Alveolar macrophage abnormalities in rabbits exposed to low concentrations of trivalent chromium

Rabbits were exposed by inhalation to a trivalent chromium compound (Cr(NO3)3) at a mean chromium concentration of 0.6 or 2.3 mg/m3 for about 4 months, 5 days/week and 6 hr/day. Light microscopic examination of the lungs revealed that both chromium levels induced a nodular intraalveolar accumulation of enlarged macrophages with granular, eosinophilic cytoplasm. Some macrophages were multinucleated and some showed advanced degenerative changes with disruption of cellular borders and nuclear pyknosis. The changes were most prominent in rabbits exposed to the high concentration and were in some areas associated with a mild interstitial infiltration of lymphocytes, neutrophils, and eosinophils. Electron microscopic examination of macrophages lavaged from the lungs revealed numerous enlarged lysosomes with membranous structures, distinct rounded inclusions, which by X-ray microanalysis were found to contain high amounts of chromium, and increased numbers of laminated inclusions probably representing ingested surfactant. The number of macrophages with a smooth surface was significantly increased. The macrophages in the lung tissue showed the same changes and in addition nodules of multinucleated "giant" cells were found. Morphometric estimation of the volume density of the type II cells did not reveal any significant differences between controls and rabbits exposed to the high concentration of chromium. In spite of the elevated number of laminated structures in the macrophages the amounts of phosphatidylcholine and 1,2-dipalmitoylphosphatidylcholine were not significantly increased in the lung. This indicates a reduced catabolism of surfactant by the alveolar macrophages.     

Lysozyme activity in ultrastructurally defined fractions of alveolar macrophages after inhalation exposure to nickel

Rabbits were exposed to 0.6 mg/m3 of nickel as NiCl2 for about one month. After exposure, alveolar macrophages were lavaged from the lung and divided into three fractions by elutriation. Laminated structures in the macrophages were related to fraction number so that the fractions with the largest cells contained the highest number of structures. The lysozyme activity decreased in unfractionated as well as in fractionated macrophages from nickel exposed rabbits. The decrease was most pronounced in the fraction with the smallest macrophages and smallest number of laminated structures. Therefore the pronounced decrease in lysozyme activity seen in this and earlier studies is not caused by the increased amount of surfactant material. Increased amount of surfactant is a hallmark of nickel inhalation exposure and the surfactant material is responsible for the morphological and metabolic effects of the macrophages. The decreased lysozyme activity is probably a direct effect of nickel on the macrophages.     

Lysozyme activity in ultrastructurally defined fractions of alveolar macrophages after inhalation exposure to nickel.

Rabbits were exposed to 0.6 mg/m3 of nickel as NiCl2 for about one month. After exposure, alveolar macrophages were lavaged from the lung and divided into three fractions by elutriation. Laminated structures in the macrophages were related to fraction number so that the fractions with the largest cells contained the highest number of structures. The lysozyme activity decreased in unfractionated as well as in fractionated macrophages from nickel exposed rabbits. The decrease was most pronounced in the fraction with the smallest macrophages and smallest number of laminated structures. Therefore the pronounced decrease in lysozyme activity seen in this and earlier studies is not caused by the increased amount of surfactant material. Increased amount of surfactant is a hallmark of nickel inhalation exposure and the surfactant material is responsible for the morphological and metabolic effects of the macrophages. The decreased lysozyme activity is probably a direct effect of nickel on the macrophages.     

Effects of nickel dust on rabbit alveolar epithelium

Eight male rabbits were exposed to metallic nickel dust, during 1 month (5 days/week, 6 hr/day) at a concentration of 1.7 mg/m³ (SD 0.5), about 40% of which was respirable. Morphometric measurements on the lungs of exposed rabbits showed about a twofold increase in volume density of type II alveolar epithelial cells compared to eight controls. This increment was about the same as the average increase in phospholipids in the lung parenchyma of the rabbits described elsewhere. The volume density of lamellar bodies within the cytoplasm of the type II cell was not significantly increased. Large amounts of laminated structures, resembling those in the alveolar type II cells, were also found free in the alveoli in the exposed rabbits.     

Lung lesions after combined inhalation of cobalt and nickel

Rabbits inhaled 0.5 mg/m3 Co2+ as CoCl2, a combination of 0.5 mg/m3 Co2+ as CoCl2 and 0.5 mg/m3 of Ni2+ as NiCl2, or filtered air (controls) for 4 months, 5 days/week, 6 hr/day. The pattern of morphological effects on lung tissue and alveolar macrophages after the simultaneous exposure to Co2+ and Ni2+ was a combination of the patterns seen after exposure to Co2+ and Ni2+ alone. However, nickel seemed to potentiate the specific effect of cobalt, i.e., the formation of noduli of type II cells. Further, the increase in phospholipids, especially in 1,2-dipalmitoylphosphatidylcholine, appeared more pronounced after the combined exposure than after the additive combination.     

Effect of iron,cobalt, and chromium dust on rabbit alveolar macrophages: A comparison with the effects of nickel dust

In a previous inhalation study, exposure of rabbits to concentrations of 0.5 to 2 mg/m³ of metallic nickel dust over a period of 4 weeks produced certain reproducible effects on their lungs. The weight and density of the lungs increased as well as the phagocytic activity and size distribution of the alveolar macrophages. Changes in the ultrastructure of the macrophages and the lung lavage fluid were also seen. In the present study, groups of four rabbits each were exposed to iron dust (0.4 and 1.4 mg/m³), cobalt dust (0.2 and 1.3 mg/m³), and chromium dust (0.6 and 3.1 mg/m³). None of these metals gave the same pattern of effects as the nickel dust. The particles, including the nickel particles, were studied with regard to morphology, surface and bulk composition, specific surface area, and solubility in fluids. The nickel on the nickel particles, together with manganese on the chromium particles were the most soluble components. Otherwise, the nickel particles did not differ substantially from the other particles.     

Rabbit lung after inhalation of soluble nickel: II. Effects on lung tissue and phospholipids

Rabbits were exposed to soluble nickel chloride, about 0.3 mg/m³ as nickel, for about 1 month (5 days/week, 6 hr/day). The upper lobe in the left lung was examined with light microscopy and samples from the left lower lobe with electron microscopy. Nodular accumulation of macrophages and laminated structures occurred in alveoli of all exposed rabbits, but otherwise no inflammatory reactions. Volume density of the alveolar epithelial type II cells was doubled due to increased cell number as well as cell volume. The tissue content of phospholipids, determined in the lower lobe of the left lung, had increased by about 40%, mainly due to elevated disaturated phosphatidylcholines. The effect pattern was almost identical to that seen earlier in inhalation experiments with metallic nickel dust, strongly indicating that nickel ions are responsible also for the changes seen after exposure to metallic nickel. This suggests that all nickel compounds may produce the pathological condition, which is similar to that seen in the disease pulmonary alveolar proteinosis.     

Toxicity of hexavalent chromium to the alveolar macrophage in vivo and in vitro

In vivo and in vitro systems were used to evaluate hexavalent chromium toxicity to alveolar macrophages. Rat alveolar macrophages were exposed to 2 micrograms calcium chromate (CaCrO4, insoluble) or 2 micrograms chromium trioxide (CrO3, soluble) in live animals, in vivo, and in tissue culture, in vitro, collected by lavage from the lung. Chemiluminescence and oxygen consumption were measured as indicators of toxicity. Trypan blue dye exclusion was used to determine macrophage viability. In vivo exposure of the macrophage to either chromium compound showed no toxic effects at a 2-micrograms dose. Macrophages exposed in tissue culture, however, had values significantly different from controls. The untreated controls for both exposure methods were compared to evaluate differences resulting from methods alone.     

Rabbit lung after combined exposure to soluble cobalt and trivalent chromium.

Eight rabbits were exposed to 0.7 +/- 0.4 mg/m3 Co2+ as CoCl2 and 1.2 +/- 0.7 mg/m3 Cr3+ as Cr(NO3)3 (group Co + Cr), eight to 0.6 +/- 0.5 mg/m3 Co2+ (group Co), and eight to filtered air (control group), for 4 months, 5 days/week, and 6 hr/day. All rabbits in group Co + Cr and group Co showed nodular aggregation of alveolar epithelial type II cells. Volume density of the type II cells was significantly higher in group Co + Cr than in group Co and the control group. There was intraalveolar macrophage accumulation in seven rabbits in group Co + Cr, one in group Co, and one in the control group. In lavage fluid the numbers of macrophages and the percentage of these cells with smooth surface and intracellular surfactant-like inclusions were more increased in group Co + Cr than in group Co as were oxidative metabolic and phagocytic activities of the macrophages. Total phospholipids, phosphatidylcholines, and especially 1,2-dipalmitoylphosphatidylcholine was markedly increased in group Co + Cr whereas only 1,2-dipalmitoylphosphatidylcholine was slightly increased in group Co. One mechanism behind the high amount of surfactant phospholipids in group Co + Cr seems to be an enhanced production of surfactant by the type II cells. Another mechanism is probably that Cr3+ reduces the capacity of alveolar macrophages to catabolize surfactant. The results imply that it is important to investigate effects of combinations of cobalt and chromium in the occupational environment.     

Analyses of chromium and nickel in human pulmonary tissue

After inhalative occupational exposure to certain compounds containing nickel and chromium (mostly over many years), an accumulation of these metals may occur in the lung tissue. This is of particular importance, both from a toxicological point of view and with regard to expert reports, since certain nickel and chromium compounds may induce lung cancers. In the context of this study, samples of pulmonary tissue from 34 deceased persons from the Bergen area (Norway) were analysed by atomic absorption spectrometry with regard to their content of chromium and nickel. The deceased comprised 21 men and 13 women. In 15 cases, death resulted from lung cancer; in the other 19 deceased, there was no indication of a malignant disease of the airways. The concentrations of nickel found in the lung tissue do not differ between patients with lung cancer and patients with healthy lungs. On the other hand, the concentration of chromium in the pulmonary tissue in the patients who had died of lung cancer and who had all been inhalative smokers, are higher (statistically significant) than in the nonsmokers or in those with healthy lungs. An accumulation of these two metals in the tumor matrix could not be detected. Both the average nickel and the average chromium concentrations were higher in the persons who had probably been exposed occupationally. Considering the present state of scientific knowledge, the aspects relevant to expert reports which result from the analyses of metals in the pulmonary tissue are discussed.     

Analyses of chromium and nickel in human pulmonary tissue

Summary After inhalative occupational exposure to certain compounds containing nickel and chromium (mostly over many years), an accumulation of these metals may occur in the lung tissue. This is of particular importance, both from a toxicological point of view and with regard to expert reports, since certain nickel and chromium compounds may induce lung cancers. In the context of this study, samples of pulmonary tissue from 34 deceased persons from the Bergen area (Norway) were analysed by atomic absorption spectrometry with regard to their content of chromium and nickel. The deceased comprised 21 men and 13 women. In 15 cases, death resulted from lung cancer; in the other 19 deceased, there was no indication of a malignant disease of the airways. The concentrations of nickel found in the lung tissue do not differ between patients with lung cancer and patients with healthy lungs. On the other hand, the concentration of chromium in the pulmonary tissue in the patients who had died of lung cancer and who had all been inhalative smokers, are higher (statistically significant) than in the nonsmokers or in those with healthy lungs. An accumulation of these two metals in the tumor matrix could not be detected. Both the average nickel and the average chromium concentrations were higher in the persons who had probably been exposed occupationally. Considering the present state of scientific knowledge, the aspects relevant to expert reports which result from the analyses of metals in the pulmonary tissue are discussed.     

Nodular accumulation of type II cells and inflammatory lesions caused by inhalation of low cobalt concentrations

Eight rabbits were exposed to 2 mg/m3 and eight to 0.4 mg/m3 of cobalt as CoCl2 for 14-16 weeks (5 days/week, 6 hr/day). Eight rabbits were used as controls. Light microscopic examination of the lungs showed nodular accumulation of alveolar type II cells in all cobalt-exposed rabbits. Abnormal macrophage reaction was observed in all eight rabbits exposed to the high dose and in five of the eight exposed to the low dose. Interstitial inflammation was present in all the rabbits exposed to the high dose and in half of the rabbits exposed to the low dose. Ultrastructural morphometric examination revealed no significant increase in the volume density of type II cells in the cobalt-exposed animals, as the nodular accumulation of these cells was balanced by an increased number of interjacent fields devoid of type II cells. In cobalt-exposed lungs, there was focal swelling of both type I and type II cells, and some of the latter lacked microvilli. The effect pattern after CoCl2 exposure was thus clearly different from those patterns seen after exposure to other toxic metals. We speculate that nodular accumulation of type II cells represents the primary lesion in CoCl2-exposed lungs and that the proliferation of such cells in interjacent areas might be suppressed by a feedback mechanism regulating surfactant production in the terminal airspaces.     

Development of an inhalation system of high melting point metal fumes and its use for exposure of rats to chromium and nickel fumes

An experimental inhalation system was developed for fumes generated from powders of high melting point metals such as chromium, nickel, manganese and iron. The system consisted of a plasma flame metal sprayer as a fume generator, a granular bed type fume collector, a fluidized bed aerosol generator, an exposure and a control chamber of a horizontal-flow type and inhalant monitoring and controlling units. Performance of the chambers was ensured by a distribution test using flyash as a test aerosol. Using this system, rats were exposed to chromium fumes for one week or to nickel fumes for two months. The exposure concentrations of the chromium and nickel fumes were 1.85 +/- 0.55 mg/m3 and 0.51 +/- 0.15 mg/m3 (mean +/- SD), near the target levels of 2 mg/m3 and 0.5 mg/m3, respectively. The mass median aerodynamic diameter and the geometric standard deviation of the chromium fumes were 2.1 microns and 2.00, respectively. Those of the nickel fumes were 3.7 microns and 1.74, respectively. Species analysis of these fume particles revealed that 26.4% of the total chromium was hexavalent and the residue was trivalent and that 1-3% of the total nickel was nickel(III) and the residue was nickel(II). Inhaled-metal concentrations in the lungs showed steady increases with the exposure periods and were within the normal range of variation. On the basis of these results, it is concluded that this system is useful for long-term inhalation experiments using high melting point metal fumes.     

Rabbit alveolar macrophages after inhalation of hexa- and trivalent chromium

Rabbits inhaled aerosols of hexavalent chromium (Na2CrO4) and trivalent chromium (Cr(NO3)3) at concentrations of 0.9 and 0.6 mg/m3 of the metal, respectively, for 4-6 weeks (5 days/week and 6 hr/day). Significantly more macrophages were obtained from the lungs of rabbits exposed to Cr(VI) but not from rabbits exposed to Cr(III) as compared with the controls. Macrophages from rabbits exposed to Cr(III) showed several conspicuous changes. About one-third of the macrophages contained round dark inclusions, 0.5-1.5 micron diameter, rich in chromium. Most cells had very large lysosomes which contained membranous fragments of different sizes surrounded by a more homogeneous matrix. Laminated inclusions similar to the lamellar bodies in the type II cells increased in number as did the percentage of cells with a smooth cell surface. Also macrophages from rabbits exposed to Cr(VI) showed morphological changes. The most pronounced one was enlarged lysosomes which contained short lamellae and electron-dense patchy inclusions. Only Cr(III) produced functional changes of the macrophages. The metabolic activity measured by reduction of nitroblue tetrazolium was increased and the phagocytic activity reduced.     

Effects of long-term nickel dust exposure on rabbit alveolar epithelium

Lungs from rabbits exposed to 1 mg/m³ metallic nickel dust for 3 and 6 months, 5 days/week, 6 hr/day, were examined by light and electron microscopy. Morphometric measurements showed threefold and twofold increases in volume density of alveolar type II cells after 3 and 6 months, respectively. The type II cells were extremely large, especially in the 3-month-exposed rabbits, and contained large amounts of lamellar bodies. Alveoli were rich in large, foamy macrophages and a granular material which previously has been found to consist mainly of phospholipid. This material probably represents an overproduction of surfactant by the type II cells. The 6-month-exposed rabbits had foci of pneumonia, suggesting a higher susceptibility to pulmonary infections, possibly due to a decreased function of alveolar macrophages.     

Investigations on the quantitative determination of nickel and chromium in human lung tissue

Summary Nickel (Ni) and some of its relatively insoluble compounds as well as chromates may be able to induce cancer in the region of the lungs, as well as in the nose and paranasal sinuses after occupational exposure. Latency periods may amount to 20 years and more. The results of recent investigations have shown that these metals cumulate in the lung tissue after inhalation of relatively insoluble chromium and nickel compounds. The quantitative detection of these heavy metals in samples of pulmonary tissue hence permits the amount of past exposure to be estimated. To establish the normal values, samples of pulmonary tissue from 30 normal subjects were investigated for chromium and nickel content. The samples were taken from different segments and lobes of the lungs, taking topographical anatomical criteria into consideration. In addition, 15 persons who had formerly been exposed to nickel and/or chromium (11 nickel refinery workers, of whom 10 had died of lung cancer, 2 stainless steel welders, 1 foundry worker, 1 electrical technician) were also investigated. From the results of 495 tissue samples from the normal group, median chromium concentrations between 130 and 280 ng/g were calculated, with median nickel concentrations of 20–40 ng/g (wet weight). If these values are related to the nickel concentrations measured in refinery workers, values 112-5,860 times higher were found. The concentrations were about 500 times higher than normal for nickel, and about 60 times higher than normal for chromium in the stainless steel welders. For the foundry workers who died of lung cancer, chromium and nickel concentrations in the normal range were calculated, with the exception of the nickel concentrations in the upper and lower lobes of the right lung. The very high nickel concentrations found in the samples of lung tissue from former nickel refinery workers should be regarded as a guideline with regard to the appraisal of the causal relationship between lung cancer and occupational exposure to relatively insoluble nickel compounds. This result is also supported by epidemiological investigations on this subgroup and must thus be considered etiologically conclusive. For the welders, chromium and nickel concentrations were found that were markedly above normal, but as yet there is no epidemiologically reliable verification for the increased occurrence of malignancies in this occupational group. On the basis of present scientific knowledge, no indications were found of relevant chromium and/or nickel exposure of the lung tissue that might be able to induce lung cancer in either foundry workers or for electric technicians.Dedicated to Professor V. Becker on his 65th birthday     

Fibronectin concentrations in lung lavage fluid after inhalation exposure to low levels of metals

Groups of rabbits were exposed by inhalation to chlorides of cobalt, nickel, and manganese as well as to tri- and hexavalent chromium at metal concentrations ranging from 0.4 to 3.9 mg/m3 for 1-4 months (5 days/week, 6 hr/day). Fibronectin content and lysozyme (muramidase) activity in lavage fluid were measured after all treatments and in alveolar macrophages after treatment with nickel chloride. In the lavage fluid no marked changes were seen in fibronectin content and lysozyme activity after exposure to tri- or hexavalent chromium or manganese. Nickel exposure significantly decreased the lysozyme activity in the lavage fluid and in the macrophages whereas the fibronectin content was unchanged in the lavage fluid and significantly increased in the macrophages. Both fibronectin content and lysozyme activity were increased markedly in the lavage fluid after cobalt exposure.     

Protective effect of selenium on lung cancer in smelter workers.

A possible protective effect of selenium against lung cancer has been indicated in recent studies. Workers in copper smelters are exposed to a combination of airborne selenium and carcinogens. In this study lung tissue concentrations of selenium, antimony, arsenic, cadmium, chromium, cobalt, lanthanum, and lead from 76 dead copper smelter workers were compared with those of 15 controls from a rural area and 10 controls from an urban area. The mean exposure time for the dead workers was 31.2 years, and the mean retirement time after the end of exposure 7.2 years. Lung cancer appeared in the workers with the lowest selenium lung tissue levels (selenium median value 71 micrograms/kg wet weight), as compared with both the controls (rural group, median value 110; urban group, median value 136) and other causes of death among the workers (median value 158). The quotient between the metals and selenium was used for comparison: a high quotient indicating a low protective effect of selenium and vice versa. The median values of the quotients between antimony, arsenic, cadmium, lanthanum, lead, chromium, and cobalt versus selenium were all numerically higher among the cases of lung cancer, the first five significantly higher (p less than 0.05) in 28 of the 35 comparisons between the lung cancer group and all other groups of smelter workers and controls. The different lung metal concentrations for each person were weighted according to their carcinogenic potency (Crx4 + Asx3 + Cdx2 + Sbx1 + Cox1 + Lax1 + Pbx1) against their corresponding selenium concentrations. From these calculations the protective effect of selenium was even more pronounced.     

Protective effect of selenium on lung cancer in smelter workers

A possible protective effect of selenium against lung cancer has been indicated in recent studies. Workers in copper smelters are exposed to a combination of airborne selenium and carcinogens. In this study lung tissue concentrations of selenium, antimony, arsenic, cadmium, chromium, cobalt, lanthanum, and lead from 76 dead copper smelter workers were compared with those of 15 controls from a rural area and 10 controls from an urban area. The mean exposure time for the dead workers was 31.2 years, and the mean retirement time after the end of exposure 7.2 years. Lung cancer appeared in the workers with the lowest selenium lung tissue levels (selenium median value 71 micrograms/kg wet weight), as compared with both the controls (rural group, median value 110; urban group, median value 136) and other causes of death among the workers (median value 158). The quotient between the metals and selenium was used for comparison: a high quotient indicating a low protective effect of selenium and vice versa. The median values of the quotients between antimony, arsenic, cadmium, lanthanum, lead, chromium, and cobalt versus selenium were all numerically higher among the cases of lung cancer, the first five significantly higher (p less than 0.05) in 28 of the 35 comparisons between the lung cancer group and all other groups of smelter workers and controls. The different lung metal concentrations for each person were weighted according to their carcinogenic potency (Crx4 + Asx3 + Cdx2 + Sbx1 + Cox1 + Lax1 + Pbx1) against their corresponding selenium concentrations. From these calculations the protective effect of selenium was even more pronounced.     

Morphologic and physiologic response of lungs to steroid and cigarette smoke: an animal model

The combined effects of cigarette smoke inhalation and hydrocortisone acetate (HCA) treatment induce prominent abnormalities in lungs of C57BL/6 male mice. These abnormalities include (1) a marked reduction of pulmonary macrophage population which is normally elevated by smoke inhalation, (2) an accumulation of surfactant and flocculent material in alveoli, (3) a decrease in alveolar space surrounded by normal septal tissue, and (4) an increase in hypertrophied alveolar parenchyma. Concomitant with altered lung morphology, lung volume and gas diffusing capacity were significantly compromised in animals subjected to smoke exposure and steroid treatment. It was found that smoke inhalation or HCA administration alone had no ill effects on the animals. The data presented indicate that manifestation of pathologic conditions resembling pulmonary fibrosis and pulmonary alveolar proteinosis is a result of cigarette smoke-drug interaction. The information reported provides a basis for an animal model which might be applicable to assessment of factors related to smoke inhalation and development of pulmonary disorders.