Increased
release of engineered nanoparticles (ENPs) from widely
used commercial products has threatened environmental health and safety,
particularly the repeated exposures to ENPs with relatively low concentration.
Herein, we studied the response of Chlorella pyrenoidesa (C. pyrenoidesa) to single and repeated exposures
to silver nanoparticles (AgNPs). Repeated exposures to AgNPs promoted
chlorophyll a and carotenoid production, and increased silver accumulation,
thus enhancing the risk of AgNPs entering the food chain. Notably,
the extracellular polymeric substances (EPS) content of the 1-AgNPs
and 3-AgNPs groups were dramatically increased by 119.1% and 151.5%,
respectively. We found that C. pyrenoidesa cells
exposed to AgNPs had several significant alterations in metabolic
process and cellular transcription. Most of the genes and metabolites
are altered in a dose-dependent manner. Compared with the control
group, single exposure had more differential genes and metabolites
than repeated exposures. 562, 1341, 4014, 227, 483, and 2409 unigenes
were differentially expressed by 1–0.5-AgNPs, 1–5-AgNPs,
1–10-AgNPs, 3–0.5-AgNPs, 3–5-AgNPs, and 3–10-AgNPs
treatment groups compared with the control. Metabolomic analyses revealed
that AgNPs altered the levels of sugars and amino acids, suggesting
that AgNPs reprogrammed carbon/nitrogen metabolism. The changes of
genes related to carbohydrate and amino acid metabolism, such as citrate
synthase (CS), isocitrate dehydrogenase (IDH1), and malate dehydrogenase
(MDH), further supported these results. These findings elucidated
the mechanism of biological responses to repeated exposures to AgNPs,
providing a new perspective on the risk assessment of nanomaterials.