Environmental and Molecular Mutagenesis 54:719^736 (2013)

Research Article

Bioassay-Directed Fractionation and Sub-Fractionation

for Mutagenicity and Chemical Analysis of Diesel
Exhaust Particles
Esra Mutlu,1,2 Sarah H.Warren,1 Peggy P. Matthews,1 Charly King,1
William P. Linak,3 Ingeborg M. Kooter,4 Judith E. Schmid,1 Jeffrey A. Ross,1
M. Ian Gilmour,1 and David M. DeMarini1*
1
National Health and Environmental Effects Research Laboratory, U.S.
Environmental Protection Agency, Research Triangle Park, North Carolina
2
Center for Environmental Medicine, Asthma and Lung Biology, University of
North Carolina, Chapel Hill, North Carolina
3
National Risk Management Research Laboratory, U.S. Environmental
Protection Agency, Research Triangle Park, North Carolina
4
Department of Environmental Health and Safety, TNO Built, Environment and
Genosciences, Utrecht, The Netherlands

Several types of diesel exhaust particles (DEPs) have
been used for toxicology studies, including a high-
organic automobile DEP (A-DEP) from Japan, and a
low-organic forklift DEP developed by the National
Institute of Standards and Technology (N-DEP). How-
ever, these DEPs were not characterized extensively
for chemical composition or sub-fractionated and
tested extensively for mutagenicity. We collected a
compressor-generated DEP (C-DEP) and character-
ized it by conducting bioassay-directed fractionation
of the extractable organics in Salmonella and corre-
lating the results by hierarchical clustering with the
concentrations of 32 polycyclic aromatic hydrocar-
bons (PAHs). Relative to A- and N-DEP, the muta-
genic potency of C-DEP was intermediate in TA100
1S9 (PAH mutagenicity) but was lowest in TA98
S9 (nitroarene mutagenicity). More than 50% of the
mass of the extractable organics of C-DEP eluted in
the nonpolar Fraction 1, and only 20% eluted in
the moderately polar Fractions 2 and 3. However,
most of the mutagenicity eluted in Fractions 2 and 3,
similar to A-DEP but different from N-DEP. HPLC-
derived mutagrams of 62 sub-fractions per fraction
confirmed that most of the mutagenicity was due to
moderately polar compounds. The diagnostic strains
identified a strong role for PAHs, nitroarenes, aro-
matic amines, and oxy-PAHs in the mutagenicity of
C-DEP. Hierarchical clustering confirmed the impor-
tance of oxy-PAHs but not that of nitroarenes. To our
knowledge this is the first use of hierarchical cluster-
ing to correlate chemical composition with the muta-
genicity of a complex mixture. The chemical analysis
and mutagenicity of C-DEP described here
makes C-DEP suitable for additional toxicological
studies. Environ. Mol. Mutagen. 54:719736,
2013. Published 2013 Wiley Periodicals, Inc.

Key words: complex mixtures; combustion emissions; Salmonella

Abbreviations: A-DEP, automobile diesel exhaust particles described in Oxy-PAHs, oxygenated polycyclic aromatic hydrocarbons; PAH(s), poly-
DeMarini et al. [2004]; C-DEP, compressor-generated diesel exhaust cyclic aromatic hydrocarbon(s)
particles; CYP1A1, an enzyme that is a member of the family of cyto-
*Correspondence to: David M. DeMarini, U.S. EPA, B105-03, RTP, NC
chrome P450s that metabolizes various xenobiotics, especially polycyclic
27711, USA.
aromatic hydrocarbons; DCM, dichloromethane; DEP(s), diesel exhaust
E-mail: demarini.david@epa.gov
particle(s); DMSO, dimethyl sulfoxide; EOM, extractable organic mate-
rial; EPA-PAHs, 16 priority PAHs identified by the U.S. Environmental Received 11 July 2013; provisionally accepted 31 July 2013; and in
Protection Agency; , the first16 PAHs listed in Table IX; HPLC, high final form 31 July 2013
performance liquid chromatography; N-DEP, NIST standard SRM 2975 DOI 10.1002/em.21812
diesel exhaust particles; Nitro-PAHs, nitroarenes; NADH (Nicotinamide
Adenine Dinucleotide plus Hydrogen); NQO1, Quinone Oxidoreductase 1; Published online 16 September 2013 in
Wiley Online Library (wileyonlinelibrary.com).

Published 2013 Wiley Periodicals, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.
Environmental and Molecular Mutagenesis. DOI 10.1002/em
720 Mutlu et al.
INTRODUCTION
Diesel exhaust particulates (DEP) are a major contribu-
tor to the particulate matter (PM) in urban air, and
chronic exposure to ambient PM is associated with
increased risks for asthma, lung cancer, and cardiovascu-
lar disease [Valavanidis et al., 2008; Lim et al., 2013].
Ambient PM is mutagenic and carcinogenic in experi-
mental systems [Claxton et al., 2004; Claxton and Wood-
all, 2007], and chronic exposure of humans to ambient air
pollution is associated with a variety of genotoxicity bio-
markers, including DNA adducts, chromosomal aberra-
tions, micronuclei, and oxidative damage to nucleobases
[Demetriou et al., 2012]. Diesel exhaust itself has been
evaluated by the International Agency for Research on
Cancer (IARC) to be a Group 1 (known) human lung car-
cinogen [Benbrahim-Tallaa et al., 2012].
DEP is comprised of a complex mixture of inorganic
and organic species. Depending on the compositions of
the fuel and lubrication oil, DEP may contain small quan-
tities (<3%) of sulfur, chlorine, and a variety of metallic
species. The remaining mass is composed of inorganic
carbon and a large variety of organic compounds. Polycy-
clic aromatic hydrocarbons (PAHs) and nitrorarenes
(nitro-PAHs) are some of the primary chemical classes in
DEP that are associated with the carcinogenicity and
mutagenicity of DEP [US EPA, 2002]. Mechanistic sup-
port for this has been shown recently by the demonstra-
tion that the human lung is able to bio-activate the
organics in DEP to produce detectable mutagenic activity
via CYP1A1 and NQO1 enzymes and that this activation
is associated with the mutagenicity of PAHs and nitro-
PAHs [Iba and Caccavale, 2013].
Although large numbers of different DEPs have been
used for toxicological research over the years, many stud-
ies of the mutagenicity of DEPs have used a standard ref-
erence material (SRM), such as SRM 2975, which was
obtained from a forklift truck and developed by the
National Institute of Standards and Technology (NIST)
[Claxton et al., 1992; Hughes et al., 1997; NIST, 2000].
Here and elsewhere [Stevens et al., 2009] we refer to this
standard as N-DEP, where the N represents NIST. In
contrast, many studies on the pulmonary toxicity of DEPs
have used an automobile-derived DEP sample (A-DEP)
[Kobayashi and Ito, 1995; Sagai et al., 1993]. Only two
studies have examined N-DEP for pulmonary effects
[Seagrave et al., 2002; Singh et al., 2004], and only one
has evaluated A-DEP for mutagenicity [DeMarini et al.,
2004]. The lack of comprehensive characterization of
these two historical DEP samples resulted in the consider-
ation for another type of DEP sample that was character-
ized chemically and toxicologically in a comprehensive
manner [Arey, 2004].
To this end, we generated a set of seven different DEP
samples using a diesel-powered compressor over a period
of time from June 2004 through September 2005 as part
of a set of studies examining the pulmonary effects of
DEP in rodents exposed by inhalation to these various
DEPs. We evaluated these DEPs for a variety of end-
points, including pulmonary inflammation and allergic
response [Ciencewicki et al., 2007; Gowdy et al., 2008;
Stevens et al., 2008, 2009], activation of pro-
inflammatory signaling in human epithelial cells [Cao
et al., 2007; Tal et al., 2008, 2010], and cardiac gene
expression [Gottipolu et al., 2009].
On the basis of these studies, as well as mutagenicity
data for these seven DEPs (D.M. DeMarini, unpublished),
we combined DEPs 3, 5, and 7 to create a single com-
pressor DEP (C-DEP). We selected these three DEPs
because of their similarity in organic carbon content and
because we had sufficient amounts of them to make a
large, 300-g sample. The resulting C-DEP had been stud-
ied for its electrophilic and redox properties [Shinyashiki
et al., 2009] and its ability to induce MMP-1 in human
bronchial epithelial cells [Li et al., 2009]. The present
report extends these studies by presenting data on
bioassay-directed fractionation of C-DEP for mutagenicity
and chemical composition.
To characterize the mutagenicity of C-DEP, we eval-
uated an organic extract of the particles for mutagenicity
in a wide variety of strains in the Salmonella mutagenic-
ity assay, and we followed this by fractionating the
extract on silica gel into four fractions of increasing
polarity; all four of these fractions were also evaluated
for mutagenicity in several strains of Salmonella. We
then sub-fractionated three of the four fractions by HPLC
using a solvent gradient of increasing polarity. We eval-
uated the 62 sub-fractions from each fraction for mutage-
nicity in three strains of Salmonella to produce
mutagrams that we compared with an HPLC chromato-
gram to identify peaks of mutagenicity that co-eluted
with various PAH standards. We analyzed the whole
extract and all four fractions for the concentrations of 32
PAHs that were pure PAHs, nitro-PAHs, or oxy-PAHs.
We then compared the chemical and mutagenicity data by
hierarchical clustering to identify those PAHs that were
associated with mutagenic activity in specific strains of
Salmonella.
MATERIALS AND METHODS
Materials and DEPs
We obtained Whatman filter paper (1001-125) from Fisher Scientific
(Pittsburgh, PA); and 0.2-mm (6874-2502) and 0.02-mm (6809-4002)
Anotop filters from GE Healthcare Biosciences, Pittsburgh, PA. We pur-
chased a Zorbax CN column (880952-705) and Zorbax CN guard col-
umn (820950-905) from Agilent Technologies, Santa Clara CA. We
obtained dichloromethane (DCM, 61005-0040), hexane (H306-1), pen-
tane (P399-4), and methanol (A452-4) from Fisher Scientific, Pittsburgh,
PA. We purchased dimethyl sulfoxide (DMSO, 154938) and silica gel

(288624, 60 A , 70230 mesh) from Sigma Aldrich, St. Louis, MO. We
procured Aroclor-induced Sprague-Dawley rat liver S9 from Moltox,
Boone, NC.
We generated C-DEP at the U.S. EPA in Research Triangle Park, NC
as described in multiple publications, including Shinyashiki et al. [2009]
where it is referred to as the DEP mix and Li et al. [2009] where it is
referred to as the DEP MIX. Briefly, we generated the various DEPs
that were combined to create C-DEP by combusting petroleum diesel in
a 30-kW (40 hp) 4-cylinder Deutz BF4M1008 diesel engine connected
to a 22.3-kW Saylor Beall 707 air compressor to provide a constant load
of 20% of the engines maximum-rated load. We attempted to main-
tain constant load and operating conditions during all seven DEP experi-
ments. However, the engine and compressor were operated outdoors
under conditions of varying temperature, humidity, and precipitation dur-
ing both summer and winter months. We chose this engine because it
was available, simple to maintain, and small enough in size to fit the
scale of our experimental facilities and exposure requirements. We chose
to operate the engine at constant load for ease of experimental reprodu-
cibility and because any choice of a load cycle for a stationary engine
would have been arbitrary. Although this engine does not include the
advanced technologies that are being used increasingly in modern
engines, it does represent an important set of legacy technologies that
are likely to remain in use for the foreseeable future.
We diluted the diesel exhaust gases 3:1 with filtered air to near ambi-
ent temperatures (35oC) and collected the DEP in a Dustex T635-9
bag house. We reverse-pulsed the Nomex felt bags periodically using
compressed air to remove the accumulated DEPs that were collected
from the hopper at the end of each day, and then we stored the collected
DEPs in sealed glass containers at 4oC. We prepared C-DEP, which was
used in the present study and in two previous studies [Li et al., 2009;
Shinyashiki et al., 2009] by mixing 100 g samples from DEPs 3, 5, and
7 in a 19-L (5 gal) plastic container using a bucket tumbler operated 25
rpm for 45 min. We dispensed the samples among a set of sterile,
brown-glass vials, flooded the vials with nitrogen gas, sealed them with
sterile caps, and stored them at 4oC.
Particle Extraction and Fractionation
We sonicated 4.63 g of particles for 45 min in 100 ml of DCM as
described by DeMarini et al. [2004] in order to extract the organics. We
filtered the resulting suspension through Whatman filter paper and fil-
tered the filtrate through a 0.2-mm Anotop filter. We sonicated insoluble
material on the filter paper again with 100 ml of DCM for 45 min and
filtered it through a 0.2-mm Anotop filter. We then combined the filtrates
and filtered them through a 0.02-mm Anotop filter; we adjusted the total
amount of extract to 150 ml of DCM. We determined the percentage of
extractable organic material (% EOM) for the DCM extract by gravimet-
ric measurement [DeMarini et al., 2004] and prepared a stock solution
of 10 mg/ml in DMSO by solvent exchange.
We fractionated 300 mg of EOM into four fractions on silica gel by
sequential elution of four solvents of increasing polarity: hexane, 75%
hexane:25% DCM, DCM, and methanol. We then determined the EOM
for each fraction and prepared stock solutions by solvent exchange at
200 mg of EOM/ml of DMSO for bioassay. This fractionation procedure
was identical to one we used previously [DeMarini et al., 2004], except
that the second fraction was eluted previously with a 50:50 mixture of
DCM and hexane.
Sub-Fractionation by Normal-Phase HPLC
We sub-fractionated fractions 2, 3, and 4 using an Agilent 1200 series
HPLC equipped with a quarternary pump and UV detector (254 nm), a
Zorbax CN column (5 mm, 4.6 x 250 mm), and a Zorbax CN guard col-
umn (5 mm, 4.6 x 12.5 mm). We used a gradient consisting of 100%
pentane (20 min, 0.5 ml/ min) followed by 100% DCM (5 min, 1 ml/
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Bioassay-Directed Fractionation of Diesel Exhaust Particles 721
min), 100% methanol (5 min, 1 ml/min) and held for 5 additional
minutes at 100% methanol. This method was similar to one we devel-
oped previously [DeMarini et al., 1992]. We collected 62 sub-fractions
(1/30 s) by an automated fraction collector. We assessed the chromato-
graphic behavior of a set of PAH standards having different polarities
and functional groups to compare the elution profiles of these standards
to the peaks of mutagenicity on the mutagrams.
PAH Analysis by Electron-Impact-Ionization Gas
Chromatography-Mass Spectrometry
We analyzed the whole extract and four fractions quantitatively for
the presence of 32 PAHs as described previously [Kooter et al., 2011].
For all but the nitro-PAHs, we analyzed the samples using GC-MS (Agi-
lent Technologies 5975C inert MSD), which was tuned in EI1 mode
using device-specific automated protocols. We performed the MS analy-
sis in selected ion-monitoring (SIM) conditions with a capillary column
AT-5ms (30 m x 0.25 mm x 0.25 lm) with a pulsed splitless injection
of 2 ll of extract. We programmed the oven as follows: 60 C for 1 min;
10 C/min; 100 C (0 min); 20 C/min; 320 C (8 min). We quantified the
PAHs by an isotope-dilution method and related the concentrations to
the added extraction standard. All 16 EPA PAHs were present in the
deuterated spiking solution; thus, we quantified each PAH relative to its
own deuterated form.
We analyzed the nitro-PAHs by GC-NCI-MS from Agilent Technolo-
gies 5975B inert XL MSD, with chemical ionization performed by meth-
ane as the reaction gas tuned with device- specific automated protocols.
We performed the MS analysis in selected ion-monitoring (SIM) condi-
tions. We used a capillary column AT-5ms (30 m x 0.25 mm x 0.25
lm) with a pulsed splitless injection of 1 ll of extract. We programmed
the oven as follows: 60 C for 1 min; 20 C/min; 320 C (8 min), and we
based the quantification of nitro-PAHs on an isotope-dilution method.
Bacterial Strains
We selected various strains for the Salmonella (Ames) mutagenicity
assay based on their sensitivity to PAHs, nitroarenes, aromatic amines,
heterocyclic amines, and compounds capable of producing oxidative
damage to DNA. The base-substitution strain TA100 [hisG46 chl-1005
(bio uvrB gal) rfa-1001 pKM1011 Fels-11 Fels-21 Gifsy-11 Gifsy-21]
[Porwollik et al., 2001] can detect PAHs in the presence of S9 [DeMar-
ini et al., 1994]; its derivative YG1042 over-expresses nitroreductase
and acetyltransferase, permitting the detection of some nitroarenes and
aromatic amines/heterocyclic amines, respectively [Hagiwara et al.,
1993]. The frameshift strain TA98 [hisD3052 chl-1008 (bio uvrB gal)
rfa-1004 pKM1011 Fels-11 Fels-21 Gifsy-11 Gifsy-21] [Porwollik
et al., 2001] detects nitroarenes and aromatic amines preferentially rela-
tive to TA100. We used derivatives of TA98 that have reduced or
enhanced abilities to detect these compounds. Thus, TA98NR has
reduced nitroreductase, which reduces the ability of this strain to detect
nitroarenes [Rosenkranz, 1981; Rosenkranz and Mermelstein, 1983].
TA98/1,8-DNP6 has reduced O-acetyltransferase, which reduces the abil-
ity of this strain to detect nitroarenes and aromatic amines/heterocyclic
amines in the presence of S9 [McCoy et al., 1983]. The other derivatives
of TA98, i.e., YG1024 [Watanabe et al., 1990], YG1021 [Watanabe
et al., 1989], and YG1041 [Hagiwara et al., 1993] over-express nitrore-
ductase and/or acetyltransferase, which enhances the ability of these
strains to detect aromatic amines/heterocyclic amines, nitroarenes, or
both, respectively.
Strain TA104 permits the recovery of mutations at both AT and GC
sites [Koch et al., 1996], whereas the other strains permit recovery of
mutations only at GC sites. Because oxidative mutagens produce a high
proportion of DNA damage at AT sites in addition to GC sites, strains
containing the allele in TA104 are sensitive to oxidative mutagens
[Levin et al., 1982]. A summary of the general functional features of
Environmental and Molecular Mutagenesis. DOI 10.1002/em
722 Mutlu et al.

TABLE I. Mutational and Enzymatic Features of Salmonella TABLE II. Interpretation of Responses in Salmonella Strains
Strains
Mutagenicity Inference
Altered enzymatic activity
Mutation TA100 1S9 > TA98 1S9 PAH-type mutagenicity because PAHs
Strain detected Nitroreductase Acetyltransferase are more mutagenic in TA100 than
in TA98 and require S9 [DeMarini
TA100 Base et al., 1994]
substitution TA98 S9 > Nitroarene-type mutagenicity because
TA98 Frameshift TA98NR S9 nitroarenes require nitroreductase for
TA98NR Frameshift # mononitroreductase mutagenicity, and some of this
TA98/1, Frameshift # activity is missing in TA98NR
8-DNP6 [Rosenkranz, 1981]
YG1024 Frameshift " TA98 S9 > Nitroarene-type mutagenicity because
YG1021 Frameshift " TA98/1,8-DNP6 S9 TA98/1,8-DNP6 is missing
YG1041 Frameshift " " acetyltransferase, which activates
YG1042 Base " " reduced nitroarenes [McCoy et al.,
substitution 1983]
TA104 Base YG1021 S9 < TA98 S9 Nitroarene-type mutagenicity because
substitution YG1021 contains additional
nitroreductase to activate nitroarenes
to mutagens [Einist o et al., 1991]
YG1024 S9 > TA98 S9 Nitroarene- and/or aromatic amine-type
these strains is shown in Table I, and a description of how we inter-
mutagenicity because YG1024 con-
preted the data from these strains is shown in Table II.
tains acetyltransferase that activates
these chemical classes to mutagens
[Einisto et al., 1991]
Mutagenicity Assays
YG1041 S9 and Greater mutagenic potencies in these
We evaluated the whole extract and four fractions for mutagenicity in YG1042 S9 strains relative to the other YG
the Salmonella mutagenicity assay by the plate-incorporation method as strains imply a role for both
described by Maron and Ames [1983]. We used S9 mix at 1 mg of S9 nitroarenes and aromatic amines/
protein/plate, and the S9 was from Aroclor-induced Sprague Dawley rat heterocyclic amines [Hagiwara et al.,
liver (Moltox, Boone, NC). We evaluated the sets of 62 sub-fractions for 1993]
mutagenicity to produce mutagrams [Lewtas et al., 1990] using a micro- TA104 Oxidative mutagenesis [Levin et al.,
suspension assay described by DeMarini et al. [1989]. Briefly, we com- 1982]
bined 50 ml of a 5X concentrate of cells grown for 16 h overnight with
50 ml of either 0.015-M sodium phosphate buffer or S9 mix in a 2-dram
vial containing 2 ml of the DMSO concentrate of sub-fraction prepared
as described above. We incubated the suspensions at 37oC for 90 min, visualize the correlation matrices by means of hierarchical clustering
placed them on ice, added 2.5 ml of molten top agar, vortexed the using Wards minimum-variance method [SAS, 2012].
samples, and then poured the contents onto minimal medium.
We included negative controls (DMSO and filter blanks) with the
experiments. In the presence of S9, the positive controls consisted of 2- RESULTS
aminoanthracene at 0.5 mg/plate for all strains. In the absence of S9, the
positive controls were 2-nitrofluorene at 3 mg/plate for TA98 and its Mutagenic Potencies of EOM and Particles
derivatives; sodium azide at 3 mg/plate for TA100 and YG1042; and
methylglyoxal at 50 mg/plate for TA104. We incubated the plates for 3 Gravimetric analysis showed that the % EOM of the
days and counted the colonies using an AccuCount 1000 colony counter organic (DCM) extract of the C-DEP was 23%. This
(Manassas, VA). means that 23% of the weight of the particles was
For the whole extract and four fractions, we performed linear regres- extractable by DCM, and it was this organic material that
sions over the linear portion of the dose-response curves to determine was subjected to mutagenicity and chemical analysis. For
the mutagenic potencies, which were expressed as revertants (rev)/mg
EOM. We defined a positive mutagenic response as a reproducible,
comparison, our previous study determined that the
dose-related response with a twofold or greater increase in revertants rel- N-DEP and A-DEP samples have EOM values of 2% and
ative to the solvent control. We performed all experiments at one plate/ 26%, respectively [DeMarini et al., 2004]. Stevens et al.
dose, and we tested all samples in two independent experiments except [2009] reported a somewhat higher % EOM for the
for some doses as noted in the data tables. A-DEP sample (69%) but had comparable values for N-
DEP (2%) and a similar C-DEP sample (19%). None of
Statistical Analyses the filter blanks was mutagenic in any of the strains (data
not shown).
We calculated Pearson product-moment correlations and Spearman
The dose-response data of the mutagenic activity of the
rank correlations in SAS v.9.3 software using the Corr procedure [SAS,
2011]. We calculated the correlations across the whole extract and four
EOM in nine strains of Salmonella are shown in Table
fractions for each pair of strains, each pair of extract/fractions, and each III, and the mutagenic potencies of the EOM and the par-
strain-extract/fraction combination. We then used SAS/JMP software to ticles (based on the 23% EOM) are shown in Table IV
 Environmental and Molecular Mutagenesis. DOI 10.1002/em
Bioassay-Directed Fractionation of Diesel Exhaust Particles 723

TABLE III. Mutagenicity of Whole Organic Extract of C-DEP TABLE IV. Mutagenic Potencies of EOM and Particles of
in Salmonella C-DEP
Rev/platea Rev/mg EOMa Rev/mg particleb
Strain EOM/plate (mg) 1S9 2S9 Strain 1S9 2S9 1S9 2S9

TA100 0 121 116 TA100 17.9 13.1 4.1 3.0

5 224 210 TA98 3.0 7.5 0.7 1.7
10 322 253
TA98NR 4.7 1.1
25 647 486
50 1009 773 TA98/1,8DNP6 3.7 0.9
100 1453b 1115b YG1021 17.1 29.3 3.9 6.7
TA98 0 39 34 YG1024 7.7 29.2 1.8 6.7
5 57 64 YG1041 18.2 67.9 4.2 15.6
10 66 95 YG1042 13.3 17.7 3.1 4.1
25 115 224 TA104 4.8 10.8 1.1 2.5
50 186 403
100 344 587b a
Data are the slopes of linear regressions calculated from the data in
TA98NR 0 45
5 72 Table I.
b
10 107 These calculated values are 23% of the rev/lg EOM because the
25 166 %EOM was 23%.
50 312
100 516
TA98/1,8DNP6 0 29
5 59
10 67
25 123
50 228
100 402
YG1021 0 41 31
5 122 167
10 197 324
25 521 840
50 878 1477
100 1502b 1966b
YG1024 0 44 21
2.5 65 99
5 71 131
10 130 331
25 234 743
YG1041 0 78 55
5 134 442
10 222 784
25 495 1768
50 975 2275b
100 1640b 2542b
YG1042 0 98 77
5 205 216
10 270 254 Fig. 1. Mutagenic potency of particles of C-DEP in various strains of
25 443 280b Salmonella; data are from Table IV.
50 672b 383b
100 743b 471b
TA104 0 249 230
5 287 254
and Figure 1. With the exception of only strain TA100,
10 315 327 the addition of S9 reduced the mutagenic potency of C-
25 397 490 DEP (Fig. 1). This implies that with the exception of
50 493 659b
100 643b 842b TA100, nitroarenes, which are direct-acting mutagens,
play an important role in the general mutagenicity of C-
a
Data are the average of two independent experiments, each having one DEP. The enhanced mutagenic potency of C-DEP in
plate per dose; thus, the data are the average of two plates per dose.
Positive control data (average rev/plate, range) are 2-aminoanthracene
TA100 upon the addition of S9 indicates the presence of
(1S9): TA100 (797, 3901300), TA98 (378, 351420), YG1021 (549, PAHs because this strain responds to PAHs, which
480637), YG1024 (1779, 15011935), YG1041 (1311, 11911389), require S9 for activation to mutagens and produce primar-
YG1042 (581, 484659), TA104 (883, 6551101); sodium azide (-S9): ily base substitutions, which is the class of mutation
TA100 (807, 728893), YG1042 (1033, 8781134); 2-nitrofluorene (- detected by TA100.
S9): TA98 (406, 309495), TA98NR (99, 80110), TA98/1,8DNP6 (68,
51105), YG1021 (2554, 24092667), YG1024 (2393, 22202600),
In the absence of S9 the particles were most mutagenic
YG1041 (2100, 19192211); and methylglyoxal (-S9): TA104 (514, in strain YG1041, which detects frameshift mutations and
460608). activates aromatic amines, heterocyclic amines, and nitro-
b
These data were not used in the linear regressions because they were arenes to mutagens. The other YG strains activate either
outside of the linear portion of the dose-response curves. the nitroarenes (YG1021) or amines (YG1024) or detect
Environmental and Molecular Mutagenesis. DOI 10.1002/em
724 Mutlu et al.
Fig. 2. Distribution of the mass of C-DEP organics across the four frac-
tions based on the mass of organics loaded onto the silica gel column and
the mass that eluted in each fraction.
base-substitution mutations (YG1042) (Table I). Interest-
ingly, the combined mutagenic potency of the particles in
YG1021, which activates the nitroarenes, and YG1024,
which activates the amines, equals that of YG1041, which
activates both (Fig. 1). The enhanced mutagenic potency
of the particles in the frameshift YG strains relative to
strain TA98 indicates that aromatic amines/heterocyclic
amines and nitroarenes are present in the organic extract
and account for some portion of the mutagenic activity of
the particles.
The higher mutagenic potency of the particles in strain
TA104 in the absence of S9 relative to the presence of
S9 suggests the presence of mutagens that cause oxidative
damage, i.e., DNA damage and consequently mutations at
AT rather than GC sites (Tables II and IV). The higher
mutagenic potency of the particles in TA100 in the
presence of S9 relative to the absence of S9 suggests
the presence of PAHs (Tables II and IV). The 3547%
reduction in mutagenic potency of the particles in strains
TA98NR and TA98/1,8-DNP6 relative to TA98 indicates
that nitroarenes and/or aromatic amines/heterocyclic
amines account for some portion of the mutagenicity of
the particles (Table IV, Fig. 1). Thus, the responses of the
particles in the various diagnostic strains used here permit
us to infer a role for PAHs, nitroarenes, aromatic/hetero-
cyclic amines, and oxidative compounds in the mutage-
nicity of C-DEP.
Mutagenic Potencies of Fractions of EOM
The distribution of the mass of the EOM across the
four fractions is shown in Figure 2, which illustrates that
50% of the mass eluted in the nonpolar hexane fraction.
Much of the remaining mass (28%) eluted in the highly
polar methanol fraction, with only 20% eluting in the
moderately polar middle two fractions (Fig. 2). The dose-
response data of the mutagenic activities of these four
fractions of EOM are shown in Table V, and the muta-
genic potencies of the fractions are shown in Table VI.
Although 50% of the mass of the EOM eluted in
Fraction 1, none of the mutagenicity did, showing that at
least half of the EOM is both nonpolar and not mutagenic
in any of the strains of Salmonella used here with or
without S9 (Table VI). The most potent fractions were
the moderately polar (middle two) fractions, with poten-
cies in the hundreds of rev/lg of EOM in many of the
YG strains, and these potencies were an order of magni-
tude greater than that of the EOM from the most polar
(methanol) fraction (Table VI). Thus, the moderately
polar fractions (Fractions 2 and 3) contained the least
amount of mass of the four fractions, but their masses
were the most mutagenic among the four fractions.
The 4-fold increased mutagenic potency of the EOM
eluting in Fraction 2 in TA100 1S9 relative to TA100
S9 suggests the presence of PAHs in this fraction;
decreased mutagenic potencies were found in Fractions 3
and 4 in TA100 with addition of S9, indicating that few
if any PAHs eluted in these fractions (Table VI). PAHs
induce primarily base-substitution mutations, and along
with TA100, YG1042 also detects this class of mutation;
consistent with this is the finding that Fraction 2 is most
potent in YG1042.
The high mutagenic potencies of Fractions 2 and 3 in
the frameshift YG strains suggest that these fractions con-
tain both nitroarenes and aromatic/heterocyclic amines.
This is confirmed by the reduced mutagenic potencies of
Fractions 2 and 3 in TA98NR, which has reduced nitrore-
ductase relative to TA98S9 (Table VI). This is also con-
firmed by the increased mutagenic potencies of Fractions
2 and 3 in YG1042 S9, which has enhanced nitroreduc-
tase, relative to TA98 S9.
The highly polar compounds in Fraction 4 were most
mutagenic in YG1041, which implies the presence of
highly polar (possibly oxygenated) nitroarenes and aro-
matic/heterocyclic amines. Fraction 4 had the lowest
mutagenic potency relative to that of Fractions 2 and 3 in
the same strains.
Comparison of the mutagenic potency of the whole
EOM with the reconstituted EOM revealed that despite
the alteration to the chemical matrix that occurred after
fractionation and upon reconstitution, the potencies of
these two EOMs were rather similar among most of the
strains (Table VI). The most notable exceptions were for
strain YG1042-S9 (17.7 vs. 38.0 rev/lg for whole vs.
reconstituted) and TA104 with or without S9, where no
mutagenicity was observed in the reconstituted sample.
Distribution of Recovered Mass and Mutagenicity of EOM
Table VII shows the incorporation of the distribution of
mass with the distribution of mutagenic potencies of the
EOM to give the distribution of recovered mutagenicity.
 Environmental and Molecular Mutagenesis. DOI 10.1002/em
Bioassay-Directed Fractionation of Diesel Exhaust Particles 725

TABLE V. Mutagenicity (Rev/Plate) of Fractions of the Organic Extract of C-DEP

1S9 -S9
EOM/
Strain plate (mg) Hexane DCM/Hex DCM Methanol Reconst. Hexane DCM/Hex DCM Methanol Reconst.

TA100 0 126 126 126 126 126 107 107 107 107 107
1 321 138
2.5 614 206 221b
5 121 983 314 163 209 120 290 415 140 156
10 137 1446a 545 196 297 135 508 698 203 176
20 135 1668a 752a 249 455 106 926 1005a 262 283
TA98 0 46 46 46 46 46 34 34 34 34 34
1 72b 54b
2.5 95 59 78b 76b
5 47 163 75 53 59 39 136 155 62 61
10 54 220 107 63 73 38 235 281 78 75
20 44 326a 204 78c 86 34 391 489 123 123
TA98NR 0 36 36 36 36 36
2.5 68b 58b
5 28 126 114 47 52
10 33 194 206 57 52
20 37 292 377 84 85
TA98/1,8DNP6 0 30 30 30 30 30
1 48b 35b
2.5 97 57
5 35 170 84 32 36
10 35 272 174 39 46
20 30 428a 242a 51c 60
YG1021 0 39 39 39 39 39 36 36 36 36 36
0.5 114b 125b
1 202 88 209 124
2.5 382 148 77b 567 269 83b
5 47 715 261 85 137 42 1010 529 106 168
10 38 1195a 453 155 218 39 1822*b 1024 166 282
20 34 1628a 880b 240 353 36 1800*a 1300a 274 522
YG1024 0 59 59 59 59 59 30 30 30 30 30
0.5 70b 61*b
1 95 70 138 144
2.5 173 110 73b 350 393 83b
5 48 339 182 91 107 27 609 734 129 182
10 50 821a 329 103 131 27 1237b 1246 230 327
20 49 1303a 727a 209 212 32 1995a 2140a 387 514a
YG1041 0 63 63 63 63 63 46 46 46 46 46
0.5 118 168
1 158 105 267 258
2.5 267 154 88 600 616 171
5 70 526 263 125 142 43 1010 1223 188 292
10 76 490 162 194 42 1808a 338 555
20 76 284 345 45 621 981
YG1042 0 102 102 102 102 102 85 85 85 85 85
0.5 222 240
1 332 184 346 226
2.5 551 263 157 310a 342 200
5 120 931 393 165 206 96 329a 382a 177 275
10 110 605 245 309 94 419a 223 329a
20 96 364b 520 91 375b 293a
TA104 0 249 249 249 249 230 230 230 230
1 321 271 244 225
2.5 404 297 317 272
5 263 591 338 262 197 439 318 227
10 250 788a 432 283 212 578 426 243
20 265 699a 530a 318 203 775a 566 253

a
Data were not used in the linear regressions because they were outside of the linear portion of the dose-response curves.
b
Data are from a single plate from a single experiment; all other data are the average of two plates, each from an independent experiment. Positive
control data (average rev/plate, range) are 2-aminoanthracene (1S9): TA100 (734, 482917), TA98 (397, 363443), YG1021 (2139, 18492305),
YG1024 (2317, 21572539), YG1041 (1319, 12551430), YG1042 (879, 786944), TA104 (883, 6551101); sodium azide (-S9): TA100 (790, 690
830), YG1042 (1005, 9291074); 2-nitrofluorene (-S9): TA98 (330, 298383), TA98NR (57, 5773), TA98/1,8DNP6 (72, 5690), YG1021 (2139,
18492305), YG1024 (2317, 21572539), YG1041 (1658, 15721759); methylglyoxal (-S9): TA104 (514, 460608).
c
Data did not quite reach 2-fold above background; however, we regressed these doses because the response was within the linear range, and limited
sample prevented testing at a higher dose.
Environmental and Molecular Mutagenesis. DOI 10.1002/em
726 Mutlu et al.

TABLE VI. Mutagenic Potencies (Rev/mg) of Fractions of EOMa the aromatic amine-type mutagenicity as indicated by the
fact that 58.2% of the mutagenicity in YG1024 S9
Fractions
eluted in this fraction. Consistent with these findings is
DCM/ the fact that most of the mutagenicity in YG1041, which
Strain Whole Hexane Hex DCM Meth Reconstituted
detects both aromatic amines and nitroarenes, eluted in
TA1001S9 17.9 0 171.2 43.2 6.1 16.5 Fraction 2.
TA100-S9 13.1 0 41.3 60.4 8.0 8.6 Only 018% of the mutagenic activity across the
TA981S9 3.0 0 17.5 17.8 1.6 2.0 strains eluted in Fraction 4, and we infer that some of the
TA98-S9 7.5 0 18.0 23.2 4.3 4.3
TA98NR-S9 4.7 0 12.8 17.5 2.4 2.3
compounds in this fraction must be highly polar deriva-
TA98/DNP6-S9 3.7 0 24.7 14.6 1.1 1.5 tives of nitroarenes (TA98 S9) and aromatic amines/het-
YG10211S9 17.1 0 133.5 41.7 10.2 15.8 erocyclic amines (YG1041 1S9) based on the
YG1021-S9 29.3 0 180.1 99.5 11.8 24.5 mutagenicity of this fraction in these two strains. Interest-
YG10241S9 7.7 0 57.2 27.7 7.4 7.6 ingly, the highly polar compounds in this fraction did not
YG1024-S9 29.2 0 121.4 122.8 17.8 30.5
YG10411S9 18.2 0 90.6 42.8 10.9 14.1
revert TA104, which reverts primarily by oxidative
YG1041-S9 67.9 0 192.5 236.8 28.8 46.9 mutagens.
YG10421S9 13.3 0 161.2 49.1 13.2 20.8 Examples comparing the percentage distribution of
YG1042-S9 17.7 0 261.0 100.8 14.1 38.0 both mass and mutagenicity using data from Table VII
TA104 1S9 4.8 0 67.7 18.1 0 0 are shown in Figure 3. PAH-type mutagenicity (TA100
TA104 S9 10.8 0 36.4 17.5 0 0
1S9) eluted primarily in Fractions 2 and 3 (Fig. 3A),
a
Data are slopes of linear regressions calculated from the data in whereas nitroarene-type mutagenicity (TA98 S9) eluted
Table V. primarily in Fractions 3 and 4 (Fig. 3B). Many other
comparisons can be made from the data in Table VII;
however, for all strains the majority of the mutagenicity
Thus, for any particular strain the resulting percentages eluted in Fractions 2 and 3, which contrasts with the elu-
represent the proportion of mutagenic activity eluting in tion of the mass, most of which eluted in Fractions 1 and
each fraction based on the mass of EOM that eluted in 4. These data illustrate the ability of the bioassay-directed
that fraction and the mutagenic potency of that mass. As fractionation to separate biologically inactive organics
noted earlier, although 50% of the mass eluted in the from biologically active ones.
nonpolar Fraction 1, this fraction was not mutagenic in A summary of the inferred classes of chemicals based
any of the strains. The moderately polar Fraction 2 con- on the mutagenicity profile in the various strains with the
tained only 6% of the mass of the whole extracted organ- chemical analysis of the 32 PAHs is shown in Table VIII.
ics, but it accounted for 2060% of the mutagenic Fraction 1 was not mutagenic, and chemical analysis
activity depending on the strain. Although the moderately showed that it contained the noncarcinogenic PAHs.
polar Fraction 3 contained only 14% of the mass of the Because of the nonpolar nature of the solvent used to
whole extracted organics, it accounted for 3364% of the elute this fraction (hexane), we infer that this fraction
mutagenic activity depending on the strain (Table VII). also contained nonpolar/nonmutagenic compounds like
The highly polar Fraction 4 contained 28% of the mass alkanes and alkenes, which are most likely from un-
but accounted for only 818% of the mutagenic activity combusted fuel and lubricating oil. Most of the 16 prior-
depending on the strain. ity EPA PAHs (the first 16 PAHs listed in Table IX)
The data in Table VII show that the majority of the eluted in Fraction 2, in which most of the PAH-type
S9-dependent base-substitution mutagenicity (TA100, mutagenicity (TA100 1S9) also eluted. Some nitroarene-
TA104, and YG1042) eluted in Fraction 2. PAHs produce type mutagenicity (TA98 S9) also eluted in Fraction 2;
primarily base-substitution mutations and require meta- however, most of this activity eluted in Fraction 3. One
bolic activation, and some of the organics in this fraction possible explanation for this apparent discrepancy is that
are likely PAHs as indicated by the fact that 57.1% of the the extracts were not analyzed for the presence of dini-
mutagenicity in TA100 1S9 was produced by this frac- troarenes, which are potent nitroarenes in DEPs, and
tion; this strain is diagnostic for PAHs. Some PAHs can because they are more polar than the mono-nitroarenes,
also cause base substitutions at AT sites, which might be they may have eluted in Fraction 3and accounted for
reflected by the fact that 61.7% of the mutagenicity in the high level of nitroarene-type mutagenicity in Fraction
TA104 1S9 is produced by Fraction 2. By contrast, most 3. Most of the oxy-PAHs eluted in Fraction 3, and some
of the nitroarene-type mutagenic activity eluted in Frac- also eluted in Fraction 4 as indicated from the sub-
tion 3 as indicated by the fact that this fraction accounted fractionation analysis. However, the limited amount of
for 58.6% of the mutagenicity in TA98 S9 and 51.8% of mutagenicity that eluted in Fraction 4 prevented us from
the mutagenicity in YG1021 -S9, which are strains diag- inferring the presence of any other chemical classes in
nostic of nitroarenes. Fraction 2 also contained most of this fraction.
 Environmental and Molecular Mutagenesis. DOI 10.1002/em
Bioassay-Directed Fractionation of Diesel Exhaust Particles 727

YG1021 YG1021 YG1024 YG1024 YG1041 YG1041 YG1042 YG1042 TA104 TA104

Calculated by multiplying the number of rev/mg of EOM for each fraction from Table VI by the number of micrograms of EOM recovered for each fraction as noted in the second column of this
0.0

0.0
47.3
52.7

100
1S9 -S9

0.0

0.0
61.7
38.3

100
0.0
46.6
41.7
11.7
100
-S9

table. These values, rev/fraction, were then expressed as a percentage relative to the sum ( ) of the recovered mass of the fractions noted in the third column of this table.
1S9

0.0
47.9
33.8
18.3
100
22.0
62.7
15.3
0.0

100
1S9 -S9

0.0
37.7
41.2
21.1
100
Distribution of recovered mutagenicity (%)a

0.0
24.8
58.2
17.0
100
1S9 -S9

36.7
41.2
22.1
0.0

100
0.0

7.7
40.4
51.8

100
-S9
TABLE VII. Distribution of Mass and Mutagenicity among Fractions of Organic Extract of C-DEP

1S9 -S9 TA98NR-S9 TA98/DNP6 1S9

0.0
48.1
34.8
17.1
100

Fig. 3. Distribution of mass and mutagenicity in strains TA100 1S9 (A)

and TA98 -S9 (B).
0.0

8.0
38.8
53.2

100

Chemical Analysis of Whole Extract and Four Fractions of

P

C-DEP
Based on our inferred role for certain chemical classes in
0.0
19.8
62.9
17.3
100

the mutagenicity of C-DEP, we analyzed the whole extract

and its four fractions for 32 PAHs that were representative
of these chemical classes, including nitro- and oxy-PAHs.
Recovery Recovered TA100 TA100 TA98 TA98

0.0
19.6
58.6
21.8
100

Despite this rather extensive analysis, the total mass identi-

fied chemically by this analysis accounted for only 0.1% of
26.4
62.3
11.3
0.0

100

the total extractable organic mass. Stevens et al. [2009] also

reported that PAHs accounted for only 0.3% of the mass of
the EOM in their DEP sample, which was similar to that of
0.0
18.9
64.1
17.0
S9

100

C-DEP. Thus, our rather extensive PAH analysis resulted in

the identification of only an extremely small portion of the
mass (%) 1S9

57.1
33.4
0.0

9.5
100

chemicals in the organic extract. Although the PAHs ana-

lyzed are potentially important in the mutagenicity of C-
DEP, they may not represent the major chemical classes
6.0
51.9

14.0
28.1
100

responsible for the observed mutagenicity of C-DEP.

The results, expressed as lg of compound/g of particle,
Mass

are shown in Figures 4A4E, which were constructed

6.1

100.7
52.3

14.1
28.3
(%)

from the data in Table IX. Of the 32 compounds

assessed, all but one (dibenzo[ah]anthracene) were found
300,000
156,750

301,950
18,200
42,200
84,800

in the whole extract. Interestingly, some PAHs eluted in

EOM
(mg)

the nonpolar Fraction 1; however, these were the noncar-

cinogenic PAHs, consistent with our finding that Fraction
DCM/H
Hexane
Sample

MeOH
Whole

DCM

1 was not mutagenic. Although we did not analyze this

P

fraction for alkanes and alkenes, the large mass (50%)

a
Environmental and Molecular Mutagenesis. DOI 10.1002/em
728 Mutlu et al.

TABLE VIII. Chemical Classes in Each Fraction Determined by Analytical Measurement of PAHs or Inferences Based on Muta-
genicity in Selected Strains of Salmonella
Analysis Fraction 1 Fraction 2 Fraction 3 Fraction 4

Measured Noncarcinogenic PAHs EPA-PAHs and Oxy-PAHs Oxy-PAHs

nitroarenes
Inferred Nonmutagenic PAHs, PAHs Nitroarenes and Oxy-PAHs
alkanes, alkenes oxidative agents

TABLE IX. Concentration of PAHs in Whole and Fractions of C-DEP

Concentration (mg/g particle)
a
PAH Whole F1 F2 F3 F4

Naphthalene 1.25 1.3 0.1 0.09 0

Acenaphthylene 0.42 0.25 0.1 0.08 0.09
Acenaphthene 0.03 0.07 0 0 0
Fluorene 1.14 0.57 0.74 0 0
Phenantherene 126.49 73.58 75.46 0.36 0
Anthracene 1.04 0.45 0.53 0.23 0.27
Fluoranthene 147.57 43.58 120.74 0.2 0
Pyrene 71.35 32.26 41.51 0.1 0
Benzo[a]anthracene 5.68 0.85 5.21 0.12 0
Chrysene 27.57 2.72 22.64 0 0
Benzo[b]fluoranthene 5.68 0.35 5.21 0 0
Benzo[k]fluoranthene 4.86 0.23 3.24 0 0
Benzo[a]pyrene 0.10 0 0.11 0 0
Indeno[123-cd]pyrene 0.70 0 0.67 0 0
Dibenzo[ah]anthracene 0.00 0 0 0 0
Benzo[ghi]perylene 0.31 0 3.02 0 0
1,4-Naphthoquinone 0.54 0 0 1.51 0.3
1-Naphthalenecarboxaldehyde 18.97 0 0.38 23.39 24.15
9-Fluorenone 16.86 0 0.23 27.92 2.49
9,10-Anthraquinone 81.08 0 3.02 98.1 1.89
1,8-Naphthalic Anhydride 84.32 0 0 339.59 10.56
9,10-Phenanthrenequinone 0.50 0 0 0 1.51
Benzanthrone 21.08 0 0 12.07 0.15
1-Pyrenecarboxaldehyde 4.14 0 0 1.89 0.15
Benz[a]anthracene-7,12-quinone 7.30 0 0.38 6.04 0.53
1-Nitronaphthalene 0.10 0 0 0 0
2-Nitronaphthalene 0.88 0 1.06 0 0
4-Nitrobiphenyl 17.51 0 0 2.34 3.77
2-Nitrofluorene 0.15 0 0 0 0.23
3-Nitrofluoranthene 0.08 0 0.23 0 0
1-Nitropyrene 94.05 0 65.65 21.21 0
6-Nitrochrysene 0.06 0 0 0 0
a
The first 16 PAHs in this list are the U.S. EPA priority PAHs.

of the extract that eluted in this fraction is assumed to
be compounds of this class coming from unburned fuel
and lubricating oil.
We inferred from the distribution of mutagenic activ-
ity (Table VII) that most of the mutagenic PAHs eluted
in Fraction 2, and chemical analysis confirmed this (Fig.
4B). Chemical analysis found that most of the nitroar-
enes also eluted in this fraction, with additional nitroar-
enes eluting in Fraction 3 (Fig. 4D). We inferred that
most of the mutagenicity due to nitroarenes eluted in
Fraction 3 (Fig. 4D), and some of this may be due to
dinitroarenes. However, we did not have any dinitroar-
enes among the standards used for the chemical analy-
sis; thus, we did not have a measure of the
concentration of dinitroarenes in the extract and
fractions.
Chemical analysis showed that most of the oxy-PAHs
eluted in Fraction 3 (Fig. 4E), which is the fraction in
 Environmental and Molecular Mutagenesis. DOI 10.1002/em
Bioassay-Directed Fractionation of Diesel Exhaust Particles 729

Fig. 4. Concentrations of the 32 PAHs analyzed in the whole extract and four fractions of C-DEP; (A) Total PAHs,
(B) 16 EPA PAHs, (C) nitro- and oxy-PAHs, (D) nitro-PAHs, and (E) oxy-PAHs.

which most of the oxidative mutagenic activity (TA104
S9) eluted (Table VII). 4-Nitrophenyl, 3-nitrofluoranthene,
9,10-phenanthrenequinone, and 1-naphthalenecarboxalde-
hyde eluted primarily in Fraction 4. Our mutagrams of
Fraction 4 showed that a peak of mutagenicity eluted
coincident with the elution of the oxy-PAH standard 1,3-
dihydroxynaphthalene (Fig. 7). Figure 5 shows examples
of the percent distribution of mass, mutagenicity in
selected strains, and PAH concentrations across the four
fractions. Again, given that the chemical analysis repre-
sents only 1% of the extractable organic mass, no direct
relationship exists between the height of any particular
PAH bar in the histogram and the heights of the mass or
mutagenicity distributions.
Correlations Between Chemical Composition
and Strain-Specific Mutagenicity
We hypothesized that if specific chemicals within the
C-DEP EOM or its fractions were mechanistically
Environmental and Molecular Mutagenesis. DOI 10.1002/em
730 Mutlu et al.
Fig. 5. Distribution of mass, mutagenicity, and PAHs across C-DEP fractions.
involved in the induction of mutations in Salmonella
exposed to them, then there should be a correlation
between the amount of those chemicals in the fraction
and the extent of mutagenicity observed. Hierarchical
clustering of the correlations between the amounts of
individual PAHs in each EOM fraction and the mutage-
nicity of the fraction in each of the Salmonella strain/acti-
vation combinations revealed several distinct clusters of
response (Fig. 6).
One of the most striking of these clusters was composed of
naphthalene, acenaphthene, acenaphthylene, 2-nitrofluorene,
9,10-phenanthroquinone, 1-naphthalenecarboxaldehyde, 6-
nitrochrysene, 1-nitronaphthalene, and 4-nitrobiphenyl. Each
of these PAHs was correlated negatively with the mutagenic
response in every Salmonella strain whether or not S9 was
present. Another cluster included phenanthrene, pyrene, fluo-
rene, and anthracene. The presence of these chemicals was
correlated negatively with mutagenicity in strain TA98 with
or without S9, as well as with mutagenicity in strains TA100,
YG1041, TA98NR, and YG1024 in the absence of S9. All
four of these PAHs are nonmutagenic in Salmonella. The
same six strain/activation combinations yielded positive corre-
lations with a group of PAHs containing the oxy-PAHs 9,10-
anthraquinone, 1,8-naphthalic anhydride, benz[a]anthracene-
7,12-quinone, 9-fluorenone, 1-pyrenecarboxaldehyde, benzan-
throne, and 1,4-naphthoquinone. Both of these groups of
PAHs were correlated only weakly with mutagenicity in the
remaining strain/activation combinations tested.
In contrast, fluoranthene, benzo[k]fluoranthene, chrysene,
2-nitronaphthalene, benzo[a]anthracene, benzo[b]fluoran-
thene, 1-nitropyrene, indeno[123-cd]pyrene, benzo[a]pyr-
ene, 3-nitrofluoranthene, and benzo[ghi]perylene comprised
a group that was positively correlated with mutagenicity in
Salmonella strains TA104, YG1021, and YG1042 with or
without S9, TA98/1,8-DNP6 without S9, and YG1024,
YG1041, and TA100 with S9; but this group correlated
only weakly with mutagenicity in the remaining strain/acti-
vation combinations studied.
Mutagenicity of Sub-Fractions of EOM
We sub-fractionated the three mutagenic fractions (Frac-
tions 2, 3, and 4) by HPLC in order to better understand
the chemical components of C-DEP that were associated
with specific types of mutagenicity based on the elution
profile of mutagenicity (mutagrams) (Fig. 7) relative to that
of a set of chemical standards (Table X, Fig. 7). Fraction 1
was not sub-fractioned because it was not mutagenic, and
previous work by us has shown that the nonmutagenic
Fraction 1 from other combustion emissions produced non-
mutagenic sub-fractionsi.e., flat mutagrams (D. DeMarini,
unpublished data). The mutagram from Fraction 2 had
PAH-type mutagenicity (TA100 1S9) that eluted at sub-
fraction 31 that was due to moderately polar compounds.
The smaller peak at sub-fraction 37 represented nitroarene-
type mutagenicity (TA98 S9), which was confirmed by
the reduced mutagenicity of this sub-fraction in TA98NR
(Fig. 7). This activity was due to moderately polar nitroar-
enes and/or aromatic amines as indicated by the elution of
the standards in this region of the mutagram.
 Environmental and Molecular Mutagenesis. DOI 10.1002/em
Bioassay-Directed Fractionation of Diesel Exhaust Particles 731

Fig. 6. Heat map of correlations between concentrations of PAHs and mutagenic potencies in various strains among the
whole extract and four fractions of C-DEP. Red represents a positive correlation; blue a negative one.

The mutagram of Fraction 3 had two distinct peaks of
mutagenicity: both PAH- and nitroarene-type activity at
sub-fraction 17 due to weakly polar compounds, and a
peak primarily of nitroarene-type mutagenicity at sub-
fraction 43 that was due to moderately polar compounds.
Moderately polar compounds accounted for the activity at
sub-fraction 43, including that of a small peak of PAH-
type mutagenicity that also eluted at this sub-fraction.
The narrow peak of mutagenicity at sub-fraction 43
implies that a single chemical class or just a few chemi-
cal classes of similar polarity eluted in this region to pro-
duce the observed mutagenicity. The scale of
mutagenicity (rev/plate) for the mutagram of Fraction 3 is
the highest of the three mutagrams, reflecting the fact that
large amounts of nitroarene-type mutagenicity eluted in
this fraction.
The mutagram of Fraction 4 had a small amount of
PAH-type mutagenicity at sub-fraction 18 that was due to
weakly polar compounds. However, the elution of moder-
ately polar compounds at sub-fraction 43 produced peaks
of both PAH-and nitroarene-type mutagenicity; an addi-
tional peak of highly polar nitroarene-type of mutagenic-
ity eluted at sub-fraction 54. Most interestingly was the
elution of mutagenicity at fraction 62 that was due to
highly polar compounds, most likely oxy-PAHs because
our oxy-PAH standard 1,3-dihydroxynaphthalene eluted
in this region.
These results provide additional insight into the chemi-
cal classes of the compounds responsible for specific
types of mutagenicity induced by C-DEP. In particular,
these data confirmed the initial fractionation data by
showing that much of the PAH-type of mutagenicity
Environmental and Molecular Mutagenesis. DOI 10.1002/em
732 Mutlu et al.

TABLE X. Elution Profile of HPLC Standardsa

Retention Polarity
Standard time (min) classification

1. Naphthalene 6.6 Aromatic nonpolar

2. Pyrene 8.7 Aromatic nonpolar
3. Benzo[a]pyrene 9.5 Aromatic nonpolar
4. Anthraquinone 11.5 Moderately polar
5. 1-Nitropyrene 16.3 Moderately polar
6. Dibenzo[aj]acridine 17.3 Moderately polar
7. Carbazole 21.2 Moderately polar
8. 1-Aminopyrene 21.4 Moderately polar
9. Acridine 25.5 Highly polar
10. 1,3-Dihydroxynaphthalene 30.8 Highly polar
a
Data from DeMarini et al. 1992.

istics different from those of the historical A-DEP and N-

Fig. 7. Mutagrams of HPLC-derived sub-fractions of Fractions 2, 3, and
4 using three strains of Salmonella. The elution profile of the standards is
noted by the arrows at the top, and the numbers refer to the standards in
Table VIII. WP, weakly polar; MP, moderately polar; HP, highly polar;
NPAHs are nitro-PAHs.
eluted in Fraction 2, and much of the nitroarene-type
mutagenicity eluted in Fraction 3. The sub-fractionation
data also showed that some oxy-PAHs also eluted in
Fraction 4; however, the majority of the ones analyzed
chemically eluted in Fraction 3.
DISCUSSION
Comparison of C-DEP to Historical DEPs
One of the goals of this research was to generate a new
sample of DEP with mutagenicity and chemical character-
DEP. Table XI shows a comparison of physical/chemical
characteristics and mutagenicity effects of these three
DEPs. The percentage of EOM of C-DEP is similar to
that of A-DEP; however, the OC/EC mass ratio and OC/
total carbon ratio of C-DEP are intermediate to that of A-
and N-DEP. The distribution of the mass of the EOM
across the fractions is similar for A- and C-DEP, but
these distributions are quite different from that of N-DEP.
Among the three DEPs the PAH-type of mutagenic activ-
ity varies by two orders of magnitude. The nitroarene-
type of mutagenic potency of A-DEP is much greater
than that of the other two DEPs. The distribution of muta-
genic activity across the fractions is somewhat similar for
all three DEPs.
A further comparison of the three DEPs is shown in
Table XII, which examines the relative mutagenic contri-
bution of various chemical classes as inferred from the
results in the diagnostic strains of Salmonella. These
comparisons show that a large percentage of the muta-
genic potency of A- and N-DEP is due to nitroarenes,
whereas a smaller percentage of the mutagenicity of C-
DEP is due to this class of compound. In contrast, A-
DEP has higher levels of PAH-type mutagenicity than do
the other DEPs.
Figure 8 shows the mutagenic potencies of the three
DEPs in six strains in the absence of S9. The mutagenic
potencies of the DEPs ranked A > N > C in TA98,
TA100, and YG1024. Thus, in general, C-DEP is less
mutagenic than the two historical DEPs. A-DEP and N-
DEP show a great decrease in mutagenicity in the
nitroreductase-deficient strains TA98NR and TA98/1,8-
DNP6 relative to that of TA98, indicating that nitroarenes
play a large role in the mutagenicity of these DEPs; in
contrast, this reduction is much less for C-DEP, indicating
that nitroarenes play a much smaller role in its mutage-
nicity. Thus, for a variety of physical/chemical character-
istics and mutagenicity, each DEP has a unique profile of
characteristics and effects.
 Environmental and Molecular Mutagenesis. DOI 10.1002/em
Bioassay-Directed Fractionation of Diesel Exhaust Particles 733

TABLE XI. Comparisons of Physical, Chemical, and Biological with an oxy-PAH standard, confirming a role for oxy-
Features of DEPs PAHs as indicated by the hierarchical clustering.
Characteristic A-DEP C-DEP N-DEP Although the data with the diagnostic strains of Salmo-
nella identify a strong role for nitroarenes in the mutage-
Physical/chemical nicity of C-DEP, the hierarchical-cluster analysis does not
% EOMa 26.3 23.0 2.0
identify the presence or absence of nitroreductase (i.e.,
OC/EC mass ratiob 5.56 0.33 0.08
OC/total carbonb 0.85 0.25 0.07 nitroarenes) as a major determinant of mutagenicity of C-
Mass distribution (%)a DEP EOM or its fractions. Instead, the hierarchical clus-
Hexane 54.8 51.9 29.1 tering identifies a strong role for the oxy-PAHs, which
Hexane/DCM 5.9 6.0 6.4 would not be expected to be acted on by nitroreductases.
DCM 6.4 14.0 6.3
The second cluster of strain/activation conditions shows
Methanol 32.9 28.1 58.2
Mutagenicitya a high degree of positive correlation for a group of chem-
PAH-type mutagenic 90.8 4.1 0.4 icals containing eight PAHs and three of the nitro-PAHs.
potency of particles This cluster includes all of those tests conducted in the
(rev/mg particles in presence of S9 with the sole exception of TA981S9,
TA100 1S9)
which clusters with the other group of strains. The pres-
Nitroarene-type 36.4 1.7 4.4
mutagenic potency of ence of S9 during the test was not sufficient to define the
particles(rev/mg cluster because tests with TA104, YG1021, and YG1042
particles in TA98 -S9) in the absence of S9 were also members of this cluster.
% of PAH-type 40.6 57.1 13.7 Interestingly, for TA100, YG1024, and YG1041, the pres-
mutagenicity in
ence of S9 shifted the pattern of response from the first
Fraction 2
% of nitroarene-type 42.1 58.6 56.3 cluster of strain/activation combinations to the second
mutagenicity in Fraction 3 cluster.
a
With respect to clusters among the chemicals, it is
Data from DeMarini et al. [2004] and current study. The solvent used
striking that there was a heterogeneous group containing
to elute Fraction 2 was 50:50 Hexane/DCM in DeMarini et al. [2004]
but was 75% hexane:25% DCM for C-DEP here. PAHs, oxy-PAHs, and nitro-PAHs that was universally
b
Data from Stevens et al [2009]; the DEP analyzed by Stevens et al. negatively correlated with mutagenicity for all of the
was not C-DEP but a mixture of some of the DEPs that were combined strain/activation combinations tested. This cluster of
to make C-DEP. chemicals includes several that are known to be muta-
genic and/or carcinogenic, including 2-nitrofluorene,
which is used frequently as a positive control for strain
Comparisons of Chemical Composition with Mutagenicity
TA98. However, there are various studies showing that
The availability of partial chemical characterization for PAHs are capable of inhibiting the activation of some
the C-DEP EOM and its four fractions, together with nitro-PAHs, such as 1-nitropyrene [Cherng et al., 1996;
mutagenicity data for each of the mixtures in a variety of Lee et al., 1994] and that co-exposure to PAHs in defined
Salmonella strains with or without S9 provided a data set mixtures of carcinogenic PAHs results in lower extents of
that permitted examination of the correlations between DNA adduction than is observed for the same PAHs indi-
patterns of mutagenic response and the prevalence of spe-  am, 2004].
vidually [Binkova and Sr
cific chemical components in the mixtures tested. Several Finally, caution must be used in interpreting the pat-
major conclusions may be drawn from this analysis. terns of correlation between chemical constituents and
It is quite clear that the various Salmonella strain/S9 mutagenicity. Although we have quantitative information
combinations segregated into two discrete clusters exhib- on a wide array of chemical constituents for the C-DEP
iting similar patterns of response to the chemicals in the EOM and four fractions, our chemical analyses account
mixtures. The first cluster of strains included TA100, for only a small amount (0.1%) of the total mass present.
TA98, YG1041, TA98NR, and YG1024, all without S9, Nonetheless, the constituents measured do include a num-
together with TA98 with S9. The mutagenicity in this ber of highly potent mutagens and carcinogens that are
cluster of strains is positively correlated with the concen- known to be associated with diesel exhaust. As illustrated
trations of a group of oxy-PAHs. These same oxy-PAHs in Table VIII, there is a remarkably good agreement
are only weakly or negatively correlated with mutagenic- between the classes of compounds detected analytically in
ity in the cluster made up of the remaining strains/activa- a specific fraction and the classes of compounds inferred
tion conditions. Both TA98 and TA98NR were members to be present in that fraction based on mutagenicity in
of this cluster, and both exhibited quite similar patterns of diagnostic strains of Salmonella.
correlation to chemical composition of the mixtures, even Further, the fact that we observed discrete clusters of
with respect to the nitro-PAHs present. The mutagram of correlation is consistent with our hypothesis that the lev-
Fraction 4 clearly shows mutagenic activity that elutes els of the measured constituents in the mixtures is to
Environmental and Molecular Mutagenesis. DOI 10.1002/em
734 Mutlu et al.

TABLE XII. Comparative Role of Nitroarenes, Arylamines, and

PAHs in the Mutagenicity of DEPs
Comparisona DEP Result Conclusion

TA98 S9 vs. A 89% # A large percentage of the

TA98NR mutagenic potency of
N 86% # A- and N-DEP is due to
C 35% # mononitroarenes; this
percentage is much less
for C-DEP.

TA98 S9 vs. A 89% # A large percentage of the

TA98/1,8-DNP6 mutagenic potency of
N 93% # A- and N-DEP is due to
C 47% # nitroarenes; this
percentage is less for
C-DEP.

TA98 vs. A 226% " A- and C-DEP contain much

YG1021 S9 more nitroarene-type of
N 7% # mutagenicity than does Fig. 8. Comparison of mutagenicity of A-DEP, N-DEP, and C-DEP;
C 394% " N-DEP. data from A-DEP and N-DEP are from DeMarini et al. [2004].

TA98 vs. A 370% " All 3 DEPs have similar More than 50% of the mass of the extractable organics of
YG1024 S9 amounts of arylamine-type C-DEP elutes in the nonpolar Fraction 1, and more than
N 441% " mutagenicity.
C 394% "
25% elutes in the highly polar Fraction 4. Although only
20% of the mass elutes in the moderately polar Frac-
TA100 1S9 A 90.5b A-DEP has 20 times more tions 2 and 3, most of the mutagenicity elutes in these
N 5.2b PAH-type mutagenicity two fractions. Although this elution profile of mutagenic-
C 4.1b than does N- or C-DEP. ity of C-DEP is similar to that of A-DEP, it is different
TA98 S9 A 36.4b A-DEP has more
from that of N-DEP, in which less mutagenicity eluted in
N 4.4b nitroarene-type the moderately polar middle fractions and more eluted in
C 1.7b mutagenicity than does the highly polar Fraction 4.
N- or C-DEP. Based on a relatively good agreement between elution
of PAHs and mutagenicity of various fractions in various
a
Data for comparisons are from Table IV for C-DEP and from DeMarini diagnostic strains, we conclude that PAHs, nitroarenes,
et al. [2004] for A- and N-DEP. aromatic amines, and oxy-PAHs are the cause of much of
b
Values are mutagenic potencies (rev/mg of particles). the mutagenicity of C-DEP. Although the hierarchical
clustering confirmed the role of oxy-PAHs, it did not con-
some extent causal of the mutational outcomes. Although firm the role for the other chemical classes, especially
we cannot provide a clear explanation for the clustering that of the nitroarenes and PAHs, likely due to the limited
of various strains with various PAHs, we think that our chemical characterization of the EOM (only 0.1% of the
analysis reflects a fundamental chemical-biological associ- mass). To our knowledge this is the first application of
ation that, at this point, we do not understand fully. hierarchical clustering to correlate chemical composition
and mutagenicity for a complex mixture and its fractions.
With further chemical characterization of the EOM, such
an analysis may provide a useful adjunct to bioassay-
CONCLUSIONS directed fractionation. In summary, we analyzed for the
These studies show that C-DEP has a unique profile of presence of 32 PAHs in the organic extract of C-DEP and
mutagenicity and chemical/physical characteristics relative its four fractions and correlated those data with extensive
to other DEPs. Compared with A- and N-DEP, the muta- mutagenicity data, making C-DEP well-characterized for
genic potency of C-DEP was intermediate in TA100 +S9 PAH concentrations and mutagenic activity. As such, C-
(PAH mutagenicity) but was lowest in TA98 -S9 (nitroar- DEP is suitable for a variety of toxicological studies.
ene mutagenicity). C-DEP also has some ability to induce
ACKNOWLEDGMENTS
oxidative-type mutagenesis; this feature has not been exam-
ined for A- or N-DEP. The authors thank Carol D. Swartz and Andrew D. Kli-
The mass distribution of C-DEP closely resembles that german for their helpful comments on this manuscript,
of A-DEP but is quite different from that of N-DEP. and L.C. Walsh for his help in experimental design and

Bioassay-Directed Fractionation of Diesel Exhaust Particles
quality assurance. They thank H. de Weerd and H.
Makarem for their help with the PAH analysis and L.
Collins and J.A. Swenberg for their help with and access
to the HPLC used for the sub-fractionation. This article
was reviewed by the National Health and Environmental
Effects Research Laboratory, U.S. Environmental Protec-
tion Agency and approved for publication. Approval does
not signify that the contents reflect the views of the
agency nor does mention of trade names or commercial
products constitute endorsement or recommendation for
use.
AUTHOR CONTRIBUTIONS
E.M., J.A.R., and D.M.D. prepared the manuscript;
S.H.W. and P.P.M. performed the mutagenicity experi-
ments; E.M. performed the extractions and fractionations
and determined the % EOMs; and C.K. generated the
samples; W.P.L. provided guidance on sample generation
and calculations of engineering parameters; I.M.K. per-
formed chemical analyses; M.I.G. oversaw the combus-
tion experiments and provided guidance on experimental
design; and J.A.R. and J.E.S. conceived and conducted
the correlative analysis and hierarchical clustering of
mutagenicity and PAH concentrations.
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