Induction of Acute Lung Injury after Intranasal Administration of Toxin Botulinum A Complex

Abstract

The inhalation of aerozolized botulinum toxin may represent a potential significant hazard to both military and civilian personnel. Since the lung is the primary target organ for inhaled toxin, the investigation reported herein was conducted to examine lung function in mice exposed to botulinum toxin A complex by intranasal route. Data includes lethality, symptomatology, measurement of respiratory function (minute ventilation, respiratory frequency, and tidal volume), and histopathology of the lungs. The clinical signs of intoxication are similar to those observed in foodborne botulism. Plethysmography revealed severe impairment of all respiratory parameters tested from 7 hours postexposure. Severe lung lesions, possibly secondary to the intoxication, were observed in mice who survived 14 days after the toxin challenge. These included intra-alveolar hemorrhage and interstitial edema. Mice immunized by the pentavalent (ABCDE) toxoid were protected against the neurotoxin (4 LD50) as revealed by the decrease of lethality and severity of nervous signs of intoxication, but not against histopathological changes in the lungs. These effects are nonspecific and require further experiments in order to specify the relationships between the pathology and the inflammatory process in the lung due to mediators such as cytokines, and possibly permanent physiological sequelae.

Introduction

Clostridium botulinum produces 7 different neurotoxins, designated A to G, which are extremely potent protein toxins. Toxin types A, B, E, and F are mainly implicated in human diseases. Foodborne botulism accounts for most botulinum intoxication cases. However, exposure to toxins A to E through aerosols or in contaminated water is seriously considered a potential threat as a warfare or bioterrorist agent (Franz et al., 1993).

Botulinum toxins inhibit the release of acetylcholine (Simpson, 1986) at neuromuscular junctions, resulting in flaccid muscle paralysis. Respiratory failure due to the paralysis of respiratory muscles is the most serious complication, resulting in death (Davis, 1993; Franz et al., 1993). These toxins also interfere with transmission at cholinergic parasympathetic terminals, producing autonomic symptoms: dry mouth and eyes, urinary retention, fluctuations of blood pressure, and impairment of autonomic control of heart rate. Recovery is slow, and the most common complaints are easy fatigue and dyspnea, although objective measurements of pulmonary function are usually normal. Many prophylactic and therapeutic countermeasures against botulism have been investigated since 1940. A new generation of botulinum vaccine is now under study: various laboratories are focused on the development of synthetic (Atassi et al., 1996; Atassi and Oshima, 1999) or recombinant vaccines against botulinum neurotoxins (Clayton et al., 1995; Gelzleichter et al., 1999; Clayton and Middlebrook, 2000). In this context, a pentavalent botulinum toxoid (PBT) directed against serotypes A to E (Siegel, 1988) is proposed to immunize specific at-risk populations, i.e., scientists in contact with botulinum toxins, and armed forces, which may be subjected to weaponized forms of the toxin. Inhalation appears to be the most likely route of exposure for the toxin if used as a terrorist weapon or warfare agent. However, this kind of poisoning seems to be particular. In testing the efficacy of vaccination against inhaled ricin in mouse (DaSilva et al., 2003), some remaining lesions in lungs were noticed even though the animals were well protected from death.

The present study was conducted in order to examine whether botulinum toxin A complex, administered by intranasal route in mice, was able to induce lung alterations even if the animals were protected against the neurotoxic effects.

Materials and Methods

Animals

Six-week-old male BALB/C mice, weighing 20–25 g at the time of exposure, were obtained from Charles River (Saint Aubain-les-Elbeuf, France). The animals were acclimatized to the laboratory conditions for at least 1 week before the experiment. They were housed 10 per cage under a 12:12-hour light/dark cycle (0600–1800 hours lights on). Food and water were available ad libitum.

The principles of Laboratory Animal Care were followed in all experiments. The experimental design of the study was approved by our institute’s Ethical Commitee.

Isolation of the Toxin A Complex

Type A neurotoxin was produced in cultures as BoNT/A-complex, a protein containing the type A neurotoxin and 6–7 nontoxic proteins. These nontoxic proteins have hemagglu-tinating activity and stabilize the neurotoxin. BoNT/A complex has been isolated from the bacterial Clostridium botulinum type A NCTC 2916 strain, according to a previously described extraction protocol (Malizio et al., 2000).

The culture was incubated for 24 hours at 37°C in anaerobic condition without any agitation in glass bottles, each containing 0.5 L TGY medium. The TGY is a broth nutritive medium containing glucose and yeast extract to support the growth of Clostridium botulinum.

It was then acidified by H₂SO₄ to pH 3.5. A heavy precipitate settled in 2–3 hours. It was collected by centrifugation (12.000 × g, 20 minutes, 4°C), washed twice with water, homogenized, and extracted with 0.05 M Na₂HPO₄/NaH₂PO₄ buffer pH 6. The solution was saturated with ammonium sulfate (35.1 g/100 ml) and stored at 4°C for 3 hours. The new precipitate was recovered by centrifugation (12,000 × g, 20 minutes, 4°C), dissolved in 0.05 M citrate buffer pH 5.5, and then dialyzed against 1 L of the same buffer for at least 18 hours at 4°C. The molecular cutoff of the dialysis tubing is 1 kDa. The solution was then centrifuged (12.000 × g, 20 minutes, 4°C) to remove insoluble material. The supernatant was loaded on a Q Sepharose Fast Flow column (25 ml, flow rate 5 ml/minute) equilibrated previously with 0.05 M citrate buffer (pH 5.5). The column was washed with the same buffer, and the collected toxin solution was saturated with solid ammonium sulfate (35.1 g/100 ml) and stored at 4°C for 3 hours. Then, the precipitate was processed as above and the supernatant was applied on a Sephacryl S-300 HR column (300 ml, flow rate 1 ml/minute), equilibrated, and then eluted with a 0.05 M citrate, 0.15 M NaCl buffer at pH 5.5. The second isolated peak contains the toxin. The toxin solution was concentrated at 20 μg/ml by ultrafiltration (Centripep Amicon 50,000 MWCO).

Toxic Activity

Mice were slightly anesthetized by an intraperitoneal injection of 100 mg/kg of ketamine. They were injected with the toxin solution into 1 nostril, using an automatic pipette (Gilson Pipetman P200, Rainin Instrument CO, Woburn, MA) fitted with a gel-loading pipette tip (Fisher Scientific, Pittsburgh, PA). The injected volume was equal to 1 μl of botulinum toxin solution per gram of body weight. Five groups of 10 mice were exposed to the botulinum complex A (from 10 to 30 ng/kg intranasal) and observed for mortality/clinical signs for up to 14 days postexposure. A probit analysis program was used to generate the dose–response curve and estimate the LD50.

Toxic Activity in Immunized Animals

The animals were divided into 2 groups: one (control; n = 10) receiving a saline solution as pretreatment and a second (vaccine group; n = 10) receiving a vaccine according the protocol shown in Figure 1. The vaccine was a Botulinum pentavalent (ABCDE) toxoid purchased from the Michigan Department of Public Health (MDPH). The product contains formalin-inactivated botulinum toxins of types A, B, C, D, and E, absorbed into aluminium phosphate. Each vial contains 0.022% formaldehyde and 1:10,000 thimerosal as preservative. Thirty days after the first injection of vaccine, animals received 4LD50 of BoNT/A complex in the same conditions as previously described for the evaluation of toxic activity.

An additional group of mice (n = 10), receiving the vaccine, was used to assess immune status. Blood samples from these animals were collected from the retro-orbital sinus, under anesthesia (ketamine-diazepam mixture), at day 0, day 7, day 15, and day 30 to measure antibody concentration.

Antibody Titration

Immune antibody response was determined by an enzyme-linked immunosorbent assay (ELISA) in 6-well microtiter plates. Preparation of inactivated toxin was obtained from a sample of purified botulinum toxin A inactivated by formalin (1%) and NaCl (0.85%). After a 2-day incubation at 4°C, the solution was dialyzed against 1 L of phosphate salt buffer pH 7.2 at 4°C for 1 day. Primary mouse antitoxin antibodies were detected with peroxidase-labeled anti-mouse IgG secondary antibody reagents (Interchim, France). The specificity of IgG was determined by the manufacture against the purified immunoglobulin. Inactivated botulinum toxin A was adsorbed onto the wells of the plates by incubation (100 ng/100 μl) overnight at room temperature. Plasma samples (1:10 dilution) were incubated in the coated plates to select toxin-specific antibodies. Dilutions were made in PBS containing bovine serum albumin (1% w/v) and Tween 20 (0.5% v/v). Each antibody layer, primary and secondary, was incubated in the plates for 1 hour at 37°C. After that, the plates were washed 3 times with PBS containing 0.05% Tween 20. Bound immunoglobulins were detected using anti-mouse immunoglobulins labeled with horseradish peroxidase enzyme (HRPO) (Interchim, France). When incubated with ABTS (azinobis-ethylbenzothiozoline sulphonic acid; Sigma) in 0.2 M citrate/phosphate buffer pH 4.3 containing H₂O₂ (0.01% v/v), a colored product was formed. The amount of mouse anti-toxin IgG was determined spectrophotometrically by the measurement of the intensity of color at 414 nm with a microplate reader. Nonspecific absorbance was eliminated by subtracting the absorbance recorded from the pooled sera of naive mice. Results were expressed as optical density (O.D.).

Signs and Symptoms

The clinical observation started at the end of the slight anesthesia status. Signs and symptoms were recorded at 2, 5, 7, 24, and 48 hours, and 7 and 15 days after toxin exposure. They included behavioral changes, locomotor activity, breathing rate, and ophthalmic status. For each of them, 3 levels of severity were defined and the symptoms were classified according to the scoring scale given in Table 1.

Breathing Electrodiagnostic

Respiratory function was assessed by the measurement of respiratory frequency, tidal volume, and minute ventilation. Mice were placed in a modified Batelle tube system. This equipment allowed flow-breathing pattern monitoring of 8 restrained animals simultaneously. A calibrated pneu-motachograph (Fleisch No. 0000, Richmond, VA, USA) and a differential pressure transducer (DP-45, Validyne, North-bridge, CA, USA) were connected to the upper part of each plethysmograph. The signal was amplified and digitized (Dell Computer Corporation, CA, USA) with an input/output AS2 card at a sampling rate of 250 HZ Software (HEM 2.1 Notocord, Croissy sur Seine, France). Data files were imported into a worksheet (Excel 5.0, Microsoft Corp., CA, USA), and the mean ± SD of all the variables, for a given time, were automatically processed.

Respiratory function was measured during 15-minute periods. Recordings were made the day before the exposure (control values) and, together with clinical observations, at 2, 5, 7, and 24 hours following the toxin challenge.

Histology

Fourteen days after toxin exposure, all surviving animals were humanely euthanized, and the lungs were removed. The tissues were fixed in formaldehyde (10%) at room temperature for a period of not less than 48 hours. After this time, each lung was trimmed to produce representative blocks from both left and right lobes, sections (6 μm) were cut from paraffin blocks and routinely stained with Harris’s hematoxylin and eosin. Histological sections were examined without knowledge of treatment, by a pathologist.

Data Analysis

Significant differences in the clinical signs scores from the control values (before exposure) on the corresponding times for each experimental group were analysed by the nonparametric paired Wilcoxon’s t-test. For each respiratory parameter in each experimental group, statistical differences between basal values and those measured at 2 hours, 5 hours, 7 hours, and 24 hours were assessed using the same test.

Results

Symptoms and Lethality

The LD50 was found to be 21.1 ng/kg with a confidence interval at 95% (13.9–32.5 ng/kg; Table 3). Up to 17.3 ng/kg, symptoms generally developed 48 hours after nasal deposition (Table 2). The onset and the severity of the symptoms together with the number of affected animals were dose-dependent. Symptoms (score 1, Table 1) typically included prostration, inactivity, piloerection, polypsnea, a refusal to eat or drink, and ptosis in some cases. The animals remained in this state until death or, more often, returned to normal behavior on day 15. Doses between 17.3 and 30 ng/kg induced more severe dose-dependent signs of intoxication (score 2). Neurologic symptoms first concerned weakness of the cranial muscles progressively generalizing to the whole musculature and affecting locomotion. Neurophtalmological signs, anorexia, and labored respiration (bradypnea) were observed 48 hours after exposure. In severe cases (4LD50) the incubation period was as short as 7 hours.

Shortly after the onset of the initial symptoms described above, weakness progressed in a paralysis of the trunk musculature (including the muscles of respiration) and the limbs. Death was preceded by a severe respiratory depression. Indeed, minute ventilation measured at 2 and 5 hours after toxin exposure did not differ from control values (Figure 3a). However, 7 hours after toxin exposure, there was a significant and dose-dependent decrease of minute ventilation. This effect was especially noticed in animals receiving the highest dose of the toxin (4LD50): 26.4 ± 13.1 versus 37.1 ± 8 ml/min. Twenty-four hours following toxin administration, the minute ventilation was greatly decreased: 5.2 ± 2 ml/min versus 52.3 ± 16.9 ml/min. This effect, 24-hour postchallenge, was correlated with a significant decrease of tidal volume (Figure 3b) and respiratory frequency (Figure 3c) by 36.6 ± 11 and 41.6 ± 18.5%, respectively, compared to the reference values.

Death occurred progressively (Table 3): at a dose of 4LD50, 20% of the animals died within 7 hours after toxin challenge. This percentage reached 50% within 24 hours and jumped to 100% over a 48-hour postexposure period.

Symptoms in Protected Animals

One week after the first injection, antitoxin IgG was detected in plasma, the mean optical density (O.D.) being 0.23 ± 0.12 (Figure 2) at day 15. one week after the first booster, the amount of IgG was enhanced by 260% (0.61 ± 0.26). At day 30, (1 day prior to botulinum neurotoxin challenge) the levels of O.D. had increased again to reach 1.4 ± 0.35.

Mice immunized by PBT vaccine and challenged with botulinum neurotoxin A (4 LD 50) were free of symptoms during the entire observation period (Table 2), excepting 1 animal showing a significant piloerection at 7 hours. No alterations of the measured respiratory parameters were detected, at least during the period considered (Figure 3). No fatal case was noticed prior to scheduled euthanasia (over a 14-day postex-posure period, Table 3).

Histology

During autopsy, visual observation revealed a significant inflammation of lung tissue. Examination of PBT-treated mice at day 14 (Figure 5) and unprotected mice that survived (Figure 6) 24 hours after exposure to toxins showed severe histopathological alterations in the lung, compared to healthy animals (Figure 4). Staining with hematoxylin and eosin revealed a thickening of the alveolar septa and perivascular areas together with a generalized spreading interstitial edema and a moderate intra-alveola/intrabronchiola hemorrhage (Figures 5 and 6). Signs of inflammation were noticed in the interstitial tissues with a marked proliferation of the macrophage cells.

Discussion

There is little reference in the literature to the cellular effects or mode action that toxins may have in the lung. No findings relevant to the effects of botulinum toxin on the respiratory tract of humans are available, because such occurrence cannot take place under normal conditions. Only 3 cases of laboratory intoxications caused by inhalation of dust that contained botulinum toxin A were reported. Very minute amounts of toxin were apparently sufficient to trigger the classical disease (Holzer, 1962) before the admittance of the patients to the hospital, where they received an antitoxin serum. They were discharged on the 9th day. The report only mentioned a subsequent slow convalescence, without further description.

Some animal experiments examined botulism by inhalation (Franz et al., 1993; Gelzleichter et al., 1999; DaSilva et al., 2003). Rhesus monkeys exposed to an approximately 2000-mouse intraperitoneal lethal dose (50 ng/kg) of aerosolized liquid botulinum toxin (Franz et al., 1993) died 2–4 days after exposure to the toxin. Clinical signs included intermittent ptosis, severe weakness, mouth breathing, serous nasal discharge, rales, salivation, and dyspnea before death. No histological change directly attributable to botulinum toxin was observed. However, mild-to-moderate subacute submucosal tracheitis, possibly secondary to the intoxication, was noticed in 2 of the 4 animals that died (Franz et al., 1993). In our experiment, signs of intoxication were rather similar. They first included piloerection and ptosis 7 hours after toxin exposure. This last symptom was generally attributed to the symmetrical cranial nerve impairment affecting the bulbar musculature resulting from a toxin-induced blockade of the voluntary motor and autonomic cholinergic junctions (Habermann and Dreyer, 1986). Then, severe muscular weakness, dysphagia, and dyspnea were observed in all the mice 24 hours after challenge. Death occurred 7 hours to 1 week after the toxin deposit.

Ventilatory failure and dyspnea due to the paralysis of respiratory muscles are usually responsible for death in botulinum intoxication in humans (Tacket and Rogawski, 1989) and characterise the ultimate symptom in the progress of pathology. Apart from clinical observations, no data were available for the evaluation of respiratory function after inhalation of botulinum toxins. In our experiment, plethysmo-graphic evaluation revealed a significant modification 7 hours after toxin exposure, although clinical examination was usually normal. Twenty-four hours after the intoxication, all pneumophysiological parameters measured (minute ventilation, tidal volume, and frequency) were affected. At this state of the disease, dyspnea was clinically observed and a few animals were already dead. These observations are in good accord with the fact that botulinum toxin A produces a more severe disease in terms of skeletal muscle weakness and need for ventilatory support.

Numerous studies have documented the ability of pentavalent botulinum toxoid (PBT) to induce protection against lethal effects (Gelzleichter et al., 1999). Our data suggest that, with regard to intranasal exposure, the antitoxin titer was sufficient to prevent all signs of intoxication when animals received 4LD50. No modifications of respiratory parameters were recorded in the PBT-treated group, suggesting that neurotransmission (mainly cholinergic) was fully protected. In this context, the recording of pneumophysiological parameters may be useful to assess immunotherapy against botulism as shown in many infectious models (De Hennezel et al., 2001).

It seems that many inhaled toxins could induce inflammatory-immune processes (Paddle, 2003) as a patho-physiological lung defense response. In the present experiment, animals exposed to the BoNT/A complex showed pulmonary histological changes with edematous lung injury. These data, together with the fact that lung pathology was also present in animals protected against neurological expression of botulism by vaccine, could suggest inflammatory processes independent of the toxic agent. Inhaled toxicants frequently induce an inflammatory response in the lungs. Such alterations were particulary observed after ricin (Rippy et al., 1991) or abrin (Griffiths et al., 1995a, 1995b) inhalation. It is well known that a number of cytokines, such as tumor necrosis factor (TNFα), are produced in response to many stimulatory agents (Van Deuren et al., 1992). The mediators are capable of interacting synergistically to modulate the intensity of the inflammatory response. As an example, adult respiratory distress syndrome (ARDS), characterized in part by severe acute hypoxemia and pulmonary edema, shows high levels of several cytokines, including TNFα, measured in bronchoalveolar lavage fluid (Suter et al., 1992). TNFα potentiates adhesion and activation of endothelial cell monolayers of polymorphonuclear neutrophils (PMN), increasing the monolayer permeability (Gibbs et al., 1990). TNFα may also directly increase pulmonary vascular permeability (Horvath et al., 1988). A previous study showed that ricin induces the release of TNFα and IL-1β by human peripheral blood mononuclear cells (Licastro et al., 1993). More recently, it was shown that ricin transcriptionally activated a large number of inflammatory mediators (DaSilva et al., 2003). This activation correlated with the increase in total protein content in bronchoalveolar lavages, suggesting increased permeability of the air–blood barrier and cytotoxicity. However, further investigation is required to determine whether there is a direct cytotoxic action of the toxin on lung cells or the recognition of the toxin as a foreign agent and the initiation of an immune-defense response. From the present investigation, a strong inflammatory response in the lungs seems to be of great consideration in botulinum toxin inhalation. A recent publication that investigated the toxicity caused by intranasal administration of Staphylococcal Enterotoxin B also reported pulmonary edema and bronchopneumonia (Savransky et al., 2003). Our histological data correlates well with this assumption.

In conclusion, the present data indicates that botulinum toxin administered by the intranasal route induces lung lesions in addition to its neurotoxic action. This pneumopathology corroborates the previous findings with ricin and staphylococal enterotoxin B. However, the relationship between the histological and physiological data needs further investigations in order to determine whether the observed effects are mainly due to the BoNT/A complex or are also shared by free BoNT/A. Futhermore, the long-term influence on pulmonary function during recovery must also be investigated.