The modification of apoptosis correlated gene, protein and protein activity in rat hippocampus induced by benzoa pyrene sub-chronic exposure / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To observe the effects of subchronic exposure to benzo[a]pyrene (B[a]P) on the mRNA and protein expression levels of apoptosis-related genes (bax, bcl-2, caspase-3, caspase-6, and caspase-9) and the activities of Caspase-3, Caspase-6, and Caspase-9 in the hippocampal neurons of rats and to investigate the neurotoxic mechanism by which B[a]P induces the apoptosis of neurons.</p><p><b>METHODS</b>Fifty-two healthy SD rat were randomly divided into five groups according to preliminary neurobehavioral test results: blank control group, solvent control group, and 1.0, 2.5, and 6.25 mg/kg B[a]P exposure groups; the rats in exposure groups were intraperitoneally injected with B[a]P every other day for 90 days. The Morris water maze was used to test the learning and memory ability of rats; flow cytometry was used to measure the apoptosis ratio of hippocampal neurons; real-time quantitative PCR and Western blot were used to measure the mRNA and protein expression levels of apoptosis-related genes; spectrophotometry was used to measure the activities of their en-coded proteins.</p><p><b>RESULTS</b>Compared with the blank control group, solvent control group, and 1.0 mg/kg B[a]P exposure group, the 2.5 and 6.25 mg/kg B[a]P exposure groups hada significantly longer mean escape latency period (P < 0.05) and a significantly increased number of times of platform crossing (P < 0.05), and the 6.25 mg/kg B[a]P exposure group had significantly lower length and percentage of time spent in the platform quadrant (P < 0.05). The early apoptosis ratio rose as the dose of B[a]P increased (P trend < 0.05); the early apoptosis ratios of 1.0, 2.5, and 6.25 mg/kg B[a]P exposure groups were significantly higher than those of blank control group and solvent control group (P < 0.05). Compared with the blank control group, solvent control group, and 1.0 and 2.5 mg/kg B[a]P exposure groups, the 6.25 mg/kg B[a]P exposure group had significantly increased Bax expression (P < 0.05) and significantly decreased Bcl-2 expression and Bcl-2/Bax ratio (P < 0.05). The 2.5 and 6.25 mg/kg B[a]P exposure groups had significantly higher expression levels of Caspase-3 and Caspase-6 than the blank control group, solvent control group, and 1.0 mg/kg B[a]P exposure group (P < 0.05). The activities of Caspase-3, Caspase-6, and Caspase-9 were significantly higher in the 2.5 and 6.25 mg/kg B[a]P exposure groups than in the blank control group and solvent control group (P < 0.05). There was a positive correlation between the activities of Caspase-3, Caspase-6, and Caspase-9 and early apoptosis ratio of hippocampal neurons in rats (r = 0.793, P = 0.019; r = 0.886, P = 0.006; r = 0.773, P = 0.025). There were no significant differences in the mRNA expression of Bax, Bcl-2, Caspase-3, Caspase-6, and Caspase-9 among these groups (P > 0.05).</p><p><b>CONCLUSION</b>Subchronic exposure to B[a]P can induce apoptosis of hippocampal neurons; its mechanism may be related to the fact that B[a]P can induce upregulated expression of Bax, inhibit expression of Bcl-2, lead to decrease in Bcl-2/Bax ratio, induce upregulated expression of Caspase-3 and Caspase-6, and cause increase in the activities of Caspase-3, Caspase-6, and Caspase-9.</p>

Effect of sodium arsenite exposure on DNA damage of rat peripheral blood lymphocytes / 中国地方病学杂志

Objective To explore the DNA damage in peripheral blood lymphocytes of rats exposed to sodium arsenite. Methods Thirty-two Wistar rats, weighing 180 - 200 g, equal male and female, were randomly divided into 4 groups, 8 in each group. Sodium arsenite 0(control) ,0.05,0.15,0.45 mg/L were given through drinking water for 30 days. Body weight and drinking water consumption were measured every day. Blood were collected and DNA damage in peripheral blood lymphocytes was examined by single cell gel electrophoresis.Results The increase of body mass[( 121.00 ± 38.57), ( 120.62 ± 42.80), ( 125.38 ± 48.68)g]and water intake [(36.9 ± 6.2), (37.9 ± 5.8), (39.3 ± 4.2)ml/d]in 0.05,0.15,0.45 mg/L sodium arsenite groups were compared with the control group[( 119.25 ± 47.27)g, (38.4 ± 5.1 )ml/d], and the difference were not significant (F = 0.040,0.828, all P ＞ 0.05). The tail ratios[46.25%(185/400) ,57.00%(228/400),64.00%(256/400)], tail lengths [(32.89 ± 17.18), (58.74 ± 36.28), (77.55 ± 35.73 ) μm]and tail moments [(6.29 ± 3.74), ( 11.20 ± 9.64),(17.30 ± 12.60)μm]in 0.05,0.15,0.45 mg/L sodium arsenite groups were significantly higher than those of the control group[39.25%(157/400), (18.73 ± 15.83),(2.61 ± 1.05)μm, all P ＜ 0.01], and the tail ratios,tail lengths and tail moments in lymphocytes increased with increased doses of arsenic concentration. Conclusions Low doses of arsenic exposure can induce DNA damage in peripheral blood lymphocytes of rats.

Effect of multidrug resistance-associated proteins 1 and 2 gene mutations on phenotype of endemic arsenic poisoning / 中国地方病学杂志

Objective To explore the effect of gene mutations of arsenic transport proteins-muhidrug resistance-associated proteins(MRP1 and MRP2)on phenotype of endemic arsenic poisoning.Methods Two hundreds and thirty-nine rural residents in 3 villages of Shuocheng Region,Shanxi Province were interviewed and examined by simple random sampling who had been lived there for 20 yearn at least.All the objects were divided into two groups on the basis of clinical examination with"The Standard Diagnosis of Endemic Arsenic Poisoning" (WT/S 211-2001):subjectives with skin lesion as a arsenic poisoning group and without skin lesion as a control group. One hundred and ninety-three blood samples were collected from each participanL Seventy-five arsenic poisoning cases and 118 controls were detected the gene mutations in the 2,17,23 exons of M RPI and the 10,18,31 exons of MRP2 by PCR-single strand conformation polymorphism (PCR-SSCP) and compared by multivariate Logistic regression model. Results Seventy-five cases and 164 controls underwent questionnaires. Age[ (58.85±11.26) vs (45.73±11.92),OR = 3.378,P ＜ 0.05],gender[male,57.3%(43/75)vs 27.4%(45/164),OR = 3.553,P＜ 0.01 ],smoking[46.7%(35/75) vs 21.3%(35/164),OR = 3.225,P ＜ 0.01 ],drinking[ 17.3%(13/75) vs 8.5% (14/164),OR = 1.836,P ＞ 0.05],vegetable and fruit intake[5.3%(4/75) vs 9.1%(15/164),OR = 0.560,P ＞ 0.05],egg and meat intake[34.7%(26/75) vs 30.5%(50/164),OR = 1.210,P ＞ 0.05],exposure of pesticide [41.3%(31/75) vs 29.3%(48/164),OR = 1.864,P ＜ 0.05] were tested by Logistic regression model. There was no gene mutation detected in the 23 exon of MRP1 and the 18 exon of MRP2. The gene mutations frequencies of the 2 exons of MRP1 in arsenic poisoning and control groups were 8.00% (6/75) and 5.93% (7/118),respectively;they were 13.33%(10/75) and 8.47%(10/118) of the 17 exons of MRP1,respectively;they were 22.67%(17/75) and 18.64%(22/118) of the 10 exons of MRP2,respectively;they were 5.33%(4/75) and 2.54%(3/118) of the 31 exons of MRP2,respectively. There was no significant difference between two groups(x2 = 0.312,1.165,0.460, 2.794,respectively,all P ＞ 0.05). After age,gender,smoking,drinking,nutritional level and exposure of pesticide being adjusted by multivariate Logistic regression model,there was no significant difference between two groups (OR = 0.803,1.892,2.388,1.098,respectively,all P ＞ 0.05). Conclusions The gene mutations of 2,17,23 exons of MRPI and the 10,18,31 exons of MRP2 may have no effect on the phenotype of endemic arsenic poisoning.

Detection and analysis of T-lymphocyte subpopulation in population exposed to high concentrations of arsenic in drinking water / 中国地方病学杂志

Objective To study the change and the significance of T-lymphocyte immune function in peripheral blood in population living in arsenic-contaminated area. Methods Fifty-three cases of patients with arsenism symptoms were selected into experimental group, inhabitants who had no chronic arsenism symptoms into control group in the endemic area of Shuocheng District, Shuozhou City, Shanxi Province in 2006. Vein blood samples were taken and analyzed with SAP assay to measure the percentage of CD3+ ,CD4+ and CD8+ T-cells. Results It was found that the percentage of CD3+, CD4+, and CD4+/CD8+ [(41.89 ± 11.58)%, (25.60 ± 9.05)% and 1.02 ± 0.41] in the experimental group was lower than that in the control group [(68.38 ± 7.23)%, (39.17± 4.28)% ,1.69 ± 0.56, t = 13.61,18.72,14.79, all P < 0.05], while there was no statistical differences of CD8+ [(25.30 ± 6.85)%] compared to the control group[(23.54 ± 8.35)%,t = 3.07,P > 0.05]. The gender-related effect of arsenic on CD4+ and CD8+ was found by multiple linear step regression analysis(t = - 3.05, - 4.30, all P < 0.05). In case group, there were no statistical differences in CD3+, CD4+, CD8+ and CD4+/CD8+[(40.65±10.06)%, (24.48 ± 6.29)%, (24.52 ± 8.16)%,0.98 ± 0.25] between males and females [(43.07±12.96)%, (26.77±3.12)%, (26.50 ±9.32)%, 1.07 ±0.41, t = - 0.76,3.05,0.30,2.10, all P > 0.05]. Conclusions The immune function of T-lymphocytes of patients with chronic arsenism has been suppressed. It is of active significance to detect T-lymphocyte subpopulation in peripheral vein in patients with chronic arsenism aiming at estimating the function of cell immune and providing early diagnosis index.

Relationship between heat shock protein 72 and DNA genetic damage in peripheral blood lymphocytes of coke oven workers / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the relationship between heat shock protein 72 (Hsp72) and DNA genetic damage in peripheral blood lymphocytes of coke oven workers and the role of Hsp72 in protection of cells from genetic damage induced by coke oven emissions.</p><p><b>METHODS</b>Two hundred and sixty-seven coke oven workers and thirty controls without occupational PAHs exposure were investigated. Benzo[a]pyrene concentrations in the ambient air individually collected were assayed by high performance liquid chromatography (HPLC). Western Blot was used to measure Hsp72 levels and Comet assay was used to evaluate DNA damage degree. Personal information was collected by questionnaire.</p><p><b>RESULTS</b>The Hsp72 level (G+/-S(G)) and olive comet tail moment (G+/-S(G) of peripheral blood lymphocytes in high-exposure workers (1.24 +/- 0.42 and 4.49 +/- 1.24) were significantly higher than those in low-exposure workers (1.01 +/- 0.35 and 2.99 +/- 1.10, P < 0.05) and control (0.85 +/- 0.34 and 2.40 +/- 1.00, P < 0.05) respectively. The Hsp72 median level of all subjects was used as the limit to divide subjects into high Hsp72 level group and low Hsp72 level group. The rate with high Hsp72 level was 36.7%, 43.1% and 58.3% in control, low exposure and high exposure workers respectively and had a rising tendency following exposure level (P = 0.003). In high Hsp72 level group Hsp72 level in high exposure workers was significantly higher than that in control (P < 0.05), and there was a rising tendency along with the increase of exposed levels. But the olive comet tail moment had no significant difference among three exposed groups (P > 0.05). In low Hsp72 level group there no difference among three exposed groups about Hsp72 levels. The olive comet tail moment in high exposure workers was significantly higher than that in low exposure workers and control (P < 0.01) and high exposure workers in Hsp72 positive group and there was a rising tendency along with the increase of exposed levels. Hsp72 levels had strong negative correlation with the olive comet tail moment (r = -0.503, P < 0.01) in high exposure workers.</p><p><b>CONCLUSION</b>The coke oven emissions can induce hsp72 expression. Hsp72 play a role of protecting cells from DNA damage induced by coke oven emissions.</p>

Influences of excision repair cross complementation group 4 genetic variations on DNA damage in lymphocytes among coke oven workers / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the relationship between excision repair cross complementation group 4 ERCC4 gene polymorphisms and DNA damage in lymphocytes of coke oven workers and controls.</p><p><b>METHODS</b>Two hundred and forty-six coke oven workers and one hundred and twenty-seven controls were recruited in the study, and peripheral vein blood was drawn after over night fasting. Comet assay was used to evaluate DNA damage, and TaqMan-MGB probes were used to analyze ERCC4 genetic variations including the three Tagged-single nucleotide polymorphisms (Tag SNPs), referred to rs744154, rs3136079 and rs31870 which were picked out from Hapmap database. Then haplotypes were reconstructed by PHASE2.0.2 software.</p><p><b>RESULTS</b>The lymphocytes Olive TM value of coke oven workers was significantly higher than that of controls (1.26+/-1.12 vs 0.52+/-0.97, P<0.01). Among coke oven workers, no significant difference was found between the Olive TM of those with different genotypes or haplotype pairs at ERCC4 gene (P>0.05). However, in the control group, the TG genotype carriers had higher Olive TM than the TT and GG genotype carriers (0.26+/-0.96 vs 0.66+/-0.98 and 0.66+/-0.51, P<0.05), and the CTG/CTG haplotype pairs carriers had the highest Olive TM (0.69+/-1.01), and no CTG haplotype carriers had the lowest Olive TM (0.25+/-0.80), and the difference was borderline (P=0.08).</p><p><b>CONCLUSION</b>The gene polymorphism at ERCC4 gene has no effects on the DNA damage of lymphocytes in coke oven workers, but the TG genotype carriers has lower DNA damage in the control. DNA damage is influenced by the interaction of genetic and environmental factors.</p>

Effects of MRP2-GSH cotransport system on hepatic arsenic metabolism in rats / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the role of multidrug resistant protein 2 (MRP2) and glutathione (GSH) cotransport system in hepatic arsenic metabolism in rats.</p><p><b>METHODS</b>Thirty healthy Wistar rats were divided randomizedly into five groups. The first group was the control group and the rats in this group were administered with normal saline. In the second, third and fourth group the rats were administered with 4, 10 and 20 mg As(+)3/kg BW of sodium arsenite respectively every other day for two weeks. The fifth group was the benzene-soluble organics (BSO) intervention group and in this group the rats were administered with 2 mmol/kg BW BSO intraperitoneally every day three days before the end of the experiment. The other treatment was the same as in other groups. All rats were sacrificed two weeks after the treatments. Arsenic contents in bile, liver and blood were detected by atomic absorption spectroscopy (AAS), and the expression of MRP2 in the membrane of hepatocyte was determined by Western-blot analysis.</p><p><b>RESULTS</b>The level of total arsenic (including organic arsenic and inorganic arsenic) in bile, liver and blood in all three different dose groups was higher than those in the control groups (P < 0.05). Arsenic levels of bile and liver were increased with intragastric arsenic dose. Blood arsenic levels were not significantly different in three different dose groups. Expression of hepatic MRP2 was increased with intragastric arsenic concentration. A positive correlation between biliary arsenic concentration and MRP2 levels was found in liver (r = 0.986, P < 0.05). For the rats pretreated with BSO, the biliary arsenic was significantly higher than that in the control group but lower than that in the high dose group; the liver and blood arsenic was higher than that in the control group and in the high dose group. Expression of MRP2 pretreated with BSO was decreased.</p><p><b>CONCLUSION</b>Sodium arsenite can induce expression of MRP2 and the up-regulation of MRP2 may play an important role in the bile secretion of arsenite and its metabolites. The function of MRP2 for transportation of arsenic and its metabolites is associated with the intracellular GSH level. BSO inhibits the synthesis of GSH, which weakens the function of the MRP2-GSH cotransport system and makes the liver arsenic increased.</p>