Biochemical studies on the toxicity of slate mine dust.

As part of a detailed experimental study of the pathogenicity of disease of slate dust workers, the early biochemical changes in rat lung from 1 to 90 days after intratracheal inoculation of slate dust of particle size below 5 micron were investigated. A severalfold increase in free cell population (initially macrophages) was elicited by the dust. The free activity of acid phosphatase tended to increase along with a break of lysosomal latency with increasing exposure period. However, actual release of enzyme activity into the acellular fraction was low. The phospholipid content varied both in cellular and acellular fractions, indicating altered turnover of membrane lipids and surfactants. At advanced periods of the study, sialic was found to be released into the acellular fraction, indicating membrane damage. Considerable decrease in glucose-6-phosphate dehydrogenase activity and free sulfhydryl content and enhanced osmotic fragility of erythrocytes were also recorded. These results indicate the potential toxicity of slate mine dust.


Introduction
The wandering cells of the lung lavage, the alveolar macrophages, are the first line of the body's defense against foreign materials and are often the first casualties of the biological effects of dusts (1,2). Their plasma membranes are damaged by the cytotoxic dusts in vitro and in vivo (3)(4)(5). Biochemical studies have also revealed that these cytotoxic dusts cause an enhanced turnover of pulmonary surfactant (6,7). The cytotoxic effects of slate dust (from Mandsaur, India) with the use of erythrocytes, in an in vitro model system, have already been described (8). In the present report, we present the cytotoxic effects and early biochemical changes in vivo in the lung lavage and in the blood of albino rats exposed to slate dust. Center (ITRC). Dust with particle size below 5,m was prepared according to the procedure described by Zaidi et al. (9).

Animal Experimentation
Female albino rats from the ITRC colony, weighing 150-200 g, were used. The dust sample and 0.15 M NaCl were sterilized separately by autoclaving at 15 lb for 15 min. Seventy adult albino rats were administered intratracheally 50 mg of dust suspended in 1 mL of 0.15 M NaCl (9). Another 70 rats received only 1 mL of 0.15 M NaCl and served as controls. The animals were maintained on a commercial pellet diet supplied by Hindustan Lever Ltd., India, and were sacrificed 1, 2, 4, 8, 16,30 and 90 days after treatment. Blood was collected from the jugular vein into heparinized centrifuged tubes. The lung lavage was collected according to Brain (10).

Treatment of the Lavage
Washings of lungs of each rat were pooled. An aliquot was taken for counting the free cell population. For separating cells from the medium, the lavage was centrifuged at 300g for 20 min in cold. The pellet was resuspended in 2.5 mL of 0.15 M NaCl. The supernatant and washings constituted the acellular fraction while the suspension of the sediment formed the cellular fraction.

Estimations
Acid Phosphata The assay system (11) contained 2.0 mL substrate mixture (0.5 g sodium (i glycerophosphate and 0.4 g sodiumdiethylbarbiturate dissolved in water, pH adjusted to 5.0 with 1N acetic acid and made up to 100 mL), 0.2 mL cell suspension and 0.3 mL water. Incubation was carried out at 370C for 30 min and reaction stopped by the addition of 1.5 mL 10% trichloroacetic acid (TCA). After centrifugation, aliquots from supernatnant were taken for orthophospate determination (12). The latency of the acid phosphatase in lysosomes was abolished by preincubation with 0.5% Triton X-100.
Slci Acid The procedure of King et al. (13) was followed for the estimation of silicic acid. Shlc Acid Sialic acid contents were determined by the method of Krantz and Lee (14).
Phospholipid& Phospholipids were extracted according to Folch et al. (15) and determined by the method of Wagner et al (16).

Studies on Blood
The heparinized blood was centrifuged and the plasma separated. The cells were washed three times with 0.15 M NaCl and suspended in the same medium to requisite concentration.
ProteiL Protein content was estimated by the method of Lowry et al. (19) with bovine serum albumin (Sigma Type V) as standard.
C(emiak All the chemicals used were either BDH, AnalaR or E. Merck, extra pure.

Statistics
All values are the result of at least six determinations per point. Standard deviations were less than 10% of the mean value.

Results and Discussion
Free Cell Population Changes in the number of free cells in the lung are shown in Figure 1. Untreated animals had between 6.017 and 9.723 x 106 free cells. Slate dust stimulated the number of free cells even after 24 hr of exposure and the effect was at its maximum after 4 days.
Thus, during the early response to a foreign body, there is enhanced assembly of wandering cells from circulation. By the end of 90 days, the increase in cell population is, however, only 2-fold. Decrease of cells with time may be indicative of the potential cytotoxic effect of the dust.

Acid Phosphatase Activity
Changes in the overt activity of acid phosphatase at different times of exposure are shown in Figure  2. Only negligible free activity of acid phosphatase was detected in the acellular fraction obtained from the control animals. Thus, in none of the animals was the activity more than 8% of the total activity. The cells were mostly undamaged and the lysosomes retained their membrane-bound latency. The small amount of free activity detected could be attributed to some membrane damage to the cells during preparation. This activity was relatively constant throughout the experiment. It can be assumed, therefore, that the changes encountered with lavage from the experimental animals represent a true physiological adaptation to stress induced by slate dust.
Lavage from dust-treated animals showed uniformly high phosphatase activity. This trend of enhanced activity persisted during the entire period of treatment.
In the cellular fraction, the increase in acid phosphatase was steady up to 8 days, but declined thereafter.. Even then, at all stages, the activity was relatively higher in the experimental group. In Figure  3, in the case of controls, the activation by 0.5% Triton-X 100 exposure was 3 ± 0.37 for the entire period. Beyond 4 days, the extent of activation tended to decrease for the experimental animals, gradually Experimental that the change in free activity was correlated with the gradual loss of latency of the cellular function. The latency is not totally broken even at the most advanced state since a doubling of activity occurs on detergent treatment. Apparently, slate dust in vivo under the conditions of the present experiment caused partial macrophage membrane damage. Thus, in spite of its high cytotoxicity and hemolytic activity, slate dust is apparently not able to break Activation is expressed as activity with Triton X-100/activity without detergent. 1

Phospholipids in Lung Lavage
The phospholipid content of cellular fraction was almost the same in both cases, except at 4 days ( Table 1). When phospholipid content was compared, it varied from 0.92 to 1.86 Mg/106 cells for controls, the minimum and maximum being at 2 days and 30 days. For the slate-treated animals, the values were in the range of 0.51 and 0.84, the two extreme values being recorded at 2 days and 16 days. Thus, phospholipid contents of macrophages vary considerably during the cellular response to slate dust; for instance, being 75% higher on day 4, presumably due to larger number of cells.
In the acellular fraction, the treated animals had only a third of the phospholipid as compared to controls at 1 and 2 days. Some absorptive removal of nonmembrane phospholipid by the dust may account for it. Subsequently, the phospholipid in this fraction was enhanced. This may be due to availability of more cells and damage to membranes, so that by 8 days it was almost similar in controls. Subsequently, the content increased dramatically and at 90 days, it was approximately 300% of control. The differences become more marked when the phospholipid contents of acellular fraction are expressed as a percent of total content of lavage. In all the stages, 80-87% of total phospholipid was in acellular fraction in controls. For the experimental group, it was distinctly lower, i.e., 58, 64, 69 and 80% at 1, 2, 4 and 8 days, respectively. Later, it increased, reaching 91 and 94% at 30 and 90 days, respectively. Initial decrease could be due to its inactivation by macrophage phospholipase (20) and later increase a defense adaptation to protect cells against damage.

Sialic Acid Contents
After 2 days, sialic acid content per 106 cells of cellular fraction decreased, indicating alerations in biomembrane function and turnover. In acellular fraction of normal lung lavage, sialic acid was not detectable. In acellular fraction of treated animals, it appears afrer 4 days of dust exposure and increased up to 90 days ( Table 2).
Since sialic acid is known to be released from membranes during cytotoxic effects of membrane damaging toxicants, this may be indicative of damage of macrophages. Removal of sialic acid could only be due to activation of sialidase (21), and this removal could make the cells even more vulnerable to damage. It will be interesting to explore the biochemical significance of this release as an index of cytotoxicity and of serum sialic acid levels in the development of slate toxicity.

Protein Content
In the cellular fraction there was an increase in protein content in experimental animals, as compared to controls, up to 4 days (Table 3). This increase of over 2-fold could only indicate additional synthesis of protein and not absorption of acellular protein on dust. The macrophages assembled for Table 2. Total sialic acid contents in the lung lavage of normal and slate dust-exposed rats. aValues are expressed as ;Ag sialic acid/10' cells in cellular fraction whereas total sialic acid content is given in acellular fraction. bNot detectable.  foreign body response could accomplish de novo synthesis of additional and new proteins to meet metabolic requirements for phagocytosis. Also, expressed in terms of whole lavage from one animal, the protein content is not merely due to increased cell populations. The maximum increase was at 4 days, 3-fold on a cell basis and 13-fold on a whole animal basis. In the acellular fraction also, there was a general increase in protein content, especially at 16 days, indicating release of cellular constituents. This is in agreement with the data on acid phosphatase and sialic acid. As with acid phosphatase, the acellular fraction contained only a small proportion of protein so that membrane damage and release is not very high.

Changes in Blood
In order to see whether the changes in lavage are reflected in circulation, some of its parameters were measured (Table 4). Erythrocyte Glu-6-P dehydrogenase tended to decrease, the maximum being 40% at 16 days. This, along with the RBC fragility in vtivo (unpublished data), shows membrane sensitivity to the hemolytic effects of slate dust (22). The reduced glutathione content in RBC and plasma proteins showed a general tendency for decline in spite of the wide fluctuation in values from sample to sample. Thus, passage through dust-laden lungs and any impact of the lung lesion on the hemopoietic system could be reflected in blood chemistry.

Conclusion
In the case of slate dust exposure, the initial foreign body response consists mainly of the collection of scavenger cells and reaches a maximum at 4 days. Subsequently, as a result of cytotoxicity and possibly any damage to the lung tissue and the hemopoietic system, the number of free cells decreases with the progress of the toxic conditions. The increase in acid phosphatase activity in the acellular fraction and the gradual decrease of activation in the cellular fraction suggest a cytotoxic ac-tion of slate dust in vivo. The release of sialic acid in the acellular fraction also indicates the alterations of biomembranes.
The above results, along with parallel studies on the hemolytic and solubility properties of slate dust (8), clearly suggest its toxicity in vivo for the pulmonary system in macrophages and in the blood. These facts and the similarity of many parameters to a cytotoxic, carcinogenic dust chrysotile, indicate that further in-depth in vivo studies are necessary to elucidate the total toxic action of slate dust and the negative impact of this agent on public and occupational health.
The above work forms part of a project entitled "Biochemical Effects of Particulate Air Pollutants on Lung," sponsored by the Environmental Protection Agency, U.S.A., and designated grant PRI-503-2. Thanks are due to Dr. Angelo Turturro in the preparation of this manuscript.