Higher Mortality Risk of Lungs Carcinoma in Vineyard Sprayers

This study investigated mortality rate of lungs carcinoma in professional vineyard sprayers. Clinical investigation was performed in  professional vineyard sprayers who had been exposed to the inhalation of the particles of Bordeaux mixture for  years on average. Bordeaux Mixture is used for prevention against mildew attacking vineyards. Th e control group was composed of  inhabitants of the same area who did not have any contact with the mentioned substance. A cytological investigation of the sputum specimens obtained from  tested inhabitants was performed. The sputum specimens were stained with standard haematoxylin-eosin method and also with special metod (rubeanic acid) to prove the incidence of copper granules in macrophages. The findings show a considerable statistical difference in the frequency of occurrence of lungs carcinoma between the group of vineyard sprayers smokers and the control group (X=.,p<.). Th e risk of lungs carcinoma in the vineyard sprayers was three times higher compared to the risk of smokers in the control group, with a statistical probability of  in the scope from .<RR<.. Mortality risk of lungs carcinoma in the professional vineyard sprayers is signifi cantly higher (p<.) compared to the risk in the control group.


Introduction
Pimentel and Marques described the case of pulmonary disease in vineyard workers and they named it "vineyard sprayer's lung". It is an occupational disease caused by inhalation of the copper sulphate solution neutralised by hydrated lime (). Th e changes in the respiratory system are caused by inhalation of the pesticide known as Bordeaux mixture (-. copper sulphate solution) which is used for spraying vineyards as a protection against mildew (). Th e roentgenogram of vineyard sprayer's lung is similar to that of alveolitis, with a tendency to develop into diff use interstitium fi brosis. Inhalation of the particles of Bordeaux mixture for a long period causes frequent chronic respiration symptoms as cough, chest constrictions and dyspnoea, along with ventilation disorder with dominant restrictive-obstructive changes (,). Experimental research, carried out on the animals exposed to the inhalation of Bordeaux mixture, caused the interalveolar heaping of macrophages, formation of granulomas and multiplication of collagen, elastic and reticular fi bres in the interstitium (). Plamenac and collaborators described alveolar macrophages with copper formations in the cytoplasm found with the workers engaged in vineyard spraying (). Pulmonary biopsy in vineyard sprayers often showed the presence of infl ammatory changes of bronchi and interstitium. Taking into the consideration clinical picture of the disease appearing after longer exposition to copper sulphate, Villard found the relation between pulmonary lesions and the development of pulmonary carcinoma (). So far professional risk of increased incidence of respiratory changes in vineyard sprayers has not been described extensively in medical literature as this type of study demands aimed cohort research through longer period. Monitoring the health in vineyard workers during a longer period encouraged us to undertake the investigation with a special focus on the mortality rate of pulmonary carcinoma in professional vineyard sprayers.

Subjects and Methods
Th e investigation was performed among the vineyard sprayers who worked in the largest vineyards in Brotnjo and Blato (  hectares of vineyards) in the Herzegovina-Neretva Canton and West Herzegovina Canton, Bosnia and Herzegovina. Bordeaux mixture is used for spraying the vineyards in the whole area. All subjects were men in the age of . on average. Th ey were divided into two groups. Th e fi rst group was composed of  workers who were for about  (SD±) years engaged in vineyard spraying. Th e control group was composed of  workers who did not get in touch with Bordeaux mixture. Th ey all had  (SD ± ) years of work experience. In this longitudinal prospective study we monitored the occurrence of lungs carcinoma in both groups, in two periods, fi rst from - and second from -. We also investigated infl uence of smoking on the incidence of lungs carcinoma in both groups. For these reasons each group was divided into sub groups of smokers and non-smokers. Taking into account professional exposure to pesticide, personal record of a patient, work experience, systematic clinical examination and chest roentgenograms all the subjects suspected to have lungs carcinoma were sent to pulmonary department for further diagnostic and therapeutic treatment. For each patient with confi rmed diagnosis of lungs cancer further therapy was performed (surgery, radiological therapy or combined chemotherapy) and his health condition was monitored until death. We made cytological analyses of sputum specimens randomly taken from  vineyard sprayers as well as from the control group. Th e only criteria for the selection had been that none of the subject had any respiratory disease of any aetiology in his case history. Sputum specimens were obtained by morning cough on three consecutive days. Only expectorated material contained pulmonary macrophages was accepted as sputum. After fi xation in  alcohol, the sputa were embedded in paraffi n and the section were strained with haematoxylin-eosin, tested for iron by Turnbull method and for copper with rubeanic acid and benzidine.

Results
In the group of  vineyard sprayers non-smokers, during the investigation  vineyard sprayers died of lungs carcinoma (mortality rate .). During the first investigation period (-)  sprayers died (mortality rate .) and during the second period (-)  sprayers died (mortality rate .). In the control group of  workers non-smokers,  workers died of lungs carcinoma (mortality rate .). In the period from -,  worker died (mortality rate .) and in the period from -,  workers died (mortality rate .) in Table . In the group of  vineyard sprayers smokers  workers died. Five workers died in the fi rst investigation period (mortality rate ) and  workers died in the second period (mortality rate .). In the control group of  smokers,  subjects died from lungs carcinoma (mortality rate .). Two subjects died in the fi rst period of investigation (mortality rate .) and  workers died ŽARKO ŠANTIĆ ET AL.: HIGHER MORTALITY RISK OF LUNGS CARCINOMA IN VINEYARD SPRAYERS in the second period (mortality rate .). Mortality rate of vineyard sprayers smokers was signifi cantly higher (.) than in the control group (.). Th e results of monitoring show that there is a signifi cant statistical diff erence in respect to the lungs carcinoma rate (X  = ., p<.) between the investigated group of workers engaged in vineyard spraying non-smokers and the control group of the workers non-smokers. Th ere is a signifi cant statistical diff erence in the height of relative risk of incidence of lungs carcinoma in the vineyard sprayers non-smokers compared to the control group (X  =., p<.). Relative risk of lungs cancer occurrence in the workers non-smokers engaged in vineyard spraying with Bordeaux Mixture is on average four times higher (.<RR<.) than in the control group. The results show that there are significant statistical diff erences in respect to the occurrence of lungs carcinoma between the group of vineyard sprayers smokers and the control group (X  =., p<.). Th e risk of lungs carcinoma was three times higher in the vineyard sprayers than in the control group of smokers with a statistical probability of  in the range .<RR<..

RESULTS OF CYTOLOGICAL INVESTIGATIONS
We made a cytological analysis of sputum specimens obtained from  randomly selected vineyard sprayers. We also analysed the same number of sputum specimens obtained from the control group in Table . We investigated  vineyard sprayers between the ages -,  of them were non-smokers and  smokers. Th e table shows increased sputum expectoration in both groups of vineyard sprayers non-smokers and smokers compared to the control group. Microscopic examination of the sputum in the non-smokers group showed the following abnormalities: abnormal columnar (irritated) cells in  () cases; squamous metaplasia of respiratory epithelium was found in  cases -in  cases () without atypical , in  () cases with ŽARKO ŠANTIĆ ET AL.: HIGHER MORTALITY RISK OF LUNGS CARCINOMA IN VINEYARD SPRAYERS atypical metaplastic cells; in  () cases copper granules were noted in the cytoplasm of the macrophages (halcophages) ( Figure ); eosonophils were found in  () cases; respiratory spirals in  () cases. In the sputum of smokers the following fi ndings were noted: abnormal (irritated) columnar cells were found in  () cases; squamous metaplasia was found in  cases, in  () cases there were no signs of atypia, while in  () cases atypical metaplastic cells were found; halcophages were found in  () cases; eosinophilia in  ( ) cases; respiratory spirals in  () cases. (Figure ) Th e control group was composed of  subjects in the age range -. Th ere were  smokers and  non-smokers. Th e following fi ndings were evident in the group of non-smokers: abnormal (irritated) cylindrical cells in  () cases; eosinophilia and respiratory spirals were found in  case ( ). Squamous metaplasia and halcophages were not evident within the group. In the group of smokers the following fi ndings were evident:  () subjects had abnormal (irritated) cyllindrical cells: squamous metaplasia was found in  cases, in  cases () without atypical and in  case () with atypical metaplastic cells; eosinophilia was found in  () cases; respiratory spirals were noted in  () cases; chalcoses were not found in this group of subjects.

Discussion
Morphological, clinical-radiological and functional changes in the lungs of vineyard sprayers are well known. Besides general symptoms, the patients also had the symptoms related to the respiratory system, in the beginning interpreted as a consequence of development of chronic bronchitis or tuberculosis, with hae-matological fi ndings almost within normal limits. Koch bacillus was not noted in the consecutive sputum that the patients produced and their tuberculin tests were negative. Th e applied therapy that included antibiotics and antituberculotics did not result with a complete regression of the disease. In some patients the radiological picture showed the presence of tumorous lungshadows, which was the indication for biopsy. In these cases pathohistological findings included: the desquamation of pneumocytes, formation of the predominantly histiocytic granulomas in the septa, individual and/or multiplied that gradually developed into fibrohyaline nodules similar to those found in silicosis (,, ). Th ere are the quotations in medical literature about the effects of Bordeaux mixture on the respiratory system that include changes in the pulmonary interstitium and bronchial ephitelium. Numerous focuses of non-specifi c bronchopneumonia, with infl ammatory cells and macrophages indicated the aff ection of bronchial parenhim, which was proved by the experiments on animals (,). At present some authors are of the opinion that copper is the aethiological agent responsible for changes in the interstitium, i.e. functional changes that occur in the cases of longer exposure to its inhalation (,). Cytological changes of the respiratory tract found in the sputum analysis are the proof of severe lesions of cyllindric ephitelium in professional vineyard sprayers. A high percentage of the sputum specimens contained desquamated, abnormal columnar bronchial cells that showed a whole spectrum of degenerative changes from non-specifi c irritation to severe cellular degenerative changes of ciliocytophoria type. Th e occurrence of numerous respiratory spirals, squamous metaplasia of the cells in the bronchial ephitelium and atypical cells