Recently there is a growing interest in determining the effects of NMs in the innermost layers of the skin (Wang et al. 2018). such as the dermis, which is mainly composed of fibroblasts. For this reason and because the toxic potential of Tb-MOF is not yet known, in this work, a first approach is presented on the toxic effect of a Tb-MOF at the cellular and epigenetic level in hFB cells. In this study, cellular toxicity in hFB was observed after treatment with 0.05 to 1.6 mg/mL Tb-MOF, exhibiting a dose-dependent response. Thus, the lowest (12.91%) and the highest (76.71%) inhibitions of cell viability were obtained with 0.05 mg/mL and 1.6 mg/mL, respectively. Similar results have been reported regarding the negative effect exerted on the viability of some human cancerous and healthy cell lines by another type of NMs based on Tb. For instance, in cancer cell lines, a study reported a decrease of approximately 10% in the viability of HeLa cells exposed to 0.1 mg/mL Eu3+-TbPO4 nanoparticles for 72 h (Di et al. 2011). Similarly, MG-63 and Saos-2 osteosarcoma cells exposed to Tb2O3 (0.001 mg/mL) nanoparticles for 48 h had a 50% reduction in cell viability. The authors demonstrated a significant increase in the level of intracellular reactive oxygen species (ROS), which they suggest could be related to cytotoxicity (Iram et al. 2016). Similar results were also reported by Setyawati et al. (2013) who observed a viability reduction of 40% in human skin fibroblasts in the presence of 0.75 mg/mL Tb-Gd2O3 nanoparticles after 24 h of exposure, suggesting that metal oxide nanoparticles could increase the mechanisms of oxidative stress and induce DNA damage and apoptosis. Our study shows the cytotoxic effect of Tb-MOF on hFB, nevertheless, if the reduction of cell viability is due to metal producing the generation of ROS and DNA damage, as proposed by some authors (Setyawati et al. 2013; Iram et al. 2016) it should still be evaluated.
On the other hand, we determined an IC50 of Tb-MOF between 0.260 ± 0.012 mg/mL (Tb concentration of 0.052 mg/mL) and 0.384 ± 0.087 mg/mL (Tb concentration of 0.077 mg/mL) on hFB. In this regard, Iram et al. (2016) showed that the presence of Tb2O3 particles had an IC50 of 0.001 mg/mL in osteosarcoma cells. This value was lower than that obtained in our work, even though the final Tb concentration in this work was lower (0.0008 mg/mL) considering the proportion in Tb2O3 particles. This indicates a higher toxicity of the Tb2O3 particles than those of Tb-MOF. However, contrasting results were obtained by Setyawati et al. (2013) regarding the viability of hFB in the presence of Tb-Gd2O3. The authors observed that 0.75 mg/mL of Tb-Gd2O3 (0.228 mg Tb/mL) induced a 40% reduction in the viability of hFB. Therefore, the contradictory results of the studies described above may indicate that the specific effects on cell lines could be due to the characteristics of the complexes of NMs and not due to the Tb concentration. Furthermore, it is important to highlight that studies have shown that same nanomaterial may have different toxic effects depending on the cell line (Heng et al. 2010), or even between the type of culture used like monocultures or co-cultures (Ventura et al. 2020). Accordingly, more studies must be conducted to determine the mechanisms by which NMs generate cytotoxicity, as well as to continue with the search for in vitro cell models that allow the implications in vivo to be inferred.
On the other hand, through inverted microscopy and FESEM-EDS analysis, it was determined that Tb-MOF (1.6 mg/mL) induced important changes in fibroblast morphology respect to the control. For instance, irregular prolongations and oval nuclei, (Supplementary Fig. 2) as well as a remarkable contraction of the cells, making them round (Fig. 1), decreased their adhesion capacity for the culture surface, which was confirmed when they were lifted when washing the culture plates. Similar results have also been obtained in osteosarcoma cells, dermal fibroblasts and L929 cells when exposed to Tb2O3 (3.73 x10-4 mg/mL), ZnO (0.5 mg/mL) and Mo (0.1 mg/mL) nanoparticles, respectively (Siddiqui et al. 2015; Iram et al. 2016; Abe et al. 2018). However, it has been proposed that morphological changes can also vary depending on the cell line and nanoparticles used (Li et al. 2012). Such losses of the specific morphological characteristics of cells have been demonstrated to be evidence of the induced toxicity of nanoparticles in cells as a stress response to the extracellular environment to which they are subjected (Iram et al. 2016; Yamaba et al. 2016). Therefore, the subtle effects of Tb-MOF on cell morphology can indirectly lead to disturbance of cell function, while severe morphological alterations, such as rounding and cell contraction, can be interpreted in terms of cell death (Zhivotovsky and Orrenius 2011).
Moreover, we showed internalization of Tb-MOF and its deposition onto the cell surface (Figs. 3, 4). It is known when NMs are deposited on cells, they can sediment which could increase their concentration on the cell surface, thus facilitating uptake by cells (Cho et al. 2011) in which, in turn, might produce a decrease in cell viability depending on the dose, as well as various metabolic and morphological effects (Plascencia-Villa et al. 2012). Thus, nanoparticles can be internalized and form agglomerates (Magdolenova et al. 2012), as well as protuberances, which can lead to cell death (Berry et al. 2003; Gupta and Curtis 2004). However, the methods of absorption and biodistribution of nanoparticles depend on the size, shape, and others physicochemical properties of nanomaterials (Kunzmann et al. 2011).
On the other hand, we showed that Tb-MOF induced a change in DNMT expression, mainly affecting DNMT3A and DMT3B, by exposing hFB cells at 0.05 − 0.02 mg/mL (Fig. 5A). In this regard, it has been reported that exposing keratinocyte cells (HaCaT) to SiO2 nanoparticles leads to a reduction of DNMT1 and DNMT3A at mRNA and protein level (Gong et al. 2010). Consistent with this finding, a downregulation of DNMT1 and − 3B was also induced by ZnO nanoparticles (0.05 mg/mL) in human embryonic kidney cells (HEK-293) (Choudhury et al. 2017). In contrast, it appears that Ag nanoparticles are able to induce the overexpression of DNMTs in neuronal cells of the mouse (HT22) (Mytych et al. 2017). In line with this result, we detected that Tb-MOF leads to upregulating DNMT3A and decrease DNMT3B expression, conjecturing that increasing in DNMT3A could arise as an adaptive cellular response to maintain the methylation state. However, it must be considered that DNMTs expression might be dependent on the nanoparticle's specific features and the type of cell to be evaluated (Pogribna and Hammons 2021). Besides, whether Tb-MOF impairs DNA methyltransferase activity (e.g DNMT3B) or only leads to dysregulation of DNMT expression require further research.
Although there are gaps in the body of knowledge regarding the epigenetic events that happen after cells are exposed to nanomaterials, it seems that loss of function of DNMT2, a DNA methyltransferase of DNA and RNA, lead to upregulate DNMT3A and DNMT3B in foreskin fibroblast cells (Lewinska et al. 2018) and all DNMTs in mouse fibroblasts (NIH3T3) by inducing a hypermethylation of DNA and RNA (Lewinska et al. 2018). However, due to the divergent activity of DNA methyltransferases (Hervouet et al. 2018), future studies need to be carried out to determine the effect of NMs on methylation and on, in principle, genes related to cell proliferation (e.g. genes involved in p53 pathway).
On the other hand, it has been also proposed that DNMTs may be involved in the adaptive response to oxidative stress, since the exposure of human embryonic lung fibroblasts (HEF) and human fetal lung fibroblasts WI-38 to hydrogen peroxide results in increased DNMTs expression (Zhang et al. 2008; Lewinska et al. 2018). These findings are relevant because there is evidence that, in general, metallic nanoparticles can increase the formation of reactive oxygen species (ROS) in skin cells, associating it with the generation of oxidative stress, an important event that affect chromatin integrity and DNA methylation (Pogribna and Hammons 2021) considered as the main cause of toxicity of nanomaterials (Dusinska et al. 2017; Pogribna and Hammons 2021). In this aspect, it would be interesting to evaluate the levels of expression of DNMT2 in fibroblast cells exposed to nanomaterials considering the sensitivity to oxidative stress and cell proliferation that has been demonstrated in human fibroblasts exposed to this type of stress (Lewinska et al. 2018).
On the other hand, TET family members (TET1, TET2 and TET3) catalyze the process of demethylation to maintain the correct balance of DNA methylation in the genome (Franchini et al. 2012). If well, that TET proteins have tumor-suppressor functions that are essential for maintaining genome integrity (Rasmussen and Helin 2016; Cimmino et al. 2017). The expression levels of TETs can vary among cells/organs, and their specific activities in many biological processes have not been elucidated (Delatte and Fuks 2013; Jiang et al. 2017). Here, we found that all Tb-MOF concentrations led to increase TETs expression levels (Fig. 5B). These results suggest an increase of 5-hmC levels in DNA associated with the increased expression of TET1, TET2 and TET3 genes (Fig. 5B). In line with our findings, it has been reported that ZnO nanoparticles induce an hypomethylation of DNA by decreasing DNA methyltransferase activity and increasing expression of TET1 and TET2 genes in HEK293 cells and human MRC5 lung fibroblast (Patil et al. 2016; Choudhury et al. 2017). Although it has been reported that there is a correlation between ROS generation and the increased expression of TETs that lead to global DNA hypomethylation (Choudhury et al. 2017), the mechanism leading to upregulation of TETs expression by TB-MOF requires to be demonstrated. Furthermore, although an increase in the expression levels of TETs has been reported, a decrease in them has also been shown, differentiated by the nanomaterial used and the model cell line (Lu et al. 2016). It indicates that further studies are warranted to clarify in greater depth the possible mechanisms governing the effects of different classes of nanometric materials, such as Tb-MOF, have had on human health.