Enzyme changes during experimental silicotic fibrosis

ENVIRONMENTAL

RESEARCH

Enzyme

25, 434-440 (1981)

Changes

during

II. Intermediary ELISKA

MIREJOVSKA,

Experimental

Metabolism

Enzymes

Silicotic

Fibrosis

of the Lungs

ARNO~T BASS, JOSEF HURYCH, JAN TEISINGER

Institute of Hygiene and Epidemiology, Centre of Industrial Hygiene Diseases, Srobdrova 48. 100 42 Prague, and Institate of Physiology, Academy of Sciences, Prague, Czechoslovakia

AND

and Occupational Czechoslovak

Received July 10, 1980 The activity of seven important enzymes of intermediary metabolism in lung tissues was monitored during the development of experimental silicosis in rats. The enzymes were triosephosphate dehydrogenase (TPDH), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), glycerol-3-phosphate NAD dehydrogenase (GPDH), hexokinase (HK), citrate synthase (CS), and hydroxyacyl-CoA dehydrogenase (HOADH). Changes were compared with the lung tissue response to nonfibrogenic corundum dust and with healthy lung tissues. In the developing silicotic lung tissue important disproportional activity changes take place in some enzymes (HK, TPDH, LDH, GPDH, HOADH); different, possibly less important changes occur after corundum application. The lung tissues of silicotic rats have an enhanced capacity for anaerobic degradation of saccharides with lactate as the end product. This finding can be related to the activation of collagen prolyl hydroxylase. The maximum changes in enzyme activities occur in the 2nd week of dust application.

INTRODUCTION The lung is a heterogenous organ in which various energy-dependent processes take place, including vessel contraction, cilia motility, bronchial secretion, and mast cell degranulation. At the level of alveoli energy is indispensable for phospholipid synthesis and surfactant secretion, the active transport of serotonin, alveolar macrophage activity, and protein biosynthesis (Fisher, 1976). Biologically active quartz dust induces deep and irreversible changes in lung tissue after a certain period following instillation of a suitable dose into the lungs of the experimental animal. Enzyme systems in silicotic lungs have as yet been little explored. Kilroe-Smith and Breyer (1960) monitored the effect of quartz inhalation on respiratory chain enzyme systems in guinea pig lungs and found reduced cytochrome-c oxidase after a period of dust action. Engelbrecht (1970) also studied the effect of quartz dust and inert carbon dust on cytochrome-c oxidase activity in lungs and blamed its inhibition on the cytotoxicity of quartz and silicic acid. Mihail et al. (1973) described reduced oxygen uptake and reduced enzyme activity of the respiratory chain after intratracheal application of mineral dusts with an admixture of Cu and quartz. They also referred to glucose metabolism disorders, estimated according to the formation of lactate and pyruvate in perfused silicotic lungs. Pavlova er al. (1978) demonstrated a much enhanced permeability of mitochondrial membranes in experimental silicosis (2-6 weeks following instillation of quartz dust) not only 434 0013-9351/81/040434-07$02.00/O Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.

INTERMEDIARY

METABOLISM

ENZYMES

OF

THE

LUNGS

435

for cations K+ and Na+ but also for NAD that escaped from mitochondria into cytoplasma. In an earlier study of Mifejovska et al. (1976) a disproportional increase was found in the activity of triosephosphate dehydrogenase, lactate dehydrogenase, and citrate synthase in a 5week experimental silicosis. The present experimental study was conducted to determine: (a) metabolic changes induced by quartz dust in the early phases of silicotic fibrosis (within 2 months after dust application), (b) possible metabolic abnormalities after phagocytosis of a dose of quartz dust in comparison to the effect of the same dose of nonfibrotic corundum dust and to healthy lung tissue. Activity of seven important enzymes of intermediary metabolism (glycolysis, the Krebs cycle, and degradation of fatty acids) was determined in the lung tissue homogenate. MATERIALS

AND METHODS

Male rats, weighing 230 g, were given a suspension of 25 mg SiO, DQ,, and/or corundum in 0.7 ml physiological solution by intratracheal instillation. In the first part of the experiment groups of six animals, each treated with SiO.,, as well as controls, were successively killed 2, 4, and 8 weeks from the day of dust application. In the second part, the rats were killed 2 and 4 weeks following quartz and corundum instillation. The extracted and weighed lungs were washed with physiological saline and cut. Part of the tissue (0.5 g) was homogenized in a medium containing 0.25 M sucrose, 0.014 M Tris-HCl buffer (pH 7.5), 50 &ml phenylmethylsulfonylfluoride, and 50 PM dithiothreitol, and the supernatant, obtained by centrifuging at lOS,OOOf:, was used for determining collagen prolyl hydroxylase (CPH) (EC 1.14.11.2) activity according to Hutton et crl. (1966), using nonhydroxylated [lJC]proline-labeled collagen as substrate. The final determination of ‘C Hyp radioactivity was done by the Juva and Prockop (1966) method. Part of the tissue (0.5 g) was homogenized and extracted with 50 mM K-Na phosphate buffer, 10 mM EDTA, and 0.1% Triton X-100, pH = 7.3. A supernatant was prepared at 15.0009 for determining hexokinase (HK) (EC 2.7.1. I), cytoplasmatic glycerol-3-phosphate NAD dehydrogenase (GPDH) (EC 1.1.1.8), triosephosphate dehydrogenase (TPDH) (EC 1.2.1.12). lactate dehydrogenase (LDH) (EC 1.1.1.27). citrate synthase (CS) (EC 4.1.3.7), malate dehydrogenase (MDH) (EC I. 1.1.37), and hydroxyacyl-CoA dehydrogenase (HOADH) (EC I. 1.1.35). For determining activity of TPDH, LDH, GPDH, MDH, and HK the method of Biicher PT cl/. (1964) was used, for CS that of Stern c’t u/. (1951), and for HOADH that of Wakil (1955), by means of an Eppendorf registration photometer (25°C). Total proteins in extracts were estimated according to Lowry ct trl. (1951), the total amount of hydroxyproline in lung tissue according to Stegemann (1958). Esteritied fatty acids were estimated according to HorejSi (1964). RESULTS

The fibrotic reaction of lung tissue after intratracheal application of 25 mg quartz dust DQ,? manifested itself by the increase of lung wet weight, total hydroxyproline, and collagen prolyl hydroxylase activity (Table 1). After 2 weeks the increase in lung wet weight was 2.7 times higher than that in the healthy control

436

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ET AL.

TABLE BIOCHEMICAL

CHANGES

IN THE LUNG

1

TISSUE DURINGTHE

DEVELOPMENT

2 weeks

Lung wet weight (g) Total Hyp (pmole/lung) CPH W

Hyp cpm/lung)

CPH (“C Hyp cpm/g)

OF EXPERIMENTAL

4 weeks

SILKOSIS

8 weeks

Dusted

Control

Dusted

Control

Dusted

Control

3.55 k 0.34 48.44 + 5.05 22,394 2 4,006 6,355 + 1,260

1.30 L 0.12 21.20 k 2.82 4,882 f 1,714 3,730 2 1,073

4.79 -r- 0.72 59.38 + 14.05 46,702 k 18,091 9,513 A 2,288

1.50 k 0.10 22.11 ” 2.78 7,611 f 2,726 5,101 k 1,810

5.65 + 0.82 101.89 + 13.07 37,533 2 6,618 6,763 i 1,607

1.72 2 0.13 30.41 k 3.72 6,704 2 2,336 3,901 2 1,295

N&e. Each value represents means + SEM of six animals. All values in the experimental groups are significantly different from controls (P < 0.01).

group, total hydroxyproline was 2.3 times higher, and CPH activity increased 4.6 times. After 8 weeks the wet weight of lungs was, in comparison to controls, 3.3 times higher, hydroxyproline 3.35 times higher, and CPH activity 5.6 times higher. Changes in the activity of six important enzymes of energy-supplying metabolism 2, 4, and 8 weeks following quartz dust application are presented in Table 2, expressed in U/g lung wet weight. At conversion of the measured activity to 100 mg extractable protein the statistical significance between control and experimental groups remained unchanged and hence is not presented. The table demonstrates a significant rise in LDH activity in all time intervals. TPDH and HK activity was significantly higher in silicotic lung tissues in the 2nd and 4th weeks. GPDH was significantly reduced (in silicotic tissues) in the 2nd and 4th weeks TABLE ACTIVITY

2 weeks

4 weeks

Enzyme

Dusted

Control

P<

TPDH

20.08 k 3.23 56.74 2 3.46 0.60 k 0.07 1.66 + 0.15 56.67 t 5.19 3.50 2 0.50

15.99 k 2.35 33.46 + 3.55 0.80 k 0.08 1.26 2 0.14 56.19 t 7.61 3.81 t 0.39

0.05

LDH GPDH HK MDH cs

2

OF SOME ENZYMES OF THE ENERGY-SUPPLYING METABOLISM DURING THE DEVELOPMENT OF EXPERIMENTAL SILICOSIS

0.01 0.01 0.01 NS NS

Dusted 28.12 ? 2.72 66.77 2 7.06 0.38 + 0.04 1.33 + 0.22 29.60 k 1.90 4.82 i 0.88

Control 22.05 2 2.08 42.83 i 5.18 0.52 k 0.07 1.03 + 0.10 30.18 t 6.48 4.05 c 0.33

IN RAT LUNGS

8 weeks P<

Dusted

0.05

25.33 + 3.26 72.42 i 13.53 1.18 4 0.22 1.55 i_ 0.19 49.34 * 7.17 2.94 t 0.47

0.01 0.01 0.05 NS NS

Control 21.62 k 2.53 49.72 k11.04 1.33 k 0.33 1.12 i 0.20 43.03 2 7.98 3.14 i 0.36

P< NS 0.01 NS NS NS NS

Note. Enzyme activities are calculated as U/g wet weight. Means ? SEM, six animals in each group; significance according to Student’s t test.

INTERMEDIARY

METABOLISM

ENZYMES

OF

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437

after dusting; in week 8 it was lower after SiO, application than in healthy tissues, but not significantly. MDH and CS activity (in silicotic lungs) was unchanged. In the second part of the experiment changes were noted in the activities of seven enzymes after instillation of an identical dose (25 mglrat) of quartz dust DQ,? and inactive corundum dust. They are demonstrated in Figs. 1 and 2 as changes in percentage of activity as found in 1 g of healthy lung tissue. The fibrotic response to the applied dust in the second part of the experiment is shown in Table 3. The silicotic lungs (Figs. 1 and 2) exhibited a significantly enhanced activity of TPDH. LDH, and HK after 2 and 4 weeks in comparison to healthy lung tissues. GPDH and HOADH activity was significantly reduced. MDH activity did not differ significantly from controls: CS increased after 2 weeks in silicosis, but decreased to normal levels after 4 weeks. In the group of rats dusted with inert corundum dust (Figs. 1 and 2) TPDH increased nonsignificantly after 2 and 4 weeks: LDH and HK increased significantly in both intervals but failed to reach the level found in the silicotic group. Changes in CS, MDH, GPDH, and HOADH activities were nonsignificant after 2 weeks. After 4 weeks activity of MDH and CS increased. DISCUSSION

Comparison of energy-supplying metabolism enzyme activities during the development of fibrotic changes in silicotic lungs and in healthy lung tissues already showed a disproportion 2 weeks after animal dusting which persisted up to the 8th week. After this period the changes disappeared and only LDH activity remained enhanced. Triosephosphate dehydrogenase (TPDH) characterizes glycolytic activity and in the first 4 weeks was significantly higher in silicotic than in healthly

q

Corundum

Cl

Quartz

x p .i 0.05 xx pc0.01

125 z 2 8 100 ,P

50 J

FIG. I. Enzyme activities in rat lungs 2 weeks after intratracheal instillation of 25 mg quartz or corundum dust. These results represent percentage of activity as found in 1 g of healthy lung tissue. Significance was assessed with Student’s t test. The height of the bars indicates the mean of eight animals. Significance between corundum and quartz groups is expressed at the outer extreme of the bars: significance between experimental groups and controls is adjacent to the 100% line.

438

MIREJOVSKA

ET AL.

zoo-

q

Corundum Cl Quartz

FIG. 2. Enzyme activities in rat lungs 4 weeks after intratracheal corundum dust. (See legend to Fig. 1.)

I p e 0.05

=I p
instillation

of 25 mg quartz or

lung tissues. Hexokinase (HK), representing the capacity for phosphorilation of monohexoses and their incorporation into metabolism, also increased by 30-38% up to the 8th week following quartz dust application. Lactate dehydrogenase (LDH) represents the capacity for mutual conversion of pyruvate to lactate. Activity of this enzyme had its peak increase already 2 weeks after (possibly even earlier) quartz dust instillation and persisted throughout the interval of 8 weeks. This finding indicates an enhanced anaerobic degradation of saccharides with high lactate production. Optimal conditions are formed in lung tissues for the activation of prolyl hydroxylase (CPH), which always signals an increased biosynthesis of collagen proteins. Lindy et al. (1970) and Mihail et al. (1973) had already demonstrated higher LDH activity in experimental silicosis. Lindy et al. (1971) found a shift in the composition of LDH isoenzymes in favor of M subunits, which is typical for anaerobic tissue metabolism during hydroxylation. The aerobic-oxidation capacity of silicotic tissues in comparison with healthy lung tissues, estimated according to the activity of citrate synthase and MDH, showed no change. TABLE BIOCHEMICAL

CHANGES

Corundum Quartz

TISSUE

DURING

3 THE DEVELOPMENT

OF EXPERIMENTAL

FIBROSIS

Lung wet weight (g)

Total Hyp (pmole/lung)

weeks 4 weeks

1.44 + 0.09

1.35 2 0.05

23.13 k 3.93 24.53 r 1.42

22.85 2 2.05 28.98 k 4.50

weeks weeks weeks weeks

2.06 2 0.09

27.75 lr 2.33

2.10 f 0.34

31.51 2 4.66

5.04 2 0.55 5.46 t 0.37

59.33 t 6.71 77.67 2 20.16

44.98 k 8.88 34.64 k 4.50 125.48 t 27.65 277.64 ” 55.47

Group Control

IN THE LUNG

Time 2

2 4 2 4

” EFA, esterified fatty acids. Nore. Each value represents means 2 SEM of seven animals.

EFA” hhng)

INTERMEDIARY

METABOLISM

ENZYMES

OF THE

LUNGS

439

Glycerol-3-phosphate dehydrogenase NAD-dependent extramitochondrial enzyme combined with gluconeogenesis of glycerolphosphate derived from lipids showed a significant decrease in the first 4 weeks. It is interesting to compare enzyme activities after application of quartz dust and nonfibrogenic corundum dust. Corundum produced a moderate increase in lung wet weight and in total hydroxyproline, lipids, and phospholipids (Table 3). After application of quartz dust and corundum there is a higher utilization of saccharides as judged from the activity of HK and TPDH even though TPDH showed a significant deviation solely in the silicotic group. Apparently the lung tissue generally covers its energy requirements for phagocytosis of dust particles in this way. In both groups the balance between pyruvate and lactate was shifted in favor of lactate: however, after phagocytosis of quartz dust the shift was much greater. The significantly higher utilization of saccharides in silicosis as opposed to application of inert corundum dust may also be attributed to oedema produced by quartz dust. Young and Knelson (1973) demonstrated experimentally a higher glucose uptake and lactate production in spontaneous pulmonary edema and in edema induced by inhalation of aerosol with NO,. The difference in metabolic abnormalities was most striking 4 weeks after quartz dust and corundum application (Fig. 2). After phagocytosis of corundum CS and MDH activity increased in lung tissues (LDH increased less). Thus tissue obtains energy more effectively by processing pyruvate in the tricarboxylic acid cycle. The greatest difference between the two processes was in the maintenance of normal GPDH and HOADH activity after application of nonfibrogenic dust. After phagocytosis of tibrogenic quartz, the activity of both enzymes was significantly reduced. HOADH is the key enzyme for degrading fatty acids, so-called poxidation. The two findings explain the generally known fact that lipidlike substances are excessively deposited in lungs in the early stages of silicosis. Even though there is the drawback in the analysis of the homogenate that changes cannot be related to individual types of lung cells, it can still be said that the metabolic response in lung tissue after application of fibrogenic and nonfibrogenie dust differs in the way by which the tissue covers its energy requirements in phagocytosis of the same amount of dust. In silicotic lung tissues the share of anaerobic saccharide degradation is unproportionally higher. The high proportion of lactate in tissue can be ascribed to the activation of prolyl hydroxylase, which is indispensable for the biosynthesis of collagen proteins. The accumulation of lipidlike substances especially in the initial stages of silicotic fibrosis is also due to the metabolically reduced degradation of fatty acids (P-oxidation). Nonfibrogenic dust significantly enhances the activity of the tricarboxylic acid cycle in the lung tissue. Thus energy is obtained more effectively than in silicotic lung tissues. ACKNOWLEDGMENTS We are grateful to Dr. K. Robock, Asbest-Institut Federal Republic Germany, for providing the quartz kovi for skillful technical assistance.

fir Arbeitsand corundum

und Umweltschutz dusts. We thank

e.V., Neuss. Mrs. J. SvihBI-

440

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ET

AL.

REFERENCES Bucher, Th., Luh, W., and Pette, D. (1964). Einfache und zusammengesetzte optische Teste mit Pyridinnukleotiden. In “Handbuch der physiologisch und pathologisch-chemischen Analyse” (Hoppe-Seyler, Thierfelder, Eds.), Bd.VI/A, pp. 292-339. Springer-Verlag BerliniHeidelbergl New York. Engelbrecht, F. M. (1970). The mechanism of silica in fibrogenesis. In “Pneumoconiosis, Proceedings of the International Conference, Johannesburg, 1969” (H. A. Shapiro, Ed.), pp. 396-403. Oxford Univ. Press, Cape Town/London. Fisher, A. B. (1976). Oxygen utilisation and energy production. 1n “The Biochemical Basis of Pulmonary Function” (R. G. Crystal, Ed.), pp. 75- 104. Dekker, New York/Basel. Hotejsi, J. (1964). An assay for esterified fatty acids. In “Chemical Analysis in Medicine” (J. Hofejsi, Ed.), pp. 394, 395. SZN, Praha. [In Czech] Hutton, J. J., Tappel, A. L., and Udenfriend, S. (1966). A rapid assay for collagen proline hydroxylase. Anal. Biochem. 16, 384-394. Juva, K., and Prockop, D. J. (1966). Modified procedure for the assay of H3 or Cl?-labeled hydroxyproline. Anal. Biochem. 15, 77-83. Kilroe-Smith, T. A., and Breyer, M. G. (1960). Activity of three respiratory enzyme systems in the guinea pig lung after inhalation of quartz dust. In “Proceedings of the Pneumoconiosis Conference, Johannesburg, 1959” (A. J. Orenstein, Ed.), pp. 461-468. Lindy, S., Kahanpaa, K., Karhunen, P., Hahne, J., and Uitto, J. (1970). Lactate dehydrogenase isoenzymes during the development of experimental fibrosis. Lab. C/in. Med. 76, 756-760. Lindy, S., Pedersen. F. B.. Turto, H. and Uitto, J. (1971). Lactate, lactate dehydrogenase. and protocollagen proline hydroxylase in rat skin autograft. Hoppe-Seyler’s Z. Physiol. Chem. 352,11131118. Lowry, O., Rosebrough, N. J., Farr, A. L., and Randale, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Bid. Chem. 193, 265-275. Mihail, G., Mihaila, D., Nestor, L., and Alexandrescu. C. (1973). Unele modificari biochimice si enzimatice in evolutia fibrozii pulmonare experimentale generate de amestecuri de pulberi minerale. Igiena 22, 275-282. Mifejovska, E., Bass, A., and Hurych, J. (1976). Changes in the enzyme activity in the silicotic lung tissue of rats Gig. Tr. No. 10, 45-47. [In Russian] Pavlova, V. I., Muchina, S. T., and Veretinskaja, I. A. (1978). The signification of the basic metabolism changes in lungs during experimental silicosis. In “Pathogenesis of Pneumoconiosis, Proceedings of the Second Symposium of the Academy of Medical Sciences USSR, Karaganda, 1976” (B. E. Altynbekov, Ed.), pp. 82-87. Academy of Medical Sciences USSR, Moscow. [In Russian] Stegemann. H. (1958). Microbestimmung von hydroxyprolin mit Chloramin-T and pdimethylaminobenzaldehyd. Hoppe-Seyler’s Z. Physiol. Chem. 311, 41-45. Stern, J. R., Shapiro, R., Stadtman, E. R., and Ochoa, S. (19.51). Enzymatic activity of citric acid. III. Reversibility and mechanism. J. Biol. Chem. 193, 703-720. Wakil, S. J. (1955). D(-)Beta-hydroxybutyryl CoA dehydrogenase. Biochim. Biophys. Acta 18, 314-315. Young, S. L., and Knelson, J. H. (1973). Increased glucose uptake by rat lung with onset ofpulmonary edema. Physiologist 16, 494-498.