REVIEW Mechanisms and measurements of nanomaterial-induced oxidative 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 the environmental and human health risks of engineered nano- materials focus on their short-term acute toxicity. However, the long-term chronic effects of nanomaterials on living systems, and in particular, on the genetic components of living systems, also warrant attention. An increasing number of nanomaterial safety studies include an assess- ment of genotoxicity as part of the overall risk evaluation. The potential of nanomaterials to directly or indirectly promote the formation of reactive oxygen species is one of the primary steps in their genotoxic repertoire. The subsequent modification of genomic DNA by reactive oxygen species could lead to the development of mutagen- esis, carcinogenesis, or other age-related diseases if the DNA damage is not repaired. This review focuses on the interactions of nanomaterials with DNA and specifically on the capacity of some nanomaterials to induce oxidative damage to DNA. A critical assessment of the analytical methodology and the potential biochemical mechanisms involved in nanomaterial induction of oxidative damage to DNA is presented, results obtained for the various studies with each nanomaterial are compared, and recommenda- tions for future research are discussed. Researchers should consider, 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 enhanced understanding of DNA damage mechanisms and (2) the verification of cellular viability before conducting genotox- icity assays to reduce the impact of fragmented DNA, formed as a consequence of cell death, on DNA damage measurements. Keywords Base lesions . Comet assay . DNA damage . Engineered nanomaterials . Genotoxicity . Toxicity Introduction Engineered or manufactured nanomaterials (ENs) are particles, fibers, tubes, spheres, rods, etc., of varied compositions that contain at least one dimension that measures 100 nm or less [1]. Partly because of their reduced sizes and larger surface area to volume ratios in comparison with microscale or macroscale materials of identical composition, ENs are typically highly surface reactive (i.e., catalytic) and show enhanced physical (i.e., higher tensile strength), chemical (i.e., persistent redox cycling) and electronic (i.e., semiconducting) properties [2, 3]. Thus, there is strong scientific and commercial interest in the development and use of ENs in fields such as engineering, agriculture, electronics, and medicine. Research- and industrial-scale developments of ENs are already proceeding at a rapid pace, with many consumer products such as athletic equipment (e.g., tennis racquets), cosmetics (e.g., suntan lotions), and laundry products (e.g., fabric softeners) already containing significant amounts of ENs (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, Biochemical Science Division, National Institute of Standards and Technology, 100 Bureau Drive, Stop 8311, Gaithersburg, MD 20899-0001, USA e-mail: [email protected]Anal Bioanal Chem (2010) 398:613–650 DOI 10.1007/s00216-010-3881-7
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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 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]
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
intratrachaelinstillation
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
616 E.J. Petersen, B.C. Nelson
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
intratrachaelinstillation
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
Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA 617
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
618 E.J. Petersen, B.C. Nelson
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
intratrachaelinstillation
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
Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA 619
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
620 E.J. Petersen, B.C. Nelson
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
Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA 621
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
622 E.J. Petersen, B.C. Nelson
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
Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA 623
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
624 E.J. Petersen, B.C. Nelson
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
Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA 625
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
626 E.J. Petersen, B.C. Nelson
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
Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA 627
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
628 E.J. Petersen, B.C. Nelson
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
Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA 629
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
630 E.J. Petersen, B.C. Nelson
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
intratrachaelinstillation
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)
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ercoileddo
uble
strand
sof
DNA
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idDNA
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2µM
0.25,0.5,
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DNA
nickingoccurred
with
and
with
outUV
light,bu
tmore
occurred
with
UV
light
Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA 631
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
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n(h)
Finding
s
Hoshino
etal.[159]
ZnS
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andQD-N
H2/OH
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anlymphom
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(WTK1)
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assay
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and2µM
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Other
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Impuritiesin
thesynthesis
processalso
caused
DNA
damage
Con
centratio
nun
itsaretypically
givenin
massof
nano
particle
pervo
lumeof
medium
unless
otherw
isespecified.
8-OH-dG8-hy
drox
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uidchromatog
raph
ywith
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idineglycosylase,SW
NTsing
le-w
alledcarbon
nano
tube,
MWNTmulti-walledcarbon
nano
tube,ELISAenzyme-lin
kedim
mun
osorbent
assay,
TEM
transm
ission
electron
microscop
y
632 E.J. Petersen, B.C. Nelson
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)
Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA 633
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-
634 E.J. Petersen, B.C. Nelson
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
Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA 635
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
636 E.J. Petersen, B.C. Nelson
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
Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA 637
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
638 E.J. Petersen, B.C. Nelson
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
Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA 639
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
640 E.J. Petersen, B.C. Nelson
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.
Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA 641
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.
642 E.J. Petersen, B.C. Nelson
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
Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA 643
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
644 E.J. Petersen, B.C. Nelson
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
Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA 645
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
646 E.J. Petersen, B.C. Nelson
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.
References
1. Nel A, Xia T, Madler L, Li N (2006) Science 311:622–6272. Li N, Xia T, Nel AE (2008) Free Radic Biol Med 44:1689–16993. Warheit DB (2008) Toxicol Sci 101:183–1854. Gonzalez L, Lison D, Kirsch-Volders M (2009) Nanotoxicology
2:252–2735. Schins RPF, van Berlo D, Wessels A, Gerloff K, Boots A,
Scherbart A, Cassee FR, Gerlofs-Nijland M, Fokkens P,Schooten FJ, Albrecht C (2009) Mutagenesis 24:24
Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA 647
6. Singh N, Manshian B, Jenkins GJS, Griffiths SM, Williams PM,Maffeis TGG, Wright CJ, Doak SH (2009) Biomaterials30:3891–3914
7. Halliwell B, Gutteridge JMC (2007) Free radicals in biology andmedicine. Oxford University Press, New York
27. Olive PL (2006) Environ Mol Mutagen 11:487–49528. Olive PL, Chan APS, Cu CS (1988) Cancer Res 48:6444–644929. Dizdaroglu M, Jaruga P, Birincioglu M, Rodriguez H (2002)
Free Radic Biol Med 32:1102–111530. Voller A, Bartlett A, Bidwell DE (1978) J Clin Pathol 31:507–
52031. Burnett R, Guichard Y, Barale E (1997) Toxicology 119:83–9332. Dorsey JG, Cooper WT (1994) Anal Chem 66:857A–867A33. Greenberg MM, Hantosi Z, Wiederholt CJ, Rithner CD (2001)
Biochemistry 40:15856–1586134. Feig DI, Sowers L, Loeb LA (1994) Proc Natl Acad Sci USA
91:6609–661335. Kreutzer DA, Essigmann JM (1998) Proc Natl Acad Sci USA
95:3578–358236. Purmal AA, Kow TW, Wallace SS (1994) Nucleic Acids Res
22:3930–393537. Wallace SS (2002) Free Radic Biol Med 33:1–14
38. Delaney MO, Wiederholt CJ, Greenberg MM (2002) AngewChem Int Ed 41:771–773
39. Wiederholt CJ, Greenberg MM (2002) J Am Chem Soc124:7278–7279
40. Dizdaroglu M, Kirkali G, Jaruga P (2008) Free Radic Biol Med45:1610–1621
41. Kalam MA, Haraguchi K, Chandani S, Loechler EL, Moriya M,Greenberg MM, Basu AK (2006) Nucleic Acids Res 34:2305–2315
42. Hurt RH, Monthioux M, Kane A (2006) Carbon 44:1028–103343. Landsiedel R, Kapp MD, Schulz M, Wiench K, Oesch F (2009)
Mutat Res Rev Mutat Res 681:241–25844. Gerloff K, Albrecht C, Boots AW, Forster I, Schins RPF (2009)
K (2007) J Physiol Pharmacol 58:461–47046. Yang H, Liu C, Yang DF, Zhang HS, Xi ZG (2009) J Appl
Toxicol 29:69–7847. Zhong BZ, Whong WZ, Ong TM (1997) Mutat Res Genet
Toxicol Environ Mutagen 393:181–18748. Jacobsen NR, Moller P, Jensen KA, Vogel U, Ladefoged O, Loft
S, Wallin H (2009) Part Fibre Toxicol 6:249. Totsuka Y, Higuchi T, Imai T, Nishikawa A, Nohmi T, Kato T,
Masuda S, Kinae N, Hiyoshi K, Ogo S, Kawanishi M, Yagi T,Ichinose T, Fukumori N, Watanabe M, Sugimura T, WakabayashiK (2009) Part Fibre Toxicol 6:23
50. Gallagher J, Sams R, Inmon J, Gelein R, Elder A, Oberdorster G,Prahalad AK (2003) Toxicol Appl Pharmacol 190:224–231
51. Kisin ER, Murray AR, Keane MJ, Shi XC, Schwegler-Berry D,Gorelik O, Arepalli S, Castranova V, Wallace WE, Kagan VE,Shvedova AA (2007) J Toxicol Environ Health Part A 70:2071–2079
52. Lindberg HK, Falck GCM, Suhonen S, Vippola M, Vanhala E,Catalan J, Savolainen K, Norppa H (2009) Toxicol Lett186:166–173
53. Pacurari M, Yin XJ, Ding M, Leonard SS, Schwegler-Berry D,Ducatman BS, Chirila M, Endo M, Castranova V, Vallyathan V(2008) Nanotoxicology 2:155–170
54. Pacurari M, Yin XJ, Zhao JS, Ding M, Leonard SS, Schwegier-Berry D, Ducatman BS, Sbarra D, Hoover MD, Castranova V,Vallyathan V (2008) Environ Health Perspect 116:1211–1217
55. Zeni O, Palumbo R, Bernini R, Zeni L, Sarti M, Scarfi MR(2008) Sensors 8:488–499
56. Zhu L, Chang DW, Dai LM, Hong YL (2007) Nano Lett7:3592–3597
57. Poland CA, Duffin R, Kinloch I, Maynard A, Wallace WAH,Seaton A, Stone V, Brown S, MacNee W, Donaldson K (2008)Nat Nanotechnol 3:423–428
58. Shvedova AA, Kisin ER, Porter D, Schulte P, Kagan VE, FadeelB, Castranova V (2009) Pharmacol Ther 121:192–204
69. Petersen EJ, Huang QG, Weber WJ Jr (2010) Environ ToxicolChem 29:1106–1112
70. Petersen EJ, Huang QG, Weber WJ Jr (2008) Environ SciTechnol 42:3090–3095
71. Petersen EJ, Huang QG, Weber WJ Jr (2008) Environ HealthPerspect 116:496–500
72. Worle-Knirsch JM, Pulskamp K, Krug HF (2006) Nano Lett6:1261–1268
73. Lin MH, Hsu TS, Yang PM, Tsai MY, Perng TP, Lin LY (2009)Int J Radiat Biol 85:214–226
74. Brown DM, Kinloch IA, Bangert U, Windle AH, Walter DM,Walker GS, Scotchford CA, Donaldson K, Stone V (2007)Carbon 45:1743–1756
75. Dhawan A, Taurozzi JS, Pandey AK, Shan WQ, Miller SM,Hashsham SA, Tarabara VV (2006) Environ Sci Technol40:7394–7401
76. Jafvert CT, Kulkarni PP (2008) Environ Sci Technol 42:5945–5950
77. Henry TB, Menn FM, Fleming JT, Wilgus J, Compton RN,Sayler GS (2007) Environ Health Perspect 115:1059–1065
78. Kovochich M, Espinasse B, Auffan M, Hotze EM, Wessel L, XiaT, Nel AE, Wiesner MR (2009) Environ Sci Technol 43:6378–6384
79. Spohn P, Hirsch C, Hasler F, Bruinink A, Krug HF, Wick P(2009) Environ Pollut 157:1134–1139
80. Bernstein R, Prat F, Foote CS (1999) J Am Chem Soc 121:464–46581. Isakovic A, Markovic Z, Todorovic-Markovic B, Nikolic N,
Vranjes-Djuric S, Mirkovic M, Dramicanin M, Harhaji L, RaicevicN, Nikolic Z, Trajkovic V (2006) Toxicol Sci 91:173–183
82. Sera N, Tokiwa H, Miyata N (1996) Carcinogenesis 17:2163–2169
83. Tokuyama H, Yamago S, Nakamura E (1993) J Am Chem Soc115:7918–919
84. Nielsen GD, Roursgaard M, Jensen KA, Poulsen SS, Larsen ST(2008) Basic Clin Pharmacol Toxicol 103:197–208
85. Moreira S, Silva NB, Almeida-Lima J, Rocha HAO, MedeirosSRB, Alves C, Gama FM (2009) Toxicol Lett 189:235–241
86. Colognato R, Bonelli A, Ponti J, Farina M, Bergamaschi E,Sabbioni E, Migliore L (2008) Mutagenesis 23:377–382
87. Ponti J, Sabbioni E, Munaro B, Broggi F, Marmorato P, FranchiniF, Colognato R, Rossi F (2009) Mutagenesis 24:439–445
88. Colvin VL (2003) Nat Biotechnol 21:1166–117089. Oberdorster G, Oberdorster E, Oberdorster J (2005) Environ
Health Perspect 113:823–83990. Papageorgiou I, Brown C, Schins R, Singh S, Newson R,
Davis S, Fisher J, Ingham E, Case CP (2007) Biomaterials28:2946–2958
91. Bhabra G, Sood A, Fisher B, Cartwright L, Saunders M, EvansWH, Surprenant A, Lopez-Castejon G, Mann S, Davis SA, HailsLA, Ingham E, Verkade P, Lane J, Heesom K, Newson R, CaseCP (2009) Nat Nanotechnol 4:876–883, http://www.nature.com/nnano/journal/v4/n12/pdf/nnano.2009.313.pdf
92. Baldwin EL, Byl JA, Osheroff N (2004) Biochemistry 43:728–735
93. Kopera E, Schwerdtle T, Hartwig A, Bal W (2004) Chem ResToxicol 17:1452–1458
94. Midander M, Cronholm P, Karlsson HL, Elihn K, Moller L,Leygraf C, Wallinder IO (2009) Small 5:389–399
101. Tsoli M, Kuhn H, Brandau W, Esche H, Schmid G (2005) Small1:841–844
102. Kang B, Mackey MA, El-Sayed MA (2010) J Am Chem Soc132:1517–1519
103. Grigg J, Tellabati A, Rhead S, Almeida GM, Higgins JA,Bowman KJ, Jones GD, Howes PB (2009) Nanotoxicology3:348–345
104. Kang JS, Yum YN, Kim JH, Song H, Jeong J, Lim YT, ChungBH, Park SN (2009) Biomol Ther 17:92–97
105. Li JJ, Zou L, Hartono D, Ong CN, Bay BH, Yung LYL (2008)Adv Mater 20:138–142
106. Chithrani BD, Ghazani AA, Chan WCW (2006) Nano Lett6:662–668
107. Chithrani BD, Chan WCW (2007) Nano Lett 7:1542–1550108. Zhang Q, Kusaka Y, Sato K (1998) J Toxicol Environ Health
Part A 53:423–438109. Pelka J, Gehrke H, Esselen M, Turk M, Crone M, Brase S,
Muller T, Blank H, Send W, Zibat V, Brenner P, Schneider R,Gerthsen D, Marko D (2009) Chem Res Toxicol 22:649–659
110. Coste F, Ober M, Carell T, Boiteux S, Zelwer C, Castaing B(2004) J Biol Chem 279:44074–44083
111. Navarro E, Piccapietra F, Wagner B, Marconi F, Kaegi R, OdzakN, Sigg L, Behra R (2008) Environ Sci Technol 42:8959–8964
112. Arakawa H, Neault JF, Tajmir-Riahi HA (2001) Biophys J81:1580–1587
113. Ahamed M, Karns M, Goodson M, Rowe J, Hussain SM, SchlagerJJ, Hong YL (2008) Toxicol Appl Pharmacol 233:404–410
114. AshaRani PV, Mun GLK, Hande MP, Valiyaveettil S (2009)ACS Nano 3:279–290
115. Kim S, Choi JE, Choi J, Chung KH, Park K, Yi J, Ryu DY(2009) Toxicol In Vitro 23:1076–1084
116. Sawosz E, Grodzik M, Zielinska M, Niemiec T, Olszanka B,Chwalibog A (2009) Arch Geflugelk 73:208–213
117. Aruoma OI, Halliwell B, Gajewski E, Dizdaroglu M (1989) JBiol Chem 264:20509–20512
118. Aruoma OI, Halliwell B, Gajewski E, Dizdaroglu M (1991)Biochem J 273:601–604
119. Gwinn MR, Leonard SS, Sargent LM, Lowry DT, McKinstryKT, Meighan T, Reynolds SH, Kashon M, Castranova V,Vallyathan V (2009) J Toxicol Environ Health Part A 72:1509–1519
120. Chen M, von Mikecz A (2005) Exp Cell Res 305:51–62121. Limbach LK, Wick P, Manser P, Grass RN, Bruinink A, Start WJ
(2007) Environ Sci Technol 41:4158–4163122. Barnes CA, Elsaesser A, Arkusz J, Smok A, Palus J, Lesniak A,
Salvati A, Hanrahan JP, de Jong WH, Dziubaltowska E, StepnikM, Rydzynski K, McKerr G, Lynch I, Dawson KA, Howard CV(2008) Nano Lett 8:3069–3074
123. Jin YH, Kannan S, Wu M, Zhao JXJ (2007) Chem Res Toxicol20:1126–1133
124. Wang JJ, Sanderson BJS, Wang H (2007) Environ Mol Mutagen48:151–157
Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA 649
130. Auffan M, Rose J, Orsiere T, Meo MD, Thill A, Zeyons O,Proux O, Masion A, Chaurand P, Spalla O, Botta A, WiesnerMR, Bottero JY (2009) Nanotoxicology 3:161–171
138. Mueller NC, Nowack B (2008) Environ Sci Technol 42:4447–4453139. Hirakawa K, Mori M, Yoshida M, Oikawa S, Kawanishi S
(2004) Free Radic Res 38:439–447140. Kemp TJ,McIntyre RA (2007) Prog React Kinet Mech 32:219–229141. Nakagawa Y, Wakuri S, Sakamoto K, Tanaka K (1997) Mutat
Res 394:125–132142. Reeves JF, Davies SJ, Dodd NJF, Jha AN (2008) Mutat Res
640:113–122143. Serpone N, Salinaro AE, Horikoshi S, Hidaka H (2006) J
Photochem Photobiol A 179:200–212144. Vevers WF, Jha AN (2008) Ecotoxicology 17:410–420145. Zhu RR, Wang SL, Chao J, Shi DL, Zhang R, Sun XY, Yao SD
(2009) Mater Sci Eng C 29:691–696146. Gopalan RC, Osman IF, Amani A, Matas MD, Anderson D
Res Commun 244:198–203168. Dizdaroglu M (1998) Free Radic Res 29:551–563169. ESCODD (2003) Free Radic Biol Med 34:1089–1099170. Park H, Grassian VH (2010) Environ Toxicol Chem 29:715–
721171. Doak SH, Griffiths SM, Manshian B, Singh N, Williams
PM, Brown AP, Jenkins GJS (2009) Mutagenesis 24:285–293
172. Sayes CM, Marchione AA, Reed KL, Warheit DB (2007) NanoLett 7:2399–2406
173. Rakiman I, Chinnadurai M, Baraneedharan U, Paul SFD,Venkatachalam P (2008) Adv Biotechnol 7:39–41