Toxicology 328 (2015) 1220

Contents lists available at ScienceDirect

Toxicology
journal homepage: www.elsevier.com/locate/toxicol

Dose-dependent effects of caffeine in human Sertoli cells metabolism

and oxidative prole: Relevance for male fertility
Tnia R. Dias a , Marco G. Alves a , Raquel L. Bernardino b , Ana D. Martins a,b ,
Ana C. Moreira b , Joaquina Silva c , Alberto Barros c,d, Mrio Sousa b,c, Branca M. Silva a, * ,
Pedro F. Oliveira a,b, *
a

CICS UBI Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilh, Portugal
Department of Microscopy, Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar (ICBAS) and Unit for Multidisciplinary Research in
Biomedicine (UMIB), University of Porto, Portugal
c
Centre for Reproductive Genetics Alberto Barros, 4100-009 Porto, Portugal
d
Department of Genetics, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal
b

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 28 October 2014
Received in revised form 3 December 2014
Accepted 4 December 2014
Available online 5 December 2014

Caffeine is a widely consumed substance present in several beverages. There is an increasing

consumption of energetic drinks, rich in caffeine, among young individuals in reproductive age. Caffeine
has been described as a modulator of cellular metabolism. Hence, we hypothesized that it alters human
Sertoli cells (hSCs) metabolism and oxidative prole, which are essential for spermatogenesis. For that
purpose, hSCs were cultured with increasing doses of caffeine (5, 50, 500 mM). Caffeine at the lowest
concentrations (5 and 50 mM) stimulated lactate production, but only hSCs exposed to 50 mM showed
increased expression of glucose transporters (GLUTs). At the highest concentration (500 mM), caffeine
stimulated LDH activity to sustain lactate production. Notably, the antioxidant capacity of hSCs decreased
in a dose-dependent manner and SCs exposed to 500 mM caffeine presented a pro-oxidant potential, with
a concurrent increase of protein oxidative damage. Hence, moderate consumption of caffeine appears to
be safe to male reproductive health since it stimulates lactate production by SCs, which can promote
germ cells survival. Nevertheless, caution should be taken by heavy consumers of energetic beverages
and food supplemented with caffeine to avoid deleterious effects in hSCs functioning and thus, abnormal
spermatogenesis.
2014 Elsevier Ireland Ltd. All rights reserved.

Keywords:
Caffeine
Sertoli cell
Spermatogenesis
Cell metabolism
Lactate
Male fertility

1. Introduction
Caffeine is one of the most widely consumed psychoactive
substances and its popularity has been attributed to its stimulant
properties. It is structurally known as 1,3,7-trimethylxanthine,

Abbreviations: 4-HNE, 4-hydroxynonenal; DNP, 2,4-dinitrophenyl; DNPH, 2,4dinitrophenylhydrazine; FBP, fructose-1,6-biphosphate; FRAP, ferric reducing
antioxidant power; FSH, follicle-stimulating hormone; GLUT1, glucose transporter
1; GLUT3, glucose transporter 3; hSCs, human Sertoli cells; LDH, lactate
dehydrogenase; MCT4, monocarboxylate transporter 4; OS, oxidative stress; PBS,
phosphatebuffered saline; PFK1, phosphofructokinase 1; PVDF, polyvinylidenediuoride; ROS, reactiveoxygenspecies; SC, Sertoli cell; TBS, Trisbuffered saline; TFA,
trifuoroaceticacid; TPTZ, 2,4,6-tripyridyl-s-triazine.
* Corresponding authors at: Health Sciences Research Centre, Faculty of Health
Sciences, University of Beira Interior, Av. Infante D. Henrique, 6201-506 Covilh,
Portugal. Tel.: +351 275329077; fax: +351 275329099.
E-mail addresses: bmcms@ubi.pt (B.M. Silva), pfobox@gmail.com (P.F. Oliveira).
http://dx.doi.org/10.1016/j.tox.2014.12.003
0300-483X/ 2014 Elsevier Ireland Ltd. All rights reserved.

naturally present in over 60 plant species, but also articially

manufactured (Barone and Roberts, 1996). The main sources of
dietary caffeine are tea leaves (Camelia sinensis) and roasted coffee
beans (Coffea Arabica and Coffea robusta), with each contributing
about equally to total caffeine intake (about 240 mg adult 1 day 1)
(Heatherley et al., 2006). While it is estimated that tea present
between 1.4 and 3.4 times less caffeine content than coffee (FSA,
2004), the total caffeine content depends on the particular leaf or
bean and on how the beverage is prepared (for review (Eteng et al.,
1997)), being that in several countries tea is consumed in higher
doses than coffee (Mukhtar et al., 1992). A daily consumption of
240300 mg of caffeine correspond to an ingestion of 37 mg
caffeine/kg of body weight in adults (Mandel, 2002). Blanchard and
Sawers (1983) demonstrated that an oral administration of 5 mg/
kg of caffeine leads to a plasma concentration of 10 mg/mL
(50 mM). Later, another study reported that the intake of 300 mg of
pure caffeine resulted also in a plasmatic concentration of

T.R. Dias et al. / Toxicology 328 (2015) 1220

approximately 50 mM (Alvi and Hammami, 2011). The caffeine

molecule is easily absorbed by humans, having approximately
100% of bioavailability when taken by oral route and reaching a
peak in the blood within 1545 min after its consumption
(Sepkowitz, 2013). After being absorbed, caffeine is distributed
to various tissues and broken down to metabolites with variable
pharmacological actions (Mandel, 2002). While the moderate
consumption of caffeine is usually seen as a relatively good
practice, there are several studies indicating that when taken in
excessive amounts may lead to various deleterious health effects
(Sepkowitz, 2013). Of particular concern is the increasing
consumption of energy drinks that are rich in caffeine and very
popular among young people (for review (Reissig et al., 2009)).
Besides, caffeine can also be found in products containing cocoa or
chocolate, as well as in several medications and dietary supplements (Andrews et al., 2007; Barone and Roberts, 1996). The major
health problems concerning caffeine and human disease include
coronary heart disease, reproductive disorders, and psychiatric
disturbances (for review (Benowitz, 1990)).
The most important mechanism of action of caffeine appears to
be the antagonism of adenosine receptors (Dunwiddie and Masino,
2001). Since caffeine has a similar molecular structure to
adenosine, with both compounds having a double bond ring
structure, caffeine has the potential to occupy adenosine receptor
sites (Fisone et al., 2004). Adenosine and its antagonists have long
been suggested to inuence the male reproductive system (Casali
et al., 2001). Several studies have demonstrated the presence of
adenosine receptors in Sertoli cells (SCs) (Rivkees 1994; Stiles et al.,
1986) and showed that these cells can be modulated by adenosine
and its analogues (Conti et al., 1989). The somatic SCs are
responsible for the functional development of the testis and
hence for the expression of the male phenotype (Mackay, 2000;
Rato et al., 2012). They also actively metabolize several substrates,
such as glucose, to ensure lactate supply to the developing germ
cells (for review (Alves et al., 2013a)). Thus, the overall metabolic
functioning of SCs is pivotal for the occurrence of normal
spermatogenesis.
Caffeine is known to increase cells metabolic rates as well as the
concentrations of free fatty acids and blood glucose (Lane, 2011;
Sinha et al., 2014). Animal studies suggest that prolonged
exposures to caffeine may affect cells metabolism, compromising
cellular homeostasis (Yokogoschi et al., 1983). Within the testis,
SCs produce lactate at high rates and any deregulation of these
processes may lead to high levels of oxidative stress (OS) and
consequently male subfertility or infertility (Aitken et al., 2010).
Interestingly, caffeine has been reported to be a protective
substance against cellular damage with benecial antioxidant
effects (Grucka-Mamczar et al., 2009). The exact mechanisms of
action of caffeine in SCs metabolism are yet to be disclosed and
there is no evidence of a clear association between caffeine, OS and
male fertility. Herein we hypothesize that caffeine can alter SCs
glycolytic and oxidative prole interfering with males reproductive potential.

13

and European Ethical Committees. The studies have been

performed according to the Declaration of Helsinki. Testicular
biopsies were obtained from patients under treatment for
recovery of male gametes and used after informed written
consent. Only cells left in the tissue culture plates after patients
treatment were used. Human SCs (hSCs) were isolated from six
testicular biopsies of men with conserved spermatogenesis,
selected from patients with anejaculation (psychological, vascular, neurologic), vasectomy or traumatic section of the vas
deferens.
2.3. Human Sertoli cell primary culture
Testicle biopsies were washed twice in HBSSf (Hanks balanced
salt solution without Ca2+ or Mg2+) through centrifugations at
500  g at room temperature, as described by Oliveira et al. (2011).
hSCs were obtained by a routine method (Oliveira et al., 2009). The
resulting cellular pellet was suspended in hSCs culture medium
(DMEM:Hams F-12 1:1, containing 15 mM HEPES, 50 U/ml
penicillin and 50 mg/ml streptomycin sulfate, 0.5 mg/ml fungizone, 50 mg/ml gentamicin and 10% heat inactivated fetal bovine
serum) and forced through a 20G needle, in order to disaggregate
large cell clusters. Then, cells were plated on Cell+ culture asks
(Sarsted, Nmbrecht, Germany) and incubated at 3033  C, 5% CO2
in air until used. After 96 h, the cultures were examined by phase
contrast microscopy and only the hSCs with contaminants below
5% were used. hSCs culture purity was determined as described
(Steger et al., 1996).
2.4. Experimental groups

2. Material and methods

hSCs were allowed to grow until reach 9095% of conuence,

and after fully washed, the culture medium was replaced by
serum-free medium (DMEM:F12 1:1, pH 7.4) supplemented with
insulintransferrinsodium selenite (ITS medium; 10 mg/ml, 5 mg/
ml, and 5 mg/ml, respectively). In order to evaluate the effect of
caffeine on SCs glycolytic and oxidative prole, four different
groups were dened: a control group without caffeine and three
other groups containing ITS medium supplemented with increasing doses of caffeine (5, 50 and 500 mM). The daily consumption of
caffeine between drinkers of caffeine-rich beverages was estimated to be of 5 mg/kg of body weight (FSA, 2004; Mandel, 2002). The
intake of such amount of caffeine leads to a plasma concentration
of about 10 mg/mL (50 mM) (Alvi and Hammami, 2011; Blanchard
and Sawers, 1983), the reason why we choose to study the effects of
50 mM of caffeine in the hSCs metabolism. Moreover, we found
pertinent to evaluate the effects of a lower concentration (5 mM)
and a higher concentration (500 mM), since caffeine content (and
hence consumption) differs signicantly amongst the various
caffeine-containing beverages and food. After the 24 h of
treatment, culture medium was collected. Then, cells were
detached from the ask using a trypsinEDTA solution, counted
with a Neubauer chamber and collected for protein extraction.
Viability evaluated by the Trypan Blue Exclusion test averaged 85
90%.

2.1. Chemicals

2.5. Proton nuclear magnetic resonance (1H NMR)

All chemicals were purchased from SigmaAldrich (St. Louis,

MO, USA) unless specically stated.

1
H NMR spectra were acquired as previously described (Alves
et al., 2011). Sodium fumarate (nal concentration of 10 mM) was
used as an internal reference (6.50 ppm) to quantify the following
metabolites present in solution (multiplet, ppm): lactate (doublet,
1.33); alanine (doublet, 1.45) and H1-a-glucose (doublet, 5.22). The
relative areas of 1H NMR resonances were quantied using the
curve-tting routine supplied with the NUTSproTM NMR spectral
analysis program (Acorn, NMR Inc., Fremont, CA, USA).

2.2. Patient selection, ethical issues and testicle tissue preparations

The patients clinical studies and testicle tissue processing was
performed at the Centre for Reproductive Genetics Alberto Barros
(Porto, Portugal) according to the Guidelines of the Local, National

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T.R. Dias et al. / Toxicology 328 (2015) 1220

2.6. Western blot

2.9. Analysis of carbonyl groups and lipid peroxidation

Total proteins were isolated from hSCs using RIPAS buffer (1x
PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF,
supplemented with 1% protease inhibitor cocktail, aprotinin and
100 mM sodium orthovanadate). Western blot was performed as
previously described (Dias et al., 2013a). The resulting membranes
were incubated overnight at 4  C with goat anti-glucose transporter 3 (GLUT3) (1:200, sc-7582, Santa Cruz Biotechnology,
Heidelberg, Germany), rabbit anti-glucose transporter 1 (GLUT1)
(1:200, sc-7903, Santa Cruz Biotechnology, Heidelberg, Germany),
or rabbit anti-phosphofructokinase 1 (PFK1) (1:500, sc-67028,
Santa Cruz Biotechnology, Heidelberg, Germany), rabbit antimonocarboxylate transporter 4 (MCT4) (1:1000, sc-50329, Santa
Cruz Biotechnology, Heidelberg, Germany), or rabbit anti-lactate
dehydrogenase (LDH) (1:10000, ab52488, Abcam, Cambridge, UK)
primary antibodies. Mouse anti-a-tubulin (1:5000, T9026, Sigma
Aldrich, St. Louis, MO, USA) was used as the protein loading control.
The immunoreactive proteins were detected separately and
visualized with goat anti-rabbit IgG-AP (1:5000, sc-2007, Santa
Cruz Biotechnology, Heidelberg, Germany), rabbit anti-goat IgG-AP
(1:5000, A4187, SigmaAldrich, St. Louis, MO, USA) or goat antimouse IgG-AP (1:5000, sc-2008, Santa Cruz Biotechnology).
Membranes were reacted with ECFTM (GE, Healthcare, Buckinghamshire, UK) and read with the BioRad FX-Pro-plus (Bio-Rad
Hemel Hempstead, UK). Densities from each band were obtained
with BIO-PROFIL Bio-1D Software from Quantity One (Vilber
Lourmat, Marne-la-Valle, France) according to standard methods
(Picado et al., 1999). The band density attained was divided by the
corresponding tubulin band intensities and expressed in fold
variation relatively to the control group.

Protein carbonyl content is commonly used as a marker for

protein oxidation while lipid peroxidation can be evaluated by
measuring some resulting aldehydic products such as 4-hydroxynonenal (4-HNE). The content of protein carbonyl groups and 4HNE in hSCs from the different experimental groups was evaluated
using the slot-blot technique and specic antibodies. For carbonyl
groups evaluation, protein samples were derivatized using 2,4dinitrophenylhydrazine (DNPH) to obtain 2,4-dinitrophenyl (DNP)
according to the method developed by Levine et al. (1990). The
slot-blot technique was performed using a Hybri-slot manifold
system (Biometra, Gttingen, Germany) and the resulting PVDF
membranes were incubated overnight (4  C) with a rabbit antiDNP (1:5000, D9656, SigmaAldrich, St. Louis, MO, USA). For lipid
peroxidation analysis, protein samples were diluted to a concentration of 0.001 mg/mL using PBS 1x to be used in the slot-blot
technique. Then, the resulting membranes were incubated
overnight (4  C) with a goat anti-4-HNE antibody (1:5000,
AB5605, Merck Millipore, Temecula, USA). Samples were visualized using rabbit anti-goat IgG-AP (1:5000, A4187, SigmaAldrich,
St. Louis, MO, USA) or goat anti-rabbit IgG-AP (1:5000, sc-2007,
Santa Cruz Biotechnology, Heidelberg, Germany), respectively.
Membranes were then reacted with ECFTM substrate (GE Healthcare, Buckinghamshire, UK) and read using a BioRad FX-Pro-plus
(Bio-Rad Hemel Hempstead, UK). Densities from each band were
quantied using the BIO-PROFIL Bio-1D Software from Quantity
One (VilberLourmat, Marne-la-Valle, France).

2.7. Lactate dehydrogenase (LDH) enzymatic assay

LDH levels were spectrophotometrically determined using a
LDH Enzymatic Assay Kit (Thermo Scientic, Waltham, MA)
according to the manufacturers instructions. In brief, 5 mg of
proteins were diluted in lysis buffer. Likewise, a blank was
prepared and boiled for 5 min at 90  C for protein denaturation.
LDH assay substrate was added to all samples in a dark
environment and left at room temperature for approximately
15 min. Then, a stop solution was used to end the enzymatic
activity and absorbance at 490 nm was measured using an Anthos
2010 microplate reader (Biochrom, Berlin, Germany). LDH
enzymatic activities were calculated as units per milligram of
protein using the molar extinction factor (e) and nal expressed as
fold variation to the control group.
2.8. Ferric reducing antioxidant power (FRAP) assay
The FRAP of the cellular pellets was determined using the
colorimetric method described by Benzie and Strain (1996). In
brief, working FRAP reagent was prepared by mixing acetate
buffer (300 mM, pH 3.6), 2,4,6-tripyridyl-s-triazine (TPTZ)
(10 mM in 40 mM HCl) and FeCl3 (20 mM) in a 10:1:1 ratio (v:
v:v). The reduction of the Fe3+TPTZ complex to a colored Fe2
+
TPTZ complex by the samples was monitored immediately after
adding the sample and 40 min later, by measuring the absorbance
at 595 nm using an Anthos 2010 microplate reader (Biochrom,
Berlin, Germany). The antioxidant potential of the samples was
determined against standards of ascorbic acid, which were
processed in the same manner as the samples. Absorbance
results were corrected by using a blank, with water instead of
sample. The changes in absorbance values of test reaction
mixtures were used to calculate FRAP value as described
elsewhere (Benzie and Strain, 1996).

2.10. Statistical analysis

Statistical signicance was assessed by one-way ANOVA,
followed by Dunn post-test using GraphPad Prism 5 (GraphPad
Software, San Diego, CA, USA). All data are presented as mean 
SEM. Differences with p < 0.05 were considered statistically
signicant.
3. Results
3.1. Caffeine (50 mM) increases glucose transporters protein
expression in human Sertoli cells
Since caffeine is known to increase cells metabolic rates
(Lane, 2011; Sinha et al., 2014), we hypothesized that it could also
alter hSCs metabolism. For that purpose, we choose key points of
hSCs metabolism starting on their primary energy substrate,
glucose. Our results showed a glucose consumption of 10.7  2.4
pmol/cell in hSCs in the control group (Fig. 1A). hSCs of the groups
exposed to 5, 50 and 500 mM of caffeine consumed 6.9  1.4,
7.0  2.6 and 12.4  3.1 pmol/cell, respectively, with no signicant
alterations relative to the control group (Fig. 1A). In these cells,
transport of glucose through the cytoplasmic membrane is
mediated by specic membrane hexose transporters (mainly
GLUT1 and GLUT3). Our results showed that exposure to 5 mM of
caffeine did not signicantly alter the protein expression levels of
GLUT1 or GLUT3 in hSCs, comparatively to the control group.
However, hSCs exposed to 50 mM of caffeine signicantly
increased GLUT1 protein levels (1.30  0.16 fold variation to
control) (Fig. 1B) and GLUT3 (1.31  0.31 fold variation to control)
(Fig. 1C) in comparison with the control group. Moreover, in cells
exposed to 50 mM of caffeine, GLUT3 protein expression was
signicantly increased relative to cells exposed to 5 mM of caffeine
(Fig. 1C). Interestingly, hSCs exposed to 500 mM of caffeine did not
present signicant differences in the protein expression levels of
GLUT1 (Fig. 1B) and GLUT3 (Fig. 1C) (1.15  0.09 and 1.10  0.25 fold
variation to control, respectively) relative to control or to the other

T.R. Dias et al. / Toxicology 328 (2015) 1220

[(Fig._1)TD$IG]

15

Fig. 1. Effect of caffeine (5, 50 and 500 mM) on glucose consumption (panel A) and glucose transporters (GLUT1 and GLUT3) expression (panels B and C) in human Sertoli cells.
The gure shows pooled data of independent experiments. Representative blots are also presented (panel D). Glucose consumption is presented in pmol/cell. Variation in
protein levels are presented as fold variation to control. Results are expressed as mean  SEM (n = 5 for each condition). Signicantly different results (P < 0.05) are indicated
as: (a) relative to control and (b) relative to 5 mM.

concentrations of caffeine. The representative blots of GLUT1 and

GLUT3 are shown in Fig. 1D.
3.2. PFK1 protein levels are decreased in human Sertoli cells exposed to
lower caffeine concentrations while exposure to the highest caffeine
concentration stimulates LDH activity
After glucose enters the cells, a key regulatory step of glycolytic
pathway is mediated by PFK1 that irreversibly converts fructose-6phosphate to fructose-1,6-bisphosphate (FBP). FBP is responsible
for the activation of the enzyme pyruvate kinase that catalyses the
irreversible conversion to pyruvate. Our results showed that the
lowest concentrations of caffeine (5 and 50 mM) decreased
PFK1 protein levels to 0.65  0.02 and 0.74  0.04 fold variation
to control, respectively (Fig. 2A). Interestingly, exposure to the
highest caffeine concentration restored PFK1 protein levels to the
control values. The formed pyruvate can then be converted to
lactate by LDH action. Our results demonstrated that LDH protein
levels were similar between groups of hSCs treated with 5 mM
(0.94  0.19 fold variation to control), 50 mM (0.90  0.15 fold
variation to control) and 500 mM (0.91  0.18 fold variation to
control) of caffeine, showing no signicant alterations comparative
to the control group (Fig. 2B).
Since LDH protein expression levels in hSCs were not altered by
caffeine action, we hypothesized that caffeine could alter the
activity of this enzyme. In fact, we veried a dose-dependent
increase in LDH activity of hSCs cultured with caffeine. The levels of
LDH enzymatic activity in groups of hSCs exposed to 5, 50 and
500 mM of caffeine were 1.26  0.16, 1.30  0.16 and 1.49  0.08 fold
variation to control, respectively (Fig. 2C). Nevertheless, only the
highest concentration of caffeine (500 mM) signicantly increased

LDH activity relative to the control group. After lactate production

by SCs, it is exported through MCT4 to be used as metabolic fuel by
developing germ cells. The MCT4 protein levels in hSCs exposed to
5, 50 and 500 mM of caffeine were 1.08  0.12, 0.95  0.10 and
0.93  0.09 fold variation to control, respectively (Fig. 2D). These
values did not reach statistical signicance when compared to the
values determined in non-exposed cells. Fig. 2E displays the
representative blots of PFK1, LDH and MCT4.
3.3. Lactate production by human Sertoli cells is stimulated by
exposure to lowest caffeine concentrations while alanine production is
only stimulated by exposure to 50 mM of caffeine
Lactate production is one of the key functions of hSCs. Nonexposed hSCs presented a lactate production of 17.6  2.4 pmol/
cell. Interestingly, exposure to 5 and 50 uM of caffeine signicantly
increased lactate production to 25.7  2.2 pmol/cell and 26.3  1.1,
respectively (Fig. 3A). However, hSCs exposed to the highest
concentration of caffeine (500 uM) did not present signicant
alterations in lactate production when compared with nonexposed cells. Alanine metabolism is closely associated with
lactate production, since pyruvate can also be converted to alanine.
Our results showed that extracellular alanine concentration in
non-exposed cells was 0.51  0.08 pmol/cell (Fig. 3B). Exposure to
5 mM of caffeine non-signicantly increased alanine production to
0.82  0.08 pmol/cell. On the other hand, there was a signicant
increase in alanine production by hSCs exposed to 50 mM of
caffeine (0.88  0.05 pmol/cell) relative to the control group
(Fig. 3B). hSCs exposed to the highest concentration of caffeine
(500 mM) produced 0.92  0.22 pmol/cell of alanine, having no
signicant differences in comparison to the other experimental

[(Fig._2)TD$IG]

16

T.R. Dias et al. / Toxicology 328 (2015) 1220

Fig. 2. Effect of caffeine (5, 50 and 500 mM) in the expression of phosphofructokinase 1 (PFK1) (panel A), lactate dehydrogenase (LDH) (panel B) and monocarboxylate
transporter 4 (MCT4) (panel D) expression as well as LDH activity (panel C) in human Sertoli cells. The gure shows pooled data of independent experiments. Representative
blots are also presented (panel E). Results are presented as fold variation to control and as mean  SEM (n = 5 for each condition). Signicantly different results (P < 0.05) are
indicated as: (a) relative to control; (b) relative to 5 mM; and (c) relative to 50 mM.

groups. We also evaluated the ratio lactate/alanine, which is often

used as an index of the redox state of the cell (Oliveira et al., 2012).
The interconversion lactatepyruvatealanine is NADH-dependent
and the NADH/NAD+ ratio can be estimated by the ratio lactate/
alanine, which is often used as a measure of the cellular redox state
(Martins et al., 2013b). Interestingly, our results showed that hSCs
exposure to any of the caffeine concentrations studied did not alter
the lactate/alanine ratio (Fig. 3C).
3.4. Normal-range dose of caffeine decreased protein oxidation and
lipid peroxidation in human Sertoli cells, while highest concentrations
of caffeine compromised cellular antioxidant potential
The high glycolytic rates observed in hSCs can lead to increased
levels of OS. Several studies have reported that caffeine consumption is associated with reduced levels of OS biomarkers (GruckaMamczar et al., 2009). This was attributed to the antioxidant
activity of caffeine (Devasagayam et al., 1996). We measured the
antioxidant potential of hSCs pellets using the FRAP assay. The
reducing power of a compound/extract serves as an indicator of its
potential antioxidant activity (FRAP value). Comparative to the
control group, which demonstrated to have 0.8  0.2 mmol of
antioxidant potential/mg of protein, hSCs exposed to 5 mM of
caffeine maintained a similar value of 0.9  0.2 mmol of antioxidant
potential/mg of protein. However, hSCs exposed to 50 mM
signicantly decreased the FRAP value to 0.6  0.1 mmol of
antioxidant potential/mg of protein when compared to nonexposed cells and the ones exposed to 5 mM of caffeine. Of note,
hSCs exposed to 500 mM of caffeine presented a negative

antioxidant potential value ( 0.4  0.2 mmol of antioxidant

potential/mg of protein), indicating a pro-oxidant effect (Fig. 4A).
Protein carbonylation and lipid peroxidation are strong
biomarkers of OS. The attack of free radicals to proteins and
membrane unsaturated fatty acids originates several products,
such as DNP and 4-HNE, respectively, which can be measured to
obtain a quantication of the cellular oxidative damages. hSCs
exposed to 5 mM of caffeine presented a signicant higher
production of protein carbonyl groups (1.38  0.11 fold variation)
comparative to the control group. In hSCs exposed to 50 mM of
caffeine there was a signicant decrease on the production of
carbonyl groups (0.85  0.12 fold variation to control) in comparison to hSCs exposed to 5 mM of caffeine. Finally, hSCs exposed to
500 mM of caffeine presented a content in carbonyl groups of
1.47  0.07 fold variation to control, which was signicantly higher
relative to non-exposed cells and cells exposed to 50 mM of
caffeine (Fig. 4B). Concerning lipid peroxidation, there were no
signicant alterations on hSCs exposed to caffeine when compared
with non-exposed cells. The lipid peroxidation values determined
in hSCs exposed to 5, 50 and 500 mM were1.12  0.07,
0.87  0.02 and 1.00  0.06 fold variation to control, respectively.
Nonetheless, there was a signicant decrease in lipid peroxidation
resultant from exposure of hSCs to 5 mM of caffeine relative to cells
exposed to 50 mM (Fig. 4C).
4. Discussion
Due to its wide consumption, caffeine potential health effects
have been a focus of several studies. Popular beverages such as

[(Fig._3)TD$IG]

T.R. Dias et al. / Toxicology 328 (2015) 1220

[(Fig._4)TD$IG]

17

Fig. 3. Effect of caffeine (5, 50 and 500 mM) in the production of lactate (panel A)
and alanine (panel B) as well as the lactate/alanine ratio (panel C) in human Sertoli
cells. Metabolites production is presented as pmol/cell. The gure shows pooled
data of independent experiments. Results are presented as mean  SEM (n = 5 for
each condition). Signicantly different results (P < 0.05) are indicated as: (a)
relative to control.

Fig. 4. Effect of caffeine (5, 50 and 500 mM) in the FRAP value (panel A) and carbonyl
groups formation (panel B) as well as lipid peroxidation (panel C) in human Sertoli
cells. FRAP value is presented in mmol antioxidant potential/mg of protein while
carbonyl groups formation and lipid peroxidation is presented as fold variation to
control. The gure shows pooled data of independent experiments. Results are
presented as mean  SEM (n = 5 for each condition). Signicantly different results
(P < 0.05) are indicated as: (a) relative to control; (b) relative to 5 mM; and (c)
relative to 50 mM.

coffee, tea and energy drinks are known to have high concentrations of caffeine (Reissig et al., 2009). The average daily
consumption of caffeine per individual was estimated to be of
5 mg/kg, reaching a concentration of 50 mM in the plasma
(Blanchard and Sawers, 1983; Chou and Benowitz, 1994).
Therefore, we tested the effect of that concentration in hSCs
glycolytic and oxidative prole to mimic the concentrations
commonly found in plasma in in vivo conditions. However, people
are commonly exposed to caffeine in lower and higher concentrations, therefore we also tested the effects of 5 and 500 mM of
caffeine. Previous studies reported that caffeine increases human
metabolic rates (Nehlig et al., 1992) and blood glucose levels
(Pizziol et al., 1998). Spermatogenesis is highly dependent on SCs
glucose metabolism, since it is the source of lactate, which is the
preferred metabolic substrate of the developing germ cells

(for review (Rato et al., 2014)). Thus, we evaluated the effect of

caffeine in glucose metabolism of these cells.
Caffeine is structurally very similar to adenosine and can bind to
adenosine receptors acting as a nonselective antagonist (Fredholm
and Lindstrm, 1999). Adenosine receptors have been identied in
SCs (Monaco and Conti, 1986). Several follicle-stimulating hormone (FSH)-stimulated actions on SCs, such as inhibin and
transferrin secretion and pyruvate metabolism, were reported to
be mediated by occupancy of A1 receptors (Conti et al., 1989;
Meroni et al., 1998). Moreover, it has been reported that adenosine
promotes lactate supply to germ cells (Galardo et al., 2010). Our
results showed that the lowest caffeine concentrations signicantly altered hSCs metabolism. Notably, exposure of hSCs to 5 mM
and 50 mM increased lactate production by these cells, without
altering signicantly glucose consumption. These results are
concomitant with previous studies showing that caffeine can

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T.R. Dias et al. / Toxicology 328 (2015) 1220

regulate glucose metabolism (Ojuka et al., 2002). Glucose uptake

by hSCs exhibits a strict hormonal regulation (Alves et al., 2013b).
Moreover, these mechanisms are modulated by a direct regulatory
effect in total GLUTs levels (Klip et al., 1994). In fact, it has been
described that hSCs have the ability to adapt their metabolism
under certain conditions. For instance, in insulin or glucose
deprivation conditions, hSCs adapt their metabolism by modulating the expression of GLUT1 and GLUT3 (Oliveira et al., 2012; Riera
et al., 2009). In our experiments, when hSCs were exposed to
50 mM of caffeine there was a signicant increase in the expression
levels of these transporters, illustrating a stimulation of glucose
uptake. Since these cells present an elevated glycolytic ux under
normal conditions (for review (Rato et al., 2014)), glucose
consumption was not signicantly altered. Nevertheless, exposure
to the lowest concentrations of caffeine (5 and 50 mM) increased
the glycolytic ux of hSCs, as demonstrated by the signicant
increase of lactate production by these cells. Since adenosine is also
known to promote lactate offer to germ cells (Galardo et al., 2010),
our results showed that caffeine at these concentrations may
mediate its effect in SCs metabolism by acting as antagonist of
adenosine receptors.
When hSCs were exposed to 500 mM of caffeine, which is a high
dose even for heavy drinkers of caffeine-rich beverages, we only
detected alterations in PFK1 levels and LDH activity. Nevertheless,
lactate production was not altered, nor glucose consumption. As
referred, SCs exhibit a metabolic plasticity under stressful
conditions (for review (Alves et al., 2013a)). When subjected to
glucose (Riera et al., 2009) or insulin (Oliveira et al., 2012)
deprivation these cells adapt their metabolism in order to sustain a
proper production of lactate, necessary for germ cell development.
Thus, our results illustrate that caffeine at high doses (500 mM)
alters SCs physiologic functions, however, these cells present some
adaptive mechanisms, such as increased PFK1 expression and LDH
activity, which allow them to maintain the same lactate production
as normal cells.
Caffeine has been reported as a protective substance against
cellular damage with benecial antioxidant effects (GruckaMamczar et al., 2009; Ouoglu et al., 2009). Compelling evidence
has shown that caffeine and adenosine receptors can modulate
cellular oxidant status (Goembiowska and Dziubina, 2012;
Prasanthi et al., 2010). Our results show that at high concentrations
(500 mM), caffeine induces a pro-oxidant environment in hSCs,
thus, explaining the requirement for the reported metabolic
adaptations in hSCs in order to sustain lactate production.
Moreover, hSCs exposed to this concentration of caffeine presented
a signicantly higher protein oxidation, as illustrated by the
signicant increase in carbonyl groups content relative to control
group and cells exposed to 50 mM. The effect of caffeine in several
cellular functions, such as reactive oxygen species (ROS) generation, have been reported to be concentration-specic (Tiwari et al.,
2014). Although caffeine is generally known for its antioxidant
properties and efciency as an hydroxyl radical scavenger in vitro
(Shi et al., 1991), our results show that at a concentration of
500 mM, caffeine induces a pro-oxidant environment in SCs that is
accompanied by an increase in proteins oxidation. The lowest dose
of caffeine tested (5 mM) did not altered the antioxidant capacity of
cells when compared to non-exposed cells, however, there was an
increase in protein oxidation, evidencing an increase of OS. hSCs
exposed to 50 mM of caffeine presented lower antioxidant capacity
than non-exposed cells and cells exposed 5 mM of caffeine.
However, hSCs from this group (50 mM of caffeine) presented
lower lipid peroxidation than non-exposed cells and lower protein
oxidation than hSCs exposed to 5 mM of caffeine. These results
support that the effects of caffeine in OS are concentrationdependent. The intermediate concentration (50 mM), which is
considered to be the regular caffeine ingestion, appears to mediate

some protective effects against OS. As discussed, caffeine can act as

a nonselective antagonist of adenosine receptors, which are
reported to control the formation of free radicals (Goembiowska
and Dziubina, 2012) and cells OS. Thus, the effects of caffeine in OS
may also be mediated by adenosine receptors, which would be also
concomitant with the changes detected in hSCs metabolism.
Concerning the effects of caffeine in the male reproductive
function, there are some conicting results. Maternal caffeine
consumption during gestation and lactation has been reported to
impair gonadal development and induce several long-term
adverse effects on the reproductive health of the offspring
(Dorostghoal et al., 2012). More specically, it leads to signicant
dose-related decreases in the testicular weight, seminiferous
tubules diameter, germinal epithelium height, testosterone levels
and sperm quality of the offspring (Dorostghoal et al., 2012).
Besides, an in vivo study with Sprague-Dawley rats demonstrated
that and oral administration of an elevated dose of 200 mg/kg of
body weight of caffeine negatively affects the histo-architecture of
the seminiferous tubules of the testis, with massive loss of
spermatogenic cells and testicular weight loss (Bassey et al., 2011).
A more recent study with the same dosage of caffeine (200 mg/kg
of body weight) also reported testicular and epididymal weight
loss as well as histological alterations, i.e., atrophic cells with
necrosis and excessive degeneration of spermatids and almost
absence of spermatozoa (Ekaluo et al., 2014). Of note, caffeine has
also been used to induce sperm capacitation, particularly with
frozen thawed spermatozoa (Funahashi and Nagai, 2001). When
human spermatozoa were incubated with caffeine, it was reported
that the rate of glycolysis was signicantly increased, without
changing ADP and ATP levels (Rees et al., 1990). Testicular
metabolism is crucial for spermatogenesis (for review (Dias
et al., 2014a; Rato et al., 2012)). The strict metabolic cooperation
established between SCs and developing germ cells is sensitive to
hormonal uctuations (Martins et al., 2013b) and several factors
(Dias et al., 2013b). More recently, we have reported that a white
tea extract, which is very rich in caffeine (for review (Dias et al.,
2013b)), is a promising antioxidant medium additive for sperm
storage at room temperature. Supplementation with a white tea
extract increased sperm antioxidant potential and decreased lipid
peroxidation, restoring spermatozoa viability to values similar to
those obtained at collection time (Dias et al., 2014b). We also
showed that a white tea extract can modulate SCs and stimulate
lactate production by these cells (Martins et al., 2013a). These
potential benets in male reproductive health were attributed to
several phytochemicals, including the high concentration of
caffeine. In fact, caffeine has been consistently reported as a
metabolic modulator. Our results show that caffeine modulates
hSCs metabolism in a dose-dependent manner. The lowest caffeine
concentration stimulates lactate production without interfering
with the glycolysis-related machinery studied. However, the
concentration of 50 mM, altered glucose transporters and increased alanine production to maintain the lactate/alanine ratio.
This ratio is very important since it reects the intracellular redox
state. Lactate has been reported to protect germ cells in vivo by
suppressing the loss of spermatocytes and spermatids in cryptorchid rats (Courtens and Plen, 1999). Thus, it appears that caffeine
at normal-range can enhance lactate production by hSCs and
promote germ cells survival. Interestingly, the most effective
concentration in preventing protein oxidation and lipid peroxidation in hSCs was 50 mM, which is a frequent concentration found in
the plasma of heavy consumers of caffeine-rich beverages.
In sum, our results show that caffeine is a modulator of
hSCs metabolism and stimulates lactate production in a dosedependent way. When exposed to a high concentration of caffeine,
hSCs present a pro-oxidant environment and crucial adaptations in
their metabolism to sustain lactate production. Moreover, it

T.R. Dias et al. / Toxicology 328 (2015) 1220

promotes proteins oxidation in hSCs. These results illustrate that

moderate consumption of caffeine appears to be safe or even
positive to the metabolic functioning of hSCs. Nevertheless,
caution should be taken by consumers of energetic beverages
and supplemented food with caffeine to avoid deleterious effects
to hSCs functioning and spermatogenesis arrest.
Conict of interest
The authors declare no competing nancial interest.
Transparency document
The Transparency document associated with this article can be
found in the online version.
Acknowledgments
This work was supported by the Fundao para a Cincia e a
Tecnologia - FCT (PTDC/QUIBIQ/121446/2010 and PEst-OE/SAU/
UI0709/2014) co-funded by Fundo Europeu de Desenvolvimento
Regional - FEDER via Programa Operacional Factores de Competitividade COMPETE/QREN. M.G. Alves (SFRH/BPD/80451/
2011) was funded by FCT. P.F. Oliveira was funded by FCT through
FSE and POPH funds (Programa Cincia 2008). UMIB was funded by
FCT (PEst-OE/SAU/UI0215/2014).
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