[Effects of 3ß-HSD gene silence or overexpression on DEHP induced oxidative damage in MCF-7 cells].

Objective: To study the oxidative damage of di- (2-ethylhexyl) phthalate (DEHP) on MCF-7 cells, and to investigate the effects of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) gene silence or overexpression on DEHP-induced oxidative damage. Methods: MCF-7 cells, 3ß-HSD gene silencing cells and 3ß-HSD gene overexpression cells were treated with different doses of DEHP (0,0.05,0.1,0.2,0.4,0.8 mmol/L) for 24h, then intracellular oxidative damage index such as MDA, SOD, GSH, GSH-PX were detected, DNA repair gene hOGG1, hMTH1 mRNA expression were tested by Q-PCR, hOGG1, hMTH1 protein expression were detected by western blot. Results: After MCF-7 cells were treated by DEHP, MDA levels increased; SOD activity, GSH content, GSH-PX activity decreased, hOGG1 and hMTH1 mRNA expression levels increased, hOGG1 and hMTH1 protein expression levels increased, the differences were statistically significant when compared with control (P<0.05 or P<0.01) . In 3ß-HSD gene silencing cells which were treated by DEHP, when compared with the same dose group of MCF-7 cells, MDA content increased, SOD activity, GSH content, GSH-PX activity decreased, hOGG1 and hMTH1 mRNA expression levels decreased, hOGG1 and hMTH1 protein expression levels decreased, the difference were statistically significant (P<0.05 or P<0.01) . In 3ß-HSD gene overexpression cells which were treated by DEHP, when compared with the same dose group of MCF-7 cells, MDA content decreased; SOD activity, GSH content, GSH-PX activity increased, of hOGG1 and hMTH1 mRNA expression levels increased, hOGG1 and hMTH1 protein expression levels increased, the difference were statistically significant (P<0.05 or P<0.01) . Conclusion: DEHP could cause oxidative damage in MCF-7 cells, induce the changes of related genes and proteins, 3ß-HSD plays an antioxidant role in the process of DEHP ox-idative damage.

[Association between expression of plasma miRNA and the risk of childhood acute lymphocytic leukemia].

Objective: To investigate the characteristics of distribution and expression profiles of plasma miRNA in childhood acute lymphocytic leukemia (cALL) patients; the association between cALL incidence risk and plasma miRNA levels; the feasibility of plasma miRNA serving as cALL diagnostic biomarker. Methods: A total of 111 pairs of newly diagnosed cALL patients and patients with fractures were collected from Shenzhen Children's Hospital, China, between January 2015 and November 2016. Age and sex of the cases and controls were 1∶ 1 matched and LNA(TM) miRNA microarray was performed using 4 pairs of cALL and controls selected from the sample population. The expression level of miRNA was validated by real time quantitative PCR. Conditional logistic regression analysis was applied to evaluate the association between miRNA expression levels and the incidence risk of cALL. The receiver operating characteristic curve (ROC) and reclassification analysis were conducted to assess the feasibility of miRNAs serving as biomarkers for cALL. Results: A total of 204 differentially expressed miRNA were screened out and let-7f-5p, miR-5100, miR-25-3p and miR-3654 were selected for validation identified according to the inclusion criteria. The expression levels of let-7f-5p, miR-5100 and miR-25-3p in the cALL patients were significantly lower than those of the controls (P<0.01). After adjusting for confounding factors, 3 miRNAs remained significantly associated with the risk of cALL (OR and 95%CI were 0.84 (0.76-0.92), 0.81 (0.73-0.90) and 0.81 (0.74-0.89), respectively. Results from both the ROC analysis and reclassification analysis showed that introduction of one or more miRNA to traditional risk factors improved the area under the curve (P<0.05) and provided additional values to diagnosis (P<0.01). Conclusion: The expression levels of let-7f-5p, miR-5100 and miR-25-3p were significantly associated with the incidence rate of cALL, and these miRNAs might serve as promising biomarkers for cALL.

[Association of exposure to environmental chemicals with risk of childhood acute lymphocytic leukemia].

Objective: This study aimed to investigate the association between exposure to environmental chemicals and the risk of childhood acute lymphocytic leukemia(cALL). Methods: A case-controlled study was conducted in Shenzhen Children's Hospital, China from January 2015 to January 2016. The cases were selected from the section of Hematology and Oncology, and the controls were selected from Orthopedics by 1∶2 matching of cases according to sex and age. A questionnaire including population data and chemical exposure characteristics was conducted on the children's parents, and urine and EDTA-blood were collected from the children. Then, we quantitatively measured the internal dose of formaldehyde(i.e., formaldehyde-human serum albumin)by enzyme-linked immunosorbent assay and the doses of metabolites benzene, toluene, and xylene(i.e., trans-muconic acid, hippuric acid, and methylhippuric acid)by high-performance liquid chromatography. Logistic regression models were used to analyze the relationships between exposure factors measured from children and their parents and cALL. Results: In the study, 71 cases(average age: 6.08±3.61 years), and 142 controls(average age: 5.91±3.57 years)were assessed; there were no differences in general demographics between two groups. The self-reported results showed that living in a home that had been painted in the past 10 years(OR=4.39, 95% CI: 1.87-10.31), maternal chemical exposure during pregnancy(OR=11.78, 95% CI: 1.65-83.88), paternal diesel or gasoline exposure(OR=8.15, 95% CI: 2.68-24.83), paternal dye exposure(OR=7.77, 95% CI: 1.52-39.67)and trash burning near the child's residence(OR=6.08, 95% CI: 1.17-31.66)were associated with increased risk of cALL. The positive detection rates of only benzene metabolites were significantly higher in cases(40/44)than controls(81/111)(χ2=5.92, P=0.021). The median formaldehyde and benzene concentrations in cases(32.120 pg/ml, 2.505 µg/gCr)were significantly higher than those in controls(18.705 pg/ml, 0.672 µg/gCr; Z values:-1.98 and-3.95, P values: 0.047 and<0.001, respectively). Multiple logistic regression analysis showed that benzene exposure(OR=1.09, 95% CI: 1.00-1.19), home painting in the past 10 years(OR=3.56, 95% CI: 1.20-10.53)and paternal diesel or gasoline exposure(OR= 3.75, 95% CI: 1.06-13.22)were associated with increased risk of cALL. Conclusion: A variety of environmental chemistry factors, such as benzene exposure, increase the risk of cALL, and further studies are warranted to explore their specific roles.

[Effects of long term and low dose Di- (2-ethylhexyl) phthalate exposure on global genome DNA methylation in HePG2 cells].

OBJECTIVE: To explore the potential effects of long-term and low-dose Di- (2-ethylhexyl) phthalate (DEHP) exposure on whole genome DNA methylation status and cytotoxicity of HePG2 cells. METHODS: HePG2 cells were exposed to 1.5、15.0 and 150.0 µmol/L DEHP for 24 hours, after continuous exposure for 20 generations, mRNA and protein expression level of DNA (cytosine-5) -methyltransferase 1 (DNMT 1) , whole genome DNA methylation, cell apoptosis levels and cell cycle were determined in both DEHP exposed cells and control cells. RESULTS: After DEHP exposure, the mRNA and protein expression levels of DNMT 1 were decreased significantly (P<0.05 or P<0.01). The genome DNA methylation levels of HePG2 cells were down-regulated along with the increasing of DEHP exposure level (15.0, 150.0 µmol/L, P<0.05 and P<0.01). In term of cell apoptosis rates, only the late-stage cell apoptosis rates of the highest DEHP dosage group (150.0 µmol/L DEHP) were observed to have a significant increase (P<0.05). There were no significant alterations in term of cell cycle. CONCLUSION: After long term and low dose DEHP exposure, the whole genome DNA methylation levels of HePG2 cells were down regulated obviously, which might be one of the most important toxic mechanism of DEHP to induce pathophysiologic changes. Meanwhile, a certain content of cell apoptosis were observed in highest dosage group of DEHP exposure, which showed cytotoxicty of DEHP. However, there are no significant effects of DEHP exposure on cell cycles.

Altered expression of hepatic metabolic enzyme and apoptosis-related gene transcripts in human hepatocytes treated with trichloroethylene.

Trichloroethylene (TCE) is a common organic solvent that has been widely used in industrial applications. Hundred cases of allergic reactions occurred after the workers were occupationally exposed to TCE in China in the past decade, but the underlying effector mechanisms of TCE remain unclear. The purpose of the present study is to examine the alteration of hepatic metabolic enzyme gene and apoptosis-related gene messenger RNA (mRNA) expression in L02 human hepatocytes (L02 cells) after treatment with TCE. L02 cells were cultured either with various doses of TCE (0.25, 0.5, 1.0 and 2.0 mmol/L) for 24 h or with a single dose of TCE (1.0 mmol/L) for different time intervals, whereas samples treated with dimethyl sulfoxide served as control. Quantitative real-time polymerase chain reaction analysis was performed to detect the mRNA expression of hepatic metabolic enzyme genes (CYP1A2, CYP3A4 and CYP2E1) and apoptosis-related genes (BAX and BAD). It was found that the transcript levels of hepatic metabolic enzyme genes and apoptosis genes including BAX and BAD were significantly increased after TCE treatment at various doses for 24 h when compared with controls. Additionally, when the cells were treated with a single dose of TCE (1.0 mmol/L) for different periods of time (3, 6, 12 and 24 h), the mRNA expression of these genes also increased significantly compared with control (p < 0.05 or p < 0.01). The conclusion of the study is that TCE could induce alteration of mRNA expression of hepatic metabolic enzyme genes and apoptosis genes, which might be implicated in the effector mechanisms of TCE cytotoxicity in vivo.

Anaphase-promoting complex/cyclosome controls HEC1 stability.

OBJECTIVE: Chromosome segregation during mitosis requires a physically large proteinaceous structure called the kinetochore to generate attachments between chromosomal DNA and spindle microtubules. It is essential for kinetochore components to be carefully regulated to guarantee successful cell division. Depletion, mutation or dysregulation of kinetochore proteins results in mitotic arrest and/or cell death. HEC1 (high expression in cancer) has been reported to be a kinetochore protein, depletion of which, by RNA interference, results in catastrophic mitotic exit. MATERIALS AND METHODS AND RESULTS: To investigate how HEC1 protein is controlled post-translation, we analysed the role of anaphase-promoting complex/cyclosome (APC/C)-Cdh1 in degradation of HEC1 protein. In this study, we show that HEC1 is an unstable protein and can be targeted by endogenous ubiquitin-proteasome system in HEK293T cells. Results of RNA interference and in vivo ubiquitination assay indicated that HEC1 could be ubiquitinated and degraded by APC/C-hCdh1 E3 ligase. The evolutionally conserved D-box at the C-terminus functioned as the degron of HEC1, destruction of which resulted in resistance to degradation mediated by APC/C-Cdh1. Overexpression of non-degradable HEC1 (D-box destroyed) induced accumulation of cyclin B protein in vivo and triggered mitotic arrest. CONCLUSION: APC/C-Cdh1 controls stability of HEC1, ensuring normal cell cycle progression.

[Activation of G protein on the membrane of TCS-sensitive cells].

Trichosanthin (TCS), a Type I Ribosome Inactivating Protein isolated from the root tuber of Trichosanthes Kirilowii M. has various biological activities including abortion induction, anti-tumor and anti-HIV. The mechanism of TCS specifically killing sensitive cells has not been studied clearly. In this study, we initially found that there exists TCS-affinity molecule on Syncytiotrophoblast cells and Jar cells. Furthermore, by [35S]GTP gamma S Binding Assay, we find that TCS can activate G protein on the membrane of TCS-sensitive cells. These results indicate that on the membrane of TCS-sensitive cells exists TCS-specific receptor.

[Study on Trichosanthin induced apoptosis of leukemia K562 cells].

Trichosanthin (TCS), an eukaryotic ribosome-inactivating protein isolated from the root tuber of Trichosanthes plant, has various biological activities including abortion induction, antitumor, and anti-HIV. In this study, cultured human leukemia K562 cells treated with trichosanthin were examined. Analysis of the cells by single laser flow cytometry showed the sub-G1 peak. DNA extracted from these cells formed a characteristic "ladder" on agarose gel electrophoresis. Under electromicroscope, typical morphological changes of apoptosis were also observed. From all of these findings, we concluded that trichosanthin was able to induce apoptosis in K562 cells.

Structure-function relationship of trichosanthin.

Trichosanthin (TCS), a 27 kDa ribosome inactivating protein extracted from the root tuber of Trichosanthes plant, was subjected to limited chymotrypsin digestion and three peptide fragments designated TCS-F1 72-247, TCS-F2 100-247, and TCS-F3 133-247 were generated. The RNA-N-glycosidase and cytotoxic activities of the TCS fragments were compared with that of inact TCS. TCS-F1 and TCS-F2 were biologically active, while TCS-F3 was completely inactive. Dose-dependent studies showed that TCS-F1 and TCS-F2 were less potent in their trophoblast cytotoxicity than intact TCS, however, full biological effect could still be obtained with a higher dosage. Based on the known three-dimensional structure of TCS, we postulate that the putative active site of TCS is located at amino acid residues 110 to 174.

[4P- syndrome (Wolf-Hirschhorn syndrome) complicated with delay onset of malignant hyperthermia: a case report].

One 8-month-old female patient, weighted 5 kg, with congenital abnormality (4P- syndrome) underwent elective cheiloplasty for cleft lip and palate. Two hours later, with smooth anesthesia and operation, a life-threatening anesthetic complication of malignant hyperthermia occurred at pediatric intensive care unit. The immediate treatments were initially hyperventilating the patient with 100% O2 and cooling the patient with ice bags. Subsequently, intravenous dantrolene 2.5 mg/kg and symptomatic supportive care were administered successfully to treat the event. Upon reviewing the articles, we found that a congenital chromosome 4P deletion abnormality complicated with a delay onset of malignant hyperthermia has not been described previously.

Structure/function relationship study of Gln156, Glu160 and Glu189 in the active site of trichosanthin.

Trichosanthin is a protein used medicinally in China for abortifacient purposes. It is also an RNA N-glycosidase which inactivates eukaryotic ribosomes by removing adenine4324 from 28S rRNA. Site-directed mutagenesis was performed to probe the role of Gln156, Glu160 and Glu189 in the active site of trichosanthin. The purified altered proteins were assayed for their potency in inhibiting in vitro protein synthesis. The data indicate Glu160 is involved in the catalytic reaction. Kinetics studies suggest the carboxylate group of Glu160 serves to stabilize the transition-state complex. Similar to ricin A, the variant [E160A]trichosanthin is more potent than [E160D]trichosanthin. This is because Glu189 serves as a back-up of the carboxylate group in case Glu160 is mutated to alanine. However, removal of Glu189 in the presence of Glu160 does not affect the ID50 value drastically. An activity of 1800-fold less than that of the wild-type protein was found when both Glu160 and Glu189 were changed to alanine, indicating that some other residues in the active site are also taken part in the lowering of energy barrier for the catalytic reaction. Although Gln156 is highly conserved in related proteins, its mutation to alanine only slightly decreases the activity, showing that this residue does not participate directly in catalysis.

Variations of gossypol susceptibility in rat spermatozoa during spermatogenesis.

Sperm collected at two epididymal regions from various rat groups force-fed with gossypol were assayed for their ATP levels and motilities to examine their response to the antifertility effect of gossypol, a yellowish polyphenolic pigment from cottonseed oil. Rats receiving gossypol administration for 2 weeks began to show a proportional decrease in spermatozoal ATP content and motility and this antifertility effect deepened with time. Recovery from the gossypol-induced ATP decrease also developed at 2 weeks after the removal of gossypol administration. This rapid development of both the inhibitory and the recovery effects in a normal 53-day spermatogenic process might have stemmed from a change of susceptibility to gossypol inhibition during spermatogenesis. A model showing a more susceptible middle stage in spermatogenesis is proposed.