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
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental toxicants found in air, water, plants, and soils which are present as volatile, semivolatile, and particulate pollutants [
The most commonly used biomarkers of PAHs exposure are metabolites of PAHs, particularly 1-hydroxypyrene (1-OHP), and PAH-DNA or protein adducts [
Biomarkers to assess exposure to PAHs at high levels are well studied, but more work is needed to validate these biomarkers when exposure occurs at low, environmental levels. Most reported biomarkers for PAHs exposure were mainly targeted against nuclear genome and proteome as well as metabolites in either serum or urine. Moreover, biomarkers as mentioned in several studies [
Enormous strides have recently been made in our understanding of the biology and pathobiology of mitochondria. Many diseases have been identified as caused by mitochondrial dysfunction, and many pharmaceuticals have been identified as previously unrecognized mitochondrial toxicants. A much smaller but growing reports indicate that mitochondria are also targeted by environmental pollutants [
In zebrafish, hundreds of genes involved in the formation of virtually every organ system have been identified by large-scale mutagenesis screening [
Therefore, this study investigated to identify new biomarkers and pathobiological role for PAHs exposure, especially BaP using targeted mitochondrial genomic and proteomic approach in cell line, peripheral blood/mesenchymal stem cell, and
Cell lines (K562, THP-1, MOLT-4, and HL-60 cells) were obtained from the American Type Culture Collection, which were cultured in RPMI 1640 medium (Gibco Laboratories, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) (see Supplementary Table 1 in Supplementary Material available online at
BaP (purity > 99%), fluoranthene (99%), pentacene (>99%), and pyrene (>99%) were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). Stock PAHs solutions were made in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) at concentration of 100
Cytotoxicity assays were carried out using the Enhanced Cell Viability Assay Kit (EZ-CyTox, Daeil Lab Service Co., Seoul, Korea) protocol. The absorbance (A450) of each well was measured using a VERSA Max microplate reader (Molecular Devices, Sunnyvale, CA, USA).
mtDNA copy number was determined according to our published protocol [
This study used a published protocol to amplify and sequence the mtDNA
Briefly, an equal amount of proteins (30
The separated proteins were excised from the gel and the gel pieces containing protein were destained with 50% acetonitrile (ACN) containing 50 mM NH4HCO3 and the gel pieces were vortexed until Coomassie Brilliant Blue was completely removed. These gel pieces were then dehydrated in 100% ACN and vacuum-dried for 20 min with SpeedVac. For the digestion, gel pieces were reduced using 10 mM dithiothreitol in 50 mM NH4HCO3 for 45 min at 56°C, followed by alkylation of cysteines with 55 mM iodoacetamide in 50 mM NH4HCO3 for 30 min in the dark. Finally, each of gel pieces was treated with 12.5 ng/
The tryptic peptides were loaded onto a fused silica microcapillary column (12 cm
The acquired LC-electrospray ionization-MS/MS fragment spectra were searched in the BioWorksBrowser (version Rev. 3.3.1 SP1, Thermo Fisher Scientific Inc.) with the SEQUEST search engines against National Center for Biotechnology Information (
Total RNA was extracted using the QIAamp RNA Blood Mini kit (Qiagen). Reverse transcription produced cDNAs using Superscript III (Applied Biosystems). The expression of poly[ADP-ribose] polymerase 1 (PARP-1) and lamin A/C (LMNA) mRNA was quantified using QuantiTect SYBR green PCR master mix (Qiagen), PARP-1 forward: 5′-GAGGAAGTAAAGGAAGCCAA-3′, PARP-1 reverse: 5′-CACAACTTCAACAGGCTCT-3′, LMNA forward: 5′-AAGCTTCGAGACCTGGAG-3′, LMNA reverse: 5′-TCCAAGAGCTTGCGGTA-3′, and
Extracted protein samples (20
For
PAH-untreated h-TERT cells showed compact cellularity with spindle shape. Cells were tightly attached to each other and to the substrate. Generally, direct exposure of PAHs such as BaP, pentacene, fluoranthene, and pyrene depressed the proliferative capacity of h-TERT cells and the cell morphology was altered in each PAH-exposure group. Cells became detached from the subsurface, and cell-to-cell attachments were lost (Figure
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.
Depending on the type of PAHs, each cell count showed different aspects. The total number of cells in the THP-1 and Molt-4 cell lines decreased 11 days after PAHs exposure. The change in the total number of cells in the THP-1 and Molt-4 cell lines decreased in a time-dependent manner. In comparison to control group, fluoranthene displayed profound significant reduction in cell count (Figures
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)).
Viability significantly decreased after two days of exposure to fluoranthene. On the third day of PAHs exposure, viability reduced remarkably in all the cells (Figures
Mitochondrial contents were increased with different pattern: mtDNA copy number was dramatically elevated after 5-day treatment of fluoranthene and pyrene in both cell line and
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
Changes of the mtDNA sequence were comprehensively studied by direct sequencing of the mtDNA control region and gene scanning for the determination of mtDNA length and heteroplasmic mutations. No alteration of mtDNA sequences was observed after direct exposure of PAHs during 7 days (Supplemental Figure 2). No alteration of mtDNA minisatellites such as 1618 poly C, 303 poly C and 514 (CA) repeat was found after PAHs exposure.
Several hundreds of cellular proteins in mitochondrial-rich cytoplasmic fraction were profoundly deregulated in comparison to control group (Figure
Summary list of identified potential biomarkers for PAHs exposure.
PAHs | Protein | Fold change |
---|---|---|
Benzopyrene | Vimentin | 19.39 |
Annexin A1 | 13.38 | |
Lamin-A/C | 10.04 | |
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 | |
|
||
Pentacene | Lamin-A/C | 6.9 |
DNA topoisomerase 2-alpha | 6.38 | |
Annexin A1 | 6.38 | |
Poly[ADP-ribose] polymerase 1 | 5.85 | |
Squalene synthase | 5.33 | |
Talin-1 | 5.33 | |
PREDICTED: u5 small nuclear ribonucleoprotein 200 kDa helicase-like, partial | 4.81 | |
|
||
Fluoranthene | Poly[ADP-ribose] polymerase 1 | 6.21 |
Elongation factor 1-gamma | 5.21 | |
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 | |
|
||
Pyrene | Talin-1 | 16.82 |
DNA topoisomerase 2-alpha | 8.17 | |
Filamin-C isoform b | 7.16 | |
E3 SUMO-protein ligase RanBP2 | 5.65 | |
CAD protein | 5.14 | |
Poly[ADP-ribose] polymerase 1 | 5.14 |
Results of PARP-1 and LMNA protein by repeat proteomic analysis.
Protein | Fold change | |
---|---|---|
First result | Second result | |
PARP-1 (accession no: 156523968) | ||
DMSO versus BaP | 3.41 | 3.58 |
DMSO versus pentacene | 5.85 | 4.31 |
DMSO versus fluoranthene | 6.21 | 5.34 |
DMSO versus pyrene | 5.14 | 3.41 |
LMNA (accession no: 27436948) | ||
DMSO versus BaP | 10.04 | 4.16 |
DMSO versus pentacene | No change | 0.97 |
DMSO versus fluoranthene | 4.50 | 3.00 |
DMSO versus pyrene | 4.14 | 1.80 |
DMSO, only DMSO-treatment as control; PARP-1, poly[ADP-ribose] polymerase 1; LMNA, lamin A/C; BaP, benzopyrene.
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.
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. This finding was confirmed using embryogenesis in zebrafish model (Figure
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.
The expression of PARP-1 protein was increased after exposure of BaP, pentacene, and fluoranthene. The LMNA proteins were increased after exposure of BaP (Figure
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
At 54 hpf, embryos treated with 400 nM BaP exhibited mild pericardial edema and showed dorsal curvature of the body axis (Figure
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).
PAHs are known genotoxic agents and induce DNA damaging effects, such as DNA adducts, DNA strand breaks, chromosomal aberrations, sister chromatid exchanges, and micronucleus formation [
In order to exert its deleterious effects, BaP must be bioactivated. The formation of BaP
In this study, a broad molecular investigation of the mitochondrial genome and proteome after PAHs exposure showed an increased mtDNA copy number, PARP-1, and LMNA protein, which could be used as biomarkers for exposure of PAHs in cell lines. PAHs directly might cause an increase in the generation of intracellular ROS, subsequently resulting in a change of the mtDNA content, and proteome. The oxidative stress induced by PAHs can lead to an increase in mitochondrial mass and mitochondrial membrane potential. The mitochondrial genome is highly susceptible to DNA damage caused by ROS and mutagens and has higher rates of mutation than does the nuclear genome. In addition, DNA damage persists longer in the mitochondrial genome. The absence of histones that provide packaging and protection of nuclear DNA and the error-prone replication and repair of mitochondrial genes all contribute to the vulnerable nature of mitochondrial DNA [
Mitochondria-rich cellular proteome was then studied to determine whether biomarkers associated with exposure of PAHs could be identified. The result showed that PARP-1 and LMNA protein might be a novel universal biomarker associated with exposure of PAHs. PARP is a monomeric protease widely present in the nuclei of most eukaryotic cells that is associated with the occurrence and development of a variety of diseases. PARP-1, the best characterized member of the PARP family, which currently comprises 18 members, is an abundant nuclear enzyme implicated in cellular responses to DNA injury provoked by genotoxic stress. PARP is involved in DNA repair and transcriptional regulation and is now recognized as a key regulator of cell survival and cell death as well as a master component of a number of transcription factors involved in tumor development and inflammation. PARP becomes activated in response to oxidative DNA damage and depletes cellular energy pools, thus leading to cellular dysfunction in various tissues. The activation of PARP may also induce various cell death processes and promotes an inflammatory response associated with multiple organ failure [
Direct exposure to PAHs induced alteration of the mitochondrial genome including increased mtDNA copy number. The proteomic analysis of the mitochondria-rich cellular fraction showed that PARP-1 and LMNA were a novel universal biomarker associated with exposure of PAHs. Thus mtDNA copy number, PARP-1, and LMNA protein might be useful biomarkers associated with PAHs toxicity and hematotoxicity.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors are thankful to the National Research Foundation of Korea (NRF) and grants funded by the Korea government (MEST) (no. 2011-0015304); the Leading Foreign Research Institute Recruitment Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) (no. 2011-0030034); and a grant from the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (no. 2013-1320070).