Chemicals: Ni(CH₃COO)₂·₂H₂O, and NH₄HCO₃ were purchased from Sigma-Aldrich.

For the synthesis of NiO NPs 50 mL of a NH₄HCO₃ 1 M solution were dropped, under ultrasound irradiation for 15 minutes at 50% of amplitude at 25°C, to a 100 mL of a Ni(CH₃COO)₂·₂H₂O 4x10^-3 M solution. The pH was maintained at 9. The high power ultrasound irradiation was carried out by an Ultrasonic processors VC750 Sonics and Materials, 20 kHz with a diameter tip of 13 mm. The precipitate was dried, calcined at 350°C for 1 h and characterized by ICP-OES, FE-SEM-EDX and XRD (31).

Animal studies were conducted in agreement with the guidelines adopted by the Italian Approved Animal Welfare Assurance (A-3143-01) and the European Communities Council Directive of November 24, 1986 (86/609/EEC). The experimental protocols were approved by the University of Perugia. Number 3 Danish Duroc pre-pubertal pigs (15 to 20 days old) underwent bilateral orchidectomy after general anaesthesia with ketamine (Ketavet 100; Intervet, Milan, Italy), at a dose of 40 mg/kg, and dexmedetomidine (Dexdomitor, Orion Corporation, Finland), at a dose of 40 g/kg, and were used as SCs donors. Specifically, pure porcine pre-pubertal SCs were isolated, and characterized according to previously established methods (27).

In detail, SCs have undergone acute (24 h) and chronic (from 1 up to 3 weeks) exposures at two subtoxic doses of NiO NPs of 1 μg/ml and 5 μg/ml according to 3-(4, 5-Dimethyl-thiazol-2-yl)22,5-diphenyl-tetrazolium bromide (MTT) assay. The control group consisted of unexposed SCs (0 NiO NPs μg/ml).

NiO NPs -treated SCs were detached by trypsin/ethylenediaminetetraacetic acid (EDTA) (Lonza, Verviers, Belgium) at 37°C for 8 min, to promote the enzymatic reaction. After washing with 1 ml Hank’s balanced salt solution (HBSS) (Sigma-Aldrich Co., St. Louis, MO, USA), samples were centrifuged at 150×g for 6 min, the supernatant was removed, the pellets were freeze-dried, and accurately weighed. Samples were dissolved by treatment with 10 ml of a mixture of sulfuric acid (H₂SO₄), (97% Sigma-Aldrich Co., St. Louis, MO, USA)/nitric acid (HNO₃ 70%), (Sigma-Aldrich Co.,St. Louis, MO, USA) (2:1). After solubilization, the obtained solutions were diluted with the EDTA solution (1:10) prior Ni2+ content determination using a Varian 700-Es series spectrometer (Agilent, Milan, Italy) in triplicate. Calibration was performed diluting a Nickel nitric acid stock solution for ICP (Sigma Aldrich, Milan, Italy) to obtain Nickel standard solutions in the 1–15-mg/ml range. The Ni2+ uptake in SCs was calculated per unit weight of freeze-dried NiO NP-treated SCs and % of the total amount added and the error expressed as SEM.

NiO NPs cytotoxicity was evaluated by the MTT (Sigma-Aldrich Co., St. Louis, MO, USA) test on unexposed and exposed SCs. Briefly NiO NPs at the concentrations of 2.5, 5, 15, 30, 45, 60 and 120 μg/ml were added to each well and cultured for additional 24 or 48 h. Then, the experiment was performed, as previously reported (28). Unexposed (0 NiO NPs μg/ml) SCs served as controls. Viability was expressed as a percentage with respect to unexposed SCs (NPs-exposed SCs ×100/unexposed SCs). The sub-toxic doses of 1 and 5 μg/ml were chosen for all subsequent experiments at 24 hours (acute exposure) and 1, 2, 3 weeks (chronic exposure) and MTT assay was performed at each experimental time-point.

Intracellular ROS were measured by treating unexposed and exposed SCs with 50 mM dichlorofluorescein diacetate (DCFHDA) (Sigma-Aldrich Co., St. Louis, MO, USA) solution in Dulbecco’s phosphate-buffered saline (D-PBS) (Sigma-Aldrich Co., St. Louis, MO, USA) at 37°C for 30 min. Fluorescence was read by using a plate reader (DTX 880 Multimode Detector, Beckman Coulter). Data were normalized for cell viability (MTT assay) and expressed as the percentage of unexposed SCs. The sensitivity of the test was confirmed by adding 30 μM hydrogen peroxide (H2O2) (30 min) on unexposed SCs as positive control.

To evaluate the oxidative DNA damage, unexposed and exposed SCs were processed in the comet assay under alkaline conditions (alkaline unwinding/alkaline electrophoresis, pH >13), basically following the original procedure (32). Briefly, SCs treated with 1 mM 4-nitroquinoline N-oxide (4NQO) (Sigma-Aldrich, Milan, Italy) for 1 h at 37°C (33) were used as positive control. At the end of treatments, the cells were detached with trypsin (Invitrogen, Milan, Italy) and collected by centrifugation (70×g, 8 min, 4°C). Then, cell pellets were gently resuspended in low-melting point agarose (Sigma-Aldrich, St. Louis, MO, USA) at 37°C, layered onto a conventional microscope slide precoated with 1% normal melting point agarose and covered with a coverslip (Knittel-Glaser, Braunschweig, Germany). Then, electrophoresis runs were then performed as previously reported (28).

The comets in each microgel were analysed (blind), at ×500 magnification with an epi-fluorescent microscope (BX41, Olympus, Tokyo, Japan), equipped with a high sensitivity black and white charge-coupled device (CCD) camera (PE2020, Pulnix, UK), under a 100-W high-pressure mercury lamp (HSH-1030-L, Ushio, Japan), using appropriate optical filters (excitation filter 510–550 nm and emission filter 590 nm). Images were elaborated by Comet Assay III software (Perceptive Instruments, UK). A total of 100 randomly selected comets (50 cells/replicate slides) were evaluated for each experimental point.

Aliquots of culture media from all the experimental groups were collected and stored at -20°C for subsequent assessment of AMH (AMH Gen II ELISA, Beckman Coulter, Webster, TX, USA) and inhibin B (inhibin B Gen II ELISA, Beckman Coulter) secretion levels as previously described (34).

AMH, inhibin B, TNF-α,IL-6, SOD1, HO-1, GHSPx and NRF2 were analysis by reverse transcriptase‐polymerase chain reaction (RT‐PCR) as previously described in Arato et al. (35) employing the primers listed in Table 1 .Total RNA was extracted using the TRIzol reagent (Sigma‐Aldrich), and quantified by reading the optical density at 260 nm. In detail, 2.5 μg of total RNA was subjected to reverse transcription (RT, Thermo Scientific) to a final volume of 20 μl. We performed the qPCR with the use of 25 ng of the cDNA obtained by RT and an SYBR Green Master Mix (Stratagene). This procedure was performed in an Mx3000P cycler (Stratagene), using FAM for detection and ROX as the reference dye. We normalized the mRNA level of each sample against β‐actin mRNA and expressed it as fold changes versus the levels in the control group.

Total protein extracts were prepared for immunoblot analysis as described in Mancuso et al. (36). Briefly, the cell extracts were separated by 4–12% SDS-PAGE, and then blotted on nitrocellulose membranes (BioRad, Hercules, CA, USA). The membranes were incubated overnight in a buffer containing 10 mM TRIS (Sigma-Aldrich Co., St. Louis, MO, USA), 0.5 M NaCl (Sigma-Aldrich Co., St. Louis, MO, USA), 1% (v/v) Tween 20 (Sigma-Aldrich Co., St. Louis, MO, USA), rabbit anti-ERK1/2 (Millipore, MA, USA, 1:2000), mouse anti-phospho- ERK1/2 (Millipore; MA, USA, 1:100), rabbit anti-JNK (Millipore; MA, USA, 1:1000), rabbit anti-phospho-JNK (Millipore; MA, USA, 1:500), rabbit anti-posphop38 (Millipore, MA, USA, 1:2000), mouse anti p38 (Millipore, MA, USA, 1:2000), anti-Akt (Cell Signalling, 1:100), rabbit anti-phospho-Akt (Cell Signalling, 1:1000), rabbit anti-phospho-NF-kB p65 antibody (AbCam, Cambridge, UK, 1:1000), rabbit anti-NF-kB p65 antibody (AbCam, Cambridge, UK, 1:1000), and mouse anti-β-actin (Sigma-Aldrich Co., St. Louis, MO, USA, 1:100) primary antibodies.

Primary antibody binding was then detected by incubating membranes for an additional 60 min in a buffer containing horseradish peroxidase conjugated anti-rabbit (Sigma-Aldrich Co., St. Louis, MO, USA,1:5000) and/or anti-mouse (Santa Cruz Biotechnology Inc., 1:5000) IgG secondary antibodies. The bands were detected by enhanced chemiluminescence and acquired by ChemiDoc imaging System (Bio-Rad, Hercules, CA, USA).

Normality analysis was performed by Shapiro–Wilk test, and statistical comparisons were analyzed using one-way ANOVA followed by Tukey’s HSD post hoc test (SigmaStat 4.0 software,Systat Software Inc., CA, USA). Values were reported as the means ± SEM of three independent experiments, each performed in triplicate. Differences were considered statistically significant at *p < 0.05, and **p < 0.001 compared to unexposed SCs (0 NiO NPs).

The synthesis of NiO NPs was performed by XRD analysis, the resulting diffractogram was characteristic of nichel oxide crystals in anatase form (JCPDS 00-001-0562) ( Figure 1A ).

A representative SEM image of NiO NPs in dry form is showed in Figure 1B , and the mean size distribution reports values of 20 ± 5 nm diameter, calculated by measuring over 100 particles in random fields of view. Results showed that NiO NPs tended to form aggregates of submicrometric dimensions. DLS analysis, confirmed some aggregation of NiO NPs in suspension. The mean hydrodynamic diameter of NiO NPs mainly distributed in a range of 100–800 nm ( Figure 1C ).

ICP-OES was used to quantify the uptake of NPs expressed as the percentage of internalized NPs and the amount of metal adsorbed per cell number (expressed as ng/10⁵), at each concentration, after 5 hours of treatment ( Figure 1S ). In the treatment after 5 hours, the percentage of internalized NPs showed a range between 1 and 3% ( Figure 1S ), where the lower treatment dosage (1µg/ml) exhibited a higher percentage of uptake than the higher dosage (5µg/ml). On the contrary, the amount absorbed and expressed as ng/10⁵ was much higher at the higher dosages (1.57 for NiO).

We could speculate that this difference between percentage and net amount absorbed was probably due to the gradual saturation of SCs, leading to a progressive slowing down in uptake as the concentration of NPs increases, further confirming literature data (37).

Other factors that could have negatively influenced the absorption of NiO particles are their marked hydrophobicity and their tendency to stick to surfaces, phenomena due to the high surface energy of these particles that causes a reduced availability for absorption by part of the SCs (38).

For the preliminary study, at 24 hours, as shown in Figure 2S , panel A, NiO NPs at low concentrations appeared to induce proliferation of SCs, probably due to an adaptive response to the increasing NPs concentration. Proliferative effects due to metal NPs have been found many times before and this has been attributed to the activation of specific pathways, such as MAP kinases (28), but many aspects remain to be elucidated. The percentage of metabolically active cells began to decline at doses of 40 μg/ml (*p< 0.05 vs. unexposed SCs), showing a LD50 of 80 μg/ml (**p<0.001 vs. unexposed SCs). Finally, a collapse in SCs viability was also identified at 100 and 120 μg/ml (**p<0.001 vs. unexposed SCs). At 48 hours, a statistically significant reduction in metabolic activity was identified, in SCs exposed to a concentration of 40 μg/ml compared to controls (*p< 0.05 vs. unexposed SCs), with a LD50 dropping to 70 μg/ml (**p<0.001 vs. unexposed SCs), followed by a drastic reduction in viability at higher concentrations ( Figure 2S ). Due to the obvious toxicity of NiO NPs, for the 3-week treatment the sub-toxic concentrations of 1 μg/ml and 5 μg/ml, were chosen, the former to simulate a mild exposure to seemingly harmless concentrations, to which humans could easily be exposed in everyday life, while the latter represents a higher dosage to allow an evaluation of possible mechanisms of toxicity.

MTT assays performed during the 3 weeks of treatment with NiO NPs showed an increase in metabolically active cells at the dosage of 5 μg/ml at 1 week ( Figure 2S panel B *p<0.05 vs. unexposed SCs). This effect could be due to a defensive mechanism put in place by the cell in response to a noxious stimulus, however it does not seem to be sufficient to preserve the cell from the statistically significant reduction in the percentage of metabolically active cells of 10% observed at the third week compared to the unexposed SCs ( Figure 2S panel B, *p<0.05 vs. unexposed SCs).

In contrast, at the concentration of 1 μg/ml the only significant effect was observed at the second week with a 10% loss of viability (*p<0.05 vs. unexposed SCs) which then recovered at the third week, demonstrating the low level of toxicity potential at this concentration, which allowed the cell to recover from the stress suffered ( Figure 2S panel B).

Morphological analysis revealed that SCs exposed to both subtoxic doses of NiO NPs did not undergo substantial changes compared with the untreated monolayer at all times of exposure, shown in the Figures 2A, D, G, J . In fact, cells exposed to NiO NPs maintained the typical squamous shape of epithelial cells with vacuoles containing lipid hormones, likely testosterone coniugated with androgen binding protein (ABP) and estradiol, well evident and abundantly distributed in the cell surface ( Figures 2B, C, E, F, H, I, K, L ).

As shown in Figure 3A , the dose of 1 μg/ml NiO NPs did not affect ROS intracellular level up to 2 weeks post exposure. On the contrary, at 3 weeks post treatment, ROS level significantly increased compared to unexposed SCs. Conversely, the dose of 5 μg/ml NiO NPs induced a significant increase of intracellular ROS amounts over time and until the end of treatment compared to unexposed SCs ( Figure 3A , **p < 0.05 **p < 0.001 vs. 0 NiO NPs). As expected, H₂O₂ (positive control) induced a significant increase in ROS intracellular levels ( Figure 3A , **p < 0.05 **p < 0.001).

The levels of oxidative DNA damage induced by NiO NPs were measured as the % of DNA in tail by the alkaline comet assay over time, after acute (24 h) and chronic exposures (from 1 to 3 weeks) to 1 and 5 μg/ml of NiO NPs. Both doses of exposure, induced a significant increase in the oxidative DNA damage over time and until the end of treatment with respect to the unexposed SCs ( Figure 3B , **p < 0.05 vs. 0 NiO NPs).

The gene expression of SOD1 significant increased at the dose of 1 μg/ml only at the third week of treatment, meanwhile at the dose of 5 μg/ml it showed a significant increase from second up to third week ( Figure 4A , *p < 0.05 and **p < 0.001 vs. 0 NiO NPs).

The gene expression of HO-1 increased at both concentrations only at the third week of NiO NPs-exposure ( Figure 4B , **p < 0.001 vs. 0 NiO NPs).

We observed a significant increase in GHSPx gene expression only at the second week at the dose of 1 μg/ml and, at first week at the dose of 5 μg/ml; with a significant reduction at 24h at the dose of 1 μg/ml ( Figure 4C , *p < 0.05 vs. 0 NiO NPs).

The gene expression of NRF2 showed a significant increase at both dose from second up to third week of treatment ( Figure 4D , *p < 0.05 and **p < 0.001 vs. 0 NiO NPs).

Exposure of SCs to both concentrations of NiO NPs induced a significant increase in AMH and inhibin B gene expression at 24 h, followed by a significant decrease after 2 week up to the third week, compared to unexposed SCs ( Figures 5A, B *p < 0.05 and **p < 0.001 vs. 0 NiO NPs).

At both concentrations, AMH and inhibin B secretion was significantly increased at 24h of treatment. The secretion of AMH was significantly decreased after dose of 1 μg/ml NiO NPs only the third week, whereas at 5 μg/ml NiO NPs, we observed a significant reduction from the second, up to the third week of treatment respect to unexposed SCs ( Figure 5C **p < 0.001 vs. 0 NiO NPs).

In contrast, inhibin B secretion was significantly decreased after exposure to both dose of treatment only at third week with respect to unexposed SCs ( Figure 5D **p < 0.001 vs. 0 NiO NPs).

We observed that NiO NPs exposure, at each concentration, induced the activation of caspase-3 at third week, with the cleavage of p19 and p17 fragments p19 kDa active fragment.

Only at the dose of 5 μg/ml NiO NPs, we demonstrated a statistical increase of both active p19 and p17 with respect to the inactive p35 fragments, expression of a more prominent apoptotic process ( Figures 6A–D **p < 0.001 vs. 0 NiO NPs).

At both concentration, the gene expression of TNF-α showed a significant increase at 24 h and during third week, ( Figures 7A , **p < 0.001 vs. 0 NiO NPs).

Moreover, IL-6 gene expression showed a significant increase at 24 h only at dose of 5 μg/ml NiO NPs, meanwhile at both concentrations, the increase resulted after week 2 up to the third week, with respect to unexposed SCs ( Figures 7B , *p < 0.05 and **p < 0.001 vs. 0 NiO NPs).

We performed Western blotting analysis to investigate the involvement of different MAPK family members (ERK1/2, JNK, p38, AKT ) and NF-kB signaling pathway after NiO NPs exposure (Figure 8A). The phosphorylation ratio of ERK1/2 showed a significant increase at both concentrations, from the second up to third week ( Figure 8B , **p < 0.001 vs. 0 NiO NPs).

The phosphorylation ratio of JNK increased at 24 h in both concentrations, with a significant increase at the second week only at the 1 μg/ml NiO NPs dose and a significant reduction at third week at the 5 μg/ml NiO NPs dose ( Figure 8C , **p < 0.001 vs. 0 NiO NPs).

The phosphorylation ratio of p38 showed a significant increase from second up to third week, at both NiO NPs doses ( Figure 8D , **p < 0.001 vs. 0 NiO NPs).

The phosphorylation ratio of AKT showed a significant increase at 24 h, second and third week, with a significant reduction only at first week at both concentrations of treatment ( Figure 8E , **p < 0.001 vs. 0 NiO NPs).

Finally, the phosphorylation ratio of p-NF-kB showed a significant increase only at first week at dose of 1 μg/ml NiO NPs, meanwhile at dose of 5 μg/ml NiO NPs, the increase resulted after 24 h and at third week of treatment compared to unexposed SCs ( Figures 8F , **p < 0.001 vs. 0 NiO NPs).