Dose-dependent effects of caffeine in human Sertoli cells metabolismand oxidative profile: Relevance for male fertility

Tânia 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, Mário Sousa b,c, Branca M. Silva a,*,Pedro F. Oliveira a,b,*aCICS – UBI – Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilhã, PortugalbDepartment of Microscopy, Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar (ICBAS) and Unit for Multidisciplinary Research inBiomedicine (UMIB), University of Porto, PortugalcCentre for Reproductive Genetics Alberto Barros, 4100-009 Porto, PortugaldDepartment of Genetics, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal

A R T I C L E I N F O

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

Keywords:CaffeineSertoli cellSpermatogenesisCell metabolismLactateMale fertility

A B S T R A C T

Caffeine is a widely consumed substance present in several beverages. There is an increasingconsumption of energetic drinks, rich in caffeine, among young individuals in reproductive age. Caffeinehas been described as a modulator of cellular metabolism. Hence, we hypothesized that it alters humanSertoli cells (hSCs) metabolism and oxidative profile, which are essential for spermatogenesis. For thatpurpose, hSCs were cultured with increasing doses of caffeine (5, 50, 500mM). Caffeine at the lowestconcentrations (5 and 50mM) stimulated lactate production, but only hSCs exposed to 50mM showedincreased expression of glucose transporters (GLUTs). At the highest concentration (500mM), caffeinestimulated LDH activity to sustain lactate production. Notably, the antioxidant capacity of hSCs decreasedin a dose-dependentmanner and SCs exposed to 500mMcaffeine presented a pro-oxidant potential, witha concurrent increase of protein oxidative damage. Hence, moderate consumption of caffeine appears tobe safe to male reproductive health since it stimulates lactate production by SCs, which can promotegerm cells survival. Nevertheless, caution should be taken by heavy consumers of energetic beveragesand food supplementedwith caffeine to avoid deleterious effects in hSCs functioning and thus, abnormalspermatogenesis.

ã 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Caffeine is one of the most widely consumed psychoactivesubstances and its popularity has been attributed to its stimulantproperties. It is structurally known as 1,3,7-trimethylxanthine,

naturally present in over 60 plant species, but also artificiallymanufactured (Barone and Roberts, 1996). The main sources ofdietary caffeine are tea leaves (Camelia sinensis) and roasted coffeebeans (Coffea Arabica and Coffea robusta), with each contributingabout equally to total caffeine intake (about 240mg adult�1 day�1)(Heatherley et al., 2006). While it is estimated that tea presentbetween 1.4 and 3.4 times less caffeine content than coffee (FSA,2004), the total caffeine content depends on the particular leaf orbean and on how the beverage is prepared (for review (Eteng et al.,1997)), being that in several countries tea is consumed in higherdoses than coffee (Mukhtar et al., 1992). A daily consumption of240–300mg of caffeine correspond to an ingestion of 3–7mgcaffeine/kg of bodyweight in adults (Mandel, 2002). Blanchard andSawers (1983) demonstrated that an oral administration of 5mg/kg of caffeine leads to a plasma concentration of 10mg/mL(50mM). Later, another study reported that the intake of 300mg ofpure caffeine resulted also in a plasmatic concentration of

Abbreviations: 4-HNE, 4-hydroxynonenal; DNP, 2,4-dinitrophenyl; DNPH, 2,4-dinitrophenylhydrazine; FBP, fructose-1,6-biphosphate; FRAP, ferric reducingantioxidant power; FSH, follicle-stimulating hormone; GLUT1, glucose transporter1; GLUT3, glucose transporter 3; hSCs, human Sertoli cells; LDH, lactatedehydrogenase; MCT4, monocarboxylate transporter 4; OS, oxidative stress; PBS,phosphatebuffered saline; PFK1, phosphofructokinase 1; PVDF, polyvinylidenedi-fluoride; ROS, reactiveoxygenspecies; SC, Sertoli cell; TBS, Tris–buffered 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 Covilhã,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.2014.12.0030300-483X/ã 2014 Elsevier Ireland Ltd. All rights reserved.

Toxicology 328 (2015) 12–20

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approximately 50mM (Alvi and Hammami, 2011). The caffeinemolecule is easily absorbed by humans, having approximately100% of bioavailability when taken by oral route and reaching apeak in the blood within 15–45min after its consumption(Sepkowitz, 2013). After being absorbed, caffeine is distributedto various tissues and broken down to metabolites with variablepharmacological actions (Mandel, 2002). While the moderateconsumption of caffeine is usually seen as a relatively goodpractice, there are several studies indicating that when taken inexcessive amounts may lead to various deleterious health effects(Sepkowitz, 2013). Of particular concern is the increasingconsumption of energy drinks that are rich in caffeine and verypopular among young people (for review (Reissig et al., 2009)).Besides, caffeine can also be found in products containing cocoa orchocolate, as well as in several medications and dietary supple-ments (Andrews et al., 2007; Barone and Roberts, 1996). The majorhealth problems concerning caffeine and human disease includecoronary heart disease, reproductive disorders, and psychiatricdisturbances (for review (Benowitz, 1990)).

The most important mechanism of action of caffeine appears tobe the antagonism of adenosine receptors (Dunwiddie andMasino,2001). Since caffeine has a similar molecular structure toadenosine, with both compounds having a double bond ringstructure, caffeine has the potential to occupy adenosine receptorsites (Fisone et al., 2004). Adenosine and its antagonists have longbeen suggested to influence the male reproductive system (Casaliet al., 2001). Several studies have demonstrated the presence ofadenosine receptors in Sertoli cells (SCs) (Rivkees 1994; Stiles et al.,1986) and showed that these cells can be modulated by adenosineand its analogues (Conti et al., 1989). The somatic SCs areresponsible for the functional development of the testis andhence 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 germcells (for review (Alves et al., 2013a)). Thus, the overall metabolicfunctioning of SCs is pivotal for the occurrence of normalspermatogenesis.

Caffeine is known to increase cells metabolic rates aswell as theconcentrations of free fatty acids and blood glucose (Lane, 2011;Sinha et al., 2014). Animal studies suggest that prolongedexposures to caffeine may affect cells metabolism, compromisingcellular homeostasis (Yokogoschi et al., 1983). Within the testis,SCs produce lactate at high rates and any deregulation of theseprocesses may lead to high levels of oxidative stress (OS) andconsequently male subfertility or infertility (Aitken et al., 2010).Interestingly, caffeine has been reported to be a protectivesubstance against cellular damage with beneficial antioxidanteffects (Grucka-Mamczar et al., 2009). The exact mechanisms ofaction of caffeine in SCs metabolism are yet to be disclosed andthere is no evidence of a clear association between caffeine, OS andmale fertility. Herein we hypothesize that caffeine can alter SCsglycolytic and oxidative profile interfering with male’s reproduc-tive potential.

2. Material and methods

2.1. Chemicals

All chemicals were purchased from Sigma–Aldrich (St. Louis,MO, USA) unless specifically stated.

2.2. Patient selection, ethical issues and testicle tissue preparations

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

and European Ethical Committees. The studies have beenperformed according to the Declaration of Helsinki. Testicularbiopsies were obtained from patients under treatment forrecovery of male gametes and used after informed writtenconsent. Only cells left in the tissue culture plates after patient’streatment were used. Human SCs (hSCs) were isolated from sixtesticular biopsies of men with conserved spermatogenesis,selected from patients with anejaculation (psychological, vascu-lar, neurologic), vasectomy or traumatic section of the vasdeferens.

2.3. Human Sertoli cell primary culture

Testicle biopsies were washed twice in HBSSf (Hanks balancedsalt solution without Ca2+ or Mg2+) through centrifugations at500� g at room temperature, as described by Oliveira et al. (2011).hSCs were obtained by a routinemethod (Oliveira et al., 2009). Theresulting cellular pellet was suspended in hSCs culture medium(DMEM:Ham’s F-12 1:1, containing 15mM HEPES, 50U/mlpenicillin and 50mg/ml streptomycin sulfate, 0.5mg/ml fungi-zone, 50mg/ml gentamicin and 10% heat inactivated fetal bovineserum) and forced through a 20G needle, in order to disaggregatelarge cell clusters. Then, cells were plated on Cell+ culture flasks(Sarsted, Nümbrecht, Germany) and incubated at 30–33 �C, 5% CO2

in air until used. After 96h, the cultures were examined by phasecontrast microscopy and only the hSCs with contaminants below5% were used. hSCs culture purity was determined as described(Steger et al., 1996).

2.4. Experimental groups

hSCs were allowed to grow until reach 90–95% of confluence,and after fully washed, the culture medium was replaced byserum-free medium (DMEM:F12 1:1, pH 7.4) supplemented withinsulin–transferrin–sodium selenite (ITSmedium; 10mg/ml, 5mg/ml, and 5mg/ml, respectively). In order to evaluate the effect ofcaffeine on SCs glycolytic and oxidative profile, four differentgroups were defined: a control group without caffeine and threeother groups containing ITS medium supplemented with increas-ing doses of caffeine (5, 50 and 500mM). The daily consumption ofcaffeine between drinkers of caffeine-rich beverages was estimat-ed to be of 5mg/kg of body weight (FSA, 2004; Mandel, 2002). Theintake of such amount of caffeine leads to a plasma concentrationof about 10mg/mL (50mM) (Alvi and Hammami, 2011; Blanchardand Sawers,1983), the reasonwhywe choose to study the effects of50mM of caffeine in the hSCs metabolism. Moreover, we foundpertinent to evaluate the effects of a lower concentration (5mM)and a higher concentration (500mM), since caffeine content (andhence consumption) differs significantly amongst the variouscaffeine-containing beverages and food. After the 24h oftreatment, culture medium was collected. Then, cells weredetached from the flask using a trypsin–EDTA solution, countedwith a Neubauer chamber and collected for protein extraction.Viability evaluated by the Trypan Blue Exclusion test averaged 85–90%.

2.5. Proton nuclear magnetic resonance (1H NMR)

1H NMR spectra were acquired as previously described (Alveset al., 2011). Sodium fumarate (final concentration of 10mM) wasused as an internal reference (6.50ppm) to quantify the followingmetabolites present in solution (multiplet, ppm): lactate (doublet,1.33); alanine (doublet,1.45) andH1-a-glucose (doublet, 5.22). Therelative areas of 1H NMR resonances were quantified using thecurve-fitting routine supplied with the NUTSproTM NMR spectralanalysis program (Acorn, NMR Inc., Fremont, CA, USA).

T.R. Dias et al. / Toxicology 328 (2015) 12–20 13

2.6. Western blot

Total proteins were isolated from hSCs using RIPAS buffer (1xPBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1mM PMSF,supplemented with 1% protease inhibitor cocktail, aprotinin and100mM sodium orthovanadate). Western blot was performed aspreviously described (Dias et al., 2013a). The resulting membraneswere incubated overnight at 4 �C with goat anti-glucose trans-porter 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 anti-monocarboxylate transporter 4 (MCT4) (1:1000, sc-50329, SantaCruz Biotechnology, Heidelberg, Germany), or rabbit anti-lactatedehydrogenase (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 andvisualized with goat anti-rabbit IgG-AP (1:5000, sc-2007, SantaCruz Biotechnology, Heidelberg, Germany), rabbit anti-goat IgG-AP(1:5000, A4187, Sigma–Aldrich, St. Louis, MO, USA) or goat anti-mouse IgG-AP (1:5000, sc-2008, Santa Cruz Biotechnology).Membranes were reacted with ECFTM (GE, Healthcare, Bucking-hamshire, UK) and read with the BioRad FX-Pro-plus (Bio-RadHemel Hempstead, UK). Densities from each band were obtainedwith BIO-PROFIL Bio-1D Software from Quantity One (VilberLourmat, Marne-la-Vallée, France) according to standard methods(Picado et al., 1999). The band density attained was divided by thecorresponding tubulin band intensities and expressed in foldvariation relatively to the control group.

2.7. Lactate dehydrogenase (LDH) enzymatic assay

LDH levels were spectrophotometrically determined using aLDH Enzymatic Assay Kit (Thermo Scientific, Waltham, MA)according to the manufacturer’s instructions. In brief, 5mg ofproteins were diluted in lysis buffer. Likewise, a blank wasprepared and boiled for 5min at 90 �C for protein denaturation.LDH assay substrate was added to all samples in a darkenvironment and left at room temperature for approximately15min. Then, a stop solution was used to end the enzymaticactivity and absorbance at 490nmwas measured using an Anthos2010 microplate reader (Biochrom, Berlin, Germany). LDHenzymatic activities were calculated as units per milligram ofprotein using the molar extinction factor (e) and final expressed asfold variation to the control group.

2.8. Ferric reducing antioxidant power (FRAP) assay

The FRAP of the cellular pellets was determined using thecolorimetric method described by Benzie and Strain (1996). Inbrief, working FRAP reagent was prepared by mixing acetatebuffer (300mM, pH 3.6), 2,4,6-tripyridyl-s-triazine (TPTZ)(10mM in 40mM HCl) and FeCl3 (20mM) 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 afteradding the sample and 40min later, by measuring the absorbanceat 595 nm using an Anthos 2010 microplate reader (Biochrom,Berlin, Germany). The antioxidant potential of the samples wasdetermined against standards of ascorbic acid, which wereprocessed in the same manner as the samples. Absorbanceresults were corrected by using a blank, with water instead ofsample. The changes in absorbance values of test reactionmixtures were used to calculate FRAP value as describedelsewhere (Benzie and Strain, 1996).

2.9. Analysis of carbonyl groups and lipid peroxidation

Protein carbonyl content is commonly used as a marker forprotein oxidation while lipid peroxidation can be evaluated bymeasuring some resulting aldehydic products such as 4-hydrox-ynonenal (4-HNE). The content of protein carbonyl groups and 4-HNE in hSCs from the different experimental groupswas evaluatedusing the slot-blot technique and specific antibodies. For carbonylgroups evaluation, protein samples were derivatized using 2,4-dinitrophenylhydrazine (DNPH) to obtain 2,4-dinitrophenyl (DNP)according to the method developed by Levine et al. (1990). Theslot-blot technique was performed using a Hybri-slot manifoldsystem (Biometra, Göttingen, Germany) and the resulting PVDFmembranes were incubated overnight (4 �C) with a rabbit anti-DNP (1:5000, D9656, Sigma–Aldrich, St. Louis, MO, USA). For lipidperoxidation analysis, protein samples were diluted to a concen-tration of 0.001mg/mL using PBS 1x to be used in the slot-blottechnique. Then, the resulting membranes were incubatedovernight (4 �C) with a goat anti-4-HNE antibody (1:5000,AB5605, Merck Millipore, Temecula, USA). Samples were visual-ized using rabbit anti-goat IgG-AP (1:5000, A4187, Sigma–Aldrich,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 Health-care, Buckinghamshire, UK) and read using a BioRad FX-Pro-plus(Bio-Rad Hemel Hempstead, UK). Densities from each band werequantified using the BIO-PROFIL Bio-1D Software from QuantityOne (VilberLourmat, Marne-la-Vallée, France).

2.10. Statistical analysis

Statistical significance was assessed by one-way ANOVA,followed by Dunn post-test using GraphPad Prism 5 (GraphPadSoftware, San Diego, CA, USA). All data are presented as mean�SEM. Differences with p<0.05 were considered statisticallysignificant.

3. Results

3.1. Caffeine (50mM) increases glucose transporters proteinexpression in human Sertoli cells

Since caffeine is known to increase cells metabolic rates(Lane, 2011; Sinha et al., 2014), we hypothesized that it could alsoalter hSCs metabolism. For that purpose, we choose key points ofhSCs metabolism starting on their primary energy substrate,glucose. Our results showed a glucose consumption of 10.7�2.4pmol/cell in hSCs in the control group (Fig. 1A). hSCs of the groupsexposed to 5, 50 and 500mM of caffeine consumed 6.9�1.4,7.0�2.6 and 12.4�3.1 pmol/cell, respectively, with no significantalterations relative to the control group (Fig. 1A). In these cells,transport of glucose through the cytoplasmic membrane ismediated by specific membrane hexose transporters (mainlyGLUT1 and GLUT3). Our results showed that exposure to 5mM ofcaffeine did not significantly alter the protein expression levels ofGLUT1 or GLUT3 in hSCs, comparatively to the control group.However, hSCs exposed to 50mM of caffeine significantlyincreased GLUT1 protein levels (1.30� 0.16 fold variation tocontrol) (Fig. 1B) and GLUT3 (1.31�0.31 fold variation to control)(Fig. 1C) in comparison with the control group. Moreover, in cellsexposed to 50mM of caffeine, GLUT3 protein expression wassignificantly increased relative to cells exposed to 5mM of caffeine(Fig. 1C). Interestingly, hSCs exposed to 500mM of caffeine did notpresent significant differences in the protein expression levels ofGLUT1 (Fig.1B) and GLUT3 (Fig.1C) (1.15�0.09 and 1.10� 0.25 foldvariation to control, respectively) relative to control or to the other

14 T.R. Dias et al. / Toxicology 328 (2015) 12–20

concentrations of caffeine. The representative blots of GLUT1 andGLUT3 are shown in Fig. 1D.

3.2. PFK1 protein levels are decreased in human Sertoli cells exposed tolower caffeine concentrations while exposure to the highest caffeineconcentration stimulates LDH activity

After glucose enters the cells, a key regulatory step of glycolyticpathway ismediated by PFK1 that irreversibly converts fructose-6-phosphate to fructose-1,6-bisphosphate (FBP). FBP is responsiblefor the activation of the enzyme pyruvate kinase that catalyses theirreversible conversion to pyruvate. Our results showed that thelowest concentrations of caffeine (5 and 50mM) decreasedPFK1 protein levels to 0.65�0.02 and 0.74�0.04 fold variationto control, respectively (Fig. 2A). Interestingly, exposure to thehighest caffeine concentration restored PFK1 protein levels to thecontrol values. The formed pyruvate can then be converted tolactate by LDH action. Our results demonstrated that LDH proteinlevels were similar between groups of hSCs treated with 5mM(0.94�0.19 fold variation to control), 50mM (0.90� 0.15 foldvariation to control) and 500mM (0.91�0.18 fold variation tocontrol) of caffeine, showing no significant alterations comparativeto the control group (Fig. 2B).

Since LDH protein expression levels in hSCs were not altered bycaffeine action, we hypothesized that caffeine could alter theactivity of this enzyme. In fact, we verified a dose-dependentincrease in LDH activity of hSCs culturedwith caffeine. The levels ofLDH enzymatic activity in groups of hSCs exposed to 5, 50 and500mMof caffeinewere 1.26�0.16,1.30� 0.16 and 1.49�0.08 foldvariation to control, respectively (Fig. 2C). Nevertheless, only thehighest concentration of caffeine (500mM) significantly increased

LDH activity relative to the control group. After lactate productionby SCs, it is exported throughMCT4 to be used as metabolic fuel bydeveloping germ cells. The MCT4 protein levels in hSCs exposed to5, 50 and 500mM of caffeine were 1.08�0.12, 0.95�0.10 and0.93�0.09 fold variation to control, respectively (Fig. 2D). Thesevalues did not reach statistical significance when compared to thevalues determined in non-exposed cells. Fig. 2E displays therepresentative blots of PFK1, LDH and MCT4.

3.3. Lactate production by human Sertoli cells is stimulated byexposure to lowest caffeine concentrations while alanine production isonly stimulated by exposure to 50mM of caffeine

Lactate production is one of the key functions of hSCs. Non-exposed hSCs presented a lactate production of 17.6�2.4 pmol/cell. Interestingly, exposure to 5 and 50uM of caffeine significantlyincreased lactate production to 25.7�2.2 pmol/cell and 26.3�1.1,respectively (Fig. 3A). However, hSCs exposed to the highestconcentration of caffeine (500uM) did not present significantalterations in lactate production when compared with non-exposed cells. Alanine metabolism is closely associated withlactate production, since pyruvate can also be converted to alanine.Our results showed that extracellular alanine concentration innon-exposed cells was 0.51�0.08pmol/cell (Fig. 3B). Exposure to5mM of caffeine non-significantly increased alanine production to0.82�0.08pmol/cell. On the other hand, there was a significantincrease in alanine production by hSCs exposed to 50mM ofcaffeine (0.88�0.05pmol/cell) relative to the control group(Fig. 3B). hSCs exposed to the highest concentration of caffeine(500mM) produced 0.92�0.22pmol/cell of alanine, having nosignificant differences in comparison to the other experimental

[(Fig._1)TD$FIG]

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

T.R. Dias et al. / Toxicology 328 (2015) 12–20 15

groups. We also evaluated the ratio lactate/alanine, which is oftenused as an index of the redox state of the cell (Oliveira et al., 2012).The interconversion lactate–pyruvate–alanine is NADH-dependentand the NADH/NAD+ ratio can be estimated by the ratio lactate/alanine, which is often used as ameasure of the cellular redox state(Martins et al., 2013b). Interestingly, our results showed that hSCsexposure to any of the caffeine concentrations studied did not alterthe lactate/alanine ratio (Fig. 3C).

3.4. “Normal-range” dose of caffeine decreased protein oxidation andlipid peroxidation in human Sertoli cells, while highest concentrationsof caffeine compromised cellular antioxidant potential

The high glycolytic rates observed in hSCs can lead to increasedlevels of OS. Several studies have reported that caffeine consump-tion is associated with reduced levels of OS biomarkers (Grucka-Mamczar et al., 2009). This was attributed to the antioxidantactivity of caffeine (Devasagayam et al., 1996). We measured theantioxidant potential of hSCs pellets using the FRAP assay. Thereducing power of a compound/extract serves as an indicator of itspotential antioxidant activity (FRAP value). Comparative to thecontrol group, which demonstrated to have 0.8�0.2mmol ofantioxidant potential/mg of protein, hSCs exposed to 5mM ofcaffeinemaintained a similar value of 0.9�0.2mmol of antioxidantpotential/mg of protein. However, hSCs exposed to 50mMsignificantly decreased the FRAP value to 0.6�0.1mmol ofantioxidant potential/mg of protein when compared to non-exposed cells and the ones exposed to 5mM of caffeine. Of note,hSCs exposed to 500mM of caffeine presented a negative

antioxidant potential value (�0.4�0.2mmol of antioxidantpotential/mg of protein), indicating a pro-oxidant effect (Fig. 4A).

Protein carbonylation and lipid peroxidation are strongbiomarkers of OS. The attack of free radicals to proteins andmembrane unsaturated fatty acids originates several products,such as DNP and 4-HNE, respectively, which can be measured toobtain a quantification of the cellular oxidative damages. hSCsexposed to 5mM of caffeine presented a significant higherproduction of protein carbonyl groups (1.38�0.11 fold variation)comparative to the control group. In hSCs exposed to 50mM ofcaffeine there was a significant decrease on the production ofcarbonyl groups (0.85�0.12 fold variation to control) in compari-son to hSCs exposed to 5mM of caffeine. Finally, hSCs exposed to500mM of caffeine presented a content in carbonyl groups of1.47�0.07 fold variation to control, which was significantly higherrelative to non-exposed cells and cells exposed to 50mM ofcaffeine (Fig. 4B). Concerning lipid peroxidation, there were nosignificant alterations on hSCs exposed to caffeinewhen comparedwith non-exposed cells. The lipid peroxidation values determinedin hSCs exposed to 5, 50 and 500mM were1.12�0.07,0.87�0.02 and 1.00� 0.06 fold variation to control, respectively.Nonetheless, there was a significant decrease in lipid peroxidationresultant from exposure of hSCs to 5mMof caffeine relative to cellsexposed to 50mM (Fig. 4C).

4. Discussion

Due to its wide consumption, caffeine potential health effectshave been a focus of several studies. Popular beverages such as

[(Fig._2)TD$FIG]

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

16 T.R. Dias et al. / Toxicology 328 (2015) 12–20

coffee, tea and energy drinks are known to have high concen-trations of caffeine (Reissig et al., 2009). The average dailyconsumption of caffeine per individual was estimated to be of5mg/kg, reaching a concentration of 50mM in the plasma(Blanchard and Sawers, 1983; Chou and Benowitz, 1994).Therefore, we tested the effect of that concentration in hSCsglycolytic and oxidative profile to mimic the concentrationscommonly found in plasma in in vivo conditions. However, peopleare commonly exposed to caffeine in lower and higher concen-trations, therefore we also tested the effects of 5 and 500mM ofcaffeine. Previous studies reported that caffeine increases humanmetabolic rates (Nehlig et al., 1992) and blood glucose levels(Pizziol et al., 1998). Spermatogenesis is highly dependent on SCsglucose metabolism, since it is the source of lactate, which is thepreferred metabolic substrate of the developing germ cells

(for review (Rato et al., 2014)). Thus, we evaluated the effect ofcaffeine in glucose metabolism of these cells.

Caffeine is structurally very similar to adenosine and can bind toadenosine receptors acting as a nonselective antagonist (Fredholmand Lindström, 1999). Adenosine receptors have been identified inSCs (Monaco and Conti, 1986). Several follicle-stimulating hor-mone (FSH)-stimulated actions on SCs, such as inhibin andtransferrin secretion and pyruvate metabolism, were reported tobe mediated by occupancy of A1 receptors (Conti et al., 1989;Meroni et al., 1998). Moreover, it has been reported that adenosinepromotes lactate supply to germ cells (Galardo et al., 2010). Ourresults showed that the lowest caffeine concentrations signifi-cantly altered hSCsmetabolism. Notably, exposure of hSCs to 5mMand 50mM increased lactate production by these cells, withoutaltering significantly glucose consumption. These results areconcomitant with previous studies showing that caffeine can

[(Fig._4)TD$FIG]

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

[(Fig._3)TD$FIG]

Fig. 3. Effect of caffeine (5, 50 and 500mM) in the production of lactate (panel A)and alanine (panel B) as well as the lactate/alanine ratio (panel C) in human Sertolicells. Metabolites production is presented as pmol/cell. The figure shows pooleddata of independent experiments. Results are presented as mean� SEM (n =5 foreach condition). Significantly different results (P<0.05) are indicated as: (a)relative to control.

T.R. Dias et al. / Toxicology 328 (2015) 12–20 17

regulate glucose metabolism (Ojuka et al., 2002). Glucose uptakeby hSCs exhibits a strict hormonal regulation (Alves et al., 2013b).Moreover, these mechanisms are modulated by a direct regulatoryeffect in total GLUTs levels (Klip et al., 1994). In fact, it has beendescribed that hSCs have the ability to adapt their metabolismunder certain conditions. For instance, in insulin or glucosedeprivation conditions, hSCs adapt their metabolism by modulat-ing the expression of GLUT1 and GLUT3 (Oliveira et al., 2012; Rieraet al., 2009). In our experiments, when hSCs were exposed to50mMof caffeine therewas a significant increase in the expressionlevels of these transporters, illustrating a stimulation of glucoseuptake. Since these cells present an elevated glycolytic flux undernormal conditions (for review (Rato et al., 2014)), glucoseconsumptionwas not significantly altered. Nevertheless, exposureto the lowest concentrations of caffeine (5 and 50mM) increasedthe glycolytic flux of hSCs, as demonstrated by the significantincrease of lactate production by these cells. Since adenosine is alsoknown to promote lactate offer to germ cells (Galardo et al., 2010),our results showed that caffeine at these concentrations maymediate its effect in SCs metabolism by acting as antagonist ofadenosine receptors.

When hSCs were exposed to 500mMof caffeine, which is a highdose even for heavy drinkers of caffeine-rich beverages, we onlydetected alterations in PFK1 levels and LDH activity. Nevertheless,lactate production was not altered, nor glucose consumption. Asreferred, SCs exhibit a metabolic plasticity under stressfulconditions (for review (Alves et al., 2013a)). When subjected toglucose (Riera et al., 2009) or insulin (Oliveira et al., 2012)deprivation these cells adapt their metabolism in order to sustain aproper production of lactate, necessary for germ cell development.Thus, our results illustrate that caffeine at high doses (500mM)alters SCs physiologic functions, however, these cells present someadaptive mechanisms, such as increased PFK1 expression and LDHactivity, which allow them tomaintain the same lactate productionas normal cells.

Caffeine has been reported as a protective substance againstcellular damage with beneficial antioxidant effects (Grucka-Mamczar et al., 2009; Ofluoglu et al., 2009). Compelling evidencehas shown that caffeine and adenosine receptors can modulatecellular oxidant status (Gołembiowska and Dziubina, 2012;Prasanthi et al., 2010). Our results show that at high concentrations(500mM), caffeine induces a pro-oxidant environment in hSCs,thus, explaining the requirement for the reported metabolicadaptations in hSCs in order to sustain lactate production.Moreover, hSCs exposed to this concentration of caffeine presenteda significantly higher protein oxidation, as illustrated by thesignificant increase in carbonyl groups content relative to controlgroup and cells exposed to 50mM. The effect of caffeine in severalcellular functions, such as reactive oxygen species (ROS) genera-tion, have been reported to be concentration-specific (Tiwari et al.,2014). Although caffeine is generally known for its antioxidantproperties and efficiency as an hydroxyl radical scavenger in vitro(Shi et al., 1991), our results show that at a concentration of500mM, caffeine induces a pro-oxidant environment in SCs that isaccompanied by an increase in proteins oxidation. The lowest doseof caffeine tested (5mM) did not altered the antioxidant capacity ofcells when compared to non-exposed cells, however, there was anincrease in protein oxidation, evidencing an increase of OS. hSCsexposed to 50mMof caffeine presented lower antioxidant capacitythan non-exposed cells and cells exposed 5mM of caffeine.However, hSCs from this group (50mM of caffeine) presentedlower lipid peroxidation than non-exposed cells and lower proteinoxidation than hSCs exposed to 5mM of caffeine. These resultssupport that the effects of caffeine in OS are concentration-dependent. The intermediate concentration (50mM), which isconsidered to be the regular caffeine ingestion, appears to mediate

some protective effects against OS. As discussed, caffeine can act asa nonselective antagonist of adenosine receptors, which arereported to control the formation of free radicals (Gołembiowskaand Dziubina, 2012) and cells OS. Thus, the effects of caffeine in OSmay also bemediated by adenosine receptors, whichwould be alsoconcomitant with the changes detected in hSCs metabolism.

Concerning the effects of caffeine in the male reproductivefunction, there are some conflicting results. Maternal caffeineconsumption during gestation and lactation has been reported toimpair gonadal development and induce several long-termadverse effects on the reproductive health of the offspring(Dorostghoal et al., 2012). More specifically, it leads to significantdose-related decreases in the testicular weight, seminiferoustubules diameter, germinal epithelium height, testosterone levelsand sperm quality of the offspring (Dorostghoal et al., 2012).Besides, an in vivo study with Sprague-Dawley rats demonstratedthat and oral administration of an elevated dose of 200mg/kg ofbody weight of caffeine negatively affects the histo-architecture ofthe seminiferous tubules of the testis, with massive loss ofspermatogenic cells and testicular weight loss (Bassey et al., 2011).A more recent study with the same dosage of caffeine (200mg/kgof body weight) also reported testicular and epididymal weightloss as well as histological alterations, i.e., atrophic cells withnecrosis and excessive degeneration of spermatids and almostabsence of spermatozoa (Ekaluo et al., 2014). Of note, caffeine hasalso been used to induce sperm capacitation, particularly withfrozen thawed spermatozoa (Funahashi and Nagai, 2001). Whenhuman spermatozoawere incubated with caffeine, it was reportedthat the rate of glycolysis was significantly increased, withoutchanging ADP and ATP levels (Rees et al., 1990). Testicularmetabolism is crucial for spermatogenesis (for review (Diaset al., 2014a; Rato et al., 2012)). The strict metabolic cooperationestablished between SCs and developing germ cells is sensitive tohormonal fluctuations (Martins et al., 2013b) and several factors(Dias et al., 2013b). More recently, we have reported that a whitetea extract, which is very rich in caffeine (for review (Dias et al.,2013b)), is a promising antioxidant medium additive for spermstorage at room temperature. Supplementation with a white teaextract increased sperm antioxidant potential and decreased lipidperoxidation, restoring spermatozoa viability to values similar tothose obtained at collection time (Dias et al., 2014b). We alsoshowed that a white tea extract can modulate SCs and stimulatelactate production by these cells (Martins et al., 2013a). Thesepotential benefits in male reproductive health were attributed toseveral phytochemicals, including the high concentration ofcaffeine. In fact, caffeine has been consistently reported as ametabolic modulator. Our results show that caffeine modulateshSCsmetabolism in a dose-dependentmanner. The lowest caffeineconcentration stimulates lactate production without interferingwith the glycolysis-related machinery studied. However, theconcentration of 50mM, altered glucose transporters and in-creased alanine production to maintain the lactate/alanine ratio.This ratio is very important since it reflects the intracellular redoxstate. Lactate has been reported to protect germ cells in vivo bysuppressing the loss of spermatocytes and spermatids in cryptor-chid rats (Courtens and Plöen, 1999). Thus, it appears that caffeineat “normal-range” can enhance lactate production by hSCs andpromote germ cells survival. Interestingly, the most effectiveconcentration in preventing protein oxidation and lipid peroxida-tion in hSCs was 50mM,which is a frequent concentration found inthe plasma of heavy consumers of caffeine-rich beverages.

In sum, our results show that caffeine is a modulator ofhSCs metabolism and stimulates lactate production in a dose-dependent way. When exposed to a high concentration of caffeine,hSCs present a pro-oxidant environment and crucial adaptations intheir metabolism to sustain lactate production. Moreover, it

18 T.R. Dias et al. / Toxicology 328 (2015) 12–20

promotes proteins oxidation in hSCs. These results illustrate thatmoderate consumption of caffeine appears to be safe or evenpositive to the metabolic functioning of hSCs. Nevertheless,caution should be taken by consumers of energetic beveragesand supplemented food with caffeine to avoid deleterious effectsto hSCs functioning and spermatogenesis arrest.

Conflict of interest

The authors declare no competing financial interest.

Transparency document

The Transparency document associated with this article can befound in the online version.

Acknowledgments

This work was supported by the “Fundação para a Ciência e aTecnologia” - FCT (PTDC/QUIBIQ/121446/2010 and PEst-OE/SAU/UI0709/2014) co-funded by Fundo Europeu de DesenvolvimentoRegional - FEDER via Programa Operacional Factores de Com-petitividade – COMPETE/QREN. M.G. Alves (SFRH/BPD/80451/2011) was funded by FCT. P.F. Oliveira was funded by FCT throughFSE and POPH funds (Programa Ciência 2008). UMIBwas funded byFCT (PEst-OE/SAU/UI0215/2014).

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