Solanesol alleviates metal oxide nanoparticles generated toxicity in human placental BeWo cells

2.1 Nanoparticles and solanesol

Chemical techniques were used to produce CuO (43 nm) and NiO (51 nm) NPs. Previous investigations have given information on the characterization and synthesis of these NPs (Ahamed et al., 2023, 2022). Solanesol (PubChem ID: 24899813) was bought from Sigma-Aldrich (St Louis, MO, USA).

2.2 Cell culture and exposure procedure

DMEM was used to cultivate human placental BeWo cells. It was supplemented with 10 % fetal bovine serum, 2 mM glutamine, 100 g/ml streptomycin, and 100 unit/ml penicillin. Cells were kept at 37 °C in an incubator that was humidified and supplied with 5 % CO₂. Grown cells were collected further cultured for additional tests at 80–85 % confluence. Before being treated to NPs and solanesol for 24 h, cells were given an overnight period to adhere to the surface of the culture flask. To prevent agglomeration before exposing cells, DMEM with varying dosages of NPs (1–200 µg/ml) and solanesol (1–200 µg/ml) was sonicated for 10 min at 40 W in a water bath sonicator.

2.3 Biochemical assays

MTT assay was conducted following the methodology previously described by Mosmann (Mosmann, 1983), with minor adjustments as outlined by Ahamed et al. (Ahamed et al., 2016). The intracellular level of ROS was measured using a microplate reader (Synergy-HT, BioTek) with the application of 2‘-7‘-dichlorodihydrofluorescein diacetate (H₂DCFDA) as described by Siddiqui et al. (Siddiqui et al., 2013). The quantification of glutathione (GSH) level was performed using Elman's method (Ellman, 1959). The activity of the glutathione peroxidase (GPx) enzyme was assessed using the experimental methods described by Rotruck et al. (Rotruck et al., 1973). The examination of mitochondrial membrane potential (MMP) was conducted using a microplate reader (Synergy-HT, BioTek) in accordance with established methods (Creed and McKenzie, 2019), with slight modifications as described by Ahamed et al. (Ahamed et al., 2021). Expression of caspase-3 gene was estimated applying real-time PCR (Applied Biosystems, Foster City, CA, USA) with the application of SYBR green. Caspase-3 enzyme was assayed using a colorimetric kit provided by BioVision (Milpitas, CA, USA). The protein content estimation in this study was conducted using Bradford's method, as described by Bradford (Bradford, 1976).

2.4 Statistical assessment

The results were analyzed using a one-way analysis of variance (ANOVA) followed by Dennett's multiple comparison tests. The significance level of p < 0.05 was accredited to the observed results. Data is reported as the mean ± standard deviation (SD) of three distinct tests (n = 3).

3.1 Nanoparticles generated cytotoxicity

Cytotoxic effects of CuO and NiO NPs in BeWo cells was tested with MTT test. The cells were exposed to various doses (ranging from 1 to 200 µg/ml) of each of these nanomaterials for a 24 h period. The MTT test is a method used to evaluate cellular metabolic activity. Data shown in Fig. 1 suggest that both types of NPs elicit a cytotoxic response in BeWo cells, with the magnitude of this response depending on the dosage administered. Nevertheless, the cytotoxicity induced by CuO NPs exhibited a somewhat greater magnitude compared to NiO NPs. The viability of cells of CuO NPs was observed to be 99 %, 96 %, 75 %, 60 %, 47 %, 34 %, 17 %, and 11 % at the corresponding doses of 1, 2, 5, 10, 25, 50, 100, and 200 µg/ml. The cell viability of NiO NPs exhibited a range of percentages, namely 99 %, 98 %, 84 %, 68 %, 52 %, 40 %, 25 %, and 19 % at the corresponding doses of 1, 2, 5, 10, 25, 50, 100, and 200 µg/ml. The cytotoxicity statistics found in this investigation align with previous publications that have indicated a correlation between the dosage of CuO and NiO nanomaterials and their cytotoxic effects in several human cell lines (De Carli et al., 2018; Fahmy et al., 2020). As an illustration, the application of CuO nanomaterials at doses ranging from 2 to 50 µg/ml for a duration of 24 h resulted in a dose-dependent manifestation of cytotoxicity in liver cells (HepG2) (Siddiqui et al., 2013). A separate investigation revealed that the presence of NiO nanostructures resulted in cytotoxicity in mouse spermatogonia cells, with the extent of harmfulness depending on the dosage administered (ranging from 10 to 320 µg/ml) over a period of 24 h (Ahamed et al., 2023).

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Fig. 1

Cytotoxic response of CuO NPs and NiO NPs in BeWo cells. * p < 0.05 vs. control.

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3.2 Cytocompatibility of solanesol

In this experiment, we conducted a more comprehensive investigation of the impact of solanesol in placental cell line. Cells were subjected to various doses (1–200 µg/ml) solanesol for a duration of 24 h, and the assessment of cytotoxicity was conducted using the MTT test. According to the data presented in Fig. 2, it can be observed that solanesol did not harm the BeWo cells at doses up to 100 µg/ml. Consistent with the findings of the current study, previous research has also demonstrated the biocompatibility of solanesol.

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Fig. 2

Cytotoxic response of solanesol in BeWo cells. * p < 0.05 vs. control.

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3.3 Solanesol alleviates nanoparticles generated cytotoxicity

In order to decide the optimal dose of solanesol for efficiently reducing the toxicity of CuO and NiO NPs in BeWo cells, we conducted the experiments where cells were exposed to a medium concentration (25 µg/ml) of both types of NPs and simultaneously co-exposed them to various safe concentrations of solanesol (1 to 100 µg/ml) for a duration of 24 h. The resulting effects were then assessed using the MTT assay. The findings indicated that the administration of solanesol at a dose of 10 µg/ml resulted highest level of mitigation against the cytotoxicity generated by both types of nanomaterials in BeWo cells (Fig. 3). Based on the obtained findings, a concentration of 10 µg/ml of solansol has been chosen for further investigation into its mechanism of action in alleviating the toxic effects generated by CuO and NiO nanomaterials (25 µg/ml) in human placental cell line.

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Fig. 3

Alleviating effect of solanesol in BeWo cells following exposure to CuO NPs and NiO NPs. * p < 0.05 vs. control. ^# p < 0.05 vs. a (NPs treated groups).

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3.4 Solanesol alleviates nanoparticles generated oxidative stress

Studies have shown that placental dysfunction and other pregnancy problems could be caused by oxidative stress in placental tissues (Onoda et al., 2017). Hence, the administration of antioxidants has the potential to safeguard placental trophoblasts against oxidative stress, potentially reducing the incidence of pregnancy problems and reproductive damage (Duhig et al., 2016). Solanesol displays excellent free radical consumption capabilities due to the existence of numerous non-conjugated double bonds (Yan et al., 2019). To illustrate the potential mechanism behind the protective properties of solanesol against the toxicity generated by NiO and CuO NPs in placental cells, various biomarkers of oxidative stress were assessed. Placental cells were exposed to metal oxide NPs (25 µg/ml) and/or solanesol (10 µg/ml) for a duration of 24 h. Data displayed in Fig. 4A suggesting the intracellular production of ROS caused by both CuO and NiO NPs was greatly reduced in the presence of solanesol. Moreover, the presence of NPs (CuO and NiO) resulted in a significant production in intracellular H₂O₂ levels, which was successfully counteracted by concurrent exposure to solanesol (Fig. 4B).

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Fig. 4

ROS (A) and H₂O₂ (B) levels in BeWo cells treated for 24 h to NPs (25 µg/ml) and/or solanesol (10 µg/ml). * p < 0.05 vs. control. ^# p < 0.05 vs. NPs treated group. SOL: solanesol.

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Solanesol is helpful during pregnancy because it has antioxidant properties that stop lipid peroxidation and keep antioxidant enzymes working (Yan et al., 2019). Here, we looked at how solanesol might slow down the reduction of antioxidants in BeWo cells induced by selected metal oxide (CuO and NiO) NPs. The findings of this study indicate that the levels of GSH and the activity of the GPx enzyme were significantly reduced in placental BeWo cells treated with both NPs. However, this depletion was effectively reversed when solanesol was co-exposed to the cells, as seen in Fig. 5A and B.

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Fig. 5

GSH level (A) and GPx enzyme activity (B) in BeWo cells treated for 24 h to NPs (25 µg/ml) and/or solanesol (10 µg/ml). * p < 0.05 vs. control. ^# p < 0.05 vs. NPs treated group. SOL: solanesol.

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3.5 Solanesol alleviates nanoparticles generated apoptosis

The processes of apoptosis are crucial factors in the impaired development of the placenta and present significant hazards to the pregnancy (Ghaneifar et al., 2020; Teng et al., 2021). Exposure to NPs like TiO₂ and polystyrene during pregnancy has been shown to cause apoptosis in trophoblasts, which can lead to problems with the placenta and poor fetal development (Ba et al., 2015; Teng et al., 2021). A study by Ebrahimzadeh Bideskan et al. (Ebrahimzadeh Bideskan et al., 2017) found that the regulation of bax and bcl-2 genes in the hippocampus of their children is affected by whether or not their mothers were exposed to TiO₂ NPs while they were pregnant or nursing. This exposure leads to apoptosis and a decrease in foetal neurogenesis. The previously reported research also indicated that exposure to CuO and NiO NPs resulted in the induction of apoptosis and oxidative stress in human placental cells and mouse spermatogonia cells, respectively (Ahamed et al., 2023, 2022). Here, potential mitigating impact of solanesol on apoptosis induced by CuO and NiO NPs in human placental cells was investigated. The assessment of caspase-3 gene expression and MMP level was employed to evaluate the observed effects. The cells were treated with CuO and NiO NPs at a concentration of 25 µg/ml, along with solanesol at a dose of 10 µg/ml, for a duration of 24 h. Results presented in Fig. 6A indicate a substantial upregulation of caspase-3 gene upon exposure to CuO and NiO NPs. It is worth noting that the concurrent exposure to solanesol had a notable mitigating effect on the activation of the caspase-3 gene produced by these metal oxide NPs. To provide additional evidence for mRNA expression results, we conducted further analysis on the enzymatic activity of caspase-3 after exposure to metal oxide NPs (CuO and NiO) and/or curcumin. In a manner consistent with the findings related to mRNA, the presence of CuO NPs led to an increased level of caspase-3 enzyme activity. However, this impact was significantly reduced when curcumin was concurrently administered, as depicted in Fig. 6B. In a study conducted by Huang et al. (Huang et al., 2014), it was observed that the administration of N-acetylcysteine, an antioxidant approved by the FDA, had a preventive effect on the induction of DNA damage and fetal brain developmental defects in pregnant mice after carbon nanotubes exposure.

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Fig. 6

Mrna expression level (a) and enzymatic activity (b) of caspase-3 gene in bewo cells treated for 24 h to NPs (25 µg/ml) and/or solanesol (10 µg/ml). * p < 0.05 vs. control. ^# p < 0.05 vs. NPs treated group. SOL: solanesol.

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The defect in mitochondria is likely to have a significant impact on the correlation between neurodevelopmental problems in the offspring and chronic maternal inflammation during pregnancy. This correlation is frequently observed in the presence of prevalent maternal conditions or circumstances, including obesity, stress, and exposure to environmental pollutants (Gyllenhammer et al., 2022). A recent study demonstrated that solanesol enhances antioxidant capacity and ameliorates mitochondrial deficits (Liu et al., 2023). Utilization of antioxidant compounds, such as solanesol, has the ability to mitigate MMP deficit, decreasing the likelihood of premature disruption of membranes, a significant contributing factor to preterm birth (Tossetta et al., 2021). This study also investigated the loss of MMP, which serves as an early apoptotic indication, in BeWo cells after being exposed to CuO and NiO NPs, as well as solanesol, for a duration of 24 h. The results presented in Fig. 7 demonstrate a considerable reduction in the MMP in BeWo cells following exposure to these metal oxide NPs. However, this impact was effectively reversed when curcumin was co-administered with the NPs. Mitochondrial dysfunction is a probable factor contributing to the correlation between neurodevelopmental disorders in offspring and chronic maternal inflammation during pregnancy. This association is frequently observed in the presence of prevalent maternal circumstances, including obesity, stress, and pollutants. The findings of our earlier investigation showed that the apoptotic effects induced by CuO NPS in placental cells were significantly mitigated with the administration of the dietary antioxidant curcumin (Ahamed et al., 2022).

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Fig. 7

MMP level in BeWo cells treated for 24 h to NPs (25 µg/ml) and/or solanesol (10 µg/ml). * p < 0.05 vs. control. ^# p < 0.05 vs. NPs treated group. SOL: solanesol.

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