Linc01588 deletion inhibits the malignant biological characteristics of hydroquinone-induced leukemic cells via miR-9-5p/SIRT1.

Leukemia caused by environmental chemical pollutants has attracted great attention, the malignant leukemic transformation model of TK6 cells induced by hydroquinone (HQ) has been previously found in our team. However, the type of leukemia corresponding to this malignant transformed cell line model needs further study and interpretation. Furthermore, the molecular mechanism of malignant proliferation of leukemic cells induced by HQ remains unclear. This study is the first to reveal the expression of aberrant genes in leukemic cells of HQ-induced malignant transformation, which may correspond to chronic lymphocytic leukemia (CLL). The expression of Linc01588, a long non-coding RNA (lncRNA), was significantly up-regulated in CLL patients and leukemic cell line model which previously described. After gain-of-function assays and loss-of-function assays, feeble cell viability, severe apoptotic phenotype and the increased secretion of TNF-α were easily observed in malignant leukemic TK6 cells with Linc01588 deletion after HQ intervention. The tumors derived from malignant TK6 cells with Linc01588 deletion inoculated subcutaneously in nude mice were smaller than controls. In CLL and its cell line model, the expression of Linc01588 and miR-9-5p, miR-9-5p and SIRT1 were negative correlation respectively in CLL and cell line model, while the expression of Linc01588 and SIRT1 were positive correlation. The dual-luciferase reporter assay showed that Linc01588 & miR-9-5p, miR-9-5p & SIRT1 could bind directly, respectively. Furthermore, knockdown of miR-9-5p successfully rescued the severe apoptotic phenotype and the increased secretion of TNF-α caused by the Linc01588 deletion, the deletion of Linc01588 in human CLL cell line MEC-2 could also inhibit malignant biological characteristics, and the phenotype caused by the deletion of Linc01588 could also be rescued after overexpression of SIRT1. Moreover, the regulation of SIRT1 expression in HQ19 cells by Linc01588 and miR-9-5 P may be related to the Akt/NF-κB pathway. In brief, Linc01588 deletion inhibits the malignant biological characteristics of HQ-induced leukemic cells via miR-9-5p/SIRT1, and it is a novel and hopeful clue for the clinical targeted therapy of CLL.

DNMT1 regulates hypermethylation and silences hsa_circ_401351 in hydroquinone-induced malignant TK6 cells.

BACKGROUND: Benzene and its metabolite hydroquinone (HQ) are widely used in daily life, and long-term exposure to benzene or HQ can induce acute myeloid leukemia (AML). Circular RNAs (circRNAs) are mostly produced by reverse splicing of gene exon mRNA precursors. The modulation of circRNA expression is connected to leukemia progression; however, the molecular mechanism is still unknown. MATERIALS AND METHODS: In this study, the cells were divided into four groups: PBS control group (PBS-TK6), TK6 malignantly transformed cells induced by 10.0 µmol/L HQ (HQ-TK6), and HQ-TK6 cells treated with 5 µmol/L 5-AzaC (DNA methyltransferase inhibitor) for 24 h (HQ + 5-AzaC). HQ-TK6 cells were treated with 200 nmol/L TSA (histone deacetylation inhibitor) for 24 h (HQ + TSA). qRT-PCR was used to identify the differential hsa_circ_401351 expression between the four groups. We further determined the hsa_circ_401351 promoter methylation level with methylation-specific PCR. DNMT1 and DNMT3b were knocked down by CRISPR/Cas9 to elucidate the specific molecular mechanism of hsa_circ_401351 in HQ-TK6 cells. CCK-8 and flow cytometry detected cell proliferation and apoptosis, respectively, after hsa_circ_401351 was overexpressed in HQ-TK6 cells. RESULTS: Compared with the PBS-TK6 group, the expression of hsa_circ_401351 was found to be lower in the HQ-TK6 group. Nevertheless, treatment with 5-AzaC or TSA increased hsa_circ_401351 expression, with the upregulation being more pronounced in the TSA group. The expression of hsa_circ_401351 in the DNMT1 knockdown group was dramatically increased by 50% compared to that in the control group, and the DNA methylation level of the hsa_circ_401351 promoter region was decreased. When hsa_circ_401351 was overexpressed, HQ-TK6 cell proliferation was significantly slowed after 48 h compared with the control group. Flow cytometry showed that cells were mainly arrested in G1 phase, and apoptosis was significantly enhanced. Similarly, qRT-PCR and Western blot data showed significant reductions in Caspase-3 mRNA and protein production, and Bcl-2 mRNA levels were also elevated. CONCLUSIONS: Overall, our research showed that elevated DNMT1 expression in HQ-TK6 cells increased methylation levels and decreased expression of the hsa_circ_401351 promoter region, limiting its ability to suppress HQ-TK6 cell growth and enhance apoptosis.

The role of GATA switch in benzene metabolite hydroquinone inhibiting erythroid differentiation in K562 cells.

The phenolic metabolite of benzene, hydroquinone (HQ), has potential risks for hematological disorders and hematotoxicity in humans. Previous studies have revealed that reactive oxygen species, DNA methylation, and histone acetylation participate in benzene metabolites inhibiting erythroid differentiation in hemin-induced K562 cells. GATA1 and GATA2 are crucial erythroid-specific transcription factors that exhibit dynamic expression patterns during erythroid differentiation. We investigated the role of GATA factors in HQ-inhibited erythroid differentiation in K562 cells. When K562 cells were induced with 40 µM hemin for 0-120 h, the mRNA and protein levels of GATA1 and GATA2 changed dynamically. After exposure to 40 µM HQ for 72 h, K562 cells were induced with 40 µM hemin for 48 h. HQ considerably reduced the percentage of hemin-induced Hb-positive cells, decreased the GATA1 mRNA, protein, and occupancy levels at α-globin and ß-globin gene clusters, and increased the GATA2 mRNA and protein levels significantly. ChIP-seq analysis revealed that HQ reduced GATA1 occupancy, and increased GATA2 occupancy at most gene loci in hemin-induced K562 cells. And GATA1 and GATA2 might play essential roles in the erythroid differentiation protein interaction network. These results elucidate that HQ decreases GATA1 occupancy and increases GATA2 occupancy at the erythroid gene loci, thereby downregulating GATA1 and upregulating GATA2 expression, which in turn modulates the expression of erythroid genes and inhibits erythroid differentiation. This partially explains the mechanism of benzene hematotoxicity.

Nrf2 affects hydroquinone-induces cell cycle arrest through the p16/pRb signaling pathway and antioxidant enzymes.

Hydroquinone (HQ), a well-known carcinogenic agent, induces oxidative stress, cell cycle arrest, apoptosis, and malignant transformation. As an antioxidant actor, the nuclear factor erythroid 2-related factor 2 (Nrf2) drives adaptive cellular protection in response to oxidative stress. The human lymphoblastoid cell line (TK6 cells) is widely used as a model for leukemia researches. In the present study, we focused on exploring whether Nrf2 regulatory cell cycle in TK6 cells upon HQ treatment and the underlying mechanisms. The results showed that the cell cycle arrest in TK6 cells induced by hydroquinone was accompanied by activation of the Nrf2 signaling pathway. We further clarified that Nrf2 loss accelerated cell cycle progression from G0/G1 to S and G2/M phases and promoted ROS production by downregulating the expression of SOD and GSH. Western blotting analysis indicated that Nrf2 regulated cell cycle progression via p16/pRb signaling pathways. Therefore, we conclude that Nrf2 is engaged in HQ-induced cell cycle arrest as well through p16/pRb and antioxidant enzymes.

Long non-coding RNA LINC01480 is activated by Foxo3a and promotes hydroquinone-induced TK6 cell apoptosis by inhibiting the PI3K/AKT pathway.

Long non-coding RNAs (lncRNAs) have been shown to play a critical role in the damage caused to the body by environmental exogenous chemicals; however, few studies have explored their effects during exposure to benzene and its metabolite, hydroquinone (HQ). An emerging lncRNA, LINC01480, was found to be associated with the immune microenvironment of some cancers, but its specific function remains unknown. Therefore, this study aimed to investigate the role of LINC01480 in HQ-induced apoptosis. The biological function of LINC01480 was investigated through gain-of-function and loss-of-function experiments. Mechanically, nuclear-cytoplasmic fractionation experiment, chromatin immunoprecipitation (ChIP), dual-luciferase reporter assay, and rescue experiments were performed. In this study, when TK6 cells were treated with HQ (0, 5, 10, and 20 µM) for 12, 24, 48, and 72 h, the expression of LINC01480 was increased in a dose-dependent manner. Meanwhile, the phosphorylation levels of PI3K and AKT decreased, and apoptosis increased. As compared to the control group, HQ-induced apoptosis was significantly reduced, and the relative survival rate of TK6 cells increased after silencing LINC01480, while overexpression of LINC01480 further sensitized TK6 cells to HQ-induced apoptotic cell death. LINC01480 negatively regulated the PI3K/AKT pathway in TK6 cells, and the apoptosis-inhibiting effect of LINC01480 silencing was reversed after inhibition of the PI3K/AKT pathway. In addition, ChIP and the dual-luciferase reporter assays showed that the transcription factor Foxo3a promoted LINC01480 transcription by directly binding to the promoter regions - 149 to - 138 of LINC01480. Moreover, short-term HQ exposure promoted the expression of Foxo3a. From these findings, we can conclude that LINC01480 is activated by Foxo3a, and promotes HQ-induced apoptosis by inhibiting the PI3K/AKT pathway, suggesting that LINC01480 might become a possible target for therapeutic intervention of HQ-induced toxicity.

Aquatic toxicity of hydroquinone and catechol following metal oxide treatment to Ceriodaphnia dubia and Pimephales promelas.

Metal oxides comprise a large group of chemicals used in water treatment to adsorb organic pollutants. The ability of titanium dioxide (TiO2) and iron (III) oxide (Fe2O3) to reduce the chronic toxicity of (phenolic) C6H6(OH)2 isomers, namely hydroquinone (HQ) and catechol (CAT) to Ceriodaphnia dubia and Pimephales promelas (less than 24 h-old) were investigated. The toxic endpoints following metal oxide treatment were compared to endpoints of untreated CAT and HQ. In chronic toxicity testing, HQ resulted in greater toxicity than CAT for both test organisms; the median lethal concentrations (LC50) for CAT were 3.66 to 12.36 mg.L-1 for C. dubia and P. promelas, respectively, while LC50 for HQ were 0.07 to 0.05 mg.L-1, respectively. Although both treated solutions presented lower toxic endpoints than those in the untreated solutions, Fe2O3 had a better potential to reduce the toxic effects of CAT and HQ than TiO2.

JNK1 activated pRb/E2F1 and inhibited p53/p21 signaling pathway is involved in hydroquinone-induced pathway malignant transformation of TK6 cells by accelerating the cell cycle progression.

Hydroquinone (HQ) is an important metabolites of benzene in the body, and it has been found to result in cellular DNA damage, mutation, cell cycle imbalance, and malignant transformation. The JNK1 signaling pathway plays an important role in DNA damage repair. In this study, we focused on whether the JNK1 signaling pathway is involved in the HQ-induced cell cycle abnormalities and the underlying mechanism. The results showed that HQ induced abnormal progression of the cell cycle and initiated the JNK1 signaling pathway. We further confirmed that JNK1 suppression decelerated the cell cycle progression through inhibiting pRb/E2F1 signaling pathway and triggering p53/p21 pathway. Therefore, we concluded that JNK1 might be involved in HQ-induced malignant transformation associated with activating pRb/E2F1 and inhibiting p53/p21 signaling pathway which resulting in accelerating the cell cycle progression.

Hsa_circ_0001944 regulates apoptosis by regulating the binding of PARP1 and HuR in leukemia and malignant transformed cells induced by hydroquinone.

Hydroquinone (HQ) is one of the major metabolites of benzene and can cause abnormal gene expression. It is a known carcinogen that alters cell cycle disruption and cell proliferation. However, its chemical mechanism remain a mystery. Circular RNAs (circRNAs) are a subtype of noncoding RNAs (ncRNAs) that play a variety of roles in biological processes. Hsa_circ_001944 expression was upregulated in 30 leukemia patients and HQ-induced malignant transformed TK6 cells. Hsa_circ_001944 silencing inhibited the growth of HQ-TK6 cells and halted the cell cycle. The silencing of hsa_circ_0001944 led to increased cell accumulation in G1 versus S phase, increased apoptosis in the sh1944 versus the shNC group, and increased levels of DNA damage (γ-H2AX), leading to cell cycle arrest. In summary, inhibition of hsa_circ_001944 restricted cell growth by inhibiting cell cycle arrest and induced growth of HQ-TK6 cells by modulating PARP1 expression. Hsa_circ_0001944 targeted HuR, which is a kind of RNA-binding protein, to control PARP1 expression via RNAinter, RBPmap, and RBPdb. Fluorescence in situ hybridization combined with immunofluorescent labeling and western blotting experiments showed that hsa_circ_001944 was able to dissociate HuR and PARP1 binding in HQ-TK6 cells, control PARP1 production, and ultimately alter the PARP1/H-Ras pathway.

Hydroquinone-induced endoplasmic reticulum stress affects TK6 cell autophagy and apoptosis via ATF6-mTOR.

Hydroquinone (HQ), one of the main active metabolites of benzene in vivo, 7is commonly used as a surrogate for benzene in in vitro studies and has been shown to be cytotoxic. The aim of this study was to investigate the role of endoplasmic reticulum stress (ERS) in HQ-induced autophagy and apoptosis in human lymphoblastoid cells (TK6) and how activating transcription factor 6 (ATF-6) is involved. We treated TK6 cells with HQ to establish a cytotoxicity model and found that HQ induced cellular ERS, autophagy and apoptosis by Western blot, flow cytometry and transmission electron microscopy. In addition, inhibition of both reactive oxygen species (ROS) and ERS inhibited cellular autophagy and apoptosis, suggesting that ERS may be induced by ROS, which in turn affects autophagy and apoptosis. Our study also found that HQ could inhibit ATF6 expression and mTOR activation. Knockdown of ATF6 enhanced autophagy and apoptosis levels and further inhibited mTOR activation; activation of ATF6 by AA147 enhanced cellular activity, suggesting that ATF6 may affect cellular autophagy and apoptosis through mTOR. In conclusion, our data suggest that ROS mediated ERS may promote autophagy and apoptosis by inhibiting ATF6-mTOR pathway after HQ treatment of TK6 cells.

Effect of mir-92a-3p on hydroquinone induced changes in human lymphoblastoid cell cycle and apoptosis.

Hydroquinone (HQ), one of the metabolites of benzene in humans, has significant hepatotoxic properties. Chronic exposure to HQ can lead to leukemia. In a previous study by this group, we constructed a model of malignant transformation of human lymphoblastoid cells (TK6) induced by chronic exposure to HQ with significant subcutaneous tumorigenic capacity in nude mice. miR-92a-3p is a tumor factor whose role in HQ-induced malignant transformation is not yet clear. In the present study, raw signal analysis and dual-luciferase reporter gene results suggested that miR-92a-3p could target and regulate TOB1, and the expression level of miR-92a-3p was significantly upregulated in the long-term HQ-induced TK6 malignant transformation model, while the anti-proliferative factor TOB1 was significantly downregulated. To investigate the mechanism behind this, we inhibited miR-92a-3p in a malignant transformation model and found a decrease in cell viability, a decrease in MMP-9 protein levels, a G2/M phase block in the cell cycle, and an upregulation of the expression of G2/M phase-related proteins cyclinB1 and CDK1. Inhibition of miR-92a-3p in combination with si-TOB1 restored cell viability, inhibited cyclin B1 and CDK1 protein levels, and attenuated the G2/M phase block. Taken together, miR-92a-3p reduced the cell proliferation rate of HQ19 and caused cell cycle arrest by targeting TOB1, which in turn contributed to the altered malignant phenotype of the cells. This study suggests that miR-92a-3p is likely to be a biomarker for long-term HQ-induced malignant transformation of TK6 and could be a potential therapeutic target for leukemia caused by long-term exposure to HQ.

PARP-1 inhibits DNMT1-mediated promoter methylation and promotes linc01132 expression in benzene-exposed workers and hydroquinone-induced malignant transformed cells.

Hydroquinone (HQ), one of the main active metabolites of benzene, can induce the abnormal expression of long non-coding RNA (lncRNA). Studies have shown that lncRNA plays an important role in the occurrence of hematologic tumors induced by benzene or HQ. However, the molecular mechanism remains to be elucidated. Here, we investigated the molecular mechanism by which poly(ADP-ribose)polymerase 1 (PARP-1) interacts with DNA methyltransferase 1 (DNMT1) to regulate promoter methylation mediated linc01132 expression in HQ-induced TK6 malignant transformed cells (HQ-MT). The results revealed that the expression of linc01132 was increased in benzene-exposed workers and HQ-MT cells. The methylation of linc01132 promoter region was inhibited. Furthermore, in HQ-MT cells treated with 5-Aza-2'-deoxycytidine (5-AzaC) (DNA methyltransferase inhibitor) or trichostatin A (TSA) (histone deacetylation inhibitor), the expression of linc01132 was increased due to the regulation of DNA promoter methylation level by inhibiting DNMT1 expression. The methylation level of linc01132 promoter was correlated negatively with the expression of linc01132 in benzene-exposed workers, indicating that DNA methylation may contribute the expression of linc01132. Knockout of DNMT1, not DNMT3b, increased the expression of linc01132 as well as the demethylation of linc01132 promoter in HQ-MT cells. It was found that by knockdown PARP-1, the expression of DNMT1 in the nucleus was increased by immunofluorescence confocal microscopy, leading to the inhibition of hypermethylation in the promoter region of linc01132. Therefore, PARP-1 inhibits DNA methyltransferase (DNMT)-mediated promoter methylation and plays a role in linc01132 expression in benzene-exposed workers or HQ-MT cells, and is associated with benzene or HQ induced leukemia progression.

PARP-1 modulates the expression of miR-223 through histone acetylation to involve in the hydroquinone-induced carcinogenesis of TK6 cells.

The upstream regulators of microRNAs were rarely reported. Hydroquinone (HQ) is the main metabolite of benzene, one of the important environmental factors contributing to leukemia and lymphoma. In HQ-induced malignant transformed TK6 (TK6-HT) cells, the expression of PARP-1 and miR-223 were upregulated. When in PARP-1 silencing TK6-HT cells, miR-223 was downregulated and the apoptotic cell number correspondingly increased. In TK6 cells treated with HQ for different terms, the expression of miR-223 and PARP-1 were dynamically observed and found to be decreased and increased, respectively. Trichostatin A could increase the expression of miR-223, then the expression of HDAC1-2 and nuclear factor kappa B were found to be increased, but that of mH2A was decreased. PARP-1 silencing inhibited the protein expression of H3Ac, mH2A, and H3K27ac. By co-immunoprecipitation experiment, PARP-1 and HDAC2 were found to form a regulatory complex. In conclusion, we demonstrated that the upregulation of PARP-1 mediated activation of acetylation to promote the transcription of miR-223 possibly via coregulating with HDAC2, an epigenetic regulation mechanism involved in cell malignant transformation resulting from long-term exposure to HQ, in which course, H3K27ac might be a specific marker for the activation of histone H3, which also gives hints for benzene exposure research.

SIRT1 regulated hexokinase-2 promoting glycolysis is involved in hydroquinone-enhanced malignant progression in human lymphoblastoid TK6 cells.

Reprogramming of cellular metabolism is a vital event during tumorigenesis. The role of glycolysis in malignant progression promoted by hydroquinone (HQ), one of the metabolic products of benzene, remains to be understood. Recently, we reported the overexpression of sirtuin 1 (SIRT1) in HQ-enhanced malignant progression of TK6 cells and hypothesized that SIRT1 might contribute to glycolysis and favor tumorigenesis. Our data showed that acute exposure of TK6 cells to HQ for 48 h inhibited glycolysis, as indicated by reduction in glucose consumption, lactate production, hexokinase activity, and the expression of SIRT1 and glycolytic enzymes, including HIF-1α, hexokinase-2 (HK-2), ENO-1, glucose transporter 1 (Glut-1), and lactic dehydrogenase A (LDHA). Knockdown of SIRT1 or inhibition of glycolysis using the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) downregulated the levels of SIRT1 and glycolytic enzymes and significantly enhanced HQ-induced cell apoptosis, although knockdown of SIRT1 or 2-DG alone had little effect on apoptosis. Furthermore, immunofluorescence and Co-IP assays demonstrated that SIRT1 regulated the expression of HK-2, and HQ treatment caused a decrease in SIRT1 and HK-2 binding to mitochondria. Importantly, we found that glycolysis was promoted with increasing HQ treatment weeks. Long-term HQ exposure increased the expression of SIRT1 and several glycolytic enzymes and promoted malignant cell progression. Moreover, compared with the PBS group, glucose consumption and lactate production increased after 10 weeks of HQ exposure, and the protein levels of SIRT1 and HK-2 were increased after 15 weeks of HQ exposure, while those of Glut-1, ENO-1, and LDHA were elevated. In addition, SIRT1 knockdown HQ 19 cells exhibited decreased lactate production, glucose consumption, glycolytic enzymes expression, cell growth, and tumor formation in nude mice. Our findings identify the high expression of SIRT1 as a strong oncogenic driver that positively regulates HK-2 and promotes glycolysis in HQ-accelerated malignant progression of TK6 cells.

Poly(ADP-ribose)polymerase-1 affects hydroquinone-induced aberrant cell cycle and apoptosis through activation of p16/pRb signaling pathway in TK6 cells.

Hydroquinone (HQ), a key metabolite of benzene, affects cell cycle and apoptosis. Poly (ADP-ribose) polymerase-1 (PARP-1) plays an important role in DNA damage repair. To explore whether PARP-1 is involved in HQ-induced cell cycle and apoptosis, we assessed the effect of PARP-1 suppression and overexpression on induction of cell cycle and apoptosis analyzed by flow cytometry analysis. We observed that HQ induced aberrant cell cycle progression and apoptosis. We further confirmed that PARP-1 suppression accelerated the cell cycle progression and inhibited cell apoptosis via inhibiting p16/pRb signal pathway after acute HQ exposure, while overexpression of PARP-1 displayed the opposite results. Therefore, we concluded that HQ-induced cell cycle and apoptosis were regulated by PARP-1 through activation of p16/pRb signaling pathway.

Products of Bisphenol A Degradation Induce Cytotoxicity in Human Erythrocytes (In Vitro).

The aim of this work has been to study the possible degradation path of BPA under the Fenton reaction, namely to determine the energetically favorable intermediate products and to compare the cytotoxicity of BPA and its intermediate products of degradation. The DFT calculations of the Gibbs free energy at M06-2X/6-311G(d,p) level of theory showed that the formation of hydroquinone was the most energetically favorable path in a water environment. To explore the cytotoxicity the erythrocytes were incubated with BPA and three intermediate products of its degradation, i.e., phenol, hydroquinone and 4-isopropylphenol, in the concentrations 5-200 µg/mL, for 1, 4 and 24 h. BPA induced the strongest hemolytic changes in erythrocytes, followed by hydroquinone, phenol and 4-isopropylphenol. In the presence of hydroquinone, the highest level of RONS was observed, whereas BPA had the weakest effect on RONS generation. In addition, hydroquinone decreased the level of GSH the most. Generally, our results suggest that a preferable BPA degradation path under a Fenton reaction should be controlled in order to avoid the formation of hydroquinone. This is applicable to the degradation of BPA during waste water treatment and during chemical degradation in sea water.

Study on the differences in sludge toxicity and microbial community structure caused by catechol, resorcinol and hydroquinone with metagenomic analysis.

The aerobic biodegradation rate, organic toxicity and microbial community structure of activated sludge acclimated by catechol, resorcinol and hydroquinone were investigated, to study the relationship between microbial structure and sludge organic toxicity caused by phenolic compounds. At the stable operation stage, the degradation rates of the dihydroxy benzenes in a single sequencing batch reactor (SBR) cycle were followed the order: resorcinol (89.71%) > hydroquinone (85.64%) > catechol (59.62%). Sludge toxicity bioassay indicated that the toxicity of sludge was catechol (45.63%) > hydroquinone (40.28%) > resorcinol (38.15%). The accumulation of secondary metabolites such as 5-10 kDa tryptophan and tyrosine protein substances caused the differential sludge toxicity. Microbial metagenomic analysis showed that the toxicity of sludge was significantly related to the microbial community structure. Thauera, Azoarcus, Pseudomonas and other Proteobacteria formed in the sludge during acclimation. Catechol group had the least dominant bacteria and loop ring opening enzyme genes (catA, dmpB, dxnF, hapD) numbers. Therefore, the degradation of catechol was the most difficult than resorcinol and hydroquinone, resulting the highest sludge toxicity.

[Effects of p16/pRb and JNK signaling pathways in hydroquinone-induced malignant transformation of TK6 cells].

Objective: To investigate the cell cycle and apoptosis in hydroquinone (HQ) -induced malignant transformation of TK6 cells and its related regulatory mechanisms. Methods: TK6 cells were exposed to 20 µmol/L HQ, 24 h/time, once a week, for 19 weeks as experimental group and TK6 cells treated with phosphate buffer (PBS) for 19 weeks was used as control group from March 2014. In regulatory mechanism research, the cells were divided into four groups: control group, experimental group, control inhibitor group and experimental inhibitor group (inhibitor groups were added 10 µmol/L P600125) . Cell cycle and apoptosis were detected by flow cytometry. The protein expression of cell cycle-related proteins and JNK signaling pathway proteins were detected by Western blot. Results: Flow cytometry showed that compared with control group, the ratio of cells in the G0/G1 phase of the experimental group was significantly decreased (P=0.001) , and the ratio of cells in the S phase was significantly increased (P=0.002) . Western blotting demonstrated that the protein expressions of p-Rb (Ser780) , E2F1, Cyclin D1, p-p16 (Ser152) , JNK1, p-JNK1 (Thr183/Tyr185) , c-jun, p-c-jun (Ser63) (P=0.015, 0.021, 0.001, 0.001, 0.005, 0.001, 0.039, 0.003) were up-regulated, while the protein expressions of Rb (P=0.048) and p16 (P=0.002) were significantly down-regulated. After exposed to SP600125, compared with experimental group, there were no significant changes in cell cycle distribution (P=0.946) and apoptosis rate (P=0.923) in experimental inhibitor group. The expression of c-jun (P=0.040) protein was down-regulated, while the expression of Rb (P=0.027) protein was up-regulated in experimental inhibitor group. Conclusion: In HQ-induced TK6 cells malignant transformation, the cell cycle is arrested in the S phase, and the p16/pRb signaling pathway is inhibited, while the JNK signaling pathway is activated. However, the activated JNK signaling pathway may not be involved in the regulation of cell cycle.

The DNMT1-associated lncRNA UCA1 was upregulated in TK6 cells transformed by long-term exposure to hydroquinone and benzene-exposed workers via DNA hypomethylation.

Exposure to benzene or its metabolite hydroquinone (HQ) is a risk factor for a series of myeloid malignancies, and long noncoding RNAs play an important role in the process of pathogenesis. Urothelial cancer-associated 1 (UCA1) functions as an oncogene in the development of acute myeloid leukemia. However, the association between DNMT1 and UCA1 with benzene or HQ exposure has not been explored. We characterized UCA1 expression in cells briefly exposed to HQ (HQ-ST cells) and HQ-induced malignantly transformed (TK6-HT cells) treated with 5-aza-2'-deoxycytidine (5-AzaC) or trichostatin A (TSA). Compared to that in control cells, UCA1 expression was increased, whereas DNMT1 was decreased in HQ-ST cells and TK6-HT cells treated with 5-AzaC or TSA. Moreover, UCA1 expression was also upregulated and positively correlated with benzene exposure time in benzene-exposed workers. Furthermore, the expression of UCA1 was negatively associated with the DNA methylation level of its promoter in benzene-exposed workers. DNMT1 rather than DNMT3b knockout in TK6-HT cells activated the expression of UCA1 by inducing its promoter hypomethylation. These results suggest that benzene or HQ exposure leads to UCA1 upregulation via DNA hypomethylation in the UCA1 promoter, which is mediated by DNMT1.

The in vitro assessment of the toxicity of volatile, oxidisable, redox-cycling compounds: phenols as an example.

Phenols are regarded as highly toxic chemicals. Their effects are difficult to study in in vitro systems because of their ambiguous fate (degradation, auto-oxidation and volatility). In the course of in vitro studies of a series of redox-cycling phenols, we found evidences of cross-contamination in several in vitro high-throughput test systems, in particular by trimethylbenzene-1, 4-diol/trimethylhydroquinone (TMHQ) and 2,6-di-tertbutyl-4-ethylphenol (DTBEP), and investigated in detail the physicochemical basis for such phenomenon and how to prevent it. TMHQ has fast degradation kinetics followed by significant diffusion rates of the resulting quinone to adjacent wells, other degradation products being able to air-diffuse as well. DTBEP showed lower degradation kinetics, but a higher diffusion rate. In both cases the in vitro toxicity was underestimated because of a decrease in concentration, in addition to cross-contamination to neighbouring wells. We identified four degradation products for TMHQ and five for DTBEP indicating that the current effects measured on cells are not only attributable to the parent phenolic compound. To overcome these drawbacks, we investigated in detail the physicochemical changes occurring in the course of the incubation and made use of gas-permeable and non-permeable plastic seals to prevent it. Diffusion was greatly prevented by the use of both plastic seals, as revealed by GC-MS analysis. Gas non-permeable plastic seals, reduced to a minimum compounds diffusion as well oxidation and did not affect the biological performance of cultured cells. Hence, no toxicological cross-contamination was observed in neighbouring wells, thus allowing a more reliable in vitro assessment of phenol-induced toxicity.

Exosomal secretion may be a self-protective mechanism of its source cells under environmental stress: A study on human bronchial epithelial cells treated with hydroquinone.

Accumulating evidence reveals that exosome plays an important role in cell-to-cell communication in both physiological and pathological processes by transferring bioactive molecules. However, the role of exosomal secretion in the adaption of its source cells to the stimuli of environmental chemicals remains elusive. In this study, we revealed that the exposure of hydroquinone (HQ; the main bioactive metabolite of benzene) to human bronchial epithelial cells (16HBE) resulted in decreased ability of cell proliferation and migration, and simultaneously DNA damage and micronuclei formation. Interestingly, when exosomal secretion of HQ treated 16HBE cells was inhibited with the inhibitor GW4869, cellular proliferation and migration were further significantly reduced; concurrently, their DNA damage and micronuclei formation were both further significantly aggravated. Herein, we conclude that exosomal secretion of 16HBE cells may be an important self-protective function against the toxic effects induced by HQ.