Data on HepG2 cells changes following exposure to cadmium sulphide quantum dots (CdS QDs).

The data included in this paper are associated with the research article entitled "Markers for toxicity to HepG2 exposed to cadmium sulphide quantum dots; damage to mitochondria" (Paesano et al.) [1]. The article concerns the cytotoxic and genotoxic effects of CdS QDs in HepG2 cells and the mechanisms involved. In this dataset, changes in expression levels of candidate genes are reported, together with details concerning synthesis and properties of CdS QDs, additional information obtained through literature survey, measures of the mitochondrial membrane potential and the glutathione redox state.

Specifications Table

Value of the data

The dataset provides a list of candidate genes useful for comparing effects of nanomaterials in other cell systems.

The data may be useful for other researchers analysing mitochondrial dysfunctionalities in stressed cells.

The literature survey may be useful for planning additional experiments on risk assessment of cadmium-based quantum dots.

Data

Table 1 shows a survey of the most recent literature on experimentation with animal cell lines and cadmium-based quantum dots. It details the main parameters which can be assessed to estimate the functionality and viability of cells and describes the main changes detected in cells. Particular reference has been made to instances of oxidative stress, apoptosis and autophagy. In addition Table 2 reports a comparison of the effects of exposure to CdS QDs and Cd2+ ions. Table 3 reports in details the experimental conditions chosen for treating HepG2 cells with CdS QDs. Table 4 lists the changes in gene expression of candidate genes upon exposure to toxic and subtoxic doses of CdS QDs. Genes are grouped according to their involvement in relevant cellular processes. Table 5 lists the changes in gene expression determined after quantitative reverse transcriptase PCR (qRT-PCR) for specific genes chosen on the basis of their involvement in relevant cellular processes.

Table 1

Literature survey detailing parameters of cell functionality determined in experiments with cadmium-based quantum dots.

Reference	Quantum dots	Dimensions	Cell line(s)	Experimental evaluation of cell parameters	Main effects or toxicity mechanisms
Al-Ali et al. [2]	Commercial, CdSe/ZnS coated with hexadecylamine, carboxyl groups or polyethylene glycol	From 4 nm to 82 nm, peak at 6 nm	THP-1 (human monocytic cell line) differentiated into macrophages	Cell viability (Relative population doubling)	No significant cytotoxicity at 24 h, some cytotoxicity at 72 h of exposure. Induction of inflammatory response. Modification of gene expression for genes of oxidative stress pathway
				Inflammatory response (ELISA analysis for cytokine, chemokine)
				Oxidative stress (Gene expression analysis)
Alomar [3]	CdS	From 53.40 nm to 5.60 nm	A549 (human lung adenocarcinoma epithelial cells)	Cell viability (MTT assay and neutral red uptake)	Loss of cell viability. Induction of oxidative stress. Reduced mitochondrial function. Induced lysosome activity. Induction of apoptosis and inflammatory response
				Mitochondrial functionality (Mitochondrial membrane potential)
				Oxidative stress (Intracellular reactive oxygen species (ROS) levels, lipid peroxidation assay, glutathione (GSH) levels, superoxide dismutase activity)
				Apoptosis (Staining of chromosomes, caspase assay, COMET assay)
				Inflammatory response (ELISA assay for release of cytokines)
Bao et al. [4]	CdSe, or CdSe core with CdS shell	6 nm	Five human cell lines: HL-7702, Bel-7402, Bel-7404, 786-O, HeLa	Cell viability (MTT assay)	Cytotoxicity increased by CdS shell, differing among cell lines. Induction of apoptosis. Toxic to mice
				Apoptosis (Flow cytometry)
				Acute toxicity (mice)
Hossain and Mukherjee [5]	CdS	3 nm	HeLa (human cell line)	Cell viability (MTT assay, microscopy)	Decrease of cell viability. Accumulation of ROS. Changes in cell and nucleus morphology
				Oxidative stress (intracellular ROS levels)
Ju et al. [6]	CdSe core with ZnS shell, uncoated or coated with polyethylene glycol	440 nm and 680 nm	HSF-42 (human skin fibroblasts)	Cell viability (MTT assay and Trypan blue exclusion)	Uncoated QDs decrease cell viability, increase foci formation and DNA damage, but without apoptosis. They induce increased ROS levels. Coated QDs do not produce effects
				DNA damage (formation of foci, Comet assay)
				Apoptosis (Western blot for caspase)
				Oxidative stress (intracellular ROS levels)
Li et al. [7]	CdTe with thioglycolic acid	Around 2 nm	NIH/3T3 (mouse cell line)	Cell viability (WST-1 assay)	Release of Cd ions. Decrease in cell viability. Changes in miRNA expression
				miRNA expression (extraction, sequencing, real time PCR analysis, Western blot for p53)
Luo et al. 2013 [8]	Commercial: Cd/Se core, shell with ZnS	From 8 nm to 12 nm (10±2 nm)	RAG (mouse renal adenocarcinoma)	Cell viability (MTT assay, Lactate Dehydrogenase activity assay to detect necrosis)	Increase in intracellular levels of reactive oxygen species. Induction of autophagy, followed by apoptosis. QDs localised in ER, endosomes, lysosomes, mitochondria. Effect on mitochondrial function. Autophagy induced by oxidative stress is seen as a defense mechanism
				Mitochondrial functionality (ATP level and membrane potential)
				Apoptosis (TUNEL staining)
				Autophagy (microscopy and protein analysis)
Manshian et al. [9]	Commercial CdSe core with ZnS shell, amine or carboxyl functional groups on the surface	From 3 nm to5 nm	Three human cell lines: BEAS-2B, HFF-1, TK6	Cell viability (flow cytometry and microscopy, Relative population doubling)	Cytotoxicity depending on cell types, BEAS-2B showed no effects. Carboxyl functionalised QDs exert genotoxic effects. Minimal increase of ROS levels. Decrease in mitochondrial functionality with carboxyl QDs. No inflammatory effect
				DNA damage (Micronuclei frequency, pancentromeric staining)
				Oxidative stress (intracellular ROS levels)
				Mitochondrial functionality (membrane potential)
				Inflammatory response (ELISA assay for cytokines)
Nguyen et al. [10]	Commercial, CdTe core, CdS shell, coated by polyacrylate	7.3±1.2 nm	HepG2 (human hepatocellular carcinoma)	Cell viability (MTT assay)	Decrease in cell viability. Swelling of mitochondria and loss of cristae. Localisation of QDs in mitochondria. Loss of membrane potential. Increase in cytosolic calcium levels. Inhibition of respiration. Decrease in ATP levels. Decrease in complexes II, III, IV, no change in complexes I and V. Activation of mitochondrial biogenesis
				Mitochondrial morphology (Transmission Electron Microscopy, TEM)
				Mitochondrial functionality (membrane potential, quantification of complexes, activity assay, oxygen consumption, ATP levels)
				Mitochondria biogenesis (ELISA for PGC-1alpha levels)
				Localisation of QDs (mitochondria enrichment)
				Oxidative stress and apoptosis (release of calcium)
Nguyen et al. [11]	Commercial, CdTe core with CdS shell coated with polymers	7.3±1.2 nm	J774A.1 (mouse macrophage), HT29 (human colon epithelium)	Cell viability (MTT assay, microscopy)	Decrease in cell viability. Morphological changes in cells, actin filaments, nucleus. No changes in inflammatory markers. Increase in the cytotoxicity of bacterial infection and alteration of immune response
				Nitric oxide production (Griess reagent)
				Inflammatory response (cytokines and chemokines assay)
Nguyen et al. [12]	Commercial, CdTe core with CdS shell coated with polymers	7.3±1.2 nm	HepG2	Cell viability (MTT assay)	Cause oxidative stress, interfere with antioxidant defenses and activate protein kinases, leading to apoptosis via both extrinsic and intrinsic pathways. Effects of CdTe-QDs were similar or greater compared to those of CdCl2 at equivalent concentrations of cadmium
				Oxidative stress (intracellular ROS levels, GSH levels, SOD and CAT activity, Nrf2 activation)
				Apoptosis (caspase activity, Annexin stain, Bcl2 and Bax levels)
Peng et al. [13]	Four types of commercial QDs, CdSe core and ZnS shell, functional groups on the surface	From 15 nm to 25 nm	HepG2 (human hepatocellular carcinoma)	Cell viability (MTT assay)	Localisation to lysosomes. No decrease in cell viability. No apoptosis. No changes in GSH levels. Upregulation of stress response genes
				Apoptosis (DNA staining)
				Oxidative stress (GSH levels)
				Stress response (analysis of gene expression)
Romoser et al. [14]	Commercial, CdSe/ZnS-COOH	15 nm	HEK (human epidermal keratinocytes), HDF (human dermal fibroblasts)	Cell viability (resazurin assay, microscopy)	Morphological changes and loss of viability. Internalisation of QDs. Upregulation of genes involved in NFkappaB pathway. Induction of inflammatory and immune response. Induction of apoptotic genes. Induction of markers for oxidative stress
				Localisation of QDs (fluorescence)
				Inflammatory response, immune response, apoptosis (gene expression analysis, Western blot, transcriptional activity of NFkappaB)
Smith et al. [15]	CdSe core, ZnS shell, coated with polymers TOPO and PMAT	12.7±0.5 nm	HepG2 (human hepatocellular carcinoma)	Localisation of QDs (fluorescence microscopy)	No effect on cell viability. No induction of GSH. No effect on oxidative stress gene markers. No induction of inflammatory response
				Cell viability (MTT assay)
				Oxidative stress (GSH levels)
				Stress response (analysis of gene expression, Western blotting)
				Inflammatory response (cytokine assay)
Tan et al. [16]	CdSe core, ZnS shell, polymer coating with carboxyl or amine groups, functionalisation	From 20 nm to 30 nm	HepG2 (human hepatocellular carcinoma), NIH/3T3 (mouse cell line)	Cell viability (microscopy, MTT assay)	Effects depend on surface charge, hydrophobicity and presence of PEG. Cytotoxicity depends on intake. QDs with PEG can reach the lysosomes. Endocytosis is mediated by clathrin
				Localisation of QDs (microscopy)
				Endocytosis (inhibitors)
Wu et al. [17]	CdTe coated with carboxyl groups	Unspecified	HepG2 (human hepatocellular carcinoma)	Apoptosis (DNA staining)	Induction of apoptosis. Increase of ROS and decrease of GSH. Induction of autophagic vacuoles. Presence of swollen mitochondria
				Oxidative stress (intracellular ROS levels, GSH levels)
				Autophagy (TEM)
Xu et al. [18]	CdTe, and nanocomposites of QDs with gambogic acid, GA-CdTe	About 5 nm	HELF (human embryonic lung fibroblasts), HepG2 (human hepatocellular carcinoma)	Cell viability (MTT assay)	Little effect on cell viability for CdTe QDs, enhanced by GA. Intake into cells. Induction of apoptosis not assessed, no changes in cell cycle phases
				Cell morphology (DAPI staining)
				QDs localisation (fluorescence microscopy)
				Apoptosis (annexin,propidium iodide, flow cytometry)
Zhang et al. [19]	CdTe	2.2 nm	AML 12 (mouse hepatocytes)	Cell viability (MTT assay)	Loss of cell viability. Decrease in ATP levels. Induction of oxidative stress. Induction of apoptosis and related genes for mitochondria-dependent pathways. Activation of Nrf2 pathway for response to ROS
				Mitochondrial functionality (ATP levels)
				Oxidative stress (intracellular ROS levels)
				Apoptosis (annexin, propidium iodide, flow cytometry, analysis of gene expression, Western blot)
Zhang et al. [20]	Commercial, CdSe core, ZnS shell, coated with PEG: neutral, acid or basic	core/shell: 6 nm (minor axis) ×12 nm (major axis)	Dendritic cells derived from PBMC (pig peripheral blood mononuclear cells)	Cell viability (flow cytometry, proliferation, cell counting, Alamar-Blue assay)	Dencritic cells intake QDs with acidic surface. Endocytosis involves clathrin and partially micropinocytosis Small loss in cell viability. Induction of oxidative stress. No induction of inflammatory response
				Endocytosis (inhibitors)
				Oxidative stress (GSH levels)
				Inflammatory response (cytokine assay)
Zhang et al. [21]	CdTe coated with 3-mercaptopropionic acid	About 4 nm	HepG2/ADM (human hepatoma, adriamycin resistant cells)	Cell viability (MTT assay, microscopy)	Decrease in cell viability, synergistic effect with drug daunorubicin through facilitated uptake. Induction of apoptosis, DNA fragmentation. Activation of caspases after release of cytochrome C. Inhibition of tumor growth in synergy with drug
				Drug uptake (electrochemical assay)
				Apoptosis (DNA staining, flow cytometry, DNA fragmentation, Western blots, TUNEL staining)
				Acute toxicity (mice)

Table 2

A comparisons of the effects of exposure to CdS QDs and Cd2+ ions.

	Gene expression	Oxidative Stress	Genotoxicity	Cell death
CdS QDs (our experiments)	Most of the induced genes belonged to one of the three major functional categories “apoptosis", "autophagy" and "stress response"	CdS QDs did not induce a rapid ROS generation, causing only mild increase in reactive oxygen species (ROS) levels	Exposure to CdS QDs generated, in HepG2 cells, a minor degree of DNA damage; after a major exposure, the extent of the damage was almost indistinguishable from that shown by the non-treated control	The data suggest that mitochondria-mediated intrinsic apoptosis pathway were activated by CdS QDs exposure, but did not arrive at the extreme point (mtDNA distruption, cell death)
Cadmium	Cadmium induces at least two types of genes: (1) genes coding for detoxifying and other cytoprotective proteins, i.e. metallothioneins, enzymes of glutathione synthesis, heat shock proteins, zinc transporter proteins; and (2) early genes response proto-oncogenes related to cell proliferation control (c-FOS, c-JUN, c-MYC, EGR-1) [22]	Cadmium induces a rapid and transient ROS generation [23], causing formation of superoxide ion and hydrogen peroxide [24]. The major toxic effects of increasing doses of Cd concentration involve decreased antioxidant enzyme levels (superoxide dismutase and glutathione peroxidase) [25]	Cadmium causes DNA fragmentation [23]	Oh and Lim [23] demonstrated that Cd-induced cell death was caspase-dependent
			Several authors report the view that genotoxicity induced by Cd is not a direct effect of the metal, but rather due to the generation of reactive oxygen free radicals and the resulting oxidative stress [26]
			Cd exhibits remarkable potential to inhibit DNA damage repair, and this has been identified as a major mechanism underlying the carcinogenic potential of Cd [27]

Table 3

Rationale of doses and time for CdS QDs treatment.

Experiment type	Treatments			Controls
	Substance	Dosesa	Timesb	Untreated	Medium+reagent assay	Medium+QDs+reagent assay	Positive
Flow cytometryd	CdS QDs	100 µg mL−1	0–30–60–240 min	X
Cytotoxicity assaye	CdS QDs	0.5–100 µg mL−1	24 h	X	X	X	Aflatoxin B
	CdSO4	Cd concentration equivalent to QDs
Gene expression analysisf	CdS QDs	3–7–14 µg mL−1	24 h	X
Genotoxicity assayg	CdS QDs	1.5 µg mL−1	1–4–24 h	X
Reactive oxygen species with DCFH-DAh	CdS QDs	3–7–14 µg mL−1	1–4 h	X	X	X	H2O2
Reactive oxygen species with microscopei	CdS QDs	14 µg mL−1	4 h	X			H2O2
Nitric oxide assayj	CdS QDs	3–7–14 µg mL−1	1–4 h	X	X	X	LPSc
Glutathione with DTNBk	CdS QDs	3–7–14 µg mL−1	24 h	X	X	X
Mitochondrial functionl	CdS QDs	14 µg mL−1	4 h	X
mtDNAm	CdS QDs	3–7–14 µg mL−1	24 h	X

The concentrations were chosen on the basis of cell viability assay. Three doses were chosen: a toxic (acute) dose, corresponding to half-maximal inhibitory concentration (IC50), a sub-toxic (subacute or upperchronic) dose (IC30) and an intermediate (chronic) dose (IC40).

The choice of treatment times includes sampling at 1 and 4 h to highlight the early cell response to CdS QDs, and 24 h to observe the late response or the recovery.

LPS=Lipopolysaccharide.

Given the small size of the QDs, the cells were treated with 100 µg mL−1 to better observe the possible variation of the parameter SS. In addition, the time-dependent uptake was monitored.

A dose-response curve was obtained by exposing the cells to doses of QDs for 24 h. This curve was used for IC50 determination.

Three doses were chosen: a toxic, corresponding to IC50, a sub-toxic (IC30) and an intermediate dose (IC40).

Since, for Comet assay, it is necessary to use a dose that does not cause cytotoxic effects, the cells were exposed to 1.5 µg mL−1 CdS QDs. The treatment times were 1 and 4 h, to highlight the early DNA damage attributed to oxidative stress, and 24 h to verify the recovery of the damage.

DCFH-DA is 2′,7′-dichlorodihydrofluorescein diacetate. Doses used are the same as in point f. Treatment times were reduced as 1 and 4 h are enough to highlight the presence of reactive oxygen species (ROS).

Because 4 h exposure to 14 µg mL−1 CdS QDs induced a large increase in ROS level, this condition was used to monitor ROS formation using fluorescence microscopy. H2O2 treatment, in conditions that give a good signal, was used as positive control.

See ROS with DCFH-DA.

DTNB is 5,5′-dithiobis(2-nitrobenzoic acid). The same conditions used for gene expression analysis were used.

Because 4 h exposure to 14 µg mL−1 CdS QDs induced a large increase in ROS level, this condition was used to analyze mitochondrial integrity.

The same conditions used for gene expression analysis were used to appreciate the possible change in the mtDNA content, following the alteration of mitochondrial function.

Table 4

Gene subjected to transcription profiling using TaqMan® Custom Array Plates. Values in bold face are statistically significant at p<0.5. Official full name and function derive from UniProt Knowledgebase (UniProtKB) (accessed in July 2016, www.uniprot.org).

Categories	Symbol	Official full name	Function	Fold change (RQ)
				3 µg mL−1	14 µg mL−1
Apoptosis	ABL1	Tyrosine-protein kinase ABL1	Non-receptor tyrosine-protein kinase that plays a role in many key processes linked to cell growth and survival such as cytoskeleton remodeling in response to extracellular stimuli, cell motility and adhesion, receptor endocytosis, autophagy, DNA damage response and apoptosis. Coordinates actin remodeling through tyrosine phosphorylation of proteins controlling cytoskeleton dynamics	0.565	1.475
	BAK1	Bcl-2 homologous antagonist/killer	In the presence of an appropriate stimulus, accelerates programmed cell death by binding to, and antagonizing the anti-apoptotic action of BCL2	1.131	1,632
	BAX	Apoptosis regulator BAX	Promotes activation of CASP3, and thereby apoptosis	1.860	2.397
	BBC3 (PUMA)	Bcl-2-binding component 3	Essential mediator of p53/TP53-dependent and p53/TP53-independent apoptosis	0.806	3.973
	CASP2	Caspase-2	Involved in the activation cascade of caspases responsible for apoptosis execution. Might function by either activating some proteins required for cell death or inactivating proteins necessary for cell survival	1.127	0.961
	CASP3	Caspase-3	Involved in the activation cascade of caspases responsible for apoptosis execution. Cleaves and activates caspase −6, −7 and −9	1.455	0.923
	CHEK2 (RAD53)	Serine/threonine-protein kinase Chk2	It is required for checkpoint-mediated cell cycle arrest, activation of DNA repair and apoptosis in response to the presence of DNA double-strand breaks. May also negatively regulate cell cycle progression during unperturbed cell cycles	1.685	1.478
	DEDD2	DNA-binding death effector domain-containing protein 2	May play a critical role in death receptor-induced apoptosis and may target CASP8 and CASP10 to the nucleus. May regulate degradation of intermediate filaments during apoptosis. May play a role in the general transcription machinery in the nucleus and might be an important regulator of the activity of GTF3C3	0.716	1.497
	DIABLO (SMAC)	Diablo homolog, mitochondrial	Promotes apoptosis by activating caspases in the cytochrome c/Apaf-1/caspase-9 pathway. Acts by opposing the inhibitory activity of inhibitor of apoptosis proteins (IAP). Inhibits the activity of BIRC6/bruce by inhibiting its binding to caspases	1.524	1.679
	HRK (BID3)	Activator of apoptosis harakiri	Promotes apoptosis	–	–
	PIDD1 (LRDD)	p53-induced death domain-containing protein 1	Promotes apoptosis downstream of the tumor suppressor as component of the DNA damage/stress response pathway that connects p53/TP53 to apoptosis	0.398	0.569
	PMAIP1 (NOXA)	Phorbol-12-myristate-13-acetate-induced protein 1	Promotes activation of caspases and apoptosis. Promotes mitochondrial membrane changes and efflux of apoptogenic proteins from the mitochondria	2.340	3.874
	PYCARD	Apoptosis-associated speck-like protein containing a CARD	Functions as key mediator in apoptosis and inflammation	2.648	3.425
	RAD21	Double-strand-break repair protein rad21 homolog	Cleavable component of the cohesin complex, involved in chromosome cohesion during cell cycle, in DNA repair, and in apoptosis	1.785	12.435
	XIAP (BIRC4)	E3 ubiquitin-protein ligase XIAP	Multi-functional protein which regulates not only caspases and apoptosis, but also modulates inflammatory signaling and immunity, copper homeostasis, mitogenic kinase signaling, cell proliferation, as well as cell invasion and metastasis. Acts as a direct caspase inhibitor	0.646	0.684
DNA repair	FANCA	Fanconi anemia group A protein	DNA repair protein that may operate in a postreplication repair or a cell cycle checkpoint function. May be involved in interstrand DNA cross-link repair and in the maintenance of normal chromosome stability	1.456	1.783
	MRE11A	MRE11 homolog A, double strand break repair nuclease	Component of the MRN complex, which plays a central role in double-strand break (DSB) repair, DNA recombination, maintenance of telomere integrity and meiosis	–	–
	MSH2	DNA mismatch repair protein Msh2	Component of the post-replicative DNA mismatch repair system (MMR). Forms two different heterodimers which bind to DNA mismatches thereby initiating DNA repair	3.320	2.310
	NBN	Nibrin	Component of the MRN complex which plays a critical role in the cellular response to DNA damage and the maintenance of chromosome integrity. The complex is involved in double-strand break repair, DNA recombination, maintenance of telomere integrity, cell cycle checkpoint control and meiosis. The complex possesses single-strand endonuclease activity and double-strand-specific 3׳-5׳ exonuclease activity, which are provided by MRE11A	3.628	1.962
	PRKDC	DNA-dependent protein kinase catalytic subunit	Serine/threonine-protein kinase that acts as a molecular sensor for DNA damage. Involved in DNA nonhomologous end joining (NHEJ) required for double-strand break repair	1.264	1.239
	RAD18	E3 ubiquitin-protein ligase RAD18	E3 ubiquitin-protein ligase involved in postreplication repair of UV-damaged DNA. Postreplication repair functions in gap-filling of a daughter strand on replication of damaged DNA. Associates to the E2 ubiquitin conjugating enzyme UBE2B to form the UBE2B-RAD18 ubiquitin ligase complex involved in mono-ubiquitination of DNA-associated PCNA on ׳Lys-164׳. Has ssDNA binding activity	1.912	1.090
	REV1	DNA repair protein REV1	Deoxycytidyl transferase involved in DNA repair. Transfers a dCMP residue from dCTP to the 3׳-end of a DNA primer in a template-dependent reaction	1.615	2.345
	RPA1	Replication protein A 70 kDa DNA-binding subunit	As part of the heterotrimeric replication protein A complex (RPA/RP-A), binds and stabilizes single-stranded DNA intermediates, which form during DNA replication or upon DNA stress. It prevents their reannealing and in parallel, recruits and activates different proteins and complexes involved in DNA metabolism. Thereby, it plays an essential role both in DNA replication and the cellular response to DNA damage	1.610	1.377
	XPA	DNA repair protein complementing XP-A cells	Involved in DNA excision repair. Initiates repair by binding to damaged sites with various affinities, depending on the photoproduct and the transcriptional state of the region	2.305	1.409
	XRCC6	X-ray repair cross-complementing protein 6e	Single-stranded DNA-dependent ATP-dependent helicase. Has a role in chromosome translocation	1.659	1.958
Cell cycle	BUB3	Mitotic checkpoint protein BUB3	Has a dual function in spindle-assembly checkpoint signaling and in promoting the establishment of correct kinetochore-microtubule (K-MT) attachments. Promotes the formation of stable end-on bipolar attachments. Necessary for kinetochore localization of BUB1	2.882	3.705
	ICMT	Protein-S-isoprenylcysteine O-methyltransferase	Catalyzes the post-translational methylation of isoprenylated C-terminal cysteine residues	0.983	0.786
	MDM2	E3 ubiquitin-protein ligase Mdm2	E3 ubiquitin-protein ligase that mediates ubiquitination of p53/TP53, leading to its degradation by the proteasome. Inhibits p53/TP53- and p73/TP73-mediated cell cycle arrest and apoptosis by binding its transcriptional activation domain	1.177	0.802
	UBAC1	Ubiquitin-associated domain-containing protein 1	Non-catalytic subunit of the KPC complex that acts as E3 ubiquitin-protein ligase. Required for poly-ubiquitination and proteasome-mediated degradation of CDKN1B during G1 phase of the cell cycle	1.840	2.490
	ATP5L	ATP synthase subunit g, mitochondrial	Mitochondrial membrane ATP synthase	6.887	3.234
	COX8A	Cytochrome C oxidase subunit 8A, mitochondrial	This protein is one of the nuclear-coded polypeptide chains of cytochrome C oxidase, the terminal oxidase in mitochondrial electron transport	2.053	2.143
	FIS1	Mitochondrial fission 1 protein	Involved in the fragmentation of the mitochondrial network and its perinuclear clustering. Plays a minor role in the recruitment and association of the fission mediator dynamin-related protein 1 (DNM1L) to the mitochondrial surface and mitochondrial fission. Can induce cytochrome c release from the mitochondrion to the cytosol, ultimately leading to apoptosis	1.158	2.059
Mitochondrial processes	MSRB2	Methionine sulfoxide reductase B2, mitochondrial	Methionine-sulfoxide reductase that specifically reduces methionine (R)-sulfoxide back to methionine. Upon oxidative stress, may play a role in the preservation of mitochondrial integrity by decreasing the intracellular reactive oxygen species build-up through its scavenging role, hence contributing to cell survival and protein maintenance	2.371	3.209
	SDHB	Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial	Iron-sulfur protein (IP) subunit of succinate dehydrogenase that is involved in complex II of the mitochondrial electron transport chain and is responsible for transferring electrons from succinate to ubiquinone (coenzyme Q)	3.340	3.911
	PRELID3A (SLMO1)	PRELI domain containing protein 3A	in vitro, the TRIAP1:PRELID3A complex mediates the transfer of phosphatidic acid (PA) between liposomes and probably functions as a PA transporter across the mitochondrion intermembrane space. Phosphatidic acid import is required for cardiolipin (CL) synthesis in the mitochondrial inner membrane	0.917	0.707
	UQCR11	Cytochrome b-c1 complex subunit 10	This is a component of the ubiquinol-cytochrome c reductase complex (complex III or cytochrome b-c1 complex), which is part of the mitochondrial respiratory chain. This protein may be closely linked to the iron-sulfur protein in the complex and function as an iron-sulfur protein binding factor	1.201	2.129
	UQCRC1	Cytochrome b-c1 complex subunit 1, mitochondrial	This is a component of the ubiquinol-cytochrome c reductase complex (complex III or cytochrome b-c1 complex), which is part of the mitochondrial respiratory chain. This protein may mediate formation of the complex between cytochromes c and c1	1.177	1.859
	UQCRC2	Cytochrome b-c1 complex subunit 2, mitochondrial	This is a component of the ubiquinol-cytochrome c reductase complex (complex III or cytochrome b-c1 complex), which is part of the mitochondrial respiratory chain. The core protein 2 is required for the assembly of the complex	5.311	4.399
	SUMO1	Small ubiquitin-related modifier 1	Ubiquitin-like protein that can be covalently attached to proteins as a monomer or a lysine-linked polymer. This plays a crucial role in a number of cellular processes such as nuclear transport, DNA replication and repair, mitosis and signal transduction	2.429	4.445
Metabolism of protein	UBQLN1	Ubiquilin 1	Plays an important role in the regulation of different protein degradation mechanisms and pathways including ubiquitin-proteasome system (UPS), autophagy and endoplasmic reticulum-associated protein degradation (ERAD) pathway. Mediates the proteasomal targeting of misfolded or accumulated proteins for degradation by binding (via UBA domain) to their polyubiquitin chains and by interacting (via ubiquitin-like domain) with the subunits of the proteasome. Involved in the regulation of macroautophagy and autophagosome formation; required for maturation of autophagy-related protein LC3 from the cytosolic form LC3-I to the membrane-bound form LC3-II and may assist in the maturation of autophagosomes to autolysosomes by mediating autophagosome-lysosome fusion	1.698	2.148
Metabolism	ADH1	Alcohol dehydrogenase 1A	Catalyzes: an alcohol + NAD+= an aldehyde or ketone + NADH	3.122	0.775
	ACSL4	Long-chain-fatty-acid–CoA ligase 4	Activation of long-chain fatty acids for both synthesis of cellular lipids, and degradation via beta-oxidation. Preferentially uses arachidonate and eicosapentaenoate as substrates.	1.137	2.912
	DHRS11a	Dehydrogenase/reductase SDR family member 11	It is a member of the large short-chain dehydrogenase/reductase (SDR) family of enzymes that metabolize steroid hormones, prostaglandins, retinoids, lipids, and xenobiotics	0.792	0.889
	GBP2	Guanylate-binding protein 2	Catalyzes the hydrolysis GTP to GMP in two consecutive cleavage reactions	5.811	17.782
	GCLC	Glutamate-cysteine ligase catalytic subunit	Involved in glutathione biosynthesis. Catalyzes: ATP+L-glutamate+L-cysteine=ADP+phosphate+gamma-L-glutamyl-L-cysteine	1.194	3.335
	MDH1	Malate dehydrogenase, cytoplasmic	Catalyzes: (S)-malate +NAD+=oxaloacetate +NADH	3.679	2.884
	MPV17	Protein Mpv17	Involved in mitochondria homeostasis. May be involved in the metabolism of reactive oxygen species and control of oxidative phosphorylation and mitochondrial DNA maintenance	1.484	1.862
	NAA25	N(alpha)-acetyltransferase 25, NatB auxiliary subunit	Non-catalytic subunit of the NatB complex which catalyzes acetylation of the N-terminal methionine residues of peptides beginning with Met-Asp-Glu. May play a role in normal cell-cycle progression	2.677	2.176
	PDHB	Pyruvate dehydrogenase E1 component subunit beta, mitochondrial	Catalyzes the overall conversion of pyruvate to acetyl-CoA and CO2, and thereby links the glycolytic pathway to the tricarboxylic cycle	1.711	2.347
	PFKP	ATP-dependent 6-phosphofructokinase, platelet type	Catalyzes the phosphorylation of D-fructose 6-phosphate to fructose 1,6-bisphosphate by ATP, the first committing step of glycolysis	1.672	3.912
	RRM1	Ribonucleoside-diphosphate reductase large subunit	Provides the precursors necessary for DNA synthesis. Catalyzes the biosynthesis of deoxyribonucleotides from the corresponding ribonucleotides	1.828	1.149
Stress response	DDIT3	DNA damage-inducible transcript 3 protein	Multifunctional transcription factor in ER stress response. Plays an essential role in the response to a wide variety of cell stresses and induces cell cycle arrest and apoptosis in response to ER stress	0.485	5.474
	DNAJB9 (HSP40)	DnaJ homolog subfamily B member 9	Involved in endoplasmic reticulum-associated degradation (ERAD) of misfolded proteins. Acts as a co-chaperone with an Hsp70 protein	2.354	1.929
	GPX3	Glutathione peroxidase 3	Protects cells and enzymes from oxidative damage, by catalyzing the reduction of hydrogen peroxide, lipid peroxides and organic hydroperoxide, by glutathione	0.769	2.162
	HSP90AA1	Heat shock protein HSP 90-alpha	Molecular chaperone that promotes the maturation, structural maintenance and proper regulation of specific target proteins involved for instance in cell cycle control and signal transduction. Undergoes a functional cycle that is linked to its ATPase activity. This cycle probably induces conformational changes in the client proteins, thereby causing their activation. Interacts dynamically with various co-chaperones that modulate its substrate recognition, ATPase cycle and chaperone function	4.360	1.074
	HSPB3	Heat shock protein beta-3	Inhibitor of actin polymerization	0.830	1.177
	HSPD1	60 kDa heat shock protein, mitochondrial	Implicated in mitochondrial protein import and macromolecular assembly. May facilitate the correct folding of imported proteins. May also prevent misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under stress conditions in the mitochondrial matrix	2.378	2.505
	HSPH1	Heat shock protein 105 kDa	Prevents the aggregation of denatured proteins in cells under severe stress, on which the ATP levels decrease markedly. Inhibits HSPA8/HSC70 ATPase and chaperone activities	1.291	0.801
	LONP1	Lon protease homolog, mitochondrial	ATP-dependent serine protease that mediates the selective degradation of misfolded, unassembled or oxidatively damaged polypeptides as well as certain short-lived regulatory proteins in the mitochondrial matrix. May also have a chaperone function in the assembly of inner membrane protein complexes. Participates in the regulation of mitochondrial gene expression and in the maintenance of the integrity of the mitochondrial genome. Binds to mitochondrial promoters and RNA in a single-stranded, site-specific, and strand-specific manner. May regulate mitochondrial DNA replication and/or gene expression using site-specific, single-stranded DNA binding to target the degradation of regulatory proteins binding to adjacent sites in mitochondrial promoters	3.157	3.041
	SIRT1	NAD-dependent protein deacetylase sirtuin-1	NAD-dependent protein deacetylase that links transcriptional regulation directly to intracellular energetics and participates in the coordination of several separated cellular functions such as cell cycle, response to DNA damage, metobolism, apoptosis and autophagy	1.008	1.838
	TXN2	Thioredoxin, mitochondrial	Has an anti-apoptotic function and plays an important role in the regulation of mitochondrial membrane potential. Could be involved in the resistance to anti-tumor agents. Possesses a dithiol-reducing activity	1.978	1.225
miRNA biogenesis	DROSHA	Ribonuclease 3	Ribonuclease III double-stranded (ds) RNA-specific endoribonuclease that is involved in the initial step of microRNA (miRNA) biogenesis	2.512	1.790
	DICER1	Endoribonuclease Dicer	Double-stranded RNA (dsRNA) endoribonuclease playing a central role in short dsRNA-mediated post-transcriptional gene silencing	1.495	0.863
	AGO2 (EIF2C2)	Protein argonaute-2	Required for RNA-mediated gene silencing (RNAi) by the RNA-induced silencing complex (RISC)	1.109	2.170
	XPO5	Exportin-5	Mediates the nuclear export of proteins bearing a double-stranded RNA binding domain (dsRBD) and double-stranded RNAs (cargos)	1.452	1.541
Gene expression	DOM3Z	Decapping and exoribonuclease protein	Ribonuclease that specifically degrades pre-mRNAs with a defective 5׳ end cap and is part of a pre-mRNA capping quality control	1.734	2.558
	METTL18	Histidine protein methyltransferase 1 homolog	Probable histidine methyltransferase	2.395	2.295
	MRPL44	39S ribosomal protein L44, mitochondrial	Component of the 39S subunit of mitochondrial ribosome. May have a function in the assembly/stability of nascent mitochondrial polypeptides exiting the ribosome	2.670	2.392
	MTIF2	Translation initiation factor IF-2, mitochondrial	One of the essential components for the initiation of protein synthesis. Protects formylmethionyl-tRNA from spontaneous hydrolysis and promotes its binding to the 30S ribosomal subunits. Also involved in the hydrolysis of GTP during the formation of the 70S ribosomal complex.	3.941	2.292
	N6AMT1	HemK methyltransferase family member 2	Heterodimeric methyltransferase that catalyzes N5-methylation of ETF1 on ׳Gln-185׳, using S-adenosyl L-methionine as methyl donor. ETF1 needs to be complexed to ERF3 in its GTP-bound form to be efficiently methylated	6.317	2.233
	RPL36AL	60S ribosomal protein L36a-like	Structural constituent of ribosome	–	–
	RPS8	40S ribosomal protein S8	Structural constituent of ribosome	2.537	4.553
	RTF1	RNA polymerase-associated protein RTF1 homolog	Component of the PAF1 complex which has multiple functions during transcription by RNA polymerase II	2.562	2.184
	SUPT3H	Transcription initiation protein SPT3 homolog	Probable transcriptional activator	4.612	8.239
	TADA1	Transcriptional adapter 1	Probably involved in transcriptional regulation	1.719	1.796
	XRN2	5׳-3׳ exoribonuclease 2	May promote the termination of transcription by RNA polymerase II. During transcription termination, cleavage at the polyadenylation site liberates a 5׳ fragment which is subsequently processed to form the mature mRNA and a 3׳ fragment which remains attached to the elongating polymerase	2.860	3.754
	YARS2	Tyrosyl-tRNA synthetase, mitochondrial	Catalyzes the attachment of tyrosine to tRNA(Tyr) in a two-step reaction: tyrosine is first activated by ATP to form Tyr-AMP and then transferred to the acceptor end of tRNA(Tyr)	3.901	3.562
	ZNF180	Zinc finger protein 180	May be involved in transcriptional regulation	1.010	0.680
Others	ACE	Angiotensin-converting enzyme	Converts angiotensin I to angiotensin II by release of the terminal His-Leu, this results in an increase of the vasoconstrictor activity of angiotensin. Also able to inactivate bradykinin, a potent vasodilator. Has also a glycosidase activity which releases GPI-anchored proteins from the membrane by cleaving the mannose linkage in the GPI moiet	–	–
	ATG3	Ubiquitin-like-conjugating enzyme ATG3	E2 conjugating enzyme required for the cytoplasm to vacuole transport, autophagy, and mitochondrial homeostasis. Responsible for the E2-like covalent binding of phosphatidylethanolamine to the C-terminal Gly of ATG8-like proteins (GABARAP or MAP1LC3A). The formation of the ATG8-phosphatidylethanolamine conjugates is essential for autophagy and for the cytoplasm to vacuole transport. Preferred substrate is MAP1LC3A	3.323	2.078
	ATP6V1E1	V-type proton ATPase subunit E 1	Subunit of the peripheral V1 complex of vacuolar ATPase essential for assembly or catalytic function. V-ATPase is responsible for acidifying a variety of intracellular compartments in eukaryotic cells	3.972	3.311
	CHAC2	Putative glutathione-specific gamma-glutamylcyclotransferase 2	Catalyzes the cleavage glutathione into 5-oxoproline and a Cys-Gly dipeptide. Acts specifically on glutathione, but not on other gamma-glutamyl peptides	2.163	0.596
	ESD	S-formylglutathione hydrolase	Serine hydrolase involved in the detoxification of formaldehyde	3.820	3.603
	FNBP1L	Formin-binding protein 1-like	Required to coordinate membrane tubulation with reorganization of the actin cytoskeleton during endocytosis. May bind to lipids such as phosphatidylinositol 4,5-bisphosphate and phosphatidylserine and promote membrane invagination and the formation of tubules	1.845	2.754
	GAD1	Glutamate decarboxylase 1	Catalyzes the production of GABA	2.746	10.255
	HEMK1	HemK methyltransferase family member 1	N5-glutamine methyltransferase responsible for the methylation of the GGQ triplet of the mitochondrial translation release factor MTRF1L	1.119	1.354
	HSPA8	Heat shock 71 kDa protein	Acts as a repressor of transcriptional activation. Inhibits the transcriptional coactivator activity of CITED1 on Smad-mediated transcription. Chaperone. Component of the PRP19-CDC5L complex that forms an integral part of the spliceosome and is required for activating pre-mRNA splicing. May have a scaffolding role in the spliceosome assembly as it contacts all other components of the core complex	0.830	1.177
	LCP1	Plastin-2	Actin-binding protein	0.342	1.324
	NEDD8	NEDD8 (Neural precursor cell expressed, developmentally down-regulated 8)	Ubiquitin-like protein which plays an important role in cell cycle control. Covalent attachment to its substrates requires prior activation by the E1 complex UBE1C-APPBP1 and linkage to the E2 enzyme UBE2M. Attachment of NEDD8 to cullins activates their associated E3 ubiquitin ligase activity, and thus promotes polyubiquitination and proteasomal degradation of cyclins and other regulatory proteins	1.866	1.834
	PEX7	Peroxisomal targeting signal 2 receptor	Binds to the N-terminal PTS2-type peroxisomal targeting signal and plays an essential role in peroxisomal protein import	7.444	2.405
	PHB2	Prohibitin-2	Acts as a mediator of transcriptional repression by nuclear hormone receptors via recruitment of histone deacetylases	1.730	1.807
	STX16	Syntaxin-16	SNARE involved in vesicular transport from the late endosomes to the trans-Golgi network	1.352	2.038
	TWF1	Twinfilin-1	Actin-binding protein involved in motile and morphological processes. Inhibits actin polymerization	3.656	2.781
Candidate endogenous	18S	Eukaryotic 18S rRNA	–
	ACTB	Actin, beta	–
	GAPDH	Glyceraldehyde-3-phosphate dehydrogenase	–

The description of gene function has been taken from OMIM® database (accessed in July 2016, www.omim.org).

Table 5

Gene subjected to transcription profiling by qRT-PCR (SYBR® Green). Values in bold face are statistically significant at p<0.5. Official full name and function derive from UniProt Knowledgebase (UniProtKB) (accessed in July 2016, www.uniprot.org).

Categories	Symbol	Official Full Name	Function	Fold Change (RQ)
				3 µg mL−1	7 µg mL−1	14 µg mL−1
Apoptosis	AIFM2	Apoptosis-inducing factor 2	Oxidoreductase, which may play a role in mediating a p53/TP53-dependent apoptosis response. Probable oxidoreductase that acts as a caspase-independent mitochondrial effector of apoptotic cell death	5.938	1.002	0.497
	CASP7	Caspase 7	Involved in the activation cascade of caspases responsible for apoptosis execution	0.448	0.501	0.349
	DAPK1	Death-associated protein kinase 1	Calcium/calmodulin-dependent serine/threonine kinase involved in multiple cellular signaling pathways that trigger cell survival, apoptosis, and autophagy. Regulates both type I apoptotic and type II autophagic cell deaths signal, depending on the cellular setting. The former is caspase-dependent, while the latter is caspase-independent and is characterized by the accumulation of autophagic vesicles	0.644	0.809	0.709
	FADD	FAS-associated death domain protein	Apoptotic adaptor molecule that recruits caspase-8 or caspase-10 to the activated Fas (CD95) or TNFR-1 receptors	5.776	0.735	0.660
	MAP3K5	Mitogen-activated protein kinase kinase kinase 5	Serine/threonine kinase which acts as an essential component of the MAP kinase signal transduction pathway. Mediates signaling for determination of cell fate such as differentiation and survival. Plays an important role in the cascades of cellular responses evoked by changes in the environment. Plays a crucial role in the apoptosis signal transduction pathway through mitochondria-dependent caspase activation	0.192	0.037	0.205
	PYCARD	Apoptosis-associated speck-like protein containing a CARD	Functions as key mediator in apoptosis and inflammation. Promotes caspase-mediated apoptosis involving predominantly caspase-8 and also caspase-9 in a probable cell type-specific manner. Involved in activation of the mitochondrial apoptotic pathway, promotes caspase-8-dependent proteolytic maturation of BID independently of FADD in certain cell types and also mediates mitochondrial translocation of BAX and activates BAX-dependent apoptosis coupled to activation of caspase−9, −2 and −3	1.270	1.510	0.793
	BAD	Bcl2-associated agonist of cell death	Promotes cell death. Successfully competes for the binding to Bcl-X(L), Bcl-2 and Bcl-W, thereby affecting the level of heterodimerization of these proteins with BAX	3.000	0.487	0.559
	BCL2	Apoptosis regulator Bcl-2	Regulates cell death by controlling the mitochondrial membrane permeability. Appears to function in a feedback loop system with caspases. Inhibits caspase activity either by preventing the release of cytochrome c from the mitochondria and/or by binding to the apoptosis-activating factor (APAF-1)	0.521	0.375	0.204
	APAF1	Apoptotic protease-activating factor 1	Oligomeric Apaf-1 mediates the cytochrome c-dependent autocatalytic activation of pro-caspase-9 (Apaf-3), leading to the activation of caspase-3 and apoptosis	5.464	0.844	0.423
	BAK1	Bcl-2 homologous antagonist/killer	In the presence of an appropriate stimulus, accelerates programmed cell death by binding to, and antagonizing the anti-apoptotic action of BCL2 or its adenovirus homolog E1B 19k protein	0.357	0.429	0.421
	BID	BH- interacting domain death agonist	The major proteolytic product p15 BID allows the release of cytochrome C	0.505	0.797	0.369
	CASP8	Caspase-8	Most upstream protease of the activation cascade of caspases responsible for the TNFRSF6/FAS mediated and TNFRSF1A induced cell death. Binding to the adapter molecule FADD recruits it to either receptor	0.473	0.684	0.326
	CASP9	Caspase-9	Involved in the activation cascade of caspases responsible for apoptosis execution. Binding of caspase-9 to Apaf-1 leads to activation of the protease which then cleaves and activates caspase-3. Promotes DNA damage-induced apoptosis in a ABL1/c-Abl-dependent manner. Proteolytically cleaves poly(ADP-ribose) polymerase	1.172	2.585	1.021
	AOX1	Aldehyde oxidase	Plays a key role in the metabolism of xenobiotics and drugs	10.629	0.807	1.169
Stress response	COX4I1	Cytochrome c oxidase subunit 4 isoform 1, mitochondrial	This protein is one of the nuclear-coded polypeptide chains of cytochrome c oxidase, the terminal oxidase in mitochondrial electron transport	1.374	1.034	1.038
	CAT	Catalase	Occurs in almost all aerobically respiring organisms and serves to protect cells from the toxic effects of hydrogen peroxide	1.705	0.714	1.873
	CYGB	Cytoglobin	May have a protective function during conditions of oxidative stress	4.563	0.514	0.509
	DHCR24	Delta(24)-sterol reductase	Catalyzes the reduction of the delta-24 double bond of sterol intermediates. Protects cells from oxidative stress by reducing caspase 3 activity during apoptosis induced by oxidative stress	3.824	0.545	0.807
	DUSP1	Dual specificity phosphatase 1	Induction by oxidative stress and heat shock. Dual specificity phosphatase that dephosphorylates MAP kinase MAPK1/ERK2 on both ׳Thr-183׳ and ׳Tyr-185׳, regulating its activity during the meiotic cell cycle	1.045	0.962	0.966
	FOXO1	Forkhead box protein O1	Transcription factor that is the main target of insulin signaling and regulates metabolic homeostasis in response to oxidative stress	1.467	0.825	1.108
	FOXO3	Forkhead box protein O3	Transcriptional activator which triggers apoptosis in the absence of survival factors, including neuronal cell death upon oxidative stress	0.986	1.076	1.354
	GPX1a	Glutathione peroxidase 1	This gene encodes a member of the glutathione peroxidase family. Glutathione peroxidase functions in the detoxification of hydrogen peroxide	1.197	1.592	1.153
	GPX4	Phospholipid hydroperoxide glutathione peroxidase, mitochondrial	Protects cells against membrane lipid peroxidation and cell death. Protects from radiation and oxidative damage	0.902	1.269	0.889
	HIF1A	Hypoxia inducible factor 1-alpha	Functions as a master transcriptional regulator of the adaptive response to hypoxia. Under hypoxic conditions, activates the transcription of over 40 genes, including erythropoietin, glucose transporters, glycolytic enzymes, vascular endothelial growth factor, HILPDA, and other genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia	0.990	1.039	2.767
	MSRA	Mitochondrial peptide methionine sulfoxide reductase	It has an important function as a repair enzyme for proteins that have been inactivated by oxidation	4.098	1.161	0.369
	OXSR1	Serine/threonine-protein kinase OSR1	Regulates downstream kinases in response to environmental stress. May also have a function in regulating the actin cytoskeleton	2.780	0.930	0.593
	PRDX1	Peroxiredoxin-1	Involved in redox regulation of the cell. Reduces peroxides with reducing equivalents provided through the thioredoxin system but not from glutaredoxin. May play an important role in eliminating peroxides generated during metabolism	0.835	0.883	0.530
	PRDX2	Peroxiredoxin-2	Involved in redox regulation of the cell. Reduces peroxides with reducing equivalents provided through the thioredoxin system. It is not able to receive electrons from glutaredoxin	0.966	1.071	1.035
	PRDX3	Thioredoxin-dependent peroxide reductase, mitochondrial	Involved in redox regulation of the cell. Protects radical-sensitive enzymes from oxidative damage by a radical-generating system	6.320	2.204	0.812
	PRDX5	Peroxiredoxin-5, mitochondrial	Reduces hydrogen peroxide and alkyl hydroperoxides with reducing equivalents provided through the thioredoxin system. Involved in intracellular redox signaling	1.424	0.541	0.527
	PRDX6	Peroxiredoxin-6	Involved in redox regulation of the cell. Can reduce H2O2 and short chain organic, fatty acid, and phospholipid hydroperoxides. May play a role in the regulation of phospholipid turnover as well as in protection against oxidative injury	1.860	0.774	0.509
	PTGS1	Prostaglandin G/H synthase 1	Converts arachidonate to prostaglandin H2 (PGH2), a committed step in prostanoid synthesis	0.285	0.144	0.816
	GBP2	Guanylate-binding protein 2	Hydrolyzes GTP to GMP in two consecutive cleavage reactions	0.901	0.868	1.959
	STK25	Serine/threonine protein kinase 25	Oxidant stress-activated serine/threonine kinase that may play a role in the response to environmental stress. Targets to the Golgi apparatus where it appears to regulate protein transport events, cell adhesion, and polarity complexes important for cell migration	0.561	0.516	0.646
	GSR	Glutathione reductase, mitochondrial	Maintains high levels of reduced glutathione in the cytosol	2.246	2.211	3.446
	GSTZ1	Maleylacetoacetate isomerase (Glutathione S-transferase zeta 1)	Bifunctional enzyme showing minimal glutathione-conjugating activity with ethacrynic acid and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and maleylacetoacetate isomerase activity. Has also low glutathione peroxidase activity with T-butyl and cumene hydroperoxides. Is able to catalyze the glutathione dependent oxygenation of dichloroacetic acid to glyoxylic acid	5.637	0.644	3.655
	GCLC	Glutamate-cysteine ligase, catalytic subunit	This protein is involved in step 1 of the subpathway that synthesizes glutathione from L-cysteine and L-glutamate	0.235	0.483	0.241
	GSS	Glutathione synthetase	This protein is involved in step 2 of the subpathway that synthesizes glutathione from L-cysteine and L-glutamate	3.095	0.467	1.625
	LONP1	Lon protease homolog, mitochondrial	ATP-dependent serine protease that mediates the selective degradation of misfolded, unassembled or oxidatively damaged polypeptides as well as certain short-lived regulatory proteins in the mitochondrial matrix. May also have a chaperone function in the assembly of inner membrane protein complexes. Participates in the regulation of mitochondrial gene expression and in the maintenance of the integrity of the mitochondrial genome. Binds to mitochondrial promoters and RNA in a single-stranded, site-specific, and strand-specific manner. May regulate mitochondrial DNA replication and/or gene expression using site-specific, single-stranded DNA binding to target the degradation of regulatory proteins binding to adjacent sites in mitochondrial promoters	0.829	1.021	0.635
	MPV17	Protein Mpv17	Involved in mitochondria homeostasis. May be involved in the metabolism of reactive oxygen species and control of oxidative phosphorylation and mitochondrial DNA maintenance	1.090	0.389	0.470
	NOS2	Nitric oxide synthase, inducible	Produces nitric oxide (NO) which is a messenger molecule with diverse functions throughout the body.	1.602	0.334	0.408
	OXR1	Oxidation resistance protein 1	May be involved in protection from oxidative damage	3.543	0.966	0.635
	SOD1	Superoxide dismutase [Cu-Zn]	Destroys radicals which are normally produced within the cells and which are toxic to biological systems	1.747	1.108	0.938
	SOD2	Superoxide dismutase [Mn], mitochondrial	Destroys superoxide anion radicals which are normally produced within the cells and which are toxic to biological systems	0.998	0.567	0.995
	TXN	Thioredoxin	Participates in various redox reactions through the reversible oxidation of its active center dithiol to a disulfide and catalyzes dithiol-disulfide exchange reactions. Plays a role in the reversible S-nitrosylation of cysteine residues in target proteins, and thereby contributes to the response to intracellular nitric oxide. Nitrosylates the active site Cys of CASP3 in response to nitric oxide (NO), and thereby inhibits caspase-3 activity	0.914	1.089	1.478
	TXNRD2	Thioredoxin reductase 2, mitochondrial	Maintains thioredoxin in a reduced state. Implicated in the defenses against oxidative stress. May play a role in redox-regulated cell signaling	1.400	0.434	0.338
	UCP2	Mitochondrial uncoupling protein 2	UCP are mitochondrial transporter proteins that create proton leaks across the inner mitochondrial membrane, thus uncoupling oxidative phosphorylation from ATP synthesis. As a result, energy is dissipated in the form of heat	0.597	0.775	0.715
Autophagy	ATG12	Ubiquitin-like protein ATG12	Ubiquitin-like protein involved in autophagy vesicles formation. Conjugation with ATG5 through a ubiquitin-like conjugating system involving also ATG7 as an E1-like activating enzyme and ATG10 as an E2-like conjugating enzyme, is essential for its function	1.600	0.581	0.747
	ATG13	Autophagy-related protein 13	Autophagy factor required for autophagosome formation and mitophagy. Target of the TOR kinase signaling pathway that regulates autophagy through the control of the phosphorylation status of ATG13 and ULK1	4.423	0.463	0.588
	ATG14	Beclin 1-associated autophagy-related key regulator	Required for both basal and inducible autophagy. Plays a role in autophagosome formation	6.869	0.821	0.561
	ATG7	Ubiquitin-like modifier-activating enzyme ATG7	E1-like activating enzyme involved in the 2 ubiquitin-like systems required for cytoplasm to vacuole transport (Cvt) and autophagy. Activates ATG12 for its conjugation with ATG5 as well as the ATG8 family proteins for their conjugation with phosphatidylethanolamine. Both systems are needed for the ATG8 association to Cvt vesicles and autophagosomes membranes. Required for autophagic death induced by caspase-8 inhibition. Required for mitophagy which contributes to regulate mitochondrial quantity and quality by eliminating the mitochondria to a basal level to fulfill cellular energy requirements and preventing excess ROS production	5.877	0.525	0.732
	GABARAP	Gamma-aminobutyric acid receptor-associated protein	Ubiquitin-like modifier that plays a role in intracellular transport of GABA(A) receptors and its interaction with the cytoskeleton. Involved in apoptosis. Involved in autophagy	1.688	0.574	2.196
	MAP1LC3A	Microtubule-associated protein 1A/1B light chain 3A	Ubiquitin-like modifier involved in formation of autophagosomal vacuoles (autophagosomes)	0.352	0.927	0.119
	MTOR	Serine/threonine-protein kinase mTOR	Serine/threonine protein kinase which is a central regulator of cellular metabolism, growth and survival in response to hormones, growth factors, nutrients, energy and stress signals. MTOR directly or indirectly regulates the phosphorylation of at least 800 proteins. Functions as part of 2 structurally and functionally distinct signaling complexes mTORC1 and mTORC2 (mTOR complex 1 and 2). mTORC1 also negatively regulates autophagy through phosphorylation of ULK1	0.700	0.914	0.779

The description of gene function has been taken from OMIM® database (accessed in July 2016, www.omim.org).

Experimental design, materials and methods

Synthesis and characterization of CdS QDs

The method1 used to synthesize CdS QDs followed Villani et al. [28], and the synthesis was performed by IMEM-CNR (Parma, Italy). X-ray diffraction (XRD) was carried out using an ARL-X’Tra device (Thermo Fisher Scientific, Waltham, MA, USA). A field emission high resolution (Scherzer resolution of ~0.19 nm) JEM-2200 FS transmission electron microscope (JEOL Ltd., Tokyo, Japan) operating at 200 kV, was used to examine QD structure. The aggregation of a group of QDs following solvent evaporation (Fig. 1a) was due to the lack of capping molecules at the QD surface. The corresponding reduced Fourier transform (FT) in the inset confirms the hexagonal structure (greenockite, P63mc) of as-synthesized CdS QDs (d=0.36 nm in agreement with standard card JCPDS no. 80-0006). The FT of the whole high resolution transmission electron microscope (HRTEM) image is presented in Fig. 1b. The expected ring feature arising from the random orientation of CdS crystals is clear, as is the overlap of (100), (002) and (101) reflections of the wurtzite structure (at high d values) due to low dimension peak broadening. Such features are in agreement with the XRD pattern shown in Fig. 1c. All peaks have been indexed according to the structure of greenockite and no other reflections arising from possible impurities are observed. A Scherrer calculation based on the FWHM (full width at half maximum) of the three main peaks produced an estimated mean size of ~6 nm. An ESEM (environmental scanning electron microscopy) Quanta 250FEG (FEI Co., Hillsboro, OR, USA) together with a QUANTAX EDS (energy-dispersive systems) XFlash® 6T detector series and the ESPRIT 2 analytical methods interface (Bruker, Berlin, Germany) was used to determine CdS QDs morphology and elemental content. Single 1 mL drops containing 80 mg L−1 CdS QDs were left to dry on an scanning electron microscopy (SEM) stub covered with carbon tape in a protected environment. Seven stubs were analysed during one round of experiments. The working parameters for SEM imaging and X-ray spectra acquisition were: pressure: 70 Pa, working distance: 9.9 mm, acceleration voltage: 20 KeV. SEM images of a CdS QDs drop at 29,750x magnification (Fig. 2a) and at 130,802x magnification (Fig. 2b) show the nanocrystals are grouped into small agglomerates of 50–100 nm. The energy-dispersive X-ray analysis (EDX) (Fig. 2c) of the point indicated by the green arrow in Fig. 2b reveals clear emission lines for Cd Lα1 and Lβ1 at 3.133 and 3.316 eV. The S Kα1 and Kβ1 lines at 2.308 and 2.464 eV are also visible.

Fig. 1

HRTEM image of ligand-free QDs assembly. (a) A CdS QDs aggregate after solvent evaporation. (b) Fourier transform analysis of the whole HRTEM image. (c) X-ray diffraction pattern.

Fig. 2

ESEM/EDX image of a 80 mg L−1 ligand-free suspension of CdS QD. The panels in (a) and (b) show SEM images at different magnifications. The green arrow in (b) indicates the point where EDX was performed. (c) EDX spectra of CdS QDs, reporting X-ray emission lines for Cd and S.

Cell culture

HepG2 cells (Fig. 3) were cultured in Dulbecco׳s Modified Eagle׳s Medium (DMEM) contained 10% (v/v) fetal bovine serum (FBS), 50 U mL−1 penicillin/streptomycin and 1% (w/v) L-glutamine in an incubator at 37 °C and 5% CO2. Cells were exposed to different doses of CdS QDs.

Fig. 3

The appearance of the cultured HepG2 cells. The bar corresponds to 500 µm.

Cytotoxicity assay

The cytotoxicity of the CdS QDs (Fig. 4) was evaluated by CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) according to the manufacturer׳s protocol. In this assay, cell viability was assumed to be proportional to the quantity of formazan generated by the reduction of MTS. Data obtained were also used in Paesano et al. [1].

Fig. 4

Cytotoxicity of CdS QDs on HepG2 cells. The inhibitory effect of various doses (0.5–100 µg mL−1) on cell viability was determined by MTS assay. The percentage reduction in viability was calculated from absorbance measurements of treated and untreated cell cultures. Data showed in figure are representative of three independent experiments, performed in triplicate.

Detection of glutathione

The cellular level of glutathione (GSH) was determined by an assay based on the conversion of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (Sigma-Aldrich) to the yellow-coloured product 2-nitro-5-thiobenzoic acid (TNB) (Fig. 5).

Fig. 5

Effect of CdS QDs on the glutathione redox state (GSH/GSSG) of HepG2 cells. Cells were exposed to either 3, 7 or 14 µg mL−1 CdS QDs for 24 h. Untreated cells cultured were run in parallel. Aflatoxin B treatment was used as positive control. Data showed in figure are representative of three independent experiments, performed in triplicate.

Assay on mitochondrial integrity

HepG2 cells were exposed to a range of CdS QDs concentrations and were subsequently stained for 30 min in 40 nM rhodamine B hexyl ester (ThermoFisher Scientific, Waltham, MA, USA), rinsed in PBS, harvested by centrifugation (800 g, 5 min) and re-suspended in PBS with 1% (v/v) FBS. The suspension was analyzed using a FC500 flow cytometer (Beckman Coulter Inc.) (Fig. 6).

Fig. 6

Effect of CdS QDs treatment on the mitochondrial membrane potential, as quantified by flow cytometry of rhodamine-stained cells. Cell were exposed to 14 µg mL−1 CdS QDs for 4 h. Rhodamine unstained [Rho(−)] and stained cells [Rho(+)] were gated and their fluorescence signal was quantified. Comparison of the two groups indicated a 12% reduction in fluorescence between the treated and untreated cells. The data shown in figure are representative of three independent experiments, performed in triplicate.

Analysis of transcriptomic data

The data reported in Table 4 were analysed to build a heat map (Fig. 7) depicting the hierarchical clustering of genes according to their expression profile. Moreover, data were summarized in a scheme that highlights the interactions between the different cellular processes involved (Fig. 8).

Fig. 7

Hierarchical clustering (heatmap) of relative transcript abundances obtained by TaqMan® Custom Array Plates in qRT-PCR. Up-regulated genes are shown in red and down- regulated ones in green. The colour intensity reflects the transcript׳s abundance. HepG2 cells were exposed for 24 h to either (1) 3 or (2) 14 µg mL−1 CdS QDs. Data showed in figure are representative of three independent experiments, each performed in triplicate for each condition.

Fig. 8

Scheme of the interaction between the intrinsic apoptosis, autophagy and stress response pathways governing the HepG2 response to CdS QDs. Up-regulation is indicated by red arrows and down-regulation by green arrows. Mitochondrial dysfunction is linked to oxidative stress, as the mitochondria are both generators of, and targets for ROS. CdS QDs may undergo autophagic sequestration and then selective compartmentalization in autophagosome, leading to a dysfunctional autophagic pathway due to the inhibition of autophagic flux. The effect could also be associated with an accumulation of ubiquitinated proteins.

Subject area	Biology
More specific subject area	Toxicogenomics, transcriptomics
Type of data	Table, image (x-ray, microscopy), text file, graph, figure
How data was acquired	Electron microscopy (JEM-2200 FS transmission electron microscope, ESEM Quanta 250FEG)
	MTS assay (CellTiter 96®AQueousOne Solution Cell Proliferation Assay)
	Real Time PCR and hierarchical clustering (TaqMan Custom Array Plates, Applied Biosystems 7900HT Fast Real-Time PCR System),
	DTNB assay [5,5′-dithiobis(2-nitrobenzoic acid)]
	Flow cytometry (FC500 flow cytometer)
Data format	Filtered and analyzed
Experimental factors	HepG2 cells were exposed to a toxic acute dose of CdS QDs corresponding to the IC50, and to two sub-toxic doses for different time periods
Experimental features	Cells were cultured in Dulbecco׳s Modified Eagle׳s Medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS), 50 U mL−1penicillin/streptomycin and 1% (w/v) L-glutamine in an incubator at 37 °C and 5% CO2
Data source location	Parma, Italy
Data accessibility	Data are available within this article