pneumoconiosis prevalence in the French collieries In the plasma parameters, fibronectin, HLE and NMEP significantly increased in all groups of coal mine ...
Int Arch Occup Environ Health ( 993) 65: 5209-5213
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Biological markers as indicators of exposure and pneumoconiotic risk: Prospective study J.M Porcher1 , C Lafuma2 , R El Nabout2 , M P Jacob2 , P Sebastien 3 , P A Borm4 , S Honnons l , and G Auburtin1 'INERIS, BP-2, 60550 Verneuil en Halatte, France 2 CNRS, URA 1460, Cr 6teil, France 3 IRSST, Montr 6al, Canada 4 University of Maastricht, The Netherlands
Summary This research is designed to evaluate a number of biological markers to estimate harmful exposure on coal miners from different mining regions in France and to relate the outcome to differences in prevalence of coal worker pneumoconiosis (CWP) between these regions Eight epidemiological groups of active and ex-miners (smokers and non-smokers) have been selected in the French collieries (North, Lorraine and Provence) according to their occupational and pneumoconiotic status The following biomarkers have been evaluated: cellularity of sputum, elementary analysis of particles in TEM/EDAX, plasma neutral metalloendopeptidase elastase type (NMEP), leucocyte elastase (HLE), fibronectin (FN) and elastin peptides Pulmonary alveolitis, expressed by sputum cellularity, is different between active workers groups but not related to the general background of pneumoconiosis prevalence in the French collieries In the plasma parameters, fibronectin, HLE and NMEP significantly increased in all groups of coal mine workers as compared to the control group, except for fibronectin parameter in Lorraine collierie The degree of increase of these parameters allow us to discriminate the different groups and suggest that plasma FN, HLE and NMEP may be considered as biological markers of chronic inhalation of coal mine dust particles The decrease of elastin peptides level in the Lorraine group alone suggests a specific alteration of elastin metabolism These parameters were not related to the development of pneumoconiosis and its degree of severity. Key words: pneumoconiosis
Biological markers Fibronectin
Coal worker's
Proteases
Sputum.
Introduction Coal workers pneumoconiosis (CWP) is a lung disorder that becomes manifest after several decades of exposure to coal mine dust and is commonly detected and classified using chest radiograms Experimental animal and clinical researchs continue to find new immunological, biochemical and molecular biology based factors involved in interstitial lung diseases and generate new methods to measure early adverse effects of inorganic dust exposure.
Several years ago the labs involved in this study set out to investigate the applicability of these new insights into research on CWP l2, 9, 14l In fact, the Maastricht group was the first one to show that Tumor Necrosis Factor (TNF) played a role in CWP in humans The approach was defined in a recent paper as "Molecular Epidemiology" l2 l. The main objective of this type of studies is not to determine the causative agent, but to appraise and use mechanisms in order to design more specific studies, e g directed to individual susceptibility and biological effects of cumulative exposure. The biological entities (enzymes, degradation products, DNA damage etc) that are used are called "biological markers", since they "mark" the (effect of) cumulative exposure, a cascade leading to early disease or disease itself. Cellularity, ultrastructural and biochemical changes of cells recovered by bronchoalveolar lavage (BAL) have shown the central role of alveolar macrophages in the pathogenesis of pneumoconiosis l 11, 16l Sputum is a non invasive means to collect pulmonary macrophages Previous work showed that the pulmonary alveolitis could be expressed in the sputum cellularity McDonald et al l12 l have studied sputum macrophages of 573 asbestos workers from 11 epidemiologic cohorts According to the different cohorts, geometric mean cellularity varied from 71 to 839 macrophages per sputum sample Moreover, Sebastien et al l14l showed that it is possible to count in light microscopy macrophages and asbestos bodies in sputum A relationship with radiographic abnormalities could be observed These two markers were a better index of past exposure to fibers than cumulative dust exposure estimated from work history Our preliminary data indicated that in coal worker pneumoconiosis (40 patients at different stages of CWP), sputum macrophages were present in higher concentration (mean cellularity = 8800 per sputum sample) and might exhibit some ultrastructural changes Our experience of coalworkers, alveolar macrophages studied by electronic microscopy shows that different types of dusts may be caracterised in sputum macrophages as well as in macrophages recovered by BAL. The plasma investigated parameters were chosen on the basis of the actual concept of fibrogenesis l4l as followed: the chronic inhalation of coal mine dust particles may induce influx of inflammatory cells (macrophages and
5210 polymorphonuclears) in the alveolar space and interstitial parenchyma The phagocytosis of inhaled more or less reactive particles is followed by the specific activation of inflammatory cells to release oxygen free radicals, proteases and mediators. The resulting unbalance between oxidizing and anti-oxidizing agents, proteases and antiproteases, the arachidonic acid metabolites, elastases and collagenases, as well as between the various cytokines and growth factors, would favor the abnormal remodelling and architecture of extra cellular matrix components This may explain the different forms of pneumoconiosis In this context, we have chosen to investigate four biological parameters, all present in the plasma: the fibronectin which is well known to play a key role in cell matrix interaction, is also involved in the recruitement and the proliferation of fibroblasts during fibrogenesis l13 l. Leucocyte elastase (HLE) which is the major serine proteinase released by activated polymorphonuclear cells, degrades most extracellular matrix components l9l. Neutral metalloendopeptidase (NMEP) which is already known as to be increased in the plasma of patients with sarcoidoses and emphysema l 8l. Elastin peptides which reflect elastin degradation l 7l. If our biological markers are valid, it would improve industrial hygiene surveys of coal mine workers Moreover, they could be easily applied to other types of occupational lung diseases occurring in mining and steel industry.
Plasma parameters
Materials and methods
Table 1
Study population
Group
Study population is composed of 474 miners and ex-miners from French collieries which exhibit important differences in coal miners pneumoconiosis (North > Lorraine > Provence) Active workers were selected by stratified random sampling according to age and work duration Retired miners from North collieries were randomly selected according to age and pneumoconiotic status Chest X-rays were reinterpreted by three trained and independent medical readers according to I LO classification Eight groups were composed: 1 control group of active Lorraine workers without mine dust exposure (C), 4 active underground coalminers groups: 3 without pneumoconiosis from Lorraine (L), Provence (P) and North (N), I with simple pneumoconiosis from North (SPN), 3 retired miners groups from North collieries without pneumoconiosis (R), with simple pneumoconiosis (SPR) and with progressive masive fibrosis (PMFR).
Sputum cellularity
Each person in the study was given a 50 ml FalconM type plastic tube containing 10 ml fixative buffer (2 5% Glutaraldehyde in sodium cacodylate buffer) This sample was blended and centrifuged (7 00 g, 30 min, 4 C) The pellet was resuspended in 2 ml of buffer and a 30 W 1 aliquot smeared between two albuminized slides, fixed by Cytoprep TM , stained using a modified Papanicolaou technique and cover slip applied After a systematic sampling of sputum samples containing any material, alveolar macrophages encountered on the two entire slides were counted and all other cell types were ignored Results were expressed in number of macrophages per sputum Elementary analysis of engulfed particles in sputum macrophages were performed in Transmission Electronic Microscopy coupled with an Energy dispersive X ray Analysis unit (TEM/EDAX).
Human plasma fibronectin content was determined by automated lazer immunonephelometry using specific human antifibronectin antibodies Human plasma leucocyte elastase content, was determined by enzyme link immuno assay (IMAC, Merck), keeping in mind that HLE is complexed with its plasma inhibitor ( 1 Proteinase inhibitor) Human neutral metalloendopeptidase activity was determined by the cleavage of the synthetic substrate succinoyl trialamine paranitroanilide at pH 7 4 Plasma elastin pepatides level was determined by ELISA assay, using anti-kapa elastin antibodies l 6l.
Analysis
Differences of markers distribution between groups were analysed by non parametric test for continuous variables and by X2 test for polychotomics variables using SPSS package.
Results Macrophages count wich is significantly increased in older age groups is not related to smoking habits Distributions (Table 1)from P, N and SPN groups are significantly different compared to controls (respectively P= O0003, O001 and 0.05 ) Active underground miners from most different groups had significant different macrophage count distributions (L vs P P=O01; L vs N P= O009; P vs SPN P=0 04) The difference between P and N is near signification (P=0 08). Macrophages counts were not significantly different between retired miners groups These differences are not modified after controlling for age. Sputum macrophages counts in the different groups Number of macrophages per sputum not detected
C L P N SPN R SPR PMFR
n S 10 14 7 6 6 6 5
18 36 67 35 38 25 25 24
< 1000 N 13 11 4 3 2 5 5 2
1000 to 4999 % 46 39 19 15 13 21 21 10
N 9 6 0 2 5 5 8 6
% 32 21 0 10 31 21 33 29
5000 + N 1 1 3 8 3 8 5 8
% 4 4 14 40 19 33 21 38
(C) control group of active Lorraine workers without mine dust exposure; (L), (P) and (N) active underground miners groups without pneumoconiosisfrom Lorraine, Provence and North collieries; (SPN) active undergroundminers groupfrom North; (SPR), (R) and (PMFR) North retired miners groups respectively with or without pneumoconiosis and with progressive massive fibrosis.
TEM/EDAX macrophage sputum analysis (Fig 1) shows the valuability of this technique for individual in situ dust analysis, unfortunately, this method is too time consuming to allow large population analysis. Results related to plasma parameters are shown in Table 2 Except for fibronectin and NMEP values in the Lorraine group of underground miners, they indicate a general increase of NMEP, HLE and fibronectin values in all the groups of underground miners, as well as of retired miners, compared
5211
Fig 1 TEM/EDAX analysis: Sputum macrophage found in a non smoker coal miner (North collierie) kaolin (a) and illite (b) will be noticed.
Table 2
Plasma parameters (NMEP nmoles hydrolysed substrate/48, HLE tg/l; Fibronection mg/i; Elastin peptides tg/1).
Group
C L P N SPN R SPR PMFR
The simultaneous presence of coal (*),
NMEP
HLE
mean
SD
N
mean
636 795 938 1214 1040 1128 1030 1060
254 584 863 794 705 769 707 915
54 63 60 47 25 44 96 45
25 20 42 57 72 81 43 55 34 73 45 64 46 82 48 07
Fibronectin
I'
N
19 83 34 41 29 65 41 23 46 74 42 72 38 90 37 46
54 63 58 44 26 44 96 44
mean 0 42 0 40 0 54 0 58 0 52 0 58 0 56 0 53
0 0 0 0 0 0 0 0
Elastin peptides
SD
N
13 15 21 23 13 18 18 16
57 63 61 47 26 44 95 44
nmean 3 81 3 00 4 12 4 04 3 77 4 54 4 33 4 03
9)
n
0 99 0 93 1 91 1 11 1 42 1 33 1 41 0 88
53 48 45 36 17 35 64 24
(C) control group of active Lorraine workers without mine dust exposure; (L), (P) and (N) active underground miners groups without pneumoconiosis from Lorraine, Provence and North collieries; (SPN) active undergroundminers groupfrom North (SPR), (R) and (PMFR) North retired miners groups respectively with or without pneumoconiosis and with progressive massive fibrosis.
5212 to the reference Lorraine group of active surface miners In the Provence group fibronectin, NMEP or HLE are significantly increased compared to the controls (respectively P=O03, P=O01, PProvence>Lorraine: Table 1). Unfortunately the hierarchical rank of these results does not reflect the prevalence of pneumoconiosis in these areas ( 1/3, 1/10 and O in North, Lorraine and Provence retired miners populations, respectively) No relation was found between macrophages counts and severity of the illness in the retired North coal miners In contrast with McDonald et al l12 l no relationship was found between macrophage counts and smoking habits Substantial variation was found in macrophage counts in daily sampling of a small subset of subjects suggesting that little reliance can be put on results from a spot sample, particularly if it is negative. Physical exercise, forced expiratory manoeuvres or saline inhalation could improve the reproducibility of sputum production, but these techniques, even non-invasive could be inacceptable for epidemiological surveys. Plasma neutral metalloendopeptidase activity, fibronectin and leukocyte elastase content are variously increased according to the different work sites, possibly as a result of differences in the composition of coal mine dust particles The increase of these parameters enables to discriminate the different groups of underground miners, so that the North group exhibits the highest increase of NMEP and fibronectin values, whereas the Lorraine groups appear as the less responsive groups. Differences are also observed regarding the intensity of the specific response of the three parameters, when compared between them, so that HLE exhibits the highest increase and fibronectin the lowest Plasma HLE appears therefore as a more sensitive biological marker to pulmonary overloading of coal mine dust particles, than fibronectin or NMEP. Finally, although fibronectin, HLE and NMEP are able to discriminate between the different groups of underground miners according to the geographic work sites, they appear unable to constitute real biological markers of pneumoconiosis (on the basis of prevalence or degree of severity) This apparently negative result allowed us to suggest that these plasma parameters could be rather considered as biological markers of injury or inflammatory reaction induced by the chronic inhalation of coal mine dust particles in underground miners and probably maintained over decades following end of exposure in retired miners For several
years it has been proposed that HLE issued from neutrophils as well as fibronectin issued from alveolar macrophages may be involved during alveolitis inflammation l3, 10l. Moreover Amrani et al l1l and Lafuma et al l 10l produced evidence showing that plasma fibronectin did behave as an accute phase reactant, more particularly following thoracic irradiation In order to valid our suggestion, we are going to investigate in all the miners groups some serum accute phase proteins known to increase during inflammation such as C reactive protein and a, proteinase inhibitor. So, we suggest that these plasma parameters cannot be considered as biological markers of pneumoconiosis (on the basis of prevalence or degree of severity) both in underground or retired miners They could be rather considered as biological markers of the inflammatory reaction induced by the chronic inhalation of coal mine dust particles in underground miners, probably maintained over decades following end of exposure in retired miners. In contrast, the decrease of elastin peptides level in the Lorraine group of underground miners suggests a specific alteration of elastin metabolism This result is in agreement with another recent epidemiological study carried out in Lorraine coal mine l5l and supporting the hypothesis that a decrease of elastin peptides level might reflect a lack of elastin breakdown or repair Further investigation is needed to test the relation of elastin peptides level to a specific test of emphysema. Acknowledgments We would like to aknowledge the medical staff and all the French collieries workers who made this study possible We gratefully thank Dr Lelong and collaborators for technical assistance in fibronectin measurement This work was supported by ECSC grant in "6th Industrial Hygiene in mines" research program.
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