Beruflich Dokumente
Kultur Dokumente
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.
LDL-cholesterol oxidation. In addition, superoxide anion neutrophils were separated using Histopaque 1077 accord-
produced by activated neutrophils can be transformed into ing to the manufacturer’s instructions (Sigma). The plasma
other more ROS such as hydroxyl radical and singlet and intermediary layer were removed and both neutrophils
oxygen. These ROS cause lipid peroxidation that is the first and erythrocytes were collected from sediment. Erythro-
step of the atherosclerosis process.11 cytes were lysed with a hemolysis solution (150 mM NH4Cl,
Békési and coworkers12 have shown that human 10 mM NaHCO3, 0.1 mM EDTA, pH 7.4) and subsequently
neutrophils treated with testosterone for 2 h show decreased centrifuged for 10 min (600 g at 48C). This procedure was
superoxide anion production, suggesting a possible anti- repeated twice for total red blood cell lyses. Thereafter,
oxidant effect. However, little is known about the effects of neutrophils were washed with PBS followed by centrifu-
testosterone on neutrophil function, damage on biomole- gation for 10 min (600 g at 48C). After centrifugation,
cules induced by oxidative stress, production of ROS and peripheral blood neutrophils were collected and maintained
regulation of antioxidant enzyme activities. The aim of this in RPMI-1640 medium supplemented with 2 mM glutamine,
study was to investigate the effect of testosterone on human 20 mM HEPES, 10% fetal calf serum, 10 U/ml penicillin,
neutrophil function. For this purpose, the following and 10 mg/ml streptomycin. The number of viable cells
measurements were performed in neutrophils after in vitro (>95% neutrophils) was determined in a Neubauer chamber
testosterone treatment: activities of superoxide dismutase under an optical microscope by Trypan blue exclusion.13
(SOD), catalase, glutathione peroxidase and glutathione
reductase, production of superoxide anion by activation of
NADPH oxidase complex, production of nitric oxide (NO), Cytotoxic effect of testosterone
content of TBARS, intracellular Ca2þ mobilization, and This assay was performed to investigate the testosterone
microbicidal and phagocytic capacities. toxicity in human neutrophils. Cells (1 106 per ml) were
cultured with different concentrations of testosterone (0, 0.1,
MATERIALS AND METHODS 1, 5, 10, 25, 50, 75, and 100 mM) for 24 h. Afterwards,
neutrophils were collected to determine the proportion of
Reagents viable cells by Trypan Blue exclusion.
All reagents for buffers were obtained from Labsynth
(Diadema, SP, Brazil). Histopaque-1077, HEPES, ethylene
glycol tetracetic acid (EGTA), ethylenediamine tetraacetic Cell culture and treatment
acid (EDTA), dihydroethidium (DHE), ionomycin, sodic Isolated neutrophils were seeded in a 6-well dishes at a
azide, albumin, nicotinamide adenine dinucleotide phos- density of 5 106 cells/well in the presence of testosterone
phate (NADPH), nicotinamide adenine dinucleotide 10 nM, 0.1 and 10 mM solubilized in DMSO. The cells were
(NADH), dimethyl sulfoxide (DMSO), 2,4-dinitrophenyl- incubated in a humidified atmosphere of 5% CO2 at 378C for
hydrazine (DNPH), 5,50 -dithiobis(2-nitrobenzoic acid) 24 h. The concentration of DMSO used was always lower
(DTNB), Triton X-100, phenazine methosulphate (PMS), than 2% of the total volume of culture medium. This
nitroblue tetrazolium (NBT), thiobarbituric acid (TBA), N- concentration of DMSO has shown not to be toxic for the
ethylmalemide (NEM), phorbol-12-myristate 13-acetate cells as found in preliminary experiments (data not shown).
(PMA), guanidine-HCL, reduced glutathione (GSH), oxi- Cells left untreated (control) or treated with DMSO (vehicle)
dized glutathione (GSSG), lipopolysaccharide (LPS), were included in all experiments. There was no difference
butylated hydroxytoluene (BHT) and hydrogen peroxide between untreated and DMSO-treated cells in all cases.
(H2O2) were obtained from Sigma Chemical Co (St Louis,
MO, USA). RPMI-1640 culture medium, lucigenin,
acetoxymethylester (Fura 2-AM) and pluronic acid were Production of ROS using lucigenin-enhanced
obtained from Molecular Probes (Eugene, OR, USA). chemiluminescence Assay
Lucigenin is extensively used to measure extracellular
superoxide content mainly produced through NADPH
Cell isolation
oxidase activation.14 Lucigenin (5 mM) was added to cells
This study was approved by the Human Ethic Committee of freshly obtained and incubated (5 105 per well) in absence
the Cruzeiro do Sul University and was performed in or in the presence of testosterone (10 nM, 0.1 and 10 mM) in
accordance with the Declaration of Helsinki. All volunteers Tyrode’s buffer (137 mM NaCl, 2.68 mM KCl, 0.49 mM
gave written informed consent before enrollment. Peripheral MgCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 5.6 mM D-
blood neutrophils obtained from non-smoking and non- glucose, and 5 mM HEPES), pH 7.4. The experiments were
obese healthy young men (age ranging from 18–30 years carried out in the presence and absence of PMA (phorbol
old) were collected by venopuncture procedure and placed myristate acetate—20 ng per well). The chemiluminescence
in vacuum/siliconized test tubes containing EDTA as response was monitored for 45 min in a luminometer (Tecan,
anticoagulant agent. Blood samples (20 ml) were diluted Salzburg, Austria). The total superoxide anion production
in the same volume of phosphate buffered saline (0.137 M during 45 min was expressed as integrated area under curve
NaCl, 2.7 mM KCl, 8.0 mM Na2HPO4, pH 7.4) solution and (AUC) analysis.
Copyright # 2010 John Wiley & Sons, Ltd. Cell Biochem Funct 2010; 28: 394–402.
396 d. p. marin ET AL.
Copyright # 2010 John Wiley & Sons, Ltd. 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.
Copyright # 2010 John Wiley & Sons, Ltd. Cell Biochem Funct 2010; 28: 394–402.
effect of testosterone on neutrophil function 399
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
Copyright # 2010 John Wiley & Sons, Ltd. Cell Biochem Funct 2010; 28: 394–402.
400 d. p. marin ET AL.
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, phils by activating NADPH-oxidase via PKC pathway acting
indicating an improvement of cell function. This increment as a diacylglycerol-analog, with no involvement of
in neutrophil phagocytosis was observed only at a intracellular [Ca2þ]i29. Superoxide anion production by
physiological range of testosterone (10 nM). High concen- activated neutrophils plays a role during microbicidal
tration of the hormone (10 mM) promoted a significant activity against pathogens. However, under certain circum-
reduction in microbicidal capacity with no effect on stances, an excess of superoxide overcomes local antiox-
phagocytosis. Similar to our results, a high dose of idant defense and leads to oxidative stress and oxidative
testosterone (35 mM) also reduced the levels of IgM in tissue injury. According to previous studies,12,30 we
splenic leucocytes,24 whereas no difference was observed at demonstrated that testosterone inhibits superoxide pro-
a lower concentration. Thus, the marked effect of duction by PMA-stimulated or non-PMA-stimulated neu-
testosterone on neutrophils seems to be found at a trophils, as assessed by DHE and lucigenin assays. This
physiological range of the hormone. Intra- and extracellular indicates a modulation of signaling events upstream
Ca2þ are necessary for efficient phagocytic and microbicidal superoxide production by testosterone. Békési et al.12 have
activities. The fusion of granules, formation of phagosome, also reported reduction of superoxide levels in human
and the process of exocytose are very sensitive to changes in neutrophils by treatment with steroids such as cortisol,
[Ca2þ]i.25 The impairment of [Ca2þ]i mobilization at estradiol, and progesterone. Androgens such as dihydrotes-
supraphysiological doses of testosterone possibly reduces tosterone have been shown to be a powerful antioxidant,
the intracellular killing of bacteria by inhibiting the release effectively preventing the formation of lipid peroxides.31
of granule proteins and ROS into the phagosome. Bennett et al.32 reported that testosterone with a keto group
Recent studies have been reported that testosterone can at the C3 position, showed a mild preventive effect
act through a nongenomic pathway in macrophages and T compared to dihydrotestosterone. In addition, leukocytes
lymphocytes.26 Nongenomic signaling of testosterone may express aromatase33 and 5a-reductase,34 and thus
involves a rapid increase of [Ca2þ]i, cAMP or activation testosterone can undergo aromatization to 17b-estradiol or
of PKC.27 In lymphocytes, testosterone rapidly raises reduction to dihydrotestosterone, respectively. Therefore,
intracellular [Ca2þ]i predominantly due to an extracellular the antioxidant properties of testosterone on human
calcium influx, whereas in macrophages the increase in neutrophils may involve several signaling pathways.
testosterone-induced calcium mobilization occurs through The reduction of superoxide anion production induced by
release from intracellular calcium stores.27 Our data testosterone is an antioxidant mechanism. In fact, decrease
demonstrates that testosterone at physiological levels of TBARS levels and damage to proteins was observed. An
rapidly raises [Ca2þ]i. Nevertheless, as a limitation of this exacerbated production of ROS by neutrophils results in an
study, we cannot state that testosterone promoted a attack on cellular components involving polyunsaturated
nongenomic signalling in human neutrophils. fatty acid residues of phospholipids, forming the final
Superoxide anion is the first ROS generated during the product of the peroxidation process being MDA.35 The
respiratory burst via reduction of molecular oxygen through superoxide anion itself does not cause lipid peroxidation.
activation of NADPH oxidase.28 PMA stimulates neutro- However, superoxide anion produced by activated neutro-
Copyright # 2010 John Wiley & Sons, Ltd. Cell Biochem Funct 2010; 28: 394–402.
effect of testosterone on neutrophil function 401
phils can be transformed into other ROS, which are able to tration does not alter nitric oxide production. The basal
oxidize lipids and proteins, for example, the LDL- levels of NO are high and further increase after stimulation
cholesterol. LDL-cholesterol in plasma and vascular with LPS and testosterone at 0.1 and 10 mM. Supraphysio-
endothelium is susceptible to oxidation, which has been logical concentrations of testosterone (0.1 and 10 mM) can
considered the main event for the development of increases NO levels characterizing an inflammatory process
atherosclerosis.10 The elevated production of free radicals and this may be due to activation of iNOS, which produces
is a major factor in the abnormal interaction of the large amounts of NO, which can act as a pro-inflammatory
neutrophils and vascular wall, contributing for the functional molecule. NO can interact with pro-inflammatory oxidants
and structural changes associated with atherosclerosis. (e.g., superoxide, hydrogen peroxide, hypochlorite) to form
Therefore, reduced physiological levels of testosterone the peroxynitrite that modify lipids and proteins. When NO
may contribute to increase the cardiovascular risk mediated concentration exceeds that of superoxide anion, NO
by oxidative stress. predominantly exhibits an inhibitory effect on oxidative
The role of androgenic steroids in the regulation of the damage.42 Lower levels of superoxide induced by testos-
antioxidant systems is still poorly understood. In the present terone treatment may mean less chance for conversion of NO
study, activities of total SOD and MnSOD, catalase and to peroxynitrite, therefore, increasing cellular levels of NO.
glutathione peroxidase were not affected in neutrophils after The anti-atherogenic properties of NO have been exten-
testosterone treatment. In agreement with our findings, no sively studied. NO presents anti-aggregators effects on
significant effects of androgens have also been reported on platelets, and antioxidant, anti-inflammatory, antiprolifera-
antioxidant enzymes in erythrocytes.36 On the other hand, a tive, and vasodilators effects on the vasculature.42
significant increase of glutathione reductase activity was Taken together, our results point out the antioxidant
observed. GSH is the most important antioxidant agent in properties of testosterone in human neutrophils as suggested
human neutrophils.37 GSH can directly detoxify ROS. by suppression of superoxide anion, and TBARS levels, and
During inactivation of oxidizing agent, there is a depletion of by the increase of nitric oxide levels, glutathione reductase
GSH and formation of GSSG (oxidized glutathione). The activity and thiol groups. Testosterone concentration taken
increase of glutathione reductase activity could elevate the as physiological concentration (10 nM), have a greater
content of GSH and, therefore, the antioxidant defense antioxidant potential than higher concentrations. Thus, the
capacity. On the other hand, increased levels of GSSG maintenance of physiological levels of testosterone con-
reflects an environment predominantly oxidative, which tributes for the redox balance in human neutrophils.
promotes formation of disulphide bridges (S-S) in proteins Reduced concentrations of testosterone in elderly and in
and impairment of their activities.38 As a consequence of the some pathological conditions may contribute for the
increase in glutathione reductase activity, which improves development of cardiovascular risk through induction of
the GSH storage, thiol groups were significantly increased oxidative stress in immune cells.
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. ACKNOWLEDGEMENTS
Consequently, in the case of an oxidant-thiol interaction, the The authors are indebted to the constant assistance of
oxidant is neutralized to a relatively lower toxic byproduct. Professor Marcelo Paes de Barros and Dr Sandra Sampaio.
Nitric oxide (NO) produced from the constitutively NO This research is supported by FAPESP and Cruzeiro do Sul
synthase (NOS) acts as an important oxidative biological University. Financial support for this work was provided by
signaling molecule in a large variety of physiological FAPESP (Fundação de Amparo a Pesquisa do Estado de São
processes, including neurotransmission, blood pressure Paulo – 07/03334-6) and Universidade Cruzeiro do Sul.
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 CONFLICT OF INTEREST
inflammatory diseases. Inducible NOS is expressed in
various cells including macrophages, neutrophils, and No conflict of interest.
epithelial cells, and it produces excessive NO during
infection, inflammation, and states of physiological stimu-
REFERENCES
lation.39 NO is generated during immune and inflammatory
responses as a toxic agent toward infectious organisms. NO 1. Kaufman JM, Vermeulen, A. The decline of androgen levels in elderly
may induce toxic reactions against other tissues of the host men and its clinical and therapeutic implications. Endocr Rev 2005; 26:
833–876.
and since it is generated at high levels in certain types of 2. Matsumoto AM. Andropause: clinical implications of the decline in
inflammation, it has been implicated as a pro-inflammatory serum testosterone levels with aging in men. J Gerontol A Biol Sci Med
agent.40 Conflicting results have been reported about Sci 2002; 57: M76–99.
regulation of NO synthesis by testosterone in immune cells. 3. Rodriguez A, Muller, DC, Metter, EJ, et al. Aging, androgens, and the
metabolic syndrome in a longitudinal study of aging. J Clin Endocrinol
Frield et al.41 found a decrease in NO synthesis induced by Metab 2007; 92: 3568–3572.
testosterone in murine macrophages. Conversely, our results 4. Vermeulen A., Androgen replacement therapy in the aging male–a
demonstrate that testosterone at a physiological concen- critical evaluation. J Clin Endocrinol Metab 2001; 86: 2380–2390.
Copyright # 2010 John Wiley & Sons, Ltd. Cell Biochem Funct 2010; 28: 394–402.
402 d. p. marin ET AL.
5. Laughlin GA, Barrett-Connor, E, Bergstrom, J., Low serum testoster- 24. Saha NR, Usami, T, Suzuki, Y., In vitro effects of steroid hormones on
one and mortality in older men. J Clin Endocrinol Metab 2008; 93: 68– IgM-secreting cells and IgM secretion in common carp (Cyprinus
75. carpio). Fish Shellfish Immunol 2004; 17: 149–158.
6. Svartberg J, von Muhlen, D, Mathiesen, E, et al. Low testosterone levels 25. Wilsson A, Lundqvist, H, Gustafsson, M, Stendahl, O., Killing of
are associated with carotid atherosclerosis in men. J Intern Med 2006; phagocytosed Staphylococcus aureus by human neutrophils requires
259: 576–582. intracellular free calcium. J Leukoc Biol 1996; 59: 902–907.
7. Webb CM, McNeill, JG, Hayward, CS, de Zeigler, D, Collins, P., 26. Benten WP, Guo, Z, Krucken, J, Wunderlich, F., Rapid effects of
Effects of testosterone on coronary vasomotor regulation in men with androgens in macrophages. Steroids 2004; 69: 585–590.
coronary heart disease. Circulation 1999; 100: 1690–1696. 27. Wunderlich F, Benten, WP, Lieberherr, M, et al. Testosterone signaling
8. Wu S, Weng, X., Therapeutic effect of andriol on serum lipids and in T cells and macrophages. Steroids 2002; 67: 535–538.
apolipoproteins in elderly male coronary heart disease patients. Chin 28. van der Vliet A., NADPH oxidases in lung biology and pathology: host
Med Sci J 1992; 7: 137–141. defense enzymes, and more. Free Radic Biol Med 2008; 44: 938–
9. Droge W., Free radicals in the physiological control of cell function. 955.
Physiol Rev 2002; 82: 47–95. 29. Granfeldt D, Samuelsson, M, Karlsson, A., Capacitative, Ca2þ influx
10. Griendling KK, Sorescu, D, Ushio-Fukai, M., NAD(P)H oxidase: role and activation of the neutrophil respiratory burst. Different regulation
in cardiovascular biology and disease. Circ Res 2000; 86: 494–501. of plasma membrane- and granule-localized NADPH-oxidase.
11. Kellogg EW, 3rd and Fridovich, I. Liposome oxidation and erythrocyte J Leukoc Biol 2002; 71: 611–617.
lysis by enzymically generated superoxide and hydrogen peroxide. 30. Bekesi G, Racz, K, Hrabak, A, et al. Systematic investigation of
J Biol Chem 1977; 252: 6721–6728. different steroid precursors with respect to their effect on superoxide
12. Bekesi G, Kakucs, R, Varbiro, S, et al. In vitro effects of different anion production by human neutrophil granulocytes. Horm Metab Res
steroid hormones on superoxide anion production of human neutrophil 2004; 36: 155–163.
granulocytes. Steroids 2000; 65: 889–894. 31. Bilinska B, Wiszniewska, B, Kosiniak-Kamysz, K, et al. Hormonal
13. Otton R, da Silva, DO, Campoio, TR, et al. Non-esterified fatty acids status of male reproductive system: androgens and estrogens in the
and human lymphocyte death: a mechanism that involves calcium testis and epididymis. In vivo and in vitro approaches. Reprod Biol
release and oxidative stress. J Endocrinol 2007; 195: 133–143. 2006; 6 Suppl 1: 43–58.
14. Hatanaka E, Levada-Pires, AC, Pithon-Curi, TC, Curi, R., Systematic 32. Bennett MJ, Albert, RH, Jez, JM, et al. Steroid recognition and
study on ROS production induced by oleic, linoleic, and gamma- regulation of hormone action: crystal structure of testosterone and
linolenic acids in human and rat neutrophils. Free Radic Biol Med NADPþ bound to 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase.
2006; 41: 1124–1132. Structure 1997; 5: 799–812.
15. Ding AH, Nathan, CF, Stuehr, DJ., Release of reactive nitrogen 33. Vottero A, Rochira, V, Capelletti, M, et al. Aromatase is differentially
intermediates and reactive oxygen intermediates from mouse peritoneal expressed in peripheral blood leukocytes from children, and adult
macrophages. Comparison of activating cytokines and evidence for female and male subjects. Eur J Endocrinol 2006; 154: 425–431.
independent production. J Immunol 1988; 141: 2407–2412. 34. Hammer F, Drescher, DG, Schneider, SB, et al. Sex steroid metabolism
16. Sampaio SC, Sousa-e-Silva, MC, Borelli, P, Curi, R, Cury, Y., Crotalus in human peripheral blood mononuclear cells changes with aging.
durissus terrificus snake venom regulates macrophage metabolism and J Clin Endocrinol Metab 2005; 90: 6283–6289.
function. J Leukoc Biol 2001; 70: 551–558. 35. Valko M, Leibfritz, D, Moncol, J, et al. Free radicals and antioxidants in
17. Otton R, Graziola, F, Hirata, MH, Curi, R, Williams, JF., Dietary fats normal physiological functions and human disease. Int J Biochem Cell
alter the activity and expression of glucose-6-phosphate dehydrogenase Biol 2007; 39: 44–84.
in rat lymphoid cells and tissues. Biochem Mol Biol Int 1998; 46: 529– 36. Massafra C, Gioia, D, De Felice, C, et al. Effects of estrogens and
536. androgens on erythrocyte antioxidant superoxide dismutase, catalase
18. Ewing JF, Janero, DR., Microplate superoxide dismutase assay employ- and glutathione peroxidase activities during the menstrual cycle.
ing a nonenzymatic superoxide generator. Anal Biochem 1995; 232: J Endocrinol 2000; 167: 447–452.
243–248. 37. Kinnula VL, Soini, Y, Kvist-Makela, K, Savolainen, ER, Koistinen, P.,
19. Aebi H., Catalase in vitro. Methods Enzymol 1984; 105: 121–126. Antioxidant defense mechanisms in human neutrophils. Antioxid Redox
20. Mannervik B., Glutathione peroxidase. Methods Enzymol 1985; 113: Signal 2002; 4: 27–34.
490–495. 38. Winterbourn CC, Hampton, MB., Thiol chemistry and specificity in
21. Fraga CG, Leibovitz, BE, Tappel, AL., Lipid peroxidation measured as redox signaling. Free Radic Biol Med 2008; 45: 549–561.
thiobarbituric acid-reactive substances in tissue slices: characterization 39. Yang CS, Yuk, JM, Jo, EK., The role of nitric oxide in mycobacterial
and comparison with homogenates and microsomes. Free Radic Biol infections. Immune Netw 2009; 9: 46–52.
Med 1988; 4: 155–161. 40. Coleman JW., Nitric oxide in immunity and inflammation. Int Immu-
22. Biteau B, Labarre, J, Toledano, MB., ATP-dependent reduction of nopharmacol 2001; 1: 1397–1406.
cysteine-sulphinic acid by S. cerevisiae sulphiredoxin. Nature 2003; 41. Friedl R, Brunner, M, Moeslinger, T, Spieckermann, PG., Testosterone
425: 980–984. inhibits expression of inducible nitric oxide synthase in murine macro-
23. Bradford MM., A rapid and sensitive method for the quantitation of phages. Life Sci 2000; 68: 417–429.
microgram quantities of protein utilizing the principle of protein-dye 42. Pacher P, Beckman, JS, Liaudet, L., Nitric oxide and peroxynitrite in
binding. Anal Biochem 1976; 72: 248–254. health and disease. Physiol Rev 2007; 87: 315–424.
Copyright # 2010 John Wiley & Sons, Ltd. Cell Biochem Funct 2010; 28: 394–402.