The involvement of serotonin in the pneumotoxicity induced by N-methylthiobenzamide

TOXICOLOGY

AND

APPLIED

93,165- 174 ( 1988)

PHARMACOLOGY

The Involvement

of Serotonin in the Pneumotoxicity by N-Methylthiobenzamide’

LESTER S. GIBBS, FENG JIAN-XING,* Department of Pharmacology 66045-2500, and *Department

and Toxicology, of Environmental

Received

May

Induced

J. TRAIGER’

AND GEORGE

University of Kansas School of Pharmacy, Lawrence, Kansas Science, Nankai University, Tainjin, People’s Republic of China

20,1987;

accepted

November

4, 1987

The Involvement of Serotonin in the Pneumotoxicity Induced by N-Methylthiobenzamide. L. S., JIAN-XING, F., AND TRAIGER, G. J. (1988). Toxicol. Appl. Pharmacol. 93, 165174. N-Methylthiobenzamide (NMTB) and a-naphthylthiourea (ANTU) are pneumotoxicants which cause pulmonary edema and hydrothorax. Recently a role was assigned to serotonin (5 hydroxytryptamine, S-HT) in the pneumotoxic response to ANTU (D. E. Mais and T. R. Bosin, 1984, Toxicol. Appl. Pharmacol. 74, 185- 194). We therefore investigated the participation of 5HT in NMTB-induced pneumotoxicity. Pulmonary clearance of 5-HT was studied after NMTB or ANTU using the rat isolated perfused lung. Lung 5-HT uptake was not depressed 5 hr after ANTU or NMTB, but was depressed 12 hr after compound administration. At both time points lungs were edematous as judged by lung wet weight to body weight ratios. Pretreatment with reserpine, a drug known to deplete 5-HT, did not affect the NMTB-induced decrease in lung 5HT uptake, but did diminish the increased lung wet weight to dry weight ratios seen after NMTB administration in rats and mice and the increased lung wet weight to body weight ratios in mice. NMTB induces a dose-dependant increase in the incorporation of j’4C]thymidine into mouse pulmonary DNA. This increase was attenuated, but not abolished, by pretreatment with reserpine. Reserpine did not alter survival time after NMTB or ANTU and did not shift the 14-day LD50 of NMTB. These data suggest that 5-HT is not a primary mediator in the pneumotoxic response to these thiono-containing compounds. 0 1988 Academic PBS, hc.

GIBBS,

Arylthiourea compounds and certain thiobenzamide derivatives are potent pneumotoxicants. The toxic signs which appear in rodents after administration of ar-naphthylthiourea (ANTU) or N-methylthiobenzamide (NMTB) are similar (Cunningham and Hurley, 1972; Cashman et al., 1982) and include pulmonary edema and hydrothorax with accompanying changes in lung morphology. A key step in the expression of ANTU- or NMTB-induced pneumotoxicity appears to involve metabolic conversion of the parent compound to an S-oxide intermediate with ’ Portions of this work were presented at the 1986 meeting of the Society of Toxicology (Toxicologist 6, 243). 2 To whom all correspondence should be sent.

subsequent covalent binding (Boyd and Neal, 1976; Gotschall et al., 1985; Penney et al., 1985). Interaction with reactive metabolites may, however, be only part of the overall toxic response of the lung to these chemicals. The lung’s ability to remove from the circulation and metabolize endogenous chemicals, such as serotonin (5-HT) (Alabaster and Bakhle, 1970), norepinephrine (Hughes et al., 1969), and prostaglandin E2 (Bakhle et al., 1977) is well established. Impairment of their pulmonary clearance may allow these vasoreactive pharmacophores to play a role in the sequence of events that leads to pulmonary hypertension (Mehendale, 1984) or lung permeability changes. For example, increased plasma concentrations of 5-HT resulting 165

0041-008X/88

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166

GIBBS, JlAN-XlNG.

from faulty uptake could lead to vasoconstriction (Reid and Rand, 1957) and permeability changes (Majno and Palade, 1961) which would allow for fluid efflux from the vasculature. Recently, 5-HT has been ascribed a role in the toxic response of the lung to ANTU and oxygen (Mais and Bosin, 1984). Because of the similarities in ANTUand NMTB-mediated pulmonary edema, we investigated a possible role for 5-HT in NMTB-induced lung injury.

METHODS Animals. Male Sprague-Dawley rats (200-250 g) were obtained from Sasco, Inc. (Omaha, NE) or from a Sascoderived breeding colony housed at the University of Kansas. Rats were caged in groups of two or three. Male mice (ICR, 20-25 g) were obtained from Sasco and caged in groups of five. All animals were housed in a temperature-controlled, 12-hr light/dark cycle environment and allowed accessto water and Purina Lab Chow ad libitum. Compound administration. Reserpine solution was diluted, when necessary, with 0.9% saline and injected at a volume of 3 ml/kg. NMTB and ANTU were injected ip as fine suspensions in corn oil (injection volume for both mice and rats was 3 ml/kg). The doses of NMTB and ANTU used in the rat isolated perfused lung experiments were 0.38 and 0.04 mmol/kg, respectively. This dose of ANTU exceeds that which has been shown to cause pulmonary edema and depressed lung 5-HT uptake (Block and Schoen, 1981). The NMTB dose was sufficient to induce pulmonary edema at the time points examined. [‘%JThymidine incorporation assay. The incorporation of [2-‘4C]thymidine (sp act 5 1 pCi/mmol) into pulmonary DNA was measured after ip administration of NMTB according to the method of Witschi and Saheb (1974). Maximum incorporation of thymidine occurred 4 days following administration of NMTB (Penney et al.. 1985). All incorporation measurements were made at this time. Results are expressed as disintegrations per minute (dpm) per milligram DNA. LDSO determinations. Four groups of four mice each were administered a single dose of NMTB (lowest dose = 0.18 mmol/kg, 0.1 log interval between doses). Cumulative mortality and daily body weights were recorded during the 14 days following injection. The moving average interpolation method of Thompson and Weil ( 1952) was used to estimate the LDSO. Determination of5HT uptakeinto the rat isolatedperfused lung (ZPL). Rats were dosed between 5 and 10 PM with a single injection of either NMTB (0.38 mmol/kg) or ANTU (0.04 mmol/kg) 5 or 12 hr prior to surgery. Control rats received an equal volume (3 ml/kg) of the

AND TRAIGER corn oil vehicle. After rats were anesthetized with sodium pentobarbital(50 mg/kg). an IPL system was used similar to that employed by Block and Schoen (198 1). Briefly, the trachea was cannulated and the lungs ventillated via a rodent respirator (Harvard Apparatus, South Natick, MA) at a rate of 60 cycles/min with a positive end expiratory pressure of 2.5 cm HZ0 and a tidal volume of 2 ml. The abdominal aorta was then severed, the chest opened, and the pulmonary artery isolated and cannulated. Warmed (37”C), carbogen-equilibrated Kreb’s bicarbonate buffer (KBB) (pH 7.4) was then perfused through the lungs at a rate of 10 ml/min. with a perfusion pressure of approximately 11 mm Hg. The isolated, perfused lungs were then placed in a water-jacketed glass chamber that was maintained at a temperature of approximately 37°C. After the lungs had perfused for 5 min, the inflow perfusion medium was switched to 0.1 pM 5-HT in KBB. A three-way stopcock allowed for this change without interruption of flow. Perfusate was then collected on ice from 0 to 5 and 5 to 10 min after perfusion medium was changed from KBB to 0.1 PM S-HT in KBB. A l.O-ml aliquot of the 5- to lo-min perfusate sample was then added with 9 1.6 ~1 0.1 N HClO,. Samples were mixed vigorously and centrifuged at approximately 30,OOOgfor 15 min at 4”C, after which 546 ~1 of the clear supematant was added to 50 ~1 of 1.O pM 3,4dihydroxybenzylamine (DHBA) in 0. I N HClO,. After thoroughly mixing, IO0 ~1 of the mixture was analyzed by high-performance liquid chromatography with electrochemical detection (HPLC-EC) (Mefford, 1981). The fraction of 5-HT taken up by the lung was determined using the equation Percentage 5-HT uptake = 5-HTi. - 5-HTou, x 1oo 5-HT,, where 5-HT,. and 5-HT,,, represent the concentrations of 5-HT in the inflow and the outflow perfusion medium, respectively. Determination of lung S-HT and 5-HZAA content. Rat lungs (either perfused or unperfused) were trimmed, rinsed in 0.9% saline, weighed, and homogenized in 10 vol of cold 0.1 N HC104 with 100 ~1 of 200 mg/ml ascorbic acid in 10% EDTA. Homogenates were then centrifuged at 30,OOOg for 10 min at 4°C. Twenty microliters of the resultant supematant was analyzed for 5-HT and 5-HIAA by HPLC-EC (Mefford, 198 I). Determination ofplatelet SHTcontent. Rats were bled by cardiac puncture and platelet-rich plasma (PRP) was obtained by centrifuging the blood at 200g for 10 min at room temperature. 5-HT content of the PRP was determined by the method of Santagostino et al. (1983). Platelet counts were determined using the Unopette Test 5806 (Becton-Dickinson VACUTAINER Systems, Rutherford, NJ) with a Coulter Counter ZM (Coulter Electronics Inc., Hialeah, FL). Determination of lung wet weight to dry weight ratios. The method of Cross et al. (1982) was used to obtain these values. Briefly, lungs were removed, rinsed in 0.9% saline, trimmed, and blotted dry. Lungs were then dried at 105°C in an oven for approximately 12 hr before weighing.

5-HT IN NMTB-INDUCED

167

LUNG DAMAGE

TABLE 1 EFFECT OF NMTB AND ANTU ON 5-HT UPTAKE AND METABOLISM AND LUNG WET WEIGHT TO BODY WEIGHT RATIOS IN RATS”

n

Percentage 5-HT uptake

Nanomoles 5-HIAA formed

Lung wet wt x IO’/ body wt

0.90 + 0.10 0.72 k 0.03 1.03 * 0.01

5.17kO.13 7.44 f 0.48’ 7.36 5 0.42’

0.45 f 0.03 0.34 + 0.05 0.30 + 0.05

5.66 + 0.23 7.61 -t 0.30’ 6.94+0.31b

5-hr postinjection time Corn oil NMTB ANTU

10 4 5

78.6 ?c 1.37 77.7 f 1.32 77.8 + 1.07 12-hr postinjection time

Corn oil NMTB ANTU

5 5 5

81.4k2.47 65.2 f 3.57’ 68.1 k4.11’

a NMTB and ANTU were administered ip at 0.38 and 0.04 mmol/kg, respectively. Data are presented as X+ SE for groups of n rats. b Significantly higher (p < 0.05) than corresponding value for rats that received vehicle only. ’ Significantly lower (p < 0.05) than corresponding value for rats that received vehicle only. Statistical methods. Differences between two group means were compared by Student’s t test (Sokal and Rohlf, I98 1). Analysis of multigroup comparisons was carried out by one-way analysis of variance. If statistically significant differences were revealed, the group means were compared by the Student-Newman-Keuls multiple-comparison procedure (Steel and Torrie, 1980). Results from all studies were considered significant at p < 0.05. Thymidine incorporation data were initially analyzed by Bartlett’s test for homogeneity of variances and found to exhibit nonequality of variances (heteroscedasticity) (Sokal and Rohlf, 1981). Common log transformation was required to obtain equality of variances so that these data could be analyzed by the methods described above. Chemicals. [2-14C]Thymidine was obtained from ICN Chemical and Radioisotope Division (Irvine, CA). Diphenylamine free base, used in the thymidine incorporation experiments, 5-HT, 5-HIAA, and DHBA were obtained from Sigma Chemical Co. (St. Louis, MO). NMTB was synthesized as previously described (Penney ef al., 1985). ANTU was obtained from Eastman Kodak Co. (Rochester, NY). Reserpine (Serpasil) was obtained from Ciba-Geigy (Summit, NJ).

HT uptake and metabolism. The uptake of 5HT and the formation of 5-HIAA were measured 5 or 12 hr after NMTB, ANTU, or corn oil (Table 1). Control values were similar to those previously reported (Gillis et al., 1978). At both 5 and 12 hr after administration of NMTB or ANTU rats presented with respiratory distress and hydrothorax. Postperfusion lung wet weight to body weight ratios of these rats were elevated at both time points. Lungs from rats treated with NMTB or ANTU 5 hr prior to termination exhibited 5-HT uptake and 5-HIAA formation which did not differ from that exhibited by lungs from control rats. However, 5-HT uptake was significantly depressed 12 hr after NMTB or ANTU. Formation of 5-HIAA was also unchanged. The unchanged 5-HT uptake which occurred 5 hr after NMTB and concurrently with pulmonary edema may have been a reflection of the redistribution of 5-HT from RESULTS perfusate to extravascular fluid. Therefore, in Efect ofNA4TB and ANTU on 5-HT Uptake, a separate IPL experiment, we measured 5HT uptake and metabolism as well as postS-HZAA Formation, and Lung Wet perfusion lung 5-HT and 5-HIAA content 5 Weight/Body Weight Ratios hr after administration of NMTB (Table 2). The rat IPL was used to determine if prior Again, prior treatment with NMTB induced treatment with NMTB affected pulmonary 5- pulmonary edema without affecting 5-HT

168

GIBBS, JIAN-XING.

AND TRAIGER

TABLE 2 EFFECT OF

NMTB ON THE DISPOSITION

OF 5-HT

IN THE ISOLATED

PERFUSED

RAT LUNG’

Group

Percentage 5-HT uptake

Nanomoles 5-HIAA formed

Lung wet wt x IO’/ body wt

Lung 5-HT (a/lung)

Lung 5-HIAA Wlung)

Corn Oil NMTB

83.6 f 1.21 85.3 f 1.00

1.24f0.14 1.36+0.11

5.3 2 0.07 7.0+0.18b

1.47 + 0.28 1.34+0.18

0.61 + 0.04 0.70 * 0.07

a Rats were terminated 5 hr after NMTB (0.38 mmol/kg). Data are presented as X+ SE for groups of three to seven rats. b Significantly higher @ < 0.05) than corresponding value for rats that received vehicle.

uptake or metabolism. Lung content of both 5-HT and 5-HIAA determined at the end of the IO-min perfusion period was also unaffected by NMTB at this time point. It therefore appeared that the perfused 5-HT was being taken up and metabolized by the lung and was not simply being redistributed to extravascular fluid. Effect ofReserpine on 5-HT Uptake, 5-HIAA Formation, and Lung Wet Weight/Body Weight Ratios after NMTB Pretreatment of rats with reserpine at a dose that markedly reduced both lung and platelet 5-HT levels (Table 3) did not affect the depressed 5-HT uptake induced by NMTB (Table 4). In agreement with Junod (1972), reserpine alone had no effect on 5-HT uptake. Reserpine did not influence the amount of 5-HIAA formed by rat isolated perfused lungs, regardless of whether rats were pretreated with NMTB. NMTB alone did not affect 5-HIAA formation. The elevated postperfusion lung wet weight/body weight ratios resulting from NMTB were not affected by reserpine. Pretreatment with reserpine, therefore, did not influence the NMTB-induced changes in the end points examined in these experiments. Eflect of Reserpine on Weight Ratios of Lungs Not Perfused with Krebs Bugler after NMTB O’Brien et al. (1985) reported that perfusion with physiologic salt solution lessened

the sensitivity of lung wet weight/dry weight ratios as an index of injury and that lung perfusion can induce mild perivascular edema. We therefore measured lung wet weight to dry weight ratios and lung wet weight to body weight ratios from rats whose lungs were not perfused with Kreb’s buffer. We also measured these parameters in mice. Reserpine pretreatment attenuated the NMTB-induced increase in rat lung wet weight to dry weight ratios (Table 5). The ratios from rats that received reserpine-NMTB were also significantly higher than ratios from control rats. Lung wet weight to body weight ratios from rats that received NMTB, regardless of the presence or absence of reserpine, were significantly different from control values. Mice treated with reserpine prior to NMTB presented with lung weight ratios that were higher than those from control mice but lower than those from mice that received TABLE 3 EFFECT OF RESERPINE ON LUNG AND PLATELET 5-HT IN RATS’

Group

Lung 5-HT h/lung)

Platelet 5-HT Q/lop platelets)

Saline Reserpine

1.87kO.14 0.86 * 0.1 I b

0.80*0.17 0.02 f 0.0 1 b

* Reserpine was administered at 3.0 mp/kg, ip, 24 hr prior to 5-HT measurements. Data are presented as X & SE for groups of five to eight rats. b Significantly lower than values for rats that received saline (p < 0.05).

5-HT IN NMTB-INDUCED

LUNG

169

DAMAGE

TABLE 4 EFFECT OF RESERPINE ON NMTB-INDUCED CHANGES IN 5-HT UPTAKE AND METABOLISM AND LUNG WET WEIGHT TO BODY WEIGHT RATIOS IN RATS’ Pretreatment compound

Challenge compound

n

Saline Saline Reserpine Reserpine

Corn oil NMTB Corn oil NMTB

10 4 4 4

Percentage 5-HT uptake 71.0 61.5 73.9 61.1

f f * +

1.37 2.22’ 1.66 l.20b

Nanomoles 5-HIAA formed

Lung wet wt x 103/ body wt

1.05 f 0.16 0.88 f 0.11 0.58 + 0.09 0.84 + 0.07

5.48 f 0.01 7.11 kO.28’ 5.23 + 0.20 6.82kO.17’

a Reserpine was administered at 3.0 m&kg, ip, 24 hr prior to NMTB (0.38 mmol/kg). Rats were terminated 12 hr after NMTB. Data are presented asX+ SE for groups of n rats. b Significantly lower (p < 0.05) than corresponding value for rats that received vehicles only or reserpine only. ’ Significantly higher (p < 0.05) than corresponding value for rats that received vehicles only or reserpine only.

NMTB alone (Table 6). Reserpine also reduced the amount of thoracic fluid present in NMTB-treated mice. Thus reserpine pretreatment reduced but did not abolish the pulmonary edema induced by NMTB in both mice and rats.

pine alone did not induce thymidine ration into pulmonary DNA.

incorpo-

Eflect of Reserpine on Lethality Induced by Thiono-Containing Compounds The acute lethality

of NMTB

is mediated

Efect of Reserpine on NMTB-Induced Stim- by pulmonary injury (Penney et al., 1985). ulation of Thymidine Incorporation We therefore examined the effect of reserpine The incorporation of [14C]thymidine into pulmonary DNA increases in a dose-related fashion in response to NMTB administration (Penney et al., 1985). Maximal incorporation occurs 4 days after NMTB. This parameter was used as an indicator of lung injury to assess the effect of reserpine on the lung’s response to NMTB. Since the pneumotoxic response to NMTB in either the rat or the mouse is both quantitatively and qualitatively similar (Cashman et al., 1982), the mouse was used to investigate this end point. Twenty-four-hour pretreatment of mice with reserpine, 3.0 mg/kg, attenuated the NMTBinduced incorporation of [14C]thymidine into mouse pulmonary DNA (Table 7). Thymidine incorporation in mice that received reserpine + NMTB was, however, significantly greater than that which occurred in vehicle-treated mice. Reserpine pretreatment therefore did not abolish lung damage. Reser-

on NMTB-induced lethality. The 14-day ip LD50 of NMTB was determined to be 0.25 mmol/kg(95% CI, 0.23-0.28 mmol/kg). This value was not affected by doses of reserpine that are known to deplete 5-HT (Table 8). Mice pretreated with reserpine (3.0 mg/kg) and challenged with a sublethal dose of NMTB (0.18 mmol/kg) died 4-6 days after receiving the NMTB. Reserpine alone did not induce mortality at a test dose of 25.0 mg/ kg. These doses of reserpine are well below its reported LD50 of 70 mg/kg (ip, mouse) (Usdin and Efron, 1972). A 20% reduction in body weights of mice occurred 24-48 hr following administration of reserpine (3.0 mg/ kg) or the combination of reserpine plus NMTB (0.18 mmol/kg) (data not shown). Reserpine also failed to alter the survival time of mice that received 0.24 or 0.60 mmol/kg NMTB (Table 9). We also investigated reserpine’s effect on ANTU-induced lethality. The 1Cday ip LD50 of ANTU was

170

GIBBS,

JIAN-XING,

AND

TABLE EFFECT OF

n 5 9 3 8

TRAIGER

5

RESERPINEPRETREATMENT ON LUNG WEIGHT RATIOS AFTER NMTB

IN RATS"

Pretreatment compound

Challenge compound

Lung wet wt x I03/body wt

Saline Saline Reserpine Reserpine

Corn oil

4.69 + 0.10

4.66 i 0.10

NMTB

7.22 f 0.256

6.18kO.17’ 4.79 iz 0.06 5.48 t 0.08’

Corn oil

5.5o-co.14

NMTB

7.27 k 0.28’

Lung wet Wdry wt

’ Reserpine was administered at 3.0 mg/kg, ip, 24 hr prior to NMTB (0.38 mmol/kg). Rats were terminated 12 hr after NMTB. Data are presented as X+ SE for groups of n rats. b Significantly different from values for rats that received vehicles only (p < 0.05). cSignificantly different from values for rats that received vehicles only or NMTB alone (p < 0.05).

determined to be 0.20 mmol/kg (95% CI, 0.15-0.26 mmol/kg). Reserpine had no effect on the survival time of mice treated with 0.3 1 mmol/kg ANTU (Table 9). This dose of ANTU results in approximately 100% mortality which occurs approximately 27 hr after dosing and thus approximates the acute lethality of 0.24 mmol/kg NMTB from a temporal standpoint. Mice treated with either ANTU or NMTB present with similar overt toxic signs, such as respiratory distress, lethargy, and piloerection. DISCUSSION Several pieces of information are presented in this study which suggest that 5-HT is not a primary mediator in the production of lung

TABLE

injury after NMTB. First, reserpine did not affect NMTB-induced lethality. The latter has been shown to correlate with pneumotoxicity (Penney et al., 1985). Second, there is an absence of a temporal correlation between pulmonary edema after NMTB and diminished lung 5-HT uptake. The latter response has been suggested to reflect damage to lung capillary endothelium (Roth et al., 1979). Third, a dose of reserpine which markedly reduced lung and platelet 5-HT levels did not affect NMTB’s depression of 5-HT uptake. Collectively, these results imply that the role of 5-HT is not pivotal in the initiation and expression of lung injury after NMTB. Presumably the key event in this process is the production of a reactive metabolite and its subsequent covalent binding to lung macromolecules (Gottschall et al., 1985).

6

EFFECTOF RESERPINEPRETREATMENT ON LUNG WEIGHT Pretreatment compound

Challenge compound

Saline Saline Reserpine Reserpine

Corn oil NMTB Corn oil NMTB

Lung wet wt X 103/ body wt 5.72 8.58 5.64 7.40

+ 0.2 1 2 0.70’ ?I 0.21 f 0.14’

RATIOS AFI’ER NMTB

Lung wet wt/ dryM 4.76 + 0.04 6.10+-0.29’

5.00? 0.15 5.50+-0.18’

IN MICE”

Thoracic fluid (ml) 0 1.18kO.13

0 0.47 f 0.22d

n Reserpine was administered at 3.0 mg/kg, ip, 24 hr prior to NMTB (0.38 mmol/kg). Mice were terminated 12 hr after NMTB. Data are presented as X+ SE for groups of five mice. b Significantly different from values for mice that received vehicles only (p i 0.05). ’ Significantly different from values for mice that received vehicles only or NMTB alone (p < 0.05). d Significantly different from values for mice that received NMTB alone (p < 0.05).

5-HT IN NMTB-INDUCED

LUNG

171

DAMAGE

TABLE 7

imum and the injury-producing processes have transpired. Since repair reflects the sum EFFECT OF RESERPINE PRETREATMENT ON NMTBINDUCED PULMONARY [‘4C]T~~~t~~~~ INCORFQRA- total of all the events occurring by various TION IN MICE’ mechanisms that have contributed to the production of injury, our finding that removal of Pretreatment Challenge dpm ‘%/mg 5-HT’s contribution diminishes the injury ren compound compound lung DNA sponse is what one might expect. The 5-HT 12 Saline Corn oil 840 + 58 from platelets which have been recruited to 10 Saline NMTB 18,470+ 5052’ the site of damage could potentiate injury by Remrpine Corn oil 780 + 77 4 Reserpine NMTB 5,355 f 927”’ 8 attracting granulocytes (Jacob et al., 1982). The products which granulocytes release are o Resetpinewas administered at 3.0 mg/kg, ip, 24 hr prior to known mediators of injury. A contributory NMTB (0.12 mmol/kg). Data are presentedasX+ SE for groups of n mice. role for 5-HT is also supported by reserpine’s bSignificantly higher than valuesfor mice that receivedvehi- suppression of ( 1) an increase in lung wet to clesonly (pcO.05). dry weight ratios after NMTB in mice and ‘Significantly lower than valuesfor mice that receivedNMTB alone (p-cO.05). rats, (2) an increase in lung wet weight to body weight ratios in NMTB-treated mice, and (3) the amount of thoracic fluid collected from On the other hand the data suggest that 5- NMTB-treated mice. These parameters reflect the amount of extravascular fluid in the lung. HT may contribute to subsequent injurious processes, i.e., those that take place after the It thus appears that 5-HT may mediate permeability changes subsequent to direct injury that initial chemical-induced damage. Reserpine allow for fluid efflux from the pulmonary vasdiminished [ 14C]thymidine incorporation into pulmonary DNA after NMTB. Thymicular compartment. dine incorporation is a measure of repair and The rat IPL has been used to demonstrate is proportional to the amount of injury that the capacity of ANTU to impair 5-HT clearhas occurred. The assay is conducted 4 days ance (Block and Schoen, 1981). When rats after dosing, a time at which repair is at a max- from our experiments were treated with equiTABLE 8 EFFECT-OFRESERPINEPRETREATMENTON THE LDSO OF NMTB IN MICE~ Pretreatment compound

Dose (mmol/kg)

Saline (No. dead/No. dosed)

0.18 0.22 0.28 0.36

018 118 618 w

Reserpine, 1.O mg/kg (No. dead/No. dosed)

Reserpine, 3.0 mg/kg (No. dead/No. dosed)

114 214

2/8

214 414

518 418 718

LD50 (mmol/kg) (95% confidence interval) 0.25 (0.23-0.28)

0.24 (0.1 s-o.3 1)

0.25 (0.1 S-0.41)

@Pretreatment compounds were administered 24 hr prior to NMTB. Cumulative mortality was recorded during the 14 days following NMTB administration.

172

GIBBS, JIAN-XING, TABLE 9

AND TRAIGER

see high amounts of radiolabeled 5-HT in the effluent due to competition for uptake by INDUCED MORTALITY IN MICE’ platelet-released 5-HT. It has been shown that platelet counts are depressed in mice as Pretreatment Challenge compound Hr to early as 1 hr after ANTU and remain decompound death (mmol/k) pressed for at least 12 hr (Mais, 1983). InSaline NMTB (0.24) 31.4 f 1.8 creases in lung 5-HT content paralleled this Reserpine NMTB (0.24) 37.8 + 2.8 peripheral thrombocytopenia. Therefore, it Saline NMTB (0.60) 14.4 zk 1.2 appears that platelets become sequestered by Reserpine NMTB (0.60) 17.0 zk 1.3 Saline ANTU (0.31) 27.1 f 5.2 the lung, and this would allow for an elevaReserpine ANTU (0.3 1) 26.5 f 3.1 tion of local 5-HT levels. This complication surrounding the use of radiolabeled 5-HT to a Reserpine was administered at 3.0 mg/kg, ip, 24 hr assess lung injury has been pointed out by prior to NMTB or ANTU. Data are presented as X+ SE Stalcup et al. (I 982). for groups of 10 mice. There was variability in 5-HIAA formation between experiments. This is probably due to changes in the HPLC-column performance. toxic doses of NMTB or ANTU 12 hr prior The time which elapsed between successive to termination, pulmonary edema and de- series of experiments may have allowed for creased 5-HT clearance were exhibited. Five alterations in retention characteristics of the hours after rats had received the same doses column packing material. We were therefore of ANTU or NMTB, 5-HT clearance was uncareful to compare 5-HIAA data only to changed, yet the lungs of the rats were edemaother data collected in the same series of extous. The uptake mechanism for 5-HT apperiments. The lability of 5-HIAA in perchlopeared to be intact at this time point because ric acid may have also contributed to the varithe lung content of 5-HT and 5-HIAA was ability in the data. It was necessary, however, unchanged by prior treatment of rats with NMTB. Thus the perfused 5-HT was not be- to use this reagent to precipitate any proteins ing lost passively to extravascular fluid. Our prior to chromatographing the samples. In several experiments reserpine was used observation of unaltered pulmonary 5-HT as a pretreatment to deplete 5-HT. We clearance with accompanying pulmonary thought that depleting 5-HT prior to the adedema is in contrast to the findings of others ministration of the toxicant would allow us for oxygen (Block and Fisher, 1977) and to better characterize 5-HT’s role in the reANTU (Block and Schoen, 1981). Results sponse of the animal to the toxicant. Reserpresented by the latter investigators were pine did not alter the survival time of mice cited as evidence that decreased lung amine after NMTB or ANTU, did not shift the clearance may serve as an indicator of pulmoLD50 of NMTB in mice, and did not affect nary edema not yet realized. depression of lung 5-HT Our IPL experiment paradigm does differ the NMTB-induced clearance in rats. Reserpine consistently infrom that employed by others in that the 5duced a weight loss in mice of approximately HT in our experiments is not radiolabeled. 20%. While a reserpine-induced cachexia Our method of analysis quantifies the entire might have rendered the animals more susamount of 5-HT, not just the exogenous raceptible to the acute lethality of NMTB or diolabeled substrate, in the perfusate. PlateANTU, we were unable to demonstrate that lets attracted to the injured lung result in high concentrations of endogenous 5-HT in the reserpine pretreatment potentiated their toxpulmonary microvasculature. If an exoge- icity. The data collected thus suggest that 5nous solution of radiolabeled 5-HT was per- HT did not play a major role in the toxic refused through such damaged lung, one could sponses described above. EFFECT OF RESERPINE ON NMTB-

AND ANTU-

5-HT IN NMTB-INDUCED

In conclusion we think that 5-HT does not play a major role very early in the course of chemical-induced pneumotoxicity. The systtm responsible for the uptake and metabolism of 5-HT in the lung appears to be functional despite lung damage and accompanying pulmonary edema. Certain of the data do suggest that 5-HT does contribute to the toxicity induced by the above chemicals. At elevated levels 5-HT could aggravate injury by attracting granulocytes or by directly imparting permeability changes on the vasculature. ACKNOWLEDGMENTS We are grateful to Dr. Narayanan Narasimhan for his help in synthesizing the N-methylthiobenzamide, Drs. Adolph Januskiewicz and Lavonne Patton for aid with the isolated perfused lung methodology, and Dr. Douglas Denney for allowing us access to the Coulter Counter. We also thank Miss Lori McKinney and Miss Sandy Miller for typing this manuscript. This work was funded in part by K. U. Biomedical Research Support Grant RR 5606.

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