Mutagenesis vol. 18 no. 5 pp. 429±438, 2003
DOI: 10.1093/mutage/geg021
DNA binding of polycyclic aromatic hydrocarbons in a human
bronchial epithelial cell line treated with diesel and gasoline particulate
extracts and benzo[a]pyrene1
Sanna K.Pohjola, Maija Lappi1, Markku Honkanen2,³,
Leena Rantanen2,³ and Kirsti Savela3
Finnish Institute of Occupational Health, Helsinki, 1Technical Research
Centre of Finland, Espoo and 2Fortum Oil and Gas Oy, Porvoo, Finland
Particulate matter of vehicle exhaust is known to contain
carcinogenic compounds such as polycyclic aromatic
hydrocarbons (PAH) and is suggested to increase lung
cancer risk in humans. This study examines the differences in diesel and gasoline-derived PAH binding to DNA
in a human bronchial epithelial cell line (BEAS-2B).
Particulate matter (PM) of gasoline exhaust was collected
from passenger cars on ®lters and semi-volatile compounds on polyurethane foam (PUF). The soluble organic
fraction (SOF) extracted from the particles was used to
expose the cells and to perform PAH analysis. Gasoline
extracts, benzo[a]pyrene (B[a]P) and reference materials
(SRM 1650 and 1587) were used to study dose-dependent
adduct formation in BEAS-2B cells. The levels of DNA
adducts were in good accord with the 10 DNA adductforming PAH concentrations analyzed in the extracts.
Gasoline extracts, SRM 1650, SRM 1587 and B[a]P
formed DNA adducts dose-dependently in BEAS-2B cells.
The time-dependent DNA adduct formation of 5.0 mM
B[a]P was lower than that of 2.5 mM B[a]P. The results of
this study indicate that reformulated and standard diesel
fuels formed about 11- and 31-fold more adducts than
gasoline, respectively, when PAH±DNA adduct levels were
calculated on an emission basis (adducts/mg PM/km),
whereas on a particulate basis (adducts/mg PM) no difference between the diesel and gasoline extracts was
observed. We conclude that the genotoxicity of diesel fuel
is based on higher particulate emission rates compared to
gasoline emission and although the concentration of PAH
compounds was higher in diesel particulate extracts, DNA
binding by the gasoline particulate-bound PAH compounds was more pronounced than that by the diesel particulate-bound PAH compounds.
Introduction
The particles present in urban air pollution are mainly derived
from diesel and gasoline fueled vehicles. Exhausts are released
straight into the breathing zone and small particles can easily
penetrate into the lungs. Vehicle exhaust has been shown to
contain many known carcinogenic compounds, such as
benzo[a]pyrene (B[a]P). Chronic exposure to particles derived
from diesel and gasoline engines causes several harmful health
effects, including increased lung cancer risk (IARC, 1989).
According to the World Health Organisation particulate matter
is linked to half a million premature deaths every year. Even
3To
short-term exposures to low levels (below 100 mg/m3) of
particulate matter (PM) in air are associated with adverse
health effects (World Health Organisation, 2000). For this
reason, the World Health Organisation cannot recommend a
guideline level for short-term average PM10 value in air.
Although the epidemiological evidence and the results from
animal tests support human carcinogenicity, at least with
respect to diesel exhaust emission, there is still no consensus
about the mechanism of carcinogenicity.
Polycyclic aromatic hydrocarbons (PAH) are the most
prominent among the genotoxic and carcinogenic agents
present in polluted urban air. Genotoxic PAH compounds are
known to react with DNA after their metabolic activation to
form DNA adducts (Conney, 1982). This initiation step is
thought to be relevant with respect to chemical carcinogenesis
(Miller, 1970; Lawley, 1989). Furthermore, DNA adducts are
useful biomarkers re¯ecting the internal dose and exposure to
PAH and nitro-PAH derived from particles. DNA adducts have
been measured in test animals (Bond et al., 1990; Gallagher
et al., 1994; Savela et al., 1995; Sato et al., 2000; Aoki et al.,
2001; Gerde et al., 2001) and in cultured human cells
(Gallagher et al., 1993; Kuljukka-Rabb et al., 2001) after
exposure to diesel particulate or urban air extracts. In human
biomonitoring studies DNA adducts have been used as
biomarkers of genotoxicity of traf®c-related pollution, taking
into account that many confounding factors, e.g. active or
passive smoking and consumption of grilled and smoked food,
may signi®cantly affect the PAH±DNA adduct levels, especially when the degree of exposure is moderate or low
(Hemminki et al., 1994; Phillips et al., 1995; Nielsen et al.,
1996; Schoket et al., 1999; Kyrtopoulos et al., 2001).
Diesel and gasoline engines emit particles and PAH
compounds due to incomplete combustion of the fuel.
Particulate mass and PAH emissions from diesel engines
are generally higher compared to those of gasoline engines
with a catalytic converter (Mi et al., 1998, 2000).
Emissions are, however, dependent on the temperature,
the test vehicles and cycles used, the fuels and also the
exhaust after-treatment technology. The concentrations of
14 PAH compounds analyzed in the soluble organic
fraction (SOF) of direct injection gasoline engine (GDI)
emissions have been shown to be 17 and 53 mg/km, when
reformulated gasoline and European standard gasoline,
respectively, were used (Kokko et al., 2000). In addition
to the mass based particle emission, the question of particle
size and number in gasoline and diesel emissions has been
raised. Particle diameters <300 nm dominate the mass
distribution of both gasoline and diesel emissions, but the
gasoline particle diameters are on average smaller than
particles found in diesel exhausts (Maricq et al., 1999;
Bunger et al., 2000).
whom correspondence should be addressed. Tel: +358 9 4747 2494; Fax: +358 9 4747 2114; Email: kirsti.savela@occuphealth.®
³Declaration
of interest: Markku Honkanen and Leena Rantanen currently work for Fortum Oil and Gas Oy
Mutagenesis vol. 18 no. 5 ã UK Environmental Mutagen Society 2003; all rights reserved.
429
S.K.Pohjola et al.
Bronchial epithelial cells are the target of inhalable particles.
Exhaust particles and particulate-bound organic compounds
have been shown to induce cytokine expression and elicit a
pro-in¯ammatory response in bronchial epithelial cells
(Bayram et al., 1998; Abe et al., 2000; Bonvallot et al.,
2001). Bronchial epithelial cells effectively express PAHactivating CYP1A1 and CYP1B1 when induced with B[a]P
and, therefore, particle-derived PAH are metabolically activated to reactive intermediates in these cells (Mollerup et al.,
2001). Phase II enzymes such as glutathione S-transferase
(GST), which detoxi®es PAH metabolites, are expressed in
bronchial cells (Mace et al., 1994), and B[a]P induces
unscheduled DNA synthesis leading to active DNA repair in
these cells (Ishikawa et al., 1984; Doolittle et al., 1985).
Hornberg et al. (1998) showed that airborne particles are able
to damage DNA of bronchial cells and the PM10 and PM2.5
fractions of the airborne particles induced sister chromatid
exchanges in a dose-dependent manner. Also, a strong linear
correlation between B[a]P concentration and DNA adduct
levels has been reported in these cells (Van Agen et al., 1997).
All these ®ndings indicate that human bronchial epithelial cell
lines are suitable for in vitro studies in order to investigate
chemical-induced lung carcinogenesis.
The aim of the present study was to analyze the metabolic
activation and DNA binding of PAH compounds derived from
diesel and gasoline particulate extracts in a human bronchial
epithelial cell line (BEAS-2B). The particulate extracts were
measured for 14 selected PAH, of which 10 were DNA adductforming compounds, and the correlation with adduct levels was
determined. Further, dose-dependent metabolic activation by
BEAS-2B cells to form PAH DNA-binding metabolites from
gasoline extracts and Standard Reference Materials (SRM)
1650 and 1587 and time-dependent DNA adduct formation by
B[a]P was studied.
Materials and methods
Chemicals and reagents
Micrococcal nuclease (from Staphylococcus aureus) was purchased from
Sigma Chemical Co. (St Louis, MO). Calf spleen phosphodiesterase was from
Calbiochem (Darmstadt, Germany) and T4 polynucleotide kinase was from
United States Biochemical (Cleveland, OH). [g-32P]ATP (7000 Ci/mmol) was
obtained from ICN Biochemicals (Costa Mesa, CA). Polyethyleneimine (PEI)±
cellulose thin layer chromatography (TLC) plates were purchased from
Macherey-Nagel (Duren, Germany). Standard Reference Materials (SRM
1650a diesel particulate matter and SRM 1587 nitrated polycyclic aromatic
hydrocarbons) were supplied by the National Institute of Standards and
Technology (NIST) (Gaithersburg, MD) and EPA 610 PAH mixture containing
16 PAH compounds was from Ehrenstorfer (Ausburg, Germany). B[a]P was
from Accu Standard (New Haven, CT). BEAS-2B cells (CRL-9609) were
obtained from the American Type Culture Collection and bronchial epithelial
cell growth medium (BEGM) from Cytotech ApS (Hùrsholm, Denmark). All
other chemicals and solvents were of analytical grade.
Emission test and particulate sampling
Exhaust particulate matter was collected from two passenger cars (Nissan
Micra 1.3, 1995 model and Mitsubishi Carisma 1.8, 2000 model) using
reference gasoline CEC-RF-02-99 (sulfur < 50 mg/kg, total aromatics
maximum 35%). The Nissan was equipped with a multipoint port fuel injection
(MPI) and the Mitsubishi with a GDI engine. Details of the particle sampling
and modi®cations of the emission tests have been previously described (Kokko
et al., 2000). Exhaust particles were collected from the Nissan at a driving
speed of 120 km/h (samples G1 and G6) and from the Mitsubishi at 50 (G3 and
G5) or 120 km/h (G4) through a dilution tunnel on Te¯on-coated glass®ber
®lters (Pall¯ex Fiber®lm T60A20, diameter 142 mm, pore size 0.3 mm). In
addition to the ®lters, polyurethane foam (PUF) was used to collect semivolatile compounds from three of the gasoline exhaust samples (G1, G3 and
G6). Filter and PUF samples were collected during the same emission tests by
positioning the PUF sampling holder downstream of the PM ®lters. In order to
430
obtain suf®cient particulate material for the biological tests, the emission tests
were repeated several times and the extracts were combined. Diesel exhaust
particles were collected from a passenger car (Toyota, indirect injection diesel
engine without any oxidative catalyst) using two reformulated fuels (RD1 and
RD2) and standard European fuel (EN97) during modi®ed ECE/EUDC tests.
Details of the diesel fuels and the test runs have been reported elsewhere
(Kuljukka et al., 1998; Isotalo et al., 2002). The particulate samples of gasoline
and diesel exhausts were extracted with dichloromethane using a Soxhlet
apparatus. To be able to use the samples in the biological tests, the
dichloromethane of SOF was exchanged for dimethylsulfoxide (DMSO)
under nitrogen.
Determination of PAH in gasoline particulate extracts
The extracts of the particulate and PUF samples were puri®ed for the PAH
analyses using high performance liquid chromatography (HPLC). PAH
concentrations in samples G1 and G1-PUF were not analyzed due to the
identical parallel emission tests performed for samples G6 and G6-PUF. The
extracts were spiked with internal standards (d10-pyrene, b,b-binaphthyl and
indeno[1,2,3-cd]¯uoranthene) and EPA 610 PAH mixture was used as the
calibration standard. The concentrations of 14 selected PAHs in the particulates
were analyzed by GC-MS(SIM) using a DB-17 capillary column (Kokko et al.,
2000).
Cell culture and treatment of BEAS-2B cells
BEAS-2B cells were maintained in 5 ml of BEGM medium at +37°C in a
humidi®ed atmosphere with 5% CO2. The basal medium (500 ml) supplemented with 26 mg bovine pituitary extract, 2.5 mg insulin, 0.25 mg human
epidermal growth factor, hydrocortisone and epinephrine, 3.25 mg triiodothyronine, 5 mg transferrin, 50 ng retinoic acid and 100 U/ml penicillin/
streptomycin was changed every 2±3 days. Cell viability of BEAS-2B cells was
determined by counting the number of living and dead cells using trypan blue
after 48 h exposure to 2.5, 5.0, 7.5 and 10.0 mM B[a]P, 150 mg SRM 1650/ml
medium and one gasoline particulate extract (200 mg/ml medium). DMSO was
used as a negative control. Cell exposures were carried out twice to nearly
con¯uent cell cultures (about 5 000 000 cells per T-25 growth bottle). Timedependent adduct formation of 2.5 and 5.0 mM B[a]P was measured during the
48 h at several time points. Cells were exposed twice to concentrations of 100
and 37±47 mg/ml medium of eight gasoline and three diesel particulate extracts,
respectively. BEAS-2B cells were exposed for 48 h to 15±150 mg/ml SRM
1650, 1±10 ml/ml SRM 1587 and 20±400 mg/ml medium G1 and G1-PUF
extracts and the dose-dependent adduct formation was detected. DMSO was
used as a negative control.
DNA isolation and 32P-post-labeling of DNA adducts
DNA was isolated from the exposed and control cells. First, cells were puri®ed
twice with phosphate-buffered saline and lysed in 1 ml of lysis buffer (50 mM
Tris±HCl pH 8.5, 1 mM EDTA, 0.5% Tween) at 55°C for 2 h with gentle
agitation. After lysis, proteinase K (0.2 mg/ml) was added and incubated at
55°C overnight until the solution was clear. RNA was digested by adding
RNase A (0.2 mg/ml) and 0.1% SDS and incubating at 37°C for 1 h. After 2 min
agitation, the proteins were precipitated with 1.7 M NaCl and centrifuged at
5000 r.p.m. for 15 min. Supernatant was removed to a new tube and DNA was
precipitated with a double volume of absolute ethanol at ±20°C for 1 h. DNA
was washed twice with 70% ethanol and dissolved in distilled water. DNA
concentrations were measured with an UV spectrophotometer (A260 nm/
A280 nm). DNA (4 mg) was analyzed for bulky aromatic DNA adducts using the
32P-post-labeling method enhanced with butanol extraction (Gupta, 1993).
DNA was digested to mononucleotides with micrococcal nuclease (0.04 U) and
spleen phosphodiesterase (0.08 U) at 37°C for 3.5 h. The modi®ed nucleotides
were enriched by butanol extraction and DNA adducts were labeled using T4
polynucleotide kinase (4.8 U) and 40 mCi [g-32P]ATP. After two-dimensional
chromatographic separation on PEI±cellulose TLC plates, DNA adducts were
visualized by autoradiography and quanti®ed based on speci®c activity of the
[g-32P]ATP (Reddy and Randerath, 1986). Each DNA sample was analyzed
three times by 32P-post-labeling assay and the average adduct levels and the
standard deviations were calculated.
Results
Autoradiograms resulting from the 32P-post-labeled DNA
adducts formed in BEAS-2B cells exposed to B[a]P, SRM
1650, SRM 1587, diesel particulate extract (RD2) and gasoline
particulate extracts (G6, G6-PUF) are shown in Figure 1A±F.
B[a]P eluted as a single spot (Figure 1A), whereas SRM 1650
and 1587 formed a diagonal radioactive zone (DRZ) of PAH
and nitro-PAH adducts (Figure 1B and C). Diesel and gasoline
DNA treated with diesel and gasoline particulate
Fig. 1. Autoradiograms of TLC maps of 32P-post-labeled DNA adducts enhanced with butanol extraction. Each panel shows the adduct pattern derived from
B[a]P (A), SRM 1650 (B), SRM 1587 (C), RD2 diesel extract (D) and G6 and G6-PUF gasoline extracts (E and F) after exposing BEAS-2B cells for 48 h.
Autoradiography was at ±70°C for 3 days.
particulate extracts formed less intense areas of DRZ than
SRMs and three to four intense spots could be observed
(Figure 1D±F). For the quantitative analysis of PAH±DNA
adducts, the area of DRZ and background was excised from
PEI±cellulose TLC plates and the radioactivity was determined
by scintillation counting.
When analyzing DNA adducts, possible loss of adducts
has been reported during the isolation and digestion of
DNA
to
2¢-deoxyribonuclease
3¢-monophosphates
(Hemminki et al., 1993). In this study we lysed the cells
and digested proteins with proteinase K during an
overnight incubation at 55°C. This is a longer treatment
at a slightly higher temperature than is usually performed
in DNA isolation (1±2 h, 37±50°C), but we used it in
order to digest all proteins and to have less background in
the post-labeling assay. In a separate experiment we have
observed that this DNA isolation procedure did not affect
human lymphocyte adduct levels compared with DNA
isolation using Qiagen genomic tips according to the
manufacturer's handbook (unpublished data).
431
S.K.Pohjola et al.
Table I. Concentrations of selected PAH compounds and B[a]P (ng/mg PM) in the gasoline and diesel extracts and the amounts of particulate matter (PM)
emitted per kilometer during the test (mg/km)
Sample
Gasoline extracts
G3
G3-PUF
G4
G5
G6
G6-PUF
Diesel
RD2
RD1
EN97
SRM 1650
14 selected PAH
(ng/mg PM)
10 DNA adduct-forming
PAH (ng/mg PM)
B[a]P
(ng/mg PM)
315.6
1277
241.6
181.0
237.3
4322
219.5
291.1
63.4
141.0
198.0
1298
2.24
3.11
0.36
2.80
15.9
12.4
1015
1266
1356
207.4a
486.2
524.2
568.4
104.0
24.5
28.3
20.4
1.33
PM emitted
(mg/km)
0.46
15.3
1.10
1.22
42.5
64.5
97.0
G1 and G1-PUF samples were not analyzed due to the identical parallel emission test performed for sample G6 and G6-PUF.
concentrations of 13 PAHs (excluding ¯uorene) according to the National Institute of Standards and Technology (NIST).
aCerti®ed
Fig. 2. Time-dependent activation of B[a]P in BEAS-2B cells. Cultures were treated with 2.5 and 5.0 mM B[a]P for 0, 3, 8, 12, 24 and 48 h and DNA was
isolated for post-labeling measurement of DNA binding.
PAH concentrations in the extracts
Table I shows the sum of 14 selected PAH compounds
(¯uorene, anthracene, phenanthrene, pyrene, ¯uoranthene,
benzo[a]anthracene, chrysene, B[a]P, benzo[e]pyrene, benzo[b]¯uoranthene, benzo[k]¯uoranthene, indeno[1,2,3-cd]pyrene, benzo[ghi]perylene and dibenz[a,h]anthracene), of which
the last 10 are DNA adduct-forming compounds (IARC, 1983;
Reddy et al., 1984; King et al., 1994; Wang et al., 1995). The
concentration of B[a]P in gasoline and diesel extracts and the
amounts of PM emitted per kilometer, including the certi®ed
concentration of 13 PAH compounds (excluding ¯uorene) in
SRM 1650 diesel extract according to the NIST, are also shown
in Table I. The PAH concentrations in diesel extracts were
analyzed earlier by Isotalo et al. (2002) and are presented here
per mg PM. Concentrations of PAH compounds per mg PM
were calculated for both ®lter and PUF samples using the PM
of ®lter samples due to the small amount of particles obtained
from PUF samples.
The highest PAH concentrations were measured in two
gasoline extracts collected on PUF. Low molecular weight
432
PAH (equal to or less than pyrene) dominated, at over 90%,
PUF samples, whereas 10 DNA adduct-forming PAH compounds represented less than 30% of the 14 PAH compounds.
In extracts obtained from the gasoline ®lters, except for sample
G4 (26%), the 10 DNA adduct-forming PAH compounds
constituted over 70% of the PAH compounds analyzed. The
concentrations of the 14 PAH compounds in diesel extracts
were 3±7 times higher than those in gasoline extracts collected
on ®lters. Diesel particulate extracts obtained from two
reformulated and standard European diesel exhaust samples
contained 41±48% of 10 DNA adduct-forming PAH compounds. The amounts of B[a]P ranged from 0.4 to 16 and from
20 to 28 ng/mg in the gasoline and diesel particulate extracts,
respectively. The certi®ed concentrations of 10 DNA adductforming PAH compounds in SRM 1650 was about 5-fold lower
and the concentration of B[a]P was nearly 20-fold lower than
in SOF obtained from the reformulated and standard diesel
exhaust particles. Gasoline fuel emitted particulate matter in
the range 0.46±15.3 mg/km. The highest emission of 15.3 mg/
km was obtained from the GDI engine (Mitsubishi) when
DNA treated with diesel and gasoline particulate
Fig. 3. Dose-dependent effect of B[a]P on DNA adducts formed in BEAS-2B cells after exposure to 0.5, 1.2, 2.5 and 5.0 mM of B[a]P for 48 h.
tested at 120 km/h (G4), while the emission of the MPI engine
(Nissan) at the same speed was only 1.22 mg/km (G6).
Reformulated diesel fuels emitted particulate matter 3- to 140fold and standard reference diesel fuel 6- to 210-fold more than
gasoline.
Cytotoxicity
The cytotoxicity of B[a]P, SRM 1650 and gasoline particulate
extracts in BEAS-2B cells was tested after 48 h exposure.
When the numbers of living and dead cells at the given
concentrations of B[a]P and particulate extracts were counted,
no decreases in the cell counts in comparison with the control
treatment were detected (data not shown).
Time- and dose-dependent adduct formation by B[a]P
Figure 2 shows the levels of DNA adducts in BEAS-2B cells at
3, 8, 12, 24 and 48 h exposure to 2.5 and 5.0 mM B[a]P. After 3,
8 and 12 h incubations DNA binding was almost at the same
level with both concentrations tested. A 4-fold increase in
adduct formation for both concentrations was obtained after 24
h incubation, increasing only slightly after 48 h. The highest
adduct levels were formed with 2.5 mM B[a]P after 48 h
exposure, which was about 1.8-fold higher than that detected
with 5.0 mM B[a]P. The dose-dependence of adduct formation
after exposure to 0.5, 1.2, 2.5 and 5.0 mM B[a]P for 48 h is
shown in Figure 3. A slight change in adduct levels from 0.5 to
1.2 mM B[a]P was observed, whereas the highest adduct levels
were formed when BEAS-2B cells were exposed to 2.5 mM. In
comparisons of the 1.2 mM dose of B[a]P with the 2.5 and 5 mM
doses, 4- to 2-fold increases in DNA binding, respectively,
were detected.
Adduct formation by Standard Reference Materials (SRM)
and gasoline extracts
DNA adducts formed as a result of treatment of BEAS-2B cells
for 48 h with two extracts of SRM were analyzed by the 32Ppost-labeling assay. SRM 1650 is an extract from a heavy
powered diesel engine containing complex mixtures of compounds including PAH and nitro-PAH compounds. SRM 1587
is a certi®ed mixture of seven nitrated polycyclic hydrocarbons
containing 2-nitro¯uorene, 9-nitroathracene, 3-nitro¯uoranthene, 1-nitropyrene, 7-nitrobenz[a]athracene, 6-nitrochrycene and 6-nitrobenzo[a]pyrene. Figure 4 shows the levels of
Fig. 4. Adduct formation in BEAS-2B cells after 48 h exposure to SRM
1650 diesel particulate matter (15±150 mg/ml) (A) and SRM 1587 nitro-PAH
compounds (1±10 ml/ml) (B). The amounts of 42 reference PAH
concentrations in SRM 1650 and seven nitro-PAH compounds in SRM 1587
for each dose are given below the bars.
433
S.K.Pohjola et al.
Fig. 5. PAH±DNA adduct formation in BEAS-2B cells after exposure to gasoline particulate extracts obtained from particles (G1) and the semi-volatile fraction
(G1-PUF) collected on a Te¯on-coated glass®ber ®lter and polyurethane foam (PUF), respectively. BEAS-2B cell cultures were treated for 48 h with DMSO, as
a negative control, and with doses of 20, 50, 100, 200 and 400 mg/ml particulate matter.
PAH and nitro-PAH adducts formed in BEAS-2B cells after
exposure to SRM 1650 (Figure 4A) and SRM 1587 (Figure 4B).
Adduct levels increased only slightly when cells were exposed
to SRM 1650 at concentrations from 15 to 150 mg/ml (from 7 to
11 adducts/108 nucleotides) and after exposure to SRM 1587 at
concentrations from 1 to 10 ml/ml (from 11 to 19 adducts/108
nucleotides). SRM 1587 formed nearly 2-fold more DNA
adducts than SRM 1650 at the concentrations tested.
PAH compounds derived from extracts of gasoline particles
(G1) and the semi-volatile fraction (G1-PUF) were shown to be
activated in BEAS-2B cells to DNA-binding metabolites
(Figure 5). The cells were exposed for 48 h to gasoline
particulate extracts at concentrations of 20, 50, 100, 200 and
400 mg PM/ml medium. The adduct levels rapidly increased
from 4 and 6 to 20 adducts/108 nucleotides when the exposure
concentration increased 5-fold, showing no increase in adduct
levels thereafter. Extracts of the semi-volatile fraction (G1PUF) formed 2-fold more adducts with the 20 and 50 mg/ml
doses than the corresponding ®lter extract (G1), but at higher
doses no differences in adduct levels were detected. After
adding the PUF extract to the cell culture at a concentration of
>50 mg/ml, the extract gradually started to precipitate, however, no precipitation of the ®lter extract was observed.
DNA adducts formed by gasoline and diesel extracts
BEAS-2B cell cultures were treated twice for 48 h with ®ve
gasoline particulate, three semi-volatile and three diesel
particulate extracts to measure PAH±DNA adducts by the
32P-post-labeling assay. Table II shows adduct levels (adducts/
108 nucleotides) which are calculated per exposure dose (37±
100 mg PM/ml), per ng 10 DNA adduct-forming PAH
compounds in the exposure dose and per mg PM emitted per
test kilometer. Table II also includes adduct levels formed by
SRM 1650 calculated per exposure dose (75 mg PM/ml) and
per ng 10 DNA adduct-forming PAH. Gasoline extract G6PUF formed the highest (291 adducts/108 nucleotides/mg PM)
and G4 the lowest level of adducts (50 adducts/108 nucleotides/
mg PM). PUF samples formed on average 2-fold more adducts
per exposure dose than gasoline ®lter samples, and diesel
434
Table II. Levels of PAH±DNA adducts (adducts/108 nucleotides) in BEAS2B cells after treatment with gasoline and diesel extracts and SRM 1650 for
48 h
Sample
Gasoline extracts
G1
G1-PUF
G3
G3-PUF
G4
G5
G6
G6-PUF
Diesel
RD2
RD1
EN97
SRM 1650
Adduct levelsa
(A)
(B)
200
199
143
149
50.4
82.7
93.2
291
1.0
0.15
0.65
0.51
0.79
0.59
0.47
0.22
65.1
44.1
84.4
141
0.13
0.08
0.15
1.4
(C)
266
264
65.6
68.6
770
91.0
114
355
2767
2845
8186
aAdduct levels were calculated (A) per mg of particulate matter (PM), (B)
per ng 10 DNA adduct-forming PAH and (C) per mg PM emitted per test
kilometer.
particulate samples formed on average 2-fold less adducts than
gasoline particulate samples. When adduct levels were calculated per ng 10 adduct-forming PAH compounds, gasoline
sample G1 formed the highest level (1.0 adducts/108 nucleotides/mg PM/ng 10 PAH) and reformulated diesel sample RD1
the lowest levels of adducts (0.1 adducts/108 nucleotides/mg
PM/ng 10 PAH). Gasoline particulate samples formed about
2.4-fold more adducts than PUF samples and 6-fold more than
diesel particulate samples, when adduct levels were calculated
per ng 10 adduct-forming PAH compounds in the exposure
dose. DNA binding of PAH compounds in SRM 1650 was
more effective than those in gasoline and diesel extracts,
showing that 2- and 11-fold more adducts were formed by
SRM than by gasoline and diesel particulate extracts, respectively. When the adduct levels were calculated taking into
DNA treated with diesel and gasoline particulate
account the dose and PM emission per test kilometer,
particulate extracts of reformulated diesels (RD2 and RD1)
formed 11-fold more and standard diesel (EN97) 31-fold more
adducts than gasoline particulate extracts. Gasoline PUF
extracts formed about the same level of adducts as the
corresponding ®lter extracts. Only gasoline sample G4 formed
a higher level of adducts than the other gasoline extracts due to
the high particulate emission rate of this sample (15.3 mg PM/
km).
Discussion
The formation of DNA adducts by PAH compounds derived
from gasoline and diesel exhaust particles and B[a]P in human
bronchial epithelial cells was studied. Dose-dependent adduct
formation by B[a]P, SRM 1650 and SRM 1587 reference
materials and DNA binding by several gasoline and diesel
extracts were also compared, with the adducted nucleotides
detected by the 32P-post-labeling assay with butanol extraction
enhancement (Gupta, 1993). The butanol enhancement procedure was used in order to be able to analyze bulky adducts,
including those formed by nitro-PAH. Nitrated PAH compounds present in diesel exhaust are mutagenic and carcinogenic and have been shown to account for about half of the
mutagenic activity of diesel emissions (Rosenkranz, 1996;
Zwirner-Baier and Neumann, 1999). Human bronchial epithelial cells have been shown to activate carcinogenic PAH such
as B[a]P and 1-nitropyrene to DNA adduct-forming metabolites (Harris et al., 1982; Jeffrey et al., 1990; Van Agen et al.,
1997).
Our results were in accord with the Mollerup et al. (2001)
study, in which they showed that cytochrome P450 CYP1A1
and CYP1B1 enzymes were expressed in BEAS-2B cells in a
dose-dependent manner, when exposed to 0.1 and 1 mM B[a]P.
Van Agen et al. (1997) also showed a strong linear correlation
between 4±1000 nM B[a]P concentrations and DNA adduct
formation in the BEAS-2B cell line, indicating a high
metabolic capacity to form benzo[a]pyrene diol epoxide
(BPDE) from parent B[a]P. In our study, the adduct levels
increased time-dependently after exposure to 2.5 and 5.0 mM
B[a]P, however, treatment of the cells with a high dose of
B[a]P (5 mM) formed fewer adducts, indicating either a loss of
viability of the cells or inhibition of the activity of cytochrome
P450 enzymes (Mollerup et al., 2001). During the 48 h
exposure to B[a]P, the adduct levels in BEAS-2B cells did not
decrease, showing that DNA-binding BPDE metabolites were
still formed. BEAS-2B cells express the p isomer of GST when
detoxifying PAH metabolites by conjugation with glutathione
(Mace et al., 1994), and the metabolites of B[a]P induce
unscheduled DNA synthesis in human bronchial epithelium,
leading to active DNA repair (Ishikawa et al., 1984; Doolittle
et al., 1985). Not only metabolic activation by phase I P450
enzymes, but also the activities of phase II detoxifying
enzymes, such as GST, and DNA repair have to be taken
into consideration when the net effect of DNA adduct
formation in bronchial epithelial cells is evaluated.
Adduct levels in BEAS-2B cells were almost 2-fold higher
after exposure to SRM 1587 than to SRM 1650. The
autoradiograms of TLC maps between these samples were
nearly identical, showing a DRZ of adduct pattern (Figure 1B
and C). The seven pure nitro-PAH compounds in SRM 1587
have more favorable conditions to form DNA-binding
metabolites in BEAS-2B cells compared with the mixture of
PAH compounds in SRM 1650 diesel particulate extract. The
reference concentration of 42 PAH compounds analyzed by the
NIST in SRM 1650, including 1-nitropyrene, was 48.3 ng in an
exposure dose of 75 mg/ml and the certi®ed concentration of
seven nitro-PAH compounds was 44.5 ng in the lowest
exposure dose of SRM 1587 (1 ml/ml). If these exposure
doses containing almost the same amounts of PAH or nitroPAH compounds are compared, the same levels (11 adducts/
108 nucleotides) of DNA adducts were formed (Figure 4).
However, SRM 1650 contains other PAH compounds than
those 42 analyzed and the effect of unidenti®ed PAH
compounds on the DNA adduct-forming potency could not
be analyzed. Nevertheless, in SRM 1650 there are many
compounds that do not react with DNA, such as ¯uorene,
including those that have as yet unknown effects on the activity
of P450 metabolizing enzymes.
The components of a complex mixture may interact with
each other to produce synergistic, antagonistic or additive
effects. Synergistic effects have been reported when the
mutagenicity of binary mixtures of B[a]P and several
polyaromatic compounds were studied (Hass et al., 1987;
Donnelly et al., 1990). Lee et al. (1994) showed that the
mutagenicity of and DNA adduct formation by a binary
mixture of 1-nitropyrene (1-NP) and B[a]P was lower than
those of and by 1-NP alone. In this study 126 ng pure B[a]P
(0.5 mM) formed 6 times more adducts than 0.2 ng B[a]P in
SRM 1650 extract (exposure dose 150 mg/ml). The same
exposure dose contained other PAH compounds, of which the
concentration of 10 DNA adduct-forming PAH compounds and
1-NP was 18.4 ng/ml. Due to the co-elution of several PAH±
DNA adducts on TLC plates, the synergistic or antagonistic
effects of DNA binding by single PAH compounds (e.g. B[a]P
and 1-NP) in complex mixtures requires further study. The
identi®cation of DNA adducts by HPLC or mass spectrometric
measurements have been shown to be promising (Carmichael
et al., 1992; Farmer and Sweetman, 1995; Savela et al., 1995;
Koganti et al., 2000).
In comparison with a human mammary carcinoma cell line
(MCF-7), the highest levels of B[a]P-related DNA adducts
(311 adducts/108 nucleotides) were formed after 24 h exposure
to 5 mM B[a]P (Kuljukka-Rabb et al., 2001), whereas in
BEAS-2B cells the highest adduct levels were found after 48 h
exposure to 2.5 mM B[a]P (395 adducts/108 nucleotides). In
BEAS-2B cells the metabolic activation of B[a]P was slower
but more intense than in MCF-7 cells, because after 24 h
exposure the adduct levels started to decrease in MCF-7 cells,
but in BEAS-2B cells the adduct levels continued to increase.
Also, DNA adducts formed by PAH compounds in SRM 1650
were 3- to 11-fold higher in BEAS-2B cells than in MCF-7
cells. This could be due to higher expression of PAHmetabolizing cytochrome P450 enzymes in bronchial epithelial
cells or less active DNA repair in BEAS-2B than in MCF-7
cells. Moreover, the DNA adducts in this study were analyzed
with butanol extraction, whereas in Kuljukka-Rabb's study, the
nuclease P1 enhancement procedure was used, which has been
shown to exclude small molecular weight aromatic adducts and
certain nitro-PAH±DNA adducts (King et al., 1994).
When BEAS-2B cells were exposed to several doses of
gasoline extracts obtained from the ®lters and PFU, higher
adduct formation was observed with the extract collected on
the PUF at doses of 20 and 50 mg/ml. When concentrations >50
mg/ml were used, the PUF samples started to precipitate in the
culture medium. The effective Soxhlet extraction could be a
435
S.K.Pohjola et al.
possible reason for this, indicating that some as yet unknown
components in the PUF were co-extracted into the SOF. Partial
precipitation of the PUF extract affects the availability of PAH
compounds to enter BEAS-2B cells. It remains to be
determined whether the components are derived from the
gasoline itself or formed during the Soxhlet extraction,
however, it was independent of pH.
Semi-volatile PAH, with molecular weights equal to or
lower than that of pyrene, have been shown to bind effectively
to the PUF (Westerholm et al., 1991). In our study, higher
concentrations of the 14 PAH and 10 DNA adduct-forming
PAH compounds were measured in the gasoline extracts
collected on PUF than those on ®lters. Gasoline particles
predominantly fall into the 10±300 nm diameter range (Maricq
et al., 1999). Due to the small particle size of gasoline exhaust,
Te¯on-coated glass®ber ®lters (pore size 300 nm) did not
adsorb all the small particles and some of them were trapped on
the PUF, situated downstream of the PM ®lter. This explains, in
addition to the semi-volatile PAH compounds, the high
concentrations of high molecular weight particulate-bound
PAH compounds in the PUF extracts. Westerholm et al. (1991)
studied the particulate- and semi-volatile-associated compounds in heavy duty diesel exhaust and they found 3-fold
higher PAH concentrations in the PUF adsorbent than in the
corresponding ®lter fraction. When comparing concentrations
of adduct-forming PAH compounds between the ®lter and PUF
samples of gasoline particulate extracts, the highest levels of 10
PAH compounds were detected in the G6-PUF sample,
whereas B[a]P concentrations did not vary between the
samples, except for gasoline ®lter sample G4, which contained
much less B[a]P than the other gasoline samples. The low
levels of carcinogenic PAH compounds in this gasoline sample
in comparison with the other samples is probably due to the
short (15 min) collection time of the particulate sample when
compared with the 40±160 min used for the collection of the
other samples. The concentrations of the 10 DNA adductforming PAH and B[a]P were higher in diesel samples than
those of gasoline (both PUF and ®lter), except for sample G6PUF. The concentrations of the 14 PAH compounds were also
3- to 7-fold higher in diesel than in gasoline extracts collected
on ®lters. This is in accord with other studies, in which PAH
compounds analyzed in diesel extracts were shown to be
between 5 and 10 times higher than those found in gasoline
extracts (Stenberg et al., 1983; Rantanen et al., 1996; Kokko
et al., 2000). It is noteworthy that the individual PAH
associated with the particulate and semi-volatile phases may
vary signi®cantly with the fuels, test cycles and collection
procedures used and the individual cars and thus caution is
warranted in the interpretation of the results. Standard diesel
fuel emitted 34 and 56% more particles in comparison with the
reformulated diesel fuels. The lowest amount of particulate
emission (0.46 mg/km) was measured when a direct injection
gasoline engine was tested at 50 km/h (G3). In a separate
emission test under similar conditions, over 2-fold higher
particulate emission was obtained from sample G5, whereas
the same engine produced particulate matter at 15.3 mg/km
when the velocity of the car was 120 km/h (G4). Therefore, if
PAH concentrations were calculated based on the emission
units (per mg PM emitted per km), all diesel fuel extracts
contained higher levels of the 14 PAH compounds (43±132 mg/
km) than gasoline extracts (0.12±5.8 mg/km). Emission rates
are in accord with the study by Durbin et al. (1999), in which
the particulate emission rates were between 0.006 and 17.2 mg/
436
km for 61 new gasoline vehicles and 9.6 and 1000 mg/km for
19 diesel vehicles.
Both gasoline and diesel exhaust extracts formed DNA
adducts in BEAS-2B cells. The adduct levels varied greatly
depending on how the results were expressed. The highest
differences between the gasoline and diesel samples were
obtained when adduct levels were calculated per mg PM
emitted per km. Reformulated and standard diesel particulate
extracts formed on average 11- and 31-fold more adducts than
gasoline particulate extracts. However, when adduct levels
were calculated per mg PM or per ng 10 adduct-forming PAH
compounds in the exposure dose, gasoline extracts formed
more adducts than diesel extracts. Therefore, gasoline
particulate extracts either contained more other DNA adductforming compounds than those 10 analyzed here or the
ef®ciency of PAH compounds to bind with DNA was greater
in gasoline than in diesel samples. PUF samples formed fewer
adducts than the corresponding ®lter samples when calculated
per ng 10 DNA adduct-forming PAH compounds, but more
when calculated per mg PM in the exposure dose. In an earlier
study we showed that DNA adduct levels were higher in calf
thymus DNA with diesel than gasoline extracts under oxidative
(+S9) and reductive (+XO) activation conditions and that
adduct levels were higher with the PUF than the ®lter extract
when adduct levels were calculated per exposure dose or per
test kilometer (Pohjola et al., 2003). It seems that PAHactivating enzymes are differently induced in BEAS-2B cells
than the enzymes in rat liver S9 and, therefore, DNA adduct
levels in these test systems are signi®cantly different.
Furthermore, when exposing cell cultures to vehicle extracts,
the ability of the soluble organic matter to penetrate into the
cells and activate DNA repair and detoxifying enzymes inside
the cells affects adduct levels. Therefore, exposing human cell
cultures to vehicle extracts rather than in vitro exposure of calf
thymus DNA can be used to assess the genotoxicity of PAH
compounds derived from vehicle exhausts.
DNA adducts calculated per mg PM correlated signi®cantly
with the sum of the 14 PAH and 10 DNA adduct-forming PAH
concentrations of gasoline and diesel extracts (R = 0.807, n = 9,
P = 0.009; R = 0.725, n = 9, P = 0.027), indicating that the postlabeling assay is an adequate method for analyzing PAHinduced DNA damage. Particulate matter emitted per kilometer
produced large differences in DNA adduct levels between the
gasoline and diesel extracts when adduct levels were calculated
on an emission basis (adducts/mg PM/km). Although diesel
extracts contained more particulate-associated PAH compounds than gasoline extracts, the DNA binding of particulate-bound PAH compounds was more effective in gasoline
than diesel extracts. Bronchial epithelial cells effectively
activated PAH compounds in diesel extracts and SRM 1650
and B[a]P to DNA-binding metabolites and thus can be used in
genotoxicity studies. BEAS-2B cells also contain nitroreductive activity, leading to the formation of high levels of adducts
derived from nitro-PAH compounds, shown to be highly
mutagenic in diesel exhaust emission.
Acknowledgements
Ms Nina Tamminen of the Finnish Institute of Occupational Health is thanked
for valuable assistance in this study. The staff of the Fortum Technology Centre
and Technical Research Centre of Finland are thanked for their technical
assistance. This study was supported by Technology Program Mobile2 of the
National Technological Agency.
DNA treated with diesel and gasoline particulate
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Received on December 24, 2002; revised on June 23, 2003;
accepted on June 30, 2003
438