Expression of Hsp27 and Hsp70 in Lymphocytes and Plasma in Healthy Workers and Coal Miners with Lung

Huazhong Univ Sci Technol [Med Sci]DoI10.1007s11596010-0441-530(4)415420,2010415Expression of Hsp27 and Hsp70 in Lymphocytes and Plasma inHealthy Workers and Coal Miners with Lung CancerHaijiao WANG(王海椒)t, Jingcai XING(那邢景才)2, Feng WANG(王峰), Wenhui HAN(韩文慧), Houma ren(任侯卯,Tangchun WU(邬堂春), Weihong CHEN(陈卫红)'Department of Occupational& Environmental Health and MOE Key Lab of Environmental and Health, School of Public Health,Tongji Medical College, Huazhong Universiry of Science and Technology. Wuhan 430030, ChinaThe Central Hospital of Xishan Coal Electricity Compary, Shanxi030053, ChinaHUazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2010Summary: In coal mines, main occupational hazard is coal-mine dust, which can cause health prob-lem including coal workers' pneumoconiosis and lung cancer. Some heat shock proteins( Hsps) havebeen reported as an acute response to a wide variety of stressful stimuli. Whether Hsps protect againstchronic environmental coal-mine dust over years is unknown. It is also interesting to know thatwhether the expression of Hsp27 and Hsp70 proteins as a marker for exposure is associated risk oflung cancer among coal miners. We investigated the association between levels of Hsp27 and Hsp70expression in lymphocytes and plasma and levels of coal-mine dust exposure in workplace or risk oflung cancer in 42 cancer-free non-coal miners, 99 cancer-free coal miners and 51 coal miners withlung cancer in Taiyuan city in China. The results showed that plasma Hsp27 levels were increased incoal miners compared to non-coal miners(P<0.01). Except high cumulative coal-mine dust exposure(OR=13. 62, 95%C1-605-30.69)and amount of smoking higher than 24 pack-year(OR-2.72, 95%Cl=1.37-5.42 ), the elevated levels of plasma Hsp70(OR=1300, 95%Cl=5. 14-32,91)and plasmaHsp27 (OR-2.97, 95%Cl-1,40-6.32)and decreased expression of Hsp70 in lymphocytes(OR=2.36, 95%CI=1. 05--531)were associated with increased risk of lung cancer. These findingssuggest that plasma Hsp27 may be a potential marker for coal-mine dust exposure. And the expresion of Hsp27 and Hsp70 levels in plasma and lymphocytes may be used as biomarkers for lung can-cer induced by occupational coal-mine dust exposure.Key words: Hsp27; Hsp70; lung cancer; flow cytometry; coal minerHeat shock proteins(Hsps) work as molecular risk of lung cancer and exposure to occupationalchaperones in various cells; they fulfill a variety of coal-mine dust. However, it remains unknown whatphysiological functions, including assisting in intracellu- molecular mechanisms underlying the pathologicallar assembly, disassembly, stabilization, and intemal process in the lung of the exposed workers. It has beentransport of intracellular proteins to maintain normal suggested that the coal-mine dust particles deposited instructure and functions of the cells-l0. In response to a the lung are important non-cellular and cellular sourceswide variety of stressful stimuli such as high temperature of reactive oxygen species(ROS)that may cause oxidaand harmful chemicals that are common to the working tive damage to the target cells and DNA. This is likelyenvironment, Hsps have been reported to be induced to due to the presence of free silica and ferrous ion that areprotect cells, tissues, and organisms against damageeffective oxidants! 3)including oxidative stress!It has been reported that the expression of Hsp27Exposure to occupational coal-mine dust is a typical and Hsp 70 proteins was detected in several types of cankind of harmful stress the coal miners experienced in cer such as prostate cancer, breast cancer, including lungtheir working environment on a daily basis. Such expo- cancer). However, it is unknown whether the pressure is a chronic process that increases dust burden in the ence of Hsp27 and Hsp70 plays any role in the developlung over time. In animal experiments, lung tumors were ment and progression of these diseases. It is well knownfound in those rats that inhaled a large amount of coal that the expression of Hsps is an acute response to varidust.In humans, individual studies of occupational ousbut this has not been studied in theworkers have shown an association between increased coal中国煤化工ty, these coal-mineHaijiaoWangE-mail:wanghaijiao2006@163.comCN MH Ghat whether the exfThese authors contributed equally to this workpression of Hsp27 and Hsp70 proteins as a marker forexposure is associated risks of lung cancer among coal416J Huazhong Univ Sci Technol [Mes]30(4:200miners1.3 Evaluation of Health Status and Lung FunctionIn a recent study, we reported that the expression of MeasurementHsp70 had a protective role against genotoxicity inducedHealth status was evaluated for all participantsby polycyclic aromatic hydrocarbons(PAHs)in the based on the information provided in the questionnaire ascoke-oven emission9. It has been shown that PAH-in- well as clinical and laboratory examination. The clinicalduced genotoxicity is associated with risk of lung cancer laboratory examination included signs, weight, height,in the coke-oven workers. Therefore, the establishment pulse, blood pressure, and hepatic function test. Lungof an association between the expression of Hsp70 and function data mainly including vital capacity (VC)andrisk of lung cancer in coal-mine workers may be impor- percentage of predicted forced expiratory volume in onetant for early detection and prevention of lung cancer in second(FEV1% pred) were determined by Hl-701 pota-this occupational population. The aims of this study were ble lung function device with the values adjusted by theto(1)explore a possible correlation between levels of height and weightcoal-mine dust exposure in the workplace and levels of 1 4 Blood Sampling and TreatmentApproximately 5 mL venous blood was collectedplasma; and(2)to assess possible association between into a heparinized tube to separate lymphocytes andlevels of Hsp 27 and Hsp70 expression and risk of lung plasma collected from each subject in the moming aftercancer in the coal-mine workerscoll-Hypaque(Biochemical Reagent Co., China). TheI MATERIALS AND METHODSlymphocytes collected were washed twice with phos-phate-buffered saline(PBS)and counted, and the numberof lymphocytes was adjusted to 1x10/mL with theIn this case-control study we recruited 51 cases of RPM1640 culture mediumcoal miners with newly diagnosed lung cancer, 99 con- 1.5 Detection of Hsp27 and Hsp70 Expression introls of cancer-free coal miners and 42 controls of can-cer-free non-coal miners. all the participants were maleCellular staining of HSPs for flow cytometry wasThe twofree control groups were identified performed according to Xiao et aA9). Briefly, lympho-through a regular health examination from the samecytes were centrifuged and washed in 0.5 mLcoal-mine general hospital, who were healthy male taining l% bovine serum albumin(BSA). After centrifuorkers without any known infections, respiratory and gation, cells were suspended in 0.3 mL of fixation solu-heart failure, and complications of other diseases such astion containing 3% paraformaldehyde and fixed for 15min at 4@C. Fixed cells were centrifuged for 5 min athypertension,diabetes and cerebral vessel disease at the 600xg, then suspended in 300 uL of diluted antibodiestime of examination. All coal miners worked at least for (1: 300 dilution of rabbit anti-human Hsp 27, 1: 500 dilu-10 years in one coal mine in Shanxi province in Midwest tion of rabbit anti-human Hsp70, 1: 500 dilution of ratChina. The operation of this coal mine is mainly under-ground digging, and the characteristics of coal ore are permeabilization solution, and incubated for 30 min atC. After washing in 0.5 mL PBS containing 1% BSAf0. 27%-7.55% and 1.37% on average. Because these the cells were incubated with 300 uL of a fluoresceinminers worked underground for more than 30 h a week, isothiocyanate-labeled(FITC) anti-rabbit or anti-rat im-they were considered exclusively exposed to coal-mine munoglobulin G(IgG)(diluted 1: 100)for another 30 mindusts without obvious exto other known toxicant(e.g. organic solvent or PAHs other than from cigarette PBS containing 1% BSA. Negative control groups formoke)in the workplace. Having given informed consent, Hsp 27 and Hsp70 were set up without anti-human Hspsthe subjects completed a standardized questionnaire that antibodies or without the secondary FITC-labelledcollected personal information, work history, life habit anti-rabbit or anti-rat IgG The stained cells were ana-(including tobacco smoking and alcohol intake)as well lyzed using a flow cytometer(FACS CALIBUR,Beck-medical history at the time of medical examination. ton Dickinson Company, USA). a total of 10 000 cellsThis study was approved by the Ethnics and Humanwere counted, and the mean fluorescence intensity wassubject Committees of the Coal-Mine General Hospital measured at 525 nm. The result was expressed as ab-and Tongji Medical College, Huazhong University of sorbance(A)valueScience and Technology( China).1.6 Sandwich ELISA for Detection of Hsp27 and1.2 Occupational Exposure DataHsp70 Levels in PlasmaData of air samplings from the workplace includedThe plasma of each sample was separated from vetotal dust and respirable dust concentrations( me/m)and nous blood and stored at -80C until analysis. Plasmapercent free silica from 1980s through 2000s, which Hsp27 and Hsp 70 levels were determined by Sandwichwere made available from the Department of Occupa- ELISA with commercially available kits( Stressgen Bio-tional Safety and Dust Control in the coal mine. The reagents, Canada). Brieffly, plattes were coated with themeasurements typically were done on a monthly basis. antilin 0. 1 mol/L carbonThe dust concentration for one job title was reported on ate中国煤化工 ncubation at4C,theaverage each year. The free silica in the coal mine dust coatwas analyzed with infrared scanner. Cumulative dust 0.05CNMHith PBs containingific binding sitesexposure was calculated by the sum of work years of one blocked by incubation with 150 uL of PBS/T containingjob title multiplying dust concentration of this job title0. 1% BSA(PBS/BSA)for I h at 37 C on a shaker. AfterHUazhongg Univ Sci Technol [Med Sci]417g, 100 uL of samples were added and the plates workplace. As a result, the coal mine dust concentrationsted for 2 h at 37 C on a shaker. Plates were were decreased to 9.30--27.00 mg/mand 14.57 mg/msix times and 100 uL of rabbit polyclonal on average. The size distribution of the dust particles wasanti-Hsp27 and-Hsp70(1/400)diluted in PBS/T con- as follows: diameter less than 2 um was 59., 2-5taining 4% mouse serum was added. After 45 min at um was 28.57%0, 5-10 um was 6. 16%, and higher than37C, the A value was determined at 490 nm with back- 10 um was 5.63%round subtraction at 620nm using a microplate reader 2.2 The Characteristics of the SubjectsCERES 900C, BIO-TEC Instruments, Inc, Belgium)The total of 42 cancer-free non-coal miners(controlThe quantity of Hsp27 and Hsp70 in plasma samples was group 1), 99 cancer-free coal miners(control group 2)estimated from the calibration curve which ranged from and 51 coal miners with lung cancer(case group)were6 to 20000 ng/mLincluded in this analysis. All subjects were male. The1.7 Statistical Analysischaracteristics of the participants were summarized inThe All data were entered into a computerized database. table 1. The case and two control groups were matchedThe continuous data were presented as xts Multivariate on age. Mean age of coal miners with lung cancer, can-logistic regression models were built for a forward stepcer-free non-coal miners and coal miners were 63.84wise selection procedure. The Students t-test and Chi 65.88 and 61.39 years, respectively. For occupationaluare test were used for comparisons between various coal mine dust exposure, the cumulative coal-mine dustcontinuous and categorical variables, respectively. Crude exposures of case group(84700-+489.74 mg/m year)and adjusted odds ratios(ORs)and 95% confidence in- were significantly higher than those of cancer-free coaltervals(CIs) were estimated in unconditional logistic miner(581. 34*332. 34 mg/m year)(P<0.01).Also, meansegression models to measure the strength of association. smoking amount(pack-year) of case group (47.12ical inferences were based on the different levels pack-year)was increased as compared with that of canof significance(P<0.05 or P<0.01). The statistical analy- cer-free groups(23. 84 and 24. 22 pack-year, respectively)sis software sas 8.02 was usec(P<0.01). For lung function test, the values of vC andFEV1% pred were significantly lower in case grot2 RESULTS(2231.96*617. 13)than those in control group(3014.71#517.04)(P<0.01)and control group2.1 Dust Concentrations and Characteristics of the (2883.79*684.35)(P<0.01). Among coal miners withWorkplacelung cancer. 31 (60.8%)had squamous carcinoma, 7Coal mine dust concentrations were higher than 30 (13.7%)had adenocarcinoma, 6(11. 8%)had small cellmg/m beforeSince 1980s, wet mining technolo- carcinoma, and 7( 13.7%)had other unspecified tumorsgies have been used to control dust concentration in theTable 1 The characteristics and coal dust exposure of the cases and controlsVariablesControl group 1Control group 2Case groupAge(±s)65.88±7963.21±7046384±8.67Dust exposure years25.27±925.3310.51Average dust concentration23.08±9933348±12.55Cumulative exposure to coal mine dust58134±33234847.00±48974(mg/m3·year)Amount of smoking(pack-year)23.84±17.5824.22±219647.12±23.79°3014.71±517.0488379±68435223196617.1352+197360.38±1998P<0.05, P<0.01 vs control group 1; P<0.01 vs control group 22.3 Expression of Hsp27 and Hsp70 in Lymphocytes (P=0.3997 and P03134). However, the levels of in-and Plasmaducible Hsp27 and Hsp70 in lymphocytes in case groupThe expression of Hsp27and Hsp70 in lymphocytes (16.94+2.73 and 17.93+4.63)were significantly dend plasma in case and two control groups were summa- creased as compared with control group I(24 19+6.30rized in table 2. The results showed that the expression of and 21.60+5. 24)(P<0. 01)or control group 2(22.94*Hsp27 or Hsp70 in lymphocytes were not significantly 10.21 and 23. 12+12.61)(P<0.01)different between case group and control groupsTable 2 The expression of Hsp27 and Hsp70 in lymphocytes and in plasma in cases and controvariablesControl group I中国煤化工24.19±6.30Hsp70 in lymphocytes21.60±524CNMHG93+4.63Plasma Hsp2791.35±45.6917048±859914631±7999Plasma Hsp702.04±3.782.44±442ll0l±12.5P<0.0l vs control group 1, P<0.01 vs control group418J Huazhong Univ Sci Technol [Med Sci] 30(4): 2010It was found that the expression of plasma Hsp27 jects intowas significantly increased in control group I and case group Igroup as compared with control group 2(91.35=45.69)asse aly,groups(table 3). Compared to controlwer expression of Hsp 27 in lymphocytesd with coal dust exposure(OR=1.40, 95%(P<0.01). The level of plasma Hsp27 in case group CI: 1.03--1.91). And lower expression of Hsp 27 and(146.31#79.99)was lower than that in control group 2 Hsp70 in lymphocytes (OR=1.94 for Hsp27 and(170.48*85.99), but without statistically significant dif- OR=1. 41 for Hsp70)and higher expression of Hsp27 andference. The altemation of plasma Hsp70 in three groups Hsp70 in plasma(OR-1. 45 for Hsp27 and OR-1. 84 foras different from that of plasma Hsp27. Increased Hsp70)were associated with increased risk of lung can-plasma Hsp70(11.01 ng/mL)was observed in case group cer For coal miners, lower expression of Hsp 27 in lym-as compared to two control groups(2.04 and 2.44 ng/mL phocytes (OR-1. 38, 95% CI: 1. 15-1.66) and higherrespectively)(P<0.01). There was no significant differ- expression of plasma Hsp27(OR=1.56, 95% CIence ghe median expression levels of Hsp in control 1.64--2.74)were associated with increased risk of lungin plasma Hsp70 between two control groups1.17-2.08) and plasma Hsp70(OR=2.12,95%C1:group l were used as the cut-off value to divide all sub- cancer.Table 3 The distribution of Hsp27 and Hsp70 in lymphocytes and plasma of case and control groupsvariablesExpression in lymphocytesExpression in plasma