Effect of Tadalafil Administration on Redox Homeostasis and Polyamine Levels in Healthy Men with High Level of Physical Activity

Background: The phosphodiesterase type 5 inhibitor (PDE5I) tadalafil, in addition to its therapeutic role, has shown antioxidant effects in different in vivo models. Supplementation with antioxidants has received interest as a suitable tool for preventing or reducing exercise-related oxidative stress, possibly leading to the improvement of sport performance in athletes. However, the use/abuse of these substances must be evaluated not only within the context of amateur sport, but especially in competitions where elite athletes are more exposed to stressful physical practice. To date, very few human studies have addressed the influence of the administration of PDE5Is on redox balance in subjects with a fitness level comparable to elite athletes; therefore, the aim of this study was to investigate for the first time whether acute ingestion of tadalafil could affect plasma markers related to cellular damage, redox homeostasis, and blood polyamines levels in healthy subjects with an elevated cardiorespiratory fitness level. Methods: Healthy male volunteers (n = 12), with a VO2max range of 40.1–56.0 mL/(kg × min), were administered with a single dose of tadalafil (20 mg). Plasma molecules related to muscle damage and redox-homeostasis, such as creatine kinase (CK), lactate dehydrogenase (LDH), total antioxidant capacity (TAC), reduced/oxidized glutathione ratio (GSH/GSSG), free thiols (FTH), antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)), as well as thiobarbituric acid reactive substances (TBARs), protein carbonyls (PrCAR), and polyamine levels (spermine (Spm) and spermidine (Spd)) were evaluated immediately before and 2, 6 and 24 hours after the acute tadalafil administration. Results: A single tadalafil administration induced an increase in CK and LDH plasma levels 24 after consumption. No effects were observed on redox homeostasis or antioxidant enzyme activities, and neither were they observed on the oxidation target molecules or polyamines levels. Conclusion: Our results show that in subjects with an elevated fitness level, a single administration of tadalafil induced a significant increase in muscle damage target without affecting plasma antioxidant status.

Recently, in addition to their role in the treatment of pathologies, in vitro and in vivo studies reported that PDE5Is (e.g., sildenafil, vardenafil, tadalafil) also possess antioxidant effects in different tissues/organs [9][10][11][12][13][14]. For instance, it has been shown that sildenafil citrate has protective effects against oxidative stress by inhibiting free radical formation and supporting antioxidant redox systems [13,15]. Interestingly, in a model of a fetal rat brain with an ischemia/reperfusion injury, tadalafil was found to be more effective than sildenafil in reducing the endogenous superoxide formation by increasing the antioxidant enzyme activity of glutathione peroxidase (GPx) [16].
Supplementation with antioxidants and/or drugs with antioxidant potential has received interest as a suitable tool for preventing or reducing exercise-related oxidative stress, possibly leading to the improvement of sport performance in athletes. Indeed, there are an increasing number of athletes (≈88%) using one or more nutritional supplements [25]. However, special attention must be given to reduce the risk which comes from the improper use of these substances, limiting their use to only specific cases/conditions. It is surprising to note that although there is much evidence to support the antioxidant effects of PDE5Is in animal models, to our knowledge, there is little information about the influence of tadalafil administration on redox balance in humans.
In 2015, our group highlighted the potential negative effects of tadalafil in healthy males [26]. In particular, we found that a prolonged exposure (repeated intake) to tadalafil increased plasma creatine kinase (CK) and lactate dehydrogenase (LDH) plasma levels, and decreased antioxidant capacity at resting conditions, thereby making subjects more susceptible to the oxidative stress induced by an exhaustive bout of exercise [26].
The purpose of this study was to investigate for the first time the effect of a single dose of tadalafil on the oxidant/antioxidant status and polyamines (PAs) levels in subjects with a cardiovascular fitness comparable to that of elite athletes and/or well-trained subjects, a group of individuals continuously exposed to exercise-induced oxidative stress.
To this end, blood sample collections were performed at different time points from tadalafil administration in a double-blind protocol to evaluate muscle damage target molecules, redox homeostasis, oxidation target molecules, antioxidant enzyme activities and PAs levels.

Subjects
The study was carried out on blood samples collected from twelve healthy men with high levels of physical activity (n = 12), with a VO 2max range of 40.1-56.0 mL/(kg × min) ( Table 1) (Di Luigi et al., 2008 [27]). All the subjects were involved in university athletic activity and in team sports (soccer, basketball, volleyball). The volunteers were non-smoker subjects who usually took no medication, anabolic agents, dietary supplementations, amino acid supplementation (e.g., arginine), vitamins and/or antioxidants. Ethical Committee's approval (Policlinico Umberto I-p.1039/08) and written informed consent were obtained. Moreover, they completed a detailed eating habits diary in which all food and drinks consumed were recorded.

Experimental Protocol
After 10 h overnight fasting and no consumption of caffeine for the prior 24 h, all the volunteers randomly received (at 8:00 a.m.) one tablet of tadalafil per os (20 mg, Cialis ® , Ely-Lilly, Indianapolis, IN, USA) in a double-blind experimental protocol ( Figure 1). No exercise, sexual intercourse, or major stress events were allowed, starting from 48 h before, and then continuing throughout, the protocol. supplementations, amino acid supplementation (e.g., arginine), vitamins and/or antioxidants. Ethical Committee's approval (Policlinico Umberto I-p.1039/08) and written informed consent were obtained. Moreover, they completed a detailed eating habits diary in which all food and drinks consumed were recorded.

Experimental Protocol
After 10 hours overnight fasting and no consumption of caffeine for the prior 24 hours, all the volunteers randomly received (at 8:00 a.m.) one tablet of tadalafil per os (20 mg, Cialis ® , Ely-Lilly, Indianapolis, IN, USA) in a double-blind experimental protocol ( Figure 1). No exercise, sexual intercourse, or major stress events were allowed, starting from 48 hours before, and then continuing throughout, the protocol. Figure 1. Flow diagram of the study. Twelve subjects were selected and then randomly assigned to the Cialis group (n = 6) or to the placebo group (n = 6) (Day 1). Then, subjects were given either Cialis (one tablet) or the placebo. Blood collections were performed at baseline (T0), and 2, 6 and 24 hours (Day 2) after administration (T2-T6-T24). After 4 weeks of washout, in the second experimental session, the volunteers were switched, and received either the placebo or Cialis, respectively. Each subject was his own control.

Biochemical Analysis
On blood samples collected, we evaluated: (1) muscle damage target molecules (creatine kinase (CK), lactate dehydrogenase (LDH)); (2) redox homeostasis (total antioxidant capacity (TAC), total glutathione (tGSH), reduced/oxidized glutathione ratio Figure 1. Flow diagram of the study. Twelve subjects were selected and then randomly assigned to the Cialis group (n = 6) or to the placebo group (n = 6) (Day 1). Then, subjects were given either Cialis (one tablet) or the placebo. Blood collections were performed at baseline (T0), and 2, 6 and 24 hours (Day 2) after administration (T2-T6-T24). After 4 weeks of washout, in the second experimental session, the volunteers were switched, and received either the placebo or Cialis, respectively. Each subject was his own control.

Materials
All chemical reagents, unless specified otherwise, were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA).

Creatine Kinase and Lactate Dehydrogenase
Plasma creatine kinase (CK) and lactate dehydrogenase (LDH) levels were measured as previously described [28] using commercial assay kits (Greiner Diagnostic GmbH, Bahlingen, Germany) following the manufacturer's instructions. Results were expressed as U Enz/L.

Total Plasma Free Thiol Determination
The free thiol (FTH) concentration of plasma samples was quantified by the Ellman assay according to Colombo et al. [30]. The molar concentration of thiols was calculated from the molar absorbance of the 2-nitro-5-thiobenzoate (TNB) anion and expressed as µmol-SH/g plasmatic proteins.

Trolox Equivalents Antioxidant Capacity (TAC)
Plasma TAC was determined spectrophotometrically, according to Ceci et al. [31]. This method is based on the reactivity of plasmatic antioxidant compounds relative to a 1 mM Trolox ® (vitamin E analog) standard.
The variation of absorbance detected was compared to those obtained using Trolox ® standards.

Thiobarbituric Acid Reactive Substances (TBARs)
TBARs level were assayed by spectrophotometric analysis [32]. The methodology measures malondialdehyde and other aldehydes produced by lipid peroxidation induced by hydroxyl free radicals. The levels of TBARs were expressed in terms of µmol/L.

Protein Carbonyls (PrCar)
Protein carbonyl levels were determined by measuring the reactivity of carbonyl derivatives with 2,4-dinitrophenylhydrazine (DNPH) as previously described [33]. Protein carbonyl content was expressed in terms of nmol/mg protein.

Enzymatic Activities
Intracellular superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities were measured as previously described [24] using commercial assay kits (Cayman Chemical Company, Ann Arbor, MI, USA) following the manufacturer's instructions. Results were expressed as units/mg of protein tested.

Statistical Analyses
The values were expressed as the means ± standard deviations (SD). Statistical analyses were performed using SPSS software (Version 21.0, IBM SPSS Statistics, Armonk, NY, USA). An a priori analysis (GPower 3.1, Heinrich-Heine-Universität Düsseldorf, Dusseldorf, Germany) was used to determine a sample size that yielded power values of 0.8 or greater. The Kolmogorov-Smirnov test was used to evaluate the variable distribution. A one-way ANOVA for repeated measures and Bonferroni post hoc analyses were used to determine significant variations over time for each parameter evaluated. A p-value <0.05 was considered statistically significant in all analysis.
No statistically significant changes were found for all parameters analyzed in the placebo group. All the data shown refer to the values found in the Cialis group.

Total Plasma Free Thiol Determination and Trolox Equivalents Antioxidant Capacity
A time-dependent analysis showed no statistically significant variation in FTH and TAC values after a single tadalafil administration (Table 3).

Oxidative Damage Analysis
Single tadalafil administration induced no statistically significant variation in plasma TBARs and PrCAR levels ( Table 4). Plasma thiobarbituric acid reactive substances (TBARs) and protein carbonyl (PrCAR) levels were measured in plasma before (T0) and 2, 6 and 24 hours (T2-T24) after tadalafil administration. Data presented are the mean ± SD, n = 12, performed in triplicate. a µmol/L; b nmol/mg.

Enzymatic Activities
A time-dependent analysis showed no statistically significant variation in plasma CAT, SOD, GPx levels and SOD/CAT and SOD/GPx ratios after a single tadalafil administration (Table 5). Plasma catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) activities and SOD/CAT and SOD/GPx ratio analysis were measured in plasma before (T0) and 2, 6 and 24 hours (T2-T24) after tadalafil administration. Data presented are the mean ± SD, n = 12, performed in triplicate, a U/mL.

Polyamines Levels
Single tadalafil administration induced no statistically significant variation in blood Spm and Spd levels and Spm/Spd ratio (Table 6). Plasma spermidine (Spd), spermine (Spm) and relative ratios (Spd/Spm) analysis were measured in blood before (T0) and 2, 6 and 24 hours (T2-T24) after tadalafil administration. Data presented are the mean ± SD, n = 12, performed in triplicate. a nmol/mL.

Discussion
The aim of this study was to assess the effects of an acute tadalafil administration on markers of muscle damage and oxidative balance in healthy men with an elevated cardiorespiratory fitness level. Indeed, all subjects have a VO 2max between 40.1 and 56.0 mL/(kg × min), corresponding for the chronological age to an excellent/superior level of cardiorespiratory fitness (Table 1).
Although the issue is still controversial, active individuals (e.g., athletes and welltrained subjects) often consume antioxidants to provide protection against oxidative stress and reduce muscular damage and fatigue symptoms [37][38][39][40].
Generally speaking, there is an increasing demand for molecules or drugs capable of managing physical and oxidative stress. This fact entails a continuous attention to the phenomenon of the unauthorized abuse of these compounds.
In this context, a great interest has shifted towards PDE5Is. Many healthy athletes who participate in sports which require endurance, and/or who compete in hypoxic conditions, misuse PDE5Is to improve performance, as these substances are not prohibited by the World Anti-Doping Agency (WADA) [41,42].
These compounds have recently showed antioxidant effects in different organs of animal models. This effect is achieved through the reduction of endogenous superoxide formation and the increase in the antioxidant enzymes' activity [9][10][11]13,15,16]. Moreover, an improved mitochondrial integrity [11,21], a decreased tissue malondialdehyde and increased SOD and GPx activity levels were found following tadalafil administration [17][18][19][20][21][22]. In a mouse model of contrast-induced nephropathy, sildenafil and tadalafil pretreatment were able to reduce nephropathy risk and reversed oxidative stress by modulating the oxidant/antioxidant balance [8].
To focus on the human model, we have demonstrated that in healthy males, a prolonged exposure to tadalafil decreases antioxidant capacity by reducing the GSH/GSSG ratio and increasing TBARs and PrCAR levels [26]. Therefore, these results suggest that the subjects have a greater susceptibility to oxidative stress. This aspect is particularly important considering that they were subjects with a high level of fitness.
To better understand the effects of tadalafil on well-trained subjects, we verified if a single dose of tadalafil increased plasma markers of muscle damage. We found that 24 hours after tadalafil ingestion, creatine kinase and lactate dehydrogenase plasma levels resulted in increased respect T0 (Table 2). These data support the hypothesis that the evidenced side effects (i.e., back pain) of the drug administration [43] could be due to its action on muscle tissue.
Subsequently, we wondered if the increase in circulating CK and LDH was paralleled by an altered redox homeostasis.
Glutathione is the primary antioxidant responsible for maintaining a reduced intracellular microenvironment. When reactive oxygen species (ROS) production is accelerated to the point that it overwhelms ROS scavenging capacity, the GSH/GSSG ratio, a well-known marker of oxidative stress, decreases, significantly reducing the antioxidant capacity [24].
In our healthy subjects, we found that a single pill substantially unaffected plasma redox homeostasis, as evidenced by glutathione levels analysis (Table 3).
An increase of 31.5 and 27.5% of GSSG were found after 6 and 24 hours with respect T0. However, this increase is not reflected in a significant decrease in the GSH/GSSG ratio, thus indicating that the buffering capacity of the plasma is able to maintain the right redox state.
In plasma, protein thiols act as a redox buffer; their concentration is much greater than those of low-molecular weight thiols. The oxidation of protein thiols to mixed disulfides is an early response to oxidative stress [44].
In our subjects, plasma free thiols remained unchanged by acute tadalafil administration, indicating that no effect on thiol/disulfide status in plasma occurred (Table 3).
Regarding the oxidative damage, the time and dose of administration of tadalafil seems to cause different effects in healthy humans. We demonstrated in a previous paper that a prolonged (two tablets with 36 hours of interval) tadalafil ingestion caused an increase in malondialdehyde (TBARs) and protein carbonylation. However, here we demonstrated that an acute dose (single administration) did not induce an increase in oxidation target molecules (Table 4).
It is known that PDE5Is modulate antioxidant enzyme activities systems. The antioxidant enzymes' superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity levels increased after administration and following a stressful stimulus [16,19,20]. Sildenafil citrate resulted in a significant increase in the erythrocyte superoxide dismutase and catalase activities in humans [15]. Moreover, recently, we have demonstrated that tadalafil may be beneficial to skeletal muscle cells by enhancing the enzymatic antioxidant system capacity in C2C12 skeletal muscle cells [24].
Nevertheless, analysis of plasma antioxidant enzyme activities revealed that, in the subjects analyzed, a single dose of tadalafil was not able to induce superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities. Hence, during acute treatment, SOD, CAT and GPx plasma levels do not reflect the presumed intracellular increase, so we can speculate that tadalafil has a putative beneficial effect for improving cellular enzymatic antioxidant system capacity without evidence at systemic level (Table 5).
Polyamines (PAs) are naturally occurring aliphatic compounds that are intrinsic constituents of all eukaryotic cells. Among these, spermine (Spm) and spermidine (Spd) play essential roles in many cellular functions [45][46][47][48] and possess different functions in the protection from reactive oxygen species [49]. Several in vitro studies have demonstrated that PAs protect cells from hydrogen peroxide-and superoxide-induced oxidative stress. Interestingly their effect seems to be distinct from that played by the most important primary antioxidant glutathione [49].
Polyamines are amines derived from arginine, which is the precursor of nitric oxide (NO) [47]. An alteration in PAs levels may affect the NO bioavailability due to the close relationship between PAs and NO metabolism [49].
In subjects who have taken a pill of tadalafil, the persistence of NO, due to phosphodiesterase type 5 (PDE5) inhibition, could determine a greater ability for this molecule to react with ROS to form toxic peroxynitrite [50]. These molecules represent the most reactive free radical species [51], able to induce a redox unbalance at rest.
It was demonstrated that PAs levels were increased in childhood obesity and correlated to markers of oxidative/nitrosative stress [52].
Interestingly, in our subjects, blood Spd and Spm levels remained unchanged by acute tadalafil administration, indicating that the increase in NO bioavailability substantially unaffected PAs metabolism (Table 6).
Finally, plasma's total antioxidant capacity, which expressed the reactivity of plasmatic antioxidant compounds in counteracting radicals, also remained unchanged. In vitro analysis of the scavenger capacity of tadalafil showed modest activity (Table 7) and, therefore, although the molecule may act for longer than other PDE5 inhibitors [6], its contribution to the plasma total antioxidant capacity may be not significant. There are few studies evaluating the effects of tadalafil on healthy athletes [27,[53][54][55]. Many studies have been conducted on subjects with pathologies or who are in conditions of extreme physiological stress [56][57][58][59][60][61][62][63]. Recently, using a similar group of subjects, we have demonstrated that a single dose of tadalafil did not substantially influence performance indicators, during a maximal standardized exercise test in a healthy athlete [27]. Moreover, the consumption did not modify the time to peak power and increased blood lactate concentrations during recovery from exercise [55]. This effect could be related to a possible effect of PDE5Is in stimulating anaerobic glycolysis [23].
The particular category of subjects used in this study could explain the results obtained here. In fact, in the athletes/well-trained subjects with the highest cardiovascular fitness levels, there is a continuous physiological adaptation responding to oxidative stress, which is especially realized in the increase in the antioxidant systems and in the control of the redox state [31,[64][65][66].

Conclusions
In the context of an increased demand for molecules or drugs that can help people to sustain physical and oxidative stress, it is especially important to know whether the use/abuse of substances may be safe or not. Although further studies are needed to clarify the molecular mechanisms behind these results, here, we showed for the first time that a single dose of tadalafil increases the plasma levels of muscle damage markers without affecting antioxidant status related molecules in healthy men with high levels of physical activity.
It must be considered that our results cannot be generalized to the entire sporting population. Indeed, we cannot exclude that in subjects with low levels of physical activity, and, therefore, with a less adapted redox system, tadalafil could have a positive effect.
Nevertheless, as with other drugs with antioxidant properties, the initial antioxidant values should be considered, as well as the possible relationship among redox biomarkers, measured before and after exercise-induced redox challenge in subjects who consumed these substances. Moreover, these data could be useful to identify the right dose, thus avoiding the possible negative effects (e.g., muscle damage) which can derive from a high dosage of these molecules. Funding: This research was funded by grants from the University of Rome "Foro Italico" ("Effects of spermine oxidase (SMOX) overexpression in skeletal muscle physiology and in pathological conditions."-CDR2.BANDO2017SS) and ("Ruolo dello stress ossidativo nella produzione di steroidi androgeni in cellule muscolari scheletriche e possibili interferenze delle fosfodiesterasi 5 sulla steroidogenesi"CUP H82F20000140001-CDR2.BANDO2020SP) to S.S. and P.S. Each author of this study further declares no relationships with the companies or manufacturers who will benefit from the results of the present study.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of Policlinico Umberto I, Rome Italy-p.1039/08. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Acknowledgments:
The authors thank Paolo Mariottini (Department of Science, "Department of Excellence 2018-2022", University of Rome "Roma Tre", 00146 Rome, Italy) and Federico Spinozzi for their precious support. Each author of this study further declares no relationships with the companies or manufacturers who will benefit from the results of the present study.

Conflicts of Interest:
The authors declare no conflict of interest.