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Species differences in release of arachidonate metabolites in response to inhaled diluted diesel exhaust
Toxicology Letters, 42 (1988) 325-332 Elsevier
325
TXL 02016
Species differences .in release of arachidonate metabolites in response to inhaled diluted diesel exhaust R.F. Henderson, R.O. McClellan
H.W. Leung*, A.G. Harmsen** and
Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute, Albuquerque, NM 87185, U.S.A. (Received 10 November 1987) (Revision received 20 April 1988) (Accepted 10 May 1988)
Key words: Aracbidonate
metabolite; Species difference; Diesel exhaust, particles; Inflammation
SUMMARY In life-span studies in CD-l mice and F344Krl rats, inhaled diluted diesel exhaust was highly fibrogenic in rats but not in mice. This was the case despite the higher lung burden, in mg soot/g lung, achieved in mice compared to rats. We tested the hypothesis that the greater fibrogenicity of the soot in rats was due in part to greater release of mediators of inflammation from alveolar cells in rats compared to mice. Female F344/rats and B6C3Fr mice were exposed for up to 17 days to diluted diesel exhaust containing 3.5 mg/m-’ of soot. The lungs of control and soot-exposed animals were lavaged after 2, 12 or 17 days of exposure. The presence of leukotriene (LT)Ba, LTG, prostaglandin (PG)Ez, PGFzu and thromboxane (TX) B2 in the lavage fluids and LTB4 and PGFa, in cultured lavage cell supernatants was determined. The total amount of each lavage fluid constituent was normalized to lung weight for species comparisons. Control rats had higher levels of TXBa (16-fold), and LTB4 (6-fold) and PGEz (2-fold) than control mice, but control mice bad higher amounts of LTG (Cfold). Control rats and mice had approximately the same amounts of PGF&g lung in bronchoalveolar lavage fluid (BALF). Rats exposed to diesel exhaust had increases in BALF PGF2, and LTB4 that were highest after 2 days of exAddress for correspondence: Dr. R.F. Henderson, Lovelace Inhalation Toxicology Research Institute, P.O. Box 5890, Albuquerque, NM 87185, U.S.A. * Current address of Dr. H.W. Leung is Syntex Corp, 3401 Hillview Ave., Palo Alto, CA 94304, U.S.A. Dr. H.W. Leung was an Associated Western Universities Faculty Research Participant during the conduct of this research. ** Current address of Dr. A.G. Harmsen is Trudeau Institute, P.O. Box 59, Saranac Lake, NY 12983, U.S.A. Abbreviations: LT, leukotriene; PG, prostaglandin; TX, throboxane; BALF, bronchoalveolar lavage fluid; AA, arachidonic acid; PMN, polymorphonuclear neutrophil; SRS, slow-reacting substance.
326
posure
and decreased
lavage fluid released consistent
thereafter.
with the hypothesis
inflammation
Mice had lesser increases
larger amounts
of LTB4 into culture
that soot-laden
than do the alveolar
rat alveolar
in both parameters. supernatants
Rat cells recovered
from
than mouse cells. The data were
cells release greater
quantities
of mediators
of
cells in mice.
INTRODUCTION
Rats exposed for up to 24 months to diluted exhaust from diesel engines [l] developed a much more severe focal pulmonary fibrosis than mice exposed for the same period, despite greater accumulation of soot/g lung in mice. The basis for the species differences in development of pulmonary fibrosis is unknown and may be due to a variety of causes. One possible contributing factor may be differences in the response of the alveolar cells to stimulation by the diesel soot particles. Stimulated macrophages and neutrophils are known to release various chemical factors that influence the course of an inflammatory response [2]. Arachidonic acid (AA) metabolites appear to be especially important in influencing the course of events [3-61. Among the lipoxygenase products of AA (called leukotrienes), leukotriene B4 (LTB4) induces a strong inflammatory response and has a potent chemotactic and chemokinetic activity toward polymorphonuclear neutrophils (PMN), eosinophils, and monocytes [3,5-71. It aggregates PMN and promotes the release of lysosomal enzymes from these cells. LTB4 also causes increased permeability of the alveolar/capillary barrier of the lung. The cysteinecontaining leukotrienes (LTC4, LTD4, LTE4) are potent bronchoconstrictors and mimic the effects of the slow-reacting substance (SRS) of anaphylaxis [3,6]. The cycle-oxygenase products of AA metabolism are also important mediators of the inflammatory response, and include prostaglandins (PG) and thromboxanes (TX) [4,5,8]. PGE2 and PGF2, alter the microvascular pressure of the lung and cause constriction of bronchial smooth muscle [5,8]. TXAz has a similar effect and promotes neutrophil adherence to endothelium, platelet aggregation, and enzyme release from platelets
[5,8]. The thromboxanes
also mediate
of the LTC4, LTD4, and LTE4. TXA2 is rapidly TXBz, which is biologically inactive. Estimates measuring the more stable TXBz.
hydrolyzed of TXAZ
the constrictive
effects
non-enzymatically are usually made
to by
The objective of the current study was to determine if there were species differences in the release of inflammatory mediators in the lungs of diesel exhaustexposed rats and mice, which might explain, in part, the differences in severity of pulmonary fibrosis induced by chronic diesel exhaust exposures in these two species. MATERIALS
AND METHODS
Thirty female F344/N rats and 60 female B6C3Fr mice were used in the study. The rats were born and raised in the barrier-maintained Inhalation Toxicology
327
Research Institute (ITRI) colony and were housed two per cage in polycarbonate cages with hardwood chip bedding and filter caps prior to being placed in conditioning chambers (Hazleton Systems, Aberdeen, MD, U.S.A.). The mice were obtained from Frederick Cancer Research Center, Frederick, MD, U.S.A., and were held in quarantine for 2 weeks prior to being placed in conditioning chambers. Feed (Lab Blox, Allied Mills, Chicago, IL, U.S.A.) and water were provided ad libitum except during exposure hours, when food was removed. Rodents were assigned to the various treatment and control groups using a table of random numbers. Rodents were placed in conditioning chambers for 2-3 weeks prior to start of diesel exposures. Thirty B6C3Fr mice and 15 F344/N rats (all females, 18-20 weeks of age, rats; 13 weeks of age, mice) were exposed 7 h/day, 5 days/week to diluted diesel exhaust containing 3.5 mg soot/m3 air. Exhaust was generated by two 1980 model, 5.7 liter Oldsmobile engines operating alternatively on the Federal Test Procedure urban driving cycle and burning a standardized certification fuel (D-2 Diesel Control Fuel, Phillips Chemical Co.) [9]. Exhaust was diluted serially with clean air to provide the desired concentrations. Particle concentrations were measured by filter samples taken daily. Ten mice and five rats were removed from the exposure after 2, 12, and 17 exposure days to determine the amount of mediators in bronchoalveolar lavage fluid (BALF). Cells recovered from BALF were also cultured and the release of mediators assayed in vitro. The day after removal from the exposure chambers, animals were anesthetized with 4% halothane in oxygen, and killed by severing the brachial arteries. The lungs with tracheas attached were removed from each animal and weighed. The lungs were lavaged two times with 0.15 M saline to obtain epithelial lining fluid and free alveolar cells as described previously [lo]. The volume of each wash was 5 ml for rats and 1 ml for mice. Tracheas were removed and weighed. The difference between the weight of the trachea and the weight of the lung plus trachea was calculated as the wet weight of the lung. Individual lavage fluid samples from the same species were pooled. Total and differential cell counts were made on BALF cells concentrated by centrifugation at 300 x g for 10 min. Cells were incubated at 37°C in RPM1 1640 at a concentration of - 4 x lo6 cells/ml for 1 h. Arachidonic acid metabolites were assayed in two cell supernatants: the lavage fluid supernatant, to determine the amount of mediators present in the bronchoalveolar lumen in vivo; and the incubation media from the cultured cells, to determine the continued release of mediators by BALF cells in vitro. The supernatants from the lavage fluid were analyzed for LTB4, LTG, PGE2, PGFz,, and TXB2 using commercially available RIA kits (New England Nuclear, Boston, MA, U.S.A. for LTG; otherwise, Seragen, Boston, MA, U.S.A.). Because of the limited amount of sample available, the supernatants from the incubated cells were analyzed only for LTB4 and PGFzol, the two metabolites that increased in BALF in response to the exposures (see Results). The data from analysis of BALF fluid were normalized by dividing the
328
A
“TTXB,
0
RAT
m
MOUSE
B ‘.’
LTB, F
0
2
”
DAYSOFEXPOSURE
DAYSOF
DAYSOFEXPOSURE
DAYS
E
r1
T
EXPOSURE
OF EXPOSUiE
0
RAT
m
MOUSE
”
DAYS
Fig. 1. Arachidonate posures
metabolites
6F
in bronchoalveolar
were 2, 12 or 17 days to diluted
EXPOSihE
lavage fluid from diesel exposed
diesel engine exhaust
containing
rats and mice. Ex-
3.5 mg soot/m3
of air. Values
are the mean + SD of five replicate assays on pooled samples from ten mice or five rats. Lavage fluid was analyzed from control animals both at the beginning and end of the 17-day experiment. Values for control
animals
are the mean of five replicates
at the start of the study and one from wet weights
rodents
from each of the two pooled at the end of the study.
to allow comparisons
between
samples,
one from rodents
Data were normalized
species.
to lung
329
total amount of each mediator recovered in the lavage fluid by the wet weight of the lung in grams to allow species comparison. RESULTS
Inhalation exposures to diesel exhaust containing 3.5 mg soot/m3 for up to 17 days did not result in any change in the mean body weight and wet lung weight of the exposed rodents. The amounts of arachidonate metabolites recovered in the lavage fluid supernatants (normalized to lung weight) are shown in Fig. 1. Control rats had higher level of PGEz, TXBz, and LTB4 than did control mice. The mediators in BALF showing an increase in response to the exposure were PGFz, (- 3-fold increase in mice, -7-fold increase in rats), and LTB4 (-3-fold peak increase in mice and rats at 2 days of exposure). With the exception of LTG, the AA metabolite concentrations in BALF from exposed rodents were higher in rats than mice. Neither the total number nor the distribution of cell types present in BALF from rats varied with exposure (Table I). Mice had a 2.4-fold increase in total BALF cells after 17 days of exposure. There were increased numbers of neutrophils after 12 days of exposure and neutrophils accounted for 20% of the total BALF cells by 17 days. Mice also had increased macrophages after 17 days. Cultured cells from the BALF of exposed rodents tended to release less PGFz, and LTB4 than cells from control animals (Table II). Cultured cells from rat BALF released more LTB4 than cells from mouse BALF.
TABLE
I
CELLULAR Exposure
CONTENT
time (days)
OF BALF FROM Total
cellsb
DIESEL
EXHAUST-EXPOSED
RODENTS”
Macrophagesb
Neutrophilsb
Mice 0 (Controls)
0.14
0.13
0.001
2
0.21
0.17
0
12
0.22
0.14
0.037
17
0.34
0.27
0.068
0.001
Rats 0 (Controls)
0.13
0.12
2
0.11
0.10
0
12
0.08
0.06
0.004
17
0.10
0.06
0
a Total cells recovered wet weight b lO%ells/g
in combined
BALF from ten mice or five rats. Values are normalized
of the lungs to allow comparisons lung.
between
species.
to the total
330
TABLE
11
IN VITRO LAVAGE Exposure
RELEASE FLUID
OF ARACHIDONIC
CELLS
time (days)
FROM
ACID
DIESEL
METABOLITES
FROM
EXHAUST-EXPOSED
PGFz,
.______. % Control
pg/ml _______
RATS
BRONCHOALVEOLAR AND MICE”
LTBd % Control
pg/ml
Mice 0 (Controls)
530-700
2
237
38
470
140
12
370 174
60
210
63
28
260
78
2800-5800
_
410
46
5800
135
12
400
45
790
18
17
390
44
560
13
17
300-370
Rats 0 (Controls) 2
380-1400
a Mean of duplicate
assays on pooled
both at the beginning duplicate from
assays
rodents
samples
from ten mice or five rats. Control
and end of the 17-day experiment.
from each of two pooled
samples,
Values for control
one from rodents
samples animals
were obtained
are the range
of
at the start of the study and one
at the end of the study.
DISCUSSION
Species differences were observed in the release of mediators of inflammation in response to inhaled diluted diesel exhaust. In general, rat BALF contained higher concentrations of AA metabolites than mouse BALF. The only AA metabolite present in higher concentrations in BALF of mice compared to rats was LTCd. In the metabolic pathway leading to formation of leukotrienes, LT& is a common precursor for LTB4 and LTC4. LTB4 is formed from LTAJ by hydrolysis of an epoxide while LTC4 is formed by conjugation of LTA4 with glutathione. One might infer from the results of the current study that the mouse lungs had greater glutathione transferase activity than the rat lungs, causing a shift in the metabolism of the precursor LTA4 toward formation of the cysteinyl leukotrienes. Such a species difference has been reported by Lorenz et al. [ll], who found a lo-fold higher level of glutathione S-transferase in mouse versus rat lung. The largest species difference was in the TXBz concentration in BALF, which did not vary much in response to the exposure in either species, but was present in rat BALF at over IO-fold higher levels than in mice. The presence of higher concentrations of this proaggregation substance in rats is consistent with the shorter partial thromboplastin time reported in rats (21 s) [12] than in mice (55 - 110 s) [13]. The two metabolites that appeared to increase in BALF in response to the exposure (LTB4 and PGF2,) increased early after initiation of the exposure and then decreased, despite continued exposure. LTB4 increased 3-fold in both species, but
331
a subsequent influx of neutrophils occurred only in mice. The reason for this is unknown, but longer exposures to diesel exhaust do result in an influx in neutrophils in both rats and mice [ 141. The sources of the arachidonate metabolites in BALF are unknown. The phagocytic cells in the aveoli as well as the alveolar [ 151and the airway [ 161epithelial cells are potential sources. The decreased amount of PGFzo, and LTB4 released from cultured phagocytic cells obtained from the exposed rodents is consistent with a depletion of these mediators from the cells or a decreased ability to form the metabolites after the release of the compounds in vivo. It could also be that it is easier to recover spent or depleted cells by lavage than the more active ones. It is unlikely that culture conditions inhibited the release of these mediators, since LTB4, which was present in lavage fluid from control animals at 6-fold higher levels in rats than in mice, was released from control cells into the culture media at - IO-fold higher levels from rat cells than mice cells. The PGFz~, which was present in BALF from control rats at approximately 1.5 times the level in mice, was also released from cultured rat cells at about 1.5 times the level released from mouse cells. In conclusion, the data demonstrate that greater amounts of AA metabolites are present in the bronchoalveolar epithelial lining fluid from rats compared to mice exposed to diluted diesel engine exhaust, and support the hypothesis that the increased fibrosis observed in rats may be, in part, related to the higher concentrations of such mediators in rat lungs. Many factors are involved in the process leading from inflammation to either normal repair or fibrosis [17]. This study addresses the initial phases of that process. Further research will be directed toward determining the relation between these initial events and later developing disease. ACKNOWLEDGEMENTS
The authors wish to acknowledge the outstanding efforts of a number of the staff of the Inhalation Toxicology Research Institute for making this study possible. We especially thank Mr. James J. Waide for the radioimmunoassay analyses. The research was sponsored by the U.S. Department of Energy’s Office of Health and Environmental Research under Contract No. DE-AC04-76EV01013 in facilities fully accredited by the American Association for the Accreditation of Laboratory Animal Care. REFERENCES
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R.A. and Leslie, C.C. (1984)
325
TXL 02016
Species differences .in release of arachidonate metabolites in response to inhaled diluted diesel exhaust R.F. Henderson, R.O. McClellan
H.W. Leung*, A.G. Harmsen** and
Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute, Albuquerque, NM 87185, U.S.A. (Received 10 November 1987) (Revision received 20 April 1988) (Accepted 10 May 1988)
Key words: Aracbidonate
metabolite; Species difference; Diesel exhaust, particles; Inflammation
SUMMARY In life-span studies in CD-l mice and F344Krl rats, inhaled diluted diesel exhaust was highly fibrogenic in rats but not in mice. This was the case despite the higher lung burden, in mg soot/g lung, achieved in mice compared to rats. We tested the hypothesis that the greater fibrogenicity of the soot in rats was due in part to greater release of mediators of inflammation from alveolar cells in rats compared to mice. Female F344/rats and B6C3Fr mice were exposed for up to 17 days to diluted diesel exhaust containing 3.5 mg/m-’ of soot. The lungs of control and soot-exposed animals were lavaged after 2, 12 or 17 days of exposure. The presence of leukotriene (LT)Ba, LTG, prostaglandin (PG)Ez, PGFzu and thromboxane (TX) B2 in the lavage fluids and LTB4 and PGFa, in cultured lavage cell supernatants was determined. The total amount of each lavage fluid constituent was normalized to lung weight for species comparisons. Control rats had higher levels of TXBa (16-fold), and LTB4 (6-fold) and PGEz (2-fold) than control mice, but control mice bad higher amounts of LTG (Cfold). Control rats and mice had approximately the same amounts of PGF&g lung in bronchoalveolar lavage fluid (BALF). Rats exposed to diesel exhaust had increases in BALF PGF2, and LTB4 that were highest after 2 days of exAddress for correspondence: Dr. R.F. Henderson, Lovelace Inhalation Toxicology Research Institute, P.O. Box 5890, Albuquerque, NM 87185, U.S.A. * Current address of Dr. H.W. Leung is Syntex Corp, 3401 Hillview Ave., Palo Alto, CA 94304, U.S.A. Dr. H.W. Leung was an Associated Western Universities Faculty Research Participant during the conduct of this research. ** Current address of Dr. A.G. Harmsen is Trudeau Institute, P.O. Box 59, Saranac Lake, NY 12983, U.S.A. Abbreviations: LT, leukotriene; PG, prostaglandin; TX, throboxane; BALF, bronchoalveolar lavage fluid; AA, arachidonic acid; PMN, polymorphonuclear neutrophil; SRS, slow-reacting substance.
326
posure
and decreased
lavage fluid released consistent
thereafter.
with the hypothesis
inflammation
Mice had lesser increases
larger amounts
of LTB4 into culture
that soot-laden
than do the alveolar
rat alveolar
in both parameters. supernatants
Rat cells recovered
from
than mouse cells. The data were
cells release greater
quantities
of mediators
of
cells in mice.
INTRODUCTION
Rats exposed for up to 24 months to diluted exhaust from diesel engines [l] developed a much more severe focal pulmonary fibrosis than mice exposed for the same period, despite greater accumulation of soot/g lung in mice. The basis for the species differences in development of pulmonary fibrosis is unknown and may be due to a variety of causes. One possible contributing factor may be differences in the response of the alveolar cells to stimulation by the diesel soot particles. Stimulated macrophages and neutrophils are known to release various chemical factors that influence the course of an inflammatory response [2]. Arachidonic acid (AA) metabolites appear to be especially important in influencing the course of events [3-61. Among the lipoxygenase products of AA (called leukotrienes), leukotriene B4 (LTB4) induces a strong inflammatory response and has a potent chemotactic and chemokinetic activity toward polymorphonuclear neutrophils (PMN), eosinophils, and monocytes [3,5-71. It aggregates PMN and promotes the release of lysosomal enzymes from these cells. LTB4 also causes increased permeability of the alveolar/capillary barrier of the lung. The cysteinecontaining leukotrienes (LTC4, LTD4, LTE4) are potent bronchoconstrictors and mimic the effects of the slow-reacting substance (SRS) of anaphylaxis [3,6]. The cycle-oxygenase products of AA metabolism are also important mediators of the inflammatory response, and include prostaglandins (PG) and thromboxanes (TX) [4,5,8]. PGE2 and PGF2, alter the microvascular pressure of the lung and cause constriction of bronchial smooth muscle [5,8]. TXAz has a similar effect and promotes neutrophil adherence to endothelium, platelet aggregation, and enzyme release from platelets
[5,8]. The thromboxanes
also mediate
of the LTC4, LTD4, and LTE4. TXA2 is rapidly TXBz, which is biologically inactive. Estimates measuring the more stable TXBz.
hydrolyzed of TXAZ
the constrictive
effects
non-enzymatically are usually made
to by
The objective of the current study was to determine if there were species differences in the release of inflammatory mediators in the lungs of diesel exhaustexposed rats and mice, which might explain, in part, the differences in severity of pulmonary fibrosis induced by chronic diesel exhaust exposures in these two species. MATERIALS
AND METHODS
Thirty female F344/N rats and 60 female B6C3Fr mice were used in the study. The rats were born and raised in the barrier-maintained Inhalation Toxicology
327
Research Institute (ITRI) colony and were housed two per cage in polycarbonate cages with hardwood chip bedding and filter caps prior to being placed in conditioning chambers (Hazleton Systems, Aberdeen, MD, U.S.A.). The mice were obtained from Frederick Cancer Research Center, Frederick, MD, U.S.A., and were held in quarantine for 2 weeks prior to being placed in conditioning chambers. Feed (Lab Blox, Allied Mills, Chicago, IL, U.S.A.) and water were provided ad libitum except during exposure hours, when food was removed. Rodents were assigned to the various treatment and control groups using a table of random numbers. Rodents were placed in conditioning chambers for 2-3 weeks prior to start of diesel exposures. Thirty B6C3Fr mice and 15 F344/N rats (all females, 18-20 weeks of age, rats; 13 weeks of age, mice) were exposed 7 h/day, 5 days/week to diluted diesel exhaust containing 3.5 mg soot/m3 air. Exhaust was generated by two 1980 model, 5.7 liter Oldsmobile engines operating alternatively on the Federal Test Procedure urban driving cycle and burning a standardized certification fuel (D-2 Diesel Control Fuel, Phillips Chemical Co.) [9]. Exhaust was diluted serially with clean air to provide the desired concentrations. Particle concentrations were measured by filter samples taken daily. Ten mice and five rats were removed from the exposure after 2, 12, and 17 exposure days to determine the amount of mediators in bronchoalveolar lavage fluid (BALF). Cells recovered from BALF were also cultured and the release of mediators assayed in vitro. The day after removal from the exposure chambers, animals were anesthetized with 4% halothane in oxygen, and killed by severing the brachial arteries. The lungs with tracheas attached were removed from each animal and weighed. The lungs were lavaged two times with 0.15 M saline to obtain epithelial lining fluid and free alveolar cells as described previously [lo]. The volume of each wash was 5 ml for rats and 1 ml for mice. Tracheas were removed and weighed. The difference between the weight of the trachea and the weight of the lung plus trachea was calculated as the wet weight of the lung. Individual lavage fluid samples from the same species were pooled. Total and differential cell counts were made on BALF cells concentrated by centrifugation at 300 x g for 10 min. Cells were incubated at 37°C in RPM1 1640 at a concentration of - 4 x lo6 cells/ml for 1 h. Arachidonic acid metabolites were assayed in two cell supernatants: the lavage fluid supernatant, to determine the amount of mediators present in the bronchoalveolar lumen in vivo; and the incubation media from the cultured cells, to determine the continued release of mediators by BALF cells in vitro. The supernatants from the lavage fluid were analyzed for LTB4, LTG, PGE2, PGFz,, and TXB2 using commercially available RIA kits (New England Nuclear, Boston, MA, U.S.A. for LTG; otherwise, Seragen, Boston, MA, U.S.A.). Because of the limited amount of sample available, the supernatants from the incubated cells were analyzed only for LTB4 and PGFzol, the two metabolites that increased in BALF in response to the exposures (see Results). The data from analysis of BALF fluid were normalized by dividing the
328
A
“TTXB,
0
RAT
m
MOUSE
B ‘.’
LTB, F
0
2
”
DAYSOFEXPOSURE
DAYSOF
DAYSOFEXPOSURE
DAYS
E
r1
T
EXPOSURE
OF EXPOSUiE
0
RAT
m
MOUSE
”
DAYS
Fig. 1. Arachidonate posures
metabolites
6F
in bronchoalveolar
were 2, 12 or 17 days to diluted
EXPOSihE
lavage fluid from diesel exposed
diesel engine exhaust
containing
rats and mice. Ex-
3.5 mg soot/m3
of air. Values
are the mean + SD of five replicate assays on pooled samples from ten mice or five rats. Lavage fluid was analyzed from control animals both at the beginning and end of the 17-day experiment. Values for control
animals
are the mean of five replicates
at the start of the study and one from wet weights
rodents
from each of the two pooled at the end of the study.
to allow comparisons
between
samples,
one from rodents
Data were normalized
species.
to lung
329
total amount of each mediator recovered in the lavage fluid by the wet weight of the lung in grams to allow species comparison. RESULTS
Inhalation exposures to diesel exhaust containing 3.5 mg soot/m3 for up to 17 days did not result in any change in the mean body weight and wet lung weight of the exposed rodents. The amounts of arachidonate metabolites recovered in the lavage fluid supernatants (normalized to lung weight) are shown in Fig. 1. Control rats had higher level of PGEz, TXBz, and LTB4 than did control mice. The mediators in BALF showing an increase in response to the exposure were PGFz, (- 3-fold increase in mice, -7-fold increase in rats), and LTB4 (-3-fold peak increase in mice and rats at 2 days of exposure). With the exception of LTG, the AA metabolite concentrations in BALF from exposed rodents were higher in rats than mice. Neither the total number nor the distribution of cell types present in BALF from rats varied with exposure (Table I). Mice had a 2.4-fold increase in total BALF cells after 17 days of exposure. There were increased numbers of neutrophils after 12 days of exposure and neutrophils accounted for 20% of the total BALF cells by 17 days. Mice also had increased macrophages after 17 days. Cultured cells from the BALF of exposed rodents tended to release less PGFz, and LTB4 than cells from control animals (Table II). Cultured cells from rat BALF released more LTB4 than cells from mouse BALF.
TABLE
I
CELLULAR Exposure
CONTENT
time (days)
OF BALF FROM Total
cellsb
DIESEL
EXHAUST-EXPOSED
RODENTS”
Macrophagesb
Neutrophilsb
Mice 0 (Controls)
0.14
0.13
0.001
2
0.21
0.17
0
12
0.22
0.14
0.037
17
0.34
0.27
0.068
0.001
Rats 0 (Controls)
0.13
0.12
2
0.11
0.10
0
12
0.08
0.06
0.004
17
0.10
0.06
0
a Total cells recovered wet weight b lO%ells/g
in combined
BALF from ten mice or five rats. Values are normalized
of the lungs to allow comparisons lung.
between
species.
to the total
330
TABLE
11
IN VITRO LAVAGE Exposure
RELEASE FLUID
OF ARACHIDONIC
CELLS
time (days)
FROM
ACID
DIESEL
METABOLITES
FROM
EXHAUST-EXPOSED
PGFz,
.______. % Control
pg/ml _______
RATS
BRONCHOALVEOLAR AND MICE”
LTBd % Control
pg/ml
Mice 0 (Controls)
530-700
2
237
38
470
140
12
370 174
60
210
63
28
260
78
2800-5800
_
410
46
5800
135
12
400
45
790
18
17
390
44
560
13
17
300-370
Rats 0 (Controls) 2
380-1400
a Mean of duplicate
assays on pooled
both at the beginning duplicate from
assays
rodents
samples
from ten mice or five rats. Control
and end of the 17-day experiment.
from each of two pooled
samples,
Values for control
one from rodents
samples animals
were obtained
are the range
of
at the start of the study and one
at the end of the study.
DISCUSSION
Species differences were observed in the release of mediators of inflammation in response to inhaled diluted diesel exhaust. In general, rat BALF contained higher concentrations of AA metabolites than mouse BALF. The only AA metabolite present in higher concentrations in BALF of mice compared to rats was LTCd. In the metabolic pathway leading to formation of leukotrienes, LT& is a common precursor for LTB4 and LTC4. LTB4 is formed from LTAJ by hydrolysis of an epoxide while LTC4 is formed by conjugation of LTA4 with glutathione. One might infer from the results of the current study that the mouse lungs had greater glutathione transferase activity than the rat lungs, causing a shift in the metabolism of the precursor LTA4 toward formation of the cysteinyl leukotrienes. Such a species difference has been reported by Lorenz et al. [ll], who found a lo-fold higher level of glutathione S-transferase in mouse versus rat lung. The largest species difference was in the TXBz concentration in BALF, which did not vary much in response to the exposure in either species, but was present in rat BALF at over IO-fold higher levels than in mice. The presence of higher concentrations of this proaggregation substance in rats is consistent with the shorter partial thromboplastin time reported in rats (21 s) [12] than in mice (55 - 110 s) [13]. The two metabolites that appeared to increase in BALF in response to the exposure (LTB4 and PGF2,) increased early after initiation of the exposure and then decreased, despite continued exposure. LTB4 increased 3-fold in both species, but
331
a subsequent influx of neutrophils occurred only in mice. The reason for this is unknown, but longer exposures to diesel exhaust do result in an influx in neutrophils in both rats and mice [ 141. The sources of the arachidonate metabolites in BALF are unknown. The phagocytic cells in the aveoli as well as the alveolar [ 151and the airway [ 161epithelial cells are potential sources. The decreased amount of PGFzo, and LTB4 released from cultured phagocytic cells obtained from the exposed rodents is consistent with a depletion of these mediators from the cells or a decreased ability to form the metabolites after the release of the compounds in vivo. It could also be that it is easier to recover spent or depleted cells by lavage than the more active ones. It is unlikely that culture conditions inhibited the release of these mediators, since LTB4, which was present in lavage fluid from control animals at 6-fold higher levels in rats than in mice, was released from control cells into the culture media at - IO-fold higher levels from rat cells than mice cells. The PGFz~, which was present in BALF from control rats at approximately 1.5 times the level in mice, was also released from cultured rat cells at about 1.5 times the level released from mouse cells. In conclusion, the data demonstrate that greater amounts of AA metabolites are present in the bronchoalveolar epithelial lining fluid from rats compared to mice exposed to diluted diesel engine exhaust, and support the hypothesis that the increased fibrosis observed in rats may be, in part, related to the higher concentrations of such mediators in rat lungs. Many factors are involved in the process leading from inflammation to either normal repair or fibrosis [17]. This study addresses the initial phases of that process. Further research will be directed toward determining the relation between these initial events and later developing disease. ACKNOWLEDGEMENTS
The authors wish to acknowledge the outstanding efforts of a number of the staff of the Inhalation Toxicology Research Institute for making this study possible. We especially thank Mr. James J. Waide for the radioimmunoassay analyses. The research was sponsored by the U.S. Department of Energy’s Office of Health and Environmental Research under Contract No. DE-AC04-76EV01013 in facilities fully accredited by the American Association for the Accreditation of Laboratory Animal Care. REFERENCES
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