- Email: [email protected]
Nonciliated bronchiolar epithelial (Clara) cell necrosis induced by organometallic carbonyl compounds
85
Toxicology Letters, 14 (1982) 85-92 Elsevier Biomedical Press
NONCILIATED BRONCHIOLAR EPITHELIAL (CLARA) CELL NECROSIS INDUCED BY ORGANOMETALLIC CARBONYL COMPOUNDS* (Pulmonary parenchymal piperonyl butoxide)
damage; methylcyc~opentadienyl
manganese tricarbonyl;
W.M. HASCHEK, P.J. HAKKINEN**, H.P. WITSCHI, R.P. HANZLIK*** and G.J. TRAIGER*** Biology Division, Oak Ridge National Laboratory, and **University of Tennessee-Oak Ridge Graduate School of Biomedicai Sciences, Oak Ridge, TN 37830; and ***Departments of Medicinal Chemistry, and Pharmacology and Toxicology, University of Kansas, Lawrence, KS 66046 (U.S.A.] (Received October 15th, 1981) (Accepted February 1lth, 1982)
SUMMARY The administration of transition metal organometallic compounds such as manganese, chromium, and iron carbonyls by the i.p. route, and nicket by inhalation (mice) or intravenously (rats), resulted in selective necrosis of the noncihated bronchiolar epithelial (Clara) cefls and variable pufmonary parenchymal damage in 3ALB/c mice and Fischer-derived rats within 24 h of administration. The pulmonary toxicity of methylcyclopentadienyl manganese tricarbonyl (MMT), a representative of this group of compounds, was enhanced by pretreatment with piperonyl butoxide (PB), an inhibitor of the mixed-function oxidase system. This finding suggests that Clara cell necrosis can result from direct toxicity and that the specificity of toxic agents for Clara cells may not be related solely to the presence of the mixed-function oxidase system.
INTRODUCTION
Administration of agents such as 4-ipomeanol, 3MF, and Ccl, produces acute necrosis of the nonciliated bronchiolar (Clara) cells of the small airways. Metabolic activation of the parent compound by the microsomal Pdso mixed-function oxidase system to a highly reactive intermediate is a key mechanism in the toxicity of these compounds [l]. While studying the pulmonary toxicity of MMT in mice [2], we
*This work has been presented, in part, by the same authors in The Toxicologist, 1 (1981) 83. Abbreviations: BHT, 3,5-di-Bert-butyi-4-hydroxytoluene; 3MF, 3-methylfuran; ~ntadienyl manganese tricarbonyl; PB, piperonyl butoxide. 0378-4274/82/0000-0000/$02.75
0 Elsevier Biomedical Press
MMT, methytcyclo-
86
observed that this compound also produced acute Clara cell necrosis. This raised the question of whether MMT would also affect the Clara cells in other species and whether other organometallic compounds would have similar effects. Results described herein show that organometallic carbonyl complexes of manganese, nickel, chromium, and iron also cause Clara cell damage in these species. MATERIALS
AND METHODS
Chemicals MMT, benzene chromium tricarbonyl, anisole chromium tricarbonyl, toluene chromium tricarbonyl, and cyclopentadienyl manganese tricarbonyl, butadiene iron tricarbonyl were purchased from Strem Chemicals (Newburyport, MA), and purified, and handled as described previously [3]. BHT was purchased from Sigma Chemical Co. (St. Louis, MO), and PB was from Pfaltz and Bauer (Stamford, CT). Nickel carbonyl was obtained from Dr. W.F. Sunderman Jr. (Farmington, CT), and 3MF was from Dr. M.R. Boyd (National Cancer Institute, Bethesda, MD). Treatment All compounds except Ni(CO), and 3MF were dissolved in corn oil and injected i.p. into female BALB/c mice and male Fischer-derived rats. Ni(CO), was injected i.v. into rats and mice were exposed by inhalation in a static inhalation chamber. Doses used are given in Table I; they are approximately half the acute LD,,
TABLE
I
CLARA
CELL
NECROSIS
PRODUCED
BY ORGANOMETALLIC i.p. dose (m&kg) Mouse
Rat
Corn oil
a
b
MMT
120
Cyclopentadienyl
Mn(CO)3
30
COMPOUNDS Clara
Treatment
8.4 10 d
Mouse
cell necrosis Rat
_
_
+++
+
++
++
+ _
+
Ni(C0)4 Benzene
Cr(CO)J
15
5
Anisole
Cr(CO)3
120
20
+++
Toluene
Cr(C0)3
120
20
++++
+ +
30
10
++++
++t
Butadiene
Fe(CO)j
aO.l ml/10
g.
bO.l ml/100 g. co.35 pi/l for 1.5 h by inhalation. d20 mg/kg
i.v.
+
87
(unpublished data). Control animals received an equal volume of corn oil i.p. or were kept in the inhalation chamber for the same length of time without the presence of the toxic inhalant. All animals were killed 24 h after treatment. In a second experiment, female mice received 1600 mg/kg of PB or an equal volume of corn oil i.p. I h before injection of 90 mg MMT/kg. In this experiment, positive controls were treated with BHT (400 mg/kg i.p.) or by exposure to 3MF (38 ~molll) for 1 h; both BHT and 3MF require metabolic activation to induce pulmonary toxicity, and animals can be protected with PB [ 1, 41. Animals were killed 24 h after treatment with MM?: and 3MF, and 3 days after BHT injection. Pathology Lungs were fixed by i-t. instillation of 10% buffered formalin. Lung sections were embedded in glycol methacrylate, cut at l-pm with a Sorvall JB4 microtome, and stained with Lee’s methylene blue-basic fuchsin for light microscopic examination. Pulmonary lesions were scored without knowledge of treatment. A scoring system of + to + + + + was used to evaluate the severity of Clara cell necrosis. Two qualitative criteria were used to determine the final score: (i) The extent of the bronchiole affected, beginning from the termination of the bronchiole, and (ii) the propo~ion of Clara cells which were necrotic or undergoing degenerative changes within the affected area. Parenchymal damage was assessed by use of a scoring system of + to + + + + based on the severity of inflammatory changes present. RESULTS
All the organometallic compounds produced selective necrosis of Clara cells in both mice and rats, with the exception of benzene Cr(CO), (Table I), which did not produce Clara cell damage in mice at a dose of 15 mg/kg. Higher doses could not be used since the animals died following severe convulsions within minutes of administration. Clara cell necrosis was more severe in the mouse than in the rat for all compounds studied except Ni(CO).+, for which the extent of damage was similar in both species. Severe Clara cell damage in the mouse seen after MMT, anisole Cr(CO),, toluene Cr(CO),, and butadiene Fe(CO), treatment consisted of necrosis of all Clara cells in the terminal bronchioles and some Clara cells in the larger, more proximal bronchioles. There was flattening of the cytoplasmic protrusions of the remaining Clara cells. The necrotic Clara cells had undergone pyknosis and sloughed into the lumen (Fig. l), leaving behind basement membrane which was either denuded or covered by flattened, abnormal-looking ciliated cells. Similar changes were seen in the rat after administration of butadiene Fe(CO&, except that the basement
Fig. 1. Bronchiole
from a mouse killed 24 h after treatment.
(a) Control,
* normal bronchiolar
nonciliated (Clara) epithelial cells. (b) Injected i.p. with 30 mg butadiene (CO)3/kg. Necrotic are sloughing into the lumen leaving behind abnormal-looking ciliated cells. Methylene fuchsin.
x 1000.
ciliated and Clara Cells blue-basic
89
Fig. 2. Bronchiole from a rat killed 24 h after treatment. (a) Control; (b) Injected i.p. with 5 mg benzene Cr(CO)3/kg. Few Clara cells are present. Several ciliated cells contain condensed cellular fragments within their cytoplasm. Methylene blue-basic fuchsin. x 1000.
90
membrane was never denuded but remained covered by virtually normal ciliated cells, presumably due to the greater proportion of ciliated cells present in this species. The least severe Clara cell damage seen in mice and rats was present only in the distal portion of the terminal bronchiole and was characterized primarily by degenerative changes and only occasionally by necrotic, sloughed cells. Degenerative changes consisted of cytoplasmic vacuolation, nuclear condensation, and flattening of Clara cells. A prominent feature seen with moderate Clara cell damage, especially in the rat, was the presence of cellular fragments, some containing chromatin, within the cytoplasm of the ciliated cells in areas of necrosis (Fig. 2) and occasionally within the adjacent Clara cells themselves. This was confirmed by electron microscopy (Haschek, unpublished observations). These cell fragments may represent either damaged cell components [5] or, more probably, phagocytosis of apoptotic bodies [6]. Both mice and rats treated with Ni(CO), had swollen, pale, vacuolated Clara cells. Parenchymal damage consisting of inflammation and necrosis was present in all animals to a variable degree. The results of pretreatment with PB on MMT are shown in Table II. Potentiation TABLE
II
PULMONARY
TOXICITY
IN THE MOUSE
WITH
AND WITHOUT
Treatment
Clara
PBa
_
_
MMTb
it
++
PB + MMT
++++
3MFc
++++
+++i _
PB + 3MF BHTd
+ _
PB + BHT
_
a1600 mg/kg
Parenchymal
WITH
PB
damage
+++ _
i.p.
b90 mg/kg
i.p.
c38 rmol/l
for 1 h by inhalation.
d400 mg/kg
cell necrosis
PRETREATMENT
i.p.
of MMT toxicity was evidenced by increased severity of pulmonary damage. Clara cell damage extended into the larger bronchioles. Moderate perivascular edema, inflammation, and focal hemorrhages were present within the parenchyma. In addition, mortality was greatly enhanced in mice treated with both PB and MMT. On the other hand, PB pretreatment completely blocked BHT-induced pulmonary parenchymal damage as well as 3MF-induced Clara cell necrosis, as described previously by Boyd et al. [7].
91
DISCUSSION
The ability of organometallic compounds to produce Clara cell damage has not been described previously. Ni(CO), is a well-known acute pulmonary toxicant [8], and MMT has been reported to produce diffuse lung damage in rats and rabbits [9-l 11. Preliminary work has indicated that several of the other organometallic compounds are pulmonary toxins in rats (Traiger, unpublished data). However, in all studies performed so far, attention has been focused on the alveolar damage caused by MMT, Ni(CO)+, and the other compounds. Our observations show that organometallic compounds may also produce epithelial lesions in the small airways. This indicates that both bronchiolar nonciliated (Clara) epithelial cells and parenchymal cells are targets in organometallic-induced pneumotoxicity. Transition metal organometallic complexes are increasingly used and have many industrial, commercial and medical applications. MMT, for example, is used as a replacement for tetraethyllead in unleaded gasolines [lo]. The potential of MMT and related agents to cause damage to airways needs to be studied further. Another interesting observation was that MMT-induced Clara cell damage in mice could not be prevented with PB pretreatment. To date, all agents which produce Clara cell necrosis, such as 4-ipomeanol, 3MF and Ccl, (all of which are non-metallic compounds), have been shown to require metabolic activation by the pulmonary mixed-function oxidase system to produce their toxic effect [l]. Although PB protected mice against 3MF- and BHT-induced lung injury, it was unable to protect mice against MMT toxicity. This suggests that metabolic activation is not a prerequisite for MMT-mediated cytotoxicity to the Clara cells. In other studies it was also concluded that MMT itself is toxic without bioactivation [l 11. The specificity of certain toxic agents for Clara cells may thus not be related solely to the presence of the mixed-function oxidase system. It is not yet clear why PB treatment actually enhances pulmonary toxicity of MMT. In the rat MMT appears to be detoxified by the P 450system primarily in the liver, with oxidation of the methyl substituent being the favored route of metabolism [12]. Following i.p. administration, much of the parent compound could be inactivated by a first-pass effect in the liver [ll]. If this process is inhibited following administration of PB, increased levels of the parent compound could remain in the circulation and reach the lungs, resulting in enhanced toxicity. In conclusion, we have shown that several organometallic compounds produce Clara cell necrosis in two animal species. For at least one compound, MMT, metabolic activation appears not to be a prerequisite for toxicity. Organometallic compounds need to be studied further to determine their potential to cause acute and possibly chronic lung damage. They might also serve as tools to study mechanisms of toxicity in small airway epithelium.
92
ACKNOWLEDGEMENT
This research was sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under contract W-7405eng-26 with the Union
Carbide
Corporation
Oak Ridge National
and by subcontract
Laboratory
3322 from the Biology
to the University
Division
of
of Tennessee.
REFERENCES
M.R. Boyd, Biochemical Crit. Rev. Toxicol., H.P.
Witschi,
damage R.P.
W.M. Haschek,
by oxygen:
Hanzlik,
pentadienyl
mechanisms
in chemical-induced
lung injury:
Roles of metabolic
activation,
7 (1980) 103-176. A.J.P.
Determining
C.E.
Harkness
manganese
Klein-Szanto
variables,
and S. Arnoldi,
tricarbonyl
and P.J. Hakkinen,
Am. Rev. Respir.
Gas chromatographic
in biological
tissues
Potentiation
of diffuse
lung
Dis., 123 (1981) 988103.
and
determination
fluids,
of methylcyclo-
J. Chromatogr.,
171 (1979)
2799283. J.P.
Kehrer
induced
and H.P.
pulmonary
S.L. Kauffman urethane
Witschi,
toxicity
Effects
and L. Herman,
exposure,
Develop.
J.F.R.
Kerr and J. Searle,
(Eds.),
Radiation
Its nature
R.B. Franklin
necrosis
a naturally
R.L.
Hackett
and
W. Moore,
dienyl manganese Hyg. Assoc.
11 R.P. 12 R.P.
hydroxytoluene-
in embryonic
mouse
neural
tube cells after
and kinetic Raven
Press,
and J.R. Mitchell, occurring
potential
role, in R.E. Meyn and H.R.
Withers
New York,
1980, pp. 367-384.
Pulmonary
bronchiolar
atmospheric
alkylation
and
Nature,
272
contaminant,
Sunderman
J.F. Stara,
tricarbonyl Toxicity
Jr.,
studies,
Pulmonary
Arch.
alveolar
Environ.
Health,
R. Miller and K.I. Campbell,
(MMT)
in rats, Environ.
reaction
to
nickel
carbonyl.
16 (1968) 349-362. Oral toxicity
of methylcyclopenta-
Res., 7 (1974) 158-168.
studies of methylcyclopentadienyl
manganese
tricarbonyl
(MMT),
Am. Ind.
J., 40 (1979) 164-167.
Hanzlik,
tricarbonyl
F.W.
and histochemical
10 R.K. Hinderer,
on butylated
53 (1980) 333-342.
1.
Ultrastructural D.K. Hysell,
changes
Research,
M.R. Boyd, C.N. Statham, (1978) 270-27
inhibitors
Pharmacol.,
17 (1968) 55-74.
in Cancer
by 3-methylfuran,
Appl.
Ultrastructural
Biol., Apoptosis:
Biology
of drug metabolism
in mice, Toxicol.
R. Stitt
and
G.J.
Traiger,
Toxic
(MMT) in rats: Role of metabolism,
Hanzlik,
P.
methylcyclopentadienyl
Bhatia,
R.
manganese
Stitt
and
tricarbonyl
effects
Toxicol.
G.J.
Traiger,
of methylcyclopentadienyl Appl.
Pharmacol.,
Biotransformation
in the rat, Drug Metab.
manganese
56 (1980) 353-360.
Dispos.,
and
excretion
8 (1980) 428-433.
of
Toxicology Letters, 14 (1982) 85-92 Elsevier Biomedical Press
NONCILIATED BRONCHIOLAR EPITHELIAL (CLARA) CELL NECROSIS INDUCED BY ORGANOMETALLIC CARBONYL COMPOUNDS* (Pulmonary parenchymal piperonyl butoxide)
damage; methylcyc~opentadienyl
manganese tricarbonyl;
W.M. HASCHEK, P.J. HAKKINEN**, H.P. WITSCHI, R.P. HANZLIK*** and G.J. TRAIGER*** Biology Division, Oak Ridge National Laboratory, and **University of Tennessee-Oak Ridge Graduate School of Biomedicai Sciences, Oak Ridge, TN 37830; and ***Departments of Medicinal Chemistry, and Pharmacology and Toxicology, University of Kansas, Lawrence, KS 66046 (U.S.A.] (Received October 15th, 1981) (Accepted February 1lth, 1982)
SUMMARY The administration of transition metal organometallic compounds such as manganese, chromium, and iron carbonyls by the i.p. route, and nicket by inhalation (mice) or intravenously (rats), resulted in selective necrosis of the noncihated bronchiolar epithelial (Clara) cefls and variable pufmonary parenchymal damage in 3ALB/c mice and Fischer-derived rats within 24 h of administration. The pulmonary toxicity of methylcyclopentadienyl manganese tricarbonyl (MMT), a representative of this group of compounds, was enhanced by pretreatment with piperonyl butoxide (PB), an inhibitor of the mixed-function oxidase system. This finding suggests that Clara cell necrosis can result from direct toxicity and that the specificity of toxic agents for Clara cells may not be related solely to the presence of the mixed-function oxidase system.
INTRODUCTION
Administration of agents such as 4-ipomeanol, 3MF, and Ccl, produces acute necrosis of the nonciliated bronchiolar (Clara) cells of the small airways. Metabolic activation of the parent compound by the microsomal Pdso mixed-function oxidase system to a highly reactive intermediate is a key mechanism in the toxicity of these compounds [l]. While studying the pulmonary toxicity of MMT in mice [2], we
*This work has been presented, in part, by the same authors in The Toxicologist, 1 (1981) 83. Abbreviations: BHT, 3,5-di-Bert-butyi-4-hydroxytoluene; 3MF, 3-methylfuran; ~ntadienyl manganese tricarbonyl; PB, piperonyl butoxide. 0378-4274/82/0000-0000/$02.75
0 Elsevier Biomedical Press
MMT, methytcyclo-
86
observed that this compound also produced acute Clara cell necrosis. This raised the question of whether MMT would also affect the Clara cells in other species and whether other organometallic compounds would have similar effects. Results described herein show that organometallic carbonyl complexes of manganese, nickel, chromium, and iron also cause Clara cell damage in these species. MATERIALS
AND METHODS
Chemicals MMT, benzene chromium tricarbonyl, anisole chromium tricarbonyl, toluene chromium tricarbonyl, and cyclopentadienyl manganese tricarbonyl, butadiene iron tricarbonyl were purchased from Strem Chemicals (Newburyport, MA), and purified, and handled as described previously [3]. BHT was purchased from Sigma Chemical Co. (St. Louis, MO), and PB was from Pfaltz and Bauer (Stamford, CT). Nickel carbonyl was obtained from Dr. W.F. Sunderman Jr. (Farmington, CT), and 3MF was from Dr. M.R. Boyd (National Cancer Institute, Bethesda, MD). Treatment All compounds except Ni(CO), and 3MF were dissolved in corn oil and injected i.p. into female BALB/c mice and male Fischer-derived rats. Ni(CO), was injected i.v. into rats and mice were exposed by inhalation in a static inhalation chamber. Doses used are given in Table I; they are approximately half the acute LD,,
TABLE
I
CLARA
CELL
NECROSIS
PRODUCED
BY ORGANOMETALLIC i.p. dose (m&kg) Mouse
Rat
Corn oil
a
b
MMT
120
Cyclopentadienyl
Mn(CO)3
30
COMPOUNDS Clara
Treatment
8.4 10 d
Mouse
cell necrosis Rat
_
_
+++
+
++
++
+ _
+
Ni(C0)4 Benzene
Cr(CO)J
15
5
Anisole
Cr(CO)3
120
20
+++
Toluene
Cr(C0)3
120
20
++++
+ +
30
10
++++
++t
Butadiene
Fe(CO)j
aO.l ml/10
g.
bO.l ml/100 g. co.35 pi/l for 1.5 h by inhalation. d20 mg/kg
i.v.
+
87
(unpublished data). Control animals received an equal volume of corn oil i.p. or were kept in the inhalation chamber for the same length of time without the presence of the toxic inhalant. All animals were killed 24 h after treatment. In a second experiment, female mice received 1600 mg/kg of PB or an equal volume of corn oil i.p. I h before injection of 90 mg MMT/kg. In this experiment, positive controls were treated with BHT (400 mg/kg i.p.) or by exposure to 3MF (38 ~molll) for 1 h; both BHT and 3MF require metabolic activation to induce pulmonary toxicity, and animals can be protected with PB [ 1, 41. Animals were killed 24 h after treatment with MM?: and 3MF, and 3 days after BHT injection. Pathology Lungs were fixed by i-t. instillation of 10% buffered formalin. Lung sections were embedded in glycol methacrylate, cut at l-pm with a Sorvall JB4 microtome, and stained with Lee’s methylene blue-basic fuchsin for light microscopic examination. Pulmonary lesions were scored without knowledge of treatment. A scoring system of + to + + + + was used to evaluate the severity of Clara cell necrosis. Two qualitative criteria were used to determine the final score: (i) The extent of the bronchiole affected, beginning from the termination of the bronchiole, and (ii) the propo~ion of Clara cells which were necrotic or undergoing degenerative changes within the affected area. Parenchymal damage was assessed by use of a scoring system of + to + + + + based on the severity of inflammatory changes present. RESULTS
All the organometallic compounds produced selective necrosis of Clara cells in both mice and rats, with the exception of benzene Cr(CO), (Table I), which did not produce Clara cell damage in mice at a dose of 15 mg/kg. Higher doses could not be used since the animals died following severe convulsions within minutes of administration. Clara cell necrosis was more severe in the mouse than in the rat for all compounds studied except Ni(CO).+, for which the extent of damage was similar in both species. Severe Clara cell damage in the mouse seen after MMT, anisole Cr(CO),, toluene Cr(CO),, and butadiene Fe(CO), treatment consisted of necrosis of all Clara cells in the terminal bronchioles and some Clara cells in the larger, more proximal bronchioles. There was flattening of the cytoplasmic protrusions of the remaining Clara cells. The necrotic Clara cells had undergone pyknosis and sloughed into the lumen (Fig. l), leaving behind basement membrane which was either denuded or covered by flattened, abnormal-looking ciliated cells. Similar changes were seen in the rat after administration of butadiene Fe(CO&, except that the basement
Fig. 1. Bronchiole
from a mouse killed 24 h after treatment.
(a) Control,
* normal bronchiolar
nonciliated (Clara) epithelial cells. (b) Injected i.p. with 30 mg butadiene (CO)3/kg. Necrotic are sloughing into the lumen leaving behind abnormal-looking ciliated cells. Methylene fuchsin.
x 1000.
ciliated and Clara Cells blue-basic
89
Fig. 2. Bronchiole from a rat killed 24 h after treatment. (a) Control; (b) Injected i.p. with 5 mg benzene Cr(CO)3/kg. Few Clara cells are present. Several ciliated cells contain condensed cellular fragments within their cytoplasm. Methylene blue-basic fuchsin. x 1000.
90
membrane was never denuded but remained covered by virtually normal ciliated cells, presumably due to the greater proportion of ciliated cells present in this species. The least severe Clara cell damage seen in mice and rats was present only in the distal portion of the terminal bronchiole and was characterized primarily by degenerative changes and only occasionally by necrotic, sloughed cells. Degenerative changes consisted of cytoplasmic vacuolation, nuclear condensation, and flattening of Clara cells. A prominent feature seen with moderate Clara cell damage, especially in the rat, was the presence of cellular fragments, some containing chromatin, within the cytoplasm of the ciliated cells in areas of necrosis (Fig. 2) and occasionally within the adjacent Clara cells themselves. This was confirmed by electron microscopy (Haschek, unpublished observations). These cell fragments may represent either damaged cell components [5] or, more probably, phagocytosis of apoptotic bodies [6]. Both mice and rats treated with Ni(CO), had swollen, pale, vacuolated Clara cells. Parenchymal damage consisting of inflammation and necrosis was present in all animals to a variable degree. The results of pretreatment with PB on MMT are shown in Table II. Potentiation TABLE
II
PULMONARY
TOXICITY
IN THE MOUSE
WITH
AND WITHOUT
Treatment
Clara
PBa
_
_
MMTb
it
++
PB + MMT
++++
3MFc
++++
+++i _
PB + 3MF BHTd
+ _
PB + BHT
_
a1600 mg/kg
Parenchymal
WITH
PB
damage
+++ _
i.p.
b90 mg/kg
i.p.
c38 rmol/l
for 1 h by inhalation.
d400 mg/kg
cell necrosis
PRETREATMENT
i.p.
of MMT toxicity was evidenced by increased severity of pulmonary damage. Clara cell damage extended into the larger bronchioles. Moderate perivascular edema, inflammation, and focal hemorrhages were present within the parenchyma. In addition, mortality was greatly enhanced in mice treated with both PB and MMT. On the other hand, PB pretreatment completely blocked BHT-induced pulmonary parenchymal damage as well as 3MF-induced Clara cell necrosis, as described previously by Boyd et al. [7].
91
DISCUSSION
The ability of organometallic compounds to produce Clara cell damage has not been described previously. Ni(CO), is a well-known acute pulmonary toxicant [8], and MMT has been reported to produce diffuse lung damage in rats and rabbits [9-l 11. Preliminary work has indicated that several of the other organometallic compounds are pulmonary toxins in rats (Traiger, unpublished data). However, in all studies performed so far, attention has been focused on the alveolar damage caused by MMT, Ni(CO)+, and the other compounds. Our observations show that organometallic compounds may also produce epithelial lesions in the small airways. This indicates that both bronchiolar nonciliated (Clara) epithelial cells and parenchymal cells are targets in organometallic-induced pneumotoxicity. Transition metal organometallic complexes are increasingly used and have many industrial, commercial and medical applications. MMT, for example, is used as a replacement for tetraethyllead in unleaded gasolines [lo]. The potential of MMT and related agents to cause damage to airways needs to be studied further. Another interesting observation was that MMT-induced Clara cell damage in mice could not be prevented with PB pretreatment. To date, all agents which produce Clara cell necrosis, such as 4-ipomeanol, 3MF and Ccl, (all of which are non-metallic compounds), have been shown to require metabolic activation by the pulmonary mixed-function oxidase system to produce their toxic effect [l]. Although PB protected mice against 3MF- and BHT-induced lung injury, it was unable to protect mice against MMT toxicity. This suggests that metabolic activation is not a prerequisite for MMT-mediated cytotoxicity to the Clara cells. In other studies it was also concluded that MMT itself is toxic without bioactivation [l 11. The specificity of certain toxic agents for Clara cells may thus not be related solely to the presence of the mixed-function oxidase system. It is not yet clear why PB treatment actually enhances pulmonary toxicity of MMT. In the rat MMT appears to be detoxified by the P 450system primarily in the liver, with oxidation of the methyl substituent being the favored route of metabolism [12]. Following i.p. administration, much of the parent compound could be inactivated by a first-pass effect in the liver [ll]. If this process is inhibited following administration of PB, increased levels of the parent compound could remain in the circulation and reach the lungs, resulting in enhanced toxicity. In conclusion, we have shown that several organometallic compounds produce Clara cell necrosis in two animal species. For at least one compound, MMT, metabolic activation appears not to be a prerequisite for toxicity. Organometallic compounds need to be studied further to determine their potential to cause acute and possibly chronic lung damage. They might also serve as tools to study mechanisms of toxicity in small airway epithelium.
92
ACKNOWLEDGEMENT
This research was sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under contract W-7405eng-26 with the Union
Carbide
Corporation
Oak Ridge National
and by subcontract
Laboratory
3322 from the Biology
to the University
Division
of
of Tennessee.
REFERENCES
M.R. Boyd, Biochemical Crit. Rev. Toxicol., H.P.
Witschi,
damage R.P.
W.M. Haschek,
by oxygen:
Hanzlik,
pentadienyl
mechanisms
in chemical-induced
lung injury:
Roles of metabolic
activation,
7 (1980) 103-176. A.J.P.
Determining
C.E.
Harkness
manganese
Klein-Szanto
variables,
and S. Arnoldi,
tricarbonyl
and P.J. Hakkinen,
Am. Rev. Respir.
Gas chromatographic
in biological
tissues
Potentiation
of diffuse
lung
Dis., 123 (1981) 988103.
and
determination
fluids,
of methylcyclo-
J. Chromatogr.,
171 (1979)
2799283. J.P.
Kehrer
induced
and H.P.
pulmonary
S.L. Kauffman urethane
Witschi,
toxicity
Effects
and L. Herman,
exposure,
Develop.
J.F.R.
Kerr and J. Searle,
(Eds.),
Radiation
Its nature
R.B. Franklin
necrosis
a naturally
R.L.
Hackett
and
W. Moore,
dienyl manganese Hyg. Assoc.
11 R.P. 12 R.P.
hydroxytoluene-
in embryonic
mouse
neural
tube cells after
and kinetic Raven
Press,
and J.R. Mitchell, occurring
potential
role, in R.E. Meyn and H.R.
Withers
New York,
1980, pp. 367-384.
Pulmonary
bronchiolar
atmospheric
alkylation
and
Nature,
272
contaminant,
Sunderman
J.F. Stara,
tricarbonyl Toxicity
Jr.,
studies,
Pulmonary
Arch.
alveolar
Environ.
Health,
R. Miller and K.I. Campbell,
(MMT)
in rats, Environ.
reaction
to
nickel
carbonyl.
16 (1968) 349-362. Oral toxicity
of methylcyclopenta-
Res., 7 (1974) 158-168.
studies of methylcyclopentadienyl
manganese
tricarbonyl
(MMT),
Am. Ind.
J., 40 (1979) 164-167.
Hanzlik,
tricarbonyl
F.W.
and histochemical
10 R.K. Hinderer,
on butylated
53 (1980) 333-342.
1.
Ultrastructural D.K. Hysell,
changes
Research,
M.R. Boyd, C.N. Statham, (1978) 270-27
inhibitors
Pharmacol.,
17 (1968) 55-74.
in Cancer
by 3-methylfuran,
Appl.
Ultrastructural
Biol., Apoptosis:
Biology
of drug metabolism
in mice, Toxicol.
R. Stitt
and
G.J.
Traiger,
Toxic
(MMT) in rats: Role of metabolism,
Hanzlik,
P.
methylcyclopentadienyl
Bhatia,
R.
manganese
Stitt
and
tricarbonyl
effects
Toxicol.
G.J.
Traiger,
of methylcyclopentadienyl Appl.
Pharmacol.,
Biotransformation
in the rat, Drug Metab.
manganese
56 (1980) 353-360.
Dispos.,
and
excretion
8 (1980) 428-433.
of