carc$$0105
Carcinogenesis vol.18 no.1 pp.167–175, 1996
Benzo[a]pyrene coated ferric oxide and aluminum oxide particles:
uptake, metabolism and DNA binding in hamster pulmonary
alveolar macrophages and tracheal epithelial cells in vitro
Jacob Cheu, Glenn Talaska, Marian Miller, Carol Rice
and David Warshawsky1
Department of Environmental Health, University of Cincinnati,
PO Box 670056, Cincinnati, OH 45267-0056, USA
1To
whom correspondence should be addressed
Ferric oxide (Fe2O3) and aluminum oxide (Al2O3) particles
are widely encountered in occupational settings. Benzo[a]
pyrene (B[a]P), a well-characterized environmental
carcinogen, is frequently adsorbed onto particles. It has
been shown that B[a]P-coated Fe2O3 particles (B[a]P2
Fe2O3) significantly increased lung tumors in the hamster
in contrast to B[a]P-coated Al2O3 (B[a]P2Al2O3) or B[a]P
alone. In order to determine the genotoxic effects of these
particles on the metabolism of B[a]P, pulmonary alveolar
macrophages (AM) from male Syrian golden hamsters were
incubated with 5 µg (19.8 nmol) B[a]P-coated respirable size
(99% ,5 µm) Fe2O3 and Al2O3 particles with loads from
0.5 to 2.0 mg. Intracellular uptake of B[a]P by AM at 24 h
was higher with B[a]P2Fe2O3 than that of B[a]P alone
(P , 0.05) or B[a]P2Al2O3 (P , 0.05). Total B[a]P metabolism was significantly greater in AM exposed to B[a]Pcoated Fe2O3 at 1.0 and 1.5 mg than in the AM exposed to
B[a]p2al2O3 (0.5, 1.0 and 1.5 mg) (P , 0.05) or B[a]P alone
(P , 0.05). Similar significant differences for Fe2O3 relative
to Al2O3 and B[a]P alone were also apparent for total
dihydrodiols, quinones and phenolic metabolites. Co-administration of 5 µg α-naphthoflavone (α-NF, an inhibitor of
cytochrome P-4501A1 and P-4501A2) and 1023 M cyclohexene oxide (CO, an inhibitor of epoxide hydrolase) significantly reduced B[a]P metabolism in B[a]P2Fe2O3 (P ,
0.05) and B[a]P2Al2O3 (P , 0.05) treated groups relative to
B[a]P alone. AM were co-cultured with hamster tracheal
epithelial cells (HTE) and treated as described above for
metabolism studies to assess the DNA binding of B[a]P metabolites in the target cells, using 32P-postlabeling techniques.
Two adducts were observed that had chromatographic
behavior similar to 7R,8S,9S-trihydroxy-10R-(N2-deoxyguanosyl-39-phosphate)-7,8,9,10-tetrahydrobenzo[a]pyrene
[(1)-anti-BPDE-dG, adduct 1, major adduct representing 70–80% of total adducts] and 7S,8R,9R-trihydroxy-10S-(N2-deoxyguanosyl-39-phosphate)-7,8,9,10tetrahydrobenzo[a]pyrene [(2)-anti-BPDE-dG, adduct 2,
representing 20–30% of total adducts]. B[a]P2Fe2O3 treatment enhanced the levels of the two B[a]P2DNA adducts in
*Abbreviations: PAH, polycyclic aromatic hydrocarbons; Fe2O3, ferric
oxide; Al2O3, aluminum oxide; B[a]P, benzo[a]pyrene; AM, pulmonary
alveolar macrophages; HTE, hamster tracheal epithelial cells; HPLC,
high performance liquid chromatography; α-NF, α-naphthoflavone; CO,
cyclohexene oxide; (1)-anti-BPDE-dG, 7R,8S,9S-trihydroxy-10R-(N2-deoxyguanosyl-39-phosphate)-7,8,9,10-tetrahydrobenzo[a]pyrene; (2)-anti-BPDEdG; 7S,8R,9R-trihydroxy-10S-(N2-deoxyguanosyl-39-phosphate)-7,8,9,10-tetrahydrobenzo[a]pyrene, B[a]P2Fe2O3, B[a]P coated ferric oxide;
B[a]P2Al2O3, B[a]P coated aluminum oxide; RAL, relative adducted
labeling.
© Oxford University Press
the HTE compared with B[a]P2Al2O3 (P , 0.05) or B[a]P
alone. The inhibitors αNF and CO significantly reduced
total adduct levels in the HTE (P , 0.05) in the B[a]P and
B[a]P2Fe2O3 treatments as well as adduct 1 and adduct 2
levels. Our data suggest that the cocarcinogenic effect of
B[a]P2Fe2O3 relative to B[a]P-coated Al2O3 can be due to:
(i) the enhancement of B[a]P metabolism in AM by Fe2O3
associated with the increased uptake of B[a]P; and (ii)
augmentation of DNA adduct formation in epithelial cells.
Introduction
Polycyclic aromatic hydrocarbons (PAHs*) are generated
through incomplete combustion, cigarette smoke and industrial
processes (1,2). It has been shown that most PAHs in the
environment are associated with particles (3,4). Particles in
the atmosphere serve as condensation nuclei for environmental
PAHs and can increase their stability by preventing photooxidative degradation of chemicals (5). Iron oxide (Fe2O3) and
aluminum oxide (Al2O3) are particles commonly encountered
in occupational settings where PAHs are also present.
Data from animal experiments indicate that Fe2O3 or Al2O3
alone are incapable of inducing tracheal bronchiogenic carcinoma in the hamster. However, when Fe2O3 is combined with
benzo[a]pyrene (B[a]P), the incidence of lung tumors was
increased significantly compared with treatment of B[a]P alone
(6,7). The increased lung cancer rate was not observed when
B[a]P was co-administered with Al2O3 (7). Results from
studies of co-exposure of B[a]P and carbon black (8), flyash
(9), asbestos (10), iron oxide (11–13) and crude air particulates
(12), indicate that the alteration of B[a]P metabolism may
play an important role in long-term particle-associated pulmonary diseases.
Cytochrome P-450 enzymes have been shown to play the
major role in metabolic activation of B[a]P (14). 7R,8S,9S,10Repoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(1)-anti-BPDE],
one of the B[a]P metabolites produced by cytochrome P-450
activation, has been shown to be more mutagenic in mammalian
cells and more tumorigenic in animals than the isomers, (1)
and (–)-syn-BPDE and (2)-anti-BPDE (15,16). DNA adducts
are regarded as an internal dosimeter of carcinogen exposure
because they reflect the net effect of competing metabolic
activation, detoxification and the action of DNA repair processes (17). Data from in vitro and in vivo experiments have
demonstrated the positive correlation between persistence of
adduct formation and cellular transformation or tumor induction (18,19).
One of the key functions of pulmonary alveolar macrophages
(AM) is to maintain the sterility of pulmonary alveoli. Particles
are not only phagocytozed but may also undergo gradual
dissolution due to the phagolysosomes of AM (20). A fraction
of adsorbed PAHs are metabolized by AM and these metabolites
may be released into the surrounding tissues (21,22). Inhaled
particles and AM are cleared by the mucociliary escalator by
167
J.Cheu et al.
Table I. Physical characteristics of particles
Particle type
Fe2O3
Al2O3
% ,stated size
µma
98.9 ,5
91.5 ø1 µm
99 ,5 µmb
80 ,1 µm
Count median
diameter
1.2
µmc
0.3 µmc
Surface area
10.8
m2/ga
198.4 m2/ga
aSized
bSized
by Micromeritics (Norcross, GA) (33).
by NIOSH (Cincinnati, OH) 1992 (33).
cSized by author (J.C.) (34).
which ~24–120 3 106 human AM and 0.75 3 106 rat AM
are removed daily (23). During movement of the mucociliary
escalator, active metabolites may have the opportunity to
contact and interact with the cells of the epithelium along the
respiratory tract. Pathological studies indicate that 90% of
human bronchiogenic carcinoma are of epithelial origin (24,25).
The goal of this study was to investigate whether selected
particles influence uptake and metabolism of B[a]P and subsequent B[a]P2DNA adduct formation. To measure the effects
of particles on the uptake and metabolism of B[a]P, AM
lavaged from Syrian golden hamsters were incubated with
B[a]P or B[a]P-coated particles. Inhibitors for cytochrome P450 activation pathways were also used to determine the
extent of enzymatic modulation. In order to determine if the
involvement of AM in overall DNA adduct formation in the
pathological target epithelial tissues, hamster tracheal epithelial
cells (HTE) were cultured with or without AM in B[a]Pcoated particles.
Materials and methods
Chemicals
Radiolabeled B[a]P, 99% pure and verified by high pressure liquid chromatography (HPLC), was purchased from Amersham (Arlington Heights, IL)
([7,10-14C]B[a]P, 29.7 mCi/mmol). Unlabeled B[a]P was purchased from
Aldrich (Milwaukee, WI), purified by recrystallization from benzene isopropanol and monitored by HPLC and UV spectroscopy. Fe2O3 (certified grade,
red anhydrous, Lot no. 78156; Fisher Scientific, Cincinnati, OH) was from
the same lot used in the hamster carcinogenicity study (26). Al2O3 (alumina
adsorption, Lot 760031; Fisher Scientific Co., Fairlawn, NJ) was used as a
comparison to Fe2O3. The B[a]P metabolite standards were supplied by the
NCI Chemical Repository, including 9,10-dihydrodiol, 4,5-dihydrodiol, 7,8dihydrodiol, 9-hydroxy, 3-hydroxy, 7-hydroxy, 4,5-quinone and 3,6-quinone.
α-Naphthoflavone (α-NF) and cyclohexene oxide (CO) were purchased from
Sigma (St Louis, MO). In order to avoid photo-oxidation, all compounds
were handled under yellow light (12,13). 7R,8S,9S-trihydroxy-10R-(N2deoxyguanosyl-39-phosphate)-7,8,9,10-tetra-hydrobenzo[a]pyrene [(1)-antiBPDE-dG] was purchased from NCI (synthesized by Dr S.Amin of the
American Health Foundation). Dr Lawrence J.Marnett of Vanderbilt University
generously provided the following chemicals: 7S,8R,9R-trihydroxy10S-(N2-deoxyguanosyl-39-phosphate)-7,8,9,10-tetrahydrobenzo[a]pyrene
[(2)-anti-BPDE-dG] and 7S,8R,9R-trihydroxy-10S-(N6-deoxyadenosyl-3phosphate)-7,8,9,10-tetrahydrobenzo[a]pyrene [(2)-anti-BPDE-dA].
Animals
Male Syrian golden hamsters, 8–10 weeks old, 120–150 g, were obtained
from SASCO Co. (Indianapolis, IN) and housed one per cage in stainless
steel racks. Hamsters were fed ad libitum up to the time of killing. Animals
were maintained in an environment of 26 6°C, 45 6 5% relative humidity,
and a 12 h light2dark cycle.
B[a]P-coated particles preparation
A mixture of the [14C]B[a]P and non-radiolabeled B[a]P (19.8 nmol, 0.5 µCi)
was evaporated to dryness under a gentle stream of nitrogen to remove the
toluene (40°C). The dried B[a]P mixture was dissolved in acetone (50 µl)
and transferred to particles. Particles coated with B[a]P were dried in a sterile
20 ml glass vial. The B[a]P-coated particles were resuspended in RPMI-1640
medium and 2.5 ml medium was applied to each AM plate. The particles
168
were coated evenly as observed by the fluorescence of B[a]P using a
fluorescence microscope; in numerous (n 5 5) microscopic fields viewed
optically using the fluorescence microscope, each contained multiple particles,
all of which were observed to be coated evenly over the surfaces in view.
Alveolar macrophage cell culture
AM were isolated by lung lavage with cold sterile saline (calcium and
magnesium free). The lavage fluid was centrifuged at 400 g at room temperature
for 10 min. The cell pellets were resuspended in RPMI-1640 medium (Sigma)
containing 0.1% gentamycin, 25 mM L-glutamine, 0.2% sodium bicarbonate
and 2 mg/ml bovine serum albumin. An aliquot of the cell suspension was
removed to determine AM concentrations using a hemocytometer. AM
(1 3 106) were plated on a 35-mm plastic culture dish (Corning Glass Works,
Corning, NY) at 37°C in a humidified atmosphere with 5% CO2. After 1.5 h
the AM were washed twice with RPMI-1640 medium (total 4 ml) to remove
unattached cells. Aliquots of 2.5 ml of fresh RPMI-1640 medium containing
different concentrations of B[a]P-coated particles were administered to the
AM. After 24 h, the medium was removed and AM were washed twice with
a total of 4 ml fresh RPMI-1640 medium. The erythrosin B dye exclusion
assay was used to determine the cytotoxicity of B[a]P-coated particles (27).
Intracellular uptake of B[a]P in AM
Aliquots of 3 3 106 AM were treated with [14C]B[a]P, [14C]B[a]P-coated
Fe2O3 or [14C]B[a]P-coated Al2O3. At 24 h, the media were removed and the
plates washed three times with a total of 3 ml deionized H2O. AM were
removed by incubation with 0.5 ml/plate trypsin-EDTA (0.05% trypsin,
0.53 mM sodium EDTA; GIBCO, Grand Island, NY) for 30 min at room
temperature, the plates washed again three times with a total of 3 ml distilled
H2O and the cellular solutions collected in 20 ml scintillation vials. Two
milliliters of 12 N NaOH were added to each vial and incubated for 24 h at
60°C for lysis of AM. Two milliliters of tert-butylhydroperoxide were then
added to each vial, which were then incubated for an additional 1 h at 60°C
for decolorization (11). Scintillation cocktail (Scintisafe plus 50%; Fisher)
was added to each vial and the samples counted by liquid scintillation
spectrometry.
B[a]P metabolism
Aliquots of AM (3 3 106) were co-incubated with [14C]B[a]P-coated particles
(0.5–2.0 mg) or B[a]P-coated particles plus inhibitors for 24 h. The inhibitors
were α-NF (5 µm/plate, an inhibitor of P-4501A1 and 1A2) and CO (103 M/
plate, an inhibitor of epoxide hydrolase) (14,28,29). After incubation, the cell
culture media were collected and the AM rinsed twice with fresh cold
(1.5 ml) RPMI 1640 medium. The washes were added to the media, then
centrifuged at 400 g for 5 min to separate particles (with cell debris) and
culture media. AM were removed from the plate by incubation wth 0.5 ml/
plate 0.05% trypsin and 0.53 mM EDTA for 30 min at room temperature.
The amounts of B[a]P and its metabolites in both media and AM were
extracted with ethyl acetate (1:1 v/v, total 15 ml). The ethyl acetate extracts
were evaporated to dryness under a gentle stream of nitrogen at 40°C. The
dried samples were reconstituted in 60 µl chloroform prior to HPLC analysis.
An HPLC chromatogram of B[a]P metabolite standards was recorded prior
to running samples. Samples prepared in chloroform were chromatographed
on a Waters HPLC with UV detector set at 254 nm under the following
conditions: Whatman ODS-2, 10 µm C-18 column 4.6 mm 3 25 cm at
ambient temperature with a flow of 1 ml/min and a methanol2water gradient
from 80–100% over a 32 min period. The gradient was 80–82% methanol for
6 min, 82–90% methanol for 3 min, 92–100% methanol for 1 min, and 100%
methanol for 22 min. All extractions were carried out under yellow lights to
avoid photodecomposition (12,13). Activities of fractions were quantified on
a Tri-Carb Packard Liquid Scintillation Spectrometer.
Co-cultivation of hamster alveolar macarophages with hamster tracheal
epithelial cells
The co-cultivation of primary hamster AM with HTE (obtained from Dr
Brooke T.Mosmann, University of Vermont) was conducted in conventional
35 mm plastic culture dishes with cellular minipore inserts (0.4 µm; Millipore
Co., MA). These inserts prevented physical contact of cells but allowed
passage of metabolites into surrounding media. Approximately 3 3 106 HTE
were plated in a 35 mm culture dish containing 2.5 ml RPMI 1640 medium
(10% newborn calf serum) at 37°C with 5% CO2 for 3–4 h. After incubation,
the unattached HTE were removed by washing the plates with fresh RPMI1640 medium (twice 2 ml/each time). AM (3 3 106) isolated from hamster
were co-cultured with HTE by placing AM on the cellular minipore insert
inside the culture dish. After 1.5 h the medium was removed and cells were
then washed with fresh RPMI-1640 medium (twice 2 ml/each time) and
administered with various concentrations of B[a]P-coated particles. After 24 h
incubation, the medium and cellular insert were removed and the plates were
washed with fresh RPMI-1640 medium (twice 2 ml/each time). HTE were
Benzo[a]pyrene uptake and metabolism
Fig. 1. HPLC chromatogram of B[a]P metabolite standards. 9,10-diol, 9,10-dihydrodiol of B[a]P; 7,8-diol, 7,8-dihydrodiol of B[a]P; 4,5-diol, 4,5-dihydrodiol
of B[a]P; 9-OH, 9-hydroxy of B[a]P; 3-OH, 3-hydroxy of B[a]P; 7-OH, 7-hydroxy of B[a]P; 4,5-Q, 4,5-quinone of B[a]P; 3,6-Q, 3,6-quinone of B[a]P.
washed, removed and collected by incubation with 0.5 ml/plate trypsin2EDTA
for 30 min at room temperature.
Isolation of DNA and DNA adduct analysis from HTE
Statistical analysis
The Wilcoxon rank-sum test or the paired t-test was used to evaluate
differences among treatment groups based on the distribution of the data (32).
Cells were centrifuged at 125 g for 5 min. The cell pellets were suspended
in 1 ml of 1% SDS, 1 mM EDTA and 24 µl 1 M Tris, pH 7.4, for
homogenization. Then 24 µl RNase A (10 mg/ml; Sigma) and 8 µl RNase T1
(5 U/ µl; Sigma) were added and the mixtures were incubated at 37°C for
1 h. Next, 60 µl of proteinase K (10 mg/ml; Sigma) was added and the sample
mixtures were incubated at 37°C for 30 min. DNA was isolated by sequential
extraction using phenol (saturated with 10 mM Tris buffer, pH 7.4), 1:1
phenol:sevag (chloroform/isoamyl alcohol, 24:1) and sevag. The DNA was
precipitated by addition of 100 µl 4 M LiCl and 10 µl glycogen (30 µg/ µl)
and an equal volume of ice-cold ethanol. The DNA was washed twice with
70% ethanol, redissolved in 1/100 SSC-1 (Sorensens sodium citrate, 1.5 mM
NaCl, 0.22 mM trisodium citrate and 1 mM EDTA) solution.
DNA concentrations were determined spectrophotometrically (30). The 32Ppostlabeling assay was performed as described by Reddy and Randerath (31)
with some modifications. DNA was hydrolyzed to 39-phosphodeoxynucleotides
with micrococcal endonuclease (0.25 U) and calf spleen phosphodiesterase
(0.001 U) at 37°C for 3 h followed by digestion with nuclease P1 (3.5 U) at
37°C for 40 min to convert unmodified nucleotides to nucleosides. The
modified nucleotides were labeled with [γ-32P]ATP (100 µCi/sample) by
incubation with polynucleotide kinase (2.8 U) for 30 min at 37°C. The
postlabeled mixtures were applied to 20 3 20 cm PEI2cellulose sheets
(Alltech, Deerfield, IL). 32P-Postlabeling analysis was conducted using a
modified four-solvent developing system described by Talaska et al. (30).
Solvents used were: D1, 0.65 M sodium phosphate (pH 6.8); D3, 3.6 M
lithium formate containing 3.5 M urea (pH 3.5); D4, 0.8 M LiCl, 0.5 M
Tris2HCl contaning 8.5 M urea (pH 8.0); and D5, 1.5 M sodium phosphate
(pH 6.0). The adducts were visualized by autoradiography at 280°C on Kodak
XAR-5 film with intensifying screens. The spots on the PEI2cellulose sheets
detected by autoradiography were excised and radioactivities were determined
by liquid scintillation spectrometry. Recoveries and phosphorylation efficiencies were not determined so the calculated levels represent minimum values.
The levels of DNA adduct were estimated using the relative adduct labeling
(RAL). The RAL is defined as CPMadducts 3 109/specific activity of
ATP 3 (3240 pmol dNTP/µg) 3 µg DNA (31).
Results
Physical characteristics of particles
Fe2O3 and Al2O3 particles contain a substantial portion within
the respirable range, as evidenced by at least 98% being
,5 µm in diameter (33; Table I). The surface areas for Al2O3
and Fe2O3 were determined to be 198.4 and 10.8 m2/g
respectively (that is, the observed diameter not adjusted by
density to derive the aerodynamic diameter). The count median
diameters for Al2O3 and Fe2O3 were 0.36 and 0.32 µm
respectively, obtained in double deionized water by suspending
particles before deposition and analyzing with a scanning
electron microscope equipped with an X-ray spectrometer and
image analysis system (33). Since the medium (RPMI 1640)
contained amino acids, glucose and serum albumin that could
increase particle agglomeration, we used optical light microscopy (34) to size the particles that had been suspended in
culture medium. Particles were allowed to settle and the liquid
removed by drying. The results indicated that after suspension
in culture medium, the count median diameter of Fe2O3 was
4-fold greater than those prepared in deionized water (from
0.32 µm to 1.2 µm), whereas Al2O3 changed only slightly
(from 0.36 µm to 0.30 µm). This indicated that certain factors
in the culture medium might enhance agglomeration of Fe2O3
particles.
Uptake studies
At 24 h, the results of B[a]P uptake in AM, the total activity
associated with the lysis of AM indicated that B[a]P2Fe2O3
169
J.Cheu et al.
Table II. Alveolar macrophagea metabolites of B[a]P-coated Fe2O3 and B[a]P-coated Al2O3
Metabolites of B[a]P
9,10-Dihydrodiold
4,5-Dihydrodiol
7,8-Dihydrodiol
Total dihydrodiol
9-Hydroxy
3,7-Hydroxy
Total hydroxy
4,5-Quinone
3,6-Quinone
Total quinone
Total metabolitesf
a3
B[a]Pb alone Dose of Fe2O3
0.0 mg
0.5 mg
1.0 mg
(n 5 4c)
(n 5 3)
(n 5 5)
5.2
1.2
2.2
8.6
1.0
2.1
3.1
0.8
2.9
3.7
15.5
6
6
6
6
6
6
6
6
6
6
6
1.0e
0.4
0.4
1.2
0.2
0.2
0.3
0.2
0.8
0.9
1.5
5.6
2.0
2.1
9.7
2.5
3.6
6.0
1.0
7.6
8.6
24.4
6
6
6
6
6
6
6
6
6
6
6
1.3e
0.3
0.1
1.2
0.3
0.4
0.6g
0.4
1.8g
2.2g
1.6g
Dose of Al2O3
1.5 mg
(n 5 3)
6.5 6 0.8
7.9 6 1.6
2.5 6 0.2g
2.2 6 0.2
2.7 6 0.3
3.3 6 0.4
11.8 6 0.7g 13.5 6 1.3g
4.2 6 0.9g
4.4 6 0.8g
8.2 6 2.2g
6.6 6 0.5g
12.7 6 2.4g 11.0 6 0.6g
1.1 6 0.2
2.1 6 0.5
7.4 6 1.0g
7.7 6 1.1g
8.5 6 1.13g 9.8 6 0.6g
33.0 6 1.9g 34.3 6 1.9g
2.0 mg
(n 5 3)
1.7
0.7
1.4
3.9
2.4
6.3
8.7
1.3
6.2
7.5
20.1
6
6
6
6
6
6
6
6
6
6
6
0.5 mg
(n 5 4)
0.1
0.1
0.1
0.2
0.1
0.4g
0.4g
0.1g
0.7g
0.9g
1.5
1.0 mg
(n 5 4)
5.2 6 0.2
2.7
0.7 6 0.1i,j 0.7
i
1.7 6 0.1
1.7
7.7 6 0.2i
5.2
0.8 6 0.1i,j 0.9
1.8 6 0.1i,j 1.9
2.7 6 0.1i,j 2.9
0.49 6 0.1i 0.4
2.1 6 0.1i,j 2.0
2.6 6 0.1i,j 2.4
13.0 6 0.3i,j 10.6
6
6
6
6
6
6
6
6
6
6
6
0.3g,i
0.2i,j
0.1i
0.2g,i,j
0.1i,j
0.1i,j
0.2i,j
0.1i
0.2i,j
0.3i,j
0.3g,i,j
1.5 mg
(n 5 4)
2.0 mg
(n 5 4)
4.8 6 0.4
0.6 6 0.1i,j
2.2 6 0.4
7.7 6 0.8i
1.0 6 0.1i,j
2.0 6 0.1i,j
3.01 6 0.2i,j
0.9 6 0.1
2.7 6 0.7i,j
3.6 6 0.3i,j
14.3 6 1.1i,j
5.0
1.1
3.3
9.5
1.6
2.8
4.4
1.2
3.5
4.7
18.6
6
6
6
6
6
6
6
6
6
6
6
0.3
0.1i,j
0.3j
0.5i
0.1i,j
0.1i
0.2h,i,j
0.1
0.3i
0.3i
0.8h,i,j
3 106 AM/plate for 24 h.
bB[a]P (19.8 nmol).
cn 5 number of plates
which represent an aliquot of pooled cells.
ethyl acetate extractable metabolites.
dSum of intracellular and extracellular
epmol metabolite 6 SE/106 AM.
fSum of dihydrodiols, phenols and quinones.
gSignificantly different when compared with B[a]P
alone (P , 0.05).
hSignificantly different when compared with 0.5 mg Al O treatment (P , 0.05).
2 3
iSignificantly different when compared with Fe O treatment at 1.0 or 1.5 mg.
2 3
jSignificantly different when compared with Fe O at 0.5 mg.
2 3
(960 6 51 pmol/106 AM) significantly increased the uptake
of B[a]P compared with B[a]P alone (307 6 36 pmol/106
AM) or B[a]P2Al2O3 particles (249 6 21 pmol/106 AM)
(P , 0.05).
Metabolism studies
The metabolites identified compared to standards were the
4,5-, 7,8- and 9,10-dihydrodiols, the 3-, 7- and 9-hydroxy and
4,5- and 3,6-quinones of B[a]P (Figure 1). The metabolism
reached a maximum at a dose of 1.5 mg Fe2O3 and decreased
at a 2.0 mg level of Fe2O3 (Table II). A similar dose-dependent
trend was observed for B[a]P2Al2O3 with the maximum
metabolism at 2.0 mg Al2O3. Total metabolism at 2.0 mg
Al2O3 was statistically different from 0.5 mg Al2O3. Total
B[a]P metabolism was significantly greater in AM exposed to
B[a]P2Fe2O3 at 1.0 mg and 1.5 mg than in the AM exposed
to B[a]P2Al2O3 (0.05 mg, 1.0 mg and 1.5 mg) (P , 0.05) or
B[a]P alone (P , 0.05) (Table II). Similar specific differences
for Fe2O3 relative to Al2O3 and B[a]P alone were also apparent
for total dihydrodiol, quinones and phenolic metabolites. The
apparent decrease in the metabolism of B[a]P at 2.0 mg Fe2O3
appears to be due to the loss of AM viability from 76 to 69%,
whereas no change of viability for Al2O3 (74–75%) was
observed at similar doses.
AM cells exposed to B[a]P2Fe2O3 (1.0 mg) generated more
4,5-dihydrodiol-B[a]P compared to cells exposed to B[a]P
alone or B[a]P2Al2O3 (Table II). At increasing doses of
Fe2O3, the production of 9,10-dihydrodiols was only increased
marginally. A significant increase of dihydrodiols was observed
in the AM cells exposed to B[a]P2Fe2O3 (1.0 and 1.5 mg)
compared with B[a]P alone. The production of 7,8-dihydrodiols
was less than that of 9,10-dihydrodiols in cells exposed with
either B[a]P2Fe2O3 or B[a]P2Al2O3 (Table II). At the levels
of 1.0 and 1.5 mg Fe2O3, the production of 3-, 7- and 9hydroxy was significantly greater than with B[a]P alone or
B[a]P2Al2O3. For the quinone metabolites, only 3,6-quinone
170
was significantly higher in AM cells exposed to B[a]P2Fe2O3
compared with B[a]P alone and B[a]P2Al2O3 (Table II).
Co-administration of α-NF (5 µm), an inhibitor of
cytochrome P-450s (1A1 and 1A2), significantly reduced the
total B[a]P metabolism in AM exposed to B[a]P2Fe2O3 and
B[a]P2Al2O3 by 64 and 55%, respectively (Figure 2). Coincubation with both CO (1023 M), an inhibitor of epoxide
hydrolase, and α-NF (5 µM), further decreased the total
metabolism of B[a]P in cells treated with B[a]P2Fe2O3 and
B[a]P2Al2O3 by 85% and 76% respectively (Figure 2). For
cells exposed to B[a]P alone, addition of both inhibitors
reduced total B[a]P metabolism by ~66%. In particular, the
addition of α-NF and CO significantly decreased the production
of dihydrodiols in the cells exposed to B[a]P alone,
B[a]P2Fe2O3 (1.0 mg) and B[a]P2Al2O3 (2.0 mg). For the
phenol metabolites, only B[a]P2Fe2O3 treatment was reduced
significantly (Figure 2).
Comparison of the metabolism of HTE with AM
In order to measure the metabolic capacity of HTE a sample
dose of B[a]P (5 µg) coated with either 1 mg Fe2O3 or 2 mg
Al2O3 was given to HTE and compared with AM under
the same conditions. The data showed that the total B[a]P
metabolism was significantly higher in AM than in HTE in
the presence of Fe2O3 particles (Figure 3). Both the production
of phenols and quinones was also significantly higher in AM
than HTE when B[a]P was coated on Fe2O3 particles. In
addition there was no significant change in B[a]P metabolism
with both HTE and AM in the presence of Al2O3 particles.
These data suggest that HTE have a different metabolic
capacity than that of AM for B[a]P. Similar data were obtained
for B[a]P alone in that AM metabolized B[a]P at a higher rate
in AM than HTE (not shown).
DNA adduct studies
Adduct pattern. 32P-Postlabeling with P1 nuclease enhancement
was performed to identify and quantify B[a]P2DNA adducts
Benzo[a]pyrene uptake and metabolism
Fig. 3. Comparative metabolism of B[a]P-coated particles between alveolar
macrophages and tracheal epithelial cells. Each bar represents the sum of
extracellular and intracellular B[a]P metabolites. B[a]P was given at a
single dose of 19.8 nmol (0.5 µCi) for all treatments. ‘Total’ is the sum of
dihydrodiols, phenols and quinones. B, B[a]P; F, Fe2O3; A, Al2O3; AM,
alveolar macrophages; HTE, tracheal epithelial cells. *Significantly different
when compared with B-F/HTE, B-A/HTE and B-A/AM.
(298 6 73), although this difference was not significant at the
0.05 level when compared with HTE exposed to B[a]P alone
(166 6 24) (Figure 6A). The total RAL for B[a]P2Fe2O3 in
HTE was higher compared with B[a]P2Al2O3 treatment (121
6 39), but this difference was not statistically significant at
P 5 0.05. Co-incubation with inhibitors reduced the total RAL
in each treatment. In particular, both inhibitors decreased total
RAL significantly in the HTE samples, which were treated
with B[a]P alone (from 166 6 24 to 44 6 11) or B[a]P2Fe2O3
(from 298 6 73 to 35 6 8) (P , 0.05). Inhibitors also reduced
the level of RAL on the samples that were treated with
B[a]P2Al2O3 (from 121 6 39 to 65 6 28), albeit with no
statistical significance (Figure 6A).
The quantitative pattern of these two adducts followed that
of total adducts in general. Both adduct 1 and adduct 2 were
significantly reduced by inhibitors in the treatment of B[a]P
or B[a]P2Fe2O3 (Figure 6B and C, P , 0.05). Adduct 1 and
adduct 2 appeared to be significantly higher in the presence
of B[a]P2Fe2O3 compared with B[a]P2Al2O3 or B[a]P alone
(P , 0.05).
Fig. 2. The effect of inhibitors on the metabolism of B[a]P in AM. Each
bar represents the sum of extracellular and intracellular B[a]P metabolites.
Culture conditions were the same as Table II. B[a]P was given at a single
dose of 19.8 nmol (0.5 µCi) for all treatments. Fe2O3, 1 mg; Al2O3, 2 mg;
α-NF, 5 µM; CO, 1023 M. ‘Total’ is the sum of dihydrodiols, phenols and
quinones. (A) B[a]P with α-NF and CO. (B) B[a]P2Fe2O3 with α-NF and
CO. (C) B[a]P2Al2O3 with α-NF and CO. *Statistically significant
compared with B[a]P (P , 0.05).
in HTE in co-culture with AM. The TLC maps in Figure 4
are representative of the autoradiographic patterns observed
from different samples analyzed. In general, there were two
main adducts in each sample. Adduct 1 and adduct 2 displayed
chromatographic behavior similar to the standards of the
(1) and (2)-enantiomers of anti-BPDE-dG (Figure 5). With
addition of the inhibitors (α-NF and CO), the adduct level
reduced significantly, yet the pattern of adducts did not change.
Quantitation of adducts. Total RAL for each sample is shown
in Figure 6. The highest level of DNA binding occurred with
HTE co-cultured with AM in the presence of B[a]P2Fe2O3
Discussion
Metabolism studies
AM are considered to be the primary defense mechanism
against inhaled particles because of their phagocytic, secretory
and migratory behavior. Several investigators have observed
the metabolic capacity of AM in several species, including
human (35), rodent (12), rabbit (12,13) and dog (8). Our data
indicate that B[a]P adsorbed onto particles is capable of being
phagocytozed (electron microscopy, data not shown) and
metabolized by AM. The B[a]P metabolites identified and
quantified in both the media and cells indicate that AM are
capable of metabolizing B[a]P on particles and releasing the
metabolites into the media.
Evidence for epoxides (4,5-oxide, 7,8-oxide and 9,10-oxide)
binding to DNA without further metabolism has been reported
(36,37). The final dihydrodiols also reflect, to some degree,
the production of epoxides. In vitro DNA binding studies
have demonstrated that 3- and 9-hydroxy B[a]P (37–40)
171
J.Cheu et al.
Fig. 4. 32P-Postlabeled B[a]P2DNA adducts present in hamster tracheal epithelial cells (3 3 106) following incubation (24 h) with AM (3 3 106) under
various conditions. B[a]P was given at a single dose of 19.8 nmol in all treatments. Inhibitors were: α-NF, 5 µM and CO, 1023 M. (A) B[a]P: OR, origin at
the lower left-hand corner of the chromatogram; chromatographic directions D3, D4 and D5. (B) B[a]P plus α-NF and CO. (C) B[a]P2Fe2O3 (1 mg). (D)
B[a]P2Fe2O3 (1 mg) plus α-NF and CO. (E) B[a]P2Al2O3 (2 mg). (F) B[a]P2Al2O3 (2 mg) plus α-NF and CO. The adducts are not corrected for their
DNA content.
and quinones (37,40) can be further converted to reactive
intermediates which bind DNA. Therefore, the greater magnitude in the overall B[a]P metabolism in the presence of Fe2O3
may pose higher potential for the interaction with DNA and
may subsequently cause adverse effects on the host in the
long-term exposure.
The enhancement of B[a]P metabolism in AM by Fe2O3
can be significantly reduced by α-NF and further reduced by
CO. A similar, although smaller, reduction of B[a]P metabolism
was seen in B[a]P2Al2O3 when either α-NF or α-NF and
CO were combined. Neither inhibitor revealed any sign of
cytotoxicity to either AM or HTE cells (data not shown).
In this study, ,1% of the total B[a]P metabolites were
water-soluble, which was consistent with our previous report
(11). It was assumed that these were conjugated B[a]P metabolites. Because of their low production, these metabolites
were not analyzed.
Bevan and Manger (41) have suggested that the main effect
of chrysotile and iron oxide in the metabolism of B[a]P from
rat microsomes is in altering rates of uptake of particle-adsorbed
B[a]P rather than enzymatic induction of aryl hydrocarbon
hydroxylase. This suggestion is consistent with our uptake
data. At 24 h, the intracellular uptake of [14C]B[a]P in AM
was significantly higher in cells exposed to B[a]P2Fe2O3
relative to cells exposed to B[a]P alone or B[a]P2Al2O3.
Phagocytosis is typically characterized by uptake of particulate
172
material .1 µm in diameter (42,43). Fe2O3 particles appeared
to agglomerate when deposited in culture medium (from
0.32 µm versus 1.2 µm), whereas Al2O3 particles remained
dispersed (0.36 µm versus 0.30 µm). Based on the criteria of
phagocytosis, the agglomeration of Fe2O3 particles may
become more ‘accessible’ for phagocytosis in the culture
condition than Al2O3. Therefore more Fe2O3 may have been
engulfed by AM thus increasing the amount of B[a]P within
the cell for metabolism. It is possible then, in addition to
increasing uptake, that Fe2O3 particles may induce P-450
enzymes or decrease the turnover rate of P-450 enzymes.
However, the only data supporting modulation is the significant
suppression of B[a]P metabolism in the presence of inhibitors,
an event which occurs independent of the particle type.
DNA binding
AM with active B[a]P metabolites can reach the tracheobronchial regions via the mucociliary escalator decrease, and
interact with epithelial DNA. It has been shown that AM,
which contained phagocytozed B[a]P2Fe2O3, migrated to
the tracheobronchial bifurcation in vivo, forming clusters of
quiescent AM. The most severe changes in the lung tissue
were observed adjacent to the AM clusters (44). Incubations
of HTE with AM increased B[a]P adduct levels around 30–
35% compared with culturing HTE alone (data not shown).
these data support the notion that the involvement of AM may
contribute to the process of pulmonary pathogenesis.
Benzo[a]pyrene uptake and metabolism
Fig. 5. Autoradiography of 32P-postlabeled B[a]P2DNA adduct standards.
(A) (1)-anti-BPDE-dG (adduct 1). (B) (2)-anti-BPDE-dG (adduct 2) and
(2)-anti-BPDE-dA (adduct 3).
Two adducts were observed following B[a]P treatments.
The major adduct [(1)-anti-BPDE-dG, adduct 1] has been
reported in numerous systems, including rodent, bacteria,
human cell cultures and explant tissues exposed to B[a]P (45–
47) and is considered to be important in the initiation of cancer
by B[a]P. Our results indicate that the level of B[a]P2DNA
adducts increased when B[a]P was coated onto Fe2O3 instead
of Al2O3 or B[a]P alone. These results corroborate and provide
a potential mechanism via increased uptake for the enhanced
carcinogenicity of these treatments in long-term studies (7).
The formation of total adducts can be significantly reduced
by addition of inhibitors (α-NF and CO). The level of adduct
2 [(2)-anti-BPDE-dG] was also decreased by co-incubation
with inhibitors. This adduct has been associated with the
peroxyl radical pathway (48,49). Initially the conversion of
parent B[a]P to (1)-7,8-dihydrodiol involves P-450 and epoxide hydrolase (14). The formation of (2)-anti-BPDE-dG is
then mediated through processes such as peroxyl radicals to
epoxidize (1)-7,8-dihydrodiol-B[a]P (48). Since earlier steps
that involve the formation of dihydrodiols were hindered by
inhibitors, the level of formation of the (2)-anti-BPDE-dG
could also be affected and reduced as we have seen. It is of
interest to note that exposure to asbestos, silica or quartz
particles is associated with the elevation of lipid peroxidation (50,51).
Fig. 6. RAL of B[a]P2DNA adducts (mean 6 SE) present in hamster
tracheal epithelial cells (HTE) (3 3 106) following incubation (24 h) with
AM (3 3 106) under various conditions. B[a]P was at the single dose
(19.8 nmol) in all treatments. Inh: inhibitors [α-NF 5 µM; CO 1023 M; F,
Fe2O3 (1 mg); A, Al2O3 (2 mg)]. *Significantly different compared with
addition of inhibitors (P , 0.05); # significantly different compared with
B[a]P or B[a]P2Al2O3 (P , 0.05). (A) Total adduct levels in HTE.
(B) Adduct 1 levels in HTE. (C) Adduct 2 levels in HTE.
Conclusion
The results presented here support the hypothesis that Fe2O3
can enhance the uptake and metabolism of B[a]P in pulmonary
AM and subsequently increase the binding of B[a]P metabolites
to the DNA of pulmonary epithelial cells. The overall enhancement of B[a]P uptake, metabolism and DNA adduct formation
might rely on several properties of Fe2O3 particles, including
particle size and their capacities to: (i) stimulate phagocytosis;
(ii) stimulate or decrease the turnover rate of P-450 enzymes;
and (iii) perturb cellular membranes to generate peroxyl
radicals. Understanding the metabolic activation of PAHs on
various types of particles is important for estimating the toxic,
mutagenic and carcinogenic potential of particle PAH coexposure in the occupational settings.
173
J.Cheu et al.
Acknowledgements
The authors thank Dr B.T.Mossman of the University of Vermont for kindly
providing the hamster tracheal epithelial cell line, Dr L.Marnett of Vanderbilt
University for his generous gift of (2)-anti-BPDE-dG/dA, Ms M.Jaeger for
her help with postlabeling experiments, R.Reilman and J.Schneider for
assistance with the HPLC analyses, S.Andringa for performance of electron
microscopy analysis, and L.Wilson for the preparation of this manuscript.
This work was supported by NIOSH grants 0H02972 (J.C.), 0H02277 (D.W.)
and NIEHS Center grant 1P30 ES06096 (G.T., M.M., C.R. and D.W.).
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Received on January 4, 1996; accepted on September 23, 1996
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