Previously we described that cadmium

Previously, we described that cadmium induces a more pronounced, but not selective, cell death on primary cholinergic neurons from basal forebrain (Del Pino et al., 2014). Degeneration of septal cholinergic neurons from basal forebrain, as happens in AD, results in memory deficits (Scheiderer et al., 2006). Thus, cholinergic neuronal loss in this region may be related with cadmium impairment of memory function, among other actions (Andersson et al., 1997). Cadmium induced basal forebrain cholinergic neuronal loss was partially mediated by selective blockade of M1R through alteration of AChE splices variants expression (Del Pino et al., 2016a), although other mechanisms should be involved. Muscarinic receptor subtypes M1-M5 are necessary to maintain cell viability and regulate learning and memory processes (Bainbridge et al., 2008; Bubser et al., 2014; Galloway et al., 2014; Zheng et al., 2012). Cadmium has been reported to downregulate M1, M2 and M4 muscarinic receptor gene expression in frontal cortex and hippocampus (Gupta et al., 2017), so cadmium could also alter other muscarinic receptors besides M1 in basal forebrain, which could induce the effect observed on cholinergic neurons. In addition, cadmium induces oxidative stress through reactive oxygen species (ROS) formation, depletion of antioxidant defenses, and reduction of antioxidant enzymes (Gonçalves et al., 2010; Luchese et al., 2007; Nemmiche, 2017; Pari and Murugavel, 2007; Thévenod, 2009). Oxidative stress has been involved in the induction of cognitive disorders and cadmium-antioxidant co-treatment could reverse cognitive disorders induced by cadmium (Gonçalves et al., 2010; Gupta et al., 2017; Maodaa et al., 2016). Oxidative stress is also able to induce muscarinic receptor activity dysfunction, AChE splice variants gene expression alteration and basal forebrain cholinergic neurons cell death (Bond et al., 2006; Del Pino et al., 2016c; Giraldo et al., 2014; Gupta et al., 2017), which could also contribute to explain the effect observed.
Materials and methods
Results
Discussion In the present work we show that cadmium induces ROS generation and lipid peroxidation in a concentration-dependent way on septal SN56 cholinergic basal forebrain neurons. In this regard, cadmium has been reported to elevate ROS levels and increase lipid peroxidation in the JNJ 5207852 dihydrochloride receptor (Gonçalves et al., 2010; Thévenod, 2009), which supports our findings. Moreover, NAC co-treatment with cadmium reversed completely the induction of oxidative stress. NAC is a potent antioxidant reported to override the oxidative stress through restoration of the pool of intracellular reduced glutathione and directly scavenge ROS, recovering cellular redox status after cadmium treatment (Gonçalves et al., 2010), which supports our results. NAC protective effect could also happen because of its action as a metal chelator (Jalilehvand et al., 2011), which could inhibit the Cd cellular uptake (Luczak and Zhitkovich, 2013), so its toxics effects. However, only ROS generation and lipid peroxidation induced by cadmium treatment were completely reversed after NAC co-treatment with Cd, while the rest of cytotoxic effects were only partially reduced. Thus, NAC protection seems to be mediated by its antioxidant effect, although the possible reduction of Cd uptake cannot be excluded as a possible contributing mechanism. Otherwise, antioxidant co-treatment with cadmium has been reported to reverse cognitive dysfunctions induced by cadmium (Gonçalves et al., 2010; Maodaa et al., 2016), suggesting this mechanism could mediate the effect observed after cadmium treatment on memory and learning processes. In addition, cadmium treatment did not affect the gene expression of M1R and M5R, nevertheless, decreased the gene expression of M2R, M3R and M4R in concentration-dependent way. Cadmium also blocked M1R in a concentration-dependent way confirming our previous results (Del Pino et al., 2016a). Antioxidant treatment partially reversed the downregulation of muscarinic receptor gene expression and the blockade of M1 receptor. Previous studies showed that cadmium treatment for 28 days blocks muscarinic receptors and decreased M1R, M2R and M4R in frontal cortex and hippocampus and antioxidant co-treatment with quercetin reversed partially this effect on muscarinic receptors (Gupta et al., 2017), which in part supports our results. The differences observed between our results and those of Gupta et al. (2017) on muscarinic receptor gene expression could be due to the differences in the region studied and because their study is based on cadmium effect after repeated exposure. As ROS may alter the conformation of the muscarinic receptors, inactivating them (Fawcett et al., 2002), antioxidant treatment could override this effect, reversing muscarinic receptors blockade; thus explaining the observed effect. Since NAC co-treatment with cadmium reversed completely the induction of oxidative stress, other mechanism should be involved on cadmium alteration of muscarinic receptors. In this regard, Aβ peptide has been reported to induce the downregulation of muscarinic M2R in the hippocampus (González et al., 2008). Cadmium has be reported to increase the production of Aβ peptides in SN56 cells (Del Pino et al., 2016b), which could mediate the effect observed in M2R and the rest of muscarinic receptors affected. Otherwise, we reported an ACh levels reduction correlated with cell death induction after cadmium (10−6-10−2M) treatment in SN56 cells, but the co-administration of ACh (10−8-10−4M) with cadmium (10−6-10−2M) did not reversed the reduction observed on cell viability (Del Pino et al., 2014), which could be due to the cadmium effects on muscarinic receptors. Muscarinic M1-4 receptors have been reported to regulate memory and learning process (Atri et al., 2004; Bainbridge et al., 2008; Bubser et al., 2014; Galloway et al., 2014; Zheng et al., 2012), thus these alterations could also mediate the cognitive disorders induced after cadmium exposure.