| BackgroundFormaldehyde (FA) is a chemical with high toxic substance, and is at second place in Priority Toxic Chemicals Control List. FA can cause a variety of toxic action after exposed through the respiratory tract or skin. FA has become one of the most important and serious indoor air pollutants for its characteristics of extensive sources, serious toxicity, high level pollution and long-term duration. It is also the important and difficult issue in environmental health and occupational health research. FA is a genotoxic chemical, which can cause DNA damage (DNA strand breaks, DNA-DNAcrosslinks, DNA-protein crosslinks, DNA adducts, RNA-formaldehyde adducts), gene mutations, chromosome breakage, sister chromatid exchange, cell transformation and inhibition of DNA repair by disrupting the genome. If DNA damage is not repaired or repaired incorrectly, it will lead to mutations or chromosomal aberrations, cell necrosis/apoptosis, or the development of cancer cells.The International Agency for Research on Cancer (IARC) reclassified FA as a known human carcinogen (Group1) in June2004. Numerous epidemiological studies have shown that many common human cancers related to long-term chronic inflammation. Chronic inflammation can lead to excessive cell proliferation and activation of the cascade, causing irreversible damage to DNA. Persistent inflammatory stimulation promote cell to the development of metastatic disease, ultimately leading to tumor growth and immune suppression. It still remain one of the hot issue that the signaling networks of DNA damage repair and inflammatory stress responses affect on the signaling pathway in tumorigenesis and the role of its mediated inflammation in tumor development. Poly(ADP-ribose)polymerase-1(PARP-1) is a sensor and monitor of DNA damage, playing a crucial role in DNA repair. In addition to DNA damage recognition, PARP-1may also be involved in inflammatory by regulating inflammatory transcription factors, cytokines, inflammatory mediators and adhesion factors. Meanwhile, the process of PARP-1is also playing an important role in regulating methylation status of genome. Therefore, this study will evaluate the role of PARP-1signal pathway in the repair of DNA damage-caused by FA from its roles in DNA repair, immune regulation and methylation. This study contributes to a deep understanding of formaldehyde-induced early health effects and having significance to preventing the harmful health effects of formaldehyde.Objective:This study used a cross-sectional design of molecular epidemiology. It mainly discusses the relationship between different levels of formaldehyde exposure and genetic damage in peripheral blood lymphocytes and immune injury in118formaldehyde-exposed workers and79non-exposed workers. Further, the study is to investigate the role of PARP-1signaling pathways in low concentrations of formaldehyde-induced DNA damage and repair in16HBE cells.Methods:We monitored ambient FA concentrations in different workplaces of a plywood factory in Shandong province, China, and determinded DNA strand breaks by comet assay (presented as Percent of DNA in the Comet Tail, TDNA%), chromosome breakage or loss by cytokinesis-block micronucleus (CBMN) assay (presented as Percent of Cytokinesis-block Micronucleus, CBMN frequencies), and blood lymphocyte subpopulations by flow cytometry, and Thl (Interferon y, IFN-y), Th2(Interleukin-4/10, IL-4and IL-10) and proinflammatory cytokines (Interleukin-6/8, IL-6, IL-8and Tumor Necrosis Factor, TNF-a) by cytometric bead array. The urine formic acid was determinded by high performance liquid chromatography, analyzing the relationship between the level of genetic damage and immune injury between formaldehyde exposure levels in peripheral blood lymphocytes with workers. The mRNA expression of PARP-1and methylation-related enzymes was detected by QT-PCR (Quantitative Real-time PCR), and the methylation status of PARP-1gene promoter detected by MassArray technology. The study was to explore the role of PARP-1and methylation-related enzymes in FA-induced epigenetic changes.We selected16HBE cell as object of study. We also determinded DNA strand breaks by comet assay (presented as TDNA%), chromosome breakage or loss by cytokinesis-block micronucleus (CBMN) assay (presented as CBMN frequencies), and reactive oxygen species by flow cytometry, and Thl (IFN-y), Th2(IL-4and IL-10) and proinflammatory cytokines (IL-、IL-8and TNF-a) by cytometric bead array. Expression of the protein in PARP-1signal pathway was detected by Western Blot, QT-PCR, and luciferase activity detection technology. We used co-immunoprecipitation method to detect interactions between proteins, and using specific chemical inhibitors and molecular inhibitor (dominant negative mutant and RNA interference technology) methods to explore the role of PARP-1pathway in low concentrations of formaldehyde-induced DNA repair. In further, we detected the expression of methylation-related enzymes, the interactions between proteins, and the methylation status of PARP-1gene promoter, to explore the mechanism of PARP-1involved in low concentrations of formaldehyde-induced epigenetic changes.Results:1. Study on genetic damage biomarker in formaldehyde exposed workers118FA-exposed workers were divided into high and low exposed group according to their FA exposure levels. The two exposed subgroups had a significant increase of formic acid compared to the control group (p=0.004and p<0.001). Compared with control, mRNA expression of PARP-1(p=0.003) was significant decrease in FA-exposed worker, however, the mRNA expression of X-ray repair cross-complementing protein-1(XRCC-1) was showed no difference.The exposed workers and controls were also well matched by age, sex, BMI, smoking status, and alcohol use. The TDNA%of peripheral blood lymphocyte in high exposed group was significant higher than control. The TDNA%of control, low exposed group and high exposed group after the work exposure was8.39±0.18%,9.63±0.23%,11.55±0.22%respectively. After pretreatment with formamidopyrimidine-DNA-glycosylase (FPG), compared with control, the TDNA%in low and high exposed group increased21.20and20.66percent (p<0.05). CBMN frequencies of control, low exposed group and high exposed group after the work exposure was2.26±0.16%o,3.04±0.23%o,4.48±0.20%o respectively. Compared with control, CBMN frequencies increased significantly in high exposed group. The increasing tendency was found for the percentage of B cells (CD19+) with the increasing level of FA exposure. Both the low and high exposure groups had a significant increase of the percentage of B cells compared to the control group (p<0.01), and the highest percentage of B cells was observed in the high exposure group. The percentage of NK cells (CD56+) was significantly higher in the low exposure group than that in the control (p=0.013). However, no significant increase of the percentage of NK cells was observed in the high exposure group compared to the control group. The percentages of total T cells (CD3+), T-cytotoxic-suppressor cells (CD8+), and T-helper-inducer cell (CD4+) were not significantly different between the control and FA exposure groups.The FA-exposed workers showed significantly higher levels of IL-10in the low exposure group and the high exposure group than that of the control (low exposure vs. control,p=0.003; and high exposure vs. control, p<0.001). The level of IL-10was also significantly higher in the high exposure group that that of the low exposure group (p=0.04). Subjects with high exposure, while not low exposure, had a higher level of IL-4than the control group (p=0.044). However, with respect to IFN-y we observed a reverse trend with significantly lower levels in the high exposure group compared to that in the control (p=0.037). The levels of IL-8were significantly lower in the low exposure group and the high exposure group than that in the control group (low exposure vs. control,p<0.001; and high exposure vs. control,p=0.007).The ratio of IFN-y/IL-4can reflect the balance state of Thl/Th2cell. The ratio of IFN-y/IL-4decreased significantly in both exposed group. The IL-6and TNF-a levels showed no significant difference among the three groups (data not shown). Correlation analysis was performed to determine the relationship between the serum levels of cytokines and formic acid (Fig.4). The levels of serum IL-10(rs=0.465, p<0.001), IL-4(rs=0.203,p=0.045), and IL-8(rs=-0.272,p=0.007) were correlated with formic acid. No significant correlations were observed between formic acid and IFN-γ.Compared with control, the mRNA expression of DNA methyltransferases-1/3a (DNMT-1and DNMT-3a) in exposed group decreased significantly, however, that of DNA methyltransferases-3b (DNMT-3b) showed no difference. The results of the methylation status of promoter regions of PARP-1showed that the methylation levels of the CpG sites selected had no significant differences among the three groups.2. The mechanism of the role of PARP-1signal pathway in FA-induced genetic effects in16HBE cell16HBE cells were treated with formaldehyde for4h, cell survival among the differ concentrations (0,10,20,40,80,160μM) were not changed significantly. While treatment with formaldehyde for24h, cell survival was reduced, with the concentration increase. The concentration greater than80μM can induce significantly decrease of cell survival in16HBE cells, and compared to the untreated group, a statistically significant difference was found. In contrast to the control group, a significant increase was found in TDNA%after FA treated for4h, in5、10and15μM FA exposure groups respectively (p<0.05), and at a peak level in10μM group. CBMN frequencies were increased in10μM FA-treated cells compared with control, but have no significant difference. Hence, we select10μM as final treatment concentration in future study.The results about protein expression of PARP-1signal pathway showed that10μM can induce overexpression of PARP-1, XRCC-1, phosphated extracellular signal-regulated protein kinase (p-ERK) and phosphated c-Jun NH2-terminal amino kinase (p-JNK), however, the expression of p-p38and P65has no changed at any detected time (1,3,6,12,24h). After treated with FA for0,12and24h respectively, the AP-1activity increased at12and24h, but no significant difference was observed. The Western Blot analysis showed that a slight increase of ATM expression was observed after addition of10μM FA for1h and3h. Moreover, FA was found to significantly increase the expression of p-p53especially. Little γ-H2AX expression was measured at any time point tested after FA exposure. And there were no difference among the groups.We explored the role of PARP-1in FA-induced DNA repair by specific chemical inhibitors and interference technology. The TDNA%increased after inhibition of PARP-1by using specific chemical inhibitors of PARP-1, PJ34. Moreover, after4h treatment with10μM FA, cells were then left to repair for2h in fresh MEM, the significant difference was also observed in the comet parameter compared with control. The DNA repair capacity is73.47%. Moreover, we investigated whether ATM was involved in FA-induced DNA repair. We used RNA interference technology to inhibit ATM. Our result showed that consistent with knocking down of ATM, p-p53expression was showed decrease. The results showed that after treatment with10μM FA for4h, there was no significant difference in the comet parameter in HBE-siATM cells compared with control. Moreover, there is no difference of DNA repair capacity between HBE-siATM cells and HBE-siGFP cells. There was increase in S phase cells upon FA treatment for24h. Introduction of the RNA Interference of ATM into HBE did not change the increase of S phase cells. While, G2/M phase cells were significantly increased after inhibition of ATM.After inhibition of PARP-1by interference technology, p-p53and p-JNK expression was showed decrease, however, no difference changes of p-ERK expression was observed. Both inhibition of JNK and ERK can reduce FA-induced PARP-1and XRCC-1up-expression. Moreover, after inhibition of JNK, p-p53expression decreased, the result was not observed after inhibition of ERK. Using co-immunoprecipitation method to detect interactions between PARP-1and XRCC-1, the results showed that FA increased the interaction between PARP-1and XRCC-1. Inhibition of JNK can impair the interaction.Furthermore, we explore the role of PARP-1signal pathway in FA-induced immune reaction.16HBE cells were treated with10μM FA for different time, NF-κB expression was not altered at each detected time. The level of IL-4, IL-6, IL-8, IL-10, TNF-α and IFN-γ were detected after treatment with FA for24h in16HBE cells. The result showed that the level of IL-4, IL-10, TNF-α and IFN-y was very low in cultured supernatant of cells. Compared with control, the level of IL-6and IL-8increased significantly. Moreover, inhibition of PARP-1impaired FA-induced the up-expression of IL-6and IL-8.16HBE cells were treated with10μM FA for2h, level of ROS was increased significantly. Inhibition of PARP-1could reduce the level of ROS, but no significant differences observed. The result implied that ROS induced by10μM was through various pathways. In order to detect the role of methylation-related enzymes and PARP-1pathway in FA-induced methylation changes, we detect the expression of methylation-related enzymes at first.10μM FA can lead to increase of DNMT-1after cells treated. Furthermore, we detected the expression of, the interactions between proteins, and the methylation status of PARP-1gene promoter, to explore the mechanism of PARP-1involved in low concentrations of formaldehyde-induced epigenetic changes. Using co-immunoprecipitation method to detect interactions between PARP-1and DNMT-1, the results showed that FA increased the interaction between PARP-1and DNMT-1. The results of the methylation status of promoter regions of PARP-1showed that the methylation levels of the CpG sites selected in the study had no significant differences between the two groups.Conclusion and Discussion:Formaldehyde exposure can cause genetic damage detected in peripheral blood lymphocytes. PARP-1is crucial for DNA injury in early stage. Because long-term repeated exposure to formaldehyde, the DNA damage is severe. Overexpression of PARP-1will reverse the regulation of PARP-1, which woud lead to further reducing the DNA repair efficiency. Such a vicious spiral cause cumulative genetic damage in peripheral blood lymphocytes, which reflecting by the increase of both TDNA%and CBMN. In addition, when DNA injury was exceed the capacity to repair, the body would shut down the DNA repair gene such as PARP-1automatically, causing PARP-1gene silenced, losing the expression function, to increase the tolerance ability of body for fault, to increase the genetic damage. The percentage of peripheral blood lymphocyte subsets can be changed after long term exposure to formaldehyde. And Th2cytokines were predominant. It is well known that the preponderance state of Th2is related to asthma and escape of tumor. So the monitoring of cytokines in peripheral blood would contribute to understand the cellular immunity and humoral immune status of workers exposed to formaldehyde, and providing theoretical basis for health protection for occupation exposure.In vitro experiments, human bronchial epithelial cells were used to verify the role of PARP-1pathway in the repair of DNA damage induced by formaldehyde. The results suggested that PARP-1pathway is not only an important protein involved in early DNA repair, it was also participated in the regulation of inflammatory reaction. In summury, the results from the vivo and vitro study implied that both short-term and long-term exposure to formaldehyde can induce different types of genetic damage and changes of immune index. The different formaldehyde exposure dose, time, test object and test conditions will cause the different results. |
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