Earthworms and Humans in Vitro: Characterizing Evolutionarily Conserved Stress and Immune Responses to Silver Nanoparticles

Abstract

Little is known about the potential threats of silver nanoparticles (AgNPs) to ecosystem health, with no detailed report existing on the stress and immune responses of soil invertebrates. Here we use earthworm primary cells, cross-referencing to human cell cultures with a particular emphasis on the conserved biological processes, and provide the first in vitro analysis of molecular and cellular toxicity mechanisms in the earthworm Eisenia fetida exposed to AgNPs (83 ± 22 nm). While we observed a clear difference in cytotoxicity of dissolved silver salt on earthworm coelomocytes and human cells (THP-1 cells, differentiated THP-1 cells and peripheral blood mononuclear cells), the coelomocytes and differentiated (macrophage-like) THP-1 cells showed a similar response to AgNPs. Intracellular accumulation of AgNPs in the coelomocytes, predominantly in a phagocytic population, was evident by several methods including transmission electron microscopy. Molecular signatures of oxidative stress and selected biomarker genes probed in a time-resolved manner suggest early regulation of oxidative stress genes and subsequent alteration of immune signaling processes following the onset of AgNP exposure in the coelomocytes and THP-1 cells. Our findings provide mechanistic clues on cellular innate immunity toward AgNPs that is likely to be evolutionarily conserved across the animal kingdom.

Nanosilver pathophysiology in earthworms: Transcriptional profiling of secretory proteins and the implication for the protein corona

“…Briefly, coelomocytes were extruded from earthworms using a physiological saline containing 2% ethanol, guaicolglyceryl-ether (a mucolytic agent) and EDTA. In accordance with the previous study, we conducted in vitro assays under isotonic conditions using an osmolarity-adjusted phosphate-buffered saline (PBS) or cell culture medium to achieve osmolarity equivalent to Lumbricus balanced salt solution (176 mOsm; for the composition see Hayashi et al, 2012). Thus, we refer to the osmolarityadjusted PBS as reduced PBS or ''R-PBS''.…”

“…Soluble silver fraction (dissolved silver complexes and free Ag + ) of NM-300K under the exposure condition (2 mg/ml, 4 h and 24 h in CCM) was determined using an ultracentrifugation method (for details see Beer et al, 2012;Foldbjerg et al, 2012;Hayashi et al, 2012). Briefly, following incubation in quadruplicate, NM-300K was ultracentrifuged at 50 000 rpm (corresponding to 70 000-135 000 Â g) for 30 min at 4 C and the supernatant was digested in 69% HNO 3 (tracemetal grade, Sigma-Aldrich).…”

The immunomodulatory effects of titanium dioxide and silver nanoparticles

“…Additionally, Ag nanoparticles induce a reduction of NO production and an upregulation of the expression of Myd88, MEKK1 and early regulation of oxidative stress genes, in both PBMCs and THP-1 cells. (Hayashi et al, 2012) Similarly, exposure for 24, 48 or 72 h to 1e25 ppm Ag nanoparticles causes a significant decrease in the cell viability of murine peritoneal macrophages, and exposure to 0.4e25 ppm of Ag nanoparticles results in a significant decline in NO production by murine macrophages (Shavandi et al, 2011). The in vitro exposure of murine macrophages to 50e100 mg/mL Ag nanoparticles results in a significant reduction in cell viability, with an investigation of intracellular localization revealing silver distributed mainly to high molecular weight proteins, and Ag nanoparticles co-localizing with lysosomes.…”

In vitronanoparticle toxicity to rat alveolar cells and coelomocytes from the earthwormLumbricus rubellus

“…Nguyen et al (2012) found that mouse macrophage cells (J774A.1) were more sensitive to CdTe-QD exposure than human colonic epithelial cells (HT29), in terms of metabolism loss, changes in morphology and rate of QD internalization. Differences in sensitivity and uptake of nanoparticles were also found between coelomocytes and a cultured cell line, in a study by Hayashi et al (2012). This study demonstrated that coelomocytes from the earthworm Eisenia fetida were more sensitive to silver nanoparticle exposure than the cultured human leukaemia (THP-1) cell line and also accumulated more silver nanoparticles.…”