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

Ann. ocrq. Hyg., Vol. 41. No. 5. pp. 51%X26, 1997 0 1997 British Occupational Hygme Smety by Elsevier Science Ltd All ri hts reserved Printed in E rest Britain 0003-4878197 517.00+0.00

Published

PII: s0003-4878(97)00014-8

A STUDY

ON LUNG TOXICITY METAL DUSTS

OF RESPIRABLE IN RATS

HARD

Z. Adamis,* E. Tatrai, K. Honma, J. Karpati and G. Ungvary National

Institute

of Occupational Health, Budapest, H-1450, of Pathology, Dokkyo Medical University, (Received

in final form

13 March

P.O. Box 22, Hungary Mibu, Tochigi, Japan

and Department

1997)

Abstract-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 viva 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. 0 1997 British Occupational Hygiene Society. Published by Elsevier Science Ltd.

INTRODUCTION

Hard metal alloys are produced in metallurgical factories. In most casesthese alloys contain tungsten carbide and cobalt as well as titanium or tantalum carbide and are used for machining tools of near-adamantine hardness (Kusaka et al., 1986). The technology of hard metal production is a complex process involving -sieving, mixing and grinding of raw materials; -preparation for pressing; -pre-sintering; -moulding; -sintering; and -machining of the finished product (International Labour Office, 1983). As is known, exposure to hard metals may give rise to respiratory diseases,for example to fibrosing alveolitis and bronchial asthma (Beth et al., 1962). The aetiology has still not been clarified and, similarly, little is known about the early inflammatory and pathological pulmonary changes. This has prompted us to study in rat lungs the effect of respirable dust samplescollected in the various stages of hard metal production in a plant in Hungary.

*Author

to whom

correspondence

should

be addressed. 515

516

Z. Adamis MATERIALS

Experimental

AND

et al. METHODS

animals

Male Sprague Dawley rats (230-260 g body weight) were used (Charles River Hungary, Isaszeg). They were divided into five groups of five animals in each. The animals were kept on standard chow diet (Charles River) and tap water ad Zibitum. Dust samples Respirable samples were collected in three stages of the manufacturing process i:n a hard metal-producing plant, that is, -finished powder for pressing (FP), -heat-treated, pre-sintered sample obtained at 800°C (PH), -wet grinding of sintered hard metal obtained by heat treatment at 1200°C G--N. For sample collection a Persometer (Hungary) was applied (Persometer is a two stage separating equipment with characteristics corresponding to the Johannesburg curve). All particles were 0.1-5.0 pm in size, with average size around 1 pm (Philips SEM 50.5 scanning electron microscope). The particles were angular and oval i:n shape. Mineralogical composition of the samples is shown in Table 1. Also the amount of material dissolved from the samples by nitric acid was determined with a Jariel Ash 9000 type ICP-AES spectrometer. The samples were kept in a 1: 1 mixture of nitric acid (approx. 35%) and distilled water in a boiling water bath for 10 mm, then incubated at room temperature for 24 h. The soluble components were then determined from the filtrate. For pulmonary lavage and biochemical experiments, quartz DQ 12 (DQ) containing 87% crystalline quartz was used as positive control (Robock, 1973). Intratracheal treatment and bronchoalveolar lavage The dust samples were suspended in sterile physiological saline, then sonicateld by ultrasound for 5 min (Soniprep 150, MSE). The rats were anaesthetised by sodium pentobarbital(40 mg per 100 g body weight). The animals were treated with dust samples of FP, PH, HM and DQ (1 and/or 3 mg per rat). In a preliminary study we administered to the animals 15 and 5 mg of dusts of FP, PH and HM (per rat). Samples were injected into the trachea of rats. All samples were suspended in 0.5 ml sterile physiological saline. The control animals were administered 0.5 ml of sterile physiological saline solution. Table

I. Mineralogical

composition of respirable hard metal industry

samples

Material

FP (%)

PH (%)

Tungsten carbide Cobalt Titanium carbide Silicium carbide Iron Sand (silica)

90 8 I 0.1 -

90 8 I 0.1 -

Analysis was carried out by a Zeiss Q 24 spectrograph. Materials and Methods.

collected

HM

in the

(%) 40 3 5 25 20 5

Abbreviations

as in

Lung

toxicity

of respirable

hard metal

dusts

517

Bronchoalveolar lavage (BAL) was performed 1, 4, 7 and 30 days after intratracheal treatment. The rats were sacrificed under ether anaesthesia and exsanguinated by cutting the renal arteries. A cannula was introduced into the trachea, the lungs left in situ were washed with Tyrode solution containing 3x5 ml sterile 4 U ml-i heparin, and massaged gently. After 2 min the fluid was withdrawn (12 ml was obtained on average), and centrifuged (at 250 g) for 10 min. The cell-free supernatant was used for further investigation. Biochemical

and histological

studies

From the washing fluid and the lung tissue, lactate dehydrogenase (LDH), acid phosphatase (AP), protein and phospholipid were determined. LDH was measured by the method of Wroblewski and La Due (1955), AP by that of Sommer (1954) protein by that of Lowry et al. (1951) and phospholipid by that of Raheja et al. (1973). All experimental results were calculated on the basis of analyses of samples taken from at least five animals (mean&SD). Significance was calculated by the multiple f-test. Histological examination: lungs and regional lymph nodes were fixed in 8% buffered neutral formalin (pH 7.4), then embedded in Paraplast (Sigma, USA) and stained with hematoxylin-eosin; with Giemsa for the analysis of cell composition, elastic van Gieson staining and Gomiiri’s silver impregnation for the detection of fibres .

RESULTS

After treatment with PH in a dose of 15 mg per rat, 7 of the 10 animals treated died within hours. On injection of a dose of 5 mg per rat, 6 out of 15 animals died the following day. The other two hard metal samples (FP and HM) given in doses of 5 mg per rat and 15 mg per rat, resp., were well tolerated by the animals. Therefore during these experiments each animal was given 1 or 3 mg, respectively. The experiment carried out 1 mg doses of the dusts proved that the acute effect of the samples collected during hard metal production was limited to the first week. Therefore, experiments with higher doses were carried out on days 4 and 7. In the BAL, on administration of 1 mg dust, LDH increased after 1 day to the effect of PH, HM and DQ. PH and DQ exceeded the control values also after days 7 and 30, while HM increased only after 30 days. The non-heat-treated sample (FP) showed roughly the same LDH activity as the control. On administration of 3 mg dust LDH activity increased after 4 days in the following order: PH>HM>DQ>FP>C After 7 days, significant increase in activity was caused only by DQ (Fig. 1). AP activity significantly increased 1 day after exposure to FP, HM and DQ. After 7 days, all dust samples exceeded the control values by 75-95%. After 30 days, significant increase was induced only by DQ (Fig. 2). Protein showed significant elevation due to DQ and PH after 7 days. After 30 days, only DQ was effective. In the case of higher dosage, protein elevated as a result of treatment with FP and PH after 4 days (Fig. 3).

518

Z. Adamis

f

Dose:

200

et al

1 mg /rat

1 day

30 days

7 days

T -,.

180 160

C

Dose:

FP

RI

HM

W

C

FP

PH

HM

3mg/rat

200

I. days

7 days

180

t

T

Txx

=eo 0 J6o

I 40 20 lJTll!l C

FP

6 w

c

m F:P

PH

HM

Fig. 1. LDH activity I, 4, 7 and 30 days after intratracheal injection of 1 and 3 mg dust in rat lung lavage fluid. C, control; abbrev. for dust samples see Materials and Methods. All values are meanfSD. Statistically significant: *P < 0.05, **P < 0.01.

DQ induced significant increase in phospholipid content both after 7 and 301 days. PH raised slightly the phospholipid level only after 7 days. On the other hand: higher dosage increased the phospholipid level due to HM after 4 days and PH, HM and DQ after 7 days (Fig. 4).

Lung

toxicity

of respirable

hard

metal

519

dusts

I

,, _ Dose: 1 mg/rat 1 day lo-

7 days

30 days

9-

07-

x

C

II-

Dose:

FP

II-i Da

PI4

c

‘P

PH

HM

W

C

FP

PH

tit4

W

3 mg/rat

lO-

L days

7 days

9-

C

Fig. 2. AP activity fluid. C, control;

FP

PH

kt.4

IX

C

FP

PI-I

HM

DQ

1, 4, 7 and 30 days after intratracheal injection of 1 and 3 mg dust in rat lung lavage abbrev. for dust samples see Materials and Methods. All values are mean&SD. Statistically significant: *P < 0.05, **P < 0.01.

Of the materials dissolvable by nitric acid, cobalt is especially noteworthy: 90% in PH, 50% in HM, while hardly 10% in the FP sample could be dissolved. From HM, iron was dissolved in highest amount and a significant amount of silicon was

520

Z. Adamis

t

Dose:

‘lmg /rat 1 day

C

et al.

FP

RI

30 days

7 days

tit4

W

C

FP

F’ti

It4

W

Jl.mI C

FP

PH

ISi

Dose : 3mg / rat

t

Ldws

7 days

T

T

ti C

FP

PH

HM

W

C

FP

PH

HM

DO

Fig. 3. Amount of protein 1, 4, 7 and 30 days after intratracheal injection of I and 3 mg dust in rat lung lavage fluid. C, control; abbrev. for dust samples see Materials and Methods. All values are meanfSD. Statistically significant: *P < 0.05, **P < 0.01.

obtained. The latter two elements (Fe, Si) enter the sample during the technological process (grinding, polishing, sand blasting, etc.), therefore FP and PH do not contain these elements (Table 2).

Lung

toxicity

of respirable

hard

metal

521

dusts

Dose: Img / rot

600

7 days

1 day

30 days

500

I

C

FP

PH

HM

m C

CFPPHHM

DO

6oo Dose: 3mg/rot L days

C

FP

PH

DQ

C

FP

Fig. 4. Amount of phospholipid 1, 4, 7 and 30 days after intratracheal lung lavage fluid. C, control; abbrev. for dust samples see Materials *SD. Statistically significant: *P < 0.05,

PH

PH

Ii4

T

7 days

HM

FP

4xx T

Ht.4

W

injection of 1 and 3 mg dust in rat and Methods. All values are mean

**P < 0.01.

522

Z. Adamis Table

2. Determination

of the acid soluble fraction hard metal industry

Material

co Cr CU Fe Mn Ni P Si Ti V Zn Total

acid soluble

Abbreviations

et (11.

fraction

as in Materials

in respirable

FP (%)

PH (X)

0.74 0.0025 0.0075 0.011 0.0007 0.0008 0.105 0.028 0.057 0.0001 0.018 0.97

7.1 0.005 0.00 I 0.028 0.0008 0.0075 0.035 0.025 0.44 0.0004 0.004 7.65

dusts of the

HM

(X)

1.7 0.014 0.0088 9.4 0.063 0.058 0.125 I .08 0.12 0.013 0.018 12.6

and Methods.

In the lungs, protein, phospholipid and AP levels did not differ from those of the controls on exposure to hard metal dust. DQ significantly increased the amount of phospholipid and AP after 30 days. Histological studies showed no pathological changes in the lungs of the control animals. After 1 day to exposure to presintered dust (PH), interstitial oedema developed with serious cellular infiltration consisting of neutrophil leukocytes and macrophages (Fig. 5). After 1 week of exposure, in addition to oedema, several leukocytes, lymphocytes and plasma cells could be observed in the interstitium and alveoli (Fig. 6). After 1 month the interstitium was thickened and in many places lymphocytic foci could be observed (Fig. 7). In the interalveolar septa and lumina of the alveoli and bronchioli, the amount of argyrophilic fibres increased (Fig. 8). No pathological changes were seen in the regional lymph nodes in the period examined. FP and HM induced essentially the same changes in the lung.

DISCUSSION

The samples studied induced various alterations in the lungs. The acute effect of PH was the most marked: upon administration, LDH activity exceeded the control value throughout the experiment, and on days 4-7 protein levels also increased. HM induced a significant increase in LDH and AP on the first day, and increased moderately the amount of phospholipid on days 4 and 7. FP was practically ineffective. DQ elicited a gradual increase in LDH, protein and phospholipid levels. Biochemical changes suitable for the assessment of lung toxicity were summarised by Lehnert (1994). He stated that LDH indicates the damage of th’e cell membrane, AP a disturbance of the mechanism of phagocytosis and the damage of phagocyte cells; protein points to the increased permeability of the alveolar-capillary barrier, while phospholipid shows the metabolic injury of Type II pneumocytes. Taking this into consideration, it may be stated that PH (heat-treated presintered sample) induces acute inflammation, damages the cell membrane and increases capillary permeability; HM (heat-treated sintered sample) produces a less

Lung

toxicity

of respirable

hard

metal

dusts

with a single intratracheal dose of 3 mg respirable pre-sintered Fig 5. Rat lung I day after treatment x160). Interstitial oedema with inflammatory infiltration (PH 1) per animal (H and E staining, interalveolar septa containing of segmented neutrophilic leukocytes and macrophages.

523

dlust in

Fig 6. Rat lung I week after exposure to a single intratracheal dose of 3 mg respirable pre-sintered dlust x160). In the lumina of the alveoli and bronchioli and the PH 1) per animal (H and E staining, interstitium, segmented neutrophilic leukocytes, lymphocytes and plasma cells are observable.

524

Z. Adamis

et al.

Fig. 7. Rat lung 1 month after-treatment with a single intratracheal dose of 3 mg respirable. pre-sintered dust (PH) per animal (H and E staining, x 160). In the thickened interalveolar septa and intraalveolarly, plasma cells and lymphocytes are seen.

Fig. 8. Rat lung 1 month after treatment with a single intratracheal dose of 3 mg respirable dust (PH) per animal (H and E staining, x320). The interalveolar septa are thickened (arrow), of the alveoli are obliterated (star).

pre-sintere:d the lumina

Lung

toxicity

of respirable

hard

metal

dusts

525

marked inflammation with characteristic cell membrane damage and a slight change in Type II pneumocyte metabolism. In the case of FP (finished powder for pressing), the examination of bronchoalveolar washing fluid revealed no damaging effect. Quartz DQ 12 gave rise to acute and subacute cytotoxic effects, described also in our previous studies (Adamis and Krass, 1991). The damaging effect of FP, PH and HM shows a correlation with the amount of the acid-soluble cobalt. The importance of cobalt in the development of hard metal lung disease was also pointed out by Harding (1950) and Schepers (1955). Harding (1950) found that cobalt produces severe acute pulmonary capillary damage in the lungs of experimental animals. Tungsten carbide, as well as a tungsten carbide/cobalt mixture in a dosage of 50 mg, produced no unexpected effect (Delahant, 1955; Harding, 1950; Schepers, 1955). Therefore, it was surprising that PH administered in doses of 5 and 15 mg, respectively caused the deaths of several rats within 24 h. Presumably, this may be due to cobalt dissolved in large amounts. Delahant (1955) also stated that cobalt often kills the animals within a time range of 15 min to 48 h after treatment. Histological investigations did not show any difference in the effect of hard metal samples examined. Fibrosing alveolitis developed by the end of the first month proved that these dusts may all induce chronic changes in the lungs. Posgay et al. (1992) observed altogether 10 cases of hard metal induced coniosis in a comprehensive retrospective longitudinal study of 30 years of a hard metal plant in Hungary. (All samples used for the experiment were collected here.) The workers were employed in different activities, according to demand. The highest morbidity rate was found in the areas of pre-sintering and grinding hard metals (four each), furthermore, there was one case recorded, both in the pressing workshop and in powder preparation. As described by Hartung et al. (1982), the individual stages of the manufacturing process are not equally hazardous. This view may, however, change in the course of time. Thus, for example: the machining of sintered material, which was previously judged to be non-hazardous, is now unanimously regarded as a hazardous process. Our investigations have proved that, on the one hand, the composition and respiratory toxicity of dusts collected in different stages of hard metal production may differ. On the other hand, the biological effect of samples of identical composition (FP, PH) is altered by the manufacturing process (heat treatment, presintering). The difference between samples was revealed by the analysis of the pulmonary washing fluid. For the study of small doses used in our experiments, this seems to be one of the most sensitive procedures. The aetiology of hard metal induced disease has still not been elucidated. According to our current knowledge, however, cobalt seems to play a significant role in the development of pathological changes. Acknowledgemen&-This Japan) and the Hungarian

work was supported Ministry of Welfare

by Koichi Honma (I-13-94/ETT).

(Dokkyo

University,

Mibu,

Tochigi,

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in virro

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