Involvement of capsaicin-sensitive nerves in paraquat-induced mortality

Chemico-Biological Interactions 116 (1998) 93–103

Involvement of capsaicin-sensitive nerves inparaquat-induced mortality

Luigi Atzori a,*, Betty Cannas a, Tinuccia Dettori a, Maria Dore a,Caterina Montaldo a, Giancarlo Ugazio b, Luigi Congiu a

a Department of Toxicology sect. General Pathology, Uni6ersity of Cagliari, Via Porcell 4,09100 Cagliari, Italy

b Department of Experimental Medicine and Oncology sect. En6ironmental Pathology,Uni6ersity of Turin, Turin, Italy

Received 16 September 1997; accepted 24 August 1998

Abstract

Paraquat (PQ), a broad spectrum herbicide, produces severe lung inflammation andnecrosis resulting in pulmonary fibrosis and respiratory failure. Tachykinins are peptidesreleased by sensory C fibers and have the ability of influencing respiratory functions andcellular proliferation. To examine whether the damage caused by PQ involves tachykinins,rats were depleted in their content of tachykinins by systemic treatment with capsaicin priorto PQ exposure. The animal subjected to this treatment showed a 3-fold higher viabilitycompared to those treated with PQ alone (75 vs 27%). Depletion of reduced glutathione(GSH) is associated with oxidative stress produced by reactive oxygen intermediates duringPQ metabolism. This is considered to be critical in the pathogenesis of lung damage by PQ.PQ treatment induced a significant depletion of GSH during the first days and a similareffect was also observed in the group of capsaicin-pretreated rats. Four weeks after PQtreatment the levels of GSH were similar to controls in rat pretreated or not with capsaicinplus PQ. This may indicate that the reduced levels of GSH may be associated to the toxicityobserved in the acute phase, but not of importance in the final PQ-induced mortality.Neutral endopeptidase (NEP) is an enzyme considered to be critical in controlling the levelsof tachykinins. Exposure of crude membrane preparations of rat lung to PQ resulted in a

* Corresponding author. Tel.: +39 70 6758390; fax: +39 70 657254; e-mail: [email protected]

0009-2797/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved.

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L. Atzori et al. / Chemico-Biological Interactions 116 (1998) 93–10394

dose-dependent inhibition of NEP activity. Since NEP inactivation may occur in lungfollowing a PQ exposure in vivo, the results indicate that during PQ intoxication a moresustained activity of tachykinins may be present, producing effects such as cell proliferation,fluid extravasation and bronchoconstriction. In conclusion, this finding supports the hypoth-esis that neuropeptides released from capsaicin-sensitive nerves could be involved in themodulation of PQ-induced lung damage. © 1998 Elsevier Science Ireland Ltd. All rightsreserved.

Keywords: Capsaicin; Glutathione; Neutral endopeptidase; Paraquat; Tachykinins

1. Introduction

Numerous toxic substances, such as bleomycin, reactive oxygen species, asbestos,butylated hydroxytoluene, ionizing radiations and paraquat (PQ), produce animalpulmonary fibrosis [1]. Among these agents, PQ is one of the most widely studiedchemicals for the development of pulmonary fibrosis in response to acute lunginjury. A single injection of PQ in rats induces a model of interstitial fibrosis similarto the one that occurs in humans. It is thought that the general events that inducePQ fibrosis include the initial damage such as membrane destruction, developmentof alveolitis, and then excessive accumulation of collagen in the lung.

PQ is a quaternary ammonium bipyridyl compound (1,1%-dimethyl-4,4%-bipyridil-ium dichloride) that is widely used as a broad spectrum contact herbicide. PQ issafe when used under the appropriate conditions, however, numerous cases ofaccidental and intentional poisoning have been reported [2]. Systemic administra-tion of this agent initiates a progression of degenerative and potentially lethallesions with intra-alveolar fibrosis which is the most characteristic feature of PQintoxication. The mechanism of PQ lung toxicity has still not been clarified eventhough cyclic single electron reduction/oxidation of the parent molecule is consid-ered to be a critical event [3]. The pathogenic effects of PQ are based on theformation of oxygen radicals followed by a cascade reaction resulting in thedestruction of the cell integrity.

The pulmonary toxicity of PQ has been described as occurring in two phases.The first one is characterized by damage and destruction of alveolar epithelial cells,edema, hemorrhage resulting in a complete denuded alveolar basement membrane.This destructive phase occurs immediately after the acute exposure to PQ and it isassociated with pulmonary edema and, frequently, the severity of the damage willresult in death. The following phase, starting after a few days, is characterized byinfiltration of miofibroblasts into the alveolar area and subsequent differentiation infibroblasts with the production of collagen, resulting in development of intra-alve-olar fibrosis.

It has been shown that PQ accumulates selectively in type-II alveolar cells. Atthese levels, the primary reaction of PQ seems to be a cycle from the oxidized to thereduced form. This oxidative stress, especially in in vitro models, may destroy

L. Atzori et al. / Chemico-Biological Interactions 116 (1998) 93–103 95

proteins, nucleic acid and polysaccharides and cause peroxidation of membranelipids, leading to cell death [3]. However, several observations indicate that this maynot be the only cause of PQ toxicity [4].

Since the pathophysiological factors, leading to PQ-induced pulmonary injury,are not entirely clear, we hypothesize that lung sensory nerves, particularly lung Cfibers, may play a role in the mechanism of damage and repair.

Neuropeptides have been shown to be located in sensory nerves under or withinthe lining of the epithelium in both human and rodents [5,6], putting them in astrategic location for the repair of airway epithelium. Conditions damaging theairway epithelium, as in the course of PQ intoxication, may release neuropeptidesinto the site of injury.

These neuropeptides, also called tachykinins, such as substance P and neu-rokinins A and B, exist in the C-fibers, also called unmyelinated fibers. In the lung,these nerve fibers are distributed in the epithelium, smooth muscle cells and bloodvessels of many mammals [7–9]. These peptides are known to have variousfunctions such as control of the bronchial tone and the vascular permeability,induction of chemotaxis and modulation of neutrophil functions, modulation of theimmune response, increase in mucus secretion, as well as stimulate growth ofsmooth muscle cells, fibroblasts, epithelial and endothelial cells [10]. All thesefunctions are expected to promote a correct repair of the cellular damage.

Levels of tachykinins are critically controlled by the enzyme neutral endopepti-dase (EC 3.4.24.11) (NEP), which cleaves and inactives many pro-inflammatorypeptides. An increased release of neuropeptides could also contribute to a pro-longed and more severe inflammatory response [11]. Exposure to oxidants has beenreported to inhibits lung NEP, so potentiating the tachykinins effect [12].

In the present study, we have investigated whether the damage caused by PQcould involve neuropeptides and found that removal of them by treating rats duringthe neonatal period with capsaicin reduced the mortality induced by PQ, withoutaffecting the glutathione (GSH) levels in the lung. Moreover, PQ inhibits theactivity of the enzyme NEP which modulates the neuropeptide-induced response.Our results indicate a possible involvement of tachykinins in PQ-induced lungdamage.

2. Materials and methods

All procedures used in this study were approved by the University of CagliariAnimal Research Committee. Sprague–Dawley rats (280–380 g) obtained fromDitta Morini (Reggio Emilia, Italy) were housed under controlled temperature(24°C) and received food and water ad libitum. Chemicals were obtained fromSigma Chemical (Milano, Italia).

To determine whether systemic treatment with capsacin would affect PQ-inducedmortality 64 rats were used with PQ alone, 52 rats pretreated with capsaicin beforereceiving PQ and 28 rats as control. In order to deplete the animals of their contentin tachykinins, at the nerve terminal level, the systemic treatment with a high dose


of capsaicin during the neonatal period was used. Twenty-four hour old rats weretreated subcutaneously with 50 mg/kg of capsaicin dissolved in 2% ethanol, 18%Tween 80 and 80% normal saline in order to deplete their content of neuropeptides.During the treatment the rats were slightly anesthetized with ether and thenexposed to pure oxygen for 5 min in order to reduce the temporary respiratorydistress caused by capsaicin administration. Prior to treatment with PQ, the animalswere tested for tachykinin depletion by dropping 50 m l of a 0.015% solution ofcapsaicin on one eye. Only rats not responding to the irritant were then used for theexperiments. After 10 weeks of capsaicin treatment the animals were exposed to PQ(30 mg/kg, intraperitoneally). Survival curve was compared to the group of ratsreceiving PQ alone.

After pentobarbital (50 mg/kg) anesthesia, rats were sacrificed and lungs pre-pared for GSH assay. Lung GSH content was determined spectrophotometrically inthe supernatant obtained after homogenizing the lungs in trichloracetic acid (6.5%)and then centrifuging for 10 min at 12000×g using Ellman’s reagent [13].

NEP was determined according to the procedure of Orlowski and Wilk [14],modified by Haxhiu-Poskurica et al. [15]. Crude preparation from rat lungs wasused as a source of NEP. The reaction mixture consisted of tissue homogenate (1:10Tris–HCl, pH 7.4), 1.25 mM substrate (glutaryl-alanine-alanine-phenylalanine-4-methoxy-2-naphthylamide), aminopeptidase M, and 50 mM Tris–HCl buffer, pH7.4. The mixture was incubated at 37°C for 60 min. Reactions were stopped by theaddition of 1 ml of 10% trichloracetic acid and then 1 ml of 0.005% (w/v) fastgarnet. Under these conditions, the hydrolysis of the substrate was proportional tothe enzyme concentration [14]. The amount of 2-naphtylamine produced during thereaction was determined by a spectrophotometer at 530 nm. The specificity of theNEP activity was assessed by carrying out the reaction in the presence of thiorphan(1 mM), a NEP inhibitor. Protein concentrations were determined by the method ofPeterson [16].

The effect of PQ and capsaicin on lung morphology was assessed by lightmicroscopy. Aliquots of the lung were fixed in buffered formalin solution, thenembedded in paraffin and processed for light microscopy. Sections were stainedwith hematoxylin-eosin.

Values shown are mean9S.E.M. The significance of differences was calculatedusing Student’s t-test. Differences were considered significant when 0.05 probabilitylevels were \0.05.

3. Results

The effect of intraperitoneally administered PQ (30 mg/kg) on rat viability isshown in Fig. 1. This concentration of PQ induces a marked mortality during thefirst 4 days with a slow and constant increase over the period of the 4 week study.Tachykinin-depleted rats, treated with PQ, showed a reduction in mortality at theend of the observation compared to rats treated with PQ alone (Fig. 1).


Lungs from PQ-treated rats with either capsaicin or saline were investigated forhistologic abnormalities after 4 weeks. Control animals, which were treated withsaline but not with PQ, had no histologic abnormalities in their lung (Fig. 2a).Lungs of PQ-treated rats showed thickening of the alveolar capillary membrane,shedding bronchiolar epithelium, and diffuse alveolar damage characterized byalveolar and interstitial edema as well as extensive interstitial thickening (Fig. 2b).In contrast animal pretreated with capsaicin before PQ showed minor lung lesions(Fig. 2d), compared to PQ alone.

Since GSH is known to play an important role in protecting cells from oxidant-induced tissue injury, the levels of this tripeptide in the lungs of animals treatedwith PQ were measured. The results in Fig. 3 indicate that pulmonary GSH levelsof rats treated with PQ were significantly decreased after 24 and 48 h compared tothat of the control. Then, the levels were normalized, and 4 weeks after PQintoxication, the content of GSH was similar to that of the control (Fig. 3). Also inthe group of tachykinin-depleted rats treated with PQ, the levels of GSH showed asignificant depletion after 24 h, but also in this group, after 4 weeks, GSH levelsreturned to control values (Fig. 4). Thus, no differences were observed between PQalone or capsaicin-pretreated plus PQ rats regarding the GSH levels.

Although it has been shown in vitro that PQ may induce peroxidation ofmembrane lipids and this effect may be important in the pathogenesis of PQtoxicity, the levels of malonaldehyde, an index of lipid peroxidation, in the lung ofrats treated with PQ did not show any increase (data not shown).

Degradation of tachykinins is controlled by the activity of the enzyme NEP. Itsactivity in washed crude membrane preparation was examined in the presence ofPQ. After exposure to various concentrations of PQ, NEP activity was inhibited ina dose-dependent manner as shown in Fig. 5.

Fig. 1. Effect of capsaicin pretreatment on PQ (30 mg/kg) induced mortality. PQ alone (——); Capsaicinpretreated plus PQ (- - -).


Fig. 2. Light micrograph of peripheral lung tissue 4 weeks after saline administration (a), PQ (b),capsaicin alone (c) and capsaicin-pretreated plus PQ (d). Hematoxylin and eosin stain. Originalmagnification: ×200.


Fig. 2. (Continued)

4. Discussion

A number of molecules have been implicated as modulating factors in lungdamage and repair. Recent evidence have indicated a role for neuropeptides,


Fig. 3. Time-course of GSH concentrations in rat lung after PQ treatment (30 mg/kg). Control (), PQalone ( ). Number of animals for each group was between four and ten. The results are expressed asmeans9S.E.M. * PB0.05.

derived from airway sensory C-fiber nerves located in the airway epithelium, indifferent cellular functions [17–20].

This study provides evidence for a possible role of neuropeptide release bycapsaicin sensitive nerve endings in the development of lung damage induced byPQ. This hypothesis is supported by the fact that capsaicin treatment of neonatalrats, which depletes the animal of the neuropeptides content, significantly protectedthem from PQ mortality.

Fig. 4. Lungs concentrations of GSH at 24 h and 28 days after PQ treatment (30 mg/kg), in ratspretreated with capsaicin or not. Saline (empty box), PQ (horizontal crossed box), capsaicin (dottedbox), capsaicin-pretreated plus PQ (vertical crossed box). Number of animals for each group wasbetween four and ten. The results are expressed as mean9S.E.M. * PB0.05.


Fig. 5. NEP activity measured in a washed crude membrane preparation from rat lung after incubation(60 min) with different PQ concentrations. Control alone (C− ); positive control with thiorphan 1 mM(C+ ). The results are expressed as mean9S.E.M. from three experiments in duplicate. * PB0.05.

The role for tachykinins in PQ-induced damage is not clear. It could bespeculated that tachykinins released from sensory nerves endings in response tocell toxicity and damage may contribute to the inflammatory response and par-ticipate with other mitogens in the proliferation of myofibroblasts and in thisway initiating the repair response. It is known that tachykinins contribute towound healing [18] and stimulate the growth of fibroblasts and epithelial cells[17,21].

NEP is a cell membrane-bound enzyme that inactivates tachykinins [15]. Dif-ferent experimental models of lung damage based on oxidant production areassociated or potentiated by inhibition of NEP [22,23]. Studies in vitro hadshown that inhibition of NEP potentiates the effects of tachykinins on cellproliferation [24]. Therefore, since PQ is capable of inhibiting the activity ofNEP, it is possible that during PQ-intoxication the effects of tachykinins aremore pronounced. Lung tissue acquires much higher concentrations of PQ thandoes the rest of the body [25]. After human exposure, in the plasma are reportedconcentration of PQ superior to 5 mM and in the urine superior to 0.5 mM, 20h after the ingestion [26]. It is difficult to evaluate the concentration of PQ atthe level of the target cells in the lung, but it has to be much higher than theone observed in the plasma. In our experiments in vitro to asses the effect of PQon NEP activity the concentration used were higher than the one observed in theplasma. We think these can be realistic concentrations in the epithelial cells ofthe lung.


A possible mechanism in capsaicin induced protection is that neonatal treatmentmay render the adult rat more resistant to the oxidative stress induced by PQ. Ourobservations do not indicate that the effect of capsaicin is dependent on changes inthe GSH content in the lung.

In vitro studies indicate that tachykinins have the capacity to cause activation ofmesenchimal cells and in this way contribute to the genesis of lung damage [20,26].The reported data that tachykinins may influence fibroblasts proliferation supportthe hypothesis that they may be of importance in the pathogenesis of subepithelialfibrosis of asthma and intra-alveolar fibrosis during interstitial lung disease.

In conclusion, our data support the hypothesis that lung tachykinins releasedfrom C fibers may have a role in PQ-induced mortality. An overexpression of theseneuropeptides caused by the NEP inactivation may be responsible for the chronicdamage, fibrosis and respiratory failure. Available and specific inhibitors of tack-ykinin receptors will help to confirm this hypothesis.

Acknowledgements

Supported by grant from MURST (ex40%), Project ‘Patologia da Tossici Ambi-entali e Occupazionali’, and MURST (60%) Italy.

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Involvement of capsaicin-sensitive nerves in paraquat-induced mortality

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