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

Abstract

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.

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.

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)).

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.

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.

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.

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.

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).