Document20

REVIEW

Mechanisms and measurements of nanomaterial-inducedoxidative damage to DNA

Elijah J. Petersen & Bryant C. Nelson

Received: 5 March 2010 /Revised: 24 May 2010 /Accepted: 26 May 2010 /Published online: 22 June 2010# US Government 2010

Abstract Many of the current investigations on theenvironmental and human health risks of engineered nano-materials focus on their short-term acute toxicity. However,the long-term chronic effects of nanomaterials on livingsystems, and in particular, on the genetic components ofliving systems, also warrant attention. An increasingnumber of nanomaterial safety studies include an assess-ment of genotoxicity as part of the overall risk evaluation.The potential of nanomaterials to directly or indirectlypromote the formation of reactive oxygen species is one ofthe primary steps in their genotoxic repertoire. Thesubsequent modification of genomic DNA by reactiveoxygen species could lead to the development of mutagen-esis, carcinogenesis, or other age-related diseases if theDNA damage is not repaired. This review focuses on theinteractions of nanomaterials with DNA and specifically onthe capacity of some nanomaterials to induce oxidativedamage to DNA. A critical assessment of the analyticalmethodology and the potential biochemical mechanismsinvolved in nanomaterial induction of oxidative damage toDNA is presented, results obtained for the various studieswith each nanomaterial are compared, and recommenda-tions for future research are discussed. Researchers shouldconsider, among other experimental recommendations, (1)the application of more chromatography-based and mass-spectrometry-based analytical techniques to the assessment

of oxidative damage to DNA to facilitate an enhancedunderstanding of DNA damage mechanisms and (2) theverification of cellular viability before conducting genotox-icity assays to reduce the impact of fragmented DNA,formed as a consequence of cell death, on DNA damagemeasurements.

Keywords Base lesions . Comet assay . DNA damage .

Engineered nanomaterials . Genotoxicity . Toxicity

Introduction

Engineered or manufactured nanomaterials (ENs) areparticles, fibers, tubes, spheres, rods, etc., of variedcompositions that contain at least one dimension thatmeasures 100 nm or less [1]. Partly because of theirreduced sizes and larger surface area to volume ratios incomparison with microscale or macroscale materials ofidentical composition, ENs are typically highly surfacereactive (i.e., catalytic) and show enhanced physical (i.e.,higher tensile strength), chemical (i.e., persistent redoxcycling) and electronic (i.e., semiconducting) properties [2,3]. Thus, there is strong scientific and commercial interestin the development and use of ENs in fields such asengineering, agriculture, electronics, and medicine.Research- and industrial-scale developments of ENs arealready proceeding at a rapid pace, with many consumerproducts such as athletic equipment (e.g., tennis racquets),cosmetics (e.g., suntan lotions), and laundry products (e.g.,fabric softeners) already containing significant amounts ofENs (for the full list of consumer products containing ENs,it is suggested that the reader visit the following Web site:http://www.nanotechproject.org/inventories/consumer/).The critical problem is that the understanding of the

Electronic supplementary material The online version of this article(doi:10.1007/s00216-010-3881-7) contains supplementary material,which is available to authorized users.

E. J. Petersen :B. C. Nelson (*)Chemical Science and Technology Laboratory, BiochemicalScience Division, National Institute of Standards and Technology,100 Bureau Drive, Stop 8311,Gaithersburg, MD 20899-0001, USAe-mail: [email protected]

Anal Bioanal Chem (2010) 398:613–650DOI 10.1007/s00216-010-3881-7

http://www.nanotechproject.org/inventories/consumer/

http://dx.doi.org/10.1007/s00216-010-3881-7

environmental health and human safety risks of ENs haslagged behind their incorporation in commercial goods. Inour opinion, comprehensive in vitro (acellular and cellular)and in vivo toxicity studies using appropriate models (cells,organisms, animals, plants) are needed to evaluate the acuteand chronic biological effects of ENs that could potentiallylead to toxicity. Most of the current studies utilize in vitromodels and focus primarily on toxicity end points dealingwith cellular viability, i.e., apoptosis, necrosis, etc. Thesetypes of studies are definitely needed, but there is a growingconsensus that complementary studies are needed that focuson the chronic biological effects of ENs such as theirpotential genotoxicity [4–6].

Understanding the long-term interactions of ENs withDNA and the mechanisms of those interactions is importantfor evaluating and predicting mutation- and cancer-relatedrisks of ENs. Traditional genotoxic end points such as genemutations (assessed via the Ames Salmonella test or thehypoxanthine phosphoribosyltransferase forward mutationassay), chromosomal damage (assessed via the micro-nuclease test), and oxidative damage to DNA [assessedvia the single cell gel electrophoresis (comet) assay] havebeen utilized with increasing frequency for the evaluationof EN-induced genotoxicity.

EN-induced oxidative stress is perhaps the most broadlydeveloped and accepted mechanism for the potential toxicactivity of ENs [1]. There are three main hypotheticalscenarios whereby ENs might induce oxidative stress andthe subsequent generation of excess intra- and extracellularreactive oxygen species (ROS) and, to a lesser extent,reactive nitrogen species that have the capacity to over-whelm a biological system’s (cell, organism, plant, animal,etc.) natural antioxidant defense mechanisms [7]. First, theENs might be inherently redox-active or have surfacefeatures/properties that catalyze redox activity, leading tooxidative stress and to generation of excess ROS. Second,the ENs might be biopersistent, meaning that once ENsenter a biological system, they do not degrade or breakdown over time but instead remain in the system, inducingsite-specific and possibly systemic inflammation. Inflam-mation initiates the recruitment of inflammatory leukocytes(monocytes, neutrophils, etc.), which then become activatedand generate excess ROS [8]. Third, the ENs might enterthe cell and physically interact with and structurally damagesubcellular organelles such as mitochondria. In such cases,the damaged mitochondria could lead to disruption of theelectron transport chain and the production of adenosinetriphosphate (ATP), which could again lead to the genera-tion of excess ROS. Many ENs have already demonstratedthe ability to directly or indirectly induce the formation ofROS in in vitro and in vivo studies [1]. The generated ROS,especially the extremely reactive hydroxyl radical (•OH),have the capability to attack DNA via several different

mechanisms to generate single-strand breaks (SSBs),double-strand breaks (DSBs), oxidatively induced basedamage, and other DNA lesions [9]. The accumulation ofSSBs and oxidatively induced base lesions can lead toDSBs, considered the most lethal type of oxidative damageto DNA [10]. Compared with other types of DNA damage,DSBs are intrinsically more difficult to repair and as little asone DSB lesion in a cell can kill the cell if the lesioninactivates a critical gene [10]. SSB and oxidativelyinduced DNA base lesions are definitely not harmless,and are known to block DNA transcription and replicationprocesses, resulting in accelerated cytotoxicity and genomicinstability [9, 11].

For the specific case of EN-induced oxidative damage toDNA, the comet assay [12–15] is the most widely utilizedmethod for evaluating the extent of genomic DNA damage(see Table 1). The principle of the assay is simple—individual cells are lysed and the DNA is subjected toagarose gel electrophoresis under either alkaline or neutralconditions, depending on whether one wants to detectpredominantly SSBs or predominantly DSBs, respectively.The DNA is then stained using a fluorescent, intercalatingdye such as ethidium bromide and the image (a comet) thatis formed comprises intact DNA and a tail consisting ofdamaged DNA strands (see Fig. 1). The comet images arecompared against controls to determine the number of DNAstrand breaks; larger comets are correlated with thepresence of more extensive strand breaks. The alkalinecomet assay can also be modified to specifically detect theoccurrence of oxidized purine base lesions and/or oxidizedpyrimidine base lesions through the incorporation of eitherpurine-specific [Escherichia coli formamidopyrimidine gly-cosylase (Fpg)] or pyrimidine-specific [(E. coli endonucle-ase III (Nth)] base excision repair (BER) enzymes in theassay protocol. These enzymes will remove the base lesionand then generate a DNA strand break at the abasic site thatcan be detected via the comet assay. The use of BERenzymes in the comet assay allows one to estimate therelative level of oxidatively induced DNA base damage, butdoes not allow identification of the specific modified DNAbase nor absolute quantification of the damage. Addition-ally, the alkaline comet assay can be used to detect alkali-labile sites (ALS), which are sites of oxidative damage thatcan be converted to strand breaks through the use ofalkaline denaturing conditions in the assay protocol. ALSinclude both apurinic and apyrimdinic sites that are formedowing to BER processes. A problem that makes comparingcomet assay results between or among laboratories difficultis that the different types of DNA damage are oftenreported in arbitrary comet score units (i.e., percentageDNA in tail, tail moment, etc.) that are not traceable to anystandard. Recently, however, it was shown that the cometassay could be calibrated through the use of ionizing

614 E.J. Petersen, B.C. Nelson

radiation [16, 17]. The calibration procedure allows thecomet score to be converted to equivalent radiation (Gy)units and for the subsequent calculation of the number oflesions per cell or the number of lesions per 106 base pairs.This development not only facilitates comparison of cometassay results between laboratories, but also has the potentialto reduce assay result variability [16].

Regarding the present review, several pertinent pointsneed to be recognized. First, it is well established that thealkaline and neutral versions of the comet assay do notsolely measure SSBs and DSBs, respectively [12–15]. Thealkaline comet assay measures both SSBs and DSBs, butpredominantly SSBs. The neutral comet assay can bespecially designed to measure solely DSBs [12], butalthough it typically measures both SSBs and DSBs, DSBsare predominately measured. Some studies that utilized thealkaline comet assay specifically reported the detection ofoxidative damage to DNA damage as SSBs, whereas otherstudies only reported the DNA damage as strand breaks.For the purpose of this review and to avoid confusionduring the assay assessments, when studies have utilizedthe alkaline comet assay, we considered the detected strandbreaks to be predominantly due to SSBs with therecognition that the strand breaks are likely to be a mixtureof SSBs and DSBs. We took a similar approach with thesingle study that utilized the neutral comet assay byreporting the strand breaks as DSBs [18]. Second, a numberof studies reported the detection of strand breaks using theFpg-modified comet assay. Many of these studies reportedand plotted the resulting oxidative damage to DNA with nomeasurement correction, when, in fact, the true DNAdamage is obtained by subtracting the strand break levelsmeasured with the Fpg-modified assay from the strandbreak levels measured with the unmodified alkaline cometassay [16]. This adjustment was made in some of thestudies on the effects of ENs [19–23].

Other assays that have been reported for the detection ofEN-induced oxidative damage to DNA in the form of DNAstrand breaks include (1) γ-H2AX assay, (2) plasmidnicking/agarose gel electrophoresis assay, and (3) alkalineprecipitation assay. The γ-H2AX assay is strictly used fordetection of DSBs and is based on the consensusphosphorylation of Ser-139 of the H2AX protein (amember of the H2 histone family) that occurs rapidly dueto the presence of DSBs [24, 25]. The phosphorylatedH2AX protein (γ-H2AX) is detected using antibodiesagainst γ-H2AX with either immunostaining or flowcytometry (see Fig. 2). A modified version of the plasmidnicking/agarose gel electrophoresis assay [26] is alsoutilized to detect SSBs and DSBs induced by the nickingaction of ENs on the DNA backbone. This assay is basedon the conversion of supercoiled (S) plasmid DNA to eithera relaxed (R) or a linear (L) form. The forms are separated

using a gel-electrophoretic platform containing an interca-lating fluorescent dye; the forms are separated on the basisof the differential electrophoretic mobilities among the S(migrates the fastest), L (migrates between S and R), and R(migrates the slowest) forms. Detection of the R form isindicative of SSBs and detection of the L form is indicativeof DSBs. The alkaline precipitation assay [27, 28] is a rapidprocedure that gives a measure of total SSBs and DSBscombined. In this procedure, cells are lysed with sodiumdodecyl sulfate and the DNA and proteins are precipitatedwith potassium chloride. The DNA that is damaged remainsin solution and therefore by taking the ratio of the amountof precipitated DNA to the amount of DNA in solution, onecan roughly estimate the fraction of strand breaks.

Assays currently utilized for evaluating EN-inducedoxidative damage to DNA in the form of DNA base lesions(excluding the previously described Fpg-modified cometassay) include (1) direct antibody assays for 8-hydroxy-2′-deoxyguanosine (8-OH-dG) and (2) liquid chromatography(LC) coupled with ultraviolet (UV) detection or LC withboth UV and electrochemical (EC) detection for thedetermination of 8-OH-dG. The modified nucleoside, 8-OH-dG, is one of the most widely recognized and utilizedbiomarkers of oxidative stress and oxidatively inducedDNA damage [29]. Many different types of biologicalcompounds, chemical agents, and ionizing radiation havebeen shown to produce ROS (usually •OH) that preferen-tially attack guanine residues (the most easily oxidizablebase) in DNA to induce the formation of 8-OH-dG as wellas other modified bases [9]. There are several studiesdescribing the use of monoclonal antibodies against 8-OH-dG, along with secondary labeled antibodies, for theevaluation of 8-OH-dG levels induced by ENs. Under thisformat, most studies utilize enzyme-linked immunosorbentassay (ELISA) [30] or immunohistochemical assay plat-forms [31] for 8-OH-dG detection and quantification. Themost specific methods for the measurement of 8-OH-dGinvolve the use of either LC/UV or LC/UV/EC methods[32] for the selective separation and specific detection of 8-OH-dG in extracted DNA samples. In these procedures,DNA extracted from either in vitro or in vivo samples isenzymatically hydrolyzed to 2′-deoxynucleosides; 8-OH-dG, in the midst of other modified and nonmodified 2′-deoxynucleosides, is detected on the basis of either its UVabsorbance or its EC oxidation properties.

In our laboratory, we employ gas chromatography/massspectrometry (GC/MS) and liquid chromatography/tandemmass spectrometry with stable isotope-dilution proceduresfor detecting and quantifying oxidatively induced DNAbase damage mediated through the interactions of ENs withcells and organisms. The use of isotope-dilution massspectrometry allows us to simultaneously identify andquantify multiple (more than 20) [29] oxidatively induced

Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA 615

Tab

le1

Sum

maryof

nano

material-indu

cedox

idativedamageto

DNA

stud

ies

Reference

Nanop

article

tested

(size)

Cell/o

rganism

tested

DNA

damage

assay

Concentratio

nsExposure

duratio

n(h)

Finding

s

Carbonnanoparticles

CarbonBlack

Gallagh

eret

al.[50]

Degussa

Printex

90(nom

inal

size

16nm

bymanufacturer)

andSterlingV

(particle

size

70nm

bymanufacturer)

Fischer

344rats(lun

gstested)by

inhalatio

n8-OH-dG

using

LC/UV/EC

1.2,

7.1,

and52

.8mg/m

3

(Printex

90)or

48.2

mg/m

3(SterlingV)

13weeks

(som

eorganism

salso

hadadditio

nal

44weeks

recovery)

Only52

.8mg/m

3of

Printex

90caused

sign

ificantly

elevated

levelsafter13

weeks

ofexposure

After

44weeks

ofrecovery,

only

7.1and52

.8mg/m

3

caused

sign

ificantly

elevated

8-OH-dG

levels

Gerloffet

al.[44]

Degussa

Printex

90(nom

inal

size

14nm

bymanufacturer)

Hum

ancarcinom

aintestinal

cells

(Caco-2cells)

Alkalinecomet

assay+/-FPG

133.3µg/mL

4Nosign

ificanteffect

with

orwith

outFPG

Jacobsen

etal.[21]

Degussa

Printex

90(nom

inal

size

14nm

bymanufacturer)

Fe1

MutaMouse

lung

epith

elialcelllin

eAlkalinecomet

assay+/-FPG

75µg/mL

3Significant

increasesobserved

with

andwith

outFPG

Jacobsen

etal.[48]

Degussa

Printex

90(nom

inal

size

14nm

bymanufacturer)

Apo

E-/-mice

(broncho

alveolar

lavage

fluidcells

tested)by

intratrachaelinstillation

Alkalinecomet

assay

54µg

3Significant

increase

observed

Karlssonet

al.[22]

Sigma-Aldrich

carbon

powder

(<30

nmby

manufacturer)

Hum

anlung

epith

elial

celllin

e(A

549)

Alkalinecomet

assay+/-FPG

2,40

,and80

µg/mL

(40and80

µg/mL

forFPG)

4Nosign

ificantincrease

inDNA

oroxidativelymodified

lesionsat

anyconcentration

Mrozet

al.[45]

Degussa

Printex

90(nom

inal

size

14nm

bymanufacturer)

tested

with

andwith

out

benzo[a]py

rene

andHub

er99

0coarse

carbon

black

(260

nmby

manufacturer)

Hum

anlung

epith

elial

celllin

e(A

549)

Alkalinecomet

assay

100µg/mL

3Significant

increase

for

nanoparticulatecarbon

black

with

andwith

out

benzo[a]py

rene

butno

tcoarse

carbon

black

Mrozet

al.[18]

Degussa

Printex

90(nom

inal

size

14nm

bymanufacturer)

tested

with

andwith

out

benzo[a]py

rene

andHub

er99

0coarse

carbon

black

(260

nmby

manufacturer)

Hum

anlung

epith

elial

celllin

e(A

549)

Alkalineandneutral

comet

assay

100µg/mL

3Significant

increase

for

nanoparticulatecarbon

black

with

andwith

out

benzo[a]py

rene

butno

tcoarse

carbon

blackfor

alkalin

ecomet

assay

Nosign

ificantincrease

for

coarse

ornanoparticulate

carbon

blackwith

neutral

comet

assay

Totsuk

aet

al.[49]

Degussa

Printex

90(nom

inal

size

14nm

bymanufacturer)

MaleC57

BL/6

Jmice

(lun

gstested)by


Alkalinecomet

assay

0.05

and0.2mg

peranim

al3,

24For

3h,

therewas

asign

ificant

increase

for0.2mgbu

tno

t0.05

mg

Nochange

for24

hcompared

with

3h


Tab

le1

(con

tinued)

Reference

Nanop

article

tested

(size)

Cell/o

rganism

tested

DNA

damage

assay

Concentratio

nsExposure

duratio

n(h)

Finding

s

Yanget

al.[46]

Nano-Inno

vatio

n(12.3±

4.1nm

,>99

.4%

carbon

purity)

Primarymou

seem

bryo

fibrob

lasts

Alkalinecomet

assay

5and10

µg/mL

24Significant

effectsat

both

concentrations

Zho

nget

al.[47]

Cabat

carbon

black(37-nm

prim

aryparticle

size

bymanufacturer)

Chinese

hamster

lung

fibrob

lasts(V

79cells)

andhu

man

embryonic

lung

fibrob

lasts

(Hel

299cells)

Alkalinecomet

assay

98,19

6,39

2,and

786µg/mL

3Noeffectsob

served

with

either

cellat

any

concentration

Carbonnanofibers

Lindb

erget

al.[52]

Sigma-Aldrich

graphitic

nanofibers

(80–20

0-nm

outerdiam

eter;30–50-nm

innerdiam

eter;5-20-µm

length;4%

metal

catalyst

bymanufacturer)

Hum

anbronchial

epith

elialcelllin

e(BEAS2B

)

Alkalinecomet

assay

3.8,

19,38

,76

,114,

228,

304,

and38

0µg/mL

24,48,72

Significant

effectsobserved

atallconcentrations

for24

hbu

ton

lyat

someconcentrations

after48

and72

h

Dose-depend

enteffectsob

served

only

afterexposure

for48

h

Moriera

etal.[85]

Cellulose

nano

fibers

(50–1,50

0-nm

length

and3–5-nm

width)

Chinese

hamster

ovarycells

Alkalinecomet

assay

100,

500,

or1,000µg/mL

48Noeffectsob

served

under

theseconcentrations

Carbonnanotubes

Folkm

annet

al.[61]

EliC

arbSWNTs

(0.9–1.7-nm

diam

eter;length

<1μm

bymanufacturer)

Fem

aleFisher344rats

(colon

mucosacells,

liver,andlung

tissues

tested)by

oral

gavage

8-OH-dG

using

LC/UV/EC

0.06

4or

0.64

mg/kg

(dispersed

incorn

oilor

salin

e)

24Significant

increasesat

both

dosesin

liver

andlung

sbu

tno

tin

thecolonmucosacells

Jacobsen

etal.[20]

EliC

arbSWNTs

(0.9–1.7

nmdiam

eter;length

<1μm

bymanufacturer)

FE1-MutaT

MMou

selung

epith

elialcelllin

eAlkalinecomet

assay+/-FPG

100µg/mL

3Nosign

ificantincrease

inthe

numberof

strand

breaks

buta

sign

ificantincrease

inthenu

mber

ofFPG

sites

Jacobsen

etal.[48]

EliC

arbSWNTs

(0.9–1.7-nm

diam

eter;length

<1μm

bymanufacturer)

Apo

E-/-mice

(broncho

alveolar

lavage

fluidcells

tested)by


Alkalinecomet

assay

54µg

3Significant

increase

observed

Karlssonet

al.[22]

Sigma-Aldrich

MWNTs

(110–170

nm×5–9μm

bymanufacturer)

Hum

anlung

epith

elial

celllin

e(A

549)

Alkalinecomet

assay+/-FPG

2,40

,and80

µg/mL

(40and80

µg/mL

forFPG)

4Significant

increase

inDNA

damageat

2,40

,and80

µg/mL

Nosign

ificantincrease

inox

idativelymod

ified

lesionsat

40or

80µg/mL

Noeffect

seen

from

soluble

fractio

nfrom

nanotubes

Kisin

etal.[51]

HipCO

SWNTs

(99.7%

carbon

bymass)

Chinese

hamster

lung

fibrob

last(V

79)cells

Alkalinecomet

assay

120,

240,

and

480µg/mL

3,24

Significant

increasesonly

at48

0μg/mLafter3hand24

0and48

0μg/mLafter24

h

Lindb

erget

al.[52]

Sigma-Aldrich

carbon

nanotubes(50%

SWNT,

40%

othernano

tubes,

1.1nm

×0.5–10

0µm

bymanufacturer)

Hum

anbronchial

epith

elialcelllin

e(BEAS2B

)

Alkalinecomet

assay

3.8,

19,38

,76

,114,

228,

304,

and38

0µg/mL

24,48,72

Significant

effectsobserved

at3.8,

228,

304,

and38

0µg/mLafter

24handat

allconcentrations

after48

and72

h


Tab

le1

(con

tinued)

Reference

Nanop

article

tested

(size)

Cell/o

rganism

tested

DNA

damage

assay

Concentratio

nsExposure

duratio

n(h)

Finding

s

Dose-depend

enteffectsob

served

after24

h

Pacurariet

al.[54]

Carbo

lexSWNTs

(0.8–2

.0-nm

diam

eter,

2–5-µm

leng

thby

manufacturer)

Normal

andmalignant

human

mesothelial

cells

H2A

X,alkalin

ecomet

assay

190and380µg/mL

24Significant

increasesin

DNA

damageat

both

concentrations

forbo

thcelltypes

Nosign

ificantincrease

inH2A

Xwas

observed

ateither

concentration

Pacurariet

al.[53]

BussanNanotech

ResearchMWNTs

(81-nm

diam

eter

and

8.19-µm

leng

th)

Normal

andmalignant

human

mesothelialcells

H2A

X,alkalin

ecomet

assay

12.5,25

,and

50µg/mL(H

2AX),

190and38

0µg/mL

(com

etassay)

24Significant

increasesin

DNA

damageob

served

at19

0and

380µg/mLforbo

thcells

with

dose–respo

nsebehavior

observed

Significant

increasesin

H2A

Xph

osph

orylationat

12.5

and

25µg/mLbu

tno

t50

µg/mL

forbo

thcells

Yanget

al.[46]

COCCSWNTs

(8-nm

diam

eter;<5-μm

length;99

.99%

carbon

purity)

Primarymou

seem

bryo

fibrob

lasts

Alkalinecomet

assay

5and10

µg/mL

24Significant

effectsat

both

concentrations

Zeniet

al.[55]

HeJiSWNTs

(1.1-nm

outerdiam

eter,50

-µm

length

bymanufacturer)

Hum

anperipheral

bloo

dlymphocytes

Alkalinecomet

assay

1,5,

and10

µg/mL

6Noeffectsob

served

Zhu

etal.[56]

Tsinghua

andNanfeng

Chemical

Group

Cooperatio

nMWNTs

Mouse

embryo

stem

cells

H2A

X100µg/mL

24Double-strand

damageobserved

Fullerenes

Dhawan

etal.[75]

C60dispersedin

water

Hum

anlymphocytes

Alkalinecomet

assay

0.02

2,0.22

,2.2,

11,

22,55

,and110µg/L

3,6

Significant

effectsconsistently

observed

atconcentrations

of2.2μg/Landhigh

erafter3and6h

C60aftersolvent

exchange

with

ethano

l0.42

,4.2,

42,21

0,42

0,1,05

0,and

2,100µg/L

3,6

Significant

effectsobserved

atconcentrations

of42

μg/Land

high

erafter3and6h

Folkm

annet

al.[61]

C6099

.9%

pure

bymanufacturer

Fem

aleFisher344

rats(colon

mucosa

cells,liv

er,andlung

tissues

tested)by

oral

gavage

8-OH-dG

using

LC/UV/EC

0.06

4or

0.64

mg/kg

(dispersed

incorn

oilor

salin

e)

24Significant

increasesat

both

concentrations

intheliv

er,

at0.64

mg/kg

inlung

,bu

tno

changesin

thecolonmucosacells

Jacobsen

etal.[20]

Sigma-Aldrich

C60

(99.9%

pure

bymanufacturer)

FE1-MutaT

MMou

selung

epith

elialcelllin

eAlkalinecomet

assay+/-FPG

100µg/mL

3Nosign

ificantincrease

inthe

numberof

strand

breaks

buta

sign

ificantincrease

inthe

numberof

FPG

sites


Tab

le1

(con

tinued)

Reference

Nanop

article

tested

(size)

Cell/o

rganism

tested

DNA

damage

assay

Concentratio

nsExposure

duratio

n(h)

Finding

s

Jacobsen

etal.[48]

Sigma-Aldrich

C60

(99.9%

pure

bymanufacturer)

Apo

E-/-mice

(broncho

alveolar

lavage

fluidcells

tested)by

intratrachael

instillation

Alkalinecomet

assay

54µg

3Nosign

ificantincrease

Totsuk

aet

al.[49]

Sigma-Aldrich

C60

(99.9%

pure

bymanufacturer)

MaleC57

Bl/6

Jmice

(lun

gstested)by


Alkalinecomet

assay

0.05

and0.2mg

peranim

al3,

24For

3h,

therewas

asign

ificant

increase

for0.2mgbu

tno

t0.05

mg

Decreasefrom

3to

24h

suggestin

gthat

repair

enzymes

may

beworking

Metal

nano

particles

Cob

altnano

particles

Colog

nato

etal.[86]

Co(100–500

nm)

Hum

anperipheral

blood

leuk

ocytes

Alkalinecomet

assay

0.59

,2.9,

and

5.9µg/mL

2Noeffectsob

served

forCo2

+

butsign

ificanteffectsob

served

at2.9and5.9µg/mLforCo

nanoparticles

Pon

tiet

al.[87]

Co(20–50

0nm

with

peak

at80

nm)

Mouse

fibrob

lastcells

(Balb/3T3)

Alkalinecomet

assay

0.05

9,0.18

,and

0.29

µg/mL

2Statistically

significantincrease

atallconcentrations

forCo

nanoparticlesandions

Conano

particlesdidno

thave

ado

se-dependent

effect

butCo

ions

did

Zhang

etal.[108]

InabataCo(10–30

nm,

compo

sedof

Coand

Co 3O4)

Plasm

idDNA

Plasm

idDNA

nicking

56,56

0,and

1,100µg/mL

8Sub

stantialDNA

damagewhich

was

decreasedwith

hydrox

ylscavengermannitol

Cobalt/chrom

ium

nanoparticles

Bhabraet

al.[91]

CoC

r(nanoparticles

29.5±6.3nm

;microparticles

2.9±1.1μm)

Hum

anBJfibrob

lasts

andBeW

ob3

0cells

(BeW

ocells

wereused

asabarrierforBJ

fibrob

lasts)

Alkalinecomet

assay,

H2A

X0.08

and0.8mg/mL

(concentratio

nfor

BeW

ocells

but

less

below

barrier)

24Significantly

elevated

DNA

damageconcentrations

observed

forfibrob

lastsforions

and

nanoparticlesat

both

concentrations

andmicroparticles

at0.8mg/mLon

ly

There

was

also

anincrease

inthe

numberof

double-strandlesionsfor

fibrob

lastsas

measuredby

H2A

Xat

both

concentrations

for

nanoparticlesandmicroparticles

CoandCrions

caused

these

effectstooforthecomet

assay

buton

lyCrions

hadthiseffect

fortheH2A

Xassay

Papageorgiouet

al.[90]

CoC

r(nanoparticles

29.5±6.3nm

;microparticles

2.9±1.1μm)

Primaryhu

man

derm

alfibrob

lasts

Alkalinecomet

assay,

8-OH-dG

byim

munohistochem

istry

3.85

×10

-6mg/mL

to77

.0mg/mLby

orders

ofmagnitude

24,72

,12

0Nanop

articlescaused

sign

ificantly

moreDNA

damagethan

microparticlesat

higher

doses

after24

h,bu

tmicroparticles

caused

moredamageafter72

h


Tab

le1

(con

tinued)

Reference

Nanop

article

tested

(size)

Cell/o

rganism

tested

DNA

damage

assay

Concentratio

nsExposure

duratio

n(h)

Finding

s

Dose-depend

entDNA

damage

after24

hfornano

particles

andmicroparticles

Papageorgiouet

al.[90]

Dose-depend

enttrendwas

less

clearfor3and5days

Microparticlesindicatedincreased

8-OH-dG

levelsat

concentrations

of3.85

and38

.5mg/mLafter

both

3and24

h,andthe

concentrations

weregenerally

larger

than

thoseforthe

nanoparticles

Nanoparticlesonly

seem

edto

increase

8-OH-dG

levels

at3.85

and38

.5mg/mL

after24

h

Cop

per

nanoparticles

Midanderet

al.[94]

<10

0nm

and

Outok

umpu

Copper

(<20

μm

bymanufacturer)

Hum

anlung

epith

elial

celllin

e(A

549)

Alkalinecomet

assay

80µg/mL

4Significantly

increasedDNA

damagefrom

Cunano

particles

butnotCumicroparticles,but

someof

thisdamagemay

befrom

thehigh

cytotoxicity

oftheCunanoparticles

Noeffectsob

served

from

dissolved

copper

fractio

nfrom

the

particles

Goldnano

particles

Grigg

etal.[103]

8±2nm

Hum

anmon

ocytecell

line(M

onoMac

6)expo

sedat

air-tissue

interface

Alkalinecomet

assay

5.4µg/cm

224

Significant

increase

observed

Jacobsen

etal.[48]

2nm

Apo

E-/-mice

(broncho

alveolar

lavage

fluidcells

tested)

byintratrachaelinstillation

Alkalinecomet

assay

0.54

µg

3Nosign

ificantincrease

Kanget

al.[104]

4,15

,10

0,and

200nm

Mouse

lymphob

lasts

(L51

78Y)

Alkalinecomet

assay

25,50,and100µg/mL

2Significant

effectsfor100-

and

200-nm

nanoparticlesat

all

concentrations

butnotfor4-

and15-nm

nanoparticles

Liet

al.[105]

20nm

Fetal

lung

fibrob

lasts

(MRC-5)

8-OH-dG

using

LC/UV/EC

0.5and1nM

(25–50

,50–100

µg/mL)

72Increase

inlesion

sat

1nM

butno

t0.5nM


Tab

le1

(con

tinued)

Reference

Nanop

article

tested

(size)

Cell/o

rganism

tested

DNA

damage

assay

Concentratio

nsExposure

duratio

n(h)

Finding

s

Iron

nano

particles

Grigg

etal.[103]

2nm

Hum

anmon

ocytecelllin

e(M

onoMac

6)andrat

alveolar

macrophages

expo

sedat

air-tissue

interface

Alkalinecomet

assay

0.51

,2.04

,5.1,

and10

.2µg/cm

224

Dose-depend

entincrease

intoxicity

with

sign

ificantincrease

only

for5.1and10

.2µg/cm

2

forMonoMac

6cells

Significant

increase

forratalveolar

macroph

ages

at10

.2µg/cm

2

Nickelnanoparticles

Zhang

etal.[108]

InabataCo(10–30

nm)

plasmid

DNA

Plasm

idDNA

nicking

56,56

0,and

1,100µg/mL

8Sub

stantialDNA

damage

was

observed

Platin

umnano

particles

Pelka

etal.[109]

Ptparticles(<

20,<10

0,and>10

0nm

)Hum

ancoloncarcinom

acelllin

e(H

T29

)Alkalinecomet

assay+/-FPG

0.00

048,

0.00

48,

0.048,

0.48,4.8,

48,48

0,and

4,800ng

/mL

3,24

Significant

increasesonly

observed

after3hfor20

-nm

particlesat

0.86

and8.6ng

/mL

andfor<10

0-nm

particlesat

8.6ng

/mLwith

andwith

outFPG

After

24h,

sign

ificantincreases

only

observed

for20

-nm

particles

and>10

0-nm

particlesat

8.6ng

/mLwith

andwith

outFPG

Nosign

ificanteffectsob

served

for>10

0-nm

particles

Silv

ernanoparticles

Ahamed

etal.[113]

25nm

with

andwith

out

polysaccharide

coating

Mouse

embryo

nicstem

andmou

seem

bryo

nic

fibrob

lastcells

H2A

X50

µg/mL

24,48,72

Increase

inphosphorylationfor

both

cells

atalltim

epo

ints

Intensity

appeared

tobe

greater

forcoated

nano

particles

AshaR

aniet

al.[114]

Starch-capped

Ag

nanoparticles(6–20nm

)Hum

anglioblastoma

cells

(U251)

and

norm

alhu

man

fibrob

lasts(IMR-90)

Alkalinecomet

assay

25,50

,10

0,20

0,and40

0µg/mL

48Concentratio

n-dependenteffects

forU25

1cells

which

were

sign

ificantafter50

µg/mL

Concentratio

n-dependent

effectsforIM

R-90cells

upto

100µg/mLand

then

nofurtherincrease

Significant

effectsat

all

concentrations

fortheIM

R-90

cells

Grigg

etal.[103]

5.5±1.5nm

Hum

anmon

ocytecell

line(M

onoMac

6)expo

sedat

air-tissue

interface

Alkalinecomet

assay

3.4µg/cm

224

Significant

increase

observed

Kim

etal.[115]

Nanop

oly(<

10-nm

diam

eter

bymanufacturer)

Hum

anhepatomacells

(HepG2)

H2A

X1and2µg/mL

24Dosedependence

observed

andincreaseddamage


Tab

le1

(con

tinued)

Reference

Nanop

article

tested

(size)

Cell/o

rganism

tested

DNA

damage

assay

Concentratio

nsExposure

duratio

n(h)

Finding

s

10mM

antio

xidant

N-acetylcysteinestopped

damagefrom

nano

particles

anddecreaseddamageto

cells

treatedwith

AgN

O3

Saw

oszet

al.[116]

Nano-Tech

(2–6

nm)

Chicken

eggs

(embryo

s)8-OH-dG

using

LC/UV/EC

15µg/egg

20days

Noincrease

inliv

erconcentrationof

8-OH-dG

Silv

er–cop

per

nanoparticles

Saw

oszet

al.[116]

Nano-Tech

(2–6

nm)

Chicken

eggs

(embryo

s)8-OH-dG

using

LC/UV/EC

15µg/egg

20days

Noincrease

inliv

erconcentrationof

8-OH-dG

Silv

er–palladium

nanoparticles

Saw

oszet

al.[116]

Nano-Tech

(2–6

nm)

Chicken

eggs

(embryo

s)8-OH-dG

using

LC/UV/EC

15µg/egg

20days

Noincrease

inliv

erconcentrationof

8-OH-dG

Metalloid

nano

particles

Germanium

nanoparticles

Lin

etal.[73]

Genano

particles

(app

roximately1–10

,20–50,

and10

0–20

0nm

indiam

eter)

Chinese

hamster

ovary

(CHO)K1cells

Alkalinecomet

assay,

H2A

X0.36

mg/mL

0,12

There

was

apparent

damageby

comet

assayafter0hforthe

Genano

particlesbu

tno

tfor

GeO

2,andsign

ificantdamage

forGenano

particlesafter12

h

Nodifference

was

observed

for

γ-H

2AX

form

ationafterGe

nanoparticle

orGeO

2additio

n,which

suggestsapotential

artifactforthecomet

assay

Effectsof

radiationafterGe

nanoparticle

additio

nwerealso

observed,andGenanoparticles

hadaradiosensitizingeffect

similarto

that

ofGeO

2

Silica

nano

particles

Barneset

al.[122]

Sinano

particles

(nom

inal

sizes30

,80

,and40

0nm

),twobatches

ofcollo

idal

nanoparticles

from

SigmaL

udox

(20-40

nm)

Mouse

embryo

fibrob

lastcelllin

e(3

T3-L1)

Alkalinecomet

assay

4and40

µg/mL

3,6,

24Nosign

ificanteffectsob

served

afteranytim

eperiod

forany

silicon

diox

ideparticle

Independ

ently

valid

ated

attwo

separate

laboratories

Gerloffet

al.[44]

SigmaSiO

2(14-nm

prim

aryparticle

size

bymanufacturer)

Hum

ancarcinom

aintestinal

cells

(Caco-2cells)

Alkalinecomet

assay+/-FPG

133.3µg/mL

4Significant

increase

observed

with

FPG

butno

twith

outFPG


Tab

le1

(con

tinued)

Reference

Nanop

article

tested

(size)

Cell/o

rganism

tested

DNA

damage

assay

Concentratio

nsExposure

duratio

n(h)

Finding

s

Jinet

al.[123

]Sinano

particles(50±3nm

)Hum

anlung

epith

elial

celllin

e(A

549)

Alkalinecomet

assay,

pulsefieldgel

electrophoresis,

agarosegel

electrophoresisusing

genomic

DNA

strands,8-OH-dG

usingwestern

blot

0.1,

1,10

,10

0,50

0µg/mL

48,72

Noeffectsob

served

atany

concentration

Lee

etal.[125]

Sigma-Aldrich

(7and

10nm

bymanufacturer)

Dap

hnia

mag

naand

Chirono

mus

ripa

rius

Alkalinecomet

assay

1µg/mL

24Nosign

ificantincrease

Wanget

al.[124]

Sigma-Aldrich

SiO

2

(7–9

nm)

Hum

anB-cell

lymphoblastoidcell

line(W

IL2-NS)

Alkalinecomet

assay

60and120µg/mL

24Significant

damagewas

notob

served

Yanget

al.[46]

Runhe

(20.2±6.4nm

;>99

.0%

purity)

Primarymou

seem

bryo

fibrob

lasts

Alkalinecomet

assay

5and10

µg/mL

24Significant

effectsat

both

concentrations

Metal

oxidenanoparticles

Aluminum

oxide

nanoparticles

Balasub

ramanyam

etal.[127

]Sigma-Aldrich

Al 2O3

(30and40

nm)

Fem

aleWistarrats

(whole

bloo

dfrom

retro-orbituswas

tested)by

gavage

Alkalinecomet

assay

500,

1,000,

and

2,000mg/kg

bygavage

4,24,48,72

Significant

effectsobserved

at1,000

and2,000mg/kg

,bu

tno

t50

0mg/kg

,after4and24

hfor

30-and40

-nm

particles

Nosign

ificanteffectsob

served

forbu

lkparticlesat

anytim

eor

concentration

Nosign

ificanteffectsob

served

foranyparticle

size

orconcentrationafter72

h

Kim

etal.[126

]Sigma-Aldrich

Al 2O3

(<50

nm)

Mouse

lymphom

acell

line(L51

78Y)and

human

bron

chial

epith

elialcells

(BEAS-2B)

Alkalinecomet

assay

1,25

0,2,50

0,and

5,000µg/mLfor

L51

78Y

cells

2Significant

damageto

L5178Y

cells

with

S-9

atall

concentrations

andonly

at2,500µg/mLwith

outS-9

68.36,

136.72

,and27

3.44

μg/mL

forBEAS-2Bcells

with

S-9

and97

.66,

195.32

,and

390.63

μg/mL

with

outS-9

Significant

damageto

BEAS-2Bcells

atall

concentrations

with

andwith

outS-9

Cerium

oxide

nanoparticles

Auffanet

al.[130]

Nano-CeO

2(Rho

dia

Chemicals;7nm

)and

micro-CeO

2(320

nm)

Normal

human

fibrob

lasts

Alkalinecomet

assay

0.00

6,0.06

,0.6,

6,60

,60

0,and

1,200μg/mL

Toxicity

consistently

observed

for

concentrations

above60

μg/mL

Nano-CeO

2was

moretoxicon

amassbasisbu

tequivalent

ona

surfacearea

basis


Tab

le1

(con

tinued)

Reference

Nanop

article

tested

(size)

Cell/o

rganism

tested

DNA

damage

assay

Concentratio

nsExposure

duratio

n(h)

Finding

s

Add

ingantio

xidant

L-ergothion

eine

decreasedDNA

damageandsign

ificantresults

only

occurred

at1,20

0μg/mL

Lee

etal.[125]

15and30

nmDap

hnia

mag

naandChirono

mus

ripa

rius

Alkalinecomet

assay

1μg/mL

24Significant

increase

for15

nmon

lyforChirono

mus

ripa

rius

andforbo

thsizesfor

Dap

hnia

mag

na

Pierscion

eket

al.[128

]10–12nm

Hum

anlens

epith

elial

celllin

e(CRL-11421)

Alkalinecomet

assay

5and10

μg/mL

24Nosign

ificanteffectsob

served

Rothen-Rutishauser

etal.[129

]CeO

2(m

eanprim

ary

particle

diam

eter

5–20

nm)

Hum

anlung

epith

elial

celllin

e(A

549)

8-OH-dG

using

oxyD

NA

assaykit

NA

(cellsexposed

inglov

ebox

exposure

setup)

10,20

,and

30minutes

Significant

increase

inpercent

ofcells

with

8-OH-dG

after20

and30

min

Copperoxidenanoparticles

Karlssonet

al.[22]

Sigma-Aldrich

CuO

(42nm

bymanufacturer)

Hum

anlung

epith

elial

celllin

e(A

549)

Alkalinecomet

assay+/-FPG

2,40

,and80

µg/mL

(40and80

µg/mL

forFPG)

4Significant

increasesin

DNA

damageat

40and80

µg/mL

butno

t2µg/mL

Highestam

ongallnanoparticles

tested

inthisstud

y

Significant

increase

inoxidative

lesionson

lyat

80µg/mL

Dose-depend

entDNA

damage

andox

idativelymod

ified

lesion

patternsob

served

Karlssonet

al.[23]

Sigma-Aldrich

CuO

(20–40

nmand

0.5–10

µm)

Hum

anlung

epith

elial

celllin

e(A

549)

Alkalinecomet

assay+/-FPG

40,80

µg/mL

4Nanop

articlescaused

sign

ificantly

moreDNA

damagethan

microparticlesat

80µg/mLand

almostat

40µg/mL(p=0.051)

Significantly

increasedDNA

damagefornano

particlesand

microparticlesat

both

concentrations

Nanop

articless

caused

sign

ificantly

moreFPG

lesion

son

lyat

80µg/mLcompared

with

thecontrol

Nosign

ificantincrease

inthe

numberof

FPG

lesions

comparedwith

controlsfor

microparticlesat

either

concentration

Midanderet

al.[94]

Sigma-Aldrich

(28nm

and2.9µm)

Hum

anlung

epith

elial

celllin

e(A

549)

Alkalinecomet

assay

80µg/mL

4Increase

inDNA

damagefrom

CuO

nano

particlesand


Tab

le1

(con

tinued)

Reference

Nanop

article

tested

(size)

Cell/o

rganism

tested

DNA

damage

assay

Concentratio

nsExposure

duratio

n(h)

Finding

s

microparticles,butsignificantly

moredamaged

from

the

nanoparticlesthan

the

microparticles

Noeffectsob

served

from

copper

fractio

ndissolvedfrom

the

particles

Iron

oxidenano

particles

Auffanet

al.[133]

Fe 2O3(m

aghemite)

(6nm

)Normal

human

fibrob

lasts

Alkalinecomet

assay

0.00

1,0.01

,0.1,

1,10

,and

100µg/mL

2,24

Nosign

ificanteffectsob

served

Bhattacharya

etal.[134]

Hem

atite

(Fe 2O3;

∼50-nm

hydrod

ynam

icradius)

Hum

andiploid

fibrob

lasts(IMR-90),

human

bron

chial

epith

elialcells

(BEAS-2B)

Alkalinecomet

assay,

8-OH-dG

usingELISA

10,25

,50

,and

250µg/mL

(com

etassay),

25and50

µg/mL

(8-O

H-dG)

24Significantly

increasedDNA

damageby

comet

assayat

50and25

0µg/mLforIM

R-90

cells

and25

0µg/mLfor

BEAS-B2cells

Nosign

ificantincrease

in8-OH-dG

was

observed

Karlssonet

al.[22]

Sigma-Aldrich

Fe 2O3

(29nm

bymanufacturer)

Hum

anlung

epith

elial

celllin

e(A

549)

Alkalinecomet

assay+/-FPG

2,40

,and80

µg/mL

(40and80

µg/mL

forFPG)

4Nosign

ificantincrease

inDNA

damage

Karlssonet

al.[22]

Sigma-Aldrich

Fe 3O4

(20–30

nmby

manufacturer)

Hum

anlung

epith

elial

celllin

e(A

549)

Alkalinecomet

assay+/-FPG

2,40

,and80

µg/mL

(40and80

µg/mL

forFPG)

4Nosign

ificantincrease

inSSBs

Significant

increase

inthenumber

ofoxidativelymodifiedlesions

at80

µg/mL

Karlssonet

al.[23]

Sigma-Aldrich

Fe 2O3

(30–60

nmand

0.15–1

µm)

Hum

anlung

epith

elial

celllin

e(A

549)

Alkalinecomet

assay+/-FPG

40and80

µg/mL

4Significant

DNA

damagefrom

microparticlesbutnot

nanoparticlesat

80µg/mL,and

therewas

asign

ificant

difference

betweenthe

twoparticle

types

Nosign

ificantincrease

inDNA

damageforeither

particle

at40

µg/mL

Nosign

ificantincrease

inthenu

mberof

oxidativelymod

ifiedlesion

sfor

either

particle

type

ateither

concentration

Karlssonet

al.[23]

Sigma-Aldrich

Fe 3O4

(20–40

nmand

0.1–0.5µm)

Hum

anlung

epith

elial

celllin

e(A

549)

Alkalinecomet

assay+/-FPG

40,80

µg/mL

4Significant

increase

inDNA

damagefrom

microparticlesat

40and80

µg/mLbu

tno

tfrom

nanoparticles

Nanop

articlescaused

sign

ificantly

moreox

idativelymod

ified

lesionsthan

microparticlesat


Tab

le1

(con

tinued)

Reference

Nanop

article

tested

(size)

Cell/o

rganism

tested

DNA

damage

assay

Concentratio

nsExposure

duratio

n(h)

Finding

s

80µg/mL,andthenu

mberwas

sign

ificantcomparedwith

controlson

lyat

80µg/mL

Magnesium

oxide

nanoparticles

Gerloffet

al.[44]

Nanoscale

Materials

MgO

(8-nm

prim

ary

particle

size

bymanufacturer)

Hum

ancarcinom

aintestinal

cells

(Caco-2cells)

Alkalinecomet

assay+/-FPG

133.3µg/mL

4Noeffect

observed

Titanium

diox

ide

nanoparticles

Bhattacharya

etal.[134]

Degussa

anatase

(91-nm

hydrod

ynam

icradius)

Hum

andiploid

fibrob

lasts(IMR-90),

human

bron

chial

epith

elialcells

(BEAS-2B)

Alkalinecomet

assay,

8-OH-dG

usingELISA

10,25

,50

,and

250µg/mL

(com

etassay),

25and50

µg/mL

(8-O

H-dG)

24Increased8-OH-dG

form

ation

at25

and50

µg/mL

forIM

R-90cells

Noeffect

onDNA

damageby

comet

assaywith

either

celllin

e

Falck

etal.[148]

Nanosized

rutile

(10um

×40

nm),

nanosizedanatase

(<25

nm),andbu

lkrutile(<

5um

)allfrom

Sigma,

allsizesfrom

manufacturer

Hum

anbronchial

epith

elialcells

(BEAS-2B)

Alkalinecomet

assay

3.8,

19,38

,76

,114,

228,

304,

and38

0μg/mL

24,48,72

Nanosized

anataseandbulk

rutile

weresimilarlyeffective,

whereas

nanosizedrutilewas

less

effective,

butno

neof

them

wereapo

tent

indu

cerof

DNA

damage

ElevatedDNA

damagewas

observed

atvariou

sconcentrations

buttrends

wereno

tclear

Dose-depend

entpatterns

wereob

served

aftersome

exposure

period

sbu

tno

tothers

andacleartrend

was

notob

served

Gerloffet

al.[44]

Degussa

P25

(20–80

nm),

Aldrich

Fine(40–30

0nm

;pure

anatase),and

TiO

2-H

AS(<

10nm

)

Hum

ancarcinom

aintestinal

cells

(Caco-2cells)

Alkalinecomet

assay+/-FPG

133.3µg/mL

4TiO

2slides

wereprocessedin

total

darkness

ornorm

allaboratory

lighting,

andlaboratory

lighting

caused

asign

ificantincrease

inDNA

damageandthenu

mberof

FPG-sensitiv

elesions

Noeffect

indark

forTiO

2of

any

size

andno

difference

among

differentsizes

Gop

alan

etal.[146]

Sigma-Aldrich

TiO

2

(anatase;40–70nm

)Hum

ansperm

and

lymphocytecells

Alkalinecomet

assay

3.73

,14

.92,

29.85,

59.7

µg/mL

0.5

Significant

increasesin

DNA

damageat

allconcentrations


Tab

le1

(con

tinued)

Reference

Nanop

article

tested

(size)

Cell/o

rganism

tested

DNA

damage

assay

Concentratio

nsExposure

duratio

n(h)

Finding

s

(cellswerespiked

inthedark,

preirradiatedwith

UV

light,or

expo

sed

toUV

light

after

nano

particle

additio

n)

forbo

thcells

undereach

condition

For

sperm

cells,no

differences

observed

asaresultof

UV

exposure

except

forpreirradiatio

nfollo

wed

byadditio

nof

3.73

µg/mLTiO

2nano

particles

For

lymphocytes,therewas

astatistically

significantincrease

from

UVexpo

sure

atthehigh

est

threedo

sescomparedwith

dark

condition

s

Gurret

al.[19]

Sigma-Aldrich

anataseat

10,20

,20

0,and

≥200

nmandKanto

Chemical

rutileat

200nm

,manufacturersizes

Hum

anbronchial

epith

elialcells

(BEAS-2B)expo

sed

intotaldarkness

Alkalinecomet

assay+/-FPG

5and10

µg/mL

prelim

inary

experiment

1In

anexperimentwith

10-nm

anataseand>200-nm

anatase,

increaseddamagewas

observed

only

at10

µg/mL

for10

-nm

particles

10µg/mLprim

ary

experiment

Significant

increase

indamage

for10

-and20

-nm

anataseand

200-nm

rutilebu

tno

tfor20

0-nm

anataseor

>200-nm

anatase

Amixture

of1:1200-nm

anatase

andrutilecaused

moredamage

than

anequivalent

totalmassof

either

ofthem

bythem

selves

Kanget

al.[149]

Degussa

P25

(25nm

bymanufacturer)

Peripheralbloo

dlymphocytes

Alkalinecomet

assay

20,50

,or

100μg/mL

6,12

,24

TiO

2caused

sign

ificantDNA

damageat

each

concentration

andeach

timepo

int

The

additio

nof

1mM

N-acetylcysteinesignificantly

decreasedDNA

damage

Karlssonet

al.[22]

Sigma-Aldrich

TiO

2

(63nm

bymanufacturer)

Hum

anlung

epith

elial

celllin

e(A

549)

Alkalinecomet

assay+/-FPG

2,40

,and80

µg/mL

(40and80

µg/mL

forFPG)

4Significant

increase

inDNA

damageon

lyat

80µg/mL

Nosign

ificantincrease

inthe

numberof

oxidatively

mod

ifiedlesions

Karlssonet

al.[23]

Sigma-Aldrich

TiO

2

(20–10

0nm

and

0.3–1µm)

Hum

anlung

epith

elial

celllin

e(A

549)

Alkalinecomet

assay+/-FPG

40,80

µg/mL

4Significantly

moreDNA

damagefrom

microparticles

than

nanoparticlesat

80µg/mL

Significant

DNA

damage

increasesat

40and80

µg/mL

formicroparticlesand

80µg/mLfornanoparticles


Tab

le1

(con

tinued)

Reference

Nanop

article

tested

(size)

Cell/o

rganism

tested

DNA

damage

assay

Concentratio

nsExposure

duratio

n(h)

Finding

s

Nosign

ificantincrease

inthe

numberof

oxidativelymod

ified

lesionsforeither

particle

type

ateither

concentration

Lee

etal.[125]

Sigma-Aldrich

(7and

20nm

bymanufacturer)

Dap

hnia

mag

naand

Chirono

mus

ripa

rius

Alkalinecomet

assay

1µg/mL

24Nosign

ificantincrease

Nakagaw

aet

al.[141]

Nippo

nAerosilp-25

(anatase,21

nm),Wako

WA

(anatase,25

5nm

),WakoWR(rutile,

255nm

),TP-3

(rutile,

420nm

),sizesby

manufacturer

Mouse

lymphom

acells

(L51

78Y)

Alkalinecomet

assay

3.1,

12.5,50

,20

0,80

0,and3,20

0µg/mL

1hdark

and

50min

with

orwith

outUV

OnlyWA

caused

DNA

damage

with

outUVirradiation

p-25

was

themostpo

tent

with

irradiationwhile

WRwas

the

least

Increasing

UVA

irradiation

energies

lead

toincreased

DNA

damageforafixed

p-25

concentration

Reeveset

al.[142]

Sigma(5

nm,anatase)

Goldfish(Carassius

auratus)

skin

cells

(GFSk-S1)

Alkalinecomet

assay

+/-FPG

and

endonu

clease

III

1,10

,and10

0µg/mL

2,24

Nosign

ificantincrease

for

endonu

clease

IIIlesion

safter24

hwith

outUVA

atanyconcentration

Significant

increase

forFPG

lesionsat

allconcentrations

after24

hwith

outUVA

Significant

increase

only

at10

0µg/mLwith

outFPG

orendonu

clease

IIIafter24

hwith

outUVA

After

2h,

sign

ificantincreases

wereob

served

with

and

with

outFPG

forUVA

only,

for10

µg/mLTiO

2on

ly,and

furthersign

ificantly

increased

forTiO

2with

UVA

Rehnet

al.[153]

Degussa

(P25

andT805)

Fem

aleWistarrats

(lun

gstested)by

intratrachealinstallatio

n

8-OH-dG

using

immunocytological

assay

0.15

,0.3,

0.6,

and

1.2mg/lung

90days

Nosign

ificanteffectsob

served

atanyconcentrationforP25

orT805

Serpo

neet

al.[143]

Manytypesof

TiO

2

(som

emod

ifiedto

passivatethesurfaceto

Plasm

idDNA

andhu

man

keratin

ocytes

Plasm

idDNA

nicking,

alkalin

ecomet

assay

50,10

0,50

0,1,00

0,5,000,

10,000

,and

20,000

µg/mL

10,20

,30

,40

,or

60min

ModifiedTiO

2didno

tcause

anincrease

inDNA

damage

afterirradiationforthe


Tab

le1

(con

tinued)

Reference

Nanoparticle

tested

(size)

Cell/o

rganism

tested

DNA

damage

assay

Concentratio

nsExposure

duratio

n(h)

Finding

s

minim

izeor

supp

ress

photoactivity

)plasmid

nickingassayor

the

comet

assaycomparedwith

controls

Unm

odifiedsamples

generally

caused

anincrease

inplasmid

DNA

damageanddamageby

comet

assayafterUVirradiation

Treatmentof

keratin

ocytes

with

TiO

2in

thedark

didno

tcause

increasedDNA

damage

Increasing

theconcentrationof

unmodifiedTiO

2increased

damagefornickingassay

butno

tin

amon

oton

icfashion

Trouilleret

al.[154

]Degussa

P25

(primary

particle

size

21nm

)C57B1/6Jpun/punmice

Alkalinecomet

assay,

8-OH-dG

using

LC/UV/EC,H2A

X

500mg/kg

(com

etassayand

8-OH-dG),50,

100,

250,

and

500mg/kg

(H2A

X)

5days

Significant

increasesin

DNA

damageby

comet

assayand

8-OH-dG

measurements

at50

0mg/kg

Significant

increasesforanu

mber

ofph

osph

orylated

cells

byγ-H

2AX

inado

se-dependent

manner

Veverset

al.[144]

Degussa

P25

(24.4±0.5nm

)Rainb

owtrou

t(O

ncorhyncus

mykiss)

gonadcells

(RTG-2)

Alkalinecomet

assay+/-FPG

5or

50µg/mL

4TiO

2caused

asign

ificantreduction

inDNA

damagewhenUVAwas

notused

Significantly

increasedDNA

damagewas

observed

inminim

alessentialmedium

with

TiO

2andUVA

WhenFPG

was

included,

only

50µg/mLwith

UVA

sign

ificantly

increased

oxidativelesions

Wanget

al.[150]

Sigma-Aldrich

TiO

2

(6–10nm

,99

%pu

rity)

Hum

anB-cell

lymphob

lastoidcell

line(W

IL2-NS)

Alkalinecomet

assay

65µg/mL

24Significant

increase

inDNA

damageob

served

Zhang

etal.[108

]InabataTiO

2(10–30

nm)

Plasm

idDNA

Plasm

idDNA

nicking

56,56

0,and

1,10

0µg/mL

8Minim

aldamageob

served

Zhu

etal.[145]

10–20nm

and50

–60nm

anatase,

50–6

0nm

rutile

Teasyplasmid

DNA

exposedto

UV

radiation

Plasm

idDNA

nicking

and8-OH-dG

byLC/UV/EC

100µg/mL

0.25

Nodamagewas

observed

after

only

UVirradiationfor

8-OH-dG

orgelelectrophoresis

Significant

increasesin

DNA

damageby

both

measureswere

observed

foreach

type

ofnano

particle


Tab

le1

(con

tinued)

Reference

Nanoparticle

tested

(size)

Cell/o

rganism

tested

DNA

damage

assay

Concentratio

nsExposure

duratio

n(h)

Finding

s

For

both

measurements,

10–2

0-nm

anatase>

50–60-nm

anatase>50–6

0-nm

rutile

Zincox

idenano

particles

Gerloffet

al.[44]

Nanoscale

Materials

(10nm

)or

Nanostructured

andAmorphousMaterials

(20nm

),bo

thsizesby

manufacturer

Hum

ancarcinom

aintestinal

cells

(Caco-2cells)

Alkalinecomet

assay+/-FPG

133.3µg/mL

4Significant

effectsob

served

with

andwith

outFPG

Different

sizesof

ZnO

particles

allcaused

DNA

damageexcept

forthelargestparticleswith

out

FPG

ataconcentration

of13

3.3µg/mL

Nosign

ificantdifferenceswere

observed

intheDNA

damage

potentialof

thevariousparticles

Gop

alan

etal.[146]

Sigma-Aldrich

ZnO

(40-70

nm)

Hum

ansperm

and

lymphocytecells

(cellswerespiked

inthedark,

preirradiatedwith

UV

light,or

exposed

toUV

light

after

nanoparticle

additio

n)

Alkalinecomet

assay

11.5,46.2,

69.4,and

92.3

µg/mL

0.5

Statistically

significantresults

wereob

served

atall

concentrations

forboth

cells

except

forthelowest

concentrationin

thedark

for

sperm

cells

For

sperm

cells,therewas

only

astatistically

significantdifference

at11.5

µg/mLforsamples

treatedwith

UV

light

compared

with

thosein

thedark

Karlssonet

al.[22]

Sigma-Aldrich

ZnO

(71nm

bymanufacturer)

Hum

anlung

epith

elial

celllin

e(A

549)

Alkalinecomet

assay+/-FPG

2,40

,and

80µg/mL

(40and

80µg/mL

forFPG)

4Significant

increase

inDNA

damageandnu

mberof

oxidativelymod

ifiedlesions

aton

ly80

µg/mL

Lin

etal.[155]

Sigma(70±13

-and

420±26

9-nm

ZnO

)Hum

anlung

epith

elial

celllin

e(A

549)

Alkalinecomet

assay

10,12,and

14µg/mL

24Nosign

ificantdifference

between

particle

sizesbu

thy

drod

ynam

icradiiweresimilarforthetwo

particlesin

thisconcentrationrang

e

Significant

effectsob

served

atallconcentrations

for

both

typesof

particles

Steep

dose

respon

seob

served


Tab

le1

(con

tinued)

Reference

Nanoparticle

tested

(size)

Cell/o

rganism

tested

DNA

damage

assay

Concentratio

nsExposure

duratio

n(h)

Finding

s

Sharm

aet

al.[156

]Sigma-Aldrich

(30nm

byTEM)

human

epidermal

celllin

e(A

431)

Alkalinecomet

assay

0.001,

0.008,

0.08

,0.8,

and

5µg/ml

6Significant

increase

observed

only

for0.8and5µg/mL

Yanget

al.[46]

Nanuo

19.6±5.8nm

(>99

.9%

purity)

Primarymou

seem

bryo

fibrob

lasts

Alkalinecomet

assay

5and10

µg/mL

24Significant

effectsat

both

concentrations

Add

ition

almetal

oxidenanoparticles

Karlssonet

al.[22]

Sigma-Aldrich

CuZ

nFe 2O4

(29nm

bymanufacturer)

Hum

anlung

epith

elial

celllin

e(A

549)

Alkalinecomet

assay+/-FPG

2,40

,80

µg/mL

(40,

80µg/mL

forFPG)

4Significant

increasesin

DNA

damageandnu

mberof

oxidativelymod

ifiedlesions

only

at40

and80

µg/mL

Quantum

dots

Anaset

al.[160]

Invitrogen

streptavidin-

functio

nalized

CdS

e–ZnS

quantum

dots

Plasm

idDNA

Plasm

idDNA

nicking+/-FPG

and

+/-endonu

clease

III

0.5nM

1DNA

damagewas

notob

served

inthedark

with

quantum

dots

butwas

observed

after

photosensitizingthequantum

dots

Sim

ilarly,

theph

otosensitized

quantum

dotswerealso

show

nto

damagepurine

andpyrimidine

bases,whereas

noeffectswere

observed

inthedark

Jacobsen

etal.[48]

American

Dye

Sou

rce

positiv

elyandnegativ

ely

chargedqu

antum

dots

(4.5–5.5

nmby

manufacturer)

ApoE-/-mice

(broncho

alveolar

lavage

fluidcells

tested)by


Alkalinecomet

assay

63µgCd

3Significant

increasesobserved

and

approxim

atelythesamevalues

forpositiv

eandnegativ

esurface

charges

Gagné

etal.[162

]CdT

equ

antum

dots(A

merican

Dye

Sou

rce)

Elliptio

complan

ata

musselstested

gills

anddigestiveglands

Alkalinecomet

assay+alkalin

eprecipitatio

nassay

1.6,

4and

8µg/mL

24Significant

increase

ingills

at1.6and4µg/mLbu

tno

t8µg/mL

Significant

increase

indigestive

glandat

4and8µg/mL

Significant

DNA

damagefor

0.5µg/mLCdfordigestive

glands

butno

tgills

Gagné

etal.[163]

CdT

ewith

cysteaminecoating,

aged

for2mon

thsor

2years

before

expo

sure

Rainb

owtrou

t(O

ncorhynchu

smykiss)

hepatocytes

Alkalinecomet

assay+alkalin

eprecipitatio

nassay

0.4,

2,10

,50

,and25

0µg/mL

48For

quantum

dotsaged

for

2mon

ths,sign

ificantincreasesin

DNA

damagefor0.4,

10,and

50µg/mLbu

tno

tfor2or

250µg/mL

Gagné

etal.[163

]For

quantum

dotsaged

for2years,

DNA

damagewas

only

sign

ificantly

increasedat

2µg/mL

For

quantum

dotsaged

for2mon

ths

only,therewas

acorrelation

betweenDNA

damageresults

and

labile

zinc/cadmium

concentrations

Green

andHow

man

[158

]Cam

bridge

BioscienceCdS

equ

antum

dots(605

biotin

conjug

ate)

Sup

ercoileddo

uble

strand

sof

DNA

Plasm

idDNA

nicking

2µM

0.25,0.5,

0.75,and1

DNA

nickingoccurred

with

and

with

outUV

light,bu

tmore

occurred

with

UV

light


base lesions (including 8-OH-dG) in individual DNAsamples. The capability and importance of monitoring theformation of 8-OH-dG is significant because of its knownmutagenic and promutagenic activity, as it can cause G→Ttransversion mutations that are found in dysfunctionalgenes associated with cancer [33]. However, there exist anumber of other oxidatively induced base lesions that areequally mutagenic, but often more difficult to accuratelydetect because of their low levels. For example, among thepyrimidine lesions, 5-hydroxycytosine and 5-hydroxyuracilare both highly mutagenic lesions, leading to C→Ttransition mutations [34–36]. Thymine glycol is anotherpyrimidine lesion that blocks DNA polymerases and is thusa lethal lesion [37]. Among the purine lesions, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-dia-mino-5-formamidopyrimidine are potentially mutagenic,and can cause G→T and A→T mutations, respectively[38–40]. In certain cases, FapyGua may even be moremutagenic than 8-OH-dG [41]. The advantages of ourmass-spectrometry-based procedures over other reportedmethods for evaluating oxidative damage to DNA bases arethe following: (1) we can obtain absolute confirmation ofthe lesion’s identity (type), (2) we can obtain absolutequantitative information on each individual lesion that istraceable to high-purity reference standards, and mostimportantly, (3) we have the capability to decipher anddescribe the mechanistic pathways by which specific ENsmight induce oxidative damage to DNA marked by theformation of DNA base lesions. In one of our presentstudies, we are utilizing GC/MS procedures to investigatethe effect of copper oxide (CuO) nanoparticles (NPs) ongenomic DNA damage in germinating plants in vivo. Thusfar, we have observed remarkable trends in terms of thetypes and levels of oxidatively induced base lesions that areformed owing to the presence and activity of the NPs. Thetrends that we currently observe seem to correlate stronglywith the NP dose and with the species and/or type of plantunder investigation (unpublished results).

Oxidatively induced DNA damage, whether it isdetected and measured as discrete strand break lesions oras DNA base lesions, is associated with biologicalmechanisms involved in the onset of carcinogenesis,mutagenesis, and premature aging in humans. Thus, it isimperative to describe and understand the mechanisms bywhich ENs could potentially mediate and/or promote thesedisease processes. Hence, this paper has two specific aims:(1) to review and assess the mechanistic details of EN-induced oxidative damage to DNA as reported in thecurrent scientific literature and (2) to review and evaluatethe predominant assays and procedures that have beenreported for measuring EN-induced oxidative damage toDNA. The review is specifically focused on studies thatinvestigated ENs that were 100 nm or smaller in at least oneT

able

1(con

tinued)

Reference

Nanoparticle

tested

(size)

Cell/o

rganism

tested

DNA

damage

assay

Concentratio

nsExposure

duratio

n(h)

Finding

s

Hoshino

etal.[159]

ZnS

-coatedCdS

equ

antum

dotscoated

with

11-m

ercaptou

ndecanoicacid

(QD-COOH),cysteamine

(QD-N

H2),or

thioglycerol

(QD-O

H)or

QD-O

H/COOH,

andQD-N

H2/OH

Hum

anlymphom

acells

(WTK1)

Alkalinecomet

assay

0.5,

1,1.5,

and2µM

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dimension and that measured and reported oxidativedamage in terms of strand break lesions and/or in terms ofoxidative base lesions. Studies on incidental NPs such asultrafines produced through combustion processes were notincluded. Additionally, one of the most frequently discussedsuggestions for EN research involves proper characterizationof the NPs [3, 6, 42, 43]. Although this review does notcover this topic in depth, the characterization methodsutilized in each study on EN-induced oxidative damage toDNA are summarized in Table S1 in the Electronicsupplementary material. It is clear that the extent of ENcharacterization spans a broad range, with many reportsproviding minimal characterization information. In ouropinion, the lack of adequate characterization data is a likelycause for the discrepancies observed among the studiesdescribed herein. The discussion will focus on five majorcategories of ENs: carbonaceous, metals, metalloids, metaloxides, and semiconducting quantum dots (Qdots).

Mechanisms and measurements

Carbon-based nanomaterials

Carbon-based (carbonaceous) ENs exist as a variety ofstructures, including tubes, spheres, particles, and fibers. Tubestructures include both single-walled carbon nanotubes(SWNTs) and multi-walled carbon nanotubes (MWNTs),spheres includeC60 fullerenes, particles include nanoparticulatecarbon black (nCB), and fibers include graphite nanofibers.

Carbon black nanoparticles

The toxicity of nCB has been investigated in seven in vitroexperiments [18, 21, 22, 44–47] and DNA damage was

typically but not always detected. A commercially availablecarbon powder (larger than 30 nm) was not found toincrease DNA strand breaks or Fpg-sensitive lesions atconcentrations up to 80 µg/mL cell medium using A549cells [22]; unless otherwise stated, all nanomaterial con-centrations for in vitro assays will have units of mass ofnanomaterial per volume of cell medium. However, Printex90 was shown to induce SSBs in A549 cells using aconcentration of 100 µg/mL [18, 45]. This result appears tobe due to the higher concentration tested by Mroz et al. [18,45], but they did not test a range of concentrations todetermine the concentration at which enhanced DNAdamage did not occur, and thus other experimental factorsin these studies could be the cause of the differing results.This same pattern of results was also observed for Fpg-sensitive sites in these studies. The lowest concentrationobserved to cause DNA damage by nCB was 5 µg/mLusing primary mouse fibroblast cells [46], whereas concen-trations as high as 786 µg/mL did not induce damage asmeasured by the alkaline comet assay in Chinese hamsterlung fibroblasts or human embryonic lung fibroblasts [47].It is surprising that these different results span 2 orders ofmagnitude and this suggests that much is yet unknownabout what characteristics of nCB are most important forinducing toxicity and to what extent cell lines differ in theirsensitivity to nCB exposure.

The toxicity of nCB has also been investigated in threein vivo studies using mice and rats, each of which studied

Fig. 2 WI-38 cells without any H2AX foci (a) and WI-38 cells withone or more foci (b–d). (Reprinted with permission from [173])

Fig. 1 Comets from the alkaline comet assay. The higher comet isrepresentative of substantial DNA damage, whereas the lower cometindicates minimal DNA damage. (Reprinted with permission fromPeggy Olive, British Columbia Cancer Agency)


inhalation exposure risks. In the two studies in which theexposure was through intratracheal installation, significantincreases in SSBs were detected using the alkaline cometassay [48, 49]. However, the concentration needed toinduce these lesions differed between the studies. SSBswere detected by Totsuka et al. [49] after addition of 200 µgper mouse but not 50 µg per mouse, whereas exposing eachmouse to 54 µg did induce DNA damage in a study byJacobsen et al. [48]. Both of these studies utilized thealkaline comet assay to detect SSBs induced by Printex 90NPs, and thus this different response could be a result of thetypes of mice tested. Gallagher et al. [50] also tested thetoxicity of Printex 90 NPs with rats but used an inhalationexposure setup. Elevated concentrations of 8-OH-dGmeasured by LC/UV detection or LC/UV/EC detectionwere detected only after the highest exposure concentrationof 52.8 mg/m3 after 13 weeks of exposure, althoughelevated concentrations of 8-OH-dG were also detected ata concentration of 7.1 mg/m3 after an additional 44-weekrecovery period in which the rats were not exposed. Thisresult suggests that the continued presence of nCB in thelungs poses a serious long-term risk.

nCB is hypothesized to cause oxidative stress and induceoxidative damage to DNA in in vitro systems on the basisof its small size and biopersistence, metal content, and/orpolycyclic aromatic hydrocarbon (PAH) content [2]. Bio-persistence relates to the small size of nCB and its capacityto enter cells, and the expected slow rate at which the NP iseliminated or excreted by the organism. Biopersistencecould lead to the induction of inflammation and activationof ROS-producing neutrophils and other inflammatory cellsas discussed previously. nCB, owing to its large surfacearea, also has the potential to adsorb transition metals (i.e.,Fe, Ni, Cu, Cr, etc.) onto its surface that could catalyzeFenton-like reactions to produce •OH. And finally, owingto its physical structure (large surface area), nCB has thecapacity to adsorb large quantities of PAHs onto its surface.PAHs can be converted to quinones via biotransformationreactions and quinones are active redox-cyclers (quinone ↔semiquinone) and generators of O2

•−. The O2•− can

dismutate to H2O2, which can react with transition metalions to produce •OH [7]. Remarkably, not a single studyinvestigated or reported nCB-induced oxidative damageto DNA based on the potential mechanisms describedabove nor did any of the studies give detailed experi-mental information on any other potential DNA damagemechanism.

Carbon nanotubes

Despite a wide range of types of carbon nanotubes (i.e.,MWNTs and SWNTs), nanotube dispersion procedures, invivo exposure procedures, and in vitro assays using various

cell types, carbon nanotubes consistently revealed thepotential for induction of DNA damage. In vitro resultsusing a variety of cell lines generally indicate DNA damageafter exposure to SWNTs or MWNTs [20, 22, 46, 51–56].These studies typically used cell lines related to the lungsgiven the serious concerns about carbon nanotubes havingeffects similar to asbestos [53, 57, 58]. DNA damage wasdetected using the alkaline comet assay both with andwithout Fpg and the γ-H2AX assay [20, 22, 46, 51–54, 56].A significant increase in SSBs was determined by thealkaline comet assay for MWNTs at the very lowconcentration of 2 µg/mL [22]. However, toxicity ofSWNTs to human peripheral blood lymphocytes was notdetected using the alkaline comet assay at SWNT concen-trations of 1, 5, and 10 µg/mL [55], and several of the otherstudies did not observe DNA damage with every assay [20,22, 54]. There was not a clear trend to the genotoxicresponses though. In a study by Jacobsen et al. [20],elevated levels of SSBs were not measured using thealkaline comet assay but the numbers of Fpg-sensitive baselesions were increased, whereas the opposite pattern wasobserved by Karlsson et al. [22]. The impact of variousfactors such as SWNTs versus MWNTs and the impact ofdifferent lengths of nanotubes have not been directlystudied. However, potentially lethal (if not repaired) DSBswere detected using the γ-H2AX assay in two studies usingMWNTs [53, 56], but DSBs were not detected in the onestudy that investigated DSBs using SWNTs [54]. On thebasis of this limited information, MWNTs may be moregenotoxic than SWNTs, but additional comparative re-search among different types of nanotubes and enhancedunderstandings of the characteristics of the nanotubes thatcause toxicity are necessary before definitive conclusionsare reached.

One potential artifact that was only tested in one of thesestudies was the potential for chemicals or metals leachedfrom the nanotubes to cause a toxic response [22].Although the soluble fraction was not shown to induceDNA damage in this study, yttrium released from SWNTshas been shown to impact the calcium ion channel oftsA201 cells [59], and SWNTs have previously been shownto leach substantial concentrations of nickel, which maycause significant toxic effects [60]. Assessing the potentialfor leached compounds to cause toxicity in future studieswould help confirm that the toxicity observed results fromexposure to the nanotubes themselves rather than fromexposure to leached catalytic metals. If metals or organicchemicals leached from the nanotubes are revealed to causetoxic responses, purifying the nanotubes to remove thesematerials may be a straightforward and important step inmitigating their potential risks.

The two in vivo studies on this topic investigated rats ormice exposed to SWNTs through oral gavage or intra-


tracheal installation, respectively [48, 61]. After exposureby oral gavage, elevated levels of 8-OH-dG were detectedusing the LC/EC method on the liver and lung but not oncolon mucosa cells after 24 h at doses of 0.064 and0.64 mg/kg body mass [61]. It is unclear though why DNAdamage was observed in some organs but not others. Thismay be a result of the biodistribution of the NPs within theorganism after exposure; different organs could be exposedto higher or lower carbon nanotube concentrations fordifferent time periods depending upon how the nanotubesare distributed within the organism and how rapidlyexcretion occurs. Previous biomedical studies indicatedthat organisms readily excrete injected modified nanotubes[62–64], but substantially different behaviors were ob-served for injected pristine SWNTs, with high massesremaining in the organisms 28 days after exposure [65].The nanotubes tested by Folkmann et al. [61] were of highpurity with regard to metal catalysts, which suggests thatpurification steps had occurred. Thus, these nanotubes mayhave acted more similarly to modified nanotubes and maybe readily excreted and accordingly have a short residencetime in the colon. It is also possible that DNA repairenzymes were stimulated in the colon mucosa cells and anypotential damage that had occurred was repaired by theconclusion of the 24-h exposure period. Similarly, de-creased DNA damage was detected in mice exposed tofullerenes by intratracheal instillation after 24 h comparedwith 3 h, which was speculated due to DNA repair [49]. Ina separate in vivo study on carbon nanotubes, mice wereexposed by intratracheal instillation [48]. Although expo-sure through oral gavage is intended to simulate toxicityafter ingestion of the NPs as would be expected for somebiomedical applications, intratracheal exposures aredesigned to test the potential impact of carbon nanotubeson organisms after inhalation. This exposure route could bean important risk for workers involved in nanomaterialmanufacturing given the concern that carbon nanotubesmay behave similarly to asbestos after inhalation [53, 57,58]. Again, significantly elevated levels of SSBs weredetected 3 h after the exposure using the alkaline cometassay. All together, these results suggest that DNA damageis a distinct possibility for organisms exposed to SWNTs.Although the potential of carbon nanotubes to induceoxidative damage to DNA in ecological receptors has notyet been investigated, past research indicates limitedabsorption and systemic distribution of carbon nanotubesafter oral ingestion, whereas significant nanotube masseshave been measured in organism guts [66–71]. Thus, invivo ecological studies should focus, when possible, onDNA damage in gut tracts, where nanotube exposure isexpected to be highest.

Carbon nanotubes have been shown to confound manycommon toxicity assays such as the 3-(4,5-dimethylthiazol-

2-yl)-2,5-diphenyltetrazolium bromide assay [72]. As such,investigators are encouraged to confirm cytotoxicity andDNA damage end points through complementary methodswhen possible. A related approach is to test cells immedi-ately after nanotube exposure using these assays. Largeobserved differences between control samples withoutnanotubes and those exposed to nanotubes only brieflywould indicate an artifact that may impact results for longernanotube exposure times. As will be discussed later forgermanium NPs (GeNPs), a significant increase in apparentoxidative damage to DNA was observed for GeNPs whenthe cells were harvested immediately after introduction ofthe GeNPs [73].

Mechanistically, both the presence of low-level transitionand heavy metal impurities within the SWNTs and theinherent biopersistence of the SWNT inside cells contributeto increased oxidative stress by the formation of ROS suchas •OH, O2

•−, and H2O2 [20, 54]. These mechanisms aresupported by the fact that even when SWNT agglomerationoccurs, ROS production is suppressed but still ongoing[20]. When transition and heavy metals are chelated, ROSformation is measurably suppressed but the presence of thenanotubes continues to stimulate ROS production [54]. The•OH radical is known to be the predominant free radicalthat attacks DNA, but none of the studies investigated orreported on this phenomenon [7]. The reported mechanismof MWNT induction of DNA damage in vitro is similar tothe mechanism ascribed to SWNTs. Oxidative damage toDNA involves the combined effects of low-level transitionmetal impurities acting as catalysts of the Fenton or Haber–Weiss reactions (producing •OH which can directly attackDNA) and the effects on persistent oxidative stress (andpersistent ROS production) when cells try to ingest MWNTthat are too large to fit inside, a phenomenon known as“frustrated phagocytosis” [74].

C60 fullerenes

The potential for fullerenes to induce oxidative damageto DNA has been studied less thoroughly than forcarbon nanotubes, with fewer than half as many paperson the subject. Overall, the potential for fullerenes todamage DNA was generally equal to or less than that ofsimilar masses of SWNTs. In in vitro experimentsutilizing the alkaline comet assay, fullerenes inducedSSBs in human lymphocytes at concentrations as low as2 μg/mL [75], but did not induce SSBs without theaddition of Fpg in a second study with mouse lungepithelial cells using a concentration of 100 µg/mL [20].The source of this discrepancy is unclear and may resultfrom the different dispersion techniques or sensitivities ofthe cell lines. Both SWNTs and C60 induced significantincreases in the number of Fpg sites but not in the number


of strand breaks after the epithelial lung cells had beenexposed for 3 h [20].

In an in vivo study that compared SWNTs and full-erenes, rats did not show elevated levels of SSBs afterfullerenes had been introduced via intratracheal installation,whereas SWNTs did significantly damage the rat DNA[48]. Additionally, fullerenes only induced a significantincrease in the 8-OH-dG levels in rat lungs after oralgavage at the highest dose (0.64 mg/kg), whereas SWNTsalso caused an effect at a lower does (0.064 mg/kg) [61]. Atthis point, a mechanistic understanding of the cause offullerene and SWNT toxicity is not available, and it isdifficult to discern why different results are exhibited inexperiments. The in vivo toxicity of fullerenes appears torelate to how the organisms are exposed to the NPs.Exposure by oral gavage yielded increased levels of 8-OH-dG at the higher-exposure concentration (0.64 mg/kg) butnot the lower concentration (0.064 mg/kg) [61], whereasintratracheal installation of 0.2 mg per animal but not0.05 mg per animal yielded a toxic response [48, 49]. In thestudy by Jacobsen et al. [48], the average mass of the micewas 18.5 g±1.4 g (N. Jacobsen, personal communication,2010), which indicates that the fullerene concentrationadded which did not induce DNA damage was approxi-mately 3 mg/kg. This value is much higher than that foundby Folkmann et al. [61] (0.64 mg/kg), but given thenumerous differences between these two studies, the sourceof the discrepancy is not clear.

Similarly to carbon nanotubes, fullerenes are veryinsoluble in water (kow=10

-6.67) and organisms will likelyonly be exposed to aggregates of these materials [76].Research has shown that the experimental approach used tomake fullerene aggregates can profoundly impact theobserved toxicity. For example, Dhawan et al. [75] foundthat aggregates prepared using an ethanol solvent exchangeresulted in fewer SSBs as measured by the alkaline cometassay than those produced via stirring in water. It is notclear at this point whether this difference stems fromdifferences in the morphology of the NP aggregates orwhether the solvent-exchange process could change thesurface characteristics of the NPs, yielding decreasedtoxicity. Although none of the studies on DNA damageby fullerenes utilized fullerenes dispersed using tetrahydro-furan (THF), a number of other studies have indicated thatthis process can yield artificially high toxicity [77–79].Observed toxicity was attributable to by-products fromthe THF procedure such as γ-butyrolactone and not thefullerene particles [77]. These findings highlight theimportance of rigorously testing for artifacts caused bythe material suspension procedures. Additionally, thisresearch suggests that care should be taken in choosingthe dispersion method, and that relevance to manufacturingprocesses or processes to which fullerenes would likely be

exposed to in the natural environment or after biologicaluptake should be considered. If manufacturers typically useTHF to disperse fullerenes for consumer products, researchto date has yielded some important concerns related to thetoxicity of this approach. But if this approach is not broadlyused by industry, significant effort has been spent trying tounderstand toxicity results borne from an artifact related tothe THF dispersion process.

The biochemical mechanism of fullerene-induced oxida-tive damage to DNA has not been thoroughly investigated.However, previous research has shown that C60 fullerenesare photosensitive compounds that are easily excited to thetriplet state via visible or UV light [80–84]. The fullerenetriplet state then undergoes an energy transfer to molecularoxygen to form singlet oxygen (1O2). The

1O2 can thenattack DNA directly and generate base lesions (preferen-tially at guanine) if the fullerene is inside the nucleus nearthe DNA [83]. Alternatively, and more likely, the veryunstable 1O2 will attack cytoplasmic or nuclear membranelipids and form lipid peroxide radicals, which are known topreferentially attack guanine residues and generate guanineradical cations [80, 82]. The continued presence (biopersis-tence) of C60 fullerenes in the cell can contribute to thefurther oxidation of 8-OH-dG to form ALS, which canresult in SSBs [80]. The detection of C60 fullerene-inducedSSBs and oxidatively induced base damage at purine basesin the two reported studies supports the probability of thepresented mechanism.

Carbon nanofibers

Firm conclusions about the potential for manufacturedgraphitic NPs to induce oxidative damage to DNA cannotbe drawn at this point because only two relevant studieshave been conducted [52, 85]. One study using cellulose-derived nanofibers showed no toxicity to Chinese hamsterovary cells even at the high concentration of 1 mg/mL [85],but commercially available graphitic nanofibers inducedDNA damage at 3.8 µg/mL after 24 h [52]. The graphiticnanofibers did have a substantial concentration of metalcatalyst remaining (4%), and this may partly account for thetoxic response. Previous studies have found that metalsleached from carbon nanotubes may cause substantialtoxicity [59], and we recommend that this potential betested in future studies. The mechanisms behind theobserved DNA damage after carbon nanofiber exposurewere not investigated [52].

Metallic nanomaterials

Metallic ENs have been manufactured in a variety ofstructural formats (rods, fibers, particles, cubes, stars, etc.),but so far, the capacity of the metallic ENs to induce


oxidative damage to DNA has only been investigated onparticulate structures. This section will cover the currentreports on cobalt, copper, gold, iron, nickel, platinum,silver, and mixed-composition particulate ENs.

Cobalt nanoparticles

Cobalt NPs (CoNPs) were shown to induce SSBs inhuman peripheral leukocytes and mouse fibroblast cellsusing the alkaline comet assay [86, 87]. Although cobaltions did not induce a significant increase in the level ofSSBs for the leukocytes [86], they caused similar butslightly fewer SSBs at similar cobalt concentrationscompared with CoNPs for mouse fibroblast cells. Theseresults indicate that the CoNP toxicity cannot be explainedsolely by toxicity of the cobalt ions. The discrepancybetween the relative toxicity of the cobalt ions betweenthese two studies likely stems from differences in the celltypes and their resistance to DNA damage given thatleukocytes were exposed to higher cobalt ion concen-trations. Although the overall toxic impact on organismsas a result of NP exposure would not change based uponthe source of those effects (ions or NPs), making thisdistinction is important. It has been postulated that theremay be nanosize toxicity effects that may stem from theunique size, high surface area, and enhanced reactivity ofNPs [88, 89]. Elucidating the extent to which NPs havethese effects is critical from a risk assessment perspective.If different effects are not observed between nanoscaleparticles and dissolved ions of the same composition, newguidelines or regulations specific to NPs are likelyunnecessary; as such, Table S1 indicates the studies thatincluded investigations of the genotoxic effects of NPsand dissolved metal ions of the same element(s). Uptakeof CoNPs by both leukocytes and fibroblasts was found tobe approximately 2 orders of magnitude larger than that ofcobalt ions [86, 87]. It is possible that CoNPs releasedsignificant quantities of cobalt ions within the cells, whichcould be the source of the observed toxicity. Futureexperiments utilizing X-ray absorption spectroscopy ortransmission electron microscopy with electron energyloss spectroscopy could potentially be utilized to deter-mine the speciation of the cobalt within the cells to gain abetter mechanistic understanding of CoNP toxicity.

Cobalt–chromium nanoparticles

The potential for cobalt–chromium (CoCr) NPs andmicroparticles to induce oxidative damage to DNA hasbeen investigated in two in vitro experiments usinghuman fibroblasts. The comparison between NPs andmicron-sized particles is important for risk assessment,because new regulations may be necessary for NPs if

these smaller particles are found to pose novel orexacerbated risks compared with larger particles of thesame chemical composition. The studies that comparedNPs with larger particles are highlighted in Table S1.CoCr NPs induced significantly higher levels of SSBs asdetermined by the alkaline comet assay at concentrationsof 3.85×10-1, 3.85, and 3.85×101mg/mL as comparedwith the microparticles after 24 h of exposure [90].Although the addition of CoCr microparticles causedsignificantly greater DNA damage than the NPs after72 h at concentrations of 3.85×10-6, 3.85×10-5, and3.85×10-1mg/mL, the DNA damage levels for the NPsat each of these concentrations was significantly less thanthat of the control, which is itself a surprising result,suggesting that the NPs may have mitigated DNA damage.This result may stem from DNA repair, given that lowermean lesion levels were observed after 3 days of exposurethan after 1 day of exposure. Overall, there was not a clearnanosize-related toxicity effect, and neither NPs normicroparticles were shown to be possess consistentlyhigher genotoxicity. Similarly, there was not a clearpattern in the relative DNA damage potential of NP andmicroparticulate CoCr for fibroblasts exposed througheither direct exposure or indirectly by adding the NPsabove an insert that contained a BeWo cell barrier [91].Interestingly, indirect exposures at concentrations of 0.08and 0.8 mg/mL for both kinds of particles inducedsignificant SSBs as detected via the alkaline comet assayand DSBs as detected via the γ-H2AX assay comparedwith the control. This result suggests that indirect effectsof NP exposure are an additional important considerationgiven that DNA damage was observed across a cellbarrier.

A partial mechanism for oxidative damage to DNA wasdeveloped and tied to the confirmed cytoplasmic uptake ofthe NPs, the subsequent intracellular corrosion of the NPs,and the final uptake of the predominant corrosion product,cobalt, but not the NPs themselves, into the nucleus [90]. Inaddition, chromium was found in the cytoplasm andnucleus, but at much lower levels. Cobalt ions (Co2+) areknown to bind to DNA, induce DNA–protein cross-linksand SSBs, and inhibit DNA repair [92, 93]. Regarding theSSBs and 8-OH-dG detected in this study, it is likely thatthe high concentration of Co2+ in the nucleus, in thepresence of H2O2, catalyzed a Fenton-like reaction andproduced •OH which attacked the DNA directly. Thesecond reported study described a mechanism by whichthe CoCr NPs generate oxidatively induced DNA damagebased not on specific ROS-inducing qualities of the NPsthemselves but on the NPs’ ability to activate intracellularsignaling pathways that indirectly lead to DNA damage[91]. The experimentally derived mechanism of oxidativelyinduced DNA damage demonstrated that NPs in contact


with a cellular wall or barrier can, just by their presence (nometal ions need cross the barrier), mediate the release ofATP. ATP can readily cross critical connexin gap junctionsin the barrier, bind to P2 receptors on cells on the other sideof the barrier, and induce oxidative damage to DNA (SSBsand DSBs). Further study of this mechanism is needed tounderstand why the presence of the CoCr NPs induces therelease of ATP.

Copper nanoparticles

In the only study on copper NPs (CuNPs), CuNPs but notreleased Cu2+ ions or copper microparticles induced SSBsas determined using the alkaline comet assay and epithelialcells at a concentration of 80 µg/mL [94]. However, thisconcentration of CuNPs was highly cytotoxic, whichsuggests that the detected DNA damage may be partly aresult of cell death and subsequent DNA fragmentation [12,15]. Nevertheless, these results for CuNPs appear torepresent a nanosize toxic effect for the CuNPs. Theauthors did not provide any information pertaining to themechanism of DNA damage induction, other than statingthat metal NPs can more easily pass through cell mem-branes compared to metal ions (i.e., Cu2+).

Gold nanoparticles

Gold is the most electronegative metal (2.54 on the Paulingscale) and a difficult metal to oxidize. By most practicalmeasures, gold is considered inert. The established view isthat gold NPs (AuNPs) should also be nontoxic (disruptiveto cellular integrity or viability) and nongenotoxic [95–100]. However, if AuNPs are sufficiently small (less than2 nm) [101] or if AuNPs can be conjugated with specificnuclear receptors [102], the particles can indeed enter thenucleus and interact directly with DNA and/or chromo-somes and induce toxicity and perhaps oxidative damage toDNA. The potential for AuNPs to induce DNA damage iscurrently unclear given the conflicting results described inthe literature. There was no increase in the level of SSBs asdetermined by the alkaline comet assay after 0.54 µgAuNPs had been administered via intratracheal installationto mice [48]. This result suggests that inhalation risks ofAuNPs at this concentration are limited. However, three invitro studies on AuNP-induced oxidative damage to DNAsuggest that AuNPs are not so harmless [103–105]. Thestudies involved the use of either citrate-stabilized orpegylated AuNPs to maintain the solubility and dispersi-bility of the NPs. Elevated levels of 8-OH-dG wereobserved in lung fibroblasts after exposure to 20-nmAuNPs at a concentration of 1 nM but not at 0.5 nM (50–100 and 25–50 µg/mL, respectively; personal communica-tion) [105]. Kang et al. [104] showed that NP size may play

an important role in the potential of NPs to induce DNAdamage. AuNPs of 100- and 200-nm size resulted inelevated levels of SSBs as determined by the alkalinecomet assay at concentrations of 25, 50, and 100 µg/mL,whereas 4- and 15-nm NPs did not have an effect at thosesame concentrations. AuNPs were also shown to induceSSBs via the alkaline comet assay with human monocytesexposed at an air–tissue interface [103]. Given thesubstantially different but important exposure conditionsused in this study, it is difficult to compare these toxicityresults with those obtained by the other studies. Overall, itappears that high AuNP concentrations on the order of50 µg/mL and higher may have the potential to induceDNA damage. One important related factor that has beenstudied to a significant extent is uptake of AuNPs by cells.Li et al. [105] indicated that their 20-nm AuNPs were invesicles clustered around the nucleus and Chithrani et al.[106] also indicated that 14-, 50-, and 74-nm AuNPs didnot enter the nucleus. The rates at which AuNPs enter andexit cells is likely an important factor and one that has beenshown to vary depending on the size and morphology ofAuNPs [106, 107]. Relating accumulation rates anddistribution within cells to toxicity results may help yielda clearer understanding of AuNP risks.

Iron nanoparticles

One study has been conducted on the potential for iron NPs(FeNPs) to induce oxidative damage to DNA [103]. FeNPswere shown to generate significantly increased levels ofSSBs in human monocytes at an air–tissue interface usingthe alkaline comet assay at concentrations of 5.1 and10.2 µg/cm2; dose-dependent effects were observed [103].The authors did not provide any information pertaining tothe mechanism of DNA damage induction.

Nickel nanoparticles

One study has been conducted on the potential for nickelNPs (NiNPs) to induce oxidative damage to DNA [108].NiNPs induced substantial damage to plasmid DNA asdetermined by agarose gel electrophoresis [108]. Noadditional information on the mechanisms of the DNAdamage was provided.

Platinum nanoparticles

In the only in vitro study on platinum NPs (PtNPs), it wassurprising that a dose–response relationship was notobserved for SSB lesions via the alkaline comet assay andthat only concentrations in the middle of the concentrationrange tested (0.1 and 1 ng/cm2) caused significant increasesin the numbers of SSBs as measured by the alkaline comet


assay [109]. This could stem from increased agglomerationat higher NP concentrations. This highlights one of the majorchallenges of nanotoxicology, namely, that NPs in solutionmay have different characteristics depending on the NPconcentration. Oxidatively modified purine base lesions werealso detected via the Fpg-modified comet assay [109].

Investigations into the mechanism for the oxidativedamage to DNA resulted in the authors finding no significantevidence of oxidative stress, i.e., no intracellular ROS, eventhough PtNPs entered the cells. On the basis of this evidenceand on preliminary evidence the report describes (but doesnot detail) demonstrating the formation of PtNP–DNAadducts, a tentative mechanism for PtNP-induced oxidativedamage to DNA appears to involve the binding of PtNPdirectly to DNA. The Fpg-modified comet assay results tendto support this possibility since it is known that the Fpg notonly repairs oxidatively induced purine lesions, but couldalso detect and repair nonbulky (e.g., methylated) purineadducts [110]. The removal of the PtNP–DNA adducts bythe action of the Fpg during the comet assay could have ledto the formation of the observed SSBs.

Silver nanoparticles

Silver NPs (AgNPs) in aqueous solutions slowly releasesilver ions (Ag+) over time and these ions may account forthe observed toxic effects [111]. Therefore, the intact AgNPand/or the released Ag+ could theoretically interact directlywith DNA if either of these species were able to cross thenuclear membrane and enter the nucleus. In fact, it hasalready been shown that Ag+ forms stable complexes withDNA at N7 of purine bases [112]. Four studies havereported AgNP-induced oxidative damage to DNA using invitro systems [103, 113–115]. Two of the studies [113, 115]reported detection of significant DSBs using the γ-H2AXassay and two studies [103, 114] reported detection ofsignificant SSBs using the alkaline comet assay. AgNPsinduced DSBs through H2AX phosphorylation even atconcentrations as low as 1 µg/mL [115]. Importantly, Kimet al. [115] also tested the toxicity of AgNO3 to assess theeffects of the silver ions, which are known to be cytotoxic.In this study the toxicity caused by AgNPs and Ag+ ionswas similar on a silver concentration basis with regard toinducing DSBs, which suggests that AgNP leaching ofsilver ions was not fully responsible for the observedtoxicity given that the NPs did not fully dissolve. Theaddition of antioxidant N-acetylcysteine prevented DSBs,thus suggesting that oxidative stress was the cause of theDNA damage. AgNPs were also shown to induce SSBs viathe alkaline comet assay with lung fibroblast (IMR-90) andhuman cancer (U251) cells at concentrations of 25 and50 µg/mL and higher, respectively [114]. The extent towhich these effects were caused by dissolution of silver

ions was not tested, and investigating such effects isstrongly encouraged for future studies. In contrast to thefour in vitro studies, the single in vivo study based on theuse of chicken embryos reported the absence of AgNP-induced oxidative damage to DNA. No significant increasesin the level of 8-OH-dG were detected via the LC/UV/ECmethod after introduction of 0.3 mL of a colloidal solutionof 50 µg/mL Ag, Ag/Cu, or Ag/Pd NPs into the embryos[116].

Three out of four in vitro studies positively confirmeduptake of the AgNPs into the cytoplasm [113–115] and twoof those confirmed the uptake of AgNPs into the nucleus[114, 115]. One study confirmed uptake of AgNPs into thecytoplasm, mitochondria, and nucleus [114] and the groupconducting this study, in addition to Kim et al. [115],performed detailed mechanistic investigations into thecause of the AgNP-induced oxidative damage to DNA.What is most interesting about this set of studies on AgNPsis the fact that significant DSB lesions were reported in halfof the studies; DSB lesions are difficult to repair and themost biologically significant DNA lesions, which suggeststhat AgNPs might possess a broad genotoxic potential. Themechanism for AgNP-induced oxidative damage to DNA,according to these reports, appears to be clearly associatedwith the presence of AgNP-induced ROS in the cytoplasm(O2

•− and H2O2) [114, 115] in combination with thepresence of structural damage to the mitochondria whichled to interruption in the mitochondrial electron transportchain and production of additional ROS [114]. The use ofAgNP dosing solutions in which all of the Ag+ had beenremoved still induced oxidative damage to DNA, indicatingthat Ag+ was not the major agent of ROS generation [114].Disruption of the mitochondrial electron transport chaininterrupts ATP synthesis and induces the formation of O2

•−.The O2

•− subsequently dismutates to H2O2, which is freelydiffusible throughout the cell and can readily pass throughthe nuclear membrane and enter the nucleus. Once in thenucleus, the H2O2 can undergo the Fenton reactioncatalyzed by Cu+ or Fe2+ ions adsorbed to DNA [117, 118].

Metalloid nanomaterials

Studies on oxidatively induced DNA damage with metalloidENs have been conducted using both the amorphous andcrystalline forms of particulate silica (SiO2) and with GeNPs.

Silica nanoparticles

Amorphous silica is an FDA-approved food additive,whereas crystalline silica is a suspected human carcinogenand is involved in the pathogenesis of silicosis [119].Previous researchers have shown that SiO2 NPs are able toenter the nucleus [120] and that these NPs do not form ROS


in either cellular or acellular systems [121]. Two recent invitro studies using amorphous SiO2 NPs [122, 123] and onein vitro study using crystalline SiO2 NPs [124] showed thatSiO2 NPs do not induce oxidative damage to DNA. On theother hand, two other in vitro studies demonstrated thatSiO2 NPs could induce oxidative damage to DNA [44, 46].For the three SiO2 NP studies in which DNA damage wasnot detected, mouse embryo fibroblast cells were exposedto a maximum NP concentration of 40 µg/mL [122], humanlung epithelial cells were exposed to a maximum NPconcentration of 500 μg/mL [123], and human B-celllymphoblastoid cells were exposed to a maximum NPconcentration of 120 µg/mL [124]. These studies allreported the absence of NP-induced oxidative DNAdamage utilizing the alkaline comet assay to test forsignificant SSB lesions [122–124]; one of these studiesalso specifically tested for oxidatively induced base lesionsusing an 8-OH-dG antibody assay and found no accumu-lation of 8-OH-dG [123]. In contrast to the previousstudies, a study based on a human carcinoma intestinal cellmodel established that SiO2 NPs could induce oxidativedamage to DNA [44]. This study utilized the Fpg-modifiedcomet assay to test for SSBs due to the accumulation ofoxidatively induced purine base lesions and found evidenceof significant SSBs, but the SSBs may have been due to theuse of NP concentrations (133.3 μg/mL) that werecytotoxic to the cells [44]. A final in vitro study reportedthe detection of significant SSBs using the alkaline cometassay [46]. This study utilized mouse embryo fibroblastsand NP concentrations of 5 and 10 µg/mL [46]. The initialmechanism of the detected DNA damage was reported tobe due to oxidative stress (glutathione depletion, superoxidedismutase inhibition, lipid peroxidation) and intracellularformation of ROS, but no other mechanistic details wereinvestigated or given. The only reported study regarding invivo effects of SiO2 NPs was performed using the aquaticorganisms Daphnia magna and Chironomus riparius. TheNPs did not induce significant SSBs as measured using thealkaline comet assay in either organism at a concentrationof 1 µg/mL [125]. The studies by Gerloff et al. [44] andYang et al. [46] did not determine the concentration atwhich effects were not observed to occur to yield lowestobserved effect concentrations, whereas Lee et al. [125] didnot determine whether damage would be observed at higherconcentrations. As such, it is impossible to assess whetherthese different results stem from the concentrations used orfrom different sensitivities of the cells or organisms to SiO2

NPs.

Germanium nanoparticles

No in vivo studies and only one in vitro cellular study havebeen conducted on GeNP-induced oxidative damage to

DNA [73]. This lone study reported the detection ofsignificant SSBs using the alkaline comet assay. Howeverthe mechanism of SSB formation was determined not to bedue to oxidative stress and the formation of ROS, but ratherdue to the binding of GeNPs directly to the DNA during thealkaline comet assay procedure. When cells were treatedwith 0.36 mg/mL of GeNPs and then immediatelyharvested, a significant increase in the numbers of SSBswas observed, but no change in DSB formation wasobserved using H2AX phosphorylation. The authors con-cluded that NP attachment directly to DNA during theassay procedure probably resulted in arbitrary DNAfragmentation. This suggests that GeNPs may cause someartifact in the alkaline comet assay. In our opinion,researchers performing nanotoxicity studies should considertesting the apparent effects immediately after NP adminis-tration to ensure that the assay itself is not confounded byan unexpected characteristic of the NP. This artifact mayalso be limited to the alkaline comet assay, which suggeststhat complementary approaches should be considered tocorroborate results if the comet assay is used.

Metal oxide nanomaterials

Metal oxide ENs exist mainly as particulate structures, butother structures do exist. This section reviews the currentmechanisms of oxidative damage to DNA induced byparticulate metal oxide structures based on aluminum,cerium, copper, iron, magnesium, titanium, zinc, and mixedmetal oxide composites.

Aluminum oxide nanoparticles

One in vitro [126] and one in vivo [127] study (using rats)have been conducted on aluminum oxide (Al2O3) NP-induced oxidative damage to DNA. Both studies reportedthe detection of significant SSBs using the alkaline cometassay. The in vitro study on Al2O3 NPs tested mouselymphoma cells and human bronchial epithelial cells withand without the addition of the S-9 metabolic activationsystem [126]. The mouse lymphoma cells showed signif-icant SSBs in the presence of S-9 at all concentrations,whereas only at 2,500 µg/mL was a significant increaseseen in the absence of S-9. However, significant SSBs weredetected at all concentrations with and without S-9 for thehuman bronchial epithelial cells. The data thus suggest thatadding S-9 increased the DNA damage to the cells, but aclear determination could not be made. In an in vivo study,rats were exposed to 500, 1,000, or 2,000 mg/kg of 30- or40-nm NPs or microparticles by oral gavage to simulaterisks as a result of ingestion exposure [127]. The 30- and40-nm NPs generally exhibited the same potential forinducing SSB lesions, with increased damage observed for


1,000 and 2,000 mg/kg after 4 and 24 h. However, nosignificant SSBs were observed after exposing the rats to500 mg/kg of the NPs or microparticles, thus suggestingthis concentration to be a threshold below which theseeffects would not be expected. Additionally, the NPs didnot induce the formation of SSBs at any of the concen-trations after 72 h. This result and the trend for decreasedDNA comet tail percentages with increased time suggestthat the rats have efficient repair enzymes to counteract theDNA damage effects of these concentrations of Al2O3 NPsor microparticles. It is also important to note that Al2O3

microparticles did not induce significant SSB lesions at anyconcentration or time point. The impact of Al3+ ions wasnot investigated nor was the rate at which dissolutionoccurred from the NPs or microparticles, which precludesdrawing a conclusion that there was a NP effect beyond NPdissolution to ions. For both of these studies, it is importantto note that the rats and cells were being exposed to veryhigh Al2O3 NPs concentrations. In comparison, Folkmannet al. [61] exposed rats to a maximum carbon nanotube orfullerene dose of 0.64 mg/kg, a concentration that is almost4 orders of magnitude less than the highest concentrationused by Balasubramanyam et al. [127]. Similarly, themouse lymphoma cells were exposed to very high NPconcentrations ranging from 0.125 to 0.5% by mass ofthe cell medium, although the bronchial epithelial cellswere exposed to concentrations 1 or 2 orders ofmagnitude smaller. In our opinion, this research high-lights the importance of determining reasonable NPexposure estimates to guide the concentration rangesused in toxicology studies. Although significant effectsmay be observed at very high concentrations, they do notnecessarily translate to these particles posing a significantrisk to humans or ecological receptors if the maximumlikely exposure concentration is several orders of magni-tude smaller. However, the lack of an effect at these highconcentrations would suggest the lack of an effect at alower concentration, unless substantial particle aggrega-tion masked the NP toxicity at the higher concentrations.And finally, neither Al2O3 NP study reported investiga-tions or experimental results related to understanding themechanisms behind the observed NP-induced damage toDNA.

Cerium oxide nanoparticles

Four studies (three in vitro and one in vivo) have beenconducted on the induction of oxidative damage to DNAvia cerium oxide (nanoceria; CeO2) NPs [125, 128–130].The genotoxicity of CeO2 NPs to cells was shown todepend on the dose. Two in vitro cellular studies reportedopposite outcomes using the alkaline comet assay: the studyby Pierscionek et al. [128] did not report significant SSBs,

whereas the study by Auffan et al. [130] reported thedetection of significant SSBs due to the redox-cyclingcapability of CeO2 NPs. Whereas Pierscionek et al. [128]did not find DNA damage to human lens epithelial cellsat 5 or 10 µg/mL, Auffan et al. [130] consistentlyobserved DNA damage to human fibroblasts at concen-trations above 60 µg/mL; Auffan et al. [130] detectedsignificant damage at 0.6 µg/mL but not at 0.006, 0.06, or6 µg/mL, thus making it difficult to determine a specificconcentration at which oxidative damage to DNA wouldnot be expected to occur. Importantly, Auffan et al. alsodiscovered that the DNA damage levels induced by CeO2

NPs and microparticles were similar when compared on asurface area basis, but that CeO2 NPs were significantlymore toxic on a mass basis. The appropriate metric thatshould be used for comparing NPs and microparticles iscurrently unclear, and in our opinion, researchers areencouraged to include both surface area and mass tofacilitate comparisons among studies. Another issuerelated to the dose metric involves whether to indicatecell exposure concentrations on a mass per volume or amass per cell dish exposure area basis. Given that NPsoften settle during the course of an experiment, Stone etal. [131] recommend using the mass per surface area asthe more relevant metric, although providing the concen-trations in both units is preferable to better enablecomparisons among studies. In the third in vitro study,human lung A549 cells showed increased oxidativedamage to DNA when they were exposed to CeO2 NPsduring a glovebox exposure setup intended to simulateexposure conditions near a CeO2 NP production facility[129]. This study utilized antibody staining for 8-OH-Gand found significant accumulation of the lesion [129].However, the mechanism for the formation of 8-OH-Gwas not investigated or reported. Nevertheless, theseresults indicate that DNA damage risks to workers shouldbe taken seriously for CeO2 NP production. In one of thevery few studies on the DNA damage risks to ecologicalorganisms, Lee et al. [125] showed that 15- and 30-nmCeO2 NPs could damage the DNA of Chironomusriparius, whereas 15-nm NPs but not 30-nm NPs damagedthe DNA of Daphnia magna; the resulting data using thealkaline comet assay showed significant formation of SSBlesions, but the mechanism of lesion formation was notinvestigated [125]. The cause for this discrepancy in theDaphnia magna data between the two different NP sizeswas not determined. But, DNA damage results correlatedwith mortality observed for the two organisms; there was astatistically significant increase in Daphnia magna mor-tality with only the 15-nm NPs and in Chironomusriparius mortality with both sizes. As such, additionalresearch is needed to elucidate the mechanisms throughwhich CeO2 NPs induce oxidative damage to DNA.


Copper oxide nanoparticles

Three studies on CuO NP-induced DNA damage in invitro cellular models have been conducted [22, 23, 94].All three studies reported detection of significant SSBlesions using the alkaline comet assay and two of thestudies specifically applied the Fpg-modified comet assayto detect significant SSBs due to the accumulation ofoxidatively modified purine lesions [22, 23]. Interestingly,all three studies noted significant oxidative damage toDNA at NP concentrations of 80 µg/mL and in eachcase the DNA damaging effects of CuO NPs werestronger than the DNA damaging effects of CuOmicron-sized particles. In all studies, CuO NPs wereconsistently and substantially more cytotoxic and in-duced significantly more oxidative damage to DNA thanadded Cu2+ ions or Cu2+ ions released from the CuO NPsthemselves. Surprisingly, the Cu2+ ions released from CuONPs were more cytotoxic than the added Cu2+ ions, butthe released Cu2+ ions did not induce significant oxidativedamage to DNA. On the basis of conclusions from all ofthe studies, the mechanisms behind the induction of DNAdamage are not clear, but it is apparent that released Cu2+

ions are not the causative factor for the in vitro oxidativestress and the resulting DNA damage. On the other hand,it is clear that CuO NPs do induce oxidative stress. Arecent study has shown that CuO NPs engage in redoxcycling, produce sustained high levels of ROS, and alterantioxidant enzyme (catalase, glutathione peroxidase)activity [132]. On the basis of this evidence, twopotential mechanisms of CuO NP-induced DNA damageemerge. Because it is known that metal ions are notgenerally effective at penetrating the cell membrane, butmetal NPs and some metal oxides such as CuO entercells at significant rates, [121, 132], it is possible thatCuO NPs enter the cell and are taken up intolysosomes. The acidic environment of the lysosomesthen causes CuO NP degradation into Cu2+ ions, whichare released into the cytoplasm and are subsequentlyreduced by O2

•− to Cu+ ions [132]. The Cu+ ions can thencatalyze the formation of •OH by reacting with H2O2; the•OH generated can then attack DNA if it is generatednear the DNA in the nucleus. The other potentialmechanism of CuO NP-induced DNA damage is basedon CuO NPs directly entering the cell and interactingwith mitochondria and inducing mitochondrial depolar-ization [23]. Mitochondrial depolarization results in lossof mitochondrial membrane potential, disruption of theelectron transport chain, and the release of ROS into thecytoplasm. Although there are no in vivo studies on CuONP-induced DNA damage, our laboratory is currentlyinvestigating the mechanisms of CuO NP-induced DNAdamage in plants using GC/MS.

Iron oxide nanoparticles

Iron oxides exist in numerous structural forms but onlythree nanoparticulate forms have been investigated for theirability to induce oxidative damage to DNA in in vitrocellular models: γ-Fe2O3 NPs (maghemite, contains Fe3+),Fe2O3 NPs (hematite, contains Fe3+), and Fe3O4 NPs(magnetite, contains both Fe2+ and Fe3+). Only one studyhas been conducted using γ-Fe2O3 NPs and the authorsreported the absence of significant SSB lesions using thealkaline comet assay [133]. This study was performed usinghuman fibroblasts at a maximum NP concentration of100 µg/mL. Three studies [22, 23, 134] have beenconducted on Fe2O3 NPs and two of the studies [22, 23]reported the absence of significant SSB lesions using boththe alkaline comet assay and the Fpg-modified comet assay.The studies that reported the absence of significant SSBlesions were conducted on human lung epithelial cells usingmaximum Fe2O3 NP concentrations of 80 µg/mL. Howev-er, the remaining Fe2O3 NP study did report the detectionof significant SSB lesions using the alkaline comet assay,but no accumulation of 8-OH-dG using ELISA [134]. Inthis study, Fe2O3 NP-induced oxidative damage to DNA inhuman diploid fibroblasts at concentrations of 50 and250 µg/mL and in human bronchial epithelial cells at250 µg/mL. These overall results suggest that the risks ofDNA damage from Fe2O3 NPs appear to be minimal exceptat very high concentrations. Thus γ-Fe2O3 NPs and Fe2O3

NPs, both of which consist of Fe3+ ions, do not appear togenerally promote oxidative damage to DNA in in vitromodels. It seems probable, on the basis of acellular ROS-induction experiments performed in the study that didreport Fe2O3 NP-induced SSB lesions, that iron oxide NPsthat consist of Fe3+ ions are unable to easily generate ROSin vitro [134]. The established mechanism of ROSproduction from iron requires the reduction of Fe3+ to Fe2+

for Fe2+ ions to catalyze the Fenton reaction and produce•OH, which can attack DNA if the •OH is near the DNA.The reduction of Fe3+ to Fe2+ is not easily achieved undernormal physiological conditions and requires specialreducing agents or a reducing environment in the cell forthe reduction to occur [132, 135]. This likely explains whyγ-Fe2O3 NPs and Fe2O3 NPs do not generally induce invitro oxidative damage to DNA. On the other hand, Fe3O4

NPs did induce the formation of SSBs in two separatestudies [22, 23]. These authors utilized the Fpg-modifiedcomet assay to detect significant SSBs due to theaccumulation of oxidatively modified purine base lesions.Lesion accumulation was only observed at high (80 µg/mL)NP concentrations. This result was not surprising becauseFe3O4 NPs contain both Fe2+ and Fe3+ ions. However, thestudies did not investigate or describe any potentialmechanisms for the observed oxidative damage to DNA.


There have been no studies investigating or reporting on thein vivo effects of any of the iron oxide NPs thus far.

Magnesium oxide nanoparticles

In the lone study on the effects of magnesium oxide (MgO)NPs, no significant increase in the numbers of SSBs wasdetected in a Caco-2 cell model utilizing both the alkalinecomet assay and the Fpg-modified comet assay [44]. Thisstudy utilized NP concentrations as high as 133.3 µg/mL.Additional research is needed using lower concentrations toyield more definitive information about the potential forMgO NPs to induce DNA damage should MgO NPsbecome widely used in consumer goods.

Titanium dioxide nanoparticles

Titanium dioxide (TiO2) NPs are semiconductors and havebeen shown to be strongly photoactive in in vitro cellmodels [136]. These NPs are perhaps the most challengingNPs to study with regard to their potential genotoxiceffects, because in addition to the typical NP challengesrelated to different sizes and aggregation states, TiO2 NPsexist in two different crystalline forms (anatase and rutile)and are photoactive. The widespread interest in the risks ofTiO2 NPs is in large part a result of the frequency withwhich they are already being used in commercial products.The estimated concentrations of these particles beingreleased into the environment are typically orders ofmagnitude larger than those for any other NPs [137, 138].

The induction of oxidative damage to DNA by TiO2 NPsis thought to stem from their inherent photoactivity andROS generation potential. The ROS generated are initiallyformed on the surface of the NPs, but when released, theROS are able to freely diffuse throughout the cellularmatrix. However, numerous in vitro studies have demon-strated oxidative damage to DNA induced by TiO2 NPsboth in the presence of UV irradiation and/or simulatedsunlight [136, 139–147] and in the absence of UVirradiation [19, 22, 23, 44, 134, 142, 143, 146, 148–150].In two studies using fish cells, TiO2 NPs in the absence ofirradiation were found to cause an increase in oxidizedpurine base lesions in one study but not in the other study[142, 144]. TiO2 NPs generated oxidatively modified DNAlesions (detected via the Fpg-modified comet assay, but notwith the Nth-modified comet assay) in the absence of UVAirradiation at concentrations of 1, 10, and 100 µg/mL usinggoldfish skin cells [142]. In the absence of either BERenzyme, significant damage was only observed at 100 µg/mL TiO2 NPs. For rainbow trout gonad cells treated withTiO2 NPs in the absence of UVA irradiation, a similarincrease in the number of oxidative lesions was notobserved with the Fpg-modified comet assay at concen-

trations of 5 and 50 µg/mL [144]. These results suggest thatTiO2 NPs in the absence of UVA irradiation may selectivelyimpact Fpg-sensitive sites, but the results are inconclusive.In the presence of UVA irradiation, significant oxidativedamage to DNA was detected under almost all conditionsfor these studies. This highlights the importance ofdetermining whether organisms exposed to TiO2 NPsaccumulate the NPs in a location (e.g., the skin) were UVlight could potentially interact with the NPs. No effectsfrom TiO2 NPs were observed in the absence of irradiationafter exposing human diploid fibroblasts, human bronchialepithelial cells, human carcinoma intestinal cells, andhuman keratinocytes using the alkaline comet assay [44,134, 143]. Conversely, TiO2 NPs were observed to induceSSBs as measured by the alkaline comet assay in theabsence of UV irradiation in human sperm and lymphocytecells and bronchial epithelial cells [19, 146]. Increases in 8-OH-dG levels were also detected for human diploid cells inthe absence of UV irradiation [134]. Different NP dosescannot explain the difference in these results given that aconcentration of 3.73 µg/mL induced SSBs in humansperm and lymphocytes [146], whereas concentration of250 µg/mL did not induce significant increases in the levelof SSBs as measured by the alkaline comet assay [134].Additionally, both of these studies used anatase TiO2 NPs,thus indicating that the type of TiO2 used cannot accountfor the difference. Although some of these differences maybe due to use of different cell lines, these results alsohighlight one of the major weaknesses of the alkaline cometassay—that it is a nonspecific assay that includes manydifferent lesions as one aggregate measurement. Having amore clear indication of the various types of lesionsaffected by nanoparticulate TiO2 could likely lead toinsights about how DNA damage occurs and why so manyof the past studies gave apparently conflicting results.

Additionally, there is one important potential artifactfor TiO2 NPs that should be considered, especially instudies utilizing the alkaline comet assay. Gerloff et al. [44]did not detect an increase in the level of SSBs when theslides for the alkaline comet assay were prepared in thedark, but they did detect significant SSBs when the slideswere prepared under normal laboratory lighting. In ouropinion, unless researchers explicitly state that all handlingsteps were conducted in the dark, results for TiO2 NPsusing the alkaline comet assay should be viewed withcaution. Even in the absence of UV irradiation, there isaccumulated evidence (detection of intracellular ROS,inhibition of intracellular ROS by ROS scavengers,activation of oxidative stress markers, etc.) from reportsthat demonstrated the formation of oxidatively inducedDNA lesions [19, 22, 23, 44, 134, 142, 146, 148–150].This may be an artifact of laboratory lighting and/or ofambient lighting for some of the studies. Nevertheless, data


from all of the reported studies indicate that there exist twoprobable mechanisms of TiO2 NP-induced oxidative damageto DNA. To derive these mechanisms, a variety of assayshave been utilized to detect and measure the presence ofsignificant SSB lesions and oxidatively modified purine baselesions, including the alkaline comet assay, Fpg-modifiedcomet assay, plasmid nicking/agarose gel electrophoresisassay, 8-OH-dG antibody ELISA and LC/EC or LC/UVmethods for 8-OH-dG [19, 22, 23, 44, 134, 136, 139–146,148–150].

The first mechanism of TiO2 NP-induced DNA damage,but not necessarily the primary mechanism, is based on thephotoactivated (via appropriately energetic light) inductionof electrons from the valence band of the TiO2 NP to theconduction band [136]. This results in the formation ofholes in the valence band and electrons in the conductionband. The hole and electron pairs can either recombine ordiffuse rapidly to the surface of the NP. At this point, twodistinct processes based on the diffusion of the holes andelectrons to the surface of the NP occur: (1) the positivelycharged holes react with (oxidize) adsorbed water orhydroxyl ions on the surface of the NP to produce •OHand (2) the electrons react with (reduce) molecular oxygento produce O2

•−. On the basis of the cellular uptake andpresence of TiO2 NPs in the cytoplasm, the short-lived •OHis unlikely to reach and enter the nucleus and attack DNA.On the other hand, O2

•− can dismutate to H2O2, which isfreely diffusible throughout the cell and can readily passthrough the nuclear membrane. Once in the nucleus, theH2O2 can undergo the Fenton reaction catalyzed by Cu+ orFe2+ ions inherently adsorbed to DNA and produce thehighly reactive •OH, which can directly attack DNA [117,118]. The second mechanism of TiO2 NP-induced oxidativedamage to DNA is also based on the photoactivation ofTiO2 NP and the production of electron–holes pairs. Thedifference is that the transition metal ion Cu2+ (orpotentially other transition metals), which is normallypresent in the cytoplasm, can be reduced to Cu+ ion bythe electron in the electron–hole pair or by the O2

•− that isformed via the reduction of molecular oxygen [139]. TheCu+ can then react with the H2O2 (or •OH) in the cytoplasmand produce copper peroxyl species which can readilydiffuse through the nuclear membrane and attack DNA. Thecrystalline form of TiO2 NPs, rutile or anatase, alsomediates which DNA damage mechanism is predominantand which type of ROS is formed. Rutile TiO2 NPs havebeen shown to produce mainly •OH and anatase TiO2 NPshave been shown to produce O2

•−, H2O2, and peroxylradicals [151]. Most recently, it was discovered thatphotoactivated TiO2 NPs generate not only •OH and O2

•−

via the mechanisms previously described, but also generatecarboxyl radical anions (CO2

•−) [152]. The CO2•− also

reacts, similarly to the electron of the photogenerated

electron–hole pairs, with adsorbed molecular oxygen onthe particle surface to generate additional O2

•−, which candismutate to H2O2 and diffuse to the nucleus.

Four in vivo studies have been conducted on TiO2 NP-induced oxidative damage to DNA [108, 125, 153, 154].Two studies reported the absence of oxidative damage toDNA [125, 153] and two studies reported the presence ofoxidative damage to DNA [108, 154]. In one study that didnot indicate DNA damage, rats were exposed to concen-trations up to 1.2 mg TiO2 per lung by intratrachealinstillation and there was no detection of significant levelsof 8-OH-dG using a polyclonal antibody staining assay andan immunohistochemical assay [153]. The second resultindicating a lack of DNA damage was from a study ofDaphnia magna and Chironomus riparius; no significantSSB lesions were detected using the alkaline comet assay[125]. However a recent study, based on the use of a mousemodel and three different assays, reported the detection ofsignificant SSB (alkaline comet assay), DSB (γ-H2AXassay), and oxidative purine base (LC/EC method for 8-OH-dG) lesions in mouse livers [154]. In this study, micewere exposed to TiO2 NPs (50 mg/kg) via oral ingestion. Theauthors concluded that the DNA damage occurred becauseof persistent inflammation combined with severe oxidativestress due to the presence of the TiO2 NPs in the mice. In thefinal study, rats were dosed (1 mg/mL) with TiO2 NPs viaintratracheal exposure and DNA strand breaks were detectedusing the plasmid nicking/agarose gel electrophoresis assay.Oxidative damage to DNA was detected, but the authorsindicated that the damage was minimal [108].

Zinc oxide nanoparticles

The induction of oxidative damage to DNA by zinc oxide(ZnO) NPs was investigated in six different in vitro cellularsystems [22, 44, 46, 146, 155, 156]. The concentration ofZnO NPs needed to cause toxicity varied among thedifferent studies, with 2 and 40 µg/mL not causing DNAdamage in one study [22], yet another study using the samecell line (A549) showed DNA damage at concentrations of10, 12, and 14 µg/mL using NPs from the samemanufacturer [155]. The discrepancy between these resultsmay stem from the exposure duration given that the cellswere exposed for either 4 h [22] or 24 h [155], respectively.These results suggest that the exposure duration may be acritical component for determining the extent to which ZnONPs could induce DNA damage in human or ecologicalexposures. Determining the elimination rates of ZnO NPs inorganisms is thus a high priority for research given thatfaster elimination causes shorter exposure durations. Allstudies on ZnO NPs used the alkaline comet assay to reportthe detection of significant SSBs. Two of the six studies[22, 44] also utilized the Fpg-modified comet assay and


reported the detection of additional SSBs due to theformation of ZnO NP-induced purine base lesions. Thesetwo studies showed that ZnO NPs can significantly increasethe level of oxidatively induced lesions when high NPconcentrations (80 μg/mL or higher) are utilized [22, 44].Even though ZnO is a semiconductor [136], ZnO NPs donot appear to behave like TiO2 NPs in the photogeneration ofROS, and Zn2+ is not a transition metal and is unlikely tocatalyze the formation of ROS via the Fenton reaction inbiological systems [46]. Two studies actually reported thedetection of intracellular ROS [46, 155], but the authors couldnot or did not determine the source/type of the ROS. Inaddition, the levels ofmarkers of oxidative stress were elevatedin four studies [44, 46, 155, 156], but none of the reportsinvestigated the mechanisms behind these findings. Eventhough excess oxidative stress appears to be a key step forthe induction of oxidative damage to DNA by ZnO NPs, theexact DNA damage mechanism has not been established.There have been no studies investigating or reporting on thein vivo effects of ZnO NPs thus far. In summary, theobserved oxidative damage to DNA at low NP concen-trations (10 μg/mL or lower) [155, 156] indicates that abetter understanding of the genotoxic risks of ZnO NPsshould be a priority.

Mixed metal oxide composite nanoparticles

No in vivo studies and only one in vitro cellular study havebeen conducted on the potential for a mixed metal oxidecomposite NP (CuZnFe2O4 NP) to induce oxidative damageto DNA [22]. This lone study reported the detection ofsignificant SSB lesions using both the alkaline comet assayand the Fpg-modified comet assay. There was not a discreteinvestigation into the mechanism of the NP-induced DNAdamage, but the study report did note that CuZnFe2O4 NP(contains Fe2+) generated significantly more DNA damagethan iron oxide NPs (Fe2O3 NPs or Fe3O4 NPs). The authorsdid not speculate, but one could extrapolate that Fe2+ ionspotentially released from CuZnFe2O4 NPs could catalyzeFenton chemistry in the nucleus and promote oxidative dam-age to DNA via the processes described earlier in this review.

Semiconductor quantum dots

Semiconductor quantum dots (Qdots) are usually composedof elements in periodic groups II–VI, III–V, or IV–VI, butso far the capacity of Qdots to induce oxidative damage toDNA has only been investigated with groups II–VI (IUPACgroups 12 and 16) elements. Determining the potentialtoxic effects of Qdots is a complex challenge as a result ofthe numerous compositions that can be synthesized withdifferent cores, shells, and surface coatings, each of whichmay impact the toxicity. Additionally, Qdots are often

synthesized using metals known to be toxic at sufficientlyhigh concentrations such as selenium and cadmium. Thus,the toxicity of weathered Qdots, which release substantialconcentrations of these ions, was found to be profoundlygreater than that of intact Qdots [157]. The long-term risksof exposure to these materials are therefore intimately relatedto environmental or biological conditions that affect Qdotstability. This section will cover the current reports on cad-mium selenide (CdSe) and cadmium telluride (CdTe) Qdots.

Cadmium selenide quantum dots

Three in vitro studies on CdSe Qdot-induced oxidativedamage to DNA have been conducted, and all three studiesutilized Qdots engineered with protective shells of zincsulfide (ZnS) [158–160]. The first study reported thedetection of significant SSB lesions induced by the CdSe–ZnS Qdots using the alkaline comet assay; however, it waseventually discovered that the Qdots were not the cause ofthe SSBs [159]. In this study, carboxylated Qdots (Qdots-COOH) but not Qdots-OH, Qdots-NH2, Qdots-OH/COOH,or Qdots-NH2/OH induced DNA damage in humanlymphoma cells [159]. These authors found that purifyingthe Qdots-COOH substantially decreased their toxicity, andthat comparable concentrations of some of the chemicalsused during the Qdots synthesis for this study causedenhanced DNA damage. The oxidative damage to DNAwas actually caused by the hydrophilic surface coating onthe Qdots. These results suggest that manufacturers may beable to substantially reduce the potential toxicity ofproducts containing Qdots by thoroughly purifying themprior to their incorporation in consumer goods. The secondstudy reported the definitive detection of CdSe–ZnS Qdot-induced oxidative damage to DNA via the plasmid nicking/agarose gel electrophoresis assay; however, nicked DNAgel bands were not identified as due to SSB or DSB lesions,but just as damaged and undamaged bands [158]. Thisstudy also investigated the effect of UV irradiation or thelack thereof on DNA damage and the authors were able toformulate some mechanistic details regarding Qdot induc-tion of DNA damage involving both photoactivated andsurface-oxide-generated ROS, but not involving releasedCd2+ (the ZnS shell inhibited the release of Cd2+ ions). Thephotoactivation of ROS was not a simple semiconductorphotoinduction of ROS as is described for TiO2 NPsbecause the ZnS shell can prevent the holes from theelectron–hole pairs from reaching the surface of the Qdotsand participating in oxidation reactions. So the only activeparticipants in the creation of ROS (in the case of Qdotswith protective shells) are photogenerated electrons whichcan reduce molecular oxygen and form O2

•−. The othermechanism put forth for the formation of ROS actuallyinvolves the ZnS protective shell and does not require


photogeneration. The ZnS shell can undergo a slow surfaceoxidative process in aqueous environments: sulfur formssulfur dioxide (SO2), which desorbs into solution and formssulfoxide radical anion (SO2

•−). In the presence of air, SO2•−

is oxidized to O2•− [161] and O2

•− dismutates to •OH,which can oxidatively damage DNA. The study reportedthe generation of ROS when the Qdots were placed in thedark and reduced levels of oxidative damage to DNA(compared with the levels observed during photoactiva-tion), but the identity of the ROS could not be ascertained.The final in vitro study also reported CdSe–ZnS Qdot-induced DNA damage under photoactivation via the use ofthe plasmid nicking/agarose gel electrophoresis assay [160].DNA strand breaks were not observed when the Qdots weretested in the dark; thus, the mechanism of DNA damage wasonce again not related to the release of Cd2+ ions. The BERenzymes Fpg and Nth were also incorporated in the experi-ments and the observation of DNA strand breaks for bothenzymes indicated that Qdots induced oxidative damage toDNA at both pyrimidine and purine base sites. A detailedmechanism for Qdot-induced oxidative damage to DNA wasgiven in the report. Initially, Qdots are photoactivated andundergo a nonradiative energy transfer to molecular oxygenwhich induces the formation of 1O2. The

1O2 then forms •OH(the mechanism for this conversion was not given by theauthors), which can attack DNA if the •OH is near the DNAin the nucleus. The authors of the study described twopossible paths of oxidative attack on DNA that •OH mighttake: (1) abstraction of a hydrogen atom from a ribosyl groupon DNA, thus creating ribosyl centered radicals—the ribose-centered radicals then cleave the ribose–phosphate backbone,which results in DNA strand breaks—and/or (2) directaddition of •OH at the C8 position of a guanine base (forexample) to produce an N7-centered guanine radical whichcan subsequently rearrange to either 8-OH-dG or FapyGuadepending on the redox microenvironment of the cell.

In summary, on the basis of the reported in vitro studies,it appears that CdSe–ZnS Qdot-induced DNA damage ismediated by either photogenerated ROS or by surface-oxide-generated ROS. Cadmium ions do not appear to havea significant role in inducing oxidative damage to DNAwhen Qdots are synthesized with a protective shell. Thusfar, there have been no studies investigating or reporting onthe in vivo effects of CdSe–ZnS Qdots related to theinduction of oxidative damage to DNA.

Cadmium telluride quantum dots

Three studies on CdTe Qdot-induced DNA damage havebeen conducted, and all three studies utilized Qdotsengineered without protective shells [48, 162, 163]. Notsurprisingly, all three studies reported detection of DNAdamage. The sole in vitro cell study utilized the alkaline

DNA precipitation assay to detect significant DNA damage(strand breaks) in rainbow trout hepatocytes that wereinduced by CdTe Qdots, but the authors did not investigatenor report any DNA damage mechanisms [163]. Theauthors studied the effects of CdTe Qdots on the hepato-cytes after the Qdots had been aged for 2 months or 2 years.Although significantly increased DNA damage was ob-served at concentrations as low as 0.4, 10, and 50 µg/mLfor Qdots aged for 2 months, DNA damage was notobserved at 2 or 250 µg/mL. This lack of a dose–responseeffect is surprising, and these results correlated with thelabile zinc/cadmium, thus suggesting that dissolution of theQdots played an important role in the observed toxicity.Results for the CdTe Qdots aged for 2 years also did notshow a dose–response effect, but these results were notcorrelated with the labile zinc/cadmium. The cause of theDNA damage potential for these aged Qdots is unclear, andfuture studies that more vigorously characterize the agedQdots are needed to unravel these effects. One in vivoecotoxicology study using the alkaline DNA precipitationassay and a freshwater mussel (Elliptio complanata) modelreported significant oxidative damage to DNA on the basisof an observed reduction in the number of DNA strandbreaks [162]. The Qdots were found to induce DNAdamage in the gills and digestive glands at low microgramper milliliter concentrations. Similar results were observedin the digestive glands but not the gills after exposure to0.5 µg/mL Cd. This suggests that these Qdots may possesssome unique toxicity to the gills. Another in vivo studyusing the alkaline comet assay and a mouse model alsoreported the detection of significant SSBs [48]. Positivelyand negatively charged CdTe Qdots induced DNA damagein mice lungs after intratracheal installation of a mass ofQdots with 63 µg Cd [48]. However neither in vivo studyinvestigated nor reported any information on DNA damagemechanisms. The DNA damage results reported for CdTeQdots without protective shells most likely were due toreleased Cd2+ ions. Cadmium(II) ions are taken up bycalcium channels in plasma membranes and Cd2+ is knownto inhibit DNA synthesis [160]. Cadmium ions are knownto induce ROS (H2O2, O2

•−, •OH) [164, 165], to interactdirectly with the major groove of DNA, and to cause DNAdamage [166, 167].

Conclusions

The following are a number of key conclusions/recommen-dations for future research on nanomaterial-induced oxida-tive damage to DNA:

1. The assays utilized to assess oxidative damage to DNAwere often not sufficiently specific to allow for athorough mechanistic understanding of how the DNA


damage was generated. The alkaline comet assay, inparticular, combines a broad range of DNA lesions intoa single measurement, yet this is the most commonlyutilized assay. For future investigations, it is recommen-ded that researchers consider using chromatography-and mass-spectrometry-based techniques, in parallelwith the alkaline comet assay, to obtain more specificand more quantitative information on individual DNAlesions. However, researchers should be aware thatchromatography-based techniques are not withoutcertain drawbacks, not least of which is the possibilityof artifactual formation/elevation the level of DNAlesions during sample preparation and analysis [168,169]. For example, molecular oxygen is often presentin buffers and solutions for preparing DNA samples foranalysis and can result in artifactual lesion formation ashas been previously demonstrated for 8-OH-dG [169].To prevent the occurrence of such lesion artifactsbefore and during chromatographic analysis, research-ers must adopt and utilize specialized sample prepara-tion procedures.

2. The extent to which the nanomaterials were character-ized in the reported studies varied greatly; some studiesonly provided characterization data obtained from themanufacturer (see Table S1). However, it is well knownthat manufacturer characterization data can be inaccu-rate and incomplete. Researchers are strongly encour-aged to characterize each nanomaterial’s physical,chemical, and electronic properties in their ownlaboratories using appropriately rigorous analyticaltechniques. Reliable nanomaterial characterization datawill promote and accelerate better interlaboratorycomparisons of genotoxicity data [3, 6, 43, 170].

3. Numerous potential experimental artifacts have beendiscussed throughout the review which can significant-ly impact the observed results. For example, GeNPsappeared to induce SSBs using the alkaline comet assayeven when the cells were harvested at time zero [73].Additionally, TiO2 NPs appeared to induce SSBs usingthe alkaline comet assay when the slides were pro-cessed in normal room light but did not induce SSBswhen the slides were processed in the dark [44]. Thereare also experimental artifacts related to synthesis by-products or environmental contaminants (i.e., leftoverheavy metal catalysts or adsorption of PAHs) promot-ing genotoxic responses in cell systems incubated withcarbon nanotubes [59]. Researchers are encouraged toexpose cells to sample filtrates after nanomaterials havebeen removed from dosing solutions to ensure that theobserved genotoxic response is due to the nanomate-rials and not due to chemicals leached from thenanomaterials. Researchers are also encouraged toassess to what extent the presence of nanomaterials in

test samples (as a consequence of incomplete nano-material removal before DNA damage analysis) influ-ences the measured genotoxic response.

4. Other researchers have noted that nanomaterials canproduce incorrect readouts in genotoxicity assays byphysically or chemically interacting with assay compo-nents [171]. For accurate determinations of in vitroDNA damage, it is essential that the cells are viablebefore, during, and after the measurement. Nonviablecells (e.g., cells undergoing apoptosis) generate frag-mented DNA and fragmented DNA is not an accuratereflection of the oxidative damage induced by thenanomaterial [12, 15].

5. For many metallic nanomaterials, it is important toassess the extent to which released ions account for theobserved genotoxic response. This would help determineto what extent the observed DNA damage stems from thenanomaterials themselves or from released ions.

6. Relatively few in vivo nanomaterial toxicity studieshave been conducted to date. Previous researchers haveobserved profoundly different toxic responses in nano-material mammalian cell studies compared with nano-material mammal studies [3, 172]. For nanomaterialgenotoxicity research, it is especially important toperform in vivo studies in order to extrapolate theobserved genotoxic responses to humans.

7. There is also a distinct lack of ecotoxicological researchin this field. Only a handful of studies have explored theextent to which nanomaterials could induce oxidativedamage to DNA in ecological receptors [125, 142, 144,162, 163], yet these organisms are expected to beexposed to significant quantities of ENs in coming years.

Acknowledgements The authors would like to thankMiral Dizdaroglu(NIST) for carefully reviewing the manuscript during its preparation andJerry Rattanong (Georgetown University) for editorial assistance.

Disclaimer Certain commercial equipment, instruments, and materi-als are identified to specify experimental procedures as completely aspossible. In no case does such identification imply a recommendationor endorsement by the NIST nor does it imply that any of thematerials, instruments, or equipment identified are necessarily the bestavailable for the purpose.

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