Acute effects of a prooxidant herbicide on the microalga Chlamydomonas reinhardtii: Screening cytotoxicity and genotoxicity endpoints.

Abstract

Since recent evidence has demonstrated that many types of chemicals exhibit oxidative and/or genotoxic potential on living organisms, reactive oxygen species (ROS) formation and DNA damage are currently the best accepted paradigms to assess the potential hazardous biological effects of a wide range of contaminants. The goal of this study was to evaluate the sensitivity of different cytotoxicity and genotoxicity responses on the model microalga Chlamydomonas reinhardtii exposed to the prooxidant herbicide paraquat. In addition to the growth endpoint, cell viability, mitochondrial membrane potential and presence of reactive oxygen species (ROS) were assayed as potential markers of cytotoxicity using flow cytometry (FCM). To study the effects of paraquat on C. reinhardtii DNA, several genotoxicity approaches were implemented for the first time in an ecotoxicological study on microalgae. Oxidative DNA base damage was analysed by measuring the oxidative DNA lesion 8-OHdG by FCM. DNA fragmentation was analysed by different methods: comet assay, and cell cycle analysis by FCM, with a particular focus on the presence of subG1-nuclei. Finally, effects on morphology of nuclei were monitored through DAPI staining. The evaluation of these endpoints showed that several physiological and biochemical parameters reacted to oxidative stress disturbances with greater sensitivity than integrative parameters such as growth rates or cell viability. The experiments revealed concentration-dependent cytotoxicity (ROS formation, depolarization of mitochondrial membrane), genotoxicity (oxidative DNA damage, DNA strand breakage, alterations in nuclear morphology), and cell cycle disturbances (subG1-nuclei, decrease of 4N population) in paraquat-treated cells. Overall, the genotoxicity results indicate that the production of ROS caused by exposure to paraquat induces oxidative DNA damage followed by DNA single- and double-strand breaks and cell cycle alterations, possibly leading to apoptosis in C. reinhardtii cells. This is supported by the observation of typical hallmarks of apoptosis, such as mitochondrial membrane depolarization, alterations in nuclear morphology and subG1 nuclei in cells exposed to the highest assayed concentrations. To our knowledge, this is the first study that provides a comprehensive analysis of oxidative DNA base damage in unicellular algal cells exposed to a prooxidant pollutant, as well as of its possible relation with other physiological effects. These results reinforce the need for additional studies on the genotoxicity of environmental pollutants on ecologically relevant organisms such as microalgae that can provide a promising basis for the characterization of potential pollutant hazards in the aquatic environment.

Toxicity of binary mixtures of pesticides to the marine microalgae Tisochrysis lutea and Skeletonema marinoi: Substance interactions and physiological impacts

“…Another consequence of photosynthesis inhibition is the potential accumulation of intracellular ROS, as previously described in many studies (Knauert and Knauer, 2008;Prado et al, 2012;Stachowski-Haberkorn et al, 2013;Esperanza et al, 2015;Dupraz et al, 2016). In the present study, significant effects, measured using either H2DCFDA or dihydroethidium, were observed after exposure to all individual substances ( Figures 3, 4 and 5A).…”

“…In addition to cell density measurements, flow cytometry can be used to analyse several morphological parameters, such as relative cell size and complexity and relative fluorescence, which is related to pigment content (Marie et al, 2005;Stachowski-Haberkorn et al, 2013;Mansano et al, 2017). Using fluorescent dyes specific to particular physiological functions, several studies have shown the usefulness of flow cytometry to indicate the toxicity of certain chemicals to microalgae (Prado et al, 2009(Prado et al, , 2012Rioboo et al, 2011;Stachowski-Haberkorn et al, 2013;Seoane et al, 2014Seoane et al, , 2017Esperanza et al, 2015;Dupraz et al, 2016;González-Pleiter et al, 2017). The most commonly investigated physiological functions in these studies are viability, general metabolic activity (often measured by non-specific esterase activity) and reactive oxygen species (ROS), whose accumulation following exposure to certain contaminants can cause oxidative stress and thus cell damage.…”

Optimization of a microplate reader method for the analysis of changes in mitochondrial membrane potential in Chlamydomonas reinhardtii cells using the fluorochrome JC-1

“…Problems with the use of JC-1 in the plant sciences may result from the fact that this dye can be hindered by the plant cell wall, which is a potential barrier for dye molecules, and fluorescence signals can be confounded by chlorophyll, which exhibits autofluorescence. So far, JC-1 staining of plant mitochondria in vivo has only been used in a few basic studies on plant cell bioenergetics, programmed cell death (PCD) investigations, or phytotoxicological experiments (Simeonova et al 2004;Poborilova et al 2013;Esperanza et al 2015). In these studies, different staining protocols and different excitation/emission wavelengths were tested, highlighting the need for this methodology to be optimized for different cell types, experimental conditions, and scientific questions.…”

Peer Review #1 of "Assessment of the ecotoxicity of urban estuarine sediment using benthic and pelagic copepod bioassays (v0.1)"

“…Other endpoints can be measured to further characterise the mechanisms of toxicity like oxidative DNA damage that can lead to genotoxicity (Esperanza et al 2015;Frenzilli et al 2009;Pellegri et al 2014). This can be measured by the comet assay, a single cell electrophoresis technique in which the DNA supercouil is relaxed and exposed electrophoresis, allowing DNA with single and double breaks to migrate towards a charged anode.…”