Inhibition of potential uptake pathways for silver nanoparticles in the estuarine snail Peringia ulvae

Mechanisms involved in the uptake of Ag NPs, and NPs in general, have been long debated within nano-ecotoxicology. In vitro studies provide evidence of the different available uptake pathways, but in vivo demonstrations are lacking. In this study, pharmacological inhibitors were employed to block specific uptake pathways that have been implicated in the transport of metal NPs and aqueous metal forms; phenamil (inhibits Na(+) channel), bafilomycin A1 (H(+) proton pump), amantadine (clathrin-mediated endocytosis), nystatin (caveolae-mediated endocytosis) and phenylarsine oxide (PAO, macropinocytosis). Peringia ulvae (snails) were exposed to 150 µg Ag L(-1) added as citrate capped Ag NPs or aqueous Ag (AgNO3) in combination with inhibitor treatment (determined by preliminary studies). Reductions in accumulated tissue burdens caused by the inhibitors were compared to control exposures (i.e. no inhibition) after 6 and 24 h. No inhibitor treatment completely eliminated the uptake of Ag in either aqueous or NP form, but all inhibitor treatments, except phenamil, significantly reduced the uptake of Ag presented as Ag NPs. Clathrin- and caveolae-mediated endocytosis appear to be mechanisms exploited by Ag NPs, with the latter pathway only active at 24 h. Inhibition of the H(+) proton pump showed that a portion of Ag NP uptake is achieved as aqueous Ag and is explained by the dissolution of the particles (∼25% in 24 h). This in vivo study demonstrates that uptake of Ag from Ag NPs is achieved by multiple pathways and that these pathways are simultaneously active.