Integrated transcriptomic and metabolomic ecotoxicity assessment of earthworms exposed to ZnO and Mn-doped ZnO nanomaterials.

INTRODUCTION

Zinc oxide (ZnO)-containing nanomaterials may elicit toxic responses in environmental organisms such as earthworms. In this study, we investigate the time-dependent gene expression and metabolomic response in earthworms exposed to ZnO and Mn-doped ZnO (ZnO:Mn) nanomaterials (both at 850 mg/kg soil) and MnCl2.

METHODS

Earthworms were exposed to ZnO nanomaterial (850 mg/kg soil) for 2, 3, 4, or 7 days, to ZnO:Mn nanomaterial (850 mg/kg soil) for 2, 4, 7, or 14 days, and to MnCl2 (45 mg/kg soil) for 14 days. Time-dependent molecular responses were characterized by transcriptomic and metabolomic analyses.

RESULTS AND DISCUSSION: Strong differential gene expression was observed after 4 and 2 days of exposure to ZnO and ZnO:Mn, respectively. Moderate differential gene expression was observed after 14 days of exposure to ZnO:Mn and MnCl2. Gene ontology (GO) enrichment analysis revealed that differentially expressed genes (DEGs) in earthworms exposed to ZnO were commonly associated with muscles and earthworm movements. Genes upregulated after 2 days of exposure to ZnO:Mn were linked to GO terms associated with microtubule-based structures mediating cellular motility. Downregulated genes were enriched to GO terms among which several were related to energy metabolism. For earthworms exposed to ZnO:Mn and MnCl2 for 14 days, few DEGs were involved in GO terms related to diverse biological implications. Aligning with the limited number of GO terms observed, metabolomic disruptions only included increased glutamate and a tendency for increased aspartate in response to MnCl2 and ZnO:Mn exposure for 14 days, respectively. Other amino acids and the oxidized-to-reduced glutathione ratio, an indicator of oxidative stress, were not affected. In summary, exposing earthworms to ZnO and ZnO:Mn nanomaterials induced a transient response in differential gene expression related to muscle biology, energy metabolism, and translation, which largely disappeared after 14 days. Combined with minimal metabolomic changes, these findings do not point to substantial long-term ecotoxic effects.