Keywords

9.1 Introduction

Nanoparticles (NPs) have been defined as materials having at least one dimension in the nanoscale (1–100 nm), bearing size-dependent physiochemical properties different than their bulk counterparts. NPs have high surface-to-volume ratio, which provide them high reactivity and physicochemical dynamicity. The advantageous properties of more complex NPs retain the potential to drawn enormous research from different fields for its application in medical diagnostics and treatments [1]. Several commercially available consumer products contains NPs, particularly in cosmetics and sunscreens [2]. NPs have also been utilized in different areas of biology and medicine including tissue engineering, drug and delivery formulations, for hyperthermia induced tumor destruction, DNA structure probes and biosensors [3,4,5]. Nonetheless, there are uncertainties that the unique properties of NPs may also pose potential health risks in the occupationally and non-occupationally exposed populations [2].

With the greater demand of NPs, there is huge ambiguity related to their potential hazards towards environment and its different trophic levels. Special concerns have been raised on NPs mediated release of metal ions, reactive oxygen species (ROS) or its direct reactivity towards biological membranes [6, 7]. A major key question persisting with NPs toxicity is whether it is linked with the general properties shared by varying NPs or is it specific to individual NPs. Considering these facts, if toxicological effects are related to shape, agglomeration or the size of NP, then similar toxicological effects can be expected for different NPs [8]. On the other hand, if NPs composition controls the interaction, then definite toxicity would be expected [9, 10]. An understanding of the toxicological effects of each NPs is critical for any prediction of their immediate and long-term risks for humans upon occupational and consumer goods exposure (Fig. 9.1).

Fig. 9.1
figure 1

Cellular interaction and uptake of natural and anthropogenic NPs

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Exposure to NPs may occur during its production via handling, aerosolization may also occur during energetic processes, such as vortexing, weighing, sonication, mixing and blending [11]. Hence, inhalation is considered a relevant route of exposure [11, 12]. Presence of the NPs in the body may induces pathophysiological changes that might contribute to the development of cancer [13], cardiovascular diseases [14], respiratory tract inflammation [15], neurodegenerative diseases [16], and many other pathologies [17]. Researchers have summarized the potential mechanisms of nanotoxicity via greater ROS level and induction of inflammatory responses such as Parkinson’s disease [18]. NPs can also alter the permeability of blood brain barrier and re-translocate from the site of deposition to other parts of the body via circulatory or lymphatic system [19]. The actual prophecy of the adversity of NPs cellular exposure and uptake are still lacking [20]. Cells can rapidly alter its transcriptomic output (gene expression profile) in response to extracellular and intracellular environmental changes, and get adapted for their survival and function. Nonetheless, biological functions and normal physiological activities can get disturbed under excessive environmental changes. Consequently, profiling of gene expression has been proven helpful in recognizing the NPs toxicity and its relevant molecular mechanism [21,22,23,24,25]. Application of toxicogenomics in NPs research can significantly contribute to unravel the mechanistic action of toxicity, parallel to traditional approaches and other omic technologies. Quantitation of mRNA transcripts in NPs treated cells not only provide the mechanistic information, rather it also provides “genetic fingerprinting” from the pattern of gene expression that the NPs elicit in vitro and in vivo. In this chapter, we have discusssed the cellular toxicity of NPs and the importance of toxicogenomics approaches in nanotechnology and NPs induced alterations in transcriptomic profile reportedly studied based on the RNA-Seq, and microarray techniques.

9.2 Toxicogenomics in Nanoparticles Research

Several biomarkers including mRNA transcripts, protein and enzyme expressions have been widely applied as health indicators for the range of human diseases, also it has been utilized to examine the xenobiotics induced changes in the health status of different model and non-model organisms [26,27,28]. The technology is based on the principle that the mRNA that codes for proteins, are expressed differently in an unexposed organism, vis-a-vis the xenobiotic exposed organisms. The quantification of genome-wide mRNA level by transcriptomic approaches involve the techniques like RNA sequencing (RNA-Seq) and oligonucleotide hybridization (microarray). Toxicogenomics, primarily comprising the hybridization technologies, has been a preferred choice in modern toxicological research. The vast data output and pathway based information represents toxicogenomics as a powerful approach, which has been used now for over decades for identifying novel mechanism of toxicity, disturbance in vital biological pathways, as well as biomarkers of toxicity [6]. In fact, the imperative benefit of toxicogenomics is the holistic approach, which provides a platform for discerning the genetic level changes from the perspective of altered pathways and networks revealing the novel mechanisms of toxicity and toxic responses (Fig. 9.2).

Fig. 9.2
figure 2

Toxicogenomic approaches for evaluation of NPs toxicity

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9.3 Nanoparticles Toxicity Analysis by RNA-seq

RNA-seq, being a novel and state-of-the art technique, allows the robust quantification of RNA transcripts on a genome-wide level [29]. Several properties of RNA-seq viz. accuracy and higher dynamic range allow detection of alternative splicing, and no preexisting knowledge of the genomic sequence makes it more advantages over microarray [29]. Consequently, the RNA-seq has recently gain entry into the field of nanotoxicology, bound to supersede microarrays in the toxicogenomics field [30]. RNA-seq can generate vast inventory of gene transcripts employing bioinformatics, DNA sequencing and sequence databases [29]. The low-abundance transcripts (approximately 30% of most transcriptomes) can easily be quantified by RNA-seq, also the technique can identify splice junctions and novel exons. RNA-seq accuracy and precision is equally comparable to quantitative real-time PCR [31, 32]. Data from various test models revealed that, about 40% changes in the protein level can be easily explained by knowing the mRNA abundance [33, 34]. Therefore, the application of RNA-seq based transcriptome profiling in NPs toxicity can provide detailed and substantial information, which can complement the results of other approaches.

The RNA-seq analysis of eukaryotic green alga Chlamydomonas reinhardtii exposed to metallic NPs (Ag, TiO2, ZnO and CdTe/CdS quantum dots) revealed specific and different effects. Of the 1.2 × 108 total reads, 5.0 × 107 (42%) mapped uniquely with a maximum of two mismatches and no deletions or insertions. Chlamydomonas reinhardtii exposed to TiO2, ZnO, Ag-NPs and quantum dots (QDs) exhibited 96, 156, 141 and 49 upregulated genes, whereas 80, 29, 86 and 55 genes were found downregulated. NPs exposure (TiO2, ZnO, and QDs) increased the levels of transcripts encoding subunits of the proteasome, suggesting proteasome inhibition, which is regarded as inducer of several major diseases, including Alzheimer’s disease, and used in chemotherapy against multiple myeloma [35]. The RNA-seq analysis of MCF-7 cells treated with fullerene derivative too exhibited strong negative effect on a number of fundamental and interconnected biological processes involved in cell growth and proliferation, mainly including mRNA transcription, protein synthesis/maturation, and cell cycle progression. Comparatively, a fullerene derivative-1 in the same study exhibited no cytotoxicity, although some pathway overlaps were observed with a another fullerene derivative-2 (Fig. 9.3).

Fig. 9.3
figure 3

(a) Structures of the two fullerenes 1 and 2. (b) Principal component analysis showing the relationship between gene expression profiles of the six fullerene-treated and of the three control samples; a compound-dependent effect is visible, with fullerene 1 leading to minor effects compared to 2, as well as a time-dependent progressive deviation from the control samples. (c) Venn diagram depicting the differentially expressed genes identified by the Kal’s Z-test on proportions common to the different experimental time points. (d) Hierarchical clustering of samples (Euclidean distance, complete linkage) based on the RNA-seq gene expression profiles (Reprinted from Toxicology, volume 314, Lucafò et al. [36], Profiling the molecular mechanism of fullerene cytotoxicity on tumor cells by RNA-seq, pages 183–192. Copyright (2013), with permission from Elsevier. See the reference list for full citation of proper credited)

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Gene ontology (GO) analysis of fullerene derivatives reveled the repression of transcriptional activators (DHX9, NR2F1, GATA4, AHR), pre-mRNA complex components (HNRNPM, WDR77, POP1) and the transcription elongator factor elongin A (TCEB3). Ribosome biogenesis was negatively affected, also the three fundamental genes of pre-rRNA (RRP9, BOP1, UTP20) responsible for the maturation and two major subunits of RNA polymerase III (POLR3B, POLR3G) were found down regulated. The down regulation of mentioned genes also repressed the protein synthesis, particularly the maturation (SEC11C, SRPRB) involved in the recognition and processing of signal peptide. In addition, molecular chaperones responsible for the folding of newly synthetized proteins, including HSP70 (HSPA1A, HSPA8) and HSP90 (HSP90AA1) were down regulated [36].

RNA-seq of nano-hybrid (made of a gold nanoparticle core and a peptide coating; P12) treated human peripheral blood mononuclear cells (PBMNC) exhibited anti-inflammatory effects. Global gene expression of PBMNC exposed to P12 revealed suppression of 233 genes upregulated by LPS stimulation, and 29 genes downregulated by LPS. Overall, ca. 40% of genes that were upregulated by LPS in human PBMNC were suppressed by P12 (Fig. 9.4).

Fig. 9.4
figure 4

Impact of anti-inflammatory nanoparticle P12 treatment on RNA-Seq transcriptome analysis of LPS-stimulated human PBMC. (a) Novel peptide-decorated nanoparticle hybrids with different surface chemistry (P12 vs. P13). (b) PCA plot. (c) Venn diagrams. (d) Differential expression profiles of top 33 genes (p < 2.6 × 10−15 and fold change >1.5) (Reprinted from Biomaterials, volume 111, Yang et al. [37], Endosomal pH modulation by peptide-gold nanoparticle hybrids enables potent anti-inflammatory activity in phagocytic immune cells, pages 90–102, Copyright (2016), with permission from Elsevier. See the reference list for full citation of proper credited)

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P12 exposure resulted in the activation of different signalling pathways including PKR, interferon, TLR, chemokine, JAK-STAT and TNF [37]. Deep sequencing-based RNA-seq analysis indicated 45 differentially expressed genes in living Hydra vulgaris, when exposed to SiO2-NPs. Among these genes, 29 transcripts were upregulated (2-fold to 25-fold) and 16 were downregulated (2.2-fold to 5-fold). The authors have concluded that a sizeable number of genes remain unknown, providing a valid source of functional information to be further investigated [38]. Fifth instar of silkworm (Bombyx mori) when exposed to TiO2-NPs exhibited differential expression of 11,268 genes in the silkworm fat body, out of which 341 genes showed significant differences, among which 138 were upregulated and 203 were downregulated (Fig. 9.5I). The GO map exhibited eleven biological processes accounting >10% of the annotated genes related to metabolic, cellular and single-organism process showing the highest percentages of annotated genes. Five cellular component subgroups >10% of annotated genes having cell, cell part and organelle showing the highest percentages of annotated genes. Three molecular functions related to binding, catalytic activity and structural molecular activity accounted for 10% or more of annotated genes have been observed in the silkworm (Fig. 9.5II). Further processing the RNA-seq data by KEGG (Kyoto Encyclopedia of Genes and Genomes) scatterplots indicated significant enrichments of all differentially expressed genes. The major affected pathways by TiO2-NPs exposure in silkworm, includes insulin signaling pathway, which mediate primarily in insect growth and development, nutrient metabolism, lipids and carbohydrates homeostasis and protein synthesis (Fig. 9.5III) [39]. Transcriptome study on the effects of Ag+ and Ag-NPs in earthworm Eisenia fetida employing RNA-seq reveled that Ag+ verses control yielded 529 and 618 downregulated and upregulated transcripts. Eisenia fetida exposed to Ag-NPs verses control exhibited 237 and 454 downregulated and upregulated transcripts, while Ag-NP verses Ag+ showed 449 and 758 downregulated and upregulated transcripts. These alterations were related with the toxicity through pathways related to sugar and protein metabolism, disruption of energy production, ribosome function, molecular stress and histones gene alteration [40].

Fig. 9.5
figure 5

(a) Statistical chart of significantly differentially expressed genes. A- represents the control group, while B- represents the experimental group. RPKM indicates the gene expression in samples. Red represents the upregulated genes in the figure; green represents downregulated genes; blue represents genes without significant differences. (b) Functional classification of significantly differentially expressed genes. A- represents the control group while B- represents the TiO2-NP treatment group. The right ordinate represents the number of genes, with the maximum value of 121 indicating that a total of 121 genes underwent GO function classification. The left vertical axis represents the percentage of genes, indicating the percentage of functional genes to all annotated genes. (c) Scatter plot of KEGG pathway enrichment statistics. A- represents control group, and B- represents experimental group. Rich factor is the ratio of numbers of differentially expressed genes annotated in this pathway term to the numbers of all genes annotated in this pathway term. Greater rich factor means greater intensiveness. Q-value is corrected P-value ranging from 0 to 1, with a lower value means greater intensiveness. Top 20 pathway terms enriched are displayed in the figure (Adopted from Figs. 9.1, 9.2, and 9.3 of Tian et al. [39] (Copyright and Permission granted from the Biology Open Journal under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed) (See the reference list for full citation of proper credited)

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9.4 Microarray Analysis of Nanoparticles Toxicity

Microarrays analysis of genome changes is based on the probe, which is a pre-prescribed set of tens of thousands of genes at once. Comparative to RNA-seq, microarray is relatively cheaper, allowing multiple comparisons across treatments or individuals within an experiment. The transcriptomic profiling using GeneChip or microarray technique allow the analysis of global gene expression in NPs exposed and unexposed test models. Exposure of retinoblastoma cell line (Y-79) with etoposide loaded NPs formulations modulated its gene activity. Microarray analysis exhibited differential expression of genes, showing upregulation of 171 genes, while 280 genes were found downregulated. The upregulated genes were mostly belonging to three groups including apoptosis, cell cycle and cell differentiation, and cell migration [41]. Time dependent (24 h and 48 h) exposure of zebrafish embryo with Ag-NPs, Ag-bulk, and Ag+ exhibited differential expression of genes in the microarray analysis. After the 24 h post fertilization, embryos treated with Ag-NPs, Ag-bulk, and Ag+ exhibited 35%, 71% and 89% of downregulated genes, while 65%, 29% and 11% genes were found upregulated. After 48 h of post fertilization 57%, 43%, 61% of genes were downregulated, and 43%, 57% and 39% genes were upregulated. The most significant over-represented in GO terms and KEGG pathways in all treatments at 24 h and 48 h were ribosome and oxidative phosphorylation. The prominent overlaps revealed that the toxicity of Ag-NP and Ag-bulk to zebrafish embryos has been predominantly associated with the toxicity of free Ag+ [25]. The microarray analysis of human THP-1 derived macrophages exposed to single walled carbon nano tubes (SWCNT) reveled statistical significance of gene expression. Authors have analyzed the abundance changes in 406 pathways archived in KEGG and BioCarta databases. Microarray data exhibited significant alterations of at least 23 different vital pathways in SWCNT treated THP-1 derived macrophages. Considering the network data, authors have suggested that SWCNT uptake into macrophages activated the nuclear factor κB (NF-κB) and activator protein 1 (AP-1), leading to oxidative stress, proinflammatory cytokines activation, recruitment of leukocytes, induction of apoptotic genes and T cells [42].

In the same connection, human macrophage cell line exposed to 5 and 100 nm Ag-NPs were analyzed for approximately 28,000 cDNA profiles using GeneChip(R). Array profile revealed the expression of 45 genes between 5 nm Ag-NPs and the control, and 30 genes between 5 and 100 nm Ag-NPs. The stress genes (HSP, HO, MT) and one cytokine gene (IL-8) were the top expressed genes in 5 nm Ag-NPs treated cells. Also, the expression of HO1, HSP-70 and MT-1E RNA was significantly increased as the log2 ratio >2.0. IL-8 was the only cytokine gene that was significantly induced among more than 70 cytokines [43]. Whole genome microarray analysis of the early gene expression changes induced by 10 and 500 nm amorphous silica NPs exhibited that the magnitude of change for the majority of genes affected were more tightly correlated with particle surface area, rather than either particle mass or number. The microarray data exhibited significant alteration of 503 and 502 genes by at least two-fold with either 10 or 500 nm silica NPs. The union of these gene sets included 753 genes, with 252 genes overlapping between the two particle size groups. Genes which were highly affected are known to play important role in lung inflammation, including Cxcl2, Ccl4 (MIP-1β) and Ccl3 (MIP-1α), along with the chemokine receptor Cxcr4. Nonetheless, gene set enrichment analysis revealed that among 1009 total biological processes, none were statistically enriched in one particle size group over the other [44]. Global gene expression of 11 nm dimercaptosuccinic acid (DMSA)-coated Fe-NPs on two cells (THP-1 and HepG2) revealed differential responses. Within ten top upregulated genes, Fe-NPs treated THP-1 cells induced maximum upregulation of Cxcl13, a humoral immune response gene. Other immune response genes upregulated were Adamdec1, Ly96, Ifi44l, Ebi3, Clec7a. While, in HepG2 cells treated with low and high doses of Fe-NPs induced maximum upregulation three genes (Ifi27, Tagln and Ifi6I) (Fig. 9.6I). Four way Venn diagram revealed that two genes (Ddx58 and Ifi27) were commonly induced by low and high doses of FeNPs in both cell lines (Fig. 9.6IIa). Eleven genes (Ddx58, Ifi44, Fbxo16, Parp9, Ifit3, Serpini1, Ifi27, Nexn, Usp25, Rg9mtd2 Ccne2) were commonly induced by low dose of FeNPs in both cell lines. On the other hand, ten genes (Tmed2, Ifi27, Ddx58, Akap12, Nampt, Narg1, Usp16, Ifi6, Col9a2, and Zcchc2) were commonly induced by high dose of Fe-NPs in THP-1 and HepG2 cells. However, no genes were commonly repressed at both doses of Fe-NPs in the cell lines (Fig. 9.6IIb). The authors also developed a heat map of top 55 genes commonly regulated in these two cell lines. The hierarchical clustering revealed that these genes were classified into four clusters. Some genes were steadily upregulated or downregulated in both types of cells, indicating cell-independent effects. Whereas some genes were inversely regulated in the two cell lines, suggesting cell-specific effects of the Fe-NPs (Fig. 9.7I). Interestingly, the authors have reported that Fe-NPs significantly enrich the hepatitis C pathway at both doses of treatments in the THP-1 cells (Fig. 9.7II) [45].

Fig. 9.6
figure 6

Panel I showing the top 10 genes with the highest expression changes in the two cell types after treatment with Fe-NPs. (a, b) Induced genes in the THP-1 cells. (c, d) Induced genes in the HepG2 cells. (e, f) Repressed genes in the THP-1 cells. (g, h) Repressed genes in the HepG2 cells. 50-up and 100-up: induced genes in cells treated with 50 μg/mL and 100 μg/mL of FeNPs, respectively; 50-down and 100-down: repressed genes in cells treated with 50 μg/mL and 100 μg/mL of FeNPs, respectively. Some uncharacterized genes with fold changes greater than the lowest fold changes in these plots are not shown in this figure. Panel II Comparison of FeRGs in the THP-1 and HepG2 cells. a Comparison of induced genes in the two cell lines. b Comparison of repressed genes in the two cell lines. Each Venn diagram is divided into four areas labeled as T-50, T-100, H-50 and H-100. T-50 and T-100, THP-1 treated with 50 μg/mL and 100 μg/mL of FeNPs, respectively. H-50 and H-100, HepG2 cells treated with 50 μg/mL and 100 μg/mL of FeNPs, respectively. The number in overlapped area represents the overlapping genes. The numbers before and after the slash represent the genes with fold changes greater than 2 and 1.5, respectively (Reused from Figs. 9.3 and 9.4 of Zhang et al. [45] (Copyright and Permission granted from the Journal of Nanobiotechnology under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited) (See the reference list for full citation of proper credited)

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Fig. 9.7
figure 7

(I) Cluster analysis of genes. Fifty-five FeRGs from the two cell lines were clustered according to their expression levels using a hierarchical clustering. The heatmap was drawn with Java TreeView. Red and green represent up- and down-regulation, respectively. The color depth reflects the expression level between −3 and +3 (marker). The numbers of genes in Clusters A to D are shown in parentheses. The fold changes of ten representative genes in four clusters are shown in the zoomed images. (II) KEGG pathway of hepatitis C in the FeNP-treated THP-1 cells. The genes in red refer to the FeRGs induced by 100 μg/mL of FeNPs. The genes in yellow refer to the FeRGs induced by both 50 μg/mL and 100 μg/mL of FeNPs. Abbreviations for the KEGG parameters can be found on the KEGG pathway webpage (Reused from Figs. 9.6 and 11 of Zhang et al. [45] (Copyright and Permission granted from the Journal of Nanobiotechnology under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited) (See the reference list for full citation of proper credited)

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Kedziorek et al. [21] have examined the genome changes in LacZ-expressing mouse NSC cell line C17.2 exposed to superparamagnetic iron oxide nanoparticles (SPIONs). Microarray analysis of 2695 probe sets, representing 1399 genes, revealed that genes belonging to molecular functions of the cell describing activities such as catalytic reactions or binding that occur at the molecular level were significantly affected by the SPIONs exposure. A significant change in 970 genes of cellular function, 299 genes of gene expression, 312 genes of developmental, 431 genes of biologic regulation and 685 genes belonging to metabolic processes, have been reported. The treated cells also exhibited significant alterations in genes belonging to secretion, transport, locomotion, reproductive processes, and establishment of localization. In addition, the expressions of heme oxygenase 1 (Hmox1) and transferrin receptor 1 (Tfrc) were repressed, while, genes responsible for detoxification (Clu, Cp, Gstm2, and Mgst1) and lysosomal function (Sulf1) were upregulated at later time points. Liu and Wang [24] have studied changes in the global gene expression of mouse macrophage (RAW264.7 cells) exposed to DMSA-Coated Fe3O4-NPs using GeneChips Mouse Genome 430 2.0 microarrays. The GO analysis revealed that several molecular functions and biological processes pertaining to metal ion transmembrane transporter activity, especially Fe ion transmembrane transporter activity and Fe ion binding, were significantly enriched in all DEGs. Tfrc, Trf, and Lcn2 genes important to iron metabolism were frequently found in the GO terms. The microarray data of lung epithelial cell line (A549) exposed to 12.1 μg/ml Ag-NPs (EC20) for 24 and 48 h exhibited altered gene regulation of more than 1000 genes (>2-fold), while considerably fewer genes responded to Ag+ (133 genes). The upregulated genes were belonging to the members of heat shock protein, metallothionein and histone families [23]. Human skin fibroblasts (HSF42) and human embryonic lung fibroblasts (IMR-90), both untransformed cells were exposed to multiwalled carbon nanotubes (MWCNTs) and multiwalled carbon nano-onions (MWCNOs) exhibited profound gene expression changes, analyzed on new generation Affymetrix HTA GeneChip system. The low doses of both materials induced expressional changes in genes of secretory pathway, protein metabolism, golgi vesicle transport, fatty acid biosynthesis, and G1/S transition of mitotic cell cycle. Also, an additional group of genes, involved in protein ubiquitination was found upregulated. Contrarily, high doses upregulated the genes in tRNA aminoacylation and amino acid metabolism pathways. Genes of inflammatory and immune response were also found upregulated [46].

BV2 microglia, which is an immortalized mouse cell line, when exposed to a mixture of anatase (70%) and rutile (30%) TiO2-NPs (Degussia P25) exhibited global gene expressional changes in the microarray. The core canonical analysis reveled that BV2 exposed to P25 upregulated the signaling processes involved in B-cell death, ERK/MAPK receptors, apoptosis, calcium, and inflammation. P25 up-regulated the inflammatory (NF-κB), cell cycling and pro-apoptotic toxicity pathways. Core analysis of P25 induced downregulation of genes exhibited alteration of adaptive change and key energy production pathways, mainly associated with hypoxia, peroxisomes, and Nrf2-mediated oxidative stress [47]. In microarray analysis, male Sprague-Dawley rats fed with Synthetic Amorphous Silica (SAS) and NM-202 (a representative nanostructured silica for OECD testing) for 24 and 84 days exhibited non significant gene alteration in jejunal epithelial samples and liver homogenates. Although, fibrosis-related gene expression was significantly affected for NM-202 treated animals after 84-days of exposure, but not for SAS treated animals [48]. Osmond-McLeod et al. [49] demonstrated the transcriptome changes by the application of NPs based sunscreens. Mice treated with both the TiO2-NPs sunscreen and UVR exposure (TiO2-NPs + UVR) showed very low levels of differential regulation, as compared to untreated mice (Control-UVR) (14 genes). Pathway analysis exhibited that three of the top 5 canonical pathways in TiO2-NPs + UVR were linked with metabolic functions (Heme Biosynthesis II; Tetrapyrrole Biosynthesis II; Mevalonate Pathway I). In addition, breast cancer regulation Stathmin1 and circadian rhythm signalling were also affected in the canonical pathways. While, ZnO-NPs sunscreen with UVR (ZnO-NPs + UVR) and ZnO-NPs sunscreen without UVR (ZnO-NPs-UVR) did not showed any transcriptome alterations [49].

HaCaT cells exposed to tungsten carbide (WC) and tungsten carbide cobalt (WC-Co) NPs exhibited whole genome transcription alterations. Fluorescence signal of microarray in all treatments revealed 1956 upregulated and 1146 downregulated differentially expressed genes, with more than two-fold expression level. HaCaT cells exposure to CoCl2 salt, as metal source of Co induced strongest changes in the gene expression (373 and 826 genes for 3 h and 3 day) followed by WC-Co (37 and 248) and WC-NPs (28 and 49), respectively. Data analysis by enrichment method exhibited the fact that differentially expressed genes were related to hypoxia, endocrine pathways, carbohydrate metabolism, and targets of several transcription factors [50]. Human lung epithelial cells (A549) exposed to silica-NPs exhibited a dose dependent response, ranging from 5 to 2258 significantly differentially modulated transcripts compared with controls, with a fold change of at least 1.5 and p-value <0.05. Canonical analysis exhibited the coagulation system and intrinsic and extrinsic prothrombin activation pathways as most highly altered. Additionally, the acute phase response, xenobiotic metabolism, TREM1 signaling pathways and oxidative stress response were altered. The authors have extended the transcriptome into exproteome to understanding the NPs effect on proteins. Heat-shock proteins (HSP70 and HSP90), detoxification enzymes such as glutathione reductase (GSR), glutathione S-transferase (GSTP1), lactate dehydrogenase (LDHA), peroxiredoxins (PRDX1, PRDX6), thioredoxin reductase (TRXR1), protein disulfide isomerase (PDIA6), and aldo-keto reductases (AKR1B1, AKR1B10, AKR1C1/C2, and AKR1C3) were found as affected proteins in the exproteome analysis [51]. Human intestinal epithelium model (Caco-2) cell line, when exposed to pristine (surface untreated) CeO2-NPs exhibited 1643 modified genes. Comparatively, the manufactured CeO2 Nanobyk™ NPs in the same study have not affected the gene regulation, while 344 and 428 modified genes were found for light (NB-DL) and acid (NB-DA) degraded CeO2 Nanobyk™ NPs. Pristine CeO2-NPs exhibited changes in the cellular growth and proliferation (274 genes) and cell death (265 genes) biological process. The canonical pathway analysis of pristine CeO2-NPs revealed that it alters the mitochondrial function through the under expression of 27 genes of complexes I, III, IV and V [52]. Fisichella et al. [53] in their previous study on pangenomic oligomicroarrays (4 × 44,000 genes) demonstrated that TiO2-STNPs have not altered the gene expression of Caco-2 cells when exposed to the highest concentration of 10 μg/ml.

Al2O3-NPs exposed human bronchial epithelial (HBE) cells significantly increased expression of 54 genes and decreased expression of 304 genes. GO analysis unravel the fact that total genes encoding proteins necessary for mitochondrial function were differentially expressed. KEGG pathway enrichment analysis of these 27 genes of mitochondrial function and neural system disease were significantly enriched. NDUFA2, NDUFS, NDUFC2, NDUFA1, NDUFA4, UQCR11 (complex III), COX7B, COX17 (complex IV) and ATP5H (complex V, F0 unit) were among the most affected genes (Fig. 9.8) [54].

Fig. 9.8
figure 8

(a) KEGG pathway enrichment analysis of mitochondria related genes. a A total of 27 mitochondria related genes were analyzed through DAVID functional annotation cluster tool. These genes mainly are involved in five KEGG pathways. (b) Oxidative phosphorylation is the most significant enrichment. b A schematic figure of the oxidative phosphorylation pathway by KEGG. mRNA microarray assay predicted up-regulated genes are stained red, and down-regulated genes are stained blue in this schematic figure (Reused from Fig. 9.2 of Li et al. [54] (Copyright and Permission granted from the Particle and Fibre Toxicology (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited). (See the reference list for full citation of proper credited)

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Global gene expression in the HepG2 cells upon 20 and 50 nm Ag-NPs treatment exhibited differential regulation of genes. After short exposure of 4 h, the 20 nm Ag-NPs induced alterations of 811 genes, out of which 649 were upregulated and 162 were downregulated. Comparatively, the 50 nm Ag-NPs treatment induced stark difference, only 21 genes were altered and all of them were upregulated. Extended exposure of 24 h did not made any massive alterations in the gene expression by both sizes of Ag-NPs. Overlapping of DEGs exhibited alterations of five common genes after 4 and 24 h of 20 nm Ag-NPs exposure, including members of the metallothionein (MT) family (MT1B, MT1F, MT1G, MT1M and MT2A). The 50 nm Ag-NPs exposure also showed four common genes of MT family, except an additional activation of SOX4 gene. Overall, MT1B and MT1M were shared by all treatment groups. Out of 108 GO terms derived from the 649 upregulated genes in 4 h 20 nm of Ag-NPs revealed 23 categories, on top listed the metabolism (47%), development (19%), protein metabolism (15%), cell differentiation (13%), biosynthesis (11%), death (9%) and cell communication (9%). The 162 downregulated genes exhibited 21 groups of GO terms enrichment, classified into 9 categories, mostly similar to upregulated GO terms, but with different percentage and orders. Within the pathways analysis, Ag-NPs affected the endocytosis, MAPK, TGF-β, p53 signalling pathways, pathways in cancer and NFR2-mediated oxidative stress response [55].

9.5 Toxicological Potential of Nanoparticles

The gene expression profiling of TiO2-NPs of varying sizes and surface properties has been reported to induce pulmonary inflammation. However, the different TiO2-NPs vary in the magnitude of the inflammatory response induced in a property-dependent manner [56]. Our recent studies demonstrated that TiO2-NPs preferentially bind in subdomains IB, IIA of HSA and minor groove of DNA [57]. TiO2-NPs might be able to enter the human stratum corneum and interact with the immune system. Silica NPs are used in the synthesis of cosmetics, foods, drugs, and printing ink tonners, on a large industrial scale. The nanotoxicity of crystalline silica causes chronic obstructive pulmonary diseases such as silicosis [58]. Silica NPs exists in the nature in many diverse forms [59, 60]. Fumed silica showed dose dependent accumulation of alveolar macrophages [61,62,63]. Cerium oxides (CeO2)-NPs are one of the most widely used types for UV protection in paints or as fuel additives [64, 65]. CeO2-NPs can be used as a scavenger of superoxide anions [66, 67]. CeO2-NPs were shown to exhibit superoxide dismutase (SOD) and catalase enzymes mimetic activities in a redox-state dependent manner. CeO2-NPs has been shown to hold the neuroprotective effects [68]. On the other hand, CeO2-NPs has been reported to be cytotoxic to human hepatoma cells [69].

Ag-NPs have antimicrobial activity and are used in food packaging material, food supplements, odour-preventing textiles, cosmetics, kitchen utensils, toys, electronics, wound dressings, and room sprays [70]. Ag-NPs released Ag ions to exert antimicrobial properties by binding to sulphur- and phosphorous containing biomolecules and also causing damage to mammalian cells [71,72,73,74,75]. The in vivo inhalation data of Ag-NPs showing varied results from a minimal inflammatory response to the presence of inflammatory lesions in the lungs [76,77,78,79]. There are studies indicating that the dose-dependent increase of Ag-NPs might stimulate toxicity in the different organs [77, 78]. It has been hypothesized that small Ag-NPs will induce more prominent pulmonary toxicity compared to larger Ag particles because of the larger deposited dose in the alveoli and the higher dissolution rate. The dissolution of Ag-NPs depends on their particle size, the pH of the solution, the ions present in the solution [80,81,82,83,84]. The in vitro results show that the dissolution rate of the 15 nm Ag-NPs will probably be higher compared to the 410 nm Ag-NPs resulting in an increased ion release. None of the studies report complete dissolution of Ag-NPs, the effects observed after exposure to Ag-NPs can be induced by the released ions, the Ag-NPs itself or a combination of both [85]. Ag ions released from Ag-NPs caused more damage inside the nucleus as compared to Ag ions released from silver nitrate [86,87,88].

The harmful effects of carbon nanotubes (CNTs) to animals and cells appeared almost a decade ago [89]. Several mechanisms of toxicity, similar to the ones linked to asbestos-exposure, have been proposed for CNTs, such as (i) association of fibres with the cell membrane causing physical damage and cell membrane malfunction, (ii) protein-fibre interaction inhibiting protein function, and (iii) induction of ROS, either directly by the CNTs themselves or indirectly through mitochondrial dysfunctions or NADPH oxidase activation induced by so-called frustrated phagocytosis in e.g. macrophages [90,91,92]. It seems probable that a combination of different mechanisms could contribute to the toxicity of CNTs, as it has been considered to be the case with asbestos [93]. Multiwalled CNTs are efficient scavengers of •OH and superoxide (•O2 −) radicals in cell-free conditions [94]. The generation of free radicals by CNTs was suggested to be related to the amount and nature of defects in the CNTs, i.e. ruptures of the graphene framework [94]. In contrast, ROS formation by single walled CNTs was observed in cell media with and without FE1-Muta™ Mouse lung epithelial cells, at intermediate levels between that of Printex 90 and C60 fullerene and correlated with the order of genotoxicity [95]. Our toxicogenomic analysis on ZnFe2O4-NPs reveled its cytotoxicity and apoptosis through ROS generation and oxidative stress via p53, survivin, bax/bcl-2 and caspase pathways in WISH cells [96]. We have also demonstrated that ZnO-NPs have the potential to induce DNA damage and alter the mitochondrial membrane potential of human lymphocytes [97]. Previously, we have reported that ZnO-QDs can induce dose dependent apoptosis induction in C2C12, HepG2 and MCF-7 cells via oxidative stress and alterations of apoptosis related genes [98, 99].

9.6 Conclusion and Future Perspective

The incessant use of NPs in various sectors and life domains may pose serious threats to ecosystem and adversely affect the living entities via interactions and accumulation of nanomaterials in the body of the organisms. Toxicogenomics approach notably contributes to our understanding of genetic changes at molecular levels. Significant change in the gene expression levels due to the NPs treatment provides information related to biochemical pathways and mechanism of action of nanotoxicants. Differential gene expression pattern may also yield molecular fingerprints of these nanotoxicants both with the in vitro and in vivo test model systems. Thus, the toxicogenomic methods have the power and potential to change nanotoxicology research landscape.