Camp. Biochem.Physiol.Vol. lOlC, No. 1, pp. 101-108,1992 Printed in Great Britain
03064492/92$5.00+ 0.00 0 1991Pergamon Press plc
THE ROLE OF PROSTAGLANDIN H SYNTHASE IN 3-METHYLINDOLE-INDUCED PNEUMOTOXICITY IN GOAT KAREN S. ACTON,* HERMAN J. BOERMANS?and TAMMYM. BRAY*$ Collaborative Program in Toxicology, *Department of Nutritional Sciences and TDepartment of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada NlG 2Wl (Telephone 519 8244120, Ext. 3752; Fax: 519 763-5902) (Received 25 March 1991)
Abstract-l. Aspirin and indomethacin were used to investigate the role of prostaglandin H synthase (PHS) system in 3-methylindole (3MI)-induced pneumotoxicity. 2. A functional test was developed to detect the inhibitory effect of oral doses of aspirin and indomethacin on PHS activity based on thromboxane B, (TXB,) production from thrombin-stimulated platelets in whole blood. 3. Goats which received oral doses of aspirin or indomethacin before administration of 3MI (0.1 g 3MI/kg body wt) showed a reduced severity in clinical signs and pathological lesions in the lung than those that received 3MI alone. 4. There was no difference in the severity of the disease between the control and the aspirin-treated animals if aspirin was given after 3MI administration. 5. The protective effect of inhibitors when administered before, but not after, 3MI dosing suggests it is the inhibition of PHS activity in activation of 3M1, not in production of prostanoids which prevented the disease process.
work has indicated that 3MI is also metabolically activated by the prostaglandin H synthase (PHS) system in ram seminal vesicles (Formosa et al., 1988) and goat lung microsomes (Formosa and Bray, 1988). The reactive intermediate formed by the PHS system was capable of covalently binding to microsomal protein which may play a part in the mechanism of cytotoxicity. In addition, the PHS system has been found to have a higher activity in the lung when compared to the MFO system (Moldeus et al., 1985), thus, it appears that the combined effects of the PHS and MFO systems may account for the tissue specificity of 3MI-induced pneumotoxicity. Non-steroidal anti-inflammatory drugs, aspirin and indomethacin, block the synthesis of prostanoids by inhibiting the cyclooxygenase enzyme of the PHS system. Aspirin causes inhibition by irreversibly acetylating a serine residue of the cyclooxygenase whereas indomethacin appears to cause inhibition by binding the cyclooxygenase at a position which reduced its affinity for the substrate (Roth and Siok, 1978; Roth et al., 1983; Kulmacz and Lands, 1985). Cyclooxygenase inhibitors are useful tools for defining the role that PHS play in the mechanism of disease processes. If the PHS is involved in the metabolic activation of the toxin, then inhibitors, when given before the toxin administration, should be able to reduce the severity of the disease. If the cause of the pneumotoxicity is only due to an increase in prostanoid production, PHS inhibitors, when given after toxin administration, should be able to prevent the disease processes. The objective of this experiment was to determine whether aspirin and indomethacin are capable of altering the clinical signs and the pathological lesions of 3MI-induced lung
INTRODUCTION
Acute bovine pulmonary edema and emphysema (ABPE) is a naturally occurring lung disease which affects cattle subjected to a sudden change in diet from poor quality forage to lush green pasture. 3-Methylindole (3M1, skatole), the main ruminal fermentation product of tryptophan after this drastic dietary change, is the causative agent of this respiratory disease (Carlson et al., 1972). The experimental administration of oral or intravenous doses of 3MI can induce the characteristic onset of pulmonary edema and interstitial emphysema in cattle (Carlson et al., 1975), sheep (Bradley et al., 1978) and goats (Dickinson et al., 1976). Other sources of 3MI are found in the feces of rat, pig and man due to the fermentation of tryptophan in the large intestine (Yoshihara and Murata, 1977; Yokoyama and Carlson, 1979). 3MI is also a component of cigarette smoke, due to the pyrolysis of tryptophan in tobacco leaves (Hoffman and Rathkamp, 1970) yet the extent of the health risk of 3MI to man has not yet been determined. The mechanism of 3MI toxicity, which is acute and specific to the lung, is still under investigation. 3MI has been shown to be activated by the cytochrome P-450 mixed-function oxidase (MFO) system to form a reactive-free radical species capable of binding to macromolecules and initiating cytotoxicty (Hanafy and Bogan, 1980; Bray and Kubow, 1985). This activation of 3MI by the MFO system alone cannot explain, however, the phenomenon of the tissue specificity of 3MI-induced pneumotoxicity. Recent $To whom correspondence
should be addressed. 101
KARENS. ACTONet al.
102
disease. The results would be useful in elucidating the by which the PHS system is involved in 3MI-induced pneumotoxicity in vivo. mechanism
MATERIAL
indometha~n dosing, as well as I and 3 hr after administration. All blood samples were subjected to the thrombin stimulation and subsequently analyzed for TXB, production by RIA.
AND METHODS
VeriJcation of inhibitory effect of aspirin and indomethacin Since dosage for aspirin or indomethacin in goats has not been well-documented, it was important to detect and verify that the inhibitor doses to be used are absorbed and at functionally adequate levels. A rapid and simple method based on thromboxane B, (TXB,) production from thrombin-stimulated platelets-in whole blood was developed to test functionally the inhibitorv effects of asnirin and indomethacin on -PHS activity -without mea&ing the plasma concentrations of inhibitors after oral administration. The procedure was based on a modification of whole blood aggregation protocols (Hwang and Donovan, 1982; Saniabadi et ai., 1983). Thrombin was obtained from Parke-Davis and Co., Ltd (Brockville, Ontario). A11 other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). The protocol for the test was as follows: 4.5 ml of blood were taken from the jugular vein using vacutainers containing OSml of 0.05 M phosphate-buffers saline (PBS), pH 7.4 with 3.2% trisodium citrate as the anticoa~lant. The &rated whole blood was subsequently diluted 1: 1 with additional PBS. A 400 ~1 aliquot of the diluted sample was transferred to a siliconized glass cuvette and p&incubated at 37°C for I min. The samule then received 25 ul of CaCl, (5 mM final concentration), and was immediately stimulated with thrombin (0.1254 units). Samples were subsequently incubated with continuous stirring for 4 min at which time 50 ~1 of aspirin (0.75 mg/ml) was added to stop any further TXB, production. Samples were allowed to clot at room temperature and were centrifuged at IOOOgfor 9 min at 4°C. Serum was collected and stored at -20°C until analyzed for TXB, by RIA. Two in uirro preliminary trials were performed to verify whether aspirin and indomethacin inhibit TXB2 production from thrombin-stimulated platelets in goat blood. In the first trial, two goats were bled, each providing three blood samples. One sample received no inhibitor whereas the others received either a low (O.O3mg/ml) or a high (O.O95mg/ml) concentration of aspirin. The low dose approximated typical plasma levels of aspirin in cattle after therapeutic dosing (Gingerich et ai., 1975), whereas the high dose was chosen to completely inhibit TXB, formation in vitro. Blood samples were prepared and analyzed for TXB, formation as described above. The second in vitro trial was similar to the aspirin study, yet indometha~n was the inhibitor used. Again, two goats were bled, providing three blood samples each. One sample received no inhibitor, whereas the other immediately received O.O7mg/ml indomethacin. Two in viuo trials were subs~uently performed to verify whether TXB, production by platelet after thrombin stimulation could be used as an in viuo functional indicator of the inhibitory effect of oral doses of aspirin or indomethacin. In the first trial, three goats were used, each receiving two doses 150 mg/ml body wt aspirin given 2 hr apart. Blood was taken from the jugular vein immediately before the first dose and 2 hr later (immediately before the second dose). A final blood sample was taken 2 hr after the second dose. In the second trial, four goats were used and divided equally into two groups. One group received an oral low dose of indomethacin (2mg/kg body wt), whereas the second received a IO-fold higher dose (20 mg/kg body wt). Doses were derived from the literature by averaging low and high indomethacin doses used for various species such as rat (Hueker et al., 1966), horse (Phillips ef ai., 1980) and man (Duggan et ui., 1972). Blood was taken immediately before
TXB, concentration was determined by the procedure of Upjohn Diagnostic Laboratories (Kalamazoo, MI) as described by Chandler and Giri (1983). Tritiated TXB, was purchased from New England Nuclear (Boston, MA). Antibody and all further reagents were obtained from Sigma Chemical Co. (St. Louis, MO). Dried TXB, extract was reconstituted in 0.1 M PBSG. Samples were analyzed in duplicate and aliquots were added to polypropylene tubes containing antibody and approx. 10,000 cpm of the tritiated prostanoid. Tubes were incubated for 1 hrat 2S”C, followed by a 4°C incubation for 20-24 hr. Unbound TXB, was separated from antibody-bound TXB, by charcoal absorption. Following centrifugation, the radioactivity of the supematant was determined by liquid scintillation counting. The cross-reactivity of the TXB, antiserum was 100% for TXB, and less than 0.1% for other prostaglandins. Experiment 1: Aspirin pre-treatment study Twelve female goats (Cadwell Farm, Drumbo, Ontario) from 5 to 12 months of age were acclimatized to their new surroundings for one week. Feed supplement (19% protein), hay and water were administered ad libitum throughout the study unless indicated. Goats were randomly divided into two groups of 6, consisting of an aspirin pretreated group and a control group. The control group received 3MI alone (0.1 g/kg body wt) whereas the aspirin group received two doses of aspirin (150 mg/kg body wt per dose) before 3MI administration. The 3MI dose has been shown to induce moderate pulmonary lesions within 72 hr (Carlson et al., 1975; Dickinson et af., 1976). All treatments were given orally in gelatin capsules. Feed was removed from both control and treatment groups 24 hr before any oral dosing. To detect the inhibitory effects of aspirin on the PHS activity, TXB, production from stimulated platelets was measured before and after aspirin administmtion. Pretreatment blood samples were taken from the jugular vein of goats from both groups immediately before dosing. Inhibitor was then given to the aspirin pre-treated group only. Two hours after aspirin administration, blood was taken again from both groups. The aspirin pretreated group then received a second dose of inhibitor to maintain the efficacy of the drug, after which 3MI was immediately given to both aspirin and control groups. A final blood sample was taken 3 hr after 3MI administration (5 hr after the first aspirin dose). Clinical signs (laboured breathing and respiration rate) were monitored throughout the duration of the study. Seventy-two hours after 3MI administration, goats were killed and lungs were removed and weighed in order to calculate the lung weight as a percent of body weight. Three lung tissue samples per goat were obtained from pre-determined areas of the right lobe for moisture content determination. The moisture content of the lung and the ratio of the whole lung weight to body wt are commonly used as indices of the severity of pulmonary lesions (Carlson ei al,, 1975; Dickinson et al., 1976; Bradley et ai., 1978). A portion of the lung samples were taken for histopathological examination. Experiment 2: Zndomethacin pre-treatment sfudy The experimental design of this experiment paralleled that of the aspirin study (Experiment 1). Twelve female goats (Thomas Farm, Orton, and Harkin Farm. Elora. Ontario) one year of age were .fed feed supplement, alfalfa pellets and water ad iib~~um. Goats were randomly divided into two groups, a control group which received 3MI (0.1 g/kg
103
PHS in 3MI-induced pneumotoxicity body wt) alone and an indomethacin pre-treated group which received two oral doses of indomethacin before 3MI administration. The lirst indomethacin dose (20 mg/kg body wt), was given 2 hr before the second dose (10 mg/kg body wt), which was halved due to the known potency of indomethacin and its undesired side-effect of diarrhea. The remaining experimental protocol for this study was the same as described in the aspirin experiment.
scale in proportion to severity (moderate, and + + + severe).
absent, + mild, + +
Statistical analysis
Data was analyzed between groups using the Student’s unpaired t-test. Significance was considered a P value less than 0.05. The evaluation of clinical signs and histopathology were expressed on an individual animal basis.
Experiment 3: Aspirin post-treatment study
Experiment 3 was designed to test the hypothesis that it is the inhibition of the activity of PHS in activation of 3M1, not the inhibition in prostanoid synthesis, that prevents the disease process. Aspirin was given 2 hr after 3MI administration. Ten female goats (Cadwell Farm, Drumbo, Ontario) from 5 to 12 months of age were randomly divided into two groups of five, consisting of a control group and an aspirin post-treated group. The control group received 3MI alone (0.1 g/kg body wt) whereas the aspirin posttreated group received two doses of aspirin (150 mg/kg body wt per dose, 2 hr apart) two hours after 3MI administration. The remaining experimental protocol for this study was the same as described in the Experiment 1. Pathology
Lung tissue from three pre-determined locations from the right lobe was lixed in 10% phosphate buffered formalin (Fisher), shaken for 24 hr and embedded in paraffin wax. Sections (5 pm thick) were stained with hematoxylin and eosin. Membrane thickness was measured with light microscopy using a micrometer scale lens. Cellularity and edema were scored semi-quantitatively with a four-point
RESULTS Inhibitory activity
The aspirin
inhibitory
effect
in
of
vitro
addition
%
z
Cl
6
*
0.095mg
1
1
2 Thrombin
3
8r
E 2
4
;
2
I 1
0
+
Goat 3
u
4
/
z
-=-
No ID
_
0.07mg ID/ml
I
I
0
0 I
1
I
I
3
4
Time (hours after 1st aspirin dose)
(b)
(b)
m4m
I
2
(units) 30 -
6
Goat 2
aspirin/ml
0
0
1 z
e
~__*______c_________--.
nd
of
and indomethacin on TXB, production by the thrombin-stimulated platelets is shown in Fig. la and b. Blood samples without aspirin or indomethacin showed increasing levels of TXBr production with increased thrombin stimulation. Samples which contained either the high aspirin dose (0.095 mg/ml) or indomethacin (0.07 mg/ml) showed no detectable levels of TXB, even with increasing concentrations of thrombin. Samples containing the low aspirin concentration (0.03 mg/ml) showed levels of TXB2 only slightly above the detectable limit. The inhibitory effect of oral doses of aspirin or indomethacin on PHS activity, as indicated by the thrombin-stimulated platelet test, is illustrated in Fig. 2a and b. Aspirin, administered orally (150 mg/kg
--o--Goat
t-
on PHS
effect of aspirin and indomethacin
I
I
2 Thrombin
3
I 4
(units)
Fig. 1. The effect of in vitro addition of aspirin (a) and indomethacin (b) on TXB, synthesis in goat blood. a: Blood samples from 2 goats received either no aspirin, 0.03 mg/ml aspirin or O.O95mg/ml aspirin and were challenged with 0.125, 0.25, 0.5, 1, 2 and 4 units of thrombin. b: Blood samples from 2 goats received either no indomethacin, or 0.07 mg/ml indomethacin and were challenged with thrombin as above. nd: indicates the concentration at which TXB, is non-detectable.
1
- -0 --
Low dose
P
High dose
2
3
Time (hours after ID dose)
Fig. 2. The effect of orally administered aspirin (a) and indomethacin (b) on TXB, synthesis in goat blood. a: Three goats were given two oral doses of 150 mg/kg aspirin at time 0 and 2 hr. The effect of oral doses of aspirin on TXB, concentration was determined at 0,2 and 4 hr after the first aspirin dose. b: Two goats were dosed with either one low dose (2.0 mg/kg body wt) or one high dose (20 mg/kg body wt) of indomethacin at time zero. The effect of oral doses of indomethacin on TXB, production was determined at 0, 1 and 3 hr after dosing. nd: indicates the concentration at which TXB, is non-detectable.
KARENS. ACTONez al.
104
body wt) inhibited TXB, synthesis to a nondetectable level 2 hr after dosing (Fig. 2a). A second dose of aspirin, given 2 hr after the first, maintained the inhibition for at least a further 2 hr. One oral high dose of indomethacin (20mg/kg body wt) inhibited TXB, production to a non-detectable level 1 and 3 hr after dosing (Fig. 2b). The low dose (2 mg/kg body wt) showed no inhibition at 1 hr, yet showed inhibition 3 hr after dosing. ClWcal signs
The cyclooxygenase inhibitor, aspirin, when administered before 3MI dosing, appeared to exert a protective effect against the development of 3MIinduced lung disease, as did indomethacin to a lesser degree (Table I). Four out of the six goats pre-treated with aspirin showed no clinical signs of respiratory distress, while the remaining two goats showed only mild-to-moderate symptoms. All of the aspirin pretreated goats survived, yet five out of the six goats treated with 3MI alone died before the end of the 72 hr experiment. In the indomethacin study all goats from both groups survived, yet only goats
receiving 3MI alone demonstrated the onset of severe clinical signs (Experiment 2, Table 1). Only one of the indomethacin-pretreated goats developed clinical signs, characterized by a delayed onset. Goats treated with indomethacin developed a mildto-moderate diarrhea commencing one day after dosing. There was no difference in the 3MI-induced pulmonary toxicity when aspirin was given after 3MI administration (Experiment 3, Table 1). Other indices of the severity of the disease, such as respiration rate, lung weight as percent of the body weight and the percent moisture content of the lung were also affected by the aspirin or indomethacin when it was given before 3ML administration (Table 2). The mean respiratory rate per min (rpm) of goats pre-treated with aspirin (43 & 21 (SD) rpm) was significantly lower than the respiration rate of goats receiving 3MI alone (82 sf: 19 rpm). The indomethacin group showed a similar trend towards a lower, more normal respiration rate (33 + 23 rpm) compared to the group receiving 3MI alone (56 t_ 35 rpm), although the values were not statistically different. However, when aspirin was
Table 1. The effect of aspirin and indomethacin 3-methyIindoIe-indu~ Goat No. Experiment ”
4 5 6
8 9 10 II 12
on clinical signs and time of death in lung disease
Onset (hour) and degree of clinical signs*
Treatment
1:Aspirin pm-treatment study 3MI 3MI 3MI 3MI 3MI 3MI
alonet alone alone alone alone alone
Aspirin Aspirin Aspirin Aspirin Aspirin Aspirin
+ + + + + +
3MIT 3MI 3MI 3MI 3MI 3MI
36, severe 12, severe 24, severe 12. severe 12, severe 24, severe
Killed at 72 hr Died at 26.5 hr Died at 68 hr Died at 21.5 hr Died at 38.5 hr Died at 69 hr
None None None None 48, mild 36, moderate
Killed Killed Killed Killed Killed Killed
at at at at at at
72 hr 72 hr 72 hr 72 hr 72 hr 72 hr
Killed Killed Killed Killed Killed Killed
at at at at at at
72 hr 72 hr 72 hr 72 hr 72 hr 72 hr
48, moderate None None None None None
Killed Killed Killed Killed Killed Killed
at at at at at at
72 hr 72 hr 72 hr 72 hr 72 hr 72 hr
24, 20, 30, 24, 36,
severe severe severe severe severe
Killed Killed Killed Killed Killed
at at at at at
72 hr 72 hr 72 hr 72 hr 72 hr
32, 24, 30, 24, 38,
severe severe severe severe severe
Died at 48 hr Killed at 72 hr Killed at 72 hr Killed at 72 hr Killed at 72 hr
Experiment 2: Indomethacin pre-treatment study None 1 3M1 alone 2 3Mt alone None 3 3M1 alone None 4 3MI alone 24. severe 24; severe 5 3MI alone 6 3MI alone 24, severe 7
8 9 10 11 12
Indomethacin Indomethacin Indomethacin Indomethacin Indomethacin Indomethacin
+ 3MI$ -t- 3MI + 3MI + 3MI + 3MI + 3MI
Experiment 3: Aspirin post-treatment 1 3MI alone 2 3MI aione 3 3MI alone 4 3MI alone 5 3MI alone 7 8 9 10 11
Manner and time of death
3MI 3MI 3MI 3MI 3MI
+ + + + +
aspirinlj aspirin aspirin aspirin aspirin
study
*Clinical signs consisted of irregular respiration rate, laboured breaths and trembling. t3MI dose (0.1 g/kg body wt) given orally. IAspirin doses (2 doses of I50 mg/kg body wt) given orally 2 hr apart before 3MI dosing @Indomethacin doses (20 mg/kg body wt + 10 mg/kg body wt) given orally 2 hr apart before 3MI dosing ItAspirin doses (2 doses of 150 mg/kg body wt) given orally 2 hr apart after 3MI dosing.
PHS
3MI-induced pneumotoxicity
in
105
Table 2. The effect of aspirin and indomethacin on clinical signs 72 hr after 3-methylindole administration Respiration rate Treatment
Experiment
Lung moisture content (%)
Lung wt/bw (%)
(rpm)
I
3MI alone* Aspirin + 3MIt:
82 f 19tli 43*21
2.3 & 0.6n 1.1 f 0.3
71.0 * 5.9qj 58.8 f 4.2
2
3MI alone Indomethacin
56 f 35 33 f 23
1.6 f 0.31 1.3 * 0.2
61.5 f 4.2 62.6 f 1.3
82k IO 78 * 23
2.4 f 0.4 2.5 + 0.5
68.2 + 2.3 61.9 + 3.1
3
+ 3MI$
3MI alone 3MI + aspirin11
l3MI dose (0.1 g/kg body wt) given orally. tMean f SD of 6 animals for Experiments 1 and 2, and 5 animals for Experiment 3. fAspirin doses (2 doses of 150 mg/kg body wt) given orally 2 hr apart before 3MI dosing. §Indomethacin doses (20 mg/kg body wt + 10 mg/kg body wt) given orally 2 hr apart before 3MI dosing. ]lAspirin doses (2 doses of 150 mg/kg body wt) given orally 2 hr apart after 3MI dosing. nstatistically significant at P i 0.05 when 3MI alone group compared to inhibitor-treated group.
given 2 hr after 3MI administration, the respiration rate was not different from its control group. Lung weight calculated as a percent of body weight (% lung wt) was also used as an index of the severity Table 3. The effect of aspirin and indomethacin
Goat No.
Edema
Experiment 1: Aspirin 3MI alone 1 2 3 4
pre-treatment
++ _ _ _ -
+ +++ ++ ++ +++ ++
_ + + + +
+ ++ + ++ ++ +
++ ++ + + ++ ++
+++ +++ ++ ++ ++ ++
+++ ++ ++ +++ +++ +++
_ _
_
_ -
+ + -
++ ++ +++ ++ +
+
_ + + + + +
+ ++ + + _ +
+++ +++ +++ +++ +
+++ +++ +++ +++ ++
++ ++ +++ +++ ++
+++ +++ +++ ++ +++
+++ ++ +++ ++ +++
+++ +++ ++ ++ +++
+ -
+ -
10 11 12
+ _ _
+ + -
Experiment 2: Indomethacin pre-treatment 3MI alone 1 _ 2 _ 3 4 5 ++ 6 ++
study
+ 3MI
9 10 11 12
_
-
stage Fibroplasia
_ _ _ _ -
Aspirin + 3MI 7 8 9
Severity of injury:
Recovery Epithelialization
+ ++ _ _ +
+ ++
Aspirin + 3MI I 8 9 10 11
in goat lung
++ + +++ ++ ++ ++
++ ++
Experiment 3: Aspirin 3MI alone 1 2 3 4 5
changes
study ++ + + _
Indomethacin I 8
on histopathological
Acute stage Cellular Hyaline membrane infiltrate
+ + +++ +++
5 6
of 3MI-induced lung disease. A heavier lung is usually indicative of tissue which has been affected by edema and increased cellularity. The average percent weight of normal, healthy lung is approx. 1.0%
post-treatment
absent,
-k-k
study
+ present (mild),
+ + present (moderate),
+ + ++ _
+ + +
_ _ + + + + + -
+ + + present (severe).
106
KAREN
S. ACTONet al.
~Dickinson et uf., 1976). Aspirin-pre-treated goats had a significantly lower percent lung weight than goats treated with 3MI alone, with respective means of 1.1 & 0.3% and 2.3 k 0.6% (Table 2). Goats which received indomethacin before 3MI treatment (1.3 + 0.2% lung wt) showed the same protective trend when compared to the group receiving 3MI alone (1.6 + O-3%), although the difference was not statistically significant (P = 0.07). Aspirin post-treated and its control group showed a similar severity of lung weight to body weight ratio (2.4 + 0.4% and 2.5 + 0.5%). Another index for the severity of pulmonary injury induced by 3MI is the percent moisture content of the lung, which measures the degree of interstitial and alveolar edema present. The percent moisture content of the lung in both the aspirin (58.8 t_ 4.2%) and indomethacin (62.6 + 1.3%) pre-treated groups was significantly lower than their respective control groups receiving 3MI alone (71.0 & 5.9% and 67.5 f 4.2%) (Table 2). However, the moisture content of aspirin post-treated group is similar to that of the control group. The gross appearance of the lungs immediately after removal illustrates a marked difference in the degree of the effect of 3MI pneumotoxicity in goats receiving 3MI alone compared to goats pre-treated with aspirin (photo not shown). The lungs of goats treated with 3MI alone were enlarged, dark red in colour, firm to the touch and displayed foam in the trachea. The lungs of goats pre-treated with aspirin were relatively normal in appearance; smaller, pink in colour and elastic to the touch.
Table 4. The in~bito~ effect of aspirin and iodomethacin prostaglandin I-I synthase activity*
Treatment
TXB, production (ng/ml blood) 0 2hr 5 hr (Time after first inhibitor dose)
on
-
Experiment 1: Aspirin pm-treatment study 3MI alone 22.2 * 3.6 22.5 rt 5.6 21.0 + 6.2 Aspirin + 3MI 21.7 + 4.1 ND NW Experiment 2: Indomethacin pm-treatment study 3MI alone 9.2 f 2 8.7+33.1 Indomethacin f 3MI 9.3 + 3.1 ND
19.1 k6.4 ND
Experiment 3: Aspirin post-treatment study 3MI alone 16.5 * 4.1 16.0 i 6.1 Aspirin + 3MI 16.1 + 3.9 ND
11.5 f 4.2 ND
*As indicated by TXB, production by thrombin stimulated platelets. tND: indicates the ~n~ntration at which TXBl is non-detec~ble.
In order to confirm that oral doses of aspirin and indomethacin were effective in inhibiting PHS activity in Experiments 1, 2 and 3, blood was analyzed from TXBr production by the thrombin-stimulated platelet PHS activity test. Table 4 shows that TXBz levels in the aspirin pre-treated or post-treated group were non-detectable 2 hr after the first aspirin dose and remained non-detectable for at least 5 hr after. The group receiving 3MI alone maintained a stable concentration of approx. 22ng TXB, per ml blood during the three sampling time periods. The doses of indometha~n a~inistered to goats in the second experiment achieved the same effect as that seen with aspirin (Table 4). DISCUSSION
Pathology Table 3 summarizes the histopathological changes which were indicative of the acute stage and the recovery stage of 3MI-induced lung disease. The acute exudative phase of 3MI toxicity was characterized by the presence of edema, hyaline membranes and cellular infiltration, whereas the proliferative recovery stage was featured by epithelialization. Diffuse alveolar edema and infiltration of erythrocytes and leukocytes were present in all goats dying acutely of 3MI toxicosis (goats 2-6). In contrast, lung from aspirin pre-treated goats showed a shift from the acute exudative stage to the proliferative phase. Only goats surviving to 72 hr (all aspirin pre-treated goats 7-12, and goat 2) reached the recovery stage as indicated by the lining of alveoli by cuboidal type II cells (epithelialization). The histopathological changes observed in the indomethacin study were similar to those observed in the aspirin study (Table 3). Although all goats in this study survived for the entire 72 hr, animals receiving 3MI alone showed acute exudative damage, whereas very little edema was seen in the indomethacin pre-treated group. The lungs of all goats showed recovery changes such as epithelialization, however, fibroplasia was more severe in animals that were not protected by indomethacin pre-treatment. The histopathological lesions of goat lungs of aspirin post-treated group is very similar to that of its control group. Aspirin has no protective effect on either the acute stage or the recovery stage of the disease development.
The results of these studies indicate that pretreatment with PHS inhibitors, especially aspirin, reduced the severity of 3MI-induced lung disease. These results are in accordance with recent in vitro work which determined that 3MI can be co-oxidized by the PHS complex in ram seminal vesicles (Formosa et al., 1988) and goat lung microsomes (Formosa and Bray, 1988). Lung cells, such as non-ciliated bronehiolar epithelial (Clara) cells and alveolar Type II cells (Sivarajah et al., 1983) contain high concentrations of PHS. 3MI is known to exert specific cytotoxic effects on Clara cells and proliferative effects on Type II cells (Huang et al., 1977; Bradley and Carlson, 1980). Thus, it appears that, in addition to MFO, this enzyme system may also be involved in the activation of 3MI. The hypothesis by Formosa and Bray (1988) and Formosa et al. (1988) that the co-oxidation of 3MI by the PHS system may be involved in 3MI-induced pneumotoxi~ity is strengthened by the current study. The proposed mechanisms of 3MI-induced lung disease are shown in Fig. 3. First, PHS, as well as MFO, was capable of activating 3MI to a reactive intermediate as indicated by the covalent binding of 3MI to microsomal protein. ESR spin trapping studies demonstrated the presence of a 3MI free radical generated from the metabolism of 3MI by horseradish peroxidase, a model system of PHS peroxidase (Formosa et al., 1988). Thus, it is likely that the cytotoxic effects such as covalent binding to tissue macromolecules and lipid peroxidation may be a
PHS in 3MLinduced pneumotoxicity
3MI NADPH, O2
-T&z
02’AA
1\
-MUI and
_I orner metabolites
Y Lipid pen Dxidation .
_+
Protein covalent binding cross linking /
\n
6
Increased ----‘--3ids
rulmonary
i”“““‘i
vasci
\
Cytotoxicity \
Fig. 3. The proposed mechanism of the pathogenesis of 3-methylindole (3MI)-induced pneumotoxicity.
consequence of the generation of a 3MI free radical by not only the well-documented MFO system (Kubow et al., 1984) but also the PHS complex. In addition to the direct tissue damage initiated by 3MI free radical, other secondary physiological responses may be seen in the target tissue as a result of altered prostanoid concentrations from the PHScatalyzed co-oxidation of 3MI (Fig. 3). Previous work showed that biosynthesis of prostaglandins was enhanced by the addition of 3MI to goat lung microsomes in vitro (Formosa and Bray, 1988), however, neither plasma nor lung prostanoid concentrations were altered by 3MI infusion in viva (Acton et al., 1990). The absence of detectable change, however, may simply indicate that the prostanoids can be rapidly degraded in the lung and plasma and lung prostanoid levels are not good indicators of altered arachidonic acid metabolism during 3MI-induced pneumotoxicity. In all experiments, edema, hyaline membranes and cellular infiltration were present in goats without inhibitor pre-treatment. These acute histopathological changes are indicative of severe damage to the alveolar-capillary membranes. Pre-treatment of aspirin and indomethacin appeared to have afforded sufficient protection against this membrane damage to permit time for proliferation of Type II epithelium and fibroblasts. Recovery of the epithelial lesion, once acute inflammation has subsided and provided there has not been fibrosis of the alveolar wall, is accomplished by transformation of Type II cell into Type I epithelium. Fibroplasia, a process of scar tissue formation, indicates that severe alveolar edema and fibrinous exudation have occurred in the lung of these animals (Said, 1973). Further indication of the protective effect of indomethacin was that fibroblast proliferation was more severe in animals that did not receive indomethacin and survived the entire 72 hr (Table 3). It is postulated that PHS inhibitors prevent
exudative damage through decreased formation of reactive 3MI metabolites. The level of the aspirin doses used in the first experiment was successful in decreasing the symptoms of 3MI-induced pneumotoxicity. One aspirin pre-treated goat (12), however, was affected by the 3MI dose, as indicated by a higher respiration rate and a heavier lung weight. The TXB2 production in the blood of this goat, unlike that of other aspirintreated animals, was not completely inhibited up to 5 hr after aspirin dosing. This indicates that this animal may not have received or absorbed all of its aspirin dose. TXB, production from thrombin-stimulated platelets in whole blood, however, appears to be a rapid and accurate test for detecting the inhibitory effect of aspirin and indomethacin in vim In a preliminary experiment, the inhibitory effect of an oral dose of indomethacin, at the same dose level as was given in the second experiment (20 mg/kg body wt), was not detected by measuring directly the plasma concentrations of PGF,,, PGE,, 6-keto PGF,, and TXB,. There was no definite decrease in any of the prostanoid concentrations measured (unpublished data). The inhibitors were given as two priming doses 2 hr and immediately before 3MI administration to focus on the effect of PHS inhibition on the initiation of the disease, as opposed to giving the inhibitor throughout the experiment. Results indicate that inhibition at this stage was important in preventing the symptoms of acute lung disease. When using prostaglandin inhibitors as a tool to manipulate disease, it is important to use more than one inhibitor. It is best to choose two compounds with different properties and structures to be sure that the response is not due to an inherent side-effect specific to one inhibitor. In this study, aspirin appeared to provide a better and more
KARENS. ACTONet al.
108
consistent protection against respiratory distress than did indomethacin. This may be due to the differences in the mechanism of inhibition of the two compounds, or it may simply be a consequence of the interfering side-effects of indomethacin as expressed by the diarrhea in all goats receiving this inhibitor. Gastrointestinal reactions have often been reported after oral administration of indomethacin in man and other species (Ferreira and Vane, 1979). Furthermore, the biological variability of the goats may also have influenced the results. Goats in the second experiment were older and less severely affected by 3M1, thus the differences between the inhibitor-pretreated group and the group receiving 3MI alone were not as great. Nevertheless, to different degrees, it was clear that both inhibitors were effective in reducing 3MI-induced pneumotoxicity. The PHS system may be an important part of the metabolic activation of 3M1, and work in conjunction with the MFO system, especially since the lung contains a high PHS activity when compared with the MFO system (Mold&us et al., 1985). The exact biochemical mechanisms of the pneumotoxicity still remains to be elucidated. Acknowledgements-The technical assistance of Pauline Bauman and Karen Beam was gratefully appreciated. Mrs Andra Williams is also acknowledged for her expert help in the preparation of this manuscript. This research was supported by NSERC and OMAF to T. M. Bray.
REFERENCES Acton K. S., Bray T. M. and Boermans H. J. (1991) Effect of 3-methylindole on the plasma and lung concentrations of prostaglandins and thromboxane B, in goats. Camp. Biochem. Physiol. (In press). Bradley B. J. and Carlson J. R. (1980) Ultrastructural pulmonary changes induced by intravenously administered 3-methvlindole in goats. Am. J. Pathol. 99.551-556. Bradley B. J., Carlson J.-R. and Dickinson E. 0. (1978) 3-Mkthylindole-induced pulmonary edema and emphysema in sheen. Am. J. Vet. Res. 39. 1355-1358. Bray T. M. and Kubow S. (1985) Involvement of free radicals in the mechanism of 3-methylindole-induced pulmonary toxicity: an example of metabolic activation in chemically induced lung disease. Environ. Health Perspect. 64, 61-67.
Carlson J. R., Dickinson E. O., Yokoyama M. T. and Bradley B. (1975) Pulmonary edema and emphysema in cattle after intraruminal and intravenous administration of 3-methylindole. Am. J. Vet. Res. 36, 1341-1347. Carlson J. R., Yokoyama M. T. and Dickinson E. 0. (1972) Induction of pulmonary edema and emphysema in cattle and goats with 3-methylindole. Science 176, 298-299. Chandler D. B. and Giri S. N. (1983) Changes in plasma concentrations of prostaglandins and plasma angiotensinconverting enzyme during bleomycin-induced lung fibrosis in hamsters. Am. Rev. Resp. Dis. 128, 71-76. Dickinson E. O., Yokoyama M. T., Carlson J. R. and Bradley B. J. (1976) Induction of pulmonary edema and emphysema in goats by intraruminal administration of 3-methylindole. Am. J. Vet. Res. 37, 667-672. Duggan D. E., Hogans A. F., Kwan K. C. and McMahon F. G. (1972) The metabolism of indomethacin in man. J. Pharmac. exp. Ther. 181, 563-575.
Ferreira S. H. and Vane J. R. (1979) Mode of action of anti-inflammatory agents which are prostaglandin synthetase inhibitors In Anti-Znflammatorv Drws - (Edited by Vane J. R. and Ferreira S.-H.), pp. 348-397. Springer, New York. Formosa P. J. and Bray T. M. (1988) Evidence for metabolism of 3-methylindole by prostaglandin H synthase and mixed-function oxidases in goat lung and liver microsomes. Biochem. Pharmac. 37, 43594366. Formosa P. J., Bray T. M. and Kubow S. (1988) Metabolism of 3-methylindole by prostaglandin H synthase in ram seminal vesicles. Can. J. Physiol. Pharmac. 66, 1524-1530.
Gingerich D. A., Baggot J. D. and Yeary R. A. (1975) Pharmacokinetics of dosage in cattle. JAVMA _ of asnirin _ 167, 945-948. Hanafy M. S. M. and Bogan J. A. (1980) The covalent binding of 3-methylindole metabolites to bovine tissue. Life Sci. 27, 1225. Hoffman D. and Rathkamp G. (1970) Quantitative determination of I-alkylindoles in cigarette smoke. Analyf. Chem. 42, 366370.
Huang T. W., Carlson J. R., Bray T. M. and Bradley B. J. (1977) 3-Methylindole-induced pulmonary injury in goats. J. Pathol. 87, 647466. Hucker H. B., Zacchei A. G., Cox S. V., Brodie D. A. and Cantwell N. H. R. (1966) Studies on the absorption, distribution and excretion of indomethacin in various species. J. Pharmac. exp. Ther. 153, 237-249. Hwang D. H. and Donovan J. (1982) In vitro and in vivo effects of vitamin E on arachidonic acid metabolism in rat platelets. J. Nutr. 112, 1233-1237. Kubow S.. Janzen E. J. and Brav T. M. (1984) Snintrapping of free radicals formed during in vitro and in kvo metabolism of 3-methylindole. J. biol. Chem. 259, 44474451. Kulmacz R. J. and Lands W. E. M. (1985) Stoichiometry and kinetics of the interaction of prostaglandin H synthase with anti-inflammatory agents. J. biol. Chem. 268, 12,572-12,578. Mold&us P., Ross D. and Larsson R. (1985) Interrelationships between xenobiotic metabolism and lipid biochemistry. Biochem. Sot. Trans. 13, 847-850. Phillips M. W., Salyer G. and Ray R. S. (1980) Equine metabolism and pharmacokinetics of indomethacin. Equine Practice 2, 45-47.
Roth G. J. and Siok C. J. (1978) Acetylation of the NH,-terminal serine of prostaglandin synthetase by asnirin. J. biol. Chem. 253. 3782-3784. Roth G. J., Machuga E. T. and 0~01s J. (1983) Isolation and covalent structure of the aspirin-modified, active-site region of prostaglandin synthetase. Biochemistry 22, 46724675.
Said S. I. (1973) The lung in relation to vasoactive hormones. Fedn. Proc. 32, 1972-1976. Saniabadi A. R., Lowe G. D. O., Forbes C. D., Prentice C. R. M. and Barbenel J. C. (1983) Platelet aggregation studies in whole human blood. Thromb. Res. 30.625432. Sivarajah K., Jones K. G., Fouts J. R., Devereux T., Shirley J. E. and Eling T. E. (1983) Prostaglandin synthetase and cytochrome P-450-dependent metabolism of benzo(a)pyrene 7,8-dihydrodiol by enriched populations of rat Clara cells and alveolar type II cells. Cancer Res. 43, 2632-2636.
Yokoyama M. T. and Carlson J. R. (1979) Microbial metabolites of tryptophan in the intestinal tract with special reference to skatole. Am. J. clin. Nutr. 32, 173-178. Yoshihara I. and Murata K. (1977) Gas chromatographic microdetermination of indole and skatole in gastrointestinal contents of domestic animals. Agric. Biol. Chem. 41, 2083-2086.
03064492/92$5.00+ 0.00 0 1991Pergamon Press plc
THE ROLE OF PROSTAGLANDIN H SYNTHASE IN 3-METHYLINDOLE-INDUCED PNEUMOTOXICITY IN GOAT KAREN S. ACTON,* HERMAN J. BOERMANS?and TAMMYM. BRAY*$ Collaborative Program in Toxicology, *Department of Nutritional Sciences and TDepartment of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada NlG 2Wl (Telephone 519 8244120, Ext. 3752; Fax: 519 763-5902) (Received 25 March 1991)
Abstract-l. Aspirin and indomethacin were used to investigate the role of prostaglandin H synthase (PHS) system in 3-methylindole (3MI)-induced pneumotoxicity. 2. A functional test was developed to detect the inhibitory effect of oral doses of aspirin and indomethacin on PHS activity based on thromboxane B, (TXB,) production from thrombin-stimulated platelets in whole blood. 3. Goats which received oral doses of aspirin or indomethacin before administration of 3MI (0.1 g 3MI/kg body wt) showed a reduced severity in clinical signs and pathological lesions in the lung than those that received 3MI alone. 4. There was no difference in the severity of the disease between the control and the aspirin-treated animals if aspirin was given after 3MI administration. 5. The protective effect of inhibitors when administered before, but not after, 3MI dosing suggests it is the inhibition of PHS activity in activation of 3M1, not in production of prostanoids which prevented the disease process.
work has indicated that 3MI is also metabolically activated by the prostaglandin H synthase (PHS) system in ram seminal vesicles (Formosa et al., 1988) and goat lung microsomes (Formosa and Bray, 1988). The reactive intermediate formed by the PHS system was capable of covalently binding to microsomal protein which may play a part in the mechanism of cytotoxicity. In addition, the PHS system has been found to have a higher activity in the lung when compared to the MFO system (Moldeus et al., 1985), thus, it appears that the combined effects of the PHS and MFO systems may account for the tissue specificity of 3MI-induced pneumotoxicity. Non-steroidal anti-inflammatory drugs, aspirin and indomethacin, block the synthesis of prostanoids by inhibiting the cyclooxygenase enzyme of the PHS system. Aspirin causes inhibition by irreversibly acetylating a serine residue of the cyclooxygenase whereas indomethacin appears to cause inhibition by binding the cyclooxygenase at a position which reduced its affinity for the substrate (Roth and Siok, 1978; Roth et al., 1983; Kulmacz and Lands, 1985). Cyclooxygenase inhibitors are useful tools for defining the role that PHS play in the mechanism of disease processes. If the PHS is involved in the metabolic activation of the toxin, then inhibitors, when given before the toxin administration, should be able to reduce the severity of the disease. If the cause of the pneumotoxicity is only due to an increase in prostanoid production, PHS inhibitors, when given after toxin administration, should be able to prevent the disease processes. The objective of this experiment was to determine whether aspirin and indomethacin are capable of altering the clinical signs and the pathological lesions of 3MI-induced lung
INTRODUCTION
Acute bovine pulmonary edema and emphysema (ABPE) is a naturally occurring lung disease which affects cattle subjected to a sudden change in diet from poor quality forage to lush green pasture. 3-Methylindole (3M1, skatole), the main ruminal fermentation product of tryptophan after this drastic dietary change, is the causative agent of this respiratory disease (Carlson et al., 1972). The experimental administration of oral or intravenous doses of 3MI can induce the characteristic onset of pulmonary edema and interstitial emphysema in cattle (Carlson et al., 1975), sheep (Bradley et al., 1978) and goats (Dickinson et al., 1976). Other sources of 3MI are found in the feces of rat, pig and man due to the fermentation of tryptophan in the large intestine (Yoshihara and Murata, 1977; Yokoyama and Carlson, 1979). 3MI is also a component of cigarette smoke, due to the pyrolysis of tryptophan in tobacco leaves (Hoffman and Rathkamp, 1970) yet the extent of the health risk of 3MI to man has not yet been determined. The mechanism of 3MI toxicity, which is acute and specific to the lung, is still under investigation. 3MI has been shown to be activated by the cytochrome P-450 mixed-function oxidase (MFO) system to form a reactive-free radical species capable of binding to macromolecules and initiating cytotoxicty (Hanafy and Bogan, 1980; Bray and Kubow, 1985). This activation of 3MI by the MFO system alone cannot explain, however, the phenomenon of the tissue specificity of 3MI-induced pneumotoxicity. Recent $To whom correspondence
should be addressed. 101
KARENS. ACTONet al.
102
disease. The results would be useful in elucidating the by which the PHS system is involved in 3MI-induced pneumotoxicity in vivo. mechanism
MATERIAL
indometha~n dosing, as well as I and 3 hr after administration. All blood samples were subjected to the thrombin stimulation and subsequently analyzed for TXB, production by RIA.
AND METHODS
VeriJcation of inhibitory effect of aspirin and indomethacin Since dosage for aspirin or indomethacin in goats has not been well-documented, it was important to detect and verify that the inhibitor doses to be used are absorbed and at functionally adequate levels. A rapid and simple method based on thromboxane B, (TXB,) production from thrombin-stimulated platelets-in whole blood was developed to test functionally the inhibitorv effects of asnirin and indomethacin on -PHS activity -without mea&ing the plasma concentrations of inhibitors after oral administration. The procedure was based on a modification of whole blood aggregation protocols (Hwang and Donovan, 1982; Saniabadi et ai., 1983). Thrombin was obtained from Parke-Davis and Co., Ltd (Brockville, Ontario). A11 other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). The protocol for the test was as follows: 4.5 ml of blood were taken from the jugular vein using vacutainers containing OSml of 0.05 M phosphate-buffers saline (PBS), pH 7.4 with 3.2% trisodium citrate as the anticoa~lant. The &rated whole blood was subsequently diluted 1: 1 with additional PBS. A 400 ~1 aliquot of the diluted sample was transferred to a siliconized glass cuvette and p&incubated at 37°C for I min. The samule then received 25 ul of CaCl, (5 mM final concentration), and was immediately stimulated with thrombin (0.1254 units). Samples were subsequently incubated with continuous stirring for 4 min at which time 50 ~1 of aspirin (0.75 mg/ml) was added to stop any further TXB, production. Samples were allowed to clot at room temperature and were centrifuged at IOOOgfor 9 min at 4°C. Serum was collected and stored at -20°C until analyzed for TXB, by RIA. Two in uirro preliminary trials were performed to verify whether aspirin and indomethacin inhibit TXB2 production from thrombin-stimulated platelets in goat blood. In the first trial, two goats were bled, each providing three blood samples. One sample received no inhibitor whereas the others received either a low (O.O3mg/ml) or a high (O.O95mg/ml) concentration of aspirin. The low dose approximated typical plasma levels of aspirin in cattle after therapeutic dosing (Gingerich et ai., 1975), whereas the high dose was chosen to completely inhibit TXB, formation in vitro. Blood samples were prepared and analyzed for TXB, formation as described above. The second in vitro trial was similar to the aspirin study, yet indometha~n was the inhibitor used. Again, two goats were bled, providing three blood samples each. One sample received no inhibitor, whereas the other immediately received O.O7mg/ml indomethacin. Two in viuo trials were subs~uently performed to verify whether TXB, production by platelet after thrombin stimulation could be used as an in viuo functional indicator of the inhibitory effect of oral doses of aspirin or indomethacin. In the first trial, three goats were used, each receiving two doses 150 mg/ml body wt aspirin given 2 hr apart. Blood was taken from the jugular vein immediately before the first dose and 2 hr later (immediately before the second dose). A final blood sample was taken 2 hr after the second dose. In the second trial, four goats were used and divided equally into two groups. One group received an oral low dose of indomethacin (2mg/kg body wt), whereas the second received a IO-fold higher dose (20 mg/kg body wt). Doses were derived from the literature by averaging low and high indomethacin doses used for various species such as rat (Hueker et al., 1966), horse (Phillips ef ai., 1980) and man (Duggan et ui., 1972). Blood was taken immediately before
TXB, concentration was determined by the procedure of Upjohn Diagnostic Laboratories (Kalamazoo, MI) as described by Chandler and Giri (1983). Tritiated TXB, was purchased from New England Nuclear (Boston, MA). Antibody and all further reagents were obtained from Sigma Chemical Co. (St. Louis, MO). Dried TXB, extract was reconstituted in 0.1 M PBSG. Samples were analyzed in duplicate and aliquots were added to polypropylene tubes containing antibody and approx. 10,000 cpm of the tritiated prostanoid. Tubes were incubated for 1 hrat 2S”C, followed by a 4°C incubation for 20-24 hr. Unbound TXB, was separated from antibody-bound TXB, by charcoal absorption. Following centrifugation, the radioactivity of the supematant was determined by liquid scintillation counting. The cross-reactivity of the TXB, antiserum was 100% for TXB, and less than 0.1% for other prostaglandins. Experiment 1: Aspirin pre-treatment study Twelve female goats (Cadwell Farm, Drumbo, Ontario) from 5 to 12 months of age were acclimatized to their new surroundings for one week. Feed supplement (19% protein), hay and water were administered ad libitum throughout the study unless indicated. Goats were randomly divided into two groups of 6, consisting of an aspirin pretreated group and a control group. The control group received 3MI alone (0.1 g/kg body wt) whereas the aspirin group received two doses of aspirin (150 mg/kg body wt per dose) before 3MI administration. The 3MI dose has been shown to induce moderate pulmonary lesions within 72 hr (Carlson et al., 1975; Dickinson et af., 1976). All treatments were given orally in gelatin capsules. Feed was removed from both control and treatment groups 24 hr before any oral dosing. To detect the inhibitory effects of aspirin on the PHS activity, TXB, production from stimulated platelets was measured before and after aspirin administmtion. Pretreatment blood samples were taken from the jugular vein of goats from both groups immediately before dosing. Inhibitor was then given to the aspirin pre-treated group only. Two hours after aspirin administration, blood was taken again from both groups. The aspirin pretreated group then received a second dose of inhibitor to maintain the efficacy of the drug, after which 3MI was immediately given to both aspirin and control groups. A final blood sample was taken 3 hr after 3MI administration (5 hr after the first aspirin dose). Clinical signs (laboured breathing and respiration rate) were monitored throughout the duration of the study. Seventy-two hours after 3MI administration, goats were killed and lungs were removed and weighed in order to calculate the lung weight as a percent of body weight. Three lung tissue samples per goat were obtained from pre-determined areas of the right lobe for moisture content determination. The moisture content of the lung and the ratio of the whole lung weight to body wt are commonly used as indices of the severity of pulmonary lesions (Carlson ei al,, 1975; Dickinson et al., 1976; Bradley et ai., 1978). A portion of the lung samples were taken for histopathological examination. Experiment 2: Zndomethacin pre-treatment sfudy The experimental design of this experiment paralleled that of the aspirin study (Experiment 1). Twelve female goats (Thomas Farm, Orton, and Harkin Farm. Elora. Ontario) one year of age were .fed feed supplement, alfalfa pellets and water ad iib~~um. Goats were randomly divided into two groups, a control group which received 3MI (0.1 g/kg
103
PHS in 3MI-induced pneumotoxicity body wt) alone and an indomethacin pre-treated group which received two oral doses of indomethacin before 3MI administration. The lirst indomethacin dose (20 mg/kg body wt), was given 2 hr before the second dose (10 mg/kg body wt), which was halved due to the known potency of indomethacin and its undesired side-effect of diarrhea. The remaining experimental protocol for this study was the same as described in the aspirin experiment.
scale in proportion to severity (moderate, and + + + severe).
absent, + mild, + +
Statistical analysis
Data was analyzed between groups using the Student’s unpaired t-test. Significance was considered a P value less than 0.05. The evaluation of clinical signs and histopathology were expressed on an individual animal basis.
Experiment 3: Aspirin post-treatment study
Experiment 3 was designed to test the hypothesis that it is the inhibition of the activity of PHS in activation of 3M1, not the inhibition in prostanoid synthesis, that prevents the disease process. Aspirin was given 2 hr after 3MI administration. Ten female goats (Cadwell Farm, Drumbo, Ontario) from 5 to 12 months of age were randomly divided into two groups of five, consisting of a control group and an aspirin post-treated group. The control group received 3MI alone (0.1 g/kg body wt) whereas the aspirin posttreated group received two doses of aspirin (150 mg/kg body wt per dose, 2 hr apart) two hours after 3MI administration. The remaining experimental protocol for this study was the same as described in the Experiment 1. Pathology
Lung tissue from three pre-determined locations from the right lobe was lixed in 10% phosphate buffered formalin (Fisher), shaken for 24 hr and embedded in paraffin wax. Sections (5 pm thick) were stained with hematoxylin and eosin. Membrane thickness was measured with light microscopy using a micrometer scale lens. Cellularity and edema were scored semi-quantitatively with a four-point
RESULTS Inhibitory activity
The aspirin
inhibitory
effect
in
of
vitro
addition
%
z
Cl
6
*
0.095mg
1
1
2 Thrombin
3
8r
E 2
4
;
2
I 1
0
+
Goat 3
u
4
/
z
-=-
No ID
_
0.07mg ID/ml
I
I
0
0 I
1
I
I
3
4
Time (hours after 1st aspirin dose)
(b)
(b)
m4m
I
2
(units) 30 -
6
Goat 2
aspirin/ml
0
0
1 z
e
~__*______c_________--.
nd
of
and indomethacin on TXB, production by the thrombin-stimulated platelets is shown in Fig. la and b. Blood samples without aspirin or indomethacin showed increasing levels of TXBr production with increased thrombin stimulation. Samples which contained either the high aspirin dose (0.095 mg/ml) or indomethacin (0.07 mg/ml) showed no detectable levels of TXB, even with increasing concentrations of thrombin. Samples containing the low aspirin concentration (0.03 mg/ml) showed levels of TXB2 only slightly above the detectable limit. The inhibitory effect of oral doses of aspirin or indomethacin on PHS activity, as indicated by the thrombin-stimulated platelet test, is illustrated in Fig. 2a and b. Aspirin, administered orally (150 mg/kg
--o--Goat
t-
on PHS
effect of aspirin and indomethacin
I
I
2 Thrombin
3
I 4
(units)
Fig. 1. The effect of in vitro addition of aspirin (a) and indomethacin (b) on TXB, synthesis in goat blood. a: Blood samples from 2 goats received either no aspirin, 0.03 mg/ml aspirin or O.O95mg/ml aspirin and were challenged with 0.125, 0.25, 0.5, 1, 2 and 4 units of thrombin. b: Blood samples from 2 goats received either no indomethacin, or 0.07 mg/ml indomethacin and were challenged with thrombin as above. nd: indicates the concentration at which TXB, is non-detectable.
1
- -0 --
Low dose
P
High dose
2
3
Time (hours after ID dose)
Fig. 2. The effect of orally administered aspirin (a) and indomethacin (b) on TXB, synthesis in goat blood. a: Three goats were given two oral doses of 150 mg/kg aspirin at time 0 and 2 hr. The effect of oral doses of aspirin on TXB, concentration was determined at 0,2 and 4 hr after the first aspirin dose. b: Two goats were dosed with either one low dose (2.0 mg/kg body wt) or one high dose (20 mg/kg body wt) of indomethacin at time zero. The effect of oral doses of indomethacin on TXB, production was determined at 0, 1 and 3 hr after dosing. nd: indicates the concentration at which TXB, is non-detectable.
KARENS. ACTONez al.
104
body wt) inhibited TXB, synthesis to a nondetectable level 2 hr after dosing (Fig. 2a). A second dose of aspirin, given 2 hr after the first, maintained the inhibition for at least a further 2 hr. One oral high dose of indomethacin (20mg/kg body wt) inhibited TXB, production to a non-detectable level 1 and 3 hr after dosing (Fig. 2b). The low dose (2 mg/kg body wt) showed no inhibition at 1 hr, yet showed inhibition 3 hr after dosing. ClWcal signs
The cyclooxygenase inhibitor, aspirin, when administered before 3MI dosing, appeared to exert a protective effect against the development of 3MIinduced lung disease, as did indomethacin to a lesser degree (Table I). Four out of the six goats pre-treated with aspirin showed no clinical signs of respiratory distress, while the remaining two goats showed only mild-to-moderate symptoms. All of the aspirin pretreated goats survived, yet five out of the six goats treated with 3MI alone died before the end of the 72 hr experiment. In the indomethacin study all goats from both groups survived, yet only goats
receiving 3MI alone demonstrated the onset of severe clinical signs (Experiment 2, Table 1). Only one of the indomethacin-pretreated goats developed clinical signs, characterized by a delayed onset. Goats treated with indomethacin developed a mildto-moderate diarrhea commencing one day after dosing. There was no difference in the 3MI-induced pulmonary toxicity when aspirin was given after 3MI administration (Experiment 3, Table 1). Other indices of the severity of the disease, such as respiration rate, lung weight as percent of the body weight and the percent moisture content of the lung were also affected by the aspirin or indomethacin when it was given before 3ML administration (Table 2). The mean respiratory rate per min (rpm) of goats pre-treated with aspirin (43 & 21 (SD) rpm) was significantly lower than the respiration rate of goats receiving 3MI alone (82 sf: 19 rpm). The indomethacin group showed a similar trend towards a lower, more normal respiration rate (33 + 23 rpm) compared to the group receiving 3MI alone (56 t_ 35 rpm), although the values were not statistically different. However, when aspirin was
Table 1. The effect of aspirin and indomethacin 3-methyIindoIe-indu~ Goat No. Experiment ”
4 5 6
8 9 10 II 12
on clinical signs and time of death in lung disease
Onset (hour) and degree of clinical signs*
Treatment
1:Aspirin pm-treatment study 3MI 3MI 3MI 3MI 3MI 3MI
alonet alone alone alone alone alone
Aspirin Aspirin Aspirin Aspirin Aspirin Aspirin
+ + + + + +
3MIT 3MI 3MI 3MI 3MI 3MI
36, severe 12, severe 24, severe 12. severe 12, severe 24, severe
Killed at 72 hr Died at 26.5 hr Died at 68 hr Died at 21.5 hr Died at 38.5 hr Died at 69 hr
None None None None 48, mild 36, moderate
Killed Killed Killed Killed Killed Killed
at at at at at at
72 hr 72 hr 72 hr 72 hr 72 hr 72 hr
Killed Killed Killed Killed Killed Killed
at at at at at at
72 hr 72 hr 72 hr 72 hr 72 hr 72 hr
48, moderate None None None None None
Killed Killed Killed Killed Killed Killed
at at at at at at
72 hr 72 hr 72 hr 72 hr 72 hr 72 hr
24, 20, 30, 24, 36,
severe severe severe severe severe
Killed Killed Killed Killed Killed
at at at at at
72 hr 72 hr 72 hr 72 hr 72 hr
32, 24, 30, 24, 38,
severe severe severe severe severe
Died at 48 hr Killed at 72 hr Killed at 72 hr Killed at 72 hr Killed at 72 hr
Experiment 2: Indomethacin pre-treatment study None 1 3M1 alone 2 3Mt alone None 3 3M1 alone None 4 3MI alone 24. severe 24; severe 5 3MI alone 6 3MI alone 24, severe 7
8 9 10 11 12
Indomethacin Indomethacin Indomethacin Indomethacin Indomethacin Indomethacin
+ 3MI$ -t- 3MI + 3MI + 3MI + 3MI + 3MI
Experiment 3: Aspirin post-treatment 1 3MI alone 2 3MI aione 3 3MI alone 4 3MI alone 5 3MI alone 7 8 9 10 11
Manner and time of death
3MI 3MI 3MI 3MI 3MI
+ + + + +
aspirinlj aspirin aspirin aspirin aspirin
study
*Clinical signs consisted of irregular respiration rate, laboured breaths and trembling. t3MI dose (0.1 g/kg body wt) given orally. IAspirin doses (2 doses of I50 mg/kg body wt) given orally 2 hr apart before 3MI dosing @Indomethacin doses (20 mg/kg body wt + 10 mg/kg body wt) given orally 2 hr apart before 3MI dosing ItAspirin doses (2 doses of 150 mg/kg body wt) given orally 2 hr apart after 3MI dosing.
PHS
3MI-induced pneumotoxicity
in
105
Table 2. The effect of aspirin and indomethacin on clinical signs 72 hr after 3-methylindole administration Respiration rate Treatment
Experiment
Lung moisture content (%)
Lung wt/bw (%)
(rpm)
I
3MI alone* Aspirin + 3MIt:
82 f 19tli 43*21
2.3 & 0.6n 1.1 f 0.3
71.0 * 5.9qj 58.8 f 4.2
2
3MI alone Indomethacin
56 f 35 33 f 23
1.6 f 0.31 1.3 * 0.2
61.5 f 4.2 62.6 f 1.3
82k IO 78 * 23
2.4 f 0.4 2.5 + 0.5
68.2 + 2.3 61.9 + 3.1
3
+ 3MI$
3MI alone 3MI + aspirin11
l3MI dose (0.1 g/kg body wt) given orally. tMean f SD of 6 animals for Experiments 1 and 2, and 5 animals for Experiment 3. fAspirin doses (2 doses of 150 mg/kg body wt) given orally 2 hr apart before 3MI dosing. §Indomethacin doses (20 mg/kg body wt + 10 mg/kg body wt) given orally 2 hr apart before 3MI dosing. ]lAspirin doses (2 doses of 150 mg/kg body wt) given orally 2 hr apart after 3MI dosing. nstatistically significant at P i 0.05 when 3MI alone group compared to inhibitor-treated group.
given 2 hr after 3MI administration, the respiration rate was not different from its control group. Lung weight calculated as a percent of body weight (% lung wt) was also used as an index of the severity Table 3. The effect of aspirin and indomethacin
Goat No.
Edema
Experiment 1: Aspirin 3MI alone 1 2 3 4
pre-treatment
++ _ _ _ -
+ +++ ++ ++ +++ ++
_ + + + +
+ ++ + ++ ++ +
++ ++ + + ++ ++
+++ +++ ++ ++ ++ ++
+++ ++ ++ +++ +++ +++
_ _
_
_ -
+ + -
++ ++ +++ ++ +
+
_ + + + + +
+ ++ + + _ +
+++ +++ +++ +++ +
+++ +++ +++ +++ ++
++ ++ +++ +++ ++
+++ +++ +++ ++ +++
+++ ++ +++ ++ +++
+++ +++ ++ ++ +++
+ -
+ -
10 11 12
+ _ _
+ + -
Experiment 2: Indomethacin pre-treatment 3MI alone 1 _ 2 _ 3 4 5 ++ 6 ++
study
+ 3MI
9 10 11 12
_
-
stage Fibroplasia
_ _ _ _ -
Aspirin + 3MI 7 8 9
Severity of injury:
Recovery Epithelialization
+ ++ _ _ +
+ ++
Aspirin + 3MI I 8 9 10 11
in goat lung
++ + +++ ++ ++ ++
++ ++
Experiment 3: Aspirin 3MI alone 1 2 3 4 5
changes
study ++ + + _
Indomethacin I 8
on histopathological
Acute stage Cellular Hyaline membrane infiltrate
+ + +++ +++
5 6
of 3MI-induced lung disease. A heavier lung is usually indicative of tissue which has been affected by edema and increased cellularity. The average percent weight of normal, healthy lung is approx. 1.0%
post-treatment
absent,
-k-k
study
+ present (mild),
+ + present (moderate),
+ + ++ _
+ + +
_ _ + + + + + -
+ + + present (severe).
106
KAREN
S. ACTONet al.
~Dickinson et uf., 1976). Aspirin-pre-treated goats had a significantly lower percent lung weight than goats treated with 3MI alone, with respective means of 1.1 & 0.3% and 2.3 k 0.6% (Table 2). Goats which received indomethacin before 3MI treatment (1.3 + 0.2% lung wt) showed the same protective trend when compared to the group receiving 3MI alone (1.6 + O-3%), although the difference was not statistically significant (P = 0.07). Aspirin post-treated and its control group showed a similar severity of lung weight to body weight ratio (2.4 + 0.4% and 2.5 + 0.5%). Another index for the severity of pulmonary injury induced by 3MI is the percent moisture content of the lung, which measures the degree of interstitial and alveolar edema present. The percent moisture content of the lung in both the aspirin (58.8 t_ 4.2%) and indomethacin (62.6 + 1.3%) pre-treated groups was significantly lower than their respective control groups receiving 3MI alone (71.0 & 5.9% and 67.5 f 4.2%) (Table 2). However, the moisture content of aspirin post-treated group is similar to that of the control group. The gross appearance of the lungs immediately after removal illustrates a marked difference in the degree of the effect of 3MI pneumotoxicity in goats receiving 3MI alone compared to goats pre-treated with aspirin (photo not shown). The lungs of goats treated with 3MI alone were enlarged, dark red in colour, firm to the touch and displayed foam in the trachea. The lungs of goats pre-treated with aspirin were relatively normal in appearance; smaller, pink in colour and elastic to the touch.
Table 4. The in~bito~ effect of aspirin and iodomethacin prostaglandin I-I synthase activity*
Treatment
TXB, production (ng/ml blood) 0 2hr 5 hr (Time after first inhibitor dose)
on
-
Experiment 1: Aspirin pm-treatment study 3MI alone 22.2 * 3.6 22.5 rt 5.6 21.0 + 6.2 Aspirin + 3MI 21.7 + 4.1 ND NW Experiment 2: Indomethacin pm-treatment study 3MI alone 9.2 f 2 8.7+33.1 Indomethacin f 3MI 9.3 + 3.1 ND
19.1 k6.4 ND
Experiment 3: Aspirin post-treatment study 3MI alone 16.5 * 4.1 16.0 i 6.1 Aspirin + 3MI 16.1 + 3.9 ND
11.5 f 4.2 ND
*As indicated by TXB, production by thrombin stimulated platelets. tND: indicates the ~n~ntration at which TXBl is non-detec~ble.
In order to confirm that oral doses of aspirin and indomethacin were effective in inhibiting PHS activity in Experiments 1, 2 and 3, blood was analyzed from TXBr production by the thrombin-stimulated platelet PHS activity test. Table 4 shows that TXBz levels in the aspirin pre-treated or post-treated group were non-detectable 2 hr after the first aspirin dose and remained non-detectable for at least 5 hr after. The group receiving 3MI alone maintained a stable concentration of approx. 22ng TXB, per ml blood during the three sampling time periods. The doses of indometha~n a~inistered to goats in the second experiment achieved the same effect as that seen with aspirin (Table 4). DISCUSSION
Pathology Table 3 summarizes the histopathological changes which were indicative of the acute stage and the recovery stage of 3MI-induced lung disease. The acute exudative phase of 3MI toxicity was characterized by the presence of edema, hyaline membranes and cellular infiltration, whereas the proliferative recovery stage was featured by epithelialization. Diffuse alveolar edema and infiltration of erythrocytes and leukocytes were present in all goats dying acutely of 3MI toxicosis (goats 2-6). In contrast, lung from aspirin pre-treated goats showed a shift from the acute exudative stage to the proliferative phase. Only goats surviving to 72 hr (all aspirin pre-treated goats 7-12, and goat 2) reached the recovery stage as indicated by the lining of alveoli by cuboidal type II cells (epithelialization). The histopathological changes observed in the indomethacin study were similar to those observed in the aspirin study (Table 3). Although all goats in this study survived for the entire 72 hr, animals receiving 3MI alone showed acute exudative damage, whereas very little edema was seen in the indomethacin pre-treated group. The lungs of all goats showed recovery changes such as epithelialization, however, fibroplasia was more severe in animals that were not protected by indomethacin pre-treatment. The histopathological lesions of goat lungs of aspirin post-treated group is very similar to that of its control group. Aspirin has no protective effect on either the acute stage or the recovery stage of the disease development.
The results of these studies indicate that pretreatment with PHS inhibitors, especially aspirin, reduced the severity of 3MI-induced lung disease. These results are in accordance with recent in vitro work which determined that 3MI can be co-oxidized by the PHS complex in ram seminal vesicles (Formosa et al., 1988) and goat lung microsomes (Formosa and Bray, 1988). Lung cells, such as non-ciliated bronehiolar epithelial (Clara) cells and alveolar Type II cells (Sivarajah et al., 1983) contain high concentrations of PHS. 3MI is known to exert specific cytotoxic effects on Clara cells and proliferative effects on Type II cells (Huang et al., 1977; Bradley and Carlson, 1980). Thus, it appears that, in addition to MFO, this enzyme system may also be involved in the activation of 3MI. The hypothesis by Formosa and Bray (1988) and Formosa et al. (1988) that the co-oxidation of 3MI by the PHS system may be involved in 3MI-induced pneumotoxi~ity is strengthened by the current study. The proposed mechanisms of 3MI-induced lung disease are shown in Fig. 3. First, PHS, as well as MFO, was capable of activating 3MI to a reactive intermediate as indicated by the covalent binding of 3MI to microsomal protein. ESR spin trapping studies demonstrated the presence of a 3MI free radical generated from the metabolism of 3MI by horseradish peroxidase, a model system of PHS peroxidase (Formosa et al., 1988). Thus, it is likely that the cytotoxic effects such as covalent binding to tissue macromolecules and lipid peroxidation may be a
PHS in 3MLinduced pneumotoxicity
3MI NADPH, O2
-T&z
02’AA
1\
-MUI and
_I orner metabolites
Y Lipid pen Dxidation .
_+
Protein covalent binding cross linking /
\n
6
Increased ----‘--3ids
rulmonary
i”“““‘i
vasci
\
Cytotoxicity \
Fig. 3. The proposed mechanism of the pathogenesis of 3-methylindole (3MI)-induced pneumotoxicity.
consequence of the generation of a 3MI free radical by not only the well-documented MFO system (Kubow et al., 1984) but also the PHS complex. In addition to the direct tissue damage initiated by 3MI free radical, other secondary physiological responses may be seen in the target tissue as a result of altered prostanoid concentrations from the PHScatalyzed co-oxidation of 3MI (Fig. 3). Previous work showed that biosynthesis of prostaglandins was enhanced by the addition of 3MI to goat lung microsomes in vitro (Formosa and Bray, 1988), however, neither plasma nor lung prostanoid concentrations were altered by 3MI infusion in viva (Acton et al., 1990). The absence of detectable change, however, may simply indicate that the prostanoids can be rapidly degraded in the lung and plasma and lung prostanoid levels are not good indicators of altered arachidonic acid metabolism during 3MI-induced pneumotoxicity. In all experiments, edema, hyaline membranes and cellular infiltration were present in goats without inhibitor pre-treatment. These acute histopathological changes are indicative of severe damage to the alveolar-capillary membranes. Pre-treatment of aspirin and indomethacin appeared to have afforded sufficient protection against this membrane damage to permit time for proliferation of Type II epithelium and fibroblasts. Recovery of the epithelial lesion, once acute inflammation has subsided and provided there has not been fibrosis of the alveolar wall, is accomplished by transformation of Type II cell into Type I epithelium. Fibroplasia, a process of scar tissue formation, indicates that severe alveolar edema and fibrinous exudation have occurred in the lung of these animals (Said, 1973). Further indication of the protective effect of indomethacin was that fibroblast proliferation was more severe in animals that did not receive indomethacin and survived the entire 72 hr (Table 3). It is postulated that PHS inhibitors prevent
exudative damage through decreased formation of reactive 3MI metabolites. The level of the aspirin doses used in the first experiment was successful in decreasing the symptoms of 3MI-induced pneumotoxicity. One aspirin pre-treated goat (12), however, was affected by the 3MI dose, as indicated by a higher respiration rate and a heavier lung weight. The TXB2 production in the blood of this goat, unlike that of other aspirintreated animals, was not completely inhibited up to 5 hr after aspirin dosing. This indicates that this animal may not have received or absorbed all of its aspirin dose. TXB, production from thrombin-stimulated platelets in whole blood, however, appears to be a rapid and accurate test for detecting the inhibitory effect of aspirin and indomethacin in vim In a preliminary experiment, the inhibitory effect of an oral dose of indomethacin, at the same dose level as was given in the second experiment (20 mg/kg body wt), was not detected by measuring directly the plasma concentrations of PGF,,, PGE,, 6-keto PGF,, and TXB,. There was no definite decrease in any of the prostanoid concentrations measured (unpublished data). The inhibitors were given as two priming doses 2 hr and immediately before 3MI administration to focus on the effect of PHS inhibition on the initiation of the disease, as opposed to giving the inhibitor throughout the experiment. Results indicate that inhibition at this stage was important in preventing the symptoms of acute lung disease. When using prostaglandin inhibitors as a tool to manipulate disease, it is important to use more than one inhibitor. It is best to choose two compounds with different properties and structures to be sure that the response is not due to an inherent side-effect specific to one inhibitor. In this study, aspirin appeared to provide a better and more
KARENS. ACTONet al.
108
consistent protection against respiratory distress than did indomethacin. This may be due to the differences in the mechanism of inhibition of the two compounds, or it may simply be a consequence of the interfering side-effects of indomethacin as expressed by the diarrhea in all goats receiving this inhibitor. Gastrointestinal reactions have often been reported after oral administration of indomethacin in man and other species (Ferreira and Vane, 1979). Furthermore, the biological variability of the goats may also have influenced the results. Goats in the second experiment were older and less severely affected by 3M1, thus the differences between the inhibitor-pretreated group and the group receiving 3MI alone were not as great. Nevertheless, to different degrees, it was clear that both inhibitors were effective in reducing 3MI-induced pneumotoxicity. The PHS system may be an important part of the metabolic activation of 3M1, and work in conjunction with the MFO system, especially since the lung contains a high PHS activity when compared with the MFO system (Mold&us et al., 1985). The exact biochemical mechanisms of the pneumotoxicity still remains to be elucidated. Acknowledgements-The technical assistance of Pauline Bauman and Karen Beam was gratefully appreciated. Mrs Andra Williams is also acknowledged for her expert help in the preparation of this manuscript. This research was supported by NSERC and OMAF to T. M. Bray.
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