By mass, metal(loid)s are the main component of PM (Cheng et al., 2016; Andrade-Oliva et al., 2020) and even from urban particulate standard reference material (E.g., SRM 1648a or NIST 1649b). Frequent elements in the PM are Zn, Fe, Pb, Mn, Ca, and Cu, among others, and they are even found in a range of concentrations comparable with the PM selected in our work (Yuang et al., 2019). Some of these essential elements are toxic to lung epithelium. Yuang and collaborators reported the viability effect from the A549 cell line exposed to metal(loid)s and found, like our results, a differential viability effect, depending on each metal(loid)s. In addition, the greatest reduction in the viability effect was observed on Fe, Cu, Mn, and Pb. Also, the co-exposure metal(loid)-CQ concentrations at a lower non-cytotoxic concentration (0.01 µM) seem to recover the cell viability. This substance, CQ, seems to exert possible protection against metal(loid) toxicity because improves the A549 cell line viability in concentrations at which metal(loids) previously decreases it. For almost all metal(loid)s recovery was evident, but to Cu co-exposure the effect was discrete.
The CQ concentration used to protect cell viability need to be tested to guarantee safety use. Our data suggest that concentrations below 0.1 µM can recover the cell viability of seven elements; in addition, this concentration did not affect biomarkers related to OxS (eg. GST and MDA). CQ and its analogs need to be tested as chemoprotective agents. Doses used for other propose are too high and administration therapeutic schemes are short to the high half-life of CQ (Kadam et al., 2016, Borba et al., 2020). Few studies that study the effect of CQ low doses, in vitro (0.2 µM in human mesenchymal stem cells) and in vivo (1.1 µg/Kg) administered in drinking water to Sprague Dawley rats for 5 weeks, reported that extended their lifetime, reduce chronic inflammation and tissue fibrosis (Li et al., 2022).
OxS is induced by the spontaneous interaction of metal(loid)s, the intrinsic metabolism, and biotransformation of drugs among other factors. The changes in GST, MDA, and AOPP were metal(loid)-dependent. The interaction metal(loid)-CQ in some cases induces more OxS, however, it is not different from single metal(loid) exposure.
GST is an inducible enzyme under xenobiotic exposure. The GST assay showed low activity in Ti and Fe co-exposure to CQ; whereas Ca, Mn, and Zn increased GST activity independent of CQ co-exposure, only the co-exposure Cu-CQ increase the activity with respect to single Cu exposure. While CQ co-exposures suggest effects on different levels and interfere with GST activity-dependent of metal(loid)s exposure. Considering that GST is involved in the detoxification and biotransformation of organic xenobiotics, and metabolites of it or the OxS metabolites (Higgins and Hayes, 2011; Fletcher et al., 2015), it has been reported that CQ chronic exposure (20 mg/Kg, one per week, 4 weeks) reduce the GSH-related enzymes such as Glutathione peroxidase in liver and kidney (Magwere et al., 1997). In a rat model of subchronic exposure to CQ (9 days, 5 mg/kg/ day) was observed a reduction in Ascorbate in the liver, and a reduction in the GSH and ascorbate in the brain and erythrocytes respect control group, with an MDA increment in all tissues (Abdel-Gayoum et al., 1992). A limitation of the GST enzymatic assay, used in the present study, is that it does not specific to the different GST isoforms and cellular locations (Higgins and Hayes, 2011).
A GSH-related enzymatic system could carry out cellular detoxification during metal(loid)s exposure, but we do not discard other mechanisms mediated by CQ exposure such as the Nrf2 pathway activation. In C2C12 a myoblast cell line exposed to 5–50 µM CQ, has been reported the over translocation of Nrf2 and the induction of Nrf2 regulated genes such as Nqo1, Hmox1 and Gsr concentration- and time-dependent (Duleh et al., 2016).
Lipoperoxidation measured as MDA, showed that CQ co-exposure with Cu, Fe, Mn, Pb, and Zn induced the increment in the MDA concentrations, single exposure to CQ and metal(loid)s by itself does not produce it. The CQ protective effect on MDA levels was only observed in CaCQ. The increment of MDA concentrations has been reported in vivo models after single and repeated exposure to CQ. However, the concentration used in the present study was lower than not inducing OxS. The co-exposure to CQ-meta(loid) could affect 1) the antioxidant-metabolic response, and 2) affect the meta(loid) intracellular distribution. The first one hypothesis could be supported by the effect on GSH content and GSH-related enzymes and the Nrf2 pathway above mentioned. The second hypothesis can be based on the CQ is a Zn ionophore able to increase Zn concentrations in the lysosome (Xue et al., 2014). Emerson et al., describe that CQ treatment in Saccharomyces cerevisiae affects metal transporters implicated in iron acquisition. CQ treatment inhibits the FeCl3 accumulation in S. cerevisiae. Iron metabolism disruption by CQ treatment has been reported in prokaryotes and eukaryotes including mammalian cells (Emerson et al., 2002). The protective effect of CQ could be explained by similar mechanisms to other metal(loid)s. In addition, CQ subchronic exposure (15 mg/kg 5×/week for 5 weeks) affect iron load, and a decrement in iron content in the liver and spleen was reported (Legssyer et al., 2003). The changes in the distribution and disponibility of divalent cations, such as Zn and Fe, can contribute to an increase in the Haber-Weiss reaction to increment the reactive oxygen species and macromolecules oxidation including lipids and proteins.
AOPP assay showed differences only in CQ co-exposures with Cu, Pb, and Ti, but in general, any difference was observed in comparison to the control. Protein oxidation is a stable biomarker of OxS, the degradation of damaged proteins depends on autophagic-lysosome- and ubiquitin-proteasome-proteolysis pathways. The increment in ubiquitin and 20s proteasome subunit in denervated muscles of CQ-treated rats was reported (Kimura et al., 2009). In addition, CQ treatment (50 mg/kg during 250) expanded the median and maximum lifespan of male mice, this effect was related to an increment in the autophagosome-like structures containing cytoplasmic organelles with changes in the proteasome activity (Doeppner et al., 2022). The discrete data of oxidation protein in the co-exposure CQ-metal(loid) could be mediated by autophagic-lysosome- and ubiquitin-proteasome-proteolysis pathways, that need further studies.
The role of SPD is related to pulmonary defense mechanisms to OxS and inflammation. SPD overexpression in mice or rats does not affect lung structure or surfactant pool and increases substantially in response to lung injury, infection, or endotoxin exposure (McCormack and Whitsett., 2002; Watson et al., 2021). The differential effect of CQ on surfactant proteins, and under metal(loid) co-exposure suggest that can affect the biogenesis of lysosome-relate organelles such as lamellar bodies in the pulmonary epithelium. It has been reported that CQ blocks the proteolytic processing of surfactant-protein C which is pH-dependent (Beers, 1996). Further studies are needed to explain the effect of CQ in lysosome-related organelles on cell survival and how it modifies metal(oid) toxicity, based on CQ induction of autophagosomes and lysosomes, and the influence of endomembrane system on metal homeostasis by the presence of metal transporters and the ion compartmentalization (Blaby-Haas and Merchant, 2014).
PM2.5 was used as a mixture of metal(loid)s representative of continuous exposure inhalation, and 3MA as a control for the main activity reported for CQ, autophagy. The PM2.5 co-exposure to 3MA or CQ induces a significant reduction in AOPP, suggesting the increment in the proteolytic pathway without changes in the GST, MDA, and SPD biomarkers, suggesting that 3MA- and CQ- co-exposures did not have interference on the OxS, GSH-related metabolism, and possibly endomembrane system through the lysosome.
Prophylactic treatments have been suggested to protect against air pollution, specifically against PM. The differential response was observed between 3MA and CQ pretreatment on MDA and SPD levels, where 3MA was not able to increment both biomarkers. This slight difference between autophagy inhibitors could be attributable to the autophagy stage inhibition, 3MA inhibits the initial stage of autophagy concerning CQ which inhibits the late stage of autophagy. It is important to highlight that 3MA, a pan-PI3K inhibitor, affects many cellular processes such as endocytosis, lysosomal acidification, and mitochondrial permeability transition, which are involved in PM toxicity. On the other hand, CQ is a lysosomotropic agent that inhibits lysosome acidification, in acidic conditions CQ is protonated and trapped into lysosomes increasing the lysosome pH and inhibiting lysosome digestion (Pasquier, 2016). Autophagy inhibitors seem that are involves lysosomal degradation that minimizes the production of reactive oxygen species.
CQ is reported that rescue the exposure cytotoxicity to paraquat. Xu and Wang (2016) reported that paraquat-induced may be through the protection of the lysosomal membrane or up-regulation of autophagy. On the other hand, another substance that has protection against the effect of PM2.5 is Astragaloside IV, a substance in traditional Chinese herbal medicine, that promotes and inhibits autophagic activity through a variety of signaling pathways and that was proposed to improve various diseases (Wang et al., 2022). The similarity in substances effects suggests that CQ might has also, a dual effect on autophagy and oxidative stress.
Chloroquine improves viability on metal(loid) toxicity and has a differential effect on OxS biomarkers. The effects apply to mixtures of metal(loid)s, such as PM2.5, and are more evident when pre-exposure to CQ and 3MA.