Dulbecco’s Modified Eagle Medium (DMEM), Dulbecco’s phosphate buffered saline (DPBS), and penicillin–streptomycin-glutamine (100 ×) were obtained from Thermo Fisher (Waltham, MA, USA). Fetal bovine serum (FBS) was obtained from ICN Biomedicals (Costa Mesa, CA, USA). HEPES buffered saline solution was purchased from Lonza Ltd. (Walkersville, MD, USA). Trypsin–EDTA solution and L-cystine dihydrochloride were purchased from Sigma Chemical Company (St. Louis, MO, USA). Amino acid-free DMEM was purchased from Nacalai Tesque (Kyoto, Japan). Sodium hydrogen carbonate (NaHCO3), hydrogen peroxide (H2O2), 100 w/v% trichloroacetic acid solution (TCA), and 1 mol/L hydrochloric acid (HCl) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Reduced glutathione (GSH) and dichloromethane were obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).
Cell culture and oxidative stress with H2O2 exposure
C2C12 myoblasts were purchased from KAC Co., Ltd. (Kyoto, Japan) and were grown at 37 °C and 5% CO2 in DMEM with 10% FBS, 1% penicillin–streptomycin-glutamine, and 1% HEPES. Myoblasts were seeded in 100-mm dishes at 1.8 × 104 cells/mL/dish. C2C12 myoblasts at approximately 80–90% confluence were differentiated into myotubes upon further incubation in 2% horse serum for 2–3 days. The procedure of the experiments was summarized in Fig. 1. To examine the effect of content of H2O2, Differentiated C2C12 cells were incubated with 1/5 DMEM (DMEM medium diluted fivefold with amino acid-free DMEM). After the incubation, the cells were exposed to 0.06 or 0.12 mM of H2O2.for 60 or 120 min (Fig. 1a). To examine the effect of Cys2 on GSH content, cells were incubated with 1/5 DMEM medium or 1/5 DMEM supplemented with 0.1, 0.3 or, 1.0 mM Cys2-HCl for 120 min (Fig. 1b). As 1/5 DMEM medium contains 0.04 mM Cys2, final concentration of Cys2 in the medium were 0.14, 0.34 or 1.04 mM. After the incubation, the cells were exposed to 0.12 mM of H2O2 ± 1 mM Cys2-HCl for 120 or 240 min to test the effect of Cys2-HCL on GSH content in the cell (Fig. 1b). Following the exposure to Cys2-HCl, the cells were rinsed with DPBS. In previous studies, the effect of NAC on oxidative stress was examined with concentrations between the range of 1.0 mM to 5.0 mM (Zhang et al. 2011; Lee et al. 2015, 2013; Aparicio-Trejo et al. 2019). Therefore, we selected the highest concentration of Cys2-HCl for our experiment to be 1.0 mM, because this value was the lowest among the values of concentrations used in previous studies (Zhang et al. 2011; Lee et al. 2015, 2013; Aparicio-Trejo et al. 2019). In addition, we selected 0.1 and 0.3 mM of Cys2-HCL to test the effect of Cys2-HCL in physiological range. Then, we tested the effect of 1 mM of Cys2 on cell viability, anti-oxidative reaction (Fig. 1c). After 120 min of incubation with 1/5 DMEM ± 1 mM Cys2-HCl, the cells were exposed to 0.12 mM of H2O2 ± 1 mM Cys2-HCl for 120 min. In addition, to test the 1 mM of Cys2 on GSH content before H2O2 exposure, the cells were collected after 120 min of incubation with 1/5 DMEM ± 1 mM Cys2-HCl (Fig. 1d).
Intracellular ATP measurements
Intracellular ATP was measured to assess cell viability (Maehara et al. 1987) using CellTiter-Glo™ (Promega Corporation, Madison, WI, USA) according to the manufacturer’s protocol. C2C12 myoblasts were seeded in 96-well plates and differentiated into myotubes. After 120 min of H2O2 exposure, the plate was equilibrated to room temperature for 30 min, and an equal volume (100 μL per well) of CellTiter-Glo™ reagent was added (Fig. 1c). Two minutes after mixing the contents of the plate, luminescence was detected using SpectraMax™ i3 (Molecular Devices Co., Sunnyvale, CA, USA).
RNA extraction and real-time qPCR
Heme oxygenase-1 (HO-1) expression was measured as typical indicator of anti-oxidative response (Kurutas 2016). Total RNA was extracted according to the manufacturer's instructions (RNeasy Mini Kit ™, Qiagen, Hilden, Germany). RNA quantification and purity were estimated by spectrophotometry at 260 nm and 280 nm. Complementary DNA was synthesized using Prime Script™ RT reagent Kit (Takara, Shiga, Japan). HO-1 expression was quantified using SYBR Green (STBR® Green Real-Time PCR Master Mix, Thermo Fisher, Waltham, MA, USA) analysis with Quant Studio™ 12 K Flex Real-Time PCR System 384 well plate (Applied Biosystems, Inc., CA, USA). The following primers were used for HO-1: forward primer, 5'-TGCAGGTGATGCTGACAGAGG -3'; reverse primer, 5'- GGGATGAGCTAGTGCTGATCTGG-3′. The relative mRNA expression was calculated using cycle threshold (Ct) values and was normalized to the Ct values of 18S ribosomal RNA (18S rRNA) (forward primer, 5'-AACGCCACTTGTCCCTCTAA-3'; reverse primer, 5'-GTGGAGCGATTTGTCTGGTT -3').
Measurement of cysteine, cystine, GSH and GSSG content
Cells were suspended in 50 μL of 10% trichloroacetic acid and mixed thoroughly following the addition of an equal amount of dichloromethane. The mixture was centrifuged at 14,000 × g for 2 min at 4 °C. Ten μL of the supernatant was diluted with 150 μL of 50 mM NaH2PO4, 1 mM 1-octanesulfonic acid sodium salt, and 2.5% (v/v) acetonitrile solution (pH 2.7). To test stability of cysteine, cystine, GSH and GSSG, the supernatant sample in the solution, the sample was analyzed after 24 h of storage at 4 degrees. As a results, the concentration was confirmed to be stable in the solution after deproteination for 24 h. Twenty μL of the sample was then separated using an Inertsil ODS-3 (5.0 μm, 3 × 150 mm column; GL Sciences Inc., Tokyo, Japan) with an HPLC system (GL-7410 pump, GL-7420 autosampler, GL-7430 oven, ED703 pulse electrochemical detector; GL Sciences Inc., Tokyo, Japan). The separation condition was previously described (Appala et al. 2016). A total of 50 mM NaH2PO4, 1 mM 1-octanesulfonic acid sodium salt, and 2.5% (v/v) acetonitrile solution (pH 2.7) were used as the mobile phase. The pump flow rate was set at 0.2 mL/min for 10 min and at 0.8 mL/min from 10 to 30 min after injection. Finally, the flow rate was maintained at 0.2 mL/min from 30 to 40 min after injection. The column oven temperature was maintained at 30 °C. The potential in the electrochemical detector was + 1800 mV. Retention time is 6.5 min for cystine, 7.5 min for cysteine, 11.3 for GSH, 20.3 for GSSG. The GSH concentration in the sample was calculated by comparing the peak area of the chromatogram with the calibration curve that was determined using a series of standard solutions with known concentrations of each substance. The concentration in the cells was calculated by multiplying the concentration of the analyzed sample by 16. The data below limit of detection (0.1 µM for cysteine, Cys2, GSH and GSSG) were expressed as N.D. As the samples had to be processed promptly when measuring glutathione-related metabolites, we did not measure necessary data (cell counts and cell weight etc.) for normalization. Therefore, the data were expressed the content per well.
Mitochondrial oxygen consumption rate
A mitochondrial stress test was then performed to assess the bioenergetic status of the cells (Brand and Nicholls 2011) (Fig. 1e). C2C12 cells were differentiated into myotubes by incubation in XFp cell culture mini plates in a CO2 incubator at 37 °C. Following a 2 h treatment of Cys2, the C2C12 myotubes were exposed to H2O2 for 60 min. Before the assay, the cells were washed twice with XF Assay medium (Seahorse Bioscience, North Billerica, MA) containing 4.5 g/L glucose, 1.0 mM sodium pyruvate, and 4.0 mM glutamine (adjusted pH to 7.35 ± 0.05), and were incubated in a CO2-free incubator at 37 °C for 60 min. Following incubation, the baseline measurement of the oxygen consumption rate (OCR) was recorded as indicator of basal oxidative metabolism, using the XFp Extracellular Flux Analyzers (Agilent Technologies, Santa Clara, CA). Next, the cells were treated with the following chemicals at the respective time durations from the beginning of the assay: oligomycin (a complex V inhibitor, final concentration 3 μM) at 20 min to induce maximal glycolytic metabolism, carbonyl cyanide- 4-(trifluoromethoxy) phenylhydrazone (FCCP) (uncoupling agent, final concentration 3 μM) at 50 min to uncouple electron transport and induce peak OCR, and antimycin A and rotenone (a complex III and I inhibitor, respectively, final concentration 0.5 μM each) at 80 min to reveal non-mitochondrial respiration. Maximal respiration after the addition of FCCP reflects the maximal capacity of the electron transport chain (Ainscow and Brand 1999). Maximal respiration rate was calculated as the maximal OCR minus the non-mitochondrial OCR determined by antimycin A and rotenone treatment.
All values are expressed as mean ± standard error. Comparison between the two groups was done using Sidak's multiple comparisons test followed by an analysis of variance test. For comparison among three groups, significant differences were determined using Tukey's multiple comparisons test or Dunnet’s multiple comparisons test. Data were analyzed using GraphPad Prism 7.04 software (GraphPad Software Inc., San Diego, CA, USA), with p < 0.05 being considered significant.