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1.
Ecotoxicol Environ Saf ; 208: 111630, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33396150

RESUMO

The non-steroidal anti-inflammatory drug diclofenac (DCF) is one of the commonly used and frequently detected drugs in water bodies, and several studies indicate its toxic effect on plants and algae. Studies performed with asynchronous Chlamydomonas reinhardtii cultures indicated that DCF inhibit the growth of population of the algae. Here, a synchronous population of C. reinhardtii, in which all cells are in the same developmental phase, is used. Following changes in cells size, photosynthetic activity and gene expression, we could compare, at the level of single cell, DCF-mediated effects with the effects caused by atrazine, a triazine herbicide that inhibits photosynthesis and triggers oxidative stress. Application of DCF and atrazine at the beginning of the cell cycle allowed us to follow the changes occurring in the cells in the subsequent stages of their development. Synchronized Chlamydomonas reinhardtii cultures (strain CC-1690, wild type) were exposed to diclofenac sodium salt (135 mg/L) or atrazine (77.6 µg/L). The cell suspension was sampled hourly (0-10 h) in the light period of the cell cycle to determine cell number and volume, photosynthetic pigment content, chlorophyll a fluorescence (OJIP test) in vivo, and selected gene expression (real-time qPCR), namely psbA, psaA, FSD1, MSD3 and APX1. The two toxicants differently influenced C. reinhardtii cells. Both substances decreased photosynthetic "vitality" (PI - performance index) of the cells, albeit for different reasons. While atrazine significantly disrupted the photosynthetic electron transport, resulting in excessive production of reactive oxygen species (ROS) and limited cell growth, DCF caused silencing of photosystem II (PSII) reaction centers, transforming them into "heat sinks", thus preventing significant ROS overproduction. Oxidative stress caused by atrazine was the probable reason for the rapid appearance of phytotoxic action soon after entering the cells, while the effects of DCF could only be seen several hours after treatment. A comparison of DCF-caused effects with the effects caused by atrazine led us to conclude that, although DCF cannot be regarded as typical photosynthetic herbicide, it exhibits an algicidal activity and can be potentially dangerous for aquatic plants and algae.


Assuntos
Chlamydomonas reinhardtii/fisiologia , Diclofenaco/toxicidade , Herbicidas/toxicidade , Fotossíntese/efeitos dos fármacos , Atrazina/metabolismo , Atrazina/toxicidade , Chlamydomonas reinhardtii/efeitos dos fármacos , Clorofila A/metabolismo , Clorófitas/metabolismo , Diclofenaco/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Herbicidas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Complexo de Proteína do Fotossistema II/metabolismo , Espécies Reativas de Oxigênio/metabolismo
2.
Ecotoxicol Environ Saf ; 208: 111646, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33396166

RESUMO

Contamination of aquatic ecosystems linked to anthropogenic activity is currently a major concern; therefore, ecotoxicological studies are needed to assess its effect on organisms. The main objective of this study was to investigate the effects of different pollutants on microalgae in search of sensitive biomarkers that can promote a common cytotoxic response regardless of the contaminant. Cultures of the freshwater microalga Chlamydomonas reinhardtii were exposed for 24 h to four chemicals, three emerging pollutants (benzophenone-3, bisphenol A and oxytetracycline) and one priority substance (atrazine). A cytometric panel was carried out to assess toxicity biomarkers including cellular growth, inherent cell properties, viability, vitality, cytoplasmic membrane potential and ROS levels. Lipid peroxidation, photosynthetic efficiency and transcriptional responses of photosynthesis- and oxidative stress-related genes using RT-qPCR were also studied. Some toxicity responses showed a similar pattern; a decrease in growth rate, vitality and photosynthetic efficiency and an increase in autofluorescence and in the number of cells with depolarised cytoplasmic membrane and were found for all chemicals tested. However, ATZ and OTC provoked a decrease in cell size, whereas BP-3 and BPA caused an increase in cell size, intracellular complexity and ROS levels and a decrease in cell viability. Assayed pollutants generally promoted an overexpression of genes related to cellular antioxidant defence system and a subexpression of photosynthesis-related genes. In addition to the traditional growth endpoint, cell vitality, autofluorescence and gene expression of catalase, glutathione peroxidase and Fe-superoxide dismutase were significantly affected for all chemicals tested, showing a common cytotoxic response. Among the tested substances, BP-3 provoked the strongest cytotoxic alterations on this microalga, pointing out that some emerging contaminants could be more harmful to organisms than priority pollutants.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Chlamydomonas reinhardtii/efeitos dos fármacos , Microalgas/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Antioxidantes/metabolismo , Chlamydomonas reinhardtii/metabolismo , Ecotoxicologia , Biomarcadores Ambientais/efeitos dos fármacos , Água Doce/química , Água Doce/microbiologia , Microalgas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos
3.
Aquat Toxicol ; 231: 105711, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33338702

RESUMO

Phytoplankton are characterized by a great phenotypic plasticity and amazing morphological variability, both playing a primary role in the acclimation to changing environments. However, there is a knowledge gap concerning the role of algal morphological plasticity in stress responses and acclimation to micropollutants. The present study aims at examining palmelloid colony formation of the green alga Chlamydomonas reinhardtii upon micropollutants exposure. Cells were exposed to four micropollutants (MPs, copper, cadmium, PFOS and paraquat) with different modes of action for a duration of 72 h. Effects of MPs on palmelloid formation, growth and physiological traits (chlorophyll fluorescence, membrane integrity and oxidative stress) were monitored by flow cytometry and fluorescence microscopy. Palmelloid formation was observed upon treatment with the four micropollutants. Number of palmelloid colonies and their size were dependent on MP concentration and exposure duration. Cells reverted to their unicellular lifestyle when colonies were harvested and inoculated in fresh medium indicating that palmelloid formation is a plastic response to micropollutants. No physiological effects of these compounds were observed in cells forming palmelloids. Palmelloid colonies accumulated lower Cd concentration than unicellular C. reinhardtii suggesting that colony formation protects the cells from MPs stress. The results show that colony formation in Chlamydomonas reinhardtii is a stress response strategy activated to face sub-lethal micropollutant concentrations.


Assuntos
Aclimatação/fisiologia , Adaptação Fisiológica , Chlamydomonas reinhardtii/anatomia & histologia , Poluentes Químicos da Água/toxicidade , Cádmio/toxicidade , Chlamydomonas reinhardtii/citologia , Chlamydomonas reinhardtii/efeitos dos fármacos , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Cobre/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Paraquat/toxicidade , Fitoplâncton
4.
Aquat Toxicol ; 224: 105502, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32480176

RESUMO

The present study examined the effect of titanium dioxide nanoparticles (nanoTiO2) and mercury (Hg) compounds on the green alga, Chlamydomonas reinhardtii. Mixtures containing nanoTiO2 of different primary sizes (5 nm, 15 nm and 20 nm), inorganic Hg (IHg) or monomethyl Hg (CH3Hg+, MeHg) were studied and compared with individual treatments. Oxidative stress and membrane damage were examined. Stability of nanoTiO2 materials in terms of hydrodynamic size and surface charge as well as Hg adsorption on different nanoTiO2 materials were characterized. The uptake of Hg compounds in the absence and presence of nanoTiO2 was also quantified. Results show that increasing concentrations of nanoTiO2 with different primary size diminished oxidative stress and membrane damage induced by high concentrations of IHg or MeHg, due to the adsorption of Hg on the nanoTiO2 aggregates and consequent decrease of cellular Hg concentrations. The observed alleviation effect of nanoTiO2 materials on Hg biouptake and toxicity was more pronounced for the materials with smaller primary size. IHg adsorbed onto the nanoTiO2 materials to a higher extent than MeHg. The present study highlights that the effects of contaminants are modulated by the co-existing engineered nanomaterials; therefore, it is essential to get a better understanding of their combined effect in the environment.


Assuntos
Chlamydomonas reinhardtii/efeitos dos fármacos , Mercúrio/toxicidade , Compostos de Metilmercúrio/toxicidade , Nanopartículas/química , Titânio/farmacologia , Poluentes Químicos da Água/toxicidade , Adsorção , Chlamydomonas reinhardtii/metabolismo , Mercúrio/metabolismo , Compostos de Metilmercúrio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Propriedades de Superfície , Titânio/química , Poluentes Químicos da Água/metabolismo
5.
Ecotoxicol Environ Saf ; 197: 110609, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32302859

RESUMO

Bisphenol A (BPA) is used throughout the world and it could enter aquatic ecosystems causing harmful effects on humans, animals and plants. The current study relies on the investigation of the toxicity of this emerging pollutant on two freshwater species from different trophic levels: the microalga Chlamydomonas reinhardtii and the clam Corbicula fluminea. After 96 h of exposure to several concentrations of BPA, the growth of C. reinhardtii was affected, being the 96 h-EC50 value for growth 30 mg L-1. The toxicity and bioaccumulation of 30 mg L-1 BPA in microalgae after 24 h of exposure were studied. Several cytotoxicity biomarkers such as vitality, oxidative stress and cytoplasmic membrane potential were altered in exposed cells and microalgae accumulated 0.16 pg BPA cell-1. Regarding C. fluminea, four treatments were established: control without BPA (C); BPA in the food (microalgae pre-exposed for 24 h to 30 mg L-1) (M); BPA in the water (7.5 mg L-1) (W); BPA in both food and water (M + W). After one month of exposure, treated bivalves showed a significantly decrease in the filtration rate and increased lipid peroxidation levels, indicating fitness reduction and oxidative damage. Furthermore, the activities of catalase, glutathione reductase, Se-dependent and total glutathione peroxidase enzymes increased significantly in W and M + W treatments with respect to the control. Clams of the M + W treatment were the most affected, indicating that the little amount of BPA bioaccumulated by microalgae could increase the damage. Emerging contaminants may accumulate in several organisms, such as microalgae, and could have negative impacts on ecosystems.


Assuntos
Compostos Benzidrílicos/toxicidade , Chlamydomonas reinhardtii/efeitos dos fármacos , Corbicula/efeitos dos fármacos , Fenóis/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Compostos Benzidrílicos/farmacocinética , Catalase/metabolismo , Chlamydomonas reinhardtii/metabolismo , Corbicula/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Microalgas/efeitos dos fármacos , Microalgas/metabolismo , Estresse Oxidativo , Fenóis/farmacocinética
6.
Ecotoxicol Environ Saf ; 196: 110552, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32259759

RESUMO

Nowadays, numerous studies have focused on the newly developed technologies for the thorough removal of tetracyclines (TCs). However, it is often ignored that the parent TCs have limited stability in aquatic environments. Thus, this study selected green alga Chlamydomonas reinhardtii with high chlorophyll content to rapidly degrade chlortetracycline (CTC) into products with low toxicity. As the results shown, the half-life times of CTC (1 × 10-6 mol/L) decreased from 10.35 h to 2.55 h by the presence of C. reinhardtii at 24±1 °C with 12/12 h dark/light cycle. The main transformation products were iso-chlortetracycline (ICTC), 4-epi-iso-chlortetracycline (EICTC), and other degradation products with lower molecular weight. The toxicity evaluation shows that the negative effects of CTC on growth rate and soluble protein content of green algae were significantly alleviated after the enhanced degradation treatment, while the generation of reactive oxygen species (ROS) and antioxidant response in algal cells returned to normal levels. The chlorophyll of algae played an important role of photosensitizer, which catalyzed the photo-induced electron/energy transfer of CTC degradation. The ROS generation of algae also was also inseparable from the enhanced degradation of CTC, especially when the chlorophyll was damaged at the high CTC concentration. Based on these results, we can better select suitable algal species to further strengthen the degradation of antibiotics and effectively reduce the environmental risk of CTC in aqueous system.


Assuntos
Antibacterianos/análise , Chlamydomonas reinhardtii/metabolismo , Clortetraciclina/análise , Poluentes Químicos da Água/análise , Antibacterianos/metabolismo , Antibacterianos/toxicidade , Antioxidantes/metabolismo , Biodegradação Ambiental , Chlamydomonas reinhardtii/efeitos dos fármacos , Clorofila/metabolismo , Clorófitas/metabolismo , Clortetraciclina/metabolismo , Clortetraciclina/toxicidade , Inativação Metabólica/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Poluentes Químicos da Água/metabolismo , Poluentes Químicos da Água/toxicidade
7.
Photosynth Res ; 143(3): 275-286, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31897856

RESUMO

The aim of this work was a comparative study of S-repleted and S-depleted photoautotrophic cultures of Chlamydomonas reinhardtii under aerobic and anoxic conditions with the main focus on PSII activity. For that we used photobioreactor with short light path connected on-line to PAM fluorometer and cultivated microalgae in twice concentrated HS medium to avoid any uncontrolled limitation by mineral elements. Photoautotrophic cultures grown under Ar + CO2 gas mixture did not reach the same Chl (a + b) concentration as control culture (grown under air + CO2). At pO2 40% of air saturation (96 µM O2), the actual quantum yield of PSII started to decrease. Under microaerobic conditions when cultures stopped growing, the most significant changes in PSII function were observed. Maximum quantum yield Fv/Fm decreased significantly along with performance index, PIabs. It was accompanied by increase of fluorescence at J point, Vj. Results indicate that microaerobic conditions are stressful for photoautotrophic cultures. Photoautotrophic cultures of microalgae under S-deprivation in aerobic or anaerobic conditions showed similar behavior as photoheterotrophic ones described earlier. However, photoautotrophic cultures during anaerobiosis establishment did not show sharp "switch off" effect of actual quantum yield. We show also that S-deprivation under air or argon as well as the growth under Ar + CO2 cause significant increase of initial rise of fluorescence, which indicates that PSII and oxygen-evolving complex might be disintegrated.


Assuntos
Processos Autotróficos , Chlamydomonas reinhardtii/metabolismo , Hidrogênio/metabolismo , Processos Fototróficos , Enxofre/deficiência , Anaerobiose/efeitos dos fármacos , Argônio/farmacologia , Atmosfera , Processos Autotróficos/efeitos dos fármacos , Técnicas de Cultura Celular por Lotes , Dióxido de Carbono/farmacologia , Chlamydomonas reinhardtii/efeitos dos fármacos , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Fluorometria , Oxigênio/metabolismo , Fotobiorreatores/microbiologia , Processos Fototróficos/efeitos dos fármacos
8.
Biochem Biophys Res Commun ; 523(4): 880-886, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-31955886

RESUMO

Metal homeostasis is essential cellular progress for cell growth. Metal ion transporters play important roles in the first line of defense to cellular metal homeostasis perturbations. NRAMP transporter family was one of the most important classes in plant cells. However, functions and substrate specificities of the NRAMP family remain unknown in Chlamydomonas reinhardtii, a model unicellular plant. In this study, we identified CrNRAMP1 as an important transporter involved in zinc and cobalt transport. Heterologous and homologous functional analyses of CrNRAMP1 showed that CrNRAMP1 plays important roles in zinc and cobalt homeostasis. The expression of CrNRAMP1 correlated with zinc or cobalt concentrations, but excluding cadmium. These results help to understand the functions and specificities of NRAMP family members in C. reinhardtii.


Assuntos
Proteínas de Algas/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cobalto/metabolismo , Zinco/metabolismo , Transporte Biológico/efeitos dos fármacos , Cádmio/toxicidade , Cátions , Chlamydomonas reinhardtii/efeitos dos fármacos , Cobalto/toxicidade , Mutação/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Zinco/toxicidade
9.
Chemosphere ; 247: 125935, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31978663

RESUMO

In this study, the effect of Chlamydomonas reinhardtii on the fate of CuO nanoparticles (CuO-NPs) in aquatic environment were investigated in terms of the colloidal stability, the free Cu2+ releasing, extracellular adsorption Cu (Cuex) and intracellular assimilation Cu (Cuin). The results showed that, with the increasing microalgal density, the absolute value of zeta potential of CuO-NPs decreased and the mean hydrodynamic diameter (MHD) became larger, leading to a better aggregation and settling behavior of CuO-NPs. The microalgae also promoted the free Cu2+ releasing, however, inhibited adsorption and assimilation of metal nanoparticles (MNPs) into microalgal cells, resulting in the reduction of the Cuex and Cuin per microalgal cell. The phenomenon was probably due to the reduced chance of contact between microalgae and MNPs. The internalization of CuO-NPs was also observed in microalgal cells by high resolution transmission electron microscope (HRTEM). Furthermore, the results of fast fourier transform (FFT)/inversed FFT (IFFT) analysis indicated that the CuO-NPs was reduced to Cu2O-NPs in the microalgae cells. The above results suggested that the microalgae can significantly affect the fate of MNPs, and subsequently, influencing the bioavailability and toxicity of MNPs in the aquatic environment.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Cobre/metabolismo , Nanopartículas/metabolismo , Poluentes Químicos da Água/metabolismo , Adsorção , Disponibilidade Biológica , Chlamydomonas reinhardtii/efeitos dos fármacos , Nanopartículas Metálicas , Microalgas , Microscopia Eletrônica de Transmissão
10.
Ecotoxicol Environ Saf ; 191: 110231, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31981954

RESUMO

Cadmium (Cd), a common environmental toxic contaminant, is easily accumulated in living organisms, leading to numerous harmful effects. Chlamydomonas reinhardtii, a unicellular eukaryotic green algae strain, is a very suitable candidate for bioremediation of Cd-contaminated water. However, for the poor resistance to Cd, application of C. reinhardtii was restricted and genes mediating Cd tolerance in C. reinhardtii remain unclear. In this paper, adaptive laboratory evolution was performed with algae constant exposure to Cd over 420-day at environmentally relevant concentrations to select C. reinhardtii strains with high tolerance to Cd. Physiological indicators, such as cell proliferation, photosynthetic pigment contents and photosynthetic activity of photosystem were detected to evaluate the Cd tolerance of selected algae strain ALE0.5. Then, whole-genome re-sequencing and transcriptome were applied to identify the genes related to Cd tolerance. Genes involved in photosynthesis (PSBP1), glutathione metabolism (CHLREDRAFT_167073, GPX5) and calcium transport (CHLREDRAFT_189266, CHLREDRAFT_191203, CHLREDRAFT_187187, CSE1) were related to Cd tolerance in C. reinhardtii. This study provides a basis for obtaining transgenic C. reinhardtii strains with high Cd tolerance used for bioremediation of Cd pollution in the future.


Assuntos
Cádmio/toxicidade , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Poluentes Químicos da Água/toxicidade , Biodegradação Ambiental , Cálcio/metabolismo , Chlamydomonas reinhardtii/efeitos dos fármacos , Glutationa/metabolismo , Redes e Vias Metabólicas/genética , Fotossíntese/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Sequenciamento Completo do Genoma
11.
Chemosphere ; 238: 124562, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31442774

RESUMO

Superparamagnetic iron oxide nanoparticles (SPION) have been widely studied for different biomedical and environmental applications. In this study we evaluated the toxicity and potential alterations of relevant physiological parameters caused to the microalga Chlamydomonas reinhardtii (C. reinhardtii) upon exposure to SPION. The results showed dose-dependent toxicity. A mechanistic study combining flow cytometry and physiological endpoints showed a toxic response consisting of a decrease in metabolic activity, increased oxidative stress and alterations in the mitochondrial membrane potential. Additionally, and due to the light absorption of SPION suspensions, we observed a significant shading effect, causing a marked decrease in photosynthetic activity. In this work, we demonstrated for the first time, the internalization of SPION by endocytosis in C. reinhardtii. These results demonstrated that SPION pose a potential risk for the environment if not managed properly.


Assuntos
Chlamydomonas reinhardtii/efeitos dos fármacos , Nanopartículas de Magnetita/toxicidade , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microalgas/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
12.
J Exp Bot ; 70(21): 6321-6336, 2019 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-31504725

RESUMO

The redox state of the plastoquinone (PQ) pool in sulfur-deprived, H2-producing Chlamydomonas reinhardtii cells was studied using single flash-induced variable fluorescence decay kinetics. During H2 production, the fluorescence decay kinetics exhibited an unusual post-illumination rise of variable fluorescence, giving a wave-like appearance. The wave showed the transient fluorescence minimum at ~60 ms after the flash, followed by a rise, reaching the transient fluorescence maximum at ~1 s after the flash, before decaying back to the initial fluorescence level. Similar wave-like fluorescence decay kinetics have been reported previously in anaerobically incubated cyanobacteria but not in green algae. From several different electron and proton transfer inhibitors used, polymyxin B, an inhibitor of type II NAD(P)H dehydrogenase (NDA2), had the effect of eliminating the fluorescence wave feature, indicating involvement of NDA2 in this phenomenon. This was further confirmed by the absence of the fluorescence wave in the Δnda2 mutant lacking NDA2. Additionally, Δnda2 mutants have also shown delayed and diminished H2 production (only 23% if compared with the wild type). Our results show that the fluorescence wave phenomenon in C. reinhardtii is observed under highly reducing conditions and is induced by the NDA2-mediated electron flow from the reduced stromal components to the PQ pool. Therefore, the fluorescence wave phenomenon is a sensitive probe for the complex network of redox reactions at the PQ pool level in the thylakoid membrane. It could be used in further characterization and improvement of the electron transfer pathways leading to H2 production in C. reinhardtii.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Hidrogênio/metabolismo , Proteínas de Algas/metabolismo , Anaerobiose , Chlamydomonas reinhardtii/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Fluorescência , Gramicidina/farmacologia , Cinética , Luz , Mitocôndrias/metabolismo , Mutação/genética , Fotossíntese/efeitos dos fármacos , Complexo de Proteína do Fotossistema I/metabolismo , Plastoquinona/metabolismo
13.
Cells ; 8(7)2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31319624

RESUMO

DNA damage is a ubiquitous threat endangering DNA integrity in all living organisms. Responses to DNA damage include, among others, induction of DNA repair and blocking of cell cycle progression in order to prevent transmission of damaged DNA to daughter cells. Here, we tested the effect of the antibiotic zeocin, inducing double stranded DNA breaks, on the cell cycle of synchronized cultures of the green alga Chlamydomonas reinhardtii. After zeocin application, DNA replication partially occurred but nuclear and cellular divisions were completely blocked. Application of zeocin combined with caffeine, known to alleviate DNA checkpoints, decreased cell viability significantly. This was probably caused by a partial overcoming of the cell cycle progression block in such cells, leading to aberrant cell divisions. The cell cycle block was accompanied by high steady state levels of mitotic cyclin-dependent kinase activity. The data indicate that DNA damage response in C. reinhardtii is connected to the cell cycle block, accompanied by increased and stabilized mitotic cyclin-dependent kinase activity.


Assuntos
Bleomicina/toxicidade , Chlamydomonas reinhardtii/efeitos dos fármacos , Citostáticos/toxicidade , Mutagênicos/toxicidade , Cafeína/farmacologia , Pontos de Checagem do Ciclo Celular , Chlamydomonas reinhardtii/genética , Quinases Ciclina-Dependentes/metabolismo , Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA de Plantas/efeitos dos fármacos
14.
Aquat Toxicol ; 214: 105259, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31352075

RESUMO

The potential of using gene expression signature as a biomarker of toxicants exposure was explored in the microalga Chlamydomonas reinhardtii exposed 2 h to mercury (Hg) as inorganic mercury (IHg) and methyl mercury (MeHg) in presence of copper (Cu) and Suwannee River Humic Acid (SRHA). Total cellular Hg (THg = IHg + MeHg) decreased in presence of SRHA for 0.7 nM IHg and 0.4 nM MeHg, but increased for 70 nM IHg exposure. In mixtures of IHg + MeHg and (IHg or MeHg) + Cu, SRHA decreased THg uptake, except for 0.7 nM IHg + 0.4 nM MeHg which was unchanged (p-value>0.05). In the absence of SRHA, 0.5 µM Cu strongly decreased intracellular THg concentration for 70 nM IHg, while it had no effect for 0.7 nM IHg and 0.4 nM MeHg. The expression of single transcripts was not correlated with measured THg uptake, but a subset of 60 transcripts showed signatures specific to the exposed metal(s) and was congruent with exposure concentration. Notably, the range of fold change values of this subset correlated with THg bioaccumulation with a two-slope pattern in line with [THg]intra/[THg]med ratios. Gene expression signature seems a promising approach to complement chemical analyses to assess bioavailability of toxicants in presence of other metals and organic matter.


Assuntos
Chlamydomonas reinhardtii/genética , Exposição Ambiental , Perfilação da Expressão Gênica , Mercúrio/toxicidade , Compostos de Metilmercúrio/toxicidade , Animais , Disponibilidade Biológica , Cádmio/toxicidade , Chlamydomonas reinhardtii/efeitos dos fármacos , Chlamydomonas reinhardtii/metabolismo , Feminino , Substâncias Húmicas/análise , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NAD/metabolismo , Oncorhynchus mykiss/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Temperatura , Poluentes Químicos da Água/toxicidade
15.
Proc Natl Acad Sci U S A ; 116(33): 16631-16640, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31358635

RESUMO

Photosystem II (PSII) undergoes frequent photooxidative damage that, if not repaired, impairs photosynthetic activity and growth. How photosynthetic organisms protect vulnerable PSII intermediate complexes during de novo assembly and repair remains poorly understood. Here, we report the genetic and biochemical characterization of chloroplast-located rubredoxin 1 (RBD1), a PSII assembly factor containing a redox-active rubredoxin domain and a single C-terminal transmembrane α-helix (TMH) domain. RBD1 is an integral thylakoid membrane protein that is enriched in stroma lamellae fractions with the rubredoxin domain exposed on the stromal side. RBD1 also interacts with PSII intermediate complexes containing cytochrome b 559 Complementation of the Chlamydomonas reinhardtii (hereafter Chlamydomonas) RBD1-deficient 2pac mutant with constructs encoding RBD1 protein truncations and site-directed mutations demonstrated that the TMH domain is essential for de novo PSII assembly, whereas the rubredoxin domain is involved in PSII repair. The rubredoxin domain exhibits a redox midpoint potential of +114 mV and is proficient in 1-electron transfers to a surrogate cytochrome c in vitro. Reduction of oxidized RBD1 is NADPH dependent and can be mediated by ferredoxin-NADP+ reductase (FNR) in vitro. We propose that RBD1 participates, together with the cytochrome b 559, in the protection of PSII intermediate complexes from photooxidative damage during de novo assembly and repair. This role of RBD1 is consistent with its evolutionary conservation among photosynthetic organisms and the fact that it is essential in photosynthetic eukaryotes.


Assuntos
Membranas Intracelulares/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Rubredoxinas/metabolismo , Tilacoides/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Chlamydomonas reinhardtii/efeitos dos fármacos , Chlamydomonas reinhardtii/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/ultraestrutura , Ferro/farmacologia , Modelos Biológicos , Oxirredução , Domínios Proteicos , Rubredoxinas/química , Tilacoides/efeitos dos fármacos , Tilacoides/ultraestrutura
16.
Sci Total Environ ; 692: 233-239, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31349164

RESUMO

Because of their biocide properties, silver nanoparticles (AgNPs) are present in numerous consumer products. The biocidal properties of AgNPs are due to both the interactions between AgNP and cell membranes and the release of dissolved silver (Ag+). Recent studies emphasized the role of different nanoparticle coatings in complexing and storing Ag+. In this study, the availability of dissolved silver in the presence of algae was assessed for three AgNPs with different silver contents (59%, 34% and 7% of total Ag), silver core sizes and casein shell thicknesses. The impact of ionic silver on the photosynthetic yield of Chlamydomonas reinhardtii was used as a proxy to estimate the amount of ionic silver toxically active during in vivo assays. The results showed that cysteine, a strong silver ligand, mitigated the toxicity of AgNPs in all cases, demonstrating the key role of Ag+ in this toxicity. The results showed that the AgNPs presenting an intermediate level of silver (34%) were 10 times more effective in terms of total mass (EC50 ten times smaller) than those presenting more (59%) or less (7%) silver. The higher toxicity was due to the higher release of Ag+ under biotic conditions due to the high surface/mass ratio of the nanoparticle silver core. Protein shells played a minor role in altering the availability of Ag+, probably acting as intermediate reservoirs. This study highlighted the utility of a very sensitive biological endpoint (i.e., algal photosynthesis) for the optimization of ionic silver delivery by nanomaterials.


Assuntos
Chlamydomonas reinhardtii/efeitos dos fármacos , Nanopartículas Metálicas/efeitos adversos , Fotossíntese/efeitos dos fármacos , Prata/efeitos adversos , Disponibilidade Biológica , Chlamydomonas reinhardtii/metabolismo , Cisteína/administração & dosagem , Relação Dose-Resposta a Droga , Íons/efeitos adversos , Substâncias Protetoras/administração & dosagem
17.
Nanotoxicology ; 13(9): 1149-1160, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31284796

RESUMO

Core-shell silver nanoparticles (NPs) consisting of an inner Ag core and successive layers of Au and Ag (Ag@Au@Ag) were used to measure the simultaneous association of Ag NPs and ionic Ag by the green alga Chlamydomonas (C.) reinhardtii. Dissolution of the inner Ag core was prevented by a gold (Au) layer, while the outer Ag layer was free to dissolve. In short-term experiments, we exposed C. reinhardtii to a range of environmentally realistic Ag concentrations added as AgNO3 or as NPs. Results provide three lines of evidence for the greater cell-association of NPs compared to dissolved Ag over the concentration range tested, assuming that cell-association comprises both uptake and adsorption. First, the cell-association rate constants (kuw) for total Ag (AgNP+D), NPs (AgNP) and AuNP were similar and 2.2-fold higher than the one from AgD exposure, suggesting predominant association of the particles over the dissolved form. Second, model calculations based on Ag fluxes suggested that only 6-33% of algal burden was from AgD. Third, the significantly lower AgNP/Au ratio measured with the algae after exposure (2.1 ± 0.1) compared to the AgNP/Au ratio of the NPs in the media (2.47 ± 0.05) suggests cell-association of NPs depleted in Ag. Core-shell NPs provide an innovative tool to understand NP behavior and to directly delineate Ag accumulation from ion and NPs in aquatic systems.


Assuntos
Chlamydomonas reinhardtii/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Ouro/química , Nanopartículas Metálicas/química , Prata/química , Solubilidade
18.
Anal Chem ; 91(15): 9590-9598, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31269797

RESUMO

The complementary use of single cell atomic mass spectrometry (MS) and ambient molecular MS allowed for the in-depth study of arsenate uptake by Chlamydomonas reinhardtii cells and of the effect this toxic metalloid species has on their lipid profile. Compared to conventional inductively coupled plasma mass spectrometry (ICP-MS) analysis, in which case hundreds of thousands of cells are digested and then analyzed, it is demonstrated that single cell (SC) ICP-MS provides uptake data that are potentially of greater biological relevance. This includes the arsenic mass distribution within the cell population, which fits to a log-normal probability function, the most frequently contained arsenic mass within the cells (1.5-1.8 fg As per cell), and the mean arsenic uptake value (ranging from 2.7 to 4.1 fg As per cell for the three arsenate incubation concentrations, that is, 15, 22.5, and 30 µg As per mL) derived from the log-normal arsenic mass distribution within the cell population. The SC approach also allows for differentiating the arsenic present in and/or adsorbed on the cells, from the arsenic present in the extracellular solution, in a single analysis. In a similar fashion, ambient molecular MS in the form of desorption easy ambient sonic spray ionization (EASI) -MS was used to rapidly profile cell membrane lipids from cells spotted directly on a glass slide. EASI-MS analysis revealed that cells grown in the presence of increasing concentrations of arsenate exhibited changes in the degree of saturation of their membrane lipids, as was observed by the increasing intensity ratio of lipids with less unsaturated acyl chains to the same type of lipids with more unsaturated fatty acid chains. Thus, indicating "homeoviscous adaptation" of extraplastidial and thylakoid cell membranes, induced by the presence of arsenate.


Assuntos
Arseniatos/metabolismo , Arseniatos/toxicidade , Chlamydomonas reinhardtii/efeitos dos fármacos , Chlamydomonas reinhardtii/metabolismo , Lipídeos/química , Espectrometria de Massas/métodos , Transporte Biológico , Metabolismo dos Lipídeos/efeitos dos fármacos , Análise de Célula Única/métodos
19.
Acta Biochim Pol ; 66(3): 257-262, 2019 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-31279329

RESUMO

Chlamydomonas reinhardtii (WT 2137) P. A. Dang. (Volvocales, Chlorophyceae) is a green microalgae serving as a suitable model in scientific research and a promising industrial biotechnology platform for production of biofuel, hydrogen and recombinant proteins. Fullerenes (C60) are allotropic carbon nanoparticles discovered in 1985 and used in biomedical studies since the early 1990s, when water solubilization methodologies were developed. Recently, surface-modified hydroxylated derivatives of fullerenes were proven to enhance algal growth and drought tolerance in plants. Here, a novel type of water-soluble [60]fullerene derivative with 12 glycine residues (GF) has been synthesized and tested for acute toxicity (up to 50 µg/ml) and as a potential biostimulant of algal growth. The effects of GF on pigment composition and growth rate of Chlamydomonas reinhardtii were systematically investigated. Our results suggest that GF was not toxic, and no negative change in the pigment content and no stress symptoms were observed. No changes in the photosynthetic parameters based on the fluorescence of chlorophyll a in Photosystem II (NPQ, Fv/Fm, Fv/F0, PI and RC/ABS) were observed. The GF had no effect on cell size and growth rate. At a concentration of 20 µg/ml, GF stimulated chlorophyll accumulation in 3-day-old cultures.


Assuntos
Chlamydomonas reinhardtii/efeitos dos fármacos , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Clorofila A/metabolismo , Clorofila/metabolismo , Fulerenos/farmacologia , Biocombustíveis , Biotecnologia/métodos , Carotenoides/metabolismo , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Fulerenos/química , Hidrogênio , Nanopartículas/química , Fotossíntese/efeitos dos fármacos , Proteínas Recombinantes , Solubilidade , Água/química
20.
Biochim Biophys Acta Bioenerg ; 1860(7): 582-590, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31226314

RESUMO

The lumenal carbonic anhydrase (CA) CAH3 from green alga Chlamydomonas reinhardtii is the only one CA identified so far in close association with the photosystem II (PSII) multi-subunit protein complex. It was proposed earlier, that CAH3 could facilitate the H+ removal from the active center of the PSII water-oxidizing complex (WOC) under the light, thereby increasing its activity. In the present work, using PSII enriched membranes from the wild type of C. reinhardtii and from the CAH3-deficient mutant cia3, we demonstrate, that the suppression of the photosynthetic activity of PSII by increased pH is more pronounced in preparations from cia3 as compared to the wild type. Experiments with CA inhibitors show that the activity of CAH3 supports the function of PSII and prevents its irreversible inactivation under light upon increased pH. The photosynthetic activity of PSII from cia3 can be restored to the wild type level upon increased pH if an excess of HCO3- is added. These findings testify that the main role of CAH3 in the vicinity of PSII is the acceleration of the HCO3- dehydration reaction. Measurements of the photoinduced electron transfer rate in PSII from water or from an artificial electron donor indicate, that CAH3 has a direct influence on the WOC function. Based on the data obtained in this work we conclude, that in vivo CA-activity of CAH3 may support the photosynthetic activity of PSII at increased pH in the thylakoid lumen and can be observed under the dark to light transition.


Assuntos
Proteínas de Algas/metabolismo , Anidrases Carbônicas/metabolismo , Chlamydomonas reinhardtii/enzimologia , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/química , Chlamydomonas reinhardtii/efeitos dos fármacos , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Transporte de Elétrons , Concentração de Íons de Hidrogênio , Fotossíntese
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