Application progress of air-liquid interface exposure technology in respiratory system toxicity induced by diesel exhaust in vitro / 环境与职业医学

Diesel exhaust (DE) is an important pollution source widely existing in the living and production environment, which is closely related to the health of the public and occupational groups. The International Agency for Research on Cancer has classified DE as a Group 1 carcinogen. Considering the negative health impacts on the respiratory system due to DE exposure in vitro, it is crucial to apply reliable test systems allowing accurate assessment of the biological effects of DE. The exposure technology of respiratory system in vitro is considered as one of the feasible measures to implement the 3R (reduce, refine, and replace) principle in animal experiments. Compared with the traditional submerged culture in vitro models, the air-liquid interface (ALI) exposure technology has the advantages including fewer influencing factors, easier exposure condition control, and shorter exposure cycle. ALI has become an important tool to study molecular events associated with physiology and pathology of respiratory system, and action modes and interactions of different cell types. Also, ALI has been increasingly widely used because it can simulate the actual processes of human respiratory system cells and/or tissues to DE exposure. This review was intended to introduce the development and advantages of ALI exposure technology, and further summarized the application progress of ALI exposure technology in studying the respiratory toxicity induced by DE exposure in vitro, so as to provide new ideas and pathways for the use of ALI exposure technology in the study of biomarkers and mechanisms of respiratory toxicity associated with DE exposure, and provide basic data to screen and promote biomarkers for exposed populations.

Assessment of Benchmark Dose in BEAS-2B Cells by Evaluating the Cell Relative Viability with Particulates in Motorcycle Exhaust / 生物医学与环境科学（英文）

Objective@#This study aimed to use an air-liquid interface (ALI) exposure system to simulate the inhalation exposure of motorcycle exhaust particulates (MEPs) and then investigate the benchmark dose (BMD) of MEPs by evaluating cell relative viability (CRV) in lung epithelial BEAS-2B cells.@*Methods@#The MEPs dose was characterized by measuring the number concentration (NC), surface area concentration (SAC), and mass concentration (MC). BEAS-2B cells were exposed to MEPs at different concentrations @*Results@#Our results reveal that BMD of NC and SAC were estimated by the best-fitting Hill model, while MC was estimated by Polynomial model. The BMDL for CRV following ALI exposure to MEPs were as follows: 364.2#/cm @*Conclusion@#These results indicate that MEPs exposure

Association of etheno-DNA adduct and DNA methylation level among workers exposed to diesel engine exhaust / 中华预防医学杂志

Objective@#To investigate the association between etheno-DNA adduct and the promoter of DNA methylation levels of cyclin dependent kinase inhibitor 2A (P16), Ras association domain family 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in workers with occupational exposure to diesel engine exhaust (DEE).@*Methods@#We recruited 124 diesel engine testing workers as DEE exposure group and 112 water pump operator in the same area as control group in Henan province in 2012 using cluster sampling. The demographic data were obtained by questionnaire survey; urine after work and venous blood samples were collected from each subject. The urinary etheno-DNA adducts were detected using UPLC-MS/MS, including 1,N6-etheno-2'-deoxyadenosine (εdA) and 3,N4-etheno-2'-deoxycytidine(εdC). The DNA methylation levels of P16, RASSF1A, and MGMT were evaluated using bisulfite-pyrosequencing assay. The percentage of methylation was expressed as the 5-methylcytosine (5mC) over the sum of cytosines (%5mC). Spearman correlation and multiple linear regression were applied to analyze the association between etheno-DNA adducts and DNA methylation of P16, RASSF1A, and MGMT.@*Results@#The median (P25-P75) of urinary εdA level was 230.00 (98.04-470.91) pmol/g creatinine in DEE exposure group, and 102.10 (49.95-194.48) creatinine in control group. The level of εdA was higher in DEE exposure group than control group (P<0.001). DNA methylation levels of P16, RASSF1A and MGMT were 2.04±0.41, 2.19 (1.94-2.51), 2.22 (1.94-2.46)%5mC in exposure group, and 2.19±0.40, 2.41 (2.11-2.67), 2.44 (2.15-2.91)%5mC in control group. DNA methylation levels were lower in exposure group (P values were 0.005, 0.002 and 0.001, respectively). Spearman correlation analysis showed that DNA methylation levels of P16, RASSF1A, and MGMT were negative associated with urinary εdA level (r values were -0.155, -0.137, and -0.198, respectively, P<0.05). No significant correlation was observed between the εdC level and any measured DNA methylation levels (P>0.05) . Multiple linear regression confirmed the negative correlation between εdA and DNA methylation levels of P16, RASSF1A, and MGMT in non-smoking group (β (95%CI) was -0.068 (-0.132--0.003), -0.082 (-0.159--0.004) and -0.048 (-0.090--0.007), P values were 0.039, 0.039 and 0.024, respectively). Moreover, εdC was negative associated with DNA methylation level of MGMT in non-smoking group (β (95%CI) was -0.094 (-0.179--0.008), P=0.032).@*Conclusion@#DEE exposure could induce the increased of εdA and decreased of DNA methylation levels of P16, RASSF1A and MGMT.

Evaluation of the viability of BEAS-2B cells exposed to gasoline engine exhaust with different particle sizes by air-liquid interface / 中华劳动卫生职业病杂志

Objective@#To evaluate the viability of gasoline engine exhaust (GEE) with different particle sizes on human lung cell line BEAS-2B in vitro by air-liquid interface (ALI) .@*Methods@#GEE were collected with a Tedlar bag and their particulate matter (PM) number, surface and mass concentration in three kind of GEE (filtered automobile exhaust, non-filtered automobile exhaust and motorcycle exhaust without three-way catalytic converter) were measured by two type of particle size spectrometer including TSI-3321 and SMPS-3938. Five groups were included, which divided into blank control group, clean air group, filtered automobile exhaust group, non-filtered automobile exhaust group and motorcycle exhaust without three-way catalytic converter group. Except the blank control group, BEAS-2B cells, cultured on the surface of Transwells, were treated with clean air or GEE by ALI method at a flow rate of 25 ml/min, 37 ℃ for 60 min in vitro. CCK-8 cytotoxicity test kit was used to determine the cell relative viability of BEAS-2B cells.@*Results@#In the filtered automobile exhaust, non-filtered automobile exhaust and motorcycle exhaust without three-way catalytic converter, high concentrations of fine particles can be detected, but the coarse particles only accounted for a small proportion, and the sequence of PM concentration was motorcycle exhaust without three-way catalytic converter group> non-filtered automobile exhaust group> filtered automobile exhaust group (P<0.001) . Compared with the clean air group, the cell relative viability in the 3 GEE-exposed groups were significantly lower (P<0.001) . Among the comparisons of GEE exposure groups with different particle size spectra, the sequence of the cell relative viability was filtered automobile exhaust group >non-filtered automobile exhaust group> motorcycle exhaust without three-way catalytic converter group (P<0.001) . When took the clean air control group as a reference, the mean of the cell relative viability in the filtered automobile exhaust group, non-filtered automobile exhaust group and motorcycle exhaust without three-way catalytic converter group, was decreased by 26.34%, 36.00% and 49.59%, respectively.@*Conclusion@#GEE with different particle size spectra could induce different levels of toxic effects to the human lung cells BEAS-2B by ALI. After lowering the concentration of particles in the GEE and using the three-way catalytic converter could obviously improve the survival rate of lung cells.

Method validation of phosphorylated histone H2AX level detection using primary cultured hepatocytes in genotoxic agent screening / 中国药理学与毒理学杂志

OBJECTIVE To establish an in vitro test method and to evaluate the genotoxicity of chemicals using primary cultured mouse hepatocytes and the changes in phosphorylated histone H2AX(γH2AX)expression levels to provide a more reliable marker of the identification of genotoxicity. METHODS Hepatocytes were isolated from BALB/c mice by an improved two-step collagenase diges?tion method and then cultured in sandwich configuration. The primary cultured hepatocytes were treat?ed with various concentrations of four known genotoxic agents bleomycin(BLM),benzo(a)pyrene〔B (a)p〕,styrene and styrene-7,8-oxide(SO)within the range of 40 μmol · L-1 and two non-genotoxic agents azathioprine(Aza)and ciclosporin A(CsA)at different time points within 24 h. The cytotoxicity induced by these toxicants was assessed by CCK-8 assay. Then,the changes in γH2AX expression levels in treated cells were determined by flow cytometry. RESULTS The four genotoxic agents could be detected and two non-genotoxic agents could not be detected by this method. The γH2AX expression level was the highest when hepatocytes were exposed to BLM and SO for 3 h,or B(a)p and styrene for 6 h(P<0.01). The production of γH2AX was 25.67,18.36,12.43 and 14.25 for the four types of genotoxic agents,respectively,and was approximately 19,13,9 and 11 times that of the vehicle control group(P<0.01)at the optimum time point and concentration. There was a significant positive corre?lation between the indicated concentrations of genotoxic chemicals and γH2AX expression levels(P<0.01). In addition,the production ofγH2AX indicated no marked increase in two non-genotoxic agents such as Aza and CsA in comparison with the control group. CONCLUSION This test method can effec?tively distinguish genotoxic agents from non-genotoxic agents,and direct genotoxic agents from indirect genotoxic agents in the absence of S9. γH2AX might be a reliable marker for the identification of the potential genotoxicity of chemicals.

Myelin protein zero and its antibody in serum as biomarkers of n-hexane-induced peripheral neuropathy and neurotoxicity effects / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Chronic exposure to n-hexane can lead to peripheral neuropathy that no effective treatment regimen could be applied presently. This study investigated whether myelin protein zero (P0) protein and its antibody could be used to distinguish n-hexane intoxication and protect workers from peripheral neuropathy.</p><p><b>METHODS</b>We compared P0 protein and its antibody among three levels of n-hexane-exposed groups, which included 18 patients with n-hexane-induced peripheral neuropathy as case group, 120 n-hexane-exposed workers as n-hexaneexposed control group, and 147 non-hexane-exposed participants used as control group. ELISA method was applied to detect P0 protein and its antibody.</p><p><b>RESULTS</b>P0 protein in serum was significantly higher in the case group and n-hexane-exposed control group in comparison with the control group (P < 0.01). Compared with the n-hexane-exposed control group, the case group also had significant increase of P0 protein (P < 0.01). After 6 months therapy, P0 protein was observed to decrease significantly in the case group (P < 0.01). The P0 antibody in serum was significantly higher in the n-hexane-exposed control group than in the control group (P < 0.01), but not significantly different between cases and controls.</p><p><b>CONCLUSIONS</b>P0 antibodies in serum may be a short-term effect biomarker for n-hexane exposure. P0 protein in serum may be an early effective biomarker for peripheral nerve neuropathy and its biological limit value needs investigation in the future study.</p>

Association between cytokines and trichloroethylene-induced hypersensitivity dermatitis / 中华预防医学杂志

<p><b>OBJECTIVE</b>To detect the cytokines levels in serums of patients with trichloroethylene-induced hypersensitivity dermatitis and explore the effect biomarkers associated with this disease.</p><p><b>METHODS</b>Twenty-two patients with TCE-induced hypersensitivity dermatitis, twenty-two healthy TCE-exposed workers from the same workshops with patients and twenty-two comparable unexposed controls were recruited in this study. Eight cytokines in serums from all subjects were detected using Liquid Suspended Biochip; the correlation among the eight cytokines including interleukin (IL)-1β (IL-1β), IL-5, IL-8, IL-10, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), macrophage chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1β (MIP-1β) and the correlation between IL-5 and eosinophil count were analyzed.</p><p><b>RESULTS</b>The medians of levels of IL-1β, IFN-γ, IL-5, IL-10, MCP-1, MIP-1β, IL-8 among patients were 0.15, 80.13, 2.95, 6.45, 83.83, 1057.90, 440.22 pg/ml, respectively, which were higher than those among the TCE-exposed workers (0.09, 16.93, 0.11, 0.07, 28.75, 241.07, 28.26 pg/ml, respectively, all P values < 0.01) and unexposed controls (0.09, 3.14, 0.11, 0.07, 25.27, 209.64, 207.34 pg/ml, respectively, all P values < 0.01). The median of level of TNF-α among the patients was 13.26 pg/ml, which was significantly higher than that among TCE-exposed workers (4.87 pg/ml, P < 0.01) but not among unexposed controls; the median of level of IL-5 among the TCE-exposed workers was 0.11 pg/ml, which was significantly higher than that among the unexposed controls (0.11 pg/ml, P < 0.01). The median of levels of IL-8 among the unexposed controls was 207.34 pg/ml, which was significantly higher than that among the TCE-exposed workers (28.26 pg/ml, P < 0.01). In case group, except for correlation of TNF-α and IFN-γ, TNF-α and IL-5, the significant positive correlations were found among any two cytokines (r(IL-1β,IFN-γ) = 0.500, r(IL-1β,TNF-α) = 0.348, r(IL-1β,MCP-1) = 0.537, r(IL-1β,MIP-1β) = 0.477, r(IL-1β,IL-8) = 0.466, r(IL-1β,IL-5) = 0.610, r(IL-1β,IL-10) = 0.626, r(IFN-γ,MCP-1) = 0.460, r(IFN-γ,MIP-1β) = 0.491, r(IFN-γ,IL-8) = 0.322, r(IFN-γ,IL-5) = 0.532, r(IFN-γ,IL-10) = 0.511, r(TNF-α,MCP-1) = 0.325, r(TNF-α,MIP-1β) = 0.283, r(TNF-α,IL-8) = 0.430, r(TNF-α,IL-10) = 0.271, r(MCP-1,MIP-1β) = 0.659, r(MCP-1,IL-8) = 0.526, r(MCP-1,IL-5) = 0.504, r(MCP-1,IL-10) = 0.614, r(MIP-1β,IL-8) = 0.601, r(MIP-1β,IL-5) = 0.451, r(MIP-1β,IL-10) = 0.579, r(IL-8,IL-5) = 0.255, r(IL-8,IL-10) = 0.403, r(IL-5,IL-10) = 0.798, all P values < 0.05). The median of level of IL-5 among the patients with high eosinophils counts was 8.92 pg/ml, which was significantly higher than that among the patients with low eosinophils counts (1.04 pg/ml, P < 0.05).</p><p><b>CONCLUSION</b>The abnormal production of IL-1β, IFN-γ, TNF-α, IL-8, MCP-1, MIP-1β, IL-5 and IL-10 was related with the pathogenesis of hypersensitivity dermatitis induced by TCE. These cytokines could be used as referential indexes in the early health surveillance and clinic disease treatment.</p>

Association between polymorphisms of metabolic genes and telomere length in workers exposed to polycyclic aromatic hydrocarbon / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the association between the polymorphisms of metabolic genes and telomere length of genomic DNA in peripheral blood of workers exposed to polycyclic aromatic hydrocarbons (PAHs).</p><p><b>METHODS</b>One hundred and forty five coke-oven workers exposed to PAHs and sixty eight non-exposed medical staffs were recruited in this study. Urinary 1-hydroxypyrene (1-OHP) served as the internal exposure dose of PAHs for all subjects. Relative telomere length (RTL) of genomic DNA in peripheral blood was used as telomere length and measured by real-time PCR. Polymorphisms of metabolic genes were detected by PCR-based methods.</p><p><b>RESULTS</b>Compared with control group, the exposure group shown a decreased RTL (1.10 +/- 0.75 vs 1.43 +/- 1.06, P < 0.05). In the coke-oven workers, after adjusting the sex, age, cigarettes per day and urinary 1-OHP, RTL (1.25 +/- 0.93) of workers with CT genotype at the CYP1A1 3801 T > C was significantly longer than that (0.93 +/- 0.51) of workers with TT genotype (P < 0.05). RTL (0.90 +/- 0.58) of individuals with the Tyr/His genotype at mEH Tyr113His was significantly shorter than that (1.24 +/- 0.90) of individuals with the Tyr/Tyr genotype (P < 0.05). RTL (1.02 +/- 0.64) of individuals with the CT genotype at AHR rs10250822 was significantly shorter than that (1.36 +/- 1.14) of individuals with the CC genotype (P < 0.05). RTL (0.93 +/- 0.54) of individuals with the AT genotype at AHR rs10247158 was significantly shorter than that (1.19 +/- 0.84) of individuals with the AA genotype (P < 0.05).</p><p><b>CONCLUSION</b>The results of present study suggested that PAHs exposure could induce the shorted RTL, CYP1A1, mEH, AHR polymorphisms might influence the change of telomere length of genomic DNA in peripheral blood of workers exposed to PAHs.</p>

Analysis of subgroups of lymphocyte in peripheral blood among dermatitis medicamentosa-like of trichloroethylene patients and healthy exposed workers / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the effects of trichloroethylene (TCE) to lymphocyte subsets among exposed workers, and explore the early immunological effect biomarkers for prevention of hypersensitivity dermatitis induced by TCE.</p><p><b>METHODS</b>Twenty-eight patients with TCE-induced hypersensitivity dermatitis, 56 healthy TCE-exposed workers from the same workshops with patients, and 28 comparable unexposed controls were recruited in this study. The total lymphocyte count and the major lymphocyte subsets including T cell, CD4(+) T cell, CD8(+) T cell, B cell, NK cell in peripheral blood were measured by Flow Cytometer analysis and Standard blood count analysis.</p><p><b>RESULTS</b>The total lymphocyte count and T cell, CD4(+) T cell, CD8(+) T cell among patients (median at 2810.00, 1846.17, 831.87, 904.05 cell counts/µl blood) were significantly increased compared with TCE-exposed workers (median at 2101.00, 1218.59, 643.87, 482.81 cell counts/µl blood, Z = -3.19, -4.96, -3.22, -4.99, P < 0.001) and unexposed controls (median at 1900.00, 1223.60, 558.60, 325.80 cell counts/µl blood, Z = -3.30, -4.46, -3.45, -5.03, P < 0.001), the NK cell and CD3(+)CD4(+)/CD3(+)CD8(+) ratio among patients (median at 255.50 cell counts/µl blood and 1.11) were significantly decreased compared with the unexposed controls (median at 642.60 cell counts/µl blood and 1.96, Z = -3.56 and -3.11, P < 0.01). Meanwhile, for the exposed workers, the CD8(+) T cell (median at 482.81 cell counts/µl blood) was significantly increased and the NK cell and CD3(+)CD4(+)/CD3(+)CD8(+) ratio (median at 318.76 cell counts/µl blood and 1.27) were significantly decreased compared with unexposed controls (median at 325.80 and 642.60 cell counts/µl blood and 1.96, Z = -2.63, -3.52, -2.29, P < 0.05).</p><p><b>CONCLUSION</b>Occupational exposure to TCE could affect the lymphocyte subsets, especially T cell and NK cell. The total lymphocyte count, T cell and CD4(+) T cell might be effect biomarkers for subjects with hypersensitivity dermatitis among TCE-exposed workers.</p>

Echocardiographic evaluation of pressure overload-induced cardiac remodeling in mice using different ultrasound machines / 南方医科大学学报

<p><b>OBJECTIVE</b>To compare the results of echocardiographic evaluation of pressure overload-induced cardiac remodeling in mice using different ultrasound machines.</p><p><b>METHODS</b>Eighteen C57 BL/6 mice were randomly divided into the sham-operated and the transverse aortic constriction (TAC) groups (n=9). Eight weeks after the operation, the cardiac function of TAC group was evaluated using Siemens ultrasonic instrument with 15L8 probe and the differences between the awake and anesthetized states were compared. The heart rate, left ventricular (LV) dimensions, systolic and diastolic functions were measured in both sham-operated and TAC groups using the Siemens ultrasonic instrument and a high-resolution ultrasonic imaging system for small animals (Vevo 770).</p><p><b>RESULTS</b>Compared with the mice in wakefulness, the anesthetized mice showed significantly decreased heart rate and LV fractional shortening (P<0.001) and markedly increased LV end diastolic diameter and LV end systolic diameter (P<0.05). Both machines sensitively detected the cardiac remodeling of TAC mice in comparison with the sham-operated group. Compared with Siemens machine, Vevo 770 provided a higher resolution of 2D and M mode echocardiography with clearer Doppler frequency image of the mitral valve flow for evaluation of the LV diastolic function.</p><p><b>CONCLUSION</b>Both machines are suitable for evaluating cardiac remodeling induced by pressure overload independent of anesthesia, though anesthesia depresses cardiac function. Vevo 770 is optimal to evaluate LV diastolic function in mice.</p>

The effect of 2,5-hexanedione on myelin protein zero expression, and its mitigation using Ginkgo biloba extract / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the role of myelin protein zero (P(0)) in 2,5-hexanedione (2,5-HD)-induced peripheral nerve injury, and the protective effect of Ginkgo biloba extract (Egb761) on 2,5-HD-induced toxic peripheral neuropathy.</p><p><b>METHODS</b>After 4 weeks of treatment with 2,5-HD at different doses (50, 100, 200, 400 mg/kg) in rats, changes in the levels of P(0) in rat sciatic nerves was investigated, and the effect of Egb761 on 2,5-HD-induced toxic peripheral neuropathy was studied.</p><p><b>RESULTS</b>The blood-nerve barrier (BNB) permeability of the sciatic nerve increased, and the expression of P(0) mRNA and P(0) protein decreased in a dose-dependent manner after treatment with 2,5-HD for 4 weeks. Pretreatment with Egb761 protected against BNB interruption, and inhibited P(0) mRNA and protein reduction during 2,5-HD treatment. Pretreatment with Egb761 significantly reduced loss of body weight (P<0.01) and mitigated gait abnormalities (2.85±0.22) induced by 400 mg/kg 2,5-HD (P<0.01). It also reduced the signs of neurotoxicity induced by 2,5-HD.</p><p><b>CONCLUSION</b>2,5-HD inhibited the expression of P(0) in a dose-dependent manner, and this may be an important mechanism by which toxic peripheral neuropathy is induced by 2,5-HD. Egb761 has a protective effect against 2,5-HD-induced peripheral neurotoxicity in rats.</p>

Hypermethylation of O(6)-methylguanine-DNA methyltransferase in human bronchial epithelial cell induced by organic extracts of coke oven emissions / 中华预防医学杂志

<p><b>OBJECTIVE</b>To elucidate the mechanism of carcinogenesis induced by coke oven emissions by investigating the cell genetic damage index and the methylation of O⁶-methylguanine-DNA methyltransferase (MGMT).</p><p><b>METHODS</b>The human bronchial epithelial cell 16HBE was treated by 1 µmol/L B(a)P for 48 h, and then was exposed continuously to either 1‰ dimethyl sulfoxide (DMSO) or organic extracts of coke oven emission (OE-COE) for five days at the concentrations of 0, 2.5, 5.0, 10.0 and 20.0 µg/ml. The methylation-specific PCR (MSP-PCR), RT-PCR and immunoblotting were applied to detect the methylation status, changes of mRNA and protein of MGMT, respectively. Single cell gel electrophoresis was used to detect DNA damage induced by OE-COE.</p><p><b>RESULTS</b>Compared with the control group (DMSO), there was a significant hypermethylation in all study groups, along with the suppression of mRNA and protein in a dose-dependent manner, and the gradation ratio of them was 1.0, 0.96, 0.96, 0.85, 0.32 and 1.0, 1.0, 1.1, 0.41, 0.52, separately. There was a significant DNA damage with a dose-effect relationship in all study groups (F = 41.22, P < 0.05), and the comet Olive tail moment was (2.98 ± 1.43), (4.76 ± 1.79), (10.09 ± 1.75), (11.38 ± 1.77), (11.67 ± 1.88). The further study found that the index of DNA damage was negatively correlated to the expression of MGMT mRNA and its protein.</p><p><b>CONCLUSION</b>The DNA damage induced by COE might be associated with the suppression of MGMT caused by its hypermethylation.</p>

Effects of high and low shear stress on vascular remodeling and endothelial vascular cell adhesion molecular-1 expression in mouse abdominal aorta / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a mouse model of abdominal aorta stenosis and analyze the alterations in the arterial wall response to high and low shear stress.</p><p><b>METHODS</b>Twenty mouse were randomized equally into 4 groups, including 3 test groups (1, 7 and 14 day groups) with surgically induced stenosis of the abdominal aorta, and a sham-operated group without stenosis. The hemodynamics and the internal diameter of the blood vessel were measured by color Doppler flow imaging. The wall shear stress was calculated by Poiseiulle hydrodynamics formula (τ(m)=η×4×V(m)/D). Pathological examination and immunohistochemistry were performed to observe the arterial morphological changes and the endothelial vascular cell adhesion molecule-1 (VCAM-1) expression. The intimal-media thickness of the aorta was measured and endothelial VCAM-1 expression analyzed quantitatively.</p><p><b>RESULTS</b>Regions of low and high flow shear stress were created upstream from the stenosis and within the stenosis, respectively. Compared with the sham-operated group, the mice with aorta stenosis showed gradually increased vascular intimal-media thickness and VCAM-1 expression intensity in the upstream aorta, but not within the regions of the stenosis.</p><p><b>CONCLUSION</b>Vascular remodeling may occur shortly after exposure to low shear stress, which plays a significant role in initiation and progression of the pathological process of atherosclerosis mediated by VCAM-1, whereas high shear stress may exert an anti-atherosclerotic effect.</p>

Molecular imaging of thrombus with microbubbles targeted to alphavbeta3-integrin using an agarose flow chamber model / 南方医科大学学报

<p><b>OBJECTIVE</b>To assess the binding ability of microbubbles targeted to alphavbeta3-integrin (MBp) for thrombus-targeted contrast-enhanced ultrasound.</p><p><b>METHODS</b>Targeted microbubbles were prepared by conjugating the monoclonal antibody against alphavbeta3-integrin to lipid shell of the microbubble via the avidin-biotin bridges. Equivalent isotype control microbubbles (MB) or targeted ultrasound microbubbles (MBp) were randomly added into the flow chamber. After a 30-min incubation with the thrombus fixed in an agarose flow chamber model, the thrombus was washed with a continuous flow of PBS solution (15 cm/s) for 2, 4, 6, 8 and 10 min, followed by thrombus imaging using contrast-enhanced ultrasound and measurement of the video intensity (VI) values of the images.</p><p><b>RESULTS</b>The VI of the thrombus in MBp group was reduced by 28%-66%, while that in control MB group was decreased by 87%-94%, and the VI values of the thrombus group were significantly greater in former group at each of the time points (P<0.05).</p><p><b>CONCLUSION</b>MBP has good targeting ability to the thrombus with resistance to the shear stress after adhesion to the thrombus. In vitro evaluation of the thrombus-binding capability of the targeted microbubble (MBp) by simulating the shear stress in vivo can be helpful for predicting the in vivo effects of ultrasonic molecular imaging using MBp.</p>

Effects of acute cerebral ischemia on cerebral perfusion: quantitative evaluation by contrast-enhanced ultrasound in dogs / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the changes of cerebral blood flow (CBF) with real-time contrast-enhanced ultrasound (CEU) in a canine model of acute cerebral ischemia.</p><p><b>METHODS</b>Cerebral perfusion was assessed in 6 dogs subjected to craniotomy with CEU at the time of 0, 30, 60, 90 and 120 min after occlusion of the left common carotid artery (LCCA). The microvascular volume (A) and blood flow velocity (beta) in the brain were measured from the time-versus-acoustic intensity plots, and the value of Axbeta were calculated. 99mTc-ECD brain single photon emission computed tomography (SPECT) was performed on the day before the experiment and at 120 min after LCCA occlusion. The radioactive counts on both sides of the cerebral cortex were calculated.</p><p><b>RESULTS</b>A significant correlation was found between Axbeta from CEU and volume of the blood flow of the CCA from Doppler flowmetry. A, beta and Axbeta values varied significantly between the different time points (P>0.001). The ipsilateral hemisphere showed a low-perfusion state while the contralateral hemisphere showed a high-perfusion state immediately after the occlusion.</p><p><b>CONCLUSIONS</b>The changes of beta is the main regulation mechanism during acute cerebral ischemia in dogs.</p>

Effect of polyethylene oxide on red blood cell velocity in rat cremaster microcirculation / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the drag-reducing effect of polyethylene oxide (PEO) on the velocity of red blood cells in rat cremaster microcirculation.</p><p><b>METHODS</b>Blood samples were collected from 6 Wistar male rats (100-110 g) via the post-orbital venous plexus. The red blood cells were separated by centrifugation and labeled by fluorescinisothiocyate (FITC). After successful establishment of cremaster model, the labeled red blood cells were injected into the jugular vein, and the microcirculation was observed and recorded under fluorescence microscope. The hemodynamic parameters and microcirculation video was recorded every 4 min since 4 min before PEO or normal saline injection. Both PEO (10 ppm) and normal saline was injected into the same rat in random sequence at a constant rate of 3.5 ml/h for 20 min followed by observation for another 20 min. The velocity of the labeled-red blood cells was determined by IPP 6.0 software.</p><p><b>RESULTS</b>Compared with normal saline, PEO significantly increased the velocity of the red blood cells in the rat cremaster microcirculation (498.7-/+182.89 microm/s vs 773.54-/+308.27 microm/s, P=0.012). No significant changes in the heart rate and arterial blood pressure were observed during the experiment (P=0.836, P=0.420).</p><p><b>CONCLUSION</b>PEO at an extremely low concentration can significantly increase the velocity of the red blood cells in rat cremaster microcirculation and produces no significant impact on heart rate and arterial blood pressure.</p>

The effect of 2,5-hexanedione on nerve growth factor in sciatic nerve of rats and VSC4.1 cell / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the effect of 2,5-hexanedione (2,5-HD) on the levels of nerve growth factor (NGF) in sciatic nerve of rats and motor-neurons.</p><p><b>METHOD</b>A total of 50 Wistar rats were randomly designed into five groups and intoxicated with 400 mgxkg(-1)xd(-1) 2,5-HD for 0, 7, 14, 21, 28 d. Immunohistochemistry and real-time PCR were used to detect the levels of NGF and NGF mRNA. Motor neuron VSC4.1 cells were administrated with 0, 2.5, 5.0, 10.0, 20.0 mmol/L 2,5-HD for 24 h and 10.0 mmol/L 2,5-HD was chosen to intoxicated VSC4.1 cells for 0, 1, 3, 6, 12, 24, 48 h respectively. Immunofluorescence technique was selected to detect the levels of NGF.</p><p><b>RESULTS</b>The NGF level in sciatic nerve of rats administrated with 400 mgxkg(-1)xd(-1) 2,5-HD showed increase tendency at begin and then decrease after exposure. The NGF mRNA level in 14 d (2(-DeltaDeltaCt)= 3.46), 21 d (2(-DeltaDeltaCt)= 5.28) and 28 d (2(-DeltaDeltaCt)= 3.10) were higher than those in 0 d (2(-DeltaDeltaCt)= 1) and 7 d (2(-DeltaDeltaCt)= 0.78). In vitro tests of VSC4.1 cells showed that NGF levels in 5.0 mmol/L (43.24 +/- 7.52), 10.0 mmol/L (43.48 +/- 10.86) and 20.0 mmol/L (63.13 +/- 10.68) were higher than those in 0 mmol/L (16.32 +/- 4.20)(q values were 19.92, 19.72, 32.78, respectively, P < 0.01) and 2.5 mmol/L (19.78 +/- 2.66) (q values were 17.50, 17.42, 30.63, respectively, P < 0.01) in 24 h and the NGF level in 20.0 mmol/L was higher than those in 5.0 mmol/L (q = 13.04, P < 0.01) and 10.0 mmol/L (q = 11.71, P < 0.01). The NGF levels of VSC4.1 cells with 10.0 mmol/L 2,5-HD in 6 h (18.66 +/- 2.89), 12 h (23.14 +/- 6.08), 24 h (27.66 +/- 6.11) and 48 h (17.25 +/- 3.05) were increased compared with that in 0 h (10.18 +/- 1.81) (q values were 9.64, 15.74, 21.76, 8.50, respectively, P < 0.01), 1 h (9.31 +/- 1.28) (q values were 10.28, 16.17, 21.95, 9.20, respectively, P < 0.01) and 3 h (10.44 +/- 2.13) (q values were 9.25, 15.24, 21.17, 8.10, respectively, P < 0.01), and NGF levels in 12 h and 24 h increased compared with those in 6 h (q values were 5.24, 10.77, respectively, P < 0.01) and 48 h (q values were 7.31, 13.26, respectively, P < 0.01).</p><p><b>CONCLUSION</b>2,5-HD could increase NGF levels in sciatic nerve of rats and motor-neurons, and the dose or time dependent effects were observed in this study.</p>

Cytotoxicity and genomic damage of benzoapyrene in gene transformed cell model / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate cytotoxicity and genotoxicity of benzo(a)pyrene (B(a)P) by 16HBE-CYP1A1 cells which are human bronchial epithelial cell with CYP1A1 transformed.</p><p><b>METHODS</b>Expression of CYP1A1 and mEH of cell models were tested by real-time quantitative polymerase chain reaction. Cells were treated with 0, 1, 5, 10 and 20 micromol/L B(a)P for 24 h. Adverse effects of B(a)P were tested by cytokinesis-block micronucleus (CBMN) cytome assays. Cytotoxicity was assessed by the nuclear division index (NDI), frequency of necrotic and apoptotic cells. Genetic damages were assessed by frequencies of CBMN, nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs).</p><p><b>RESULTS</b>High levels of CYP1A1 and mEH were found in 16HBE-CYP1A1 cells (relative mRNA content was 7.8 x 10(-4) and 0.030 respectively). In 16HBE-CYP1A1 cells, NDI were decreased in 1, 5, 10 and 20 micromol/L B(a)P treated groups, 1.92 +/- 0.04, 1.71 +/- 0.01, 1.61 +/- 0.04, and 1.41 +/- 0.01, respectively; and lower than control group (2.08 +/- 0.03). Compared with control group ((82.67 +/- 6.66)%), the binucleated cells ratios were decreased, (76.33 +/- 3.51)%, (66.33 +/- 0.58)%, (51.67 +/- 1.53)% and (39.0 +/- 1.0)% respectively.Necrotic cells ratios were (1.93 +/- 0.42)%, (2.20 +/- 0.53)%, (8.07 +/- 0.90)% and (15.27 +/- 2.80)%, respectively, higher than control group ((0.47 +/- 0.11)%). The differences were significant (F values were 899.94, 303.33, 240.87, P < 0.01). Apoptotic cells were increased at lower groups and decreased to normal at higher groups treated by B(a)P. They were (1.20 +/- 0.53)%, (2.00 +/- 0.20)%, (1.47 +/- 0.12)%, (1.20 +/- 0.00)% and (1.20 +/- 0.00)%, respectively. Analysis on biomarkers of genetic damage, the significant dose-effect relationship were observed in NPBs and NBUDs (F values were 50.23, 121.09, P < 0.01, respectively). Frequencies of NPBs were (4.67 +/- 2.89) per thousand, (7.33 +/- 1.53) per thousand, (10.67 +/- 2.08) per thousand and (11.00 +/- 1.00) per thousand respectively. Frequencies of NBUDs were (2.33 +/- 0.58) per thousand, (4.00 +/- 1.00) per thousand, (5.00 +/- 1.00) per thousand, and (7.67 +/- 1.16) per thousand respectively. However, the dose-relationship of CBMN last only to 10 micromol/L B(a)P treated groups in 16HBE-CYP1A1 cells, and frequencies of CBMN were (8.33 +/- 3.21) per thousand, (14.67 +/- 1.15) per thousand, respectively. Frequency of CBMN was (16.67 +/- 2.88) per thousand in 20 micromol/L B(a)P treated group, lower than 10 micromol/L B(a)P treated group ((17.67 +/- 2.08) per thousand). In 16HBEV control cells, the cytotoxicity was found only in higher B(a)P treated groups and frequencies of CBMN, NPBs and NBUDs were increased also. While no significant differences were observed between 5, 10, 20 micromol/L B(a)P treated groups (they were (6.37 +/- 2.08) per thousand, (9.33 +/- 1.52) per thousand, (9.33 +/- 3.21) per thousand; (4.33 +/- 1.53) per thousand, (6.00 +/- 2.65) per thousand, (5.33 +/- 1.53) per thousand and (2.33 +/- 0.58) per thousand, (3.33 +/- 1.16) per thousand, (3.67 +/- 1.16) per thousand, respectively).</p><p><b>CONCLUSIONS</b>The genetic damages were more severe after treated with activated B(a)P, which may be induced by decreased NDI, increased necrotic cells and inhibition of apoptosis.</p>

Association between telomere length and occupational polycyclic aromatic hydrocarbons exposure / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the association between polycyclic aromatic hydrocarbons (PAHs) exposure and telomere length (TL), so as to investigate the effective biomarkers to evaluate the genetic damage in peripheral blood of workers exposed to PAHs.</p><p><b>METHODS</b>The exposure group consisted of 145 coke-oven workers (including 30 top-oven workers, 76 side-oven workers and 39 bottom-oven workers), and the non-exposure control group comprised 68 medical staffs. At 6 hours after the weekend duty shift, the samples of urine and 1 ml venous blood were collected from each subject. Airborne benzene-soluble matter (BSM) and particulate-phase B(a)P in the working environment of coke-oven and controls were sampled and analyzed. The concentration of urinary 1-hydroxypyrene (1-OHPyr) was determined. A real-time PCR method was used to determine the relative telomere length (RTL) of genomic DNA in peripheral blood. The relationship between the RTL and external exposure of PAHs, the potential factors which might have influence on TL were analyzed.</p><p><b>RESULTS</b>The medians of air BSM and particulate-phase B(a)P were higher in coke-oven (BSM: 328.6 µg/m(3); B(a)P: 926.9 ng/m(3)) than those in control working environment (BSM:97.8 µg/m(3); B(a)P: 49.1 ng/m(3)). The level of 1-OHPyr among coke-oven workers was significantly higher than that of non-exposed group (12.2 µmol/mol Cr vs 0.7 µmol/mol Cr; t = 26.971, P < 0.01). RTL in coke-oven workers were significantly shorter than those of controls (1.10 ± 0.75 vs 1.43 ± 1.06; t = 2.263, P = 0.026), and after adjusting for cigarettes per day and urinary 1-OHPyr, the significant difference was still observed (F(adju) = 5.496, P(adju) = 0.020). Stratification analysis found that RTL among the male and non-drinking groups in coke-oven workers were shorter than those the same sex and alcohol using status in controls (1.08 ± 0.73 vs 1.51 ± 1.10, F = 9.212, P = 0.003; 0.96 ± 0.38 vs 1.26 ± 0.46, F = 6.484, P = 0.012). Significant correlation between RTL and age was found (r = -0.284, P = 0.019) in non-exposure group.</p><p><b>CONCLUSION</b>PAH-exposure has effect on TL of genomic DNA in peripheral blood, which is mainly observed in the male and non-drinking groups between PAH-exposed workers and controls. It indicates that TL of genomic DNA in peripheral blood might be an effective biomarker as PAH-induced genetic damage.</p>

Evaluation of tumor angiogenesis using microbubbles conjugated with RGD peptides and contrast enhanced ultrasound / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To assess the feasibility of usage of microbubbles conjugated with RGD peptides and contrast enhanced ultrasound (CEU) in detection of tumor angiogenesis.</p><p><b>METHODS</b>Lipid microbubbles (MB) were prepared, and the RGD peptides were covalently conjugated to the lipid shell of MB (MB(RGD)). Six nude mice with tumor created by dorsal inoculation of HepG2 tumor cells were used as the test group. Six nude mice without tumor were served as the control group. 10 minutes after bolus injection of MB and MB(RGD) randomly (30 min interval) via a tail vein catheter, CEU was performed on the tumors of the test group and the thigh skeletal muscles of control group. The video intensity (VI) of tumors and the skeletal muscles were measured. The tumors and the skeletal muscles were harvested for immunohistochemical examination.</p><p><b>RESULTS</b>Only a slight contrast enhancement of the tumor was seen with MB, and the VI was 5.33 ± 1.71. While a remarkable enhancement of the tumor was observed after injection of MB(RGD). The VI was up to 17.03 ± 3.58, 3.18 folds higher as compared with that obtained by injection of MB (P < 0.05). As expected, there were no obvious contrast enhancement of the skeletal muscles with both MB(RGD) and MB. There was a high expression of αvβ3-integrin in tumor neovascular endothelium, however, no apparent expression of αvβ3-integrin was observed in the skeletal muscle vascular endothelium.</p><p><b>CONCLUSION</b>CEU with MB(RGD) can be used to effectively evaluate the angiogenesis of tumors, and it may greatly contribute to the early judgement of the nature of tumor.</p>