Toxicology, 8 (1977) 177--184 © Elsevier/North-Holland Scientific Publishers, Ltd.
E A R L Y BIOCHEMICAL RESPONSE OF P U L M O N A R Y TISSUE TO MANGANESE DIOXIDE
JASWANT SINGH, J.L. KAW and S.H. ZAIDI Industrial Toxicology Research Centre, Mahatma Gandhi Marg, Post Box No. 80, Lucknow-226001 (India)
(Received December 7th, 1976) (Revision received April 13th, 1977) (Accepted April 28th, 1977)
SUMMARY
The biochemical response of pulmonary tissue to MnO2 dust burden of 30 days duration was studied in rats. The activities of enzymes from isolated fractions of rat lungs were not significantly altered, even though manganese content was increased significantly in tissues remote from lungs, indicating translocation of the dust from its intrapulmonary location.
INTRODUCTION
Pathogenicity due to manganese dust has been known since 1837 when Couper observed the first case of manganese poisoning among manganese ore crushers. The metal causes neurological and sexual disturbances [1--5]. The lesions, though predominantly extrapulmonary, have been reported in the lungs [6--8] which serve as portals for the entry of dust into the system [9--111. The role of manganese dioxide (MnO2) in the production of human or experimental pneumoconiosis is little understood. Most of the results from such studies have been derived from observations extending over prolonged periods [6,8,11--13]. Zaidi et al. [14] showed experimentally that manganese by itself does not cause marked pulmonary changes though in combination with Candida albicans collagenous fibrosis of the lung results at an early stage. In the present experiments attempts have been made to correlate the early histopathological and biochemical alterations in the lungs following a dust burden of manganese dioxide. This would help to explore whether MnO2 produces any early pneumoconiotic lesions, correlated with biochemical changes as elicited by silica or asbestos [ 15,16].
177
MATERIALS AND METHODS
Dust Manganese dioxide dust {Particle size < 5 ~mol) was prepared according to Zaidi et al. [12]. Details of the size distribution and chemical composition of the dust used in the present experiment are the same as described by one of us [12]. Animals Ten adult male albino rats, Wistar strain weighing 150 +- 5 g were inoculated intratracheally with 50 mg of dust suspension in I ml saline according to the method described by Zaidi [17]. An equal number of control animals, belonging to the same strain and having comparable body weight to those of the experimental group, were similarly inoculated with 1 ml saline. The animals were housed in air-conditioned rooms and maintained on a standard pellet diet (Protein 24%, lipids 4%, crude fibre 4%, phosphorus 0.6%, calcium 1%, ash content 8%, total caloric value 3.2 Calories/g; Hindustan Liver Ltd., India) and given water ad libitum to drink. Animals were killed 30 days post-inoculation by ex-sanguination. The lungs of the dead animals were removed, freed of hilar structures and cleansed of adhering blood and other extraneous material by washing in chilled physiological saline and subsequent blotting on a Whatman No. 1 filter paper. One part of the lung was processed for the assay of enzyme activities and determination of manganese content and the other part used for histopathological studies. Manganese was also estimated in other tissues like brain, liver, pancrease, kidney, testis and blood to assess the degree of manganese mobilization from the intrapulmonary location. Preparation o f lung homogenate and mitochondria The procedure employed for homogenizing lung and obtaining mitochondrial and post-mitochondrial fractions is the same as reported earlier [i51. Assay o f enzymes Whole homogenate. Glutamic-oxaloacetate transaminase (EC 2.6.1.1), glutamic pyruvate transaminase (EC 2.6.1.2), alkaline phosphatase (EC 3.1.3.1) acid phosphatase (EC 3.1.3.2) and 5'-nucleotidase (EC 3.1.3.5) were assayed in the whole homogenate according to Wootton [18]. Post-mitochondrial supernatant. The activity of phospho-glucomutase (EC 2.7.5.1), lactate dehydrogenase (EC 1.1.1.27), aldolase (EC 4.1.2.7) and malic dehydrogenase (EC 1.1.1.37) was determined respectively by the methods of Sutherland [19], Kornberg [20], Beck [21] and Ochoa [22]. Mitochondrial fraction. The enzyme activities of adenosine triphosphatase (EC 3.6.1.3) [23], cytochrome c oxidase (EC 1.9.3.1) [24], diaphorase (EC 1.6.4.3) [25], NADH~ytochrome c reductase (EC 1.6.99.3) [26], succinic dehydrogenase (EC 1.3.99.1) [27] and malic dehydrogenase (EC 178
1.1.1.37) [22] were determined using isolated mitochondrial fraction. Protein estimation. Protein was estimated in the TCA precipitate by the m e t h o d of Lowry et al. [28] using bovine serum albumin as standard. Estimation of manganese. For analysis of manganese, the samples were prepared by the wet digestion procedure [29]. The resulting carbon free residue was dissolved in 0.1 N HC1 and read at 279.5 nm on a Perkin-Elmer303 atomic absorption spectrophotometer. The standard solution of manganese was similarly prepared using MnSO4 • 4H20 of analytical quality for comparison. Histopathological method. Pieces of lung tissue were fixed in 10% formal saline and 5 pm thick paraffin sections were cut. Following deparaffinization and dehydration, the sections were stained with hematoxylin-eosin and silver impregnated for reticulin and collagen [30]. Statistical methods. The test described by Fisher [31] was employed to calculate the statistical significance between control and experimental values. P values less than 0.05 were considered to be significant. Recovery of protein. The protein content estimated from whole homogenate, mitochondrial and post-mitochondrial supernatant fractions remained unchanged (Table I). Enzyme assay. The data for the activity of the enzymes measured from whole homogenate and isolated fractions are given in Table II. Among the enzymes of the whole homogenate the activity of only GPT and alkaline phosphatase decreased by 19 and 27% whereas the activity of 5'-nucleotidase, GOT and acid phosphatase remained unaltered. Among the mitochondrial enzymes the activity of succinic dehydrogenase and NADHcytochrome c reductase increased. The activity of other enzymes assayed, viz., Mg2+-activated adenosine triphosphatase, cytochrome c oxidase, diaphorase and malic dehydrogenase remained unaffected. Among the enzymes of post-mitochondrial supernatant (11 000 g), phosphoglucomutase
TABLE I DISTRIBUTION OF PROTEIN IN THE LUNG OF NORMAL AND MnO2 INOCULATED RATS Data p r e s e n t e d
are t h e m e a n v a l u e f r o m six a n i m a l s + S . E .
Protein content (mg/g fresh tissue)
Whole homogenate Mitochondrial fraction Post-mitochondrial supernatant
Control
Experimental
90.4 + 2.53
96.4 ~ 2.02
9.2 ± 0.29
1 0 . 0 -+ 0 . 4 4
53.4 ± 1.21
52.2 • 0.97
179
T A B L E II C H A N G E S IN E N Z Y M E A C T I V I T Y O F THE L U N G IN N O R M A L A N D M n O 2 INOCULATED RATS D a t a e x p r e s s e d are t h e m e a n o f six a n i m a l s ± S.E. Enzyme activity a Control
Experimental
Whole h o m o g e n a t e Glutamic-oxaioacetate transaminase Glutamic-pyruvate transaminase Acid p h o s p h a t a s e Alkaline p h o s p hatase 5"Nucleotidase
132 82 9 110 4.2
± ± -~ ± ~
11.7 4.0 1.0 2.1 0.2
130 ± 67 ± 10 ± 80 -+ 3.0±
9.1 1.0 0.4 10.0 b 0.1
59 398 83 49 93 492
± 4.7 ± 27.1 ± 8.0 ± 4.0 ± 6.1 ± 28.0
72 400 103 55 89 526
± 9.0 ± 18.1 ± 12.0 b ± 2.2 ± 6.0 ± 26.0
772 844 192 87
± 40.0 + 24.0 ± 17.0 ± 5.0
796 873 295 69
± 41.0 ± 40.0 * 21.0 b ± 1.0 c
M i t o c h o n d r i a l fraction Succinic dehydrogenase Mg~ ÷ - A d e n o s i n e t r i p h o s p h a t a s e NADH-cytochrome c reductase Cytochrome c oxidase Diaphorase Malic d e h y d r o g e n a s e
Pos t-m itoch ondrial superna tan t Lactic d e h y d r o g e n a s e Malic d e h y d r o g e n a s e Phosphoglucomutase Aldolase
ananomoles of the respective substrate transformed/min/mg b p < 0.02. cp
E A R L Y BIOCHEMICAL RESPONSE OF P U L M O N A R Y TISSUE TO MANGANESE DIOXIDE
JASWANT SINGH, J.L. KAW and S.H. ZAIDI Industrial Toxicology Research Centre, Mahatma Gandhi Marg, Post Box No. 80, Lucknow-226001 (India)
(Received December 7th, 1976) (Revision received April 13th, 1977) (Accepted April 28th, 1977)
SUMMARY
The biochemical response of pulmonary tissue to MnO2 dust burden of 30 days duration was studied in rats. The activities of enzymes from isolated fractions of rat lungs were not significantly altered, even though manganese content was increased significantly in tissues remote from lungs, indicating translocation of the dust from its intrapulmonary location.
INTRODUCTION
Pathogenicity due to manganese dust has been known since 1837 when Couper observed the first case of manganese poisoning among manganese ore crushers. The metal causes neurological and sexual disturbances [1--5]. The lesions, though predominantly extrapulmonary, have been reported in the lungs [6--8] which serve as portals for the entry of dust into the system [9--111. The role of manganese dioxide (MnO2) in the production of human or experimental pneumoconiosis is little understood. Most of the results from such studies have been derived from observations extending over prolonged periods [6,8,11--13]. Zaidi et al. [14] showed experimentally that manganese by itself does not cause marked pulmonary changes though in combination with Candida albicans collagenous fibrosis of the lung results at an early stage. In the present experiments attempts have been made to correlate the early histopathological and biochemical alterations in the lungs following a dust burden of manganese dioxide. This would help to explore whether MnO2 produces any early pneumoconiotic lesions, correlated with biochemical changes as elicited by silica or asbestos [ 15,16].
177
MATERIALS AND METHODS
Dust Manganese dioxide dust {Particle size < 5 ~mol) was prepared according to Zaidi et al. [12]. Details of the size distribution and chemical composition of the dust used in the present experiment are the same as described by one of us [12]. Animals Ten adult male albino rats, Wistar strain weighing 150 +- 5 g were inoculated intratracheally with 50 mg of dust suspension in I ml saline according to the method described by Zaidi [17]. An equal number of control animals, belonging to the same strain and having comparable body weight to those of the experimental group, were similarly inoculated with 1 ml saline. The animals were housed in air-conditioned rooms and maintained on a standard pellet diet (Protein 24%, lipids 4%, crude fibre 4%, phosphorus 0.6%, calcium 1%, ash content 8%, total caloric value 3.2 Calories/g; Hindustan Liver Ltd., India) and given water ad libitum to drink. Animals were killed 30 days post-inoculation by ex-sanguination. The lungs of the dead animals were removed, freed of hilar structures and cleansed of adhering blood and other extraneous material by washing in chilled physiological saline and subsequent blotting on a Whatman No. 1 filter paper. One part of the lung was processed for the assay of enzyme activities and determination of manganese content and the other part used for histopathological studies. Manganese was also estimated in other tissues like brain, liver, pancrease, kidney, testis and blood to assess the degree of manganese mobilization from the intrapulmonary location. Preparation o f lung homogenate and mitochondria The procedure employed for homogenizing lung and obtaining mitochondrial and post-mitochondrial fractions is the same as reported earlier [i51. Assay o f enzymes Whole homogenate. Glutamic-oxaloacetate transaminase (EC 2.6.1.1), glutamic pyruvate transaminase (EC 2.6.1.2), alkaline phosphatase (EC 3.1.3.1) acid phosphatase (EC 3.1.3.2) and 5'-nucleotidase (EC 3.1.3.5) were assayed in the whole homogenate according to Wootton [18]. Post-mitochondrial supernatant. The activity of phospho-glucomutase (EC 2.7.5.1), lactate dehydrogenase (EC 1.1.1.27), aldolase (EC 4.1.2.7) and malic dehydrogenase (EC 1.1.1.37) was determined respectively by the methods of Sutherland [19], Kornberg [20], Beck [21] and Ochoa [22]. Mitochondrial fraction. The enzyme activities of adenosine triphosphatase (EC 3.6.1.3) [23], cytochrome c oxidase (EC 1.9.3.1) [24], diaphorase (EC 1.6.4.3) [25], NADH~ytochrome c reductase (EC 1.6.99.3) [26], succinic dehydrogenase (EC 1.3.99.1) [27] and malic dehydrogenase (EC 178
1.1.1.37) [22] were determined using isolated mitochondrial fraction. Protein estimation. Protein was estimated in the TCA precipitate by the m e t h o d of Lowry et al. [28] using bovine serum albumin as standard. Estimation of manganese. For analysis of manganese, the samples were prepared by the wet digestion procedure [29]. The resulting carbon free residue was dissolved in 0.1 N HC1 and read at 279.5 nm on a Perkin-Elmer303 atomic absorption spectrophotometer. The standard solution of manganese was similarly prepared using MnSO4 • 4H20 of analytical quality for comparison. Histopathological method. Pieces of lung tissue were fixed in 10% formal saline and 5 pm thick paraffin sections were cut. Following deparaffinization and dehydration, the sections were stained with hematoxylin-eosin and silver impregnated for reticulin and collagen [30]. Statistical methods. The test described by Fisher [31] was employed to calculate the statistical significance between control and experimental values. P values less than 0.05 were considered to be significant. Recovery of protein. The protein content estimated from whole homogenate, mitochondrial and post-mitochondrial supernatant fractions remained unchanged (Table I). Enzyme assay. The data for the activity of the enzymes measured from whole homogenate and isolated fractions are given in Table II. Among the enzymes of the whole homogenate the activity of only GPT and alkaline phosphatase decreased by 19 and 27% whereas the activity of 5'-nucleotidase, GOT and acid phosphatase remained unaltered. Among the mitochondrial enzymes the activity of succinic dehydrogenase and NADHcytochrome c reductase increased. The activity of other enzymes assayed, viz., Mg2+-activated adenosine triphosphatase, cytochrome c oxidase, diaphorase and malic dehydrogenase remained unaffected. Among the enzymes of post-mitochondrial supernatant (11 000 g), phosphoglucomutase
TABLE I DISTRIBUTION OF PROTEIN IN THE LUNG OF NORMAL AND MnO2 INOCULATED RATS Data p r e s e n t e d
are t h e m e a n v a l u e f r o m six a n i m a l s + S . E .
Protein content (mg/g fresh tissue)
Whole homogenate Mitochondrial fraction Post-mitochondrial supernatant
Control
Experimental
90.4 + 2.53
96.4 ~ 2.02
9.2 ± 0.29
1 0 . 0 -+ 0 . 4 4
53.4 ± 1.21
52.2 • 0.97
179
T A B L E II C H A N G E S IN E N Z Y M E A C T I V I T Y O F THE L U N G IN N O R M A L A N D M n O 2 INOCULATED RATS D a t a e x p r e s s e d are t h e m e a n o f six a n i m a l s ± S.E. Enzyme activity a Control
Experimental
Whole h o m o g e n a t e Glutamic-oxaioacetate transaminase Glutamic-pyruvate transaminase Acid p h o s p h a t a s e Alkaline p h o s p hatase 5"Nucleotidase
132 82 9 110 4.2
± ± -~ ± ~
11.7 4.0 1.0 2.1 0.2
130 ± 67 ± 10 ± 80 -+ 3.0±
9.1 1.0 0.4 10.0 b 0.1
59 398 83 49 93 492
± 4.7 ± 27.1 ± 8.0 ± 4.0 ± 6.1 ± 28.0
72 400 103 55 89 526
± 9.0 ± 18.1 ± 12.0 b ± 2.2 ± 6.0 ± 26.0
772 844 192 87
± 40.0 + 24.0 ± 17.0 ± 5.0
796 873 295 69
± 41.0 ± 40.0 * 21.0 b ± 1.0 c
M i t o c h o n d r i a l fraction Succinic dehydrogenase Mg~ ÷ - A d e n o s i n e t r i p h o s p h a t a s e NADH-cytochrome c reductase Cytochrome c oxidase Diaphorase Malic d e h y d r o g e n a s e
Pos t-m itoch ondrial superna tan t Lactic d e h y d r o g e n a s e Malic d e h y d r o g e n a s e Phosphoglucomutase Aldolase
ananomoles of the respective substrate transformed/min/mg b p < 0.02. cp
