Comparative in vitro toxicity of cadmium and lead on redox cycling in type II pneumocytes.

Both cadmium and lead have pulmonary toxicity: cadmium can cause lung cancer, fibrosis and emphysema; lead can induce a moderate interstitial pulmonary fibrosis. Both metals give rise to depletion of glutathione and depletion of the protein-bound sulfhydryl groups, and lead to the production of reactive oxygen species. In the primary culture of type II pneumocytes, which is one of the most important cell groups from the aspect of glutathione metabolism and thus redox balance, the effect of cadmium chloride and lead nitrate upon the enzymes of the glutathione cycle, upon superoxide dismutase and upon the structure of type II pneumocytes was examined. Depending on the concentration, cadmium inhibited each of these parameters, whereas lead nitrate significantly increased the activity of glutathione reductase while inhibiting other parameters. Both metals induced damage of the membranes of type II cells, depending on the concentration, although cadmium caused significantly more damage than lead. The data obtained suggest that both substances cause an imbalance in the redox cycle and diversely affect the function and membrane structure of type II pneumocytes.

Combined pulmonary toxicity of cadmium chloride and sodium diethyldithiocarbamate.

The pulmonary toxicity of sodium diethyldithiocarbamate and cadmium chloride, each separately and in combination, was compared in Sprague-Dawley rats after single intratracheal instillation in sequential experiments by chemical, immunological and morphological methods. With combined exposure, the cadmium content of the lungs increased permanently relative to that of the lungs of just cadmium-treated animals. Immunoglobulin levels of the whole blood did not change, whereas in bronchoalveolar lavage the IgA and IgG levels increased significantly. Morphological changes were characteristic of the effects of cadmium but were more extensive and more serious than in the case of cadmium administration alone: by the end of the first month, interstitial fibrosis, emphysema and injury of membranes of type I pneumocytes developed and hypertrophy and loss of microvilli in type II pneumocytes were detectable. These results showed that although dithiocarbamates as chelating agents are suitable for the removal of cadmium from organisms, they alter the redistribution of cadmium within the organism, thereby increasing the cadmium content in the lungs, and structural changes are more serious than observed upon cadmium exposure alone.

Study of inflammatory responses to crocidolite and basalt wool in the rat lung.

The subacute effects of crocidolite and basalt wool dusts were studied by nmeans of biochemical, morphological. and histological methods 1 and .3 mo after intrabronchial instillation. The cell count, protein and phospholipid contents, and lactate dehydrogenase (LDH) activity were determined in the bronchoalveolar lavage (BAL). Both types of fibers induced a prolonged inflammatory reaction in the lung. All the parameters studied in the experimental groups were more markedly elevated after 3 mo. Relative to the control, the protein and LDH values were increased three- to fivefold, the phospholipid content twofold, and the number of free cells in the BAL exceeded the control level up to ninefold. The inflammatory responses to crocidolite and basalt wool in the lung did not differ significantly. In spite of this, basalt wool is recoinmended as an asbestos substitute, as the use of this man-nade fiber may result in a significantly lower release of dust than that from crocidolite.

Effects of sodium diethyldithiocarbamate on type II pulmonary epithelial cells in vitro.

Dithiocarbamates (DDTC) are chemicals widely used in the form of pesticides, therapeutic and chelating agents, and scavengers. Since DDTC interfere with SH, Cu, and Zn enzymes due to chelating properties, it was of interest to clarify, in primary culture of type II alveolar pneumocytes, the effect of this compound upon enzymes of glutathione cycle, Cu, Zn-superoxide dismutase, and the membrane structure of cells. DDTC significantly inhibited the activity of superoxide dismutase and the activity of gamma-glutamyl transpeptidase, glutathione reductase, and alkaline phosphatase, whereas an increase in the activity of glutathione peroxidase was found. The membranes of pneumocytes type II were injured. Data show that DDTC adversely affected type II pneumocyte function and structure.

Characterisation of lectin binding patterns of mouse bronchiolar and rat alveolar epithelial cells in culture.

Lung epithelial cell differentiation pathways remain unclear. This is due in part to the plasticity of these cells and the lack of markers which accurately reflect their differentiation status. The aim of this study was to determine if lectin binding properties are useful determinants of functional differentiation status in vitro. Mouse Clara cells were cultured for 5 days. During this time, no alteration in differentiation was evident by electron microscopy. No significant alteration in binding reactivity of Bauhinia purpurea (BPA), Maclura pomifera (MPA), Concanavalin A, Wheat germ or Helix pomatia lectins occurred in cultures compared with Clara cells in mouse lung tissue. In contrast, nitrotetrazolium blue reductase activity and CC10 expression declined in culture. Rat type II cells were cultured for 8 days. Between days 0 and 4, the number of type II cells identified by electron microscopy was constant at 70-80%, decreasing to 8% by day 6. In contrast, by day 4, only 42% cells retained alkaline phosphatase activity. BPA and MPA reactivity was altered at day 0 and day 4 respectively, compared with cells in situ. Therefore, the reactivity of lectins analysed here does not reflect functional differentiation status of cultured mouse Clara cells. However, BPA and MPA reactivity may be a sensitive indicator of alterations in rat type II cell differentiation in vitro.

In vitro and in vivo tests for determination of the pathogenicity of quartz, diatomaceous earth, mordenite and clinoptilolite.

The effects of samples of crystalline quartz, diatomaceous earth, mordenite and clinoptilolite were investigated in vitro (as concerns erythrocyte haemolysis and lactate dehydrogenase (LDH) release from peritoneal macrophages) and in vivo (on LDH, protein and phospholipids in rat bronchoalveolar lavage (BAL), and phospholipids in rat lung tissue). The respirable mineral samples were instilled intratracheally. Determinations in the BAL were carried out after 15, 60 and 180 days, and in the lung tissue after 90, 180 and 360 days. Quartz DQ and quartz FQ induced acute, subacute and chronic inflammation and progressive fibrosis. However, due to the Al2O3 contamination on the surface of the particles quartz FQ caused a delayed response in vivo. Diatomaceous earth produced acute/subacute inflammation that gradually became more moderate after 60 days. Clinoptilolite was inert, whereas the other zeolite sample, mordenite, was cytotoxic in vivo. The reason for this was presumably the needle and rod-shaped particles in the mordenite samples. The investigation revealed that different in vitro and in vivo methods canprovide valuable data concerning the pulmonary toxicity of minerals.

Effects of lead(II) nitrate and a dithiocarbamate fungicide on the rat lung.

The pulmonary toxicity of two potential environmental pollutants was studied in rats 1, 7 and 30 days after a single intratracheal instillation of lead nitrate and Dithane M-45 (mancoceb), either individually or in various combinations. The cell count, protein, phospholipids and lactate dehydrogenase level were determined in the bronchoalveolar lavage fluid, as were the protein, phospholipids and acid phosphatase contents in the lung tissue. Lead nitrate and Dithane M-45 induced acute inflammation reactions with different features. The effects of mixtures of lead nitrate and Dithane M-45 were found to be different from those of the individual components.

Surface changes in type II pneumocytes isolated from rats during the cultivation.

Type II cells isolated from the rat lung were maintained in culture for 8 days. The activity of alkaline phosphatase and lectin binding properties were studied. The alkaline phosphatase activity and the number of lamellar bodies were continually decreasing during the studied time period. The profile of lectin binding (Maclura pomifera and Ricinus communis) did not change during the cultivation.

Combined pulmonary toxicity of diethyldithiocarbamate and lead (II) oxide in rats.

The pulmonary toxicity of sodium diethyldithiocarbamate and lead(II) oxide alone or in combination was studied in rats after a single intratracheal instillation. The lead content in the lungs and the whole blood was determined and it has been found that the clearance of lead from the lung was delayed by dithiocarbamate complex formation, which probably had a role in increased IgA levels in the bronchoalveolar fluid and the induction of local immune response. The combined exposure gave rise to calcium deposits in the lungs both extra- and intracellularly after 1 month of exposure. Both separate and combined exposure invoked permanent injury in membranes or dystrophic changes in the cytoplasm of pneumocytes, which may initiate and generate a series of events leading to fibrosing alveolitis.

A study on lung toxicity of respirable hard metal dusts in rats.

The aetiology of hard metal lung disease has not been clarified so far. The pulmonary toxicity of respirable dusts collected in a hard metal factory was studied in vivo in rats. The effect of the samples was examined 1, 4, 7 and 30 days after single intratracheal injection. Lactate dehydrogenase (LDH), acid phosphatase (AP), protein and phospholipid were determined in cellfree bronchoalveolar lavage (BAL) and lung tissue. The lungs and regional lymph nodes were processed histologically. Lung toxicity of the samples collected during hard metal production varied. Samples containing considerable amount of cobalt dissolved upon acid treatment were found to induce inflammation. It has been established that the biological effect of samples of identical composition is changed by heat treatment and pre-sintering. Our examinations seem to prove that cobalt plays a prominent role in the development of pathological alterations.

In vitro and in vivo assessment of the pulmonary toxicity of cellulose.

The lung-damaging effect of intratracheally administered cellulose was studied by biochemical and histological methods. Cell count, protein, phospholipid, lactate dehydrogenase and acid phosphatase were determined in bronchoalveolar lavage fluid 1, 3 and 7 days after intratracheal instillation. Histological tests were performed after days 1, 3 and 30. In vitro, cellulose did not damage the macrophage cells. In vivo, interstitial oedema as well as the initial signs of inflammation could be detected in the lung after the first day. Inflammation after 1 week could be noted, partly interstitial and partly intra-alveolar and intrabronchial. In the bronchoalveolar lavage fluid, protein, lactate dehydrogenase, acid phosphatase, phospholipid and cell count were enhanced after days 1 and 3. After 1 month, the developing bronchioalveolitis is fibrous in character. Contrary to the in vivo study, cellulose did not damage rat peritoneal macrophages.

In vivo pulmonary toxicity of cellulose in rats.

Cellulose after a single intratracheal dose (15 mg per animal) brought about fibrosing granulomatous alveobronchiolitis and an increase of IgA production in the bronchoalveolar lavage. Fibrosing alveolitis showed moderate progression as a function of time. With different morphological methods, injury of type I pneumocytes and the incomplete repair of type II pneumocytes were detected. The damage of the alveolar epithelium initiated and activated a series of processes that led to definite pulmonary alterations: pulmonary fibrosis leading to the disintegration of the alveolo-capillary morphological functional unit.

The pulmonary toxicity of cinnamon dust in rats.

The histopathological effect of a single intratracheal dose of respirable cinnamon dust, cinnamon dust extract, and cellulose dust on the lungs of rats was studied sequentially one, seven days and one month after treatment. Exposure to respirable cinnamon and cellulose dusts resulted in alveobronchiolitis at the end of the first and seventh day, and fibrotic changes by the end of the first month. As the extract of cinnamon dust caused no histopathological alterations, it is assumed that the cellulose content of cinnamon dust was responsible for the histological reactions.

Role of cellulose in wood dust-induced fibrosing alveo-bronchiolitis in rat.

Our experiments suggest that in the development of plant dust-induced fibrosing alveobronchiolitis--Scadding's fibrosing alveolitis--the cellulose content of plant dusts has a decisive aetiological role. Namely, the wood dust (pine) and the cellulose induced morphologically identical granulomatous inflammation and fibrosis, whereas the fibre-free extract of wood dust did not cause pathological changes in the lungs. The induction of H2O2 and superoxide anion production, shown in vitro in leucocytes, probably has an important role in the development of fibrosis.

On the structural differences in the membranes between ciliated bronchial and non-ciliated lymphoepithelium in rats.

The membrane structure of bronchial ciliated epithelium and lymphoepithelium was studied in rats by lectin histochemistry. The lymphoepithelium, contrary to bronchial ciliated epithelium, did not contain terminal beta-D-galactose residues. Moreover L-fucose and beta-D-Gal (1-3)-D-GalNAc residues, being masked, could be visualized only after enzymatic digestion of terminal sialic acid. These structural differences in membranes provide a basis for the different functions of bronchial lymphoepithelium.

Studies on the cytotoxicity of ceramic respirable dusts using in vitro and in vivo test systems.

The effect of a number of ceramic raw materials and airborne samples in workplaces has been investigated in vitro [erythrocyte haemolysis, macrophage TTC (2,3,5-triphenyl-tetrazolium chloride) reduction and LDH (lactate dehydrogenase) activity] and in vivo (protein, LDH and phospholipid in cell-free bronchopulmonary lavage). In the in vitro experiments described we were able to distinguish between the dusts causing different types of reaction in the lung. Dusts classified as 'quartz type' in vitro were also cytotoxic in vivo. Dusts have also been divided into three groups according to their degree of hazardous nature, taking into consideration the time before an inflammatory reaction is detected and the degree of inflammatory response as characterized by biochemical parameters. One feature of the test systems which is of practical significance is that in vitro experiments require only 15 mg of dust, and in vivo experiments only 100 mg.

Biological effects of the respirable dusts from ore mines.

The biological effects of ore dusts were examined on peritoneal macrophages in vitro and on rat lung in vivo. The methylene blue adsorption of the dust samples, cytotoxicity, and phospholipid and hydroxyproline content of the lung were determined. It was realized that the examined ore samples had cytotoxic effects and from the in vitro results one can get information about the type of damage to be expected in the lung tissue. It is supposed that the examined ore dusts are less dangerous and slower acting than quartz, but they cause progressive fibrosis. The in vivo biochemical experiments do not give information about the type of damage to be expected in lung tissue; and the data on ore, bentonite, and coal do not differ from each other. To determine the typical biochemical changes caused by quartz, we obtain the most valuable information by determination of lung weight and phospholipid, phosphatidylcholine, and hydroxyproline content.

Experimental pneumoconiosis due to dusts of ore mines. II.

The effect of dead rock dusts from a Hungarian ore mine was studied in long term experiments. Histological changes caused by intratracheally introduced respirable mixed rock patterns (Porphyry, Enargite, Scarnic grained- and drill-cuttings) were examined and compared with standard DQ12 quartz samples 3, 6, 12 and 20 months after treatment. All three dusts proved to be fibrogenic, although in different extent. In the case of Porphyry rock patterns also storage type reaction developed. In the case of Scarnic dusts chronic pneumonitis, focal condensation of the reticular network and very mild fibrosis could be observed. The described changes have confirmed that there is a close connection between the destruction of macrophages and the fibrogenic effect. The free quartz content of the dusts, the amount and ratio of "protective" metal oxides (Al-, Ca-, K-, Mg-, Na-oxides) may be responsible for the differences in the histological reaction and the progression of fibrosis.

Occupational exposure to organophosphorus insecticides and synthetic pyrethroid.

Dermal and respiratory exposure to pirimiphosmethyl, dimethoate and permethrin were determined for applicators and operators in greenhouse tomato spraying operations. Dermal exposure is several times higher than the degree of respiratory exposure. Dermal exposure in terms of different parts of the body shows significant differences. For applicators the exposure of hands, arms and legs is the greatest, and the operators are the most exposed on their hands and to a small extent on legs. This fact should be taken into account when providing the workers with suitable protective clothing. The carefully selected technology of spraying also has great significance in decreasing the degree of exposure. Because of the chronic toxicity of dimethoate, all possible methods should be taken to reduce exposure.

Short term in vivo method for prediction of the fibrogenic effect of different mineral dusts.

The effect of intratracheal introduction of different metal and mineral dusts and the change in activity of pulmonary acidic phosphatase have been studied as a function of time (72 h, 2 weeks, 1, 12, 20 months). The activity and localization of acid phosphatase were compared with the degree of pulmonary damage caused by dusts. The degree of fibrosis was determined on the basis of the composition of cells and fibres, according to Belt and King's classification. Due to the membrane-damaging effect of DQ 12 silica and mixed dusts (enargite and porphyry rock dusts) an increase in acid phosphatase activity of macrophages could be observed at the end of the first month. At the same time non-fibrogenic or only mild fibrogenic dusts (bentonite, corundum, scarnic rock dust) caused a decrease or disappearance of tissue acid phosphatase activity. It has been stated that there is a very close correlation between the change in pulmonary acidic phosphatase activity and the progression of pulmonary fibrosis due to exposure to mineral dusts. The above investigations have been most useful in predicting the subsequent effect of rock patterns, emphasizing at the same time the importance of in vivo long term experiments.