Research Article

Genotoxicity, Cytotoxicity, and Reactive Oxygen SpeciesInduced by Single-Walled CarbonNanotubes and C60Fullerenes in the FE1-MutaTMMouse Lung Epithelial Cells

Nicklas Raun Jacobsen,1 Giulio Pojana,2 Paul White,3 Peter M�ller,4

Corey Alexander Cohn,1 Karen Smith Korsholm,5 Ulla Vogel,1

Antonio Marcomini,2 Steffen Loft,4 and H�kanWallin1*1National Research Center for the Working Environment,

DK-2100 Copenhagen Ø, Denmark2Department of Environmental Sciences, University Ca’Foscari of Venice,

30123 Venice, Italy3Mutagenesis Section, Safe Environments Program, Healthy Environments and

Consumer Safety Branch, Health Canada, Ottawa, Canada4Department of Environmental Health, University of Copenhagen,

1014 Copenhagen K, Denmark5Department of Infectious Disease Immunology, Statens Serum Institut,

DK-2300 Copenhagen S, Denmark

Viability, cell cycle effects, genotoxicity, reactive ox-ygen species production, and mutagenicity of C60

fullerenes (C60) and single-walled carbon nanotubes(SWCNT) were assessed in the FE1-MutaTMMouselung epithelial cell line. None of these particlesinduced cell death within 24 hr at doses between 0and 200 lg/ml or during long-term subculture ex-posure (576 hr) at 100 lg/ml, as determined bytwo different assays. However, cell proliferationwas slower with SWCNT exposure and a largerfraction of the cells were in the G1 phase. Exposureto carbon black resulted in the greatest reactive ox-ygen species generation followed by SWCNT andC60 in both cellular and cell-free particle suspen-

sions. C60 and SWCNT did not increase the levelof strand breaks, but significantly increased thelevel of FPG sensitive sites/oxidized purines (22and 56%, respectively) determined by the cometassay. The mutant frequency in the cII gene wasunaffected by 576 hr of exposure to either 100lg/ml C60 or SWCNT when compared with con-trol incubations, whereas we have previouslyreported that carbon black and diesel exhaust par-ticles induce mutations using an identical exposurescenario. These results indicate that SWCNT andC60 are less genotoxic in vitro than carbon blackand diesel exhaust particles. Environ. Mol. Muta-gen. 49:476–487, 2008. VVC 2008 Wiley-Liss, Inc.

Key words: comet assay; FE1-MML; in vitro; mutation; oxidative damage

INTRODUCTION

Engineered nanomaterials are increasingly incorporated

into consumer products, but the potential for toxicity and

lack of knowledge of these products have brought nano-

toxicology to the forefront of toxicological research

[Oberdorster et al., 2005]. There is increasing concern

that exposure to some engineered nanoparticles may lead

to adverse health effects, particularly in the elderly or

people with compromised cardiovascular or respiratory

Abbreviations: BET, Brunauer, Emmett, and Teller; B[a]P, benzo[a]-pyrene; C60, fullerene C60; CB, carbon black; DCF, 20,70-dichloro-fluorescein; DCFH, 20,70-dichlorodihydrofluorescin; DCFH-DA, 20,70-dichlorodihydrofluorescein diacetate; DLS, dynamic light scattering;FE1-MML, FE1-MutaTMMouse lung; FPG, formamidopyrimidine[fapy]-DNA glycosylase; ICP-MS, inductively coupled plasma-massspectrometry; LDH, lactate dehydrogenase; ROS, reactive oxygenspecies; SWCNT, single-walled carbon nanotubes; TEM, transmissionelectron microscopy.

Grant sponsor: European Union; Grant number: FP6-012912, NEST; Grant

sponsor: The Danish Research Council; Grant number: 2052-03-0016;

Grant sponsors: Air Pollution in a Life Time Health Perspective, Aase og

Ejnar Danielsens Fond, Marie Curie Incoming International Fellowship.

*Correspondence to: Hakan Wallin, National Research Center for the

Working Environment, Lersø Parkalle 105, DK-2100 Copenhagen Ø,

Denmark. E-mail: HWA@nrcwe.dk

Received 13 October 2007; provisionally accepted 8 April 2008; and in

final form 9 April 2008

DOI 10.1002/em.20406

Published online 8 July 2008 in Wiley InterScience (www.interscience.

wiley.com).

VVC 2008Wiley-Liss, Inc.

Environmental andMolecular Mutagenesis 49:476^487 (2008)

systems [Gwinn and Vallyathan, 2006]. Single-walled car-

bon nanotubes (SWCNT) and C60 fullerenes (C60) are

expected to be among the most widely used engineered

nanomaterials, and material scientists have envisioned

their use within many applications (e.g., disease treat-

ment, electronics, composite materials) [Bosi et al., 2003;

Lam et al., 2006; Harrison and Atala, 2007]. The broad

range of applications will result in increased levels of pro-

duction, human exposure, and release in the environment.

The extent to which these materials will be accepted by

consumers and regulatory authorities will depend on their

safety.

The physicochemical properties of engineered nano-

particles differ greatly from particles with equivalent

composition but larger size. There is also a great varia-

tion in the quality and purity of the materials available

on the market. Thus, evaluating the toxicity of engi-

neered nanoparticles cannot rely on extrapolation of tox-

icity data from larger particles and comparison of

results across research laboratories can be difficult. Sev-

eral studies have assessed the in vitro and in vivo toxic-

ity of similar particles as examined in this study. Pub-

lished data on the in vitro toxicity of SWCNT are

inconsistent with the majority of the publications report-

ing a minor degree of cytotoxicity [Shvedova et al.,

2003; Cui et al., 2005; Manna et al., 2005; Fiorito et al.,

2006; Garibaldi et al., 2006; Kagan et al., 2006; Tian

et al., 2006; Worle-Knirsch et al., 2006; Davoren et al.,

2007; Kisin et al., 2007; Pulskamp et al., 2007]. For

example, in one paper, highly purified SWCNT were

reported not to be cytotoxic in either human or murine

macrophages. They were taken up to a very limited

extent by human macrophages and they did not affect

the release of nitrous oxide, a key inflammatory media-

tor, by murine macrophage cells. The authors therefore

speculated that the toxicity published in other reports

was due to the presence of graphite or metals within the

SWCNT preparations [Fiorito et al., 2006]. In addition

to a direct particle effect, epithelial cells may also be

secondarily affected by oxidative bursts induced by

phagocytic cells in tissues. The experiments carried out

here will not reflect such a secondary effect. The pub-

lished data on the toxicity of C60 in vitro are also incon-

sistent, but the majority conclude that C60 are not cyto-

toxic or genotoxic [Moussa et al., 1997; Sayes et al.,

2004; Fiorito et al., 2006; Jia et al., 2005; Sayes et al.,

2005; Isakovic et al., 2006; Mori et al., 2006; Porter

et al., 2006]. Exceptions include the observations of Isa-

kovic et al. [2006], who noted severe toxicity after 24

hr of <1 lg C60/ml in mouse fibrosarcoma, and rat and

human glioma cell lines. They also reported a rapid gen-

eration of reactive oxygen species (ROS) induced necro-

sis, which was blocked by N-acetylcysteine. C60 also

induced strand breaks in human lymphocytes DNA

[Dhawan et al., 2006].

We have recently reported that carbon black (CB)

[Jacobsen et al., 2007] and diesel exhaust particles

(SRM 1650b) [Jacobsen et al., 2008] induced mutations

at the cII loci in the FE1-MutaTMMouse lung epithelial

(FE1-MML) cell line following the same experimental

protocol as we report here. These cells contain a shuttle

vector that facilitates the analysis of mutations at the cIItransgenes. Thus the cell line, which was derived from

alveolar epithelium by spontaneous immortalization, is a

convenient tool for assessing the potential mutagenic

and other adverse effects in pulmonary tissues. The

aforementioned study of CB also focused on endpoints

such as cell viability, proliferation, genotoxicity and

mutagenicity. C60, SWCNT, and CB are all nanosized

particles that, in principle, consist of pure carbon. C60 is

a perfect spherical carbon molecule; SWCNT consists

of tubes of a single layer of graphite and CB consists of

amorphous carbon particles. Because CB is an animal

carcinogen we were concerned about the paucity of in-

formation on the mechanisms of carcinogenicity and

toxicity of carbonaceous nanoparticles. In this study, we

compared the toxicity of C60 and SWCNT to that of CB

in the FE1-MML cell line, and also examined the gener-

ation of ROS to determine the role of ROS as a possible

genotoxic mechanism.

MATERIALS ANDMETHODS

Particle

Pure C60 (99.9%) was purchased through Sigma-Aldrich (Brøndby,

Denmark) (Prod. 572500). Carbonaceous particles such as C60 consist

of agglomerates of spherical or fibrous primary particles. The declared

primary particle size of C60 was 0.7 nm. The EliCarb1 SWCNT was

purchased as a dry powder from Thomas Swan (Consett, UK). They

declared a primary particle cross-sectional diameter was 0.9–1.7 nm

and an average length was <1 lm. The CB examined (i.e., Printex 90)

was a gift from Evonik Degussa GmbH (Frankfurt, Germany). The

declared primary particle size was 14 nm.

Particle Characterization

The surface area and the pore size distribution were determined after

the materials were pretreated under high vacuum at 3008C for 2 hr. N2

isotherm adsorption/desorption and pore size were determined on an

ASAP2000 BET (Micrometrics, NH) at an adsorption temperature of

21968C. The surface area was determined by the Brunauer, Emmett,

and Teller (BET) [Brunauer et al., 1938] method and the mesopore-size

distributions were calculated from adsorption isotherms [Barret et al.,

1951] by the Halsey equation for nitrogen [Halsey, 1948].

The PAH content in Soxhlet extracts was determined by high per-

formance liquid chromatography (HPLC) with fluorescence detection

as we have described previously [Jacobsen et al., 2007]. The elemen-

tal composition was determined in raw particle material on a NA

1500 NCS elemental analyzer (Fisons Instruments, Valencia, CA), or

by digesting 10 mg of the particles in a microwave oven with 3 ml of

HNO3 and 9 ml of H2SO4 at 2508C for 60 min. The resulting solution

was evaporated to dryness and reconstituted in 25 ml demineralized

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Mutagenicity of SWCNTor C60 in FE1-MMLCell Line 477

MilliQ water, which was analyzed by inductively coupled plasma-

mass spectrometry (ICP-MS) on an Elan 6100 ICP-MS (Perkin-Elmer,

MA).

The shape and size distributions of the aqueous suspensions were

determined after 1 mg of each material (C60, SWCNT, CB) was sus-

pended in 10 ml of MilliQ water and sonicated for 1 hr at 150 W using

a 24 kHz ultrasonic processor (UP200S, Hielscher Ultrasonics GmbH,

Stuttgart, Germany) equipped with a 3-mm-diameter microtip and acous-

tic power density of 460 W/cm2. The materials were sonicated in glass

beakers placed in an ice bath with a pulse cycle of 0.7 sec on and 0.3

sec off to avoid sample heating. The dispersions were analyzed by trans-

mission electron microscopy (TEM) and dynamic light scattering (DLS)

analysis. TEM measurements were performed on a Jeol 3010 instrument

(Jeol, Tokyo, Japan) operating at 300 kV and equipped with a Gatan

slow-scan CCD camera. For each sample, a drop of the suspension was

deposited on a copper grid covered by a carbon film and the water was

allowed to evaporate at room temperature. The hydrodynamic radius,

and the particle size distribution in terms of mass, number and volume

was determined by DLS on a Nicomp 380 DLS Submicron Particle Sizer

(PSS, CA) with 1,024 correlation channels equipped with a 15 mW He-

Ne laser diode at 632.8 nm and a photodiode detector set at a 908 angle.The size distribution was also analyzed within cell media. Particle

suspensions of 100 lg/ml of either SWCNT or C60 in cell media were

generated as described below in section ‘‘Particle exposure media’’

and analyzed by DLS in a Nano Zetasizer (Malvern Instruments, UK).

Theoretically, the system allows for sizing of particles between 0.6

and 6,000 nm hydrodynamic diameter. For particle sizing, the Malvern

DLS uses a 4 mW, 633 nm He-Ne Class I Laser and measures the

back scattered light intensity at 1738 with an Avalanche photodiode

QE detector. The optical data were recorded and recalculated for high-

resolution size distribution using the Dispersion Technology Software

v. 5.0 (Malvern Instruments). Samples were measured in disposable

polystyrene cuvettes containing a minimum of 0.2 ml sample. Size dis-

tributions were calculated by the DTS software using a refractive

index of 2.02 and absorption coefficient of 2 for CB and SWCNT and

a refractive index of 1 and absorption coefficient of 0.5 for C60. Parti-

cle suspensions were analyzed unfiltered and following filtration (5,

3.1, 0.8, and 0.22 lm filters) and compared with unfiltered pure media.

Five measurements each consisting of 12–16 scans were conducted for

each filtration.

FE1MutaTMMouse Lung Epithelial Cell Line

The development and characterization of the FE1-MML epithelial cell

line established from the transgenic MutaTMMouse [Myhr, 1991] have

been described previously [White et al., 2003]. The cells were cultured

in DMEM F12 (1:1) medium (Invitrogen, Taastrup, Denmark) supple-

mented with 2% (v/v) heat inactivated fetal bovine serum (Gibco, Invi-

trogen, Taastrup, Denmark), 100 U/ml Penicillin G, 100 lg/ml strepto-

mycin (Gibco, Invitrogen), 2 mM L-glutamine (Merck, Sigma-Aldrich,

Brøndby, Denmark), and 1 ng/ml murine epidermal growth factor

(Roche, Hvidovre, Denmark).

Particle ExposureMedia

Approximately 11 mg of dry autoclaved C60 or SWCNT were soni-

cated for three rounds of 8 min in a 10-ml glass beaker on an ice-bath

after the addition of 2 ml medium before each of three sonication steps.

The sonicator (Branson Sonifier S-450D, Branson Ultrasonics, Danbury,

CT) was equipped with a disruptor horn (model no.: 101-147-037) and

operated under the following settings: each sonication round was 8 min,

10% amplitude, and alternating pulses of 10 sec on and off. The sonica-

tor was on for a total of 12 min. The sonicated suspensions were diluted

to a final concentration of 100 lg/ml and were added to the cells imme-

diately thereafter. Suspensions were prepared freshly before every

exposure.

Cytotoxicity

Cytotoxicity was tested in FE1-MML cells at 60–70% confluence

(these were single passage experiments). The cells were incubated in

duplicate with 0, 20, 40, 60, 80, 100, or 200 lg/ml of either C60 or

SWCNT in Petri dishes for 24 hr. Agglomerates were detected as precip-

itates on the cells by visual inspection and light microscopy. However,

the agglomerates appeared not to attach to the cells. Smaller agglomer-

ates, not detected by visual inspection, may be kept in suspension for

extended time and thus only slowly reach the cells. The medium was

collected and frozen at 2808C for lactate dehydrogenase (LDH) analysis

(see later) and the living and dead cells were counted using a Nucleo-

Counter (ChemoMetec A/S, Allerød, Denmark).

Cytotoxicity was also monitored in experiments in which the cell pop-

ulations were repeatedly reseeded and exposed to particles (long-term

subcultures) (see the section ‘‘FE1-MML mutagenicity analysis’’). The

materials (n 5 10) and media controls (n 5 13) were tested during all

eight exposure rounds and the media were collected and frozen at

2808C. The cytotoxicity was determined by LDH activity (Cytotoxic-

ity Detection Kit1, Roche, Hvidovre, Denmark) in thawed media as

recommended by the manufacturer (see Jacobsen et al. [2007] for more

detail). Also at each passage, 6–9 samples were randomly selected

(i.e., 2–3 for each exposure) and counted using the NucleoCounter

live/dead assay; the cell numbers were used to determine reseeding

density (Fig. 1B). Each plate was also inspected by microscope after

each exposure round.

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Fig. 1. Total cell counts following exposure of (A) 60–70% confluent

cells for 24 hr (mean of 2 6 SEM), and (B) following 1–9 rounds of 72

hr exposures for either particles (100 lg/ml) or control media (mean of

2 to 3 6 SEM).

478 Jacobsen et al.

Cell Cycle Analysis

Effects on cell cycle progression, measured as DNA content, were

determined by flow cytometry [Coligan et al., 1998]. Dishes seeded with

300,000 cells underwent one round of exposure to pure medium,

SWCNT (100 lg/ml), or C60 (100 lg/ml) and the cells were analyzed at

seeding (t 5 224 hr), immediately before exposure (t 5 0 hr), and after

24, 48, and 72 hr of exposure. The experiment was performed on two

separate occasions, and on both occasions 4–6 replicates were analyzed

for each exposure and each time point. The harvested cells were resus-

pended in sample buffer (1 g of glucose/liter of PBS), centrifuged sev-

eral times (10 min, 400g, 48C) and resuspended in fresh sample buffer.

After the last centrifugation, the supernatant was removed and the cells

were vigorously vortexed while slowly adding 1 ml of 70% ethanol at

2208C. The samples were stored at 58C until analysis. Prior to flow

cytometric analysis, the samples were centrifuged (5 min, 1,800g, 48C)and the ethanol was removed. The cells were then resuspended in 250 llstaining solution with 1,000 U RNAse A (Sigma-Aldrich, Brøndby, Den-

mark), 10 ml sample buffer, and 0.5 ml propidium iodide (Sigma-

Aldrich, Brøndby, Denmark, 1 mg/ml), and incubated in the dark on a

rocking platform for 30 min at room temperature. Within 2 hr the cells

were acquired on a BD FACSCanto. The cells were gated according to

the forward-scatter height-to-area profile, and the forward-side scatter

profile ensured that mainly single intact cells were included in the

analysis. A minimum of 10,000 cells were acquired per sample. The

degree of propidium iodide staining was used to determine the ratio of

cells in the G1/G0 (low DNA content), S (intermediate), and G2/mito-

sis (high) phase according to the Watson (Pragmatic) model using

FlowJo’s Cell Cycle Platform (Tree Star, Olten, Switzerland).

Determination of ROS Generating Ability

The effect of the materials on ROS generation was assessed both with

FE1-MML cells and in a cell-free environment. 20,70-dichlorofluoresceindiacetate (DCFH-DA) (Invitrogen) was used for detection of intracellular

ROS production. DCFH-DA is hydrolyzed within cells to nonfluorescent

20,70-dichlorodihydrofluorescin (DCFH) which is unable to leave the

cells. DCFH can be oxidized to fluorescent 20,70-dichlorofluorescein(DCF) by hydroxyl radicals, peroxynitrite, and nitric oxide [LeBel et al.,

1992; Rota et al., 1999]. The DCFH-DA was chemically hydrolyzed to

DCFH in alkaline solution for ROS determinations in cell free media as

described by LeBel et al. [1992]. Fifty thousand FE1-MML cells were

seeded in each well in a 96-well plate and were incubated for 24 hr. The

DCFH-DA solution was diluted to 10 lM in Hank’s balanced saline so-

lution (without phenol) and added to the cells. After 1 hr the media were

removed by inverting the plate on sterile gauze. The cells were exposed

for 3 hr in particle suspensions. Parallel experiments using DCFH were

performed in cell-free environment.

Particle suspensions were prepared as described above except that,

instead of cell medium, Hank’s buffered saline solution was used and

the sonicator pause was reduced to 5 sec. All dilutions were sonicated

for an additional 2 min immediately before use. The cells were incubated

either with CB (2.08, 6.25, or 18.75 lg/ml), C60, or SWCNT (2.78, 8.33,

25 lg/ml) in four replicates within one experiment. The highest of these

concentrations, 18.75 and 25 lg/ml, equal to 11.7 and 15.6 lg/cm2, were

almost identical to the particle mass of particles/surface area in the other

experiments (i.e., 11.3 and 15 lg/cm2, respectively). We also tested ROS

generation with 75 lg/ml CB and 100 lg/ml SWCNT and C60 in cell

free media. DCF was determined at kex 5 490 and kem 5 520 nm on a

fluorescence spectrophotometer (Victor Wallac-2 1420, Perkin Elmer,

Denmark). H2O2 and horseradish peroxidase were used as positive con-

trols and the results are given as H2O2 equivalents. The following equa-

tion converts fluorescence to H2O2 concentration: [H2O2] nM 5 0.0004

3 fluorescence 2 86.

DNADamage

DNA damage (strand breaks and formamidopyrimidine [fapy]-DNA

glycosylase (FPG) sites) was determined by the comet assay [Møller

et al., 2003; Jacobsen et al., 2007]. The net level of FPG sensitive sites

was obtained as the difference in score between parallel slides treated

with FPG enzyme and buffer. Data were from four sets of experiments

each containing five replicates. Five hundred thousand cells were grown

to �50% confluence, the media were changed and the cells exposed for

3 hr to control media or media containing either 100 lg/ml C60 or

SWCNT in 10 ml medium. The cells were then washed in PBS, trypsi-

nized, and resuspended in media (40% media, 50% fetal calf serum, and

10% DMSO) and frozen to 2808C in isopropanol containers. Positive

controls were A549 lung epithelial cells treated with 1 lM of photosen-

sitizer Ro19-8022 (a generous gift from Hoffmann-La Roche, Basel,

Switzerland) and irradiated for 4 min with a 500-W halogen lamp ele-

vated 33 cm above the cells according to the protocol described by

Guarnieri et al. [2008]. Upon irradiation Ro19-8022 produces singlet ox-

ygen, which induces purine modifications, i.e., 8-oxo-7,8-dihydro-20-deoxyguanosine, and only few direct strand breaks.

FE1-MML Mutagenicity Analysis

The exposure setup has been described previously [Jacobsen et al.,

2007]. Briefly, Petri dishes (Nunc, Biotech Line, Denmark) were seeded

with 300,000 cells (passage 15) in 10 ml growth medium and exposed to

either control medium (n 5 13) or 100 lg/ml of C60 or SWCNT (n 510). We estimated that the selected dose would not cause marked cyto-

toxicity even following long term exposure. The cells were incubated in

the dark at 378C and 5% CO2 with or without the test particles for a

total of eight exposure rounds, yielding a total exposure time of 576 hr

(8 3 72 hr). The cumulative dose applied was 8 mg (8 passages 3 10

ml of media 3 100 lg particles/ml). After the eighth passage the plates

were washed thoroughly with PBS to remove excess particles and the

cells were trypsinized and reseeded without the test materials. The cells

were harvested after 72 hr and stored at 2808C. As positive control we

reanalyzed DNA from cells exposed to 0.1 lg benzo[a]pyrene (B[a]P)

per ml medium for five rounds and pure medium for the remaining three

rounds [Jacobsen et al., 2007].

DNA Purification and cII Mutation Analysis

Approximately 4.9 3 106 frozen cells were suspended in lysis buffer

and the DNA purified using RecoverEase DNA isolation kits (Stratagene,

La Jolla, CA). Additionally, DNA was purified by phenol/chloroform

extraction [White et al., 2003] to ensure packaging and scoring. The

DNA preparation (8 ll) was packaged with Transpack packing extract

(Stratagene) and the phage preparation used to infect Escherichia coli

G1250 (hfl2). DNA from cells exposed to B[a]P [Jacobsen et al., 2007]

was included as a positive control. Phage with mutations that inactivate

the cII locus were identified by plaque formation under selective growth

conditions (i.e., 248C). The total number of infectious phages was deter-

mined by plaque formation under non-selective growth conditions (i.e.,

378C) according to the manufacturer’s protocol (k Select-cIITM Mutation

Detection System for the Big Blue Rodents, Stratagene). Only experi-

ments with more than 50,000 plaques were included in the analyses and

the determinations of mutant frequency were based on totals of 0.5–2

million screened plaques.

Statistics

All results were analyzed by Students’ t-test with Bonferonni correc-

tion for multiple testing because the main outcome was the statistically

significant difference between particle and unexposed cell cultures. Thus,

the statistical significance level was P < 0.025. Each Petri dish in paral-

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Mutagenicity of SWCNTor C60 in FE1-MMLCell Line 479

lel exposures was considered as one observation in the analysis of flow

cytometry data and mutant frequency data. The comet assay results were

analyzed by paired Students’ t-tests because the experimental analysis

was designed to minimize the day-to-day variation by having parallel

samples of particle exposed and unexposed samples in the same batch of

analysis. The geometric mean of strand breaks and FPG sites of five rep-

licates were considered as one observation and the experiments were

repeated for 4 days (n 5 4). The statistical analysis was performed in

Excel 2002 for Windows.

RESULTS

Particle Characterization

We determined some important physical and chemical

properties of CB, C60, and SWCNT (Table I). The surface

area of the C60 was considerably smaller than that of the

other materials. This is reflected by the fact that C60 forms

well-organized tight crystal structures. In contrast,

SWCNT forms loose hollow spaghetti-like structures and

CB forms chain-like and spherical agglomerates with large

voids. This was also reflected by the absence of pores and

the relatively larger particle size of C60 in water (Table I).

The size distribution of SWCNT in water could not be

determined by DLS or TEM due to severe agglomeration.

The SWCNT were the only particles that contained transi-

tion metals determined by ICP-MS (Table I). Despite treat-

ment in concentrated mineral acids at 2508C, only about

90% of CB and SWCNT were digested. Therefore the

exact elemental composition may be slightly underesti-

mated. We did not detect any organic contamination (i.e.,

PAHs) in C60 and this agrees with the purity declared by

the manufacturer. We detected 417 ng/g of the US Envi-

ronmental Protection Agency EPA priority PAH [Williams

et al., 2002] in SWCNT and one-fifth of this in CB. Com-

pared with particles sampled from typical combustion

processes, the particles contain little PAH.

Cell Viability and Proliferation

The viability of C60- and SWCNT-treated FE1-MML

cells was assessed following 24 hr of incubation at seven

concentrations ranging from 0 to 200 lg/ml (referred to

as ‘‘single passage experiments’’ in the ‘‘Materials and

Methods’’ section). The fraction of live cells was 98–99%

as determined by NucleoCounter, and 91–94% as deter-

mined by LDH measurement at all concentrations. We

noted that there were, on average, 12% (4–29%) fewer

cells harvested from the dishes with SWCNT when com-

pared with control or C60 exposed dishes (Fig. 1A).

We also measured the fraction of live FE1-MML cells fol-

lowing each of the eight rounds of 72-hr exposures, and fol-

lowing the final washing (referred to as ‘‘long-term subcul-

tures’’ in the ‘‘Materials and Methods’’ section). Through all

rounds, the viability was between 95 and 99% determined by

NucleoCounter, and 90–96% as determined by LDH meas-

urements (data not shown). We also noted that the number of

harvested cells was lower from the dishes treated with

SWCNT after each of the eight exposure rounds. Thus, with

SWCNT there were, on average, 45% fewer cells (2.7 mil-

lion cells harvested cells) compared with controls (4.9 mil-

Environmental and Molecular Mutagenesis. DOI 10.1002/em

TABLE I. Particle Characteristics: (A) Physical and Chemical Characterization and (B) PAH Content of CB, C60, and SWCNT

CB C60 SWCNT

A

BET surface area (m2/g) 338 6 3 <20 731 6 2

Average pore size (nm) 60 0 15

ICP-MS >99% Ca >99.9% C �95% C,a 2% Fe,

<0.001% Co, Ni, Mnb

Elemental analysis 0.82% N, 0.01% H N, H <LOD N, H <LOD

Avg. particle size in MilliQ (nm)c 30 and 410 150 and 700 ND

Avg. particle size in media (nm)d 98 and 153 311 32–60,e 280–417,e and 2,356

B

Acenaphtene <LOD <LOD 133 ng/g

Phenanthrene 18 ng/g <LOD 79 ng/g

Flouranthene 19 ng/g <LOD 87 ng/g

Fluorene <LOD <LOD 65 ng/g

Pyrene 22 ng/g <LOD 53 ng/g

Benzo[a]anthracene 4 ng/g <LOD <LOD

Chrysene 12 ng/g <LOD <LOD

For details see the ‘‘Materials and Methods’’ section.

ND: not determined; <LOD: below the limit of detection (0.3–2 ng/g).aCarbon content was determined by the suppliers (Dr. Zimmermann [Evonik DegussaGmbH] and Russel Clark [Thomas Swan&Co. Ltd.]) [Personal communica-

tion with Dr. Zimmermann (Evonik Degussa GmbH) and Russel Clark (Thomas Swan&Co. Ltd.)].bContaminants frequently detected in SWCNT.cTwomodes of particles were detected by DLS. The values are the arithmetic means of the distributions. Results were supported by TEM imaging.dData analysis revealed that C60 and SWCNT suspensions contained very few particles above detection limit (>6 lm). A 7.7 nm mode was additionally

detected in C60 and SWCNT suspensions but not in CB suspensions. This mode correlates with a peak determined for pure cell media.eSeveral different peaks were determined within the noted range dependent on the filter used.

480 Jacobsen et al.

lion cells) (Fig. 1B). Because we did not detect increased cell

death, this was likely an effect of SWCNT on cell prolifera-

tion. After the final exposure round without SWCNT, the

same cell numbers were retrieved from the SWCNT and the

control dishes. Therefore the SWCNT effect on cell prolifer-

ation seems to be reversible.

Cell Cycle Analysis

The distribution of cell populations in the cell cycle

phases were determined by evaluating the DNA content

of the cells by flow cytometry. As shown in Figure 2, the

SWCNT-treated cell populations contained significantly

fewer cells in the S/G2 phases, and more cells in G1 after

24 hr compared with controls, and this effect was even

more prominent after 48 hr. Together, these results sug-

gest that SWCNT increase the fraction of cells arrested in

G1. After 72 hr of treatment, the controls and C60

exposed cells showed a decreased rate of division due to

near confluence. However, the SWCNT-exposed cells

still had a significantly higher proportion of cells in G1

and a lower proportion in S phase and G2 phase. The

difference between control cells and C60 treated cells

was not statistically significant. Examples of flow cyto-

metric analysis of selected representative samples are

shown in Figure 3.

ROS Producing Ability

The ability of the materials to produce ROS both

within cells and in cell-free solution was determined after

3 hr by DCF fluorescence. CB caused an increasing

response with increasing mass dose, both outside and

within the cells (Fig. 4). SWCNT had a very similar

response at concentrations less than 25 lg/ml, but the

ROS generation was lower at 25 and 100 lg/ml. Because

of experimental limitations, we only tested particle con-

centrations (in lg/ml) up to a quarter of those used in the

long-term subcultures. However by mass/plate-surface

area, or mass/cell, the exposures were identical (Table II).

In cell-free medium, ROS production from C60 was

less than that from the other particles, and there was no

dose response relationship (Fig. 3). ROS production was,

however, elevated above background. We also tested

higher concentrations in a cell free environment and

found that 75 lg CB/ml produced 3.2 million fluorescent

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Fig. 2. Flow cytometric analysis of propidium iodine stained FE1-MML

cells going through one exposure round. Cells were harvested at five dif-

ferent times during the exposure. The numbers of cells in % are shown

for each of the cell cycle phases (G1, S, and G2/M). (A) Cell population

at the seeding of 300,000 cells from a near confluent flask and at expo-

sure start following 24 hr of settling. Exposed and unexposed cells were

also analyzed following (B) 24 hr, (C) 48 hr, and (D) 72 hr. Each col-

umn represents the mean and SD of two experiments with 8–12 repli-

cates. A minimum of 10,000 cells were analyzed from each sample.

Bonferonni corrected significance: *P < 0.025; **P < 0.005; ***P <

0.0001; aData set was log transformed due to unequal variance and to

fulfill the criteria for the statistical analysis.

Mutagenicity of SWCNTor C60 in FE1-MMLCell Line 481

units (1,194 nM H2O2 reaction equivalents), whereas 100

lg/ml of both SWCNT and C60 resulted in a low response

of 0.15 million (below detection limit of H2O2 reaction

equivalents).

Comet Assay

DNA damage was determined by the comet assay. As

illustrated in Figure 5, neither C60 nor SWCNT induced

strand breaks. Both particles, however, resulted in oxida-

tion of purines as detected by generation of FPG sensitive

sites. C60 exposure increased the level of FPG sensitive

sites by 1.22-fold (P 5 0.017), whereas SWCNT

increased the level by 1.56-fold (P 5 0.017). Because of

the short exposure time, it is unlikely that the observed

difference in levels of FPG sites between SWCNT- and

C60-exposed cells was caused by differences in cell cycle.

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Fig. 3. Raw data generated by flow cytometric analysis of selected rep-

resentative samples stained with propidium iodide. The samples shown

were either at seeding (A) or seeded FE1-MML cells allowed to settle

for 24 hr before exposed to control media (B), C60 (C), or SWCNT (D)

for 48 hr. The three phases G1, S, and G2 are illustrated in B. A mini-

mum of 10,000 cells were analyzed from each sample. The degree of

propidium iodide incorporation into the DNA of the cells was detected

in the phycoerythrin-area (PE-A) channel of a FACSCanto and the per-

centage of cells in the different phases were calculated using the Watson

model.

Fig. 4. ROS production measured by DCF in a cell-free environment

(filled columns) or within FE1 MutaTMMouse lung epithelial cells (open

columns) following 3 hr of incubation. The environment was stimulated

with different mass loadings of CB, SWCNT, and C60. Each bar repre-

sents the mean and SD of four replicates within one experiment. The

blank contains no test particles.

482 Jacobsen et al.

cII Mutant Frequency

The mutant frequency was slightly decreased for both

the C60 (0.92-fold; 95% CI 0.74–1.10) and SWCNT-

exposed cells (0.95-fold; 95% CI 0.65–1.25), but the

change was not statistically significant (Table III).

DISCUSSION

The production of engineered nanomaterials is rapidly

expanding. There is potential for human exposure and

environmental release in all phases of the life-cycle of

nanomaterials. We are concerned about the poorly charac-

terized toxicity of many nanoparticles and that the under-

standing of the fundamental principles of the toxicity of

insoluble particles is incomplete. In this report we have

examined the ability of SWCNT and C60 to affect cell vi-

ability, proliferation, ROS producing ability, genotoxicity,

and mutagenicity.

In the last decade many scientific papers have dealt

with the toxicity of nanoparticles, both in vitro and in

vivo. Results for SWCNT are inconsistent with the major-

ity of in vitro publications showing weak or no effects. In

this study we did not detect any effects on cell death at

concentrations up to 200 lg/ml for 24 hr, or during long-

Environmental and Molecular Mutagenesis. DOI 10.1002/em

TABLE II. Exposure Dose by Volume, Surface Area, and Cell Number

Petri dishes (66 cm2, 10 ml),

mutant frequency analysis

Petri dishes (66 cm2, 10 ml),

comet assay 96-well plates (0.32 cm2, 200 ll), ROS production

100 lg/ml 100 lg/ml 2.08 2.78 6.25 8.33 18.75 25 75 100 (lg/ml)

15 lg/cm2 15 lg/cm2 1.3 1.7 3.9 5.2 11.7 15.6 46.9 62.5 (lg/cm2)

213–3,333a lg/106 cells �500b lg/106 cells 8c 11c 25c 33c 75c 100c 300c 400c (lg/106 cells)

For example, in the comet assay a value of 100 lg/ml equals 15 lg/cm2 and 500 lg/106 cells.aValues correspond to the end (4.7 million cells) and the beginning (0.3 million cells) of the exposure, respectively.bOn the basis of 2 million cell per dish.cOn the basis of 50,000 cells per well.

Fig. 5. DNA strand breaks and FPG-sensitive sites in FE1 MutaTM

Mouse lung epithelial cells following 3 hr of incubation with either 100

lg C60 or SWCNT /ml. Values are shown as mean 6 SEM for 20

experiments conducted over 4 days. N 5 4. (*) indicates statistical sig-

nificance using paired t-tests and Bonferonni adjusted significance P <

0.025. The level of DNA damage in positive control samples

(Ro19-8022) were 11.5 6 1.8 and 178.5 6 16.9 arbitrary units (mean 6SEM) for the strand breaks and FPG-sensitive sites, respectively.

TABLE III. The Effect of C60 or SWCNT Exposure on the cIIMutant Frequency in FE1 MutaTMMouse cells

cII

Sample Pfu screened MF 3 1025 Avg. MF 6 SD

Positive control 1 486,000 151.2 151.2

Negative control 1 1,477,500 17.7 14.2 6 2.8

2 1,702,500 14.5

3 1,075,000 13.8

4 2,100,000 11.8

5 1,232,500 10.2

6 1,500,000 11.5

7 2,025,000 14.5

8 1,581,250 14.0

9 1,335,000 18.5

10 1,050,000 13.1

11 1,029,375 19.2

12 970,000 12.0

13 671,250 13.9

C60 1 1,188,750 14.1 13.1 6 3.8

2 1,055,000 13.8 P 5 0.43

3 1,800,000 5.8

4 1,187,500 13.6

5 1,147,500 12.9

6 1,900,000 11.5

7 1,545,000 11.7

8 2,335,000 15.6

9 1,162,000 20.7

10 1,766,250 11.4

SWCNT 1 1,048,750 15.8 13.5 6 4.0

2 2,297,500 12.8 P 5 0.64

3 1,737,500 13.2

4 1,180,000 20.2

5 1,955,000 14.6

6 1,056,250 8.5

7 1,456,250 13.5

8 1,247,500 8.1

9 1,503,750 18.7

10 1,222,500 9.9

MF: mutant frequency; Pfu: total number of plaque forming units

screened.

Cells for positive control were exposed to benzo[a]pyrene as described

in the ‘‘Methods and Materials’’ section. Values shown are individual

mutant frequencies as well as mean 6 SD.

Mutagenicity of SWCNTor C60 in FE1-MMLCell Line 483

term exposure to 100 lg/ml. This result is consistent with

a several published papers. For example, no effect on via-

bility of rat macrophages was observed following expo-

sure to unrefined or refined (8% and 2.5% metal, respec-

tively) SWCNT up to 100 lg/ml (62.5 lg/cm2). However,

increased production of ROS was detected in the unre-

fined SWCNT only, but this was attributed to the higher

concentrations of metals [Pulskamp et al., 2007]. Refined

(2.5% metal) and unrefined SWCNT (8% metal) were

tested at a concentration of 50 lg/ml on a human lung

epithelial cell line using several different assays without

cytotoxic response [Worle-Knirsch et al., 2006]. Davoren

et al. [2007] detected no effect of doses up to 200 lg/ml

unrefined SWCNT, containing 10% iron, in a human lung

epithelial cell line, whereas SWCNT at 400 or 800 lg/ml

was associated with cytotoxicity. A few investigators [Cui

et al., 2005; Manna et al., 2005; Tian et al., 2006] have

reported severe effects on cell viability, measured by the

MTT assay, of SWCNT on cultured mammalian cells.

However, the reliability of in vitro toxicity data with the

MTT assay has been questioned due to the apparent inter-

action of SWCNT with the assay reagent [Worle-Knirsch

et al., 2006]. Nevertheless, these studies documented other

negative effects such as increased intracellular ROS pro-

duction, increased activation of NF-jB in human kerati-

nocytes [Manna et al., 2005], decreased adhesive ability,

and induction of G1 arrest and apoptosis in human

embryo kidney cells [Cui et al., 2005]. The properties of

SWCNT differ markedly between manufacturers and even

between batches. Unfortunately, the physical and chemi-

cal properties, purity and metal content were not well

described in many reports on in vitro testing of SWCNT.

In this study we found 45% fewer cells on dishes with

SWCNT-treated cells compared with controls following

each round of exposure. We also demonstrated that cells

exposed to SWCNT grow markedly slower. We showed a

marked decline of cells in S/G2 phase and the difference

was significant at all time points (24, 48, and 72 hr). These

results suggest that SWCNT induces cell cycle arrest (G1

block). It has been reported previously that SWCNT slow

down cell proliferation. For example Cui et al. published

similar results and their detailed analysis showed a

decrease of markers associated with G1 to S transition

(e.g., cdk2, cdk4, and cyclin A, E, and D3) as well as

markers associated with S, G2, and M phase. They also

showed induction of several markers of apoptosis and an

increase in fraction of cells with less DNA than in G1-cells

(apoptotic cells). These indications of apoptosis increased

with exposure time and concentration [Cui et al., 2005].

Garibaldi et al. also reported proliferation effects on a rat

cardiac muscle cell line. Cell viability and the frequency of

apoptosis was unaffected by 3 days of culture at 200 lg/ml, but SWCNT exposed cells had a limited ability to pro-

liferate after reseeding, exhibited differences in shape, and

had a higher frequency of cell death. The cells eventually

recovered and the authors believed this to be a physical

effect rather than a chemical effect [Garibaldi et al., 2006].

The aforementioned studies examined SWCNT contain-

ing less than 10% iron and this underscores the fact that

toxic in vitro effects reported in other publications may

have been elicited by contaminants such as graphite or

metal in the SWCNT preparations. For example, it has

been reported that in vitro exposure of human epidermal

keratinocytes to SWCNT containing 30% iron resulted in

loss of viability (from 60 lg/ml, 18 hr), oxidative stress,

decrease in GSH and vitamin E, and morphological

changes [Shvedova et al., 2003]. It is likely that the toxic-

ity of SWCNT is strongly influenced by the iron content,

because redox cycling between di- and trivalent iron spe-

cies associated with particles can lead to the formation of

ROS [Cohn et al., 2006]. In this context it is especially

relevant that iron can catalyze the production of ROS

within cells and in biological fluids.

In vivo experiments with rodents treated with SWCNT

published to date appear more consistent than in vitro

studies, with four papers reporting pathological responses

including granulomas, wall thickening, and fibrotic lesions

following pulmonary exposure [Lam et al., 2004; Warheit

et al., 2004; Shvedova et al., 2005; Mangum et al., 2006].

Such effects resemble those produced by dangerous fi-

brous materials (e.g., asbestos) [Donaldson and Tran,

2004].

The literature on in vitro exposure of C60 indicates a

very different toxicity. The majority of the published

papers report little or no response in a variety of cell lines

and at doses as high as 226 lg/cm2 [Moussa et al., 1997;

Jia et al., 2005; Fiorito et al., 2006; Porter et al., 2006].

In this study we did not find a significant effect of C60 on

viability following exposures up to 30 lg/cm2 (200 lg/ml) for 24 hr or 15 lg/cm2 (100 lg/ml) for a total of 576

hr and no effect on cell cycle and proliferation. This is

consistent with the aforementioned publications. Two

groups have presented results that are in stark contrast to

these negative results. Severe toxicity (LC50 5 2–50 lg/ml), membrane damage, and lipid peroxidation has been

observed in human dermal fibroblasts, human liver carci-

noma cells, and neuronal human astrocytes [Sayes et al.,

2004, 2005]. Another group showed even higher toxicity

(LC50 � 0.5 lg/ml) toward mouse fibrosarcoma and rat

and human glioma cell lines. Seventy-five percent of the

cells died within 6 hr following exposure of mouse fibro-

sarcoma to 1 lg/ml C60. Again, it was shown that the tox-

icity likely stemmed from ROS production [Isakovic

et al., 2006]. It should be noted that both of these groups

used THF to dissolve C60 in water, whereas THF was not

used in three of the four publications showing no toxicity.

Interestingly, THF also seems to affect the toxicity of C60

in animals. None of the C60 rodent experiments used THF

and all but one of these [Tsuchiya et al., 1996] failed to

show toxicity [Nelson et al., 1993; Satoh et al., 1995;

Environmental and Molecular Mutagenesis. DOI 10.1002/em

484 Jacobsen et al.

Moussa et al., 1996; Gharbi et al., 2005; Sayes et al.,

2007; Baker et al., 2008], even following intraperitoneal

delivery of up to 2 g/kg. When C60 suspended in THF

was used to expose largemouth bass, lipid peroxidation in

the brain tissue and depletion of GSH in gills was demon-

strated [Oberdorster, 2004]. In another study, fathead min-

now and daphnia were exposed to C60 dispersed in THF

or water. The C60/THF dispersion was 40 times more

toxic to daphnia than the C60/water dispersion and it

killed all fathead minnows, whereas there was no mortal-

ity with the water dispersion sample [Zhu et al., 2006].

Recently, it has been shown that a highly toxic

oxidization product of THF (g-butyrolactone) is formed in

C60-THF suspensions which may be the cause for the

augmented toxicity of C60 [Henry et al., 2007].

Our results showed that ROS production was highest

with CB and SWCNT and far less with C60, and this

applied both within cells and in cell-free media. We

detected a decrease in ROS production at high SWCNT

concentrations. Agglomeration of SWCNT may explain

why we measured this decrease. We suspect that high

agglomeration may lead to an increased tendency for sur-

face-produced ROS to react with another part of the parti-

cle surface instead of the probe. If the decrease was due to

assay interference, similar levels of oxidized bases would

be measured by the comet assay following CB and

SWCNT exposure. However, the present results, and data

from a previous study [Jacobsen et al., 2007], indicate that

CB induces more damage to DNA, as measured by both

oxidized bases and strand breaks, than SWCNT and C60 do

at similar concentrations. Moreover, we did not observe

any induction of mutations by SWCNT or C60. The induc-

tion of FPG sites corresponds to 250 and 630 lesions per

diploid cell for the treatment with C60 and SWCNT,

respectively, using the calibration curve and assumptions

previously reported [Møller et al., 2004]. This induction in

the level of FPG sites is similar to the increases that we

have observed in mononuclear blood cells of humans fol-

lowing exposure to air pollution particles in the urban air

[Vinzents et al., 2005]. Additionally Kisin et al. recently

showed that acid treated nanotubes (0.23% Fe) did induce

significant levels of strand breaks. However, this was only

detected at cytotoxic exposure concentrations. Exposure

concentrations below cytotoxicity did not result in

increased levels of strand breaks. Moreover, Kisin et al.

[2007] were not able to detect increased mutation rates in

the Ames assay, nor did they detect increased levels of

micronucleated cells. Earlier examinations of C60 failed to

show induction of bacterial mutations in four Salmonella

strains, or an increase in the frequency of chromosomal

aberrations in cultured Chinese hamster lung cells [Mori

et al., 2006]. Nevertheless, others have shown that both

water- and ethanol-dissolved C60 were able to increase

genotoxicity in human lymphocytes as measured by the

comet assay [Dhawan et al., 2006].

The differences between the genotoxicity of SWCNT,

C60, and CB may be partly due to differences in particle

sizes. CB is the smallest particle when dispersed in water

or media (Table I) and, by ocular inspection it is readily

suspended in biological media. In media the size of C60

aggregates were 311 nm. The SWCNT also formed aggre-

gates, as determined by the presence of several stable

peaks from 32 to 2,356 nm. C60 and SWCNT suspensions

also had a small particle fraction in the micrometer range

(>6 lm). Thus, CB may have an increased ability to

enter cells and organelles. Using TEM Worle-Knirsch

et al. [2006] did find evidence of numerous bundles of

SWCNT, several hundred nm in size, located primarily

within endosomes in a human lung epithelial cell line.

However, this was not observed in another study using

the same cell line [Davoren et al., 2007]. A study by Fior-

ito et al. [2006] did show uptake of SWCNT in human

macrophages, although at a lesser rate than fullerenes and

graphite. None of these studies tested size distribution

within the media suspensions.

PAHs are the most prominent among the genotoxic

agents present in the tested particles. However, we only

detected 75 and 417 ng/g of the 16 EPA priority PAHs in

CB and SWCNT, respectively, which is very low com-

pared with levels found on combustion particles. This

suggests that PAH content has little importance on toxic-

ity. However, we are aware that the organic fraction

may contain small amounts of other more potent PAH

(e.g., nitro PAHs or hydroxy-PAHs) or other substances

not included within the EPA list. Although diesel

exhaust particles contain more than 3,000-fold more

PAH than CB, we recently reported that CB and diesel

exhaust particles are similarly potently mutagenic and

DNA damaging, and we concluded that the mechanisms

for the genotoxicity of the two particles differs [Jacobsen

et al., 2008].

In summary, we draw the following conclusions from

our study: (1) SWCNT were unable to increase cII mutant

frequency or the level of strand breaks, but did increase

the level of FPG labile sites by 1.56-fold compared with

controls; and ROS production was similar to CB at low

doses; (2) C60 did not affect the cII mutant frequency, or

the level of strand breaks, but did increase the level of

FPG labile sites by 1.22-fold, and ROS production was

only slightly elevated above background levels; and (3)

the mutagenicity and genotoxicity of the nanoparticles

correlated best with pore size, the concentration of inor-

ganic elements and, to a lesser extent, with PAH content

or surface area.

The data presented here and those from other in vitro

experiments indicate that SWCNT are not very toxic.

This is in contrast to the results from rodent experiments.

We therefore recommend that future toxicological

research on nanotubes should focus on understanding

mechanisms or toxicity within animals.

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Mutagenicity of SWCNTor C60 in FE1-MMLCell Line 485

ACKNOWLEDGMENTS

The technical assistance from Birgitte Korsholm, Keld

Alstrup Jensen (NRCWE), Stefano Zuin, and Davide Val-

lotto (University of Venice) is gratefully appreciated.

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Accepted by—M. Honma

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Mutagenicity of SWCNTor C60 in FE1-MMLCell Line 487