Lixing Huang

Benzo[a]pyrene exposure influences the cardiac development and the expression of cardiovascular relative genes in zebrafish (Danio rerio) embryos

It is reported that the most abundant polycyclic aromatic hydrocarbons (PAHs) in weathered crude oils are cardiotoxic. However, the action mechanism of PAHs on vertebrate cardiovascular development and disease is unclear. In the present study, the cardiac morphology and functioning of zebrafish embryos exposed to benzo[a]pyrene [B(a)P], as a high-ring PAHs, for 72 h were observed and determined. The results showed that B(a)P exposure resulted in cardiac developmental defects in zebrafish embryos. Significant changes in expression level of multiple genes potentially critical for regulating the B(a)P-induced cardiovascular developmental defects were also found. A gene network regulating cardiac development perturbed by B(a)P exposure was identified and established by computational analysis and employment of some databases. The information from the network could provide a clue for further mechanistic studies explaining molecular events regulating B(a)P-mediated cardiovascular defects and consequences.

Low-level pyrene exposure causes cardiac toxicity in zebrafish (Danio rerio) embryos.

It is widely accepted that the most abundant polycyclic aromatic hydrocarbons (PAHs) in weathered crude oils is cardiotoxic. Although PAHs toxic endpoints show strong correlation with the aryl hydrocarbon receptor (AhR), a ligand activated transcription factor, and is thought to be a potent inducer of cytochrome P4501A, the action mechanism of PAHs on vertebrate cardiovascular development and disease is unclear. Herein, we address the cardiac developmental effects of exposure to the weak AhR agonist pyrene on the early life-stages of zebrafish. Embryos were exposed to 0, 0.05, 0.5, 5, and 50 nmol/L pyrene up to 72h post-fertilization (hpf). Pyrene-treated embryos showed dose-dependent heart abnormalities, such as pericardial edema and cardiac looping defects. Changes in AhR1a, AhR1b, AhR2, and Cyp1A expression were assessed by real-time RT-PCR. The results showed that low-level pyrene failed to alter these genes expression. However, the homeodomain transcription factor Nkx2.5, which plays an essential role in the development of the cardiovascular system, was down-regulated in a dose-dependent manner by pyrene exposure. The bone morphogenetic protein 2b (Bmp2b), which has been identified as the upstream gene of Nkx2.5, also was inhibited in a dose-dependent manner after treatment with pyrene. Taken together, these data indicated that embryonic exposure of zebrafish to low-level environmental pyrene disrupt normal cardiac development and alter expression of defective cardiac differentiation related genes.

Phenanthrene causes ocular developmental toxicity in zebrafish embryos and the possible mechanisms involved

Recent studies show that polycyclic aromatic hydrocarbons (PAHs) may be a candidate cause of developmental defects of the retina, but the mechanism is still unclear. We evaluated the mechanism(s) underlying PAH-induced retinal development defects due to exposure to environmental concentrations of Phenanthrene (Phe) in zebrafish. We found that exposure to environmental concentrations of Phe caused obvious morphological changes, developmental retardation, apoptosis, and reduction of cell proliferation in the retina. Our results indicated that Phe could cause visual system developmental defects. Phe exposure up-regulated aryl hydrocarbon receptor (AhR) and microphthalmia-associated transcription factor (Mtif) expression, and down-regulated zinc finger E-box binding homeobox 1 (Zeb1) and paired box 6 (Pax6). Moreover, we demonstrated that AhR was a repressor of Zeb1. We propose that Phes ocular toxicity is mediated by up-regulating AhR, which then down-regulates Zeb1, in turn inducing Mitf expression while inhibiting Pax6 expression.

Phenanthrene exposure produces cardiac defects during embryo development of zebrafish (Danio rerio) through activation of MMP-9

Phenanthrene (Phe) is one of the most abundant polycyclic aromatic hydrocarbons in the aquatic environment as a result of human activities. It is widely accepted that Phe has cardiotoxic effects. Even so, knowledge concerning the mechanism(s) of cardiac development toxicity is still limited. In this study, we exposed zebrafish embryos to environmentally relevant concentrations of Phe and then investigated its cardiotoxic effects and the mechanism(s) involved. Some cardiac morphogenetic defects, which was characterized by an abnormally looped and enlarged heart, dilated and thinner ventricular wall, and increased interstitial fibrosis, were observed in the Phe treated groups. The mRNA and protein expression levels of matrix metalloproteinase-9 (MMP-9), as well as the MMP-9 activity, were induced. Moreover, during co-treatment of the zebrafish embryos with MMP-9 inhibitor, the cardiac defects caused by Phe were attenuated. In addition, Phe exposure led to an up-regulation of transforming growth factor β (TGF-β), which plays a crucial role in mediating cardiac fibrosis. Taken together, our data indicated that the exposure to Phe of zebrafish embryos disrupted normal cardiac development, and that the cardiac defects induced by Phe were mediated by the MMP-9, while TGF-β was also involved in these cardiac defects.

Use of toxicogenomics to predict the potential toxic effect of Benzo(a)pyrene on zebrafish embryos: Ocular developmental toxicity

Benzo(a)pyrene (BaP) is a representative polycyclic aromatic hydrocarbon (PAH), which is ubiquitous in the environment. The toxic effects of BaP on fish embryos have been described in detail, but some potentially toxic effects of BaP might have been neglected owing to the limitations of traditional techniques. In the present research, global transcriptional patterns were used to study the potentially toxic effects of BaP, as well as its underlying toxicological mechanisms. The expression levels of multiple genes were significantly changed by BaP exposure. The results of ontology assignments and cluster analysis showed that BaP could affect the processes of photoreceptor maintenance and phototransduction. We also conducted an experiment on phototactic response and found that larvae exposed to BaP displayed a decreasing response to light. In addition, BaP exposure decreased the cellular density of the ganglion cell layer (GCL) significantly. These results suggested that BaP exposure induced visual system developmental defects and dysfunction by perturbation of photoreceptor development related genes. Our results were helpful for an understanding of the toxicity of BaP. This study also indicated that microarray analysis was effective for predicting the potential toxicity of chemicals with high sensitivity and accuracy.

Phenanthrene exposure induces cardiac hypertrophy via reducing miR-133a expression by DNA methylation.

Growing evidence indicates that there is an emerging link between environmental pollution and cardiac hypertrophy, while the mechanism is unclear. The objective of this study was to examine whether phenanthrene (Phe) could cause cardiac hypertrophy, and elucidate the molecular mechanisms involved. We found that: 1) Phe exposure increased the heart weight and cardiomyocyte size of rats; 2) Phe exposure led to enlarged cell size, and increased protein synthesis in H9C2 cells; 3) Phe exposure induced important markers of cardiac hypertrophy, such as atrial natriuretic peptide, B-type natriuretic peptide, and c-Myc in H9C2 cells and rat hearts; 4) Phe exposure perturbed miR-133a, CdC42 and RhoA, which were key regulators of cardiac hypertrophy, in H9C2 cells and rat hearts; 5) Phe exposure induced DNA methyltransferases (DNMTs) in H9C2 cells and rat hearts; 6) Phe exposure led to methylation of CpG sites within the miR-133a locus and reduced miR-133a expression in H9C2 cells; 7) DNMT inhibition and miR-133a overexpression could both alleviate the enlargement of cell size and perturbation of CdC42 and RhoA caused by Phe exposure. These results indicated that Phe could induce cardiomyocyte hypertrophy in the rat and H9C2 cells. The mechanism might involve reducing miR-133a expression by DNA methylation.

Toxicogenomic analysis in the combined effect of tributyltin and benzo[a]pyrene on the development of zebrafish embryos.

There is a growing recognition that the toxic effects of chemical mixtures are been an important issue in toxicological sciences. Tributyltin (TBT) and benzo[a]pyrene (BaP) are widespread pollutants that occur simultaneously in the aquatic environments. This study was designed to examine comprehensively the combined effects of TBT and BaP on zebrafish (Danio rerio) embryos using toxicogenomic approach combined with biochemical detection and morphological analysis, and tried to gain insight into the mechanisms underlying the combined effects of TBT and BaP. The results of toxicogenomic data indicated that: (1) TBT cotreatment rescued the embryos from decreased hatching ratio caused by BaP alone, while the alteration of gene expression (in this article the phrase gene expression is used as a synonym to gene transcription, although in is acknowledged that gene expression can also be regulated by, e.g., translation and mRNA or protein stability) relative to zebrafish hatching in the BaP groups was resumed by the cotreatment with TBT; (2) BaP cotreatment decreased TBT-mediated dorsal curvature, and alleviated the perturbation of Notch pathway caused by TBT alone; (3) cotreatment with TBT decreased BaP-mediated bradycardia, which might be due to that TBT cotreatment alleviated the perturbation in expression of genes related to cardiac muscle cell development and calcium handling caused by BaP alone; 4) TBT cotreatment brought an antagonistic effect on the BaP-mediated oxidative stress and DNA damage. These results suggested that toxicogenomic approach was available for analyzing combined toxicity with high sensitivity and accuracy, which might improve our understanding and predictability for the combined effects of chemicals.

Exposure to low dose benzo[a]pyrene during early life stages causes symptoms similar to cardiac hypertrophy in adult zebrafish

Growing evidence indicates that polycyclic aromatic hydrocarbons (PAHs) can lead to cardiac hypertrophy and recent research indicates that exposure to low dose crude oil during early embryonic development may lead to impacts on heart health at later life stages. The aim of this study was to evaluate whether exposure during early life stages to low dose benzo[a]pyrene (BaP), as a high-ring PAH, would lead to cardiac hypertrophy at later life stages. Zebrafish were exposed to low dose BaP until 96 hpf, then transferred to clean water and maintained for a year before histological and molecular biological analysis. Our results showed that exposure to low level BaP during early life stages increased heart weight to body weight ratios and deposited collagen in the heart of adult zebrafish. ANP, BNP and c-Myc were also induced in the heart of adult zebrafish by BaP. These results proved that low level BaP exposure during early life stages caused symptoms similar to cardiac hypertrophy in adult zebrafish. Our results displayed an elevated expression of CdC42, RhoA, p-ERK1, 2 and Rac1. Therefore, the mechanism of the cardiac hypertrophy caused by BaP exposure during early life stages may be through inducing the expression of CdC42, RhoA and Rac1, together with activating ERK1, 2.

A Novel Effect of Polychlorinated Biphenyls: Impairment of the Tight Junctions in the Mouse Epididymis

Polychlorinated biphenyls (PCBs) exhibit a broad range of adverse biological effects, including reproductive toxicity. However, the mechanisms by which PCBs disrupt the epididymis remain obscure. We analyzed the gene expression profile in mice epididymis exposed to PCBs (Aroclor 1254) at doses comparable to human exposure using a cDNA microarray. Differentially expressed genes were involved in a variety of function categories and biological pathways, including GTP binding, nucleosome assembly, and ribosome and protein disulfide isomerase. The differentially expressed genes related to GTP binding were highly enriched. The abundance of GTP binding proteins related to tight junctions being reduced and the phosphorylation level of their downstream effectors were impaired after exposure to PCBs. The results of tracer studies demonstrated that the permeability of blood-epididymis barrier was increased by PCB exposure. In addition, PCB exposure also disrupted the expression of the tight junction proteins, zonula occludens-1 and occludin. We demonstrated for the first time that exposure to PCBs at doses relevant for the general population was able to affect the blood-epididymis barrier in mice through altering GTP binding and tight junction proteins. Our results provided a novel insight into the molecular mechanisms linking PCB exposure to sperm maturation.

Transcriptome Analysis of Male and Female Sebastiscus marmoratus

Background The rockfish Sebastiscus marmoratus, which is widely distributed in the East Sea and the South Sea of China, is a sensitive model for the toxic effects and mechanisms of marine contaminants. To gain a global view of the molecular mechanism(s) whereby gene expression may influence sexual dimorphism in S. marmoratus, and to develop a database for further toxicological studies, we performed a large-scale transcriptome study. Methodology/Principal Findings The Illumina DNA sequencing platform was employed to obtain 27,559,578 and 25,821,126 reads from two cDNA libraries generated from adult male and female S. marmoratus, respectively. Transcriptome de novo assembly was carried out with the short reads assembling program–SOAPdenovo. The reads assembled into 78,675 unigenes, of which 38,677 showed homology to existing protein sequences. Clusters of orthologous groups, gene orthology, and the Kyoto Encyclopedia of Genes and Genomes annotations were performed to better understand the functions of these unigenes. There were 1,209 potential sex differentially expressed unigenes, with 1,049 predicted to be differentially expressed in females and 160 in males. Fifteen randomly chosen unigenes were confirmed using real-time PCR as female or male predominantly expressed genes. Conclusions/Significance This is the first report of an annotated transcriptome of S. marmoratus and identification of sex differentially expressed genes. These data will be of interest to researchers using this model. This work also provides an archive for future studies in molecular mechanisms of sexual dimorphism and evolution and can be used in comparative studies of other fish.