The widespread and ever-rising amount of plastic litter is closely linked to the increasing levels of MNPLs found in all environmental compartments. These tiny particles (< 5 mm) are ubiquitously distributed in soil, air, freshwater, and marine ecosystems from where they easily enter the trophic chain . As a result, humans are believed to be mainly exposed to MNPLs via the ingestion of contaminated food or water, but also through other routes such as inhalation or dermal deposition . Under this potential broad human exposure scenario, urgent hazard assessment is required.
Great efforts are being addressed to the understanding of the environmental and ecotoxicological effects of these emergent contaminants. However, studies based on mammal and human models focusing on the characterization of the MNPLs’ impact on human health are still limited . Among those available in the literature, it has been fairly described that MNPLs significantly internalize cells and translocate through physiological barriers [7, 8]; however, whether this uptake results in a biological impact is not sufficiently clear. While some authors describe a lack of cytotoxic and cytostatic effects [37, 38], others have reported MNPL-induced ROS production and pro-inflammatory responses in vitro, as well as mild histological lesions and metabolic disorders in rodent systems . Regardless of these disparities, overall, MNPLs are considered to have low acute toxicity. Nonetheless, much remains to be unveiled in terms of MNPLs’ long-term effects and their role as carriers of different environmental pollutants, which is now attracting attention as a potential toxicological risk associated with MNPLs exposure.
In this context, our work contributes to the field in two ways: (1) by the analysis of the impact of MNPLs and arsenic (co)exposures under a long-term exposure scenario; and (2) by the establishment of a model in which the already damaged genetic background of cells may render them more susceptible to the alterations induced by MNPLs, allowing the detection of typically unnoticed mild effects.
Our selected co-contaminants of study are AsIII and PSNPLs, as representative legacy and emergent contaminants, respectively. They share a ubiquitous environmental distribution although both have major implications in terms of human exposure via the intake of contaminated water and, at a lesser proportion, inhalation. As a result, primary target organs (gastrointestinal and respiratory tracts) are potentially affected by both contaminants [40, 41]. Thus, these coexisting contaminants could have a joint impact on human health. Indeed, there is accumulating evidence hinting at MNPLs/arsenic interaction. Arsenic adsorption onto MNPLs has already been reported in debris samples collected from the open sea . Besides, laboratory studies have confirmed arsenic adsorption onto polystyrene microplastic particles , and polytetrafluoroethylene microparticles . Accordingly, our data demonstrate that arsenic adsorbs onto single PS particles, and it can form PSNPLs/AsIII aggregates (see Figure 3A). Although the proportion of interactions within our samples are not quantifiable with the use of TEM/EDX, the physical interaction is observed and, thus, the generation of a certain number of PSNPLs/AsIII complexes could induce differential effects compared to those of the addition of the arsenic and MNPLs as independent compounds.
Aiming to test the long-term impact of the PSNPLs/AsIII complexes, and to demonstrate whether the effects induced by arsenic exposure are exacerbated under a co-exposure scenario, we have evaluated endpoints regarding genotoxicity and carcinogenicity after the chronic (co)exposure of AsTC, our selected in vitro model. The used AsTC derive from MEF cells previously demonstrated to be sensitive to oxidative stress which is closely linked with genotoxicity, genomic and chromosomal instability, and the eventual transformation driven by 30-weeks of chronic arsenic exposure [30, 31]. We assume the compromised genetic background of these cells can be helpful to make more evident the biological impact of MNPLs and their co-exposures.
The high cellular uptake of PSNPLs in our system (see Figures 2A and 2B), led us to consider whether the PSNPLs/AsIII interaction would translate into an increased arsenic bioavailability and a higher internalization rate. This phenomenon has already been described upon in vitro co-exposures to arsenic and nanoparticles (NPs) such as TiO2NPs and SiO2NPs [42, 43], and after the combined exposure to PSNPLs and AgNPs . However, as shown in Figure 3B, the levels of arsenic internalized in AsTC remained stable with increasing doses of PSNPLs. Therefore, the remarkable genotoxic/oncogenic effects observed upon PSNPLs/AsIII co-exposure are not due to the PSNPL-mediated facilitation of AsIII uptake, but rather due to potential alterations induced by PSNPLs/AsIII at the molecular level.
Among those notable effects of arsenic and MNPLs (co)exposure in our system, we have found a significant induction of both total and oxidative DNA damage. Arsenic is a well-known genotoxic compound and one of its most studied mechanisms of action is the induction of ROS and oxidative stress . In addition, plenty of those studies reporting adverse effects of MNPLs have detected increased ROS levels and DNA damage after short-term exposures [10–12, 46]. Concordantly, with the analysis of the long-term effects of the exposure to PSNPLs, AsIII, and PSNPLs/AsIII we found a significant increase in the total and oxidative DNA damage when compared with passage-matched AsTC (see Figure 4). Interestingly, the oxidative damage derived from the PSNPLs/AsIII co-exposure is significantly higher than that observed after single exposures (see Figure 4B). This effect of contaminant mixtures enhancing arsenic-induced oxidative stress and genotoxicity has also been recently reported after short-term exposures when analyzing the impact of the co-exposure to AsIII/TiO2NPs , AsIII/SiO2NPs , and AsIII/polystyrene microplastics . Therefore, our results and those from other groups contribute to support the existence of positive interactions between contaminants.
Remarkably, the positive interaction between AsIII and PSNPLs also adds to the aggressiveness of the arsenic-induced oncogenic phenotype. Arsenic capacity to drive in vitro carcinogenicity is well established , while this potential aspect of MNPLs' long-term impact has not been explored up to date. In in vitro studies, the usefulness of a battery of cancer hallmarks to assess the transformed status of cells has been proposed . These include morphological changes, accelerated proliferation, secretome alterations, metastatic potential, and deregulation of the differentiation status. The measure of these features has been proven useful to assess arsenic-induced carcinogenesis before [50–52]. In the present study passage-matched AsTC that, as previously mentioned meet all endpoints, display an evident transformed phenotype . Interestingly, according to our data, these hallmarks remain unchanged for AsTC subjected to 12 weeks of PSNPLs or AsIII single exposure but are significantly enhanced under PSNPLs/AsIII co-exposure settings. The increment on the proportion of spindle-like cells within the population (see Figure 5A and B), and especially the dramatically increased capacity of cells to grow independently of anchorage (see Figure 6A), migrate (see Figure 6B), and invade (see Figure 6C) confirm the co-exposure-mediated acquisition of a further aggressive transformed phenotype. To better characterize the oncogenic features of the (co)exposed AsTC, we evaluated their tumorsphere-forming ability as a marker of the stemness status in our cell population. Different studies have linked the conversion of non-stem cells to cancer stem cells with carcinogenesis , and, specifically, with arsenic-induced carcinogenesis [54–57]. However, we did not find significant differences under the different exposure scenarios tested, thus stemness induction seems not to be the mechanism by which PSNPLs/AsIII promotes tumor aggressiveness. Taken together, these findings highlight the urgent need to explore MNPLs’ long-term effects and their potential role as co-carcinogens with other environmental pollutants.