Marc Martel

Asbestos Exposure Dose - Bronchoalveolar Milieu Response in Asbestos Workers and the Sheep Model: Evidences of a Threshold for Chrysotile-Induced Fibrosis

Epidemiological studies of asbestos workers have proposed a linear dose-response relationship between asbestos cumulative exposure indices and the incidence of asbestos-related lung diseases. However, for chrysotile, several studies have not observed such a linear relationship in low exposure workers. To further study the relationship of chrysotile exposure dose - lung tissue response, we designed in the sheep model one experiment of one exposure at variable intensity and one experiment with variable rates of individual exposures. In the first study, 6 groups of 6 sheep were exposed to either 0, 1, 10, 25, 50 or 100 mg chrysotile in 100 ml saline and lung lavage analyses carried out at day 0, 12, 24, 40 and 60 after. Histopathology was done at day 60. In the second experiment, 4 groups of 12 sheep were exposed to 100 ml saline every week or every two weeks, 100 mg chrysotile in 100 ml saline every week or every two weeks. In the second experiment, lung damage was assessed by changes in vital capacity (VC), lung compliance (Cst) and profusion of chest radiographic opacities (PO). On BAL in the control sheep, there were small and transient early increases in total cells/ml, macrophages/ml, neutrophils/ml, lactate dehydrogenase (LDH)/ml, alkaline phosphatase (AK)/ml, beta-glucuronidase (beta G)/ml and procollagen lll/ml. In the 1, 10, 25, and 50 mg asbestos sheep groups, there were no significant differences. However, in the 100 mg asbestos sheep, there were significant sustained increases in total cells/ml (3-6 times controls), macrophages/ml (2-6 times controls), neutrophils/ml (60-600 times controls), LDH/ml (5-10 times controls), AK/ml (1.5-2 times controls), beta G/ml (3-5 times controls), procollagen lll/ml (2 times controls) and IgG (1.5-3 times controls). These data were correlated with histopathological findings. In the sheep exposed weekly to chrysotile, significant changes in VC, Cst and PO occurred at mean cumulative exposure dose of 1.2 g whereas in the sheep exposed every 2 weeks, similar changes in VC, Cst and PO occurred at the cumulative exposure dose of 3.6 g (P less than 0.05). In conclusion, our data suggest that there is a threshold for chrysotile-induced fibrosis. This level cannot be considered adequately in term of cumulative exposure dose but must take into account intensity and rate of individual exposures.

Heterogeneous distribution of alkaline phosphatase and γ-glutamyl transpeptidase in the mouse nephron

In the mouse nephron, ALP and gamma-GT were found to be heterogeneously distributed along the proximal tubule. For both enzymes, 4 large categories of tubules could be recognized on the basis of the enzymatic activity: intense; intermediate; weak; negative. The localization of ALP and gamma-GT was in opposite gradient along the proximal tubule and it apparently corresponded to the 3 sequential segments S1, S2, and S3. In fact, S1 could be identified with certainty because this first portion was often seen attached to the renal corpuscle. This segment displayed a very intense ALP activity (category 1), but a weak one for gamma-GT (category 3). Intermediate tubules for ALP and gamma-GT activities (category 2) seemingly were parts of S2. Those tubules where ALP activity was weak (category 3) while that of gamma-GT was intense (category 1) probably belonged to S3. As a result, it becomes possible to clearly distinguish the segments S1, S2, and S3, not only on a structural and biochemical basis but as well by the localization of brush border enzymes. Distal tubules showed no enzyme activity (category 4). In other respects, the presence of ALP and gamma-GT on the parietal layer of Bowmans capsule strongly suggests that these tall cylindrical cells are morphologically and enzymatically identical to those of the S1 segment, and that they might have similar functional roles.

Effect of aluminum inhalation on alveolar phospholipid profiles in experimental silicosis

In the sheep tracheal lobe model of silicosis, we have recently reported that total phospholipid, lecithin, and phosphatidylglycerol levels were elevated in lung lavage. To investigate further this observation, we obtained complete phospholipid profiles of lung lavage in 10 sheep exposed to saline only (Sa group), 10 sheep exposed to aluminum lactate inhalation only (Al group), 10 sheep exposed to 100 mg Minusil-5 in saline followed by monthly saline inhalation (Si group), and 10 sheep exposed to 100 mg Minusil-5 in saline followed by monthly aluminum lactate inhalation (Si-Al group). The following phospholipid components were measured: total phospholipids, phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylcholine, disaturated phosphatidylcholine, sphingomyelin, and lyso-phosphatidylcholine. All values were comparable in the Sa group, Al group, and Si-Al group. In the Si group, there was a significant increase in total phospholipid to approximately 200% of the control values. The phospholipid profile of this group demonstrated an increase in all of the phospholipid components with some enrichment of the fraction of PG, PE, and PI. We concluded that lung exposure to silica dust significantly increases the concentration of phospholipids in the alveoli. This increase is of a large spectrum of alveolar phospholipids and is completely suppressed by aluminum lactate inhalation.

The lung biological activity of american attapulgite

Attapulgite is a fibrous mineral industrially consumed at the rate of over a million tons per year but the biological activity of the material is not fully known. To evaluate the in vivo toxicity of the fibrous material, we exposed the tracheal lobe of 16 sheep to a single exposure of either 100 ml saline, 100 mg UICC asbestos fibers in 100 ml saline, 100 mg short asbestos fibers in 100 ml saline, or 100 mg attapulgite in 100 ml saline. The animals were studied by bronchoalveolar lavage (BAL) at Days 2, 12, 24, 40, and 60 and by autopsy at Day 60. In the saline-exposed sheep, BAL and lung histology did not change. In the UICC asbestos-exposed animals, we reproduced the BAL changes previously reported (R. Bégin, M. Rola-Pleszczynski, S. Massé, Y. Berthiaume, and G. Drapeau, 1985, Environ. Res. 36, 389-404). In the short asbestos-exposed sheep, there were no significant BAL changes. In the attapulgite sheep, we found significant and sustained increases in total BAL cells (X2, P less than 0.05), macrophages (X2, P less than 0.05), neutrophils (X5, P less than 0.01), fibronectin (X2-3, P less than 0.05), lactate dehydrogenase (X2, P less than 0.05), beta-glucuronidase (X2, P less than 0.05), but BAL cellularity returned to control levels by Day 60 whereas in the UICC asbestos-exposed sheep, it remained significantly above control. Lung histology demonstrated the characteristic peribronchiolar fibrosing alveolitis in the UICC asbestos-exposed sheep, whereas macrophagic alveolitis with minimal airway distortion was seen in the short asbestos-exposed sheep and in all of the attapulgite-exposed sheep but three which had typical peribronchiolar alveolitis quite similar to that observed in UICC-exposed sheep, but of lower intensity. In conclusion, the study documents that attapulgite is not an inert material for lung tissue; it does produce a macrophagic and neutrophilic alveolitis in the early inflammatory phase of lung reaction with peribronchiolar involvement in some sheep. Whether or not this initial attapulgite induced alveolitis leads to lung fibrosis remains to be evaluated in a longer follow-up study of attapulgite-exposed animals.

Bronchoalveolar and Lung Tissue Analyses in Asbestos-exposed Humans and Sheep

Can the human risk of pneumoconiosis be predicted on the basis of animal exposure to industrial dusts? To bring newer insights into this current issue, we have compared bronchoalveolar lavage (BAL) and lung tissue analyses in long term asbestos workers and sheep exposed once in the tracheal lobe to either saline, latex beads, or 100 mg of Canadian chrysotile UICC asbestos fibers. In the sheep, BAL and lung tissue were obtained sequentially up to 8 months after initial exposure.

Enzyme activities of lung lavage in asbestosis.

We analyzed lung lavage supernatant for amylase, lactate dehydrogenase (LD), alkaline phosphatase (ALP), beta-glucuronidase (beta G), and albumin, and a differential count was made of the cellular component of lung lavage in 18 normal controls and 36 long-term asbestos workers of the mines and mills of Québec. The men were concomitantly evaluated by the usual clinical, radiological, functional parameters and 67Ga lung scan. In 7 workers without asbestosis and normal 67Ga scan, lavage enzyme activities, albumin and cell counts were comparable to those of controls. Of 9 without sufficient criteria for asbestosis but increased 67Ga lung uptake, cell counts documented significant increases in the mean number of macrophages (X 2), lymphocytes (X 2) and neutrophils (X 3). Supernatant analyses showed significant increases in amylase (X 4-5), LD (X 2.5), ALP (X 1.5) and beta G (X 2-4). These changes were comparable to those in the lavage of workers with well established asbestosis except that in the latter, the lymphocyte count was slightly lower but the neutrophil count higher (p less than 0.05). These data document that enzyme activities of lung lavage can differentiate asbestos workers with early or late asbestosis from controls and asbestos workers without disease.

Elevated histamine content of lung lavage in human asbestosis

Increased histamine levels in lung lavage have been previously reported in humans with idiopathic fibrosing alveolitis and mast cell proliferation was documented on lung tissue. Similarly, mast cell proliferation has been documented in experimental asbestosis. To evaluate the relevance of these observations to human asbestosis, we performed bronchoalveolar lavage in 10 normal volunteers (group N) and 22 long-term asbestos workers from the mines and mills of Quebec. The 22 asbestos workers were evaluated by standard clinical, functional, radiographic, and gallium-67 lung uptake tests. Five did not have any abnormality suggestive of asbestosis and constituted group A. The 6 in group B were without sufficient criteria for well-established asbestosis, but their lung pressure-volume curve was rigid and67Ga lung uptake was increased; the 11 in group C had well-established asbestosis. In addition to standard parameters of BAL cellularity and biochemistry, we measured histamine levels by fluorometric method. In normals, histamine levels were 1.0 ± 0.3 ng/ml, in group A 1.25 ± 0.37 ng/ml (NS), in group B 2.26 ± 0.86 ng/ml (P < 0.05 B vs A or N), and in group C it was 2.65 ± 0.44 ng/ml (P < 0.05 C vs A or N). BAL eosinophils were absent in normals and group A, 272 ± 136/ml in group B, and 764 ± 350 in group C (P < 0.05 for groups B and C vs normals and group A), but did not correlate with histamine content of BAL. This study provides evidence of an elevated content of histamine in the bronchoalveolar milieu of patients with early and late asbestosis. This finding is of interest in relation to the potential role of the mast cell in the disease process.

Simultaneous visualization of alkaline phosphatase and γ-glutamyl transpeptidase in kidney sections

An histochemical method is presented to simultaneously localize, for the first time, alkaline phosphatase (ALP) and gamma-glutamyltranspeptidase (gamma-GT) in the kidney. The reaction product of ALP activity appears as a dark brown precipitate of lead sulfide, while a bright red copper chelate of an azo dye (Fast blue BBN salt) final product indicates sites of gamma-GT activity. The amalgamation of Mayaharas (ALP) and Rutenbergs (gamma-GT) techniques resulted in the demonstration of various categories of kidney tubules, according to the staining reaction of the cell brush borders: Black tubules where ALP predominates; Intermediate tubules showing a mixture of brown and red precipitates; Red tubules indicating a prevalence of gamma-GT activity; Negative tubules. A possible relation might exist between the staining characteristics observed and the different proximal tubule segments, thus allowing their distinction. In addition, this technique has the advantage to permit the concomitant study of ALP and gamma-GT distribution on the same tissue section instead of serial sections, so reducing the number of manipulations and observations as well as the amount of tissue required.

Identification of proximal tubule segments in the mouse nephron by simultaneous visualization of alkaline phosphatase and γ-glutamyl transpeptidase

ALP and gamma-GT are 2 brush border enzymes that can be individually demonstrated on adjacent sections by the histochemical methods of Mayahara (ALP) and Rutenberg (gamma-GT). On the basis of each enzyme activity, it was possible to recognize different categories of tubules in the mouse nephron. In fact, both enzymes were heterogeneously distributed along the proximal tubule, but in opposite gradients. The various staining intensities probably corresponded to proximal segmentation, but were sometimes difficult to evaluate. A technique was perfected to localize both enzymes in the same tissue section. Since each enzyme produced a distinct type of colored precipitates (ALP: black, gamma-GT: red), 4 categories of tubules could be identified, according to staining characteristics: 1. black tubules where ALP activity was predominant, corresponded to S1 segments, 2. black and red tubules where the 2 activities were about equivalent, were considered as parts of S2, 3. red ones where gamma-GT activity was high, were identified as portions of S3, 4. negative tubules where no activity was apparent, represented distal and straight collecting tubules. In addition to economize time and tissue, this simple technique permits to easily estimate variations in enzyme activities that probably correspond to structural and functional differences in the segments of the proximal tubule.