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A comparison of the effects of copper nanoparticles and copper sulfate on Phaeodactylum tricornutum physiology and transcription
Accepted Manuscript Title: A comparison of the effects of copper nanoparticles and copper sulfate on Phaeodactylum tricornutum physiology and transcription Authors: Youchao Zhu, Jiahui Xu, Tao Lu, Meng Zhang, Mingjing Ke, Zhengwei Fu, Xiangliang Pan, Haifeng Qian PII: DOI: Reference:
S1382-6689(17)30254-5 http://dx.doi.org/10.1016/j.etap.2017.08.029 ENVTOX 2864
To appear in:
Environmental Toxicology and Pharmacology
Received date: Accepted date:
23-6-2017 28-8-2017
Please cite this article as: Zhu, Youchao, Xu, Jiahui, Lu, Tao, Zhang, Meng, Ke, Mingjing, Fu, Zhengwei, Pan, Xiangliang, Qian, Haifeng, A comparison of the effects of copper nanoparticles and copper sulfate on Phaeodactylum tricornutum physiology and transcription.Environmental Toxicology and Pharmacology http://dx.doi.org/10.1016/j.etap.2017.08.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A comparison of the effects of copper nanoparticles and copper sulfate on Phaeodactylum tricornutum physiology and transcription Youchao Zhu†, Jiahui Xu‡, Tao Lu†, Meng Zhang†, Mingjing Ke†, Zhengwei Fu‡‡, Xiangliang Pan†, Haifeng Qian†,*
†
College of Environment, Zhejiang University of Technology, Hangzhou 310032, P.
R. of China ‡
Department of Food Science and Technology, Zhejiang University of Technology,
Hangzhou 310032, P. R. of China ‡‡
College of Biotechnology and Bioengineering, Zhejiang University of Technology,
Hangzhou 310032, P. R. of China This manuscript has been thoroughly edited by a native English speaker from an editing company (Boston Professional Group (BPG) Editing). Editing Certificate will be provided upon request.
*Correspondence Author: Tel.:+86 5718832 0742, Fax: +86 571 8832 0599, E-mail address: [email protected]
1
Graphical abstract
Research highlights ► The effect of CuNPs on P. tricornutum was studied at the level of gene transcription. ► The toxicity between CuNPs and CuSO4 under same concentrations was compared. ► An experiment of the effect of salinity on DLS and zeta potential of CuNPs in culture medium was conducted ► The high dissolved Cu explained the similar toxic responses between CuNPs and CuSO4 treatments.
Abstract: Copper nanoparticles (CuNPs) have been used in a broad range of applications. However, they are inevitably released into the marine environment, making it necessary to evaluate their potential effects on marine phytoplankton. In this study, the short-term (96 h) effects of CuNPs and CuSO4 on Phacodactylum tricornutum growth, photosynthesis, reactive oxygen species production and transcription were assessed. It was found that high concentrations (40 μM) of CuNPs and CuSO4 significantly inhibited the growth, photosynthesis and induced oxidative 2
stress of P. tricornutum, while lower concentrations caused a hormetic response as indicated by a slight stimulation in algal growth. The high percentage of dissolved Cu (78-100%) in culture medium suggested that the dissolved Cu was the main driver of toxicity during CuNPs treatment. The algal cells upregulated electron transport chainrelated genes to produce more energy and restore photosynthesis after 96 h of treatment with CuNPs and CuSO4. This study delineates the cellular mechanism behind the toxicity of CuNPs and CuSO4 on marine diatoms. Keywords: Phaeodactylum tricornutum; Copper nanoparticles; Oxidative stress; Gene transcription
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Introduction During the last decade, nanoparticles (NPs) have been widely used in numerous applications, including in medicine, consumer products, food, personal products, and energy conservation (Mornet et al. 2004; Tiede et al. 2008; Vance et al. 2015). NPs have unique physical, chemical, and biological properties compared to their bulk counterparts due to their large surface area and quantum effects derived from being very small particles (Stampoulis et al. 2009); one dimension of these NPs ranges from1 to 100 nm (Hochella 2002). These characteristics endow NPs with stronger catalytic activity (Narayanan and El-Sayed 2005). Copper nanoparticles (CuNPs) are highly reactive (Reinhard et al. 1998), which makes them strongly antibacterial (Ruparelia et al. 2008), and have been widely used as nucleating agents, metal catalysts and desulfurizers, including in printing, lithium ion battery manufacturing and production of biomaterials (Cui et al. 2016; Kang et al. 2010; Wang et al. 2015; Wu et al. 2016). As a result of their extensive use in these applications, CuNPs have inevitably been released from industrial sewage into the natural aquatic ecosystem, causing unwanted effects on aquatic microorganisms, animals, and phytoplanktons. A number of studies have shown that CuNPs are toxic to some microorganisms and phytoplanktons. For example, exposure to CuNPs inhibits the growth of three Lemnaceae species to varying degrees, where there is a 50% effective concentration (EC50) of 1.15 mg L-1 for Spirodela polyrhiza, 0.84 mg L-1 for Lemna minor and 0.64 mg L-1 for Wolffia arrhiza (L. Song et al. 2015). CuNPs have also been shown to significantly inhibit the photosynthetic yield of Chlamydomonas reinhardtii (Muller et al. 2016). In addition, previous studies found that CuNPs induce oxidative stress that causes protein, DNA, and cell membrane damage, and ultimately causes cell growth 4
inhibition and death (H. Chen et al. 2015; F. F. Li et al. 2013). Moreover, it is widely recognized that metallic ions can be released from metallic NP suspensions (Griffitt et al. 2008). CuNPs dissolve and release Cu ions into culture medium, potentially increasing the Cu ion concentration, which needs to be considered when evaluating CuNPs toxicity (F. M. Li et al. 2015). It is controversial whether CuNPs toxicity is nano-specific or from Cu ion release (Kaweeteerawat et al. 2015). In addition, NP toxicity against phytoplankton can be influenced by the physical behavior of the NPs, such as aggregation and adsorption in culture medium (Cheloni et al. 2016; Gilbert et al. 2009). To address whether the toxicity is specific to the NPs or their components, it is critical to conduct direct comparisons between NPs and their ionic form under comparable conditions. In this study, the marine diatom Phaeodactylum tricornutum was selected as a test organism with which to evaluate the toxicity of CuNPs. To better assess CuNPs toxicity, CuSO4 treatment was included in this study when comparing the toxicity of CuNPs and Cu ions, where the total Cu concentrations were kept consistent at 0, 10, and 40 μM. In an effort to delineate the mechanisms behind CuNPs toxicity, a number of variables, i.e. growth, pigment production, photosynthesis, enzymatic activity, and transcription levels, were evaluated and compared under different treatment conditions to distinguish between the effects of CuNPs and Cu ions at comparable concentrations.
1. Materials and methods
1.1. Algal cultures and growth conditions 5
The marine diatom Phaeodactylum tricornutum (FACHB-863) was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences. P. tricornutum was cultured at 22 ± 0.5℃ in 150 mL sterile Erdschreiber’s medium (without soil extract, initial pH = 7.8) in 250 mL Erlenmeyer flasks under cool-white fluorescence (≈54 E m−2 s−1) on a 12/12 h light/dark cycle. The Erlenmeyer flasks and other experimental materials were soaked in nitric acid (10% v/v) for 24 h and then rinsed at least 7 times with Milli-Q water (resistance of 18.25 MΩ·cm). The algal growth was measured at an optical density of 680 (OD680) using a spectrophotometer. The regression equation generated from cell density (y × 105·mL-1) and OD680 (x) was y = 100 x + 1.12 (R2 = 99.08) (Wei et al. 2014).
1.2. CuNPs preparation and characterization CuNPs (diam:10-30nm; purity: 99.9%) were obtained from Shanghai Huzheng Nano Technology Co. LTD (AGS-WMB1000C, Shanghai, China) and suspended in Milli-Q water by sonicating (20 kHz and 100 W bath at 25℃) for 30 min using an ultrasonic cleaner. CuNPs size distribution and stability at 100 mg·L-1 in culture medium were evaluated based on dynamic light scattering (DLS) and Zeta Potential using a NanoBrook OmnirParticle Sizer and Zeta Potential Analyzer (Brookhaven Instruments Corporation, Holtsville, NY, USA) at salinities of 0, 1.19, 2.38, 4.77, 9.54, and 38.15‰. Culture medium containing 10 and 40 μM CuNPs was filtered through a 50kDa filter by centrifuging at 4,852 r/min for 15 min to remove the CuNPs from suspension. These filtrates were then acidified with 2% HNO3 and the concentration of dissolved Cu in the culture medium was quantified using an Atomic Absorption Spectrometer (PE-AA800, USA). The DLS, Zeta Potential, and dissolved 6
Cu were measured at 1, 24, 48, 72, and 96 h after CuNPs were added into culture medium.
1.3 Growth inhibition analysis Before each experiment, the cells were grown until they reached the exponential growth phase, the initial cell density upon starting treatment was approximately 5.12 × 105 cells mL-1. The CuNPs suspension and CuSO4 solution were added into the cultures at final concentrations of 0, 10, 20, 40, and 80 μM. Inhibition of growth by treatment with CuNPs and Cu ions for 24, 48, 72 and 96 h was calculated using the following equation: % inhibition =100×(1-ODexposed/ODcontrol) Where OD680exposed refers to the CuNPs- or CuSO4-treated group and the OD680control to the control group. % inhibition is the inhibition of cell yield after exposure to CuNPs and CuSO4 for the indicated periods of time. EC50 was calculated using the regression equation, which was generated from the growth inhibition curves at 48 and 96 h. Four replicates were tested for each concentration.
1.4 Photosynthetic pigment analysis The amount of photosynthetic pigments, such as chlorophyll a, chlorophyll b, and carotenoids, was determined to analyze the photosynthetic capacity of P. tricornutum after exposure to CuNPs and CuSO4 for 48 and 96 h. These pigments were extracted by adding 5 mL acetone (90% v/v) to 40 mL control or treated P. tricornutum samples, centrifuging at 7,000 r/min for 5 min at 4°C, storing at 4°C for at least 24 h, and then quantifying at wavelengths of 440, 630, 644, 647, 662, 664 and 750 nm. 7
Calculations were performed as described by (Jeffrey and Humphrey 1975).
1.5 Quantification of enzymatic activity and reactive oxygen species (ROS) After exposure to CuNPs or CuSO4 for 48 or 96 h, 40 mL of algal culture was centrifuged at 7,000 r/min for 5 min at 4°C, and then an extraction was performed using 1 mL phosphate buffer saline (PBS). Protein concentrations were determined using bicinchoninic acid (BCA) (BCA protein kit, Sangon Company, China). The enzymatic activity and concentrations of the antioxidants glutathione (GSH), peroxidase (POD) and superoxide dismutase (SOD), as well as the oxidant marker MDA, were measured using reagent kits (Beyotime Institute of Biotechnology, Haimen, China). The fluorescence was read in a fluorescence microplate reader (BioTEK, USA). ROS was measured using the fluorescent probe DCFH-DA as described in (H. Song et al. 2017). DCFH-DA is non-fluorescent and passes easily through the cell membrane. However, once inside a cell, it is hydrolyzed by intracellular esterases to form DCFH, which cannot pass through the cell membrane and is oxidized by ROS to generate fluorescent DCF. Therefore, ROS levels can be measured using fluorescent intensity.
1.6 RNA extraction, reverse transcription, and real-time quantitative PCR Respiratory and photosynthetic electron transport chain and cell division-related genes were amplified and quantified by real-time quantitative PCR (qRT-PCR) using an Eppendorf Master Cycler®ep RealPlex4 system (Wesseling-Berzdorf, Germany). After exposure to 0, 10 and 40 μM CuNPs or CuSO4, 40 mL of algal culture was centrifuged at 7,000 r/min for 5 min at 4℃. Total RNA was extracted from the algal 8
cells using RNAiso (Takara Bio, Dalian, China) according to the manufacturer’s instructions. RNA was reverse-transcribed into cDNA using a reverse transcriptase kit (Toyobo, Tokyo, Japan). qRT-PCR was conducted in an Eppendorf Master Cycler ep RealPlex4 (Wesseling-Berzdorf, Germany) using SYBR Green PCR reagents (Toyobo, Tokyo, Japan) as described in our previous report (S. Chen et al. 2017). For analysis, 18S rDNA was used as a housekeeping gene against which to normalize other genes and the 2−ΔΔCt method was used to calculate relative gene transcription levels (Livak and Schmittgen 2001). Three genes that encode proteins related to the respiratory electron transport chain, nad5, cox3, and atpA, which encode the NADH dehydrogenase subunit (complex I), cytochrome c oxidase subunit (complex IV), and ATP synthase, respectively, were selected. Four photosynthesis electron transport chain-related genes, psbD, psaB, petF, and rbcL, which encode proteins in the photosystem I (PS I) and photosystem II (PS II) reaction centers, were chosen. Two genes related to the synthesis of iron-sulfur cluster proteins that function in the sulfur assimilation (SUF) and iron sulfur cluster (ISC) system, sufS and iscU, respectively, as well as a cell division-related gene (ftsH), were also followed.
1.7. Data analysis Each experiment had four replicate groups for each concentration. The results were calculated as mean ± standard error of the mean (SEM) and statistically significant differences were evaluated using ANOVA and t-tests using the StatView 5.0 program, where p<0.05 was considered significant.
2 Results 9
2.1 Rate of CuNPs dissolution in culture medium Within the first hour, the Cu dissolved in the culture medium reached approximately 71% and 86% of the initial CuNPs concentrations at 40 and 10 µM, respectively (Fig. 1a). This ultimately stabilized at 78% for 40 µM from 24 to 96 h, and had almost completely dissolved for 10 µM after 24 h. The DLS analysis of the CuNPs in culture medium of different salinities is shown in Fig. 1b. The nanoparticle sizes ranged from 2 to 6 µm after 1 h in culture medium, and became too unstable to measure by DLS due to high ionic strength after 72 h with the largest observed being over 10 um. However, the scattering behavior of the CuNPs in ultrapure water was relatively stable, thus, the NPs remained approximately 1 μm in size until 96 h. As shown in Fig. 1c, the zeta potential also displayed a salinity-dependent trend. The zeta potential hovered around 0 mV in the original culture medium and the absolute values increased as the salinity decreased. The CuNPs were the most stable in ultrapure water with a zeta potential of approximately 60 mV.
2.2 Effects of CuNPs and CuSO4 treatment on algal growth P. tricornutum growth was slightly stimulated by 48 h of treatment with low concentrations (less than or equal to 10 µM) of CuNPs and CuSO4, but then was slightly inhibited after 96 h of treatment (Fig. 2). Higher concentrations (20, 40, and 80 µM) of CuNPs and CuSO4 significantly inhibited P. tricornutum growth with increasing inhibition observed as the length of exposure increased. The EC50s for 48 h of treatment of P. tricornutum with CuNPs and CuSO4 were 115.7 μM (95% CI [105.3, 126.1]) and 98.9 μM (95% CI [89.7, 108.2]), respectively. The 96 h EC50s of 10
CuNPs and CuSO4 on P. tricornutum were 65.1 μM (95% CI [59.4, 70.8]) and 59.9 μM (95% CI [54.1, 65.7]), respectively.
2.3 Effects of CuNPs and CuSO4 treatment on chlorophyll content As shown in Fig. 3, the amounts of the photosynthetic pigments, including chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids, all significantly decreased after treatment with 40 μM of CuNPs and CuSO4. After exposure to CuNPs for 48 h, the Chl a, Chl b, and carotenoid content decreased by 41.4%, 47.2%, and 44.3% of the control, respectively. These pigments decreased after 48 h of CuSO4 treatment, although less so than from CuNPs, by 45.5%, 54.6%, and 51.4% of the control for Chl a, Chl b, and carotenoids, respectively. After 96 h of treatment, Chl a, Chl b, and carotenoids were 76.7%, 83.6% and 76.1% of the control, respectively, following CuNPs treatment, and inhibition was slightly higher from CuSO4 treatment. Interestingly, all three pigments slightly increased after treatment with a lower concentration (10 μM) of CuSO4 and CuNPs.
2.4 Effects of CuNPs and CuSO4 treatment on ROS accumulation and antioxidant enzyme activity We measured the generation of ROS after treatment with Cu ions and CuNPs for 48 and 96 h. After 48 h, the algal cells had accumulated high concentrations of ROS in all treatment groups. As shown in Fig. 4, ROS production after treatment with 10 and 40 μM CuNPs increased by 1.43- and 2.53-fold of the control, and with 10 and 40 μM CuSO4, there was an increase of 2.72- and 3.73-fold of the control, respectively. After 96 h exposure to 10 μM CuNPs and CuSO4, ROS production returned to levels 11
approximately equivalent to the control, but ROS levels remained high at 2.75- and 3.44-fold of the control after treatment with 40 μM CuNPs and CuSO4, respectively. The GSH content decreased significantly to 77.6% and 30.1% of the control when treated with 10 and 40 μM CuNPs for 48 h, and 64.8% and 22.1% after 10 and 40 μM CuSO4, respectively (Fig 5a). After 96 h, the GSH content of those treated groups recovered to 83.0%, 42.6%, and 94.4%, 30.9% of the control, respectively. The SOD activity significantly increased to 2.08- and 2.13-fold of the control after exposure to 40 μM CuNPs and CuSO4, respectively (Fig. 5b), and remained high after 96 h. After treatment with 10 μM CuNPs and CuSO4, the SOD activity was only slightly increased at both timepoints assessed. The POD activity did not change significantly after treatment with 10 μM CuNPs and CuSO4 at either timepoint, but significantly increased after 48 h in 40 μM CuNPs and CuSO4, reaching 4.53- and 9.71-fold of the control, respectively. At 96 h, the POD activity dropped to 1.49- and 1.75-fold of the control in 40 μM of CuNPs and CuSO4, respectively (Fig 5c). In addition, the levels of MDA, an indicator of lipid peroxidation as a marker of oxidative stress, were quantified. After 48 h, the MDA content did not significantly vary under low concentrations (10 μM) of CuNPs or CuSO4, but increased significantly (1.44- and 1.73-fold of the control, respectively) at a higher concentration of 40 μM of CuNPs and CuSO4 (Fig. 5d). However, after 96 h of treatment, the MDA content recovered and only increased significantly after treatment with 40 μM CuSO4.
2.5 Effect of CuNPs and CuSO4 on transcription As shown in Fig. 6, the transcription levels of 7 respiratory and photosynthetic electron transport chain-related genes, cox3, nad5, atpA, psaB, petF, rbcL, and psbD, 12
did not change after 48 h of treatment with 10 μM CuNPs and CuSO4. After 96 h, there was anupregulation of the transcription levels of all these genes to cope with CuNPs- and CuSO4-induced stress. Two genes encoding proteins involved in ironsulfur cluster synthesis, sufS and IscU, were both upregulated after 48 h and 96 h in 10 μM CuNPs and CuSO4. The cell division-related gene ftsH was downregulated after treatment for 48 h and typically recovered by 96 h of treatment.
3. Discussion It has been well-established that a number of NPs can inhibit the growth of marine diatoms (Galletti et al. 2016; J. J. Li et al. 2017). In the present study, we found CuNPs at a concentration of 40 μM inhibited P. tricornutum growth, while a lower concentration of 10 μM slightly stimulated growth (Fig. 2). This phenomenon is supported by a study by (Mykhaylenko and Zolotareva 2017), where low concentrations (0.67-4 mg L−1) of CuNPs stimulated an increase in Chlorella vulgaris biomass of approximately 20%. The hormetic dose-response observed in this present study has been recognized as a slight overcompensation following an initial disruption in homeostasis that represents the repair process modestly overshooting the original homeostatic set point (Iavicoli et al. 2010). Lipid-soluble pigments in photosystem I (PS I) and photosystem II (PS II) are critical to plant photosynthesis. Cu may interfere with the biosynthesis of the photosynthetic machinery by altering the pigments and proteins composing the photosynthetic membranes (Pradhan et al. 2015). CuNPs caused an observable decrease in chlorophyll and carotenoid content when treating with 40 μM (Fig. 3), demonstrating that CuNPs inhibit photosynthesis, probably by causing a decrease in antenna size of 13
the photosynthetic reaction center complexes that influences pigment synthesis (Björkman 1981). By contrast, the amount of pigment increased when algae were treated with 10 μM of both CuNPs and CuSO4, which is similar to a study published by (Pradhan et al. 2015). It has been well-demonstrated that metal compounds induce intracellular ROS production in plants and algae (F. M. Li et al. 2015; Qian et al. 2013; Qian et al. 2016). Under normal conditions, the cell maintains a balance between the production and removal of ROS; however, under heavy metal stress, an excess of oxygenated substances is generated that causes membrane lipid peroxidation and protein degradation (Gill and Tuteja 2010). Once the balance between ROS production and elimination is disrupted, the enzymatic clearance system is activated to remove excess ROS in cells. Therefore, we speculated that SOD, POD and GSH content may be altered to deal with excessive ROS. It was found that excessive ROS was produced in all treatment groups after 48 h of treatment, and ROS levels remained high until 96 h. These results are consistent with work by (Bai et al. 2015). SOD catalyzes O2- into H2O2 and O2 through the Haber-Weiss reaction, thereby directly reducing the negative impact O2- has on the cell membrane. The increased SOD levels (Fig. 5b) aided in coping with excessive O2-, while POD decomposed H2O2 into harmless H2O and O2. The increased POD levels (Fig. 5c) after Cu treatment suggest that the subsequent stage of the intracellular ROS scavenging system was activated. However, the decrease in GSH resulted in less H2O2 degradation due to the direct involvement of GSH in the removal of free radicals from plant cells (Fig. 5a). Furthermore, the increase observed in MDA content (Fig. 5d) indicates that the antioxidant enzymes failed to completely eradicate ROS-mediated effects. There was surplus ROS still 14
interacting with the cell membrane, causing an overproduction in MDA and increase in cell membrane permeability that eventually led to cell death (Wei et al. 2014). The decrease in the transcription of photosynthesis-related genes (Fig. 6a) suggests that a decrease in the rate of photosynthetic electron transport slows the synthesis of ATP and NADPH, and further slows down CO2 assimilation (Cid et al. 1995). The downregulation of a gene in the Calvin cycle, rbcL, also indicates inhibition of CO2 assimilation (Fig. 6b). The decrease in the transcription of respiration-related genes implies a slower rate of energy metabolism and formation of macromolecules, inhibiting the growth of algae as a result (Wei et al. 2014). After 96 h of exposure to CuNPs and CuSO4, the levels of the photosynthesis and respiration-related gene transcripts began to return to control levels or, in some cases, increased (Fig. 6c). This suggests there was some compensation for the damage incurred by CuNPs as an adaptive mechanism after 96 h of treatment. These results are similar to those in a study by (Moisset et al. 2015), where diuron caused a downregulation in the transcription of several photosynthetic metabolism and mitochondrial metabolismrelated genes in three major freshwater diatom species after 6 h of exposure. However, by the 2nd day, the transcripts had returned to control levels and were upregulated on the 7th or 14th day. However, there are some discrepancies between these results and those gathered by (Wei et al. 2014) after exposure to 40 μM CuSO4, which could be caused by differences in the culture mediums. In this present study, the culture medium was optimized and did not contain soil extracts. The soil extracts in Erdschreiber’ s medium used in the study by (Wei et al. 2014) could contain an undefined quantity of high affinity Cu binding ligands and/or a high concentration of ethylenediaminetetraacetic acid (EDTA); both of these can decrease the concentration 15
of bioavailable Cu and, thus, its toxicity. Furthermore, the transcription levels after 48 h exposure to low concentrations of CuNPs and CuSO4 (10 μM) did not decrease and were upregulated at earlier timepoints than from 40 μM CuNPs. This shows that P. tricornutum was less affected by low concentrations of CuNPs and CuSO4, underwent less stress, and mobilized mechanisms to adjust to this stress rapidly. In addition, transcripts of the cell division-related gene ftsH decreased after exposure to 40 μM CuNPs and CuSO4, suggesting cell division of P. tricornutum was inhibited, likely due to a lower energy supply. But the gene transcription of cell division intended to be upregulated at a later timepoint in order to boost newer cells. Interestingly, it was found that iron-sulfur cluster assembly and transport-related genes (IscU and sufS) were upregulated after 48 and 96 h exposure to CuNPs and CuSO4. Specifically, IscU was upregulated almost 6-fold the control after exposure to 40 μM CuNPs. That may be because the ISC and the SUF can be inhibited by H2O2 and O2, where IscS-IscU is more susceptible than SufS-SufE to oxidative modification by H2O2 (Dai and Outten 2012). Therefore, P. tricornutum may be upregulating IscU more than sufS to cope with excess H2O2 by creating more synthetic iron-sulfur clusters in order to maintain active cell metabolism, increase the rate of electron transport, and provide more energy for cells. However, this may cause persistent generation of ROS (Wei et al. 2014), which is in accord with the high levels of ROS noted after 48 and 96 h treatment with CuNPs and CuSO4. It could easily be concluded that the toxicity to P. tricornutum of CuSO4 was not notably greater than CuNPs at equivalent concentrations in this study. However, these results contradicted many studies that concluded metallic ions have greater toxicity to aquatic organisms than the corresponding metal-containing NPs (Qian et al. 2013). 16
Therefore, we speculated that the dissolution rate of CuNPs was responsible for this result. The culture medium had a high pH and ionic strength that likely increased the dissolution rate of NPs in this study, similar to observations by (Miao et al. 2010). Indeed, when the Cu dissolved in culture medium was analyzed, it was found that the dissolution rate stabilized at 78% after 24 h treatment with 40 μM CuNPs, while at 10 μM CuNPs, the dissolution rate reached almost 100%. Such a high rate of dissolution in culture medium suggests the toxicity observed in this study was mostly derived from the dissolved Cu rather than the CuNPs themselves. In addition, CuNPs aggregation in the culture medium may also affect toxicity. Since the majority of the CuNPs had dissolved in the culture medium at 10 and 40 μM concentrations, the DLS and zeta potential were unable to be measured due to the rather low sample count rate. Therefore, the DLS and zeta potential studies were conducted at a CuNPs concentration of 1.56 mM. CuNPs in culture medium with a high salt concentration aggregated rapidly within a few hours (Fig. 1b). The CuNPs were larger in the high salinity medium than the lower salinity medium, indicating that salinity was a significant factor for the stability of CuNPs in suspension in culture medium. The zeta potential was also more unstable in the culture medium than in lower salinity medium (Fig. 1c). The aggregation of CuNPs was greater in salt water than in fresh water, because the high ionic strength of the former compresses the electric double layer surrounding the suspended CuNPs, reducing the repulsive forces between them and increasing the tendency to aggregate (Batley et al. 2013; Bian et al. 2011). In this case, homo-aggregation, where CuNPs aggregate with one another, may be more effective than hetero-aggregation, where CuNPs aggregate with cells. This may result in less available suspended CuNPs to the cells and thereby alleviate CuNPs 17
toxicity (J. J. Li et al. 2017).
4. Conclusion A high concentration of CuNPs notably inhibits P. tricornutum growth and photosynthesis and can induce a large amount of ROS that causes destruction to the cell membrane. Concurrently, the antioxidant defense system is activated, but is unable to completely eliminate the accumulated ROS. The algal cells upregulated almost all genes involved in the redox transport chain in response, which typically provides energy for the cells and promotes cell growth. In this study, it was found the dissolved Cu was responsible for the toxicity of CuNPs for P. Tricornutum.
Funding: This work was supported by the National Natural Science Foundation of China (CN) [grant numbers 2157, 7128].
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List of figures Fig. 1 (a) Percentage Cu ions in culture medium released from 10 and 40 µM CuNPs. (b) Hydrodynamic diameters and (c) zeta potential of 1.56 mM CuNPs in mediums with different salinities (0-38.15‰, where 0‰ refers to ultrapure water and 38.1% to the original cultural medium). Values are presented as mean ± SD (n = 3).
Fig. 2 Percent inhibition of P. tricornutum growth by different concentrations of (a) CuNPs and (b) CuSO4. Values are presented as mean ± SD (n = 4).
Fig. 3 Changes in pigment content (Chl a, Chl b, and Carotenoids) after exposure to 10 or 40 μM CuNPs for (a) 48 and (b) 96 h, and 10 or 40 μM CuSO4 for (c) 48 and (d) 96 h. Different letters represent statistically significant differences relative to each other, where p<0.05 is considered significant. Values are presented as mean ± SE (n = 4). Fig. 4 The effect of exposure to CuNPs (10 and 40 μM) and CuSO4 (10 and 40 μM) for 48 and 96 h on intracellular reactive oxygen species (ROS) production. Different letters represent statistically significant differences relative to each other, where p<0.05 is considered significant. Values are presented as mean ± SE (n = 4).
Fig. 5 The effect of exposure to different concentrations of CuNPs and CuSO4 for 48 and 96 h on enzymatic activity. Changes in (a) glutathione peptide (GSH), (b) superoxide dismutase (SOD), (c) peroxidase (POD), and (d) malondialdehyde (MDA) content. Different letters represent statistically significant differences relative to each other, where p<0.05 is considered significant. Values are presented as mean ± SE (n = 4).
Fig. 6 Effect of exposure to CuNPs and CuSO4 on the transcription of genes in Phaeodactylum tricornutum. Effects on the respiratory electron transport chain and synthesis of iron-sulfur cluster protein-related genes after (a) 48 and (b) 96 h, as well as the photosynthetic electron transport chain and cell division-related genes at (c) 48 and (d) 96 h. Different letters represent statistically significant differences relative to 25
each other, where p< 0.05 is considered significant. Values are presented as mean ± SE (n = 4).
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S1382-6689(17)30254-5 http://dx.doi.org/10.1016/j.etap.2017.08.029 ENVTOX 2864
To appear in:
Environmental Toxicology and Pharmacology
Received date: Accepted date:
23-6-2017 28-8-2017
Please cite this article as: Zhu, Youchao, Xu, Jiahui, Lu, Tao, Zhang, Meng, Ke, Mingjing, Fu, Zhengwei, Pan, Xiangliang, Qian, Haifeng, A comparison of the effects of copper nanoparticles and copper sulfate on Phaeodactylum tricornutum physiology and transcription.Environmental Toxicology and Pharmacology http://dx.doi.org/10.1016/j.etap.2017.08.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A comparison of the effects of copper nanoparticles and copper sulfate on Phaeodactylum tricornutum physiology and transcription Youchao Zhu†, Jiahui Xu‡, Tao Lu†, Meng Zhang†, Mingjing Ke†, Zhengwei Fu‡‡, Xiangliang Pan†, Haifeng Qian†,*
†
College of Environment, Zhejiang University of Technology, Hangzhou 310032, P.
R. of China ‡
Department of Food Science and Technology, Zhejiang University of Technology,
Hangzhou 310032, P. R. of China ‡‡
College of Biotechnology and Bioengineering, Zhejiang University of Technology,
Hangzhou 310032, P. R. of China This manuscript has been thoroughly edited by a native English speaker from an editing company (Boston Professional Group (BPG) Editing). Editing Certificate will be provided upon request.
*Correspondence Author: Tel.:+86 5718832 0742, Fax: +86 571 8832 0599, E-mail address: [email protected]
1
Graphical abstract
Research highlights ► The effect of CuNPs on P. tricornutum was studied at the level of gene transcription. ► The toxicity between CuNPs and CuSO4 under same concentrations was compared. ► An experiment of the effect of salinity on DLS and zeta potential of CuNPs in culture medium was conducted ► The high dissolved Cu explained the similar toxic responses between CuNPs and CuSO4 treatments.
Abstract: Copper nanoparticles (CuNPs) have been used in a broad range of applications. However, they are inevitably released into the marine environment, making it necessary to evaluate their potential effects on marine phytoplankton. In this study, the short-term (96 h) effects of CuNPs and CuSO4 on Phacodactylum tricornutum growth, photosynthesis, reactive oxygen species production and transcription were assessed. It was found that high concentrations (40 μM) of CuNPs and CuSO4 significantly inhibited the growth, photosynthesis and induced oxidative 2
stress of P. tricornutum, while lower concentrations caused a hormetic response as indicated by a slight stimulation in algal growth. The high percentage of dissolved Cu (78-100%) in culture medium suggested that the dissolved Cu was the main driver of toxicity during CuNPs treatment. The algal cells upregulated electron transport chainrelated genes to produce more energy and restore photosynthesis after 96 h of treatment with CuNPs and CuSO4. This study delineates the cellular mechanism behind the toxicity of CuNPs and CuSO4 on marine diatoms. Keywords: Phaeodactylum tricornutum; Copper nanoparticles; Oxidative stress; Gene transcription
3
Introduction During the last decade, nanoparticles (NPs) have been widely used in numerous applications, including in medicine, consumer products, food, personal products, and energy conservation (Mornet et al. 2004; Tiede et al. 2008; Vance et al. 2015). NPs have unique physical, chemical, and biological properties compared to their bulk counterparts due to their large surface area and quantum effects derived from being very small particles (Stampoulis et al. 2009); one dimension of these NPs ranges from1 to 100 nm (Hochella 2002). These characteristics endow NPs with stronger catalytic activity (Narayanan and El-Sayed 2005). Copper nanoparticles (CuNPs) are highly reactive (Reinhard et al. 1998), which makes them strongly antibacterial (Ruparelia et al. 2008), and have been widely used as nucleating agents, metal catalysts and desulfurizers, including in printing, lithium ion battery manufacturing and production of biomaterials (Cui et al. 2016; Kang et al. 2010; Wang et al. 2015; Wu et al. 2016). As a result of their extensive use in these applications, CuNPs have inevitably been released from industrial sewage into the natural aquatic ecosystem, causing unwanted effects on aquatic microorganisms, animals, and phytoplanktons. A number of studies have shown that CuNPs are toxic to some microorganisms and phytoplanktons. For example, exposure to CuNPs inhibits the growth of three Lemnaceae species to varying degrees, where there is a 50% effective concentration (EC50) of 1.15 mg L-1 for Spirodela polyrhiza, 0.84 mg L-1 for Lemna minor and 0.64 mg L-1 for Wolffia arrhiza (L. Song et al. 2015). CuNPs have also been shown to significantly inhibit the photosynthetic yield of Chlamydomonas reinhardtii (Muller et al. 2016). In addition, previous studies found that CuNPs induce oxidative stress that causes protein, DNA, and cell membrane damage, and ultimately causes cell growth 4
inhibition and death (H. Chen et al. 2015; F. F. Li et al. 2013). Moreover, it is widely recognized that metallic ions can be released from metallic NP suspensions (Griffitt et al. 2008). CuNPs dissolve and release Cu ions into culture medium, potentially increasing the Cu ion concentration, which needs to be considered when evaluating CuNPs toxicity (F. M. Li et al. 2015). It is controversial whether CuNPs toxicity is nano-specific or from Cu ion release (Kaweeteerawat et al. 2015). In addition, NP toxicity against phytoplankton can be influenced by the physical behavior of the NPs, such as aggregation and adsorption in culture medium (Cheloni et al. 2016; Gilbert et al. 2009). To address whether the toxicity is specific to the NPs or their components, it is critical to conduct direct comparisons between NPs and their ionic form under comparable conditions. In this study, the marine diatom Phaeodactylum tricornutum was selected as a test organism with which to evaluate the toxicity of CuNPs. To better assess CuNPs toxicity, CuSO4 treatment was included in this study when comparing the toxicity of CuNPs and Cu ions, where the total Cu concentrations were kept consistent at 0, 10, and 40 μM. In an effort to delineate the mechanisms behind CuNPs toxicity, a number of variables, i.e. growth, pigment production, photosynthesis, enzymatic activity, and transcription levels, were evaluated and compared under different treatment conditions to distinguish between the effects of CuNPs and Cu ions at comparable concentrations.
1. Materials and methods
1.1. Algal cultures and growth conditions 5
The marine diatom Phaeodactylum tricornutum (FACHB-863) was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences. P. tricornutum was cultured at 22 ± 0.5℃ in 150 mL sterile Erdschreiber’s medium (without soil extract, initial pH = 7.8) in 250 mL Erlenmeyer flasks under cool-white fluorescence (≈54 E m−2 s−1) on a 12/12 h light/dark cycle. The Erlenmeyer flasks and other experimental materials were soaked in nitric acid (10% v/v) for 24 h and then rinsed at least 7 times with Milli-Q water (resistance of 18.25 MΩ·cm). The algal growth was measured at an optical density of 680 (OD680) using a spectrophotometer. The regression equation generated from cell density (y × 105·mL-1) and OD680 (x) was y = 100 x + 1.12 (R2 = 99.08) (Wei et al. 2014).
1.2. CuNPs preparation and characterization CuNPs (diam:10-30nm; purity: 99.9%) were obtained from Shanghai Huzheng Nano Technology Co. LTD (AGS-WMB1000C, Shanghai, China) and suspended in Milli-Q water by sonicating (20 kHz and 100 W bath at 25℃) for 30 min using an ultrasonic cleaner. CuNPs size distribution and stability at 100 mg·L-1 in culture medium were evaluated based on dynamic light scattering (DLS) and Zeta Potential using a NanoBrook OmnirParticle Sizer and Zeta Potential Analyzer (Brookhaven Instruments Corporation, Holtsville, NY, USA) at salinities of 0, 1.19, 2.38, 4.77, 9.54, and 38.15‰. Culture medium containing 10 and 40 μM CuNPs was filtered through a 50kDa filter by centrifuging at 4,852 r/min for 15 min to remove the CuNPs from suspension. These filtrates were then acidified with 2% HNO3 and the concentration of dissolved Cu in the culture medium was quantified using an Atomic Absorption Spectrometer (PE-AA800, USA). The DLS, Zeta Potential, and dissolved 6
Cu were measured at 1, 24, 48, 72, and 96 h after CuNPs were added into culture medium.
1.3 Growth inhibition analysis Before each experiment, the cells were grown until they reached the exponential growth phase, the initial cell density upon starting treatment was approximately 5.12 × 105 cells mL-1. The CuNPs suspension and CuSO4 solution were added into the cultures at final concentrations of 0, 10, 20, 40, and 80 μM. Inhibition of growth by treatment with CuNPs and Cu ions for 24, 48, 72 and 96 h was calculated using the following equation: % inhibition =100×(1-ODexposed/ODcontrol) Where OD680exposed refers to the CuNPs- or CuSO4-treated group and the OD680control to the control group. % inhibition is the inhibition of cell yield after exposure to CuNPs and CuSO4 for the indicated periods of time. EC50 was calculated using the regression equation, which was generated from the growth inhibition curves at 48 and 96 h. Four replicates were tested for each concentration.
1.4 Photosynthetic pigment analysis The amount of photosynthetic pigments, such as chlorophyll a, chlorophyll b, and carotenoids, was determined to analyze the photosynthetic capacity of P. tricornutum after exposure to CuNPs and CuSO4 for 48 and 96 h. These pigments were extracted by adding 5 mL acetone (90% v/v) to 40 mL control or treated P. tricornutum samples, centrifuging at 7,000 r/min for 5 min at 4°C, storing at 4°C for at least 24 h, and then quantifying at wavelengths of 440, 630, 644, 647, 662, 664 and 750 nm. 7
Calculations were performed as described by (Jeffrey and Humphrey 1975).
1.5 Quantification of enzymatic activity and reactive oxygen species (ROS) After exposure to CuNPs or CuSO4 for 48 or 96 h, 40 mL of algal culture was centrifuged at 7,000 r/min for 5 min at 4°C, and then an extraction was performed using 1 mL phosphate buffer saline (PBS). Protein concentrations were determined using bicinchoninic acid (BCA) (BCA protein kit, Sangon Company, China). The enzymatic activity and concentrations of the antioxidants glutathione (GSH), peroxidase (POD) and superoxide dismutase (SOD), as well as the oxidant marker MDA, were measured using reagent kits (Beyotime Institute of Biotechnology, Haimen, China). The fluorescence was read in a fluorescence microplate reader (BioTEK, USA). ROS was measured using the fluorescent probe DCFH-DA as described in (H. Song et al. 2017). DCFH-DA is non-fluorescent and passes easily through the cell membrane. However, once inside a cell, it is hydrolyzed by intracellular esterases to form DCFH, which cannot pass through the cell membrane and is oxidized by ROS to generate fluorescent DCF. Therefore, ROS levels can be measured using fluorescent intensity.
1.6 RNA extraction, reverse transcription, and real-time quantitative PCR Respiratory and photosynthetic electron transport chain and cell division-related genes were amplified and quantified by real-time quantitative PCR (qRT-PCR) using an Eppendorf Master Cycler®ep RealPlex4 system (Wesseling-Berzdorf, Germany). After exposure to 0, 10 and 40 μM CuNPs or CuSO4, 40 mL of algal culture was centrifuged at 7,000 r/min for 5 min at 4℃. Total RNA was extracted from the algal 8
cells using RNAiso (Takara Bio, Dalian, China) according to the manufacturer’s instructions. RNA was reverse-transcribed into cDNA using a reverse transcriptase kit (Toyobo, Tokyo, Japan). qRT-PCR was conducted in an Eppendorf Master Cycler ep RealPlex4 (Wesseling-Berzdorf, Germany) using SYBR Green PCR reagents (Toyobo, Tokyo, Japan) as described in our previous report (S. Chen et al. 2017). For analysis, 18S rDNA was used as a housekeeping gene against which to normalize other genes and the 2−ΔΔCt method was used to calculate relative gene transcription levels (Livak and Schmittgen 2001). Three genes that encode proteins related to the respiratory electron transport chain, nad5, cox3, and atpA, which encode the NADH dehydrogenase subunit (complex I), cytochrome c oxidase subunit (complex IV), and ATP synthase, respectively, were selected. Four photosynthesis electron transport chain-related genes, psbD, psaB, petF, and rbcL, which encode proteins in the photosystem I (PS I) and photosystem II (PS II) reaction centers, were chosen. Two genes related to the synthesis of iron-sulfur cluster proteins that function in the sulfur assimilation (SUF) and iron sulfur cluster (ISC) system, sufS and iscU, respectively, as well as a cell division-related gene (ftsH), were also followed.
1.7. Data analysis Each experiment had four replicate groups for each concentration. The results were calculated as mean ± standard error of the mean (SEM) and statistically significant differences were evaluated using ANOVA and t-tests using the StatView 5.0 program, where p<0.05 was considered significant.
2 Results 9
2.1 Rate of CuNPs dissolution in culture medium Within the first hour, the Cu dissolved in the culture medium reached approximately 71% and 86% of the initial CuNPs concentrations at 40 and 10 µM, respectively (Fig. 1a). This ultimately stabilized at 78% for 40 µM from 24 to 96 h, and had almost completely dissolved for 10 µM after 24 h. The DLS analysis of the CuNPs in culture medium of different salinities is shown in Fig. 1b. The nanoparticle sizes ranged from 2 to 6 µm after 1 h in culture medium, and became too unstable to measure by DLS due to high ionic strength after 72 h with the largest observed being over 10 um. However, the scattering behavior of the CuNPs in ultrapure water was relatively stable, thus, the NPs remained approximately 1 μm in size until 96 h. As shown in Fig. 1c, the zeta potential also displayed a salinity-dependent trend. The zeta potential hovered around 0 mV in the original culture medium and the absolute values increased as the salinity decreased. The CuNPs were the most stable in ultrapure water with a zeta potential of approximately 60 mV.
2.2 Effects of CuNPs and CuSO4 treatment on algal growth P. tricornutum growth was slightly stimulated by 48 h of treatment with low concentrations (less than or equal to 10 µM) of CuNPs and CuSO4, but then was slightly inhibited after 96 h of treatment (Fig. 2). Higher concentrations (20, 40, and 80 µM) of CuNPs and CuSO4 significantly inhibited P. tricornutum growth with increasing inhibition observed as the length of exposure increased. The EC50s for 48 h of treatment of P. tricornutum with CuNPs and CuSO4 were 115.7 μM (95% CI [105.3, 126.1]) and 98.9 μM (95% CI [89.7, 108.2]), respectively. The 96 h EC50s of 10
CuNPs and CuSO4 on P. tricornutum were 65.1 μM (95% CI [59.4, 70.8]) and 59.9 μM (95% CI [54.1, 65.7]), respectively.
2.3 Effects of CuNPs and CuSO4 treatment on chlorophyll content As shown in Fig. 3, the amounts of the photosynthetic pigments, including chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids, all significantly decreased after treatment with 40 μM of CuNPs and CuSO4. After exposure to CuNPs for 48 h, the Chl a, Chl b, and carotenoid content decreased by 41.4%, 47.2%, and 44.3% of the control, respectively. These pigments decreased after 48 h of CuSO4 treatment, although less so than from CuNPs, by 45.5%, 54.6%, and 51.4% of the control for Chl a, Chl b, and carotenoids, respectively. After 96 h of treatment, Chl a, Chl b, and carotenoids were 76.7%, 83.6% and 76.1% of the control, respectively, following CuNPs treatment, and inhibition was slightly higher from CuSO4 treatment. Interestingly, all three pigments slightly increased after treatment with a lower concentration (10 μM) of CuSO4 and CuNPs.
2.4 Effects of CuNPs and CuSO4 treatment on ROS accumulation and antioxidant enzyme activity We measured the generation of ROS after treatment with Cu ions and CuNPs for 48 and 96 h. After 48 h, the algal cells had accumulated high concentrations of ROS in all treatment groups. As shown in Fig. 4, ROS production after treatment with 10 and 40 μM CuNPs increased by 1.43- and 2.53-fold of the control, and with 10 and 40 μM CuSO4, there was an increase of 2.72- and 3.73-fold of the control, respectively. After 96 h exposure to 10 μM CuNPs and CuSO4, ROS production returned to levels 11
approximately equivalent to the control, but ROS levels remained high at 2.75- and 3.44-fold of the control after treatment with 40 μM CuNPs and CuSO4, respectively. The GSH content decreased significantly to 77.6% and 30.1% of the control when treated with 10 and 40 μM CuNPs for 48 h, and 64.8% and 22.1% after 10 and 40 μM CuSO4, respectively (Fig 5a). After 96 h, the GSH content of those treated groups recovered to 83.0%, 42.6%, and 94.4%, 30.9% of the control, respectively. The SOD activity significantly increased to 2.08- and 2.13-fold of the control after exposure to 40 μM CuNPs and CuSO4, respectively (Fig. 5b), and remained high after 96 h. After treatment with 10 μM CuNPs and CuSO4, the SOD activity was only slightly increased at both timepoints assessed. The POD activity did not change significantly after treatment with 10 μM CuNPs and CuSO4 at either timepoint, but significantly increased after 48 h in 40 μM CuNPs and CuSO4, reaching 4.53- and 9.71-fold of the control, respectively. At 96 h, the POD activity dropped to 1.49- and 1.75-fold of the control in 40 μM of CuNPs and CuSO4, respectively (Fig 5c). In addition, the levels of MDA, an indicator of lipid peroxidation as a marker of oxidative stress, were quantified. After 48 h, the MDA content did not significantly vary under low concentrations (10 μM) of CuNPs or CuSO4, but increased significantly (1.44- and 1.73-fold of the control, respectively) at a higher concentration of 40 μM of CuNPs and CuSO4 (Fig. 5d). However, after 96 h of treatment, the MDA content recovered and only increased significantly after treatment with 40 μM CuSO4.
2.5 Effect of CuNPs and CuSO4 on transcription As shown in Fig. 6, the transcription levels of 7 respiratory and photosynthetic electron transport chain-related genes, cox3, nad5, atpA, psaB, petF, rbcL, and psbD, 12
did not change after 48 h of treatment with 10 μM CuNPs and CuSO4. After 96 h, there was anupregulation of the transcription levels of all these genes to cope with CuNPs- and CuSO4-induced stress. Two genes encoding proteins involved in ironsulfur cluster synthesis, sufS and IscU, were both upregulated after 48 h and 96 h in 10 μM CuNPs and CuSO4. The cell division-related gene ftsH was downregulated after treatment for 48 h and typically recovered by 96 h of treatment.
3. Discussion It has been well-established that a number of NPs can inhibit the growth of marine diatoms (Galletti et al. 2016; J. J. Li et al. 2017). In the present study, we found CuNPs at a concentration of 40 μM inhibited P. tricornutum growth, while a lower concentration of 10 μM slightly stimulated growth (Fig. 2). This phenomenon is supported by a study by (Mykhaylenko and Zolotareva 2017), where low concentrations (0.67-4 mg L−1) of CuNPs stimulated an increase in Chlorella vulgaris biomass of approximately 20%. The hormetic dose-response observed in this present study has been recognized as a slight overcompensation following an initial disruption in homeostasis that represents the repair process modestly overshooting the original homeostatic set point (Iavicoli et al. 2010). Lipid-soluble pigments in photosystem I (PS I) and photosystem II (PS II) are critical to plant photosynthesis. Cu may interfere with the biosynthesis of the photosynthetic machinery by altering the pigments and proteins composing the photosynthetic membranes (Pradhan et al. 2015). CuNPs caused an observable decrease in chlorophyll and carotenoid content when treating with 40 μM (Fig. 3), demonstrating that CuNPs inhibit photosynthesis, probably by causing a decrease in antenna size of 13
the photosynthetic reaction center complexes that influences pigment synthesis (Björkman 1981). By contrast, the amount of pigment increased when algae were treated with 10 μM of both CuNPs and CuSO4, which is similar to a study published by (Pradhan et al. 2015). It has been well-demonstrated that metal compounds induce intracellular ROS production in plants and algae (F. M. Li et al. 2015; Qian et al. 2013; Qian et al. 2016). Under normal conditions, the cell maintains a balance between the production and removal of ROS; however, under heavy metal stress, an excess of oxygenated substances is generated that causes membrane lipid peroxidation and protein degradation (Gill and Tuteja 2010). Once the balance between ROS production and elimination is disrupted, the enzymatic clearance system is activated to remove excess ROS in cells. Therefore, we speculated that SOD, POD and GSH content may be altered to deal with excessive ROS. It was found that excessive ROS was produced in all treatment groups after 48 h of treatment, and ROS levels remained high until 96 h. These results are consistent with work by (Bai et al. 2015). SOD catalyzes O2- into H2O2 and O2 through the Haber-Weiss reaction, thereby directly reducing the negative impact O2- has on the cell membrane. The increased SOD levels (Fig. 5b) aided in coping with excessive O2-, while POD decomposed H2O2 into harmless H2O and O2. The increased POD levels (Fig. 5c) after Cu treatment suggest that the subsequent stage of the intracellular ROS scavenging system was activated. However, the decrease in GSH resulted in less H2O2 degradation due to the direct involvement of GSH in the removal of free radicals from plant cells (Fig. 5a). Furthermore, the increase observed in MDA content (Fig. 5d) indicates that the antioxidant enzymes failed to completely eradicate ROS-mediated effects. There was surplus ROS still 14
interacting with the cell membrane, causing an overproduction in MDA and increase in cell membrane permeability that eventually led to cell death (Wei et al. 2014). The decrease in the transcription of photosynthesis-related genes (Fig. 6a) suggests that a decrease in the rate of photosynthetic electron transport slows the synthesis of ATP and NADPH, and further slows down CO2 assimilation (Cid et al. 1995). The downregulation of a gene in the Calvin cycle, rbcL, also indicates inhibition of CO2 assimilation (Fig. 6b). The decrease in the transcription of respiration-related genes implies a slower rate of energy metabolism and formation of macromolecules, inhibiting the growth of algae as a result (Wei et al. 2014). After 96 h of exposure to CuNPs and CuSO4, the levels of the photosynthesis and respiration-related gene transcripts began to return to control levels or, in some cases, increased (Fig. 6c). This suggests there was some compensation for the damage incurred by CuNPs as an adaptive mechanism after 96 h of treatment. These results are similar to those in a study by (Moisset et al. 2015), where diuron caused a downregulation in the transcription of several photosynthetic metabolism and mitochondrial metabolismrelated genes in three major freshwater diatom species after 6 h of exposure. However, by the 2nd day, the transcripts had returned to control levels and were upregulated on the 7th or 14th day. However, there are some discrepancies between these results and those gathered by (Wei et al. 2014) after exposure to 40 μM CuSO4, which could be caused by differences in the culture mediums. In this present study, the culture medium was optimized and did not contain soil extracts. The soil extracts in Erdschreiber’ s medium used in the study by (Wei et al. 2014) could contain an undefined quantity of high affinity Cu binding ligands and/or a high concentration of ethylenediaminetetraacetic acid (EDTA); both of these can decrease the concentration 15
of bioavailable Cu and, thus, its toxicity. Furthermore, the transcription levels after 48 h exposure to low concentrations of CuNPs and CuSO4 (10 μM) did not decrease and were upregulated at earlier timepoints than from 40 μM CuNPs. This shows that P. tricornutum was less affected by low concentrations of CuNPs and CuSO4, underwent less stress, and mobilized mechanisms to adjust to this stress rapidly. In addition, transcripts of the cell division-related gene ftsH decreased after exposure to 40 μM CuNPs and CuSO4, suggesting cell division of P. tricornutum was inhibited, likely due to a lower energy supply. But the gene transcription of cell division intended to be upregulated at a later timepoint in order to boost newer cells. Interestingly, it was found that iron-sulfur cluster assembly and transport-related genes (IscU and sufS) were upregulated after 48 and 96 h exposure to CuNPs and CuSO4. Specifically, IscU was upregulated almost 6-fold the control after exposure to 40 μM CuNPs. That may be because the ISC and the SUF can be inhibited by H2O2 and O2, where IscS-IscU is more susceptible than SufS-SufE to oxidative modification by H2O2 (Dai and Outten 2012). Therefore, P. tricornutum may be upregulating IscU more than sufS to cope with excess H2O2 by creating more synthetic iron-sulfur clusters in order to maintain active cell metabolism, increase the rate of electron transport, and provide more energy for cells. However, this may cause persistent generation of ROS (Wei et al. 2014), which is in accord with the high levels of ROS noted after 48 and 96 h treatment with CuNPs and CuSO4. It could easily be concluded that the toxicity to P. tricornutum of CuSO4 was not notably greater than CuNPs at equivalent concentrations in this study. However, these results contradicted many studies that concluded metallic ions have greater toxicity to aquatic organisms than the corresponding metal-containing NPs (Qian et al. 2013). 16
Therefore, we speculated that the dissolution rate of CuNPs was responsible for this result. The culture medium had a high pH and ionic strength that likely increased the dissolution rate of NPs in this study, similar to observations by (Miao et al. 2010). Indeed, when the Cu dissolved in culture medium was analyzed, it was found that the dissolution rate stabilized at 78% after 24 h treatment with 40 μM CuNPs, while at 10 μM CuNPs, the dissolution rate reached almost 100%. Such a high rate of dissolution in culture medium suggests the toxicity observed in this study was mostly derived from the dissolved Cu rather than the CuNPs themselves. In addition, CuNPs aggregation in the culture medium may also affect toxicity. Since the majority of the CuNPs had dissolved in the culture medium at 10 and 40 μM concentrations, the DLS and zeta potential were unable to be measured due to the rather low sample count rate. Therefore, the DLS and zeta potential studies were conducted at a CuNPs concentration of 1.56 mM. CuNPs in culture medium with a high salt concentration aggregated rapidly within a few hours (Fig. 1b). The CuNPs were larger in the high salinity medium than the lower salinity medium, indicating that salinity was a significant factor for the stability of CuNPs in suspension in culture medium. The zeta potential was also more unstable in the culture medium than in lower salinity medium (Fig. 1c). The aggregation of CuNPs was greater in salt water than in fresh water, because the high ionic strength of the former compresses the electric double layer surrounding the suspended CuNPs, reducing the repulsive forces between them and increasing the tendency to aggregate (Batley et al. 2013; Bian et al. 2011). In this case, homo-aggregation, where CuNPs aggregate with one another, may be more effective than hetero-aggregation, where CuNPs aggregate with cells. This may result in less available suspended CuNPs to the cells and thereby alleviate CuNPs 17
toxicity (J. J. Li et al. 2017).
4. Conclusion A high concentration of CuNPs notably inhibits P. tricornutum growth and photosynthesis and can induce a large amount of ROS that causes destruction to the cell membrane. Concurrently, the antioxidant defense system is activated, but is unable to completely eliminate the accumulated ROS. The algal cells upregulated almost all genes involved in the redox transport chain in response, which typically provides energy for the cells and promotes cell growth. In this study, it was found the dissolved Cu was responsible for the toxicity of CuNPs for P. Tricornutum.
Funding: This work was supported by the National Natural Science Foundation of China (CN) [grant numbers 2157, 7128].
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List of figures Fig. 1 (a) Percentage Cu ions in culture medium released from 10 and 40 µM CuNPs. (b) Hydrodynamic diameters and (c) zeta potential of 1.56 mM CuNPs in mediums with different salinities (0-38.15‰, where 0‰ refers to ultrapure water and 38.1% to the original cultural medium). Values are presented as mean ± SD (n = 3).
Fig. 2 Percent inhibition of P. tricornutum growth by different concentrations of (a) CuNPs and (b) CuSO4. Values are presented as mean ± SD (n = 4).
Fig. 3 Changes in pigment content (Chl a, Chl b, and Carotenoids) after exposure to 10 or 40 μM CuNPs for (a) 48 and (b) 96 h, and 10 or 40 μM CuSO4 for (c) 48 and (d) 96 h. Different letters represent statistically significant differences relative to each other, where p<0.05 is considered significant. Values are presented as mean ± SE (n = 4). Fig. 4 The effect of exposure to CuNPs (10 and 40 μM) and CuSO4 (10 and 40 μM) for 48 and 96 h on intracellular reactive oxygen species (ROS) production. Different letters represent statistically significant differences relative to each other, where p<0.05 is considered significant. Values are presented as mean ± SE (n = 4).
Fig. 5 The effect of exposure to different concentrations of CuNPs and CuSO4 for 48 and 96 h on enzymatic activity. Changes in (a) glutathione peptide (GSH), (b) superoxide dismutase (SOD), (c) peroxidase (POD), and (d) malondialdehyde (MDA) content. Different letters represent statistically significant differences relative to each other, where p<0.05 is considered significant. Values are presented as mean ± SE (n = 4).
Fig. 6 Effect of exposure to CuNPs and CuSO4 on the transcription of genes in Phaeodactylum tricornutum. Effects on the respiratory electron transport chain and synthesis of iron-sulfur cluster protein-related genes after (a) 48 and (b) 96 h, as well as the photosynthetic electron transport chain and cell division-related genes at (c) 48 and (d) 96 h. Different letters represent statistically significant differences relative to 25
each other, where p< 0.05 is considered significant. Values are presented as mean ± SE (n = 4).
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