Non-steroidal anti-inflammatory drugs (NSAIDs), such as diclofenac (DCF), form a significant group of environmental contaminants. When the toxic effects of DCF on plants are analyzed, authors often focus on photosynthesis, while
mitochondrial respiration is usually overlooked. Therefore, an in vivo investigation of plant mitochondria functioning under DCF treatment is needed. In the present work, we decided to use the green alga Chlamydomonas reinhardtii as a model organism. Methods: Synchronous cultures of Chlamydomonas reinhardtii strain CC-1690 were treated with DCF at a concentration of 135.5 mg × L−1, corresponding to the toxicological value EC50/24. To assess the effects of short-term exposure to DCF on mitochondrial activity, oxygen consumption rate, mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (mtROS) production were analyzed. To inhibit cytochrome c oxidase or alternative oxidase activity, potassium cyanide (KCN) or salicylhydroxamic acid (SHAM) were used, respectively. Moreover, the cell’s structure organization was analyzed using confocal microscopy and transmission electron microscopy.

These results published in PeerJ 12:e18005 DOI 10.7717/peerj.18005

Population density and cell volume: 

The number and volume of cells were estimated using an electronic particle counter (Beckman Coulter Z2) run by dedicated software. The obtained data ranged from 1535600 to 2037150 for the number of cells measurement and from 68.25 to 539.61 for the volume of cells measurement  

Mitochondrial respiration, mitochondrial membrane potential (MMP) and mitochondrial ROS (mtROS) assessment:

Oxygen consumption rate was determined with a Clark-type oxygen electrode after 6h of DCF treatment (6h after the start of the light period of the cell cycle). Before measurement, the cultures were darkened for 30 min to stop photosynthesis and all further steps were performed under dim light or in darkness. 1 mL of cell suspension (about 1.5×10*6 cells/mL) was sampled directly from the culture vessel and placed in a measuring chamber of Oxyview. The cell suspension was stirred continuously, and the oxygen consumption rate measurements were carried out at 30°C in total darkness. The oxygen consumption rate was expressed in nmol O2 and recalculated per 1 million cells (nmol O2/L ×10*6 cells/min). The obtained data ranged from 13.31 to 32.90 for the oxygen consumption rate measurement.
Cells suspension (15 mL) taken from the culture vessel was placed in a black Falcon tube and incubated with 0.5 mM KCN 10 or with 3 mM SHAM 30 for 5 min at 30°C. After the incubation oxygen consumption rate was measured as described above.

Stock solutions of the fluorochromes were prepared in DMSO. The final fluorochrome concentration in the incubation mixture was 0.5 µM for Mito Tracker™ Orange (MtO) and 3 µM for JC-1, respectively. The final concentration of DMSO in the incubation mixture did not exceed 0.1% (v/v). 
Before the sampling, the culture vessels were darkened for 30 min to stop photosynthetic processes. MMP and mtROS were assessed according to, with modification: cells were pelleted by centrifugation for 5 min at 460 g in black test tubes and resuspended in HSM (heated to 30°C) to obtain 5×106 cells×mL-1 for MtO-staining and to obtain 1×106 cells×mL-1 for JC-1-staining, respectively. The measurement of MMP or mtROS was performed after incubation with the appropriate fluorochrome (20 min for JC-1 and 45 min for MtO) in black 98-well plates, using the spectrofluorometer Varioskan Flash Microplate Reader (Thermo Fisher Scientific, USA). The excitation wavelength for JC-1 was 488 nm and waves of emission were 538 and 596 nm (for monomers and oligomers of fluorochrome, respectively). The excitation wavelength for MTO was 551 nm and the wave of emission was 576 nm.The obtained data ranged from 8.17 to 24.53 and from 0.72 to 1.74 for the MMP or mtROS measurement, respectively.

For confocal microscopy examination, the cell suspensions were centrifuged, and the pellet (about 1 × 106 cells × mL−1) was resuspended in a small volume of HSM and incubated with 3 µM JC-1. Cells stained with JC-1 were fixed with 4% paraformaldehyde in HEPES buffer for 1 h at RT. Cells were visualized with Leica STELLARIS 5 WLL confocal microscopy with Lightning module using FITC/TRITC ex/em. The photos presented are maximum projections taken from z-stacks with Lightning deconvolution combining signals from the green-fluorescent JC-1 monomers (absorption/ emission maxima ~514/529 nm) and the red-fluorescent J-aggregates (emission maximum 590 nm). The mean fluorescence intensity of 10 representative cells of each experimental variant was quantified in triplicate using Leica Application Suite X. To visualize both J-monomers and J-aggregates combined with autofluorescence of chlorophyll in untreated and DCF-treated cells, 488 nm laser line excitation was used. Monomers were visualized at 536 nm, J-aggregates at 581 nm and chlorophyll at 676 nm emission wavelength for pinhole Airy calculation. Presented photos are maximum intensity projections of z-stacks taken with Lightning deconvolution module.

Electron microscope examination:

For electron microscope examination, cells were sampled after 24h of incubation with DCF in a final concentration of 135.5 mg/L, or without toxicant (the density of cells was about 8-10×10*6 cells/mL). Cultures were fixed overnight with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.2. Post-fixed with 2% osmium tetroxide (Agar) in 0.1 M sodium cacodylate buffer. Then dehydrated with increasing concentration of alcohol. After that infiltrated and embedded in Epon 812 resin. Ultrasections (approximately 65 nm) were cut on Leica UC7. Sections were stained with uranyl acetate and lead citrate. They were examined using a Tecnai Spirit BioTWIN transmission electron microscope (FEI).