Hindawi Publishing Corporation

BioMed Research International

Volume 2014, Article ID 605135, 12 pages
http://dx.doi.org/10.1155/2014/605135

Research Article
Profiling of Biomarkers for the Exposure of
Polycyclic Aromatic Hydrocarbons: Lamin-A/C Isoform 3,
Poly[ADP-ribose] Polymerase 1, and Mitochondria Copy
Number Are Identified as Universal Biomarkers

Hwan-Young Kim,1,2 Hye-Ran Kim,2,3 Min-Gu Kang,1 Nguyen Thi Dai Trang,2
Hee-Jo Baek,4 Jae-Dong Moon,4 Jong-Hee Shin,1 Soon-Pal Suh,1 Dong-Wook Ryang,1
Hoon Kook,4 and Myung-Geun Shin1,2,4
1
Department of Laboratory Medicine, Chonnam National University Medical School and Chonnam National University
Hwasun Hospital, 160 Ilsimri, Hwasun-eup, Hwasun-gun, Jeollanam-do 519-809, Republic of Korea
2
Brain Korea 21 Plus Project, Chonnam National University Medical School, Gwangju, Republic of Korea
3
Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
4
Environment Health Center for Childhood Leukemia and Cancer, Chonnam National University Hwasun Hospital,
Hwasun, Republic of Korea

Correspondence should be addressed to Myung-Geun Shin; [email protected]

Received 28 February 2014; Revised 2 June 2014; Accepted 4 June 2014; Published 10 July 2014

Academic Editor: Giulio Mengozzi

Copyright © 2014 Hwan-Young Kim et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

This study investigated the profiling of polycyclic aromatic hydrocarbon- (PAH-) induced genotoxicity in cell lines and zebrafish.
Each type of cells displayed different proportionality of apoptosis. Mitochondrial DNA (mtDNA) copy number was dramatically
elevated after 5-day treatment of fluoranthene and pyrene. The notable deregulated proteins for PAHs exposure were displayed as
follows: lamin-A/C isoform 3 and annexin A1 for benzopyrene; lamin-A/C isoform 3 and DNA topoisomerase 2-alpha for pentacene;
poly[ADP-ribose] polymerase 1 (PARP-1) for fluoranthene; and talin-1 and DNA topoisomerase 2-alpha for pyrene. Among them,
lamin-A/C isoform 3 and PARP-1 were further confirmed using mRNA and protein expression study. Obvious morphological
abnormalities including curved backbone and cardiomegaly in zebrafish were observed in the 54 hpf with more than 400 nM of
benzopyrene. In conclusion, the change of mitochondrial genome (increased mtDNA copy number) was closely associated with
PAH exposure in cell lines and mesenchymal stem cells. Lamin-A/C isoform 3, talin-1, and annexin A1 were identified as universal
biomarkers for PAHs exposure. Zebrafish, specifically at embryo stage, showed suitable in vivo model for monitoring PAHs exposure
to hematopoietic tissue and other organs.

1. Introduction with PAHs exposure such as carcinogenesis and endocrine

disruption. Although the adverse effects of individual PAHs
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous are not exactly alike, the United States Environmental Pro-
environmental toxicants found in air, water, plants, and soils tection Agency (EPA) has designated 32 PAHs compounds
which are present as volatile, semivolatile, and particulate as priority pollutants (http://www.epa.gov/). The toxicity of
pollutants [1]. PAHs have been of increasing concern in the PAHs is structure dependent. Benzopyrene (BaP) among
human health field due to their wide-spread dispersion in 32 PAH compounds is notable for being the first chemical
the environment and the adverse health effects associated carcinogen to be discovered [2, 3].
2 BioMed Research International

The most commonly used biomarkers of PAHs exposure Previous published protocol was used for the isolation and
are metabolites of PAHs, particularly 1-hydroxypyrene (1- characterization of bone marrow-derived mesenchymal
OHP), and PAH-DNA or protein adducts [3]. 1-OHP is the stem cells (h-TERT) [17]. For in vitro cell line study, cells
principal product of pyrene metabolism, representing 90% were cultured and maintained in RPMI media containing
of its metabolites [4]. Following inhalation, the half-life of 10% fetal bovine serum and four types of PAHs such as BaP,
1-OHP is on average about 18–20 hours [5–7]. Pyrene is the pentacene, fluoranthene, and pyrene were added in the cell
only known precursor of 1-OHP [8]; it forms a consistent pro- culture media with 100 𝜇M concentration.
portion of higher molecular weight PAHs in the environment
[9]. Main analytical methods employed to measure 1-OHP 2.2. Chemicals. BaP (purity > 99%), fluoranthene (99%),
are high performance liquid chromatography (LC) combined pentacene (>99%), and pyrene (>99%) were purchased from
with fluorescence detection and gas chromatography with Sigma (Sigma-Aldrich, St. Louis, MO, USA). Stock PAHs
mass spectrometry [10, 11]. solutions were made in dimethyl sulfoxide (DMSO) (Sigma-
Biomarkers to assess exposure to PAHs at high levels Aldrich) at concentration of 100 𝜇M.
are well studied, but more work is needed to validate these
biomarkers when exposure occurs at low, environmental 2.3. Cytotoxicity Assay. Cytotoxicity assays were carried out
levels. Most reported biomarkers for PAHs exposure were using the Enhanced Cell Viability Assay Kit (EZ-CyTox, Daeil
mainly targeted against nuclear genome and proteome as well Lab Service Co., Seoul, Korea) protocol. The absorbance
as metabolites in either serum or urine. Moreover, biomarkers (A450) of each well was measured using a VERSA Max
as mentioned in several studies [3] did not reflect PAHs microplate reader (Molecular Devices, Sunnyvale, CA, USA).
exposure sensitively in genomic and proteomic level.
Enormous strides have recently been made in our
understanding of the biology and pathobiology of mito- 2.4. Determination of mtDNA Copy Number. mtDNA copy
chondria. Many diseases have been identified as caused by number was determined according to our published protocol
mitochondrial dysfunction, and many pharmaceuticals have [13]. For in vitro model study, the purified PCR product of
been identified as previously unrecognized mitochondrial cytochrome b (Cytb) gene was inserted into pGEM-T easy
toxicants. A much smaller but growing reports indicate that vector and E. coli JM 109 cells (Promega, Madison, WI, USA)
mitochondria are also targeted by environmental pollutants were transformed in order to obtain recombinant plasmids.
[12]. Past evidence had indicated that the mtDNA repair The mtDNA copy number was calculated using the following
capacity is limited and that the proximity of mtDNA to sites formula: [𝑋 𝜇g/𝜇L plasmid DNA/4419 (plasmid length) ×
of reactive oxygen species generation suggested that mtDNA 660] × 6.022 × 1023 = 𝑌 molecules/𝜇L, where 𝑋 represents
may be more susceptible to mutation than nuclear DNA. Our the concentration of plasmid DNA and 𝑌 represents copy
laboratory has recently reported that hnRNP protein and the number. For in vivo model study, a mixture of 25 𝜇L con-
change of mitochondrial genome are recognized as novel and taining 12.5 𝜇L 2x QuantiTect SYBR green PCR master
useful markers for benzene exposure [13]. Moreover, there is mix (Qiagen, Valencia, CA, USA), 400 𝜇M Cytb primers
currently a paucity of data on the direct effects of PAHs in forward (5󸀠 -TTCTGAGGGGCCACAGTAAT-3󸀠 ) and reverse
primary hematopoietic cells and various cell lines. (5󸀠 -GGGGTTATTTGATCCGGTTT-3󸀠 ), and 50 ng of total
In zebrafish, hundreds of genes involved in the formation DNA were used for the PCR with the CFX96 real-time system
of virtually every organ system have been identified by large- (Bio-Rad, Hercules, CA, USA). For PCR, 95∘ C for 15 minutes
scale mutagenesis screening [14]. Consequently, the pheno- was followed by 35 cycles of 20 seconds at 94∘ C, 30 seconds
types resulting from loss of gene function through mutation at 52∘ C, 30 seconds at 72∘ C, and a melting reaction with a
can be compared to malformations resulting from embryonic decrease of 1∘ C per cycle between 72∘ C and 92∘ C.
exposure to contaminants. This “chemical genetic” approach
has been used recently to identify specific mechanisms of 2.5. Direct Sequencing of mtDNA Control Region. This study
developmental toxicity [15, 16]. used a published protocol to amplify and sequence the
Therefore, this study investigated to identify new bio- mtDNA control region gene and minisatellites (303 poly C,
markers and pathobiological role for PAHs exposure, espe- 16189 poly C and 514 (CA) repeat) [13]. The mtDNA sequen-
cially BaP using targeted mitochondrial genomic and pro- ces obtained were analyzed using the Revised Cambridge
teomic approach in cell line, peripheral blood/mesenchy- Reference Sequence (http://www.mitomap.org/), Blast2 pro-
mal stem cell, and in vivo zebrafish model. gram (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi),
and the MitoAnalyzer (http://www.cstl.nist.gov/biotech/str-
2. Materials and Methods base/mitoanalyzer.html) to identify mtDNA aberrations.

2.1. Reagents and Cell Lines. Cell lines (K562, THP-1, MOLT- 2.6. Proteomic Assay of Mitochondria-Rich Cellular Fraction
4, and HL-60 cells) were obtained from the American Type
Culture Collection, which were cultured in RPMI 1640 2.6.1. One-Dimensional SDS-Polyacrylamide Gel Electrophore-
medium (Gibco Laboratories, Grand Island, NY, USA) sis. Briefly, an equal amount of proteins (30 𝜇g) was then sep-
supplemented with 10% fetal bovine serum (Gibco) arated on NuPAGE 4–12% Bis-Tris Gel (Invitrogen; Carlsbad
(see Supplementary Table 1 in Supplementary Material CA, USA). After separation, the gel was stained with GelCode
available online at http://dx.doi.org/ 10.1155/2014/605135). Blue Stain Reagent (Thermo scientific) and the blue-stained
BioMed Research International 3

gel lanes were removed by manual cutting. Each blue-stained (Applied Biosystems). The expression of poly[ADP-ribose]
gel lane was separately cut into 5 slices. Each of these gel slices polymerase 1 (PARP-1) and lamin A/C (LMNA) mRNA was
was then further cut into sizes of ∼1 mm3 and transferred to quantified using QuantiTect SYBR green PCR master mix
a clean 1.5 mL tube. (Qiagen), PARP-1 forward: 5󸀠 -GAGGAAGTAAAGGAA-
GCCAA-3󸀠 , PARP-1 reverse: 5󸀠 -CACAACTTCAACAGG-
2.6.2. Enzymatic In-Gel Digestion. The separated proteins CTCT-3󸀠 , LMNA forward: 5󸀠 -AAGCTTCGAGACCTG-
were excised from the gel and the gel pieces containing pro- GAG-3󸀠 , LMNA reverse: 5󸀠 -TCCAAGAGCTTGCGGTA-3󸀠 ,
tein were destained with 50% acetonitrile (ACN) containing and 𝛽-actin mRNA as a normalization control. The ΔΔCt
50 mM NH4 HCO3 and the gel pieces were vortexed until method was used to calculate relative changes in gene
Coomassie Brilliant Blue was completely removed. These gel expression determined by real-time quantitative PCR using
pieces were then dehydrated in 100% ACN and vacuum-dried CFX96 (Bio-Rad). Normalization was achieved using Ct
for 20 min with SpeedVac. For the digestion, gel pieces were values of PARP-1 and LMNA mRNA from PAHs-treated cells
reduced using 10 mM dithiothreitol in 50 mM NH4 HCO3 and 𝛽-actin mRNA. The ΔCtcalibrator value (mean PARP-1 and
for 45 min at 56∘ C, followed by alkylation of cysteines with LMNA—mean 𝛽 actin) was obtained from the mean Ct value
55 mM iodoacetamide in 50 mM NH4 HCO3 for 30 min in the of PARP-1 and LMNA mRNA and 𝛽-actin mRNA from the
dark. Finally, each of gel pieces was treated with 12.5 ng/𝜇L control cells (𝑛 = 10). The ΔΔCt value was calculated as ΔCt
sequencing grade modified trypsin (Promega) in 50 mM minus ΔCtcalibrator . The final relative quantification of PARP-1
NH4 HCO3 buffer (pH 7.8) at 37∘ C overnight. Following and LMNA mRNA was expressed as ΔΔCt.
digestion, tryptic peptides were extracted with 5% formic
acid in 50% ACN solution at room temperature for 20 min. 2.8. Western Blot. Extracted protein samples (20 𝜇g per well)
The supernatants were collected and dried with SpeedVac. were separated on a 12% SDS-Bis-Tris polyacrylamide gel.
Resuspended samples in 0.1% formic acid were purified and After transfer, the nitrocellulose membrane was incubated
concentrated using C18 ZipTips (Millipore, Billerica, MA, over night with 10 mL of primary antibodies against PARP-
USA) before mass spectrometry (MS) analysis. 1, LMNA (Santa Cruz Biotechnology, Delaware Avenue, CA,
USA), and 𝛽-actin (Santa Cruz Biotechnology) at 4∘ C. The
2.6.3. Nano-LC-Electrospray Ionization-MS/MS Analysis. The membrane was then incubated with the appropriate goat anti-
tryptic peptides were loaded onto a fused silica microcapil- mouse IgG antibody (1 : 1000) (Jackson ImmunoResearch
lary column (12 cm × 75 𝜇m) packed with C18 reversed phase Laboratories, West Grove, PA, USA) to detect biotinylated
resin (5 𝜇m, 200 Å). LC separation was conducted under a protein markers in 10 mL of blocking buffer with gentle
linear gradient as follows: a 3–40% solvent B (ACN contain- agitation for 1 hour at room temperature. The proteins
ing 0.1% formic acid) gradient (solvent A; DW containing were visualized using a chemiluminescence detection system
0.1% formic acid), with a flow rate of 250 nL/min, for 60 (Amersham ECL system, London, UK).
minutes. The column was directly connected to linear trap
quadropole linear ion-trap mass spectrometer (Finnigan, 2.9. In Vivo Study. For in vivo model study, zebrafish embryos
San Jose, CA, USA) equipped with a nanoelectrospray ion 30 h after fertilization (hpf) were exposed to BaP at concen-
source. The electrospray voltage was set at 1.95 kV, and the trations of 200, 400, 600, 800, and 1000 nM. Seventy embryos
threshold for switching from MS to MS/MS was 500. The were cultured in 40 mL of BaP solution in each petri dish,
normalized collision energy for MS/MS was 35% of main and there were three replicates for each of the five treat-
radio frequency amplitude and the duration of activation ments. Embryos were collected at 54 hpf, 78 hpf, and 102 hpf.
was 30 ms. All spectra were acquired in data-dependent scan Embryos were maintained under the same temperature and
mode. Each full MS scan was followed by five MS/MS scans pH conditions for the duration of experiments.
corresponding to the range from the most intense to the
fifth intense peaks of full MS scan. Repeat count of peak
3. Results
for dynamic exclusion was 1, and its repeat duration was
30 seconds. The dynamic exclusion duration was set for 180 3.1. The Change of Cell Morphology. PAH-untreated h-TERT
seconds and the width of exclusion mass was ±1.5 Da. cells showed compact cellularity with spindle shape. Cells
were tightly attached to each other and to the substrate.
2.6.4. Database Searching and Validation. The acquired Generally, direct exposure of PAHs such as BaP, pentacene,
LC-electrospray ionization-MS/MS fragment spectra were fluoranthene, and pyrene depressed the proliferative capacity
searched in the BioWorksBrowser (version Rev. 3.3.1 SP1, of h-TERT cells and the cell morphology was altered in
Thermo Fisher Scientific Inc.) with the SEQUEST search each PAH-exposure group. Cells became detached from the
engines against National Center for Biotechnology Informa- subsurface, and cell-to-cell attachments were lost (Figure 1).
tion (http://www.ncbi.nlm.nih.gov/) nonredundant human
database. 3.2. The Change of Total Cell Counts. Depending on the type
of PAHs, each cell count showed different aspects. The total
2.7. Quantitative mRNA Expression Study. Total RNA was number of cells in the THP-1 and Molt-4 cell lines decreased
extracted using the QIAamp RNA Blood Mini kit (Qiagen). 11 days after PAHs exposure. The change in the total number
Reverse transcription produced cDNAs using Superscript III of cells in the THP-1 and Molt-4 cell lines decreased in
4 BioMed Research International

1 day 4 days 7 days 10 days 14 days

BaP

Pentacene

Fluoranthene

Pyrene

DMSO

Normal

Figure 1: Morphological change of human mesenchymal stem (h-TERT) cells after PAHs exposure. PAH-untreated cells (DMSO and normal)
showed compact cellularity with spindle shape. h-TERT cells were tightly attached to each other and to the substrate. Generally, direct exposure
of PAHs depressed the proliferative capacity of h-TERT cells with a thread-like or round shape and loose cell-to-cell attachment. Each PAHs
compound showed different cytotoxic effect. DMSO and normal indicated only DMSO-treatment and culture solution itself (no treatment
of PAHs and DMSO), respectively.

a time-dependent manner. In comparison to control group, 3.4. Increased mtDNA Copy Number. Mitochondrial contents
fluoranthene displayed profound significant reduction in cell were increased with different pattern: mtDNA copy number
count (Figures 2(a) and 2(b)). The change in the total cell was dramatically elevated after 5-day treatment of fluoran-
count for the THP-1 and Molt-4 cell lines had a similar thene and pyrene in both cell line and in vivo zebrafish model.
pattern after PAHs exposure. Cytotoxicity study carried out mtDNA copy numbers were generally increased after PAHs
the experiment with 100 𝜇M of PAHs after selecting the exposure in a dose and time-dependent manner in the cell
minimum concentration that is poisonous to cells. lines. These findings suggested that loss of compensatory
ability in response to high levels of oxidative stress was
3.3. Viability and Apoptosis. Viability significantly decreased induced by high concentrations of PAHs (Figure 3).
after two days of exposure to fluoranthene. On the third day
of PAHs exposure, viability reduced remarkably in all the
cells (Figures 2(c) and 2(d)). Each type of cell lines displayed 3.5. Sequence Alteration of mtDNA Control Region. Changes
different proportionality of apoptosis. Several hundreds of of the mtDNA sequence were comprehensively studied
PAHs exposure biomarkers were identified in comparison to by direct sequencing of the mtDNA control region and
control group (Supplemental Figure 1). gene scanning for the determination of mtDNA length and
BioMed Research International 5

THP-1 Molt-4

Benzopyrene Benzopyrene

Pentacene Pentacene

Fluoranthene Fluoranthene

Pyrene Pyrene

DMSO DMSO

0 1 2 3 0.0 0.2 0.4 0.6 0.8 1.0

Cell count (×106 ) Cell count (×106 )
16 days 2 days 16 days 2 days
11 days 1 day 11 days 1 day
4 days 4 days
(a) (b)
THP-1 Molt-4
150 110

100
100
Viability (%)

Viability (%)

90

80
50

70

0 60
8 hr 24 hr 2 days 3 days 6 days 8 hr 24 hr 2 days 3 days 6 days

Benzopyrene Pyrene Benzopyrene Pyrene

Pentacene DMSO Pentacene DMSO
Fluoranthene Fluoranthene
(c) (d)

Figure 2: The change of cell count and viability after PAHs exposure in THP-1 and Molt-4 cell line. Depending on the type of PAHs, each cell
count showed different aspects. In comparison to DMSO treated (0.1%) group, fluoranthene displayed profound significant reduction in cell
count, especially in THP-1 and Molt-4 cell line ((a) and (b)). Viability was significantly decreased after fluoranthene exposure for two days.
On the third day of PAHs exposure, viability was reduced remarkably in both cell lines ((c) and (d)).

heteroplasmic mutations. No alteration of mtDNA sequences 3.7. Confirmation of Mitochondrial Protein Markers
was observed after direct exposure of PAHs during 7 days
(Supplemental Figure 2). No alteration of mtDNA minisatel- 3.7.1. Increased mRNA Expression of PARP-1 and LMNA Gene.
lites such as 1618 poly C, 303 poly C and 514 (CA) repeat was mRNA expression of PARP-1 and LMNA gene was generally
found after PAHs exposure. increased in THP-1 and h-TERT cell lines after exposure
of PAHs with different pattern. This finding was confirmed
using embryogenesis in zebrafish model (Figure 5).
3.6. Mitochondrial Protein Markers. Several hundreds of
cellular proteins in mitochondrial-rich cytoplasmic fraction 3.7.2. Increased Protein Expression of PARP-1 and LMNA
were profoundly deregulated in comparison to control group Gene. The expression of PARP-1 protein was increased after
(Figure 4). The notable deregulated proteins for PAHs expo- exposure of BaP, pentacene, and fluoranthene. The LMNA
sure were displayed as follows: LMNA and annexin A1 for proteins were increased after exposure of BaP (Figure 6).
BaP; LMNA and DNA topoisomerase 2-alpha for pentacene;
PARP-1 for fluoranthene; and talin-1 and DNA topoisomerase 3.8. Morphological Abnormalities of Zebrafish. At 54 hpf,
2-alpha for pyrene (Tables 1 and 2). embryos treated with 400 nM BaP exhibited mild pericardial
6 BioMed Research International

×104
5

CYTB/𝛽-actin
3

0
1 2 3 4 5 7
(day)
Benzopyrene Pyrene
Pentacene DMSO
Fluoranthene
(a)
54 hpf 78 hpf
4 3

3
2
CYTB/𝛽-actin

CYTB/𝛽-actin

1
1

0 0
Normal DMSO 400 nM 600 nM 800 nM 1 𝜇M Normal DMSO 400 nM 600 nM 800 nM
(b)

Figure 3: The change of mtDNA copy number after PAHs exposure. mtDNA copy number was increased after exposure of PAHs with
different pattern in THP-1 cell line (a) and in vivo zebrafish model (b). mtDNA copy number was dramatically elevated after 5-day treatment
of fluoranthene and pyrene in both THP-1 cell line and in vivo zebrafish model. hpf, hours per fertilization in zebrafish; normal, no treatment
group; and DMSO, only DMSO (0.1%) treated group.

edema and showed dorsal curvature of the body axis widely in different industrial activities and job titles, ranging
(Figure 7). Dorsal curvature was more severe by higher from 0.1 to 48 000 ng/m3 [21–23]. In smokers, BaP levels
concentration of BaP as edema accumulated. Notably, eye and range from 0.5 to 7.8 𝜇g/100 cigarettes when exposure is from
jaw growths were similarly reduced by BaP treatment. mainstream smoke and from 2.5 to 19.9 𝜇g/100 cigarettes
when it comes from side-stream smoke. Levels from passive
4. Discussion smoking are lower, ranging from 0.0028 to 0.76 𝜇g/m3 of BaP
[24]. Besides occupational exposure, dietary intake seems
PAHs are known genotoxic agents and induce DNA dam- to be the most important source of PAHs in nonsmokers
aging effects, such as DNA adducts, DNA strand breaks, [24, 25]. There is a high variation in atmospheric PAHs levels
chromosomal aberrations, sister chromatid exchanges, and across geographical areas with BaP concentrations ranging
micronucleus formation [18]. The main sources of human from 0.01 to 100 ng/m3 BaP [26]. Airborne PAHs are usually
exposure to PAHs are occupation, passive and active smok- analyzed by gas chromatography/mass spectrometry [27,
ing, and food, water, and air pollution [19]. The total intake 28] or high performance LC [29–31], mostly from particles
of carcinogenic PAHs in the general population has been collected in a filter after extraction with organic solvents.
estimated to be 3 𝜇g/day [20]. Levels of occupational exposure In order to exert its deleterious effects, BaP must be
of BaP, which is one of the main PAHs compounds, vary bioactivated. The formation of BaP o-quinones has been
BioMed Research International 7

Benzopyrene Fluoranthene
Cell adhesion, 0.4 Cell
communication, Cell adhesion, 0.6
System process, 2.6 Apoptosis, 1.8 System process, 2.3 Apoptosis, 2.3
5.3 Cell
Response to Transport, 6.4 communication , Cell cycle, 7.0
Transport, Cell cycle, 7.7 Response to
stimulus, 2.8 stimulus, 5.3 7.0
9.0 Cellular
Reproduction, 0.4 Reproduction, 1.2 Cellular
component component
organization, organization,
6.3 4.7
Cellular process,
Metabolic process,
15.8 Cellular process,
36.8 Metabolic process, 16.4
32.7

Development
process, 5.7
Generation of
Development
Localization, 0.2 Homeostatic precursor
process, 7.0
Immune system process, 0.2 metabolites and Immune system Homeostatic
process, 3.5 energy, 1.5 process, 6.4 process, 0.6

Pentacene Pyrene
Cell
Apoptosis, 1.2 Cell
Cell communication,
System process, 2.0 Cell adhesion, 1.4 communication, System process, 2.7 adhesion,
5.4
1.5
Apoptosis, 1.1 6.0 Response to Cell cycle, 6.6
Response to Cell cycle, 5.5 stimulus, 1.7 Transport, Cellular
stimulus, 2.0 Cellular 12.0 component
Transport, Reproduction, 1.0
Reproduction, 0.6 component organization,
10.9
organization, 6.4
5.2
Cellular process,
Cellular process, 16.5
16.4 Metabolic process,
Metabolic process, 34.6
38.8

Development
Development process, 5.9
process, 5.5
Generation of
Homeostatic Homeostatic
Generation of precursor
process, 0.6 Localization, 0.2 Immune system process, 0.2 metabolites and
precursor
Localization, 0.3 metabolites and process, 2.5 energy, 1.5
Immune system
energy, 1.4
process, 2.3

(a)
Benzopyrene Fluoranthene
Translation regulator
Translation regulator
Structural molecular activity, 2.7
activity, 11.4 activity, 1.3 Transcription Antioxidant
Transcription Transporter
Transporter activity, 2.7 activity, 0.9
Motor activity, 0.3 regulator activity, 3.4 Structural molecular regulator
activity, 4.4 activity, 10.0 activity, 6.4
Receptor activity, 1.0
Receptor
Ion channel activity, 2.7
Binding, 33.6
activity, 0.7 Binding, 39.3 Motor
activity, 0.9

Ion channel
Enzyme activity, 1.8
regulator Catalytic activity, 34.9 Catalytic activity, 34.5
activity, 3.4
Enzyme
regulator
activity, 3.6

Pentacene Pyrene
Translation
Translation regulator Transcription regulator Transporter
Structural activity, 3.4
activity, 2.0 regulator activity, 2.1
molecular activity, 4.8
activity, 10.2 Transcription Transporter Antioxidant
Motor regulator activity, 5.1 Structural activity, 0.7
activity, 0.4 activity, 3.9 molecular
activity, 12.3
Receptor Receptor
activity, 2.0 activity, 1.7 Binding, 37.3
Ion channel Binding, 36.5
Motor
activity, 1.6 activity, 1.7

Catalytic activity, 31.5

Catalytic activity, 36.5 Ion channel
activity, 0.7

Enzyme Enzyme
regulator regulator
activity, 2.0 activity, 3.8

(b)

Figure 4: Functional grouping of potential candidate biomarkers for PAHs exposure. Identified potential biomarkers were categorized as their
biological process (a) and molecular functions (b). These candidate biomarkers for PAHs exposure were isolated using proteomic analysis of
mitochondria-rich cellular fraction in THP-1 cell line.

described as one of the BaP activation pathways. Cytochrome metabolized by the cytochrome P4501A, the microsomal
P4501A (CYP1A) is able to produce BaP-7, 8 diol that is epoxide hydrolase, and the glutathione-S-transferase 𝛼 [33].
further oxidized to BaP-7, 8-dione by AKR1A1 [32]. BaP binds AhR is a ligand-activated transcription factor involved in
to and activates the aryl hydrocarbon receptor (AhR), being the regulation of biological responses to planar aromatic
8 BioMed Research International

Table 1: Summary list of identified potential biomarkers for PAHs exposure.

PAHs Protein Fold change

Vimentin 19.39
Annexin A1 13.38
Lamin-A/C 10.04
Benzopyrene NADPH: adrenodoxin oxidoreductase, mitochondrial isoform 2 precursor 5.3
Squalene synthase 5.3
Heterogeneous nuclear ribonucleoproteins A2/B1 isoform B1 4.82
T-complex protein 1 subunit theta 4.35
Talin-1 4.35
Lamin-A/C 6.9
DNA topoisomerase 2-alpha 6.38
Annexin A1 6.38
Pentacene Poly[ADP-ribose] polymerase 1 5.85
Squalene synthase 5.33
Talin-1 5.33
PREDICTED: u5 small nuclear ribonucleoprotein 200 kDa helicase-like, 4.81
partial
Poly[ADP-ribose] polymerase 1 6.21
Elongation factor 1-gamma 5.21
Fluoranthene Heat shock 70 kDa protein 1A/1B 5.21
Heterogeneous nuclear ribonucleoproteins A2/B1 isoform B1 5.21
Probable ATP-dependent RNA helicase DDX5 5.21
T-complex protein 1 subunit theta 5.21
Talin-1 16.82
DNA topoisomerase 2-alpha 8.17
Pyrene Filamin-C isoform b 7.16
E3 SUMO-protein ligase RanBP2 5.65
CAD protein 5.14
Poly[ADP-ribose] polymerase 1 5.14

Table 2: Results of PARP-1 and LMNA protein by repeat proteomic hydrocarbons. AhR ligands have been generally classified
analysis. into two categories, synthetic or naturally occurring. The first
ligands to be discovered were synthetic and members of halo-
Fold change
Protein genated aromatic hydrocarbons. Naturally occurring com-
First result Second result pounds that have been identified as ligands of AhR include
PARP-1 (accession no: 156523968) derivatives of tryptophan [34, 35]. The major contributors to
air PAHs in the urban and suburban atmosphere are mobile
DMSO versus BaP 3.41 3.58
sources from diesel and gasoline engines. Emissions from
DMSO versus pentacene 5.85 4.31 these sources contain mainly benzo(g,h,i)perylene, pyrene,
DMSO versus fluoranthene 6.21 5.34 fluoranthene, and phenanthrene [36], so that measuring
5.14 3.41 only BaP as an index substance may result in exposure
DMSO versus pyrene
underestimation [3].
LMNA (accession no: 27436948) In this study, a broad molecular investigation of the
DMSO versus BaP 10.04 4.16 mitochondrial genome and proteome after PAHs exposure
DMSO versus pentacene No change 0.97 showed an increased mtDNA copy number, PARP-1, and
LMNA protein, which could be used as biomarkers for
DMSO versus fluoranthene 4.50 3.00
exposure of PAHs in cell lines. PAHs directly might cause an
DMSO versus pyrene 4.14 1.80 increase in the generation of intracellular ROS, subsequently
DMSO, only DMSO-treatment as control; PARP-1, poly[ADP-ribose] poly- resulting in a change of the mtDNA content, and proteome.
merase 1; LMNA, lamin A/C; BaP, benzopyrene. The oxidative stress induced by PAHs can lead to an increase
BioMed Research International 9

PARP-1 LMNA
2.0 2.0

Expression (ΔΔ Cq ) 1.5 1.5

Expression (ΔΔ Cq )
1.0 1.0

0.5 0.5

0.0 0.0
Normal

DMSO

BaP

Fluoranthene

Pyrene

Normal

DMSO

BaP

Fluoranthene

Pyrene
Pentacene

Pentacene
5 PARP-1 20 LMNA

4
Expression (ΔΔ Cq )

Expression (ΔΔ Cq )
15
3
10
2
5
1

0 0
Normal

DMSO

BaP

Fluoranthene

Pyrene

Normal

DMSO

BaP

Fluoranthene

Pyrene
Pentacene

Pentacene
54 hpf 78 hpf
3 Vimentin 5 Vimentin

4
Expression (ΔΔ Cq )
Expression (ΔΔ Cq )

2
3

2
1
1

0 0
Normal

DMSO

200 nM

400 nM

600 nM

800 nM

1000 nM

Normal

DMSO

200 nM

400 nM

600 nM

800 nM

1000 nM

Figure 5: mRNA expression study of candidate biomarker genes. mRNA expression of PARP-1 and LMNA gene was generally increased in
THP-1 and h-TERT cell lines after exposure of PAHs with different pattern. Normal, no treatment group; DMSO, only DMSO (0.1%) treated
group.

in mitochondrial mass and mitochondrial membrane poten- copy number was increased. The increase of mtDNA copy
tial. The mitochondrial genome is highly susceptible to DNA number was thought to compensate for declining respiratory
damage caused by ROS and mutagens and has higher rates of function during the oxidative stress after PAH exposure.
mutation than does the nuclear genome. In addition, DNA Mitochondria-rich cellular proteome was then studied
damage persists longer in the mitochondrial genome. The to determine whether biomarkers associated with exposure
absence of histones that provide packaging and protection of of PAHs could be identified. The result showed that PARP-
nuclear DNA and the error-prone replication and repair of 1 and LMNA protein might be a novel universal biomarker
mitochondrial genes all contribute to the vulnerable nature associated with exposure of PAHs. PARP is a monomeric
of mitochondrial DNA [37]. Therefore, the present study protease widely present in the nuclei of most eukaryotic cells
targeted the mitochondrial genome and proteome to identify that is associated with the occurrence and development of a
biomarkers associated with PAH exposure. The results of the variety of diseases. PARP-1, the best characterized member of
present study showed that, after PAHs exposure, mtDNA the PARP family, which currently comprises 18 members, is
10 BioMed Research International

Fluoranthene
Pentacene

Normal
DMSO
Pyrene
BaP
PARP-1 116 K

LMNA 69/62 K

GAPDH 37 K

Figure 6: Confirmation of PARP-1 and LMNA biomarkers using Western blot. The expression of PARP-1 was remarkably increased after
exposure of BaP, pentacene, and fluoranthene (100 𝜇M concentration). LMNA protein was highly expressed after BaP exposure. Normal, no
treatment group; DMSO, only DMSO (0.1%) treated group; K, kilodalton.

Normal DMSO 200 nM

400 nM 600 nM 800 nM

Figure 7: Morphological abnormalities in the general shape of zebrafish after BaP exposure. Obvious morphological abnormalities including
curved backbone (arrow) were developed after exposure of more than 400 nM concentration of BaP during the embryogenesis (54 hours per
fertilization).

an abundant nuclear enzyme implicated in cellular responses which can lead to increased expression of NF-𝜅B-dependent
to DNA injury provoked by genotoxic stress. PARP is genes such as oncogenes, cell adhesion molecules, matrix
involved in DNA repair and transcriptional regulation and metalloproteinases, and growth factors [39]. Inhibition of
is now recognized as a key regulator of cell survival and cell PARP activity is protective in a wide range of inflammatory
death as well as a master component of a number of and ischemia-reperfusion-associated diseases, including car-
transcription factors involved in tumor development and diovascular diseases, diabetes, rheumatoid arthritis, endo-
inflammation. PARP becomes activated in response to oxida- toxic shock, and stroke [38]. LMNA, nuclear intermediate
tive DNA damage and depletes cellular energy pools, thus filament proteins, is a basic component of the nuclear lamina.
leading to cellular dysfunction in various tissues. The acti- Mutations in LMNA are associated with a broad range of
vation of PARP may also induce various cell death pro- laminopathies, congenital diseases affecting tissue regenera-
cesses and promotes an inflammatory response associated tion, and homeostasis. This study showed global profiling of
with multiple organ failure [38]. It is known to activate toxic changes of PAHs in cell lines, h-TERT, and zebrafish
nuclear factor-𝜅B (NF-𝜅B) through a variety of pathways, model. The change of mitochondrial genome (increased
BioMed Research International 11

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The authors declare that there is no conflict of interests pational and Environmental Health, vol. 74, no. 7, pp. 505–513,
regarding the publication of this paper. 2001.
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