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.
Humans ; Hydroquinones/toxicity* ; MAP Kinase Signaling System ; Cell Cycle ; Cell Transformation, Neoplastic ; Apoptosis

OBJECTIVE: To investigate the regulatory effect and mechanism of DNA methyltransferase 3A (DNMT3a) in hydroquinone-induced hematopoietic stem cell toxicity. METHODS: Cells (HSPC-1) were divided into 4 groups, that is A: normal HSPC-1; B: HQ-intervented HSPC-1; C: group B + pcDNA3 empty vector; D: group B + pcDNA3- DNMT3a. RT-qPCR and Western blot were used to detect the expression levels of DNMT3a and PARP-1 mRNA and protein, respectively. Cell morphology was observe; Cell viability and apoptosis rate of HSPC-1 were detected by MTT and flow cytometry, respectively. RESULTS: Compared with group A, the expression levels of DNMT3a mRNA and protein in HSPC-1 of group B were decreased, while PARP-1 mRNA and protein were increased (P<0.05); there was no significant difference in the above indexes between group C and group B; compared with group B, the expression levels of DNMT3a mRNA and protein showed increased, while PARP-1 mRNA and protein were decreased significantly in cells of group D transfected with DNMT3a (P<0.05). Cells in each group were transfected with DNMT3a and cultured for 24 h, HSPC-1 in group A showed high density growth and mononuclear fusion growth, while the number of HSPC-1 in group B and C decreased and grew slowly. Compared with group B and C, the cell growth rate of group D was accelerated. The MTT analysis showed that cell viability of HSPC-1 in group B were lower than that of group A at 24 h, 48 h and 72 h (P<0.05); after transfected with DNMT3a, the cell viability of HSPC-1 in group D were higher than that of group B at 24 h, 48 h and 72 h (P<0.05). The apoptosis rate of cells in group B was significantly higher than that of group A (P<0.001), while the apoptosis rate in group D was lower than that of group B (P<0.001). CONCLUSION DNMT3a may be involved in the damage of hematopoietic stem cells induced by hydroquinone, which may be related to the regulation of PARP-1 activity by hydroquinone-inhibited DNMT3a.
Apoptosis ; Cell Proliferation ; DNA Methyltransferase 3A ; Hematopoietic Stem Cells/drug effects* ; Humans ; Hydroquinones/toxicity* ; Poly (ADP-Ribose) Polymerase-1 ; RNA, Messenger/metabolism*

The molecular mechanism of DNA damage induced by hydroquinone (HQ) remains unclear. Poly(ADP-ribose) polymerase-1 (PARP-1) usually works as a DNA damage sensor, and hence, it is possible that PARP-1 is involved in the DNA damage response induced by HQ. In TK6 cells treated with HQ, PARP activity as well as the expression of apoptosis antagonizing transcription factor (AATF), PARP-1, and phosphorylated H2AX (γ-H2AX) were maximum at 0.5 h, 6 h, 3 h, and 3 h, respectively. To explore the detailed mechanisms underlying the prompt DNA repair reaction, the above indicators were investigated in PARP-1-silenced cells. PARP activity and expression of AATF and PARP-1 decreased to 36%, 32%, and 33%, respectively, in the cells; however, γ-H2AX expression increased to 265%. Co-immunoprecipitation (co-IP) assays were employed to determine whether PARP-1 and AATF formed protein complexes. The interaction between these proteins together with the results from IP assays and confocal microscopy indicated that poly(ADP-ribosyl)ation (PARylation) regulated AATF expression. In conclusion, PARP-1 was involved in the DNA damage repair induced by HQ via increasing the accumulation of AATF through PARylation.
Antioxidants ; toxicity ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Cell Line ; DNA Damage ; drug effects ; Gene Expression Regulation ; drug effects ; Gene Silencing ; Histones ; genetics ; metabolism ; Humans ; Hydroquinones ; toxicity ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; genetics ; metabolism ; Protein Transport ; Repressor Proteins ; genetics ; metabolism

OBJECTIVETo investigate the DNA methylation changes induced by hydroquinone (HQ) in human bronchial epithelial cells and to explore the role of poly (ADP-ribose) polymerase-l (PARP-l) in this process.

METHODSHuman bronchial epithelial 16HBE cells and PARP-l-deficient 16HBE cells (16HBE-shPARP-l cells) were exposed to HQ (10, 20, 40, 60, and 80 µmol/L) for 48h, while control cells were treated with an equal volume of PBS solution. The changes in genomic DNA methylation were investigated by high-performance capillary electrophoresis, and the expression levels of PARP-l and DNA methyltransferase 1 (DNMT1) were measured.

RESULTSThe percentages of methylated DNA of overall genome (mCpG%) in 16HBE and 16HBE-shPARP-l cells were 4.89%±0.07% and 9.53%±0.51%, respectively; after treatment with 5-aza-2'-deoxycytidine for 72 h, mCpG% decreased to 3.07±0.12% and 6.34%±0.3%, respectively. The one-way analysis of variance revealed significant differences in mCpG% between the cells exposed to different concentrations of HQ in both 16HBE and 16HBE-shPARP-l groups (F = 61.25, P < 0.01; F = 60.36, P < 0.01). For 16HBE cells treated with HQ (10, 20, 40, 60, and 80 µmol/L), the mRNA expression levels of PARP-1 were 145.0%, 159.0%, 169.0%, 215.0%, and 236.0%, respectively, compared with those in the control group, with significant differences (P < 0.01 for all); for 16HBE-shPARP-l cells treated with HQ (10, 20, 40, 60, and 80 µmol/L), the mRNA expression levels of PARP-l were 170.0%, 223.0%, 264.0%, 327.0%, and 320.0%, respectively, compared with those in the control group, with significant differences (P < 0.01 for all). When the dose of HQ reached 20, 40, 60, and 80 µmol/L, the mRNA expression levels of DNMT1 in 16HBE group were 114.0%, 126.0%, 136.0%, and 162.0%, respectively, compared with those in the control group, with significant differences (P < 0.01 for all); when the dose of HQ reached 10, 20, 40, 60, and 80 µmol/L, the mRNA expression levels of DNMT1 in the 16HBE-shPARP-l group were 141.0%, 165.2%, 186.9%, 202.1%, and 217.3%, respectively, compared with those in the control group, with significant differences (P < 0.01 for all).

CONCLUSIONHQ can induce hypomethylation in 16HBE cells, and PARP-1 can regulate DNA methylation in 16HBE cells by influencing the expression and activity of DNMT1.

Cells, Cultured ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Damage ; DNA Methylation ; Epithelial Cells ; metabolism ; Humans ; Hydroquinones ; toxicity ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism

OBJECTIVETo explore the effect and mechanism of DNA polymerase β expression level on cell apoptosis and mitochondrial membrane potential induced by hydroquinone.

METHODSPolβ wild-type cells (polβ+/+), polβ overexpressed cells (polβ oe) and polβ null cells (polβ-/-) were applied as a model cell system, The effect of cell apoptosis and mitochondrial membrane potential induced by different doses of hydroquinone were analyzed by flow cytometry. The ROS and ·OH assay kit were used to examine the cellular ROS and ·OH level. The activity of cellular SOD and GSH-Px were tested by Chemiluminescence method after exposed to different concentrations of hydroquinone.

RESULTSWith the dose of hydroquinone increased, the rate of apoptosis and falling of mitochondrial membrane potential (ΔΨm) in cells were increased compared with the control. When compared with polβ+/+ cells, the rate of apoptosis in polβ-/- cells exposed to 20.00, 40.00, 80.00 µmol/L hydroquinone increased and the rate of apoptosis in polβ oe cells exposed to 10.00, 20.00, 40.00, 80.00 µmol/L hydroquinone decreased (P < 0.05). Compared with polβ+/+ cells (20.60% ± 0.57%, 37.95% ± 0.64%, 44.50% ± 1.27%, 57.55% ± 1.06%), the rate of cell which undergone mitochondrial depolarization in polβ-/- cells treated with 10.00, 20.00, 40.00, 80.00 µmol/L hydroquinone (33.60% ± 1.55%, 46.05% ± 1.77%, 52.75% ± 2.05%, 75.20% ± 0.56%) increased. The rate of cell which undergone mitochondrial depolarization in polβ oe cells exposed to 10.00, 20.00, 40.00, 80.00 µmol/L hydroquinone (16.05% ± 1.20%, 29.80% ± 1.21%, 35.15% ± 1.06%, 53.80% ± 0.85%) decreased (P < 0.05). When compared with polβ+/+ cells, fluorescent intensity of polβ-/- cells treated with different dosages of hydroquinone increased, while which of polβ oe cells decreased (P < 0.05). Compared with polβ+/+ cells, ·OH level of polβ-/- cells treated with 20.00, 40.00 µmol/L hydroquinone significantly enhanced, while which of polβ oe cells decreased sharply (P < 0.05). Under the same concentrations of hydroquinone, the activity of SOD and GSH-Px were decreased most rapidly in polβ-/- cells. The activity of SOD and GSH-Px in polβ oe cells decreased slower than in the polβ-/- cells.

CONCLUSIONHydroquinone could induced apoptosis by the generation of ROS and decrease of ΔΨm; polβ could protect cells from apoptosis induced by hydroquinone through decrease of ROS level and depolarization of mitochondria.

Animals ; Apoptosis ; drug effects ; Cells, Cultured ; DNA Polymerase beta ; metabolism ; Hydroquinones ; toxicity ; Membrane Potential, Mitochondrial ; drug effects ; Mice

OBJECTIVETo investigate the protective effects of the tert-butylhydroquinone (tBHQ) pretreatment on neurotoxicity and oxidative stress induced by paraquat (PQ) in PC12 cells.

METHODSCytotoxicity of PC12 cells was measured by MTT assay, following the PC12 cells treatment with different concentrations of 100, 300 micromol/L PQ for 24 h and 48 h. PC12 cells were pretreated with or without 40 micromol/L tBHQ for 4 h, PC12 cells were exposed to PQ at the doses of 0, 100, 300 micromol/L for 24 h and 48 h, respectively. The viability of PC12 cells was measured by MTT assay, the apoptosis rates of PC12 cells were detected by flow cytometry (FCM) and the malondialdehyde (MDA) levels of PC12 cells were examine by thiobarbituric acid (TBA) method.

RESULTSWhen the exposure doses of PQ were 100 and 300 micromol/L for 24 h, the viability of PC12 cells pretreated with tBHQ was significantly higher than that of PC12 cells only exposed to PQ (P < 0.05 or P < 0.01). When the exposure dose of PQ was 100 micromol/L for 48 h, the viability of PC12 cells pretreated with tBHQ was significantly higher than that of PC12 cells only exposed to PQ (P < 0.01). When the exposure doses of PQ were 100 and 300 micromol/L for 24 h, the apoptosis rates and MDA levels of PC12 cells pretreated with tBHQ were significantly lower than those of PC12 cells only exposed to PQ (P < 0.05 or P < 0.01).

CONCLUSIONStBHQ pretreatment can reduce the cytotoxicity, apoptosis and oxidative stress induced by PQ in PC12 cells.

Animals ; Apoptosis ; drug effects ; Cell Survival ; drug effects ; Hydroquinones ; pharmacology ; Oxidative Stress ; drug effects ; PC12 Cells ; Paraquat ; toxicity ; Rats ; Reactive Oxygen Species ; analysis

OBJECTIVETo investigate the effects of hydroquinone (HQ) on expression of topoisomerase IIα (TOPOIIα) in human bone marrow mononuclear cells, and to explore the role and possible regulatory mechanism of TOPOIIα involved in toxicity of HQ to hematopoietic cells.

METHODSAfter human bone marrow mononuclear cells were exposed to 50 µmol/L HQ (used the cells which were exposed to sterile distilled water as control); the activity of TOPOII was measured by TOPOII assay kit; the expression levels of TOPOIIα mRNA and protein were detected by RT-PCR technique and Western blotting method respectively; the chromatin immunoprecipitation (ChIP) assay was carried out to study the possible mechanism of TOPOIIα expression changes.

RESULTS(1) The activity of TOPOII was inhibited obviously; the protein and mRNA expression of TOPOIIα were 0.017 ± 0.029 and 0.610 ± 0.128, significantly lower than that in the control with the significant difference (P < 0.01) after treated with HQ for 10 h; (2) The decreased content of TOPOIIα was associated with descended level of histone H4 acetylation than in the control, from 1.198 ± 0.056 to 0.324 ± 0.229, with the significant difference (P < 0.01), without accompanied descended level of histone H3 acetylation, from 1.253 ± 0.045 to 1.177 ± 0.025 (P > 0.05); (3) TOPOIIα mRNA expression decreased gradually after HQ processing, and the chemical modification (histone H4 acetylation) of TOPOIIα promoter happened prior to the mRNA expression.

CONCLUSIONHQ could repress the expression of TOPOIIα in human bone marrow mononuclear cells; the change of histone chemical modification plays an important role in the benzene's hematopoietic toxicity.

Acetylation ; Adult ; Antigens, Neoplasm ; metabolism ; Bone Marrow Cells ; drug effects ; metabolism ; Cells, Cultured ; DNA Topoisomerases, Type II ; metabolism ; DNA-Binding Proteins ; metabolism ; Female ; Histones ; metabolism ; Humans ; Hydroquinones ; toxicity ; Male ; Young Adult

OBJECTIVETo investigate the effects of hydroquinone (HQ) on expression of ubiquitin-ligating enzyme Rad18 in human hepatic cells (L-02), and to explore the role and possible mechanism of Rad18 involved in toxicity of HQ to hepatic cells.

METHODSAfter L-02 hepatic cells were exposed to HQ with various concentrations (0, 5, 10, 20, 40, 80 and 160 micromol/L) for 24 h, cell survival rate was measured by MTT assay; DNA impairment was evaluated by single cell gel electrophoresis (SCGE); The expression levels of Rad18 mRNA and protein were detected by Real-time fluorescent quantitative polymerase chain reaction (QPCR) technique and Western blot method respectively.

RESULTSHQ with concentration from 0 to 80 micromol/L had little effect on survival rate of L-02 (P > 0.05); Whereas the survival rate in the group of 160 micromol/L was significantly lower than in the control with the significant difference (P < 0.01) after treated with HQ for 24 h; The higher dose of HQ presented, the more degrees of olive tail moment (OTM) were produced and a dose-dependent relationship was shown. HQ in a low concentration (0 to approximately 40 micromol/L) could induce increase in the expression of Rad18 mRNA and protein which was in proportion to the increment of HQ concentration; the expression of Rad18 mRNA was enhanced increasingly, while the expression of Rad18 protein unchanged basically once the concentration of HQ exceeded 40 micromol/L; Besides, there was a positive correlation between OTM and the expression level of Rad18 mRNA (r = 0.919, P < 0.01).

CONCLUSIONHQ could regulate up the expression of Rad18 in L-02 hepatic cells.

Cell Survival ; drug effects ; Cells, Cultured ; DNA Damage ; drug effects ; DNA-Binding Proteins ; metabolism ; Hepatocytes ; drug effects ; enzymology ; Humans ; Hydroquinones ; toxicity ; Ubiquitin-Protein Ligases

OBJECTIVETo explore the effects of hydroquinone (HQ) on reactive oxygen species (ROS) generation, antioxydase activities and the expression of human 8-oxo-guanine DNA glycosylase (hOGG1) mRNA in human A549 lung adenocarcinoma cell strains.

METHODSA549 cells were treated with different concentrations of HQ. Cell survival was determined by methyl thiazolyl tetrazolium (MTT). Changes of ROS were detected by fluorescent probe. The contents of malonaldehyde and activities of antioxydase were determined through colorimetry. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the level of hOGG1 mRNA.

RESULTSWith the increased concentration of HQ, the findings were as follows. (1) The absorbance value of A549 cell decreased. There was significant difference between 160 micromol/L (0.584+/-0.098) and 320 micromol/L (0.328+/-0.066) of HQ (q=5.56 and 9.07, P<0.05) with the control group (0.989+/-0.150), and the cell survival rate were less than 80%. (2) The ROS in A549 cell increased. 40 micromol/L (39.80+/-4.15) and 80 micromol/L (101.99+/-9.45) had statistical significance (q=10.74 and 30.32, P<0.05) with the control group (5.71+/-0.50). (3) It was found that the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) decreased and malonaldehyde (MDA) increased. Compared with the control group [(25.62+/-0.28) U/mg prot and (38.97+/-2.61) U/mg prot], the activities of SOD and GSH-Px had a significant decrease (q=12.17 and 8.78, P<0.05) in 80 micromol/L [(22.93+/-0.56) U/mg prot and (25.60+/-2.31) U/mg prot]. And MDA had a significant increase (q=10.90 and 15.49, P<0.05) in 40 micromol/L [(1.07+/-0.01) nmol/mg prot] and 80 micromol/L [(1.19+/-0.08) nmol/mg prot] as compared with the control group [(0.77+/-0.04) nmol/mg prot]. The decrease of SOD (r=-0.95, F=20.00, P=0.04) and GSH-Px activities (r=-0.99, F=115.48, P=0.01) and the increase of MDA contents (r=0.96, F=21.31, P=0.04) all had a dose-response relationship. (4) RT-PCR results showed that the expression of hOGG1 mRNA decreased. The significant difference was observed between the expression of hOGG1 mRNA in 80 micromol/L (0.478+/-0.017) (q=11.70, P<0.05) with the control group (0.715+/-0.038).

CONCLUSIONThis study suggests that HQ could induce oxidative damage and changes of the expression of hOGG1 mRNA in A549 cells.

Cell Line, Tumor ; DNA Glycosylases ; genetics ; Down-Regulation ; Gene Expression ; Gene Expression Regulation, Enzymologic ; drug effects ; Humans ; Hydroquinones ; toxicity ; RNA, Messenger ; genetics

OBJECTIVETo investigate the protective effect of the tert-butylhydroquinone (tBHQ) on PC12 cells from neurotoxicity induced by manganese.

METHODSCytotoxicity of PC12 cells was measured by MTT assay, following the PC12 cells treatment with different concentrations of MnCl₂ (300, 600, 900 μmol/L) for 24, 48 or 72 h. PC12 cells were pretreated with 40 μmol/L tBHQ for 12 h, followed by the treatment of 600 micromol/L or 300 μmol/L MnCl₂ for 72 h. Cytotoxicity of PC12 cells was measured by MTT assay, and cell apoptosis was examined by the method of Annexin V-FITC/PI in flow cytometry (FCM).

RESULTSThe proliferation of PC12 cells treated with 300, 600, 900 μmol/L MnCl2 was suppressed in the dose dependent pattern (P < 0.01). Proliferation of PC12 cells treated with 600 μmol/L MnCl₂ was suppressed to 40% of that in control group (P < 0.01), but the proliferation rate of PC12 cell pretreated with 40 μmol/L tBHQ was 180% of that in control group (P < 0.01). Apoptotic rate of PC12 cells treated with 300 micromol/L MnCl₂ was higher than the vehicle control group (P < 0.01). Apoptotic rate of 40 μmol/L tBHQ pretreatment followed by 300 μmol/L MnCl₂ treatment was lower than that of MnCl2 treatment group (P < 0.01). The inhibition rate of apoptosis was 61%.

CONCLUSIONSManganese may suppress PC12 cells proliferation and induce apoptosis. tBHQ can reduce PC12 cells proliferation suppressed by manganese and attenuate the apoptosis induced by manganese.

Animals ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Drug Antagonism ; Hydroquinones ; pharmacology ; Manganese ; toxicity ; PC12 Cells ; drug effects ; Rats