Gene expression changes in plants and microorganisms exposed to nanomaterials

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ScienceDirect Gene expression changes in plants and microorganisms exposed to nanomaterials Benoit Van Aken Unique properties of nanomaterials allow them to interact unexpectedly with biological systems. Analysis of the transcriptional response (change in gene expression) in exposed organisms constitutes a powerful approach for understanding the mechanisms of toxicity and molecular responses in cells exposed to nanomaterials. Transcriptional analyses have been conducted to study the effects of nanomaterials on humans, mammalian models, and other organisms important for the ecosystem. The present article reviews recent gene expression studies conducted to understand the effects of nanomaterials on plants and bacteria. As plants and bacteria are essential components of the food chain and/or play a central role in nutrient cycling and biodegradation, their interactions with nanomaterials have important implications for the environment and public health. Address Civil and Environmental Engineering, Temple University, Philadelphia, PA 19122, USA Corresponding author: Van Aken, Benoit ([email protected])

Current Opinion in Biotechnology 2015, 33:206–219 This review comes from a themed issue on Environmental biotechnology Edited by Spiros N Agathos and Nico Boon For a complete overview see the Issue and the Editorial Available online 29th March 2015 http://dx.doi.org/10.1016/j.copbio.2015.03.005 0958-1669/# 2015 Elsevier Ltd. All rights reserved.

Introduction Nanomaterials (NMs) are engineered structures with at least one dimension less than 100 nm. Because of their small size and large surface area, NMs exhibit unique physicochemical properties leading to their utilization in an increasing number of products and processes, ranging from self-sterilizing fabrics to hyperthermic destruction of tumor cells [1,2]. However, unique properties making NMs attractive for technological applications also cause them to interact with biological systems. Nanosized materials have been shown to enter living organisms, spread through organs and tissues, and exert various toxic effects at the cellular level, including membrane disruption, protein inactivation, DNA damage, Current Opinion in Biotechnology 2015, 33:206–219

disruption of energy transfer, formation of reactive oxygen species (ROS), and release of toxic substances [1–6] (see below). Human exposure to NMs may occur in the workplace (e.g., NM manufacturing industry), from NM release from customer products (e.g., NM-containing paints, fabrics, and cosmetics), or through ingestion of NM-contaminated food and water (e.g., plants growing on NM-contaminated soil). Besides occupational and consumer exposure, NMs have recently raised ecological concerns because of their probable dispersion into the environment [1,6,7,8]. Engineered NMs may enter the environment through intentional releases, such as the use of zero-valent iron (ZVI) nanoparticles (NPs) for groundwater remediation, or unintentional releases, such as discharge from production facilities and loss from customer products. Released NMs may then cycle through different environmental compartments and eventually contaminate living organisms [6]. Limited information is available regarding the fate of NMs in the environment and more research is needed to understand the migration, aggregation, dissolution, transformation, and interaction of NMs with organic matter in complex systems [6,9]. Similarly, little is known about the potential impact of NMs on specific ecological processes, such as nutrient cycling, plant–bacteria interactions, or mycorrhizal symbiosis [10]. Environmental regulation of nanomaterials has been slow to emerge, because of the late recognition of their environmental impact and lack of characterization of their toxic effects [11,12]. There is therefore a critical need for collecting more data about the ecotoxicity of NMs to support further regulatory efforts. As with other contaminants, the ecotoxicological assessment of NMs is frequently hindered by the low level of environmental exposure and multiplicity of potential cellular targets. Gene expression analyses for understanding the molecular mechanisms of toxicity (i.e., toxicogenomics) may help capture the cellular impact of contaminants even at very low dose [2,9,13–15]. Although the toxicity of NMs to human cell lines, mammalian models, and ecological indicator organisms (e.g., algae, fishes) has recently been the subject of intense investigation, their effects on plants and microorganisms have received less attention. As plants constitute the base of the terrestrial food chain, their exposure to nanomaterials may have important implications for agriculture and human health [1,6,8,16]. Similarly, the effects of NMs on bacteria may have consequences for the ecosystem (e.g., nutrient cycling) and environmental biotechnologies (e.g., wastewater treatment, biodegradation) [17]. www.sciencedirect.com

Plants and bacteria exposed to metal nanoparticles Van Aken 207

The present contribution reviews recent published studies reporting gene expression changes in plants and bacteria exposed to NMs.

Properties and toxicity of nanomaterials Specific interactions of NMs with biological systems originate mainly from their small size, large surface area, and intrinsic reactivity [2–5,17]. NM sizes fall within the scale of macromolecules (e.g., proteins, nucleic acids) and cellular organelles (e.g., ribosomes, mitochondria), resulting in unanticipated biological activities [18]. Besides specific nano-size effects, metal-based nanoparticles (MNPs) exhibit toxicity associated with the metal constituent(s). In short, NMs can interact with biological systems through (1) chemical, (2) mechanical, (3) catalytic, and (4) surface effects (Figure 1) [19]: 1. Chemical effects: Because of their large surface area, MNPs and quantum dots (QDs) may dissolve quickly in biological fluids as compared with their bulk counterparts, releasing toxic ions that interact with biomolecules and/or cause redox imbalances. For instance, the toxicity of silver (Ag) NPs is believed to be related, among other specific nanosized effects, to the release of silver ions (Ag+), which bind strongly to functional groups of proteins (e.g., sulfhydryl groups) causing alteration or inhibition of their activity [6]. Other metals, such as copper (Cu) and iron (Fe), react with oxygen and hydrogen peroxide to produce the hydroxyl radical (OH), which can damage most organic molecules, including proteins, lipids, and DNA [6,19]. 2. Mechanical effects: NMs — especially carbon-based nanomaterials (CNMs) — may act mechanically by direct association with cellular structures and by Figure 1

Release of toxic ions

Nanomaterials Ag

Oxidative damage to membrane

+

Cu

+

Cd

2+

Metal binding to proteins

ROS

Ag Endocytosis

Ag

+

+

Cu Fe

+

Ag

+

Ag

++

+

ROS

Generation of ROS ROS

Excessive catalysis

Membrane disruption, puncture

Oxidative damage to proteins Oxidative damage to DNA Membrane coating Current Opinion in Biotechnology

Observed and suggested mechanisms of action of nanomaterials (NMs) on cells.Source: Adapted from Rico et al. [8], Hajipour et al. [24], and Suresh et al. [25]. www.sciencedirect.com

clogging pores in walls and membranes. For instance, exposure to Escherichia coli cells to single-walled carbon nanotubes (SWNTs) was shown to affect the membrane integrity, resulting in cytoplasmic material leaking [20]. Aggregation of titanium dioxide (TiO2) NPs on root surfaces of exposed maize seedlings (Zea mays) reduced the root hydraulic conductivity, resulting in hydric stress affecting the plant development [21]. 3. Catalytic effects: Unlike soluble metal ions that are frequently bounds to specific chelators and/or compartmentalized in the cell, metal species at the surface of NPs may not be subjected to cellular regulation, while retaining catalytic activity, resulting in excessive generation of modified metabolites. For instance, the ‘ultrahigh chemical reactivity’ of Cu NPs, leading to excessive accumulation of alkalescent substances and resulting metabolic alkalosis, was shown to be responsible for their in vitro and in vivo toxicity [22]. 4. Surface effects: Negatively charged NM surfaces can bind positive functional groups, leading again to protein inactivation [19]. Metal oxide NPs frequently carry surface hydroxyl groups that are negatively charged at physiological pH and can covalently bind positive functional groups of proteins, such as sulfhydryl groups in cysteine residues. Such interactions are responsible for the specific affinity of MNPs for certain human proteins, including peroxiredoxin, annexin, and ribosomal proteins [19]. On the basis of the current knowledge, it seems that NM toxicity originates largely from the release of toxic metals (MNPs and QDs) and the generation of ROS (MNPs, QDs, and CNMs). In addition to these mechanisms, CNMs may exert mechanical effects on cell membranes. The modes of action of NMs on cellular structures are multiple, complex, and still poorly understood, especially considering the variety of NM sizes, shapes, compositions, and atomic arrangements. For instance, the cytotoxicity of SWNTs on E. coli was shown to increase with the proportion of metallic versus semi-conductor forms [23]. The mechanisms of toxicity of NMs toward microorganisms have been investigated primarily because of the potential use of NMs as antimicrobial agents [24,25]. Reported inhibitory concentrations of NMs toward bacteria range from 2.5 mg L 1 to >500 mg L 1, depending on the type of NMs and the bacterial species [24]. Bondarenko et al. [18] reviewed the toxicity of MNPs (Ag, copper oxide — CuO, and zinc oxide — ZnO NPs) in various organisms, including bacteria, algae, fishes, and mammalian cells. Although comparison between toxicological data recorded with different organisms and under different conditions has to be interpreted with caution, the authors estimated that bacteria were among the most tolerant groups — presumably due to metal resistance mechanisms (e.g., cation efflux systems) (Table 1). Current Opinion in Biotechnology 2015, 33:206–219

208 Environmental biotechnology

Table 1 Summary of toxicity data of three types of metal-based nanoparticles (MNPs) for different groups of organisms Group of organisms

Bacteria Plants Algae Fishes Mammalian cells

Median MICa for bacteria, median EC/ LC50a for algae, fishes, and mammalian cells, and median LOAELa for plants, mg L 1 (number of referencesb for plants and number of datab for other organisms) Ag NPs

CuO NPs

ZnO NPs

7.10 (46) 250 (5) 0.36 (17) 1.36 (17) 11.3 (25)

200 (13) 50 (1) 2.8 (5) 100 (1) 25 (21)

500 (15) 500 (6) 0.08 (5) 3.0 (4) 43 (25)

Refs c

Gene expression changes in bacteria

[18] [8,21] [18] [18] [18]

a

MIC, minimum inhibitory concentration; EC50, effect concentration 50%; LC50, lethal concentration 50%; LOAEL, lowest-observed-adverse-effect level. b Number of references or data sets used to derive the median values. c Refs [8,21] are review articles from which the values presented in the table were obtained (plants), Ref [18] provided directly the presented values (all other organisms).

The different modes of action of NMs that are described above are applicable to all living organisms, including bacterial cells (for review, see Refs [18,24,26,27]). However, the most frequently described or postulated toxic mechanisms of NMs on bacteria include oxidative stress (generation of ROS) [24,26,27], membrane disruption [24,27], DNA damage [18], inhibition of membrane electron transfer [24], and binding functional groups in macromolecules [24]. Prior research has shown that NMs enter plant tissues, presumably via pores in the cell wall and endocytosis pathways, and may be translocated through the vascular system [1,8,19,21]. Most studies on the phytotoxicity of NMs have been based on observable parameters, such as germination rate and biomass growth, revealing both positive (low exposure) and negative effects (high exposure) [1,8,21,28,29,30]. As for bacteria, all potential modes of action of NMs listed above are susceptible to occur in plants (for review, see Refs [1,8,19,21,25]). Nevertheless, the most commonly described or suggested mechanisms of NM toxicity in plants include physical surface coating and/or pore clogging (reducing hydraulic transfer and nutrient uptake) [19], oxidative stress (generation of ROS) [1,19,21], DNA damage [8], toxicity of dissolved metals [19,21], reduction of photosynthetic activity [1], and disruption of rhizospheric microbial communities [21].

Gene expression changes in response to nanoparticle exposure Gene expression analysis has been widely used to study the molecular bases of the toxicity of environmental contaminants — including NMs — in a variety of organisms. Gene-specific methods, such as reverse-transcription Current Opinion in Biotechnology 2015, 33:206–219

real-time (quantitative) PCR (RT-qPCR), are typically conducted on a limited suite of genes with identified responses and/or functions. High-throughput methods, such as DNA microarrays, although less quantitative than RT-qPCR, allow capturing the transcriptional response at the whole-genome scale [2,13,14].

Because of the widespread utilization of Ag as antimicrobial agent, many transcriptional studies conducted on NM-exposed bacteria have focused on Ag NPs (Table 2). Transcriptional analyses based on RT-qPCR or microarray have revealed a rather consistent pattern of gene expression in response to Ag NP exposure. For instance, two recent whole-genome microarray studies from Nagy et al. [26] and McQuillan and Shaw [31] focusing on the effects of Ag NPs on the model bacteria, E. coli, showed differential expression of genes involved in the regulation of oxidative balance, sulfur metabolism, and homeostasis of Ag, Cu, and Fe. Another investigation indicated that E. coli exposure to Ag NPs resulted in changes in the expression of genes involved in Cu homeostasis (Cu stress response) [32]. Using a different approach, Radzig et al. [33] observed that E. coli mutants deficient in genes involved in DNA oxidative damage and porin synthesis were less resistant to Ag NPs than wild type strains, suggesting effects through disruption of the redox balance and water homeostasis. Although Ag NP toxicity has been attributed by some authors solely to the release of Ag+ ions [26], McQuillan et al. [32] reported that Ag NP dissolution resulted in Ag+ concentration in the medium too low to explain the observed cell inhibition. In their microarray study, the same authors observed that the majority of differentially expressed genes were uniquely regulated by either Ag NPs or soluble Ag+, leading them to suggest that NPs delivered Ag+ directly at the membrane, resulting in higher cellular concentration [31]. Studies conducted on the antimicrobial effect of other MNPs have similarly shown expression changes in genes involved in oxidative stress and metal homeostasis (Table 2). An expression microarray analysis conducted on E. coli exposed to cerium oxide (CeO2) NPs revealed up-regulation of various oxidoreductases, suggesting oxidative stress, disruption of cellular respiration, and Fe deficiency [34]. Investigating the effects of ZnO NPs on Campylobacter jejuni, Xie et al. [27] observed up-regulation of oxidative and general stress response genes. Another study from Yang et al. [35] reported that both QDs and their dissolved metal constituents up-regulated metal efflux transporters and oxidative stress response genes in the model bacterium, Pseudomonas aeruginosa. Up-regulation of genes involved in response to oxidative stress (e.g., catalases, peroxidases) indicates that MNPs act on cells through the generation of ROS. Metals can transfer electrons to O2, forming ROS, such as superoxide www.sciencedirect.com

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Table 2 Summary of selected gene expression studies on the effects of nanomaterials (NMs) on bacteria and plants Nano-materials (NMs)

Species or group of organisms

NM size and properties

Concentration applied

Embedded in zeolite membrane

Release of 20 mg L Ag+ in 48 h

Ag NPs

E. coli

Diameter 142  20 nm

500–1000 mg L

Ag NPs

E. coli

Diameter 8.3 nm

4–150 mg L

Ag NPs

N. Europaea, A. vinelandii, R. etli, A. lipoferum, P. stutzeri

Diameter 35.4  5.1 nm

Ag NPs

E. coli (wild type and 4000 single gene deletion mutants)

Citrate-coated: diameter 25.5  8.2 nm, 45.0  18.0 nm, 66.5  25.0 nm; PVPcoated: diameter 168  85 nm; BPEIcoated: diameter 85.5  52.0 nm

10% MICc (2.5  10 3 mg L 1 for N. europaea), 60% of MICc (15  10 3 mg L 1 for N. europaea) 0.2–100 mg L 1

Ag NPs

E. coli

Diameter 142  20 nm

40 mg L

Au NPs

E. coli

4,6-Diamino-pyrimidine thiol-modified Au NPS

10 mg L 1 (for transcriptomic analyses)

CdO NPs

E. coli

Diameter 3 nm

0 120 mg L

CdS NPs

E. coli

Diameter 3 nm

0–50 mg L

1

1

1

1

Cell death related with Ag+ release

Inhibition of cell growth, association with cell wall, interaction with cell membranes Inhibition of cell growth, inhibition of biofilm formation Inhibition of cell growth

Inhibition of cell growth

Inhibition of cell growth, association with cell surface

1

L

Antibacterial activity, collapse of membrane potential, decrease of ATPase activity and ATP content, chemotaxis [+] Inhibition of cell growth, morphological changes, surface damage, generation of ROS Inhibition of cell growth, morphological changes, surface damage, generation of ROS

Gene expressionb: upregulation [+], downregulation [ ] Antioxidant (glutaredoxin, thioredoxin) [+], metal transport [+], metal reduction (multicopper oxidase) [+], ATPase pumps [+], antibiotic resistance [+], ferrochelatase [+], iron transporter [ ] Cu stress response [+/ ]

Refs

[26]

[32]

[33] Ammonia monooxygenase N. Europaea [+] at 10% of MIC, [ ] at 60% of MIC c

[10]

‘Ag-specific’ genes: response to Cu toxicity, oxidative stress, DNA damage, bacterial flagellar motor activity; ‘NP-specific’ genes: assembly of outer membrane lipopolysaccharides (LPS), quinone binding, ubiquinone synthesis (defense against ROS) Heat shock response [+], Fe– S assembly systems [+], Fe and SO42 homeostasis [+], redox stress response [+], copper homeostasis [+] ATP synthases [ ], peroxidase [ ], alkyl hydroperoxidase [ ], ribosomal protein (inhibition of tRNA-binding) [ ] Cell division proteins (septum formation) [ ]

[42]

Cell division proteins (septum formation) [ ]

[31]

[40]

[37]

[38]

Plants and bacteria exposed to metal nanoparticles Van Aken 209

Current Opinion in Biotechnology 2015, 33:206–219

Gene expression studies in bacteria Ag NPs E. coli, S. aureus (E. coli only for microarrays)

Physiological effects a

Nano-materials (NMs)

Species or group of organisms

NM size and properties

Concentration applied

1

Physiological effects a

CeO2 NPs

E. coli, S. oneidensis, B. subtilis (E. coli only for microarrays)

Diameter 6  3.5 to 40  10 nm

50–150 mg L

Poly-styrene NPs

E. coli (wild type and 4000 single gene deletion mutants)

Cationic (aminofunctionalized), diameter 56.9  2.1 nm

1–500 mg L

Carboxyl QDs (Cd, Zn, Se)

P. aeruginosa

Diameter 7.4  1.1 nm

20 nM (for transcriptomic analyses)

No growth inhibition <500 nM, extracellular precipitation of metal NPs

Coated QDs

N. Europaea, A. vinelandii, R. etli, A. lipoferum, P. stutzeri

PEI QDs: 1–200 nM; PMAO QDs: 50–800 nM

Inhibition of cell growth

TiO2 NPs

Soil bacteria

Polyethylenimine-coated (PEI) QDs: diameter 41.6  3.4 nm; Polymaleic anhydridealt-1-octadecene-coated (PMAO) QDs: diameter 32.6  2.7 nm Diameter 20–30 nm (in suspension 194 nm)

ZnO NPs

E. coli, C. jejuni, S. enterica (C. jejuni only for RT-qPCR) P. chlororaphis O6

ZnO NPs, CuO NPs www.sciencedirect.com

Zero-valent Fe (ZVI) NPs

P. stutzeri

2000 mg kg

Inhibition of cell growth

1

Inhibition of cell growth (IC50 = 158 mg L 1)

1

1

Diameter 30 nm

25–100 mg L

CuO: diameters <50 nm; ZnO: diameter <100 nm

CuO 200 mg Cu L 1; ZnO 500 mg Cu L 1

Sodium polyacrylic acidcoated ZVI NPs, diameter 50 nm

1000–5000 mg L

1

Change bacterial community structure: reduction of nitrogen fixers and methane oxidizers, increase of species involved in biodegradation of persistent organic pollutants Inhibition of cell growth, morphological changes, cytoplasmic leakage Increase of cellular level pyoverdine (PVD) by ZnO, decrease of cellular level of PVD by CuO Inhibition of cell growth, damage to cell wall

Gene expressionb: upregulation [+], downregulation [ ] Cytochrome d terminal oxidases [+], nitrite reductase [+], succinate dehydrogenase cytochrome b terminal oxidases [ ] (indicative of oxidative stress, disruption of cellular respiration, and iron deficiency) Lipopolysaccharide biosynthetic pathway, outer membrane transport channels, ubiquinone biosynthetic pathways, flagellar movement, and DNA repair systems Metal efflux transporters [+], superoxide dismutase (oxidative stress) [+], antibiotic resistance [+] Nitrogenases [+], nitrite/ nitrate reductase [+], ammonia monooxygenase [+] at low concentrations of PEI QDs and [ ] at high concentration PEI QDs

Refs

[34]

[41]

[35]

[44]

[48]

Oxidative stress [+], general stress response [+]

[27]

Inner membrane PVD transporter gene [ ] by CuO

[39]

Catalase (oxidative stress) [+], proteomic analysis: membrane proteins [ ], response to intracellular oxidative stress [+]

[48]

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Table 2 (Continued )

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1

5 mg L

Non-modified and carboxylated-SWNTs, diameter 1–2 nm, length 0.5–2 mm

0, 10, 50 mg L

Diameter 1–2 nm, length 5–15 mm

219 mg L

Gene expression studies in plants Nanosized Ag-silica A. thaliana hybrid complex

Diameter 20 nm

1–100 mg L

Ag NPs

A. thaliana

Diameter 20 nm

1–20 mg L 1, 5 mg L (for transcriptomic analyses)

Ag NPs

A. thaliana

Diameter 20 nm

>0.5 mg L 1 (for transcriptomic analyses)

Ag NPs

A. thaliana

Diameter 20 nm

0.2, 0.5, 1.0 mg L

1

Ag NPs

A. thaliana

Diameter 20 nm

0.2, 0.5, 1.0 mg L

1

E. coli

SWNTs

Marine bacteria: S. pomeroyi, O. lumbeijerinckii, V. splendidus, V. gigantis Denitrifying bacterium: P. denitrificans

SWNTs

SWNTs

Activated sludge

0–200 mg L

1

1

1

Reduction of viability, reduction of metabolic activity

Sigma factor [+], higher with SWNTs than MWNTs

[20]

Growth inhibition, bactericidal effect

Sigma E factor (stress response) [+], sigma S factor (membrane integrity [no effect]

[43]

Inhibition of growth and nitrate reduction

Ribonucleotide reductase (DNA damage) [+], nitrate reductase [ ], genes involved in energy metabolism [ ]

[45]

Change in bacterial community structure: reduction of Sphingomonadaceae 1

1

Increased growth at low dose and decreased growth at high doses, change in leaf morphology, increased pathogen resistance Increase of plant biomass at low dose, reduction of plant biomass at high dose

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Inhibition of growth, reduction of root elongation, disruption of thylakoid membrane, decrease of chlorophyll content Reduction of chlorophyll, increase of anthocyanin, lipid peroxidation, ROS production, changes in mitochondrial membrane potential No significant morphological changes reported

[47]

Pathogenesis-related (PR) gene (systemic acquired resistance-SAR) [+]

[53]

Response to metals [+], response to oxidative stress [+], thalianol pathway [+] (defense), response to pathogens [ ], response to hormonal stimuli [ ], regulation of organ size [ ] Antioxidant [+], oxidative stress response (superoxide dismutase, catalase) [+], water homeostasis (aquaporins) [+] Sulfur assimilation [+], glutathione biosynthesis [+], glutathione S-transferase [+], glutathione reductase [+]

[29]

Proliferating cell nuclear antigen (PCNA) [+], DNA mismatch repair — MMR (oxidative damage to cell components) [+]

[52]

[50]

[51]

Plants and bacteria exposed to metal nanoparticles Van Aken 211

SWNTs: diameter 0.9 nm, length 2 mm MWNTs: diameter 30 nm, length 70 mm Carboxylated-SWNTs, diameter 2–20 nm, length 0.3–1.4 mm

SWNTs, MWNTs

Nano-materials (NMs)

Species or group of organisms

NM size and properties

Physiological effects a

Concentration applied

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Ag NPs

Wheat (T. aestivum)

Diameter 10 nm

Ag NPs

Broad bean (V. faba)

Diameter 60 nm

12.5, 25, 50, 100 mg L

Al2O3 NPs

Tobacco (N. tabacum)

Not specified

1000–10 000 mg L

CeO2 NPs, In2O3 NPs

A. thaliana

CeO2 NPs: diameter 10– 30 nm; In2O3 NPs: 20–70 nm

250–5000 mg L

CeO2 NPs, ZnO NPs

Soybean (G. max)

0, 500, 1000, 2000, 4000 mg L 1

Fullerene soot (FS), TiO2 NPs, ZnO NPs

A. thaliana

CeO2 NPs: diameter 7 nm, ZnO NPs: diameter 8 nm ZnO: diameter <100 nm; TiO2 (anastase: rutile 80:20): diameter <150 nm; FS (7% fullerene soot, 76% C60, 22% C70)

TiO2 NPs

A. thaliana

Diameter 155 nm

2500 mg L leaves

TiO2 NPs

Tobacco (N. tabacum)

Diameter <25 nm

1000–50 000 mg L

100 mg L

1

1

1

1

1

sprayed on

1

Reduction of shoot and root length, increase of root branching, accumulation of Ag in shoots, increase of oxidized glutathione Genotoxicity: increase of chromosomal aberrations and micronuclei, decrease of mitotic index Dose-dependent inhibition of plant growth, reduction of biomass, root length, and leaf count CeO2: Increase of biomass at low dose and decrease at high dose, reduction of chlorophyll, increase of anthocyanin, lipid peroxidation at high dose; In2O3: No significant effect Genotoxicity: DNA damage, mutations, deletions, homologous recombination Reduction of plant biomass with ZnO and FS NPs, not with TiO2 NPs

Increased chloroplastic light absorption, increased lightharvesting complex II (LHCII), regulation of light distribution between photosystems, acceleration of light energy conversion, water photolysis, and oxygen evolution Dose-dependent reduction of germination, inhibition of plant growth, reduction of biomass and root length

Gene expressionb: upregulation [+], downregulation [ ] Gene encoding metallothionein (metal ion sequestration) [+]

Refs

[58]

[61]

MicroRNA (miRNA) [+] (implication in abiotic stress response)

[59]

CeO2 and In2O3: Stress response [+], sulfur assimilation [+], glutathione synthesis [+]

[54]

[66]

ZnO and FS: Abiotic and biotic stress response [+] (oxidative stress, draught, pathogens), biosynthesis [ ], cell organization [ ], electron transport [ ], energy pathways (indicative of response to toxic stress) [ ] Light-harvesting complex II (LHCII) b gene [+]

[49]

MicroRNA (miRNA) [+] (implication in abiotic stress response)

[60]

[55]

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Table 2 (Continued )

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1

A. thaliana

Diameter 5 nm

0–150 mg L

Graphene oxide NPs

A. thaliana

Diameter 40–50 nm

0.01–1 mg L

SWNTs

A. thaliana, rice (O. sativa)

Diameter 1–2 nm, length 5–30 mm

5, 25, 100 mg L

SWNTs

Maize (Z. mays)

Diameter 1–2 nm, length 30 mm

20 mg L

MWNTs

Tobacco (N. tabacum)

Diameter 20 nm, length 0.5–1 mm

0.1–500 mg L

MWNTs

Barley (H. vulgare), soybean (G. max), maize (Z. mays)

Diameter 15–40 nm, length >1 mm

50, 100, 200 mg L

a b c

Inhibition of germination and plant growth (wild-type)

1

Inhibition of development of plant seedlings in the presence of polyethylene glycol (PEG)induced (drought) and NaClinduced (salt) stress Negative effects on A. thaliana and rice protoplast cells, including cell death, DNA damage, and generation of ROS Enhancement of growth of seminal roots, inhibition of growth of hair roots

1

1

1

Enhancement of growth over a wide range of concentrations (5– 500 mg L 1) 1

Enhancement of germination and plant growth

The physiological effects presented are usually limited to those listed in the abstract of the referred articles. The gene differentially expressed presented are usually limited to those listed in the abstract of the referred articles. MIC, minimum inhibitory concentration.

Defense response [+], SAR [+], pathogenesis [+]; tolerance of mutant line atnp01: synthesis of storage and lipid transport proteins (stress response), tolerance of mutant line atnp02: MYB transcription factor superfamily (development and metabolism regulation, response to stress) Root development [ ], response to drought and/or salt stress [+]

[56]

Ascorbate peroxidase and superoxide dismutase (ROSscavenging) [+]

[62]

Development of each type of seminal roots [+], development of hair roots [ ], acetyltransferase (HAT) (histone H3) [+] Cell division (CycB), cell wall formation (extensin — NtLRX1), and water transport (aquaporin — NtPIP1) [+] Water channel proteins [+]

[65]

[57]

[63]

[64]

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CaS QDs

214 Environmental biotechnology

(O2 ), hydrogen peroxide (H2O2), and hydroxyl radical (OH), which are known to damage cellular components [36]. Similarly, up-regulation of genes involved in sulfur metabolism probably indicates the biosynthesis of antioxidant molecules (e.g., glutathione) capable of neutralizing ROS [26,31]. Up-regulation of genes involved in transport and oxidation/reduction of Ag and Cu (isoelectronic metals) may reflect an attempt to decrease intracellular level of toxic ions. Differential expression of genes involved in Fe metabolism may be a response to oxidative stress by preventing Fenton’s reaction [26]. Besides oxidative stress response and metal homeostasis, other cellular processes have been shown to be affected by MNPs. Hossain and Mukherjee [37,38] reported that both CdS and CdO NPs inhibited septum formation in E. coli during the mitotic cycle, which was associated with down-regulation of two important cell division proteins. Exposure of the plant growth-promoting bacterium, P. chlororaphis O6, to ZnO and CuO NPs caused changes in cellular levels of pyoverdine (PVD — siderophore) and expression of an inner membrane PVD transporter gene, suggesting that NPs could affect plant–microorganism interactions [39]. A few publications have then reported that exposure of bacteria (E. coli and P. aeruginosa) to MNPs induced antibiotic resistance genes, suggesting stimulation of general stress defense mechanisms [26,35]. MNPs may also affect bacterial cells through the specific activity of coating molecules. For instance, a study from Cui et al. [40] showed that 4,6-diaminopyrimidine sulfhydrylmodified gold (Au) NPs exhibited antimicrobial activity against E. coli cells mostly related to the surface-modifying molecule, causing down-regulation of a F-type ATP synthase (disruption of membrane potential), peroxidase and hydroperoxide reductases (oxidative stress response), and a ribosomal protein S10 (proteosynthesis). Ivask et al. [41,42] used a genome-wide library of 4000 E. coli single-gene deletion mutants to identify genes involved in the cell response to NMs. In a first study, exposure to cationic polystyrene NPs was shown to inhibit microbial growth in the parent E. coli strain. Detection of NP-sensitive deletion mutants allowed identifying several gene clusters involved in lipopolysaccharide (LPS) and ubiquinone biosynthesis, membrane transport, and DNA repair, suggesting toxic mechanisms through outer membrane destabilization and ROS production [41]. Using a similar approach, the authors then investigated the molecular response of E. coli to coated Ag NPs with different size and surface charges [42]. Although all Ag NPs inhibited the microbial growth, cationic (branched polyethylene imine-coated) Ag NPs exhibited a higher toxicity. Screening the library of deletion mutants, the authors identified ‘Ag-specific’ genes (i.e., responsive to Ag NPs and Ag+) and ‘NP-specific’ genes (i.e., responsive to a particular NP type). ‘Ag-specific’ Current Opinion in Biotechnology 2015, 33:206–219

genes were involved in stress response (e.g., oxidative stress, DNA damage) and ‘NP-specific’ genes were involved in the synthesis of outer membrane LPS, known to protect the cell against small cationic molecules. A subset of genes responsive only to cationic Ag NPs were involved in defense against ROS generated at the cell surface. Several studies have demonstrated changes in gene expression in bacteria exposed to CNMs. Kang et al. [20] reported that E. coli exposure to SWNTs resulted in reduction of viability and metabolic activity, loss of membrane integrity, and leaking of cellular material, which were more severe than the changes caused by multiwalled carbon nanotubes (MWNTs). Similarly, exposure to SWNTs induced higher overexpression of sigma transcription factors than exposure to MWNTs — suggesting further that SWNTs impose a higher stress on the cells. A series of model marine bacteria (i.e., Silicibacter pomeroyi, Oceanospirillum beijerinckii, Vibrio splendidus, and Vibrio gigantis) exposed to carboxylated SWNTs showed differential responses, ranging from growth inhibition to bactericidal effect [43]. Gene expression analysis conducted using RT-qPCR in the most SWNT-sensitive species, S. pomeroyi, revealed induction of genes involved in general stress response (e.g., sigma E factor), while genes involved in preservation of membrane integrity (i.e., sigma S factor) were unaffected, suggesting that SWNTs act on the cells through oxidative stress rather than membrane damage. Besides cellular toxicity, a limited number of studies have focused on the environmental impact of NMs. In order to investigate the effect of NMs on nitrogen cycling, Yang et al. [10,44] examined the growth inhibition and expression profile of selected genes in model nitrogen-metabolizing bacteria exposed to coated QDs and Ag NPs. Results showed that the nitrifier, Nitrosomonas europaea, was generally more susceptible to both kinds of NMs than the denitrifier, Pseudomonas stutzeri, and the nitrogen fixer, Azotobacter vinelandii. Exposure to sublethal doses of QDs (cationic) resulted in up-regulation of most nitrogen-metabolic genes in the three strains (e.g., ammonia monooxygenase, nitrite/nitrate reductase, nitrogenase) [44]. On the other hand, ammonia monooxygenase of N. europaea was down-regulated by high dose of Ag NPs [15]. These results collectively suggest that ammonia oxidation is the most susceptible nitrogencycling process to NM contamination. Exposure to carboxyl-modified SWNTs inhibited the growth and nitrate reduction in the model denitrifying bacterium, Paracoccus denitrificans. These effects were associated with the up-regulation of ribonucleotide reductase, indicative of potential DNA damage, and the down-regulation of nitrate reductase and other genes involved in the energy metabolism, which was consistent with the inhibition of nitrate reduction [45]. Another publication reported that www.sciencedirect.com

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zero-valent iron (ZVI) NPs applied for the purpose of environmental remediation impacted the soil bacterium, P. stutzeri, causing up-regulation of various oxidative stress response genes [46]. Besides inducing changes in gene expression, NMs were shown to affect the microbial community structure in different environments. For instance, using automated ribosomal intergenic spacer analysis (ARISA), Goyal et al. [47] observed that SWNTs differentially impacted major bacterial groups in activated sludge, with higher reduction of Sphingomonadaceae strains relative to other families. More recently, Ge et al. [48] used metagenomic pyrosequencing to study the effects of TiO2 NPs on the bacterial community in soil microcosms, showing that NPs resulted in overall decrease of the bacterial diversity and differentially impacted different functional groups, such as reducing the number of species involved in nitrogen fixation and methane oxidation, and increasing the number of species involved in biodegradation of persistent organic pollutants. Gene expression changes in plants

Transcriptional studies on the effects of NMs on plants — mostly conducted with the model species, Arabidopsis thaliana — revealed primarily changes in gene expression in relation with biotic and abiotic stimuli (Table 2). For instance, using whole-genome microarray analysis, Landa et al. [49] reported that exposure of A. thaliana to ZnO NPs and fullerene soot (FS) (100 mg L 1) — but not TiO2 NPs — caused phytotoxic effects (biomass reduction) associated with up-regulation of genes involved in biotic and abiotic stress responses (e.g., oxidative stress, draught, pathogens) and downregulation of genes involved in cell biosynthesis, cell organization, electron transport, and energy pathways (indicative of general cellular stress). In another microarray study, Kaveh et al. [29] showed that A. thaliana exposure to Ag NPs caused increase of plant growth at low dose (2.5 mg L 1) and decrease at higher dose (5 mg L 1). Exposure to moderate toxic level (5 mg L 1) 1) resulted in up-regulation of genes involved in response to metal and oxidative stress and down-regulation of genes involved in response to pathogens and hormonal stimuli. A significant overlap was observed between genes differentially expressed in response to Ag NPs and soluble Ag+, suggesting that Ag NP-induced stress originated partly from silver toxicity and partly from specific nanosize effects. Other studies on A. thaliana exposed to Ag NPs similarly reported induction of genes involved in response to oxidative stress [50–52], water homeostasis [50], oxidative damage to DNA [51,52], and systemic acquired resistance (SAR) [53]. Other reports indicated that NMs impacted also photosynthetic pathways. For instance, Ma et al. [54] investigated the effects of cerium oxide (CeO2) and indium oxide (In2O3) NPs on A. thaliana, showing that high levels www.sciencedirect.com

of CeO2 — but not In2O3 — NPs (1000 mg L 1) negatively impacted plant growth and chlorophyll production. Exposure to both types of NPs also caused induction of genes involved in glutathione metabolism (oxidative stress response) and metal stress response. In an attempt to understand the positive effect of TiO2 NPs on plant growth, Ze et al. [55] analyzed the photosynthetic efficiency in exposed A. thaliana plants. Plant exposure to TiO2 NPs induced a light-harvesting complex II (LHCII) gene, resulting in higher LHCII content in the thylakoid membrane and increased light absorption efficiency in the chloroplast. Marmiroli et al. [56] investigated the transcriptomic response of A. thaliana exposed to cadmium sulfide (CdS) QDs in wild type and tolerant dissociation (Ds) transposition-induced mutant lines. CdS QDs inhibited germination and plant growth in wild-type plants, which was not caused by the release of Cd2+ ions. In all lines, exposure to sublethal levels of QDs resulted in overexpression of genes involved in defense response, SAR, and pathogenesis. The tolerance of one mutant line (atnp01) was associated with up-regulation of genes involved in the synthesis of storage and lipid transport proteins (playing a role in stress response). The tolerance of the second mutant line (atnp02) was suggested to be associated with a member of the MYB transcription factor superfamily known to be involved in development and metabolism regulation, and in response to biotic and abiotic stress. A study has recently showed that CNMs could increase the susceptibility of plants to other abiotic stresses: Wang et al. [57] reported that graphene oxide NPs negatively affected the development of A. thaliana seedlings, but only in the presence of polyethylene glycol (PEG)-induced (drought) and NaCl-induced (salt) stress. Gene expression using RT-qPCR showed that the combination of graphene oxide with PEG or NaCl treatment resulted in down-regulation of genes involved in root development and induction of genes involved in response to drought and/or salt stress. Few reports have been published on the transcriptomic response of other plant species exposed to NMs (Table 2). Exposure of wheat (Triticum aestivum) to Ag NPs resulted in reduction of the plant biomass and accumulation of oxidized glutathione (indicative of oxidative stress) [58]. In addition, overexpression of a metallothionein gene suggested that the plant responded to Ag NPs by metal ion sequestration. Two studies conducted with the model plant, tobacco (Nicotiana tabacum), showed that exposure to aluminum oxide (Al2O3) and TiO2 NPs resulted in a range of phytotoxic effects (e.g., reduction of germination rate and plant biomass) associated with up-regulation of a suite of microRNAs (miRNAs) [59,60]. Small endogenous noncoding RNAs are gene regulators known to be involved in plant development and tolerance to abiotic Current Opinion in Biotechnology 2015, 33:206–219

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stresses, including drought, salinity, cold, and heavy metals.

suggesting that MNPs acts on cells by generating ROS, which are known to interact with DNA [67].

Several studies have been published on the positive and negative effects of CNMs on different plant species. Shen et al. [61] reported that exposure of A. thaliana and rice (Oryza sativa) protoplast cells to SWNTs resulted in negative effects, including cell death, DNA damage, and generation of ROS. The involvement of ROS was confirmed by up-regulation of ROS-scavenging genes in exposed A. thaliana leaf cell cultures, including ascorbate peroxidase and superoxide dismutase. Khodakovskaya et al. [62] reported that exposure of tobacco cell (N. tabacum) cultures to MWNTs significantly enhanced the growth over a wide range of concentrations and induced several genes involved in cell division (cell cycle-CycB), cell wall formation (extensin — NtLRX1), and water transport (aquaporin — NtPIP1). Similarly, Lahiani et al. [63] reported that exposure of three crop species, including barley (Hordeum vulgare), soybean (Glycin max), and maize (Zea mays), to MWNTs enhanced germination and plant growth, which was associated with overexpression of genes encoding different types of water channel proteins. Although the authors concluded that MWNTs could be used as exogenous growth regulators for crop species, this practice may raise concerns because of potential food contamination by NMs. Similarly, exposure to SWNTs was reported to enhance growth of seminal roots, while inhibiting growth of hair roots in maize plants (Z. mais), which was supported by the up-regulation and down-regulation of genes involved in the development of each type of roots, respectively [64]. Interestingly, SWNTs also up-regulated the expression of acetyltransferase (HAT) genes involved in histone H3 modification, suggesting that, as with other types of stresses, SWNTs initiate a global response regulated at the epigenetic level.

Conclusions Gene expression analyses are typically conducted to complement morphological, physiological, and/or proteomic investigations and determine whether observed changes are reflected at the transcriptional level [3,13]. However, in many instances, gene expression analysis may provide additional specific information that cannot be obtained directly from other approaches. For instance, NMs frequently interfere with multiple cellular targets, making difficult to identify specific toxic mechanisms based solely on morphological changes or biochemical assays [6,19]. On the other hand, toxicological studies on NMs have been frequently conducted through acute testing (high-dose exposure over a short period of time), even though environmental exposure is better captured through chronic toxicity testing (low-dose exposure over a long period of time). Gene expression changes have been shown to be induced by very low levels of contaminants and constitute therefore promising biomarkers for the detection of chronic toxicity potentially associated with NMs [68]. In addition, high-throughput transcriptomic analysis allows identifying unique expression patterns — or ‘genetic fingerprinting’ — informing on potential toxicity pathways and modes of action [13].

In contrast with what was observed with bacteria, plant exposure to NMs seems to induce a wider molecular response, affecting multiple transcription factors and genes involved in various cellular stresses (referred to as ‘biotic and abiotic stimuli’), which may reflect the existence of more complex and networked transcriptional pathways in eukaryotic multicellular organisms.

Although often considered collectively as a single class of contaminants, engineered NMs exhibit today a wide variety of sizes, shapes, compositions, atomic structures, coatings, and redox activities, resulting in a multiplicity of interactions with different biological systems. Potential applications of NMs in consumer products and medicine are calling for NMs generating lesser ecological impacts. The design of NMs with lower toxicity therefore requires understanding better the structure–activity relationships, which is being achieved using high-throughput toxicity screening methods [9]. Gene expression studies are expected to contribute to the development and validation of genomic biomarkers suitable for high-throughput screening technologies. Unique gene expression patterns may further help identify the modes of action and structure–activity relationships.

Although not directly focusing on gene expression, a few studies reported the genotoxic effects of NMs on plants. Using random polymorphic DNA assay (RPDA), Lo´pezMoreno et al. [65] showed that soybean (G. max) exposure to high levels of ZnO and CeO2 NPs resulted in new bands, indicative of DNA damage, mutations, deletions, and/or homologous recombinations. Similarly, Patlolla et al. [66] reported that Ag NPs were genotoxic to broad bean plants (Vicia faba), as evidenced by the NP-induced increase of chromosomal aberrations and micronuclei, and decrease of the mitotic index. These observations are consistent with other biochemical and molecular studies

Despite its great promises for understanding further the modes of action of NMs on biological systems, highthroughput toxicogenomics can lead to unclear results. Genome-wide gene expression analyses typically reveal cascades of transcriptional reactions and sets of co-regulated genes, which do not necessarily provide evidence of causative relationships between toxic stimuli and transcriptional responses. For instance, MNPs are shown to stimulate genes of the sulfur metabolism, which may reflect either biosynthesis of antioxidant species (e.g., glutathione) or higher turnover of sulfur-containing proteins inactivated by metal binding [26]. As another example, ROS are

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Thomas CR, George S, Horst AM, Ji ZX, Miller RJ, PeraltaVidea JR, Xia TA, Pokhrel S, Madler L, Gardea-Torresdey JL et al.: Nanomaterials in the environment: from materials to highthroughput screening to organisms. ACS Nano 2011, 5:13-20.

constitutively generated by multiple cellular processes and play an important role as secondary messengers. Induction of ROS and genes involved in response to oxidative stress upon exposure to MNs indicates either the direct generation of ROS by NMs or the indirect signaling of cellular stresses [36,69]. Gene expression analyses are therefore most meaningful when completed with relevant morphological, physiological, and/or proteomic investigations.

9.

High-throughput gene expression analyses will certainly continue to play a key role in understanding the mechanisms of cellular toxicity of NMs, as well as other environmental contaminants. However, meaningful comparison between experiments currently will require uniformizing gene expression analysis platforms and data processing [13]. As increasing numbers of transcriptomic datasets are published, there is a critical need for more integrated and accessible databases allowing investigators to perform transcriptional data mining. Similarly, conventional approaches for drawing molecular maps and transcriptional networks (e.g., gene clustering methods) will need to be replaced by more advanced system biology approaches [70].

12. Auffan M, Rose J, Bottero JY, Lowry GV, Jolivet JP, Wiesner MR: Towards a definition of inorganic nanoparticles from an environmental, health and safety perspective. Nat Nanotechnol 2009, 4:634-641.

Acknowledgements This work was supported by a U.S. Department of Agriculture (USDA) — National Institute of Food and Agriculture (NIFA) grant (award number: 2012-67009-19982).

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Current Opinion in Biotechnology 2015, 33:206–219