Yu Kong

A common carcinogen benzo[a]pyrene causes p53 overexpression in mouse cervix via DNA damage

Benzo[a]pyrene (BaP) is cytotoxic and/or genotoxic to lung, stomach and skin tissue in the body. However, the effect of BaP on cervical tissue remains unclear. The present study detected DNA damage and the expression of the p53 gene in BaP-induced cervical tissue in female mice. Animals were intraperitoneally injected and orally gavaged with BaP at the doses of 2.5, 5, and 10mg/kg twice a week for 14 weeks. The single-cell gel electrophoresis (SCGE) assay was used to detect the DNA damage. Immunohistochemistry (IHC) and in situ hybridization (ISH) were used to detect the expression of p53 protein and p53 mRNA, respectively. The results showed that BaP induced a significant and dose-dependent increase of the number of cells with DNA damaged and the tail length as well as Comet tail moment in cervical tissue. The expression level of p53 protein and mRNA was increased. The results demonstrate that BaP may show toxic effect on the cervix by increasing DNA damage and the expression of the p53 gene.

Studies on Renaturation with Simultaneous Purification of Recombinant Human Proinsulin from E. coli with High Performance Hydrophobic Interaction Chromatography

Abstract The renaturation with simultaneous purification of recombinant human proinsulin (rh‐proinsulin) expressed in E. coli by high performance hydrophobic interaction chromatography (HPHIC) was investigated. The result indicates that the reduced/denatured rh‐proinsulin, extracted with 8.0 mol L−1 urea solution in the presence of β‐mercaptoethanol can be renatured and purified, simultaneously, in 45 min with HPHIC, resulting in the purity and mass recovery being more than 90% and 94%, respectively. The disulfide bonds of rh‐proinsulin can correctly form on the HPHIC column without the presence of reduced and oxidized glutathione (GSH, GSSG). The renaturation efficiency of rh‐proinsulin with HPHIC was tested by enzyme cleavage in order to obtain insulin. The result was also confirmed with RPLC, SDS–PAGE, and MALDI‐TOF, respectively. The renatured and purified rh‐proinsulin can directly be enzyme‐cleaved in the collected fraction containing the rh‐proinsulin. Thus, the technology for the renatured and purified rh‐proinsulin is very simple and fast.

Cardioprotective effect of total paeony glycosides against isoprenaline-induced myocardial ischemia in rats.

Paeoniae radix is a traditional Chinese medicinal herb for treating some diseases; important components are total paeony glycosides (TPGs), an approved drug by the State Food and Drug Administration (SFDA) for the therapy of rheumatoid arthritis (RA). We firstly reported myocardial benefits of TPGs previously, and the present study is to further investigate the underlying mechanisms for preventing oxidative damage in cardiomyopathy. We measured the capacity of TPGs to scavenge free radicals in vitro. Then 60 SD rats were randomly divided into five groups: (1) a normal control group, (2) an isoprenaline (ISO)-induced myocardial ischemic model group, (3) a TPG treatment group (TPGs 269.4 mg/kg delivered by intragastric administration for 3 days before ISO administration and TPGs 449 mg/kg delivered for 3 days after ISO administration), (4) a TPG therapy group (TPGs 449 mg/kg delivered for 3 days after ISO administration), and (5) a positive control group (propranolol 15 mg/kg for 3 days after ISO administration). The ISO-induced myocardial ischemic model was established by subcutaneous injection of 1mg/kg/8h ISO (2 times). The activities of myocardial enzymes, including glutamic oxaloacetic transaminase (GOT), creatine kinase (CK), lactate dehydrogenase (LDH), antioxidant enzyme superoxide dismutase (SOD) as well as the content of lipid peroxidation product malondialdehyde (MDA) were detected. We found that TPGs potently eliminated hydroxyl radicals and superoxide in vitro using ESR assays. Compared with model rats, TPG treatment, TPG therapy and the positive control treatment exhibited significantly reduced activities of GOT, LDH, and CK (p < 0.01), increased activity of SOD (p < 0.01) and lower levels of MDA (p < 0.05). More interestingly, the protective effect of TPG treatment was even better than that of propranolol. These results suggest that TPGs significantly ameliorate ISO-induced myocardial ischemia and their action might be through reducing oxidative stress in ischemic myocardium.

Assay of melamine in milk products with a pH‐mediated stacking technique in capillary electrophoresis

A pH-mediated stacking method in capillary electrophoresis as an assay for low concentrations of melamine in milk products was established. Real samples were treated with acetone and sodium acetate and injected directly after centrifugation and filtration. Several experimental factors, such as buffer pH, buffer concentration, sample matrix, injection/sweeping ratio, sweeping time/voltages, separation voltages, as well as sample pretreatment, which affected stacking and separation, were investigated and optimized. Under the selected condition, a low LOD of 0.01 μmol/L (S/N = 5) and a wide range of linearity of 0.01∼1.0 μmol/L could be easily achieved with a good reproducibility (RSDs < 5.8% for both migration time and peak area) and an acceptable recovery of 94.0∼103.2% (for milk, infant formula, yogurt, and milk products). The proposed method was suitable for routine assay of melamine in real milk samples.

Impact of AhR, CYP1A1 and GSTM1 Genetic Polymorphisms on TP53 R273G Mutations in Individuals Exposed to Polycyclic Aromatic Hydrocarbons

This study was to undertaken to investigate the impacts of AhR, CYP1A1, GSTM1 genetic polymorphisms on the R273G mutation in exon 8 of the tumor suppressor p53 gene (TP53) among polycyclic aromatic hydrocarbons (PAHs) exposed to coke-oven workers. One hundred thirteen workers exposed to PAH and 82 control workers were recruited. We genotyped for polymorphisms in the AhR, CYP1A1, GSTM1, and TP53 R273G mutation in blood by PCR methods, and determined the levels of 1-hydroxypyrene as PAH exposure marker in urine using the high pressure liquid chromatography assay. We found that the distribution of alcohol users and the urinary excretion of 1-OHP in the exposed workers were significantly higher than that of the control workers (p=0.004, p<0.001, respectively). Significant differences were observed in the p53 genotype distributions of smoking subjects (p=0.01, 95%CI: 1.23-6.01) and PAH exposure (p=0.008, 95%CI: 1.24-4.48), respectively. Further, significant differences were observed in the p53 exon 8 mutations for the genetic polymorphisms of Lys/Arg for AhR (p=0.02, 95%CI: 0.70-15.86), Val/Val for CYP1A1 (p=0.04, 95%CI: 0.98-19.09) and null for GSTM1 (p=0.02, 95%CI: 1.19-6.26), respectively. Our findings indicated that polymorphisms of PAH metabolic genes, such as AhR, CYP1A1, GSTM1 polymorphisms may interact with p53 genetic variants and may contribute to PAH related cancers.

Stacking and Analysis of Melamine in Milk Products with Acetonitrile-Salt Stacking Technique in Capillary Electrophoresis

Melamine was measured in real milk products with capillary electrophoresis (CE) based on acetonitrile-salt stacking (ASS) method. Real milk samples were deproteinized with acetonitrile at a final concentration of 60% (v/v) and then injected hydrodynamically at 50 mBar for 40.0 s. The optimized buffer contains 80.0 mmol/L pH 2.8 phosphates. Melamine could be detected within 20.0 min at +10 kV with a low limit of detection (LOD) of 0.03 μmol/L. Satisfactory reproducibility (inter- and intraday RSD% both for migration time and peak area was lower than 5.0%) and a wide linearity range of 0.05 μmol/L ~ 10.0 μmol/L were achieved. The proposed method was suitable for routine assay of MEL in real milk samples that was subjected to a simple treatment step.

Disturbance of Bcl-2, Bax, Caspase-3, Ki-67 and C-myc expression in acute and subchronic exposure to benzo(a)pyrene in cervix.

Epidemiological studies have demonstrated that cigarette smoking is an important cofactor or an independent risk factor for the development of cervical cancer. Benzo(a)pyrene (BaP) is one of the most potent tobacco smoke carcinogens in tobacco smoke. BaP induced DNA damage and over expression in p53 cervical tissue of mice as demonstrated in our previous study. Here we present the findings of exposure to BaP on the expression of Bcl-2, C-myc, Ki-67, Caspase-3 and Bax genes in mouse cervix. Acute intraperitoneal administration of BaP (12.5, 25, 50, 100mg/kg body weight) to ICR female mice induced a significant increase in Bcl-2, C-myc, Ki-67 mRNA and protein level till 72h except in Bcl-2 at 24h with 12.5, 25, 50mg/kg as well as at 48h with 12.5mg/kg body weight post treatment. A significant increase was also seen in Caspase-3 and Bax mRNA and protein level with peak level at 24h and gradual decrease till 72h, however, the expression of caspase-3 increased while that of Bax decreased with increasing dose of Bap after 24h. In sub chronic intraperitoneal and oral gavage administration of BaP (2.5, 5, 10mg/kg body weight), similar significant increase was observed for all the examined genes as compared to the control and vehicle groups, however the expression of Bax decreased in a dose dependent manner. The findings of this study will help in further understanding the molecular mechanism of BaP induced carcinogenesis of cervical cancer.

Synergistic Increase of Oxidative Stress and Tumor Markers in PAH-Exposed Workers

In this study, we investigated oxidative stress and tumor marker levels of polycyclic aromatic hydrocarbons (PAHs) in 136 coke oven workers and in 60 control subjects, and evaluated the correlation between oxidative stress and tumor marker levels. Questionnaires on basic demographic information were also administered. Significant differences in employment time and percentages of alcohol drinkers were observed between the control and exposed groups. PAH exposure was assessed using urinary 1-hydroxy-pyrene (1-OHP) levels and was found to be significantly higher in workers than in the controls. Significant differences (P<0.001) of MDA, GST, LDH, NSE, Cyfra21-1, and of SCC and TNF-a (P<0.0001 and P<0.05, P<0.001, respectively) levels were observed among controls and coke-oven workers, except for bottom coke oven workers. Associations between age and risk of increased TNF-a, smoking and increased GST activities, and drinking with increased MDA concentrations, were marginal (P=0.055, P=0.048, P=0.057, respectively). The association between smoking with MDA (P=0.004), NSE (P=0.005), SCC (P=0.004) and TNF-a (P<0.001), and drinking with TNF-a levels was significant (P=0.012). In addition, a significant positive correlation between oxidative stress and tumor markers was found in the present study. These results suggest that a synergistic increase of oxidative stress and tumor markers induced by PAHs may play a role in toxic responses for PAHs in coke oven workers.

Lymphocyte Oxidative Stress/Genotoxic Effects Are Related to Serum IgG and IgA Levels in Coke Oven Workers

We investigated oxidative stress/genotoxic effects levels, immunoglobulin levels, polycyclic aromatic hydrocarbons (PAHs) levels exposed in 126 coke oven workers and in 78 control subjects, and evaluated the association between oxidative stress/genotoxic effects levels and immunoglobulin levels. Significant differences were observed in biomarkers, including 1-hydroxypyrene levels, employment time, percentages of alcohol drinkers, MDA, 8-OHdG levels, CTL levels and CTM, MN, CA frequency, and IgG, IgA levels between the control and exposed groups. Slightly higher 1-OHP levels in smoking users were observed. For the dose-response relationship of IgG, IgA, IgM, and IgE by 1-OHP, each one percentage increase in urinary 1-OHP generates a 0.109%, 0.472%, 0.051%, and 0.067% decrease in control group and generates a 0.312%, 0.538%, 0.062%, and 0.071% decrease in exposed group, respectively. Except for age, alcohol and smoking status, IgM, and IgE, a significant correlation in urinary 1-OHP and other biomarkers in the total population was observed. Additionally, a significant negative correlation in genotoxic/oxidative damage biomarkers of MDA, 8-OH-dG, CTL levels, and immunoglobins of IgG and IgA levels, especially in coke oven workers, was found. These data suggest that oxidative stress/DNA damage induced by PAHs may play a role in toxic responses for PAHs in immunological functions.

Studies on the refolding of the reduced-denatured insulin with size exclusion chromatography

The refolding of the reduced-denatured insulin from bovine pancreas was investigated with the size exclusion chromatography (SEC). It was shown that the reduced-denatured insulin originally denatured with 7.0 mol L−1 guanidine hydrochloride (GuHCI) or 8.0 mol L−1 urea could not be refolded with a non-oxidized mobile phase. Although the oxidized and reduced glutathione (GSSG and GSH) were employed in the oxidized mobile phase, the reduced-denatured insulin still could not be renatured. However, in the presence of 2.0 mol Lt-1 urea in the oxidized mobile phase employed, the reduced-denatured insulin can be refolded with SEC, and the aggregation of denatured insulin can be diminished by urea. In addition, the disulfide exchange of reduced-denatured insulin also can be accelerated with GSSG/GSH in the oxidized mobile phase. The three disulfide bridges of insulin were formed correctly and the reduced-unfolded insulin can be renatured completely. The results were further tested with reversed-phase liquid chromatography (RPLC) and hydrophobic interaction chromatography (HIC).