Nuclear factor erythroid 2-related factor 2 rescues the oxidative stress induced by di- N -butylphthalate in testicular Leydig cells

Cell culture

Mouse Leydig TM3 cell line was obtained from the cell bank of Chinese Academy of Science (Shanghai, China). TM3 cells were cultured in Dulbecco’s modified Eagle’s and Ham’s F12 (DME/F12 1:1, vol/vol) medium (PAA, Germany) and 2.5% foetal bovine serum (PAA). DBP (Sigma-Aldrich; St Louis, Missouri, USA) was applied at 100 mg/L in all single-dose assays.

Quantitative real-time reverse transcription polymerase chain reaction

Total RNA was extracted with RNAiso reagent (TaKaRa, Dalian, China). RNAs were then converted to complementary DNAs (cDNAs) with PrimeScript™ RT Reagent Kit (TaKaRa). Primers for mouse Nrf2, HO-1, NQO1, glutamate–cysteine ligase catalytic subunit (Gclc), glutamate–cysteine ligase regulatory subunit (Gclm), glutathione S-transferase mu 1 (GSTM1), peroxiredoxin 6 (Prx6) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were well established. PCR procedure was run with SYBR Green Premix Ex Taq™ (TaKaRa) in 20 μL system on ABI 7500n (Applied Biosystem, Forster City, California, USA). For each sample, the average value of threshold cycle was normalized to GAPDH level with the formula 2 − Δ Δ C t . Results were thus presented by expressional fold over control.

Western blotting

Total protein of lysates was extracted and equal protein amount of 25 μg was loaded onto 10% sodium dodecyl sulphate polyacrylamide gel for electrophoresis. Gels were subsequently transferred to nitrocellulose membrane. The membranes were blockaded for 1 h with 5% non-fat milk. Primary antibodies of Nrf2, HO-1, NQO1, Gclc, Gclm, GSTM1, Prx6 and Tubulin were then added, and the membranes were kept at 4°C overnight. Corresponding secondary antibodies were applied followed by horseradish peroxidase application.

Measurements of 8-oxo-dGuo, GSH and ROS

Liquid chromatography–mass spectrometry was used to measure concentrations of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo) and glutathione (GSH). Total GSH and oxidized GSH (GSSG) concentrations were calculated from a standard curve based on GSSG (Cayman Chemical, Ann Arbor, Michigan, USA) according to the GSH assay kit and normalized to protein concentration. Net intracellular accumulation of ROS was measured with fluorescent probe species. ⁹ After 2 h of treatment with antioxidants, cells were processed with Hank’s balanced salt solution (HBSS) (Gibco, Carlsbad, California, USA) and loaded with dihydrodichlorofluorescein diacetate. After incubation, cells were washed twice with HBSS, suspended in complete medium and examined under a microscope. The number of dichlorofluorescein -stained cells was calculated.

Luciferase reporter assay

A detailed protocol was described previously by Tanigawa et al. ⁹ Briefly, the oligonucleotides corresponding to the ARE of the mouse NQO1 gene were synthesized. Wild-type (WT) and Nrf2 knockdown (KD) Leydig cells were cultured for 24 h and were then transfected with ARE plasmid encoding firefly luciferase and Renilla luciferase using the Effectene Transfection Reagent kit (Qiagen, Hilden, Germany) or Lipofectamine 2000 (Invitrogen, Grand Island, New York, USA) using the manufacturer’s protocols. After 24 h of incubation, the cells were incubated in the presence or absence of 12-O-tetradecanoylphorbol-13-acetate (TPA; 10 μM), sulphoraphane (SFN; 5 μM) in dimethyl sulphoxide as control. Luciferase activities were normalized to transfection efficiency indicated by Renilla expression, and ARE transcription activity was expressed as fold change in relation to control.

Generation of Nrf2-shRNA and Nrf2-lentivirus

Fifth passage TM3 cells were allowed to adhere for 24 h before transfection. Using Lipofectamine 2000, cells were transfected with plasmids-containing short hairpin RNA (shRNA) against mouse Nrf2 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA). Two shRNAs were generated together with a control shRNA to optimize efficiency. At 48 h post-infection, puromycin (1:5000) was added for selection. Cells were cultured for 2 weeks for generation of stable lines, and efficiencies were further assessed by Western blotting. Generation of Nrf2-bearing lentivirus was carried out according to well-established protocols. Briefly, the Nrf2 expression virus was cloned by digesting Nrf2 from percutaneous coronary intervention-Nrf2 and then half-blunt ligating it into the Lv control displacing control site. Production and titration of the virus were previously described by Hurttila et al. ¹⁰

CV assay

Leydig cells were seeded in 96-well plates at a density of 2500 cells/well and were treated with 10 μM of DBP and control respectively for 0, 24, 48 and 72 h. Crystal violet (CV) assay was applied at each time point. After medium was gently removed, cells were fixed with 10% formalin for 5 min and were then stained with 0.05% CV for 30 min. After two washes, straight methanol was applied and plates were read at absorbance of 540 nm. Normalization in panel (a) was made to absorbance of control at 0 h.

Measurement of testosterone level

Cells were treated with luteinizing hormone (LH), and testosterone content was detected by mouse testosterone enzyme-linked immunosorbent assay kit (Solarbio, Shanghai, China) according to the manufacturer’s protocol. Briefly, cells were stimulated by LH (100 ng/mL) for 12 h and media was collected. Samples were diluted serially, added to 96-well plates, and kept at room temperature for 12 min. After sequential applications of two working reagents, samples were treated with substrates and were kept in darkness for colour development. Optical densities were read at 450 nm of absorbance. Data were plotted as percentage of change compared with the control.

Statistical analysis

All assays were repeated at least three times and were presented as mean ± standard deviation. The two-tailed Student’s t test was used for comparisons between two groups. The value of p < 0.05 was considered statistically significant.

DBP and Nrf2-KD induces ROS in testicular Leydig cells

As DBP is an established toxin inducing oxidative stress, we compared ROS levels in Leydig cells treated either by DBP or by shRNA of Nrf2, a well-established transcription factor with antioxidative activity. The synthesized shRNAs as well as lentiviral vector for Nrf2 overexpression showed satisfactory efficacy in altering messenger RNA (mRNA) expression (Figure 1(a)) and protein levels (Figure 1(b)) in testicular Leydig cells compared with the control. DBP significantly increased ROS level in Leydig cells to a similar extent of Nrf2-KD (Figure 1(c)). Administration of TPA further increased ROS levels of control, DBP and Nrf2-KD groups (Figure 1(c)). Exposure to ROS inhibitor, carbon monoxide-releasing molecule (CORM) abolished the induction ROS by TPA (Figure 1(c)). ROS levels between cells treated with DBP and Nrf2-KD remained unchanged despite TPA or CORM exposures, indicating that loss of Nrf2 could be necessary to reduce ROS induced by DBP.

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Figure 1.

DBP induces intracellular ROS. Nrf2 shRNAs and lentiviral vectors showed satisfactory efficiency in mRNA (a) and protein level (b). Intracellular ROS levels were measured using DCF staining (c). Determination of the concentration of total GSH in cells treated and untreated with TPA (10 μM) for 24 h (d). Determination of the ratio of GSH/GSSG (e) and 8-oxo-dGuo levels in Leydig cells (f). Data are presented as mean ± SD (n = 4). DBP: di-N-butylphthalate; ROS: reactive oxygen species; Nrf2: nuclear factor erythroid 2-related factor 2; shRNA: short hairpin ribonucleic acid; mRNA: messenger RNA; DCF: dichlorofluorescein; GSH: glutathione; TPA: 12-O-tetradecanoylphorbol-13-acetate; GSSG: oxidized glutathione; 8-oxo-dGuo: 8-oxo-7,8-dihydro-2′-deoxyguanosine. **p < 0.01.

In order to better profile ROS state, we then measured intracellular redox level in terms of the ratio of reduced GSH to GSSG. Total GSH was significantly increased by DPB and Nrf2-KD, and the level was further increased with the presence of TPA (Figure 2(d)). Nonetheless, the GSH/GSSG ratio was significantly reduced in cells treated with DBP and Nrf2-KD and was further decreased (Figure 2(d)). Consistently, effects between DBP and Nrf2-KD remained statistically insignificant. The redox profile indicated that DBP induced a similarly more oxidized intracellular milieu in Leydig cells to Nrf2-KD. We next looked at the level of 8-oxo-dGuo, a major product of DNA oxidation. Likewise, DBP and Nrf2-KD significantly increased 8-oxo-dGuo level, which was further elevated by TPA, with, still, insignificant changes between DBP and Nrf2-KD (Figure 1(f)). We thus concluded that DBP induced ROS in testicular Leydig cells to a similar extent of Nrf2-KD, which indicated that Nrf2 could play a critical role in the DBP-induced intracellular oxidative stress.

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Figure 2.

DBP induces increased Nrf2. Real-time qRT-PCR showing DBP-induced changes in Nrf2 and Nrf2-targeted antioxidant genes (a). Western blotting revealing Nrf2 protein change following DBP stimulation. (b) Relative NQO1 promoter reporter activity in Leydig cells with and without Nrf2-KD in response to DBP treatment over time (c) and at different doses (d). Protein level change of Nrf2 and its target genes with different Nrf2 status and DBP treatment (e). DBP: di-N-butylphthalate; Nrf2: nuclear factor erythroid 2-related factor 2; qRT-PCR: quantitative real-time reverse transcription polymerase chain reaction; NQO1: nicotinamide adenine dinucleotide phosphate quinone oxidoreductase 1.

DBP induced upregulation of Nrf2

We next hypothesized that endogenous Nrf2 could be increased in the presence of DBP to active downstream antioxidative responses. We first measured expressions of a series of Nrf2 responsive genes with qRT-PCR. We found that Nrf2 expression was significantly increased in response to DBP, together with upregulation of its target genes of HO-1, NQO-1 and Prx6 (Figure 2(a)). Protein levels of Nrf2 corresponded to expression change of mRNA (Figure 2(b)). In order to elucidate the role of Nrf2 in the antioxidative response to DBP, we constructed a luciferase reporter containing ARE derived from NQO1 promoter. As expected, ARE activity of Nrf2-KD Leydig cells was substantially decreased and responded to prolonged DBP stimulation to a much less fold change in comparison with the controls (Figure 2(c)). Using the same luciferase reporter, we also examined responses between Nrf-WT and Nrf-KD cells to different doses of DBP, which induced increased ARE promoter activity of Nrf2-KD cells to a much less extant compared with the controls (Figure 2(d)). The uncompromised ARE activity in the presence of both WT Nrf2 and DBP indicated that DBP did not impair the binding ability of Nrf2 to ARE elements.

We thus further investigated whether replenish or overexpression of Nrf2 in Leydig cells could implement target antioxidant genes in the presence of DBP. Using lentivirus delivered Nrf2 plasmid, we found that overexpression of Nrf2, in the presence of DBP, substantially upregulated HO-1, NQO1 and Prx6, which was in accordance with qRT-PCR results (Figure 2(e)). This effect could not be simulated by CORM, in line with results shown in Figures 1(c) to (f) and 2(e). We thus concluded that in order to keep the DBP-induced oxidative stress low, Nrf2 overexpression was an optimal modality surpassing the effect of ROS inhibitor.

Upregulation of Nrf2 by SFN restores the oxidative stress by DBP

Based on the results aforementioned, we hypothesized that oxidative stress by DBP could be restored by Nrf2 upregulation. We thus used SFN, a critical antioxidant that induced nuclear translocation of Nrf2 and ARE transactivation. Here we showed that SFN increased Nrf2 level comparable with lentiviral delivery (Figure 3(a)). We then examined intracellular ROS level and 8-oxo-dGuo. We found that SFN almost completely restored oxidative stress caused by DBP in Leydig cells (Figure 3(b) to (c)).

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Figure 3.

(a) Western blotting showing Nrf2 level treated by SFN and lentiviral overexpression. Determination of intracellular ROS (b) and 8-oxo-dGuo levels (c) in Leydig cells treated with DBP and DBP + SFN. Proliferation curves showing effects of DBP alone and with SFN on Leydig cells (d). ELISA detected testosterone levels in the culturing media of Leydig cells stimulated by LH between different treatment groups (e). Nrf2: nuclear factor erythroid 2-related factor 2; SFN: sulforaphane; 8-oxo-dGuo: 8-oxo-7,8-dihydro-2′-deoxyguanosine; DBP: di-N-butylphthalate; ELISA: enzyme-linked immunosorbent assay; LH: luteinizing hormone.

We next investigated whether salvage of ROS stress led to recovery of functions of Leydig cells. We first examined cell proliferative ability using CV assay and found that at 72 h of treatment, DBP significantly lowered proliferation compared with the control and DBP + SFN, indicating that SNF could recover cell proliferation declination induced by DBP (Figure 3(d)). One of the most critical biological activities of testicular Leydig cells was to produce testosterone in response to follicle-stimulating hormone (FSH) stimulation. We then measured testosterone level in the medium of cells stimulated by exogenous FSH. In comparison with the control, DBP induced significantly decreased hormone production, whilst SFN restored the impairment back to normal (Figure 3(e)). We therefore conclude that SFN, via induction of Nrf2 activity, antagonized the oxidative stress induced by DBP and restored cellular function.