Engineered Nanomaterial Activity at the Organelle Level: Impacts on

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Perspective

Engineered nanomaterial activity at the organelle level: impacts on the chloroplasts and mitochondria Luca Pagano, Elena Maestri, Marina Caldara, Jason C. White, Nelson Marmiroli, and Marta Marmiroli ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b02046 • Publication Date (Web): 28 Aug 2018 Downloaded from http://pubs.acs.org on August 30, 2018

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ACS Sustainable Chemistry & Engineering

Engineered nanomaterial activity at the organelle level: impacts on the chloroplasts and mitochondria

Luca Pagano,1 Elena Maestri,1 Marina Caldara,1 Jason C. White,2 Nelson Marmiroli,1,* Marta Marmiroli1

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Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parco Area delle Scienze 11/A, 43124 Parma, Italy.

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The Connecticut Agricultural Experiment Station, 123 Huntington Street, 06504 New Haven, CT, USA.

* [email protected]. Phone: +39 0521 905606.

Abstract

One of the challenges potentially limiting the continued widespread commercial development and application of engineered nanomaterials (ENMs) is the still perceived lack of knowledge on their potential toxic effects. Although evidence has been accumulating on the biological effects of ENMs at the level of cells, tissues, and organisms, wide differences in design make the results so far obtained not easily comparable. More importantly, risk assessment procedures are not sufficiently harmonized. Experimental data from assays involving fungi, plants, and animals have shown that mitochondria and chloroplasts are primary targets of metal-based ENMs. To provide a unifying picture of the molecular mechanisms of nanomaterial action, the aim of this perspective paper is to examine critically the current literature in this area: instances of mitochondrial and chloroplastic involvement in ENMs response are evaluated to describe the interplay between nuclear and organelle genomes observed in different organisms. This paper highlights critical parameters to consider when designing sustainable ENMs and suggests a standardized set of endpoints that can be sought when assessing the impact of ENMs exposure on environmental and human health.

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Keywords: Engineered Nanomaterials, Mitochondrion, Chloroplast, Safe by design, Risk Assessment.

Introduction

Nanotechnology has the potential to positively impact the lives of all people .1,2 Because of their unique physico-chemical properties and activity, ENMs have found use in a wide range of applications,3 from chemistry4 to cosmetics;5 from agriculture6-8 to food;9,10 from mechanical engineering11 to electronics and communications.12 With the widespread use of these new technologies, safety concerns have been raised for both public and environmental health.13-16 Works addressing the effects and responses to ENMs exposure have been entering the literature for some time, involving multiple models ranging from plants to animals,17-19 as well as considering potential trophic transfer within food chains20,21 and interactions with soil and the natural organic matter (NOM).22 Many questions regarding the mechanistic basis of toxicity and the ultimate risk of these materials remain unanswered. More advanced and detailed investigations are needed and should focus on: i) the physico-chemical form of ENMs within biota;23 ii) the oxidative potential of ENMs;24 iii) the effect of coating and functionalization,25 iv) dosimetry,26 v) the interaction with cellular components and biomolecules (e.g. corona protein),27 vi) impacts on soil microbiome28 and vii) quantitative models aimed at describing and predicting the relationships between ENMs structure and activity (QSAR).29 Moreover, the importance of robust experimental design to avoid artifacts and misinterpretations of ENMs testing, including addressing the relevance of exposure concentrations, cannot be underestimated.30,31 Although the current plant nanotoxicology literature is developing rapidly, results are difficult to harmonize because of differences in conditions, concentration, exposure duration, and medium used. There is also significant interest in devising


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alternative testing strategies (ATS) and new models to replace, reduce and refine traditional platforms that are not appropriate for ENMs.32 In cellular assays with fungi, plants and humans, the involvement of organelles such as mitochondria and chloroplasts in maintaining primary metabolic functions and in ROS scavenging appear to be a crucial part of the response to metal based ENMs exposure.33-36 Specifically, mitochondria show a high level of conservation in response to ENMs, ranging from simpler eukaryotic models (such as the baker’s yeast Saccharomyces cerevisiae) to higher eukaryotes such as dicot plants (e.g. Arabidopsis thaliana (L.) Heynh) and human cells. The results are particularly evident when using Gene Ontology (GO) bioinformatics tools to characterize biological processes and molecular pathways. For chloroplasts, evidence of the conservation of ENM response across different plant species has also been reported.35,37,38 On the other hand, mitochondria and chloroplasts may become important intracellular targets during the ENM exposure, leading to perturbations in the metabolic functionality and cellular detoxification processes.33,35 This perspective considered the involvement of mitochondria and chloroplasts in the ENMs exposure, for a mechanistic explanation of the physiological and molecular responses, assessed by genomic, transcriptomic and proteomic endpoints. This study provides a robust set of assays to test chloroplast and mitochondrial integrity and functionality, with the aim of contributing to standardization of methods for the evolution and the assessment of risk related to ENM exposure.

Methods and strategies to assess mitochondrial and chloroplast integrity and functionality

In the last decade, new concerns related to ENMs exposure to biota, and discussion over the methods of study to be used,39 have emerged. In particular, the role of mitochondria and chloroplasts is of great interest given their primary metabolic and energetic function, as well as their scavenging of

reactive oxygen species (ROS) and biosynthesis of secondary metabolites (in ACS Paragon Plus Environment

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plants). To provide a comprehensive picture of mitochondrial and chloroplast response to ENMs treatment, including an assessment of the structural and functional implications at the organelle level, a set of robust methods and techniques gleaned from the recent literature is reviewed below. The assays are assessed with a top-down approach, focusing on the capacity to give a perspective on the general morphological, functional and molecular effects in the two organelles. The panel of assays takes into account parameters such as the integrity of the organelle, the membrane potential, and some relevant enzymatic functions. All of these can be summarized as “phenomic” analyses as they represent the expression of the phenotypic traits of the organisms studied. Molecular analyses consider mitochondrial and chloroplastic genome integrity (mtDNA, cpDNA), and organism response during the ENMs exposure at transcriptomic and proteomic levels. An approach known as X-omics is used for in vivo experiments, and data are analyzed by extrapolating whole organisms organelle involvement in ENMs response to the cellular context. The methods for mitochondrial work are largely taken from the eukaryote Saccharomyces cerevisiae and from different human cell lines. For plants, mitochondrial and chloroplast methods are largely derived from Arabidopsis thaliana, and also from some widely used crop species (e.g. soybean, tomato). This fact may explain the more limited use of assays for chloroplast as compared with mitochondria for ENMs treatments. Importantly, many of the protocols reported can likely be adapted to different species or to the different experimental conditions.

Morphological, physiological and biochemical assays for mitochondria and chloroplasts

Morphological measurements obtained by Transmission Electron Microscopy (TEM) can be applied both to mitochondria and chloroplasts, providing information on organelle size, number (per cell), shape, and ultrastructure.40-42 Mitochondria chains structure, membrane integrity, and membrane potential (∆ψ) can be evaluated using several fluorescent dyes, including staining with


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Rhodamine or Tetramethylrhodamine ethyl ester (TMRE),34,40,43 or by fluorescent proteins specifically targeted to these organelles.44 On the physiological side, mitochondrial activity can be investigated by a range of colorimetric/fluorescent assays that measure the major functions such as the cellular respiration by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),43 or by oxygen consumption using an oxygraph. Furthermore, oxidative stress can be monitored by measuring total ROS and/or nitric oxide (NO) concentration and/or the glutathione redox state. ROS can be monitored by 2',7'dichlorofluorescin diacetate (DCFDA), which can be oxidized to fluorescent dichlorofluorescein in the presence of ROS.45,46 Nitric oxide production is related to NO synthase; the activity of the latter has been monitored in the past by measuring the conversion of radioactive arginine to citrulline. In addition, a highly sensitive colorimetric method focused on the enzymatic conversion of nitrate to nitrite, followed by quantitation using Griess Reagent can be used.47 Glutathione oxidation states (GSH/GSSG+GSH) can be evaluated biochemically by oxidation of GSH with 5,5’-dithiobis-2nitrobenzoic acid (DTNB) to GSSG and then with the formation of fluorescent 2-nitro-5thiobenzoic acid (TNB).48 Mitochondrial measurements in plants have generally been focused on the functionality of cellular respiration and ROS production; these parameters can be difficult to discriminate because of the presence and contribution of the chloroplast to energy production and ROS scavenging. For chloroplasts, the main physiological measurements that have been reported are the photosynthetic efficiency in term of measurements for chlorophyll a, b and carotenoids concentrations,49 as well as the chloroplast oxygen production and photochemical activity.50 Abscissic acid (ABA) and indole3-acetic acid (IAA) production can also be assessed41 to quantify hormones production in response to stimuli.

Genomic, transcriptomic and proteomic analysis


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Genomic tools for target identification use different strategies depending on the organism being studied. For example, the extensive genomic knowledge of the model organism S. cerevisiae enables the use of barcoded collections of about 7000 deletion mutants which cover all loci. This can provide a powerful platform to obtain information on specific targeted genes involved in the response to stimuli or genes that are physically or functionally involved in biological processes.51 For more complex genomes, such as that of A. thaliana, additional tools were developed to specifically or casually target genes across the entire length of the genome. Tools that have been already used for this type of analysis are T-DNA or transposon tagging.52,53 Depending on the extension and position that the inserted element occupies on the plant genome, these techniques can either suppress (knock-out), or modify (knock-in) target gene expression. The integrity of mtDNA and cpDNA can be assessed by an absolute quantification assay using Real Time quantitative Polymerase Chain Reaction (RT qPCR). Selection of mutagenized lines can also be improved by using new genome editing based methodologies (e.g. CRISPR-Cas9)54 that can produce even single nucleotide modifications in targeted genes of interest. Methods available for transcriptomic analyses of mitochondrion and chloroplast include those widely used in RNA quantitation such as microarray55 and RNAseq.56 The microarray technique is based on the signal obtained by the homology of a fluorescent probes generated by RNA to the probe sequence attached on the microarray surface, whereas RNAseq provides the complete sequencing of the RNA population present in the sample analyzed. The two techniques possess both strength and weaknesses related to physical and operational limits of sample preparation protocols, devices and the bioinformatic tools required for data analyses.55,56 Microarray analyses produce a relative quantification of the RNA sample, and require further validation of the results by orthogonal assays (e.g. Real Time qPCR); this confirmation step is not typically necessary for RNAseq techniques. Proteomics is based on protein separation techniques according to the different physicochemical properties of the biomolecules of interest (e.g. isoelectric point, protein mass, ACS Paragon Plus Environment

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hydrophobicity),57 followed by mass fingerprinting through mass spectrometry (MS)-based techniques. The results produced can be qualitative (presence/absence), semi-quantitative or quantitative depending on the analytical platform used.58 Isobaric tags such as iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) and TMT (Tandem Mass Tag) have been used to provide quantitative levels of protein abundance.59,60 Through the combined use of phenomic, genomic, transcriptomic and proteomic data, it is possible to provide a more mechanistic and comprehensive understanding of the cellular response to ENMs exposure and more specifically, the role of mitochondrion and chloroplast in this response.

The mitochondrial role in ENMs response

Mitochondrion, as the core of metabolic processes in the eukaryotic cell, has the essential role in ATP production by oxidative phosphorylation, providing energy for all cellular processes through oxidation of carbohydrates or fatty acids. The process produces an electro-chemical gradient across the mitochondrial inner membrane, thereby generating a proton motive force that supports inter-membrane ATPases, transport substrates or ions, and produces heat. Exposure to ENMs can interfere with mitochondrial function generating different symptoms of stress in the different models considered.

Mitochondrial phenomics

Several physiological experiments have shown how mitochondria can be affected by ENMs exposure. Zheng et al.61 reported the negative effect of CdSe/ZnS core/shell QDs on human keratinocytes using fluorescent microscopy, flow cytometry, and TEM. The authors assessed the differences in surface coatings and charges against cellular uptake, ROS generation, cytotoxicity, ACS Paragon Plus Environment

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and mitochondrial targeting derived from the QDs functionalization. Li et al.40 reported that the presence of ZnO NPs (10 mg mL-1) in rat liver cells resulted in an alterations of the mitochondria ultrastructure, collapse of the membrane potential, swelling, decreased respiration rate, permeabilisation of the inner membrane, release of cytochrome c, and generation of ROS. Similar results with regard to mechanisms of action and morphological effects were partially confirmed by Zhang et al.62 in mutant strains of Saccharomyces cerevisiae using a slower dissolving ZnO NPs. Similarly, Siddiqui et al.43 showed that CuO NPs (2–50 µg mL-1) induced dose-dependent cytotoxicity in HepG2 cells that was mediated by ROS generation and oxidative stress, and lead directly to a mitochondrial-mediated cell apoptosis. Tang et al.63 described how CdTe QDs (19 µM) can induce mitochondrial dysfunction in Danio rerio (zebrafish) liver cells. Pasquali et al.34 reported that in Saccharomyces cerevisiae, exposure to 75-150 mg L−1 CdS QDs increased the levels of ROS and modified the redox state by decreasing the levels of reduced/total glutathione (GSH/GSSG+GSH); in addition, there was a reduction of both oxygen consumption and cytochrome c abundance, with a consequent disruption of membrane potential and modification of organelle morphology. Interestingly, some of these phenotypic changes are consistent with observations on selected tolerant knock-out mutants identified by Marmiroli et al.64 indicating the protective role of this organelle in the response to ENM exposure. Results are summarized in Figures 1 and 2, in order to correlate all the analyses performed34,64 and to extrapolate the major components of the response. Furthermore, physiological measurements (e.g. NO, ROS, mtDNA integrity) related to mitochondrial function in HepG2 cells exposed to CdS QDs was conducted to highlight potential biomarkers of exposure and effect associated with QDs or other metal based ENMs.65,66 At concentrations ranging from 3 to 14 µg mL−1, CdS QDs induce only minor damage to genomic DNA but do trigger the mitochondria-mediated intrinsic apoptotic pathway, primarily because of the increased ROS production.65,66 Separately, Maurer and Meyer33 reported mitochondria-targeted specific effects of Ag-based ENMs which resulted in a loss of membrane potential, inhibition of the ACS Paragon Plus Environment

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oxidative phosphorylation enzymes, and changes in calcium sequestration within mitochondria. Literature reported the conservation of mitochondrion physiological/biochemical effects of ENMs in the different organisms considered, this might reflect a preservation in the molecular mechanisms related to the ENMs response.

Molecular response in yeast and animal systems

A number of molecular studies have been published that investigate S. cerevisiae34,64 and human cells65 exposure to different ENMs by evaluating a panel of genes and pathways involved in mitochondrial response. Marmiroli et al.64 worked with yeast exposed to CdS QDs treatment (200 mg L-1); the authors isolated 112 sensitive and 114 resistant mutants from the haploid collection of deletion strains, focusing on genes whose deletion was involved in abiotic stress response. Complementary analysis of these mutants with a genomic library led to the identification of three primary genes of interest: HSC82, which encodes a heat shock 90 protein;67 ALD3, whose gene product is a cytoplasmic aldehyde dehydrogenase;68 and DSK2, which is involved in protein ubiquitination.69 Some of the other genes that were altered by CdS QDs response were also observed to be involved in the response to other types of ENMs. For example, TRK1 is a gene encoding for a structural component of the Trk1p–Trk2p complex K+ transport system;70 Smith et al.71 observed that a ∆trk1 mutant was resistant to functionalized Au NPs treatment. Strtak et al.72 grew S. cerevisiae for 24-days (for 100 generations) in presence of CdTe QDs at half the minimum inhibitory concentration (MIC50) of the wild type strain; the authors noted that this exposure led to the selection of resistant ∆bul1 mutated strains that showed a higher fitness in presence of QDs. BUL1, like DSK2, has an important role in polyubiquitination in yeast;73 specifically, it is involved in mediating the transfer of the ubiquitin. Interestingly, BUL1 was found to also be involved in the response to CdS QDs exposure.64 Concerning mitochondrial organization, genes isolated by the mutant screening are mainly involved in inner mitochondrial membrane and metabolic functions: i) ACS Paragon Plus Environment

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SOM1 encodes for a peptidase subunit involved in protein processing in the intermembrane space;74 ii) PHB2 is a subunit of a chaperone complex and mitochondrial segregation;75 iii) HMI1 is an ATP-dependent helicase required in maintaining mtDNA integrity;76 iv) POR2 is a component of a voltage-dependent anion channel;77 while among the genes involved in mitochondrial metabolism, were isolated v) PDB1 encoding a subunit of the pyruvate dehydrogenase complex,78 vi) UPS2 involved in phospholipid metabolism79 and vii) PIM1 involved in protein metabolism.80 These genes might be an interesting starting point for determination of putative biomarkers of ENMs exposure, in yeast. Pasquali et al.34 analyzed the transcriptomic response of yeast to CdS QDs, specifically focusing on mitochondrial functionality. The authors coupled the transcriptomic results with morphological and physiological parameters such as cytochrome function, ROS production and mtDNA integrity by measuring the induction of respiratory deficient (RD) phenotypes. Interestingly, there were correlations between the cellular transcriptomic profile and the phenotypes of the isolated (sensitive and tolerant) mutants, showing gene both involved in mitochondrial organization and maintenance, as reported in Figure 3a and in the network (Figure 4). The importance of mitochondria in CdS QDs response was also confirmed in the human cell lines HepG265 where the authors observed the up-regulation of several genes involved in mitochondrial functions or strictly associated with the mitochondrial functionality: AIFM2 and APAF1 were involved in the mitochondrion-mediated intrinsic apoptotic pathway.81 Separately, the genes involved in oxidative stress response, OXR1, AOX1 are associated with protection from ROS and the regulation of ROS homeostasis, respectively; in addition, ATG3 and ATG7 were involved in membrane nucleation and autophagosome formation.65 Other possible biomarkers specific for mitochondria function are LONP1, mitochondrial matrix protein that belongs to the Lon family of ATP-dependent proteases, which mediates the selective degradation of misfolded, unassembled or oxidatively damaged polypeptides;82 and HSPD1, a mitochondrial heat shock protein.83 The effects of exposure to a low concentration of QDs on HepG2 and S. cerevisiae mtDNA was negligible.34,65 ACS Paragon Plus Environment

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The mechanisms underlying CdS QDs response in HepG2 cells, as confirmed by RNAseq, are shown in Figure 5, which is taken from Paesano et al.66 These findings align with what observed in human breast adenocarcinoma MCF-7 cell lines exposed CdTe QDs (~5 × 109 QDs cell-1).45 ROSinduced activation of the mitochondrion-mediated intrinsic apoptotic pathway was also observed in mice exposed to 2 µg ml-1 of Cu2O NPs.46 Several examples of quantitative proteomics combined with physiological and genomic analyses in model organisms treated with ENMs that showed mitochondrial implications, have been reported in the literature. For example, Rong-Mullins et al.84 observed that S. cerevisiae treated with Cu NPs possessed an over-abundance of 19 metal transporters (e.g. CUP1 or FIT2) that are known to regulate Cu response and Fe levels of Sod2, a mitochondrial superoxide dismutase. Gioria et al.85 reported DNA damage, inflammation, oxidative stress, and mitochondrial injury in human Caco-2 cells exposed to Au NP at 300 µM.

Mitochondrial molecular response in plants

The mitochondrial involvement of plants in response to ENMs exposure shows a clear conservation of mechanisms with yeast.35 Particularly, GO classifications of mitochondrial organization and response to stress/stimuli are redundant across the different species (Figure 3a) and are represented in the network in Figure 6. For example, At3g23990 (HSP60), which is involved in mitochondrial organization and response to several stimuli (heat shock, heavy metals),86 is up-regulated in A. thaliana exposed to TiO2 NPs but down-regulated with ZnO NPs treatment. In addition, At4g39460 (SAMC1), a S-adenosylmethionine transmembrane transporter involved in transport to organelles and their organization,87 was up-regulated by TiO2 NPs exposure. Regarding genes related to response to stimuli, response to metal (bivalent) ions could be significant as metal oxides release part of the ions; the following genes are modulated upon exposure to TiO2, ZnO and CuO NPs: At1g17290 alanine aminotransferase (AlaAT1)88 is involved in response to cadmium ions ACS Paragon Plus Environment

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and to hypoxia; At1g48030, At3g17240, and lipoamide dehydrogenases (mtLPD1, mtLPD2)89 are involved in response to cadmium ions and light; At1g63940, a monodehydroascorbate reductase (MDAR6),90 is involved in response to cadmium ions and to cold; and At4g37910 is a mitochondrial heat shock protein 70-1 (mtHsc70-1)91 that is involved in protein folding, response to cadmium ion and to salt stress. In the cases of ZnO and CuO NPs, the effects can likely be explained by the ion release21,92,93 but the mechanism remains unclear for TiO2. A possible explanation for the effects of TiO2 may be related to the different conditions used for transcriptomic analyses (e.g. early/longterm response).35 Interestingly, genes such as At5g62520 (SRO5), which is involved in ROS scavenging and response to salt stress,94 was up-regulated in all the treatments considered (CdS QDs, ZnO, CuO, TiO2 and Ag NPs), with the exception of CeO2 NPs. Notably, mitochondrial bivalent metal ions, fatty acid and ammonium transporters seem to have a major role in ENM response (Figure 3).

Chloroplast involvement in the ENMs exposure

Alterations in photosynthetic activity have been commonly reported as an indicator of plant physiological response to ENMs exposure.95 The existing literature clearly suggests that chloroplasts and mitochondria play a pivotal role in ENMs response and detoxification, but they may also be a primary targets for nanotoxicity. Oxidative stress sensing and signaling pathways within chloroplasts are well known.96 For example, Wang et al.37 demonstrated direct evidence of ENMs impacts on the two photosystems and on the thylakoid electron transport chain.

Morphological, physiological and biochemical effects in chloroplasts


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The physiological effects of ENM exposure differ not only with particle type but also with the plant species studied.38,49 This may determine positive, neutral or negative effects depending on the species studied, the physiological assay, and the type of molecular analysis.35,38,97 For example, Ag NPs (10-15 nm) at concentrations higher than 500 mg L-1, significantly reduced chlorophyll content in tomato (Solanum lycopersicum L).98 Trujillo-Reyes et al.99 observed that a 15-day exposure of Lactuca sativa L. to core-shell Fe/Fe3O4 reduced the chlorophyll a, b content in the plant tissues; a similar response was noted for A. thaliana treated with 72 µM of Fe2O3 NPs for different exposure durations (7 to 56 days).100 Nair et al.101 showed that a 21-day treatment of CuO NPs (50 nm) at concentrations higher than 100 mg L-1 negatively affected photosynthetic pigment content in mung bean (Vigna radiata (L.) Wilczek). Marmiroli et al.102 demonstrated that 14-day exposure to 80 mg L-1 of CdS QDs (5 nm) significantly decreased the chlorophyll production in wild type A. thaliana (accession Ler-0). Exposure to NP CeO2 (25 nm) produced either negative, positive or neutral effects of pigments, depending on plant species and concentrations used: treatments with concentrations higher than 1000 mg L-1 in A. thaliana103 or 500 mg L-1 in rice (Oryza sativa L.)104 significantly reduced the chlorophyll content. Conversely, Rossi et al.105 observed an increase in chlorophyll content in soybean (Glycine max L.) exposed to 500 mg L-1 CeO2 NPs, suggesting enhanced photosynthetic efficiency upon ENM treatment, also reported by Cao et al.,106 coupled with increased levels photorespiration and maximum carboxylation rate. Zhao et al.107 reported an absence of effects on chlorophyll content or chloroplastic functionality for cucumber (Cucumis sativus L.) exposed to 400-800 mg L-1 ZnO NPs in a life cycle study. Rui et al.108 reported increased root length, plant height, biomass, and chlorophyll content of peanut plants (Arachis hypogaea L.) exposed to Fe2O3 NPs. Li et al.109 reported that the chlorophyll content of rice was increased by treatment with molybdenum sulfide nanosheets. Photosynthetic activity, as a whole function related to the chloroplast, becomes a strong indicator of the stress induced by ENMs presence in plants. Activity of ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) is significantly affected by ENMs ACS Paragon Plus Environment

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exposure: Jiang et al.110 reported that a 10 mg L-1 Ag NPs treatment on the model aquatic higher plant Spirodela polyrhiza affected photosynthetic activity, inhibited Photosystem II, and reduced the activity of RuBisCo (-37%). On the other hand, it is remarkable that treatment of zucchini plants with 1.5-7.5 mg L-1 SiO2 NPs (12 nm) mitigates the effects of salt stress by increasing gas exchange and chlorophyll fluorescence parameters, such as photosynthetic rate (enhancing the photosynthetic pigments synthesis and Photosystem II activity), transpiration rate, and the rate of electron transport.111 Therefore, there are accumulating evidences that ENMs may stimulate plant photosynthesis, as reported also by Shweta et al.,112 Tarafdar et al.,113 Pradhan et al.,114 cited among others for brevity.

Genomic, transcriptomic, and proteomic evidence

Chloroplast response to ENMs can be assessed also with molecular strategies, able to highlight the correlations between the physiological and the molecular effects related to the ENMs exposure. CdS QD resistant A. thaliana phenotypes that had been obtained from an Ac/Ds mutagenized line were exposed to a CdS QDs concentration that was lethal for the wild type;102 the authors showed the involvement of genes related to stress response (At1g13880, ELM2), and seed reserve protein content, as well as in [4Fe-4S]-cluster-containing complex assembly in the chloroplasts (At3g24430, HCF101). Ruotolo et al.35 used a high-throughput analysis to consider all the genes involved in the ENMs response in A. thaliana. The authors focused on genes located on chloroplastic genome and those located on genomic DNA whose gene products showed a chloroplastic localization or function; several GO categories were commonly represented and reflected the main pathways involved, including metabolic processes, response to stimuli and transporters (Figure 3b). Using this information as a starting point, it is possible to assemble a network of gene interactions for the chloroplast (Figure 7). The common metabolic processes ACS Paragon Plus Environment

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highlight several genes involved in branched-chain amino acid metabolism, as well as fatty acid and secondary metabolite metabolism. The leucine biosynthetic genes At1g31180 (IMD3), At2g43100 (IPMI2), and At3g58990 (IPMI1) are strongly down-regulated. Similarly, fatty acid metabolism genes At2g30200, At2g29980 (FAD3), and At4g15440 are generally down-regulated, with the exception being TiO2 NP exposure. Importantly, the above processes are impacted by many types of stressors, including salinity and ROS, or in response to karrikins, a family of seed germination regulators produced by the carbonization of plant tissues.115,116 Concerning GO categories related to stimuli response, several genes involved in ENMs exposure/effects are known to be triggered by multiple stressors (e.g. ROS, light, heavy metals and salts), suggesting that ENMs exposure may cause damage in the plant at cellular and tissue levels.92 Among the genes belonging to this class, At2g15620 (NIR), a gene involved in reduction of nitrite and in response to salt stress,117 was down-regulated upon NP ZnO, CuO and CdS QDs exposure. At3g47860 (CHL), a chloroplastic lipocalin involved in oxidative stress response,118 is up-regulated by TiO2 NPs and CdS QDs and down-regulated by CuO treatment. At4g25100 (FSD1), a gene that encodes for a Fe-Superoxide dismutase that is involved in response to oxidative stress and to bivalent ions,119 is down-regulated by NP CuO and ZnO treatments. Interestingly, the effects of ZnO, CuO and CdS QDs seem to be more similar among them than to other types of ENMs (e.g. CeO2, TiO2), possibly due to a higher rate of dissolution from these metal oxides or to their intrinsic physico-chemical properties.35,120 Photosynthetic genes are a representative GO class modulated by ENMs exposure.35 Wang et al.37 reported that in A. thaliana, the thylakoid membrane transport chain involved in the transmission of the electrons between photosystems II and I, specifically the cytochrome b6f, was a sensitive target for CuO NPs. Pagano et al.38 demonstrated that several genes encoding for structural components of the chloroplast, as well as cytochrome b6f, can serve as biomarkers of exposure for metal based-ENMs, not only in model organisms but also in crop species such as zucchini (Cucurbita pepo L.) and tomato (Solanum lycopersicum L.). Another good biomarker is ACS Paragon Plus Environment

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AtCg00590, named ORF31 in A. thaliana, whose sequence is highly preserved across the plants. Sulfate assimilation is also related to chloroplastic response to abiotic and biotic stress.121 The tripeptide glutathione is widely known to be critical in ROS detoxification and as a substrate for the synthesis of protective molecules such as phytochelatins and glucosinolates, whose biosynthesis is regulated by genes such as At3g22890 (APS1) and At4g39940 (APK2).121 Importantly, these genes are known to be significantly affected upon exposure to NP Ag, Ti and CdS QDs.35 A proteomics-based comparison obtained by analyzing the over- and under-abundant proteins of A. thaliana122 partially confirmed the transcriptomic analyses, including the chloroplastic involvement in the response to CdS QDs. Approximately, half of the proteomic changes correlate with the transcriptional modulation, which was mainly triggered in response to oxidative stress generated by ROS production. Confirmation of these results were also highlighted utilizing more advanced quantitative proteomic techniques with different nanomaterials (e.g. CeO2)123 and isobaric tag analyses.

Similarities and differences across organelle ENMs response

Response to ENMs in mitochondrial and chloroplast shows a certain level of conservation, not necessarily in terms of orthologous genes regulated across the different species, but certainly in the main processes involved (Figures 3, 8). This may depend by several parameters that need to be taken into account during experiments: i) the differential (positive or negative) effects of the ENMs tested; ii) the sensitivity of the organism to the ENMs due to the ratio biomass/ENM concentration at the treatment (directly linked to physiology of the organisms, or to the growth stage in which the treatment is performed); iii) the experimental condition (e.g. ENM concentration, duration of exposure, medium used); iv) the analytical platforms used (e.g. different instrument sensitivity,


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bioinformatics tools with insufficient sensitivity to characterize the biological information obtained). Although the modes of action of the different ENMs are still under investigation, by comparing the mitochondrial and chloroplast response, it is possible to highlight common genes and pathways (Figures 8a, 9). The primary response to ENM exposure is related to oxidative stress but, it remains unclear if the generation of intracellular ROS derives directly from damage at level of mitochondrial and chloroplast membranes, specifically at the level of electron transport chain. Several GO classes are conserved and with similar mechanisms of activation between the two organelles (with organism differences taken in account): response to stressors (chemicals, metal ions), and carboxylic acid and alpha amino acid metabolic processes are the most common categories represented (Figures 8b, 9). Study of GO classes related to the metal stress response are abundant in literature, both from the physiological and molecular perspective and across different model organisms.35,92,95,124-126 GO classes related to metabolic processes as a function of stress are also interesting given the importance to energy production via electron transfer through protein based-transport chains.115,127 GO classes involving sulfur compound transport and related metabolic processes such as S-glycosides compounds deserve particular mention.121 These processes are known to be redundant in plants for both organelles. Ruotolo et al.35 reported that glucosinolates may be considered as a potential storage for sulfur in S-deficient conditions that are common to metal-based ENMs exposure. In addition, mitochondrial transporters seem to be particularly affected by ENMs treatments across different species. Van Aken et al.128 noted that mitochondrial carrier proteins are over-represented among ENM-stress responsive genes, suggesting that stress may induce an increase in metabolites transport to the mitochondrion. Genes encoding these carriers contain several regulatory elements involved in triggering mitochondrial stress response, which was also reported in yeast.129 Interestingly, two genes were modulated both in the chloroplast and mitochondrion: At2g15620 (NIR1) gene that encodes for a nitrite reductase involved in nitrate assimilation in low ACS Paragon Plus Environment

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nitrate conditions,130 and At2g40300 (FER4) encoding for ferritin, which is essential to protect cells against oxidative damage.131 NIR1 modulation is often coupled with 14-3-3s protein regulation that are known to be triggered under conditions of nitrogen and sulphur starvation;132 14-3-3s family protein modulation was also observed during CdS QDs exposure by both transcriptomic and proteomic techniques.102 Furthermore, 14-3-3s proteins are involved in mitochondrial and chloroplast ATPase regulation.133 Studies that consider common processes or effects across different species and different ENMs will be particularly important and new tools that help to highlight and/or characterize the response at both the physiological and the molecular level are needed. Pagano et al.38 developed putative molecular biomarkers from transcriptomic studies in Arabidopsis thaliana exposed to CdS QDs and showed that these markers were responsive to many types of metal-based ENMs in crops, although the magnitude of response varied somewhat with ENMs type and plant species. Combining these omic results with the commonly used physiological tests92,95 and plant metal uptake studies134 will enable a more mechanistic understanding of the ENMs exposure/effects. This type of work can be considered a first step toward uncovering the specific functions involved, and more specifically the regulation of the biological processes associated to ENMs exposure by studying not only gene expression at the level of transcription, but also at the level of gene products85,122,123 and metabolites.135,136

Conclusions

The applications of nanotechnology will result in exposure at many levels of biological complexity: the targeting of specific organisms (e.g. through nanopesticides and nanofertilizers),137 specific organelles (e.g. mitochondrion and chloroplast)138 or specific macromolecules (e.g. in drug delivery),139,140 and risk assessment procedures to establish the environmental and human health ACS Paragon Plus Environment

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safety of ENMs are of critical importance.141 In this context, the development of new methods for the isolation of biomarkers of exposure, effect and susceptibility,142,143 coupled with the new and advanced analytical techniques like single cell inductively coupled plasma mass spectrometry (scICP-MS)144 or synchrotron radiation-based µ-Fourier transformed infrared spectroscopy (SR µFTIR),145 may be functional tools to give new insight on ENMs properties, form and applications in the cellular (and intracellular) environment. An increased awareness on the safety and sustainability of ENMs may certainly speed up the development of better products selectively assembled to be “safe by design”: a concept refering to products that may not alter the cellular redox-equilibrium, and their reactivity, and whose target specificity and safety can be predictable over long time periods.146,147 The existence of extranuclear targets such as mitochondria and chloroplasts increases the possibilities for nanotechnology applications, but also increases the risks related to their use. For example, in humans there can be impacts of several mitochondrial diseases such as cancer, cardiovascular diseases, diabetes, neurological disorders that await for new treatment regimens.148150

Nanomedicine-based therapeutics can be a possible answer to these types of diseases, in terms of

nanoplatforms for drug delivery, photodynamic therapy and gene therapy.151 In plants (crop plants in particular) there is the need of treating diseases and pests. In this context, evidences of a dual effect in suppressing diseases and in enhancing the plant viability by Cu-based ENMs treatments are already reported in literature,152 as well as the effects on pollen morphology and allergenicity.153 These observations, testified by the evidences reported at the physiological and molecular level, can be considered as a new achievement in the comprehension of mitochondrial and chloroplast role in the ENMs response. These findings might be also functional for a more accurate risk assessment of environmental and health safety investigations, and in providing safer by design efforts.

Acknowledgements


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NM, EM and LP acknowledge the support of the project INTENSE, grant no. 652515. MC and MM acknowledge the support of FIL (“Fondi Locali per la Ricerca”), provided by University of Parma. JCW acknowledges USDA NIFA AFRI 2011-67006-30181, USDA Hatch CONH00145, and USDA CONH00147.

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Synopsis This paper examines the involvement of mitochondria and chloroplasts in ENMs response and highlights critical features and parameters to consider for the sustainable application of ENMs.

Figure captions Figure 1. Mitochondrial chains formation in yeast expressing mt-RFP (mitochondrial localized red fluorescent protein) and staining with rhodamine for mitochondrial membrane potential.34 When yeast were grown without ENMs treatment (in this case CdS QDs), both techniques allowed observation of mitochondria chains. The addition of the CdS QDs destroyed these chains. Punctuated mitochondria were observed upon transformation with mt-RFP; whereas the drop of the mitochondrial membrane potential did not allow the binding of rhodamine, but did allow a staining of the endoplasmic reticulum. The percentage of cells presenting mitochondrial chains is reported on top left. The addition of CdS QDs decreases almost 3-fold the presence of these structures. The bar corresponds to 5 µm. Figure 2. Principal Component Analysis (PCA) and proportion of the variance related to the physiological parameters assessed: growth phenotype (GP), from Marmiroli et al.64; duplication time (DT); gene expression related to wild type strain of the genes that were deleted in the resistant mutants (GE); ROS production; ratio between redox states of glutathione (GSR); mitochondrial chains derived by the treatment with CdS QDs (MC). The major components in the system are related to the redox state of glutathione (43.3% of the total variance) and duplication time/growth phenotype (21.7% of the total variance). Figure 3. (a) Mitochondrial GO classes (P-value lower than 10-5) involved ENMs response (data from Pasquali et al.34, partially reviewed in Ruotolo et al.35, from Paesano et al.66 (b) Chloroplast GO classes (P-value lower than 10-5) involved ENMs response (data partially reviewed in Ruotolo ACS Paragon Plus Environment

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et al.).35 The number of genes recognized for each GO class is reported in green for HepG2 cells, in red for Saccharomyces cerevisiae and in blue for Arabidopsis thaliana, respectively. Figure 4. Network of interaction of the mitochondrial genes involved in QDs response in Saccharomyces cerevisiae (data from Pasquali et al.)34. Genetic and physical interaction of the query genes (indicated with lines of different thickness) are represented in green and orange. Purple and yellow represent respectively the mitochondrial localization (when reported in literature). Figure 5. Schematics of the interaction between intrinsic apoptosis, autophagy and stress response pathways involved in the HepG2 cells response to CdS QDs. Up-regulation and down-regulation are indicated respectively as red and green arrows. [Reprinted from: Paesano et al., (2017)66; Copyright © 2017 Paesano, Perotti, Buschini, Carubbi, Marmiroli, Maestri, Iannotta and Marmiroli; licensee Elsevier].

Figure 6. Network of interaction of the mitochondrial genes involved in ENMs response in Arabidopsis thaliana (data from the review Ruotolo et al.)35. Physical interaction and colocalization of the query genes (indicated with lines of different thickness) are represented in red and blue. Purple and yellow represent respectively the mitochondrial localization (when reported in literature).

Figure 7. Network of interaction of the chloroplastic genes involved in ENMs response in Arabidopsis thaliana (data from the review Ruotolo et al.)35. Co-localization and predicted interactions of the query genes (indicated with lines of different thickness) are represented in blue and orange. Purple represents chloroplast localization (when reported in literature).

Figure 8. (a) Venn’s diagram related to the A. thaliana mitochondrial and chloroplast genes involved in the ENMs response, highlighting the genes commonly modulated in the two organelles. ACS Paragon Plus Environment

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(b) GO classes highlighted in A. thaliana mitochondrial and chloroplast response. Common GO categories are showed with the red arrows.

Figure 9. Principal and common molecular mechanisms that involve the mitochondrion (a) and chloroplast (b) highlighted by the analyses using genomic, transcriptomic and proteomic techniques.


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Biographies

Dr. Luca Pagano is a researcher at the Dept. of Chemistry, Life Sciences and Environmental Sustainability of the University of Parma. Dr. Pagano received his Ph.D. in Biotechnology from the University of Parma in 2014. His research activities are mainly focused on engineered nanomaterial risk assessment and ecotoxicological impact on model organisms and crop species.

Prof. Elena Maestri is Full Professor of Biology in the Department of Chemistry, Life Sciences and Environmental Sustainability of the University of Parma. Prof. Maestri received her Ph.D. in Genetics in 1989. Her work is in environmental biotechnology and phytoremediation, with a recent focus on emerging contaminants and risks for humans and the environments. Since 2016 she is the coordinator of the BSc in Biotechnology. Her teaching activities are in the field of Applied Biology.


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Dr. Marina Caldara is a researcher at the Dept. of Chemistry, Life Sciences and Environmental Sustainability of the University of Parma. Dr. Caldara received her Ph.D. in Bioscience Engineering from the Vrije Universitetit Brussel in 2007. Her research activities are mainly focused on studying effects of bioactive molecules in microorganisms, including engineered nanomaterials.

Dr. Jason C. White is Vice Director and Chief Analytical Chemist at the Connecticut Agricultural Experiment Station (CAES). Dr. White received his Ph.D. in Environmental Toxicology from Cornell University in 1997. After one year as a Post-Doctoral Associate at CAES, Dr. White joined the Department of Soil and Water in 1998. In 2009, he assumed the Department Head position in Analytical Chemistry and in 2013, was also appointed as Vice Director. The CAES Analytical Chemistry department provides sample analysis to all other state agencies, and also participates in the FDA Food Emergency Response Network (FERN) Chemistry Cooperative Agreement Program (cCAP). Dr. White also has research programs in two separate areas; nanomaterial contamination of agricultural crops, as well as the phytoremediation of soils contaminated with persistent organic pollutants. Dr. White also has Adjunct Faculty Appointments at the University of Texas-El Paso, University of Massachusetts, the University of New Haven, and Post University.


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Prof. Nelson Marmiroli is Full Professor in the Department of Chemistry, Life Sciences and Environmental Sustainability of the University of Parma. Prof. Marmiroli coordinates a team that includes full professors, associate professors, research associates and technicians. His current research topics are focused on: application of environmental biotechnologies for sustainability; phytoremediation, bioremediation, emerging contaminants (nanomaterials), interaction of plants with pollutants, genetic and molecular bases of genotype-environment interactions in different organisms (proteomic, genomic, transcriptomic analyses); molecular traceability of food supply chains for food safety and authenticity protection, coexistence of genetically modified plants with non modified plants.

Dr. Marta Marmiroli is a researcher at the Department of Chemistry, Life Sciences and Environmental Sustainability of the University of Parma. Dr. Marmiroli received her Ph.D. in Biotechnology from the University of Parma in 2002. Her main research activities are focused on environmental biotechnology and phytoremediation, with a recent interest on engineered nanomaterials contamination and related risks for humans and the environments.


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Synopsis This paper examines the involvement of mitochondria and chloroplasts in ENMs response and highlights critical features and parameters to consider for the sustainable application of ENMs.

84x47mm (300 x 300 DPI)



Figure 1. Mitochondrial chains formation in yeast expressing mt-RFP (mitochondrial localized red fluorescent protein) and staining with rhodamine for mitochondrial membrane potential.34 When yeast were grown without ENMs treatment (in this case CdS QDs), both techniques allowed observation of mitochondria chains. The addition of the CdS QDs destroyed these chains. Punctuated mitochondria were observed upon transformation with mt-RFP; whereas the drop of the mitochondrial membrane potential did not allow the binding of rhodamine, but did allow a staining of the endoplasmic reticulum. The percentage of cells presenting mitochondrial chains is reported on top left. The addition of CdS QDs decreases almost 3-fold the presence of these structures. The bar corresponds to 5 µm. 124x110mm (300 x 300 DPI)


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Figure 2. Principal Component Analysis (PCA) and proportion of the variance related to the physiological parameters assessed: growth phenotype (GP), from Marmiroli et al.64; duplication time (DT); gene expression related to wild type strain of the genes that were deleted in the resistant mutants (GE); ROS production; ratio between redox states of glutathione (GSR); mitochondrial chains derived by the treatment with CdS QDs (MC). The major components in the system are related to the redox state of glutathione (43.3% of the total variance) and duplication time/growth phenotype (21.7% of the total variance). 124x134mm (300 x 300 DPI)



Figure 3. (a) Mitochondrial GO classes (P-value lower than 10-5) involved ENMs response (data from Pasquali et al.34, partially reviewed in Ruotolo et al.35, from Paesano et al.66 (b) Chloroplast GO classes (Pvalue lower than 10-5) involved ENMs response (data partially reviewed in Ruotolo et al.).35 The number of genes recognized for each GO class is reported in green for HepG2 cells, in red for Saccharomyces cerevisiae and in blue for Arabidopsis thaliana, respectively. 279x470mm (300 x 300 DPI)


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Figure 4. Network of interaction of the mitochondrial genes involved in QDs response in Saccharomyces cerevisiae (data from Pasquali et al.)34. Genetic and physical interaction of the query genes (indicated with lines of different thickness) are represented in green and orange. Purple and yellow represent respectively the mitochondrial localization (when reported in literature). 299x305mm (300 x 300 DPI)



Figure 5. Schematics of the interaction between intrinsic apoptosis, autophagy and stress response pathways involved in the HepG2 cells response to CdS QDs. Up-regulation and down-regulation are indicated respectively as red and green arrows. [Reprinted from: Paesano et al., (2017)66; Copyright © 2017 Paesano, Perotti, Buschini, Carubbi, Marmiroli, Maestri, Iannotta and Marmiroli; licensee Elsevier]. 189x133mm (300 x 300 DPI)


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Figure 6. Network of interaction of the mitochondrial genes involved in ENMs response in Arabidopsis thaliana (data from the review Ruotolo et al.)35. Physical interaction and co-localization of the query genes (indicated with lines of different thickness) are represented in red and blue. Purple and yellow represent respectively the mitochondrial localization (when reported in literature). 250x283mm (300 x 300 DPI)



Figure 7. Network of interaction of the chloroplastic genes involved in ENMs response in Arabidopsis thaliana (data from the review Ruotolo et al.)35. Co-localization and predicted interactions of the query genes (indicated with lines of different thickness) are represented in blue and orange. Purple represents chloroplast localization (when reported in literature). 350x281mm (300 x 300 DPI)


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Figure 8. (a) Venn’s diagram related to the A. thaliana mitochondrial and chloroplast genes involved in the ENMs response, highlighting the genes commonly modulated in the two organelles. (b) GO classes highlighted in A. thaliana mitochondrial and chloroplast response. Common GO categories are showed with the red arrows. 350x408mm (300 x 300 DPI)



Figure 9. Principal and common molecular mechanisms that involve the mitochondrion (a) and chloroplast (b) highlighted by the analyses using genomic, transcriptomic and proteomic techniques. 290x449mm (300 x 300 DPI)


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