Effect of taurine associated gold nanoparticles on oxidative stress in muscle of mice exposed to overuse model

Muscle overuse and its consequent muscle damage has no cure. Therefore, the present study aimed to investigate the regulatory role of tau-AuNPs on muscle recovery of muscle overuse model. The animals (Male Swiss mice) were randomly divided into four groups: Control (Ctr; n=6); tau-AuNPs (n=6); overuse (n=6); and overuse plus tauAuNPs (n=6). Exercise sessions were performed for 21 consecutive days, and one exercise model was applied daily in the following sequence: low intensity, moderate intensity, and high intensity. The mice were then sacrifi ced. The quadriceps muscles were surgically removed for subsequent biochemical analysis (oxidative stress parameters, DNA damage markers and muscle differentiation protein). The overuse group signifi cantly increased the oxidative stress parameters and DNA damage markers, whereas tauAuNPs signifi cantly decreased the oxidative stress parameters in the overuse animal model. However, there were no signifi cant differences observed between overuse group and overuse plus tau-AuNPs administrated group in relation to DNA damage markers including DNA damage frequency and index levels when compared to control and tauAuNPs groups. Muscle differentiation protein Myf-5 was increased in the overuse plus tau-AuNPs administration group when compared to control group. In conclusion, tauAuNPs had signifi cant effect on reducing oxidative stress parameters and increasing myogenic regulatory protein Myf-5 in the overuse group. However, it did not have signifi cant effect on reducing DNA damage.


INTRODUCTION
The repetitive microtrauma in relation to volume and intensity of the physical exercise can fail the muscle adaptation potentiality is named overuse caused injury. This has a serious consequence in terms of negatively promoting number of cellular and biochemical alterations in the muscular system (DiFiori et al. 2014). In general, muscle adaptation needs sufficient recovery time, but in case of overuse muscles do not get enough recovery time, leads reactive oxygen species production (ROS) and further ROS induced damages in the cellular and subcellular components of muscles (Thirupathi et al. 2018). Since ROS carry out some of the major signaling for muscle improvements including adaptation and plasticity, it is important to maintain the redox balance (Brown & Griendling 2015, Powers et al. 2011. Number of exogenous antioxidants have been used to balance the muscle redox system (Ostjen et al. 2018, Silva et al. 2010, Sakellariou et al. 2016. In a recent work, our group have shown that taurine (tau) supplementation modulates various cellular remodeling parameters after overuse-induced muscle damage, and these positive effects may be related to its antioxidant capacity (Thirupathi et al. 2018). Tau is a thiol based amino acid synthesized through cysteine metabolism; and can also be obtained exogenously. Although it does not participate in the structure of proteins and enzymes, tau has an important regulatory function in calcium fluxes, DNA protection (Thirupathi et al. 2018), in the stabilization of cell membrane structure (Schaffer et al. 2010), and in the regulation of inflammatory mediators (da Silva et al. 2014, Nakajima et al. 2010). In addition, the tau supplementation has a positive effect on redox homeostasis in the skeletal muscle (McLeay et al. 2017, et al. 2017, Seidel et al. 2018). However, the accuracy of tau`s antioxidant property in the cellular level is inconclusive and limited.
Due to versatile characteristics of gold nanoparticles (AuNPs), it can be used for a wide range of biomedical applications. The binding potential between the analytes and the AuNPs can facilitate the physiochemical properties of AuNPs which has a profound effect on biological situation. Previous study has reported that thiol association with AuNPs improved the redox system (Paula et al. 2015). This association might be exerting antioxidant and anti-inflammatory action for improving the redox system (Victor et al. 2012) and the AuNPs also have high reactive centres, which can bind strongly to thiol groups of intracellular compounds (Levy et al. 2004). In addition, AuNPs can penetrate into cells and carry biologically active substances (Jin et al. 2010, Arvizo et al. 2010. Since tau and AuNPs possessing cellular regulatory properties and biological functions, it is believed that the association of both molecules can decrease the oxidative stress, and further consequent DNA damage, and increase the degree of cellular protection for facilitating the process of muscle repair, particularly in muscle injury conditions.

Preparation of Tau-AuNPs
Tau synthetic 99% was acquired from Sigma-Aldrich (T0625). AuNPs were prepared by reduction of tetrachloroauric acid (Sigma-Aldrich -St. Louis, MO, USA) with sodium citrate (Nuclear Diadema, SP, Brazil) and characterized as previously described (Paula et al. 2015). Briefly, 35 uL of HAuCl 4 was diluted in 100 mL of water and the solution was harmed until 90 o C under magnetic stirring. Then, 5 mL of sodium citrate (10 mmol/L) was added, and the system maintained under reflux and stirring at 700 rpm for 20 min. The interaction between AuNPs and tau was evaluated by UV-vis spectroscopy via surface plasmon resonance (SPR) band using a Shimadzu instrument model UV-1800 (Shimadzu Corp., Kyoto, Japan). Briefly, 0.1 mL of tau at 0.10 mol / L, 0.25 mol / L, and 0.50 mol / L aliquots were added to 1 mL of AuNPs solution and the UV-vis spectra registered. Tau-AuNPs FTIR spectra were recorded employing a Fourier transform infrared Shimadzu IRAffinity-1S. In this case, a solution of tau-AuNPs was deposited on the surface of the sample holder, and then it was kept in a desiccator under reduced pressure until complete evaporation of the water.

Animals
Male Swiss mice (30-35 g) were obtained from our own breeding colony. The animals were housed according to the experimental design to a cage, on a 12 h light/dark cycle (lights on at 07:00), with free access to food (Nuvilab CR1, Nuvital Nutrientes S/A, Brazil) and water. All experimental procedures were performed in accordance with the Brazilian Guidelines for the Care and Use of Animals for Scientific and Didactic purposes (DOU 27/5/13, MCTI, p.7), and the local ethics committee approved the study. The animals were randomly divided into four groups: Control (Ctr; n=6); tau-AuNPs (n=6); overuse (n=6) and overuse plus tau-AuNPs (n=6).

Overuse model
All the animals were habituated on a ninechannel, motor-driven treadmill at a speed of 10 m min−1 for 10 min/day for one week to reduce the stress of a new environment. The mice did not receive any stimuli to run. The overuse model consisted of three different exercise types: low intensity (60 minutes at a speed of 13 m.min-1, no incline), moderate intensity (60 minutes at a speed of 17 m. min1, no incline), and high intensity (inclination -16% until exhaustion at a speed of 17 m.min-1-eccentric exercise). The exhaustion was considered by the inability of the animals to maintain a continuous rhythm (remained for 30 seconds or more at the bottom of lane run). Exercise sessions were performed for 21 consecutive days, and one exercise model was applied daily in the following sequence: low intensity, moderate intensity, and high intensity.

Tau-AuNPs administration
100 µL of Tau-AuNPs were administered subcutaneously on the back of the animals immediately after the high-intensity exercise.

Sample preparation
Twenty-four hours after the last exercise session, twenty microliters of blood were collected from the tail of the animal to determine the DNA damage. After this proceeding, the animals were killed by decapitation and the right quadriceps (central portion) were surgically removed and immediately processed or aliquoted and stored at -70°C for subsequent biochemical analysis. The central portion (50mg) of left quadriceps was immediately homogenized in specific buffer containing 1% Triton X-100, 100 mMTris (pH 7.4), 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10mM ethylenediaminetetraacetic acid (EDTA), 10mM sodium vanadate, 2mM phenylmethylsulfonyl fluoride (PMSF), and 0.1 mg/ml aprotinin at 4°C with Polytron MR 2100 (Kinematica, Switzerland). The homogenate was centrifuged at 11,000 rpm for 30 minutes at 4°C. The concentration of total proteins was determined from the supernatant (Bradford 1976). The proteins were resuspended and stored in Laemmli buffer containing 100 mmol/L dithiothreitol (DTT) for further immunoblotting assay with specific antibodies.

Oxidative stress parameters
Oxidized intracellular 2',7'-dichlorofluorescein (DCF) levels were monitored in samples incubated with 2',7'-dichlorodihydrofluorescein (DCFH). The formation of the oxidized fluorescent derivative was monitored at excitation and emission wavelengths of 488 and 525 nm, respectively, using a fluorescence spectrophotometer instruments. The malondialdehyde (MDA) concentrations in the tissue samples were determined by reverse phase high performance liquid chromatography (Prominence, Shimadzu Corporation, Japan), using a column Ascentis® C18 -250 x 2.1 mm, 5 μm (Supelco, Sigma-Aldrich) using a thiobarbituric acid derivatization (Grotto et al. 2007). A standard curve was prepared using malondialdehyde tetrabutylammonium salt at concentrations ranging from 0.5 to 5.0µM. Total thiol content (sulfhydryl) was determined using DTNB oxidation as a reference. The reaction was initiated by adding 30 µl of 10 mM DTNB to phosphate-buffered saline. Following 30 min of incubation at room temperature, the absorbance at 412 nm was measured and the amount of TNB formed was calculated (equivalent to the thiol group content), as previously described (Ellman 1959).

Comet assay
Comet assays were performed under alkaline conditions (Tice et al. 2000) and visual scores were classified (Collins et al. 1997). The blood sample was placed in cold PBS and minced with a syringe plunger to obtain a fine cell suspension. Aliquots (20 μL) were embedded in low melting agarose (0.75%, w/v; 80 μL). These mixtures were then deposited onto microscope slides, which were precoated with normal melting point agarose (1.5%, w/v) and furnished with coverslips (two slides per sample). The slides were briefly placed on ice and the cover slip was carefully removed. The base slides were immersed in freshly prepared lysis solution (2.5 M NaCl, 100 mM EDTA, and 10 mMTris, pH = 10.0-10.5), before being immersed for 20 min in freshly prepared alkaline buffer (290 mMNaOH, 1 mM EDTA, pH > 13). Next, electrophoresis experiments (15 min/290 mA; 25 V; 0.7 V/cm) were performed using the same buffer. All these steps were carried out in the minimal indirect light. Following electrophoresis, the slides were neutralized with 400 mMTris (pH = 7.5) and stained with ethidium bromide solution (10 mg/mL). Damage indexes (DI) were calculated by visually separating the cells into five classes according to their tail size (0 = no tails to 4 = maximumlength tails). An individual DI was thus obtained for each sample and subsequently for each group studied. The group DIs ranged between 0 (completely undamaged = 100 cells × 0) and 400 (maximum damage = 100 cells × 4). The damage frequency (DF in %) was calculated for each sample, based on the number of cells with tails compared to those without. Visual scores for a Comet assay are a reliable evaluation method and usually agree closely with computer-based image analysis methods. All slides were coded for blind analysis.

Protein content
Protein levels were measured in all samples using the Bradford method (Bradford 1976) which is based on an absorbance (595 nm) shift of the dye Coomassie brilliant blue G-250 in which the red form of the dye is converted into its bluer form upon binding to the protein in the sample. Protein standards were obtained by diluting a stock solution of bovine serum albumin. Linear regression was used to determine the actual protein concentration of each sample.

Western Blot
250 μg of protein per sample were applied on polyacrylamide gel (SDS-PAGE). Electrophoresis was performed in a Mini-PROTEAN® Tetra electrophoresis system (Bio-Rad, Hercules, CA, USA), with electrophoresis buffer solution. Proteins separated on SDS-PAGE were transferred to the nitrocellulose membrane using the Mini Trans-Blot® Electrophoretic Transfer Cell (Bio-Rad) equipment. Nitrocellulose membranes containing the transferred proteins were incubated in blocking solution for 2 hours at room temperature to decrease non-specific protein binding. Membranes were then incubated with specific primary antibodies, anti-Myf-5 acquired from Cell Signaling Biotechnology (Beverly, MA, USA) under constant and overnight stirring at 4°C. Original membranes were reblotted with β-actin as the control protein and then incubated in solution with peroxidase conjugated secondary antibody for 2 hours at room temperature. Then, membranes were incubated for 2 minutes with enzymatic substrate and exposed to the RX film in a developing cassette. Intensity and area of the bands were captured using a scanner (HP G2710), quantified through the Scion Image program (Scion Corporation, Frederick, MD, USA).

Statistical analysis
All data are presented as mean ± SEM and differences between groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey post-hoc tests. P<0.05 was considered statistically significant. All statistical analyses were performed using the Graph Pad Prism 7 software.

Tau-AuNPs interaction
The electronic spectra of AuNPs and tau-AuNPs are presented in figure 1a and show a surface plasmon resonant (SPR) band with maximum absorption at 525 nm, typical of spherical gold nanoparticles. As can be seen, the addition of tau at different concentrations did not affect the SPR band. The UV-vis spectra of the AuNPs remained unchanged even after 12 h of addition of tau, regardless of the concentration of tau, indicating the stability of the solutions. Warming solutions also showed no effect. Finally, it was not possible to synthesize AuNPs replacing sodium citrate and using tau as a reducing and stabilizing agent. The figure1b shows the vibrational spectra of tau and tau-AuNPs obtained by FT-IR (Fourier transform infrared spectroscopy) in the ATR mode and shows that all vibrational modes of tau are preserved after association with AuNPs.

Oxidative stress indicators
Both DCFH oxidation ( Figure 2a) and MDA level ( Figure 2b) were significantly increased in the overuse group when compared to control, whereas combined Tau-AuNPs decreased the ROS generation and lipid peroxidation in the overuse group. Sulfhydryl content was decreased in the overuse group when compared to control group, but the administration of Tau-AuNPs did not significantly reverse these alterations in skeletal muscle (Figure 2c).

DNA damage indicators
The frequency and index of DNA damage in blood were used as genotoxicity parameters. As we expected, the overuse group had high level of DNA frequency ( Figure 3a) and index ( Figure  3b), but there were no significant differences observed in both parameters of Tau-AuNPs administration group when compared to overuse group.

Muscle differentiation protein
Muscle differentiation proteins are important markers for finding muscle damage and repairing. We analysed the myf-5 protein is an important muscle differentiation protein. The level of muscle differentiation protein myf-5 was significantly decreased in the overuse group; whereas the administration of Tau-AuNPs increased the myf-5 level significantly when compared to control and overuse groups ( Figure  4).

DISCUSSION
The interactions of thiol-AuNPs at the cellular level can modulate several biological processes such as inflammation, oxidative stress, tissue repair, and has now become the focus of several research groups. Tau or 2-aminoethanesulfonic acid, is an amino acid of molecular formula C2H7NO3S which contains the functional groups -NH2 and -SO3H. The metal atoms in the surface of gold nanoparticles can interact with donor-acceptor species or ligands in a similar way as the related metal complexes (Grove & Karpowicz 2017, Toma et al. 2010. Previously, we reported the interaction between AuNPs and NAC (N-acetylcysteine), and its further biological ANAND THIRUPATHI et al.

TAURINE ASSOCIATED GOLD NANOPARTICLES ON OXIDATIVE STRESS
An Acad Bras Cienc (2021) 93(2) e20191450 6 | 12 effects. NAC present a -SH group, while tau present a -SO 3H group, which explains the difference in the UV-vis spectrum of the NAC-AuNPs when compared to those of the tau-AuNPs. Here, we analysed the tau associated gold nanoparticle in reducing oxidative stress, and DNA damage induced by muscle overuse. Our results revealed an important role of the association of these molecules in mechanisms that regulate the recovery process after muscle injury. Although the interaction between tau-AuNPs, based on the UV-vis and FTIR spectra, is not so evident, it is noticeable that the concomitant administration of tau-AuNPs have a biological effect, and capable of altering several biochemical parameters, as shown by the results of this study. In addition, the maintenance of the vibrational modes in the FTIR spectrum corroborates the UV-vis results, and in agreement with reported previously by Grove and Karpowicz (Grove & Karpowicz 2017).
Muscular systems are the primary site for metabolic insults and adaptation. Regular exercise with optimum level has beneficial effects on muscles, but unaccustomed exercise with higher intensity has deleterious effect on muscles including ROS production and consequent muscle damage. Several studies have observed that tau scavenge the ROS and regulate the antioxidant defense mechanism (Jong et al. 2012, Zhang et al. 2014. However, its role in the cellular level as antioxidants is still debatable. Therefore, we attempted to use first time for the association of tau-AuNPs as ROS scavenger to counteract the ROS during muscle overuse. As we expected, overuse animal with tau-AuNPs decreased the ROS production. The possible way for scavenge the overuse induced ROS by tau-AuNPs is diverse the electrons from electron transport chain to oxygen molecule, and AuNPs can facilitate the entrance of tau into mitochondria (Daniel & Astruc 2004). Our previous study found that tau regulates the mitochondrial complexes in order to reduce the ROS production in the overuse muscles (Thirupathi et al. 2018). This study supports our previous study, and AuNPs might facilitate the tau`s entrance at the mitochondrial level.
Low levels of ROS production are crucial in the skeletal muscle since it is involved to carry out number of signaling during muscle adaptation and repairing. However, higher Figure 2. DCFH oxidation (a) and oxidative damage in the lipids (b) and thiol groups (c) of the quadriceps muscles of mice submitted to muscle overuse and tau-AuNPs administration. The results are expressed as the mean and SEM and were analyzed by one-way ANOVA followed by the post-hoc Bonferroni test. *p≤0.05 compared to the control; #p≤0.05 compared to the overuse group. All statistical analyses were performed using Graph Pad Prism 7 software. Groups: control (Ctrl), tau-AuNPs, overuse (Ov), overuse plus tau-AuNPs (Ov+Tau-AuNPS). levels of ROS production, on the other hand, can damage the components of cellular organs such as proteins and lipids resulting to changes in the muscular functions. Our group have reported that experimental overuse increased the ROS generation and subsequent oxidative damage (Thirupathi et al. 2018). The present study found that there was a significant difference in the overuse group that were not administrated with tau-AuNPs. However, tau-AuNPs administrated group did not show significant improvement in increasing sufhydryl level when compared to control. The possible reason is that the higher bound activity of AuNPs to thiol group of internal sulfur containing amino acids including homocysteine and cysteine may have disturbed the sulfhydryl levels (Bürgi 2015, Fenoglio et al. 2008). Moreover, the MDA level, a by-product of lipid peroxidation process, was increased with overuse muscles, whereas Tau-AuNPs decreased the MDA level. This result suggests a role of tau in the stabilization of muscle cells membrane from ROS insults. In addition, AuNPs have shown to dismutate superoxide radicals (Cao et al. 2011). AuNPs with other compounds increased the twofold higher radical scavenging activity (Martín et al. 2010). The present results corroborated with the above findings that tau-AuNPs increased the ROS scavenging activity by reducing MDA level.
It is well established that increased ROS generation can lead to DNA damage. Tau has been reported to decrease the ROS generation through different mechanisms. However, the role of tau in protecting DNA from ROS induced oxidative damage is limited. We first time reported that tau-AuNPs role on protecting DNA damage in the overuse model. As we expected, the overuse group had increased index and frequency of DNA damage. However, unexpectedly overuse group administrated with Tau-AuNPs did not reduce the DNA damage frequency and index. Previous studies have reported that administration of tau decreased the DNA damage by reducing oxidative damages (Thirupathi et al. 2018, Sugiura et al. 2013. In contrast, AuNPs administration increased the DNA damage (Cardoso et al. 2014). However, the damage caused by AuNPs is depending on the size and administration duration of the nanoparticles (Cardoso et al. 2014, Berbeco et al. 2012. Our study corroborates with the above findings that tau alone had significant effect on reducing DNA damage, whereas the combination of Tau-AuNPs did not have significant The DNA damage frequency (a) and index (b) were expressed as the mean and SEM and were analyzed by one-way ANOVA followed by the post-hoc Bonferroni test. *p≤0.05 compared to the control and Tau-AuNPs. All statistical analyses were performed using Graph Pad Prism 7 software. Groups: control (Ctrl), tau-AuNPs, overuse (Ov), overuse plus tau-AuNPS (Ov+TauAuNPS).
improvements in the protection of DNA damage in the overuse model. The possible reason is that the AuNP may mitigate the effect of tau at the nuclear level for inducing DNA damage. However, different administration duration and size of the nanoparticles can impact the DNA damage.
The overuse of muscle and its consequent biochemical alterations are significantly influenced the muscle regeneration ability. The Myf-5 is an important muscle development protein belongs to myogenic regulatory factors. It is highly expressed in the activated muscle stem cells in early response to muscle injury. Studies have observed that tau plays a major role in the muscle regeneration through modulating calcium signaling pathways (Miyazaki et al. 2013) and regulating inflammatory mediators (Zhao et al. 2018, Nam et al. 2017. However, its role in regulating myogenic program is not well established. In this study, we reported first time that the role of tau-AuNPs on muscle recovery proteins in muscle overuse model. Recent study reported that AuNPs enhance the myogenic differentiation through p38 MAPK and promote the skeletal muscle regeneration (Ge et al. 2018). A study reported that tau mediated cytoprotection in the skeletal muscle (Uozumi et al. 2006). Our study supports with the above findings that tau-AuNPs increased the level of Myf-5 protein in the overuse model.

CONCLUSIONS
In conclusion, Tau-AuNPs can efficiently overcome the oxidative damage induced by muscle overuse. In addition, Tau-AuNPs influences the myogenic regulatory protein Myf-5 for improving muscle recovery of overuse model. However, Tau-AuNPs in reducing DNA damage was not significantly altered in the overuse animals, and this can be the limitation of this study. Overall, our results revealed an important role of the association of these molecules in mechanisms that regulate the muscle recovery.