Increased damage of exon 5 of p53 gene in workers from an arsenic plant.

Mutagenesis is a multistage process. Substitution mutations can be induced by base modified through alteration of pairing property. Mutations of exon 5 and 8 of p53 gene have been found in most arsenicosis patients with precarcinomas and carcinomas, but never in arsenicosis individuals without precarcinomas and carcinomas. This study investigates whether base modification exists in exon 5 and 8 of p53 gene, and explores the dose-effect relationship between damage of exon 5 of p53 gene and urinary arsenic. Concentrations of urinary 8-hydroxydeoxyguanine (8-OHdG) are analyzed to identify the occurrence of DNA damage. The real-time PCR developed by Sikorsky et al. is applied to detect base modification in exon 5 and 8 of p53 gene for apparently healthy participants. Our results show that the mean total arsenic concentrations of two exposed groups from an arsenic plant are significantly elevated compared with the control group, and the damage level of exon 5 of the high-exposed group is significantly higher than that of the control group, but which does not happen in exon 8. The closely correlation between the damage index of exon 5 and urinary organic arsenic concentration are found. Concentration of 8-OHdG of the high-exposed group is significantly higher than that of the control group. These results imply that base modification in exon 5 of p53 gene can be induced by arsenic. In addition, our study suggests that the damage level of exon 5 is a useful biomarker to assess adverse health effect levels caused by chronic exposure to arsenic.

[Study on the association between urinary organic arsenic and 8-hydroxydeoxyguanine in workers exposed to arsenic].

OBJECTIVE: To evaluate the association between metabolism of arsenic and DNA oxidative damage in workers in a arsenic mill. METHODS: Urinary organic arsenic and 8-hydroxydeoxyguanine were detected in 37 workers highly exposed to arsenic and 16 administrative and logistic staff with mild exposure in a arsenic mill in Yunnan province, and also 28 local people who did not have the exposure in the near past time. The correlation between metabolism of arsenic and DNA oxidative damage was evaluated. RESULTS: The urinary organic arsenic concentration was respectively (0.48 +/- 0.37) mg/L and (0.08 +/- 0.05) mg/L for men with high and low exposure, and was respectively 0.11 mg/L and (0.30 +/- 0.24) mg/ L for women with high and low exposure, while it was lower than 0.02 mg/L in the controls. Urinary 8-hydroxydeoxyguanine concentration was (18.07 +/- 11.68) micromol/mol creatinine, (11.79 +/- 8.25) micromol/mol creatinine, (10.07 +/- 3.04) micromol/mol creatinine for the males with high and low exposure and of controls, respectively, (P < 0.05), and it was 84.35 micromol/mol creatinine, (21.27 +/- 5.89) micromol/mol creatinine, (14.43 +/- 2.58) micromol/mol creatinine for females with high and low exposure and of controls, respectively. The female workers exposed to arsenic had higher urinary 8-hydroxydeoxyguanine levels than males did (P < 0.05). The increased tendencies of urinary 8-hydroxydeoxyguanine levels with the organic arsenic concentration were found in workers (r(s) = 0.279, P = 0.019). CONCLUSION: Occupational individuals exposed to arsenic have obvious DNA oxidative damage, which is more severe in females. The difference of metabolism of arsenic may play a key role.

[Changes of the certain genotoxicities in workers occupationally exposed to arsenic].

OBJECTIVE: To study the changes of genotoxicities of workers occupationally exposed to arsenic in a mill of Yunnan Province. METHODS: The micronucleated cell and micronucleus frequency, comet rate and tail length of lymphocyte in comet assay from peripheral blood, urinary total and organic arsenic were detected and evaluated in 40 workers (exposure group), and also local people who did not contact with any poison obviously (control group). RESULTS: The micronucleated cell and micronucleus frequencies, the comet rate and tail length of lymphocyte in exposure groups were all more higher than those of controls (P < 0.01). The urinary total and organic arsenic concentrations in exposure groups were more higher than those of controls (all lower than 0.02mg/L for controls). The increased tendencies of micronucleated cells and micronucleus frequencies with the products of organic arsenic concentrations and service length were found in exposure groups(r(s) = 0.356, P = 0.024, r(s) = 0.347, P = 0.028). CONCLUSION indicated that arsenic could induce the damage of chromosome and DNA in lymphocyte of peripheral blood in workers occupationally exposed to arsenic.

[The mechanisms and effects of lutein on inducing the cell differentiation of human esophagus cancer EC9706].

OBJECTIVE: The purpose of the study was to explore the effects and molecular mechanisms of lutein on the differentiation of esophagus cancer EC9706 cell. METHODS: EC9706 cells were seeded in 1640 medium before the addition of test compounds. The respective test compound was added in fresh medium and the control cell received the vehicle (DMSO) or Fluorouracil. The proliferation and cell cycle of EC9706 were determined by MTT assay and flow cytometry, respectively. The change in cytomorphology was investigated by using HE staining. Proliferation and differentiation cells were checked and observed by methyl green-pyronine staining. The protein expression of cyclin D1 was detected by immunohistochemistry. RESULTS: Compared with the DMSO control group, the proliferation of the EC9706 cells treated with lutein (100 microg/mL and 150 microg/mL) could markedly be decreased and the cell cycle was blocked at G0/G1 phage which caused significant changes in the cytomorphology of EC9706 cell line, and the cell malignant degree tended to drop down, the protein expression of cyclin D1 was also down-regulated significantly. CONCLUSION: Lutein can inhibit the proliferation of EC9706 cell, and promote the cancer cell differentiation. cyclin D1 may be involved in cell proliferation and differentiation events in esophageal cancer EC9706 cell, which is regulated by lutein.

[Real-time PCR used to detect p53 gene damage in workers exposed to arsenic].

OBJECTIVE: To evaluate the relationship between the metabolism of arsenic and the damage of exon 5 and 8 of p53 gene from workers in a arsenic mill, and with real-time PCR technique, to establish the method probing the gene-specific DNA damage in people. METHODS: By real-time PCR, the damages of exon 5 and 8 of p53 gene were probed in 37 workers exposed highly to, 16 manager and logistic employees exposed less to an arsenic mill in Yunnan province, and also 25 local people who did not contact with any white arsenic in near past time. At the same time, the urinary total and organic arsenic of workers were detected. The correlation between metabolism of arsenic and damage of p53 gene was evaluated. RESULTS: Total urinary arsenic concentrations were (1.18 +/- 0.76) mg/L and (0.32 +/- 0.28) mg/L for high and low exposed male workers, and 0.23 mg/L, (0.53 +/- 0. 30) mg/L for high and low exposed females. Organic urinary arsenic concentrations were (0.48 +/- 0.37) mg/L and (0.08 +/- 0.05) mg/L for high and low exposed males, and 0.11 mg/L, (0.30 +/- 0.24) mg/L for high and low exposed females. The total and organic urinary arsenic of high exposed group was higher than that of control male (P < 0.05), all in control group were lower than 0.02 mg/L for reference. The Ct relative value of exon 5 of p53 gene in high exposed group was higher than that in control male (P < 0.05), and the increased tendency of Ct relative value of exon 5 of p53 gene was found in workers with organic arsenic concentration going up (r(s) = 0.355, P = 0.011). The Ct relative value of exon 8 of p53 gene in low exposed group was higher than that in control male (P < 0.05), but the difference between high exposed and low exposed or reference's was not obvious (P > 0.05). CONCLUSION: The damages in exon 5 and 8 of p53 gene in workers exposed to arsenic may be induced. The metabolism of arsenic may be very important in the damage of exon 5. It is feasible for real-time PCR technique used to detect gene-specific DNA damage in people.

[Effects and mechanism of lutein on apoptosis of esophageal carcinoma EC9706 cells].

OBJECTIVE: To study the effects of lutein on apoptosis and its mechanism. METHOD: The cells of human esophageal carcinoma EC9706 were grown in RPMI medium containing 10% bovine serum and were treated with lutein at 100 microg x mL(-1) concentration. Flow cytometry was employed to investigate the effects of lutein on cell apoptosis of EC9706 cells. Histochemistry was performed to determine apoptosis-related protein expresion. RESULT: Flow cytometry analyses revealed that lutein increased EC9706 cell apoptosis ratio when treated with lutein 100 microg x mL(-1) at 96 h. Lutein decreased the expression of Bcl-2 protein and increased the expression of Bax protein in EC9706 cells. CONCLUSION: Lutein could inhibit mitosis and stimulate apoptosis of EC9706 cells. The apoptotic effect may result from the down-regulation of expression of Bcl-2 and up-regulation expression of Bax.

[Study on rat's DNA damage of p53 induced by chlorinated acetic acids of drinking water disinfection by-products].

OBJECTIVE: To study on the DNA damage of p53 induced by dichloroacetic acid(DCA) and trichloroacetic acid( TCA), approve their genotoxicity and discuss molecular mechanism of their carcinogenic action. METHODS: Administered SD rats with DCA or TCA by i.p. injection, extracted DNA from rat's liver, and then used RDPCR to detect DNA damage of exon 70f p53 gene. RESULTS: Two hybridization bands were detected in treated group induced by DCA. It was indicated that DCA can result in DNA damage of exon 7 of p53 gene of rat's liver tissue, and there were two broken sites. It was not detected damage of exon 7 of p53 gene of rat induced by TCA. CONCLUSION: There may be the relationship between DCA carcinogenic action and the damage of p53. The result that the damage of p53 of target tissue detected by RDPCR was consistent well with rat carcinogenic test.

[Study of rat's p53 gene damage and organ specificity induced by benzidine].

OBJECTIVE: Studying the main target organs and the genetic toxicity mechanism of benzidine. METHODS: SD rats were given benzidine i.p. injection. DNA was extracted from rat's liver, kidney, lung and bladders. Then ss probes RDPCR was used to detect the damaged DNA of exon 7 of p53 gene. RESULTS: Hybridization bands were found in liver, bladder and lung tissues after ss probes RDPCR, while no hybridization bands were found in kidney tissues. CONCLUSION: The result indicates that benzidine can cause the DNA lesions of exon 7 of p53 gene and its major target organs are liver, bladder and lung. The toxicity mechanism of benzidine is probably related to p53 gene damage.

[Blood leptin, orexins and NPY levels and their relations in obese children].

OBJECTIVE: To investigate the concentration levels of leptin, orexins and neuropeptide Y (NPY) in the blood of obese children, and to analysed the relationship between these substances. METHODS: RIA methods were used to measure the concentrations of leptin, orexinA, orexinB, and NPY in the blood of 98 obese children [BMI: male (29.24 +/- 1.87) kg/mZ, female (28.12 +/- 2.30) kg/m2] and in 104 normal children [BMI: male (20.49 +/- 1.95) kg/m2, female (19.59 +/- 1.51) kg/m2] as the control group. RESULTS: The leptin concentrations in obese children [male (26.00 +/- 14.66) ng/mL; female (33.59 +/- 14.63) ng/mL] were higher than those in the control group [male (6.65 +/- 44.49) ng/mL; female [10.48 +/- 5.52) ng/mL P < 0.013]. The concentrations of plasma orexinA in obes children [male (3.23 +/- 1.86) pg/mL; female (3.38 +/- 1.80) pg/mL] were lower than those in the control group [male (4.52 +/- 1.52) pg/mL; female (4.71 +/- 1.53) pg/mL P < 0.05]; Negative correlations between leptin and NPY were noted in the obesity group (r = -0.302) and the control group (r = -0.310, P < 0.01), while the slopes in the two groups were different (control group -2.969; obese group -0.809). A positive correlation between NPY and orexinA was noted (r = 0.207, P < 0.05). The fluctuation range of orexinA in obese children was markedly narrowed when compared with that in the control group. CONCLUSION: The concentration level of peripheral orexinA and leptin in the obese and non-obese children change inversely. The obesity in children correlates with the concentrations of orexinA, leptin, NPY as well as with their interactions.

Mutation analysis of HOXD13 gene in a Chinese pedigree with synpolydactyly.

OBJECTIVE: To study the clinical features and to identify homeobox D13 (HOXD13) gene mutation of the affected individuals in a Chinese synpolydactyly (SPD) kindred. METHODS: Clinical data and peripheral blood samples of SPD family members were obtained through field investigation. For every member of this pedigreeìthe fragment containing mutational hot spots of HOXD13 was amplified by PCR for mutation screening. To examine whether there is any other mutation within coding sequence of HOXD13, exon 1 and exon 2 of HOXD13 were also amplified by PCR. All the amplified fragments were electrophoresed on 2% agarose gels and then the mutant fragments were electrophoresed on 5% polyacrylamide gels to be separated. Purified PCR products of normal and selected mutant alleles were directly sequenced. RESULTS: Comparing the HOXD13 coding sequence of the affected individuals with HOXD13 sequence in the GenBank and with that of the unaffected, an inserted segment coding 8 alanine residues within HOXD13 was found segregating with the disorder. This mutation is also termed polyalanine expansion. The 8-alanine expansion can be interpreted as a reduplication of normal alanines 5-12. CONCLUSION: The results suggest that synpolydactyly in this kindred may be caused by polyalanine expansion in HOXD13.

[Detecting the DNA damage of N-ras gene induced by potassium dichromate].

OBJECTIVE: Applying RDPCR to detect the DNA damage of N-ras gene induced by Potassium dichromate METHODS: Preparing single-stranded probes of exon 1 of N-ras gene in human. The genomic DNA was treated with Potassium dichromate, then amplified by RDPCR and detected by Southern hybridization with the probe. RESULTS: The clear hybridized bands can be seen in the position which is induced by Potassium dichromate on the dose of 100 micromol/L, but can't be detected on the dose over 1000 mol/L. CONCLUSION: It indicates that Potassium dichromate can cause the DNA damage of N-ras gene, which should be the key point of its carcinogenesis mechanisms.

[Application with single-stranded probes in randomized terminal linker-dependent PCR].

OBJECTIVE: Preparing single-stranded (ss) probes in place of double-stranded (ds) probes to improve the hybridization efficiency and specificity of randomized terminal linker-dependent PCR (RDPCR). METHODS: Using asymmetric PCR and single-primer PCR to prepare ss probes of extron 7 of p53 gene in rat, then comparing the results hybridized with ss probes and ds probes. RESULTS: Preparation of ss probes by asymmetric PCR and single-primer PCR gets success. Hybridization results showed that the ss probes could get better signals and less noise than ds probes. CONCLUSION: In comparison with ds probes, the application of ss probes can increase the hybridization sensitivity and specificity of RDPCR.

[Establishment and characterization of the lung cancer A549 cell stains with down-regulated expressed HOGG1 genes].

OBJECTIVE: To establish lung cancer A549 cell strains with down-regulated expressed HOGG1 genes and study its biological characteristics. METHODS: The eukaryotic expression vectors with genes of hammerhead ribozyme targeting human 8-oxoguanine DNA glycosylase 1 (HOGG1) mRNA were transfected into A549 cells by liposmes, and then were screened by G418 till stable cell strains were constructed. The positive recombinants were identified by RT-PCR to amplify NEO genes. The efficacy of inhibition was tested by RT-PCR. Furthermore, morphology, survival curves, cell cycle, cloning efficiency grow in the soft agar and the activity of superoxide dismutase (SOD) were investigated. RESULTS: The HOGG1-down-regulated expressed A549 cell strains were obtained after G418 selection and NEO genes identification. The mRNA expression level of HOGG1 genes in the down-regulated expressed cells named A549-R were decreased 61.5% than in the untransfected cells A549 (P < 0.05). There was no significant difference between A549-R cells and A549 cells in morphology, the population doubling time and cell cycle. The numbers of clones in the soft agar of A549-R cells was decreased nearly to 50% than of A549 cells (P < 0.05), but the activity of SOD was increased obviously (P < 0.05). CONCLUSION: The lung cancer A549 cell strains with down-regulated expressed HOGG1 genes by silencing and incision of ribozyme were successfully established. Its biological characteristics have no significant changes in most of indexes.

[Establishing a method to detect the DNA damage of N-ras gene].

OBJECTIVE: Applying RDPCR to detect the DNA damage of N-ras gene in human. METHODS: Single-primer PCR was used to prepare single strand (ss) probes of extron 1 of N-ras gene in human. The genomic DNA was digested completely by restriction endonuclease, then amplified by RDPCR and detected by Southern hybridization with the probe. RESULTS: The ss probes were successfully prepared by single-primer PCR. The hybridized bands were clearly seen in the expected migration positions. CONCLUSION: The result shows that the method to detect the damaged position of N-ras gene has been established, which would be helpful to further studies on chemical carcinogenesis and on the prevention of tumor.

Randomized terminal linker-dependent PCR: a versatile and sensitive method for detection of DNA damage.

OBJECTIVE: To design and develop a novel, sensitive and versatile method for in vivo foot printing and studies of DNA damage, such as DNA adducts and strand breaks. METHODS: Starting with mammalian genomic DNA, single-stranded products were made by repeated primer extension, these products were ligated to a double-stranded linker having a randomized 3' overhang, and used for PCR. DNA breaks in p53 gene produced by restriction endonuclease AfaI were detected by using this new method followed by Southern hybridization with DIG-labeled probe. RESULTS: This randomized terminal linker-dependent PCR (RDPCR) method could generate band signals many-fold stronger than conventional ligation-mediated PCR (LMPCR), and it was more rapid, convenient and accurate than the terminal transferase-dependent PCR (TDPCR). CONCLUSION: DNA strand breakage can be detected sensitively in the gene level by RDPCR. Any lesion that blocks primer extension should be detectable.

[Epidemiological analysis of syndactyly in Chinese perinatals].

OBJECTIVE: To study the epidemiological features of syndactyly (SD) in Chinese perinatals. METHODS: Data were collected through Chinese Birth Defects Monitoring Network, a hospital-based congenital malformation registry system. From 1987 through 2001 (except 1994, 1995), all live or still births with 28 weeks of gestation or more born in participating hospitals were assessed within 7 days after delivery. RESULTS: Totally 2311 perinatals with SD were identified among 7 478 746 births, and 57.2% of them were in association with other anomalies. The overall prevalence rate of SD was 3.09/10 000, the rate of isolated SD and associated SD was 1.32/10 000, and 1.77/10 000 respectively. The prevalence rates in urban and rural areas, in male and female births were 3.22/10 000 and 2.79/10 000, 3.42/10 000 and 2.59/10 000 respectively. An increasing trend was found during that period. The perinatal fatality rate of SD was 20.7%, that of isolated form was 5.7%, while that of associated form was 31.9%. The proportion of SD occurring in right side was the same as that in left side, and the proportion of SD in upper limbs equaled to that in lower limbs. CONCLUSIONS: The prevalence rate of SD in Chinese perinatals was similar to that reported in foreign literatures. Associated form of SD was more frequently seen. The prevalence of SD in urban areas was higher than in rural areas. Male excess was identified in both isolated and associated forms of SD. No selective predominance was observed either by affected side or by affected limbs.

[Improvement of the silver-stain and it's test and verification in comet assay].

In order to find out a better silver-stain method so that to replace EB-stain in comet assay, the selected silver-stain methods were tested via determining fixing, coloring, displaying, fading, and testing the pigmentation temperature. When it was optimized, it was used in testing the DNA damage by two types of compounds, namely potassium dichromate (K2Cr2O7) and 1,2-dichloroethane (DCE) to two kind of cells, namely spleen cells of mouse and human outer lymphocyte, and, the sensitivity was compared with that of EB-stain. The results showed that the modified silver-stain was the first successful non-fluorescent dye method in comet assay that can dye clearly, permanently, with a light background and good contrast, and it was easy to manipulate and it was cheap. The stain can detect the DNA damage by compounds to cells and has the same positive threshold like that in EB-stain. It can even dye the small fragments that EB-stain cannot. It is suggested that in comet assay, the silver-stain can substitute the EB-stain completely.

[Applying randomized terminal linker-dependent PCR to detect the DNA damage of p53 gene in rat].

In order to use the technique of Randomized Terminal Linker-dependent PCR(RDPCR) to detect DNA lesions of specific gene in vivo, rats were administered with potassium dichromate by i.p. injection and genomic DNA of lung were extracted. Single-stranded products were made by repeated primer extension, these products were ligated to a linker and thus amplified by primer P2 and PL. The final PCR products were detected by electrophoresis and Southern hybridization with DIG-labeled probe. The results showed that two hybridization bands for extron 7 of p53 gene were found with dosage of 20.0 mg/kg and 40.0 mg/kg indicating the DNA lesions of extron 7 of p53 gene, by potassium dichromate and there were two DNA lesion cites. The findings from this study provide evidences both for further investigation of mutational mechanism of hexavalent chromium and expansion on using RDPCR in toxicology.

[Influence of placenta on fetal hypoxia in intrahepatic cholestasis of pregnancy].

OBJECTIVE: To determine the influence of placenta on fetal hypoxia in intrahepatic cholestasis of pregnancy (ICP). METHODS: The transfer of oxygen across the placental membranes in ICP group (n = 7) was compared with that of controls (n = 8) by dual perfusion of the human placental lobule in vitro. RESULTS: The oxygen consumption and the volume loss of perfusate from the fetal circuit (< 5 ml/h) of placental lobule from women with ICP are similar to controls. The rate of oxygen transfer across the placental membranes in ICP was similar to controls, too (P < 0.05, Power > 0.08). These findings suggest that the transfer of oxygen across the placental membrane in ICP is normal value. CONCLUSION: The placenta in ICP has not direct impact on the fetal oxygenation just leads to insufficiency of placental oxygen reserve resulting from a reduction in the size of the intervilous space.