Linking toxicity and adaptive responses across the transcriptome, proteome, and phenotype of Chlamydomonas reinhardtii exposed to silver

Abstract

Understanding mechanistic and cellular events underlying a toxicological outcome allows the prediction of impact of environmental stressors to organisms living in different habitats. A systems-based approach aids in characterizing molecular events, and thereby the cellular pathways that have been perturbed. However, mapping only adverse outcomes of a toxicant falls short of describing the stress or adaptive response that is mounted to maintain homeostasis on perturbations and may confer resistance to the toxic insult. Silver is a potential threat to aquatic organisms because of the increasing use of silver-based nanomaterials, which release free silver ions. The effects of silver were investigated at the transcriptome, proteome, and cellular levels of Chlamydomonas reinhardtii. The cells instigate a fast transcriptome and proteome response, including perturbations in copper transport system and detoxification mechanisms. Silver causes an initial toxic insult, which leads to a plummeting of ATP and photosynthesis and damage because of oxidative stress. In response, the cells mount a defense response to combat oxidative stress and to eliminate silver via efflux transporters. From the analysis of the perturbations of the cell's functions, we derived a detailed mechanistic understanding of temporal dynamics of toxicity and adaptive response pathways for C. reinhardtii exposed to silver.

Keywords: adaptive pathway; adverse outcome pathway; algae; systems biology; toxicity response.

Fig. 1.

Uptake and transport of silver in C. reinhardtii. Intracellular concentrations of silver (A) on exposure to 100 and 200 nM, and (Inset) to 500-nM exposure. No intracellular silver was quantifiable in C. reinhardtii exposed to 10 nM silver and is therefore not shown in the graph. Heat map of Cu transporters (B) in C. reinhardtii exposed to silver for varying durations with each box representing a protein at the transcriptome and proteome level, green being down-regulated and red up-regulated. The SD is shown in the figures. Atx1, antioxidant 1, copper chaperone; Cox2A, subunit 2A of cytochrome oxidase; Ctr1 and -3, copper transporter 1 and 3; Fox1, Ferroxidase; Pcy1, Plastocyanin. For the molecular responses, algae exposed to 500 nM Ag⁺ were not analyzed (see Methods).

Fig. 2.

Regulation of functional pathways in C. reinhardtii at physiological (A, C, E, G) and molecular (B, D, F, H) levels. Photosynthesis (A, B); lipid peroxidation compared with the control (C) and oxidative stress response (D); growth (E, F); ATP content (G) and synthesis (H). In the heat maps each square represents a protein, with green being down-regulated and red up-regulated. For the molecular responses, algae exposed to 500 nM Ag⁺ were not analyzed (see Methods).

Fig. 3.

Molecular and physiological changes of lipid synthesis in C. reinhardtii exposed to silver for 1 h. Regulation of proteins (A) and lipid bodies containing precursors of lipids stained green with Nile red (B). The chloroplasts autoflourescence is seen as red. (Scale bars in B, 10 µm.) For the molecular responses, algae exposed to 500 nM Ag⁺ were not analyzed (see Methods).

Fig. 4.

The toxicity and adaptive response pathways, as derived from linking transcriptome and proteome responses to physiological effects. (A) Schematic representation of biological pathways in C. reinhardtii affected by Ag⁺. (B) Schematic representation of the toxicity pathway. (C) Schematic representation of the adaptive-response pathway.