Erratum

This article refers to:

Genotoxicity of engineered nanomaterials: A critical review

The publishers would like to apologise for an error that occurred in the publication of Nanotoxicology, December 2008; 2(4): 252–274.

Genotoxicity of engineered nanomaterials: A critical review LAETITIA GONZALEZ¹, DOMINIQUE LISON², & MICHELINE KIRSCH-VOLDERS¹

Tables I–III and Figure 2 contained some errors and should have appeared as below:

Figure 2.  Summary of the available studies conducted with the comet assay (A) or the micronucleus test (B). Results are categorized on the basis of chemical nature of the ENMs and their mean primary particle size. Statistically significant genotoxic effects or absence of genotoxic effects are represented as ▪ or □, respectively. Comet assay data on specific lesions detected by enzymatic treatment with formamidopyrimidine DNA-glycosylase (fpg) and endonuclease III (EndoIII) are included. The numbers in brackets refer to the individual studies reviewed in Table II.

Table I.  An overview of the genotoxicity endpoints that can be assessed in function of the cellular targets or functionality potentially affected by ENMs.

	Genotoxicity endpoints
					MN
Potential genotoxicity targets/functions	DNA adducts	Gene mutations	DNA strand breakage (Comet)	CA	C−	C+	Cytokinesis block	Centrosome aberrations	Examples
DNA	+	+	+	+	+				Auffan et al. (2006); Gurr et al. (2005); Jacobsen et al. (2007); Jin et al. (2007); Kim et al. (2006); Maenosono et al. (2007); Mroz et al. (2008); Muller et al. (2008); Niwa and Iwai (2006); Papageorgiou et al. (2007); Park et al. (2007); Rahman et al. (2002); Reeves et al. (2008); Theogaraj et al. (2007); Wang et al. (2007); Warheit et al. (2007).
Non DNA targets:
Nucleosomes			?	?	?
Cytoskeleton:
actin/myosin							+
tubulin depolymerisation						+			Muller et al. (2008); Papageorgiou et al. (2007); Rahman et al. (2002).
delayed cellular traficking	+	+	+	+	+
Centrosomes								+
DNA repair	+	+	+	+	+				Jin et al. (2007)
Other key proteins:
Zn finger (e.g., p53, DNA repair)	+	+	+	+	+	+
SH groups (e.g., tubulin)	+	+	+	+	+	+
Metallothionein	+	+	+	+	+	+

Examples of publications are given according to the investigated cellular targets. CA, chromosome aberrations; MN, micronucleus assay; C−, centromere–negative micronuclei; C+, centromere–positive micronuclei after FISH with pancentromeric probes.

Table II.  Summary of available in vitro and in vivo genotoxicity studies with ENMs (see text for selection procedure).

NP type			Cell type	Test	Top dose	Concentration/dose With or without FCS	Result	Reference
In vitro studies
Carbon
Carbon black
14 nm			A549	Alkaline Comet assay	LDH release, Trypan blue	100 µg/ml 3 h -FCS	+	Mroz et al. (2008) [1]
				Neutral Comet assay			−
			FE1 MML (Muta^TM mouse lung epithelial cells)	Alkaline Comet assay	LDH (92,8% cell viability after 24h)	75 µg/ml 3 h +FCS	+	Jacobsen et al. (2007) [2]
				Alkaline Comet assay +fpg			+
				Mutant frequency		75 µg/ml-3 passages of 72 h +FCS	+ (cII gene and LacZ gene)
14nm (BaP- coated)			A549	Alkaline Comet assay	LDH release,	100 µg/ml 3 h -FCS	+	Mroz et al. (2008) [1]
				Neutral Comet assay	Trypan blue		−
260 nm				Alkaline Comet assay	LDH release,	100 µg/ml 3 h -FCS	+	Mroz et al. (2008) [1]
				Neutral Comet assay	Trypan blue		−
Hydroxyl fullerene (C60(OH)24)
7.1 nm			CHO	MN	LDN, cell counting	10–1000 pg/ml–80 days +FCS	+ (10 pg/ml)	Niwa and Iwai (2006) [3]
			Hela	MN		10–1000 pg/ml–80 days +FCS	+ (1000 pg/ml)
			HEK293	MN		10–1000 pg/ml–80 days +FCS	+ (100 pg/ml)
MWCNT
(11.3 nm/ 0.7µm)			RLE	CBMN	LDH, MTT, apoptosis	10–50 µg/ml–24 h +FCS	+ (10 µg/ml)	Muller et al. (2008) [4]
			MCF-7	CBMN		10–50 µg/ml–48 h +FCS	+ (10 µg/ml; Cen+/Cen-)
MWCNT			Aprt+/- 3C4 ES cells	Mutant frequency	apoptosis	5 µg/ml–12 days +FCS	(+)	Zhu et al. (2007) [5]
Cerium oxide
9 nm			Salmonella typhimurium	Ames test	Maximum recommended dose level OECD 471 guideline	50–5000 µg/plate	–	Park et al. (2007)[6]
320 nm							−
Cobalt
CoCr
29.5 nm			Primary human dermal fibroblasts	Alkaline Comet assay	MTT and LDH but top dose at 0% cell viability for Comet and 8-OH-dG, 50% for CBMN	5×10^−4–5000 µm^3/cell +FCS 1 day 3 days	+ (0,005 µm^3/cell)	Papageorgiou et al. (2007) [7]
				CBMN		5–500 µm^3/cell (12 h/12 h) +FCS	+ (5×10^-4 µm^3/cell)
				8-OH-dG		50–5000 µm^3/cell +FCS	+ (50 µm^3/cell)
						3 h	−
						24 h	(+)
2.904 µm				Alkaline Comet assay	MTT and LDH but top dose at 0% cell viability for Comet and 8-OH-dG, 50% for CBMN	5×10^−4–5000 µm^3/cell +FCS 1 day	+ (5×10^-4 µm^3/cell)
						3 days	+ (0,005 µm^3/cell)
						5 days	+ (5×10^−4 µm^3/cell)
				CBMN		5–500 µm^3/cell (12 h/12 h) +FCS	+ (5 µm^3/cell)
				8-OH-dG		50–5000 µm^3/cell +FCS
						3 h	(+)
						24 h	(+)
29.5 nm vs. 2.904 µm				Alkaline Comet assay			n-size>µ-size
				CBMN			n-size=µ-size
				BN C+/C-			n-size>µ-size
				MONO C+/C-			n-size>µ-size
				8-OH-dG			?
Polyvinylpyrolidone-stabilized cobalt ferrite NPs (RITC labelled with silica shell) 50nm			Salmonella typhimurium	Ames assay Salmonella mutagenicity assay		0,25–1 mg /plate–48 h	+ (0,5 mg/ plate, but not reproducible)	Kim et al. (2006) [8]
			CHL	CA		0,25–1 mg/ml-6 h FCS?	–
Iron
DMSA-coated Fe2O3 20 nm			Primary human dermal fibroblasts	Alkaline Comet assay	WST-1 assay	10^−6–10^−1 mg/ml (0,001–100 µg/ml) +FCS		(Auffan et al. 2006) [9]
						2 h	–
						24 h	–
Tetramethylammonium hydroxide (TMAOH) capped FePt 9 nm			Salmonella typhimurium/E. coli	Ames test	Maximum recommended dose level OECD 471 guideline	78,1–5000 µg/plate–48 h	– (except S. typhimurium TA 100–S9 (+))	(Maenosono et al. 2007) [10]
Silica
SiO2
7.21 nm			WIL2-NS	Alkaline Comet assay	MTT, population growth, apoptosis	60 and 120 µg/ml-6, 24, 48 h FCS?	–	(Wang et al. 2007b) [11]
				CBMN	CBPI	30–120 µg/ml FCS?	-
						6 h +26 h	+ (60 µg/ml)
						24 h +26 h	+ (30 µg/ml)
						48 h +26 h	+ (60 µg/ml)
				HPRT mutation assay		30–120 µg/ml 24 h FCS?	(120 µg/ml)
50 nm			A549	8-OH-dG	MTT, Trypan blue, apoptosis	0,1–500 µg/ml +FCS		(Jin et al. 2007) [12]
						48 h	–
						72 h	–
				Comet assay		48 h	–
						72 h	–
Titaniumdioxide
6.6 nm			WIL2-NS	Alkaline Comet assay (data not shown)	MTT, population growth, apoptosis	26–130 µg/ml–6, 24, 48 h +FCS	+ (65 µg/ml-24 h)	Wang et al. 2007a) [13]
				CBMN	CBPI	26–130 µg/ml +FCS
						6 h +26 h	+ (65 µg/ml)
						24 h +26 h	+ (65 µg/ml)
						48 h +26 h	+ (26 µg/ml)
				HPRT mutation assay (data not shown)		26–130 µg/ml–6, 24, 48 h +FCS	+ (65 µg/ml-24 h)
≤20 nm			SHE fibroblasts	MN +Kinetochore staining (data not shown)	Cytotoxicity increased >10 µg/cm^2 (data not shown)	0,5–10 µg/cm^2 FCS?		(Rahman et al. 2002) [14]
						12 h	–
						24 h	+ (1 µg/cm^2)
						48 h	+ (5 µg/cm^2)
						66 h	+ (1 µg/cm^2)
						72 h	+ (1 µg/cm^2)
>200 nm			SHE fibroblasts	MN	Cytotoxicity increased >10 µg/cm^2 (data not shown)	0,5–10 µg/cm^2 FCS? (12, 24, 48, 66, 72 h)	–
Anatase- TiO2
5 nm			GFSk-S1 cells	Comet assay	NRR assay	10 µg/ml–2 h +FCS	+	(Reeves et al. 2008) [15]
						10 µg/ml +UVA–2 h +FCS	+
						1–100 µg/ml–24 h +FCS	+ (100 µg/ml)
				Comet assay +fpg		10 µg/ml–2 h +FCS	+
						10 µg/ml +UVA–2 h +FCS	+
						1–100 µg/ml–24 h +FCS	+ (1 µg/ml)
				Comet assay +EndoIII		1–100 µg/ml–24 h +FCS	−
10 nm			BEAS-2B	Alkaline comet assay (w/o/with fpg)	MTT IC50 3 days=6,5 µg/ml	10 µg/ml–1 h +FCS	+/−	(Gurr et al. 2005) [16]
				CBMN		10 µg/ml–24 h +FCS	+
20 nm			BEAS-2B	Alkaline comet assay (w/o/with fpg)	MTT (50% after 3 days<10 µg/ml Apoptosis	10 µg/ml–1 h +FCS	+/−	(Gurr et al. 2005) [16]
60 nm (37% Al, 12–18% SiO2-coat)			CHO-WBL	Chromosome aberrations	Cell count	1715–5000 µg/ml (3 h+17 h recovery) with and w/o UV -FCS	−	(Theogaraj et al. 2007) [17]
200 nm			BEAS-2B	Alkaline comet assay (w/o/with fpg)	MTT (50% after 3 days<10 µg/ml	10 µg/ml–1 h +FCS	−/−	(Gurr et al. 2005) [16]
				CBMN		10 µg/ml–24 h +FCS	+
>200 nm			BEAS-2B	Alkaline comet assay (w/o/with fpg)	MTT (50% after 3 days<10 µg/ml	10 µg/ml–1 h +FCS	−/−	(Gurr et al. 2005) [16]
				CBMN	Apoptosis	10 µg/ml–24 h	−
Rutile TiO2
14–22 nm (Al-coat and with or w/o organic coat)			CHO-WBL	Chromosome aberrations	Cell count	348,1–5000 µg/ml (3 h+17 h recovery) with and w/o UV -FCS	–	(Theogaraj et al. 2007) [17]
200 nm			BEAS-2B	Alkaline comet assay (w/o/with fpg)	MTT (50% after 3 days<10 µg/ml	10 µg/ml–1 h +FCS	+/−	(Gurr et al. 2005) [16]
				CBMN		10 µg/ml–24 h +FCS
Rutile and anatase TiO2
21 nm (20%/80% coated and non-coated)			CHO-WBL	Chromosome aberrations	Cell count	209,7–800 µg/ml (3 h+17 h recovery) with and w/o UV -FCS	–	(Theogaraj et al. 2007) [17]
200nm (50%/50%)			BEAS-2B	Alkaline comet assay with fpg	MTT (50% after 3 days<10 µg/ml	10 µg/ml–1 h +FCS	+ (synergy)	(Gurr et al. 2005) [16]
140 nm (79%/21%)			Salmonella typhimurium/ E. coli	Ames test (data not shown)	Test not mentioned	100–5000 µg/plate	−	(Warheit et al. 2007) [18]
			CHO	Chromosome aberrations (data not shown)		25–2500 µg/ml–4, 20 h-FCS	−
In vivo studies
Carbon
MWCNT (11.3 nm/ 0.7µm)	Intratracheal instillation	Female Wistar rats	AT-II cells	CBMN	Data on inflammation	0.5 and 2 mg/rat-72 h	+ (2 mg/rat)	Muller et al. (2008) [4]
Iron
Magnetoliposomes 14 nm?	Endovenous injection	Female Swiss mice	Anucleated polychromatophylic erythrocytes Female Swiss mice	MN	/?	100 µl of 1,8×10^15 particles/ml (12, 24, 48 h)	(+) 24 h	Garcia et al. (2002)[19]
Polaspartic acid coated Fe
8.5 nm	Endovenous injection	Female Swiss mice	Anucleated polychromatophylic erythrocytes	MN		50 µl of 0,6×10^16 particles/ml		Sadeghiani et al. (2005)[20]
						1 day	+
						7 days	−
						15 days	−
						30 days	−
						50 µl of 1,6×10^16 particles/ml	+
						1 day	+
						7 days	+
						15 days	−
						30 days	−
Titaniumdioxide
20 nm (hydrophilic and hydrophobic surface)	Intratracheal instillation	Female Wistar rats	Lung parenchyma cells	8-oxo-gua		0,15–1,2 mg/ml 90 days	(+)	Rehn et al. (2003) [21]

Information on the cell type used, the applied genotoxicity test, the method with which the top dose was determined, the concentration range, exposure time, presence or absence of foetal calf serum and the results are given. Results are represented as+when statistically significant effects were observed, as (+) when no statistically significance was determined but a positive trend was visible and as–when no genotoxic effect was detected. Concentrations between brackets in the results column correspond to the lowest statistically significant concentration. A549, human lung carcinoma cell line; Aprt+/− 3C4 ES cells, a mouse embryonic stem cell line; BaP, benzo(a)pyrene; BEAS-2B, human bronchial epithelial cells; BN, binucleate cells; C-, centromere–negative micronuclei; C+, centromere–positive micronuclei after FISH with pancentromeric probes; CBMN, cytokinesis-block micronucleus assay; CBPI, cytokinesis-block proliferation index; CHL, Chinese hamster lung fibroblast cells; CHO, Chinese hamster ovarian cells; CHO-WBL, Chinese hamster ovarian cell subclone; FCS, foetal calf serum; GFSk-S1, primary cell line developed from goldfish skin; HEK293, human embryonic kidney cell line; Hela, human cervical cancer cell line; LDH, lactate dehydrogenase; MCF-7, human breast cancer cell line; MN, micronucleus assay; MONO, mononucleate cells; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MWCNT, multi-walled carbon nanotubes; NRR assay, neutral red lysosomal retention assay; SHE, Syrian hamster embryo; RLE, rat lung epithelial cells; WIL2-NS, a human lymphoblastoid cell line; WST-1 assay, cytotoxicity assay based on the metabolic activity of cells.

Table III.  Evaluation of the available literature on in vitro and in vivo genotoxicity of ENMs based on the proposed minimal criteria for nanotoxicology and genotoxicology.

			Minimal criteria for nanotoxicology							Minimal criteria for genotoxicology
NP type	Test	Reference	Method of synthesis and /or preparation	Particle size and distribution	Specific surface area	Aggregation status in relevant media	Composition/relevant coatings	Crystal structure/ crystallinity	Purity of the sample	Adequate top concentration	Cell toxicity	Positive control	Negative (solvent) control	replicate cultures	Repeat experiment	Adequate cells	Dose-effect
In vitro studies
Carbon
Carbon black
14 nm	Alkaline Comet assay		P	M						✓	✓	✓	✓	?	?	✓
	Neutral Comet assay	Mroz et al. (2008)	P	M						✓	✓	✓	✓	?	?	✓
14 nm	Alkaline Comet assay	Jacobsen et al. (2007)	✓	M	✓			✓	✓	✓	✓	✓	✓	✓	✓	✓
	Alkaline Comet assay +fpg		✓	M	✓				✓	✓	✓	✓	✓	✓	✓	✓
	Gene mutation frequency		✓	M	✓				✓	✓	✓	✓	✓	✓	✓	✓
14 nm BaP	Alkaline Comet assay	Mroz et al. (2008)	P	M			✓	✓		✓	✓	✓	✓	?	?	✓
	Neutral Comet assay		P	M			✓			✓	✓	✓	✓	?	?	✓
260 nm	Alkaline Comet assay		P	M						✓	✓	✓	✓	?	?	✓
	Neutral Comet assay		P	M						✓	✓	✓	✓	?	?	✓
Hydroxyl fullerene (C60(OH)24)7.1 nm
	MN	Niwa and Iwai, (2006)	?	✓						✓	✓		✓	?	?	✓	✓
	MN		?	✓						✓	✓		✓	?	?	✓	✓
	MN		?	✓						✓	✓		✓	?	?	✓	✓
MWCNT
(11.3 nm/0.7µm)	CBMN	Muller et al. (2008)	✓	✓	✓	✓	✓	na	✓	✓	✓	✓	✓	✓	✓	✓	✓
	CBMN		✓	✓	✓	(DLS)	✓	na	✓	✓	✓		✓	✓		✓	✓
MWCNT	Aprt+/- 3C4 ES cells	Zhu et al. (2007)	✓			✓	✓	na	✓	✓	✓		✓	✓		✓
Cerium oxide
9 nm	Ames test	Park et al. (2007)		✓ (TEM,BET)	✓ (BET)		✓ (XPS,EDX)	✓ (XRD)	✓ (EDX)	✓	✓	✓	✓	✓	?	✓	✓
320 nm				✓	✓		✓	✓	✓	✓	✓	✓	✓	✓	?	✓	✓
Cobalt
CoCr
29.5 nm	Alkaline Comet assay	Papageorgiou et al. (2007)	✓	✓						✓	✓		✓	✓		✓	✓
	CBMN		✓	✓						✓	✓		✓	✓		✓	✓
	8-OH-dG		✓	✓						✓	✓		✓	?		✓	✓
2.904 µm	Alkaline Comet assay		✓	✓			✓		✓	✓	✓		✓	✓		✓	✓
	CBMN		✓	✓			✓		✓	✓	✓		✓	✓		✓	✓
	8-OH-dG		✓	✓			✓		✓	✓	✓		✓	?		✓	✓
Polyvinylpyrolidone-stabilized cobalt ferrite NPs (RITC labelled with silica shell) 50nm	Ames assay Salmonella mutagenicity assay	Kim et al. (2006)	✓	✓ Size (TEM)			✓					✓	✓	✓	✓	✓	✓
	CA		✓	✓ (TEM)			✓					✓	✓	?	?	✓	✓
Iron
DMSA-coated Fe2O3 20 nm	Alkaline Comet assay	Auffan et al. (2006)	✓	✓	✓	✓	✓	✓	✓		✓	✓	✓	✓		✓	✓
Tetramethylammonium hydroxide (TMAOH) capped FePt 9 nm	Ames test	Maenosono et al. (2007)	✓	✓Before capping			✓	✓Before capping	✓	✓		✓	✓	✓		✓	✓
Silica
SiO2
7.21 nm	Alkaline Comet assay	Wang et al. (2007b)	P	✓					M	✓	✓	✓	✓		✓	✓	✓
	CBMN		P	✓					M	✓	✓	✓	✓		✓	✓	✓
	HPRT mutation assay		P	✓					M	✓	✓	✓	✓		✓	✓	✓
50 nm	Alkaline comet assay	Jin et al. (2007)	✓	✓						✓	✓		✓	?	✓	✓	✓
	8-OH-dG		✓	✓						✓	✓	✓	✓	?	?	✓	✓
Titaniumdioxide
6.6 nm	Alkaline comet assay	Wang et al. (2007a)	P	✓					M	✓	✓		✓		✓	✓	✓
	CBMN		P	✓					M	✓	✓		✓		✓	✓	✓
	HPRT mutation assay		P
≤ 20 nm	MN	Rahman et al. (2002)	P						M	✓	✓		✓		✓	✓	✓
> 200 nm	MN		P							?	✓		✓	✓	✓	✓	✓
Anatase- TiO2
5 nm	Comet assay	Reeves et al. (2008)	M/✓	M						✓	✓	✓	✓	✓	✓	✓	✓
	Comet assay +fpg		M/✓	M						✓	✓	✓	✓	✓	✓	✓	✓
	Comet assay +EndoIII		M/✓	M						✓	✓	✓	✓	✓	✓	✓	✓
10 nm	Alkaline comet assay (w/o/with fpg)	Gurr et al. (2005)	P	M				✓		✓	✓		✓			✓	✓
	CBMN		P	M				✓		✓	✓		✓			✓	✓
20 nm	Alkaline comet assay (w/o/with fpg)		P	M				✓		✓	✓		✓			✓	✓
60 nm (37% Al, 12–18% SiO2-coat)	Chromosome aberrations	Theogaraj et al. (2007)	P	✓			✓	✓	✓	✓	✓	✓	✓	✓		✓	✓
200 nm	Alkaline comet assay (w/o/with fpg)	Gurr et al. (2005)	P	M				✓		✓	✓		✓			✓	✓
	CBMN		P	M				✓		✓	✓		✓			✓	✓
> 200 nm	Alkaline comet assay (w/o/with fpg)		P	M				✓		✓	✓		✓			✓	✓
	CBMN		P	M				✓		✓	✓		✓			✓	✓
Rutile TiO2
14–22 nm (Al-coat and with or w/o organic coat)	Chromosome aberrations	Theogaraj et al. (2007)	P	✓			✓	✓	✓	✓	✓	✓	✓	✓		✓	✓
200 nm	Alkaline comet assay (w/o/with fpg)	Gurr et al. (2005)	P	M				✓		✓	✓		✓			✓	✓
	CBMN		P	M				✓		✓	✓		✓			✓	✓
Rutile and anatase TiO2
21nm (20%/80% coated and non-coated)	Chromosome aberrations	Theogaraj et al. (2007)	P	✓			✓	✓	✓	✓	✓	✓	✓	✓		✓	✓
200 nm (50%/50%)	Alkaline comet assay with fpg	Gurr et al. (2005)	P	M				✓		✓	✓		✓			✓	✓
140 nm (79%/21%	Ames test	Warheit et al. (2007)	M/✓	✓	✓	✓	✓	✓	✓	✓	Data not shown	✓	✓			✓	✓
	Chromosome aberrations									✓		✓	✓			✓	✓
In vivo rodent studies
Carbon
MWCNT (11.3 nm/0.7 µm)	CBMN	Muller et al. (2008)	✓	✓	✓	✓	✓	na	✓	✓	✓	✓	✓	✓		✓	✓
Iron
Magnetoliposomes 14 nm	MN	Garcia et al. (2002)	✓	✓ size			✓			?	✓		Not treated	na		✓
Polaspartic acid coated Fe
8.5 nm	MN	Sadeghiani et al. (2005)	✓	✓ size			✓			?	✓		Not treated	na		✓	✓
Titaniumdioxide
20 nm (hydrophilic and hydrophobic surface)	8-oxo-gua	Rehn et al. (2003)	M/✓	✓ size		✓					✓	✓	✓			✓	✓

Publications are categorized based on the chemical nature and the size of ENMs. ✓, criterion is satisfied;?, unclear; M, manufacturer information included; na, not applicable; P, preparation details included in the publication.