High-throughput tool to discriminate effects of NMs (Cu-NPs, Cu-nanowires, CuNO₃, and Cu salt aged): transcriptomics in Enchytraeus crypticus

Abstract

The current testing of nanomaterials (NMs) via standard toxicity tests does not cover many of the NMs specificities. One of the recommendations lays on understanding the mechanisms of action, as these can help predicting long-term effects and safe-by-design production. In the present study, we used the high-throughput gene expression tool, developed for Enchytraeus crypticus (4 × 44k Agilent microarray), to study the effects of exposure to several copper (Cu) forms. The Cu treatments included two NMs (spherical and wires) and two copper-salt treatments (CuNO₃ spiked and Cu salt field historical contamination). To relate gene expression with higher effect level, testing was done with reproduction effect concentrations (EC₂₀, EC₅₀), using 3 and 7 days as exposure periods. Results showed that time plays a major role in the transcriptomic response, most of it occurring after 3 days. Analysis of gene expression profiles showed that Cu-salt-aged and Cu-nanowires (Nwires) differed from CuNO₃ and Cu-nanoparticles (NPs). Functional analysis revealed specific mechanisms: Cu-NPs uniquely affected senescence and cuticle pattern formation, which can result from the contact of the NPs with the worms’ tegument. Cu-Nwires affected reproduction via male gamete generation and hermaphrodite genitalia development. CuNO₃ affected neurotransmission and locomotory behavior, both of which can be related with avoidance response. Cu salt-aged uniquely affected phagocytosis and reproductive system development (via different mechanisms than Cu-Nwires). For the first time for Cu (nano)materials, the adverse outcome pathways (AOPs) drafted here provide an overview for common and unique effects per material and linkage with apical effects.

Keywords:

• Nanomaterials
• high-throughput
• toxicogenomics
• mechanisms of response
• adverse outcome pathway

Disclosure statement

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.

Additional information

Funding

This study was supported by the European Commission EU-FP7 SUN (G.A. No. 604305), MARINA (G.A. No. 263215) and MODERN (Ref. 309314- 2). By funding FEDER through COMPETE Programa Operacional Factores de Competitividade, and by National funding through FCT-Fundação para a Ciência e Tecnologia, within the research project NANOkA FCOMP-01-0124- FEDER-008944 (Ref. FCT PTDC/BIA-BEC/103716/2008), project POCI-01-0145-FEDER-007679 (FCT ref. UID/CTM/50011/2013), project NM_OREO (POCI-01-0145-FEDER-016771, PTDC/AAG-MAA/4084/2014), through the post-doc grant to Susana Gomes (SFRH/BPD/95775/2013) and CESAM (UID/AMB/50017/2013 - POCI-01-0145-FEDER-007638), to FCT/MCTES through national funds (PIDDAC), and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020.