Modification Of Response Protein Polymorphisms On Occurrence Of Silicosis

Silicosis has long been one of the most serious and prevalent occupational healthproblems in China for its high morbidity and mortality. Although their pathogenesis remainsincompletely understood, some evidence suggest the participation of cytokines produced byAMs and oxidative stress induced by silica leading to the fibroblast proliferation andextracellular matrix(ECM) deposition. The heat stress proteins (HSPs) can be induced andplay important role for the cell protection as a result of various mechanical or chemicalstresses. A little is known about relation between HSPs and silicosis,the aim of this study is toexplore the roles of response proteins on occurrence of silicosis by in vitro experiments_andmolecular epidemiology.Sprague-Dawley (SD) rat alveolar macrophages (AMs) and human embryonic lungfibroblasts (HELF) was formed into an in vitro model. The mRNA levels of heat stressproteins(HSPs), such as HSP70-1, HSP70-2,HSP70-hom, HSP27, metallothionein (MT)1A,MT2A, haemoxygenase-1(HO-1) and the reported factors related to the synthesis ordegradation of ECM, metalloproteinases-9(MMP9) and tissue inhibitor ofmetalloproteinases-1(TIMP1) of HELF stimulated by supernatants of AMs exposed to silicawere measured by RT-PCR technology. At the same time, identification of proteins of treatedHELF was conducted by two-dimensional polyacrylamide gel electrophoresis(2-DE) andmatrix assisted laser desorption-ionization-time of flight massspectrometry(MALDI-TOF-MS). One of identified proteins,HSP27,was further validated byWestern blot assay.It was shown that there is good dose-effect and time-effect relationships betweenHSP70-1, HSP70-2, HSP70-hom, HSP27, HO-1, MMP9 and TIMP1 mRNA levels andconcentration of silica and supernatants exposure time (P＜0.05). Different response proteinshad their own induction dynamic characters, and the time they reach the peak expression wasdiversity. It is not found dose-effect relationship between the MT1A, MT2A mRNA levels andthe concentration of silica, while they can be induced by the supernatants(P＞0.05). MMP9,TIMP1 mRNA can be obviously induced and MMP9 mRNA levels increased earlier thanthose of TIMP1, but TIMP1 increased more obviously than MMP9 with time continuing.Several proteins in induced HELF were identified by 2-DE-MS: Peroxiredoxinâ…¡ isoform b, heat shock protein 27, a protein similar to calreticulin isoform 3, a protein similar tocalreticulin isoform 5. HSP27 was further validated by western blot and the results showedthat it increased with the concentration of silica increasing. The change tendency ofHSP27mRNA and protein expression was consistent.Epidemiologic investigation: Case-control study was applied in our study. Weinvestigated 519 workers from two large coal plants in Southwestern China, including 225patients with silicosis (â… : 134;â…¡, 73;â…¢, 18) and 294 control miners(self-reported to beHan descent, the primary ethnic group in China) with similar coal mine dust-exposurehistories but without apparent pulmonary disease or inflammation. Job exposure matrix(JEM)was used to assess the exposure to accumulated dust. Uniform Questionnaire was applied toevery subject for collecting the personal information including cigarette smoking, alcoholconsumption, occupational health habit, cognition of occupational hazard, psychological statusand nutrition status. ELISA method was used to determine Serum MMP9/TIMP1 in 174nonsmokers (41 healthy controls and 133 workers). PCR-RFLP-was used to genotype 519workers by MMP9(C-1562T),HSP70-1(G190C),HSP70-2(A1267G) andHSP70-hom(T2437C).It was found that JEM (Arithmetic mean, AM) in the cases was significantly higher thanthat in the control miners by one-way ANOVA (â… : 105.4±46.9;â…¡: 114.3±40.3;â…¢:92.3±34.3; p＜0.01). Silicosis category has positive correlation to JEM (P＜0.01). Althougheach index of dust exposure was under the occupational exposure limit (OEL), the mean dustconcentration of some job categories were over it. At some workplace, the total dustconcentrations reached 30%.The measurement of pulmonary ventilation function showed that each lung function index,including FVC, FEV1.0 and FEV1.0/FVC (measured value and predicted value), weresignificantly lower in the silicosis group than in the control miners (p＜0.05) and degradedfollowing the increase of JEM category (p＜0.05). The prevalence of lung dysfunction categorywas trended as follows by Chi-square test: Restricted ventilation dysfunction＞Obstructedventilation dysfunction＞Mixed ventilation dysfunction. The odds ratio (OR) of lungdysfunction in the workers was obtained by Logistic regression analysis and listed as follows,â…¡+â…¢silicosis (12.557),â… category silicosis (4.863), BI＜400(2.971), BI 600～(2.504),JEM150～(2.390), BI 400～(2.300), chronic bronchitis (1.715).Serum MMP9 in the 64 control miners was significantly higher than that in the 41 healthycontrols by Rank test (p＜0.05).There was no such relationship in serum TIMP1 (P＞0.05). Asthe silicosis category increased, the concentrations of serum MMP9 and TIMP1 were also increased, but the ratio of MMP9/TIMP1 was decreased, both of these end points weresignificantly higher in each silicosis category than that in the healthy controls (p＜0.05).Compared with control miners in the adjusted model considering the JEM category and agecategory, TIMP1 level in I silicosis patients was significantly higher (p＜0.05), MMP9/TIMP1ratio in theâ…¡silicosis patients was significantly higher (p<0.05), MMP9 and TIMP1 level intheâ…¢silicosis patients were significantly higher (p＜0.05), but MMP9/TIMP1 ratio in theâ…¢silicosis patients were significantly lower (p＜0.01)The frequency of HSPT0-2 1267G was significant different in each silicosis category(P=0.046), moreâ…¡andâ…¢patients carried G allele than that those with I silicosis category andcontrol miners ((P＜0.05). The frequency of HSP70-hom(T2437C) C/C carriers in theâ…¡andâ…¢silicosis patients was higher than that in control miners andâ… silicosis patients (8.8% vs2.4%å’Œ3%). Adjusted by JEM category and BI category, silicosis risk in the homozygous major allelecarriers was significantly higher than that in the heterozygous allele carriers and homozygousminor allele carriers(P＜0.05).The distribution of MMP9-1562C/C carriers and HSP70-2G carriers were marginallydifferent betweenâ…¡andâ…¢silicosis patients andâ… silicosis and control miners (P=0.087). Inthe subgroup with JEM more than 150, the frequency of HSP70-hom2437C carriers was alsomarginally different in the silicosis group compared with the control miners (P=0.094). Nointeraction was found between cigarette smoking and genotypes. HSP70-1(190),HSP70-2(1267) and HSP70-hom(2437) were highly linked with each other by LDA software.Haplotype analysis by PHASE analysis soft found that G-A-T and G-G-T haplotypes of thesethree SNPs was more frequent in theâ…¡andâ…¡category silicosis compared to theâ… silicosisand controls(P＜0.05).In conclusion, the expression of HSP70-1, HSP70-2,HSP70-hom, HSP27, HO-1 andMMP9/TIMP1 mRNA of HELF could be induced by AMs supernatants exposed to SiO₂,which displayed a clear dose-effect and time-effect relationships. They had differentcharacteristics of induction, which might be caused by the differences of the expression ofregular sequence in the genes. There were phase differences in the expression of differentresponse protein, which could protect the cells in turn. The phase difference of the expressionof MMP9/TIMP1 mRNA indicated MMP9 expression and activity increased in early phase,and ECM degraded. The appearance of TIMP1 inhibited the activity of MMP9, and theMMP9/TIMP imbalance appeared, which prompted collagen deposition. HSP27,Peroxiredoxinâ…¡and calreticulin had a closed correlation with oxidative-stress induced bySiO₂. In order to prevent silicosis, we not only control the dust concentration in the workplace,but also improve personal protection and decrease the dust exposure at the same time. We alsoneed to enhance occupational health education and health promotion in miners, and identifysusceptible population (HSP70-hom2437C C/C genotype, G-A-T or G-G-T haplotype carriersof HSP70-1, HSP70-2, HSP70-hom). Struggle for the WHO goal of eliminating silicosis.