ToxSci Advance Access originally published online on March 28, 2008
Toxicological Sciences 2008 104(1):198-209; doi:10.1093/toxsci/kfn061
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Sulfur-Containing Malodorant Vapors Enhance Responsiveness to the Sensory Irritant Capsaicin
Toxicology Program, Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, Connecticut 06269
1 To whom correspondence should be addressed at Toxicology Program, Department of Pharmaceutical Sciences, 69 N. Eagleville Rd, U-3092, University of Connecticut, Storrs, CT 06269-3092. Fax: (860) 486-5792. E-mail: john.morris{at}uconn.edu.
Received February 7, 2008; accepted March 13, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The nose is innervated with both odor responsive olfactory (cranial nerve I) and irritant responsive trigeminal (cranial nerve V) nerves. The nature and extent of any interactions between these two nerves is poorly understood. The aim of the current study was to determine if two sulfur-containing malodorants, ethyl sulfide and t-butyl sulfide, modulated responsiveness to the trigeminal C fiber stimulant capsaicin using female C57Bl/6J mice as an experimental model. Cessation or marked slowing of flow at the onset of each expiration (termed braking) was used as a biomarker for trigeminal nerve stimulation. Aerosolized capsaicin solution (100 µg/ml) increased the time of braking from baseline levels of 8 ms to an average of 69 ms. At an exposure concentration of 100 ppm the malodorants induced only a minimal time of braking response (< 35 ms); the time of braking response in animals exposed to either malodorant plus capsaicin was 2.5-fold greater than in animals exposed to capsaicin alone (p < 0.01). In a subsequent experiment the time of breaking response to capsaicin was doubled (281 vs. 146 ms) by concomitant exposure to a no effect level of ethyl sulfide (11 ppm) and the modulation of capsaicin responsiveness was nearly abolished by inclusion of the adenosine antagonist theophylline in the aerosol formulation (time of braking 184 ms, p
0.05 compared with capsaicin alone). These results suggest trigeminal nerve responsiveness is enhanced by exposure to malodorants through a theophylline-sensitive paracrine signaling pathway between olfactory and trigeminal nerves. Key Words: sensory irritation; inhalation toxicology; volatile organic compound.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The nose is a sensory organ, innervated with olfactory nerves specialized for odor perception and trigeminal nerves for irritant perception. These sensory pathways are mediated by the olfactory (cranial nerve I) and the trigeminal (cranial nerve V) nerve, respectively (Shusterman, 2002
). Most volatile organic compounds stimulate both nerves, albeit with differing concentration-response relationships (Dalton, 2003; Hummel and Livermore, 2002). Discrimination of the role of these nerves in the perception of irritation is confounded by the fact that perceived irritation may actually be reflective of odor intolerance or annoyance rather than direct trigeminal nerve stimulation (Dalton, 2003). Much evidence suggests the existence of interactions between the trigeminal and olfactory nerves. It has been shown that the presence of odors increases the sensitivity of trigeminal nerves (Ianilli et al., 2006; Kobal and Hummel, 1998; Livermore et al., 1992) and individuals with self-reported odor intolerance have augmented sensitivity to the sensory irritant capsaicin (Johansson et al., 2006). Moreover, individuals with olfactory dysfunction exhibit decreased sensitivity of the trigeminal nerve to irritants (Hummel et al., 2003) with the diminished sensitivity being specific for nasal compared with cutaneous trigeminal afferents (Frasnelli et al., 2006). These observations suggest a significant interplay exists between olfactory and trigeminal nerves. The mechanisms for such interactions are not fully understood and may reflect learned behaviors that are cued by odorants (Dalton, 2003; Hummel and Livermore, 2002), as well as local physiologic, central nervous system neural integrative and/or other cognitive/emotional pathways (Dalton, 2003; Frasnelli and Hummel, in press; Hummel and Livermore, 2002; Johansson et al., 2006).
Stimulation of nasal trigeminal nerves may result in the nasal sensory irritation response. In humans this is characterized by the sensation of tingling, pain and/or irritation and can lead to avoidance behavior (Dalton, 2003; Nielsen et al., 2007; Shusterman, 2002). Sensory irritation is the primary complaint about poor indoor air quality (Hall et al., 1993; Hodgson, 2002) and forms the basis for approximately one-half of the occupational exposure guidelines in the United States (Schapper, 1993). The sensory irritation response in animal models has been extensively reviewed (Alarie, 1973; Bos et al., 1992; Bos et al., 2002; Nielsen, 1991; Nielsen et al., 2007). In animal models, this response is characterized by a pause at the onset of expiration and decreased breathing rate (Alarie, 1973). This is termed "braking" (Vijayaraghavan et al., 1993) and results from glottis closure and increased laryngeal resistance, followed by rapid exhalation (Alarie, 1973; Vijayaraghavan et al., 1993). This response has been well characterized in mice (Alarie, 1981; Vijayaraghavan et al., 1993), thus, mice were used as the animal model for the current experiments.
Irritants are commonly used as crowd control agents and malodorants have potential for this use as well (Salem et al., 2006). Little is known about toxicological interactions that may result from combined exposures to these agents. Were a physiological interaction between odorants and irritants to occur it would be of toxicological relevance because individuals may be exposed to both agents simultaneously. In rats and mice, exposure to strong odorants results in olfactory cellular stress, as indicated by induction of heat shock protein in olfactory mucosal sustentacular cells (Carr, 2005; Carr et al., 2001; Hegg and Lucero, 2006). The response is characterized by induction of a selective subset of heat shock proteins, including HSP25, and appears to be greatest in olfactory mucosa that lines the nasal air stream lines (Carr et al., 2001; Kimbell et al., 1993; Lee et al., 2005; Moulin et al., 2002). This response is odorant specific, being induced by some odorants such as lemon, peppermint (r-carvone), and propyl mercaptan, but not others such as butyric acid and skatole (Carr et al., 2001). Interestingly, HSP25 is also induced in respiratory mucosa of the dorsal nasal cavity that is adjacent to olfactory mucosa (Hegg and Lucero, 2006). Because adenosine triphosphate (ATP) is known to be released from stressed cells, the role of paracrine purinergic signaling in stress protein induction has been examined (Hegg and Lucero, 2006). In vitro studies with olfactory mucosal slices revealed that both ATP and odorant (r-carvone) induced HSP25 and that the induction by either agent was diminished by inclusion of the ATP P2 receptor antagonist suramin. Similar results were observed in vivo, with odorant (r-carvone or hepatanal)-induced HSP25 expression being blocked by pretreatment with ATP P2 receptor antagonists (Hegg and Lucero, 2006). These studies provide evidence that odorants induce physiological changes within the nose, including the release of the purine ATP.
Purinergic signaling pathways are well characterized in a variety of organs including the bladder and the gut (Bertrand, 2003; Burnstock, 2001; Schiebert and Zsembery, 2003). ATP can stimulate sensory nerves through P2 receptors, or can be catabolized by ubiquitous extracellular ATPases and 5-nucleotidases to form adenosine, which can act through the A1, A2a, A2b, or A3 receptors (Fredholm et al., 2001). Pulmonary sensory nerves of the rodent are activated by the purines ATP and/or adenosine (Hong et al., 1998; Kollarik et al., 2003). In humans a common side effect of intravenous adenosine agonist therapy is dyspnea and chest tightness, suggesting respiratory tract sensory nerves of the human are responsive to this purinergic mediator as well (Burki et al., 2005). In the rat respiratory tract, adenosine can act as a neuromodulator, sensitizing C fibers to capsaicin (Gu et al., 2003). Recent studies in our laboratory have examined the contribution of purinergic signaling pathways to stimulation of nasal sensory trigeminal nerves by inspired irritants in the mouse. These studies revealed that nasal trigeminal nerves are stimulated by ATP and adenosine, and that trigeminal nerve stimulation by the irritants acetic acid and styrene is diminished by pretreatment with the adenosine receptor antagonist theophylline (Vaughan et al., 2006). It is not known if adenosine was acting via direct stimulation of trigeminal nerves or via modulation of C fiber responsiveness to irritants similar to that reported for the rat (Gu et al., 2003).
The observations that odorants may cause ATP release in olfactory mucosa and that purinergic pathways for stimulation of nasal trigeminal nerves exist, suggests a potential physiological mechanism for interactions between odorants and irritants. The current research examined this possibility using a mouse model. The overall objective of the current research was to determine if sulfur-based malodorants modulated the sensory irritation response to the known irritant capsaicin. Changes in breathing pattern (decreased breathing frequency and time of braking during exhalation, Vijayaraghavan et al., 1993) were used as quantitative measures of the sensory irritant response. Capsaicin was selected as the sensory irritant because it is a common crowd control agent and because it is without odor and is known to act specifically through the TRPV1 receptor which is expressed primarily on C fibers (Caterina et al., 2000; Kollarik et al., 2003; Szallasi and Blumberg, 1999). Our previous studies have shown that the sensory irritation response to capsaicin is absent in TRPV1 –/– knockout mice (Symanowicz et al., 2004). Ethyl sulfide and t-butyl sulfide were selected as malodorants because they are potent, with odor detection limits in the low ppb range (1–10 ppb, Patte et al., 1975). Specific goals were to: (1) characterize the sensory irritation response, if any, to high concentration of ethyl sulfide and t-butyl sulfide; (2) determine if a toxicological interaction occurred relative to induction of sensory irritation by coexposure to capsaicin and the malodorants; and (3) examine the potential role of adenosine signaling pathways in any observed interaction by including the adenosine receptor antagonist, theophylline (Fredholm et al., 2001; Vaughan et al., 2006), in the capsaicin aerosol formulation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals and reagents.
As in our previous studies (Morris et al., 2003
; Symanowicz et al., 2004; Vaughan et al., 2006), C57Bl/6J female mice, obtained from Jackson Laboratories (Bar Harbor, ME), were used in all studies. Animals were 5–6 weeks of age at purchase and were housed over hardwood shavings (Sani-Chip Dry, P.J. Murphy Forest Products, Montville, NJ) in animal rooms maintained at 22–25°C with a 12-h light-dark cycle (lights on at 6:30 A.M.). Food (Lab Diet, PMI Nutrition International, Brentwood, MO) and tap water were provided ad libitum. Animals were acclimated for at least 1 week prior to use and were used within 8 weeks of arrival. Thus, the female mice were 7–14 weeks of age at the time of use; at this time body weights averaged 
20 g. All protocols were approved by the University of Connecticut Institutional Animal Care and Use Committee. Ethyl sulfide (reagent grade, 98% purity), t-butyl sulfide (reagent grade, 99% purity), capsaicin and theophylline were obtained from Sigma-Aldrich (St Louis, MO). All other reagents were obtained from local suppliers and were of the highest purity available.
Experimental designs.
The first experiments were aimed at determining if ethyl sulfide and t-butyl sulfide initiated the sensory irritation response as measured by a decrease in breathing frequency (Alarie, 1973, 1981, ; Bos et al., 1992, 2002). Toward this end mice were challenged with 100 ppm of either compound. Mice exposed to clean air served as controls. The malodorant concentration was approximately 20,000-fold higher than the odor threshold and was selected on the basis of ease of generation of the atmospheres. Because little is known about the breathing pattern response to these malodorants, other breathing parameters, including end expiratory pause (a measure of pulmonary irritation) and peak expiratory flow (normalized to tidal volume, a measure of airway obstruction) were measured as well (Vijayaraghavan et al., 1993). The goal of the second series of experiments was to determine if either malodorant modulated the sensory irritation response capsaicin, which acts primarily through C fibers in the mouse (Caterina et al., 2000; Kollarik et al., 2003; Szallasi and Blumberg, 1999). The concentration response for capsaicin-induced sensory irritation was first characterized using aerosols of 0 (vehicle), 50, 100, 200, or 300 µg/ml capsaicin to aid in interpretation of the malodorant interaction study. For this interaction study mice were exposed to 100 µg/ml capsaicin aerosol with concomitant exposure to 100 ppm ethyl sulfide, 100 ppm t-butyl sulfide or clean air (control). This experiment was designed to reveal a potentiation of the response to capsaicin by the malodorants.
The aim of the third series of experiments was to determine if any potentiation occurred at lower odorant concentrations and whether adenosine signaling plays a role in the potentiation. A target malodorant concentration of 12 ppm was used to reflect a no effect level based on pilot studies. To examine the role of adenosine signaling the adenosine receptor antagonist theophylline (Fredholm et al., 2001; Vaughan et al., 2006) was included in the capsaicin (or vehicle) aerosol formulation. A theophylline concentration of 2 mg/ml was used; pilot experiments revealed this was sufficient to diminish the sensory irritation response to adenosine aerosol (data not shown). This experiment focused on ethyl sulfide, the more potent of the two malodorants. Mice were exposed to capsaicin (100 µg/ml) or vehicle aerosol, with or without theophylline, with concomitant exposure to clean air (control) or ethyl sulfide. In addition, mice were exposed to ethyl sulfide or theophylline aerosol alone to determine if either altered breathing parameters.
Exposure protocols.
Spontaneously breathing mice were exposed to malodorants and/or irritants and respiratory parameters were monitored in a Buxco double plethysmograph (Buxco, Inc., Sharon, CT) using the Buxco noninvasive mechanics software. Animals were restrained in the plethysmograph by a latex collar, but were not anesthetized. Mice were able to withdraw their heads from the head space (exposure chamber) side of the plethysmograph at any time but did not. Multiple breathing patterns were analyzed in the spontaneously breathing mice including: breathing frequency, tidal volume, the duration of any pause at the end of expiration, and peak expiratory flow. These values were obtained by the Buxco software by analysis of the transducer-derived pressure signals from the plethysmograph. Because sensory irritation in the mouse is characterized by a decreased breathing frequency due to braking at the onset of expiration (Alarie, 1973; Vijayaraghavan et al., 1993), the current study focused on these parameters. The Buxco signal was reprocessed to provide time of braking at the onset of expiration. This was measured by determining the time require to achieved 15% of the peak expiratory flow for each breath. The 15% cut-off was selected on the basis of a signal to noise evaluation of the data. Identical results were obtained if 10, 20, or 25% cut-off values were used. (It is important to note that these measures differed from those used by Vijayaraghavan et al., 1993, who used cutoffs based on 25 and 75% of tidal volume, rather than cutoffs based on peak flow as we did. Thus, although trends can be compared across studies, direct comparisons of the data, particularly in control animals are not possible.)
Mice were exposed while in the plethysmograph by drawing clean or irritant-laden air into the head space at a flow rate of 0.6 l/min. Air temperature ranged between 22 and 25°C and relative humidity averaged 50%. After a 10-min acclimatization period, a 10-min baseline exposure to clean air commenced followed by a 15-min exposure to irritant. The exposure was initiated by switching from the clean air line to the irritant-laden air line. In control animals this was mimicked by switching from one clean air line to another clean air line. Breathing parameters were collected during the baseline and exposure periods. One-minute average values were recorded and used for statistical analysis (Alarie, 1981). Plethsymograph head space air samples were drawn during exposure and analyzed for irritant concentration as described below.
Irritant generation and analysis.
Ethyl sulfide and t-butyl sulfide atmospheres were generated by flash evaporation. Airborne concentrations were measured by gas chromatography using a Varian Model 3800 gas chromatograph (Sugar Land, TX) equipped with a gas sampling valve (0.5 ml loops), DB-WAX column (Agilent Technologies, Santa Clara, CA) and flame ionization detection. Standard curves were generated by evaporation of known amounts of the liquid malodorants in a glass container and sampling of the air with the sample train used for plethysmograph sampling.
Aerosols were generated with a Lovelace nebulizer. Particle size averaged 2.2 µm mass median aerodynamic diameter (
g = 2.4, Mercer impactor). The vehicle for nebulization was 5:1.7:0.05 saline:diethyleneglycol monoethyl ether: ethanol. Solutions of capsaicin in ethanol were substituted for the ethanol to generate capsaicin aerosols. Aerosol concentrations were measured by drawing plethysmograph head space air through a 0.2-µm pore filter, elution with mobile phase and analysis of capsaicin content by HPLC (Varian Model 2450, C18 column) with ultraviolet detection (210 nm) using a mobile phase of 1:1 acetonitrile:water and/or by determination with a TSI, Inc., (Shoreview, MN) Model 4250 aerosol monitor.
Data analysis.
Data are presented as mean ± SEM unless otherwise indicated. Breathing pattern responses (breathing frequency, time of braking, etc.) were analyzed either by following the time course of the response during the baseline and exposure and/or by comparing the average time of braking throughout the entire exposure. Data were log transformed as appropriate to correct for heteroscedasticity.
A repeated measures ANOVA (with the repeated measure being time) was performed on each animal group to determine if the time of braking differed during exposure compared with baseline as revealed by a statistically significant effect of time. If appropriate this was followed by Newman-Keuls tests to determine at which times breathing frequencies were significantly different from baseline. To determine if coexposure or pharmacological manipulations altered any responses, breathing pattern data obtained during the exposure period only were compared among groups by repeated measures ANOVA with one factor being exposure group (e.g., vehicle vs. capsaicin aerosol) and the other factor being the repeated measure, time. (Each animal was exposed only once, thus these represent between animal comparisons.) To simplify analysis, the average response over the entire exposure was calculated and then compared among groups by ANOVA. (Because the exposure times were identical for all groups these average response data are equivalent to an area under the curve comparison.) If a significant difference was observed, Newman-Keuls test was then used for individual group comparisons. All statistical calculations were performed with Statistica Software (StatSoft, Tulsa, OK). A p value of 0.05 or less was required for statistical significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sensory Irritation Response to Malodorants
Shown in Figure 1 are the breathing frequency (Fig. 1A) and time of braking (Fig. 1B) values during baseline and exposure to clean air, or target concentrations of 100 ppm ethyl sulfide or 100 ppm t-butyl sulfide. Breathing parameters were also recorded during the first three minutes postexposure to assess the reversibility of any responses. Ethyl sulfide and t-butyl sulfide concentrations averaged 101 and 112 ppm, respectively. Repeated measures ANOVA revealed a significant effect of time for the ethyl sulfide and t-butyl sulfide groups, but not for the air control group. As can be seen, breathing frequency was slightly increased over baseline during minute 1 of exposure for both malodorant groups (p < 0.05, Newman-Keuls test). The same trend was observed for the air controls, but did not attain statistical significance (these data are also provided in Table 1). Interestingly, the breathing frequency was also increased over baseline levels during the first minute after the exposure (when the malodorant was removed). This achieved significance for the t-butyl sulfide group (p < 0.05 Newman-Keuls test), but not the ethyl sulfide group. At no time was the breathing frequency in any group significantly lower during exposure than during the baseline period. The minimum 1 min average breathing frequency during exposure averaged 88.6 ± 3.6%, 89.4 ± 6.3%, and 93.2 ± 4.5%, respectively, in the air, 100 ppm ethyl sulfide and 100 ppm t-butyl sulfide groups. (Baseline breathing frequency was 262 breaths per minute.)
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Shown in Figure 1B are the time of braking responses (see also Table 1). Repeated measure ANOVA revealed a significant effect of exposure time for the ethyl sulfide (p < 0.001) and t-butyl sulfide (p < 0.001), but not air (p = 0.16) groups. Newman-Keuls revealed the time of braking was significantly elevated over baseline during minutes 1 and 2 of ethyl sulfide and minute 2 of t-butyl sulfide exposure. The maximum time of braking averaged 8 ± 3, 56 ± 25, and 34 ± 23 ms in the air, ethyl sulfide, and t-butyl sulfide groups, respectively. The maximal time of braking in the two malodorant groups differed from the air group but not each other (p < 0.05, ANOVA followed by Newman-Keuls test). The mean time of braking throughout the exposure in the air, ethyl sulfide and t-butyl sulfide groups averaged 4 ± 1, 27 ± 19, and 11 ± 7 ms, respectively. These did not differ significantly from each other (p = 0.4, ANOVA), likely due to the transient nature of the response to the malodorants.
The Buxco software also provides measures of tidal volume, peak expiratory flow (normalized to tidal volume) and end expiratory pause (Table 1). Time of braking is shown in the table for comparative purposes. Tidal volumes averaged 0.2 ml and were not significantly altered during exposure to air or t-butyl sulfide, but were decreased to 0.17 ml/min during minute 1 of exposure to ethyl sulfide (p < 0.05, repeated measures ANOVA followed by Newman-Keuls test). Peak expiratory flow (normalized to tidal volume) averaged 13–14 ml/s/ml during baseline was not altered during exposure to air. During the first minute of exposure to ethyl sulfide or t-butyl sulfide the peak expiratory flow to tidal volume ratio was significantly increased to 21 and 18 ml/sec/ml, respectively (p < 0.05 compared with baseline in each case, repeated measures ANOVA followed by Newman-Keuls test). These changes may be a reflection of the more rapid breathing that occurred during minute 1 of exposure (see Fig. 1A). The duration of the end expiratory pause ranged between 7 and 10 ms and was not influenced by exposure to either malodorant.
Ethyl sulfide appeared to be the more potent of the two malodorants as evidenced by the greater time of braking and peak expiratory flow responses observed for this vapor. To better characterize the responses to this vapor, groups of mice were exposed to target concentrations of either 20 or 300 ppm ES; measured concentrations were 19 and 287 ppm, respectively. Breathing frequencies were not decreased below baseline in either group (p > 0.05, repeated measures ANOVA followed by Newman-Keuls test). The maximal time of braking response averaged 31 ± 9 and 55 ± 4 ms in the 20 and 300 ppm groups and the mean time of braking throughout the exposure averaged, 10 ± 2 and 33 ± 5 ms, respectively. Comparison of these responses to those observed at 100 ppm (see above) suggests that 100–300 ppm represents maximal response concentrations and/or that the concentration-response curve is very shallow.
Modulation of Sensory Irritation by Malodorants
To determine if the presence of malodorants modulated the response to the sensory irritant capsaicin, animals were exposed to capsaicin (or vehicle) aerosol with and without simultaneous exposure to ethyl sulfide or t-butyl sulfide. This study also included concentration-response studies on capsaicin to aid in interpretation of the response pattern. Based on pilot studies, capsaicin concentrations of 50, 100, 200, or 300 µg/ml in the nebulizer fluid were used to provide concentration-response data for moderate response levels. The aim was not to perform a full concentration-response study as would be needed to determine RD50. Measured airborne capsaicin concentrations were 0.2, 0.5, 1.0, and 1.4 µg/l, respectively, in the 50, 100, 200, and 300 µg/ml groups. Target concentrations for the malodorants were 100 ppm to match the previous experiment; measured concentrations averaged 88 ppm for ethyl sulfide and 100 ppm for t-butyl sulfide. The capsaicin concentration response and capsaicin plus malodorant studies were performed simultaneously to minimize animal use.
Concentration-response relationships for the breathing frequency and mean time of braking response to capsaicin aerosols are shown in Figures 2A and 2B, respectively. (A typical time course in the time of braking response is provided below.) Capsaicin induced a concentration-dependent increase, with times of braking being significantly greater than baseline in the 50, 100, 200, and 300 µg/ml groups. The time of braking response to 200 and 300 µg/ml capsaicin aerosol were 2.5- and 3.0-fold higher than that to 100 µg/ml. Breathing frequencies were significantly reduced by the high concentrations of capsaicin indicating capsaicin induced the full sensory irritation response. Specifically, the minimal breathing frequency in the 200 and 300 µg/ml groups averaged 60 and 61% of baseline (p < 0.05 ANOVA followed by Newman-Keuls test). The minimal breathing frequencies in the 100 µg/ml, 50 µg/ml, or vehicle groups averaged 85% or higher of baseline and did not differ significantly from baseline. Thus, in the 50 and 100 µg/ml groups a significant increase in time of braking occurred without a statistically significant decrease in breathing frequency.
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Shown in Figure 3 are the time courses of the time of braking responses in animals exposed to 100 µg/ml capsaicin aerosol with or without coexposure to 100 ppm ethyl sulfide (because the experiment was performed simultaneously with the concentration-response study, these are the same animals as 100 µg/ml group in Fig. 2). Repeated measures ANOVA revealed a significant effect of time for both groups (p < 0.001) indicating an expiratory braking response occurred. The response to capsaicin plus ethyl sulfide was significantly greater than the response to capsaicin alone (p < 0.05, repeated measures ANOVA).
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Shown in Figure 4 are the average time of braking responses throughout the exposure in animals exposed to vehicle or capsaicin aerosol, with or without ethyl sulfide or t-butyl sulfide coexposure. The response differed among groups (p < 0.0001, ANOVA). Although the time of braking responses in the vehicle aerosol plus ethyl sulfide or t-butyl sulfide groups were higher than the vehicle aerosol group the differences were not statistically significant (p > 0.10). The average values in the vehicle, vehicle-ethyl sulfide and vehicle-t-butyl sulfide groups were 18, 34, and 38 ms, respectively. The average time of braking values in the capsaicin-air, capsaicin-ethyl sulfide and capsaicin-t-butyl sulfide groups were 69, 162, and 169 ms, respectively (p < 0.05, Newman-Keuls test). The increase in response was greater than can be explained by the slight, nonstatistically significant, response to the malodorants alone.
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A similar pattern was seen with the breathing frequency responses, however the data are difficult to interpret because breathing frequencies were elevated above 100% of baseline in the vehicle aerosol groups. For example, during minute 1 of exposure, breathing frequency averaged 113, 124, and 120% of baseline in the vehicle, vehicle-ethyl sulfide and vehicle-t-butyl sulfide groups, respectively, compared with 105, 62, and 61% of baseline in the capsaicin-air, capsaicin-ethyl sulfide, and capsaicin-t-butyl sulfide groups. The values in the latter two groups differed from all others at the p < 0.05 level (Newman-Keuls test).
Low Concentration Malodorant and the Role of Adenosine Signaling
These experiments were aimed at determining if the modulation of the sensory irritation response to capsaicin could be observed at a no effect concentration of malodorant (12 ppm) and assessing the potential role of adenosine signaling pathways in this process. The more potent of the malodorants, ethyl sulfide (see Fig. 1), was used in these studies. To examine the potential contribution of adenosine signaling pathways the adenosine receptor antagonist, theophylline, was included in the aerosol formulation at a concentration of 2 mg/ml.
The first study tested whether ethyl sulfide, the vehicle aerosol or theophylline altered breathing parameters. Ethyl sulfide concentrations averaged 14 ppm. Shown in Figure 5 are the time of braking responses in animals exposed to vehicle aerosol, vehicle aerosol which included 2 mg/ml theophylline and vehicle aerosol with ethyl sulfide. A slight increase in time of braking was seen in all three groups (p < 0.05 repeated measures ANOVA). Two factor repeated measures ANOVA revealed a significant effect of time (p < 0.001), but did not detect a difference among the three groups (p = 0.9) or a statistical interaction between time and exposure group (p = 0.2). The average times of braking throughout the exposure in the vehicle, vehicle plus theophylline and vehicle with coexposure to ethyl sulfide groups were 36, 40, and 33 ms, respectively. Breathing frequencies were not statistically different from baseline levels during exposure (p = 0.4) and were similar in the three groups indicating that neither theophylline nor ethyl sulfide altered this parameter.
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Shown in Figure 6 are the average times of braking responses in the capsaicin-ethyl sulfide-theophylline experiment. Ethyl sulfide concentrations averaged 11 ppm. As can be seen, capsaicin increased the expiratory braking response over that of vehicle aerosol (Fig. 5) and the response was similar in animals exposed to capsaicin or capsaicin plus theophylline aerosol (time of braking of 146 and 145 ms, respectively). The response to capsaicin and ethyl sulfide was significantly higher than in the capsaicin-air group (281 vs. 146 ms). Theophylline significantly attenuated the response to the mixture of capsaicin and ethyl sulfide (184 ms compared with 281 ms). This attenuation was apparent in the first minute of exposure (data not shown). Shown in Table 2 are the breathing pattern data from minute 1 of exposure in these mice. Also included are the vehicle control data. Breathing frequency was significantly increased in the first minute of exposure (compared with baseline) in the vehicle aerosol groups with identical responses being observed in all three vehicle aerosol groups. In the capsaicin aerosol groups, breathing frequency was significantly decreased compared with the vehicle aerosol groups. Similar patterns were seen with the breathing frequency and time of braking responses, viz., theophylline significantly attenuated the response to the capsaicin-ethyl sulfide mixture, but was without effect on the response to capsaicin alone. Tidal volume, peak expiratory flow and time of pause were similar in all groups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sensory irritation is defined and measured in mice as a decrease in breathing frequency due to expiratory braking (Alarie, 1972, 1981
; Bos et al., 1992, 2002). Early expiratory braking is caused by closure of the glottis followed by rapid exhalation when the glottis opens (Vijayaraghavan et al., 1993). The malodorants ethyl sulfide and t-butyl sulfide elicited only small transient increases in times of braking at the onset of exposure but did not cause decreased in breathing frequency. Because breathing frequency did not change, perhaps exhalation after glottal opening was sufficiently rapid to compensate for the increases in time of braking. This is supported by the observation that peak expiratory flow rates (normalized to tidal volume) were also increased at the onset of exposure. This was observed in those groups in which the time of braking averaged 60 ms or less (ethyl sulfide, t-butyl sulfide, vehicle aerosol and 50 or 100 µg/ml capsaicin groups). Breathing frequencies were significantly reduced in those groups in which the time of braking exceeded 100 ms (200 or 300 µg/ml capsaicin or capsaicin with concomitant malodorant), suggesting that rapid exhalation after glottal opening cannot compensate for braking responses of this duration.
That the malodorants did not cause a significant decrease in breathing frequency suggests the malodorants were not eliciting the full sensory irritation response as operationally defined by Alarie (1973), despite the fact that they slightly stimulated trigeminal nerves as indicated by the transient increase in time of braking. The ethyl sulfide and t-butyl sulfide exposure concentrations (100 ppm) were approximately 20,000-fold higher than their odor threshold of roughly 5 ppb (Patte et al., 1975). Thus, even at 20,000 times the odor threshold these compounds did not diminish breathing frequencies in mice. An absence of the sensory irritation response in mice exposed to strong odorants has been reported previously (Alarie, 1966, 1973). Interestingly, a transient increase in breathing frequency occurred at the onset of exposure to either malodorant. This likely represents a generalized response to a change in the environment because: (1) a similar (although not statistically significant) pattern was seen in control animals, and (2) the same pattern of increased breathing frequency was seen at the end of the exposure when the malodorant was removed. Similar patterns have been observed previously (Alarie, 1973). Pulmonary irritation, caused by stimulation of vagal nerves, is characterized by a pause at the end of expiration (Vijayaraghavan et al., 1993). Neither malodorant (nor vehicle aerosol) induces this response.
The current results partially characterize the concentration-response relationship for ethyl sulfide; the response at 300 ppm being no higher than that 100 ppm. In the presence of vehicle aerosol, 14 ppm ethyl sulfide was without effect on time of braking (Fig. 5), suggesting this is a no effect level for the malodorant with respect to the time of braking response. The 14 ppm exposure level is roughly 2000 times the odor threshold for this malodorant (Patte et al., 1975) suggesting the apparent absence of trigeminal sensory nerve stimulation even at concentrations well above the odor threshold. This concentration-response pattern is similar to that for a variety of compounds (Dalton, 2003), specifically that the odor threshold is considerably lower than the irritancy threshold.
Capsaicin is a known irritant that acts through the TRPV1 receptor that is expressed in the mouse on sensory C fibers (Caterina et al., 2000; Jia and Lee, in press; Kollarik et al., 2003; Szallasi and Blumberg, 1999). Our current and previous studies (Symanowicz et al., 2004) confirm that capsaicin aerosols causes the sensory irritation response in C57Bl/6J mice. The breathing frequency response to capsaicin (63% of baseline for 300 µg/ml capsaicin) is similar to that observed previously at the same capsaicin concentration (61% of baseline, Symanowicz et al., 2004). Our previous studies have shown the sensory irritation response is absent in TRPV1 –/– knockout mice, confirming the role for this receptor in mediating the sensory irritation response to this irritant (Symanowicz et al., 2004). Capsaicin is highly lipophilic, thus, the inclusion of diethyleneglycolmonoethyl ether and ethanol in the nebulization fluid was necessary to keep the capsaicin in solution during the nebulization. The vehicle aerosol itself produced a slight expiratory braking response (average time of braking of 35 ms) but no expiratory pause. The lack of expiratory pause indicates pulmonary irritation did not occur (Alarie, 1973; Vijayaraghavan et al., 1993). Capsaicin aerosols produced concentration-dependent increases in the time of braking response with average time of braking exceeding 200 ms observed in the 300 µg/ml group. The concentration response appears somewhat steep, with the response at 200 and 300 µg/ml being 2.5- and 3.0-fold greater than that observed at 100 µg/ml.
Although 100 ppm ethyl sulfide or t-butyl sulfide produced a minimal increase in the time of braking (Fig. 1B), the time of braking response to coexposures of these malodorants with 100 µg/ml capsaicin was 2.4-fold greater than the response to capsaicin alone (Fig. 4), suggesting a potentiation occurred. Because the concentration-response relationship for ethyl sulfide appeared to be very shallow it seems unlikely that the increased response during the coexposures reflected an enhancement of the ethyl sulfide response per se. In contrast, the response to 200 µg/ml capsaicin aerosol was 2.5-fold greater than the response to 100 µg/ml, suggesting that an approximate doubling of the responsiveness to capsaicin by the malodorants would explain the pattern that was observed. This conclusion is further supported by the study with 11 ppm ethyl sulfide in which ethyl sulfide enhanced the responsiveness to capsaicin at a concentration that was without effect on the response to vehicle aerosol (Fig. 5). This result confirms a potentiation occurred. This conclusion is drawn irrespective of whether an additive or multiplicative model for the interaction is assumed.
Ethyl sulfide and t-butyl sulfide enhancement of the capsaicin response was fully apparent within 1 min of the onset of exposure, therefore, it is unlikely that the effect is due to cytotoxicity of these compounds. The compounds might act on mast or other cells to initiate the potentiation. Because the concentrations of the malodorants ranged between 2000–20,000 times their odor thresholds it is possible that the interaction was due to their malodorant properties. Moreover, clinical studies have suggested an interplay between odorants and irritants, with enhanced irritant sensitivity being reported in the presence of odorants (Kobal and Hummel, 1998; Livermore et al., 1992) and decreased sensitivity being reported in subjects with olfactory dysfunction (Frasnelli et al., 2006). The mechanisms for such interactions are unknown. It is thought that conditioned behavior to odorant cues may be important in the human (Dalton, 2003; Hummel and Livermore, 2002) as well as other psychological factors and/or physiological interactions (Dalton, 2003; Frasnelli and Hummel, in press). In the current study, responses in each mouse were measured during the first exposure of that animal to odorant, thus the observed responses cannot be reflective of conditioned behaviors. (Conditioned responses to odorants might occur in the mouse, but were not the subject of this experiment.) The current results suggest a physiologically based interaction may exist between olfactory and trigeminal nerves.
Inclusion of theophylline in the aerosol formulation nearly abolished the potentiation of the sensory irritation response to capsaicin by ethyl sulfide without altering the responsiveness to capsaicin alone (in the absence of ethyl sulfide, Fig. 6). Were theophylline to be acting nonspecifically on nasal physiological parameters to reduce responsiveness, for example by altering aerosol deposition within the nose, it would be expected to diminish the response any irritant. That it was without effect on the response to vehicle or capsaicin aerosol suggests it acts specifically on the pathways involved in the potentiation process. A similar observation has been made relative to the adenosine potentiation of vagal nerve responsiveness to capsaicin (Gu et al., 2003). In contrast to its lack of effect on the response to capsaicin, theophylline has been shown to diminish the sensory irritation responses to acetic acid and styrene (Vaughan et al., 2006). This indicates that the contribution of theophylline-sensitive pathways to the sensory irritant response may be irritant specific. The reasons for this specificity are not known, however, both acetic acid and styrene stimulate A
and C fibers (Morris et al., 2003; Symanowicz et al., 2004), whereas capsaicin stimulates predominantly, if not exclusively, C fibers (Caterina et al, 2000; Kollarik et al., 2003; Symanowicz et al., 2004; Szallasi and Blumberg, 1999). Theophylline is a central stimulant and systemically administered theophylline increases breathing frequency in the mouse (Vaughan et al., 2006). However, central nervous system stimulation of theophylline does not appear to be involved in its effects in the current study because breathing frequency was not altered by aerosolized theophylline (Table 2). Moreover, the attenuation of the ethyl sulfide potentiation of capsaicin responsiveness by theophylline was apparent within the first minute of exposure; it seems unlikely that sufficient theophylline could be absorbed and distributed to the central nervous system within this time frame. Thus, it appears that theophylline is acting locally within the nose and therefore, the potentiation of capsaicin responsiveness represents a local nasally mediated phenomenon.
Theophylline is a potent adenosine receptor antagonist but has many other actions as well including inhibition of phosphodiseterase (Barnes, 2003; Fredholm et al., 2001). Thus, the current experiments do not conclusively implicate adenosine signaling as a critical pathway for the potentiation of capsaicin by ethyl sulfide. However, adenosine has previously been shown to enhance vagal responsiveness to capsaicin (Gu et al., 2003) providing biological feasibility to the potential role of adenosine signaling. Moreover, it has been shown that a variety of odorants stimulate ATP release from olfactory mucosa at physiologically significant levels as indicated by the induction of sustentacular cell stress proteins in vitro and in vivo (Carr, 2005; Carr et al., 2001; Hegg and Lucero, 2006). It is known that ATP can be rapidly converted to adenosine extracellularly (Fredholm et al., 2001) That odorants stimulate ATP release (Hegg and Lucero, 2006), and that adenosine sensitizes sensory nerves to capsaicin (Gu et al., 2003), in combination with the current observation that malodorant potentiation of capsaicin responsiveness is attenuated by theophylline, all serve to add support to the conclusion that adenosine-based nasal paracrine signaling pathways may exist between the olfactory and trigeminal nerves.
The cellular mechanism(s) through which adenosine might potentiate the response to capsaicin are not known. It could be acting through multicellular pathways. For example, adenosine might act by stimulating mast cells to release mediators which sensitize sensory nerves (Meade et al., 2001; Palosa et al., 2002). Whatever pathways are involved, they likely include C fibers because capsaicin is a C fiber agonist (Caterina et al., 2000; Jia and Lee, in press; Kollarik et al., 2003; Szallasi and Blumberg, 1999). Adenosine might act by sensitizing C fibers to all irritants and/or by interacting specifically with the TRPV1 receptor to enhance its responsiveness. The latter does not seem likely however, because adenosine acts as an inhibitor of TRPV1 function in vitro (Puntambekar et al., 2004) and, in the rat, adenosine potentiates vagal responses not only to capsaicin, but also to lactic acid and lung hyperinflation (Gu et al., 2003). It is not known if this phenomenon is specific to the sulfur-based malodorants ethyl sulfide and t-butyl sulfide or if it is more widespread. It can be noted however that using stress protein induction as a biomarker, it appears that a variety of odorants induce ATP release in nasal mucosa (Carr, 2005; Carr et al., 2001). Further studies are needed to examine these possibilities.
In summary, an interaction exists between the malodorants ethyl sulfide and t-butyl sulfide and the sensory irritant capsaicin, with the malodorants greatly increasing the response to capsaicin. This interaction was attenuated by simultaneous administration of theophylline suggesting an possible role for paracrine adenosine-based signaling mechanism in this interaction. Such interactions may be particularly relevant in evaluating the safety of combined exposures to capsaicin and malodorants, as might occur in crowd control. More generally, it is known that individuals who report intolerance to odors also report sensitivity to irritants (Dalton, 2003; Johansson et al., 2006) and that strong odors can trigger asthma attacks in some individuals (Shusterman, 2002). The current results may provide a physiological basis for these observations.
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ACKNOWLEDGMENTS |
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The expert technical assistance of Barbro Simmons is gratefully acknowledged, as are the helpful suggestions of Melanie Collins, M.D.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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