Toxicity of nickel ions and comprehensive analysis of nickel ion-associated gene expression profiles in THP-1 cells

The aim of the present study was to explore the toxic effects and underlying mechanisms of nickel ions during therapeutic nickel-based alloy-treatment in congenital heart disease by investigating the metal-induced cytotoxicity to the human monocyte-derived macrophage cell line THP-1. THP-1 cells were treated with NiCl2·6H2O (25, 50, 100, 200, 400 and 800 µM) for 24, 48 and 72 h, respectively. MTT was applied to detect THP-1 cell proliferation following NiCl2 treatment. Apoptosis of THP-1 cells was quantified using flow cytometry. Illumina sequencing was used for screening the associated genes, whose mRNA expression levels were further confirmed by quantitative real-time polymerase chain reaction. High concentrations of nickel ions had a significant suppressive effect on cell proliferation at the three concentrations investigated (200, 400 and 800 µM). Treatment with nickel ions (25–400 µM) for 48 h reduced cell viability in a dose-dependent manner. The mRNA expression levels of RELB, FIGF, SPI-1, CXCL16 and CRLF2 were significantly increased following nickel treatment. The results of the present study suggested that nickel ions exert toxic effects on THP-1 cell growth, which may indicate toxicity of the nickel ion during treatment of congenital heart disease. The identification of genes modified by the toxic effects of nickel on THP-1 cells (EPOR, RELB, FIGF, SPI-1, TGF-β1, CXCL16 and CRLF2) may aid in the development of interventional measures for the treatment/prevention of nickel ion-associated toxic effects during the treatment of congenital heart disease.


Introduction
Congenital heart disease is the most common type of congenital malformation (1). Only certain types of congenital heart disease are able to recover naturally, and others present with increased severity and frequency of complications with increasing age (2). Interventional therapy is one of the therapeutic methods used for congenital heart disease (2). Blocking cardiac defects with metal occluders of different shapes is a commonly used surgical alternative, with the nickel-chromium (Ni-Cr) alloy occluder being most widely used in the clinical setting (3).
The clinical application of the Ni-Cr alloy occluder is a result of advances in medical technology and material sciences, and that of a process of continuous research and improvement. Use of the Ni-Cr alloy occluder avoids the risks and trauma of open-heart surgery, allowing short hospital stays and fast recovery for patients (4). However, long-term nickel-based alloy implantation has been increasingly documented to be associated with biological effects, including cytotoxicity and genotoxicity, eliciting tissue inflammation and potential sensitization, thus raising questions over its bio-safety (5). In previous experiments following-up children receiving an atrial septal defect occluder implantation, it was observed that the nickel concentration in the blood was significantly increased 24 h and one month subsequent to surgery (Zhang et al; unpublished results). Other studies have also reported that the Ni-Cr alloy occluder implantation results in the release of the nickel ion into the chambers of the heart, which may result in cell damage due to local and systemic toxicity (6) and lead to headaches, dyspnea and other unpleasant symptoms (7,8).
Being the 'first line of defense', macrophages are essential determinants of implant biocompatibility (9). Pro-inflammatory studies have observed that during the first six weeks following implantation, macrophages interact

Toxicity of nickel ions and comprehensive analysis of nickel ion-associated gene expression profiles in THP-1 cells
heavily with the prosthetic device (10). Therefore, the present study aimed to investigate the cytotoxicity of nickel ions on THP-1, a human monocytic cell line derived from the peripheral blood, and to explore the underlying mechanism of the toxicity of the nickel alloy in the heart during its application for congenital heart disease. The present study additionally aimed to provide further insights for physicians and patients on the clinical use and adverse effects of nickel alloy implantation in congenital heart disease and various additional associated diseases. Cytotoxicity analysis. Cells were plated on six-well plates and were allowed to adhere. Following different treatments for 48 h, cells from each group (5x10 5 cells/group re-suspended in 0.5 ml ice-cold RPMI 1640) were transferred into a clean centrifuge tube and incubated with 1.25 µl Hoechst 33342 for 15 min at room temperature in the dark to stain the nuclei of all cells. Subsequently, the plates were centrifuged at 2,000 x g for 5 min and the supernatants were discarded. Cells were then re-suspended in 0.5 ml ice-cold RPMI 1640 prior to the addition of 10 µl PI (5 µg/ml) for staining of the dead cells. The samples were kept on ice in the dark and immediately analyzed using a FACSCalibur Cell flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Cytotoxicity was expressed as the percentage of dead cells (PI-positive) relative to the total number of cells.

Materials
RNA-sequencing assay. THP-1 cells were cultured with different concentrations of NiCl 2 ·6H 2 O (25, 50 or 100 µM) for 1 h following starvation for 48 h. Total RNA was prepared by the acid phenol method using TRIzol reagent in accordance with the manufacturer's instructions. Areas containing THP-1 RNAs were sent to Beijing Genomics Institution (Shenzhen, China) for Illumina sequencing.

Pathway analysis of differentially expressed genes.
Pathways were constructed using the Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/) database using the Web-based GEne SeT Analysis Toolkit (http://bioinfo.vanderbilt.edu/webgestalt/) to identify differential networks and core regulators involved in the nickel-induced cytotoxic response. The analysis completed was limited to categories with P<0.05.
Quantitative polymerase chain reaction (qPCR). PCR was performed using the total RNA (1 µg) prepared for the RNA-sequencing assay with the primers listed in Table I
Confirmation of cytotoxicity-associated genes. The authenticity and reliability of the cytotoxicity-associated genes was then verified by specifically examining the expression levels of ten congenital heart disease-associated genes, including EPOR, As presented in Fig. 3, RELB, FIGF, SPI-1, TGF-β1, CXCL16 and CRLF2 through qPCR analysis. RELB, FIGF, SPI-1, CXCL16 and CRLF2 mRNA expression levels of nickel-treated cells presented were significantly increased compared with those in the control group (P<0.01). TGF-β1 and EPOR mRNA expression levels were also significantly increased following nickel challenge (P<0.05).

Discussion
The present study investigated the cytotoxic effects of nickel ions on THP-1 cells and the possible mechanisms involved. The results suggested that nickel ions exerted a growth-inhibitory effect on THP-1 cells and in particular, it was demonstrated that the toxicity of nickel ions to THP-1 cells may be associated with the differential expression of certain genes, including EPOR, RELB, FIGF, SPI-1, TGF-β1, CXCL16 and CRLF2.
In the present study, it was observed that the proliferation of THP-1 cells gradually increased following stimulation for a short period of time with low concentrations (25-100 µM) of nickel ions, which may be associated with the incompletely compromised ability of the cells to resist and metabolize the insult. However, with the increases in the nickel ion concentration and incubation time, a rapid decline in the number of live cells was observed. The cells began to die when incubated with high concentrations of nickel ions (200-800 µM). Cell viability analysis suggested that the concentration and incubation time with the nickel ions may be two important indicators of the toxic effect of nickel. Thus, correct assessment of nickel ion toxicity requires to comprehensively take the concentration and time period of its use into account.
Multiple studies have examined the association between nickel exposure and blood and heart diseases (11,12). The toxicity of the nickel ion to the heart has become a focus of research in recent years since nickel-associated products are being increasingly used in heart disease therapy (13). Accumulating evidence has demonstrated that nickel exposure negatively regulates cardiac function (14,15). The results from a previous study indicated that heart rate variability (a predictor for arrhythmias, mortality risk and severity of illness) was regulated by nickel-induced oxidative-inflammatory responses (14). Thus, it is suggested that nickel may cause certain cardio-regulatory responses and may induce varying responses during systolic and diastolic phases (15).
Although the phenomenon of nickel-induced cardiotoxicity has raised increasing concerns, only few studies are available on the mechanism of nickel ion-induced toxicity to the heart during its use in congenital heart disease (16). Previous studies have demonstrated that congenital heart disease is regulated and affected by multiple genes and pathways, including EPOR, SPI-1, p38/MAPK and TGF-β1 (17)(18)(19)(20). It remains to be fully elucidated whether there is an association between nickel toxicity and congenital heart disease, and if so, what the associated genes and pathways are. In the present study, the expression levels of EPOR, RELB, FIGF, SPI-1, TGF-β1, CXCL16 and CRLF2 were observed to be significantly  PRKACB, CAMK2D, PDC, CNGA4, CNGB1, OR2V1, CYorf17,  OR11L1, OR10A2, OR52L1, OR52K1, OR6V1, OR2T33,  DAPL1, OR6K3, OR13G1   Taste transduction  1.22x10 -3  PRKACB, TAS2R10, TAS2R50, TAS2R4, TAS2R46, GRM4,  TAS2R43, TAS2R13, TAS2R60, TAS2R3, GNG13, TAS2R39   RNA transport  1.61x10 -3  EIF4G2, XPO1, TPR, TMEM48, PNN, NUP205, DDX39B,  PRMT5, NUP155, NUP160, NCBP1, POP1, NUP50, GEMIN5,  NUP107, NUP88, NUP54, NXT2, NXF3, NUPL1, THOC1  increased subsequent to nickel ion stimulation in THP-1 cells. Nickel can also generate cytotoxic reactive oxygen species through TGF-β1 activation (20). CRLF2 gene alterations have been observed to correlate with poor prognosis in Japanese BCR-ABL1-negative high-risk B-cell precursor acute lymphoblastic leukemia (21), which suggests that the downregulation of CRLF2 may block the inflammatory effect of nickel ions. FIGF was detected in adult lung and heart tissues and was observed to exhibit mitogenic activity on fibroblasts (22,23). CXCL16 appears to have a role in the homing of CD4(+) T cells in acute and chronic rejection models of heart allotransplantation (24). Nickel ion stimulation exerted differential effects on these heart disease-associated genes, which may indicate that the toxicity mechanism of the nickel ion may be regulated by these genes. Further studies, including loss-of-function analysis by RNA inference and in vivo experiments, are required to fully elucidate the mechanistic significance of these genes and pathways in the observed nickel cytotoxicity.
In conclusion, the present study demonstrated that the toxicity of the nickel ion to THP-1 cells may be controlled by or is associated with certain genes, including EPOR, RELB, FIGF, SPI-1, TGF-β1, CXCL16 and CRLF2. These observations provided insight and advance the understanding of the genetic basis of nickel ion-induced toxicity in its therapeutic use for congenital heart disease.