Download Altered Gene Expression Patterns in MCF-7

Research Article
Altered Gene Expression Patterns in MCF-7 Cells Induced
by the Urban Dust Particulate Complex Mixture
Standard Reference Material 1649a
1
2
1
1
Brinda Mahadevan, Channa Keshava, Tamara Musafia-Jeknic, Arta Pecaj, Ainsley Weston,
1
and William M. Baird
2
1
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon and 2Toxicology and Molecular
Biology Branch, National Institute for Occupational Safety and Health, CDC, Morgantown, West Virginia
Abstract
Human exposures to polycyclic aromatic hydrocarbon (PAH)
occur in complex mixtures. Here, gene expression patterns
were investigated using standard reference material (SRM)
1649a (urban dust). MCF-7 cells were exposed to SRM 1649a
alone or SRM 1649a with either benzo[a]pyrene (BP) or
dibenzo[a,l]pyrene (DBP) for 24 hours. Global analyses of the
gene expression data revealed alterations of 41 RNA transcripts
with at least 2-fold change (signal log ratio V 1 or z 1) in
response to SRM 1649a exposure. Increase in expression of
cytochrome P450 (CYP) genes was observed in response to BP
exposure (CYP1A1 and CYP1B1; signal log ratio of 4.7 and 2.5,
respectively). An additive induction of CYP1A1 and CYP1B1
was observed with cotreatment of SRM 1649a and BP. On the
contrary, no change in gene expression of CYP1A1 and CYP1B1
was observed when the cells were exposed to DBP. Furthermore, to study the effect of complex PAH mixtures on the
metabolic activation of carcinogenic PAH to DNA-binding
derivatives and to relate this with gene expression studies,
PAH-DNA adduct formation was determined. SRM 1649a
decreased the total level of BP-DNA adducts in comparison
with BP alone. No significant difference in adduct levels was
observed in response to either DBP alone or in combination
with SRM 1649a. These results provide a transcriptional
signature for chemical carcinogen exposure; in addition, they
suggest a major factor in carcinogenic activity of PAH within
complex mixtures is their ability to promote or inhibit the
activation of carcinogenic PAH by the induction of CYP
enzymes. (Cancer Res 2005; 65(4): 1251-8)
Introduction
Ambient air contains dust particles that are complex, potentially
genotoxic mixtures that pose danger to human health (1). Air
pollution exposure resulting from the incomplete combustion of
organic matter is suspected of contributing to cancer risk (2, 3).
Recently, it has been shown that reduction of exposure to
particulate air pollution lowers the risk of heritable mutations in
mice with implications for health risks to future generations (4, 5).
Because air pollutants are composed of >2,800 chemicals (6), their
synergistic, antagonistic or additive effects may be important
factors in human carcinogenesis (7). Studies have shown that the
main contributors to genotoxicity are polycyclic aromatic hydrocarbon (PAH) and its derivatives (8–10).
Chemical analysis of complex mixtures in ambient air is difficult
because several biologically active components occur at low
concentrations. The National Institute of Standards and Technology have developed a number of natural matrix standard reference
materials (SRM) since the early 1980s for the determination of
organic contaminants in environmental matrices (11). The first
particle-based, environmental natural matrix SRM developed by
the National Institute of Standards and Technology for organic
contaminants was SRM 1649 (urban dust/organics) which was
issued in 1982 (12, 13). SRM 1649a is the same particulate material
that was issued previously in 1982 as SRM 1649 (14); this material
has been rebottled and reanalyzed to provide updated certified
values as well as certified, reference, and information values for
additional constituents. For our studies, we used SRM 1649a in
addition to representative examples of carcinogenic PAHs,
benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) to understand
the metabolic activation and DNA binding of these PAHs within
complex mixtures.
It is understood that exposure to PAHs can target the expression
of regulatory proteins involved in activation, detoxification and
DNA repair, thus altering the ability of the cells to maintain genomic
integrity. Jeffy et al. (15) have suggested that exposure to PAHs may
be a predisposing factor in the etiology of sporadic breast cancer by
disrupting the expression of BRCA-1, a tumor suppressor gene.
Identification of genes whose expression is specifically modified by
exposure to complex mixtures of PAH would provide a better
understanding of their mechanisms of action and allow development of sensitive and specific biomarkers of both exposure and
susceptibility for use in both mechanistic laboratory and epidemiology studies. Therefore, in the current work we used high density
oligonucleotide microarray to compare the effects of the complex
environmental mixture SRM 1649a on gene expression, metabolic
activation and PAH-DNA binding of BP and DBP in human
mammary carcinoma derived MCF-7 cells in culture. Here we
focused on cytochrome P450 (CYP) CYP1A1 and CYP1B1, which
account for PAH metabolism, and also confirmed the expression of
these relevant genes by real-time PCR. To study the relationship of
gene expression to DNA damage, we also determined the DNA
adduct formation and protein expression in MCF-7 cells exposed to
SRM 1649a alone and in combination with BP or DBP.
Materials and Methods
Note: Brinda Mahadevan and Channa Keshava contributed equally to this work.
Requests for reprints: William M. Baird, Department of Environmental and
Molecular Toxicology, Oregon State University, 1007 Agricultural and Life Sciences,
Corvallis, OR. Phone: 541-737-1886; Fax: 541-737-0497; E-mail: [email protected].
I2005 American Association for Cancer Research.
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Cell Culture and Treatment. The MCF-7 cells (obtained from the
Karmanos Cancer Center, Detroit, MI) were cultured in a 75-cm2 flask
(Corning, Corning, NY) in a 1:1 mixture of F-12 Nutrient Mixture and DMEM
(Life Technologies, Grand Island, NY). The medium was supplemented with
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10% fetal bovine serum (Intergen, Purchase, NY), containing 15 mmol/L
HEPES buffer and antibiotics (200 units/mL penicillin, 200 Ag/mL
streptomycin, and 25 Ag/mL ampicillin) at 37jC with 5% CO2. Cell cultures
were subcultured at a ratio of 1:4 when the cells covered the entire surface
of the flask.
All cell culture flasks, in which the cells covered f70% of the surface,
were replenished with fresh medium (20 mL) 24 hours before treatment.
SRM 1649a was obtained from the National Institute of Standards and
Technology (Gaithersburg, MD) and had a unit of SRM in a bottle
containing 2.5 g of atmospheric particulate material (14). A detailed
description and chemical composition of SRM 1649a is available under
http://patapsco.nist.gov/srmcatalog/certificates/1649a.pdf. SRM 1649a
treatments in cell culture were done using a dose of 400 Ag in 50 AL of
DMSO. The cells were treated with either solvent alone as a control (DMSO,
75 AL), SRM 1649a (400 Ag), BP (20.2 Ag), SRM 1649a (400 Ag) plus BP
(20.2 Ag), DBP (0.2 Ag), or SRM 1649a (400 Ag) plus DBP (0.2 Ag). The
concentrations were based on previous studies in our laboratory, which
indicated that these doses gave detectable levels of DNA binding without
causing excessive toxicity to the cells (16). The cells were harvested after
24 hours of continuous exposure.
High-Density Oligonucleotide Array Expression Analysis. Total RNA
was isolated from treated and control cultures of MCF-7 cells using RNeasy
Kit (Qiagen, Valencia, CA) and subsequent steps leading to hybridization
and scanning of the human genome U133A arrays were done according to
the DNA microarray manufacturer’s instructions (Affymetrix, Santa Clara,
CA). Briefly, total RNA (10 Ag) was used for the preparation of cDNA
using a T7-poly T primer and the reverse transcriptase Superscript II
(Life Technologies, Carlsbad, CA). Labeled cRNA transcript was generated
from the cDNA sample by an in vitro transcription reaction using Enzo
Bioarray high yield RNA transcript labeling kit (Enzo Diagnostic, Farmingdale, NY). The labeled cRNA transcript was purified (RNeasy, Qiagen)
and each cRNA sample (20 Ag) was fragmented by mild alkaline treatment
(94jC, 35 min). Each fragmented cRNA sample (15 Ag) was hybridized to
U133A human genome arrays, representing >22,000 genes (45jC, 16 hours;
in rotisserie oven set at 60 rpm). Subsequent washing and staining of
the probe arrays was done using the GeneChip fluidics station protocol
EukGE-WS2. Furthermore, probe arrays were scanned using the Affymetrix
Gene Array 2500 Scanner. The differential gene expression data analysis was
done using Microarray Suite 5.0 software. Two biological replicates were
used for every treatment. A signal log ratio (SLR) of one was considered
equivalent to 2-fold change in gene expression.
Quantitative Real-time PCR Analysis. Relative quantitation with realtime, reverse-transcriptase PCR (RT-PCR) was done for both CYP1A1 and
CYP1B1 using an ABI PRISM 7700 Sequence Detection System (PE Applied
Biosystems, Foster City, CA), according to manufacturer’s instructions. The
cDNA prepared for microarray analysis was diluted 1:50 and 1.5 AL was
used as template to perform RT-PCR in a 50-AL reaction using 20
assays-on-demand gene expression primers and probes for both CYP1A1
(Hs00153120_m1/X02612), CYP1B1 (Hs00164383_m1/U03688) and house
keeping gene GAPDH (Hs99999908w; PE Applied Biosystems/Genbank).
RT-PCR consisted of initial denaturing for 10 minutes at 95jC, 40 cycles
of 95jC for 15 seconds, and 50jC for 1 minute. Each sample was assayed
in quadruplicates and the cycle threshold (C T) values were normalized
to the house keeping gene GAPDH and the fold change was calculated
using 2 DDCT method (17).
DNA Isolation. A standard DNA isolation protocol was used (18). Briefly,
cell culture samples were homogenized in a glass homogenizer containing
EDTA, SDS buffer [10 mmol/L Tris, 1 mmol/L Na2EDTA, and 1% SDS (w/v),
pH 8]. The homogenates were treated with RNase, DNase-free (50 units/mL;
Boehringer-Mannheim Co., Indianapolis, IN) and RNase T1 (1,000 units/mL;
Boehringer-Mannheim) at 37jC for 1 hour, followed by treatment with
proteinase K (500 Ag/mL; Sigma Chemical, St. Louis, MO) at 37jC for 1 hour.
The DNA was extracted with equal volumes of Tris-equilibrated phenol
(Boehringer-Mannheim) followed by extraction with 1:1 volume of Trisequilibrated phenol and chloroform/isoamyl alcohol (24:1) and then with
equal volumes of chloroform/isoamyl alcohol (24:1). The aqueous layer was
treated with 1/10 volume of 5 mol/L NaCl and twice the volume of cold 100%
Cancer Res 2005; 65: (4). February 15, 2005
ethanol to precipitate the DNA which was then dissolved in double-distilled
water; its concentration was determined by UV absorbance at 260 nm.
33
P-Postlabeling of DNA Adducts. Post-labeling was carried out as
described previously (19). Briefly, 10 Ag DNA isolated from MCF-7 cells
after treatment were digested with nuclease P1 and prostatic acid
phosphatase, post-labeled with [g-33P]ATP (3,000 Ci/mmol), cleaved to
adducted mononucleotides with snake venom phosphodiesterase I, and
prepurified with a Sep-Pak C18 cartridge (Waters, Milford, MA).
Subsequent separation by analytic high performance liquid chromatography (HPLC-Varian system equipped with two pumps and an autosampler;
Varian Systems, CA) was carried out using a C18 reverse-phase column
(5 Am Ultrasphere ODS, 4.6 250 mm). Adducts were resolved by elution
at 1 ml/min with 0.1 mol/L ammonium phosphate (pH 5.5, solvent A) and
100% HPLC grade methanol (solvent B). The elution gradient was as
follows: 44% to 60% solvent B over 40 minutes, 60% to 80% solvent B over
10 minutes, isocratic elution at 80% solvent B over 10 minutes, and 80% to
44% B over 5 minutes. The radiolabeled nucleotides were detected by an
online radioisotope flow detector (Packard Instruments, Downers Grove,
IL), and the level of DNA binding was calculated based on the labeling
efficiency of a [3H]B[a]P-7,8-dihydrodiol 9,10-epoxide standard (20). Three
independent sets of the post-labeling reaction were carried out for every
sample treated, to determine the total PAH-DNA adduct levels.
Microsome Isolation. Microsomes were prepared as described previously (21) with minor modifications. Briefly, cell culture samples were
homogenized with a steel homogenizer in microsomal homogenization
buffer (0.25 mol/L K2HPO4, 0.15 mol/L KCl, 10 mmol/L EDTA, and 0.25
mmol/L phenylmethylsulfonyl fluoride) and were centrifuged at 15,000 g
for 20 minutes at 4jC. The supernatant was centrifuged at 100,000 g for
90 minutes at 4jC, and the pellet was resuspended in microsome dilution
buffer (0.1 mol/L KH2PO4, 20% glycerol, 10 mmol/L EDTA, 0.1 mmol/L DTT,
and 0.25 mmol/L phenylmethylsulfonyl fluoride). The protein concentration
was spectrophotometrically determined at 562 nm using the Bicinchoninic
acid colorimetric assay (Pierce, Rockford, IL).
Ethoxyresorufin O-Deethylation Assay. Fifty micrograms of microsomal protein were added to 200 Amol/L 7-ethoxyresorufin (Sigma
Chemical) in 0.1 mol/L Tris-HCl (pH 7.8) buffer and added in a black 96well plate (E&K Scientific, Campbell, CA). NADPH (Sigma Chemical) was
added to each well and the fluorescence was measured with a Spectra MAX
Gemini plate reader (Molecular Devices, Sunnyvale, CA). The excitation
wavelength was 535 nm, emission 585 nm and the assay was monitored over
10 minutes. Each sample was assayed in triplicate and the amount of
resorufin produced was calculated from the fluorescence of a known
concentration of resorufin.
Western Blots. Microsomal proteins were separated by SDS-PAGE on a
7.5% READY gel (Bio-Rad, Hercules, CA). Microsomal proteins (50 Ag) were
diluted with loading buffer [0.625 mol/L Tris-base (pH 6.8), 2% SDS, 10%
glycerol, 5% 2-mercaptoethanol, and 0.001% bromophenol blue]. Proteins
were denatured by boiling for 3 minutes, followed by rapid cooling on ice.
Following gel electrophoresis at 200 V for 1 hour, the microsomal proteins
were transferred to nitrocellulose using a mini Trans-Blot electrophoretic
transfer cell (Bio-Rad,) at 100 V for 45 minutes. Uniformity of sample
loading was confirmed by staining the nitrocellulose membrane with
Ponceau S (Sigma Chemical). The membrane was washed thrice with PBS-T
[PBS with 0.3% (w/v) Tween 20] for 5 minutes each and blocked with
1:3 Nap-Sure blocker (Geno Technology, St. Louis, MO): PBS-T for 1 hour on
a shaker. After three further washes with Nap-Sure: PBS-T (1:7), the
membrane was incubated with the primary antibody for 2 hours and was
washed again with Nap-Sure blocker: PBS-T (1:7). Human CYP1A1 was
detected by a rabbit polyclonal CYP1A1 antibody (1:1500) prepared against
purified recombinant human CYP1A1 protein. The antibody against human
CYP1A1 was a gift from Dr. F.P. Guengerich (Center for Molecular
Toxicology, Vanderbilt University School of Medicine, Nashville, TN).
Dr. C. Marcus (Department of Pharmacology and Toxicology, University
of New Mexico, Albuquerque, NM) prepared and provided the human
CYP1B1 rabbit polyclonal antibody (1:1,000). The immunoreactive proteins
were detected by incubating the membrane with peroxidase-conjugated
anti-rabbit IgG (1:30,000 for CYP1A1 and 1:20,000 for CYP1B1) for
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30 minutes. After three washes with Nap-Sure blocker: PBS-T (1:7), the
immunoreactive proteins were observed using an enhanced chemiluminescence detection method, as described by the manufacturer (Amersham Life
Science, Arlington Heights, IL). Microsomal protein (10 Ag) from V79 cells
expressing either human CYP1A1 or CYP1B1 (18) was used as a positive
control in the immunoblots.
Statistical Analysis. Correlation coefficients were determined according
to the method of Pearson using Excel (22).
Results
Altered Gene Expression in MCF-7 Cells in Response
to Complex Mixture SRM 1649a
Exposure of MCF-7 cells to BP, DBP, SRM 1649a, BP plus SRM
1649a, and DBP plus SRM 1649a resulted in altered gene expression
patterns in many RNA species. Global gene expression analysis (at
least 2-fold change SLR V 1 or z 1) revealed 129, 41, and 39 genes
that were altered in response to BP, SRM 1649a, and DBP,
respectively. The data reported here with regard to microarray
studies were based on 2-fold up-regulated or down-regulated genes
as being significant (P V 0.05).
Functional Classification of Differentially Expressed
Genes
The differentially expressed genes were classified based on their
function and selected genes that were altered on exposure to BP,
SRM 1649a, and DBP are listed in Tables 1, 2 and 3 respectively
(with a short functional description). The genes are arranged with
their short name/gene symbol, so they can be easily looked up and
linked to the text.
Genes involved in cell cycle, proliferation/apoptosis, DNA
repair and tumor suppression. On exposure to BP, 8 of 10 genes
were categorized as those involved in cell cycle regulation/
apoptosis and DNA repair. Whereas SRM 1649a exposure induced
three genes, DBP influenced only 2 of 10 genes categorized here.
Most genes that were induced or repressed were either directly or
indirectly involved in cell cycle regulation (CDKN1A, BAX,
BCL2L11, PPARG, OKL38, CYR61, and DUSP1) and or apoptosis.
Nuclear accumulation of p53 and the transcriptional induction of
various target genes such as CDKN1A has been shown previously
on human cells exposed to BP, DBP, or its ultimate metabolite (23,
24). Peroxisome proliferative activated receptor g (PPARG) was upregulated on exposure to DBP or SRM 1649a. Two important genes
Table 1. Selected genes that were induced at least SLR of z 1 (2-fold) in response to BP exposure
Genbank
accession no.
Gene
symbol
Gene title
Treatment
BP
NM_000499
CYP1A1
NM_001924
GADD45A
NM_015516
E2IG4
NM_000104
CYP1B1
NM_002083
NM_001353
GPX2
AKR1C1
U05598
AKR1C2
NM_000463
UGT1A10
NM_015869
PPARG
NM_000389
CDKN1A
NM_000120
U19599
X06399
EPHX1
BAX
CYP2B6 NS
AA629050
NM_001065
BCL2L11 NS
TNFRSF1A
NM_000107
DDB2
NM_013370
NM_000903
OKL38
NQO1
Cytochrome P450, family 1,
subfamily A, polypeptide 1
Growth arrest and
DNA-damage-inducible, a
Hypothetical protein,
estradiol-induced
Cytochrome P450, family 1,
subfamily B, polypeptide 1
Glutathione peroxidase 2
Aldo-keto reductase family 1,
member C1
Aldo-keto reductase family 1,
member C2
UDP glycosyltransferase 1 family,
polypeptide A10
Peroxisome proliferative activated
receptor, g
Cyclin-dependent kinase
inhibitor 1A (p21, Cip1)
Epoxide hydrolase 1
BCL2-associated X protein
Cytochrome P450, family 2,
subfamily B, polypeptide 6
BCL2-like 11 (apoptosis facilitator)
Tumor necrosis factor receptor
superfamily, member 1A
Damage-specific DNA
binding protein 2
Pregnancy-induced growth inhibitor
NADPH dehydrogenase, quinone
1649a
DBP
4.7 F 0.28
0.6 F 0.21
0.7 F 0.63
3.1 F 0.00
1.1 F 0.91
3.0 F 0.21
2.3 F 0.35
0.5 F 0.14
0.3 F 0.00
2.5 F 0.28
1.8 F 0.07
0.2 F 0.21
2.1 F 0.14
1.7 F 0.07
0.7 F 0.07
0.1 F 0.00
0.6 F 0.21
0.2 F 0.00
1.6 F 0.07
0.2 F 0.07
0.0 F 0.00
1.7 F 0.14
0.6 F 0.14
0.5 F 0.07
1.7 F 0.21
1.2 F 0.14
0.9 F 0.07
1.5 F 0.00
0.2 F 0.14
1.5 F 0.28
1.4 F 0.07
1.3 F 0.00
1.1 F 0.14
0.1 F 0.14
0.4 F 0.77
0.4 F 0.14
0.3 F 0.07
0.6 F 0.35
0.3 F 0.2
1.3 F 0.21
1.1 F 0.00
1.1 F 0.49
0.3 F 0.14
0.2 F 0.21
0.2 F 0.14
1.0 F 0.00
0.0 F 0.14
1.2 F 0.21
1.0 F 0.00
2.0 F 0.07
0.2 F 0.07
0.4 F 0.07
0.0 F 0.14
0.2 F 0.28
NOTE: Selected genes that were induced by at least a SLR of z 1 in response to BP treatment. The data represents mean F SD where n = 2 arrays per
treatment. All genes were significantly altered (P V 0.05) on exposure to SRM 1649a except for those indicated as not significant (NS) following the gene
symbol.
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Table 2. Selected genes that were induced at least SLR of z 1 (2-fold) in response to SRM 1649a exposure
Genbank
accession no.
Gene
symbol
Gene title
Treatment
1649a
NM_000104
CYP1B1
M92934
BC004490
CTGF
FOS
NM_001554
NM_004417
BF594446
CYR61
DUSP1
MCL1
U73936
JAG1 NS
NM_015869
PPARG
BG491844
JUN
AI091047
SLC2A1
Cytochrome P450, family 1,
subfamily B, polypeptide 1
Connective tissue growth factor
v-fos FBJ murine osteosarcoma
viral oncogene homologue
Cysteine-rich, angiogenic inducer, 61
Dual specificity phosphatase 1
Myeloid cell leukemia sequence 1
(BCL2 related)
Jagged 1 (Alagille syndrome) involved
in cell communication
Peroxisome proliferative
activated receptor, g
v-jun sarcoma virus 17 oncogene
homologue (avian)
Solute carrier family 2
( facilitated glucose
transporter), member 1
BP
DBP
1.8 F 0.07
2.5 F 0.28
0.2 F 0.21
2.0 F 0.70
1.7 F 0.28
0.6 F 0.49
0.3 F 0.21
1.8 F 0.91
0.9 F 1.20
1.3 F 0.49
1.2 F 0.42
1.2 F 0.28
0.1 F 0.28
0.1 F 0.07
0.5 F 1.06
1.0 F 0.77
0.9 F 0.77
0.8 F 0.14
2.0 F 0.84
2.0 F 0.99
2.6 F 0.35
1.2 F 0.14
1.7 F 0.21
0.9 F 0.07
1.0 F 0.00
0.3 F 0.14
0.4 F 0.28
1.7 F 0.00
0.3 F 0.92
0.8 F 0.00
NOTE: Selected genes that were induced by at least a SLR of z 1 in response to SRM 1649a treatment. The data represents mean F SD where n = 2
arrays per treatment. All genes were significantly altered (P V 0.05) on exposure to SRM 1649a except for those indicated as not significant (NS)
following the gene symbol.
linked to DNA repair (GADD45A and DDB2) were observed to be
up-regulated on exposure to BP and DBP, whereas an increase in
the expression of the mitochondrial tumor suppressor gene MTSG1
was observed on DBP treatment. DDB2 being a nucleotide excision
repair protein (25) substantiates its increased expression in our
study, on exposure to BP and DBP.
Genes involved in immune response, cell structure/adhesion, transport, and receptor/inhibitor. Two genes (TNFRSF6
and E21G4) were up-regulated on exposure to BP, whereas three
unique genes involved in cell structure/adhesion and transport
(CTGF, JAG1, and SLC2A1) respectively, increased in expression on
exposure to SRM 1649a. DBP and SRM 1649a exposure indicated an
increase in the expression of connective tissue growth factor
(CTGF), whereas tissue factor pathway inhibitor (TFPI2) and tumor
necrosis factor receptor (TNFRSF6) showed increased expression
on exposure to DBP.
Oncogenes. Proto-oncogenes (RAB6, FOS, JUN, and MCL1) were
particularly up-regulated on exposure to SRM 1649a and DBP but
expressed relatively low on exposure to BP.
Genes involved in metabolism. Several of the genes known to
be involved in the metabolism of PAHs (CYP1A1, CYP1B1, AKR1C1,
AKR1C2, NQO1, EPHX1, UGT1A1, CYP2B6, and GPX2) were found
Table 3. Selected genes that were induced at least (2-fold) SLR of z 1 in response to DBP exposure
Genbank
accession no.
Gene
symbol
Gene title
Treatment
DBP
NM_001924
M92934
NM_000389
AA164751
L27624
BE552421
NM_000107
NM_002869
L08599
GADD45A
CTGF
CDKN1A
TNFRSF6
TFPI2
MTSG1
DDB2
RAB6A
CDH1
Growth arrest and DNA damage–inducible, a
Connective tissue growth factor
Cyclin-dependent kinase inhibitor 1A (p21, Cip1)
Tumor necrosis factor receptor superfamily, member 6
Tissue factor pathway inhibitor 2
Mitochondrial tumor suppressor gene 1
Damage-specific DNA binding protein 2
RAB6A, member RAS oncogene family
Cadherin 1, type 1, E-cadherin (epithelial)
3.0
1.8
1.5
1.3
1.2
1.2
1.2
1.1
1.0
F
F
F
F
F
F
F
F
F
1649a
0.21
0.92
0.28
0.14
0.14
0.21
0.21
0.07
0.07
1.1
2.0
0.2
0.7
0.7
1.1
0.0
1.0
0.8
F
F
F
F
F
F
F
F
F
BP
0.92
0.71
0.14
0.71
0.07
0.49
0.14
0.28
0.07
3.1
0.6
1.5
1.2
1.5
1.1
1.0
0.3
0.3
F
F
F
F
F
F
F
F
F
0.00
0.49
0.00
0.35
0.21
0.49
0.00
0.78
0.92
NOTE: Selected genes that were induced by a SLR of z 1 in response to DBP treatment. The data represents mean F SD where n = 2 arrays per
treatment. All genes were significantly altered (P V 0.05) on exposure to DBP.
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Urban Dust Exposure Alters Gene Expression
to be up-regulated especially on exposure to BP, whereas CYP1B1
levels increased on exposure to SRM 1649a. It has been shown (26)
that CYP1A1 and CYP1B1 are key enzymes involved in the
metabolism of the two carcinogenic PAH (BP and DBP) used in
this study. AKR1C1 and AKR1C2 have also been shown to be
involved in the diversion of PAH trans-dihydrodiols from diolepoxides to o-quinones (27, 28) that may thus provide an
alternative pathway of PAH activation.
Quinone oxidoreductase (NQO1) is known to catalyze the
activation of some environmental procarcinogens present in
tobacco smoke, and the genotypes of NQO1 was recently shown to
play an important role in the development of smoking related
bladder cancer (29). BP being a carcinogenic PAH present in
environmental tobacco smoke it was no surprise to see NQO1 upregulated in our study on cells exposed to BP. Another enzyme
epoxide hydrolase (EPHX) is known to play a dual role in the
detoxification and activation of tobacco procarcinogens and two
polymorphic variants were found to confer lower and higher activity
to the enzyme (30). Analysis of a hospital-based case study of newly
diagnosed lung cancer patients for polymorphisms affecting the
enzyme activity indicated that genetically reduced microsomal
epoxide hydrolase activity may be protective against lung cancer
(31). The epoxide hydrolase gene (EPHX1) was up-regulated in our
study on exposure to BP which provides confirmation of the ability of
EPHX to hydrolate PAH epoxy metabolites to the carcinogenic PAH
diol-epoxides (32). Here, we have further analyzed and focused on
the altered transcripts of CYP1A1 and CYP1B1.
Figure 2. Formation of DNA adducts (average pmol/mg) in MCF-7 cells after
24 hours of exposure to BP, SRM 1649a, SRM 1649a plus BP, DBP, and SRM
1649a plus DBP. Three independent sets of post-labeling reactions were carried
out for every sample treated, to determine the total PAH-DNA adduct levels.
Error bars represent SD of triplicate samples analyzed.
A significant increase in the expression of CYP1A1 was observed
in MCF-7 cells exposed to BP plus SRM 1649a in comparison to
SRM 1649a alone (Fig. 1A). On the contrary, DBP plus SRM 1649a
did not show significant increase in CYP1A1 expression when
compared with 1649a. The array data was validated with RT-PCR,
and correlated with microarray data with regard to the transcript
detection for CYP1A1 (r = 0.95). A similar pattern in the expression
of CYP1B1 was also observed where there was an increase of
CYP1B1 expression when treated with BP plus SRM 1649a in
comparison to SRM 1649a alone (Fig. 1B). DBP plus SRM 1649a did
not show a significant increase in CYP1B1 expression in
comparison to SRM 1649a. This expression analysis of CYP1B1
was also validated by RT-PCR that correlated well with the
transcript detection for CYP1B1 (r = 0.97).
DNA Adduct Formation in MCF-7 Cells in Culture
The effect of the complex mixture SRM 1649a on DNA adduct
formation in human mammary carcinoma MCF-7 cells was
compared with BP and DBP both individually and in combination
with SRM 1649a. The cells were treated for 24 hours, and the DNA
adducts were 33P-postlabeled and analyzed by HPLC. MCF-7 cells
treated with DMSO (vehicle control) or SRM 1649a alone did not
exhibit any DNA binding. Quantitative analysis of the total average
levels of DNA binding is illustrated in Fig. 3. Treatment with a
combination of BP and SRM 1649a indicated a 2-fold decrease in
DNA binding in comparison to BP alone. On the other hand,
treatment with a combination of DBP and SRM 1649a did not show
any significant difference in the formation of DNA adducts (Fig. 2).
Cytochrome P450 Expression
Figure 1. Gene expression levels of CYP1A1 (A) and CYP1B1 (B ) following
24 hours exposure to BP, SRM 1649a, SRM 1649a plus BP, DBP, and SRM
1649a plus DBP. Data represents mean F SD (n = 2 arrays per treatment).
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The ethoxyresorufin O-deethylation assay was done using
microsomes from MCF-7 cells exposed to PAHs and SRM 1649a.
The effect of SRM 1649a on CYP induction as measured by the
ethoxyresorufin O-deethylation assay is shown in Table 4. Both
DMSO- and DBP-treated samples exhibited background levels of
CYP activity. Treatment with SRM 1649a, although revealed CYP
induction, a combination treatment of BP plus SRM 1649a and DBP
plus SRM 1649a indicated no additive increase in CYP activity, in
comparison to BP or DBP alone (Table 4).
Western analyses were done to examine the ability of the
complex mixture SRM 1649a to induce the expression of human
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CYP 1A1 and CYP 1B1 proteins in MCF-7 cells (Fig. 3). Treatment
with DMSO or DBP alone did not express either CYP 1A1 or CYP
1B1 proteins (Fig. 3A and B). Whereas BP and SRM 1649a
expressed both proteins CYP 1A1 and CYP 1B1, an additive
increase in the expression of both proteins was evident in the
treatment with BP plus 1649a (Fig. 3A and B).
Table 4. Effect of SRM 1649a on CYP enzyme activity
Discussion
Testing complex mixtures presents a formidable scientific
problem, because most recently available toxicologic data have
been obtained from single substance studies and are not simply
applicable to mixtures of chemicals. Although there are no special
strategies and standard protocols available for determining toxic
and genotoxic effects of complex mixtures, the fundamental
concepts of evaluation are the same as those for single substances
(33). Furthermore, toxicologic as well as genotoxic evaluations of
complex mixtures are complicated by the potential for the additive,
antagonistic or synergistic component interactions. Therefore, in
our study, we have documented the effects of two different
carcinogenic PAHs (BP, moderately potent; DBP, extremely potent),
the complex mixture SRM 1649a and the results of their
interactions relative to the individual compound or mixture alone
particularly with regard to two PAH metabolism genes CYP1A1 and
CYP1B1.
Several research organizations have conducted animal carcinogenicity bioassays on specific chemicals and has identified
many of them as mammary carcinogens (ref. 34; including PAHs
and nitro-PAHs) that show evidence of genotoxicity. Gammon et
al. (35) have assessed PAH exposure by measuring PAH-DNA
adducts and although they observed a lack of a relationship
between PAH-DNA adducts and exposures to grilled food and
tobacco smoke as primary sources of PAHs, other sources such
as air pollution may be more important (36). Given the modest
relative risks of environmental factors, associated with breast
cancer, discovery of a risk would be valuable if that could save
many thousands of lives (36). Recent studies have shown
Figure 3. Detection of CYP1A1 (A ) and CYP1B1 (B ) proteins by Western blot.
Actin antibody was used to detect the actin protein expression, which was
used as loading control. Microsomal protein (10 Ag) from V79 cells expressing
either human CYP1A1 or CYP1B1 was used as a positive control in the
immunoblots.
Cancer Res 2005; 65: (4). February 15, 2005
Treatment
CYP enzyme activity
(pmol per min per mg)
DMSO (vehicle control)
BP
SRM 1649a
BP + SRM 1649a
DBP
DBP + SRM 1649a
0.18
1.43
0.71
1.31
0.22
0.10
F
F
F
F
F
F
0.21
0.90
0.17
1.1
0.14
0.14
NOTE: The effect of SRM 1649a on CYP enzyme activity (pmol per min
per mg) after 24 hours exposure compared with BP and DBP alone or
in combination with SRM 1649a. Microsomes were isolated from
MCF-7 cells treated with SRM 1649a, BP, DBP, SRM 1649a plus BP, and
SRM 1649a plus DBP and the CYP enzyme activity was determined by
EROD assay as described in Materials and Methods. An average of four
individual experiments is presented as a simple mean F SD.
evidence that biologically effective amounts of PAH compounds
associated with particulate matter were transferred to the
intracellular environment and elicited the activation of various
genes (37, 38).
The selection of a human mammary carcinoma derived cell line
in this study reflects our interest in breast cancer and or pulmonary
health effects that are potentially attributable to exposure to urban
dust. We discuss here two genes (CTGF and CYR61) that indicated
increased expression on exposure to SRM 1649a (Table 2) in our
study and their possible link to breast cancer. Connective tissue
growth factor (CTGF) is known to be a potent angiogenic factor.
The results obtained by Frazier and Grotendorst (39) suggest that
cancer stroma formation involves induction of fibroproliferative
growth factors such as CTGF. Recent studies in samples from
primary breast tumors suggest that CTGF and CYR61 may play a
role in the progression of breast cancer and might serve as a
valuable tool for monitoring tumor status in breast cancer patients
(40). Tsai et al. (41) have further shown that CYR61 is a tumorpromoting factor and a key regulator of breast cancer progression.
SRM 1649a in our study increased the expression of CTGF and
CYR61, which is the first report connecting urban dust particulate
and genes involved in breast cancer progression. Overall, although
there is some overlap in the genes modified between different PAHs
and SRM 1649a, these data suggest that each PAH modifies
expression of a largely unique set of genes that may be
characteristic of each treatment.
PAHs can induce tumor promotion through several, likely
combinatorial, mechanisms including negative effects on transcription of tumor-suppressor genes (42), transcriptional activation
of proto-oncogenes (43), and activation of enzymes that oxidize
procarcinogenic PAHs to reactive metabolites (44). To date, no
mutations in the BRCA-1 gene have been identified in sporadic
breast cancers, whereas the expression levels of BRCA-1 in breast
tumors are lower than those observed in normal mammary tissue
(45). Recent work by Jeffy et al. (46) suggest that BP, a carcinogenic
PAH found in tobacco smoke, environmental pollution, and foods
may contribute to breast carcinogenesis through the inhibition of
the tumor suppressor gene BRCA-1. Under the experimental
conditions used in our study, the global gene expression analysis
revealed no alterations in the tumor suppressor gene BRCA-1;
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Urban Dust Exposure Alters Gene Expression
however, Jeffy et al. (15) in a study of BRCA-1 expression in MCF-7
cells, did see a decrease after 72 hours of BP treatment.
Here, we have mainly focused on the alteration of two
important genes CYP1A1 and CYP1B1 known to be involved in
the metabolism of PAHs (26) in MCF-7 cells treated with BP,
SRM 1649a, DBP and combination treatments of BP plus SRM
1649a and DBP plus 1649a. The data obtained on several other
genes that were altered by >2-fold on exposure to combination
treatments (BP plus SRM 1649a and DBP plus SRM 1649a) will
be reported in a separate article in view of the specific nature of
this present discussion. Bartosiewicz et al. (47) have shown that
administration of BP to mice up-regulated CYP1A1 and CYP1A2
genes. The gene expression data in our study using the human
U133A microarray representing >22,000 genes indicated expression (>2-fold) of CYP1A1 and CYP1B1 in cells treated with BP,
DBP, and SRM 1649a (Fig. 1A and B). An additive increase in
expression of CYP1A1 and CYP1B1 was noted only in cells
treated with BP plus SRM 1649a (Fig. 1A and B). Shimada and
Fujii-Kuriyama (26) have shown that BP and other carcinogenic
PAHs, such as dibenz[a,h]anthracene, benzo[b]fluoranthene and
DBP induce CYP1B1 as well as CYP1A1 in liver and lung of AhR
(+/+) mice, but not in those of AhR( / ) mice. Also, CYP1A1
and CYP1B1 enzymes having similar, but not identical, substrate
specificity towards various PAHs (26) which would explain the
significant increase in CYP1A1 and CYP1B1 genes in cells treated
with BP and SRM 1649a. The gene expression data correlates
directly with the CYP1A1 activity as measured by ethoxyresorufin
O-deethylation assay and the protein expression levels of both
CYP1A1 and CYP1B1 as determined by Western blot (Table 4;
Fig. 3).
In our study, relating CYP1A1 or CYP1B1 gene and protein
induction levels (Figs. 1A and B and 3A and B) to metabolism of
the PAH forming DNA adducts (Fig. 2) in cells treated with SRM
1649a in comparison to SRM 1649a plus BP did not account for
the observed decrease in PAH-DNA adduct level. A similar
outcome was observed in an in vivo study with SRM 1597
derived from coal tar (16). Although SRM 1649a did induce the
CYP1A1 and CYP1B1 gene and protein, the induction levels were
not higher than those calculated in cells treated with SRM 1649a
plus BP or BP alone. These results suggest that the CYP enzymes
metabolized many of the various PAH in SRM 1649a to nonDNA-binding metabolities.
However, the decrease in DNA adducts formed in cells treated
with SRM 1649a plus BP may indicate the ability of PAH within
mixtures to competitively inhibit the activation of carcinogenic
PAH. Recently, Binkova and Sram (23) showed that, in human
diploid lung fibroblasts, BP-diol epoxide adduct levels were up to
5-fold lower in an artificial carcinogenic PAH mixture and up to
10-fold lower in an environmental mixture and they attribute this
decrease in adduct formation to competition for the metabolic
References
1. Lazarova M, Slamenova D. Genotoxic effects of a
complex mixture adsorbed onto ambient air particles
on human cells in vitro ; the effects of vitamins E and C.
Mutat Res 2004;557:167–75.
2. Hemminki K, Pershagen G. Cancer risk of air
pollution: epidemiological evidence. Environ Health
Perspect 1994;102 Suppl 4:187–92.
3. Perera FP. Molecular epidemiology: insights into
cancer susceptibility, risk assessment, and prevention.
J Natl Cancer Inst 1996;88:496–509.
www.aacrjournals.org
activation enzymes, their saturation and/or inactivation by PAH
metabolites. It has also been shown previously that the covalent
binding of different dibenzopyrenes to DNA as binary mixtures
formed relatively lower amounts of DNA adducts when compared
with DBP in skin and lungs (48). Similar inhibition or reduction in
PAH mixture induced tumor formation has also been reported
(16, 48). Competitive inhibition of the activation of PAH could
occur in several ways. One is simply competitive inhibition of the
CYP1A1 metabolism of the PAH, another is that the metabolism
of the PAH could be shifted to produce metabolites not capable
of being activated to DNA binding intermediates such as phenols.
Conducting biochemical experiments with SRM 1649a to
understand classic Michaelis-Menten kinetics would clearly
elucidate the competitive inhibition of the CYP enzymes that
may be operating.
Another mechanism by which SRM 1649a may be affecting the
formation of DNA adducts when cotreated with BP is by altering
the global gene expression pattern (Table 2). Cells treated with
SRM 1649a in comparison to BP exhibited an increase in the
expression of oncogenes (FOS and JUN) and a decrease in tumor
suppressor gene (CDKN1A; Tables 2 and 3). These responses
would make it possible for increased tumor formation allowing
for a decrease in adduct formation in the cotreated SRM 1649a
plus BP group compared with BP alone. Although our studies
with SRM 1649a were done using cells in culture, the results are
in good agreement with a published animal study using SRM 1597
(16) where a decrease in adduct formation in the cotreated SRM
1597 plus BP group compared with BP alone was observed in
spite of a similar tumorigenic response between the two
treatment groups.
In conclusion, the microarray results from our study shows a
broad spectrum of PAH-specific patterns of expression among the
genes examined. Future work will focus on using the broad
spectrum genes expressed to explore basic mechanisms of PAH
and/or urban dust particulate toxicity and to generate new
hypotheses. Also, the gene response patterns may shed new light
on the mechanisms of toxic chemical mixture-induced human
diseases and may also be useful for development of molecular
biomarkers of exposure and/or effect in mechanistic, epidemiologic, and risk assessment studies.
Acknowledgments
Received 7/8/2004; revised 11/12/2004; accepted 12/5/2004.
Grant support: National Cancer Institute CA 28825, DHHS and National Institute
of Environmental Health Sciences grant 5T32 ES07060-23 (B. Mahadevan).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Tuan Nguyen for the preparation of figures and Jennifer Atkin for adduct
analysis.
4. Somers CM, McCarry BE, Malek F, Quinn JS. Reduction
of particulate air pollution lowers the risk of heritable
mutations in mice. Science 2004;304:1008–10.
5. Samet JM, DeMarini DM, Malling HV. Biomedicine. Do
airborne particles induce heritable mutations? Science
2004;304:971–2.
6. Lewtas J, Gallagher J. Complex mixtures of urban air
pollutants: identification and comparative assessment
of mutagenic and tumorigenic chemicals and emission
sources. IARC Sci Publ 1990;252–60.
7. Donelly K, Brown KW, Anderson VS, Barbee GC,
Safe SH. Metabolism and bacterial mutagenicity of
binary mixtures of benzo[a]pyrene and polychlorinated
aromatic hydrocarbons. Environ Mol Mutagen 1990;16:
238–45.
8. Cerna M, Pastorkova A, Vrbikova V, et al. Mutagenicity
monitoring of airborne particulate matter (PM10) in the
Czech Republic. Mutat Res 1999;444:373–86.
9. Wendy Hsiao W, Mo ZY, Fang M, Shi X, Wang F.
Cytotoxicity of PM2,5 and PM2,5-10 ambient air
pollutants assessed by the MTT and the comet assays.
Mutat Res 2000;471:45–55.
10. Binkova B, Vesely D, Vesela D, et al. Genotoxicity
and embryotoxicity of urban air particulate matter
1257
Cancer Res 2005; 65: (4). February 15, 2005
Downloaded from cancerres.aacrjournals.org on August 2, 2017. © 2005 American Association for Cancer
Research.
Cancer Research
collected during winter and summer period in two
different districts of the Czech Republic. Mutat Res
1999;440:45–58.
11. Wise SA SM. Reference materials for environmental
analysis: making and using them. In: Clement R, Siu M,
editors. Boca Raton (FL): Lewis Publishers; 1997.
p. 143–86.
12. May WE, Benner BA, Wise SA, Schuetzle D, Lewtas J.
Standard reference materials for chemical and biological studies of complex environmental samples. Mutat
Res 1992;276:11–22.
13. Wise SA, Sander LC, Schantz MM, Hays MJ, Benner
BA. Polycyclic Aromat. Compd 2000;13:419–56.
14. National Institute of Standards and Technology.
Certificate of analysis, standard reference material
(SRM) 1649, urban dust/organics (revised January 27,
1992). Gaithersburg (MD): National Institute of Standards and Technology;1982.
15. Jeffy BD, Chirnomas RB, Chen EJ, et al. Activation
of the aromatic hydrocarbon receptor pathway is not
sufficient for transcriptional repression of BRCA-1:
requirements for metabolism of benzo[a]pyrene to
7r,8t-dihydroxy-9t,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. Cancer Res 2002;62:113–21.
16. Marston CP, Pereira C, Ferguson J, et al. Effect of a
complex environmental mixture from coal tar containing polycyclic aromatic hydrocarbons (PAH) on
the tumor initiation, PAH-DNA binding and metabolic
activation of carcinogenic PAH in mouse epidermis.
Carcinogenesis 2001;22:1077–86.
17. Livak KJ, Schmittgen TD. Analysis of relative gene
expression data using real-time quantitative PCR and
the 2(-D D C(T)) method. Methods 2001;25:402–8.
18. Luch A, Coffing SL, Tang YM, et al. Stable expression
of human cytochrome P450 1B1 in V79 Chinese
hamster cells and metabolically catalyzed DNA adduct
formation of dibenzo[a ,l]pyrene. Chem Res Toxicol
1998;11:686–95.
19. Ralston SL, Seidel A, Luch A, et al. Stereoselective
activation of dibenzo[a,l]pyrene to ( )-anti
(11R,12S,13S,14R)- and (+)-syn(11S,12R,13S,14R)-11,12diol-13,14- epoxides which bind extensively to deoxyadenosine residues of DNA in the human mammary
carcinoma cell line MCF-7. Carcinogenesis 1995;16:
2899–907.
20. Lau HH, Baird WM. Detection and identification of
benzo[a ]pyrene-DNA adducts by [35S]phosphorothioate labeling and HPLC. Carcinogenesis 1991;12:
885–93.
21. Otto S, Marcus C, Pidgeon C, Jefcoate CR. A novel
adrenocorticotropin-inducible cytochrome P 450
from rat microsomes catalyzes polycyclic aromatic
hydrocarbon metabolism. Endocrinology 1991;129:
970–82.
22. Saunders DB. RGT, association and prediction. 2nd
ed. In: Dolan J, Langan C, editors. Basic and clinical
Cancer Res 2005; 65: (4). February 15, 2005
biostatistics. Norwalk (CT): Appleton and Lange;
1994;162–87.
23. Binkova B, Sram RJ. The genotoxic effect of
carcinogenic PAHs, their artificial and environmental
mixtures (EOM) on human diploid lung fibroblasts.
Mutat Res 2004;547:109–21.
24. Mahadevan B, Luch A, Seidel A, et al. Effects of
the ( )-anti-11R ,12S -dihydrodiol 13S ,14R -epoxide
of dibenzo[a ,l]pyrene on DNA adduct formation and
cell cycle arrest in human diploid fibroblasts. Carcinogenesis 2001;22:161–9.
25. Hartman AR, Ford JM. BRCA1 induces DNA damage
recognition factors and enhances nucleotide excision
repair. Nat Genet 2002;32:180–4.
26. Shimada T, Fujii-Kuriyama Y. Metabolic activation
of polycyclic aromatic hydrocarbons to carcinogens
by cytochromes P 450 1A1 and 1B1. Cancer Sci 2004;
95:1–6.
27. Palackal NT, Lee SH, Harvey RG, et al. Activation of
polycyclic aromatic hydrocarbon trans-dihydrodiol
proximate carcinogens by human aldo-keto reductase
(AKR1C) enzymes and their functional overexpression
in human lung carcinoma (A549) cells. J Biol Chem
2002;277:24799–808.
28. Burczynski ME, Lin HK, Penning TM. Isoformspecific induction of a human aldo-keto reductase by
polycyclic aromatic hydrocarbons (PAHs), electrophiles, and oxidative stress: implications for the
alternative pathway of PAH activation catalyzed by
human dihydrodiol dehydrogenase. Cancer Res 1999;
59:607–14.
29. Choi JY, Lee KM, Cho SH, et al. CYP2E1 and NQO1
genotypes, smoking and bladder cancer. Pharmacogenetics 2003;13:349–55.
30. Hassett C, Aicher L, Sidhu JS, Omiecinski CJ. Human
microsomal epoxide hydrolase: genetic polymorphism
and functional expression in vitro of amino acid
variants. Hum Mol Genet 1994;3:421–8.
31. Gsur A, Zidek T, Schnattinger K, et al. Association of
microsomal epoxide hydrolase polymorphisms and
lung cancer risk. Br J Cancer 2003;89:702–6.
32. Georgiadis P, Demopoulos NA, Topinka J, et al.
Impact of phase I or phase II enzyme polymorphisms
on lymphocyte DNA adducts in subjects exposed to
urban air pollution and environmental tobacco smoke.
Toxicol Lett 2004;149:269–80.
33. Yang RS, Hong HL, Boorman GA. Toxicology of
chemical mixtures: experimental approaches, underlying concepts, and some results. Toxicol Lett 1989;49:
183–97.
34. Wolff MS, Collman GW, Barrett JC, Huff J. Breast
cancer and environmental risk factors: epidemiological and experimental findings. Annu Rev Pharmacol
Toxicol 1996;36:573–96.
35. Gammon MD, Santella RM, Neugut AI, et al.
Environmental toxins and breast cancer on Long
1258
Island. I. Polycyclic aromatic hydrocarbon DNA
adducts. Cancer Epidemiol Biomarkers Prev 2002;11:
677–85.
36. Brody JG, Rudel RA. Environmental pollutants and
breast cancer. Environ Health Perspect 2003;111:
1007–19.
37. Vincent R, Goegan P, Johnson G, Brook JR,
Kumarathasan P, Bouthillier L, Burnett RT. Regulation of promoter-CAT stress genes in HepG2 cells by
suspensions of particles from ambient air. Fundam
Appl Toxicol 1997;39:18–32.
38. Thomson E, Goegan P, Kumarathasan P, Vincent R.
Air pollutants increase gene expression of the
vasoconstrictor endothelin-1 in the lungs. Biochim
Biophys Acta 2004;1689:75–82.
39. Frazier KS, Grotendorst GR. Expression of connective tissue growth factor mRNA in the fibrous stroma
of mammary tumors. Int J Biochem Cell Biol 1997;29:
153–61.
40. Xie D, Nakachi K, Wang H, et al. Elevated levels of
connective tissue growth factor, WISP-1, and CYR61 in
primary breast cancers associated with more advanced features. Cancer Res 2001;61:8917–23.
41. Tsai MS, Bogart DF, Castaneda JM, et al. Cyr61
promotes breast tumorigenesis and cancer progression. Oncogene 2002;21:8178–85.
42. Denissenko MF, Venkatachalam S, Ma YH, Wani AA.
Site-specific induction and repair of benzo[a ]pyrene
diol epoxide DNA damage in human H-ras protooncogene as revealed by restriction cleavage inhibition. Mutat Res 1996;363:27–42.
43. Bral CM, Ramos KS. Identification of benzo[a]pyrene-inducible cis-acting elements within c-Ha-ras
transcriptional regulatory sequences. Mol Pharmacol
1997;52:974–82.
44. Aust AE, Antczak MR, Maher VM, McCormick JJ.
Identifying human cells capable of metabolizing
various classes of carcinogens. J Supramol Struct Cell
Biochem 1981;16:269–79.
45. Thompson ME, Jensen RA, Obermiller PS, Paige PS,
Holt DL. Decreased expression of BRCA1 accelerates growth and is often present during sporadic breast cancer progression. Nat Genet 1995;9:
444–50.
46. Jeffy BD, Chirnomas RB, Romagnolo DF.
Epigenetics of breast cancer: polycyclic aromatic
hydrocarbons as risk factors. Environ Mol Mutagen
2002;39:235–44.
47. Bartosiewicz M, Penn S, Buckpitt A. Applications of
gene arrays in environmental toxicology: fingerprints
of gene regulation associated with cadmium chloride,
benzo(a)pyrene, and trichloroethylene. Environ Health
Perspect 2001;109:71–4.
48. Hughes NC, Phillips DH. Covalent binding of
dibenzpyrenes and benzo[a]pyrene to DNA: evidence
for synergistic and inhibitory interactions when
applied in combination to mouse skin. Carcinogenesis
1990;11:1611–9.
www.aacrjournals.org
Downloaded from cancerres.aacrjournals.org on August 2, 2017. © 2005 American Association for Cancer
Research.
Altered Gene Expression Patterns in MCF-7 Cells Induced by
the Urban Dust Particulate Complex Mixture Standard
Reference Material 1649a
Brinda Mahadevan, Channa Keshava, Tamara Musafia-Jeknic, et al.
Cancer Res 2005;65:1251-1258.
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