levels).8
Reactive oxygen species (ROS) are involved in physio-
logical and pathological conditions such as aging and
atherosclerosis.9 ROS are produced in human neutrophils,
mainly through the respiratory burst to kill internalized
pathogens. In addition, ROS can regulate enzyme activities,
signal transcription, and gene expression.9 Atherosclerosis is
a multifactor disease characterized by inflammatory,
metabolic, and hemodynamic processes. Among the
mechanisms involved, the oxidation of LDL-cholesterol
plays an important part. 10 Activated neutrophils can
contribute for the development of this process increasing
production of ROS through NADPH oxidase activation in
the site of vascular damage and by releasing the
mieloperoxidase (MPO) enzyme. MPO pronounces the
toxicity of superoxide anion and produces a more potent
oxidant agent, the hypochlorous acid, contributing for

Received 3 December 2009

Revised 26 February 2010
Copyright # 2010 John Wiley & Sons, Ltd. Accepted 6 April 2010
 effect of testosterone on neutrophil function
LDL-cholesterol oxidation. In addition, superoxide anion
produced by activated neutrophils can be transformed into
other more ROS such as hydroxyl radical and singlet
oxygen. These ROS cause lipid peroxidation that is the first
step of the atherosclerosis process.11
Békési and coworkers12 have shown that human
neutrophils treated with testosterone for 2 h show decreased
superoxide anion production, suggesting a possible anti-
oxidant effect. However, little is known about the effects of
testosterone on neutrophil function, damage on biomole-
cules induced by oxidative stress, production of ROS and
regulation of antioxidant enzyme activities. The aim of this
study was to investigate the effect of testosterone on human
neutrophil function. For this purpose, the following
measurements were performed in neutrophils after in vitro
testosterone treatment: activities of superoxide dismutase
(SOD), catalase, glutathione peroxidase and glutathione
reductase, production of superoxide anion by activation of
NADPH oxidase complex, production of nitric oxide (NO),
content of TBARS, intracellular Ca2þ mobilization, and
microbicidal and phagocytic capacities.
MATERIALS AND METHODS
Reagents
All reagents for buffers were obtained from Labsynth
(Diadema, SP, Brazil). Histopaque-1077, HEPES, ethylene
glycol tetracetic acid (EGTA), ethylenediamine tetraacetic
acid (EDTA), dihydroethidium (DHE), ionomycin, sodic
azide, albumin, nicotinamide adenine dinucleotide phos-
phate (NADPH), nicotinamide adenine dinucleotide
(NADH), dimethyl sulfoxide (DMSO), 2,4-dinitrophenyl-
hydrazine (DNPH), 5,50 -dithiobis(2-nitrobenzoic acid)
(DTNB), Triton X-100, phenazine methosulphate (PMS),
nitroblue tetrazolium (NBT), thiobarbituric acid (TBA), N-
ethylmalemide (NEM), phorbol-12-myristate 13-acetate
(PMA), guanidine-HCL, reduced glutathione (GSH), oxi-
dized glutathione (GSSG), lipopolysaccharide (LPS),
butylated hydroxytoluene (BHT) and hydrogen peroxide
(H2O2) were obtained from Sigma Chemical Co (St Louis,
MO, USA). RPMI-1640 culture medium, lucigenin,
acetoxymethylester (Fura 2-AM) and pluronic acid were
obtained from Molecular Probes (Eugene, OR, USA).
Cell isolation
This study was approved by the Human Ethic Committee of
the Cruzeiro do Sul University and was performed in
accordance with the Declaration of Helsinki. All volunteers
gave written informed consent before enrollment. Peripheral
blood neutrophils obtained from non-smoking and non-
obese healthy young men (age ranging from 18–30 years
old) were collected by venopuncture procedure and placed
in vacuum/siliconized test tubes containing EDTA as
anticoagulant agent. Blood samples (20 ml) were diluted
in the same volume of phosphate buffered saline (0.137 M
NaCl, 2.7 mM KCl, 8.0 mM Na2HPO4, pH 7.4) solution and
Copyright # 2010 John Wiley & Sons, Ltd.
395
neutrophils were separated using Histopaque 1077 accord-
ing to the manufacturer’s instructions (Sigma). The plasma
and intermediary layer were removed and both neutrophils
and erythrocytes were collected from sediment. Erythro-
cytes were lysed with a hemolysis solution (150 mM NH4Cl,
10 mM NaHCO3, 0.1 mM EDTA, pH 7.4) and subsequently
centrifuged for 10 min (600 g at 48C). This procedure was
repeated twice for total red blood cell lyses. Thereafter,
neutrophils were washed with PBS followed by centrifu-
gation for 10 min (600 g at 48C). After centrifugation,
peripheral blood neutrophils were collected and maintained
in RPMI-1640 medium supplemented with 2 mM glutamine,
20 mM HEPES, 10% fetal calf serum, 10 U/ml penicillin,
and 10 mg/ml streptomycin. The number of viable cells
(>95% neutrophils) was determined in a Neubauer chamber
under an optical microscope by Trypan blue exclusion.13
Cytotoxic effect of testosterone
This assay was performed to investigate the testosterone
toxicity in human neutrophils. Cells (1
106 per ml) were
cultured with different concentrations of testosterone (0, 0.1,
1, 5, 10, 25, 50, 75, and 100 mM) for 24 h. Afterwards,
neutrophils were collected to determine the proportion of
viable cells by Trypan Blue exclusion.
Cell culture and treatment
Isolated neutrophils were seeded in a 6-well dishes at a
density of 5
106 cells/well in the presence of testosterone
10 nM, 0.1 and 10 mM solubilized in DMSO. The cells were
incubated in a humidified atmosphere of 5% CO2 at 378C for
24 h. The concentration of DMSO used was always lower
than 2% of the total volume of culture medium. This
concentration of DMSO has shown not to be toxic for the
cells as found in preliminary experiments (data not shown).
Cells left untreated (control) or treated with DMSO (vehicle)
were included in all experiments. There was no difference
between untreated and DMSO-treated cells in all cases.
Production of ROS using lucigenin-enhanced
chemiluminescence Assay
Lucigenin is extensively used to measure extracellular
superoxide content mainly produced through NADPH
oxidase activation.14 Lucigenin (5 mM) was added to cells
freshly obtained and incubated (5
105 per well) in absence
or in the presence of testosterone (10 nM, 0.1 and 10 mM) in
Tyrode’s buffer (137 mM NaCl, 2.68 mM KCl, 0.49 mM
MgCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 5.6 mM D-
glucose, and 5 mM HEPES), pH 7.4. The experiments were
carried out in the presence and absence of PMA (phorbol
myristate acetate—20 ng per well). The chemiluminescence
response was monitored for 45 min in a luminometer (Tecan,
Salzburg, Austria). The total superoxide anion production
during 45 min was expressed as integrated area under curve
(AUC) analysis.
Cell Biochem Funct 2010; 28: 394–402.
396 d. p. marin
Measurement of ROS by using dihydroethidium
DHE was used for the fluorimetric measurement of
intracellular superoxide content. DHE is a lipophilic probe
that readily diffuses across cell membranes. Once inside the
cell, DHE is rapidly oxidized to ethidium (a red fluorescent
compound) by superoxide and H2O2 (in the presence of
peroxidase). Ethidium is trapped in the nucleus by
intercalating into DNA, leading to an increase of ethidium
fluorescence. The cells (5
105 per well) were pre-loaded
with DHE (5 mM) by incubation at room temperature in the
dark for 15 min. Afterwards, the cells were treated with
testosterone at 10 nM, 0.1 and 10 mM in Tyrode’s buffer and
analyzed in a fluorimeter (Tecan, Salzburg, Austria). The
assay was carried out in the presence and absence of PMA
(20 ng per well). Fluorescence was measured at 396 nm
of excitation wavelength and at 590 nm of emission
wavelength.
Nitric oxide production
Nitric oxide production was measured according to Ding
et al.15 through nitrite determination. Nitric oxide is rapidly
converted to nitrite in aqueous solution and, therefore, total
nitrite can be used as indicator of nitric oxide concentration.
Neutrophils (1
106 per well) were incubated with
testosterone (10 nM, 0.1 and 10 mM) and LPS (10 mg per
well) for 4 h. Afterwards, spectrophotometric analysis of
total nitrite in the supernatants was performed by using
Griess’s reagent (1% sulfanilic acid, 0.1% N-1-naphthyl-
ethylenediamine dihydrochloride). Absorbance was
measured at 550 nm and nitrite concentration was deter-
mined using sodium nitrite as standard.
Phagocytic and microbicidal capacities
Candida albicans (kindly given by Dr Sandra Cocuzzo
Sampaio from the Butantan Institute, São Paulo, Brazil)
were cultured in Sabouraud agar and transferred onto fresh
agar 24 h prior to the killing assay. Candida cells were
harvested with phosphate-buffer and counted in a Neubauer
chamber. Under these conditions, sufficient numbers of
Candida cells can be obtained without germ tubes and
hyphae. Cell viability was checked by trypan blue exclusion.
Candida albicans (1
106/ml) were opsonized with 2.5%
normal human serum, for 60 min at 378C, prior to each
assay. Neutrophils (1
106 cells per ml) were treated with
testosterone for 3 h at 378C in an atmosphere containing 5%
CO2 in the presence of Candida albicans. After incubation,
homogenates were centrifuged and stained with Rosenfeld
stain. For phagocytic and microbicidal capacity assays,
different scores were given to the number of neutrophils that
had internalized and killed no Candida albican cells (
0); 1
or 2 (
1); 3 or 4 (
2); >4 cells (
3). The indexes of
microbicidal and phagocytic capacity were calculated by the
sum of the scores obtained in each experiment, as previously
suggested.16
Copyright # 2010 John Wiley & Sons, Ltd.
ET AL.
Preparation of homogenates
Pellets from neutrophil culture (5
106) were mixed with
1.0 ml of 50 mM phosphate buffer, briefly vortexed and
sonicated in a Vibra Cell (Connecticut, USA) as previously
described.17 A centrifugation step was included (10 000
g,
10 min, at 48C) in order to eliminate debris from the crude
homogenate; the supernatant was then used for further
analysis. The extracts for enzyme assays and oxidative lesions
were prepared with a 50 mM phosphate buffer (pH 7.4).
Assay of superoxide dismutase activity (SOD)
The activity of SOD was measured according to the method
described by Ewing and Janero.18 The complete reaction
buffer for total SOD assay consisted of 50 mM phosphate
buffer, pH 7.4, containing 0.1 mM EDTA, 50 mM nitro-
bluetetrazolium (NBT), 78 mM NADH, and 3.3 mM phena-
zine metasulphate (PMS) used as O2 generator. Discrimi-
nation between Cu/Zn SOD and MnSOD was achieved by
using NaCN to inhibit the Cu/Zn SOD. The absorbance was
continuously monitored at 560 nm over 2 min as an index of
NBT reduction in the kinetic mode.
Assay of catalase activity
The activity of catalase was measured according to the
method of Aebi19 by monitoring the initial rate of
disappearance of H2O2 (initial concentration, 10 mM) at
240 nm (e ¼ 0.071 mM1 cm1). The complete reaction
mixture for catalase activity assay consisted of 0.1 mM
phosphate buffer, pH 7.4, and 10 mM H2O2. For the assay,
900 ml phosphate buffer, 50 ml H2O2 solution and 50 ml
sample were added in a 1 ml cuvette. The reaction was
initiated by addition of H2O2, and the absorbance was
monitored over 2 min at 240 nm.
Assay of glutathione peroxidase activity (GPx)
The activity of GPx was measured according to the method
of Mannervik.20 The enzyme activity was determined by
using 2.5 U/ml glutathione reductase, 10 mM GSH, 250 mM
sodic azide (as catalase inhibitor), and reduced form of
1.2 mM b-NADPH in the presence of 4.8 mM tert-buthyl
hydroperoxide used as substrate. The oxidation of NADPH
was monitored at 340 nm during 2 min in 0.2 M phosphate
buffer, pH 7.4, in a Pharmacia Biotech spectrophotometer
(model Ultrospec 3000).
Assay of glutathione reductase activity
The activity of glutathione reductase was determined using
the method described by Mannervik.20 The enzyme activity
was assayed by using 3.6 mM NADPH and oxidized
glutathione 10 mM. The oxidation of NADPH was
monitored in 0.2 M phosphate buffer, pH 7.4, at 340 nm
during 2 min.
Cell Biochem Funct 2010; 28: 394–402.
 effect of testosterone on neutrophil function 397

Parameters of oxidative damage Statistical analysis. The results are expressed as mean
 SEM and (n) is the number of experiments (at least 3).
Content of TBARS. To evaluate the extension of lipid
ANOVA was employed to detect differences among the
peroxidation induced during testosterone treatment, levels of
groups followed by the Tukey’s post-test (INStat; Graph Pad
lipid oxidation products were measured using TBARS
Software, San Diego, CA, U.S.A.).
(TBA-reactive substances) in cell homogenates as pre-
viously described.21 After extraction, BHT (solubilised in
4% ethanol solution) was added to sample to stop oxidation RESULTS
reactions. To detect the colored adducts, each sample was
Cell viability
incubated at 1008C for 15 min with phosphate buffer
(50 mM pH 7.4), 0.375% TBA in 0.25 M HCl and 2% Triton The viability of human neutrophils was not affected by
X-100. After reaching the room temperature, absorbance of treatment with testosterone at concentrations from 0.1 to
the solutions was measured at 535 nm. A standard curve was 50 mM. However, at 75 and 100 mM testosterone concen-
performed using 10 mM malondialdehyde (MDA). trations, the cell viability was decreased by 40% and 60%,
respectively (R2 ¼ 0.90) (Figure 1).
Thiol groups. Thiol groups were measured as indicators of
amino acid oxidation in the total protein fraction. The higher
content of protein sulfhydryl groups (-SH) is an indicator of Intra- and extracellular superoxide content
a less oxidizing environment. Homogenate was obtained by
precipitation with 20% trichloracetic acid solution in ice. Appropriate controls were carried out containing testoster-
After washing once with 0.3 M HClO4, 5 mM EDTA and one plus DHE or lucigenin and PMA in the assay medium
0.06% bipyridine solution, and twice with the mixtured 1:1 without cells. Testosterone did not directly affect DHE and
ethyl acetate:ethanol (v/v), the protein precipitate for thiol lucigenin ROS-detecting system. Intracellular superoxide
determination assay was dried at room temperature to content was determined through oxidation of DHE that
remove traces of organic solvent. The pellet was sub- readily diffuses across the cell membrane. Testosterone
sequently dissolved in 500 ml 6 M guanidine:HCl and strongly inhibited basal and PMA-stimulated superoxide
reduced thiol groups were detected by formation of colored production by neutrophils. The inhibitory effect of
adducts upon reaction with 4 mM 5.5-dithio-bis(2-nitro- testosterone by non PMA-stimulated neutrophils was of
benzoic acid) solution (DTNB). A control treatment with 42% and 34%, respectively, for 10 nM and 0.1 mM. In PMA-
10 mM N-ethylmaleimide solution—a specific thiol-block- stimulated cells, testosterone treatment promoted a signifi-
ing compound—was introduced to remove non-specific cant reduction in intracellular superoxide production by
DTNB bonds from other organic groups present in the 14%, 15% and 20% for testosterone at 10 nM, 0.1 and
samples. The absorbance at 412 nm of DTNB-treated 10 mM, respectively when compared with the control group
samples was calculated using GSH as standard.22 (Figure 2A). Kinetic monitoring of extracellular superoxide
levels was the slowest event that occurred within 45 min
Fluorescent measurement of intracellular calcium concen- after treatment of the cells with testosterone and PMA
tration [Ca2 þ ]i. Changes in cytosolic calcium levels were (Figure 2B). Extracellular superoxide levels were signifi-
fluorimetrically monitored using the calcium-sensitive cantly decreased (AUC p < 0.05) by testosterone at all
probe Fura 2-AM as previously described.13 The loading
period for Fura 2-AM (5 mM) was 1 h at 378C in cells
suspended (1
106/ml) in Tyrode’s solution. Afterwards,
cells were washed and treated with testosterone (10 nM, 0.1
and 10 mM). Intracellular [Ca2þ]i was monitored for 60 min
and fluorescence of Fura 2-AM was measured in 300 ml
aliquots using a spectrofluorimeter (Tecan, Salzburg,
Austria) at 510 nm emission wavelength and excitation
wavelengths alternating between 340 and 380 nm. Trans-
formation of the fluorescent signal for [Ca2þ]i was
performed by calibration with ionomycin (100 mM, maxi-
mum concentration) followed by EGTA addition (60 mM,
minimum concentration) according to the Grynkiewicz
equation, using the Kd of 224 nM (according to the
Molecular Probes catalog). The total calcium mobilization
during 60 min was expressed as integrated AUC analysis.
Figure 1. Effect of testosterone on human neutrophils viability. Neutro-
Protein determination. Protein content of cell homogenates phils were treated with testosterone at 0, 0.1, 1, 5, 10, 25, 50, 75, and
was determined by the method of Bradford,23 using BSA as 100 mM for 24 h. The values are presented as mean  SEM of six exper-
standard. iments. p < 0.05, for comparison with the control group

Copyright # 2010 John Wiley & Sons, Ltd. Cell Biochem Funct 2010; 28: 394–402.
398 d. p. marin ET AL.

Figure 2. Superoxide production by testosterone-treated human neutrophils. PMA-stimulation of superoxide anion production measured in human
neutrophils (5
105 per well) acutely treated with testosterone (T) at 10 nM, 0.1 and 10 mM. (A) Dihydroethidium (5 mM) was used to measure intracellular
superoxide content. (B) Extracellular superoxide content was assayed using lucigenin (5 mM). The values are presented as mean þ SEM of ten determinations. 
p < 0.05 as compared with the control group

concentrations (63%, 55% and 56% for 10 nM, 0.1 and Enzyme activities
10 mM, respectively) as compared to the control group.
The enzymatic antioxidant defense of the human neutrophils
was represented by total and manganese superoxide
dismutase, catalase, and glutathione peroxidase and
Production of nitric oxide
Incubation of LPS-stimulated neutrophils with testosterone
for 4 h caused a marked increase of nitric oxide production
by 58% and 68%, respectively, for testosterone at 0.1
and10 mM (Figure 3). On the other hand, no significant
alteration of nitric oxide production was found after
incubation of cells with 10 nM testosterone.

Phagocytic and microbicidal capacities

No significant changes were found in phagocytic capacity of
neutrophils treated with testosterone at concentrations of 0.1
and 10 mM. However, 10 nM testosterone promoted an
increment of about 36% as compared to the control group
(Figure 4A). Microbicidal capacity of neutrophils was
decreased by 46% at 10 mM testosterone when compared to
control cells (Figure 4B).
Figure 3. Production of nitric oxide by testosterone-treated human neu-
trophils. Production of nitric oxide was measured in human neutrophils
(1
106 per ml) treated with testosterone at 10 nM, 0.1 and 10 mM. LPS-
stimulated (10 mg per well) nitric oxide production after 4 h treatment. The
values are presented as mean þ SEM of five determinations.  p < 0.05 as
compared to the control group

Copyright # 2010 John Wiley & Sons, Ltd. Cell Biochem Funct 2010; 28: 394–402.
 effect of testosterone on neutrophil function 399

Figure 5. Maximal glutathione reductase activity after 24 h culture of

human neutrophils (5
106 per ml) with testosterone at 10 nM, 0.1 and
10 mM. The values are presented as mean þ SEM of seven determinations.

p < 0.05 as compared with the control group. #p < 0.05 as compared with
testosterone 0.1 and 10 mM

DISCUSSION
The primary functions of neutrophils in the innate immune
response are to phagocyte and kill invading microbial
pathogens through a potent antimicrobial arsenal generated
during the respiratory burst. In the present investigation,
Figure 4. Phagocytic and microbicidal capacities of testosterone-treated the treatment of human neutrophils with testosterone
human neutrophils. (A) Score of phagocytic and (B) microbicidal capacities
of human neutrophils (1
106 per ml) after 3 h incubation with Candida
albicans (1
106 per ml) and testosterone (10 nM, 0.1 and 10 mM). The
values are presented as mean þ SEM of four determinations.  p < 0.05 as
compared with the control group

reductase activities. Total and MnSOD, catalase and

glutathione peroxidase activities were not changed by
testosterone treatment. On the other hand, glutathione
reductase activity was increased by 3.9-, 2.2-, and 2.4- fold
for 10 nM, 0.1 and 10 mM, respectively, compared to the
control group (Figure 5).

Indicators of oxidative stress

Testosterone at 10 nM and 0.1 mM reduced by 63% and 58%,
respectively, the TBARS levels of neutrophils after 24 h
culture (Figure 6A). In contrast, levels of thiol groups were
increased by 42%, 68% and 60% for 10 nM, 0.1 and 10 mM,
respectively, when compared with the control group
(Figure 6B).

Intracellular calcium mobilization

Testosterone induced markedly changes in neutrophil
intracellular calcium mobilization during 60 min as com-
pared with the control group (Figure 7). Neutrophils treated
with testosterone at 10 nM showed increased total [Ca2þ]i
release by 153%. In contrast, a significant decrease by 17%
(AUC p < 0.05) was observed for [Ca2þ]i in neutrophils
treated with testosterone at 10 mM.
Figure 6. Oxidative lesions in biomolecules. Oxidative lesions in biomo-
lecules of human neutrophils (1
107 per ml) after 24 h culture with
testosterone 10 nM, 0.1 and 10 mM. (A) Concentration of MDA as lipoper-
oxidation marker, (B) concentration of thiol group. The values are presented
as mean þ SEM of six determinations.  p < 0.05 as compared with the
control group

Copyright # 2010 John Wiley & Sons, Ltd. Cell Biochem Funct 2010; 28: 394–402.
400
Figure 7. Kinetic of calcium release [Ca2þ]i in testosterone-treated human neutrophils. Kinetic of calcium release ([Ca2þ]i; nM) in human neutrophils. Cells
(1
106 per well) were previously incubated with Fura 2-AM (5 mM) for 1 h and then acutely treated with testosterone 10 nM, 0.1 and 10 mM. The values are
presented as mean of six determinations
exhibited a significant increment in phagocytic capacity,
indicating an improvement of cell function. This increment
in neutrophil phagocytosis was observed only at a
physiological range of testosterone (10 nM). High concen-
tration of the hormone (10 mM) promoted a significant
reduction in microbicidal capacity with no effect on
phagocytosis. Similar to our results, a high dose of
testosterone (35 mM) also reduced the levels of IgM in
splenic leucocytes,24 whereas no difference was observed at
a lower concentration. Thus, the marked effect of
testosterone on neutrophils seems to be found at a
physiological range of the hormone. Intra- and extracellular
Ca2þ are necessary for efficient phagocytic and microbicidal
activities. The fusion of granules, formation of phagosome,
and the process of exocytose are very sensitive to changes in
[Ca2þ]i.25 The impairment of [Ca2þ]i mobilization at
supraphysiological doses of testosterone possibly reduces
the intracellular killing of bacteria by inhibiting the release
of granule proteins and ROS into the phagosome.
Recent studies have been reported that testosterone can
act through a nongenomic pathway in macrophages and T
lymphocytes.26 Nongenomic signaling of testosterone
involves a rapid increase of [Ca2þ]i, cAMP or activation
of PKC.27 In lymphocytes, testosterone rapidly raises
intracellular [Ca2þ]i predominantly due to an extracellular
calcium influx, whereas in macrophages the increase in
testosterone-induced calcium mobilization occurs through
release from intracellular calcium stores.27 Our data
demonstrates that testosterone at physiological levels
rapidly raises [Ca2þ]i. Nevertheless, as a limitation of this
study, we cannot state that testosterone promoted a
nongenomic signalling in human neutrophils.
Superoxide anion is the first ROS generated during the
respiratory burst via reduction of molecular oxygen through
activation of NADPH oxidase.28 PMA stimulates neutro-
Copyright # 2010 John Wiley & Sons, Ltd.
d. p. marin ET AL.
phils by activating NADPH-oxidase via PKC pathway acting
as a diacylglycerol-analog, with no involvement of
intracellular [Ca2þ]i29. Superoxide anion production by
activated neutrophils plays a role during microbicidal
activity against pathogens. However, under certain circum-
stances, an excess of superoxide overcomes local antiox-
idant defense and leads to oxidative stress and oxidative
tissue injury. According to previous studies,12,30 we
demonstrated that testosterone inhibits superoxide pro-
duction by PMA-stimulated or non-PMA-stimulated neu-
trophils, as assessed by DHE and lucigenin assays. This
indicates a modulation of signaling events upstream
superoxide production by testosterone. Békési et al.12 have
also reported reduction of superoxide levels in human
neutrophils by treatment with steroids such as cortisol,
estradiol, and progesterone. Androgens such as dihydrotes-
tosterone have been shown to be a powerful antioxidant,
effectively preventing the formation of lipid peroxides.31
Bennett et al.32 reported that testosterone with a keto group
at the C3 position, showed a mild preventive effect
compared to dihydrotestosterone. In addition, leukocytes
may express aromatase33 and 5a-reductase,34 and thus
testosterone can undergo aromatization to 17b-estradiol or
reduction to dihydrotestosterone, respectively. Therefore,
the antioxidant properties of testosterone on human
neutrophils may involve several signaling pathways.
The reduction of superoxide anion production induced by
testosterone is an antioxidant mechanism. In fact, decrease
of TBARS levels and damage to proteins was observed. An
exacerbated production of ROS by neutrophils results in an
attack on cellular components involving polyunsaturated
fatty acid residues of phospholipids, forming the final
product of the peroxidation process being MDA.35 The
superoxide anion itself does not cause lipid peroxidation.
However, superoxide anion produced by activated neutro-
Cell Biochem Funct 2010; 28: 394–402.
 effect of testosterone on neutrophil function
phils can be transformed into other ROS, which are able to
oxidize lipids and proteins, for example, the LDL-
cholesterol. LDL-cholesterol in plasma and vascular
endothelium is susceptible to oxidation, which has been
considered the main event for the development of
atherosclerosis.10 The elevated production of free radicals
is a major factor in the abnormal interaction of the
neutrophils and vascular wall, contributing for the functional
and structural changes associated with atherosclerosis.
Therefore, reduced physiological levels of testosterone
may contribute to increase the cardiovascular risk mediated
by oxidative stress.
The role of androgenic steroids in the regulation of the
antioxidant systems is still poorly understood. In the present
study, activities of total SOD and MnSOD, catalase and
glutathione peroxidase were not affected in neutrophils after
testosterone treatment. In agreement with our findings, no
significant effects of androgens have also been reported on
antioxidant enzymes in erythrocytes.36 On the other hand, a
significant increase of glutathione reductase activity was
observed. GSH is the most important antioxidant agent in
human neutrophils.37 GSH can directly detoxify ROS.
During inactivation of oxidizing agent, there is a depletion of
GSH and formation of GSSG (oxidized glutathione). The
increase of glutathione reductase activity could elevate the
content of GSH and, therefore, the antioxidant defense
capacity. On the other hand, increased levels of GSSG
reflects an environment predominantly oxidative, which
promotes formation of disulphide bridges (S-S) in proteins
and impairment of their activities.38 As a consequence of the
increase in glutathione reductase activity, which improves
the GSH storage, thiol groups were significantly increased
by testosterone treatment. An important feature of most
thiols is that they can act as reducing agents. ROS have a
strong tendency to transfer electrons to other species.
Consequently, in the case of an oxidant-thiol interaction, the
oxidant is neutralized to a relatively lower toxic byproduct.
Nitric oxide (NO) produced from the constitutively NO
synthase (NOS) acts as an important oxidative biological
signaling molecule in a large variety of physiological
processes, including neurotransmission, blood pressure
regulation, and immune regulation.35 Despite these import-
ant physiological functions, NO is also a well-known toxic
agent and is thought to promote a number of chronic
inflammatory diseases. Inducible NOS is expressed in
various cells including macrophages, neutrophils, and
epithelial cells, and it produces excessive NO during
infection, inflammation, and states of physiological stimu-
lation.39 NO is generated during immune and inflammatory
responses as a toxic agent toward infectious organisms. NO
may induce toxic reactions against other tissues of the host
and since it is generated at high levels in certain types of
inflammation, it has been implicated as a pro-inflammatory
agent.40 Conflicting results have been reported about
regulation of NO synthesis by testosterone in immune cells.
Frield et al.41 found a decrease in NO synthesis induced by
testosterone in murine macrophages. Conversely, our results
demonstrate that testosterone at a physiological concen-
Copyright # 2010 John Wiley & Sons, Ltd.
401
tration does not alter nitric oxide production. The basal
levels of NO are high and further increase after stimulation
with LPS and testosterone at 0.1 and 10 mM. Supraphysio-
logical concentrations of testosterone (0.1 and 10 mM) can
increases NO levels characterizing an inflammatory process
and this may be due to activation of iNOS, which produces
large amounts of NO, which can act as a pro-inflammatory
molecule. NO can interact with pro-inflammatory oxidants
(e.g., superoxide, hydrogen peroxide, hypochlorite) to form
the peroxynitrite that modify lipids and proteins. When NO
concentration exceeds that of superoxide anion, NO
predominantly exhibits an inhibitory effect on oxidative
damage.42 Lower levels of superoxide induced by testos-
terone treatment may mean less chance for conversion of NO
to peroxynitrite, therefore, increasing cellular levels of NO.
The anti-atherogenic properties of NO have been exten-
sively studied. NO presents anti-aggregators effects on
platelets, and antioxidant, anti-inflammatory, antiprolifera-
tive, and vasodilators effects on the vasculature.42
Taken together, our results point out the antioxidant
properties of testosterone in human neutrophils as suggested
by suppression of superoxide anion, and TBARS levels, and
by the increase of nitric oxide levels, glutathione reductase
activity and thiol groups. Testosterone concentration taken
as physiological concentration (10 nM), have a greater
antioxidant potential than higher concentrations. Thus, the
maintenance of physiological levels of testosterone con-
tributes for the redox balance in human neutrophils.
Reduced concentrations of testosterone in elderly and in
some pathological conditions may contribute for the
development of cardiovascular risk through induction of
oxidative stress in immune cells.
ACKNOWLEDGEMENTS
The authors are indebted to the constant assistance of
Professor Marcelo Paes de Barros and Dr Sandra Sampaio.
This research is supported by FAPESP and Cruzeiro do Sul
University. Financial support for this work was provided by
FAPESP (Fundação de Amparo a Pesquisa do Estado de São
Paulo – 07/03334-6) and Universidade Cruzeiro do Sul.
CONFLICT OF INTEREST
No conflict of interest.
REFERENCES
1. Kaufman JM, Vermeulen, A. The decline of androgen levels in elderly
men and its clinical and therapeutic implications. Endocr Rev 2005; 26:
833–876.
2. Matsumoto AM. Andropause: clinical implications of the decline in
serum testosterone levels with aging in men. J Gerontol A Biol Sci Med
Sci 2002; 57: M76–99.
3. Rodriguez A, Muller, DC, Metter, EJ, et al. Aging, androgens, and the
metabolic syndrome in a longitudinal study of aging. J Clin Endocrinol
Metab 2007; 92: 3568–3572.
4. Vermeulen A., Androgen replacement therapy in the aging male–a
critical evaluation. J Clin Endocrinol Metab 2001; 86: 2380–2390.
Cell Biochem Funct 2010; 28: 394–402.
402 d. p. marin
5. Laughlin GA, Barrett-Connor, E, Bergstrom, J., Low serum testoster-
one and mortality in older men. J Clin Endocrinol Metab 2008; 93: 68–
75.
6. Svartberg J, von Muhlen, D, Mathiesen, E, et al. Low testosterone levels
are associated with carotid atherosclerosis in men. J Intern Med 2006;
259: 576–582.
7. Webb CM, McNeill, JG, Hayward, CS, de Zeigler, D, Collins, P.,
Effects of testosterone on coronary vasomotor regulation in men with
coronary heart disease. Circulation 1999; 100: 1690–1696.
8. Wu S, Weng, X., Therapeutic effect of andriol on serum lipids and
apolipoproteins in elderly male coronary heart disease patients. Chin
Med Sci J 1992; 7: 137–141.
9. Droge W., Free radicals in the physiological control of cell function.
Physiol Rev 2002; 82: 47–95.
10. Griendling KK, Sorescu, D, Ushio-Fukai, M., NAD(P)H oxidase: role
in cardiovascular biology and disease. Circ Res 2000; 86: 494–501.
11. Kellogg EW, 3rd and Fridovich, I. Liposome oxidation and erythrocyte
lysis by enzymically generated superoxide and hydrogen peroxide.
J Biol Chem 1977; 252: 6721–6728.
12. Bekesi G, Kakucs, R, Varbiro, S, et al. In vitro effects of different
steroid hormones on superoxide anion production of human neutrophil
granulocytes. Steroids 2000; 65: 889–894.
13. Otton R, da Silva, DO, Campoio, TR, et al. Non-esterified fatty acids
and human lymphocyte death: a mechanism that involves calcium
release and oxidative stress. J Endocrinol 2007; 195: 133–143.
14. Hatanaka E, Levada-Pires, AC, Pithon-Curi, TC, Curi, R., Systematic
study on ROS production induced by oleic, linoleic, and gamma-
linolenic acids in human and rat neutrophils. Free Radic Biol Med
2006; 41: 1124–1132.
15. Ding AH, Nathan, CF, Stuehr, DJ., Release of reactive nitrogen
intermediates and reactive oxygen intermediates from mouse peritoneal
macrophages. Comparison of activating cytokines and evidence for
independent production. J Immunol 1988; 141: 2407–2412.
16. Sampaio SC, Sousa-e-Silva, MC, Borelli, P, Curi, R, Cury, Y., Crotalus
durissus terrificus snake venom regulates macrophage metabolism and
function. J Leukoc Biol 2001; 70: 551–558.
17. Otton R, Graziola, F, Hirata, MH, Curi, R, Williams, JF., Dietary fats
alter the activity and expression of glucose-6-phosphate dehydrogenase
in rat lymphoid cells and tissues. Biochem Mol Biol Int 1998; 46: 529–
536.
18. Ewing JF, Janero, DR., Microplate superoxide dismutase assay employ-
ing a nonenzymatic superoxide generator. Anal Biochem 1995; 232:
243–248.
19. Aebi H., Catalase in vitro. Methods Enzymol 1984; 105: 121–126.
20. Mannervik B., Glutathione peroxidase. Methods Enzymol 1985; 113:
490–495.
21. Fraga CG, Leibovitz, BE, Tappel, AL., Lipid peroxidation measured as
thiobarbituric acid-reactive substances in tissue slices: characterization
and comparison with homogenates and microsomes. Free Radic Biol
Med 1988; 4: 155–161.
22. Biteau B, Labarre, J, Toledano, MB., ATP-dependent reduction of
cysteine-sulphinic acid by S. cerevisiae sulphiredoxin. Nature 2003;
425: 980–984.
23. Bradford MM., A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem 1976; 72: 248–254.
Copyright # 2010 John Wiley & Sons, Ltd.
ET AL.
24. Saha NR, Usami, T, Suzuki, Y., In vitro effects of steroid hormones on
IgM-secreting cells and IgM secretion in common carp (Cyprinus
carpio). Fish Shellfish Immunol 2004; 17: 149–158.
25. Wilsson A, Lundqvist, H, Gustafsson, M, Stendahl, O., Killing of
phagocytosed Staphylococcus aureus by human neutrophils requires
intracellular free calcium. J Leukoc Biol 1996; 59: 902–907.
26. Benten WP, Guo, Z, Krucken, J, Wunderlich, F., Rapid effects of
androgens in macrophages. Steroids 2004; 69: 585–590.
27. Wunderlich F, Benten, WP, Lieberherr, M, et al. Testosterone signaling
in T cells and macrophages. Steroids 2002; 67: 535–538.
28. van der Vliet A., NADPH oxidases in lung biology and pathology: host
defense enzymes, and more. Free Radic Biol Med 2008; 44: 938–
955.
29. Granfeldt D, Samuelsson, M, Karlsson, A., Capacitative, Ca2þ influx
and activation of the neutrophil respiratory burst. Different regulation
of plasma membrane- and granule-localized NADPH-oxidase.
J Leukoc Biol 2002; 71: 611–617.
30. Bekesi G, Racz, K, Hrabak, A, et al. Systematic investigation of
different steroid precursors with respect to their effect on superoxide
anion production by human neutrophil granulocytes. Horm Metab Res
2004; 36: 155–163.
31. Bilinska B, Wiszniewska, B, Kosiniak-Kamysz, K, et al. Hormonal
status of male reproductive system: androgens and estrogens in the
testis and epididymis. In vivo and in vitro approaches. Reprod Biol
2006; 6 Suppl 1: 43–58.
32. Bennett MJ, Albert, RH, Jez, JM, et al. Steroid recognition and
regulation of hormone action: crystal structure of testosterone and
NADPþ bound to 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase.
Structure 1997; 5: 799–812.
33. Vottero A, Rochira, V, Capelletti, M, et al. Aromatase is differentially
expressed in peripheral blood leukocytes from children, and adult
female and male subjects. Eur J Endocrinol 2006; 154: 425–431.
34. Hammer F, Drescher, DG, Schneider, SB, et al. Sex steroid metabolism
in human peripheral blood mononuclear cells changes with aging.
J Clin Endocrinol Metab 2005; 90: 6283–6289.
35. Valko M, Leibfritz, D, Moncol, J, et al. Free radicals and antioxidants in
normal physiological functions and human disease. Int J Biochem Cell
Biol 2007; 39: 44–84.
36. Massafra C, Gioia, D, De Felice, C, et al. Effects of estrogens and
androgens on erythrocyte antioxidant superoxide dismutase, catalase
and glutathione peroxidase activities during the menstrual cycle.
J Endocrinol 2000; 167: 447–452.
37. Kinnula VL, Soini, Y, Kvist-Makela, K, Savolainen, ER, Koistinen, P.,
Antioxidant defense mechanisms in human neutrophils. Antioxid Redox
Signal 2002; 4: 27–34.
38. Winterbourn CC, Hampton, MB., Thiol chemistry and specificity in
redox signaling. Free Radic Biol Med 2008; 45: 549–561.
39. Yang CS, Yuk, JM, Jo, EK., The role of nitric oxide in mycobacterial
infections. Immune Netw 2009; 9: 46–52.
40. Coleman JW., Nitric oxide in immunity and inflammation. Int Immu-
nopharmacol 2001; 1: 1397–1406.
41. Friedl R, Brunner, M, Moeslinger, T, Spieckermann, PG., Testosterone
inhibits expression of inducible nitric oxide synthase in murine macro-
phages. Life Sci 2000; 68: 417–429.
42. Pacher P, Beckman, JS, Liaudet, L., Nitric oxide and peroxynitrite in
health and disease. Physiol Rev 2007; 87: 315–424.
Cell Biochem Funct 2010; 28: 394–402.