Biological effects of inorganic arsenic on primary cultures of rat astrocytes

Abstract

Abstract. It is well established that inorganic arsenic induces neurotoxic effects and neurological defects in humans and laboratory animals. The cellular and molecular mechanisms of its actions, however, remain elusive. Herein we report the effects of arsenite (NaAsO 2 ) on primary cultures of rat astrocytes. Cells underwent induction of heat shock protein 70 only at the highest doses of inorganic arsenic (30 and 60 μM), suggesting a high threshold to respond to stress. We also investigated arsenic genotoxicity with the comet assay. Interestingly, although cells treated with 10 μM arsenite for 24 h maintained >70% viability, with respect to untreated cells, high DNA damage was already observed. Since arsenic is not known to be a direct-acting genotoxic agent, we investigated the possibility that its effects are due, in astrocytes as well, to ROS formation, as already described for other cell types. However, FACS analysis after CM-H 2 DCFDA staining did not evidence any significant increase of ROS production while, on the contrary, at the highest arsenite concentrations used, ROS production decreased. Concordantly, we found that, if most cells in the culture are still alive (i.e. up to 10 μM arsenite), they show a treatment-dependent increase in the concentration of SOD1. On the other hand, SOD2 concentration did not change. Finally, we found that astrocytes also synthesize PIPPin, an RNA-binding protein, the concentration of which was recently reported to change in response to stress induced by cadmium. Here we also report that, in cells exposed to high doses of arsenite, an anti-PIPPin antibody-positive faster migrating protein appears.

Update of the risk assessment of inorganic arsenic in food

“…The standard alkaline comet assay, that allows to detect single (SSB) and double‐strand breaks (DSB) and alkali‐labile sites (apurinic/apyrimidinic sites), has been used in a variety of cells in culture of rodent or human origin, either primary or transformed. In general, arsenite and arsenate were confirmed as good inducers of DNA breaks and alkali‐labile sites at subtoxic concentrations (Benhusein et al, 2016 ; Catanzaro et al, 2010 ; Jiang et al, 2013 ; Li et al, 2014 ; Naranmandura, Carew, et al, 2011 ; Singh et al, 2011 ; Xu, McClain, et al, 2016 ). The induction of oxidative DNA base damage by iAs was further supported by studies where the formamidopyrimidine‐DNA glycosylase (Fpg)‐modified comet assay was associated with the use of mouse cell lines defective in the DNA glycosylase specific for removal of 8‐oxoguanine, OGG1 (Bach et al., 2014 , 2015 ).…”

“…The source and mechanisms of ROS production by iAs have been addressed in some studies (Catanzaro et al., 2010 ; Ebert et al., 2014 ; Li et al., 2014 ; Naranmandura, Carew, et al., 2011 ). In general, long‐term cell exposure and/or high concentrations of sodium arsenite were required to measure a significant increase in the levels of intracellular ROS.…”