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Reduction in pulmonary and hepatic respiratory cytochrome contents by fly ash inhalation in rats
Toxicology
15
Letters, 49 (1989) 15-20
Elsevier
TOXLET
02212
Reduction in pulmonary and hepatic respiratory cytochrome contents by fly ash inhalation in rats
S.S. Chauhan,
S.R. Tyagi*, R.K. Kapoor**
qf Biochemistry.
Department (Received
10 April 1989)
(Accepted
31 May 1989)
V.P. Chest Institute,
Key words; Fly ash inhalation;
Mitochondrial
and U.K. Misra***
University of Delhi, Delhi 110007 (India)
cytochromes;
NADH-oxidase;
Lungs; Liver; [Rats]
SUMMARY Exposure activity.
of rats to fly ash for 15 days, 6 hours daily, inhibited
The content
fly-ash-exposed affecting
of cytochrome
group.
However,
the cytochrome
both organs
b and cytochromes
a+a,
in liver, fly ash exposure
b content,
indicating
pulmonary
and hepatic
was significantly reduced
a tissue-specific
NADH-oxidase
lower in the lungs of the
the cytochrome
a+a,
effect. Mitochondrial
level without
protein
content
in
was the same in both groups.
INTRODUCTION
Energy acquired by living cells is conserved in the form of adenosine triphosphate (ATP), which serves as an ‘energy currency’ in powering the other functions of cells. In the mitochondria of all cells with nuclei, electrons donated by organic molecules are passed from NADH or FADH2 to 02 by a series of electron carriers, and in this process ATP is generated [ 11. Mitochondrial cytochromes constitute a major portion
*Post-doctoral U.S.A.
Fellow, Department
**Post-doctoral
of Biochemistry,
Emory University
School of Medicine,
Atlanta,
GA,
Fellow.
***Professor and Head, Department of Biochemistry, V.P. Chest Institute, Delhi, India. At present: Visiting Professor, University of North Carolina at Chapel Hill, Department of Pharmacology, 1130, FLOB, Chapel Hill, NC 27514. U.S.A. Address Cancer
for correspondence:
Institute,
Laboratory
S.S. Chauhan, of Molecular
Visiting Biology,
Fellow, Building
National
Institutes
37 Room
2E22,
U.S.A. Tel. (301)496-6970
037%4274/89/$3,50
@ 1989 Elsevier Science Publishers
B.V. (Biomedical
Division)
of Health, Bethesda,
National
MD
20892,
16
of the electron transport chain, and hence their amount and proportions may be important for the efficient synthesis of ATP. Fly ash is a major environmental particulate pollutant containing polycyclic aromatic hydrocarbons and toxic trace elements [2]. Fly ash particles remain air-borne from a few hours to months, depending upon their aerodynamic size [2]. In vivo and in vitro cytotoxicity of these particles has been demonstrated [3, 41. After deposition in the lungs, fly ash and/or its chemical species are translocated to the extrapulmonary organs [5, 61. Exposure of rats to fly ash caused histological lesions in the lung and liver, reduced red and white blood cell numbers, and decreased hemoglobin content [4, 71. Fly ash also induced hepatic and pulmonary mixed-function oxidases in rats [8,9). Wilkins et al. [lo] reported lower ATP content in the lungs of rats exposed to fly ash. However, these workers did not attribute any reason for fly-ash-induced reduction in ATP content. The present study was therefore planned to investigate the effect of coal fly ash inhalation on mitochondrial cytochrome content and on NADH-oxidase activity, and hence on ATP synthesis in the rat lung and liver. MATERIALS
AND METHODS
Materials
Bovine serum albumin, NADH, EDTA, sodium deoxycholate, glycerol, ~,~,~,~~-tetramethy1 p-phenyldiamine (TMPD) and dithiothreitol were purchased from Sigma Chemical Co., St. Louis, MO. Sodium dithionite and sucrose were obtained from the British Drug Houses, India. Collection offly ash ami generation
of its aerosols
Fly ash was collected in bulk from the electrostatic precipitator of Inderprasth Thermal Power Station, New Delhi, during operation when bituminous coal was burned. It was passed through a 400-mesh (40 pm) stainless steel sieve. The shape of the fly ash particles so obtained was defined by scanning electron microscopy. The majority of the particles were spherical (both solid and hollow) and exhibited microcrystal deposition on their surfaces. Other particles were triangular, rod-shaped, or irregular in shape. The sieved fly ash was used for the generation of aerosols in the inhalation chamber. Approximately 58 % of the sieved fly ash particles were of respirable size, being 5 pm or less in diameter. The larger particles were trapped in a vessel through which the aerosol passed before entering the inhalation chamber. Therefore the concentration of respirable particles increased up to 68% in the inhalation chamber. Details of the inhalation chamber, the chemical composition of fly ash, and the generation of aerosols have been described elsewhere [4]. and exposure of animals to fly ash aerosols Male Wistar strain rats weighing 200-250 g were obtained from V.P. Chest Institute’s Small Animal Facility and maintained as described earlier [4]_ Maintenance
17
Rats were randomly divided into two groups and housed individually at all times throughout the experiment. One group was exposed 6 hours daily for 15 consecutive days to a fly ash concentration of 0.27 +O.Ol mg/l {mean f SE). The fly ash concentration in the inhalation chamber was monitored regularly. A group of control rats was exposed to filtered clean air for the same period of time under identical conditions. Sacrifice of animals and isolation of mitochondria On day 16, 1 day after the last tly ash exposure, overnight-fasted rats were anesthetized by intraperitoneal injection of pentobarbital (60 mg/kg body wt.). Lungs and livers were perfused in situ with ice-cold normal saline until they turned white. Subsequent processing of the tissues was done at 4°C. Mitochondria were isolated according to the method of Ritter and Malejka-Giganti [ 1l]. The tissues were cleaned, wiped and weighed, and a 20% homogenate was prepared in 0.25 M sucrose solution with a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 600 x g at 4°C in a Sorvall RC-SB centrifuge for 10 minutes. The supernatant was transferred to another centrifuge tube and the pellet was suspended in a small volume of the same solution, homogenized briefly and centrifuged. The combined supernatants were again centrifuged at 16 000 x g for 20 minutes to obtain a mitochondrial pellet. The mitochondria so obtained were washed and suspended in buffer. Estimation of NADH oxidase and respiratory c~tochrome~ Mitochondrial protein was estimated by the method of Lowry et al. 1121.NADH oxidase activity in mitochondria was estimated by the method of Mackler [13]. The mitochondrial pellet was suspended in 596 sucrose solution. The incubation mixture contained 0.1 k NADH, 0.005 M EDTA, 0.2 M phosphate buffer (pH 7.5) and mitochondrial protein in a final volume of 1.Oml. The reaction was started by the addition of enzyme, then the decrease in optical density at 340 nm was measured in a Beckman DU spectrophotometer. An extinction coefficient of 6.22 nM-‘-cm-’ for NADH was used to calculate the amount of NADH oxidized. The amounts of pulmonary and hepatic respiratory cytochromes were estimated according to the method of Rieske [ 141 and Gazzoti et al. [ 151. The mitochondrial pellet was suspended in 10 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol, 2 mM MgCl2 and 2 mM dithiothreitol. The assay mixture consisted of 1.O ml of enzyme protein (5-6 mg), 1.O ml of 1.O% deoxycholate, 0.45 ml of 200 mM phosphate buffer (pH 7.4), and 0.05 ml of I mM KsFe(CN)b. The assay mixture was divided equally into two cuvettes which were referred to as the reference and sample cuvettes. The contents of the sample cuvette were stirred with a few grains of sodium dithionite and the reduced-oxidized spectrum for cytochrome c and cytochromes a + a3 was recorded in the range of 500-650 nm. The contents of the reference cuvette were then stirred with a few grains of TMPD and the spectrum for cytochrome b was recorded in the range of 550-600
18
nm. The spectra were recorded in a doubIe-beam Shimadzu sp~trophotometer (Model UV-240). The amounts of the cytochromes were calculated from the difference in absorbance between the reduced and oxidized spectrums. The results were statistically analyzed using Student’s t-test. RESULTS
AND DISCUSSION
Fly ash inhalation 6 hours daily for 15 days did not affect the body weight or liver weight of rats, but significantly increased the lung weight as compared to control group rats (data not given). No change in mitochondrial protein content was noticed in the lungs or liver of the fly ash group as compared to the controls (Table I). NADH-oxidase activity in both organs was observed to be significantly lower in the fly ash group than in control animals (Table I). Similarly, fly ash inhalation significantly decreased the content of cytochrome b and cytochromes a+a3 in the lungs (Table II). However, only cytochrome at-a3 content was significantly lower in the livers of fly ash exposed rats compared with the control group (Table II). Fly ash inhalation did not have any effect on cytochrome c content in any of the organs studied (Table II). NADH-oxidase was observed to be reduced in the lungs and livers of experimental animals (Table I). Because NADH-oxidase is the first enzyme of the electron transport chain, the flow of electrons to the electron transport chain may have thus been obstructed. Cytochromes 6, c and a+a3 are the electron carriers in the mitochondrial electron transport chain. Reduction in the levels of cytochromes b and at-a3 in the lungs of experimental animals suggests that fly ash interferes with the flow of elec-
TABLE EFFECT
I OF FLY ASH INHALATION
NADH-OXIDASE
ON PULMONARY
AND
HEPATIC
MITOCHONDRIAL
IN RATS Control
Fly-ash-exposed
Lungs Mitochondrial
protein
NADH-oxidase oxidized/mg
(nmol NADH protein/min)
(mg/g tissue)
7.30+0.50
7.43 k 0.42
6.00 kO.98
3.77i0.24a
Liver
Mitochondrial
protein
NADH-oxidase oxidized/mg
(mg/g tissue)
14.06+ 1.20
12.902
1.19
(nmol NADH 12.21+ 1.93
protein/min)
*Values significantly
different
from control
Values are the mean &- SE from 6 animals
at 5 % level. in each group.
6.87 +0.50a
19 TABLE II EFFECT OF FLY ASH INHALATION TOCHROMES IN RATS
ON PULMONARY
AND HEPATIC RESPIRATORY
Control
Fly-ash-exposed
0.35 & 0.06 0.18,0.04 0.37t_o.O4
0.19*0.02= 0.21+0.02 0.26+0.01”
0.26 & 0.03 0.12,0.01 0.25f0.02
0.25 f 0.02 0.10+0.01 0.08 *o.o03a
CY-
Lungs
Cytochrome b Cytochrome c Cytochromes ata3 Liver
Cytochrome b Cytochrome c Cytochromes ~+a-,
+‘alues significantly different from control at 5 % level. Values are expressed in nmol cytochrome/mg protein. Values are the mean 5 SE from 6 animals in each group.
trons at these steps (Table II). After deposition in the lungs, fly ash and its chemical species are translocated to the liver and other extrapulmonary organs [4-71. In liver, only terminal cytochromes a+a3 were affected by translocated fly ash, indicating that the effect of inhaled fly ash is different on lung and liver (Table II). Since the relatively smaller amount of translocated fly ash (or its chemical species) that reached the liver decreased cytochromes a+~ but not cytochrome b, it is likely that cytochromes a+~ are more susceptible than cytochrome b to fly-ash-induced reduction. These results reveal that fly ash inhalation reduces the entry of electrons into the electron transport chain by reducing NADH-oxidase, and decreases the efficiency of electron transfer by de&easing the levels of mitochondrial cytochromes (Tables I and II). A decrease in either the cytochromes or NADH-oxidase can be expected to decrease the levels of ATP in lung and liver of by-ash-exposed animals. Wilkins et al. [IO] also observed a decrease in lung ATP levels by fly ash exposure. The metals lead and chromium have also been reported to decrease 02 consumption and ATP levels in rat brain and thymocytes, respectively [ 16, 171. Similarly, Garrett et al. [18] reported decreased ATP content in fly-ash-treated rabbit alveolar macrophages and Chinese hamster ovary cells. Inhibition of NADH-oxidase would lead to an increased level of NADH and thus increase NADPH formation by transhydrogenase reactions. An increased supply of NADPH favors the induction of mixed-function oxidase activity [19]. We have previously described the induction of mixed-function oxidases by fly ash inhalation in the lung and liver of rats [9]. The present study thus suggests that fly ash inhalation may decrease ATP levels by inhibiting NADH-oxidase and reducing the levels of respiratory cytochromes.
20
ACKNOWLEDGEMENTS
This study was supported by a grant from the Department of the Environment, Government of India, New Delhi. S.S.C. is grateful to the Indian Council of Scientific and Industrial Research for financial assistance. The generous help of Drs. S.M. Smith and K.V. Chin in the preparation
of the manuscript
is also acknowledged.
REFERENCES
I Hinkle, E.E. and McCarty, 2 Natusch,
D.F.S.
P.E. (1978) How cells make ATP. Sci. Am. 238, 104123.
and Wallace.
J.R. (1974) Urban
aerosol
toxicity:
influence
of particle
size. Science
184.6955699. 3 Schiff. L.J., Byrne, M.M. and Graham, hum in vivo and in vitro. J. Toxicol. 4 Chauhan.
S.S.. Chaudhary,
fly ash in rats. Environ. 5 Srivastava.
Health
V.K., Narayan,
changes
in hamster
tracheal
epithe-
8,431448.
S. and Misra,
U.K.
(1987) Cytotoxicity
of inhaled
coal
Res. 43, I-12.
V.K., Sengupta,
fly ash in various
J.A. (1981) Fly ash induced
Environ.
organs
S., Kumar,
R. and Misra,
of rats at various
periods
U.K. (1984) Distribution
after exposure.
J. Environ.
of metals of inhaled
Sci. Health
19,663-672.
6 Srivastava, V.K., Chauhan. S.S., Srivastava, P.K., Kumar. V. and Misra, U.K. (1986) Fetal translocation and metabolism of PAH obtained from coal fly ash given intratracheally to pregnant rats. J. Toxicol. Environ. 7 Srivastava,
Health
18,459469.
P.K., Chaudhary,
ical and pathological
V.K., Chauhan,
S.S., Srivastava,
V.K. and Misra, U.K. (1984) Biochem-
effects of fly ash on lung, liver and blood of rats. Arch. Environ.
Contam.
Toxi-
col. 13.441452. 8 Chauhan.
S.S., Srivastava,
coal fly ash polycyclic
P.K., Srivastava,
aromatic
rat lung and liver by vitamin 9 Chauhan,
A and citrate.
S.S., Singh. S.K. and Misra,
P-450 species by coal fly ash inhalation 10 Wilkins,
E., Wilkins,
of the lactating
O.H..
Rosebrough,
Folin phenol reagent. 13 Mackler.
J. Toxicol.
Environ.
U.K. (1989) Induction in rats. Toxicology
study. J. Environ.
I1 Ritter, C.L. and Malejka-Giganti. 12 Lowry,
V. and Misra,
and metal induced
Health
N.J..
of
activity
in
17, 357-363.
of pulmonary
and hepatic
cytochrome
interaction
of lung surfactant
with
17, 169-186.
Pharmacol.
oxidase
in the mammary
gland and liver
3 1, 239-247.
Farr, A.L. and Randall,
R.J. (1951) Protein
measurement
with the
193,265-275.
oxidase of heart muscle. Meth. Enzymol.
14 Rieske, J.S. (1967) The quantitative
oxidase
56.955105.
D. (1982) Mixed function
rat. Biochem.
J. Biol. Chem.
B. (1967) DPNH
Sci. Health
U.K. (1986) Inhibition
mixed function
M.G. and Seodi. 0. (1982) A physicochemical
fly ash: a preliminary microsomes
V.K.. Kumar.
hydrocarbons
determination
of mitochondrial
10,261-263. hemoprotein.
Meth. Enzymol.
10,
488493. 15 Gazzoti.
P., Malmstrom,
K. and Crompton,
M. (1980) In: E. Carafoli
brane Biochemistry. Springer-Verlag, Berlin, p. 62. 16 Dumas. P., Gueldry, D., Loireau. A., Chomard, P., Buthieau,
(1985)Effect of lead intoxication
on properties
of mitochondria
A.M.,
and G. Somenza Austissier
in brain of young
(Eds.), Mem-
and Nicole.
C.R.S.
rats. Sot. Biol. Ses.
Fil. 179, 1755183. 17 Lazzarini.
A., Luciani,
S., Beltrame,
M. and Arslan,
(iii) on energy charge and O2 consumption 18 Garrett,
N.E., Campbell,
bit alveolar
macrophages
terials. II. Particles
P. (1985) Effect of chromium
in rat thymocytes.
J.A., Stack, H.F., Waters, and Chinese hamster
from coal related processes.
Chem. Biol. Interact.
(vi) and chromium 53,273-281.
M.D. and Letwas, J. (1981) The utilization
ovary cells for evaluation Environ.
of toxicity
of particulate
of rabma-
Res. 24, 366376.
19 Forti. G.C.. Paolini. M.. Hreha, P., Corci. C., Biagi, G.L. and Bronzetti, G. (1984) NADPH generating system: influence on microsomal monooxygenase stability during incubation for the liver microsomal assay with rat and mouse S-9 fraction.
Mutat.
Res. 129,281-297.
15
Letters, 49 (1989) 15-20
Elsevier
TOXLET
02212
Reduction in pulmonary and hepatic respiratory cytochrome contents by fly ash inhalation in rats
S.S. Chauhan,
S.R. Tyagi*, R.K. Kapoor**
qf Biochemistry.
Department (Received
10 April 1989)
(Accepted
31 May 1989)
V.P. Chest Institute,
Key words; Fly ash inhalation;
Mitochondrial
and U.K. Misra***
University of Delhi, Delhi 110007 (India)
cytochromes;
NADH-oxidase;
Lungs; Liver; [Rats]
SUMMARY Exposure activity.
of rats to fly ash for 15 days, 6 hours daily, inhibited
The content
fly-ash-exposed affecting
of cytochrome
group.
However,
the cytochrome
both organs
b and cytochromes
a+a,
in liver, fly ash exposure
b content,
indicating
pulmonary
and hepatic
was significantly reduced
a tissue-specific
NADH-oxidase
lower in the lungs of the
the cytochrome
a+a,
effect. Mitochondrial
level without
protein
content
in
was the same in both groups.
INTRODUCTION
Energy acquired by living cells is conserved in the form of adenosine triphosphate (ATP), which serves as an ‘energy currency’ in powering the other functions of cells. In the mitochondria of all cells with nuclei, electrons donated by organic molecules are passed from NADH or FADH2 to 02 by a series of electron carriers, and in this process ATP is generated [ 11. Mitochondrial cytochromes constitute a major portion
*Post-doctoral U.S.A.
Fellow, Department
**Post-doctoral
of Biochemistry,
Emory University
School of Medicine,
Atlanta,
GA,
Fellow.
***Professor and Head, Department of Biochemistry, V.P. Chest Institute, Delhi, India. At present: Visiting Professor, University of North Carolina at Chapel Hill, Department of Pharmacology, 1130, FLOB, Chapel Hill, NC 27514. U.S.A. Address Cancer
for correspondence:
Institute,
Laboratory
S.S. Chauhan, of Molecular
Visiting Biology,
Fellow, Building
National
Institutes
37 Room
2E22,
U.S.A. Tel. (301)496-6970
037%4274/89/$3,50
@ 1989 Elsevier Science Publishers
B.V. (Biomedical
Division)
of Health, Bethesda,
National
MD
20892,
16
of the electron transport chain, and hence their amount and proportions may be important for the efficient synthesis of ATP. Fly ash is a major environmental particulate pollutant containing polycyclic aromatic hydrocarbons and toxic trace elements [2]. Fly ash particles remain air-borne from a few hours to months, depending upon their aerodynamic size [2]. In vivo and in vitro cytotoxicity of these particles has been demonstrated [3, 41. After deposition in the lungs, fly ash and/or its chemical species are translocated to the extrapulmonary organs [5, 61. Exposure of rats to fly ash caused histological lesions in the lung and liver, reduced red and white blood cell numbers, and decreased hemoglobin content [4, 71. Fly ash also induced hepatic and pulmonary mixed-function oxidases in rats [8,9). Wilkins et al. [lo] reported lower ATP content in the lungs of rats exposed to fly ash. However, these workers did not attribute any reason for fly-ash-induced reduction in ATP content. The present study was therefore planned to investigate the effect of coal fly ash inhalation on mitochondrial cytochrome content and on NADH-oxidase activity, and hence on ATP synthesis in the rat lung and liver. MATERIALS
AND METHODS
Materials
Bovine serum albumin, NADH, EDTA, sodium deoxycholate, glycerol, ~,~,~,~~-tetramethy1 p-phenyldiamine (TMPD) and dithiothreitol were purchased from Sigma Chemical Co., St. Louis, MO. Sodium dithionite and sucrose were obtained from the British Drug Houses, India. Collection offly ash ami generation
of its aerosols
Fly ash was collected in bulk from the electrostatic precipitator of Inderprasth Thermal Power Station, New Delhi, during operation when bituminous coal was burned. It was passed through a 400-mesh (40 pm) stainless steel sieve. The shape of the fly ash particles so obtained was defined by scanning electron microscopy. The majority of the particles were spherical (both solid and hollow) and exhibited microcrystal deposition on their surfaces. Other particles were triangular, rod-shaped, or irregular in shape. The sieved fly ash was used for the generation of aerosols in the inhalation chamber. Approximately 58 % of the sieved fly ash particles were of respirable size, being 5 pm or less in diameter. The larger particles were trapped in a vessel through which the aerosol passed before entering the inhalation chamber. Therefore the concentration of respirable particles increased up to 68% in the inhalation chamber. Details of the inhalation chamber, the chemical composition of fly ash, and the generation of aerosols have been described elsewhere [4]. and exposure of animals to fly ash aerosols Male Wistar strain rats weighing 200-250 g were obtained from V.P. Chest Institute’s Small Animal Facility and maintained as described earlier [4]_ Maintenance
17
Rats were randomly divided into two groups and housed individually at all times throughout the experiment. One group was exposed 6 hours daily for 15 consecutive days to a fly ash concentration of 0.27 +O.Ol mg/l {mean f SE). The fly ash concentration in the inhalation chamber was monitored regularly. A group of control rats was exposed to filtered clean air for the same period of time under identical conditions. Sacrifice of animals and isolation of mitochondria On day 16, 1 day after the last tly ash exposure, overnight-fasted rats were anesthetized by intraperitoneal injection of pentobarbital (60 mg/kg body wt.). Lungs and livers were perfused in situ with ice-cold normal saline until they turned white. Subsequent processing of the tissues was done at 4°C. Mitochondria were isolated according to the method of Ritter and Malejka-Giganti [ 1l]. The tissues were cleaned, wiped and weighed, and a 20% homogenate was prepared in 0.25 M sucrose solution with a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 600 x g at 4°C in a Sorvall RC-SB centrifuge for 10 minutes. The supernatant was transferred to another centrifuge tube and the pellet was suspended in a small volume of the same solution, homogenized briefly and centrifuged. The combined supernatants were again centrifuged at 16 000 x g for 20 minutes to obtain a mitochondrial pellet. The mitochondria so obtained were washed and suspended in buffer. Estimation of NADH oxidase and respiratory c~tochrome~ Mitochondrial protein was estimated by the method of Lowry et al. 1121.NADH oxidase activity in mitochondria was estimated by the method of Mackler [13]. The mitochondrial pellet was suspended in 596 sucrose solution. The incubation mixture contained 0.1 k NADH, 0.005 M EDTA, 0.2 M phosphate buffer (pH 7.5) and mitochondrial protein in a final volume of 1.Oml. The reaction was started by the addition of enzyme, then the decrease in optical density at 340 nm was measured in a Beckman DU spectrophotometer. An extinction coefficient of 6.22 nM-‘-cm-’ for NADH was used to calculate the amount of NADH oxidized. The amounts of pulmonary and hepatic respiratory cytochromes were estimated according to the method of Rieske [ 141 and Gazzoti et al. [ 151. The mitochondrial pellet was suspended in 10 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol, 2 mM MgCl2 and 2 mM dithiothreitol. The assay mixture consisted of 1.O ml of enzyme protein (5-6 mg), 1.O ml of 1.O% deoxycholate, 0.45 ml of 200 mM phosphate buffer (pH 7.4), and 0.05 ml of I mM KsFe(CN)b. The assay mixture was divided equally into two cuvettes which were referred to as the reference and sample cuvettes. The contents of the sample cuvette were stirred with a few grains of sodium dithionite and the reduced-oxidized spectrum for cytochrome c and cytochromes a + a3 was recorded in the range of 500-650 nm. The contents of the reference cuvette were then stirred with a few grains of TMPD and the spectrum for cytochrome b was recorded in the range of 550-600
18
nm. The spectra were recorded in a doubIe-beam Shimadzu sp~trophotometer (Model UV-240). The amounts of the cytochromes were calculated from the difference in absorbance between the reduced and oxidized spectrums. The results were statistically analyzed using Student’s t-test. RESULTS
AND DISCUSSION
Fly ash inhalation 6 hours daily for 15 days did not affect the body weight or liver weight of rats, but significantly increased the lung weight as compared to control group rats (data not given). No change in mitochondrial protein content was noticed in the lungs or liver of the fly ash group as compared to the controls (Table I). NADH-oxidase activity in both organs was observed to be significantly lower in the fly ash group than in control animals (Table I). Similarly, fly ash inhalation significantly decreased the content of cytochrome b and cytochromes a+a3 in the lungs (Table II). However, only cytochrome at-a3 content was significantly lower in the livers of fly ash exposed rats compared with the control group (Table II). Fly ash inhalation did not have any effect on cytochrome c content in any of the organs studied (Table II). NADH-oxidase was observed to be reduced in the lungs and livers of experimental animals (Table I). Because NADH-oxidase is the first enzyme of the electron transport chain, the flow of electrons to the electron transport chain may have thus been obstructed. Cytochromes 6, c and a+a3 are the electron carriers in the mitochondrial electron transport chain. Reduction in the levels of cytochromes b and at-a3 in the lungs of experimental animals suggests that fly ash interferes with the flow of elec-
TABLE EFFECT
I OF FLY ASH INHALATION
NADH-OXIDASE
ON PULMONARY
AND
HEPATIC
MITOCHONDRIAL
IN RATS Control
Fly-ash-exposed
Lungs Mitochondrial
protein
NADH-oxidase oxidized/mg
(nmol NADH protein/min)
(mg/g tissue)
7.30+0.50
7.43 k 0.42
6.00 kO.98
3.77i0.24a
Liver
Mitochondrial
protein
NADH-oxidase oxidized/mg
(mg/g tissue)
14.06+ 1.20
12.902
1.19
(nmol NADH 12.21+ 1.93
protein/min)
*Values significantly
different
from control
Values are the mean &- SE from 6 animals
at 5 % level. in each group.
6.87 +0.50a
19 TABLE II EFFECT OF FLY ASH INHALATION TOCHROMES IN RATS
ON PULMONARY
AND HEPATIC RESPIRATORY
Control
Fly-ash-exposed
0.35 & 0.06 0.18,0.04 0.37t_o.O4
0.19*0.02= 0.21+0.02 0.26+0.01”
0.26 & 0.03 0.12,0.01 0.25f0.02
0.25 f 0.02 0.10+0.01 0.08 *o.o03a
CY-
Lungs
Cytochrome b Cytochrome c Cytochromes ata3 Liver
Cytochrome b Cytochrome c Cytochromes ~+a-,
+‘alues significantly different from control at 5 % level. Values are expressed in nmol cytochrome/mg protein. Values are the mean 5 SE from 6 animals in each group.
trons at these steps (Table II). After deposition in the lungs, fly ash and its chemical species are translocated to the liver and other extrapulmonary organs [4-71. In liver, only terminal cytochromes a+a3 were affected by translocated fly ash, indicating that the effect of inhaled fly ash is different on lung and liver (Table II). Since the relatively smaller amount of translocated fly ash (or its chemical species) that reached the liver decreased cytochromes a+~ but not cytochrome b, it is likely that cytochromes a+~ are more susceptible than cytochrome b to fly-ash-induced reduction. These results reveal that fly ash inhalation reduces the entry of electrons into the electron transport chain by reducing NADH-oxidase, and decreases the efficiency of electron transfer by de&easing the levels of mitochondrial cytochromes (Tables I and II). A decrease in either the cytochromes or NADH-oxidase can be expected to decrease the levels of ATP in lung and liver of by-ash-exposed animals. Wilkins et al. [IO] also observed a decrease in lung ATP levels by fly ash exposure. The metals lead and chromium have also been reported to decrease 02 consumption and ATP levels in rat brain and thymocytes, respectively [ 16, 171. Similarly, Garrett et al. [18] reported decreased ATP content in fly-ash-treated rabbit alveolar macrophages and Chinese hamster ovary cells. Inhibition of NADH-oxidase would lead to an increased level of NADH and thus increase NADPH formation by transhydrogenase reactions. An increased supply of NADPH favors the induction of mixed-function oxidase activity [19]. We have previously described the induction of mixed-function oxidases by fly ash inhalation in the lung and liver of rats [9]. The present study thus suggests that fly ash inhalation may decrease ATP levels by inhibiting NADH-oxidase and reducing the levels of respiratory cytochromes.
20
ACKNOWLEDGEMENTS
This study was supported by a grant from the Department of the Environment, Government of India, New Delhi. S.S.C. is grateful to the Indian Council of Scientific and Industrial Research for financial assistance. The generous help of Drs. S.M. Smith and K.V. Chin in the preparation
of the manuscript
is also acknowledged.
REFERENCES
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