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Molecular ecotoxicology of nanosilver guided using in-vitro prognosis

by Yuya Hayashi, Lars-Henrik Heckmann, Péter Engelmann, Herman Autrup, Duncan S. Sutherland, Janeck J. Scott-Fordsmand of the iNano-School, Department of BioScience - Terrestrial Ecology, Department of Public Health - Institute of Environmental and Occupational Medicine, Aarhus University (Denmark)

ABSTRACT

To study the molecular and cellular basis of silver nanoparticle (AgNP) toxicity, we here used a recently established in vitro model of earthworm coelomocytes in comparison to the conventional in vivo molecular ecotoxicology approach. Compared to the latter where the test organisms are exposed to NPs of interest held in an environmental matrix, in vitro models benefit from the ease of controlling exposure conditions in a defined set of biochemical milieus that NPs may encounter. The AgNPs tested in the present study originated from the same source, but to enhance the colloidal stability in the in vitro test media the NPs were pre-treated with serum proteins. In addition to physical characterisation of AgNPs, the active silver ion fraction was measured (in serum-supplemented cell culture medium and in soil pore-water). Using flow cytometry and atomic absorption spectrophotometry, we show that a specific population of coelomocytes can accumulate AgNPs in vitro and that silver uptake from the soil environment was evident at the organismal level (measured as total accumulated silver). For the comparison of mode-of-action of AgNPs, we have selected several biomarker genes within stress and immune pathways and assessed their transcriptional interplay at a non-lethal concentration of AgNPs over time in vitro (1-6 hours) and in vivo (1-14 days). In vitro dataset displayed a transition from oxidative stress responses to immune signalling during the 6 hours exposure period. Similarly, a characteristic clustering pattern of oxidative stress response genes was observed in early time points of the in vivo dataset, with stronger association to immune genes at a later time point. Hence, our finding depicts a general response pattern initiating with regulation of stress genes and a subsequent shift to immune genes. This study suggests that AgNPs are likely to be recognised by the earthworm immune system gradually altering the host immune status as the exposure persists.